SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR

MEDICAL APPLICATIONS

By

SANGYUP KIM

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2005

Copyright 2005

by

Sangyup Kim

To all who made this work possible

ACKNOWLEDGMENTS

I owe what I am today and this moment to many people without whom I could not

have been here First of all I would like to express my sincere and deepest appreciation

to my parents wife Mikyung daughters Heehyung and Erin whose love patience and

support helped me pursue my dream I gratefully acknowledge my advisor Dr H El-

Shall who gave me an opportunity to become part of an exciting and beneficial research

project and exert myself in the research work His guidance knowledge and stimulating

scientific and critical thinking have contributed to my success to a great extent and

inspired me to dedicate myself to research work I especially appreciate Dr R Partch as a

co-advisor who has helped me conduct experiments with valuable discussion for my

research from my first year I am greatly thankful to Dr C McDonald whose idea and

valuable experimental advice and discussion in the area of polymer chemistry and

membrane technology helped cultivate my interest in this area and were vital to

performing this research Without his contribution this work could not have been

achieved I also thank my doctoral committee members Dr R Singh Dr A Zaman and

Dr B Koopman for appreciated comments

Financial support and equipment use for research from the Particle Engineering

Research Center at the University of Florida were vital in continuing my study for more

than 4 years My sincere thanks are due to my former advisor Dr Kiryoung Ha in

Keimyung University who helped me open my eyes and broaden my thought and

introduced me into a limitless polymer field I would like to also thank to past and current

iv

members in Dr El-Shallrsquos research group for their assistance advice and support I

sincerely appreciate Stephen for the assistance of thesis writing

v

TABLE OF CONTENTS page

ACKNOWLEDGMENTS iv

LIST OF TABLES viii

LIST OF FIGURES x

1 INTRODUCTION 1

2 BACKGROUND AND LITERATURE SURVEY5

21 The Significance of End Stage Renal Disease (ESRD)5 22 Hemodialysis (HD) Treatment 6 23 Advances in Membrane Technology 8 24 Sorbent Technology16 25 Limitation of Current Hemodialysis Treatment 19 26 Latex Particles 20

261 The Components for Emulsion Polymerization 21 262 Particle Nucleation 21 263 Types of Processes for Emulsion Polymerization 23 264 Chemistry of Emulsion Polymerization 24 265 Seed Emulsion Polymerization25 266 Polystyrene Latex Particles 26 267 Various Applications of Latex Particles28

27 Proteins 30 271 Interaction Forces between Proteins33 272 β -Microglobulin (β M)2 2 35 273 Serum Albumin 36

28 Protein Adsorption38 281 Interaction between Protein Molecule and Latex Particle38

29 Hypothesis for Toxin Removal39 291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size 40 292 Toxin Removal by Selective Adsorption on Engineered Latex Particles 41

3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY 44

31 Materials 44 32 Latex Particle Preparation46

321 Preparation of Seed Latex Particles47

vi

322 Preparation of Seeded Latex Particles 48 323 Preparation of Core Shell Latex Particles 48 324 Purification of Synthesized Latex Particles48

33 Characterization49 331 Degree of Conversion49 332 Fourier Transform Infrared (FTIR) Spectroscopy49 333 Quasielastic Light Scattering (QELS) 50 334 Field Emission-Scanning Electron Microscopy (FE-SEM) 50 335 Zeta Potential Measurement 50 336 Protein Adsorption51 337 Blood Biocompatibility by Hemolysis Test 53

4 RESULTS AND DISCUSSION55

41 Polymerization of Latex Particles55 411 Polystyrene Seed Latex Particles55 412 The Growth of Polystyrene (PS) Seed Latex Particles61 413 Core Shell Latex Particles 71

42 Characterization of Latex Particles75 421 Fourier Transform Infrared Spectroscopy (FTIR)75 422 Zeta Potential Measurements 75

43 Protein Adsorption Study 85 431 Adsorption Isotherm87 432 Adsorbed Layer Thickness 103 433 Gibbs Free Energy of Protein Adsorption 104 434 Kinetics of Adsorption 107

44 Blood Compatibility 112

5 SUMMARY OF RESULTS CONCLUSION AND RECOMMENDATION FOR FUTURE WORK115

51 Summary of Results115 52 Conclusions121 53 Recommendation for Future Work121

LIST OF REFERENCES122

BIOGRAPHICAL SKETCH 141

vii

LIST OF TABLES

Table page 4-1 The polymerization recipe of polystyrene (PS) seed particles 57

4-2 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles less than 500nm in size 63

4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size64

4-4 The preparation recipe of PS core with various shell latex particles72

4-5 The isoelectric point (IEP) of common proteins 83

4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model 93

4-7 The equilibrium concentration values (q ) of BSA adsorption on PSPMMA particles calculated the Langmuir-Freundlich isotherm model

m 10096

4-8 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model

90 1098

4-9 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model

75 25100

4-10 The equilibrium concentration values of β M adsorption calculated the Langmuir-Freundlich isotherm model

2102

4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37 Co 104

4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37 Co 104

4-13 The values of Gibbs free energy change of BSA adsorption in phosphate buffer (PB) at 37 Co 105

4-14 The values of Gibbs free energy change of BSA adsorption in phosphate buffered saline (PBS) at 37 Co 105

viii

4-15 The values of Gibbs free energy change of β M adsorption in phosphate buffere (PB) at 37 C and pH 74

2o 106

4-16 Fitting parameters of second-order kinetic model 111

ix

LIST OF FIGURES

Figure page 1-2 Stereo drawing of the αndashcarbon backbone of β M2 2

2-1 An image of the location and cross section of a human kidney 5

2-2 A schematic draw of the hemodialysis route7

2-3 Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes 11

2-4 The chemical structure of polysulfone (PSf) 12

2-5 The chemical structure of polyamide (PA)13

2-6 Chemical structure of polyacrylonitrile (PAN) 14

2-7 Emulsion polymerization system24

2-8 L-α-amino acid 31

2-9 Ribbon diagram of human β M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ)

236

2-10 Secondary structure of human serum albumin (HSA) with sub-domains 37

2-11 The relationship between pore size and particle size in body-centered cubic and closed packed arrays41

2-12 Schematic representation of the protein adsorption on the core shell latex particles at pH 7442

3-1 Chemical structure of main chemicals45

3-2 Experimental setup for semi-continuous emulsion polymerization 46

3-3 The particle preparation scheme in various types and size ranges of latex particles 47

3-4 Schematic of the procedure for a protein adsorption test 52

3-5 Separation of RBC from whole blood by centrifuge process53

x

3-6 Schematic of the procedure for hemolysis test 54

4-1 Schematic representation of semi-continuous seed latex particles preparation and growth56

4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS (B) PS (C) PS (D) PSS259 S233 S207 S181 59

4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm 65

4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm 68

4-5 Dependence of the particle size on the surfactant to monomer ratio70

4-6 Schematic of core shell latex particle structures 71

4-7 Scanning Electron Micrograph of latex particles (A) PS (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA

100

90 10 75 25 73

4-8 FTIR spectra of polymerized latex particles (A) bare polystyrene (PS) (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA100 90 10 75 25 76

4-9 Schematic representation of ion distribution near a positively charged surface 78

4-10 Schematic representation of zeta potential measurement79

4-11 Zeta potential of PS latex particles at 25 Co 80

4-12 Zeta potential of PSPMMA latex particles at 25 C100o 81

4-13 Zeta potential of PSPMMA PAA latex particles at 25 C90 10o 82

4-14 Zeta potential of PSPMMA PAA latex particles at 25 C75 25o 82

4-15 The decay of surface potential with distance from surface in various electrolyte concentrations (1) low (2) intermediate (3) high 84

4-16 High-affinity adsorption isotherm of typical flexible polymer on solid surface 86

4-17 Overall schematic representation of the protein adsorption on the synthesized latex particles86

4-18 The dependence of UV absorbance on BSA concentration (A) 10mgml (B) 06mgml (C) 02mgml87

4-19 Standard curve of net absorbance vs BSA sample concentration 88

xi

4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37 C in phosphate buffer mediao 89

4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37 Co 90

4-22 Adsorption isotherm of BSA adsorption 37 C in phosphate buffered saline (PBS) on PS latex particles

o

91

4-23 Conformation of bovine serum albumin (BSA) on latex particles92

4-24 Adsorption isotherm of BSA 37 C in PB on PSPMMA core shell latex particles

o100

94

4-25 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA core shell latex particles

o100

95

4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA PAA core shell latex particles

o90 10

97

4-27 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA PAA core shell latex particles

o90 10

98

4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA PAA latex particleso75 25 99

4-29 Adsorption isotherm of BSA 37 C in PBS on PSPMMA PAA core shell latex particles

o75 25

99

4-30 Adsorption isotherm of β M onto PS and PSPMMA latex particles in PB at 37 C and pH 74

2 100o 101

4-31 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74

2 90 10o 101

4-32 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74

2 75 25o 102

4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37 C and pH 74

o

107

4-34 The kinetics of protein adsorption in PB on PS latex particles at 37 C and pH 74

o

109

4-35 The kinetics of protein adsorption in PB on PSPMMA latex particles at 37 C and pH 74

100o

109

4-36 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74

90 10o 110

xii

4-37 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74

75 25o 110

4-38 Image of red blood cells 113

4-39 Hemolysis caused by latex particles n=5114

xiii

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS

By

Sangyup Kim

December 2005

Chair Hassan El-Shall Major Department Materials Science and Engineering

Latex particles with well-defined colloidal and surface characteristics have received

increasing attention due to their useful applications in many areas especially as solid

phase supports in numerous biological applications such as immunoassay DNA

diagnostic cell separation and drug delivery carrier Hemodialysis membrane using

these particles would be another potential application for the advanced separation

treatment for patients with end stage renal disease (ESRD) It is desirable to remove

middle molecular weight proteins with minimal removal of other proteins such as

albumin Thus it is necessary to understand the fundamental interactions between the

particles and blood proteins to maximize the performance of these membranes This

improvement will have significant economic and health impact

The objective of this study is to synthesize polymeric latex particles of specific

functionality to achieve the desired selective separation of target proteins from the human

blood Semi-continuous seed emulsion polymerization was used to prepare monodisperse

xiv

polystyrene seed particles ranging from 126plusmn75 to 216plusmn53 nm in size which are then

enlarged by about 800nm Surfactant amount played a key role in controlling the latex

particle size Negatively charged latex particles with a different hydrophobicity were

prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid

monomer The prepared polymeric particles include bare polystyrene (PS) particles less

hydrophobic PS core and PMMA shell particles and more hydrophilic PS core and

PMMA-co-PAA shell latex particles with a 370nm mean diameter SEM light scattering

and zeta potential measurements were used to characterize particle size and surface

properties Adsorption isotherms of two proteins bovine serum albumin (BSA) and β2-

microglobulin (β2M) on latex particles were obtained as a function of pH and ionic

strength using the bicinchoninic acid (BCA) assay method The Langmuir-Freundlich

adsorption model was used to determine the adsorption amount of protein at equilibrium

The thickness of adsorbed BSA layer on latex particles was obtained in order to

investigate the adsorption orientation such as end-on or side-on mode Adsorption

kinetics experiments for both proteins and all latex particles were also performed The

adsorption kinetic constant determined from the Langmuir-Freundlich adsorption

isotherm model was used to calculate Gibbs free energy of adsorption to compare the

competitive adsorption of BSA and β2M Hemolysis tests were performed to investigate

the blood compatibility of synthesized latex particles PSPMMA90PAA10 and

PSPMMA75PAA25 core shell latex particles had desirable material properties with not

only a large amount and high rate of selective β2M adsorption over BSA but also high

blood compatibility showing less than 3 hemolysis

xv

CHAPTER 1 INTRODUCTION

End stage renal disease (ESRD) is a chronic condition in which kidney function is

impaired to the extent that the patientrsquos survival requires removal of toxins from the

blood by dialysis therapy or kidney transplantation The National Kidney Foundation

estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF] The

number of people with ESRD is rapidly increasing in the United States with

approximately 96 295 incidents and 406 081 prevalent patients including 292 215 on

dialysis and 113 866 with a functioning graft in 2001 It is projected that there will be

more than 22 million ESRD patients by 2030 [USRDS 2003] The expenditure for the

ESRD treatment program had reached $228 billion 64 of the Medicare budget in

2001 Due in part to the limited availability of kidneys for transplantation hemodialysis

is the primary clinical treatment for the patients with ESRD

The central element of a hemodialysis instrument is the semipermeable membrane

that allows for selective transport of low molecular weight biological metabolites less

than 5000 Da such as urea and creatinine as well as excess water and electrolytes [Baker

2004] from the blood One limitation of current dialysis technologies is the inability to

efficiently remove middle molecular weight toxins such as β2-Microglobulin (β2M) and

interleukin 6 (IL-6)

β2M is a causative protein of dialysis-related amyloidosis (DRA) a disease arising

in patients with chronic kidney failure as a serious complication of long-term

hemodialysis treatment [Gejyo et al 1985] β2M deposition in tissue is the primary cause

1

2

of destructive arthritis and carpal tunnel syndrome [Vincent et al 1992 Drueke 2000]

The β2M structure is shown in Figure 1-2

Figure 1-2 Stereo drawing of the αndashcarbon backbone of β2M [Becker et al 1985]

Although attempts have been made to increase the efficiency of middle molecular

weight toxin removal by changes in the membrane pore size and the use of innovative

materials to adsorb these toxins [Samtleben et al 1996 Ronco et al 2001] removal

efficiency is not as high as those achieved by a normal healthy kidney Traditional

membranes have a number of processing and performance limitations [Westhuyzen et al

1992 Leypoldt et al 1998] such as a restricted choice of surface chemistries and limited

control of porosity The development of novel engineering membrane technology is

needed to remove middle molecule toxins

Polymeric latex particles have received increasing attention in medical application

areas especially as solid phase supports in biological applications [Piskin et al 1994]

Examples of these applications include immunoassay [Chen et al 2003 Radomske-

Galant et al 2003] DNA diagnostic [Elaїssari et al 1998] cell separation drug delivery

carrier [Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] etc This is because of

3

the well-defined colloidal and surface characteristics of the particles By using a seed

emulsion polymerization method it is possible to synthesize monodisperse latex particles

with various particle size ranges and surface chemistry Functionalized core-shell latex

particles can be introduced by multi-step emulsion polymerization Core particles are

synthesized in the first stage of the polymerization and the functional monomer is added

in the second stage This is done without any emulsifier addition to prevent the

production of new homopolymer particles [Keusch et al 1973] The core-shell particles

are useful in a broad range of applications because of their improved physical and

chemical properties over their single-component counterparts [Lu et al 1996 Nelliappan

et al 1997] Through the development of a hemodialysis membrane using monodisperse

latex particles improvements in advanced separation treatment for patients with end

stage renal disease (ESRD) can be realized This requires the maximum removal of

middle molecular weight proteins with minimal removal of other beneficial proteins such

as albumin Thus an understanding of the fundamental interactions between the particles

and biopolymers is vital to maximize the performance of this membrane technology

The field of material science and biotechnology is based on fundamental chemistry

has developed over the past three decades into todayrsquos powerful discipline that enables

the development of advanced technical devices for pharmaceutical and biomedical

applications This novel and highly interdisciplinary field is closely associated with both

the physical and chemical properties of organic and inorganic particles [Niemeyer 2001]

Hemodialysis membrane using these particles would lead to improvements in the

advanced separation treatment for patients with end stage renal disease (ESRD) The

interdisciplinary nature of this approach enables a more complete understanding of the

4

phenomena of protein adsorption and the material properties necessary for selective

separation This innovative approach to membrane fabrication has the potential of making

inexpensive highly efficient membranes for both industrial and specialized separation

processes

The goal of this study is to prepare polymeric latex particles with tailored

properties to maximize separation of toxin molecules and to investigate the fundamental

interactions between the particles and molecules in the biological system in order to

optimize the performance of a membrane material for these applications

Polymeric latex particles were synthesized with specific functionality in an attempt

to achieve selective separation Seeded emulsion polymerization was used to synthesize

functionalized monodisperse latex particles in various sizes Negatively charged

hydrophobic polystyrene latex particles were synthesized by the same method Core shell

latex particles PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25 were also

synthesized to differentiate the degree of hydrophobicity of particles Scanning Electron

Micrograph (SEM) and light scattering measurements were used to characterize particle

size and shape and zeta potential measurements were conducted to measure the electrical

surface property of synthesized particles Adsorption isotherms of target proteins bovine

serum albumin (BSA) and β2M on latex particles were obtained as a function of pH

ionic strength and protein concentrations using the BCA assay method Adsorption

kinetics for both proteins on the latex particles were also measured Finally hemolysis

tests were run to determine the biocompatibility of polymer latex particles with human

blood This research will be described in more detail in chapters 3 and 4

CHAPTER 2 BACKGROUND AND LITERATURE SURVEY

21 The Significance of End Stage Renal Disease (ESRD)

The kidneys are responsible for removing excess fluid minerals and wastes from

the blood regulating electrolyte balance and blood pressure and the stimulation of red

blood cell production They also produce hormones such as erythropoietin (EPO) and

calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005 Kurbel

et al 2003] EPO acts on the bone marrow to increase the production of red blood cell in

case of bleeding or moving to high altitudes Calcitriol mainly acts on both the cells of

the intestine to promote the absorption of calcium from food and also bone to mobilize

calcium from the bone to the blood When kidneys fail harmful waste builds up blood

pressure rises and body retains excess fluid The body also does not make enough red

blood cells When this happens treatment is needed to replace the function of failed

kidneys

Figure 2-1 An image of the location and cross section of a human kidney

5

6

The National Kidney Foundation estimates that over 20 million Americans had

chronic kidney disease in 2002 [NKF] Chronic renal disease is a gradual and progressive

loss of the function unlike acute renal disease where sudden reversible failure of kidney

function occurs Chronic renal failure usually takes place over a number of years as the

internal structures of the kidney are slowly damaged In the early stages there can be no

symptoms In fact progression may be so gradual that symptoms do not occur until

kidney function is less than one-tenth that of a normal health kidney If left untreated

chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal

Disease (ESRD) ESRD is a rapidly growing heath-care problem in the United States In

2001 approximately 96295 incidents and 406081 prevalent patients were diagnosed

with ESRD including 292215 patients on dialysis and 113866 patients with a

functioning graft [USRDS 2003] The projected number of patients with ESRD is

expected to exceed 22 million patients by 2030 with much of this growth being driven by

the increasing prevalence of major contributing factors such as diabetes and high blood

pressure [USRDS 2003] A great extent of ESRD program cost and Medicare budget

have been spent in 2001 Due in part to a limited availability of kidneys for

transplantation hemodialysis (HD) is the primary method of treatment for ESRD and is

currently used for approximately 61 of US ESRD patients

22 Hemodialysis (HD) Treatment

HD removes toxins from the body by extracorporeal circulation of the blood

through a semipermeable membrane referred to as a dialyzer The toxins are removed

primarily by diffusion across the membrane to a dialysate solution which is circulated on

the opposite side of the membrane The cleaned blood is then returned to the blood

stream A surgically constructed vascular access connects the extracorporeal circuit to the

7

patientrsquos system Treatments are almost always performed three times per week in

specially equipped dialysis facilities for 3-4 hours per each treatment Figure 2-2 shows

the scheme of the hemodialysis route The critical element of a HD instrument is the

semipermeable membrane which allows for selective transport of low molecular weight

biological metabolites from blood

igure 2-2 A schematic draw of the hemodialysis route

ESRD patients who undergo dialysis therapy often experience several other

problems associated with the treatment such as anemia fatigue bone problems joint

problems itching sleep disorders and restless legs Anemia is common in patient with

kidney disease because the kidneys produce the hormone erythropoietin (EPO) which

stimulates the bone marrow to produce red blood cells Diseased kidneys often do not

produce enough EPO to stimulate the bone marrow to make a sufficient amount of red

blood cells This leads to bone disease referred to as renal osteodystrophy and causes

bones to become thin and weak or malformed and can affect both children and adults

Older patients and women who have gone through menopause are at greater risk for this

disease Uremic toxins which cannot be remove from the blood by the current dialyzer

membranes can lead to itching This problem can also be related to high levels of

F

8

parathyroid hormone (PTH) Dialysis patients can also experience day-night revers

is they have insomnia at night and sleep during the day This can be related to possible

nerve damage in the body and a chemical imbalance in the blood due to the excess toxin

Oxidative stress is a problem for patients on maintenance dialysis This problem is due to

an imbalance between pro- and antioxidant factors [Roselaar et al 1995 Cristol et al

1994] Oxidative stress affects oxidation of low-density lipoproteins which are the main

factor for atherogenesis [Huysmans et al 1998] and is also involved in the development

of malignancies and diabetes mellitus [Rice-Evans et al 1993] In order to reduce

antioxidant defense dialysis is needed to contribute to help stimulate free radical

production or eliminate antioxidants Dialysis-related amyloidosis (DRA) is also a

common and serious problem for people who have been on dialysis for more than 5

years DRA develops when proteins in the blood deposit on joints and tendons causi

pain stiffness and fluid in the joint as is the case with arthritis Normally the healthy

kidneys can filter out these proteins but dialysis filters are not as effective

23 Advances in Membrane Technology

al that

s

ng

Dialysis for blood of ESRD

Hemo filtration

s

ysis using a cellulose based membrane occurred

1913 John Abel [1990] from the Johns Hopkins Medical School described a method

purification is widely used in the treatment

dialysis (HD) techniques use a semi-permeable membrane to replace the

role of the kidney The membranes used in HD can be broadly classified into those based

on cellulose and those manufactured from synthetic copolymers These membranes come

in various shapes such as sheets tubular structures or hollow fiber arrangements The

hollow fiber type is the most popular and is incorporated into over 800 different device

in world wide [Ronco et al 2001]

The first attempt at blood dial

9

where sing

e

om

ysis was performed until the mid 1960 The basic

molec

rial in

een

benzyl)

was used for the membrane with

hepar

by the blood of a living animal may be submitted to dialysis outside the body u

a membrane based on cellulose and returned to the natural circulation without exposur

to air infection by microorganisms or any alteration that would necessarily be prejudicial

to life This same technique is still used to today however the device used has been

modified over the years as better membranes were developed and the anti-coagulant

heparin has become available

Cellulose membranes have been widely used for the treatment of renal failure fr

1928 when the first human dial

ular structure of cellulose is made of a long chain containing hydroxyl (OH)

groups The realization that such groups imparted undesirable qualities on the mate

respect to blood contact behavior was discovered in the early 1970s and since has b

the focus of development These modified cellulose membranes used the partial

substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt

to reduce their negative effect The result is a molecular mosaic of hydrophobic (

and hydrophilic (hydroxyl and cellulose) regions

Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients

with acute renal failure in 1943 Cellophane tubing

in as the anticoagulant For the next 17 years hemodialysis therapy was performed

by this method but only for the patients with acute reversible renal failure Vascular

access required repeated surgical insertions of cannulas (slender tubes) into an artery and

vein and limited the number of treatments for a patient could receive in order to

minimize the amount of vascular damage

10

Initially the need for dialysis in patients with acute renal failure was determ

mainly by the development of signs and sym

ined

ptoms of uremia After dialysis some time

might

et al

ent improvement In 1960 the development of a shunt [Quinton et al

1960]

fone (PSf) polyamide (PA) and polyacrylonitrile (PAN) by phase inversion or

precip

lidone

e

elapse before uremic manifestations returned to warrant a sequential treatment of

dialysis Many patients with acute renal failure secondary to accidental or surgical

trauma were hypercatabolic but the interdialytic interval might be prolonged because of

anorexia or use of a low-protein diet However Teschan and his coworkers [Obrien

1959] showed that patient well-being and survival were improved by what they termed

prophylactic daily hemodiaysis or administration of the treatment before the patient

again became sick with uremia Their report in 1959 was the first description of daily

hemodialysis

Development of membrane accessories such as a shunt has also been an area of

focus for treatm

a flexible polytetrafluoroethylene (PTFE or Teflonreg) tubing made many more

hemodialysis treatments possible for chronic kidney failure patients PTFE has a non-

stick surface and is relative biocompatibility leading to minimized blood clotting in the

shunt

Synthetic membranes are prepared from engineered thermoplastics such as

polysul

itation of a blended mixture resulting in the formation of asymmetric and

anisotropic structures Figure 2-3 shows a fiber type of the Polyflux S membrane

consisting of poluamide (PA) polyacrylethersulfone (PAES) and polyvinylpyrro

(PVP) with the integral three-layer structure The skin layer on the inside fiber typ

membrane contacts blood and has a very high surface porosity and a narrow pore size

11

distribution This layer constitutes the discriminating barrier deciding the permeabili

and solute retention properties of the membrane The skin layer is supported by thick

sponge-type structure larger pores providing mechanical strength and very low

hydrodynamic resistance

ty

Figure 2-3 Cross-sectional SEM image view of the Polyflux S (polyamide +

polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch et al 1998]

n

[Malchesky 2004] because of its thermal stability mechanical strength and chemical

inertn ciated

ls

m

PSf is a widely used membrane material for the hemodialysis applicatio

ess According to a report from the National Surveillance of Dialysis-Asso

Disease (NSDAD) in the US over 70 of hemodialysis membranes were PSf based

[Bowry 2002] This is most likely because PSf has many advantages over other materia

This synthetic polymer is one of few materials that can withstand sterilization by stea

ethylene oxide and γ-radiation PSf membrane can be prepared by conventional

immersion precipitation methods into many different shapes including porous hollow

12

fiber or flat sheet hemodialysis membranes The material also has a high permeab

low molecular weight proteins and solute and high endotoxin retention The chemical

structure of PSf is shown in Figure 2-4

ility to

Figure 2-4 The chemical structure of polysulfone (PSf)

There is one major disadvantage to PSf The hydrophobic nature of the PSf causes

ment alternative pathway

leadin

s not

3

et al

in

O acrylate

serious complications through the activation of the comple

g to the adsorption of serum proteins onto the membranes [Singh et al 2003]

Anticoagulants are added during dialysis therapy to avoid blood clotting but this doe

completely eliminate the problem In order to overcome this disadvantage of the PSf

membrane various studies have been performed to change the materialrsquos surface

properties These investigations include hydrophilic polymer coating [Brink et al 199

Kim et al 1988 Higuchi et al 2003] layer grafting onto PSf membrane [Wavhal

2002 Song et al 2000 Pieracci et al 2002 Mok et al1994] and chemical reaction of

hydrophilic components onto the membrane surface [Higuchi et al 1990 1991 1993

Blanco et al 2001 Nabe et al 1997 Guiver et al 1993] Hydrophilic monomers 2-

hydroxy-ethylmethacrylate (HEMA) acrylic acid (AA) and methacrylic acid (MMA)

have also been grafted onto PSf membrane to increase flux and Bovine Serum Album

(BSA) retention [Ulbricht et al 1996] Hancock et al [2000] synthesized

polysulfonepoly(ethylene oxide) (PEO) block copolymers to improve the resistance to

platelet adhesion Kim et al [2003] also studied blending a sulfonated PE

diblock copolymer into PSf in order to reduce platelet adhesion and enhance

13

biocompatibility PEO is a commonly used biomaterial due to its excellent resistance

protein adsorption and inherent biocompatibility [Harris 1992] Kim et al [20

a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf

membrane with an ultra-thin skin layer The polymer had an entrapped diblock

copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-SO

to

05] studied

ate

lic graft

l)

e of thier

aromatic

orand

3 acryl

and a hydrophobic block of octadecylacrylate (OA) Molecular dynamic (MD)

simulations were performed as a function of copolymer density to optimize interfacial

structure information McMurry [2004] developed a strategy using an amphiphi

copolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glyco

When compared to unmodified PSf these graft copolymer and resulting blend

membranes are found to hold promise for biomedical device applications

Polyamide (PA) membranes have also been used for hemodialysis becaus

mechanical strength in both wet and dry conditions Polyamide consists of

aliphatic monomers with amide bonding (-CONH-) also known as a peptide bond

The basic amide bond in polyamide is shown in Figure 2-5 R1 and R2 can be either

aromatic or aliphatic linkage group

C N

H

O

~ R R1 2~C N

H

O

~ R R1 2~

Figure 2-5 The chemical structure of polyamide (PA)

Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane

ompatible technique due to the

use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of

and concluded that PA hemofiltration was a highly bioc

14

the ac

e

e

is

n

is a semi-crystalline polymer and the mechanical properties

strong

2

CN

tivated anaphylatoxins and β2-Microglobulin (β2M) The PA based membrane

Polyfluxreg (manufactured by Gambro GmbH Germany) blended with polyamide

polyarylethersulfone and polyvinylpyrrolidone (PVP) was able to clean small molecules

such as urea dreatinine and phosphate as well as decrease β2M amount by 502

[Hoenich et al 2002] Due to the non-selectivity of the membrane removal of thes

unwanted materials also led to the undesirable loss of beneficial proteins during therapy

Meier et al [2000] evaluated different immune parameters using a modified cellulos

low-flux hemophan and synthetic high-flux PA membrane during a 1 year period in

chronic hemodialysis patients They found that the 1-year immunological evaluation of

hemodiaysis membrane biocompatibility was associated with changes in the pattern of

chronic T-cell actiovation

Polyacrylonitrile (PAN) is another commonly used membrane material because it

inherently hydrophilic and has been commercialized for ultrafiltration and microfiltratio

[Scharnagl et al 2001] PAN

ly depend on the crystalline structures The chemical structure of PAN is shown in

figure 2-6

CH CH

n2

CN

CH CH n

Chemical structure of polyacrylonitrile (PAN)

The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming

agent gives PAN membranes more flexible processing parameters and increased

e has been optimized through

copolymerization with many other vinyl monomers including glycidyl methacrylate

Figure 2-6

performance [Jung et al 2005] PAN membrane performanc

15

[Godj

acid

for

for

a non-

solub this

in

oid underneath the inner

surface of membrane decreased by increasing the amount of solvent DMSA in the

evargova et al 1999 Hicke et al 2002] N-vinylimidazole [Godjevargova et al

2000] hydroxyl ethyl methacrylate [Ray et al 1999 Bhat et al 2000] methacrylic

[Ray et al 1999] vinyl pyrrolidone [Ray et al 1999] acrylic acid [Trotta et al 2002]

acrylamide [Musale et al 1997] and vinylchloride [Broadhead et al 1998] These

monomers provide a reactive group for enzyme immobilization improved mechanical

strength solvent-resistance pervaporation permeation flux anti-fouling and bio-

compatibility Because of this PAN-based copolymer membranes have great potential

the treatment of hemodialysis in an artificial kidney This material can also be used

other applications like the treatment of wastewater the production of ultra-pure water

biocatalysis together with separation and methanol separation by pervaporation

Lin and his coworker [2004] studied the modification of PAN based dialyzer

membranes to increase the hemocompatibility by the immobilization of chitosan and

heparin conjugates on the surface of the PAN membrane When a foreign material is

exposed to blood plasma proteins are adsorbed clotting factors are activated and

le fibrin network or thrombus is formatted [Goosen et al 1980] The result of

research was that the biocompatible chitosan polymer and a blood anticoagulant hepar

prevented blood clotting They showed prolonged coagulation time reduced platelet

adsorption thrombus formation and protein adsorption

Nie et al [2004] studied PAN-based ultrafiltration hollow-fiber membranes

(UHFMs) In order to improve the membrane performance acrylonitrile (AN) was

copolymerized with other functional monomers such as maleic anhydride and α-allyl

glucoside They found that the number and size of macrov

16

intern erum

and

d a

rface The second step is the

transf after

yjak et

embrane

1978 Malchesky et al 1978] has been further developed to increase the efficiency of

al coagulant The water flux of the UHFMs also decreased while the bovine s

albumin rejection increased minutely Godjevargova et al [1992] modified the PAN

based membrane with hydroxylamine and diethylaminoethylmethacrylate to improve

membrane dialysis properties Formed functional groups like primary amine oxime

tertiary amine groups provided the membrane with more hydrophilic properties an

substantial increase in the permeability of the membranes

The wide use of filtration in practice is limited by membrane fouling Solute

molecules deposit on and in the membrane in the process of filtration causing dramatic

reduction in flux through the membrane Fouling occurs mostly in the filtration of

proteins Three kinetic steps are involved in the fouling of UF membranes according to

Nisson [1990] The first step is the transfer of solute to the su

er of solute into the membrane until it either finally adsorbs or passes through

a set of adsorption-desorption events The third step includes surface binding

accompanied by structural rearrangement in the adsorbed state [Ko et al 1993] Br

al [1998] studied the surface modification of a commercially available PAN membrane

to develop superior filtration properties with less fouling by proteins The PAN

membrane was immersed in excess NaOH solution to convert some of the surface nitrile

groups into carboxylic groups by the hydrolysis process This modified PAN m

was not so severely fouled in the Bovine Serum Albumin (BSA) filtration test The pore

size however decreased during the hydrolysis process leading to a significant reduction

in flux and made the membrane less productive in the ultrafiltration (UF) mode

24 Sorbent Technology

Over the last three decades sorbent technology [Castino et al 1976 Korshak et al

17

dialysis or replace it for the treatment of ESRD Sorbents remove solutes from solution

through specific or nonspecific adsorption depending on both the nature of the solute and

the sorbent Specific adsorption contains tailored ligands or antibodies with high

selectivity for target molecules een used in autoimmune

disord

and

e

al 1996] various resins [Ronco et al 2001]

album

e

as

Specific adsorbents have b

ers such as idiopathic thrombocytopenic purpura [Snyder et al 1992] and for the

removal of lipids in familial hyper cholesterolemia [Bosch et al 1999] Nonspecific

adsorbents such as charcoal and resins attract target molecules through various forces

including hydrophobic interactions ionic (or electrostatic) attraction hydrogen bonding

and van der Waals interactions

New dialysate with sorbents has become an accepted modification of dialysis

sorbent hemoperfusion is gaining ground as a valuable addition to dialysis especially as

new sorbents are developed [Winchester et al 2001] Hemoperfusion is defined as th

removal of toxins or metabolites from circulation by the passing of blood within a

suitable extracorpoteal circuit over semipermeable microcapsules containing adsorbents

such as activated charcoal [Samtleben et

in-conjugated agarose etc Novel adsorptive carbons with larger pore diameters

have been synthesized for potential clinical use [Mikhalovsky 1989] Newly recognized

uremic toxins [Dhondt et al 2000 Haag-Weber et al 2000] have resulted in several

investigations on alternatives to standard or high-flux hemodialysis to remove thes

molecules These methods include hemodiafilteration with [de Francisco et al 2000] or

without [Ward et al 2000 Takenaka et al 2001] dialysate regeneration using sorbents

well as hemoperfusion using such adsorbents as charcoal and resins

18

Kolarz et al [1989 1995] studied the hyper-crosslinked sorbent prepared from

styrene and divinylbenzene (DVB) for a hemoperfusion application They found that the

pore structure of a swelling sorbent was changed by additional crosslinking with α αrsquo-

dichloro-p-xylene in the presence of a tin chloride catalyst and in a di

chloroethane

soluti and

en

decyl groups that attract β2M through a hydrophobic interaction The

adsor

of

5]

d

penet

on They also realized that the hemocompatibility was useful for nemoperfusion

could be imparted to the sorbents by introducing sulfonyl groups at a concentration of

about 02mmolg

A special polymeric adsorbing material (BM-010 from Kaneka Japan) has be

investigated by another group [Furuyoshi et al 1991] for the selective removal of β2M

from the blood of dialysis partients The adsorbent consists of porous cellulose beads

modified with hexa

ption capacity of this material is 1mg of β2M per 1ml of adsorbent Using a

hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a

high-flux hemodialyzer several small clinical trials were performed During 4-5 hours

treatment about 210mg of β2M were removed thus reducing the concentration in the

blood by 60-70 of the initial level [Nakazawa et al 1993 Gejyo et al 1993 199

RenalTech developed a hemoperfusion device BetaSorbreg containing the hydrate

cross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure

designed to remove molecules between 4 and 30 kDa [Winchester et al 2002] In this

case solute molecules are separated according to their size based on their ability to

rate the porous network of the beaded sorbents The resin beads were prepared with

a blood compatible coating and confirmed to be biocompatible in vivo in animals

[Cowgill et al 2001]

19

25 Limitation of Current Hemodialysis Treatment

Hemodialysis is a widely used life-sustaining treatment for patients with ESRD

However it does not replace all of the complex functions of a normal healthy kidne

a result patients on dia

y As

lysis still suffer from a range of problems including infection

accelerated cardio ion anemia

chron

e

se middle

alysis

sessio

s

f

vascular disease high blood pressure chronic malnutrit

ic joint and back pain and a considerably shortened life span One significant

limitation of the current dialysis technology is the inability to efficiently remove larger

toxic molecules This is mainly because of the broad pore size distribution reducing th

selective removal of toxins and unsatisfied biocompatibility causing lots of

complications such as inflammation blood clotting calcification infection etc

Dialysis purifies the patients blood by efficiently removing small molecules like

salts urea and excess water However as toxic molecules increase in size their removal

rate by hemodialysis substantially declines Typically only 10 - 40 of the

molecular weight toxins (300-15000 Da) are removed from the blood during a di

n [Vanholder et al 1995] These toxins then reach an abnormally high level and

begin to damage the body One such toxin β2M causes destructive arthritis and carpal

tunnel syndrome by joining together like the links of chain to form a few very large

molecules and deposit damaging the surrounding tissues [Lonnemann et al 2002] This i

also a main cause of mortality for long-term dialysis patients Other middle molecule

toxins appear to inhibit the immune system and may play a significant role in the high

susceptibility to infections in dialysis patients Still others are believed to impair the

functioning of several other body systems such as the hematopoietic and other endocrine

systems This may contribute to accelerated cardiovascular disease the leading cause o

20

death among dialysis patients as well as clinical malnutrition which affects up to 50 of

this patient population

Over the last decade polymeric dialysis membranes have been developed to

increase the capacity for removing middle molecular weight toxins by changing the pore

size of dialyzer membranes and using new materials that adsorb these toxins for

impro se

styrene made during World War II in the United

States The Dow chemical compan anufacturer of polystyrene

includ

d of

on

rug

ntrol of

ved removal characteristics However removal efficiency is not as high as tho

achieved by a normal healthy kidney

26 Latex Particles

The first synthetic polymer synthesized using emulsion polymerization was a

rubber composed of 13-butadiene and

y has been a major m

ing latex which they used in paint formulations The theory of emulsion

polymerization in which a surfactant is used was established by Harkins [1948] and by

Smith and Ewart [1948] By 1956 the technology was complete including the metho

building larger diameter particles from smaller ones The product by this emulsi

polymerization is referred to as latex a colloidal dispersion of polymer particles in water

medium [Odian 1991] Latexes are currently undergoing extensive research and

development for a broad range of areas including adhesives inks paints coatings d

delivery systems medical assay kits gloves paper coatings floor polish films carpet

backing and foam mattresses to cosmetics The relatively well known and easy co

the emulsion process is one of main advantages for these applications Therefore

polymeric latex particle prepared by emulsion polymerization can be a candidate for the

medical applications because of the easy control of the particle size and morphology as

well as flexible surface chemistry to be required

21

261 The Components for Emulsion Polymerization

The main components for emulsion polymerization process are the monomer a

dispersing medium a surfactant and an initiator Available monomers are styrene

sing medium is usually water

which

ticles

n

ation To

monomer droplets but in micelles because the initiators are

insolu le

and

olution

butadiene methylmethacylate acryl acid etc The disper

will maintain a low solution viscosity provide good heat transfer and allow

transfer of the monomers from the monomer droplets into micelles and growing par

surrounded by surfactants respectively The surfactant (or emulsifier) has both

hydrophilic and hydrophobic segments Its main functions is to provide the nucleatio

sites for particles and aid in the colloidal stability of the growing particles Initiators are

water-soluble inorganic salts which dissociate into radicals to initiate polymeriz

control the molecular weight a chain transfer agent such as mercaptan may be present

262 Particle Nucleation

Free radicals are produced by dissociation of initiators at the rate on the order of

1013 radicals per milliliter per second in the water phase The location of the

polymerization is not in the

ble in the organic monomer droplets Such initiators are referred to as oil-insolub

initiators This is one of the big differences between emulsion polymerization

suspension polymerization where initiators are oil-soluble and the reaction occurs in the

monomer droplets Because the monomer droplets have a much smaller total surface area

they do not compete effectively with micelles to capture the radicals produced in s

It is in the micelles that the oil soluble monomer and water soluble initiator meet and is

favored as the reaction site because of the high monomer concentration compared to that

in the monomer droplets As polymerization proceeds the micelles grow by the addition

of monomer from the aqueous solution whose concentration is refilled by dissolution of

22

monomer from the monomer droplets There are three types of particles in the emulsion

system monomer droplets inactive micelles in which polymerization is not occurring

and active micelles in which polymerization is occurring referred to as growing polymer

particles

The mechanism of particle nucleation occurs by two simultaneous processes

micellar nucleation and homogeneous nucleation Micellar nucleation is the entry of

radicals ei

ther primary or oligomeric radicals formed by solution polymerization from

the aq ed

the

r

s

n

is coagulation with other particles rather than

polymerization of monomer The driving force for coagulation of precursor particles

ueous phase into the micelles In homogeneous nucleation solution-polymeriz

oligomeric radicals are becoming insoluble and precipitating onto themselves or onto

oligomers whose propagation has ended [Fitch et al 1969] The relative levels of micella

and homogeneous nucleation are variable with the water solubility of the monomer and

the surfactant concentration Homogeneous nucleation is favored for monomers with

higher water solubility and low surfactant concentration and micellar nucleation is

favored for monomers with low water solubility and high surfactant concentration It has

also been shown that homogeneous nucleation occurs in systems where the surfactant

concentration is below CMC [Roe 1968] A highly water insoluble monomer such a

styrene [Hansen et al 1979 Ugelstad et al 1979] has probably created by micellar

nucleation while a water soluble monomer such as vinyl acetate [Zollars 1979] has bee

formed by homogeneous nucleation

A third latex formation reaction mechanism has been proposed referred to as

coagulative nucleation In this reaction the major growth process for the first-formed

polymer particles (precursor particles)

23

sever les

yer

es

r a

l

the beginning of the polymerization As soon as the initiator is

gins with the formation and

growt

and

ved

added at one time during the later stages of the polymerization prior to complete

al nanometers in size is their relative instability compared to larger sized partic

The small size of a precursor particle with its high curvature of the electrical double la

permits the low surface charge density and high colloidal instability Once the particl

become large enough in size maintaining the high colloidal stability there is no longe

driving force for coagulation and further growth of particles takes place only by the

polymerization process

263 Types of Processes for Emulsion Polymerization

There are three types of production processes used in emulsion polymerization

batch semi-continuous (or semi-batch) and continuous In the batch type process al

components are added at

added and the temperature is increased polymerization be

h of latex particles at the same time There is no further process control possible

once the polymerization is started In the semi-continuous emulsion polymerization

process one or more components can be added continuously Various profiles of particle

nucleation and growth can be generated from different orders of component addition

during polymerization There are advantages to this process such as control of the

polymerization rate the particle number colloidal stability copolymer composition

particle morphology In the continuous process the emulsion polymerization components

are fed continuously into the reaction vessel while the product is simultaneously remo

at the same rate High production rate steady heat removal and uniform quality of

latexes are advantages of the continuous polymerization processes

Other available methods include the intermittent addition and shot addition of one

or more of the components In the shot addition process the additional components are

24

conversion of the main monomer This method has been used successfully to develo

water-soluble functional monomers such as sodium styrene sulfonat

p

e [Kim et al 1989]

264

lsion

Chemistry of Emulsion Polymerization

Emulsion polymerization is one type of free radical polymerization and can be

divided into three distinct stages initiation propagation and termination The emu

polymerization system is shown in Figure 2-7

Aqueous phaseAqueous phase

Micelle withmonomer

I Rbull

Emulsifier

Polymer particle swollenWith monomer

Monomerdroplet

I Rbull

Micelle withmonomer

I Rbull

Emulsifier

Polymer particle swollenWith monomer

MonomerdropletMonomerdroplet

I Rbull

Figure 2-7 Emulsion polymerization system [Radicals (Rmiddot) are created from initiators

(I) Monomer is transferred from larger monomer droplet into micelles by emulsifier Initiated polymer particle by radicals is keep growing until monomers are all consumed The reaction is performed in aqueous media]

In the initiation stage free radicals are created from an initiator by heat or an

ultraviolet radiation source The initiator with either peroxides groups (-O-O-) such as

odium persulfate or azo groups (-N=N-) such as azobisisobutyronitrile is commonly

used for em t

with the m er

chain ere

s

ulsion polymerization The primary free radicals created from initiator reac

onomer for initiation of polymerization In the propagation stage the polym

grows by monomer addition to the active center a free radical reactive site Th

are two possible modes of propagations head-to-head addition and head-to-tail addition

25

The head-to-tail mode is the predominant configuration of the polymer chain a result o

steric hindrance of the substitute bulky group In the termination stage polymer chain

growth is terminated by either coupling of two growing chains forming one polymer

molecule or transferring a hydrogen atom (dispropotionation) from one growing chain to

another forming two polymer molecules one having a saturated end group and the other

with an unsaturated end-group

265 Seed Emulsion Polymerization

Polymer latex particles have received increasing interest because of the versatili

of the many applications heterophase polymerization processes like emulsion dispersion

micro-emulsion seeded emulsion precipitation etc Especially to prepare well-defined

microspheres having monodispe

f

ty

rse and various particle sizes as well as surface group

ed particles prepared by emulsion polymerization

and e

s

hological design In

seede to control

n

functionalilties it is necessary to use se

nlarge them to a desired size in the further stages of reactions

Polymeric particles are required to have a uniform particle size in many

applications such as chromatography where they are used as a packing material

Morphological control of latex particle is also important for many practical application

[Schmidt 1972] Seed emulsion polymerization (or two-stage emulsion polymerization) is

a useful method to achieve both monodisperse particle size and morp

d emulsion polymerization [Gilbert 1995] preformed lsquoseedrsquo latex is used

the number of particles present in the final latex The advantage of seeded emulsio

polymerization is that the poorly reproducible process of particle nucleation can be

bypassed so that the number concentration of particles is constant and known Various

mechanisms have been proposed for the growth of latex particles [Ugelstad et al 1980

Okubo et al 1992] using this polymerization technique The initial seed particle

26

preparation step is well known and relatively easy to perform because at the relative

small particle sizes (02 to 05 micron) the particle growth process can be readily

controlled by the use of an emulsifier Enough emulsifier is used to prevent coagulation

but not enough to cause the formation of new particles As the particles are grown to

larger sizes in successive seeding steps it becomes increasingly difficult to mainta

stable uncoagulated emulsion without forming new particles and thereby destroying

monodisperisty of the latex Recently there have been several reports concerning t

reaction kinetics of seed emulsion polymerization and the development of latex

morphologies over the course of the reaction [Chern et al 1990 Delacal et al 1990 L

et al 1995 Lee 2000 2002]

266 Polystyrene Latex Particles

A number of papers have described the synthesis of the polystyrene latex particl

bearing various functional surface groups such as carboxyl [Lee et al 2000 Tun

2002 Reb et al 2000] hydroxyl [Tamai et al 1989] marcapto [Nilson 1989] epoxy

[Shimizu et al 2000 Luo et al

ly

in a

he

ee

es

cel et al

2004] acetal [Izquierdo et al 2004 Santos et al 1997]

ometyl [Izquierdo et al 2004 Park et al 2001

Sarob

h the

d

thymine [Dahman et al 2003] chlor

e et al 1998] amine [Counsin et al 1994 Ganachaud et al 1997 Ganachaud et al

1995 Miraballes-Martinez et al 2000 2001 Anna et al 2005] ester [Nagai et al 1999]

etc In order to produce these functionalized particles different methods for particle

preparation must be used The polymer emulsion with core-shell morphology of latex

particles is one of them This is a multistep emulsion polymerization process in whic

polystyrene ldquocorerdquo particle is synthesized in the first stage and the functional monomer is

added in the second stage of the polymerization without any emulsifier postfeeding to

prevent the production of new homopolymer particles thus forming the functionalize

27

polymer ldquoshellrdquo on the ldquocorerdquo particle [Keusch et al 1973] There are requirements to

limit secondary nucleation and encourage core-shell formation in seeded emulsion

polymerization including the addition of smaller seed particles at high solid content to

increase particle surface area low surfactant concentration to prevent formation of

micelles and the semi-continuous addition of monomer to create a starved-feed conditio

and keep the monomer concentration low There are some advantages [Hergeth et al

1989] of dispersions with polymeric core-shell particles First it is possible to modi

interfacial properties of polymer particles in the aqueous phase by the addition of only

very small amounts of a modifying agent during the last period of the reaction Thus

these core-shell particles are useful in a broad range of applications since they always

exhibit improved physical and chemical properties over their single-component

counterparts [Lu et al 1996 Nelliappan et al 1997] In this way the improvement of

surface properties of such dispersions is straightforward and inexpensive The other is

that polymers with a core-shell structure are perfect model systems for investigating th

material properties of polymer blends and composites because of their regular

distribution of one polymer inside a matrix polymer and because of the simple sp

geometry of the system

Their properties usually depend on the structures of latex particles Chen and his

coworkers [Chen et al 1991 1992 1993] reported the morphological development of

core shell latex particles of polystyrenepoly(methyl methacrylate) during

polymerization Before the research by Chen and his coworker Min et al [Min et al

1983] reported the morph

n

fy the

e

herical

ological development of core shell latex of polystyrene

(PS)polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the

28

additi

of

ity of graft

ing reagents Thus protein adsorption on polymeric solid surfaces has

st are microspheres defined as ldquofine

polym can be

n

r was

containing antigen urine serum etc The latex will become agglutinated and visibly

on method of PS They found that the percentage of grafting PS to the PBA was

greatest for the batch reaction and the PBA-PS core-shell particles with a high degree

grafting remained spherical upon aging test because of the emulsifying abil

copolymer

267 Various Applications of Latex Particles

Latex particles are applicable to a wide range of areas such as biomedical

applications [Piskin et al 1994] especially as the solid phase such as in immunoassays

[Chern et al 2003 Radomske-Galant et al 2003] DNA diagnostic drug delivery carriers

[Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] blood cell separations and

column pack

become a center of attention Of particular intere

er particles having diameters in the range of 01 to several micronsrdquo which

used as functional tools by themselves or by coupling with biocompounds Singer and

Plotz [1956] firstly studied microsphere or latex agglutination test (LATrsquos) by using

monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support o

which the biomolecules were going to adsorb The biomolecule adsorption howeve

limited by possible desorption of the adsorbed species or loss of specific activity of the

complex formed Since this work was published the latex particle applications for

immunoassay have been rapidly and widely studied and developed

Latex agglutination test or latex immunoassays start with tiny spherical latex

particles with a uniform diameter and similar surface properties The particles are coated

with antibodies (sensitized) through the hydrophobic interaction of portions of the protein

with the PS surface of the particles If sensitized particles are mixed with a sample

29

agglomerate Latex tests are inexpensive as compared with the other techniques [Ba

1988]

ngs

sed

tion Rheumatoid factor (RF) in different age subpopulations has also been

evalu

studied a

latex

Unipath [Percival 1996] has manufactured a range of immunoassay materials ba

on a chromatographic principle and deeply colored latex particles for use in the home and

clinical environments The latex particles which are already sensitized with a

monoclonal antibody can detect any antigens that bound to the surface of the latex

particles Some detectable pollutants include estrogen mimics which induce

abnormalities in the reproductive system of male fishes and lead to a total or partial male

feminiza

ated according to a patientrsquos clinical status by using a rapid slide latex agglutination

test for qualitative and semiquantitative measurement in human serum along with latex

immunoassay method [Onen et al 1998] Magalhaes and his coworkers [2004]

diagnostic method of contamination of male fishes by estrogen mimics using the

production of vitellogenin (VTG) as a biomarker This was based on a reverse

agglutination test developed with monoclonal antibodies specific to this biomarker

Premstaller and his coworkers [Premstaller et al 2000 2001] have prepared a porous

poly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient

chromatographic separation of biomelecules such as proteins and nucleic acids They

used a porogen a mixture of tetrahydrofuran and decanol to fabricate a micropellicular

PS-DVB backbone Legido-Quigley and his colleagues [2004] have developed the

monolith column to obtain further chromatographic functionality to the column by

introducing chloromethylstyrene in place of styrene into the polymer mixture

30

Core shell type monodisperse polymer colloids have been synthesized by Sarobe

and Forcada [1998] with chloromethyl functionality in order to improve the biomolecu

adsorption through a two-step emulsion polymerization process They investigated the

functonalized particles by optimizing the experimental parameters of the functional

monomer including reaction temperature the amount and type of redox initiator sys

used the type of addition of the initiator system and the use of washing They c

le

tem

oncluded

that th

sis

serve as separation

chann

of

usually

along the polypeptide chain which contain positive or negative charges [Norde 1998]

e relation between the amount of iron sulfate and the persulfatebisulfite system

added should be controlled to obtain monodisperse particles and prevent the premature

coagulation of the polymer particles during the polymerization

A semi-continuous emulsion polymerization technique for latex particle synthe

was performed by McDonald and other researchers [Ramakrishnan et al 2004 Steve et

al 1999] They introduced a variety of particles sizes compositions morphologies and

surface modifications to fabricate latex composite membranes (LCMs) Arraying and

stabilizing latex particles on the surface of a microporous substrate form narrowly

distributed interstitial pores formed between the particles which

els They investigated the membrane performance using gas fluxes water

permeability and the retention characterization of dextran molecules From these tests

they concluded that the narrow discriminating layer made of the latex particles leads to a

highly efficient composite membrane

27 Proteins

Proteins are natural polyamides comprised of about 20 different α-amino acids

varying hydrophobicity [Norde 1998] Proteins are more or less amphiphilic and

highly surface active because of the number of amino acid residues in the side groups

31

Proteins are polymers of L-α-amino acids [Solomon and Fryhle 2000] The α refers to a

carbon with a primary amine a carboxylic acid a hydrogen and a variable side-chain

group designated as R Carbon atoms rent groups are asymmetric and can

exhib l

s

e

on

with four diffe

it two different spatial arrangements (L and D configurations) due to the tetrahedra

nature of the bonds The L refers to one of these two possible configurations Amino

acids of the D-configuration are not found in natural proteins and do not participate in

biological reactions Figure 2-8 shows the chiral carbon in 3-D as the L isomer Protein

consist of twenty different amino acids differentiated by their side-chain groups [Norde

1998] The side-chain groups have different chemical properties such as polarity charg

and size and influence the chemical properties of proteins as well as determine the

overall structure of the protein For instance the polar amino acids tend to be on the

outside of the protein when they interact with water and the nonpolar amino acids are

the inside forming a hydrophobic core

Figure 2-8 L-α-amino acid

The covalent linkage between two amino acids is known as a peptide bond A

peptide bond is formed when the amino group of one amino acid reacts with the carboxyl

group of another amino acid to form an amide bond through the elimination of water

This arrangement gives the protein chain a polarity such that one end will have a free

amino group called the N-terminus and the other end will have a free carboxyl group

called the C-terminus [Solomon and Fryhle 2000] Peptide bonds tend to be planar and

32

give the polypeptide backbone rigidity Rotation can still occur around both of the α-

carbon bonds resulting in a polypeptide backbone with different potential conformations

relative to the positions of the R groups Although many conformations are theoretically

possible interactions between the R-groups will limit the number of potential

nd to form a single functional conformation In other words

the conformation or shape of the protein is

α-helix

can interact with other

secondary structures within the sam

conformations and proteins te

due to the interactions of the chain side

groups with one another and with the polypeptide backbone The interactions can be

between amino acids that are close together as in a poly-peptide between groups that are

further apart as in amino acids or even on between groups on different polypeptides all

together These different types of interactions are often discussed in terms of primary

secondary tertiary and quaternary protein structure

The primary amino acid sequence and positions of disulfide bonds strongly

influence the overall structure of protein [Norde 1986] For example certain side-chains

will promote hydrogen-bonding between neighboring amino acids of the polypeptide

backbone resulting in secondary structures such as β-sheets or α-helices In the

conformation the peptide backbone takes on a spiral staircase shape that is stabilized by

H-bonds between carbonyl and amide groups of every fourth amino acid residue This

restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure

Certain amino acids promote the formation of either α-helices or β-sheets due to the

nature of the side-chain groups Some side chain groups may prevent the formation of

secondary structures and result in a more flexible polypeptide backbone which is often

called the random coil conformation These secondary structures

e polypeptide to form motifs or domains (ie a

33

tertiary structure) A motif is a common combination of secondary structures and a

domain is a portion of a protein that folds independently Many proteins are composed o

multiple subunits and therefore exhibit quaternary structures

271 Interaction Forces between Proteins

Proteins in aqueous solution acquire compact ordered conformations In such a

compact conformation the movement along the polypeptide chain is severely restrict

implying a low conformational entropy The compact structure is possible only if

interactions within the protein molecule and interactions between the protein molecule

and its environment are sufficiently favorable to compensate for the low conformational

entropy [Malmsten 1998] Protein adsorption study is often focused on structural

rearrangements in the protein molecules because of its significance to the biological

functioning of the molecules and the important role such rearrangements play in the

mechanism of the adsorption process Knowledge of the majo

f

ed

r interaction forces that act

structures helps to understand the behavior

of pro

arge

a

ded

between protein chains and control the protein

teins at interface These forces include Coulomb interaction hydrogen bonds

hydrophobic interaction and van der Waals interactions

Coulombic interaction Most of the amino acid residues carrying electric ch

are located at the aqueous boundary of the protein molecule An the isoelectric point

(IEP) of the protein where the positive and negative charges are more or less evenly

distributed over the protein molecule intramolecular electrostatic attraction makes

compact structure favorable to proteins Deviation to either more positive or more

negative charge however leads to intramolecular repulsion and encourages an expan

structure Tanford [1967] calculated the electrostatic Gibbs energy for both a compact

impenetrable spherical molecule (protein) and a loose solvent-permeated spherical

34

molecule (protein) over which the charge is spread out From the results he found th

the repulsion force was reduced at higher ionic strength d

at

ue to the screening action of ion

ble

e

the

d the

tween

dehyd

e

ent

oles

tein

Hydrogen bond Most hydrogen bonds in proteins form between amide and

carbonyl groups of the polypeptide backbone [Malmsten 1998] The number of availa

hydrogen bonds involving peptide units is therefore far greater than that involving sid

chains Because α-helices and β-sheets are aligned more or less parallel to each other

interchain hydrogen bonds enforce each other Kresheck and Klotz [1969] examine

role of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between

peptide units do not stabilize a compact structure of protein However because the

peptide chain is shielded from water due to other interactions hydrogen bonding be

peptide groups do stabilize α-helical and β-sheet structures

Hydrophobic interaction Hydrophobic interaction refers to the spontaneous

ration and subsequent aggregation of non-polar components in an aqueous

environment In aqueous solutions of proteins the various non-polar amino acid residues

will be found in the interior of the molecule thus shielded from water The

intermolecular hydrophobic interaction for the stability of a compact protein structure

was first recognized by Kauzmann [1959] If all the hydrophobic residues are buried in

the interior and all the hydrophilic residues are at the outermost border of the molecul

intramolecular hydrophobic interaction would cause a compact protein structure

However geometrical and other types of interactions generally cause a fraction of the

hydrophobic residues to be exposed to the aqueous environm

Van der Waals interaction The mutual interaction between ionic groups dip

and induced dipoles in a protein molecule cannot be established as long as the pro

35

structure is not known in great detail Moreover the surrounding aqueous medium also

contains dipoles and ions that compete for participation in the interactions w

the protein molecule Dispersion interactions favor a compact structure However

because the Hamaker constant for proteins is only a little larger than that of water the

resulting effect is relatively small [Nir 1977]

272 β

ith groups of

rgiles et al 1996

Floeg

) is degraded [Floege 2001] Renal

β2M from the serum resulting in an increase in β2M

conce well

the

)

a

al

2-Microglobulin (β2M)

The protein β2M is of particular interest because it is involved in the human

disorder dialysis-related amyloidosis (DRA) [Geiyo et al 1985 A

e 2001] DRA is a complication in end stage renal failure patients who have been on

dialysis for more than 5 years [Bardin et al 1986 Drueke 2000] DRA develops when

proteins in the blood deposit on joints and tendons causing pain stiffness and fluid in

the joints as is the case with arthritis In vivo β2M is present as the non-polymorphic

light chain of the class I major histocompatibility complex (MHC-I) As part of its

normal catabolic cycle β2M dissociates from the MHC-I complex and is transported in

the serum to the kidney where the majority (95

failure disrupts the clearance of

ntration by up to 60-fold [Floege 2001] By a mechanism that is currently not

understood β2M then self-associates into amyloid fibrils and typically accumulates in

musculoskeletal system [Homma et al 1989] Analysis of ex vivo material has shown

that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRA

is present as of full-length wild-type β2M although significant amounts (~20-30) of

truncated or modified forms of the protein are also present [Floege 2001 Bellotti et al

1998] Figure 2-9 shows the ribbon diagram of human β2M Native β2M consists of

single chain of 100 amino acid residues and has a seven stranded β-sandwich fold typic

36

of the immunoglobulin superfamily [Saper et al 1991 Trinh et al 2002] β2M was fi

isolated from human urine and characterized by Berggard et al [1980] in1968 The

normal serum concentration of β

rst

re

f

e

2M is 10 to 25 mgL It is a small globular protein with

a molecular weight of 118 kDa a Strokes radius of 16Aring and a negative charge under

physiological conditions (isoelectric point IEP = 57) β2M contains two β-sheets that a

held together by a single disulphide bridge between the cysteines in positions 25 and 81

[Berggard et al 1980 Parker et al 1982 Cunningham et al 1973] β2M cannot be

removed completely by current dialysis techniques but through a better understanding o

the structure and interaction forces that lead to this structure it will be possible to mor

efficiently remove this problematic protein

Figure 2-9 Ribbon diagram of human β M taken from the crystal structure of the protein

bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khal 2000]

273 Serum Albumin

Serum albumin is the most abundant protein found in plasma and is typically

present in the blood at a concentration of 35~

2an et

50gL According to extensive studies about

its physiological and pharmacological properties albumin has a high affinity to a very

wide range of materials such as electrolytes (Cu+2 Zn+2) fatty acids amino acids

metabolites and many drug compounds [Fehske et al 1981 Kraghhansen 1981 Putnam

37

1984 Peters 1985] The most important physiological role of the protein is therefore to

bring such solutes in the bloodstream to their target organs as well as to maintain the pH

and osmotic pressure of the plasma Bovine serum albumin (BSA) is an ellipsoidal

protein with the dimensions of 140 X 40 X 40Aring [Peter 1985] The primary structure is a

single helical polypeptide of 66 kDa (IEP = 47) with 585 residues containing 17 pairs of

as a soft an

rfaces [Kondo et al 1991 Norde et al 1992 Soderquist et al

1980 ikely

is

disulfide bridges and one free cysteine [Dugaiczyk et al 1982] BSA has been classified

d flexible protein because it has a great tendency to change its conformation

on adsorption to solid su

Carter and Ho 1994] and consists of three homologous domains (I-III) most l

derived through gene multiplication [Brown 1976] Each domain is composed of A and B

sub-domains [He et al 1992] The secondary structure of human serum albumin (HSA)

shown in Figure 2-10

Figure 2-10 Secondary structure of human serum albumin (HSA) with sub-domains

[Zunszain et al 2003]

HSA has the same structure domains with the serum albumin from other species

such as BSA [Brown 1976] Although all three domains of the albumin molecule have

similar three-dimensional structures their assembly is highly asymmetric [Sugio et al

38

1999] Domains I and II are almost perpendicular to each other to form a T-shaped

assembly in which the tail of subdomain IIA is attached to the interface region between

sub-domains IA and IB by hydrophobic interactions and hydrogen bonds In contrast

domain III protrudes from sub-domain IIB at a 45ordm angle to form the Y-shaped assembly

for domains II and III Domain III interacts only with sub-domain IIB These features

make the albumin molecule heart-shaped

28 Protein Adsorption

Protein adsorption studies date back to the 1930rsquos At the beginning these studies

mainly focused on the determination of the molecular weight electrophoretic and

ral

rearrangem

adsorption and biocompatibility of the sorbent materials were investigated [Norde 1986]

There are interaction forces at the interfaces between protein molecules and latex

particles These forces are mainly divided into the following groups hydrophobic

interaction ionic interaction hydrogen bonding and van der Waals interaction [Andrade

1985]

Hydrophobic interaction It is known that hydrophobic interaction has a major

role in protein adsorption phenomena The adsorption of proteins on the low charged

latex particles occurs by this interaction force Generally monomers such as styrene offer

hydrophobic surfaces th

by this interaction force is maximum at the isoelectric point (IEP) of the protein and the

strength [Suzawa et al 1980 1982 Shirahama et al 1989 Kondo et al 1992] By the

chromatographic applications Later the adsorption mechanism especially the structu

ents was studied Recently the studies of the relation between protein

281 Interaction between Protein Molecule and Latex Particle

at protein molecules adsorb to The amount of adsorbed protein

pH at maximum adsorption shifts to a more acidic region with an increase in ionic

39

reports [Suzawa et al 1980 1982 Lee et al 1988] protein adsorption was greater

hydrophobic surface than on a hydrophilic one implying that hydrophobic interaction is

on a

s

ydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA)

Ionic

ely

t

ces

n force is operative over small

distan e

ble

n

nt dialysis membrane therapy for end stage renal disease (ESRD)

one of most dominant forces in protein adsorption

Ionic interaction Negatively charged latex particles have ionic functional

groups on their surfaces such as salts of sulfonic and carboxylic acid Sulfate group

originate from an initiator such as sodium persulfate and carboxylic groups originate

from a h

bonds are formed between the negative charges of these latex particles and positive

surface charges of protein molecules The conventional low-charged latex particles rar

form these ionic bonds

Hydrogen bonding Hydrogen bond is a strong secondary interatomic bond tha

exists between a bound hydrogen atom (its unscreened proton) and the electrons of

adjacent atoms [Callister 1999] Protein can be adsorbed on hydrophilic polar surfa

through hydrogen bonding Hydrogen bonds are frequently formed between hydroxyl-

carbonyl or amide-hydroxyl Hydroxyl-hydroxyl or amide-hydroxyl bonds are also

formed in protein adsorption

Van der Waals interaction This interactio

ces only when water has been excluded and the two non-polar groups come clos

to each other Lewinrsquos calculation showed that the van der Waals interaction is negligi

compared with the forces involved in the entropy increases ie hydrophobic interactio

[Lewin 1974]

29 Hypothesis for Toxin Removal

As mentioned earlier insufficient removal of middle molecular range toxins is a

major drawback of curre

40

patien

is

y

ese

haracteristics over the last decade However removal

effici

ing a novel membrane design composed of an assembly of engineered

polymeric latex particles s ions Important factors

will i

e

ith a

is established it is

expec

e

ts This may cause destructive arthritis and carpal tunnel syndrome inhibit the

immune system and accelerate cardiovascular disease leading to death among dialys

patients In order to overcome these complications artificial dialysis membranes have

been developed to increase the capacity for removing middle molecular weight toxins b

changing the pore size of dialysis membranes and using new materials that adsorb th

toxins for improved removal c

ency is not as high as those achieved by a normal healthy kidney

With the knowledge obtained from all above literatures the following hypothesis

has been established for the development of a membrane for the successful removal of

the target protein β2-Microglobulin (β2M) without the removal of serum albumin This

will be done us

ynthesized to predetermined specificat

nclude pore size and surface chemistry

291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size

The packing of monodispersed spherical particles can lead to the formation of

porous layers suitable for use as filters and membranes [Hsieh et al 1991] The pore siz

is defined as the largest spherical particles that can pass through the interstitial spaces

It is well known that monodispersed spherical particles can be obtained from the

seed emulsion polymerization method Many commercially available latex particles are

synthesized by this technique and are inexpensive When the defect free membrane w

monodisperse pore size distribution based on a particular particle array

ted to outperform the traditional hemodialysis membrane limitation which is a

broad range of pore size distribution When spherical particles pack in regular crystallin

arrays a number of packing geometries such as hexagonal closest packing cubic closest

41

packing and body-centered cubic packing are possible The pore size of the array

depends on these packing geometries as well as particle size Theoretical pore size can be

calculated from the simple geometry of the arr

ay Figure 2-11 shows the relationship

between particle size and pore size

0001

001

01

001 01 1 10

Por

e

1

10

microm

) S

ize

(

Particle Diameter (microm)

Body-Centered Cubic ArrayClosest Packed Array

0001

Siz

e (

Particle Diameter (microm)

001

01

001 01 1 10

Por

e

1

10

microm

)

Body-Centered Cubic ArrayClosest Packed ArrayBody-Centered Cubic ArrayClosest Packed Array

and closed packed arrays [Steve et al 1999]

McDonald and his coworkers [Ramakrishnan et al 2004 Steve et al 1999] have

described the fabrication of latex composite membranes and their performance in

previous papers It is from this prior research and knowledge gained from conversations

with Dr McDonald that this current research has been initiated [McDonald 2003] The

focus of this thesis is the extension of McDonaldrsquos research in the area of composite

membranes to medical application such as dialysis membranes

292 Toxin Removal by Selective Adsorption on Engineered Latex Particles

The flexibility in latex surface properties is a significant advantage to this

materialrsquos ability to separate proteins since the selectivity is strongly dependent on the

charge interactions of both the protein and the latex particles under the conditions of

Figure 2-11 The relationship between pore size and particle size in body-centered cubic

42

separation [Menon et al 1999 Chun et al 2002] First all sorbents were designed to

have negative surface properties because most plasma proteins in blood are negative and

should not be removed with charge interaction between proteins and sorbents at

physiological condition (pH74) It was reported that a negatively charged surface was

more blood compatible than a positive one [Srinivasan et al 1971] The toxin protein β2-

Microglobulin (β2M) can be selectively adsorbed on the surface of latex particles by the

design of a suitable hydrophobic portioned surface where only β2M protein can be

anchored with hydrophobic interactions Albumin adsorption on the latex particle is not

side-on m

adsorption of β M protein on the engineered latex particles

allowed because charge repulsion is more dominant than hydrophobic interaction with

ode The figure 2-12 shows the schematic representation of the selective

2

PMMAPAAShell

COO-

OSO3-

OSO3-

PSCore

COO-

-

-

-

-

-

-

Albumin-

Chargerepulsion

--

-

- -

- -

- --

Hydrophobic

β2-Microglobulin

2

Interaction

β -Microglobulin

PMMAPAAShell

COO-

-

-

-

-

-

-

--

-

-

-

-

-

Albumin-

Chargerepulsion

OSO3-

OSO3-

PSCore

COO-

--

-

- -

--

-

- -

- -

- --

- -

- --

Hydrophobic

β2-Microglobulin

2

particles at pH 74

Interaction

β -Microglobulin

Figure 2-12 Schematic representation of the protein adsorption on the core shell latex

43

Hydrophilichydrophobic microdomain structures were proven to be more blood

compatible [Mori et al 1982 Higuchi et al 1993 Deppisch et al 1998] The

optimization of suitable hydrophobic to hydrophilic ratio is also important for the

biocompatibility The monomers such as styrene (St) methyl methacrylate (MMA) and

acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in

emulsion polymerization process

In summary the background and fundamental literature survey about the history

material properties and limitation of hemodialysis membrane latex particle preparation

surface chemistry and manufacturing process target proteins and protein adsorption and

finally the hypothesis for a toxin removal with high separation efficiency have been

suggested This research focuses on such hypothesis and the materials and

characterization methodology for achievement of suggested hypotheses are described in

the next chapter

CHAPTER 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY

As mentioned earlier the goal of this study is to prepare polymeric latex particles

with tailored properties to maximize separation of toxin molecules and to investigate

fundamental interactions between the applied particles and molecules in the

system to optimize the membrane performance for hemodialysis applications Polymeric

latex particles wit

the

biological

h monodisperse size distribution to obtain uniform pore size and

various size ranges to utilize membrane construction are necessary Surface engineering

with various combination of hydrophobichydrophilic domain on the surface of latex

particles is expected to affect the removal of target protein by selective adsorption and to

improve the biocompatibility of membrane Therefore the materials and characterization

methodology are addressed in this chapter

31 Materials

Styrene (St) monomer used for a seed and a core particle was purchased from

Fisher Scientific and used without any other purification process Acrylic acid (AA) and

methyl methacrylate (MMA) monomers introduced for shell formation were purchased

from Fisher Scientific and used without any other purification process Sodium persulfate

(SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as

received Divinylbenzene (DVB) crossliking agent was purchased from Aldrich The

anionic surfactant Aerosol regMA80-I [sodium di(13-dimethylbutyl) sulfosuccinate] was

kindly donated by Cytec 78-80 Aerosol regMA80-I is mixed with isopropanol and

water Its critical micelle concentration (CMC) is 245 mM (119) Ion exchange resin

44

45

AGreg 501-x8 Resin (20-50 mesh) was purchased from Bio-Rad Laboratories Inc It is a

mixed-bed resin with H+ and OH- as the ionic forms and is 8 crosslinked Bovine serum

albumin (BSA) (heat shock treated) was purchas Fisher Scientific The isoelectric

point of t lin

(β2M)

as

gma

ed from

he BSA is 47-49 and the molecular weight is 66300Da β2-Microglobu

was purchase from ICN Biomedicals Inc and was separated from patients with

chronic renal disease and lyophilized from ammonium bicarbonate The molecular

weight by SDS-PAGE was approximately 12000Da BSA and β2M proteins were used

received without further purification process In order to investigate the protein

adsorption evaluation bicinchoninic acid protein assay kits were purchased from Si

(Cat BCA-1) and Pierce Biotechnology (Cat 23225) The chemical structures for the

main chemicals are shown in Figure 3-1

CH2 CH

Styrene (Mw = 10415)

CH2 CHCH2 CH

Styrene (Mw = 10415)

CH2 CH

Acrylic acid (Mw = 7206)

C

OH

O

CH2 CH

Acrylic acid (Mw = 7206)

C

OH

O

CH CH2

C

OCH3

O

CH

CH CH3

2

C

OCH3

O

CH3

Methyl methacrylate (Mw = 10012)Methyl methacrylate (Mw = 10012)

CH2 CH

Divinylbenzene (Mw = 1302)

CH CH2

CH2 CH

Divinylbenzene (Mw = 1302)

CH CH2CH CH

+(Na) -(O3S)

CH -C-O-CH-CH -CH-CH3

O

2 2 3

O CH CH3

CH3 CH3

CH-C-O-CH-CH2-CH-CH3

Anionic surfactant (MA80-I) (Mw = 3885)

+(Na) -(O3S)

CH -C-O-CH-CH -CH-CH3

O

2 2 3

O CH CH3

CH3 CH3

CH-C-O-CH-CH2-CH-CH3

Anionic surfactant (MA80-I) (Mw = 3885)2

+(Na)-(O) S OO

O O

- +

Initiator sodium persulfate (Mw = 23811)

O SO

(O) (Na)+ -

O O

- +(Na) (O) S OO

O SO

(O) (Na)+ -

O O

- +

Initiator sodium persulfate (Mw = 23811) Figure 3-1 Chemical structure of main chemicals

(Na) (O) S OO

O SO

(O) (Na)

46

32 Latex Particle Preparation

The experimental set up and the particle preparation schemes for various types and

size ranges of latex particles are shown in Figure 3-2 and 3-3 In the experimental setup a

mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at

end of glass stirrer The agitation rate is precisely adjusted by controller motor Paar glas

reactor with 1L volume is partially in silicon oil bath on hot plate The reactor has fo

openings for a stirrer a thermometer a nitrogen gas inlet and a reactant feeding lnlet

The reactants for latex polymerization are fed by precisely controlled by meterin

The particle preparation is described in each latex preparation section

the

s

ur

g pump

Figure 3-2 Experimental setup for semi-continuous emulsion polymerization

1 Motor 2 Mechanical stirrer bar 3 Thermometer 4 Inlet of N2 5 Inlet for initiator and monomer 6 Glass reactor (l L) 7 Oil bath 8 Hot plate 9 Pump for initiator 10 Pump for monomer 11 Tubes for reactant feed

47

Styrene monomer

Bare PS

PSPMMA100

PSPMMA90PAA10

PSPMMA P75 AA25

Gr

win

ticle

size

PS seedparticles

og

par

Styrene monomer

Bare PS

PSPMMA100

PSPMMA90PAA10

PSPMMA P75 AA25

Gr

win

ticle

size

PS seedparticles

og

par

Figure 3-3 The particle preparation scheme in various types and size ranges of latex particles

321 Preparation of Seed Latex Particles

Polystyrene seed particles were synthesized through a typical semi-continuous

emulsion polymerization Polystyrene latex particles were prepared in a four necked Paar

glass vessel equipped with a mechanical glass stirrer thermometer glass funnel for

nitrogen gas purge and a tube for monomer feeding The reactor was placed in a silicon

oil bath on a hot plate for homogenous and stable tempe he vessel was

firstly charged with de-ionized water the emulsifier so itiator

sodium bicarbonate (SBC) styrene (St) monomer divinylbenzene (DVB) monomer

under nitrogen gas atmosphere and then heated to 72plusmn2 as

llowed to continue for another hour Second reactants St and DVB monomers SPS

initiator and SBC were fed separately using fine fluid metering pumps (Model RHSY

Fluid metering INC NY) under a nitrogen gas atmosphere for 2h After complete feeding

rature control T

dium persulfate (SPS) in

oC for 1h The reaction w

a

48

of all reactants the reaction was continued for another hour at elevated temperature 80plusmn1

oC and then cooled to room temperature by removal of the oil bath

322 Preparation of Seeded Latex Particles

The particle growth was achieved by seeded continuous emulsion polymerization

The vessel was charged with the polystyrene seeds and de-ionized water to make desired

solid content and heated up to 72plusmn2 oC for 1h under nitrogen gas atmosphere Then St

and DVB monomers and solution of SPS and SBC in de-ionized water were continuously

added using metering pump with a precisely controlled feed rate After feeding all

reactants the reaction was continued for three hours keeping temperature of 80plusmn2 oC and

then the reaction vessel was cooled down to room temperature

Monodisperse spherical polystyrene seeds with a 280nm mean diameter were used

e-polymerized polystyrene seeded emulsion

was charged in a 500m

323 Preparation of Core Shell Latex Particles

for core shell structured latex particle The pr

l glass flask under a nitrogen gas atmosphere De-ionized water

was added to achieve the desired solid content The emulsion was heated to 80plusmn2oC for

10 hour Monomers initiator and SBC to form the shell were added by fluid metering

pumps (LAB PUMP JRRHSY model Fluid metering Inc USA) with fine controlled

feed rate Flow rate can be adjusted by control ring graduated in 450 division from 0 to

100 flow which is in the range of 10mlmin respectively The reaction was sustained

for additional 30 hours at 90plusmn2oC then cooled down to room temperature

324 Purification of Synthesized Latex Particles

Synthesized latex particles were purified with an ion exchange resin 200g (10

ww polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin

and stirred for 40 minute to remove unreacted monomers initiators and other impurities

49

Then the emulsion was diluted with DI water to a specific degree of solid content as

needed

33 Characterization

331

solid

termined

oscopy

d

hours

Dried latex (10mg) was mixed with

250 m on

Degree of Conversion

The degree of monomer to polymer conversion was determined gravimetrically

following the procedure described by Lee et al [1995] before the latex cleaning process

The synthesized emulsion raw materials were weighed and dried in a conventional oven

at 120oC for 30min to evaporate any unreacted monomers and water The remaining

was weighed and the degree of conversion was determined This process was repeated

three times and a mean value of the degree of conversion was de

332 Fourier Transform Infrared (FTIR) Spectr

A molecule can absorb only select frequencies (energy) of infrared radiation which

match the natural vibration frequencies The energy absorbed serves to increase the

amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]

Only those bonds which have a dipole moment that changes as a function of time can

absorb the infrared radiation The range of wavelengths for infrared radiation is between

25 microm (4000 cm-1) and 25 microm (400cm-1)

The synthesis of the latex particles such as polystyrene (PS) homopolymer and

PSPMMA PSPMMA90PAA10 PSPMMA75PAA25 core-shell copolymers were verifie

with FTIR Purified latex particles were dried in vacuum oven at 40oC for 24

before analysis in the FTIR (Nicolet Magma USA)

g of KBr which had also been dried in vacuum oven at 120oC for 3h Transmissi

spectra using the drift mode were plotted with 128 scans and 4cm-1 resolution

50

333 Quasielastic Light Scattering (QELS)

Latex particle size was measured by the Brookhaven ZetaPlus particle size

analyzer The solid concentration as 0001 (wv) This

ation spectroscopy (PCS) of quasielastically scattered light

For th s

ed using a FE-SEM (JEOL

JSM-

ith

ent

01

us zeta-

potential analyzer It measures the electrophoretic mobility velocity of charged colloidal

of latex particles used w

instrument uses photon correl

is technique time dependent interference patterns of light scattered from particle

in the sample cell are analyzed The interference pattern changes due to Brownian

motion giving rise to fluctuations in scattering intensity The frequency of these

fluctuations depends on the particlersquos diffusion constant that is inversely proportional to

the particle diameter

334 Field Emission-Scanning Electron Microscopy (FE-SEM)

Particle size and surface morphology were characteriz

6335F Japan) A diluted suspension of latex particles in water was dropped onto a

silicon wafer at room temperature and allowed to dry The sample was then coated w

the thinnest layer of carbon needed to obtain the required conductivity for this instrum

Secondary electron image mode was used with a 15KV of accelerating voltage The

magnification range used was between 10000X and 70000X

335 Zeta Potential Measurement

Synthesized and purified latex particles were diluted with de-ionized water to 0

wt and adjusted six different pH values 205 345 481 565 643 and 747 These

particle suspensions were then transferred to standard cuvettes for zeta potential

measurement At least two runs of ten measurements were taken for each sample and

averaged

The zeta potential measurement was carried out using Brookhaven ZetaPl

51

particles in solution and calculate zeta-potential by using the Smoluchowski equation

The f quency

gnitude

umin (BSA) as a standard model protein and β2-Microglobulin

uffer (PB) and

phosp

5

h the selected protein

e 005 01 03 05 and 07 mgml and those for

β2M

d at

teins in the supernatant after the centrifugation process using the

bicinc t

requency of laser light passing through the sample and is compared to the fre

of a reference beam The shift in the frequency called a Doppler shift and the ma

of the shift correspond to the polarity and the magnitude of the electrophoretic mobility

respectively The zeta potential is calculated from the solution conditions and the

measured mobility

336 Protein Adsorption

Protein adsorption experiments of the synthesized latex particles were performed

with bovine serum alb

(β2M) as a target protein in two types of buffer solution phosphate b

hate buffered saline (PBS) To make 5 mM of PB solution 0345g of sodium

phosphate monobasic (NaH2PO4H2O) was dissolve in 500ml of de-ionized water To

make PBS solution 0345g of sodium phosphate monobasic (NaH2PO4H2O) and 4178g

(143mM) of sodium chloride (NaCl) were dissolved in 500ml of de-ionized water The

synthesized latex particles were diluted with each buffer to have a solids content of 0

(ww) adjusted to pH values of 32 48 and 74 and mixed wit

BSA concentrations were chosen to b

were 0015 0030 0045 and 0060mgml The mixture was gently rotated using

the shaker Labquakereg (BarnsteadThermolyne Model 4002110 USA) with 8 RPM in

the incubator at 37oC for 12 hours before the latex-protein mixture was centrifuge

13000 rpm for 15min The amount of protein adsorbed was determined by quantifying

the free pro

honinic acid (BCA) assay method [Lowry et al 1951 Smith et al 1985 Baptista e

al 2003 Wiehelman et al 1988 Brown et al 1989] BCA assay kits consist of Reagent

52

A containing bicinchoninic acid sodium carbonate sodium tartrate and sodium

bicarbonate in 01N NaOH with pH=1125 and Reagent B containing 4 (wv) copper

(II) sulfate pentahydrate The BCA working reagent was prepared by mixing 50 parts of

Reagent A with 1 part of Reagent B 100microl of protein supernatant was then mixed with

2ml of BCA working reagent in a UV cuvette Incubation was allowed to continue

at room temperature

further

until color developed about 2h The absorbance at 562nm was

pectrophotometer (Perkin-Elmer Lambda 800 USA)

Unkn

Separate the p rticles in centrifuge mix supernatant with BCA reagents

measured using a UV-VIS s

own concentration of sample protein was determined by comparison to a standard

of known protein concentrations The adsorbed amount per unit surface area was

determined by the mass balance of the protein after adsorption process The simple

schematic of the procedure for a protein adsorption test is shown in Figure 3-4

Incubate mixture with agitation for 12hrs at 37oC

aand develop the color by incubation for 2hrs at room temperature

Suspend latex particles in aqueous buffer media

Add the dissolved target protein

Measure color intensity using UVVis spectroscopy at 562nm

Suspend latex particles in aqueous buffer media

Separate the p rticles in centrifuge mix supernatant with BCA reagents

Add the dissolved target protein

Incubate mixture with agitation for 12hrs at 37oC

aand develop the color by incubation for 2hrs at room temperature

Measure color intensity using UVVis spectroscopy at 562nm

Figure 3-4 Schematic of the procedure for a protein adsorption test

53

337 Blood Biocompatibility by Hemolysis Test

Hemolysis is the destruction of red blood cells which leads to the release of

hemoglobin from within the red blood cells into the blood plasma Hemolysis testing is

used to evaluate blood compatibility of the latex particles and the damaged to the red

blood cells can be determined by monitoring amount of hemoglobin released The red

blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutes

Figure 3-5 shows the phase separated blood with plasma white blood cells and red

cells after centrifugation process Separated RBSs were then washed with isotonic

phosphate buffer solution (PBS) at pH 74 to remove debris and serum protein Th

process was repeated 3 times

blood

is

Figure 3-5 Separation of RBC from whole blood by centrifuge process

Prepared latex particles were re-dispersed in PBS by sonification to obtain

homogeneously dispersed latex particles 100microl of the mixture of red blood cell (3 parts)

and PBS (11parts) was added to 1ml of 05 (ww) particle suspension PBS was used as

a negative control resulting 0 hemolysis and DI water used as a positive control to

produce 100 hemoglobin released by completely destroyed RBCs The mixture was

incubated in water bath with gentle shaking for 30 minutes at 37oC and then centrifuged

at 1500 rpm for 15 minutes 100microl of the supernatant was mixed with 2ml of the mixture

) (EtOHHCl = 2005 ww) to prevent the

White blood cells

Plasma

Red blood cells

of ethanol (99) and hydrochloric acid (37

54

precipitation of hemoglobin In order to remove remaining particles the mixture was

centri fuged again with 13000 RPM at room temperature The supernatant was then

transferred into the UV cuvette The amount of hemoglobin release was determined by

monitoring the UV absorbance at a wavelength of 397nm The schematic of the

procedure for hemolysis test is shown in Figure 3-6

Separate red blood cells (RBCs) from blood

Mix RBCs with phosphate buffer solution (PBS)

Wash and re-suspend RBC with PBS repeat three times

Add la l of RBC and incu in at 37oC

tex particle suspension into 100mbate sample with agitation for 30m

Separate particle ith EtOHHCl solutions and mix the supernatant w

Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm

Separate red blood cells (RBCs) from bloodSeparate red blood cells (RBCs) from blood

Mix RBCs with phosphate buffer solution (PBS)Mix RBCs with phosphate buffer solution (PBS)

Wash and re-suspend RBC with PBS repeat three timesWash and re-suspend RBC with PBS repeat three times

Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC

Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC

Separate particles and mix the ith EtOHHCl solution supernatant wSeparate particles and mix the ith EtOHHCl solution supernatant w

Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nmEvaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm

Figure 3-6 Schematic of the procedure for hemolysis test

In summary a description of the experimental materials and characterization

methodology has been explained In the next chapter the results and discussion of the

data obtained from these experiments is addressed

CHAPTER 4 RESULTS AND DISCUSSION

41 Polymerization of Latex Particles

411 Polystyrene Seed Latex Particles

Polymer particles have many different applications including spacer particles

lubricants packing materials for chromatography standard particles and diagnostic

drugs All applications strongly require these particles to have a uniform particle size

Uniform particle size is the first requirement for a latex composite membrane formed by

particle arrays with the interstitial spaces serving as pores for size discriminations [Jons

et al 1999 Ramakrishnan et al 2004] Although suspension polymerization has been

mainly used as one of conventional polymerization methods to prepare uniform particles

the uniformity of the recovered particles is insufficient for membrane use There is

another method known as seed emulsion polymerization where a vinyl monomer is

absorbed into fine monodiserse seed particles and polymerization causes the monomer to

crease the sizes of the seed particles uniformly In order to obtain monodisperse larger

particles by this m

particles and polymerization of the monomer is repeated

In this study cross-linked polystyrene seed latex particles were firstly synthesized

in the presence of styrene monomer divinylbenzene crosslink agent sodium persulfate

initiator sodium bicarbonate buffer and the Aerosol regMA80-I [sodium di(13-

dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion

polymerization process in which one or more components can be added continuously

in

ethod the procedure of absorption of monomer into fine polymer

55

56

Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and

buffer are continuously added to enlarge the seed latex particles Various profiles of

particle nucleation and growth can be gene different orders of component

addition during polymerizati f this process such as easy

control of the polymeriza particle number

and particle morphology The main goal in

this process is to avoid secondary particle

rated from

on There are several advantages o

tion rate monodisperse particle size and

colloidal stability copolymer composition

formation leading to a monodisperse particle

size distribution Figure 4-1 shows the schematic representation of seed latex particle

preparation and growth

Monomer droplet

Seed particles Amount of surfactantsis variable

Enlarged final Add monomers

PS

PS

PSs4

particles Number of particlesare constant

Seed code

PSs1

s2

s3

Monomer droplet

Seed particles Amount of surfactantsis variable

Enlarged final Add monomers

PS

PS

PSs4

tion The

particles Number of particlesare constant

Seed code

PSs1

s2

s3

Figure 4-1 Schematic representation of semi-continuous seed latex particles preparation and growth

After the first synthesis of a small size of crosslinked polystyrene (PS) seed

particles stabilized by surfactants styrene DVB and initiator are continuously fed into

the system to enlarge the seed latex particles maintaining a narrow size distribu

recipes and reaction conditions for synthesizing these seed particles are summarized in

57

Table 4-1 The original recipe for latex particle preparation from McDonald was

modified for this emulsion system [McDonald 2003]

Table 4-1 The polymerization recipe of polystyrene (PS) seed particles Latex label PSS259 PSS233 PSS207 PSS181

Temperature (oC) 75 plusmn 5 75 plusmn 5 75 plusmn 5 75 plusmn 5

Divinylbenzene (g) 052 052 052 052

MA 80 (g) 259 233 207 181

NaHCO

Watera (g) 1315 1315 1315 1315

Styrene (g) 9948 9948 9948 9948

112 112 112 112

Na2S2O8 (g) 190 190 190 190

Waterb (g) 200 200 200 200

Reaction time (min) 140 140 140 140

Solids content 396 396 396 396

Mean particle diameter (nm) 126plusmn75 171plusmn39 182plusmn41 216plusmn53

Conversion () 965 979 957 971

3 (g)

Four different seed latex particles were synthesized based on surfactant amount in

order to determine the optimum condition for stable and monodisperse latex particles

The emulsion polymerization of latex particles has to be carried out in a narrow range of

surfactant causes flocculation or phase separation leading to broad particle

size distribution and an unstable emulsion sy

surfactant concentration where particles are stable [Nestor et al 2005] A wide range of

concentrations

stem Therefore the range of surfactant

amount was varied from 181g to 259g in increments of 026g in each latex label Other

reactants amounts were kept constant Watera is the initial charge amount and waterb

represents the amount of aqueous solution containing dissolved initiator and buffer

58

which are fed continuously after the seed particle creation The extent of monome

polymer conversion was obtained by gravimetric calcu

r to

lation and was more than 95 at

cripts such as

S207 and S181 i the surf mount added for the seed latex

nversion indi perce experi obtain

content divided by the theoretically estimated solid content and was calculated by the

all seed particles labeled PSS259 PSS233 PSS207 and PSS181 Labeled subs

S259 S233 ndicate actant a

preparation The co cates the ntage of mentally ed solid

following formula

100 colids () times=entsoliatedntentoalExperimentConversion

n the experimenta content is the weight of olid rema after

mulsion at 120oC f in and the estimated solid content is th ht

lated from the recipe

rti as di each t load d

mple of ea pension iluted by (ww)

deionized water and the mean particle diameter was determined using the Brookhaven

ZetaPlus particle size analyzer From these results the seed latex particle size increased

as the amount of surfactant decreased The amount of emulsifier also affects the

polymerization process by changing the number of micelles and their size It is known

that large amount of emulsifier produces larger numbers of smaller sized particles [Odian

1991] and was corroborated in the current work

These synthesized seed latex particles should be of narrow size distribution in order

to obtain monodisperse larger particles The SEM image showing the particle

morphology and size of a sample of polystyrene seed particles can be seen in the Figure

4-2

contdEstim (4-1)

In this equatio l solid the s ining

evaporation of e or 30m e weig

of monomers calcu

As expected the seed pa cle size w fferent for surfactan ing in see

emulsion system A sa ch sus was d 0001 with

59

Figure 4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)

PSS259 (B) PSS233 (C) PSS207 (D) PSS181 (subscripts indicate the amount of surfactant added)

(A)

(B)

60

(C)

(D)

Figure 4-2 Continued

61

s seen in the image the seed particles are very smooth and spherical The

uniformity in the particle size distribution of these PS seed latex particles can also be

seen in the SEM image From this data we concluded that seed particles synthesized in

the presence of a surfactant with the chosen amounts are suitable for use to prepare larger

particles with high size uniformity and smooth spherical morphology

412 The Growth of Polystyrene (PS) Seed Latex Particles

Seeded emulsion polymerization has been conducted for several decades and

various mechanisms have been proposed Gracio et al [1970] suggested that the growing

polymer particles consist of an expanding polymer-rich core surrounded by a monomer-

rich shell with the outer shell providing a major locus of polymerization Seeded

mulsion polymerization is commonly used for preparing latex particles less than 1microm in

size [Cha et al 1995 Zou et al 1990 1992 Gandhi et al 1990 Park et al 1990] Such

latexes can be obtained from the growth of pre-prepared seed particles The seeds

introduced in this process serve as nucleation sites for particle growth By first swelling

the seed latex particles with additional monomer to the desired size polymerization can

then be initiated Several size ranges of monodisperse polystyrene (PS) latex particles

were prepared by this multistep seeded emulsion polymerization method For the

fabrication of particle-based membranes monodisperse latex particles with a variety of

size ranges are required

In this study seed particles from 171nm to 470nm in mean diameter and with a

highly uniform size distribution were prepared and used Watera is the initial charge

mount and waterb represents the amount aqueous solution containing dissolved initiator

and buffer that is continuously fed into system Feed time is precisely controlled by

metering pump The solid contents were maintained between 199 and 300 After

A

e

a

62

polym d as

es

ere 923 8402

n of these latex particles

are sh

ss

erization under these conditions the conversions of the latex particles labele

PS258 PS320 PS370 and PS410 were 952 957 946 and 964 respectively The recip

used in seeded emulsion polymerization to form the particles less than 500nm in mean

diameter are shown at the Table 4-2 The conversions of the latex particles larger than

500nm in mean diameter and labeled as PS525 PS585 PS640 and PS790 w

723 and 969 respectively The recipes used in the preparatio

own at the Table 4-3

A simple calculation can be used to predict the ultimate size that a latex particle can

reach after ending the polymerization Letrsquos suppose that seed density (ρs) and grown

particle density (ρgp) are the same The following equation is obtained from the ma

balance (m = ρV) where m and V are mass and volume of a particle respectively

gp

gp

s

s

Vm

Vm

In this equation m

= (4-2)

be

s and mgp are the weight of seed and total weight of seed and

added monomers respectively and Vs and Vgp are the volume of seed and grown

particle respectively

If we know the mass of monomer to be added the ultimate particle size can

calculated from the equation 4-3

3

2s D m ⎠⎝+ χ

where D

1s D m

⎟⎟⎞

⎜⎜⎛

= (4-3)

the

in

s and Dgp are the diameters for seed and grown particle respectively and χ is

weight of the monomer added Crosslinked PS latex particles with a size range from

258plusmn112 ~ 410plusmn117 in diameter polymerized by the recipes in Table 4-2 are shown

the SEM images in Figure 4-3

63

Table 4-2 Continuous addition emulsion polymerization recipe for growing polystyrene

Latex label PS

(PS) latex particles less than 500nm in size

258 PS320 PS370 PS410

Initial charge

Seed latex (g) 1000171 1000216 1000216 1000320

Watera (g) 1470 2500 1984 2600

06 07 06 06

Fee

Initiator stream

2 2 8

Feed time (min) 0-167 0-140 0-100 0-92

Solid content 284 300 300 200

Mean particle diameter (nm) 258plusmn112 320plusmn154 182plusmn71 410plusmn117

Conv

Monomer continuous

Styrene (g) 800 1200 800 600

Divinylbenzene (g)

d time (min) 0-163 0-110 0-75 0-82

Na S O (g) 07 065 05 05

NaHCO3 (g) 07 065 05 05

Waterb (g) 750 1200 200 370

ersion () 952 957 946 964 The solid content of seed latex for PS258 PS320 PS 370 and PS410 are 392 399 399 anrespectively

d 300

64

Table 4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size

Latex label PS525 PS585 PS640 PS790

Initial charge

Seed latex (g) 1000390 1000470 1000470 1000320

Watera (g) 1000 520 1200 360

Monomer continuous

Styrene (g) 290 200 400 173

Divinylbenzene (g) 02 014

03 009

Feed time (min) 0-80 0-40 10-76 5-65

Initiator stream

Na2S2O8 (g) 02 014 03 012

NaHCO3 (g) 02 014 03 009

Waterb (g) 200 120 400 200

Feed time (min) 0-85 0-46 0-77 0-75

Solids content 199 200 200 201

Mean particle diameter (nm) 525plusmn151 585plusmn186 640plusmn178 790plusmn693

Conversion () 923 8402 723 969 The solid content of seed latex for PS525 PS585 PS 640 and PS790 are 200 182 193 and 173 respectively

65

Figure 4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)

370nm (D) 410nm

(A)

(B)

66

(C)

(D)

Figure 4-3 Continued

67

article size was well matched to that estimated by the geometrical calculation

From this it can be concluded that the added monomer was consumed for polymer

particle growth with a high degree of conversion From the SEM characterization newly

nucleated particles were not seen as indicated by the narrow particle size distribution

The particles less than 500nm in mean diameter were very spherical in shape with a

highly uniform particle size distribution As the particles were grown larger than 500nm

mean diameter however they became of non-spherical shape with an uneven surface

This irregularity of particle surface can be attributed to the non-homogeneous monomer

swelling into the shell of a growing polymer which can be controlled by the factors such

as temperature agitation speed initiator feeding rate and surfactant amount required to

stabilize the colloidal system The SEM image of latex particles larger than 500nm mean

iameter is shown in Figure 4-4 The particle size distribution is broader than that of the

s particles These particles can however still be used for membrane construction

as a support layer rather than skin layer The support layer does not necessarily have to be

as highly monodisperse as the skin layer which require pores of high uniformity for the

selective removal of toxins

As mentioned earlier the amount of surfactant as well as monomer in an emulsion

system is the primary determinant of the particle diameter [Odian 1991] Anionic

surfactants are generally recommended at the level of 02-30 wt based on the amount

of water [Odian 1991] The critical micelle concentration (CMC) of Aerosolreg MA 80-I

anionic surfactant is 119 The surfactant loading in this emulsion system ranged from

the 121 for SS181 to 171 for SS259

P

d

maller

68

Figure 4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm

(C) 640nm (D) 790nm

(A)

(B)

69

(C)

(D)

Figure 4-4 Continued

70

The surfactant to monomer ratio refers to the value of the surfactant amount

divided by monomer amount after polymerization ends The surfactant amount is the

weight of the surfactant contained in emulsion and the monomer amount is the total

weight of the monomer added for polymerization to form the ultimate polymer particles

in emulsion As the surfactant to monomer ratio decreases mean particle diameter

increases as described by Odian [1991] Generally lower surfactant concentration forms

fewer micelles resulting in larger particle size [Evans et al 1999] However there is a

plateau in particle mean diameter between a surfactant to monomer ratio of 0005 and

0013 This is due to low polymerization conversion of latex particles from monomer

leading to a smaller particle size than expected This is not a factor of surfactant amount

The dependence of the particle size on the amount of surfactant is shown in Figure 4-5

0

200

400

600

800

1000

0000 0005 0010 0015 0020 0025 0030

Surfactant to monomer ratio (ww)

Part

icle

mea

n di

amet

er (n

m)

Figure 4-5 Dependence of the particle size on the surfactant to monomer ratio

71

413

e

f bare

microdomain structures is important for biocompatibility [Mori et al 1982 Higuchi et al

1993 Deppisch et al 1998] Figure 4-6 shows the schematic of core shell latex particle

structure The recipe for core shell structures is shown in Table 4-4

Core Shell Latex Particles

The latex particles shown in Figure 4-3 (A) and labeled as S258 in Table 4-2 wer

used as seeds to prepare core-shell latex particles because of their high particle

uniformity and smooth surface morphology Methyl methacrylate (MMA) and acrylic

acid (AA) monomers were introduced to increase surface hydrophilicity over that o

polystyrene particles AA is a more hydrophilic monomer than MMA because of the

carboxyl acid group in AA is more favorable for hydrogen boding with water The

surface carboxyl groups on PAA may have many promising applications in biomedical

and biochemical fields [Kang et al 2005] MMA monomer is less hydrophobic than

styrene PMMA is known to more biocompatible than PS but still PMMA is hydrophobic

The optimization of suitable hydrophobic to hydrophilic ratios and control of

Polystyreneseed particle

Core shelllatex particles

PSPMMA100

PSPMMA90PAA10

PSPMMA75PAA25

Polystyreneseed particle

Core shelllatex particles

PSPMMA100

PSPMMA90PAA10

Figure 4-6 Schematic of core shell latex particle structures

PSPMMA75PAA25

72

Table 4-4 The preparation recipe of PS core with various shell latex particles Latex 25 label PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA

Initial charge

PS seed latex (g) 500 500 500a

Monomer continuous

Methly methacylate (g) 320 288 240

Acrylic acid (g) 00 32 80

Initiator stream

Na

258 258 258

Water (g) 1570 1570 1570

Feed time (min) 0-120 0-50 0-60

b 200

Feed time (min) 0-122 0-60 0-65

952

2S2O8 (g) 02 02 02

NaHCO3 (g) 02 02 02

Water (g) 200 200

Solids content 183 183 183

Mean particle diameter (nm) 370plusmn186 370plusmn178 370plusmn191

Conversion () 955 924

Three types of core shell structures were prepared PMMA75PAA25 shell is a

copolymer consisting of the PMMA to PAA ratio of 75 to 25 by weight

PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90 to 10 by

weight PMMA100 is the PMMA homopolymer shell on PS core The particle size of

these core-shell particles as well as PS latex particle was prepared to be about 370nm in

mean diameter and are characterized to determine their zeta potential protein adsorption

and biocompatibility The image of SEM of PS and core shell particles is shown at Figure

4-7 The synthesized latex particles had a high uniformity in size and a smooth spherical

surface morphology

73

Figure 4-7 Scanning

Electron Micrograph of latex particles (A) PS (B) PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMMA75PAA25

(A)

(B)

74

(C)

(D)

Figure 4-7 Continued

75

42 Characterization of Latex Particles

421 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectroscopy has proven to be a powerful tool to characterize polymeric

materials A molecule can absorb only select frequencies (energy) of infrared radiation

which match the natural vibration frequencies The energy absorbed serves to increase

the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]

Only those bonds which have a dipole moment that changes as a function of time can

absorb infrared radiation The FTIR spectra of polymerized latex particles made during

this work are shown in Figure 4-8 There are number of characteristic peaks for PS latex

particles [Bhutto et al 2003 Li at al 2005] Aromatic C-H stretching vibration is shown

at 3002 cm-1 and 3103 cm-1 and aliphatic C-H asymmetrical and symmetrical stretching

shown at 2900 cm-1and 2850 cm-1 respectively There is an additional carbonyl (C=O)

adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some

amount of either or both PMMA and PAA The broad OH group peak at 3400 cm-1

appear for particles containing some amount of PAA and has intensity dependent on the

amount of PAA in the shell For example the peak intensity of OH groups for

PSPMMA75PAA25 is greater than that of PSPMMA90PAA10 since there is a higher AA

monomer content in PSPMMA75PAA25 than in PSPMMA90PAA10 This OH peak is not

seen for PS and PS core PMMA shell particles because of the absence of OH groups on

PS and PMMA polymer chains

422 Zeta Potential Measurements

The electrical surface property was characterized by zeta potential measurements of

tex particles Zeta potential is an important and useful indicator of surface charge which

can be used to predict and control the stability of colloidal systems The greater the zeta

is

la

76

1520002500300035004000

Wavenumbers (cm-1)

li

Re

ativ

e

Tra

nsm

ttanc

e

Figure 4-8 FTIR spectra of polymerized latex particles (A) ba

PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMM

(B)

(C) P

(D) P

C

C

OCH3

O

CH3

XCH2

CH2 n

CH

CH2 C

C

OCH3

O

XCH2 CH

OC

CH3

Y

OH

(A) Bare PS

500100000

re polystyrene (PS) (B) A75PAA25

PSPMMA100

SPMMA90PAA10

SPMMA75PAA25

77

potential the more likely the suspension is to be stable because the charged particles repel

one another and thus overcome the natural tendency to aggregate The measurement of

zeta potential is often the key ontrol of the interaction of proteins

with solid surfaces such as latex particles

Zeta potential (ζ) is the elec at exists at the shear plane of a particle

which is some small distance from the surface [Myers 1999] The zeta potential of the

particles can be determined by measuring the mobility of the particles in an applied

electric field termed electr its response to an alternating electric

field Colloidal particles dispersed in lu ically charged due to their ionic

characteristics and dipolar attributes The development of a net charge at the particle

surface affects the distribution of ions in the neighboring ng in

an increased concentration of counter ions to the surface Each particle dispersed in a

ns called fixed layer or Stern layer

Outside th compositions of ions of opposite polarities

form ble layer is form

particle-liquid interface This double layer may be considered to consist of two parts an

inner region which inclu nd relatively strongly to the surface and an outer or

diffuse region in which the ion distribution is determined by a balance of electrostatic

forces and random thermal motion The potential in this region therefore decays with the

distance from the surface until at a certain distance it becomes zero Figure 4-9 shows a

schematic representation of a typical ion distribution near a positively charged surface

The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the

to understanding and c

trical potential th

ophoretic mobility or

a so tion are electr

interfacial region resulti

solution is surrounded by oppositely charged io

e Stern layer there are varying

ed in the region of the ing a cloud-like area Thus an electrical dou

des ions bou

center of those ions which are effectively adsorbed to the surface [Hunter 1981] The

78

extent of ion adsorption is determined by electrical and other long-range interactions

between the individual ions and surface of particles The ions outside of the Stern layer

form the diffuse double layer also referred to as the Gouy-Chapman layer [Burns and

Zydney 2000]

Shear plane

Stern layer

ψζ

0

ψs

ψShear plane

Stern layer

ψζ

0

ψs

ψ

t

controlled electric field is applied via electrodes immersed in a sample suspension and

Figure 4-9 Schematic representation of ion distribution near a positively charged surface

Zeta potential is a function of the surface charge of a particle any adsorbed layer a

the interface and the nature and composition of the surrounding medium in which the

particle is suspended The principle of determining zeta potential is very simple A

79

this causes the charged particles to move towards the electrode of opposite polarity

Viscous forces acting upon the moving particle tend to oppose this motion and the

equilibrium is rapidly established between the effects of the electrostatic attraction and

the viscosity dra

g The particle therefore reaches a constant terminal velocity

Figure 4-10 Schematic representation of zeta potential measurement(source

httpnitioncomenproductszeecom_shtm)

Because protein adsorption mechanisms are very complex and hard to explain in

biological systems protein adsorption is generally studied by using more ideal systems

consisting of one or more well characterized proteins a well-characterized adsorbent and

a well-defined aqueous solution Even so small changes in the experimental conditions

such as pH ionic strength and temperature generate totally different results Therefore

two media systems phosphate buffer (PB) and phosphate buffered saline (PBS) were

chosen to be used for the zeta potential measurements and protein adsorption study PB is

a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4H2O) to

n a constant pH PBS is used to keep the water concentration inside and outside of

the cell balanced If the water concentration is unbalanced the cell risks bursting or

shriveling up because of a phenomenon called osmosis PBS is a solution with the 5 mM

of monobasic sodium phosphate (NaH2PO4H2O) and 143 mM of sodium chloride (NaCl)

in water

maintai

80

The results of zeta potential measurement at room temperature (25plusmn1oC) as

function of pH and ionic strength (different media PB and PBS) for synthesized lat

particles are seen in Figure 4-11 Zeta potential values of polystyrene (PS) latex particle

at the Figure 4-9 are negative between -291 mV and -599 mV in PB and betwe

ex

s

en -203

mV and -278 mV in PBS at all pH ranges These negative zeta potential values of PS

latex particles are due to the sulfate groups [Vandenhu et al 1970] originated from

persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer

chain where polymerization was initiated

-100

-80

-60

-40

-20

0

pH

Zta

pte

ntl

ζ

20

40

1 2 3 4 5 6 7 8 9

eo

ia (m

V)

PB (5mM)

PBS (5mM +143mM)

en -

Figure 4-11 Zeta potential of PS latex particles at 25oC

The zeta potential measurements of PSPMMA100 core shell latex particles as seen

in Figure 4-12 were also negative with a range of -28 and -505 mV in PB and betwe

143 mV and -186 mV in PBS at the pH 21-78 These negative values are also due to

sulfate groups on the particle surface

81

-100

-80

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

Zea

pote

nti

l ζ

(mV

pH

ta

)

PB (5mM)

PBS (5mM + 143mM)

Figure 4-12 Zeta potential of PSPMMA100 latex particles at 25oC

The zeta potential for PSPMMA90PAA10 and PSPMMA75PAA25 was also

negative as seen in Figure 4-13 and 4-14 The zeta potential value for the

PSPMMA90PAA10 were between -367 mV and -678 mV in PB medium and between -

147 mV and -193 mV in PBS both at 25oC In case of the PSPMMA75PAA25 core shell

particles zeta potential values were between -291 mV and -520 mV in PB and between

-115 mV and -210 mV in PBS media Sulfate groups in PSPMMA75PAA25 and

PSPMMA90PAA10 core shell particles contribute to the negative zeta potential values

participate in forming a

negat

cid is expressed by the term pKa which is the pH

at which an acid is 50 dissociated However the contribution of the carboxylic acid

Carboxylic groups from acrylic acid monomer also would

ive surface charge of the latex particles at a pH greater than 70 because the pKa

value for the carboxylic acid is between 40 and 60 pH range [Ottewill et al 1967] This

indicates that carboxylic acid dissociation begins at a pH between pH 46-60 and

increases with pH The strength of an a

82

group to negative zeta potential values of PSPMMA-PAA core shell particles was not

detectable because there was no significant difference in zeta potential values for

synthesized latex particles at a pH greater than 70

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

Zeta

pot

entia

l ζ

(mV

)

PB (5mM)PBS (5mM + 143mM)

-80

-100

pH

Figure 4-13 Zeta potential of PSPMMA90PAA10 latex particles at 25oC

-100

-80

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

pH

Ze p

ont

i ζ

V)

tate

al (m

PB (5mM)PBS (5mM + 143mM)

Figure 4-14 Zeta potential of PSPMMA75PAA25 latex particles at 25oC

83

The zeta potential plots and values were similar for all latex particles This may be

due to the similar initiator density on the latex surface The initiator concentrations

monomers for shell polymerization were the same fo

to

r all core shell latex particles (062

ww from Table 4-4) A similar concentration (055 ww) of initiator to monomer was

added to prepare bare PS latex particles used in zeta potential measurements

Since the zeta potential values of all synthesized latex particles were negative

between pH=20 and pH=78 the isoelecric point (IEP) of these latex particles would be

less than pH=20 Sulfates have a pKa in the range of 10 to 20 [James 1985] indicating

that the sulfate group the conjugate base of a strong acid is protonated (-C-OSO3H) at

less than pH 20 This means that the synthesized latex particles are all negative at the

physiological pH Most serum proteins have a negative surface charge in blood

therefore when the negative latex particles are applied to blood stream fatal

tein is

avoided by charge repulsion in the blood Table 4-5 shows the common blood proteins

and their isoelectric points (IEP)

Table 4-5 The isoelectric point (IEP) of common proteins Serum proteins Isoelectric point (IEP)

complication by coagulation between applied latex particles and serum pro s

Albumin 48 α1-Globulin 20 α2-Microglobulin 54 γ1-Globulin 58 Heptoglobin 41 Hemoglobin 72 β1-lipoprotein 54 Immunoglobulin G (Ig G) 73 Fibrinogen 58 (source httpwwwfleshandbonescomreadingroompdf1085pdf

httpwwwmedalorgvisitorwww5CActive5Cch135Cch13015Cch130105aspx )

84

The absolute zeta potential values of the latex particles in phosphate buffer (PB)

was higher than that in phosphate buffered saline (PBS) This is because high

concentration of sodium and chloride electrolytes in PBS compressed the electrical

double layer causing rapid decay of surface potential Figure 4-15 shows that the

thickness of the electrical double layer is inversely proportional to the concentration of

electrolyte in the system

Surf

ace

Pote

ntia

l ψ

1κ3

10

1e

1κ 1κ

(1)(3) (2)

2 1 Z

Surf

ace

Pote

ntia

l ψ

1κ3

10

1e

1κ 1κ

(1)(3) (2)

Figure 4-15 The decay of surface potential with distance from surface in various

electrolyte concentrations (1) low (2) intermediate (3) high [Myers 1999]

2 1 Z

The electrical potential in the solution surrounding the particle surface falls off

the Debye-Huckel

yers 1999]

exponentially with distance (Z) from the surface according to

approximation [M

z) (- exp 0 κψψ = (4-4)

e reciprocal of the thickness of the electrical double layer referred to as the

The potential fallen off by a factor e The theoretical equation for the

ickness 1κ is

where κ is th

Debye length of 1

double layer th

21

22i

0

ekT 1

⎟⎟⎠

⎜⎜⎝

=sum ii zcεε

κ

⎞⎛ (4-5)

85

where

tration of

y was performed to measure the protein adsorption on the

synthesized latex particles Many physical and chemical processes occur at the boundary

between two phases and adsorption is one of the fundamental surface phenomena [Oscik

1982] This phenomenon is driven by the change in the concentration of the components

at the interface with respect to their concentrations in the bulk phase There are two

adsorption processes physical adsorption occurring when non-balanced physical forces

appear at the boundary of the phases and chemical adsorption or chemisorption

t the

interface [J

n leads

ational entropy hence adsorption takes place only if the

loss in conformational entropy is compensated

ter adsorption Cp The initial part of the

isotherm merges with the Γ-axis because at low polymer concentration the protein has a

high affinity for the surface and all of the polymer is adsorbed until the solid surface is

saturated

ε0 is the permittivity of a vacuum of free space ε is the permittivity of a medium e

is the relative permittivity or dielectric constant of the medium c is the concen

an ion (i) z is the valency of an ion (i) k is Boltzmannrsquos constant T is absolute

temperature (K)

43 Protein Adsorption Study

The adsorption stud

occurring when atoms and molecules from adjacent phases form chemical bonds a

aroniec et al 1988]

Flexible proteins in solution possess high conformational entropy as a result of the

various states each of the many segments in the protein chain can have Adsorptio

to a reduction of this conform

by sufficient attraction between polymer

segment and solid surface [Martin 1998] Isotherm shape of high affinity adsorption

between polymer and solid is shown in Figure 4-16 where the adsorbed mass Γ is plotted

against the polymer concentration in solution af

86

Figur

de

arboxyl

e 4-16 High-affinity adsorption isotherm of typical flexible polymer on solid

surface

In order to investigate the adsorption of blood proteins latex particle properties

such as electrical surface charge hydrophobicity and environmental conditions like pH

ionic strength and temperature are considered as experimental factors [Arai and Nor

1990] Figure 4-17 shows the overall schematic representation of the protein adsorption

on the synthesized latex particles with the functional groups such as sulfate and c

groups

CP

Γ

PMMAPAAShell

PS or PMMAShell OSO3

-

OSO3-

OSO3-

OSO3-

PS

-O3SO

PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

Protein

NH3+

+

+H3N

+

COO-

Hydrophobic interaction

NH3

COO-

H3N

COO-

-OOC

Ionic interaction

Hydrophobic interaction

Hydrogen bonding

Ionic interaction

PMMAPAAShellOSO3

-

OSO3-

OSO3-

OSO3-

PS or PMMAShell -O3SOOSO3

-

PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

-O3SO

OSO3-

OSO3-

OSO3-

PS PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

Protein

NH3+

+

+H3N

+

COO-NH3

COO-

H3N

COO-

-OOC

NH3+

+

+H3N

+

COO-

Hydrophobic interaction

NH3

COO-

H3N

COO-

-OOC

Ionic interaction

Hydrophobic interaction

Hydrogen bonding

Figure 4-17 Overall schematic representation of the protein adsorption on the

Ionic interaction

synthesized latex particles

The surface functional groups as well as suitable surface hydrophobicity

hydrophilicity of latex particles are closely related to protein adsorption property As

indicated at Figure 4-17 various interaction forces such as hydrophobic interaction ionic

87

interaction hydrogen interaction and van der Waals interaction [Andrade 1985] exist

between protein molecule and the solid surface of latex particle

431 Adsorption Isotherm

The protein adsorption isotherm is generally defined as the relationship between the

amount of protein adsorbed by a solid (latex particle) at a constant temperature and as a

ine

the amount of protein adsorbed onto latex particles To evaluate the equilibrium

conce s

x

function of the concentration of protein Adsorption isotherms were plotted to determ

ntration of target proteins a calibration curve is firstly needed Figure 4-18 show

the UV absorbance as a factor of bovine serum albumin (BSA) concentrations The UV

absorbance of the known protein concentration was measured at 562nm which is the λma

for a protein treated by a bicinchoninic acid (BCA) assay

0

02

04

06

08

500 520 540 560 580 600

Wavelength (nm)

Abs

orba

nce

Figur gml (B)

06mgml (C) 02mgml

(A)

(C)

(B)

Max562nm

e 4-18 The dependence of UV absorbance on BSA concentration (A) 10m

Figure 4-19 shows the standard calibration curve used in adsorption isotherm

experiment of protein onto latex particles The net absorbance of each known protein

88

concentration was plotted and then a linear equation passing through a concentration of

zero protein concentration was obtained This equation is applied

to determine the

orbed on the synthesized latex particles The amount of free

protei

eported

trations (005 01 03 05

and 07mgml) were used to generate this curve as well as the adsorption isotherm

unknown protein amount ads

n was also determined using UV light at the 280nm wavelength however a

distinguishable UV intensity was too low to determine a precise value of protein

adsorption

The bicinchninic acid (BCA) assay technique [Smith et al 1985] has been r

for high efficiency of protein evaluation and was used for determination of protein

adsorption [Tangpasuthadol et al 2003 Williams et al 2003] This technique has many

advantages such as high sensitivity easy of use color complex stability and less

susceptibility to any detergents Five different BSA concen

y = 00R2 =

004741x 09939

00

01

02

03

04

0 200 400 600 800

BSA Standard (microgml)

Net

Abs

orba

nce

at 5

62nm

Figure 4-19 Standard curve of net absorbance vs BSA sample concentration

89

All isotherms were fitted to the Langmuir-Freundlich isotherms using the nonlinea

regression method Two adsorption isotherm models the Langmuir type isotherm a

Langmuir-Freundlich combination type isotherm were compared As seen the Figure 4-

20 The Langmuir isotherm model deviated further from the experimental data Th

all isotherms were fitted to the Langmuir-Freundlich isotherms [Yoon et al 1996

al 1998] usi

r

nd the

erefore

Lee et

ng the nonlinear regression method with following equation

n

n

eq

eqm

kC

kC1

1

1+

where q is the amount of adsorbed BSA per unit surface area q

qq = (4-6)

stant

were between 045 and 055 and hence n was fixed to its average 05 Actual qm values

were evaluated after fixing the n value

m is the adsorbed amount

in equilibrium Ceq is the equilibrium concentration of BSA k is the adsorption con

and n is the exponential factor for a heterogeneous system n values are empirical and

usually from zero to no more than unity [Jaekel 2002] The values of n in our system

00

10

20

30

0 02 04 06 08

C (mgml)eq

q (m

gm

2 )

pH 32Langmuir-Freundlich modelLangmuir model

Figure 4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on

polystyrene latex particles at 37oC in phosphate buffer media

90

The BSA protein adsorption on the synthesized latex particles was performed at

three pH levels 32 48 74 in both phosphate buffer (PB) and phosphate buffered saline

(PBS) at physiological temperature 37oC These pH levels correspond to an acidic pH

lower than the isoelectric point (IEP) of BSA the pH at the IEP and an alkaline pH

higher than the IEP of BSA respectively The IEP corresponds to the pH at which the

amino acid becomes a zwitterion Here its overall electrostatic charge is neutral and the

charge expressed by the basic amino group (-NH2 -NH3+) of the amino acid is equal to

the charge expressed by the acidic carboxyl group (-COOH -COO ) The IEP of BSA is

-

pH 48 and indicates that the side chain of the amino acids of BSA contains a greater

amount of acidic functional groups than that of basic functional groups At pH 32 and

74 BSA has a positive and negative charge respectively

Figure 4-21 and 4-22 show adsorption isotherms of BSA onto polystyrene (PS)

latex particles

00

10

20

30

40

0 02 04 06 08

Ceq (mgml)

q (m

gm

2 )

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in

phosphate buffer (PB) at 37oC

91

00

10

30

40

0 02 04 06 08

Ceq (mgml)

q (m

gm

2

20

)

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

(PBS) on PS latex particles

At the IEP of BSA the adsorbed amount (q

Figure 4-22 Adsorption isotherm of BSA adsorption 37oC in phosphate buffered saline

onto PS at equilibrium was

maxim

um at the IEP of BSA This is because the BSA molecules form the most

compact structures at the IEP eliminating the repulsion force between proteins A higher

number of BSA molecules can adsorb on a given surface area by hydrophobic interaction

when in this compact structure This observation agrees well with reported data by other

researchers who also indicated that the maximum adsorption from aqueous protein

solution was observed at the IEP [Mularen 1954 Watkins et al 1977 Oh at el 2000]

There was no significant difference of adsorbed BSA amount in PB and in PBS media at

pH 48 The conformational alteration of BSA was not affected significantly by the ionic

strength of the two solutions There was evidence of the effect of the electrostatic

interaction to some extent even at the IEP since the amount of BSA adsorbed in PBS is

slightly hig

m) of BSA

um indicating that hydrophobic interaction between latex particle and BSA

protein is maxim

her than in PB at this pH

92

The adsorbed amount of protein is shown to decrease at a pH 32 and 74 The

decrease in adsorption efficiency when deviating from IEP of BSA is a result of the

increase in conformational size of the protein molecules and the lateral electrostatic

repulsions between adjacent adsorbed BSA molecules Figure 4-23 shows a schematic of

possible conformational changes at each pH where hydrophobic interaction always exists

between non-polar portion of a protein and hydrophobic portion of solid surface

- - --- ---Latex surface

Zeta potential

BSA(+)

(-)

48 7432pH

(0)

+++ + ++

--+

++-

- - --- ---Latex surface

Zeta poten pH(0)

+++ + ++

--

tial

BSA(+)

(-)

48 7432

+

++-

ming

4

s

al blood Table 4-6 shows the calculated results of

Figure 4-23 Conformation of bovine serum albumin (BSA) on latex particles (Assuthat charge repulsion reduces the effect of the hydrophobic interaction at pH 74)

In addition the adsorbed amount of BSA at pH 32 was larger than that at pH 74

and still less than that at 48 This is because there is the electrostatic interaction between

the positive BSA molecules and negative PS latex particles at pH 32 and electrostatic

repulsion is dominant between negative BSA and negative PS latex particles at pH 7

The amount adsorbed increased with increasing ionic strength in pH 32 and 74 This i

because the high concentration of electrolytes reduces the electrostatic repulsion between

BSA and PS latex particle It should be noted that the BSA concentration used in these

experiments was lower than that found in normal human blood This is because the

equilibrium BSA adsorption onto the latex particles is actually reached at a much lower

concentration than that found in norm

93

adsor rm

ein

leading to a compact conformation of BSA There are two main forces affecting

adsorption amount at an acidic pH of 32 They include the ionic attraction between the

negative latex particle and the positive protein and lateral repulsion between positive

proteins The smallest amount of BSA adsorption was observed at a pH of 74 At this

point the charge repulsion between negatively charged particles and protein molecules

exists the h

of cha

bed BSA amount at equilibrium from the Langmuir-Freundich adsorption isothe

equation

In summary the hydrophobic interaction between PS latex particle and BSA

protein exists and is the dominant mechanism for the adsorption process Conformational

changes of BSA at each pH also affected the adsorption amount Adsorption was highest

at pH 48 because at this pH there is the least amount of ionic repulsion in the prot

and the lateral repulsion between proteins dominant adsorption even though there always

ydrophobic interaction between the solid latex particle and protein The effect

rge repulsion was reduced by the high concentration of electrolytes in PBS medium

Table 4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2) 32 174 48 299 Phosphate buffer 74 136 32 260 48 283 Phosphate buffered saline 74 207

The adsorption isotherms of BSA on PSPMMA core shell particles are shown in

Figure 4-24 in phosphate buffer (PB) and in Figure 4-25 in phosphate buffered saline

(PBS) The adsorbed BSA amount was calculated using the Langmuir-Freundlich

100

94

adsorption isotherm equation and the amount listed in Table 4-7 The trend seen in the

adsorption results was very similar to the case of PS latex The overall adsorption process

was d rticle

rs

hat

0

ominated by hydrophobic interactions between the PSPMMA100 core shell pa

and BSA proteins At pH 48 the adsorbed amount (qm) of BSA on PSPMMA100 core

shell particles was 221 mgm2 in PB and 237 mgm2 in PBS and are the maximum

values recorded among the three pH levels 32 48 and 74 Suzawa and his co-worke

[1982] have obtained similar results This can be also explained by the compact

conformation of BSA molecules at the IEP of BSA which lead to a high protein

population on the solid surface At a pH of 32 and 74 BSA adsorption was less than t

at pH 48 because of the protein conformation change There was a small increase in

adsorbed BSA at pH 32 because of the charge attraction between negative PSPMMA10

core shell particles and positive BSA At pH 74 however the adsorption of BSA was

further reduced by charge repulsion between the negative PSPMMA100 core shell

particles and the negative BSA proteins

000 02 04 06 08

Ceq (mgml)

10

20q (m

gm

302 )

40

50pH=32pH=48pH=74Mode 8)l (pH 4Mode 2)l (pH 3Mode 4)

Figure 4-24 Adsorption isotherm of BSA 37oC in PB on PSPMMA core shell latex

l (pH 7

100

particles

95

The adsorbed amount of BSA increased more rapidly in PBS with increasing ioni

strength at pH 74 This shows that the low concentration of electrolytes promotes

electrostatic repulsion between negative BSA and negative PSPMMA

c

on

x

l 1980

100 core shell

particle As the ion strength of PBS is increased however the high concentration of

electrolyte leads to less electrostatic repulsion promoting more rapid protein adsorpti

By comparing the amount of adsorbed protein between bare PS latex particles and

PSPMMA100 core shell particles the amount of protein adsorption on the PS late

particles is greater than that on the PSPMMA100 core-shell particles [Suzawa et a

1982 Lee et al 1988]

00

10

20

30

40

m2 )

0 02 04 06 08

Ceq (mgml)

q (m

g

pH=32pH=48pH=74Model (pH 2) 3Model (pH 48)Model (pH 74)

Figure 4-25 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA100 core shell

latex particles

96

Table particles calculated the Langmuir-Freundlich isotherm model

4-7 The equilibrium concentration values (qm) of BSA adsorption on PSPMMA100

Media pH qm (mgm ) 2

32 140

48 221 Phosphate buffer

74 130

32 169

48 237 Phosphate buffered saline

74 162

The BSA adsorption mechanism and trend on PSPMMA core shell particles was

very similar to that of the PS latex particle Unlike the case of PS and PSPMMA100 latex

particles the amount of BSA adsorbed onto PSPMMA90PAA10 and PSPMMA75PAA25

core shell particles was higher at pH 32 than pH 48 and 74 Figure 4-26 through 4-29

shows the adsorption isotherms of BSA on PSPMMA90PAA10 and PSPMMA75PAA25

core shell particles The amount of BSA adsorbed increased at pH 32 and pH 48 as the

hydrophilicity was developed by the addition of PAA On the contrary the adsorbed

amount of BSA was dramatically decreased at pH 74 The adsorption amount of BSA on

PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles at equilibrium is listed in

Table 4-8 and 4-9 respectively

Carboxylic acid as well as sulfate groups are distributed on the surface of

PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles and seem to largely affect

the BSA adsorption The isotherms showed very strong pH dependence to the BSA

adsorption onto these core shell latex particles The carboxylic groups (COOH) may

especially enhance the BSA adsorption prominently at pH 32 by hydrogen bonding with

protein molecules The pKa value for the PAA is 40-60 and a good indication that the

97

carboxylic acid groups are protonated at pH 32[Gebhardt et al 1983] The main

adsorption interaction force at pH 32 was generated by hydrogen bonding as well as

charge attraction between latex particle protein even th the conformational

repulsion between proteins results in a reduction of the protein

adsorption at this pH The amount of BSA adsorbed also increased as the PAA amount in

eased The a tion of BSA w inimal however less

than 07 mgm2 at pH 74 Here the carboxylic groups are ionized which increases the

charge repulsion by ionized carboxylic groups (COO-) resulting in PSPMMA-PAA latex

particle

and ough

change and charge

the core shell particles incr dsorp as m

s with much lower protein adsorption

00

20

40

q (m

g2 )

60

m

pH=32pH=48pH=74Model (pH 32)Model (pH 48)

0 02 04 06 08

Ceq (mgml)

Model (pH 74)

o shell

Figure 4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA90PAA10 corelatex particles

98

00

40

60

80

0 02 04

C (mgm

mm

2 )

20

06 08

eq l)

q (

g

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-27 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA90PAA10 core

shell latex particles

Table 4-8 The equilibrium concentration values of BSA adsorption on PSPMMA90PAA10 particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2)

32 361

48 283 Phosphate buffer

74 043

32 519

48 226 Phosphate buffered saline

74 043

99

00

20

40

60

80

100

0 02 04 06 08Ceq (mgml)

q (m

gm

2 )

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

oFigure 4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA75PAA25 latex particles

00

100

20

40

60

80

0 02 04 06 08Ceq (mgml)

q (

m2 )

pH=32pH=48pH=74

mg

Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-29 Adsorption isotherm of BSA 37oC in PBS on PSPMMA75PAA25 core shell

latex particles

100

Table 4-9 The equilibrium concentration values of BSA adsorption on PSPMMA75PAA25 particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2)

32 1083

48 662 Phosphate buffer (PB)

74 075

32 1077

48 681 Phosphate buffered saline (PBS)

74 057

The adsorption of β2-microglobulin (β2M) was determined under one set of

onditions in phosphate buffer at 37oC and pH 74 because of the limited amount of β2M

protein The adsorption isotherms of the β2M protein on the synthesized latex particles

are shown in Figure 4-30 4-31 and 4-32 The β2M protein concentrations for adsorption

isotherm experiment were chosen to be 0015 0030 0045 and 0060mgml since the

β2M concentration for the patients with renal failure can be elevated to similar

concentrations about 005mgml or more up to 50 times the normal level [Nissenson et

al 1995] There were steep initial slopes of the adsorption isotherm curves of β2M on

latex particles in all isotherms indicating a high affinity adsorption type It seems that the

hydrophobic interaction is enough for the adsorption process of β2M proteins to occur on

the latex particles even though charge repulsion exists between protein and latex particle

at pH 74 The complete plateau regions were not seen for all isotherms indicating that

β2M proteins were not saturated at this level and there are still available sites for β2M

c

adsorption on latex particles The initial slope of the β2M isotherms for PS was steeper

101

than that of any of the other latex particle PSPMMA100 PSPMMA90PAA10 and

PSPMMA 75PAA25

00

02

0 0002 0004 0006 0008

04

06

10

Ceq (mgml)

q (m

2 )

08

gm

PSPSPMMA100Model (PS)Model (PSPMMA100)

Figure 4-30 Adsorption isotherm of β2M onto PS and PSPMMA100 latex particles in PB

at 37 C and pH 74 o

00

02

04

06

08

10

0 0002 0004 0006 0008C (mgml)

q (m

m2 )

eq

g

PS

PSPMMA90PAA10

Model (PS)

Model (PSPMMA90PAA10)

Figure 4-31 Adsorption isotherm of β2M onto PS and PSPMMA90PAA10 latex particles

in PB at 37oC and pH 74

102

00

02

04

06

08

10

0 0002 0004 0006 0008Ceq (mgml)

q (m

gm

2 )

PS

PSPMMA75PAA25

Model (PS)Model (PSPMMA75PAA25)

Figure 4-32 Adsorption isotherm of β2M onto PS and PSPMMA75PAA25 latex particles

in PB at 37oC and pH 74

The equilibrium concentration values (qm) of β2M adsorption on latex particles are

calculated and listed in Table 4-10 The difference between the maximum amounts of

β2M adsorption on all the latex particles was not significantly different and their values

were between 069 and 08mgm2

Table 4-10 The equilibrium concentration values of β2M adsorption calculated the Langmuir-Freundlich isotherm model

Latex particles qm (mgm2)

PS 080

PSPMMA100 069

PSPMMA90PAA10 072

PSPMMA75PAA25 072

103

432 Adsorbed Layer Thickness

There are several possible explanations for large amount of BSA adsorbed at acidic

pH levels including the end-on adsorption pattern of BSA the flexibility of BSA

molecules the tilting of protein molecules due to the asymmetry of the charge

distribution or multilayer formation [Peula et al 1993] The adsorbed BSA thickness (δ)

can be calculated by the following equation [Chiu et al 1978] This equation was also

cited again by other researchers [Shirahama et al 1985]

BSA

mqρπ

δbullbull

=33

(4-7)

where π

33 is the packing factor qm is the plateau value of the adsorbed am

ρBSA is the hich corresponds to the reciprocal of its known

partial specific volume The layer thickness (δ) of BSA proteins on synthesized latex

partic

he IEP [Shirahama et al 1985] Compared to the

hydrodynamic dimensions of BSA 140 times 40 times 40 Aring3 [Fair et al 1980 Peter 1985] these

nd

ode It is unnecessary ultilayering because thickness do

exceed 140 Aring The layer thickness (δ) of BSA proteins on synthesized latex particles

e red saline (PBS) was ran 28 ~ 83 Aring at 37oC and pH 48 These

lso listed in Table 4-1

ount and

density of BSA molecule w

les in phosphate buffer (PB) ranged from 27 - 81 Aring at 37oC and pH 48 These

calculated values are listed in Table 4-11 Only δ values at pH 48 the isoelectric point

(IEP) of BSA are important because intra- and intermolecular electrostatic repulsion of

protein molecules is minimized at t

values of layer thickness indicate that the BSA molecules adsorb by side-on (40Aring) a

end-on (140Aring) m to consider m

not

in phosphat buffe ged of

calculated values are a 2

104

Table 4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37oC pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

32 21 17 44 133

48 37 27 35 81

74 17 16 5 9

Table 4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37oC

pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

32 32 21 64 132

48 35 29

74 26 20 5 7

28 83

1n

1n

neqm

So k is closely related to the initial slope of the isotherm showing the affinity

between BSA and latex particles [Yoon et al 1996] A key point in the characterization

433 Gibbs Free Energy of Protein Adsorption

In the Langmuir isotherm model k represents the ratio of adsorption to the

desorption rate constants This definition is not applicable to the Langmuir-Freundlich

isotherm model but its meaning is similar [Yoon et al 1996] In this sense k is closely

related to the affinity between protein molecules and latex particles When the

equilibrium concentration Ceq reaches zero the value of Ceq will be very small (1gtgt

kCeq ) and then the equation 4-6 becomes

(4-8)

of adsorption processes on solid-liquid interfaces is to determine the change in Gibbsrsquos

free energy (∆Gdeg) one of the corresponding parameters for the thermodynamic driving

forces during adsorption The traditional method to evaluate adsorption thermodynamic

kCqq 1=

105

constants is based on measurement of adsorption isotherms under static conditions

[Malm ten 1998] The Langmuir-Freundlich ads lf

easily found from Then this enables calculation of the free-

energy change ∆G during adsorption expressed as

(4-9)

where R is the general gas constant (R = 8314Jmiddotmol K ) and T is the absolute

tem ture Gibb e energy A P

listed in Table 4-13 and 4-14 respectively As the ∆Gdegads ore negativ e

pro adsorption on a solid substra ill be more favorable

e of BSA adsorption in phosphate o

pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

s orption equilibrium constant k can be

a slope of the isotherm

degads

lfads kRTG lnminus=∆ o

-1 -1

pera s fre change of BS adsorption values in B and PBS are

values are m e th

tein te w

Table 4-13 The values of Gibbs free energy changbuffer (PB) at 37 C

32 -2411 -2726 -2846 -2805

48 -2352 -2383 -2198 -1972 ∆Go

80

ads (kJmol)

74 -2452 -888 -324 -2

Table 4-14 The values of Gibbs free energy change of BSA adsorption in phosphate

pH PS PSPMMAbuffered saline (PBS) at 37oC

100 PSPMMA90PAA10 PSPMMA75PAA25

32 -2315 -2620 -2312 -2683

48 -2149 -2299 -2090 -2138 ∆Go

(kJmads ol)

74 -2430 -1992 -258 -263

Table 4-15 shows the calculated values of Gibbs free energy change of β M

adsorption on synthesized latex particles PSPMMA PAA core shell latex particle has

2

75 25

106

largest negative Gibbs free energy of β2

2

M adsorption indicating largest affinity betw

β

een

2M adsorption in phosphate buffere (PB) at 37 C and pH74

PS PSPMMA100

M and latex particle

Table 4-15 The values of Gibbs free energy change of βo

PSPMMA90PAA10 PSPMMA75PAA25

∆Go03 ads

(kJmol) -3626 -3466 -3659 -39

The Gibbs free energy of β2M was much more negative in phosphate buffer (PB)

than that of BSA in physiological condition (37 C and pH 74) o from the Table 4-13 and

the Figure 4-31 This indicates that β2M molecules more readily adsorb on latex particles

BSA This can be explained by the rate of diffusion and accessibility of β2M molecules to

the latex particle ier and faster for β2M m an BSA because of

size (molec ight) erical shape molecules It o be

due to the higher hydrophobic force and less electrostatic repulsion that contribute to the

adsorption of β2M molecules not as prevalent in the case of BSA because the IEP of β2M

(57) is clo H 74) than that of BSA (48) causing β2M to

be less electro cally lled p ro

can be seen that the ∆ β2M adsorption onto the synth article

increasing wi hy icity o x particles s the ∆Goads of the BSA

molecules is decreasing or constant This phenomenon supports the theory that the

select lizing

than BSA and that the affinity of β2M for the latex particle is much larger than that of

surface It is eas olecules th

the smaller ular we and sph of β2M may als

ser to physiological condition (p

stati repe from the latex articles than BSA F m Figure 4-33 it

Goads of esized latex p s is

th the drophil f the late wherea

ive adsorption of β2M molecules over BSA molecules can be enhanced by uti

more hydrophilic PSPMMA90PAA10 and PSPMMA75PAA25 core shell latex particles

107

-60

-50

1 2 3 4

-40∆Gad

s

-30

10

0

o (k

Jm

o-

-20l)

β2-Microglobulin (β2M)Bovine serum albumin (BSA)

Figure 4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37

and pH 74

434 Kinetics of Adsorption

to

oC

Protein adsorption is a complex process known to have a large biological impact

and is currently not well understood quantitatively because of extreme sensitivity to pH

the concentration of other electrolytes and molecules present temperature and many

other factors that can change in the physiological system Accurate knowledge of the

adsorption kinetics under a given set of conditions is a prerequisite for elucidating the

mechanisms of many fundamental biological processes to be predicted at the molecular

level [Regner 1987] This adsorption kinetics approach is used in protein adsorption

studies because of the uncertainty related to the time needed for the interfacial proteins

reach the equilibrium It is generally accepted that the process of protein adsorption is

comprised of the following steps a) transport toward the interface b) attachment at the

interface c) eventual structural rearrangements in the adsorbed state d) detachment from

PS PSPMMA100 PSPMMA90PAA10PSPMMA75PAA25

108

the interface and transport away from the interface Each of these steps can affect the

overall rate of the adsorption process

The quantitative analysis of the protein adsorption kinetics requires that the protein

amount adsorbed is known as a function of time The kinetics tests were carried out in

phosphate buffer (PB) at 37oC and pH 74 a condition similar to physiological condition

The protein concentrations chosen were 07mgml for BSA and 006mgml for β2M

which are the maximal concentrations used in an adsorption isotherm test A time-based

evaluation of the adsorption was made by measuring the concentration of protein in

solution at different incubation times The results are shown below from Figure 4-34 to 4-

37 The adsorption kinetics of proteins onto th cond-e latex particles is expressed as a se

order model [Oumlzacar 2003] shown by the following equation

2)( tet qqk

dtdq

minus= (4-10)

Integrating above equation and applying the boundary conditions yields

ee

t tq

+=

1 (4-11)

where q

qkq

t

2

2

2

the previous adsorption isotherm results representing good reproducibility of adsorption

t and qe stand for the amount (mgm ) of protein adsorbed at time t and at

equilibrium respectively k is the equilibrium rate constant of second-order adsorption

(m mghr) and t is the incubation time (hr)

The fitting parameters calculated by using the equation 4-11 are listed in table 4-16

From this data it is seen that the amount of BSA adsorbed at equilibrium was higher than

that of β2M on PS and PSPMMA100 latex particles but less than that of β2M on

PSPMMA90PAA10 and PSPMMA75PAA25 latex particles These results are similar to

109

00

05

15

20

0 2 4 6 8 10 12 14

Time (hr)

rot

n ad

sorb

d (

10

25

Pei

em

gm

2

BSA2nd order model (BSA))β2M2nd order model (β2M)

Figure 4-34 The kinetics of protein adsorption in PB on PS latex particles at 37oC and

pH 74

00

05

10

15

20

25

30

0 2 4 6 8 10 12 14

Time (hr)

Prte

inds

oo

arb

ed (

mg

m2 )

BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-35 The kinetics of protein adsorption in PB on PSPMMA100 latex partic

37les at

oC and pH 74

110

00

03

06

09

12

15

0 2 4 6 8 10 12 14

Time (hr)

Prot

ein

adso

rbed

(m

gm

2 ) BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-36 The kinetics of protein adsorption in PB on PSPMMA90PAA10 latex

particles o at 37 C and pH 74

00

03

06

09

12

15

0 2 4 6 8 10 12 14

Time (hr)

BS

A ad

sorb

ed (

mg

m2 )

BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-37 The kinetics of protein adsorption in PB on PSPMMA75PAA25 latex

particles at 37oC and pH 74

111

test The variation of β2M equilibrium adsorption amount was nearly negligible for all

latex particles The plateau region is reached earlier for β2M than BSA on all latex

particles and the K values (equilibrium rate constant of second order adsorption) for the

β2M adsorption process onto latex particles are much larger than for BSA adsorption

These values indicate that the β2M molecules have a higher adsorption rate onto the latex

particles than the BSA molecules

Table 4-16 Fitting parameters of second-order kinetic model BSA β2M

Latex particles qe (mgm2) K (hr-1) qe (mgm2) K ( -1) hr

PS 144 67 074 7549

PSPMMA100 175 353 076 4438

PSPMMA90PAA10 046 97 075 5695

PSPMMA75PAA25 060 179 075 5168 K = (kqe) hr-1

There are several explanations for the phenomena observed in these experiments

First the smaller sizes of β2M molecules allow easier diffusion and a close approach to

the latex substrate Second a reduced number of hydrophobic domains on the latex

particle surface replaced by sulfate groups (-OSO3-) and ionized carboxylic groups

(COO-) are not favorable for larger BSA molecules Third the conformational change by

intra- and inter-molecular repulsion would be faster in β2M due to smaller molecular

weight Finally the ionic repulsion between β2M and the latex particles is less than that

condition (pH 74) than IEP (48) of BSA

between BSA and latex particles because IEP (57) of β2M is closer to the tested

112

44 Blood Compatibility

Blood compatibility can be defined as the property of a material or device tha

permits it to function in contact with blood without inducing any adverse reactions

Successful functioning of blood contacting biomedical materials or devices in contact

with blood strongly depends on the extent to which they damage blood during operation

High shear stresses turbulence rela

t

tively long contact times and cellular impact with

artificial surfaces are most common events experienced by blood in contact with artificial

1978 Winslow and et al

1993] Therefore before any materia e placed into the human b

biocompatibility testing must be the first performed in the view of safety The hemolysis

s a method for pr ary screening of biomaterials to determine

h blood a er circulating body fluid The tests estimat

oglobin released from th blood cell d e induced

foreign material

There are several types of blood cells in our body but the ones that make blood red

are erythrocytes or red blood cells (RBCs) Red blood cells (erythrocytes) are the most

numerous type in the blood These cells are average about 48-54 million per cubic

millimeter (mm3 same as a microliter (microl)) of blood but these values can vary over a

larger range depending on such factors as health and altitude RBCs are red because they

contain hemoglobin which is a protein that contains iron When blood circulates through

lungs oxygen binds to the hemoglobin and is then delivered to all the parts of body

RBCs are made in the bone marrow They start out as a nucleus like other cells but

when they fill up with hemoglobin the nucleus becomes smaller until it disappears

Without a nucleus RBCs are fragile and live only about 120 days In a normal healthy

devises and can cause hemolysis or thrombosis [Bernhard et al

ls can b ody

test serves a elimin the

biocompatibility wit nd oth e the

concentration of hem e red amag by a

113

body about 2 million red blood ne marrow produces new

ones j ing

dy

le

cells die per second But bo

ust as fast RBCs are about 75 micrometer across and are responsible for carry

oxygen salts and other organic substances to the cells and tissues throughout our bo

In humans and most mammals the cells are concave (indented in the middle) and flexib

enough to squeeze through even the tiniest of blood vessels the capillaries

Figure 4-38 Image of red blood cells

(Sourcehttpwwwpbrchawaiiedubemfmicroangelarbc1htm)

ch

100 90 10 75 25 f

RBCs are among the first cells that come into contact with a foreign material

injected in the blood Therefore a hemolysis assay gives information about the

biocompatibility in the case of an in vivo application The concentrations of prepared

latex particles used for the tests in this research are 05 25 and 50 (ww) and the

incubation time under moderate shaking was 30 minutes at 37oC Test results are shown

in Figure 4-39 The amount of blood cells that ruptured when in contact with the PS latex

particles was largely increased from about 30 to 91 as the solid content of PS

increased from 05 to 50 However hemolysis by the core shell latex particles su

as PSPMMA PSPMMA PAA and PSPMMA PAA was less than 35 O

114

particular interest was that hemolysis caused by PSPMMA PAA particles at 50

solid concentration was less than 02 hemolysis the lowest value among the latex

particles The low hemolysis value indicates that the PSPMMA PAA core shell latex

particles are the most blood compatible at all solid contents Other core shell particles

such as PSPMMA and PSPMMA PAA also had excellent blood comp

90 10

90 10

100 75 25 atibility with

low RBS breakage

01 2 3

20

40

60

80

100

Hem

olys

is (

)

052550

Figure 4-39 He

PS PSPMMA100 PSPMMA PAA90 10

molysis caused by latex particles n=5

4PSPMMA75PAA25

CHAPTER 5 SUMMARY OF RESULTS CONCLUSIONS AND RECOMMENDATION FOR

FUTURE WORK

51 Summary of Results

The goal of this study was to synthesize monodisperse polymeric latex particles

with tailored properties and to investigate the fundamental interactions between the

synthesized latex particles and target proteins An understanding of the fundamental

mechanism of selective adsorption is strongly required in order to maximize the

separation performance of membranes made of these materials so that it may applicable

to hemodialysis therapy for end stage renal disease (ESRD) patients For the achievement

of above research goal several investigations have been performed Analysis of the

obtained data leads to several conclusions as discussed below

Monodisperse polystyrene seed latex particles were synthesized with the size

crosslinking agent to make the latex particles more hydrodynamically stable The

conversion of monomer to polymer was over 95 and seed latex particles are spherical in

shape and have smooth surface From these small seeds bigger latex particles as large as

800nm were synthesized using a semi-continuous seeded emulsion polymerization

process The particles with a mean diameter less than 500nm are very spherical in shape

and highly uniform in particle size distribution However as the particles grow larger

than 500nm in mean diameter they became non-spherical with an uneven surface This

irregularity of particle surface can be attributed to the non-homogeneous monomer

ranges between 126plusmn75 and 216plusmn53 nm Divinylbenzene (DVB) was used as a

115

116

swelling into the shell of a growing polymer which can be controlled by the factors such

as temperature agitation speed initiator feeding rate and surfactant amount required to

stabilize the colloidal system It was determ rfactant to monomer ratio

dec ic

acid (AA) monomers were intro rticle surfaces more

hydro

to

ting of a

orm

ration is seen between

3002

0 cm-

0cm-1

-1

03

ined that as the su

reased the mean particle diameter increased Methyl methacrylate (MMA) and acryl

duced to make the latex pa

philic than bare polystyrene particles The prepared core shell latex particles were

made of PMMA100 PMMA90PAA10 and PMMA75PAA25 PMMA100 is the PMMA

homopolymer shell on a PS core PMMA90PAA10 shell is a copolymer with a PMMA

PAA ratio of 90 to 10 by weight PMMA75PAA25 shell is a copolymer consis

PMMA to PAA ratio of 75 to 25 by weight The particle size of these core-shell

particles were prepared to be about 370nm in mean diameter

The successful synthesis of latex particles was confirmed using Fourier Transf

Infrared spectroscopy (FTIR) The C-H aromatic stretching vib

cm-1 and 3103 cm-1 for PS latex particles The C-H asymmetrical stretching and

symmetrical stretching vibration peaks of CH2 for all latex particles is seen at 290

1and 2850 cm-1 respectively There is a carbonyl (C=O) characteristic band at 173

wavenumber for PSPMMA core-shell latex particles The broad OH group peak at

3400 cm

100

can be seen in the spectroscopy of the particle with PMMAPAA shell

(PSPMMA PAA and PSPMMA PAA ) The peak intensity of OH group of

PSPMMA PAA was larger than that of PSPMMA PAA relatively due to more AA

monomer content in PSPMMA PAA than in PSPMMA PAA

In the zeta potential measurements polystyrene (PS) latex particles had negative

values between -291 mV and -599 mV in the phosphate buffer (PB) and between -2

90 10 75 25

75 25 90 10

75 25 90 10

117

mV and -278 mV in the phosphate buffered saline (PBS) at 25oC and pH 21-78 These

negative zeta potential values for PS latex particles are due to ionization of the sulfate

groups (-OSO3-) originated from the initiator Zeta potential values of the PSPMMA

core shell latex particles were also negative between -28 mV and -505 mV in PB and

between -143 mV and -186 mV in PBS at pH 21-78 Negative values are also

generated by dissociation of sulfate groups on a particle surface The zeta potential value

of PSPMMA PAA and PSPMMA PAA were of negative values between -36

and -678 mV in PB medium and -147 mV and -193 mV in PBS medium at 25

100

s

7 mV

MMA75PAA25 and

PSPM

otein

g the

l change of BSA at

each w

90 10 75 25

oC For

PSPMMA PAA core shell particles zeta potential values were between -291 mV and

-520 mV in PB media and between -115 mV and -210 mV in PBS media Sulfate

groups contributed to the negative zeta potential values in PSP

75 25

MA90PAA10 core shell particles Carboxylic groups from polyacrylic acid also

contribute to the negative surface charge for the latex particles at greater than pH 70

however the degree of their contribution was not detectable from the zeta potential

measurements The absolute zeta potential values of the synthesized latex particles in PB

were higher than that in PBS This is because of the high concentration of sodium

chloride electrolyte in PBS that compress electrical double layer thickness

The bicinchoninic acid (BCA) assay technique was used to determine the pr

adsorption and all isotherms were fitted to the Langmuir-Freundlich isotherms usin

nonlinear regression method The hydrophobic interaction between PS latex particle and

BSA protein was dominant for the adsorption process Conformationa

pH also affected the adsorption amount which was highest at pH 48 because of lo

ionic repulsion making compact conformation of BSA The next larger amount of BSA

118

adsorbed onto PS was at pH 32 There were two main forces affecting the amount of

adsorption at acidic pH values One is ionic attraction between negative latex particle a

positive protein The other is lateral repulsion between positive proteins The smallest

amount of BSA adsorption was at pH 74 where charge repulsion between negative

particles and proteins is present at this pH Lateral repulsion between proteins wa

dominant for protein adsorption even though there always exists the hydrophobic

interaction between solid latex particle and protein The charge repulsion was reduced by

the high concentration of electrolytes in the PBS medium The BSA adsorption process

on PSPMMA core shell particles was very similar to the case of PS latex particle where

hydrophobic interaction between latex particle and protein was dominant However t

adsorbed BSA amount was less than that of PS in PBS The adsorbed amount of BS

PSPMMA

nd

s

he

A on

an

ed

n The main

adsor

90PAA10 and PSPMMA75PAA25 core shell particles was higher at pH 32 th

pH 48 and 74 The adsorbed BSA amount became higher at pH 32 and pH 48 as the

hydrophilicity was increased by the higher PAA amount On the contrary the adsorb

amount of BSA was dramatically decreased at pH 74 Carboxylic acid groups as well as

the sulfate groups are distributed on the surface of PSPMMA90PAA10 and

PSPMMA75PAA25 core shell particles and largely affect the BSA adsorptio

ption interaction forces at pH 32 was generated by hydrogen bonding as well as

charge attraction between latex particle and protein even when the conformational change

and charge repulsion between proteins reduce the adsorption of protein at this pH The

adsorbed BSA amount was also increased as the amount of PAA increases However the

adsorption of BSA was minimal (less than 07 mgm2) at pH 74 where the carboxylic

groups are ionized leading to more enhancement of charge repulsion by ionization of the

119

carboxylic groups (COO-) Because of this the PSPMMA-PAA latex particles have a

much lower protein adsorption

There were steep initial slopes for adsorption isotherms of β

in

s

there

M

r

e

es

o

2M on latex particles

all isotherms indicating a high affinity type adsorption The complete plateau region

were not seen for all isotherms indicating that β2M proteins were not saturated and

are still available sites for β2M adsorption on latex particles The initial slope of β2

isotherms for PS was steeper than for the other latex particles PSPMMA100

PSPMMA90PAA10 and PSPMMA75PAA25

The layer thickness (δ) of BSA proteins on synthesized latex particles in phosphate

buffer (PB) was in the range of 27 - 81 Aring at 37 C and pH 48 Only the δ values at pH

48 the isoelectric point (IEP) of BSA are important because intra- and inter-molecular

electrostatic repulsion of the protein molecules is minimized at the IEP Compared to the

hydrodynamic dimensions of BSA 140 times 40 times 40 Aring these values of the adsorbed laye

thickness indicates that the BSA molecules adsorb by the side-on (40Aring) and end-on mod

(140Aring) Consideration of the multi-layering is unnecessary because the adsorbed

thickness of BSA do not exceed 140 Aring

The Gibbs free energy of β

o

3

2M was more negative in phosphate buffer (PB) than

that of BSA in physiological condition (37 C and pH 74) indicating that βo2M molecul

are more favorably adsorbed on the latex particles than BSA and that the affinity of β2M

was much larger than BSA This may be explained by the rate of diffusion and

accessibility of β2M molecules being easier and faster than that for BSA because of

smaller size (or weight) and spherical shape of β2M molecules It may also be possible t

explain that more hydrophobic forces (less electrostatic repulsion) contributes to

120

adsorption of β2M than to that of BSA because the IEP of β2M (57) is closer to

physiological condition (pH 74) than that of BSA (pH 48) The selective adsorption of

β2M m

in β2M

larger than in BSA adsorption indicating

that fa

an that between BSA and latex particles because

IEP (

ch as

Other

olecules over BSA molecules can be enhanced by developing more hydrophilic

PSPMMA90PAA25 and PSPMMA75PAA25 core shell latex particles

The adsorption kinetics of proteins on latex particles was expressed as a second-

order model The plateau region for β2M was reached earlier than BSA on all latex

particles and the K values (equilibrium rate constant of 2nd order adsorption)

adsorption process on latex particles were much

ster adsorption rate of β2M molecules than that of the BSA molecules There are

several explanations for these phenomena First the smaller size of β2M molecules can

more easily diffuse and approach to the latex substrate Second enhanced hydrophilic

domain on the latex particle surface by sulfate groups (SO4-) and ionized carboxylic

groups (COO-) is not favorable for larger BSA molecules but is for smaller β2M to be

anchored Third the conformational change by intra- and inter-molecular repulsion

would be faster in β2M due to lower molecular weight Finally the ionic repulsion

between β2M and latex particles is less th

57) of β2M is closer to the tested condition (pH 74) than IEP (48) of BSA

In the biocompatibility test the number of red blood cells (RBCs) ruptured by PS

latex particles ranged from 30 to 91 as the solid content of PS increased from 05 to

50 respectively However hemolysis of the other core shell latex particles su

PSPMMA100 PSPMMA90PAA10 and PSPMMA75PAA25 were less than 35 The

PSPMMA90PAA10 particles at all applied solid concentrations had less than 02

hemolysis and was the most blood compatible among the prepared latex particles

121

core shell particles such as PSPMMA100 and PSPMMA75PAA25 also had excelle

compatibility with low RBC breakage

52 Conclusions

The major conclusions from this study include

nt blood

bull microglobulin (β2M) over that of the bovine serum albumin (BSA) at pH 74

was achieved by controlling the PMMA and PAA ratio to optimize

e particles posses the most desirable requirements for use in a human dialysis

ns

icles

olecule

bull

Using engineered core shell latex particles selective adsorption of β2

physiological condition was demonstrated

bull The rate of adsorption of the β2M was also much higher than that of BSA This

hydrophobichydrophilic microdomain size

bull The core shell particles also exhibited excellent biocompatibility with blood Thes

membrane

53 Recommendation for Future Work

With the results and conclusions obtained through this study several suggestio

may be given for future work as follows

bull Competitive adsorption tests of whole blood proteins on synthesized latex partunder physiological condition

bull Preparation of latex particles with optimum hydrophobic hydrophilic microdomains to maximize selective adsorption and removal of toxin molecules

bull Membrane fabrication by suitable pore size design to discriminate a toxin mby size sieving as a function of particle layers

Evaluation of membrane performance such as flow rate in water and in simulated blood fluid (SBF)

bull Application for hemodialysis as well as other technologies such as a water remediation

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al of Medicine 21 888-892

s

ilure 25 419-430

o

of protein using bicinchoninic acid Analytical Biochemistry 150 76-85

Smith on polymerization Journal of Chemical Physics 16 592-599

histocompatibility antigen HLA-A2 at 26Ǻ resolution Journal of Molecular Biology 219 277-319

chloromethyl functionality Colloids and Surfaces a-Physicochemical and Engineering Aspects 135 293-297

(1998) Functionalized monodisperse particles with chloromethyl groups for thcovalent coupling of proteins Macromolecules 31 4282-4287

and microfiltration Desalination 139 191-198

3 673 133

Hatakeyama M Ohba R Hatori H Yoshikawa T Suzuki F Oomori A Tanaka H Kawaguchi H Watan

18 877-881

acrylic acid copolymer latex Colloid and Polymer Science 263 141-146

hama H Suzuki K and Suzawa T (1989) Bovine hemoglobin adsorption opolymer lattices Journal of Colloid and Interface Science 129 4

Singer J M and Plotz C M (1956) Latex Fixation Test 1 application to the serologicdiagnosis of rheumatoid arthritis American Journ

Singh N P Bansal R Thakur A Kohli R Bansal R C and Agarwal S K (2003)Effect of membrane composition on cytokine production and clinical symptomduring hemodialysis a crossover study Renal Fa

Smith P K Krohn R I Hermanson G T Mallia A K Gartner F H ProvenzanM D Fujimoto E K Goeke N M Olson B J and Klenk D C (1985) Measurement

W V and Ewart R H (1948) Kinetics of emulsi

138

Snyder H W Cochran S K Balint J P Bertram J H Mittelman A Guthrie T Hand Jones F R (1992) Experience with protein α-immunoadsorption in treatmentresistan

t adult immune thrombocytopenic purpura Blood 79 2237-2245

Solomon T W Fryhle C B (2000) Organic chemistry 7th Ed John Wiley amp Sons

Song Y Q Sheng J Wei M and Yuan X B (2000) Surface modification of polysulfone membranes by low-temperature plasma-graft poly(ethylene glycol)

Sriniv characterizations of polymer with thier antithrombogenic characteristics Marcel Dekker New York

Suzaw ovine serum albumin on synthetic polymer lattices Journal of Colloid and Interface Science 78 266-268

Suzawa T Shirahama H and Fujimoto T (1982) Adsorption of bovine serum albumin onto homo-polymer and co-polymer lattices Journal of Colloid and

Taken itness of

9 10-14

412

Tanfo New York

Tangp ion of chitosan films- effects of hydrophobicity on protein adsorption Carbohydrate

Trinh S E (2002) Crystal structure of monomeric human beta 2 microglobulin reveals clues to its

Trotta F Drioli E Baggiani C and Lacopo D (2002) Molecular imprinted polymeric membrane for naringin recognition Journal of Membrane Science 201

Soderquist M E and Walton A G (1980) Structural changes in proteins adsorbed on polymer surfaces Journal of Colloid and Interface Science 75 386-397

New York

onto polysulfone membranes Journal of Applied Polymer Science 78 979-985

asan S Sawyer P N (1971) Correlation of the surface charge

a T and Murakami T (1980) Adsorption of b

Interface Science 86 144-150

aka T Itaya Y Tsuchiya Y Kobayashi K and Suzuki H (2001) Fbiocompatible high-flux hemodiafiltration for dialysis-related amyloidosis BloodPurification 1

Tamai H Hasegawa M and Suzawa T (1989) Surface characterization of hydrophilic functional polymer latex particles Journal of Applied Polymer Science 38 403-

rd C (1967) Physical chemistry of macromolecules John Wiley amp Sons

asuthadol V Pongchaisirikul N and Hoven V P (2003) Surface modificat

Research 338 937-942

C H Smith D P Kalverda A P Phillips S E V and Radford

amyloidogenic properties Proceedings of the National Academy of Sciences of the United States of America 99 9771-9776

77-84

139

Tuncel A Tuncel M Ergun B Alagoz C and Bahar T (2002) Carboxyl carrying large uniform latex particles Colloids and Surfaces a-Physicochemical and Engineering Aspects 197 79-94

Ugelstad J Kaggerud K H Fitch R M (1980) Polymer colloid II Plenum New York

USRDS (2003) United State Renal Date System (USRDS) 2003 annual report

Van Noordwijk J (2001) Dialyzing for life the development of the artificial kidney

Vandpolymerisation of styrene from characterisation of polymer end groups Chemical

Vanholder R Desmet R Vogeleere P and Ringoir S (1995) Middle molecules

1992)

f the American Society of Nephrology 11 2344-2350

38

anes by low temperature plasma induced graft polymerization Journal of Membrane Science 209 255-269

Westhssing with renalin on serum beta2 microglobulin and

complement activation in hemodialysis patients American Journal of Nephrology

Wiec

formation Analytical Biochemistry 175 231-237

WilliaAn evaluation of protein assays for quantitative determination of drugs

Journal of Biochemical and Biophysical Methods 57 45-55

Kluwer Academic Dordrecht

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and Process Engineering 51 89

toxicity and removal by hemodialysis and related strategies Artificial Organs 19 1120-1125

Vincent C Chanard J Caudwell V Lavaud S Wong T and Revillard J P (Kinetics of I-125 beta2 microglobulin turnover in dialyzed patients Kidney International 42 1434-1443

Ward R A Schmidt S Hullin J Hillebrand G F and Samtleben W (2000) A comparison of on-line hemodiafiltration and high-flux hemodialysis A prospectiveclinical study Journal o

Watkins R W and Robertson C R (1977) Total internal reflection technique for examination of protein sdsorption Journal of Biomedical Materials Research 11915-9

Wavhal D S and Fisher E R (2002) Hydrophilic modification of polyethersulfone membr

uyzen J Foreman K Battistutta D Saltissi D and Fleming S J (1992) Effect of dialyzer reproce

12 29-36

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ms K M Arthur S J Burrell G Kelly F Phillips D W and Marshall T (2003)

140

Winchester J F Ronco C Brady J A Cowgill L D Salsberg J Yousha E Choquette M Albright R Clemmer J Davankov V Tsyurupa M PavlL Pavlov M Cohen G Horl W

ova Gotch F and Levin N W (2002) The next

step from high-flux dialysis application of sorbent technology Blood Purification

Winchester J P Ronco J A Brendolan A Davankov V Tsyurupa M Pavlova L 2001)

oglobin

Yang Q and Yang C Z (2000) Formation of an

Yoon J Y Park H Y Kim J H and Kim W S (1996) Adsorption of BSA on highly carboxylated microspheres- quantitative effects of surface functional groups

Zollars R L (1979) Kinetics of the emulsion polymerization of vinyl acetate Journal of

Zou oss-

Zou D Ma S Guan R Park M Sun L Aklonis J J and Salovey R (1992)

20 81-86

Clemme J Polaschegg H D Muller T E La Greca G Levin N W (Rationale for combined hemoperfusionhemodialysis in uremia Contribution of Nephrology 133 174-179

Winslow R M Vandergriff K D Motterlini R (1993) Mechanism of hemtoxicity Thrombosis and Haemostasis 70 36-41

S C Ge H X Hu Y Jiang Xpositively charged poly(butyl cyanoacrylate) nanoparticles stabilized with chitosColloid and Polymer Science 278 285-292

and interaction forces Journal of Colloid and Interface Science 177 613-620

Applied Polymer Science 24 1353-1370

D Derlich V Gandhi K Park M Sun L Kriz D Lee Y D Kim G Aklonis J J and Salovey R (1990) Model filled polymers 1 synthesis of crlinked monodisperse polystyrene beads Journal of Polymer Science Part A Polymer Chemistry 28 1909-1921

Model filled polymers 5 synthesis of cross-linked monodisperse polymethacrylate beads Journal of Polymer Science Part A Polymer Chemistry 30 137-144

Korea on August 7 1968 He obtained a Bachelor of Science degree in Chemistry from

Keim d graduate school and earned a

resear n Korea Institute of Science and

Coati e

ia

Busin

BIOGRAPHICAL SKETCH

Sangyup Kim was born and raised in the small town of Sung-Joo Kyungbook

yung University (Daegu Korea) in 1993 He entere

Master of Science degree in Chemical engineering in 1995 He worked as a student

cher for 2 years at Textile Polymer Division i

Technology (KIST) in Seoul Korea Next he worked over a year at the Automobile

ng Division in DongJoo-PPG in Korea In August 2001 he began studying for th

degree of Doctor of Philosophy in materials science and engineering at the University of

Florida in Gainesville After getting his PhD he plans to work at the Digital Med

ess Division of Samsung Electronics in Korea starting in January 2006

141

Copyright 2005

by

Sangyup Kim

To all who made this work possible

ACKNOWLEDGMENTS

I owe what I am today and this moment to many people without whom I could not

have been here First of all I would like to express my sincere and deepest appreciation

to my parents wife Mikyung daughters Heehyung and Erin whose love patience and

support helped me pursue my dream I gratefully acknowledge my advisor Dr H El-

Shall who gave me an opportunity to become part of an exciting and beneficial research

project and exert myself in the research work His guidance knowledge and stimulating

scientific and critical thinking have contributed to my success to a great extent and

inspired me to dedicate myself to research work I especially appreciate Dr R Partch as a

co-advisor who has helped me conduct experiments with valuable discussion for my

research from my first year I am greatly thankful to Dr C McDonald whose idea and

valuable experimental advice and discussion in the area of polymer chemistry and

membrane technology helped cultivate my interest in this area and were vital to

performing this research Without his contribution this work could not have been

achieved I also thank my doctoral committee members Dr R Singh Dr A Zaman and

Dr B Koopman for appreciated comments

Financial support and equipment use for research from the Particle Engineering

Research Center at the University of Florida were vital in continuing my study for more

than 4 years My sincere thanks are due to my former advisor Dr Kiryoung Ha in

Keimyung University who helped me open my eyes and broaden my thought and

introduced me into a limitless polymer field I would like to also thank to past and current

iv

members in Dr El-Shallrsquos research group for their assistance advice and support I

sincerely appreciate Stephen for the assistance of thesis writing

v

TABLE OF CONTENTS page

ACKNOWLEDGMENTS iv

LIST OF TABLES viii

LIST OF FIGURES x

1 INTRODUCTION 1

2 BACKGROUND AND LITERATURE SURVEY5

21 The Significance of End Stage Renal Disease (ESRD)5 22 Hemodialysis (HD) Treatment 6 23 Advances in Membrane Technology 8 24 Sorbent Technology16 25 Limitation of Current Hemodialysis Treatment 19 26 Latex Particles 20

261 The Components for Emulsion Polymerization 21 262 Particle Nucleation 21 263 Types of Processes for Emulsion Polymerization 23 264 Chemistry of Emulsion Polymerization 24 265 Seed Emulsion Polymerization25 266 Polystyrene Latex Particles 26 267 Various Applications of Latex Particles28

27 Proteins 30 271 Interaction Forces between Proteins33 272 β -Microglobulin (β M)2 2 35 273 Serum Albumin 36

28 Protein Adsorption38 281 Interaction between Protein Molecule and Latex Particle38

29 Hypothesis for Toxin Removal39 291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size 40 292 Toxin Removal by Selective Adsorption on Engineered Latex Particles 41

3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY 44

31 Materials 44 32 Latex Particle Preparation46

321 Preparation of Seed Latex Particles47

vi

322 Preparation of Seeded Latex Particles 48 323 Preparation of Core Shell Latex Particles 48 324 Purification of Synthesized Latex Particles48

33 Characterization49 331 Degree of Conversion49 332 Fourier Transform Infrared (FTIR) Spectroscopy49 333 Quasielastic Light Scattering (QELS) 50 334 Field Emission-Scanning Electron Microscopy (FE-SEM) 50 335 Zeta Potential Measurement 50 336 Protein Adsorption51 337 Blood Biocompatibility by Hemolysis Test 53

4 RESULTS AND DISCUSSION55

41 Polymerization of Latex Particles55 411 Polystyrene Seed Latex Particles55 412 The Growth of Polystyrene (PS) Seed Latex Particles61 413 Core Shell Latex Particles 71

42 Characterization of Latex Particles75 421 Fourier Transform Infrared Spectroscopy (FTIR)75 422 Zeta Potential Measurements 75

43 Protein Adsorption Study 85 431 Adsorption Isotherm87 432 Adsorbed Layer Thickness 103 433 Gibbs Free Energy of Protein Adsorption 104 434 Kinetics of Adsorption 107

44 Blood Compatibility 112

5 SUMMARY OF RESULTS CONCLUSION AND RECOMMENDATION FOR FUTURE WORK115

51 Summary of Results115 52 Conclusions121 53 Recommendation for Future Work121

LIST OF REFERENCES122

BIOGRAPHICAL SKETCH 141

vii

LIST OF TABLES

Table page 4-1 The polymerization recipe of polystyrene (PS) seed particles 57

4-2 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles less than 500nm in size 63

4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size64

4-4 The preparation recipe of PS core with various shell latex particles72

4-5 The isoelectric point (IEP) of common proteins 83

4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model 93

4-7 The equilibrium concentration values (q ) of BSA adsorption on PSPMMA particles calculated the Langmuir-Freundlich isotherm model

m 10096

4-8 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model

90 1098

4-9 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model

75 25100

4-10 The equilibrium concentration values of β M adsorption calculated the Langmuir-Freundlich isotherm model

2102

4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37 Co 104

4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37 Co 104

4-13 The values of Gibbs free energy change of BSA adsorption in phosphate buffer (PB) at 37 Co 105

4-14 The values of Gibbs free energy change of BSA adsorption in phosphate buffered saline (PBS) at 37 Co 105

viii

4-15 The values of Gibbs free energy change of β M adsorption in phosphate buffere (PB) at 37 C and pH 74

2o 106

4-16 Fitting parameters of second-order kinetic model 111

ix

LIST OF FIGURES

Figure page 1-2 Stereo drawing of the αndashcarbon backbone of β M2 2

2-1 An image of the location and cross section of a human kidney 5

2-2 A schematic draw of the hemodialysis route7

2-3 Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes 11

2-4 The chemical structure of polysulfone (PSf) 12

2-5 The chemical structure of polyamide (PA)13

2-6 Chemical structure of polyacrylonitrile (PAN) 14

2-7 Emulsion polymerization system24

2-8 L-α-amino acid 31

2-9 Ribbon diagram of human β M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ)

236

2-10 Secondary structure of human serum albumin (HSA) with sub-domains 37

2-11 The relationship between pore size and particle size in body-centered cubic and closed packed arrays41

2-12 Schematic representation of the protein adsorption on the core shell latex particles at pH 7442

3-1 Chemical structure of main chemicals45

3-2 Experimental setup for semi-continuous emulsion polymerization 46

3-3 The particle preparation scheme in various types and size ranges of latex particles 47

3-4 Schematic of the procedure for a protein adsorption test 52

3-5 Separation of RBC from whole blood by centrifuge process53

x

3-6 Schematic of the procedure for hemolysis test 54

4-1 Schematic representation of semi-continuous seed latex particles preparation and growth56

4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS (B) PS (C) PS (D) PSS259 S233 S207 S181 59

4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm 65

4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm 68

4-5 Dependence of the particle size on the surfactant to monomer ratio70

4-6 Schematic of core shell latex particle structures 71

4-7 Scanning Electron Micrograph of latex particles (A) PS (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA

100

90 10 75 25 73

4-8 FTIR spectra of polymerized latex particles (A) bare polystyrene (PS) (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA100 90 10 75 25 76

4-9 Schematic representation of ion distribution near a positively charged surface 78

4-10 Schematic representation of zeta potential measurement79

4-11 Zeta potential of PS latex particles at 25 Co 80

4-12 Zeta potential of PSPMMA latex particles at 25 C100o 81

4-13 Zeta potential of PSPMMA PAA latex particles at 25 C90 10o 82

4-14 Zeta potential of PSPMMA PAA latex particles at 25 C75 25o 82

4-15 The decay of surface potential with distance from surface in various electrolyte concentrations (1) low (2) intermediate (3) high 84

4-16 High-affinity adsorption isotherm of typical flexible polymer on solid surface 86

4-17 Overall schematic representation of the protein adsorption on the synthesized latex particles86

4-18 The dependence of UV absorbance on BSA concentration (A) 10mgml (B) 06mgml (C) 02mgml87

4-19 Standard curve of net absorbance vs BSA sample concentration 88

xi

4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37 C in phosphate buffer mediao 89

4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37 Co 90

4-22 Adsorption isotherm of BSA adsorption 37 C in phosphate buffered saline (PBS) on PS latex particles

o

91

4-23 Conformation of bovine serum albumin (BSA) on latex particles92

4-24 Adsorption isotherm of BSA 37 C in PB on PSPMMA core shell latex particles

o100

94

4-25 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA core shell latex particles

o100

95

4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA PAA core shell latex particles

o90 10

97

4-27 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA PAA core shell latex particles

o90 10

98

4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA PAA latex particleso75 25 99

4-29 Adsorption isotherm of BSA 37 C in PBS on PSPMMA PAA core shell latex particles

o75 25

99

4-30 Adsorption isotherm of β M onto PS and PSPMMA latex particles in PB at 37 C and pH 74

2 100o 101

4-31 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74

2 90 10o 101

4-32 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74

2 75 25o 102

4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37 C and pH 74

o

107

4-34 The kinetics of protein adsorption in PB on PS latex particles at 37 C and pH 74

o

109

4-35 The kinetics of protein adsorption in PB on PSPMMA latex particles at 37 C and pH 74

100o

109

4-36 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74

90 10o 110

xii

4-37 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74

75 25o 110

4-38 Image of red blood cells 113

4-39 Hemolysis caused by latex particles n=5114

xiii

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS

By

Sangyup Kim

December 2005

Chair Hassan El-Shall Major Department Materials Science and Engineering

Latex particles with well-defined colloidal and surface characteristics have received

increasing attention due to their useful applications in many areas especially as solid

phase supports in numerous biological applications such as immunoassay DNA

diagnostic cell separation and drug delivery carrier Hemodialysis membrane using

these particles would be another potential application for the advanced separation

treatment for patients with end stage renal disease (ESRD) It is desirable to remove

middle molecular weight proteins with minimal removal of other proteins such as

albumin Thus it is necessary to understand the fundamental interactions between the

particles and blood proteins to maximize the performance of these membranes This

improvement will have significant economic and health impact

The objective of this study is to synthesize polymeric latex particles of specific

functionality to achieve the desired selective separation of target proteins from the human

blood Semi-continuous seed emulsion polymerization was used to prepare monodisperse

xiv

polystyrene seed particles ranging from 126plusmn75 to 216plusmn53 nm in size which are then

enlarged by about 800nm Surfactant amount played a key role in controlling the latex

particle size Negatively charged latex particles with a different hydrophobicity were

prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid

monomer The prepared polymeric particles include bare polystyrene (PS) particles less

hydrophobic PS core and PMMA shell particles and more hydrophilic PS core and

PMMA-co-PAA shell latex particles with a 370nm mean diameter SEM light scattering

and zeta potential measurements were used to characterize particle size and surface

properties Adsorption isotherms of two proteins bovine serum albumin (BSA) and β2-

microglobulin (β2M) on latex particles were obtained as a function of pH and ionic

strength using the bicinchoninic acid (BCA) assay method The Langmuir-Freundlich

adsorption model was used to determine the adsorption amount of protein at equilibrium

The thickness of adsorbed BSA layer on latex particles was obtained in order to

investigate the adsorption orientation such as end-on or side-on mode Adsorption

kinetics experiments for both proteins and all latex particles were also performed The

adsorption kinetic constant determined from the Langmuir-Freundlich adsorption

isotherm model was used to calculate Gibbs free energy of adsorption to compare the

competitive adsorption of BSA and β2M Hemolysis tests were performed to investigate

the blood compatibility of synthesized latex particles PSPMMA90PAA10 and

PSPMMA75PAA25 core shell latex particles had desirable material properties with not

only a large amount and high rate of selective β2M adsorption over BSA but also high

blood compatibility showing less than 3 hemolysis

xv

CHAPTER 1 INTRODUCTION

End stage renal disease (ESRD) is a chronic condition in which kidney function is

impaired to the extent that the patientrsquos survival requires removal of toxins from the

blood by dialysis therapy or kidney transplantation The National Kidney Foundation

estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF] The

number of people with ESRD is rapidly increasing in the United States with

approximately 96 295 incidents and 406 081 prevalent patients including 292 215 on

dialysis and 113 866 with a functioning graft in 2001 It is projected that there will be

more than 22 million ESRD patients by 2030 [USRDS 2003] The expenditure for the

ESRD treatment program had reached $228 billion 64 of the Medicare budget in

2001 Due in part to the limited availability of kidneys for transplantation hemodialysis

is the primary clinical treatment for the patients with ESRD

The central element of a hemodialysis instrument is the semipermeable membrane

that allows for selective transport of low molecular weight biological metabolites less

than 5000 Da such as urea and creatinine as well as excess water and electrolytes [Baker

2004] from the blood One limitation of current dialysis technologies is the inability to

efficiently remove middle molecular weight toxins such as β2-Microglobulin (β2M) and

interleukin 6 (IL-6)

β2M is a causative protein of dialysis-related amyloidosis (DRA) a disease arising

in patients with chronic kidney failure as a serious complication of long-term

hemodialysis treatment [Gejyo et al 1985] β2M deposition in tissue is the primary cause

1

2

of destructive arthritis and carpal tunnel syndrome [Vincent et al 1992 Drueke 2000]

The β2M structure is shown in Figure 1-2

Figure 1-2 Stereo drawing of the αndashcarbon backbone of β2M [Becker et al 1985]

Although attempts have been made to increase the efficiency of middle molecular

weight toxin removal by changes in the membrane pore size and the use of innovative

materials to adsorb these toxins [Samtleben et al 1996 Ronco et al 2001] removal

efficiency is not as high as those achieved by a normal healthy kidney Traditional

membranes have a number of processing and performance limitations [Westhuyzen et al

1992 Leypoldt et al 1998] such as a restricted choice of surface chemistries and limited

control of porosity The development of novel engineering membrane technology is

needed to remove middle molecule toxins

Polymeric latex particles have received increasing attention in medical application

areas especially as solid phase supports in biological applications [Piskin et al 1994]

Examples of these applications include immunoassay [Chen et al 2003 Radomske-

Galant et al 2003] DNA diagnostic [Elaїssari et al 1998] cell separation drug delivery

carrier [Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] etc This is because of

3

the well-defined colloidal and surface characteristics of the particles By using a seed

emulsion polymerization method it is possible to synthesize monodisperse latex particles

with various particle size ranges and surface chemistry Functionalized core-shell latex

particles can be introduced by multi-step emulsion polymerization Core particles are

synthesized in the first stage of the polymerization and the functional monomer is added

in the second stage This is done without any emulsifier addition to prevent the

production of new homopolymer particles [Keusch et al 1973] The core-shell particles

are useful in a broad range of applications because of their improved physical and

chemical properties over their single-component counterparts [Lu et al 1996 Nelliappan

et al 1997] Through the development of a hemodialysis membrane using monodisperse

latex particles improvements in advanced separation treatment for patients with end

stage renal disease (ESRD) can be realized This requires the maximum removal of

middle molecular weight proteins with minimal removal of other beneficial proteins such

as albumin Thus an understanding of the fundamental interactions between the particles

and biopolymers is vital to maximize the performance of this membrane technology

The field of material science and biotechnology is based on fundamental chemistry

has developed over the past three decades into todayrsquos powerful discipline that enables

the development of advanced technical devices for pharmaceutical and biomedical

applications This novel and highly interdisciplinary field is closely associated with both

the physical and chemical properties of organic and inorganic particles [Niemeyer 2001]

Hemodialysis membrane using these particles would lead to improvements in the

advanced separation treatment for patients with end stage renal disease (ESRD) The

interdisciplinary nature of this approach enables a more complete understanding of the

4

phenomena of protein adsorption and the material properties necessary for selective

separation This innovative approach to membrane fabrication has the potential of making

inexpensive highly efficient membranes for both industrial and specialized separation

processes

The goal of this study is to prepare polymeric latex particles with tailored

properties to maximize separation of toxin molecules and to investigate the fundamental

interactions between the particles and molecules in the biological system in order to

optimize the performance of a membrane material for these applications

Polymeric latex particles were synthesized with specific functionality in an attempt

to achieve selective separation Seeded emulsion polymerization was used to synthesize

functionalized monodisperse latex particles in various sizes Negatively charged

hydrophobic polystyrene latex particles were synthesized by the same method Core shell

latex particles PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25 were also

synthesized to differentiate the degree of hydrophobicity of particles Scanning Electron

Micrograph (SEM) and light scattering measurements were used to characterize particle

size and shape and zeta potential measurements were conducted to measure the electrical

surface property of synthesized particles Adsorption isotherms of target proteins bovine

serum albumin (BSA) and β2M on latex particles were obtained as a function of pH

ionic strength and protein concentrations using the BCA assay method Adsorption

kinetics for both proteins on the latex particles were also measured Finally hemolysis

tests were run to determine the biocompatibility of polymer latex particles with human

blood This research will be described in more detail in chapters 3 and 4

CHAPTER 2 BACKGROUND AND LITERATURE SURVEY

21 The Significance of End Stage Renal Disease (ESRD)

The kidneys are responsible for removing excess fluid minerals and wastes from

the blood regulating electrolyte balance and blood pressure and the stimulation of red

blood cell production They also produce hormones such as erythropoietin (EPO) and

calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005 Kurbel

et al 2003] EPO acts on the bone marrow to increase the production of red blood cell in

case of bleeding or moving to high altitudes Calcitriol mainly acts on both the cells of

the intestine to promote the absorption of calcium from food and also bone to mobilize

calcium from the bone to the blood When kidneys fail harmful waste builds up blood

pressure rises and body retains excess fluid The body also does not make enough red

blood cells When this happens treatment is needed to replace the function of failed

kidneys

Figure 2-1 An image of the location and cross section of a human kidney

5

6

The National Kidney Foundation estimates that over 20 million Americans had

chronic kidney disease in 2002 [NKF] Chronic renal disease is a gradual and progressive

loss of the function unlike acute renal disease where sudden reversible failure of kidney

function occurs Chronic renal failure usually takes place over a number of years as the

internal structures of the kidney are slowly damaged In the early stages there can be no

symptoms In fact progression may be so gradual that symptoms do not occur until

kidney function is less than one-tenth that of a normal health kidney If left untreated

chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal

Disease (ESRD) ESRD is a rapidly growing heath-care problem in the United States In

2001 approximately 96295 incidents and 406081 prevalent patients were diagnosed

with ESRD including 292215 patients on dialysis and 113866 patients with a

functioning graft [USRDS 2003] The projected number of patients with ESRD is

expected to exceed 22 million patients by 2030 with much of this growth being driven by

the increasing prevalence of major contributing factors such as diabetes and high blood

pressure [USRDS 2003] A great extent of ESRD program cost and Medicare budget

have been spent in 2001 Due in part to a limited availability of kidneys for

transplantation hemodialysis (HD) is the primary method of treatment for ESRD and is

currently used for approximately 61 of US ESRD patients

22 Hemodialysis (HD) Treatment

HD removes toxins from the body by extracorporeal circulation of the blood

through a semipermeable membrane referred to as a dialyzer The toxins are removed

primarily by diffusion across the membrane to a dialysate solution which is circulated on

the opposite side of the membrane The cleaned blood is then returned to the blood

stream A surgically constructed vascular access connects the extracorporeal circuit to the

7

patientrsquos system Treatments are almost always performed three times per week in

specially equipped dialysis facilities for 3-4 hours per each treatment Figure 2-2 shows

the scheme of the hemodialysis route The critical element of a HD instrument is the

semipermeable membrane which allows for selective transport of low molecular weight

biological metabolites from blood

igure 2-2 A schematic draw of the hemodialysis route

ESRD patients who undergo dialysis therapy often experience several other

problems associated with the treatment such as anemia fatigue bone problems joint

problems itching sleep disorders and restless legs Anemia is common in patient with

kidney disease because the kidneys produce the hormone erythropoietin (EPO) which

stimulates the bone marrow to produce red blood cells Diseased kidneys often do not

produce enough EPO to stimulate the bone marrow to make a sufficient amount of red

blood cells This leads to bone disease referred to as renal osteodystrophy and causes

bones to become thin and weak or malformed and can affect both children and adults

Older patients and women who have gone through menopause are at greater risk for this

disease Uremic toxins which cannot be remove from the blood by the current dialyzer

membranes can lead to itching This problem can also be related to high levels of

F

8

parathyroid hormone (PTH) Dialysis patients can also experience day-night revers

is they have insomnia at night and sleep during the day This can be related to possible

nerve damage in the body and a chemical imbalance in the blood due to the excess toxin

Oxidative stress is a problem for patients on maintenance dialysis This problem is due to

an imbalance between pro- and antioxidant factors [Roselaar et al 1995 Cristol et al

1994] Oxidative stress affects oxidation of low-density lipoproteins which are the main

factor for atherogenesis [Huysmans et al 1998] and is also involved in the development

of malignancies and diabetes mellitus [Rice-Evans et al 1993] In order to reduce

antioxidant defense dialysis is needed to contribute to help stimulate free radical

production or eliminate antioxidants Dialysis-related amyloidosis (DRA) is also a

common and serious problem for people who have been on dialysis for more than 5

years DRA develops when proteins in the blood deposit on joints and tendons causi

pain stiffness and fluid in the joint as is the case with arthritis Normally the healthy

kidneys can filter out these proteins but dialysis filters are not as effective

23 Advances in Membrane Technology

al that

s

ng

Dialysis for blood of ESRD

Hemo filtration

s

ysis using a cellulose based membrane occurred

1913 John Abel [1990] from the Johns Hopkins Medical School described a method

purification is widely used in the treatment

dialysis (HD) techniques use a semi-permeable membrane to replace the

role of the kidney The membranes used in HD can be broadly classified into those based

on cellulose and those manufactured from synthetic copolymers These membranes come

in various shapes such as sheets tubular structures or hollow fiber arrangements The

hollow fiber type is the most popular and is incorporated into over 800 different device

in world wide [Ronco et al 2001]

The first attempt at blood dial

9

where sing

e

om

ysis was performed until the mid 1960 The basic

molec

rial in

een

benzyl)

was used for the membrane with

hepar

by the blood of a living animal may be submitted to dialysis outside the body u

a membrane based on cellulose and returned to the natural circulation without exposur

to air infection by microorganisms or any alteration that would necessarily be prejudicial

to life This same technique is still used to today however the device used has been

modified over the years as better membranes were developed and the anti-coagulant

heparin has become available

Cellulose membranes have been widely used for the treatment of renal failure fr

1928 when the first human dial

ular structure of cellulose is made of a long chain containing hydroxyl (OH)

groups The realization that such groups imparted undesirable qualities on the mate

respect to blood contact behavior was discovered in the early 1970s and since has b

the focus of development These modified cellulose membranes used the partial

substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt

to reduce their negative effect The result is a molecular mosaic of hydrophobic (

and hydrophilic (hydroxyl and cellulose) regions

Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients

with acute renal failure in 1943 Cellophane tubing

in as the anticoagulant For the next 17 years hemodialysis therapy was performed

by this method but only for the patients with acute reversible renal failure Vascular

access required repeated surgical insertions of cannulas (slender tubes) into an artery and

vein and limited the number of treatments for a patient could receive in order to

minimize the amount of vascular damage

10

Initially the need for dialysis in patients with acute renal failure was determ

mainly by the development of signs and sym

ined

ptoms of uremia After dialysis some time

might

et al

ent improvement In 1960 the development of a shunt [Quinton et al

1960]

fone (PSf) polyamide (PA) and polyacrylonitrile (PAN) by phase inversion or

precip

lidone

e

elapse before uremic manifestations returned to warrant a sequential treatment of

dialysis Many patients with acute renal failure secondary to accidental or surgical

trauma were hypercatabolic but the interdialytic interval might be prolonged because of

anorexia or use of a low-protein diet However Teschan and his coworkers [Obrien

1959] showed that patient well-being and survival were improved by what they termed

prophylactic daily hemodiaysis or administration of the treatment before the patient

again became sick with uremia Their report in 1959 was the first description of daily

hemodialysis

Development of membrane accessories such as a shunt has also been an area of

focus for treatm

a flexible polytetrafluoroethylene (PTFE or Teflonreg) tubing made many more

hemodialysis treatments possible for chronic kidney failure patients PTFE has a non-

stick surface and is relative biocompatibility leading to minimized blood clotting in the

shunt

Synthetic membranes are prepared from engineered thermoplastics such as

polysul

itation of a blended mixture resulting in the formation of asymmetric and

anisotropic structures Figure 2-3 shows a fiber type of the Polyflux S membrane

consisting of poluamide (PA) polyacrylethersulfone (PAES) and polyvinylpyrro

(PVP) with the integral three-layer structure The skin layer on the inside fiber typ

membrane contacts blood and has a very high surface porosity and a narrow pore size

11

distribution This layer constitutes the discriminating barrier deciding the permeabili

and solute retention properties of the membrane The skin layer is supported by thick

sponge-type structure larger pores providing mechanical strength and very low

hydrodynamic resistance

ty

Figure 2-3 Cross-sectional SEM image view of the Polyflux S (polyamide +

polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch et al 1998]

n

[Malchesky 2004] because of its thermal stability mechanical strength and chemical

inertn ciated

ls

m

PSf is a widely used membrane material for the hemodialysis applicatio

ess According to a report from the National Surveillance of Dialysis-Asso

Disease (NSDAD) in the US over 70 of hemodialysis membranes were PSf based

[Bowry 2002] This is most likely because PSf has many advantages over other materia

This synthetic polymer is one of few materials that can withstand sterilization by stea

ethylene oxide and γ-radiation PSf membrane can be prepared by conventional

immersion precipitation methods into many different shapes including porous hollow

12

fiber or flat sheet hemodialysis membranes The material also has a high permeab

low molecular weight proteins and solute and high endotoxin retention The chemical

structure of PSf is shown in Figure 2-4

ility to

Figure 2-4 The chemical structure of polysulfone (PSf)

There is one major disadvantage to PSf The hydrophobic nature of the PSf causes

ment alternative pathway

leadin

s not

3

et al

in

O acrylate

serious complications through the activation of the comple

g to the adsorption of serum proteins onto the membranes [Singh et al 2003]

Anticoagulants are added during dialysis therapy to avoid blood clotting but this doe

completely eliminate the problem In order to overcome this disadvantage of the PSf

membrane various studies have been performed to change the materialrsquos surface

properties These investigations include hydrophilic polymer coating [Brink et al 199

Kim et al 1988 Higuchi et al 2003] layer grafting onto PSf membrane [Wavhal

2002 Song et al 2000 Pieracci et al 2002 Mok et al1994] and chemical reaction of

hydrophilic components onto the membrane surface [Higuchi et al 1990 1991 1993

Blanco et al 2001 Nabe et al 1997 Guiver et al 1993] Hydrophilic monomers 2-

hydroxy-ethylmethacrylate (HEMA) acrylic acid (AA) and methacrylic acid (MMA)

have also been grafted onto PSf membrane to increase flux and Bovine Serum Album

(BSA) retention [Ulbricht et al 1996] Hancock et al [2000] synthesized

polysulfonepoly(ethylene oxide) (PEO) block copolymers to improve the resistance to

platelet adhesion Kim et al [2003] also studied blending a sulfonated PE

diblock copolymer into PSf in order to reduce platelet adhesion and enhance

13

biocompatibility PEO is a commonly used biomaterial due to its excellent resistance

protein adsorption and inherent biocompatibility [Harris 1992] Kim et al [20

a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf

membrane with an ultra-thin skin layer The polymer had an entrapped diblock

copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-SO

to

05] studied

ate

lic graft

l)

e of thier

aromatic

orand

3 acryl

and a hydrophobic block of octadecylacrylate (OA) Molecular dynamic (MD)

simulations were performed as a function of copolymer density to optimize interfacial

structure information McMurry [2004] developed a strategy using an amphiphi

copolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glyco

When compared to unmodified PSf these graft copolymer and resulting blend

membranes are found to hold promise for biomedical device applications

Polyamide (PA) membranes have also been used for hemodialysis becaus

mechanical strength in both wet and dry conditions Polyamide consists of

aliphatic monomers with amide bonding (-CONH-) also known as a peptide bond

The basic amide bond in polyamide is shown in Figure 2-5 R1 and R2 can be either

aromatic or aliphatic linkage group

C N

H

O

~ R R1 2~C N

H

O

~ R R1 2~

Figure 2-5 The chemical structure of polyamide (PA)

Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane

ompatible technique due to the

use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of

and concluded that PA hemofiltration was a highly bioc

14

the ac

e

e

is

n

is a semi-crystalline polymer and the mechanical properties

strong

2

CN

tivated anaphylatoxins and β2-Microglobulin (β2M) The PA based membrane

Polyfluxreg (manufactured by Gambro GmbH Germany) blended with polyamide

polyarylethersulfone and polyvinylpyrrolidone (PVP) was able to clean small molecules

such as urea dreatinine and phosphate as well as decrease β2M amount by 502

[Hoenich et al 2002] Due to the non-selectivity of the membrane removal of thes

unwanted materials also led to the undesirable loss of beneficial proteins during therapy

Meier et al [2000] evaluated different immune parameters using a modified cellulos

low-flux hemophan and synthetic high-flux PA membrane during a 1 year period in

chronic hemodialysis patients They found that the 1-year immunological evaluation of

hemodiaysis membrane biocompatibility was associated with changes in the pattern of

chronic T-cell actiovation

Polyacrylonitrile (PAN) is another commonly used membrane material because it

inherently hydrophilic and has been commercialized for ultrafiltration and microfiltratio

[Scharnagl et al 2001] PAN

ly depend on the crystalline structures The chemical structure of PAN is shown in

figure 2-6

CH CH

n2

CN

CH CH n

Chemical structure of polyacrylonitrile (PAN)

The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming

agent gives PAN membranes more flexible processing parameters and increased

e has been optimized through

copolymerization with many other vinyl monomers including glycidyl methacrylate

Figure 2-6

performance [Jung et al 2005] PAN membrane performanc

15

[Godj

acid

for

for

a non-

solub this

in

oid underneath the inner

surface of membrane decreased by increasing the amount of solvent DMSA in the

evargova et al 1999 Hicke et al 2002] N-vinylimidazole [Godjevargova et al

2000] hydroxyl ethyl methacrylate [Ray et al 1999 Bhat et al 2000] methacrylic

[Ray et al 1999] vinyl pyrrolidone [Ray et al 1999] acrylic acid [Trotta et al 2002]

acrylamide [Musale et al 1997] and vinylchloride [Broadhead et al 1998] These

monomers provide a reactive group for enzyme immobilization improved mechanical

strength solvent-resistance pervaporation permeation flux anti-fouling and bio-

compatibility Because of this PAN-based copolymer membranes have great potential

the treatment of hemodialysis in an artificial kidney This material can also be used

other applications like the treatment of wastewater the production of ultra-pure water

biocatalysis together with separation and methanol separation by pervaporation

Lin and his coworker [2004] studied the modification of PAN based dialyzer

membranes to increase the hemocompatibility by the immobilization of chitosan and

heparin conjugates on the surface of the PAN membrane When a foreign material is

exposed to blood plasma proteins are adsorbed clotting factors are activated and

le fibrin network or thrombus is formatted [Goosen et al 1980] The result of

research was that the biocompatible chitosan polymer and a blood anticoagulant hepar

prevented blood clotting They showed prolonged coagulation time reduced platelet

adsorption thrombus formation and protein adsorption

Nie et al [2004] studied PAN-based ultrafiltration hollow-fiber membranes

(UHFMs) In order to improve the membrane performance acrylonitrile (AN) was

copolymerized with other functional monomers such as maleic anhydride and α-allyl

glucoside They found that the number and size of macrov

16

intern erum

and

d a

rface The second step is the

transf after

yjak et

embrane

1978 Malchesky et al 1978] has been further developed to increase the efficiency of

al coagulant The water flux of the UHFMs also decreased while the bovine s

albumin rejection increased minutely Godjevargova et al [1992] modified the PAN

based membrane with hydroxylamine and diethylaminoethylmethacrylate to improve

membrane dialysis properties Formed functional groups like primary amine oxime

tertiary amine groups provided the membrane with more hydrophilic properties an

substantial increase in the permeability of the membranes

The wide use of filtration in practice is limited by membrane fouling Solute

molecules deposit on and in the membrane in the process of filtration causing dramatic

reduction in flux through the membrane Fouling occurs mostly in the filtration of

proteins Three kinetic steps are involved in the fouling of UF membranes according to

Nisson [1990] The first step is the transfer of solute to the su

er of solute into the membrane until it either finally adsorbs or passes through

a set of adsorption-desorption events The third step includes surface binding

accompanied by structural rearrangement in the adsorbed state [Ko et al 1993] Br

al [1998] studied the surface modification of a commercially available PAN membrane

to develop superior filtration properties with less fouling by proteins The PAN

membrane was immersed in excess NaOH solution to convert some of the surface nitrile

groups into carboxylic groups by the hydrolysis process This modified PAN m

was not so severely fouled in the Bovine Serum Albumin (BSA) filtration test The pore

size however decreased during the hydrolysis process leading to a significant reduction

in flux and made the membrane less productive in the ultrafiltration (UF) mode

24 Sorbent Technology

Over the last three decades sorbent technology [Castino et al 1976 Korshak et al

17

dialysis or replace it for the treatment of ESRD Sorbents remove solutes from solution

through specific or nonspecific adsorption depending on both the nature of the solute and

the sorbent Specific adsorption contains tailored ligands or antibodies with high

selectivity for target molecules een used in autoimmune

disord

and

e

al 1996] various resins [Ronco et al 2001]

album

e

as

Specific adsorbents have b

ers such as idiopathic thrombocytopenic purpura [Snyder et al 1992] and for the

removal of lipids in familial hyper cholesterolemia [Bosch et al 1999] Nonspecific

adsorbents such as charcoal and resins attract target molecules through various forces

including hydrophobic interactions ionic (or electrostatic) attraction hydrogen bonding

and van der Waals interactions

New dialysate with sorbents has become an accepted modification of dialysis

sorbent hemoperfusion is gaining ground as a valuable addition to dialysis especially as

new sorbents are developed [Winchester et al 2001] Hemoperfusion is defined as th

removal of toxins or metabolites from circulation by the passing of blood within a

suitable extracorpoteal circuit over semipermeable microcapsules containing adsorbents

such as activated charcoal [Samtleben et

in-conjugated agarose etc Novel adsorptive carbons with larger pore diameters

have been synthesized for potential clinical use [Mikhalovsky 1989] Newly recognized

uremic toxins [Dhondt et al 2000 Haag-Weber et al 2000] have resulted in several

investigations on alternatives to standard or high-flux hemodialysis to remove thes

molecules These methods include hemodiafilteration with [de Francisco et al 2000] or

without [Ward et al 2000 Takenaka et al 2001] dialysate regeneration using sorbents

well as hemoperfusion using such adsorbents as charcoal and resins

18

Kolarz et al [1989 1995] studied the hyper-crosslinked sorbent prepared from

styrene and divinylbenzene (DVB) for a hemoperfusion application They found that the

pore structure of a swelling sorbent was changed by additional crosslinking with α αrsquo-

dichloro-p-xylene in the presence of a tin chloride catalyst and in a di

chloroethane

soluti and

en

decyl groups that attract β2M through a hydrophobic interaction The

adsor

of

5]

d

penet

on They also realized that the hemocompatibility was useful for nemoperfusion

could be imparted to the sorbents by introducing sulfonyl groups at a concentration of

about 02mmolg

A special polymeric adsorbing material (BM-010 from Kaneka Japan) has be

investigated by another group [Furuyoshi et al 1991] for the selective removal of β2M

from the blood of dialysis partients The adsorbent consists of porous cellulose beads

modified with hexa

ption capacity of this material is 1mg of β2M per 1ml of adsorbent Using a

hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a

high-flux hemodialyzer several small clinical trials were performed During 4-5 hours

treatment about 210mg of β2M were removed thus reducing the concentration in the

blood by 60-70 of the initial level [Nakazawa et al 1993 Gejyo et al 1993 199

RenalTech developed a hemoperfusion device BetaSorbreg containing the hydrate

cross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure

designed to remove molecules between 4 and 30 kDa [Winchester et al 2002] In this

case solute molecules are separated according to their size based on their ability to

rate the porous network of the beaded sorbents The resin beads were prepared with

a blood compatible coating and confirmed to be biocompatible in vivo in animals

[Cowgill et al 2001]

19

25 Limitation of Current Hemodialysis Treatment

Hemodialysis is a widely used life-sustaining treatment for patients with ESRD

However it does not replace all of the complex functions of a normal healthy kidne

a result patients on dia

y As

lysis still suffer from a range of problems including infection

accelerated cardio ion anemia

chron

e

se middle

alysis

sessio

s

f

vascular disease high blood pressure chronic malnutrit

ic joint and back pain and a considerably shortened life span One significant

limitation of the current dialysis technology is the inability to efficiently remove larger

toxic molecules This is mainly because of the broad pore size distribution reducing th

selective removal of toxins and unsatisfied biocompatibility causing lots of

complications such as inflammation blood clotting calcification infection etc

Dialysis purifies the patients blood by efficiently removing small molecules like

salts urea and excess water However as toxic molecules increase in size their removal

rate by hemodialysis substantially declines Typically only 10 - 40 of the

molecular weight toxins (300-15000 Da) are removed from the blood during a di

n [Vanholder et al 1995] These toxins then reach an abnormally high level and

begin to damage the body One such toxin β2M causes destructive arthritis and carpal

tunnel syndrome by joining together like the links of chain to form a few very large

molecules and deposit damaging the surrounding tissues [Lonnemann et al 2002] This i

also a main cause of mortality for long-term dialysis patients Other middle molecule

toxins appear to inhibit the immune system and may play a significant role in the high

susceptibility to infections in dialysis patients Still others are believed to impair the

functioning of several other body systems such as the hematopoietic and other endocrine

systems This may contribute to accelerated cardiovascular disease the leading cause o

20

death among dialysis patients as well as clinical malnutrition which affects up to 50 of

this patient population

Over the last decade polymeric dialysis membranes have been developed to

increase the capacity for removing middle molecular weight toxins by changing the pore

size of dialyzer membranes and using new materials that adsorb these toxins for

impro se

styrene made during World War II in the United

States The Dow chemical compan anufacturer of polystyrene

includ

d of

on

rug

ntrol of

ved removal characteristics However removal efficiency is not as high as tho

achieved by a normal healthy kidney

26 Latex Particles

The first synthetic polymer synthesized using emulsion polymerization was a

rubber composed of 13-butadiene and

y has been a major m

ing latex which they used in paint formulations The theory of emulsion

polymerization in which a surfactant is used was established by Harkins [1948] and by

Smith and Ewart [1948] By 1956 the technology was complete including the metho

building larger diameter particles from smaller ones The product by this emulsi

polymerization is referred to as latex a colloidal dispersion of polymer particles in water

medium [Odian 1991] Latexes are currently undergoing extensive research and

development for a broad range of areas including adhesives inks paints coatings d

delivery systems medical assay kits gloves paper coatings floor polish films carpet

backing and foam mattresses to cosmetics The relatively well known and easy co

the emulsion process is one of main advantages for these applications Therefore

polymeric latex particle prepared by emulsion polymerization can be a candidate for the

medical applications because of the easy control of the particle size and morphology as

well as flexible surface chemistry to be required

21

261 The Components for Emulsion Polymerization

The main components for emulsion polymerization process are the monomer a

dispersing medium a surfactant and an initiator Available monomers are styrene

sing medium is usually water

which

ticles

n

ation To

monomer droplets but in micelles because the initiators are

insolu le

and

olution

butadiene methylmethacylate acryl acid etc The disper

will maintain a low solution viscosity provide good heat transfer and allow

transfer of the monomers from the monomer droplets into micelles and growing par

surrounded by surfactants respectively The surfactant (or emulsifier) has both

hydrophilic and hydrophobic segments Its main functions is to provide the nucleatio

sites for particles and aid in the colloidal stability of the growing particles Initiators are

water-soluble inorganic salts which dissociate into radicals to initiate polymeriz

control the molecular weight a chain transfer agent such as mercaptan may be present

262 Particle Nucleation

Free radicals are produced by dissociation of initiators at the rate on the order of

1013 radicals per milliliter per second in the water phase The location of the

polymerization is not in the

ble in the organic monomer droplets Such initiators are referred to as oil-insolub

initiators This is one of the big differences between emulsion polymerization

suspension polymerization where initiators are oil-soluble and the reaction occurs in the

monomer droplets Because the monomer droplets have a much smaller total surface area

they do not compete effectively with micelles to capture the radicals produced in s

It is in the micelles that the oil soluble monomer and water soluble initiator meet and is

favored as the reaction site because of the high monomer concentration compared to that

in the monomer droplets As polymerization proceeds the micelles grow by the addition

of monomer from the aqueous solution whose concentration is refilled by dissolution of

22

monomer from the monomer droplets There are three types of particles in the emulsion

system monomer droplets inactive micelles in which polymerization is not occurring

and active micelles in which polymerization is occurring referred to as growing polymer

particles

The mechanism of particle nucleation occurs by two simultaneous processes

micellar nucleation and homogeneous nucleation Micellar nucleation is the entry of

radicals ei

ther primary or oligomeric radicals formed by solution polymerization from

the aq ed

the

r

s

n

is coagulation with other particles rather than

polymerization of monomer The driving force for coagulation of precursor particles

ueous phase into the micelles In homogeneous nucleation solution-polymeriz

oligomeric radicals are becoming insoluble and precipitating onto themselves or onto

oligomers whose propagation has ended [Fitch et al 1969] The relative levels of micella

and homogeneous nucleation are variable with the water solubility of the monomer and

the surfactant concentration Homogeneous nucleation is favored for monomers with

higher water solubility and low surfactant concentration and micellar nucleation is

favored for monomers with low water solubility and high surfactant concentration It has

also been shown that homogeneous nucleation occurs in systems where the surfactant

concentration is below CMC [Roe 1968] A highly water insoluble monomer such a

styrene [Hansen et al 1979 Ugelstad et al 1979] has probably created by micellar

nucleation while a water soluble monomer such as vinyl acetate [Zollars 1979] has bee

formed by homogeneous nucleation

A third latex formation reaction mechanism has been proposed referred to as

coagulative nucleation In this reaction the major growth process for the first-formed

polymer particles (precursor particles)

23

sever les

yer

es

r a

l

the beginning of the polymerization As soon as the initiator is

gins with the formation and

growt

and

ved

added at one time during the later stages of the polymerization prior to complete

al nanometers in size is their relative instability compared to larger sized partic

The small size of a precursor particle with its high curvature of the electrical double la

permits the low surface charge density and high colloidal instability Once the particl

become large enough in size maintaining the high colloidal stability there is no longe

driving force for coagulation and further growth of particles takes place only by the

polymerization process

263 Types of Processes for Emulsion Polymerization

There are three types of production processes used in emulsion polymerization

batch semi-continuous (or semi-batch) and continuous In the batch type process al

components are added at

added and the temperature is increased polymerization be

h of latex particles at the same time There is no further process control possible

once the polymerization is started In the semi-continuous emulsion polymerization

process one or more components can be added continuously Various profiles of particle

nucleation and growth can be generated from different orders of component addition

during polymerization There are advantages to this process such as control of the

polymerization rate the particle number colloidal stability copolymer composition

particle morphology In the continuous process the emulsion polymerization components

are fed continuously into the reaction vessel while the product is simultaneously remo

at the same rate High production rate steady heat removal and uniform quality of

latexes are advantages of the continuous polymerization processes

Other available methods include the intermittent addition and shot addition of one

or more of the components In the shot addition process the additional components are

24

conversion of the main monomer This method has been used successfully to develo

water-soluble functional monomers such as sodium styrene sulfonat

p

e [Kim et al 1989]

264

lsion

Chemistry of Emulsion Polymerization

Emulsion polymerization is one type of free radical polymerization and can be

divided into three distinct stages initiation propagation and termination The emu

polymerization system is shown in Figure 2-7

Aqueous phaseAqueous phase

Micelle withmonomer

I Rbull

Emulsifier

Polymer particle swollenWith monomer

Monomerdroplet

I Rbull

Micelle withmonomer

I Rbull

Emulsifier

Polymer particle swollenWith monomer

MonomerdropletMonomerdroplet

I Rbull

Figure 2-7 Emulsion polymerization system [Radicals (Rmiddot) are created from initiators

(I) Monomer is transferred from larger monomer droplet into micelles by emulsifier Initiated polymer particle by radicals is keep growing until monomers are all consumed The reaction is performed in aqueous media]

In the initiation stage free radicals are created from an initiator by heat or an

ultraviolet radiation source The initiator with either peroxides groups (-O-O-) such as

odium persulfate or azo groups (-N=N-) such as azobisisobutyronitrile is commonly

used for em t

with the m er

chain ere

s

ulsion polymerization The primary free radicals created from initiator reac

onomer for initiation of polymerization In the propagation stage the polym

grows by monomer addition to the active center a free radical reactive site Th

are two possible modes of propagations head-to-head addition and head-to-tail addition

25

The head-to-tail mode is the predominant configuration of the polymer chain a result o

steric hindrance of the substitute bulky group In the termination stage polymer chain

growth is terminated by either coupling of two growing chains forming one polymer

molecule or transferring a hydrogen atom (dispropotionation) from one growing chain to

another forming two polymer molecules one having a saturated end group and the other

with an unsaturated end-group

265 Seed Emulsion Polymerization

Polymer latex particles have received increasing interest because of the versatili

of the many applications heterophase polymerization processes like emulsion dispersion

micro-emulsion seeded emulsion precipitation etc Especially to prepare well-defined

microspheres having monodispe

f

ty

rse and various particle sizes as well as surface group

ed particles prepared by emulsion polymerization

and e

s

hological design In

seede to control

n

functionalilties it is necessary to use se

nlarge them to a desired size in the further stages of reactions

Polymeric particles are required to have a uniform particle size in many

applications such as chromatography where they are used as a packing material

Morphological control of latex particle is also important for many practical application

[Schmidt 1972] Seed emulsion polymerization (or two-stage emulsion polymerization) is

a useful method to achieve both monodisperse particle size and morp

d emulsion polymerization [Gilbert 1995] preformed lsquoseedrsquo latex is used

the number of particles present in the final latex The advantage of seeded emulsio

polymerization is that the poorly reproducible process of particle nucleation can be

bypassed so that the number concentration of particles is constant and known Various

mechanisms have been proposed for the growth of latex particles [Ugelstad et al 1980

Okubo et al 1992] using this polymerization technique The initial seed particle

26

preparation step is well known and relatively easy to perform because at the relative

small particle sizes (02 to 05 micron) the particle growth process can be readily

controlled by the use of an emulsifier Enough emulsifier is used to prevent coagulation

but not enough to cause the formation of new particles As the particles are grown to

larger sizes in successive seeding steps it becomes increasingly difficult to mainta

stable uncoagulated emulsion without forming new particles and thereby destroying

monodisperisty of the latex Recently there have been several reports concerning t

reaction kinetics of seed emulsion polymerization and the development of latex

morphologies over the course of the reaction [Chern et al 1990 Delacal et al 1990 L

et al 1995 Lee 2000 2002]

266 Polystyrene Latex Particles

A number of papers have described the synthesis of the polystyrene latex particl

bearing various functional surface groups such as carboxyl [Lee et al 2000 Tun

2002 Reb et al 2000] hydroxyl [Tamai et al 1989] marcapto [Nilson 1989] epoxy

[Shimizu et al 2000 Luo et al

ly

in a

he

ee

es

cel et al

2004] acetal [Izquierdo et al 2004 Santos et al 1997]

ometyl [Izquierdo et al 2004 Park et al 2001

Sarob

h the

d

thymine [Dahman et al 2003] chlor

e et al 1998] amine [Counsin et al 1994 Ganachaud et al 1997 Ganachaud et al

1995 Miraballes-Martinez et al 2000 2001 Anna et al 2005] ester [Nagai et al 1999]

etc In order to produce these functionalized particles different methods for particle

preparation must be used The polymer emulsion with core-shell morphology of latex

particles is one of them This is a multistep emulsion polymerization process in whic

polystyrene ldquocorerdquo particle is synthesized in the first stage and the functional monomer is

added in the second stage of the polymerization without any emulsifier postfeeding to

prevent the production of new homopolymer particles thus forming the functionalize

27

polymer ldquoshellrdquo on the ldquocorerdquo particle [Keusch et al 1973] There are requirements to

limit secondary nucleation and encourage core-shell formation in seeded emulsion

polymerization including the addition of smaller seed particles at high solid content to

increase particle surface area low surfactant concentration to prevent formation of

micelles and the semi-continuous addition of monomer to create a starved-feed conditio

and keep the monomer concentration low There are some advantages [Hergeth et al

1989] of dispersions with polymeric core-shell particles First it is possible to modi

interfacial properties of polymer particles in the aqueous phase by the addition of only

very small amounts of a modifying agent during the last period of the reaction Thus

these core-shell particles are useful in a broad range of applications since they always

exhibit improved physical and chemical properties over their single-component

counterparts [Lu et al 1996 Nelliappan et al 1997] In this way the improvement of

surface properties of such dispersions is straightforward and inexpensive The other is

that polymers with a core-shell structure are perfect model systems for investigating th

material properties of polymer blends and composites because of their regular

distribution of one polymer inside a matrix polymer and because of the simple sp

geometry of the system

Their properties usually depend on the structures of latex particles Chen and his

coworkers [Chen et al 1991 1992 1993] reported the morphological development of

core shell latex particles of polystyrenepoly(methyl methacrylate) during

polymerization Before the research by Chen and his coworker Min et al [Min et al

1983] reported the morph

n

fy the

e

herical

ological development of core shell latex of polystyrene

(PS)polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the

28

additi

of

ity of graft

ing reagents Thus protein adsorption on polymeric solid surfaces has

st are microspheres defined as ldquofine

polym can be

n

r was

containing antigen urine serum etc The latex will become agglutinated and visibly

on method of PS They found that the percentage of grafting PS to the PBA was

greatest for the batch reaction and the PBA-PS core-shell particles with a high degree

grafting remained spherical upon aging test because of the emulsifying abil

copolymer

267 Various Applications of Latex Particles

Latex particles are applicable to a wide range of areas such as biomedical

applications [Piskin et al 1994] especially as the solid phase such as in immunoassays

[Chern et al 2003 Radomske-Galant et al 2003] DNA diagnostic drug delivery carriers

[Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] blood cell separations and

column pack

become a center of attention Of particular intere

er particles having diameters in the range of 01 to several micronsrdquo which

used as functional tools by themselves or by coupling with biocompounds Singer and

Plotz [1956] firstly studied microsphere or latex agglutination test (LATrsquos) by using

monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support o

which the biomolecules were going to adsorb The biomolecule adsorption howeve

limited by possible desorption of the adsorbed species or loss of specific activity of the

complex formed Since this work was published the latex particle applications for

immunoassay have been rapidly and widely studied and developed

Latex agglutination test or latex immunoassays start with tiny spherical latex

particles with a uniform diameter and similar surface properties The particles are coated

with antibodies (sensitized) through the hydrophobic interaction of portions of the protein

with the PS surface of the particles If sensitized particles are mixed with a sample

29

agglomerate Latex tests are inexpensive as compared with the other techniques [Ba

1988]

ngs

sed

tion Rheumatoid factor (RF) in different age subpopulations has also been

evalu

studied a

latex

Unipath [Percival 1996] has manufactured a range of immunoassay materials ba

on a chromatographic principle and deeply colored latex particles for use in the home and

clinical environments The latex particles which are already sensitized with a

monoclonal antibody can detect any antigens that bound to the surface of the latex

particles Some detectable pollutants include estrogen mimics which induce

abnormalities in the reproductive system of male fishes and lead to a total or partial male

feminiza

ated according to a patientrsquos clinical status by using a rapid slide latex agglutination

test for qualitative and semiquantitative measurement in human serum along with latex

immunoassay method [Onen et al 1998] Magalhaes and his coworkers [2004]

diagnostic method of contamination of male fishes by estrogen mimics using the

production of vitellogenin (VTG) as a biomarker This was based on a reverse

agglutination test developed with monoclonal antibodies specific to this biomarker

Premstaller and his coworkers [Premstaller et al 2000 2001] have prepared a porous

poly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient

chromatographic separation of biomelecules such as proteins and nucleic acids They

used a porogen a mixture of tetrahydrofuran and decanol to fabricate a micropellicular

PS-DVB backbone Legido-Quigley and his colleagues [2004] have developed the

monolith column to obtain further chromatographic functionality to the column by

introducing chloromethylstyrene in place of styrene into the polymer mixture

30

Core shell type monodisperse polymer colloids have been synthesized by Sarobe

and Forcada [1998] with chloromethyl functionality in order to improve the biomolecu

adsorption through a two-step emulsion polymerization process They investigated the

functonalized particles by optimizing the experimental parameters of the functional

monomer including reaction temperature the amount and type of redox initiator sys

used the type of addition of the initiator system and the use of washing They c

le

tem

oncluded

that th

sis

serve as separation

chann

of

usually

along the polypeptide chain which contain positive or negative charges [Norde 1998]

e relation between the amount of iron sulfate and the persulfatebisulfite system

added should be controlled to obtain monodisperse particles and prevent the premature

coagulation of the polymer particles during the polymerization

A semi-continuous emulsion polymerization technique for latex particle synthe

was performed by McDonald and other researchers [Ramakrishnan et al 2004 Steve et

al 1999] They introduced a variety of particles sizes compositions morphologies and

surface modifications to fabricate latex composite membranes (LCMs) Arraying and

stabilizing latex particles on the surface of a microporous substrate form narrowly

distributed interstitial pores formed between the particles which

els They investigated the membrane performance using gas fluxes water

permeability and the retention characterization of dextran molecules From these tests

they concluded that the narrow discriminating layer made of the latex particles leads to a

highly efficient composite membrane

27 Proteins

Proteins are natural polyamides comprised of about 20 different α-amino acids

varying hydrophobicity [Norde 1998] Proteins are more or less amphiphilic and

highly surface active because of the number of amino acid residues in the side groups

31

Proteins are polymers of L-α-amino acids [Solomon and Fryhle 2000] The α refers to a

carbon with a primary amine a carboxylic acid a hydrogen and a variable side-chain

group designated as R Carbon atoms rent groups are asymmetric and can

exhib l

s

e

on

with four diffe

it two different spatial arrangements (L and D configurations) due to the tetrahedra

nature of the bonds The L refers to one of these two possible configurations Amino

acids of the D-configuration are not found in natural proteins and do not participate in

biological reactions Figure 2-8 shows the chiral carbon in 3-D as the L isomer Protein

consist of twenty different amino acids differentiated by their side-chain groups [Norde

1998] The side-chain groups have different chemical properties such as polarity charg

and size and influence the chemical properties of proteins as well as determine the

overall structure of the protein For instance the polar amino acids tend to be on the

outside of the protein when they interact with water and the nonpolar amino acids are

the inside forming a hydrophobic core

Figure 2-8 L-α-amino acid

The covalent linkage between two amino acids is known as a peptide bond A

peptide bond is formed when the amino group of one amino acid reacts with the carboxyl

group of another amino acid to form an amide bond through the elimination of water

This arrangement gives the protein chain a polarity such that one end will have a free

amino group called the N-terminus and the other end will have a free carboxyl group

called the C-terminus [Solomon and Fryhle 2000] Peptide bonds tend to be planar and

32

give the polypeptide backbone rigidity Rotation can still occur around both of the α-

carbon bonds resulting in a polypeptide backbone with different potential conformations

relative to the positions of the R groups Although many conformations are theoretically

possible interactions between the R-groups will limit the number of potential

nd to form a single functional conformation In other words

the conformation or shape of the protein is

α-helix

can interact with other

secondary structures within the sam

conformations and proteins te

due to the interactions of the chain side

groups with one another and with the polypeptide backbone The interactions can be

between amino acids that are close together as in a poly-peptide between groups that are

further apart as in amino acids or even on between groups on different polypeptides all

together These different types of interactions are often discussed in terms of primary

secondary tertiary and quaternary protein structure

The primary amino acid sequence and positions of disulfide bonds strongly

influence the overall structure of protein [Norde 1986] For example certain side-chains

will promote hydrogen-bonding between neighboring amino acids of the polypeptide

backbone resulting in secondary structures such as β-sheets or α-helices In the

conformation the peptide backbone takes on a spiral staircase shape that is stabilized by

H-bonds between carbonyl and amide groups of every fourth amino acid residue This

restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure

Certain amino acids promote the formation of either α-helices or β-sheets due to the

nature of the side-chain groups Some side chain groups may prevent the formation of

secondary structures and result in a more flexible polypeptide backbone which is often

called the random coil conformation These secondary structures

e polypeptide to form motifs or domains (ie a

33

tertiary structure) A motif is a common combination of secondary structures and a

domain is a portion of a protein that folds independently Many proteins are composed o

multiple subunits and therefore exhibit quaternary structures

271 Interaction Forces between Proteins

Proteins in aqueous solution acquire compact ordered conformations In such a

compact conformation the movement along the polypeptide chain is severely restrict

implying a low conformational entropy The compact structure is possible only if

interactions within the protein molecule and interactions between the protein molecule

and its environment are sufficiently favorable to compensate for the low conformational

entropy [Malmsten 1998] Protein adsorption study is often focused on structural

rearrangements in the protein molecules because of its significance to the biological

functioning of the molecules and the important role such rearrangements play in the

mechanism of the adsorption process Knowledge of the majo

f

ed

r interaction forces that act

structures helps to understand the behavior

of pro

arge

a

ded

between protein chains and control the protein

teins at interface These forces include Coulomb interaction hydrogen bonds

hydrophobic interaction and van der Waals interactions

Coulombic interaction Most of the amino acid residues carrying electric ch

are located at the aqueous boundary of the protein molecule An the isoelectric point

(IEP) of the protein where the positive and negative charges are more or less evenly

distributed over the protein molecule intramolecular electrostatic attraction makes

compact structure favorable to proteins Deviation to either more positive or more

negative charge however leads to intramolecular repulsion and encourages an expan

structure Tanford [1967] calculated the electrostatic Gibbs energy for both a compact

impenetrable spherical molecule (protein) and a loose solvent-permeated spherical

34

molecule (protein) over which the charge is spread out From the results he found th

the repulsion force was reduced at higher ionic strength d

at

ue to the screening action of ion

ble

e

the

d the

tween

dehyd

e

ent

oles

tein

Hydrogen bond Most hydrogen bonds in proteins form between amide and

carbonyl groups of the polypeptide backbone [Malmsten 1998] The number of availa

hydrogen bonds involving peptide units is therefore far greater than that involving sid

chains Because α-helices and β-sheets are aligned more or less parallel to each other

interchain hydrogen bonds enforce each other Kresheck and Klotz [1969] examine

role of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between

peptide units do not stabilize a compact structure of protein However because the

peptide chain is shielded from water due to other interactions hydrogen bonding be

peptide groups do stabilize α-helical and β-sheet structures

Hydrophobic interaction Hydrophobic interaction refers to the spontaneous

ration and subsequent aggregation of non-polar components in an aqueous

environment In aqueous solutions of proteins the various non-polar amino acid residues

will be found in the interior of the molecule thus shielded from water The

intermolecular hydrophobic interaction for the stability of a compact protein structure

was first recognized by Kauzmann [1959] If all the hydrophobic residues are buried in

the interior and all the hydrophilic residues are at the outermost border of the molecul

intramolecular hydrophobic interaction would cause a compact protein structure

However geometrical and other types of interactions generally cause a fraction of the

hydrophobic residues to be exposed to the aqueous environm

Van der Waals interaction The mutual interaction between ionic groups dip

and induced dipoles in a protein molecule cannot be established as long as the pro

35

structure is not known in great detail Moreover the surrounding aqueous medium also

contains dipoles and ions that compete for participation in the interactions w

the protein molecule Dispersion interactions favor a compact structure However

because the Hamaker constant for proteins is only a little larger than that of water the

resulting effect is relatively small [Nir 1977]

272 β

ith groups of

rgiles et al 1996

Floeg

) is degraded [Floege 2001] Renal

β2M from the serum resulting in an increase in β2M

conce well

the

)

a

al

2-Microglobulin (β2M)

The protein β2M is of particular interest because it is involved in the human

disorder dialysis-related amyloidosis (DRA) [Geiyo et al 1985 A

e 2001] DRA is a complication in end stage renal failure patients who have been on

dialysis for more than 5 years [Bardin et al 1986 Drueke 2000] DRA develops when

proteins in the blood deposit on joints and tendons causing pain stiffness and fluid in

the joints as is the case with arthritis In vivo β2M is present as the non-polymorphic

light chain of the class I major histocompatibility complex (MHC-I) As part of its

normal catabolic cycle β2M dissociates from the MHC-I complex and is transported in

the serum to the kidney where the majority (95

failure disrupts the clearance of

ntration by up to 60-fold [Floege 2001] By a mechanism that is currently not

understood β2M then self-associates into amyloid fibrils and typically accumulates in

musculoskeletal system [Homma et al 1989] Analysis of ex vivo material has shown

that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRA

is present as of full-length wild-type β2M although significant amounts (~20-30) of

truncated or modified forms of the protein are also present [Floege 2001 Bellotti et al

1998] Figure 2-9 shows the ribbon diagram of human β2M Native β2M consists of

single chain of 100 amino acid residues and has a seven stranded β-sandwich fold typic

36

of the immunoglobulin superfamily [Saper et al 1991 Trinh et al 2002] β2M was fi

isolated from human urine and characterized by Berggard et al [1980] in1968 The

normal serum concentration of β

rst

re

f

e

2M is 10 to 25 mgL It is a small globular protein with

a molecular weight of 118 kDa a Strokes radius of 16Aring and a negative charge under

physiological conditions (isoelectric point IEP = 57) β2M contains two β-sheets that a

held together by a single disulphide bridge between the cysteines in positions 25 and 81

[Berggard et al 1980 Parker et al 1982 Cunningham et al 1973] β2M cannot be

removed completely by current dialysis techniques but through a better understanding o

the structure and interaction forces that lead to this structure it will be possible to mor

efficiently remove this problematic protein

Figure 2-9 Ribbon diagram of human β M taken from the crystal structure of the protein

bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khal 2000]

273 Serum Albumin

Serum albumin is the most abundant protein found in plasma and is typically

present in the blood at a concentration of 35~

2an et

50gL According to extensive studies about

its physiological and pharmacological properties albumin has a high affinity to a very

wide range of materials such as electrolytes (Cu+2 Zn+2) fatty acids amino acids

metabolites and many drug compounds [Fehske et al 1981 Kraghhansen 1981 Putnam

37

1984 Peters 1985] The most important physiological role of the protein is therefore to

bring such solutes in the bloodstream to their target organs as well as to maintain the pH

and osmotic pressure of the plasma Bovine serum albumin (BSA) is an ellipsoidal

protein with the dimensions of 140 X 40 X 40Aring [Peter 1985] The primary structure is a

single helical polypeptide of 66 kDa (IEP = 47) with 585 residues containing 17 pairs of

as a soft an

rfaces [Kondo et al 1991 Norde et al 1992 Soderquist et al

1980 ikely

is

disulfide bridges and one free cysteine [Dugaiczyk et al 1982] BSA has been classified

d flexible protein because it has a great tendency to change its conformation

on adsorption to solid su

Carter and Ho 1994] and consists of three homologous domains (I-III) most l

derived through gene multiplication [Brown 1976] Each domain is composed of A and B

sub-domains [He et al 1992] The secondary structure of human serum albumin (HSA)

shown in Figure 2-10

Figure 2-10 Secondary structure of human serum albumin (HSA) with sub-domains

[Zunszain et al 2003]

HSA has the same structure domains with the serum albumin from other species

such as BSA [Brown 1976] Although all three domains of the albumin molecule have

similar three-dimensional structures their assembly is highly asymmetric [Sugio et al

38

1999] Domains I and II are almost perpendicular to each other to form a T-shaped

assembly in which the tail of subdomain IIA is attached to the interface region between

sub-domains IA and IB by hydrophobic interactions and hydrogen bonds In contrast

domain III protrudes from sub-domain IIB at a 45ordm angle to form the Y-shaped assembly

for domains II and III Domain III interacts only with sub-domain IIB These features

make the albumin molecule heart-shaped

28 Protein Adsorption

Protein adsorption studies date back to the 1930rsquos At the beginning these studies

mainly focused on the determination of the molecular weight electrophoretic and

ral

rearrangem

adsorption and biocompatibility of the sorbent materials were investigated [Norde 1986]

There are interaction forces at the interfaces between protein molecules and latex

particles These forces are mainly divided into the following groups hydrophobic

interaction ionic interaction hydrogen bonding and van der Waals interaction [Andrade

1985]

Hydrophobic interaction It is known that hydrophobic interaction has a major

role in protein adsorption phenomena The adsorption of proteins on the low charged

latex particles occurs by this interaction force Generally monomers such as styrene offer

hydrophobic surfaces th

by this interaction force is maximum at the isoelectric point (IEP) of the protein and the

strength [Suzawa et al 1980 1982 Shirahama et al 1989 Kondo et al 1992] By the

chromatographic applications Later the adsorption mechanism especially the structu

ents was studied Recently the studies of the relation between protein

281 Interaction between Protein Molecule and Latex Particle

at protein molecules adsorb to The amount of adsorbed protein

pH at maximum adsorption shifts to a more acidic region with an increase in ionic

39

reports [Suzawa et al 1980 1982 Lee et al 1988] protein adsorption was greater

hydrophobic surface than on a hydrophilic one implying that hydrophobic interaction is

on a

s

ydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA)

Ionic

ely

t

ces

n force is operative over small

distan e

ble

n

nt dialysis membrane therapy for end stage renal disease (ESRD)

one of most dominant forces in protein adsorption

Ionic interaction Negatively charged latex particles have ionic functional

groups on their surfaces such as salts of sulfonic and carboxylic acid Sulfate group

originate from an initiator such as sodium persulfate and carboxylic groups originate

from a h

bonds are formed between the negative charges of these latex particles and positive

surface charges of protein molecules The conventional low-charged latex particles rar

form these ionic bonds

Hydrogen bonding Hydrogen bond is a strong secondary interatomic bond tha

exists between a bound hydrogen atom (its unscreened proton) and the electrons of

adjacent atoms [Callister 1999] Protein can be adsorbed on hydrophilic polar surfa

through hydrogen bonding Hydrogen bonds are frequently formed between hydroxyl-

carbonyl or amide-hydroxyl Hydroxyl-hydroxyl or amide-hydroxyl bonds are also

formed in protein adsorption

Van der Waals interaction This interactio

ces only when water has been excluded and the two non-polar groups come clos

to each other Lewinrsquos calculation showed that the van der Waals interaction is negligi

compared with the forces involved in the entropy increases ie hydrophobic interactio

[Lewin 1974]

29 Hypothesis for Toxin Removal

As mentioned earlier insufficient removal of middle molecular range toxins is a

major drawback of curre

40

patien

is

y

ese

haracteristics over the last decade However removal

effici

ing a novel membrane design composed of an assembly of engineered

polymeric latex particles s ions Important factors

will i

e

ith a

is established it is

expec

e

ts This may cause destructive arthritis and carpal tunnel syndrome inhibit the

immune system and accelerate cardiovascular disease leading to death among dialys

patients In order to overcome these complications artificial dialysis membranes have

been developed to increase the capacity for removing middle molecular weight toxins b

changing the pore size of dialysis membranes and using new materials that adsorb th

toxins for improved removal c

ency is not as high as those achieved by a normal healthy kidney

With the knowledge obtained from all above literatures the following hypothesis

has been established for the development of a membrane for the successful removal of

the target protein β2-Microglobulin (β2M) without the removal of serum albumin This

will be done us

ynthesized to predetermined specificat

nclude pore size and surface chemistry

291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size

The packing of monodispersed spherical particles can lead to the formation of

porous layers suitable for use as filters and membranes [Hsieh et al 1991] The pore siz

is defined as the largest spherical particles that can pass through the interstitial spaces

It is well known that monodispersed spherical particles can be obtained from the

seed emulsion polymerization method Many commercially available latex particles are

synthesized by this technique and are inexpensive When the defect free membrane w

monodisperse pore size distribution based on a particular particle array

ted to outperform the traditional hemodialysis membrane limitation which is a

broad range of pore size distribution When spherical particles pack in regular crystallin

arrays a number of packing geometries such as hexagonal closest packing cubic closest

41

packing and body-centered cubic packing are possible The pore size of the array

depends on these packing geometries as well as particle size Theoretical pore size can be

calculated from the simple geometry of the arr

ay Figure 2-11 shows the relationship

between particle size and pore size

0001

001

01

001 01 1 10

Por

e

1

10

microm

) S

ize

(

Particle Diameter (microm)

Body-Centered Cubic ArrayClosest Packed Array

0001

Siz

e (

Particle Diameter (microm)

001

01

001 01 1 10

Por

e

1

10

microm

)

Body-Centered Cubic ArrayClosest Packed ArrayBody-Centered Cubic ArrayClosest Packed Array

and closed packed arrays [Steve et al 1999]

McDonald and his coworkers [Ramakrishnan et al 2004 Steve et al 1999] have

described the fabrication of latex composite membranes and their performance in

previous papers It is from this prior research and knowledge gained from conversations

with Dr McDonald that this current research has been initiated [McDonald 2003] The

focus of this thesis is the extension of McDonaldrsquos research in the area of composite

membranes to medical application such as dialysis membranes

292 Toxin Removal by Selective Adsorption on Engineered Latex Particles

The flexibility in latex surface properties is a significant advantage to this

materialrsquos ability to separate proteins since the selectivity is strongly dependent on the

charge interactions of both the protein and the latex particles under the conditions of

Figure 2-11 The relationship between pore size and particle size in body-centered cubic

42

separation [Menon et al 1999 Chun et al 2002] First all sorbents were designed to

have negative surface properties because most plasma proteins in blood are negative and

should not be removed with charge interaction between proteins and sorbents at

physiological condition (pH74) It was reported that a negatively charged surface was

more blood compatible than a positive one [Srinivasan et al 1971] The toxin protein β2-

Microglobulin (β2M) can be selectively adsorbed on the surface of latex particles by the

design of a suitable hydrophobic portioned surface where only β2M protein can be

anchored with hydrophobic interactions Albumin adsorption on the latex particle is not

side-on m

adsorption of β M protein on the engineered latex particles

allowed because charge repulsion is more dominant than hydrophobic interaction with

ode The figure 2-12 shows the schematic representation of the selective

2

PMMAPAAShell

COO-

OSO3-

OSO3-

PSCore

COO-

-

-

-

-

-

-

Albumin-

Chargerepulsion

--

-

- -

- -

- --

Hydrophobic

β2-Microglobulin

2

Interaction

β -Microglobulin

PMMAPAAShell

COO-

-

-

-

-

-

-

--

-

-

-

-

-

Albumin-

Chargerepulsion

OSO3-

OSO3-

PSCore

COO-

--

-

- -

--

-

- -

- -

- --

- -

- --

Hydrophobic

β2-Microglobulin

2

particles at pH 74

Interaction

β -Microglobulin

Figure 2-12 Schematic representation of the protein adsorption on the core shell latex

43

Hydrophilichydrophobic microdomain structures were proven to be more blood

compatible [Mori et al 1982 Higuchi et al 1993 Deppisch et al 1998] The

optimization of suitable hydrophobic to hydrophilic ratio is also important for the

biocompatibility The monomers such as styrene (St) methyl methacrylate (MMA) and

acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in

emulsion polymerization process

In summary the background and fundamental literature survey about the history

material properties and limitation of hemodialysis membrane latex particle preparation

surface chemistry and manufacturing process target proteins and protein adsorption and

finally the hypothesis for a toxin removal with high separation efficiency have been

suggested This research focuses on such hypothesis and the materials and

characterization methodology for achievement of suggested hypotheses are described in

the next chapter

CHAPTER 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY

As mentioned earlier the goal of this study is to prepare polymeric latex particles

with tailored properties to maximize separation of toxin molecules and to investigate

fundamental interactions between the applied particles and molecules in the

system to optimize the membrane performance for hemodialysis applications Polymeric

latex particles wit

the

biological

h monodisperse size distribution to obtain uniform pore size and

various size ranges to utilize membrane construction are necessary Surface engineering

with various combination of hydrophobichydrophilic domain on the surface of latex

particles is expected to affect the removal of target protein by selective adsorption and to

improve the biocompatibility of membrane Therefore the materials and characterization

methodology are addressed in this chapter

31 Materials

Styrene (St) monomer used for a seed and a core particle was purchased from

Fisher Scientific and used without any other purification process Acrylic acid (AA) and

methyl methacrylate (MMA) monomers introduced for shell formation were purchased

from Fisher Scientific and used without any other purification process Sodium persulfate

(SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as

received Divinylbenzene (DVB) crossliking agent was purchased from Aldrich The

anionic surfactant Aerosol regMA80-I [sodium di(13-dimethylbutyl) sulfosuccinate] was

kindly donated by Cytec 78-80 Aerosol regMA80-I is mixed with isopropanol and

water Its critical micelle concentration (CMC) is 245 mM (119) Ion exchange resin

44

45

AGreg 501-x8 Resin (20-50 mesh) was purchased from Bio-Rad Laboratories Inc It is a

mixed-bed resin with H+ and OH- as the ionic forms and is 8 crosslinked Bovine serum

albumin (BSA) (heat shock treated) was purchas Fisher Scientific The isoelectric

point of t lin

(β2M)

as

gma

ed from

he BSA is 47-49 and the molecular weight is 66300Da β2-Microglobu

was purchase from ICN Biomedicals Inc and was separated from patients with

chronic renal disease and lyophilized from ammonium bicarbonate The molecular

weight by SDS-PAGE was approximately 12000Da BSA and β2M proteins were used

received without further purification process In order to investigate the protein

adsorption evaluation bicinchoninic acid protein assay kits were purchased from Si

(Cat BCA-1) and Pierce Biotechnology (Cat 23225) The chemical structures for the

main chemicals are shown in Figure 3-1

CH2 CH

Styrene (Mw = 10415)

CH2 CHCH2 CH

Styrene (Mw = 10415)

CH2 CH

Acrylic acid (Mw = 7206)

C

OH

O

CH2 CH

Acrylic acid (Mw = 7206)

C

OH

O

CH CH2

C

OCH3

O

CH

CH CH3

2

C

OCH3

O

CH3

Methyl methacrylate (Mw = 10012)Methyl methacrylate (Mw = 10012)

CH2 CH

Divinylbenzene (Mw = 1302)

CH CH2

CH2 CH

Divinylbenzene (Mw = 1302)

CH CH2CH CH

+(Na) -(O3S)

CH -C-O-CH-CH -CH-CH3

O

2 2 3

O CH CH3

CH3 CH3

CH-C-O-CH-CH2-CH-CH3

Anionic surfactant (MA80-I) (Mw = 3885)

+(Na) -(O3S)

CH -C-O-CH-CH -CH-CH3

O

2 2 3

O CH CH3

CH3 CH3

CH-C-O-CH-CH2-CH-CH3

Anionic surfactant (MA80-I) (Mw = 3885)2

+(Na)-(O) S OO

O O

- +

Initiator sodium persulfate (Mw = 23811)

O SO

(O) (Na)+ -

O O

- +(Na) (O) S OO

O SO

(O) (Na)+ -

O O

- +

Initiator sodium persulfate (Mw = 23811) Figure 3-1 Chemical structure of main chemicals

(Na) (O) S OO

O SO

(O) (Na)

46

32 Latex Particle Preparation

The experimental set up and the particle preparation schemes for various types and

size ranges of latex particles are shown in Figure 3-2 and 3-3 In the experimental setup a

mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at

end of glass stirrer The agitation rate is precisely adjusted by controller motor Paar glas

reactor with 1L volume is partially in silicon oil bath on hot plate The reactor has fo

openings for a stirrer a thermometer a nitrogen gas inlet and a reactant feeding lnlet

The reactants for latex polymerization are fed by precisely controlled by meterin

The particle preparation is described in each latex preparation section

the

s

ur

g pump

Figure 3-2 Experimental setup for semi-continuous emulsion polymerization

1 Motor 2 Mechanical stirrer bar 3 Thermometer 4 Inlet of N2 5 Inlet for initiator and monomer 6 Glass reactor (l L) 7 Oil bath 8 Hot plate 9 Pump for initiator 10 Pump for monomer 11 Tubes for reactant feed

47

Styrene monomer

Bare PS

PSPMMA100

PSPMMA90PAA10

PSPMMA P75 AA25

Gr

win

ticle

size

PS seedparticles

og

par

Styrene monomer

Bare PS

PSPMMA100

PSPMMA90PAA10

PSPMMA P75 AA25

Gr

win

ticle

size

PS seedparticles

og

par

Figure 3-3 The particle preparation scheme in various types and size ranges of latex particles

321 Preparation of Seed Latex Particles

Polystyrene seed particles were synthesized through a typical semi-continuous

emulsion polymerization Polystyrene latex particles were prepared in a four necked Paar

glass vessel equipped with a mechanical glass stirrer thermometer glass funnel for

nitrogen gas purge and a tube for monomer feeding The reactor was placed in a silicon

oil bath on a hot plate for homogenous and stable tempe he vessel was

firstly charged with de-ionized water the emulsifier so itiator

sodium bicarbonate (SBC) styrene (St) monomer divinylbenzene (DVB) monomer

under nitrogen gas atmosphere and then heated to 72plusmn2 as

llowed to continue for another hour Second reactants St and DVB monomers SPS

initiator and SBC were fed separately using fine fluid metering pumps (Model RHSY

Fluid metering INC NY) under a nitrogen gas atmosphere for 2h After complete feeding

rature control T

dium persulfate (SPS) in

oC for 1h The reaction w

a

48

of all reactants the reaction was continued for another hour at elevated temperature 80plusmn1

oC and then cooled to room temperature by removal of the oil bath

322 Preparation of Seeded Latex Particles

The particle growth was achieved by seeded continuous emulsion polymerization

The vessel was charged with the polystyrene seeds and de-ionized water to make desired

solid content and heated up to 72plusmn2 oC for 1h under nitrogen gas atmosphere Then St

and DVB monomers and solution of SPS and SBC in de-ionized water were continuously

added using metering pump with a precisely controlled feed rate After feeding all

reactants the reaction was continued for three hours keeping temperature of 80plusmn2 oC and

then the reaction vessel was cooled down to room temperature

Monodisperse spherical polystyrene seeds with a 280nm mean diameter were used

e-polymerized polystyrene seeded emulsion

was charged in a 500m

323 Preparation of Core Shell Latex Particles

for core shell structured latex particle The pr

l glass flask under a nitrogen gas atmosphere De-ionized water

was added to achieve the desired solid content The emulsion was heated to 80plusmn2oC for

10 hour Monomers initiator and SBC to form the shell were added by fluid metering

pumps (LAB PUMP JRRHSY model Fluid metering Inc USA) with fine controlled

feed rate Flow rate can be adjusted by control ring graduated in 450 division from 0 to

100 flow which is in the range of 10mlmin respectively The reaction was sustained

for additional 30 hours at 90plusmn2oC then cooled down to room temperature

324 Purification of Synthesized Latex Particles

Synthesized latex particles were purified with an ion exchange resin 200g (10

ww polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin

and stirred for 40 minute to remove unreacted monomers initiators and other impurities

49

Then the emulsion was diluted with DI water to a specific degree of solid content as

needed

33 Characterization

331

solid

termined

oscopy

d

hours

Dried latex (10mg) was mixed with

250 m on

Degree of Conversion

The degree of monomer to polymer conversion was determined gravimetrically

following the procedure described by Lee et al [1995] before the latex cleaning process

The synthesized emulsion raw materials were weighed and dried in a conventional oven

at 120oC for 30min to evaporate any unreacted monomers and water The remaining

was weighed and the degree of conversion was determined This process was repeated

three times and a mean value of the degree of conversion was de

332 Fourier Transform Infrared (FTIR) Spectr

A molecule can absorb only select frequencies (energy) of infrared radiation which

match the natural vibration frequencies The energy absorbed serves to increase the

amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]

Only those bonds which have a dipole moment that changes as a function of time can

absorb the infrared radiation The range of wavelengths for infrared radiation is between

25 microm (4000 cm-1) and 25 microm (400cm-1)

The synthesis of the latex particles such as polystyrene (PS) homopolymer and

PSPMMA PSPMMA90PAA10 PSPMMA75PAA25 core-shell copolymers were verifie

with FTIR Purified latex particles were dried in vacuum oven at 40oC for 24

before analysis in the FTIR (Nicolet Magma USA)

g of KBr which had also been dried in vacuum oven at 120oC for 3h Transmissi

spectra using the drift mode were plotted with 128 scans and 4cm-1 resolution

50

333 Quasielastic Light Scattering (QELS)

Latex particle size was measured by the Brookhaven ZetaPlus particle size

analyzer The solid concentration as 0001 (wv) This

ation spectroscopy (PCS) of quasielastically scattered light

For th s

ed using a FE-SEM (JEOL

JSM-

ith

ent

01

us zeta-

potential analyzer It measures the electrophoretic mobility velocity of charged colloidal

of latex particles used w

instrument uses photon correl

is technique time dependent interference patterns of light scattered from particle

in the sample cell are analyzed The interference pattern changes due to Brownian

motion giving rise to fluctuations in scattering intensity The frequency of these

fluctuations depends on the particlersquos diffusion constant that is inversely proportional to

the particle diameter

334 Field Emission-Scanning Electron Microscopy (FE-SEM)

Particle size and surface morphology were characteriz

6335F Japan) A diluted suspension of latex particles in water was dropped onto a

silicon wafer at room temperature and allowed to dry The sample was then coated w

the thinnest layer of carbon needed to obtain the required conductivity for this instrum

Secondary electron image mode was used with a 15KV of accelerating voltage The

magnification range used was between 10000X and 70000X

335 Zeta Potential Measurement

Synthesized and purified latex particles were diluted with de-ionized water to 0

wt and adjusted six different pH values 205 345 481 565 643 and 747 These

particle suspensions were then transferred to standard cuvettes for zeta potential

measurement At least two runs of ten measurements were taken for each sample and

averaged

The zeta potential measurement was carried out using Brookhaven ZetaPl

51

particles in solution and calculate zeta-potential by using the Smoluchowski equation

The f quency

gnitude

umin (BSA) as a standard model protein and β2-Microglobulin

uffer (PB) and

phosp

5

h the selected protein

e 005 01 03 05 and 07 mgml and those for

β2M

d at

teins in the supernatant after the centrifugation process using the

bicinc t

requency of laser light passing through the sample and is compared to the fre

of a reference beam The shift in the frequency called a Doppler shift and the ma

of the shift correspond to the polarity and the magnitude of the electrophoretic mobility

respectively The zeta potential is calculated from the solution conditions and the

measured mobility

336 Protein Adsorption

Protein adsorption experiments of the synthesized latex particles were performed

with bovine serum alb

(β2M) as a target protein in two types of buffer solution phosphate b

hate buffered saline (PBS) To make 5 mM of PB solution 0345g of sodium

phosphate monobasic (NaH2PO4H2O) was dissolve in 500ml of de-ionized water To

make PBS solution 0345g of sodium phosphate monobasic (NaH2PO4H2O) and 4178g

(143mM) of sodium chloride (NaCl) were dissolved in 500ml of de-ionized water The

synthesized latex particles were diluted with each buffer to have a solids content of 0

(ww) adjusted to pH values of 32 48 and 74 and mixed wit

BSA concentrations were chosen to b

were 0015 0030 0045 and 0060mgml The mixture was gently rotated using

the shaker Labquakereg (BarnsteadThermolyne Model 4002110 USA) with 8 RPM in

the incubator at 37oC for 12 hours before the latex-protein mixture was centrifuge

13000 rpm for 15min The amount of protein adsorbed was determined by quantifying

the free pro

honinic acid (BCA) assay method [Lowry et al 1951 Smith et al 1985 Baptista e

al 2003 Wiehelman et al 1988 Brown et al 1989] BCA assay kits consist of Reagent

52

A containing bicinchoninic acid sodium carbonate sodium tartrate and sodium

bicarbonate in 01N NaOH with pH=1125 and Reagent B containing 4 (wv) copper

(II) sulfate pentahydrate The BCA working reagent was prepared by mixing 50 parts of

Reagent A with 1 part of Reagent B 100microl of protein supernatant was then mixed with

2ml of BCA working reagent in a UV cuvette Incubation was allowed to continue

at room temperature

further

until color developed about 2h The absorbance at 562nm was

pectrophotometer (Perkin-Elmer Lambda 800 USA)

Unkn

Separate the p rticles in centrifuge mix supernatant with BCA reagents

measured using a UV-VIS s

own concentration of sample protein was determined by comparison to a standard

of known protein concentrations The adsorbed amount per unit surface area was

determined by the mass balance of the protein after adsorption process The simple

schematic of the procedure for a protein adsorption test is shown in Figure 3-4

Incubate mixture with agitation for 12hrs at 37oC

aand develop the color by incubation for 2hrs at room temperature

Suspend latex particles in aqueous buffer media

Add the dissolved target protein

Measure color intensity using UVVis spectroscopy at 562nm

Suspend latex particles in aqueous buffer media

Separate the p rticles in centrifuge mix supernatant with BCA reagents

Add the dissolved target protein

Incubate mixture with agitation for 12hrs at 37oC

aand develop the color by incubation for 2hrs at room temperature

Measure color intensity using UVVis spectroscopy at 562nm

Figure 3-4 Schematic of the procedure for a protein adsorption test

53

337 Blood Biocompatibility by Hemolysis Test

Hemolysis is the destruction of red blood cells which leads to the release of

hemoglobin from within the red blood cells into the blood plasma Hemolysis testing is

used to evaluate blood compatibility of the latex particles and the damaged to the red

blood cells can be determined by monitoring amount of hemoglobin released The red

blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutes

Figure 3-5 shows the phase separated blood with plasma white blood cells and red

cells after centrifugation process Separated RBSs were then washed with isotonic

phosphate buffer solution (PBS) at pH 74 to remove debris and serum protein Th

process was repeated 3 times

blood

is

Figure 3-5 Separation of RBC from whole blood by centrifuge process

Prepared latex particles were re-dispersed in PBS by sonification to obtain

homogeneously dispersed latex particles 100microl of the mixture of red blood cell (3 parts)

and PBS (11parts) was added to 1ml of 05 (ww) particle suspension PBS was used as

a negative control resulting 0 hemolysis and DI water used as a positive control to

produce 100 hemoglobin released by completely destroyed RBCs The mixture was

incubated in water bath with gentle shaking for 30 minutes at 37oC and then centrifuged

at 1500 rpm for 15 minutes 100microl of the supernatant was mixed with 2ml of the mixture

) (EtOHHCl = 2005 ww) to prevent the

White blood cells

Plasma

Red blood cells

of ethanol (99) and hydrochloric acid (37

54

precipitation of hemoglobin In order to remove remaining particles the mixture was

centri fuged again with 13000 RPM at room temperature The supernatant was then

transferred into the UV cuvette The amount of hemoglobin release was determined by

monitoring the UV absorbance at a wavelength of 397nm The schematic of the

procedure for hemolysis test is shown in Figure 3-6

Separate red blood cells (RBCs) from blood

Mix RBCs with phosphate buffer solution (PBS)

Wash and re-suspend RBC with PBS repeat three times

Add la l of RBC and incu in at 37oC

tex particle suspension into 100mbate sample with agitation for 30m

Separate particle ith EtOHHCl solutions and mix the supernatant w

Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm

Separate red blood cells (RBCs) from bloodSeparate red blood cells (RBCs) from blood

Mix RBCs with phosphate buffer solution (PBS)Mix RBCs with phosphate buffer solution (PBS)

Wash and re-suspend RBC with PBS repeat three timesWash and re-suspend RBC with PBS repeat three times

Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC

Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC

Separate particles and mix the ith EtOHHCl solution supernatant wSeparate particles and mix the ith EtOHHCl solution supernatant w

Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nmEvaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm

Figure 3-6 Schematic of the procedure for hemolysis test

In summary a description of the experimental materials and characterization

methodology has been explained In the next chapter the results and discussion of the

data obtained from these experiments is addressed

CHAPTER 4 RESULTS AND DISCUSSION

41 Polymerization of Latex Particles

411 Polystyrene Seed Latex Particles

Polymer particles have many different applications including spacer particles

lubricants packing materials for chromatography standard particles and diagnostic

drugs All applications strongly require these particles to have a uniform particle size

Uniform particle size is the first requirement for a latex composite membrane formed by

particle arrays with the interstitial spaces serving as pores for size discriminations [Jons

et al 1999 Ramakrishnan et al 2004] Although suspension polymerization has been

mainly used as one of conventional polymerization methods to prepare uniform particles

the uniformity of the recovered particles is insufficient for membrane use There is

another method known as seed emulsion polymerization where a vinyl monomer is

absorbed into fine monodiserse seed particles and polymerization causes the monomer to

crease the sizes of the seed particles uniformly In order to obtain monodisperse larger

particles by this m

particles and polymerization of the monomer is repeated

In this study cross-linked polystyrene seed latex particles were firstly synthesized

in the presence of styrene monomer divinylbenzene crosslink agent sodium persulfate

initiator sodium bicarbonate buffer and the Aerosol regMA80-I [sodium di(13-

dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion

polymerization process in which one or more components can be added continuously

in

ethod the procedure of absorption of monomer into fine polymer

55

56

Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and

buffer are continuously added to enlarge the seed latex particles Various profiles of

particle nucleation and growth can be gene different orders of component

addition during polymerizati f this process such as easy

control of the polymeriza particle number

and particle morphology The main goal in

this process is to avoid secondary particle

rated from

on There are several advantages o

tion rate monodisperse particle size and

colloidal stability copolymer composition

formation leading to a monodisperse particle

size distribution Figure 4-1 shows the schematic representation of seed latex particle

preparation and growth

Monomer droplet

Seed particles Amount of surfactantsis variable

Enlarged final Add monomers

PS

PS

PSs4

particles Number of particlesare constant

Seed code

PSs1

s2

s3

Monomer droplet

Seed particles Amount of surfactantsis variable

Enlarged final Add monomers

PS

PS

PSs4

tion The

particles Number of particlesare constant

Seed code

PSs1

s2

s3

Figure 4-1 Schematic representation of semi-continuous seed latex particles preparation and growth

After the first synthesis of a small size of crosslinked polystyrene (PS) seed

particles stabilized by surfactants styrene DVB and initiator are continuously fed into

the system to enlarge the seed latex particles maintaining a narrow size distribu

recipes and reaction conditions for synthesizing these seed particles are summarized in

57

Table 4-1 The original recipe for latex particle preparation from McDonald was

modified for this emulsion system [McDonald 2003]

Table 4-1 The polymerization recipe of polystyrene (PS) seed particles Latex label PSS259 PSS233 PSS207 PSS181

Temperature (oC) 75 plusmn 5 75 plusmn 5 75 plusmn 5 75 plusmn 5

Divinylbenzene (g) 052 052 052 052

MA 80 (g) 259 233 207 181

NaHCO

Watera (g) 1315 1315 1315 1315

Styrene (g) 9948 9948 9948 9948

112 112 112 112

Na2S2O8 (g) 190 190 190 190

Waterb (g) 200 200 200 200

Reaction time (min) 140 140 140 140

Solids content 396 396 396 396

Mean particle diameter (nm) 126plusmn75 171plusmn39 182plusmn41 216plusmn53

Conversion () 965 979 957 971

3 (g)

Four different seed latex particles were synthesized based on surfactant amount in

order to determine the optimum condition for stable and monodisperse latex particles

The emulsion polymerization of latex particles has to be carried out in a narrow range of

surfactant causes flocculation or phase separation leading to broad particle

size distribution and an unstable emulsion sy

surfactant concentration where particles are stable [Nestor et al 2005] A wide range of

concentrations

stem Therefore the range of surfactant

amount was varied from 181g to 259g in increments of 026g in each latex label Other

reactants amounts were kept constant Watera is the initial charge amount and waterb

represents the amount of aqueous solution containing dissolved initiator and buffer

58

which are fed continuously after the seed particle creation The extent of monome

polymer conversion was obtained by gravimetric calcu

r to

lation and was more than 95 at

cripts such as

S207 and S181 i the surf mount added for the seed latex

nversion indi perce experi obtain

content divided by the theoretically estimated solid content and was calculated by the

all seed particles labeled PSS259 PSS233 PSS207 and PSS181 Labeled subs

S259 S233 ndicate actant a

preparation The co cates the ntage of mentally ed solid

following formula

100 colids () times=entsoliatedntentoalExperimentConversion

n the experimenta content is the weight of olid rema after

mulsion at 120oC f in and the estimated solid content is th ht

lated from the recipe

rti as di each t load d

mple of ea pension iluted by (ww)

deionized water and the mean particle diameter was determined using the Brookhaven

ZetaPlus particle size analyzer From these results the seed latex particle size increased

as the amount of surfactant decreased The amount of emulsifier also affects the

polymerization process by changing the number of micelles and their size It is known

that large amount of emulsifier produces larger numbers of smaller sized particles [Odian

1991] and was corroborated in the current work

These synthesized seed latex particles should be of narrow size distribution in order

to obtain monodisperse larger particles The SEM image showing the particle

morphology and size of a sample of polystyrene seed particles can be seen in the Figure

4-2

contdEstim (4-1)

In this equatio l solid the s ining

evaporation of e or 30m e weig

of monomers calcu

As expected the seed pa cle size w fferent for surfactan ing in see

emulsion system A sa ch sus was d 0001 with

59

Figure 4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)

PSS259 (B) PSS233 (C) PSS207 (D) PSS181 (subscripts indicate the amount of surfactant added)

(A)

(B)

60

(C)

(D)

Figure 4-2 Continued

61

s seen in the image the seed particles are very smooth and spherical The

uniformity in the particle size distribution of these PS seed latex particles can also be

seen in the SEM image From this data we concluded that seed particles synthesized in

the presence of a surfactant with the chosen amounts are suitable for use to prepare larger

particles with high size uniformity and smooth spherical morphology

412 The Growth of Polystyrene (PS) Seed Latex Particles

Seeded emulsion polymerization has been conducted for several decades and

various mechanisms have been proposed Gracio et al [1970] suggested that the growing

polymer particles consist of an expanding polymer-rich core surrounded by a monomer-

rich shell with the outer shell providing a major locus of polymerization Seeded

mulsion polymerization is commonly used for preparing latex particles less than 1microm in

size [Cha et al 1995 Zou et al 1990 1992 Gandhi et al 1990 Park et al 1990] Such

latexes can be obtained from the growth of pre-prepared seed particles The seeds

introduced in this process serve as nucleation sites for particle growth By first swelling

the seed latex particles with additional monomer to the desired size polymerization can

then be initiated Several size ranges of monodisperse polystyrene (PS) latex particles

were prepared by this multistep seeded emulsion polymerization method For the

fabrication of particle-based membranes monodisperse latex particles with a variety of

size ranges are required

In this study seed particles from 171nm to 470nm in mean diameter and with a

highly uniform size distribution were prepared and used Watera is the initial charge

mount and waterb represents the amount aqueous solution containing dissolved initiator

and buffer that is continuously fed into system Feed time is precisely controlled by

metering pump The solid contents were maintained between 199 and 300 After

A

e

a

62

polym d as

es

ere 923 8402

n of these latex particles

are sh

ss

erization under these conditions the conversions of the latex particles labele

PS258 PS320 PS370 and PS410 were 952 957 946 and 964 respectively The recip

used in seeded emulsion polymerization to form the particles less than 500nm in mean

diameter are shown at the Table 4-2 The conversions of the latex particles larger than

500nm in mean diameter and labeled as PS525 PS585 PS640 and PS790 w

723 and 969 respectively The recipes used in the preparatio

own at the Table 4-3

A simple calculation can be used to predict the ultimate size that a latex particle can

reach after ending the polymerization Letrsquos suppose that seed density (ρs) and grown

particle density (ρgp) are the same The following equation is obtained from the ma

balance (m = ρV) where m and V are mass and volume of a particle respectively

gp

gp

s

s

Vm

Vm

In this equation m

= (4-2)

be

s and mgp are the weight of seed and total weight of seed and

added monomers respectively and Vs and Vgp are the volume of seed and grown

particle respectively

If we know the mass of monomer to be added the ultimate particle size can

calculated from the equation 4-3

3

2s D m ⎠⎝+ χ

where D

1s D m

⎟⎟⎞

⎜⎜⎛

= (4-3)

the

in

s and Dgp are the diameters for seed and grown particle respectively and χ is

weight of the monomer added Crosslinked PS latex particles with a size range from

258plusmn112 ~ 410plusmn117 in diameter polymerized by the recipes in Table 4-2 are shown

the SEM images in Figure 4-3

63

Table 4-2 Continuous addition emulsion polymerization recipe for growing polystyrene

Latex label PS

(PS) latex particles less than 500nm in size

258 PS320 PS370 PS410

Initial charge

Seed latex (g) 1000171 1000216 1000216 1000320

Watera (g) 1470 2500 1984 2600

06 07 06 06

Fee

Initiator stream

2 2 8

Feed time (min) 0-167 0-140 0-100 0-92

Solid content 284 300 300 200

Mean particle diameter (nm) 258plusmn112 320plusmn154 182plusmn71 410plusmn117

Conv

Monomer continuous

Styrene (g) 800 1200 800 600

Divinylbenzene (g)

d time (min) 0-163 0-110 0-75 0-82

Na S O (g) 07 065 05 05

NaHCO3 (g) 07 065 05 05

Waterb (g) 750 1200 200 370

ersion () 952 957 946 964 The solid content of seed latex for PS258 PS320 PS 370 and PS410 are 392 399 399 anrespectively

d 300

64

Table 4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size

Latex label PS525 PS585 PS640 PS790

Initial charge

Seed latex (g) 1000390 1000470 1000470 1000320

Watera (g) 1000 520 1200 360

Monomer continuous

Styrene (g) 290 200 400 173

Divinylbenzene (g) 02 014

03 009

Feed time (min) 0-80 0-40 10-76 5-65

Initiator stream

Na2S2O8 (g) 02 014 03 012

NaHCO3 (g) 02 014 03 009

Waterb (g) 200 120 400 200

Feed time (min) 0-85 0-46 0-77 0-75

Solids content 199 200 200 201

Mean particle diameter (nm) 525plusmn151 585plusmn186 640plusmn178 790plusmn693

Conversion () 923 8402 723 969 The solid content of seed latex for PS525 PS585 PS 640 and PS790 are 200 182 193 and 173 respectively

65

Figure 4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)

370nm (D) 410nm

(A)

(B)

66

(C)

(D)

Figure 4-3 Continued

67

article size was well matched to that estimated by the geometrical calculation

From this it can be concluded that the added monomer was consumed for polymer

particle growth with a high degree of conversion From the SEM characterization newly

nucleated particles were not seen as indicated by the narrow particle size distribution

The particles less than 500nm in mean diameter were very spherical in shape with a

highly uniform particle size distribution As the particles were grown larger than 500nm

mean diameter however they became of non-spherical shape with an uneven surface

This irregularity of particle surface can be attributed to the non-homogeneous monomer

swelling into the shell of a growing polymer which can be controlled by the factors such

as temperature agitation speed initiator feeding rate and surfactant amount required to

stabilize the colloidal system The SEM image of latex particles larger than 500nm mean

iameter is shown in Figure 4-4 The particle size distribution is broader than that of the

s particles These particles can however still be used for membrane construction

as a support layer rather than skin layer The support layer does not necessarily have to be

as highly monodisperse as the skin layer which require pores of high uniformity for the

selective removal of toxins

As mentioned earlier the amount of surfactant as well as monomer in an emulsion

system is the primary determinant of the particle diameter [Odian 1991] Anionic

surfactants are generally recommended at the level of 02-30 wt based on the amount

of water [Odian 1991] The critical micelle concentration (CMC) of Aerosolreg MA 80-I

anionic surfactant is 119 The surfactant loading in this emulsion system ranged from

the 121 for SS181 to 171 for SS259

P

d

maller

68

Figure 4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm

(C) 640nm (D) 790nm

(A)

(B)

69

(C)

(D)

Figure 4-4 Continued

70

The surfactant to monomer ratio refers to the value of the surfactant amount

divided by monomer amount after polymerization ends The surfactant amount is the

weight of the surfactant contained in emulsion and the monomer amount is the total

weight of the monomer added for polymerization to form the ultimate polymer particles

in emulsion As the surfactant to monomer ratio decreases mean particle diameter

increases as described by Odian [1991] Generally lower surfactant concentration forms

fewer micelles resulting in larger particle size [Evans et al 1999] However there is a

plateau in particle mean diameter between a surfactant to monomer ratio of 0005 and

0013 This is due to low polymerization conversion of latex particles from monomer

leading to a smaller particle size than expected This is not a factor of surfactant amount

The dependence of the particle size on the amount of surfactant is shown in Figure 4-5

0

200

400

600

800

1000

0000 0005 0010 0015 0020 0025 0030

Surfactant to monomer ratio (ww)

Part

icle

mea

n di

amet

er (n

m)

Figure 4-5 Dependence of the particle size on the surfactant to monomer ratio

71

413

e

f bare

microdomain structures is important for biocompatibility [Mori et al 1982 Higuchi et al

1993 Deppisch et al 1998] Figure 4-6 shows the schematic of core shell latex particle

structure The recipe for core shell structures is shown in Table 4-4

Core Shell Latex Particles

The latex particles shown in Figure 4-3 (A) and labeled as S258 in Table 4-2 wer

used as seeds to prepare core-shell latex particles because of their high particle

uniformity and smooth surface morphology Methyl methacrylate (MMA) and acrylic

acid (AA) monomers were introduced to increase surface hydrophilicity over that o

polystyrene particles AA is a more hydrophilic monomer than MMA because of the

carboxyl acid group in AA is more favorable for hydrogen boding with water The

surface carboxyl groups on PAA may have many promising applications in biomedical

and biochemical fields [Kang et al 2005] MMA monomer is less hydrophobic than

styrene PMMA is known to more biocompatible than PS but still PMMA is hydrophobic

The optimization of suitable hydrophobic to hydrophilic ratios and control of

Polystyreneseed particle

Core shelllatex particles

PSPMMA100

PSPMMA90PAA10

PSPMMA75PAA25

Polystyreneseed particle

Core shelllatex particles

PSPMMA100

PSPMMA90PAA10

Figure 4-6 Schematic of core shell latex particle structures

PSPMMA75PAA25

72

Table 4-4 The preparation recipe of PS core with various shell latex particles Latex 25 label PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA

Initial charge

PS seed latex (g) 500 500 500a

Monomer continuous

Methly methacylate (g) 320 288 240

Acrylic acid (g) 00 32 80

Initiator stream

Na

258 258 258

Water (g) 1570 1570 1570

Feed time (min) 0-120 0-50 0-60

b 200

Feed time (min) 0-122 0-60 0-65

952

2S2O8 (g) 02 02 02

NaHCO3 (g) 02 02 02

Water (g) 200 200

Solids content 183 183 183

Mean particle diameter (nm) 370plusmn186 370plusmn178 370plusmn191

Conversion () 955 924

Three types of core shell structures were prepared PMMA75PAA25 shell is a

copolymer consisting of the PMMA to PAA ratio of 75 to 25 by weight

PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90 to 10 by

weight PMMA100 is the PMMA homopolymer shell on PS core The particle size of

these core-shell particles as well as PS latex particle was prepared to be about 370nm in

mean diameter and are characterized to determine their zeta potential protein adsorption

and biocompatibility The image of SEM of PS and core shell particles is shown at Figure

4-7 The synthesized latex particles had a high uniformity in size and a smooth spherical

surface morphology

73

Figure 4-7 Scanning

Electron Micrograph of latex particles (A) PS (B) PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMMA75PAA25

(A)

(B)

74

(C)

(D)

Figure 4-7 Continued

75

42 Characterization of Latex Particles

421 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectroscopy has proven to be a powerful tool to characterize polymeric

materials A molecule can absorb only select frequencies (energy) of infrared radiation

which match the natural vibration frequencies The energy absorbed serves to increase

the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]

Only those bonds which have a dipole moment that changes as a function of time can

absorb infrared radiation The FTIR spectra of polymerized latex particles made during

this work are shown in Figure 4-8 There are number of characteristic peaks for PS latex

particles [Bhutto et al 2003 Li at al 2005] Aromatic C-H stretching vibration is shown

at 3002 cm-1 and 3103 cm-1 and aliphatic C-H asymmetrical and symmetrical stretching

shown at 2900 cm-1and 2850 cm-1 respectively There is an additional carbonyl (C=O)

adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some

amount of either or both PMMA and PAA The broad OH group peak at 3400 cm-1

appear for particles containing some amount of PAA and has intensity dependent on the

amount of PAA in the shell For example the peak intensity of OH groups for

PSPMMA75PAA25 is greater than that of PSPMMA90PAA10 since there is a higher AA

monomer content in PSPMMA75PAA25 than in PSPMMA90PAA10 This OH peak is not

seen for PS and PS core PMMA shell particles because of the absence of OH groups on

PS and PMMA polymer chains

422 Zeta Potential Measurements

The electrical surface property was characterized by zeta potential measurements of

tex particles Zeta potential is an important and useful indicator of surface charge which

can be used to predict and control the stability of colloidal systems The greater the zeta

is

la

76

1520002500300035004000

Wavenumbers (cm-1)

li

Re

ativ

e

Tra

nsm

ttanc

e

Figure 4-8 FTIR spectra of polymerized latex particles (A) ba

PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMM

(B)

(C) P

(D) P

C

C

OCH3

O

CH3

XCH2

CH2 n

CH

CH2 C

C

OCH3

O

XCH2 CH

OC

CH3

Y

OH

(A) Bare PS

500100000

re polystyrene (PS) (B) A75PAA25

PSPMMA100

SPMMA90PAA10

SPMMA75PAA25

77

potential the more likely the suspension is to be stable because the charged particles repel

one another and thus overcome the natural tendency to aggregate The measurement of

zeta potential is often the key ontrol of the interaction of proteins

with solid surfaces such as latex particles

Zeta potential (ζ) is the elec at exists at the shear plane of a particle

which is some small distance from the surface [Myers 1999] The zeta potential of the

particles can be determined by measuring the mobility of the particles in an applied

electric field termed electr its response to an alternating electric

field Colloidal particles dispersed in lu ically charged due to their ionic

characteristics and dipolar attributes The development of a net charge at the particle

surface affects the distribution of ions in the neighboring ng in

an increased concentration of counter ions to the surface Each particle dispersed in a

ns called fixed layer or Stern layer

Outside th compositions of ions of opposite polarities

form ble layer is form

particle-liquid interface This double layer may be considered to consist of two parts an

inner region which inclu nd relatively strongly to the surface and an outer or

diffuse region in which the ion distribution is determined by a balance of electrostatic

forces and random thermal motion The potential in this region therefore decays with the

distance from the surface until at a certain distance it becomes zero Figure 4-9 shows a

schematic representation of a typical ion distribution near a positively charged surface

The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the

to understanding and c

trical potential th

ophoretic mobility or

a so tion are electr

interfacial region resulti

solution is surrounded by oppositely charged io

e Stern layer there are varying

ed in the region of the ing a cloud-like area Thus an electrical dou

des ions bou

center of those ions which are effectively adsorbed to the surface [Hunter 1981] The

78

extent of ion adsorption is determined by electrical and other long-range interactions

between the individual ions and surface of particles The ions outside of the Stern layer

form the diffuse double layer also referred to as the Gouy-Chapman layer [Burns and

Zydney 2000]

Shear plane

Stern layer

ψζ

0

ψs

ψShear plane

Stern layer

ψζ

0

ψs

ψ

t

controlled electric field is applied via electrodes immersed in a sample suspension and

Figure 4-9 Schematic representation of ion distribution near a positively charged surface

Zeta potential is a function of the surface charge of a particle any adsorbed layer a

the interface and the nature and composition of the surrounding medium in which the

particle is suspended The principle of determining zeta potential is very simple A

79

this causes the charged particles to move towards the electrode of opposite polarity

Viscous forces acting upon the moving particle tend to oppose this motion and the

equilibrium is rapidly established between the effects of the electrostatic attraction and

the viscosity dra

g The particle therefore reaches a constant terminal velocity

Figure 4-10 Schematic representation of zeta potential measurement(source

httpnitioncomenproductszeecom_shtm)

Because protein adsorption mechanisms are very complex and hard to explain in

biological systems protein adsorption is generally studied by using more ideal systems

consisting of one or more well characterized proteins a well-characterized adsorbent and

a well-defined aqueous solution Even so small changes in the experimental conditions

such as pH ionic strength and temperature generate totally different results Therefore

two media systems phosphate buffer (PB) and phosphate buffered saline (PBS) were

chosen to be used for the zeta potential measurements and protein adsorption study PB is

a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4H2O) to

n a constant pH PBS is used to keep the water concentration inside and outside of

the cell balanced If the water concentration is unbalanced the cell risks bursting or

shriveling up because of a phenomenon called osmosis PBS is a solution with the 5 mM

of monobasic sodium phosphate (NaH2PO4H2O) and 143 mM of sodium chloride (NaCl)

in water

maintai

80

The results of zeta potential measurement at room temperature (25plusmn1oC) as

function of pH and ionic strength (different media PB and PBS) for synthesized lat

particles are seen in Figure 4-11 Zeta potential values of polystyrene (PS) latex particle

at the Figure 4-9 are negative between -291 mV and -599 mV in PB and betwe

ex

s

en -203

mV and -278 mV in PBS at all pH ranges These negative zeta potential values of PS

latex particles are due to the sulfate groups [Vandenhu et al 1970] originated from

persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer

chain where polymerization was initiated

-100

-80

-60

-40

-20

0

pH

Zta

pte

ntl

ζ

20

40

1 2 3 4 5 6 7 8 9

eo

ia (m

V)

PB (5mM)

PBS (5mM +143mM)

en -

Figure 4-11 Zeta potential of PS latex particles at 25oC

The zeta potential measurements of PSPMMA100 core shell latex particles as seen

in Figure 4-12 were also negative with a range of -28 and -505 mV in PB and betwe

143 mV and -186 mV in PBS at the pH 21-78 These negative values are also due to

sulfate groups on the particle surface

81

-100

-80

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

Zea

pote

nti

l ζ

(mV

pH

ta

)

PB (5mM)

PBS (5mM + 143mM)

Figure 4-12 Zeta potential of PSPMMA100 latex particles at 25oC

The zeta potential for PSPMMA90PAA10 and PSPMMA75PAA25 was also

negative as seen in Figure 4-13 and 4-14 The zeta potential value for the

PSPMMA90PAA10 were between -367 mV and -678 mV in PB medium and between -

147 mV and -193 mV in PBS both at 25oC In case of the PSPMMA75PAA25 core shell

particles zeta potential values were between -291 mV and -520 mV in PB and between

-115 mV and -210 mV in PBS media Sulfate groups in PSPMMA75PAA25 and

PSPMMA90PAA10 core shell particles contribute to the negative zeta potential values

participate in forming a

negat

cid is expressed by the term pKa which is the pH

at which an acid is 50 dissociated However the contribution of the carboxylic acid

Carboxylic groups from acrylic acid monomer also would

ive surface charge of the latex particles at a pH greater than 70 because the pKa

value for the carboxylic acid is between 40 and 60 pH range [Ottewill et al 1967] This

indicates that carboxylic acid dissociation begins at a pH between pH 46-60 and

increases with pH The strength of an a

82

group to negative zeta potential values of PSPMMA-PAA core shell particles was not

detectable because there was no significant difference in zeta potential values for

synthesized latex particles at a pH greater than 70

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

Zeta

pot

entia

l ζ

(mV

)

PB (5mM)PBS (5mM + 143mM)

-80

-100

pH

Figure 4-13 Zeta potential of PSPMMA90PAA10 latex particles at 25oC

-100

-80

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

pH

Ze p

ont

i ζ

V)

tate

al (m

PB (5mM)PBS (5mM + 143mM)

Figure 4-14 Zeta potential of PSPMMA75PAA25 latex particles at 25oC

83

The zeta potential plots and values were similar for all latex particles This may be

due to the similar initiator density on the latex surface The initiator concentrations

monomers for shell polymerization were the same fo

to

r all core shell latex particles (062

ww from Table 4-4) A similar concentration (055 ww) of initiator to monomer was

added to prepare bare PS latex particles used in zeta potential measurements

Since the zeta potential values of all synthesized latex particles were negative

between pH=20 and pH=78 the isoelecric point (IEP) of these latex particles would be

less than pH=20 Sulfates have a pKa in the range of 10 to 20 [James 1985] indicating

that the sulfate group the conjugate base of a strong acid is protonated (-C-OSO3H) at

less than pH 20 This means that the synthesized latex particles are all negative at the

physiological pH Most serum proteins have a negative surface charge in blood

therefore when the negative latex particles are applied to blood stream fatal

tein is

avoided by charge repulsion in the blood Table 4-5 shows the common blood proteins

and their isoelectric points (IEP)

Table 4-5 The isoelectric point (IEP) of common proteins Serum proteins Isoelectric point (IEP)

complication by coagulation between applied latex particles and serum pro s

Albumin 48 α1-Globulin 20 α2-Microglobulin 54 γ1-Globulin 58 Heptoglobin 41 Hemoglobin 72 β1-lipoprotein 54 Immunoglobulin G (Ig G) 73 Fibrinogen 58 (source httpwwwfleshandbonescomreadingroompdf1085pdf

httpwwwmedalorgvisitorwww5CActive5Cch135Cch13015Cch130105aspx )

84

The absolute zeta potential values of the latex particles in phosphate buffer (PB)

was higher than that in phosphate buffered saline (PBS) This is because high

concentration of sodium and chloride electrolytes in PBS compressed the electrical

double layer causing rapid decay of surface potential Figure 4-15 shows that the

thickness of the electrical double layer is inversely proportional to the concentration of

electrolyte in the system

Surf

ace

Pote

ntia

l ψ

1κ3

10

1e

1κ 1κ

(1)(3) (2)

2 1 Z

Surf

ace

Pote

ntia

l ψ

1κ3

10

1e

1κ 1κ

(1)(3) (2)

Figure 4-15 The decay of surface potential with distance from surface in various

electrolyte concentrations (1) low (2) intermediate (3) high [Myers 1999]

2 1 Z

The electrical potential in the solution surrounding the particle surface falls off

the Debye-Huckel

yers 1999]

exponentially with distance (Z) from the surface according to

approximation [M

z) (- exp 0 κψψ = (4-4)

e reciprocal of the thickness of the electrical double layer referred to as the

The potential fallen off by a factor e The theoretical equation for the

ickness 1κ is

where κ is th

Debye length of 1

double layer th

21

22i

0

ekT 1

⎟⎟⎠

⎜⎜⎝

=sum ii zcεε

κ

⎞⎛ (4-5)

85

where

tration of

y was performed to measure the protein adsorption on the

synthesized latex particles Many physical and chemical processes occur at the boundary

between two phases and adsorption is one of the fundamental surface phenomena [Oscik

1982] This phenomenon is driven by the change in the concentration of the components

at the interface with respect to their concentrations in the bulk phase There are two

adsorption processes physical adsorption occurring when non-balanced physical forces

appear at the boundary of the phases and chemical adsorption or chemisorption

t the

interface [J

n leads

ational entropy hence adsorption takes place only if the

loss in conformational entropy is compensated

ter adsorption Cp The initial part of the

isotherm merges with the Γ-axis because at low polymer concentration the protein has a

high affinity for the surface and all of the polymer is adsorbed until the solid surface is

saturated

ε0 is the permittivity of a vacuum of free space ε is the permittivity of a medium e

is the relative permittivity or dielectric constant of the medium c is the concen

an ion (i) z is the valency of an ion (i) k is Boltzmannrsquos constant T is absolute

temperature (K)

43 Protein Adsorption Study

The adsorption stud

occurring when atoms and molecules from adjacent phases form chemical bonds a

aroniec et al 1988]

Flexible proteins in solution possess high conformational entropy as a result of the

various states each of the many segments in the protein chain can have Adsorptio

to a reduction of this conform

by sufficient attraction between polymer

segment and solid surface [Martin 1998] Isotherm shape of high affinity adsorption

between polymer and solid is shown in Figure 4-16 where the adsorbed mass Γ is plotted

against the polymer concentration in solution af

86

Figur

de

arboxyl

e 4-16 High-affinity adsorption isotherm of typical flexible polymer on solid

surface

In order to investigate the adsorption of blood proteins latex particle properties

such as electrical surface charge hydrophobicity and environmental conditions like pH

ionic strength and temperature are considered as experimental factors [Arai and Nor

1990] Figure 4-17 shows the overall schematic representation of the protein adsorption

on the synthesized latex particles with the functional groups such as sulfate and c

groups

CP

Γ

PMMAPAAShell

PS or PMMAShell OSO3

-

OSO3-

OSO3-

OSO3-

PS

-O3SO

PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

Protein

NH3+

+

+H3N

+

COO-

Hydrophobic interaction

NH3

COO-

H3N

COO-

-OOC

Ionic interaction

Hydrophobic interaction

Hydrogen bonding

Ionic interaction

PMMAPAAShellOSO3

-

OSO3-

OSO3-

OSO3-

PS or PMMAShell -O3SOOSO3

-

PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

-O3SO

OSO3-

OSO3-

OSO3-

PS PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

Protein

NH3+

+

+H3N

+

COO-NH3

COO-

H3N

COO-

-OOC

NH3+

+

+H3N

+

COO-

Hydrophobic interaction

NH3

COO-

H3N

COO-

-OOC

Ionic interaction

Hydrophobic interaction

Hydrogen bonding

Figure 4-17 Overall schematic representation of the protein adsorption on the

Ionic interaction

synthesized latex particles

The surface functional groups as well as suitable surface hydrophobicity

hydrophilicity of latex particles are closely related to protein adsorption property As

indicated at Figure 4-17 various interaction forces such as hydrophobic interaction ionic

87

interaction hydrogen interaction and van der Waals interaction [Andrade 1985] exist

between protein molecule and the solid surface of latex particle

431 Adsorption Isotherm

The protein adsorption isotherm is generally defined as the relationship between the

amount of protein adsorbed by a solid (latex particle) at a constant temperature and as a

ine

the amount of protein adsorbed onto latex particles To evaluate the equilibrium

conce s

x

function of the concentration of protein Adsorption isotherms were plotted to determ

ntration of target proteins a calibration curve is firstly needed Figure 4-18 show

the UV absorbance as a factor of bovine serum albumin (BSA) concentrations The UV

absorbance of the known protein concentration was measured at 562nm which is the λma

for a protein treated by a bicinchoninic acid (BCA) assay

0

02

04

06

08

500 520 540 560 580 600

Wavelength (nm)

Abs

orba

nce

Figur gml (B)

06mgml (C) 02mgml

(A)

(C)

(B)

Max562nm

e 4-18 The dependence of UV absorbance on BSA concentration (A) 10m

Figure 4-19 shows the standard calibration curve used in adsorption isotherm

experiment of protein onto latex particles The net absorbance of each known protein

88

concentration was plotted and then a linear equation passing through a concentration of

zero protein concentration was obtained This equation is applied

to determine the

orbed on the synthesized latex particles The amount of free

protei

eported

trations (005 01 03 05

and 07mgml) were used to generate this curve as well as the adsorption isotherm

unknown protein amount ads

n was also determined using UV light at the 280nm wavelength however a

distinguishable UV intensity was too low to determine a precise value of protein

adsorption

The bicinchninic acid (BCA) assay technique [Smith et al 1985] has been r

for high efficiency of protein evaluation and was used for determination of protein

adsorption [Tangpasuthadol et al 2003 Williams et al 2003] This technique has many

advantages such as high sensitivity easy of use color complex stability and less

susceptibility to any detergents Five different BSA concen

y = 00R2 =

004741x 09939

00

01

02

03

04

0 200 400 600 800

BSA Standard (microgml)

Net

Abs

orba

nce

at 5

62nm

Figure 4-19 Standard curve of net absorbance vs BSA sample concentration

89

All isotherms were fitted to the Langmuir-Freundlich isotherms using the nonlinea

regression method Two adsorption isotherm models the Langmuir type isotherm a

Langmuir-Freundlich combination type isotherm were compared As seen the Figure 4-

20 The Langmuir isotherm model deviated further from the experimental data Th

all isotherms were fitted to the Langmuir-Freundlich isotherms [Yoon et al 1996

al 1998] usi

r

nd the

erefore

Lee et

ng the nonlinear regression method with following equation

n

n

eq

eqm

kC

kC1

1

1+

where q is the amount of adsorbed BSA per unit surface area q

qq = (4-6)

stant

were between 045 and 055 and hence n was fixed to its average 05 Actual qm values

were evaluated after fixing the n value

m is the adsorbed amount

in equilibrium Ceq is the equilibrium concentration of BSA k is the adsorption con

and n is the exponential factor for a heterogeneous system n values are empirical and

usually from zero to no more than unity [Jaekel 2002] The values of n in our system

00

10

20

30

0 02 04 06 08

C (mgml)eq

q (m

gm

2 )

pH 32Langmuir-Freundlich modelLangmuir model

Figure 4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on

polystyrene latex particles at 37oC in phosphate buffer media

90

The BSA protein adsorption on the synthesized latex particles was performed at

three pH levels 32 48 74 in both phosphate buffer (PB) and phosphate buffered saline

(PBS) at physiological temperature 37oC These pH levels correspond to an acidic pH

lower than the isoelectric point (IEP) of BSA the pH at the IEP and an alkaline pH

higher than the IEP of BSA respectively The IEP corresponds to the pH at which the

amino acid becomes a zwitterion Here its overall electrostatic charge is neutral and the

charge expressed by the basic amino group (-NH2 -NH3+) of the amino acid is equal to

the charge expressed by the acidic carboxyl group (-COOH -COO ) The IEP of BSA is

-

pH 48 and indicates that the side chain of the amino acids of BSA contains a greater

amount of acidic functional groups than that of basic functional groups At pH 32 and

74 BSA has a positive and negative charge respectively

Figure 4-21 and 4-22 show adsorption isotherms of BSA onto polystyrene (PS)

latex particles

00

10

20

30

40

0 02 04 06 08

Ceq (mgml)

q (m

gm

2 )

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in

phosphate buffer (PB) at 37oC

91

00

10

30

40

0 02 04 06 08

Ceq (mgml)

q (m

gm

2

20

)

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

(PBS) on PS latex particles

At the IEP of BSA the adsorbed amount (q

Figure 4-22 Adsorption isotherm of BSA adsorption 37oC in phosphate buffered saline

onto PS at equilibrium was

maxim

um at the IEP of BSA This is because the BSA molecules form the most

compact structures at the IEP eliminating the repulsion force between proteins A higher

number of BSA molecules can adsorb on a given surface area by hydrophobic interaction

when in this compact structure This observation agrees well with reported data by other

researchers who also indicated that the maximum adsorption from aqueous protein

solution was observed at the IEP [Mularen 1954 Watkins et al 1977 Oh at el 2000]

There was no significant difference of adsorbed BSA amount in PB and in PBS media at

pH 48 The conformational alteration of BSA was not affected significantly by the ionic

strength of the two solutions There was evidence of the effect of the electrostatic

interaction to some extent even at the IEP since the amount of BSA adsorbed in PBS is

slightly hig

m) of BSA

um indicating that hydrophobic interaction between latex particle and BSA

protein is maxim

her than in PB at this pH

92

The adsorbed amount of protein is shown to decrease at a pH 32 and 74 The

decrease in adsorption efficiency when deviating from IEP of BSA is a result of the

increase in conformational size of the protein molecules and the lateral electrostatic

repulsions between adjacent adsorbed BSA molecules Figure 4-23 shows a schematic of

possible conformational changes at each pH where hydrophobic interaction always exists

between non-polar portion of a protein and hydrophobic portion of solid surface

- - --- ---Latex surface

Zeta potential

BSA(+)

(-)

48 7432pH

(0)

+++ + ++

--+

++-

- - --- ---Latex surface

Zeta poten pH(0)

+++ + ++

--

tial

BSA(+)

(-)

48 7432

+

++-

ming

4

s

al blood Table 4-6 shows the calculated results of

Figure 4-23 Conformation of bovine serum albumin (BSA) on latex particles (Assuthat charge repulsion reduces the effect of the hydrophobic interaction at pH 74)

In addition the adsorbed amount of BSA at pH 32 was larger than that at pH 74

and still less than that at 48 This is because there is the electrostatic interaction between

the positive BSA molecules and negative PS latex particles at pH 32 and electrostatic

repulsion is dominant between negative BSA and negative PS latex particles at pH 7

The amount adsorbed increased with increasing ionic strength in pH 32 and 74 This i

because the high concentration of electrolytes reduces the electrostatic repulsion between

BSA and PS latex particle It should be noted that the BSA concentration used in these

experiments was lower than that found in normal human blood This is because the

equilibrium BSA adsorption onto the latex particles is actually reached at a much lower

concentration than that found in norm

93

adsor rm

ein

leading to a compact conformation of BSA There are two main forces affecting

adsorption amount at an acidic pH of 32 They include the ionic attraction between the

negative latex particle and the positive protein and lateral repulsion between positive

proteins The smallest amount of BSA adsorption was observed at a pH of 74 At this

point the charge repulsion between negatively charged particles and protein molecules

exists the h

of cha

bed BSA amount at equilibrium from the Langmuir-Freundich adsorption isothe

equation

In summary the hydrophobic interaction between PS latex particle and BSA

protein exists and is the dominant mechanism for the adsorption process Conformational

changes of BSA at each pH also affected the adsorption amount Adsorption was highest

at pH 48 because at this pH there is the least amount of ionic repulsion in the prot

and the lateral repulsion between proteins dominant adsorption even though there always

ydrophobic interaction between the solid latex particle and protein The effect

rge repulsion was reduced by the high concentration of electrolytes in PBS medium

Table 4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2) 32 174 48 299 Phosphate buffer 74 136 32 260 48 283 Phosphate buffered saline 74 207

The adsorption isotherms of BSA on PSPMMA core shell particles are shown in

Figure 4-24 in phosphate buffer (PB) and in Figure 4-25 in phosphate buffered saline

(PBS) The adsorbed BSA amount was calculated using the Langmuir-Freundlich

100

94

adsorption isotherm equation and the amount listed in Table 4-7 The trend seen in the

adsorption results was very similar to the case of PS latex The overall adsorption process

was d rticle

rs

hat

0

ominated by hydrophobic interactions between the PSPMMA100 core shell pa

and BSA proteins At pH 48 the adsorbed amount (qm) of BSA on PSPMMA100 core

shell particles was 221 mgm2 in PB and 237 mgm2 in PBS and are the maximum

values recorded among the three pH levels 32 48 and 74 Suzawa and his co-worke

[1982] have obtained similar results This can be also explained by the compact

conformation of BSA molecules at the IEP of BSA which lead to a high protein

population on the solid surface At a pH of 32 and 74 BSA adsorption was less than t

at pH 48 because of the protein conformation change There was a small increase in

adsorbed BSA at pH 32 because of the charge attraction between negative PSPMMA10

core shell particles and positive BSA At pH 74 however the adsorption of BSA was

further reduced by charge repulsion between the negative PSPMMA100 core shell

particles and the negative BSA proteins

000 02 04 06 08

Ceq (mgml)

10

20q (m

gm

302 )

40

50pH=32pH=48pH=74Mode 8)l (pH 4Mode 2)l (pH 3Mode 4)

Figure 4-24 Adsorption isotherm of BSA 37oC in PB on PSPMMA core shell latex

l (pH 7

100

particles

95

The adsorbed amount of BSA increased more rapidly in PBS with increasing ioni

strength at pH 74 This shows that the low concentration of electrolytes promotes

electrostatic repulsion between negative BSA and negative PSPMMA

c

on

x

l 1980

100 core shell

particle As the ion strength of PBS is increased however the high concentration of

electrolyte leads to less electrostatic repulsion promoting more rapid protein adsorpti

By comparing the amount of adsorbed protein between bare PS latex particles and

PSPMMA100 core shell particles the amount of protein adsorption on the PS late

particles is greater than that on the PSPMMA100 core-shell particles [Suzawa et a

1982 Lee et al 1988]

00

10

20

30

40

m2 )

0 02 04 06 08

Ceq (mgml)

q (m

g

pH=32pH=48pH=74Model (pH 2) 3Model (pH 48)Model (pH 74)

Figure 4-25 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA100 core shell

latex particles

96

Table particles calculated the Langmuir-Freundlich isotherm model

4-7 The equilibrium concentration values (qm) of BSA adsorption on PSPMMA100

Media pH qm (mgm ) 2

32 140

48 221 Phosphate buffer

74 130

32 169

48 237 Phosphate buffered saline

74 162

The BSA adsorption mechanism and trend on PSPMMA core shell particles was

very similar to that of the PS latex particle Unlike the case of PS and PSPMMA100 latex

particles the amount of BSA adsorbed onto PSPMMA90PAA10 and PSPMMA75PAA25

core shell particles was higher at pH 32 than pH 48 and 74 Figure 4-26 through 4-29

shows the adsorption isotherms of BSA on PSPMMA90PAA10 and PSPMMA75PAA25

core shell particles The amount of BSA adsorbed increased at pH 32 and pH 48 as the

hydrophilicity was developed by the addition of PAA On the contrary the adsorbed

amount of BSA was dramatically decreased at pH 74 The adsorption amount of BSA on

PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles at equilibrium is listed in

Table 4-8 and 4-9 respectively

Carboxylic acid as well as sulfate groups are distributed on the surface of

PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles and seem to largely affect

the BSA adsorption The isotherms showed very strong pH dependence to the BSA

adsorption onto these core shell latex particles The carboxylic groups (COOH) may

especially enhance the BSA adsorption prominently at pH 32 by hydrogen bonding with

protein molecules The pKa value for the PAA is 40-60 and a good indication that the

97

carboxylic acid groups are protonated at pH 32[Gebhardt et al 1983] The main

adsorption interaction force at pH 32 was generated by hydrogen bonding as well as

charge attraction between latex particle protein even th the conformational

repulsion between proteins results in a reduction of the protein

adsorption at this pH The amount of BSA adsorbed also increased as the PAA amount in

eased The a tion of BSA w inimal however less

than 07 mgm2 at pH 74 Here the carboxylic groups are ionized which increases the

charge repulsion by ionized carboxylic groups (COO-) resulting in PSPMMA-PAA latex

particle

and ough

change and charge

the core shell particles incr dsorp as m

s with much lower protein adsorption

00

20

40

q (m

g2 )

60

m

pH=32pH=48pH=74Model (pH 32)Model (pH 48)

0 02 04 06 08

Ceq (mgml)

Model (pH 74)

o shell

Figure 4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA90PAA10 corelatex particles

98

00

40

60

80

0 02 04

C (mgm

mm

2 )

20

06 08

eq l)

q (

g

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-27 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA90PAA10 core

shell latex particles

Table 4-8 The equilibrium concentration values of BSA adsorption on PSPMMA90PAA10 particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2)

32 361

48 283 Phosphate buffer

74 043

32 519

48 226 Phosphate buffered saline

74 043

99

00

20

40

60

80

100

0 02 04 06 08Ceq (mgml)

q (m

gm

2 )

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

oFigure 4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA75PAA25 latex particles

00

100

20

40

60

80

0 02 04 06 08Ceq (mgml)

q (

m2 )

pH=32pH=48pH=74

mg

Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-29 Adsorption isotherm of BSA 37oC in PBS on PSPMMA75PAA25 core shell

latex particles

100

Table 4-9 The equilibrium concentration values of BSA adsorption on PSPMMA75PAA25 particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2)

32 1083

48 662 Phosphate buffer (PB)

74 075

32 1077

48 681 Phosphate buffered saline (PBS)

74 057

The adsorption of β2-microglobulin (β2M) was determined under one set of

onditions in phosphate buffer at 37oC and pH 74 because of the limited amount of β2M

protein The adsorption isotherms of the β2M protein on the synthesized latex particles

are shown in Figure 4-30 4-31 and 4-32 The β2M protein concentrations for adsorption

isotherm experiment were chosen to be 0015 0030 0045 and 0060mgml since the

β2M concentration for the patients with renal failure can be elevated to similar

concentrations about 005mgml or more up to 50 times the normal level [Nissenson et

al 1995] There were steep initial slopes of the adsorption isotherm curves of β2M on

latex particles in all isotherms indicating a high affinity adsorption type It seems that the

hydrophobic interaction is enough for the adsorption process of β2M proteins to occur on

the latex particles even though charge repulsion exists between protein and latex particle

at pH 74 The complete plateau regions were not seen for all isotherms indicating that

β2M proteins were not saturated at this level and there are still available sites for β2M

c

adsorption on latex particles The initial slope of the β2M isotherms for PS was steeper

101

than that of any of the other latex particle PSPMMA100 PSPMMA90PAA10 and

PSPMMA 75PAA25

00

02

0 0002 0004 0006 0008

04

06

10

Ceq (mgml)

q (m

2 )

08

gm

PSPSPMMA100Model (PS)Model (PSPMMA100)

Figure 4-30 Adsorption isotherm of β2M onto PS and PSPMMA100 latex particles in PB

at 37 C and pH 74 o

00

02

04

06

08

10

0 0002 0004 0006 0008C (mgml)

q (m

m2 )

eq

g

PS

PSPMMA90PAA10

Model (PS)

Model (PSPMMA90PAA10)

Figure 4-31 Adsorption isotherm of β2M onto PS and PSPMMA90PAA10 latex particles

in PB at 37oC and pH 74

102

00

02

04

06

08

10

0 0002 0004 0006 0008Ceq (mgml)

q (m

gm

2 )

PS

PSPMMA75PAA25

Model (PS)Model (PSPMMA75PAA25)

Figure 4-32 Adsorption isotherm of β2M onto PS and PSPMMA75PAA25 latex particles

in PB at 37oC and pH 74

The equilibrium concentration values (qm) of β2M adsorption on latex particles are

calculated and listed in Table 4-10 The difference between the maximum amounts of

β2M adsorption on all the latex particles was not significantly different and their values

were between 069 and 08mgm2

Table 4-10 The equilibrium concentration values of β2M adsorption calculated the Langmuir-Freundlich isotherm model

Latex particles qm (mgm2)

PS 080

PSPMMA100 069

PSPMMA90PAA10 072

PSPMMA75PAA25 072

103

432 Adsorbed Layer Thickness

There are several possible explanations for large amount of BSA adsorbed at acidic

pH levels including the end-on adsorption pattern of BSA the flexibility of BSA

molecules the tilting of protein molecules due to the asymmetry of the charge

distribution or multilayer formation [Peula et al 1993] The adsorbed BSA thickness (δ)

can be calculated by the following equation [Chiu et al 1978] This equation was also

cited again by other researchers [Shirahama et al 1985]

BSA

mqρπ

δbullbull

=33

(4-7)

where π

33 is the packing factor qm is the plateau value of the adsorbed am

ρBSA is the hich corresponds to the reciprocal of its known

partial specific volume The layer thickness (δ) of BSA proteins on synthesized latex

partic

he IEP [Shirahama et al 1985] Compared to the

hydrodynamic dimensions of BSA 140 times 40 times 40 Aring3 [Fair et al 1980 Peter 1985] these

nd

ode It is unnecessary ultilayering because thickness do

exceed 140 Aring The layer thickness (δ) of BSA proteins on synthesized latex particles

e red saline (PBS) was ran 28 ~ 83 Aring at 37oC and pH 48 These

lso listed in Table 4-1

ount and

density of BSA molecule w

les in phosphate buffer (PB) ranged from 27 - 81 Aring at 37oC and pH 48 These

calculated values are listed in Table 4-11 Only δ values at pH 48 the isoelectric point

(IEP) of BSA are important because intra- and intermolecular electrostatic repulsion of

protein molecules is minimized at t

values of layer thickness indicate that the BSA molecules adsorb by side-on (40Aring) a

end-on (140Aring) m to consider m

not

in phosphat buffe ged of

calculated values are a 2

104

Table 4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37oC pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

32 21 17 44 133

48 37 27 35 81

74 17 16 5 9

Table 4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37oC

pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

32 32 21 64 132

48 35 29

74 26 20 5 7

28 83

1n

1n

neqm

So k is closely related to the initial slope of the isotherm showing the affinity

between BSA and latex particles [Yoon et al 1996] A key point in the characterization

433 Gibbs Free Energy of Protein Adsorption

In the Langmuir isotherm model k represents the ratio of adsorption to the

desorption rate constants This definition is not applicable to the Langmuir-Freundlich

isotherm model but its meaning is similar [Yoon et al 1996] In this sense k is closely

related to the affinity between protein molecules and latex particles When the

equilibrium concentration Ceq reaches zero the value of Ceq will be very small (1gtgt

kCeq ) and then the equation 4-6 becomes

(4-8)

of adsorption processes on solid-liquid interfaces is to determine the change in Gibbsrsquos

free energy (∆Gdeg) one of the corresponding parameters for the thermodynamic driving

forces during adsorption The traditional method to evaluate adsorption thermodynamic

kCqq 1=

105

constants is based on measurement of adsorption isotherms under static conditions

[Malm ten 1998] The Langmuir-Freundlich ads lf

easily found from Then this enables calculation of the free-

energy change ∆G during adsorption expressed as

(4-9)

where R is the general gas constant (R = 8314Jmiddotmol K ) and T is the absolute

tem ture Gibb e energy A P

listed in Table 4-13 and 4-14 respectively As the ∆Gdegads ore negativ e

pro adsorption on a solid substra ill be more favorable

e of BSA adsorption in phosphate o

pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

s orption equilibrium constant k can be

a slope of the isotherm

degads

lfads kRTG lnminus=∆ o

-1 -1

pera s fre change of BS adsorption values in B and PBS are

values are m e th

tein te w

Table 4-13 The values of Gibbs free energy changbuffer (PB) at 37 C

32 -2411 -2726 -2846 -2805

48 -2352 -2383 -2198 -1972 ∆Go

80

ads (kJmol)

74 -2452 -888 -324 -2

Table 4-14 The values of Gibbs free energy change of BSA adsorption in phosphate

pH PS PSPMMAbuffered saline (PBS) at 37oC

100 PSPMMA90PAA10 PSPMMA75PAA25

32 -2315 -2620 -2312 -2683

48 -2149 -2299 -2090 -2138 ∆Go

(kJmads ol)

74 -2430 -1992 -258 -263

Table 4-15 shows the calculated values of Gibbs free energy change of β M

adsorption on synthesized latex particles PSPMMA PAA core shell latex particle has

2

75 25

106

largest negative Gibbs free energy of β2

2

M adsorption indicating largest affinity betw

β

een

2M adsorption in phosphate buffere (PB) at 37 C and pH74

PS PSPMMA100

M and latex particle

Table 4-15 The values of Gibbs free energy change of βo

PSPMMA90PAA10 PSPMMA75PAA25

∆Go03 ads

(kJmol) -3626 -3466 -3659 -39

The Gibbs free energy of β2M was much more negative in phosphate buffer (PB)

than that of BSA in physiological condition (37 C and pH 74) o from the Table 4-13 and

the Figure 4-31 This indicates that β2M molecules more readily adsorb on latex particles

BSA This can be explained by the rate of diffusion and accessibility of β2M molecules to

the latex particle ier and faster for β2M m an BSA because of

size (molec ight) erical shape molecules It o be

due to the higher hydrophobic force and less electrostatic repulsion that contribute to the

adsorption of β2M molecules not as prevalent in the case of BSA because the IEP of β2M

(57) is clo H 74) than that of BSA (48) causing β2M to

be less electro cally lled p ro

can be seen that the ∆ β2M adsorption onto the synth article

increasing wi hy icity o x particles s the ∆Goads of the BSA

molecules is decreasing or constant This phenomenon supports the theory that the

select lizing

than BSA and that the affinity of β2M for the latex particle is much larger than that of

surface It is eas olecules th

the smaller ular we and sph of β2M may als

ser to physiological condition (p

stati repe from the latex articles than BSA F m Figure 4-33 it

Goads of esized latex p s is

th the drophil f the late wherea

ive adsorption of β2M molecules over BSA molecules can be enhanced by uti

more hydrophilic PSPMMA90PAA10 and PSPMMA75PAA25 core shell latex particles

107

-60

-50

1 2 3 4

-40∆Gad

s

-30

10

0

o (k

Jm

o-

-20l)

β2-Microglobulin (β2M)Bovine serum albumin (BSA)

Figure 4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37

and pH 74

434 Kinetics of Adsorption

to

oC

Protein adsorption is a complex process known to have a large biological impact

and is currently not well understood quantitatively because of extreme sensitivity to pH

the concentration of other electrolytes and molecules present temperature and many

other factors that can change in the physiological system Accurate knowledge of the

adsorption kinetics under a given set of conditions is a prerequisite for elucidating the

mechanisms of many fundamental biological processes to be predicted at the molecular

level [Regner 1987] This adsorption kinetics approach is used in protein adsorption

studies because of the uncertainty related to the time needed for the interfacial proteins

reach the equilibrium It is generally accepted that the process of protein adsorption is

comprised of the following steps a) transport toward the interface b) attachment at the

interface c) eventual structural rearrangements in the adsorbed state d) detachment from

PS PSPMMA100 PSPMMA90PAA10PSPMMA75PAA25

108

the interface and transport away from the interface Each of these steps can affect the

overall rate of the adsorption process

The quantitative analysis of the protein adsorption kinetics requires that the protein

amount adsorbed is known as a function of time The kinetics tests were carried out in

phosphate buffer (PB) at 37oC and pH 74 a condition similar to physiological condition

The protein concentrations chosen were 07mgml for BSA and 006mgml for β2M

which are the maximal concentrations used in an adsorption isotherm test A time-based

evaluation of the adsorption was made by measuring the concentration of protein in

solution at different incubation times The results are shown below from Figure 4-34 to 4-

37 The adsorption kinetics of proteins onto th cond-e latex particles is expressed as a se

order model [Oumlzacar 2003] shown by the following equation

2)( tet qqk

dtdq

minus= (4-10)

Integrating above equation and applying the boundary conditions yields

ee

t tq

+=

1 (4-11)

where q

qkq

t

2

2

2

the previous adsorption isotherm results representing good reproducibility of adsorption

t and qe stand for the amount (mgm ) of protein adsorbed at time t and at

equilibrium respectively k is the equilibrium rate constant of second-order adsorption

(m mghr) and t is the incubation time (hr)

The fitting parameters calculated by using the equation 4-11 are listed in table 4-16

From this data it is seen that the amount of BSA adsorbed at equilibrium was higher than

that of β2M on PS and PSPMMA100 latex particles but less than that of β2M on

PSPMMA90PAA10 and PSPMMA75PAA25 latex particles These results are similar to

109

00

05

15

20

0 2 4 6 8 10 12 14

Time (hr)

rot

n ad

sorb

d (

10

25

Pei

em

gm

2

BSA2nd order model (BSA))β2M2nd order model (β2M)

Figure 4-34 The kinetics of protein adsorption in PB on PS latex particles at 37oC and

pH 74

00

05

10

15

20

25

30

0 2 4 6 8 10 12 14

Time (hr)

Prte

inds

oo

arb

ed (

mg

m2 )

BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-35 The kinetics of protein adsorption in PB on PSPMMA100 latex partic

37les at

oC and pH 74

110

00

03

06

09

12

15

0 2 4 6 8 10 12 14

Time (hr)

Prot

ein

adso

rbed

(m

gm

2 ) BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-36 The kinetics of protein adsorption in PB on PSPMMA90PAA10 latex

particles o at 37 C and pH 74

00

03

06

09

12

15

0 2 4 6 8 10 12 14

Time (hr)

BS

A ad

sorb

ed (

mg

m2 )

BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-37 The kinetics of protein adsorption in PB on PSPMMA75PAA25 latex

particles at 37oC and pH 74

111

test The variation of β2M equilibrium adsorption amount was nearly negligible for all

latex particles The plateau region is reached earlier for β2M than BSA on all latex

particles and the K values (equilibrium rate constant of second order adsorption) for the

β2M adsorption process onto latex particles are much larger than for BSA adsorption

These values indicate that the β2M molecules have a higher adsorption rate onto the latex

particles than the BSA molecules

Table 4-16 Fitting parameters of second-order kinetic model BSA β2M

Latex particles qe (mgm2) K (hr-1) qe (mgm2) K ( -1) hr

PS 144 67 074 7549

PSPMMA100 175 353 076 4438

PSPMMA90PAA10 046 97 075 5695

PSPMMA75PAA25 060 179 075 5168 K = (kqe) hr-1

There are several explanations for the phenomena observed in these experiments

First the smaller sizes of β2M molecules allow easier diffusion and a close approach to

the latex substrate Second a reduced number of hydrophobic domains on the latex

particle surface replaced by sulfate groups (-OSO3-) and ionized carboxylic groups

(COO-) are not favorable for larger BSA molecules Third the conformational change by

intra- and inter-molecular repulsion would be faster in β2M due to smaller molecular

weight Finally the ionic repulsion between β2M and the latex particles is less than that

condition (pH 74) than IEP (48) of BSA

between BSA and latex particles because IEP (57) of β2M is closer to the tested

112

44 Blood Compatibility

Blood compatibility can be defined as the property of a material or device tha

permits it to function in contact with blood without inducing any adverse reactions

Successful functioning of blood contacting biomedical materials or devices in contact

with blood strongly depends on the extent to which they damage blood during operation

High shear stresses turbulence rela

t

tively long contact times and cellular impact with

artificial surfaces are most common events experienced by blood in contact with artificial

1978 Winslow and et al

1993] Therefore before any materia e placed into the human b

biocompatibility testing must be the first performed in the view of safety The hemolysis

s a method for pr ary screening of biomaterials to determine

h blood a er circulating body fluid The tests estimat

oglobin released from th blood cell d e induced

foreign material

There are several types of blood cells in our body but the ones that make blood red

are erythrocytes or red blood cells (RBCs) Red blood cells (erythrocytes) are the most

numerous type in the blood These cells are average about 48-54 million per cubic

millimeter (mm3 same as a microliter (microl)) of blood but these values can vary over a

larger range depending on such factors as health and altitude RBCs are red because they

contain hemoglobin which is a protein that contains iron When blood circulates through

lungs oxygen binds to the hemoglobin and is then delivered to all the parts of body

RBCs are made in the bone marrow They start out as a nucleus like other cells but

when they fill up with hemoglobin the nucleus becomes smaller until it disappears

Without a nucleus RBCs are fragile and live only about 120 days In a normal healthy

devises and can cause hemolysis or thrombosis [Bernhard et al

ls can b ody

test serves a elimin the

biocompatibility wit nd oth e the

concentration of hem e red amag by a

113

body about 2 million red blood ne marrow produces new

ones j ing

dy

le

cells die per second But bo

ust as fast RBCs are about 75 micrometer across and are responsible for carry

oxygen salts and other organic substances to the cells and tissues throughout our bo

In humans and most mammals the cells are concave (indented in the middle) and flexib

enough to squeeze through even the tiniest of blood vessels the capillaries

Figure 4-38 Image of red blood cells

(Sourcehttpwwwpbrchawaiiedubemfmicroangelarbc1htm)

ch

100 90 10 75 25 f

RBCs are among the first cells that come into contact with a foreign material

injected in the blood Therefore a hemolysis assay gives information about the

biocompatibility in the case of an in vivo application The concentrations of prepared

latex particles used for the tests in this research are 05 25 and 50 (ww) and the

incubation time under moderate shaking was 30 minutes at 37oC Test results are shown

in Figure 4-39 The amount of blood cells that ruptured when in contact with the PS latex

particles was largely increased from about 30 to 91 as the solid content of PS

increased from 05 to 50 However hemolysis by the core shell latex particles su

as PSPMMA PSPMMA PAA and PSPMMA PAA was less than 35 O

114

particular interest was that hemolysis caused by PSPMMA PAA particles at 50

solid concentration was less than 02 hemolysis the lowest value among the latex

particles The low hemolysis value indicates that the PSPMMA PAA core shell latex

particles are the most blood compatible at all solid contents Other core shell particles

such as PSPMMA and PSPMMA PAA also had excellent blood comp

90 10

90 10

100 75 25 atibility with

low RBS breakage

01 2 3

20

40

60

80

100

Hem

olys

is (

)

052550

Figure 4-39 He

PS PSPMMA100 PSPMMA PAA90 10

molysis caused by latex particles n=5

4PSPMMA75PAA25

CHAPTER 5 SUMMARY OF RESULTS CONCLUSIONS AND RECOMMENDATION FOR

FUTURE WORK

51 Summary of Results

The goal of this study was to synthesize monodisperse polymeric latex particles

with tailored properties and to investigate the fundamental interactions between the

synthesized latex particles and target proteins An understanding of the fundamental

mechanism of selective adsorption is strongly required in order to maximize the

separation performance of membranes made of these materials so that it may applicable

to hemodialysis therapy for end stage renal disease (ESRD) patients For the achievement

of above research goal several investigations have been performed Analysis of the

obtained data leads to several conclusions as discussed below

Monodisperse polystyrene seed latex particles were synthesized with the size

crosslinking agent to make the latex particles more hydrodynamically stable The

conversion of monomer to polymer was over 95 and seed latex particles are spherical in

shape and have smooth surface From these small seeds bigger latex particles as large as

800nm were synthesized using a semi-continuous seeded emulsion polymerization

process The particles with a mean diameter less than 500nm are very spherical in shape

and highly uniform in particle size distribution However as the particles grow larger

than 500nm in mean diameter they became non-spherical with an uneven surface This

irregularity of particle surface can be attributed to the non-homogeneous monomer

ranges between 126plusmn75 and 216plusmn53 nm Divinylbenzene (DVB) was used as a

115

116

swelling into the shell of a growing polymer which can be controlled by the factors such

as temperature agitation speed initiator feeding rate and surfactant amount required to

stabilize the colloidal system It was determ rfactant to monomer ratio

dec ic

acid (AA) monomers were intro rticle surfaces more

hydro

to

ting of a

orm

ration is seen between

3002

0 cm-

0cm-1

-1

03

ined that as the su

reased the mean particle diameter increased Methyl methacrylate (MMA) and acryl

duced to make the latex pa

philic than bare polystyrene particles The prepared core shell latex particles were

made of PMMA100 PMMA90PAA10 and PMMA75PAA25 PMMA100 is the PMMA

homopolymer shell on a PS core PMMA90PAA10 shell is a copolymer with a PMMA

PAA ratio of 90 to 10 by weight PMMA75PAA25 shell is a copolymer consis

PMMA to PAA ratio of 75 to 25 by weight The particle size of these core-shell

particles were prepared to be about 370nm in mean diameter

The successful synthesis of latex particles was confirmed using Fourier Transf

Infrared spectroscopy (FTIR) The C-H aromatic stretching vib

cm-1 and 3103 cm-1 for PS latex particles The C-H asymmetrical stretching and

symmetrical stretching vibration peaks of CH2 for all latex particles is seen at 290

1and 2850 cm-1 respectively There is a carbonyl (C=O) characteristic band at 173

wavenumber for PSPMMA core-shell latex particles The broad OH group peak at

3400 cm

100

can be seen in the spectroscopy of the particle with PMMAPAA shell

(PSPMMA PAA and PSPMMA PAA ) The peak intensity of OH group of

PSPMMA PAA was larger than that of PSPMMA PAA relatively due to more AA

monomer content in PSPMMA PAA than in PSPMMA PAA

In the zeta potential measurements polystyrene (PS) latex particles had negative

values between -291 mV and -599 mV in the phosphate buffer (PB) and between -2

90 10 75 25

75 25 90 10

75 25 90 10

117

mV and -278 mV in the phosphate buffered saline (PBS) at 25oC and pH 21-78 These

negative zeta potential values for PS latex particles are due to ionization of the sulfate

groups (-OSO3-) originated from the initiator Zeta potential values of the PSPMMA

core shell latex particles were also negative between -28 mV and -505 mV in PB and

between -143 mV and -186 mV in PBS at pH 21-78 Negative values are also

generated by dissociation of sulfate groups on a particle surface The zeta potential value

of PSPMMA PAA and PSPMMA PAA were of negative values between -36

and -678 mV in PB medium and -147 mV and -193 mV in PBS medium at 25

100

s

7 mV

MMA75PAA25 and

PSPM

otein

g the

l change of BSA at

each w

90 10 75 25

oC For

PSPMMA PAA core shell particles zeta potential values were between -291 mV and

-520 mV in PB media and between -115 mV and -210 mV in PBS media Sulfate

groups contributed to the negative zeta potential values in PSP

75 25

MA90PAA10 core shell particles Carboxylic groups from polyacrylic acid also

contribute to the negative surface charge for the latex particles at greater than pH 70

however the degree of their contribution was not detectable from the zeta potential

measurements The absolute zeta potential values of the synthesized latex particles in PB

were higher than that in PBS This is because of the high concentration of sodium

chloride electrolyte in PBS that compress electrical double layer thickness

The bicinchoninic acid (BCA) assay technique was used to determine the pr

adsorption and all isotherms were fitted to the Langmuir-Freundlich isotherms usin

nonlinear regression method The hydrophobic interaction between PS latex particle and

BSA protein was dominant for the adsorption process Conformationa

pH also affected the adsorption amount which was highest at pH 48 because of lo

ionic repulsion making compact conformation of BSA The next larger amount of BSA

118

adsorbed onto PS was at pH 32 There were two main forces affecting the amount of

adsorption at acidic pH values One is ionic attraction between negative latex particle a

positive protein The other is lateral repulsion between positive proteins The smallest

amount of BSA adsorption was at pH 74 where charge repulsion between negative

particles and proteins is present at this pH Lateral repulsion between proteins wa

dominant for protein adsorption even though there always exists the hydrophobic

interaction between solid latex particle and protein The charge repulsion was reduced by

the high concentration of electrolytes in the PBS medium The BSA adsorption process

on PSPMMA core shell particles was very similar to the case of PS latex particle where

hydrophobic interaction between latex particle and protein was dominant However t

adsorbed BSA amount was less than that of PS in PBS The adsorbed amount of BS

PSPMMA

nd

s

he

A on

an

ed

n The main

adsor

90PAA10 and PSPMMA75PAA25 core shell particles was higher at pH 32 th

pH 48 and 74 The adsorbed BSA amount became higher at pH 32 and pH 48 as the

hydrophilicity was increased by the higher PAA amount On the contrary the adsorb

amount of BSA was dramatically decreased at pH 74 Carboxylic acid groups as well as

the sulfate groups are distributed on the surface of PSPMMA90PAA10 and

PSPMMA75PAA25 core shell particles and largely affect the BSA adsorptio

ption interaction forces at pH 32 was generated by hydrogen bonding as well as

charge attraction between latex particle and protein even when the conformational change

and charge repulsion between proteins reduce the adsorption of protein at this pH The

adsorbed BSA amount was also increased as the amount of PAA increases However the

adsorption of BSA was minimal (less than 07 mgm2) at pH 74 where the carboxylic

groups are ionized leading to more enhancement of charge repulsion by ionization of the

119

carboxylic groups (COO-) Because of this the PSPMMA-PAA latex particles have a

much lower protein adsorption

There were steep initial slopes for adsorption isotherms of β

in

s

there

M

r

e

es

o

2M on latex particles

all isotherms indicating a high affinity type adsorption The complete plateau region

were not seen for all isotherms indicating that β2M proteins were not saturated and

are still available sites for β2M adsorption on latex particles The initial slope of β2

isotherms for PS was steeper than for the other latex particles PSPMMA100

PSPMMA90PAA10 and PSPMMA75PAA25

The layer thickness (δ) of BSA proteins on synthesized latex particles in phosphate

buffer (PB) was in the range of 27 - 81 Aring at 37 C and pH 48 Only the δ values at pH

48 the isoelectric point (IEP) of BSA are important because intra- and inter-molecular

electrostatic repulsion of the protein molecules is minimized at the IEP Compared to the

hydrodynamic dimensions of BSA 140 times 40 times 40 Aring these values of the adsorbed laye

thickness indicates that the BSA molecules adsorb by the side-on (40Aring) and end-on mod

(140Aring) Consideration of the multi-layering is unnecessary because the adsorbed

thickness of BSA do not exceed 140 Aring

The Gibbs free energy of β

o

3

2M was more negative in phosphate buffer (PB) than

that of BSA in physiological condition (37 C and pH 74) indicating that βo2M molecul

are more favorably adsorbed on the latex particles than BSA and that the affinity of β2M

was much larger than BSA This may be explained by the rate of diffusion and

accessibility of β2M molecules being easier and faster than that for BSA because of

smaller size (or weight) and spherical shape of β2M molecules It may also be possible t

explain that more hydrophobic forces (less electrostatic repulsion) contributes to

120

adsorption of β2M than to that of BSA because the IEP of β2M (57) is closer to

physiological condition (pH 74) than that of BSA (pH 48) The selective adsorption of

β2M m

in β2M

larger than in BSA adsorption indicating

that fa

an that between BSA and latex particles because

IEP (

ch as

Other

olecules over BSA molecules can be enhanced by developing more hydrophilic

PSPMMA90PAA25 and PSPMMA75PAA25 core shell latex particles

The adsorption kinetics of proteins on latex particles was expressed as a second-

order model The plateau region for β2M was reached earlier than BSA on all latex

particles and the K values (equilibrium rate constant of 2nd order adsorption)

adsorption process on latex particles were much

ster adsorption rate of β2M molecules than that of the BSA molecules There are

several explanations for these phenomena First the smaller size of β2M molecules can

more easily diffuse and approach to the latex substrate Second enhanced hydrophilic

domain on the latex particle surface by sulfate groups (SO4-) and ionized carboxylic

groups (COO-) is not favorable for larger BSA molecules but is for smaller β2M to be

anchored Third the conformational change by intra- and inter-molecular repulsion

would be faster in β2M due to lower molecular weight Finally the ionic repulsion

between β2M and latex particles is less th

57) of β2M is closer to the tested condition (pH 74) than IEP (48) of BSA

In the biocompatibility test the number of red blood cells (RBCs) ruptured by PS

latex particles ranged from 30 to 91 as the solid content of PS increased from 05 to

50 respectively However hemolysis of the other core shell latex particles su

PSPMMA100 PSPMMA90PAA10 and PSPMMA75PAA25 were less than 35 The

PSPMMA90PAA10 particles at all applied solid concentrations had less than 02

hemolysis and was the most blood compatible among the prepared latex particles

121

core shell particles such as PSPMMA100 and PSPMMA75PAA25 also had excelle

compatibility with low RBC breakage

52 Conclusions

The major conclusions from this study include

nt blood

bull microglobulin (β2M) over that of the bovine serum albumin (BSA) at pH 74

was achieved by controlling the PMMA and PAA ratio to optimize

e particles posses the most desirable requirements for use in a human dialysis

ns

icles

olecule

bull

Using engineered core shell latex particles selective adsorption of β2

physiological condition was demonstrated

bull The rate of adsorption of the β2M was also much higher than that of BSA This

hydrophobichydrophilic microdomain size

bull The core shell particles also exhibited excellent biocompatibility with blood Thes

membrane

53 Recommendation for Future Work

With the results and conclusions obtained through this study several suggestio

may be given for future work as follows

bull Competitive adsorption tests of whole blood proteins on synthesized latex partunder physiological condition

bull Preparation of latex particles with optimum hydrophobic hydrophilic microdomains to maximize selective adsorption and removal of toxin molecules

bull Membrane fabrication by suitable pore size design to discriminate a toxin mby size sieving as a function of particle layers

Evaluation of membrane performance such as flow rate in water and in simulated blood fluid (SBF)

bull Application for hemodialysis as well as other technologies such as a water remediation

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Korea on August 7 1968 He obtained a Bachelor of Science degree in Chemistry from

Keim d graduate school and earned a

resear n Korea Institute of Science and

Coati e

ia

Busin

BIOGRAPHICAL SKETCH

Sangyup Kim was born and raised in the small town of Sung-Joo Kyungbook

yung University (Daegu Korea) in 1993 He entere

Master of Science degree in Chemical engineering in 1995 He worked as a student

cher for 2 years at Textile Polymer Division i

Technology (KIST) in Seoul Korea Next he worked over a year at the Automobile

ng Division in DongJoo-PPG in Korea In August 2001 he began studying for th

degree of Doctor of Philosophy in materials science and engineering at the University of

Florida in Gainesville After getting his PhD he plans to work at the Digital Med

ess Division of Samsung Electronics in Korea starting in January 2006

141

To all who made this work possible

ACKNOWLEDGMENTS

I owe what I am today and this moment to many people without whom I could not

have been here First of all I would like to express my sincere and deepest appreciation

to my parents wife Mikyung daughters Heehyung and Erin whose love patience and

support helped me pursue my dream I gratefully acknowledge my advisor Dr H El-

Shall who gave me an opportunity to become part of an exciting and beneficial research

project and exert myself in the research work His guidance knowledge and stimulating

scientific and critical thinking have contributed to my success to a great extent and

inspired me to dedicate myself to research work I especially appreciate Dr R Partch as a

co-advisor who has helped me conduct experiments with valuable discussion for my

research from my first year I am greatly thankful to Dr C McDonald whose idea and

valuable experimental advice and discussion in the area of polymer chemistry and

membrane technology helped cultivate my interest in this area and were vital to

performing this research Without his contribution this work could not have been

achieved I also thank my doctoral committee members Dr R Singh Dr A Zaman and

Dr B Koopman for appreciated comments

Financial support and equipment use for research from the Particle Engineering

Research Center at the University of Florida were vital in continuing my study for more

than 4 years My sincere thanks are due to my former advisor Dr Kiryoung Ha in

Keimyung University who helped me open my eyes and broaden my thought and

introduced me into a limitless polymer field I would like to also thank to past and current

iv

members in Dr El-Shallrsquos research group for their assistance advice and support I

sincerely appreciate Stephen for the assistance of thesis writing

v

TABLE OF CONTENTS page

ACKNOWLEDGMENTS iv

LIST OF TABLES viii

LIST OF FIGURES x

1 INTRODUCTION 1

2 BACKGROUND AND LITERATURE SURVEY5

21 The Significance of End Stage Renal Disease (ESRD)5 22 Hemodialysis (HD) Treatment 6 23 Advances in Membrane Technology 8 24 Sorbent Technology16 25 Limitation of Current Hemodialysis Treatment 19 26 Latex Particles 20

261 The Components for Emulsion Polymerization 21 262 Particle Nucleation 21 263 Types of Processes for Emulsion Polymerization 23 264 Chemistry of Emulsion Polymerization 24 265 Seed Emulsion Polymerization25 266 Polystyrene Latex Particles 26 267 Various Applications of Latex Particles28

27 Proteins 30 271 Interaction Forces between Proteins33 272 β -Microglobulin (β M)2 2 35 273 Serum Albumin 36

28 Protein Adsorption38 281 Interaction between Protein Molecule and Latex Particle38

29 Hypothesis for Toxin Removal39 291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size 40 292 Toxin Removal by Selective Adsorption on Engineered Latex Particles 41

3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY 44

31 Materials 44 32 Latex Particle Preparation46

321 Preparation of Seed Latex Particles47

vi

322 Preparation of Seeded Latex Particles 48 323 Preparation of Core Shell Latex Particles 48 324 Purification of Synthesized Latex Particles48

33 Characterization49 331 Degree of Conversion49 332 Fourier Transform Infrared (FTIR) Spectroscopy49 333 Quasielastic Light Scattering (QELS) 50 334 Field Emission-Scanning Electron Microscopy (FE-SEM) 50 335 Zeta Potential Measurement 50 336 Protein Adsorption51 337 Blood Biocompatibility by Hemolysis Test 53

4 RESULTS AND DISCUSSION55

41 Polymerization of Latex Particles55 411 Polystyrene Seed Latex Particles55 412 The Growth of Polystyrene (PS) Seed Latex Particles61 413 Core Shell Latex Particles 71

42 Characterization of Latex Particles75 421 Fourier Transform Infrared Spectroscopy (FTIR)75 422 Zeta Potential Measurements 75

43 Protein Adsorption Study 85 431 Adsorption Isotherm87 432 Adsorbed Layer Thickness 103 433 Gibbs Free Energy of Protein Adsorption 104 434 Kinetics of Adsorption 107

44 Blood Compatibility 112

5 SUMMARY OF RESULTS CONCLUSION AND RECOMMENDATION FOR FUTURE WORK115

51 Summary of Results115 52 Conclusions121 53 Recommendation for Future Work121

LIST OF REFERENCES122

BIOGRAPHICAL SKETCH 141

vii

LIST OF TABLES

Table page 4-1 The polymerization recipe of polystyrene (PS) seed particles 57

4-2 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles less than 500nm in size 63

4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size64

4-4 The preparation recipe of PS core with various shell latex particles72

4-5 The isoelectric point (IEP) of common proteins 83

4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model 93

4-7 The equilibrium concentration values (q ) of BSA adsorption on PSPMMA particles calculated the Langmuir-Freundlich isotherm model

m 10096

4-8 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model

90 1098

4-9 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model

75 25100

4-10 The equilibrium concentration values of β M adsorption calculated the Langmuir-Freundlich isotherm model

2102

4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37 Co 104

4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37 Co 104

4-13 The values of Gibbs free energy change of BSA adsorption in phosphate buffer (PB) at 37 Co 105

4-14 The values of Gibbs free energy change of BSA adsorption in phosphate buffered saline (PBS) at 37 Co 105

viii

4-15 The values of Gibbs free energy change of β M adsorption in phosphate buffere (PB) at 37 C and pH 74

2o 106

4-16 Fitting parameters of second-order kinetic model 111

ix

LIST OF FIGURES

Figure page 1-2 Stereo drawing of the αndashcarbon backbone of β M2 2

2-1 An image of the location and cross section of a human kidney 5

2-2 A schematic draw of the hemodialysis route7

2-3 Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes 11

2-4 The chemical structure of polysulfone (PSf) 12

2-5 The chemical structure of polyamide (PA)13

2-6 Chemical structure of polyacrylonitrile (PAN) 14

2-7 Emulsion polymerization system24

2-8 L-α-amino acid 31

2-9 Ribbon diagram of human β M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ)

236

2-10 Secondary structure of human serum albumin (HSA) with sub-domains 37

2-11 The relationship between pore size and particle size in body-centered cubic and closed packed arrays41

2-12 Schematic representation of the protein adsorption on the core shell latex particles at pH 7442

3-1 Chemical structure of main chemicals45

3-2 Experimental setup for semi-continuous emulsion polymerization 46

3-3 The particle preparation scheme in various types and size ranges of latex particles 47

3-4 Schematic of the procedure for a protein adsorption test 52

3-5 Separation of RBC from whole blood by centrifuge process53

x

3-6 Schematic of the procedure for hemolysis test 54

4-1 Schematic representation of semi-continuous seed latex particles preparation and growth56

4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS (B) PS (C) PS (D) PSS259 S233 S207 S181 59

4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm 65

4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm 68

4-5 Dependence of the particle size on the surfactant to monomer ratio70

4-6 Schematic of core shell latex particle structures 71

4-7 Scanning Electron Micrograph of latex particles (A) PS (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA

100

90 10 75 25 73

4-8 FTIR spectra of polymerized latex particles (A) bare polystyrene (PS) (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA100 90 10 75 25 76

4-9 Schematic representation of ion distribution near a positively charged surface 78

4-10 Schematic representation of zeta potential measurement79

4-11 Zeta potential of PS latex particles at 25 Co 80

4-12 Zeta potential of PSPMMA latex particles at 25 C100o 81

4-13 Zeta potential of PSPMMA PAA latex particles at 25 C90 10o 82

4-14 Zeta potential of PSPMMA PAA latex particles at 25 C75 25o 82

4-15 The decay of surface potential with distance from surface in various electrolyte concentrations (1) low (2) intermediate (3) high 84

4-16 High-affinity adsorption isotherm of typical flexible polymer on solid surface 86

4-17 Overall schematic representation of the protein adsorption on the synthesized latex particles86

4-18 The dependence of UV absorbance on BSA concentration (A) 10mgml (B) 06mgml (C) 02mgml87

4-19 Standard curve of net absorbance vs BSA sample concentration 88

xi

4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37 C in phosphate buffer mediao 89

4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37 Co 90

4-22 Adsorption isotherm of BSA adsorption 37 C in phosphate buffered saline (PBS) on PS latex particles

o

91

4-23 Conformation of bovine serum albumin (BSA) on latex particles92

4-24 Adsorption isotherm of BSA 37 C in PB on PSPMMA core shell latex particles

o100

94

4-25 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA core shell latex particles

o100

95

4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA PAA core shell latex particles

o90 10

97

4-27 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA PAA core shell latex particles

o90 10

98

4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA PAA latex particleso75 25 99

4-29 Adsorption isotherm of BSA 37 C in PBS on PSPMMA PAA core shell latex particles

o75 25

99

4-30 Adsorption isotherm of β M onto PS and PSPMMA latex particles in PB at 37 C and pH 74

2 100o 101

4-31 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74

2 90 10o 101

4-32 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74

2 75 25o 102

4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37 C and pH 74

o

107

4-34 The kinetics of protein adsorption in PB on PS latex particles at 37 C and pH 74

o

109

4-35 The kinetics of protein adsorption in PB on PSPMMA latex particles at 37 C and pH 74

100o

109

4-36 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74

90 10o 110

xii

4-37 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74

75 25o 110

4-38 Image of red blood cells 113

4-39 Hemolysis caused by latex particles n=5114

xiii

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS

By

Sangyup Kim

December 2005

Chair Hassan El-Shall Major Department Materials Science and Engineering

Latex particles with well-defined colloidal and surface characteristics have received

increasing attention due to their useful applications in many areas especially as solid

phase supports in numerous biological applications such as immunoassay DNA

diagnostic cell separation and drug delivery carrier Hemodialysis membrane using

these particles would be another potential application for the advanced separation

treatment for patients with end stage renal disease (ESRD) It is desirable to remove

middle molecular weight proteins with minimal removal of other proteins such as

albumin Thus it is necessary to understand the fundamental interactions between the

particles and blood proteins to maximize the performance of these membranes This

improvement will have significant economic and health impact

The objective of this study is to synthesize polymeric latex particles of specific

functionality to achieve the desired selective separation of target proteins from the human

blood Semi-continuous seed emulsion polymerization was used to prepare monodisperse

xiv

polystyrene seed particles ranging from 126plusmn75 to 216plusmn53 nm in size which are then

enlarged by about 800nm Surfactant amount played a key role in controlling the latex

particle size Negatively charged latex particles with a different hydrophobicity were

prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid

monomer The prepared polymeric particles include bare polystyrene (PS) particles less

hydrophobic PS core and PMMA shell particles and more hydrophilic PS core and

PMMA-co-PAA shell latex particles with a 370nm mean diameter SEM light scattering

and zeta potential measurements were used to characterize particle size and surface

properties Adsorption isotherms of two proteins bovine serum albumin (BSA) and β2-

microglobulin (β2M) on latex particles were obtained as a function of pH and ionic

strength using the bicinchoninic acid (BCA) assay method The Langmuir-Freundlich

adsorption model was used to determine the adsorption amount of protein at equilibrium

The thickness of adsorbed BSA layer on latex particles was obtained in order to

investigate the adsorption orientation such as end-on or side-on mode Adsorption

kinetics experiments for both proteins and all latex particles were also performed The

adsorption kinetic constant determined from the Langmuir-Freundlich adsorption

isotherm model was used to calculate Gibbs free energy of adsorption to compare the

competitive adsorption of BSA and β2M Hemolysis tests were performed to investigate

the blood compatibility of synthesized latex particles PSPMMA90PAA10 and

PSPMMA75PAA25 core shell latex particles had desirable material properties with not

only a large amount and high rate of selective β2M adsorption over BSA but also high

blood compatibility showing less than 3 hemolysis

xv

CHAPTER 1 INTRODUCTION

End stage renal disease (ESRD) is a chronic condition in which kidney function is

impaired to the extent that the patientrsquos survival requires removal of toxins from the

blood by dialysis therapy or kidney transplantation The National Kidney Foundation

estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF] The

number of people with ESRD is rapidly increasing in the United States with

approximately 96 295 incidents and 406 081 prevalent patients including 292 215 on

dialysis and 113 866 with a functioning graft in 2001 It is projected that there will be

more than 22 million ESRD patients by 2030 [USRDS 2003] The expenditure for the

ESRD treatment program had reached $228 billion 64 of the Medicare budget in

2001 Due in part to the limited availability of kidneys for transplantation hemodialysis

is the primary clinical treatment for the patients with ESRD

The central element of a hemodialysis instrument is the semipermeable membrane

that allows for selective transport of low molecular weight biological metabolites less

than 5000 Da such as urea and creatinine as well as excess water and electrolytes [Baker

2004] from the blood One limitation of current dialysis technologies is the inability to

efficiently remove middle molecular weight toxins such as β2-Microglobulin (β2M) and

interleukin 6 (IL-6)

β2M is a causative protein of dialysis-related amyloidosis (DRA) a disease arising

in patients with chronic kidney failure as a serious complication of long-term

hemodialysis treatment [Gejyo et al 1985] β2M deposition in tissue is the primary cause

1

2

of destructive arthritis and carpal tunnel syndrome [Vincent et al 1992 Drueke 2000]

The β2M structure is shown in Figure 1-2

Figure 1-2 Stereo drawing of the αndashcarbon backbone of β2M [Becker et al 1985]

Although attempts have been made to increase the efficiency of middle molecular

weight toxin removal by changes in the membrane pore size and the use of innovative

materials to adsorb these toxins [Samtleben et al 1996 Ronco et al 2001] removal

efficiency is not as high as those achieved by a normal healthy kidney Traditional

membranes have a number of processing and performance limitations [Westhuyzen et al

1992 Leypoldt et al 1998] such as a restricted choice of surface chemistries and limited

control of porosity The development of novel engineering membrane technology is

needed to remove middle molecule toxins

Polymeric latex particles have received increasing attention in medical application

areas especially as solid phase supports in biological applications [Piskin et al 1994]

Examples of these applications include immunoassay [Chen et al 2003 Radomske-

Galant et al 2003] DNA diagnostic [Elaїssari et al 1998] cell separation drug delivery

carrier [Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] etc This is because of

3

the well-defined colloidal and surface characteristics of the particles By using a seed

emulsion polymerization method it is possible to synthesize monodisperse latex particles

with various particle size ranges and surface chemistry Functionalized core-shell latex

particles can be introduced by multi-step emulsion polymerization Core particles are

synthesized in the first stage of the polymerization and the functional monomer is added

in the second stage This is done without any emulsifier addition to prevent the

production of new homopolymer particles [Keusch et al 1973] The core-shell particles

are useful in a broad range of applications because of their improved physical and

chemical properties over their single-component counterparts [Lu et al 1996 Nelliappan

et al 1997] Through the development of a hemodialysis membrane using monodisperse

latex particles improvements in advanced separation treatment for patients with end

stage renal disease (ESRD) can be realized This requires the maximum removal of

middle molecular weight proteins with minimal removal of other beneficial proteins such

as albumin Thus an understanding of the fundamental interactions between the particles

and biopolymers is vital to maximize the performance of this membrane technology

The field of material science and biotechnology is based on fundamental chemistry

has developed over the past three decades into todayrsquos powerful discipline that enables

the development of advanced technical devices for pharmaceutical and biomedical

applications This novel and highly interdisciplinary field is closely associated with both

the physical and chemical properties of organic and inorganic particles [Niemeyer 2001]

Hemodialysis membrane using these particles would lead to improvements in the

advanced separation treatment for patients with end stage renal disease (ESRD) The

interdisciplinary nature of this approach enables a more complete understanding of the

4

phenomena of protein adsorption and the material properties necessary for selective

separation This innovative approach to membrane fabrication has the potential of making

inexpensive highly efficient membranes for both industrial and specialized separation

processes

The goal of this study is to prepare polymeric latex particles with tailored

properties to maximize separation of toxin molecules and to investigate the fundamental

interactions between the particles and molecules in the biological system in order to

optimize the performance of a membrane material for these applications

Polymeric latex particles were synthesized with specific functionality in an attempt

to achieve selective separation Seeded emulsion polymerization was used to synthesize

functionalized monodisperse latex particles in various sizes Negatively charged

hydrophobic polystyrene latex particles were synthesized by the same method Core shell

latex particles PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25 were also

synthesized to differentiate the degree of hydrophobicity of particles Scanning Electron

Micrograph (SEM) and light scattering measurements were used to characterize particle

size and shape and zeta potential measurements were conducted to measure the electrical

surface property of synthesized particles Adsorption isotherms of target proteins bovine

serum albumin (BSA) and β2M on latex particles were obtained as a function of pH

ionic strength and protein concentrations using the BCA assay method Adsorption

kinetics for both proteins on the latex particles were also measured Finally hemolysis

tests were run to determine the biocompatibility of polymer latex particles with human

blood This research will be described in more detail in chapters 3 and 4

CHAPTER 2 BACKGROUND AND LITERATURE SURVEY

21 The Significance of End Stage Renal Disease (ESRD)

The kidneys are responsible for removing excess fluid minerals and wastes from

the blood regulating electrolyte balance and blood pressure and the stimulation of red

blood cell production They also produce hormones such as erythropoietin (EPO) and

calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005 Kurbel

et al 2003] EPO acts on the bone marrow to increase the production of red blood cell in

case of bleeding or moving to high altitudes Calcitriol mainly acts on both the cells of

the intestine to promote the absorption of calcium from food and also bone to mobilize

calcium from the bone to the blood When kidneys fail harmful waste builds up blood

pressure rises and body retains excess fluid The body also does not make enough red

blood cells When this happens treatment is needed to replace the function of failed

kidneys

Figure 2-1 An image of the location and cross section of a human kidney

5

6

The National Kidney Foundation estimates that over 20 million Americans had

chronic kidney disease in 2002 [NKF] Chronic renal disease is a gradual and progressive

loss of the function unlike acute renal disease where sudden reversible failure of kidney

function occurs Chronic renal failure usually takes place over a number of years as the

internal structures of the kidney are slowly damaged In the early stages there can be no

symptoms In fact progression may be so gradual that symptoms do not occur until

kidney function is less than one-tenth that of a normal health kidney If left untreated

chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal

Disease (ESRD) ESRD is a rapidly growing heath-care problem in the United States In

2001 approximately 96295 incidents and 406081 prevalent patients were diagnosed

with ESRD including 292215 patients on dialysis and 113866 patients with a

functioning graft [USRDS 2003] The projected number of patients with ESRD is

expected to exceed 22 million patients by 2030 with much of this growth being driven by

the increasing prevalence of major contributing factors such as diabetes and high blood

pressure [USRDS 2003] A great extent of ESRD program cost and Medicare budget

have been spent in 2001 Due in part to a limited availability of kidneys for

transplantation hemodialysis (HD) is the primary method of treatment for ESRD and is

currently used for approximately 61 of US ESRD patients

22 Hemodialysis (HD) Treatment

HD removes toxins from the body by extracorporeal circulation of the blood

through a semipermeable membrane referred to as a dialyzer The toxins are removed

primarily by diffusion across the membrane to a dialysate solution which is circulated on

the opposite side of the membrane The cleaned blood is then returned to the blood

stream A surgically constructed vascular access connects the extracorporeal circuit to the

7

patientrsquos system Treatments are almost always performed three times per week in

specially equipped dialysis facilities for 3-4 hours per each treatment Figure 2-2 shows

the scheme of the hemodialysis route The critical element of a HD instrument is the

semipermeable membrane which allows for selective transport of low molecular weight

biological metabolites from blood

igure 2-2 A schematic draw of the hemodialysis route

ESRD patients who undergo dialysis therapy often experience several other

problems associated with the treatment such as anemia fatigue bone problems joint

problems itching sleep disorders and restless legs Anemia is common in patient with

kidney disease because the kidneys produce the hormone erythropoietin (EPO) which

stimulates the bone marrow to produce red blood cells Diseased kidneys often do not

produce enough EPO to stimulate the bone marrow to make a sufficient amount of red

blood cells This leads to bone disease referred to as renal osteodystrophy and causes

bones to become thin and weak or malformed and can affect both children and adults

Older patients and women who have gone through menopause are at greater risk for this

disease Uremic toxins which cannot be remove from the blood by the current dialyzer

membranes can lead to itching This problem can also be related to high levels of

F

8

parathyroid hormone (PTH) Dialysis patients can also experience day-night revers

is they have insomnia at night and sleep during the day This can be related to possible

nerve damage in the body and a chemical imbalance in the blood due to the excess toxin

Oxidative stress is a problem for patients on maintenance dialysis This problem is due to

an imbalance between pro- and antioxidant factors [Roselaar et al 1995 Cristol et al

1994] Oxidative stress affects oxidation of low-density lipoproteins which are the main

factor for atherogenesis [Huysmans et al 1998] and is also involved in the development

of malignancies and diabetes mellitus [Rice-Evans et al 1993] In order to reduce

antioxidant defense dialysis is needed to contribute to help stimulate free radical

production or eliminate antioxidants Dialysis-related amyloidosis (DRA) is also a

common and serious problem for people who have been on dialysis for more than 5

years DRA develops when proteins in the blood deposit on joints and tendons causi

pain stiffness and fluid in the joint as is the case with arthritis Normally the healthy

kidneys can filter out these proteins but dialysis filters are not as effective

23 Advances in Membrane Technology

al that

s

ng

Dialysis for blood of ESRD

Hemo filtration

s

ysis using a cellulose based membrane occurred

1913 John Abel [1990] from the Johns Hopkins Medical School described a method

purification is widely used in the treatment

dialysis (HD) techniques use a semi-permeable membrane to replace the

role of the kidney The membranes used in HD can be broadly classified into those based

on cellulose and those manufactured from synthetic copolymers These membranes come

in various shapes such as sheets tubular structures or hollow fiber arrangements The

hollow fiber type is the most popular and is incorporated into over 800 different device

in world wide [Ronco et al 2001]

The first attempt at blood dial

9

where sing

e

om

ysis was performed until the mid 1960 The basic

molec

rial in

een

benzyl)

was used for the membrane with

hepar

by the blood of a living animal may be submitted to dialysis outside the body u

a membrane based on cellulose and returned to the natural circulation without exposur

to air infection by microorganisms or any alteration that would necessarily be prejudicial

to life This same technique is still used to today however the device used has been

modified over the years as better membranes were developed and the anti-coagulant

heparin has become available

Cellulose membranes have been widely used for the treatment of renal failure fr

1928 when the first human dial

ular structure of cellulose is made of a long chain containing hydroxyl (OH)

groups The realization that such groups imparted undesirable qualities on the mate

respect to blood contact behavior was discovered in the early 1970s and since has b

the focus of development These modified cellulose membranes used the partial

substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt

to reduce their negative effect The result is a molecular mosaic of hydrophobic (

and hydrophilic (hydroxyl and cellulose) regions

Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients

with acute renal failure in 1943 Cellophane tubing

in as the anticoagulant For the next 17 years hemodialysis therapy was performed

by this method but only for the patients with acute reversible renal failure Vascular

access required repeated surgical insertions of cannulas (slender tubes) into an artery and

vein and limited the number of treatments for a patient could receive in order to

minimize the amount of vascular damage

10

Initially the need for dialysis in patients with acute renal failure was determ

mainly by the development of signs and sym

ined

ptoms of uremia After dialysis some time

might

et al

ent improvement In 1960 the development of a shunt [Quinton et al

1960]

fone (PSf) polyamide (PA) and polyacrylonitrile (PAN) by phase inversion or

precip

lidone

e

elapse before uremic manifestations returned to warrant a sequential treatment of

dialysis Many patients with acute renal failure secondary to accidental or surgical

trauma were hypercatabolic but the interdialytic interval might be prolonged because of

anorexia or use of a low-protein diet However Teschan and his coworkers [Obrien

1959] showed that patient well-being and survival were improved by what they termed

prophylactic daily hemodiaysis or administration of the treatment before the patient

again became sick with uremia Their report in 1959 was the first description of daily

hemodialysis

Development of membrane accessories such as a shunt has also been an area of

focus for treatm

a flexible polytetrafluoroethylene (PTFE or Teflonreg) tubing made many more

hemodialysis treatments possible for chronic kidney failure patients PTFE has a non-

stick surface and is relative biocompatibility leading to minimized blood clotting in the

shunt

Synthetic membranes are prepared from engineered thermoplastics such as

polysul

itation of a blended mixture resulting in the formation of asymmetric and

anisotropic structures Figure 2-3 shows a fiber type of the Polyflux S membrane

consisting of poluamide (PA) polyacrylethersulfone (PAES) and polyvinylpyrro

(PVP) with the integral three-layer structure The skin layer on the inside fiber typ

membrane contacts blood and has a very high surface porosity and a narrow pore size

11

distribution This layer constitutes the discriminating barrier deciding the permeabili

and solute retention properties of the membrane The skin layer is supported by thick

sponge-type structure larger pores providing mechanical strength and very low

hydrodynamic resistance

ty

Figure 2-3 Cross-sectional SEM image view of the Polyflux S (polyamide +

polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch et al 1998]

n

[Malchesky 2004] because of its thermal stability mechanical strength and chemical

inertn ciated

ls

m

PSf is a widely used membrane material for the hemodialysis applicatio

ess According to a report from the National Surveillance of Dialysis-Asso

Disease (NSDAD) in the US over 70 of hemodialysis membranes were PSf based

[Bowry 2002] This is most likely because PSf has many advantages over other materia

This synthetic polymer is one of few materials that can withstand sterilization by stea

ethylene oxide and γ-radiation PSf membrane can be prepared by conventional

immersion precipitation methods into many different shapes including porous hollow

12

fiber or flat sheet hemodialysis membranes The material also has a high permeab

low molecular weight proteins and solute and high endotoxin retention The chemical

structure of PSf is shown in Figure 2-4

ility to

Figure 2-4 The chemical structure of polysulfone (PSf)

There is one major disadvantage to PSf The hydrophobic nature of the PSf causes

ment alternative pathway

leadin

s not

3

et al

in

O acrylate

serious complications through the activation of the comple

g to the adsorption of serum proteins onto the membranes [Singh et al 2003]

Anticoagulants are added during dialysis therapy to avoid blood clotting but this doe

completely eliminate the problem In order to overcome this disadvantage of the PSf

membrane various studies have been performed to change the materialrsquos surface

properties These investigations include hydrophilic polymer coating [Brink et al 199

Kim et al 1988 Higuchi et al 2003] layer grafting onto PSf membrane [Wavhal

2002 Song et al 2000 Pieracci et al 2002 Mok et al1994] and chemical reaction of

hydrophilic components onto the membrane surface [Higuchi et al 1990 1991 1993

Blanco et al 2001 Nabe et al 1997 Guiver et al 1993] Hydrophilic monomers 2-

hydroxy-ethylmethacrylate (HEMA) acrylic acid (AA) and methacrylic acid (MMA)

have also been grafted onto PSf membrane to increase flux and Bovine Serum Album

(BSA) retention [Ulbricht et al 1996] Hancock et al [2000] synthesized

polysulfonepoly(ethylene oxide) (PEO) block copolymers to improve the resistance to

platelet adhesion Kim et al [2003] also studied blending a sulfonated PE

diblock copolymer into PSf in order to reduce platelet adhesion and enhance

13

biocompatibility PEO is a commonly used biomaterial due to its excellent resistance

protein adsorption and inherent biocompatibility [Harris 1992] Kim et al [20

a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf

membrane with an ultra-thin skin layer The polymer had an entrapped diblock

copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-SO

to

05] studied

ate

lic graft

l)

e of thier

aromatic

orand

3 acryl

and a hydrophobic block of octadecylacrylate (OA) Molecular dynamic (MD)

simulations were performed as a function of copolymer density to optimize interfacial

structure information McMurry [2004] developed a strategy using an amphiphi

copolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glyco

When compared to unmodified PSf these graft copolymer and resulting blend

membranes are found to hold promise for biomedical device applications

Polyamide (PA) membranes have also been used for hemodialysis becaus

mechanical strength in both wet and dry conditions Polyamide consists of

aliphatic monomers with amide bonding (-CONH-) also known as a peptide bond

The basic amide bond in polyamide is shown in Figure 2-5 R1 and R2 can be either

aromatic or aliphatic linkage group

C N

H

O

~ R R1 2~C N

H

O

~ R R1 2~

Figure 2-5 The chemical structure of polyamide (PA)

Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane

ompatible technique due to the

use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of

and concluded that PA hemofiltration was a highly bioc

14

the ac

e

e

is

n

is a semi-crystalline polymer and the mechanical properties

strong

2

CN

tivated anaphylatoxins and β2-Microglobulin (β2M) The PA based membrane

Polyfluxreg (manufactured by Gambro GmbH Germany) blended with polyamide

polyarylethersulfone and polyvinylpyrrolidone (PVP) was able to clean small molecules

such as urea dreatinine and phosphate as well as decrease β2M amount by 502

[Hoenich et al 2002] Due to the non-selectivity of the membrane removal of thes

unwanted materials also led to the undesirable loss of beneficial proteins during therapy

Meier et al [2000] evaluated different immune parameters using a modified cellulos

low-flux hemophan and synthetic high-flux PA membrane during a 1 year period in

chronic hemodialysis patients They found that the 1-year immunological evaluation of

hemodiaysis membrane biocompatibility was associated with changes in the pattern of

chronic T-cell actiovation

Polyacrylonitrile (PAN) is another commonly used membrane material because it

inherently hydrophilic and has been commercialized for ultrafiltration and microfiltratio

[Scharnagl et al 2001] PAN

ly depend on the crystalline structures The chemical structure of PAN is shown in

figure 2-6

CH CH

n2

CN

CH CH n

Chemical structure of polyacrylonitrile (PAN)

The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming

agent gives PAN membranes more flexible processing parameters and increased

e has been optimized through

copolymerization with many other vinyl monomers including glycidyl methacrylate

Figure 2-6

performance [Jung et al 2005] PAN membrane performanc

15

[Godj

acid

for

for

a non-

solub this

in

oid underneath the inner

surface of membrane decreased by increasing the amount of solvent DMSA in the

evargova et al 1999 Hicke et al 2002] N-vinylimidazole [Godjevargova et al

2000] hydroxyl ethyl methacrylate [Ray et al 1999 Bhat et al 2000] methacrylic

[Ray et al 1999] vinyl pyrrolidone [Ray et al 1999] acrylic acid [Trotta et al 2002]

acrylamide [Musale et al 1997] and vinylchloride [Broadhead et al 1998] These

monomers provide a reactive group for enzyme immobilization improved mechanical

strength solvent-resistance pervaporation permeation flux anti-fouling and bio-

compatibility Because of this PAN-based copolymer membranes have great potential

the treatment of hemodialysis in an artificial kidney This material can also be used

other applications like the treatment of wastewater the production of ultra-pure water

biocatalysis together with separation and methanol separation by pervaporation

Lin and his coworker [2004] studied the modification of PAN based dialyzer

membranes to increase the hemocompatibility by the immobilization of chitosan and

heparin conjugates on the surface of the PAN membrane When a foreign material is

exposed to blood plasma proteins are adsorbed clotting factors are activated and

le fibrin network or thrombus is formatted [Goosen et al 1980] The result of

research was that the biocompatible chitosan polymer and a blood anticoagulant hepar

prevented blood clotting They showed prolonged coagulation time reduced platelet

adsorption thrombus formation and protein adsorption

Nie et al [2004] studied PAN-based ultrafiltration hollow-fiber membranes

(UHFMs) In order to improve the membrane performance acrylonitrile (AN) was

copolymerized with other functional monomers such as maleic anhydride and α-allyl

glucoside They found that the number and size of macrov

16

intern erum

and

d a

rface The second step is the

transf after

yjak et

embrane

1978 Malchesky et al 1978] has been further developed to increase the efficiency of

al coagulant The water flux of the UHFMs also decreased while the bovine s

albumin rejection increased minutely Godjevargova et al [1992] modified the PAN

based membrane with hydroxylamine and diethylaminoethylmethacrylate to improve

membrane dialysis properties Formed functional groups like primary amine oxime

tertiary amine groups provided the membrane with more hydrophilic properties an

substantial increase in the permeability of the membranes

The wide use of filtration in practice is limited by membrane fouling Solute

molecules deposit on and in the membrane in the process of filtration causing dramatic

reduction in flux through the membrane Fouling occurs mostly in the filtration of

proteins Three kinetic steps are involved in the fouling of UF membranes according to

Nisson [1990] The first step is the transfer of solute to the su

er of solute into the membrane until it either finally adsorbs or passes through

a set of adsorption-desorption events The third step includes surface binding

accompanied by structural rearrangement in the adsorbed state [Ko et al 1993] Br

al [1998] studied the surface modification of a commercially available PAN membrane

to develop superior filtration properties with less fouling by proteins The PAN

membrane was immersed in excess NaOH solution to convert some of the surface nitrile

groups into carboxylic groups by the hydrolysis process This modified PAN m

was not so severely fouled in the Bovine Serum Albumin (BSA) filtration test The pore

size however decreased during the hydrolysis process leading to a significant reduction

in flux and made the membrane less productive in the ultrafiltration (UF) mode

24 Sorbent Technology

Over the last three decades sorbent technology [Castino et al 1976 Korshak et al

17

dialysis or replace it for the treatment of ESRD Sorbents remove solutes from solution

through specific or nonspecific adsorption depending on both the nature of the solute and

the sorbent Specific adsorption contains tailored ligands or antibodies with high

selectivity for target molecules een used in autoimmune

disord

and

e

al 1996] various resins [Ronco et al 2001]

album

e

as

Specific adsorbents have b

ers such as idiopathic thrombocytopenic purpura [Snyder et al 1992] and for the

removal of lipids in familial hyper cholesterolemia [Bosch et al 1999] Nonspecific

adsorbents such as charcoal and resins attract target molecules through various forces

including hydrophobic interactions ionic (or electrostatic) attraction hydrogen bonding

and van der Waals interactions

New dialysate with sorbents has become an accepted modification of dialysis

sorbent hemoperfusion is gaining ground as a valuable addition to dialysis especially as

new sorbents are developed [Winchester et al 2001] Hemoperfusion is defined as th

removal of toxins or metabolites from circulation by the passing of blood within a

suitable extracorpoteal circuit over semipermeable microcapsules containing adsorbents

such as activated charcoal [Samtleben et

in-conjugated agarose etc Novel adsorptive carbons with larger pore diameters

have been synthesized for potential clinical use [Mikhalovsky 1989] Newly recognized

uremic toxins [Dhondt et al 2000 Haag-Weber et al 2000] have resulted in several

investigations on alternatives to standard or high-flux hemodialysis to remove thes

molecules These methods include hemodiafilteration with [de Francisco et al 2000] or

without [Ward et al 2000 Takenaka et al 2001] dialysate regeneration using sorbents

well as hemoperfusion using such adsorbents as charcoal and resins

18

Kolarz et al [1989 1995] studied the hyper-crosslinked sorbent prepared from

styrene and divinylbenzene (DVB) for a hemoperfusion application They found that the

pore structure of a swelling sorbent was changed by additional crosslinking with α αrsquo-

dichloro-p-xylene in the presence of a tin chloride catalyst and in a di

chloroethane

soluti and

en

decyl groups that attract β2M through a hydrophobic interaction The

adsor

of

5]

d

penet

on They also realized that the hemocompatibility was useful for nemoperfusion

could be imparted to the sorbents by introducing sulfonyl groups at a concentration of

about 02mmolg

A special polymeric adsorbing material (BM-010 from Kaneka Japan) has be

investigated by another group [Furuyoshi et al 1991] for the selective removal of β2M

from the blood of dialysis partients The adsorbent consists of porous cellulose beads

modified with hexa

ption capacity of this material is 1mg of β2M per 1ml of adsorbent Using a

hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a

high-flux hemodialyzer several small clinical trials were performed During 4-5 hours

treatment about 210mg of β2M were removed thus reducing the concentration in the

blood by 60-70 of the initial level [Nakazawa et al 1993 Gejyo et al 1993 199

RenalTech developed a hemoperfusion device BetaSorbreg containing the hydrate

cross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure

designed to remove molecules between 4 and 30 kDa [Winchester et al 2002] In this

case solute molecules are separated according to their size based on their ability to

rate the porous network of the beaded sorbents The resin beads were prepared with

a blood compatible coating and confirmed to be biocompatible in vivo in animals

[Cowgill et al 2001]

19

25 Limitation of Current Hemodialysis Treatment

Hemodialysis is a widely used life-sustaining treatment for patients with ESRD

However it does not replace all of the complex functions of a normal healthy kidne

a result patients on dia

y As

lysis still suffer from a range of problems including infection

accelerated cardio ion anemia

chron

e

se middle

alysis

sessio

s

f

vascular disease high blood pressure chronic malnutrit

ic joint and back pain and a considerably shortened life span One significant

limitation of the current dialysis technology is the inability to efficiently remove larger

toxic molecules This is mainly because of the broad pore size distribution reducing th

selective removal of toxins and unsatisfied biocompatibility causing lots of

complications such as inflammation blood clotting calcification infection etc

Dialysis purifies the patients blood by efficiently removing small molecules like

salts urea and excess water However as toxic molecules increase in size their removal

rate by hemodialysis substantially declines Typically only 10 - 40 of the

molecular weight toxins (300-15000 Da) are removed from the blood during a di

n [Vanholder et al 1995] These toxins then reach an abnormally high level and

begin to damage the body One such toxin β2M causes destructive arthritis and carpal

tunnel syndrome by joining together like the links of chain to form a few very large

molecules and deposit damaging the surrounding tissues [Lonnemann et al 2002] This i

also a main cause of mortality for long-term dialysis patients Other middle molecule

toxins appear to inhibit the immune system and may play a significant role in the high

susceptibility to infections in dialysis patients Still others are believed to impair the

functioning of several other body systems such as the hematopoietic and other endocrine

systems This may contribute to accelerated cardiovascular disease the leading cause o

20

death among dialysis patients as well as clinical malnutrition which affects up to 50 of

this patient population

Over the last decade polymeric dialysis membranes have been developed to

increase the capacity for removing middle molecular weight toxins by changing the pore

size of dialyzer membranes and using new materials that adsorb these toxins for

impro se

styrene made during World War II in the United

States The Dow chemical compan anufacturer of polystyrene

includ

d of

on

rug

ntrol of

ved removal characteristics However removal efficiency is not as high as tho

achieved by a normal healthy kidney

26 Latex Particles

The first synthetic polymer synthesized using emulsion polymerization was a

rubber composed of 13-butadiene and

y has been a major m

ing latex which they used in paint formulations The theory of emulsion

polymerization in which a surfactant is used was established by Harkins [1948] and by

Smith and Ewart [1948] By 1956 the technology was complete including the metho

building larger diameter particles from smaller ones The product by this emulsi

polymerization is referred to as latex a colloidal dispersion of polymer particles in water

medium [Odian 1991] Latexes are currently undergoing extensive research and

development for a broad range of areas including adhesives inks paints coatings d

delivery systems medical assay kits gloves paper coatings floor polish films carpet

backing and foam mattresses to cosmetics The relatively well known and easy co

the emulsion process is one of main advantages for these applications Therefore

polymeric latex particle prepared by emulsion polymerization can be a candidate for the

medical applications because of the easy control of the particle size and morphology as

well as flexible surface chemistry to be required

21

261 The Components for Emulsion Polymerization

The main components for emulsion polymerization process are the monomer a

dispersing medium a surfactant and an initiator Available monomers are styrene

sing medium is usually water

which

ticles

n

ation To

monomer droplets but in micelles because the initiators are

insolu le

and

olution

butadiene methylmethacylate acryl acid etc The disper

will maintain a low solution viscosity provide good heat transfer and allow

transfer of the monomers from the monomer droplets into micelles and growing par

surrounded by surfactants respectively The surfactant (or emulsifier) has both

hydrophilic and hydrophobic segments Its main functions is to provide the nucleatio

sites for particles and aid in the colloidal stability of the growing particles Initiators are

water-soluble inorganic salts which dissociate into radicals to initiate polymeriz

control the molecular weight a chain transfer agent such as mercaptan may be present

262 Particle Nucleation

Free radicals are produced by dissociation of initiators at the rate on the order of

1013 radicals per milliliter per second in the water phase The location of the

polymerization is not in the

ble in the organic monomer droplets Such initiators are referred to as oil-insolub

initiators This is one of the big differences between emulsion polymerization

suspension polymerization where initiators are oil-soluble and the reaction occurs in the

monomer droplets Because the monomer droplets have a much smaller total surface area

they do not compete effectively with micelles to capture the radicals produced in s

It is in the micelles that the oil soluble monomer and water soluble initiator meet and is

favored as the reaction site because of the high monomer concentration compared to that

in the monomer droplets As polymerization proceeds the micelles grow by the addition

of monomer from the aqueous solution whose concentration is refilled by dissolution of

22

monomer from the monomer droplets There are three types of particles in the emulsion

system monomer droplets inactive micelles in which polymerization is not occurring

and active micelles in which polymerization is occurring referred to as growing polymer

particles

The mechanism of particle nucleation occurs by two simultaneous processes

micellar nucleation and homogeneous nucleation Micellar nucleation is the entry of

radicals ei

ther primary or oligomeric radicals formed by solution polymerization from

the aq ed

the

r

s

n

is coagulation with other particles rather than

polymerization of monomer The driving force for coagulation of precursor particles

ueous phase into the micelles In homogeneous nucleation solution-polymeriz

oligomeric radicals are becoming insoluble and precipitating onto themselves or onto

oligomers whose propagation has ended [Fitch et al 1969] The relative levels of micella

and homogeneous nucleation are variable with the water solubility of the monomer and

the surfactant concentration Homogeneous nucleation is favored for monomers with

higher water solubility and low surfactant concentration and micellar nucleation is

favored for monomers with low water solubility and high surfactant concentration It has

also been shown that homogeneous nucleation occurs in systems where the surfactant

concentration is below CMC [Roe 1968] A highly water insoluble monomer such a

styrene [Hansen et al 1979 Ugelstad et al 1979] has probably created by micellar

nucleation while a water soluble monomer such as vinyl acetate [Zollars 1979] has bee

formed by homogeneous nucleation

A third latex formation reaction mechanism has been proposed referred to as

coagulative nucleation In this reaction the major growth process for the first-formed

polymer particles (precursor particles)

23

sever les

yer

es

r a

l

the beginning of the polymerization As soon as the initiator is

gins with the formation and

growt

and

ved

added at one time during the later stages of the polymerization prior to complete

al nanometers in size is their relative instability compared to larger sized partic

The small size of a precursor particle with its high curvature of the electrical double la

permits the low surface charge density and high colloidal instability Once the particl

become large enough in size maintaining the high colloidal stability there is no longe

driving force for coagulation and further growth of particles takes place only by the

polymerization process

263 Types of Processes for Emulsion Polymerization

There are three types of production processes used in emulsion polymerization

batch semi-continuous (or semi-batch) and continuous In the batch type process al

components are added at

added and the temperature is increased polymerization be

h of latex particles at the same time There is no further process control possible

once the polymerization is started In the semi-continuous emulsion polymerization

process one or more components can be added continuously Various profiles of particle

nucleation and growth can be generated from different orders of component addition

during polymerization There are advantages to this process such as control of the

polymerization rate the particle number colloidal stability copolymer composition

particle morphology In the continuous process the emulsion polymerization components

are fed continuously into the reaction vessel while the product is simultaneously remo

at the same rate High production rate steady heat removal and uniform quality of

latexes are advantages of the continuous polymerization processes

Other available methods include the intermittent addition and shot addition of one

or more of the components In the shot addition process the additional components are

24

conversion of the main monomer This method has been used successfully to develo

water-soluble functional monomers such as sodium styrene sulfonat

p

e [Kim et al 1989]

264

lsion

Chemistry of Emulsion Polymerization

Emulsion polymerization is one type of free radical polymerization and can be

divided into three distinct stages initiation propagation and termination The emu

polymerization system is shown in Figure 2-7

Aqueous phaseAqueous phase

Micelle withmonomer

I Rbull

Emulsifier

Polymer particle swollenWith monomer

Monomerdroplet

I Rbull

Micelle withmonomer

I Rbull

Emulsifier

Polymer particle swollenWith monomer

MonomerdropletMonomerdroplet

I Rbull

Figure 2-7 Emulsion polymerization system [Radicals (Rmiddot) are created from initiators

(I) Monomer is transferred from larger monomer droplet into micelles by emulsifier Initiated polymer particle by radicals is keep growing until monomers are all consumed The reaction is performed in aqueous media]

In the initiation stage free radicals are created from an initiator by heat or an

ultraviolet radiation source The initiator with either peroxides groups (-O-O-) such as

odium persulfate or azo groups (-N=N-) such as azobisisobutyronitrile is commonly

used for em t

with the m er

chain ere

s

ulsion polymerization The primary free radicals created from initiator reac

onomer for initiation of polymerization In the propagation stage the polym

grows by monomer addition to the active center a free radical reactive site Th

are two possible modes of propagations head-to-head addition and head-to-tail addition

25

The head-to-tail mode is the predominant configuration of the polymer chain a result o

steric hindrance of the substitute bulky group In the termination stage polymer chain

growth is terminated by either coupling of two growing chains forming one polymer

molecule or transferring a hydrogen atom (dispropotionation) from one growing chain to

another forming two polymer molecules one having a saturated end group and the other

with an unsaturated end-group

265 Seed Emulsion Polymerization

Polymer latex particles have received increasing interest because of the versatili

of the many applications heterophase polymerization processes like emulsion dispersion

micro-emulsion seeded emulsion precipitation etc Especially to prepare well-defined

microspheres having monodispe

f

ty

rse and various particle sizes as well as surface group

ed particles prepared by emulsion polymerization

and e

s

hological design In

seede to control

n

functionalilties it is necessary to use se

nlarge them to a desired size in the further stages of reactions

Polymeric particles are required to have a uniform particle size in many

applications such as chromatography where they are used as a packing material

Morphological control of latex particle is also important for many practical application

[Schmidt 1972] Seed emulsion polymerization (or two-stage emulsion polymerization) is

a useful method to achieve both monodisperse particle size and morp

d emulsion polymerization [Gilbert 1995] preformed lsquoseedrsquo latex is used

the number of particles present in the final latex The advantage of seeded emulsio

polymerization is that the poorly reproducible process of particle nucleation can be

bypassed so that the number concentration of particles is constant and known Various

mechanisms have been proposed for the growth of latex particles [Ugelstad et al 1980

Okubo et al 1992] using this polymerization technique The initial seed particle

26

preparation step is well known and relatively easy to perform because at the relative

small particle sizes (02 to 05 micron) the particle growth process can be readily

controlled by the use of an emulsifier Enough emulsifier is used to prevent coagulation

but not enough to cause the formation of new particles As the particles are grown to

larger sizes in successive seeding steps it becomes increasingly difficult to mainta

stable uncoagulated emulsion without forming new particles and thereby destroying

monodisperisty of the latex Recently there have been several reports concerning t

reaction kinetics of seed emulsion polymerization and the development of latex

morphologies over the course of the reaction [Chern et al 1990 Delacal et al 1990 L

et al 1995 Lee 2000 2002]

266 Polystyrene Latex Particles

A number of papers have described the synthesis of the polystyrene latex particl

bearing various functional surface groups such as carboxyl [Lee et al 2000 Tun

2002 Reb et al 2000] hydroxyl [Tamai et al 1989] marcapto [Nilson 1989] epoxy

[Shimizu et al 2000 Luo et al

ly

in a

he

ee

es

cel et al

2004] acetal [Izquierdo et al 2004 Santos et al 1997]

ometyl [Izquierdo et al 2004 Park et al 2001

Sarob

h the

d

thymine [Dahman et al 2003] chlor

e et al 1998] amine [Counsin et al 1994 Ganachaud et al 1997 Ganachaud et al

1995 Miraballes-Martinez et al 2000 2001 Anna et al 2005] ester [Nagai et al 1999]

etc In order to produce these functionalized particles different methods for particle

preparation must be used The polymer emulsion with core-shell morphology of latex

particles is one of them This is a multistep emulsion polymerization process in whic

polystyrene ldquocorerdquo particle is synthesized in the first stage and the functional monomer is

added in the second stage of the polymerization without any emulsifier postfeeding to

prevent the production of new homopolymer particles thus forming the functionalize

27

polymer ldquoshellrdquo on the ldquocorerdquo particle [Keusch et al 1973] There are requirements to

limit secondary nucleation and encourage core-shell formation in seeded emulsion

polymerization including the addition of smaller seed particles at high solid content to

increase particle surface area low surfactant concentration to prevent formation of

micelles and the semi-continuous addition of monomer to create a starved-feed conditio

and keep the monomer concentration low There are some advantages [Hergeth et al

1989] of dispersions with polymeric core-shell particles First it is possible to modi

interfacial properties of polymer particles in the aqueous phase by the addition of only

very small amounts of a modifying agent during the last period of the reaction Thus

these core-shell particles are useful in a broad range of applications since they always

exhibit improved physical and chemical properties over their single-component

counterparts [Lu et al 1996 Nelliappan et al 1997] In this way the improvement of

surface properties of such dispersions is straightforward and inexpensive The other is

that polymers with a core-shell structure are perfect model systems for investigating th

material properties of polymer blends and composites because of their regular

distribution of one polymer inside a matrix polymer and because of the simple sp

geometry of the system

Their properties usually depend on the structures of latex particles Chen and his

coworkers [Chen et al 1991 1992 1993] reported the morphological development of

core shell latex particles of polystyrenepoly(methyl methacrylate) during

polymerization Before the research by Chen and his coworker Min et al [Min et al

1983] reported the morph

n

fy the

e

herical

ological development of core shell latex of polystyrene

(PS)polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the

28

additi

of

ity of graft

ing reagents Thus protein adsorption on polymeric solid surfaces has

st are microspheres defined as ldquofine

polym can be

n

r was

containing antigen urine serum etc The latex will become agglutinated and visibly

on method of PS They found that the percentage of grafting PS to the PBA was

greatest for the batch reaction and the PBA-PS core-shell particles with a high degree

grafting remained spherical upon aging test because of the emulsifying abil

copolymer

267 Various Applications of Latex Particles

Latex particles are applicable to a wide range of areas such as biomedical

applications [Piskin et al 1994] especially as the solid phase such as in immunoassays

[Chern et al 2003 Radomske-Galant et al 2003] DNA diagnostic drug delivery carriers

[Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] blood cell separations and

column pack

become a center of attention Of particular intere

er particles having diameters in the range of 01 to several micronsrdquo which

used as functional tools by themselves or by coupling with biocompounds Singer and

Plotz [1956] firstly studied microsphere or latex agglutination test (LATrsquos) by using

monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support o

which the biomolecules were going to adsorb The biomolecule adsorption howeve

limited by possible desorption of the adsorbed species or loss of specific activity of the

complex formed Since this work was published the latex particle applications for

immunoassay have been rapidly and widely studied and developed

Latex agglutination test or latex immunoassays start with tiny spherical latex

particles with a uniform diameter and similar surface properties The particles are coated

with antibodies (sensitized) through the hydrophobic interaction of portions of the protein

with the PS surface of the particles If sensitized particles are mixed with a sample

29

agglomerate Latex tests are inexpensive as compared with the other techniques [Ba

1988]

ngs

sed

tion Rheumatoid factor (RF) in different age subpopulations has also been

evalu

studied a

latex

Unipath [Percival 1996] has manufactured a range of immunoassay materials ba

on a chromatographic principle and deeply colored latex particles for use in the home and

clinical environments The latex particles which are already sensitized with a

monoclonal antibody can detect any antigens that bound to the surface of the latex

particles Some detectable pollutants include estrogen mimics which induce

abnormalities in the reproductive system of male fishes and lead to a total or partial male

feminiza

ated according to a patientrsquos clinical status by using a rapid slide latex agglutination

test for qualitative and semiquantitative measurement in human serum along with latex

immunoassay method [Onen et al 1998] Magalhaes and his coworkers [2004]

diagnostic method of contamination of male fishes by estrogen mimics using the

production of vitellogenin (VTG) as a biomarker This was based on a reverse

agglutination test developed with monoclonal antibodies specific to this biomarker

Premstaller and his coworkers [Premstaller et al 2000 2001] have prepared a porous

poly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient

chromatographic separation of biomelecules such as proteins and nucleic acids They

used a porogen a mixture of tetrahydrofuran and decanol to fabricate a micropellicular

PS-DVB backbone Legido-Quigley and his colleagues [2004] have developed the

monolith column to obtain further chromatographic functionality to the column by

introducing chloromethylstyrene in place of styrene into the polymer mixture

30

Core shell type monodisperse polymer colloids have been synthesized by Sarobe

and Forcada [1998] with chloromethyl functionality in order to improve the biomolecu

adsorption through a two-step emulsion polymerization process They investigated the

functonalized particles by optimizing the experimental parameters of the functional

monomer including reaction temperature the amount and type of redox initiator sys

used the type of addition of the initiator system and the use of washing They c

le

tem

oncluded

that th

sis

serve as separation

chann

of

usually

along the polypeptide chain which contain positive or negative charges [Norde 1998]

e relation between the amount of iron sulfate and the persulfatebisulfite system

added should be controlled to obtain monodisperse particles and prevent the premature

coagulation of the polymer particles during the polymerization

A semi-continuous emulsion polymerization technique for latex particle synthe

was performed by McDonald and other researchers [Ramakrishnan et al 2004 Steve et

al 1999] They introduced a variety of particles sizes compositions morphologies and

surface modifications to fabricate latex composite membranes (LCMs) Arraying and

stabilizing latex particles on the surface of a microporous substrate form narrowly

distributed interstitial pores formed between the particles which

els They investigated the membrane performance using gas fluxes water

permeability and the retention characterization of dextran molecules From these tests

they concluded that the narrow discriminating layer made of the latex particles leads to a

highly efficient composite membrane

27 Proteins

Proteins are natural polyamides comprised of about 20 different α-amino acids

varying hydrophobicity [Norde 1998] Proteins are more or less amphiphilic and

highly surface active because of the number of amino acid residues in the side groups

31

Proteins are polymers of L-α-amino acids [Solomon and Fryhle 2000] The α refers to a

carbon with a primary amine a carboxylic acid a hydrogen and a variable side-chain

group designated as R Carbon atoms rent groups are asymmetric and can

exhib l

s

e

on

with four diffe

it two different spatial arrangements (L and D configurations) due to the tetrahedra

nature of the bonds The L refers to one of these two possible configurations Amino

acids of the D-configuration are not found in natural proteins and do not participate in

biological reactions Figure 2-8 shows the chiral carbon in 3-D as the L isomer Protein

consist of twenty different amino acids differentiated by their side-chain groups [Norde

1998] The side-chain groups have different chemical properties such as polarity charg

and size and influence the chemical properties of proteins as well as determine the

overall structure of the protein For instance the polar amino acids tend to be on the

outside of the protein when they interact with water and the nonpolar amino acids are

the inside forming a hydrophobic core

Figure 2-8 L-α-amino acid

The covalent linkage between two amino acids is known as a peptide bond A

peptide bond is formed when the amino group of one amino acid reacts with the carboxyl

group of another amino acid to form an amide bond through the elimination of water

This arrangement gives the protein chain a polarity such that one end will have a free

amino group called the N-terminus and the other end will have a free carboxyl group

called the C-terminus [Solomon and Fryhle 2000] Peptide bonds tend to be planar and

32

give the polypeptide backbone rigidity Rotation can still occur around both of the α-

carbon bonds resulting in a polypeptide backbone with different potential conformations

relative to the positions of the R groups Although many conformations are theoretically

possible interactions between the R-groups will limit the number of potential

nd to form a single functional conformation In other words

the conformation or shape of the protein is

α-helix

can interact with other

secondary structures within the sam

conformations and proteins te

due to the interactions of the chain side

groups with one another and with the polypeptide backbone The interactions can be

between amino acids that are close together as in a poly-peptide between groups that are

further apart as in amino acids or even on between groups on different polypeptides all

together These different types of interactions are often discussed in terms of primary

secondary tertiary and quaternary protein structure

The primary amino acid sequence and positions of disulfide bonds strongly

influence the overall structure of protein [Norde 1986] For example certain side-chains

will promote hydrogen-bonding between neighboring amino acids of the polypeptide

backbone resulting in secondary structures such as β-sheets or α-helices In the

conformation the peptide backbone takes on a spiral staircase shape that is stabilized by

H-bonds between carbonyl and amide groups of every fourth amino acid residue This

restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure

Certain amino acids promote the formation of either α-helices or β-sheets due to the

nature of the side-chain groups Some side chain groups may prevent the formation of

secondary structures and result in a more flexible polypeptide backbone which is often

called the random coil conformation These secondary structures

e polypeptide to form motifs or domains (ie a

33

tertiary structure) A motif is a common combination of secondary structures and a

domain is a portion of a protein that folds independently Many proteins are composed o

multiple subunits and therefore exhibit quaternary structures

271 Interaction Forces between Proteins

Proteins in aqueous solution acquire compact ordered conformations In such a

compact conformation the movement along the polypeptide chain is severely restrict

implying a low conformational entropy The compact structure is possible only if

interactions within the protein molecule and interactions between the protein molecule

and its environment are sufficiently favorable to compensate for the low conformational

entropy [Malmsten 1998] Protein adsorption study is often focused on structural

rearrangements in the protein molecules because of its significance to the biological

functioning of the molecules and the important role such rearrangements play in the

mechanism of the adsorption process Knowledge of the majo

f

ed

r interaction forces that act

structures helps to understand the behavior

of pro

arge

a

ded

between protein chains and control the protein

teins at interface These forces include Coulomb interaction hydrogen bonds

hydrophobic interaction and van der Waals interactions

Coulombic interaction Most of the amino acid residues carrying electric ch

are located at the aqueous boundary of the protein molecule An the isoelectric point

(IEP) of the protein where the positive and negative charges are more or less evenly

distributed over the protein molecule intramolecular electrostatic attraction makes

compact structure favorable to proteins Deviation to either more positive or more

negative charge however leads to intramolecular repulsion and encourages an expan

structure Tanford [1967] calculated the electrostatic Gibbs energy for both a compact

impenetrable spherical molecule (protein) and a loose solvent-permeated spherical

34

molecule (protein) over which the charge is spread out From the results he found th

the repulsion force was reduced at higher ionic strength d

at

ue to the screening action of ion

ble

e

the

d the

tween

dehyd

e

ent

oles

tein

Hydrogen bond Most hydrogen bonds in proteins form between amide and

carbonyl groups of the polypeptide backbone [Malmsten 1998] The number of availa

hydrogen bonds involving peptide units is therefore far greater than that involving sid

chains Because α-helices and β-sheets are aligned more or less parallel to each other

interchain hydrogen bonds enforce each other Kresheck and Klotz [1969] examine

role of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between

peptide units do not stabilize a compact structure of protein However because the

peptide chain is shielded from water due to other interactions hydrogen bonding be

peptide groups do stabilize α-helical and β-sheet structures

Hydrophobic interaction Hydrophobic interaction refers to the spontaneous

ration and subsequent aggregation of non-polar components in an aqueous

environment In aqueous solutions of proteins the various non-polar amino acid residues

will be found in the interior of the molecule thus shielded from water The

intermolecular hydrophobic interaction for the stability of a compact protein structure

was first recognized by Kauzmann [1959] If all the hydrophobic residues are buried in

the interior and all the hydrophilic residues are at the outermost border of the molecul

intramolecular hydrophobic interaction would cause a compact protein structure

However geometrical and other types of interactions generally cause a fraction of the

hydrophobic residues to be exposed to the aqueous environm

Van der Waals interaction The mutual interaction between ionic groups dip

and induced dipoles in a protein molecule cannot be established as long as the pro

35

structure is not known in great detail Moreover the surrounding aqueous medium also

contains dipoles and ions that compete for participation in the interactions w

the protein molecule Dispersion interactions favor a compact structure However

because the Hamaker constant for proteins is only a little larger than that of water the

resulting effect is relatively small [Nir 1977]

272 β

ith groups of

rgiles et al 1996

Floeg

) is degraded [Floege 2001] Renal

β2M from the serum resulting in an increase in β2M

conce well

the

)

a

al

2-Microglobulin (β2M)

The protein β2M is of particular interest because it is involved in the human

disorder dialysis-related amyloidosis (DRA) [Geiyo et al 1985 A

e 2001] DRA is a complication in end stage renal failure patients who have been on

dialysis for more than 5 years [Bardin et al 1986 Drueke 2000] DRA develops when

proteins in the blood deposit on joints and tendons causing pain stiffness and fluid in

the joints as is the case with arthritis In vivo β2M is present as the non-polymorphic

light chain of the class I major histocompatibility complex (MHC-I) As part of its

normal catabolic cycle β2M dissociates from the MHC-I complex and is transported in

the serum to the kidney where the majority (95

failure disrupts the clearance of

ntration by up to 60-fold [Floege 2001] By a mechanism that is currently not

understood β2M then self-associates into amyloid fibrils and typically accumulates in

musculoskeletal system [Homma et al 1989] Analysis of ex vivo material has shown

that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRA

is present as of full-length wild-type β2M although significant amounts (~20-30) of

truncated or modified forms of the protein are also present [Floege 2001 Bellotti et al

1998] Figure 2-9 shows the ribbon diagram of human β2M Native β2M consists of

single chain of 100 amino acid residues and has a seven stranded β-sandwich fold typic

36

of the immunoglobulin superfamily [Saper et al 1991 Trinh et al 2002] β2M was fi

isolated from human urine and characterized by Berggard et al [1980] in1968 The

normal serum concentration of β

rst

re

f

e

2M is 10 to 25 mgL It is a small globular protein with

a molecular weight of 118 kDa a Strokes radius of 16Aring and a negative charge under

physiological conditions (isoelectric point IEP = 57) β2M contains two β-sheets that a

held together by a single disulphide bridge between the cysteines in positions 25 and 81

[Berggard et al 1980 Parker et al 1982 Cunningham et al 1973] β2M cannot be

removed completely by current dialysis techniques but through a better understanding o

the structure and interaction forces that lead to this structure it will be possible to mor

efficiently remove this problematic protein

Figure 2-9 Ribbon diagram of human β M taken from the crystal structure of the protein

bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khal 2000]

273 Serum Albumin

Serum albumin is the most abundant protein found in plasma and is typically

present in the blood at a concentration of 35~

2an et

50gL According to extensive studies about

its physiological and pharmacological properties albumin has a high affinity to a very

wide range of materials such as electrolytes (Cu+2 Zn+2) fatty acids amino acids

metabolites and many drug compounds [Fehske et al 1981 Kraghhansen 1981 Putnam

37

1984 Peters 1985] The most important physiological role of the protein is therefore to

bring such solutes in the bloodstream to their target organs as well as to maintain the pH

and osmotic pressure of the plasma Bovine serum albumin (BSA) is an ellipsoidal

protein with the dimensions of 140 X 40 X 40Aring [Peter 1985] The primary structure is a

single helical polypeptide of 66 kDa (IEP = 47) with 585 residues containing 17 pairs of

as a soft an

rfaces [Kondo et al 1991 Norde et al 1992 Soderquist et al

1980 ikely

is

disulfide bridges and one free cysteine [Dugaiczyk et al 1982] BSA has been classified

d flexible protein because it has a great tendency to change its conformation

on adsorption to solid su

Carter and Ho 1994] and consists of three homologous domains (I-III) most l

derived through gene multiplication [Brown 1976] Each domain is composed of A and B

sub-domains [He et al 1992] The secondary structure of human serum albumin (HSA)

shown in Figure 2-10

Figure 2-10 Secondary structure of human serum albumin (HSA) with sub-domains

[Zunszain et al 2003]

HSA has the same structure domains with the serum albumin from other species

such as BSA [Brown 1976] Although all three domains of the albumin molecule have

similar three-dimensional structures their assembly is highly asymmetric [Sugio et al

38

1999] Domains I and II are almost perpendicular to each other to form a T-shaped

assembly in which the tail of subdomain IIA is attached to the interface region between

sub-domains IA and IB by hydrophobic interactions and hydrogen bonds In contrast

domain III protrudes from sub-domain IIB at a 45ordm angle to form the Y-shaped assembly

for domains II and III Domain III interacts only with sub-domain IIB These features

make the albumin molecule heart-shaped

28 Protein Adsorption

Protein adsorption studies date back to the 1930rsquos At the beginning these studies

mainly focused on the determination of the molecular weight electrophoretic and

ral

rearrangem

adsorption and biocompatibility of the sorbent materials were investigated [Norde 1986]

There are interaction forces at the interfaces between protein molecules and latex

particles These forces are mainly divided into the following groups hydrophobic

interaction ionic interaction hydrogen bonding and van der Waals interaction [Andrade

1985]

Hydrophobic interaction It is known that hydrophobic interaction has a major

role in protein adsorption phenomena The adsorption of proteins on the low charged

latex particles occurs by this interaction force Generally monomers such as styrene offer

hydrophobic surfaces th

by this interaction force is maximum at the isoelectric point (IEP) of the protein and the

strength [Suzawa et al 1980 1982 Shirahama et al 1989 Kondo et al 1992] By the

chromatographic applications Later the adsorption mechanism especially the structu

ents was studied Recently the studies of the relation between protein

281 Interaction between Protein Molecule and Latex Particle

at protein molecules adsorb to The amount of adsorbed protein

pH at maximum adsorption shifts to a more acidic region with an increase in ionic

39

reports [Suzawa et al 1980 1982 Lee et al 1988] protein adsorption was greater

hydrophobic surface than on a hydrophilic one implying that hydrophobic interaction is

on a

s

ydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA)

Ionic

ely

t

ces

n force is operative over small

distan e

ble

n

nt dialysis membrane therapy for end stage renal disease (ESRD)

one of most dominant forces in protein adsorption

Ionic interaction Negatively charged latex particles have ionic functional

groups on their surfaces such as salts of sulfonic and carboxylic acid Sulfate group

originate from an initiator such as sodium persulfate and carboxylic groups originate

from a h

bonds are formed between the negative charges of these latex particles and positive

surface charges of protein molecules The conventional low-charged latex particles rar

form these ionic bonds

Hydrogen bonding Hydrogen bond is a strong secondary interatomic bond tha

exists between a bound hydrogen atom (its unscreened proton) and the electrons of

adjacent atoms [Callister 1999] Protein can be adsorbed on hydrophilic polar surfa

through hydrogen bonding Hydrogen bonds are frequently formed between hydroxyl-

carbonyl or amide-hydroxyl Hydroxyl-hydroxyl or amide-hydroxyl bonds are also

formed in protein adsorption

Van der Waals interaction This interactio

ces only when water has been excluded and the two non-polar groups come clos

to each other Lewinrsquos calculation showed that the van der Waals interaction is negligi

compared with the forces involved in the entropy increases ie hydrophobic interactio

[Lewin 1974]

29 Hypothesis for Toxin Removal

As mentioned earlier insufficient removal of middle molecular range toxins is a

major drawback of curre

40

patien

is

y

ese

haracteristics over the last decade However removal

effici

ing a novel membrane design composed of an assembly of engineered

polymeric latex particles s ions Important factors

will i

e

ith a

is established it is

expec

e

ts This may cause destructive arthritis and carpal tunnel syndrome inhibit the

immune system and accelerate cardiovascular disease leading to death among dialys

patients In order to overcome these complications artificial dialysis membranes have

been developed to increase the capacity for removing middle molecular weight toxins b

changing the pore size of dialysis membranes and using new materials that adsorb th

toxins for improved removal c

ency is not as high as those achieved by a normal healthy kidney

With the knowledge obtained from all above literatures the following hypothesis

has been established for the development of a membrane for the successful removal of

the target protein β2-Microglobulin (β2M) without the removal of serum albumin This

will be done us

ynthesized to predetermined specificat

nclude pore size and surface chemistry

291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size

The packing of monodispersed spherical particles can lead to the formation of

porous layers suitable for use as filters and membranes [Hsieh et al 1991] The pore siz

is defined as the largest spherical particles that can pass through the interstitial spaces

It is well known that monodispersed spherical particles can be obtained from the

seed emulsion polymerization method Many commercially available latex particles are

synthesized by this technique and are inexpensive When the defect free membrane w

monodisperse pore size distribution based on a particular particle array

ted to outperform the traditional hemodialysis membrane limitation which is a

broad range of pore size distribution When spherical particles pack in regular crystallin

arrays a number of packing geometries such as hexagonal closest packing cubic closest

41

packing and body-centered cubic packing are possible The pore size of the array

depends on these packing geometries as well as particle size Theoretical pore size can be

calculated from the simple geometry of the arr

ay Figure 2-11 shows the relationship

between particle size and pore size

0001

001

01

001 01 1 10

Por

e

1

10

microm

) S

ize

(

Particle Diameter (microm)

Body-Centered Cubic ArrayClosest Packed Array

0001

Siz

e (

Particle Diameter (microm)

001

01

001 01 1 10

Por

e

1

10

microm

)

Body-Centered Cubic ArrayClosest Packed ArrayBody-Centered Cubic ArrayClosest Packed Array

and closed packed arrays [Steve et al 1999]

McDonald and his coworkers [Ramakrishnan et al 2004 Steve et al 1999] have

described the fabrication of latex composite membranes and their performance in

previous papers It is from this prior research and knowledge gained from conversations

with Dr McDonald that this current research has been initiated [McDonald 2003] The

focus of this thesis is the extension of McDonaldrsquos research in the area of composite

membranes to medical application such as dialysis membranes

292 Toxin Removal by Selective Adsorption on Engineered Latex Particles

The flexibility in latex surface properties is a significant advantage to this

materialrsquos ability to separate proteins since the selectivity is strongly dependent on the

charge interactions of both the protein and the latex particles under the conditions of

Figure 2-11 The relationship between pore size and particle size in body-centered cubic

42

separation [Menon et al 1999 Chun et al 2002] First all sorbents were designed to

have negative surface properties because most plasma proteins in blood are negative and

should not be removed with charge interaction between proteins and sorbents at

physiological condition (pH74) It was reported that a negatively charged surface was

more blood compatible than a positive one [Srinivasan et al 1971] The toxin protein β2-

Microglobulin (β2M) can be selectively adsorbed on the surface of latex particles by the

design of a suitable hydrophobic portioned surface where only β2M protein can be

anchored with hydrophobic interactions Albumin adsorption on the latex particle is not

side-on m

adsorption of β M protein on the engineered latex particles

allowed because charge repulsion is more dominant than hydrophobic interaction with

ode The figure 2-12 shows the schematic representation of the selective

2

PMMAPAAShell

COO-

OSO3-

OSO3-

PSCore

COO-

-

-

-

-

-

-

Albumin-

Chargerepulsion

--

-

- -

- -

- --

Hydrophobic

β2-Microglobulin

2

Interaction

β -Microglobulin

PMMAPAAShell

COO-

-

-

-

-

-

-

--

-

-

-

-

-

Albumin-

Chargerepulsion

OSO3-

OSO3-

PSCore

COO-

--

-

- -

--

-

- -

- -

- --

- -

- --

Hydrophobic

β2-Microglobulin

2

particles at pH 74

Interaction

β -Microglobulin

Figure 2-12 Schematic representation of the protein adsorption on the core shell latex

43

Hydrophilichydrophobic microdomain structures were proven to be more blood

compatible [Mori et al 1982 Higuchi et al 1993 Deppisch et al 1998] The

optimization of suitable hydrophobic to hydrophilic ratio is also important for the

biocompatibility The monomers such as styrene (St) methyl methacrylate (MMA) and

acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in

emulsion polymerization process

In summary the background and fundamental literature survey about the history

material properties and limitation of hemodialysis membrane latex particle preparation

surface chemistry and manufacturing process target proteins and protein adsorption and

finally the hypothesis for a toxin removal with high separation efficiency have been

suggested This research focuses on such hypothesis and the materials and

characterization methodology for achievement of suggested hypotheses are described in

the next chapter

CHAPTER 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY

As mentioned earlier the goal of this study is to prepare polymeric latex particles

with tailored properties to maximize separation of toxin molecules and to investigate

fundamental interactions between the applied particles and molecules in the

system to optimize the membrane performance for hemodialysis applications Polymeric

latex particles wit

the

biological

h monodisperse size distribution to obtain uniform pore size and

various size ranges to utilize membrane construction are necessary Surface engineering

with various combination of hydrophobichydrophilic domain on the surface of latex

particles is expected to affect the removal of target protein by selective adsorption and to

improve the biocompatibility of membrane Therefore the materials and characterization

methodology are addressed in this chapter

31 Materials

Styrene (St) monomer used for a seed and a core particle was purchased from

Fisher Scientific and used without any other purification process Acrylic acid (AA) and

methyl methacrylate (MMA) monomers introduced for shell formation were purchased

from Fisher Scientific and used without any other purification process Sodium persulfate

(SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as

received Divinylbenzene (DVB) crossliking agent was purchased from Aldrich The

anionic surfactant Aerosol regMA80-I [sodium di(13-dimethylbutyl) sulfosuccinate] was

kindly donated by Cytec 78-80 Aerosol regMA80-I is mixed with isopropanol and

water Its critical micelle concentration (CMC) is 245 mM (119) Ion exchange resin

44

45

AGreg 501-x8 Resin (20-50 mesh) was purchased from Bio-Rad Laboratories Inc It is a

mixed-bed resin with H+ and OH- as the ionic forms and is 8 crosslinked Bovine serum

albumin (BSA) (heat shock treated) was purchas Fisher Scientific The isoelectric

point of t lin

(β2M)

as

gma

ed from

he BSA is 47-49 and the molecular weight is 66300Da β2-Microglobu

was purchase from ICN Biomedicals Inc and was separated from patients with

chronic renal disease and lyophilized from ammonium bicarbonate The molecular

weight by SDS-PAGE was approximately 12000Da BSA and β2M proteins were used

received without further purification process In order to investigate the protein

adsorption evaluation bicinchoninic acid protein assay kits were purchased from Si

(Cat BCA-1) and Pierce Biotechnology (Cat 23225) The chemical structures for the

main chemicals are shown in Figure 3-1

CH2 CH

Styrene (Mw = 10415)

CH2 CHCH2 CH

Styrene (Mw = 10415)

CH2 CH

Acrylic acid (Mw = 7206)

C

OH

O

CH2 CH

Acrylic acid (Mw = 7206)

C

OH

O

CH CH2

C

OCH3

O

CH

CH CH3

2

C

OCH3

O

CH3

Methyl methacrylate (Mw = 10012)Methyl methacrylate (Mw = 10012)

CH2 CH

Divinylbenzene (Mw = 1302)

CH CH2

CH2 CH

Divinylbenzene (Mw = 1302)

CH CH2CH CH

+(Na) -(O3S)

CH -C-O-CH-CH -CH-CH3

O

2 2 3

O CH CH3

CH3 CH3

CH-C-O-CH-CH2-CH-CH3

Anionic surfactant (MA80-I) (Mw = 3885)

+(Na) -(O3S)

CH -C-O-CH-CH -CH-CH3

O

2 2 3

O CH CH3

CH3 CH3

CH-C-O-CH-CH2-CH-CH3

Anionic surfactant (MA80-I) (Mw = 3885)2

+(Na)-(O) S OO

O O

- +

Initiator sodium persulfate (Mw = 23811)

O SO

(O) (Na)+ -

O O

- +(Na) (O) S OO

O SO

(O) (Na)+ -

O O

- +

Initiator sodium persulfate (Mw = 23811) Figure 3-1 Chemical structure of main chemicals

(Na) (O) S OO

O SO

(O) (Na)

46

32 Latex Particle Preparation

The experimental set up and the particle preparation schemes for various types and

size ranges of latex particles are shown in Figure 3-2 and 3-3 In the experimental setup a

mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at

end of glass stirrer The agitation rate is precisely adjusted by controller motor Paar glas

reactor with 1L volume is partially in silicon oil bath on hot plate The reactor has fo

openings for a stirrer a thermometer a nitrogen gas inlet and a reactant feeding lnlet

The reactants for latex polymerization are fed by precisely controlled by meterin

The particle preparation is described in each latex preparation section

the

s

ur

g pump

Figure 3-2 Experimental setup for semi-continuous emulsion polymerization

1 Motor 2 Mechanical stirrer bar 3 Thermometer 4 Inlet of N2 5 Inlet for initiator and monomer 6 Glass reactor (l L) 7 Oil bath 8 Hot plate 9 Pump for initiator 10 Pump for monomer 11 Tubes for reactant feed

47

Styrene monomer

Bare PS

PSPMMA100

PSPMMA90PAA10

PSPMMA P75 AA25

Gr

win

ticle

size

PS seedparticles

og

par

Styrene monomer

Bare PS

PSPMMA100

PSPMMA90PAA10

PSPMMA P75 AA25

Gr

win

ticle

size

PS seedparticles

og

par

Figure 3-3 The particle preparation scheme in various types and size ranges of latex particles

321 Preparation of Seed Latex Particles

Polystyrene seed particles were synthesized through a typical semi-continuous

emulsion polymerization Polystyrene latex particles were prepared in a four necked Paar

glass vessel equipped with a mechanical glass stirrer thermometer glass funnel for

nitrogen gas purge and a tube for monomer feeding The reactor was placed in a silicon

oil bath on a hot plate for homogenous and stable tempe he vessel was

firstly charged with de-ionized water the emulsifier so itiator

sodium bicarbonate (SBC) styrene (St) monomer divinylbenzene (DVB) monomer

under nitrogen gas atmosphere and then heated to 72plusmn2 as

llowed to continue for another hour Second reactants St and DVB monomers SPS

initiator and SBC were fed separately using fine fluid metering pumps (Model RHSY

Fluid metering INC NY) under a nitrogen gas atmosphere for 2h After complete feeding

rature control T

dium persulfate (SPS) in

oC for 1h The reaction w

a

48

of all reactants the reaction was continued for another hour at elevated temperature 80plusmn1

oC and then cooled to room temperature by removal of the oil bath

322 Preparation of Seeded Latex Particles

The particle growth was achieved by seeded continuous emulsion polymerization

The vessel was charged with the polystyrene seeds and de-ionized water to make desired

solid content and heated up to 72plusmn2 oC for 1h under nitrogen gas atmosphere Then St

and DVB monomers and solution of SPS and SBC in de-ionized water were continuously

added using metering pump with a precisely controlled feed rate After feeding all

reactants the reaction was continued for three hours keeping temperature of 80plusmn2 oC and

then the reaction vessel was cooled down to room temperature

Monodisperse spherical polystyrene seeds with a 280nm mean diameter were used

e-polymerized polystyrene seeded emulsion

was charged in a 500m

323 Preparation of Core Shell Latex Particles

for core shell structured latex particle The pr

l glass flask under a nitrogen gas atmosphere De-ionized water

was added to achieve the desired solid content The emulsion was heated to 80plusmn2oC for

10 hour Monomers initiator and SBC to form the shell were added by fluid metering

pumps (LAB PUMP JRRHSY model Fluid metering Inc USA) with fine controlled

feed rate Flow rate can be adjusted by control ring graduated in 450 division from 0 to

100 flow which is in the range of 10mlmin respectively The reaction was sustained

for additional 30 hours at 90plusmn2oC then cooled down to room temperature

324 Purification of Synthesized Latex Particles

Synthesized latex particles were purified with an ion exchange resin 200g (10

ww polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin

and stirred for 40 minute to remove unreacted monomers initiators and other impurities

49

Then the emulsion was diluted with DI water to a specific degree of solid content as

needed

33 Characterization

331

solid

termined

oscopy

d

hours

Dried latex (10mg) was mixed with

250 m on

Degree of Conversion

The degree of monomer to polymer conversion was determined gravimetrically

following the procedure described by Lee et al [1995] before the latex cleaning process

The synthesized emulsion raw materials were weighed and dried in a conventional oven

at 120oC for 30min to evaporate any unreacted monomers and water The remaining

was weighed and the degree of conversion was determined This process was repeated

three times and a mean value of the degree of conversion was de

332 Fourier Transform Infrared (FTIR) Spectr

A molecule can absorb only select frequencies (energy) of infrared radiation which

match the natural vibration frequencies The energy absorbed serves to increase the

amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]

Only those bonds which have a dipole moment that changes as a function of time can

absorb the infrared radiation The range of wavelengths for infrared radiation is between

25 microm (4000 cm-1) and 25 microm (400cm-1)

The synthesis of the latex particles such as polystyrene (PS) homopolymer and

PSPMMA PSPMMA90PAA10 PSPMMA75PAA25 core-shell copolymers were verifie

with FTIR Purified latex particles were dried in vacuum oven at 40oC for 24

before analysis in the FTIR (Nicolet Magma USA)

g of KBr which had also been dried in vacuum oven at 120oC for 3h Transmissi

spectra using the drift mode were plotted with 128 scans and 4cm-1 resolution

50

333 Quasielastic Light Scattering (QELS)

Latex particle size was measured by the Brookhaven ZetaPlus particle size

analyzer The solid concentration as 0001 (wv) This

ation spectroscopy (PCS) of quasielastically scattered light

For th s

ed using a FE-SEM (JEOL

JSM-

ith

ent

01

us zeta-

potential analyzer It measures the electrophoretic mobility velocity of charged colloidal

of latex particles used w

instrument uses photon correl

is technique time dependent interference patterns of light scattered from particle

in the sample cell are analyzed The interference pattern changes due to Brownian

motion giving rise to fluctuations in scattering intensity The frequency of these

fluctuations depends on the particlersquos diffusion constant that is inversely proportional to

the particle diameter

334 Field Emission-Scanning Electron Microscopy (FE-SEM)

Particle size and surface morphology were characteriz

6335F Japan) A diluted suspension of latex particles in water was dropped onto a

silicon wafer at room temperature and allowed to dry The sample was then coated w

the thinnest layer of carbon needed to obtain the required conductivity for this instrum

Secondary electron image mode was used with a 15KV of accelerating voltage The

magnification range used was between 10000X and 70000X

335 Zeta Potential Measurement

Synthesized and purified latex particles were diluted with de-ionized water to 0

wt and adjusted six different pH values 205 345 481 565 643 and 747 These

particle suspensions were then transferred to standard cuvettes for zeta potential

measurement At least two runs of ten measurements were taken for each sample and

averaged

The zeta potential measurement was carried out using Brookhaven ZetaPl

51

particles in solution and calculate zeta-potential by using the Smoluchowski equation

The f quency

gnitude

umin (BSA) as a standard model protein and β2-Microglobulin

uffer (PB) and

phosp

5

h the selected protein

e 005 01 03 05 and 07 mgml and those for

β2M

d at

teins in the supernatant after the centrifugation process using the

bicinc t

requency of laser light passing through the sample and is compared to the fre

of a reference beam The shift in the frequency called a Doppler shift and the ma

of the shift correspond to the polarity and the magnitude of the electrophoretic mobility

respectively The zeta potential is calculated from the solution conditions and the

measured mobility

336 Protein Adsorption

Protein adsorption experiments of the synthesized latex particles were performed

with bovine serum alb

(β2M) as a target protein in two types of buffer solution phosphate b

hate buffered saline (PBS) To make 5 mM of PB solution 0345g of sodium

phosphate monobasic (NaH2PO4H2O) was dissolve in 500ml of de-ionized water To

make PBS solution 0345g of sodium phosphate monobasic (NaH2PO4H2O) and 4178g

(143mM) of sodium chloride (NaCl) were dissolved in 500ml of de-ionized water The

synthesized latex particles were diluted with each buffer to have a solids content of 0

(ww) adjusted to pH values of 32 48 and 74 and mixed wit

BSA concentrations were chosen to b

were 0015 0030 0045 and 0060mgml The mixture was gently rotated using

the shaker Labquakereg (BarnsteadThermolyne Model 4002110 USA) with 8 RPM in

the incubator at 37oC for 12 hours before the latex-protein mixture was centrifuge

13000 rpm for 15min The amount of protein adsorbed was determined by quantifying

the free pro

honinic acid (BCA) assay method [Lowry et al 1951 Smith et al 1985 Baptista e

al 2003 Wiehelman et al 1988 Brown et al 1989] BCA assay kits consist of Reagent

52

A containing bicinchoninic acid sodium carbonate sodium tartrate and sodium

bicarbonate in 01N NaOH with pH=1125 and Reagent B containing 4 (wv) copper

(II) sulfate pentahydrate The BCA working reagent was prepared by mixing 50 parts of

Reagent A with 1 part of Reagent B 100microl of protein supernatant was then mixed with

2ml of BCA working reagent in a UV cuvette Incubation was allowed to continue

at room temperature

further

until color developed about 2h The absorbance at 562nm was

pectrophotometer (Perkin-Elmer Lambda 800 USA)

Unkn

Separate the p rticles in centrifuge mix supernatant with BCA reagents

measured using a UV-VIS s

own concentration of sample protein was determined by comparison to a standard

of known protein concentrations The adsorbed amount per unit surface area was

determined by the mass balance of the protein after adsorption process The simple

schematic of the procedure for a protein adsorption test is shown in Figure 3-4

Incubate mixture with agitation for 12hrs at 37oC

aand develop the color by incubation for 2hrs at room temperature

Suspend latex particles in aqueous buffer media

Add the dissolved target protein

Measure color intensity using UVVis spectroscopy at 562nm

Suspend latex particles in aqueous buffer media

Separate the p rticles in centrifuge mix supernatant with BCA reagents

Add the dissolved target protein

Incubate mixture with agitation for 12hrs at 37oC

aand develop the color by incubation for 2hrs at room temperature

Measure color intensity using UVVis spectroscopy at 562nm

Figure 3-4 Schematic of the procedure for a protein adsorption test

53

337 Blood Biocompatibility by Hemolysis Test

Hemolysis is the destruction of red blood cells which leads to the release of

hemoglobin from within the red blood cells into the blood plasma Hemolysis testing is

used to evaluate blood compatibility of the latex particles and the damaged to the red

blood cells can be determined by monitoring amount of hemoglobin released The red

blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutes

Figure 3-5 shows the phase separated blood with plasma white blood cells and red

cells after centrifugation process Separated RBSs were then washed with isotonic

phosphate buffer solution (PBS) at pH 74 to remove debris and serum protein Th

process was repeated 3 times

blood

is

Figure 3-5 Separation of RBC from whole blood by centrifuge process

Prepared latex particles were re-dispersed in PBS by sonification to obtain

homogeneously dispersed latex particles 100microl of the mixture of red blood cell (3 parts)

and PBS (11parts) was added to 1ml of 05 (ww) particle suspension PBS was used as

a negative control resulting 0 hemolysis and DI water used as a positive control to

produce 100 hemoglobin released by completely destroyed RBCs The mixture was

incubated in water bath with gentle shaking for 30 minutes at 37oC and then centrifuged

at 1500 rpm for 15 minutes 100microl of the supernatant was mixed with 2ml of the mixture

) (EtOHHCl = 2005 ww) to prevent the

White blood cells

Plasma

Red blood cells

of ethanol (99) and hydrochloric acid (37

54

precipitation of hemoglobin In order to remove remaining particles the mixture was

centri fuged again with 13000 RPM at room temperature The supernatant was then

transferred into the UV cuvette The amount of hemoglobin release was determined by

monitoring the UV absorbance at a wavelength of 397nm The schematic of the

procedure for hemolysis test is shown in Figure 3-6

Separate red blood cells (RBCs) from blood

Mix RBCs with phosphate buffer solution (PBS)

Wash and re-suspend RBC with PBS repeat three times

Add la l of RBC and incu in at 37oC

tex particle suspension into 100mbate sample with agitation for 30m

Separate particle ith EtOHHCl solutions and mix the supernatant w

Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm

Separate red blood cells (RBCs) from bloodSeparate red blood cells (RBCs) from blood

Mix RBCs with phosphate buffer solution (PBS)Mix RBCs with phosphate buffer solution (PBS)

Wash and re-suspend RBC with PBS repeat three timesWash and re-suspend RBC with PBS repeat three times

Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC

Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC

Separate particles and mix the ith EtOHHCl solution supernatant wSeparate particles and mix the ith EtOHHCl solution supernatant w

Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nmEvaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm

Figure 3-6 Schematic of the procedure for hemolysis test

In summary a description of the experimental materials and characterization

methodology has been explained In the next chapter the results and discussion of the

data obtained from these experiments is addressed

CHAPTER 4 RESULTS AND DISCUSSION

41 Polymerization of Latex Particles

411 Polystyrene Seed Latex Particles

Polymer particles have many different applications including spacer particles

lubricants packing materials for chromatography standard particles and diagnostic

drugs All applications strongly require these particles to have a uniform particle size

Uniform particle size is the first requirement for a latex composite membrane formed by

particle arrays with the interstitial spaces serving as pores for size discriminations [Jons

et al 1999 Ramakrishnan et al 2004] Although suspension polymerization has been

mainly used as one of conventional polymerization methods to prepare uniform particles

the uniformity of the recovered particles is insufficient for membrane use There is

another method known as seed emulsion polymerization where a vinyl monomer is

absorbed into fine monodiserse seed particles and polymerization causes the monomer to

crease the sizes of the seed particles uniformly In order to obtain monodisperse larger

particles by this m

particles and polymerization of the monomer is repeated

In this study cross-linked polystyrene seed latex particles were firstly synthesized

in the presence of styrene monomer divinylbenzene crosslink agent sodium persulfate

initiator sodium bicarbonate buffer and the Aerosol regMA80-I [sodium di(13-

dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion

polymerization process in which one or more components can be added continuously

in

ethod the procedure of absorption of monomer into fine polymer

55

56

Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and

buffer are continuously added to enlarge the seed latex particles Various profiles of

particle nucleation and growth can be gene different orders of component

addition during polymerizati f this process such as easy

control of the polymeriza particle number

and particle morphology The main goal in

this process is to avoid secondary particle

rated from

on There are several advantages o

tion rate monodisperse particle size and

colloidal stability copolymer composition

formation leading to a monodisperse particle

size distribution Figure 4-1 shows the schematic representation of seed latex particle

preparation and growth

Monomer droplet

Seed particles Amount of surfactantsis variable

Enlarged final Add monomers

PS

PS

PSs4

particles Number of particlesare constant

Seed code

PSs1

s2

s3

Monomer droplet

Seed particles Amount of surfactantsis variable

Enlarged final Add monomers

PS

PS

PSs4

tion The

particles Number of particlesare constant

Seed code

PSs1

s2

s3

Figure 4-1 Schematic representation of semi-continuous seed latex particles preparation and growth

After the first synthesis of a small size of crosslinked polystyrene (PS) seed

particles stabilized by surfactants styrene DVB and initiator are continuously fed into

the system to enlarge the seed latex particles maintaining a narrow size distribu

recipes and reaction conditions for synthesizing these seed particles are summarized in

57

Table 4-1 The original recipe for latex particle preparation from McDonald was

modified for this emulsion system [McDonald 2003]

Table 4-1 The polymerization recipe of polystyrene (PS) seed particles Latex label PSS259 PSS233 PSS207 PSS181

Temperature (oC) 75 plusmn 5 75 plusmn 5 75 plusmn 5 75 plusmn 5

Divinylbenzene (g) 052 052 052 052

MA 80 (g) 259 233 207 181

NaHCO

Watera (g) 1315 1315 1315 1315

Styrene (g) 9948 9948 9948 9948

112 112 112 112

Na2S2O8 (g) 190 190 190 190

Waterb (g) 200 200 200 200

Reaction time (min) 140 140 140 140

Solids content 396 396 396 396

Mean particle diameter (nm) 126plusmn75 171plusmn39 182plusmn41 216plusmn53

Conversion () 965 979 957 971

3 (g)

Four different seed latex particles were synthesized based on surfactant amount in

order to determine the optimum condition for stable and monodisperse latex particles

The emulsion polymerization of latex particles has to be carried out in a narrow range of

surfactant causes flocculation or phase separation leading to broad particle

size distribution and an unstable emulsion sy

surfactant concentration where particles are stable [Nestor et al 2005] A wide range of

concentrations

stem Therefore the range of surfactant

amount was varied from 181g to 259g in increments of 026g in each latex label Other

reactants amounts were kept constant Watera is the initial charge amount and waterb

represents the amount of aqueous solution containing dissolved initiator and buffer

58

which are fed continuously after the seed particle creation The extent of monome

polymer conversion was obtained by gravimetric calcu

r to

lation and was more than 95 at

cripts such as

S207 and S181 i the surf mount added for the seed latex

nversion indi perce experi obtain

content divided by the theoretically estimated solid content and was calculated by the

all seed particles labeled PSS259 PSS233 PSS207 and PSS181 Labeled subs

S259 S233 ndicate actant a

preparation The co cates the ntage of mentally ed solid

following formula

100 colids () times=entsoliatedntentoalExperimentConversion

n the experimenta content is the weight of olid rema after

mulsion at 120oC f in and the estimated solid content is th ht

lated from the recipe

rti as di each t load d

mple of ea pension iluted by (ww)

deionized water and the mean particle diameter was determined using the Brookhaven

ZetaPlus particle size analyzer From these results the seed latex particle size increased

as the amount of surfactant decreased The amount of emulsifier also affects the

polymerization process by changing the number of micelles and their size It is known

that large amount of emulsifier produces larger numbers of smaller sized particles [Odian

1991] and was corroborated in the current work

These synthesized seed latex particles should be of narrow size distribution in order

to obtain monodisperse larger particles The SEM image showing the particle

morphology and size of a sample of polystyrene seed particles can be seen in the Figure

4-2

contdEstim (4-1)

In this equatio l solid the s ining

evaporation of e or 30m e weig

of monomers calcu

As expected the seed pa cle size w fferent for surfactan ing in see

emulsion system A sa ch sus was d 0001 with

59

Figure 4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)

PSS259 (B) PSS233 (C) PSS207 (D) PSS181 (subscripts indicate the amount of surfactant added)

(A)

(B)

60

(C)

(D)

Figure 4-2 Continued

61

s seen in the image the seed particles are very smooth and spherical The

uniformity in the particle size distribution of these PS seed latex particles can also be

seen in the SEM image From this data we concluded that seed particles synthesized in

the presence of a surfactant with the chosen amounts are suitable for use to prepare larger

particles with high size uniformity and smooth spherical morphology

412 The Growth of Polystyrene (PS) Seed Latex Particles

Seeded emulsion polymerization has been conducted for several decades and

various mechanisms have been proposed Gracio et al [1970] suggested that the growing

polymer particles consist of an expanding polymer-rich core surrounded by a monomer-

rich shell with the outer shell providing a major locus of polymerization Seeded

mulsion polymerization is commonly used for preparing latex particles less than 1microm in

size [Cha et al 1995 Zou et al 1990 1992 Gandhi et al 1990 Park et al 1990] Such

latexes can be obtained from the growth of pre-prepared seed particles The seeds

introduced in this process serve as nucleation sites for particle growth By first swelling

the seed latex particles with additional monomer to the desired size polymerization can

then be initiated Several size ranges of monodisperse polystyrene (PS) latex particles

were prepared by this multistep seeded emulsion polymerization method For the

fabrication of particle-based membranes monodisperse latex particles with a variety of

size ranges are required

In this study seed particles from 171nm to 470nm in mean diameter and with a

highly uniform size distribution were prepared and used Watera is the initial charge

mount and waterb represents the amount aqueous solution containing dissolved initiator

and buffer that is continuously fed into system Feed time is precisely controlled by

metering pump The solid contents were maintained between 199 and 300 After

A

e

a

62

polym d as

es

ere 923 8402

n of these latex particles

are sh

ss

erization under these conditions the conversions of the latex particles labele

PS258 PS320 PS370 and PS410 were 952 957 946 and 964 respectively The recip

used in seeded emulsion polymerization to form the particles less than 500nm in mean

diameter are shown at the Table 4-2 The conversions of the latex particles larger than

500nm in mean diameter and labeled as PS525 PS585 PS640 and PS790 w

723 and 969 respectively The recipes used in the preparatio

own at the Table 4-3

A simple calculation can be used to predict the ultimate size that a latex particle can

reach after ending the polymerization Letrsquos suppose that seed density (ρs) and grown

particle density (ρgp) are the same The following equation is obtained from the ma

balance (m = ρV) where m and V are mass and volume of a particle respectively

gp

gp

s

s

Vm

Vm

In this equation m

= (4-2)

be

s and mgp are the weight of seed and total weight of seed and

added monomers respectively and Vs and Vgp are the volume of seed and grown

particle respectively

If we know the mass of monomer to be added the ultimate particle size can

calculated from the equation 4-3

3

2s D m ⎠⎝+ χ

where D

1s D m

⎟⎟⎞

⎜⎜⎛

= (4-3)

the

in

s and Dgp are the diameters for seed and grown particle respectively and χ is

weight of the monomer added Crosslinked PS latex particles with a size range from

258plusmn112 ~ 410plusmn117 in diameter polymerized by the recipes in Table 4-2 are shown

the SEM images in Figure 4-3

63

Table 4-2 Continuous addition emulsion polymerization recipe for growing polystyrene

Latex label PS

(PS) latex particles less than 500nm in size

258 PS320 PS370 PS410

Initial charge

Seed latex (g) 1000171 1000216 1000216 1000320

Watera (g) 1470 2500 1984 2600

06 07 06 06

Fee

Initiator stream

2 2 8

Feed time (min) 0-167 0-140 0-100 0-92

Solid content 284 300 300 200

Mean particle diameter (nm) 258plusmn112 320plusmn154 182plusmn71 410plusmn117

Conv

Monomer continuous

Styrene (g) 800 1200 800 600

Divinylbenzene (g)

d time (min) 0-163 0-110 0-75 0-82

Na S O (g) 07 065 05 05

NaHCO3 (g) 07 065 05 05

Waterb (g) 750 1200 200 370

ersion () 952 957 946 964 The solid content of seed latex for PS258 PS320 PS 370 and PS410 are 392 399 399 anrespectively

d 300

64

Table 4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size

Latex label PS525 PS585 PS640 PS790

Initial charge

Seed latex (g) 1000390 1000470 1000470 1000320

Watera (g) 1000 520 1200 360

Monomer continuous

Styrene (g) 290 200 400 173

Divinylbenzene (g) 02 014

03 009

Feed time (min) 0-80 0-40 10-76 5-65

Initiator stream

Na2S2O8 (g) 02 014 03 012

NaHCO3 (g) 02 014 03 009

Waterb (g) 200 120 400 200

Feed time (min) 0-85 0-46 0-77 0-75

Solids content 199 200 200 201

Mean particle diameter (nm) 525plusmn151 585plusmn186 640plusmn178 790plusmn693

Conversion () 923 8402 723 969 The solid content of seed latex for PS525 PS585 PS 640 and PS790 are 200 182 193 and 173 respectively

65

Figure 4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)

370nm (D) 410nm

(A)

(B)

66

(C)

(D)

Figure 4-3 Continued

67

article size was well matched to that estimated by the geometrical calculation

From this it can be concluded that the added monomer was consumed for polymer

particle growth with a high degree of conversion From the SEM characterization newly

nucleated particles were not seen as indicated by the narrow particle size distribution

The particles less than 500nm in mean diameter were very spherical in shape with a

highly uniform particle size distribution As the particles were grown larger than 500nm

mean diameter however they became of non-spherical shape with an uneven surface

This irregularity of particle surface can be attributed to the non-homogeneous monomer

swelling into the shell of a growing polymer which can be controlled by the factors such

as temperature agitation speed initiator feeding rate and surfactant amount required to

stabilize the colloidal system The SEM image of latex particles larger than 500nm mean

iameter is shown in Figure 4-4 The particle size distribution is broader than that of the

s particles These particles can however still be used for membrane construction

as a support layer rather than skin layer The support layer does not necessarily have to be

as highly monodisperse as the skin layer which require pores of high uniformity for the

selective removal of toxins

As mentioned earlier the amount of surfactant as well as monomer in an emulsion

system is the primary determinant of the particle diameter [Odian 1991] Anionic

surfactants are generally recommended at the level of 02-30 wt based on the amount

of water [Odian 1991] The critical micelle concentration (CMC) of Aerosolreg MA 80-I

anionic surfactant is 119 The surfactant loading in this emulsion system ranged from

the 121 for SS181 to 171 for SS259

P

d

maller

68

Figure 4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm

(C) 640nm (D) 790nm

(A)

(B)

69

(C)

(D)

Figure 4-4 Continued

70

The surfactant to monomer ratio refers to the value of the surfactant amount

divided by monomer amount after polymerization ends The surfactant amount is the

weight of the surfactant contained in emulsion and the monomer amount is the total

weight of the monomer added for polymerization to form the ultimate polymer particles

in emulsion As the surfactant to monomer ratio decreases mean particle diameter

increases as described by Odian [1991] Generally lower surfactant concentration forms

fewer micelles resulting in larger particle size [Evans et al 1999] However there is a

plateau in particle mean diameter between a surfactant to monomer ratio of 0005 and

0013 This is due to low polymerization conversion of latex particles from monomer

leading to a smaller particle size than expected This is not a factor of surfactant amount

The dependence of the particle size on the amount of surfactant is shown in Figure 4-5

0

200

400

600

800

1000

0000 0005 0010 0015 0020 0025 0030

Surfactant to monomer ratio (ww)

Part

icle

mea

n di

amet

er (n

m)

Figure 4-5 Dependence of the particle size on the surfactant to monomer ratio

71

413

e

f bare

microdomain structures is important for biocompatibility [Mori et al 1982 Higuchi et al

1993 Deppisch et al 1998] Figure 4-6 shows the schematic of core shell latex particle

structure The recipe for core shell structures is shown in Table 4-4

Core Shell Latex Particles

The latex particles shown in Figure 4-3 (A) and labeled as S258 in Table 4-2 wer

used as seeds to prepare core-shell latex particles because of their high particle

uniformity and smooth surface morphology Methyl methacrylate (MMA) and acrylic

acid (AA) monomers were introduced to increase surface hydrophilicity over that o

polystyrene particles AA is a more hydrophilic monomer than MMA because of the

carboxyl acid group in AA is more favorable for hydrogen boding with water The

surface carboxyl groups on PAA may have many promising applications in biomedical

and biochemical fields [Kang et al 2005] MMA monomer is less hydrophobic than

styrene PMMA is known to more biocompatible than PS but still PMMA is hydrophobic

The optimization of suitable hydrophobic to hydrophilic ratios and control of

Polystyreneseed particle

Core shelllatex particles

PSPMMA100

PSPMMA90PAA10

PSPMMA75PAA25

Polystyreneseed particle

Core shelllatex particles

PSPMMA100

PSPMMA90PAA10

Figure 4-6 Schematic of core shell latex particle structures

PSPMMA75PAA25

72

Table 4-4 The preparation recipe of PS core with various shell latex particles Latex 25 label PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA

Initial charge

PS seed latex (g) 500 500 500a

Monomer continuous

Methly methacylate (g) 320 288 240

Acrylic acid (g) 00 32 80

Initiator stream

Na

258 258 258

Water (g) 1570 1570 1570

Feed time (min) 0-120 0-50 0-60

b 200

Feed time (min) 0-122 0-60 0-65

952

2S2O8 (g) 02 02 02

NaHCO3 (g) 02 02 02

Water (g) 200 200

Solids content 183 183 183

Mean particle diameter (nm) 370plusmn186 370plusmn178 370plusmn191

Conversion () 955 924

Three types of core shell structures were prepared PMMA75PAA25 shell is a

copolymer consisting of the PMMA to PAA ratio of 75 to 25 by weight

PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90 to 10 by

weight PMMA100 is the PMMA homopolymer shell on PS core The particle size of

these core-shell particles as well as PS latex particle was prepared to be about 370nm in

mean diameter and are characterized to determine their zeta potential protein adsorption

and biocompatibility The image of SEM of PS and core shell particles is shown at Figure

4-7 The synthesized latex particles had a high uniformity in size and a smooth spherical

surface morphology

73

Figure 4-7 Scanning

Electron Micrograph of latex particles (A) PS (B) PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMMA75PAA25

(A)

(B)

74

(C)

(D)

Figure 4-7 Continued

75

42 Characterization of Latex Particles

421 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectroscopy has proven to be a powerful tool to characterize polymeric

materials A molecule can absorb only select frequencies (energy) of infrared radiation

which match the natural vibration frequencies The energy absorbed serves to increase

the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]

Only those bonds which have a dipole moment that changes as a function of time can

absorb infrared radiation The FTIR spectra of polymerized latex particles made during

this work are shown in Figure 4-8 There are number of characteristic peaks for PS latex

particles [Bhutto et al 2003 Li at al 2005] Aromatic C-H stretching vibration is shown

at 3002 cm-1 and 3103 cm-1 and aliphatic C-H asymmetrical and symmetrical stretching

shown at 2900 cm-1and 2850 cm-1 respectively There is an additional carbonyl (C=O)

adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some

amount of either or both PMMA and PAA The broad OH group peak at 3400 cm-1

appear for particles containing some amount of PAA and has intensity dependent on the

amount of PAA in the shell For example the peak intensity of OH groups for

PSPMMA75PAA25 is greater than that of PSPMMA90PAA10 since there is a higher AA

monomer content in PSPMMA75PAA25 than in PSPMMA90PAA10 This OH peak is not

seen for PS and PS core PMMA shell particles because of the absence of OH groups on

PS and PMMA polymer chains

422 Zeta Potential Measurements

The electrical surface property was characterized by zeta potential measurements of

tex particles Zeta potential is an important and useful indicator of surface charge which

can be used to predict and control the stability of colloidal systems The greater the zeta

is

la

76

1520002500300035004000

Wavenumbers (cm-1)

li

Re

ativ

e

Tra

nsm

ttanc

e

Figure 4-8 FTIR spectra of polymerized latex particles (A) ba

PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMM

(B)

(C) P

(D) P

C

C

OCH3

O

CH3

XCH2

CH2 n

CH

CH2 C

C

OCH3

O

XCH2 CH

OC

CH3

Y

OH

(A) Bare PS

500100000

re polystyrene (PS) (B) A75PAA25

PSPMMA100

SPMMA90PAA10

SPMMA75PAA25

77

potential the more likely the suspension is to be stable because the charged particles repel

one another and thus overcome the natural tendency to aggregate The measurement of

zeta potential is often the key ontrol of the interaction of proteins

with solid surfaces such as latex particles

Zeta potential (ζ) is the elec at exists at the shear plane of a particle

which is some small distance from the surface [Myers 1999] The zeta potential of the

particles can be determined by measuring the mobility of the particles in an applied

electric field termed electr its response to an alternating electric

field Colloidal particles dispersed in lu ically charged due to their ionic

characteristics and dipolar attributes The development of a net charge at the particle

surface affects the distribution of ions in the neighboring ng in

an increased concentration of counter ions to the surface Each particle dispersed in a

ns called fixed layer or Stern layer

Outside th compositions of ions of opposite polarities

form ble layer is form

particle-liquid interface This double layer may be considered to consist of two parts an

inner region which inclu nd relatively strongly to the surface and an outer or

diffuse region in which the ion distribution is determined by a balance of electrostatic

forces and random thermal motion The potential in this region therefore decays with the

distance from the surface until at a certain distance it becomes zero Figure 4-9 shows a

schematic representation of a typical ion distribution near a positively charged surface

The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the

to understanding and c

trical potential th

ophoretic mobility or

a so tion are electr

interfacial region resulti

solution is surrounded by oppositely charged io

e Stern layer there are varying

ed in the region of the ing a cloud-like area Thus an electrical dou

des ions bou

center of those ions which are effectively adsorbed to the surface [Hunter 1981] The

78

extent of ion adsorption is determined by electrical and other long-range interactions

between the individual ions and surface of particles The ions outside of the Stern layer

form the diffuse double layer also referred to as the Gouy-Chapman layer [Burns and

Zydney 2000]

Shear plane

Stern layer

ψζ

0

ψs

ψShear plane

Stern layer

ψζ

0

ψs

ψ

t

controlled electric field is applied via electrodes immersed in a sample suspension and

Figure 4-9 Schematic representation of ion distribution near a positively charged surface

Zeta potential is a function of the surface charge of a particle any adsorbed layer a

the interface and the nature and composition of the surrounding medium in which the

particle is suspended The principle of determining zeta potential is very simple A

79

this causes the charged particles to move towards the electrode of opposite polarity

Viscous forces acting upon the moving particle tend to oppose this motion and the

equilibrium is rapidly established between the effects of the electrostatic attraction and

the viscosity dra

g The particle therefore reaches a constant terminal velocity

Figure 4-10 Schematic representation of zeta potential measurement(source

httpnitioncomenproductszeecom_shtm)

Because protein adsorption mechanisms are very complex and hard to explain in

biological systems protein adsorption is generally studied by using more ideal systems

consisting of one or more well characterized proteins a well-characterized adsorbent and

a well-defined aqueous solution Even so small changes in the experimental conditions

such as pH ionic strength and temperature generate totally different results Therefore

two media systems phosphate buffer (PB) and phosphate buffered saline (PBS) were

chosen to be used for the zeta potential measurements and protein adsorption study PB is

a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4H2O) to

n a constant pH PBS is used to keep the water concentration inside and outside of

the cell balanced If the water concentration is unbalanced the cell risks bursting or

shriveling up because of a phenomenon called osmosis PBS is a solution with the 5 mM

of monobasic sodium phosphate (NaH2PO4H2O) and 143 mM of sodium chloride (NaCl)

in water

maintai

80

The results of zeta potential measurement at room temperature (25plusmn1oC) as

function of pH and ionic strength (different media PB and PBS) for synthesized lat

particles are seen in Figure 4-11 Zeta potential values of polystyrene (PS) latex particle

at the Figure 4-9 are negative between -291 mV and -599 mV in PB and betwe

ex

s

en -203

mV and -278 mV in PBS at all pH ranges These negative zeta potential values of PS

latex particles are due to the sulfate groups [Vandenhu et al 1970] originated from

persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer

chain where polymerization was initiated

-100

-80

-60

-40

-20

0

pH

Zta

pte

ntl

ζ

20

40

1 2 3 4 5 6 7 8 9

eo

ia (m

V)

PB (5mM)

PBS (5mM +143mM)

en -

Figure 4-11 Zeta potential of PS latex particles at 25oC

The zeta potential measurements of PSPMMA100 core shell latex particles as seen

in Figure 4-12 were also negative with a range of -28 and -505 mV in PB and betwe

143 mV and -186 mV in PBS at the pH 21-78 These negative values are also due to

sulfate groups on the particle surface

81

-100

-80

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

Zea

pote

nti

l ζ

(mV

pH

ta

)

PB (5mM)

PBS (5mM + 143mM)

Figure 4-12 Zeta potential of PSPMMA100 latex particles at 25oC

The zeta potential for PSPMMA90PAA10 and PSPMMA75PAA25 was also

negative as seen in Figure 4-13 and 4-14 The zeta potential value for the

PSPMMA90PAA10 were between -367 mV and -678 mV in PB medium and between -

147 mV and -193 mV in PBS both at 25oC In case of the PSPMMA75PAA25 core shell

particles zeta potential values were between -291 mV and -520 mV in PB and between

-115 mV and -210 mV in PBS media Sulfate groups in PSPMMA75PAA25 and

PSPMMA90PAA10 core shell particles contribute to the negative zeta potential values

participate in forming a

negat

cid is expressed by the term pKa which is the pH

at which an acid is 50 dissociated However the contribution of the carboxylic acid

Carboxylic groups from acrylic acid monomer also would

ive surface charge of the latex particles at a pH greater than 70 because the pKa

value for the carboxylic acid is between 40 and 60 pH range [Ottewill et al 1967] This

indicates that carboxylic acid dissociation begins at a pH between pH 46-60 and

increases with pH The strength of an a

82

group to negative zeta potential values of PSPMMA-PAA core shell particles was not

detectable because there was no significant difference in zeta potential values for

synthesized latex particles at a pH greater than 70

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

Zeta

pot

entia

l ζ

(mV

)

PB (5mM)PBS (5mM + 143mM)

-80

-100

pH

Figure 4-13 Zeta potential of PSPMMA90PAA10 latex particles at 25oC

-100

-80

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

pH

Ze p

ont

i ζ

V)

tate

al (m

PB (5mM)PBS (5mM + 143mM)

Figure 4-14 Zeta potential of PSPMMA75PAA25 latex particles at 25oC

83

The zeta potential plots and values were similar for all latex particles This may be

due to the similar initiator density on the latex surface The initiator concentrations

monomers for shell polymerization were the same fo

to

r all core shell latex particles (062

ww from Table 4-4) A similar concentration (055 ww) of initiator to monomer was

added to prepare bare PS latex particles used in zeta potential measurements

Since the zeta potential values of all synthesized latex particles were negative

between pH=20 and pH=78 the isoelecric point (IEP) of these latex particles would be

less than pH=20 Sulfates have a pKa in the range of 10 to 20 [James 1985] indicating

that the sulfate group the conjugate base of a strong acid is protonated (-C-OSO3H) at

less than pH 20 This means that the synthesized latex particles are all negative at the

physiological pH Most serum proteins have a negative surface charge in blood

therefore when the negative latex particles are applied to blood stream fatal

tein is

avoided by charge repulsion in the blood Table 4-5 shows the common blood proteins

and their isoelectric points (IEP)

Table 4-5 The isoelectric point (IEP) of common proteins Serum proteins Isoelectric point (IEP)

complication by coagulation between applied latex particles and serum pro s

Albumin 48 α1-Globulin 20 α2-Microglobulin 54 γ1-Globulin 58 Heptoglobin 41 Hemoglobin 72 β1-lipoprotein 54 Immunoglobulin G (Ig G) 73 Fibrinogen 58 (source httpwwwfleshandbonescomreadingroompdf1085pdf

httpwwwmedalorgvisitorwww5CActive5Cch135Cch13015Cch130105aspx )

84

The absolute zeta potential values of the latex particles in phosphate buffer (PB)

was higher than that in phosphate buffered saline (PBS) This is because high

concentration of sodium and chloride electrolytes in PBS compressed the electrical

double layer causing rapid decay of surface potential Figure 4-15 shows that the

thickness of the electrical double layer is inversely proportional to the concentration of

electrolyte in the system

Surf

ace

Pote

ntia

l ψ

1κ3

10

1e

1κ 1κ

(1)(3) (2)

2 1 Z

Surf

ace

Pote

ntia

l ψ

1κ3

10

1e

1κ 1κ

(1)(3) (2)

Figure 4-15 The decay of surface potential with distance from surface in various

electrolyte concentrations (1) low (2) intermediate (3) high [Myers 1999]

2 1 Z

The electrical potential in the solution surrounding the particle surface falls off

the Debye-Huckel

yers 1999]

exponentially with distance (Z) from the surface according to

approximation [M

z) (- exp 0 κψψ = (4-4)

e reciprocal of the thickness of the electrical double layer referred to as the

The potential fallen off by a factor e The theoretical equation for the

ickness 1κ is

where κ is th

Debye length of 1

double layer th

21

22i

0

ekT 1

⎟⎟⎠

⎜⎜⎝

=sum ii zcεε

κ

⎞⎛ (4-5)

85

where

tration of

y was performed to measure the protein adsorption on the

synthesized latex particles Many physical and chemical processes occur at the boundary

between two phases and adsorption is one of the fundamental surface phenomena [Oscik

1982] This phenomenon is driven by the change in the concentration of the components

at the interface with respect to their concentrations in the bulk phase There are two

adsorption processes physical adsorption occurring when non-balanced physical forces

appear at the boundary of the phases and chemical adsorption or chemisorption

t the

interface [J

n leads

ational entropy hence adsorption takes place only if the

loss in conformational entropy is compensated

ter adsorption Cp The initial part of the

isotherm merges with the Γ-axis because at low polymer concentration the protein has a

high affinity for the surface and all of the polymer is adsorbed until the solid surface is

saturated

ε0 is the permittivity of a vacuum of free space ε is the permittivity of a medium e

is the relative permittivity or dielectric constant of the medium c is the concen

an ion (i) z is the valency of an ion (i) k is Boltzmannrsquos constant T is absolute

temperature (K)

43 Protein Adsorption Study

The adsorption stud

occurring when atoms and molecules from adjacent phases form chemical bonds a

aroniec et al 1988]

Flexible proteins in solution possess high conformational entropy as a result of the

various states each of the many segments in the protein chain can have Adsorptio

to a reduction of this conform

by sufficient attraction between polymer

segment and solid surface [Martin 1998] Isotherm shape of high affinity adsorption

between polymer and solid is shown in Figure 4-16 where the adsorbed mass Γ is plotted

against the polymer concentration in solution af

86

Figur

de

arboxyl

e 4-16 High-affinity adsorption isotherm of typical flexible polymer on solid

surface

In order to investigate the adsorption of blood proteins latex particle properties

such as electrical surface charge hydrophobicity and environmental conditions like pH

ionic strength and temperature are considered as experimental factors [Arai and Nor

1990] Figure 4-17 shows the overall schematic representation of the protein adsorption

on the synthesized latex particles with the functional groups such as sulfate and c

groups

CP

Γ

PMMAPAAShell

PS or PMMAShell OSO3

-

OSO3-

OSO3-

OSO3-

PS

-O3SO

PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

Protein

NH3+

+

+H3N

+

COO-

Hydrophobic interaction

NH3

COO-

H3N

COO-

-OOC

Ionic interaction

Hydrophobic interaction

Hydrogen bonding

Ionic interaction

PMMAPAAShellOSO3

-

OSO3-

OSO3-

OSO3-

PS or PMMAShell -O3SOOSO3

-

PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

-O3SO

OSO3-

OSO3-

OSO3-

PS PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

Protein

NH3+

+

+H3N

+

COO-NH3

COO-

H3N

COO-

-OOC

NH3+

+

+H3N

+

COO-

Hydrophobic interaction

NH3

COO-

H3N

COO-

-OOC

Ionic interaction

Hydrophobic interaction

Hydrogen bonding

Figure 4-17 Overall schematic representation of the protein adsorption on the

Ionic interaction

synthesized latex particles

The surface functional groups as well as suitable surface hydrophobicity

hydrophilicity of latex particles are closely related to protein adsorption property As

indicated at Figure 4-17 various interaction forces such as hydrophobic interaction ionic

87

interaction hydrogen interaction and van der Waals interaction [Andrade 1985] exist

between protein molecule and the solid surface of latex particle

431 Adsorption Isotherm

The protein adsorption isotherm is generally defined as the relationship between the

amount of protein adsorbed by a solid (latex particle) at a constant temperature and as a

ine

the amount of protein adsorbed onto latex particles To evaluate the equilibrium

conce s

x

function of the concentration of protein Adsorption isotherms were plotted to determ

ntration of target proteins a calibration curve is firstly needed Figure 4-18 show

the UV absorbance as a factor of bovine serum albumin (BSA) concentrations The UV

absorbance of the known protein concentration was measured at 562nm which is the λma

for a protein treated by a bicinchoninic acid (BCA) assay

0

02

04

06

08

500 520 540 560 580 600

Wavelength (nm)

Abs

orba

nce

Figur gml (B)

06mgml (C) 02mgml

(A)

(C)

(B)

Max562nm

e 4-18 The dependence of UV absorbance on BSA concentration (A) 10m

Figure 4-19 shows the standard calibration curve used in adsorption isotherm

experiment of protein onto latex particles The net absorbance of each known protein

88

concentration was plotted and then a linear equation passing through a concentration of

zero protein concentration was obtained This equation is applied

to determine the

orbed on the synthesized latex particles The amount of free

protei

eported

trations (005 01 03 05

and 07mgml) were used to generate this curve as well as the adsorption isotherm

unknown protein amount ads

n was also determined using UV light at the 280nm wavelength however a

distinguishable UV intensity was too low to determine a precise value of protein

adsorption

The bicinchninic acid (BCA) assay technique [Smith et al 1985] has been r

for high efficiency of protein evaluation and was used for determination of protein

adsorption [Tangpasuthadol et al 2003 Williams et al 2003] This technique has many

advantages such as high sensitivity easy of use color complex stability and less

susceptibility to any detergents Five different BSA concen

y = 00R2 =

004741x 09939

00

01

02

03

04

0 200 400 600 800

BSA Standard (microgml)

Net

Abs

orba

nce

at 5

62nm

Figure 4-19 Standard curve of net absorbance vs BSA sample concentration

89

All isotherms were fitted to the Langmuir-Freundlich isotherms using the nonlinea

regression method Two adsorption isotherm models the Langmuir type isotherm a

Langmuir-Freundlich combination type isotherm were compared As seen the Figure 4-

20 The Langmuir isotherm model deviated further from the experimental data Th

all isotherms were fitted to the Langmuir-Freundlich isotherms [Yoon et al 1996

al 1998] usi

r

nd the

erefore

Lee et

ng the nonlinear regression method with following equation

n

n

eq

eqm

kC

kC1

1

1+

where q is the amount of adsorbed BSA per unit surface area q

qq = (4-6)

stant

were between 045 and 055 and hence n was fixed to its average 05 Actual qm values

were evaluated after fixing the n value

m is the adsorbed amount

in equilibrium Ceq is the equilibrium concentration of BSA k is the adsorption con

and n is the exponential factor for a heterogeneous system n values are empirical and

usually from zero to no more than unity [Jaekel 2002] The values of n in our system

00

10

20

30

0 02 04 06 08

C (mgml)eq

q (m

gm

2 )

pH 32Langmuir-Freundlich modelLangmuir model

Figure 4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on

polystyrene latex particles at 37oC in phosphate buffer media

90

The BSA protein adsorption on the synthesized latex particles was performed at

three pH levels 32 48 74 in both phosphate buffer (PB) and phosphate buffered saline

(PBS) at physiological temperature 37oC These pH levels correspond to an acidic pH

lower than the isoelectric point (IEP) of BSA the pH at the IEP and an alkaline pH

higher than the IEP of BSA respectively The IEP corresponds to the pH at which the

amino acid becomes a zwitterion Here its overall electrostatic charge is neutral and the

charge expressed by the basic amino group (-NH2 -NH3+) of the amino acid is equal to

the charge expressed by the acidic carboxyl group (-COOH -COO ) The IEP of BSA is

-

pH 48 and indicates that the side chain of the amino acids of BSA contains a greater

amount of acidic functional groups than that of basic functional groups At pH 32 and

74 BSA has a positive and negative charge respectively

Figure 4-21 and 4-22 show adsorption isotherms of BSA onto polystyrene (PS)

latex particles

00

10

20

30

40

0 02 04 06 08

Ceq (mgml)

q (m

gm

2 )

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in

phosphate buffer (PB) at 37oC

91

00

10

30

40

0 02 04 06 08

Ceq (mgml)

q (m

gm

2

20

)

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

(PBS) on PS latex particles

At the IEP of BSA the adsorbed amount (q

Figure 4-22 Adsorption isotherm of BSA adsorption 37oC in phosphate buffered saline

onto PS at equilibrium was

maxim

um at the IEP of BSA This is because the BSA molecules form the most

compact structures at the IEP eliminating the repulsion force between proteins A higher

number of BSA molecules can adsorb on a given surface area by hydrophobic interaction

when in this compact structure This observation agrees well with reported data by other

researchers who also indicated that the maximum adsorption from aqueous protein

solution was observed at the IEP [Mularen 1954 Watkins et al 1977 Oh at el 2000]

There was no significant difference of adsorbed BSA amount in PB and in PBS media at

pH 48 The conformational alteration of BSA was not affected significantly by the ionic

strength of the two solutions There was evidence of the effect of the electrostatic

interaction to some extent even at the IEP since the amount of BSA adsorbed in PBS is

slightly hig

m) of BSA

um indicating that hydrophobic interaction between latex particle and BSA

protein is maxim

her than in PB at this pH

92

The adsorbed amount of protein is shown to decrease at a pH 32 and 74 The

decrease in adsorption efficiency when deviating from IEP of BSA is a result of the

increase in conformational size of the protein molecules and the lateral electrostatic

repulsions between adjacent adsorbed BSA molecules Figure 4-23 shows a schematic of

possible conformational changes at each pH where hydrophobic interaction always exists

between non-polar portion of a protein and hydrophobic portion of solid surface

- - --- ---Latex surface

Zeta potential

BSA(+)

(-)

48 7432pH

(0)

+++ + ++

--+

++-

- - --- ---Latex surface

Zeta poten pH(0)

+++ + ++

--

tial

BSA(+)

(-)

48 7432

+

++-

ming

4

s

al blood Table 4-6 shows the calculated results of

Figure 4-23 Conformation of bovine serum albumin (BSA) on latex particles (Assuthat charge repulsion reduces the effect of the hydrophobic interaction at pH 74)

In addition the adsorbed amount of BSA at pH 32 was larger than that at pH 74

and still less than that at 48 This is because there is the electrostatic interaction between

the positive BSA molecules and negative PS latex particles at pH 32 and electrostatic

repulsion is dominant between negative BSA and negative PS latex particles at pH 7

The amount adsorbed increased with increasing ionic strength in pH 32 and 74 This i

because the high concentration of electrolytes reduces the electrostatic repulsion between

BSA and PS latex particle It should be noted that the BSA concentration used in these

experiments was lower than that found in normal human blood This is because the

equilibrium BSA adsorption onto the latex particles is actually reached at a much lower

concentration than that found in norm

93

adsor rm

ein

leading to a compact conformation of BSA There are two main forces affecting

adsorption amount at an acidic pH of 32 They include the ionic attraction between the

negative latex particle and the positive protein and lateral repulsion between positive

proteins The smallest amount of BSA adsorption was observed at a pH of 74 At this

point the charge repulsion between negatively charged particles and protein molecules

exists the h

of cha

bed BSA amount at equilibrium from the Langmuir-Freundich adsorption isothe

equation

In summary the hydrophobic interaction between PS latex particle and BSA

protein exists and is the dominant mechanism for the adsorption process Conformational

changes of BSA at each pH also affected the adsorption amount Adsorption was highest

at pH 48 because at this pH there is the least amount of ionic repulsion in the prot

and the lateral repulsion between proteins dominant adsorption even though there always

ydrophobic interaction between the solid latex particle and protein The effect

rge repulsion was reduced by the high concentration of electrolytes in PBS medium

Table 4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2) 32 174 48 299 Phosphate buffer 74 136 32 260 48 283 Phosphate buffered saline 74 207

The adsorption isotherms of BSA on PSPMMA core shell particles are shown in

Figure 4-24 in phosphate buffer (PB) and in Figure 4-25 in phosphate buffered saline

(PBS) The adsorbed BSA amount was calculated using the Langmuir-Freundlich

100

94

adsorption isotherm equation and the amount listed in Table 4-7 The trend seen in the

adsorption results was very similar to the case of PS latex The overall adsorption process

was d rticle

rs

hat

0

ominated by hydrophobic interactions between the PSPMMA100 core shell pa

and BSA proteins At pH 48 the adsorbed amount (qm) of BSA on PSPMMA100 core

shell particles was 221 mgm2 in PB and 237 mgm2 in PBS and are the maximum

values recorded among the three pH levels 32 48 and 74 Suzawa and his co-worke

[1982] have obtained similar results This can be also explained by the compact

conformation of BSA molecules at the IEP of BSA which lead to a high protein

population on the solid surface At a pH of 32 and 74 BSA adsorption was less than t

at pH 48 because of the protein conformation change There was a small increase in

adsorbed BSA at pH 32 because of the charge attraction between negative PSPMMA10

core shell particles and positive BSA At pH 74 however the adsorption of BSA was

further reduced by charge repulsion between the negative PSPMMA100 core shell

particles and the negative BSA proteins

000 02 04 06 08

Ceq (mgml)

10

20q (m

gm

302 )

40

50pH=32pH=48pH=74Mode 8)l (pH 4Mode 2)l (pH 3Mode 4)

Figure 4-24 Adsorption isotherm of BSA 37oC in PB on PSPMMA core shell latex

l (pH 7

100

particles

95

The adsorbed amount of BSA increased more rapidly in PBS with increasing ioni

strength at pH 74 This shows that the low concentration of electrolytes promotes

electrostatic repulsion between negative BSA and negative PSPMMA

c

on

x

l 1980

100 core shell

particle As the ion strength of PBS is increased however the high concentration of

electrolyte leads to less electrostatic repulsion promoting more rapid protein adsorpti

By comparing the amount of adsorbed protein between bare PS latex particles and

PSPMMA100 core shell particles the amount of protein adsorption on the PS late

particles is greater than that on the PSPMMA100 core-shell particles [Suzawa et a

1982 Lee et al 1988]

00

10

20

30

40

m2 )

0 02 04 06 08

Ceq (mgml)

q (m

g

pH=32pH=48pH=74Model (pH 2) 3Model (pH 48)Model (pH 74)

Figure 4-25 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA100 core shell

latex particles

96

Table particles calculated the Langmuir-Freundlich isotherm model

4-7 The equilibrium concentration values (qm) of BSA adsorption on PSPMMA100

Media pH qm (mgm ) 2

32 140

48 221 Phosphate buffer

74 130

32 169

48 237 Phosphate buffered saline

74 162

The BSA adsorption mechanism and trend on PSPMMA core shell particles was

very similar to that of the PS latex particle Unlike the case of PS and PSPMMA100 latex

particles the amount of BSA adsorbed onto PSPMMA90PAA10 and PSPMMA75PAA25

core shell particles was higher at pH 32 than pH 48 and 74 Figure 4-26 through 4-29

shows the adsorption isotherms of BSA on PSPMMA90PAA10 and PSPMMA75PAA25

core shell particles The amount of BSA adsorbed increased at pH 32 and pH 48 as the

hydrophilicity was developed by the addition of PAA On the contrary the adsorbed

amount of BSA was dramatically decreased at pH 74 The adsorption amount of BSA on

PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles at equilibrium is listed in

Table 4-8 and 4-9 respectively

Carboxylic acid as well as sulfate groups are distributed on the surface of

PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles and seem to largely affect

the BSA adsorption The isotherms showed very strong pH dependence to the BSA

adsorption onto these core shell latex particles The carboxylic groups (COOH) may

especially enhance the BSA adsorption prominently at pH 32 by hydrogen bonding with

protein molecules The pKa value for the PAA is 40-60 and a good indication that the

97

carboxylic acid groups are protonated at pH 32[Gebhardt et al 1983] The main

adsorption interaction force at pH 32 was generated by hydrogen bonding as well as

charge attraction between latex particle protein even th the conformational

repulsion between proteins results in a reduction of the protein

adsorption at this pH The amount of BSA adsorbed also increased as the PAA amount in

eased The a tion of BSA w inimal however less

than 07 mgm2 at pH 74 Here the carboxylic groups are ionized which increases the

charge repulsion by ionized carboxylic groups (COO-) resulting in PSPMMA-PAA latex

particle

and ough

change and charge

the core shell particles incr dsorp as m

s with much lower protein adsorption

00

20

40

q (m

g2 )

60

m

pH=32pH=48pH=74Model (pH 32)Model (pH 48)

0 02 04 06 08

Ceq (mgml)

Model (pH 74)

o shell

Figure 4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA90PAA10 corelatex particles

98

00

40

60

80

0 02 04

C (mgm

mm

2 )

20

06 08

eq l)

q (

g

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-27 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA90PAA10 core

shell latex particles

Table 4-8 The equilibrium concentration values of BSA adsorption on PSPMMA90PAA10 particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2)

32 361

48 283 Phosphate buffer

74 043

32 519

48 226 Phosphate buffered saline

74 043

99

00

20

40

60

80

100

0 02 04 06 08Ceq (mgml)

q (m

gm

2 )

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

oFigure 4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA75PAA25 latex particles

00

100

20

40

60

80

0 02 04 06 08Ceq (mgml)

q (

m2 )

pH=32pH=48pH=74

mg

Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-29 Adsorption isotherm of BSA 37oC in PBS on PSPMMA75PAA25 core shell

latex particles

100

Table 4-9 The equilibrium concentration values of BSA adsorption on PSPMMA75PAA25 particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2)

32 1083

48 662 Phosphate buffer (PB)

74 075

32 1077

48 681 Phosphate buffered saline (PBS)

74 057

The adsorption of β2-microglobulin (β2M) was determined under one set of

onditions in phosphate buffer at 37oC and pH 74 because of the limited amount of β2M

protein The adsorption isotherms of the β2M protein on the synthesized latex particles

are shown in Figure 4-30 4-31 and 4-32 The β2M protein concentrations for adsorption

isotherm experiment were chosen to be 0015 0030 0045 and 0060mgml since the

β2M concentration for the patients with renal failure can be elevated to similar

concentrations about 005mgml or more up to 50 times the normal level [Nissenson et

al 1995] There were steep initial slopes of the adsorption isotherm curves of β2M on

latex particles in all isotherms indicating a high affinity adsorption type It seems that the

hydrophobic interaction is enough for the adsorption process of β2M proteins to occur on

the latex particles even though charge repulsion exists between protein and latex particle

at pH 74 The complete plateau regions were not seen for all isotherms indicating that

β2M proteins were not saturated at this level and there are still available sites for β2M

c

adsorption on latex particles The initial slope of the β2M isotherms for PS was steeper

101

than that of any of the other latex particle PSPMMA100 PSPMMA90PAA10 and

PSPMMA 75PAA25

00

02

0 0002 0004 0006 0008

04

06

10

Ceq (mgml)

q (m

2 )

08

gm

PSPSPMMA100Model (PS)Model (PSPMMA100)

Figure 4-30 Adsorption isotherm of β2M onto PS and PSPMMA100 latex particles in PB

at 37 C and pH 74 o

00

02

04

06

08

10

0 0002 0004 0006 0008C (mgml)

q (m

m2 )

eq

g

PS

PSPMMA90PAA10

Model (PS)

Model (PSPMMA90PAA10)

Figure 4-31 Adsorption isotherm of β2M onto PS and PSPMMA90PAA10 latex particles

in PB at 37oC and pH 74

102

00

02

04

06

08

10

0 0002 0004 0006 0008Ceq (mgml)

q (m

gm

2 )

PS

PSPMMA75PAA25

Model (PS)Model (PSPMMA75PAA25)

Figure 4-32 Adsorption isotherm of β2M onto PS and PSPMMA75PAA25 latex particles

in PB at 37oC and pH 74

The equilibrium concentration values (qm) of β2M adsorption on latex particles are

calculated and listed in Table 4-10 The difference between the maximum amounts of

β2M adsorption on all the latex particles was not significantly different and their values

were between 069 and 08mgm2

Table 4-10 The equilibrium concentration values of β2M adsorption calculated the Langmuir-Freundlich isotherm model

Latex particles qm (mgm2)

PS 080

PSPMMA100 069

PSPMMA90PAA10 072

PSPMMA75PAA25 072

103

432 Adsorbed Layer Thickness

There are several possible explanations for large amount of BSA adsorbed at acidic

pH levels including the end-on adsorption pattern of BSA the flexibility of BSA

molecules the tilting of protein molecules due to the asymmetry of the charge

distribution or multilayer formation [Peula et al 1993] The adsorbed BSA thickness (δ)

can be calculated by the following equation [Chiu et al 1978] This equation was also

cited again by other researchers [Shirahama et al 1985]

BSA

mqρπ

δbullbull

=33

(4-7)

where π

33 is the packing factor qm is the plateau value of the adsorbed am

ρBSA is the hich corresponds to the reciprocal of its known

partial specific volume The layer thickness (δ) of BSA proteins on synthesized latex

partic

he IEP [Shirahama et al 1985] Compared to the

hydrodynamic dimensions of BSA 140 times 40 times 40 Aring3 [Fair et al 1980 Peter 1985] these

nd

ode It is unnecessary ultilayering because thickness do

exceed 140 Aring The layer thickness (δ) of BSA proteins on synthesized latex particles

e red saline (PBS) was ran 28 ~ 83 Aring at 37oC and pH 48 These

lso listed in Table 4-1

ount and

density of BSA molecule w

les in phosphate buffer (PB) ranged from 27 - 81 Aring at 37oC and pH 48 These

calculated values are listed in Table 4-11 Only δ values at pH 48 the isoelectric point

(IEP) of BSA are important because intra- and intermolecular electrostatic repulsion of

protein molecules is minimized at t

values of layer thickness indicate that the BSA molecules adsorb by side-on (40Aring) a

end-on (140Aring) m to consider m

not

in phosphat buffe ged of

calculated values are a 2

104

Table 4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37oC pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

32 21 17 44 133

48 37 27 35 81

74 17 16 5 9

Table 4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37oC

pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

32 32 21 64 132

48 35 29

74 26 20 5 7

28 83

1n

1n

neqm

So k is closely related to the initial slope of the isotherm showing the affinity

between BSA and latex particles [Yoon et al 1996] A key point in the characterization

433 Gibbs Free Energy of Protein Adsorption

In the Langmuir isotherm model k represents the ratio of adsorption to the

desorption rate constants This definition is not applicable to the Langmuir-Freundlich

isotherm model but its meaning is similar [Yoon et al 1996] In this sense k is closely

related to the affinity between protein molecules and latex particles When the

equilibrium concentration Ceq reaches zero the value of Ceq will be very small (1gtgt

kCeq ) and then the equation 4-6 becomes

(4-8)

of adsorption processes on solid-liquid interfaces is to determine the change in Gibbsrsquos

free energy (∆Gdeg) one of the corresponding parameters for the thermodynamic driving

forces during adsorption The traditional method to evaluate adsorption thermodynamic

kCqq 1=

105

constants is based on measurement of adsorption isotherms under static conditions

[Malm ten 1998] The Langmuir-Freundlich ads lf

easily found from Then this enables calculation of the free-

energy change ∆G during adsorption expressed as

(4-9)

where R is the general gas constant (R = 8314Jmiddotmol K ) and T is the absolute

tem ture Gibb e energy A P

listed in Table 4-13 and 4-14 respectively As the ∆Gdegads ore negativ e

pro adsorption on a solid substra ill be more favorable

e of BSA adsorption in phosphate o

pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

s orption equilibrium constant k can be

a slope of the isotherm

degads

lfads kRTG lnminus=∆ o

-1 -1

pera s fre change of BS adsorption values in B and PBS are

values are m e th

tein te w

Table 4-13 The values of Gibbs free energy changbuffer (PB) at 37 C

32 -2411 -2726 -2846 -2805

48 -2352 -2383 -2198 -1972 ∆Go

80

ads (kJmol)

74 -2452 -888 -324 -2

Table 4-14 The values of Gibbs free energy change of BSA adsorption in phosphate

pH PS PSPMMAbuffered saline (PBS) at 37oC

100 PSPMMA90PAA10 PSPMMA75PAA25

32 -2315 -2620 -2312 -2683

48 -2149 -2299 -2090 -2138 ∆Go

(kJmads ol)

74 -2430 -1992 -258 -263

Table 4-15 shows the calculated values of Gibbs free energy change of β M

adsorption on synthesized latex particles PSPMMA PAA core shell latex particle has

2

75 25

106

largest negative Gibbs free energy of β2

2

M adsorption indicating largest affinity betw

β

een

2M adsorption in phosphate buffere (PB) at 37 C and pH74

PS PSPMMA100

M and latex particle

Table 4-15 The values of Gibbs free energy change of βo

PSPMMA90PAA10 PSPMMA75PAA25

∆Go03 ads

(kJmol) -3626 -3466 -3659 -39

The Gibbs free energy of β2M was much more negative in phosphate buffer (PB)

than that of BSA in physiological condition (37 C and pH 74) o from the Table 4-13 and

the Figure 4-31 This indicates that β2M molecules more readily adsorb on latex particles

BSA This can be explained by the rate of diffusion and accessibility of β2M molecules to

the latex particle ier and faster for β2M m an BSA because of

size (molec ight) erical shape molecules It o be

due to the higher hydrophobic force and less electrostatic repulsion that contribute to the

adsorption of β2M molecules not as prevalent in the case of BSA because the IEP of β2M

(57) is clo H 74) than that of BSA (48) causing β2M to

be less electro cally lled p ro

can be seen that the ∆ β2M adsorption onto the synth article

increasing wi hy icity o x particles s the ∆Goads of the BSA

molecules is decreasing or constant This phenomenon supports the theory that the

select lizing

than BSA and that the affinity of β2M for the latex particle is much larger than that of

surface It is eas olecules th

the smaller ular we and sph of β2M may als

ser to physiological condition (p

stati repe from the latex articles than BSA F m Figure 4-33 it

Goads of esized latex p s is

th the drophil f the late wherea

ive adsorption of β2M molecules over BSA molecules can be enhanced by uti

more hydrophilic PSPMMA90PAA10 and PSPMMA75PAA25 core shell latex particles

107

-60

-50

1 2 3 4

-40∆Gad

s

-30

10

0

o (k

Jm

o-

-20l)

β2-Microglobulin (β2M)Bovine serum albumin (BSA)

Figure 4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37

and pH 74

434 Kinetics of Adsorption

to

oC

Protein adsorption is a complex process known to have a large biological impact

and is currently not well understood quantitatively because of extreme sensitivity to pH

the concentration of other electrolytes and molecules present temperature and many

other factors that can change in the physiological system Accurate knowledge of the

adsorption kinetics under a given set of conditions is a prerequisite for elucidating the

mechanisms of many fundamental biological processes to be predicted at the molecular

level [Regner 1987] This adsorption kinetics approach is used in protein adsorption

studies because of the uncertainty related to the time needed for the interfacial proteins

reach the equilibrium It is generally accepted that the process of protein adsorption is

comprised of the following steps a) transport toward the interface b) attachment at the

interface c) eventual structural rearrangements in the adsorbed state d) detachment from

PS PSPMMA100 PSPMMA90PAA10PSPMMA75PAA25

108

the interface and transport away from the interface Each of these steps can affect the

overall rate of the adsorption process

The quantitative analysis of the protein adsorption kinetics requires that the protein

amount adsorbed is known as a function of time The kinetics tests were carried out in

phosphate buffer (PB) at 37oC and pH 74 a condition similar to physiological condition

The protein concentrations chosen were 07mgml for BSA and 006mgml for β2M

which are the maximal concentrations used in an adsorption isotherm test A time-based

evaluation of the adsorption was made by measuring the concentration of protein in

solution at different incubation times The results are shown below from Figure 4-34 to 4-

37 The adsorption kinetics of proteins onto th cond-e latex particles is expressed as a se

order model [Oumlzacar 2003] shown by the following equation

2)( tet qqk

dtdq

minus= (4-10)

Integrating above equation and applying the boundary conditions yields

ee

t tq

+=

1 (4-11)

where q

qkq

t

2

2

2

the previous adsorption isotherm results representing good reproducibility of adsorption

t and qe stand for the amount (mgm ) of protein adsorbed at time t and at

equilibrium respectively k is the equilibrium rate constant of second-order adsorption

(m mghr) and t is the incubation time (hr)

The fitting parameters calculated by using the equation 4-11 are listed in table 4-16

From this data it is seen that the amount of BSA adsorbed at equilibrium was higher than

that of β2M on PS and PSPMMA100 latex particles but less than that of β2M on

PSPMMA90PAA10 and PSPMMA75PAA25 latex particles These results are similar to

109

00

05

15

20

0 2 4 6 8 10 12 14

Time (hr)

rot

n ad

sorb

d (

10

25

Pei

em

gm

2

BSA2nd order model (BSA))β2M2nd order model (β2M)

Figure 4-34 The kinetics of protein adsorption in PB on PS latex particles at 37oC and

pH 74

00

05

10

15

20

25

30

0 2 4 6 8 10 12 14

Time (hr)

Prte

inds

oo

arb

ed (

mg

m2 )

BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-35 The kinetics of protein adsorption in PB on PSPMMA100 latex partic

37les at

oC and pH 74

110

00

03

06

09

12

15

0 2 4 6 8 10 12 14

Time (hr)

Prot

ein

adso

rbed

(m

gm

2 ) BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-36 The kinetics of protein adsorption in PB on PSPMMA90PAA10 latex

particles o at 37 C and pH 74

00

03

06

09

12

15

0 2 4 6 8 10 12 14

Time (hr)

BS

A ad

sorb

ed (

mg

m2 )

BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-37 The kinetics of protein adsorption in PB on PSPMMA75PAA25 latex

particles at 37oC and pH 74

111

test The variation of β2M equilibrium adsorption amount was nearly negligible for all

latex particles The plateau region is reached earlier for β2M than BSA on all latex

particles and the K values (equilibrium rate constant of second order adsorption) for the

β2M adsorption process onto latex particles are much larger than for BSA adsorption

These values indicate that the β2M molecules have a higher adsorption rate onto the latex

particles than the BSA molecules

Table 4-16 Fitting parameters of second-order kinetic model BSA β2M

Latex particles qe (mgm2) K (hr-1) qe (mgm2) K ( -1) hr

PS 144 67 074 7549

PSPMMA100 175 353 076 4438

PSPMMA90PAA10 046 97 075 5695

PSPMMA75PAA25 060 179 075 5168 K = (kqe) hr-1

There are several explanations for the phenomena observed in these experiments

First the smaller sizes of β2M molecules allow easier diffusion and a close approach to

the latex substrate Second a reduced number of hydrophobic domains on the latex

particle surface replaced by sulfate groups (-OSO3-) and ionized carboxylic groups

(COO-) are not favorable for larger BSA molecules Third the conformational change by

intra- and inter-molecular repulsion would be faster in β2M due to smaller molecular

weight Finally the ionic repulsion between β2M and the latex particles is less than that

condition (pH 74) than IEP (48) of BSA

between BSA and latex particles because IEP (57) of β2M is closer to the tested

112

44 Blood Compatibility

Blood compatibility can be defined as the property of a material or device tha

permits it to function in contact with blood without inducing any adverse reactions

Successful functioning of blood contacting biomedical materials or devices in contact

with blood strongly depends on the extent to which they damage blood during operation

High shear stresses turbulence rela

t

tively long contact times and cellular impact with

artificial surfaces are most common events experienced by blood in contact with artificial

1978 Winslow and et al

1993] Therefore before any materia e placed into the human b

biocompatibility testing must be the first performed in the view of safety The hemolysis

s a method for pr ary screening of biomaterials to determine

h blood a er circulating body fluid The tests estimat

oglobin released from th blood cell d e induced

foreign material

There are several types of blood cells in our body but the ones that make blood red

are erythrocytes or red blood cells (RBCs) Red blood cells (erythrocytes) are the most

numerous type in the blood These cells are average about 48-54 million per cubic

millimeter (mm3 same as a microliter (microl)) of blood but these values can vary over a

larger range depending on such factors as health and altitude RBCs are red because they

contain hemoglobin which is a protein that contains iron When blood circulates through

lungs oxygen binds to the hemoglobin and is then delivered to all the parts of body

RBCs are made in the bone marrow They start out as a nucleus like other cells but

when they fill up with hemoglobin the nucleus becomes smaller until it disappears

Without a nucleus RBCs are fragile and live only about 120 days In a normal healthy

devises and can cause hemolysis or thrombosis [Bernhard et al

ls can b ody

test serves a elimin the

biocompatibility wit nd oth e the

concentration of hem e red amag by a

113

body about 2 million red blood ne marrow produces new

ones j ing

dy

le

cells die per second But bo

ust as fast RBCs are about 75 micrometer across and are responsible for carry

oxygen salts and other organic substances to the cells and tissues throughout our bo

In humans and most mammals the cells are concave (indented in the middle) and flexib

enough to squeeze through even the tiniest of blood vessels the capillaries

Figure 4-38 Image of red blood cells

(Sourcehttpwwwpbrchawaiiedubemfmicroangelarbc1htm)

ch

100 90 10 75 25 f

RBCs are among the first cells that come into contact with a foreign material

injected in the blood Therefore a hemolysis assay gives information about the

biocompatibility in the case of an in vivo application The concentrations of prepared

latex particles used for the tests in this research are 05 25 and 50 (ww) and the

incubation time under moderate shaking was 30 minutes at 37oC Test results are shown

in Figure 4-39 The amount of blood cells that ruptured when in contact with the PS latex

particles was largely increased from about 30 to 91 as the solid content of PS

increased from 05 to 50 However hemolysis by the core shell latex particles su

as PSPMMA PSPMMA PAA and PSPMMA PAA was less than 35 O

114

particular interest was that hemolysis caused by PSPMMA PAA particles at 50

solid concentration was less than 02 hemolysis the lowest value among the latex

particles The low hemolysis value indicates that the PSPMMA PAA core shell latex

particles are the most blood compatible at all solid contents Other core shell particles

such as PSPMMA and PSPMMA PAA also had excellent blood comp

90 10

90 10

100 75 25 atibility with

low RBS breakage

01 2 3

20

40

60

80

100

Hem

olys

is (

)

052550

Figure 4-39 He

PS PSPMMA100 PSPMMA PAA90 10

molysis caused by latex particles n=5

4PSPMMA75PAA25

CHAPTER 5 SUMMARY OF RESULTS CONCLUSIONS AND RECOMMENDATION FOR

FUTURE WORK

51 Summary of Results

The goal of this study was to synthesize monodisperse polymeric latex particles

with tailored properties and to investigate the fundamental interactions between the

synthesized latex particles and target proteins An understanding of the fundamental

mechanism of selective adsorption is strongly required in order to maximize the

separation performance of membranes made of these materials so that it may applicable

to hemodialysis therapy for end stage renal disease (ESRD) patients For the achievement

of above research goal several investigations have been performed Analysis of the

obtained data leads to several conclusions as discussed below

Monodisperse polystyrene seed latex particles were synthesized with the size

crosslinking agent to make the latex particles more hydrodynamically stable The

conversion of monomer to polymer was over 95 and seed latex particles are spherical in

shape and have smooth surface From these small seeds bigger latex particles as large as

800nm were synthesized using a semi-continuous seeded emulsion polymerization

process The particles with a mean diameter less than 500nm are very spherical in shape

and highly uniform in particle size distribution However as the particles grow larger

than 500nm in mean diameter they became non-spherical with an uneven surface This

irregularity of particle surface can be attributed to the non-homogeneous monomer

ranges between 126plusmn75 and 216plusmn53 nm Divinylbenzene (DVB) was used as a

115

116

swelling into the shell of a growing polymer which can be controlled by the factors such

as temperature agitation speed initiator feeding rate and surfactant amount required to

stabilize the colloidal system It was determ rfactant to monomer ratio

dec ic

acid (AA) monomers were intro rticle surfaces more

hydro

to

ting of a

orm

ration is seen between

3002

0 cm-

0cm-1

-1

03

ined that as the su

reased the mean particle diameter increased Methyl methacrylate (MMA) and acryl

duced to make the latex pa

philic than bare polystyrene particles The prepared core shell latex particles were

made of PMMA100 PMMA90PAA10 and PMMA75PAA25 PMMA100 is the PMMA

homopolymer shell on a PS core PMMA90PAA10 shell is a copolymer with a PMMA

PAA ratio of 90 to 10 by weight PMMA75PAA25 shell is a copolymer consis

PMMA to PAA ratio of 75 to 25 by weight The particle size of these core-shell

particles were prepared to be about 370nm in mean diameter

The successful synthesis of latex particles was confirmed using Fourier Transf

Infrared spectroscopy (FTIR) The C-H aromatic stretching vib

cm-1 and 3103 cm-1 for PS latex particles The C-H asymmetrical stretching and

symmetrical stretching vibration peaks of CH2 for all latex particles is seen at 290

1and 2850 cm-1 respectively There is a carbonyl (C=O) characteristic band at 173

wavenumber for PSPMMA core-shell latex particles The broad OH group peak at

3400 cm

100

can be seen in the spectroscopy of the particle with PMMAPAA shell

(PSPMMA PAA and PSPMMA PAA ) The peak intensity of OH group of

PSPMMA PAA was larger than that of PSPMMA PAA relatively due to more AA

monomer content in PSPMMA PAA than in PSPMMA PAA

In the zeta potential measurements polystyrene (PS) latex particles had negative

values between -291 mV and -599 mV in the phosphate buffer (PB) and between -2

90 10 75 25

75 25 90 10

75 25 90 10

117

mV and -278 mV in the phosphate buffered saline (PBS) at 25oC and pH 21-78 These

negative zeta potential values for PS latex particles are due to ionization of the sulfate

groups (-OSO3-) originated from the initiator Zeta potential values of the PSPMMA

core shell latex particles were also negative between -28 mV and -505 mV in PB and

between -143 mV and -186 mV in PBS at pH 21-78 Negative values are also

generated by dissociation of sulfate groups on a particle surface The zeta potential value

of PSPMMA PAA and PSPMMA PAA were of negative values between -36

and -678 mV in PB medium and -147 mV and -193 mV in PBS medium at 25

100

s

7 mV

MMA75PAA25 and

PSPM

otein

g the

l change of BSA at

each w

90 10 75 25

oC For

PSPMMA PAA core shell particles zeta potential values were between -291 mV and

-520 mV in PB media and between -115 mV and -210 mV in PBS media Sulfate

groups contributed to the negative zeta potential values in PSP

75 25

MA90PAA10 core shell particles Carboxylic groups from polyacrylic acid also

contribute to the negative surface charge for the latex particles at greater than pH 70

however the degree of their contribution was not detectable from the zeta potential

measurements The absolute zeta potential values of the synthesized latex particles in PB

were higher than that in PBS This is because of the high concentration of sodium

chloride electrolyte in PBS that compress electrical double layer thickness

The bicinchoninic acid (BCA) assay technique was used to determine the pr

adsorption and all isotherms were fitted to the Langmuir-Freundlich isotherms usin

nonlinear regression method The hydrophobic interaction between PS latex particle and

BSA protein was dominant for the adsorption process Conformationa

pH also affected the adsorption amount which was highest at pH 48 because of lo

ionic repulsion making compact conformation of BSA The next larger amount of BSA

118

adsorbed onto PS was at pH 32 There were two main forces affecting the amount of

adsorption at acidic pH values One is ionic attraction between negative latex particle a

positive protein The other is lateral repulsion between positive proteins The smallest

amount of BSA adsorption was at pH 74 where charge repulsion between negative

particles and proteins is present at this pH Lateral repulsion between proteins wa

dominant for protein adsorption even though there always exists the hydrophobic

interaction between solid latex particle and protein The charge repulsion was reduced by

the high concentration of electrolytes in the PBS medium The BSA adsorption process

on PSPMMA core shell particles was very similar to the case of PS latex particle where

hydrophobic interaction between latex particle and protein was dominant However t

adsorbed BSA amount was less than that of PS in PBS The adsorbed amount of BS

PSPMMA

nd

s

he

A on

an

ed

n The main

adsor

90PAA10 and PSPMMA75PAA25 core shell particles was higher at pH 32 th

pH 48 and 74 The adsorbed BSA amount became higher at pH 32 and pH 48 as the

hydrophilicity was increased by the higher PAA amount On the contrary the adsorb

amount of BSA was dramatically decreased at pH 74 Carboxylic acid groups as well as

the sulfate groups are distributed on the surface of PSPMMA90PAA10 and

PSPMMA75PAA25 core shell particles and largely affect the BSA adsorptio

ption interaction forces at pH 32 was generated by hydrogen bonding as well as

charge attraction between latex particle and protein even when the conformational change

and charge repulsion between proteins reduce the adsorption of protein at this pH The

adsorbed BSA amount was also increased as the amount of PAA increases However the

adsorption of BSA was minimal (less than 07 mgm2) at pH 74 where the carboxylic

groups are ionized leading to more enhancement of charge repulsion by ionization of the

119

carboxylic groups (COO-) Because of this the PSPMMA-PAA latex particles have a

much lower protein adsorption

There were steep initial slopes for adsorption isotherms of β

in

s

there

M

r

e

es

o

2M on latex particles

all isotherms indicating a high affinity type adsorption The complete plateau region

were not seen for all isotherms indicating that β2M proteins were not saturated and

are still available sites for β2M adsorption on latex particles The initial slope of β2

isotherms for PS was steeper than for the other latex particles PSPMMA100

PSPMMA90PAA10 and PSPMMA75PAA25

The layer thickness (δ) of BSA proteins on synthesized latex particles in phosphate

buffer (PB) was in the range of 27 - 81 Aring at 37 C and pH 48 Only the δ values at pH

48 the isoelectric point (IEP) of BSA are important because intra- and inter-molecular

electrostatic repulsion of the protein molecules is minimized at the IEP Compared to the

hydrodynamic dimensions of BSA 140 times 40 times 40 Aring these values of the adsorbed laye

thickness indicates that the BSA molecules adsorb by the side-on (40Aring) and end-on mod

(140Aring) Consideration of the multi-layering is unnecessary because the adsorbed

thickness of BSA do not exceed 140 Aring

The Gibbs free energy of β

o

3

2M was more negative in phosphate buffer (PB) than

that of BSA in physiological condition (37 C and pH 74) indicating that βo2M molecul

are more favorably adsorbed on the latex particles than BSA and that the affinity of β2M

was much larger than BSA This may be explained by the rate of diffusion and

accessibility of β2M molecules being easier and faster than that for BSA because of

smaller size (or weight) and spherical shape of β2M molecules It may also be possible t

explain that more hydrophobic forces (less electrostatic repulsion) contributes to

120

adsorption of β2M than to that of BSA because the IEP of β2M (57) is closer to

physiological condition (pH 74) than that of BSA (pH 48) The selective adsorption of

β2M m

in β2M

larger than in BSA adsorption indicating

that fa

an that between BSA and latex particles because

IEP (

ch as

Other

olecules over BSA molecules can be enhanced by developing more hydrophilic

PSPMMA90PAA25 and PSPMMA75PAA25 core shell latex particles

The adsorption kinetics of proteins on latex particles was expressed as a second-

order model The plateau region for β2M was reached earlier than BSA on all latex

particles and the K values (equilibrium rate constant of 2nd order adsorption)

adsorption process on latex particles were much

ster adsorption rate of β2M molecules than that of the BSA molecules There are

several explanations for these phenomena First the smaller size of β2M molecules can

more easily diffuse and approach to the latex substrate Second enhanced hydrophilic

domain on the latex particle surface by sulfate groups (SO4-) and ionized carboxylic

groups (COO-) is not favorable for larger BSA molecules but is for smaller β2M to be

anchored Third the conformational change by intra- and inter-molecular repulsion

would be faster in β2M due to lower molecular weight Finally the ionic repulsion

between β2M and latex particles is less th

57) of β2M is closer to the tested condition (pH 74) than IEP (48) of BSA

In the biocompatibility test the number of red blood cells (RBCs) ruptured by PS

latex particles ranged from 30 to 91 as the solid content of PS increased from 05 to

50 respectively However hemolysis of the other core shell latex particles su

PSPMMA100 PSPMMA90PAA10 and PSPMMA75PAA25 were less than 35 The

PSPMMA90PAA10 particles at all applied solid concentrations had less than 02

hemolysis and was the most blood compatible among the prepared latex particles

121

core shell particles such as PSPMMA100 and PSPMMA75PAA25 also had excelle

compatibility with low RBC breakage

52 Conclusions

The major conclusions from this study include

nt blood

bull microglobulin (β2M) over that of the bovine serum albumin (BSA) at pH 74

was achieved by controlling the PMMA and PAA ratio to optimize

e particles posses the most desirable requirements for use in a human dialysis

ns

icles

olecule

bull

Using engineered core shell latex particles selective adsorption of β2

physiological condition was demonstrated

bull The rate of adsorption of the β2M was also much higher than that of BSA This

hydrophobichydrophilic microdomain size

bull The core shell particles also exhibited excellent biocompatibility with blood Thes

membrane

53 Recommendation for Future Work

With the results and conclusions obtained through this study several suggestio

may be given for future work as follows

bull Competitive adsorption tests of whole blood proteins on synthesized latex partunder physiological condition

bull Preparation of latex particles with optimum hydrophobic hydrophilic microdomains to maximize selective adsorption and removal of toxin molecules

bull Membrane fabrication by suitable pore size design to discriminate a toxin mby size sieving as a function of particle layers

Evaluation of membrane performance such as flow rate in water and in simulated blood fluid (SBF)

bull Application for hemodialysis as well as other technologies such as a water remediation

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Korea on August 7 1968 He obtained a Bachelor of Science degree in Chemistry from

Keim d graduate school and earned a

resear n Korea Institute of Science and

Coati e

ia

Busin

BIOGRAPHICAL SKETCH

Sangyup Kim was born and raised in the small town of Sung-Joo Kyungbook

yung University (Daegu Korea) in 1993 He entere

Master of Science degree in Chemical engineering in 1995 He worked as a student

cher for 2 years at Textile Polymer Division i

Technology (KIST) in Seoul Korea Next he worked over a year at the Automobile

ng Division in DongJoo-PPG in Korea In August 2001 he began studying for th

degree of Doctor of Philosophy in materials science and engineering at the University of

Florida in Gainesville After getting his PhD he plans to work at the Digital Med

ess Division of Samsung Electronics in Korea starting in January 2006

141

ACKNOWLEDGMENTS

I owe what I am today and this moment to many people without whom I could not

have been here First of all I would like to express my sincere and deepest appreciation

to my parents wife Mikyung daughters Heehyung and Erin whose love patience and

support helped me pursue my dream I gratefully acknowledge my advisor Dr H El-

Shall who gave me an opportunity to become part of an exciting and beneficial research

project and exert myself in the research work His guidance knowledge and stimulating

scientific and critical thinking have contributed to my success to a great extent and

inspired me to dedicate myself to research work I especially appreciate Dr R Partch as a

co-advisor who has helped me conduct experiments with valuable discussion for my

research from my first year I am greatly thankful to Dr C McDonald whose idea and

valuable experimental advice and discussion in the area of polymer chemistry and

membrane technology helped cultivate my interest in this area and were vital to

performing this research Without his contribution this work could not have been

achieved I also thank my doctoral committee members Dr R Singh Dr A Zaman and

Dr B Koopman for appreciated comments

Financial support and equipment use for research from the Particle Engineering

Research Center at the University of Florida were vital in continuing my study for more

than 4 years My sincere thanks are due to my former advisor Dr Kiryoung Ha in

Keimyung University who helped me open my eyes and broaden my thought and

introduced me into a limitless polymer field I would like to also thank to past and current

iv

members in Dr El-Shallrsquos research group for their assistance advice and support I

sincerely appreciate Stephen for the assistance of thesis writing

v

TABLE OF CONTENTS page

ACKNOWLEDGMENTS iv

LIST OF TABLES viii

LIST OF FIGURES x

1 INTRODUCTION 1

2 BACKGROUND AND LITERATURE SURVEY5

21 The Significance of End Stage Renal Disease (ESRD)5 22 Hemodialysis (HD) Treatment 6 23 Advances in Membrane Technology 8 24 Sorbent Technology16 25 Limitation of Current Hemodialysis Treatment 19 26 Latex Particles 20

261 The Components for Emulsion Polymerization 21 262 Particle Nucleation 21 263 Types of Processes for Emulsion Polymerization 23 264 Chemistry of Emulsion Polymerization 24 265 Seed Emulsion Polymerization25 266 Polystyrene Latex Particles 26 267 Various Applications of Latex Particles28

27 Proteins 30 271 Interaction Forces between Proteins33 272 β -Microglobulin (β M)2 2 35 273 Serum Albumin 36

28 Protein Adsorption38 281 Interaction between Protein Molecule and Latex Particle38

29 Hypothesis for Toxin Removal39 291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size 40 292 Toxin Removal by Selective Adsorption on Engineered Latex Particles 41

3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY 44

31 Materials 44 32 Latex Particle Preparation46

321 Preparation of Seed Latex Particles47

vi

322 Preparation of Seeded Latex Particles 48 323 Preparation of Core Shell Latex Particles 48 324 Purification of Synthesized Latex Particles48

33 Characterization49 331 Degree of Conversion49 332 Fourier Transform Infrared (FTIR) Spectroscopy49 333 Quasielastic Light Scattering (QELS) 50 334 Field Emission-Scanning Electron Microscopy (FE-SEM) 50 335 Zeta Potential Measurement 50 336 Protein Adsorption51 337 Blood Biocompatibility by Hemolysis Test 53

4 RESULTS AND DISCUSSION55

41 Polymerization of Latex Particles55 411 Polystyrene Seed Latex Particles55 412 The Growth of Polystyrene (PS) Seed Latex Particles61 413 Core Shell Latex Particles 71

42 Characterization of Latex Particles75 421 Fourier Transform Infrared Spectroscopy (FTIR)75 422 Zeta Potential Measurements 75

43 Protein Adsorption Study 85 431 Adsorption Isotherm87 432 Adsorbed Layer Thickness 103 433 Gibbs Free Energy of Protein Adsorption 104 434 Kinetics of Adsorption 107

44 Blood Compatibility 112

5 SUMMARY OF RESULTS CONCLUSION AND RECOMMENDATION FOR FUTURE WORK115

51 Summary of Results115 52 Conclusions121 53 Recommendation for Future Work121

LIST OF REFERENCES122

BIOGRAPHICAL SKETCH 141

vii

LIST OF TABLES

Table page 4-1 The polymerization recipe of polystyrene (PS) seed particles 57

4-2 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles less than 500nm in size 63

4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size64

4-4 The preparation recipe of PS core with various shell latex particles72

4-5 The isoelectric point (IEP) of common proteins 83

4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model 93

4-7 The equilibrium concentration values (q ) of BSA adsorption on PSPMMA particles calculated the Langmuir-Freundlich isotherm model

m 10096

4-8 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model

90 1098

4-9 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model

75 25100

4-10 The equilibrium concentration values of β M adsorption calculated the Langmuir-Freundlich isotherm model

2102

4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37 Co 104

4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37 Co 104

4-13 The values of Gibbs free energy change of BSA adsorption in phosphate buffer (PB) at 37 Co 105

4-14 The values of Gibbs free energy change of BSA adsorption in phosphate buffered saline (PBS) at 37 Co 105

viii

4-15 The values of Gibbs free energy change of β M adsorption in phosphate buffere (PB) at 37 C and pH 74

2o 106

4-16 Fitting parameters of second-order kinetic model 111

ix

LIST OF FIGURES

Figure page 1-2 Stereo drawing of the αndashcarbon backbone of β M2 2

2-1 An image of the location and cross section of a human kidney 5

2-2 A schematic draw of the hemodialysis route7

2-3 Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes 11

2-4 The chemical structure of polysulfone (PSf) 12

2-5 The chemical structure of polyamide (PA)13

2-6 Chemical structure of polyacrylonitrile (PAN) 14

2-7 Emulsion polymerization system24

2-8 L-α-amino acid 31

2-9 Ribbon diagram of human β M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ)

236

2-10 Secondary structure of human serum albumin (HSA) with sub-domains 37

2-11 The relationship between pore size and particle size in body-centered cubic and closed packed arrays41

2-12 Schematic representation of the protein adsorption on the core shell latex particles at pH 7442

3-1 Chemical structure of main chemicals45

3-2 Experimental setup for semi-continuous emulsion polymerization 46

3-3 The particle preparation scheme in various types and size ranges of latex particles 47

3-4 Schematic of the procedure for a protein adsorption test 52

3-5 Separation of RBC from whole blood by centrifuge process53

x

3-6 Schematic of the procedure for hemolysis test 54

4-1 Schematic representation of semi-continuous seed latex particles preparation and growth56

4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS (B) PS (C) PS (D) PSS259 S233 S207 S181 59

4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm 65

4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm 68

4-5 Dependence of the particle size on the surfactant to monomer ratio70

4-6 Schematic of core shell latex particle structures 71

4-7 Scanning Electron Micrograph of latex particles (A) PS (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA

100

90 10 75 25 73

4-8 FTIR spectra of polymerized latex particles (A) bare polystyrene (PS) (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA100 90 10 75 25 76

4-9 Schematic representation of ion distribution near a positively charged surface 78

4-10 Schematic representation of zeta potential measurement79

4-11 Zeta potential of PS latex particles at 25 Co 80

4-12 Zeta potential of PSPMMA latex particles at 25 C100o 81

4-13 Zeta potential of PSPMMA PAA latex particles at 25 C90 10o 82

4-14 Zeta potential of PSPMMA PAA latex particles at 25 C75 25o 82

4-15 The decay of surface potential with distance from surface in various electrolyte concentrations (1) low (2) intermediate (3) high 84

4-16 High-affinity adsorption isotherm of typical flexible polymer on solid surface 86

4-17 Overall schematic representation of the protein adsorption on the synthesized latex particles86

4-18 The dependence of UV absorbance on BSA concentration (A) 10mgml (B) 06mgml (C) 02mgml87

4-19 Standard curve of net absorbance vs BSA sample concentration 88

xi

4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37 C in phosphate buffer mediao 89

4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37 Co 90

4-22 Adsorption isotherm of BSA adsorption 37 C in phosphate buffered saline (PBS) on PS latex particles

o

91

4-23 Conformation of bovine serum albumin (BSA) on latex particles92

4-24 Adsorption isotherm of BSA 37 C in PB on PSPMMA core shell latex particles

o100

94

4-25 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA core shell latex particles

o100

95

4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA PAA core shell latex particles

o90 10

97

4-27 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA PAA core shell latex particles

o90 10

98

4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA PAA latex particleso75 25 99

4-29 Adsorption isotherm of BSA 37 C in PBS on PSPMMA PAA core shell latex particles

o75 25

99

4-30 Adsorption isotherm of β M onto PS and PSPMMA latex particles in PB at 37 C and pH 74

2 100o 101

4-31 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74

2 90 10o 101

4-32 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74

2 75 25o 102

4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37 C and pH 74

o

107

4-34 The kinetics of protein adsorption in PB on PS latex particles at 37 C and pH 74

o

109

4-35 The kinetics of protein adsorption in PB on PSPMMA latex particles at 37 C and pH 74

100o

109

4-36 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74

90 10o 110

xii

4-37 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74

75 25o 110

4-38 Image of red blood cells 113

4-39 Hemolysis caused by latex particles n=5114

xiii

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS

By

Sangyup Kim

December 2005

Chair Hassan El-Shall Major Department Materials Science and Engineering

Latex particles with well-defined colloidal and surface characteristics have received

increasing attention due to their useful applications in many areas especially as solid

phase supports in numerous biological applications such as immunoassay DNA

diagnostic cell separation and drug delivery carrier Hemodialysis membrane using

these particles would be another potential application for the advanced separation

treatment for patients with end stage renal disease (ESRD) It is desirable to remove

middle molecular weight proteins with minimal removal of other proteins such as

albumin Thus it is necessary to understand the fundamental interactions between the

particles and blood proteins to maximize the performance of these membranes This

improvement will have significant economic and health impact

The objective of this study is to synthesize polymeric latex particles of specific

functionality to achieve the desired selective separation of target proteins from the human

blood Semi-continuous seed emulsion polymerization was used to prepare monodisperse

xiv

polystyrene seed particles ranging from 126plusmn75 to 216plusmn53 nm in size which are then

enlarged by about 800nm Surfactant amount played a key role in controlling the latex

particle size Negatively charged latex particles with a different hydrophobicity were

prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid

monomer The prepared polymeric particles include bare polystyrene (PS) particles less

hydrophobic PS core and PMMA shell particles and more hydrophilic PS core and

PMMA-co-PAA shell latex particles with a 370nm mean diameter SEM light scattering

and zeta potential measurements were used to characterize particle size and surface

properties Adsorption isotherms of two proteins bovine serum albumin (BSA) and β2-

microglobulin (β2M) on latex particles were obtained as a function of pH and ionic

strength using the bicinchoninic acid (BCA) assay method The Langmuir-Freundlich

adsorption model was used to determine the adsorption amount of protein at equilibrium

The thickness of adsorbed BSA layer on latex particles was obtained in order to

investigate the adsorption orientation such as end-on or side-on mode Adsorption

kinetics experiments for both proteins and all latex particles were also performed The

adsorption kinetic constant determined from the Langmuir-Freundlich adsorption

isotherm model was used to calculate Gibbs free energy of adsorption to compare the

competitive adsorption of BSA and β2M Hemolysis tests were performed to investigate

the blood compatibility of synthesized latex particles PSPMMA90PAA10 and

PSPMMA75PAA25 core shell latex particles had desirable material properties with not

only a large amount and high rate of selective β2M adsorption over BSA but also high

blood compatibility showing less than 3 hemolysis

xv

CHAPTER 1 INTRODUCTION

End stage renal disease (ESRD) is a chronic condition in which kidney function is

impaired to the extent that the patientrsquos survival requires removal of toxins from the

blood by dialysis therapy or kidney transplantation The National Kidney Foundation

estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF] The

number of people with ESRD is rapidly increasing in the United States with

approximately 96 295 incidents and 406 081 prevalent patients including 292 215 on

dialysis and 113 866 with a functioning graft in 2001 It is projected that there will be

more than 22 million ESRD patients by 2030 [USRDS 2003] The expenditure for the

ESRD treatment program had reached $228 billion 64 of the Medicare budget in

2001 Due in part to the limited availability of kidneys for transplantation hemodialysis

is the primary clinical treatment for the patients with ESRD

The central element of a hemodialysis instrument is the semipermeable membrane

that allows for selective transport of low molecular weight biological metabolites less

than 5000 Da such as urea and creatinine as well as excess water and electrolytes [Baker

2004] from the blood One limitation of current dialysis technologies is the inability to

efficiently remove middle molecular weight toxins such as β2-Microglobulin (β2M) and

interleukin 6 (IL-6)

β2M is a causative protein of dialysis-related amyloidosis (DRA) a disease arising

in patients with chronic kidney failure as a serious complication of long-term

hemodialysis treatment [Gejyo et al 1985] β2M deposition in tissue is the primary cause

1

2

of destructive arthritis and carpal tunnel syndrome [Vincent et al 1992 Drueke 2000]

The β2M structure is shown in Figure 1-2

Figure 1-2 Stereo drawing of the αndashcarbon backbone of β2M [Becker et al 1985]

Although attempts have been made to increase the efficiency of middle molecular

weight toxin removal by changes in the membrane pore size and the use of innovative

materials to adsorb these toxins [Samtleben et al 1996 Ronco et al 2001] removal

efficiency is not as high as those achieved by a normal healthy kidney Traditional

membranes have a number of processing and performance limitations [Westhuyzen et al

1992 Leypoldt et al 1998] such as a restricted choice of surface chemistries and limited

control of porosity The development of novel engineering membrane technology is

needed to remove middle molecule toxins

Polymeric latex particles have received increasing attention in medical application

areas especially as solid phase supports in biological applications [Piskin et al 1994]

Examples of these applications include immunoassay [Chen et al 2003 Radomske-

Galant et al 2003] DNA diagnostic [Elaїssari et al 1998] cell separation drug delivery

carrier [Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] etc This is because of

3

the well-defined colloidal and surface characteristics of the particles By using a seed

emulsion polymerization method it is possible to synthesize monodisperse latex particles

with various particle size ranges and surface chemistry Functionalized core-shell latex

particles can be introduced by multi-step emulsion polymerization Core particles are

synthesized in the first stage of the polymerization and the functional monomer is added

in the second stage This is done without any emulsifier addition to prevent the

production of new homopolymer particles [Keusch et al 1973] The core-shell particles

are useful in a broad range of applications because of their improved physical and

chemical properties over their single-component counterparts [Lu et al 1996 Nelliappan

et al 1997] Through the development of a hemodialysis membrane using monodisperse

latex particles improvements in advanced separation treatment for patients with end

stage renal disease (ESRD) can be realized This requires the maximum removal of

middle molecular weight proteins with minimal removal of other beneficial proteins such

as albumin Thus an understanding of the fundamental interactions between the particles

and biopolymers is vital to maximize the performance of this membrane technology

The field of material science and biotechnology is based on fundamental chemistry

has developed over the past three decades into todayrsquos powerful discipline that enables

the development of advanced technical devices for pharmaceutical and biomedical

applications This novel and highly interdisciplinary field is closely associated with both

the physical and chemical properties of organic and inorganic particles [Niemeyer 2001]

Hemodialysis membrane using these particles would lead to improvements in the

advanced separation treatment for patients with end stage renal disease (ESRD) The

interdisciplinary nature of this approach enables a more complete understanding of the

4

phenomena of protein adsorption and the material properties necessary for selective

separation This innovative approach to membrane fabrication has the potential of making

inexpensive highly efficient membranes for both industrial and specialized separation

processes

The goal of this study is to prepare polymeric latex particles with tailored

properties to maximize separation of toxin molecules and to investigate the fundamental

interactions between the particles and molecules in the biological system in order to

optimize the performance of a membrane material for these applications

Polymeric latex particles were synthesized with specific functionality in an attempt

to achieve selective separation Seeded emulsion polymerization was used to synthesize

functionalized monodisperse latex particles in various sizes Negatively charged

hydrophobic polystyrene latex particles were synthesized by the same method Core shell

latex particles PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25 were also

synthesized to differentiate the degree of hydrophobicity of particles Scanning Electron

Micrograph (SEM) and light scattering measurements were used to characterize particle

size and shape and zeta potential measurements were conducted to measure the electrical

surface property of synthesized particles Adsorption isotherms of target proteins bovine

serum albumin (BSA) and β2M on latex particles were obtained as a function of pH

ionic strength and protein concentrations using the BCA assay method Adsorption

kinetics for both proteins on the latex particles were also measured Finally hemolysis

tests were run to determine the biocompatibility of polymer latex particles with human

blood This research will be described in more detail in chapters 3 and 4

CHAPTER 2 BACKGROUND AND LITERATURE SURVEY

21 The Significance of End Stage Renal Disease (ESRD)

The kidneys are responsible for removing excess fluid minerals and wastes from

the blood regulating electrolyte balance and blood pressure and the stimulation of red

blood cell production They also produce hormones such as erythropoietin (EPO) and

calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005 Kurbel

et al 2003] EPO acts on the bone marrow to increase the production of red blood cell in

case of bleeding or moving to high altitudes Calcitriol mainly acts on both the cells of

the intestine to promote the absorption of calcium from food and also bone to mobilize

calcium from the bone to the blood When kidneys fail harmful waste builds up blood

pressure rises and body retains excess fluid The body also does not make enough red

blood cells When this happens treatment is needed to replace the function of failed

kidneys

Figure 2-1 An image of the location and cross section of a human kidney

5

6

The National Kidney Foundation estimates that over 20 million Americans had

chronic kidney disease in 2002 [NKF] Chronic renal disease is a gradual and progressive

loss of the function unlike acute renal disease where sudden reversible failure of kidney

function occurs Chronic renal failure usually takes place over a number of years as the

internal structures of the kidney are slowly damaged In the early stages there can be no

symptoms In fact progression may be so gradual that symptoms do not occur until

kidney function is less than one-tenth that of a normal health kidney If left untreated

chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal

Disease (ESRD) ESRD is a rapidly growing heath-care problem in the United States In

2001 approximately 96295 incidents and 406081 prevalent patients were diagnosed

with ESRD including 292215 patients on dialysis and 113866 patients with a

functioning graft [USRDS 2003] The projected number of patients with ESRD is

expected to exceed 22 million patients by 2030 with much of this growth being driven by

the increasing prevalence of major contributing factors such as diabetes and high blood

pressure [USRDS 2003] A great extent of ESRD program cost and Medicare budget

have been spent in 2001 Due in part to a limited availability of kidneys for

transplantation hemodialysis (HD) is the primary method of treatment for ESRD and is

currently used for approximately 61 of US ESRD patients

22 Hemodialysis (HD) Treatment

HD removes toxins from the body by extracorporeal circulation of the blood

through a semipermeable membrane referred to as a dialyzer The toxins are removed

primarily by diffusion across the membrane to a dialysate solution which is circulated on

the opposite side of the membrane The cleaned blood is then returned to the blood

stream A surgically constructed vascular access connects the extracorporeal circuit to the

7

patientrsquos system Treatments are almost always performed three times per week in

specially equipped dialysis facilities for 3-4 hours per each treatment Figure 2-2 shows

the scheme of the hemodialysis route The critical element of a HD instrument is the

semipermeable membrane which allows for selective transport of low molecular weight

biological metabolites from blood

igure 2-2 A schematic draw of the hemodialysis route

ESRD patients who undergo dialysis therapy often experience several other

problems associated with the treatment such as anemia fatigue bone problems joint

problems itching sleep disorders and restless legs Anemia is common in patient with

kidney disease because the kidneys produce the hormone erythropoietin (EPO) which

stimulates the bone marrow to produce red blood cells Diseased kidneys often do not

produce enough EPO to stimulate the bone marrow to make a sufficient amount of red

blood cells This leads to bone disease referred to as renal osteodystrophy and causes

bones to become thin and weak or malformed and can affect both children and adults

Older patients and women who have gone through menopause are at greater risk for this

disease Uremic toxins which cannot be remove from the blood by the current dialyzer

membranes can lead to itching This problem can also be related to high levels of

F

8

parathyroid hormone (PTH) Dialysis patients can also experience day-night revers

is they have insomnia at night and sleep during the day This can be related to possible

nerve damage in the body and a chemical imbalance in the blood due to the excess toxin

Oxidative stress is a problem for patients on maintenance dialysis This problem is due to

an imbalance between pro- and antioxidant factors [Roselaar et al 1995 Cristol et al

1994] Oxidative stress affects oxidation of low-density lipoproteins which are the main

factor for atherogenesis [Huysmans et al 1998] and is also involved in the development

of malignancies and diabetes mellitus [Rice-Evans et al 1993] In order to reduce

antioxidant defense dialysis is needed to contribute to help stimulate free radical

production or eliminate antioxidants Dialysis-related amyloidosis (DRA) is also a

common and serious problem for people who have been on dialysis for more than 5

years DRA develops when proteins in the blood deposit on joints and tendons causi

pain stiffness and fluid in the joint as is the case with arthritis Normally the healthy

kidneys can filter out these proteins but dialysis filters are not as effective

23 Advances in Membrane Technology

al that

s

ng

Dialysis for blood of ESRD

Hemo filtration

s

ysis using a cellulose based membrane occurred

1913 John Abel [1990] from the Johns Hopkins Medical School described a method

purification is widely used in the treatment

dialysis (HD) techniques use a semi-permeable membrane to replace the

role of the kidney The membranes used in HD can be broadly classified into those based

on cellulose and those manufactured from synthetic copolymers These membranes come

in various shapes such as sheets tubular structures or hollow fiber arrangements The

hollow fiber type is the most popular and is incorporated into over 800 different device

in world wide [Ronco et al 2001]

The first attempt at blood dial

9

where sing

e

om

ysis was performed until the mid 1960 The basic

molec

rial in

een

benzyl)

was used for the membrane with

hepar

by the blood of a living animal may be submitted to dialysis outside the body u

a membrane based on cellulose and returned to the natural circulation without exposur

to air infection by microorganisms or any alteration that would necessarily be prejudicial

to life This same technique is still used to today however the device used has been

modified over the years as better membranes were developed and the anti-coagulant

heparin has become available

Cellulose membranes have been widely used for the treatment of renal failure fr

1928 when the first human dial

ular structure of cellulose is made of a long chain containing hydroxyl (OH)

groups The realization that such groups imparted undesirable qualities on the mate

respect to blood contact behavior was discovered in the early 1970s and since has b

the focus of development These modified cellulose membranes used the partial

substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt

to reduce their negative effect The result is a molecular mosaic of hydrophobic (

and hydrophilic (hydroxyl and cellulose) regions

Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients

with acute renal failure in 1943 Cellophane tubing

in as the anticoagulant For the next 17 years hemodialysis therapy was performed

by this method but only for the patients with acute reversible renal failure Vascular

access required repeated surgical insertions of cannulas (slender tubes) into an artery and

vein and limited the number of treatments for a patient could receive in order to

minimize the amount of vascular damage

10

Initially the need for dialysis in patients with acute renal failure was determ

mainly by the development of signs and sym

ined

ptoms of uremia After dialysis some time

might

et al

ent improvement In 1960 the development of a shunt [Quinton et al

1960]

fone (PSf) polyamide (PA) and polyacrylonitrile (PAN) by phase inversion or

precip

lidone

e

elapse before uremic manifestations returned to warrant a sequential treatment of

dialysis Many patients with acute renal failure secondary to accidental or surgical

trauma were hypercatabolic but the interdialytic interval might be prolonged because of

anorexia or use of a low-protein diet However Teschan and his coworkers [Obrien

1959] showed that patient well-being and survival were improved by what they termed

prophylactic daily hemodiaysis or administration of the treatment before the patient

again became sick with uremia Their report in 1959 was the first description of daily

hemodialysis

Development of membrane accessories such as a shunt has also been an area of

focus for treatm

a flexible polytetrafluoroethylene (PTFE or Teflonreg) tubing made many more

hemodialysis treatments possible for chronic kidney failure patients PTFE has a non-

stick surface and is relative biocompatibility leading to minimized blood clotting in the

shunt

Synthetic membranes are prepared from engineered thermoplastics such as

polysul

itation of a blended mixture resulting in the formation of asymmetric and

anisotropic structures Figure 2-3 shows a fiber type of the Polyflux S membrane

consisting of poluamide (PA) polyacrylethersulfone (PAES) and polyvinylpyrro

(PVP) with the integral three-layer structure The skin layer on the inside fiber typ

membrane contacts blood and has a very high surface porosity and a narrow pore size

11

distribution This layer constitutes the discriminating barrier deciding the permeabili

and solute retention properties of the membrane The skin layer is supported by thick

sponge-type structure larger pores providing mechanical strength and very low

hydrodynamic resistance

ty

Figure 2-3 Cross-sectional SEM image view of the Polyflux S (polyamide +

polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch et al 1998]

n

[Malchesky 2004] because of its thermal stability mechanical strength and chemical

inertn ciated

ls

m

PSf is a widely used membrane material for the hemodialysis applicatio

ess According to a report from the National Surveillance of Dialysis-Asso

Disease (NSDAD) in the US over 70 of hemodialysis membranes were PSf based

[Bowry 2002] This is most likely because PSf has many advantages over other materia

This synthetic polymer is one of few materials that can withstand sterilization by stea

ethylene oxide and γ-radiation PSf membrane can be prepared by conventional

immersion precipitation methods into many different shapes including porous hollow

12

fiber or flat sheet hemodialysis membranes The material also has a high permeab

low molecular weight proteins and solute and high endotoxin retention The chemical

structure of PSf is shown in Figure 2-4

ility to

Figure 2-4 The chemical structure of polysulfone (PSf)

There is one major disadvantage to PSf The hydrophobic nature of the PSf causes

ment alternative pathway

leadin

s not

3

et al

in

O acrylate

serious complications through the activation of the comple

g to the adsorption of serum proteins onto the membranes [Singh et al 2003]

Anticoagulants are added during dialysis therapy to avoid blood clotting but this doe

completely eliminate the problem In order to overcome this disadvantage of the PSf

membrane various studies have been performed to change the materialrsquos surface

properties These investigations include hydrophilic polymer coating [Brink et al 199

Kim et al 1988 Higuchi et al 2003] layer grafting onto PSf membrane [Wavhal

2002 Song et al 2000 Pieracci et al 2002 Mok et al1994] and chemical reaction of

hydrophilic components onto the membrane surface [Higuchi et al 1990 1991 1993

Blanco et al 2001 Nabe et al 1997 Guiver et al 1993] Hydrophilic monomers 2-

hydroxy-ethylmethacrylate (HEMA) acrylic acid (AA) and methacrylic acid (MMA)

have also been grafted onto PSf membrane to increase flux and Bovine Serum Album

(BSA) retention [Ulbricht et al 1996] Hancock et al [2000] synthesized

polysulfonepoly(ethylene oxide) (PEO) block copolymers to improve the resistance to

platelet adhesion Kim et al [2003] also studied blending a sulfonated PE

diblock copolymer into PSf in order to reduce platelet adhesion and enhance

13

biocompatibility PEO is a commonly used biomaterial due to its excellent resistance

protein adsorption and inherent biocompatibility [Harris 1992] Kim et al [20

a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf

membrane with an ultra-thin skin layer The polymer had an entrapped diblock

copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-SO

to

05] studied

ate

lic graft

l)

e of thier

aromatic

orand

3 acryl

and a hydrophobic block of octadecylacrylate (OA) Molecular dynamic (MD)

simulations were performed as a function of copolymer density to optimize interfacial

structure information McMurry [2004] developed a strategy using an amphiphi

copolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glyco

When compared to unmodified PSf these graft copolymer and resulting blend

membranes are found to hold promise for biomedical device applications

Polyamide (PA) membranes have also been used for hemodialysis becaus

mechanical strength in both wet and dry conditions Polyamide consists of

aliphatic monomers with amide bonding (-CONH-) also known as a peptide bond

The basic amide bond in polyamide is shown in Figure 2-5 R1 and R2 can be either

aromatic or aliphatic linkage group

C N

H

O

~ R R1 2~C N

H

O

~ R R1 2~

Figure 2-5 The chemical structure of polyamide (PA)

Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane

ompatible technique due to the

use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of

and concluded that PA hemofiltration was a highly bioc

14

the ac

e

e

is

n

is a semi-crystalline polymer and the mechanical properties

strong

2

CN

tivated anaphylatoxins and β2-Microglobulin (β2M) The PA based membrane

Polyfluxreg (manufactured by Gambro GmbH Germany) blended with polyamide

polyarylethersulfone and polyvinylpyrrolidone (PVP) was able to clean small molecules

such as urea dreatinine and phosphate as well as decrease β2M amount by 502

[Hoenich et al 2002] Due to the non-selectivity of the membrane removal of thes

unwanted materials also led to the undesirable loss of beneficial proteins during therapy

Meier et al [2000] evaluated different immune parameters using a modified cellulos

low-flux hemophan and synthetic high-flux PA membrane during a 1 year period in

chronic hemodialysis patients They found that the 1-year immunological evaluation of

hemodiaysis membrane biocompatibility was associated with changes in the pattern of

chronic T-cell actiovation

Polyacrylonitrile (PAN) is another commonly used membrane material because it

inherently hydrophilic and has been commercialized for ultrafiltration and microfiltratio

[Scharnagl et al 2001] PAN

ly depend on the crystalline structures The chemical structure of PAN is shown in

figure 2-6

CH CH

n2

CN

CH CH n

Chemical structure of polyacrylonitrile (PAN)

The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming

agent gives PAN membranes more flexible processing parameters and increased

e has been optimized through

copolymerization with many other vinyl monomers including glycidyl methacrylate

Figure 2-6

performance [Jung et al 2005] PAN membrane performanc

15

[Godj

acid

for

for

a non-

solub this

in

oid underneath the inner

surface of membrane decreased by increasing the amount of solvent DMSA in the

evargova et al 1999 Hicke et al 2002] N-vinylimidazole [Godjevargova et al

2000] hydroxyl ethyl methacrylate [Ray et al 1999 Bhat et al 2000] methacrylic

[Ray et al 1999] vinyl pyrrolidone [Ray et al 1999] acrylic acid [Trotta et al 2002]

acrylamide [Musale et al 1997] and vinylchloride [Broadhead et al 1998] These

monomers provide a reactive group for enzyme immobilization improved mechanical

strength solvent-resistance pervaporation permeation flux anti-fouling and bio-

compatibility Because of this PAN-based copolymer membranes have great potential

the treatment of hemodialysis in an artificial kidney This material can also be used

other applications like the treatment of wastewater the production of ultra-pure water

biocatalysis together with separation and methanol separation by pervaporation

Lin and his coworker [2004] studied the modification of PAN based dialyzer

membranes to increase the hemocompatibility by the immobilization of chitosan and

heparin conjugates on the surface of the PAN membrane When a foreign material is

exposed to blood plasma proteins are adsorbed clotting factors are activated and

le fibrin network or thrombus is formatted [Goosen et al 1980] The result of

research was that the biocompatible chitosan polymer and a blood anticoagulant hepar

prevented blood clotting They showed prolonged coagulation time reduced platelet

adsorption thrombus formation and protein adsorption

Nie et al [2004] studied PAN-based ultrafiltration hollow-fiber membranes

(UHFMs) In order to improve the membrane performance acrylonitrile (AN) was

copolymerized with other functional monomers such as maleic anhydride and α-allyl

glucoside They found that the number and size of macrov

16

intern erum

and

d a

rface The second step is the

transf after

yjak et

embrane

1978 Malchesky et al 1978] has been further developed to increase the efficiency of

al coagulant The water flux of the UHFMs also decreased while the bovine s

albumin rejection increased minutely Godjevargova et al [1992] modified the PAN

based membrane with hydroxylamine and diethylaminoethylmethacrylate to improve

membrane dialysis properties Formed functional groups like primary amine oxime

tertiary amine groups provided the membrane with more hydrophilic properties an

substantial increase in the permeability of the membranes

The wide use of filtration in practice is limited by membrane fouling Solute

molecules deposit on and in the membrane in the process of filtration causing dramatic

reduction in flux through the membrane Fouling occurs mostly in the filtration of

proteins Three kinetic steps are involved in the fouling of UF membranes according to

Nisson [1990] The first step is the transfer of solute to the su

er of solute into the membrane until it either finally adsorbs or passes through

a set of adsorption-desorption events The third step includes surface binding

accompanied by structural rearrangement in the adsorbed state [Ko et al 1993] Br

al [1998] studied the surface modification of a commercially available PAN membrane

to develop superior filtration properties with less fouling by proteins The PAN

membrane was immersed in excess NaOH solution to convert some of the surface nitrile

groups into carboxylic groups by the hydrolysis process This modified PAN m

was not so severely fouled in the Bovine Serum Albumin (BSA) filtration test The pore

size however decreased during the hydrolysis process leading to a significant reduction

in flux and made the membrane less productive in the ultrafiltration (UF) mode

24 Sorbent Technology

Over the last three decades sorbent technology [Castino et al 1976 Korshak et al

17

dialysis or replace it for the treatment of ESRD Sorbents remove solutes from solution

through specific or nonspecific adsorption depending on both the nature of the solute and

the sorbent Specific adsorption contains tailored ligands or antibodies with high

selectivity for target molecules een used in autoimmune

disord

and

e

al 1996] various resins [Ronco et al 2001]

album

e

as

Specific adsorbents have b

ers such as idiopathic thrombocytopenic purpura [Snyder et al 1992] and for the

removal of lipids in familial hyper cholesterolemia [Bosch et al 1999] Nonspecific

adsorbents such as charcoal and resins attract target molecules through various forces

including hydrophobic interactions ionic (or electrostatic) attraction hydrogen bonding

and van der Waals interactions

New dialysate with sorbents has become an accepted modification of dialysis

sorbent hemoperfusion is gaining ground as a valuable addition to dialysis especially as

new sorbents are developed [Winchester et al 2001] Hemoperfusion is defined as th

removal of toxins or metabolites from circulation by the passing of blood within a

suitable extracorpoteal circuit over semipermeable microcapsules containing adsorbents

such as activated charcoal [Samtleben et

in-conjugated agarose etc Novel adsorptive carbons with larger pore diameters

have been synthesized for potential clinical use [Mikhalovsky 1989] Newly recognized

uremic toxins [Dhondt et al 2000 Haag-Weber et al 2000] have resulted in several

investigations on alternatives to standard or high-flux hemodialysis to remove thes

molecules These methods include hemodiafilteration with [de Francisco et al 2000] or

without [Ward et al 2000 Takenaka et al 2001] dialysate regeneration using sorbents

well as hemoperfusion using such adsorbents as charcoal and resins

18

Kolarz et al [1989 1995] studied the hyper-crosslinked sorbent prepared from

styrene and divinylbenzene (DVB) for a hemoperfusion application They found that the

pore structure of a swelling sorbent was changed by additional crosslinking with α αrsquo-

dichloro-p-xylene in the presence of a tin chloride catalyst and in a di

chloroethane

soluti and

en

decyl groups that attract β2M through a hydrophobic interaction The

adsor

of

5]

d

penet

on They also realized that the hemocompatibility was useful for nemoperfusion

could be imparted to the sorbents by introducing sulfonyl groups at a concentration of

about 02mmolg

A special polymeric adsorbing material (BM-010 from Kaneka Japan) has be

investigated by another group [Furuyoshi et al 1991] for the selective removal of β2M

from the blood of dialysis partients The adsorbent consists of porous cellulose beads

modified with hexa

ption capacity of this material is 1mg of β2M per 1ml of adsorbent Using a

hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a

high-flux hemodialyzer several small clinical trials were performed During 4-5 hours

treatment about 210mg of β2M were removed thus reducing the concentration in the

blood by 60-70 of the initial level [Nakazawa et al 1993 Gejyo et al 1993 199

RenalTech developed a hemoperfusion device BetaSorbreg containing the hydrate

cross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure

designed to remove molecules between 4 and 30 kDa [Winchester et al 2002] In this

case solute molecules are separated according to their size based on their ability to

rate the porous network of the beaded sorbents The resin beads were prepared with

a blood compatible coating and confirmed to be biocompatible in vivo in animals

[Cowgill et al 2001]

19

25 Limitation of Current Hemodialysis Treatment

Hemodialysis is a widely used life-sustaining treatment for patients with ESRD

However it does not replace all of the complex functions of a normal healthy kidne

a result patients on dia

y As

lysis still suffer from a range of problems including infection

accelerated cardio ion anemia

chron

e

se middle

alysis

sessio

s

f

vascular disease high blood pressure chronic malnutrit

ic joint and back pain and a considerably shortened life span One significant

limitation of the current dialysis technology is the inability to efficiently remove larger

toxic molecules This is mainly because of the broad pore size distribution reducing th

selective removal of toxins and unsatisfied biocompatibility causing lots of

complications such as inflammation blood clotting calcification infection etc

Dialysis purifies the patients blood by efficiently removing small molecules like

salts urea and excess water However as toxic molecules increase in size their removal

rate by hemodialysis substantially declines Typically only 10 - 40 of the

molecular weight toxins (300-15000 Da) are removed from the blood during a di

n [Vanholder et al 1995] These toxins then reach an abnormally high level and

begin to damage the body One such toxin β2M causes destructive arthritis and carpal

tunnel syndrome by joining together like the links of chain to form a few very large

molecules and deposit damaging the surrounding tissues [Lonnemann et al 2002] This i

also a main cause of mortality for long-term dialysis patients Other middle molecule

toxins appear to inhibit the immune system and may play a significant role in the high

susceptibility to infections in dialysis patients Still others are believed to impair the

functioning of several other body systems such as the hematopoietic and other endocrine

systems This may contribute to accelerated cardiovascular disease the leading cause o

20

death among dialysis patients as well as clinical malnutrition which affects up to 50 of

this patient population

Over the last decade polymeric dialysis membranes have been developed to

increase the capacity for removing middle molecular weight toxins by changing the pore

size of dialyzer membranes and using new materials that adsorb these toxins for

impro se

styrene made during World War II in the United

States The Dow chemical compan anufacturer of polystyrene

includ

d of

on

rug

ntrol of

ved removal characteristics However removal efficiency is not as high as tho

achieved by a normal healthy kidney

26 Latex Particles

The first synthetic polymer synthesized using emulsion polymerization was a

rubber composed of 13-butadiene and

y has been a major m

ing latex which they used in paint formulations The theory of emulsion

polymerization in which a surfactant is used was established by Harkins [1948] and by

Smith and Ewart [1948] By 1956 the technology was complete including the metho

building larger diameter particles from smaller ones The product by this emulsi

polymerization is referred to as latex a colloidal dispersion of polymer particles in water

medium [Odian 1991] Latexes are currently undergoing extensive research and

development for a broad range of areas including adhesives inks paints coatings d

delivery systems medical assay kits gloves paper coatings floor polish films carpet

backing and foam mattresses to cosmetics The relatively well known and easy co

the emulsion process is one of main advantages for these applications Therefore

polymeric latex particle prepared by emulsion polymerization can be a candidate for the

medical applications because of the easy control of the particle size and morphology as

well as flexible surface chemistry to be required

21

261 The Components for Emulsion Polymerization

The main components for emulsion polymerization process are the monomer a

dispersing medium a surfactant and an initiator Available monomers are styrene

sing medium is usually water

which

ticles

n

ation To

monomer droplets but in micelles because the initiators are

insolu le

and

olution

butadiene methylmethacylate acryl acid etc The disper

will maintain a low solution viscosity provide good heat transfer and allow

transfer of the monomers from the monomer droplets into micelles and growing par

surrounded by surfactants respectively The surfactant (or emulsifier) has both

hydrophilic and hydrophobic segments Its main functions is to provide the nucleatio

sites for particles and aid in the colloidal stability of the growing particles Initiators are

water-soluble inorganic salts which dissociate into radicals to initiate polymeriz

control the molecular weight a chain transfer agent such as mercaptan may be present

262 Particle Nucleation

Free radicals are produced by dissociation of initiators at the rate on the order of

1013 radicals per milliliter per second in the water phase The location of the

polymerization is not in the

ble in the organic monomer droplets Such initiators are referred to as oil-insolub

initiators This is one of the big differences between emulsion polymerization

suspension polymerization where initiators are oil-soluble and the reaction occurs in the

monomer droplets Because the monomer droplets have a much smaller total surface area

they do not compete effectively with micelles to capture the radicals produced in s

It is in the micelles that the oil soluble monomer and water soluble initiator meet and is

favored as the reaction site because of the high monomer concentration compared to that

in the monomer droplets As polymerization proceeds the micelles grow by the addition

of monomer from the aqueous solution whose concentration is refilled by dissolution of

22

monomer from the monomer droplets There are three types of particles in the emulsion

system monomer droplets inactive micelles in which polymerization is not occurring

and active micelles in which polymerization is occurring referred to as growing polymer

particles

The mechanism of particle nucleation occurs by two simultaneous processes

micellar nucleation and homogeneous nucleation Micellar nucleation is the entry of

radicals ei

ther primary or oligomeric radicals formed by solution polymerization from

the aq ed

the

r

s

n

is coagulation with other particles rather than

polymerization of monomer The driving force for coagulation of precursor particles

ueous phase into the micelles In homogeneous nucleation solution-polymeriz

oligomeric radicals are becoming insoluble and precipitating onto themselves or onto

oligomers whose propagation has ended [Fitch et al 1969] The relative levels of micella

and homogeneous nucleation are variable with the water solubility of the monomer and

the surfactant concentration Homogeneous nucleation is favored for monomers with

higher water solubility and low surfactant concentration and micellar nucleation is

favored for monomers with low water solubility and high surfactant concentration It has

also been shown that homogeneous nucleation occurs in systems where the surfactant

concentration is below CMC [Roe 1968] A highly water insoluble monomer such a

styrene [Hansen et al 1979 Ugelstad et al 1979] has probably created by micellar

nucleation while a water soluble monomer such as vinyl acetate [Zollars 1979] has bee

formed by homogeneous nucleation

A third latex formation reaction mechanism has been proposed referred to as

coagulative nucleation In this reaction the major growth process for the first-formed

polymer particles (precursor particles)

23

sever les

yer

es

r a

l

the beginning of the polymerization As soon as the initiator is

gins with the formation and

growt

and

ved

added at one time during the later stages of the polymerization prior to complete

al nanometers in size is their relative instability compared to larger sized partic

The small size of a precursor particle with its high curvature of the electrical double la

permits the low surface charge density and high colloidal instability Once the particl

become large enough in size maintaining the high colloidal stability there is no longe

driving force for coagulation and further growth of particles takes place only by the

polymerization process

263 Types of Processes for Emulsion Polymerization

There are three types of production processes used in emulsion polymerization

batch semi-continuous (or semi-batch) and continuous In the batch type process al

components are added at

added and the temperature is increased polymerization be

h of latex particles at the same time There is no further process control possible

once the polymerization is started In the semi-continuous emulsion polymerization

process one or more components can be added continuously Various profiles of particle

nucleation and growth can be generated from different orders of component addition

during polymerization There are advantages to this process such as control of the

polymerization rate the particle number colloidal stability copolymer composition

particle morphology In the continuous process the emulsion polymerization components

are fed continuously into the reaction vessel while the product is simultaneously remo

at the same rate High production rate steady heat removal and uniform quality of

latexes are advantages of the continuous polymerization processes

Other available methods include the intermittent addition and shot addition of one

or more of the components In the shot addition process the additional components are

24

conversion of the main monomer This method has been used successfully to develo

water-soluble functional monomers such as sodium styrene sulfonat

p

e [Kim et al 1989]

264

lsion

Chemistry of Emulsion Polymerization

Emulsion polymerization is one type of free radical polymerization and can be

divided into three distinct stages initiation propagation and termination The emu

polymerization system is shown in Figure 2-7

Aqueous phaseAqueous phase

Micelle withmonomer

I Rbull

Emulsifier

Polymer particle swollenWith monomer

Monomerdroplet

I Rbull

Micelle withmonomer

I Rbull

Emulsifier

Polymer particle swollenWith monomer

MonomerdropletMonomerdroplet

I Rbull

Figure 2-7 Emulsion polymerization system [Radicals (Rmiddot) are created from initiators

(I) Monomer is transferred from larger monomer droplet into micelles by emulsifier Initiated polymer particle by radicals is keep growing until monomers are all consumed The reaction is performed in aqueous media]

In the initiation stage free radicals are created from an initiator by heat or an

ultraviolet radiation source The initiator with either peroxides groups (-O-O-) such as

odium persulfate or azo groups (-N=N-) such as azobisisobutyronitrile is commonly

used for em t

with the m er

chain ere

s

ulsion polymerization The primary free radicals created from initiator reac

onomer for initiation of polymerization In the propagation stage the polym

grows by monomer addition to the active center a free radical reactive site Th

are two possible modes of propagations head-to-head addition and head-to-tail addition

25

The head-to-tail mode is the predominant configuration of the polymer chain a result o

steric hindrance of the substitute bulky group In the termination stage polymer chain

growth is terminated by either coupling of two growing chains forming one polymer

molecule or transferring a hydrogen atom (dispropotionation) from one growing chain to

another forming two polymer molecules one having a saturated end group and the other

with an unsaturated end-group

265 Seed Emulsion Polymerization

Polymer latex particles have received increasing interest because of the versatili

of the many applications heterophase polymerization processes like emulsion dispersion

micro-emulsion seeded emulsion precipitation etc Especially to prepare well-defined

microspheres having monodispe

f

ty

rse and various particle sizes as well as surface group

ed particles prepared by emulsion polymerization

and e

s

hological design In

seede to control

n

functionalilties it is necessary to use se

nlarge them to a desired size in the further stages of reactions

Polymeric particles are required to have a uniform particle size in many

applications such as chromatography where they are used as a packing material

Morphological control of latex particle is also important for many practical application

[Schmidt 1972] Seed emulsion polymerization (or two-stage emulsion polymerization) is

a useful method to achieve both monodisperse particle size and morp

d emulsion polymerization [Gilbert 1995] preformed lsquoseedrsquo latex is used

the number of particles present in the final latex The advantage of seeded emulsio

polymerization is that the poorly reproducible process of particle nucleation can be

bypassed so that the number concentration of particles is constant and known Various

mechanisms have been proposed for the growth of latex particles [Ugelstad et al 1980

Okubo et al 1992] using this polymerization technique The initial seed particle

26

preparation step is well known and relatively easy to perform because at the relative

small particle sizes (02 to 05 micron) the particle growth process can be readily

controlled by the use of an emulsifier Enough emulsifier is used to prevent coagulation

but not enough to cause the formation of new particles As the particles are grown to

larger sizes in successive seeding steps it becomes increasingly difficult to mainta

stable uncoagulated emulsion without forming new particles and thereby destroying

monodisperisty of the latex Recently there have been several reports concerning t

reaction kinetics of seed emulsion polymerization and the development of latex

morphologies over the course of the reaction [Chern et al 1990 Delacal et al 1990 L

et al 1995 Lee 2000 2002]

266 Polystyrene Latex Particles

A number of papers have described the synthesis of the polystyrene latex particl

bearing various functional surface groups such as carboxyl [Lee et al 2000 Tun

2002 Reb et al 2000] hydroxyl [Tamai et al 1989] marcapto [Nilson 1989] epoxy

[Shimizu et al 2000 Luo et al

ly

in a

he

ee

es

cel et al

2004] acetal [Izquierdo et al 2004 Santos et al 1997]

ometyl [Izquierdo et al 2004 Park et al 2001

Sarob

h the

d

thymine [Dahman et al 2003] chlor

e et al 1998] amine [Counsin et al 1994 Ganachaud et al 1997 Ganachaud et al

1995 Miraballes-Martinez et al 2000 2001 Anna et al 2005] ester [Nagai et al 1999]

etc In order to produce these functionalized particles different methods for particle

preparation must be used The polymer emulsion with core-shell morphology of latex

particles is one of them This is a multistep emulsion polymerization process in whic

polystyrene ldquocorerdquo particle is synthesized in the first stage and the functional monomer is

added in the second stage of the polymerization without any emulsifier postfeeding to

prevent the production of new homopolymer particles thus forming the functionalize

27

polymer ldquoshellrdquo on the ldquocorerdquo particle [Keusch et al 1973] There are requirements to

limit secondary nucleation and encourage core-shell formation in seeded emulsion

polymerization including the addition of smaller seed particles at high solid content to

increase particle surface area low surfactant concentration to prevent formation of

micelles and the semi-continuous addition of monomer to create a starved-feed conditio

and keep the monomer concentration low There are some advantages [Hergeth et al

1989] of dispersions with polymeric core-shell particles First it is possible to modi

interfacial properties of polymer particles in the aqueous phase by the addition of only

very small amounts of a modifying agent during the last period of the reaction Thus

these core-shell particles are useful in a broad range of applications since they always

exhibit improved physical and chemical properties over their single-component

counterparts [Lu et al 1996 Nelliappan et al 1997] In this way the improvement of

surface properties of such dispersions is straightforward and inexpensive The other is

that polymers with a core-shell structure are perfect model systems for investigating th

material properties of polymer blends and composites because of their regular

distribution of one polymer inside a matrix polymer and because of the simple sp

geometry of the system

Their properties usually depend on the structures of latex particles Chen and his

coworkers [Chen et al 1991 1992 1993] reported the morphological development of

core shell latex particles of polystyrenepoly(methyl methacrylate) during

polymerization Before the research by Chen and his coworker Min et al [Min et al

1983] reported the morph

n

fy the

e

herical

ological development of core shell latex of polystyrene

(PS)polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the

28

additi

of

ity of graft

ing reagents Thus protein adsorption on polymeric solid surfaces has

st are microspheres defined as ldquofine

polym can be

n

r was

containing antigen urine serum etc The latex will become agglutinated and visibly

on method of PS They found that the percentage of grafting PS to the PBA was

greatest for the batch reaction and the PBA-PS core-shell particles with a high degree

grafting remained spherical upon aging test because of the emulsifying abil

copolymer

267 Various Applications of Latex Particles

Latex particles are applicable to a wide range of areas such as biomedical

applications [Piskin et al 1994] especially as the solid phase such as in immunoassays

[Chern et al 2003 Radomske-Galant et al 2003] DNA diagnostic drug delivery carriers

[Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] blood cell separations and

column pack

become a center of attention Of particular intere

er particles having diameters in the range of 01 to several micronsrdquo which

used as functional tools by themselves or by coupling with biocompounds Singer and

Plotz [1956] firstly studied microsphere or latex agglutination test (LATrsquos) by using

monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support o

which the biomolecules were going to adsorb The biomolecule adsorption howeve

limited by possible desorption of the adsorbed species or loss of specific activity of the

complex formed Since this work was published the latex particle applications for

immunoassay have been rapidly and widely studied and developed

Latex agglutination test or latex immunoassays start with tiny spherical latex

particles with a uniform diameter and similar surface properties The particles are coated

with antibodies (sensitized) through the hydrophobic interaction of portions of the protein

with the PS surface of the particles If sensitized particles are mixed with a sample

29

agglomerate Latex tests are inexpensive as compared with the other techniques [Ba

1988]

ngs

sed

tion Rheumatoid factor (RF) in different age subpopulations has also been

evalu

studied a

latex

Unipath [Percival 1996] has manufactured a range of immunoassay materials ba

on a chromatographic principle and deeply colored latex particles for use in the home and

clinical environments The latex particles which are already sensitized with a

monoclonal antibody can detect any antigens that bound to the surface of the latex

particles Some detectable pollutants include estrogen mimics which induce

abnormalities in the reproductive system of male fishes and lead to a total or partial male

feminiza

ated according to a patientrsquos clinical status by using a rapid slide latex agglutination

test for qualitative and semiquantitative measurement in human serum along with latex

immunoassay method [Onen et al 1998] Magalhaes and his coworkers [2004]

diagnostic method of contamination of male fishes by estrogen mimics using the

production of vitellogenin (VTG) as a biomarker This was based on a reverse

agglutination test developed with monoclonal antibodies specific to this biomarker

Premstaller and his coworkers [Premstaller et al 2000 2001] have prepared a porous

poly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient

chromatographic separation of biomelecules such as proteins and nucleic acids They

used a porogen a mixture of tetrahydrofuran and decanol to fabricate a micropellicular

PS-DVB backbone Legido-Quigley and his colleagues [2004] have developed the

monolith column to obtain further chromatographic functionality to the column by

introducing chloromethylstyrene in place of styrene into the polymer mixture

30

Core shell type monodisperse polymer colloids have been synthesized by Sarobe

and Forcada [1998] with chloromethyl functionality in order to improve the biomolecu

adsorption through a two-step emulsion polymerization process They investigated the

functonalized particles by optimizing the experimental parameters of the functional

monomer including reaction temperature the amount and type of redox initiator sys

used the type of addition of the initiator system and the use of washing They c

le

tem

oncluded

that th

sis

serve as separation

chann

of

usually

along the polypeptide chain which contain positive or negative charges [Norde 1998]

e relation between the amount of iron sulfate and the persulfatebisulfite system

added should be controlled to obtain monodisperse particles and prevent the premature

coagulation of the polymer particles during the polymerization

A semi-continuous emulsion polymerization technique for latex particle synthe

was performed by McDonald and other researchers [Ramakrishnan et al 2004 Steve et

al 1999] They introduced a variety of particles sizes compositions morphologies and

surface modifications to fabricate latex composite membranes (LCMs) Arraying and

stabilizing latex particles on the surface of a microporous substrate form narrowly

distributed interstitial pores formed between the particles which

els They investigated the membrane performance using gas fluxes water

permeability and the retention characterization of dextran molecules From these tests

they concluded that the narrow discriminating layer made of the latex particles leads to a

highly efficient composite membrane

27 Proteins

Proteins are natural polyamides comprised of about 20 different α-amino acids

varying hydrophobicity [Norde 1998] Proteins are more or less amphiphilic and

highly surface active because of the number of amino acid residues in the side groups

31

Proteins are polymers of L-α-amino acids [Solomon and Fryhle 2000] The α refers to a

carbon with a primary amine a carboxylic acid a hydrogen and a variable side-chain

group designated as R Carbon atoms rent groups are asymmetric and can

exhib l

s

e

on

with four diffe

it two different spatial arrangements (L and D configurations) due to the tetrahedra

nature of the bonds The L refers to one of these two possible configurations Amino

acids of the D-configuration are not found in natural proteins and do not participate in

biological reactions Figure 2-8 shows the chiral carbon in 3-D as the L isomer Protein

consist of twenty different amino acids differentiated by their side-chain groups [Norde

1998] The side-chain groups have different chemical properties such as polarity charg

and size and influence the chemical properties of proteins as well as determine the

overall structure of the protein For instance the polar amino acids tend to be on the

outside of the protein when they interact with water and the nonpolar amino acids are

the inside forming a hydrophobic core

Figure 2-8 L-α-amino acid

The covalent linkage between two amino acids is known as a peptide bond A

peptide bond is formed when the amino group of one amino acid reacts with the carboxyl

group of another amino acid to form an amide bond through the elimination of water

This arrangement gives the protein chain a polarity such that one end will have a free

amino group called the N-terminus and the other end will have a free carboxyl group

called the C-terminus [Solomon and Fryhle 2000] Peptide bonds tend to be planar and

32

give the polypeptide backbone rigidity Rotation can still occur around both of the α-

carbon bonds resulting in a polypeptide backbone with different potential conformations

relative to the positions of the R groups Although many conformations are theoretically

possible interactions between the R-groups will limit the number of potential

nd to form a single functional conformation In other words

the conformation or shape of the protein is

α-helix

can interact with other

secondary structures within the sam

conformations and proteins te

due to the interactions of the chain side

groups with one another and with the polypeptide backbone The interactions can be

between amino acids that are close together as in a poly-peptide between groups that are

further apart as in amino acids or even on between groups on different polypeptides all

together These different types of interactions are often discussed in terms of primary

secondary tertiary and quaternary protein structure

The primary amino acid sequence and positions of disulfide bonds strongly

influence the overall structure of protein [Norde 1986] For example certain side-chains

will promote hydrogen-bonding between neighboring amino acids of the polypeptide

backbone resulting in secondary structures such as β-sheets or α-helices In the

conformation the peptide backbone takes on a spiral staircase shape that is stabilized by

H-bonds between carbonyl and amide groups of every fourth amino acid residue This

restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure

Certain amino acids promote the formation of either α-helices or β-sheets due to the

nature of the side-chain groups Some side chain groups may prevent the formation of

secondary structures and result in a more flexible polypeptide backbone which is often

called the random coil conformation These secondary structures

e polypeptide to form motifs or domains (ie a

33

tertiary structure) A motif is a common combination of secondary structures and a

domain is a portion of a protein that folds independently Many proteins are composed o

multiple subunits and therefore exhibit quaternary structures

271 Interaction Forces between Proteins

Proteins in aqueous solution acquire compact ordered conformations In such a

compact conformation the movement along the polypeptide chain is severely restrict

implying a low conformational entropy The compact structure is possible only if

interactions within the protein molecule and interactions between the protein molecule

and its environment are sufficiently favorable to compensate for the low conformational

entropy [Malmsten 1998] Protein adsorption study is often focused on structural

rearrangements in the protein molecules because of its significance to the biological

functioning of the molecules and the important role such rearrangements play in the

mechanism of the adsorption process Knowledge of the majo

f

ed

r interaction forces that act

structures helps to understand the behavior

of pro

arge

a

ded

between protein chains and control the protein

teins at interface These forces include Coulomb interaction hydrogen bonds

hydrophobic interaction and van der Waals interactions

Coulombic interaction Most of the amino acid residues carrying electric ch

are located at the aqueous boundary of the protein molecule An the isoelectric point

(IEP) of the protein where the positive and negative charges are more or less evenly

distributed over the protein molecule intramolecular electrostatic attraction makes

compact structure favorable to proteins Deviation to either more positive or more

negative charge however leads to intramolecular repulsion and encourages an expan

structure Tanford [1967] calculated the electrostatic Gibbs energy for both a compact

impenetrable spherical molecule (protein) and a loose solvent-permeated spherical

34

molecule (protein) over which the charge is spread out From the results he found th

the repulsion force was reduced at higher ionic strength d

at

ue to the screening action of ion

ble

e

the

d the

tween

dehyd

e

ent

oles

tein

Hydrogen bond Most hydrogen bonds in proteins form between amide and

carbonyl groups of the polypeptide backbone [Malmsten 1998] The number of availa

hydrogen bonds involving peptide units is therefore far greater than that involving sid

chains Because α-helices and β-sheets are aligned more or less parallel to each other

interchain hydrogen bonds enforce each other Kresheck and Klotz [1969] examine

role of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between

peptide units do not stabilize a compact structure of protein However because the

peptide chain is shielded from water due to other interactions hydrogen bonding be

peptide groups do stabilize α-helical and β-sheet structures

Hydrophobic interaction Hydrophobic interaction refers to the spontaneous

ration and subsequent aggregation of non-polar components in an aqueous

environment In aqueous solutions of proteins the various non-polar amino acid residues

will be found in the interior of the molecule thus shielded from water The

intermolecular hydrophobic interaction for the stability of a compact protein structure

was first recognized by Kauzmann [1959] If all the hydrophobic residues are buried in

the interior and all the hydrophilic residues are at the outermost border of the molecul

intramolecular hydrophobic interaction would cause a compact protein structure

However geometrical and other types of interactions generally cause a fraction of the

hydrophobic residues to be exposed to the aqueous environm

Van der Waals interaction The mutual interaction between ionic groups dip

and induced dipoles in a protein molecule cannot be established as long as the pro

35

structure is not known in great detail Moreover the surrounding aqueous medium also

contains dipoles and ions that compete for participation in the interactions w

the protein molecule Dispersion interactions favor a compact structure However

because the Hamaker constant for proteins is only a little larger than that of water the

resulting effect is relatively small [Nir 1977]

272 β

ith groups of

rgiles et al 1996

Floeg

) is degraded [Floege 2001] Renal

β2M from the serum resulting in an increase in β2M

conce well

the

)

a

al

2-Microglobulin (β2M)

The protein β2M is of particular interest because it is involved in the human

disorder dialysis-related amyloidosis (DRA) [Geiyo et al 1985 A

e 2001] DRA is a complication in end stage renal failure patients who have been on

dialysis for more than 5 years [Bardin et al 1986 Drueke 2000] DRA develops when

proteins in the blood deposit on joints and tendons causing pain stiffness and fluid in

the joints as is the case with arthritis In vivo β2M is present as the non-polymorphic

light chain of the class I major histocompatibility complex (MHC-I) As part of its

normal catabolic cycle β2M dissociates from the MHC-I complex and is transported in

the serum to the kidney where the majority (95

failure disrupts the clearance of

ntration by up to 60-fold [Floege 2001] By a mechanism that is currently not

understood β2M then self-associates into amyloid fibrils and typically accumulates in

musculoskeletal system [Homma et al 1989] Analysis of ex vivo material has shown

that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRA

is present as of full-length wild-type β2M although significant amounts (~20-30) of

truncated or modified forms of the protein are also present [Floege 2001 Bellotti et al

1998] Figure 2-9 shows the ribbon diagram of human β2M Native β2M consists of

single chain of 100 amino acid residues and has a seven stranded β-sandwich fold typic

36

of the immunoglobulin superfamily [Saper et al 1991 Trinh et al 2002] β2M was fi

isolated from human urine and characterized by Berggard et al [1980] in1968 The

normal serum concentration of β

rst

re

f

e

2M is 10 to 25 mgL It is a small globular protein with

a molecular weight of 118 kDa a Strokes radius of 16Aring and a negative charge under

physiological conditions (isoelectric point IEP = 57) β2M contains two β-sheets that a

held together by a single disulphide bridge between the cysteines in positions 25 and 81

[Berggard et al 1980 Parker et al 1982 Cunningham et al 1973] β2M cannot be

removed completely by current dialysis techniques but through a better understanding o

the structure and interaction forces that lead to this structure it will be possible to mor

efficiently remove this problematic protein

Figure 2-9 Ribbon diagram of human β M taken from the crystal structure of the protein

bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khal 2000]

273 Serum Albumin

Serum albumin is the most abundant protein found in plasma and is typically

present in the blood at a concentration of 35~

2an et

50gL According to extensive studies about

its physiological and pharmacological properties albumin has a high affinity to a very

wide range of materials such as electrolytes (Cu+2 Zn+2) fatty acids amino acids

metabolites and many drug compounds [Fehske et al 1981 Kraghhansen 1981 Putnam

37

1984 Peters 1985] The most important physiological role of the protein is therefore to

bring such solutes in the bloodstream to their target organs as well as to maintain the pH

and osmotic pressure of the plasma Bovine serum albumin (BSA) is an ellipsoidal

protein with the dimensions of 140 X 40 X 40Aring [Peter 1985] The primary structure is a

single helical polypeptide of 66 kDa (IEP = 47) with 585 residues containing 17 pairs of

as a soft an

rfaces [Kondo et al 1991 Norde et al 1992 Soderquist et al

1980 ikely

is

disulfide bridges and one free cysteine [Dugaiczyk et al 1982] BSA has been classified

d flexible protein because it has a great tendency to change its conformation

on adsorption to solid su

Carter and Ho 1994] and consists of three homologous domains (I-III) most l

derived through gene multiplication [Brown 1976] Each domain is composed of A and B

sub-domains [He et al 1992] The secondary structure of human serum albumin (HSA)

shown in Figure 2-10

Figure 2-10 Secondary structure of human serum albumin (HSA) with sub-domains

[Zunszain et al 2003]

HSA has the same structure domains with the serum albumin from other species

such as BSA [Brown 1976] Although all three domains of the albumin molecule have

similar three-dimensional structures their assembly is highly asymmetric [Sugio et al

38

1999] Domains I and II are almost perpendicular to each other to form a T-shaped

assembly in which the tail of subdomain IIA is attached to the interface region between

sub-domains IA and IB by hydrophobic interactions and hydrogen bonds In contrast

domain III protrudes from sub-domain IIB at a 45ordm angle to form the Y-shaped assembly

for domains II and III Domain III interacts only with sub-domain IIB These features

make the albumin molecule heart-shaped

28 Protein Adsorption

Protein adsorption studies date back to the 1930rsquos At the beginning these studies

mainly focused on the determination of the molecular weight electrophoretic and

ral

rearrangem

adsorption and biocompatibility of the sorbent materials were investigated [Norde 1986]

There are interaction forces at the interfaces between protein molecules and latex

particles These forces are mainly divided into the following groups hydrophobic

interaction ionic interaction hydrogen bonding and van der Waals interaction [Andrade

1985]

Hydrophobic interaction It is known that hydrophobic interaction has a major

role in protein adsorption phenomena The adsorption of proteins on the low charged

latex particles occurs by this interaction force Generally monomers such as styrene offer

hydrophobic surfaces th

by this interaction force is maximum at the isoelectric point (IEP) of the protein and the

strength [Suzawa et al 1980 1982 Shirahama et al 1989 Kondo et al 1992] By the

chromatographic applications Later the adsorption mechanism especially the structu

ents was studied Recently the studies of the relation between protein

281 Interaction between Protein Molecule and Latex Particle

at protein molecules adsorb to The amount of adsorbed protein

pH at maximum adsorption shifts to a more acidic region with an increase in ionic

39

reports [Suzawa et al 1980 1982 Lee et al 1988] protein adsorption was greater

hydrophobic surface than on a hydrophilic one implying that hydrophobic interaction is

on a

s

ydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA)

Ionic

ely

t

ces

n force is operative over small

distan e

ble

n

nt dialysis membrane therapy for end stage renal disease (ESRD)

one of most dominant forces in protein adsorption

Ionic interaction Negatively charged latex particles have ionic functional

groups on their surfaces such as salts of sulfonic and carboxylic acid Sulfate group

originate from an initiator such as sodium persulfate and carboxylic groups originate

from a h

bonds are formed between the negative charges of these latex particles and positive

surface charges of protein molecules The conventional low-charged latex particles rar

form these ionic bonds

Hydrogen bonding Hydrogen bond is a strong secondary interatomic bond tha

exists between a bound hydrogen atom (its unscreened proton) and the electrons of

adjacent atoms [Callister 1999] Protein can be adsorbed on hydrophilic polar surfa

through hydrogen bonding Hydrogen bonds are frequently formed between hydroxyl-

carbonyl or amide-hydroxyl Hydroxyl-hydroxyl or amide-hydroxyl bonds are also

formed in protein adsorption

Van der Waals interaction This interactio

ces only when water has been excluded and the two non-polar groups come clos

to each other Lewinrsquos calculation showed that the van der Waals interaction is negligi

compared with the forces involved in the entropy increases ie hydrophobic interactio

[Lewin 1974]

29 Hypothesis for Toxin Removal

As mentioned earlier insufficient removal of middle molecular range toxins is a

major drawback of curre

40

patien

is

y

ese

haracteristics over the last decade However removal

effici

ing a novel membrane design composed of an assembly of engineered

polymeric latex particles s ions Important factors

will i

e

ith a

is established it is

expec

e

ts This may cause destructive arthritis and carpal tunnel syndrome inhibit the

immune system and accelerate cardiovascular disease leading to death among dialys

patients In order to overcome these complications artificial dialysis membranes have

been developed to increase the capacity for removing middle molecular weight toxins b

changing the pore size of dialysis membranes and using new materials that adsorb th

toxins for improved removal c

ency is not as high as those achieved by a normal healthy kidney

With the knowledge obtained from all above literatures the following hypothesis

has been established for the development of a membrane for the successful removal of

the target protein β2-Microglobulin (β2M) without the removal of serum albumin This

will be done us

ynthesized to predetermined specificat

nclude pore size and surface chemistry

291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size

The packing of monodispersed spherical particles can lead to the formation of

porous layers suitable for use as filters and membranes [Hsieh et al 1991] The pore siz

is defined as the largest spherical particles that can pass through the interstitial spaces

It is well known that monodispersed spherical particles can be obtained from the

seed emulsion polymerization method Many commercially available latex particles are

synthesized by this technique and are inexpensive When the defect free membrane w

monodisperse pore size distribution based on a particular particle array

ted to outperform the traditional hemodialysis membrane limitation which is a

broad range of pore size distribution When spherical particles pack in regular crystallin

arrays a number of packing geometries such as hexagonal closest packing cubic closest

41

packing and body-centered cubic packing are possible The pore size of the array

depends on these packing geometries as well as particle size Theoretical pore size can be

calculated from the simple geometry of the arr

ay Figure 2-11 shows the relationship

between particle size and pore size

0001

001

01

001 01 1 10

Por

e

1

10

microm

) S

ize

(

Particle Diameter (microm)

Body-Centered Cubic ArrayClosest Packed Array

0001

Siz

e (

Particle Diameter (microm)

001

01

001 01 1 10

Por

e

1

10

microm

)

Body-Centered Cubic ArrayClosest Packed ArrayBody-Centered Cubic ArrayClosest Packed Array

and closed packed arrays [Steve et al 1999]

McDonald and his coworkers [Ramakrishnan et al 2004 Steve et al 1999] have

described the fabrication of latex composite membranes and their performance in

previous papers It is from this prior research and knowledge gained from conversations

with Dr McDonald that this current research has been initiated [McDonald 2003] The

focus of this thesis is the extension of McDonaldrsquos research in the area of composite

membranes to medical application such as dialysis membranes

292 Toxin Removal by Selective Adsorption on Engineered Latex Particles

The flexibility in latex surface properties is a significant advantage to this

materialrsquos ability to separate proteins since the selectivity is strongly dependent on the

charge interactions of both the protein and the latex particles under the conditions of

Figure 2-11 The relationship between pore size and particle size in body-centered cubic

42

separation [Menon et al 1999 Chun et al 2002] First all sorbents were designed to

have negative surface properties because most plasma proteins in blood are negative and

should not be removed with charge interaction between proteins and sorbents at

physiological condition (pH74) It was reported that a negatively charged surface was

more blood compatible than a positive one [Srinivasan et al 1971] The toxin protein β2-

Microglobulin (β2M) can be selectively adsorbed on the surface of latex particles by the

design of a suitable hydrophobic portioned surface where only β2M protein can be

anchored with hydrophobic interactions Albumin adsorption on the latex particle is not

side-on m

adsorption of β M protein on the engineered latex particles

allowed because charge repulsion is more dominant than hydrophobic interaction with

ode The figure 2-12 shows the schematic representation of the selective

2

PMMAPAAShell

COO-

OSO3-

OSO3-

PSCore

COO-

-

-

-

-

-

-

Albumin-

Chargerepulsion

--

-

- -

- -

- --

Hydrophobic

β2-Microglobulin

2

Interaction

β -Microglobulin

PMMAPAAShell

COO-

-

-

-

-

-

-

--

-

-

-

-

-

Albumin-

Chargerepulsion

OSO3-

OSO3-

PSCore

COO-

--

-

- -

--

-

- -

- -

- --

- -

- --

Hydrophobic

β2-Microglobulin

2

particles at pH 74

Interaction

β -Microglobulin

Figure 2-12 Schematic representation of the protein adsorption on the core shell latex

43

Hydrophilichydrophobic microdomain structures were proven to be more blood

compatible [Mori et al 1982 Higuchi et al 1993 Deppisch et al 1998] The

optimization of suitable hydrophobic to hydrophilic ratio is also important for the

biocompatibility The monomers such as styrene (St) methyl methacrylate (MMA) and

acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in

emulsion polymerization process

In summary the background and fundamental literature survey about the history

material properties and limitation of hemodialysis membrane latex particle preparation

surface chemistry and manufacturing process target proteins and protein adsorption and

finally the hypothesis for a toxin removal with high separation efficiency have been

suggested This research focuses on such hypothesis and the materials and

characterization methodology for achievement of suggested hypotheses are described in

the next chapter

CHAPTER 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY

As mentioned earlier the goal of this study is to prepare polymeric latex particles

with tailored properties to maximize separation of toxin molecules and to investigate

fundamental interactions between the applied particles and molecules in the

system to optimize the membrane performance for hemodialysis applications Polymeric

latex particles wit

the

biological

h monodisperse size distribution to obtain uniform pore size and

various size ranges to utilize membrane construction are necessary Surface engineering

with various combination of hydrophobichydrophilic domain on the surface of latex

particles is expected to affect the removal of target protein by selective adsorption and to

improve the biocompatibility of membrane Therefore the materials and characterization

methodology are addressed in this chapter

31 Materials

Styrene (St) monomer used for a seed and a core particle was purchased from

Fisher Scientific and used without any other purification process Acrylic acid (AA) and

methyl methacrylate (MMA) monomers introduced for shell formation were purchased

from Fisher Scientific and used without any other purification process Sodium persulfate

(SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as

received Divinylbenzene (DVB) crossliking agent was purchased from Aldrich The

anionic surfactant Aerosol regMA80-I [sodium di(13-dimethylbutyl) sulfosuccinate] was

kindly donated by Cytec 78-80 Aerosol regMA80-I is mixed with isopropanol and

water Its critical micelle concentration (CMC) is 245 mM (119) Ion exchange resin

44

45

AGreg 501-x8 Resin (20-50 mesh) was purchased from Bio-Rad Laboratories Inc It is a

mixed-bed resin with H+ and OH- as the ionic forms and is 8 crosslinked Bovine serum

albumin (BSA) (heat shock treated) was purchas Fisher Scientific The isoelectric

point of t lin

(β2M)

as

gma

ed from

he BSA is 47-49 and the molecular weight is 66300Da β2-Microglobu

was purchase from ICN Biomedicals Inc and was separated from patients with

chronic renal disease and lyophilized from ammonium bicarbonate The molecular

weight by SDS-PAGE was approximately 12000Da BSA and β2M proteins were used

received without further purification process In order to investigate the protein

adsorption evaluation bicinchoninic acid protein assay kits were purchased from Si

(Cat BCA-1) and Pierce Biotechnology (Cat 23225) The chemical structures for the

main chemicals are shown in Figure 3-1

CH2 CH

Styrene (Mw = 10415)

CH2 CHCH2 CH

Styrene (Mw = 10415)

CH2 CH

Acrylic acid (Mw = 7206)

C

OH

O

CH2 CH

Acrylic acid (Mw = 7206)

C

OH

O

CH CH2

C

OCH3

O

CH

CH CH3

2

C

OCH3

O

CH3

Methyl methacrylate (Mw = 10012)Methyl methacrylate (Mw = 10012)

CH2 CH

Divinylbenzene (Mw = 1302)

CH CH2

CH2 CH

Divinylbenzene (Mw = 1302)

CH CH2CH CH

+(Na) -(O3S)

CH -C-O-CH-CH -CH-CH3

O

2 2 3

O CH CH3

CH3 CH3

CH-C-O-CH-CH2-CH-CH3

Anionic surfactant (MA80-I) (Mw = 3885)

+(Na) -(O3S)

CH -C-O-CH-CH -CH-CH3

O

2 2 3

O CH CH3

CH3 CH3

CH-C-O-CH-CH2-CH-CH3

Anionic surfactant (MA80-I) (Mw = 3885)2

+(Na)-(O) S OO

O O

- +

Initiator sodium persulfate (Mw = 23811)

O SO

(O) (Na)+ -

O O

- +(Na) (O) S OO

O SO

(O) (Na)+ -

O O

- +

Initiator sodium persulfate (Mw = 23811) Figure 3-1 Chemical structure of main chemicals

(Na) (O) S OO

O SO

(O) (Na)

46

32 Latex Particle Preparation

The experimental set up and the particle preparation schemes for various types and

size ranges of latex particles are shown in Figure 3-2 and 3-3 In the experimental setup a

mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at

end of glass stirrer The agitation rate is precisely adjusted by controller motor Paar glas

reactor with 1L volume is partially in silicon oil bath on hot plate The reactor has fo

openings for a stirrer a thermometer a nitrogen gas inlet and a reactant feeding lnlet

The reactants for latex polymerization are fed by precisely controlled by meterin

The particle preparation is described in each latex preparation section

the

s

ur

g pump

Figure 3-2 Experimental setup for semi-continuous emulsion polymerization

1 Motor 2 Mechanical stirrer bar 3 Thermometer 4 Inlet of N2 5 Inlet for initiator and monomer 6 Glass reactor (l L) 7 Oil bath 8 Hot plate 9 Pump for initiator 10 Pump for monomer 11 Tubes for reactant feed

47

Styrene monomer

Bare PS

PSPMMA100

PSPMMA90PAA10

PSPMMA P75 AA25

Gr

win

ticle

size

PS seedparticles

og

par

Styrene monomer

Bare PS

PSPMMA100

PSPMMA90PAA10

PSPMMA P75 AA25

Gr

win

ticle

size

PS seedparticles

og

par

Figure 3-3 The particle preparation scheme in various types and size ranges of latex particles

321 Preparation of Seed Latex Particles

Polystyrene seed particles were synthesized through a typical semi-continuous

emulsion polymerization Polystyrene latex particles were prepared in a four necked Paar

glass vessel equipped with a mechanical glass stirrer thermometer glass funnel for

nitrogen gas purge and a tube for monomer feeding The reactor was placed in a silicon

oil bath on a hot plate for homogenous and stable tempe he vessel was

firstly charged with de-ionized water the emulsifier so itiator

sodium bicarbonate (SBC) styrene (St) monomer divinylbenzene (DVB) monomer

under nitrogen gas atmosphere and then heated to 72plusmn2 as

llowed to continue for another hour Second reactants St and DVB monomers SPS

initiator and SBC were fed separately using fine fluid metering pumps (Model RHSY

Fluid metering INC NY) under a nitrogen gas atmosphere for 2h After complete feeding

rature control T

dium persulfate (SPS) in

oC for 1h The reaction w

a

48

of all reactants the reaction was continued for another hour at elevated temperature 80plusmn1

oC and then cooled to room temperature by removal of the oil bath

322 Preparation of Seeded Latex Particles

The particle growth was achieved by seeded continuous emulsion polymerization

The vessel was charged with the polystyrene seeds and de-ionized water to make desired

solid content and heated up to 72plusmn2 oC for 1h under nitrogen gas atmosphere Then St

and DVB monomers and solution of SPS and SBC in de-ionized water were continuously

added using metering pump with a precisely controlled feed rate After feeding all

reactants the reaction was continued for three hours keeping temperature of 80plusmn2 oC and

then the reaction vessel was cooled down to room temperature

Monodisperse spherical polystyrene seeds with a 280nm mean diameter were used

e-polymerized polystyrene seeded emulsion

was charged in a 500m

323 Preparation of Core Shell Latex Particles

for core shell structured latex particle The pr

l glass flask under a nitrogen gas atmosphere De-ionized water

was added to achieve the desired solid content The emulsion was heated to 80plusmn2oC for

10 hour Monomers initiator and SBC to form the shell were added by fluid metering

pumps (LAB PUMP JRRHSY model Fluid metering Inc USA) with fine controlled

feed rate Flow rate can be adjusted by control ring graduated in 450 division from 0 to

100 flow which is in the range of 10mlmin respectively The reaction was sustained

for additional 30 hours at 90plusmn2oC then cooled down to room temperature

324 Purification of Synthesized Latex Particles

Synthesized latex particles were purified with an ion exchange resin 200g (10

ww polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin

and stirred for 40 minute to remove unreacted monomers initiators and other impurities

49

Then the emulsion was diluted with DI water to a specific degree of solid content as

needed

33 Characterization

331

solid

termined

oscopy

d

hours

Dried latex (10mg) was mixed with

250 m on

Degree of Conversion

The degree of monomer to polymer conversion was determined gravimetrically

following the procedure described by Lee et al [1995] before the latex cleaning process

The synthesized emulsion raw materials were weighed and dried in a conventional oven

at 120oC for 30min to evaporate any unreacted monomers and water The remaining

was weighed and the degree of conversion was determined This process was repeated

three times and a mean value of the degree of conversion was de

332 Fourier Transform Infrared (FTIR) Spectr

A molecule can absorb only select frequencies (energy) of infrared radiation which

match the natural vibration frequencies The energy absorbed serves to increase the

amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]

Only those bonds which have a dipole moment that changes as a function of time can

absorb the infrared radiation The range of wavelengths for infrared radiation is between

25 microm (4000 cm-1) and 25 microm (400cm-1)

The synthesis of the latex particles such as polystyrene (PS) homopolymer and

PSPMMA PSPMMA90PAA10 PSPMMA75PAA25 core-shell copolymers were verifie

with FTIR Purified latex particles were dried in vacuum oven at 40oC for 24

before analysis in the FTIR (Nicolet Magma USA)

g of KBr which had also been dried in vacuum oven at 120oC for 3h Transmissi

spectra using the drift mode were plotted with 128 scans and 4cm-1 resolution

50

333 Quasielastic Light Scattering (QELS)

Latex particle size was measured by the Brookhaven ZetaPlus particle size

analyzer The solid concentration as 0001 (wv) This

ation spectroscopy (PCS) of quasielastically scattered light

For th s

ed using a FE-SEM (JEOL

JSM-

ith

ent

01

us zeta-

potential analyzer It measures the electrophoretic mobility velocity of charged colloidal

of latex particles used w

instrument uses photon correl

is technique time dependent interference patterns of light scattered from particle

in the sample cell are analyzed The interference pattern changes due to Brownian

motion giving rise to fluctuations in scattering intensity The frequency of these

fluctuations depends on the particlersquos diffusion constant that is inversely proportional to

the particle diameter

334 Field Emission-Scanning Electron Microscopy (FE-SEM)

Particle size and surface morphology were characteriz

6335F Japan) A diluted suspension of latex particles in water was dropped onto a

silicon wafer at room temperature and allowed to dry The sample was then coated w

the thinnest layer of carbon needed to obtain the required conductivity for this instrum

Secondary electron image mode was used with a 15KV of accelerating voltage The

magnification range used was between 10000X and 70000X

335 Zeta Potential Measurement

Synthesized and purified latex particles were diluted with de-ionized water to 0

wt and adjusted six different pH values 205 345 481 565 643 and 747 These

particle suspensions were then transferred to standard cuvettes for zeta potential

measurement At least two runs of ten measurements were taken for each sample and

averaged

The zeta potential measurement was carried out using Brookhaven ZetaPl

51

particles in solution and calculate zeta-potential by using the Smoluchowski equation

The f quency

gnitude

umin (BSA) as a standard model protein and β2-Microglobulin

uffer (PB) and

phosp

5

h the selected protein

e 005 01 03 05 and 07 mgml and those for

β2M

d at

teins in the supernatant after the centrifugation process using the

bicinc t

requency of laser light passing through the sample and is compared to the fre

of a reference beam The shift in the frequency called a Doppler shift and the ma

of the shift correspond to the polarity and the magnitude of the electrophoretic mobility

respectively The zeta potential is calculated from the solution conditions and the

measured mobility

336 Protein Adsorption

Protein adsorption experiments of the synthesized latex particles were performed

with bovine serum alb

(β2M) as a target protein in two types of buffer solution phosphate b

hate buffered saline (PBS) To make 5 mM of PB solution 0345g of sodium

phosphate monobasic (NaH2PO4H2O) was dissolve in 500ml of de-ionized water To

make PBS solution 0345g of sodium phosphate monobasic (NaH2PO4H2O) and 4178g

(143mM) of sodium chloride (NaCl) were dissolved in 500ml of de-ionized water The

synthesized latex particles were diluted with each buffer to have a solids content of 0

(ww) adjusted to pH values of 32 48 and 74 and mixed wit

BSA concentrations were chosen to b

were 0015 0030 0045 and 0060mgml The mixture was gently rotated using

the shaker Labquakereg (BarnsteadThermolyne Model 4002110 USA) with 8 RPM in

the incubator at 37oC for 12 hours before the latex-protein mixture was centrifuge

13000 rpm for 15min The amount of protein adsorbed was determined by quantifying

the free pro

honinic acid (BCA) assay method [Lowry et al 1951 Smith et al 1985 Baptista e

al 2003 Wiehelman et al 1988 Brown et al 1989] BCA assay kits consist of Reagent

52

A containing bicinchoninic acid sodium carbonate sodium tartrate and sodium

bicarbonate in 01N NaOH with pH=1125 and Reagent B containing 4 (wv) copper

(II) sulfate pentahydrate The BCA working reagent was prepared by mixing 50 parts of

Reagent A with 1 part of Reagent B 100microl of protein supernatant was then mixed with

2ml of BCA working reagent in a UV cuvette Incubation was allowed to continue

at room temperature

further

until color developed about 2h The absorbance at 562nm was

pectrophotometer (Perkin-Elmer Lambda 800 USA)

Unkn

Separate the p rticles in centrifuge mix supernatant with BCA reagents

measured using a UV-VIS s

own concentration of sample protein was determined by comparison to a standard

of known protein concentrations The adsorbed amount per unit surface area was

determined by the mass balance of the protein after adsorption process The simple

schematic of the procedure for a protein adsorption test is shown in Figure 3-4

Incubate mixture with agitation for 12hrs at 37oC

aand develop the color by incubation for 2hrs at room temperature

Suspend latex particles in aqueous buffer media

Add the dissolved target protein

Measure color intensity using UVVis spectroscopy at 562nm

Suspend latex particles in aqueous buffer media

Separate the p rticles in centrifuge mix supernatant with BCA reagents

Add the dissolved target protein

Incubate mixture with agitation for 12hrs at 37oC

aand develop the color by incubation for 2hrs at room temperature

Measure color intensity using UVVis spectroscopy at 562nm

Figure 3-4 Schematic of the procedure for a protein adsorption test

53

337 Blood Biocompatibility by Hemolysis Test

Hemolysis is the destruction of red blood cells which leads to the release of

hemoglobin from within the red blood cells into the blood plasma Hemolysis testing is

used to evaluate blood compatibility of the latex particles and the damaged to the red

blood cells can be determined by monitoring amount of hemoglobin released The red

blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutes

Figure 3-5 shows the phase separated blood with plasma white blood cells and red

cells after centrifugation process Separated RBSs were then washed with isotonic

phosphate buffer solution (PBS) at pH 74 to remove debris and serum protein Th

process was repeated 3 times

blood

is

Figure 3-5 Separation of RBC from whole blood by centrifuge process

Prepared latex particles were re-dispersed in PBS by sonification to obtain

homogeneously dispersed latex particles 100microl of the mixture of red blood cell (3 parts)

and PBS (11parts) was added to 1ml of 05 (ww) particle suspension PBS was used as

a negative control resulting 0 hemolysis and DI water used as a positive control to

produce 100 hemoglobin released by completely destroyed RBCs The mixture was

incubated in water bath with gentle shaking for 30 minutes at 37oC and then centrifuged

at 1500 rpm for 15 minutes 100microl of the supernatant was mixed with 2ml of the mixture

) (EtOHHCl = 2005 ww) to prevent the

White blood cells

Plasma

Red blood cells

of ethanol (99) and hydrochloric acid (37

54

precipitation of hemoglobin In order to remove remaining particles the mixture was

centri fuged again with 13000 RPM at room temperature The supernatant was then

transferred into the UV cuvette The amount of hemoglobin release was determined by

monitoring the UV absorbance at a wavelength of 397nm The schematic of the

procedure for hemolysis test is shown in Figure 3-6

Separate red blood cells (RBCs) from blood

Mix RBCs with phosphate buffer solution (PBS)

Wash and re-suspend RBC with PBS repeat three times

Add la l of RBC and incu in at 37oC

tex particle suspension into 100mbate sample with agitation for 30m

Separate particle ith EtOHHCl solutions and mix the supernatant w

Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm

Separate red blood cells (RBCs) from bloodSeparate red blood cells (RBCs) from blood

Mix RBCs with phosphate buffer solution (PBS)Mix RBCs with phosphate buffer solution (PBS)

Wash and re-suspend RBC with PBS repeat three timesWash and re-suspend RBC with PBS repeat three times

Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC

Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC

Separate particles and mix the ith EtOHHCl solution supernatant wSeparate particles and mix the ith EtOHHCl solution supernatant w

Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nmEvaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm

Figure 3-6 Schematic of the procedure for hemolysis test

In summary a description of the experimental materials and characterization

methodology has been explained In the next chapter the results and discussion of the

data obtained from these experiments is addressed

CHAPTER 4 RESULTS AND DISCUSSION

41 Polymerization of Latex Particles

411 Polystyrene Seed Latex Particles

Polymer particles have many different applications including spacer particles

lubricants packing materials for chromatography standard particles and diagnostic

drugs All applications strongly require these particles to have a uniform particle size

Uniform particle size is the first requirement for a latex composite membrane formed by

particle arrays with the interstitial spaces serving as pores for size discriminations [Jons

et al 1999 Ramakrishnan et al 2004] Although suspension polymerization has been

mainly used as one of conventional polymerization methods to prepare uniform particles

the uniformity of the recovered particles is insufficient for membrane use There is

another method known as seed emulsion polymerization where a vinyl monomer is

absorbed into fine monodiserse seed particles and polymerization causes the monomer to

crease the sizes of the seed particles uniformly In order to obtain monodisperse larger

particles by this m

particles and polymerization of the monomer is repeated

In this study cross-linked polystyrene seed latex particles were firstly synthesized

in the presence of styrene monomer divinylbenzene crosslink agent sodium persulfate

initiator sodium bicarbonate buffer and the Aerosol regMA80-I [sodium di(13-

dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion

polymerization process in which one or more components can be added continuously

in

ethod the procedure of absorption of monomer into fine polymer

55

56

Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and

buffer are continuously added to enlarge the seed latex particles Various profiles of

particle nucleation and growth can be gene different orders of component

addition during polymerizati f this process such as easy

control of the polymeriza particle number

and particle morphology The main goal in

this process is to avoid secondary particle

rated from

on There are several advantages o

tion rate monodisperse particle size and

colloidal stability copolymer composition

formation leading to a monodisperse particle

size distribution Figure 4-1 shows the schematic representation of seed latex particle

preparation and growth

Monomer droplet

Seed particles Amount of surfactantsis variable

Enlarged final Add monomers

PS

PS

PSs4

particles Number of particlesare constant

Seed code

PSs1

s2

s3

Monomer droplet

Seed particles Amount of surfactantsis variable

Enlarged final Add monomers

PS

PS

PSs4

tion The

particles Number of particlesare constant

Seed code

PSs1

s2

s3

Figure 4-1 Schematic representation of semi-continuous seed latex particles preparation and growth

After the first synthesis of a small size of crosslinked polystyrene (PS) seed

particles stabilized by surfactants styrene DVB and initiator are continuously fed into

the system to enlarge the seed latex particles maintaining a narrow size distribu

recipes and reaction conditions for synthesizing these seed particles are summarized in

57

Table 4-1 The original recipe for latex particle preparation from McDonald was

modified for this emulsion system [McDonald 2003]

Table 4-1 The polymerization recipe of polystyrene (PS) seed particles Latex label PSS259 PSS233 PSS207 PSS181

Temperature (oC) 75 plusmn 5 75 plusmn 5 75 plusmn 5 75 plusmn 5

Divinylbenzene (g) 052 052 052 052

MA 80 (g) 259 233 207 181

NaHCO

Watera (g) 1315 1315 1315 1315

Styrene (g) 9948 9948 9948 9948

112 112 112 112

Na2S2O8 (g) 190 190 190 190

Waterb (g) 200 200 200 200

Reaction time (min) 140 140 140 140

Solids content 396 396 396 396

Mean particle diameter (nm) 126plusmn75 171plusmn39 182plusmn41 216plusmn53

Conversion () 965 979 957 971

3 (g)

Four different seed latex particles were synthesized based on surfactant amount in

order to determine the optimum condition for stable and monodisperse latex particles

The emulsion polymerization of latex particles has to be carried out in a narrow range of

surfactant causes flocculation or phase separation leading to broad particle

size distribution and an unstable emulsion sy

surfactant concentration where particles are stable [Nestor et al 2005] A wide range of

concentrations

stem Therefore the range of surfactant

amount was varied from 181g to 259g in increments of 026g in each latex label Other

reactants amounts were kept constant Watera is the initial charge amount and waterb

represents the amount of aqueous solution containing dissolved initiator and buffer

58

which are fed continuously after the seed particle creation The extent of monome

polymer conversion was obtained by gravimetric calcu

r to

lation and was more than 95 at

cripts such as

S207 and S181 i the surf mount added for the seed latex

nversion indi perce experi obtain

content divided by the theoretically estimated solid content and was calculated by the

all seed particles labeled PSS259 PSS233 PSS207 and PSS181 Labeled subs

S259 S233 ndicate actant a

preparation The co cates the ntage of mentally ed solid

following formula

100 colids () times=entsoliatedntentoalExperimentConversion

n the experimenta content is the weight of olid rema after

mulsion at 120oC f in and the estimated solid content is th ht

lated from the recipe

rti as di each t load d

mple of ea pension iluted by (ww)

deionized water and the mean particle diameter was determined using the Brookhaven

ZetaPlus particle size analyzer From these results the seed latex particle size increased

as the amount of surfactant decreased The amount of emulsifier also affects the

polymerization process by changing the number of micelles and their size It is known

that large amount of emulsifier produces larger numbers of smaller sized particles [Odian

1991] and was corroborated in the current work

These synthesized seed latex particles should be of narrow size distribution in order

to obtain monodisperse larger particles The SEM image showing the particle

morphology and size of a sample of polystyrene seed particles can be seen in the Figure

4-2

contdEstim (4-1)

In this equatio l solid the s ining

evaporation of e or 30m e weig

of monomers calcu

As expected the seed pa cle size w fferent for surfactan ing in see

emulsion system A sa ch sus was d 0001 with

59

Figure 4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)

PSS259 (B) PSS233 (C) PSS207 (D) PSS181 (subscripts indicate the amount of surfactant added)

(A)

(B)

60

(C)

(D)

Figure 4-2 Continued

61

s seen in the image the seed particles are very smooth and spherical The

uniformity in the particle size distribution of these PS seed latex particles can also be

seen in the SEM image From this data we concluded that seed particles synthesized in

the presence of a surfactant with the chosen amounts are suitable for use to prepare larger

particles with high size uniformity and smooth spherical morphology

412 The Growth of Polystyrene (PS) Seed Latex Particles

Seeded emulsion polymerization has been conducted for several decades and

various mechanisms have been proposed Gracio et al [1970] suggested that the growing

polymer particles consist of an expanding polymer-rich core surrounded by a monomer-

rich shell with the outer shell providing a major locus of polymerization Seeded

mulsion polymerization is commonly used for preparing latex particles less than 1microm in

size [Cha et al 1995 Zou et al 1990 1992 Gandhi et al 1990 Park et al 1990] Such

latexes can be obtained from the growth of pre-prepared seed particles The seeds

introduced in this process serve as nucleation sites for particle growth By first swelling

the seed latex particles with additional monomer to the desired size polymerization can

then be initiated Several size ranges of monodisperse polystyrene (PS) latex particles

were prepared by this multistep seeded emulsion polymerization method For the

fabrication of particle-based membranes monodisperse latex particles with a variety of

size ranges are required

In this study seed particles from 171nm to 470nm in mean diameter and with a

highly uniform size distribution were prepared and used Watera is the initial charge

mount and waterb represents the amount aqueous solution containing dissolved initiator

and buffer that is continuously fed into system Feed time is precisely controlled by

metering pump The solid contents were maintained between 199 and 300 After

A

e

a

62

polym d as

es

ere 923 8402

n of these latex particles

are sh

ss

erization under these conditions the conversions of the latex particles labele

PS258 PS320 PS370 and PS410 were 952 957 946 and 964 respectively The recip

used in seeded emulsion polymerization to form the particles less than 500nm in mean

diameter are shown at the Table 4-2 The conversions of the latex particles larger than

500nm in mean diameter and labeled as PS525 PS585 PS640 and PS790 w

723 and 969 respectively The recipes used in the preparatio

own at the Table 4-3

A simple calculation can be used to predict the ultimate size that a latex particle can

reach after ending the polymerization Letrsquos suppose that seed density (ρs) and grown

particle density (ρgp) are the same The following equation is obtained from the ma

balance (m = ρV) where m and V are mass and volume of a particle respectively

gp

gp

s

s

Vm

Vm

In this equation m

= (4-2)

be

s and mgp are the weight of seed and total weight of seed and

added monomers respectively and Vs and Vgp are the volume of seed and grown

particle respectively

If we know the mass of monomer to be added the ultimate particle size can

calculated from the equation 4-3

3

2s D m ⎠⎝+ χ

where D

1s D m

⎟⎟⎞

⎜⎜⎛

= (4-3)

the

in

s and Dgp are the diameters for seed and grown particle respectively and χ is

weight of the monomer added Crosslinked PS latex particles with a size range from

258plusmn112 ~ 410plusmn117 in diameter polymerized by the recipes in Table 4-2 are shown

the SEM images in Figure 4-3

63

Table 4-2 Continuous addition emulsion polymerization recipe for growing polystyrene

Latex label PS

(PS) latex particles less than 500nm in size

258 PS320 PS370 PS410

Initial charge

Seed latex (g) 1000171 1000216 1000216 1000320

Watera (g) 1470 2500 1984 2600

06 07 06 06

Fee

Initiator stream

2 2 8

Feed time (min) 0-167 0-140 0-100 0-92

Solid content 284 300 300 200

Mean particle diameter (nm) 258plusmn112 320plusmn154 182plusmn71 410plusmn117

Conv

Monomer continuous

Styrene (g) 800 1200 800 600

Divinylbenzene (g)

d time (min) 0-163 0-110 0-75 0-82

Na S O (g) 07 065 05 05

NaHCO3 (g) 07 065 05 05

Waterb (g) 750 1200 200 370

ersion () 952 957 946 964 The solid content of seed latex for PS258 PS320 PS 370 and PS410 are 392 399 399 anrespectively

d 300

64

Table 4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size

Latex label PS525 PS585 PS640 PS790

Initial charge

Seed latex (g) 1000390 1000470 1000470 1000320

Watera (g) 1000 520 1200 360

Monomer continuous

Styrene (g) 290 200 400 173

Divinylbenzene (g) 02 014

03 009

Feed time (min) 0-80 0-40 10-76 5-65

Initiator stream

Na2S2O8 (g) 02 014 03 012

NaHCO3 (g) 02 014 03 009

Waterb (g) 200 120 400 200

Feed time (min) 0-85 0-46 0-77 0-75

Solids content 199 200 200 201

Mean particle diameter (nm) 525plusmn151 585plusmn186 640plusmn178 790plusmn693

Conversion () 923 8402 723 969 The solid content of seed latex for PS525 PS585 PS 640 and PS790 are 200 182 193 and 173 respectively

65

Figure 4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)

370nm (D) 410nm

(A)

(B)

66

(C)

(D)

Figure 4-3 Continued

67

article size was well matched to that estimated by the geometrical calculation

From this it can be concluded that the added monomer was consumed for polymer

particle growth with a high degree of conversion From the SEM characterization newly

nucleated particles were not seen as indicated by the narrow particle size distribution

The particles less than 500nm in mean diameter were very spherical in shape with a

highly uniform particle size distribution As the particles were grown larger than 500nm

mean diameter however they became of non-spherical shape with an uneven surface

This irregularity of particle surface can be attributed to the non-homogeneous monomer

swelling into the shell of a growing polymer which can be controlled by the factors such

as temperature agitation speed initiator feeding rate and surfactant amount required to

stabilize the colloidal system The SEM image of latex particles larger than 500nm mean

iameter is shown in Figure 4-4 The particle size distribution is broader than that of the

s particles These particles can however still be used for membrane construction

as a support layer rather than skin layer The support layer does not necessarily have to be

as highly monodisperse as the skin layer which require pores of high uniformity for the

selective removal of toxins

As mentioned earlier the amount of surfactant as well as monomer in an emulsion

system is the primary determinant of the particle diameter [Odian 1991] Anionic

surfactants are generally recommended at the level of 02-30 wt based on the amount

of water [Odian 1991] The critical micelle concentration (CMC) of Aerosolreg MA 80-I

anionic surfactant is 119 The surfactant loading in this emulsion system ranged from

the 121 for SS181 to 171 for SS259

P

d

maller

68

Figure 4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm

(C) 640nm (D) 790nm

(A)

(B)

69

(C)

(D)

Figure 4-4 Continued

70

The surfactant to monomer ratio refers to the value of the surfactant amount

divided by monomer amount after polymerization ends The surfactant amount is the

weight of the surfactant contained in emulsion and the monomer amount is the total

weight of the monomer added for polymerization to form the ultimate polymer particles

in emulsion As the surfactant to monomer ratio decreases mean particle diameter

increases as described by Odian [1991] Generally lower surfactant concentration forms

fewer micelles resulting in larger particle size [Evans et al 1999] However there is a

plateau in particle mean diameter between a surfactant to monomer ratio of 0005 and

0013 This is due to low polymerization conversion of latex particles from monomer

leading to a smaller particle size than expected This is not a factor of surfactant amount

The dependence of the particle size on the amount of surfactant is shown in Figure 4-5

0

200

400

600

800

1000

0000 0005 0010 0015 0020 0025 0030

Surfactant to monomer ratio (ww)

Part

icle

mea

n di

amet

er (n

m)

Figure 4-5 Dependence of the particle size on the surfactant to monomer ratio

71

413

e

f bare

microdomain structures is important for biocompatibility [Mori et al 1982 Higuchi et al

1993 Deppisch et al 1998] Figure 4-6 shows the schematic of core shell latex particle

structure The recipe for core shell structures is shown in Table 4-4

Core Shell Latex Particles

The latex particles shown in Figure 4-3 (A) and labeled as S258 in Table 4-2 wer

used as seeds to prepare core-shell latex particles because of their high particle

uniformity and smooth surface morphology Methyl methacrylate (MMA) and acrylic

acid (AA) monomers were introduced to increase surface hydrophilicity over that o

polystyrene particles AA is a more hydrophilic monomer than MMA because of the

carboxyl acid group in AA is more favorable for hydrogen boding with water The

surface carboxyl groups on PAA may have many promising applications in biomedical

and biochemical fields [Kang et al 2005] MMA monomer is less hydrophobic than

styrene PMMA is known to more biocompatible than PS but still PMMA is hydrophobic

The optimization of suitable hydrophobic to hydrophilic ratios and control of

Polystyreneseed particle

Core shelllatex particles

PSPMMA100

PSPMMA90PAA10

PSPMMA75PAA25

Polystyreneseed particle

Core shelllatex particles

PSPMMA100

PSPMMA90PAA10

Figure 4-6 Schematic of core shell latex particle structures

PSPMMA75PAA25

72

Table 4-4 The preparation recipe of PS core with various shell latex particles Latex 25 label PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA

Initial charge

PS seed latex (g) 500 500 500a

Monomer continuous

Methly methacylate (g) 320 288 240

Acrylic acid (g) 00 32 80

Initiator stream

Na

258 258 258

Water (g) 1570 1570 1570

Feed time (min) 0-120 0-50 0-60

b 200

Feed time (min) 0-122 0-60 0-65

952

2S2O8 (g) 02 02 02

NaHCO3 (g) 02 02 02

Water (g) 200 200

Solids content 183 183 183

Mean particle diameter (nm) 370plusmn186 370plusmn178 370plusmn191

Conversion () 955 924

Three types of core shell structures were prepared PMMA75PAA25 shell is a

copolymer consisting of the PMMA to PAA ratio of 75 to 25 by weight

PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90 to 10 by

weight PMMA100 is the PMMA homopolymer shell on PS core The particle size of

these core-shell particles as well as PS latex particle was prepared to be about 370nm in

mean diameter and are characterized to determine their zeta potential protein adsorption

and biocompatibility The image of SEM of PS and core shell particles is shown at Figure

4-7 The synthesized latex particles had a high uniformity in size and a smooth spherical

surface morphology

73

Figure 4-7 Scanning

Electron Micrograph of latex particles (A) PS (B) PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMMA75PAA25

(A)

(B)

74

(C)

(D)

Figure 4-7 Continued

75

42 Characterization of Latex Particles

421 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectroscopy has proven to be a powerful tool to characterize polymeric

materials A molecule can absorb only select frequencies (energy) of infrared radiation

which match the natural vibration frequencies The energy absorbed serves to increase

the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]

Only those bonds which have a dipole moment that changes as a function of time can

absorb infrared radiation The FTIR spectra of polymerized latex particles made during

this work are shown in Figure 4-8 There are number of characteristic peaks for PS latex

particles [Bhutto et al 2003 Li at al 2005] Aromatic C-H stretching vibration is shown

at 3002 cm-1 and 3103 cm-1 and aliphatic C-H asymmetrical and symmetrical stretching

shown at 2900 cm-1and 2850 cm-1 respectively There is an additional carbonyl (C=O)

adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some

amount of either or both PMMA and PAA The broad OH group peak at 3400 cm-1

appear for particles containing some amount of PAA and has intensity dependent on the

amount of PAA in the shell For example the peak intensity of OH groups for

PSPMMA75PAA25 is greater than that of PSPMMA90PAA10 since there is a higher AA

monomer content in PSPMMA75PAA25 than in PSPMMA90PAA10 This OH peak is not

seen for PS and PS core PMMA shell particles because of the absence of OH groups on

PS and PMMA polymer chains

422 Zeta Potential Measurements

The electrical surface property was characterized by zeta potential measurements of

tex particles Zeta potential is an important and useful indicator of surface charge which

can be used to predict and control the stability of colloidal systems The greater the zeta

is

la

76

1520002500300035004000

Wavenumbers (cm-1)

li

Re

ativ

e

Tra

nsm

ttanc

e

Figure 4-8 FTIR spectra of polymerized latex particles (A) ba

PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMM

(B)

(C) P

(D) P

C

C

OCH3

O

CH3

XCH2

CH2 n

CH

CH2 C

C

OCH3

O

XCH2 CH

OC

CH3

Y

OH

(A) Bare PS

500100000

re polystyrene (PS) (B) A75PAA25

PSPMMA100

SPMMA90PAA10

SPMMA75PAA25

77

potential the more likely the suspension is to be stable because the charged particles repel

one another and thus overcome the natural tendency to aggregate The measurement of

zeta potential is often the key ontrol of the interaction of proteins

with solid surfaces such as latex particles

Zeta potential (ζ) is the elec at exists at the shear plane of a particle

which is some small distance from the surface [Myers 1999] The zeta potential of the

particles can be determined by measuring the mobility of the particles in an applied

electric field termed electr its response to an alternating electric

field Colloidal particles dispersed in lu ically charged due to their ionic

characteristics and dipolar attributes The development of a net charge at the particle

surface affects the distribution of ions in the neighboring ng in

an increased concentration of counter ions to the surface Each particle dispersed in a

ns called fixed layer or Stern layer

Outside th compositions of ions of opposite polarities

form ble layer is form

particle-liquid interface This double layer may be considered to consist of two parts an

inner region which inclu nd relatively strongly to the surface and an outer or

diffuse region in which the ion distribution is determined by a balance of electrostatic

forces and random thermal motion The potential in this region therefore decays with the

distance from the surface until at a certain distance it becomes zero Figure 4-9 shows a

schematic representation of a typical ion distribution near a positively charged surface

The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the

to understanding and c

trical potential th

ophoretic mobility or

a so tion are electr

interfacial region resulti

solution is surrounded by oppositely charged io

e Stern layer there are varying

ed in the region of the ing a cloud-like area Thus an electrical dou

des ions bou

center of those ions which are effectively adsorbed to the surface [Hunter 1981] The

78

extent of ion adsorption is determined by electrical and other long-range interactions

between the individual ions and surface of particles The ions outside of the Stern layer

form the diffuse double layer also referred to as the Gouy-Chapman layer [Burns and

Zydney 2000]

Shear plane

Stern layer

ψζ

0

ψs

ψShear plane

Stern layer

ψζ

0

ψs

ψ

t

controlled electric field is applied via electrodes immersed in a sample suspension and

Figure 4-9 Schematic representation of ion distribution near a positively charged surface

Zeta potential is a function of the surface charge of a particle any adsorbed layer a

the interface and the nature and composition of the surrounding medium in which the

particle is suspended The principle of determining zeta potential is very simple A

79

this causes the charged particles to move towards the electrode of opposite polarity

Viscous forces acting upon the moving particle tend to oppose this motion and the

equilibrium is rapidly established between the effects of the electrostatic attraction and

the viscosity dra

g The particle therefore reaches a constant terminal velocity

Figure 4-10 Schematic representation of zeta potential measurement(source

httpnitioncomenproductszeecom_shtm)

Because protein adsorption mechanisms are very complex and hard to explain in

biological systems protein adsorption is generally studied by using more ideal systems

consisting of one or more well characterized proteins a well-characterized adsorbent and

a well-defined aqueous solution Even so small changes in the experimental conditions

such as pH ionic strength and temperature generate totally different results Therefore

two media systems phosphate buffer (PB) and phosphate buffered saline (PBS) were

chosen to be used for the zeta potential measurements and protein adsorption study PB is

a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4H2O) to

n a constant pH PBS is used to keep the water concentration inside and outside of

the cell balanced If the water concentration is unbalanced the cell risks bursting or

shriveling up because of a phenomenon called osmosis PBS is a solution with the 5 mM

of monobasic sodium phosphate (NaH2PO4H2O) and 143 mM of sodium chloride (NaCl)

in water

maintai

80

The results of zeta potential measurement at room temperature (25plusmn1oC) as

function of pH and ionic strength (different media PB and PBS) for synthesized lat

particles are seen in Figure 4-11 Zeta potential values of polystyrene (PS) latex particle

at the Figure 4-9 are negative between -291 mV and -599 mV in PB and betwe

ex

s

en -203

mV and -278 mV in PBS at all pH ranges These negative zeta potential values of PS

latex particles are due to the sulfate groups [Vandenhu et al 1970] originated from

persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer

chain where polymerization was initiated

-100

-80

-60

-40

-20

0

pH

Zta

pte

ntl

ζ

20

40

1 2 3 4 5 6 7 8 9

eo

ia (m

V)

PB (5mM)

PBS (5mM +143mM)

en -

Figure 4-11 Zeta potential of PS latex particles at 25oC

The zeta potential measurements of PSPMMA100 core shell latex particles as seen

in Figure 4-12 were also negative with a range of -28 and -505 mV in PB and betwe

143 mV and -186 mV in PBS at the pH 21-78 These negative values are also due to

sulfate groups on the particle surface

81

-100

-80

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

Zea

pote

nti

l ζ

(mV

pH

ta

)

PB (5mM)

PBS (5mM + 143mM)

Figure 4-12 Zeta potential of PSPMMA100 latex particles at 25oC

The zeta potential for PSPMMA90PAA10 and PSPMMA75PAA25 was also

negative as seen in Figure 4-13 and 4-14 The zeta potential value for the

PSPMMA90PAA10 were between -367 mV and -678 mV in PB medium and between -

147 mV and -193 mV in PBS both at 25oC In case of the PSPMMA75PAA25 core shell

particles zeta potential values were between -291 mV and -520 mV in PB and between

-115 mV and -210 mV in PBS media Sulfate groups in PSPMMA75PAA25 and

PSPMMA90PAA10 core shell particles contribute to the negative zeta potential values

participate in forming a

negat

cid is expressed by the term pKa which is the pH

at which an acid is 50 dissociated However the contribution of the carboxylic acid

Carboxylic groups from acrylic acid monomer also would

ive surface charge of the latex particles at a pH greater than 70 because the pKa

value for the carboxylic acid is between 40 and 60 pH range [Ottewill et al 1967] This

indicates that carboxylic acid dissociation begins at a pH between pH 46-60 and

increases with pH The strength of an a

82

group to negative zeta potential values of PSPMMA-PAA core shell particles was not

detectable because there was no significant difference in zeta potential values for

synthesized latex particles at a pH greater than 70

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

Zeta

pot

entia

l ζ

(mV

)

PB (5mM)PBS (5mM + 143mM)

-80

-100

pH

Figure 4-13 Zeta potential of PSPMMA90PAA10 latex particles at 25oC

-100

-80

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

pH

Ze p

ont

i ζ

V)

tate

al (m

PB (5mM)PBS (5mM + 143mM)

Figure 4-14 Zeta potential of PSPMMA75PAA25 latex particles at 25oC

83

The zeta potential plots and values were similar for all latex particles This may be

due to the similar initiator density on the latex surface The initiator concentrations

monomers for shell polymerization were the same fo

to

r all core shell latex particles (062

ww from Table 4-4) A similar concentration (055 ww) of initiator to monomer was

added to prepare bare PS latex particles used in zeta potential measurements

Since the zeta potential values of all synthesized latex particles were negative

between pH=20 and pH=78 the isoelecric point (IEP) of these latex particles would be

less than pH=20 Sulfates have a pKa in the range of 10 to 20 [James 1985] indicating

that the sulfate group the conjugate base of a strong acid is protonated (-C-OSO3H) at

less than pH 20 This means that the synthesized latex particles are all negative at the

physiological pH Most serum proteins have a negative surface charge in blood

therefore when the negative latex particles are applied to blood stream fatal

tein is

avoided by charge repulsion in the blood Table 4-5 shows the common blood proteins

and their isoelectric points (IEP)

Table 4-5 The isoelectric point (IEP) of common proteins Serum proteins Isoelectric point (IEP)

complication by coagulation between applied latex particles and serum pro s

Albumin 48 α1-Globulin 20 α2-Microglobulin 54 γ1-Globulin 58 Heptoglobin 41 Hemoglobin 72 β1-lipoprotein 54 Immunoglobulin G (Ig G) 73 Fibrinogen 58 (source httpwwwfleshandbonescomreadingroompdf1085pdf

httpwwwmedalorgvisitorwww5CActive5Cch135Cch13015Cch130105aspx )

84

The absolute zeta potential values of the latex particles in phosphate buffer (PB)

was higher than that in phosphate buffered saline (PBS) This is because high

concentration of sodium and chloride electrolytes in PBS compressed the electrical

double layer causing rapid decay of surface potential Figure 4-15 shows that the

thickness of the electrical double layer is inversely proportional to the concentration of

electrolyte in the system

Surf

ace

Pote

ntia

l ψ

1κ3

10

1e

1κ 1κ

(1)(3) (2)

2 1 Z

Surf

ace

Pote

ntia

l ψ

1κ3

10

1e

1κ 1κ

(1)(3) (2)

Figure 4-15 The decay of surface potential with distance from surface in various

electrolyte concentrations (1) low (2) intermediate (3) high [Myers 1999]

2 1 Z

The electrical potential in the solution surrounding the particle surface falls off

the Debye-Huckel

yers 1999]

exponentially with distance (Z) from the surface according to

approximation [M

z) (- exp 0 κψψ = (4-4)

e reciprocal of the thickness of the electrical double layer referred to as the

The potential fallen off by a factor e The theoretical equation for the

ickness 1κ is

where κ is th

Debye length of 1

double layer th

21

22i

0

ekT 1

⎟⎟⎠

⎜⎜⎝

=sum ii zcεε

κ

⎞⎛ (4-5)

85

where

tration of

y was performed to measure the protein adsorption on the

synthesized latex particles Many physical and chemical processes occur at the boundary

between two phases and adsorption is one of the fundamental surface phenomena [Oscik

1982] This phenomenon is driven by the change in the concentration of the components

at the interface with respect to their concentrations in the bulk phase There are two

adsorption processes physical adsorption occurring when non-balanced physical forces

appear at the boundary of the phases and chemical adsorption or chemisorption

t the

interface [J

n leads

ational entropy hence adsorption takes place only if the

loss in conformational entropy is compensated

ter adsorption Cp The initial part of the

isotherm merges with the Γ-axis because at low polymer concentration the protein has a

high affinity for the surface and all of the polymer is adsorbed until the solid surface is

saturated

ε0 is the permittivity of a vacuum of free space ε is the permittivity of a medium e

is the relative permittivity or dielectric constant of the medium c is the concen

an ion (i) z is the valency of an ion (i) k is Boltzmannrsquos constant T is absolute

temperature (K)

43 Protein Adsorption Study

The adsorption stud

occurring when atoms and molecules from adjacent phases form chemical bonds a

aroniec et al 1988]

Flexible proteins in solution possess high conformational entropy as a result of the

various states each of the many segments in the protein chain can have Adsorptio

to a reduction of this conform

by sufficient attraction between polymer

segment and solid surface [Martin 1998] Isotherm shape of high affinity adsorption

between polymer and solid is shown in Figure 4-16 where the adsorbed mass Γ is plotted

against the polymer concentration in solution af

86

Figur

de

arboxyl

e 4-16 High-affinity adsorption isotherm of typical flexible polymer on solid

surface

In order to investigate the adsorption of blood proteins latex particle properties

such as electrical surface charge hydrophobicity and environmental conditions like pH

ionic strength and temperature are considered as experimental factors [Arai and Nor

1990] Figure 4-17 shows the overall schematic representation of the protein adsorption

on the synthesized latex particles with the functional groups such as sulfate and c

groups

CP

Γ

PMMAPAAShell

PS or PMMAShell OSO3

-

OSO3-

OSO3-

OSO3-

PS

-O3SO

PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

Protein

NH3+

+

+H3N

+

COO-

Hydrophobic interaction

NH3

COO-

H3N

COO-

-OOC

Ionic interaction

Hydrophobic interaction

Hydrogen bonding

Ionic interaction

PMMAPAAShellOSO3

-

OSO3-

OSO3-

OSO3-

PS or PMMAShell -O3SOOSO3

-

PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

-O3SO

OSO3-

OSO3-

OSO3-

PS PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

Protein

NH3+

+

+H3N

+

COO-NH3

COO-

H3N

COO-

-OOC

NH3+

+

+H3N

+

COO-

Hydrophobic interaction

NH3

COO-

H3N

COO-

-OOC

Ionic interaction

Hydrophobic interaction

Hydrogen bonding

Figure 4-17 Overall schematic representation of the protein adsorption on the

Ionic interaction

synthesized latex particles

The surface functional groups as well as suitable surface hydrophobicity

hydrophilicity of latex particles are closely related to protein adsorption property As

indicated at Figure 4-17 various interaction forces such as hydrophobic interaction ionic

87

interaction hydrogen interaction and van der Waals interaction [Andrade 1985] exist

between protein molecule and the solid surface of latex particle

431 Adsorption Isotherm

The protein adsorption isotherm is generally defined as the relationship between the

amount of protein adsorbed by a solid (latex particle) at a constant temperature and as a

ine

the amount of protein adsorbed onto latex particles To evaluate the equilibrium

conce s

x

function of the concentration of protein Adsorption isotherms were plotted to determ

ntration of target proteins a calibration curve is firstly needed Figure 4-18 show

the UV absorbance as a factor of bovine serum albumin (BSA) concentrations The UV

absorbance of the known protein concentration was measured at 562nm which is the λma

for a protein treated by a bicinchoninic acid (BCA) assay

0

02

04

06

08

500 520 540 560 580 600

Wavelength (nm)

Abs

orba

nce

Figur gml (B)

06mgml (C) 02mgml

(A)

(C)

(B)

Max562nm

e 4-18 The dependence of UV absorbance on BSA concentration (A) 10m

Figure 4-19 shows the standard calibration curve used in adsorption isotherm

experiment of protein onto latex particles The net absorbance of each known protein

88

concentration was plotted and then a linear equation passing through a concentration of

zero protein concentration was obtained This equation is applied

to determine the

orbed on the synthesized latex particles The amount of free

protei

eported

trations (005 01 03 05

and 07mgml) were used to generate this curve as well as the adsorption isotherm

unknown protein amount ads

n was also determined using UV light at the 280nm wavelength however a

distinguishable UV intensity was too low to determine a precise value of protein

adsorption

The bicinchninic acid (BCA) assay technique [Smith et al 1985] has been r

for high efficiency of protein evaluation and was used for determination of protein

adsorption [Tangpasuthadol et al 2003 Williams et al 2003] This technique has many

advantages such as high sensitivity easy of use color complex stability and less

susceptibility to any detergents Five different BSA concen

y = 00R2 =

004741x 09939

00

01

02

03

04

0 200 400 600 800

BSA Standard (microgml)

Net

Abs

orba

nce

at 5

62nm

Figure 4-19 Standard curve of net absorbance vs BSA sample concentration

89

All isotherms were fitted to the Langmuir-Freundlich isotherms using the nonlinea

regression method Two adsorption isotherm models the Langmuir type isotherm a

Langmuir-Freundlich combination type isotherm were compared As seen the Figure 4-

20 The Langmuir isotherm model deviated further from the experimental data Th

all isotherms were fitted to the Langmuir-Freundlich isotherms [Yoon et al 1996

al 1998] usi

r

nd the

erefore

Lee et

ng the nonlinear regression method with following equation

n

n

eq

eqm

kC

kC1

1

1+

where q is the amount of adsorbed BSA per unit surface area q

qq = (4-6)

stant

were between 045 and 055 and hence n was fixed to its average 05 Actual qm values

were evaluated after fixing the n value

m is the adsorbed amount

in equilibrium Ceq is the equilibrium concentration of BSA k is the adsorption con

and n is the exponential factor for a heterogeneous system n values are empirical and

usually from zero to no more than unity [Jaekel 2002] The values of n in our system

00

10

20

30

0 02 04 06 08

C (mgml)eq

q (m

gm

2 )

pH 32Langmuir-Freundlich modelLangmuir model

Figure 4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on

polystyrene latex particles at 37oC in phosphate buffer media

90

The BSA protein adsorption on the synthesized latex particles was performed at

three pH levels 32 48 74 in both phosphate buffer (PB) and phosphate buffered saline

(PBS) at physiological temperature 37oC These pH levels correspond to an acidic pH

lower than the isoelectric point (IEP) of BSA the pH at the IEP and an alkaline pH

higher than the IEP of BSA respectively The IEP corresponds to the pH at which the

amino acid becomes a zwitterion Here its overall electrostatic charge is neutral and the

charge expressed by the basic amino group (-NH2 -NH3+) of the amino acid is equal to

the charge expressed by the acidic carboxyl group (-COOH -COO ) The IEP of BSA is

-

pH 48 and indicates that the side chain of the amino acids of BSA contains a greater

amount of acidic functional groups than that of basic functional groups At pH 32 and

74 BSA has a positive and negative charge respectively

Figure 4-21 and 4-22 show adsorption isotherms of BSA onto polystyrene (PS)

latex particles

00

10

20

30

40

0 02 04 06 08

Ceq (mgml)

q (m

gm

2 )

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in

phosphate buffer (PB) at 37oC

91

00

10

30

40

0 02 04 06 08

Ceq (mgml)

q (m

gm

2

20

)

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

(PBS) on PS latex particles

At the IEP of BSA the adsorbed amount (q

Figure 4-22 Adsorption isotherm of BSA adsorption 37oC in phosphate buffered saline

onto PS at equilibrium was

maxim

um at the IEP of BSA This is because the BSA molecules form the most

compact structures at the IEP eliminating the repulsion force between proteins A higher

number of BSA molecules can adsorb on a given surface area by hydrophobic interaction

when in this compact structure This observation agrees well with reported data by other

researchers who also indicated that the maximum adsorption from aqueous protein

solution was observed at the IEP [Mularen 1954 Watkins et al 1977 Oh at el 2000]

There was no significant difference of adsorbed BSA amount in PB and in PBS media at

pH 48 The conformational alteration of BSA was not affected significantly by the ionic

strength of the two solutions There was evidence of the effect of the electrostatic

interaction to some extent even at the IEP since the amount of BSA adsorbed in PBS is

slightly hig

m) of BSA

um indicating that hydrophobic interaction between latex particle and BSA

protein is maxim

her than in PB at this pH

92

The adsorbed amount of protein is shown to decrease at a pH 32 and 74 The

decrease in adsorption efficiency when deviating from IEP of BSA is a result of the

increase in conformational size of the protein molecules and the lateral electrostatic

repulsions between adjacent adsorbed BSA molecules Figure 4-23 shows a schematic of

possible conformational changes at each pH where hydrophobic interaction always exists

between non-polar portion of a protein and hydrophobic portion of solid surface

- - --- ---Latex surface

Zeta potential

BSA(+)

(-)

48 7432pH

(0)

+++ + ++

--+

++-

- - --- ---Latex surface

Zeta poten pH(0)

+++ + ++

--

tial

BSA(+)

(-)

48 7432

+

++-

ming

4

s

al blood Table 4-6 shows the calculated results of

Figure 4-23 Conformation of bovine serum albumin (BSA) on latex particles (Assuthat charge repulsion reduces the effect of the hydrophobic interaction at pH 74)

In addition the adsorbed amount of BSA at pH 32 was larger than that at pH 74

and still less than that at 48 This is because there is the electrostatic interaction between

the positive BSA molecules and negative PS latex particles at pH 32 and electrostatic

repulsion is dominant between negative BSA and negative PS latex particles at pH 7

The amount adsorbed increased with increasing ionic strength in pH 32 and 74 This i

because the high concentration of electrolytes reduces the electrostatic repulsion between

BSA and PS latex particle It should be noted that the BSA concentration used in these

experiments was lower than that found in normal human blood This is because the

equilibrium BSA adsorption onto the latex particles is actually reached at a much lower

concentration than that found in norm

93

adsor rm

ein

leading to a compact conformation of BSA There are two main forces affecting

adsorption amount at an acidic pH of 32 They include the ionic attraction between the

negative latex particle and the positive protein and lateral repulsion between positive

proteins The smallest amount of BSA adsorption was observed at a pH of 74 At this

point the charge repulsion between negatively charged particles and protein molecules

exists the h

of cha

bed BSA amount at equilibrium from the Langmuir-Freundich adsorption isothe

equation

In summary the hydrophobic interaction between PS latex particle and BSA

protein exists and is the dominant mechanism for the adsorption process Conformational

changes of BSA at each pH also affected the adsorption amount Adsorption was highest

at pH 48 because at this pH there is the least amount of ionic repulsion in the prot

and the lateral repulsion between proteins dominant adsorption even though there always

ydrophobic interaction between the solid latex particle and protein The effect

rge repulsion was reduced by the high concentration of electrolytes in PBS medium

Table 4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2) 32 174 48 299 Phosphate buffer 74 136 32 260 48 283 Phosphate buffered saline 74 207

The adsorption isotherms of BSA on PSPMMA core shell particles are shown in

Figure 4-24 in phosphate buffer (PB) and in Figure 4-25 in phosphate buffered saline

(PBS) The adsorbed BSA amount was calculated using the Langmuir-Freundlich

100

94

adsorption isotherm equation and the amount listed in Table 4-7 The trend seen in the

adsorption results was very similar to the case of PS latex The overall adsorption process

was d rticle

rs

hat

0

ominated by hydrophobic interactions between the PSPMMA100 core shell pa

and BSA proteins At pH 48 the adsorbed amount (qm) of BSA on PSPMMA100 core

shell particles was 221 mgm2 in PB and 237 mgm2 in PBS and are the maximum

values recorded among the three pH levels 32 48 and 74 Suzawa and his co-worke

[1982] have obtained similar results This can be also explained by the compact

conformation of BSA molecules at the IEP of BSA which lead to a high protein

population on the solid surface At a pH of 32 and 74 BSA adsorption was less than t

at pH 48 because of the protein conformation change There was a small increase in

adsorbed BSA at pH 32 because of the charge attraction between negative PSPMMA10

core shell particles and positive BSA At pH 74 however the adsorption of BSA was

further reduced by charge repulsion between the negative PSPMMA100 core shell

particles and the negative BSA proteins

000 02 04 06 08

Ceq (mgml)

10

20q (m

gm

302 )

40

50pH=32pH=48pH=74Mode 8)l (pH 4Mode 2)l (pH 3Mode 4)

Figure 4-24 Adsorption isotherm of BSA 37oC in PB on PSPMMA core shell latex

l (pH 7

100

particles

95

The adsorbed amount of BSA increased more rapidly in PBS with increasing ioni

strength at pH 74 This shows that the low concentration of electrolytes promotes

electrostatic repulsion between negative BSA and negative PSPMMA

c

on

x

l 1980

100 core shell

particle As the ion strength of PBS is increased however the high concentration of

electrolyte leads to less electrostatic repulsion promoting more rapid protein adsorpti

By comparing the amount of adsorbed protein between bare PS latex particles and

PSPMMA100 core shell particles the amount of protein adsorption on the PS late

particles is greater than that on the PSPMMA100 core-shell particles [Suzawa et a

1982 Lee et al 1988]

00

10

20

30

40

m2 )

0 02 04 06 08

Ceq (mgml)

q (m

g

pH=32pH=48pH=74Model (pH 2) 3Model (pH 48)Model (pH 74)

Figure 4-25 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA100 core shell

latex particles

96

Table particles calculated the Langmuir-Freundlich isotherm model

4-7 The equilibrium concentration values (qm) of BSA adsorption on PSPMMA100

Media pH qm (mgm ) 2

32 140

48 221 Phosphate buffer

74 130

32 169

48 237 Phosphate buffered saline

74 162

The BSA adsorption mechanism and trend on PSPMMA core shell particles was

very similar to that of the PS latex particle Unlike the case of PS and PSPMMA100 latex

particles the amount of BSA adsorbed onto PSPMMA90PAA10 and PSPMMA75PAA25

core shell particles was higher at pH 32 than pH 48 and 74 Figure 4-26 through 4-29

shows the adsorption isotherms of BSA on PSPMMA90PAA10 and PSPMMA75PAA25

core shell particles The amount of BSA adsorbed increased at pH 32 and pH 48 as the

hydrophilicity was developed by the addition of PAA On the contrary the adsorbed

amount of BSA was dramatically decreased at pH 74 The adsorption amount of BSA on

PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles at equilibrium is listed in

Table 4-8 and 4-9 respectively

Carboxylic acid as well as sulfate groups are distributed on the surface of

PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles and seem to largely affect

the BSA adsorption The isotherms showed very strong pH dependence to the BSA

adsorption onto these core shell latex particles The carboxylic groups (COOH) may

especially enhance the BSA adsorption prominently at pH 32 by hydrogen bonding with

protein molecules The pKa value for the PAA is 40-60 and a good indication that the

97

carboxylic acid groups are protonated at pH 32[Gebhardt et al 1983] The main

adsorption interaction force at pH 32 was generated by hydrogen bonding as well as

charge attraction between latex particle protein even th the conformational

repulsion between proteins results in a reduction of the protein

adsorption at this pH The amount of BSA adsorbed also increased as the PAA amount in

eased The a tion of BSA w inimal however less

than 07 mgm2 at pH 74 Here the carboxylic groups are ionized which increases the

charge repulsion by ionized carboxylic groups (COO-) resulting in PSPMMA-PAA latex

particle

and ough

change and charge

the core shell particles incr dsorp as m

s with much lower protein adsorption

00

20

40

q (m

g2 )

60

m

pH=32pH=48pH=74Model (pH 32)Model (pH 48)

0 02 04 06 08

Ceq (mgml)

Model (pH 74)

o shell

Figure 4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA90PAA10 corelatex particles

98

00

40

60

80

0 02 04

C (mgm

mm

2 )

20

06 08

eq l)

q (

g

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-27 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA90PAA10 core

shell latex particles

Table 4-8 The equilibrium concentration values of BSA adsorption on PSPMMA90PAA10 particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2)

32 361

48 283 Phosphate buffer

74 043

32 519

48 226 Phosphate buffered saline

74 043

99

00

20

40

60

80

100

0 02 04 06 08Ceq (mgml)

q (m

gm

2 )

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

oFigure 4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA75PAA25 latex particles

00

100

20

40

60

80

0 02 04 06 08Ceq (mgml)

q (

m2 )

pH=32pH=48pH=74

mg

Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-29 Adsorption isotherm of BSA 37oC in PBS on PSPMMA75PAA25 core shell

latex particles

100

Table 4-9 The equilibrium concentration values of BSA adsorption on PSPMMA75PAA25 particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2)

32 1083

48 662 Phosphate buffer (PB)

74 075

32 1077

48 681 Phosphate buffered saline (PBS)

74 057

The adsorption of β2-microglobulin (β2M) was determined under one set of

onditions in phosphate buffer at 37oC and pH 74 because of the limited amount of β2M

protein The adsorption isotherms of the β2M protein on the synthesized latex particles

are shown in Figure 4-30 4-31 and 4-32 The β2M protein concentrations for adsorption

isotherm experiment were chosen to be 0015 0030 0045 and 0060mgml since the

β2M concentration for the patients with renal failure can be elevated to similar

concentrations about 005mgml or more up to 50 times the normal level [Nissenson et

al 1995] There were steep initial slopes of the adsorption isotherm curves of β2M on

latex particles in all isotherms indicating a high affinity adsorption type It seems that the

hydrophobic interaction is enough for the adsorption process of β2M proteins to occur on

the latex particles even though charge repulsion exists between protein and latex particle

at pH 74 The complete plateau regions were not seen for all isotherms indicating that

β2M proteins were not saturated at this level and there are still available sites for β2M

c

adsorption on latex particles The initial slope of the β2M isotherms for PS was steeper

101

than that of any of the other latex particle PSPMMA100 PSPMMA90PAA10 and

PSPMMA 75PAA25

00

02

0 0002 0004 0006 0008

04

06

10

Ceq (mgml)

q (m

2 )

08

gm

PSPSPMMA100Model (PS)Model (PSPMMA100)

Figure 4-30 Adsorption isotherm of β2M onto PS and PSPMMA100 latex particles in PB

at 37 C and pH 74 o

00

02

04

06

08

10

0 0002 0004 0006 0008C (mgml)

q (m

m2 )

eq

g

PS

PSPMMA90PAA10

Model (PS)

Model (PSPMMA90PAA10)

Figure 4-31 Adsorption isotherm of β2M onto PS and PSPMMA90PAA10 latex particles

in PB at 37oC and pH 74

102

00

02

04

06

08

10

0 0002 0004 0006 0008Ceq (mgml)

q (m

gm

2 )

PS

PSPMMA75PAA25

Model (PS)Model (PSPMMA75PAA25)

Figure 4-32 Adsorption isotherm of β2M onto PS and PSPMMA75PAA25 latex particles

in PB at 37oC and pH 74

The equilibrium concentration values (qm) of β2M adsorption on latex particles are

calculated and listed in Table 4-10 The difference between the maximum amounts of

β2M adsorption on all the latex particles was not significantly different and their values

were between 069 and 08mgm2

Table 4-10 The equilibrium concentration values of β2M adsorption calculated the Langmuir-Freundlich isotherm model

Latex particles qm (mgm2)

PS 080

PSPMMA100 069

PSPMMA90PAA10 072

PSPMMA75PAA25 072

103

432 Adsorbed Layer Thickness

There are several possible explanations for large amount of BSA adsorbed at acidic

pH levels including the end-on adsorption pattern of BSA the flexibility of BSA

molecules the tilting of protein molecules due to the asymmetry of the charge

distribution or multilayer formation [Peula et al 1993] The adsorbed BSA thickness (δ)

can be calculated by the following equation [Chiu et al 1978] This equation was also

cited again by other researchers [Shirahama et al 1985]

BSA

mqρπ

δbullbull

=33

(4-7)

where π

33 is the packing factor qm is the plateau value of the adsorbed am

ρBSA is the hich corresponds to the reciprocal of its known

partial specific volume The layer thickness (δ) of BSA proteins on synthesized latex

partic

he IEP [Shirahama et al 1985] Compared to the

hydrodynamic dimensions of BSA 140 times 40 times 40 Aring3 [Fair et al 1980 Peter 1985] these

nd

ode It is unnecessary ultilayering because thickness do

exceed 140 Aring The layer thickness (δ) of BSA proteins on synthesized latex particles

e red saline (PBS) was ran 28 ~ 83 Aring at 37oC and pH 48 These

lso listed in Table 4-1

ount and

density of BSA molecule w

les in phosphate buffer (PB) ranged from 27 - 81 Aring at 37oC and pH 48 These

calculated values are listed in Table 4-11 Only δ values at pH 48 the isoelectric point

(IEP) of BSA are important because intra- and intermolecular electrostatic repulsion of

protein molecules is minimized at t

values of layer thickness indicate that the BSA molecules adsorb by side-on (40Aring) a

end-on (140Aring) m to consider m

not

in phosphat buffe ged of

calculated values are a 2

104

Table 4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37oC pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

32 21 17 44 133

48 37 27 35 81

74 17 16 5 9

Table 4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37oC

pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

32 32 21 64 132

48 35 29

74 26 20 5 7

28 83

1n

1n

neqm

So k is closely related to the initial slope of the isotherm showing the affinity

between BSA and latex particles [Yoon et al 1996] A key point in the characterization

433 Gibbs Free Energy of Protein Adsorption

In the Langmuir isotherm model k represents the ratio of adsorption to the

desorption rate constants This definition is not applicable to the Langmuir-Freundlich

isotherm model but its meaning is similar [Yoon et al 1996] In this sense k is closely

related to the affinity between protein molecules and latex particles When the

equilibrium concentration Ceq reaches zero the value of Ceq will be very small (1gtgt

kCeq ) and then the equation 4-6 becomes

(4-8)

of adsorption processes on solid-liquid interfaces is to determine the change in Gibbsrsquos

free energy (∆Gdeg) one of the corresponding parameters for the thermodynamic driving

forces during adsorption The traditional method to evaluate adsorption thermodynamic

kCqq 1=

105

constants is based on measurement of adsorption isotherms under static conditions

[Malm ten 1998] The Langmuir-Freundlich ads lf

easily found from Then this enables calculation of the free-

energy change ∆G during adsorption expressed as

(4-9)

where R is the general gas constant (R = 8314Jmiddotmol K ) and T is the absolute

tem ture Gibb e energy A P

listed in Table 4-13 and 4-14 respectively As the ∆Gdegads ore negativ e

pro adsorption on a solid substra ill be more favorable

e of BSA adsorption in phosphate o

pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

s orption equilibrium constant k can be

a slope of the isotherm

degads

lfads kRTG lnminus=∆ o

-1 -1

pera s fre change of BS adsorption values in B and PBS are

values are m e th

tein te w

Table 4-13 The values of Gibbs free energy changbuffer (PB) at 37 C

32 -2411 -2726 -2846 -2805

48 -2352 -2383 -2198 -1972 ∆Go

80

ads (kJmol)

74 -2452 -888 -324 -2

Table 4-14 The values of Gibbs free energy change of BSA adsorption in phosphate

pH PS PSPMMAbuffered saline (PBS) at 37oC

100 PSPMMA90PAA10 PSPMMA75PAA25

32 -2315 -2620 -2312 -2683

48 -2149 -2299 -2090 -2138 ∆Go

(kJmads ol)

74 -2430 -1992 -258 -263

Table 4-15 shows the calculated values of Gibbs free energy change of β M

adsorption on synthesized latex particles PSPMMA PAA core shell latex particle has

2

75 25

106

largest negative Gibbs free energy of β2

2

M adsorption indicating largest affinity betw

β

een

2M adsorption in phosphate buffere (PB) at 37 C and pH74

PS PSPMMA100

M and latex particle

Table 4-15 The values of Gibbs free energy change of βo

PSPMMA90PAA10 PSPMMA75PAA25

∆Go03 ads

(kJmol) -3626 -3466 -3659 -39

The Gibbs free energy of β2M was much more negative in phosphate buffer (PB)

than that of BSA in physiological condition (37 C and pH 74) o from the Table 4-13 and

the Figure 4-31 This indicates that β2M molecules more readily adsorb on latex particles

BSA This can be explained by the rate of diffusion and accessibility of β2M molecules to

the latex particle ier and faster for β2M m an BSA because of

size (molec ight) erical shape molecules It o be

due to the higher hydrophobic force and less electrostatic repulsion that contribute to the

adsorption of β2M molecules not as prevalent in the case of BSA because the IEP of β2M

(57) is clo H 74) than that of BSA (48) causing β2M to

be less electro cally lled p ro

can be seen that the ∆ β2M adsorption onto the synth article

increasing wi hy icity o x particles s the ∆Goads of the BSA

molecules is decreasing or constant This phenomenon supports the theory that the

select lizing

than BSA and that the affinity of β2M for the latex particle is much larger than that of

surface It is eas olecules th

the smaller ular we and sph of β2M may als

ser to physiological condition (p

stati repe from the latex articles than BSA F m Figure 4-33 it

Goads of esized latex p s is

th the drophil f the late wherea

ive adsorption of β2M molecules over BSA molecules can be enhanced by uti

more hydrophilic PSPMMA90PAA10 and PSPMMA75PAA25 core shell latex particles

107

-60

-50

1 2 3 4

-40∆Gad

s

-30

10

0

o (k

Jm

o-

-20l)

β2-Microglobulin (β2M)Bovine serum albumin (BSA)

Figure 4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37

and pH 74

434 Kinetics of Adsorption

to

oC

Protein adsorption is a complex process known to have a large biological impact

and is currently not well understood quantitatively because of extreme sensitivity to pH

the concentration of other electrolytes and molecules present temperature and many

other factors that can change in the physiological system Accurate knowledge of the

adsorption kinetics under a given set of conditions is a prerequisite for elucidating the

mechanisms of many fundamental biological processes to be predicted at the molecular

level [Regner 1987] This adsorption kinetics approach is used in protein adsorption

studies because of the uncertainty related to the time needed for the interfacial proteins

reach the equilibrium It is generally accepted that the process of protein adsorption is

comprised of the following steps a) transport toward the interface b) attachment at the

interface c) eventual structural rearrangements in the adsorbed state d) detachment from

PS PSPMMA100 PSPMMA90PAA10PSPMMA75PAA25

108

the interface and transport away from the interface Each of these steps can affect the

overall rate of the adsorption process

The quantitative analysis of the protein adsorption kinetics requires that the protein

amount adsorbed is known as a function of time The kinetics tests were carried out in

phosphate buffer (PB) at 37oC and pH 74 a condition similar to physiological condition

The protein concentrations chosen were 07mgml for BSA and 006mgml for β2M

which are the maximal concentrations used in an adsorption isotherm test A time-based

evaluation of the adsorption was made by measuring the concentration of protein in

solution at different incubation times The results are shown below from Figure 4-34 to 4-

37 The adsorption kinetics of proteins onto th cond-e latex particles is expressed as a se

order model [Oumlzacar 2003] shown by the following equation

2)( tet qqk

dtdq

minus= (4-10)

Integrating above equation and applying the boundary conditions yields

ee

t tq

+=

1 (4-11)

where q

qkq

t

2

2

2

the previous adsorption isotherm results representing good reproducibility of adsorption

t and qe stand for the amount (mgm ) of protein adsorbed at time t and at

equilibrium respectively k is the equilibrium rate constant of second-order adsorption

(m mghr) and t is the incubation time (hr)

The fitting parameters calculated by using the equation 4-11 are listed in table 4-16

From this data it is seen that the amount of BSA adsorbed at equilibrium was higher than

that of β2M on PS and PSPMMA100 latex particles but less than that of β2M on

PSPMMA90PAA10 and PSPMMA75PAA25 latex particles These results are similar to

109

00

05

15

20

0 2 4 6 8 10 12 14

Time (hr)

rot

n ad

sorb

d (

10

25

Pei

em

gm

2

BSA2nd order model (BSA))β2M2nd order model (β2M)

Figure 4-34 The kinetics of protein adsorption in PB on PS latex particles at 37oC and

pH 74

00

05

10

15

20

25

30

0 2 4 6 8 10 12 14

Time (hr)

Prte

inds

oo

arb

ed (

mg

m2 )

BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-35 The kinetics of protein adsorption in PB on PSPMMA100 latex partic

37les at

oC and pH 74

110

00

03

06

09

12

15

0 2 4 6 8 10 12 14

Time (hr)

Prot

ein

adso

rbed

(m

gm

2 ) BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-36 The kinetics of protein adsorption in PB on PSPMMA90PAA10 latex

particles o at 37 C and pH 74

00

03

06

09

12

15

0 2 4 6 8 10 12 14

Time (hr)

BS

A ad

sorb

ed (

mg

m2 )

BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-37 The kinetics of protein adsorption in PB on PSPMMA75PAA25 latex

particles at 37oC and pH 74

111

test The variation of β2M equilibrium adsorption amount was nearly negligible for all

latex particles The plateau region is reached earlier for β2M than BSA on all latex

particles and the K values (equilibrium rate constant of second order adsorption) for the

β2M adsorption process onto latex particles are much larger than for BSA adsorption

These values indicate that the β2M molecules have a higher adsorption rate onto the latex

particles than the BSA molecules

Table 4-16 Fitting parameters of second-order kinetic model BSA β2M

Latex particles qe (mgm2) K (hr-1) qe (mgm2) K ( -1) hr

PS 144 67 074 7549

PSPMMA100 175 353 076 4438

PSPMMA90PAA10 046 97 075 5695

PSPMMA75PAA25 060 179 075 5168 K = (kqe) hr-1

There are several explanations for the phenomena observed in these experiments

First the smaller sizes of β2M molecules allow easier diffusion and a close approach to

the latex substrate Second a reduced number of hydrophobic domains on the latex

particle surface replaced by sulfate groups (-OSO3-) and ionized carboxylic groups

(COO-) are not favorable for larger BSA molecules Third the conformational change by

intra- and inter-molecular repulsion would be faster in β2M due to smaller molecular

weight Finally the ionic repulsion between β2M and the latex particles is less than that

condition (pH 74) than IEP (48) of BSA

between BSA and latex particles because IEP (57) of β2M is closer to the tested

112

44 Blood Compatibility

Blood compatibility can be defined as the property of a material or device tha

permits it to function in contact with blood without inducing any adverse reactions

Successful functioning of blood contacting biomedical materials or devices in contact

with blood strongly depends on the extent to which they damage blood during operation

High shear stresses turbulence rela

t

tively long contact times and cellular impact with

artificial surfaces are most common events experienced by blood in contact with artificial

1978 Winslow and et al

1993] Therefore before any materia e placed into the human b

biocompatibility testing must be the first performed in the view of safety The hemolysis

s a method for pr ary screening of biomaterials to determine

h blood a er circulating body fluid The tests estimat

oglobin released from th blood cell d e induced

foreign material

There are several types of blood cells in our body but the ones that make blood red

are erythrocytes or red blood cells (RBCs) Red blood cells (erythrocytes) are the most

numerous type in the blood These cells are average about 48-54 million per cubic

millimeter (mm3 same as a microliter (microl)) of blood but these values can vary over a

larger range depending on such factors as health and altitude RBCs are red because they

contain hemoglobin which is a protein that contains iron When blood circulates through

lungs oxygen binds to the hemoglobin and is then delivered to all the parts of body

RBCs are made in the bone marrow They start out as a nucleus like other cells but

when they fill up with hemoglobin the nucleus becomes smaller until it disappears

Without a nucleus RBCs are fragile and live only about 120 days In a normal healthy

devises and can cause hemolysis or thrombosis [Bernhard et al

ls can b ody

test serves a elimin the

biocompatibility wit nd oth e the

concentration of hem e red amag by a

113

body about 2 million red blood ne marrow produces new

ones j ing

dy

le

cells die per second But bo

ust as fast RBCs are about 75 micrometer across and are responsible for carry

oxygen salts and other organic substances to the cells and tissues throughout our bo

In humans and most mammals the cells are concave (indented in the middle) and flexib

enough to squeeze through even the tiniest of blood vessels the capillaries

Figure 4-38 Image of red blood cells

(Sourcehttpwwwpbrchawaiiedubemfmicroangelarbc1htm)

ch

100 90 10 75 25 f

RBCs are among the first cells that come into contact with a foreign material

injected in the blood Therefore a hemolysis assay gives information about the

biocompatibility in the case of an in vivo application The concentrations of prepared

latex particles used for the tests in this research are 05 25 and 50 (ww) and the

incubation time under moderate shaking was 30 minutes at 37oC Test results are shown

in Figure 4-39 The amount of blood cells that ruptured when in contact with the PS latex

particles was largely increased from about 30 to 91 as the solid content of PS

increased from 05 to 50 However hemolysis by the core shell latex particles su

as PSPMMA PSPMMA PAA and PSPMMA PAA was less than 35 O

114

particular interest was that hemolysis caused by PSPMMA PAA particles at 50

solid concentration was less than 02 hemolysis the lowest value among the latex

particles The low hemolysis value indicates that the PSPMMA PAA core shell latex

particles are the most blood compatible at all solid contents Other core shell particles

such as PSPMMA and PSPMMA PAA also had excellent blood comp

90 10

90 10

100 75 25 atibility with

low RBS breakage

01 2 3

20

40

60

80

100

Hem

olys

is (

)

052550

Figure 4-39 He

PS PSPMMA100 PSPMMA PAA90 10

molysis caused by latex particles n=5

4PSPMMA75PAA25

CHAPTER 5 SUMMARY OF RESULTS CONCLUSIONS AND RECOMMENDATION FOR

FUTURE WORK

51 Summary of Results

The goal of this study was to synthesize monodisperse polymeric latex particles

with tailored properties and to investigate the fundamental interactions between the

synthesized latex particles and target proteins An understanding of the fundamental

mechanism of selective adsorption is strongly required in order to maximize the

separation performance of membranes made of these materials so that it may applicable

to hemodialysis therapy for end stage renal disease (ESRD) patients For the achievement

of above research goal several investigations have been performed Analysis of the

obtained data leads to several conclusions as discussed below

Monodisperse polystyrene seed latex particles were synthesized with the size

crosslinking agent to make the latex particles more hydrodynamically stable The

conversion of monomer to polymer was over 95 and seed latex particles are spherical in

shape and have smooth surface From these small seeds bigger latex particles as large as

800nm were synthesized using a semi-continuous seeded emulsion polymerization

process The particles with a mean diameter less than 500nm are very spherical in shape

and highly uniform in particle size distribution However as the particles grow larger

than 500nm in mean diameter they became non-spherical with an uneven surface This

irregularity of particle surface can be attributed to the non-homogeneous monomer

ranges between 126plusmn75 and 216plusmn53 nm Divinylbenzene (DVB) was used as a

115

116

swelling into the shell of a growing polymer which can be controlled by the factors such

as temperature agitation speed initiator feeding rate and surfactant amount required to

stabilize the colloidal system It was determ rfactant to monomer ratio

dec ic

acid (AA) monomers were intro rticle surfaces more

hydro

to

ting of a

orm

ration is seen between

3002

0 cm-

0cm-1

-1

03

ined that as the su

reased the mean particle diameter increased Methyl methacrylate (MMA) and acryl

duced to make the latex pa

philic than bare polystyrene particles The prepared core shell latex particles were

made of PMMA100 PMMA90PAA10 and PMMA75PAA25 PMMA100 is the PMMA

homopolymer shell on a PS core PMMA90PAA10 shell is a copolymer with a PMMA

PAA ratio of 90 to 10 by weight PMMA75PAA25 shell is a copolymer consis

PMMA to PAA ratio of 75 to 25 by weight The particle size of these core-shell

particles were prepared to be about 370nm in mean diameter

The successful synthesis of latex particles was confirmed using Fourier Transf

Infrared spectroscopy (FTIR) The C-H aromatic stretching vib

cm-1 and 3103 cm-1 for PS latex particles The C-H asymmetrical stretching and

symmetrical stretching vibration peaks of CH2 for all latex particles is seen at 290

1and 2850 cm-1 respectively There is a carbonyl (C=O) characteristic band at 173

wavenumber for PSPMMA core-shell latex particles The broad OH group peak at

3400 cm

100

can be seen in the spectroscopy of the particle with PMMAPAA shell

(PSPMMA PAA and PSPMMA PAA ) The peak intensity of OH group of

PSPMMA PAA was larger than that of PSPMMA PAA relatively due to more AA

monomer content in PSPMMA PAA than in PSPMMA PAA

In the zeta potential measurements polystyrene (PS) latex particles had negative

values between -291 mV and -599 mV in the phosphate buffer (PB) and between -2

90 10 75 25

75 25 90 10

75 25 90 10

117

mV and -278 mV in the phosphate buffered saline (PBS) at 25oC and pH 21-78 These

negative zeta potential values for PS latex particles are due to ionization of the sulfate

groups (-OSO3-) originated from the initiator Zeta potential values of the PSPMMA

core shell latex particles were also negative between -28 mV and -505 mV in PB and

between -143 mV and -186 mV in PBS at pH 21-78 Negative values are also

generated by dissociation of sulfate groups on a particle surface The zeta potential value

of PSPMMA PAA and PSPMMA PAA were of negative values between -36

and -678 mV in PB medium and -147 mV and -193 mV in PBS medium at 25

100

s

7 mV

MMA75PAA25 and

PSPM

otein

g the

l change of BSA at

each w

90 10 75 25

oC For

PSPMMA PAA core shell particles zeta potential values were between -291 mV and

-520 mV in PB media and between -115 mV and -210 mV in PBS media Sulfate

groups contributed to the negative zeta potential values in PSP

75 25

MA90PAA10 core shell particles Carboxylic groups from polyacrylic acid also

contribute to the negative surface charge for the latex particles at greater than pH 70

however the degree of their contribution was not detectable from the zeta potential

measurements The absolute zeta potential values of the synthesized latex particles in PB

were higher than that in PBS This is because of the high concentration of sodium

chloride electrolyte in PBS that compress electrical double layer thickness

The bicinchoninic acid (BCA) assay technique was used to determine the pr

adsorption and all isotherms were fitted to the Langmuir-Freundlich isotherms usin

nonlinear regression method The hydrophobic interaction between PS latex particle and

BSA protein was dominant for the adsorption process Conformationa

pH also affected the adsorption amount which was highest at pH 48 because of lo

ionic repulsion making compact conformation of BSA The next larger amount of BSA

118

adsorbed onto PS was at pH 32 There were two main forces affecting the amount of

adsorption at acidic pH values One is ionic attraction between negative latex particle a

positive protein The other is lateral repulsion between positive proteins The smallest

amount of BSA adsorption was at pH 74 where charge repulsion between negative

particles and proteins is present at this pH Lateral repulsion between proteins wa

dominant for protein adsorption even though there always exists the hydrophobic

interaction between solid latex particle and protein The charge repulsion was reduced by

the high concentration of electrolytes in the PBS medium The BSA adsorption process

on PSPMMA core shell particles was very similar to the case of PS latex particle where

hydrophobic interaction between latex particle and protein was dominant However t

adsorbed BSA amount was less than that of PS in PBS The adsorbed amount of BS

PSPMMA

nd

s

he

A on

an

ed

n The main

adsor

90PAA10 and PSPMMA75PAA25 core shell particles was higher at pH 32 th

pH 48 and 74 The adsorbed BSA amount became higher at pH 32 and pH 48 as the

hydrophilicity was increased by the higher PAA amount On the contrary the adsorb

amount of BSA was dramatically decreased at pH 74 Carboxylic acid groups as well as

the sulfate groups are distributed on the surface of PSPMMA90PAA10 and

PSPMMA75PAA25 core shell particles and largely affect the BSA adsorptio

ption interaction forces at pH 32 was generated by hydrogen bonding as well as

charge attraction between latex particle and protein even when the conformational change

and charge repulsion between proteins reduce the adsorption of protein at this pH The

adsorbed BSA amount was also increased as the amount of PAA increases However the

adsorption of BSA was minimal (less than 07 mgm2) at pH 74 where the carboxylic

groups are ionized leading to more enhancement of charge repulsion by ionization of the

119

carboxylic groups (COO-) Because of this the PSPMMA-PAA latex particles have a

much lower protein adsorption

There were steep initial slopes for adsorption isotherms of β

in

s

there

M

r

e

es

o

2M on latex particles

all isotherms indicating a high affinity type adsorption The complete plateau region

were not seen for all isotherms indicating that β2M proteins were not saturated and

are still available sites for β2M adsorption on latex particles The initial slope of β2

isotherms for PS was steeper than for the other latex particles PSPMMA100

PSPMMA90PAA10 and PSPMMA75PAA25

The layer thickness (δ) of BSA proteins on synthesized latex particles in phosphate

buffer (PB) was in the range of 27 - 81 Aring at 37 C and pH 48 Only the δ values at pH

48 the isoelectric point (IEP) of BSA are important because intra- and inter-molecular

electrostatic repulsion of the protein molecules is minimized at the IEP Compared to the

hydrodynamic dimensions of BSA 140 times 40 times 40 Aring these values of the adsorbed laye

thickness indicates that the BSA molecules adsorb by the side-on (40Aring) and end-on mod

(140Aring) Consideration of the multi-layering is unnecessary because the adsorbed

thickness of BSA do not exceed 140 Aring

The Gibbs free energy of β

o

3

2M was more negative in phosphate buffer (PB) than

that of BSA in physiological condition (37 C and pH 74) indicating that βo2M molecul

are more favorably adsorbed on the latex particles than BSA and that the affinity of β2M

was much larger than BSA This may be explained by the rate of diffusion and

accessibility of β2M molecules being easier and faster than that for BSA because of

smaller size (or weight) and spherical shape of β2M molecules It may also be possible t

explain that more hydrophobic forces (less electrostatic repulsion) contributes to

120

adsorption of β2M than to that of BSA because the IEP of β2M (57) is closer to

physiological condition (pH 74) than that of BSA (pH 48) The selective adsorption of

β2M m

in β2M

larger than in BSA adsorption indicating

that fa

an that between BSA and latex particles because

IEP (

ch as

Other

olecules over BSA molecules can be enhanced by developing more hydrophilic

PSPMMA90PAA25 and PSPMMA75PAA25 core shell latex particles

The adsorption kinetics of proteins on latex particles was expressed as a second-

order model The plateau region for β2M was reached earlier than BSA on all latex

particles and the K values (equilibrium rate constant of 2nd order adsorption)

adsorption process on latex particles were much

ster adsorption rate of β2M molecules than that of the BSA molecules There are

several explanations for these phenomena First the smaller size of β2M molecules can

more easily diffuse and approach to the latex substrate Second enhanced hydrophilic

domain on the latex particle surface by sulfate groups (SO4-) and ionized carboxylic

groups (COO-) is not favorable for larger BSA molecules but is for smaller β2M to be

anchored Third the conformational change by intra- and inter-molecular repulsion

would be faster in β2M due to lower molecular weight Finally the ionic repulsion

between β2M and latex particles is less th

57) of β2M is closer to the tested condition (pH 74) than IEP (48) of BSA

In the biocompatibility test the number of red blood cells (RBCs) ruptured by PS

latex particles ranged from 30 to 91 as the solid content of PS increased from 05 to

50 respectively However hemolysis of the other core shell latex particles su

PSPMMA100 PSPMMA90PAA10 and PSPMMA75PAA25 were less than 35 The

PSPMMA90PAA10 particles at all applied solid concentrations had less than 02

hemolysis and was the most blood compatible among the prepared latex particles

121

core shell particles such as PSPMMA100 and PSPMMA75PAA25 also had excelle

compatibility with low RBC breakage

52 Conclusions

The major conclusions from this study include

nt blood

bull microglobulin (β2M) over that of the bovine serum albumin (BSA) at pH 74

was achieved by controlling the PMMA and PAA ratio to optimize

e particles posses the most desirable requirements for use in a human dialysis

ns

icles

olecule

bull

Using engineered core shell latex particles selective adsorption of β2

physiological condition was demonstrated

bull The rate of adsorption of the β2M was also much higher than that of BSA This

hydrophobichydrophilic microdomain size

bull The core shell particles also exhibited excellent biocompatibility with blood Thes

membrane

53 Recommendation for Future Work

With the results and conclusions obtained through this study several suggestio

may be given for future work as follows

bull Competitive adsorption tests of whole blood proteins on synthesized latex partunder physiological condition

bull Preparation of latex particles with optimum hydrophobic hydrophilic microdomains to maximize selective adsorption and removal of toxin molecules

bull Membrane fabrication by suitable pore size design to discriminate a toxin mby size sieving as a function of particle layers

Evaluation of membrane performance such as flow rate in water and in simulated blood fluid (SBF)

bull Application for hemodialysis as well as other technologies such as a water remediation

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Vincent C Chanard J Caudwell V Lavaud S Wong T and Revillard J P (Kinetics of I-125 beta2 microglobulin turnover in dialyzed patients Kidney International 42 1434-1443

Ward R A Schmidt S Hullin J Hillebrand G F and Samtleben W (2000) A comparison of on-line hemodiafiltration and high-flux hemodialysis A prospectiveclinical study Journal o

Watkins R W and Robertson C R (1977) Total internal reflection technique for examination of protein sdsorption Journal of Biomedical Materials Research 11915-9

Wavhal D S and Fisher E R (2002) Hydrophilic modification of polyethersulfone membr

uyzen J Foreman K Battistutta D Saltissi D and Fleming S J (1992) Effect of dialyzer reproce

12 29-36

helman K J Braun R D and Fitzpatrick J D (1988) Investigation of the bicinchoninic acid protein assay Identification of the groups responsible for color

ms K M Arthur S J Burrell G Kelly F Phillips D W and Marshall T (2003)

140

Winchester J F Ronco C Brady J A Cowgill L D Salsberg J Yousha E Choquette M Albright R Clemmer J Davankov V Tsyurupa M PavlL Pavlov M Cohen G Horl W

ova Gotch F and Levin N W (2002) The next

step from high-flux dialysis application of sorbent technology Blood Purification

Winchester J P Ronco J A Brendolan A Davankov V Tsyurupa M Pavlova L 2001)

oglobin

Yang Q and Yang C Z (2000) Formation of an

Yoon J Y Park H Y Kim J H and Kim W S (1996) Adsorption of BSA on highly carboxylated microspheres- quantitative effects of surface functional groups

Zollars R L (1979) Kinetics of the emulsion polymerization of vinyl acetate Journal of

Zou oss-

Zou D Ma S Guan R Park M Sun L Aklonis J J and Salovey R (1992)

20 81-86

Clemme J Polaschegg H D Muller T E La Greca G Levin N W (Rationale for combined hemoperfusionhemodialysis in uremia Contribution of Nephrology 133 174-179

Winslow R M Vandergriff K D Motterlini R (1993) Mechanism of hemtoxicity Thrombosis and Haemostasis 70 36-41

S C Ge H X Hu Y Jiang Xpositively charged poly(butyl cyanoacrylate) nanoparticles stabilized with chitosColloid and Polymer Science 278 285-292

and interaction forces Journal of Colloid and Interface Science 177 613-620

Applied Polymer Science 24 1353-1370

D Derlich V Gandhi K Park M Sun L Kriz D Lee Y D Kim G Aklonis J J and Salovey R (1990) Model filled polymers 1 synthesis of crlinked monodisperse polystyrene beads Journal of Polymer Science Part A Polymer Chemistry 28 1909-1921

Model filled polymers 5 synthesis of cross-linked monodisperse polymethacrylate beads Journal of Polymer Science Part A Polymer Chemistry 30 137-144

Korea on August 7 1968 He obtained a Bachelor of Science degree in Chemistry from

Keim d graduate school and earned a

resear n Korea Institute of Science and

Coati e

ia

Busin

BIOGRAPHICAL SKETCH

Sangyup Kim was born and raised in the small town of Sung-Joo Kyungbook

yung University (Daegu Korea) in 1993 He entere

Master of Science degree in Chemical engineering in 1995 He worked as a student

cher for 2 years at Textile Polymer Division i

Technology (KIST) in Seoul Korea Next he worked over a year at the Automobile

ng Division in DongJoo-PPG in Korea In August 2001 he began studying for th

degree of Doctor of Philosophy in materials science and engineering at the University of

Florida in Gainesville After getting his PhD he plans to work at the Digital Med

ess Division of Samsung Electronics in Korea starting in January 2006

141

members in Dr El-Shallrsquos research group for their assistance advice and support I

sincerely appreciate Stephen for the assistance of thesis writing

v

TABLE OF CONTENTS page

ACKNOWLEDGMENTS iv

LIST OF TABLES viii

LIST OF FIGURES x

1 INTRODUCTION 1

2 BACKGROUND AND LITERATURE SURVEY5

21 The Significance of End Stage Renal Disease (ESRD)5 22 Hemodialysis (HD) Treatment 6 23 Advances in Membrane Technology 8 24 Sorbent Technology16 25 Limitation of Current Hemodialysis Treatment 19 26 Latex Particles 20

261 The Components for Emulsion Polymerization 21 262 Particle Nucleation 21 263 Types of Processes for Emulsion Polymerization 23 264 Chemistry of Emulsion Polymerization 24 265 Seed Emulsion Polymerization25 266 Polystyrene Latex Particles 26 267 Various Applications of Latex Particles28

27 Proteins 30 271 Interaction Forces between Proteins33 272 β -Microglobulin (β M)2 2 35 273 Serum Albumin 36

28 Protein Adsorption38 281 Interaction between Protein Molecule and Latex Particle38

29 Hypothesis for Toxin Removal39 291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size 40 292 Toxin Removal by Selective Adsorption on Engineered Latex Particles 41

3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY 44

31 Materials 44 32 Latex Particle Preparation46

321 Preparation of Seed Latex Particles47

vi

322 Preparation of Seeded Latex Particles 48 323 Preparation of Core Shell Latex Particles 48 324 Purification of Synthesized Latex Particles48

33 Characterization49 331 Degree of Conversion49 332 Fourier Transform Infrared (FTIR) Spectroscopy49 333 Quasielastic Light Scattering (QELS) 50 334 Field Emission-Scanning Electron Microscopy (FE-SEM) 50 335 Zeta Potential Measurement 50 336 Protein Adsorption51 337 Blood Biocompatibility by Hemolysis Test 53

4 RESULTS AND DISCUSSION55

41 Polymerization of Latex Particles55 411 Polystyrene Seed Latex Particles55 412 The Growth of Polystyrene (PS) Seed Latex Particles61 413 Core Shell Latex Particles 71

42 Characterization of Latex Particles75 421 Fourier Transform Infrared Spectroscopy (FTIR)75 422 Zeta Potential Measurements 75

43 Protein Adsorption Study 85 431 Adsorption Isotherm87 432 Adsorbed Layer Thickness 103 433 Gibbs Free Energy of Protein Adsorption 104 434 Kinetics of Adsorption 107

44 Blood Compatibility 112

5 SUMMARY OF RESULTS CONCLUSION AND RECOMMENDATION FOR FUTURE WORK115

51 Summary of Results115 52 Conclusions121 53 Recommendation for Future Work121

LIST OF REFERENCES122

BIOGRAPHICAL SKETCH 141

vii

LIST OF TABLES

Table page 4-1 The polymerization recipe of polystyrene (PS) seed particles 57

4-2 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles less than 500nm in size 63

4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size64

4-4 The preparation recipe of PS core with various shell latex particles72

4-5 The isoelectric point (IEP) of common proteins 83

4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model 93

4-7 The equilibrium concentration values (q ) of BSA adsorption on PSPMMA particles calculated the Langmuir-Freundlich isotherm model

m 10096

4-8 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model

90 1098

4-9 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model

75 25100

4-10 The equilibrium concentration values of β M adsorption calculated the Langmuir-Freundlich isotherm model

2102

4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37 Co 104

4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37 Co 104

4-13 The values of Gibbs free energy change of BSA adsorption in phosphate buffer (PB) at 37 Co 105

4-14 The values of Gibbs free energy change of BSA adsorption in phosphate buffered saline (PBS) at 37 Co 105

viii

4-15 The values of Gibbs free energy change of β M adsorption in phosphate buffere (PB) at 37 C and pH 74

2o 106

4-16 Fitting parameters of second-order kinetic model 111

ix

LIST OF FIGURES

Figure page 1-2 Stereo drawing of the αndashcarbon backbone of β M2 2

2-1 An image of the location and cross section of a human kidney 5

2-2 A schematic draw of the hemodialysis route7

2-3 Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes 11

2-4 The chemical structure of polysulfone (PSf) 12

2-5 The chemical structure of polyamide (PA)13

2-6 Chemical structure of polyacrylonitrile (PAN) 14

2-7 Emulsion polymerization system24

2-8 L-α-amino acid 31

2-9 Ribbon diagram of human β M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ)

236

2-10 Secondary structure of human serum albumin (HSA) with sub-domains 37

2-11 The relationship between pore size and particle size in body-centered cubic and closed packed arrays41

2-12 Schematic representation of the protein adsorption on the core shell latex particles at pH 7442

3-1 Chemical structure of main chemicals45

3-2 Experimental setup for semi-continuous emulsion polymerization 46

3-3 The particle preparation scheme in various types and size ranges of latex particles 47

3-4 Schematic of the procedure for a protein adsorption test 52

3-5 Separation of RBC from whole blood by centrifuge process53

x

3-6 Schematic of the procedure for hemolysis test 54

4-1 Schematic representation of semi-continuous seed latex particles preparation and growth56

4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS (B) PS (C) PS (D) PSS259 S233 S207 S181 59

4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm 65

4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm 68

4-5 Dependence of the particle size on the surfactant to monomer ratio70

4-6 Schematic of core shell latex particle structures 71

4-7 Scanning Electron Micrograph of latex particles (A) PS (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA

100

90 10 75 25 73

4-8 FTIR spectra of polymerized latex particles (A) bare polystyrene (PS) (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA100 90 10 75 25 76

4-9 Schematic representation of ion distribution near a positively charged surface 78

4-10 Schematic representation of zeta potential measurement79

4-11 Zeta potential of PS latex particles at 25 Co 80

4-12 Zeta potential of PSPMMA latex particles at 25 C100o 81

4-13 Zeta potential of PSPMMA PAA latex particles at 25 C90 10o 82

4-14 Zeta potential of PSPMMA PAA latex particles at 25 C75 25o 82

4-15 The decay of surface potential with distance from surface in various electrolyte concentrations (1) low (2) intermediate (3) high 84

4-16 High-affinity adsorption isotherm of typical flexible polymer on solid surface 86

4-17 Overall schematic representation of the protein adsorption on the synthesized latex particles86

4-18 The dependence of UV absorbance on BSA concentration (A) 10mgml (B) 06mgml (C) 02mgml87

4-19 Standard curve of net absorbance vs BSA sample concentration 88

xi

4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37 C in phosphate buffer mediao 89

4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37 Co 90

4-22 Adsorption isotherm of BSA adsorption 37 C in phosphate buffered saline (PBS) on PS latex particles

o

91

4-23 Conformation of bovine serum albumin (BSA) on latex particles92

4-24 Adsorption isotherm of BSA 37 C in PB on PSPMMA core shell latex particles

o100

94

4-25 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA core shell latex particles

o100

95

4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA PAA core shell latex particles

o90 10

97

4-27 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA PAA core shell latex particles

o90 10

98

4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA PAA latex particleso75 25 99

4-29 Adsorption isotherm of BSA 37 C in PBS on PSPMMA PAA core shell latex particles

o75 25

99

4-30 Adsorption isotherm of β M onto PS and PSPMMA latex particles in PB at 37 C and pH 74

2 100o 101

4-31 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74

2 90 10o 101

4-32 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74

2 75 25o 102

4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37 C and pH 74

o

107

4-34 The kinetics of protein adsorption in PB on PS latex particles at 37 C and pH 74

o

109

4-35 The kinetics of protein adsorption in PB on PSPMMA latex particles at 37 C and pH 74

100o

109

4-36 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74

90 10o 110

xii

4-37 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74

75 25o 110

4-38 Image of red blood cells 113

4-39 Hemolysis caused by latex particles n=5114

xiii

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS

By

Sangyup Kim

December 2005

Chair Hassan El-Shall Major Department Materials Science and Engineering

Latex particles with well-defined colloidal and surface characteristics have received

increasing attention due to their useful applications in many areas especially as solid

phase supports in numerous biological applications such as immunoassay DNA

diagnostic cell separation and drug delivery carrier Hemodialysis membrane using

these particles would be another potential application for the advanced separation

treatment for patients with end stage renal disease (ESRD) It is desirable to remove

middle molecular weight proteins with minimal removal of other proteins such as

albumin Thus it is necessary to understand the fundamental interactions between the

particles and blood proteins to maximize the performance of these membranes This

improvement will have significant economic and health impact

The objective of this study is to synthesize polymeric latex particles of specific

functionality to achieve the desired selective separation of target proteins from the human

blood Semi-continuous seed emulsion polymerization was used to prepare monodisperse

xiv

polystyrene seed particles ranging from 126plusmn75 to 216plusmn53 nm in size which are then

enlarged by about 800nm Surfactant amount played a key role in controlling the latex

particle size Negatively charged latex particles with a different hydrophobicity were

prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid

monomer The prepared polymeric particles include bare polystyrene (PS) particles less

hydrophobic PS core and PMMA shell particles and more hydrophilic PS core and

PMMA-co-PAA shell latex particles with a 370nm mean diameter SEM light scattering

and zeta potential measurements were used to characterize particle size and surface

properties Adsorption isotherms of two proteins bovine serum albumin (BSA) and β2-

microglobulin (β2M) on latex particles were obtained as a function of pH and ionic

strength using the bicinchoninic acid (BCA) assay method The Langmuir-Freundlich

adsorption model was used to determine the adsorption amount of protein at equilibrium

The thickness of adsorbed BSA layer on latex particles was obtained in order to

investigate the adsorption orientation such as end-on or side-on mode Adsorption

kinetics experiments for both proteins and all latex particles were also performed The

adsorption kinetic constant determined from the Langmuir-Freundlich adsorption

isotherm model was used to calculate Gibbs free energy of adsorption to compare the

competitive adsorption of BSA and β2M Hemolysis tests were performed to investigate

the blood compatibility of synthesized latex particles PSPMMA90PAA10 and

PSPMMA75PAA25 core shell latex particles had desirable material properties with not

only a large amount and high rate of selective β2M adsorption over BSA but also high

blood compatibility showing less than 3 hemolysis

xv

CHAPTER 1 INTRODUCTION

End stage renal disease (ESRD) is a chronic condition in which kidney function is

impaired to the extent that the patientrsquos survival requires removal of toxins from the

blood by dialysis therapy or kidney transplantation The National Kidney Foundation

estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF] The

number of people with ESRD is rapidly increasing in the United States with

approximately 96 295 incidents and 406 081 prevalent patients including 292 215 on

dialysis and 113 866 with a functioning graft in 2001 It is projected that there will be

more than 22 million ESRD patients by 2030 [USRDS 2003] The expenditure for the

ESRD treatment program had reached $228 billion 64 of the Medicare budget in

2001 Due in part to the limited availability of kidneys for transplantation hemodialysis

is the primary clinical treatment for the patients with ESRD

The central element of a hemodialysis instrument is the semipermeable membrane

that allows for selective transport of low molecular weight biological metabolites less

than 5000 Da such as urea and creatinine as well as excess water and electrolytes [Baker

2004] from the blood One limitation of current dialysis technologies is the inability to

efficiently remove middle molecular weight toxins such as β2-Microglobulin (β2M) and

interleukin 6 (IL-6)

β2M is a causative protein of dialysis-related amyloidosis (DRA) a disease arising

in patients with chronic kidney failure as a serious complication of long-term

hemodialysis treatment [Gejyo et al 1985] β2M deposition in tissue is the primary cause

1

2

of destructive arthritis and carpal tunnel syndrome [Vincent et al 1992 Drueke 2000]

The β2M structure is shown in Figure 1-2

Figure 1-2 Stereo drawing of the αndashcarbon backbone of β2M [Becker et al 1985]

Although attempts have been made to increase the efficiency of middle molecular

weight toxin removal by changes in the membrane pore size and the use of innovative

materials to adsorb these toxins [Samtleben et al 1996 Ronco et al 2001] removal

efficiency is not as high as those achieved by a normal healthy kidney Traditional

membranes have a number of processing and performance limitations [Westhuyzen et al

1992 Leypoldt et al 1998] such as a restricted choice of surface chemistries and limited

control of porosity The development of novel engineering membrane technology is

needed to remove middle molecule toxins

Polymeric latex particles have received increasing attention in medical application

areas especially as solid phase supports in biological applications [Piskin et al 1994]

Examples of these applications include immunoassay [Chen et al 2003 Radomske-

Galant et al 2003] DNA diagnostic [Elaїssari et al 1998] cell separation drug delivery

carrier [Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] etc This is because of

3

the well-defined colloidal and surface characteristics of the particles By using a seed

emulsion polymerization method it is possible to synthesize monodisperse latex particles

with various particle size ranges and surface chemistry Functionalized core-shell latex

particles can be introduced by multi-step emulsion polymerization Core particles are

synthesized in the first stage of the polymerization and the functional monomer is added

in the second stage This is done without any emulsifier addition to prevent the

production of new homopolymer particles [Keusch et al 1973] The core-shell particles

are useful in a broad range of applications because of their improved physical and

chemical properties over their single-component counterparts [Lu et al 1996 Nelliappan

et al 1997] Through the development of a hemodialysis membrane using monodisperse

latex particles improvements in advanced separation treatment for patients with end

stage renal disease (ESRD) can be realized This requires the maximum removal of

middle molecular weight proteins with minimal removal of other beneficial proteins such

as albumin Thus an understanding of the fundamental interactions between the particles

and biopolymers is vital to maximize the performance of this membrane technology

The field of material science and biotechnology is based on fundamental chemistry

has developed over the past three decades into todayrsquos powerful discipline that enables

the development of advanced technical devices for pharmaceutical and biomedical

applications This novel and highly interdisciplinary field is closely associated with both

the physical and chemical properties of organic and inorganic particles [Niemeyer 2001]

Hemodialysis membrane using these particles would lead to improvements in the

advanced separation treatment for patients with end stage renal disease (ESRD) The

interdisciplinary nature of this approach enables a more complete understanding of the

4

phenomena of protein adsorption and the material properties necessary for selective

separation This innovative approach to membrane fabrication has the potential of making

inexpensive highly efficient membranes for both industrial and specialized separation

processes

The goal of this study is to prepare polymeric latex particles with tailored

properties to maximize separation of toxin molecules and to investigate the fundamental

interactions between the particles and molecules in the biological system in order to

optimize the performance of a membrane material for these applications

Polymeric latex particles were synthesized with specific functionality in an attempt

to achieve selective separation Seeded emulsion polymerization was used to synthesize

functionalized monodisperse latex particles in various sizes Negatively charged

hydrophobic polystyrene latex particles were synthesized by the same method Core shell

latex particles PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25 were also

synthesized to differentiate the degree of hydrophobicity of particles Scanning Electron

Micrograph (SEM) and light scattering measurements were used to characterize particle

size and shape and zeta potential measurements were conducted to measure the electrical

surface property of synthesized particles Adsorption isotherms of target proteins bovine

serum albumin (BSA) and β2M on latex particles were obtained as a function of pH

ionic strength and protein concentrations using the BCA assay method Adsorption

kinetics for both proteins on the latex particles were also measured Finally hemolysis

tests were run to determine the biocompatibility of polymer latex particles with human

blood This research will be described in more detail in chapters 3 and 4

CHAPTER 2 BACKGROUND AND LITERATURE SURVEY

21 The Significance of End Stage Renal Disease (ESRD)

The kidneys are responsible for removing excess fluid minerals and wastes from

the blood regulating electrolyte balance and blood pressure and the stimulation of red

blood cell production They also produce hormones such as erythropoietin (EPO) and

calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005 Kurbel

et al 2003] EPO acts on the bone marrow to increase the production of red blood cell in

case of bleeding or moving to high altitudes Calcitriol mainly acts on both the cells of

the intestine to promote the absorption of calcium from food and also bone to mobilize

calcium from the bone to the blood When kidneys fail harmful waste builds up blood

pressure rises and body retains excess fluid The body also does not make enough red

blood cells When this happens treatment is needed to replace the function of failed

kidneys

Figure 2-1 An image of the location and cross section of a human kidney

5

6

The National Kidney Foundation estimates that over 20 million Americans had

chronic kidney disease in 2002 [NKF] Chronic renal disease is a gradual and progressive

loss of the function unlike acute renal disease where sudden reversible failure of kidney

function occurs Chronic renal failure usually takes place over a number of years as the

internal structures of the kidney are slowly damaged In the early stages there can be no

symptoms In fact progression may be so gradual that symptoms do not occur until

kidney function is less than one-tenth that of a normal health kidney If left untreated

chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal

Disease (ESRD) ESRD is a rapidly growing heath-care problem in the United States In

2001 approximately 96295 incidents and 406081 prevalent patients were diagnosed

with ESRD including 292215 patients on dialysis and 113866 patients with a

functioning graft [USRDS 2003] The projected number of patients with ESRD is

expected to exceed 22 million patients by 2030 with much of this growth being driven by

the increasing prevalence of major contributing factors such as diabetes and high blood

pressure [USRDS 2003] A great extent of ESRD program cost and Medicare budget

have been spent in 2001 Due in part to a limited availability of kidneys for

transplantation hemodialysis (HD) is the primary method of treatment for ESRD and is

currently used for approximately 61 of US ESRD patients

22 Hemodialysis (HD) Treatment

HD removes toxins from the body by extracorporeal circulation of the blood

through a semipermeable membrane referred to as a dialyzer The toxins are removed

primarily by diffusion across the membrane to a dialysate solution which is circulated on

the opposite side of the membrane The cleaned blood is then returned to the blood

stream A surgically constructed vascular access connects the extracorporeal circuit to the

7

patientrsquos system Treatments are almost always performed three times per week in

specially equipped dialysis facilities for 3-4 hours per each treatment Figure 2-2 shows

the scheme of the hemodialysis route The critical element of a HD instrument is the

semipermeable membrane which allows for selective transport of low molecular weight

biological metabolites from blood

igure 2-2 A schematic draw of the hemodialysis route

ESRD patients who undergo dialysis therapy often experience several other

problems associated with the treatment such as anemia fatigue bone problems joint

problems itching sleep disorders and restless legs Anemia is common in patient with

kidney disease because the kidneys produce the hormone erythropoietin (EPO) which

stimulates the bone marrow to produce red blood cells Diseased kidneys often do not

produce enough EPO to stimulate the bone marrow to make a sufficient amount of red

blood cells This leads to bone disease referred to as renal osteodystrophy and causes

bones to become thin and weak or malformed and can affect both children and adults

Older patients and women who have gone through menopause are at greater risk for this

disease Uremic toxins which cannot be remove from the blood by the current dialyzer

membranes can lead to itching This problem can also be related to high levels of

F

8

parathyroid hormone (PTH) Dialysis patients can also experience day-night revers

is they have insomnia at night and sleep during the day This can be related to possible

nerve damage in the body and a chemical imbalance in the blood due to the excess toxin

Oxidative stress is a problem for patients on maintenance dialysis This problem is due to

an imbalance between pro- and antioxidant factors [Roselaar et al 1995 Cristol et al

1994] Oxidative stress affects oxidation of low-density lipoproteins which are the main

factor for atherogenesis [Huysmans et al 1998] and is also involved in the development

of malignancies and diabetes mellitus [Rice-Evans et al 1993] In order to reduce

antioxidant defense dialysis is needed to contribute to help stimulate free radical

production or eliminate antioxidants Dialysis-related amyloidosis (DRA) is also a

common and serious problem for people who have been on dialysis for more than 5

years DRA develops when proteins in the blood deposit on joints and tendons causi

pain stiffness and fluid in the joint as is the case with arthritis Normally the healthy

kidneys can filter out these proteins but dialysis filters are not as effective

23 Advances in Membrane Technology

al that

s

ng

Dialysis for blood of ESRD

Hemo filtration

s

ysis using a cellulose based membrane occurred

1913 John Abel [1990] from the Johns Hopkins Medical School described a method

purification is widely used in the treatment

dialysis (HD) techniques use a semi-permeable membrane to replace the

role of the kidney The membranes used in HD can be broadly classified into those based

on cellulose and those manufactured from synthetic copolymers These membranes come

in various shapes such as sheets tubular structures or hollow fiber arrangements The

hollow fiber type is the most popular and is incorporated into over 800 different device

in world wide [Ronco et al 2001]

The first attempt at blood dial

9

where sing

e

om

ysis was performed until the mid 1960 The basic

molec

rial in

een

benzyl)

was used for the membrane with

hepar

by the blood of a living animal may be submitted to dialysis outside the body u

a membrane based on cellulose and returned to the natural circulation without exposur

to air infection by microorganisms or any alteration that would necessarily be prejudicial

to life This same technique is still used to today however the device used has been

modified over the years as better membranes were developed and the anti-coagulant

heparin has become available

Cellulose membranes have been widely used for the treatment of renal failure fr

1928 when the first human dial

ular structure of cellulose is made of a long chain containing hydroxyl (OH)

groups The realization that such groups imparted undesirable qualities on the mate

respect to blood contact behavior was discovered in the early 1970s and since has b

the focus of development These modified cellulose membranes used the partial

substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt

to reduce their negative effect The result is a molecular mosaic of hydrophobic (

and hydrophilic (hydroxyl and cellulose) regions

Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients

with acute renal failure in 1943 Cellophane tubing

in as the anticoagulant For the next 17 years hemodialysis therapy was performed

by this method but only for the patients with acute reversible renal failure Vascular

access required repeated surgical insertions of cannulas (slender tubes) into an artery and

vein and limited the number of treatments for a patient could receive in order to

minimize the amount of vascular damage

10

Initially the need for dialysis in patients with acute renal failure was determ

mainly by the development of signs and sym

ined

ptoms of uremia After dialysis some time

might

et al

ent improvement In 1960 the development of a shunt [Quinton et al

1960]

fone (PSf) polyamide (PA) and polyacrylonitrile (PAN) by phase inversion or

precip

lidone

e

elapse before uremic manifestations returned to warrant a sequential treatment of

dialysis Many patients with acute renal failure secondary to accidental or surgical

trauma were hypercatabolic but the interdialytic interval might be prolonged because of

anorexia or use of a low-protein diet However Teschan and his coworkers [Obrien

1959] showed that patient well-being and survival were improved by what they termed

prophylactic daily hemodiaysis or administration of the treatment before the patient

again became sick with uremia Their report in 1959 was the first description of daily

hemodialysis

Development of membrane accessories such as a shunt has also been an area of

focus for treatm

a flexible polytetrafluoroethylene (PTFE or Teflonreg) tubing made many more

hemodialysis treatments possible for chronic kidney failure patients PTFE has a non-

stick surface and is relative biocompatibility leading to minimized blood clotting in the

shunt

Synthetic membranes are prepared from engineered thermoplastics such as

polysul

itation of a blended mixture resulting in the formation of asymmetric and

anisotropic structures Figure 2-3 shows a fiber type of the Polyflux S membrane

consisting of poluamide (PA) polyacrylethersulfone (PAES) and polyvinylpyrro

(PVP) with the integral three-layer structure The skin layer on the inside fiber typ

membrane contacts blood and has a very high surface porosity and a narrow pore size

11

distribution This layer constitutes the discriminating barrier deciding the permeabili

and solute retention properties of the membrane The skin layer is supported by thick

sponge-type structure larger pores providing mechanical strength and very low

hydrodynamic resistance

ty

Figure 2-3 Cross-sectional SEM image view of the Polyflux S (polyamide +

polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch et al 1998]

n

[Malchesky 2004] because of its thermal stability mechanical strength and chemical

inertn ciated

ls

m

PSf is a widely used membrane material for the hemodialysis applicatio

ess According to a report from the National Surveillance of Dialysis-Asso

Disease (NSDAD) in the US over 70 of hemodialysis membranes were PSf based

[Bowry 2002] This is most likely because PSf has many advantages over other materia

This synthetic polymer is one of few materials that can withstand sterilization by stea

ethylene oxide and γ-radiation PSf membrane can be prepared by conventional

immersion precipitation methods into many different shapes including porous hollow

12

fiber or flat sheet hemodialysis membranes The material also has a high permeab

low molecular weight proteins and solute and high endotoxin retention The chemical

structure of PSf is shown in Figure 2-4

ility to

Figure 2-4 The chemical structure of polysulfone (PSf)

There is one major disadvantage to PSf The hydrophobic nature of the PSf causes

ment alternative pathway

leadin

s not

3

et al

in

O acrylate

serious complications through the activation of the comple

g to the adsorption of serum proteins onto the membranes [Singh et al 2003]

Anticoagulants are added during dialysis therapy to avoid blood clotting but this doe

completely eliminate the problem In order to overcome this disadvantage of the PSf

membrane various studies have been performed to change the materialrsquos surface

properties These investigations include hydrophilic polymer coating [Brink et al 199

Kim et al 1988 Higuchi et al 2003] layer grafting onto PSf membrane [Wavhal

2002 Song et al 2000 Pieracci et al 2002 Mok et al1994] and chemical reaction of

hydrophilic components onto the membrane surface [Higuchi et al 1990 1991 1993

Blanco et al 2001 Nabe et al 1997 Guiver et al 1993] Hydrophilic monomers 2-

hydroxy-ethylmethacrylate (HEMA) acrylic acid (AA) and methacrylic acid (MMA)

have also been grafted onto PSf membrane to increase flux and Bovine Serum Album

(BSA) retention [Ulbricht et al 1996] Hancock et al [2000] synthesized

polysulfonepoly(ethylene oxide) (PEO) block copolymers to improve the resistance to

platelet adhesion Kim et al [2003] also studied blending a sulfonated PE

diblock copolymer into PSf in order to reduce platelet adhesion and enhance

13

biocompatibility PEO is a commonly used biomaterial due to its excellent resistance

protein adsorption and inherent biocompatibility [Harris 1992] Kim et al [20

a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf

membrane with an ultra-thin skin layer The polymer had an entrapped diblock

copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-SO

to

05] studied

ate

lic graft

l)

e of thier

aromatic

orand

3 acryl

and a hydrophobic block of octadecylacrylate (OA) Molecular dynamic (MD)

simulations were performed as a function of copolymer density to optimize interfacial

structure information McMurry [2004] developed a strategy using an amphiphi

copolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glyco

When compared to unmodified PSf these graft copolymer and resulting blend

membranes are found to hold promise for biomedical device applications

Polyamide (PA) membranes have also been used for hemodialysis becaus

mechanical strength in both wet and dry conditions Polyamide consists of

aliphatic monomers with amide bonding (-CONH-) also known as a peptide bond

The basic amide bond in polyamide is shown in Figure 2-5 R1 and R2 can be either

aromatic or aliphatic linkage group

C N

H

O

~ R R1 2~C N

H

O

~ R R1 2~

Figure 2-5 The chemical structure of polyamide (PA)

Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane

ompatible technique due to the

use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of

and concluded that PA hemofiltration was a highly bioc

14

the ac

e

e

is

n

is a semi-crystalline polymer and the mechanical properties

strong

2

CN

tivated anaphylatoxins and β2-Microglobulin (β2M) The PA based membrane

Polyfluxreg (manufactured by Gambro GmbH Germany) blended with polyamide

polyarylethersulfone and polyvinylpyrrolidone (PVP) was able to clean small molecules

such as urea dreatinine and phosphate as well as decrease β2M amount by 502

[Hoenich et al 2002] Due to the non-selectivity of the membrane removal of thes

unwanted materials also led to the undesirable loss of beneficial proteins during therapy

Meier et al [2000] evaluated different immune parameters using a modified cellulos

low-flux hemophan and synthetic high-flux PA membrane during a 1 year period in

chronic hemodialysis patients They found that the 1-year immunological evaluation of

hemodiaysis membrane biocompatibility was associated with changes in the pattern of

chronic T-cell actiovation

Polyacrylonitrile (PAN) is another commonly used membrane material because it

inherently hydrophilic and has been commercialized for ultrafiltration and microfiltratio

[Scharnagl et al 2001] PAN

ly depend on the crystalline structures The chemical structure of PAN is shown in

figure 2-6

CH CH

n2

CN

CH CH n

Chemical structure of polyacrylonitrile (PAN)

The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming

agent gives PAN membranes more flexible processing parameters and increased

e has been optimized through

copolymerization with many other vinyl monomers including glycidyl methacrylate

Figure 2-6

performance [Jung et al 2005] PAN membrane performanc

15

[Godj

acid

for

for

a non-

solub this

in

oid underneath the inner

surface of membrane decreased by increasing the amount of solvent DMSA in the

evargova et al 1999 Hicke et al 2002] N-vinylimidazole [Godjevargova et al

2000] hydroxyl ethyl methacrylate [Ray et al 1999 Bhat et al 2000] methacrylic

[Ray et al 1999] vinyl pyrrolidone [Ray et al 1999] acrylic acid [Trotta et al 2002]

acrylamide [Musale et al 1997] and vinylchloride [Broadhead et al 1998] These

monomers provide a reactive group for enzyme immobilization improved mechanical

strength solvent-resistance pervaporation permeation flux anti-fouling and bio-

compatibility Because of this PAN-based copolymer membranes have great potential

the treatment of hemodialysis in an artificial kidney This material can also be used

other applications like the treatment of wastewater the production of ultra-pure water

biocatalysis together with separation and methanol separation by pervaporation

Lin and his coworker [2004] studied the modification of PAN based dialyzer

membranes to increase the hemocompatibility by the immobilization of chitosan and

heparin conjugates on the surface of the PAN membrane When a foreign material is

exposed to blood plasma proteins are adsorbed clotting factors are activated and

le fibrin network or thrombus is formatted [Goosen et al 1980] The result of

research was that the biocompatible chitosan polymer and a blood anticoagulant hepar

prevented blood clotting They showed prolonged coagulation time reduced platelet

adsorption thrombus formation and protein adsorption

Nie et al [2004] studied PAN-based ultrafiltration hollow-fiber membranes

(UHFMs) In order to improve the membrane performance acrylonitrile (AN) was

copolymerized with other functional monomers such as maleic anhydride and α-allyl

glucoside They found that the number and size of macrov

16

intern erum

and

d a

rface The second step is the

transf after

yjak et

embrane

1978 Malchesky et al 1978] has been further developed to increase the efficiency of

al coagulant The water flux of the UHFMs also decreased while the bovine s

albumin rejection increased minutely Godjevargova et al [1992] modified the PAN

based membrane with hydroxylamine and diethylaminoethylmethacrylate to improve

membrane dialysis properties Formed functional groups like primary amine oxime

tertiary amine groups provided the membrane with more hydrophilic properties an

substantial increase in the permeability of the membranes

The wide use of filtration in practice is limited by membrane fouling Solute

molecules deposit on and in the membrane in the process of filtration causing dramatic

reduction in flux through the membrane Fouling occurs mostly in the filtration of

proteins Three kinetic steps are involved in the fouling of UF membranes according to

Nisson [1990] The first step is the transfer of solute to the su

er of solute into the membrane until it either finally adsorbs or passes through

a set of adsorption-desorption events The third step includes surface binding

accompanied by structural rearrangement in the adsorbed state [Ko et al 1993] Br

al [1998] studied the surface modification of a commercially available PAN membrane

to develop superior filtration properties with less fouling by proteins The PAN

membrane was immersed in excess NaOH solution to convert some of the surface nitrile

groups into carboxylic groups by the hydrolysis process This modified PAN m

was not so severely fouled in the Bovine Serum Albumin (BSA) filtration test The pore

size however decreased during the hydrolysis process leading to a significant reduction

in flux and made the membrane less productive in the ultrafiltration (UF) mode

24 Sorbent Technology

Over the last three decades sorbent technology [Castino et al 1976 Korshak et al

17

dialysis or replace it for the treatment of ESRD Sorbents remove solutes from solution

through specific or nonspecific adsorption depending on both the nature of the solute and

the sorbent Specific adsorption contains tailored ligands or antibodies with high

selectivity for target molecules een used in autoimmune

disord

and

e

al 1996] various resins [Ronco et al 2001]

album

e

as

Specific adsorbents have b

ers such as idiopathic thrombocytopenic purpura [Snyder et al 1992] and for the

removal of lipids in familial hyper cholesterolemia [Bosch et al 1999] Nonspecific

adsorbents such as charcoal and resins attract target molecules through various forces

including hydrophobic interactions ionic (or electrostatic) attraction hydrogen bonding

and van der Waals interactions

New dialysate with sorbents has become an accepted modification of dialysis

sorbent hemoperfusion is gaining ground as a valuable addition to dialysis especially as

new sorbents are developed [Winchester et al 2001] Hemoperfusion is defined as th

removal of toxins or metabolites from circulation by the passing of blood within a

suitable extracorpoteal circuit over semipermeable microcapsules containing adsorbents

such as activated charcoal [Samtleben et

in-conjugated agarose etc Novel adsorptive carbons with larger pore diameters

have been synthesized for potential clinical use [Mikhalovsky 1989] Newly recognized

uremic toxins [Dhondt et al 2000 Haag-Weber et al 2000] have resulted in several

investigations on alternatives to standard or high-flux hemodialysis to remove thes

molecules These methods include hemodiafilteration with [de Francisco et al 2000] or

without [Ward et al 2000 Takenaka et al 2001] dialysate regeneration using sorbents

well as hemoperfusion using such adsorbents as charcoal and resins

18

Kolarz et al [1989 1995] studied the hyper-crosslinked sorbent prepared from

styrene and divinylbenzene (DVB) for a hemoperfusion application They found that the

pore structure of a swelling sorbent was changed by additional crosslinking with α αrsquo-

dichloro-p-xylene in the presence of a tin chloride catalyst and in a di

chloroethane

soluti and

en

decyl groups that attract β2M through a hydrophobic interaction The

adsor

of

5]

d

penet

on They also realized that the hemocompatibility was useful for nemoperfusion

could be imparted to the sorbents by introducing sulfonyl groups at a concentration of

about 02mmolg

A special polymeric adsorbing material (BM-010 from Kaneka Japan) has be

investigated by another group [Furuyoshi et al 1991] for the selective removal of β2M

from the blood of dialysis partients The adsorbent consists of porous cellulose beads

modified with hexa

ption capacity of this material is 1mg of β2M per 1ml of adsorbent Using a

hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a

high-flux hemodialyzer several small clinical trials were performed During 4-5 hours

treatment about 210mg of β2M were removed thus reducing the concentration in the

blood by 60-70 of the initial level [Nakazawa et al 1993 Gejyo et al 1993 199

RenalTech developed a hemoperfusion device BetaSorbreg containing the hydrate

cross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure

designed to remove molecules between 4 and 30 kDa [Winchester et al 2002] In this

case solute molecules are separated according to their size based on their ability to

rate the porous network of the beaded sorbents The resin beads were prepared with

a blood compatible coating and confirmed to be biocompatible in vivo in animals

[Cowgill et al 2001]

19

25 Limitation of Current Hemodialysis Treatment

Hemodialysis is a widely used life-sustaining treatment for patients with ESRD

However it does not replace all of the complex functions of a normal healthy kidne

a result patients on dia

y As

lysis still suffer from a range of problems including infection

accelerated cardio ion anemia

chron

e

se middle

alysis

sessio

s

f

vascular disease high blood pressure chronic malnutrit

ic joint and back pain and a considerably shortened life span One significant

limitation of the current dialysis technology is the inability to efficiently remove larger

toxic molecules This is mainly because of the broad pore size distribution reducing th

selective removal of toxins and unsatisfied biocompatibility causing lots of

complications such as inflammation blood clotting calcification infection etc

Dialysis purifies the patients blood by efficiently removing small molecules like

salts urea and excess water However as toxic molecules increase in size their removal

rate by hemodialysis substantially declines Typically only 10 - 40 of the

molecular weight toxins (300-15000 Da) are removed from the blood during a di

n [Vanholder et al 1995] These toxins then reach an abnormally high level and

begin to damage the body One such toxin β2M causes destructive arthritis and carpal

tunnel syndrome by joining together like the links of chain to form a few very large

molecules and deposit damaging the surrounding tissues [Lonnemann et al 2002] This i

also a main cause of mortality for long-term dialysis patients Other middle molecule

toxins appear to inhibit the immune system and may play a significant role in the high

susceptibility to infections in dialysis patients Still others are believed to impair the

functioning of several other body systems such as the hematopoietic and other endocrine

systems This may contribute to accelerated cardiovascular disease the leading cause o

20

death among dialysis patients as well as clinical malnutrition which affects up to 50 of

this patient population

Over the last decade polymeric dialysis membranes have been developed to

increase the capacity for removing middle molecular weight toxins by changing the pore

size of dialyzer membranes and using new materials that adsorb these toxins for

impro se

styrene made during World War II in the United

States The Dow chemical compan anufacturer of polystyrene

includ

d of

on

rug

ntrol of

ved removal characteristics However removal efficiency is not as high as tho

achieved by a normal healthy kidney

26 Latex Particles

The first synthetic polymer synthesized using emulsion polymerization was a

rubber composed of 13-butadiene and

y has been a major m

ing latex which they used in paint formulations The theory of emulsion

polymerization in which a surfactant is used was established by Harkins [1948] and by

Smith and Ewart [1948] By 1956 the technology was complete including the metho

building larger diameter particles from smaller ones The product by this emulsi

polymerization is referred to as latex a colloidal dispersion of polymer particles in water

medium [Odian 1991] Latexes are currently undergoing extensive research and

development for a broad range of areas including adhesives inks paints coatings d

delivery systems medical assay kits gloves paper coatings floor polish films carpet

backing and foam mattresses to cosmetics The relatively well known and easy co

the emulsion process is one of main advantages for these applications Therefore

polymeric latex particle prepared by emulsion polymerization can be a candidate for the

medical applications because of the easy control of the particle size and morphology as

well as flexible surface chemistry to be required

21

261 The Components for Emulsion Polymerization

The main components for emulsion polymerization process are the monomer a

dispersing medium a surfactant and an initiator Available monomers are styrene

sing medium is usually water

which

ticles

n

ation To

monomer droplets but in micelles because the initiators are

insolu le

and

olution

butadiene methylmethacylate acryl acid etc The disper

will maintain a low solution viscosity provide good heat transfer and allow

transfer of the monomers from the monomer droplets into micelles and growing par

surrounded by surfactants respectively The surfactant (or emulsifier) has both

hydrophilic and hydrophobic segments Its main functions is to provide the nucleatio

sites for particles and aid in the colloidal stability of the growing particles Initiators are

water-soluble inorganic salts which dissociate into radicals to initiate polymeriz

control the molecular weight a chain transfer agent such as mercaptan may be present

262 Particle Nucleation

Free radicals are produced by dissociation of initiators at the rate on the order of

1013 radicals per milliliter per second in the water phase The location of the

polymerization is not in the

ble in the organic monomer droplets Such initiators are referred to as oil-insolub

initiators This is one of the big differences between emulsion polymerization

suspension polymerization where initiators are oil-soluble and the reaction occurs in the

monomer droplets Because the monomer droplets have a much smaller total surface area

they do not compete effectively with micelles to capture the radicals produced in s

It is in the micelles that the oil soluble monomer and water soluble initiator meet and is

favored as the reaction site because of the high monomer concentration compared to that

in the monomer droplets As polymerization proceeds the micelles grow by the addition

of monomer from the aqueous solution whose concentration is refilled by dissolution of

22

monomer from the monomer droplets There are three types of particles in the emulsion

system monomer droplets inactive micelles in which polymerization is not occurring

and active micelles in which polymerization is occurring referred to as growing polymer

particles

The mechanism of particle nucleation occurs by two simultaneous processes

micellar nucleation and homogeneous nucleation Micellar nucleation is the entry of

radicals ei

ther primary or oligomeric radicals formed by solution polymerization from

the aq ed

the

r

s

n

is coagulation with other particles rather than

polymerization of monomer The driving force for coagulation of precursor particles

ueous phase into the micelles In homogeneous nucleation solution-polymeriz

oligomeric radicals are becoming insoluble and precipitating onto themselves or onto

oligomers whose propagation has ended [Fitch et al 1969] The relative levels of micella

and homogeneous nucleation are variable with the water solubility of the monomer and

the surfactant concentration Homogeneous nucleation is favored for monomers with

higher water solubility and low surfactant concentration and micellar nucleation is

favored for monomers with low water solubility and high surfactant concentration It has

also been shown that homogeneous nucleation occurs in systems where the surfactant

concentration is below CMC [Roe 1968] A highly water insoluble monomer such a

styrene [Hansen et al 1979 Ugelstad et al 1979] has probably created by micellar

nucleation while a water soluble monomer such as vinyl acetate [Zollars 1979] has bee

formed by homogeneous nucleation

A third latex formation reaction mechanism has been proposed referred to as

coagulative nucleation In this reaction the major growth process for the first-formed

polymer particles (precursor particles)

23

sever les

yer

es

r a

l

the beginning of the polymerization As soon as the initiator is

gins with the formation and

growt

and

ved

added at one time during the later stages of the polymerization prior to complete

al nanometers in size is their relative instability compared to larger sized partic

The small size of a precursor particle with its high curvature of the electrical double la

permits the low surface charge density and high colloidal instability Once the particl

become large enough in size maintaining the high colloidal stability there is no longe

driving force for coagulation and further growth of particles takes place only by the

polymerization process

263 Types of Processes for Emulsion Polymerization

There are three types of production processes used in emulsion polymerization

batch semi-continuous (or semi-batch) and continuous In the batch type process al

components are added at

added and the temperature is increased polymerization be

h of latex particles at the same time There is no further process control possible

once the polymerization is started In the semi-continuous emulsion polymerization

process one or more components can be added continuously Various profiles of particle

nucleation and growth can be generated from different orders of component addition

during polymerization There are advantages to this process such as control of the

polymerization rate the particle number colloidal stability copolymer composition

particle morphology In the continuous process the emulsion polymerization components

are fed continuously into the reaction vessel while the product is simultaneously remo

at the same rate High production rate steady heat removal and uniform quality of

latexes are advantages of the continuous polymerization processes

Other available methods include the intermittent addition and shot addition of one

or more of the components In the shot addition process the additional components are

24

conversion of the main monomer This method has been used successfully to develo

water-soluble functional monomers such as sodium styrene sulfonat

p

e [Kim et al 1989]

264

lsion

Chemistry of Emulsion Polymerization

Emulsion polymerization is one type of free radical polymerization and can be

divided into three distinct stages initiation propagation and termination The emu

polymerization system is shown in Figure 2-7

Aqueous phaseAqueous phase

Micelle withmonomer

I Rbull

Emulsifier

Polymer particle swollenWith monomer

Monomerdroplet

I Rbull

Micelle withmonomer

I Rbull

Emulsifier

Polymer particle swollenWith monomer

MonomerdropletMonomerdroplet

I Rbull

Figure 2-7 Emulsion polymerization system [Radicals (Rmiddot) are created from initiators

(I) Monomer is transferred from larger monomer droplet into micelles by emulsifier Initiated polymer particle by radicals is keep growing until monomers are all consumed The reaction is performed in aqueous media]

In the initiation stage free radicals are created from an initiator by heat or an

ultraviolet radiation source The initiator with either peroxides groups (-O-O-) such as

odium persulfate or azo groups (-N=N-) such as azobisisobutyronitrile is commonly

used for em t

with the m er

chain ere

s

ulsion polymerization The primary free radicals created from initiator reac

onomer for initiation of polymerization In the propagation stage the polym

grows by monomer addition to the active center a free radical reactive site Th

are two possible modes of propagations head-to-head addition and head-to-tail addition

25

The head-to-tail mode is the predominant configuration of the polymer chain a result o

steric hindrance of the substitute bulky group In the termination stage polymer chain

growth is terminated by either coupling of two growing chains forming one polymer

molecule or transferring a hydrogen atom (dispropotionation) from one growing chain to

another forming two polymer molecules one having a saturated end group and the other

with an unsaturated end-group

265 Seed Emulsion Polymerization

Polymer latex particles have received increasing interest because of the versatili

of the many applications heterophase polymerization processes like emulsion dispersion

micro-emulsion seeded emulsion precipitation etc Especially to prepare well-defined

microspheres having monodispe

f

ty

rse and various particle sizes as well as surface group

ed particles prepared by emulsion polymerization

and e

s

hological design In

seede to control

n

functionalilties it is necessary to use se

nlarge them to a desired size in the further stages of reactions

Polymeric particles are required to have a uniform particle size in many

applications such as chromatography where they are used as a packing material

Morphological control of latex particle is also important for many practical application

[Schmidt 1972] Seed emulsion polymerization (or two-stage emulsion polymerization) is

a useful method to achieve both monodisperse particle size and morp

d emulsion polymerization [Gilbert 1995] preformed lsquoseedrsquo latex is used

the number of particles present in the final latex The advantage of seeded emulsio

polymerization is that the poorly reproducible process of particle nucleation can be

bypassed so that the number concentration of particles is constant and known Various

mechanisms have been proposed for the growth of latex particles [Ugelstad et al 1980

Okubo et al 1992] using this polymerization technique The initial seed particle

26

preparation step is well known and relatively easy to perform because at the relative

small particle sizes (02 to 05 micron) the particle growth process can be readily

controlled by the use of an emulsifier Enough emulsifier is used to prevent coagulation

but not enough to cause the formation of new particles As the particles are grown to

larger sizes in successive seeding steps it becomes increasingly difficult to mainta

stable uncoagulated emulsion without forming new particles and thereby destroying

monodisperisty of the latex Recently there have been several reports concerning t

reaction kinetics of seed emulsion polymerization and the development of latex

morphologies over the course of the reaction [Chern et al 1990 Delacal et al 1990 L

et al 1995 Lee 2000 2002]

266 Polystyrene Latex Particles

A number of papers have described the synthesis of the polystyrene latex particl

bearing various functional surface groups such as carboxyl [Lee et al 2000 Tun

2002 Reb et al 2000] hydroxyl [Tamai et al 1989] marcapto [Nilson 1989] epoxy

[Shimizu et al 2000 Luo et al

ly

in a

he

ee

es

cel et al

2004] acetal [Izquierdo et al 2004 Santos et al 1997]

ometyl [Izquierdo et al 2004 Park et al 2001

Sarob

h the

d

thymine [Dahman et al 2003] chlor

e et al 1998] amine [Counsin et al 1994 Ganachaud et al 1997 Ganachaud et al

1995 Miraballes-Martinez et al 2000 2001 Anna et al 2005] ester [Nagai et al 1999]

etc In order to produce these functionalized particles different methods for particle

preparation must be used The polymer emulsion with core-shell morphology of latex

particles is one of them This is a multistep emulsion polymerization process in whic

polystyrene ldquocorerdquo particle is synthesized in the first stage and the functional monomer is

added in the second stage of the polymerization without any emulsifier postfeeding to

prevent the production of new homopolymer particles thus forming the functionalize

27

polymer ldquoshellrdquo on the ldquocorerdquo particle [Keusch et al 1973] There are requirements to

limit secondary nucleation and encourage core-shell formation in seeded emulsion

polymerization including the addition of smaller seed particles at high solid content to

increase particle surface area low surfactant concentration to prevent formation of

micelles and the semi-continuous addition of monomer to create a starved-feed conditio

and keep the monomer concentration low There are some advantages [Hergeth et al

1989] of dispersions with polymeric core-shell particles First it is possible to modi

interfacial properties of polymer particles in the aqueous phase by the addition of only

very small amounts of a modifying agent during the last period of the reaction Thus

these core-shell particles are useful in a broad range of applications since they always

exhibit improved physical and chemical properties over their single-component

counterparts [Lu et al 1996 Nelliappan et al 1997] In this way the improvement of

surface properties of such dispersions is straightforward and inexpensive The other is

that polymers with a core-shell structure are perfect model systems for investigating th

material properties of polymer blends and composites because of their regular

distribution of one polymer inside a matrix polymer and because of the simple sp

geometry of the system

Their properties usually depend on the structures of latex particles Chen and his

coworkers [Chen et al 1991 1992 1993] reported the morphological development of

core shell latex particles of polystyrenepoly(methyl methacrylate) during

polymerization Before the research by Chen and his coworker Min et al [Min et al

1983] reported the morph

n

fy the

e

herical

ological development of core shell latex of polystyrene

(PS)polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the

28

additi

of

ity of graft

ing reagents Thus protein adsorption on polymeric solid surfaces has

st are microspheres defined as ldquofine

polym can be

n

r was

containing antigen urine serum etc The latex will become agglutinated and visibly

on method of PS They found that the percentage of grafting PS to the PBA was

greatest for the batch reaction and the PBA-PS core-shell particles with a high degree

grafting remained spherical upon aging test because of the emulsifying abil

copolymer

267 Various Applications of Latex Particles

Latex particles are applicable to a wide range of areas such as biomedical

applications [Piskin et al 1994] especially as the solid phase such as in immunoassays

[Chern et al 2003 Radomske-Galant et al 2003] DNA diagnostic drug delivery carriers

[Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] blood cell separations and

column pack

become a center of attention Of particular intere

er particles having diameters in the range of 01 to several micronsrdquo which

used as functional tools by themselves or by coupling with biocompounds Singer and

Plotz [1956] firstly studied microsphere or latex agglutination test (LATrsquos) by using

monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support o

which the biomolecules were going to adsorb The biomolecule adsorption howeve

limited by possible desorption of the adsorbed species or loss of specific activity of the

complex formed Since this work was published the latex particle applications for

immunoassay have been rapidly and widely studied and developed

Latex agglutination test or latex immunoassays start with tiny spherical latex

particles with a uniform diameter and similar surface properties The particles are coated

with antibodies (sensitized) through the hydrophobic interaction of portions of the protein

with the PS surface of the particles If sensitized particles are mixed with a sample

29

agglomerate Latex tests are inexpensive as compared with the other techniques [Ba

1988]

ngs

sed

tion Rheumatoid factor (RF) in different age subpopulations has also been

evalu

studied a

latex

Unipath [Percival 1996] has manufactured a range of immunoassay materials ba

on a chromatographic principle and deeply colored latex particles for use in the home and

clinical environments The latex particles which are already sensitized with a

monoclonal antibody can detect any antigens that bound to the surface of the latex

particles Some detectable pollutants include estrogen mimics which induce

abnormalities in the reproductive system of male fishes and lead to a total or partial male

feminiza

ated according to a patientrsquos clinical status by using a rapid slide latex agglutination

test for qualitative and semiquantitative measurement in human serum along with latex

immunoassay method [Onen et al 1998] Magalhaes and his coworkers [2004]

diagnostic method of contamination of male fishes by estrogen mimics using the

production of vitellogenin (VTG) as a biomarker This was based on a reverse

agglutination test developed with monoclonal antibodies specific to this biomarker

Premstaller and his coworkers [Premstaller et al 2000 2001] have prepared a porous

poly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient

chromatographic separation of biomelecules such as proteins and nucleic acids They

used a porogen a mixture of tetrahydrofuran and decanol to fabricate a micropellicular

PS-DVB backbone Legido-Quigley and his colleagues [2004] have developed the

monolith column to obtain further chromatographic functionality to the column by

introducing chloromethylstyrene in place of styrene into the polymer mixture

30

Core shell type monodisperse polymer colloids have been synthesized by Sarobe

and Forcada [1998] with chloromethyl functionality in order to improve the biomolecu

adsorption through a two-step emulsion polymerization process They investigated the

functonalized particles by optimizing the experimental parameters of the functional

monomer including reaction temperature the amount and type of redox initiator sys

used the type of addition of the initiator system and the use of washing They c

le

tem

oncluded

that th

sis

serve as separation

chann

of

usually

along the polypeptide chain which contain positive or negative charges [Norde 1998]

e relation between the amount of iron sulfate and the persulfatebisulfite system

added should be controlled to obtain monodisperse particles and prevent the premature

coagulation of the polymer particles during the polymerization

A semi-continuous emulsion polymerization technique for latex particle synthe

was performed by McDonald and other researchers [Ramakrishnan et al 2004 Steve et

al 1999] They introduced a variety of particles sizes compositions morphologies and

surface modifications to fabricate latex composite membranes (LCMs) Arraying and

stabilizing latex particles on the surface of a microporous substrate form narrowly

distributed interstitial pores formed between the particles which

els They investigated the membrane performance using gas fluxes water

permeability and the retention characterization of dextran molecules From these tests

they concluded that the narrow discriminating layer made of the latex particles leads to a

highly efficient composite membrane

27 Proteins

Proteins are natural polyamides comprised of about 20 different α-amino acids

varying hydrophobicity [Norde 1998] Proteins are more or less amphiphilic and

highly surface active because of the number of amino acid residues in the side groups

31

Proteins are polymers of L-α-amino acids [Solomon and Fryhle 2000] The α refers to a

carbon with a primary amine a carboxylic acid a hydrogen and a variable side-chain

group designated as R Carbon atoms rent groups are asymmetric and can

exhib l

s

e

on

with four diffe

it two different spatial arrangements (L and D configurations) due to the tetrahedra

nature of the bonds The L refers to one of these two possible configurations Amino

acids of the D-configuration are not found in natural proteins and do not participate in

biological reactions Figure 2-8 shows the chiral carbon in 3-D as the L isomer Protein

consist of twenty different amino acids differentiated by their side-chain groups [Norde

1998] The side-chain groups have different chemical properties such as polarity charg

and size and influence the chemical properties of proteins as well as determine the

overall structure of the protein For instance the polar amino acids tend to be on the

outside of the protein when they interact with water and the nonpolar amino acids are

the inside forming a hydrophobic core

Figure 2-8 L-α-amino acid

The covalent linkage between two amino acids is known as a peptide bond A

peptide bond is formed when the amino group of one amino acid reacts with the carboxyl

group of another amino acid to form an amide bond through the elimination of water

This arrangement gives the protein chain a polarity such that one end will have a free

amino group called the N-terminus and the other end will have a free carboxyl group

called the C-terminus [Solomon and Fryhle 2000] Peptide bonds tend to be planar and

32

give the polypeptide backbone rigidity Rotation can still occur around both of the α-

carbon bonds resulting in a polypeptide backbone with different potential conformations

relative to the positions of the R groups Although many conformations are theoretically

possible interactions between the R-groups will limit the number of potential

nd to form a single functional conformation In other words

the conformation or shape of the protein is

α-helix

can interact with other

secondary structures within the sam

conformations and proteins te

due to the interactions of the chain side

groups with one another and with the polypeptide backbone The interactions can be

between amino acids that are close together as in a poly-peptide between groups that are

further apart as in amino acids or even on between groups on different polypeptides all

together These different types of interactions are often discussed in terms of primary

secondary tertiary and quaternary protein structure

The primary amino acid sequence and positions of disulfide bonds strongly

influence the overall structure of protein [Norde 1986] For example certain side-chains

will promote hydrogen-bonding between neighboring amino acids of the polypeptide

backbone resulting in secondary structures such as β-sheets or α-helices In the

conformation the peptide backbone takes on a spiral staircase shape that is stabilized by

H-bonds between carbonyl and amide groups of every fourth amino acid residue This

restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure

Certain amino acids promote the formation of either α-helices or β-sheets due to the

nature of the side-chain groups Some side chain groups may prevent the formation of

secondary structures and result in a more flexible polypeptide backbone which is often

called the random coil conformation These secondary structures

e polypeptide to form motifs or domains (ie a

33

tertiary structure) A motif is a common combination of secondary structures and a

domain is a portion of a protein that folds independently Many proteins are composed o

multiple subunits and therefore exhibit quaternary structures

271 Interaction Forces between Proteins

Proteins in aqueous solution acquire compact ordered conformations In such a

compact conformation the movement along the polypeptide chain is severely restrict

implying a low conformational entropy The compact structure is possible only if

interactions within the protein molecule and interactions between the protein molecule

and its environment are sufficiently favorable to compensate for the low conformational

entropy [Malmsten 1998] Protein adsorption study is often focused on structural

rearrangements in the protein molecules because of its significance to the biological

functioning of the molecules and the important role such rearrangements play in the

mechanism of the adsorption process Knowledge of the majo

f

ed

r interaction forces that act

structures helps to understand the behavior

of pro

arge

a

ded

between protein chains and control the protein

teins at interface These forces include Coulomb interaction hydrogen bonds

hydrophobic interaction and van der Waals interactions

Coulombic interaction Most of the amino acid residues carrying electric ch

are located at the aqueous boundary of the protein molecule An the isoelectric point

(IEP) of the protein where the positive and negative charges are more or less evenly

distributed over the protein molecule intramolecular electrostatic attraction makes

compact structure favorable to proteins Deviation to either more positive or more

negative charge however leads to intramolecular repulsion and encourages an expan

structure Tanford [1967] calculated the electrostatic Gibbs energy for both a compact

impenetrable spherical molecule (protein) and a loose solvent-permeated spherical

34

molecule (protein) over which the charge is spread out From the results he found th

the repulsion force was reduced at higher ionic strength d

at

ue to the screening action of ion

ble

e

the

d the

tween

dehyd

e

ent

oles

tein

Hydrogen bond Most hydrogen bonds in proteins form between amide and

carbonyl groups of the polypeptide backbone [Malmsten 1998] The number of availa

hydrogen bonds involving peptide units is therefore far greater than that involving sid

chains Because α-helices and β-sheets are aligned more or less parallel to each other

interchain hydrogen bonds enforce each other Kresheck and Klotz [1969] examine

role of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between

peptide units do not stabilize a compact structure of protein However because the

peptide chain is shielded from water due to other interactions hydrogen bonding be

peptide groups do stabilize α-helical and β-sheet structures

Hydrophobic interaction Hydrophobic interaction refers to the spontaneous

ration and subsequent aggregation of non-polar components in an aqueous

environment In aqueous solutions of proteins the various non-polar amino acid residues

will be found in the interior of the molecule thus shielded from water The

intermolecular hydrophobic interaction for the stability of a compact protein structure

was first recognized by Kauzmann [1959] If all the hydrophobic residues are buried in

the interior and all the hydrophilic residues are at the outermost border of the molecul

intramolecular hydrophobic interaction would cause a compact protein structure

However geometrical and other types of interactions generally cause a fraction of the

hydrophobic residues to be exposed to the aqueous environm

Van der Waals interaction The mutual interaction between ionic groups dip

and induced dipoles in a protein molecule cannot be established as long as the pro

35

structure is not known in great detail Moreover the surrounding aqueous medium also

contains dipoles and ions that compete for participation in the interactions w

the protein molecule Dispersion interactions favor a compact structure However

because the Hamaker constant for proteins is only a little larger than that of water the

resulting effect is relatively small [Nir 1977]

272 β

ith groups of

rgiles et al 1996

Floeg

) is degraded [Floege 2001] Renal

β2M from the serum resulting in an increase in β2M

conce well

the

)

a

al

2-Microglobulin (β2M)

The protein β2M is of particular interest because it is involved in the human

disorder dialysis-related amyloidosis (DRA) [Geiyo et al 1985 A

e 2001] DRA is a complication in end stage renal failure patients who have been on

dialysis for more than 5 years [Bardin et al 1986 Drueke 2000] DRA develops when

proteins in the blood deposit on joints and tendons causing pain stiffness and fluid in

the joints as is the case with arthritis In vivo β2M is present as the non-polymorphic

light chain of the class I major histocompatibility complex (MHC-I) As part of its

normal catabolic cycle β2M dissociates from the MHC-I complex and is transported in

the serum to the kidney where the majority (95

failure disrupts the clearance of

ntration by up to 60-fold [Floege 2001] By a mechanism that is currently not

understood β2M then self-associates into amyloid fibrils and typically accumulates in

musculoskeletal system [Homma et al 1989] Analysis of ex vivo material has shown

that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRA

is present as of full-length wild-type β2M although significant amounts (~20-30) of

truncated or modified forms of the protein are also present [Floege 2001 Bellotti et al

1998] Figure 2-9 shows the ribbon diagram of human β2M Native β2M consists of

single chain of 100 amino acid residues and has a seven stranded β-sandwich fold typic

36

of the immunoglobulin superfamily [Saper et al 1991 Trinh et al 2002] β2M was fi

isolated from human urine and characterized by Berggard et al [1980] in1968 The

normal serum concentration of β

rst

re

f

e

2M is 10 to 25 mgL It is a small globular protein with

a molecular weight of 118 kDa a Strokes radius of 16Aring and a negative charge under

physiological conditions (isoelectric point IEP = 57) β2M contains two β-sheets that a

held together by a single disulphide bridge between the cysteines in positions 25 and 81

[Berggard et al 1980 Parker et al 1982 Cunningham et al 1973] β2M cannot be

removed completely by current dialysis techniques but through a better understanding o

the structure and interaction forces that lead to this structure it will be possible to mor

efficiently remove this problematic protein

Figure 2-9 Ribbon diagram of human β M taken from the crystal structure of the protein

bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khal 2000]

273 Serum Albumin

Serum albumin is the most abundant protein found in plasma and is typically

present in the blood at a concentration of 35~

2an et

50gL According to extensive studies about

its physiological and pharmacological properties albumin has a high affinity to a very

wide range of materials such as electrolytes (Cu+2 Zn+2) fatty acids amino acids

metabolites and many drug compounds [Fehske et al 1981 Kraghhansen 1981 Putnam

37

1984 Peters 1985] The most important physiological role of the protein is therefore to

bring such solutes in the bloodstream to their target organs as well as to maintain the pH

and osmotic pressure of the plasma Bovine serum albumin (BSA) is an ellipsoidal

protein with the dimensions of 140 X 40 X 40Aring [Peter 1985] The primary structure is a

single helical polypeptide of 66 kDa (IEP = 47) with 585 residues containing 17 pairs of

as a soft an

rfaces [Kondo et al 1991 Norde et al 1992 Soderquist et al

1980 ikely

is

disulfide bridges and one free cysteine [Dugaiczyk et al 1982] BSA has been classified

d flexible protein because it has a great tendency to change its conformation

on adsorption to solid su

Carter and Ho 1994] and consists of three homologous domains (I-III) most l

derived through gene multiplication [Brown 1976] Each domain is composed of A and B

sub-domains [He et al 1992] The secondary structure of human serum albumin (HSA)

shown in Figure 2-10

Figure 2-10 Secondary structure of human serum albumin (HSA) with sub-domains

[Zunszain et al 2003]

HSA has the same structure domains with the serum albumin from other species

such as BSA [Brown 1976] Although all three domains of the albumin molecule have

similar three-dimensional structures their assembly is highly asymmetric [Sugio et al

38

1999] Domains I and II are almost perpendicular to each other to form a T-shaped

assembly in which the tail of subdomain IIA is attached to the interface region between

sub-domains IA and IB by hydrophobic interactions and hydrogen bonds In contrast

domain III protrudes from sub-domain IIB at a 45ordm angle to form the Y-shaped assembly

for domains II and III Domain III interacts only with sub-domain IIB These features

make the albumin molecule heart-shaped

28 Protein Adsorption

Protein adsorption studies date back to the 1930rsquos At the beginning these studies

mainly focused on the determination of the molecular weight electrophoretic and

ral

rearrangem

adsorption and biocompatibility of the sorbent materials were investigated [Norde 1986]

There are interaction forces at the interfaces between protein molecules and latex

particles These forces are mainly divided into the following groups hydrophobic

interaction ionic interaction hydrogen bonding and van der Waals interaction [Andrade

1985]

Hydrophobic interaction It is known that hydrophobic interaction has a major

role in protein adsorption phenomena The adsorption of proteins on the low charged

latex particles occurs by this interaction force Generally monomers such as styrene offer

hydrophobic surfaces th

by this interaction force is maximum at the isoelectric point (IEP) of the protein and the

strength [Suzawa et al 1980 1982 Shirahama et al 1989 Kondo et al 1992] By the

chromatographic applications Later the adsorption mechanism especially the structu

ents was studied Recently the studies of the relation between protein

281 Interaction between Protein Molecule and Latex Particle

at protein molecules adsorb to The amount of adsorbed protein

pH at maximum adsorption shifts to a more acidic region with an increase in ionic

39

reports [Suzawa et al 1980 1982 Lee et al 1988] protein adsorption was greater

hydrophobic surface than on a hydrophilic one implying that hydrophobic interaction is

on a

s

ydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA)

Ionic

ely

t

ces

n force is operative over small

distan e

ble

n

nt dialysis membrane therapy for end stage renal disease (ESRD)

one of most dominant forces in protein adsorption

Ionic interaction Negatively charged latex particles have ionic functional

groups on their surfaces such as salts of sulfonic and carboxylic acid Sulfate group

originate from an initiator such as sodium persulfate and carboxylic groups originate

from a h

bonds are formed between the negative charges of these latex particles and positive

surface charges of protein molecules The conventional low-charged latex particles rar

form these ionic bonds

Hydrogen bonding Hydrogen bond is a strong secondary interatomic bond tha

exists between a bound hydrogen atom (its unscreened proton) and the electrons of

adjacent atoms [Callister 1999] Protein can be adsorbed on hydrophilic polar surfa

through hydrogen bonding Hydrogen bonds are frequently formed between hydroxyl-

carbonyl or amide-hydroxyl Hydroxyl-hydroxyl or amide-hydroxyl bonds are also

formed in protein adsorption

Van der Waals interaction This interactio

ces only when water has been excluded and the two non-polar groups come clos

to each other Lewinrsquos calculation showed that the van der Waals interaction is negligi

compared with the forces involved in the entropy increases ie hydrophobic interactio

[Lewin 1974]

29 Hypothesis for Toxin Removal

As mentioned earlier insufficient removal of middle molecular range toxins is a

major drawback of curre

40

patien

is

y

ese

haracteristics over the last decade However removal

effici

ing a novel membrane design composed of an assembly of engineered

polymeric latex particles s ions Important factors

will i

e

ith a

is established it is

expec

e

ts This may cause destructive arthritis and carpal tunnel syndrome inhibit the

immune system and accelerate cardiovascular disease leading to death among dialys

patients In order to overcome these complications artificial dialysis membranes have

been developed to increase the capacity for removing middle molecular weight toxins b

changing the pore size of dialysis membranes and using new materials that adsorb th

toxins for improved removal c

ency is not as high as those achieved by a normal healthy kidney

With the knowledge obtained from all above literatures the following hypothesis

has been established for the development of a membrane for the successful removal of

the target protein β2-Microglobulin (β2M) without the removal of serum albumin This

will be done us

ynthesized to predetermined specificat

nclude pore size and surface chemistry

291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size

The packing of monodispersed spherical particles can lead to the formation of

porous layers suitable for use as filters and membranes [Hsieh et al 1991] The pore siz

is defined as the largest spherical particles that can pass through the interstitial spaces

It is well known that monodispersed spherical particles can be obtained from the

seed emulsion polymerization method Many commercially available latex particles are

synthesized by this technique and are inexpensive When the defect free membrane w

monodisperse pore size distribution based on a particular particle array

ted to outperform the traditional hemodialysis membrane limitation which is a

broad range of pore size distribution When spherical particles pack in regular crystallin

arrays a number of packing geometries such as hexagonal closest packing cubic closest

41

packing and body-centered cubic packing are possible The pore size of the array

depends on these packing geometries as well as particle size Theoretical pore size can be

calculated from the simple geometry of the arr

ay Figure 2-11 shows the relationship

between particle size and pore size

0001

001

01

001 01 1 10

Por

e

1

10

microm

) S

ize

(

Particle Diameter (microm)

Body-Centered Cubic ArrayClosest Packed Array

0001

Siz

e (

Particle Diameter (microm)

001

01

001 01 1 10

Por

e

1

10

microm

)

Body-Centered Cubic ArrayClosest Packed ArrayBody-Centered Cubic ArrayClosest Packed Array

and closed packed arrays [Steve et al 1999]

McDonald and his coworkers [Ramakrishnan et al 2004 Steve et al 1999] have

described the fabrication of latex composite membranes and their performance in

previous papers It is from this prior research and knowledge gained from conversations

with Dr McDonald that this current research has been initiated [McDonald 2003] The

focus of this thesis is the extension of McDonaldrsquos research in the area of composite

membranes to medical application such as dialysis membranes

292 Toxin Removal by Selective Adsorption on Engineered Latex Particles

The flexibility in latex surface properties is a significant advantage to this

materialrsquos ability to separate proteins since the selectivity is strongly dependent on the

charge interactions of both the protein and the latex particles under the conditions of

Figure 2-11 The relationship between pore size and particle size in body-centered cubic

42

separation [Menon et al 1999 Chun et al 2002] First all sorbents were designed to

have negative surface properties because most plasma proteins in blood are negative and

should not be removed with charge interaction between proteins and sorbents at

physiological condition (pH74) It was reported that a negatively charged surface was

more blood compatible than a positive one [Srinivasan et al 1971] The toxin protein β2-

Microglobulin (β2M) can be selectively adsorbed on the surface of latex particles by the

design of a suitable hydrophobic portioned surface where only β2M protein can be

anchored with hydrophobic interactions Albumin adsorption on the latex particle is not

side-on m

adsorption of β M protein on the engineered latex particles

allowed because charge repulsion is more dominant than hydrophobic interaction with

ode The figure 2-12 shows the schematic representation of the selective

2

PMMAPAAShell

COO-

OSO3-

OSO3-

PSCore

COO-

-

-

-

-

-

-

Albumin-

Chargerepulsion

--

-

- -

- -

- --

Hydrophobic

β2-Microglobulin

2

Interaction

β -Microglobulin

PMMAPAAShell

COO-

-

-

-

-

-

-

--

-

-

-

-

-

Albumin-

Chargerepulsion

OSO3-

OSO3-

PSCore

COO-

--

-

- -

--

-

- -

- -

- --

- -

- --

Hydrophobic

β2-Microglobulin

2

particles at pH 74

Interaction

β -Microglobulin

Figure 2-12 Schematic representation of the protein adsorption on the core shell latex

43

Hydrophilichydrophobic microdomain structures were proven to be more blood

compatible [Mori et al 1982 Higuchi et al 1993 Deppisch et al 1998] The

optimization of suitable hydrophobic to hydrophilic ratio is also important for the

biocompatibility The monomers such as styrene (St) methyl methacrylate (MMA) and

acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in

emulsion polymerization process

In summary the background and fundamental literature survey about the history

material properties and limitation of hemodialysis membrane latex particle preparation

surface chemistry and manufacturing process target proteins and protein adsorption and

finally the hypothesis for a toxin removal with high separation efficiency have been

suggested This research focuses on such hypothesis and the materials and

characterization methodology for achievement of suggested hypotheses are described in

the next chapter

CHAPTER 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY

As mentioned earlier the goal of this study is to prepare polymeric latex particles

with tailored properties to maximize separation of toxin molecules and to investigate

fundamental interactions between the applied particles and molecules in the

system to optimize the membrane performance for hemodialysis applications Polymeric

latex particles wit

the

biological

h monodisperse size distribution to obtain uniform pore size and

various size ranges to utilize membrane construction are necessary Surface engineering

with various combination of hydrophobichydrophilic domain on the surface of latex

particles is expected to affect the removal of target protein by selective adsorption and to

improve the biocompatibility of membrane Therefore the materials and characterization

methodology are addressed in this chapter

31 Materials

Styrene (St) monomer used for a seed and a core particle was purchased from

Fisher Scientific and used without any other purification process Acrylic acid (AA) and

methyl methacrylate (MMA) monomers introduced for shell formation were purchased

from Fisher Scientific and used without any other purification process Sodium persulfate

(SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as

received Divinylbenzene (DVB) crossliking agent was purchased from Aldrich The

anionic surfactant Aerosol regMA80-I [sodium di(13-dimethylbutyl) sulfosuccinate] was

kindly donated by Cytec 78-80 Aerosol regMA80-I is mixed with isopropanol and

water Its critical micelle concentration (CMC) is 245 mM (119) Ion exchange resin

44

45

AGreg 501-x8 Resin (20-50 mesh) was purchased from Bio-Rad Laboratories Inc It is a

mixed-bed resin with H+ and OH- as the ionic forms and is 8 crosslinked Bovine serum

albumin (BSA) (heat shock treated) was purchas Fisher Scientific The isoelectric

point of t lin

(β2M)

as

gma

ed from

he BSA is 47-49 and the molecular weight is 66300Da β2-Microglobu

was purchase from ICN Biomedicals Inc and was separated from patients with

chronic renal disease and lyophilized from ammonium bicarbonate The molecular

weight by SDS-PAGE was approximately 12000Da BSA and β2M proteins were used

received without further purification process In order to investigate the protein

adsorption evaluation bicinchoninic acid protein assay kits were purchased from Si

(Cat BCA-1) and Pierce Biotechnology (Cat 23225) The chemical structures for the

main chemicals are shown in Figure 3-1

CH2 CH

Styrene (Mw = 10415)

CH2 CHCH2 CH

Styrene (Mw = 10415)

CH2 CH

Acrylic acid (Mw = 7206)

C

OH

O

CH2 CH

Acrylic acid (Mw = 7206)

C

OH

O

CH CH2

C

OCH3

O

CH

CH CH3

2

C

OCH3

O

CH3

Methyl methacrylate (Mw = 10012)Methyl methacrylate (Mw = 10012)

CH2 CH

Divinylbenzene (Mw = 1302)

CH CH2

CH2 CH

Divinylbenzene (Mw = 1302)

CH CH2CH CH

+(Na) -(O3S)

CH -C-O-CH-CH -CH-CH3

O

2 2 3

O CH CH3

CH3 CH3

CH-C-O-CH-CH2-CH-CH3

Anionic surfactant (MA80-I) (Mw = 3885)

+(Na) -(O3S)

CH -C-O-CH-CH -CH-CH3

O

2 2 3

O CH CH3

CH3 CH3

CH-C-O-CH-CH2-CH-CH3

Anionic surfactant (MA80-I) (Mw = 3885)2

+(Na)-(O) S OO

O O

- +

Initiator sodium persulfate (Mw = 23811)

O SO

(O) (Na)+ -

O O

- +(Na) (O) S OO

O SO

(O) (Na)+ -

O O

- +

Initiator sodium persulfate (Mw = 23811) Figure 3-1 Chemical structure of main chemicals

(Na) (O) S OO

O SO

(O) (Na)

46

32 Latex Particle Preparation

The experimental set up and the particle preparation schemes for various types and

size ranges of latex particles are shown in Figure 3-2 and 3-3 In the experimental setup a

mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at

end of glass stirrer The agitation rate is precisely adjusted by controller motor Paar glas

reactor with 1L volume is partially in silicon oil bath on hot plate The reactor has fo

openings for a stirrer a thermometer a nitrogen gas inlet and a reactant feeding lnlet

The reactants for latex polymerization are fed by precisely controlled by meterin

The particle preparation is described in each latex preparation section

the

s

ur

g pump

Figure 3-2 Experimental setup for semi-continuous emulsion polymerization

1 Motor 2 Mechanical stirrer bar 3 Thermometer 4 Inlet of N2 5 Inlet for initiator and monomer 6 Glass reactor (l L) 7 Oil bath 8 Hot plate 9 Pump for initiator 10 Pump for monomer 11 Tubes for reactant feed

47

Styrene monomer

Bare PS

PSPMMA100

PSPMMA90PAA10

PSPMMA P75 AA25

Gr

win

ticle

size

PS seedparticles

og

par

Styrene monomer

Bare PS

PSPMMA100

PSPMMA90PAA10

PSPMMA P75 AA25

Gr

win

ticle

size

PS seedparticles

og

par

Figure 3-3 The particle preparation scheme in various types and size ranges of latex particles

321 Preparation of Seed Latex Particles

Polystyrene seed particles were synthesized through a typical semi-continuous

emulsion polymerization Polystyrene latex particles were prepared in a four necked Paar

glass vessel equipped with a mechanical glass stirrer thermometer glass funnel for

nitrogen gas purge and a tube for monomer feeding The reactor was placed in a silicon

oil bath on a hot plate for homogenous and stable tempe he vessel was

firstly charged with de-ionized water the emulsifier so itiator

sodium bicarbonate (SBC) styrene (St) monomer divinylbenzene (DVB) monomer

under nitrogen gas atmosphere and then heated to 72plusmn2 as

llowed to continue for another hour Second reactants St and DVB monomers SPS

initiator and SBC were fed separately using fine fluid metering pumps (Model RHSY

Fluid metering INC NY) under a nitrogen gas atmosphere for 2h After complete feeding

rature control T

dium persulfate (SPS) in

oC for 1h The reaction w

a

48

of all reactants the reaction was continued for another hour at elevated temperature 80plusmn1

oC and then cooled to room temperature by removal of the oil bath

322 Preparation of Seeded Latex Particles

The particle growth was achieved by seeded continuous emulsion polymerization

The vessel was charged with the polystyrene seeds and de-ionized water to make desired

solid content and heated up to 72plusmn2 oC for 1h under nitrogen gas atmosphere Then St

and DVB monomers and solution of SPS and SBC in de-ionized water were continuously

added using metering pump with a precisely controlled feed rate After feeding all

reactants the reaction was continued for three hours keeping temperature of 80plusmn2 oC and

then the reaction vessel was cooled down to room temperature

Monodisperse spherical polystyrene seeds with a 280nm mean diameter were used

e-polymerized polystyrene seeded emulsion

was charged in a 500m

323 Preparation of Core Shell Latex Particles

for core shell structured latex particle The pr

l glass flask under a nitrogen gas atmosphere De-ionized water

was added to achieve the desired solid content The emulsion was heated to 80plusmn2oC for

10 hour Monomers initiator and SBC to form the shell were added by fluid metering

pumps (LAB PUMP JRRHSY model Fluid metering Inc USA) with fine controlled

feed rate Flow rate can be adjusted by control ring graduated in 450 division from 0 to

100 flow which is in the range of 10mlmin respectively The reaction was sustained

for additional 30 hours at 90plusmn2oC then cooled down to room temperature

324 Purification of Synthesized Latex Particles

Synthesized latex particles were purified with an ion exchange resin 200g (10

ww polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin

and stirred for 40 minute to remove unreacted monomers initiators and other impurities

49

Then the emulsion was diluted with DI water to a specific degree of solid content as

needed

33 Characterization

331

solid

termined

oscopy

d

hours

Dried latex (10mg) was mixed with

250 m on

Degree of Conversion

The degree of monomer to polymer conversion was determined gravimetrically

following the procedure described by Lee et al [1995] before the latex cleaning process

The synthesized emulsion raw materials were weighed and dried in a conventional oven

at 120oC for 30min to evaporate any unreacted monomers and water The remaining

was weighed and the degree of conversion was determined This process was repeated

three times and a mean value of the degree of conversion was de

332 Fourier Transform Infrared (FTIR) Spectr

A molecule can absorb only select frequencies (energy) of infrared radiation which

match the natural vibration frequencies The energy absorbed serves to increase the

amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]

Only those bonds which have a dipole moment that changes as a function of time can

absorb the infrared radiation The range of wavelengths for infrared radiation is between

25 microm (4000 cm-1) and 25 microm (400cm-1)

The synthesis of the latex particles such as polystyrene (PS) homopolymer and

PSPMMA PSPMMA90PAA10 PSPMMA75PAA25 core-shell copolymers were verifie

with FTIR Purified latex particles were dried in vacuum oven at 40oC for 24

before analysis in the FTIR (Nicolet Magma USA)

g of KBr which had also been dried in vacuum oven at 120oC for 3h Transmissi

spectra using the drift mode were plotted with 128 scans and 4cm-1 resolution

50

333 Quasielastic Light Scattering (QELS)

Latex particle size was measured by the Brookhaven ZetaPlus particle size

analyzer The solid concentration as 0001 (wv) This

ation spectroscopy (PCS) of quasielastically scattered light

For th s

ed using a FE-SEM (JEOL

JSM-

ith

ent

01

us zeta-

potential analyzer It measures the electrophoretic mobility velocity of charged colloidal

of latex particles used w

instrument uses photon correl

is technique time dependent interference patterns of light scattered from particle

in the sample cell are analyzed The interference pattern changes due to Brownian

motion giving rise to fluctuations in scattering intensity The frequency of these

fluctuations depends on the particlersquos diffusion constant that is inversely proportional to

the particle diameter

334 Field Emission-Scanning Electron Microscopy (FE-SEM)

Particle size and surface morphology were characteriz

6335F Japan) A diluted suspension of latex particles in water was dropped onto a

silicon wafer at room temperature and allowed to dry The sample was then coated w

the thinnest layer of carbon needed to obtain the required conductivity for this instrum

Secondary electron image mode was used with a 15KV of accelerating voltage The

magnification range used was between 10000X and 70000X

335 Zeta Potential Measurement

Synthesized and purified latex particles were diluted with de-ionized water to 0

wt and adjusted six different pH values 205 345 481 565 643 and 747 These

particle suspensions were then transferred to standard cuvettes for zeta potential

measurement At least two runs of ten measurements were taken for each sample and

averaged

The zeta potential measurement was carried out using Brookhaven ZetaPl

51

particles in solution and calculate zeta-potential by using the Smoluchowski equation

The f quency

gnitude

umin (BSA) as a standard model protein and β2-Microglobulin

uffer (PB) and

phosp

5

h the selected protein

e 005 01 03 05 and 07 mgml and those for

β2M

d at

teins in the supernatant after the centrifugation process using the

bicinc t

requency of laser light passing through the sample and is compared to the fre

of a reference beam The shift in the frequency called a Doppler shift and the ma

of the shift correspond to the polarity and the magnitude of the electrophoretic mobility

respectively The zeta potential is calculated from the solution conditions and the

measured mobility

336 Protein Adsorption

Protein adsorption experiments of the synthesized latex particles were performed

with bovine serum alb

(β2M) as a target protein in two types of buffer solution phosphate b

hate buffered saline (PBS) To make 5 mM of PB solution 0345g of sodium

phosphate monobasic (NaH2PO4H2O) was dissolve in 500ml of de-ionized water To

make PBS solution 0345g of sodium phosphate monobasic (NaH2PO4H2O) and 4178g

(143mM) of sodium chloride (NaCl) were dissolved in 500ml of de-ionized water The

synthesized latex particles were diluted with each buffer to have a solids content of 0

(ww) adjusted to pH values of 32 48 and 74 and mixed wit

BSA concentrations were chosen to b

were 0015 0030 0045 and 0060mgml The mixture was gently rotated using

the shaker Labquakereg (BarnsteadThermolyne Model 4002110 USA) with 8 RPM in

the incubator at 37oC for 12 hours before the latex-protein mixture was centrifuge

13000 rpm for 15min The amount of protein adsorbed was determined by quantifying

the free pro

honinic acid (BCA) assay method [Lowry et al 1951 Smith et al 1985 Baptista e

al 2003 Wiehelman et al 1988 Brown et al 1989] BCA assay kits consist of Reagent

52

A containing bicinchoninic acid sodium carbonate sodium tartrate and sodium

bicarbonate in 01N NaOH with pH=1125 and Reagent B containing 4 (wv) copper

(II) sulfate pentahydrate The BCA working reagent was prepared by mixing 50 parts of

Reagent A with 1 part of Reagent B 100microl of protein supernatant was then mixed with

2ml of BCA working reagent in a UV cuvette Incubation was allowed to continue

at room temperature

further

until color developed about 2h The absorbance at 562nm was

pectrophotometer (Perkin-Elmer Lambda 800 USA)

Unkn

Separate the p rticles in centrifuge mix supernatant with BCA reagents

measured using a UV-VIS s

own concentration of sample protein was determined by comparison to a standard

of known protein concentrations The adsorbed amount per unit surface area was

determined by the mass balance of the protein after adsorption process The simple

schematic of the procedure for a protein adsorption test is shown in Figure 3-4

Incubate mixture with agitation for 12hrs at 37oC

aand develop the color by incubation for 2hrs at room temperature

Suspend latex particles in aqueous buffer media

Add the dissolved target protein

Measure color intensity using UVVis spectroscopy at 562nm

Suspend latex particles in aqueous buffer media

Separate the p rticles in centrifuge mix supernatant with BCA reagents

Add the dissolved target protein

Incubate mixture with agitation for 12hrs at 37oC

aand develop the color by incubation for 2hrs at room temperature

Measure color intensity using UVVis spectroscopy at 562nm

Figure 3-4 Schematic of the procedure for a protein adsorption test

53

337 Blood Biocompatibility by Hemolysis Test

Hemolysis is the destruction of red blood cells which leads to the release of

hemoglobin from within the red blood cells into the blood plasma Hemolysis testing is

used to evaluate blood compatibility of the latex particles and the damaged to the red

blood cells can be determined by monitoring amount of hemoglobin released The red

blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutes

Figure 3-5 shows the phase separated blood with plasma white blood cells and red

cells after centrifugation process Separated RBSs were then washed with isotonic

phosphate buffer solution (PBS) at pH 74 to remove debris and serum protein Th

process was repeated 3 times

blood

is

Figure 3-5 Separation of RBC from whole blood by centrifuge process

Prepared latex particles were re-dispersed in PBS by sonification to obtain

homogeneously dispersed latex particles 100microl of the mixture of red blood cell (3 parts)

and PBS (11parts) was added to 1ml of 05 (ww) particle suspension PBS was used as

a negative control resulting 0 hemolysis and DI water used as a positive control to

produce 100 hemoglobin released by completely destroyed RBCs The mixture was

incubated in water bath with gentle shaking for 30 minutes at 37oC and then centrifuged

at 1500 rpm for 15 minutes 100microl of the supernatant was mixed with 2ml of the mixture

) (EtOHHCl = 2005 ww) to prevent the

White blood cells

Plasma

Red blood cells

of ethanol (99) and hydrochloric acid (37

54

precipitation of hemoglobin In order to remove remaining particles the mixture was

centri fuged again with 13000 RPM at room temperature The supernatant was then

transferred into the UV cuvette The amount of hemoglobin release was determined by

monitoring the UV absorbance at a wavelength of 397nm The schematic of the

procedure for hemolysis test is shown in Figure 3-6

Separate red blood cells (RBCs) from blood

Mix RBCs with phosphate buffer solution (PBS)

Wash and re-suspend RBC with PBS repeat three times

Add la l of RBC and incu in at 37oC

tex particle suspension into 100mbate sample with agitation for 30m

Separate particle ith EtOHHCl solutions and mix the supernatant w

Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm

Separate red blood cells (RBCs) from bloodSeparate red blood cells (RBCs) from blood

Mix RBCs with phosphate buffer solution (PBS)Mix RBCs with phosphate buffer solution (PBS)

Wash and re-suspend RBC with PBS repeat three timesWash and re-suspend RBC with PBS repeat three times

Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC

Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC

Separate particles and mix the ith EtOHHCl solution supernatant wSeparate particles and mix the ith EtOHHCl solution supernatant w

Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nmEvaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm

Figure 3-6 Schematic of the procedure for hemolysis test

In summary a description of the experimental materials and characterization

methodology has been explained In the next chapter the results and discussion of the

data obtained from these experiments is addressed

CHAPTER 4 RESULTS AND DISCUSSION

41 Polymerization of Latex Particles

411 Polystyrene Seed Latex Particles

Polymer particles have many different applications including spacer particles

lubricants packing materials for chromatography standard particles and diagnostic

drugs All applications strongly require these particles to have a uniform particle size

Uniform particle size is the first requirement for a latex composite membrane formed by

particle arrays with the interstitial spaces serving as pores for size discriminations [Jons

et al 1999 Ramakrishnan et al 2004] Although suspension polymerization has been

mainly used as one of conventional polymerization methods to prepare uniform particles

the uniformity of the recovered particles is insufficient for membrane use There is

another method known as seed emulsion polymerization where a vinyl monomer is

absorbed into fine monodiserse seed particles and polymerization causes the monomer to

crease the sizes of the seed particles uniformly In order to obtain monodisperse larger

particles by this m

particles and polymerization of the monomer is repeated

In this study cross-linked polystyrene seed latex particles were firstly synthesized

in the presence of styrene monomer divinylbenzene crosslink agent sodium persulfate

initiator sodium bicarbonate buffer and the Aerosol regMA80-I [sodium di(13-

dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion

polymerization process in which one or more components can be added continuously

in

ethod the procedure of absorption of monomer into fine polymer

55

56

Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and

buffer are continuously added to enlarge the seed latex particles Various profiles of

particle nucleation and growth can be gene different orders of component

addition during polymerizati f this process such as easy

control of the polymeriza particle number

and particle morphology The main goal in

this process is to avoid secondary particle

rated from

on There are several advantages o

tion rate monodisperse particle size and

colloidal stability copolymer composition

formation leading to a monodisperse particle

size distribution Figure 4-1 shows the schematic representation of seed latex particle

preparation and growth

Monomer droplet

Seed particles Amount of surfactantsis variable

Enlarged final Add monomers

PS

PS

PSs4

particles Number of particlesare constant

Seed code

PSs1

s2

s3

Monomer droplet

Seed particles Amount of surfactantsis variable

Enlarged final Add monomers

PS

PS

PSs4

tion The

particles Number of particlesare constant

Seed code

PSs1

s2

s3

Figure 4-1 Schematic representation of semi-continuous seed latex particles preparation and growth

After the first synthesis of a small size of crosslinked polystyrene (PS) seed

particles stabilized by surfactants styrene DVB and initiator are continuously fed into

the system to enlarge the seed latex particles maintaining a narrow size distribu

recipes and reaction conditions for synthesizing these seed particles are summarized in

57

Table 4-1 The original recipe for latex particle preparation from McDonald was

modified for this emulsion system [McDonald 2003]

Table 4-1 The polymerization recipe of polystyrene (PS) seed particles Latex label PSS259 PSS233 PSS207 PSS181

Temperature (oC) 75 plusmn 5 75 plusmn 5 75 plusmn 5 75 plusmn 5

Divinylbenzene (g) 052 052 052 052

MA 80 (g) 259 233 207 181

NaHCO

Watera (g) 1315 1315 1315 1315

Styrene (g) 9948 9948 9948 9948

112 112 112 112

Na2S2O8 (g) 190 190 190 190

Waterb (g) 200 200 200 200

Reaction time (min) 140 140 140 140

Solids content 396 396 396 396

Mean particle diameter (nm) 126plusmn75 171plusmn39 182plusmn41 216plusmn53

Conversion () 965 979 957 971

3 (g)

Four different seed latex particles were synthesized based on surfactant amount in

order to determine the optimum condition for stable and monodisperse latex particles

The emulsion polymerization of latex particles has to be carried out in a narrow range of

surfactant causes flocculation or phase separation leading to broad particle

size distribution and an unstable emulsion sy

surfactant concentration where particles are stable [Nestor et al 2005] A wide range of

concentrations

stem Therefore the range of surfactant

amount was varied from 181g to 259g in increments of 026g in each latex label Other

reactants amounts were kept constant Watera is the initial charge amount and waterb

represents the amount of aqueous solution containing dissolved initiator and buffer

58

which are fed continuously after the seed particle creation The extent of monome

polymer conversion was obtained by gravimetric calcu

r to

lation and was more than 95 at

cripts such as

S207 and S181 i the surf mount added for the seed latex

nversion indi perce experi obtain

content divided by the theoretically estimated solid content and was calculated by the

all seed particles labeled PSS259 PSS233 PSS207 and PSS181 Labeled subs

S259 S233 ndicate actant a

preparation The co cates the ntage of mentally ed solid

following formula

100 colids () times=entsoliatedntentoalExperimentConversion

n the experimenta content is the weight of olid rema after

mulsion at 120oC f in and the estimated solid content is th ht

lated from the recipe

rti as di each t load d

mple of ea pension iluted by (ww)

deionized water and the mean particle diameter was determined using the Brookhaven

ZetaPlus particle size analyzer From these results the seed latex particle size increased

as the amount of surfactant decreased The amount of emulsifier also affects the

polymerization process by changing the number of micelles and their size It is known

that large amount of emulsifier produces larger numbers of smaller sized particles [Odian

1991] and was corroborated in the current work

These synthesized seed latex particles should be of narrow size distribution in order

to obtain monodisperse larger particles The SEM image showing the particle

morphology and size of a sample of polystyrene seed particles can be seen in the Figure

4-2

contdEstim (4-1)

In this equatio l solid the s ining

evaporation of e or 30m e weig

of monomers calcu

As expected the seed pa cle size w fferent for surfactan ing in see

emulsion system A sa ch sus was d 0001 with

59

Figure 4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)

PSS259 (B) PSS233 (C) PSS207 (D) PSS181 (subscripts indicate the amount of surfactant added)

(A)

(B)

60

(C)

(D)

Figure 4-2 Continued

61

s seen in the image the seed particles are very smooth and spherical The

uniformity in the particle size distribution of these PS seed latex particles can also be

seen in the SEM image From this data we concluded that seed particles synthesized in

the presence of a surfactant with the chosen amounts are suitable for use to prepare larger

particles with high size uniformity and smooth spherical morphology

412 The Growth of Polystyrene (PS) Seed Latex Particles

Seeded emulsion polymerization has been conducted for several decades and

various mechanisms have been proposed Gracio et al [1970] suggested that the growing

polymer particles consist of an expanding polymer-rich core surrounded by a monomer-

rich shell with the outer shell providing a major locus of polymerization Seeded

mulsion polymerization is commonly used for preparing latex particles less than 1microm in

size [Cha et al 1995 Zou et al 1990 1992 Gandhi et al 1990 Park et al 1990] Such

latexes can be obtained from the growth of pre-prepared seed particles The seeds

introduced in this process serve as nucleation sites for particle growth By first swelling

the seed latex particles with additional monomer to the desired size polymerization can

then be initiated Several size ranges of monodisperse polystyrene (PS) latex particles

were prepared by this multistep seeded emulsion polymerization method For the

fabrication of particle-based membranes monodisperse latex particles with a variety of

size ranges are required

In this study seed particles from 171nm to 470nm in mean diameter and with a

highly uniform size distribution were prepared and used Watera is the initial charge

mount and waterb represents the amount aqueous solution containing dissolved initiator

and buffer that is continuously fed into system Feed time is precisely controlled by

metering pump The solid contents were maintained between 199 and 300 After

A

e

a

62

polym d as

es

ere 923 8402

n of these latex particles

are sh

ss

erization under these conditions the conversions of the latex particles labele

PS258 PS320 PS370 and PS410 were 952 957 946 and 964 respectively The recip

used in seeded emulsion polymerization to form the particles less than 500nm in mean

diameter are shown at the Table 4-2 The conversions of the latex particles larger than

500nm in mean diameter and labeled as PS525 PS585 PS640 and PS790 w

723 and 969 respectively The recipes used in the preparatio

own at the Table 4-3

A simple calculation can be used to predict the ultimate size that a latex particle can

reach after ending the polymerization Letrsquos suppose that seed density (ρs) and grown

particle density (ρgp) are the same The following equation is obtained from the ma

balance (m = ρV) where m and V are mass and volume of a particle respectively

gp

gp

s

s

Vm

Vm

In this equation m

= (4-2)

be

s and mgp are the weight of seed and total weight of seed and

added monomers respectively and Vs and Vgp are the volume of seed and grown

particle respectively

If we know the mass of monomer to be added the ultimate particle size can

calculated from the equation 4-3

3

2s D m ⎠⎝+ χ

where D

1s D m

⎟⎟⎞

⎜⎜⎛

= (4-3)

the

in

s and Dgp are the diameters for seed and grown particle respectively and χ is

weight of the monomer added Crosslinked PS latex particles with a size range from

258plusmn112 ~ 410plusmn117 in diameter polymerized by the recipes in Table 4-2 are shown

the SEM images in Figure 4-3

63

Table 4-2 Continuous addition emulsion polymerization recipe for growing polystyrene

Latex label PS

(PS) latex particles less than 500nm in size

258 PS320 PS370 PS410

Initial charge

Seed latex (g) 1000171 1000216 1000216 1000320

Watera (g) 1470 2500 1984 2600

06 07 06 06

Fee

Initiator stream

2 2 8

Feed time (min) 0-167 0-140 0-100 0-92

Solid content 284 300 300 200

Mean particle diameter (nm) 258plusmn112 320plusmn154 182plusmn71 410plusmn117

Conv

Monomer continuous

Styrene (g) 800 1200 800 600

Divinylbenzene (g)

d time (min) 0-163 0-110 0-75 0-82

Na S O (g) 07 065 05 05

NaHCO3 (g) 07 065 05 05

Waterb (g) 750 1200 200 370

ersion () 952 957 946 964 The solid content of seed latex for PS258 PS320 PS 370 and PS410 are 392 399 399 anrespectively

d 300

64

Table 4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size

Latex label PS525 PS585 PS640 PS790

Initial charge

Seed latex (g) 1000390 1000470 1000470 1000320

Watera (g) 1000 520 1200 360

Monomer continuous

Styrene (g) 290 200 400 173

Divinylbenzene (g) 02 014

03 009

Feed time (min) 0-80 0-40 10-76 5-65

Initiator stream

Na2S2O8 (g) 02 014 03 012

NaHCO3 (g) 02 014 03 009

Waterb (g) 200 120 400 200

Feed time (min) 0-85 0-46 0-77 0-75

Solids content 199 200 200 201

Mean particle diameter (nm) 525plusmn151 585plusmn186 640plusmn178 790plusmn693

Conversion () 923 8402 723 969 The solid content of seed latex for PS525 PS585 PS 640 and PS790 are 200 182 193 and 173 respectively

65

Figure 4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)

370nm (D) 410nm

(A)

(B)

66

(C)

(D)

Figure 4-3 Continued

67

article size was well matched to that estimated by the geometrical calculation

From this it can be concluded that the added monomer was consumed for polymer

particle growth with a high degree of conversion From the SEM characterization newly

nucleated particles were not seen as indicated by the narrow particle size distribution

The particles less than 500nm in mean diameter were very spherical in shape with a

highly uniform particle size distribution As the particles were grown larger than 500nm

mean diameter however they became of non-spherical shape with an uneven surface

This irregularity of particle surface can be attributed to the non-homogeneous monomer

swelling into the shell of a growing polymer which can be controlled by the factors such

as temperature agitation speed initiator feeding rate and surfactant amount required to

stabilize the colloidal system The SEM image of latex particles larger than 500nm mean

iameter is shown in Figure 4-4 The particle size distribution is broader than that of the

s particles These particles can however still be used for membrane construction

as a support layer rather than skin layer The support layer does not necessarily have to be

as highly monodisperse as the skin layer which require pores of high uniformity for the

selective removal of toxins

As mentioned earlier the amount of surfactant as well as monomer in an emulsion

system is the primary determinant of the particle diameter [Odian 1991] Anionic

surfactants are generally recommended at the level of 02-30 wt based on the amount

of water [Odian 1991] The critical micelle concentration (CMC) of Aerosolreg MA 80-I

anionic surfactant is 119 The surfactant loading in this emulsion system ranged from

the 121 for SS181 to 171 for SS259

P

d

maller

68

Figure 4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm

(C) 640nm (D) 790nm

(A)

(B)

69

(C)

(D)

Figure 4-4 Continued

70

The surfactant to monomer ratio refers to the value of the surfactant amount

divided by monomer amount after polymerization ends The surfactant amount is the

weight of the surfactant contained in emulsion and the monomer amount is the total

weight of the monomer added for polymerization to form the ultimate polymer particles

in emulsion As the surfactant to monomer ratio decreases mean particle diameter

increases as described by Odian [1991] Generally lower surfactant concentration forms

fewer micelles resulting in larger particle size [Evans et al 1999] However there is a

plateau in particle mean diameter between a surfactant to monomer ratio of 0005 and

0013 This is due to low polymerization conversion of latex particles from monomer

leading to a smaller particle size than expected This is not a factor of surfactant amount

The dependence of the particle size on the amount of surfactant is shown in Figure 4-5

0

200

400

600

800

1000

0000 0005 0010 0015 0020 0025 0030

Surfactant to monomer ratio (ww)

Part

icle

mea

n di

amet

er (n

m)

Figure 4-5 Dependence of the particle size on the surfactant to monomer ratio

71

413

e

f bare

microdomain structures is important for biocompatibility [Mori et al 1982 Higuchi et al

1993 Deppisch et al 1998] Figure 4-6 shows the schematic of core shell latex particle

structure The recipe for core shell structures is shown in Table 4-4

Core Shell Latex Particles

The latex particles shown in Figure 4-3 (A) and labeled as S258 in Table 4-2 wer

used as seeds to prepare core-shell latex particles because of their high particle

uniformity and smooth surface morphology Methyl methacrylate (MMA) and acrylic

acid (AA) monomers were introduced to increase surface hydrophilicity over that o

polystyrene particles AA is a more hydrophilic monomer than MMA because of the

carboxyl acid group in AA is more favorable for hydrogen boding with water The

surface carboxyl groups on PAA may have many promising applications in biomedical

and biochemical fields [Kang et al 2005] MMA monomer is less hydrophobic than

styrene PMMA is known to more biocompatible than PS but still PMMA is hydrophobic

The optimization of suitable hydrophobic to hydrophilic ratios and control of

Polystyreneseed particle

Core shelllatex particles

PSPMMA100

PSPMMA90PAA10

PSPMMA75PAA25

Polystyreneseed particle

Core shelllatex particles

PSPMMA100

PSPMMA90PAA10

Figure 4-6 Schematic of core shell latex particle structures

PSPMMA75PAA25

72

Table 4-4 The preparation recipe of PS core with various shell latex particles Latex 25 label PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA

Initial charge

PS seed latex (g) 500 500 500a

Monomer continuous

Methly methacylate (g) 320 288 240

Acrylic acid (g) 00 32 80

Initiator stream

Na

258 258 258

Water (g) 1570 1570 1570

Feed time (min) 0-120 0-50 0-60

b 200

Feed time (min) 0-122 0-60 0-65

952

2S2O8 (g) 02 02 02

NaHCO3 (g) 02 02 02

Water (g) 200 200

Solids content 183 183 183

Mean particle diameter (nm) 370plusmn186 370plusmn178 370plusmn191

Conversion () 955 924

Three types of core shell structures were prepared PMMA75PAA25 shell is a

copolymer consisting of the PMMA to PAA ratio of 75 to 25 by weight

PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90 to 10 by

weight PMMA100 is the PMMA homopolymer shell on PS core The particle size of

these core-shell particles as well as PS latex particle was prepared to be about 370nm in

mean diameter and are characterized to determine their zeta potential protein adsorption

and biocompatibility The image of SEM of PS and core shell particles is shown at Figure

4-7 The synthesized latex particles had a high uniformity in size and a smooth spherical

surface morphology

73

Figure 4-7 Scanning

Electron Micrograph of latex particles (A) PS (B) PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMMA75PAA25

(A)

(B)

74

(C)

(D)

Figure 4-7 Continued

75

42 Characterization of Latex Particles

421 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectroscopy has proven to be a powerful tool to characterize polymeric

materials A molecule can absorb only select frequencies (energy) of infrared radiation

which match the natural vibration frequencies The energy absorbed serves to increase

the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]

Only those bonds which have a dipole moment that changes as a function of time can

absorb infrared radiation The FTIR spectra of polymerized latex particles made during

this work are shown in Figure 4-8 There are number of characteristic peaks for PS latex

particles [Bhutto et al 2003 Li at al 2005] Aromatic C-H stretching vibration is shown

at 3002 cm-1 and 3103 cm-1 and aliphatic C-H asymmetrical and symmetrical stretching

shown at 2900 cm-1and 2850 cm-1 respectively There is an additional carbonyl (C=O)

adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some

amount of either or both PMMA and PAA The broad OH group peak at 3400 cm-1

appear for particles containing some amount of PAA and has intensity dependent on the

amount of PAA in the shell For example the peak intensity of OH groups for

PSPMMA75PAA25 is greater than that of PSPMMA90PAA10 since there is a higher AA

monomer content in PSPMMA75PAA25 than in PSPMMA90PAA10 This OH peak is not

seen for PS and PS core PMMA shell particles because of the absence of OH groups on

PS and PMMA polymer chains

422 Zeta Potential Measurements

The electrical surface property was characterized by zeta potential measurements of

tex particles Zeta potential is an important and useful indicator of surface charge which

can be used to predict and control the stability of colloidal systems The greater the zeta

is

la

76

1520002500300035004000

Wavenumbers (cm-1)

li

Re

ativ

e

Tra

nsm

ttanc

e

Figure 4-8 FTIR spectra of polymerized latex particles (A) ba

PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMM

(B)

(C) P

(D) P

C

C

OCH3

O

CH3

XCH2

CH2 n

CH

CH2 C

C

OCH3

O

XCH2 CH

OC

CH3

Y

OH

(A) Bare PS

500100000

re polystyrene (PS) (B) A75PAA25

PSPMMA100

SPMMA90PAA10

SPMMA75PAA25

77

potential the more likely the suspension is to be stable because the charged particles repel

one another and thus overcome the natural tendency to aggregate The measurement of

zeta potential is often the key ontrol of the interaction of proteins

with solid surfaces such as latex particles

Zeta potential (ζ) is the elec at exists at the shear plane of a particle

which is some small distance from the surface [Myers 1999] The zeta potential of the

particles can be determined by measuring the mobility of the particles in an applied

electric field termed electr its response to an alternating electric

field Colloidal particles dispersed in lu ically charged due to their ionic

characteristics and dipolar attributes The development of a net charge at the particle

surface affects the distribution of ions in the neighboring ng in

an increased concentration of counter ions to the surface Each particle dispersed in a

ns called fixed layer or Stern layer

Outside th compositions of ions of opposite polarities

form ble layer is form

particle-liquid interface This double layer may be considered to consist of two parts an

inner region which inclu nd relatively strongly to the surface and an outer or

diffuse region in which the ion distribution is determined by a balance of electrostatic

forces and random thermal motion The potential in this region therefore decays with the

distance from the surface until at a certain distance it becomes zero Figure 4-9 shows a

schematic representation of a typical ion distribution near a positively charged surface

The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the

to understanding and c

trical potential th

ophoretic mobility or

a so tion are electr

interfacial region resulti

solution is surrounded by oppositely charged io

e Stern layer there are varying

ed in the region of the ing a cloud-like area Thus an electrical dou

des ions bou

center of those ions which are effectively adsorbed to the surface [Hunter 1981] The

78

extent of ion adsorption is determined by electrical and other long-range interactions

between the individual ions and surface of particles The ions outside of the Stern layer

form the diffuse double layer also referred to as the Gouy-Chapman layer [Burns and

Zydney 2000]

Shear plane

Stern layer

ψζ

0

ψs

ψShear plane

Stern layer

ψζ

0

ψs

ψ

t

controlled electric field is applied via electrodes immersed in a sample suspension and

Figure 4-9 Schematic representation of ion distribution near a positively charged surface

Zeta potential is a function of the surface charge of a particle any adsorbed layer a

the interface and the nature and composition of the surrounding medium in which the

particle is suspended The principle of determining zeta potential is very simple A

79

this causes the charged particles to move towards the electrode of opposite polarity

Viscous forces acting upon the moving particle tend to oppose this motion and the

equilibrium is rapidly established between the effects of the electrostatic attraction and

the viscosity dra

g The particle therefore reaches a constant terminal velocity

Figure 4-10 Schematic representation of zeta potential measurement(source

httpnitioncomenproductszeecom_shtm)

Because protein adsorption mechanisms are very complex and hard to explain in

biological systems protein adsorption is generally studied by using more ideal systems

consisting of one or more well characterized proteins a well-characterized adsorbent and

a well-defined aqueous solution Even so small changes in the experimental conditions

such as pH ionic strength and temperature generate totally different results Therefore

two media systems phosphate buffer (PB) and phosphate buffered saline (PBS) were

chosen to be used for the zeta potential measurements and protein adsorption study PB is

a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4H2O) to

n a constant pH PBS is used to keep the water concentration inside and outside of

the cell balanced If the water concentration is unbalanced the cell risks bursting or

shriveling up because of a phenomenon called osmosis PBS is a solution with the 5 mM

of monobasic sodium phosphate (NaH2PO4H2O) and 143 mM of sodium chloride (NaCl)

in water

maintai

80

The results of zeta potential measurement at room temperature (25plusmn1oC) as

function of pH and ionic strength (different media PB and PBS) for synthesized lat

particles are seen in Figure 4-11 Zeta potential values of polystyrene (PS) latex particle

at the Figure 4-9 are negative between -291 mV and -599 mV in PB and betwe

ex

s

en -203

mV and -278 mV in PBS at all pH ranges These negative zeta potential values of PS

latex particles are due to the sulfate groups [Vandenhu et al 1970] originated from

persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer

chain where polymerization was initiated

-100

-80

-60

-40

-20

0

pH

Zta

pte

ntl

ζ

20

40

1 2 3 4 5 6 7 8 9

eo

ia (m

V)

PB (5mM)

PBS (5mM +143mM)

en -

Figure 4-11 Zeta potential of PS latex particles at 25oC

The zeta potential measurements of PSPMMA100 core shell latex particles as seen

in Figure 4-12 were also negative with a range of -28 and -505 mV in PB and betwe

143 mV and -186 mV in PBS at the pH 21-78 These negative values are also due to

sulfate groups on the particle surface

81

-100

-80

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

Zea

pote

nti

l ζ

(mV

pH

ta

)

PB (5mM)

PBS (5mM + 143mM)

Figure 4-12 Zeta potential of PSPMMA100 latex particles at 25oC

The zeta potential for PSPMMA90PAA10 and PSPMMA75PAA25 was also

negative as seen in Figure 4-13 and 4-14 The zeta potential value for the

PSPMMA90PAA10 were between -367 mV and -678 mV in PB medium and between -

147 mV and -193 mV in PBS both at 25oC In case of the PSPMMA75PAA25 core shell

particles zeta potential values were between -291 mV and -520 mV in PB and between

-115 mV and -210 mV in PBS media Sulfate groups in PSPMMA75PAA25 and

PSPMMA90PAA10 core shell particles contribute to the negative zeta potential values

participate in forming a

negat

cid is expressed by the term pKa which is the pH

at which an acid is 50 dissociated However the contribution of the carboxylic acid

Carboxylic groups from acrylic acid monomer also would

ive surface charge of the latex particles at a pH greater than 70 because the pKa

value for the carboxylic acid is between 40 and 60 pH range [Ottewill et al 1967] This

indicates that carboxylic acid dissociation begins at a pH between pH 46-60 and

increases with pH The strength of an a

82

group to negative zeta potential values of PSPMMA-PAA core shell particles was not

detectable because there was no significant difference in zeta potential values for

synthesized latex particles at a pH greater than 70

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

Zeta

pot

entia

l ζ

(mV

)

PB (5mM)PBS (5mM + 143mM)

-80

-100

pH

Figure 4-13 Zeta potential of PSPMMA90PAA10 latex particles at 25oC

-100

-80

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

pH

Ze p

ont

i ζ

V)

tate

al (m

PB (5mM)PBS (5mM + 143mM)

Figure 4-14 Zeta potential of PSPMMA75PAA25 latex particles at 25oC

83

The zeta potential plots and values were similar for all latex particles This may be

due to the similar initiator density on the latex surface The initiator concentrations

monomers for shell polymerization were the same fo

to

r all core shell latex particles (062

ww from Table 4-4) A similar concentration (055 ww) of initiator to monomer was

added to prepare bare PS latex particles used in zeta potential measurements

Since the zeta potential values of all synthesized latex particles were negative

between pH=20 and pH=78 the isoelecric point (IEP) of these latex particles would be

less than pH=20 Sulfates have a pKa in the range of 10 to 20 [James 1985] indicating

that the sulfate group the conjugate base of a strong acid is protonated (-C-OSO3H) at

less than pH 20 This means that the synthesized latex particles are all negative at the

physiological pH Most serum proteins have a negative surface charge in blood

therefore when the negative latex particles are applied to blood stream fatal

tein is

avoided by charge repulsion in the blood Table 4-5 shows the common blood proteins

and their isoelectric points (IEP)

Table 4-5 The isoelectric point (IEP) of common proteins Serum proteins Isoelectric point (IEP)

complication by coagulation between applied latex particles and serum pro s

Albumin 48 α1-Globulin 20 α2-Microglobulin 54 γ1-Globulin 58 Heptoglobin 41 Hemoglobin 72 β1-lipoprotein 54 Immunoglobulin G (Ig G) 73 Fibrinogen 58 (source httpwwwfleshandbonescomreadingroompdf1085pdf

httpwwwmedalorgvisitorwww5CActive5Cch135Cch13015Cch130105aspx )

84

The absolute zeta potential values of the latex particles in phosphate buffer (PB)

was higher than that in phosphate buffered saline (PBS) This is because high

concentration of sodium and chloride electrolytes in PBS compressed the electrical

double layer causing rapid decay of surface potential Figure 4-15 shows that the

thickness of the electrical double layer is inversely proportional to the concentration of

electrolyte in the system

Surf

ace

Pote

ntia

l ψ

1κ3

10

1e

1κ 1κ

(1)(3) (2)

2 1 Z

Surf

ace

Pote

ntia

l ψ

1κ3

10

1e

1κ 1κ

(1)(3) (2)

Figure 4-15 The decay of surface potential with distance from surface in various

electrolyte concentrations (1) low (2) intermediate (3) high [Myers 1999]

2 1 Z

The electrical potential in the solution surrounding the particle surface falls off

the Debye-Huckel

yers 1999]

exponentially with distance (Z) from the surface according to

approximation [M

z) (- exp 0 κψψ = (4-4)

e reciprocal of the thickness of the electrical double layer referred to as the

The potential fallen off by a factor e The theoretical equation for the

ickness 1κ is

where κ is th

Debye length of 1

double layer th

21

22i

0

ekT 1

⎟⎟⎠

⎜⎜⎝

=sum ii zcεε

κ

⎞⎛ (4-5)

85

where

tration of

y was performed to measure the protein adsorption on the

synthesized latex particles Many physical and chemical processes occur at the boundary

between two phases and adsorption is one of the fundamental surface phenomena [Oscik

1982] This phenomenon is driven by the change in the concentration of the components

at the interface with respect to their concentrations in the bulk phase There are two

adsorption processes physical adsorption occurring when non-balanced physical forces

appear at the boundary of the phases and chemical adsorption or chemisorption

t the

interface [J

n leads

ational entropy hence adsorption takes place only if the

loss in conformational entropy is compensated

ter adsorption Cp The initial part of the

isotherm merges with the Γ-axis because at low polymer concentration the protein has a

high affinity for the surface and all of the polymer is adsorbed until the solid surface is

saturated

ε0 is the permittivity of a vacuum of free space ε is the permittivity of a medium e

is the relative permittivity or dielectric constant of the medium c is the concen

an ion (i) z is the valency of an ion (i) k is Boltzmannrsquos constant T is absolute

temperature (K)

43 Protein Adsorption Study

The adsorption stud

occurring when atoms and molecules from adjacent phases form chemical bonds a

aroniec et al 1988]

Flexible proteins in solution possess high conformational entropy as a result of the

various states each of the many segments in the protein chain can have Adsorptio

to a reduction of this conform

by sufficient attraction between polymer

segment and solid surface [Martin 1998] Isotherm shape of high affinity adsorption

between polymer and solid is shown in Figure 4-16 where the adsorbed mass Γ is plotted

against the polymer concentration in solution af

86

Figur

de

arboxyl

e 4-16 High-affinity adsorption isotherm of typical flexible polymer on solid

surface

In order to investigate the adsorption of blood proteins latex particle properties

such as electrical surface charge hydrophobicity and environmental conditions like pH

ionic strength and temperature are considered as experimental factors [Arai and Nor

1990] Figure 4-17 shows the overall schematic representation of the protein adsorption

on the synthesized latex particles with the functional groups such as sulfate and c

groups

CP

Γ

PMMAPAAShell

PS or PMMAShell OSO3

-

OSO3-

OSO3-

OSO3-

PS

-O3SO

PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

Protein

NH3+

+

+H3N

+

COO-

Hydrophobic interaction

NH3

COO-

H3N

COO-

-OOC

Ionic interaction

Hydrophobic interaction

Hydrogen bonding

Ionic interaction

PMMAPAAShellOSO3

-

OSO3-

OSO3-

OSO3-

PS or PMMAShell -O3SOOSO3

-

PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

-O3SO

OSO3-

OSO3-

OSO3-

PS PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

Protein

NH3+

+

+H3N

+

COO-NH3

COO-

H3N

COO-

-OOC

NH3+

+

+H3N

+

COO-

Hydrophobic interaction

NH3

COO-

H3N

COO-

-OOC

Ionic interaction

Hydrophobic interaction

Hydrogen bonding

Figure 4-17 Overall schematic representation of the protein adsorption on the

Ionic interaction

synthesized latex particles

The surface functional groups as well as suitable surface hydrophobicity

hydrophilicity of latex particles are closely related to protein adsorption property As

indicated at Figure 4-17 various interaction forces such as hydrophobic interaction ionic

87

interaction hydrogen interaction and van der Waals interaction [Andrade 1985] exist

between protein molecule and the solid surface of latex particle

431 Adsorption Isotherm

The protein adsorption isotherm is generally defined as the relationship between the

amount of protein adsorbed by a solid (latex particle) at a constant temperature and as a

ine

the amount of protein adsorbed onto latex particles To evaluate the equilibrium

conce s

x

function of the concentration of protein Adsorption isotherms were plotted to determ

ntration of target proteins a calibration curve is firstly needed Figure 4-18 show

the UV absorbance as a factor of bovine serum albumin (BSA) concentrations The UV

absorbance of the known protein concentration was measured at 562nm which is the λma

for a protein treated by a bicinchoninic acid (BCA) assay

0

02

04

06

08

500 520 540 560 580 600

Wavelength (nm)

Abs

orba

nce

Figur gml (B)

06mgml (C) 02mgml

(A)

(C)

(B)

Max562nm

e 4-18 The dependence of UV absorbance on BSA concentration (A) 10m

Figure 4-19 shows the standard calibration curve used in adsorption isotherm

experiment of protein onto latex particles The net absorbance of each known protein

88

concentration was plotted and then a linear equation passing through a concentration of

zero protein concentration was obtained This equation is applied

to determine the

orbed on the synthesized latex particles The amount of free

protei

eported

trations (005 01 03 05

and 07mgml) were used to generate this curve as well as the adsorption isotherm

unknown protein amount ads

n was also determined using UV light at the 280nm wavelength however a

distinguishable UV intensity was too low to determine a precise value of protein

adsorption

The bicinchninic acid (BCA) assay technique [Smith et al 1985] has been r

for high efficiency of protein evaluation and was used for determination of protein

adsorption [Tangpasuthadol et al 2003 Williams et al 2003] This technique has many

advantages such as high sensitivity easy of use color complex stability and less

susceptibility to any detergents Five different BSA concen

y = 00R2 =

004741x 09939

00

01

02

03

04

0 200 400 600 800

BSA Standard (microgml)

Net

Abs

orba

nce

at 5

62nm

Figure 4-19 Standard curve of net absorbance vs BSA sample concentration

89

All isotherms were fitted to the Langmuir-Freundlich isotherms using the nonlinea

regression method Two adsorption isotherm models the Langmuir type isotherm a

Langmuir-Freundlich combination type isotherm were compared As seen the Figure 4-

20 The Langmuir isotherm model deviated further from the experimental data Th

all isotherms were fitted to the Langmuir-Freundlich isotherms [Yoon et al 1996

al 1998] usi

r

nd the

erefore

Lee et

ng the nonlinear regression method with following equation

n

n

eq

eqm

kC

kC1

1

1+

where q is the amount of adsorbed BSA per unit surface area q

qq = (4-6)

stant

were between 045 and 055 and hence n was fixed to its average 05 Actual qm values

were evaluated after fixing the n value

m is the adsorbed amount

in equilibrium Ceq is the equilibrium concentration of BSA k is the adsorption con

and n is the exponential factor for a heterogeneous system n values are empirical and

usually from zero to no more than unity [Jaekel 2002] The values of n in our system

00

10

20

30

0 02 04 06 08

C (mgml)eq

q (m

gm

2 )

pH 32Langmuir-Freundlich modelLangmuir model

Figure 4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on

polystyrene latex particles at 37oC in phosphate buffer media

90

The BSA protein adsorption on the synthesized latex particles was performed at

three pH levels 32 48 74 in both phosphate buffer (PB) and phosphate buffered saline

(PBS) at physiological temperature 37oC These pH levels correspond to an acidic pH

lower than the isoelectric point (IEP) of BSA the pH at the IEP and an alkaline pH

higher than the IEP of BSA respectively The IEP corresponds to the pH at which the

amino acid becomes a zwitterion Here its overall electrostatic charge is neutral and the

charge expressed by the basic amino group (-NH2 -NH3+) of the amino acid is equal to

the charge expressed by the acidic carboxyl group (-COOH -COO ) The IEP of BSA is

-

pH 48 and indicates that the side chain of the amino acids of BSA contains a greater

amount of acidic functional groups than that of basic functional groups At pH 32 and

74 BSA has a positive and negative charge respectively

Figure 4-21 and 4-22 show adsorption isotherms of BSA onto polystyrene (PS)

latex particles

00

10

20

30

40

0 02 04 06 08

Ceq (mgml)

q (m

gm

2 )

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in

phosphate buffer (PB) at 37oC

91

00

10

30

40

0 02 04 06 08

Ceq (mgml)

q (m

gm

2

20

)

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

(PBS) on PS latex particles

At the IEP of BSA the adsorbed amount (q

Figure 4-22 Adsorption isotherm of BSA adsorption 37oC in phosphate buffered saline

onto PS at equilibrium was

maxim

um at the IEP of BSA This is because the BSA molecules form the most

compact structures at the IEP eliminating the repulsion force between proteins A higher

number of BSA molecules can adsorb on a given surface area by hydrophobic interaction

when in this compact structure This observation agrees well with reported data by other

researchers who also indicated that the maximum adsorption from aqueous protein

solution was observed at the IEP [Mularen 1954 Watkins et al 1977 Oh at el 2000]

There was no significant difference of adsorbed BSA amount in PB and in PBS media at

pH 48 The conformational alteration of BSA was not affected significantly by the ionic

strength of the two solutions There was evidence of the effect of the electrostatic

interaction to some extent even at the IEP since the amount of BSA adsorbed in PBS is

slightly hig

m) of BSA

um indicating that hydrophobic interaction between latex particle and BSA

protein is maxim

her than in PB at this pH

92

The adsorbed amount of protein is shown to decrease at a pH 32 and 74 The

decrease in adsorption efficiency when deviating from IEP of BSA is a result of the

increase in conformational size of the protein molecules and the lateral electrostatic

repulsions between adjacent adsorbed BSA molecules Figure 4-23 shows a schematic of

possible conformational changes at each pH where hydrophobic interaction always exists

between non-polar portion of a protein and hydrophobic portion of solid surface

- - --- ---Latex surface

Zeta potential

BSA(+)

(-)

48 7432pH

(0)

+++ + ++

--+

++-

- - --- ---Latex surface

Zeta poten pH(0)

+++ + ++

--

tial

BSA(+)

(-)

48 7432

+

++-

ming

4

s

al blood Table 4-6 shows the calculated results of

Figure 4-23 Conformation of bovine serum albumin (BSA) on latex particles (Assuthat charge repulsion reduces the effect of the hydrophobic interaction at pH 74)

In addition the adsorbed amount of BSA at pH 32 was larger than that at pH 74

and still less than that at 48 This is because there is the electrostatic interaction between

the positive BSA molecules and negative PS latex particles at pH 32 and electrostatic

repulsion is dominant between negative BSA and negative PS latex particles at pH 7

The amount adsorbed increased with increasing ionic strength in pH 32 and 74 This i

because the high concentration of electrolytes reduces the electrostatic repulsion between

BSA and PS latex particle It should be noted that the BSA concentration used in these

experiments was lower than that found in normal human blood This is because the

equilibrium BSA adsorption onto the latex particles is actually reached at a much lower

concentration than that found in norm

93

adsor rm

ein

leading to a compact conformation of BSA There are two main forces affecting

adsorption amount at an acidic pH of 32 They include the ionic attraction between the

negative latex particle and the positive protein and lateral repulsion between positive

proteins The smallest amount of BSA adsorption was observed at a pH of 74 At this

point the charge repulsion between negatively charged particles and protein molecules

exists the h

of cha

bed BSA amount at equilibrium from the Langmuir-Freundich adsorption isothe

equation

In summary the hydrophobic interaction between PS latex particle and BSA

protein exists and is the dominant mechanism for the adsorption process Conformational

changes of BSA at each pH also affected the adsorption amount Adsorption was highest

at pH 48 because at this pH there is the least amount of ionic repulsion in the prot

and the lateral repulsion between proteins dominant adsorption even though there always

ydrophobic interaction between the solid latex particle and protein The effect

rge repulsion was reduced by the high concentration of electrolytes in PBS medium

Table 4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2) 32 174 48 299 Phosphate buffer 74 136 32 260 48 283 Phosphate buffered saline 74 207

The adsorption isotherms of BSA on PSPMMA core shell particles are shown in

Figure 4-24 in phosphate buffer (PB) and in Figure 4-25 in phosphate buffered saline

(PBS) The adsorbed BSA amount was calculated using the Langmuir-Freundlich

100

94

adsorption isotherm equation and the amount listed in Table 4-7 The trend seen in the

adsorption results was very similar to the case of PS latex The overall adsorption process

was d rticle

rs

hat

0

ominated by hydrophobic interactions between the PSPMMA100 core shell pa

and BSA proteins At pH 48 the adsorbed amount (qm) of BSA on PSPMMA100 core

shell particles was 221 mgm2 in PB and 237 mgm2 in PBS and are the maximum

values recorded among the three pH levels 32 48 and 74 Suzawa and his co-worke

[1982] have obtained similar results This can be also explained by the compact

conformation of BSA molecules at the IEP of BSA which lead to a high protein

population on the solid surface At a pH of 32 and 74 BSA adsorption was less than t

at pH 48 because of the protein conformation change There was a small increase in

adsorbed BSA at pH 32 because of the charge attraction between negative PSPMMA10

core shell particles and positive BSA At pH 74 however the adsorption of BSA was

further reduced by charge repulsion between the negative PSPMMA100 core shell

particles and the negative BSA proteins

000 02 04 06 08

Ceq (mgml)

10

20q (m

gm

302 )

40

50pH=32pH=48pH=74Mode 8)l (pH 4Mode 2)l (pH 3Mode 4)

Figure 4-24 Adsorption isotherm of BSA 37oC in PB on PSPMMA core shell latex

l (pH 7

100

particles

95

The adsorbed amount of BSA increased more rapidly in PBS with increasing ioni

strength at pH 74 This shows that the low concentration of electrolytes promotes

electrostatic repulsion between negative BSA and negative PSPMMA

c

on

x

l 1980

100 core shell

particle As the ion strength of PBS is increased however the high concentration of

electrolyte leads to less electrostatic repulsion promoting more rapid protein adsorpti

By comparing the amount of adsorbed protein between bare PS latex particles and

PSPMMA100 core shell particles the amount of protein adsorption on the PS late

particles is greater than that on the PSPMMA100 core-shell particles [Suzawa et a

1982 Lee et al 1988]

00

10

20

30

40

m2 )

0 02 04 06 08

Ceq (mgml)

q (m

g

pH=32pH=48pH=74Model (pH 2) 3Model (pH 48)Model (pH 74)

Figure 4-25 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA100 core shell

latex particles

96

Table particles calculated the Langmuir-Freundlich isotherm model

4-7 The equilibrium concentration values (qm) of BSA adsorption on PSPMMA100

Media pH qm (mgm ) 2

32 140

48 221 Phosphate buffer

74 130

32 169

48 237 Phosphate buffered saline

74 162

The BSA adsorption mechanism and trend on PSPMMA core shell particles was

very similar to that of the PS latex particle Unlike the case of PS and PSPMMA100 latex

particles the amount of BSA adsorbed onto PSPMMA90PAA10 and PSPMMA75PAA25

core shell particles was higher at pH 32 than pH 48 and 74 Figure 4-26 through 4-29

shows the adsorption isotherms of BSA on PSPMMA90PAA10 and PSPMMA75PAA25

core shell particles The amount of BSA adsorbed increased at pH 32 and pH 48 as the

hydrophilicity was developed by the addition of PAA On the contrary the adsorbed

amount of BSA was dramatically decreased at pH 74 The adsorption amount of BSA on

PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles at equilibrium is listed in

Table 4-8 and 4-9 respectively

Carboxylic acid as well as sulfate groups are distributed on the surface of

PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles and seem to largely affect

the BSA adsorption The isotherms showed very strong pH dependence to the BSA

adsorption onto these core shell latex particles The carboxylic groups (COOH) may

especially enhance the BSA adsorption prominently at pH 32 by hydrogen bonding with

protein molecules The pKa value for the PAA is 40-60 and a good indication that the

97

carboxylic acid groups are protonated at pH 32[Gebhardt et al 1983] The main

adsorption interaction force at pH 32 was generated by hydrogen bonding as well as

charge attraction between latex particle protein even th the conformational

repulsion between proteins results in a reduction of the protein

adsorption at this pH The amount of BSA adsorbed also increased as the PAA amount in

eased The a tion of BSA w inimal however less

than 07 mgm2 at pH 74 Here the carboxylic groups are ionized which increases the

charge repulsion by ionized carboxylic groups (COO-) resulting in PSPMMA-PAA latex

particle

and ough

change and charge

the core shell particles incr dsorp as m

s with much lower protein adsorption

00

20

40

q (m

g2 )

60

m

pH=32pH=48pH=74Model (pH 32)Model (pH 48)

0 02 04 06 08

Ceq (mgml)

Model (pH 74)

o shell

Figure 4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA90PAA10 corelatex particles

98

00

40

60

80

0 02 04

C (mgm

mm

2 )

20

06 08

eq l)

q (

g

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-27 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA90PAA10 core

shell latex particles

Table 4-8 The equilibrium concentration values of BSA adsorption on PSPMMA90PAA10 particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2)

32 361

48 283 Phosphate buffer

74 043

32 519

48 226 Phosphate buffered saline

74 043

99

00

20

40

60

80

100

0 02 04 06 08Ceq (mgml)

q (m

gm

2 )

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

oFigure 4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA75PAA25 latex particles

00

100

20

40

60

80

0 02 04 06 08Ceq (mgml)

q (

m2 )

pH=32pH=48pH=74

mg

Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-29 Adsorption isotherm of BSA 37oC in PBS on PSPMMA75PAA25 core shell

latex particles

100

Table 4-9 The equilibrium concentration values of BSA adsorption on PSPMMA75PAA25 particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2)

32 1083

48 662 Phosphate buffer (PB)

74 075

32 1077

48 681 Phosphate buffered saline (PBS)

74 057

The adsorption of β2-microglobulin (β2M) was determined under one set of

onditions in phosphate buffer at 37oC and pH 74 because of the limited amount of β2M

protein The adsorption isotherms of the β2M protein on the synthesized latex particles

are shown in Figure 4-30 4-31 and 4-32 The β2M protein concentrations for adsorption

isotherm experiment were chosen to be 0015 0030 0045 and 0060mgml since the

β2M concentration for the patients with renal failure can be elevated to similar

concentrations about 005mgml or more up to 50 times the normal level [Nissenson et

al 1995] There were steep initial slopes of the adsorption isotherm curves of β2M on

latex particles in all isotherms indicating a high affinity adsorption type It seems that the

hydrophobic interaction is enough for the adsorption process of β2M proteins to occur on

the latex particles even though charge repulsion exists between protein and latex particle

at pH 74 The complete plateau regions were not seen for all isotherms indicating that

β2M proteins were not saturated at this level and there are still available sites for β2M

c

adsorption on latex particles The initial slope of the β2M isotherms for PS was steeper

101

than that of any of the other latex particle PSPMMA100 PSPMMA90PAA10 and

PSPMMA 75PAA25

00

02

0 0002 0004 0006 0008

04

06

10

Ceq (mgml)

q (m

2 )

08

gm

PSPSPMMA100Model (PS)Model (PSPMMA100)

Figure 4-30 Adsorption isotherm of β2M onto PS and PSPMMA100 latex particles in PB

at 37 C and pH 74 o

00

02

04

06

08

10

0 0002 0004 0006 0008C (mgml)

q (m

m2 )

eq

g

PS

PSPMMA90PAA10

Model (PS)

Model (PSPMMA90PAA10)

Figure 4-31 Adsorption isotherm of β2M onto PS and PSPMMA90PAA10 latex particles

in PB at 37oC and pH 74

102

00

02

04

06

08

10

0 0002 0004 0006 0008Ceq (mgml)

q (m

gm

2 )

PS

PSPMMA75PAA25

Model (PS)Model (PSPMMA75PAA25)

Figure 4-32 Adsorption isotherm of β2M onto PS and PSPMMA75PAA25 latex particles

in PB at 37oC and pH 74

The equilibrium concentration values (qm) of β2M adsorption on latex particles are

calculated and listed in Table 4-10 The difference between the maximum amounts of

β2M adsorption on all the latex particles was not significantly different and their values

were between 069 and 08mgm2

Table 4-10 The equilibrium concentration values of β2M adsorption calculated the Langmuir-Freundlich isotherm model

Latex particles qm (mgm2)

PS 080

PSPMMA100 069

PSPMMA90PAA10 072

PSPMMA75PAA25 072

103

432 Adsorbed Layer Thickness

There are several possible explanations for large amount of BSA adsorbed at acidic

pH levels including the end-on adsorption pattern of BSA the flexibility of BSA

molecules the tilting of protein molecules due to the asymmetry of the charge

distribution or multilayer formation [Peula et al 1993] The adsorbed BSA thickness (δ)

can be calculated by the following equation [Chiu et al 1978] This equation was also

cited again by other researchers [Shirahama et al 1985]

BSA

mqρπ

δbullbull

=33

(4-7)

where π

33 is the packing factor qm is the plateau value of the adsorbed am

ρBSA is the hich corresponds to the reciprocal of its known

partial specific volume The layer thickness (δ) of BSA proteins on synthesized latex

partic

he IEP [Shirahama et al 1985] Compared to the

hydrodynamic dimensions of BSA 140 times 40 times 40 Aring3 [Fair et al 1980 Peter 1985] these

nd

ode It is unnecessary ultilayering because thickness do

exceed 140 Aring The layer thickness (δ) of BSA proteins on synthesized latex particles

e red saline (PBS) was ran 28 ~ 83 Aring at 37oC and pH 48 These

lso listed in Table 4-1

ount and

density of BSA molecule w

les in phosphate buffer (PB) ranged from 27 - 81 Aring at 37oC and pH 48 These

calculated values are listed in Table 4-11 Only δ values at pH 48 the isoelectric point

(IEP) of BSA are important because intra- and intermolecular electrostatic repulsion of

protein molecules is minimized at t

values of layer thickness indicate that the BSA molecules adsorb by side-on (40Aring) a

end-on (140Aring) m to consider m

not

in phosphat buffe ged of

calculated values are a 2

104

Table 4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37oC pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

32 21 17 44 133

48 37 27 35 81

74 17 16 5 9

Table 4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37oC

pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

32 32 21 64 132

48 35 29

74 26 20 5 7

28 83

1n

1n

neqm

So k is closely related to the initial slope of the isotherm showing the affinity

between BSA and latex particles [Yoon et al 1996] A key point in the characterization

433 Gibbs Free Energy of Protein Adsorption

In the Langmuir isotherm model k represents the ratio of adsorption to the

desorption rate constants This definition is not applicable to the Langmuir-Freundlich

isotherm model but its meaning is similar [Yoon et al 1996] In this sense k is closely

related to the affinity between protein molecules and latex particles When the

equilibrium concentration Ceq reaches zero the value of Ceq will be very small (1gtgt

kCeq ) and then the equation 4-6 becomes

(4-8)

of adsorption processes on solid-liquid interfaces is to determine the change in Gibbsrsquos

free energy (∆Gdeg) one of the corresponding parameters for the thermodynamic driving

forces during adsorption The traditional method to evaluate adsorption thermodynamic

kCqq 1=

105

constants is based on measurement of adsorption isotherms under static conditions

[Malm ten 1998] The Langmuir-Freundlich ads lf

easily found from Then this enables calculation of the free-

energy change ∆G during adsorption expressed as

(4-9)

where R is the general gas constant (R = 8314Jmiddotmol K ) and T is the absolute

tem ture Gibb e energy A P

listed in Table 4-13 and 4-14 respectively As the ∆Gdegads ore negativ e

pro adsorption on a solid substra ill be more favorable

e of BSA adsorption in phosphate o

pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

s orption equilibrium constant k can be

a slope of the isotherm

degads

lfads kRTG lnminus=∆ o

-1 -1

pera s fre change of BS adsorption values in B and PBS are

values are m e th

tein te w

Table 4-13 The values of Gibbs free energy changbuffer (PB) at 37 C

32 -2411 -2726 -2846 -2805

48 -2352 -2383 -2198 -1972 ∆Go

80

ads (kJmol)

74 -2452 -888 -324 -2

Table 4-14 The values of Gibbs free energy change of BSA adsorption in phosphate

pH PS PSPMMAbuffered saline (PBS) at 37oC

100 PSPMMA90PAA10 PSPMMA75PAA25

32 -2315 -2620 -2312 -2683

48 -2149 -2299 -2090 -2138 ∆Go

(kJmads ol)

74 -2430 -1992 -258 -263

Table 4-15 shows the calculated values of Gibbs free energy change of β M

adsorption on synthesized latex particles PSPMMA PAA core shell latex particle has

2

75 25

106

largest negative Gibbs free energy of β2

2

M adsorption indicating largest affinity betw

β

een

2M adsorption in phosphate buffere (PB) at 37 C and pH74

PS PSPMMA100

M and latex particle

Table 4-15 The values of Gibbs free energy change of βo

PSPMMA90PAA10 PSPMMA75PAA25

∆Go03 ads

(kJmol) -3626 -3466 -3659 -39

The Gibbs free energy of β2M was much more negative in phosphate buffer (PB)

than that of BSA in physiological condition (37 C and pH 74) o from the Table 4-13 and

the Figure 4-31 This indicates that β2M molecules more readily adsorb on latex particles

BSA This can be explained by the rate of diffusion and accessibility of β2M molecules to

the latex particle ier and faster for β2M m an BSA because of

size (molec ight) erical shape molecules It o be

due to the higher hydrophobic force and less electrostatic repulsion that contribute to the

adsorption of β2M molecules not as prevalent in the case of BSA because the IEP of β2M

(57) is clo H 74) than that of BSA (48) causing β2M to

be less electro cally lled p ro

can be seen that the ∆ β2M adsorption onto the synth article

increasing wi hy icity o x particles s the ∆Goads of the BSA

molecules is decreasing or constant This phenomenon supports the theory that the

select lizing

than BSA and that the affinity of β2M for the latex particle is much larger than that of

surface It is eas olecules th

the smaller ular we and sph of β2M may als

ser to physiological condition (p

stati repe from the latex articles than BSA F m Figure 4-33 it

Goads of esized latex p s is

th the drophil f the late wherea

ive adsorption of β2M molecules over BSA molecules can be enhanced by uti

more hydrophilic PSPMMA90PAA10 and PSPMMA75PAA25 core shell latex particles

107

-60

-50

1 2 3 4

-40∆Gad

s

-30

10

0

o (k

Jm

o-

-20l)

β2-Microglobulin (β2M)Bovine serum albumin (BSA)

Figure 4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37

and pH 74

434 Kinetics of Adsorption

to

oC

Protein adsorption is a complex process known to have a large biological impact

and is currently not well understood quantitatively because of extreme sensitivity to pH

the concentration of other electrolytes and molecules present temperature and many

other factors that can change in the physiological system Accurate knowledge of the

adsorption kinetics under a given set of conditions is a prerequisite for elucidating the

mechanisms of many fundamental biological processes to be predicted at the molecular

level [Regner 1987] This adsorption kinetics approach is used in protein adsorption

studies because of the uncertainty related to the time needed for the interfacial proteins

reach the equilibrium It is generally accepted that the process of protein adsorption is

comprised of the following steps a) transport toward the interface b) attachment at the

interface c) eventual structural rearrangements in the adsorbed state d) detachment from

PS PSPMMA100 PSPMMA90PAA10PSPMMA75PAA25

108

the interface and transport away from the interface Each of these steps can affect the

overall rate of the adsorption process

The quantitative analysis of the protein adsorption kinetics requires that the protein

amount adsorbed is known as a function of time The kinetics tests were carried out in

phosphate buffer (PB) at 37oC and pH 74 a condition similar to physiological condition

The protein concentrations chosen were 07mgml for BSA and 006mgml for β2M

which are the maximal concentrations used in an adsorption isotherm test A time-based

evaluation of the adsorption was made by measuring the concentration of protein in

solution at different incubation times The results are shown below from Figure 4-34 to 4-

37 The adsorption kinetics of proteins onto th cond-e latex particles is expressed as a se

order model [Oumlzacar 2003] shown by the following equation

2)( tet qqk

dtdq

minus= (4-10)

Integrating above equation and applying the boundary conditions yields

ee

t tq

+=

1 (4-11)

where q

qkq

t

2

2

2

the previous adsorption isotherm results representing good reproducibility of adsorption

t and qe stand for the amount (mgm ) of protein adsorbed at time t and at

equilibrium respectively k is the equilibrium rate constant of second-order adsorption

(m mghr) and t is the incubation time (hr)

The fitting parameters calculated by using the equation 4-11 are listed in table 4-16

From this data it is seen that the amount of BSA adsorbed at equilibrium was higher than

that of β2M on PS and PSPMMA100 latex particles but less than that of β2M on

PSPMMA90PAA10 and PSPMMA75PAA25 latex particles These results are similar to

109

00

05

15

20

0 2 4 6 8 10 12 14

Time (hr)

rot

n ad

sorb

d (

10

25

Pei

em

gm

2

BSA2nd order model (BSA))β2M2nd order model (β2M)

Figure 4-34 The kinetics of protein adsorption in PB on PS latex particles at 37oC and

pH 74

00

05

10

15

20

25

30

0 2 4 6 8 10 12 14

Time (hr)

Prte

inds

oo

arb

ed (

mg

m2 )

BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-35 The kinetics of protein adsorption in PB on PSPMMA100 latex partic

37les at

oC and pH 74

110

00

03

06

09

12

15

0 2 4 6 8 10 12 14

Time (hr)

Prot

ein

adso

rbed

(m

gm

2 ) BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-36 The kinetics of protein adsorption in PB on PSPMMA90PAA10 latex

particles o at 37 C and pH 74

00

03

06

09

12

15

0 2 4 6 8 10 12 14

Time (hr)

BS

A ad

sorb

ed (

mg

m2 )

BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-37 The kinetics of protein adsorption in PB on PSPMMA75PAA25 latex

particles at 37oC and pH 74

111

test The variation of β2M equilibrium adsorption amount was nearly negligible for all

latex particles The plateau region is reached earlier for β2M than BSA on all latex

particles and the K values (equilibrium rate constant of second order adsorption) for the

β2M adsorption process onto latex particles are much larger than for BSA adsorption

These values indicate that the β2M molecules have a higher adsorption rate onto the latex

particles than the BSA molecules

Table 4-16 Fitting parameters of second-order kinetic model BSA β2M

Latex particles qe (mgm2) K (hr-1) qe (mgm2) K ( -1) hr

PS 144 67 074 7549

PSPMMA100 175 353 076 4438

PSPMMA90PAA10 046 97 075 5695

PSPMMA75PAA25 060 179 075 5168 K = (kqe) hr-1

There are several explanations for the phenomena observed in these experiments

First the smaller sizes of β2M molecules allow easier diffusion and a close approach to

the latex substrate Second a reduced number of hydrophobic domains on the latex

particle surface replaced by sulfate groups (-OSO3-) and ionized carboxylic groups

(COO-) are not favorable for larger BSA molecules Third the conformational change by

intra- and inter-molecular repulsion would be faster in β2M due to smaller molecular

weight Finally the ionic repulsion between β2M and the latex particles is less than that

condition (pH 74) than IEP (48) of BSA

between BSA and latex particles because IEP (57) of β2M is closer to the tested

112

44 Blood Compatibility

Blood compatibility can be defined as the property of a material or device tha

permits it to function in contact with blood without inducing any adverse reactions

Successful functioning of blood contacting biomedical materials or devices in contact

with blood strongly depends on the extent to which they damage blood during operation

High shear stresses turbulence rela

t

tively long contact times and cellular impact with

artificial surfaces are most common events experienced by blood in contact with artificial

1978 Winslow and et al

1993] Therefore before any materia e placed into the human b

biocompatibility testing must be the first performed in the view of safety The hemolysis

s a method for pr ary screening of biomaterials to determine

h blood a er circulating body fluid The tests estimat

oglobin released from th blood cell d e induced

foreign material

There are several types of blood cells in our body but the ones that make blood red

are erythrocytes or red blood cells (RBCs) Red blood cells (erythrocytes) are the most

numerous type in the blood These cells are average about 48-54 million per cubic

millimeter (mm3 same as a microliter (microl)) of blood but these values can vary over a

larger range depending on such factors as health and altitude RBCs are red because they

contain hemoglobin which is a protein that contains iron When blood circulates through

lungs oxygen binds to the hemoglobin and is then delivered to all the parts of body

RBCs are made in the bone marrow They start out as a nucleus like other cells but

when they fill up with hemoglobin the nucleus becomes smaller until it disappears

Without a nucleus RBCs are fragile and live only about 120 days In a normal healthy

devises and can cause hemolysis or thrombosis [Bernhard et al

ls can b ody

test serves a elimin the

biocompatibility wit nd oth e the

concentration of hem e red amag by a

113

body about 2 million red blood ne marrow produces new

ones j ing

dy

le

cells die per second But bo

ust as fast RBCs are about 75 micrometer across and are responsible for carry

oxygen salts and other organic substances to the cells and tissues throughout our bo

In humans and most mammals the cells are concave (indented in the middle) and flexib

enough to squeeze through even the tiniest of blood vessels the capillaries

Figure 4-38 Image of red blood cells

(Sourcehttpwwwpbrchawaiiedubemfmicroangelarbc1htm)

ch

100 90 10 75 25 f

RBCs are among the first cells that come into contact with a foreign material

injected in the blood Therefore a hemolysis assay gives information about the

biocompatibility in the case of an in vivo application The concentrations of prepared

latex particles used for the tests in this research are 05 25 and 50 (ww) and the

incubation time under moderate shaking was 30 minutes at 37oC Test results are shown

in Figure 4-39 The amount of blood cells that ruptured when in contact with the PS latex

particles was largely increased from about 30 to 91 as the solid content of PS

increased from 05 to 50 However hemolysis by the core shell latex particles su

as PSPMMA PSPMMA PAA and PSPMMA PAA was less than 35 O

114

particular interest was that hemolysis caused by PSPMMA PAA particles at 50

solid concentration was less than 02 hemolysis the lowest value among the latex

particles The low hemolysis value indicates that the PSPMMA PAA core shell latex

particles are the most blood compatible at all solid contents Other core shell particles

such as PSPMMA and PSPMMA PAA also had excellent blood comp

90 10

90 10

100 75 25 atibility with

low RBS breakage

01 2 3

20

40

60

80

100

Hem

olys

is (

)

052550

Figure 4-39 He

PS PSPMMA100 PSPMMA PAA90 10

molysis caused by latex particles n=5

4PSPMMA75PAA25

CHAPTER 5 SUMMARY OF RESULTS CONCLUSIONS AND RECOMMENDATION FOR

FUTURE WORK

51 Summary of Results

The goal of this study was to synthesize monodisperse polymeric latex particles

with tailored properties and to investigate the fundamental interactions between the

synthesized latex particles and target proteins An understanding of the fundamental

mechanism of selective adsorption is strongly required in order to maximize the

separation performance of membranes made of these materials so that it may applicable

to hemodialysis therapy for end stage renal disease (ESRD) patients For the achievement

of above research goal several investigations have been performed Analysis of the

obtained data leads to several conclusions as discussed below

Monodisperse polystyrene seed latex particles were synthesized with the size

crosslinking agent to make the latex particles more hydrodynamically stable The

conversion of monomer to polymer was over 95 and seed latex particles are spherical in

shape and have smooth surface From these small seeds bigger latex particles as large as

800nm were synthesized using a semi-continuous seeded emulsion polymerization

process The particles with a mean diameter less than 500nm are very spherical in shape

and highly uniform in particle size distribution However as the particles grow larger

than 500nm in mean diameter they became non-spherical with an uneven surface This

irregularity of particle surface can be attributed to the non-homogeneous monomer

ranges between 126plusmn75 and 216plusmn53 nm Divinylbenzene (DVB) was used as a

115

116

swelling into the shell of a growing polymer which can be controlled by the factors such

as temperature agitation speed initiator feeding rate and surfactant amount required to

stabilize the colloidal system It was determ rfactant to monomer ratio

dec ic

acid (AA) monomers were intro rticle surfaces more

hydro

to

ting of a

orm

ration is seen between

3002

0 cm-

0cm-1

-1

03

ined that as the su

reased the mean particle diameter increased Methyl methacrylate (MMA) and acryl

duced to make the latex pa

philic than bare polystyrene particles The prepared core shell latex particles were

made of PMMA100 PMMA90PAA10 and PMMA75PAA25 PMMA100 is the PMMA

homopolymer shell on a PS core PMMA90PAA10 shell is a copolymer with a PMMA

PAA ratio of 90 to 10 by weight PMMA75PAA25 shell is a copolymer consis

PMMA to PAA ratio of 75 to 25 by weight The particle size of these core-shell

particles were prepared to be about 370nm in mean diameter

The successful synthesis of latex particles was confirmed using Fourier Transf

Infrared spectroscopy (FTIR) The C-H aromatic stretching vib

cm-1 and 3103 cm-1 for PS latex particles The C-H asymmetrical stretching and

symmetrical stretching vibration peaks of CH2 for all latex particles is seen at 290

1and 2850 cm-1 respectively There is a carbonyl (C=O) characteristic band at 173

wavenumber for PSPMMA core-shell latex particles The broad OH group peak at

3400 cm

100

can be seen in the spectroscopy of the particle with PMMAPAA shell

(PSPMMA PAA and PSPMMA PAA ) The peak intensity of OH group of

PSPMMA PAA was larger than that of PSPMMA PAA relatively due to more AA

monomer content in PSPMMA PAA than in PSPMMA PAA

In the zeta potential measurements polystyrene (PS) latex particles had negative

values between -291 mV and -599 mV in the phosphate buffer (PB) and between -2

90 10 75 25

75 25 90 10

75 25 90 10

117

mV and -278 mV in the phosphate buffered saline (PBS) at 25oC and pH 21-78 These

negative zeta potential values for PS latex particles are due to ionization of the sulfate

groups (-OSO3-) originated from the initiator Zeta potential values of the PSPMMA

core shell latex particles were also negative between -28 mV and -505 mV in PB and

between -143 mV and -186 mV in PBS at pH 21-78 Negative values are also

generated by dissociation of sulfate groups on a particle surface The zeta potential value

of PSPMMA PAA and PSPMMA PAA were of negative values between -36

and -678 mV in PB medium and -147 mV and -193 mV in PBS medium at 25

100

s

7 mV

MMA75PAA25 and

PSPM

otein

g the

l change of BSA at

each w

90 10 75 25

oC For

PSPMMA PAA core shell particles zeta potential values were between -291 mV and

-520 mV in PB media and between -115 mV and -210 mV in PBS media Sulfate

groups contributed to the negative zeta potential values in PSP

75 25

MA90PAA10 core shell particles Carboxylic groups from polyacrylic acid also

contribute to the negative surface charge for the latex particles at greater than pH 70

however the degree of their contribution was not detectable from the zeta potential

measurements The absolute zeta potential values of the synthesized latex particles in PB

were higher than that in PBS This is because of the high concentration of sodium

chloride electrolyte in PBS that compress electrical double layer thickness

The bicinchoninic acid (BCA) assay technique was used to determine the pr

adsorption and all isotherms were fitted to the Langmuir-Freundlich isotherms usin

nonlinear regression method The hydrophobic interaction between PS latex particle and

BSA protein was dominant for the adsorption process Conformationa

pH also affected the adsorption amount which was highest at pH 48 because of lo

ionic repulsion making compact conformation of BSA The next larger amount of BSA

118

adsorbed onto PS was at pH 32 There were two main forces affecting the amount of

adsorption at acidic pH values One is ionic attraction between negative latex particle a

positive protein The other is lateral repulsion between positive proteins The smallest

amount of BSA adsorption was at pH 74 where charge repulsion between negative

particles and proteins is present at this pH Lateral repulsion between proteins wa

dominant for protein adsorption even though there always exists the hydrophobic

interaction between solid latex particle and protein The charge repulsion was reduced by

the high concentration of electrolytes in the PBS medium The BSA adsorption process

on PSPMMA core shell particles was very similar to the case of PS latex particle where

hydrophobic interaction between latex particle and protein was dominant However t

adsorbed BSA amount was less than that of PS in PBS The adsorbed amount of BS

PSPMMA

nd

s

he

A on

an

ed

n The main

adsor

90PAA10 and PSPMMA75PAA25 core shell particles was higher at pH 32 th

pH 48 and 74 The adsorbed BSA amount became higher at pH 32 and pH 48 as the

hydrophilicity was increased by the higher PAA amount On the contrary the adsorb

amount of BSA was dramatically decreased at pH 74 Carboxylic acid groups as well as

the sulfate groups are distributed on the surface of PSPMMA90PAA10 and

PSPMMA75PAA25 core shell particles and largely affect the BSA adsorptio

ption interaction forces at pH 32 was generated by hydrogen bonding as well as

charge attraction between latex particle and protein even when the conformational change

and charge repulsion between proteins reduce the adsorption of protein at this pH The

adsorbed BSA amount was also increased as the amount of PAA increases However the

adsorption of BSA was minimal (less than 07 mgm2) at pH 74 where the carboxylic

groups are ionized leading to more enhancement of charge repulsion by ionization of the

119

carboxylic groups (COO-) Because of this the PSPMMA-PAA latex particles have a

much lower protein adsorption

There were steep initial slopes for adsorption isotherms of β

in

s

there

M

r

e

es

o

2M on latex particles

all isotherms indicating a high affinity type adsorption The complete plateau region

were not seen for all isotherms indicating that β2M proteins were not saturated and

are still available sites for β2M adsorption on latex particles The initial slope of β2

isotherms for PS was steeper than for the other latex particles PSPMMA100

PSPMMA90PAA10 and PSPMMA75PAA25

The layer thickness (δ) of BSA proteins on synthesized latex particles in phosphate

buffer (PB) was in the range of 27 - 81 Aring at 37 C and pH 48 Only the δ values at pH

48 the isoelectric point (IEP) of BSA are important because intra- and inter-molecular

electrostatic repulsion of the protein molecules is minimized at the IEP Compared to the

hydrodynamic dimensions of BSA 140 times 40 times 40 Aring these values of the adsorbed laye

thickness indicates that the BSA molecules adsorb by the side-on (40Aring) and end-on mod

(140Aring) Consideration of the multi-layering is unnecessary because the adsorbed

thickness of BSA do not exceed 140 Aring

The Gibbs free energy of β

o

3

2M was more negative in phosphate buffer (PB) than

that of BSA in physiological condition (37 C and pH 74) indicating that βo2M molecul

are more favorably adsorbed on the latex particles than BSA and that the affinity of β2M

was much larger than BSA This may be explained by the rate of diffusion and

accessibility of β2M molecules being easier and faster than that for BSA because of

smaller size (or weight) and spherical shape of β2M molecules It may also be possible t

explain that more hydrophobic forces (less electrostatic repulsion) contributes to

120

adsorption of β2M than to that of BSA because the IEP of β2M (57) is closer to

physiological condition (pH 74) than that of BSA (pH 48) The selective adsorption of

β2M m

in β2M

larger than in BSA adsorption indicating

that fa

an that between BSA and latex particles because

IEP (

ch as

Other

olecules over BSA molecules can be enhanced by developing more hydrophilic

PSPMMA90PAA25 and PSPMMA75PAA25 core shell latex particles

The adsorption kinetics of proteins on latex particles was expressed as a second-

order model The plateau region for β2M was reached earlier than BSA on all latex

particles and the K values (equilibrium rate constant of 2nd order adsorption)

adsorption process on latex particles were much

ster adsorption rate of β2M molecules than that of the BSA molecules There are

several explanations for these phenomena First the smaller size of β2M molecules can

more easily diffuse and approach to the latex substrate Second enhanced hydrophilic

domain on the latex particle surface by sulfate groups (SO4-) and ionized carboxylic

groups (COO-) is not favorable for larger BSA molecules but is for smaller β2M to be

anchored Third the conformational change by intra- and inter-molecular repulsion

would be faster in β2M due to lower molecular weight Finally the ionic repulsion

between β2M and latex particles is less th

57) of β2M is closer to the tested condition (pH 74) than IEP (48) of BSA

In the biocompatibility test the number of red blood cells (RBCs) ruptured by PS

latex particles ranged from 30 to 91 as the solid content of PS increased from 05 to

50 respectively However hemolysis of the other core shell latex particles su

PSPMMA100 PSPMMA90PAA10 and PSPMMA75PAA25 were less than 35 The

PSPMMA90PAA10 particles at all applied solid concentrations had less than 02

hemolysis and was the most blood compatible among the prepared latex particles

121

core shell particles such as PSPMMA100 and PSPMMA75PAA25 also had excelle

compatibility with low RBC breakage

52 Conclusions

The major conclusions from this study include

nt blood

bull microglobulin (β2M) over that of the bovine serum albumin (BSA) at pH 74

was achieved by controlling the PMMA and PAA ratio to optimize

e particles posses the most desirable requirements for use in a human dialysis

ns

icles

olecule

bull

Using engineered core shell latex particles selective adsorption of β2

physiological condition was demonstrated

bull The rate of adsorption of the β2M was also much higher than that of BSA This

hydrophobichydrophilic microdomain size

bull The core shell particles also exhibited excellent biocompatibility with blood Thes

membrane

53 Recommendation for Future Work

With the results and conclusions obtained through this study several suggestio

may be given for future work as follows

bull Competitive adsorption tests of whole blood proteins on synthesized latex partunder physiological condition

bull Preparation of latex particles with optimum hydrophobic hydrophilic microdomains to maximize selective adsorption and removal of toxin molecules

bull Membrane fabrication by suitable pore size design to discriminate a toxin mby size sieving as a function of particle layers

Evaluation of membrane performance such as flow rate in water and in simulated blood fluid (SBF)

bull Application for hemodialysis as well as other technologies such as a water remediation

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usible

dra

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An de J D (1985) Surface and interfacial aspects of biomedical polymer Plenum New York

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s L B (1988) Latex agglutination tests American Clinical Laboratoty News 7 2026

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n T Zingraff J Kuntz D Drueke T (1986) Dialysis related amyloidosis

r J W and Reeke G N (1985) 3 Dimension

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krishnan S McDonald C J Prudhomme R K and Carbeck J D (2004) Latex composite membranes structure and pro

S K Sawant S B Joshi J B and Pangarkar V G (1999) Methanol semembranes for separation of methanol-ethylene glycol mixtures by pe

P Margarit-Puri K Klapper M and Mullen K (2000) Polymerizable and

polymerization Macromolecules 33 7718-7723

CRC Baca Raton (FL)

Riceevans C A and Diplock A T (1993) Current status of antioxidant therapy FRadical Biology and Medicine 15 77-96

C P (1968a) Surface chemistry asp

C P (1968b) Surface chemistry relationships in emulsion polymerization Industrial and Engi

Ronco C Brendolan A Winchester J F Golds E Clemmer J Polaschegg H D Muller T E La Greca G and Levin N W (2001) First clinical experience wan adjunctive hemoperfusion device designed specifically to remove beta(2)

o C Ghezzi P M Hoenich N A Delfino P G (2001) Membranes and filters for hemodialysis Database 2001 Karger Basel

R (1995) Detection of oxidants in uremic plasma by electron spin resonance spectroscopy Kidney International 48 199-206

eben W Blumenstein M Bosch T Lysaght M J and Schmidt B (199

20 408-413

Samtleben W Gurland H J Lysaght M J Winchester J F (1996) Plasma exchanand hemoperfusion Kluwer Academic Dordrecht

137

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or

Saper M A Bjorkman P J and Wiley D C (1991) Refined structure of the human

Sarobe J and Forcada J (1998) Synthesis of monodisperse polymer particles with

Sarobe J Molina-Bolivar J A Forcada J Galisteo F and Hidalgo-Alvarez R e

Scharnagl N and Buschatz H (2001) Polyacrylonitrile (PAN) membranes for ultra-

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Shimizu N Sugimoto K Tang J W Nishi T Sato I Hiramoto M Aizawa S

abe H and Handa H (2000) high-performance affinity beads for identifying drug receptors Nature Biotechnology

Shirahama H and Suzawa T (1985) Adsorption of bovine serum albumin onto styrene

Shira nto 83-490

al of Medicine 21 888-892

s

ilure 25 419-430

o

of protein using bicinchoninic acid Analytical Biochemistry 150 76-85

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Hatakeyama M Ohba R Hatori H Yoshikawa T Suzuki F Oomori A Tanaka H Kawaguchi H Watan

18 877-881

acrylic acid copolymer latex Colloid and Polymer Science 263 141-146

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Smith P K Krohn R I Hermanson G T Mallia A K Gartner F H ProvenzanM D Fujimoto E K Goeke N M Olson B J and Klenk D C (1985) Measurement

W V and Ewart R H (1948) Kinetics of emulsi

138

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t adult immune thrombocytopenic purpura Blood 79 2237-2245

Solomon T W Fryhle C B (2000) Organic chemistry 7th Ed John Wiley amp Sons

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Sriniv characterizations of polymer with thier antithrombogenic characteristics Marcel Dekker New York

Suzaw ovine serum albumin on synthetic polymer lattices Journal of Colloid and Interface Science 78 266-268

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Taken itness of

9 10-14

412

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Tangp ion of chitosan films- effects of hydrophobicity on protein adsorption Carbohydrate

Trinh S E (2002) Crystal structure of monomeric human beta 2 microglobulin reveals clues to its

Trotta F Drioli E Baggiani C and Lacopo D (2002) Molecular imprinted polymeric membrane for naringin recognition Journal of Membrane Science 201

Soderquist M E and Walton A G (1980) Structural changes in proteins adsorbed on polymer surfaces Journal of Colloid and Interface Science 75 386-397

New York

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Tamai H Hasegawa M and Suzawa T (1989) Surface characterization of hydrophilic functional polymer latex particles Journal of Applied Polymer Science 38 403-

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Research 338 937-942

C H Smith D P Kalverda A P Phillips S E V and Radford

amyloidogenic properties Proceedings of the National Academy of Sciences of the United States of America 99 9771-9776

77-84

139

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1992)

f the American Society of Nephrology 11 2344-2350

38

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Wiec

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Watkins R W and Robertson C R (1977) Total internal reflection technique for examination of protein sdsorption Journal of Biomedical Materials Research 11915-9

Wavhal D S and Fisher E R (2002) Hydrophilic modification of polyethersulfone membr

uyzen J Foreman K Battistutta D Saltissi D and Fleming S J (1992) Effect of dialyzer reproce

12 29-36

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ms K M Arthur S J Burrell G Kelly F Phillips D W and Marshall T (2003)

140

Winchester J F Ronco C Brady J A Cowgill L D Salsberg J Yousha E Choquette M Albright R Clemmer J Davankov V Tsyurupa M PavlL Pavlov M Cohen G Horl W

ova Gotch F and Levin N W (2002) The next

step from high-flux dialysis application of sorbent technology Blood Purification

Winchester J P Ronco J A Brendolan A Davankov V Tsyurupa M Pavlova L 2001)

oglobin

Yang Q and Yang C Z (2000) Formation of an

Yoon J Y Park H Y Kim J H and Kim W S (1996) Adsorption of BSA on highly carboxylated microspheres- quantitative effects of surface functional groups

Zollars R L (1979) Kinetics of the emulsion polymerization of vinyl acetate Journal of

Zou oss-

Zou D Ma S Guan R Park M Sun L Aklonis J J and Salovey R (1992)

20 81-86

Clemme J Polaschegg H D Muller T E La Greca G Levin N W (Rationale for combined hemoperfusionhemodialysis in uremia Contribution of Nephrology 133 174-179

Winslow R M Vandergriff K D Motterlini R (1993) Mechanism of hemtoxicity Thrombosis and Haemostasis 70 36-41

S C Ge H X Hu Y Jiang Xpositively charged poly(butyl cyanoacrylate) nanoparticles stabilized with chitosColloid and Polymer Science 278 285-292

and interaction forces Journal of Colloid and Interface Science 177 613-620

Applied Polymer Science 24 1353-1370

D Derlich V Gandhi K Park M Sun L Kriz D Lee Y D Kim G Aklonis J J and Salovey R (1990) Model filled polymers 1 synthesis of crlinked monodisperse polystyrene beads Journal of Polymer Science Part A Polymer Chemistry 28 1909-1921

Model filled polymers 5 synthesis of cross-linked monodisperse polymethacrylate beads Journal of Polymer Science Part A Polymer Chemistry 30 137-144

Korea on August 7 1968 He obtained a Bachelor of Science degree in Chemistry from

Keim d graduate school and earned a

resear n Korea Institute of Science and

Coati e

ia

Busin

BIOGRAPHICAL SKETCH

Sangyup Kim was born and raised in the small town of Sung-Joo Kyungbook

yung University (Daegu Korea) in 1993 He entere

Master of Science degree in Chemical engineering in 1995 He worked as a student

cher for 2 years at Textile Polymer Division i

Technology (KIST) in Seoul Korea Next he worked over a year at the Automobile

ng Division in DongJoo-PPG in Korea In August 2001 he began studying for th

degree of Doctor of Philosophy in materials science and engineering at the University of

Florida in Gainesville After getting his PhD he plans to work at the Digital Med

ess Division of Samsung Electronics in Korea starting in January 2006

141

TABLE OF CONTENTS page

ACKNOWLEDGMENTS iv

LIST OF TABLES viii

LIST OF FIGURES x

1 INTRODUCTION 1

2 BACKGROUND AND LITERATURE SURVEY5

21 The Significance of End Stage Renal Disease (ESRD)5 22 Hemodialysis (HD) Treatment 6 23 Advances in Membrane Technology 8 24 Sorbent Technology16 25 Limitation of Current Hemodialysis Treatment 19 26 Latex Particles 20

261 The Components for Emulsion Polymerization 21 262 Particle Nucleation 21 263 Types of Processes for Emulsion Polymerization 23 264 Chemistry of Emulsion Polymerization 24 265 Seed Emulsion Polymerization25 266 Polystyrene Latex Particles 26 267 Various Applications of Latex Particles28

27 Proteins 30 271 Interaction Forces between Proteins33 272 β -Microglobulin (β M)2 2 35 273 Serum Albumin 36

28 Protein Adsorption38 281 Interaction between Protein Molecule and Latex Particle38

29 Hypothesis for Toxin Removal39 291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size 40 292 Toxin Removal by Selective Adsorption on Engineered Latex Particles 41

3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY 44

31 Materials 44 32 Latex Particle Preparation46

321 Preparation of Seed Latex Particles47

vi

322 Preparation of Seeded Latex Particles 48 323 Preparation of Core Shell Latex Particles 48 324 Purification of Synthesized Latex Particles48

33 Characterization49 331 Degree of Conversion49 332 Fourier Transform Infrared (FTIR) Spectroscopy49 333 Quasielastic Light Scattering (QELS) 50 334 Field Emission-Scanning Electron Microscopy (FE-SEM) 50 335 Zeta Potential Measurement 50 336 Protein Adsorption51 337 Blood Biocompatibility by Hemolysis Test 53

4 RESULTS AND DISCUSSION55

41 Polymerization of Latex Particles55 411 Polystyrene Seed Latex Particles55 412 The Growth of Polystyrene (PS) Seed Latex Particles61 413 Core Shell Latex Particles 71

42 Characterization of Latex Particles75 421 Fourier Transform Infrared Spectroscopy (FTIR)75 422 Zeta Potential Measurements 75

43 Protein Adsorption Study 85 431 Adsorption Isotherm87 432 Adsorbed Layer Thickness 103 433 Gibbs Free Energy of Protein Adsorption 104 434 Kinetics of Adsorption 107

44 Blood Compatibility 112

5 SUMMARY OF RESULTS CONCLUSION AND RECOMMENDATION FOR FUTURE WORK115

51 Summary of Results115 52 Conclusions121 53 Recommendation for Future Work121

LIST OF REFERENCES122

BIOGRAPHICAL SKETCH 141

vii

LIST OF TABLES

Table page 4-1 The polymerization recipe of polystyrene (PS) seed particles 57

4-2 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles less than 500nm in size 63

4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size64

4-4 The preparation recipe of PS core with various shell latex particles72

4-5 The isoelectric point (IEP) of common proteins 83

4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model 93

4-7 The equilibrium concentration values (q ) of BSA adsorption on PSPMMA particles calculated the Langmuir-Freundlich isotherm model

m 10096

4-8 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model

90 1098

4-9 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model

75 25100

4-10 The equilibrium concentration values of β M adsorption calculated the Langmuir-Freundlich isotherm model

2102

4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37 Co 104

4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37 Co 104

4-13 The values of Gibbs free energy change of BSA adsorption in phosphate buffer (PB) at 37 Co 105

4-14 The values of Gibbs free energy change of BSA adsorption in phosphate buffered saline (PBS) at 37 Co 105

viii

4-15 The values of Gibbs free energy change of β M adsorption in phosphate buffere (PB) at 37 C and pH 74

2o 106

4-16 Fitting parameters of second-order kinetic model 111

ix

LIST OF FIGURES

Figure page 1-2 Stereo drawing of the αndashcarbon backbone of β M2 2

2-1 An image of the location and cross section of a human kidney 5

2-2 A schematic draw of the hemodialysis route7

2-3 Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes 11

2-4 The chemical structure of polysulfone (PSf) 12

2-5 The chemical structure of polyamide (PA)13

2-6 Chemical structure of polyacrylonitrile (PAN) 14

2-7 Emulsion polymerization system24

2-8 L-α-amino acid 31

2-9 Ribbon diagram of human β M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ)

236

2-10 Secondary structure of human serum albumin (HSA) with sub-domains 37

2-11 The relationship between pore size and particle size in body-centered cubic and closed packed arrays41

2-12 Schematic representation of the protein adsorption on the core shell latex particles at pH 7442

3-1 Chemical structure of main chemicals45

3-2 Experimental setup for semi-continuous emulsion polymerization 46

3-3 The particle preparation scheme in various types and size ranges of latex particles 47

3-4 Schematic of the procedure for a protein adsorption test 52

3-5 Separation of RBC from whole blood by centrifuge process53

x

3-6 Schematic of the procedure for hemolysis test 54

4-1 Schematic representation of semi-continuous seed latex particles preparation and growth56

4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS (B) PS (C) PS (D) PSS259 S233 S207 S181 59

4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm 65

4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm 68

4-5 Dependence of the particle size on the surfactant to monomer ratio70

4-6 Schematic of core shell latex particle structures 71

4-7 Scanning Electron Micrograph of latex particles (A) PS (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA

100

90 10 75 25 73

4-8 FTIR spectra of polymerized latex particles (A) bare polystyrene (PS) (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA100 90 10 75 25 76

4-9 Schematic representation of ion distribution near a positively charged surface 78

4-10 Schematic representation of zeta potential measurement79

4-11 Zeta potential of PS latex particles at 25 Co 80

4-12 Zeta potential of PSPMMA latex particles at 25 C100o 81

4-13 Zeta potential of PSPMMA PAA latex particles at 25 C90 10o 82

4-14 Zeta potential of PSPMMA PAA latex particles at 25 C75 25o 82

4-15 The decay of surface potential with distance from surface in various electrolyte concentrations (1) low (2) intermediate (3) high 84

4-16 High-affinity adsorption isotherm of typical flexible polymer on solid surface 86

4-17 Overall schematic representation of the protein adsorption on the synthesized latex particles86

4-18 The dependence of UV absorbance on BSA concentration (A) 10mgml (B) 06mgml (C) 02mgml87

4-19 Standard curve of net absorbance vs BSA sample concentration 88

xi

4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37 C in phosphate buffer mediao 89

4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37 Co 90

4-22 Adsorption isotherm of BSA adsorption 37 C in phosphate buffered saline (PBS) on PS latex particles

o

91

4-23 Conformation of bovine serum albumin (BSA) on latex particles92

4-24 Adsorption isotherm of BSA 37 C in PB on PSPMMA core shell latex particles

o100

94

4-25 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA core shell latex particles

o100

95

4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA PAA core shell latex particles

o90 10

97

4-27 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA PAA core shell latex particles

o90 10

98

4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA PAA latex particleso75 25 99

4-29 Adsorption isotherm of BSA 37 C in PBS on PSPMMA PAA core shell latex particles

o75 25

99

4-30 Adsorption isotherm of β M onto PS and PSPMMA latex particles in PB at 37 C and pH 74

2 100o 101

4-31 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74

2 90 10o 101

4-32 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74

2 75 25o 102

4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37 C and pH 74

o

107

4-34 The kinetics of protein adsorption in PB on PS latex particles at 37 C and pH 74

o

109

4-35 The kinetics of protein adsorption in PB on PSPMMA latex particles at 37 C and pH 74

100o

109

4-36 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74

90 10o 110

xii

4-37 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74

75 25o 110

4-38 Image of red blood cells 113

4-39 Hemolysis caused by latex particles n=5114

xiii

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS

By

Sangyup Kim

December 2005

Chair Hassan El-Shall Major Department Materials Science and Engineering

Latex particles with well-defined colloidal and surface characteristics have received

increasing attention due to their useful applications in many areas especially as solid

phase supports in numerous biological applications such as immunoassay DNA

diagnostic cell separation and drug delivery carrier Hemodialysis membrane using

these particles would be another potential application for the advanced separation

treatment for patients with end stage renal disease (ESRD) It is desirable to remove

middle molecular weight proteins with minimal removal of other proteins such as

albumin Thus it is necessary to understand the fundamental interactions between the

particles and blood proteins to maximize the performance of these membranes This

improvement will have significant economic and health impact

The objective of this study is to synthesize polymeric latex particles of specific

functionality to achieve the desired selective separation of target proteins from the human

blood Semi-continuous seed emulsion polymerization was used to prepare monodisperse

xiv

polystyrene seed particles ranging from 126plusmn75 to 216plusmn53 nm in size which are then

enlarged by about 800nm Surfactant amount played a key role in controlling the latex

particle size Negatively charged latex particles with a different hydrophobicity were

prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid

monomer The prepared polymeric particles include bare polystyrene (PS) particles less

hydrophobic PS core and PMMA shell particles and more hydrophilic PS core and

PMMA-co-PAA shell latex particles with a 370nm mean diameter SEM light scattering

and zeta potential measurements were used to characterize particle size and surface

properties Adsorption isotherms of two proteins bovine serum albumin (BSA) and β2-

microglobulin (β2M) on latex particles were obtained as a function of pH and ionic

strength using the bicinchoninic acid (BCA) assay method The Langmuir-Freundlich

adsorption model was used to determine the adsorption amount of protein at equilibrium

The thickness of adsorbed BSA layer on latex particles was obtained in order to

investigate the adsorption orientation such as end-on or side-on mode Adsorption

kinetics experiments for both proteins and all latex particles were also performed The

adsorption kinetic constant determined from the Langmuir-Freundlich adsorption

isotherm model was used to calculate Gibbs free energy of adsorption to compare the

competitive adsorption of BSA and β2M Hemolysis tests were performed to investigate

the blood compatibility of synthesized latex particles PSPMMA90PAA10 and

PSPMMA75PAA25 core shell latex particles had desirable material properties with not

only a large amount and high rate of selective β2M adsorption over BSA but also high

blood compatibility showing less than 3 hemolysis

xv

CHAPTER 1 INTRODUCTION

End stage renal disease (ESRD) is a chronic condition in which kidney function is

impaired to the extent that the patientrsquos survival requires removal of toxins from the

blood by dialysis therapy or kidney transplantation The National Kidney Foundation

estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF] The

number of people with ESRD is rapidly increasing in the United States with

approximately 96 295 incidents and 406 081 prevalent patients including 292 215 on

dialysis and 113 866 with a functioning graft in 2001 It is projected that there will be

more than 22 million ESRD patients by 2030 [USRDS 2003] The expenditure for the

ESRD treatment program had reached $228 billion 64 of the Medicare budget in

2001 Due in part to the limited availability of kidneys for transplantation hemodialysis

is the primary clinical treatment for the patients with ESRD

The central element of a hemodialysis instrument is the semipermeable membrane

that allows for selective transport of low molecular weight biological metabolites less

than 5000 Da such as urea and creatinine as well as excess water and electrolytes [Baker

2004] from the blood One limitation of current dialysis technologies is the inability to

efficiently remove middle molecular weight toxins such as β2-Microglobulin (β2M) and

interleukin 6 (IL-6)

β2M is a causative protein of dialysis-related amyloidosis (DRA) a disease arising

in patients with chronic kidney failure as a serious complication of long-term

hemodialysis treatment [Gejyo et al 1985] β2M deposition in tissue is the primary cause

1

2

of destructive arthritis and carpal tunnel syndrome [Vincent et al 1992 Drueke 2000]

The β2M structure is shown in Figure 1-2

Figure 1-2 Stereo drawing of the αndashcarbon backbone of β2M [Becker et al 1985]

Although attempts have been made to increase the efficiency of middle molecular

weight toxin removal by changes in the membrane pore size and the use of innovative

materials to adsorb these toxins [Samtleben et al 1996 Ronco et al 2001] removal

efficiency is not as high as those achieved by a normal healthy kidney Traditional

membranes have a number of processing and performance limitations [Westhuyzen et al

1992 Leypoldt et al 1998] such as a restricted choice of surface chemistries and limited

control of porosity The development of novel engineering membrane technology is

needed to remove middle molecule toxins

Polymeric latex particles have received increasing attention in medical application

areas especially as solid phase supports in biological applications [Piskin et al 1994]

Examples of these applications include immunoassay [Chen et al 2003 Radomske-

Galant et al 2003] DNA diagnostic [Elaїssari et al 1998] cell separation drug delivery

carrier [Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] etc This is because of

3

the well-defined colloidal and surface characteristics of the particles By using a seed

emulsion polymerization method it is possible to synthesize monodisperse latex particles

with various particle size ranges and surface chemistry Functionalized core-shell latex

particles can be introduced by multi-step emulsion polymerization Core particles are

synthesized in the first stage of the polymerization and the functional monomer is added

in the second stage This is done without any emulsifier addition to prevent the

production of new homopolymer particles [Keusch et al 1973] The core-shell particles

are useful in a broad range of applications because of their improved physical and

chemical properties over their single-component counterparts [Lu et al 1996 Nelliappan

et al 1997] Through the development of a hemodialysis membrane using monodisperse

latex particles improvements in advanced separation treatment for patients with end

stage renal disease (ESRD) can be realized This requires the maximum removal of

middle molecular weight proteins with minimal removal of other beneficial proteins such

as albumin Thus an understanding of the fundamental interactions between the particles

and biopolymers is vital to maximize the performance of this membrane technology

The field of material science and biotechnology is based on fundamental chemistry

has developed over the past three decades into todayrsquos powerful discipline that enables

the development of advanced technical devices for pharmaceutical and biomedical

applications This novel and highly interdisciplinary field is closely associated with both

the physical and chemical properties of organic and inorganic particles [Niemeyer 2001]

Hemodialysis membrane using these particles would lead to improvements in the

advanced separation treatment for patients with end stage renal disease (ESRD) The

interdisciplinary nature of this approach enables a more complete understanding of the

4

phenomena of protein adsorption and the material properties necessary for selective

separation This innovative approach to membrane fabrication has the potential of making

inexpensive highly efficient membranes for both industrial and specialized separation

processes

The goal of this study is to prepare polymeric latex particles with tailored

properties to maximize separation of toxin molecules and to investigate the fundamental

interactions between the particles and molecules in the biological system in order to

optimize the performance of a membrane material for these applications

Polymeric latex particles were synthesized with specific functionality in an attempt

to achieve selective separation Seeded emulsion polymerization was used to synthesize

functionalized monodisperse latex particles in various sizes Negatively charged

hydrophobic polystyrene latex particles were synthesized by the same method Core shell

latex particles PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25 were also

synthesized to differentiate the degree of hydrophobicity of particles Scanning Electron

Micrograph (SEM) and light scattering measurements were used to characterize particle

size and shape and zeta potential measurements were conducted to measure the electrical

surface property of synthesized particles Adsorption isotherms of target proteins bovine

serum albumin (BSA) and β2M on latex particles were obtained as a function of pH

ionic strength and protein concentrations using the BCA assay method Adsorption

kinetics for both proteins on the latex particles were also measured Finally hemolysis

tests were run to determine the biocompatibility of polymer latex particles with human

blood This research will be described in more detail in chapters 3 and 4

CHAPTER 2 BACKGROUND AND LITERATURE SURVEY

21 The Significance of End Stage Renal Disease (ESRD)

The kidneys are responsible for removing excess fluid minerals and wastes from

the blood regulating electrolyte balance and blood pressure and the stimulation of red

blood cell production They also produce hormones such as erythropoietin (EPO) and

calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005 Kurbel

et al 2003] EPO acts on the bone marrow to increase the production of red blood cell in

case of bleeding or moving to high altitudes Calcitriol mainly acts on both the cells of

the intestine to promote the absorption of calcium from food and also bone to mobilize

calcium from the bone to the blood When kidneys fail harmful waste builds up blood

pressure rises and body retains excess fluid The body also does not make enough red

blood cells When this happens treatment is needed to replace the function of failed

kidneys

Figure 2-1 An image of the location and cross section of a human kidney

5

6

The National Kidney Foundation estimates that over 20 million Americans had

chronic kidney disease in 2002 [NKF] Chronic renal disease is a gradual and progressive

loss of the function unlike acute renal disease where sudden reversible failure of kidney

function occurs Chronic renal failure usually takes place over a number of years as the

internal structures of the kidney are slowly damaged In the early stages there can be no

symptoms In fact progression may be so gradual that symptoms do not occur until

kidney function is less than one-tenth that of a normal health kidney If left untreated

chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal

Disease (ESRD) ESRD is a rapidly growing heath-care problem in the United States In

2001 approximately 96295 incidents and 406081 prevalent patients were diagnosed

with ESRD including 292215 patients on dialysis and 113866 patients with a

functioning graft [USRDS 2003] The projected number of patients with ESRD is

expected to exceed 22 million patients by 2030 with much of this growth being driven by

the increasing prevalence of major contributing factors such as diabetes and high blood

pressure [USRDS 2003] A great extent of ESRD program cost and Medicare budget

have been spent in 2001 Due in part to a limited availability of kidneys for

transplantation hemodialysis (HD) is the primary method of treatment for ESRD and is

currently used for approximately 61 of US ESRD patients

22 Hemodialysis (HD) Treatment

HD removes toxins from the body by extracorporeal circulation of the blood

through a semipermeable membrane referred to as a dialyzer The toxins are removed

primarily by diffusion across the membrane to a dialysate solution which is circulated on

the opposite side of the membrane The cleaned blood is then returned to the blood

stream A surgically constructed vascular access connects the extracorporeal circuit to the

7

patientrsquos system Treatments are almost always performed three times per week in

specially equipped dialysis facilities for 3-4 hours per each treatment Figure 2-2 shows

the scheme of the hemodialysis route The critical element of a HD instrument is the

semipermeable membrane which allows for selective transport of low molecular weight

biological metabolites from blood

igure 2-2 A schematic draw of the hemodialysis route

ESRD patients who undergo dialysis therapy often experience several other

problems associated with the treatment such as anemia fatigue bone problems joint

problems itching sleep disorders and restless legs Anemia is common in patient with

kidney disease because the kidneys produce the hormone erythropoietin (EPO) which

stimulates the bone marrow to produce red blood cells Diseased kidneys often do not

produce enough EPO to stimulate the bone marrow to make a sufficient amount of red

blood cells This leads to bone disease referred to as renal osteodystrophy and causes

bones to become thin and weak or malformed and can affect both children and adults

Older patients and women who have gone through menopause are at greater risk for this

disease Uremic toxins which cannot be remove from the blood by the current dialyzer

membranes can lead to itching This problem can also be related to high levels of

F

8

parathyroid hormone (PTH) Dialysis patients can also experience day-night revers

is they have insomnia at night and sleep during the day This can be related to possible

nerve damage in the body and a chemical imbalance in the blood due to the excess toxin

Oxidative stress is a problem for patients on maintenance dialysis This problem is due to

an imbalance between pro- and antioxidant factors [Roselaar et al 1995 Cristol et al

1994] Oxidative stress affects oxidation of low-density lipoproteins which are the main

factor for atherogenesis [Huysmans et al 1998] and is also involved in the development

of malignancies and diabetes mellitus [Rice-Evans et al 1993] In order to reduce

antioxidant defense dialysis is needed to contribute to help stimulate free radical

production or eliminate antioxidants Dialysis-related amyloidosis (DRA) is also a

common and serious problem for people who have been on dialysis for more than 5

years DRA develops when proteins in the blood deposit on joints and tendons causi

pain stiffness and fluid in the joint as is the case with arthritis Normally the healthy

kidneys can filter out these proteins but dialysis filters are not as effective

23 Advances in Membrane Technology

al that

s

ng

Dialysis for blood of ESRD

Hemo filtration

s

ysis using a cellulose based membrane occurred

1913 John Abel [1990] from the Johns Hopkins Medical School described a method

purification is widely used in the treatment

dialysis (HD) techniques use a semi-permeable membrane to replace the

role of the kidney The membranes used in HD can be broadly classified into those based

on cellulose and those manufactured from synthetic copolymers These membranes come

in various shapes such as sheets tubular structures or hollow fiber arrangements The

hollow fiber type is the most popular and is incorporated into over 800 different device

in world wide [Ronco et al 2001]

The first attempt at blood dial

9

where sing

e

om

ysis was performed until the mid 1960 The basic

molec

rial in

een

benzyl)

was used for the membrane with

hepar

by the blood of a living animal may be submitted to dialysis outside the body u

a membrane based on cellulose and returned to the natural circulation without exposur

to air infection by microorganisms or any alteration that would necessarily be prejudicial

to life This same technique is still used to today however the device used has been

modified over the years as better membranes were developed and the anti-coagulant

heparin has become available

Cellulose membranes have been widely used for the treatment of renal failure fr

1928 when the first human dial

ular structure of cellulose is made of a long chain containing hydroxyl (OH)

groups The realization that such groups imparted undesirable qualities on the mate

respect to blood contact behavior was discovered in the early 1970s and since has b

the focus of development These modified cellulose membranes used the partial

substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt

to reduce their negative effect The result is a molecular mosaic of hydrophobic (

and hydrophilic (hydroxyl and cellulose) regions

Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients

with acute renal failure in 1943 Cellophane tubing

in as the anticoagulant For the next 17 years hemodialysis therapy was performed

by this method but only for the patients with acute reversible renal failure Vascular

access required repeated surgical insertions of cannulas (slender tubes) into an artery and

vein and limited the number of treatments for a patient could receive in order to

minimize the amount of vascular damage

10

Initially the need for dialysis in patients with acute renal failure was determ

mainly by the development of signs and sym

ined

ptoms of uremia After dialysis some time

might

et al

ent improvement In 1960 the development of a shunt [Quinton et al

1960]

fone (PSf) polyamide (PA) and polyacrylonitrile (PAN) by phase inversion or

precip

lidone

e

elapse before uremic manifestations returned to warrant a sequential treatment of

dialysis Many patients with acute renal failure secondary to accidental or surgical

trauma were hypercatabolic but the interdialytic interval might be prolonged because of

anorexia or use of a low-protein diet However Teschan and his coworkers [Obrien

1959] showed that patient well-being and survival were improved by what they termed

prophylactic daily hemodiaysis or administration of the treatment before the patient

again became sick with uremia Their report in 1959 was the first description of daily

hemodialysis

Development of membrane accessories such as a shunt has also been an area of

focus for treatm

a flexible polytetrafluoroethylene (PTFE or Teflonreg) tubing made many more

hemodialysis treatments possible for chronic kidney failure patients PTFE has a non-

stick surface and is relative biocompatibility leading to minimized blood clotting in the

shunt

Synthetic membranes are prepared from engineered thermoplastics such as

polysul

itation of a blended mixture resulting in the formation of asymmetric and

anisotropic structures Figure 2-3 shows a fiber type of the Polyflux S membrane

consisting of poluamide (PA) polyacrylethersulfone (PAES) and polyvinylpyrro

(PVP) with the integral three-layer structure The skin layer on the inside fiber typ

membrane contacts blood and has a very high surface porosity and a narrow pore size

11

distribution This layer constitutes the discriminating barrier deciding the permeabili

and solute retention properties of the membrane The skin layer is supported by thick

sponge-type structure larger pores providing mechanical strength and very low

hydrodynamic resistance

ty

Figure 2-3 Cross-sectional SEM image view of the Polyflux S (polyamide +

polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch et al 1998]

n

[Malchesky 2004] because of its thermal stability mechanical strength and chemical

inertn ciated

ls

m

PSf is a widely used membrane material for the hemodialysis applicatio

ess According to a report from the National Surveillance of Dialysis-Asso

Disease (NSDAD) in the US over 70 of hemodialysis membranes were PSf based

[Bowry 2002] This is most likely because PSf has many advantages over other materia

This synthetic polymer is one of few materials that can withstand sterilization by stea

ethylene oxide and γ-radiation PSf membrane can be prepared by conventional

immersion precipitation methods into many different shapes including porous hollow

12

fiber or flat sheet hemodialysis membranes The material also has a high permeab

low molecular weight proteins and solute and high endotoxin retention The chemical

structure of PSf is shown in Figure 2-4

ility to

Figure 2-4 The chemical structure of polysulfone (PSf)

There is one major disadvantage to PSf The hydrophobic nature of the PSf causes

ment alternative pathway

leadin

s not

3

et al

in

O acrylate

serious complications through the activation of the comple

g to the adsorption of serum proteins onto the membranes [Singh et al 2003]

Anticoagulants are added during dialysis therapy to avoid blood clotting but this doe

completely eliminate the problem In order to overcome this disadvantage of the PSf

membrane various studies have been performed to change the materialrsquos surface

properties These investigations include hydrophilic polymer coating [Brink et al 199

Kim et al 1988 Higuchi et al 2003] layer grafting onto PSf membrane [Wavhal

2002 Song et al 2000 Pieracci et al 2002 Mok et al1994] and chemical reaction of

hydrophilic components onto the membrane surface [Higuchi et al 1990 1991 1993

Blanco et al 2001 Nabe et al 1997 Guiver et al 1993] Hydrophilic monomers 2-

hydroxy-ethylmethacrylate (HEMA) acrylic acid (AA) and methacrylic acid (MMA)

have also been grafted onto PSf membrane to increase flux and Bovine Serum Album

(BSA) retention [Ulbricht et al 1996] Hancock et al [2000] synthesized

polysulfonepoly(ethylene oxide) (PEO) block copolymers to improve the resistance to

platelet adhesion Kim et al [2003] also studied blending a sulfonated PE

diblock copolymer into PSf in order to reduce platelet adhesion and enhance

13

biocompatibility PEO is a commonly used biomaterial due to its excellent resistance

protein adsorption and inherent biocompatibility [Harris 1992] Kim et al [20

a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf

membrane with an ultra-thin skin layer The polymer had an entrapped diblock

copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-SO

to

05] studied

ate

lic graft

l)

e of thier

aromatic

orand

3 acryl

and a hydrophobic block of octadecylacrylate (OA) Molecular dynamic (MD)

simulations were performed as a function of copolymer density to optimize interfacial

structure information McMurry [2004] developed a strategy using an amphiphi

copolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glyco

When compared to unmodified PSf these graft copolymer and resulting blend

membranes are found to hold promise for biomedical device applications

Polyamide (PA) membranes have also been used for hemodialysis becaus

mechanical strength in both wet and dry conditions Polyamide consists of

aliphatic monomers with amide bonding (-CONH-) also known as a peptide bond

The basic amide bond in polyamide is shown in Figure 2-5 R1 and R2 can be either

aromatic or aliphatic linkage group

C N

H

O

~ R R1 2~C N

H

O

~ R R1 2~

Figure 2-5 The chemical structure of polyamide (PA)

Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane

ompatible technique due to the

use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of

and concluded that PA hemofiltration was a highly bioc

14

the ac

e

e

is

n

is a semi-crystalline polymer and the mechanical properties

strong

2

CN

tivated anaphylatoxins and β2-Microglobulin (β2M) The PA based membrane

Polyfluxreg (manufactured by Gambro GmbH Germany) blended with polyamide

polyarylethersulfone and polyvinylpyrrolidone (PVP) was able to clean small molecules

such as urea dreatinine and phosphate as well as decrease β2M amount by 502

[Hoenich et al 2002] Due to the non-selectivity of the membrane removal of thes

unwanted materials also led to the undesirable loss of beneficial proteins during therapy

Meier et al [2000] evaluated different immune parameters using a modified cellulos

low-flux hemophan and synthetic high-flux PA membrane during a 1 year period in

chronic hemodialysis patients They found that the 1-year immunological evaluation of

hemodiaysis membrane biocompatibility was associated with changes in the pattern of

chronic T-cell actiovation

Polyacrylonitrile (PAN) is another commonly used membrane material because it

inherently hydrophilic and has been commercialized for ultrafiltration and microfiltratio

[Scharnagl et al 2001] PAN

ly depend on the crystalline structures The chemical structure of PAN is shown in

figure 2-6

CH CH

n2

CN

CH CH n

Chemical structure of polyacrylonitrile (PAN)

The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming

agent gives PAN membranes more flexible processing parameters and increased

e has been optimized through

copolymerization with many other vinyl monomers including glycidyl methacrylate

Figure 2-6

performance [Jung et al 2005] PAN membrane performanc

15

[Godj

acid

for

for

a non-

solub this

in

oid underneath the inner

surface of membrane decreased by increasing the amount of solvent DMSA in the

evargova et al 1999 Hicke et al 2002] N-vinylimidazole [Godjevargova et al

2000] hydroxyl ethyl methacrylate [Ray et al 1999 Bhat et al 2000] methacrylic

[Ray et al 1999] vinyl pyrrolidone [Ray et al 1999] acrylic acid [Trotta et al 2002]

acrylamide [Musale et al 1997] and vinylchloride [Broadhead et al 1998] These

monomers provide a reactive group for enzyme immobilization improved mechanical

strength solvent-resistance pervaporation permeation flux anti-fouling and bio-

compatibility Because of this PAN-based copolymer membranes have great potential

the treatment of hemodialysis in an artificial kidney This material can also be used

other applications like the treatment of wastewater the production of ultra-pure water

biocatalysis together with separation and methanol separation by pervaporation

Lin and his coworker [2004] studied the modification of PAN based dialyzer

membranes to increase the hemocompatibility by the immobilization of chitosan and

heparin conjugates on the surface of the PAN membrane When a foreign material is

exposed to blood plasma proteins are adsorbed clotting factors are activated and

le fibrin network or thrombus is formatted [Goosen et al 1980] The result of

research was that the biocompatible chitosan polymer and a blood anticoagulant hepar

prevented blood clotting They showed prolonged coagulation time reduced platelet

adsorption thrombus formation and protein adsorption

Nie et al [2004] studied PAN-based ultrafiltration hollow-fiber membranes

(UHFMs) In order to improve the membrane performance acrylonitrile (AN) was

copolymerized with other functional monomers such as maleic anhydride and α-allyl

glucoside They found that the number and size of macrov

16

intern erum

and

d a

rface The second step is the

transf after

yjak et

embrane

1978 Malchesky et al 1978] has been further developed to increase the efficiency of

al coagulant The water flux of the UHFMs also decreased while the bovine s

albumin rejection increased minutely Godjevargova et al [1992] modified the PAN

based membrane with hydroxylamine and diethylaminoethylmethacrylate to improve

membrane dialysis properties Formed functional groups like primary amine oxime

tertiary amine groups provided the membrane with more hydrophilic properties an

substantial increase in the permeability of the membranes

The wide use of filtration in practice is limited by membrane fouling Solute

molecules deposit on and in the membrane in the process of filtration causing dramatic

reduction in flux through the membrane Fouling occurs mostly in the filtration of

proteins Three kinetic steps are involved in the fouling of UF membranes according to

Nisson [1990] The first step is the transfer of solute to the su

er of solute into the membrane until it either finally adsorbs or passes through

a set of adsorption-desorption events The third step includes surface binding

accompanied by structural rearrangement in the adsorbed state [Ko et al 1993] Br

al [1998] studied the surface modification of a commercially available PAN membrane

to develop superior filtration properties with less fouling by proteins The PAN

membrane was immersed in excess NaOH solution to convert some of the surface nitrile

groups into carboxylic groups by the hydrolysis process This modified PAN m

was not so severely fouled in the Bovine Serum Albumin (BSA) filtration test The pore

size however decreased during the hydrolysis process leading to a significant reduction

in flux and made the membrane less productive in the ultrafiltration (UF) mode

24 Sorbent Technology

Over the last three decades sorbent technology [Castino et al 1976 Korshak et al

17

dialysis or replace it for the treatment of ESRD Sorbents remove solutes from solution

through specific or nonspecific adsorption depending on both the nature of the solute and

the sorbent Specific adsorption contains tailored ligands or antibodies with high

selectivity for target molecules een used in autoimmune

disord

and

e

al 1996] various resins [Ronco et al 2001]

album

e

as

Specific adsorbents have b

ers such as idiopathic thrombocytopenic purpura [Snyder et al 1992] and for the

removal of lipids in familial hyper cholesterolemia [Bosch et al 1999] Nonspecific

adsorbents such as charcoal and resins attract target molecules through various forces

including hydrophobic interactions ionic (or electrostatic) attraction hydrogen bonding

and van der Waals interactions

New dialysate with sorbents has become an accepted modification of dialysis

sorbent hemoperfusion is gaining ground as a valuable addition to dialysis especially as

new sorbents are developed [Winchester et al 2001] Hemoperfusion is defined as th

removal of toxins or metabolites from circulation by the passing of blood within a

suitable extracorpoteal circuit over semipermeable microcapsules containing adsorbents

such as activated charcoal [Samtleben et

in-conjugated agarose etc Novel adsorptive carbons with larger pore diameters

have been synthesized for potential clinical use [Mikhalovsky 1989] Newly recognized

uremic toxins [Dhondt et al 2000 Haag-Weber et al 2000] have resulted in several

investigations on alternatives to standard or high-flux hemodialysis to remove thes

molecules These methods include hemodiafilteration with [de Francisco et al 2000] or

without [Ward et al 2000 Takenaka et al 2001] dialysate regeneration using sorbents

well as hemoperfusion using such adsorbents as charcoal and resins

18

Kolarz et al [1989 1995] studied the hyper-crosslinked sorbent prepared from

styrene and divinylbenzene (DVB) for a hemoperfusion application They found that the

pore structure of a swelling sorbent was changed by additional crosslinking with α αrsquo-

dichloro-p-xylene in the presence of a tin chloride catalyst and in a di

chloroethane

soluti and

en

decyl groups that attract β2M through a hydrophobic interaction The

adsor

of

5]

d

penet

on They also realized that the hemocompatibility was useful for nemoperfusion

could be imparted to the sorbents by introducing sulfonyl groups at a concentration of

about 02mmolg

A special polymeric adsorbing material (BM-010 from Kaneka Japan) has be

investigated by another group [Furuyoshi et al 1991] for the selective removal of β2M

from the blood of dialysis partients The adsorbent consists of porous cellulose beads

modified with hexa

ption capacity of this material is 1mg of β2M per 1ml of adsorbent Using a

hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a

high-flux hemodialyzer several small clinical trials were performed During 4-5 hours

treatment about 210mg of β2M were removed thus reducing the concentration in the

blood by 60-70 of the initial level [Nakazawa et al 1993 Gejyo et al 1993 199

RenalTech developed a hemoperfusion device BetaSorbreg containing the hydrate

cross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure

designed to remove molecules between 4 and 30 kDa [Winchester et al 2002] In this

case solute molecules are separated according to their size based on their ability to

rate the porous network of the beaded sorbents The resin beads were prepared with

a blood compatible coating and confirmed to be biocompatible in vivo in animals

[Cowgill et al 2001]

19

25 Limitation of Current Hemodialysis Treatment

Hemodialysis is a widely used life-sustaining treatment for patients with ESRD

However it does not replace all of the complex functions of a normal healthy kidne

a result patients on dia

y As

lysis still suffer from a range of problems including infection

accelerated cardio ion anemia

chron

e

se middle

alysis

sessio

s

f

vascular disease high blood pressure chronic malnutrit

ic joint and back pain and a considerably shortened life span One significant

limitation of the current dialysis technology is the inability to efficiently remove larger

toxic molecules This is mainly because of the broad pore size distribution reducing th

selective removal of toxins and unsatisfied biocompatibility causing lots of

complications such as inflammation blood clotting calcification infection etc

Dialysis purifies the patients blood by efficiently removing small molecules like

salts urea and excess water However as toxic molecules increase in size their removal

rate by hemodialysis substantially declines Typically only 10 - 40 of the

molecular weight toxins (300-15000 Da) are removed from the blood during a di

n [Vanholder et al 1995] These toxins then reach an abnormally high level and

begin to damage the body One such toxin β2M causes destructive arthritis and carpal

tunnel syndrome by joining together like the links of chain to form a few very large

molecules and deposit damaging the surrounding tissues [Lonnemann et al 2002] This i

also a main cause of mortality for long-term dialysis patients Other middle molecule

toxins appear to inhibit the immune system and may play a significant role in the high

susceptibility to infections in dialysis patients Still others are believed to impair the

functioning of several other body systems such as the hematopoietic and other endocrine

systems This may contribute to accelerated cardiovascular disease the leading cause o

20

death among dialysis patients as well as clinical malnutrition which affects up to 50 of

this patient population

Over the last decade polymeric dialysis membranes have been developed to

increase the capacity for removing middle molecular weight toxins by changing the pore

size of dialyzer membranes and using new materials that adsorb these toxins for

impro se

styrene made during World War II in the United

States The Dow chemical compan anufacturer of polystyrene

includ

d of

on

rug

ntrol of

ved removal characteristics However removal efficiency is not as high as tho

achieved by a normal healthy kidney

26 Latex Particles

The first synthetic polymer synthesized using emulsion polymerization was a

rubber composed of 13-butadiene and

y has been a major m

ing latex which they used in paint formulations The theory of emulsion

polymerization in which a surfactant is used was established by Harkins [1948] and by

Smith and Ewart [1948] By 1956 the technology was complete including the metho

building larger diameter particles from smaller ones The product by this emulsi

polymerization is referred to as latex a colloidal dispersion of polymer particles in water

medium [Odian 1991] Latexes are currently undergoing extensive research and

development for a broad range of areas including adhesives inks paints coatings d

delivery systems medical assay kits gloves paper coatings floor polish films carpet

backing and foam mattresses to cosmetics The relatively well known and easy co

the emulsion process is one of main advantages for these applications Therefore

polymeric latex particle prepared by emulsion polymerization can be a candidate for the

medical applications because of the easy control of the particle size and morphology as

well as flexible surface chemistry to be required

21

261 The Components for Emulsion Polymerization

The main components for emulsion polymerization process are the monomer a

dispersing medium a surfactant and an initiator Available monomers are styrene

sing medium is usually water

which

ticles

n

ation To

monomer droplets but in micelles because the initiators are

insolu le

and

olution

butadiene methylmethacylate acryl acid etc The disper

will maintain a low solution viscosity provide good heat transfer and allow

transfer of the monomers from the monomer droplets into micelles and growing par

surrounded by surfactants respectively The surfactant (or emulsifier) has both

hydrophilic and hydrophobic segments Its main functions is to provide the nucleatio

sites for particles and aid in the colloidal stability of the growing particles Initiators are

water-soluble inorganic salts which dissociate into radicals to initiate polymeriz

control the molecular weight a chain transfer agent such as mercaptan may be present

262 Particle Nucleation

Free radicals are produced by dissociation of initiators at the rate on the order of

1013 radicals per milliliter per second in the water phase The location of the

polymerization is not in the

ble in the organic monomer droplets Such initiators are referred to as oil-insolub

initiators This is one of the big differences between emulsion polymerization

suspension polymerization where initiators are oil-soluble and the reaction occurs in the

monomer droplets Because the monomer droplets have a much smaller total surface area

they do not compete effectively with micelles to capture the radicals produced in s

It is in the micelles that the oil soluble monomer and water soluble initiator meet and is

favored as the reaction site because of the high monomer concentration compared to that

in the monomer droplets As polymerization proceeds the micelles grow by the addition

of monomer from the aqueous solution whose concentration is refilled by dissolution of

22

monomer from the monomer droplets There are three types of particles in the emulsion

system monomer droplets inactive micelles in which polymerization is not occurring

and active micelles in which polymerization is occurring referred to as growing polymer

particles

The mechanism of particle nucleation occurs by two simultaneous processes

micellar nucleation and homogeneous nucleation Micellar nucleation is the entry of

radicals ei

ther primary or oligomeric radicals formed by solution polymerization from

the aq ed

the

r

s

n

is coagulation with other particles rather than

polymerization of monomer The driving force for coagulation of precursor particles

ueous phase into the micelles In homogeneous nucleation solution-polymeriz

oligomeric radicals are becoming insoluble and precipitating onto themselves or onto

oligomers whose propagation has ended [Fitch et al 1969] The relative levels of micella

and homogeneous nucleation are variable with the water solubility of the monomer and

the surfactant concentration Homogeneous nucleation is favored for monomers with

higher water solubility and low surfactant concentration and micellar nucleation is

favored for monomers with low water solubility and high surfactant concentration It has

also been shown that homogeneous nucleation occurs in systems where the surfactant

concentration is below CMC [Roe 1968] A highly water insoluble monomer such a

styrene [Hansen et al 1979 Ugelstad et al 1979] has probably created by micellar

nucleation while a water soluble monomer such as vinyl acetate [Zollars 1979] has bee

formed by homogeneous nucleation

A third latex formation reaction mechanism has been proposed referred to as

coagulative nucleation In this reaction the major growth process for the first-formed

polymer particles (precursor particles)

23

sever les

yer

es

r a

l

the beginning of the polymerization As soon as the initiator is

gins with the formation and

growt

and

ved

added at one time during the later stages of the polymerization prior to complete

al nanometers in size is their relative instability compared to larger sized partic

The small size of a precursor particle with its high curvature of the electrical double la

permits the low surface charge density and high colloidal instability Once the particl

become large enough in size maintaining the high colloidal stability there is no longe

driving force for coagulation and further growth of particles takes place only by the

polymerization process

263 Types of Processes for Emulsion Polymerization

There are three types of production processes used in emulsion polymerization

batch semi-continuous (or semi-batch) and continuous In the batch type process al

components are added at

added and the temperature is increased polymerization be

h of latex particles at the same time There is no further process control possible

once the polymerization is started In the semi-continuous emulsion polymerization

process one or more components can be added continuously Various profiles of particle

nucleation and growth can be generated from different orders of component addition

during polymerization There are advantages to this process such as control of the

polymerization rate the particle number colloidal stability copolymer composition

particle morphology In the continuous process the emulsion polymerization components

are fed continuously into the reaction vessel while the product is simultaneously remo

at the same rate High production rate steady heat removal and uniform quality of

latexes are advantages of the continuous polymerization processes

Other available methods include the intermittent addition and shot addition of one

or more of the components In the shot addition process the additional components are

24

conversion of the main monomer This method has been used successfully to develo

water-soluble functional monomers such as sodium styrene sulfonat

p

e [Kim et al 1989]

264

lsion

Chemistry of Emulsion Polymerization

Emulsion polymerization is one type of free radical polymerization and can be

divided into three distinct stages initiation propagation and termination The emu

polymerization system is shown in Figure 2-7

Aqueous phaseAqueous phase

Micelle withmonomer

I Rbull

Emulsifier

Polymer particle swollenWith monomer

Monomerdroplet

I Rbull

Micelle withmonomer

I Rbull

Emulsifier

Polymer particle swollenWith monomer

MonomerdropletMonomerdroplet

I Rbull

Figure 2-7 Emulsion polymerization system [Radicals (Rmiddot) are created from initiators

(I) Monomer is transferred from larger monomer droplet into micelles by emulsifier Initiated polymer particle by radicals is keep growing until monomers are all consumed The reaction is performed in aqueous media]

In the initiation stage free radicals are created from an initiator by heat or an

ultraviolet radiation source The initiator with either peroxides groups (-O-O-) such as

odium persulfate or azo groups (-N=N-) such as azobisisobutyronitrile is commonly

used for em t

with the m er

chain ere

s

ulsion polymerization The primary free radicals created from initiator reac

onomer for initiation of polymerization In the propagation stage the polym

grows by monomer addition to the active center a free radical reactive site Th

are two possible modes of propagations head-to-head addition and head-to-tail addition

25

The head-to-tail mode is the predominant configuration of the polymer chain a result o

steric hindrance of the substitute bulky group In the termination stage polymer chain

growth is terminated by either coupling of two growing chains forming one polymer

molecule or transferring a hydrogen atom (dispropotionation) from one growing chain to

another forming two polymer molecules one having a saturated end group and the other

with an unsaturated end-group

265 Seed Emulsion Polymerization

Polymer latex particles have received increasing interest because of the versatili

of the many applications heterophase polymerization processes like emulsion dispersion

micro-emulsion seeded emulsion precipitation etc Especially to prepare well-defined

microspheres having monodispe

f

ty

rse and various particle sizes as well as surface group

ed particles prepared by emulsion polymerization

and e

s

hological design In

seede to control

n

functionalilties it is necessary to use se

nlarge them to a desired size in the further stages of reactions

Polymeric particles are required to have a uniform particle size in many

applications such as chromatography where they are used as a packing material

Morphological control of latex particle is also important for many practical application

[Schmidt 1972] Seed emulsion polymerization (or two-stage emulsion polymerization) is

a useful method to achieve both monodisperse particle size and morp

d emulsion polymerization [Gilbert 1995] preformed lsquoseedrsquo latex is used

the number of particles present in the final latex The advantage of seeded emulsio

polymerization is that the poorly reproducible process of particle nucleation can be

bypassed so that the number concentration of particles is constant and known Various

mechanisms have been proposed for the growth of latex particles [Ugelstad et al 1980

Okubo et al 1992] using this polymerization technique The initial seed particle

26

preparation step is well known and relatively easy to perform because at the relative

small particle sizes (02 to 05 micron) the particle growth process can be readily

controlled by the use of an emulsifier Enough emulsifier is used to prevent coagulation

but not enough to cause the formation of new particles As the particles are grown to

larger sizes in successive seeding steps it becomes increasingly difficult to mainta

stable uncoagulated emulsion without forming new particles and thereby destroying

monodisperisty of the latex Recently there have been several reports concerning t

reaction kinetics of seed emulsion polymerization and the development of latex

morphologies over the course of the reaction [Chern et al 1990 Delacal et al 1990 L

et al 1995 Lee 2000 2002]

266 Polystyrene Latex Particles

A number of papers have described the synthesis of the polystyrene latex particl

bearing various functional surface groups such as carboxyl [Lee et al 2000 Tun

2002 Reb et al 2000] hydroxyl [Tamai et al 1989] marcapto [Nilson 1989] epoxy

[Shimizu et al 2000 Luo et al

ly

in a

he

ee

es

cel et al

2004] acetal [Izquierdo et al 2004 Santos et al 1997]

ometyl [Izquierdo et al 2004 Park et al 2001

Sarob

h the

d

thymine [Dahman et al 2003] chlor

e et al 1998] amine [Counsin et al 1994 Ganachaud et al 1997 Ganachaud et al

1995 Miraballes-Martinez et al 2000 2001 Anna et al 2005] ester [Nagai et al 1999]

etc In order to produce these functionalized particles different methods for particle

preparation must be used The polymer emulsion with core-shell morphology of latex

particles is one of them This is a multistep emulsion polymerization process in whic

polystyrene ldquocorerdquo particle is synthesized in the first stage and the functional monomer is

added in the second stage of the polymerization without any emulsifier postfeeding to

prevent the production of new homopolymer particles thus forming the functionalize

27

polymer ldquoshellrdquo on the ldquocorerdquo particle [Keusch et al 1973] There are requirements to

limit secondary nucleation and encourage core-shell formation in seeded emulsion

polymerization including the addition of smaller seed particles at high solid content to

increase particle surface area low surfactant concentration to prevent formation of

micelles and the semi-continuous addition of monomer to create a starved-feed conditio

and keep the monomer concentration low There are some advantages [Hergeth et al

1989] of dispersions with polymeric core-shell particles First it is possible to modi

interfacial properties of polymer particles in the aqueous phase by the addition of only

very small amounts of a modifying agent during the last period of the reaction Thus

these core-shell particles are useful in a broad range of applications since they always

exhibit improved physical and chemical properties over their single-component

counterparts [Lu et al 1996 Nelliappan et al 1997] In this way the improvement of

surface properties of such dispersions is straightforward and inexpensive The other is

that polymers with a core-shell structure are perfect model systems for investigating th

material properties of polymer blends and composites because of their regular

distribution of one polymer inside a matrix polymer and because of the simple sp

geometry of the system

Their properties usually depend on the structures of latex particles Chen and his

coworkers [Chen et al 1991 1992 1993] reported the morphological development of

core shell latex particles of polystyrenepoly(methyl methacrylate) during

polymerization Before the research by Chen and his coworker Min et al [Min et al

1983] reported the morph

n

fy the

e

herical

ological development of core shell latex of polystyrene

(PS)polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the

28

additi

of

ity of graft

ing reagents Thus protein adsorption on polymeric solid surfaces has

st are microspheres defined as ldquofine

polym can be

n

r was

containing antigen urine serum etc The latex will become agglutinated and visibly

on method of PS They found that the percentage of grafting PS to the PBA was

greatest for the batch reaction and the PBA-PS core-shell particles with a high degree

grafting remained spherical upon aging test because of the emulsifying abil

copolymer

267 Various Applications of Latex Particles

Latex particles are applicable to a wide range of areas such as biomedical

applications [Piskin et al 1994] especially as the solid phase such as in immunoassays

[Chern et al 2003 Radomske-Galant et al 2003] DNA diagnostic drug delivery carriers

[Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] blood cell separations and

column pack

become a center of attention Of particular intere

er particles having diameters in the range of 01 to several micronsrdquo which

used as functional tools by themselves or by coupling with biocompounds Singer and

Plotz [1956] firstly studied microsphere or latex agglutination test (LATrsquos) by using

monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support o

which the biomolecules were going to adsorb The biomolecule adsorption howeve

limited by possible desorption of the adsorbed species or loss of specific activity of the

complex formed Since this work was published the latex particle applications for

immunoassay have been rapidly and widely studied and developed

Latex agglutination test or latex immunoassays start with tiny spherical latex

particles with a uniform diameter and similar surface properties The particles are coated

with antibodies (sensitized) through the hydrophobic interaction of portions of the protein

with the PS surface of the particles If sensitized particles are mixed with a sample

29

agglomerate Latex tests are inexpensive as compared with the other techniques [Ba

1988]

ngs

sed

tion Rheumatoid factor (RF) in different age subpopulations has also been

evalu

studied a

latex

Unipath [Percival 1996] has manufactured a range of immunoassay materials ba

on a chromatographic principle and deeply colored latex particles for use in the home and

clinical environments The latex particles which are already sensitized with a

monoclonal antibody can detect any antigens that bound to the surface of the latex

particles Some detectable pollutants include estrogen mimics which induce

abnormalities in the reproductive system of male fishes and lead to a total or partial male

feminiza

ated according to a patientrsquos clinical status by using a rapid slide latex agglutination

test for qualitative and semiquantitative measurement in human serum along with latex

immunoassay method [Onen et al 1998] Magalhaes and his coworkers [2004]

diagnostic method of contamination of male fishes by estrogen mimics using the

production of vitellogenin (VTG) as a biomarker This was based on a reverse

agglutination test developed with monoclonal antibodies specific to this biomarker

Premstaller and his coworkers [Premstaller et al 2000 2001] have prepared a porous

poly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient

chromatographic separation of biomelecules such as proteins and nucleic acids They

used a porogen a mixture of tetrahydrofuran and decanol to fabricate a micropellicular

PS-DVB backbone Legido-Quigley and his colleagues [2004] have developed the

monolith column to obtain further chromatographic functionality to the column by

introducing chloromethylstyrene in place of styrene into the polymer mixture

30

Core shell type monodisperse polymer colloids have been synthesized by Sarobe

and Forcada [1998] with chloromethyl functionality in order to improve the biomolecu

adsorption through a two-step emulsion polymerization process They investigated the

functonalized particles by optimizing the experimental parameters of the functional

monomer including reaction temperature the amount and type of redox initiator sys

used the type of addition of the initiator system and the use of washing They c

le

tem

oncluded

that th

sis

serve as separation

chann

of

usually

along the polypeptide chain which contain positive or negative charges [Norde 1998]

e relation between the amount of iron sulfate and the persulfatebisulfite system

added should be controlled to obtain monodisperse particles and prevent the premature

coagulation of the polymer particles during the polymerization

A semi-continuous emulsion polymerization technique for latex particle synthe

was performed by McDonald and other researchers [Ramakrishnan et al 2004 Steve et

al 1999] They introduced a variety of particles sizes compositions morphologies and

surface modifications to fabricate latex composite membranes (LCMs) Arraying and

stabilizing latex particles on the surface of a microporous substrate form narrowly

distributed interstitial pores formed between the particles which

els They investigated the membrane performance using gas fluxes water

permeability and the retention characterization of dextran molecules From these tests

they concluded that the narrow discriminating layer made of the latex particles leads to a

highly efficient composite membrane

27 Proteins

Proteins are natural polyamides comprised of about 20 different α-amino acids

varying hydrophobicity [Norde 1998] Proteins are more or less amphiphilic and

highly surface active because of the number of amino acid residues in the side groups

31

Proteins are polymers of L-α-amino acids [Solomon and Fryhle 2000] The α refers to a

carbon with a primary amine a carboxylic acid a hydrogen and a variable side-chain

group designated as R Carbon atoms rent groups are asymmetric and can

exhib l

s

e

on

with four diffe

it two different spatial arrangements (L and D configurations) due to the tetrahedra

nature of the bonds The L refers to one of these two possible configurations Amino

acids of the D-configuration are not found in natural proteins and do not participate in

biological reactions Figure 2-8 shows the chiral carbon in 3-D as the L isomer Protein

consist of twenty different amino acids differentiated by their side-chain groups [Norde

1998] The side-chain groups have different chemical properties such as polarity charg

and size and influence the chemical properties of proteins as well as determine the

overall structure of the protein For instance the polar amino acids tend to be on the

outside of the protein when they interact with water and the nonpolar amino acids are

the inside forming a hydrophobic core

Figure 2-8 L-α-amino acid

The covalent linkage between two amino acids is known as a peptide bond A

peptide bond is formed when the amino group of one amino acid reacts with the carboxyl

group of another amino acid to form an amide bond through the elimination of water

This arrangement gives the protein chain a polarity such that one end will have a free

amino group called the N-terminus and the other end will have a free carboxyl group

called the C-terminus [Solomon and Fryhle 2000] Peptide bonds tend to be planar and

32

give the polypeptide backbone rigidity Rotation can still occur around both of the α-

carbon bonds resulting in a polypeptide backbone with different potential conformations

relative to the positions of the R groups Although many conformations are theoretically

possible interactions between the R-groups will limit the number of potential

nd to form a single functional conformation In other words

the conformation or shape of the protein is

α-helix

can interact with other

secondary structures within the sam

conformations and proteins te

due to the interactions of the chain side

groups with one another and with the polypeptide backbone The interactions can be

between amino acids that are close together as in a poly-peptide between groups that are

further apart as in amino acids or even on between groups on different polypeptides all

together These different types of interactions are often discussed in terms of primary

secondary tertiary and quaternary protein structure

The primary amino acid sequence and positions of disulfide bonds strongly

influence the overall structure of protein [Norde 1986] For example certain side-chains

will promote hydrogen-bonding between neighboring amino acids of the polypeptide

backbone resulting in secondary structures such as β-sheets or α-helices In the

conformation the peptide backbone takes on a spiral staircase shape that is stabilized by

H-bonds between carbonyl and amide groups of every fourth amino acid residue This

restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure

Certain amino acids promote the formation of either α-helices or β-sheets due to the

nature of the side-chain groups Some side chain groups may prevent the formation of

secondary structures and result in a more flexible polypeptide backbone which is often

called the random coil conformation These secondary structures

e polypeptide to form motifs or domains (ie a

33

tertiary structure) A motif is a common combination of secondary structures and a

domain is a portion of a protein that folds independently Many proteins are composed o

multiple subunits and therefore exhibit quaternary structures

271 Interaction Forces between Proteins

Proteins in aqueous solution acquire compact ordered conformations In such a

compact conformation the movement along the polypeptide chain is severely restrict

implying a low conformational entropy The compact structure is possible only if

interactions within the protein molecule and interactions between the protein molecule

and its environment are sufficiently favorable to compensate for the low conformational

entropy [Malmsten 1998] Protein adsorption study is often focused on structural

rearrangements in the protein molecules because of its significance to the biological

functioning of the molecules and the important role such rearrangements play in the

mechanism of the adsorption process Knowledge of the majo

f

ed

r interaction forces that act

structures helps to understand the behavior

of pro

arge

a

ded

between protein chains and control the protein

teins at interface These forces include Coulomb interaction hydrogen bonds

hydrophobic interaction and van der Waals interactions

Coulombic interaction Most of the amino acid residues carrying electric ch

are located at the aqueous boundary of the protein molecule An the isoelectric point

(IEP) of the protein where the positive and negative charges are more or less evenly

distributed over the protein molecule intramolecular electrostatic attraction makes

compact structure favorable to proteins Deviation to either more positive or more

negative charge however leads to intramolecular repulsion and encourages an expan

structure Tanford [1967] calculated the electrostatic Gibbs energy for both a compact

impenetrable spherical molecule (protein) and a loose solvent-permeated spherical

34

molecule (protein) over which the charge is spread out From the results he found th

the repulsion force was reduced at higher ionic strength d

at

ue to the screening action of ion

ble

e

the

d the

tween

dehyd

e

ent

oles

tein

Hydrogen bond Most hydrogen bonds in proteins form between amide and

carbonyl groups of the polypeptide backbone [Malmsten 1998] The number of availa

hydrogen bonds involving peptide units is therefore far greater than that involving sid

chains Because α-helices and β-sheets are aligned more or less parallel to each other

interchain hydrogen bonds enforce each other Kresheck and Klotz [1969] examine

role of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between

peptide units do not stabilize a compact structure of protein However because the

peptide chain is shielded from water due to other interactions hydrogen bonding be

peptide groups do stabilize α-helical and β-sheet structures

Hydrophobic interaction Hydrophobic interaction refers to the spontaneous

ration and subsequent aggregation of non-polar components in an aqueous

environment In aqueous solutions of proteins the various non-polar amino acid residues

will be found in the interior of the molecule thus shielded from water The

intermolecular hydrophobic interaction for the stability of a compact protein structure

was first recognized by Kauzmann [1959] If all the hydrophobic residues are buried in

the interior and all the hydrophilic residues are at the outermost border of the molecul

intramolecular hydrophobic interaction would cause a compact protein structure

However geometrical and other types of interactions generally cause a fraction of the

hydrophobic residues to be exposed to the aqueous environm

Van der Waals interaction The mutual interaction between ionic groups dip

and induced dipoles in a protein molecule cannot be established as long as the pro

35

structure is not known in great detail Moreover the surrounding aqueous medium also

contains dipoles and ions that compete for participation in the interactions w

the protein molecule Dispersion interactions favor a compact structure However

because the Hamaker constant for proteins is only a little larger than that of water the

resulting effect is relatively small [Nir 1977]

272 β

ith groups of

rgiles et al 1996

Floeg

) is degraded [Floege 2001] Renal

β2M from the serum resulting in an increase in β2M

conce well

the

)

a

al

2-Microglobulin (β2M)

The protein β2M is of particular interest because it is involved in the human

disorder dialysis-related amyloidosis (DRA) [Geiyo et al 1985 A

e 2001] DRA is a complication in end stage renal failure patients who have been on

dialysis for more than 5 years [Bardin et al 1986 Drueke 2000] DRA develops when

proteins in the blood deposit on joints and tendons causing pain stiffness and fluid in

the joints as is the case with arthritis In vivo β2M is present as the non-polymorphic

light chain of the class I major histocompatibility complex (MHC-I) As part of its

normal catabolic cycle β2M dissociates from the MHC-I complex and is transported in

the serum to the kidney where the majority (95

failure disrupts the clearance of

ntration by up to 60-fold [Floege 2001] By a mechanism that is currently not

understood β2M then self-associates into amyloid fibrils and typically accumulates in

musculoskeletal system [Homma et al 1989] Analysis of ex vivo material has shown

that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRA

is present as of full-length wild-type β2M although significant amounts (~20-30) of

truncated or modified forms of the protein are also present [Floege 2001 Bellotti et al

1998] Figure 2-9 shows the ribbon diagram of human β2M Native β2M consists of

single chain of 100 amino acid residues and has a seven stranded β-sandwich fold typic

36

of the immunoglobulin superfamily [Saper et al 1991 Trinh et al 2002] β2M was fi

isolated from human urine and characterized by Berggard et al [1980] in1968 The

normal serum concentration of β

rst

re

f

e

2M is 10 to 25 mgL It is a small globular protein with

a molecular weight of 118 kDa a Strokes radius of 16Aring and a negative charge under

physiological conditions (isoelectric point IEP = 57) β2M contains two β-sheets that a

held together by a single disulphide bridge between the cysteines in positions 25 and 81

[Berggard et al 1980 Parker et al 1982 Cunningham et al 1973] β2M cannot be

removed completely by current dialysis techniques but through a better understanding o

the structure and interaction forces that lead to this structure it will be possible to mor

efficiently remove this problematic protein

Figure 2-9 Ribbon diagram of human β M taken from the crystal structure of the protein

bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khal 2000]

273 Serum Albumin

Serum albumin is the most abundant protein found in plasma and is typically

present in the blood at a concentration of 35~

2an et

50gL According to extensive studies about

its physiological and pharmacological properties albumin has a high affinity to a very

wide range of materials such as electrolytes (Cu+2 Zn+2) fatty acids amino acids

metabolites and many drug compounds [Fehske et al 1981 Kraghhansen 1981 Putnam

37

1984 Peters 1985] The most important physiological role of the protein is therefore to

bring such solutes in the bloodstream to their target organs as well as to maintain the pH

and osmotic pressure of the plasma Bovine serum albumin (BSA) is an ellipsoidal

protein with the dimensions of 140 X 40 X 40Aring [Peter 1985] The primary structure is a

single helical polypeptide of 66 kDa (IEP = 47) with 585 residues containing 17 pairs of

as a soft an

rfaces [Kondo et al 1991 Norde et al 1992 Soderquist et al

1980 ikely

is

disulfide bridges and one free cysteine [Dugaiczyk et al 1982] BSA has been classified

d flexible protein because it has a great tendency to change its conformation

on adsorption to solid su

Carter and Ho 1994] and consists of three homologous domains (I-III) most l

derived through gene multiplication [Brown 1976] Each domain is composed of A and B

sub-domains [He et al 1992] The secondary structure of human serum albumin (HSA)

shown in Figure 2-10

Figure 2-10 Secondary structure of human serum albumin (HSA) with sub-domains

[Zunszain et al 2003]

HSA has the same structure domains with the serum albumin from other species

such as BSA [Brown 1976] Although all three domains of the albumin molecule have

similar three-dimensional structures their assembly is highly asymmetric [Sugio et al

38

1999] Domains I and II are almost perpendicular to each other to form a T-shaped

assembly in which the tail of subdomain IIA is attached to the interface region between

sub-domains IA and IB by hydrophobic interactions and hydrogen bonds In contrast

domain III protrudes from sub-domain IIB at a 45ordm angle to form the Y-shaped assembly

for domains II and III Domain III interacts only with sub-domain IIB These features

make the albumin molecule heart-shaped

28 Protein Adsorption

Protein adsorption studies date back to the 1930rsquos At the beginning these studies

mainly focused on the determination of the molecular weight electrophoretic and

ral

rearrangem

adsorption and biocompatibility of the sorbent materials were investigated [Norde 1986]

There are interaction forces at the interfaces between protein molecules and latex

particles These forces are mainly divided into the following groups hydrophobic

interaction ionic interaction hydrogen bonding and van der Waals interaction [Andrade

1985]

Hydrophobic interaction It is known that hydrophobic interaction has a major

role in protein adsorption phenomena The adsorption of proteins on the low charged

latex particles occurs by this interaction force Generally monomers such as styrene offer

hydrophobic surfaces th

by this interaction force is maximum at the isoelectric point (IEP) of the protein and the

strength [Suzawa et al 1980 1982 Shirahama et al 1989 Kondo et al 1992] By the

chromatographic applications Later the adsorption mechanism especially the structu

ents was studied Recently the studies of the relation between protein

281 Interaction between Protein Molecule and Latex Particle

at protein molecules adsorb to The amount of adsorbed protein

pH at maximum adsorption shifts to a more acidic region with an increase in ionic

39

reports [Suzawa et al 1980 1982 Lee et al 1988] protein adsorption was greater

hydrophobic surface than on a hydrophilic one implying that hydrophobic interaction is

on a

s

ydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA)

Ionic

ely

t

ces

n force is operative over small

distan e

ble

n

nt dialysis membrane therapy for end stage renal disease (ESRD)

one of most dominant forces in protein adsorption

Ionic interaction Negatively charged latex particles have ionic functional

groups on their surfaces such as salts of sulfonic and carboxylic acid Sulfate group

originate from an initiator such as sodium persulfate and carboxylic groups originate

from a h

bonds are formed between the negative charges of these latex particles and positive

surface charges of protein molecules The conventional low-charged latex particles rar

form these ionic bonds

Hydrogen bonding Hydrogen bond is a strong secondary interatomic bond tha

exists between a bound hydrogen atom (its unscreened proton) and the electrons of

adjacent atoms [Callister 1999] Protein can be adsorbed on hydrophilic polar surfa

through hydrogen bonding Hydrogen bonds are frequently formed between hydroxyl-

carbonyl or amide-hydroxyl Hydroxyl-hydroxyl or amide-hydroxyl bonds are also

formed in protein adsorption

Van der Waals interaction This interactio

ces only when water has been excluded and the two non-polar groups come clos

to each other Lewinrsquos calculation showed that the van der Waals interaction is negligi

compared with the forces involved in the entropy increases ie hydrophobic interactio

[Lewin 1974]

29 Hypothesis for Toxin Removal

As mentioned earlier insufficient removal of middle molecular range toxins is a

major drawback of curre

40

patien

is

y

ese

haracteristics over the last decade However removal

effici

ing a novel membrane design composed of an assembly of engineered

polymeric latex particles s ions Important factors

will i

e

ith a

is established it is

expec

e

ts This may cause destructive arthritis and carpal tunnel syndrome inhibit the

immune system and accelerate cardiovascular disease leading to death among dialys

patients In order to overcome these complications artificial dialysis membranes have

been developed to increase the capacity for removing middle molecular weight toxins b

changing the pore size of dialysis membranes and using new materials that adsorb th

toxins for improved removal c

ency is not as high as those achieved by a normal healthy kidney

With the knowledge obtained from all above literatures the following hypothesis

has been established for the development of a membrane for the successful removal of

the target protein β2-Microglobulin (β2M) without the removal of serum albumin This

will be done us

ynthesized to predetermined specificat

nclude pore size and surface chemistry

291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size

The packing of monodispersed spherical particles can lead to the formation of

porous layers suitable for use as filters and membranes [Hsieh et al 1991] The pore siz

is defined as the largest spherical particles that can pass through the interstitial spaces

It is well known that monodispersed spherical particles can be obtained from the

seed emulsion polymerization method Many commercially available latex particles are

synthesized by this technique and are inexpensive When the defect free membrane w

monodisperse pore size distribution based on a particular particle array

ted to outperform the traditional hemodialysis membrane limitation which is a

broad range of pore size distribution When spherical particles pack in regular crystallin

arrays a number of packing geometries such as hexagonal closest packing cubic closest

41

packing and body-centered cubic packing are possible The pore size of the array

depends on these packing geometries as well as particle size Theoretical pore size can be

calculated from the simple geometry of the arr

ay Figure 2-11 shows the relationship

between particle size and pore size

0001

001

01

001 01 1 10

Por

e

1

10

microm

) S

ize

(

Particle Diameter (microm)

Body-Centered Cubic ArrayClosest Packed Array

0001

Siz

e (

Particle Diameter (microm)

001

01

001 01 1 10

Por

e

1

10

microm

)

Body-Centered Cubic ArrayClosest Packed ArrayBody-Centered Cubic ArrayClosest Packed Array

and closed packed arrays [Steve et al 1999]

McDonald and his coworkers [Ramakrishnan et al 2004 Steve et al 1999] have

described the fabrication of latex composite membranes and their performance in

previous papers It is from this prior research and knowledge gained from conversations

with Dr McDonald that this current research has been initiated [McDonald 2003] The

focus of this thesis is the extension of McDonaldrsquos research in the area of composite

membranes to medical application such as dialysis membranes

292 Toxin Removal by Selective Adsorption on Engineered Latex Particles

The flexibility in latex surface properties is a significant advantage to this

materialrsquos ability to separate proteins since the selectivity is strongly dependent on the

charge interactions of both the protein and the latex particles under the conditions of

Figure 2-11 The relationship between pore size and particle size in body-centered cubic

42

separation [Menon et al 1999 Chun et al 2002] First all sorbents were designed to

have negative surface properties because most plasma proteins in blood are negative and

should not be removed with charge interaction between proteins and sorbents at

physiological condition (pH74) It was reported that a negatively charged surface was

more blood compatible than a positive one [Srinivasan et al 1971] The toxin protein β2-

Microglobulin (β2M) can be selectively adsorbed on the surface of latex particles by the

design of a suitable hydrophobic portioned surface where only β2M protein can be

anchored with hydrophobic interactions Albumin adsorption on the latex particle is not

side-on m

adsorption of β M protein on the engineered latex particles

allowed because charge repulsion is more dominant than hydrophobic interaction with

ode The figure 2-12 shows the schematic representation of the selective

2

PMMAPAAShell

COO-

OSO3-

OSO3-

PSCore

COO-

-

-

-

-

-

-

Albumin-

Chargerepulsion

--

-

- -

- -

- --

Hydrophobic

β2-Microglobulin

2

Interaction

β -Microglobulin

PMMAPAAShell

COO-

-

-

-

-

-

-

--

-

-

-

-

-

Albumin-

Chargerepulsion

OSO3-

OSO3-

PSCore

COO-

--

-

- -

--

-

- -

- -

- --

- -

- --

Hydrophobic

β2-Microglobulin

2

particles at pH 74

Interaction

β -Microglobulin

Figure 2-12 Schematic representation of the protein adsorption on the core shell latex

43

Hydrophilichydrophobic microdomain structures were proven to be more blood

compatible [Mori et al 1982 Higuchi et al 1993 Deppisch et al 1998] The

optimization of suitable hydrophobic to hydrophilic ratio is also important for the

biocompatibility The monomers such as styrene (St) methyl methacrylate (MMA) and

acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in

emulsion polymerization process

In summary the background and fundamental literature survey about the history

material properties and limitation of hemodialysis membrane latex particle preparation

surface chemistry and manufacturing process target proteins and protein adsorption and

finally the hypothesis for a toxin removal with high separation efficiency have been

suggested This research focuses on such hypothesis and the materials and

characterization methodology for achievement of suggested hypotheses are described in

the next chapter

CHAPTER 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY

As mentioned earlier the goal of this study is to prepare polymeric latex particles

with tailored properties to maximize separation of toxin molecules and to investigate

fundamental interactions between the applied particles and molecules in the

system to optimize the membrane performance for hemodialysis applications Polymeric

latex particles wit

the

biological

h monodisperse size distribution to obtain uniform pore size and

various size ranges to utilize membrane construction are necessary Surface engineering

with various combination of hydrophobichydrophilic domain on the surface of latex

particles is expected to affect the removal of target protein by selective adsorption and to

improve the biocompatibility of membrane Therefore the materials and characterization

methodology are addressed in this chapter

31 Materials

Styrene (St) monomer used for a seed and a core particle was purchased from

Fisher Scientific and used without any other purification process Acrylic acid (AA) and

methyl methacrylate (MMA) monomers introduced for shell formation were purchased

from Fisher Scientific and used without any other purification process Sodium persulfate

(SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as

received Divinylbenzene (DVB) crossliking agent was purchased from Aldrich The

anionic surfactant Aerosol regMA80-I [sodium di(13-dimethylbutyl) sulfosuccinate] was

kindly donated by Cytec 78-80 Aerosol regMA80-I is mixed with isopropanol and

water Its critical micelle concentration (CMC) is 245 mM (119) Ion exchange resin

44

45

AGreg 501-x8 Resin (20-50 mesh) was purchased from Bio-Rad Laboratories Inc It is a

mixed-bed resin with H+ and OH- as the ionic forms and is 8 crosslinked Bovine serum

albumin (BSA) (heat shock treated) was purchas Fisher Scientific The isoelectric

point of t lin

(β2M)

as

gma

ed from

he BSA is 47-49 and the molecular weight is 66300Da β2-Microglobu

was purchase from ICN Biomedicals Inc and was separated from patients with

chronic renal disease and lyophilized from ammonium bicarbonate The molecular

weight by SDS-PAGE was approximately 12000Da BSA and β2M proteins were used

received without further purification process In order to investigate the protein

adsorption evaluation bicinchoninic acid protein assay kits were purchased from Si

(Cat BCA-1) and Pierce Biotechnology (Cat 23225) The chemical structures for the

main chemicals are shown in Figure 3-1

CH2 CH

Styrene (Mw = 10415)

CH2 CHCH2 CH

Styrene (Mw = 10415)

CH2 CH

Acrylic acid (Mw = 7206)

C

OH

O

CH2 CH

Acrylic acid (Mw = 7206)

C

OH

O

CH CH2

C

OCH3

O

CH

CH CH3

2

C

OCH3

O

CH3

Methyl methacrylate (Mw = 10012)Methyl methacrylate (Mw = 10012)

CH2 CH

Divinylbenzene (Mw = 1302)

CH CH2

CH2 CH

Divinylbenzene (Mw = 1302)

CH CH2CH CH

+(Na) -(O3S)

CH -C-O-CH-CH -CH-CH3

O

2 2 3

O CH CH3

CH3 CH3

CH-C-O-CH-CH2-CH-CH3

Anionic surfactant (MA80-I) (Mw = 3885)

+(Na) -(O3S)

CH -C-O-CH-CH -CH-CH3

O

2 2 3

O CH CH3

CH3 CH3

CH-C-O-CH-CH2-CH-CH3

Anionic surfactant (MA80-I) (Mw = 3885)2

+(Na)-(O) S OO

O O

- +

Initiator sodium persulfate (Mw = 23811)

O SO

(O) (Na)+ -

O O

- +(Na) (O) S OO

O SO

(O) (Na)+ -

O O

- +

Initiator sodium persulfate (Mw = 23811) Figure 3-1 Chemical structure of main chemicals

(Na) (O) S OO

O SO

(O) (Na)

46

32 Latex Particle Preparation

The experimental set up and the particle preparation schemes for various types and

size ranges of latex particles are shown in Figure 3-2 and 3-3 In the experimental setup a

mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at

end of glass stirrer The agitation rate is precisely adjusted by controller motor Paar glas

reactor with 1L volume is partially in silicon oil bath on hot plate The reactor has fo

openings for a stirrer a thermometer a nitrogen gas inlet and a reactant feeding lnlet

The reactants for latex polymerization are fed by precisely controlled by meterin

The particle preparation is described in each latex preparation section

the

s

ur

g pump

Figure 3-2 Experimental setup for semi-continuous emulsion polymerization

1 Motor 2 Mechanical stirrer bar 3 Thermometer 4 Inlet of N2 5 Inlet for initiator and monomer 6 Glass reactor (l L) 7 Oil bath 8 Hot plate 9 Pump for initiator 10 Pump for monomer 11 Tubes for reactant feed

47

Styrene monomer

Bare PS

PSPMMA100

PSPMMA90PAA10

PSPMMA P75 AA25

Gr

win

ticle

size

PS seedparticles

og

par

Styrene monomer

Bare PS

PSPMMA100

PSPMMA90PAA10

PSPMMA P75 AA25

Gr

win

ticle

size

PS seedparticles

og

par

Figure 3-3 The particle preparation scheme in various types and size ranges of latex particles

321 Preparation of Seed Latex Particles

Polystyrene seed particles were synthesized through a typical semi-continuous

emulsion polymerization Polystyrene latex particles were prepared in a four necked Paar

glass vessel equipped with a mechanical glass stirrer thermometer glass funnel for

nitrogen gas purge and a tube for monomer feeding The reactor was placed in a silicon

oil bath on a hot plate for homogenous and stable tempe he vessel was

firstly charged with de-ionized water the emulsifier so itiator

sodium bicarbonate (SBC) styrene (St) monomer divinylbenzene (DVB) monomer

under nitrogen gas atmosphere and then heated to 72plusmn2 as

llowed to continue for another hour Second reactants St and DVB monomers SPS

initiator and SBC were fed separately using fine fluid metering pumps (Model RHSY

Fluid metering INC NY) under a nitrogen gas atmosphere for 2h After complete feeding

rature control T

dium persulfate (SPS) in

oC for 1h The reaction w

a

48

of all reactants the reaction was continued for another hour at elevated temperature 80plusmn1

oC and then cooled to room temperature by removal of the oil bath

322 Preparation of Seeded Latex Particles

The particle growth was achieved by seeded continuous emulsion polymerization

The vessel was charged with the polystyrene seeds and de-ionized water to make desired

solid content and heated up to 72plusmn2 oC for 1h under nitrogen gas atmosphere Then St

and DVB monomers and solution of SPS and SBC in de-ionized water were continuously

added using metering pump with a precisely controlled feed rate After feeding all

reactants the reaction was continued for three hours keeping temperature of 80plusmn2 oC and

then the reaction vessel was cooled down to room temperature

Monodisperse spherical polystyrene seeds with a 280nm mean diameter were used

e-polymerized polystyrene seeded emulsion

was charged in a 500m

323 Preparation of Core Shell Latex Particles

for core shell structured latex particle The pr

l glass flask under a nitrogen gas atmosphere De-ionized water

was added to achieve the desired solid content The emulsion was heated to 80plusmn2oC for

10 hour Monomers initiator and SBC to form the shell were added by fluid metering

pumps (LAB PUMP JRRHSY model Fluid metering Inc USA) with fine controlled

feed rate Flow rate can be adjusted by control ring graduated in 450 division from 0 to

100 flow which is in the range of 10mlmin respectively The reaction was sustained

for additional 30 hours at 90plusmn2oC then cooled down to room temperature

324 Purification of Synthesized Latex Particles

Synthesized latex particles were purified with an ion exchange resin 200g (10

ww polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin

and stirred for 40 minute to remove unreacted monomers initiators and other impurities

49

Then the emulsion was diluted with DI water to a specific degree of solid content as

needed

33 Characterization

331

solid

termined

oscopy

d

hours

Dried latex (10mg) was mixed with

250 m on

Degree of Conversion

The degree of monomer to polymer conversion was determined gravimetrically

following the procedure described by Lee et al [1995] before the latex cleaning process

The synthesized emulsion raw materials were weighed and dried in a conventional oven

at 120oC for 30min to evaporate any unreacted monomers and water The remaining

was weighed and the degree of conversion was determined This process was repeated

three times and a mean value of the degree of conversion was de

332 Fourier Transform Infrared (FTIR) Spectr

A molecule can absorb only select frequencies (energy) of infrared radiation which

match the natural vibration frequencies The energy absorbed serves to increase the

amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]

Only those bonds which have a dipole moment that changes as a function of time can

absorb the infrared radiation The range of wavelengths for infrared radiation is between

25 microm (4000 cm-1) and 25 microm (400cm-1)

The synthesis of the latex particles such as polystyrene (PS) homopolymer and

PSPMMA PSPMMA90PAA10 PSPMMA75PAA25 core-shell copolymers were verifie

with FTIR Purified latex particles were dried in vacuum oven at 40oC for 24

before analysis in the FTIR (Nicolet Magma USA)

g of KBr which had also been dried in vacuum oven at 120oC for 3h Transmissi

spectra using the drift mode were plotted with 128 scans and 4cm-1 resolution

50

333 Quasielastic Light Scattering (QELS)

Latex particle size was measured by the Brookhaven ZetaPlus particle size

analyzer The solid concentration as 0001 (wv) This

ation spectroscopy (PCS) of quasielastically scattered light

For th s

ed using a FE-SEM (JEOL

JSM-

ith

ent

01

us zeta-

potential analyzer It measures the electrophoretic mobility velocity of charged colloidal

of latex particles used w

instrument uses photon correl

is technique time dependent interference patterns of light scattered from particle

in the sample cell are analyzed The interference pattern changes due to Brownian

motion giving rise to fluctuations in scattering intensity The frequency of these

fluctuations depends on the particlersquos diffusion constant that is inversely proportional to

the particle diameter

334 Field Emission-Scanning Electron Microscopy (FE-SEM)

Particle size and surface morphology were characteriz

6335F Japan) A diluted suspension of latex particles in water was dropped onto a

silicon wafer at room temperature and allowed to dry The sample was then coated w

the thinnest layer of carbon needed to obtain the required conductivity for this instrum

Secondary electron image mode was used with a 15KV of accelerating voltage The

magnification range used was between 10000X and 70000X

335 Zeta Potential Measurement

Synthesized and purified latex particles were diluted with de-ionized water to 0

wt and adjusted six different pH values 205 345 481 565 643 and 747 These

particle suspensions were then transferred to standard cuvettes for zeta potential

measurement At least two runs of ten measurements were taken for each sample and

averaged

The zeta potential measurement was carried out using Brookhaven ZetaPl

51

particles in solution and calculate zeta-potential by using the Smoluchowski equation

The f quency

gnitude

umin (BSA) as a standard model protein and β2-Microglobulin

uffer (PB) and

phosp

5

h the selected protein

e 005 01 03 05 and 07 mgml and those for

β2M

d at

teins in the supernatant after the centrifugation process using the

bicinc t

requency of laser light passing through the sample and is compared to the fre

of a reference beam The shift in the frequency called a Doppler shift and the ma

of the shift correspond to the polarity and the magnitude of the electrophoretic mobility

respectively The zeta potential is calculated from the solution conditions and the

measured mobility

336 Protein Adsorption

Protein adsorption experiments of the synthesized latex particles were performed

with bovine serum alb

(β2M) as a target protein in two types of buffer solution phosphate b

hate buffered saline (PBS) To make 5 mM of PB solution 0345g of sodium

phosphate monobasic (NaH2PO4H2O) was dissolve in 500ml of de-ionized water To

make PBS solution 0345g of sodium phosphate monobasic (NaH2PO4H2O) and 4178g

(143mM) of sodium chloride (NaCl) were dissolved in 500ml of de-ionized water The

synthesized latex particles were diluted with each buffer to have a solids content of 0

(ww) adjusted to pH values of 32 48 and 74 and mixed wit

BSA concentrations were chosen to b

were 0015 0030 0045 and 0060mgml The mixture was gently rotated using

the shaker Labquakereg (BarnsteadThermolyne Model 4002110 USA) with 8 RPM in

the incubator at 37oC for 12 hours before the latex-protein mixture was centrifuge

13000 rpm for 15min The amount of protein adsorbed was determined by quantifying

the free pro

honinic acid (BCA) assay method [Lowry et al 1951 Smith et al 1985 Baptista e

al 2003 Wiehelman et al 1988 Brown et al 1989] BCA assay kits consist of Reagent

52

A containing bicinchoninic acid sodium carbonate sodium tartrate and sodium

bicarbonate in 01N NaOH with pH=1125 and Reagent B containing 4 (wv) copper

(II) sulfate pentahydrate The BCA working reagent was prepared by mixing 50 parts of

Reagent A with 1 part of Reagent B 100microl of protein supernatant was then mixed with

2ml of BCA working reagent in a UV cuvette Incubation was allowed to continue

at room temperature

further

until color developed about 2h The absorbance at 562nm was

pectrophotometer (Perkin-Elmer Lambda 800 USA)

Unkn

Separate the p rticles in centrifuge mix supernatant with BCA reagents

measured using a UV-VIS s

own concentration of sample protein was determined by comparison to a standard

of known protein concentrations The adsorbed amount per unit surface area was

determined by the mass balance of the protein after adsorption process The simple

schematic of the procedure for a protein adsorption test is shown in Figure 3-4

Incubate mixture with agitation for 12hrs at 37oC

aand develop the color by incubation for 2hrs at room temperature

Suspend latex particles in aqueous buffer media

Add the dissolved target protein

Measure color intensity using UVVis spectroscopy at 562nm

Suspend latex particles in aqueous buffer media

Separate the p rticles in centrifuge mix supernatant with BCA reagents

Add the dissolved target protein

Incubate mixture with agitation for 12hrs at 37oC

aand develop the color by incubation for 2hrs at room temperature

Measure color intensity using UVVis spectroscopy at 562nm

Figure 3-4 Schematic of the procedure for a protein adsorption test

53

337 Blood Biocompatibility by Hemolysis Test

Hemolysis is the destruction of red blood cells which leads to the release of

hemoglobin from within the red blood cells into the blood plasma Hemolysis testing is

used to evaluate blood compatibility of the latex particles and the damaged to the red

blood cells can be determined by monitoring amount of hemoglobin released The red

blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutes

Figure 3-5 shows the phase separated blood with plasma white blood cells and red

cells after centrifugation process Separated RBSs were then washed with isotonic

phosphate buffer solution (PBS) at pH 74 to remove debris and serum protein Th

process was repeated 3 times

blood

is

Figure 3-5 Separation of RBC from whole blood by centrifuge process

Prepared latex particles were re-dispersed in PBS by sonification to obtain

homogeneously dispersed latex particles 100microl of the mixture of red blood cell (3 parts)

and PBS (11parts) was added to 1ml of 05 (ww) particle suspension PBS was used as

a negative control resulting 0 hemolysis and DI water used as a positive control to

produce 100 hemoglobin released by completely destroyed RBCs The mixture was

incubated in water bath with gentle shaking for 30 minutes at 37oC and then centrifuged

at 1500 rpm for 15 minutes 100microl of the supernatant was mixed with 2ml of the mixture

) (EtOHHCl = 2005 ww) to prevent the

White blood cells

Plasma

Red blood cells

of ethanol (99) and hydrochloric acid (37

54

precipitation of hemoglobin In order to remove remaining particles the mixture was

centri fuged again with 13000 RPM at room temperature The supernatant was then

transferred into the UV cuvette The amount of hemoglobin release was determined by

monitoring the UV absorbance at a wavelength of 397nm The schematic of the

procedure for hemolysis test is shown in Figure 3-6

Separate red blood cells (RBCs) from blood

Mix RBCs with phosphate buffer solution (PBS)

Wash and re-suspend RBC with PBS repeat three times

Add la l of RBC and incu in at 37oC

tex particle suspension into 100mbate sample with agitation for 30m

Separate particle ith EtOHHCl solutions and mix the supernatant w

Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm

Separate red blood cells (RBCs) from bloodSeparate red blood cells (RBCs) from blood

Mix RBCs with phosphate buffer solution (PBS)Mix RBCs with phosphate buffer solution (PBS)

Wash and re-suspend RBC with PBS repeat three timesWash and re-suspend RBC with PBS repeat three times

Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC

Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC

Separate particles and mix the ith EtOHHCl solution supernatant wSeparate particles and mix the ith EtOHHCl solution supernatant w

Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nmEvaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm

Figure 3-6 Schematic of the procedure for hemolysis test

In summary a description of the experimental materials and characterization

methodology has been explained In the next chapter the results and discussion of the

data obtained from these experiments is addressed

CHAPTER 4 RESULTS AND DISCUSSION

41 Polymerization of Latex Particles

411 Polystyrene Seed Latex Particles

Polymer particles have many different applications including spacer particles

lubricants packing materials for chromatography standard particles and diagnostic

drugs All applications strongly require these particles to have a uniform particle size

Uniform particle size is the first requirement for a latex composite membrane formed by

particle arrays with the interstitial spaces serving as pores for size discriminations [Jons

et al 1999 Ramakrishnan et al 2004] Although suspension polymerization has been

mainly used as one of conventional polymerization methods to prepare uniform particles

the uniformity of the recovered particles is insufficient for membrane use There is

another method known as seed emulsion polymerization where a vinyl monomer is

absorbed into fine monodiserse seed particles and polymerization causes the monomer to

crease the sizes of the seed particles uniformly In order to obtain monodisperse larger

particles by this m

particles and polymerization of the monomer is repeated

In this study cross-linked polystyrene seed latex particles were firstly synthesized

in the presence of styrene monomer divinylbenzene crosslink agent sodium persulfate

initiator sodium bicarbonate buffer and the Aerosol regMA80-I [sodium di(13-

dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion

polymerization process in which one or more components can be added continuously

in

ethod the procedure of absorption of monomer into fine polymer

55

56

Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and

buffer are continuously added to enlarge the seed latex particles Various profiles of

particle nucleation and growth can be gene different orders of component

addition during polymerizati f this process such as easy

control of the polymeriza particle number

and particle morphology The main goal in

this process is to avoid secondary particle

rated from

on There are several advantages o

tion rate monodisperse particle size and

colloidal stability copolymer composition

formation leading to a monodisperse particle

size distribution Figure 4-1 shows the schematic representation of seed latex particle

preparation and growth

Monomer droplet

Seed particles Amount of surfactantsis variable

Enlarged final Add monomers

PS

PS

PSs4

particles Number of particlesare constant

Seed code

PSs1

s2

s3

Monomer droplet

Seed particles Amount of surfactantsis variable

Enlarged final Add monomers

PS

PS

PSs4

tion The

particles Number of particlesare constant

Seed code

PSs1

s2

s3

Figure 4-1 Schematic representation of semi-continuous seed latex particles preparation and growth

After the first synthesis of a small size of crosslinked polystyrene (PS) seed

particles stabilized by surfactants styrene DVB and initiator are continuously fed into

the system to enlarge the seed latex particles maintaining a narrow size distribu

recipes and reaction conditions for synthesizing these seed particles are summarized in

57

Table 4-1 The original recipe for latex particle preparation from McDonald was

modified for this emulsion system [McDonald 2003]

Table 4-1 The polymerization recipe of polystyrene (PS) seed particles Latex label PSS259 PSS233 PSS207 PSS181

Temperature (oC) 75 plusmn 5 75 plusmn 5 75 plusmn 5 75 plusmn 5

Divinylbenzene (g) 052 052 052 052

MA 80 (g) 259 233 207 181

NaHCO

Watera (g) 1315 1315 1315 1315

Styrene (g) 9948 9948 9948 9948

112 112 112 112

Na2S2O8 (g) 190 190 190 190

Waterb (g) 200 200 200 200

Reaction time (min) 140 140 140 140

Solids content 396 396 396 396

Mean particle diameter (nm) 126plusmn75 171plusmn39 182plusmn41 216plusmn53

Conversion () 965 979 957 971

3 (g)

Four different seed latex particles were synthesized based on surfactant amount in

order to determine the optimum condition for stable and monodisperse latex particles

The emulsion polymerization of latex particles has to be carried out in a narrow range of

surfactant causes flocculation or phase separation leading to broad particle

size distribution and an unstable emulsion sy

surfactant concentration where particles are stable [Nestor et al 2005] A wide range of

concentrations

stem Therefore the range of surfactant

amount was varied from 181g to 259g in increments of 026g in each latex label Other

reactants amounts were kept constant Watera is the initial charge amount and waterb

represents the amount of aqueous solution containing dissolved initiator and buffer

58

which are fed continuously after the seed particle creation The extent of monome

polymer conversion was obtained by gravimetric calcu

r to

lation and was more than 95 at

cripts such as

S207 and S181 i the surf mount added for the seed latex

nversion indi perce experi obtain

content divided by the theoretically estimated solid content and was calculated by the

all seed particles labeled PSS259 PSS233 PSS207 and PSS181 Labeled subs

S259 S233 ndicate actant a

preparation The co cates the ntage of mentally ed solid

following formula

100 colids () times=entsoliatedntentoalExperimentConversion

n the experimenta content is the weight of olid rema after

mulsion at 120oC f in and the estimated solid content is th ht

lated from the recipe

rti as di each t load d

mple of ea pension iluted by (ww)

deionized water and the mean particle diameter was determined using the Brookhaven

ZetaPlus particle size analyzer From these results the seed latex particle size increased

as the amount of surfactant decreased The amount of emulsifier also affects the

polymerization process by changing the number of micelles and their size It is known

that large amount of emulsifier produces larger numbers of smaller sized particles [Odian

1991] and was corroborated in the current work

These synthesized seed latex particles should be of narrow size distribution in order

to obtain monodisperse larger particles The SEM image showing the particle

morphology and size of a sample of polystyrene seed particles can be seen in the Figure

4-2

contdEstim (4-1)

In this equatio l solid the s ining

evaporation of e or 30m e weig

of monomers calcu

As expected the seed pa cle size w fferent for surfactan ing in see

emulsion system A sa ch sus was d 0001 with

59

Figure 4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)

PSS259 (B) PSS233 (C) PSS207 (D) PSS181 (subscripts indicate the amount of surfactant added)

(A)

(B)

60

(C)

(D)

Figure 4-2 Continued

61

s seen in the image the seed particles are very smooth and spherical The

uniformity in the particle size distribution of these PS seed latex particles can also be

seen in the SEM image From this data we concluded that seed particles synthesized in

the presence of a surfactant with the chosen amounts are suitable for use to prepare larger

particles with high size uniformity and smooth spherical morphology

412 The Growth of Polystyrene (PS) Seed Latex Particles

Seeded emulsion polymerization has been conducted for several decades and

various mechanisms have been proposed Gracio et al [1970] suggested that the growing

polymer particles consist of an expanding polymer-rich core surrounded by a monomer-

rich shell with the outer shell providing a major locus of polymerization Seeded

mulsion polymerization is commonly used for preparing latex particles less than 1microm in

size [Cha et al 1995 Zou et al 1990 1992 Gandhi et al 1990 Park et al 1990] Such

latexes can be obtained from the growth of pre-prepared seed particles The seeds

introduced in this process serve as nucleation sites for particle growth By first swelling

the seed latex particles with additional monomer to the desired size polymerization can

then be initiated Several size ranges of monodisperse polystyrene (PS) latex particles

were prepared by this multistep seeded emulsion polymerization method For the

fabrication of particle-based membranes monodisperse latex particles with a variety of

size ranges are required

In this study seed particles from 171nm to 470nm in mean diameter and with a

highly uniform size distribution were prepared and used Watera is the initial charge

mount and waterb represents the amount aqueous solution containing dissolved initiator

and buffer that is continuously fed into system Feed time is precisely controlled by

metering pump The solid contents were maintained between 199 and 300 After

A

e

a

62

polym d as

es

ere 923 8402

n of these latex particles

are sh

ss

erization under these conditions the conversions of the latex particles labele

PS258 PS320 PS370 and PS410 were 952 957 946 and 964 respectively The recip

used in seeded emulsion polymerization to form the particles less than 500nm in mean

diameter are shown at the Table 4-2 The conversions of the latex particles larger than

500nm in mean diameter and labeled as PS525 PS585 PS640 and PS790 w

723 and 969 respectively The recipes used in the preparatio

own at the Table 4-3

A simple calculation can be used to predict the ultimate size that a latex particle can

reach after ending the polymerization Letrsquos suppose that seed density (ρs) and grown

particle density (ρgp) are the same The following equation is obtained from the ma

balance (m = ρV) where m and V are mass and volume of a particle respectively

gp

gp

s

s

Vm

Vm

In this equation m

= (4-2)

be

s and mgp are the weight of seed and total weight of seed and

added monomers respectively and Vs and Vgp are the volume of seed and grown

particle respectively

If we know the mass of monomer to be added the ultimate particle size can

calculated from the equation 4-3

3

2s D m ⎠⎝+ χ

where D

1s D m

⎟⎟⎞

⎜⎜⎛

= (4-3)

the

in

s and Dgp are the diameters for seed and grown particle respectively and χ is

weight of the monomer added Crosslinked PS latex particles with a size range from

258plusmn112 ~ 410plusmn117 in diameter polymerized by the recipes in Table 4-2 are shown

the SEM images in Figure 4-3

63

Table 4-2 Continuous addition emulsion polymerization recipe for growing polystyrene

Latex label PS

(PS) latex particles less than 500nm in size

258 PS320 PS370 PS410

Initial charge

Seed latex (g) 1000171 1000216 1000216 1000320

Watera (g) 1470 2500 1984 2600

06 07 06 06

Fee

Initiator stream

2 2 8

Feed time (min) 0-167 0-140 0-100 0-92

Solid content 284 300 300 200

Mean particle diameter (nm) 258plusmn112 320plusmn154 182plusmn71 410plusmn117

Conv

Monomer continuous

Styrene (g) 800 1200 800 600

Divinylbenzene (g)

d time (min) 0-163 0-110 0-75 0-82

Na S O (g) 07 065 05 05

NaHCO3 (g) 07 065 05 05

Waterb (g) 750 1200 200 370

ersion () 952 957 946 964 The solid content of seed latex for PS258 PS320 PS 370 and PS410 are 392 399 399 anrespectively

d 300

64

Table 4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size

Latex label PS525 PS585 PS640 PS790

Initial charge

Seed latex (g) 1000390 1000470 1000470 1000320

Watera (g) 1000 520 1200 360

Monomer continuous

Styrene (g) 290 200 400 173

Divinylbenzene (g) 02 014

03 009

Feed time (min) 0-80 0-40 10-76 5-65

Initiator stream

Na2S2O8 (g) 02 014 03 012

NaHCO3 (g) 02 014 03 009

Waterb (g) 200 120 400 200

Feed time (min) 0-85 0-46 0-77 0-75

Solids content 199 200 200 201

Mean particle diameter (nm) 525plusmn151 585plusmn186 640plusmn178 790plusmn693

Conversion () 923 8402 723 969 The solid content of seed latex for PS525 PS585 PS 640 and PS790 are 200 182 193 and 173 respectively

65

Figure 4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)

370nm (D) 410nm

(A)

(B)

66

(C)

(D)

Figure 4-3 Continued

67

article size was well matched to that estimated by the geometrical calculation

From this it can be concluded that the added monomer was consumed for polymer

particle growth with a high degree of conversion From the SEM characterization newly

nucleated particles were not seen as indicated by the narrow particle size distribution

The particles less than 500nm in mean diameter were very spherical in shape with a

highly uniform particle size distribution As the particles were grown larger than 500nm

mean diameter however they became of non-spherical shape with an uneven surface

This irregularity of particle surface can be attributed to the non-homogeneous monomer

swelling into the shell of a growing polymer which can be controlled by the factors such

as temperature agitation speed initiator feeding rate and surfactant amount required to

stabilize the colloidal system The SEM image of latex particles larger than 500nm mean

iameter is shown in Figure 4-4 The particle size distribution is broader than that of the

s particles These particles can however still be used for membrane construction

as a support layer rather than skin layer The support layer does not necessarily have to be

as highly monodisperse as the skin layer which require pores of high uniformity for the

selective removal of toxins

As mentioned earlier the amount of surfactant as well as monomer in an emulsion

system is the primary determinant of the particle diameter [Odian 1991] Anionic

surfactants are generally recommended at the level of 02-30 wt based on the amount

of water [Odian 1991] The critical micelle concentration (CMC) of Aerosolreg MA 80-I

anionic surfactant is 119 The surfactant loading in this emulsion system ranged from

the 121 for SS181 to 171 for SS259

P

d

maller

68

Figure 4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm

(C) 640nm (D) 790nm

(A)

(B)

69

(C)

(D)

Figure 4-4 Continued

70

The surfactant to monomer ratio refers to the value of the surfactant amount

divided by monomer amount after polymerization ends The surfactant amount is the

weight of the surfactant contained in emulsion and the monomer amount is the total

weight of the monomer added for polymerization to form the ultimate polymer particles

in emulsion As the surfactant to monomer ratio decreases mean particle diameter

increases as described by Odian [1991] Generally lower surfactant concentration forms

fewer micelles resulting in larger particle size [Evans et al 1999] However there is a

plateau in particle mean diameter between a surfactant to monomer ratio of 0005 and

0013 This is due to low polymerization conversion of latex particles from monomer

leading to a smaller particle size than expected This is not a factor of surfactant amount

The dependence of the particle size on the amount of surfactant is shown in Figure 4-5

0

200

400

600

800

1000

0000 0005 0010 0015 0020 0025 0030

Surfactant to monomer ratio (ww)

Part

icle

mea

n di

amet

er (n

m)

Figure 4-5 Dependence of the particle size on the surfactant to monomer ratio

71

413

e

f bare

microdomain structures is important for biocompatibility [Mori et al 1982 Higuchi et al

1993 Deppisch et al 1998] Figure 4-6 shows the schematic of core shell latex particle

structure The recipe for core shell structures is shown in Table 4-4

Core Shell Latex Particles

The latex particles shown in Figure 4-3 (A) and labeled as S258 in Table 4-2 wer

used as seeds to prepare core-shell latex particles because of their high particle

uniformity and smooth surface morphology Methyl methacrylate (MMA) and acrylic

acid (AA) monomers were introduced to increase surface hydrophilicity over that o

polystyrene particles AA is a more hydrophilic monomer than MMA because of the

carboxyl acid group in AA is more favorable for hydrogen boding with water The

surface carboxyl groups on PAA may have many promising applications in biomedical

and biochemical fields [Kang et al 2005] MMA monomer is less hydrophobic than

styrene PMMA is known to more biocompatible than PS but still PMMA is hydrophobic

The optimization of suitable hydrophobic to hydrophilic ratios and control of

Polystyreneseed particle

Core shelllatex particles

PSPMMA100

PSPMMA90PAA10

PSPMMA75PAA25

Polystyreneseed particle

Core shelllatex particles

PSPMMA100

PSPMMA90PAA10

Figure 4-6 Schematic of core shell latex particle structures

PSPMMA75PAA25

72

Table 4-4 The preparation recipe of PS core with various shell latex particles Latex 25 label PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA

Initial charge

PS seed latex (g) 500 500 500a

Monomer continuous

Methly methacylate (g) 320 288 240

Acrylic acid (g) 00 32 80

Initiator stream

Na

258 258 258

Water (g) 1570 1570 1570

Feed time (min) 0-120 0-50 0-60

b 200

Feed time (min) 0-122 0-60 0-65

952

2S2O8 (g) 02 02 02

NaHCO3 (g) 02 02 02

Water (g) 200 200

Solids content 183 183 183

Mean particle diameter (nm) 370plusmn186 370plusmn178 370plusmn191

Conversion () 955 924

Three types of core shell structures were prepared PMMA75PAA25 shell is a

copolymer consisting of the PMMA to PAA ratio of 75 to 25 by weight

PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90 to 10 by

weight PMMA100 is the PMMA homopolymer shell on PS core The particle size of

these core-shell particles as well as PS latex particle was prepared to be about 370nm in

mean diameter and are characterized to determine their zeta potential protein adsorption

and biocompatibility The image of SEM of PS and core shell particles is shown at Figure

4-7 The synthesized latex particles had a high uniformity in size and a smooth spherical

surface morphology

73

Figure 4-7 Scanning

Electron Micrograph of latex particles (A) PS (B) PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMMA75PAA25

(A)

(B)

74

(C)

(D)

Figure 4-7 Continued

75

42 Characterization of Latex Particles

421 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectroscopy has proven to be a powerful tool to characterize polymeric

materials A molecule can absorb only select frequencies (energy) of infrared radiation

which match the natural vibration frequencies The energy absorbed serves to increase

the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]

Only those bonds which have a dipole moment that changes as a function of time can

absorb infrared radiation The FTIR spectra of polymerized latex particles made during

this work are shown in Figure 4-8 There are number of characteristic peaks for PS latex

particles [Bhutto et al 2003 Li at al 2005] Aromatic C-H stretching vibration is shown

at 3002 cm-1 and 3103 cm-1 and aliphatic C-H asymmetrical and symmetrical stretching

shown at 2900 cm-1and 2850 cm-1 respectively There is an additional carbonyl (C=O)

adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some

amount of either or both PMMA and PAA The broad OH group peak at 3400 cm-1

appear for particles containing some amount of PAA and has intensity dependent on the

amount of PAA in the shell For example the peak intensity of OH groups for

PSPMMA75PAA25 is greater than that of PSPMMA90PAA10 since there is a higher AA

monomer content in PSPMMA75PAA25 than in PSPMMA90PAA10 This OH peak is not

seen for PS and PS core PMMA shell particles because of the absence of OH groups on

PS and PMMA polymer chains

422 Zeta Potential Measurements

The electrical surface property was characterized by zeta potential measurements of

tex particles Zeta potential is an important and useful indicator of surface charge which

can be used to predict and control the stability of colloidal systems The greater the zeta

is

la

76

1520002500300035004000

Wavenumbers (cm-1)

li

Re

ativ

e

Tra

nsm

ttanc

e

Figure 4-8 FTIR spectra of polymerized latex particles (A) ba

PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMM

(B)

(C) P

(D) P

C

C

OCH3

O

CH3

XCH2

CH2 n

CH

CH2 C

C

OCH3

O

XCH2 CH

OC

CH3

Y

OH

(A) Bare PS

500100000

re polystyrene (PS) (B) A75PAA25

PSPMMA100

SPMMA90PAA10

SPMMA75PAA25

77

potential the more likely the suspension is to be stable because the charged particles repel

one another and thus overcome the natural tendency to aggregate The measurement of

zeta potential is often the key ontrol of the interaction of proteins

with solid surfaces such as latex particles

Zeta potential (ζ) is the elec at exists at the shear plane of a particle

which is some small distance from the surface [Myers 1999] The zeta potential of the

particles can be determined by measuring the mobility of the particles in an applied

electric field termed electr its response to an alternating electric

field Colloidal particles dispersed in lu ically charged due to their ionic

characteristics and dipolar attributes The development of a net charge at the particle

surface affects the distribution of ions in the neighboring ng in

an increased concentration of counter ions to the surface Each particle dispersed in a

ns called fixed layer or Stern layer

Outside th compositions of ions of opposite polarities

form ble layer is form

particle-liquid interface This double layer may be considered to consist of two parts an

inner region which inclu nd relatively strongly to the surface and an outer or

diffuse region in which the ion distribution is determined by a balance of electrostatic

forces and random thermal motion The potential in this region therefore decays with the

distance from the surface until at a certain distance it becomes zero Figure 4-9 shows a

schematic representation of a typical ion distribution near a positively charged surface

The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the

to understanding and c

trical potential th

ophoretic mobility or

a so tion are electr

interfacial region resulti

solution is surrounded by oppositely charged io

e Stern layer there are varying

ed in the region of the ing a cloud-like area Thus an electrical dou

des ions bou

center of those ions which are effectively adsorbed to the surface [Hunter 1981] The

78

extent of ion adsorption is determined by electrical and other long-range interactions

between the individual ions and surface of particles The ions outside of the Stern layer

form the diffuse double layer also referred to as the Gouy-Chapman layer [Burns and

Zydney 2000]

Shear plane

Stern layer

ψζ

0

ψs

ψShear plane

Stern layer

ψζ

0

ψs

ψ

t

controlled electric field is applied via electrodes immersed in a sample suspension and

Figure 4-9 Schematic representation of ion distribution near a positively charged surface

Zeta potential is a function of the surface charge of a particle any adsorbed layer a

the interface and the nature and composition of the surrounding medium in which the

particle is suspended The principle of determining zeta potential is very simple A

79

this causes the charged particles to move towards the electrode of opposite polarity

Viscous forces acting upon the moving particle tend to oppose this motion and the

equilibrium is rapidly established between the effects of the electrostatic attraction and

the viscosity dra

g The particle therefore reaches a constant terminal velocity

Figure 4-10 Schematic representation of zeta potential measurement(source

httpnitioncomenproductszeecom_shtm)

Because protein adsorption mechanisms are very complex and hard to explain in

biological systems protein adsorption is generally studied by using more ideal systems

consisting of one or more well characterized proteins a well-characterized adsorbent and

a well-defined aqueous solution Even so small changes in the experimental conditions

such as pH ionic strength and temperature generate totally different results Therefore

two media systems phosphate buffer (PB) and phosphate buffered saline (PBS) were

chosen to be used for the zeta potential measurements and protein adsorption study PB is

a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4H2O) to

n a constant pH PBS is used to keep the water concentration inside and outside of

the cell balanced If the water concentration is unbalanced the cell risks bursting or

shriveling up because of a phenomenon called osmosis PBS is a solution with the 5 mM

of monobasic sodium phosphate (NaH2PO4H2O) and 143 mM of sodium chloride (NaCl)

in water

maintai

80

The results of zeta potential measurement at room temperature (25plusmn1oC) as

function of pH and ionic strength (different media PB and PBS) for synthesized lat

particles are seen in Figure 4-11 Zeta potential values of polystyrene (PS) latex particle

at the Figure 4-9 are negative between -291 mV and -599 mV in PB and betwe

ex

s

en -203

mV and -278 mV in PBS at all pH ranges These negative zeta potential values of PS

latex particles are due to the sulfate groups [Vandenhu et al 1970] originated from

persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer

chain where polymerization was initiated

-100

-80

-60

-40

-20

0

pH

Zta

pte

ntl

ζ

20

40

1 2 3 4 5 6 7 8 9

eo

ia (m

V)

PB (5mM)

PBS (5mM +143mM)

en -

Figure 4-11 Zeta potential of PS latex particles at 25oC

The zeta potential measurements of PSPMMA100 core shell latex particles as seen

in Figure 4-12 were also negative with a range of -28 and -505 mV in PB and betwe

143 mV and -186 mV in PBS at the pH 21-78 These negative values are also due to

sulfate groups on the particle surface

81

-100

-80

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

Zea

pote

nti

l ζ

(mV

pH

ta

)

PB (5mM)

PBS (5mM + 143mM)

Figure 4-12 Zeta potential of PSPMMA100 latex particles at 25oC

The zeta potential for PSPMMA90PAA10 and PSPMMA75PAA25 was also

negative as seen in Figure 4-13 and 4-14 The zeta potential value for the

PSPMMA90PAA10 were between -367 mV and -678 mV in PB medium and between -

147 mV and -193 mV in PBS both at 25oC In case of the PSPMMA75PAA25 core shell

particles zeta potential values were between -291 mV and -520 mV in PB and between

-115 mV and -210 mV in PBS media Sulfate groups in PSPMMA75PAA25 and

PSPMMA90PAA10 core shell particles contribute to the negative zeta potential values

participate in forming a

negat

cid is expressed by the term pKa which is the pH

at which an acid is 50 dissociated However the contribution of the carboxylic acid

Carboxylic groups from acrylic acid monomer also would

ive surface charge of the latex particles at a pH greater than 70 because the pKa

value for the carboxylic acid is between 40 and 60 pH range [Ottewill et al 1967] This

indicates that carboxylic acid dissociation begins at a pH between pH 46-60 and

increases with pH The strength of an a

82

group to negative zeta potential values of PSPMMA-PAA core shell particles was not

detectable because there was no significant difference in zeta potential values for

synthesized latex particles at a pH greater than 70

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

Zeta

pot

entia

l ζ

(mV

)

PB (5mM)PBS (5mM + 143mM)

-80

-100

pH

Figure 4-13 Zeta potential of PSPMMA90PAA10 latex particles at 25oC

-100

-80

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

pH

Ze p

ont

i ζ

V)

tate

al (m

PB (5mM)PBS (5mM + 143mM)

Figure 4-14 Zeta potential of PSPMMA75PAA25 latex particles at 25oC

83

The zeta potential plots and values were similar for all latex particles This may be

due to the similar initiator density on the latex surface The initiator concentrations

monomers for shell polymerization were the same fo

to

r all core shell latex particles (062

ww from Table 4-4) A similar concentration (055 ww) of initiator to monomer was

added to prepare bare PS latex particles used in zeta potential measurements

Since the zeta potential values of all synthesized latex particles were negative

between pH=20 and pH=78 the isoelecric point (IEP) of these latex particles would be

less than pH=20 Sulfates have a pKa in the range of 10 to 20 [James 1985] indicating

that the sulfate group the conjugate base of a strong acid is protonated (-C-OSO3H) at

less than pH 20 This means that the synthesized latex particles are all negative at the

physiological pH Most serum proteins have a negative surface charge in blood

therefore when the negative latex particles are applied to blood stream fatal

tein is

avoided by charge repulsion in the blood Table 4-5 shows the common blood proteins

and their isoelectric points (IEP)

Table 4-5 The isoelectric point (IEP) of common proteins Serum proteins Isoelectric point (IEP)

complication by coagulation between applied latex particles and serum pro s

Albumin 48 α1-Globulin 20 α2-Microglobulin 54 γ1-Globulin 58 Heptoglobin 41 Hemoglobin 72 β1-lipoprotein 54 Immunoglobulin G (Ig G) 73 Fibrinogen 58 (source httpwwwfleshandbonescomreadingroompdf1085pdf

httpwwwmedalorgvisitorwww5CActive5Cch135Cch13015Cch130105aspx )

84

The absolute zeta potential values of the latex particles in phosphate buffer (PB)

was higher than that in phosphate buffered saline (PBS) This is because high

concentration of sodium and chloride electrolytes in PBS compressed the electrical

double layer causing rapid decay of surface potential Figure 4-15 shows that the

thickness of the electrical double layer is inversely proportional to the concentration of

electrolyte in the system

Surf

ace

Pote

ntia

l ψ

1κ3

10

1e

1κ 1κ

(1)(3) (2)

2 1 Z

Surf

ace

Pote

ntia

l ψ

1κ3

10

1e

1κ 1κ

(1)(3) (2)

Figure 4-15 The decay of surface potential with distance from surface in various

electrolyte concentrations (1) low (2) intermediate (3) high [Myers 1999]

2 1 Z

The electrical potential in the solution surrounding the particle surface falls off

the Debye-Huckel

yers 1999]

exponentially with distance (Z) from the surface according to

approximation [M

z) (- exp 0 κψψ = (4-4)

e reciprocal of the thickness of the electrical double layer referred to as the

The potential fallen off by a factor e The theoretical equation for the

ickness 1κ is

where κ is th

Debye length of 1

double layer th

21

22i

0

ekT 1

⎟⎟⎠

⎜⎜⎝

=sum ii zcεε

κ

⎞⎛ (4-5)

85

where

tration of

y was performed to measure the protein adsorption on the

synthesized latex particles Many physical and chemical processes occur at the boundary

between two phases and adsorption is one of the fundamental surface phenomena [Oscik

1982] This phenomenon is driven by the change in the concentration of the components

at the interface with respect to their concentrations in the bulk phase There are two

adsorption processes physical adsorption occurring when non-balanced physical forces

appear at the boundary of the phases and chemical adsorption or chemisorption

t the

interface [J

n leads

ational entropy hence adsorption takes place only if the

loss in conformational entropy is compensated

ter adsorption Cp The initial part of the

isotherm merges with the Γ-axis because at low polymer concentration the protein has a

high affinity for the surface and all of the polymer is adsorbed until the solid surface is

saturated

ε0 is the permittivity of a vacuum of free space ε is the permittivity of a medium e

is the relative permittivity or dielectric constant of the medium c is the concen

an ion (i) z is the valency of an ion (i) k is Boltzmannrsquos constant T is absolute

temperature (K)

43 Protein Adsorption Study

The adsorption stud

occurring when atoms and molecules from adjacent phases form chemical bonds a

aroniec et al 1988]

Flexible proteins in solution possess high conformational entropy as a result of the

various states each of the many segments in the protein chain can have Adsorptio

to a reduction of this conform

by sufficient attraction between polymer

segment and solid surface [Martin 1998] Isotherm shape of high affinity adsorption

between polymer and solid is shown in Figure 4-16 where the adsorbed mass Γ is plotted

against the polymer concentration in solution af

86

Figur

de

arboxyl

e 4-16 High-affinity adsorption isotherm of typical flexible polymer on solid

surface

In order to investigate the adsorption of blood proteins latex particle properties

such as electrical surface charge hydrophobicity and environmental conditions like pH

ionic strength and temperature are considered as experimental factors [Arai and Nor

1990] Figure 4-17 shows the overall schematic representation of the protein adsorption

on the synthesized latex particles with the functional groups such as sulfate and c

groups

CP

Γ

PMMAPAAShell

PS or PMMAShell OSO3

-

OSO3-

OSO3-

OSO3-

PS

-O3SO

PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

Protein

NH3+

+

+H3N

+

COO-

Hydrophobic interaction

NH3

COO-

H3N

COO-

-OOC

Ionic interaction

Hydrophobic interaction

Hydrogen bonding

Ionic interaction

PMMAPAAShellOSO3

-

OSO3-

OSO3-

OSO3-

PS or PMMAShell -O3SOOSO3

-

PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

-O3SO

OSO3-

OSO3-

OSO3-

PS PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

Protein

NH3+

+

+H3N

+

COO-NH3

COO-

H3N

COO-

-OOC

NH3+

+

+H3N

+

COO-

Hydrophobic interaction

NH3

COO-

H3N

COO-

-OOC

Ionic interaction

Hydrophobic interaction

Hydrogen bonding

Figure 4-17 Overall schematic representation of the protein adsorption on the

Ionic interaction

synthesized latex particles

The surface functional groups as well as suitable surface hydrophobicity

hydrophilicity of latex particles are closely related to protein adsorption property As

indicated at Figure 4-17 various interaction forces such as hydrophobic interaction ionic

87

interaction hydrogen interaction and van der Waals interaction [Andrade 1985] exist

between protein molecule and the solid surface of latex particle

431 Adsorption Isotherm

The protein adsorption isotherm is generally defined as the relationship between the

amount of protein adsorbed by a solid (latex particle) at a constant temperature and as a

ine

the amount of protein adsorbed onto latex particles To evaluate the equilibrium

conce s

x

function of the concentration of protein Adsorption isotherms were plotted to determ

ntration of target proteins a calibration curve is firstly needed Figure 4-18 show

the UV absorbance as a factor of bovine serum albumin (BSA) concentrations The UV

absorbance of the known protein concentration was measured at 562nm which is the λma

for a protein treated by a bicinchoninic acid (BCA) assay

0

02

04

06

08

500 520 540 560 580 600

Wavelength (nm)

Abs

orba

nce

Figur gml (B)

06mgml (C) 02mgml

(A)

(C)

(B)

Max562nm

e 4-18 The dependence of UV absorbance on BSA concentration (A) 10m

Figure 4-19 shows the standard calibration curve used in adsorption isotherm

experiment of protein onto latex particles The net absorbance of each known protein

88

concentration was plotted and then a linear equation passing through a concentration of

zero protein concentration was obtained This equation is applied

to determine the

orbed on the synthesized latex particles The amount of free

protei

eported

trations (005 01 03 05

and 07mgml) were used to generate this curve as well as the adsorption isotherm

unknown protein amount ads

n was also determined using UV light at the 280nm wavelength however a

distinguishable UV intensity was too low to determine a precise value of protein

adsorption

The bicinchninic acid (BCA) assay technique [Smith et al 1985] has been r

for high efficiency of protein evaluation and was used for determination of protein

adsorption [Tangpasuthadol et al 2003 Williams et al 2003] This technique has many

advantages such as high sensitivity easy of use color complex stability and less

susceptibility to any detergents Five different BSA concen

y = 00R2 =

004741x 09939

00

01

02

03

04

0 200 400 600 800

BSA Standard (microgml)

Net

Abs

orba

nce

at 5

62nm

Figure 4-19 Standard curve of net absorbance vs BSA sample concentration

89

All isotherms were fitted to the Langmuir-Freundlich isotherms using the nonlinea

regression method Two adsorption isotherm models the Langmuir type isotherm a

Langmuir-Freundlich combination type isotherm were compared As seen the Figure 4-

20 The Langmuir isotherm model deviated further from the experimental data Th

all isotherms were fitted to the Langmuir-Freundlich isotherms [Yoon et al 1996

al 1998] usi

r

nd the

erefore

Lee et

ng the nonlinear regression method with following equation

n

n

eq

eqm

kC

kC1

1

1+

where q is the amount of adsorbed BSA per unit surface area q

qq = (4-6)

stant

were between 045 and 055 and hence n was fixed to its average 05 Actual qm values

were evaluated after fixing the n value

m is the adsorbed amount

in equilibrium Ceq is the equilibrium concentration of BSA k is the adsorption con

and n is the exponential factor for a heterogeneous system n values are empirical and

usually from zero to no more than unity [Jaekel 2002] The values of n in our system

00

10

20

30

0 02 04 06 08

C (mgml)eq

q (m

gm

2 )

pH 32Langmuir-Freundlich modelLangmuir model

Figure 4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on

polystyrene latex particles at 37oC in phosphate buffer media

90

The BSA protein adsorption on the synthesized latex particles was performed at

three pH levels 32 48 74 in both phosphate buffer (PB) and phosphate buffered saline

(PBS) at physiological temperature 37oC These pH levels correspond to an acidic pH

lower than the isoelectric point (IEP) of BSA the pH at the IEP and an alkaline pH

higher than the IEP of BSA respectively The IEP corresponds to the pH at which the

amino acid becomes a zwitterion Here its overall electrostatic charge is neutral and the

charge expressed by the basic amino group (-NH2 -NH3+) of the amino acid is equal to

the charge expressed by the acidic carboxyl group (-COOH -COO ) The IEP of BSA is

-

pH 48 and indicates that the side chain of the amino acids of BSA contains a greater

amount of acidic functional groups than that of basic functional groups At pH 32 and

74 BSA has a positive and negative charge respectively

Figure 4-21 and 4-22 show adsorption isotherms of BSA onto polystyrene (PS)

latex particles

00

10

20

30

40

0 02 04 06 08

Ceq (mgml)

q (m

gm

2 )

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in

phosphate buffer (PB) at 37oC

91

00

10

30

40

0 02 04 06 08

Ceq (mgml)

q (m

gm

2

20

)

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

(PBS) on PS latex particles

At the IEP of BSA the adsorbed amount (q

Figure 4-22 Adsorption isotherm of BSA adsorption 37oC in phosphate buffered saline

onto PS at equilibrium was

maxim

um at the IEP of BSA This is because the BSA molecules form the most

compact structures at the IEP eliminating the repulsion force between proteins A higher

number of BSA molecules can adsorb on a given surface area by hydrophobic interaction

when in this compact structure This observation agrees well with reported data by other

researchers who also indicated that the maximum adsorption from aqueous protein

solution was observed at the IEP [Mularen 1954 Watkins et al 1977 Oh at el 2000]

There was no significant difference of adsorbed BSA amount in PB and in PBS media at

pH 48 The conformational alteration of BSA was not affected significantly by the ionic

strength of the two solutions There was evidence of the effect of the electrostatic

interaction to some extent even at the IEP since the amount of BSA adsorbed in PBS is

slightly hig

m) of BSA

um indicating that hydrophobic interaction between latex particle and BSA

protein is maxim

her than in PB at this pH

92

The adsorbed amount of protein is shown to decrease at a pH 32 and 74 The

decrease in adsorption efficiency when deviating from IEP of BSA is a result of the

increase in conformational size of the protein molecules and the lateral electrostatic

repulsions between adjacent adsorbed BSA molecules Figure 4-23 shows a schematic of

possible conformational changes at each pH where hydrophobic interaction always exists

between non-polar portion of a protein and hydrophobic portion of solid surface

- - --- ---Latex surface

Zeta potential

BSA(+)

(-)

48 7432pH

(0)

+++ + ++

--+

++-

- - --- ---Latex surface

Zeta poten pH(0)

+++ + ++

--

tial

BSA(+)

(-)

48 7432

+

++-

ming

4

s

al blood Table 4-6 shows the calculated results of

Figure 4-23 Conformation of bovine serum albumin (BSA) on latex particles (Assuthat charge repulsion reduces the effect of the hydrophobic interaction at pH 74)

In addition the adsorbed amount of BSA at pH 32 was larger than that at pH 74

and still less than that at 48 This is because there is the electrostatic interaction between

the positive BSA molecules and negative PS latex particles at pH 32 and electrostatic

repulsion is dominant between negative BSA and negative PS latex particles at pH 7

The amount adsorbed increased with increasing ionic strength in pH 32 and 74 This i

because the high concentration of electrolytes reduces the electrostatic repulsion between

BSA and PS latex particle It should be noted that the BSA concentration used in these

experiments was lower than that found in normal human blood This is because the

equilibrium BSA adsorption onto the latex particles is actually reached at a much lower

concentration than that found in norm

93

adsor rm

ein

leading to a compact conformation of BSA There are two main forces affecting

adsorption amount at an acidic pH of 32 They include the ionic attraction between the

negative latex particle and the positive protein and lateral repulsion between positive

proteins The smallest amount of BSA adsorption was observed at a pH of 74 At this

point the charge repulsion between negatively charged particles and protein molecules

exists the h

of cha

bed BSA amount at equilibrium from the Langmuir-Freundich adsorption isothe

equation

In summary the hydrophobic interaction between PS latex particle and BSA

protein exists and is the dominant mechanism for the adsorption process Conformational

changes of BSA at each pH also affected the adsorption amount Adsorption was highest

at pH 48 because at this pH there is the least amount of ionic repulsion in the prot

and the lateral repulsion between proteins dominant adsorption even though there always

ydrophobic interaction between the solid latex particle and protein The effect

rge repulsion was reduced by the high concentration of electrolytes in PBS medium

Table 4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2) 32 174 48 299 Phosphate buffer 74 136 32 260 48 283 Phosphate buffered saline 74 207

The adsorption isotherms of BSA on PSPMMA core shell particles are shown in

Figure 4-24 in phosphate buffer (PB) and in Figure 4-25 in phosphate buffered saline

(PBS) The adsorbed BSA amount was calculated using the Langmuir-Freundlich

100

94

adsorption isotherm equation and the amount listed in Table 4-7 The trend seen in the

adsorption results was very similar to the case of PS latex The overall adsorption process

was d rticle

rs

hat

0

ominated by hydrophobic interactions between the PSPMMA100 core shell pa

and BSA proteins At pH 48 the adsorbed amount (qm) of BSA on PSPMMA100 core

shell particles was 221 mgm2 in PB and 237 mgm2 in PBS and are the maximum

values recorded among the three pH levels 32 48 and 74 Suzawa and his co-worke

[1982] have obtained similar results This can be also explained by the compact

conformation of BSA molecules at the IEP of BSA which lead to a high protein

population on the solid surface At a pH of 32 and 74 BSA adsorption was less than t

at pH 48 because of the protein conformation change There was a small increase in

adsorbed BSA at pH 32 because of the charge attraction between negative PSPMMA10

core shell particles and positive BSA At pH 74 however the adsorption of BSA was

further reduced by charge repulsion between the negative PSPMMA100 core shell

particles and the negative BSA proteins

000 02 04 06 08

Ceq (mgml)

10

20q (m

gm

302 )

40

50pH=32pH=48pH=74Mode 8)l (pH 4Mode 2)l (pH 3Mode 4)

Figure 4-24 Adsorption isotherm of BSA 37oC in PB on PSPMMA core shell latex

l (pH 7

100

particles

95

The adsorbed amount of BSA increased more rapidly in PBS with increasing ioni

strength at pH 74 This shows that the low concentration of electrolytes promotes

electrostatic repulsion between negative BSA and negative PSPMMA

c

on

x

l 1980

100 core shell

particle As the ion strength of PBS is increased however the high concentration of

electrolyte leads to less electrostatic repulsion promoting more rapid protein adsorpti

By comparing the amount of adsorbed protein between bare PS latex particles and

PSPMMA100 core shell particles the amount of protein adsorption on the PS late

particles is greater than that on the PSPMMA100 core-shell particles [Suzawa et a

1982 Lee et al 1988]

00

10

20

30

40

m2 )

0 02 04 06 08

Ceq (mgml)

q (m

g

pH=32pH=48pH=74Model (pH 2) 3Model (pH 48)Model (pH 74)

Figure 4-25 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA100 core shell

latex particles

96

Table particles calculated the Langmuir-Freundlich isotherm model

4-7 The equilibrium concentration values (qm) of BSA adsorption on PSPMMA100

Media pH qm (mgm ) 2

32 140

48 221 Phosphate buffer

74 130

32 169

48 237 Phosphate buffered saline

74 162

The BSA adsorption mechanism and trend on PSPMMA core shell particles was

very similar to that of the PS latex particle Unlike the case of PS and PSPMMA100 latex

particles the amount of BSA adsorbed onto PSPMMA90PAA10 and PSPMMA75PAA25

core shell particles was higher at pH 32 than pH 48 and 74 Figure 4-26 through 4-29

shows the adsorption isotherms of BSA on PSPMMA90PAA10 and PSPMMA75PAA25

core shell particles The amount of BSA adsorbed increased at pH 32 and pH 48 as the

hydrophilicity was developed by the addition of PAA On the contrary the adsorbed

amount of BSA was dramatically decreased at pH 74 The adsorption amount of BSA on

PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles at equilibrium is listed in

Table 4-8 and 4-9 respectively

Carboxylic acid as well as sulfate groups are distributed on the surface of

PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles and seem to largely affect

the BSA adsorption The isotherms showed very strong pH dependence to the BSA

adsorption onto these core shell latex particles The carboxylic groups (COOH) may

especially enhance the BSA adsorption prominently at pH 32 by hydrogen bonding with

protein molecules The pKa value for the PAA is 40-60 and a good indication that the

97

carboxylic acid groups are protonated at pH 32[Gebhardt et al 1983] The main

adsorption interaction force at pH 32 was generated by hydrogen bonding as well as

charge attraction between latex particle protein even th the conformational

repulsion between proteins results in a reduction of the protein

adsorption at this pH The amount of BSA adsorbed also increased as the PAA amount in

eased The a tion of BSA w inimal however less

than 07 mgm2 at pH 74 Here the carboxylic groups are ionized which increases the

charge repulsion by ionized carboxylic groups (COO-) resulting in PSPMMA-PAA latex

particle

and ough

change and charge

the core shell particles incr dsorp as m

s with much lower protein adsorption

00

20

40

q (m

g2 )

60

m

pH=32pH=48pH=74Model (pH 32)Model (pH 48)

0 02 04 06 08

Ceq (mgml)

Model (pH 74)

o shell

Figure 4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA90PAA10 corelatex particles

98

00

40

60

80

0 02 04

C (mgm

mm

2 )

20

06 08

eq l)

q (

g

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-27 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA90PAA10 core

shell latex particles

Table 4-8 The equilibrium concentration values of BSA adsorption on PSPMMA90PAA10 particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2)

32 361

48 283 Phosphate buffer

74 043

32 519

48 226 Phosphate buffered saline

74 043

99

00

20

40

60

80

100

0 02 04 06 08Ceq (mgml)

q (m

gm

2 )

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

oFigure 4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA75PAA25 latex particles

00

100

20

40

60

80

0 02 04 06 08Ceq (mgml)

q (

m2 )

pH=32pH=48pH=74

mg

Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-29 Adsorption isotherm of BSA 37oC in PBS on PSPMMA75PAA25 core shell

latex particles

100

Table 4-9 The equilibrium concentration values of BSA adsorption on PSPMMA75PAA25 particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2)

32 1083

48 662 Phosphate buffer (PB)

74 075

32 1077

48 681 Phosphate buffered saline (PBS)

74 057

The adsorption of β2-microglobulin (β2M) was determined under one set of

onditions in phosphate buffer at 37oC and pH 74 because of the limited amount of β2M

protein The adsorption isotherms of the β2M protein on the synthesized latex particles

are shown in Figure 4-30 4-31 and 4-32 The β2M protein concentrations for adsorption

isotherm experiment were chosen to be 0015 0030 0045 and 0060mgml since the

β2M concentration for the patients with renal failure can be elevated to similar

concentrations about 005mgml or more up to 50 times the normal level [Nissenson et

al 1995] There were steep initial slopes of the adsorption isotherm curves of β2M on

latex particles in all isotherms indicating a high affinity adsorption type It seems that the

hydrophobic interaction is enough for the adsorption process of β2M proteins to occur on

the latex particles even though charge repulsion exists between protein and latex particle

at pH 74 The complete plateau regions were not seen for all isotherms indicating that

β2M proteins were not saturated at this level and there are still available sites for β2M

c

adsorption on latex particles The initial slope of the β2M isotherms for PS was steeper

101

than that of any of the other latex particle PSPMMA100 PSPMMA90PAA10 and

PSPMMA 75PAA25

00

02

0 0002 0004 0006 0008

04

06

10

Ceq (mgml)

q (m

2 )

08

gm

PSPSPMMA100Model (PS)Model (PSPMMA100)

Figure 4-30 Adsorption isotherm of β2M onto PS and PSPMMA100 latex particles in PB

at 37 C and pH 74 o

00

02

04

06

08

10

0 0002 0004 0006 0008C (mgml)

q (m

m2 )

eq

g

PS

PSPMMA90PAA10

Model (PS)

Model (PSPMMA90PAA10)

Figure 4-31 Adsorption isotherm of β2M onto PS and PSPMMA90PAA10 latex particles

in PB at 37oC and pH 74

102

00

02

04

06

08

10

0 0002 0004 0006 0008Ceq (mgml)

q (m

gm

2 )

PS

PSPMMA75PAA25

Model (PS)Model (PSPMMA75PAA25)

Figure 4-32 Adsorption isotherm of β2M onto PS and PSPMMA75PAA25 latex particles

in PB at 37oC and pH 74

The equilibrium concentration values (qm) of β2M adsorption on latex particles are

calculated and listed in Table 4-10 The difference between the maximum amounts of

β2M adsorption on all the latex particles was not significantly different and their values

were between 069 and 08mgm2

Table 4-10 The equilibrium concentration values of β2M adsorption calculated the Langmuir-Freundlich isotherm model

Latex particles qm (mgm2)

PS 080

PSPMMA100 069

PSPMMA90PAA10 072

PSPMMA75PAA25 072

103

432 Adsorbed Layer Thickness

There are several possible explanations for large amount of BSA adsorbed at acidic

pH levels including the end-on adsorption pattern of BSA the flexibility of BSA

molecules the tilting of protein molecules due to the asymmetry of the charge

distribution or multilayer formation [Peula et al 1993] The adsorbed BSA thickness (δ)

can be calculated by the following equation [Chiu et al 1978] This equation was also

cited again by other researchers [Shirahama et al 1985]

BSA

mqρπ

δbullbull

=33

(4-7)

where π

33 is the packing factor qm is the plateau value of the adsorbed am

ρBSA is the hich corresponds to the reciprocal of its known

partial specific volume The layer thickness (δ) of BSA proteins on synthesized latex

partic

he IEP [Shirahama et al 1985] Compared to the

hydrodynamic dimensions of BSA 140 times 40 times 40 Aring3 [Fair et al 1980 Peter 1985] these

nd

ode It is unnecessary ultilayering because thickness do

exceed 140 Aring The layer thickness (δ) of BSA proteins on synthesized latex particles

e red saline (PBS) was ran 28 ~ 83 Aring at 37oC and pH 48 These

lso listed in Table 4-1

ount and

density of BSA molecule w

les in phosphate buffer (PB) ranged from 27 - 81 Aring at 37oC and pH 48 These

calculated values are listed in Table 4-11 Only δ values at pH 48 the isoelectric point

(IEP) of BSA are important because intra- and intermolecular electrostatic repulsion of

protein molecules is minimized at t

values of layer thickness indicate that the BSA molecules adsorb by side-on (40Aring) a

end-on (140Aring) m to consider m

not

in phosphat buffe ged of

calculated values are a 2

104

Table 4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37oC pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

32 21 17 44 133

48 37 27 35 81

74 17 16 5 9

Table 4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37oC

pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

32 32 21 64 132

48 35 29

74 26 20 5 7

28 83

1n

1n

neqm

So k is closely related to the initial slope of the isotherm showing the affinity

between BSA and latex particles [Yoon et al 1996] A key point in the characterization

433 Gibbs Free Energy of Protein Adsorption

In the Langmuir isotherm model k represents the ratio of adsorption to the

desorption rate constants This definition is not applicable to the Langmuir-Freundlich

isotherm model but its meaning is similar [Yoon et al 1996] In this sense k is closely

related to the affinity between protein molecules and latex particles When the

equilibrium concentration Ceq reaches zero the value of Ceq will be very small (1gtgt

kCeq ) and then the equation 4-6 becomes

(4-8)

of adsorption processes on solid-liquid interfaces is to determine the change in Gibbsrsquos

free energy (∆Gdeg) one of the corresponding parameters for the thermodynamic driving

forces during adsorption The traditional method to evaluate adsorption thermodynamic

kCqq 1=

105

constants is based on measurement of adsorption isotherms under static conditions

[Malm ten 1998] The Langmuir-Freundlich ads lf

easily found from Then this enables calculation of the free-

energy change ∆G during adsorption expressed as

(4-9)

where R is the general gas constant (R = 8314Jmiddotmol K ) and T is the absolute

tem ture Gibb e energy A P

listed in Table 4-13 and 4-14 respectively As the ∆Gdegads ore negativ e

pro adsorption on a solid substra ill be more favorable

e of BSA adsorption in phosphate o

pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

s orption equilibrium constant k can be

a slope of the isotherm

degads

lfads kRTG lnminus=∆ o

-1 -1

pera s fre change of BS adsorption values in B and PBS are

values are m e th

tein te w

Table 4-13 The values of Gibbs free energy changbuffer (PB) at 37 C

32 -2411 -2726 -2846 -2805

48 -2352 -2383 -2198 -1972 ∆Go

80

ads (kJmol)

74 -2452 -888 -324 -2

Table 4-14 The values of Gibbs free energy change of BSA adsorption in phosphate

pH PS PSPMMAbuffered saline (PBS) at 37oC

100 PSPMMA90PAA10 PSPMMA75PAA25

32 -2315 -2620 -2312 -2683

48 -2149 -2299 -2090 -2138 ∆Go

(kJmads ol)

74 -2430 -1992 -258 -263

Table 4-15 shows the calculated values of Gibbs free energy change of β M

adsorption on synthesized latex particles PSPMMA PAA core shell latex particle has

2

75 25

106

largest negative Gibbs free energy of β2

2

M adsorption indicating largest affinity betw

β

een

2M adsorption in phosphate buffere (PB) at 37 C and pH74

PS PSPMMA100

M and latex particle

Table 4-15 The values of Gibbs free energy change of βo

PSPMMA90PAA10 PSPMMA75PAA25

∆Go03 ads

(kJmol) -3626 -3466 -3659 -39

The Gibbs free energy of β2M was much more negative in phosphate buffer (PB)

than that of BSA in physiological condition (37 C and pH 74) o from the Table 4-13 and

the Figure 4-31 This indicates that β2M molecules more readily adsorb on latex particles

BSA This can be explained by the rate of diffusion and accessibility of β2M molecules to

the latex particle ier and faster for β2M m an BSA because of

size (molec ight) erical shape molecules It o be

due to the higher hydrophobic force and less electrostatic repulsion that contribute to the

adsorption of β2M molecules not as prevalent in the case of BSA because the IEP of β2M

(57) is clo H 74) than that of BSA (48) causing β2M to

be less electro cally lled p ro

can be seen that the ∆ β2M adsorption onto the synth article

increasing wi hy icity o x particles s the ∆Goads of the BSA

molecules is decreasing or constant This phenomenon supports the theory that the

select lizing

than BSA and that the affinity of β2M for the latex particle is much larger than that of

surface It is eas olecules th

the smaller ular we and sph of β2M may als

ser to physiological condition (p

stati repe from the latex articles than BSA F m Figure 4-33 it

Goads of esized latex p s is

th the drophil f the late wherea

ive adsorption of β2M molecules over BSA molecules can be enhanced by uti

more hydrophilic PSPMMA90PAA10 and PSPMMA75PAA25 core shell latex particles

107

-60

-50

1 2 3 4

-40∆Gad

s

-30

10

0

o (k

Jm

o-

-20l)

β2-Microglobulin (β2M)Bovine serum albumin (BSA)

Figure 4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37

and pH 74

434 Kinetics of Adsorption

to

oC

Protein adsorption is a complex process known to have a large biological impact

and is currently not well understood quantitatively because of extreme sensitivity to pH

the concentration of other electrolytes and molecules present temperature and many

other factors that can change in the physiological system Accurate knowledge of the

adsorption kinetics under a given set of conditions is a prerequisite for elucidating the

mechanisms of many fundamental biological processes to be predicted at the molecular

level [Regner 1987] This adsorption kinetics approach is used in protein adsorption

studies because of the uncertainty related to the time needed for the interfacial proteins

reach the equilibrium It is generally accepted that the process of protein adsorption is

comprised of the following steps a) transport toward the interface b) attachment at the

interface c) eventual structural rearrangements in the adsorbed state d) detachment from

PS PSPMMA100 PSPMMA90PAA10PSPMMA75PAA25

108

the interface and transport away from the interface Each of these steps can affect the

overall rate of the adsorption process

The quantitative analysis of the protein adsorption kinetics requires that the protein

amount adsorbed is known as a function of time The kinetics tests were carried out in

phosphate buffer (PB) at 37oC and pH 74 a condition similar to physiological condition

The protein concentrations chosen were 07mgml for BSA and 006mgml for β2M

which are the maximal concentrations used in an adsorption isotherm test A time-based

evaluation of the adsorption was made by measuring the concentration of protein in

solution at different incubation times The results are shown below from Figure 4-34 to 4-

37 The adsorption kinetics of proteins onto th cond-e latex particles is expressed as a se

order model [Oumlzacar 2003] shown by the following equation

2)( tet qqk

dtdq

minus= (4-10)

Integrating above equation and applying the boundary conditions yields

ee

t tq

+=

1 (4-11)

where q

qkq

t

2

2

2

the previous adsorption isotherm results representing good reproducibility of adsorption

t and qe stand for the amount (mgm ) of protein adsorbed at time t and at

equilibrium respectively k is the equilibrium rate constant of second-order adsorption

(m mghr) and t is the incubation time (hr)

The fitting parameters calculated by using the equation 4-11 are listed in table 4-16

From this data it is seen that the amount of BSA adsorbed at equilibrium was higher than

that of β2M on PS and PSPMMA100 latex particles but less than that of β2M on

PSPMMA90PAA10 and PSPMMA75PAA25 latex particles These results are similar to

109

00

05

15

20

0 2 4 6 8 10 12 14

Time (hr)

rot

n ad

sorb

d (

10

25

Pei

em

gm

2

BSA2nd order model (BSA))β2M2nd order model (β2M)

Figure 4-34 The kinetics of protein adsorption in PB on PS latex particles at 37oC and

pH 74

00

05

10

15

20

25

30

0 2 4 6 8 10 12 14

Time (hr)

Prte

inds

oo

arb

ed (

mg

m2 )

BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-35 The kinetics of protein adsorption in PB on PSPMMA100 latex partic

37les at

oC and pH 74

110

00

03

06

09

12

15

0 2 4 6 8 10 12 14

Time (hr)

Prot

ein

adso

rbed

(m

gm

2 ) BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-36 The kinetics of protein adsorption in PB on PSPMMA90PAA10 latex

particles o at 37 C and pH 74

00

03

06

09

12

15

0 2 4 6 8 10 12 14

Time (hr)

BS

A ad

sorb

ed (

mg

m2 )

BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-37 The kinetics of protein adsorption in PB on PSPMMA75PAA25 latex

particles at 37oC and pH 74

111

test The variation of β2M equilibrium adsorption amount was nearly negligible for all

latex particles The plateau region is reached earlier for β2M than BSA on all latex

particles and the K values (equilibrium rate constant of second order adsorption) for the

β2M adsorption process onto latex particles are much larger than for BSA adsorption

These values indicate that the β2M molecules have a higher adsorption rate onto the latex

particles than the BSA molecules

Table 4-16 Fitting parameters of second-order kinetic model BSA β2M

Latex particles qe (mgm2) K (hr-1) qe (mgm2) K ( -1) hr

PS 144 67 074 7549

PSPMMA100 175 353 076 4438

PSPMMA90PAA10 046 97 075 5695

PSPMMA75PAA25 060 179 075 5168 K = (kqe) hr-1

There are several explanations for the phenomena observed in these experiments

First the smaller sizes of β2M molecules allow easier diffusion and a close approach to

the latex substrate Second a reduced number of hydrophobic domains on the latex

particle surface replaced by sulfate groups (-OSO3-) and ionized carboxylic groups

(COO-) are not favorable for larger BSA molecules Third the conformational change by

intra- and inter-molecular repulsion would be faster in β2M due to smaller molecular

weight Finally the ionic repulsion between β2M and the latex particles is less than that

condition (pH 74) than IEP (48) of BSA

between BSA and latex particles because IEP (57) of β2M is closer to the tested

112

44 Blood Compatibility

Blood compatibility can be defined as the property of a material or device tha

permits it to function in contact with blood without inducing any adverse reactions

Successful functioning of blood contacting biomedical materials or devices in contact

with blood strongly depends on the extent to which they damage blood during operation

High shear stresses turbulence rela

t

tively long contact times and cellular impact with

artificial surfaces are most common events experienced by blood in contact with artificial

1978 Winslow and et al

1993] Therefore before any materia e placed into the human b

biocompatibility testing must be the first performed in the view of safety The hemolysis

s a method for pr ary screening of biomaterials to determine

h blood a er circulating body fluid The tests estimat

oglobin released from th blood cell d e induced

foreign material

There are several types of blood cells in our body but the ones that make blood red

are erythrocytes or red blood cells (RBCs) Red blood cells (erythrocytes) are the most

numerous type in the blood These cells are average about 48-54 million per cubic

millimeter (mm3 same as a microliter (microl)) of blood but these values can vary over a

larger range depending on such factors as health and altitude RBCs are red because they

contain hemoglobin which is a protein that contains iron When blood circulates through

lungs oxygen binds to the hemoglobin and is then delivered to all the parts of body

RBCs are made in the bone marrow They start out as a nucleus like other cells but

when they fill up with hemoglobin the nucleus becomes smaller until it disappears

Without a nucleus RBCs are fragile and live only about 120 days In a normal healthy

devises and can cause hemolysis or thrombosis [Bernhard et al

ls can b ody

test serves a elimin the

biocompatibility wit nd oth e the

concentration of hem e red amag by a

113

body about 2 million red blood ne marrow produces new

ones j ing

dy

le

cells die per second But bo

ust as fast RBCs are about 75 micrometer across and are responsible for carry

oxygen salts and other organic substances to the cells and tissues throughout our bo

In humans and most mammals the cells are concave (indented in the middle) and flexib

enough to squeeze through even the tiniest of blood vessels the capillaries

Figure 4-38 Image of red blood cells

(Sourcehttpwwwpbrchawaiiedubemfmicroangelarbc1htm)

ch

100 90 10 75 25 f

RBCs are among the first cells that come into contact with a foreign material

injected in the blood Therefore a hemolysis assay gives information about the

biocompatibility in the case of an in vivo application The concentrations of prepared

latex particles used for the tests in this research are 05 25 and 50 (ww) and the

incubation time under moderate shaking was 30 minutes at 37oC Test results are shown

in Figure 4-39 The amount of blood cells that ruptured when in contact with the PS latex

particles was largely increased from about 30 to 91 as the solid content of PS

increased from 05 to 50 However hemolysis by the core shell latex particles su

as PSPMMA PSPMMA PAA and PSPMMA PAA was less than 35 O

114

particular interest was that hemolysis caused by PSPMMA PAA particles at 50

solid concentration was less than 02 hemolysis the lowest value among the latex

particles The low hemolysis value indicates that the PSPMMA PAA core shell latex

particles are the most blood compatible at all solid contents Other core shell particles

such as PSPMMA and PSPMMA PAA also had excellent blood comp

90 10

90 10

100 75 25 atibility with

low RBS breakage

01 2 3

20

40

60

80

100

Hem

olys

is (

)

052550

Figure 4-39 He

PS PSPMMA100 PSPMMA PAA90 10

molysis caused by latex particles n=5

4PSPMMA75PAA25

CHAPTER 5 SUMMARY OF RESULTS CONCLUSIONS AND RECOMMENDATION FOR

FUTURE WORK

51 Summary of Results

The goal of this study was to synthesize monodisperse polymeric latex particles

with tailored properties and to investigate the fundamental interactions between the

synthesized latex particles and target proteins An understanding of the fundamental

mechanism of selective adsorption is strongly required in order to maximize the

separation performance of membranes made of these materials so that it may applicable

to hemodialysis therapy for end stage renal disease (ESRD) patients For the achievement

of above research goal several investigations have been performed Analysis of the

obtained data leads to several conclusions as discussed below

Monodisperse polystyrene seed latex particles were synthesized with the size

crosslinking agent to make the latex particles more hydrodynamically stable The

conversion of monomer to polymer was over 95 and seed latex particles are spherical in

shape and have smooth surface From these small seeds bigger latex particles as large as

800nm were synthesized using a semi-continuous seeded emulsion polymerization

process The particles with a mean diameter less than 500nm are very spherical in shape

and highly uniform in particle size distribution However as the particles grow larger

than 500nm in mean diameter they became non-spherical with an uneven surface This

irregularity of particle surface can be attributed to the non-homogeneous monomer

ranges between 126plusmn75 and 216plusmn53 nm Divinylbenzene (DVB) was used as a

115

116

swelling into the shell of a growing polymer which can be controlled by the factors such

as temperature agitation speed initiator feeding rate and surfactant amount required to

stabilize the colloidal system It was determ rfactant to monomer ratio

dec ic

acid (AA) monomers were intro rticle surfaces more

hydro

to

ting of a

orm

ration is seen between

3002

0 cm-

0cm-1

-1

03

ined that as the su

reased the mean particle diameter increased Methyl methacrylate (MMA) and acryl

duced to make the latex pa

philic than bare polystyrene particles The prepared core shell latex particles were

made of PMMA100 PMMA90PAA10 and PMMA75PAA25 PMMA100 is the PMMA

homopolymer shell on a PS core PMMA90PAA10 shell is a copolymer with a PMMA

PAA ratio of 90 to 10 by weight PMMA75PAA25 shell is a copolymer consis

PMMA to PAA ratio of 75 to 25 by weight The particle size of these core-shell

particles were prepared to be about 370nm in mean diameter

The successful synthesis of latex particles was confirmed using Fourier Transf

Infrared spectroscopy (FTIR) The C-H aromatic stretching vib

cm-1 and 3103 cm-1 for PS latex particles The C-H asymmetrical stretching and

symmetrical stretching vibration peaks of CH2 for all latex particles is seen at 290

1and 2850 cm-1 respectively There is a carbonyl (C=O) characteristic band at 173

wavenumber for PSPMMA core-shell latex particles The broad OH group peak at

3400 cm

100

can be seen in the spectroscopy of the particle with PMMAPAA shell

(PSPMMA PAA and PSPMMA PAA ) The peak intensity of OH group of

PSPMMA PAA was larger than that of PSPMMA PAA relatively due to more AA

monomer content in PSPMMA PAA than in PSPMMA PAA

In the zeta potential measurements polystyrene (PS) latex particles had negative

values between -291 mV and -599 mV in the phosphate buffer (PB) and between -2

90 10 75 25

75 25 90 10

75 25 90 10

117

mV and -278 mV in the phosphate buffered saline (PBS) at 25oC and pH 21-78 These

negative zeta potential values for PS latex particles are due to ionization of the sulfate

groups (-OSO3-) originated from the initiator Zeta potential values of the PSPMMA

core shell latex particles were also negative between -28 mV and -505 mV in PB and

between -143 mV and -186 mV in PBS at pH 21-78 Negative values are also

generated by dissociation of sulfate groups on a particle surface The zeta potential value

of PSPMMA PAA and PSPMMA PAA were of negative values between -36

and -678 mV in PB medium and -147 mV and -193 mV in PBS medium at 25

100

s

7 mV

MMA75PAA25 and

PSPM

otein

g the

l change of BSA at

each w

90 10 75 25

oC For

PSPMMA PAA core shell particles zeta potential values were between -291 mV and

-520 mV in PB media and between -115 mV and -210 mV in PBS media Sulfate

groups contributed to the negative zeta potential values in PSP

75 25

MA90PAA10 core shell particles Carboxylic groups from polyacrylic acid also

contribute to the negative surface charge for the latex particles at greater than pH 70

however the degree of their contribution was not detectable from the zeta potential

measurements The absolute zeta potential values of the synthesized latex particles in PB

were higher than that in PBS This is because of the high concentration of sodium

chloride electrolyte in PBS that compress electrical double layer thickness

The bicinchoninic acid (BCA) assay technique was used to determine the pr

adsorption and all isotherms were fitted to the Langmuir-Freundlich isotherms usin

nonlinear regression method The hydrophobic interaction between PS latex particle and

BSA protein was dominant for the adsorption process Conformationa

pH also affected the adsorption amount which was highest at pH 48 because of lo

ionic repulsion making compact conformation of BSA The next larger amount of BSA

118

adsorbed onto PS was at pH 32 There were two main forces affecting the amount of

adsorption at acidic pH values One is ionic attraction between negative latex particle a

positive protein The other is lateral repulsion between positive proteins The smallest

amount of BSA adsorption was at pH 74 where charge repulsion between negative

particles and proteins is present at this pH Lateral repulsion between proteins wa

dominant for protein adsorption even though there always exists the hydrophobic

interaction between solid latex particle and protein The charge repulsion was reduced by

the high concentration of electrolytes in the PBS medium The BSA adsorption process

on PSPMMA core shell particles was very similar to the case of PS latex particle where

hydrophobic interaction between latex particle and protein was dominant However t

adsorbed BSA amount was less than that of PS in PBS The adsorbed amount of BS

PSPMMA

nd

s

he

A on

an

ed

n The main

adsor

90PAA10 and PSPMMA75PAA25 core shell particles was higher at pH 32 th

pH 48 and 74 The adsorbed BSA amount became higher at pH 32 and pH 48 as the

hydrophilicity was increased by the higher PAA amount On the contrary the adsorb

amount of BSA was dramatically decreased at pH 74 Carboxylic acid groups as well as

the sulfate groups are distributed on the surface of PSPMMA90PAA10 and

PSPMMA75PAA25 core shell particles and largely affect the BSA adsorptio

ption interaction forces at pH 32 was generated by hydrogen bonding as well as

charge attraction between latex particle and protein even when the conformational change

and charge repulsion between proteins reduce the adsorption of protein at this pH The

adsorbed BSA amount was also increased as the amount of PAA increases However the

adsorption of BSA was minimal (less than 07 mgm2) at pH 74 where the carboxylic

groups are ionized leading to more enhancement of charge repulsion by ionization of the

119

carboxylic groups (COO-) Because of this the PSPMMA-PAA latex particles have a

much lower protein adsorption

There were steep initial slopes for adsorption isotherms of β

in

s

there

M

r

e

es

o

2M on latex particles

all isotherms indicating a high affinity type adsorption The complete plateau region

were not seen for all isotherms indicating that β2M proteins were not saturated and

are still available sites for β2M adsorption on latex particles The initial slope of β2

isotherms for PS was steeper than for the other latex particles PSPMMA100

PSPMMA90PAA10 and PSPMMA75PAA25

The layer thickness (δ) of BSA proteins on synthesized latex particles in phosphate

buffer (PB) was in the range of 27 - 81 Aring at 37 C and pH 48 Only the δ values at pH

48 the isoelectric point (IEP) of BSA are important because intra- and inter-molecular

electrostatic repulsion of the protein molecules is minimized at the IEP Compared to the

hydrodynamic dimensions of BSA 140 times 40 times 40 Aring these values of the adsorbed laye

thickness indicates that the BSA molecules adsorb by the side-on (40Aring) and end-on mod

(140Aring) Consideration of the multi-layering is unnecessary because the adsorbed

thickness of BSA do not exceed 140 Aring

The Gibbs free energy of β

o

3

2M was more negative in phosphate buffer (PB) than

that of BSA in physiological condition (37 C and pH 74) indicating that βo2M molecul

are more favorably adsorbed on the latex particles than BSA and that the affinity of β2M

was much larger than BSA This may be explained by the rate of diffusion and

accessibility of β2M molecules being easier and faster than that for BSA because of

smaller size (or weight) and spherical shape of β2M molecules It may also be possible t

explain that more hydrophobic forces (less electrostatic repulsion) contributes to

120

adsorption of β2M than to that of BSA because the IEP of β2M (57) is closer to

physiological condition (pH 74) than that of BSA (pH 48) The selective adsorption of

β2M m

in β2M

larger than in BSA adsorption indicating

that fa

an that between BSA and latex particles because

IEP (

ch as

Other

olecules over BSA molecules can be enhanced by developing more hydrophilic

PSPMMA90PAA25 and PSPMMA75PAA25 core shell latex particles

The adsorption kinetics of proteins on latex particles was expressed as a second-

order model The plateau region for β2M was reached earlier than BSA on all latex

particles and the K values (equilibrium rate constant of 2nd order adsorption)

adsorption process on latex particles were much

ster adsorption rate of β2M molecules than that of the BSA molecules There are

several explanations for these phenomena First the smaller size of β2M molecules can

more easily diffuse and approach to the latex substrate Second enhanced hydrophilic

domain on the latex particle surface by sulfate groups (SO4-) and ionized carboxylic

groups (COO-) is not favorable for larger BSA molecules but is for smaller β2M to be

anchored Third the conformational change by intra- and inter-molecular repulsion

would be faster in β2M due to lower molecular weight Finally the ionic repulsion

between β2M and latex particles is less th

57) of β2M is closer to the tested condition (pH 74) than IEP (48) of BSA

In the biocompatibility test the number of red blood cells (RBCs) ruptured by PS

latex particles ranged from 30 to 91 as the solid content of PS increased from 05 to

50 respectively However hemolysis of the other core shell latex particles su

PSPMMA100 PSPMMA90PAA10 and PSPMMA75PAA25 were less than 35 The

PSPMMA90PAA10 particles at all applied solid concentrations had less than 02

hemolysis and was the most blood compatible among the prepared latex particles

121

core shell particles such as PSPMMA100 and PSPMMA75PAA25 also had excelle

compatibility with low RBC breakage

52 Conclusions

The major conclusions from this study include

nt blood

bull microglobulin (β2M) over that of the bovine serum albumin (BSA) at pH 74

was achieved by controlling the PMMA and PAA ratio to optimize

e particles posses the most desirable requirements for use in a human dialysis

ns

icles

olecule

bull

Using engineered core shell latex particles selective adsorption of β2

physiological condition was demonstrated

bull The rate of adsorption of the β2M was also much higher than that of BSA This

hydrophobichydrophilic microdomain size

bull The core shell particles also exhibited excellent biocompatibility with blood Thes

membrane

53 Recommendation for Future Work

With the results and conclusions obtained through this study several suggestio

may be given for future work as follows

bull Competitive adsorption tests of whole blood proteins on synthesized latex partunder physiological condition

bull Preparation of latex particles with optimum hydrophobic hydrophilic microdomains to maximize selective adsorption and removal of toxin molecules

bull Membrane fabrication by suitable pore size design to discriminate a toxin mby size sieving as a function of particle layers

Evaluation of membrane performance such as flow rate in water and in simulated blood fluid (SBF)

bull Application for hemodialysis as well as other technologies such as a water remediation

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38

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140

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Zou oss-

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20 81-86

Clemme J Polaschegg H D Muller T E La Greca G Levin N W (Rationale for combined hemoperfusionhemodialysis in uremia Contribution of Nephrology 133 174-179

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Model filled polymers 5 synthesis of cross-linked monodisperse polymethacrylate beads Journal of Polymer Science Part A Polymer Chemistry 30 137-144

Korea on August 7 1968 He obtained a Bachelor of Science degree in Chemistry from

Keim d graduate school and earned a

resear n Korea Institute of Science and

Coati e

ia

Busin

BIOGRAPHICAL SKETCH

Sangyup Kim was born and raised in the small town of Sung-Joo Kyungbook

yung University (Daegu Korea) in 1993 He entere

Master of Science degree in Chemical engineering in 1995 He worked as a student

cher for 2 years at Textile Polymer Division i

Technology (KIST) in Seoul Korea Next he worked over a year at the Automobile

ng Division in DongJoo-PPG in Korea In August 2001 he began studying for th

degree of Doctor of Philosophy in materials science and engineering at the University of

Florida in Gainesville After getting his PhD he plans to work at the Digital Med

ess Division of Samsung Electronics in Korea starting in January 2006

141

322 Preparation of Seeded Latex Particles 48 323 Preparation of Core Shell Latex Particles 48 324 Purification of Synthesized Latex Particles48

33 Characterization49 331 Degree of Conversion49 332 Fourier Transform Infrared (FTIR) Spectroscopy49 333 Quasielastic Light Scattering (QELS) 50 334 Field Emission-Scanning Electron Microscopy (FE-SEM) 50 335 Zeta Potential Measurement 50 336 Protein Adsorption51 337 Blood Biocompatibility by Hemolysis Test 53

4 RESULTS AND DISCUSSION55

41 Polymerization of Latex Particles55 411 Polystyrene Seed Latex Particles55 412 The Growth of Polystyrene (PS) Seed Latex Particles61 413 Core Shell Latex Particles 71

42 Characterization of Latex Particles75 421 Fourier Transform Infrared Spectroscopy (FTIR)75 422 Zeta Potential Measurements 75

43 Protein Adsorption Study 85 431 Adsorption Isotherm87 432 Adsorbed Layer Thickness 103 433 Gibbs Free Energy of Protein Adsorption 104 434 Kinetics of Adsorption 107

44 Blood Compatibility 112

5 SUMMARY OF RESULTS CONCLUSION AND RECOMMENDATION FOR FUTURE WORK115

51 Summary of Results115 52 Conclusions121 53 Recommendation for Future Work121

LIST OF REFERENCES122

BIOGRAPHICAL SKETCH 141

vii

LIST OF TABLES

Table page 4-1 The polymerization recipe of polystyrene (PS) seed particles 57

4-2 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles less than 500nm in size 63

4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size64

4-4 The preparation recipe of PS core with various shell latex particles72

4-5 The isoelectric point (IEP) of common proteins 83

4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model 93

4-7 The equilibrium concentration values (q ) of BSA adsorption on PSPMMA particles calculated the Langmuir-Freundlich isotherm model

m 10096

4-8 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model

90 1098

4-9 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model

75 25100

4-10 The equilibrium concentration values of β M adsorption calculated the Langmuir-Freundlich isotherm model

2102

4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37 Co 104

4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37 Co 104

4-13 The values of Gibbs free energy change of BSA adsorption in phosphate buffer (PB) at 37 Co 105

4-14 The values of Gibbs free energy change of BSA adsorption in phosphate buffered saline (PBS) at 37 Co 105

viii

4-15 The values of Gibbs free energy change of β M adsorption in phosphate buffere (PB) at 37 C and pH 74

2o 106

4-16 Fitting parameters of second-order kinetic model 111

ix

LIST OF FIGURES

Figure page 1-2 Stereo drawing of the αndashcarbon backbone of β M2 2

2-1 An image of the location and cross section of a human kidney 5

2-2 A schematic draw of the hemodialysis route7

2-3 Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes 11

2-4 The chemical structure of polysulfone (PSf) 12

2-5 The chemical structure of polyamide (PA)13

2-6 Chemical structure of polyacrylonitrile (PAN) 14

2-7 Emulsion polymerization system24

2-8 L-α-amino acid 31

2-9 Ribbon diagram of human β M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ)

236

2-10 Secondary structure of human serum albumin (HSA) with sub-domains 37

2-11 The relationship between pore size and particle size in body-centered cubic and closed packed arrays41

2-12 Schematic representation of the protein adsorption on the core shell latex particles at pH 7442

3-1 Chemical structure of main chemicals45

3-2 Experimental setup for semi-continuous emulsion polymerization 46

3-3 The particle preparation scheme in various types and size ranges of latex particles 47

3-4 Schematic of the procedure for a protein adsorption test 52

3-5 Separation of RBC from whole blood by centrifuge process53

x

3-6 Schematic of the procedure for hemolysis test 54

4-1 Schematic representation of semi-continuous seed latex particles preparation and growth56

4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS (B) PS (C) PS (D) PSS259 S233 S207 S181 59

4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm 65

4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm 68

4-5 Dependence of the particle size on the surfactant to monomer ratio70

4-6 Schematic of core shell latex particle structures 71

4-7 Scanning Electron Micrograph of latex particles (A) PS (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA

100

90 10 75 25 73

4-8 FTIR spectra of polymerized latex particles (A) bare polystyrene (PS) (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA100 90 10 75 25 76

4-9 Schematic representation of ion distribution near a positively charged surface 78

4-10 Schematic representation of zeta potential measurement79

4-11 Zeta potential of PS latex particles at 25 Co 80

4-12 Zeta potential of PSPMMA latex particles at 25 C100o 81

4-13 Zeta potential of PSPMMA PAA latex particles at 25 C90 10o 82

4-14 Zeta potential of PSPMMA PAA latex particles at 25 C75 25o 82

4-15 The decay of surface potential with distance from surface in various electrolyte concentrations (1) low (2) intermediate (3) high 84

4-16 High-affinity adsorption isotherm of typical flexible polymer on solid surface 86

4-17 Overall schematic representation of the protein adsorption on the synthesized latex particles86

4-18 The dependence of UV absorbance on BSA concentration (A) 10mgml (B) 06mgml (C) 02mgml87

4-19 Standard curve of net absorbance vs BSA sample concentration 88

xi

4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37 C in phosphate buffer mediao 89

4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37 Co 90

4-22 Adsorption isotherm of BSA adsorption 37 C in phosphate buffered saline (PBS) on PS latex particles

o

91

4-23 Conformation of bovine serum albumin (BSA) on latex particles92

4-24 Adsorption isotherm of BSA 37 C in PB on PSPMMA core shell latex particles

o100

94

4-25 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA core shell latex particles

o100

95

4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA PAA core shell latex particles

o90 10

97

4-27 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA PAA core shell latex particles

o90 10

98

4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA PAA latex particleso75 25 99

4-29 Adsorption isotherm of BSA 37 C in PBS on PSPMMA PAA core shell latex particles

o75 25

99

4-30 Adsorption isotherm of β M onto PS and PSPMMA latex particles in PB at 37 C and pH 74

2 100o 101

4-31 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74

2 90 10o 101

4-32 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74

2 75 25o 102

4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37 C and pH 74

o

107

4-34 The kinetics of protein adsorption in PB on PS latex particles at 37 C and pH 74

o

109

4-35 The kinetics of protein adsorption in PB on PSPMMA latex particles at 37 C and pH 74

100o

109

4-36 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74

90 10o 110

xii

4-37 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74

75 25o 110

4-38 Image of red blood cells 113

4-39 Hemolysis caused by latex particles n=5114

xiii

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS

By

Sangyup Kim

December 2005

Chair Hassan El-Shall Major Department Materials Science and Engineering

Latex particles with well-defined colloidal and surface characteristics have received

increasing attention due to their useful applications in many areas especially as solid

phase supports in numerous biological applications such as immunoassay DNA

diagnostic cell separation and drug delivery carrier Hemodialysis membrane using

these particles would be another potential application for the advanced separation

treatment for patients with end stage renal disease (ESRD) It is desirable to remove

middle molecular weight proteins with minimal removal of other proteins such as

albumin Thus it is necessary to understand the fundamental interactions between the

particles and blood proteins to maximize the performance of these membranes This

improvement will have significant economic and health impact

The objective of this study is to synthesize polymeric latex particles of specific

functionality to achieve the desired selective separation of target proteins from the human

blood Semi-continuous seed emulsion polymerization was used to prepare monodisperse

xiv

polystyrene seed particles ranging from 126plusmn75 to 216plusmn53 nm in size which are then

enlarged by about 800nm Surfactant amount played a key role in controlling the latex

particle size Negatively charged latex particles with a different hydrophobicity were

prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid

monomer The prepared polymeric particles include bare polystyrene (PS) particles less

hydrophobic PS core and PMMA shell particles and more hydrophilic PS core and

PMMA-co-PAA shell latex particles with a 370nm mean diameter SEM light scattering

and zeta potential measurements were used to characterize particle size and surface

properties Adsorption isotherms of two proteins bovine serum albumin (BSA) and β2-

microglobulin (β2M) on latex particles were obtained as a function of pH and ionic

strength using the bicinchoninic acid (BCA) assay method The Langmuir-Freundlich

adsorption model was used to determine the adsorption amount of protein at equilibrium

The thickness of adsorbed BSA layer on latex particles was obtained in order to

investigate the adsorption orientation such as end-on or side-on mode Adsorption

kinetics experiments for both proteins and all latex particles were also performed The

adsorption kinetic constant determined from the Langmuir-Freundlich adsorption

isotherm model was used to calculate Gibbs free energy of adsorption to compare the

competitive adsorption of BSA and β2M Hemolysis tests were performed to investigate

the blood compatibility of synthesized latex particles PSPMMA90PAA10 and

PSPMMA75PAA25 core shell latex particles had desirable material properties with not

only a large amount and high rate of selective β2M adsorption over BSA but also high

blood compatibility showing less than 3 hemolysis

xv

CHAPTER 1 INTRODUCTION

End stage renal disease (ESRD) is a chronic condition in which kidney function is

impaired to the extent that the patientrsquos survival requires removal of toxins from the

blood by dialysis therapy or kidney transplantation The National Kidney Foundation

estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF] The

number of people with ESRD is rapidly increasing in the United States with

approximately 96 295 incidents and 406 081 prevalent patients including 292 215 on

dialysis and 113 866 with a functioning graft in 2001 It is projected that there will be

more than 22 million ESRD patients by 2030 [USRDS 2003] The expenditure for the

ESRD treatment program had reached $228 billion 64 of the Medicare budget in

2001 Due in part to the limited availability of kidneys for transplantation hemodialysis

is the primary clinical treatment for the patients with ESRD

The central element of a hemodialysis instrument is the semipermeable membrane

that allows for selective transport of low molecular weight biological metabolites less

than 5000 Da such as urea and creatinine as well as excess water and electrolytes [Baker

2004] from the blood One limitation of current dialysis technologies is the inability to

efficiently remove middle molecular weight toxins such as β2-Microglobulin (β2M) and

interleukin 6 (IL-6)

β2M is a causative protein of dialysis-related amyloidosis (DRA) a disease arising

in patients with chronic kidney failure as a serious complication of long-term

hemodialysis treatment [Gejyo et al 1985] β2M deposition in tissue is the primary cause

1

2

of destructive arthritis and carpal tunnel syndrome [Vincent et al 1992 Drueke 2000]

The β2M structure is shown in Figure 1-2

Figure 1-2 Stereo drawing of the αndashcarbon backbone of β2M [Becker et al 1985]

Although attempts have been made to increase the efficiency of middle molecular

weight toxin removal by changes in the membrane pore size and the use of innovative

materials to adsorb these toxins [Samtleben et al 1996 Ronco et al 2001] removal

efficiency is not as high as those achieved by a normal healthy kidney Traditional

membranes have a number of processing and performance limitations [Westhuyzen et al

1992 Leypoldt et al 1998] such as a restricted choice of surface chemistries and limited

control of porosity The development of novel engineering membrane technology is

needed to remove middle molecule toxins

Polymeric latex particles have received increasing attention in medical application

areas especially as solid phase supports in biological applications [Piskin et al 1994]

Examples of these applications include immunoassay [Chen et al 2003 Radomske-

Galant et al 2003] DNA diagnostic [Elaїssari et al 1998] cell separation drug delivery

carrier [Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] etc This is because of

3

the well-defined colloidal and surface characteristics of the particles By using a seed

emulsion polymerization method it is possible to synthesize monodisperse latex particles

with various particle size ranges and surface chemistry Functionalized core-shell latex

particles can be introduced by multi-step emulsion polymerization Core particles are

synthesized in the first stage of the polymerization and the functional monomer is added

in the second stage This is done without any emulsifier addition to prevent the

production of new homopolymer particles [Keusch et al 1973] The core-shell particles

are useful in a broad range of applications because of their improved physical and

chemical properties over their single-component counterparts [Lu et al 1996 Nelliappan

et al 1997] Through the development of a hemodialysis membrane using monodisperse

latex particles improvements in advanced separation treatment for patients with end

stage renal disease (ESRD) can be realized This requires the maximum removal of

middle molecular weight proteins with minimal removal of other beneficial proteins such

as albumin Thus an understanding of the fundamental interactions between the particles

and biopolymers is vital to maximize the performance of this membrane technology

The field of material science and biotechnology is based on fundamental chemistry

has developed over the past three decades into todayrsquos powerful discipline that enables

the development of advanced technical devices for pharmaceutical and biomedical

applications This novel and highly interdisciplinary field is closely associated with both

the physical and chemical properties of organic and inorganic particles [Niemeyer 2001]

Hemodialysis membrane using these particles would lead to improvements in the

advanced separation treatment for patients with end stage renal disease (ESRD) The

interdisciplinary nature of this approach enables a more complete understanding of the

4

phenomena of protein adsorption and the material properties necessary for selective

separation This innovative approach to membrane fabrication has the potential of making

inexpensive highly efficient membranes for both industrial and specialized separation

processes

The goal of this study is to prepare polymeric latex particles with tailored

properties to maximize separation of toxin molecules and to investigate the fundamental

interactions between the particles and molecules in the biological system in order to

optimize the performance of a membrane material for these applications

Polymeric latex particles were synthesized with specific functionality in an attempt

to achieve selective separation Seeded emulsion polymerization was used to synthesize

functionalized monodisperse latex particles in various sizes Negatively charged

hydrophobic polystyrene latex particles were synthesized by the same method Core shell

latex particles PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25 were also

synthesized to differentiate the degree of hydrophobicity of particles Scanning Electron

Micrograph (SEM) and light scattering measurements were used to characterize particle

size and shape and zeta potential measurements were conducted to measure the electrical

surface property of synthesized particles Adsorption isotherms of target proteins bovine

serum albumin (BSA) and β2M on latex particles were obtained as a function of pH

ionic strength and protein concentrations using the BCA assay method Adsorption

kinetics for both proteins on the latex particles were also measured Finally hemolysis

tests were run to determine the biocompatibility of polymer latex particles with human

blood This research will be described in more detail in chapters 3 and 4

CHAPTER 2 BACKGROUND AND LITERATURE SURVEY

21 The Significance of End Stage Renal Disease (ESRD)

The kidneys are responsible for removing excess fluid minerals and wastes from

the blood regulating electrolyte balance and blood pressure and the stimulation of red

blood cell production They also produce hormones such as erythropoietin (EPO) and

calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005 Kurbel

et al 2003] EPO acts on the bone marrow to increase the production of red blood cell in

case of bleeding or moving to high altitudes Calcitriol mainly acts on both the cells of

the intestine to promote the absorption of calcium from food and also bone to mobilize

calcium from the bone to the blood When kidneys fail harmful waste builds up blood

pressure rises and body retains excess fluid The body also does not make enough red

blood cells When this happens treatment is needed to replace the function of failed

kidneys

Figure 2-1 An image of the location and cross section of a human kidney

5

6

The National Kidney Foundation estimates that over 20 million Americans had

chronic kidney disease in 2002 [NKF] Chronic renal disease is a gradual and progressive

loss of the function unlike acute renal disease where sudden reversible failure of kidney

function occurs Chronic renal failure usually takes place over a number of years as the

internal structures of the kidney are slowly damaged In the early stages there can be no

symptoms In fact progression may be so gradual that symptoms do not occur until

kidney function is less than one-tenth that of a normal health kidney If left untreated

chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal

Disease (ESRD) ESRD is a rapidly growing heath-care problem in the United States In

2001 approximately 96295 incidents and 406081 prevalent patients were diagnosed

with ESRD including 292215 patients on dialysis and 113866 patients with a

functioning graft [USRDS 2003] The projected number of patients with ESRD is

expected to exceed 22 million patients by 2030 with much of this growth being driven by

the increasing prevalence of major contributing factors such as diabetes and high blood

pressure [USRDS 2003] A great extent of ESRD program cost and Medicare budget

have been spent in 2001 Due in part to a limited availability of kidneys for

transplantation hemodialysis (HD) is the primary method of treatment for ESRD and is

currently used for approximately 61 of US ESRD patients

22 Hemodialysis (HD) Treatment

HD removes toxins from the body by extracorporeal circulation of the blood

through a semipermeable membrane referred to as a dialyzer The toxins are removed

primarily by diffusion across the membrane to a dialysate solution which is circulated on

the opposite side of the membrane The cleaned blood is then returned to the blood

stream A surgically constructed vascular access connects the extracorporeal circuit to the

7

patientrsquos system Treatments are almost always performed three times per week in

specially equipped dialysis facilities for 3-4 hours per each treatment Figure 2-2 shows

the scheme of the hemodialysis route The critical element of a HD instrument is the

semipermeable membrane which allows for selective transport of low molecular weight

biological metabolites from blood

igure 2-2 A schematic draw of the hemodialysis route

ESRD patients who undergo dialysis therapy often experience several other

problems associated with the treatment such as anemia fatigue bone problems joint

problems itching sleep disorders and restless legs Anemia is common in patient with

kidney disease because the kidneys produce the hormone erythropoietin (EPO) which

stimulates the bone marrow to produce red blood cells Diseased kidneys often do not

produce enough EPO to stimulate the bone marrow to make a sufficient amount of red

blood cells This leads to bone disease referred to as renal osteodystrophy and causes

bones to become thin and weak or malformed and can affect both children and adults

Older patients and women who have gone through menopause are at greater risk for this

disease Uremic toxins which cannot be remove from the blood by the current dialyzer

membranes can lead to itching This problem can also be related to high levels of

F

8

parathyroid hormone (PTH) Dialysis patients can also experience day-night revers

is they have insomnia at night and sleep during the day This can be related to possible

nerve damage in the body and a chemical imbalance in the blood due to the excess toxin

Oxidative stress is a problem for patients on maintenance dialysis This problem is due to

an imbalance between pro- and antioxidant factors [Roselaar et al 1995 Cristol et al

1994] Oxidative stress affects oxidation of low-density lipoproteins which are the main

factor for atherogenesis [Huysmans et al 1998] and is also involved in the development

of malignancies and diabetes mellitus [Rice-Evans et al 1993] In order to reduce

antioxidant defense dialysis is needed to contribute to help stimulate free radical

production or eliminate antioxidants Dialysis-related amyloidosis (DRA) is also a

common and serious problem for people who have been on dialysis for more than 5

years DRA develops when proteins in the blood deposit on joints and tendons causi

pain stiffness and fluid in the joint as is the case with arthritis Normally the healthy

kidneys can filter out these proteins but dialysis filters are not as effective

23 Advances in Membrane Technology

al that

s

ng

Dialysis for blood of ESRD

Hemo filtration

s

ysis using a cellulose based membrane occurred

1913 John Abel [1990] from the Johns Hopkins Medical School described a method

purification is widely used in the treatment

dialysis (HD) techniques use a semi-permeable membrane to replace the

role of the kidney The membranes used in HD can be broadly classified into those based

on cellulose and those manufactured from synthetic copolymers These membranes come

in various shapes such as sheets tubular structures or hollow fiber arrangements The

hollow fiber type is the most popular and is incorporated into over 800 different device

in world wide [Ronco et al 2001]

The first attempt at blood dial

9

where sing

e

om

ysis was performed until the mid 1960 The basic

molec

rial in

een

benzyl)

was used for the membrane with

hepar

by the blood of a living animal may be submitted to dialysis outside the body u

a membrane based on cellulose and returned to the natural circulation without exposur

to air infection by microorganisms or any alteration that would necessarily be prejudicial

to life This same technique is still used to today however the device used has been

modified over the years as better membranes were developed and the anti-coagulant

heparin has become available

Cellulose membranes have been widely used for the treatment of renal failure fr

1928 when the first human dial

ular structure of cellulose is made of a long chain containing hydroxyl (OH)

groups The realization that such groups imparted undesirable qualities on the mate

respect to blood contact behavior was discovered in the early 1970s and since has b

the focus of development These modified cellulose membranes used the partial

substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt

to reduce their negative effect The result is a molecular mosaic of hydrophobic (

and hydrophilic (hydroxyl and cellulose) regions

Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients

with acute renal failure in 1943 Cellophane tubing

in as the anticoagulant For the next 17 years hemodialysis therapy was performed

by this method but only for the patients with acute reversible renal failure Vascular

access required repeated surgical insertions of cannulas (slender tubes) into an artery and

vein and limited the number of treatments for a patient could receive in order to

minimize the amount of vascular damage

10

Initially the need for dialysis in patients with acute renal failure was determ

mainly by the development of signs and sym

ined

ptoms of uremia After dialysis some time

might

et al

ent improvement In 1960 the development of a shunt [Quinton et al

1960]

fone (PSf) polyamide (PA) and polyacrylonitrile (PAN) by phase inversion or

precip

lidone

e

elapse before uremic manifestations returned to warrant a sequential treatment of

dialysis Many patients with acute renal failure secondary to accidental or surgical

trauma were hypercatabolic but the interdialytic interval might be prolonged because of

anorexia or use of a low-protein diet However Teschan and his coworkers [Obrien

1959] showed that patient well-being and survival were improved by what they termed

prophylactic daily hemodiaysis or administration of the treatment before the patient

again became sick with uremia Their report in 1959 was the first description of daily

hemodialysis

Development of membrane accessories such as a shunt has also been an area of

focus for treatm

a flexible polytetrafluoroethylene (PTFE or Teflonreg) tubing made many more

hemodialysis treatments possible for chronic kidney failure patients PTFE has a non-

stick surface and is relative biocompatibility leading to minimized blood clotting in the

shunt

Synthetic membranes are prepared from engineered thermoplastics such as

polysul

itation of a blended mixture resulting in the formation of asymmetric and

anisotropic structures Figure 2-3 shows a fiber type of the Polyflux S membrane

consisting of poluamide (PA) polyacrylethersulfone (PAES) and polyvinylpyrro

(PVP) with the integral three-layer structure The skin layer on the inside fiber typ

membrane contacts blood and has a very high surface porosity and a narrow pore size

11

distribution This layer constitutes the discriminating barrier deciding the permeabili

and solute retention properties of the membrane The skin layer is supported by thick

sponge-type structure larger pores providing mechanical strength and very low

hydrodynamic resistance

ty

Figure 2-3 Cross-sectional SEM image view of the Polyflux S (polyamide +

polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch et al 1998]

n

[Malchesky 2004] because of its thermal stability mechanical strength and chemical

inertn ciated

ls

m

PSf is a widely used membrane material for the hemodialysis applicatio

ess According to a report from the National Surveillance of Dialysis-Asso

Disease (NSDAD) in the US over 70 of hemodialysis membranes were PSf based

[Bowry 2002] This is most likely because PSf has many advantages over other materia

This synthetic polymer is one of few materials that can withstand sterilization by stea

ethylene oxide and γ-radiation PSf membrane can be prepared by conventional

immersion precipitation methods into many different shapes including porous hollow

12

fiber or flat sheet hemodialysis membranes The material also has a high permeab

low molecular weight proteins and solute and high endotoxin retention The chemical

structure of PSf is shown in Figure 2-4

ility to

Figure 2-4 The chemical structure of polysulfone (PSf)

There is one major disadvantage to PSf The hydrophobic nature of the PSf causes

ment alternative pathway

leadin

s not

3

et al

in

O acrylate

serious complications through the activation of the comple

g to the adsorption of serum proteins onto the membranes [Singh et al 2003]

Anticoagulants are added during dialysis therapy to avoid blood clotting but this doe

completely eliminate the problem In order to overcome this disadvantage of the PSf

membrane various studies have been performed to change the materialrsquos surface

properties These investigations include hydrophilic polymer coating [Brink et al 199

Kim et al 1988 Higuchi et al 2003] layer grafting onto PSf membrane [Wavhal

2002 Song et al 2000 Pieracci et al 2002 Mok et al1994] and chemical reaction of

hydrophilic components onto the membrane surface [Higuchi et al 1990 1991 1993

Blanco et al 2001 Nabe et al 1997 Guiver et al 1993] Hydrophilic monomers 2-

hydroxy-ethylmethacrylate (HEMA) acrylic acid (AA) and methacrylic acid (MMA)

have also been grafted onto PSf membrane to increase flux and Bovine Serum Album

(BSA) retention [Ulbricht et al 1996] Hancock et al [2000] synthesized

polysulfonepoly(ethylene oxide) (PEO) block copolymers to improve the resistance to

platelet adhesion Kim et al [2003] also studied blending a sulfonated PE

diblock copolymer into PSf in order to reduce platelet adhesion and enhance

13

biocompatibility PEO is a commonly used biomaterial due to its excellent resistance

protein adsorption and inherent biocompatibility [Harris 1992] Kim et al [20

a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf

membrane with an ultra-thin skin layer The polymer had an entrapped diblock

copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-SO

to

05] studied

ate

lic graft

l)

e of thier

aromatic

orand

3 acryl

and a hydrophobic block of octadecylacrylate (OA) Molecular dynamic (MD)

simulations were performed as a function of copolymer density to optimize interfacial

structure information McMurry [2004] developed a strategy using an amphiphi

copolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glyco

When compared to unmodified PSf these graft copolymer and resulting blend

membranes are found to hold promise for biomedical device applications

Polyamide (PA) membranes have also been used for hemodialysis becaus

mechanical strength in both wet and dry conditions Polyamide consists of

aliphatic monomers with amide bonding (-CONH-) also known as a peptide bond

The basic amide bond in polyamide is shown in Figure 2-5 R1 and R2 can be either

aromatic or aliphatic linkage group

C N

H

O

~ R R1 2~C N

H

O

~ R R1 2~

Figure 2-5 The chemical structure of polyamide (PA)

Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane

ompatible technique due to the

use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of

and concluded that PA hemofiltration was a highly bioc

14

the ac

e

e

is

n

is a semi-crystalline polymer and the mechanical properties

strong

2

CN

tivated anaphylatoxins and β2-Microglobulin (β2M) The PA based membrane

Polyfluxreg (manufactured by Gambro GmbH Germany) blended with polyamide

polyarylethersulfone and polyvinylpyrrolidone (PVP) was able to clean small molecules

such as urea dreatinine and phosphate as well as decrease β2M amount by 502

[Hoenich et al 2002] Due to the non-selectivity of the membrane removal of thes

unwanted materials also led to the undesirable loss of beneficial proteins during therapy

Meier et al [2000] evaluated different immune parameters using a modified cellulos

low-flux hemophan and synthetic high-flux PA membrane during a 1 year period in

chronic hemodialysis patients They found that the 1-year immunological evaluation of

hemodiaysis membrane biocompatibility was associated with changes in the pattern of

chronic T-cell actiovation

Polyacrylonitrile (PAN) is another commonly used membrane material because it

inherently hydrophilic and has been commercialized for ultrafiltration and microfiltratio

[Scharnagl et al 2001] PAN

ly depend on the crystalline structures The chemical structure of PAN is shown in

figure 2-6

CH CH

n2

CN

CH CH n

Chemical structure of polyacrylonitrile (PAN)

The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming

agent gives PAN membranes more flexible processing parameters and increased

e has been optimized through

copolymerization with many other vinyl monomers including glycidyl methacrylate

Figure 2-6

performance [Jung et al 2005] PAN membrane performanc

15

[Godj

acid

for

for

a non-

solub this

in

oid underneath the inner

surface of membrane decreased by increasing the amount of solvent DMSA in the

evargova et al 1999 Hicke et al 2002] N-vinylimidazole [Godjevargova et al

2000] hydroxyl ethyl methacrylate [Ray et al 1999 Bhat et al 2000] methacrylic

[Ray et al 1999] vinyl pyrrolidone [Ray et al 1999] acrylic acid [Trotta et al 2002]

acrylamide [Musale et al 1997] and vinylchloride [Broadhead et al 1998] These

monomers provide a reactive group for enzyme immobilization improved mechanical

strength solvent-resistance pervaporation permeation flux anti-fouling and bio-

compatibility Because of this PAN-based copolymer membranes have great potential

the treatment of hemodialysis in an artificial kidney This material can also be used

other applications like the treatment of wastewater the production of ultra-pure water

biocatalysis together with separation and methanol separation by pervaporation

Lin and his coworker [2004] studied the modification of PAN based dialyzer

membranes to increase the hemocompatibility by the immobilization of chitosan and

heparin conjugates on the surface of the PAN membrane When a foreign material is

exposed to blood plasma proteins are adsorbed clotting factors are activated and

le fibrin network or thrombus is formatted [Goosen et al 1980] The result of

research was that the biocompatible chitosan polymer and a blood anticoagulant hepar

prevented blood clotting They showed prolonged coagulation time reduced platelet

adsorption thrombus formation and protein adsorption

Nie et al [2004] studied PAN-based ultrafiltration hollow-fiber membranes

(UHFMs) In order to improve the membrane performance acrylonitrile (AN) was

copolymerized with other functional monomers such as maleic anhydride and α-allyl

glucoside They found that the number and size of macrov

16

intern erum

and

d a

rface The second step is the

transf after

yjak et

embrane

1978 Malchesky et al 1978] has been further developed to increase the efficiency of

al coagulant The water flux of the UHFMs also decreased while the bovine s

albumin rejection increased minutely Godjevargova et al [1992] modified the PAN

based membrane with hydroxylamine and diethylaminoethylmethacrylate to improve

membrane dialysis properties Formed functional groups like primary amine oxime

tertiary amine groups provided the membrane with more hydrophilic properties an

substantial increase in the permeability of the membranes

The wide use of filtration in practice is limited by membrane fouling Solute

molecules deposit on and in the membrane in the process of filtration causing dramatic

reduction in flux through the membrane Fouling occurs mostly in the filtration of

proteins Three kinetic steps are involved in the fouling of UF membranes according to

Nisson [1990] The first step is the transfer of solute to the su

er of solute into the membrane until it either finally adsorbs or passes through

a set of adsorption-desorption events The third step includes surface binding

accompanied by structural rearrangement in the adsorbed state [Ko et al 1993] Br

al [1998] studied the surface modification of a commercially available PAN membrane

to develop superior filtration properties with less fouling by proteins The PAN

membrane was immersed in excess NaOH solution to convert some of the surface nitrile

groups into carboxylic groups by the hydrolysis process This modified PAN m

was not so severely fouled in the Bovine Serum Albumin (BSA) filtration test The pore

size however decreased during the hydrolysis process leading to a significant reduction

in flux and made the membrane less productive in the ultrafiltration (UF) mode

24 Sorbent Technology

Over the last three decades sorbent technology [Castino et al 1976 Korshak et al

17

dialysis or replace it for the treatment of ESRD Sorbents remove solutes from solution

through specific or nonspecific adsorption depending on both the nature of the solute and

the sorbent Specific adsorption contains tailored ligands or antibodies with high

selectivity for target molecules een used in autoimmune

disord

and

e

al 1996] various resins [Ronco et al 2001]

album

e

as

Specific adsorbents have b

ers such as idiopathic thrombocytopenic purpura [Snyder et al 1992] and for the

removal of lipids in familial hyper cholesterolemia [Bosch et al 1999] Nonspecific

adsorbents such as charcoal and resins attract target molecules through various forces

including hydrophobic interactions ionic (or electrostatic) attraction hydrogen bonding

and van der Waals interactions

New dialysate with sorbents has become an accepted modification of dialysis

sorbent hemoperfusion is gaining ground as a valuable addition to dialysis especially as

new sorbents are developed [Winchester et al 2001] Hemoperfusion is defined as th

removal of toxins or metabolites from circulation by the passing of blood within a

suitable extracorpoteal circuit over semipermeable microcapsules containing adsorbents

such as activated charcoal [Samtleben et

in-conjugated agarose etc Novel adsorptive carbons with larger pore diameters

have been synthesized for potential clinical use [Mikhalovsky 1989] Newly recognized

uremic toxins [Dhondt et al 2000 Haag-Weber et al 2000] have resulted in several

investigations on alternatives to standard or high-flux hemodialysis to remove thes

molecules These methods include hemodiafilteration with [de Francisco et al 2000] or

without [Ward et al 2000 Takenaka et al 2001] dialysate regeneration using sorbents

well as hemoperfusion using such adsorbents as charcoal and resins

18

Kolarz et al [1989 1995] studied the hyper-crosslinked sorbent prepared from

styrene and divinylbenzene (DVB) for a hemoperfusion application They found that the

pore structure of a swelling sorbent was changed by additional crosslinking with α αrsquo-

dichloro-p-xylene in the presence of a tin chloride catalyst and in a di

chloroethane

soluti and

en

decyl groups that attract β2M through a hydrophobic interaction The

adsor

of

5]

d

penet

on They also realized that the hemocompatibility was useful for nemoperfusion

could be imparted to the sorbents by introducing sulfonyl groups at a concentration of

about 02mmolg

A special polymeric adsorbing material (BM-010 from Kaneka Japan) has be

investigated by another group [Furuyoshi et al 1991] for the selective removal of β2M

from the blood of dialysis partients The adsorbent consists of porous cellulose beads

modified with hexa

ption capacity of this material is 1mg of β2M per 1ml of adsorbent Using a

hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a

high-flux hemodialyzer several small clinical trials were performed During 4-5 hours

treatment about 210mg of β2M were removed thus reducing the concentration in the

blood by 60-70 of the initial level [Nakazawa et al 1993 Gejyo et al 1993 199

RenalTech developed a hemoperfusion device BetaSorbreg containing the hydrate

cross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure

designed to remove molecules between 4 and 30 kDa [Winchester et al 2002] In this

case solute molecules are separated according to their size based on their ability to

rate the porous network of the beaded sorbents The resin beads were prepared with

a blood compatible coating and confirmed to be biocompatible in vivo in animals

[Cowgill et al 2001]

19

25 Limitation of Current Hemodialysis Treatment

Hemodialysis is a widely used life-sustaining treatment for patients with ESRD

However it does not replace all of the complex functions of a normal healthy kidne

a result patients on dia

y As

lysis still suffer from a range of problems including infection

accelerated cardio ion anemia

chron

e

se middle

alysis

sessio

s

f

vascular disease high blood pressure chronic malnutrit

ic joint and back pain and a considerably shortened life span One significant

limitation of the current dialysis technology is the inability to efficiently remove larger

toxic molecules This is mainly because of the broad pore size distribution reducing th

selective removal of toxins and unsatisfied biocompatibility causing lots of

complications such as inflammation blood clotting calcification infection etc

Dialysis purifies the patients blood by efficiently removing small molecules like

salts urea and excess water However as toxic molecules increase in size their removal

rate by hemodialysis substantially declines Typically only 10 - 40 of the

molecular weight toxins (300-15000 Da) are removed from the blood during a di

n [Vanholder et al 1995] These toxins then reach an abnormally high level and

begin to damage the body One such toxin β2M causes destructive arthritis and carpal

tunnel syndrome by joining together like the links of chain to form a few very large

molecules and deposit damaging the surrounding tissues [Lonnemann et al 2002] This i

also a main cause of mortality for long-term dialysis patients Other middle molecule

toxins appear to inhibit the immune system and may play a significant role in the high

susceptibility to infections in dialysis patients Still others are believed to impair the

functioning of several other body systems such as the hematopoietic and other endocrine

systems This may contribute to accelerated cardiovascular disease the leading cause o

20

death among dialysis patients as well as clinical malnutrition which affects up to 50 of

this patient population

Over the last decade polymeric dialysis membranes have been developed to

increase the capacity for removing middle molecular weight toxins by changing the pore

size of dialyzer membranes and using new materials that adsorb these toxins for

impro se

styrene made during World War II in the United

States The Dow chemical compan anufacturer of polystyrene

includ

d of

on

rug

ntrol of

ved removal characteristics However removal efficiency is not as high as tho

achieved by a normal healthy kidney

26 Latex Particles

The first synthetic polymer synthesized using emulsion polymerization was a

rubber composed of 13-butadiene and

y has been a major m

ing latex which they used in paint formulations The theory of emulsion

polymerization in which a surfactant is used was established by Harkins [1948] and by

Smith and Ewart [1948] By 1956 the technology was complete including the metho

building larger diameter particles from smaller ones The product by this emulsi

polymerization is referred to as latex a colloidal dispersion of polymer particles in water

medium [Odian 1991] Latexes are currently undergoing extensive research and

development for a broad range of areas including adhesives inks paints coatings d

delivery systems medical assay kits gloves paper coatings floor polish films carpet

backing and foam mattresses to cosmetics The relatively well known and easy co

the emulsion process is one of main advantages for these applications Therefore

polymeric latex particle prepared by emulsion polymerization can be a candidate for the

medical applications because of the easy control of the particle size and morphology as

well as flexible surface chemistry to be required

21

261 The Components for Emulsion Polymerization

The main components for emulsion polymerization process are the monomer a

dispersing medium a surfactant and an initiator Available monomers are styrene

sing medium is usually water

which

ticles

n

ation To

monomer droplets but in micelles because the initiators are

insolu le

and

olution

butadiene methylmethacylate acryl acid etc The disper

will maintain a low solution viscosity provide good heat transfer and allow

transfer of the monomers from the monomer droplets into micelles and growing par

surrounded by surfactants respectively The surfactant (or emulsifier) has both

hydrophilic and hydrophobic segments Its main functions is to provide the nucleatio

sites for particles and aid in the colloidal stability of the growing particles Initiators are

water-soluble inorganic salts which dissociate into radicals to initiate polymeriz

control the molecular weight a chain transfer agent such as mercaptan may be present

262 Particle Nucleation

Free radicals are produced by dissociation of initiators at the rate on the order of

1013 radicals per milliliter per second in the water phase The location of the

polymerization is not in the

ble in the organic monomer droplets Such initiators are referred to as oil-insolub

initiators This is one of the big differences between emulsion polymerization

suspension polymerization where initiators are oil-soluble and the reaction occurs in the

monomer droplets Because the monomer droplets have a much smaller total surface area

they do not compete effectively with micelles to capture the radicals produced in s

It is in the micelles that the oil soluble monomer and water soluble initiator meet and is

favored as the reaction site because of the high monomer concentration compared to that

in the monomer droplets As polymerization proceeds the micelles grow by the addition

of monomer from the aqueous solution whose concentration is refilled by dissolution of

22

monomer from the monomer droplets There are three types of particles in the emulsion

system monomer droplets inactive micelles in which polymerization is not occurring

and active micelles in which polymerization is occurring referred to as growing polymer

particles

The mechanism of particle nucleation occurs by two simultaneous processes

micellar nucleation and homogeneous nucleation Micellar nucleation is the entry of

radicals ei

ther primary or oligomeric radicals formed by solution polymerization from

the aq ed

the

r

s

n

is coagulation with other particles rather than

polymerization of monomer The driving force for coagulation of precursor particles

ueous phase into the micelles In homogeneous nucleation solution-polymeriz

oligomeric radicals are becoming insoluble and precipitating onto themselves or onto

oligomers whose propagation has ended [Fitch et al 1969] The relative levels of micella

and homogeneous nucleation are variable with the water solubility of the monomer and

the surfactant concentration Homogeneous nucleation is favored for monomers with

higher water solubility and low surfactant concentration and micellar nucleation is

favored for monomers with low water solubility and high surfactant concentration It has

also been shown that homogeneous nucleation occurs in systems where the surfactant

concentration is below CMC [Roe 1968] A highly water insoluble monomer such a

styrene [Hansen et al 1979 Ugelstad et al 1979] has probably created by micellar

nucleation while a water soluble monomer such as vinyl acetate [Zollars 1979] has bee

formed by homogeneous nucleation

A third latex formation reaction mechanism has been proposed referred to as

coagulative nucleation In this reaction the major growth process for the first-formed

polymer particles (precursor particles)

23

sever les

yer

es

r a

l

the beginning of the polymerization As soon as the initiator is

gins with the formation and

growt

and

ved

added at one time during the later stages of the polymerization prior to complete

al nanometers in size is their relative instability compared to larger sized partic

The small size of a precursor particle with its high curvature of the electrical double la

permits the low surface charge density and high colloidal instability Once the particl

become large enough in size maintaining the high colloidal stability there is no longe

driving force for coagulation and further growth of particles takes place only by the

polymerization process

263 Types of Processes for Emulsion Polymerization

There are three types of production processes used in emulsion polymerization

batch semi-continuous (or semi-batch) and continuous In the batch type process al

components are added at

added and the temperature is increased polymerization be

h of latex particles at the same time There is no further process control possible

once the polymerization is started In the semi-continuous emulsion polymerization

process one or more components can be added continuously Various profiles of particle

nucleation and growth can be generated from different orders of component addition

during polymerization There are advantages to this process such as control of the

polymerization rate the particle number colloidal stability copolymer composition

particle morphology In the continuous process the emulsion polymerization components

are fed continuously into the reaction vessel while the product is simultaneously remo

at the same rate High production rate steady heat removal and uniform quality of

latexes are advantages of the continuous polymerization processes

Other available methods include the intermittent addition and shot addition of one

or more of the components In the shot addition process the additional components are

24

conversion of the main monomer This method has been used successfully to develo

water-soluble functional monomers such as sodium styrene sulfonat

p

e [Kim et al 1989]

264

lsion

Chemistry of Emulsion Polymerization

Emulsion polymerization is one type of free radical polymerization and can be

divided into three distinct stages initiation propagation and termination The emu

polymerization system is shown in Figure 2-7

Aqueous phaseAqueous phase

Micelle withmonomer

I Rbull

Emulsifier

Polymer particle swollenWith monomer

Monomerdroplet

I Rbull

Micelle withmonomer

I Rbull

Emulsifier

Polymer particle swollenWith monomer

MonomerdropletMonomerdroplet

I Rbull

Figure 2-7 Emulsion polymerization system [Radicals (Rmiddot) are created from initiators

(I) Monomer is transferred from larger monomer droplet into micelles by emulsifier Initiated polymer particle by radicals is keep growing until monomers are all consumed The reaction is performed in aqueous media]

In the initiation stage free radicals are created from an initiator by heat or an

ultraviolet radiation source The initiator with either peroxides groups (-O-O-) such as

odium persulfate or azo groups (-N=N-) such as azobisisobutyronitrile is commonly

used for em t

with the m er

chain ere

s

ulsion polymerization The primary free radicals created from initiator reac

onomer for initiation of polymerization In the propagation stage the polym

grows by monomer addition to the active center a free radical reactive site Th

are two possible modes of propagations head-to-head addition and head-to-tail addition

25

The head-to-tail mode is the predominant configuration of the polymer chain a result o

steric hindrance of the substitute bulky group In the termination stage polymer chain

growth is terminated by either coupling of two growing chains forming one polymer

molecule or transferring a hydrogen atom (dispropotionation) from one growing chain to

another forming two polymer molecules one having a saturated end group and the other

with an unsaturated end-group

265 Seed Emulsion Polymerization

Polymer latex particles have received increasing interest because of the versatili

of the many applications heterophase polymerization processes like emulsion dispersion

micro-emulsion seeded emulsion precipitation etc Especially to prepare well-defined

microspheres having monodispe

f

ty

rse and various particle sizes as well as surface group

ed particles prepared by emulsion polymerization

and e

s

hological design In

seede to control

n

functionalilties it is necessary to use se

nlarge them to a desired size in the further stages of reactions

Polymeric particles are required to have a uniform particle size in many

applications such as chromatography where they are used as a packing material

Morphological control of latex particle is also important for many practical application

[Schmidt 1972] Seed emulsion polymerization (or two-stage emulsion polymerization) is

a useful method to achieve both monodisperse particle size and morp

d emulsion polymerization [Gilbert 1995] preformed lsquoseedrsquo latex is used

the number of particles present in the final latex The advantage of seeded emulsio

polymerization is that the poorly reproducible process of particle nucleation can be

bypassed so that the number concentration of particles is constant and known Various

mechanisms have been proposed for the growth of latex particles [Ugelstad et al 1980

Okubo et al 1992] using this polymerization technique The initial seed particle

26

preparation step is well known and relatively easy to perform because at the relative

small particle sizes (02 to 05 micron) the particle growth process can be readily

controlled by the use of an emulsifier Enough emulsifier is used to prevent coagulation

but not enough to cause the formation of new particles As the particles are grown to

larger sizes in successive seeding steps it becomes increasingly difficult to mainta

stable uncoagulated emulsion without forming new particles and thereby destroying

monodisperisty of the latex Recently there have been several reports concerning t

reaction kinetics of seed emulsion polymerization and the development of latex

morphologies over the course of the reaction [Chern et al 1990 Delacal et al 1990 L

et al 1995 Lee 2000 2002]

266 Polystyrene Latex Particles

A number of papers have described the synthesis of the polystyrene latex particl

bearing various functional surface groups such as carboxyl [Lee et al 2000 Tun

2002 Reb et al 2000] hydroxyl [Tamai et al 1989] marcapto [Nilson 1989] epoxy

[Shimizu et al 2000 Luo et al

ly

in a

he

ee

es

cel et al

2004] acetal [Izquierdo et al 2004 Santos et al 1997]

ometyl [Izquierdo et al 2004 Park et al 2001

Sarob

h the

d

thymine [Dahman et al 2003] chlor

e et al 1998] amine [Counsin et al 1994 Ganachaud et al 1997 Ganachaud et al

1995 Miraballes-Martinez et al 2000 2001 Anna et al 2005] ester [Nagai et al 1999]

etc In order to produce these functionalized particles different methods for particle

preparation must be used The polymer emulsion with core-shell morphology of latex

particles is one of them This is a multistep emulsion polymerization process in whic

polystyrene ldquocorerdquo particle is synthesized in the first stage and the functional monomer is

added in the second stage of the polymerization without any emulsifier postfeeding to

prevent the production of new homopolymer particles thus forming the functionalize

27

polymer ldquoshellrdquo on the ldquocorerdquo particle [Keusch et al 1973] There are requirements to

limit secondary nucleation and encourage core-shell formation in seeded emulsion

polymerization including the addition of smaller seed particles at high solid content to

increase particle surface area low surfactant concentration to prevent formation of

micelles and the semi-continuous addition of monomer to create a starved-feed conditio

and keep the monomer concentration low There are some advantages [Hergeth et al

1989] of dispersions with polymeric core-shell particles First it is possible to modi

interfacial properties of polymer particles in the aqueous phase by the addition of only

very small amounts of a modifying agent during the last period of the reaction Thus

these core-shell particles are useful in a broad range of applications since they always

exhibit improved physical and chemical properties over their single-component

counterparts [Lu et al 1996 Nelliappan et al 1997] In this way the improvement of

surface properties of such dispersions is straightforward and inexpensive The other is

that polymers with a core-shell structure are perfect model systems for investigating th

material properties of polymer blends and composites because of their regular

distribution of one polymer inside a matrix polymer and because of the simple sp

geometry of the system

Their properties usually depend on the structures of latex particles Chen and his

coworkers [Chen et al 1991 1992 1993] reported the morphological development of

core shell latex particles of polystyrenepoly(methyl methacrylate) during

polymerization Before the research by Chen and his coworker Min et al [Min et al

1983] reported the morph

n

fy the

e

herical

ological development of core shell latex of polystyrene

(PS)polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the

28

additi

of

ity of graft

ing reagents Thus protein adsorption on polymeric solid surfaces has

st are microspheres defined as ldquofine

polym can be

n

r was

containing antigen urine serum etc The latex will become agglutinated and visibly

on method of PS They found that the percentage of grafting PS to the PBA was

greatest for the batch reaction and the PBA-PS core-shell particles with a high degree

grafting remained spherical upon aging test because of the emulsifying abil

copolymer

267 Various Applications of Latex Particles

Latex particles are applicable to a wide range of areas such as biomedical

applications [Piskin et al 1994] especially as the solid phase such as in immunoassays

[Chern et al 2003 Radomske-Galant et al 2003] DNA diagnostic drug delivery carriers

[Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] blood cell separations and

column pack

become a center of attention Of particular intere

er particles having diameters in the range of 01 to several micronsrdquo which

used as functional tools by themselves or by coupling with biocompounds Singer and

Plotz [1956] firstly studied microsphere or latex agglutination test (LATrsquos) by using

monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support o

which the biomolecules were going to adsorb The biomolecule adsorption howeve

limited by possible desorption of the adsorbed species or loss of specific activity of the

complex formed Since this work was published the latex particle applications for

immunoassay have been rapidly and widely studied and developed

Latex agglutination test or latex immunoassays start with tiny spherical latex

particles with a uniform diameter and similar surface properties The particles are coated

with antibodies (sensitized) through the hydrophobic interaction of portions of the protein

with the PS surface of the particles If sensitized particles are mixed with a sample

29

agglomerate Latex tests are inexpensive as compared with the other techniques [Ba

1988]

ngs

sed

tion Rheumatoid factor (RF) in different age subpopulations has also been

evalu

studied a

latex

Unipath [Percival 1996] has manufactured a range of immunoassay materials ba

on a chromatographic principle and deeply colored latex particles for use in the home and

clinical environments The latex particles which are already sensitized with a

monoclonal antibody can detect any antigens that bound to the surface of the latex

particles Some detectable pollutants include estrogen mimics which induce

abnormalities in the reproductive system of male fishes and lead to a total or partial male

feminiza

ated according to a patientrsquos clinical status by using a rapid slide latex agglutination

test for qualitative and semiquantitative measurement in human serum along with latex

immunoassay method [Onen et al 1998] Magalhaes and his coworkers [2004]

diagnostic method of contamination of male fishes by estrogen mimics using the

production of vitellogenin (VTG) as a biomarker This was based on a reverse

agglutination test developed with monoclonal antibodies specific to this biomarker

Premstaller and his coworkers [Premstaller et al 2000 2001] have prepared a porous

poly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient

chromatographic separation of biomelecules such as proteins and nucleic acids They

used a porogen a mixture of tetrahydrofuran and decanol to fabricate a micropellicular

PS-DVB backbone Legido-Quigley and his colleagues [2004] have developed the

monolith column to obtain further chromatographic functionality to the column by

introducing chloromethylstyrene in place of styrene into the polymer mixture

30

Core shell type monodisperse polymer colloids have been synthesized by Sarobe

and Forcada [1998] with chloromethyl functionality in order to improve the biomolecu

adsorption through a two-step emulsion polymerization process They investigated the

functonalized particles by optimizing the experimental parameters of the functional

monomer including reaction temperature the amount and type of redox initiator sys

used the type of addition of the initiator system and the use of washing They c

le

tem

oncluded

that th

sis

serve as separation

chann

of

usually

along the polypeptide chain which contain positive or negative charges [Norde 1998]

e relation between the amount of iron sulfate and the persulfatebisulfite system

added should be controlled to obtain monodisperse particles and prevent the premature

coagulation of the polymer particles during the polymerization

A semi-continuous emulsion polymerization technique for latex particle synthe

was performed by McDonald and other researchers [Ramakrishnan et al 2004 Steve et

al 1999] They introduced a variety of particles sizes compositions morphologies and

surface modifications to fabricate latex composite membranes (LCMs) Arraying and

stabilizing latex particles on the surface of a microporous substrate form narrowly

distributed interstitial pores formed between the particles which

els They investigated the membrane performance using gas fluxes water

permeability and the retention characterization of dextran molecules From these tests

they concluded that the narrow discriminating layer made of the latex particles leads to a

highly efficient composite membrane

27 Proteins

Proteins are natural polyamides comprised of about 20 different α-amino acids

varying hydrophobicity [Norde 1998] Proteins are more or less amphiphilic and

highly surface active because of the number of amino acid residues in the side groups

31

Proteins are polymers of L-α-amino acids [Solomon and Fryhle 2000] The α refers to a

carbon with a primary amine a carboxylic acid a hydrogen and a variable side-chain

group designated as R Carbon atoms rent groups are asymmetric and can

exhib l

s

e

on

with four diffe

it two different spatial arrangements (L and D configurations) due to the tetrahedra

nature of the bonds The L refers to one of these two possible configurations Amino

acids of the D-configuration are not found in natural proteins and do not participate in

biological reactions Figure 2-8 shows the chiral carbon in 3-D as the L isomer Protein

consist of twenty different amino acids differentiated by their side-chain groups [Norde

1998] The side-chain groups have different chemical properties such as polarity charg

and size and influence the chemical properties of proteins as well as determine the

overall structure of the protein For instance the polar amino acids tend to be on the

outside of the protein when they interact with water and the nonpolar amino acids are

the inside forming a hydrophobic core

Figure 2-8 L-α-amino acid

The covalent linkage between two amino acids is known as a peptide bond A

peptide bond is formed when the amino group of one amino acid reacts with the carboxyl

group of another amino acid to form an amide bond through the elimination of water

This arrangement gives the protein chain a polarity such that one end will have a free

amino group called the N-terminus and the other end will have a free carboxyl group

called the C-terminus [Solomon and Fryhle 2000] Peptide bonds tend to be planar and

32

give the polypeptide backbone rigidity Rotation can still occur around both of the α-

carbon bonds resulting in a polypeptide backbone with different potential conformations

relative to the positions of the R groups Although many conformations are theoretically

possible interactions between the R-groups will limit the number of potential

nd to form a single functional conformation In other words

the conformation or shape of the protein is

α-helix

can interact with other

secondary structures within the sam

conformations and proteins te

due to the interactions of the chain side

groups with one another and with the polypeptide backbone The interactions can be

between amino acids that are close together as in a poly-peptide between groups that are

further apart as in amino acids or even on between groups on different polypeptides all

together These different types of interactions are often discussed in terms of primary

secondary tertiary and quaternary protein structure

The primary amino acid sequence and positions of disulfide bonds strongly

influence the overall structure of protein [Norde 1986] For example certain side-chains

will promote hydrogen-bonding between neighboring amino acids of the polypeptide

backbone resulting in secondary structures such as β-sheets or α-helices In the

conformation the peptide backbone takes on a spiral staircase shape that is stabilized by

H-bonds between carbonyl and amide groups of every fourth amino acid residue This

restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure

Certain amino acids promote the formation of either α-helices or β-sheets due to the

nature of the side-chain groups Some side chain groups may prevent the formation of

secondary structures and result in a more flexible polypeptide backbone which is often

called the random coil conformation These secondary structures

e polypeptide to form motifs or domains (ie a

33

tertiary structure) A motif is a common combination of secondary structures and a

domain is a portion of a protein that folds independently Many proteins are composed o

multiple subunits and therefore exhibit quaternary structures

271 Interaction Forces between Proteins

Proteins in aqueous solution acquire compact ordered conformations In such a

compact conformation the movement along the polypeptide chain is severely restrict

implying a low conformational entropy The compact structure is possible only if

interactions within the protein molecule and interactions between the protein molecule

and its environment are sufficiently favorable to compensate for the low conformational

entropy [Malmsten 1998] Protein adsorption study is often focused on structural

rearrangements in the protein molecules because of its significance to the biological

functioning of the molecules and the important role such rearrangements play in the

mechanism of the adsorption process Knowledge of the majo

f

ed

r interaction forces that act

structures helps to understand the behavior

of pro

arge

a

ded

between protein chains and control the protein

teins at interface These forces include Coulomb interaction hydrogen bonds

hydrophobic interaction and van der Waals interactions

Coulombic interaction Most of the amino acid residues carrying electric ch

are located at the aqueous boundary of the protein molecule An the isoelectric point

(IEP) of the protein where the positive and negative charges are more or less evenly

distributed over the protein molecule intramolecular electrostatic attraction makes

compact structure favorable to proteins Deviation to either more positive or more

negative charge however leads to intramolecular repulsion and encourages an expan

structure Tanford [1967] calculated the electrostatic Gibbs energy for both a compact

impenetrable spherical molecule (protein) and a loose solvent-permeated spherical

34

molecule (protein) over which the charge is spread out From the results he found th

the repulsion force was reduced at higher ionic strength d

at

ue to the screening action of ion

ble

e

the

d the

tween

dehyd

e

ent

oles

tein

Hydrogen bond Most hydrogen bonds in proteins form between amide and

carbonyl groups of the polypeptide backbone [Malmsten 1998] The number of availa

hydrogen bonds involving peptide units is therefore far greater than that involving sid

chains Because α-helices and β-sheets are aligned more or less parallel to each other

interchain hydrogen bonds enforce each other Kresheck and Klotz [1969] examine

role of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between

peptide units do not stabilize a compact structure of protein However because the

peptide chain is shielded from water due to other interactions hydrogen bonding be

peptide groups do stabilize α-helical and β-sheet structures

Hydrophobic interaction Hydrophobic interaction refers to the spontaneous

ration and subsequent aggregation of non-polar components in an aqueous

environment In aqueous solutions of proteins the various non-polar amino acid residues

will be found in the interior of the molecule thus shielded from water The

intermolecular hydrophobic interaction for the stability of a compact protein structure

was first recognized by Kauzmann [1959] If all the hydrophobic residues are buried in

the interior and all the hydrophilic residues are at the outermost border of the molecul

intramolecular hydrophobic interaction would cause a compact protein structure

However geometrical and other types of interactions generally cause a fraction of the

hydrophobic residues to be exposed to the aqueous environm

Van der Waals interaction The mutual interaction between ionic groups dip

and induced dipoles in a protein molecule cannot be established as long as the pro

35

structure is not known in great detail Moreover the surrounding aqueous medium also

contains dipoles and ions that compete for participation in the interactions w

the protein molecule Dispersion interactions favor a compact structure However

because the Hamaker constant for proteins is only a little larger than that of water the

resulting effect is relatively small [Nir 1977]

272 β

ith groups of

rgiles et al 1996

Floeg

) is degraded [Floege 2001] Renal

β2M from the serum resulting in an increase in β2M

conce well

the

)

a

al

2-Microglobulin (β2M)

The protein β2M is of particular interest because it is involved in the human

disorder dialysis-related amyloidosis (DRA) [Geiyo et al 1985 A

e 2001] DRA is a complication in end stage renal failure patients who have been on

dialysis for more than 5 years [Bardin et al 1986 Drueke 2000] DRA develops when

proteins in the blood deposit on joints and tendons causing pain stiffness and fluid in

the joints as is the case with arthritis In vivo β2M is present as the non-polymorphic

light chain of the class I major histocompatibility complex (MHC-I) As part of its

normal catabolic cycle β2M dissociates from the MHC-I complex and is transported in

the serum to the kidney where the majority (95

failure disrupts the clearance of

ntration by up to 60-fold [Floege 2001] By a mechanism that is currently not

understood β2M then self-associates into amyloid fibrils and typically accumulates in

musculoskeletal system [Homma et al 1989] Analysis of ex vivo material has shown

that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRA

is present as of full-length wild-type β2M although significant amounts (~20-30) of

truncated or modified forms of the protein are also present [Floege 2001 Bellotti et al

1998] Figure 2-9 shows the ribbon diagram of human β2M Native β2M consists of

single chain of 100 amino acid residues and has a seven stranded β-sandwich fold typic

36

of the immunoglobulin superfamily [Saper et al 1991 Trinh et al 2002] β2M was fi

isolated from human urine and characterized by Berggard et al [1980] in1968 The

normal serum concentration of β

rst

re

f

e

2M is 10 to 25 mgL It is a small globular protein with

a molecular weight of 118 kDa a Strokes radius of 16Aring and a negative charge under

physiological conditions (isoelectric point IEP = 57) β2M contains two β-sheets that a

held together by a single disulphide bridge between the cysteines in positions 25 and 81

[Berggard et al 1980 Parker et al 1982 Cunningham et al 1973] β2M cannot be

removed completely by current dialysis techniques but through a better understanding o

the structure and interaction forces that lead to this structure it will be possible to mor

efficiently remove this problematic protein

Figure 2-9 Ribbon diagram of human β M taken from the crystal structure of the protein

bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khal 2000]

273 Serum Albumin

Serum albumin is the most abundant protein found in plasma and is typically

present in the blood at a concentration of 35~

2an et

50gL According to extensive studies about

its physiological and pharmacological properties albumin has a high affinity to a very

wide range of materials such as electrolytes (Cu+2 Zn+2) fatty acids amino acids

metabolites and many drug compounds [Fehske et al 1981 Kraghhansen 1981 Putnam

37

1984 Peters 1985] The most important physiological role of the protein is therefore to

bring such solutes in the bloodstream to their target organs as well as to maintain the pH

and osmotic pressure of the plasma Bovine serum albumin (BSA) is an ellipsoidal

protein with the dimensions of 140 X 40 X 40Aring [Peter 1985] The primary structure is a

single helical polypeptide of 66 kDa (IEP = 47) with 585 residues containing 17 pairs of

as a soft an

rfaces [Kondo et al 1991 Norde et al 1992 Soderquist et al

1980 ikely

is

disulfide bridges and one free cysteine [Dugaiczyk et al 1982] BSA has been classified

d flexible protein because it has a great tendency to change its conformation

on adsorption to solid su

Carter and Ho 1994] and consists of three homologous domains (I-III) most l

derived through gene multiplication [Brown 1976] Each domain is composed of A and B

sub-domains [He et al 1992] The secondary structure of human serum albumin (HSA)

shown in Figure 2-10

Figure 2-10 Secondary structure of human serum albumin (HSA) with sub-domains

[Zunszain et al 2003]

HSA has the same structure domains with the serum albumin from other species

such as BSA [Brown 1976] Although all three domains of the albumin molecule have

similar three-dimensional structures their assembly is highly asymmetric [Sugio et al

38

1999] Domains I and II are almost perpendicular to each other to form a T-shaped

assembly in which the tail of subdomain IIA is attached to the interface region between

sub-domains IA and IB by hydrophobic interactions and hydrogen bonds In contrast

domain III protrudes from sub-domain IIB at a 45ordm angle to form the Y-shaped assembly

for domains II and III Domain III interacts only with sub-domain IIB These features

make the albumin molecule heart-shaped

28 Protein Adsorption

Protein adsorption studies date back to the 1930rsquos At the beginning these studies

mainly focused on the determination of the molecular weight electrophoretic and

ral

rearrangem

adsorption and biocompatibility of the sorbent materials were investigated [Norde 1986]

There are interaction forces at the interfaces between protein molecules and latex

particles These forces are mainly divided into the following groups hydrophobic

interaction ionic interaction hydrogen bonding and van der Waals interaction [Andrade

1985]

Hydrophobic interaction It is known that hydrophobic interaction has a major

role in protein adsorption phenomena The adsorption of proteins on the low charged

latex particles occurs by this interaction force Generally monomers such as styrene offer

hydrophobic surfaces th

by this interaction force is maximum at the isoelectric point (IEP) of the protein and the

strength [Suzawa et al 1980 1982 Shirahama et al 1989 Kondo et al 1992] By the

chromatographic applications Later the adsorption mechanism especially the structu

ents was studied Recently the studies of the relation between protein

281 Interaction between Protein Molecule and Latex Particle

at protein molecules adsorb to The amount of adsorbed protein

pH at maximum adsorption shifts to a more acidic region with an increase in ionic

39

reports [Suzawa et al 1980 1982 Lee et al 1988] protein adsorption was greater

hydrophobic surface than on a hydrophilic one implying that hydrophobic interaction is

on a

s

ydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA)

Ionic

ely

t

ces

n force is operative over small

distan e

ble

n

nt dialysis membrane therapy for end stage renal disease (ESRD)

one of most dominant forces in protein adsorption

Ionic interaction Negatively charged latex particles have ionic functional

groups on their surfaces such as salts of sulfonic and carboxylic acid Sulfate group

originate from an initiator such as sodium persulfate and carboxylic groups originate

from a h

bonds are formed between the negative charges of these latex particles and positive

surface charges of protein molecules The conventional low-charged latex particles rar

form these ionic bonds

Hydrogen bonding Hydrogen bond is a strong secondary interatomic bond tha

exists between a bound hydrogen atom (its unscreened proton) and the electrons of

adjacent atoms [Callister 1999] Protein can be adsorbed on hydrophilic polar surfa

through hydrogen bonding Hydrogen bonds are frequently formed between hydroxyl-

carbonyl or amide-hydroxyl Hydroxyl-hydroxyl or amide-hydroxyl bonds are also

formed in protein adsorption

Van der Waals interaction This interactio

ces only when water has been excluded and the two non-polar groups come clos

to each other Lewinrsquos calculation showed that the van der Waals interaction is negligi

compared with the forces involved in the entropy increases ie hydrophobic interactio

[Lewin 1974]

29 Hypothesis for Toxin Removal

As mentioned earlier insufficient removal of middle molecular range toxins is a

major drawback of curre

40

patien

is

y

ese

haracteristics over the last decade However removal

effici

ing a novel membrane design composed of an assembly of engineered

polymeric latex particles s ions Important factors

will i

e

ith a

is established it is

expec

e

ts This may cause destructive arthritis and carpal tunnel syndrome inhibit the

immune system and accelerate cardiovascular disease leading to death among dialys

patients In order to overcome these complications artificial dialysis membranes have

been developed to increase the capacity for removing middle molecular weight toxins b

changing the pore size of dialysis membranes and using new materials that adsorb th

toxins for improved removal c

ency is not as high as those achieved by a normal healthy kidney

With the knowledge obtained from all above literatures the following hypothesis

has been established for the development of a membrane for the successful removal of

the target protein β2-Microglobulin (β2M) without the removal of serum albumin This

will be done us

ynthesized to predetermined specificat

nclude pore size and surface chemistry

291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size

The packing of monodispersed spherical particles can lead to the formation of

porous layers suitable for use as filters and membranes [Hsieh et al 1991] The pore siz

is defined as the largest spherical particles that can pass through the interstitial spaces

It is well known that monodispersed spherical particles can be obtained from the

seed emulsion polymerization method Many commercially available latex particles are

synthesized by this technique and are inexpensive When the defect free membrane w

monodisperse pore size distribution based on a particular particle array

ted to outperform the traditional hemodialysis membrane limitation which is a

broad range of pore size distribution When spherical particles pack in regular crystallin

arrays a number of packing geometries such as hexagonal closest packing cubic closest

41

packing and body-centered cubic packing are possible The pore size of the array

depends on these packing geometries as well as particle size Theoretical pore size can be

calculated from the simple geometry of the arr

ay Figure 2-11 shows the relationship

between particle size and pore size

0001

001

01

001 01 1 10

Por

e

1

10

microm

) S

ize

(

Particle Diameter (microm)

Body-Centered Cubic ArrayClosest Packed Array

0001

Siz

e (

Particle Diameter (microm)

001

01

001 01 1 10

Por

e

1

10

microm

)

Body-Centered Cubic ArrayClosest Packed ArrayBody-Centered Cubic ArrayClosest Packed Array

and closed packed arrays [Steve et al 1999]

McDonald and his coworkers [Ramakrishnan et al 2004 Steve et al 1999] have

described the fabrication of latex composite membranes and their performance in

previous papers It is from this prior research and knowledge gained from conversations

with Dr McDonald that this current research has been initiated [McDonald 2003] The

focus of this thesis is the extension of McDonaldrsquos research in the area of composite

membranes to medical application such as dialysis membranes

292 Toxin Removal by Selective Adsorption on Engineered Latex Particles

The flexibility in latex surface properties is a significant advantage to this

materialrsquos ability to separate proteins since the selectivity is strongly dependent on the

charge interactions of both the protein and the latex particles under the conditions of

Figure 2-11 The relationship between pore size and particle size in body-centered cubic

42

separation [Menon et al 1999 Chun et al 2002] First all sorbents were designed to

have negative surface properties because most plasma proteins in blood are negative and

should not be removed with charge interaction between proteins and sorbents at

physiological condition (pH74) It was reported that a negatively charged surface was

more blood compatible than a positive one [Srinivasan et al 1971] The toxin protein β2-

Microglobulin (β2M) can be selectively adsorbed on the surface of latex particles by the

design of a suitable hydrophobic portioned surface where only β2M protein can be

anchored with hydrophobic interactions Albumin adsorption on the latex particle is not

side-on m

adsorption of β M protein on the engineered latex particles

allowed because charge repulsion is more dominant than hydrophobic interaction with

ode The figure 2-12 shows the schematic representation of the selective

2

PMMAPAAShell

COO-

OSO3-

OSO3-

PSCore

COO-

-

-

-

-

-

-

Albumin-

Chargerepulsion

--

-

- -

- -

- --

Hydrophobic

β2-Microglobulin

2

Interaction

β -Microglobulin

PMMAPAAShell

COO-

-

-

-

-

-

-

--

-

-

-

-

-

Albumin-

Chargerepulsion

OSO3-

OSO3-

PSCore

COO-

--

-

- -

--

-

- -

- -

- --

- -

- --

Hydrophobic

β2-Microglobulin

2

particles at pH 74

Interaction

β -Microglobulin

Figure 2-12 Schematic representation of the protein adsorption on the core shell latex

43

Hydrophilichydrophobic microdomain structures were proven to be more blood

compatible [Mori et al 1982 Higuchi et al 1993 Deppisch et al 1998] The

optimization of suitable hydrophobic to hydrophilic ratio is also important for the

biocompatibility The monomers such as styrene (St) methyl methacrylate (MMA) and

acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in

emulsion polymerization process

In summary the background and fundamental literature survey about the history

material properties and limitation of hemodialysis membrane latex particle preparation

surface chemistry and manufacturing process target proteins and protein adsorption and

finally the hypothesis for a toxin removal with high separation efficiency have been

suggested This research focuses on such hypothesis and the materials and

characterization methodology for achievement of suggested hypotheses are described in

the next chapter

CHAPTER 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY

As mentioned earlier the goal of this study is to prepare polymeric latex particles

with tailored properties to maximize separation of toxin molecules and to investigate

fundamental interactions between the applied particles and molecules in the

system to optimize the membrane performance for hemodialysis applications Polymeric

latex particles wit

the

biological

h monodisperse size distribution to obtain uniform pore size and

various size ranges to utilize membrane construction are necessary Surface engineering

with various combination of hydrophobichydrophilic domain on the surface of latex

particles is expected to affect the removal of target protein by selective adsorption and to

improve the biocompatibility of membrane Therefore the materials and characterization

methodology are addressed in this chapter

31 Materials

Styrene (St) monomer used for a seed and a core particle was purchased from

Fisher Scientific and used without any other purification process Acrylic acid (AA) and

methyl methacrylate (MMA) monomers introduced for shell formation were purchased

from Fisher Scientific and used without any other purification process Sodium persulfate

(SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as

received Divinylbenzene (DVB) crossliking agent was purchased from Aldrich The

anionic surfactant Aerosol regMA80-I [sodium di(13-dimethylbutyl) sulfosuccinate] was

kindly donated by Cytec 78-80 Aerosol regMA80-I is mixed with isopropanol and

water Its critical micelle concentration (CMC) is 245 mM (119) Ion exchange resin

44

45

AGreg 501-x8 Resin (20-50 mesh) was purchased from Bio-Rad Laboratories Inc It is a

mixed-bed resin with H+ and OH- as the ionic forms and is 8 crosslinked Bovine serum

albumin (BSA) (heat shock treated) was purchas Fisher Scientific The isoelectric

point of t lin

(β2M)

as

gma

ed from

he BSA is 47-49 and the molecular weight is 66300Da β2-Microglobu

was purchase from ICN Biomedicals Inc and was separated from patients with

chronic renal disease and lyophilized from ammonium bicarbonate The molecular

weight by SDS-PAGE was approximately 12000Da BSA and β2M proteins were used

received without further purification process In order to investigate the protein

adsorption evaluation bicinchoninic acid protein assay kits were purchased from Si

(Cat BCA-1) and Pierce Biotechnology (Cat 23225) The chemical structures for the

main chemicals are shown in Figure 3-1

CH2 CH

Styrene (Mw = 10415)

CH2 CHCH2 CH

Styrene (Mw = 10415)

CH2 CH

Acrylic acid (Mw = 7206)

C

OH

O

CH2 CH

Acrylic acid (Mw = 7206)

C

OH

O

CH CH2

C

OCH3

O

CH

CH CH3

2

C

OCH3

O

CH3

Methyl methacrylate (Mw = 10012)Methyl methacrylate (Mw = 10012)

CH2 CH

Divinylbenzene (Mw = 1302)

CH CH2

CH2 CH

Divinylbenzene (Mw = 1302)

CH CH2CH CH

+(Na) -(O3S)

CH -C-O-CH-CH -CH-CH3

O

2 2 3

O CH CH3

CH3 CH3

CH-C-O-CH-CH2-CH-CH3

Anionic surfactant (MA80-I) (Mw = 3885)

+(Na) -(O3S)

CH -C-O-CH-CH -CH-CH3

O

2 2 3

O CH CH3

CH3 CH3

CH-C-O-CH-CH2-CH-CH3

Anionic surfactant (MA80-I) (Mw = 3885)2

+(Na)-(O) S OO

O O

- +

Initiator sodium persulfate (Mw = 23811)

O SO

(O) (Na)+ -

O O

- +(Na) (O) S OO

O SO

(O) (Na)+ -

O O

- +

Initiator sodium persulfate (Mw = 23811) Figure 3-1 Chemical structure of main chemicals

(Na) (O) S OO

O SO

(O) (Na)

46

32 Latex Particle Preparation

The experimental set up and the particle preparation schemes for various types and

size ranges of latex particles are shown in Figure 3-2 and 3-3 In the experimental setup a

mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at

end of glass stirrer The agitation rate is precisely adjusted by controller motor Paar glas

reactor with 1L volume is partially in silicon oil bath on hot plate The reactor has fo

openings for a stirrer a thermometer a nitrogen gas inlet and a reactant feeding lnlet

The reactants for latex polymerization are fed by precisely controlled by meterin

The particle preparation is described in each latex preparation section

the

s

ur

g pump

Figure 3-2 Experimental setup for semi-continuous emulsion polymerization

1 Motor 2 Mechanical stirrer bar 3 Thermometer 4 Inlet of N2 5 Inlet for initiator and monomer 6 Glass reactor (l L) 7 Oil bath 8 Hot plate 9 Pump for initiator 10 Pump for monomer 11 Tubes for reactant feed

47

Styrene monomer

Bare PS

PSPMMA100

PSPMMA90PAA10

PSPMMA P75 AA25

Gr

win

ticle

size

PS seedparticles

og

par

Styrene monomer

Bare PS

PSPMMA100

PSPMMA90PAA10

PSPMMA P75 AA25

Gr

win

ticle

size

PS seedparticles

og

par

Figure 3-3 The particle preparation scheme in various types and size ranges of latex particles

321 Preparation of Seed Latex Particles

Polystyrene seed particles were synthesized through a typical semi-continuous

emulsion polymerization Polystyrene latex particles were prepared in a four necked Paar

glass vessel equipped with a mechanical glass stirrer thermometer glass funnel for

nitrogen gas purge and a tube for monomer feeding The reactor was placed in a silicon

oil bath on a hot plate for homogenous and stable tempe he vessel was

firstly charged with de-ionized water the emulsifier so itiator

sodium bicarbonate (SBC) styrene (St) monomer divinylbenzene (DVB) monomer

under nitrogen gas atmosphere and then heated to 72plusmn2 as

llowed to continue for another hour Second reactants St and DVB monomers SPS

initiator and SBC were fed separately using fine fluid metering pumps (Model RHSY

Fluid metering INC NY) under a nitrogen gas atmosphere for 2h After complete feeding

rature control T

dium persulfate (SPS) in

oC for 1h The reaction w

a

48

of all reactants the reaction was continued for another hour at elevated temperature 80plusmn1

oC and then cooled to room temperature by removal of the oil bath

322 Preparation of Seeded Latex Particles

The particle growth was achieved by seeded continuous emulsion polymerization

The vessel was charged with the polystyrene seeds and de-ionized water to make desired

solid content and heated up to 72plusmn2 oC for 1h under nitrogen gas atmosphere Then St

and DVB monomers and solution of SPS and SBC in de-ionized water were continuously

added using metering pump with a precisely controlled feed rate After feeding all

reactants the reaction was continued for three hours keeping temperature of 80plusmn2 oC and

then the reaction vessel was cooled down to room temperature

Monodisperse spherical polystyrene seeds with a 280nm mean diameter were used

e-polymerized polystyrene seeded emulsion

was charged in a 500m

323 Preparation of Core Shell Latex Particles

for core shell structured latex particle The pr

l glass flask under a nitrogen gas atmosphere De-ionized water

was added to achieve the desired solid content The emulsion was heated to 80plusmn2oC for

10 hour Monomers initiator and SBC to form the shell were added by fluid metering

pumps (LAB PUMP JRRHSY model Fluid metering Inc USA) with fine controlled

feed rate Flow rate can be adjusted by control ring graduated in 450 division from 0 to

100 flow which is in the range of 10mlmin respectively The reaction was sustained

for additional 30 hours at 90plusmn2oC then cooled down to room temperature

324 Purification of Synthesized Latex Particles

Synthesized latex particles were purified with an ion exchange resin 200g (10

ww polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin

and stirred for 40 minute to remove unreacted monomers initiators and other impurities

49

Then the emulsion was diluted with DI water to a specific degree of solid content as

needed

33 Characterization

331

solid

termined

oscopy

d

hours

Dried latex (10mg) was mixed with

250 m on

Degree of Conversion

The degree of monomer to polymer conversion was determined gravimetrically

following the procedure described by Lee et al [1995] before the latex cleaning process

The synthesized emulsion raw materials were weighed and dried in a conventional oven

at 120oC for 30min to evaporate any unreacted monomers and water The remaining

was weighed and the degree of conversion was determined This process was repeated

three times and a mean value of the degree of conversion was de

332 Fourier Transform Infrared (FTIR) Spectr

A molecule can absorb only select frequencies (energy) of infrared radiation which

match the natural vibration frequencies The energy absorbed serves to increase the

amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]

Only those bonds which have a dipole moment that changes as a function of time can

absorb the infrared radiation The range of wavelengths for infrared radiation is between

25 microm (4000 cm-1) and 25 microm (400cm-1)

The synthesis of the latex particles such as polystyrene (PS) homopolymer and

PSPMMA PSPMMA90PAA10 PSPMMA75PAA25 core-shell copolymers were verifie

with FTIR Purified latex particles were dried in vacuum oven at 40oC for 24

before analysis in the FTIR (Nicolet Magma USA)

g of KBr which had also been dried in vacuum oven at 120oC for 3h Transmissi

spectra using the drift mode were plotted with 128 scans and 4cm-1 resolution

50

333 Quasielastic Light Scattering (QELS)

Latex particle size was measured by the Brookhaven ZetaPlus particle size

analyzer The solid concentration as 0001 (wv) This

ation spectroscopy (PCS) of quasielastically scattered light

For th s

ed using a FE-SEM (JEOL

JSM-

ith

ent

01

us zeta-

potential analyzer It measures the electrophoretic mobility velocity of charged colloidal

of latex particles used w

instrument uses photon correl

is technique time dependent interference patterns of light scattered from particle

in the sample cell are analyzed The interference pattern changes due to Brownian

motion giving rise to fluctuations in scattering intensity The frequency of these

fluctuations depends on the particlersquos diffusion constant that is inversely proportional to

the particle diameter

334 Field Emission-Scanning Electron Microscopy (FE-SEM)

Particle size and surface morphology were characteriz

6335F Japan) A diluted suspension of latex particles in water was dropped onto a

silicon wafer at room temperature and allowed to dry The sample was then coated w

the thinnest layer of carbon needed to obtain the required conductivity for this instrum

Secondary electron image mode was used with a 15KV of accelerating voltage The

magnification range used was between 10000X and 70000X

335 Zeta Potential Measurement

Synthesized and purified latex particles were diluted with de-ionized water to 0

wt and adjusted six different pH values 205 345 481 565 643 and 747 These

particle suspensions were then transferred to standard cuvettes for zeta potential

measurement At least two runs of ten measurements were taken for each sample and

averaged

The zeta potential measurement was carried out using Brookhaven ZetaPl

51

particles in solution and calculate zeta-potential by using the Smoluchowski equation

The f quency

gnitude

umin (BSA) as a standard model protein and β2-Microglobulin

uffer (PB) and

phosp

5

h the selected protein

e 005 01 03 05 and 07 mgml and those for

β2M

d at

teins in the supernatant after the centrifugation process using the

bicinc t

requency of laser light passing through the sample and is compared to the fre

of a reference beam The shift in the frequency called a Doppler shift and the ma

of the shift correspond to the polarity and the magnitude of the electrophoretic mobility

respectively The zeta potential is calculated from the solution conditions and the

measured mobility

336 Protein Adsorption

Protein adsorption experiments of the synthesized latex particles were performed

with bovine serum alb

(β2M) as a target protein in two types of buffer solution phosphate b

hate buffered saline (PBS) To make 5 mM of PB solution 0345g of sodium

phosphate monobasic (NaH2PO4H2O) was dissolve in 500ml of de-ionized water To

make PBS solution 0345g of sodium phosphate monobasic (NaH2PO4H2O) and 4178g

(143mM) of sodium chloride (NaCl) were dissolved in 500ml of de-ionized water The

synthesized latex particles were diluted with each buffer to have a solids content of 0

(ww) adjusted to pH values of 32 48 and 74 and mixed wit

BSA concentrations were chosen to b

were 0015 0030 0045 and 0060mgml The mixture was gently rotated using

the shaker Labquakereg (BarnsteadThermolyne Model 4002110 USA) with 8 RPM in

the incubator at 37oC for 12 hours before the latex-protein mixture was centrifuge

13000 rpm for 15min The amount of protein adsorbed was determined by quantifying

the free pro

honinic acid (BCA) assay method [Lowry et al 1951 Smith et al 1985 Baptista e

al 2003 Wiehelman et al 1988 Brown et al 1989] BCA assay kits consist of Reagent

52

A containing bicinchoninic acid sodium carbonate sodium tartrate and sodium

bicarbonate in 01N NaOH with pH=1125 and Reagent B containing 4 (wv) copper

(II) sulfate pentahydrate The BCA working reagent was prepared by mixing 50 parts of

Reagent A with 1 part of Reagent B 100microl of protein supernatant was then mixed with

2ml of BCA working reagent in a UV cuvette Incubation was allowed to continue

at room temperature

further

until color developed about 2h The absorbance at 562nm was

pectrophotometer (Perkin-Elmer Lambda 800 USA)

Unkn

Separate the p rticles in centrifuge mix supernatant with BCA reagents

measured using a UV-VIS s

own concentration of sample protein was determined by comparison to a standard

of known protein concentrations The adsorbed amount per unit surface area was

determined by the mass balance of the protein after adsorption process The simple

schematic of the procedure for a protein adsorption test is shown in Figure 3-4

Incubate mixture with agitation for 12hrs at 37oC

aand develop the color by incubation for 2hrs at room temperature

Suspend latex particles in aqueous buffer media

Add the dissolved target protein

Measure color intensity using UVVis spectroscopy at 562nm

Suspend latex particles in aqueous buffer media

Separate the p rticles in centrifuge mix supernatant with BCA reagents

Add the dissolved target protein

Incubate mixture with agitation for 12hrs at 37oC

aand develop the color by incubation for 2hrs at room temperature

Measure color intensity using UVVis spectroscopy at 562nm

Figure 3-4 Schematic of the procedure for a protein adsorption test

53

337 Blood Biocompatibility by Hemolysis Test

Hemolysis is the destruction of red blood cells which leads to the release of

hemoglobin from within the red blood cells into the blood plasma Hemolysis testing is

used to evaluate blood compatibility of the latex particles and the damaged to the red

blood cells can be determined by monitoring amount of hemoglobin released The red

blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutes

Figure 3-5 shows the phase separated blood with plasma white blood cells and red

cells after centrifugation process Separated RBSs were then washed with isotonic

phosphate buffer solution (PBS) at pH 74 to remove debris and serum protein Th

process was repeated 3 times

blood

is

Figure 3-5 Separation of RBC from whole blood by centrifuge process

Prepared latex particles were re-dispersed in PBS by sonification to obtain

homogeneously dispersed latex particles 100microl of the mixture of red blood cell (3 parts)

and PBS (11parts) was added to 1ml of 05 (ww) particle suspension PBS was used as

a negative control resulting 0 hemolysis and DI water used as a positive control to

produce 100 hemoglobin released by completely destroyed RBCs The mixture was

incubated in water bath with gentle shaking for 30 minutes at 37oC and then centrifuged

at 1500 rpm for 15 minutes 100microl of the supernatant was mixed with 2ml of the mixture

) (EtOHHCl = 2005 ww) to prevent the

White blood cells

Plasma

Red blood cells

of ethanol (99) and hydrochloric acid (37

54

precipitation of hemoglobin In order to remove remaining particles the mixture was

centri fuged again with 13000 RPM at room temperature The supernatant was then

transferred into the UV cuvette The amount of hemoglobin release was determined by

monitoring the UV absorbance at a wavelength of 397nm The schematic of the

procedure for hemolysis test is shown in Figure 3-6

Separate red blood cells (RBCs) from blood

Mix RBCs with phosphate buffer solution (PBS)

Wash and re-suspend RBC with PBS repeat three times

Add la l of RBC and incu in at 37oC

tex particle suspension into 100mbate sample with agitation for 30m

Separate particle ith EtOHHCl solutions and mix the supernatant w

Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm

Separate red blood cells (RBCs) from bloodSeparate red blood cells (RBCs) from blood

Mix RBCs with phosphate buffer solution (PBS)Mix RBCs with phosphate buffer solution (PBS)

Wash and re-suspend RBC with PBS repeat three timesWash and re-suspend RBC with PBS repeat three times

Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC

Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC

Separate particles and mix the ith EtOHHCl solution supernatant wSeparate particles and mix the ith EtOHHCl solution supernatant w

Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nmEvaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm

Figure 3-6 Schematic of the procedure for hemolysis test

In summary a description of the experimental materials and characterization

methodology has been explained In the next chapter the results and discussion of the

data obtained from these experiments is addressed

CHAPTER 4 RESULTS AND DISCUSSION

41 Polymerization of Latex Particles

411 Polystyrene Seed Latex Particles

Polymer particles have many different applications including spacer particles

lubricants packing materials for chromatography standard particles and diagnostic

drugs All applications strongly require these particles to have a uniform particle size

Uniform particle size is the first requirement for a latex composite membrane formed by

particle arrays with the interstitial spaces serving as pores for size discriminations [Jons

et al 1999 Ramakrishnan et al 2004] Although suspension polymerization has been

mainly used as one of conventional polymerization methods to prepare uniform particles

the uniformity of the recovered particles is insufficient for membrane use There is

another method known as seed emulsion polymerization where a vinyl monomer is

absorbed into fine monodiserse seed particles and polymerization causes the monomer to

crease the sizes of the seed particles uniformly In order to obtain monodisperse larger

particles by this m

particles and polymerization of the monomer is repeated

In this study cross-linked polystyrene seed latex particles were firstly synthesized

in the presence of styrene monomer divinylbenzene crosslink agent sodium persulfate

initiator sodium bicarbonate buffer and the Aerosol regMA80-I [sodium di(13-

dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion

polymerization process in which one or more components can be added continuously

in

ethod the procedure of absorption of monomer into fine polymer

55

56

Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and

buffer are continuously added to enlarge the seed latex particles Various profiles of

particle nucleation and growth can be gene different orders of component

addition during polymerizati f this process such as easy

control of the polymeriza particle number

and particle morphology The main goal in

this process is to avoid secondary particle

rated from

on There are several advantages o

tion rate monodisperse particle size and

colloidal stability copolymer composition

formation leading to a monodisperse particle

size distribution Figure 4-1 shows the schematic representation of seed latex particle

preparation and growth

Monomer droplet

Seed particles Amount of surfactantsis variable

Enlarged final Add monomers

PS

PS

PSs4

particles Number of particlesare constant

Seed code

PSs1

s2

s3

Monomer droplet

Seed particles Amount of surfactantsis variable

Enlarged final Add monomers

PS

PS

PSs4

tion The

particles Number of particlesare constant

Seed code

PSs1

s2

s3

Figure 4-1 Schematic representation of semi-continuous seed latex particles preparation and growth

After the first synthesis of a small size of crosslinked polystyrene (PS) seed

particles stabilized by surfactants styrene DVB and initiator are continuously fed into

the system to enlarge the seed latex particles maintaining a narrow size distribu

recipes and reaction conditions for synthesizing these seed particles are summarized in

57

Table 4-1 The original recipe for latex particle preparation from McDonald was

modified for this emulsion system [McDonald 2003]

Table 4-1 The polymerization recipe of polystyrene (PS) seed particles Latex label PSS259 PSS233 PSS207 PSS181

Temperature (oC) 75 plusmn 5 75 plusmn 5 75 plusmn 5 75 plusmn 5

Divinylbenzene (g) 052 052 052 052

MA 80 (g) 259 233 207 181

NaHCO

Watera (g) 1315 1315 1315 1315

Styrene (g) 9948 9948 9948 9948

112 112 112 112

Na2S2O8 (g) 190 190 190 190

Waterb (g) 200 200 200 200

Reaction time (min) 140 140 140 140

Solids content 396 396 396 396

Mean particle diameter (nm) 126plusmn75 171plusmn39 182plusmn41 216plusmn53

Conversion () 965 979 957 971

3 (g)

Four different seed latex particles were synthesized based on surfactant amount in

order to determine the optimum condition for stable and monodisperse latex particles

The emulsion polymerization of latex particles has to be carried out in a narrow range of

surfactant causes flocculation or phase separation leading to broad particle

size distribution and an unstable emulsion sy

surfactant concentration where particles are stable [Nestor et al 2005] A wide range of

concentrations

stem Therefore the range of surfactant

amount was varied from 181g to 259g in increments of 026g in each latex label Other

reactants amounts were kept constant Watera is the initial charge amount and waterb

represents the amount of aqueous solution containing dissolved initiator and buffer

58

which are fed continuously after the seed particle creation The extent of monome

polymer conversion was obtained by gravimetric calcu

r to

lation and was more than 95 at

cripts such as

S207 and S181 i the surf mount added for the seed latex

nversion indi perce experi obtain

content divided by the theoretically estimated solid content and was calculated by the

all seed particles labeled PSS259 PSS233 PSS207 and PSS181 Labeled subs

S259 S233 ndicate actant a

preparation The co cates the ntage of mentally ed solid

following formula

100 colids () times=entsoliatedntentoalExperimentConversion

n the experimenta content is the weight of olid rema after

mulsion at 120oC f in and the estimated solid content is th ht

lated from the recipe

rti as di each t load d

mple of ea pension iluted by (ww)

deionized water and the mean particle diameter was determined using the Brookhaven

ZetaPlus particle size analyzer From these results the seed latex particle size increased

as the amount of surfactant decreased The amount of emulsifier also affects the

polymerization process by changing the number of micelles and their size It is known

that large amount of emulsifier produces larger numbers of smaller sized particles [Odian

1991] and was corroborated in the current work

These synthesized seed latex particles should be of narrow size distribution in order

to obtain monodisperse larger particles The SEM image showing the particle

morphology and size of a sample of polystyrene seed particles can be seen in the Figure

4-2

contdEstim (4-1)

In this equatio l solid the s ining

evaporation of e or 30m e weig

of monomers calcu

As expected the seed pa cle size w fferent for surfactan ing in see

emulsion system A sa ch sus was d 0001 with

59

Figure 4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)

PSS259 (B) PSS233 (C) PSS207 (D) PSS181 (subscripts indicate the amount of surfactant added)

(A)

(B)

60

(C)

(D)

Figure 4-2 Continued

61

s seen in the image the seed particles are very smooth and spherical The

uniformity in the particle size distribution of these PS seed latex particles can also be

seen in the SEM image From this data we concluded that seed particles synthesized in

the presence of a surfactant with the chosen amounts are suitable for use to prepare larger

particles with high size uniformity and smooth spherical morphology

412 The Growth of Polystyrene (PS) Seed Latex Particles

Seeded emulsion polymerization has been conducted for several decades and

various mechanisms have been proposed Gracio et al [1970] suggested that the growing

polymer particles consist of an expanding polymer-rich core surrounded by a monomer-

rich shell with the outer shell providing a major locus of polymerization Seeded

mulsion polymerization is commonly used for preparing latex particles less than 1microm in

size [Cha et al 1995 Zou et al 1990 1992 Gandhi et al 1990 Park et al 1990] Such

latexes can be obtained from the growth of pre-prepared seed particles The seeds

introduced in this process serve as nucleation sites for particle growth By first swelling

the seed latex particles with additional monomer to the desired size polymerization can

then be initiated Several size ranges of monodisperse polystyrene (PS) latex particles

were prepared by this multistep seeded emulsion polymerization method For the

fabrication of particle-based membranes monodisperse latex particles with a variety of

size ranges are required

In this study seed particles from 171nm to 470nm in mean diameter and with a

highly uniform size distribution were prepared and used Watera is the initial charge

mount and waterb represents the amount aqueous solution containing dissolved initiator

and buffer that is continuously fed into system Feed time is precisely controlled by

metering pump The solid contents were maintained between 199 and 300 After

A

e

a

62

polym d as

es

ere 923 8402

n of these latex particles

are sh

ss

erization under these conditions the conversions of the latex particles labele

PS258 PS320 PS370 and PS410 were 952 957 946 and 964 respectively The recip

used in seeded emulsion polymerization to form the particles less than 500nm in mean

diameter are shown at the Table 4-2 The conversions of the latex particles larger than

500nm in mean diameter and labeled as PS525 PS585 PS640 and PS790 w

723 and 969 respectively The recipes used in the preparatio

own at the Table 4-3

A simple calculation can be used to predict the ultimate size that a latex particle can

reach after ending the polymerization Letrsquos suppose that seed density (ρs) and grown

particle density (ρgp) are the same The following equation is obtained from the ma

balance (m = ρV) where m and V are mass and volume of a particle respectively

gp

gp

s

s

Vm

Vm

In this equation m

= (4-2)

be

s and mgp are the weight of seed and total weight of seed and

added monomers respectively and Vs and Vgp are the volume of seed and grown

particle respectively

If we know the mass of monomer to be added the ultimate particle size can

calculated from the equation 4-3

3

2s D m ⎠⎝+ χ

where D

1s D m

⎟⎟⎞

⎜⎜⎛

= (4-3)

the

in

s and Dgp are the diameters for seed and grown particle respectively and χ is

weight of the monomer added Crosslinked PS latex particles with a size range from

258plusmn112 ~ 410plusmn117 in diameter polymerized by the recipes in Table 4-2 are shown

the SEM images in Figure 4-3

63

Table 4-2 Continuous addition emulsion polymerization recipe for growing polystyrene

Latex label PS

(PS) latex particles less than 500nm in size

258 PS320 PS370 PS410

Initial charge

Seed latex (g) 1000171 1000216 1000216 1000320

Watera (g) 1470 2500 1984 2600

06 07 06 06

Fee

Initiator stream

2 2 8

Feed time (min) 0-167 0-140 0-100 0-92

Solid content 284 300 300 200

Mean particle diameter (nm) 258plusmn112 320plusmn154 182plusmn71 410plusmn117

Conv

Monomer continuous

Styrene (g) 800 1200 800 600

Divinylbenzene (g)

d time (min) 0-163 0-110 0-75 0-82

Na S O (g) 07 065 05 05

NaHCO3 (g) 07 065 05 05

Waterb (g) 750 1200 200 370

ersion () 952 957 946 964 The solid content of seed latex for PS258 PS320 PS 370 and PS410 are 392 399 399 anrespectively

d 300

64

Table 4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size

Latex label PS525 PS585 PS640 PS790

Initial charge

Seed latex (g) 1000390 1000470 1000470 1000320

Watera (g) 1000 520 1200 360

Monomer continuous

Styrene (g) 290 200 400 173

Divinylbenzene (g) 02 014

03 009

Feed time (min) 0-80 0-40 10-76 5-65

Initiator stream

Na2S2O8 (g) 02 014 03 012

NaHCO3 (g) 02 014 03 009

Waterb (g) 200 120 400 200

Feed time (min) 0-85 0-46 0-77 0-75

Solids content 199 200 200 201

Mean particle diameter (nm) 525plusmn151 585plusmn186 640plusmn178 790plusmn693

Conversion () 923 8402 723 969 The solid content of seed latex for PS525 PS585 PS 640 and PS790 are 200 182 193 and 173 respectively

65

Figure 4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)

370nm (D) 410nm

(A)

(B)

66

(C)

(D)

Figure 4-3 Continued

67

article size was well matched to that estimated by the geometrical calculation

From this it can be concluded that the added monomer was consumed for polymer

particle growth with a high degree of conversion From the SEM characterization newly

nucleated particles were not seen as indicated by the narrow particle size distribution

The particles less than 500nm in mean diameter were very spherical in shape with a

highly uniform particle size distribution As the particles were grown larger than 500nm

mean diameter however they became of non-spherical shape with an uneven surface

This irregularity of particle surface can be attributed to the non-homogeneous monomer

swelling into the shell of a growing polymer which can be controlled by the factors such

as temperature agitation speed initiator feeding rate and surfactant amount required to

stabilize the colloidal system The SEM image of latex particles larger than 500nm mean

iameter is shown in Figure 4-4 The particle size distribution is broader than that of the

s particles These particles can however still be used for membrane construction

as a support layer rather than skin layer The support layer does not necessarily have to be

as highly monodisperse as the skin layer which require pores of high uniformity for the

selective removal of toxins

As mentioned earlier the amount of surfactant as well as monomer in an emulsion

system is the primary determinant of the particle diameter [Odian 1991] Anionic

surfactants are generally recommended at the level of 02-30 wt based on the amount

of water [Odian 1991] The critical micelle concentration (CMC) of Aerosolreg MA 80-I

anionic surfactant is 119 The surfactant loading in this emulsion system ranged from

the 121 for SS181 to 171 for SS259

P

d

maller

68

Figure 4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm

(C) 640nm (D) 790nm

(A)

(B)

69

(C)

(D)

Figure 4-4 Continued

70

The surfactant to monomer ratio refers to the value of the surfactant amount

divided by monomer amount after polymerization ends The surfactant amount is the

weight of the surfactant contained in emulsion and the monomer amount is the total

weight of the monomer added for polymerization to form the ultimate polymer particles

in emulsion As the surfactant to monomer ratio decreases mean particle diameter

increases as described by Odian [1991] Generally lower surfactant concentration forms

fewer micelles resulting in larger particle size [Evans et al 1999] However there is a

plateau in particle mean diameter between a surfactant to monomer ratio of 0005 and

0013 This is due to low polymerization conversion of latex particles from monomer

leading to a smaller particle size than expected This is not a factor of surfactant amount

The dependence of the particle size on the amount of surfactant is shown in Figure 4-5

0

200

400

600

800

1000

0000 0005 0010 0015 0020 0025 0030

Surfactant to monomer ratio (ww)

Part

icle

mea

n di

amet

er (n

m)

Figure 4-5 Dependence of the particle size on the surfactant to monomer ratio

71

413

e

f bare

microdomain structures is important for biocompatibility [Mori et al 1982 Higuchi et al

1993 Deppisch et al 1998] Figure 4-6 shows the schematic of core shell latex particle

structure The recipe for core shell structures is shown in Table 4-4

Core Shell Latex Particles

The latex particles shown in Figure 4-3 (A) and labeled as S258 in Table 4-2 wer

used as seeds to prepare core-shell latex particles because of their high particle

uniformity and smooth surface morphology Methyl methacrylate (MMA) and acrylic

acid (AA) monomers were introduced to increase surface hydrophilicity over that o

polystyrene particles AA is a more hydrophilic monomer than MMA because of the

carboxyl acid group in AA is more favorable for hydrogen boding with water The

surface carboxyl groups on PAA may have many promising applications in biomedical

and biochemical fields [Kang et al 2005] MMA monomer is less hydrophobic than

styrene PMMA is known to more biocompatible than PS but still PMMA is hydrophobic

The optimization of suitable hydrophobic to hydrophilic ratios and control of

Polystyreneseed particle

Core shelllatex particles

PSPMMA100

PSPMMA90PAA10

PSPMMA75PAA25

Polystyreneseed particle

Core shelllatex particles

PSPMMA100

PSPMMA90PAA10

Figure 4-6 Schematic of core shell latex particle structures

PSPMMA75PAA25

72

Table 4-4 The preparation recipe of PS core with various shell latex particles Latex 25 label PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA

Initial charge

PS seed latex (g) 500 500 500a

Monomer continuous

Methly methacylate (g) 320 288 240

Acrylic acid (g) 00 32 80

Initiator stream

Na

258 258 258

Water (g) 1570 1570 1570

Feed time (min) 0-120 0-50 0-60

b 200

Feed time (min) 0-122 0-60 0-65

952

2S2O8 (g) 02 02 02

NaHCO3 (g) 02 02 02

Water (g) 200 200

Solids content 183 183 183

Mean particle diameter (nm) 370plusmn186 370plusmn178 370plusmn191

Conversion () 955 924

Three types of core shell structures were prepared PMMA75PAA25 shell is a

copolymer consisting of the PMMA to PAA ratio of 75 to 25 by weight

PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90 to 10 by

weight PMMA100 is the PMMA homopolymer shell on PS core The particle size of

these core-shell particles as well as PS latex particle was prepared to be about 370nm in

mean diameter and are characterized to determine their zeta potential protein adsorption

and biocompatibility The image of SEM of PS and core shell particles is shown at Figure

4-7 The synthesized latex particles had a high uniformity in size and a smooth spherical

surface morphology

73

Figure 4-7 Scanning

Electron Micrograph of latex particles (A) PS (B) PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMMA75PAA25

(A)

(B)

74

(C)

(D)

Figure 4-7 Continued

75

42 Characterization of Latex Particles

421 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectroscopy has proven to be a powerful tool to characterize polymeric

materials A molecule can absorb only select frequencies (energy) of infrared radiation

which match the natural vibration frequencies The energy absorbed serves to increase

the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]

Only those bonds which have a dipole moment that changes as a function of time can

absorb infrared radiation The FTIR spectra of polymerized latex particles made during

this work are shown in Figure 4-8 There are number of characteristic peaks for PS latex

particles [Bhutto et al 2003 Li at al 2005] Aromatic C-H stretching vibration is shown

at 3002 cm-1 and 3103 cm-1 and aliphatic C-H asymmetrical and symmetrical stretching

shown at 2900 cm-1and 2850 cm-1 respectively There is an additional carbonyl (C=O)

adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some

amount of either or both PMMA and PAA The broad OH group peak at 3400 cm-1

appear for particles containing some amount of PAA and has intensity dependent on the

amount of PAA in the shell For example the peak intensity of OH groups for

PSPMMA75PAA25 is greater than that of PSPMMA90PAA10 since there is a higher AA

monomer content in PSPMMA75PAA25 than in PSPMMA90PAA10 This OH peak is not

seen for PS and PS core PMMA shell particles because of the absence of OH groups on

PS and PMMA polymer chains

422 Zeta Potential Measurements

The electrical surface property was characterized by zeta potential measurements of

tex particles Zeta potential is an important and useful indicator of surface charge which

can be used to predict and control the stability of colloidal systems The greater the zeta

is

la

76

1520002500300035004000

Wavenumbers (cm-1)

li

Re

ativ

e

Tra

nsm

ttanc

e

Figure 4-8 FTIR spectra of polymerized latex particles (A) ba

PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMM

(B)

(C) P

(D) P

C

C

OCH3

O

CH3

XCH2

CH2 n

CH

CH2 C

C

OCH3

O

XCH2 CH

OC

CH3

Y

OH

(A) Bare PS

500100000

re polystyrene (PS) (B) A75PAA25

PSPMMA100

SPMMA90PAA10

SPMMA75PAA25

77

potential the more likely the suspension is to be stable because the charged particles repel

one another and thus overcome the natural tendency to aggregate The measurement of

zeta potential is often the key ontrol of the interaction of proteins

with solid surfaces such as latex particles

Zeta potential (ζ) is the elec at exists at the shear plane of a particle

which is some small distance from the surface [Myers 1999] The zeta potential of the

particles can be determined by measuring the mobility of the particles in an applied

electric field termed electr its response to an alternating electric

field Colloidal particles dispersed in lu ically charged due to their ionic

characteristics and dipolar attributes The development of a net charge at the particle

surface affects the distribution of ions in the neighboring ng in

an increased concentration of counter ions to the surface Each particle dispersed in a

ns called fixed layer or Stern layer

Outside th compositions of ions of opposite polarities

form ble layer is form

particle-liquid interface This double layer may be considered to consist of two parts an

inner region which inclu nd relatively strongly to the surface and an outer or

diffuse region in which the ion distribution is determined by a balance of electrostatic

forces and random thermal motion The potential in this region therefore decays with the

distance from the surface until at a certain distance it becomes zero Figure 4-9 shows a

schematic representation of a typical ion distribution near a positively charged surface

The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the

to understanding and c

trical potential th

ophoretic mobility or

a so tion are electr

interfacial region resulti

solution is surrounded by oppositely charged io

e Stern layer there are varying

ed in the region of the ing a cloud-like area Thus an electrical dou

des ions bou

center of those ions which are effectively adsorbed to the surface [Hunter 1981] The

78

extent of ion adsorption is determined by electrical and other long-range interactions

between the individual ions and surface of particles The ions outside of the Stern layer

form the diffuse double layer also referred to as the Gouy-Chapman layer [Burns and

Zydney 2000]

Shear plane

Stern layer

ψζ

0

ψs

ψShear plane

Stern layer

ψζ

0

ψs

ψ

t

controlled electric field is applied via electrodes immersed in a sample suspension and

Figure 4-9 Schematic representation of ion distribution near a positively charged surface

Zeta potential is a function of the surface charge of a particle any adsorbed layer a

the interface and the nature and composition of the surrounding medium in which the

particle is suspended The principle of determining zeta potential is very simple A

79

this causes the charged particles to move towards the electrode of opposite polarity

Viscous forces acting upon the moving particle tend to oppose this motion and the

equilibrium is rapidly established between the effects of the electrostatic attraction and

the viscosity dra

g The particle therefore reaches a constant terminal velocity

Figure 4-10 Schematic representation of zeta potential measurement(source

httpnitioncomenproductszeecom_shtm)

Because protein adsorption mechanisms are very complex and hard to explain in

biological systems protein adsorption is generally studied by using more ideal systems

consisting of one or more well characterized proteins a well-characterized adsorbent and

a well-defined aqueous solution Even so small changes in the experimental conditions

such as pH ionic strength and temperature generate totally different results Therefore

two media systems phosphate buffer (PB) and phosphate buffered saline (PBS) were

chosen to be used for the zeta potential measurements and protein adsorption study PB is

a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4H2O) to

n a constant pH PBS is used to keep the water concentration inside and outside of

the cell balanced If the water concentration is unbalanced the cell risks bursting or

shriveling up because of a phenomenon called osmosis PBS is a solution with the 5 mM

of monobasic sodium phosphate (NaH2PO4H2O) and 143 mM of sodium chloride (NaCl)

in water

maintai

80

The results of zeta potential measurement at room temperature (25plusmn1oC) as

function of pH and ionic strength (different media PB and PBS) for synthesized lat

particles are seen in Figure 4-11 Zeta potential values of polystyrene (PS) latex particle

at the Figure 4-9 are negative between -291 mV and -599 mV in PB and betwe

ex

s

en -203

mV and -278 mV in PBS at all pH ranges These negative zeta potential values of PS

latex particles are due to the sulfate groups [Vandenhu et al 1970] originated from

persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer

chain where polymerization was initiated

-100

-80

-60

-40

-20

0

pH

Zta

pte

ntl

ζ

20

40

1 2 3 4 5 6 7 8 9

eo

ia (m

V)

PB (5mM)

PBS (5mM +143mM)

en -

Figure 4-11 Zeta potential of PS latex particles at 25oC

The zeta potential measurements of PSPMMA100 core shell latex particles as seen

in Figure 4-12 were also negative with a range of -28 and -505 mV in PB and betwe

143 mV and -186 mV in PBS at the pH 21-78 These negative values are also due to

sulfate groups on the particle surface

81

-100

-80

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

Zea

pote

nti

l ζ

(mV

pH

ta

)

PB (5mM)

PBS (5mM + 143mM)

Figure 4-12 Zeta potential of PSPMMA100 latex particles at 25oC

The zeta potential for PSPMMA90PAA10 and PSPMMA75PAA25 was also

negative as seen in Figure 4-13 and 4-14 The zeta potential value for the

PSPMMA90PAA10 were between -367 mV and -678 mV in PB medium and between -

147 mV and -193 mV in PBS both at 25oC In case of the PSPMMA75PAA25 core shell

particles zeta potential values were between -291 mV and -520 mV in PB and between

-115 mV and -210 mV in PBS media Sulfate groups in PSPMMA75PAA25 and

PSPMMA90PAA10 core shell particles contribute to the negative zeta potential values

participate in forming a

negat

cid is expressed by the term pKa which is the pH

at which an acid is 50 dissociated However the contribution of the carboxylic acid

Carboxylic groups from acrylic acid monomer also would

ive surface charge of the latex particles at a pH greater than 70 because the pKa

value for the carboxylic acid is between 40 and 60 pH range [Ottewill et al 1967] This

indicates that carboxylic acid dissociation begins at a pH between pH 46-60 and

increases with pH The strength of an a

82

group to negative zeta potential values of PSPMMA-PAA core shell particles was not

detectable because there was no significant difference in zeta potential values for

synthesized latex particles at a pH greater than 70

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

Zeta

pot

entia

l ζ

(mV

)

PB (5mM)PBS (5mM + 143mM)

-80

-100

pH

Figure 4-13 Zeta potential of PSPMMA90PAA10 latex particles at 25oC

-100

-80

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

pH

Ze p

ont

i ζ

V)

tate

al (m

PB (5mM)PBS (5mM + 143mM)

Figure 4-14 Zeta potential of PSPMMA75PAA25 latex particles at 25oC

83

The zeta potential plots and values were similar for all latex particles This may be

due to the similar initiator density on the latex surface The initiator concentrations

monomers for shell polymerization were the same fo

to

r all core shell latex particles (062

ww from Table 4-4) A similar concentration (055 ww) of initiator to monomer was

added to prepare bare PS latex particles used in zeta potential measurements

Since the zeta potential values of all synthesized latex particles were negative

between pH=20 and pH=78 the isoelecric point (IEP) of these latex particles would be

less than pH=20 Sulfates have a pKa in the range of 10 to 20 [James 1985] indicating

that the sulfate group the conjugate base of a strong acid is protonated (-C-OSO3H) at

less than pH 20 This means that the synthesized latex particles are all negative at the

physiological pH Most serum proteins have a negative surface charge in blood

therefore when the negative latex particles are applied to blood stream fatal

tein is

avoided by charge repulsion in the blood Table 4-5 shows the common blood proteins

and their isoelectric points (IEP)

Table 4-5 The isoelectric point (IEP) of common proteins Serum proteins Isoelectric point (IEP)

complication by coagulation between applied latex particles and serum pro s

Albumin 48 α1-Globulin 20 α2-Microglobulin 54 γ1-Globulin 58 Heptoglobin 41 Hemoglobin 72 β1-lipoprotein 54 Immunoglobulin G (Ig G) 73 Fibrinogen 58 (source httpwwwfleshandbonescomreadingroompdf1085pdf

httpwwwmedalorgvisitorwww5CActive5Cch135Cch13015Cch130105aspx )

84

The absolute zeta potential values of the latex particles in phosphate buffer (PB)

was higher than that in phosphate buffered saline (PBS) This is because high

concentration of sodium and chloride electrolytes in PBS compressed the electrical

double layer causing rapid decay of surface potential Figure 4-15 shows that the

thickness of the electrical double layer is inversely proportional to the concentration of

electrolyte in the system

Surf

ace

Pote

ntia

l ψ

1κ3

10

1e

1κ 1κ

(1)(3) (2)

2 1 Z

Surf

ace

Pote

ntia

l ψ

1κ3

10

1e

1κ 1κ

(1)(3) (2)

Figure 4-15 The decay of surface potential with distance from surface in various

electrolyte concentrations (1) low (2) intermediate (3) high [Myers 1999]

2 1 Z

The electrical potential in the solution surrounding the particle surface falls off

the Debye-Huckel

yers 1999]

exponentially with distance (Z) from the surface according to

approximation [M

z) (- exp 0 κψψ = (4-4)

e reciprocal of the thickness of the electrical double layer referred to as the

The potential fallen off by a factor e The theoretical equation for the

ickness 1κ is

where κ is th

Debye length of 1

double layer th

21

22i

0

ekT 1

⎟⎟⎠

⎜⎜⎝

=sum ii zcεε

κ

⎞⎛ (4-5)

85

where

tration of

y was performed to measure the protein adsorption on the

synthesized latex particles Many physical and chemical processes occur at the boundary

between two phases and adsorption is one of the fundamental surface phenomena [Oscik

1982] This phenomenon is driven by the change in the concentration of the components

at the interface with respect to their concentrations in the bulk phase There are two

adsorption processes physical adsorption occurring when non-balanced physical forces

appear at the boundary of the phases and chemical adsorption or chemisorption

t the

interface [J

n leads

ational entropy hence adsorption takes place only if the

loss in conformational entropy is compensated

ter adsorption Cp The initial part of the

isotherm merges with the Γ-axis because at low polymer concentration the protein has a

high affinity for the surface and all of the polymer is adsorbed until the solid surface is

saturated

ε0 is the permittivity of a vacuum of free space ε is the permittivity of a medium e

is the relative permittivity or dielectric constant of the medium c is the concen

an ion (i) z is the valency of an ion (i) k is Boltzmannrsquos constant T is absolute

temperature (K)

43 Protein Adsorption Study

The adsorption stud

occurring when atoms and molecules from adjacent phases form chemical bonds a

aroniec et al 1988]

Flexible proteins in solution possess high conformational entropy as a result of the

various states each of the many segments in the protein chain can have Adsorptio

to a reduction of this conform

by sufficient attraction between polymer

segment and solid surface [Martin 1998] Isotherm shape of high affinity adsorption

between polymer and solid is shown in Figure 4-16 where the adsorbed mass Γ is plotted

against the polymer concentration in solution af

86

Figur

de

arboxyl

e 4-16 High-affinity adsorption isotherm of typical flexible polymer on solid

surface

In order to investigate the adsorption of blood proteins latex particle properties

such as electrical surface charge hydrophobicity and environmental conditions like pH

ionic strength and temperature are considered as experimental factors [Arai and Nor

1990] Figure 4-17 shows the overall schematic representation of the protein adsorption

on the synthesized latex particles with the functional groups such as sulfate and c

groups

CP

Γ

PMMAPAAShell

PS or PMMAShell OSO3

-

OSO3-

OSO3-

OSO3-

PS

-O3SO

PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

Protein

NH3+

+

+H3N

+

COO-

Hydrophobic interaction

NH3

COO-

H3N

COO-

-OOC

Ionic interaction

Hydrophobic interaction

Hydrogen bonding

Ionic interaction

PMMAPAAShellOSO3

-

OSO3-

OSO3-

OSO3-

PS or PMMAShell -O3SOOSO3

-

PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

-O3SO

OSO3-

OSO3-

OSO3-

PS PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

Protein

NH3+

+

+H3N

+

COO-NH3

COO-

H3N

COO-

-OOC

NH3+

+

+H3N

+

COO-

Hydrophobic interaction

NH3

COO-

H3N

COO-

-OOC

Ionic interaction

Hydrophobic interaction

Hydrogen bonding

Figure 4-17 Overall schematic representation of the protein adsorption on the

Ionic interaction

synthesized latex particles

The surface functional groups as well as suitable surface hydrophobicity

hydrophilicity of latex particles are closely related to protein adsorption property As

indicated at Figure 4-17 various interaction forces such as hydrophobic interaction ionic

87

interaction hydrogen interaction and van der Waals interaction [Andrade 1985] exist

between protein molecule and the solid surface of latex particle

431 Adsorption Isotherm

The protein adsorption isotherm is generally defined as the relationship between the

amount of protein adsorbed by a solid (latex particle) at a constant temperature and as a

ine

the amount of protein adsorbed onto latex particles To evaluate the equilibrium

conce s

x

function of the concentration of protein Adsorption isotherms were plotted to determ

ntration of target proteins a calibration curve is firstly needed Figure 4-18 show

the UV absorbance as a factor of bovine serum albumin (BSA) concentrations The UV

absorbance of the known protein concentration was measured at 562nm which is the λma

for a protein treated by a bicinchoninic acid (BCA) assay

0

02

04

06

08

500 520 540 560 580 600

Wavelength (nm)

Abs

orba

nce

Figur gml (B)

06mgml (C) 02mgml

(A)

(C)

(B)

Max562nm

e 4-18 The dependence of UV absorbance on BSA concentration (A) 10m

Figure 4-19 shows the standard calibration curve used in adsorption isotherm

experiment of protein onto latex particles The net absorbance of each known protein

88

concentration was plotted and then a linear equation passing through a concentration of

zero protein concentration was obtained This equation is applied

to determine the

orbed on the synthesized latex particles The amount of free

protei

eported

trations (005 01 03 05

and 07mgml) were used to generate this curve as well as the adsorption isotherm

unknown protein amount ads

n was also determined using UV light at the 280nm wavelength however a

distinguishable UV intensity was too low to determine a precise value of protein

adsorption

The bicinchninic acid (BCA) assay technique [Smith et al 1985] has been r

for high efficiency of protein evaluation and was used for determination of protein

adsorption [Tangpasuthadol et al 2003 Williams et al 2003] This technique has many

advantages such as high sensitivity easy of use color complex stability and less

susceptibility to any detergents Five different BSA concen

y = 00R2 =

004741x 09939

00

01

02

03

04

0 200 400 600 800

BSA Standard (microgml)

Net

Abs

orba

nce

at 5

62nm

Figure 4-19 Standard curve of net absorbance vs BSA sample concentration

89

All isotherms were fitted to the Langmuir-Freundlich isotherms using the nonlinea

regression method Two adsorption isotherm models the Langmuir type isotherm a

Langmuir-Freundlich combination type isotherm were compared As seen the Figure 4-

20 The Langmuir isotherm model deviated further from the experimental data Th

all isotherms were fitted to the Langmuir-Freundlich isotherms [Yoon et al 1996

al 1998] usi

r

nd the

erefore

Lee et

ng the nonlinear regression method with following equation

n

n

eq

eqm

kC

kC1

1

1+

where q is the amount of adsorbed BSA per unit surface area q

qq = (4-6)

stant

were between 045 and 055 and hence n was fixed to its average 05 Actual qm values

were evaluated after fixing the n value

m is the adsorbed amount

in equilibrium Ceq is the equilibrium concentration of BSA k is the adsorption con

and n is the exponential factor for a heterogeneous system n values are empirical and

usually from zero to no more than unity [Jaekel 2002] The values of n in our system

00

10

20

30

0 02 04 06 08

C (mgml)eq

q (m

gm

2 )

pH 32Langmuir-Freundlich modelLangmuir model

Figure 4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on

polystyrene latex particles at 37oC in phosphate buffer media

90

The BSA protein adsorption on the synthesized latex particles was performed at

three pH levels 32 48 74 in both phosphate buffer (PB) and phosphate buffered saline

(PBS) at physiological temperature 37oC These pH levels correspond to an acidic pH

lower than the isoelectric point (IEP) of BSA the pH at the IEP and an alkaline pH

higher than the IEP of BSA respectively The IEP corresponds to the pH at which the

amino acid becomes a zwitterion Here its overall electrostatic charge is neutral and the

charge expressed by the basic amino group (-NH2 -NH3+) of the amino acid is equal to

the charge expressed by the acidic carboxyl group (-COOH -COO ) The IEP of BSA is

-

pH 48 and indicates that the side chain of the amino acids of BSA contains a greater

amount of acidic functional groups than that of basic functional groups At pH 32 and

74 BSA has a positive and negative charge respectively

Figure 4-21 and 4-22 show adsorption isotherms of BSA onto polystyrene (PS)

latex particles

00

10

20

30

40

0 02 04 06 08

Ceq (mgml)

q (m

gm

2 )

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in

phosphate buffer (PB) at 37oC

91

00

10

30

40

0 02 04 06 08

Ceq (mgml)

q (m

gm

2

20

)

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

(PBS) on PS latex particles

At the IEP of BSA the adsorbed amount (q

Figure 4-22 Adsorption isotherm of BSA adsorption 37oC in phosphate buffered saline

onto PS at equilibrium was

maxim

um at the IEP of BSA This is because the BSA molecules form the most

compact structures at the IEP eliminating the repulsion force between proteins A higher

number of BSA molecules can adsorb on a given surface area by hydrophobic interaction

when in this compact structure This observation agrees well with reported data by other

researchers who also indicated that the maximum adsorption from aqueous protein

solution was observed at the IEP [Mularen 1954 Watkins et al 1977 Oh at el 2000]

There was no significant difference of adsorbed BSA amount in PB and in PBS media at

pH 48 The conformational alteration of BSA was not affected significantly by the ionic

strength of the two solutions There was evidence of the effect of the electrostatic

interaction to some extent even at the IEP since the amount of BSA adsorbed in PBS is

slightly hig

m) of BSA

um indicating that hydrophobic interaction between latex particle and BSA

protein is maxim

her than in PB at this pH

92

The adsorbed amount of protein is shown to decrease at a pH 32 and 74 The

decrease in adsorption efficiency when deviating from IEP of BSA is a result of the

increase in conformational size of the protein molecules and the lateral electrostatic

repulsions between adjacent adsorbed BSA molecules Figure 4-23 shows a schematic of

possible conformational changes at each pH where hydrophobic interaction always exists

between non-polar portion of a protein and hydrophobic portion of solid surface

- - --- ---Latex surface

Zeta potential

BSA(+)

(-)

48 7432pH

(0)

+++ + ++

--+

++-

- - --- ---Latex surface

Zeta poten pH(0)

+++ + ++

--

tial

BSA(+)

(-)

48 7432

+

++-

ming

4

s

al blood Table 4-6 shows the calculated results of

Figure 4-23 Conformation of bovine serum albumin (BSA) on latex particles (Assuthat charge repulsion reduces the effect of the hydrophobic interaction at pH 74)

In addition the adsorbed amount of BSA at pH 32 was larger than that at pH 74

and still less than that at 48 This is because there is the electrostatic interaction between

the positive BSA molecules and negative PS latex particles at pH 32 and electrostatic

repulsion is dominant between negative BSA and negative PS latex particles at pH 7

The amount adsorbed increased with increasing ionic strength in pH 32 and 74 This i

because the high concentration of electrolytes reduces the electrostatic repulsion between

BSA and PS latex particle It should be noted that the BSA concentration used in these

experiments was lower than that found in normal human blood This is because the

equilibrium BSA adsorption onto the latex particles is actually reached at a much lower

concentration than that found in norm

93

adsor rm

ein

leading to a compact conformation of BSA There are two main forces affecting

adsorption amount at an acidic pH of 32 They include the ionic attraction between the

negative latex particle and the positive protein and lateral repulsion between positive

proteins The smallest amount of BSA adsorption was observed at a pH of 74 At this

point the charge repulsion between negatively charged particles and protein molecules

exists the h

of cha

bed BSA amount at equilibrium from the Langmuir-Freundich adsorption isothe

equation

In summary the hydrophobic interaction between PS latex particle and BSA

protein exists and is the dominant mechanism for the adsorption process Conformational

changes of BSA at each pH also affected the adsorption amount Adsorption was highest

at pH 48 because at this pH there is the least amount of ionic repulsion in the prot

and the lateral repulsion between proteins dominant adsorption even though there always

ydrophobic interaction between the solid latex particle and protein The effect

rge repulsion was reduced by the high concentration of electrolytes in PBS medium

Table 4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2) 32 174 48 299 Phosphate buffer 74 136 32 260 48 283 Phosphate buffered saline 74 207

The adsorption isotherms of BSA on PSPMMA core shell particles are shown in

Figure 4-24 in phosphate buffer (PB) and in Figure 4-25 in phosphate buffered saline

(PBS) The adsorbed BSA amount was calculated using the Langmuir-Freundlich

100

94

adsorption isotherm equation and the amount listed in Table 4-7 The trend seen in the

adsorption results was very similar to the case of PS latex The overall adsorption process

was d rticle

rs

hat

0

ominated by hydrophobic interactions between the PSPMMA100 core shell pa

and BSA proteins At pH 48 the adsorbed amount (qm) of BSA on PSPMMA100 core

shell particles was 221 mgm2 in PB and 237 mgm2 in PBS and are the maximum

values recorded among the three pH levels 32 48 and 74 Suzawa and his co-worke

[1982] have obtained similar results This can be also explained by the compact

conformation of BSA molecules at the IEP of BSA which lead to a high protein

population on the solid surface At a pH of 32 and 74 BSA adsorption was less than t

at pH 48 because of the protein conformation change There was a small increase in

adsorbed BSA at pH 32 because of the charge attraction between negative PSPMMA10

core shell particles and positive BSA At pH 74 however the adsorption of BSA was

further reduced by charge repulsion between the negative PSPMMA100 core shell

particles and the negative BSA proteins

000 02 04 06 08

Ceq (mgml)

10

20q (m

gm

302 )

40

50pH=32pH=48pH=74Mode 8)l (pH 4Mode 2)l (pH 3Mode 4)

Figure 4-24 Adsorption isotherm of BSA 37oC in PB on PSPMMA core shell latex

l (pH 7

100

particles

95

The adsorbed amount of BSA increased more rapidly in PBS with increasing ioni

strength at pH 74 This shows that the low concentration of electrolytes promotes

electrostatic repulsion between negative BSA and negative PSPMMA

c

on

x

l 1980

100 core shell

particle As the ion strength of PBS is increased however the high concentration of

electrolyte leads to less electrostatic repulsion promoting more rapid protein adsorpti

By comparing the amount of adsorbed protein between bare PS latex particles and

PSPMMA100 core shell particles the amount of protein adsorption on the PS late

particles is greater than that on the PSPMMA100 core-shell particles [Suzawa et a

1982 Lee et al 1988]

00

10

20

30

40

m2 )

0 02 04 06 08

Ceq (mgml)

q (m

g

pH=32pH=48pH=74Model (pH 2) 3Model (pH 48)Model (pH 74)

Figure 4-25 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA100 core shell

latex particles

96

Table particles calculated the Langmuir-Freundlich isotherm model

4-7 The equilibrium concentration values (qm) of BSA adsorption on PSPMMA100

Media pH qm (mgm ) 2

32 140

48 221 Phosphate buffer

74 130

32 169

48 237 Phosphate buffered saline

74 162

The BSA adsorption mechanism and trend on PSPMMA core shell particles was

very similar to that of the PS latex particle Unlike the case of PS and PSPMMA100 latex

particles the amount of BSA adsorbed onto PSPMMA90PAA10 and PSPMMA75PAA25

core shell particles was higher at pH 32 than pH 48 and 74 Figure 4-26 through 4-29

shows the adsorption isotherms of BSA on PSPMMA90PAA10 and PSPMMA75PAA25

core shell particles The amount of BSA adsorbed increased at pH 32 and pH 48 as the

hydrophilicity was developed by the addition of PAA On the contrary the adsorbed

amount of BSA was dramatically decreased at pH 74 The adsorption amount of BSA on

PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles at equilibrium is listed in

Table 4-8 and 4-9 respectively

Carboxylic acid as well as sulfate groups are distributed on the surface of

PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles and seem to largely affect

the BSA adsorption The isotherms showed very strong pH dependence to the BSA

adsorption onto these core shell latex particles The carboxylic groups (COOH) may

especially enhance the BSA adsorption prominently at pH 32 by hydrogen bonding with

protein molecules The pKa value for the PAA is 40-60 and a good indication that the

97

carboxylic acid groups are protonated at pH 32[Gebhardt et al 1983] The main

adsorption interaction force at pH 32 was generated by hydrogen bonding as well as

charge attraction between latex particle protein even th the conformational

repulsion between proteins results in a reduction of the protein

adsorption at this pH The amount of BSA adsorbed also increased as the PAA amount in

eased The a tion of BSA w inimal however less

than 07 mgm2 at pH 74 Here the carboxylic groups are ionized which increases the

charge repulsion by ionized carboxylic groups (COO-) resulting in PSPMMA-PAA latex

particle

and ough

change and charge

the core shell particles incr dsorp as m

s with much lower protein adsorption

00

20

40

q (m

g2 )

60

m

pH=32pH=48pH=74Model (pH 32)Model (pH 48)

0 02 04 06 08

Ceq (mgml)

Model (pH 74)

o shell

Figure 4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA90PAA10 corelatex particles

98

00

40

60

80

0 02 04

C (mgm

mm

2 )

20

06 08

eq l)

q (

g

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-27 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA90PAA10 core

shell latex particles

Table 4-8 The equilibrium concentration values of BSA adsorption on PSPMMA90PAA10 particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2)

32 361

48 283 Phosphate buffer

74 043

32 519

48 226 Phosphate buffered saline

74 043

99

00

20

40

60

80

100

0 02 04 06 08Ceq (mgml)

q (m

gm

2 )

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

oFigure 4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA75PAA25 latex particles

00

100

20

40

60

80

0 02 04 06 08Ceq (mgml)

q (

m2 )

pH=32pH=48pH=74

mg

Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-29 Adsorption isotherm of BSA 37oC in PBS on PSPMMA75PAA25 core shell

latex particles

100

Table 4-9 The equilibrium concentration values of BSA adsorption on PSPMMA75PAA25 particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2)

32 1083

48 662 Phosphate buffer (PB)

74 075

32 1077

48 681 Phosphate buffered saline (PBS)

74 057

The adsorption of β2-microglobulin (β2M) was determined under one set of

onditions in phosphate buffer at 37oC and pH 74 because of the limited amount of β2M

protein The adsorption isotherms of the β2M protein on the synthesized latex particles

are shown in Figure 4-30 4-31 and 4-32 The β2M protein concentrations for adsorption

isotherm experiment were chosen to be 0015 0030 0045 and 0060mgml since the

β2M concentration for the patients with renal failure can be elevated to similar

concentrations about 005mgml or more up to 50 times the normal level [Nissenson et

al 1995] There were steep initial slopes of the adsorption isotherm curves of β2M on

latex particles in all isotherms indicating a high affinity adsorption type It seems that the

hydrophobic interaction is enough for the adsorption process of β2M proteins to occur on

the latex particles even though charge repulsion exists between protein and latex particle

at pH 74 The complete plateau regions were not seen for all isotherms indicating that

β2M proteins were not saturated at this level and there are still available sites for β2M

c

adsorption on latex particles The initial slope of the β2M isotherms for PS was steeper

101

than that of any of the other latex particle PSPMMA100 PSPMMA90PAA10 and

PSPMMA 75PAA25

00

02

0 0002 0004 0006 0008

04

06

10

Ceq (mgml)

q (m

2 )

08

gm

PSPSPMMA100Model (PS)Model (PSPMMA100)

Figure 4-30 Adsorption isotherm of β2M onto PS and PSPMMA100 latex particles in PB

at 37 C and pH 74 o

00

02

04

06

08

10

0 0002 0004 0006 0008C (mgml)

q (m

m2 )

eq

g

PS

PSPMMA90PAA10

Model (PS)

Model (PSPMMA90PAA10)

Figure 4-31 Adsorption isotherm of β2M onto PS and PSPMMA90PAA10 latex particles

in PB at 37oC and pH 74

102

00

02

04

06

08

10

0 0002 0004 0006 0008Ceq (mgml)

q (m

gm

2 )

PS

PSPMMA75PAA25

Model (PS)Model (PSPMMA75PAA25)

Figure 4-32 Adsorption isotherm of β2M onto PS and PSPMMA75PAA25 latex particles

in PB at 37oC and pH 74

The equilibrium concentration values (qm) of β2M adsorption on latex particles are

calculated and listed in Table 4-10 The difference between the maximum amounts of

β2M adsorption on all the latex particles was not significantly different and their values

were between 069 and 08mgm2

Table 4-10 The equilibrium concentration values of β2M adsorption calculated the Langmuir-Freundlich isotherm model

Latex particles qm (mgm2)

PS 080

PSPMMA100 069

PSPMMA90PAA10 072

PSPMMA75PAA25 072

103

432 Adsorbed Layer Thickness

There are several possible explanations for large amount of BSA adsorbed at acidic

pH levels including the end-on adsorption pattern of BSA the flexibility of BSA

molecules the tilting of protein molecules due to the asymmetry of the charge

distribution or multilayer formation [Peula et al 1993] The adsorbed BSA thickness (δ)

can be calculated by the following equation [Chiu et al 1978] This equation was also

cited again by other researchers [Shirahama et al 1985]

BSA

mqρπ

δbullbull

=33

(4-7)

where π

33 is the packing factor qm is the plateau value of the adsorbed am

ρBSA is the hich corresponds to the reciprocal of its known

partial specific volume The layer thickness (δ) of BSA proteins on synthesized latex

partic

he IEP [Shirahama et al 1985] Compared to the

hydrodynamic dimensions of BSA 140 times 40 times 40 Aring3 [Fair et al 1980 Peter 1985] these

nd

ode It is unnecessary ultilayering because thickness do

exceed 140 Aring The layer thickness (δ) of BSA proteins on synthesized latex particles

e red saline (PBS) was ran 28 ~ 83 Aring at 37oC and pH 48 These

lso listed in Table 4-1

ount and

density of BSA molecule w

les in phosphate buffer (PB) ranged from 27 - 81 Aring at 37oC and pH 48 These

calculated values are listed in Table 4-11 Only δ values at pH 48 the isoelectric point

(IEP) of BSA are important because intra- and intermolecular electrostatic repulsion of

protein molecules is minimized at t

values of layer thickness indicate that the BSA molecules adsorb by side-on (40Aring) a

end-on (140Aring) m to consider m

not

in phosphat buffe ged of

calculated values are a 2

104

Table 4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37oC pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

32 21 17 44 133

48 37 27 35 81

74 17 16 5 9

Table 4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37oC

pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

32 32 21 64 132

48 35 29

74 26 20 5 7

28 83

1n

1n

neqm

So k is closely related to the initial slope of the isotherm showing the affinity

between BSA and latex particles [Yoon et al 1996] A key point in the characterization

433 Gibbs Free Energy of Protein Adsorption

In the Langmuir isotherm model k represents the ratio of adsorption to the

desorption rate constants This definition is not applicable to the Langmuir-Freundlich

isotherm model but its meaning is similar [Yoon et al 1996] In this sense k is closely

related to the affinity between protein molecules and latex particles When the

equilibrium concentration Ceq reaches zero the value of Ceq will be very small (1gtgt

kCeq ) and then the equation 4-6 becomes

(4-8)

of adsorption processes on solid-liquid interfaces is to determine the change in Gibbsrsquos

free energy (∆Gdeg) one of the corresponding parameters for the thermodynamic driving

forces during adsorption The traditional method to evaluate adsorption thermodynamic

kCqq 1=

105

constants is based on measurement of adsorption isotherms under static conditions

[Malm ten 1998] The Langmuir-Freundlich ads lf

easily found from Then this enables calculation of the free-

energy change ∆G during adsorption expressed as

(4-9)

where R is the general gas constant (R = 8314Jmiddotmol K ) and T is the absolute

tem ture Gibb e energy A P

listed in Table 4-13 and 4-14 respectively As the ∆Gdegads ore negativ e

pro adsorption on a solid substra ill be more favorable

e of BSA adsorption in phosphate o

pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

s orption equilibrium constant k can be

a slope of the isotherm

degads

lfads kRTG lnminus=∆ o

-1 -1

pera s fre change of BS adsorption values in B and PBS are

values are m e th

tein te w

Table 4-13 The values of Gibbs free energy changbuffer (PB) at 37 C

32 -2411 -2726 -2846 -2805

48 -2352 -2383 -2198 -1972 ∆Go

80

ads (kJmol)

74 -2452 -888 -324 -2

Table 4-14 The values of Gibbs free energy change of BSA adsorption in phosphate

pH PS PSPMMAbuffered saline (PBS) at 37oC

100 PSPMMA90PAA10 PSPMMA75PAA25

32 -2315 -2620 -2312 -2683

48 -2149 -2299 -2090 -2138 ∆Go

(kJmads ol)

74 -2430 -1992 -258 -263

Table 4-15 shows the calculated values of Gibbs free energy change of β M

adsorption on synthesized latex particles PSPMMA PAA core shell latex particle has

2

75 25

106

largest negative Gibbs free energy of β2

2

M adsorption indicating largest affinity betw

β

een

2M adsorption in phosphate buffere (PB) at 37 C and pH74

PS PSPMMA100

M and latex particle

Table 4-15 The values of Gibbs free energy change of βo

PSPMMA90PAA10 PSPMMA75PAA25

∆Go03 ads

(kJmol) -3626 -3466 -3659 -39

The Gibbs free energy of β2M was much more negative in phosphate buffer (PB)

than that of BSA in physiological condition (37 C and pH 74) o from the Table 4-13 and

the Figure 4-31 This indicates that β2M molecules more readily adsorb on latex particles

BSA This can be explained by the rate of diffusion and accessibility of β2M molecules to

the latex particle ier and faster for β2M m an BSA because of

size (molec ight) erical shape molecules It o be

due to the higher hydrophobic force and less electrostatic repulsion that contribute to the

adsorption of β2M molecules not as prevalent in the case of BSA because the IEP of β2M

(57) is clo H 74) than that of BSA (48) causing β2M to

be less electro cally lled p ro

can be seen that the ∆ β2M adsorption onto the synth article

increasing wi hy icity o x particles s the ∆Goads of the BSA

molecules is decreasing or constant This phenomenon supports the theory that the

select lizing

than BSA and that the affinity of β2M for the latex particle is much larger than that of

surface It is eas olecules th

the smaller ular we and sph of β2M may als

ser to physiological condition (p

stati repe from the latex articles than BSA F m Figure 4-33 it

Goads of esized latex p s is

th the drophil f the late wherea

ive adsorption of β2M molecules over BSA molecules can be enhanced by uti

more hydrophilic PSPMMA90PAA10 and PSPMMA75PAA25 core shell latex particles

107

-60

-50

1 2 3 4

-40∆Gad

s

-30

10

0

o (k

Jm

o-

-20l)

β2-Microglobulin (β2M)Bovine serum albumin (BSA)

Figure 4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37

and pH 74

434 Kinetics of Adsorption

to

oC

Protein adsorption is a complex process known to have a large biological impact

and is currently not well understood quantitatively because of extreme sensitivity to pH

the concentration of other electrolytes and molecules present temperature and many

other factors that can change in the physiological system Accurate knowledge of the

adsorption kinetics under a given set of conditions is a prerequisite for elucidating the

mechanisms of many fundamental biological processes to be predicted at the molecular

level [Regner 1987] This adsorption kinetics approach is used in protein adsorption

studies because of the uncertainty related to the time needed for the interfacial proteins

reach the equilibrium It is generally accepted that the process of protein adsorption is

comprised of the following steps a) transport toward the interface b) attachment at the

interface c) eventual structural rearrangements in the adsorbed state d) detachment from

PS PSPMMA100 PSPMMA90PAA10PSPMMA75PAA25

108

the interface and transport away from the interface Each of these steps can affect the

overall rate of the adsorption process

The quantitative analysis of the protein adsorption kinetics requires that the protein

amount adsorbed is known as a function of time The kinetics tests were carried out in

phosphate buffer (PB) at 37oC and pH 74 a condition similar to physiological condition

The protein concentrations chosen were 07mgml for BSA and 006mgml for β2M

which are the maximal concentrations used in an adsorption isotherm test A time-based

evaluation of the adsorption was made by measuring the concentration of protein in

solution at different incubation times The results are shown below from Figure 4-34 to 4-

37 The adsorption kinetics of proteins onto th cond-e latex particles is expressed as a se

order model [Oumlzacar 2003] shown by the following equation

2)( tet qqk

dtdq

minus= (4-10)

Integrating above equation and applying the boundary conditions yields

ee

t tq

+=

1 (4-11)

where q

qkq

t

2

2

2

the previous adsorption isotherm results representing good reproducibility of adsorption

t and qe stand for the amount (mgm ) of protein adsorbed at time t and at

equilibrium respectively k is the equilibrium rate constant of second-order adsorption

(m mghr) and t is the incubation time (hr)

The fitting parameters calculated by using the equation 4-11 are listed in table 4-16

From this data it is seen that the amount of BSA adsorbed at equilibrium was higher than

that of β2M on PS and PSPMMA100 latex particles but less than that of β2M on

PSPMMA90PAA10 and PSPMMA75PAA25 latex particles These results are similar to

109

00

05

15

20

0 2 4 6 8 10 12 14

Time (hr)

rot

n ad

sorb

d (

10

25

Pei

em

gm

2

BSA2nd order model (BSA))β2M2nd order model (β2M)

Figure 4-34 The kinetics of protein adsorption in PB on PS latex particles at 37oC and

pH 74

00

05

10

15

20

25

30

0 2 4 6 8 10 12 14

Time (hr)

Prte

inds

oo

arb

ed (

mg

m2 )

BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-35 The kinetics of protein adsorption in PB on PSPMMA100 latex partic

37les at

oC and pH 74

110

00

03

06

09

12

15

0 2 4 6 8 10 12 14

Time (hr)

Prot

ein

adso

rbed

(m

gm

2 ) BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-36 The kinetics of protein adsorption in PB on PSPMMA90PAA10 latex

particles o at 37 C and pH 74

00

03

06

09

12

15

0 2 4 6 8 10 12 14

Time (hr)

BS

A ad

sorb

ed (

mg

m2 )

BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-37 The kinetics of protein adsorption in PB on PSPMMA75PAA25 latex

particles at 37oC and pH 74

111

test The variation of β2M equilibrium adsorption amount was nearly negligible for all

latex particles The plateau region is reached earlier for β2M than BSA on all latex

particles and the K values (equilibrium rate constant of second order adsorption) for the

β2M adsorption process onto latex particles are much larger than for BSA adsorption

These values indicate that the β2M molecules have a higher adsorption rate onto the latex

particles than the BSA molecules

Table 4-16 Fitting parameters of second-order kinetic model BSA β2M

Latex particles qe (mgm2) K (hr-1) qe (mgm2) K ( -1) hr

PS 144 67 074 7549

PSPMMA100 175 353 076 4438

PSPMMA90PAA10 046 97 075 5695

PSPMMA75PAA25 060 179 075 5168 K = (kqe) hr-1

There are several explanations for the phenomena observed in these experiments

First the smaller sizes of β2M molecules allow easier diffusion and a close approach to

the latex substrate Second a reduced number of hydrophobic domains on the latex

particle surface replaced by sulfate groups (-OSO3-) and ionized carboxylic groups

(COO-) are not favorable for larger BSA molecules Third the conformational change by

intra- and inter-molecular repulsion would be faster in β2M due to smaller molecular

weight Finally the ionic repulsion between β2M and the latex particles is less than that

condition (pH 74) than IEP (48) of BSA

between BSA and latex particles because IEP (57) of β2M is closer to the tested

112

44 Blood Compatibility

Blood compatibility can be defined as the property of a material or device tha

permits it to function in contact with blood without inducing any adverse reactions

Successful functioning of blood contacting biomedical materials or devices in contact

with blood strongly depends on the extent to which they damage blood during operation

High shear stresses turbulence rela

t

tively long contact times and cellular impact with

artificial surfaces are most common events experienced by blood in contact with artificial

1978 Winslow and et al

1993] Therefore before any materia e placed into the human b

biocompatibility testing must be the first performed in the view of safety The hemolysis

s a method for pr ary screening of biomaterials to determine

h blood a er circulating body fluid The tests estimat

oglobin released from th blood cell d e induced

foreign material

There are several types of blood cells in our body but the ones that make blood red

are erythrocytes or red blood cells (RBCs) Red blood cells (erythrocytes) are the most

numerous type in the blood These cells are average about 48-54 million per cubic

millimeter (mm3 same as a microliter (microl)) of blood but these values can vary over a

larger range depending on such factors as health and altitude RBCs are red because they

contain hemoglobin which is a protein that contains iron When blood circulates through

lungs oxygen binds to the hemoglobin and is then delivered to all the parts of body

RBCs are made in the bone marrow They start out as a nucleus like other cells but

when they fill up with hemoglobin the nucleus becomes smaller until it disappears

Without a nucleus RBCs are fragile and live only about 120 days In a normal healthy

devises and can cause hemolysis or thrombosis [Bernhard et al

ls can b ody

test serves a elimin the

biocompatibility wit nd oth e the

concentration of hem e red amag by a

113

body about 2 million red blood ne marrow produces new

ones j ing

dy

le

cells die per second But bo

ust as fast RBCs are about 75 micrometer across and are responsible for carry

oxygen salts and other organic substances to the cells and tissues throughout our bo

In humans and most mammals the cells are concave (indented in the middle) and flexib

enough to squeeze through even the tiniest of blood vessels the capillaries

Figure 4-38 Image of red blood cells

(Sourcehttpwwwpbrchawaiiedubemfmicroangelarbc1htm)

ch

100 90 10 75 25 f

RBCs are among the first cells that come into contact with a foreign material

injected in the blood Therefore a hemolysis assay gives information about the

biocompatibility in the case of an in vivo application The concentrations of prepared

latex particles used for the tests in this research are 05 25 and 50 (ww) and the

incubation time under moderate shaking was 30 minutes at 37oC Test results are shown

in Figure 4-39 The amount of blood cells that ruptured when in contact with the PS latex

particles was largely increased from about 30 to 91 as the solid content of PS

increased from 05 to 50 However hemolysis by the core shell latex particles su

as PSPMMA PSPMMA PAA and PSPMMA PAA was less than 35 O

114

particular interest was that hemolysis caused by PSPMMA PAA particles at 50

solid concentration was less than 02 hemolysis the lowest value among the latex

particles The low hemolysis value indicates that the PSPMMA PAA core shell latex

particles are the most blood compatible at all solid contents Other core shell particles

such as PSPMMA and PSPMMA PAA also had excellent blood comp

90 10

90 10

100 75 25 atibility with

low RBS breakage

01 2 3

20

40

60

80

100

Hem

olys

is (

)

052550

Figure 4-39 He

PS PSPMMA100 PSPMMA PAA90 10

molysis caused by latex particles n=5

4PSPMMA75PAA25

CHAPTER 5 SUMMARY OF RESULTS CONCLUSIONS AND RECOMMENDATION FOR

FUTURE WORK

51 Summary of Results

The goal of this study was to synthesize monodisperse polymeric latex particles

with tailored properties and to investigate the fundamental interactions between the

synthesized latex particles and target proteins An understanding of the fundamental

mechanism of selective adsorption is strongly required in order to maximize the

separation performance of membranes made of these materials so that it may applicable

to hemodialysis therapy for end stage renal disease (ESRD) patients For the achievement

of above research goal several investigations have been performed Analysis of the

obtained data leads to several conclusions as discussed below

Monodisperse polystyrene seed latex particles were synthesized with the size

crosslinking agent to make the latex particles more hydrodynamically stable The

conversion of monomer to polymer was over 95 and seed latex particles are spherical in

shape and have smooth surface From these small seeds bigger latex particles as large as

800nm were synthesized using a semi-continuous seeded emulsion polymerization

process The particles with a mean diameter less than 500nm are very spherical in shape

and highly uniform in particle size distribution However as the particles grow larger

than 500nm in mean diameter they became non-spherical with an uneven surface This

irregularity of particle surface can be attributed to the non-homogeneous monomer

ranges between 126plusmn75 and 216plusmn53 nm Divinylbenzene (DVB) was used as a

115

116

swelling into the shell of a growing polymer which can be controlled by the factors such

as temperature agitation speed initiator feeding rate and surfactant amount required to

stabilize the colloidal system It was determ rfactant to monomer ratio

dec ic

acid (AA) monomers were intro rticle surfaces more

hydro

to

ting of a

orm

ration is seen between

3002

0 cm-

0cm-1

-1

03

ined that as the su

reased the mean particle diameter increased Methyl methacrylate (MMA) and acryl

duced to make the latex pa

philic than bare polystyrene particles The prepared core shell latex particles were

made of PMMA100 PMMA90PAA10 and PMMA75PAA25 PMMA100 is the PMMA

homopolymer shell on a PS core PMMA90PAA10 shell is a copolymer with a PMMA

PAA ratio of 90 to 10 by weight PMMA75PAA25 shell is a copolymer consis

PMMA to PAA ratio of 75 to 25 by weight The particle size of these core-shell

particles were prepared to be about 370nm in mean diameter

The successful synthesis of latex particles was confirmed using Fourier Transf

Infrared spectroscopy (FTIR) The C-H aromatic stretching vib

cm-1 and 3103 cm-1 for PS latex particles The C-H asymmetrical stretching and

symmetrical stretching vibration peaks of CH2 for all latex particles is seen at 290

1and 2850 cm-1 respectively There is a carbonyl (C=O) characteristic band at 173

wavenumber for PSPMMA core-shell latex particles The broad OH group peak at

3400 cm

100

can be seen in the spectroscopy of the particle with PMMAPAA shell

(PSPMMA PAA and PSPMMA PAA ) The peak intensity of OH group of

PSPMMA PAA was larger than that of PSPMMA PAA relatively due to more AA

monomer content in PSPMMA PAA than in PSPMMA PAA

In the zeta potential measurements polystyrene (PS) latex particles had negative

values between -291 mV and -599 mV in the phosphate buffer (PB) and between -2

90 10 75 25

75 25 90 10

75 25 90 10

117

mV and -278 mV in the phosphate buffered saline (PBS) at 25oC and pH 21-78 These

negative zeta potential values for PS latex particles are due to ionization of the sulfate

groups (-OSO3-) originated from the initiator Zeta potential values of the PSPMMA

core shell latex particles were also negative between -28 mV and -505 mV in PB and

between -143 mV and -186 mV in PBS at pH 21-78 Negative values are also

generated by dissociation of sulfate groups on a particle surface The zeta potential value

of PSPMMA PAA and PSPMMA PAA were of negative values between -36

and -678 mV in PB medium and -147 mV and -193 mV in PBS medium at 25

100

s

7 mV

MMA75PAA25 and

PSPM

otein

g the

l change of BSA at

each w

90 10 75 25

oC For

PSPMMA PAA core shell particles zeta potential values were between -291 mV and

-520 mV in PB media and between -115 mV and -210 mV in PBS media Sulfate

groups contributed to the negative zeta potential values in PSP

75 25

MA90PAA10 core shell particles Carboxylic groups from polyacrylic acid also

contribute to the negative surface charge for the latex particles at greater than pH 70

however the degree of their contribution was not detectable from the zeta potential

measurements The absolute zeta potential values of the synthesized latex particles in PB

were higher than that in PBS This is because of the high concentration of sodium

chloride electrolyte in PBS that compress electrical double layer thickness

The bicinchoninic acid (BCA) assay technique was used to determine the pr

adsorption and all isotherms were fitted to the Langmuir-Freundlich isotherms usin

nonlinear regression method The hydrophobic interaction between PS latex particle and

BSA protein was dominant for the adsorption process Conformationa

pH also affected the adsorption amount which was highest at pH 48 because of lo

ionic repulsion making compact conformation of BSA The next larger amount of BSA

118

adsorbed onto PS was at pH 32 There were two main forces affecting the amount of

adsorption at acidic pH values One is ionic attraction between negative latex particle a

positive protein The other is lateral repulsion between positive proteins The smallest

amount of BSA adsorption was at pH 74 where charge repulsion between negative

particles and proteins is present at this pH Lateral repulsion between proteins wa

dominant for protein adsorption even though there always exists the hydrophobic

interaction between solid latex particle and protein The charge repulsion was reduced by

the high concentration of electrolytes in the PBS medium The BSA adsorption process

on PSPMMA core shell particles was very similar to the case of PS latex particle where

hydrophobic interaction between latex particle and protein was dominant However t

adsorbed BSA amount was less than that of PS in PBS The adsorbed amount of BS

PSPMMA

nd

s

he

A on

an

ed

n The main

adsor

90PAA10 and PSPMMA75PAA25 core shell particles was higher at pH 32 th

pH 48 and 74 The adsorbed BSA amount became higher at pH 32 and pH 48 as the

hydrophilicity was increased by the higher PAA amount On the contrary the adsorb

amount of BSA was dramatically decreased at pH 74 Carboxylic acid groups as well as

the sulfate groups are distributed on the surface of PSPMMA90PAA10 and

PSPMMA75PAA25 core shell particles and largely affect the BSA adsorptio

ption interaction forces at pH 32 was generated by hydrogen bonding as well as

charge attraction between latex particle and protein even when the conformational change

and charge repulsion between proteins reduce the adsorption of protein at this pH The

adsorbed BSA amount was also increased as the amount of PAA increases However the

adsorption of BSA was minimal (less than 07 mgm2) at pH 74 where the carboxylic

groups are ionized leading to more enhancement of charge repulsion by ionization of the

119

carboxylic groups (COO-) Because of this the PSPMMA-PAA latex particles have a

much lower protein adsorption

There were steep initial slopes for adsorption isotherms of β

in

s

there

M

r

e

es

o

2M on latex particles

all isotherms indicating a high affinity type adsorption The complete plateau region

were not seen for all isotherms indicating that β2M proteins were not saturated and

are still available sites for β2M adsorption on latex particles The initial slope of β2

isotherms for PS was steeper than for the other latex particles PSPMMA100

PSPMMA90PAA10 and PSPMMA75PAA25

The layer thickness (δ) of BSA proteins on synthesized latex particles in phosphate

buffer (PB) was in the range of 27 - 81 Aring at 37 C and pH 48 Only the δ values at pH

48 the isoelectric point (IEP) of BSA are important because intra- and inter-molecular

electrostatic repulsion of the protein molecules is minimized at the IEP Compared to the

hydrodynamic dimensions of BSA 140 times 40 times 40 Aring these values of the adsorbed laye

thickness indicates that the BSA molecules adsorb by the side-on (40Aring) and end-on mod

(140Aring) Consideration of the multi-layering is unnecessary because the adsorbed

thickness of BSA do not exceed 140 Aring

The Gibbs free energy of β

o

3

2M was more negative in phosphate buffer (PB) than

that of BSA in physiological condition (37 C and pH 74) indicating that βo2M molecul

are more favorably adsorbed on the latex particles than BSA and that the affinity of β2M

was much larger than BSA This may be explained by the rate of diffusion and

accessibility of β2M molecules being easier and faster than that for BSA because of

smaller size (or weight) and spherical shape of β2M molecules It may also be possible t

explain that more hydrophobic forces (less electrostatic repulsion) contributes to

120

adsorption of β2M than to that of BSA because the IEP of β2M (57) is closer to

physiological condition (pH 74) than that of BSA (pH 48) The selective adsorption of

β2M m

in β2M

larger than in BSA adsorption indicating

that fa

an that between BSA and latex particles because

IEP (

ch as

Other

olecules over BSA molecules can be enhanced by developing more hydrophilic

PSPMMA90PAA25 and PSPMMA75PAA25 core shell latex particles

The adsorption kinetics of proteins on latex particles was expressed as a second-

order model The plateau region for β2M was reached earlier than BSA on all latex

particles and the K values (equilibrium rate constant of 2nd order adsorption)

adsorption process on latex particles were much

ster adsorption rate of β2M molecules than that of the BSA molecules There are

several explanations for these phenomena First the smaller size of β2M molecules can

more easily diffuse and approach to the latex substrate Second enhanced hydrophilic

domain on the latex particle surface by sulfate groups (SO4-) and ionized carboxylic

groups (COO-) is not favorable for larger BSA molecules but is for smaller β2M to be

anchored Third the conformational change by intra- and inter-molecular repulsion

would be faster in β2M due to lower molecular weight Finally the ionic repulsion

between β2M and latex particles is less th

57) of β2M is closer to the tested condition (pH 74) than IEP (48) of BSA

In the biocompatibility test the number of red blood cells (RBCs) ruptured by PS

latex particles ranged from 30 to 91 as the solid content of PS increased from 05 to

50 respectively However hemolysis of the other core shell latex particles su

PSPMMA100 PSPMMA90PAA10 and PSPMMA75PAA25 were less than 35 The

PSPMMA90PAA10 particles at all applied solid concentrations had less than 02

hemolysis and was the most blood compatible among the prepared latex particles

121

core shell particles such as PSPMMA100 and PSPMMA75PAA25 also had excelle

compatibility with low RBC breakage

52 Conclusions

The major conclusions from this study include

nt blood

bull microglobulin (β2M) over that of the bovine serum albumin (BSA) at pH 74

was achieved by controlling the PMMA and PAA ratio to optimize

e particles posses the most desirable requirements for use in a human dialysis

ns

icles

olecule

bull

Using engineered core shell latex particles selective adsorption of β2

physiological condition was demonstrated

bull The rate of adsorption of the β2M was also much higher than that of BSA This

hydrophobichydrophilic microdomain size

bull The core shell particles also exhibited excellent biocompatibility with blood Thes

membrane

53 Recommendation for Future Work

With the results and conclusions obtained through this study several suggestio

may be given for future work as follows

bull Competitive adsorption tests of whole blood proteins on synthesized latex partunder physiological condition

bull Preparation of latex particles with optimum hydrophobic hydrophilic microdomains to maximize selective adsorption and removal of toxin molecules

bull Membrane fabrication by suitable pore size design to discriminate a toxin mby size sieving as a function of particle layers

Evaluation of membrane performance such as flow rate in water and in simulated blood fluid (SBF)

bull Application for hemodialysis as well as other technologies such as a water remediation

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Korea on August 7 1968 He obtained a Bachelor of Science degree in Chemistry from

Keim d graduate school and earned a

resear n Korea Institute of Science and

Coati e

ia

Busin

BIOGRAPHICAL SKETCH

Sangyup Kim was born and raised in the small town of Sung-Joo Kyungbook

yung University (Daegu Korea) in 1993 He entere

Master of Science degree in Chemical engineering in 1995 He worked as a student

cher for 2 years at Textile Polymer Division i

Technology (KIST) in Seoul Korea Next he worked over a year at the Automobile

ng Division in DongJoo-PPG in Korea In August 2001 he began studying for th

degree of Doctor of Philosophy in materials science and engineering at the University of

Florida in Gainesville After getting his PhD he plans to work at the Digital Med

ess Division of Samsung Electronics in Korea starting in January 2006

141

LIST OF TABLES

Table page 4-1 The polymerization recipe of polystyrene (PS) seed particles 57

4-2 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles less than 500nm in size 63

4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size64

4-4 The preparation recipe of PS core with various shell latex particles72

4-5 The isoelectric point (IEP) of common proteins 83

4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model 93

4-7 The equilibrium concentration values (q ) of BSA adsorption on PSPMMA particles calculated the Langmuir-Freundlich isotherm model

m 10096

4-8 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model

90 1098

4-9 The equilibrium concentration values of BSA adsorption on PSPMMA PAA particles calculated the Langmuir-Freundlich isotherm model

75 25100

4-10 The equilibrium concentration values of β M adsorption calculated the Langmuir-Freundlich isotherm model

2102

4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37 Co 104

4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37 Co 104

4-13 The values of Gibbs free energy change of BSA adsorption in phosphate buffer (PB) at 37 Co 105

4-14 The values of Gibbs free energy change of BSA adsorption in phosphate buffered saline (PBS) at 37 Co 105

viii

4-15 The values of Gibbs free energy change of β M adsorption in phosphate buffere (PB) at 37 C and pH 74

2o 106

4-16 Fitting parameters of second-order kinetic model 111

ix

LIST OF FIGURES

Figure page 1-2 Stereo drawing of the αndashcarbon backbone of β M2 2

2-1 An image of the location and cross section of a human kidney 5

2-2 A schematic draw of the hemodialysis route7

2-3 Cross-sectional SEM image view of the Polyflux S (polyamide + polyacrylethersulfone + polyvinypyrollidone) dialysis membranes 11

2-4 The chemical structure of polysulfone (PSf) 12

2-5 The chemical structure of polyamide (PA)13

2-6 Chemical structure of polyacrylonitrile (PAN) 14

2-7 Emulsion polymerization system24

2-8 L-α-amino acid 31

2-9 Ribbon diagram of human β M taken from the crystal structure of the protein bound to the heavy chain of the MHC class I complex (PDB 1DUZ)

236

2-10 Secondary structure of human serum albumin (HSA) with sub-domains 37

2-11 The relationship between pore size and particle size in body-centered cubic and closed packed arrays41

2-12 Schematic representation of the protein adsorption on the core shell latex particles at pH 7442

3-1 Chemical structure of main chemicals45

3-2 Experimental setup for semi-continuous emulsion polymerization 46

3-3 The particle preparation scheme in various types and size ranges of latex particles 47

3-4 Schematic of the procedure for a protein adsorption test 52

3-5 Separation of RBC from whole blood by centrifuge process53

x

3-6 Schematic of the procedure for hemolysis test 54

4-1 Schematic representation of semi-continuous seed latex particles preparation and growth56

4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A) PS (B) PS (C) PS (D) PSS259 S233 S207 S181 59

4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C) 370nm (D) 410nm 65

4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm (C) 640nm (D) 790nm 68

4-5 Dependence of the particle size on the surfactant to monomer ratio70

4-6 Schematic of core shell latex particle structures 71

4-7 Scanning Electron Micrograph of latex particles (A) PS (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA

100

90 10 75 25 73

4-8 FTIR spectra of polymerized latex particles (A) bare polystyrene (PS) (B) PSPMMA (C) PSPMMA PAA (D) PSPMMA PAA100 90 10 75 25 76

4-9 Schematic representation of ion distribution near a positively charged surface 78

4-10 Schematic representation of zeta potential measurement79

4-11 Zeta potential of PS latex particles at 25 Co 80

4-12 Zeta potential of PSPMMA latex particles at 25 C100o 81

4-13 Zeta potential of PSPMMA PAA latex particles at 25 C90 10o 82

4-14 Zeta potential of PSPMMA PAA latex particles at 25 C75 25o 82

4-15 The decay of surface potential with distance from surface in various electrolyte concentrations (1) low (2) intermediate (3) high 84

4-16 High-affinity adsorption isotherm of typical flexible polymer on solid surface 86

4-17 Overall schematic representation of the protein adsorption on the synthesized latex particles86

4-18 The dependence of UV absorbance on BSA concentration (A) 10mgml (B) 06mgml (C) 02mgml87

4-19 Standard curve of net absorbance vs BSA sample concentration 88

xi

4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on polystyrene latex particles at 37 C in phosphate buffer mediao 89

4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in phosphate buffer (PB) at 37 Co 90

4-22 Adsorption isotherm of BSA adsorption 37 C in phosphate buffered saline (PBS) on PS latex particles

o

91

4-23 Conformation of bovine serum albumin (BSA) on latex particles92

4-24 Adsorption isotherm of BSA 37 C in PB on PSPMMA core shell latex particles

o100

94

4-25 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA core shell latex particles

o100

95

4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA PAA core shell latex particles

o90 10

97

4-27 Adsorption isotherm of BSA at 37 C in PBS on PSPMMA PAA core shell latex particles

o90 10

98

4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA PAA latex particleso75 25 99

4-29 Adsorption isotherm of BSA 37 C in PBS on PSPMMA PAA core shell latex particles

o75 25

99

4-30 Adsorption isotherm of β M onto PS and PSPMMA latex particles in PB at 37 C and pH 74

2 100o 101

4-31 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74

2 90 10o 101

4-32 Adsorption isotherm of β M onto PS and PSPMMA PAA latex particles in PB at 37 C and pH 74

2 75 25o 102

4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37 C and pH 74

o

107

4-34 The kinetics of protein adsorption in PB on PS latex particles at 37 C and pH 74

o

109

4-35 The kinetics of protein adsorption in PB on PSPMMA latex particles at 37 C and pH 74

100o

109

4-36 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74

90 10o 110

xii

4-37 The kinetics of protein adsorption in PB on PSPMMA PAA latex particles at 37 C and pH 74

75 25o 110

4-38 Image of red blood cells 113

4-39 Hemolysis caused by latex particles n=5114

xiii

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

SYNTHESIS AND ENGINEERING OF POLYMERIC LATEX PARTICLES FOR MEDICAL APPLICATIONS

By

Sangyup Kim

December 2005

Chair Hassan El-Shall Major Department Materials Science and Engineering

Latex particles with well-defined colloidal and surface characteristics have received

increasing attention due to their useful applications in many areas especially as solid

phase supports in numerous biological applications such as immunoassay DNA

diagnostic cell separation and drug delivery carrier Hemodialysis membrane using

these particles would be another potential application for the advanced separation

treatment for patients with end stage renal disease (ESRD) It is desirable to remove

middle molecular weight proteins with minimal removal of other proteins such as

albumin Thus it is necessary to understand the fundamental interactions between the

particles and blood proteins to maximize the performance of these membranes This

improvement will have significant economic and health impact

The objective of this study is to synthesize polymeric latex particles of specific

functionality to achieve the desired selective separation of target proteins from the human

blood Semi-continuous seed emulsion polymerization was used to prepare monodisperse

xiv

polystyrene seed particles ranging from 126plusmn75 to 216plusmn53 nm in size which are then

enlarged by about 800nm Surfactant amount played a key role in controlling the latex

particle size Negatively charged latex particles with a different hydrophobicity were

prepared by introduction of a sodium persulfate initiator and hydrophilic acrylic acid

monomer The prepared polymeric particles include bare polystyrene (PS) particles less

hydrophobic PS core and PMMA shell particles and more hydrophilic PS core and

PMMA-co-PAA shell latex particles with a 370nm mean diameter SEM light scattering

and zeta potential measurements were used to characterize particle size and surface

properties Adsorption isotherms of two proteins bovine serum albumin (BSA) and β2-

microglobulin (β2M) on latex particles were obtained as a function of pH and ionic

strength using the bicinchoninic acid (BCA) assay method The Langmuir-Freundlich

adsorption model was used to determine the adsorption amount of protein at equilibrium

The thickness of adsorbed BSA layer on latex particles was obtained in order to

investigate the adsorption orientation such as end-on or side-on mode Adsorption

kinetics experiments for both proteins and all latex particles were also performed The

adsorption kinetic constant determined from the Langmuir-Freundlich adsorption

isotherm model was used to calculate Gibbs free energy of adsorption to compare the

competitive adsorption of BSA and β2M Hemolysis tests were performed to investigate

the blood compatibility of synthesized latex particles PSPMMA90PAA10 and

PSPMMA75PAA25 core shell latex particles had desirable material properties with not

only a large amount and high rate of selective β2M adsorption over BSA but also high

blood compatibility showing less than 3 hemolysis

xv

CHAPTER 1 INTRODUCTION

End stage renal disease (ESRD) is a chronic condition in which kidney function is

impaired to the extent that the patientrsquos survival requires removal of toxins from the

blood by dialysis therapy or kidney transplantation The National Kidney Foundation

estimates that over 20 million Americans had chronic kidney disease in 2002 [NKF] The

number of people with ESRD is rapidly increasing in the United States with

approximately 96 295 incidents and 406 081 prevalent patients including 292 215 on

dialysis and 113 866 with a functioning graft in 2001 It is projected that there will be

more than 22 million ESRD patients by 2030 [USRDS 2003] The expenditure for the

ESRD treatment program had reached $228 billion 64 of the Medicare budget in

2001 Due in part to the limited availability of kidneys for transplantation hemodialysis

is the primary clinical treatment for the patients with ESRD

The central element of a hemodialysis instrument is the semipermeable membrane

that allows for selective transport of low molecular weight biological metabolites less

than 5000 Da such as urea and creatinine as well as excess water and electrolytes [Baker

2004] from the blood One limitation of current dialysis technologies is the inability to

efficiently remove middle molecular weight toxins such as β2-Microglobulin (β2M) and

interleukin 6 (IL-6)

β2M is a causative protein of dialysis-related amyloidosis (DRA) a disease arising

in patients with chronic kidney failure as a serious complication of long-term

hemodialysis treatment [Gejyo et al 1985] β2M deposition in tissue is the primary cause

1

2

of destructive arthritis and carpal tunnel syndrome [Vincent et al 1992 Drueke 2000]

The β2M structure is shown in Figure 1-2

Figure 1-2 Stereo drawing of the αndashcarbon backbone of β2M [Becker et al 1985]

Although attempts have been made to increase the efficiency of middle molecular

weight toxin removal by changes in the membrane pore size and the use of innovative

materials to adsorb these toxins [Samtleben et al 1996 Ronco et al 2001] removal

efficiency is not as high as those achieved by a normal healthy kidney Traditional

membranes have a number of processing and performance limitations [Westhuyzen et al

1992 Leypoldt et al 1998] such as a restricted choice of surface chemistries and limited

control of porosity The development of novel engineering membrane technology is

needed to remove middle molecule toxins

Polymeric latex particles have received increasing attention in medical application

areas especially as solid phase supports in biological applications [Piskin et al 1994]

Examples of these applications include immunoassay [Chen et al 2003 Radomske-

Galant et al 2003] DNA diagnostic [Elaїssari et al 1998] cell separation drug delivery

carrier [Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] etc This is because of

3

the well-defined colloidal and surface characteristics of the particles By using a seed

emulsion polymerization method it is possible to synthesize monodisperse latex particles

with various particle size ranges and surface chemistry Functionalized core-shell latex

particles can be introduced by multi-step emulsion polymerization Core particles are

synthesized in the first stage of the polymerization and the functional monomer is added

in the second stage This is done without any emulsifier addition to prevent the

production of new homopolymer particles [Keusch et al 1973] The core-shell particles

are useful in a broad range of applications because of their improved physical and

chemical properties over their single-component counterparts [Lu et al 1996 Nelliappan

et al 1997] Through the development of a hemodialysis membrane using monodisperse

latex particles improvements in advanced separation treatment for patients with end

stage renal disease (ESRD) can be realized This requires the maximum removal of

middle molecular weight proteins with minimal removal of other beneficial proteins such

as albumin Thus an understanding of the fundamental interactions between the particles

and biopolymers is vital to maximize the performance of this membrane technology

The field of material science and biotechnology is based on fundamental chemistry

has developed over the past three decades into todayrsquos powerful discipline that enables

the development of advanced technical devices for pharmaceutical and biomedical

applications This novel and highly interdisciplinary field is closely associated with both

the physical and chemical properties of organic and inorganic particles [Niemeyer 2001]

Hemodialysis membrane using these particles would lead to improvements in the

advanced separation treatment for patients with end stage renal disease (ESRD) The

interdisciplinary nature of this approach enables a more complete understanding of the

4

phenomena of protein adsorption and the material properties necessary for selective

separation This innovative approach to membrane fabrication has the potential of making

inexpensive highly efficient membranes for both industrial and specialized separation

processes

The goal of this study is to prepare polymeric latex particles with tailored

properties to maximize separation of toxin molecules and to investigate the fundamental

interactions between the particles and molecules in the biological system in order to

optimize the performance of a membrane material for these applications

Polymeric latex particles were synthesized with specific functionality in an attempt

to achieve selective separation Seeded emulsion polymerization was used to synthesize

functionalized monodisperse latex particles in various sizes Negatively charged

hydrophobic polystyrene latex particles were synthesized by the same method Core shell

latex particles PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25 were also

synthesized to differentiate the degree of hydrophobicity of particles Scanning Electron

Micrograph (SEM) and light scattering measurements were used to characterize particle

size and shape and zeta potential measurements were conducted to measure the electrical

surface property of synthesized particles Adsorption isotherms of target proteins bovine

serum albumin (BSA) and β2M on latex particles were obtained as a function of pH

ionic strength and protein concentrations using the BCA assay method Adsorption

kinetics for both proteins on the latex particles were also measured Finally hemolysis

tests were run to determine the biocompatibility of polymer latex particles with human

blood This research will be described in more detail in chapters 3 and 4

CHAPTER 2 BACKGROUND AND LITERATURE SURVEY

21 The Significance of End Stage Renal Disease (ESRD)

The kidneys are responsible for removing excess fluid minerals and wastes from

the blood regulating electrolyte balance and blood pressure and the stimulation of red

blood cell production They also produce hormones such as erythropoietin (EPO) and

calcitriol that keep bones strong and blood healthy [Casadevall and Rossert 2005 Kurbel

et al 2003] EPO acts on the bone marrow to increase the production of red blood cell in

case of bleeding or moving to high altitudes Calcitriol mainly acts on both the cells of

the intestine to promote the absorption of calcium from food and also bone to mobilize

calcium from the bone to the blood When kidneys fail harmful waste builds up blood

pressure rises and body retains excess fluid The body also does not make enough red

blood cells When this happens treatment is needed to replace the function of failed

kidneys

Figure 2-1 An image of the location and cross section of a human kidney

5

6

The National Kidney Foundation estimates that over 20 million Americans had

chronic kidney disease in 2002 [NKF] Chronic renal disease is a gradual and progressive

loss of the function unlike acute renal disease where sudden reversible failure of kidney

function occurs Chronic renal failure usually takes place over a number of years as the

internal structures of the kidney are slowly damaged In the early stages there can be no

symptoms In fact progression may be so gradual that symptoms do not occur until

kidney function is less than one-tenth that of a normal health kidney If left untreated

chronic kidney disease may ultimately lead to kidney failure known as End Stage Renal

Disease (ESRD) ESRD is a rapidly growing heath-care problem in the United States In

2001 approximately 96295 incidents and 406081 prevalent patients were diagnosed

with ESRD including 292215 patients on dialysis and 113866 patients with a

functioning graft [USRDS 2003] The projected number of patients with ESRD is

expected to exceed 22 million patients by 2030 with much of this growth being driven by

the increasing prevalence of major contributing factors such as diabetes and high blood

pressure [USRDS 2003] A great extent of ESRD program cost and Medicare budget

have been spent in 2001 Due in part to a limited availability of kidneys for

transplantation hemodialysis (HD) is the primary method of treatment for ESRD and is

currently used for approximately 61 of US ESRD patients

22 Hemodialysis (HD) Treatment

HD removes toxins from the body by extracorporeal circulation of the blood

through a semipermeable membrane referred to as a dialyzer The toxins are removed

primarily by diffusion across the membrane to a dialysate solution which is circulated on

the opposite side of the membrane The cleaned blood is then returned to the blood

stream A surgically constructed vascular access connects the extracorporeal circuit to the

7

patientrsquos system Treatments are almost always performed three times per week in

specially equipped dialysis facilities for 3-4 hours per each treatment Figure 2-2 shows

the scheme of the hemodialysis route The critical element of a HD instrument is the

semipermeable membrane which allows for selective transport of low molecular weight

biological metabolites from blood

igure 2-2 A schematic draw of the hemodialysis route

ESRD patients who undergo dialysis therapy often experience several other

problems associated with the treatment such as anemia fatigue bone problems joint

problems itching sleep disorders and restless legs Anemia is common in patient with

kidney disease because the kidneys produce the hormone erythropoietin (EPO) which

stimulates the bone marrow to produce red blood cells Diseased kidneys often do not

produce enough EPO to stimulate the bone marrow to make a sufficient amount of red

blood cells This leads to bone disease referred to as renal osteodystrophy and causes

bones to become thin and weak or malformed and can affect both children and adults

Older patients and women who have gone through menopause are at greater risk for this

disease Uremic toxins which cannot be remove from the blood by the current dialyzer

membranes can lead to itching This problem can also be related to high levels of

F

8

parathyroid hormone (PTH) Dialysis patients can also experience day-night revers

is they have insomnia at night and sleep during the day This can be related to possible

nerve damage in the body and a chemical imbalance in the blood due to the excess toxin

Oxidative stress is a problem for patients on maintenance dialysis This problem is due to

an imbalance between pro- and antioxidant factors [Roselaar et al 1995 Cristol et al

1994] Oxidative stress affects oxidation of low-density lipoproteins which are the main

factor for atherogenesis [Huysmans et al 1998] and is also involved in the development

of malignancies and diabetes mellitus [Rice-Evans et al 1993] In order to reduce

antioxidant defense dialysis is needed to contribute to help stimulate free radical

production or eliminate antioxidants Dialysis-related amyloidosis (DRA) is also a

common and serious problem for people who have been on dialysis for more than 5

years DRA develops when proteins in the blood deposit on joints and tendons causi

pain stiffness and fluid in the joint as is the case with arthritis Normally the healthy

kidneys can filter out these proteins but dialysis filters are not as effective

23 Advances in Membrane Technology

al that

s

ng

Dialysis for blood of ESRD

Hemo filtration

s

ysis using a cellulose based membrane occurred

1913 John Abel [1990] from the Johns Hopkins Medical School described a method

purification is widely used in the treatment

dialysis (HD) techniques use a semi-permeable membrane to replace the

role of the kidney The membranes used in HD can be broadly classified into those based

on cellulose and those manufactured from synthetic copolymers These membranes come

in various shapes such as sheets tubular structures or hollow fiber arrangements The

hollow fiber type is the most popular and is incorporated into over 800 different device

in world wide [Ronco et al 2001]

The first attempt at blood dial

9

where sing

e

om

ysis was performed until the mid 1960 The basic

molec

rial in

een

benzyl)

was used for the membrane with

hepar

by the blood of a living animal may be submitted to dialysis outside the body u

a membrane based on cellulose and returned to the natural circulation without exposur

to air infection by microorganisms or any alteration that would necessarily be prejudicial

to life This same technique is still used to today however the device used has been

modified over the years as better membranes were developed and the anti-coagulant

heparin has become available

Cellulose membranes have been widely used for the treatment of renal failure fr

1928 when the first human dial

ular structure of cellulose is made of a long chain containing hydroxyl (OH)

groups The realization that such groups imparted undesirable qualities on the mate

respect to blood contact behavior was discovered in the early 1970s and since has b

the focus of development These modified cellulose membranes used the partial

substitution of benzyl groups to replace the proton of the hydroxyl groups in an attempt

to reduce their negative effect The result is a molecular mosaic of hydrophobic (

and hydrophilic (hydroxyl and cellulose) regions

Kolff [Van Noordwijk 2001] studied the rotating drum artificial kidney for patients

with acute renal failure in 1943 Cellophane tubing

in as the anticoagulant For the next 17 years hemodialysis therapy was performed

by this method but only for the patients with acute reversible renal failure Vascular

access required repeated surgical insertions of cannulas (slender tubes) into an artery and

vein and limited the number of treatments for a patient could receive in order to

minimize the amount of vascular damage

10

Initially the need for dialysis in patients with acute renal failure was determ

mainly by the development of signs and sym

ined

ptoms of uremia After dialysis some time

might

et al

ent improvement In 1960 the development of a shunt [Quinton et al

1960]

fone (PSf) polyamide (PA) and polyacrylonitrile (PAN) by phase inversion or

precip

lidone

e

elapse before uremic manifestations returned to warrant a sequential treatment of

dialysis Many patients with acute renal failure secondary to accidental or surgical

trauma were hypercatabolic but the interdialytic interval might be prolonged because of

anorexia or use of a low-protein diet However Teschan and his coworkers [Obrien

1959] showed that patient well-being and survival were improved by what they termed

prophylactic daily hemodiaysis or administration of the treatment before the patient

again became sick with uremia Their report in 1959 was the first description of daily

hemodialysis

Development of membrane accessories such as a shunt has also been an area of

focus for treatm

a flexible polytetrafluoroethylene (PTFE or Teflonreg) tubing made many more

hemodialysis treatments possible for chronic kidney failure patients PTFE has a non-

stick surface and is relative biocompatibility leading to minimized blood clotting in the

shunt

Synthetic membranes are prepared from engineered thermoplastics such as

polysul

itation of a blended mixture resulting in the formation of asymmetric and

anisotropic structures Figure 2-3 shows a fiber type of the Polyflux S membrane

consisting of poluamide (PA) polyacrylethersulfone (PAES) and polyvinylpyrro

(PVP) with the integral three-layer structure The skin layer on the inside fiber typ

membrane contacts blood and has a very high surface porosity and a narrow pore size

11

distribution This layer constitutes the discriminating barrier deciding the permeabili

and solute retention properties of the membrane The skin layer is supported by thick

sponge-type structure larger pores providing mechanical strength and very low

hydrodynamic resistance

ty

Figure 2-3 Cross-sectional SEM image view of the Polyflux S (polyamide +

polyacrylethersulfone + polyvinypyrollidone) dialysis membranes [Deppisch et al 1998]

n

[Malchesky 2004] because of its thermal stability mechanical strength and chemical

inertn ciated

ls

m

PSf is a widely used membrane material for the hemodialysis applicatio

ess According to a report from the National Surveillance of Dialysis-Asso

Disease (NSDAD) in the US over 70 of hemodialysis membranes were PSf based

[Bowry 2002] This is most likely because PSf has many advantages over other materia

This synthetic polymer is one of few materials that can withstand sterilization by stea

ethylene oxide and γ-radiation PSf membrane can be prepared by conventional

immersion precipitation methods into many different shapes including porous hollow

12

fiber or flat sheet hemodialysis membranes The material also has a high permeab

low molecular weight proteins and solute and high endotoxin retention The chemical

structure of PSf is shown in Figure 2-4

ility to

Figure 2-4 The chemical structure of polysulfone (PSf)

There is one major disadvantage to PSf The hydrophobic nature of the PSf causes

ment alternative pathway

leadin

s not

3

et al

in

O acrylate

serious complications through the activation of the comple

g to the adsorption of serum proteins onto the membranes [Singh et al 2003]

Anticoagulants are added during dialysis therapy to avoid blood clotting but this doe

completely eliminate the problem In order to overcome this disadvantage of the PSf

membrane various studies have been performed to change the materialrsquos surface

properties These investigations include hydrophilic polymer coating [Brink et al 199

Kim et al 1988 Higuchi et al 2003] layer grafting onto PSf membrane [Wavhal

2002 Song et al 2000 Pieracci et al 2002 Mok et al1994] and chemical reaction of

hydrophilic components onto the membrane surface [Higuchi et al 1990 1991 1993

Blanco et al 2001 Nabe et al 1997 Guiver et al 1993] Hydrophilic monomers 2-

hydroxy-ethylmethacrylate (HEMA) acrylic acid (AA) and methacrylic acid (MMA)

have also been grafted onto PSf membrane to increase flux and Bovine Serum Album

(BSA) retention [Ulbricht et al 1996] Hancock et al [2000] synthesized

polysulfonepoly(ethylene oxide) (PEO) block copolymers to improve the resistance to

platelet adhesion Kim et al [2003] also studied blending a sulfonated PE

diblock copolymer into PSf in order to reduce platelet adhesion and enhance

13

biocompatibility PEO is a commonly used biomaterial due to its excellent resistance

protein adsorption and inherent biocompatibility [Harris 1992] Kim et al [20

a self-transformable copolymer to enhance the hydrophilicity of an asymmetric PSf

membrane with an ultra-thin skin layer The polymer had an entrapped diblock

copolymer containing a hydrophilic block of poly (ethylene glycol) (PEG)-SO

to

05] studied

ate

lic graft

l)

e of thier

aromatic

orand

3 acryl

and a hydrophobic block of octadecylacrylate (OA) Molecular dynamic (MD)

simulations were performed as a function of copolymer density to optimize interfacial

structure information McMurry [2004] developed a strategy using an amphiphi

copolymer added to PSf membranes by introducing polysulfone-g-poly (ethylene glyco

When compared to unmodified PSf these graft copolymer and resulting blend

membranes are found to hold promise for biomedical device applications

Polyamide (PA) membranes have also been used for hemodialysis becaus

mechanical strength in both wet and dry conditions Polyamide consists of

aliphatic monomers with amide bonding (-CONH-) also known as a peptide bond

The basic amide bond in polyamide is shown in Figure 2-5 R1 and R2 can be either

aromatic or aliphatic linkage group

C N

H

O

~ R R1 2~C N

H

O

~ R R1 2~

Figure 2-5 The chemical structure of polyamide (PA)

Panichi and co-workers [1998] evaluated the biocompatibility of the PA membrane

ompatible technique due to the

use of a synthetic membrane with a sterile re-infusion fluid and the convective removal of

and concluded that PA hemofiltration was a highly bioc

14

the ac

e

e

is

n

is a semi-crystalline polymer and the mechanical properties

strong

2

CN

tivated anaphylatoxins and β2-Microglobulin (β2M) The PA based membrane

Polyfluxreg (manufactured by Gambro GmbH Germany) blended with polyamide

polyarylethersulfone and polyvinylpyrrolidone (PVP) was able to clean small molecules

such as urea dreatinine and phosphate as well as decrease β2M amount by 502

[Hoenich et al 2002] Due to the non-selectivity of the membrane removal of thes

unwanted materials also led to the undesirable loss of beneficial proteins during therapy

Meier et al [2000] evaluated different immune parameters using a modified cellulos

low-flux hemophan and synthetic high-flux PA membrane during a 1 year period in

chronic hemodialysis patients They found that the 1-year immunological evaluation of

hemodiaysis membrane biocompatibility was associated with changes in the pattern of

chronic T-cell actiovation

Polyacrylonitrile (PAN) is another commonly used membrane material because it

inherently hydrophilic and has been commercialized for ultrafiltration and microfiltratio

[Scharnagl et al 2001] PAN

ly depend on the crystalline structures The chemical structure of PAN is shown in

figure 2-6

CH CH

n2

CN

CH CH n

Chemical structure of polyacrylonitrile (PAN)

The addition of additives such as polyvinylpyrrolidone (PVP) as a pore forming

agent gives PAN membranes more flexible processing parameters and increased

e has been optimized through

copolymerization with many other vinyl monomers including glycidyl methacrylate

Figure 2-6

performance [Jung et al 2005] PAN membrane performanc

15

[Godj

acid

for

for

a non-

solub this

in

oid underneath the inner

surface of membrane decreased by increasing the amount of solvent DMSA in the

evargova et al 1999 Hicke et al 2002] N-vinylimidazole [Godjevargova et al

2000] hydroxyl ethyl methacrylate [Ray et al 1999 Bhat et al 2000] methacrylic

[Ray et al 1999] vinyl pyrrolidone [Ray et al 1999] acrylic acid [Trotta et al 2002]

acrylamide [Musale et al 1997] and vinylchloride [Broadhead et al 1998] These

monomers provide a reactive group for enzyme immobilization improved mechanical

strength solvent-resistance pervaporation permeation flux anti-fouling and bio-

compatibility Because of this PAN-based copolymer membranes have great potential

the treatment of hemodialysis in an artificial kidney This material can also be used

other applications like the treatment of wastewater the production of ultra-pure water

biocatalysis together with separation and methanol separation by pervaporation

Lin and his coworker [2004] studied the modification of PAN based dialyzer

membranes to increase the hemocompatibility by the immobilization of chitosan and

heparin conjugates on the surface of the PAN membrane When a foreign material is

exposed to blood plasma proteins are adsorbed clotting factors are activated and

le fibrin network or thrombus is formatted [Goosen et al 1980] The result of

research was that the biocompatible chitosan polymer and a blood anticoagulant hepar

prevented blood clotting They showed prolonged coagulation time reduced platelet

adsorption thrombus formation and protein adsorption

Nie et al [2004] studied PAN-based ultrafiltration hollow-fiber membranes

(UHFMs) In order to improve the membrane performance acrylonitrile (AN) was

copolymerized with other functional monomers such as maleic anhydride and α-allyl

glucoside They found that the number and size of macrov

16

intern erum

and

d a

rface The second step is the

transf after

yjak et

embrane

1978 Malchesky et al 1978] has been further developed to increase the efficiency of

al coagulant The water flux of the UHFMs also decreased while the bovine s

albumin rejection increased minutely Godjevargova et al [1992] modified the PAN

based membrane with hydroxylamine and diethylaminoethylmethacrylate to improve

membrane dialysis properties Formed functional groups like primary amine oxime

tertiary amine groups provided the membrane with more hydrophilic properties an

substantial increase in the permeability of the membranes

The wide use of filtration in practice is limited by membrane fouling Solute

molecules deposit on and in the membrane in the process of filtration causing dramatic

reduction in flux through the membrane Fouling occurs mostly in the filtration of

proteins Three kinetic steps are involved in the fouling of UF membranes according to

Nisson [1990] The first step is the transfer of solute to the su

er of solute into the membrane until it either finally adsorbs or passes through

a set of adsorption-desorption events The third step includes surface binding

accompanied by structural rearrangement in the adsorbed state [Ko et al 1993] Br

al [1998] studied the surface modification of a commercially available PAN membrane

to develop superior filtration properties with less fouling by proteins The PAN

membrane was immersed in excess NaOH solution to convert some of the surface nitrile

groups into carboxylic groups by the hydrolysis process This modified PAN m

was not so severely fouled in the Bovine Serum Albumin (BSA) filtration test The pore

size however decreased during the hydrolysis process leading to a significant reduction

in flux and made the membrane less productive in the ultrafiltration (UF) mode

24 Sorbent Technology

Over the last three decades sorbent technology [Castino et al 1976 Korshak et al

17

dialysis or replace it for the treatment of ESRD Sorbents remove solutes from solution

through specific or nonspecific adsorption depending on both the nature of the solute and

the sorbent Specific adsorption contains tailored ligands or antibodies with high

selectivity for target molecules een used in autoimmune

disord

and

e

al 1996] various resins [Ronco et al 2001]

album

e

as

Specific adsorbents have b

ers such as idiopathic thrombocytopenic purpura [Snyder et al 1992] and for the

removal of lipids in familial hyper cholesterolemia [Bosch et al 1999] Nonspecific

adsorbents such as charcoal and resins attract target molecules through various forces

including hydrophobic interactions ionic (or electrostatic) attraction hydrogen bonding

and van der Waals interactions

New dialysate with sorbents has become an accepted modification of dialysis

sorbent hemoperfusion is gaining ground as a valuable addition to dialysis especially as

new sorbents are developed [Winchester et al 2001] Hemoperfusion is defined as th

removal of toxins or metabolites from circulation by the passing of blood within a

suitable extracorpoteal circuit over semipermeable microcapsules containing adsorbents

such as activated charcoal [Samtleben et

in-conjugated agarose etc Novel adsorptive carbons with larger pore diameters

have been synthesized for potential clinical use [Mikhalovsky 1989] Newly recognized

uremic toxins [Dhondt et al 2000 Haag-Weber et al 2000] have resulted in several

investigations on alternatives to standard or high-flux hemodialysis to remove thes

molecules These methods include hemodiafilteration with [de Francisco et al 2000] or

without [Ward et al 2000 Takenaka et al 2001] dialysate regeneration using sorbents

well as hemoperfusion using such adsorbents as charcoal and resins

18

Kolarz et al [1989 1995] studied the hyper-crosslinked sorbent prepared from

styrene and divinylbenzene (DVB) for a hemoperfusion application They found that the

pore structure of a swelling sorbent was changed by additional crosslinking with α αrsquo-

dichloro-p-xylene in the presence of a tin chloride catalyst and in a di

chloroethane

soluti and

en

decyl groups that attract β2M through a hydrophobic interaction The

adsor

of

5]

d

penet

on They also realized that the hemocompatibility was useful for nemoperfusion

could be imparted to the sorbents by introducing sulfonyl groups at a concentration of

about 02mmolg

A special polymeric adsorbing material (BM-010 from Kaneka Japan) has be

investigated by another group [Furuyoshi et al 1991] for the selective removal of β2M

from the blood of dialysis partients The adsorbent consists of porous cellulose beads

modified with hexa

ption capacity of this material is 1mg of β2M per 1ml of adsorbent Using a

hemoperfusion cartridge containing 350ml of these cellulose beads in sequence with a

high-flux hemodialyzer several small clinical trials were performed During 4-5 hours

treatment about 210mg of β2M were removed thus reducing the concentration in the

blood by 60-70 of the initial level [Nakazawa et al 1993 Gejyo et al 1993 199

RenalTech developed a hemoperfusion device BetaSorbreg containing the hydrate

cross-linked polystyrene (PS) divinylbenzene (DVB) resin sorbents with a pore structure

designed to remove molecules between 4 and 30 kDa [Winchester et al 2002] In this

case solute molecules are separated according to their size based on their ability to

rate the porous network of the beaded sorbents The resin beads were prepared with

a blood compatible coating and confirmed to be biocompatible in vivo in animals

[Cowgill et al 2001]

19

25 Limitation of Current Hemodialysis Treatment

Hemodialysis is a widely used life-sustaining treatment for patients with ESRD

However it does not replace all of the complex functions of a normal healthy kidne

a result patients on dia

y As

lysis still suffer from a range of problems including infection

accelerated cardio ion anemia

chron

e

se middle

alysis

sessio

s

f

vascular disease high blood pressure chronic malnutrit

ic joint and back pain and a considerably shortened life span One significant

limitation of the current dialysis technology is the inability to efficiently remove larger

toxic molecules This is mainly because of the broad pore size distribution reducing th

selective removal of toxins and unsatisfied biocompatibility causing lots of

complications such as inflammation blood clotting calcification infection etc

Dialysis purifies the patients blood by efficiently removing small molecules like

salts urea and excess water However as toxic molecules increase in size their removal

rate by hemodialysis substantially declines Typically only 10 - 40 of the

molecular weight toxins (300-15000 Da) are removed from the blood during a di

n [Vanholder et al 1995] These toxins then reach an abnormally high level and

begin to damage the body One such toxin β2M causes destructive arthritis and carpal

tunnel syndrome by joining together like the links of chain to form a few very large

molecules and deposit damaging the surrounding tissues [Lonnemann et al 2002] This i

also a main cause of mortality for long-term dialysis patients Other middle molecule

toxins appear to inhibit the immune system and may play a significant role in the high

susceptibility to infections in dialysis patients Still others are believed to impair the

functioning of several other body systems such as the hematopoietic and other endocrine

systems This may contribute to accelerated cardiovascular disease the leading cause o

20

death among dialysis patients as well as clinical malnutrition which affects up to 50 of

this patient population

Over the last decade polymeric dialysis membranes have been developed to

increase the capacity for removing middle molecular weight toxins by changing the pore

size of dialyzer membranes and using new materials that adsorb these toxins for

impro se

styrene made during World War II in the United

States The Dow chemical compan anufacturer of polystyrene

includ

d of

on

rug

ntrol of

ved removal characteristics However removal efficiency is not as high as tho

achieved by a normal healthy kidney

26 Latex Particles

The first synthetic polymer synthesized using emulsion polymerization was a

rubber composed of 13-butadiene and

y has been a major m

ing latex which they used in paint formulations The theory of emulsion

polymerization in which a surfactant is used was established by Harkins [1948] and by

Smith and Ewart [1948] By 1956 the technology was complete including the metho

building larger diameter particles from smaller ones The product by this emulsi

polymerization is referred to as latex a colloidal dispersion of polymer particles in water

medium [Odian 1991] Latexes are currently undergoing extensive research and

development for a broad range of areas including adhesives inks paints coatings d

delivery systems medical assay kits gloves paper coatings floor polish films carpet

backing and foam mattresses to cosmetics The relatively well known and easy co

the emulsion process is one of main advantages for these applications Therefore

polymeric latex particle prepared by emulsion polymerization can be a candidate for the

medical applications because of the easy control of the particle size and morphology as

well as flexible surface chemistry to be required

21

261 The Components for Emulsion Polymerization

The main components for emulsion polymerization process are the monomer a

dispersing medium a surfactant and an initiator Available monomers are styrene

sing medium is usually water

which

ticles

n

ation To

monomer droplets but in micelles because the initiators are

insolu le

and

olution

butadiene methylmethacylate acryl acid etc The disper

will maintain a low solution viscosity provide good heat transfer and allow

transfer of the monomers from the monomer droplets into micelles and growing par

surrounded by surfactants respectively The surfactant (or emulsifier) has both

hydrophilic and hydrophobic segments Its main functions is to provide the nucleatio

sites for particles and aid in the colloidal stability of the growing particles Initiators are

water-soluble inorganic salts which dissociate into radicals to initiate polymeriz

control the molecular weight a chain transfer agent such as mercaptan may be present

262 Particle Nucleation

Free radicals are produced by dissociation of initiators at the rate on the order of

1013 radicals per milliliter per second in the water phase The location of the

polymerization is not in the

ble in the organic monomer droplets Such initiators are referred to as oil-insolub

initiators This is one of the big differences between emulsion polymerization

suspension polymerization where initiators are oil-soluble and the reaction occurs in the

monomer droplets Because the monomer droplets have a much smaller total surface area

they do not compete effectively with micelles to capture the radicals produced in s

It is in the micelles that the oil soluble monomer and water soluble initiator meet and is

favored as the reaction site because of the high monomer concentration compared to that

in the monomer droplets As polymerization proceeds the micelles grow by the addition

of monomer from the aqueous solution whose concentration is refilled by dissolution of

22

monomer from the monomer droplets There are three types of particles in the emulsion

system monomer droplets inactive micelles in which polymerization is not occurring

and active micelles in which polymerization is occurring referred to as growing polymer

particles

The mechanism of particle nucleation occurs by two simultaneous processes

micellar nucleation and homogeneous nucleation Micellar nucleation is the entry of

radicals ei

ther primary or oligomeric radicals formed by solution polymerization from

the aq ed

the

r

s

n

is coagulation with other particles rather than

polymerization of monomer The driving force for coagulation of precursor particles

ueous phase into the micelles In homogeneous nucleation solution-polymeriz

oligomeric radicals are becoming insoluble and precipitating onto themselves or onto

oligomers whose propagation has ended [Fitch et al 1969] The relative levels of micella

and homogeneous nucleation are variable with the water solubility of the monomer and

the surfactant concentration Homogeneous nucleation is favored for monomers with

higher water solubility and low surfactant concentration and micellar nucleation is

favored for monomers with low water solubility and high surfactant concentration It has

also been shown that homogeneous nucleation occurs in systems where the surfactant

concentration is below CMC [Roe 1968] A highly water insoluble monomer such a

styrene [Hansen et al 1979 Ugelstad et al 1979] has probably created by micellar

nucleation while a water soluble monomer such as vinyl acetate [Zollars 1979] has bee

formed by homogeneous nucleation

A third latex formation reaction mechanism has been proposed referred to as

coagulative nucleation In this reaction the major growth process for the first-formed

polymer particles (precursor particles)

23

sever les

yer

es

r a

l

the beginning of the polymerization As soon as the initiator is

gins with the formation and

growt

and

ved

added at one time during the later stages of the polymerization prior to complete

al nanometers in size is their relative instability compared to larger sized partic

The small size of a precursor particle with its high curvature of the electrical double la

permits the low surface charge density and high colloidal instability Once the particl

become large enough in size maintaining the high colloidal stability there is no longe

driving force for coagulation and further growth of particles takes place only by the

polymerization process

263 Types of Processes for Emulsion Polymerization

There are three types of production processes used in emulsion polymerization

batch semi-continuous (or semi-batch) and continuous In the batch type process al

components are added at

added and the temperature is increased polymerization be

h of latex particles at the same time There is no further process control possible

once the polymerization is started In the semi-continuous emulsion polymerization

process one or more components can be added continuously Various profiles of particle

nucleation and growth can be generated from different orders of component addition

during polymerization There are advantages to this process such as control of the

polymerization rate the particle number colloidal stability copolymer composition

particle morphology In the continuous process the emulsion polymerization components

are fed continuously into the reaction vessel while the product is simultaneously remo

at the same rate High production rate steady heat removal and uniform quality of

latexes are advantages of the continuous polymerization processes

Other available methods include the intermittent addition and shot addition of one

or more of the components In the shot addition process the additional components are

24

conversion of the main monomer This method has been used successfully to develo

water-soluble functional monomers such as sodium styrene sulfonat

p

e [Kim et al 1989]

264

lsion

Chemistry of Emulsion Polymerization

Emulsion polymerization is one type of free radical polymerization and can be

divided into three distinct stages initiation propagation and termination The emu

polymerization system is shown in Figure 2-7

Aqueous phaseAqueous phase

Micelle withmonomer

I Rbull

Emulsifier

Polymer particle swollenWith monomer

Monomerdroplet

I Rbull

Micelle withmonomer

I Rbull

Emulsifier

Polymer particle swollenWith monomer

MonomerdropletMonomerdroplet

I Rbull

Figure 2-7 Emulsion polymerization system [Radicals (Rmiddot) are created from initiators

(I) Monomer is transferred from larger monomer droplet into micelles by emulsifier Initiated polymer particle by radicals is keep growing until monomers are all consumed The reaction is performed in aqueous media]

In the initiation stage free radicals are created from an initiator by heat or an

ultraviolet radiation source The initiator with either peroxides groups (-O-O-) such as

odium persulfate or azo groups (-N=N-) such as azobisisobutyronitrile is commonly

used for em t

with the m er

chain ere

s

ulsion polymerization The primary free radicals created from initiator reac

onomer for initiation of polymerization In the propagation stage the polym

grows by monomer addition to the active center a free radical reactive site Th

are two possible modes of propagations head-to-head addition and head-to-tail addition

25

The head-to-tail mode is the predominant configuration of the polymer chain a result o

steric hindrance of the substitute bulky group In the termination stage polymer chain

growth is terminated by either coupling of two growing chains forming one polymer

molecule or transferring a hydrogen atom (dispropotionation) from one growing chain to

another forming two polymer molecules one having a saturated end group and the other

with an unsaturated end-group

265 Seed Emulsion Polymerization

Polymer latex particles have received increasing interest because of the versatili

of the many applications heterophase polymerization processes like emulsion dispersion

micro-emulsion seeded emulsion precipitation etc Especially to prepare well-defined

microspheres having monodispe

f

ty

rse and various particle sizes as well as surface group

ed particles prepared by emulsion polymerization

and e

s

hological design In

seede to control

n

functionalilties it is necessary to use se

nlarge them to a desired size in the further stages of reactions

Polymeric particles are required to have a uniform particle size in many

applications such as chromatography where they are used as a packing material

Morphological control of latex particle is also important for many practical application

[Schmidt 1972] Seed emulsion polymerization (or two-stage emulsion polymerization) is

a useful method to achieve both monodisperse particle size and morp

d emulsion polymerization [Gilbert 1995] preformed lsquoseedrsquo latex is used

the number of particles present in the final latex The advantage of seeded emulsio

polymerization is that the poorly reproducible process of particle nucleation can be

bypassed so that the number concentration of particles is constant and known Various

mechanisms have been proposed for the growth of latex particles [Ugelstad et al 1980

Okubo et al 1992] using this polymerization technique The initial seed particle

26

preparation step is well known and relatively easy to perform because at the relative

small particle sizes (02 to 05 micron) the particle growth process can be readily

controlled by the use of an emulsifier Enough emulsifier is used to prevent coagulation

but not enough to cause the formation of new particles As the particles are grown to

larger sizes in successive seeding steps it becomes increasingly difficult to mainta

stable uncoagulated emulsion without forming new particles and thereby destroying

monodisperisty of the latex Recently there have been several reports concerning t

reaction kinetics of seed emulsion polymerization and the development of latex

morphologies over the course of the reaction [Chern et al 1990 Delacal et al 1990 L

et al 1995 Lee 2000 2002]

266 Polystyrene Latex Particles

A number of papers have described the synthesis of the polystyrene latex particl

bearing various functional surface groups such as carboxyl [Lee et al 2000 Tun

2002 Reb et al 2000] hydroxyl [Tamai et al 1989] marcapto [Nilson 1989] epoxy

[Shimizu et al 2000 Luo et al

ly

in a

he

ee

es

cel et al

2004] acetal [Izquierdo et al 2004 Santos et al 1997]

ometyl [Izquierdo et al 2004 Park et al 2001

Sarob

h the

d

thymine [Dahman et al 2003] chlor

e et al 1998] amine [Counsin et al 1994 Ganachaud et al 1997 Ganachaud et al

1995 Miraballes-Martinez et al 2000 2001 Anna et al 2005] ester [Nagai et al 1999]

etc In order to produce these functionalized particles different methods for particle

preparation must be used The polymer emulsion with core-shell morphology of latex

particles is one of them This is a multistep emulsion polymerization process in whic

polystyrene ldquocorerdquo particle is synthesized in the first stage and the functional monomer is

added in the second stage of the polymerization without any emulsifier postfeeding to

prevent the production of new homopolymer particles thus forming the functionalize

27

polymer ldquoshellrdquo on the ldquocorerdquo particle [Keusch et al 1973] There are requirements to

limit secondary nucleation and encourage core-shell formation in seeded emulsion

polymerization including the addition of smaller seed particles at high solid content to

increase particle surface area low surfactant concentration to prevent formation of

micelles and the semi-continuous addition of monomer to create a starved-feed conditio

and keep the monomer concentration low There are some advantages [Hergeth et al

1989] of dispersions with polymeric core-shell particles First it is possible to modi

interfacial properties of polymer particles in the aqueous phase by the addition of only

very small amounts of a modifying agent during the last period of the reaction Thus

these core-shell particles are useful in a broad range of applications since they always

exhibit improved physical and chemical properties over their single-component

counterparts [Lu et al 1996 Nelliappan et al 1997] In this way the improvement of

surface properties of such dispersions is straightforward and inexpensive The other is

that polymers with a core-shell structure are perfect model systems for investigating th

material properties of polymer blends and composites because of their regular

distribution of one polymer inside a matrix polymer and because of the simple sp

geometry of the system

Their properties usually depend on the structures of latex particles Chen and his

coworkers [Chen et al 1991 1992 1993] reported the morphological development of

core shell latex particles of polystyrenepoly(methyl methacrylate) during

polymerization Before the research by Chen and his coworker Min et al [Min et al

1983] reported the morph

n

fy the

e

herical

ological development of core shell latex of polystyrene

(PS)polybuthyl acrylateat (PBA) by seeded emulsion polymerization as a function of the

28

additi

of

ity of graft

ing reagents Thus protein adsorption on polymeric solid surfaces has

st are microspheres defined as ldquofine

polym can be

n

r was

containing antigen urine serum etc The latex will become agglutinated and visibly

on method of PS They found that the percentage of grafting PS to the PBA was

greatest for the batch reaction and the PBA-PS core-shell particles with a high degree

grafting remained spherical upon aging test because of the emulsifying abil

copolymer

267 Various Applications of Latex Particles

Latex particles are applicable to a wide range of areas such as biomedical

applications [Piskin et al 1994] especially as the solid phase such as in immunoassays

[Chern et al 2003 Radomske-Galant et al 2003] DNA diagnostic drug delivery carriers

[Luck et al 1998 Kurisawa et al 1995 Yang et al 2000] blood cell separations and

column pack

become a center of attention Of particular intere

er particles having diameters in the range of 01 to several micronsrdquo which

used as functional tools by themselves or by coupling with biocompounds Singer and

Plotz [1956] firstly studied microsphere or latex agglutination test (LATrsquos) by using

monodisperse polystyrene (PS) and polyvinyltoluene polymer particles as the support o

which the biomolecules were going to adsorb The biomolecule adsorption howeve

limited by possible desorption of the adsorbed species or loss of specific activity of the

complex formed Since this work was published the latex particle applications for

immunoassay have been rapidly and widely studied and developed

Latex agglutination test or latex immunoassays start with tiny spherical latex

particles with a uniform diameter and similar surface properties The particles are coated

with antibodies (sensitized) through the hydrophobic interaction of portions of the protein

with the PS surface of the particles If sensitized particles are mixed with a sample

29

agglomerate Latex tests are inexpensive as compared with the other techniques [Ba

1988]

ngs

sed

tion Rheumatoid factor (RF) in different age subpopulations has also been

evalu

studied a

latex

Unipath [Percival 1996] has manufactured a range of immunoassay materials ba

on a chromatographic principle and deeply colored latex particles for use in the home and

clinical environments The latex particles which are already sensitized with a

monoclonal antibody can detect any antigens that bound to the surface of the latex

particles Some detectable pollutants include estrogen mimics which induce

abnormalities in the reproductive system of male fishes and lead to a total or partial male

feminiza

ated according to a patientrsquos clinical status by using a rapid slide latex agglutination

test for qualitative and semiquantitative measurement in human serum along with latex

immunoassay method [Onen et al 1998] Magalhaes and his coworkers [2004]

diagnostic method of contamination of male fishes by estrogen mimics using the

production of vitellogenin (VTG) as a biomarker This was based on a reverse

agglutination test developed with monoclonal antibodies specific to this biomarker

Premstaller and his coworkers [Premstaller et al 2000 2001] have prepared a porous

poly(styrene-divinylbenzene) (PS-DVB) polymer monolith to use for highly efficient

chromatographic separation of biomelecules such as proteins and nucleic acids They

used a porogen a mixture of tetrahydrofuran and decanol to fabricate a micropellicular

PS-DVB backbone Legido-Quigley and his colleagues [2004] have developed the

monolith column to obtain further chromatographic functionality to the column by

introducing chloromethylstyrene in place of styrene into the polymer mixture

30

Core shell type monodisperse polymer colloids have been synthesized by Sarobe

and Forcada [1998] with chloromethyl functionality in order to improve the biomolecu

adsorption through a two-step emulsion polymerization process They investigated the

functonalized particles by optimizing the experimental parameters of the functional

monomer including reaction temperature the amount and type of redox initiator sys

used the type of addition of the initiator system and the use of washing They c

le

tem

oncluded

that th

sis

serve as separation

chann

of

usually

along the polypeptide chain which contain positive or negative charges [Norde 1998]

e relation between the amount of iron sulfate and the persulfatebisulfite system

added should be controlled to obtain monodisperse particles and prevent the premature

coagulation of the polymer particles during the polymerization

A semi-continuous emulsion polymerization technique for latex particle synthe

was performed by McDonald and other researchers [Ramakrishnan et al 2004 Steve et

al 1999] They introduced a variety of particles sizes compositions morphologies and

surface modifications to fabricate latex composite membranes (LCMs) Arraying and

stabilizing latex particles on the surface of a microporous substrate form narrowly

distributed interstitial pores formed between the particles which

els They investigated the membrane performance using gas fluxes water

permeability and the retention characterization of dextran molecules From these tests

they concluded that the narrow discriminating layer made of the latex particles leads to a

highly efficient composite membrane

27 Proteins

Proteins are natural polyamides comprised of about 20 different α-amino acids

varying hydrophobicity [Norde 1998] Proteins are more or less amphiphilic and

highly surface active because of the number of amino acid residues in the side groups

31

Proteins are polymers of L-α-amino acids [Solomon and Fryhle 2000] The α refers to a

carbon with a primary amine a carboxylic acid a hydrogen and a variable side-chain

group designated as R Carbon atoms rent groups are asymmetric and can

exhib l

s

e

on

with four diffe

it two different spatial arrangements (L and D configurations) due to the tetrahedra

nature of the bonds The L refers to one of these two possible configurations Amino

acids of the D-configuration are not found in natural proteins and do not participate in

biological reactions Figure 2-8 shows the chiral carbon in 3-D as the L isomer Protein

consist of twenty different amino acids differentiated by their side-chain groups [Norde

1998] The side-chain groups have different chemical properties such as polarity charg

and size and influence the chemical properties of proteins as well as determine the

overall structure of the protein For instance the polar amino acids tend to be on the

outside of the protein when they interact with water and the nonpolar amino acids are

the inside forming a hydrophobic core

Figure 2-8 L-α-amino acid

The covalent linkage between two amino acids is known as a peptide bond A

peptide bond is formed when the amino group of one amino acid reacts with the carboxyl

group of another amino acid to form an amide bond through the elimination of water

This arrangement gives the protein chain a polarity such that one end will have a free

amino group called the N-terminus and the other end will have a free carboxyl group

called the C-terminus [Solomon and Fryhle 2000] Peptide bonds tend to be planar and

32

give the polypeptide backbone rigidity Rotation can still occur around both of the α-

carbon bonds resulting in a polypeptide backbone with different potential conformations

relative to the positions of the R groups Although many conformations are theoretically

possible interactions between the R-groups will limit the number of potential

nd to form a single functional conformation In other words

the conformation or shape of the protein is

α-helix

can interact with other

secondary structures within the sam

conformations and proteins te

due to the interactions of the chain side

groups with one another and with the polypeptide backbone The interactions can be

between amino acids that are close together as in a poly-peptide between groups that are

further apart as in amino acids or even on between groups on different polypeptides all

together These different types of interactions are often discussed in terms of primary

secondary tertiary and quaternary protein structure

The primary amino acid sequence and positions of disulfide bonds strongly

influence the overall structure of protein [Norde 1986] For example certain side-chains

will promote hydrogen-bonding between neighboring amino acids of the polypeptide

backbone resulting in secondary structures such as β-sheets or α-helices In the

conformation the peptide backbone takes on a spiral staircase shape that is stabilized by

H-bonds between carbonyl and amide groups of every fourth amino acid residue This

restricts the rotation of the bonds in the peptide backbone resulting in a rigid structure

Certain amino acids promote the formation of either α-helices or β-sheets due to the

nature of the side-chain groups Some side chain groups may prevent the formation of

secondary structures and result in a more flexible polypeptide backbone which is often

called the random coil conformation These secondary structures

e polypeptide to form motifs or domains (ie a

33

tertiary structure) A motif is a common combination of secondary structures and a

domain is a portion of a protein that folds independently Many proteins are composed o

multiple subunits and therefore exhibit quaternary structures

271 Interaction Forces between Proteins

Proteins in aqueous solution acquire compact ordered conformations In such a

compact conformation the movement along the polypeptide chain is severely restrict

implying a low conformational entropy The compact structure is possible only if

interactions within the protein molecule and interactions between the protein molecule

and its environment are sufficiently favorable to compensate for the low conformational

entropy [Malmsten 1998] Protein adsorption study is often focused on structural

rearrangements in the protein molecules because of its significance to the biological

functioning of the molecules and the important role such rearrangements play in the

mechanism of the adsorption process Knowledge of the majo

f

ed

r interaction forces that act

structures helps to understand the behavior

of pro

arge

a

ded

between protein chains and control the protein

teins at interface These forces include Coulomb interaction hydrogen bonds

hydrophobic interaction and van der Waals interactions

Coulombic interaction Most of the amino acid residues carrying electric ch

are located at the aqueous boundary of the protein molecule An the isoelectric point

(IEP) of the protein where the positive and negative charges are more or less evenly

distributed over the protein molecule intramolecular electrostatic attraction makes

compact structure favorable to proteins Deviation to either more positive or more

negative charge however leads to intramolecular repulsion and encourages an expan

structure Tanford [1967] calculated the electrostatic Gibbs energy for both a compact

impenetrable spherical molecule (protein) and a loose solvent-permeated spherical

34

molecule (protein) over which the charge is spread out From the results he found th

the repulsion force was reduced at higher ionic strength d

at

ue to the screening action of ion

ble

e

the

d the

tween

dehyd

e

ent

oles

tein

Hydrogen bond Most hydrogen bonds in proteins form between amide and

carbonyl groups of the polypeptide backbone [Malmsten 1998] The number of availa

hydrogen bonds involving peptide units is therefore far greater than that involving sid

chains Because α-helices and β-sheets are aligned more or less parallel to each other

interchain hydrogen bonds enforce each other Kresheck and Klotz [1969] examine

role of peptide-peptide hydrogen bonds and concluded that hydrogen bonds between

peptide units do not stabilize a compact structure of protein However because the

peptide chain is shielded from water due to other interactions hydrogen bonding be

peptide groups do stabilize α-helical and β-sheet structures

Hydrophobic interaction Hydrophobic interaction refers to the spontaneous

ration and subsequent aggregation of non-polar components in an aqueous

environment In aqueous solutions of proteins the various non-polar amino acid residues

will be found in the interior of the molecule thus shielded from water The

intermolecular hydrophobic interaction for the stability of a compact protein structure

was first recognized by Kauzmann [1959] If all the hydrophobic residues are buried in

the interior and all the hydrophilic residues are at the outermost border of the molecul

intramolecular hydrophobic interaction would cause a compact protein structure

However geometrical and other types of interactions generally cause a fraction of the

hydrophobic residues to be exposed to the aqueous environm

Van der Waals interaction The mutual interaction between ionic groups dip

and induced dipoles in a protein molecule cannot be established as long as the pro

35

structure is not known in great detail Moreover the surrounding aqueous medium also

contains dipoles and ions that compete for participation in the interactions w

the protein molecule Dispersion interactions favor a compact structure However

because the Hamaker constant for proteins is only a little larger than that of water the

resulting effect is relatively small [Nir 1977]

272 β

ith groups of

rgiles et al 1996

Floeg

) is degraded [Floege 2001] Renal

β2M from the serum resulting in an increase in β2M

conce well

the

)

a

al

2-Microglobulin (β2M)

The protein β2M is of particular interest because it is involved in the human

disorder dialysis-related amyloidosis (DRA) [Geiyo et al 1985 A

e 2001] DRA is a complication in end stage renal failure patients who have been on

dialysis for more than 5 years [Bardin et al 1986 Drueke 2000] DRA develops when

proteins in the blood deposit on joints and tendons causing pain stiffness and fluid in

the joints as is the case with arthritis In vivo β2M is present as the non-polymorphic

light chain of the class I major histocompatibility complex (MHC-I) As part of its

normal catabolic cycle β2M dissociates from the MHC-I complex and is transported in

the serum to the kidney where the majority (95

failure disrupts the clearance of

ntration by up to 60-fold [Floege 2001] By a mechanism that is currently not

understood β2M then self-associates into amyloid fibrils and typically accumulates in

musculoskeletal system [Homma et al 1989] Analysis of ex vivo material has shown

that the majority of amyloid fibrils in patients with Dialysis Related Amiloidosis (DRA

is present as of full-length wild-type β2M although significant amounts (~20-30) of

truncated or modified forms of the protein are also present [Floege 2001 Bellotti et al

1998] Figure 2-9 shows the ribbon diagram of human β2M Native β2M consists of

single chain of 100 amino acid residues and has a seven stranded β-sandwich fold typic

36

of the immunoglobulin superfamily [Saper et al 1991 Trinh et al 2002] β2M was fi

isolated from human urine and characterized by Berggard et al [1980] in1968 The

normal serum concentration of β

rst

re

f

e

2M is 10 to 25 mgL It is a small globular protein with

a molecular weight of 118 kDa a Strokes radius of 16Aring and a negative charge under

physiological conditions (isoelectric point IEP = 57) β2M contains two β-sheets that a

held together by a single disulphide bridge between the cysteines in positions 25 and 81

[Berggard et al 1980 Parker et al 1982 Cunningham et al 1973] β2M cannot be

removed completely by current dialysis techniques but through a better understanding o

the structure and interaction forces that lead to this structure it will be possible to mor

efficiently remove this problematic protein

Figure 2-9 Ribbon diagram of human β M taken from the crystal structure of the protein

bound to the heavy chain of the MHC class I complex (PDB 1DUZ) [Khal 2000]

273 Serum Albumin

Serum albumin is the most abundant protein found in plasma and is typically

present in the blood at a concentration of 35~

2an et

50gL According to extensive studies about

its physiological and pharmacological properties albumin has a high affinity to a very

wide range of materials such as electrolytes (Cu+2 Zn+2) fatty acids amino acids

metabolites and many drug compounds [Fehske et al 1981 Kraghhansen 1981 Putnam

37

1984 Peters 1985] The most important physiological role of the protein is therefore to

bring such solutes in the bloodstream to their target organs as well as to maintain the pH

and osmotic pressure of the plasma Bovine serum albumin (BSA) is an ellipsoidal

protein with the dimensions of 140 X 40 X 40Aring [Peter 1985] The primary structure is a

single helical polypeptide of 66 kDa (IEP = 47) with 585 residues containing 17 pairs of

as a soft an

rfaces [Kondo et al 1991 Norde et al 1992 Soderquist et al

1980 ikely

is

disulfide bridges and one free cysteine [Dugaiczyk et al 1982] BSA has been classified

d flexible protein because it has a great tendency to change its conformation

on adsorption to solid su

Carter and Ho 1994] and consists of three homologous domains (I-III) most l

derived through gene multiplication [Brown 1976] Each domain is composed of A and B

sub-domains [He et al 1992] The secondary structure of human serum albumin (HSA)

shown in Figure 2-10

Figure 2-10 Secondary structure of human serum albumin (HSA) with sub-domains

[Zunszain et al 2003]

HSA has the same structure domains with the serum albumin from other species

such as BSA [Brown 1976] Although all three domains of the albumin molecule have

similar three-dimensional structures their assembly is highly asymmetric [Sugio et al

38

1999] Domains I and II are almost perpendicular to each other to form a T-shaped

assembly in which the tail of subdomain IIA is attached to the interface region between

sub-domains IA and IB by hydrophobic interactions and hydrogen bonds In contrast

domain III protrudes from sub-domain IIB at a 45ordm angle to form the Y-shaped assembly

for domains II and III Domain III interacts only with sub-domain IIB These features

make the albumin molecule heart-shaped

28 Protein Adsorption

Protein adsorption studies date back to the 1930rsquos At the beginning these studies

mainly focused on the determination of the molecular weight electrophoretic and

ral

rearrangem

adsorption and biocompatibility of the sorbent materials were investigated [Norde 1986]

There are interaction forces at the interfaces between protein molecules and latex

particles These forces are mainly divided into the following groups hydrophobic

interaction ionic interaction hydrogen bonding and van der Waals interaction [Andrade

1985]

Hydrophobic interaction It is known that hydrophobic interaction has a major

role in protein adsorption phenomena The adsorption of proteins on the low charged

latex particles occurs by this interaction force Generally monomers such as styrene offer

hydrophobic surfaces th

by this interaction force is maximum at the isoelectric point (IEP) of the protein and the

strength [Suzawa et al 1980 1982 Shirahama et al 1989 Kondo et al 1992] By the

chromatographic applications Later the adsorption mechanism especially the structu

ents was studied Recently the studies of the relation between protein

281 Interaction between Protein Molecule and Latex Particle

at protein molecules adsorb to The amount of adsorbed protein

pH at maximum adsorption shifts to a more acidic region with an increase in ionic

39

reports [Suzawa et al 1980 1982 Lee et al 1988] protein adsorption was greater

hydrophobic surface than on a hydrophilic one implying that hydrophobic interaction is

on a

s

ydrophilic compound such as acrylic acid (AA) or methacrylic acid (MAA)

Ionic

ely

t

ces

n force is operative over small

distan e

ble

n

nt dialysis membrane therapy for end stage renal disease (ESRD)

one of most dominant forces in protein adsorption

Ionic interaction Negatively charged latex particles have ionic functional

groups on their surfaces such as salts of sulfonic and carboxylic acid Sulfate group

originate from an initiator such as sodium persulfate and carboxylic groups originate

from a h

bonds are formed between the negative charges of these latex particles and positive

surface charges of protein molecules The conventional low-charged latex particles rar

form these ionic bonds

Hydrogen bonding Hydrogen bond is a strong secondary interatomic bond tha

exists between a bound hydrogen atom (its unscreened proton) and the electrons of

adjacent atoms [Callister 1999] Protein can be adsorbed on hydrophilic polar surfa

through hydrogen bonding Hydrogen bonds are frequently formed between hydroxyl-

carbonyl or amide-hydroxyl Hydroxyl-hydroxyl or amide-hydroxyl bonds are also

formed in protein adsorption

Van der Waals interaction This interactio

ces only when water has been excluded and the two non-polar groups come clos

to each other Lewinrsquos calculation showed that the van der Waals interaction is negligi

compared with the forces involved in the entropy increases ie hydrophobic interactio

[Lewin 1974]

29 Hypothesis for Toxin Removal

As mentioned earlier insufficient removal of middle molecular range toxins is a

major drawback of curre

40

patien

is

y

ese

haracteristics over the last decade However removal

effici

ing a novel membrane design composed of an assembly of engineered

polymeric latex particles s ions Important factors

will i

e

ith a

is established it is

expec

e

ts This may cause destructive arthritis and carpal tunnel syndrome inhibit the

immune system and accelerate cardiovascular disease leading to death among dialys

patients In order to overcome these complications artificial dialysis membranes have

been developed to increase the capacity for removing middle molecular weight toxins b

changing the pore size of dialysis membranes and using new materials that adsorb th

toxins for improved removal c

ency is not as high as those achieved by a normal healthy kidney

With the knowledge obtained from all above literatures the following hypothesis

has been established for the development of a membrane for the successful removal of

the target protein β2-Microglobulin (β2M) without the removal of serum albumin This

will be done us

ynthesized to predetermined specificat

nclude pore size and surface chemistry

291 Toxin Removal by Size Sieving Based on Monodispersed Pore Size

The packing of monodispersed spherical particles can lead to the formation of

porous layers suitable for use as filters and membranes [Hsieh et al 1991] The pore siz

is defined as the largest spherical particles that can pass through the interstitial spaces

It is well known that monodispersed spherical particles can be obtained from the

seed emulsion polymerization method Many commercially available latex particles are

synthesized by this technique and are inexpensive When the defect free membrane w

monodisperse pore size distribution based on a particular particle array

ted to outperform the traditional hemodialysis membrane limitation which is a

broad range of pore size distribution When spherical particles pack in regular crystallin

arrays a number of packing geometries such as hexagonal closest packing cubic closest

41

packing and body-centered cubic packing are possible The pore size of the array

depends on these packing geometries as well as particle size Theoretical pore size can be

calculated from the simple geometry of the arr

ay Figure 2-11 shows the relationship

between particle size and pore size

0001

001

01

001 01 1 10

Por

e

1

10

microm

) S

ize

(

Particle Diameter (microm)

Body-Centered Cubic ArrayClosest Packed Array

0001

Siz

e (

Particle Diameter (microm)

001

01

001 01 1 10

Por

e

1

10

microm

)

Body-Centered Cubic ArrayClosest Packed ArrayBody-Centered Cubic ArrayClosest Packed Array

and closed packed arrays [Steve et al 1999]

McDonald and his coworkers [Ramakrishnan et al 2004 Steve et al 1999] have

described the fabrication of latex composite membranes and their performance in

previous papers It is from this prior research and knowledge gained from conversations

with Dr McDonald that this current research has been initiated [McDonald 2003] The

focus of this thesis is the extension of McDonaldrsquos research in the area of composite

membranes to medical application such as dialysis membranes

292 Toxin Removal by Selective Adsorption on Engineered Latex Particles

The flexibility in latex surface properties is a significant advantage to this

materialrsquos ability to separate proteins since the selectivity is strongly dependent on the

charge interactions of both the protein and the latex particles under the conditions of

Figure 2-11 The relationship between pore size and particle size in body-centered cubic

42

separation [Menon et al 1999 Chun et al 2002] First all sorbents were designed to

have negative surface properties because most plasma proteins in blood are negative and

should not be removed with charge interaction between proteins and sorbents at

physiological condition (pH74) It was reported that a negatively charged surface was

more blood compatible than a positive one [Srinivasan et al 1971] The toxin protein β2-

Microglobulin (β2M) can be selectively adsorbed on the surface of latex particles by the

design of a suitable hydrophobic portioned surface where only β2M protein can be

anchored with hydrophobic interactions Albumin adsorption on the latex particle is not

side-on m

adsorption of β M protein on the engineered latex particles

allowed because charge repulsion is more dominant than hydrophobic interaction with

ode The figure 2-12 shows the schematic representation of the selective

2

PMMAPAAShell

COO-

OSO3-

OSO3-

PSCore

COO-

-

-

-

-

-

-

Albumin-

Chargerepulsion

--

-

- -

- -

- --

Hydrophobic

β2-Microglobulin

2

Interaction

β -Microglobulin

PMMAPAAShell

COO-

-

-

-

-

-

-

--

-

-

-

-

-

Albumin-

Chargerepulsion

OSO3-

OSO3-

PSCore

COO-

--

-

- -

--

-

- -

- -

- --

- -

- --

Hydrophobic

β2-Microglobulin

2

particles at pH 74

Interaction

β -Microglobulin

Figure 2-12 Schematic representation of the protein adsorption on the core shell latex

43

Hydrophilichydrophobic microdomain structures were proven to be more blood

compatible [Mori et al 1982 Higuchi et al 1993 Deppisch et al 1998] The

optimization of suitable hydrophobic to hydrophilic ratio is also important for the

biocompatibility The monomers such as styrene (St) methyl methacrylate (MMA) and

acrylic acid (AA) are widely used hydrophobic and hydrophilic monomer models in

emulsion polymerization process

In summary the background and fundamental literature survey about the history

material properties and limitation of hemodialysis membrane latex particle preparation

surface chemistry and manufacturing process target proteins and protein adsorption and

finally the hypothesis for a toxin removal with high separation efficiency have been

suggested This research focuses on such hypothesis and the materials and

characterization methodology for achievement of suggested hypotheses are described in

the next chapter

CHAPTER 3 EXPERIMENTAL AND CHARACTERIZATION METHODOLOGY

As mentioned earlier the goal of this study is to prepare polymeric latex particles

with tailored properties to maximize separation of toxin molecules and to investigate

fundamental interactions between the applied particles and molecules in the

system to optimize the membrane performance for hemodialysis applications Polymeric

latex particles wit

the

biological

h monodisperse size distribution to obtain uniform pore size and

various size ranges to utilize membrane construction are necessary Surface engineering

with various combination of hydrophobichydrophilic domain on the surface of latex

particles is expected to affect the removal of target protein by selective adsorption and to

improve the biocompatibility of membrane Therefore the materials and characterization

methodology are addressed in this chapter

31 Materials

Styrene (St) monomer used for a seed and a core particle was purchased from

Fisher Scientific and used without any other purification process Acrylic acid (AA) and

methyl methacrylate (MMA) monomers introduced for shell formation were purchased

from Fisher Scientific and used without any other purification process Sodium persulfate

(SPS) and sodium bicarbonate (SBC) were obtained from Fishers Scientific and used as

received Divinylbenzene (DVB) crossliking agent was purchased from Aldrich The

anionic surfactant Aerosol regMA80-I [sodium di(13-dimethylbutyl) sulfosuccinate] was

kindly donated by Cytec 78-80 Aerosol regMA80-I is mixed with isopropanol and

water Its critical micelle concentration (CMC) is 245 mM (119) Ion exchange resin

44

45

AGreg 501-x8 Resin (20-50 mesh) was purchased from Bio-Rad Laboratories Inc It is a

mixed-bed resin with H+ and OH- as the ionic forms and is 8 crosslinked Bovine serum

albumin (BSA) (heat shock treated) was purchas Fisher Scientific The isoelectric

point of t lin

(β2M)

as

gma

ed from

he BSA is 47-49 and the molecular weight is 66300Da β2-Microglobu

was purchase from ICN Biomedicals Inc and was separated from patients with

chronic renal disease and lyophilized from ammonium bicarbonate The molecular

weight by SDS-PAGE was approximately 12000Da BSA and β2M proteins were used

received without further purification process In order to investigate the protein

adsorption evaluation bicinchoninic acid protein assay kits were purchased from Si

(Cat BCA-1) and Pierce Biotechnology (Cat 23225) The chemical structures for the

main chemicals are shown in Figure 3-1

CH2 CH

Styrene (Mw = 10415)

CH2 CHCH2 CH

Styrene (Mw = 10415)

CH2 CH

Acrylic acid (Mw = 7206)

C

OH

O

CH2 CH

Acrylic acid (Mw = 7206)

C

OH

O

CH CH2

C

OCH3

O

CH

CH CH3

2

C

OCH3

O

CH3

Methyl methacrylate (Mw = 10012)Methyl methacrylate (Mw = 10012)

CH2 CH

Divinylbenzene (Mw = 1302)

CH CH2

CH2 CH

Divinylbenzene (Mw = 1302)

CH CH2CH CH

+(Na) -(O3S)

CH -C-O-CH-CH -CH-CH3

O

2 2 3

O CH CH3

CH3 CH3

CH-C-O-CH-CH2-CH-CH3

Anionic surfactant (MA80-I) (Mw = 3885)

+(Na) -(O3S)

CH -C-O-CH-CH -CH-CH3

O

2 2 3

O CH CH3

CH3 CH3

CH-C-O-CH-CH2-CH-CH3

Anionic surfactant (MA80-I) (Mw = 3885)2

+(Na)-(O) S OO

O O

- +

Initiator sodium persulfate (Mw = 23811)

O SO

(O) (Na)+ -

O O

- +(Na) (O) S OO

O SO

(O) (Na)+ -

O O

- +

Initiator sodium persulfate (Mw = 23811) Figure 3-1 Chemical structure of main chemicals

(Na) (O) S OO

O SO

(O) (Na)

46

32 Latex Particle Preparation

The experimental set up and the particle preparation schemes for various types and

size ranges of latex particles are shown in Figure 3-2 and 3-3 In the experimental setup a

mechanical glass stirrer is connected from a motor and Teflon stirrer bar is located at

end of glass stirrer The agitation rate is precisely adjusted by controller motor Paar glas

reactor with 1L volume is partially in silicon oil bath on hot plate The reactor has fo

openings for a stirrer a thermometer a nitrogen gas inlet and a reactant feeding lnlet

The reactants for latex polymerization are fed by precisely controlled by meterin

The particle preparation is described in each latex preparation section

the

s

ur

g pump

Figure 3-2 Experimental setup for semi-continuous emulsion polymerization

1 Motor 2 Mechanical stirrer bar 3 Thermometer 4 Inlet of N2 5 Inlet for initiator and monomer 6 Glass reactor (l L) 7 Oil bath 8 Hot plate 9 Pump for initiator 10 Pump for monomer 11 Tubes for reactant feed

47

Styrene monomer

Bare PS

PSPMMA100

PSPMMA90PAA10

PSPMMA P75 AA25

Gr

win

ticle

size

PS seedparticles

og

par

Styrene monomer

Bare PS

PSPMMA100

PSPMMA90PAA10

PSPMMA P75 AA25

Gr

win

ticle

size

PS seedparticles

og

par

Figure 3-3 The particle preparation scheme in various types and size ranges of latex particles

321 Preparation of Seed Latex Particles

Polystyrene seed particles were synthesized through a typical semi-continuous

emulsion polymerization Polystyrene latex particles were prepared in a four necked Paar

glass vessel equipped with a mechanical glass stirrer thermometer glass funnel for

nitrogen gas purge and a tube for monomer feeding The reactor was placed in a silicon

oil bath on a hot plate for homogenous and stable tempe he vessel was

firstly charged with de-ionized water the emulsifier so itiator

sodium bicarbonate (SBC) styrene (St) monomer divinylbenzene (DVB) monomer

under nitrogen gas atmosphere and then heated to 72plusmn2 as

llowed to continue for another hour Second reactants St and DVB monomers SPS

initiator and SBC were fed separately using fine fluid metering pumps (Model RHSY

Fluid metering INC NY) under a nitrogen gas atmosphere for 2h After complete feeding

rature control T

dium persulfate (SPS) in

oC for 1h The reaction w

a

48

of all reactants the reaction was continued for another hour at elevated temperature 80plusmn1

oC and then cooled to room temperature by removal of the oil bath

322 Preparation of Seeded Latex Particles

The particle growth was achieved by seeded continuous emulsion polymerization

The vessel was charged with the polystyrene seeds and de-ionized water to make desired

solid content and heated up to 72plusmn2 oC for 1h under nitrogen gas atmosphere Then St

and DVB monomers and solution of SPS and SBC in de-ionized water were continuously

added using metering pump with a precisely controlled feed rate After feeding all

reactants the reaction was continued for three hours keeping temperature of 80plusmn2 oC and

then the reaction vessel was cooled down to room temperature

Monodisperse spherical polystyrene seeds with a 280nm mean diameter were used

e-polymerized polystyrene seeded emulsion

was charged in a 500m

323 Preparation of Core Shell Latex Particles

for core shell structured latex particle The pr

l glass flask under a nitrogen gas atmosphere De-ionized water

was added to achieve the desired solid content The emulsion was heated to 80plusmn2oC for

10 hour Monomers initiator and SBC to form the shell were added by fluid metering

pumps (LAB PUMP JRRHSY model Fluid metering Inc USA) with fine controlled

feed rate Flow rate can be adjusted by control ring graduated in 450 division from 0 to

100 flow which is in the range of 10mlmin respectively The reaction was sustained

for additional 30 hours at 90plusmn2oC then cooled down to room temperature

324 Purification of Synthesized Latex Particles

Synthesized latex particles were purified with an ion exchange resin 200g (10

ww polystyrene solid content) of emulsion were mixed with 4g of ion exchange resin

and stirred for 40 minute to remove unreacted monomers initiators and other impurities

49

Then the emulsion was diluted with DI water to a specific degree of solid content as

needed

33 Characterization

331

solid

termined

oscopy

d

hours

Dried latex (10mg) was mixed with

250 m on

Degree of Conversion

The degree of monomer to polymer conversion was determined gravimetrically

following the procedure described by Lee et al [1995] before the latex cleaning process

The synthesized emulsion raw materials were weighed and dried in a conventional oven

at 120oC for 30min to evaporate any unreacted monomers and water The remaining

was weighed and the degree of conversion was determined This process was repeated

three times and a mean value of the degree of conversion was de

332 Fourier Transform Infrared (FTIR) Spectr

A molecule can absorb only select frequencies (energy) of infrared radiation which

match the natural vibration frequencies The energy absorbed serves to increase the

amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]

Only those bonds which have a dipole moment that changes as a function of time can

absorb the infrared radiation The range of wavelengths for infrared radiation is between

25 microm (4000 cm-1) and 25 microm (400cm-1)

The synthesis of the latex particles such as polystyrene (PS) homopolymer and

PSPMMA PSPMMA90PAA10 PSPMMA75PAA25 core-shell copolymers were verifie

with FTIR Purified latex particles were dried in vacuum oven at 40oC for 24

before analysis in the FTIR (Nicolet Magma USA)

g of KBr which had also been dried in vacuum oven at 120oC for 3h Transmissi

spectra using the drift mode were plotted with 128 scans and 4cm-1 resolution

50

333 Quasielastic Light Scattering (QELS)

Latex particle size was measured by the Brookhaven ZetaPlus particle size

analyzer The solid concentration as 0001 (wv) This

ation spectroscopy (PCS) of quasielastically scattered light

For th s

ed using a FE-SEM (JEOL

JSM-

ith

ent

01

us zeta-

potential analyzer It measures the electrophoretic mobility velocity of charged colloidal

of latex particles used w

instrument uses photon correl

is technique time dependent interference patterns of light scattered from particle

in the sample cell are analyzed The interference pattern changes due to Brownian

motion giving rise to fluctuations in scattering intensity The frequency of these

fluctuations depends on the particlersquos diffusion constant that is inversely proportional to

the particle diameter

334 Field Emission-Scanning Electron Microscopy (FE-SEM)

Particle size and surface morphology were characteriz

6335F Japan) A diluted suspension of latex particles in water was dropped onto a

silicon wafer at room temperature and allowed to dry The sample was then coated w

the thinnest layer of carbon needed to obtain the required conductivity for this instrum

Secondary electron image mode was used with a 15KV of accelerating voltage The

magnification range used was between 10000X and 70000X

335 Zeta Potential Measurement

Synthesized and purified latex particles were diluted with de-ionized water to 0

wt and adjusted six different pH values 205 345 481 565 643 and 747 These

particle suspensions were then transferred to standard cuvettes for zeta potential

measurement At least two runs of ten measurements were taken for each sample and

averaged

The zeta potential measurement was carried out using Brookhaven ZetaPl

51

particles in solution and calculate zeta-potential by using the Smoluchowski equation

The f quency

gnitude

umin (BSA) as a standard model protein and β2-Microglobulin

uffer (PB) and

phosp

5

h the selected protein

e 005 01 03 05 and 07 mgml and those for

β2M

d at

teins in the supernatant after the centrifugation process using the

bicinc t

requency of laser light passing through the sample and is compared to the fre

of a reference beam The shift in the frequency called a Doppler shift and the ma

of the shift correspond to the polarity and the magnitude of the electrophoretic mobility

respectively The zeta potential is calculated from the solution conditions and the

measured mobility

336 Protein Adsorption

Protein adsorption experiments of the synthesized latex particles were performed

with bovine serum alb

(β2M) as a target protein in two types of buffer solution phosphate b

hate buffered saline (PBS) To make 5 mM of PB solution 0345g of sodium

phosphate monobasic (NaH2PO4H2O) was dissolve in 500ml of de-ionized water To

make PBS solution 0345g of sodium phosphate monobasic (NaH2PO4H2O) and 4178g

(143mM) of sodium chloride (NaCl) were dissolved in 500ml of de-ionized water The

synthesized latex particles were diluted with each buffer to have a solids content of 0

(ww) adjusted to pH values of 32 48 and 74 and mixed wit

BSA concentrations were chosen to b

were 0015 0030 0045 and 0060mgml The mixture was gently rotated using

the shaker Labquakereg (BarnsteadThermolyne Model 4002110 USA) with 8 RPM in

the incubator at 37oC for 12 hours before the latex-protein mixture was centrifuge

13000 rpm for 15min The amount of protein adsorbed was determined by quantifying

the free pro

honinic acid (BCA) assay method [Lowry et al 1951 Smith et al 1985 Baptista e

al 2003 Wiehelman et al 1988 Brown et al 1989] BCA assay kits consist of Reagent

52

A containing bicinchoninic acid sodium carbonate sodium tartrate and sodium

bicarbonate in 01N NaOH with pH=1125 and Reagent B containing 4 (wv) copper

(II) sulfate pentahydrate The BCA working reagent was prepared by mixing 50 parts of

Reagent A with 1 part of Reagent B 100microl of protein supernatant was then mixed with

2ml of BCA working reagent in a UV cuvette Incubation was allowed to continue

at room temperature

further

until color developed about 2h The absorbance at 562nm was

pectrophotometer (Perkin-Elmer Lambda 800 USA)

Unkn

Separate the p rticles in centrifuge mix supernatant with BCA reagents

measured using a UV-VIS s

own concentration of sample protein was determined by comparison to a standard

of known protein concentrations The adsorbed amount per unit surface area was

determined by the mass balance of the protein after adsorption process The simple

schematic of the procedure for a protein adsorption test is shown in Figure 3-4

Incubate mixture with agitation for 12hrs at 37oC

aand develop the color by incubation for 2hrs at room temperature

Suspend latex particles in aqueous buffer media

Add the dissolved target protein

Measure color intensity using UVVis spectroscopy at 562nm

Suspend latex particles in aqueous buffer media

Separate the p rticles in centrifuge mix supernatant with BCA reagents

Add the dissolved target protein

Incubate mixture with agitation for 12hrs at 37oC

aand develop the color by incubation for 2hrs at room temperature

Measure color intensity using UVVis spectroscopy at 562nm

Figure 3-4 Schematic of the procedure for a protein adsorption test

53

337 Blood Biocompatibility by Hemolysis Test

Hemolysis is the destruction of red blood cells which leads to the release of

hemoglobin from within the red blood cells into the blood plasma Hemolysis testing is

used to evaluate blood compatibility of the latex particles and the damaged to the red

blood cells can be determined by monitoring amount of hemoglobin released The red

blood cells (RBCs) were separated by centrifuging blood at 1500 rpm for 15 minutes

Figure 3-5 shows the phase separated blood with plasma white blood cells and red

cells after centrifugation process Separated RBSs were then washed with isotonic

phosphate buffer solution (PBS) at pH 74 to remove debris and serum protein Th

process was repeated 3 times

blood

is

Figure 3-5 Separation of RBC from whole blood by centrifuge process

Prepared latex particles were re-dispersed in PBS by sonification to obtain

homogeneously dispersed latex particles 100microl of the mixture of red blood cell (3 parts)

and PBS (11parts) was added to 1ml of 05 (ww) particle suspension PBS was used as

a negative control resulting 0 hemolysis and DI water used as a positive control to

produce 100 hemoglobin released by completely destroyed RBCs The mixture was

incubated in water bath with gentle shaking for 30 minutes at 37oC and then centrifuged

at 1500 rpm for 15 minutes 100microl of the supernatant was mixed with 2ml of the mixture

) (EtOHHCl = 2005 ww) to prevent the

White blood cells

Plasma

Red blood cells

of ethanol (99) and hydrochloric acid (37

54

precipitation of hemoglobin In order to remove remaining particles the mixture was

centri fuged again with 13000 RPM at room temperature The supernatant was then

transferred into the UV cuvette The amount of hemoglobin release was determined by

monitoring the UV absorbance at a wavelength of 397nm The schematic of the

procedure for hemolysis test is shown in Figure 3-6

Separate red blood cells (RBCs) from blood

Mix RBCs with phosphate buffer solution (PBS)

Wash and re-suspend RBC with PBS repeat three times

Add la l of RBC and incu in at 37oC

tex particle suspension into 100mbate sample with agitation for 30m

Separate particle ith EtOHHCl solutions and mix the supernatant w

Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm

Separate red blood cells (RBCs) from bloodSeparate red blood cells (RBCs) from blood

Mix RBCs with phosphate buffer solution (PBS)Mix RBCs with phosphate buffer solution (PBS)

Wash and re-suspend RBC with PBS repeat three timesWash and re-suspend RBC with PBS repeat three times

Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC

Add latex particle suspension into 100ml of RBC and incubate sample with agitation for 30min at 37oC

Separate particles and mix the ith EtOHHCl solution supernatant wSeparate particles and mix the ith EtOHHCl solution supernatant w

Evaluate free hemoglobin in the solution using UVVis spectroscopy at 397nmEvaluate free hemoglobin in the solution using UVVis spectroscopy at 397nm

Figure 3-6 Schematic of the procedure for hemolysis test

In summary a description of the experimental materials and characterization

methodology has been explained In the next chapter the results and discussion of the

data obtained from these experiments is addressed

CHAPTER 4 RESULTS AND DISCUSSION

41 Polymerization of Latex Particles

411 Polystyrene Seed Latex Particles

Polymer particles have many different applications including spacer particles

lubricants packing materials for chromatography standard particles and diagnostic

drugs All applications strongly require these particles to have a uniform particle size

Uniform particle size is the first requirement for a latex composite membrane formed by

particle arrays with the interstitial spaces serving as pores for size discriminations [Jons

et al 1999 Ramakrishnan et al 2004] Although suspension polymerization has been

mainly used as one of conventional polymerization methods to prepare uniform particles

the uniformity of the recovered particles is insufficient for membrane use There is

another method known as seed emulsion polymerization where a vinyl monomer is

absorbed into fine monodiserse seed particles and polymerization causes the monomer to

crease the sizes of the seed particles uniformly In order to obtain monodisperse larger

particles by this m

particles and polymerization of the monomer is repeated

In this study cross-linked polystyrene seed latex particles were firstly synthesized

in the presence of styrene monomer divinylbenzene crosslink agent sodium persulfate

initiator sodium bicarbonate buffer and the Aerosol regMA80-I [sodium di(13-

dimethylbutyl) sulfosuccinate] surfactant using a batch type semi-continuous emulsion

polymerization process in which one or more components can be added continuously

in

ethod the procedure of absorption of monomer into fine polymer

55

56

Styrene (St) monomer and divinylbenzene (DVB) crosslink agent as well as initiator and

buffer are continuously added to enlarge the seed latex particles Various profiles of

particle nucleation and growth can be gene different orders of component

addition during polymerizati f this process such as easy

control of the polymeriza particle number

and particle morphology The main goal in

this process is to avoid secondary particle

rated from

on There are several advantages o

tion rate monodisperse particle size and

colloidal stability copolymer composition

formation leading to a monodisperse particle

size distribution Figure 4-1 shows the schematic representation of seed latex particle

preparation and growth

Monomer droplet

Seed particles Amount of surfactantsis variable

Enlarged final Add monomers

PS

PS

PSs4

particles Number of particlesare constant

Seed code

PSs1

s2

s3

Monomer droplet

Seed particles Amount of surfactantsis variable

Enlarged final Add monomers

PS

PS

PSs4

tion The

particles Number of particlesare constant

Seed code

PSs1

s2

s3

Figure 4-1 Schematic representation of semi-continuous seed latex particles preparation and growth

After the first synthesis of a small size of crosslinked polystyrene (PS) seed

particles stabilized by surfactants styrene DVB and initiator are continuously fed into

the system to enlarge the seed latex particles maintaining a narrow size distribu

recipes and reaction conditions for synthesizing these seed particles are summarized in

57

Table 4-1 The original recipe for latex particle preparation from McDonald was

modified for this emulsion system [McDonald 2003]

Table 4-1 The polymerization recipe of polystyrene (PS) seed particles Latex label PSS259 PSS233 PSS207 PSS181

Temperature (oC) 75 plusmn 5 75 plusmn 5 75 plusmn 5 75 plusmn 5

Divinylbenzene (g) 052 052 052 052

MA 80 (g) 259 233 207 181

NaHCO

Watera (g) 1315 1315 1315 1315

Styrene (g) 9948 9948 9948 9948

112 112 112 112

Na2S2O8 (g) 190 190 190 190

Waterb (g) 200 200 200 200

Reaction time (min) 140 140 140 140

Solids content 396 396 396 396

Mean particle diameter (nm) 126plusmn75 171plusmn39 182plusmn41 216plusmn53

Conversion () 965 979 957 971

3 (g)

Four different seed latex particles were synthesized based on surfactant amount in

order to determine the optimum condition for stable and monodisperse latex particles

The emulsion polymerization of latex particles has to be carried out in a narrow range of

surfactant causes flocculation or phase separation leading to broad particle

size distribution and an unstable emulsion sy

surfactant concentration where particles are stable [Nestor et al 2005] A wide range of

concentrations

stem Therefore the range of surfactant

amount was varied from 181g to 259g in increments of 026g in each latex label Other

reactants amounts were kept constant Watera is the initial charge amount and waterb

represents the amount of aqueous solution containing dissolved initiator and buffer

58

which are fed continuously after the seed particle creation The extent of monome

polymer conversion was obtained by gravimetric calcu

r to

lation and was more than 95 at

cripts such as

S207 and S181 i the surf mount added for the seed latex

nversion indi perce experi obtain

content divided by the theoretically estimated solid content and was calculated by the

all seed particles labeled PSS259 PSS233 PSS207 and PSS181 Labeled subs

S259 S233 ndicate actant a

preparation The co cates the ntage of mentally ed solid

following formula

100 colids () times=entsoliatedntentoalExperimentConversion

n the experimenta content is the weight of olid rema after

mulsion at 120oC f in and the estimated solid content is th ht

lated from the recipe

rti as di each t load d

mple of ea pension iluted by (ww)

deionized water and the mean particle diameter was determined using the Brookhaven

ZetaPlus particle size analyzer From these results the seed latex particle size increased

as the amount of surfactant decreased The amount of emulsifier also affects the

polymerization process by changing the number of micelles and their size It is known

that large amount of emulsifier produces larger numbers of smaller sized particles [Odian

1991] and was corroborated in the current work

These synthesized seed latex particles should be of narrow size distribution in order

to obtain monodisperse larger particles The SEM image showing the particle

morphology and size of a sample of polystyrene seed particles can be seen in the Figure

4-2

contdEstim (4-1)

In this equatio l solid the s ining

evaporation of e or 30m e weig

of monomers calcu

As expected the seed pa cle size w fferent for surfactan ing in see

emulsion system A sa ch sus was d 0001 with

59

Figure 4-2 Scanning Electron Micrograph (SEM) of polystyrene seed latex particles (A)

PSS259 (B) PSS233 (C) PSS207 (D) PSS181 (subscripts indicate the amount of surfactant added)

(A)

(B)

60

(C)

(D)

Figure 4-2 Continued

61

s seen in the image the seed particles are very smooth and spherical The

uniformity in the particle size distribution of these PS seed latex particles can also be

seen in the SEM image From this data we concluded that seed particles synthesized in

the presence of a surfactant with the chosen amounts are suitable for use to prepare larger

particles with high size uniformity and smooth spherical morphology

412 The Growth of Polystyrene (PS) Seed Latex Particles

Seeded emulsion polymerization has been conducted for several decades and

various mechanisms have been proposed Gracio et al [1970] suggested that the growing

polymer particles consist of an expanding polymer-rich core surrounded by a monomer-

rich shell with the outer shell providing a major locus of polymerization Seeded

mulsion polymerization is commonly used for preparing latex particles less than 1microm in

size [Cha et al 1995 Zou et al 1990 1992 Gandhi et al 1990 Park et al 1990] Such

latexes can be obtained from the growth of pre-prepared seed particles The seeds

introduced in this process serve as nucleation sites for particle growth By first swelling

the seed latex particles with additional monomer to the desired size polymerization can

then be initiated Several size ranges of monodisperse polystyrene (PS) latex particles

were prepared by this multistep seeded emulsion polymerization method For the

fabrication of particle-based membranes monodisperse latex particles with a variety of

size ranges are required

In this study seed particles from 171nm to 470nm in mean diameter and with a

highly uniform size distribution were prepared and used Watera is the initial charge

mount and waterb represents the amount aqueous solution containing dissolved initiator

and buffer that is continuously fed into system Feed time is precisely controlled by

metering pump The solid contents were maintained between 199 and 300 After

A

e

a

62

polym d as

es

ere 923 8402

n of these latex particles

are sh

ss

erization under these conditions the conversions of the latex particles labele

PS258 PS320 PS370 and PS410 were 952 957 946 and 964 respectively The recip

used in seeded emulsion polymerization to form the particles less than 500nm in mean

diameter are shown at the Table 4-2 The conversions of the latex particles larger than

500nm in mean diameter and labeled as PS525 PS585 PS640 and PS790 w

723 and 969 respectively The recipes used in the preparatio

own at the Table 4-3

A simple calculation can be used to predict the ultimate size that a latex particle can

reach after ending the polymerization Letrsquos suppose that seed density (ρs) and grown

particle density (ρgp) are the same The following equation is obtained from the ma

balance (m = ρV) where m and V are mass and volume of a particle respectively

gp

gp

s

s

Vm

Vm

In this equation m

= (4-2)

be

s and mgp are the weight of seed and total weight of seed and

added monomers respectively and Vs and Vgp are the volume of seed and grown

particle respectively

If we know the mass of monomer to be added the ultimate particle size can

calculated from the equation 4-3

3

2s D m ⎠⎝+ χ

where D

1s D m

⎟⎟⎞

⎜⎜⎛

= (4-3)

the

in

s and Dgp are the diameters for seed and grown particle respectively and χ is

weight of the monomer added Crosslinked PS latex particles with a size range from

258plusmn112 ~ 410plusmn117 in diameter polymerized by the recipes in Table 4-2 are shown

the SEM images in Figure 4-3

63

Table 4-2 Continuous addition emulsion polymerization recipe for growing polystyrene

Latex label PS

(PS) latex particles less than 500nm in size

258 PS320 PS370 PS410

Initial charge

Seed latex (g) 1000171 1000216 1000216 1000320

Watera (g) 1470 2500 1984 2600

06 07 06 06

Fee

Initiator stream

2 2 8

Feed time (min) 0-167 0-140 0-100 0-92

Solid content 284 300 300 200

Mean particle diameter (nm) 258plusmn112 320plusmn154 182plusmn71 410plusmn117

Conv

Monomer continuous

Styrene (g) 800 1200 800 600

Divinylbenzene (g)

d time (min) 0-163 0-110 0-75 0-82

Na S O (g) 07 065 05 05

NaHCO3 (g) 07 065 05 05

Waterb (g) 750 1200 200 370

ersion () 952 957 946 964 The solid content of seed latex for PS258 PS320 PS 370 and PS410 are 392 399 399 anrespectively

d 300

64

Table 4-3 Continuous addition emulsion polymerization recipe for growing polystyrene (PS) latex particles larger than 500nm in size

Latex label PS525 PS585 PS640 PS790

Initial charge

Seed latex (g) 1000390 1000470 1000470 1000320

Watera (g) 1000 520 1200 360

Monomer continuous

Styrene (g) 290 200 400 173

Divinylbenzene (g) 02 014

03 009

Feed time (min) 0-80 0-40 10-76 5-65

Initiator stream

Na2S2O8 (g) 02 014 03 012

NaHCO3 (g) 02 014 03 009

Waterb (g) 200 120 400 200

Feed time (min) 0-85 0-46 0-77 0-75

Solids content 199 200 200 201

Mean particle diameter (nm) 525plusmn151 585plusmn186 640plusmn178 790plusmn693

Conversion () 923 8402 723 969 The solid content of seed latex for PS525 PS585 PS 640 and PS790 are 200 182 193 and 173 respectively

65

Figure 4-3 SEM of PS latex particles less than 500nm in size (A) 258nm (B) 320nm (C)

370nm (D) 410nm

(A)

(B)

66

(C)

(D)

Figure 4-3 Continued

67

article size was well matched to that estimated by the geometrical calculation

From this it can be concluded that the added monomer was consumed for polymer

particle growth with a high degree of conversion From the SEM characterization newly

nucleated particles were not seen as indicated by the narrow particle size distribution

The particles less than 500nm in mean diameter were very spherical in shape with a

highly uniform particle size distribution As the particles were grown larger than 500nm

mean diameter however they became of non-spherical shape with an uneven surface

This irregularity of particle surface can be attributed to the non-homogeneous monomer

swelling into the shell of a growing polymer which can be controlled by the factors such

as temperature agitation speed initiator feeding rate and surfactant amount required to

stabilize the colloidal system The SEM image of latex particles larger than 500nm mean

iameter is shown in Figure 4-4 The particle size distribution is broader than that of the

s particles These particles can however still be used for membrane construction

as a support layer rather than skin layer The support layer does not necessarily have to be

as highly monodisperse as the skin layer which require pores of high uniformity for the

selective removal of toxins

As mentioned earlier the amount of surfactant as well as monomer in an emulsion

system is the primary determinant of the particle diameter [Odian 1991] Anionic

surfactants are generally recommended at the level of 02-30 wt based on the amount

of water [Odian 1991] The critical micelle concentration (CMC) of Aerosolreg MA 80-I

anionic surfactant is 119 The surfactant loading in this emulsion system ranged from

the 121 for SS181 to 171 for SS259

P

d

maller

68

Figure 4-4 SEM of PS latex particles larger than 500nm in size (A) 525nm (B) 585nm

(C) 640nm (D) 790nm

(A)

(B)

69

(C)

(D)

Figure 4-4 Continued

70

The surfactant to monomer ratio refers to the value of the surfactant amount

divided by monomer amount after polymerization ends The surfactant amount is the

weight of the surfactant contained in emulsion and the monomer amount is the total

weight of the monomer added for polymerization to form the ultimate polymer particles

in emulsion As the surfactant to monomer ratio decreases mean particle diameter

increases as described by Odian [1991] Generally lower surfactant concentration forms

fewer micelles resulting in larger particle size [Evans et al 1999] However there is a

plateau in particle mean diameter between a surfactant to monomer ratio of 0005 and

0013 This is due to low polymerization conversion of latex particles from monomer

leading to a smaller particle size than expected This is not a factor of surfactant amount

The dependence of the particle size on the amount of surfactant is shown in Figure 4-5

0

200

400

600

800

1000

0000 0005 0010 0015 0020 0025 0030

Surfactant to monomer ratio (ww)

Part

icle

mea

n di

amet

er (n

m)

Figure 4-5 Dependence of the particle size on the surfactant to monomer ratio

71

413

e

f bare

microdomain structures is important for biocompatibility [Mori et al 1982 Higuchi et al

1993 Deppisch et al 1998] Figure 4-6 shows the schematic of core shell latex particle

structure The recipe for core shell structures is shown in Table 4-4

Core Shell Latex Particles

The latex particles shown in Figure 4-3 (A) and labeled as S258 in Table 4-2 wer

used as seeds to prepare core-shell latex particles because of their high particle

uniformity and smooth surface morphology Methyl methacrylate (MMA) and acrylic

acid (AA) monomers were introduced to increase surface hydrophilicity over that o

polystyrene particles AA is a more hydrophilic monomer than MMA because of the

carboxyl acid group in AA is more favorable for hydrogen boding with water The

surface carboxyl groups on PAA may have many promising applications in biomedical

and biochemical fields [Kang et al 2005] MMA monomer is less hydrophobic than

styrene PMMA is known to more biocompatible than PS but still PMMA is hydrophobic

The optimization of suitable hydrophobic to hydrophilic ratios and control of

Polystyreneseed particle

Core shelllatex particles

PSPMMA100

PSPMMA90PAA10

PSPMMA75PAA25

Polystyreneseed particle

Core shelllatex particles

PSPMMA100

PSPMMA90PAA10

Figure 4-6 Schematic of core shell latex particle structures

PSPMMA75PAA25

72

Table 4-4 The preparation recipe of PS core with various shell latex particles Latex 25 label PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA

Initial charge

PS seed latex (g) 500 500 500a

Monomer continuous

Methly methacylate (g) 320 288 240

Acrylic acid (g) 00 32 80

Initiator stream

Na

258 258 258

Water (g) 1570 1570 1570

Feed time (min) 0-120 0-50 0-60

b 200

Feed time (min) 0-122 0-60 0-65

952

2S2O8 (g) 02 02 02

NaHCO3 (g) 02 02 02

Water (g) 200 200

Solids content 183 183 183

Mean particle diameter (nm) 370plusmn186 370plusmn178 370plusmn191

Conversion () 955 924

Three types of core shell structures were prepared PMMA75PAA25 shell is a

copolymer consisting of the PMMA to PAA ratio of 75 to 25 by weight

PMMA90PAA10 shell is a copolymer with the PMMA to PAA ratio of 90 to 10 by

weight PMMA100 is the PMMA homopolymer shell on PS core The particle size of

these core-shell particles as well as PS latex particle was prepared to be about 370nm in

mean diameter and are characterized to determine their zeta potential protein adsorption

and biocompatibility The image of SEM of PS and core shell particles is shown at Figure

4-7 The synthesized latex particles had a high uniformity in size and a smooth spherical

surface morphology

73

Figure 4-7 Scanning

Electron Micrograph of latex particles (A) PS (B) PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMMA75PAA25

(A)

(B)

74

(C)

(D)

Figure 4-7 Continued

75

42 Characterization of Latex Particles

421 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectroscopy has proven to be a powerful tool to characterize polymeric

materials A molecule can absorb only select frequencies (energy) of infrared radiation

which match the natural vibration frequencies The energy absorbed serves to increase

the amplitude of the vibrational motions of the bonds in the molecule [Pavin et al 1996]

Only those bonds which have a dipole moment that changes as a function of time can

absorb infrared radiation The FTIR spectra of polymerized latex particles made during

this work are shown in Figure 4-8 There are number of characteristic peaks for PS latex

particles [Bhutto et al 2003 Li at al 2005] Aromatic C-H stretching vibration is shown

at 3002 cm-1 and 3103 cm-1 and aliphatic C-H asymmetrical and symmetrical stretching

shown at 2900 cm-1and 2850 cm-1 respectively There is an additional carbonyl (C=O)

adsorption at 1730cm-1 wavenumber for all core shell latex particles containing some

amount of either or both PMMA and PAA The broad OH group peak at 3400 cm-1

appear for particles containing some amount of PAA and has intensity dependent on the

amount of PAA in the shell For example the peak intensity of OH groups for

PSPMMA75PAA25 is greater than that of PSPMMA90PAA10 since there is a higher AA

monomer content in PSPMMA75PAA25 than in PSPMMA90PAA10 This OH peak is not

seen for PS and PS core PMMA shell particles because of the absence of OH groups on

PS and PMMA polymer chains

422 Zeta Potential Measurements

The electrical surface property was characterized by zeta potential measurements of

tex particles Zeta potential is an important and useful indicator of surface charge which

can be used to predict and control the stability of colloidal systems The greater the zeta

is

la

76

1520002500300035004000

Wavenumbers (cm-1)

li

Re

ativ

e

Tra

nsm

ttanc

e

Figure 4-8 FTIR spectra of polymerized latex particles (A) ba

PSPMMA100 (C) PSPMMA90PAA10 (D) PSPMM

(B)

(C) P

(D) P

C

C

OCH3

O

CH3

XCH2

CH2 n

CH

CH2 C

C

OCH3

O

XCH2 CH

OC

CH3

Y

OH

(A) Bare PS

500100000

re polystyrene (PS) (B) A75PAA25

PSPMMA100

SPMMA90PAA10

SPMMA75PAA25

77

potential the more likely the suspension is to be stable because the charged particles repel

one another and thus overcome the natural tendency to aggregate The measurement of

zeta potential is often the key ontrol of the interaction of proteins

with solid surfaces such as latex particles

Zeta potential (ζ) is the elec at exists at the shear plane of a particle

which is some small distance from the surface [Myers 1999] The zeta potential of the

particles can be determined by measuring the mobility of the particles in an applied

electric field termed electr its response to an alternating electric

field Colloidal particles dispersed in lu ically charged due to their ionic

characteristics and dipolar attributes The development of a net charge at the particle

surface affects the distribution of ions in the neighboring ng in

an increased concentration of counter ions to the surface Each particle dispersed in a

ns called fixed layer or Stern layer

Outside th compositions of ions of opposite polarities

form ble layer is form

particle-liquid interface This double layer may be considered to consist of two parts an

inner region which inclu nd relatively strongly to the surface and an outer or

diffuse region in which the ion distribution is determined by a balance of electrostatic

forces and random thermal motion The potential in this region therefore decays with the

distance from the surface until at a certain distance it becomes zero Figure 4-9 shows a

schematic representation of a typical ion distribution near a positively charged surface

The Stern surface (also referred to as the outer Helmholtz plane) is drawn through the

to understanding and c

trical potential th

ophoretic mobility or

a so tion are electr

interfacial region resulti

solution is surrounded by oppositely charged io

e Stern layer there are varying

ed in the region of the ing a cloud-like area Thus an electrical dou

des ions bou

center of those ions which are effectively adsorbed to the surface [Hunter 1981] The

78

extent of ion adsorption is determined by electrical and other long-range interactions

between the individual ions and surface of particles The ions outside of the Stern layer

form the diffuse double layer also referred to as the Gouy-Chapman layer [Burns and

Zydney 2000]

Shear plane

Stern layer

ψζ

0

ψs

ψShear plane

Stern layer

ψζ

0

ψs

ψ

t

controlled electric field is applied via electrodes immersed in a sample suspension and

Figure 4-9 Schematic representation of ion distribution near a positively charged surface

Zeta potential is a function of the surface charge of a particle any adsorbed layer a

the interface and the nature and composition of the surrounding medium in which the

particle is suspended The principle of determining zeta potential is very simple A

79

this causes the charged particles to move towards the electrode of opposite polarity

Viscous forces acting upon the moving particle tend to oppose this motion and the

equilibrium is rapidly established between the effects of the electrostatic attraction and

the viscosity dra

g The particle therefore reaches a constant terminal velocity

Figure 4-10 Schematic representation of zeta potential measurement(source

httpnitioncomenproductszeecom_shtm)

Because protein adsorption mechanisms are very complex and hard to explain in

biological systems protein adsorption is generally studied by using more ideal systems

consisting of one or more well characterized proteins a well-characterized adsorbent and

a well-defined aqueous solution Even so small changes in the experimental conditions

such as pH ionic strength and temperature generate totally different results Therefore

two media systems phosphate buffer (PB) and phosphate buffered saline (PBS) were

chosen to be used for the zeta potential measurements and protein adsorption study PB is

a simple aqueous system of 5 mM monobasic sodium phosphate (NaH2PO4H2O) to

n a constant pH PBS is used to keep the water concentration inside and outside of

the cell balanced If the water concentration is unbalanced the cell risks bursting or

shriveling up because of a phenomenon called osmosis PBS is a solution with the 5 mM

of monobasic sodium phosphate (NaH2PO4H2O) and 143 mM of sodium chloride (NaCl)

in water

maintai

80

The results of zeta potential measurement at room temperature (25plusmn1oC) as

function of pH and ionic strength (different media PB and PBS) for synthesized lat

particles are seen in Figure 4-11 Zeta potential values of polystyrene (PS) latex particle

at the Figure 4-9 are negative between -291 mV and -599 mV in PB and betwe

ex

s

en -203

mV and -278 mV in PBS at all pH ranges These negative zeta potential values of PS

latex particles are due to the sulfate groups [Vandenhu et al 1970] originated from

persulfate initiators attached via an oxygen bridge (-C-O-SO3-) at the end of polymer

chain where polymerization was initiated

-100

-80

-60

-40

-20

0

pH

Zta

pte

ntl

ζ

20

40

1 2 3 4 5 6 7 8 9

eo

ia (m

V)

PB (5mM)

PBS (5mM +143mM)

en -

Figure 4-11 Zeta potential of PS latex particles at 25oC

The zeta potential measurements of PSPMMA100 core shell latex particles as seen

in Figure 4-12 were also negative with a range of -28 and -505 mV in PB and betwe

143 mV and -186 mV in PBS at the pH 21-78 These negative values are also due to

sulfate groups on the particle surface

81

-100

-80

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

Zea

pote

nti

l ζ

(mV

pH

ta

)

PB (5mM)

PBS (5mM + 143mM)

Figure 4-12 Zeta potential of PSPMMA100 latex particles at 25oC

The zeta potential for PSPMMA90PAA10 and PSPMMA75PAA25 was also

negative as seen in Figure 4-13 and 4-14 The zeta potential value for the

PSPMMA90PAA10 were between -367 mV and -678 mV in PB medium and between -

147 mV and -193 mV in PBS both at 25oC In case of the PSPMMA75PAA25 core shell

particles zeta potential values were between -291 mV and -520 mV in PB and between

-115 mV and -210 mV in PBS media Sulfate groups in PSPMMA75PAA25 and

PSPMMA90PAA10 core shell particles contribute to the negative zeta potential values

participate in forming a

negat

cid is expressed by the term pKa which is the pH

at which an acid is 50 dissociated However the contribution of the carboxylic acid

Carboxylic groups from acrylic acid monomer also would

ive surface charge of the latex particles at a pH greater than 70 because the pKa

value for the carboxylic acid is between 40 and 60 pH range [Ottewill et al 1967] This

indicates that carboxylic acid dissociation begins at a pH between pH 46-60 and

increases with pH The strength of an a

82

group to negative zeta potential values of PSPMMA-PAA core shell particles was not

detectable because there was no significant difference in zeta potential values for

synthesized latex particles at a pH greater than 70

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

Zeta

pot

entia

l ζ

(mV

)

PB (5mM)PBS (5mM + 143mM)

-80

-100

pH

Figure 4-13 Zeta potential of PSPMMA90PAA10 latex particles at 25oC

-100

-80

-60

-40

-20

0

20

40

1 2 3 4 5 6 7 8 9

pH

Ze p

ont

i ζ

V)

tate

al (m

PB (5mM)PBS (5mM + 143mM)

Figure 4-14 Zeta potential of PSPMMA75PAA25 latex particles at 25oC

83

The zeta potential plots and values were similar for all latex particles This may be

due to the similar initiator density on the latex surface The initiator concentrations

monomers for shell polymerization were the same fo

to

r all core shell latex particles (062

ww from Table 4-4) A similar concentration (055 ww) of initiator to monomer was

added to prepare bare PS latex particles used in zeta potential measurements

Since the zeta potential values of all synthesized latex particles were negative

between pH=20 and pH=78 the isoelecric point (IEP) of these latex particles would be

less than pH=20 Sulfates have a pKa in the range of 10 to 20 [James 1985] indicating

that the sulfate group the conjugate base of a strong acid is protonated (-C-OSO3H) at

less than pH 20 This means that the synthesized latex particles are all negative at the

physiological pH Most serum proteins have a negative surface charge in blood

therefore when the negative latex particles are applied to blood stream fatal

tein is

avoided by charge repulsion in the blood Table 4-5 shows the common blood proteins

and their isoelectric points (IEP)

Table 4-5 The isoelectric point (IEP) of common proteins Serum proteins Isoelectric point (IEP)

complication by coagulation between applied latex particles and serum pro s

Albumin 48 α1-Globulin 20 α2-Microglobulin 54 γ1-Globulin 58 Heptoglobin 41 Hemoglobin 72 β1-lipoprotein 54 Immunoglobulin G (Ig G) 73 Fibrinogen 58 (source httpwwwfleshandbonescomreadingroompdf1085pdf

httpwwwmedalorgvisitorwww5CActive5Cch135Cch13015Cch130105aspx )

84

The absolute zeta potential values of the latex particles in phosphate buffer (PB)

was higher than that in phosphate buffered saline (PBS) This is because high

concentration of sodium and chloride electrolytes in PBS compressed the electrical

double layer causing rapid decay of surface potential Figure 4-15 shows that the

thickness of the electrical double layer is inversely proportional to the concentration of

electrolyte in the system

Surf

ace

Pote

ntia

l ψ

1κ3

10

1e

1κ 1κ

(1)(3) (2)

2 1 Z

Surf

ace

Pote

ntia

l ψ

1κ3

10

1e

1κ 1κ

(1)(3) (2)

Figure 4-15 The decay of surface potential with distance from surface in various

electrolyte concentrations (1) low (2) intermediate (3) high [Myers 1999]

2 1 Z

The electrical potential in the solution surrounding the particle surface falls off

the Debye-Huckel

yers 1999]

exponentially with distance (Z) from the surface according to

approximation [M

z) (- exp 0 κψψ = (4-4)

e reciprocal of the thickness of the electrical double layer referred to as the

The potential fallen off by a factor e The theoretical equation for the

ickness 1κ is

where κ is th

Debye length of 1

double layer th

21

22i

0

ekT 1

⎟⎟⎠

⎜⎜⎝

=sum ii zcεε

κ

⎞⎛ (4-5)

85

where

tration of

y was performed to measure the protein adsorption on the

synthesized latex particles Many physical and chemical processes occur at the boundary

between two phases and adsorption is one of the fundamental surface phenomena [Oscik

1982] This phenomenon is driven by the change in the concentration of the components

at the interface with respect to their concentrations in the bulk phase There are two

adsorption processes physical adsorption occurring when non-balanced physical forces

appear at the boundary of the phases and chemical adsorption or chemisorption

t the

interface [J

n leads

ational entropy hence adsorption takes place only if the

loss in conformational entropy is compensated

ter adsorption Cp The initial part of the

isotherm merges with the Γ-axis because at low polymer concentration the protein has a

high affinity for the surface and all of the polymer is adsorbed until the solid surface is

saturated

ε0 is the permittivity of a vacuum of free space ε is the permittivity of a medium e

is the relative permittivity or dielectric constant of the medium c is the concen

an ion (i) z is the valency of an ion (i) k is Boltzmannrsquos constant T is absolute

temperature (K)

43 Protein Adsorption Study

The adsorption stud

occurring when atoms and molecules from adjacent phases form chemical bonds a

aroniec et al 1988]

Flexible proteins in solution possess high conformational entropy as a result of the

various states each of the many segments in the protein chain can have Adsorptio

to a reduction of this conform

by sufficient attraction between polymer

segment and solid surface [Martin 1998] Isotherm shape of high affinity adsorption

between polymer and solid is shown in Figure 4-16 where the adsorbed mass Γ is plotted

against the polymer concentration in solution af

86

Figur

de

arboxyl

e 4-16 High-affinity adsorption isotherm of typical flexible polymer on solid

surface

In order to investigate the adsorption of blood proteins latex particle properties

such as electrical surface charge hydrophobicity and environmental conditions like pH

ionic strength and temperature are considered as experimental factors [Arai and Nor

1990] Figure 4-17 shows the overall schematic representation of the protein adsorption

on the synthesized latex particles with the functional groups such as sulfate and c

groups

CP

Γ

PMMAPAAShell

PS or PMMAShell OSO3

-

OSO3-

OSO3-

OSO3-

PS

-O3SO

PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

Protein

NH3+

+

+H3N

+

COO-

Hydrophobic interaction

NH3

COO-

H3N

COO-

-OOC

Ionic interaction

Hydrophobic interaction

Hydrogen bonding

Ionic interaction

PMMAPAAShellOSO3

-

OSO3-

OSO3-

OSO3-

PS or PMMAShell -O3SOOSO3

-

PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

-O3SO

OSO3-

OSO3-

OSO3-

PS PSCore

HOOC

-O3SO

O3SO

3

HOOC

HOOC

-

-O SO

Protein

NH3+

+

+H3N

+

COO-NH3

COO-

H3N

COO-

-OOC

NH3+

+

+H3N

+

COO-

Hydrophobic interaction

NH3

COO-

H3N

COO-

-OOC

Ionic interaction

Hydrophobic interaction

Hydrogen bonding

Figure 4-17 Overall schematic representation of the protein adsorption on the

Ionic interaction

synthesized latex particles

The surface functional groups as well as suitable surface hydrophobicity

hydrophilicity of latex particles are closely related to protein adsorption property As

indicated at Figure 4-17 various interaction forces such as hydrophobic interaction ionic

87

interaction hydrogen interaction and van der Waals interaction [Andrade 1985] exist

between protein molecule and the solid surface of latex particle

431 Adsorption Isotherm

The protein adsorption isotherm is generally defined as the relationship between the

amount of protein adsorbed by a solid (latex particle) at a constant temperature and as a

ine

the amount of protein adsorbed onto latex particles To evaluate the equilibrium

conce s

x

function of the concentration of protein Adsorption isotherms were plotted to determ

ntration of target proteins a calibration curve is firstly needed Figure 4-18 show

the UV absorbance as a factor of bovine serum albumin (BSA) concentrations The UV

absorbance of the known protein concentration was measured at 562nm which is the λma

for a protein treated by a bicinchoninic acid (BCA) assay

0

02

04

06

08

500 520 540 560 580 600

Wavelength (nm)

Abs

orba

nce

Figur gml (B)

06mgml (C) 02mgml

(A)

(C)

(B)

Max562nm

e 4-18 The dependence of UV absorbance on BSA concentration (A) 10m

Figure 4-19 shows the standard calibration curve used in adsorption isotherm

experiment of protein onto latex particles The net absorbance of each known protein

88

concentration was plotted and then a linear equation passing through a concentration of

zero protein concentration was obtained This equation is applied

to determine the

orbed on the synthesized latex particles The amount of free

protei

eported

trations (005 01 03 05

and 07mgml) were used to generate this curve as well as the adsorption isotherm

unknown protein amount ads

n was also determined using UV light at the 280nm wavelength however a

distinguishable UV intensity was too low to determine a precise value of protein

adsorption

The bicinchninic acid (BCA) assay technique [Smith et al 1985] has been r

for high efficiency of protein evaluation and was used for determination of protein

adsorption [Tangpasuthadol et al 2003 Williams et al 2003] This technique has many

advantages such as high sensitivity easy of use color complex stability and less

susceptibility to any detergents Five different BSA concen

y = 00R2 =

004741x 09939

00

01

02

03

04

0 200 400 600 800

BSA Standard (microgml)

Net

Abs

orba

nce

at 5

62nm

Figure 4-19 Standard curve of net absorbance vs BSA sample concentration

89

All isotherms were fitted to the Langmuir-Freundlich isotherms using the nonlinea

regression method Two adsorption isotherm models the Langmuir type isotherm a

Langmuir-Freundlich combination type isotherm were compared As seen the Figure 4-

20 The Langmuir isotherm model deviated further from the experimental data Th

all isotherms were fitted to the Langmuir-Freundlich isotherms [Yoon et al 1996

al 1998] usi

r

nd the

erefore

Lee et

ng the nonlinear regression method with following equation

n

n

eq

eqm

kC

kC1

1

1+

where q is the amount of adsorbed BSA per unit surface area q

qq = (4-6)

stant

were between 045 and 055 and hence n was fixed to its average 05 Actual qm values

were evaluated after fixing the n value

m is the adsorbed amount

in equilibrium Ceq is the equilibrium concentration of BSA k is the adsorption con

and n is the exponential factor for a heterogeneous system n values are empirical and

usually from zero to no more than unity [Jaekel 2002] The values of n in our system

00

10

20

30

0 02 04 06 08

C (mgml)eq

q (m

gm

2 )

pH 32Langmuir-Freundlich modelLangmuir model

Figure 4-20 Fitted models for adsorption isotherm of bovine serum albumin (BSA) on

polystyrene latex particles at 37oC in phosphate buffer media

90

The BSA protein adsorption on the synthesized latex particles was performed at

three pH levels 32 48 74 in both phosphate buffer (PB) and phosphate buffered saline

(PBS) at physiological temperature 37oC These pH levels correspond to an acidic pH

lower than the isoelectric point (IEP) of BSA the pH at the IEP and an alkaline pH

higher than the IEP of BSA respectively The IEP corresponds to the pH at which the

amino acid becomes a zwitterion Here its overall electrostatic charge is neutral and the

charge expressed by the basic amino group (-NH2 -NH3+) of the amino acid is equal to

the charge expressed by the acidic carboxyl group (-COOH -COO ) The IEP of BSA is

-

pH 48 and indicates that the side chain of the amino acids of BSA contains a greater

amount of acidic functional groups than that of basic functional groups At pH 32 and

74 BSA has a positive and negative charge respectively

Figure 4-21 and 4-22 show adsorption isotherms of BSA onto polystyrene (PS)

latex particles

00

10

20

30

40

0 02 04 06 08

Ceq (mgml)

q (m

gm

2 )

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-21 Adsorption isotherm of BSA on polystyrene (PS) latex particles in

phosphate buffer (PB) at 37oC

91

00

10

30

40

0 02 04 06 08

Ceq (mgml)

q (m

gm

2

20

)

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

(PBS) on PS latex particles

At the IEP of BSA the adsorbed amount (q

Figure 4-22 Adsorption isotherm of BSA adsorption 37oC in phosphate buffered saline

onto PS at equilibrium was

maxim

um at the IEP of BSA This is because the BSA molecules form the most

compact structures at the IEP eliminating the repulsion force between proteins A higher

number of BSA molecules can adsorb on a given surface area by hydrophobic interaction

when in this compact structure This observation agrees well with reported data by other

researchers who also indicated that the maximum adsorption from aqueous protein

solution was observed at the IEP [Mularen 1954 Watkins et al 1977 Oh at el 2000]

There was no significant difference of adsorbed BSA amount in PB and in PBS media at

pH 48 The conformational alteration of BSA was not affected significantly by the ionic

strength of the two solutions There was evidence of the effect of the electrostatic

interaction to some extent even at the IEP since the amount of BSA adsorbed in PBS is

slightly hig

m) of BSA

um indicating that hydrophobic interaction between latex particle and BSA

protein is maxim

her than in PB at this pH

92

The adsorbed amount of protein is shown to decrease at a pH 32 and 74 The

decrease in adsorption efficiency when deviating from IEP of BSA is a result of the

increase in conformational size of the protein molecules and the lateral electrostatic

repulsions between adjacent adsorbed BSA molecules Figure 4-23 shows a schematic of

possible conformational changes at each pH where hydrophobic interaction always exists

between non-polar portion of a protein and hydrophobic portion of solid surface

- - --- ---Latex surface

Zeta potential

BSA(+)

(-)

48 7432pH

(0)

+++ + ++

--+

++-

- - --- ---Latex surface

Zeta poten pH(0)

+++ + ++

--

tial

BSA(+)

(-)

48 7432

+

++-

ming

4

s

al blood Table 4-6 shows the calculated results of

Figure 4-23 Conformation of bovine serum albumin (BSA) on latex particles (Assuthat charge repulsion reduces the effect of the hydrophobic interaction at pH 74)

In addition the adsorbed amount of BSA at pH 32 was larger than that at pH 74

and still less than that at 48 This is because there is the electrostatic interaction between

the positive BSA molecules and negative PS latex particles at pH 32 and electrostatic

repulsion is dominant between negative BSA and negative PS latex particles at pH 7

The amount adsorbed increased with increasing ionic strength in pH 32 and 74 This i

because the high concentration of electrolytes reduces the electrostatic repulsion between

BSA and PS latex particle It should be noted that the BSA concentration used in these

experiments was lower than that found in normal human blood This is because the

equilibrium BSA adsorption onto the latex particles is actually reached at a much lower

concentration than that found in norm

93

adsor rm

ein

leading to a compact conformation of BSA There are two main forces affecting

adsorption amount at an acidic pH of 32 They include the ionic attraction between the

negative latex particle and the positive protein and lateral repulsion between positive

proteins The smallest amount of BSA adsorption was observed at a pH of 74 At this

point the charge repulsion between negatively charged particles and protein molecules

exists the h

of cha

bed BSA amount at equilibrium from the Langmuir-Freundich adsorption isothe

equation

In summary the hydrophobic interaction between PS latex particle and BSA

protein exists and is the dominant mechanism for the adsorption process Conformational

changes of BSA at each pH also affected the adsorption amount Adsorption was highest

at pH 48 because at this pH there is the least amount of ionic repulsion in the prot

and the lateral repulsion between proteins dominant adsorption even though there always

ydrophobic interaction between the solid latex particle and protein The effect

rge repulsion was reduced by the high concentration of electrolytes in PBS medium

Table 4-6 The equilibrium concentration values of BSA adsorption on polystyrene (PS) latex particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2) 32 174 48 299 Phosphate buffer 74 136 32 260 48 283 Phosphate buffered saline 74 207

The adsorption isotherms of BSA on PSPMMA core shell particles are shown in

Figure 4-24 in phosphate buffer (PB) and in Figure 4-25 in phosphate buffered saline

(PBS) The adsorbed BSA amount was calculated using the Langmuir-Freundlich

100

94

adsorption isotherm equation and the amount listed in Table 4-7 The trend seen in the

adsorption results was very similar to the case of PS latex The overall adsorption process

was d rticle

rs

hat

0

ominated by hydrophobic interactions between the PSPMMA100 core shell pa

and BSA proteins At pH 48 the adsorbed amount (qm) of BSA on PSPMMA100 core

shell particles was 221 mgm2 in PB and 237 mgm2 in PBS and are the maximum

values recorded among the three pH levels 32 48 and 74 Suzawa and his co-worke

[1982] have obtained similar results This can be also explained by the compact

conformation of BSA molecules at the IEP of BSA which lead to a high protein

population on the solid surface At a pH of 32 and 74 BSA adsorption was less than t

at pH 48 because of the protein conformation change There was a small increase in

adsorbed BSA at pH 32 because of the charge attraction between negative PSPMMA10

core shell particles and positive BSA At pH 74 however the adsorption of BSA was

further reduced by charge repulsion between the negative PSPMMA100 core shell

particles and the negative BSA proteins

000 02 04 06 08

Ceq (mgml)

10

20q (m

gm

302 )

40

50pH=32pH=48pH=74Mode 8)l (pH 4Mode 2)l (pH 3Mode 4)

Figure 4-24 Adsorption isotherm of BSA 37oC in PB on PSPMMA core shell latex

l (pH 7

100

particles

95

The adsorbed amount of BSA increased more rapidly in PBS with increasing ioni

strength at pH 74 This shows that the low concentration of electrolytes promotes

electrostatic repulsion between negative BSA and negative PSPMMA

c

on

x

l 1980

100 core shell

particle As the ion strength of PBS is increased however the high concentration of

electrolyte leads to less electrostatic repulsion promoting more rapid protein adsorpti

By comparing the amount of adsorbed protein between bare PS latex particles and

PSPMMA100 core shell particles the amount of protein adsorption on the PS late

particles is greater than that on the PSPMMA100 core-shell particles [Suzawa et a

1982 Lee et al 1988]

00

10

20

30

40

m2 )

0 02 04 06 08

Ceq (mgml)

q (m

g

pH=32pH=48pH=74Model (pH 2) 3Model (pH 48)Model (pH 74)

Figure 4-25 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA100 core shell

latex particles

96

Table particles calculated the Langmuir-Freundlich isotherm model

4-7 The equilibrium concentration values (qm) of BSA adsorption on PSPMMA100

Media pH qm (mgm ) 2

32 140

48 221 Phosphate buffer

74 130

32 169

48 237 Phosphate buffered saline

74 162

The BSA adsorption mechanism and trend on PSPMMA core shell particles was

very similar to that of the PS latex particle Unlike the case of PS and PSPMMA100 latex

particles the amount of BSA adsorbed onto PSPMMA90PAA10 and PSPMMA75PAA25

core shell particles was higher at pH 32 than pH 48 and 74 Figure 4-26 through 4-29

shows the adsorption isotherms of BSA on PSPMMA90PAA10 and PSPMMA75PAA25

core shell particles The amount of BSA adsorbed increased at pH 32 and pH 48 as the

hydrophilicity was developed by the addition of PAA On the contrary the adsorbed

amount of BSA was dramatically decreased at pH 74 The adsorption amount of BSA on

PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles at equilibrium is listed in

Table 4-8 and 4-9 respectively

Carboxylic acid as well as sulfate groups are distributed on the surface of

PSPMMA90PAA10 and PSPMMA75PAA25 core shell particles and seem to largely affect

the BSA adsorption The isotherms showed very strong pH dependence to the BSA

adsorption onto these core shell latex particles The carboxylic groups (COOH) may

especially enhance the BSA adsorption prominently at pH 32 by hydrogen bonding with

protein molecules The pKa value for the PAA is 40-60 and a good indication that the

97

carboxylic acid groups are protonated at pH 32[Gebhardt et al 1983] The main

adsorption interaction force at pH 32 was generated by hydrogen bonding as well as

charge attraction between latex particle protein even th the conformational

repulsion between proteins results in a reduction of the protein

adsorption at this pH The amount of BSA adsorbed also increased as the PAA amount in

eased The a tion of BSA w inimal however less

than 07 mgm2 at pH 74 Here the carboxylic groups are ionized which increases the

charge repulsion by ionized carboxylic groups (COO-) resulting in PSPMMA-PAA latex

particle

and ough

change and charge

the core shell particles incr dsorp as m

s with much lower protein adsorption

00

20

40

q (m

g2 )

60

m

pH=32pH=48pH=74Model (pH 32)Model (pH 48)

0 02 04 06 08

Ceq (mgml)

Model (pH 74)

o shell

Figure 4-26 Adsorption isotherm of BSA at 37 C in PB on PSPMMA90PAA10 corelatex particles

98

00

40

60

80

0 02 04

C (mgm

mm

2 )

20

06 08

eq l)

q (

g

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-27 Adsorption isotherm of BSA at 37oC in PBS on PSPMMA90PAA10 core

shell latex particles

Table 4-8 The equilibrium concentration values of BSA adsorption on PSPMMA90PAA10 particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2)

32 361

48 283 Phosphate buffer

74 043

32 519

48 226 Phosphate buffered saline

74 043

99

00

20

40

60

80

100

0 02 04 06 08Ceq (mgml)

q (m

gm

2 )

pH=32pH=48pH=74Model (pH 32)Model (pH 48)Model (pH 74)

oFigure 4-28 Adsorption isotherm of BSA 37 C in PB on PSPMMA75PAA25 latex particles

00

100

20

40

60

80

0 02 04 06 08Ceq (mgml)

q (

m2 )

pH=32pH=48pH=74

mg

Model (pH 32)Model (pH 48)Model (pH 74)

Figure 4-29 Adsorption isotherm of BSA 37oC in PBS on PSPMMA75PAA25 core shell

latex particles

100

Table 4-9 The equilibrium concentration values of BSA adsorption on PSPMMA75PAA25 particles calculated the Langmuir-Freundlich isotherm model

Media pH qm (mgm2)

32 1083

48 662 Phosphate buffer (PB)

74 075

32 1077

48 681 Phosphate buffered saline (PBS)

74 057

The adsorption of β2-microglobulin (β2M) was determined under one set of

onditions in phosphate buffer at 37oC and pH 74 because of the limited amount of β2M

protein The adsorption isotherms of the β2M protein on the synthesized latex particles

are shown in Figure 4-30 4-31 and 4-32 The β2M protein concentrations for adsorption

isotherm experiment were chosen to be 0015 0030 0045 and 0060mgml since the

β2M concentration for the patients with renal failure can be elevated to similar

concentrations about 005mgml or more up to 50 times the normal level [Nissenson et

al 1995] There were steep initial slopes of the adsorption isotherm curves of β2M on

latex particles in all isotherms indicating a high affinity adsorption type It seems that the

hydrophobic interaction is enough for the adsorption process of β2M proteins to occur on

the latex particles even though charge repulsion exists between protein and latex particle

at pH 74 The complete plateau regions were not seen for all isotherms indicating that

β2M proteins were not saturated at this level and there are still available sites for β2M

c

adsorption on latex particles The initial slope of the β2M isotherms for PS was steeper

101

than that of any of the other latex particle PSPMMA100 PSPMMA90PAA10 and

PSPMMA 75PAA25

00

02

0 0002 0004 0006 0008

04

06

10

Ceq (mgml)

q (m

2 )

08

gm

PSPSPMMA100Model (PS)Model (PSPMMA100)

Figure 4-30 Adsorption isotherm of β2M onto PS and PSPMMA100 latex particles in PB

at 37 C and pH 74 o

00

02

04

06

08

10

0 0002 0004 0006 0008C (mgml)

q (m

m2 )

eq

g

PS

PSPMMA90PAA10

Model (PS)

Model (PSPMMA90PAA10)

Figure 4-31 Adsorption isotherm of β2M onto PS and PSPMMA90PAA10 latex particles

in PB at 37oC and pH 74

102

00

02

04

06

08

10

0 0002 0004 0006 0008Ceq (mgml)

q (m

gm

2 )

PS

PSPMMA75PAA25

Model (PS)Model (PSPMMA75PAA25)

Figure 4-32 Adsorption isotherm of β2M onto PS and PSPMMA75PAA25 latex particles

in PB at 37oC and pH 74

The equilibrium concentration values (qm) of β2M adsorption on latex particles are

calculated and listed in Table 4-10 The difference between the maximum amounts of

β2M adsorption on all the latex particles was not significantly different and their values

were between 069 and 08mgm2

Table 4-10 The equilibrium concentration values of β2M adsorption calculated the Langmuir-Freundlich isotherm model

Latex particles qm (mgm2)

PS 080

PSPMMA100 069

PSPMMA90PAA10 072

PSPMMA75PAA25 072

103

432 Adsorbed Layer Thickness

There are several possible explanations for large amount of BSA adsorbed at acidic

pH levels including the end-on adsorption pattern of BSA the flexibility of BSA

molecules the tilting of protein molecules due to the asymmetry of the charge

distribution or multilayer formation [Peula et al 1993] The adsorbed BSA thickness (δ)

can be calculated by the following equation [Chiu et al 1978] This equation was also

cited again by other researchers [Shirahama et al 1985]

BSA

mqρπ

δbullbull

=33

(4-7)

where π

33 is the packing factor qm is the plateau value of the adsorbed am

ρBSA is the hich corresponds to the reciprocal of its known

partial specific volume The layer thickness (δ) of BSA proteins on synthesized latex

partic

he IEP [Shirahama et al 1985] Compared to the

hydrodynamic dimensions of BSA 140 times 40 times 40 Aring3 [Fair et al 1980 Peter 1985] these

nd

ode It is unnecessary ultilayering because thickness do

exceed 140 Aring The layer thickness (δ) of BSA proteins on synthesized latex particles

e red saline (PBS) was ran 28 ~ 83 Aring at 37oC and pH 48 These

lso listed in Table 4-1

ount and

density of BSA molecule w

les in phosphate buffer (PB) ranged from 27 - 81 Aring at 37oC and pH 48 These

calculated values are listed in Table 4-11 Only δ values at pH 48 the isoelectric point

(IEP) of BSA are important because intra- and intermolecular electrostatic repulsion of

protein molecules is minimized at t

values of layer thickness indicate that the BSA molecules adsorb by side-on (40Aring) a

end-on (140Aring) m to consider m

not

in phosphat buffe ged of

calculated values are a 2

104

Table 4-11 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffer (PB) at 37oC pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

32 21 17 44 133

48 37 27 35 81

74 17 16 5 9

Table 4-12 Absorbed BSA layer thickness (Aring) of BSA in phosphate buffered saline (PBS) at 37oC

pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

32 32 21 64 132

48 35 29

74 26 20 5 7

28 83

1n

1n

neqm

So k is closely related to the initial slope of the isotherm showing the affinity

between BSA and latex particles [Yoon et al 1996] A key point in the characterization

433 Gibbs Free Energy of Protein Adsorption

In the Langmuir isotherm model k represents the ratio of adsorption to the

desorption rate constants This definition is not applicable to the Langmuir-Freundlich

isotherm model but its meaning is similar [Yoon et al 1996] In this sense k is closely

related to the affinity between protein molecules and latex particles When the

equilibrium concentration Ceq reaches zero the value of Ceq will be very small (1gtgt

kCeq ) and then the equation 4-6 becomes

(4-8)

of adsorption processes on solid-liquid interfaces is to determine the change in Gibbsrsquos

free energy (∆Gdeg) one of the corresponding parameters for the thermodynamic driving

forces during adsorption The traditional method to evaluate adsorption thermodynamic

kCqq 1=

105

constants is based on measurement of adsorption isotherms under static conditions

[Malm ten 1998] The Langmuir-Freundlich ads lf

easily found from Then this enables calculation of the free-

energy change ∆G during adsorption expressed as

(4-9)

where R is the general gas constant (R = 8314Jmiddotmol K ) and T is the absolute

tem ture Gibb e energy A P

listed in Table 4-13 and 4-14 respectively As the ∆Gdegads ore negativ e

pro adsorption on a solid substra ill be more favorable

e of BSA adsorption in phosphate o

pH PS PSPMMA100 PSPMMA90PAA10 PSPMMA75PAA25

s orption equilibrium constant k can be

a slope of the isotherm

degads

lfads kRTG lnminus=∆ o

-1 -1

pera s fre change of BS adsorption values in B and PBS are

values are m e th

tein te w

Table 4-13 The values of Gibbs free energy changbuffer (PB) at 37 C

32 -2411 -2726 -2846 -2805

48 -2352 -2383 -2198 -1972 ∆Go

80

ads (kJmol)

74 -2452 -888 -324 -2

Table 4-14 The values of Gibbs free energy change of BSA adsorption in phosphate

pH PS PSPMMAbuffered saline (PBS) at 37oC

100 PSPMMA90PAA10 PSPMMA75PAA25

32 -2315 -2620 -2312 -2683

48 -2149 -2299 -2090 -2138 ∆Go

(kJmads ol)

74 -2430 -1992 -258 -263

Table 4-15 shows the calculated values of Gibbs free energy change of β M

adsorption on synthesized latex particles PSPMMA PAA core shell latex particle has

2

75 25

106

largest negative Gibbs free energy of β2

2

M adsorption indicating largest affinity betw

β

een

2M adsorption in phosphate buffere (PB) at 37 C and pH74

PS PSPMMA100

M and latex particle

Table 4-15 The values of Gibbs free energy change of βo

PSPMMA90PAA10 PSPMMA75PAA25

∆Go03 ads

(kJmol) -3626 -3466 -3659 -39

The Gibbs free energy of β2M was much more negative in phosphate buffer (PB)

than that of BSA in physiological condition (37 C and pH 74) o from the Table 4-13 and

the Figure 4-31 This indicates that β2M molecules more readily adsorb on latex particles

BSA This can be explained by the rate of diffusion and accessibility of β2M molecules to

the latex particle ier and faster for β2M m an BSA because of

size (molec ight) erical shape molecules It o be

due to the higher hydrophobic force and less electrostatic repulsion that contribute to the

adsorption of β2M molecules not as prevalent in the case of BSA because the IEP of β2M

(57) is clo H 74) than that of BSA (48) causing β2M to

be less electro cally lled p ro

can be seen that the ∆ β2M adsorption onto the synth article

increasing wi hy icity o x particles s the ∆Goads of the BSA

molecules is decreasing or constant This phenomenon supports the theory that the

select lizing

than BSA and that the affinity of β2M for the latex particle is much larger than that of

surface It is eas olecules th

the smaller ular we and sph of β2M may als

ser to physiological condition (p

stati repe from the latex articles than BSA F m Figure 4-33 it

Goads of esized latex p s is

th the drophil f the late wherea

ive adsorption of β2M molecules over BSA molecules can be enhanced by uti

more hydrophilic PSPMMA90PAA10 and PSPMMA75PAA25 core shell latex particles

107

-60

-50

1 2 3 4

-40∆Gad

s

-30

10

0

o (k

Jm

o-

-20l)

β2-Microglobulin (β2M)Bovine serum albumin (BSA)

Figure 4-33 Gibbs free energy of adsorption of proteins on latex particles in PB at 37

and pH 74

434 Kinetics of Adsorption

to

oC

Protein adsorption is a complex process known to have a large biological impact

and is currently not well understood quantitatively because of extreme sensitivity to pH

the concentration of other electrolytes and molecules present temperature and many

other factors that can change in the physiological system Accurate knowledge of the

adsorption kinetics under a given set of conditions is a prerequisite for elucidating the

mechanisms of many fundamental biological processes to be predicted at the molecular

level [Regner 1987] This adsorption kinetics approach is used in protein adsorption

studies because of the uncertainty related to the time needed for the interfacial proteins

reach the equilibrium It is generally accepted that the process of protein adsorption is

comprised of the following steps a) transport toward the interface b) attachment at the

interface c) eventual structural rearrangements in the adsorbed state d) detachment from

PS PSPMMA100 PSPMMA90PAA10PSPMMA75PAA25

108

the interface and transport away from the interface Each of these steps can affect the

overall rate of the adsorption process

The quantitative analysis of the protein adsorption kinetics requires that the protein

amount adsorbed is known as a function of time The kinetics tests were carried out in

phosphate buffer (PB) at 37oC and pH 74 a condition similar to physiological condition

The protein concentrations chosen were 07mgml for BSA and 006mgml for β2M

which are the maximal concentrations used in an adsorption isotherm test A time-based

evaluation of the adsorption was made by measuring the concentration of protein in

solution at different incubation times The results are shown below from Figure 4-34 to 4-

37 The adsorption kinetics of proteins onto th cond-e latex particles is expressed as a se

order model [Oumlzacar 2003] shown by the following equation

2)( tet qqk

dtdq

minus= (4-10)

Integrating above equation and applying the boundary conditions yields

ee

t tq

+=

1 (4-11)

where q

qkq

t

2

2

2

the previous adsorption isotherm results representing good reproducibility of adsorption

t and qe stand for the amount (mgm ) of protein adsorbed at time t and at

equilibrium respectively k is the equilibrium rate constant of second-order adsorption

(m mghr) and t is the incubation time (hr)

The fitting parameters calculated by using the equation 4-11 are listed in table 4-16

From this data it is seen that the amount of BSA adsorbed at equilibrium was higher than

that of β2M on PS and PSPMMA100 latex particles but less than that of β2M on

PSPMMA90PAA10 and PSPMMA75PAA25 latex particles These results are similar to

109

00

05

15

20

0 2 4 6 8 10 12 14

Time (hr)

rot

n ad

sorb

d (

10

25

Pei

em

gm

2

BSA2nd order model (BSA))β2M2nd order model (β2M)

Figure 4-34 The kinetics of protein adsorption in PB on PS latex particles at 37oC and

pH 74

00

05

10

15

20

25

30

0 2 4 6 8 10 12 14

Time (hr)

Prte

inds

oo

arb

ed (

mg

m2 )

BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-35 The kinetics of protein adsorption in PB on PSPMMA100 latex partic

37les at

oC and pH 74

110

00

03

06

09

12

15

0 2 4 6 8 10 12 14

Time (hr)

Prot

ein

adso

rbed

(m

gm

2 ) BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-36 The kinetics of protein adsorption in PB on PSPMMA90PAA10 latex

particles o at 37 C and pH 74

00

03

06

09

12

15

0 2 4 6 8 10 12 14

Time (hr)

BS

A ad

sorb

ed (

mg

m2 )

BSA2nd order model (BSA)β2M2nd order model (β2M)

Figure 4-37 The kinetics of protein adsorption in PB on PSPMMA75PAA25 latex

particles at 37oC and pH 74

111

test The variation of β2M equilibrium adsorption amount was nearly negligible for all

latex particles The plateau region is reached earlier for β2M than BSA on all latex

particles and the K values (equilibrium rate constant of second order adsorption) for the

β2M adsorption process onto latex particles are much larger than for BSA adsorption

These values indicate that the β2M molecules have a higher adsorption rate onto the latex

particles than the BSA molecules

Table 4-16 Fitting parameters of second-order kinetic model BSA β2M

Latex particles qe (mgm2) K (hr-1) qe (mgm2) K ( -1) hr

PS 144 67 074 7549

PSPMMA100 175 353 076 4438

PSPMMA90PAA10 046 97 075 5695

PSPMMA75PAA25 060 179 075 5168 K = (kqe) hr-1

There are several explanations for the phenomena observed in these experiments

First the smaller sizes of β2M molecules allow easier diffusion and a close approach to

the latex substrate Second a reduced number of hydrophobic domains on the latex

particle surface replaced by sulfate groups (-OSO3-) and ionized carboxylic groups

(COO-) are not favorable for larger BSA molecules Third the conformational change by

intra- and inter-molecular repulsion would be faster in β2M due to smaller molecular

weight Finally the ionic repulsion between β2M and the latex particles is less than that

condition (pH 74) than IEP (48) of BSA

between BSA and latex particles because IEP (57) of β2M is closer to the tested

112

44 Blood Compatibility

Blood compatibility can be defined as the property of a material or device tha

permits it to function in contact with blood without inducing any adverse reactions

Successful functioning of blood contacting biomedical materials or devices in contact

with blood strongly depends on the extent to which they damage blood during operation

High shear stresses turbulence rela

t

tively long contact times and cellular impact with

artificial surfaces are most common events experienced by blood in contact with artificial

1978 Winslow and et al

1993] Therefore before any materia e placed into the human b

biocompatibility testing must be the first performed in the view of safety The hemolysis

s a method for pr ary screening of biomaterials to determine

h blood a er circulating body fluid The tests estimat

oglobin released from th blood cell d e induced

foreign material

There are several types of blood cells in our body but the ones that make blood red

are erythrocytes or red blood cells (RBCs) Red blood cells (erythrocytes) are the most

numerous type in the blood These cells are average about 48-54 million per cubic

millimeter (mm3 same as a microliter (microl)) of blood but these values can vary over a

larger range depending on such factors as health and altitude RBCs are red because they

contain hemoglobin which is a protein that contains iron When blood circulates through

lungs oxygen binds to the hemoglobin and is then delivered to all the parts of body

RBCs are made in the bone marrow They start out as a nucleus like other cells but

when they fill up with hemoglobin the nucleus becomes smaller until it disappears

Without a nucleus RBCs are fragile and live only about 120 days In a normal healthy

devises and can cause hemolysis or thrombosis [Bernhard et al

ls can b ody

test serves a elimin the

biocompatibility wit nd oth e the

concentration of hem e red amag by a

113

body about 2 million red blood ne marrow produces new

ones j ing

dy

le

cells die per second But bo

ust as fast RBCs are about 75 micrometer across and are responsible for carry

oxygen salts and other organic substances to the cells and tissues throughout our bo

In humans and most mammals the cells are concave (indented in the middle) and flexib

enough to squeeze through even the tiniest of blood vessels the capillaries

Figure 4-38 Image of red blood cells

(Sourcehttpwwwpbrchawaiiedubemfmicroangelarbc1htm)

ch

100 90 10 75 25 f

RBCs are among the first cells that come into contact with a foreign material

injected in the blood Therefore a hemolysis assay gives information about the

biocompatibility in the case of an in vivo application The concentrations of prepared

latex particles used for the tests in this research are 05 25 and 50 (ww) and the

incubation time under moderate shaking was 30 minutes at 37oC Test results are shown

in Figure 4-39 The amount of blood cells that ruptured when in contact with the PS latex

particles was largely increased from about 30 to 91 as the solid content of PS

increased from 05 to 50 However hemolysis by the core shell latex particles su

as PSPMMA PSPMMA PAA and PSPMMA PAA was less than 35 O

114

particular interest was that hemolysis caused by PSPMMA PAA particles at 50

solid concentration was less than 02 hemolysis the lowest value among the latex

particles The low hemolysis value indicates that the PSPMMA PAA core shell latex

particles are the most blood compatible at all solid contents Other core shell particles

such as PSPMMA and PSPMMA PAA also had excellent blood comp

90 10

90 10

100 75 25 atibility with

low RBS breakage

01 2 3

20

40

60

80

100

Hem

olys

is (

)

052550

Figure 4-39 He

PS PSPMMA100 PSPMMA PAA90 10

molysis caused by latex particles n=5

4PSPMMA75PAA25

CHAPTER 5 SUMMARY OF RESULTS CONCLUSIONS AND RECOMMENDATION FOR

FUTURE WORK

51 Summary of Results

The goal of this study was to synthesize monodisperse polymeric latex particles

with tailored properties and to investigate the fundamental interactions between the

synthesized latex particles and target proteins An understanding of the fundamental

mechanism of selective adsorption is strongly required in order to maximize the

separation performance of membranes made of these materials so that it may applicable

to hemodialysis therapy for end stage renal disease (ESRD) patients For the achievement

of above research goal several investigations have been performed Analysis of the

obtained data leads to several conclusions as discussed below

Monodisperse polystyrene seed latex particles were synthesized with the size

crosslinking agent to make the latex particles more hydrodynamically stable The

conversion of monomer to polymer was over 95 and seed latex particles are spherical in

shape and have smooth surface From these small seeds bigger latex particles as large as

800nm were synthesized using a semi-continuous seeded emulsion polymerization

process The particles with a mean diameter less than 500nm are very spherical in shape

and highly uniform in particle size distribution However as the particles grow larger

than 500nm in mean diameter they became non-spherical with an uneven surface This

irregularity of particle surface can be attributed to the non-homogeneous monomer

ranges between 126plusmn75 and 216plusmn53 nm Divinylbenzene (DVB) was used as a

115

116

swelling into the shell of a growing polymer which can be controlled by the factors such

as temperature agitation speed initiator feeding rate and surfactant amount required to

stabilize the colloidal system It was determ rfactant to monomer ratio

dec ic

acid (AA) monomers were intro rticle surfaces more

hydro

to

ting of a

orm

ration is seen between

3002

0 cm-

0cm-1

-1

03

ined that as the su

reased the mean particle diameter increased Methyl methacrylate (MMA) and acryl

duced to make the latex pa

philic than bare polystyrene particles The prepared core shell latex particles were

made of PMMA100 PMMA90PAA10 and PMMA75PAA25 PMMA100 is the PMMA

homopolymer shell on a PS core PMMA90PAA10 shell is a copolymer with a PMMA

PAA ratio of 90 to 10 by weight PMMA75PAA25 shell is a copolymer consis

PMMA to PAA ratio of 75 to 25 by weight The particle size of these core-shell

particles were prepared to be about 370nm in mean diameter

The successful synthesis of latex particles was confirmed using Fourier Transf

Infrared spectroscopy (FTIR) The C-H aromatic stretching vib

cm-1 and 3103 cm-1 for PS latex particles The C-H asymmetrical stretching and

symmetrical stretching vibration peaks of CH2 for all latex particles is seen at 290

1and 2850 cm-1 respectively There is a carbonyl (C=O) characteristic band at 173

wavenumber for PSPMMA core-shell latex particles The broad OH group peak at

3400 cm

100

can be seen in the spectroscopy of the particle with PMMAPAA shell

(PSPMMA PAA and PSPMMA PAA ) The peak intensity of OH group of

PSPMMA PAA was larger than that of PSPMMA PAA relatively due to more AA

monomer content in PSPMMA PAA than in PSPMMA PAA

In the zeta potential measurements polystyrene (PS) latex particles had negative

values between -291 mV and -599 mV in the phosphate buffer (PB) and between -2

90 10 75 25

75 25 90 10

75 25 90 10

117

mV and -278 mV in the phosphate buffered saline (PBS) at 25oC and pH 21-78 These

negative zeta potential values for PS latex particles are due to ionization of the sulfate

groups (-OSO3-) originated from the initiator Zeta potential values of the PSPMMA

core shell latex particles were also negative between -28 mV and -505 mV in PB and

between -143 mV and -186 mV in PBS at pH 21-78 Negative values are also

generated by dissociation of sulfate groups on a particle surface The zeta potential value

of PSPMMA PAA and PSPMMA PAA were of negative values between -36

and -678 mV in PB medium and -147 mV and -193 mV in PBS medium at 25

100

s

7 mV

MMA75PAA25 and

PSPM

otein

g the

l change of BSA at

each w

90 10 75 25

oC For

PSPMMA PAA core shell particles zeta potential values were between -291 mV and

-520 mV in PB media and between -115 mV and -210 mV in PBS media Sulfate

groups contributed to the negative zeta potential values in PSP

75 25

MA90PAA10 core shell particles Carboxylic groups from polyacrylic acid also

contribute to the negative surface charge for the latex particles at greater than pH 70

however the degree of their contribution was not detectable from the zeta potential

measurements The absolute zeta potential values of the synthesized latex particles in PB

were higher than that in PBS This is because of the high concentration of sodium

chloride electrolyte in PBS that compress electrical double layer thickness

The bicinchoninic acid (BCA) assay technique was used to determine the pr

adsorption and all isotherms were fitted to the Langmuir-Freundlich isotherms usin

nonlinear regression method The hydrophobic interaction between PS latex particle and

BSA protein was dominant for the adsorption process Conformationa

pH also affected the adsorption amount which was highest at pH 48 because of lo

ionic repulsion making compact conformation of BSA The next larger amount of BSA

118

adsorbed onto PS was at pH 32 There were two main forces affecting the amount of

adsorption at acidic pH values One is ionic attraction between negative latex particle a

positive protein The other is lateral repulsion between positive proteins The smallest

amount of BSA adsorption was at pH 74 where charge repulsion between negative

particles and proteins is present at this pH Lateral repulsion between proteins wa

dominant for protein adsorption even though there always exists the hydrophobic

interaction between solid latex particle and protein The charge repulsion was reduced by

the high concentration of electrolytes in the PBS medium The BSA adsorption process

on PSPMMA core shell particles was very similar to the case of PS latex particle where

hydrophobic interaction between latex particle and protein was dominant However t

adsorbed BSA amount was less than that of PS in PBS The adsorbed amount of BS

PSPMMA

nd

s

he

A on

an

ed

n The main

adsor

90PAA10 and PSPMMA75PAA25 core shell particles was higher at pH 32 th

pH 48 and 74 The adsorbed BSA amount became higher at pH 32 and pH 48 as the

hydrophilicity was increased by the higher PAA amount On the contrary the adsorb

amount of BSA was dramatically decreased at pH 74 Carboxylic acid groups as well as

the sulfate groups are distributed on the surface of PSPMMA90PAA10 and

PSPMMA75PAA25 core shell particles and largely affect the BSA adsorptio

ption interaction forces at pH 32 was generated by hydrogen bonding as well as

charge attraction between latex particle and protein even when the conformational change

and charge repulsion between proteins reduce the adsorption of protein at this pH The

adsorbed BSA amount was also increased as the amount of PAA increases However the

adsorption of BSA was minimal (less than 07 mgm2) at pH 74 where the carboxylic

groups are ionized leading to more enhancement of charge repulsion by ionization of the

119

carboxylic groups (COO-) Because of this the PSPMMA-PAA latex particles have a

much lower protein adsorption

There were steep initial slopes for adsorption isotherms of β

in

s

there

M

r

e

es

o

2M on latex particles

all isotherms indicating a high affinity type adsorption The complete plateau region

were not seen for all isotherms indicating that β2M proteins were not saturated and

are still available sites for β2M adsorption on latex particles The initial slope of β2

isotherms for PS was steeper than for the other latex particles PSPMMA100

PSPMMA90PAA10 and PSPMMA75PAA25

The layer thickness (δ) of BSA proteins on synthesized latex particles in phosphate

buffer (PB) was in the range of 27 - 81 Aring at 37 C and pH 48 Only the δ values at pH

48 the isoelectric point (IEP) of BSA are important because intra- and inter-molecular

electrostatic repulsion of the protein molecules is minimized at the IEP Compared to the

hydrodynamic dimensions of BSA 140 times 40 times 40 Aring these values of the adsorbed laye

thickness indicates that the BSA molecules adsorb by the side-on (40Aring) and end-on mod

(140Aring) Consideration of the multi-layering is unnecessary because the adsorbed

thickness of BSA do not exceed 140 Aring

The Gibbs free energy of β

o

3

2M was more negative in phosphate buffer (PB) than

that of BSA in physiological condition (37 C and pH 74) indicating that βo2M molecul

are more favorably adsorbed on the latex particles than BSA and that the affinity of β2M

was much larger than BSA This may be explained by the rate of diffusion and

accessibility of β2M molecules being easier and faster than that for BSA because of

smaller size (or weight) and spherical shape of β2M molecules It may also be possible t

explain that more hydrophobic forces (less electrostatic repulsion) contributes to

120

adsorption of β2M than to that of BSA because the IEP of β2M (57) is closer to

physiological condition (pH 74) than that of BSA (pH 48) The selective adsorption of

β2M m

in β2M

larger than in BSA adsorption indicating

that fa

an that between BSA and latex particles because

IEP (

ch as

Other

olecules over BSA molecules can be enhanced by developing more hydrophilic

PSPMMA90PAA25 and PSPMMA75PAA25 core shell latex particles

The adsorption kinetics of proteins on latex particles was expressed as a second-

order model The plateau region for β2M was reached earlier than BSA on all latex

particles and the K values (equilibrium rate constant of 2nd order adsorption)

adsorption process on latex particles were much

ster adsorption rate of β2M molecules than that of the BSA molecules There are

several explanations for these phenomena First the smaller size of β2M molecules can

more easily diffuse and approach to the latex substrate Second enhanced hydrophilic

domain on the latex particle surface by sulfate groups (SO4-) and ionized carboxylic

groups (COO-) is not favorable for larger BSA molecules but is for smaller β2M to be

anchored Third the conformational change by intra- and inter-molecular repulsion

would be faster in β2M due to lower molecular weight Finally the ionic repulsion

between β2M and latex particles is less th

57) of β2M is closer to the tested condition (pH 74) than IEP (48) of BSA

In the biocompatibility test the number of red blood cells (RBCs) ruptured by PS

latex particles ranged from 30 to 91 as the solid content of PS increased from 05 to

50 respectively However hemolysis of the other core shell latex particles su

PSPMMA100 PSPMMA90PAA10 and PSPMMA75PAA25 were less than 35 The

PSPMMA90PAA10 particles at all applied solid concentrations had less than 02

hemolysis and was the most blood compatible among the prepared latex particles

121

core shell particles such as PSPMMA100 and PSPMMA75PAA25 also had excelle

compatibility with low RBC breakage

52 Conclusions

The major conclusions from this study include

nt blood

bull microglobulin (β2M) over that of the bovine serum albumin (BSA) at pH 74

was achieved by controlling the PMMA and PAA ratio to optimize

e particles posses the most desirable requirements for use in a human dialysis

ns

icles

olecule

bull

Using engineered core shell latex particles selective adsorption of β2

physiological condition was demonstrated

bull The rate of adsorption of the β2M was also much higher than that of BSA This

hydrophobichydrophilic microdomain size

bull The core shell particles also exhibited excellent biocompatibility with blood Thes

membrane

53 Recommendation for Future Work

With the results and conclusions obtained through this study several suggestio

may be given for future work as follows

bull Competitive adsorption tests of whole blood proteins on synthesized latex partunder physiological condition

bull Preparation of latex particles with optimum hydrophobic hydrophilic microdomains to maximize selective adsorption and removal of toxin molecules

bull Membrane fabrication by suitable pore size design to discriminate a toxin mby size sieving as a function of particle layers

Evaluation of membrane performance such as flow rate in water and in simulated blood fluid (SBF)

bull Application for hemodialysis as well as other technologies such as a water remediation

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Korea on August 7 1968 He obtained a Bachelor of Science degree in Chemistry from

Keim d graduate school and earned a

resear n Korea Institute of Science and

Coati e

ia

Busin

BIOGRAPHICAL SKETCH

Sangyup Kim was born and raised in the small town of Sung-Joo Kyungbook

yung University (Daegu Korea) in 1993 He entere

Master of Science degree in Chemical engineering in 1995 He worked as a student

cher for 2 years at Textile Polymer Division i

Technology (KIST) in Seoul Korea Next he worked over a year at the Automobile

ng Division in DongJoo-PPG in Korea In August 2001 he began studying for th

degree of Doctor of Philosophy in materials science and engineering at the University of

Florida in Gainesville After getting his PhD he plans to work at the Digital Med

ess Division of Samsung Electronics in Korea starting in January 2006

141