Conferring materials with antibacterial properties 1

2.2.2 Incorporation of antibacterial agents 2.2.3 Functionalizing surfaces with antibacterial agents 1416172.3 Surface Functionalization 2.3.1 Surface graft copolymerization 2.3.2 Self-a

CONFERRING MATERIALS WITH ANTIBACTERIAL

PROPERTIES

SHI ZHILONG

(B.E., M.S BUCT)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL & BIOMOLECULAR

ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE

First of all, I would like to express my cordial gratitude to my supervisors, Prof Neoh Koon Gee and Prof Kang En-Tang, for their guidance, advice, support and encouragement throughout the long period of this research work I have learnt invaluable knowledge from them on how to do research work and how to enjoy in doing research Their enthusiasm, sincerity and dedication to scientific research have greatly impressed me and will benefit me in my future career

I would like to thank all my colleagues in the laboratory and the technicians of the Department of Chemical and Biomolecular Engineering for their kind help and assistance In particular, thanks are due to Dr Cen Lian, Dr Li Yali, Mr Hu Feixiong for their helpful advice It is my great pleasure to work with all of them The financial support provided by the National University of Singapore is greatly appreciated

Finally, I would like to express my deepest gratitude and indebtedness to my parents for their constant concern and support Special thanks to my wife Zhang Nan for her love, patience and encouragement

2.2.2 Incorporation of antibacterial agents

2.2.3 Functionalizing surfaces with antibacterial agents

1416172.3 Surface Functionalization

2.3.1 Surface graft copolymerization

2.3.2 Self-assembly polyelectrolyte multilayers

232326

Chapter 3 SURFACE FUNCTIONALIZATION OF POLYMERIC

SUBSTRATE TO ACHIEVE ANTIBACTERIAL PROPERTIES

28

3.1 Antibacterial Activity of Polymeric Substrate with Surface

Grafted Viologen Moieties

3.2 Surface Grafted Viologen for Precipitation of Silver Nanoparticles

and Their Combined Bactericidal Activities

51

Chapter 4 ANTIBACTERIAL AND ADSORPTION

CHARACTERISTICS OF ACTIVATED CARBON

FUNCTIONALIZED WITH QUATERNARY AMMONIUM

Chapter 5 IN VITRO ANTIBACTERIAL AND CYTOTOXICITY

ASSAY OF MULTILAYERED POLYELECTROLYTE

FUNCTIONALIZED STAINLESS STEEL

Chapter 6 NOVEL STRATEGIES FOR CONFERRING

ANTIBACTERIAL PROPERTIES TO BONE CEMENT

115

6.1 Antibacterial and Mechanical Properties of Bone Cement

Impregnated with Chitosan Nanoparticles

6.2 Antibacterial and Mechanical Properties of Bone Cement

Containing a Monomer or Polymer with Norfloxacin Moieties 142

Chapter 8 RECOMMENDATIONS FOR FUTURE STUDY 165 REFERENCES 169 LIST OF PUBLICATIONS 190

Biofilms are complex communities of surface attached microorganisms, comprising either single or multiple species Biofilms are found ubiquitously on virtually all natural, medical, and industrial settings where bacteria exist, and they can cause device-related infections, bacterial drug resistance and microbial-induced corrosion In this thesis, different approaches of surface and bulk functionalization to confer materials with antibacterial properties to combat biofilm were developed depending on the materials of interest At the same time, other important properties of the materials such as mechanical property and cytotoxicity were investigated after the functionalization process

Surface modification techniques were developed for the functionalization of poly(ethylene terephthalate) (PET) film with viologen moieties, N, N'-disubstituted-4, 4'-bipyridinium, using graft copolymerization The antibacterial property of the

viologen graft copolymerized PET film was assayed against Escherichia coli (E coli, a

Gram-negative bacterium) Further, silver nanoparticles can be deposited on the modified film through the photoinduced reduction capability of the viologen moieties

in silver salt solution The size and distribution of the silver nanoparticles with varying reaction time were investigated The combined antibacterial effect of viologen and silver nanoparticles was assessed The stability of the modified film was carried out after aging in a weathering chamber

Two types of quaternary ammonium group were also successfully covalently coupled

on the activated carbon (AC) using wet chemistry method Both types of

functionalized ACs show highly effective antibacterial activities against E coli and

Staphylococcus aureus (S aureus, a Gram-positive bacterium) Furthermore, the

functionalized ACs can be used in repeated antibacterial applications with little loss in efficacy Using phenol as a model compound, the adsorption capacity of the different ACs was also investigated

A simple and versatile method was then developed to confer stainless steel (SS) with antibacterial property via the alternate deposition of quaternized polyethylenimine (PEI) or quaternized PEI-silver complex and poly(acrylic acid) (PAA) The

antibacterial activity was assessed using E coli and S aureus The inhibition of

bacterial growth on the surface of functionalized SS was investigated using fluorescence microscopy after staining with a combination dye The cytotoxicity of the functionalized SS towards mammalian cells was evaluated by the MTT assay

Two new strategies were developed to confer antibacterial properties to bone cement using bulk modification The first strategy employs chitosan nanoparticles which may

be further derivatized with quaternary ammonium groups, while the second uses norfloxacin moieties, as one of the components in the commercial poly(methyl

methacrylate) bone cement Antibacterial assay using S aureus and Staphylococcus

epidermidis (S epidermidis) was carried out The results showed promising

bactericidal activities The studies also addressed the issue of mechanical property and cytotoxicity of the bone cement after functionalization

EDX Energy dispersive X-ray spectroscopy

E coli Escherichia coli

FE-SEM Field emission scanning electron microscopy

FTIR Fourier transform infrared

GMA Glycidyl methacrylate

HVV N-hexyl-N′-(4-vinylbenzyl)-4,4′-bipyridinium bromide chloride ICP Induced coupled plasma

MTT 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide NOR Norfloxacin

PAA Poly(acrylic acid)

PEI Polyethylenimine

PET Poly(ethylene terephthalate)

PMMA Poly(methyl methacrylate)

PVP Poly(4-vinyl pyridine)

PBS Phosphate buffer solution

QAS 3-(trimethoxysilyl)- propyldimethyloctadecylammonium chloride

S aureus Staphylococcus aureus

S epidermidis Staphylococcus epidermidis

SEM Scanning electron microscopy

LIST OF FIGURES

Figure 3.1 Scheme of synthesis of HVV

Figure 3.2 Schematic illustration of the surface functionalization of PET films

with HVV

Figure 3.3 FTIR spectrum of the as-synthesized HVV powder

Figure 3.4 XPS: (a) N 1s, (b) Cl 2p, and (c) Br 3d core-level spectra of as-

synthesized HVV powder

Figure 3.5 XPS wide scan, N 1s and C 1s core-level spectra (a, b, c) of pristine

PET film, wide scan, N 1s and C 1s core-level spectra (d, e, f) of PET film after HVV graft-copolymerization using 40 wt% HVV monomer concentration

Figure 3.6 UV-visible absorption spectra of PET film graft copolymerized with

HVV (using 40 wt% HVV monomer concentration): base spectrum, after 10 min irradiation (0 min spectrum) and bleaching in air (10 min spectrum)

Figure 3.7 Optical micrographs of (a) pristine PET and (b) HVV graft-

copolymerized PET surface after exposure to waterborne E coli and

subsequent incubation in solid growth agar for 24h，respectively

Figure 3.8 Scanning electron micrographs of (a) pristine PET film and (b) PET

film after HVV graft-copolymerization after exposure to waterborne

E coli and subsequently incubated with solid growth agar for 24h,

respectively

Figure 3.9 Effect of HVV monomer concentration on (a) surface concentration of

N+ , and (b) antibacterial activity of the HVV graft-copolymerized PET

For the antibacterial test, the film was in contact with the E coli in PBS

solution for 18h

Figure 3.10 Survival ratio of E coli cells in PBS at 37 oC as a function of time in

contact with the different substrates: (a) pristine PET, (b) HVV graft copolymerized PET (40 wt% HVV monomer used in graft copolymerization) and (c) HVV graft copolymerization PET after 10min UV irradiation and then left to bleach in air The cell number was determined by spread plate method

various time periods

Figure 3.13 XPS Ag 3d core-level spectrum of the HVVN-PET film after reaction

with a 200 mg/l AgNO3 solution for 15 min

Figure 3.14 UV-visible absorption spectra of the HVV-PET films before and after

reaction with a 200 mg/l AgNO3 solution under UV irradiation for different periods of time

Figure 3.15 Scanning electron micrographs of the HVV-PET films before and after

reaction with a 200 mg/l AgNO3 solution for (a) 15 min, and (b) 30 min under UV irradiation

Figure 3.16 Viable E coli cell number as a function of time in contact with the

different substrates: Pristine PET (█), VBV-PET (◆), HVVN-PET (●), HVVN-PET after reaction in AgNO3 for 30 min (▼) and HVVN-PET after reaction in AgNO3 for 15 min (▲) The cell number was determined by the surface-spread method

Figure 3.17 Comparison of antibacterial effect of the silver coated HVVN-PET film

before and after aging in a weathering chamber for 48 h The silver coated HVV-PET film was obtained after 15 min photoinduced reaction in a 200 mg/l AgNO3 solution

Figure 4.1 Schematic representation of the two routes for the functionalization of

AC with quaternary ammonium groups

Figure 4.2 FTIR spectra of (a) AC and (b) AC-COOH

Figure 4.3 XPS wide scan of AC (a), Q-AC (b) and P-AC (c); N 1s core-level

spectra of AC (d), Q-AC (e) and P-AC (f)

Figure 4.4 FE-SEM micrographs of AC (a), Q-AC (b) and P-AC (c)

Figure 4.5 Viable E coli cell number (a) and viable S aureus cell number (b) as a

function of time in contact with the different substrates: Control (■),

AC (●), Q-AC (Œ), and P-AC (▲) The cell number was determined by the surface spread method

Figure 4.6 Repeated antibacterial assay using 100 mg of P-AC in contact with 30

ml E coli suspension (107 cells/ml)

Figure 4.7 Adsorption isotherms of phenol on AC (●), Q-AC (Œ) and P-AC (▲)

Figure 4.8 Pore size distributions of AC, Q-AC and P-AC

Figure 5.1 Variation in water contact angle of PAA/q-PEI multilayers as a function

of the number of layers deposited on stainless steel Even numbers represent films with PAA as the outermost layer whereas odd numbers denote q-PEI as the outermost layer

Figure 5.2 XPS wide scan spectrum (a) of pristine stainless steel, wide scan (b)

and Ag 3d core-level spectra (c) of stainless steel after 5 bilayers of PAA/q-PEI-Ag deposition

Figure 5.3 Scanning electron micrographs of the PAA/q-PEI-Ag

Figure 5.4 Number of viable E coli (a) and S aureus (b) cells in PBS at 37oC as a

function of time in contact with the different substrates The cell number was determined by surface-spread method

Figure 5.5 Optical densities (at 600 nm) of broth containing S aureus after

different periods in contact with the substrates, which were first

immersed in an S aureus suspension of 107 cells/ml for 2h before transferring to the broth

Figure 5.6 Fluorescence microscopy images of pristine stainless steel under green

filter (a), PAA/q-PEI functionalized stainless steel under red filter (b) and green filter (c), PAA/q-PEI-Ag functionalized stainless steel under red filter (d), after immersion in a PBS suspension of 108 cells/ml of S

aureus for 5 h

Figure 5.7 Cytotoxicity of the pristine and functionalized stainless steel substrates

relative to the non-toxic control (growth culture medium) Triton X-100 serves as the toxic control

Figure 5.8 Release of Ag from (a) PAA/q-PEI-Ag functionalized stainless steel

and (b) PAA/q-PEI-Ag functionalized stainless steel after heating at

120 oC for 3 h in vacuum The substrates were immersed in PBS at

37oC with stirring at 100 rpm

Figure 5.9 Optical densities (at 600 nm) of broth containing S aureus after

different periods in contact with “aged” PAA/q-PEI-Ag functionalized substrates: (a) aging carried out by immersing the substrate in PBS for

21 days at 37 oC, (b) aging was carried out as in (a) but after the substrate has been heated at 120 oC for 3 h in vacuum The “aged”

substrates were first immersed in an S aureus suspension of 107

cells/ml for 2h before transferring to the broth The results of pristine stainless steel are represented here for comparison

Figure 6.3 FE-SEM images of CS NP ((a) and (b)) and QCS NP (c)

Figure 6.4 Young’s modulus of original and CS (30%), CS, CS NP and QCS

NP-loaded Smartset plain (a) and gentamicin-loaded (b) bone cements With the exception of CS (30%), the other substrates were mixed at a weight ratio of CS or QCS to PMMA bone cement powder of 15% (#),

(*) denote significant differences (P < 0.05) compared with original

bone cement which is freshly prepared or after 3 weeks in PBS, respectively

Figure 6.5 Bending modulus of original and CS (30%), CS, CS NP and QCS

NP-loaded Smartset plain (a) and gentamicin-loaded (b) bone cements With the exception of CS (30%), the other substrates were mixed at a weight ratio of CS or QCS to PMMA bone cement powder of 15%

Figure 6.6 Number of viable adherent S aureus (a) and S epidermidis (b) cells on

the different substrates based on Smartset bone cement without gentamicin

Figure 6.7 Number of viable adherent S aureus (a) and S epidermidis (b) cells on

the different substrates based on Smartset bone cement with gentamicin

Figure 6.8 Fluorescence microscopy images of Smartset bone cement, and CS, CS

NP and QCS NP-loaded bone cement substrates under green filter (a, c,

e, g) and red filter (b, d, f, h), respectively, after immersion in bacterial suspension (108 S aureus cells/ml, for 3 h at 37 oC)

Figure 6.9 Schematic representation of synthesis of M-NOR and P-NOR

Figure 6.10 FTIR spectrum of (a) NOR and (b) M-NOR powders

Figure 6.11 Scanning electron micrographs and EDX of (a) original, (b) M-NOR, (c)

P-NOR and gentamicin-loaded CMW Smartset bone cements For (b) and (c), the weight ratio of M-NOR or P-NOR to PMMA bone cement powder is 15%

Figure 6.12 Young’s modulus (a) and bending modulus (b) of original, NOR,

M-NOR, P-NOR and gentamicin-loaded bone cements NOR, M-NOR

or P-NOR were mixed with PMMA bone cement powder at a weight ratio of either 4% or 15% as indicated on the X-axis (#), (*) denote

significant differences (P < 0.05) compared with original bone cement

which is freshly prepared or after 3 weeks in PBS, respectively

Figure 6.13 Number of viable adherent S aureus (a) and S epidermidis (b) cells on

the different substrates based on Smartset bone cement

Table 4.1 Characteristics of ACs before and after functionalization

Table 5.1 Surface composition of stainless steel before and after functionalization

as determined from XPS analysis

Table 6.1 Composition of the bone cements used

Table 6.2 Characteristics of CS NP and QCS NP

Table 6.3 Number of viable adherent S aureus and S epidermidis cells (CFU/cm2)

on the different substrates based on Palacos bone cement

Table 6.4 Cytotoxicity assay of 3T3 cells on the different bone cements

Table 6.5 Antibiotic release rate and surface composition of freshly prepared and

aged bone cements

Table 6.6 Cytotoxicity assay of 3T3 cells on the different bone cements

CHAPTER 1 INTRODUCTION

INTRODUCTION

The rise of multidrug-resistant pathogens and recalcitrance of biofilm infections presents a formidable challenge to combating infectious diseases Hence, there is an ever-growing need for the prevention of bacterial infections in the hospital

colonized by bacteria, they require modification to render them antibacterial and hence unable to transmit bacterial infections The main research focus of this project is to modify materials using surface and bulk functionalization such that antibacterial property is conferred to these materials The techniques developed in this project have potential applications in the development of materials for use where bacterial contamination and infection controls are required, including but not limited to water treatment, medical devices (bone and dental materials), textile manufacturing, biofouling, antimicrobial filters and food packaging

The choice of materials in any application is closely related to the desired performance The materials selected in this project include polymeric and inorganic materials which are used as biomaterials and in industries The different surface and bulk modifications were developed according to the material properties The specific aims of this study were as follows:

1) To develop a technique of covalently immobilizing quaternary ammonium salt on polymeric and inorganic materials and explore the antibacterial effect of these materials

Chapter 1

2) To develop a simple and versatile method to functionalize stainless steel to confer

it with antibacterial property and investigate the cytotoxicity of the functionalized substrates

3) To develop new strategies to introduce or enhance antibacterial activities in bone cement which is a biomaterial used in orthopaedic surgery

In Chapter 2, biofilm and biofilm-related infections will be reviewed In addition, the methods to combat biomaterials-related infections and surface functionalization methods will be reviewed respectively The first part of Chapter 3 describes a surface modification technique involving an asymmetric viologen, N-hexyl-N′-(4-vinylbenzyl) -4,4′-bipyridinium bromide chloride (HVV), which was synthesized and graft copolymerized onto commercial PET films The surface graft concentration of HVV

on the poly(ethylene terephthalate) (PET) film is easily controlled by varying the monomer concentration used in the UV-induced graft copolymerization process The HVV surface functionalized PET film functions as a smart window whose transmittance is reduced upon exposure to light Concomitantly, the film possesses

antibacterial activity, as shown by its bactericidal effect on E coli The antibacterial

activity depends on the concentration of pyridinium groups on the surface and a surface concentration of 25 nmol/cm2 on PET has been shown to be highly effective in killing the bacteria In the second part of Chapter 3, it is shown that the antibacterial activity can be enhanced by silver nanoparticles which can be deposited on the surface

of the viologen-PET film through photoinduced reduction of the silver ions in salt solution The size and distribution of the silver nanoparticles can be varied by

after prolonged immersion in phosphate buffer solution and after aging in a weathering chamber

The functionalization of activated carbon (AC) with two types of quaternary ammonium groups to achieve antibacterial property is described in Chapter 4 The first route utilized covalently coupled 3-(trimethoxysilyl)-propyldimethyloctadecyl- ammonium chloride (QAS) on the AC surface while the second route employed a polycation, poly(vinyl-N-hexylpyridinium bromide) The successful attachment of these two types of quaternary ammonium was indicated by FTIR and XPS analyses Both types of functionalized ACs show highly effective antibacterial activities against

E coli and S aureus Furthermore, the functionalized ACs can be used in repeated

antibacterial applications with little loss in efficacy Using phenol as a model compound, the adsorption capacity of the different ACs was also investigated The different degrees of decrease in the adsorptive capacity of the two types of functionalized ACs can be related to the changes in the surface area and pore size distribution arising from the different functionalization routes

Chapter 5 describes a new strategy for confering stainless steel with antibacterial property via the alternate deposition of quaternized polyethylenimine (PEI) or quaternized polyethylenimine-silver complex and poly(acrylic acid) (PAA) The success of the deposition of the polyelectrolyte multilayers (PEM) and its chemical nature was investigated by static water contact angle and XPS respectively The

antibacterial activity was assessed using E coli, and S aureus The inhibition of E coli and S aureus growth on the surface of functionalized films was clearly shown using the LIVE/DEAD Baclight bacterial viability kits and fluorescence microscopy The

Chapter 1

cytotoxicity of the PEM to mammalian cells, evaluated by the MTT assay, was shown

to be minimal and long term antibacterial efficacy can be maintained These results indicate new possibilities for the use of such easily built and functionalized architectures for the functionalization of surfaces of implanted medical devices

Bacterial infection of poly(methyl methacrylate) (PMMA) bone cements remains a significant complication following total joint replacements, with infection rates ranging from 1% to 3% despite strict antiseptic operative procedures Antibiotics such

as gentamicin have been loaded into bone cement as one of the measures against prosthesis-related infection However, the protective effect of the antibiotic-loaded cement against bacterial infection is generally lost if bacterial contact with the implant occurs after a delay of several weeks because of the decreased concentration of the released antibiotics Hence, it would be desirable to formulate new bone cements which can prevent biofilm formation even after extended time without these accompanying problems In Chapter 6 two new strategies which we have developed to confer antibacterial properties to bone cement are described The first strategy employs chitosan nanoparticles which may be further derivatized with quaternary ammonium groups, while the second uses norfloxacin-containing monomer, as one of the components in the commercial PMMA bone cement The chitosan nanoparticles or norfloxacin moieties can be used to confer antibacterial properties to plain bone cement or to further enhance the effects of antibiotic-loaded cement These modified cements offered two distinct advantages: high mechanical strength even with an additive to bone cement powder loading of 15%, and long lasting antibacterial effect

Chapter 7 gives the overall conclusion of the present work and Chapter 8 gives recommendations for further work

CHAPTER 2 LITERATURE SURVEY

2.1 Biofilm

Biofilms are complex communities of surface-attached microorganisms, comprising either single or multiple species (Costerton et al., 1995; Bryers, 2000; O'Toole et al., 2000; Stoodley et al., 2002) The biofilm concept was first developed and articulated in environment microbiology (Costerton et al., 1978) Over the past few decades, there has been a growing realization that bacteria in most environments are not found in a unicellular, planktonic (free-living) form such as those typically studied in the laboratory, but exist predominantly in multi-cellular surface attached communities called biofilms This realization has spurred much research into the physical and chemical properties of biofilms, the characterization of their morphology, and the mechanisms of their development

Biofilms are found ubiquitously in virtually all natural, medical, and industrial settings where bacteria exist They can form in almost any hydrated environment that has the proper nutrient conditions, and can develop on a wide variety of abiotic hydrophobic and hydrophilic surfaces, including glass, metals, and polymer (Verheyen et al., 1993; Marshall, 1994; An and Friedman, 1998) The formation of biofilms involves events of

a continual dynamic sequence, which has generally been divided into four development stages: bacterial growth as planktonic cells is the first stage, followed by the transportation to a surface or interface In the second stage, bacterial interaction occurs in a reversible manner (O'Toole and Kolter, 1998) The third stage of biofilm formation is the irreversible attachment of bacteria and formation of microcolonies through specific (adhesions) and non-specific interactions (hydrogen bonds, van der Waals forces and hydrophobic interactions) with the surface (Characklis, 1990) The fourth stage involves the dispersal and detanchment of bacteria from the mature

Chapter 2

biofilm Bacteria growing within biofilms present different properties that significantly distinguish them from the corresponding planktonic ones, namely protection from the extreme conditions in the surrounding environment; differences in phenotypic expression and growth characteristics; cooperation between organisms either in monotypic or in mixed populations in utilizing nutrients; and intercellular communication (Jass et al., 2003) In addition to the intrinsic characteristics of bacteria and material surface, many environmental factors including nutrient sources and local conditions (pH, osmolarity, temperature, oxygen, surface properties and hydrodynamic conditions) can exert great effects on the biofilm formation These factors can influence which species will be able to colonize and form biofilms and the maximum biofilm thickness and density (Stoodley et al., 1999)

Within biofilms, bacterial species demonstrate cooperative behaviour and can subsequently differentiate further to exhibit complex multi-cellular behaviour Bacteria may be susceptible to harsh environmental conditions, and growing within complex communities has been shown to offer protection (Costerton et al., 1999) The biofilms most often encountered include dental plaque and the slime on surfaces within both natural and man-made water systems, including domestic water supplies and drains It

is only recently that biofilms have been implicated in many medical conditions and infections (Gristina, 1987; Donlan, 2001) With the increasing use of invasive medical procedures, infections involving biofilms form an important consideration as a risk factor for complications postoperatively Many persistent and chronic infections, such

as endocarditis, osteomyelitis, periodontitis, otitis media and biliary tract infections, have also been attributed to bacterial biofilms (Costerton et al 1999) The modern-day

lifestyle provides more opportunities for biofilms to cause problems to mankind The focus of the next section is on the review of biomedical device-related infections

Chapter 2

2.2 Preventative Strategies for Device-related Infections

Biomaterials are generally substances other than food or drugs contained in the therapeutic or diagnostic system that are in contact with tissue or biological fluids (Peppas and Langer, 1994) The insertion of indwelling or implanted medical devices made from biomaterials, such as prosthetic heart valves, cardiac pacemakers, total joint replacement or other orthopaedic devices, dental implants, intravascular catheters and renal dialysis shunts, has become an indispensable part of modern medical practices (Hanker and Giammara, 1988; Angelova and Hunkeler, 1999; Anderson et al., 2004; Langer and Tirrell, 2004) In spite of many advances in biomaterials, strict aseptic conditions during the surgical process and systemic administration of antibiotics, the incidence of infections caused by bacteria is still high (Stamm, 1978; Sugarman, 1986; Donlan, 2001; Parsek and Singh, 2003; Campoccia et al., 2006, Bryers and Ratner, 2006)

Bacterial colonization of the biomaterial surfaces can result in biofilms This term was introduced into medical microbiology when Marrie et al (1982) examined the surfaces

of devices that had failed because of bacterial infection It has been shown that biofilm grown cells can become 1000-fold more resistant to the effects of antibiotics than their planktonic counterparts (Hoyle and Costerton, 1991; Stewart and Costerton, 2001; Levy and Marshall, 2004) Biofilms show resistance to a wide range of antibiotics (including ampicillin, strepotomycin, gentamicin and many others) and biocide oxidants such as ozone, chlorine and iodine At the same time, biofilms can modulate the host’s immune response The combination of these two factors, host-mediated immune modulation and antibacterial resistance, makes them extremely difficult to

biofilms are not implicated Consequently, the only treatment often left to the clinician

is to remove the infected implant, followed by a prolonged period of intensive antibiotic therapy

According to the US National Institutes of Heath, biofilms cause over 80% of infections in the body In the US, about 90,000 people die each year from nosocomial infections (Weinstein, 1998) Of the 2.6 million orthopedic implants inserted into humans annually in the United States, approximately 112,000 (4.3%) become infected (Darouiche, 2004) The annual infection rate for cardiovascular implants is even higher (7.4%) When considering all indwelling devices, the number of implant-associated infections approaches approximately 1 million per year Perhaps of equal concern, antibiotics administered systemically are showing lower efficacy against implant-associated infections As a result, implant removal and/or amputation are increasingly more prevalent In addition to human pain and suffering, device-related infections present a significant economic burden to society Estimates of the direct medical costs associated with such infections exceed $3 billion annually in the U.S alone The number of device-associated infections will continue to rise as more patients receive biomedical implants From 1996 to 2001, the number of hip and knee joint replacements increased by 14% (Deyo el at 2004) The majority of these implant procedures were performed on patients 65 years of age and older (Moore et al.1991) The worldwide increase in life expectancy and advances in medical technology will lead to greater demand for medical implants and a rising number of implant-associated infections Prevention of medical device-related infections remains a major dilemma in the delivery of quality medical care, and the problem causes high rates of mortality and morbidity and significant increases in health care costs (Schierholz and Beuth, 2001;

Chapter 2

Darouiche, 2004) Although the removal of some implants may not be a serious problem medically, the incidental effects on the patient’s well-being and morale may

be severe and hinder recovery significantly, in addition to increased financial costs

Despite considerable advances in our understanding, there have been few new effective treatments against biofilms (Ceri et al., 1999) Antibiotics have been used successfully where the infection has been recognized at early onset, before a mature biofilm is developed However, the use of long-term prophylactic antibiotics is not recommended, as they have been associated with the proliferation of resistant microorganisms, even though it is a general practice that prophylactic antibiotics are given in many hospitals at the time of surgery A number of new strategies (Klueh et al., 2000; Tiller et al., 2001; Tew et al., 2002; Hume et al., 2004) have been developed

by researchers to overcome the problems encountered in biofilm control in vitro These alternative strategies may result in reduced use of antibiotics, hence reducing cost outlay and the potential development of antibiotic-resistant microorganisms

The prevention and control of biomaterial-centered infections has been the subject of many investigations Functionalization of biomaterials for preventing biomaterials- centered infections through killing the bacteria on/near the surface is an important method in recent research Compared with other methods, such as systemic administration of antibiotics and strictly non-septic conditions in surgical process, surface functionalization is a relatively straight-forward strategy to confer antibacterial property to biomaterial surfaces Ideally, this can be achieved without compromising the bulk property of biomaterials Another advantage is that it can prevent

cannot reach A variety of such techniques have been developed to inhibit biomaterials-centered infections, which can be broadly classified into three categories: development of antiadhesive surfaces, incorporation of antibacterial agents and functionalized surfaces with antibacterial agents

2.2.1 Development of antiadhesive surfaces

Several research groups have tried to modify devices with new surface properties that would lead to a reduction of bacterial adhesion Bridgett et al (1992) studied the

adherence of three isolates of S epidermidis to polystyrene surfaces that were modified

with a copolymer of poly(ethylene oxide) and poly(propylene oxide) A substantial reduction in bacterial adhesion was achieved Similar results were found by Desai et al

(1992), who investigated the adhesion of S epidermidis, S aureus and P aeruginosa

to polymers that were surface-modified with poly(ethylene oxide) They observed reductions in adherent bacteria of between 70% and 95% compared with the untreated polymer A photochemical coating of polymer was used by Dunkirk el al (1991), demonstrating that the coating reduced adhesion of a variety of bacterial strains Tebbs

et al (1994) compared the adherence of five S epidermidis strains to a polyurethane

catheter Adhesion of three strains to the coated catheters was considerably reduced

By radiation or glow discharge techniques, Jasen el al (1987) developed a polyurethane surface covalently bonded with 2-hydroxymethylmethacrylate which

shows a reduced adhesion of S epidermidis

More recent research work on surface modification of polymeric biomaterials to prevent bacterial adhesion involved the use of sulfonated poly(ethylene oxide) as surfactant in a polyurethane (Han et al., 1998) or the introduction of

Chapter 2

glycerophosphorylcholine as a chain extender in polyurethane (Baumgartner et al., 1997) Both approaches lead to increased water uptake and low bacterial adhesion Overviews on experimental research on the surface modification of materials and on binding macromolecules such as albumin to surfaces in order to prevent bacterial adherence can be found elsewhere (An et al., 2000; Kohnen and Jansen, 2000)

The previously described approaches, which aim at the modification of the surface properties of materials to create ‘zero’ adhesion Due to the complex nature of bacteria-materials interactions, it is unlikely that the adhesion of bacteria can be completely prevented by antiadhesive surface modification alone (Mittelman, 1996) Modifying device surfaces to become more hydrophilic may reduce the adherence of some bacterial strains, but may also increase the adherence of some other strains (Hogt

et al., 1983) In an experimental study that investigated the relationship between bacterial adhesion and the free surface enthalpy of adhesion of a large number of different modified polymers, Jansen and Kohnen (1995) demonstrated that it is impossible to develop a surface that shows an absolute bacterial ‘zero’ adherence in vitro Jansen and Kohnen (1995) demonstrated that it is impossible to develop a surface that shows an absolute bacterial ‘zero’ adherence in vitro However, Kingshott

et al (2003) have shown virtually zero bacterial adherence to hydrophilic PEG hydrogel layers that function as steric barrier coating Such antiadhesive surfaces seem promising since cell adhesive motifs such as RGD peptide can be incorporated in the hydrogel layers to enhance tissue integration (Harris et al., 2004) The interesting aspect of this strategy is that the modified substrate exhibits selective biointeractiveness, adhesive to cells but inhibiting bacteria, a property which is

2.2.2 Incorporation of antibacterial agents

The loading of medical devices with antimicrobial agents either for therapeutic or preventive purposes has a long tradition The best known polymer drug delivery systems are the PMMA-gentamicin bone cement and the PMMA-gentamicin beads used for treatment of bone and soft tissue infections (Welch, 1978; Marcinko, 1985; Hendriks et al., 2004) The main principle of such devices is that an antibacterial agent

is incorporated into the interior of the devices If such a device comes into contact with

an aqueous environment, release of the drug into the near vicinity occurs The amount

of the antibacterial agent released is influenced by the processing parameters, loading dose, applied technique, molecular size of the drug and the physico-chemical properties of the device A high antibacterial concentration is reached initially in the very near vicinity of the device surface, mostly exceeding the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of susceptible bacteria Most of such materials exhibit a release pattern according to first-order kinetics, which an initially high drug release and subsequent exponential decrease of the drug

Jasen and Peters (1991) have investigated the incorporation of flucloxacillin, clindamycin and ciprofloxacin into polyurethane and demonstrated a considerable

reduction of the in vitro adherence of S epidermidis The most promising development

in this field in the last few years was a catheter using a combination of chlorhexidine and silver sulfadiazine This catheter became available about 10 years ago (ArrowGard, Arrow International, USA) A synergistic effect of chlorhexidine and sulfadiazine has been shown in vitro (Quesnel et al., 1978) A disadvantage of all of these approaches may result from the risk for development of resistance against the antibacterial agents

Chapter 2

This approach, however, is not without its technical hurdles Care must be taken to ensure that impregnation of the medical device does not alter its desired physicochemical properties For example, catheters have desired degrees of lubriciousness, persistence length, and compatibility with host tissue that cannot be radically altered without impairing their utility In addition, each device must be loaded with enough of the antimicrobial agent in question such that the catheter releases its antimicrobial payload at bactericidal or bacteriostatic concentrations for the lifetime of the device

2.2.3 Functionalized surfaces with antibacterial agents

Device surfaces functionalized with antibacterial agents have significant advantages They can incorporate a large variety of agents on the surface and the existing devices can be modified easily and inexpensively without changing the device bulk properties Coating with antibacterial agents have shown promising results and appear to increase biocompatibility and resist the adhesion of the bacteria on device surfaces The current surface-treatment techniques will be reviewed as follow:

Direct deposition of silver

Silver has been reported to have the highest antibacterial activity and low toxicity compared with other metals (Feng et al., 2000; Furno et al., 2004; Gosheger et al., 2004) Silver has also extensively been used to prevent device-related infections Some researchers have applied silver directly onto a device surface through physical methods With the release of silver ions, bacteria on/near the surface can be killed, which has resulted in reduction of biomaterials-centered infections

McLean et al (1993) reported that silver-copper surface films, sputter-coated onto materials, show antibacterial activity against biofilm formation Sioshansi et al (1994) used the technique of ion implantation to deposit silver-based coatings on a silicone rubber which thereafter demonstrated antibacterial activity Recently, Woodyard et al (1996) compared the antibacterial effects of several silver-treated catheters through ion-beam-assisted deposition Their findings suggest that silver-treated catheters, in particular, do have inhibitory effect on bacteria and may be valuable in lowering the risk of infections A newly developed biopolymer by Milder (1999) is the so-called

“oligodynamic iontophoresis-enhanced” material The polymer was impregnated with silver, platinum and carbon particles The silver and platinum particles acted as electrodes in battery-like chemistry, releasing a steady flow of silver which provided

an effective colonization resistance By controlling the particle size and concentration, different silver release profiles can be achieved which could be successful in the prevention of infection In more recent research, Devanas et al (2002) applied an ion beam technique with low implantation energy for the formation of silver nanoparticles

on the surface of polymers that exhibited an improved effect on antibacterial activities

Although silver deposition has resulted in some successful applications, it has been reported that high silver concentration can cause neurotoxical complications in the human body, which certainly limits the applications of this technique (Vik et al., 1985) Another disadvantage is that its effectiveness would vanish after the complete release

of silver in the long-term application

et al (2000) developed a technique where silver iodide was bound as sub-micron particles by a positively charged nitrogen functional group on immobilized polybiguanide surface Results showed that the treated surfaces prevent the growth of bacteria

Entrapping antibacterial agents

Entrapping antibacterial agents in a polymer matrix applied on device surfaces has shown promising results in some clinical studies Polymer matrices can be formed either by in situ crosslinking of prepolymers or applying non-reactive polymer coatings Polymer matrices with antibacterial agents could be used directly to construct

a device or applied as a thin layer of coating on surfaces Several researchers have studied silver sulfadiazine and chlorhexidine in a polyurethane matrix applied to central venous catheters for preventing device-related infections (Bach et al., 1993; Greenfeld et al., 1995) Clinical studies on the performance of the catheters have shown inconsistent outcomes (Civetta et al., 1996; Maki et al., 1997)

Covalent immobilization of quaternary ammonium on surfaces

The methods described above focus on the technologies that provide biomaterials with antibacterial agents or antibiotics which leach out to inhibit bacterial growth on/near the surface However, these approaches, while certainly useful, are quite limited because of the fact that the antibacterial agent is free to leave the surface This has serious adverse effects on the durability and useful life of the treated material, though

it does not appear to impact marketability Another serious problem with leaching-based technologies is that the compounds released into the environment at sub lethal concentrations have the effect of increasing drug resistance throughout the microbial realm The appearance rate of antibiotic resistant bacteria is currently a great concern among the medical community

To address the deficiencies inherent in the leaching type of antibacterial surfaces, one new route for creating antibacterial surfaces is covalent grafting with long hydrophobic polycationic chains such as quaternary amine (Tiller et al., 2001) In this approach, no antibacterial agents are leached from the surface, providing long term protection against bacterial infections and reducing the risk of developing antibiotics-resistant bacterial strains, as the concentration of surface antibacterial groups is constantly above the minimal inhibitory concentration

Among the polycations, quaternary amine is used most widely (Thorsteinsson et al., 2003) Some researchers have investigated antibacterial activity of quaternary amine in solutions Nakagawa et al (1982) reported that soluble alkyl quaternary amine can kill bacteria Li et al (2000) studied the antibacterial activity of pyridinium-type quaternary amine Their findings show that quaternary amines exhibit many unique

Chapter 2

properties and are used as effective antibacterial agents in solutions But the mechanism of these cationic disinfectants is still not very clear It is typically described

as proceeding in the following steps:

Adsorption onto the bacterial cell surface

Diffusion through the cell wall

Binding to the cytoplasmic membrane

Disruption and disintegration of the cytoplasmic membrane

Release of electrolytes such as potassium ions and phosphate from the cell

Release of nucleic materials such as DNA and RNA

Death of the cell

It has been realized that quaternary amines attached to surfaces can also kill bacteria upon contact Isquith et al (1972) were the first to report antibacterial activity of alkyl

quaternary amines coated glass and cotton towards S faecalis and E coli Then

Flemming et al (2000) also found good antibacterial activity of polyurethanes

functionalized with methyl and ethyl quaternary amines towards S aureus Gottenbos

et al (2002) also investigated antibacterial activity of silicone rubber functionalized with alkyl quaternary amines Whereas antibacterial effects were observed in vitro, the antibacterial effects was not broad-spectrum, displaying only a modest reduction in the viability of attached Gram-negative organisms and showing limited efficacy when rigorously examined in vivo They also proposed that immobilized alkyl quaternary amines have the same disinfection mechanism as those in solution More recently, Tiller et al (2001, 2002) described methods for treating flat surfaces such as glass, high-density polyethylene, low-density polyethylene, polypropylene, and nylon, poly(ethylene terephthalate) with poly(4-vinyl-pyridine) modified with pendant quaternary ammonium salts The antibacterial properties of these materials were

amine, it was able to kill bacteria that were resistant to other types of cationic antibacterials This same group has also demonstrated the utility of immobilizing antibacterial polymers on fabrics and polyolefins The active material for all of the above biocidal surfaces was synthesized by either classical free radical polymerization

or simple coupling reactions and then applied to an activated surface Chains of six carbon units in length were one of the most effective

The existing approaches of covalent grafting of quaternary amine on surfaces, however, suffer from significant limitations Firstly, the existing approaches involves a number

of chemical reaction steps and complicated and expensive protocols Thus, it is difficult to control quaternary amine concentration, which is a key factor to inhibit biomaterials-centered infections and at the same time prevent toxicity to the human body (Danese, 2002) Hence, a simple and high efficiency method for surface functionalization would be of great value in preventing biomaterials infections Secondly, the antibacterial effect and broad spectrum bactericidal ability is still not sufficient for actual applications Thirdly, much research has been done on alkyl and pyridine-type quaternary amine, little research is reported on the viologen-type quaternary amine, which has been demonstrated to have high antibacterial effect (Avram et al., 2001)

Chapter 2

2.3 Surface Functionalization

Surface functionalization of materials to obtain desired properties has become an important research area in the industry Many improvements have been made in developing surface treatments to alter the chemical and physical properties of material surfaces without affecting bulk properties

2.3.1 Surface graft copolymerization

Surface grafting is one of the most commonly performed means to change the surface properties of materials for such applications as biomaterials, membranes, and adhesives In surface graft polymerization, the modification is achieved by grafting suitable macromolecular chains on the surface of materials through covalent bonding The key advantage of these techniques is that the surface of the materials can be modified or tailored to acquire very distinctive properties such as hydrophilicity, hydrophobicity, biocompatibility and adhesive properties through the choice of different grafting monomers, while maintaining the substrate properties It also ensures

an easy and controllable introduction of graft chains with a high density and exact localization onto the surface Compared with physically coated polymer chains, the covalent attachment of the grafted chains onto a material surface avoids their desorption and maintains long-term chemical stability of the introduced chains All these aspects give the rationale for applying the grafting process for surface modification

Surface grafting commonly includes two steps: surface activation and graft polymerization Because of the absence of chemically reactive functional groups on

on them that can generate further grafting processes The generated peroxides and hydroxyperoxides (and minor functional groups such as carbonyl and carboxylic groups), in particular, are capable of initiating radical polymerization of vinyl monomers, resulting in surface-grafted polymer chains In practice, UV, high-energy electron, γ-ray irradiation, plasma treatment, ozone exposure, are commonly used (Chapiro and Jendrychowskabonamour, 1980; Peeling and Clark, 1983; Suzuki et al., 1986; Masuoka et al., 1989; Allmer et al., 1990)

Plasma discharge method

Plasma can be broadly defined as a gas containing charged and neutral species, including some of the following: electrons, positive ions, negative ions, radicals, atoms and molecules Plasma treatment involves reactions between gas-phase species, reactions between gas-phase species and surface species, and reactions between surface species Inert gas plasmas, such as helium, neon and argon, have been used for etching purposes Free radicals are formed at the surface and the surface is activated during the subsequent exposure to the atmosphere These radicals may be stable for long durations, thereby having an opportunity to react It is easy and convenient to use plasma treatment to introduce initiators onto substrate surfaces

The plasma technique has been applied to surface modification involving the grafting

of functional groups (Gupta et al., 2001), metallization (Wang et al., 2002), immobilization of proteins and biological molecules (Hayat et al., 1992), antibacterial coating (Gray et al., 2003) and so on

Chapter 2

UV irradiation method

Ultraviolet (UV) energy has been extensively applied for surface graft polymerization

of polymers with the aid of a photoinitiator or a photosensitizer, such as benzophenone (Ranby, 1992) During UV irradiation, the photoinitiators are excited to a high-energy state which promotes abstraction of hydrogen from the polymer substrate Thus, radicals are generated on the polymer surface for the subsequent initiation of graft copolymerization

UV-induced surface grafting can be performed by one-step (mutual irradiation grafting)

or two-step (pre-activation grafting) methods One-step method or mutual irradiation grafting, which is performed by irradiating the polymer in the presence of a solvent containing a monomer and a photosensitizer (Wright, 1967; Ogiwara et al., 1981) (Fouassier et al., 1989), is very efficient, and surface activation and graft copolymerization are achieved simultaneously In 1990, a novel process was developed for continuous photoinitiated graft polymerization of acrylamide and acrylic acid onto the surface of high-density polyethylene tape film, presoaked in a solution containing monomer and initiator under nitrogen atmosphere (Yao and Ranby, 1990) However, the one-step method cannot totally eliminate homopolymerization initiated by free radicals formed during irradiation of the monomer In addition, the one-step method requires the addition of a photosensitizer that, if remaining on the sample after the treatment, can promote light-induced degradation or can be leached out in the surrounding environment A photo-induced living graft copolymerization method consisting of two steps was then investigated by Ma et al (2000) In the first step, benzophenone abstracts hydrogen from the substrate to generate surface radicals,

the subsequent step, the monomer solutions are added onto the active substrate, and the surface initiators initiate the graft polymerization under UV irradation In this method, graft density and graft polymer chain length can be controlled independently since initiator formation and graft polymerization occur independently in the successive steps In addition, the method substantially eliminates formation of undesired homopolymer and crosslinked or branched polymer Another two-step method can be used to avoid introducing photoinitiator In this case, the polymer can be pre-activated

by UV, plasma, ozone, high-energy electron or γ-ray irradiation exposure to produce peroxide groups on the substrate Graft copolymerization is subsequently initiated by UV-irradiation with the substrate in contact with a monomer

2.3.2 Self-assembled polyelectrolyte multilayers

Polyelectrolyte are macromolecules that have a large number of charged or chargeable groups when dissolved in polar solvents, primarily water (Dautzenberg, 1994) Polyelectrolytes are typically weak polyacids or polybases Upon dissolution of polyelectrolytes in water, they dissociate into macromolecular ions and counterions The effective charge of each segment is controlled by the pH value of the solution (Netz and Andelman, 2003) The characteristic aspect of polyelectrolytes is the high expansion or stretching of the polyion chain owing to the strong electrostatic repulsion between equally charged segments of the chain (De Gennes et al., 1976)

Over the past several years, Decherhave developed a layer-by-layer deposition method based on a simple and versatile method for preparing supported multilayer thin films (Decher and Hong, 1991; Decher, 1997) These and other types of organic thin films show promise in applications such as sensors, friction reducing coatings, integrated