Nanosized selenium: a novel platform technology to prevent ... · As an important category of bacterial infections, healthcare-associated infections (HAIs) are considered as an increasing

Nanosized Selenium: A Novel Platform Technology to Prevent

Bacterial Infections

A Dissertation Presented

By

Qi Wang

To

The Department of Bioengineering

In partial fulfillment of the requirements

For the degree of

Doctor of Philosophy

In the field of Bioengineering

Northeastern University

Boston, Massachusetts

May 2015

ii

ABSTRACT

As an important category of bacterial infections, healthcare-associated

infections (HAIs) are considered as an increasing threat to the safety and

health of patients worldwide. HAIs lead to extended hospital stays,

contribute to increased medical costs, and are a significant cause of

morbidity and mortality. In the United States, infections encountered in

the hospital or a health care facility affect more than 1.7 million patients,

cost $35.7 billion to $45 billion, and contribute to 88,000 deaths in

hospitals annually.

The most conventional and widely accepted method to fight against

bacterial infections is using antibiotics. However, because of the

widespread and sometimes inappropriate use of antibiotics, many strains

of bacteria have rapidly developed antibiotic resistance. Those new,

stronger bacteria pose serious, worldwide threats to public health and

welfare. In 2014, the World Health Organization (WHO) reported

antibiotic resistance as a global serious threat that is no longer a

prediction for the future and it has the potential to affect anyone, of any

age, in any country.

The most effective strategy to prevent antibiotic resistance is

iii

minimizing the use of antibiotics. In recent years, nanomaterials have

been investigated as one of the potential substitutes of antibiotics. As a

result of their vastly increased ratio of surface area to volume,

nanomaterials will likely exert a stronger interaction with bacteria which

may affect bacterial growth and propagation. A major concern of most

existing antibacterial nanomaterials, like silver nanoparticles, is their

potential toxicity. But selenium is a non-metallic material and a required

nutrition for the human body, which is recommended by the FDA at a 53

to 60 μg daily intake. Nanosized selenium is considered to be healthier

and less toxic compared with many metal-based nanomaterials due to the

generation of reactive oxygen species from metals, especially heavy

metals.

Therefore, the objectives of this dissertation were to synthesize

selenium nanoparticles, characterize nanosized selenium coatings on

various materials, test the effectiveness of selenium coated materials at

inhibiting bacteria growth and biofilm formation and investigate the

mechanisms of how selenium nanoparticles inhibit bacteria growth. The

nanosized selenium coated materials showed significant and continuous

inhibitions to bacteria growth by up to 92.5% without using any

iv

antibiotics. The work performed in this dissertation presents a novel

platform technology based on nanosized selenium to inhibit bacterial

infections on various materials, which demonstrates the strong potential

applications of nanosized selenium as an antibacterial agent in hospital

environments and healthcare settings.

v

VITA

Qi Wang was born on September 24, 1988 in Loudi, Hunan Province, a

newly developing city in the central south of China. Before coming to the

United States for graduate studies, he received his Bachelor’s degree in

Chemical Physics at the University of Science and Technology of China

(USTC) in 2010. In his first two years in United States, he completed his

Master of Science degree in Chemistry at Brown University. Then and

now he has been working on nanomaterials, especially selenium

nanoparticles, for antibacterial applications in Prof. Thomas Webster's

Nanomedicine Lab. He has published 5 journal papers, 1 book chapter

and more than 6 conference proceedings as first author. He has presented

his research at over 20 national and international conferences. He has also

mentored 5 undergraduates and master students in their research projects.

His research is now being commercialized by a company called Senstruct.

After finish his PhD, he plans to work as a professional engineer in the

area of biomedical research and development.

vi

AC KNOWLEDGEMENTS

First of all, I would like to thank my graduate advisor, Dr. Thomas J.

Webster, for his guidance, mentorship and support to my research through

my 5 years of academia at Brown University and Northeastern University.

The lessons I have learned from him have made me a better biomedical

engineer.

I want to thank Dr. Justin Seil and Mr. Anson Leong for leading me into

nanosized selenium as a thesis research topic area and their help with my

initial experiments as a part of this work. I also would like to thank my

committee members, Dr. Rebecca Carrier and Dr. Shashi Murthy, for their

constructive suggestions and criticisms that have greatly improved the

quality of my work.

I would like to thank Dr. Edgar Goluch and Dr. Anand Asthagiri for

generously providing their lab space and resources for my research during

the first year of my career at Northeastern. Dr. Geoffrey Williams, Dr.

Joserph Orchardo, Dr. Vera Fonseca, Dr. Philip Larese-Casanova, and Mr.

vii

William Fowle are also acknowledged for helping me with various

facilities and instruments to accomplish my research. I would also like to

thank Northeastern University for funding my research.

I would like to thank all of the lovely lab members from the

Nanomedicine Lab for their support and making me enjoy my stay in the

laboratory. I also thank my best friend Daoling Huang for helping me to

overcome so many challenges in the process of perusing my PhD degree.

Most of all, I want to thank my parents who gave birth to me and

encouraged me to choose career path I am taking today. I thank the other

members of my family for being a source of inspiration for me and for

being a part of my life.

viii

TABLE OF CONTENTS

Abstract......................................................................................................ii

Vita....................................................................... .....................................v

Acknowledgements...................................................................................vi

Table of Contents....................................................................................viii

List of Tables..........................................................................................xiv

List of Illustrations..................................................................................xv

Chapter 1

Introduction.............................................................................................1

1.1 Bacterial and Biofilm Infections...............................................1

1.2 Antibacterial and Anti-infection Strategies...............................5

1.3 Nanomaterials as a Novel Antibacterial Approach.................12

1.4 Selenium and Nanosized Selenium........................................16

1.5 Objectives...............................................................................20

1.6 References..............................................................................21

Chapter 2

Selenium Nanoparticle Coatings for Preventing Biofilm Formation

ix

on Polycarbonate Medical Devices........................................................38

2.1 Introduction............................................................................38

2.2 Materials and Methods..........................................................42

2.3 Results....................................................................................46

2.3.1 SEM Images, AFM Images and AAS Results...............46

2.3.2 Bacterial Assays.............................................................51

2.4 Discussions.............................................................................56

2.5 Conclusions............................................................................58

2.6 References..............................................................................59

Chapter 3

Inhibition of Various Gram-positive and Gram-negative Bacteria

Growth on Paper Towels by Selenium Nanoparticles........................64

3.1 Introduction............................................................................64

3.2 Materials and Methods...........................................................70

3.2.1 Synthesis of Selenium Nanoparticle Coatings...............70

3.2.2 Characterization of Coated Paper Towels......................72

3.2.3 Mechanical Tests...........................................................73

3.2.4 Bacterial Inhibitory Tests...............................................74

3.2.5 Mechanism of Action: Protein Adsorption Assays........76

x

3.2.6 Release of Selenium......................................................77

3.2.7 Statistics.........................................................................78

3.3 Results and Discussion...........................................................79

3.3.1 Synthesis and Characterization of Selenium

Nanoparticles………………………………………………..79

3.3.2 Mechanical Tests...........................................................84

3.3.3 Bacterial Inhibitory Tests...............................................86

3.3.4 Mechanism of Action: Protein Adsorption Assays........94

3.3.5 Release of Selenium......................................................95

3.4 Conclusions............................................................................97

3.5 References..............................................................................98

Chapter 4

Red Selenium Nanoparticles and Gray Selenium Nanorods as

Antibacterial Agents for PEEK Medical Devices..............................106

4.1 Introduction..........................................................................106

4.2 Materials and Methods.........................................................112

4.2.1 Preparation of Selenium Coated PEEK.......................112

4.2.2 SEM and EDS of PEEK with Selenium Coatings.......114

4.2.3 Contact Angle Measurements......................................115

xi

4.2.4 Bacterial Inhibitory Tests.............................................116

4.2.5 Statistics.......................................................................118

4.3 Results and Discussion.........................................................119

4.3.1 PEEK Coated with Selenium Nanoparticles...............119

4.3.2 SEM and EDS of PEEK with Selenium Coatings.......120

4.3.3 Contact Angles.............................................................125

4.4.4 Bacterial Inhibitory Tests.............................................127

4.4 Conclusions..........................................................................131

4.5 References............................................................................132

Chapter 5

Antibacterial Selenium Quantum Dots and Nanoparticles Produced

by UV/VIS/NIR Pulsed Nanosecond Laser Ablation.........................138

5.1 Introduction..........................................................................138

5.2 Experiments and Results......................................................140

5.2.1 Preparation of Selenium Nanoparticles by Ablation...140

5.2.2 Characterization of Selenium Nanoparticles...............142

5.2.3 Generation of Selenium Quantum Dots.......................150

5.2.4 Antibacterial Tests.......................................................153

5.3 Conclusions..........................................................................155

xii

5.4 References............................................................................156

Chapter 6

Inhibited Growth of Pseudomonas aeruginosa by Dextran- and

Polyacrylic Acid-coated Ceria Nanoparticles.......................161

6.1 Introduction..........................................................................161


6 . 2 . 1 S yn t h e s i s a n d C h a r a c t e r i z a t i o n o f C e r i a

Nanoparticles…………………………..…………………..164

6.2.2 Bacterial Assays...........................................................166

6.2.3 Statistics.......................................................................167

6.3 Results..................................................................................168

6.3.1 Characterization of Ceria Nanoparticles……………..168


6.4 Conclusions..........................................................................174

6.5 References............................................................................175

Chapter 7

Investigation of Antibacterial Properties of ZTA and Silicon Nitride

Implants Compared with Titanium………………………………....181

7.1 Introduction..........................................................................181

xiii


7.2.1 Materials Characterization………………….………..184


7.3 Results..................................................................................187

7.3.1 Materials Characterization…………………………..187


7.4 Conclusions..........................................................................193

7.5 References............................................................................193

Chapter 8

Conclusions and Future Outlook........................................................199

Appendix...............................................................................................204

xiv

LIST OF TABLES

Table 2-1. Amount of selenium coated on polycarbonate surfaces

determined using AAS........................................................................48

Table 2-2. Difference in percentage of viable bacteria numbers on

nanosized selenium coated polycarbonate compared with control

groups (uncoated polycarbonate)........................................................52

Table 2-3. Log difference of viable bacteria numbers on nanosized

selenium coated polycarbonate compared with control groups

(uncoated polycarbonate) ..................................................................53

Table 3-1. Maximum load (the load at the moment of complete breakage)

of paper towels in tension tests...........................................................86

Table 3-2. The release of selenium from the selenium coated paper

towels..................................................................................................97

Table 4-1. Contact angles on three types of PEEK samples coated with

and without selenium……………….…………...............................127

Table 5-1. Summary of the laser parameters used, the corresponding size

and zeta potential of the selenium nanoparticles obtained…...……147

xv

LIST OF FIGURES

Figure 1-1. The biofilm life cycle...............................................................4

Figure 1-2. Common sites of biofilm infection..........................................5

Figure 1-3. Mean frequency of healthcare worker contact for 28 surfaces

in an intensive care unit........................................................................9

Figure 1-4. Mechanisms of nano-scaled silver interacting with bacteria.14

Figure 1-5. Major mechanistic injury responses induced by common

nanomaterials………………………..……………………………....16

Figure 1-6. Black/gray, glassy amorphous and red amorphous

selenium……………………….……………………………………17

Figure 2-1. SEM images of selenium coated polycarbonate samples

before and after a tape test..................................................................48

Figure 2-2. AFM images and RMS roughness of polycarbonate

surface.................................................................................................50

Figure 2-3. Bacteria (S. aureus) growth on the surface of polycarbonate.

Polycarbonate samples were treated with bacteria in 0.03% TSB

(Tryptic Soy Broth) and were incubated for 24, 48 or 72 hours…….52

Figure 2-4. S. aureus densities on various amounts of coated selenium on

xvi

the surface of polycarbonate...............................................................54

Figure 3-1. Light image of selenium coated and uncoated paper

towels..................................................................................................80

Figure 3-2. SEM images of selenium coated (image a) and uncoated

paper (image b) towel samples at a magnification of 8.0 K…….......81

Figure 3-3. Scanning electron microscopy images of paper towels.........82

Figure 3-4. AFM images and RMS roughness of paper towel surfaces...83

Figure 3-5. Tension tests for paper towels in various conditions.............85

Figure 3-6. The growth of Staphylococcus aureus on the surface of paper

towels..................................................................................................88

Figure 3-7. The growth of Staphylococcus epidermidis on the surface of

paper towels........................................................................................89

Figure 3-8. The growth of Pseudomonas aeruginosa on the surface of

paper towels........................................................................................91

Figure 3-9. The growth of E. coli on the surface of paper towels............93

Figure 3-10. The amount of adsorbed total proteins on selenium coated

and uncoated paper towels..................................................................95

Figure 4-1. Light images of (a) uncoated PEEK, (b) PEEK coated with

selenium nanoparticles (red coated PEEK), and (c) PEEK coated with

xvii

selenium nanoparticles and with a heating treatment (gray coated

PEEK)………………………….………………………………….120

Figure 4-2. Scanning electron microscopy images of: (a) uncoated PEEK,

(b) PEEK coated with selenium nanoparticles, (c) PEEK coated with

selenium nanoparticles and with a heating treatment……………..122

Figure 4-3. Energy-dispersive X-ray spectroscopic analyses of (a) a spot

of selenium nanoparticles on PEEK, (b) a blank area without

nanoparticles on PEEK, (c) a spot of selenium nanorods on PEEK,

and (d) a blank area without nanorods on PEEK……………......…124

Figure 4-4. Contact angle measurements on (a) uncoated PEEK, (b) red

selenium nanoparticles coated PEEK, and (c) gray selenium

nanorods coated PEEK…………………………………...….......126

Figure 4-5. The growth of P. aeruginosa biofilm on red selenium coated

PEEK, gray selenium coated PEEK and uncoated PEEK…………129

Figure 5-1. Diagram of generating selenium nanoparticles by ablation.142

Figure 5-2. TEM images and diffraction pattern of selenium nanoparticles

after the first set of irradiation at 532 nm in de-ionized water, before

any centrifugation and any filtration………………………...….…143

Figure 5-3. DLS spectra of the centrifuged and filtrated colloidal solution

xviii

of selenium nanoparticles after the first and second set of irradiations

(time=15 min, f=10Hz) in de-ionized water and ethanol……….…145

Figure 5-4. TEM images of selenium nanoparticles obtained in a)

de-ionized water and b) ethanol……………………………..……..147

Figure 5-5. HRTEM images of selenium quantum dots synthesized during

the second set of irradiation by a) UV irradiation in de-ionized water,

b) visible irradiation in de-ionized water, c) UV irradiation in ethanol

and d) visible irradiation in ethanol……………………...………...152

Figure 5-6. The growth of E. coli with a treatment of selenium

nanoparticles…………………………………………………….....155

Figure 6-1. Characterization of dextran-coated ceria nanoparticles…...169

Figure 6-2. Inhibited P. aeruginosa growth in the presence of ceria

nanoparticles at three time points (1, 6, and 24 hours). The volume

ratio of bacterial solution and ceria nanoparticles is 10:1………....171


nanoparticles at three time points (1, 6, and 24 hours). The volume

ratio of bacterial solution and ceria nanoparticles is 5:1………......171

Figure 7-1. SEM images of (a) titanium and (b) as-fired silicon nitride at

a magnification of 10k.……….........................................................188

xix

Figure 7-2. AFM images and RMS (root mean square, scan area =

10um×10um) roughness of (a) titanium and (b) ZTA………..........189

Figure 7-3. The growth of Pseudomonas aeruginosa on silicon nitride,

ZTA and titanium, measured by the method of colony forming

units..………....................................................................................190

Figure 7-4. The growth of Staphylococcus aureus on silicon nitride, ZTA

and titanium, measured by the method of crystal violet staining.....192

Figure A1-1. SEM images of selenium nanoparticle coated isoplast before

(a) and after (b) the tape test.............................................................205

Figure A1-2. SEM images of selenium nanoparticle coated PVC (a) and

uncoated PVC (b)…………………………………………….........205

Figure A1-3. SEM images of selenium nanoparticle coated on the surface

of a commercially available keyboard (a) and the uncoated surface of

the keyboard (b)……………………………………...……….........206


of the back side of a “Windows Surface” tablet (a) and the uncoated

surface of the table (b)……………………………….……….........206

Figure A1-5. SEM images of selenium nanoparticle coated glass (a) and

uncoated glass (b)…………………………………………….........207

xx

Figure A1-6. SEM images of stainless steel……………………...........209

Figure A1-7. SEM images of (a) uncoated polyurethane and (b) selenium

coated polyurethane by spray coating.……………………….........210

Figure A1-8. SEM images of selenium nanoparticle coated paper towels

after 5 minutes of sonication treatments..…………………...…......211

Figure A2-1. SEM images of flat and porous titanium………………...212

Figure A2-2. SEM images of flat titanium after being treated with

acid………………………………………………………………...213

Figure A2-3. SEM images of porous titanium after being treated with

acid………………………………………………………………...214

1

Chapter 1

Introduction

1.1 Bacterial and Biofilm Infections

Bacteria are single-celled prokaryotic microorganisms. There are

thousands of different types of bacteria and they present in every

conceivable environment all over the earth.1 Bacteria have a number of

shapes, ranging from spheres to rods and spirals. They are

broadly classified by their color after a Gram stain is applied.

Gram-positive bacteria stain blue, while Gram-negative bacteria stain

pink. Gram-positive bacteria possess a thick cell wall containing many

layers of peptidoglycan and teichoic acids. Gram-negative bacteria have

thinner cell walls but surrounded by a second lipid membrane containing

a substance known as lipopolysaccharide (LPS), a highly inflammatory

chemical that is responsible for much of the toxicity of Gram-negative

bacteria and provokes an immune response in the human body.2

The human body normally contains trillions of individual bacteria, which

outnumber the mammalian cells of the body by approximately 10 to 1.3

2

The vast majority of the bacteria in the human body are harmless and

some are beneficial, such as helping break down food in the intestine.

Only a few types of bacteria are pathogenic and cause infectious diseases

by producing harmful toxins or invading tissues. Examples of bacteria

that cause infections include Streptococcus, Pseudomonas,

Staphylococcus, and Escherichia coli. These bacteria cause bacterial

infections such as pneumonia, ear infections, diarrhea, urinary tract

infections, skin disorders and so on. Bacterial infections can result in mild

to life-threatening illnesses that require immediate intervention or

treatment.

As an important category of bacterial infections, healthcare-associated

infections (HAIs) are considered an increasing threat to safety and health

of patient in the United States.4 HAIs are infections that patients develop

during the course of receiving healthcare treatment for other conditions.

HAIs lead to extended hospital stays, contribute to increased medical

costs, and are a significant cause of morbidity and mortality. In the United

States, infections encountered in the hospital or a health care facility

affect more than 1.7 million patients, cost $35.7 billion to $45 billion, and

3

contribute to 88,000 deaths in hospitals annually.5-7

Catheter-associated

urinary tract infections are the most common HAIs, followed by surgical

site infections, bloodstream infections and pneumonia. Many cases of

HAIs result from the contamination of medical devices in the healthcare

setting, which have been associated with biofilm growth. It has been

reported by the National Institutes of Health that 80% of all chronic

infections are due to biofilms.8,9

A biofilm is an aggregate of bacteria in which bacterial cells adhere to

each other on a wet or moist surface. Biofilms may form on living or

non-living surfaces and can be prevalent in natural, industrial and hospital

settings.10,11

The formation of a biofilm (as shown in Figure 1-1) begins

with the attachment of free-floating bacteria to the surface. Along with

the generation of exopolysaccharide, the attachment of bacteria becomes

irreversible. As the bacteria propagate quickly, the biofilm structure

develops and becomes more complicated. At the last stage of biofilm

growth, the bacteria release into the environment and contaminate other

surfaces.

4

Figure 1-1. The biofilm life cycle.12

1: individual cells populate the

surface. 2: EPS (exopolysaccharide) is produced and attachment becomes

irreversible. 3 & 4: biofilm architecture develops and matures. 5: single

cells are released from the biofilm.

Biofilms are considered easy to form but hard to treat, which can cause

wide-spread infections13

in the human body; for example, through

catheter infections, infections on inert surfaces of artificial implants14

and

the formation of dental plaques15

(Figure 1-2). Statistics show that

biofilms are involved in an estimated 80% of all infections.16

These

biofilm infections can be serious and hard to treat because the

development of the biofilm structure may allow for bacteria to be

increasingly antibiotic resistant, because the bacteria in the biofilm is held

together and protected by a matrix of EPS (extracellular polymeric

5

substance or exopolysaccharide). This matrix protects bacteria cells

within it and facilitates communication among them through biochemical

signals, resulting in their increased resistance to detergents and antibiotics.

In some cases, the effect of antibiotic resistance can be increased a

thousand-fold.17

Thus, bacteria biofilms are frequently related with

persistent infections18

in the human body.

Figure 1-2. Common sites of biofilm infection.12

Once bacteria enter the

circulatory system, they can spread to any moist surface of the human

body.

1.2 Antibacterial and Anti-infection Strategies

The most conventional and widely accepted method to fight against

bacterial infections is using antibiotics. After their developments in the

6

1940s,19

antibiotics offered physicians a powerful tool to fight against

bacterial infections which has saved millions of human lives. There are

generally two types of antibiotics, bactericidal antibiotics and

bacteriostatic antibiotics. Bactericidals kill bacteria while bacteriostatics

just prevent bacteria from dividing. They are also classified based on their

mechanism of action when targeting bacteria functions or growth

processes. For example, penicillins and polymyxins target the bacterial

cell wall or cell membrane; lipiarmycins and sulfonamides interfere with

essential bacterial enzymes; and lincosamides and macrolides target

protein synthesis, which are usually a mechanism for bacteriostatics.20,21

In the recent years, more and more antibiotics have been indentified and

are being used to treat patients. However, because of the widespread and

sometimes inappropriate use of antibiotics, many strains of bacteria have

rapidly developed antibiotic resistance.22

Antibiotic resistance is

developed in bacteria strains through a process of natural selection. New,

stronger bacteria not killed by antibiotics pose serious, worldwide threat

to public health and welfare. It has also become a significant challenge to

researchers. The WHO (World Health Organization) recently reported

7

antibiotic resistance as a global serious threat that is no longer a

prediction for the future but is now reality. It has the potential to affect

anyone, of any age, in any country.23

The most typical example of

antibiotic resistance is MRSA (Methicillin-resistance Staphylococcus

aureus) which means any strain of Staphylococcus aureus that is resistant

to a group of antibiotics including penicillin, methicillin, dicloxacillin,

and oxacillin. Other examples include Escherichia coli with resistance to

third-generation cephalosporins and to fluoroquinolones, Enterococcus

with resistance to penicillin and vancomycin, and so on.24

One of the

most important action recommended by the WHO to fight against

antibiotic resistant bacteria is preventing infections from happening in the

first place, which could be achieved through better hygiene, access to

clean water, infection control in health-care facilities, and vaccination to

reduce the need for antibiotics.23

Another approach is to discover

potential alternatives to antibiotics, such as bacteriophages.

Bacteriophages are tiny viruses that infect bacteria. They can replicate in

a bacterium and make enzymes that dissolves the wall of the bacteria,

which enables them to leave the bacterium and infect other bacteria.25

Bacteriophage have been considered as a possible therapy against

8

antibiotic-resistant strains for many bacteria.26

Moreover, with significant

advances in nanotechnology, nanomaterials have also been widely

investigated for their potential ability to kill bacteria under some

circumstances. This will be further discussed in the next part of this

dissertation.

In a nosocomial environment, preventing infections often starts with

sterilization of the patient’s skin or surgeon’s hands which are reservoirs

for bacterial contamination.27

The immediate vicinity of patients has been

considered to play an important role in the transmission of nosocomial

pathogens. Studies have showed that “high-touch” (i.e., frequently

touched) surfaces in the immediate vicinity of patients may be a reservoir

for nosocomial bacteria causing wide-spread infections.28,29

Figure 1-3

shows surfaces that are frequently touched in nosocomial environments.

Some nosocomial pathogens, such as methicillin-resistant Staphylococcus

aureus, vancomycin-resistant Enterococci, Acinetobacter baumannii and

Escherichia coli, have been shown to persist on those surfaces for several

days to several months.30

Regarding environmental infection control in

healthcare facilities, it is also recommended by the Healthcare Infection

9

Control Practices Advisory Committee and the Centers for Disease

Control, to clean and disinfect high-tough surfaces (such as bed rails,

doorknobs and light switches, and surfaces around toilets in patients’

rooms) on a more frequent basis than minimal-touch surfaces.31

Figure 1-3. Mean frequency of healthcare worker contact for 28 surfaces

in an intensive care unit.32

ABHR = alcohol based hand rub, SCD =

sequential compression device.

The antimicrobial agents used to destroy pathogenic microorganisms on

those surfaces are called disinfectants. The two types of disinfectants that

10

are most frequently used are alcohols and oxidizing agents. In the

healthcare setting, alcohols, usually ethanol or isopropanol, have

generally underrated germicidal characteristics.33

The optimum

bactericidal concentration is 60%-90% alcohol in water and their activity

drops sharply when diluted below a 50% concentration.34

As

disinfectants, they are not corrosive and easy to prepare, but can be a fire

hazard and have no or limited residual activity making extended exposure

difficult to achieve due to evaporation. They also have limited activity in

the presence of an organic material. In addition, alcohols cannot destroy

bacterial spores so they are considered as an intermediate level

disinfectant. The FDA (Food and Drug Administration) has not cleared

any liquid chemical sterilant or high-level disinfectant with alcohol as the

main active ingredient. Alcohols are not recommended for sterilizing

medical and surgical materials principally because they lack sporicidal

action and they cannot penetrate protein-rich materials.35

Fatal

postoperative wound infections with Clostridium have occurred when

alcohols were used to sterilize surgical instruments contaminated with

bacterial spores.36

11

Oxidizing agents, such as hypochlorites and hydrogen peroxide, are also

widely used as disinfectants. The most prevalent hypochlorite products

are aqueous solutions of 5.25%-6.15% sodium hypochlorite, usually

called household bleach. It works as an disinfectant by oxidizing the cell

membrane of microorganisms, which results in a loss of structure and

leads to cell lysis and death. The advantages of sodium hypochlorite

include that it has a broad spectrum of antimicrobial activity, does not

leave toxic residues, is unaffected by water hardness, is inexpensive and

fast acting.37

However, there are also some limitations where using

sodium hypochlorite as a disinfectant. At the concentration used in

household bleach (5.25-6.15%), sodium hypochlorite can produce ocular

irritation or oropharyngeal, esophageal, and gastric burns.38,39

Other

disadvantages of hypochlorites include corrosiveness to some metals,

inactivation by organic matter, discoloring of fabrics, and the release of

toxic chlorine gas when mixed with ammonia or acid that is used in

household cleaning agents.40,41

Therefore, due to the limitations of

conventional disinfection strategies, it is necessary and significant to

develop other antimicrobial methods used in nosocomial environments.

12

1.3 Nanomaterials as a Novel Antibacterial Approach

An nanomaterial is usually defined as having at least one dimension with

morphological features in the nanoscale, specifically between 1 nm and

100 nm. In recent years, nanomaterials have drawn increasing attention

from many researchers because these materials exhibit special properties

stemming from their nanoscale dimensions, which are different compared

with conventional micron or bulk materials. These special properties

enable nanomaterials to be used for various applications including drug

delivery42-44

, tissue engineering45,46

, diagnostics47-49

, energy storage50,51

,

novel semiconductor52

, catalysis53-55

, construction56

and so on.

An important feature of nanomaterials or nanoparticles is their vastly

increased ratio of surface area to volume, which allows for potentially

increased interactions between nanomaterials and biological targets, such

as mammalian cells and bacteria.46

As a result, nanomaterials will likely

exert a stronger interaction with bacteria which may affect their growth

and propagation. Thus, various nanomaterials have been developed and

studied for their potential antibacterial applications.

13

The most widely investigated antibacterial nanomaterials to date are

silver nanoparticles and silver-based materials.58

Silver nanoparticles

have proved to be an effective antimicrobial agent against bacteria,

viruses and other eukaryotic micro-organisms.58,59

Silver nanoparticles of

relatively small sizes (1~10 nm) can interact with the bacteria cells

directly.60

They interact with and kill bacteria through two possible

mechanisms.61

As shown in Figure 1-4, one mechanism is that silver

nanoparticles binds to the bacterial cell wall and cell membrane and

interacts with thiol group compounds found in the respiratory enzymes of

bacteria, thus inhibiting the respiration process.62

Silver forms stable

S-Ag bonds with thiol group compounds and participate in catalytic

oxidation reactions resulting in the formation of disulfide bonds

(R-S-S-R). In another mechanism, silver nanoparticles penetrate inside

the bacteria and interact with DNA molecules because they release

reactive oxygen species (ROS) and silver ions. DNA molecules turn into

a condensed form (only when DNA molecules are in a relaxed state, do

they replicate effectively) and lose their replication ability leading to cell

death.63,64

Besides silver nanoparticles, many other nanostructured

materials, such as copper containing materials65,66

, zinc oxide

14

nanoparticles67

, gold nanoparticles68

, carbon nanotubes69

, polymer films70

and so on, have also been studied for antibacterial applications.

Figure 1-4. Mechanisms of nano-scaled silver interacting with bacteria.57

The biggest concern for using nanomaterials for biomedical applications

is their potential toxicity. As summarized in Figure1-5, nanomaterials can

result in injury responses through various mechanisms or pathways, such

as producing reactive oxygen species (ROS) that can interact and destroy

the structure of many proteins and DNA in healthy cells.72,73

In a clinical

study, using silver nanoparticle coated dressings for the treatment of

15

burns has led to abnormal elevation of blood liver levels and argyria (blue

or gray discoloration of the skin).74

Silver nanoparticles and some other

nanomaterials have also been shown to be cytotoxic to alveolar

macrophage cells as well as epithelial lung cells.75

The toxicity of

nanomaterials is usually affected by their composition, however, other

physicochemical properties, such as size, surface chemistry, shape,

protein absorption gradient and surface roughness, also play a crucial role

in determining the toxicity of nanomaterials.76

But, for most

nanomaterials, their interactions with bacteria or mammalian cells remain

largely unknown and there is not a material that is well-established and

widely accepted for antibacterial purposes. In this dissertation, a novel

nanomaterial, selenium nanoparticles, are investigated for their potential

antibacterial applications.

16

Figure 1-5. Major mechanistic injury responses induced by common

nanomaterials.71

A) Oxidative stress; B) dissolution and release of toxic

metal ions; C) cationic injury to surface membrane and organelles; D)

pro-fibrogenic responses to CNT; E) inflammasomeactivation by

long-aspect-ratio materials; F) photoactivation and influence of bandgap;

G) Zebrafish embryo hatching interference; H) cell membrane lysis by

surface reactivity.

1.4 Selenium and Nanosized Selenium

Selenium belongs to the group of metalloids from the chalcogen family in

the periodic table of elements. It exists in a range of oxidation states from

17

+6 to -2. The most common selenium compounds are selenide, selenite

and selenate with oxidation states of -2, +4, and +6, respectively.

Selenium has various allotropic forms (as shown in Figure 1-6), such as

the red amorphous form, black vitreous form, three (α, β, γ) of red

crystalline monoclinic forms and the gray/black crystalline hexagonal

form.77-80

Commercially, selenium is produced as a byproduct of copper

refining. It is used in electronics, glass, ceramics, steel and pigment

manufacturing.81

Selenium salts are toxic in large amounts, but trace

amounts are necessary for cellular function in many organisms. Selenium

is naturally found in humans and animals as a part of selenoproteins,

which play an important role in antioxidant defense systems, thyroid

hormone metabolism and redox control of cell reactions.82

Figure 1-6. Black/gray, glassy amorphous and red amorphous selenium.83

18

Previously, selenium and its compounds were studied for reducing or

preventing cancer. It has been shown that high levels of selenium in the

blood (~154 μg/ml) are correlated with reduced numbers of cancers

including pancreatic, gastric, lung, nasopharyngeal, breast, uterine,

respiratory, digestive and gynecological84

and people in areas of low soil

selenium (lower than 0.05 ppm) and people with decreased plasma

selenium levels (below 128 ng/ml) have higher cancer incidence and/or

cancer mortality.85,86

Many in vitro studies also demonstrated the

inhibitory effects of selenium on the growth of many cancerous cell

lines.87-89

However, the mechanisms of selenium-related chemoprevention

are complex and remain largely unknown.90

What is more, other researchers provided evidence of the antibacterial

properties of many selenium compounds. For example,

selenium-enriched probiotics have been shown to strongly inhibit the

growth of pathogenic E. coli in vivo and in vitro.91

The synthesized

organoselenium compounds were shown to be as effective as penicillin in

inhibiting S. aureus growth in solution in vitro.92

One of the possible

19

mechanisms of antibacterial properties of selenium is due to the ability of

selenium to generate superoxide radicals and to catalyze oxidation of

intracellular thiol resulting in thiol depletion that leads to cell death.93

Recently, as the organic forms of selenium have been studied for its

biological effects, elemental selenium nanoparticles have also drawn

some attention.94

Various methods of synthesizing selenium nanoparticles

have been reported.95-101

As selenium compounds, selenium nanoparticles

have also drawn a great deal of attention as promising cancer therapeutic

agents and drugs carriers.102,103

In addition, because of the small size and

higher ratio of surface area to volume compared to conventional selenium

materials, selenium nanoparticles are believed to have different possible

mechanisms against bacterial growth and biofilm formation, such as a

change in hydrophobicity of a surface preventing bacteria from

attachment.104

Importantly, abundant evidence also supports that

nanosized selenium has better biocompatibility, bioefficacy and lower

toxicity compared with inorganic and organic selenocompounds.94,103,105

20

1.5 Objectives

The objectives of this thesis are to synthesize selenium nanoparticles,

characterize nanosized selenium coatings, test the effectiveness of

selenium coated materials at inhibiting various bacteria growth and

biofilm formation, and investigate the mechanism(s) of how selenium

nanoparticles inhibit bacteria growth. Chapter 2 describes introducing

antibacterial properties to polycarbonate medical devices using selenium

nanoparticles. Chapter 3 describes studies concerning how to inhibit

several types of bacterial growth and biofilm formation on normal paper

towels. Chapter 4 explains how red selenium nanoparticles and gray

selenium nanorods work as antibacterial agents for PEEK (polyether

ether ketone) medical device. Chapter 5 evaluates the antibacterial effects

of selenium quantum dots and nanoparticles produced by UV/VIS/NIR

pulsed manosecond laser ablation. Chapter 6 describes the inhibited

growth of Pseudomonas aeruginosa by dextran- and polyacrylic

acid-coated ceria nanoparticles. Chapter 7 evaluates the antibacterial

properties of an implant material, silicon nitride. Lastly, based on our

studies, the conclusions of this chapter presents the overall promise of

nanosized selenium for preventing bacterial infection with some future

21

outlook for this research area to advance.

1.6 References

1. Whitman WB, Coleman DC, Wiebe WJ. Prokaryotes: the unseen

majority. Proceedings of the National Academy of Sciences of the

United States of America. Jun 9 1998;95(12):6578-6583.

2. Madigan MT, Martinko JM, Brock TD. Brock biology of

microorganisms. 11th ed. Upper Saddle River, NJ: Pearson

Prentice Hall; 2006.

3. Sears CL. A dynamic partnership: Celebrating our gut flora.

Anaerobe. Oct 2005;11(5):247-251.

4. Weinstein RA. SHEA consensus panel report: a smooth takeoff.

Society for Healthcare Epidemiology of America. Infection control

and hospital epidemiology : the official journal of the Society of

Hospital Epidemiologists of America. Feb 1998;19(2):91-93.

5. Malone DL, Genuit T, Tracy JK, Gannon C, Napolitano LM.

Surgical site infections: reanalysis of risk factors. The Journal of

surgical research. Mar 2002;103(1):89-95.

6. Klevens RM, Edwards JR, Richards CL, Jr., et al. Estimating

22

health care-associated infections and deaths in U.S. hospitals, 2002.

Public Health Rep. Mar-Apr 2007;122(2):160-166.

7. Scott RD. The direct medical costs of healthcare-associated

infections in U.S. hospitals and the benefits of prevention. In:

Prevention CfDCa, ed. Atlanta, GA2009.

8. Raffa RB, Iannuzzo JR, Levine DR, et al. Bacterial communication

("quorum sensing") via ligands and receptors: a novel

pharmacologic target for the design of antibiotic drugs. The

Journal of pharmacology and experimental therapeutics. Feb

2005;312(2):417-423.

9. Wolcott RD, Ehrlich GD. Biofilms and chronic infections. Jama.

Jun 11 2008;299(22):2682-2684.

10. Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms:

From the natural environment to infectious diseases. Nat Rev

Microbiol. Feb 2004;2(2):95-108.

11. Lear G LG. Microbial Biofilms: Current Research and Applications.

Wymondham NR180JA: Caister Academic Press; 2012.

12. Alfred B. Cunningham JEL, and Rockford J. Ross. Biofilm

hypertextbook: Montana State University Center for Biofilm

23

Engineering; 2011.

13. Costerton JW. Introduction to biofilm. Int J Antimicrob Ag. May

1999;11(3-4):217-221.

14. Auler ME, Morreira D, Rodrigues FFO, et al. Biofilm formation on

intrauterine devices in patients with recurrent vulvovaginal

candidiasis. Med Mycol. Feb 2010;48(1):211-216.

15. Rogers AH. Molecular Oral Microbiology. Wymondham NR18

0JA: Caister Academic Press; 2008.

16. Research on microbial biofilms (PA-03-047): NIH, National Heart,

Lung, and Blood Institute; 2002.

17. Stewart PS, Costerton JW. Antibiotic resistance of bacteria in

biofilms. Lancet. Jul 14 2001;358(9276):135-138.

18. James P. Nataro SC-R. Persistent bacterial infections. Washington

DC: ASM Press; 2000.

19. Waksman SA. What Is an Antibiotic or an Antibiotic Substance.

Mycologia. 1947;39(5):565-569.

20. Finberg RW, Moellering RC, Tally FP, et al. The importance of

bactericidal drugs: future directions in infectious disease. Clinical

infectious diseases : an official publication of the Infectious

24

Diseases Society of America. Nov 1 2004;39(9):1314-1320.

21. Cunha BA. Antibiotic essentials. Royal Oak, Mich. Sudbury, Mass.:

Physicians' Press Jones & Bartlett Learning; 2002.

22. Khachatourians GG. Agricultural use of antibiotics and the

evolution and transfer of antibiotic-resistant bacteria. Can Med

Assoc J. Nov 3 1998;159(9):1129-1136.

23. WHO. Antimicrobial resistance: global report on surveillance. 20

Avenue Appia, 1211 Geneva 27, Switzerland. April 2014.

24. Hidron AI, Edwards JR, Patel J. Antimicrobial-resistant pathogens

associated with healthcare-associated infections: annual summary

of data reported to the National Healthcare Safety Network at the

Centers for Disease Control and Prevention, 2006-2007 (vol 29, pg

996, 2008). Infect Cont Hosp Ep. Jan 2009;30(1).

25. Duckworth DH. "Who discovered bacteriophage?". Bacteriological

reviews. Dec 1976;40(4):793-802.

26. Keen EC. Phage therapy: concept to cure. Frontiers in

microbiology. 2012;3:238.

27. Lina G, Boutite F, Tristan A, Bes M, Etienne J, Vandenesch F.

Bacterial competition for human nasal cavity colonization: role of

25

Staphylococcal agr alleles. Applied and environmental

microbiology. Jan 2003;69(1):18-23.

28. Pittet D, Allegranzi B, Sax H, et al. Evidence-based model for hand

transmission during patient care and the role of improved practices.

The Lancet. Infectious diseases. Oct 2006;6(10):641-652.

29. Hayden MK, Blom DW, Lyle EA, Moore CG, Weinstein RA. Risk

of hand or glove contamination after contact with patients

colonized with vancomycin-resistant enterococcus or the colonized

patients' environment. Infection control and hospital epidemiology :

the official journal of the Society of Hospital Epidemiologists of

America. Feb 2008;29(2):149-154.

30. Kramer A, Schwebke I, Kampf G. How long do nosocomial

pathogens persist on inanimate surfaces? A systematic review. Bmc

Infect Dis. Aug 16 2006;6.

31. Sehulster L, Chinn RY. Guidelines for environmental infection

control in health-care facilities. Recommendations of CDC and the

Healthcare Infection Control Practices Advisory Committee

(HICPAC). MMWR. Recommendations and reports : Morbidity

and mortality weekly report. Recommendations and reports /

26

Centers for Disease Control. Jun 6 2003;52(RR-10):1-42.

32. Huslage K, Rutala WA, Sickbert-Bennett E, Weber DJ. A

quantitative approach to defining "high-touch" surfaces in hospitals.

Infection control and hospital epidemiology : the official journal of

the Society of Hospital Epidemiologists of America. Aug

2010;31(8):850-853.

33. Spaulding EH. Alcohol as a Surgical Disinfectant. AORN

Journal.2(5):67-71.

34. Morton HE. The Relationship of Concentration and Germicidal

Efficiency of Ethyl Alcohol. Ann Ny Acad Sci.

1950;53(1):191-196.

35. Block SS. Disinfection, sterilization, and preservation. 5th ed.

Philadelphia, PA: Lippincott Williams & Wilkins; 2001.

36. Nye RN, Mallory TB. A note on the fallacy of using alcohol for the

sterilization for surgical instruments. Boston Med Surg J. Jul-Dec

1923;189:561-563.

37. Rutala WA, Weber DJ. Uses of inorganic hypochlorite (bleach) in

health-care facilities. Clin Microbiol Rev. Oct 1997;10(4):597-&.

38. Ingram TA. Response of the Human Eye to Accidental Exposure to

27

Sodium-Hypochlorite. J Endodont. May 1990;16(5):235-238.

39. Haag JR, Gieser RG. Effects of Swimming Pool Water on the

Cornea. Jama-J Am Med Assoc. 1983;249(18):2507-2508.

40. Mrvos R, Dean BS, Krenzelok EP. Home Exposures to Chlorine

Chloramine Gas - Review of 216 Cases. Southern Med J. Jun

1993;86(6):654-657.

41. Reisz GR, Gammon RS. Toxic Pneumonitis from Mixing

Household Cleaners. Chest. Jan 1986;89(1):49-52.

42. Allen C, Maysinger D, Eisenberg A. Nano-engineering block

copolymer aggregates for drug delivery. Colloid Surface B. Nov

1999;16(1-4):3-27.

43. Sun XM, Liu Z, Welsher K, et al. Nano-Graphene Oxide for

Cellular Imaging and Drug Delivery. Nano Res. Sep

2008;1(3):203-212.

44. Portney NG, Ozkan M. Nano-oncology: drug delivery, imaging,

and sensing. Anal Bioanal Chem. Feb 2006;384(3):620-630.

45. Zhang SG. Fabrication of novel biomaterials through molecular

self-assembly. Nat Biotechnol. Oct 2003;21(10):1171-1178.

46. Goldberg M, Langer R, Jia XQ. Nanostructured materials for

28

applications in drug delivery and tissue engineering. J Biomat

Sci-Polym E. Mar 2007;18(3):241-268.

47. Michalet X, Pinaud FF, Bentolila LA, et al. Quantum dots for live

cells, in vivo imaging, and diagnostics. Science. Jan 28

2005;307(5709):538-544.

48. Klostranec JM, Xiang Q, Farcas GA, et al. Convergence of

quantum dot barcodes with microfluidics and signal processing for

multiplexed high-throughput infectious disease diagnostics. Nano

Lett. Sep 2007;7(9):2812-2818.

49. Smith AM, Dave S, Nie SM, True L, Gao XH. Multicolor quantum

dots for molecular diagnostics of cancer. Expert Rev Mol Diagn.

Mar 2006;6(2):231-244.

50. Nozik AJ. Quantum dot solar cells. Physica E. Apr

2002;14(1-2):115-120.

51. Robel I, Subramanian V, Kuno M, Kamat PV. Quantum dot solar

cells. Harvesting light energy with CdSe nanocrystals molecularly

linked to mesoscopic TiO2 films. J Am Chem Soc. Feb 22

2006;128(7):2385-2393.

52. Waldner J-B. Nanocomputers and swarm intelligence. London

29

Hoboken, NJ: ISTE; John Wiley; 2008.

53. Joo SH, Choi SJ, Oh I, et al. Ordered nanoporous arrays of carbon

supporting high dispersions of platinum nanoparticles (vol 412, pg

169, 2001). Nature. Nov 22 2001;414(6862):470-470.

54. Crooks RM, Zhao MQ, Sun L, Chechik V, Yeung LK.

Dendrimer-encapsulated metal nanoparticles: Synthesis,

characterization, and applications to catalysis. Accounts Chem Res.

Mar 2001;34(3):181-190.

55. Astruc D, Lu F, Aranzaes JR. Nanoparticles as recyclable catalysts:

The frontier between homogeneous and heterogeneous catalysis.

Angew Chem Int Edit. 2005;44(48):7852-7872.

56. Brockmann T, Fontana P, Meng B, Muller U. Nanotechnology in

construction engineering. Beton- Stahlbetonbau. Jul

2008;103(7):446-454.

57. Marambio-Jones C, Hoek EMV. A review of the antibacterial

effects of silver nanomaterials and potential implications for human

health and the environment. J Nanopart Res. Jun

2010;12(5):1531-1551.

58. Rai M, Yadav A, Gade A. Silver nanoparticles as a new generation

30

of antimicrobials. Biotechnology advances. Jan-Feb

2009;27(1):76-83.

59. Gong P, Li HM, He XX, et al. Preparation and antibacterial activity

of Fe3O4@Ag nanoparticles. Nanotechnology. Jul 18 2007;18(28).

60. Morones JR, Elechiguerra JL, Camacho A, et al. The bactericidal

effect of silver nanoparticles. Nanotechnology. Oct

2005;16(10):2346-2353.

61. H.Y Song KKK, I.H Oh, B.T Lee. Fabrication of Silver

Nanoparticles and Their Antimicrobial Mechanisms. European

Cells and Materials. 2006;11:58.

62. Klasen HJ. A historical review of the use of silver in the treatment

of burns. II. Renewed interest for silver. Burns. Mar

2000;26(2):131-138.

63. Liau SY, Read DC, Pugh WJ, Furr JR, Russell AD. Interaction of

silver nitrate with readily identifiable groups: relationship to the

antibacterial action of silver ions. Lett Appl Microbiol. Oct

1997;25(4):279-283.

64. Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN, Kim JO. A

mechanistic study of the antibacterial effect of silver ions on

31

Escherichia coli and Staphylococcus aureus. Journal of biomedical

materials research. Dec 15 2000;52(4):662-668.

65. McLean RJ, Hussain AA, Sayer M, Vincent PJ, Hughes DJ, Smith

TJ. Antibacterial activity of multilayer silver-copper surface films

on catheter material. Canadian journal of microbiology. Sep

1993;39(9):895-899.

66. Mulligan AM, Wilson M, Knowles JC. The effect of increasing

copper content in phosphate-based glasses on biofilms of

Streptococcus sanguis. Biomaterials. May 2003;24(10):1797-1807.

67. Li JH, Hong RY, Li MY, Li HZ, Zheng Y, Ding J. Effects of ZnO

nanoparticles on the mechanical and antibacterial properties of

polyurethane coatings. Prog Org Coat. Mar 2009;64(4):504-509.

68. Gu HW, Ho PL, Tong E, Wang L, Xu B. Presenting vancomycin on

nanoparticles to enhance antimicrobial activities. Nano Lett. Sep

2003;3(9):1261-1263.

69. Kang S, Mauter MS, Elimelech M. Microbial Cytotoxicity of

Carbon-Based Nanomaterials: Implications for River Water and

Wastewater Effluent. Environ Sci Technol. Apr 1

2009;43(7):2648-2653.

32

70. Martin TP, Kooi SE, Chang SH, Sedransk KL, Gleason KK.

Initiated chemical vapor deposition of antimicrobial polymer

coatings. Biomaterials. Feb 2007;28(6):909-915.

71. Nel A, Xia T, Meng H, et al. Nanomaterial Toxicity Testing in the

21st Century: Use of a Predictive Toxicological Approach and

High-Throughput Screening. Accounts Chem Res. Mar 19

2013;46(3):607-621.

72. Halliwell B, Gutteridge JMC. Free radicals in biology and

medicine. 4th ed. Oxford ; New York: Oxford University Press;

2007.

73. Oberdorster G, Oberdorster E, Oberdorster J. Nanotoxicology: An

emerging discipline evolving from studies of ultrafine particles.

Environmental health perspectives. Jul 2005;113(7):823-839.

74. Trop M, Novak M, Rodl S, Hellbom B, Kroell W, Goessler W.

Silver coated dressing Acticoat caused raised liver enzymes and

argyria-like symptoms in burn patient. J Trauma. Mar

2006;60(3):648-652.

75. Soto K, Garza KM, Murr LE. Cytotoxic effects of aggregated

nanomaterials. Acta Biomater. May 2007;3(3):351-358.

33

76. Sharifi S, Behzadi S, Laurent S, Forrest ML, Stroeve P, Mahmoudi

M. Toxicity of nanomaterials. Chem Soc Rev.

2012;41(6):2323-2343.

77. Takahashi T. Comparative X-Ray-Photoemission Study of

Monoclinic, Trigonal, and Amorphous Selenium. Phys Rev B.

1982;26(10):5963-5964.

78. Cherin P, Unger P. Refinement of Crystal-Structure of

Alpha-Monoclinic Se. Acta Crystall B-Stru. 1972;B

28(Jan15):313-&.

79. Cherin P, Unger P. Crystal Structure of Trigonal Selenium. Inorg

Chem. 1967;6(8):1589-&.

80. Foss O, Janickis V. Crystal-Structure of Gamma-Monoclinic

Selenium. J Chem Soc Dalton. 1980(4):624-627.

81. Barceloux DG. Selenium. J Toxicol-Clin Toxic.

1999;37(2):145-172.

82. L M. Minerals in human nutrition. Amsterdam: Elsevier; 2003.

83. Black, glassy amorphous (with thin layer of grey selenium) and red

amorphous selenium. http://en.wikipedia.org/wiki/Selenium.

84. Navarro-Alarcon M, Lopez-Martinez MC. Essentiality of selenium

34

in the human body: relationship with different diseases. Sci Total

Environ. Apr 17 2000;249(1-3):347-371.

85. Clark LC, Cantor KP, Allaway WH. Selenium in Forage Crops and

Cancer Mortality in United-States Counties. Arch Environ Health.

Jan-Feb 1991;46(1):37-42.

86. Combs GF, Gray WP. Chemopreventive agents: Selenium.

Pharmacol Therapeut. Sep 1998;79(3):179-192.

87. Lu JX, Jiang C, Kaeck M, Ganther H, Ip C, Thompson H. Cellular

and Metabolic Effects of Triphenylselenonium Chloride in a

Mammary Cell-Culture Model. Carcinogenesis. Mar

1995;16(3):513-517.

88. Redman C, Scott JA, Baines AT, et al. Inhibitory effect of

selenomethionine on the growth of three selected human tumor cell

lines. Cancer Lett. Mar 13 1998;125(1-2):103-110.

89. Kaeck M, Lu JX, Strange R, Ip C, Ganther HE, Thompson HJ.

Differential induction of growth arrest inducible genes by selenium

compounds. Biochem Pharmacol. Apr 4 1997;53(7):921-926.

90. Combs GF. Impact of selenium and cancer-prevention findings on

the nutrition-health paradigm. Nutr Cancer. 2001;40(1):6-11.

35

91. Yang J, Huang K, Qin S, Wu X, Zhao Z, Chen F. Antibacterial

action of selenium-enriched probiotics against pathogenic

Escherichia coli. Digestive diseases and sciences. Feb

2009;54(2):246-254.

92. Pietka-Ottlik M, Wojtowicz-Mlochowska H, Kolodziejczyk K,

Piasecki E, Mlochowski J. New organoselenium compounds active

against pathogenic bacteria, fungi and viruses. Chemical &

pharmaceutical bulletin. Oct 2008;56(10):1423-1427.

93. Sieber FB, WI, US), G ¹̈nther, Wolfgang H. H. (West Chester, PA,

US), Daziano, Jean-pierre (Marseilles, FR), Krieg-kowald,

Marianne (Barrington, RI, US), Bousbaa, Jamal (Durham, GB),

Bula, Raymond J. (Cross Plains, WI, US),, Inventor; MCW

Research Foundation, Inc. (Milwaukee, WI, US), assignee. Method

of making, and the use of cytotoxic agents containing elemental

selenium2007.

94. Zhang JS, Wang XF, Xu TW. Elemental selenium at nano size

(Nano-Se) as a potential chemopreventive agent with reduced risk

of selenium toxicity: Comparison with Se-methylselenocysteine in

mice. Toxicol Sci. Jan 2008;101(1):22-31.

36

95. Jiang ZY, Xie ZX, Xie SY, Zhang XH, Huang RB, Zheng LS. High

purity trigonal selenium nanorods growth via laser ablation under

controlled temperature. Chem Phys Lett. Jan 17

2003;368(3-4):425-429.

96. An CH, Wang ST. Diameter-selected synthesis of single crystalline

trigonal selenium nanowires. Mater Chem Phys. Feb 15

2007;101(2-3):357-361.

97. Jiang XC, Kemal L, Yu AB. Silver-induced growth of selenium

nanowires in aqueous solution. Mater Lett. May

2007;61(11-12):2584-2588.

98. Lee S, Kwon C, Park B, Jung S. Synthesis of selenium nanowires

morphologically directed by Shinorhizobial oligosaccharides.

Carbohyd Res. Jul 6 2009;344(10):1230-1234.

99. Zhang X, Xie Y, Xu F, Liu XH, Zhang SY, Tian XB. Room

temperature rapid growth of monocrystalline selenium nanowires

in a polymer buffer system. Solid State Sci. Mar

2003;5(3):525-527.

100. Zhu YJ, Hu XL. Preparation of powders of selenium nanorods and

nanowires by microwave-polyol method. Mater Lett. Mar

37

2004;58(7-8):1234-1236.

101. Lin ZH, Wang CRC. Evidence on the size-dependent absorption

spectral evolution of selenium nanoparticles. Mater Chem Phys.

Aug 15 2005;92(2-3):591-594.

102. Liu W, Li XL, Wong YS, et al. Selenium Nanoparticles as a Carrier

of 5-Fluorouracil to Achieve Anticancer Synergism. Acs Nano. Aug

2012;6(8):6578-6591.

103. Kong L, Yuan Q, Zhu H, et al. The suppression of prostate LNCaP

cancer cells growth by Selenium nanoparticles through

Akt/Mdm2/AR controlled apoptosis. Biomaterials. Sep

2011;32(27):6515-6522.

104. Tran PA, Sarin L, Hurt RH, Webster TJ. Differential effects of

nanoselenium doping on healthy and cancerous osteoblasts in

coculture on titanium. International journal of nanomedicine.

2010;5:351-358.

105. Weekley CM, Aitken JB, Musgrave IF, Harris HH.

Methylselenocysteine Treatment Leads to Diselenide Formation in

Human Cancer Cells: Evidence from X-ray Absorption

Spectroscopy Studies. Biochemistry. Jan 24 2012;51(3):736-738.

38

Chapter 2

Nanosized Selenium for Preventing Biofilm Formation on

Polycarbonate Medical Devices

2.1 Introduction

An implanted medical device provides a surface for bacteria to attach to

and multiply in a patient's body, resulting in the formation of a biofilm.

A bacterial biofilm is an aggregate of one or more types of bacteria in a

hydrated polymeric-like matrix.1 Once formed, this polymeric matrix

works as a shield to prevent drugs from penetrating the biofilm. Thus,

bacteria in biofilms are more tolerant to antibiotic therapies than

planktonic bacteria, making these pharmaceutical treatments less

effective or even completely ineffective.2-5

Moreover, outer bacteria that

break off from a biofilm can enter and attack the rest of the body, causing

wide spread infection. Clearly, preventing biofilm formation is critical in

the fight against healthcare-associated infections (or HAIs)

6 and currently

no consistent solution exists.

39

S. aureus (Staphylococcus aureus) is one of the bacterium commonly

found in numerous infections. These infections can be serious when they

occur on surgical wounds, in the bloodstream, or in the lungs. Each year,

there are 11 million outpatient/emergency room visits and 464,000

hospital admissions in the U.S. alone due to S. aureus infections.7 S.

aureus biofilms have been found on a wide range of medical devices

including prosthetic heart valves, central venous catheters, urinary

catheters, orthopedic prostheses, penile prostheses, contact lenses,

endocarditis, otitis media, osteomyelitis, and sinusitis.8 These biofilms are

easy to form but hard to treat. Therefore, it is significant to develop a

method to prevent bacteria from attaching on the surface of today’s

medical devices.

One of the most widely publicized antibacterial coatings has been with

silver to prevent biofilm formation (such as using a pure silver coating,

silver-palladium alloys, and polymers coated with silver).9-12

Besides

silver coatings, copper and its alloys have also been considered as a

promising material for biofilm inhibition.13-15

However, when it comes to

medical devices, one major problem with both silver and copper coatings

40

is their potential toxicity to mammalian cells due to the generation of

reactive oxygen species since neither of these elements are part of our

recommended daily nutrition.16,17

Unlike other antibacterial materials used in the healthcare industry,

selenium is a naturally occurring micronutrient needed for a healthy life

and is recommended for daily intake by the FDA. In human beings,

nutrition from selenium is achieved from 25 selenoproteins or enzymes

with selenocysteine at their active center,18

which are fundamental for a

human’s antioxidant defense system and other processes. Importantly, a

low selenium intake (less than 40 g per day) has been associated with an

increased risk of mortality, poor immune function, and cognitive

decline.19

In previous studies, selenium has been investigated for various medical

applications such as anticancer applications20,21

and as a potential

orthopedic implant material.22

There are also studies showing that

particulate selenium and its compounds can inhibit bacteria growth

(specifically, S. aureus)23

and biofilm formation.24

Specifically, selenium

41

nanoparticles demonstrated an effectiveness towards inhibiting the

growth of planktonic S. aureus in solution by up to 60 times compared

with no treatment,23

however, as mentioned, inhibiting bacteria in

biofilms is even harder. Along this line, inhibition of P. aeruginosa and S.

aureus biofilms was recently reported when using an organo-selenium

compound,24

but the coating only contained 0.2% selenium and the

substrate coated was cellulose, which has a rough surface and fibrous

structure not commonly used in medical devices. In addition, the process

used to coat the organo-selenium compound on cellulose discs was

complicated including polymerization, curing, and treating with

glutathione (GSH) before use; processes that would be difficult to

implement for numerous medical devices, especially catheters.

Therefore, the objective of this study was for the first time to coat

selenium nanoparticles on polycarbonate medical devices using a novel,

easy and quick precipitation reaction. Further, such materials were tested

for their effectiveness to kill bacteria and/or prevent bacteria from

attaching. In doing so, this study revealed a novel, easy to use, and

cost-effective nano-selenium based coating method to prevent S. aureus

42

biofilm formation.

2.2 Materials and Methods

2.2.1 Materials

Selenium nanoparticles were synthesized through a simple reaction

between glutathione (reduced form, GSH) (97%, TCI America, Portland,

OR) and sodium selenite (99%, Alfa Aesar, Ward Hill, MA)

(glutathione:sodium selenite = 4:1 molar mixture) and at the same time

were coated on the surface of one popular medical device polymer

(polycarbonate films, McMASTER-CARR, 85585k82, cut into round

films 7.01mm in diameter and 0.13mm in thickness). Sodium hydroxide

(NaOH) was added to bring the pH of the solution to the alkaline regimen

to start the reaction. As the size of selenium nanoparticles on the surface

is influenced by the concentration of NaOH and the coating time, two

coating conditions were used in this study. One was using a 0.5M NaOH

solution for 30 seconds and the other one was using a 1.0M NaOH

solution for 60 seconds. Selenium nanoparticles were formed

immediately following the addition of NaOH as visualized by a color

change of the reactant solution form clear white to clear red. The coated

43

substrates were rinsed in deionized water three times to stop the coating

reaction and to remove the free, non-adherent, selenium nanoparticles.

2.2.2 Materials Characterization

After coating, tape tests (D3359-09e2: Standard Test Methods for

Measuring Adhesion by Tape Test)25

were used to test the strength of

adhesion of the selenium nanoparticles on the substrate surfaces. SEM

(Scanning Electron Microscope, HITACHI 2700) images of the substrate

surfaces were taken before and after the adhesion tests to determine

coating strength and the distribution of selenium nanoparticles. Before

scanning the surface of polycarbonate under SEM, the films were coated

with a 2nm gold layer using a Sputter Coater (EMITECH K550, Emitech

Ltd.) to make them conductive. The coverage of selenium nanoparticles

on the polycarbonate surface was analyzed and calculated based on SEM

images using ImageJ (Wayne Rasband). An AFM (Atomic Force

Microscope, MFP3D, Asylum Research, sharp tipped cantilever, K =

0.06N/M, Contact Mode) was used to demonstrate that the coated

selenium nanoparticles increased the surface area and surface roughness

of polycarbonate. Lastly, the coated samples before and after a tape test

44

were treated in 1mL aqua regia for 30 minutes to dissolve all the selenium

into solution. After treatment, the solutions were collected in glass vials

separately and then boiled to remove all the liquid. 5 mL of 2% nitric acid

was added into each vial to dissolve the residue. After about 24 hours, the

solutions were measured by AAS (Atomic Absorption Spectroscopy,

Furnace, AA600) to determine the amount of selenium in each solution.

SEM images of the treated films were taken to confirm that all the coated

selenium was removed by aqua regia. Then, the amount of selenium on

every sample was calculated using AAS. Measurements were completed

in triplicate for each of the coating conditions.

2.2.3 Bacterial Assays

For the bacteria experiments, a bacteria cell line of S. aureus was

obtained in freeze-dried form from the American Type Culture Collection

(catalog number 25923). The cells were propagated in 30 mg/mL of

tryptic soy broth (TSB). A bacteria solution was prepared at a

concentration of 106 bacteria/ml, which was assessed by measuring the

optical density of the bacterial solution using a standard curve correlating

optical densities and bacterial concentrations. The optical densities were

45

measured at 562nm using a SpectraMax M5 plate reader (Molecular

Devices, Sunnyvale, CA). Selenium coated samples were rinsed with

75% ethanol for 20 minutes for sterilization purposes and were left in the

sterile petri dishes for 30 minutes to completely dry. Then, the samples

were transferred into a 24-well plate and treated with the prepared

bacterial solutions (106 bacteria/ml) and cultured for either 24, 48 or 72

hours in an incubator (37°, humidified, 5% CO2). For those samples that

were cultured for 48 and 72 hours, the media was changed with 1mL

sterile and fresh TSB (0.3mg/mL) every 24 hours. After the treatment, the

samples were rinsed with a PBS (phosphate buffered saline) solution

twice and placed into 1.5ml microfuge tubes with 1ml of PBS. These

tubes were shaken at 3000 rpm for 15 minutes on a vortex mixer to

release the bacteria attached on the surface into the solution. Solutions

with bacteria were spread on agar plates and bacteria colonies were

counted after 18 hours of incubation.

2.2.4 Statistics

Bacterial tests were conducted in triplicate and repeated three times. Data

were collected and the significant differences were assessed with the

46

probability associated with a one-tailed Student's t-test. Statistical

analyses were performed using Microsoft Excel (Redmond, WA).

2.3 Results

2.3.1 SEM Images, AFM Images and AAS Results

SEM images of the selenium coated polycarbonate surfaces showed that

the selenium nanoparticles were spherical and were well distributed on

the polymer surface (Figure 2-1). Most of the nanoparticles were

approximately 50-100nm in diameter. Nanoparticles coated with a

condition of 1.0M NaOH for 60s were larger than those with the

condition of 0.5M NaOH for 30s, since greater amounts of NaOH

increased the rate of the reaction resulting in larger selenium

nanoparticles. The concentration of selenium nanoparticles on the

polycarbonate surfaces were 19.34g/m2 and 20.95g/m

2 for the 0.5M

NaOH for 30s condition and for the 1.0M NaOH for 60s condition,

respectively. After the tape test, some of the selenium nanoparticles came

off and parts of the surface were not covered by selenium. Tape tests

removed about 50% and 75% of the selenium nanoparticles for the 0.5M

NaOH for 30s condition and for the 1.0M NaOH for 60s condition,

47

respectively, according to AAS results (Table 2-1), which was also

demonstrated by the coverage analysis from the four SEM images (see

the caption for Figure 2-1). This result indicated that selenium

nanoparticles with larger sizes might have less adhesion to an underlying

substrate than smaller selenium nanoparticles. However, the selenium

coated surfaces provided a desirable large surface area, which is

important for increasing the exposure of selenium to bacteria to maximize

inhibition.

48

Figure 2-1. SEM images of selenium coated polycarbonate samples

before and after a tape test. (a) 0.5M NaOH, coating for 30 seconds and

before a tape test. (b) 0.5M NaOH, coating for 30 seconds and after a tape

test. (c) 1.0M NaOH, coating for 60 seconds and before a tape test. (d)

1.0M NaOH, coating for 60 seconds and after a tape test. The coverage of

selenium nanoparticles on the surface for image (a), (b), (c), (d) are

11.74%, 6.94%, 11.38%, 5.88% respectively.

Table 2-1. Amount of selenium coated on polycarbonate surfaces

determined using AAS.

From the AFM images (Figure 2-2), the RMS (root mean square, scan

area = 10 m×10 m) roughness for the selenium coated surface at the

condition of 0.5M NaOH for 30s (image a) and with the condition of

1.0M NaOH for 60s (image c) were 45.997nm and 53.084nm,

Amount of

selenium

0.5M NaOH, 30s 1.0M NaOH, 60s

Before tape test 19.34 g/m2 20.95 g/m

2

After tape test 9.22 g/m2 5.09 g/m

2

49

respectively; after the tape test, the RMS roughness decreased to

21.731nm (image b) and 34.925nm (image d) for those two coating

conditions. The results were consistent with the SEM images and AAS

data because parts of the coated selenium nanoparticles were removed

from the surface by the tape test, resulting in a decrease in roughness. The

RMS roughness of the uncoated surface (image e) was 14.898nm, which

was much smaller than the selenium coated polycarbonate samples. So,

there was a significant increase in roughness, therefore surface area, after

coating with selenium nanoparticles.

50


10um×10um) roughness of polycarbonate surface. (a) Selenium coated

with 0.5M NaOH for 30s, RMS roughness = 45.997nm. (b) Selenium

coated with 0.5M NaOH for 30s, after tape test, RMS roughness =

21.731nm. (c) Selenium coated with 1.0M NaOH for 60s, RMS

51

roughness = 53.084nm. (d) Selenium coated with 1.0M NaOH for 60s,

after tape test, RMS roughness = 34.925nm. (e) Uncoated sample, RMS

roughness = 14.898nm.


Most strikingly, all of the selenium coated samples showed a higher

effectiveness at inhibiting bacteria growth on the polycarbonate surface

than the uncoated surface (Figure 2-3). Compared to uncoated samples,

more than 91%, 15% and 73% of the bacteria (compared with uncoated

polycarbonate) died or had been removed after 24, 48 and 72 hours on the

selenium coated polycarbonate (Table 2-2). The logarithmic difference

between control groups and selenium coated samples were over 1.05,

0.07 and 0.60 for incubation after 24, 48 and 72 hours, respectively

(Table 2-3).

52

Figure 2-3. Bacteria (S. aureus) growth on the surface of polycabonate.

Polycarbonate samples were treated with bacteria (S. aureus) in 0.03%

TSB (Tryptic Soy Broth) and were incubated for 24, 48 or 72 hours. The

media was changed with 0.03% TSB every 24 hours for those samples

incubated with 48 hours or 72 hours. The control group is uncoated

polycarbonate. bf = before tape test; aft = after tape test. Data=Mean ±

standard deviation by mean, n=3; *p<0.05 compared with control group

(uncoated polycarbonate) after 24 hours; **p<0.004 compared with

control group after 48 hours; ***p<0.02 compared with control group

after 72 hours.

Table 2-2. Difference in percentage of viable bacteria numbers on

nanosized selenium coated polycarbonate compared with control groups

(uncoated polycarbonate). bf = before tape test; aft = after tape test.

53

%

Difference

0.5M, 30s,

bf

0.5M, 30s,

aft

1.0M, 60s,

bf

1.0M, 60s,

aft

24 hours 5.75 7.49 8.88 7.36

48 hours 48.50 63.43 29.87 85.00

72 hours 23.47 26.96 14.45 13.70

Table 2-3. Log difference of viable bacteria numbers on nanosized

selenium coated polycarbonate compared with control groups (uncoated

polycarbonate). Log difference = log(numbers of viable bacteria for

control group) - log(numbers of viable bacteria for coated samples). bf =

before tape test; aft = after tape test.

Log

difference

0.5M, 30s,

bf

0.5M, 30s,

aft

1.0M, 60s,

bf

1.0M, 60s,

aft

24 hours 1.24 1.13 1.05 1.13

48 hours 0.31 0.20 0.52 0.07

72 hours 0.63 0.60 0.84 0.86

However, results did not show any significant difference at inhibiting

bacteria growth between samples coated with the various amounts of

54

selenium when the samples were incubated for 24 hours (Figure 2-4). The

reason might be that a 24 hour bacteria culture time was not long enough

to show such differences between the coated samples or even the lowest

concentration (5.09 g/m2) of selenium was not significant enough to stop

bacteria growth.

Figure 2-4. S. aureus densities on various amounts of coated selenium on

the surface of polycarbonate. The polycarbonate films were cultured in a

bacteria solution (106 bacteria/ml) for either 24 hours, 48 hours or 72

hours. The media was changed with 0.03% TSB every 24 hours.

But when the selenium coated samples were cultured in bacteria solutions

for 48 hours, the inhibition of bacteria growth was consistent with the

amount of selenium on the polycarbonate surface (Figures 2-3 and 2-4).

55

Thus, after 48 hours of bacteria culture time, the polycarbonate surface

with more selenium had a stronger ability to prevent bacteria from

growing on the surface.

Lastly, the difference in bacteria growth among the samples with various

amounts of coated selenium disappeared after the selenium coated

samples were cultured with bacteria for 72 hours (Figures 2-3 and 2-4).

However, the selenium coated samples still significantly inhibited

bacteria growth compared to the control. After 24 hours of treatment, the

bacteria density on the uncoated polycarbonate was almost constant,

around 12000 cells/mL, which meant that the uncoated polycarbonate

surface was saturated by the attached bacteria and most likely, bacteria

started forming a biofilm. But for selenium coated samples, the results

after 24 hours of treatment showed that they highly inhibited biofilm

formation. Although the bacteria numbers increased after 48 hours, the

selenium coated samples finally inhibited biofilm formation by at least

73% compared to the uncoated polycarbonate after 72 hours of culture.

Clearly, to fully inhibit bacteria, the concentration, size, and coverage of

selenium on polycarbonate needs to optimized.

56

2.4 Discussions

Nanoparticles have been widely investigated for various medical

applications because of their high surface-to-volume ratios and their

smaller size compared with conventional micron-size particles.26

The

high surface area of nanoparticles provides more sites for interacting with

biological entities and for functionalization with other bioactive

molecules, such as anticancer and antibacterial drugs.23

As for the present

studies, the nanostructured selenium increased the surface area available

to interact with and kill bacteria, while also changing the surface

morphology to ultimately inhibit the attachment of bacteria.

Although a wide range of nanoparticles have been created and

investigated for various applications, such as iron oxide nanoparticles,

carbon nanotubes, silver and gold nanoparticles, only a few studies have

reported the effectiveness of nanoparticles at inhibiting biofilm formation,

in particular S. aureus. Nanostructured selenium as a novel nano-scale

natural material remains largely unexplored in this respect. However,

there have been studies reporting the synthesis of selenium nanoparticles

57

and their biological effects toward mammalian cells in vitro.27,28

It was

demonstrated in those studies that nanostructured selenium had a 7-fold

lower acute toxicity than sodium selenite in mice showing less

pro-oxidative effects than selenite, as measured by cell growth.28

Selenium nanoparticles have also been shown effective at preventing the

growth of planktonic S. aureus23

rather than the formation of biofilms.

In a possible mechanism towards inhibiting bacteria, nanostructured

selenium may serve as a catalyst, oxidizing thiol groups, and reducing

oxygen to superoxide.29

As thiol is an essential substance for bacteria cell

function, selenium can inhibit bacteria by depleting their thiol levels. This

intracellular thiol depletion mechanism is significant because healthy

cells are more resilient to this effect than bacteria cells. Moreover, the

nano features of the selenium coating and the change in hydrophobicity

that may have resulted from coating polycarbonate with selenium

nanoparticles30

may also serve as an important role in inhibiting biofilm

formation. However, the mechanism of selenium inhibited bacteria

growth in biofilms is likely complicated and further studies are certainly

required.

58

In summary, the potential ability of nanostructured selenium to inhibit

biofilm formation was investigated in this study. Based on the results of

bacteria assays after 24, 48 and 72 hours, the selenium coating did

significantly decrease the number of bacteria attached on the surface, thus,

inhibiting biofilm formation on polycarbonate. Importantly, all this was

accomplished without using antibiotics.

2.5 Conclusions

Overall, it was demonstrated that selenium nanoparticles were

precipitated on polycarbonate medical devices using a simple fast

reaction (occurring within 60 seconds) which strongly inhibited the

growth of bacteria (S. aureus) on the surface after 24 and 72 hours by at

least 91% and 73%, respectively, compared to uncoated polycarbonate

surfaces. This result suggests that nanostructured selenium maybe an

effective way to prevent S. aureus infections and biofilm formation. More

in-depth and long-term studies with different kinds of bacteria and

substrates should be implemented to achieve a better understanding of

such novel antibacterial properties of selenium.

59

2.6 References

1. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: A

common cause of persistent infections. Science. May 21

1999;284(5418):1318-1322.

2. Costerton JW. Introduction to biofilm. Int J Antimicrob Ag. May

1999;11(3-4):217-221.

3. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR,

Lappinscott HM. Microbial Biofilms. Annu Rev Microbiol.

1995;49:711-745.

4. Zottola EA, Sasahara KC. Microbial Biofilms in the

Food-Processing Industry - Should They Be a Concern. Int J Food

Microbiol. Oct 1994;23(2):125-148.

5. Darouiche RO. Current concepts - Treatment of infections

associated with surgical implants. New Engl J Med. Apr 1

2004;350(14):1422-1429.

6. Maurette P, Sfa CAMR. To err is human: building a safer health

system. Ann Fr Anesth. Jun 2002;21(6):453-454.

7. Martinez LR, Han G, Chacko M, et al. Antimicrobial and Healing

60

Efficacy of Sustained Release Nitric Oxide Nanoparticles Against

Staphylococcus Aureus Skin Infection. J Invest Dermatol. Oct

2009;129(10):2463-2469.

8. Singh R, Ray P, Das A, Sharma M. Role of persisters and

small-colony variants in antibiotic resistance of planktonic and

biofilm-associated Staphylococcus aureus: an in vitro study. J Med

Microbiol. Aug 2009;58(8):1067-1073.

9. Chiang WC, Schroll C, Hilbert LR, Moller P, Tolker-Nielsen T.

Silver-Palladium Surfaces Inhibit Biofilm Formation. Appl Environ

Microb. Mar 2009;75(6):1674-1678.

10. Jansen B, Kohnen W. Prevention of Biofilm Formation by Polymer

Modification. J Ind Microbiol. Oct 1995;15(4):391-396.

11. Secinti KD, Ozalp H, Attar A, Sargon MF. Nanoparticle silver ion

coatings inhibit biofilm formation on titanium implants. J Clin

Neurosci. Mar 2011;18(3):391-395.

12. Liao YA, Wang YQ, Feng XX, Wang WC, Xu FJ, Zhang LQ.

Antibacterial surfaces through dopamine functionalization and

silver nanoparticle immobilization. Mater Chem Phys. Jun 1

2010;121(3):534-540.

61

13. Mclean RJC, Hussain AA, Sayer M, Vincent PJ, Hughes DJ, Smith

TJN. Antibacterial Activity of Multilayer Silver Copper

Surface-Films on Catheter Material. Can J Microbiol. Sep

1993;39(9):895-899.

14. Qin ZQ, Zhang JD, Hu YF, et al. Organic compounds inhibiting S.

epidermidis adhesion and biofilm formation. Ultramicroscopy. Jul

2009;109(8):881-888.




16. Brewer GJ. Copper toxicity in the general population. Clin

Neurophysiol. Apr 2010;121(4):459-460.

17. Foldbjerg R, Olesen P, Hougaard M, Dang DA, Hoffmann HJ,

Autrup H. PVP-coated silver nanoparticles and silver ions induce

reactive oxygen species, apoptosis and necrosis in THP-1

monocytes. Toxicol Lett. Oct 28 2009;190(2):156-162.

18. Kryukov GV, Castellano S, Novoselov SV, et al. Characterization

of mammalian selenoproteomes. Science. May 30

2003;300(5624):1439-1443.

62

19. Rayman MP. Selenium and human health. Lancet. Mar 31

2012;379(9822):1256-1268.

20. Rayman MP. Selenium in cancer prevention: a review of the

evidence and mechanism of action. P Nutr Soc. Nov

2005;64(4):527-542.

21. Clark LC, Combs GF, Turnbull BW, et al. Effects of selenium

supplementation for cancer prevention in patients with carcinoma

of the skin a randomized controlled trial - A randomized controlled

trial. Jama-J Am Med Assoc. Dec 25 1996;276(24):1957-1963.

22. Perla V, Webster TJ. Better osteoblast adhesion on nanoparticulate

selenium - A promising orthopedic implant material. J Biomed

Mater Res A. Nov 1 2005;75A(2):356-364.

23. Tran PA, Webster TJ. Selenium nanoparticles inhibit

Staphylococcus aureus growth. Int J Nanomed. 2011;6.

24. Tran PL, Hammond AA, Mosley T, et al. Organoselenium Coating

on Cellulose Inhibits the Formation of Biofilms by Pseudomonas

aeruginosa and Staphylococcus aureus. Appl Environ Microb. Jun 1

2009;75(11):3586-3592.

25. KL M. Adhesion measurement: Recent progress, unsolved

63

problems, and prospects: American Society for Testing and

Materials; 1978.

26. Poole CP, Owens FJ. Introduction to nanotechnology. Hoboken,

N.J.: John Wiley; 2003.

27. Zhang SY, Zhang J, Wang HY, Chen HY. Synthesis of selenium

nanoparticles in the presence of polysaccharides. Mater Lett. Aug

2004;58(21):2590-2594.

28. Zhang JS, Gao XY, Zhang LD, Bao YP. Biological effects of a

nano red elemental selenium. Biofactors. 2001;15(1):27-38.

29. Spallholz JE, Shriver BJ, Reid TW. Dimethyldiselenide and

methylseleninic acid generate superoxide in an in vitro

chemiluminescence assay in the presence of glutathione:

Implications for the anticarcinogenic activity of

L-selenomethionine and L-Se-methylselenocysteine. Nutr Cancer.

2001;40(1):34-41.



coculture on titanium. Int J Nanomed. 2010;5:351-358.

64

Chapter 3

Inhibition of Various Gram-positive and Gram-negative Bacteria

Growth on Paper Towels by Selenium Nanoparticles

3.1 Introduction

Bacterial contaminations are frequently found on paper products

worldwide, involving various strains from the genera Bacillus,

Staphylococcus, Pseudomonas and Enterobacter.1-3

These bacteria

contribute to the formation of biofilm in machinery, resulting in

contamination or even corrosion on paper products. In the hospital

environment, hand washing has been identified as the most significant

manners towards preventing the spread of microbial infections,4,5

with

hand drying as the critical last stage of the hand washing process. Among

the three most frequently used methods to dry hands (hot air dryers, cloth

towels and paper towels), paper towels have been recognized as the most

hygienic method.6-8

However, in some circumstances, such as for paper

towels hanging in the splashing zone or those used for cleaning surfaces,

they have been considered as potential sources of bacteria

contaminations,9,10

especially in a nosocomial environment.

65

Previously, studies evaluated the potential bacterial contamination of

unused paper towels.11-13

In a hand wash experiment, participants who

washed their hands with water, regular or antibacterial soap followed by

drying with paper towels, surprisingly had more bacteria on their hands

after washing than before, which indicated a possible bacterial

transmission from paper towels.12

It was further demonstrated that a

zig-zag transfer of bacteria between paper towel dispensers and hands

could take place if either one is contaminated.13

In addition, there are also

concerns for bacterial contamination on numerous paper products, for

example, filter paper in water and air purifying systems, and wrapping

paper used in the food industry.14,15

Filter papers are commonly covered

by a thick layer of bacterial biofilm and need frequent replacement.

Microbial contamination is one of the major reasons causing food

spoilage during food storage, especially when the foods are packaged and

stored for a long period of time. Actually, developing effective,

antimicrobial packaging materials for controlling microbial growth in/on

food is now an active area of research .16,17

66

Due to the porous structure of fibers in all paper products, such materials

are prone to bacteria growth and, thus, are sources for continual

contamination. Besides, this porous structure provides an environment

that favors the attachment of bacteria and makes it more difficult to kill

bacteria once forming a biofilm. One of the most promising approaches

towards preventing infections is coating paper products with

antimicrobial materials. For example, Wenbing Hu and his colleagues

reported that introducing antibacterial properties to filter paper by coating

the paper with graphene oxide, showed about a 70% inhibition to

Escherichia coli growth after 2 hours. However, the graphene-based

paper had mild cytotoxicity resulting in 20% of healthy mammalian A945

cell death after 2 hours.18

Kalyani Chule et al. studied the antibacterial

activities of ZnO nanoparticle coated paper19

and results showed a

significant decrease in bacteria counts after 24 hours. Besides ZnO

nanoparticles, silver nanoparticles have also been loaded on filter paper

for antibacterial purposes, processing strong antibacterial properties.20

But one major problem for ZnO, silver nanoparticles and other

metal-based materials is their toxicity to healthy cells due to the

generation of reactive oxygen species.21,22

Those materials may result in

67

severe health problems when such coated paper products are used for

food wrapping or clinical applications.

Selenium and its compounds have been widely used in electronics,

glasses, ceramics, steel and pigment manufacturing.23

But due to toxicity

concerns, not until recently has selenium been considered as material for

biomedical applications. Selenium is found naturally in humans and

animals as a critical element in selenoproteins, which play an important

role in antioxidant defense systems, thyroid hormone metabolism and

redox control of cell reactions.24

Because of this, selenium and selenium

nanoparticles have been receiving dramatically increasing attention in

several biomedical applications. For example, it has been shown that high

levels of selenium in the blood (~154 μg/ml) correlated with a reduced

number of cancer occurrence including pancreatic, gastric, lung,

nasopharyngeal, breast, uterine, respiratory, digestive and gynecological

cancer25

and many in vitro and in vivo studies have demonstrated the

inhibitory effects of selenium on the activity of various cancer cells.25-29

Selenium nanoparticles have also been considered as a biocompatible

68

anticancer orthopedic implant material due to the increased adhesion of

healthy osteoblasts on nanostructured selenium.30

However, in terms of potential antibacterial applications, selenium is a

novel material that has not been widely explored. There are a few studies

concerning the antibacterial properties of selenium or its compounds. For

example, selenium-enriched probiotics have been shown to strongly

inhibit the growth of pathogenic E. coli in vivo and in vitro.31

The

synthesized organoselenium compounds were shown to be as effective as

penicillin at inhibiting Staphylococcus aureus growth in solution in

vitro.32

In addition, the growth of planktonic Staphylococcus aureus was

strongly inhibited in the presence of selenium nanoparticles.33

Actually, many kinds of other nanomaterials, including silver

nanoparticles, zinc oxide nanoparticles, copper nanoparticles and so on,

have been studied to provide a novel strategy to eradicate bacterial

infections, especially for various medical device applications.34-37

But a

major problem for these metal-based nanoparticles has been their toxicity

to healthy mammalian cells due to their generation of reactive oxygen

69

species,21,22,38

which could result in severe health problems when used as

medical devices or clinical applications. Compared with the above

mentioned materials, selenium is expected to be healthier and less toxic to

humans and the environment. For an adult, selenium is recommended by

the FDA to be consumed at 53-60 g per day and is the nutrition for 25

selenoproteins with selenocrysteine at their active center.39

Moreover,

studies have demonstrated that selenium and selenium nanoparticles can

improve healthy human cell growth.30,40

Therefore, in this chapter, selenium nanoparticles were used as a coating

material to introduce antibacterial properties to paper towels. Selenium

nanoparticles were coated on paper towel surfaces through a quick

precipitation reaction (happening within 60 seconds). In addition, their

effectiveness at preventing biofilm formation was tested in bacterial

assays involving four diverse types of bacteria. The results showed that

the selenium coatings significantly inhibited all bacterial activities on

paper towels. Coating with nanosized selenium successfully introduced

antibacterial properties to paper towels, revealing a promising

70

selenium-based method to decrease the presence and spreading of

numerous human infections.


3.2.1 Synthesis of Selenium Nanoparticle Coatings

Selenium nanoparticles were synthesized and coated on paper towels

(MB550A Hand Towel, Tork Advanced, cut into round chips, 7.00mm in

diameter) through a simple and quick precipitation reaction. The reaction

of the synthesis was:

which involves glutathione (reduced form, GSH) (97%, TCI America,

Portland, OR) and sodium selenite (99%, Alfa Aesar, Ward Hill, MA)

mixed at a 4:1 molar ratio.

The paper towel samples were first washed with 75% ethanol followed by

rinsing with deionized water five times and then transferred into a flask

containing a mixed solution of glutathione and sodium selenite. Sodium

hydroxide was added into the mixture of glutathione and sodium selenite

to bring the pH of the solution into the alkaline regimen to initiate the

71

reaction at room temperature. Selenium nanoparticles were formed and

precipitated on the surface of paper towels immediately following the

addition of NaOH as visualized by a color change of the reactant solution

from colorless to clear red. The paper towel samples were coated for 30s

under a 200 rpm (round per minute) agitation to ensure a uniform coating.

The coated substrates were rinsed in deionized water five times and

soaked in water for 24 hours to remove the free, non-adherent, selenium

nanoparticles and remaining reactants. Then, the selenium coated paper

towels were left in petri dishes for several days to completely dry before

being used in any further experiments or tests.

The uncoated samples that were used as a control group in the following

experiments were prepared by treating paper towels with the same

procedure for selenium coated paper towel samples described above

except that sodium hydroxide was not added so that the reaction did not

occur.

72

3.2.2 Characterization of Coated Paper Towels

SEM (Scanning Electron Microscope, HITACHI S-4800) images of the

paper towel surfaces were taken to determine the size, coverage and

distribution of selenium nanoparticles. Before scanning the surface of a

paper towel under SEM, the samples were coated with a 5 nm platinum

layer using a sputter coater (Cressington 208 HR, Cressington Scientific

Instruments Ltd.) to make the samples conductive. The coverage of

selenium nanoparticles on the paper towel surface was analyzed and

calculated based on the SEM images using ImageJ (Wayne Rasband). An

AFM (Atomic Force Microscope, MFP3D, Asylum Research, sharp

tipped cantilever, K = 0.06N/M, Contact Mode) was used to demonstrate

that there was an increase in surface roughness on paper towel samples

after being coated with selenium nanoparticles.

The coated samples were treated in 1mL aqua regia for 30 minutes to

dissolve all the selenium into solution. After treatment, the solutions were

collected in glass vials separately and then boiled to remove all the liquid.

5 mL of 2% nitric acid was added into each vial to dissolve the residue,

which contained all the selenium from the solution. After about 24 hours,

73

the solutions were measured with AAS (Atomic Absorption Spectroscopy,

Furnace, AA600) to determine the concentration of selenium in each

solution. SEM images of the treated films were taken to confirm that all

the coated selenium was removed by aqua regia. Then, the amount of

selenium on every sample was calculated based on the AAS results.

Measurements were completed in triplicate for both blank control

samples (uncoated paper towels) and selenium coated paper towel

samples.

3.2.3 Mechanical Tests

A MTESTQuattro materials testing system (ADMET, Norwood, MA)

was developed according to ASTM and ISO standards. The system was

used to measure the mechanical properties of selenium coated and

uncoated paper towels. The samples were prepared by cutting the paper

towels into 18.00mm×40.00mm rectangle pieces and then coating them

with selenium nanoparticles using the procedure described above.

Tension tests were performed by stretching the paper towel until complete

breakage occurred. The size of tension loaded area was 18.00mm width

by 30.00mm length (the stretching direction). The tension tests were

74

performed for both completely dry and wet (saturated by DI water) paper

towels separately. Load and stress data were recorded automatically by

the ADMET software. The values of maximum stress were calculated

based on the data and the loaded area (18.00 mm×30.00 mm) of the

tested samples.

3.2.4 Bacterial Inhibitory Tests

Four bacterial cell lines were involved in the tests to evaluate the

growth of various types of bacteria on selenium coated and uncoated

paper towels. The bacterial cell lines, Staphylococcus aureus (catalog

number 25923), Pseudomonas aeruginosa (catalog number 27853),

Escherichia coli (catalog number 25922) and Staphylococcus epidermidis

(catalog number 35984), were obtained in freeze-dried form from the

American Type Culture Collection (Manassas, VA). Each strain of

bacteria was propagated in 30 mg/mL tryptic soy broth (TSB) for 20

hours. A bacteria solution was prepared using 0.3 mg/mL TSB at a




75


Devices, Sunnyvale, CA).

Selenium coated and uncoated paper towel samples were rinsed in

separate petri dishes with 75% ethanol for 20 minutes for sterilization

purposes and were left in the sterile petri dishes for 30 minutes to

completely dry. Then, the samples were transferred into a 48-well plate

and rinsed with sterile 0.3 mg/mL TSB twice to remove any possible

ethanol residue. Each sample was treated separately with 1 mL of the

prepared bacterial solutions (106 bacteria/ml) and cultured for either 24,

48 or 72 hours in an incubator (37°C, humidified, 5% CO2). For those

samples that were cultured for 48 and 72 hours, the media was changed

with 1 mL of sterile and fresh TSB every 24 hours. After the treatment,

the samples were rinsed with a PBS (phosphate buffered saline) solution

twice and placed into 1.5ml microfuge tubes with 1ml of PBS. These

microfuge tubes were shaken at 3000 rpm for 15 minutes on a vortex

mixer to completely release the bacteria attached on the surface into the

solution. Solutions with bacteria from each sample were spread on agar

plates and bacteria colonies were counted after about 20 hours of

76

incubation. The bacterial inhibitory tests on the four types of bacteria

strains were conducted individually and identically.

3.2.5 Mechanism of Action: Protein Adsorption Assays

The total protein adsorption on the selenium coated and uncoated paper

towel samples was measured using a Piece BCA Protein Assay Kit

(catalog number 23227, Piece Biotechnology, Thermo Scientific,

Rockford, IL). The paper towel samples were rinsed with 75% ethanol for

20 minutes for sterilization purposes. After being left in sterile petri

dishes for 30 minutes to completely dry, the samples were transferred into

a 48-well plate and rinsed with sterile PBS twice. Each sample was

treated separately with 1 mL of sterile TSB and then cultured for 24, 48

or 72 hours in an incubator (37°C, humidified, 5% CO2). For those



the samples were transferred to a new 48-well plate and washed with a

cold (~4°C) PBS solution twice to remove non-adsorbed proteins in the

media. Then, each sample was treated with 0.5 mL RIPA buffer (catalog

number 89900, Piece Biotechnology, Thermo Scientific, Rockford, IL)

77

for 10 minutes, swirling the plate occasionally at the same time. After the

treatment, the solution in each well was transferred to a microcentrifuge

tube and stored in a -80°C freezer for further analysis. The concentration

of proteins in solution gathered after 24, 48 and 72 hours were measured

following the manufacturer’s instructions for the Pierce BCA Protein

Assay Kit, in which the sample to work reagent ratio and incubation time

were modified compared with the standard protocol to achieve a lower

minimum detection level. BSA (bovine serum albumin) standards for

plotting a standard curve were also treated in the same way as the

samples. The concentration of protein adsorbed on each sample was

calculated based on the standard curve.

3.2.6 Release of Selenium

The release of selenium from the surface of coated paper towels were

measured using ICP-MS (inductively coupled plasma mass spectrometry,

Bruker Aurora M90). To measure the total amount of Se on the surface of

coated paper towels, 1 mL of 10 M NaOH was added with each sample (7

mm in diameter, as described in 3.2.1) into a 10 mL vial. The vial was

placed standing for 3 days to allow all coated selenium to be completely

78

dissolved by NaOH. Then, the solution was diluted 100 times using DI

water before ICP measurements. Paper towel samples without Se coating

were also treated with the same procedure and the solution was also

collected for ICP measurements. In order to measure the release of the

selenium from the paper towel samples, both coated and uncoated

samples were treated individually with 1 mL sterile TSB in a 37 °C

incubator for 24, 48 and 72 hours, which are the same time scales as the

treatments for the bacterial assays. The media for each sample was

collected every 24 hours and replaced with 1 mL of fresh TSB. The

collected solutions were diluted 10 times using DI water before ICP

measurements.

3.2.7 Statistics

All bacterial inhibitory tests and protein adsorption assays were

conducted in triplicate and repeated three times. Data were collected and

the significant differences were assessed with the probability associated

with a one-tailed Student's t-test. Statistical analyses were performed

using Microsoft Excel (Redmond, WA).

79

3.3 Results and Discussion

3.3.1 Synthesis and Characterization of Selenium Nanoparticles

After coated with selenium nanoparticles, the color of the paper towel

surfaces changed from white to light red (Figure 3-1). The red color

indicated the formation of selenium nanoparticles as one of the selenium

allotropes is red41

. The coated paper towels can maintain its red

appearance for at least 12 months at room temperature, although the most

stable form of selenium is considered as a gray color crystal and this gray

selenium can be formed by the mild heating of other allotropes. The

nano-sized red selenium has a high biological activity in terms of cell

proliferation, enzyme induction and protection of human cells against

damage from free radicals. It also has a much lower acute toxicity

compared with sodium selenite.42

80

Figure 3-1. Light image of selenium coated and uncoated paper towels.

The three pieces on the top are paper towels without selenium coating.

The three on the bottom are paper towels coated with selenium

nanoparticles.

The SEM images of the selenium coated paper towels (image a) and

uncoated paper towels (image b) are shown in Figure 3-2 and Figure 3-3.

As shown in image a, the selenium nanoparticles were well distributed on

the fibrous structure of the paper towels and covered almost all parts of

the surface. Image b showed a paper towel surface without coatings, onto

which no particles were observed. From image c in Figure 3-3, which is a

selenium coated sample, observed under higher magnification, the

81

diameters of the selenium particles were measured and the size

distribution was found to be between 50 and 100 nm. The surface

coverage of selenium nanoparticles was 4.36% according to

measurements by ImageJ.

Figure 3-2. SEM images of selenium coated (image a) and uncoated

paper (image b) towel samples at a magnification of 8.0 K.

82

Figure 3-3. Scanning electron microscopy images of: (a) a paper towel

with a nano-selenium coating at a 3.5 k magnification, (b) a paper towel

83

without a selenium coating at a 3.5 k magnification, and (c) the paper

towel in image a at a 10.0 k magnification.

The AFM images in Figure 3-4 showed that the RMS (root mean square,

scan area = 10 m×10 m) roughness of the paper towel surface

increased from 15.89nm (Figure 3-4, image a) to 31.14nm (Figure 3-4,

image b) after coated with selenium nanoparticles. The surface roughness

was significantly increased after coated the paper towels with selenium

nanoparticles.

Figure 3-4. AFM images and RMS (root mean square, area = 10 m×10

m) roughness of paper towel surfaces. Image (a) is the surface of

selenium coated paper towel with RMS roughness = 31.14 nm. Image (b)

is the surface of uncoated paper towel with RMS roughness =15.89 nm.

84

According to AAS results, the concentration of the selenium

nanoparticles on the coated paper towel surface was 69.00 g/m2. This

concentration is about 4 times larger than concentration of selenium on

the coated polycarbonate surfaces under the same coating conditions as

mentioned in chapter 2. The reason was that the fibrous structure of the

paper towel significantly increased surface area to allow for more

selenium nanoparticle deposition. Compared with the flat surfaces of

most other materials, the surface of paper towels with a fibrous structure

had a larger surface area, resulting in increased deposition and adhesion

of selenium nanoparticles. Overall, the selenium nanoparticles were

successfully synthesized and coated on the paper towels using a simple

quick reaction (within a minute), and the selenium coatings can retain its

red appearance for a long time period (over a year) at room temperature.

3.3.2 Mechanical Tests

In the mechanical tension tests, the maximum stress of the uncoated and

selenium coated paper towels were 27.87 kN/m2 and 27.79 kN/m

2 (or

2046 g and 2040 g stretching loads), respectively, as shown in Figure 3

85

and Table 1. After the samples were saturated with DI water, the

maximum stresses are 2.969 kN/m2 and 2.901 kN/m

2 (or 218 g and 213 g

loads) for the wet paper towel samples with and without selenium

coatings, respectively. Therefore, in both dry and wet conditions, there

was no significant change in terms of the mechanical properties for the

paper towels after coated with selenium nanoparticles.

Figure 3-5. Tension tests for paper towels in various conditions: a. dry

and uncoated; b. dry and selenium nanoparticle coated; c. wet and

uncoated; d. wet and selenium nanoparticle coated.

86

Table 3-1. Values of maximum stress (the stress at the moment of

complete breakage) for each paper towel sample in the tension tests.

Maximum stress (kN/m2) Without coating Selenium coated

Dry 27.87 27.79

Wet 2.901 2.969


The results of bacteria assays involving Staphylococcus aureus (S.

aureus), Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E.

coli) and Staphylococcus epidermidis (S. epidermidis) showed a high

effectiveness for the selenium coated paper towels at inhibiting bacteria

growth. As seen in Figure 3-6, the selenium coated paper towels had

88.6%, 88.9% and 88.8% less bacteria (S. aureus) attached than the

uncoated paper towels after 24, 48 and 72 hours, respectively. Moreover,

from the 24 hour culture time to the 48 hour culture time, there was an

increase in bacteria numbers on uncoated paper towel samples, but was

constant to the 72 hours culture time, implying that the uncoated paper

87

towel was saturated by bacteria after 48 hours of treatment. In contrast,

the bacteria numbers on the selenium coated paper towels remained at a

low level not increasing from 24 to 48 to 72 hours, indicating successful

inhibition to the growth of S. aureus. In the bacterial assays involving S.

aureus on polycarbonate samples in chapter 2, there were distinct

increases in bacteria numbers from 24 hours to 48 hours on all the coated

samples, which indicated that after 48 hours the bacteria growth was not

completely inhibited by the selenium coatings on polycarbonate.

However, Figure 3-6 showed that the selenium coatings on paper towel

samples almost completely inhibited bacterial growth after 48 treatment.

The reason was that the amount of coated selenium nanoparticles on

polycarbonate samples was much smaller than the amount on paper towel

samples. Thus, the surface attached with more selenium nanoparticles

revealed a stronger ability to prevent the formation of S. aureus biofilms,

especially when the bacteria in the biofilm propagating quickly.

88

Figure 3-6. The growth of Staphylococcus aureus on the surface of paper

towels. The control group is the uncoated paper towels. Data=Mean ±

standard deviation, n=3; *p<0.02 compared with the control group

(uncoated paper) after 24 hours; **p<0.002 compared with control group

after 48 hours; ***p<0.002 compared with the control group after 72

hours.

A similar inhibitory activity was observed in the bacterial assays

involving another gram-positive bacterium, S. epidermidis. As shown in

Figure 3-7, the selenium coated paper towels had 90.66%, 92.50% and

92.16% less bacteria attached compared with the uncoated paper towels

after 24, 48 and 72 hours, respectively. From 24 hours to 48 hours, there

was a large increase in the numbers of bacteria, which means the bacteria

propagated quickly during this period on paper towels without a selenium

89

coating. However, on the surface of selenium coated paper towels, there

was not an obvious increase in the bacteria numbers from 24 hours to 48

hours. In addition, after incubation for 48 hours and 72 hours, the

uncoated paper towel was saturated by bacteria (S. epidermidis),

indicating the formation of a matured biofilm, while the selenium coated

paper towels still had a significantly reduced number of bacteria after 72

hours. Overall, the numbers of S. epidermidis on the selenium coated

paper towels remained at a low level not increasing from 24 to 48 to 72

hours, indicating successful inhibition to the growth and biofilm

formation of S. epidermidis.

Figure 3-7. The growth of Staphylococcus epidermidis on the surface of

paper towels. There was more than a 90% inhibition of bacteria growth

compared with control groups. Data=mean ± standard deviation, n=3; *,

**, ***p<0.05 compared with the control group (uncoated paper) after

either 24, 48 or 72 hours, respectively.

90

Results also showed a significant inhibition of P. aeruginosa on selenium

coated paper towels. Specifically, the results in Figure 3-8 showed a

successful inhibition of the growth of P. aeruginosa by 55% and 84%

after 48 or 72 hours, respectively, onto the surface of selenium coated

paper towels. But after 24 hours of incubation, there was no significant

decrease in bacteria numbers observed on selenium coated paper towels

compared with uncoated paper towels. The reason may be that P.

aeruginosa are gram-negative bacteria that have an extra layer of

bacterial outer membrane (made of lipopolysaccharide and protein) as a

part of their cell wall. Thus, compared with S. aureus and S. epidermidis,

which are gram-positive bacteria that only have the plasma membrane in

their cell wall but don’t have the bacterial outer membrane, it took longer

for selenium to penetrate into and interact with P. aeruginosa; thus, it

took a longer time for the gram-negative bacteria to be killed. However,

the total amount of P. aeruginosa increased from 24 to 48 to 72 hours on

paper towels without selenium coatings, while on the surface of selenium

coated paper towels, the amount of P. aeruginosa decreased from 24

hours to 48 hours and further decreased dramatically from 48 hours to 72

91

hours. Therefore, the activity of P. aeruginosa was successfully inhibited

on selenium coated paper towels and the inhibition increased as the time

of treatment increased. Future studies will be helpful to investigate higher

concentrations of selenium to decrease P. aeruginosa growth over long

time periods.

Figure 3-8. The growth of Pseudomonas aeruginosa on the surface of

paper towels. Data=mean ± standard deviation, n=3; *, **p<0.05

compared with the control group (uncoated paper) after either 48 or 72

hours, respectively.

The results of the bacterial inhibitory assays with E. coli are shown in

Figure 3-9. The selenium coated paper towels inhibited E. coli growth by

about 50%~60% compared with the uncoated paper towels after

treatment for 24 hours, 48 hours and 72 hours. As shown in Figure 3-6

and Figure 3-7, a saturation of S. epidermidis and P. aeruginosa growth

92

on the uncoated paper towel surfaces was observed after incubation for

48 hours. But for E. coli, after 24 hours of incubation, the bacteria

saturated the surface of paper towels not coated with selenium and

formed a mature biofilm. The reason for this could be that E. coli

propagates much faster than the other two bacteria as the re-generation

time of E. coli is only 15~20 minutes under optimal conditions in the

laboratory. Moreover, although the growth of E. coli was significantly

inhibited, the percentage of bacteria population reduction was not as

much as the reduction for S. aureus and S. epidermidis. Because E.coli is

also a gram-negative bacterium, its outer membrane can prevent the

interaction between the selenium nanoparticles or the selenium element

and the bacteria cells. Overall, compared with the uncoated paper towels,

the selenium coated paper towels showed a significant inhibition to the

growth of E. coli continuously from 24 to 72 hours.

93

Figure 3-9. The growth of E. coli on the surface of paper towels.

Data=mean ± standard deviation, n=3; *, **, ***p<0.05 compared with

the control group (uncoated paper) after either 24, 48 or 72 hours,

respectively.

In conclusion, the growth of S. aureus, S. epidermidis, P. aeruginosa and

E.coli on paper towels was successfully inhibited after coated with

selenium nanoparticles. The selenium particles when coated on paper

towels were extremely effective and continuously inhibited (about a 90 %

reduction) the growth of gram-positive bacteria including S. aureus and S.

epidermidis after 24, 48 or 72 hours. From gram-negative bacteria like P.

aeruginosa and E. coli, the selenium coated paper towels also

significantly inhibited bacterial growth after either 48 or 72 hours of

incubation. However, it took a longer time for P. aeruginosa and E. coli

94

to be influenced by selenium nanoparticles and the overall reduction of

the bacteria population was less than that observed for the gram-positive

bacteria, which means that the activities of gram-negative bacteria were

more difficult to slow by selenium nanoparticle coatings on the surface of

paper towels. However, further studies will investigate higher

concentrations or altered selenium nanoparticle sizes to maximally

decrease gram-negative bacteria growth.

3.3.4 Mechanism of Action: Protein Adsorption Assays

Based on the results from protein adsorption assays as shown in Figure

3-10, the selenium nanoparticle coated paper towels significantly

increased protein adsorption over uncoated paper towels after a treatment

in TSB for either 24, 48 or 72 hours. After coating the paper towels with

selenium nanoparticles, there was an increase in the surface area and

nanoscale roughness, which allowed more proteins to adsorb to the

surface of the paper towels. The increased protein adsorption might play

an important role in inhibiting bacteria growth on the selenium coated

paper towels, because those proteins could interact with bacteria cell

membranes and prevent bacteria cells from attaching to the surface. This

95

could eventually prevent bacteria from forming biofilms, thus, inhibiting

bacteria growth on the surface. Further mechanistic studies are necessary

to achieve a better understanding of how selenium nanoparticle coatings

inhibit the growth of various bacteria on the surface of paper towels.

Figure 3-10. The amount of adsorbed total proteins on selenium coated

and uncoated paper towels. Data=mean ± standard deviation, n=3; *, **,

***p<0.05 compared with the paper towels without coating for a

treatment after either 24, 48 or 72 hours.

3.3.5 Release of Selenium

The table 3-2 below shows the release of selenium from the selenium

nanoparticle coated paper towels, which was measured by ICP-MS.

96

About 0.94% of coated selenium was dissolved into media after first 24

hours. Another 0.51% was dissolved in the following 24 hours and an

even lower amount (0.45%) was released into solution during the third

day of treatment. As observed from the antibacterial tests, the paper

towels coated with selenium nanoparticles significantly inhibited bacteria

growth from 24 hours to 72 hours (except the test with E. coli for 24

hours). During this 3-day period, there was only 2 percent of selenium

released from the surface, which means that low selenium concentrations

between 15 ppb to 35 ppb were effective enough to inhibit bacterial

growth. As less than 1% of the selenium coatings were released during

the first 24 hours, the selenium coatings could remain in an aqueous

environment for more than 100 days as a minimum (although most likely

longer since selenium release decreased with time). Thus, the nanosized

selenium coatings on the paper towels surface could retain its potential

antibacterial properties for at least several months.

97

Table 3-2. The release of selenium from the selenium coated paper towels.

Results are the average of three samples.

Release of Se Total 1st day 2nd day 3rd day

Se concentration (ppb) 3663.8±151.8 34.47±3.45 18.68±1.93 16.31±1.52

Percentage N/A 0.94% 0.51% 0.45%

3.4 Conclusions

Selenium nanoparticles were synthesized and coated on the surface of

paper towels through a simple and quick precipitation process which

happened within one minute. Impressively, compared with the paper

towels without selenium coatings, the selenium coated paper towels

significantly inhibited the growth of S. aureus, S. epidermidis, P.

aeruginosa and E. coli after 24, 48 or 72 hours. There was significant and

continuous bacteria inhibition with about a 90% reduction from 24 to 72

hours in the growth of gram-positive bacteria including S. aureus and S.

epidermidis. The selenium coated paper towels showed a relatively lower

effectiveness at inhibiting gram-negative bacteria like P. aeruginosa and

E. coli with about a 57% and 84% reduction, respectively, after 72 hours

98

of treatment. In addition, there were larger amounts of proteins adsorbed

on the surface of selenium coated paper towels compared with uncoated

paper towels, which might be an important reason why selenium

nanoparticle coatings can inhibit diverse bacterial growth on the surface

of paper towels. Therefore, this study suggested that coating paper

products with selenium nanoparticles may be an effective way to decrease

various gram-positive and gram-negative bacteria growth on paper

products, which might be used for potentially important applications for

antimicrobial purposes in the food packaging industry and in clinical

environments.

3.5 REFERENCES

1. Desjardins E, Beaulieu C. Identification of bacteria contaminating

pulp and a paper machine in a Canadian paper mill. J Ind Microbiol

Biot. Mar 2003;30(3):141-145.

2. Namjoshi K, Johnson S, Montello P, Pullman GS. Survey of

bacterial populations present in US-produced linerboard with high

recycle content. J Appl Microbiol. Feb 2010;108(2):416-427.

3. Oqvist CK, Kurola J, Pakarinen J, et al. Prokaryotic microbiota of

99

recycled paper mills with low or zero effluent. J Ind Microbiol Biot.

Oct 2008;35(10):1165-1173.

4. Garner JS, Favero MS. Cdc Guideline for Handwashing and

Hospital Environmental-Control, 1985. Infect Cont Hosp Ep. Apr

1986;7(4):231-243.

5. McGuckin M. Improving handwashing in hospitals: a patient

education and empowerment program. LDI issue brief. Nov

2001;7(3):1-4.

6. Boyce JM PD. Draft guideline for hand hygiene in healthcare

settings. In: CDC/HICPAC, ed. Vol 512002.

7. Blackmore M. The Journal of Infection Control Nursing.

Hand-drying methods. Nursing times. Sep 16-22

1987;83(37):71-74.

8. Guzewich J RM. White paper: evaluation of risks related to

microbiological contamination of ready-to-eat food by food

preparation workers and the effectiveness of interventions to

minimise those risks. In: Administration FaD, ed. College park

(MD)September 1999.

9. Hattula JL, Stevens PE. A descriptive study of the handwashing

100

environment in a long-term care facility. Clinical nursing research.

Nov 1997;6(4):363-374.

10. Harrison WA, Griffith CJ, Ayers T, Michaels B. Bacterial transfer

and cross-contamination potential associated with paper-towel

dispensing. American journal of infection control. Nov

2003;31(7):387-391.

11. Griffith CJ, Malik R, Cooper RA, Looker N, Michaels B.

Environmental surface cleanliness and the potential for

contamination during handwashing. American journal of infection

control. Apr 2003;31(2):93-96.

12. Gendron LM, Trudel L, Moineau S, Duchaine C. Evaluation of

bacterial contaminants found on unused paper towels and possible

postcontamination after handwashing: A pilot study. American

journal of infection control. Mar 2012;40(2):E5-E9.

13. Harrison WA, Griffith CJ, Ayers T, Michaels B. Bacterial transfer

and cross-contamination potential associated with paper-towel

dispensing. American journal of infection control. Nov

2003;31(7):387-391.

14. Yokota H, Tanabe K, Sezaki M, et al. Arsenic contamination of

101

ground and pond water and water purification system using pond

water in Bangladesh. Eng Geol. Jun 2001;60(1-4):323-331.

15. Rodriguez A, Batlle R, Nerin C. The use of natural essential oils as

antimicrobial solutions in paper packaging. Part II. Prog Org Coat.

Aug 2007;60(1):33-38.

16. Balasubramanian A, Yam K, Chikindas ML. Antimicrobial

packaging: potential vs. reality—a review. Journal of Applied

Packaging Research. October 2009;3(4):193-221.

17. Garces LOE, Nerin, Puerta Cristina DE. LA. UN. (ES), Inventor;

Artibal S. A. (ES), assignee. Antimicrobial packaging based on the

use of natural extracts and the process to obtain this

packaging2006.

18. Hu WB, Peng C, Luo WJ, et al. Graphene-Based Antibacterial

Paper. Acs Nano. Jul 2010;4(7):4317-4323.

19. Ghule K, Ghule AV, Chen BJ, Ling YC. Preparation and

characterization of ZnO nanoparticles coated paper and its

antibacterial activity study. Green Chem. 2006;8(12):1034-1041.

20. Tankhiwale R, Bajpai SK. Graft copolymerization onto

cellulose-based filter paper and its further development as silver

102

nanoparticles loaded antibacterial food-packaging material. Colloid

Surface B. Mar 1 2009;69(2):164-168.

21. Xia T, Kovochich M, Liong M, et al. Comparison of the

Mechanism of Toxicity of Zinc Oxide and Cerium Oxide

Nanoparticles Based on Dissolution and Oxidative Stress

Properties. Acs Nano. Oct 2008;2(10):2121-2134.

22. Foldbjerg R, Olesen P, Hougaard M, Dang DA, Hoffmann HJ,

Autrup H. PVP-coated silver nanoparticles and silver ions induce

reactive oxygen species, apoptosis and necrosis in THP-1

monocytes. Toxicol Lett. Oct 28 2009;190(2):156-162.

23. Barceloux DG. Selenium. J Toxicol-Clin Toxic.

1999;37(2):145-172.

24. McDowell LR. Minerals in Animal and Human Nutrition (Second

Edition): Elsevier B.V.; 2003.

25. Navarro-Alarcon M, Lopez-Martinez MC. Essentiality of selenium

in the human body: relationship with different diseases. Sci Total

Environ. Apr 17 2000;249(1-3):347-371.

26. Redman C, Scott JA, Baines AT, et al. Inhibitory effect of

selenomethionine on the growth of three selected human tumor cell

103

lines. Cancer Lett. Mar 13 1998;125(1-2):103-110.

27. Kaeck M, Lu J, Strange R, Ip C, Ganther HE, Thompson HJ.

Differential induction of growth arrest inducible genes by selenium

compounds. Biochem Pharmacol. Apr 4 1997;53(7):921-926.

28. Huang Y, He L, Liu W, et al. Selective cellular uptake and

induction of apoptosis of cancer-targeted selenium nanoparticles.

Biomaterials. Sep 2013;34(29):7106-7116.




2011;32(27):6515-6522.


selenium - A promising orthopedic implant material. Journal of

Biomedical Materials Research Part A. Nov 1

2005;75A(2):356-364.

31. Yang JJ, Huang KH, Qin SY, Wu XS, Zhao ZP, Chen F.

Antibacterial Action of Selenium-Enriched Probiotics Against

Pathogenic Escherichia coli. Digestive diseases and sciences. Feb

2009;54(2):246-254.

104

32. Pietka-Ottlik M, Wojtowicz-Mlociiowska H, Kolodziejczyk K,

Piasecki E, Mlochowski J. New organoselenium compounds active

against pathogenic bacteria, fungi and viruses. Chemical &

pharmaceutical bulletin. Oct 2008;56(10):1423-1427.


Staphylococcus aureus growth. International journal of

nanomedicine. 2011;6.


of antimicrobials. Biotechnology advances. Jan-Feb

2009;27(1):76-83.

35. Ruparelia JP, Chatteriee AK, Duttagupta SP, Mukherji S. Strain

specificity in antimicrobial activity of silver and copper

nanoparticles. Acta Biomater. May 2008;4(3):707-716.

36. Zhang LL, Jiang YH, Ding YL, Povey M, York D. Investigation

into the antibacterial behaviour of suspensions of ZnO

nanoparticles (ZnO nanofluids). J Nanopart Res. Jun

2007;9(3):479-489.

37. Taylor EN, Kummer KM, Durmus NG, Leuba K, Tarquinio KM,

Webster TJ. Superparamagnetic Iron Oxide Nanoparticles (SPION)

105

for the Treatment of Antibiotic-Resistant Biofilms. Small. Oct 8

2012;8(19):3016-3027.

38. AshaRani PV, Mun GLK, Hande MP, Valiyaveettil S. Cytotoxicity

and Genotoxicity of Silver Nanoparticles in Human Cells. Acs

Nano. Feb 2009;3(2):279-290.



2003;300(5624):1439-1443.

40. Tran P, Webster TJ. Enhanced osteoblast adhesion on

nanostructured selenium compacts for anti-cancer orthopedic

applications. International journal of nanomedicine.

2008;3(3):391-396.

41. Greenwood NN, Earnshaw A. Chemistry of the elements. 2nd ed.

Oxford ; Boston: Butterworth-Heinemann; 1997.



106

Chapter 4

Red Selenium Nanoparticles and Gray Selenium Nanorods as

Antibacterial Agents for PEEK Medical Devices

4.1 Introduction

Polyetheretherketone (PEEK) is a polymer that has been used, or at least

intended to, in many different biomedical applications such as spinal

cages1, craniomaxillofacial implants

2, artificial heart valves

3, dental

applications4, bone implants

5, and so on. The major concern about this

material, and many other polymeric materials used for medical

implantation, is that they can be infected by bacteria. These infections are

usually complicated and hard to treat due to many factors such as various

bacteria species attaching to PEEK, quick growth of bacteria, the

immediate presence of serum proteins adsorbed to PEEK surfaces

mediating such bacteria adhesion, fluid flow around the implant which

provides for constant nutrients to bacteria, host immune system variables,

and many other complications6.

Clearly, device associated infections are very complicated because when

107

an implant becomes colonized by bacteria, significant patient morbidity

can follow7. The reason is that adherent bacteria are resistant to the host

immune system and systemic antibiotic therapy, since the implant

provides a niche for bacteria. Thus, the bacteria can grow on an implant

and form a biofilm over its surface, causing oral infections, inflammatory

reactions, destruction of the adjacent tissue, and implant loosening or

even detachment. Moreover, these bacterial contaminations can lead to

poor tissue (such as bone) healing.7

The treatment of such implant-related infections requires extensive

repeated surgery, implant extrication, extended duration of systemic

antibiotic therapy, tissue loss, substantial cost, suffering, and disability2,6

.

In the case of spinal instrumentation, these load-bearing implants cannot

always be removed, and so the bacterial infection, which occurs in 6% of

these patients, cannot frequently be fully eliminated.8 For hip

replacements, deep infections occur at 1.63% within 2 years of surgery.9

Moreover, the percentage of deep prosthetic infections for total knee

arthroplasty (TKA) and total hip arthroplasty (THA) procedures have

been reported to be up to 18%.10

This infection is also the most common

108

reason for repeated surgery after an otherwise successful prosthetic joint

replacement2. Finally, treatment costs related to infected orthopedic

implants can range from 1.52 to 1.76 times the cost of the original

surgery.11

Because of all the reasons mentioned above, many strategies to modify

PEEK implant surfaces have been developed and evaluated to prevent or

limit implant-associated infections. There are two different approaches in

these strategies. First, anti-adhesive surfaces are created to prevent or

limit the initial adhesion of bacteria to the surface of the implants. The

second approach is the incorporation of antimicrobial agents into the

polymeric implant materials.

Examples of anti-adhesive surfaces are material surfaces treated by

oxygen plasma. Oxygen plasma has been investigated to prevent the

initial adhesion of bacteria by increasing the surface energy of PEEK

when adding functional groups onto the polymer surface. However,

Rochford et al. showed that it does not affect bacterial adhesion in a

109

pre-operative model and there is still a need for further investigation in a

post-operative model including determining the effect of the oxygen

plasma treatment on the immune response.2 The surface of PEEK has also

been modified by photoinduction and self-initiated graft polymerization

with 2-methacryloyloxyethyl phosphorylcholine (MPC; PMPC-grafted

PEEK). Previous research by Tateishi et al. reported that bacteria

(specifically, E. coli) adhered to the surface of PMPC-grafted PEEK at a

significantly reduced number, less than 1%, however, this result is limited

because of a short period of treatment time (1 hour) in the tests.3,12

Thus,

it has not been possible to capture an accurate in vivo assessment of this

antibacterial strategy.

Another method to improve the antibacterial properties of PEEK is by

coating or incorporating PEEK with hydroxyapatite (HA). Wang et al.

have tried to modify smooth and rough PEEK surfaces with

nano-fluorohydroxyapatite (nano-FHA) since nano-FHA has been used

successfully for bone regeneration.13

The results of their study showed

that a few bacteria cells (S. mutans) were detected on the

PEEK/nano-FHA compared to the pristine PEEK after 4 hours of bacteria

110

exposure. After 24 hours, there was a reduction of S. mutans by 86.5% on

the surface of PEEK/nano-FHA compared to the bare PEEK.13

However,

the growth of S. mutans on PEEK was not detected after 24 hours and S.

mutans is a gram-positive bacterium that is commonly found in oral

infections but not found in many other infections which occur on PEEK

medical devices.

Silver additives have become ideal candidates for the development of

infection-resistant PEEK composites. However, initial investigations

performed by Jaekel showed that incorporation of up to 5 weight percent

of bactericidal silver agents in the PEEK matrix caused no significant

reduction in adherent bacteria with both injection-molded and machined

surfaces.14

The experiment was repeated and after 24 hours of bacterial

incubation, no significant differences could be observed between

Ag-composites and unfilled PEEK controls. It was concluded in that

study that silver was not effective at decreasing bacteria growth at the

concentrations tested and the method by which the silver was

incorporated.14

Therefore, the present Ag incorporation techniques need

to be optimized and other approaches need to be developed to improve

111

the antimicrobial properties of PEEK implants. Such studies also clearly

highlight the sensitivity of introducing Ag into PEEK to obtain

antibacterial properties.

Compared with many other nanoparticles used to modify the surfaces of

medical devices (such as silver nanoparticles, zinc oxide nanoparticles,

and copper oxide nanoparticles, etc.15-17

), selenium nanoparticles, as a

nonmetal material, have been considered as a healthier and less toxic

biomaterial for surface coatings.18,19

Previously, nanosized selenium has

also been studied for anticancer applications20-22

and investigated for

preventing various bacterial infections.23-26

However, nanosized selenium

has never been used with PEEK to impose antibacterial properties.

In addition, red selenium and gray selenium are two elemental forms of

selenium. The red form of selenium can be transformed into the gray

form with mild heating as the gray selenium is thermodynamically more

stable.27

A few studies have reported that red selenium is biological

active,28,29

while almost no studies have investigated the potential

bioactivity of gray selenium. Therefore, in this study, selenium

112

nanoparticles were tested for the first time as a coating material for PEEK

medical devices to inhibit bacterial growth. Two types of nanosized

selenium, red selenium nanoparticles and gray selenium nanorods, were

successfully coated on PEEK medical devices. The results of bacterial

tests showed that both red and gray nano sized selenium coatings

inhibited the growth and biofilm formation of P. aeruginosa (a bacterium

responsible for numerous medical device infections) on the surface of

PEEK medical devices. Therefore, this study demonstrated that coating

PEEK with selenium nanoparticles could be a viable approach to improve

the antibacterial properties of PEEK medical devices.


4.2.1 Preparation of Selenium Coated PEEK

Selenium nanoparticles were synthesized through a simple and quick

precipitation reaction and were precipitated on the surface of PEEK

(PEEK Optima, Invibio, Lancashire, UK). The synthesis of selenium

nanoparticles involved a reaction between glutathione (reduced form,

GSH) (97%, TCI America, Portland, OR) and sodium selenite (99%, Alfa

Aesar, Ward Hill, MA) that were mixed at a 4:1 molar ratio. The PEEK

113

samples were first washed with 75% ethanol followed by rinsing with

deionized water three times and then transferred into a flask containing a

mixed solution of glutathione and sodium selenite. Sodium hydroxide

was added into the mixture of glutathione and sodium selenite to bring

the pH of the solution into the alkaline regimen to initiate the reaction at

room temperature. Selenium nanoparticles were formed and precipitated

on the surface of PEEK immediately following the addition of NaOH as

visualized by a color change of the reactant solution from colorless to

clear red. The PEEK samples were treated in the reactants for 60 seconds

under 200 rpm (rounds per minute) of agitation to ensure a uniform

coating. The coated PEEK samples were rinsed in deionized water five

times and soaked in deionized water for 24 hours to remove the free,

non-adherent, selenium nanoparticles and possible remaining reactants.

Then, the selenium nanoparticle coated PEEK samples were left in petri

dishes for several days to dry before being used in any further

experiments.

A group of coated PEEK samples were further heated in a vacuum oven

(Lindberg Blue M, Thermo Scientific) for 6 days at a temperature of

114

100 °C to create gray selenium coated PEEK. The samples were turned

over after 3 days to ensure uniform heating on both sides. Both gray and

red selenium coated PEEK medical devices were left at room temperature

for more than six months to see whether there was any color change. The

uncoated PEEK samples that were used as controls in the following

experiments or tests were prepared by soaking PEEK in the mixture of

glutathione and sodium selenite, but sodium hydroxide was not added

into the mixture so that the reaction did not occur. Then, the uncoated

PEEK samples were treated using the same procedure as described above

for the selenium coated PEEK.

4.2.2 SEM and EDS of PEEK with Selenium Coatings

In order to determine the size, coverage and distribution of the coated

selenium nanoparticles, the surfaces of selenium coated PEEK were

characterized by scanning electron microscope (SEM) using a HITACHI

S-4800 field emission scanning electron microscope (Hitachi High

Technologies America, Inc., Schaumburg, IL). Before scanning the

surface of PEEK using SEM, the samples were coated with a 5 nm

platinum layer using a sputter coater (Cressington 208 HR, Cressington

115

Scientific Instruments Ltd., Watford, United Kingdom) to make the

PEEK samples conductive.

Energy-dispersive X-ray spectroscopy (EDS) was used to measure the

elemental composition on the surface of the PEEK samples after coating.

The EDS instrument was operated based on the SEM system mentioned

above and an acceleration voltage of 20 kV was used in the

measurements. The measurements were taken at various spots on the

PEEK surface (both with and without nanosized selenium coverage).

4.2.3 Contact Angle Measurement

Water contact angles on the PEEK surfaces before and after being coated

with selenium nanoparticles were measured using a Pioneer 800 Contact

Angle Measurement System (Surface Electro Optics Co., Ltd., South

Korea). The pictures were taken and the contact angles were analyzed

automatically by the Surfaceware 8 program. The height between the

needle and the surface was 1.7 mm and the volume of the water drop was

15 μL. The contact angle was measured 5 seconds after the drop was

116

placed on the surface and the tests were conducted at a temperature of 20

ºC.


Bacterial inhibition tests using crystal violet assays were conducted to

evaluate the growth of bacteria on selenium coated and uncoated PEEK

medical devices. The bacteria cell line, Pseudomonas aeruginosa (catalog

number 27853), was obtained in freeze-dried form from the American

Type Culture Collection (Manassas, VA). The bacteria strain was

propagated in 30 mg/mL tryptic soy broth (TSB) for 20 hours. A bacteria

solution of P. aeruginosa was prepared using 0.3 mg/mL TSB at a





Devices, Sunnyvale, CA).

The PEEK samples with or without selenium coatings were rinsed in

separate Petri dishes with 75% ethanol for 20 minutes for sterilization

117

purposes and were left in the sterile petri dishes for 30 minutes to dry

them completely. Then, the samples were transferred to a 24-well plate

and rinsed with sterile TSB (30 mg/mL) to remove any possible ethanol

residue. Each sample was treated separately with 1 mL of the prepared

bacterial solutions (106 bacteria/ml) and they were cultured for either 24,

48 or 72 hours in an incubator (37°C, humidified, 5% CO2). For those



the solution in each well was removed and each sample was rinsed with a

PBS (phosphate buffered saline) solution three times, then the samples

were transferred into a new 24-well plate.

After turning off the light in the laminar flow hood, 0.5 mL of a 0.1%

crystal violet solution was added into each well and incubated for 15

minutes to stain the bacterial biofilm formed on the PEEK samples. The

remaining staining solution was removed and each sample was washed

with sterile PBS extensively (rinsing with 1 mL PBS three times) to

remove any access crystal violet. The plate was air dried at room

temperature for one day to let the stained samples dry completely. Then, 1

118

mL of 100% ethanol was added into each well to dissolve the crystal

violet dye. After letting the well plate sit for 2 minutes, the solution from

each well was transferred into a 96 well plate and the optical density was

measured at 562 nm using a SpectraMax M5 plate reader.

In the bacterial inhibition tests, the treatment of each sample was

conducted individually and identically. Another group of coated and

uncoated PEEK samples were included as blank controls. They were

treated with a sterile TSB solution without bacteria and were also stained

using the same process as described above. The optical density of the

blank control group was deducted from the optical density of bacteria

treated PEEK samples to calculate the absorbency from the bacterial

biofilm formed on PEEK surfaces. The growth of bacterial biofilm was

quantified based on the absorbency.

4.2.5 Statistics

All bacterial inhibitory tests were conducted in triplicate and repeated

three times. Data were collected and the significant differences were

assessed with the probability associated with a one-tailed Student's t-test.

119

Statistical analyses were performed using Microsoft Excel (Redmond,

WA).

4.3 Results and Discussion

4.3.1 PEEK Coated with Selenium Nanoparticles

After the coating process as described in 4.2.1, the color of the PEEK

samples changed from light tan (Figure 4-1, image a) to light red (Figure

4-1, image b). The red color indicated the formation of selenium

nanoparticles on the surfaces of PEEK. The selenium coated PEEK could

maintain its red appearance for several months at room temperature. After

heated at 100 °C for 6 days, the red PEEK in image b turned into the gray

PEEK as shown in image c in Figure 4-1. During the heat treatment, a

gray color gradually appeared as the red color decayed on the PEEK

surface. The color changed from red to gray indicating the formation of

gray selenium on the PEEK surfaces, because the gray selenium allotrope

is the most stable form of selenium and can be formed by the mild

heating of the red allotrope. Both red and gray PEEK could retain its

color for at least six months at room temperature.

120

Figure 4-1. Light images of (a) uncoated PEEK, (b) PEEK coated with

selenium nanoparticles (red coated PEEK), and (c) PEEK coated with

selenium nanoparticles and with heating treatment (gray coated PEEK).

4.3.2 SEM and EDS of PEEK

As observed form the SEM image (Figure 4-2, image b) of red coated

PEEK, the PEEK surface was coated with a large amount of selenium

nanoparticles. These nanoparticles were well distributed on the PEEK

surface and covered almost all parts of the polymer surface. The

diameters of the coated nanoparticles were measured and the size

121

distribution was found to be between 50 and 100 nm. Image a in Figure

4-2 was the surface of a PEEK sample without coatings, onto which no

nanoparticle was observed. As shown the SEM image (image c in Figure

4-2) of gray coated PEEK (formed by heating red coated PEEK at 100 °C

for 6 days), there were rod-shaped selenium clusters observed on the

surface and all of the selenium nanoparticles from the red coated PEEK

(image b) disappeared. The length of these nanorods was from 1000 nm

to 3000 nm and the width was from 100 nm to 200 nm.

122

Figure 4-2. Scanning electron microscopy images of: (a) uncoated PEEK,

123

(b) PEEK coated with selenium nanoparticles, (c) PEEK coated with

selenium nanoparticles and with a heating treatment. Magnification =

10.0 k and acceleration voltage = 5.0 kV.

According to the light images (Figure 4-1) and SEM images (Figure 4-2)

of red selenium coated PEEK before and after the heat treatment, it is

likely that the red selenium nanoparticles were transferred into gray

selenium nanorods during the heat treatment. In order to confirm that

both attached nanoparticles and nanorods were consist of selenium

element, EDS analyses were taken on various spots on the PEEK surfaces.

As shown in Figure 4-3 (a), a strong peak in the spectra demonstrated the

existence of selenium element on the spot with a nanoparticle attached.

But on a blank spot in Figure 4-3 (b), no selenium element was detected.

Thus, the nanoparticles attached on the PEEK surfaces were made of

selenium element. Similar peaks were also observed in Figure 4-3 (c) and

(d) and the only difference from spectra (c) to spectra (d) is the

disappearance of the selenium peak, which indicated that the gray

nanorodes formed by heating the red selenium nanoparticles

124

125

Figure 4-3. Energy-dispersive X-ray spectroscopic analyses of (a) a spot

with visualization of nanoparticle on red PEEK, (b) a blank area without

nanoparticle on red PEEK, (c) a spot with visualization of nanorodes on

gray PEEK, and (d) a blank area without nanorodes on gray PEEK.

4.3.3 Contact Angles

The results of water contact angle measurements of on the three types of

PEEK surfaces were shown in Figure 4-4 and Table 4-1 below. The water

contact angle on the uncoated PEEK was 68.47°. The red nanoparticles

coated and gray nanorodes coated PEEK had a contact angle of 78.14°

and 76.98°, respectively. Both were larger than the uncoated PEEK,

which indicated that the PEEK surface became more hydrophobic after

coated with selenium nanoparticles or nanorodes. The contact angle of

red selenium coated PEEK and gray selenium coated PEEK was similar.

Thus, there was not a significant change in the hydrophilicity of the

selenium coated PEEK after the coated red selenium nanoparticles was

heated and transferred into gray selenium nanorodes.

126

Figure 4-4. Contact angle measurements of (a) uncoated PEEK, (b) red

selenium nanoparticles coated PEEK, and (c) gray selenium nanorodes

coated PEEK.

127

Table 4-1. Contact angles of three types of PEEK samples.

Sample number Uncoated PEEK Red PEEK Gray PEEK

1 68.72° 76.78° 77.26°

2 67.10° 78.78° 77.78°

3 69.59° 78.87° 75.91°

Average 68.47°±1.26° 78.14°±1.18° 76.98±0.97°


As seen in Figure 4-4, the results of bacteria assays involving

Pseudomonas aeruginosa (P. aeruginosa) showed that either red

selenium nanoparticles coated paper towels or gray selenium nanorodes

coated paper towels had a high effectiveness at inhibiting bacterial

biofilm growth from 1 day to 3 days. The crystal violet absorbance in the

graph was the optical density of crystal violet from the bacteria treated

PEEK sample deducted by the optical density of crystal violet from the

blank control group treated with sterile tryptic soy broth. In first day of

the treatment, the biofilm was at the stage of initial attachment and it took

time for the bacteria to adapt to the microenvironment on the surfaces.

128

There was not too much biofilm attached on all 3 types of PEEK surfaces,

but a larger biofilm mass was still detected on the uncoated PEEK than

red nanoparticles or gray nanorodes coated PEEK. From 1 day to 2 days,

the amount of the biofilm increased quickly for all the PEEK samples,

which indicated the biofilm began to mature and bacteria propagated

rapidly in the biofilm. After the second day, there was significantly more

biofilm attached on the uncoated PEEK than the red selenium or gray

selenium coated PEEK. From 2 days to 3 days, the biofilm was almost

saturated by bacteria and there were not large increases in the total

amount of the biofilm. However, in the third day of the treatment, the

total amount of biofilm increased more on the surface of uncoated PEEK

than the two selenium coated PEEK, so after the third day, the red and

gray PEEK revealed more significant inhibitions to the growth of P.

aeruginosa biofilm than the inhibition after the second day. Besides, no

significant difference in the biofilm growth was observed between the red

selenium nanoparticles coated PEEK and the gray selenium nanorodes

coated PEEK at the first day, while after three days, the red PEEK

showed a little higher inhibition to the biofilm growth. The reason could

be that there were more nano-scale features on the red PEEK than the

129

gray PEEK, which might result in a more active interaction between the

coated nanoparticles and the bacterial cells. Overall, both types of

nanosized selenium coated PEEK successfully inhibited the formation of

P. aeruginosa biofilm compared with PEEK without selenium coating.

Figure 4-5. The growth of P. aeruginosa biofilm on red selenium coated

PEEK, gray selenium coated PEEK and uncoated PEEK. Data=mean ±

standard deviation, n=3; *p<0.05 compared with red PEEK after

treatment of the same time period. **p<0.05 compared with gray PEEK

after treatment of the same time period.

As mentioned previously, P. aeruginosa are gram-negative bacteria that

have an extra layer of bacterial outer membrane as a part of their cell wall,

130

which results in that it might take longer for selenium element or

selenium nanoclusters to penetrate into and interact with P. aeruginosa.

Thus, it may be expected that the red nanoparticles or gray nanorodes

coatings on the surface of the PEEK will show a stronger inhibition to

gram-positive bacteria, like S. aureus. Further bacterial inhibitory tests on

PEEK medical devices involving other bacteria stains will be helpful to

explore more of the antibacterial properties of red or gray selenium

coated PEEK.

In addition to preventing bacterial infections on PEEK, red selenium

nanoparticle coatings demonstrated improved healthy bone cell

(osteoblast) growth while inhibiting cancerous bone cell (osteosarcoma)

growth.19,30

Selenium is also an important component for selenoproteins

in the human body that were found to be fundamental for the human

antioxidant defense system31

and were recently proven to be an essential

transporter for generating healthy bone.32

Therefore, coating PEEK with

red selenium nanoparticles may potentially improve its antibacterial

without being toxic to healthy cells (especially bone cells), which are

both of vital importance for PEEK, especially PEEK medical devices

131

used in the design for orthopedic and spinal devices.

4.4 Conclusions

In conclusion, red selenium nanoparticles were synthesized and coated on

the surface of PEEK medical devices here. By heating the coated PEEK

samples, the red selenium nanoparticles were successfully transferred to

gray selenium nanorods, which were shown to have antibacterial

properties for the first time here. Both red and gray coatings on PEEK

medical devices were stable for at least six months at room temperature.

Importantly, compared with the PEEK without selenium coatings, the red

or gray selenium coated PEEK significantly inhibited the growth of P.

aeruginosa after 1, 2 and 3 days. The inhibition of bacterial biofilm

growth was more and more significant from 1 to 3 days. In addition, the

red or gray selenium coatings on the PEEK surfaces decreased the

hydrophilicity of the PEEK medical devices, which might play an

important role in inhibiting the growth of a bacterial biofilm. Therefore,

this study suggested that coating PEEK medical devices with red

selenium nanoparticles or gray selenium nanorods might be an effective

way to decrease the growth of bacterial biofilm on those medical devices,

132

which could have potential applications for preventing infections on

various medical devices used in healthcare environments. Moreover,

coupled with previous studies that have demonstrated that red selenium

increases healthy bone cell growth, the present results demonstrate the

promise of red selenium nanoparticle coatings on PEEK for numerous

orthopedic and spinal applications.

4.5 REFERENCES

1. Toth JM, Wang M, Estes BT, Scifert JL, Seim HB, Turner AS.

Polyetheretherketone as a biomaterial for spinal applications.

Biomaterials. Jan 2006;27(3):324-334.

2. Rochford ETJ, Poulsson AHC, Varela JS, Lezuo P, Richards RG,

Moriarty TF. Bacterial adhesion to orthopaedic implant materials

and a novel oxygen plasma modified PEEK surface. Colloid

Surface B. Jan 1 2014;113:213-222.

3. Tateishi T, Kyomoto M, Kakinoki S, Yamaoka T, Ishihara K.

Reduced platelets and bacteria adhesion on poly(ether ether ketone)

by photoinduced and self-initiated graft polymerization of

2-methacryloyloxyethyl phosphorylcholine. J Biomed Mater Res A.

133

May 2014;102(5):1342-1349.

4. Stawarczyk B, Beuer F, Wimmer T, et al. Polyetheretherketone-A

suitable material for fixed dental prostheses? J Biomed Mater Res

B. Oct 2013;101(7):1209-1216.

5. Sagomonyants KB, Jarman-Smith ML, Devine JN, Aronow MS,

Gronowicz GA. The in vitro response of human osteoblasts to

polyetheretherketone (PEEK) substrates compared to commercially

pure titanium. Biomaterials. Apr 2008;29(11):1563-1572.

6. Kurtz SM, Plastics Design Library. PEEK biomaterials handbook.

1st ed. Oxford UK ; Waltham, MA: William Andrew; 2012.

7. Gorth DJ, Puckett S, Ercan B, Webster TJ, Rahaman M, Bal BS.

Decreased bacteria activity on Si3N4 surfaces compared with

PEEK or titanium. International journal of nanomedicine.

2012;7:4829-4840.

8. Collins I, Wilson-MacDonald J, Chami G, et al. The diagnosis and

management of infection following instrumented spinal fusion. Eur

Spine J. Mar 2008;17(3):445-450.

9. Ong KL, Kurtz SM, Lau E, Bozic KJ, Berry DJ, Parvizi J.

Prosthetic Joint Infection Risk After Total Hip Arthroplasty in the

134

Medicare Population. J Arthroplasty. Sep 2009;24(6):105-109.

10. Matar WY, Jafari SM, Restrepo C, Austin M, Purtill JJ, Parvizi J.

Preventing Infection in Total Joint Arthroplasty. J Bone Joint Surg

Am. Dec 1 2010;92A:36-46.

11. Kurtz SM, Lau E, Schmier J, Ong KL, Zhao K, Parvizi J. Infection

burden for hip and knee arthroplasty in the United States. J

Arthroplasty. Oct 2008;23(7):984-991.

12. Kyomoto M, Moro T, Takatori Y, Kawaguchi H, Nakamura K,

Ishihara K. Self-initiated surface grafting with

poly(2-methacryloyloxyethyl phosphorylcholine) on

poly(ether-ether-ketone). Biomaterials. Feb 2010;31(6):1017-1024.

13. Wang LX, He S, Wu XM, et al.

Polyetheretherketone/nano-fluorohydroxyapatite composite with

antimicrobial activity and osseointegration properties. Biomaterials.

Aug 2014;35(25):6758-6775.

14. Jaekel DJ. Development and Fabrication of Silver Composite

PEEK to Prevent Microbial Attachment and Periprosthetic

Infection Biomedical Engineering, Drexel University; 2012.


135

of antimicrobials. Biotechnol Adv. Jan-Feb 2009;27(1):76-83.




17. Li JH, Hong RY, Li MY, Li HZ, Zheng Y, Ding J. Effects of ZnO

nanoparticles on the mechanical and antibacterial properties of

polyurethane coatings. Prog Org Coat. Mar 2009;64(4):504-509.


selenium - A promising orthopedic implant material. J Biomed

Mater Res A. Nov 1 2005;75A(2):356-364.

19. Tran P, Webster TJ. Enhanced osteoblast adhesion on

nanostructured selenium compacts for anti-cancer orthopedic

applications. Int J Nanomed. 2008;3(3):391-396.

20. Huang YY, He LZ, Liu W, et al. Selective cellular uptake and

induction of apoptosis of cancer-targeted selenium nanoparticles.

Biomaterials. Sep 2013;34(29):7106-7116.

21. Sun DD, Liu YN, Yu QQ, et al. Inhibition of tumor growth and

vasculature and fluorescence imaging using functiOnalized

ruthenium-thiol protected selenium nanoparticles. Biomaterials.

136

Feb 2014;35(5):1572-1583.




2011;32(27):6515-6522.


Staphylococcus aureus growth. Int J Nanomed. 2011;6.

24. Wang Q, Webster TJ. Short communication: inhibiting biofilm

formation on paper towels through the use of selenium

nanoparticles coatings. Int J Nanomed. 2013;8:407-411.

25. Tran PA, Webster TJ. Antimicrobial selenium nanoparticle coatings

on polymeric medical devices. Nanotechnology. Apr 19

2013;24(15).

26. Wang Q, Webster TJ. Nanostructured selenium for preventing

biofilm formation on polycarbonate medical devices. J Biomed

Mater Res A. Dec 2012;100A(12):3205-3210.

27. Greenwood NN, Earnshaw A. Chemistry of the elements. 2nd ed.

Oxford ; Boston: Butterworth-Heinemann; 1997.


137


29. Domokos-Szabolcsy E, Marton L, Sztrik A, Babka B, Prokisch J,

Fari M. Accumulation of red elemental selenium nanoparticles and

their biological effects in Nicotinia tabacum. Plant Growth Regul.

Dec 2012;68(3):525-531.



coculture on titanium. Int J Nanomed. 2010;5:351-358.



2003;300(5624):1439-1443.

32. Pietschmann N, Rijntjes E, Hoeg A, et al. Selenoprotein P is the

essential selenium transporter for bones. Metallomics.

2014;6(5):1043-1049.

138

Chapter 5

Antibacterial Selenium Quantum Dots and Nanoparticles Produced

by UV/VIS/NIR Pulsed Nanosecond Laser Ablation

5.1 Introduction

Selenium has been defined by the Materials Research Society and the

American Physical Society as an energy critical element meaning that this

element is required for emerging sustainable energy sources that might

encounter supply disruptions1,2

. Selenium belongs to the oxygen family

(group 16 in the periodic table), it is then not oxidized in air and insoluble

in water. It is a p-type semiconductor having a direct energy bandgap

around 1.7 eV, it is therefore used in CuInSe2 (CIS) solar cells3 and

quantum dot (CdSe) solar cells4. Moreover, selenium has also very

interesting biological assets, as anti-cancer5,6

and anti-bacterial

properties7,8

. The goal of this chapter is to report the synthesis of

selenium by pulsed laser ablation in liquids (PLAL) and characterization

of very pure selenium nanoparticles and quantum dots that can be use

either for photovoltaic or biological applications.

139

The ability to produce nanoparticles free of any surface contamination is

very challenging especially for bio-medical applications. The advantages

of PLAL synthesis compared to the other conventional methods are the

simplicity of the set-up and the surface purity of the synthesized

nanoparticles (absence of unnecessary adducts and byproducts)9. Indeed,

selenium nanoparticles are generally synthesized by chemical

methods10-12

. Only three papers in the literature report the synthesis of

selenium nanoparticles by pulsed laser ablation in liquids. In 2010, Singh

et al.13

used a Nd:YAG laser at 1064 nm, 10 ns pulse duration, 10 Hz

repetition rate. In 2012, Kuzmin et al.14

used a copper vapor laser at 511

nm and 578 nm, 15 ns pulse duration, 15 kHz repetition rate. In 2013,

Van Overschelde et al.15

used a KrF excimer laser at 248 nm, 6 ns pulse

duration, 50 Hz repetition rate. They all used water as solvent and

irradiated selenium powder, ingot and pellet respectively.

In this chapter, selenium powders were irradiated using three different

wavelengths, a near-infra-red wavelength at 1064 nm (fundamental beam,

λ), a visible wavelength at 532 nm (second harmonic beam, λ/2) and an

ultra-violet wavelength at 355 nm (third harmonic beam, λ/3), all issued

140

from the same Nd:YAG laser source. Their effects on the particle size

distribution were studied by dynamic light scattering and transmission

electronic microscopy, revealing then the production of selenium

quantum dots (size < 4 nm) by photo-fragmentation. It was found that the

crystallinity of the nanoparticles depends on their size. The zeta-potential

measurement revealled that the colloidal solutions produced in de-ionized

water were stable while the ones synthesized in ethanol agglomerate. The

concentration of selenium was measured using ICP-MS. The antibacterial

effect of selenium nanostructures were analyzed on E. Coli bacteria.

Finally, selenium quantum dots produced by this method could also be

useful for quantum dots solar cells.

5.2 Experiments and Results

5.2.1 Preparation of Selenium Nanoparticles by Ablation

Selenium is known to have several different allotropic structures: trigonal,

monoclinic (α-, β-), cubic (α-, β-), rhombohedric, ortho-rhombic and

amorphous16

; however, the most common allotropic phases of selenium

are trigonal, α-monoclinic and amorphous. Raman spectroscopy and

X-Ray Diffraction were performed on a Horiba-Yvon Jobin spectrometer

141

(iHR 320) using a red laser at 785 nm, and on a Rigaku Ultima IV

diffractometer working at 40 kV, 44 mA, respectively. Results of both the

Raman spectroscopy and X-Ray Diffraction on the pure selenium powder

(99.999%, ~200 mesh) purchased from Alfa Aesar reveals the trigonal

(hexagonal) structure of selenium

The experimental setting for ablation is shown in Figure 5-1. Pure

selenium powder has been placed at the bottom of the glass cuvette and

then filled with the liquid (de-ionized water or ethanol 99.5%) until the

top of the cuvette (~4 ml). As the selenium’s density (4.28 g/cm3) is

higher than the one of water (1g/cm3) and the one of ethanol (0.79 g/cm

3),

selenium is then found at the bottom of the cuvette, requiring a magnetic

stirrer to mix selenium with the solvent. A magnetic stirrer is used at 1000

rpm/minute to assure a uniform dispersion of selenium powder into the

solution during the irradiation. The laser beam is then focused in the

middle of the solution. The laser used in this experiment is a Nd:YAG

laser from EKSPLA NT342B with a pulse duration of 3.6 ns and a

repetition rate of 10 Hz, each pulse having a top hat profile. Energy of the

laser was monitored during all the duration of the experiment and kept

142

constant at ~23±1 mJ/pulse. Upon focusing this pulse energy corresponds

to a fluence of ~2 J/cm2.

Figure 5-1. Diagram of generating selenium nanoparticles by ablation.

During the first set of irradiations, the solution was irradiated at a given

wavelength over a 15 minute interval at a 10Hz repetition rate. A

red-brick color solution was produced either in water or ethanol,

indicating the presence of amorphous nanoparticles as already noted by

Ref.15

; while the solution becomes colorless after centrifugation at 10000

rpm for 10 minutes leaving in suspension only the smallest nanoparticles.

5.2.2 Characterization of Selenium Nanoparticles

From Transmission Electronic Microscope (TEM) observations, carried

143

out on a JEOL 2010F microscope operating at 200 kV, the diffraction

pattern of the biggest nanoparticles shows an amorphous structure while

the smallest ones exhibit a crystalline structure, as shown in Figure 5-2.

This means that the nanoparticles of selenium grow so quickly after the

irradiation pulse that the vast majority of them do not have time to

crystallize due to the rapid quenching led by the surrounding liquid media.

Then, most of these big particles settled down progressively by gravity

after the stop of the magnetic stirrer and for sure after the centrifugation

treatment.

Figure 5-2. TEM images and diffraction pattern of selenium

144

nanoparticles after the first set of irradiation at 532 nm in de-ionized

water, before any centrifugation and any filtration. a) Spherical particle

around 300 nm, b) Spherical particle around ~150 nm, c) Spherical

particle around 80 nm, d), e) and f) are the diffraction pattern of image a),

b) and c) respectively.

To refine the size distribution of the selenium nanoparticles produced, the

solutions were also filtrated through a 200 nm pore size filter to be easily

measurable by Dynamic Light Scattering (Zetasizer Nano ZS from

Malvern Instruments). The particle size distributions of the selenium

colloidal solutions measured by DLS in de-ionized water and ethanol are

shown on Figures 5-2a and 5-2b after the first set of irradiations. The

particles sizes synthesized in de-ionized water are all around ~100 nm

whatever the wavelength used, except for the infra-red irradiation where

smaller nanoparticles (~20 nm) are also produced. In ethanol, the

situation is quite different, very small particles (<10 nm) and big

nanoparticles (~ 200nm) are produced at the same time. The

polydispersity is then much higher in ethanol compared to de-ionized

water.

145

Figure 5-3. DLS spectra of the centrifuged and filtrated colloidal solution

of selenium nanoparticles after the first and second set of irradiations

(time=15 min, f=10Hz) in de-ionized water and ethanol. a) first set of

irradiations in DI water; b) first set of irradiations in ethanol; c) second

set of irradiations in DI water; d) second set of irradiations in ethanol.

The solution was centrifuged at 10000 rpm during 10 minutes after each

set of irradiations (time=15 min, f=10Hz) and then filtrated through a 200

nm pore size filter. Then, the solution was analyzed by DLS. The insets

indicate the zeta potential of each sample.

146

The stability of the colloidal solutions was evaluated by the measurement

of the zeta potential, which is the electrical potential surrounding the

particle. It is clear from the insets of Figure 5-3 that the particles

synthesized in de-ionized water are stable while the ones in ethanol are

not and tend to flocculate. Indeed, the zeta potential indicates the degree

of repulsion between adjacent particles. If the zeta potential is higher than

30 mV, then the solution is considered as stable which is mostly the case

in de-ionized water except for the solutions synthesized with the infra-red

wavelength. At the contrary, if the zeta potential has a value below 30 mV,

the solution is unstable and the particles flocculate which is the case in

ethanol. Due to flocculation, the particle size distributions obtained in

ethanol are more poly-dispersed compared to the ones obtained in

de-ionized water (Figure 5-3). From TEM images carried out at low

magnification on a JEOL 1230 microscope operating at 120 kV (as shown

in Figure 5-4), we clearly see the difference between the two solvents,

nanoparticles are quite dispersed in water while they flocculate in ethanol

and form a cluster of particles which is detected by the DLS as the size

distribution around 200 nm (Figure 5-3b). The instability of the

nanoparticles synthesized in ethanol has already been noted by Werner et

147

al.17

who investigated the synthesis of silver nanoparticles in different

primary alcohols.

Figure 5-4. TEM images of selenium nanoparticles obtained in a)

de-ionized water and b) ethanol.

Table 5-1. Summary of the laser parameters used, the corresponding size

and zeta potential of selenium nanoparticles obtained. The energy at all

wavelengths has been fixed to ~23±1 mJ/pulse corresponding to a fluence

of ~2 J/cm2 and the laser repetition rate was 10 Hz.

Liquid Samples* Wavelength

(nm)

Size

determined

by DLS

(nm)

Zeta

potential

determined

by DLS

148

(mV)

DI water 1a 355 98±13 -40±12

2a 532 118±15 -30±10

3a 1064 148±32

(83%)

22±3 (17%)

-12±5

DI water 1b 355 64±9 -24±6

2b 532 62±12 -26±7

3b 1064 107±3 -16±7

Ethanol 4a 355 183±16

(51%)

2.4±0.3

(27%)

0.6±0.1

(22%)

12±24

5a 532 275±20 2±14

149

(58%)

2.0±0.2

(42%)

6a 1064 209±29

(49%)

3.2±0.5

(35%)

0.7±0.1

(16%)

-14±13

Ethanol 4b 355 178±13

(83%)

2±17 (17%)

3±16

5b 532 255±28

(94%)

0.7±0.1 (6%)

1±13

6b 1064 196±38 -2±15

*“a” means first set of irradiation i.e. these solutions have been irradiated

during 15 minutes and then centrifuged during 10 minutes at 10000 rpm

and finally filtrated through a 200 nm pore size filter, “b” means

150

secondary set of irradiations i.e. these solutions have been irradiated a

first time during 15 minutes and then centrifuged during 10 minutes at

10000 rpm and irradiated again during 15 minutes and centrifuged a last

time during 10 minutes at 10000 rpm and finally filtrated through a 200

nm pore size filter .

5.2.3 Generation of Selenium Quantum Dots

To reduce the polydispersity, the solutions were irradiated a second time

for 15 minutes at 10 Hz and then centrifuged again at 10000 rpm for 10

minutes and filtrated through a 200 nm pore size filter. From Figure 5-3c,

the smallest nanoparticles (~60 nm) synthesized in de-ionized water were

obtained with the UV and visible wavelengths. This can be explained by

photo-fragmentation, selenium absorbs below ~730 nm, i.e. the UV and

visible wavelengths used in our experiment, and is therefore

photo-fragmented; while the synthesis using the NIR wavelength only

produced nanoparticles by thermal ablation. Therefore, the particle size

decreases with decreasing wavelength. From Figure 5-3d, the particles

synthesized in ethanol are poly-dispersed whatever the wavelength used.

Furthermore, they are also bigger compared to the ones synthesized in

151

water except for the UV and visible wavelength where a significant

percentage (~17% and 6%, respectively) of particles below 3 nm are

produced. These nanoparticles are quantum dots of selenium; indeed, the

exact Bohr radius of selenium has been calculate to be around 1.16 nm18

and experimentally the quantum confinement limit has been determined

around 3.75 ± 0.15 nm13

by the shift of the surface plasmon resonance

peak. Actually, a strong blue shift occurs on this peak below the

confinement limit. This is the first time in the literature that selenium

quantum dots have been synthesized using ultra-violet and visible

wavelengths. The synthesis of selenium quantum dots by infra-red

wavelength was already reported in 2010 by Singh et al. 13

.

High Resolution Transmission Electron Microscopy (HRTEM)

experiments were also carried out on a JEOL 2010F microscope

operating at 200 kV. Quantum dots are efficiently synthesized in water

and ethanol by using the UV and visible wavelength (Figure 5-5). Indeed,

the laser mechanism inducing size reduction can be either thermal

ablation or photo-fragmentation depending on the irradiation wavelength.

For UV and visible wavelengths, the size reduction of selenium particles

152

is due to photo-fragmentation while for NIR wavelength, it is due to

thermal ablation. From the diffraction patterns shown in Figure 5-5 as

insets, the quantum dots are crystalline and adopt the trigonal (hexagonal)

structure of selenium. Therefore, the crystal structure of the initial powder

is conserved even after the irradiation.

Figure 5-5. HRTEM images of selenium quantum dots synthesized

153

during the second set of irradiation by a) UV irradiation in de-ionized

water, b) visible irradiation in de-ionized water, c) UV irradiation in

ethanol and d) visible irradiation in ethanol. In the right upper corner, as

an inset, the FFT pattern of the quantum dots is shown.

5.2.4 Antibacterial Tests

The antibacterial test was conducted on E. Coli bacteria by using the most

stable and less dispersed selenium nanoparticles, obtained after the

second irradiation in de-ionized water (samples 1b, 2b, 3b). The samples

used the antibacterial test had to be the same concentration. To measure

the selenium concentrations of the prepared samples, 1 mL of each

solution was dissolved using 10 M NaOH and then diluted 100 times

before ICP (Bruker Aurora M90) measurements. The concentrations of

samples 1b, 2b are 2.1 ppm and 1.35 ppm, respectively, while the

concentration of sample 3b was undetectable. Therefore, sample 3b was

not considered for the antibacterial test and sample 1b was diluted to the

same concentration as sample 2b. A red selenium nanoparticle sample

that previously showed significant inhibition to bacterial growth19,20

was

also included in the bacterial tests as a comparison. These samples have

154

been sterilized by UV light irradiation. Then, 0.02 ml of each solution

was mixed with 0.18 ml of bacteria solution (E. coli, 106 bacteria/ml) in

96-well plates. Incubation was running for either 4, 8 or 24 hours. The

bacteria numbers were calculated by measuring the absorbency curves of

the samples registered at 562 nm and compared with the standard

absorbency curve of E. coli. As shown in Figure 5-6 below, all selenium

samples show significant inhibition to the growth of E. coli after 8 hours

and 24 hours. The percentages of inhibited bacterial growth were not very

high, and the reason could be that the samples prepared and used in the

bacterial treatment had a very low concentration (only 0.135 ppm), which

is much lower than the concentrations of selenium used in the bacterial

assays in the chapter 3. Even though, the selenium nanoparticles and

quantum dots produced by laser ablation showed promising properties to

prevent bacterial growth.

155

Figure 5-6. The growth of E. coli with a treatment of selenium

nanoparticles. Data=Mean ± standard deviation, n=3; *p<0.05 compared

with the control group (DI water) after either 4, 8 or 24 hours.

5.3 Conclusions

In conclusion, nanosecond laser irradiation provides enough energy

density (~2 J/cm2) to melt selenium powder whatever the wavelength

used. Size-reduction efficiency in PLAL increases by decreasing

wavelength because of the increase in photon energy. Amorphization or

crystallization of the molten selenium will depend on the size of the

particle. Nanoparticles with size above ~300 nm are amorphous while

156

below ~ 80 nm they are crystalline. Thus, contrary to what is generally

believed, the nanoparticles produced by laser ablation in liquids are not

only amorphous but exhibit a size-dependent crystallinity behavior. The

centrifugation process and the second set of irradiations are an essential

part of the synthesis process to obtain a well-defined mono-dispersed size

distribution. In terms of stability, de-ionized water has to be preferred as

solvent compared to ethanol due to agglomeration. Quantum dots have

also been obtained by irradiating selenium powder in de-ionized water

and ethanol using the ultra-violet or visible wavelength. Furthermore,

quantum dots adopt the trigonal structure of selenium. To conclude,

selenium nanoparticles can be used for various biomedical applications21

,

including antibacterial applications, while selenium quantum dots can be

used for optoelectronics applications like for example in quantum dot

solar cells22

.

5.3 References

1. Jaffe R, Price J, Ceder G, et al. Energy Critical Elements: Securing

Materials for Emerging Technologies. POPA Reports.

2011;February:1-28.

157

2. Hurd AJ, Kelley RL, Eggert RG, Lee M-H. Energy-critical

elements for sustainable development. MRS Bulletin.

2012;37:405-410.

3. Guo Q, Kim SJ, Kar M, et al. Development of CuInSe2

Nanocrystal and Nanoring Inks for Low-Cost Solar Cells. Nano

Letters. 2008;8:2982-2987.

4. Robel I, Subramanian V, Kuno M, Kamat PV. Quantum Dot Solar

Cells. Harvesting Light Energy with CdSe Nanocrystals

Molecularly Linked to Mesoscopic TiO2 Films. Journal of the

American Chemical Society. 2006;128:2385-2393.

5. Sun D, Liu Y, Yu Q, et al. Inhibition of tumor growth and

vasculature and fluorescence imaging using functionalized

ruthenium-thiol protected selenium nanoparticles. Biomaterials.

2014;35:1572-1583.

6. Ramamurthy CH, Sampath KS, Arunkumar P, et al. Green

synthesis and characterization of selenium nanoparticles and its

augmented cytotoxicity with doxorubicin on cancer cells.

Bioprocess and Biosystems Engineering 2013;36:1131-1139.

7. Tran PA, Webster TJ. Antimicrobial selenium nanoparticle coatings

158

on polymeric medical devices. Nanotechnology. Apr 19

2013;24(15).



nanoparticles coatings. Int J Nanomed. 2013;8:407-411.

9. Yang G. Laser Ablation in Liquids: Principles and Applications in

the Preparation of Nanomaterials: Pan Stanford Publishing; 2012.

10. Gao X, Zhang J, Zhang L. Hollow Sphere Selenium Nanoparticles:

Their In-Vitro Anti- Hydroxyl Radical Effect. Advanced Materials.

2002;14:290-293.

11. Chen L, Wen YM. The role of bacterial biofilm in persistent

infections and control strategies. Int J Oral Sci. Apr

2011;3(2):66-73.

12. Kumar A, Sevonkaev I, Goia DV. Synthesis of selenium particles

with various morphologies. Journal of Colloid and Interface

Science. 2014;416:119-123.

13. Singh SC, Mishra SK, Srivastava RK, Gopal R. Optical Properties

of Selenium Quantum Dots Produced with Laser Irradiation of

Water Suspended Se Nanoparticles. Journal of Physical Chemistry

159

C. 2010;114:17374-17384.

14. Kuzmin PG, Shafeev GA, Voronov VV, et al. Bioavailable

nanoparticles obtained in laser ablation of a selenium target in

water. Quantum Electronics. 2012;42:1042-1044.

15. Van Overschelde O, Guisbiers G, Snyders S. Green synthesis of

selenium nanoparticles by excimer pulsed laser ablation in water.

APL Materials. 2013;1:042114.

16. Minaev VS, Timoshenkov SP, Kalugin VV. Structural and phase

transformations in condensed selenium. Journal of Optoelectronics

and Advanced Materials. 2005;7:1717-1741.

17. Werner D, Hashimoto S, Tomita T, Matsuo S, Makita Y.

Examination of Silver Nanoparticles Fabrication by Pulsed Laser

Ablation of Flakes in Primary Alcohols. Journal of Physical

Chemistry C. 2008;112:1321-1329.

18. Tutihasi S, Chen I. Optical Properties and Band Structure of

Trigonal Selenium. Physical Review. 1967;158:623-630.

19. Wang Q, Webster TJ. Nanostructured selenium for preventing

biofilm formation on polycarbonate medical devices. Journal of

Biomedical Materials Research Part A. Dec

160

2012;100A(12):3205-3210.



nanoparticles coatings. International journal of nanomedicine.

2013;8:407-411.

21. Rayman MP. Selenium and human health. Lancet. Mar 31

2012;379(9822):1256-1268.

22. Kamat PV. Quantum Dot Solar Cells. The Next Big Thing in

Photovoltaics. J Phys Chem Lett. Mar 21 2013;4(6):908-918.

161

Chapter 6

Inhibited Growth of Pseudomonas aeruginosa by Dextran and

Polyacrylic acid Coated Ceria Nanoparticles

6.1 Introduction

Among rare earth elements (the fifteen lanthanides along with scandium

and yttrium), cerium is the most abundant element at 66.5 ppm in the

Earth’s crust compared to that of copper (60 ppm) or tin (2.3 ppm).1,2

As a

highly abundant material, ceria (or cerium oxide, CeO2) is

technologically important due to its wide-range of applications, for

example in catalysts for the elimination of toxic auto-exhaust gases,3,4

oxygen sensors,5,6

fuel cells,7-9

electrochromic thin-films10,11

and so on.

Upon the transition from a macro-sized particle into a nanocrystalline

particle, ceria significantly changes its physicochemical properties to

usually possess a high density of nanocrystalline ceria interfaces.12

Thus,

nanostructured ceria has attracted extensive attention in such applications

due to improvements in redox properties, transport properties, and high

surface to volume ratios.

162

However, although ceria nanoparticles have been studied for numerous

applications in traditional science and engineering, there have been

almost no studies regarding the potential biomedical applications of ceria

until recently when researchers demonstrated that ceria nanoparticles

possess antioxidant properties at physiological pH values. The ability of

these nanoparticles to act as an antioxidant lies in their ability to

reversibly switch from Ce3+

to Ce4+

.13

In recent studies, this antioxidative

ability enables the application of ceria nanoparticles to protect against

radiation damage, oxidative stress and inflammation.14-16

For example,

Tsai et al. reported that free radical scavenging can be achieved in murine

insulinoma βTC-tet cells by the introduction of crystalline ceria

nanoparticles.17

Moreover, in previous studies, due to safety concerns for biomedical

applications, the toxicity of ZnO, CeO2 (ceria) and TiO2 was compared

based on dissolution and oxidative stress properties.18,19

The results

showed that ceria nanoparticles can suppress the generation of ROS

(reactive oxygen species) production and induce cellular resistance to an

163

exogenous source of oxidative stress to protect cells from oxidant injury.

However, to further minimize the potential toxicity of ceria nanoparticles,

the synthesis of biocompatible dextran-coated ceria nanoparticles and its

enhanced stability in aqueous solution was also reported.15

To build off of these exciting medical applications of ceria nanoparticles,

the present study investigated for the first time the influence of ceria

nanoparticles on bacteria growth. Pseudomonas aeruginosa (P.

aeruginosa) is an aerobic Gram-negative bacterium that is an important

cause of various infections especially in hospitals. Hospitalized patients

may be colonized with P. aeruginosa on admission or during their

hospital stay and P. aeruginosa can be isolated from nearly any

conceivable source within hospitals.20,21

It counts for 11-13.8% of all

nosocomial infections when a microbiological isolate is identifiable.22

Infections caused by P. aeruginosa are not only common,23,24

but are also

associated with high morbidity and mortality when compared with other

bacterial pathogens.25,26

An additional concern involves the increased

antimicrobial resistance of nosocomial P. aeruginosa isolates.27

Even

more troublesome, P. aeruginosa attaches to biotic and abiotic surfaces

164

forming a biofilm. Once formed, the P. aeruginosa infections are much

more difficult to treat due to the formation of exopolysaccharide (EPS).

Biofilms of P. aeruginosa have been found on numerous medical devices

including central venous catheters, contact lenses, mechanical heart

valves, intrauterine devices, and indwelling urinary catheters.28,29

Thus, it

is significant to develop active and biocompatible molecules (that do not

rely on antibiotics that bacteria may develop a resistance towards) that

kill bacteria at early stages of infections before biofilm formation.

Therefore, in this chapter, for the first time, we investigated the

effectiveness of ceria nanoparticles on influencing/inhibiting bacterial

growth (specifically, P. aeruginosa). The results showed that the ceria

nanoparticles significantly inhibited the growth of P. aeruginosa,

revealing a promising application of ceria nanoparticles to prevent

bacterial infections that should be further explored.


6.2.1 Synthesis and Characterization of Ceria Nanoparticles

165

The corresponding dextran or polyacrylic acid coated ceria nanoparticles

were synthesized according to previously described procedures.15,30,31

Briefly, a solution of cerium (III) nitrate (1.0 M, Aldrich, 99%) in water

(5.0 mL) was mixed with an aqueous solution of either polyacrylic acid

(PAA, 0.5 M, Sigma) or dextran (10 kDa,1.0 M, Sigma). The resulting

mixtures were then added to an ammonium hydroxide solution (30.0 mL,

30 %, Sigma Aldrich) under continuous stirring for 24-h at room

temperature. The preparation was then centrifuged at 4000 rpm for two

30-minute cycles to settle down any debris and large agglomerates. The

supernatant solution was then purified from free polymers and other

reagents and then concentrated using a 30K Amicon cell (Millipore Inc.).

The final dextran and polyacrylic acid coated nanoceria preparation were

stored at a 1.5 mg Ce/mL concentration in DI water until further used.

The various polymer-coated nanoceria preparations were characterized by

dynamic light scattering to determine the nanoparticles size and zeta

potential to determine nanoparticle's surface charge (Malvern

DLS/Zetasizer). FT-IR experiments were performed on vacuum dried

166

samples to verify the surface functionalities on nanoparticles (Perkin

Elmer Spectrum 100 FT-IR spectrometer).


A bacteria cell line of Pseudomonas aeruginosa (P. aeruginosa) was

obtained in freeze-dried form from the American Type Culture Collection

(catalog number 27853; Manassas, VA, USA). The bacterial cells were

propagated in 30 mg/mL tryptic soy broth (TSB) (catalog number 22092,

Sigma-Aldrich, St. Louis, MO, USA) for 24 hours in an incubator (37°C,

humidified, 5% carbon dioxide). When the second passage of bacteria

reached its stationary phase, the second passage was frozen in one part

TSB and one part 40% sterile glycerol. Before bacterial seeding, a sterile

10 μL loop was used to withdraw bacteria from the prepared frozen stock

and streaked onto a TSB agar plate, and the TSB agar plate was incubated

for 20 hours to form single bacterial colonies. Then, a single colony was

collected using a sterile loop and was incubated in a 15 mL sterile test

tube containing 3 mL of TSB for 20 hours. The bacterial solution was

diluted to a concentration of 107 bacteria/mL, which was assessed by

measuring the optical density of the bacterial solution using a standard

167

curve correlating optical densities and bacterial concentrations. The

optical densities were measured at 562 nm using a SpectraMax M5 plate

reader (Molecular Devices, Sunnyvale, CA).

Two concentrations of either dextran or polyacrylic acid coated ceria

nanoparticles were tested against bacterial growth. The solutions of ceria

nanoparticles were mixed with bacterial solutions (107 bacteria/mL) at a

1:10 or 1:5 volume ratio and cultured for either 1, 6 or 24 hours in the

incubator. The solutions of sterile TSB were mixed with bacterial

solutions as blank controls. After the treatment, the solutions with

bacteria were diluted with a PBS (catalog number P4417, Sigma-Aldrich,

St. Louis, MO, USA) solution and were spread on agar plates and

bacteria colonies were counted after 18 hours of incubation.

6.2.3 Statistics

Bacterial assays were conducted in triplicate and repeated three times.

Data were collected and differences were assessed with the probability

associated with a one-tailed Student's t-test. Statistical analyses were

performed using Microsoft Excel 2010 (Microsoft Corp., Redmond, WA).

168

6. 3 Results

6.3.1 Characterization of Ceria Nanoparticles

The ceria nanoparticle preparations used in this study were aqueous

suspension of nanoparticles coated with either polyacrylic acid or dextran.

The synthetic procedure used to make these nanoparticles is highly

reproducible, yielding nanoparticles with a cerium oxide core of 3-4 nm

by TEM as shown in Figure 6-1. Overall hydrodynamic diameter of the

polyacrylic acid coated nanoceria was 5 nm while the size for dextran

coated cerium oxide nanoparticles was 14 nm. XPS analysis showed the

presence of Ce+3

and Ce+4

in the nanoceria preparation (Figure 6-1D).

Confirmation of polymer coating was accessed by FT-IR as previously

described15,30,31

and by zeta potential, confirming the presence of a highly

negative charged polymer coating for the polyacrylic acid coated ceria

nanoparticles ( ζ = -45 mV) and a lower negative charge (close to neutral)

for the dextran coated ceria nanoparticles (ζ= -2 mV).

169

Figure 6-1. Characterization of dextran-coated ceria nanoparticles. A)

TEM image with corresponding SAED pattern (inset). B) Representative

HRTEM image of a single crystal. C) XRD image showing the presence

of (111), (220), (311), and (331) planes, typical of an fcc crystal. D) XPS

analysis showing the presence of Ce+3

and Ce+4

in the nanoceria

preparation. E) DLS analysis showing a monodisperse nanoparticle

preparation with an average hydrodynamic diameter of 10 nm. F)

Photograph showing a 1) 10.0, 2) 20.0, 3) 30.0, and 4) 40.0mM aqueous

solutions of dextran-coated nanoparticles.15

170


As shown in Figures 6-2 and 6-3, the ceria nanoparticles did not show

significant inhibition to P. aeruginosa growth compared with TSB

(tryptic soy broth) controls after 1 hour of treatment. However, after 6

hours of treatment, both concentrations of the dextran coated ceria

nanoparticles significantly inhibited the growth of P. aeruginosa by about

20% compared with TSB controls (p < 0.05). The polyacrylic acid coated

ceria nanoparticles showed significant inhibition of P. aeruginosa growth

only for the higher concentration (bacterial solution:ceria nanoparticles =

5:1volume ratio).

171


nanoparticles at three time points (1, 6, and 24 hours). The volume ratio

of bacterial solution (107 bacteria/mL) and ceria nanoparticles is 10:1.

Data is represented as mean ± standard deviation, n=3; *p < 0.01

compared with the TSB (tryptic soy broth) control group after 24 hours of

treatment. **p < 0.05 compared with the TSB (tryptic soy broth) control

group after 6 hours of treatment. Dex = dextran and PAA = polyacrylic

acid.


nanoparticles at three time points (1, 6, and 24 hours). The volume ratio

172

of bacterial solution (107 bacteria/mL) and ceria nanoparticles is 5:1. Data

is represented as mean ± standard deviation, n=3; *p < 0.01 compared

with the TSB (tryptic soy broth) control group after 24 hours of treatment.

**p < 0.05 compared with the TSB (tryptic soy broth) control group after

6 hours of treatment. Dex = dextran and PAA = polyacrylic acid.

Most importantly, the growth of P. aeruginosa was strongly inhibited in

the presence of dextran or polyacrylic acid coated ceria nanoparticles

after 24 hours compared with TSB controls (p < 0.01). When the volume

ratio of bacterial solution to ceria nanoparticles was 10:1, P. aeruginosa

growth was inhibited by 40.12% and 26.88% in the presence of dextran

and polyacrylic acid coated ceria nanoparticles, respectively, compared

with the TSB controls. When the concentration of ceria nanoparticles

doubled, the inhibition of P. aeruginosa growth reached 55.41% and

36.44% for dextran and polyacrylic acid coated ceria nanoparticles,

respectively. Critically, all of this inhibition occurred without the use of

antibiotics, only when using the ceria nanoparticles themselves.

Based on the results of bacterial assays, the inhibition to P. aeruginosa

173

growth by ceria nanoparticles was more significant as treatment time

increased to 24 hours. In addition, the dextran coated ceria nanoparticles

showed stronger inhibition to bacterial growth than that of polyacrylic

acid coated ceria nanoparticles, especially after treatment for 6 and 24

hours. One possible reason for dextran coating and PAA coating could

affect the bacterial growth differently is that the size of dextran coated

ceria nanoparticles is 14 nm and the size of PAA coated ceria

nanoparticles is 5 nm, which results in that they interact with bacterial

cells differently. Moreover, as expected, from 6 hours to 24 hours, the

bacteria propagated quickly in the TSB control, but the bacteria numbers

decreased even more during this time period for the treatment with both

kinds of ceria nanoparticles.

Importantly, the mechanism of how ceria nanoparticles prevent bacterial

growth remains unexplored. Since ceria nanoparticles have been shown to

suppress the generation of ROS (reactive oxygen species)19

, its

antibacterial properties demonstrated here for the first time could be

different from many other nanoparticles (such as zinc oxide nanoparticles,

silver nanoparticles and so on) that are believed to kill bacterial mainly

174

through the generation of ROS.

Although requiring additional investigation, the possible mechanism for

the antibacterial action of the coated CeO2 nanoparticles formulated here

could be that the dextran and PAA coatings interacted with specific

proteins on the bacterial cell membranes to alter the permeability of the

cell membranes, thus, killing the bacterial cells. Moreover, it is possible

that the dextran or PAA coated CeO2 nanoparticles studied here may have

promoted the adsorption, from the TSB, of proteins that are known to

inhibit bacteria growth (such as fibronectin, mucin, vitronectin, etc).32

Future studies should also investigate how universal such bacterial

inhibition is, by exposing such coated CeO2 nanoparticles to other

bacteria (such as Staphylococcus aureus and Escherichia coli).

6.4 Conclusions

In conclusion, the growth of P. aeruginosa was significantly inhibited in

the presence of either dextran or polyacrylic acid coated ceria

nanoparticles after 24 hours. The effectiveness of bacterial growth

inhibition reached 55.41% for dextran coated ceria nanoparticles

175

compared with TSB control. This study revealed for the first time that the

characterized ceria nanoparticles could potentially serve as a novel

antibacterial agent for biomedical applications. Further studies, such as

longer term treatment and elucidating the mechanism of action, are

important to achieve a better understanding and application of ceria in

numerous antibacterial applications.

6.5 References

1. Ahrens TJ. Global Earth Physics: A Handbook of Physical

Constants. Washington, DC: American Geophysical Union; 1995.

2. Lide D. CRC Handbook of Chemistry and Physics. 88th edn ed.

Boca Raton, FL: CRC Publishing Co.; 2007.

3. Trovarelli A. Catalytic properties of ceria and CeO2-containing

materials. Catal Rev. 1996;38(4):439-520.

4. Kaspar J, Fornasiero P, Graziani M. Use of CeO2-based oxides in

the three-way catalysis. Catal Today. Apr 29 1999;50(2):285-298.

5. Beie HJ, Gnorich A. Oxygen Gas Sensors Based on Ceo2 Thick

and Thin-Films. Sensor Actuat B-Chem. Jun 1991;4(3-4):393-399.

6. Jasinski P, Suzuki T, Anderson HU. Nanocrystalline undoped ceria

176

oxygen sensor. Sensor Actuat B-Chem. Oct 15 2003;95(1-3):73-77.

7. Park SD, Vohs JM, Gorte RJ. Direct oxidation of hydrocarbons in a

solid-oxide fuel cell. Nature. Mar 16 2000;404(6775):265-267.

8. Sun CW, Hui R, Roller J. Cathode materials for solid oxide fuel

cells: a review. J Solid State Electr. Jul 2010;14(7):1125-1144.

9. Sun CW, Stimming U. Recent anode advances in solid oxide fuel

cells. J Power Sources. Sep 27 2007;171(2):247-260.

10. Porqueras I, Person C, Corbella C, Vives M, Pinyol A, Bertan E.

Characteristics of e-beam deposited electrochromic CeO2 thin

films. Solid State Ionics. Dec 2003;165(1-4):131-137.

11. Ozer N. Optical properties and electrochromic characterization of

sol-gel deposited ceria films. Sol Energ Mat Sol C. Jun

2001;68(3-4):391-400.

12. Ivanov VK, Usatenko AV, Shcherbakov AB. Antioxidant activity of

nanocrystalline ceria to anthocyanins. Russ J Inorg Chem+. Oct

2009;54(10):1522-1527.

13. Tarnuzzer RW, Colon J, Patil S, Seal S. Vacancy engineered ceria

nanostructures for protection from radiation-induced cellular

damage. Nano Lett. Dec 2005;5(12):2573-2577.

177

14. Das M, Patil S, Bhargava N, et al. Auto-catalytic ceria

nanoparticles offer neuroprotection to adult rat spinal cord neurons.

Biomaterials. Apr 2007;28(10):1918-1925.

15. Perez JM, Asati A, Nath S, Kaittanis C. Synthesis of biocompatible

dextran-coated nanoceria with pH-dependent antioxidant properties.

Small. May 2008;4(5):552-556.

16. Lee SS, Zhu HG, Contreras EQ, Prakash A, Puppala HL, Colvin

VL. High Temperature Decomposition of Cerium Precursors To

Form Ceria Nanocrystal Libraries for Biological Applications.

Chem Mater. Feb 14 2012;24(3):424-432.

17. Tsai YY, Oca-Cossio J, Agering K, et al. Novel synthesis of cerium

oxide nanoparticles for free radical scavenging. Nanomedicine-Uk.

Jun 2007;2(3):325-332.

18. Nel A, Xia T, Madler L, Li N. Toxic potential of materials at the

nanolevel. Science. Feb 3 2006;311(5761):622-627.

19. Xia T, Kovochich M, Liong M, et al. Comparison of the

Mechanism of Toxicity of Zinc Oxide and Cerium Oxide

Nanoparticles Based on Dissolution and Oxidative Stress

Properties. Acs Nano. Oct 2008;2(10):2121-2134.

178

20. Bonten MJM, Bergmans DCJJ, Speijer H, Stobberingh EE.

Characteristics of polyclonal endemicity of Pseudomonas

aeruginosa colonization in intensive care units - Implications for

infection control. American journal of respiratory and critical care

medicine. Oct 1999;160(4):1212-1219.

21. Pirnay JP, De Vos D, Cochez C, et al. Molecular epidemiology of

Pseudomonas aeruginosa colonization in a burn unit: Persistence of

a multidrug-resistant clone and a silver sulfadiazine-resistant clone.

J Clin Microbiol. Mar 2003;41(3):1192-1202.

22. Lizioli A, Privitera G, Alliata E, et al. Prevalence of nosocomial

infections in Italy: result from the Lombardy survey in 2000.

Journal of Hospital Infection. Jun 2003;54(2):141-148.

23. Rello J, Ollendorf DA, Oster G, et al. Epidemiology and outcomes

of ventilator-associated pneumonia in a large US database. Chest.

Dec 2002;122(6):2115-2121.

24. Kollef MH, Shorr A, Tabak YP, Gupta V, Liu LZ, Johannes RS.

Epidemiology and outcomes of health-care-associated pneumonia -

Results from a large US database of culture-positive pneumonia.

Chest. Dec 2005;128(6):3854-3862.

179

25. Osmon S, Ward S, Fraser VJ, Kollef MH. Hospital mortality for

patients with bacteremia due to Staphylococcus aureus or

Pseudomonas aeruginosa. Chest. Feb 2004;125(2):607-616.

26. Harbarth S, Ferriere K, Hugonnet S, Ricou B, Suter P, Pittet D.

Epidemiology and prognostic determinants of bloodstream

infections in surgical intensive care. Arch Surg-Chicago. Dec

2002;137(12):1353-1359.

27. Gaynes R, Edwards JR, Infections NN. Overview of nosocomial

infections caused by gram-negative bacilli. Clinical Infectious

Diseases. Sep 15 2005;41(6):848-854.

28. Donlan RM, Costerton JW. Biofilms: Survival mechanisms of

clinically relevant microorganisms. Clin Microbiol Rev. Apr

2002;15(2):167-+.



1999;284(5418):1318-1322.

30. Asati A, Santra S, Kaittanis C, Nath S, Perez JM. Oxidase-Like

Activity of Polymer-Coated Cerium Oxide Nanoparticles. Angew

Chem Int Edit. 2009;48(13):2308-2312.

180

31. Asati A, Santra S, Kaittanis C, Perez JM.

Surface-Charge-Dependent Cell Localization and Cytotoxicity of

Cerium Oxide Nanoparticles. Acs Nano. Sep 2010;4(9):5321-5331.

32. Kawakubo M, Ito Y, Okimura Y, et al. Natural antibiotic function

of a human gastric mucin against Helicobacter pylori infection.

Science. Aug 13 2004;305(5686):1003-1006.

181

Chapter 7

Investigation of Antibacterial Properties of ZTA and Silicon Nitride

Implants Compared with Titanium

7.1 Introduction

As is well known, bacteria can easily form biofilms when they attach to a

artificial implant surfaces. A bacterial biofilm is an aggregate of one or

more types of bacteria in a hydrated polymeric matrix.1 An implanted

medical device provides a surface for bacteria to attach and multiply in

the patient's body, resulting in the formation of biofilms. Once formed,

the polymeric matrix works as a shield to prevent drugs from penetrating

inside the biofilm. Moreover, the outer cells that break off from the

biofilm can enter and attack the body, causing wide spread infections.

Preventing biofilm formation is critical in the fight against healthcare

associated infections (or HAIs).2

Pseudomonas aeruginosa is one of the bacterium commonly found in

numerous infections. It is an effective and common opportunistic

pathogen of humans, causing serious infections in cystic fibrosis,

182

intensive care, burn and immunocompromised patients.3,4

Pseudomonas

aeruginosa have been implicated during chronic infection of cystic

fibrosis patients.5,6

In addition to chronic infection, P. aeruginosa biofilms

contribute to morbidity of patients with medical implants including

catheters7, prosthetics

8 and stainless steel implants

9. Therefore, it is

significant to develop a material to prevent bacterial growth on the

surface of implant medical devices.

Titanium is the most established implant material in recent years. About

one million patients worldwide are treated every year for replacement of

arthritic or damaged hips and knees.10,11

Although it is considered as one

of the few materials that naturally meet every requirement for

implantation in the human body, bacterial infections is a severe problem

for titanium being used as implant materials.

As novel ceramics materials for biomedical applications, both ZTA

(zirconia toughened alumina) and Si3N4 (silicon nitride) have been

recently investigated as potential implant materials.12-19

ZTA is a ceramic

material comprising alumina and zirconia. ZTA components are

183

comprised of an alumina-rich composition (about 20% zirconia and 80%

alumina) where zirconia is evenly dispersed in the alumina matrix. These

ceramics exhibit superior strength and toughness compared to

conventional alumina and zirconia.20

These ceramic properties have made

ZTA a potential advancement of current clinically available orthopedic

biomaterials. Studies have showed the promise of ZTA being used as an

orthopedic implant material.21-23

Silicon nitride is a chemical compound of the elements silicon and

nitrogen and has been used in many industrial applications. Due to its

hydrophilic and microtextured surface, silicon nitride has better materials

strength, durability and reliability than conventional implant materials,

such as PEEK and titanium.24,25

Polished and porous implants made of

silicon nitride have shown encouraging outcomes in spine and

maxillofacial surgery.26,27

However, the antibacterial properties of either

ZTA or silicon nitride have not been widely studied, and the resistance to

bacterial infection is an important and desirable property for implant

materials.

184

Thus, the objective of this chapter is to investigate the antibacterial

properties of the novel implant materials, silicon nitride and ZTA,

compared with titanium. The results of this study showed that ZTA

significantly inhibited the growth of Pseudomonas aeruginosa and

Staphylococcus aureus compared with the widely used implant material,

titanium, which suggested that ZTA could be a potential implant material

with antibacterial functions and could be used as an alternative to

titanium. Moreover, silicon nitride also successfully inhibited the growth

of Staphylococcus aureus biofilm compared with titanium.



The ceramics used in this study included medical grades of silicon nitride

and ZTA, and both are provided by Amedica Corporation, Salt Lake City,

UT. The silicon nitride an “as-fired” surface that has nanostructured

features. Titanium samples used in this study are ASTM grade 4 titanium

from Fisher Scientific, Continental Steel & Tube Co. Fort Lauderdale, FL.

The morphology of all the surfaces was characterized using a SEM

(Scanning Electron Microscope, HITACHI 2700). Before scanning the

185

surface of polycarbonate under SEM, the films were coated with a 2 nm

gold layer using a Sputter Coater (EMITECH K550, Emitech Ltd.) to

make them conductive. An AFM (Atomic Force Microscope, MFP3D,

Asylum Research, sharp tipped cantilever, K = 0.06N/M, Tapping Mode)

was used to measure the surface roughness of the materials.


For sterilization purpose, all samples were exposed under ultraviolet light

for 24 hours on all sides. In the bacteria experiments, bacteria cell lines of

Pseudomonas aeruginosa and Staphylococcus aureus were obtained in

freeze-dried form from the American Type Culture Collection (catalog

number 27853). The cells were propagated in 30 mg/ml of Tryptic soy

broth (TSB). A bacteria solution was prepared at a concentration of 106

bacteria/ml, which was assessed by measuring the optical density of the

bacterial solution at 562 nm using a standard curve correlating optical

densities and bacterial concentrations. The ZTA, silicon nitride and

titanium samples were treated with bacterial solutions in a 24-well plate

and cultured for either 24, 48 or 72 hours in an incubator (37°, humidified,

5% CO2). The media was changed with fresh and sterile 0.03% TSB

186

every 24 hours for those samples incubated with 48 hours or 72 hours.

Then, the samples were put in 15ml microfuge tubes with 3ml of PBS.

These tubes were sonicated for 10 minutes to release the bacteria attached

on the surface into the solution. Solutions with bacteria were spread on

agar plates and bacteria colonies were counted after 18 hours of

incubation.

Besides the colony counting method mentioned above, the crystal violet

assays were also used to test the growth of bacteria. After the ZTA,

silicon nitride and titanium samples were treated with bacteria solution

for 24, 48 or 72 hours, for each sample, 1 mL bacterial solution with both

planktonic and attached bacteria will be stained with 0.5 mL 0.1% crystal

violet solution for 15 minutes in a 1.5 mL centrifuge tube. Then the

centrifuge tubes will be centrifuged with 10000 g for 5 minutes. The

supernatant solution was removed leaving only the bacteria pellet in the

tube. The pellet was washed with PBS (phosphate buffered saline) once

followed with a centrifuge for 5 minutes under 10000 g. After removing

the supernatant solution again, 1 mL 100% ethanol was added into each

tube to dissolve the crystal violet stain in the bacteria. Then the

187

absorbency of solutions in each tube was measured at 562nm using a

SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA).

Bacterial tests were conducted in triplicate and repeated three times. Data

were collected and the significant differences were assessed with the

probability associated with a one-tailed Student's t-test. Statistical

analyses were performed using Microsoft Excel (Redmond, WA).

7. 3 Results


SEM images of the surfaces of titanium and as-fired silicon nitride were

illustrated in Figure 7-1. The SEM image of titanium showed a typical

micron-rough surface of machined materials. The as-fired silicon nitride

had nanostructured surface features with larger total surface area

compared with titanium. The as-fired silicon nitride surface morphology

reflected the natural condition of this material subsequent to densification

by sintering and hot-isostatic pressing.28

The surface was composed of

randomly oriented acicular protruding grains typically less than 1μm in

cross-section, yielding a unique nanotexture. Individual hexagonal grains

188

of silicon nitride have definitive linear facets (in cross-section) and sharp

corners at the termination of the acicular grains, which are typically less

than 100 nm. These unique nano-features provided a desirable large

surface area, which might play a role in interaction with bacteria.

Figure 7-1. SEM images of (a) titanium and (b) as-fired silicon nitride at

a magnification of 10k.

The AFM images of titanium and ZTA are shown in the Figure 7-2 and

the surface roughness measurements were derived from the AFM images.

The titanium surface had randomly microsized features with a RMS (root

mean square, scan area = 10 m×10 m) roughness of 393.9 nm. The

ZTA surface showed more uniform nanoscale patterns with a much higher

RMS roughness, which is 490.8 nm. These results showed that Ti and

189

ZTA have markedly different surface topographies that might affect

bacterial adhesion.


10um×10um) roughness of (a) titanium and (b) ZTA. RMS roughness of

titatnium = 393.9 nm. RMS roughness of ZTA = 490.8 nm.


As shown in Figure 7-3, the ZTA samples a showed significant inhibition

to the growth of Pseudomonas aeruginosa from 24 hours to 72 hours

compared with titanium samples. There are about 50% less bacteria

adhesion on the surface of ZTA than titanium after 24 hours. After 48

hours, the ZTA samples inhibited the growth of Pseudomonas aeruginosa

by 46% compared with the titanium samples. After 72 hours, the ZTA

190

samples still showed a significantly decreased bacterial growth by 45%

compared with titanium samples. However, there was no significant

difference in the growth of Pseudomonas aeruginosa on the surface of

silicon nitride and titanium, although there are relatively less bacteria

attached on silicon nitride than titanium. Thus, the results demonstrated

that there is a continuous and significant prevention to Pseudomonas

aeruginosa growth on the surface of ZTA from 24 hours to 72 hours.

Figure 7-3. The growth of Pseudomonas aeruginosa on silicon nitride,

ZTA and titanium, measured by the method of colony forming units.

Data=Mean ± standard deviation, n=3; *p<0.05 compared with titanium

samples after 24 hours; **p<0.05 compared with titanium samples after

48 hours; ***p<0.05 compared with titanium samples after 72 hours. SN

= silicon nitride.

191

In another bacterial inhibitory test, the growth of Staphylococcus aureus

biofilm after either 24, 48 or 72 hours on silicon nitride, ZTA and

titanium was also measured using crystal violet assays. As shown in

Figure 7-4, the titanium samples had more bacteria attached compared

with silicon nitride or ZTA samples. Particularly, there was some decrease

in bacteria numbers for silicon nitride compared with titanium after 24

hours, but the differences between these two were not significant.

However, the silicon nitride surface showed significantly less bacterial

growth than titanium surface after 48 and 72 hours, which means silicon

nitride inhibited the growth of Staphylococcus aureus biofilm compared

with titanium. Moreover, less crystal violet absorbance was observed for

ZTA compared with titanium after incubation of 24, 48 and 72 hours.

From 24 hours to 72 hours (Figure 7-4), ZTA surface showed a

continuously significant inhibition to the growth of Staphylococcus

aureus biofilm compared with titanium surface. Overall, less bacterial

activity of Staphylococcus aureus was manifested on both silicon nitride

and ZTA than on titanium, probably because of the surface chemistry and

nanostructure differences between these materials. The results of the

192

bacterial tests showed that ZTA effectively inhibited the growth of both

gram-positive (Staphylococcus aureus) and gram-negative (Pseudomonas

aeruginosa) bacteria after 1, 2 and 3 days compared with titanium, which

demonstrated that ZTA might be considered as a potential alternative of

titanium for implant material.

Figure 7-4. The growth of Staphylococcus aureus on silicon nitride, ZTA

and titanium, measured by the method of crystal violet staining.

Data=Mean ± standard deviation, n=3; *p<0.05 compared with titanium

samples after 24 hours; **p<0.05 compared with titanium samples after

48 hours; ***p<0.05 compared with titanium samples after 72 hours. SN

= silicon nitride.

193

7.4 Conclusions

In this study, it was showed that ZTA and silicon nitride as novel ceramic

materials strongly inhibited the growth of gram-positive bacteria

(Staphylococcus aureus) on the surface compared with titanium. ZTA

surface also showed a significant inhibition to the growth of

gram-negative bacteria (Pseudomonas aeruginosa) compared with

titanium after 24, 48 and 72 hours. Because the substantially lower loads

of Staphylococcus aureus and Pseudomonas aeruginosa were measured

on ZTA surfaces than titanium, for up to 72 hours of incubation, using

ZTA as an implant material might be an effective way to prevent

infections on implant medical devices. Therefore, this study suggested

that ZTA with advantages of preventing infections might serve as a

promising alternative of titanium for biomedical applications.

7.5 References



1999;284(5418):1318-1322.

2. Maurette P, Sfa CAMR. To err is human: building a safer health

194

system. Ann Fr Anesth. Jun 2002;21(6):453-454.

3. Bodey GP, Bolivar R, Fainstein V, Jadeja L. Infections Caused by

Pseudomonas-Aeruginosa. Rev Infect Dis. 1983;5(2):279-313.

4. Costerton JW. Cystic fibrosis pathogenesis and the role of biofilms

in persistent infection. Trends Microbiol. Feb 2001;9(2):50-52.

5. O'Toole G, Kaplan HB, Kolter R. Biofilm formation as microbial

development. Annu Rev Microbiol. 2000;54:49-79.

6. Singh PK, Schaefer AL, Parsek MR, Moninger TO, Welsh MJ,

Greenberg EP. Quorum-sensing signals indicate that cystic fibrosis

lungs are infected with bacterial biofilms. Nature. Oct 12

2000;407(6805):762-764.

7. Khaled GH, Finkelstein FO, Carey HB, Wardlaw SC, Kliger AS,

Edberg SC. Method for studying development of colonization and

infection of dialysis catheters. Advances in peritoneal dialysis.

Conference on Peritoneal Dialysis. 2001;17:163-171.

8. McNeil SA, Nordstrom-Lerner L, Malani PN, Zervos M, Kauffman

CA. Outbreak of sternal surgical site infections due to

Pseudomonas aeruginosa traced to a scrub nurse with

onychomycosis. Clin Infect Dis. Aug 1 2001;33(3):317-323.

195

9. Traverso C, De Feo F, Messas-Kapal A, et al. Long term effect on

IOP of a stainless steel glaucoma drainage implant (Ex-PRESS) in

combined surgery with phacoemulsification (vol 89, pg 423, 2005).

Brit J Ophthalmol. May 2005;89(5):645-645.

10. Pohler OEM. Unalloyed titanium for implants in bone surgery.

Injury. Dec 2000;31:S7-S13.

11. Harlan N. Titanium bone implants. Mater Technol. Sep

2000;15(3):185-187.

12. Zhou YS, Ohashi M, Tomita N, Ikeuchi K, Takashima K. Study on

the possibility of silicon nitride silicon nitride as a material for hip

prostheses. Mat Sci Eng C-Biomim. Dec 1997;5(2):125-129.

13. Clarke IC, Manaka M, Green DD, et al. Current status of zirconia

used in total hip implants. J Bone Joint Surg Am. 2003;85A:73-84.

14. Bal BS, Rahaman MN. Orthopedic applications of silicon nitride

ceramics. Acta Biomater. Aug 2012;8(8):2889-2898.

15. Silva CCGE, Konig B, Carbonari MJ, Yoshimoto M, Allegrini S,

Bressiani JC. Bone growth around silicon nitride implants - An

evaluation by scanning electron microscopy. Mater Charact. Sep

2008;59(9):1339-1341.

196

16. Masonis JL, Bourne RB, Ries MD, McCalden RW, Salehi A,

Kelman DC. Zirconia femoral head fractures - A clinical and

retrieval analysis. J Arthroplasty. Oct 2004;19(7):898-905.

17. Chevalier J. What future for zirconia as a biomaterial?

Biomaterials. Feb 2006;27(4):535-543.

18. Neumann A, Reske T, Held M, Jahnke K, Ragoss C, Maier HR.

Comparative investigation of the biocompatibility of various

silicon nitride ceramic qualities in vitro. J Mater Sci-Mater M. Oct

2004;15(10):1135-1140.

19. Amaral M, Costa MA, Lopes MA, Silva RF, Santos JD, Fernandes

MH. Si3N4-bioglass composites stimulate the proliferation of

MG63 osteoblast-like cells and support the osteogenic

differentiation of human bone marrow cells. Biomaterials. Dec

2002;23(24):4897-4906.

20. Huet R, Sakona A, Kurtz SM. Strength and reliability of alumina

ceramic femoral heads: Review of design, testing, and retrieval

analysis. J Mech Behav Biomed. Apr 2011;4(3):476-483.

21. Lombardi AV, Berend KR, Seng BE, Clarke IC, Adams JB. Delta

Ceramic-on-Alumina Ceramic Articulation in Primary THA

197

Prospective, Randomized FDA-IDE Study and Retrieval Analysis.

Clin Orthop Relat R. Feb 2010;468(2):367-374.

22. Hamilton WG, McAuley JP, Dennis DA, Murphy JA, Blumenfeld

TJ, Politi J. THA With Delta Ceramic on Ceramic Results of a

Multicenter Investigational Device Exemption Trial (vol 10, doi #

10.1007/s11999-009-1091-4, 2010). Clin Orthop Relat R. Mar

2010;468(3):909-909.

23. Callaghan JJ, Liu SS. Ceramic on crosslinked polyethylene in total

hip replacement: any better than metal on crosslinked polyethylene?

The Iowa orthopaedic journal. 2009;29:1-4.

24. Mazzocchi M, Bellosi A. On the possibility of silicon nitride as a

ceramic for structural orthopaedic implants. Part I: processing,

microstructure, mechanical properties, cytotoxicity. J Mater

Sci-Mater M. Aug 2008;19(8):2881-2887.

25. Arafat A, Schroen K, de Smet LCPM, Sudholter EJR, Zuilhof H.

Tailor-made functionalization of silicon nitride surfaces. J Am

Chem Soc. Jul 21 2004;126(28):8600-8601.

26. Neumann A, Unkel C, Werry C, et al. Prototype of a silicon nitride

ceramic-based miniplate osteofixation system for the midface.

198

Otolaryngology--head and neck surgery : official journal of

American Academy of Otolaryngology-Head and Neck Surgery.

Jun 2006;134(6):923-930.

27. Bal BS, Rahaman MN. Orthopedic applications of silicon nitride

ceramics. Acta Biomater. Aug 2012;8(8):2889-2898.

28. Kim SS, Baik SG. Hot Isostatic Pressing of Sintered Silicon-Nitride.

J Am Ceram Soc. Jul 1991;74(7):1735-1738.

199

Chapter 8

Conclusions and Future Outlook

The studies performed in this dissertation presented an approach based on

nanosized selenium to inhibit bacterial infections on several types of

materials, which demonstrated the potential applications of nanosized

selenium as an antibacterial agent in surface coatings.

Specifically, nanosized selenium was successfully synthesized and coated

on various substrates including polymeric medical devices, paper towels,

and metallic materials through a simple fast precipitation method. The

properties of nanosized selenium coatings, such as size, distribution,

coverage, composition, and change of surface geography, were fully

characterized using techniques such as a standard tape test, scanning

electron microscopy, energy-dispersive X-ray spectroscopy, atomic force

microscopy, contact angle measurement, and so on. The concentrations of

the selenium coatings were measured using atomic absorption

spectroscopy and inductively coupled plasma mass spectrometry. Most

importantly, the nanosized selenium coatings significantly decreased the

200

growth of various gram-positive and gram-negative bacteria, thus

inhibiting bacterial biofilm formation on these substrates. These

antibacterial effects with inhibitory percentages of up to 91% were

achieved without using any antibiotics. This dissertation suggests that

nanosized selenium maybe used as an effective antibacterial agent for

helping medical devices and healthcare facilities fighting against

infections.

Several aspects in this work need to be further investigated in the future

to achieve a better understanding of the antibacterial properties of

nanosized selenium and to expand a wider range of applications of

nanosized selenium as an antibacterial agent.

Particularly, for different materials, the reaction to synthesize and coat

nanosized selenium needs to be modified. Several parameters, such as the

concentrations of each reactant, temperature for the reaction, speed of

agitation during the reaction, and time for the coating process, should be

optimized to prepare selenium coatings that are more uniform, having

stronger adhesion and more desired concentration. Another way to think

201

about conducting the reaction in practice is doing a spray coating on the

surface of the targeted material. A few primary results were included in

the Appendix of this dissertation. With a precise control and balanced

spray, this could be a more efficient way, compared with the existing

aqueous phase reaction, to coat the surfaces of some materials with

nanosized selenium and introduce antibacterial properties to these

materials. The spray coating method could also potentially use the two

reactants more effectively, which can save the costs of synthesizing

selenium nanoparticles and is a very important aspect for commercial

applications.

Moreover, this nanosized selenium based antibacterial approach should

be applied to other materials that are also widely used for various medical

devices and healthcare products, such as polyvinyl chloride, co-polyesters,

titanium, silicone, stainless steel and so on. The surface properties, such

as roughness, hydrophilicity, chemical composition, and surface charges,

could be remarkably different between each kind of these materials, thus,

the interaction between bacteria and the nanosized selenium coated

material surface might be dramatically varied. These factors may cause

202

difficulties to use the nanosized selenium based antibacterial technology

on various materials

In addition, the anti-infective properties should be further tested on other

bacterial species and even other microorganisms. Because compared with

Staphyloccocus aureus, Staphylococcus epidermidis, Pseudomonas

aeruginosa and Escherichia coli used in this project, other bacterial

species or other microorganisms such as fungus and virus, may have

significantly different sizes, surface proteins, cell membranes and other

properties, which may reveal different interactions with nanosized

selenium. Further studies, such as gene expression tests, mammalian cell

assays, selenium uptake measurements, should be implemented to

achieve a better understanding of the antibacterial effects of nanosized

selenium and access its potential toxicity to human and environment.

Lastly, but not the least, in vivo studies in animals such as mice, should be

implemented to further investigate the antibacterial properties and safety

concentrations of nanosized selenium for biomedical applications.

Specifically, the released amount and distribution of selenium

203

nanoparticles in the animals should be measured. The lifetime of released

selenium nanoparticles or how long it takes for the clearance of selenium

nanoparticles in body of the animals should be accessed. It is also of vital

importance to determine the effectiveness of the nanosized selenium at

inhibiting various infections in the in vivo situation.

Overall, the work concerning nanosized selenium described in this

dissertation established a novel platform technology to prevent bacteria

and biofilm infections. This technology may be a significant contribution

to the field of nanomedicine, especially improving the strategies of

fighting against infections in various hospital environments and

healthcare settings.

204

Appendix

A1. Selenium Coatings on Surfaces of Various Materials

In addition to the materials that were coated with nanosized selenium and

studied on their antibacterial properties, various other material substrates

were successfully coated with selenium nanoparticles. The SEM images

of these substrates were included below to demonstrate the nanosized

selenium coatings on these materials. Besides polycarbonate and PEEK

mentioned the previous chapters, selenium nanoparticles were also coated

on other polymeric materials that were used for medial devices, such as

isoplast and PVC, as shown in Figure A1-1 and Figure A1-2. In order to

introduce antibacterial properties to the “high-touch” (frequently touched)

surfaces around us, nanosized selenium was coated on the surfaces of

computer keyboard and tablet, which were both real commercial products

that people use almost every day. Figure A1-3 and Figure A1-4 showed

that the surfaces of computer keyboard and tablet were fully covered with

selenium nanoparticles compared with the uncoated ones. In Figure A1-5,

glass, as other frequently used material, was also coated with selenium

nanoparticles. The glass surface was fully and evenly covered by

205

selenium nanoparticles.

Figure A1-1. SEM images of selenium nanoparticle coated isoplast

before (a) and after (b) the tape test as mentioned in Chapter 2.

Figure A1-2. SEM images of selenium nanoparticle coated PVC (a) and

uncoated PVC (b).

206


of a commercially available keyboard (a) and the uncoated surface of the

keyboard (b).


of the back side of a “Windows Surface” tablet (a) and the uncoated

surface of the table (b).

207

Figure A1-5. SEM images of selenium nanoparticle coated glass (a) and

uncoated glass (b).

Moreover, nanosized selenium was also coated on stainless steel as

shown in Figure A1-6(a), because stainless steel is a metallic material that

widely used and often touched in healthcare environment, such as being

used for medical supply carts and bed rails in the patient’s room. Besides

the normal aqueous phase reaction to synthesize and coat nanosized

selenium on the surface of materials, another spray coating method was

used to coat selenium nanoparticles on stainless steel. In this method, the

two reactants, sodium selenite and glutathione, were put in two spray

bottles separately and then the reactants were sprayed one by one to the

surface of stainless steel. The result of spray coating on stainless steel

was shown in Figure A1-6(b). The formation of nanosized selenium was

208

observed after the coating process. However, this coating method was not

working quite successful for polyurethane as shown in Figure A1-7. No

much particle-like feature was observed after spray coating. The reason

might be the hydrophobicity of polyurethane that prevented the reactants

from evenly distributed on the surface. Further studies are important to

modify this coating process so that it can work for more types of

materials. In many circumstances, the spray coating method could be an

easier approach to coat nanosized selenium and introduce antibacterial

property to material surfaces than the conventional aqueous phase

reaction.

209

Figure A1-6. SEM images of stainless steel that were (a) coated with

nanosized selenium by the aqueous phase reaction; (b) coated with

nanosized selenium by spray coating; (c) without selenium coatings.

210

Figure A1-7. SEM images of (a) uncoated polyurethane and (b) selenium

coated polyurethane by spray coating.

In order to test the adhesion of selenium nanoparticle coatings on paper

towels, the selenium coated paper towels were sonicated for 5 minutes in

DI water. Then, SEM images (Figure A1-8) of the sonicated paper towels

were taken under two magnifications. It was observed from the SEM

images that there were still a lot of selenium nanoparticles attached on the

surface of paper towels after the sonication, which means that the most of

the nanoszied selenium coatings were strongly attached to the paper

towels and hard to be removed by washing process, like sonication.

211

Figure A1-8. SEM images of selenium nanoparticle coated paper towels

after 5 minutes of sonication treatments. (a) magnification = 3k; (b)

magnification = 20k.

A2. Surface Treatment of Flat and Porous Titanium Implants

Commercially available bone implant materials, flat and porous titanium

from Stryker Corporation, Kalamazoo, Michigan, were treated with nitric

acid to modify the surface features. Both flat and porous titanium were

treated with 1 M, 5 M or10 M nitric acid separately. In each concentration

of nitric acid, both flat and porous titanium were treated for 5, 30 or 60

minutes. SEM images of titanium samples were taken before and after

acid treatments using a Scanning Electron Microscope, HITACHI 2700.

The SEM images of the flat and porous titanium without acid treatment

212

are in Figure A2-1. The SEM images of flat titanium after being treated

with acid are in Figure A2-2. The SEM images of porous titanium after

being treated with acid are in Figure A2-3.

Figure A2-1. SEM images of (a) untreated flat titanium at a

magnification of 50; (b) untreated flat titanium at a magnification of 10k;

(c) untreated porous titanium at a magnification of 50; (d) untreated

porous titanium at a magnification of 10k.

213

Figure A2-2. SEM images of flat titanium after being treated with acid.

(a) 1M nitric acid, treat for 5 minutes; (b) 1M nitric acid, treat for 30

minutes; (c) 1M nitric acid, treat for 60 minutes; (d) 5M nitric acid, treat

for 5 minutes; (e) 5M nitric acid, treat for 30 minutes; (f) 1M nitric acid,

treat for 60 minutes; (g) 10M nitric acid, treat for 5 minutes; (h) 10M

nitric acid, treat for 30 minutes; (i) 10M nitric acid, treat for 60 minutes.

214

Figure A2-3. SEM images of porous titanium after being treated with

acid. (a) 1M nitric acid, treat for 5 minutes; (b) 1M nitric acid, treat for 30

minutes; (c) 1M nitric acid, treat for 60 minutes; (d) 5M nitric acid, treat

for 5 minutes; (e) 5M nitric acid, treat for 30 minutes; (f) 1M nitric acid,

treat for 60 minutes; (g) 10M nitric acid, treat for 5 minutes; (h) 10M

nitric acid, treat for 30 minutes; (i) 10M nitric acid, treat for 60 minutes.

Documents

Nanosized selenium: a novel platform technology to prevent ... · As an important category of bacterial infections, healthcare-associated infections (HAIs) are considered as an increasing