Novel Polysaccharide Based Polymers and Nanoparticles for Controlled Drug Delivery and

Biomedical Imaging

by

Alireza Shalviri

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Graduate Department of Pharmaceutical Sciences University of Toronto

© Copyright by Alireza Shalviri (2012)

ii

Novel Polysaccharide Based Polymers and Nanoparticles for Controlled Delivery of Drugs and

Imaging Agents

Alireza Shalviri

Doctor of Philosophy

Graduate Department of Pharmaceutical Sciences

University of Toronto

2012

Abstract

The use of polysaccharides as building blocks in the development of drugs and contrast agents

delivery systems is rapidly growing. This can be attributed to the outstanding virtues of

polysaccharides such as biocompatibility, biodegradability, upgradability, multiple reacting

groups and low cost. The focus of this thesis was to develop and characterize novel starch based

hydrogels and nanoparticles for delivery of drugs and imaging agents. To this end, two different

systems were developed. The first system includes polymer and nanoparticles prepared by graft

polymerization of polymethacrylic acid and polysorbate 80 onto starch. This starch based

platform nanotechnology was developed using the design principles based on the

pathophysiology of breast cancer, with applications in both medical imaging and breast cancer

chemotherapy. The nanoparticles exhibited a high degree of doxorubicin loading as well as

sustained pH dependent release of the drug. The drug loaded nanoparticles were significantly

more effective against multidrug resistant human breast cancer cells compared to free

doxorubicin. Systemic administration of the starch based nanoparticles co-loaded with

doxorubicin and a near infrared fluorescent probe allowed for non-invasive real time monitoring

of the nanoparticles biodistribution, tumor accumulation, and clearance. Systemic administration

iii

of the clinically relevant doses of the drug loaded particles to a mouse model of breast cancer

significantly enhanced therapeutic efficacy while minimizing side effects compared to free

doxorubicin. A novel, starch based magnetic resonance imaging (MRI) contrast agent with good

in vitro and in vivo tolerability was formulated which exhibited superior signal enhancement in

tumor and vasculature. The second system is a co-polymeric hydrogel of starch and xanthan gum

with adjustable swelling and permeation properties. The hydrogels exhibited excellent film

forming capability, and appeared to be particularly useful in controlled delivery applications of

larger molecular size compounds. The starch based hydrogels, polymers and nanoparticles

developed in this work have shown great potentials for controlled drug delivery and biomedical

imaging applications.

iv

Acknowledgments

I would like to thank my supervisor Dr. Shirley Wu for believing in me as well as for her

kindness, leadership, patience, and continuous support. She always believed in my abilities and

pushed me to be the best I could be; for this I will always remain thankful to her. Without her

mentorship this thesis would have not been possible.

I am greatly appreciative to Drs. Ping Lee, Tigran Chalikian and Edgar Acosta for taking the

time to attend my committee meetings and giving me insightful guidance and recommendations.

I also thank Dr. Warren Foltz, Dr. Andrew Rauth, and Dr. Heiko Heerklotz for their continuous

support and guidance throughout my thesis.

My sincere thanks to all my colleagues who helped me, especially Ping Cai. I also extend my

gratitude to those not mentioned here who have taken part, small or large, in making this work

possible.

I am grateful to the Ontario Graduate Scholarship Program, Natural Sciences and Engineering

Research Council of Canada, BioPotato network (Co-leaders: Drs. Helen Tai and Yvan

Pelletier), Agricultural Bioproducts Innovation Program (ABIP) of Agriculture & Agri-Food

Canada, CIHR/CBCRA, University of Toronto and Leslie Dan Faculty of Pharmacy for

scholarships and research funding.

I thank my wife, Mana, for always being there for me. Without her love, care and undivided

attention I would not have achieved any of this.

I thank my parents and my sister for their belief in my abilities and constant moral and financial

support. I owe all my success to them. Finally, I thank the mice. Without them meaningful

innovation would not be possible.

v

Table of Contents

Acknowledgments.......................................................................................................................... iv

Table of Contents .............................................................................................................................v

List of Tables ................................................................................................................................ xii

List of Figures .............................................................................................................................. xiii

List of Abbreviations ................................................................................................................... xxi

Chapter 1. Introduction ....................................................................................................................1

1.1 Breast Cancer .......................................................................................................................2

1.1.1 Epidemiology ...........................................................................................................2

1.1.2 Breast Cancer Cells and Tumor Microenvironment ................................................2

1.1.3 Doxorubicin: a Potent Drug for Breast Cancer Chemotherapy ...............................5

1.1.4 Barriers to Cancer Chemotherapy ............................................................................7

1.1.5 Nanoparticulate Systems in Cancer Therapy .........................................................13

1.1.6 Nanoparticles as Theranostics in Cancer ...............................................................19

1.2 Polysaccharides in Drug Delivery .....................................................................................22

1.2.1 Starch .....................................................................................................................23

1.2.2 Xanthan gum ..........................................................................................................26

1.3 Biomedical Imaging ...........................................................................................................27

1.3.1 In vivo Fluorescence Imaging ................................................................................27

1.3.2 Magnetic Resonance Imaging ................................................................................30

1.4 Drug Delivery to the Brain ................................................................................................40

1.4.1 Brain Anatomy .......................................................................................................41

1.4.2 Strategies to Enhance Drug Delivery to the Brain .................................................43

1.5 Goal for this work ..............................................................................................................47

1.6 Synopsis .............................................................................................................................48

vi

Chapter 2. Design of pH-responsive Nanoparticles of Terpolymer of Poly(methacrylic acid),

Polysorbate 80 and Starch for Delivery of Doxorubicin ...........................................................51

2.1 Abstract ..............................................................................................................................52

2.2 Introduction ........................................................................................................................53

2.3 Materials and Methods .......................................................................................................55

2.3.1 Materials ................................................................................................................55

2.3.2 Synthesis of PMAA-PS 80-g-St Nanoparticles .....................................................55

2.3.3 FTIR and 1H NMR Spectroscopy ..........................................................................56

2.3.4 Examination of the Nanoparticles with TEM ........................................................57

2.3.5 Determination of Particle Size and Surface Charge ..............................................57

2.3.6 Titration Studies .....................................................................................................57

2.4 Results and Discussion ......................................................................................................58

2.4.1 PMAA-PS 80-g-St Nanoparticles were Synthesized Using a Simple One-pot Method ...................................................................................................................58

2.4.2 Polymer Composition of the Nanoparticles ...........................................................60

2.4.3 Nanoparticles Size and Morphology ......................................................................64

2.4.4 PMAA-PS 80-g-St Nanoparticles Show pH-responsive Swelling in Physiological pH Range .........................................................................................67

2.4.5 Properties of Carboxylic Acid Groups in the Nanoparticles .................................68

2.4.6 Effect of Processing Parameters on Particle size and pH Sensitivity ....................71

2.5 Conclusions ........................................................................................................................74

2.6 Acknowledgements ............................................................................................................74

Chapter 3. pH Dependent Doxorubicin Release by Nanoparticles Based on Terpolymer of

Poly(Methacrylic Acid), Polysorbate 80, and Starch for Overcoming Multi-drug

Resistance in Breast Cancer Cells .............................................................................................75

3.1 Abstract ..............................................................................................................................76

3.2 Introduction ........................................................................................................................77

3.3 Materials and Methods .......................................................................................................80

vii

3.3.1 Materials ................................................................................................................80

3.3.2 Cell Maintenance ...................................................................................................80

3.3.3 Synthesis of PMAA-PS 80-g-St Nanoparticles .....................................................81

3.3.4 Fourier Transform Infrared Spectroscopy .............................................................81

3.3.5 Isothermal Titration Calorimetry (ITC) .................................................................81

3.3.6 Dynamic Light Scattering ......................................................................................82

3.3.7 Transmission Electron Microscopy .......................................................................82

3.3.8 Drug Loading Studies ............................................................................................83

3.3.9 X-ray Powder Diffraction (XRPD) ........................................................................84

3.3.10 In vitro Drug Release .............................................................................................84

3.3.11 Cell Uptake Studies Using Fluorescence Microscopy ...........................................84

3.3.12 Cellular Uptake of Nanoparticles by Flow Cytometry ..........................................85

3.3.13 In vitro Assessment of Anticancer Efficacy of Dox-loaded Nanoparticles ...........86

3.4 Results ................................................................................................................................86

3.4.1 Properties of PMAA-PS 80-g-St Nanoparticles and Their High Capability of Efficiently Loading Dox without Loss of Colloidal Stability ................................86

3.4.2 FTIR, XRD and ITC Experiments Revealed Strong Ionic Interaction between the Nanoparticles and Dox .....................................................................................88

3.4.3 The Nanoparticles Exhibited Sustained and pH Dependent Release of Dox in vitro ........................................................................................................................95

3.4.4 Substantial Cellular Uptake of the Nanoparticles Evidenced by Fluorescence Microscopy, TEM and Flow Cytometry ................................................................96

3.4.5 The Dox Loaded Nanoparticles Were Significantly More Effective Against

MDR1 Cells than Free Dox .................................................................................101

3.5 Discussion ........................................................................................................................103

3.6 Conclusions ......................................................................................................................105

3.7 Acknowledgements ..........................................................................................................105

viii

Chapter 4. Evaluation of New Multifunctional Nanoparticles based on Terpolymer of

Polymethacrylic acid, Polysorbate 80, and Starch as a Theranostic Nanoplatform for

Simultaneous in vivo Imaging and Treatment of Breast Cancer .............................................106

4.1 Abstract ............................................................................................................................107

4.2 Introduction ......................................................................................................................108

4.3 Materials and Methods .....................................................................................................110

4.3.1 Materials ..............................................................................................................110

4.3.2 Preparation of Dual Mode Nanoparticles ............................................................111

4.3.3 Dynamic Light Scattering, Electrophoretic Mobility Measurements, and Transmission Electron Microscopy .....................................................................113

4.3.4 Drug Release Studies ...........................................................................................113

4.3.5 Cell Lines .............................................................................................................114

4.3.6 Animal Model and In vivo Treatment Protocol in Tumor Bearing Mice ............114

4.3.7 Real Time In vivo and Ex-vivo Near-infrared Fluorescent Imaging ....................115

4.3.8 Ex-vivo Tumor Fluorescence Microscopy ...........................................................117

4.4 Results ..............................................................................................................................118

4.4.1 Properties of the Nanoparticles ............................................................................118

4.4.2 Distribution and Tumor Accumulation of the PF-NPs and SA-NPs in Whole Animals In vivo ....................................................................................................122

4.4.3 Real Time Pharmocokinetics of Nanoparticles in Tumor Tissue ........................125

4.4.4 Organ Distribution of the Nanoparticles Determined by Ex Vivo Imaging ........126

4.4.5 Microscopic Imaging of Tumor tissue Demonstrated Extravasation of the Nanoparticles in the Tumor .................................................................................128

4.4.6 Anti-tumor Efficacy of the Nanoparticles in a Murine Breast Cancer Tumor Model ...................................................................................................................130

4.4.7 Preliminarily Assessment of Toxicity of the Nanoparticles ................................132

4.5 Discussion ........................................................................................................................134

4.6 Conclusions ......................................................................................................................136

4.7 Acknowledgments............................................................................................................137

ix

Chapter 5. A New Starch-based Polymeric MRI Contrast Agent with Superior Signal

Enhancement in Blood and Tumor .........................................................................................138

5.1 Abstract ............................................................................................................................139

5.2 Introduction ......................................................................................................................140

5.3 Materials and Methods .....................................................................................................142

5.3.2 Cell line and Maintenance ...................................................................................142

5.3.3 Preparation of the Gd3+

Loaded PMAA-PS 80-g-St-DTPA Polymer (PolyGd) ..143

5.3.4 Confirmation of DTPA Conjugation to Starch ....................................................144

5.3.5 Determination of DTPA Content and Binding Affinity of Gd3+

to St-DTPA .....145

5.3.6 Determination of Cytotoxicity of PolyGd............................................................145

5.3.7 Comparison of the Relaxivity Properties of PolyGd and Omniscan® in vitro ....146

5.3.8 Experimental Animals and Induction of Subcutaneous Breast Tumors ..............146

5.3.9 Determination of In vivo MRI Contrast Enhancement of PolyGd in Mice .........147

5.3.10 Validation of Whole-body Gd3+ Distribution by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) ........................................................148

5.4 Results and Discussion ....................................................................................................148

5.4.1 Confirmation of DTPA Conjugation to Starch ....................................................148

5.4.2 Determination of DTPA Content and Binding Affinity of Gd3+ to St-DTPA .....154

5.4.3 PolyGd Exhibited Lower Cytotoxicity than Free Gd3+ ........................................155

5.4.4 PolyGd showed much higher relaxivity than Omniscan®...................................156

5.4.5 In vivo MRI Contrast Enhancement of PolyGd in Mice ......................................159

5.4.6 Biodistribution and Clearance of the PolyGd from the Body ..............................167

5.5 Conclusions ......................................................................................................................168

5.6 Acknowledgments............................................................................................................169

Chapter 6. Evaluating the Capability of Novel Starch Based Nanoparticles for Controlled

Delivery of Drugs and Imaging Agents to the Brain ..............................................................170

6.1 Abstract ............................................................................................................................171

x

6.2 Introduction ......................................................................................................................172

6.3 Materials and Methods .....................................................................................................174

6.3.1 Materials ..............................................................................................................174

6.3.2 Synthesis and Preparation of the PMAA-Ps 80-g-St Polymer and

Nanoparticles .......................................................................................................175

6.3.3 Physicochemical Characterization of the PMAA-PS 80-g-St Polymer and Nanoparticles .......................................................................................................175

6.3.4 Time-of-Flight-Secondary Ion Mass Spectrometry .............................................176

6.3.5 Animal Studies .....................................................................................................176

6.3.6 In vivo Magnetic Resonance Imaging (MRI).......................................................176

6.3.7 Ex-vivo Fluorescence Imaging of the Brain .........................................................177

6.3.8 Fluorescence Microscopy ....................................................................................178

6.4 Results ..............................................................................................................................179

6.4.1 Properties of PMAA-PS 80-g-St Polymer and Nanoparticles .............................179

6.4.2 Brain Accumulation of the PMAA-PS 80-g-St Nanoparticles at Macroscopic

and Microscopic Levels .......................................................................................184

6.5 Conclusions ......................................................................................................................189

Chapter 7. Novel Modified Starch-xanthan Gum Hydrogels for Controlled Drug Delivery:

Synthesis and Characterization ...............................................................................................190

7.1 Abstract ............................................................................................................................191

7.2 Introduction ......................................................................................................................192

7.3 Materials and Methods .....................................................................................................193

7.3.1 Chemicals .............................................................................................................193

7.3.2 Synthesis of Cross-linked Fully Gelatinized Starch and Xanthan Gum Hydrogels .............................................................................................................193

7.3.3 Examination of Film Morphology .......................................................................194

7.3.4 Confirmation of Cross-linking by Fourier Transformed Infrared Spectroscopy .194

7.3.5 Solid State 31P NMR Spectroscopy......................................................................194

xi

7.3.6 Study of the Swelling Behavior of Cross-linked Starch-xanthan Gum Polymer .195

7.3.7 Determination of Gel Mesh Size..........................................................................195

7.3.8 Permeability Studies ............................................................................................197

7.3.9 Statistical Analysis ...............................................................................................198

7.4 Results and Discussion ....................................................................................................198

7.4.1 Morphology..........................................................................................................198

7.4.2 Cross-linking of Starch-Xanthan Gum with STMP was Confirmed ...................199

7.4.3 Swelling Kinetics and Equilibrium Swelling Ratio .............................................205

7.4.4 Effect of STMP and Xanthan gum Concentration on the Film Swelling ............206

7.4.5 Effect of Medium pH and Buffer salts on the Film Swelling ..............................209

7.4.6 Mesh Size of the Modified Starch-Xanthan Gum Gels .......................................210

7.4.7 Permeability Studies ............................................................................................212

7.5 Conclusion .......................................................................................................................215

7.6 Acknowledgements ..........................................................................................................216

Chapter 8. Conclusions and Future Perspectives .........................................................................217

8.1 Overall Conclusions and Original Contributions of This Thesis .....................................217

8.2 Limitation of the Work and Future Directions ................................................................222

8.2.1 Biodegradation, Body Clearance, and Biocompatibility of the PMAA-PS 80-

g-St Nanoparticles ................................................................................................222

8.2.2 Utility of the PMAA-PS 80-g-St Polymer and Nanoparticles as a Dual Model Imaging Probe ......................................................................................................223

8.2.3 Active Targeting ..................................................................................................224

8.2.4 Delivery of Dual Agents by the Nanoparticles ....................................................225

8.2.5 In vivo assessment of PMAA-PS 80-g-St for drug delivery to the CNS .............226

8.2.6 Optimal Polymerization Method for Making More Uniform Polymers .................227

8.2.6 In Vivo Assessment of Efficacy in Multidrug Resistant Tumor Model ..............227

Bibliography ................................................................................................................................228

xii

List of Tables

Table 1.1 Examples of drug-nanoparticles system for delivery to the brain ................................ 44

Table 2.1 Nanoparticle preparation recipes and the polymer composition.. ................................ 60

Table 2.2 Intensity-weighted hydrodynamic diameter of nanoparticles with different feed molar

ratio of MAA/St in 0.15 M PBS of various pH.. .......................................................... 65

Table 3.1 Characterization of the drug-loaded nanoparticles. The effect of drug loading on the

particles size and surface charge is investigated.. ......................................................... 87

Table 4.1 Summary of physicochemical properties of SA-NPs and PF-NPs ............................. 118

Table 4.2 Tumor associated pharmacokinetic data derived from the tumor average fluorescence

intensity versus time curve for SA-NPs and PF-NPs.. ............................................... 126

Table 5.1 Gd3+

content, molecular weight, and r1 for Omniscan® and PolyGd. The r1 were

measured in saline at 3 and 7 T.. ................................................................................. 158

Table 7.1 Equilibrium swelling ratio of films with 10% xanthan gum and various cross-linker

STMP levels.. .............................................................................................................. 206

Table 7.2 Equilibrium volume swelling and gel mesh size of starch-xanthan gum gels containing

10% xanthan gum and varying concentrations of cross-linker (STMP).. ................... 212

Table 7.3 Equilibrium swelling ratio of films in 0.15M phosphate buffer of pH=7.4 and

permeability of vitamin B12 across modified starch-xanthan gum films of various

compositions. .............................................................................................................. 213

Table 7.4 Permeability of macromolecules and drugs of various molecular weights and charges

across the starch-xanthan gum gel film containing 10% XG and 5% STMP in 0.15 M

phosphate buffer of pH=7.4.. ...................................................................................... 214

Table 7.5 Permeability of two weakly acidic drugs across starch-xanthan gum films containing

10%XG and 5% STMP in pH 2 and 7.4 buffer solutions with ionic strength of 0.15M.

.................................................................................................................................... 215

xiii

List of Figures

Figure 1.1 Chemical structure of Doxorubicin ............................................................................... 6

Figure 1.2 Factors leading to MDR in cancer. One of the leading causes of treatment failure in

cancer is the onset of resistant disease. MDR can be divided into two broad categories:

Cellular resistance and non-cellular resistance. ............................................................ 11

Figure 1.3 Passive targeting of the tumor by EPR. The EPR effect is the balance of enhanced

tumor permeability with poor tumor interstitial fluid drainage, resulting in the selective

uptake and retention of nanoparticles in the tumor tissue. ........................................... 14

Figure 1.4 Drug loaded nanoparticles can overcome MDR cancer cells. Endocytosis of the drug

loaded nanoparticles in membrane bound vesicles protects the drug from the action of

the membrane efflux pumps. The nanoparticles release the drug deep inside the cell

and the drug can gain access to its cellular target site (e.g. DNA) ............................... 14

Figure 1.5 Chemical structure of starch ........................................................................................ 24

Figure 1.6 Xanthan gum chemical structure ................................................................................. 27

Figure 1.7 Schematic comparison of fluorophores in the visible spectrum versus the near infrared

for deep in vivo fluorescent imaging. Fluorophores in the visible regions are limited by

poor penetration of the excitation photon or the poor penetration of the emission

photons preventing the detection of fluorescent signal in vivo. Near infrared photons

can provide deep tissue penetration. ............................................................................. 29

Figure 2.1 Schematic reaction of starch grafting and terpolymer formation. FTIR spectra of

Starch, PMAA-PS 80, and PMAA-PS 80-g-St. ............................................................................ 61

Figure 2.2 1H NMR spectra of A) PS 80, B) Starch, C) PMAA-PS 80, D) PMAA-PS 80-g-St-2 in

0.05M NaOD ................................................................................................................ 63

Figure 2.3 A) Intensity-weighted hydrodynamic diameter of the PMAA-PS 80-g-St-2

nanoparticles in 0.15 M pH 7.4 PBS. The particles showed a narrow size distribution.

B) TEM images of PMAA-PS 80-g-St-2 in 0.15 M PBS of pH=7.4.. ......................... 66

xiv

Figure 2.4 A) Relative diameter vs. pH for the nanoparticles with different feed molar ratio of

MAA/St in 0.15 M PBS. DpH/D4 represents particles diameter at different pH relative

to pH 4. B) Effect of pH on surface charge for particles of various MAA/St molar

ratio.. ............................................................................................................................. 68

Figure 2.5 A) Potentiometric titration curves. Empty triangles represent the uncorrected

potentiometric titation curve for PMAA-PS 80-g-St-2 latex dispersion. Filled circles

represent the titration curve after correction. Empty circles show the blank titration

curve. The arrow represents the equivalence point. The equivalence points are used to

calculate the MAA contents in various nanoparticle batches. B) Variation in the

apparent dissociation constant (pKa) as a function of the degree of ionization (α) for

nanoparticles of different starch and MAA contents. ................................................... 70

Figure 2.6 Effect of A) SDS, B) PS 80, C) total monomer concentration, D) cross-linker molar

ratio on particle size and pH sensitivity.. ...................................................................... 73

Figure 3.1 A) Number-weighted Gaussian distribution of PMAA-PS 80-g-St nanoparticles

loaded with doxorubicin (LC=33%) in 0.15 M phosphate buffer at pH 7.4, B)

Transmission electron micrograph of doxorubicin loaded nanoparticles (LC=33%)... 88

Figure 3.2 FTIR spectra of A) PMAA-PS 80-g-St nanoparticles, B) Dox, C) Dox loaded PMAA-

PS 80-g-St nanoparticles.. ............................................................................................. 89

Figure 3.3 XRD spectrum of A) Doxorubicin in native form, B) PMAA-PS 80-g-St

nanoparticles, C) Doxorubicin loaded nanoparticles (LC=50%), D) doxorubicin loaded

nanoparticles (LC=50%) after 6 months storage at room temperature.. ....................... 91

Figure 3.4 A) The blank differential enthalpy curves of titrating 8.5 mM doxorubicin into buffers

of various pH. B) Differential enthalpy curves of titrating 8.5 mM doxorubicin into

0.1mg/ml PMAA-PS 80-g-St nanoparticles in buffers at different pH. The ionic

strength was kept constant at 0.15M by addition of NaCl. C) The blank differential

enthalpy curves of titrating 8.5 mM doxorubicin into DDIW with different NaCl

contents, D) Differential enthalpy curves of titrating 8.5 mM doxorubicin into

0.1mg/ml PMAA-PS 80-g-St nanoparticles in DDIW with different NaCl contents. .. 94

xv

Figure 3.5 Effect of pH on kinetics of doxorubicin release from the naoparticles with drug

loading content of 33% at 37 ºC. The release of free doxorubicin from the dialysis bag

was used as control. For each buffer system the ionic strength was kept constant at

0.15 M by adding NaCl. ................................................................................................ 95

Figure 3.6 A) Fluorescence microscopy image of MDA-MB435/LCC6 cells with and without

(control) 4 hr incubation with fluorescent nanoparticles at final concentration of 0.25

mg/ml. Nuclei were stained with Hoechst 33342 and visualized with DAPI filter, cell

membranes were stained with Vybrant™DiI and visualized with Cy3 filter, and NPs

were labelled fluoresceinamine isomer I and visualized with FITC filter. Optical slices

were taken every 2 µm from the uppermost and lowermost regions of the cell, allowing

for selection of an image at approximately the midpoint of the nucleus. B) TEM

micrographs of MDA-MB435/LCC6 cells treated with 0.25 mg/ml PMAA-PS 80-g-St

NPs for 4 hrs. The nanoparticles were loaded with gadolinium (metal) and appear as

electron dense deposits. ................................................................................................ 98

Figure 3.7 Flow cytometry histograms for MDA-MB435/LCC6 cells showing the effect of

incubation time and temperature on particle uptake. The cells were incubated with

fluorescent labelled naoparticles at the final nanoparticle concentration of 0.25 mg/ml.

A) MDA-MB435/WT (1) untreated cells at 37 ºC, (2) 1 hr incubation at 37 ºC, (3) 4

hrs incubation at 37 ºC, (4) 24 hrs incubation at 37 ºC. B) MDA-MB435/WT (1)

untreated cells, (2) 1 hr incubation at 4ºC, (3) 1hr incubation at 37 ºC. C) MDA-MB

435/MDR1 (1) untreated cells, (2) 1 hr incubation at 37 ºC, (3) 4 hrs incubation at 37

ºC, (4) 24 hrs incubation at 37 ºC. D) MDA-MB435/MDR1 (1) untreated cells, (2) 1 hr

incubation at 4ºC, (3) 1 hr incubation at 37 ºC.. ......................................................... 100

Figure 3.8 Determination of the response of MDA-MB435/LCC6 cell types to free doxorubicin

and doxorubicin loaded nanoparticles by MTT assay. (A-B) Cell viability of MDA-

MB435/LCC6/WT (n=3) cells after exposure to increasing concentrations of blank

nanoparticles (blank NPs), free doxorubicin and doxorubicin loaded nanoparticles

(Dox-NPs) for 24 hrs (A) and 48 hours (B). (C-D) Cell viability of

MDA435/LCC6/MDR1 (n=3) cells after exposure to increasing concentrations of

blank NPs, free doxorubicin and Dox-NPs for 24 hrs (C) and 48 hrs (D). Cells with no

xvi

treatment and incubated with blank nanoparticles were used as control for free drug

and drug loaded nanoparticle respectively. Cell viability is expressed as the percent of

control for each treatment group. ................................................................................ 102

Figure 4.1 A) Schematic diagram of PF-NPs and SA-NPs and the reaction scheme for

conjugation of the NIR dye and loading of Dox. B) Chemical structure of the PMAA-

PS 80-g-St polymer. .................................................................................................... 119

Figure 4.2 Size distribution and shapes of A) SA-NPs and B) PF-NPs, as determined by dynamic

light scattering (DLS) and transmission electron microscopy (TEM), respectively. . 120

Figure 4.3 Drug release kinetics from the SA-NPs (LC=21.1%) and PF-NPs (LC=49.7%). Drug

release was measured in 0.15 M Tris/NaCl buffer, pH=7.4, at 37 ºC. The release of free

doxorubicin from the dialysis bag was used as control. ............................................. 121

Figure 4.4 A) Whole animal real time biodistribution and tumor targeting of SA-NPs and PF-

NPs in mice bearing an orthotopic breast tumor model. Nanoparticle-associated

fluorescence was determined prior to intravenous injection (baseline), and then at

various hours following nanoparticles injection up to 14 days. B) Time-dependent

excretion profiles of SA-NPs and PF-NPs from the whole body (left) and tumor (right).

The fluorescence intensity for the region of interest was recorded as average radiant

efficiency.. .................................................................................................................. 124

Figure 4.5 Quantitative results of tissue distribution and tumor accumulation for SA-NPs and PF-

NPs. Ratio of the relative fluorescence intensity in major organs, tumor, and blood as a

function of time after intravenous injection of nanoparticles, compared to normal

major organs and tumors not injected with NIR dye conjugated-nanoparticles.. ....... 127

Figure 4.6 Microscopic distribution of the nanoparticles within the tumor. Fluorescent signal

observed in tumor tissue treated with A) vehicle only; B) FITC-labeled SA-NPs; C)

FITC-labeled PF-NPs. Tumors arising from orthotopically implanted EMT6/WT cells

were allowed to grow for 8 days prior to injection of nanoparticles. Four hours

following nanoparticle introduction, animals were sacrificed and the distribution of

particles assessed within both core and peripheral regions. ....................................... 129

xvii

Figure 4.7 Anti-tumour activity of starch based nanoparticles in EMT6/WT tumor bearing mice.

Tumor cells were implanted orthotopicly on day zero. Mice were treated with 5%

dextrose (n=2×4), free Dox (n=8), PF-NPs (n=2×4), and SA-NPs (n=2×3) at a dose of

2×10 mg/kg equivalent to Dox on day 8 and 15. A) Tumor volume up to day 62. Each

curve represents one animal. B) Kaplan Meier survival curves for 5% dextrose, free

Dox, PF-NPs, and SA-NPs. The trend in survival curves were significantly different

(p=0.0033, Mantel Cox).............................................................................................. 131

Figure 4.8 Time profiles of body weight of tumor-bearing mice treated with 5% dextrose

(n=2×4), free Dox (n=2×4) , PF-NPs (n=2×4), and SA-NPs (n=2×3) at a dose of 2×10

mg/kg equivalent to Dox. Balb/c mice were inoculated with EMT6/WT tumor in the

mammary fat pad and received treatment on day 8 and 15 post inoculation. Each curve

represents one animal. ................................................................................................. 132

Figure 4.9 Histological sections of lung, kidney, liver, and heart of tumor-bearing mice treated

with 5% dextrose, free Dox, PF-NPs, and SA-NPs at a dose of 2×10 mg/kg equivalent

to Dox. The samples were not collected at the same time after treatment, but rather

collected after euthanasia of the animals as necessitated by tumor size end point.

Sections were stained with H&E and observed under a light microscope. ................ 134

Figure 5.1 A) Reaction of starch with DTPA-bisanhydride to form St-DTPA by direct acylation

of starch hydroxyl groups. DTPA-bisanhydride can react with one starch molecule to

form St-DTPA or two starch molecules to form ST-DTPA-St. B) Possible chemical

structures of PolyGd. .................................................................................................. 150

Figure 5.2 FTIR spectra of A) starch, B) DTPA, C) St-DTPA.. ................................................ 152

Figure 5.3 1H NMR spectra of A) DTPA, B) starch, C) St-DTPA. Major peaks for DTPA and

starch have been assigned on the molecular schemes.. ............................................... 153

Figure 5.4 Normalized differential heat (NDH) curves titrating 2 mM Gd3+

into 0.1 mg/ml

aqueous buffer solution of St or St-DTPA at 25 ºC and pH 5.6. Titration of Gd3+

into

starch did not produce any heat while titration into St-DTPA produced a large

endothermic heat indicating binding of Gd3+

to St-DTPA. Assuming one Gd3+

ion

xviii

binds to one DTPA molecule, the amount of covalently bound DTPA to starch is

calculated at the titration end point (inflection point) indicated by an arrow. ............ 155

Figure 5.5 The toxicity of saline, blank polymer, PolyGd, and free Gd3+

to rat hepatocytes in

culture exposed for 30 min, 60 min, 120 min, or 240 min. “%live” represents the

percent of hepatocytes excluding trypan blue.. ........................................................... 156

Figure 5.6 Coronal T1-weighted (3D-FLASH, TE/TR 3/25 msec, flip angle 20º) whole body

images of Balb/c mice injected with Omniscan® (0.1 mmol/kg Gd3+

) and PolyGd

(0.025 mmol/kg Gd3+

). At one fourth the dose of Omniscan®, the PolyGd produces a

much higher contrast over an extended period of time in the cardiovascular system. 160

Figure 5.7 Quantitative MRI of whole-body distribution: A) R1 maps of Balb/c mice injected

with PolyGd (0.025 mmol/kg Gd+3

). B) Change in relaxation rates, ∆R1, of left

ventricular blood, liver, bladder, and kidneys for Omniscan® (0.1 mmol/Kg Gd3+

) and

PolyGd (0.025 mmol/Kg Gd3+

) overtime relative to baseline. The Gd3+

loaded polymer

causes a much higher increase in blood relaxation rate for an extended period of time

compared to Omniscan®.. .......................................................................................... 162

Figure 5.8 MR angiography: A) MIP angiogram displaying contrast enhancement of (1) whole

body and (2) neck and head regions, obtained prior to and at 15 minutes following

PolyGd injection at 0.025 mmol/kg Gd3+

. B) Kinetics of vascular signal to noise (S/N)

ratio and contrast to-noise (C/N) ratio measured from the inferior vena cava in whole-

body angiograms.. ....................................................................................................... 164

Figure 5.9 Tumor distribution of PolyGd (0.025 mmol/kg Gd3+

): A) T1-weighted images (1) and

the corresponding R1 maps (2). B) Time course of ∆R1 in tumor periphery and tumor

core, displaying elevated tumor R1 even 48 hours after contrast agent injection.. ..... 166

Figure 5.10 Biodistribution, elimination and tumor accumulation of the PolyGd (0.025 mmol/kg

Gd3+

) in tumor bearing Balb/c mice. The Gd3+

content was determined using ICP-

AES.. ........................................................................................................................... 168

Figure 6.1 A) 1H NMR spectra of 1) PS 80, 2) PMAA-PS 80-g-St in 0.1M NaOD. B) polymer

composition and physical properties of the PMAA-PS 80-g-St nanoparticles. For size

xix

measurements, the particles were disperse in PBS pH of 7.4 and ionic strength of 150

mM. For ξ-potential measurements PBS buffers of 7.4 and ionic strength of 10 mM

was used ...................................................................................................................... 180

Figure 6.2 A) Schematic diagram of self-assembly of PMAA-PS 80-g-St terpolymer into

nanoparticles upon complexation with Gd3+

and conjugation of the fluorescent

moieties. B) TEM images of the self-assembled nanoparticles in water. ................... 182

Figure 6.3 Negative TOF-SIMS spectra of PS 80, PMAA-g-St, and PMAA-PS 80-g-St, in the

m/z range of 0 to 300 atomic mass units. ................................................................... 183

Figure 6.4 Quantitative MRI of brain distribution: A) R1 maps of Balb/c mice (n=3) injected with

Gd3+

loaded PMAA-PS 80-g-St nanoparticles (0.05 mmol/kg Gd+3

). B) Longitudinal

relaxation rates (R1) of sagittal sinus, ventricles, cortex, and sub-cortex for Gd3+

loaded

PMAA-PS 80-g-St-DTPA polymer overtime.. ........................................................... 185

Figure 6.5 Qualitative and quantitative results of brain distribution and accumulation for PMAA-

PS 80-g-St nanoparticles. A) Ex-vivo near infrared fluorescence images of the whole

brain. Ratio of the relative fluorescence intensity in brain as a function of time after

intravenous injection of nanoparticles compared to normal brain not injected with

nanoparticles. B) Fluorescence microscopy image of perfused mice brains 45 minutes

following iv administration of saline, PMAA-g-St, and PMAA-PS 80-g-St. The

particles can be detected in the perivascular regions of the brain capillaries for samples

treated with PMAA-PS 80-g-St nanoparticles. ........................................................... 188

Figure 7.1 SEM images of (A) surface (left-bottom corner) and cross section (right-top corner),

(B) surface, (C) cross section of cross-linked starch-xanthan gum film containing 10%

xanthan gum and 5% STMP. ...................................................................................... 199

Figure 7.2 FTIR spectra of (A) pure starch, (B) starch reacted with 20% STMP, (C) pure xanthan

gum, (D) xanthan gum reacted with 20% STMP, (E) physical mixture of starch and

xanthan gum, (F) physical mixture of starch, xanthan gum, and 20% STMP, and (G)

mixture of starch and xanthan gum reacted with 20% STMP .................................... 201

xx

Figure 7.3 Schematic representation of cross-linking reaction of starch and xanthan gum with

sodium trimethaphosphate (STMP) ............................................................................ 202

Figure 7.4 A) 31

P NMR spectra of (I) pure starch, (II) starch reacted with 5% STMP, (III) pure

xanthan gum, (IV) xanthan gum reacted with 5% STMP. B) 31

P NMR spectra of (I)

starch-xanthan gum without STMP, (II) STMP, (III) physical mixture of starch,

xanthan gum and STMP, (IV) starch and 5% xanthan gum reacted with 5% STMP. C)

31P NMR spectra of (I) starch and 10% xanthan gum reacted with 2% STMP, (II)

starch and 10% xanthan gum reacted with 5% STMP, (III) starch and 10% xanthan

gum reacted with 20% STMP. .................................................................................... 205

Figure 7.5 Swelling kinetics of cross-linked starch-xanthan gum films containing 5% xanthan

gum and 2% or 10% STMP in 0.15M phosphate buffer of pH=7.4 ........................... 207

Figure 7.6 Equilibrium swelling behavior of modified starch xanthan gum films of various

compositions with respect to change in (A) xanthan gum concentration in DDIW (B)

pH with constant ionic strength of 0.15M. ................................................................. 208

Figure 7.7 Relative diffusion coefficients of molecular probes as a function of molecular radius

across modified starch-xanthan gum films with various STMP levels. ..................... 211

Figure 7.8 A plot of Y/Q-1 as a function of the hydration factor (Q-1)-1

for starch-xanthan gum

hydrogels of various compositions. The slope of the line is equal to the scale factor, Y.

.................................................................................................................................... 212

xxi

List of Abbreviations

1H-NMR hydrogen nuclear magnetic resonance spectroscopy

31P-NMR phosphorous nuclear magnetic resonance spectroscopy

AcAn acetic anhydride

ALT alanine aminotransferase

α-MEM alpha-modified minimal essential medium

ANOVA analysis of variance

ATP adenosine-5’-triphosphate

AUC area under the curve

BBB blood brain barrier

CCAC Canada council on animal care

CK creatine kinase

CO2 carbon dioxide

D2O deuterated water

DAPI 4’,6-diamino-2-phenylindole

DDIW distilled deionized water

DLS dynamic light scattering

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

Dox doxorubicin

DTPA diethylenetriaminepenta acetic acid

DTPA-bis-An diethylenetriaminepenta acetic acid bisanhydride

EDC N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride

EE encapsulation efficiency

xxii

EPR enhanced permeability and retention effect

EtOH ethanol

FA fluoresceinamine isomer I

FBS fetal bovine serum

FDA food and drug administration

FITC fluorescein isothiocyanate

FTIR Fourier transform infrared spectroscopy

Gd gadolinium

GD growth tumor delay

GSH glutathione

H&E hematoxylin and eosin

HCl hydrochloric acid

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HF 750 HiLyte Fluor TM

750 hydrazide

IC50 inhibitory concentration for 50% effect

ITC isothermal titration calorimetry

Kel elimination rate constant

KPS potassium persulfate

LDH lactate dehydrogenase

λem emission wavelength

λex excitation wavelength

LC loading content

LRP lung resistance protein

LV left ventricle

MAA methacrylic acid

MBA N,N′-Methylenebisacrylamide

xxiii

MDR multidrug resistance

MPS mononuclear phagocytic system

MRI magnetic resonance imaging

MRP1 multidrug resistance protein 1

MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide

NADPH nicotinamide adenine dinucleotide phosphate

NaOH sodium hydroxide

NHS N-Hydroxysuccinimide

NIR near-infrared

NPs nanoparticles

PBS phosphate buffered saline

PEG polyethylene glycol

PET positron emission tomography

PF-NPs preformed nanoparticles

Pgp P-glycoprotein

PMAA polymethacrylic acid

PMAA-PS 80-g-St polymethacylic acid grafted starch

PS 80 polysorbate 80

Py pyridine

Ri relaxation rate

ri relaxivity

ROS reactive oxygen species

S/V surface area to volume ratio

SA-NPs self-assembled nanoparticles

SD standard deviation

SDS sodium dodecyl sulphate

xxiv

SEM standard error of the mean

St starch

STMP sodium trimethaphosphate

STS sodium thiosulfate

t1/2 half life

TEM transmission electron microscopy

v/v volume by volume

w/v weight by volume

XG xanthan gum

XRPD x-ray powder diffraction

ξ zeta potential

1

Chapter 1. Introduction

2

1.1 Breast Cancer

1.1.1 Epidemiology

Cancer is a serious and prevalent health problem that affects us all. There were 12,661,000 new

cases of cancer in 2008, and 7,564,000 people had died from previous cancer diseases [1]. In

2011, 177,800 Canadians were newly diagnosed with cancer, and cancer related deaths

accounted for 29.3% of total deaths in Canada [2].

Breast cancer is the most prevalent form of cancer among women. There were 1,384,000 new

cases in 2008, making up 23% of all women’s cancer [1]. With 458,000 deaths worldwide, breast

cancer is considered the most frequent cause of cancer related death in women. According to

Canadian Cancer Society, there were 23,600 new cases of breast cancer in 2011, making it the

most common cancer in Canadian women. [2]. Although the mortality rates are on a gradual

decline in most developed countries since 1990, mainly due to better screening techniques and

more effective treatment strategies, the disease claimed the lives of 5,100 Canadian women,

comprising 14.8% of total cancer deaths, second only to lung cancer [2].

1.1.2 Breast Cancer Cells and Tumor Microenvironment

Breast cancer, as in other solid cancers, is fundamentally characterized by continuous and

uncontrolled growth. Many researchers share the view that tumorigenesis proceeds via a process

analogous to Darwinian evolution, in which a succession of genetic changes, each conferring one

or another type of growth advantage, leads to the progressive conversion of normal human cells

into cancer cells. Hanahan and Weinberg have proposed that six essential alterations in cell

physiology enabled by the genetic instability result in malignant growth [3, 4]. These include

continuous proliferation independent of cells’ microenvironment, acquired resistance to anti-

proliferative signals, evasion of apoptosis, ability to replicate without limits, ability to induce

3

angiogenesis, and the ability of some cancer cells to undergo metastasis. These acquired

physiological alterations generally apply to solid tumors, including breast cancer.

Breast tumors are not merely an accumulation of neoplastic cells, but rather a complex tissue

with blood vessels, stromal cells, infiltrating immune-competent cells, and a differentiated

extracellular matrix [3-5]. All these cell types interact with each other to build a unique tumor

microenvironment. This heterogeneous mass grows until it reaches an approximate volume of 2

mm3, beyond which the diffusion of nutrients and oxygen cannot take place and areas of hypoxia

and acidosis develop [6, 7]. As a result, the tumor contains interspersed regions of well

oxygenated (pO2 > 2.5 mmHg) and poorly oxygenated (pO2 ≤ 2.5 mmHg), or hypoxic, tissue

heterogeneously distributed throughout the tumor mass. Cancer cells of a breast tumor can adapt

to thrive in areas of low oxygen concentrations that would otherwise induce normal cell death [8,

9]. Normal cells fulfill 90% of their energy requirements through the Krebs cycle which uses

pyruvate formed from glycolysis in a series of reactions that donate electrons via NADH and

FADH2 to the respiratory chain complexes in mitochondria [8]. This high efficiency glucose

metabolism requires oxygen. With limited oxygen, such as with muscles that have undergone

prolonged exercise, pyruvate is not used in the Kreb’s cycle and is converted into lactic acid by

lactate dehydrogenase (LDH) in a process termed anaerobic glycolysis. This process can also

produce cellular energy but with poor efficiency. Warburg was the first one to report that even in

the presence of oxygen, 50% of tumor ATP is produced through glycolytic catabolism [8]. This

switch from high efficiency aerobic oxidative phosphorylation to low efficiency anaerobic

glycolysis for cellular chemical energy production in tumors is known as the Warburg effect [8,

9]. Through reduction of the oxygen consumption, the Warburg effect enhances cancer cell

survival in hypoxic tumor microenvironment by decreasing the oxygen-starved fraction of the

tumor distal from blood vessels, and by reducing the production of the reactive oxygen species

4

(ROS) that are the by-product of electron transport in the mitochondria during the Kreb cycle

[10]. Due to cytotoxic nature of the ROS, reduction in their production by the Warburg effect

provides cancer cells with a survival advantage in hypoxic tumor tissues [11].

Tumor growth beyond 2 mm3 is angiogenesis dependent. Angiogenesis is defined as the process

of formation of the new blood vessels. The hypoxic nature of the tumor environment activates a

series of hypoxia-sensitive transcription factors in cancer cells, stromal fibroblasts, and tumor

associated macrophages. These cells all work in harmony to generate new tumor blood vessels

[12-14]. Formation of new vascular network by angiogenesis supply the tumor with oxygen and

nutrients required to support its continued growth. However, tumor vasculature is significantly

different in terms of its structure and physiology from healthy blood vessels. Many tumors reveal

chaotic networks of tortuous and distended veins, venules and venous capillaries along with

intertwining capillaries branching from arterioles and veins [15, 16]. Irregularities of vascular

wall structures in tumors have also been described with walls composed of a mosaic of cancer

and endothelial cells which leads to widened interendothelial junctions and numerous endothelial

fenestrations with the net effect of leaky blood vessels unique to tumor neovasculature [17].

These blood vessels are highly permeable to the extravasation of therapeutic macromolecules

and small colloidal particles [18, 19].

The resulting intratumoral circulation is characterized by tortuous microvessels lacking the

normal hierarchical arrangement of arterioles, capillaries and venules. Within this altered

microenvironment, blood flow is sluggish with unstable rheology, anomalous and generally

stagnant [15, 16]. As a result, these characteristics lead to heterogeneous perfusion with hypoxia

and acidity in low-flow regions. In fact, hypoxia is a pathophysiological property of breast

tumors, with up to 60% of the tumor existing in a hypoxic state [7].

5

Accumulation of lactic acid in the tumor microenvironment coupled with insufficient blood

supply and poor lymphatic drainage results in acidic pH states in the solid tumor

microenvironment. Although there is a distribution, in vivo pH measurements, made by needle

type microelectrodes on human patients having various solid tumors (adenocarcinoma, squamous

cell carcinoma, soft tissue sarcoma, and malignant melanoma) in readily accessible areas (limbs,

neck, or chest wall), show the mean pH value to be 6.9 with values as low as 5.7 being reported

for some tumors [20, 21]. Increasingly, it has been proposed by many researchers that the mildly

acidic tumor environment can be exploited to achieve high local drug concentrations and to

minimize overall systemic exposure [22, 23] . The use of pH responsive carrier systems in the

delivery of chemotherapeutics will be discussed in section 1.1.5.3.

1.1.3 Doxorubicin: a Potent Drug for Breast Cancer Chemotherapy

Doxorubicin (Dox), Figure 1.1, is a chemotherapeutic agent which belongs to the anthracycline

antibiotics family. The drug has been widely adopted as a first line chemotherapy agent, most

often in combination with other agents, for the treatment of various types of cancer including

hematological malignancies, carcinomas, and sarcomas [24, 25]. Its widespread use in the

treatment of breast cancer has been recognized as one of the reasons for the decreased mortality

rates of the disease [26]. A number of mechanisms have been proposed to account for the broad-

spectrum anticancer activity of Dox. These include: (1) DNA intercalation by its planar three-

ring structure, (2) poisoning topoisomerase II enhancing DNA strand breaks, (3) generation of

free radicals, (4) possible disruption of the cell membrane functionality [27-29]. The cell nucleus

is the main cellular target of Dox even though involvement of mitochondria in Dox cytotoxicity

such as mitochondria-mediated apoptosis has also been observed [30]. It is likely that multiple

pathways are used by Dox to inflict damage to cancer cells.

6

Dox is commonly administered intravenously in the form of commercially available injections

Adriamycin® and Rubex® for maintaining the therapeutic levels in blood. In addition, two

PEGylated liposomal formulations of Dox, Doxil® and Caelyx®, are also available.

Dox administration in breast cancer chemotherapy is often limited by serious systemic side

effects such as myelosuppression and congestive heart failure [31-33]. The Dox induced

cardiomyopathy appears to be cumulative limiting the maximum lifetime dose of the drug in

humans to 450 mg/m2 [33]. The detailed mechanism of cardiotoxicity is not fully understood;

however, it is believed that Ca2+

activated ATPase, cAMP, and lipid peroxidation are involved,

as well as the ability of Dox to produce free radicals. Myocardium cells seem to have an

enhanced sensitivity to these effects leading to cardiomyopathy, and decreased cardiac

ventricular ejection fraction [29, 31, 32, 34].

Although Dox is highly effective in a variety of non-resistant breast cancer cell lines, its efficacy

is significantly reduced in multi-drug resistant (MDR) sub-cell lines such as murine

EMT6/AR1.0 and human MDA435/LCC6/MDR1. Hence Dox is an excellent compound for

evaluation of novel MDR-reversal approaches.

Figure 1.1 Chemical structure of Doxorubicin

7

1.1.4 Barriers to Cancer Chemotherapy

1.1.4.1 Drug Resistance

Drug resistance is one of the major obstacles in successful and effective treatment of breast

cancer. The underlying causes of drug resistance are complex and multi-factorial providing the

cancer cells with many ways to survive cancer chemotherapy. In general, the mechanisms of

drug resistance can be classified into non-cellular resistance and cellular resistance. These two

mechanisms are described in more detail below.

Non-cellular resistance: Poor efficacy in classical chemotherapy often is associated with the

unique anatomical or physiological features of the solid tumors. It is a well-known fact that

multi-cellular spheroids of tumor cells are more resistant to anticancer agents than the

corresponding monolayer cultures [35]. Solid tumors are also found to be less sensitive to

chemotherapy than malignancies consisting of individual cells, e.g. leukemia. As discussed in the

previous section, the tumor environment and blood vessel architecture is unique and

characterized by hypoxic regions, acidic microenvironment, sluggish, and inhomogeneous blood

circulation. Most blood vessels inside tumor are highly disorganized as they take tortuous turns

and many of these twisted blood vessels near the center of tumor, are crushed due to the irregular

growth of tumor in a confined space. Some sites in the tumor are thus far away from the blood

supply making it difficult for drug molecules, especially those with larger molecular weight and

lipophilicity, to reach these sites [36]. In addition, due to the elevated vascular permeability of

tumor vessels along with the absence of a functional lymphatic network, in this confined space,

interstitial fluid is collected more abundantly than in normal tissue, creating another impediment

to drug distribution inside tumor mass. The accumulation of this fluid leads to elevated

interstitial fluid pressure that decreases from the core toward the periphery of the tumor [37]. The

8

altered composition of extracellular fluid, associated with increased interstitial pressure and

sluggish blood flow hinders drug distribution and therefore the efficacy of chemotherapy.

As discussed previously poor blood supply and excessive growth leads to development of

hypoxic regions in tumors. Hypoxic cells may become quiescent and resistant to cytotoxic agents

that target proliferating cells [38]. The reduction in oxygen levels also compromises the anti-

cancer efficacy of some drugs such as Dox which is known to be much more effective in

oxygenated regions of the tumor by generating ROS species through the redox cycling of its

quinine nucleus [28]. Hypoxia, especially chronic hypoxia, can promote metabolic changes and

genomic instability of cancer cells, further enabling the acquisition of random mutations and

driving the malignancy of the tumor [38, 39]. Furthermore, the acidic environment of the tumor

due to production of lactic acid may potentially deactivate weak base drugs such as doxorubicin

[40].

In summary, non-cellular resistance is one of the major causes of poor efficacy of conventional

chemotherapeutic agents in solid tumors as only a limited fraction of the systemically

administered anticancer drug can reach and penetrate the target tumor site and remain active.

Cellular resistance: The cancer cells may still survive despite the significant levels of drug in

the tumor. Multidrug resistance (MDR) is a major barrier to effective treatment of cancer. This is

due to the ability of cancer cells to effectively neutralize the cytotoxicity of classical agents such

as Dox. MDR can be acquired following failed rounds of drug therapy, or it can be innate, pre-

programmed into the genetic code of the cancer cells [40, 41, 42]. In addition, the drug resistance

in MDR cells is rarely specific to one drug and generally develops as cross-resistance to a range

of structurally and functionally unrelated compounds [43]. Hydrophobic, amphiphathic drugs

such as vinca alkaloids (vincristine, vinblastine), taxanes (paclitaxel, docetaxel),

9

epipodophyllotoxins (etoposide, teniposide), anthracyclines (doxorubicin, daunorubicin) are

more frequently associated with MDR phenomena. MDR is often attributed to cell membrane

drug transport proteins, metabolic pathways, and intracellular targets common to cancer

chemotherapeutics [41, 43, 44].

Over-expression of ATP Binding Cassette (ABC) efflux pumps is one of the major mechanisms

of the MDR. P-glycoprotein (P-gp) and Multidrug Resistance Protein 1 (MRP1) are two main

ABC efflux pumps that are over-expressed in drug resistant breast cancers [45, 46]. Like all

ABC family members, both P-gp and MRP1 utilize the energy released from ATP hydrolysis to

transport drug molecules in the cell membrane to the outside. P-gp and MRP1 works in concert

to effectively decrease the intracellular concentration of the drugs before they become active and

reach their cellular target [41]. P-gp preferentially transports neutral or mildly cationic

compounds while MRP1 is more effective against lipophilic anions (e.g. glucuronate or

glutathione conjugates) [45]. The expression of both pumps is controlled by hypoxia-inducible

transcription factors, and is responsive to changes in intracellular redox status and ROS

production [47, 48]. Hence, the hypoxic nature of the tumor microenvironment as well as the

redox cycling associated with certain cytotoxic drugs such as Dox can upregulate the expression

of these plasma membrane efflux pumps induce acquired MDR. It has been suggested that

certain non-ionic surfactants (e.g. polysorbate 80), and block co-polymers (e.g. PluoronicTM P85)

may inhibit the P-gp efflux pumps [49]. In addition to P-gp and MRP1, breast cancer resistance

(BCRP) has also been reported in some resistant breast cancer cells [45]. More than one of the

mentioned membrane-associated drug transports may be present in the same cancer cell and

render the cell even more resistant to chemotherapy.

10

Alternatively, cancer cells may become resistant by sequestration of drugs in cytoplasmic

vesicles. Using fluorescence microscopy and taking advantage of the fluorosence properties of

Dox, Beyer et al. have demonstrated that the subcellular distribution of the drug following cell

uptake was significantly different between chemosensitive and MDR cell lines with higher

accumulation of the drug in the cytosolic vesicles being observed in MDR cells [50]. By

preventing the drug from interacting with its cellular targets, MDR cancer cells can effectively

neutralize the chemotherapeutic agents.

MDR can also be the result of cell ability to evade apoptosis. Apoptosis is the process of

programmed cell death or cell suicide that can be elicited by a number of stimuli such as DNA

damage caused by certain anticancer agents (e.g doxorubicin and cisplatin) [51]. The apoptotic

pathway involves highly organized and specific signal transduction that is negatively or

positively regulated by anti-apoptotic factors and pro-apoptotic factors. Pakunlu et al.

demonstrated that Bc12, an anti-apoptotic factor, is upregulated in Dox resistant tumor cells [52].

By avoiding the occurrence of apoptosis, cancer cells become less sensitive to certain

chemotherapeutic agents.

Some drug-resistance cancer cells were also found to have more active drug detoxification

systems. Glutathione (GSH) and glutathione-S-transferase act to sequester a number of

anticancer agents and are overexpressed in MDR cells [42]. An increase in intracellular GSH

facilitates the removal of the drug-GSH conjugates from the cell via MRP1 which is also

upregulated in MDR cells [48].

Cancer cells may also protect themselves from the action of cytotoxic drugs by alteration in drug

targets themselves. By altering the conformation of the drug target or masking the drug docking

site through post-translational modification, the efficiency with which the target is modified or

11

its function disrupted by the drug is decreased, rendering the cell resistant to the action of the

drug [41]. For example, altered tubulin structure may compromise the effectiveness of anti-

microtubule agents [53].

Many anticancer drugs exert their action by inflecting damage to DNA. The resistant cells often

modify their DNA repair mechanisms through upregulation of the DNA repair proteins [44].

Increased repair of Dox-DNA adducts would decrease the rate of induced DNA lesions and

promote the restitution of DNA structure, overcoming Dox genotoxicity. In addition, DNA

damage signalling pathways responsible for relaying the information regarding genotoxicity to

effectors of apoptosis are shut down, rendering the cells resistant to the cytotoxic drugs [41, 44].

As discussed, clinical drug resistance is a multi-factorial phenomenon, and the development of

MDR phenotype in cancer patients may involve any combination of the several physiological

alterations outlined above (Figure 1.2). While MDR may be acquired through different

mechanisms in different patients, the development of multidrug resistance is almost universal.

Figure 1.2 Factors leading to MDR in cancer. One of the leading causes of treatment failure in

cancer is the onset of resistant disease. MDR can be divided into two broad categories: Cellular

resistance and non-cellular resistance.

Factors leading to multi-drug resistance

Cellular resistance

Drug efflux pumps

up-regulation

Intracellular Sequestration

Drug detoxification

enzymes

up-regulation

Increased DNA repair

capacity

Anti-apoptotic proteins

up-regulation

Drug targets down-

regulation

Non-cellular resistance

Tumor Hypoxia

and acidity

Sluggish blood flow

Low intratumoral

drug concentration

Elevated interstitial

fluid pressure

12

1.1.4.2 Systemic Side Effects

Chemotherapeutics are often non-specific to cancer cells. A systemically administered cytotoxic

drug causes toxicity in both normal and cancer cells. As a result, a major limitation of cancer

chemotherapy is the systemic toxicity of the agents which ultimately influences their therapeutic

efficacy. While most of the classical chemotherapeutics such as Dox are more toxic towards

highly proliferating cells, these drugs will still induce toxicity in other cell populations due to

their ability to produce ROS through redox cycling reactions [28]. In addition to cancer cells,

other cells in the body such as the cells of gastrointestinal tract, hair follicles, and bone marrow

are continually proliferating in an adult, reducing the selectivity of classical chemotherapy.

Hence, some side effects such as nausea, vomiting, alopecia, and myelosuppression are common

for all cytotoxic drugs. While other side effects are more drug specific. For example, Dox

administration is associated with irreversible cardiotoxicity that is attributed to its major phase I

metabolite doxorubicinol, limiting the maximum allowable lifetime dose of the drug [54, 55]. It

has been suggested that Dox induces cardiomyopathy and congestive heart failure by interfering

with the sarcoplasmic reticulum. Co-administration of the cardio-protectants or the use of

anthracycline derivatives with lower demonstrated cardiotoxicity have been attempted by

different researchers [56-58]. However, these approaches have often led to reduced anti-cancer

efficacy, preventing effective anti-cancer outcomes.

Because anticancer drugs often show a steep dose-response curve, it is has been suggested by

some clinicians that aggressive chemotherapy is associated with greater therapeutic efficacy [59],

but high doses of chemotherapeutics are generally associated with high levels of acute and

chronic toxicity. In order to improve therapeutic efficacy, the drug associated systemic toxicity

needs to be reduced. This may be achieved through limiting the drug exposure to the healthy

13

cells while increasing the drug concentration in the cancer cells. This rational forms the basis for

the use of nanotechnology as drug delivery vector for cancer chemotherapy.

1.1.5 Nanoparticulate Systems in Cancer Therapy

Despite outstanding progress in the field of cancer biology and understanding the fundamentals

of the disease, challenges remain in our ability to translate our understanding of fundamental

cancer biology to the clinic. Classical chemotherapies suffer many challenges which include

dose limiting systemic toxicity, hypoxia, relatively low intra-tumoral drug levels, and MDR

phenotypes of cancer cells, often limiting their therapeutic potentials. Over the past two decades,

nanotechnology-based approaches have emerged as an exciting field with promises to remedy

these limitations. First, a well-designed nanoparticulate system with optimum size and

circulation half-life would facilitate controlled and tumor specific drug accumulation and release,

thus reducing the systemic toxicity that is often associated with classical chemotherapeutics and

increasing the possibility of more aggressive chemotherapy and possibly better clinical outcome

[60]. Second, the encapsulated drug is protected from the harsh environments of the body, drug

metabolizing enzymes, and extensive binding to serum proteins while in the circulation leading

to improved therapeutic efficacy [61]. Third, due to leaky nature of the tumor vasculature and its

poor lymphatic drainage, drug carriers with optimum size (50-300 nm) and prolonged blood

circulation will preferentially accumulate in the tumor, a phenomenon known as Enhanced

Permeation and Retention effect (EPR) (Figure 1.3) [18, 19]. Finally, the sub-micron size of the

drug carriers affords the ability to enter deep within the cell and overcome the MDR efflux pump

mechanisms which are currently one of the major causes of treatment failure in the clinic (Figure

1.4) [60, 62-67]. By designing an optimum nanoparticulate drug delivery system, the right drug

can be delivered to the right site in the body at the right time.

14

Figure 1.3 Passive targeting of the tumor by EPR. The EPR effect is the balance of enhanced

tumor permeability with poor tumor interstitial fluid drainage, resulting in the selective uptake

and retention of nanoparticles in the tumor tissue [18, 19].

Figure 1.4 Drug loaded nanoparticles can overcome MDR cancer cells. Endocytosis of the drug

loaded nanoparticles in membrane bound vesicles protects the drug from the action of the

membrane efflux pumps. The nanoparticles release the drug deep inside the cell and the drug can

gain access to its cellular target site (e.g. DNA)

Enhanced Permeability & Retention Effect (EPR)

1. Tumor-associated leaky vasculature2. Poor lymphatic drainage

Tumor

Neovasculature

Loose

Interendothelial

Junctions

(Fenestrations)

Blood

Flow

Lymph

Flow Tumor

~ < 300 nm

Blood

pH =7.4

Lysosomal

pH < 6

Tumor

pH < 7Drug

Dox efflux by

P-gp pumps

Leaky tumor

vasculatureActive uptake of NPs

by tumor cells

Dox

15

1.1.5.1 Requirements of an Ideal Nanoparticulate Drug Delivery System for Cancer Chemotherapy

Ideally, a drug carrier should have as many of the following properties as possible: (1) good

biocompatibility profile, (2) biodegradable with non-toxic degradation products, (3) convenient,

cost-effective, and reproducible preparation, (4) ability to efficiently load the drug at high

contents, (5) controlled and tumor specific drug release kinetics, (6) optimum size and

circulation half-life, (7) passive and/or active tumor targeting capabilities, and (8)

“upgradability”, i.e., allowing further surface modifications.

As mentioned previously, chemotherapeutics typically show a steep dose-response curve. To

ensure therapeutic success sufficiently high dose intensity has to be used. In other words,

reasonably high drug loading capacity and sufficient drug release at the diseased site are required

or else an unreasonably large quantity of the drug carrier has to be administered for effective

cancer treatment [68]. Cytotoxic drugs generally do not discriminate between cancer cells and

healthy cells in the body; hence, an ideal drug delivery system should exhibit minimal non-

specific drug release while in the circulation followed by increased release rate upon

accumulation in the tumor. Size, blood circulation time, and colloidal stability of the

nanoparticles are all important characteristics of the drug carrier. Nanoparticles which are

smaller than 10 nm are rapidly cleared by the renal route preventing adequate time for tumor

accumulation, while passive targeting by the EPR effect is significantly reduced with particles

larger than 300 nm [69, 70]. The size range of 50-200 nm has found to be optimal in promoting

the passive targeting of the nano-carriers to the tumor site. However, it has to be pointed out that

the range of acceptable nanoparticle sizes for optimized chemotherapy is highly material

dependent and will change from polymeric to inorganic to lipid based formulations. The long

circulation of the nanoparticulate drug delivery system is required to increase the number of

16

exposures of tumor tissue to the drug delivery system, promoting the passive tumor targeting by

EPR effect. In fact, it is believed that EPR effect-mediated passive tumor uptake is optimized

with circulation times of at least 4 hours [61, 70]. Surface grafting of some hydrophilic polymers

to the nanoparticles can prolong the circulation half-life of nanoparticles, and will be reviewed in

the next section.

1.1.5.2 Surface Modification to Prolong Nanoparticles Blood Circulation

Plasma proteins, including immunoglobulins and complement proteins readily bind to foreign

matters in the blood [61, 70]. These plasma proteins, or opsonins, effectively, tag the foreign

particles for removal from the circulation by resident tissue macrophages of the liver, lymph, and

spleen, known as the mononuclear phagocytic system (MPS) [71]. The removal of opsonised

nanoparticles from the systemic circulation reduces the longevity of nanoparticle circulation and

hence compromises its therapeutic efficacy. The surface of the nanoparticles can be modified to

prevent this rapid uptake producing long circulating or “stealth” particles which promote both

passive and/or active targeting. Vonarbourg et al. have reviewed the general conditions for the

stealthiness of colloidal drug carriers [72]. These include having a small size, with a neutral and

hydrophilic surface, as well as a thick, well-anchored and flexible coating.

The use of hydrophilic polymer coatings to nanoparticulates is a common practice to achieve a

long circulation time. The most commonly used polymer for this purpose is poly(ethylene

glycol) (PEG) [73]. It is generally accepted that the PEGylation of the particle surface prevents

opsonisation by enhancing the steric repulsion between nanoparticles’ surfaces and blood

components, and by forming an inert, impenetrable polymer layer over the surface of the

nanoparticles with the effectiveness mainly depending on the degree of surface coverage and

molar mass [74]. Additionally, it has been suggested that the PEG coats allow the selective

17

adsorption of some serum proteins that are sometimes known as dysopsonins, i.e. plasma

components which are believed to prevent opsonization. In other words, it may be the selective

adsorption, and not the prevention of adsorption, that alters the pharmacokinetics and fate of

nanoparticles [75, 76]. Nevertheless, it is the general consensus that the surface PEGylation of

the nanoparticles prolongs their blood circulation.

However, PEG is not biodegradable; also, chemical attachment of additional groups such as

targeting moieties, and/or metal chelators, to PEGylated surfaces is difficult and involves

complicated reaction schemes mostly due to the absence of reactive groups on the PEGylated

surfaces. This is one of the reasons why polysaccharide coatings have been considered as an

alternative to the PEG coatings. Additionally, oligo and polysaccharides may achieve active

targeting per se since they have specific receptors in certain cells or tissues [77]. Moreover,

polysaccharides display well-documented biocompatibilities and biodegradabilities, which are

the desired basic characteristics for polymers used as biomaterials. Polysaccharides have been

suggested as biocompatible polymer coatings for nanoparticles. For example, heparin coating of

poly(methyl methacrylate), PMMA, nanoparticles exhibited an increased circulation half-life of

5 h compared to only a few minutes for the bare nanoparticles [78]. Similarly, coating of

superparamagnetic iron oxide nanoparticles (SPIONs) with dextran increased their half-life up to

4.5 h [79]. However, there have been reports of hypersensitivity reactions upon administration of

dextran in the clinic [80]. Hydroxyethyl starch (HES) is currently investigated at the industrial

level as a biodegradable substitute for PEG, so that HESylation of proteins could substitute

PEGylation [81]. In one study poly(lactic-co-glycolic acid), PLGA, nanoparticles stabilized with

HES exhibited reduced human serum albumin (HSA) and fibrinogen (FBG) adsorption, and

uptake by phagocytic cells in vitro [82].

18

In summary, the polysaccharide coating may provide steric protection against protein adsorption

and macrophage uptake. Additionally, as polysaccharides offer many available reactive groups,

active targeting could be obtained by grafting ligands onto the nanoparticle surface. Considering

the very large variety of polysaccharides in nature, one could imagine the wide array of surface-

engineered nanoparticles adapted to a given therapeutic and monitoring purpose.

1.1.5.3 pH-responsive Nanoparticles in Cancer Chemotherapy

As discussed previously, tumor environment is acidic with average pH of 6.9 but