Current Developments in Antimicrobial Surface Coatings for Biomedical Applications

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Current Developments in Antimicrobial Surface Coatings for Biomedical Applications

J.J.T.M. Swartjes1, P.K. Sharma1, T.G. van Kooten*,1, H.C. van der Mei1, M. Mahmoudi2,

H.J. Busscher1 and E.T.J. Rochford1

1University of Groningen and University Medical Center Groningen, Department of Biomedical Engineering, P.O. Box

196, 9700 AD Groningen, The Netherlands; 2Department of Nanotechnology and Nanotechnology Research Center,

Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

Abstract: Bacterial adhesion and subsequent biofilm formation on material surfaces represent a serious problem in soci-ety from both an economical and health perspective. Surface coating approaches to prevent bacterial adhesion and biofilm formation are of increased importance due to the increasing prevalence of antibiotic resistant bacterial strains. Effective antimicrobial surface coatings can be based on an anti-adhesive principle that prevents bacteria to adhere, or on bacteri-cidal strategies, killing organisms either before or after contact is made with the surface. Many strategies, however, im-plement a multi-functional approach that incorporates both of these mechanisms. For anti-adhesive strategies, the use of polymer chains, or hydrogels is preferred, although recently a new class of super-hydrophobic surfaces has been described which demonstrate improved anti-adhesive activity. In addition, bacterial killing can be achieved using antimicrobial pep-tides, antibiotics, chitosan or enzymes directly bound, tethered through spacer-molecules or encased in biodegradable ma-trices, nanoparticles and quaternary ammonium compounds. Notwithstanding the ubiquitous nature of the problem of mi-crobial colonization of material surfaces, this review focuses on the recent developments in antimicrobial surface coatings with respect to biomaterial implants and devices. In this biomedical arena, to rank the different coating strategies in order of increasing efficacy is impossible, since this depends on the clinical application aimed for and whether expectations are short- or long term. Considering that the era of antibiotics to control infectious biofilms will eventually come to an end, the future for biofilm control on biomaterial implants and devices is likely with surface-associated modifications that are non-antibiotic related.

Keywords: Antibacterial, antibiotics, antimicrobial peptides, bacterial adhesion, nanoparticles, quaternary ammonium com-pounds, surface coating.

1. INTRODUCTION

Bacterial adhesion and subsequent biofilm formation on material surfaces represent a serious problem in society from both an economical and health perspective [1-3]. Biofilms formed in industrial settings like pipelines, water treatment plants, heat exchangers and on ship hulls, contribute to de-creased efficiency accompanied with huge increases in oper-ating costs. Furthermore, microbial adhesion and growth on food processing equipment, but even more so on medical devices and implants, can cause serious complications to human health. Whereas mechanical removal of biofilms in industrial settings is expensive, but usually effective, in medical applications removal represents a last resort solu-tion. Biofilm detachment and mechanical removal from bio-material-associated infections means extensive debridement and high-risk revision surgery accompanied by increased risks of further infectious complications. Treatment and pre-vention methods include the use of antibiotics, but the low

*Address correspondence to this author at the Department of Biomedical Engineering, University Medical Center Groningen (FB40), Antonius Deus-inglaan 1, 9713 AV, Groningen, The Netherlands; Tel: +31-50-3633122; Fax: +31-50-3633159; E-mail: [email protected]

sensitivity of bacteria to antibiotics induced by the biofilm mode of growth, together with the increasing number of multi-resistant strains, makes their use currently less effec-tive than it has ever been [4-6].

As an alternative to the use of antibiotics to prevent bac-teria from causing infection or to treat established biofilms, the development of new materials or surface coatings that prevent viable bacteria from adhering has been the center of attention in many studies [7-9]. Since the first step of bacte-ria in developing into a highly resistant biofilm, is to adhere firmly onto a surface, interfering with this step can reduce infection risks. This is achieved not only by preventing biofilm formation, but also by maintaining bacteria in their planktonic, non-adhering state, which means these pathogens are more sensitive to antibiotics and clearance by the im-mune system. For decades, the method of choice to create these so-called anti-adhesive coatings has been the modifica-tion of surfaces with polymer brushes [10, 11]. When suffi-ciently long polymer chains are grafted to a surface at a high enough surface density, a steric barrier is created which can prevent adhesion of proteins and bacteria. Polyethylene gly-col (PEG) was one of the first polymers used to this end and demonstrated log reductions in adhesion of both proteins and

2 Current Medicinal Chemistry, 2015, Vol. 22, No. 1 Swartjes et al.

bacterial cells. This led to a period in which several variants of PEG-based brush coatings were designed and evaluated [11-13]. However, the use of polymer brushes never achieves complete adhesion prevention and the few bacteria that do manage to adhere still demonstrate the capacity of growing into a biofilm [11].

To date, the original thought that rather simple polymer brushes would be sufficient for preventing implants and medical devices to become colonized by bacteria has been surpassed by the realization that multiple functionalities, including tissue integrative ones, need to be combined in one surface coating in order to effectively prevent implant and device colonization [14]. Additionally, the diverse range of implants applied in the clinical setting requires the design of any future antimicrobial coating to be carefully matched to the intended application. For example, the requirements of a coating for a short-term catheter differ dramatically from those of a permanent hip implant. Consequently, during the design process a number of variables must be considered, the first of which is the duration of the coating efficacy. Micro-organisms can be encountered pre-operatively from wound contamination, peri-operatively from the operating room or contaminated equipment and post-operatively over the life-time of the implant via hematogenous seeding [15]. For a short term implant these three contamination mechanisms can be treated similarly; however, for long term implants a compromise needs to be made for the duration of protection afforded by the coating: simply over the early high risk pe-riod or also the long-term low risk period. A second consid-eration is whether the mechanism applied should release antimicrobials or present the active component bound to the surface. The release of antimicrobial compounds is benefi-cial as it not only kills microorganisms associated with the implant surface directly, but also any susceptible pathogens in the surrounding area. However there is a caveat, the re-lease profiles of such coatings are often difficult to effec-tively control and often inappropriate concentrations of an-timicrobials are released. For many of these coatings an ini-tial massive burst of the active component is delivered fol-lowed by a longer period of diminishing release. It is during this latter phase that bacteria may be exposed to sub-inhibitory concentrations of antimicrobials which is condu-cive to the development of resistance and therefore may ren-der the coating ineffective [16].

In this review, we aim to provide an overview of the cur-rent developments in antimicrobial surface coatings for use in the biomedical field, over the past few years. An overview

will be given of the main types of antimicrobial strategies for surface coating: use of antimicrobial peptides (AMPs), anti-biotics, enzymes, nanoparticles (NPs), quaternary ammo-nium compounds (QACs), anti-adhesive polymers, super hydrophobic coatings and chitosan based strategies. (Fig. 1) shows four example strategies for creating antimicrobial surface coatings, e.g. surface immobilization of antimicrobi-als, surface coatings designed to release antimicrobial into the surrounding, hydrogel or other matrix structures contain-ing bound antimicrobials and antimicrobials tethered to a surface through spacer-molecules.

2. OVERVIEW OF ANTIMICROBIAL SURFACE

COATINGS

2.1. Antibiotics

The most commonly used antimicrobials are antibiotics, of which penicillin is perhaps the most well-known and one of the earliest to be applied in a medical setting. In the dec-ades after its discovery, manufacturing methods were simpli-fied and new formulations were discovered, making the use of antibiotics widespread [6]. The dark side of the wide availability and use of antibiotics turned out to be the rise of bacterial strains that had developed resistance against one or more antibiotic agents. Methicillin resistant Staphylococcus aureus for example is one of the most notorious among these strains and although the name suggests it to be only resistant to methicillin, in reality the resistance profile is often not just restricted to methicillin [17]. Despite the rise of resistant strains, antibiotics are still widely used and subject of new research to develop antibacterial coatings for a number of reasons. Firstly, because of the relative ease of translating techniques involving currently used antibiotics to new gen-erations of antibiotics. Secondly, because developments in surface coating technology permit controlled localized re-lease which decreases the risk of bacterial resistance devel-opment compared to systemic administration [18]. However, sub-inhibitory concentrations of antibiotics remain to form a high risk factor in antibiotic releasing materials and coatings [16].

Clinically, the application of antibiotic releasing hy-droxyapatite (HAP) is common practice in orthopedic sur-gery; bone implants are often coated with antibiotic releasing HAP to prevent infection while at the same time promoting bone ingrowth [19, 20]. Belcarz et al. modified HAP by ad-dition of -1,3-glucan, creating an elastic composite coating that was able to bind antibiotics by ionic interactions and

Fig. (1). Schematic presentation of four example strategies for antimicrobial surface coating of materials. Combinations of strategies to achieve optimum results are often applied.

Antimicrobial Coatings for Biomedical Applications Current Medicinal Chemistry, 2015, Vol. 22, No. 1 3

released the majority of the drug during the first 48 h, with a very short period of drug release at sub-inhibitory concentra-tions [21]. Avoiding the problem of release of sub-inhibitory concentrations was approached in a different way by Noble et al., who created a poly(2-hydroxyethyl methacrylate) (pHEMA) polymeric monolith and added a self-assembled multilayer (SAM) coating of long methylene chains [22]. Addition of ciprofloxacin resulted in an antibiotic releasing coating that could be switched “on” and “off” by using ultra-sound. After application of ultrasound the methylene chains re-organized to a relatively impermeable self-assembled coating stopping the release of antibiotic. Although a small amount of background release was observed, the applied system is a promising way of delivering antibiotics on-demand.

In addition, release of antibiotics by coating degradation is possible by using degradable polymers such as poly(D,L-lactide), poly( -caprolactone) or poly(trimethylene carbon-ate) [23-25]. Combining different degradable polymers into a multilayer system offers the opportunity to include multiple antibiotics that allow modulation of the release profile per antibiotic [24] and additionally degradable surfaces may be inherently resistant to infection [26]. An alternative method to obtain multilayer systems has been described by Shukla and co-workers who applied tetra-layers of (poly-2-dextran sulfate/vancomycin/dextran sulfate) by spray coating [27]. To this end, a vacuum was applied to the back of a porous gelatin surface and each individual layer was sprayed on, followed by a rinsing step. The tetra-layer system on gelatin sponges showed more linear release kinetics compared to flat substrates, expanding the time of release by 100 h. Addition-ally, hydrolytically degradable polyelectrolyte multilayers manufactured by Wong et al. consisted of a non-degradable bactericidal base bilayer of N,N-dodecyl,methyl-poly (ethyl-eneimine) (DMLPEI) and poly(acrylic acid) (PAA) on plasma-etched silicon topped with the degradable gentamicin sulfate (GS) containing top layer. This top layer consisted of (PAA/GS/PAA) tetra-layers in which hydrolytically degrad-able poly( -amino-ester) was included [28]. These films showed high burst release of gentamicin in the first hours, while the bactericidal base-coating prevented bacterial colo-nization of substrates by S. aureus for up to two weeks.

In contrast to release coatings, surface binding of antibi-otic agents creates a high local concentration, minimizing the risk of bacterial exposure to sub-inhibitory concentrations and thereby reduces the risk of resistance. Current immobili-zation studies focus mainly on binding of vancomycin, which is considered to be a last resort in treatment of infec-tions caused by multi-resistant bacterial strains [29]. Since the working mechanism of vancomycin requires penetration of the cell wall, surface tethering is generally performed by including spacers that allow for a certain degree of freedom to penetrate the cell wall. Jose et al. used a double ami-noethoxyethoxyacetate linker combined with a 3-aminopropyltriethoxysilane modified titanium surface, to provide a vancomycin surface distance of about 4 nm [30]. Surface coating of titanium particles confirmed that the van-comycin-surface distance was sufficient to retain antimicro-bial activity, reducing S. aureus colony-forming units by 88% over two hours, while repeated exposure to bacterial suspensions did not alter the antimicrobial activity. Swanson et al. passivated titanium surfaces to increase the amount of

hydroxide groups, which were then changed to amine func-tional groups through a 3-aminopropyl-triethoxysilane reac-tion. The amine functional groups were converted to alde-hyde groups via a glutaraldehyde reaction and were bonded to the amine functional group of chitosan. This layer was used to subsequently promote the binding of a chitosan-vancomycin mixture, creating a surface coating capable of giving a zone of inhibition for S. aureus similar to the use of standard freely soluble vancomycin [31].

With increased antibiotic resistance, focus on alternative antimicrobial therapeutics is gaining, but controlled antibi-otic therapy by means of surface coating remains an enticing topic. Not only to maintain the current last-resort antibiotics such as vancomycin, but also to be able to responsibly use new, future formulations of antibiotics in order to avoid de-velopment of resistant strains shortly after they are intro-duced.

2.2. Antimicrobial Peptides

The concept of using peptides against microbial attack is not a recent development and in fact has been employed by nature, as shown by the antimicrobial peptides that are part of the innate immune system [32, 33]. The current aug-mented attention in science towards the use of AMPs in an-timicrobial applications is largely due to their broad antimi-crobial spectrum which includes both Gram-positive and Gram-negative bacteria and even viruses [34, 35] with rela-tively little induction of resistance among its target organ-isms [36, 37]. Additional to the broad range of susceptible microorganisms, AMPs are effective against strains that have developed a high degree of antibiotic resistance; for exam-ple, methicillin resistant S. aureus [38]. AMPs generally have an overall positive charge and contain a large portion of hydrophobic residues. Their antibacterial activity comes from association with the negatively charged bacterial cell wall, after which hydrophobic interactions of accumulated AMPs disrupt the cell wall [35]. Together these characteris-tics mean that AMPs are well suited for incorporation in sur-face coatings and therefore this area of research has obtained the attention of many investigations.

Built-up from a variety of amino-acids, AMPs are suit-able for surface attachment by various coupling mechanisms [39]. The primary amine-groups associated with most amino acids can be used to directly couple AMPs to activated sur-faces containing aldehyde, carboxyl or NHS modifications [34, 40, 41]. N-terminal coupling of AMPs to gold has been achieved by modification of the surface using 11-mercaptoundecanoic acid (MUA), which is subsequently activated using EDC and NHS. After this, the amine group of the AMP can react with the activated carboxylic terminal of the established MUA monolayer (see Fig. 2) [42]. The stability of this approach is emphasized by Humblot et al. who demonstrated a 40% reduction in bacterial adhesion after six months of storage at 4°C, including exposure of the coated surfaces to four bacterial adhesion assays during this six months period [43]. Coupling of the AMP gramicidin A onto gold has also been successfully achieved by modifying gold surfaces using cystamine, which was then allowed to react with the aldehyde functional group at the NH2 terminus of gramicidin A, formed by natural formylation [44].


Direct and rather uncomplicated coupling of AMPs is preferable and possible on e.g. gold surfaces as well as tita-nium [45], but coating of implants for orthopedic applica-tions might require additional surface modification to make the implant more suitable to fulfill its function inside the body. Calcium phosphate (CaP) has been known to enhance bone growth on orthopedic implants and a system in which an AMP (Tet213) was added by absorbing it into micro-porous CaP coated titanium showed high antimicrobial activ-ity against Pseudomonas aeruginosa [46]. In another study, the antimicrobial peptide HHC-36 was incorporated into a multilayer system of CaP on TiO2 nanotubes [47]. The tita-nium nanotubes were loaded with AMPs using vacuum-assisted physical adsorption, while the CaP was loaded by applying an AMP solution in ethanol and letting it dry in air. For a better controlled release profile a phospholipid layer was added on top of the CaP, to create a bio-inspired cell membrane. The modified surfaces showed sufficient release of AMPs to kill S. aureus and P. aeruginosa, while os-teoblast-like cells were able to attach to the implants and no cytotoxicity against these cells was observed after five days. The difference of these AMP loaded surfaces compared to directly coupled AMPs by the aforementioned surface chem-istry is that rather than killing bacteria upon contact, the AMPs are released and bacteria in the vicinity of the surface are killed before they can adhere.

An additional method to load AMPs onto the surface of materials from which they then are released and kill bacteria close-by, is to apply hydrogels with incorporated AMPs. This mechanism has been applied by immersion of a dry poly(2-hydroxyethyl methacrylate) or poly(methacrylic acid) (PMAA) hydrogel in solutions containing the desired AMP [48, 49]. Further to hydrogel coatings that release their AMP load, AMPs can also be attached to the surface of, or within, the hydrogel, employing contact killing combined with the anti-adhesiveness that some hydrogels exert [50]. PEG based hydrogels containing AMPs have been prepared by mixing photo-polymerizable epsilon-poly-L-lysine-graft-methacryla- mide with poly(ethylene glycol) diacrylate and dimethyl-acrylamide followed by UV treatment [51]. These hydrogels were attached to fluoroalkyl fumarate copolymer disks by plasma-UV induced surface grafting polymerization; after

argon plasma treatment of the surface the hydrogel precursor solution was cross-linked by UV exposure. These hydrogel modified surfaces subsequently demonstrated 1 to 6-log re-ductions in adhering microorganisms for six different strains (Escherichia coli, P. aeruginosa, Serratia marcescens, S. aureus, Candida albicans, Fusarium solani) demonstrating the potential of these coatings.

Alternatively, surface tethering of AMPs using larger polymer chains, offer the non-adhesive advantage of brush-like structures, while at the same time allowing freedom of movement for AMPs to optimize their efficacy. The in-creased efficacy offered by more mobile adhesion of AMPs has been demonstrated by comparing cathelin LL37 directly coupled to epoxy-silanized titanium surfaces with attachment including a PEG spacer (using -amino- -carboxy-PEG), and is supported by the observation that immobilization of AMPs reduces their activity compared to free soluble pep-tides [52, 53]. However, the efficacy of AMPs depends on the appropriate chain length and the AMP used. For exam-ple, some AMPs require the penetration of bacterial cell walls to function; if a short chain length prevents this the AMP is rendered ineffective.

Shalev et al. used a bio-inspired approach by depositing a polydopamine layer on several kinds of surfaces and subse-quently coupling an ultra-short lipopeptide to the formed polydopamine layer, which resulted in a non-leaching coat-ing of covalently coupled AMPs with high killing efficiency against E. coli and S. aureus [54]. In a study using a similar coating approach, a catechol derivative was used to attach a double amine-functionalized PEG linker to titanium surfaces after which Magainin I, a well-known AMP, was attached. By combining anti-adhesive with antimicrobial properties in this way, reductions in bacterial adhesion of more than 90% were achieved [55].

AMPs offer high antimicrobial efficiency and the wide variety of possibilities to incorporate them into surface coat-ings demonstrates the relative ease by which they can be chemically modified. However, for future applications and surface coating development based on the antimicrobial properties of peptides, it is important to consider the mode of action of the desired coating. Releasing coatings can deplete

Fig. (2). Scheme showing magainin I immobilization on gold by 11-mercaptoundecanoic acid and 6-mercaptohexanol modification (1:3 ratio) of the surface, followed by esterification using NHS/EDC and ultimately coupling of magainin I. Adapted from [43] and reprinted with permission.


rapidly if AMPs are released too quickly, while for surface tethered AMPs the efficacy can largely depend on the chain length of the spacer molecule [56]. Although most AMPs are considered biocompatible, and indeed do not show any direct toxicity to eukaryotic cells, worries are expressed because of their resemblance to some eukaryotic signaling peptides [35] and possible hemolytic effects [57]. This alternative form of toxicity by mimicking host peptides could potentially induce unwanted cell responses and requires additional attention, before the use of AMPs can be considered completely safe.

2.3. Antibacterial Enzymes

The use of enzymes is common in detergents, industrial processes and the food industry. Considered as non-toxic bioactive non-fouling compounds, enzymes are being recog-nized as a valuable source for production of antimicrobial surface coatings [58]. The biocompatibility of enzymes is evident due to the natural source of these agents and pres-ence in the human body. Enzymes serve as catalysts for chemical reactions, increasing the rate and efficiency at which they take place by lowering the activation energy of the reaction. Regarding the adhesion of bacteria, enzymes can either interfere with the adhesion mechanism used by bacteria to adhere to a surface, or they can kill bacteria. Kill-ing is achieved by catalyzing hydrolysis of parts of the pep-tidoglycan cell wall, leading to lysis of the cell. Whilst inter-ference with the adhesion mechanism can be achieved by enzymatic degradation or rearrangement of molecules, or molecular-assemblies, essential for adhesion, e.g. extracellu-lar DNA (eDNA), adhesive proteins or carbohydrates.

Retaining enzymatic activity is a pre-requisite for any ef-fective enzyme surface coatings; however, this can be diffi-cult to achieve. Although most enzymes demonstrate optimal efficacy at physiological conditions, stability beyond these conditions can be limited. Additionally, the conformational structure of an enzyme is of key importance for their activity to ensure optimal accessibility to the active site. Providing flexibility of the enzyme is one way to keep its activity after surface immobilization, as Muszanska et al. demonstrated by using poly-ethylene oxide (PEO) to attach lysozyme to sili-cone rubber [59]. To this end, Pluronic F-127 (PEO99-PPO65-PEO99) was modified to change the PEO hydroxyl end-groups into aldehyde functionalities, which reacted with the amine groups of lysozyme from chicken egg white. The hydrophobic polypropyleneoxide (PPO) backbone of the Pluronic induced the formation of micelles which were ad-sorbed to hydrophobic silicone rubber surfaces, creating a polymer brush with lysozyme functionalities. They showed that lysozyme functionalization of 1% of the Pluronic pre-served the anti-adhesive properties of the brush against Ba-cillus subtilis whilst the lysozyme remained active, based on the increased fraction of dead bacteria. Yuan et al. used PEG and lysozyme in a “grafting from” approach, by dopamine mediated coating of a terminal alkyl halide initiator on stain-less steel surfaces, followed by surface-initiated atom trans-fer radical polymerization (ATRP) of PEG-monomethacrylate, after which lysozyme was coupled to the chain end of PEG branches using 1,1`-carbonyldiimidazole as a bio-functional linker [60]. Because of the broad-spectrum of lysozyme as an antimicrobial, it has been exten-sively used in many more types of surface coatings, includ-

ing layer-by-layer assembly based on electrostatic interac-tions [61-63], immobilization using Fischer carbine complex [64] and in mesoporous release systems [65].

As an important component of extracellular polymeric substances, eDNA was shown to be vital for bacterial adhe-sion as well as biofilm formation in several bacterial strains [66]. Swartjes et al. demonstrated that enzymatic cleavage of eDNA by a functional DNase I surface coating was effective in disrupting the extracellular polymeric substances of bacte-ria, and yielded a reduction in adhering bacteria of 99% for P. aeruginosa and 95% for S. aureus, while 14 h biofilms formed by these strains were reduced to thicknesses of 0.2 and 3 m, respectively [67]. By applying polydopamine as an intermedi-ate coupling layer on polymethylmethacrylate, DNase I was bound by Michael addition reactions, yielding a DNase I coat-ing (see Fig. 3) that retained its ability to degrade DNA for at least 14 h without leakage of active enzyme.

Fig. (3). Formation of polydopamine films on polymethylmethacry-late (PMMA) and attachment of DNase I by Michael addition reac-tion. Reprinted with permission from [67].

A glycoside hydrolase called dispersin B (DspB), which cleaves poly-N-acetylglucosamine polysaccharides, is an-other example of an enzyme known to disturb biofilm forma-tion by specifically attacking an extracellular polymeric sub-stances component necessary for biofilm formation [68]. Pavlukhina et al. showed that a DspB loaded coating was able to reduce Staphylococcus epidermidis surface coverage by 98% [69]. PMAA surface hydrogels on silicon wafers were prepared by depositing bilayers of PMAA/ poly(allylamine hydrochloride) (PAH) which were cross-linked using glutaraldehyde. Incorporation of DspB was per-formed overnight by submersion in a 0.5 mg mL-1 solution and the resulting coating showed complete retention of DspB at a wide pH range. In another study, DspB was “grafted onto” surfaces by using a poly(dimethylaminoethyl methacrylate) with quinone functionalities as a glue layer on top of which five bilayers of PAH together with oxidized dopamine moieties (Pox(mDOPA)) were cross-linked [70]. By applying a top layer of Pox(mDOPA), the surface was then rendered active towards grafting of DspB. Coating of DspB by this method decreased the number of viable S. epi-dermidis bacteria in 24 h old biofilms by 97%.


Several enzymes that are able to interfere with bacterial adhesion have been coated and whether they attack and kill bacteria or whether they target essential parts of the adhesion mechanism, their enzymatic activity can reduce adhesion and proliferation. Essential to the efficacy of surface coating en-zymes is to retain their full, or at least most of their function. The examples highlighted here show that several approaches are possible and enzymes can be used in combination with other anti-adhesive coatings, like polymer brushes, to in-crease the overall effect.

2.4. Nanoparticles

When the size of certain materials reaches the nano-scale, the chemical, electrical, mechanical and optical properties can change completely compared to the bulk material. Nanoparticles (NPs) have been known to possess antibacte-rial properties for quite some time and besides for their ef-fects in solution, NPs have been applied in surface coatings and release systems. Whereas most antibacterial agents, such as antibiotics, are developed for a specific target within bac-teria, e.g. the cell wall or vital components in the cytoplasm, NPs were generally designed for other applications and more or less serendipitously found to have properties which make them suitable against bacterial adhesion and growth. Even though in many cases the exact mechanisms of NP toxicity against bacteria are not fully understood, it is clear that in some cases NPs are able to attach to the bacterial cell wall by electrostatic interactions and disrupt the cell membrane [55, 56]. Another general mechanism of bacterial toxicity by NPs is through the generation of reactive oxygen species which induces oxidative stress by free radical formation [57]. A more extensive overview of the killing mechanism by differ-ent kinds of NPs for several strains of bacteria is presented by Mahmoudi et al. [71].

The bactericidal effect of silver is known for many years and silver-ions and silver-based materials have been used as disinfectants and as an antimicrobial in paints [72]. When considering antibacterial NPs, silver is still most abundantly represented, although other metals are increas-ingly being studied. Direct immobilization of silver NPs (Ag-NPs) to glass can be achieved by modification of glass surfaces with aminopropyl-triethoxysilane and placing it in a colloidal suspension of Ag-NPs afterwards. The survival of S. epidermdis on these Ag-NP modified glass surfaces after 24 h incubation at 37°C was 105 times lower than that on control glass surfaces. Chen et al. have successfully incorporated Ag-NP into layered double hydroxides (LDHs) on titanium plates [73]. The nanoporous Mg-Al LDHs resulting from hydrothermal attachment to Ti were immersed in AgNO3 solution at 100°C, resulting in the formation of Ag-NPs on the LDH covered surface. Trans-mission electron microscopy images revealed that the Ag-NPs were well dispersed on the surface and that the major-ity of the particles was in the range of 5-20 nm. Experi-ments on their antibacterial properties showed 99% reduc-tion in the number of adhering organisms after 3 h of expo-sure to bacterial suspensions of E. coli, P. aeruginosa, S. aureus and B. subtilis, even after 4 runs with the same coat-ing, showing that the coating was stable and retained its antibacterial activity. The high temperature and aggressive way of coating makes it suitable only for metals and other

hard materials. Coating of polymeric materials can require a more delicate approach and is more easily achieved using polymer based coatings. A N-vinylpyrrollidinone and n-butyl methacrylate based hydrophilic surface coating has been described by Stevens et al., who embedded both Ag-NPs and heparin in the coating, designed for application on central venous catheters [74]. The embedded Ag-NPs were found to have a bactericidal effect against several S. aureus strains, even enhanced by the presence of heparin which at the same time improved the non-thrombogenic behavior of the coating. Another way of coating medically relevant polymer materials is given by Huda et al. where coopera-tive electrostatic adsorption was used. In this system, NPs stabilized with SAMs of -functionalized alkane thiols, were given opposite charges using N,N,N-trimethyl(11-mercaptoundecyl)-ammonium chloride (positive charges) and mercaptoundecanoic acid (negative charges) and de-posited on polypropylene and polyvinylchloride. The ad-sorbed NP coatings showed antibacterial effects due to the release of Ag+ ions and were stable for several months [75]. A release-system based coating has been described by Liu et al. who used poly(lactic-co-glycolic) acid (PLGA) as a degradable reservoir for Ag-NPs [76]. Stainless steel was dip-coated by immersing it three times in 17.5% (w/v) PLGA in chloroform containing spherical Ag-NPs of 20-40 nm diameter for 30 s and incubating for 12 h at 37°C. A 2% silver containing PLGA coating not only inhibited growth of S. aureus and P. aeruginosa in vitro, but using a rat femoral canal model they observed no sign of bacterial survival around the coated implant after 8 weeks. In addi-tion, at the same time the coated implants significantly im-proved the generation of bone.

Next to silver, other metal NPs used for antibacterial sur-face coatings include Cu or CuO. Akhavan et al. used a sol-gel procedure to synthesize silica thin films containing cop-per-based NPs on soda lime glass substrates [77]. Depending on the temperature of the subsequent heat treatment step, the films contained either mainly CuO (reduction at 300°C) or mainly Cu (reduction at 600°C) NPs. Bactericidal effects of the coating were tested against E. coli and it showed that use of the CuO-NPs decreased bacterial killing in absolute num-bers, however, when the antibacterial activity was normal-ized by its Cu/Si ratio, heat treatment actually improved the antibacterial activity. They concluded that Cu-NPs were a stronger antibacterial material compared to CuO-NPs, due to increased photo-inactivation of bacteria. Cu-NPs coated cel-lulose films have also been demonstrated to have a bacteri-cidal effect [78]. By dissolving cotton linter in aqueous cu-prammonium and casting it on glass substrates followed by exposure to 10 wt% NaOH aqueous solution for 10 min, Cu/cellulose coatings were produced. Subsequent placement of the film in 0.3 M NaBH4 aqueous solution at 5°C for 5 h resulted in Cu/cellulose nanocomposite films, containing Cu-NPs with a mean size of 47.5 nm, which completely killed S. aureus and E. coli bacteria in suspension within 1 h.

Rai et al. have used the antibiotic cefaclor as both a re-ducing agent for tetrachloroauric(III) acid (HAuCl4) as well as a capping agent for the resulting gold NPs (22-52 nm di-ameter) [79]. Coating on glass surfaces was achieved using PEI, which resulted in extremely stable coatings even at


highly acidic (pH 3) and alkaline (pH 10) conditions. Coat-ings were effective in completely eradicating S. aureus and E. coli from suspension within 6 h and binding of cefaclor to gold NPs lowered the minimum inhibition concentration (MIC) from 50 mg mL-1 to 10 mg mL-1, showing the benefi-cial effect of antibiotic coating to gold NP.

Besides metals, silica NPs have been applied as antibac-terial surface coatings as well. However, since silica NPs do not display any known form of antibacterial activity, they require addition of components offering them such proper-ties. One strategy is to coat silica NPs with a quaternary ammonium cationic surfactant, as was achieved by Botequim et al., who used didodecyldimethylammonium bromide (DDAB) [80]. In their work, silica NPs of either 8 or 80 nm in size were coated with DDAB and subsequently coated to glass coverslips using dopamine hydrochloride as a coupling agent. Coated substrates showed antibacterial activity by completely preventing the adhesion of living cells of C. albi-cans, E. coli, and S. aureus, after 6 h incubation with 1 105, 1 103 and 1 106 cells mL-1, respectively.

This large collection of studies demonstrates the extent of the field and the wide range of methods available to apply these ultra-small particles to fight bacterial adhesion and biofilm formation. However, with new methods also come new restrictions. A point of concern for the use of nanoparti-cles in antibacterial surface coatings is the quick assembly of a layer of adsorbed proteins on the nanoparticle surface, called the protein corona, when exposed to bodily fluids [81-83]. The protein corona can partly obstruct functional mole-cules on the nanoparticle surface and reduce the overall de-sired effect, requiring a higher dose to achieve the same net effect as would be achieved in the absence of these surface associated proteins.

Whether applied on their own, or in combination with other antimicrobial compounds, some NPs display excellent antibacterial properties. Initially, worries were expressed towards the toxicity against human cells and tissue, as for example seen in amino-modified polystyrene nanoparticles [84], but most studies incorporate these concerns into their experimental set-up and seldom find any negative effects on proliferation or adhesion of mammalian cells. Nevertheless, it is important to continue taking this aspect in consideration.

2.5. Quaternary Ammonium Compounds

The general chemical structure of quaternary ammonium compounds (QACs) is represented by R1R2R3R4N

+ X (Fig. 4), in which R depicts a hydrogen atom, an alkyl group or an alkyl group with other functional groups, and X represents an anion [85]. The efficacy of QACs towards killing of bac-teria has turned out to be greatly dependent on whether the positive charge density in a coating exceeds the required threshold of 1015 N+ cm 2 [86, 87] and on the length of the alkyl chain. Generally, when the alkyl chain length falls be-low 4, or above 18, the antimicrobial effects are almost com-pletely diminished [85, 88]. The chain length dependence of the efficacy towards the antibacterial properties is related to the mechanism by which QACs inhibit or kill bacterial cells in solution, but it is uncertain whether this mechanism also prevails for QACs immobilized on a surface. Generally it is assumed that the positively charged quaternary nitrogen of a

QAC molecule is strongly attracted to the negative cell wall of bacteria interacting with negatively charged phospholipid head groups. Once the QAC molecule becomes associated with the cell wall, the hydrophobic alkyl tail of the QAC becomes incorporated into the hydrophobic bacterial cell membrane. When the concentration of QACs in the cell membrane becomes high enough, this causes disruption of the cell membrane with subsequent leakage of the of the bac-terial cytosol, resulting in lysis of the cell [85].

N

R1

R2

R3

R4

X

Fig. (4). The general structure of a quaternary ammonium ion. R can represent a hydrogen atom or an alkyl group that can be substi-tuted with other functional groups and X represents the anion.

Since the antimicrobial activity of QACs is mainly ex-pressed by incorporation into the bacterial cell membrane, QAC surface coatings require a certain degree of freedom for the molecule, similar to antibiotics and enzymes as previ-ously discussed. Polymer mediated surface tethering is one way to achieve sufficient flexibility for QACs to retain their antimicrobial properties. Hyperbranched polyurea coatings have shown to be an effective way of tethering PEI to silicon substrates, after which amino groups of the PEI coating could be converted into hydrophobic, poly-cationic species by a consecutive two-step alkylation process [89]. Fabricated coatings were more hydrophobic than the underlying silicon and showed to have a charge density of 1015 N+ cm 2, above the required threshold positive charge density, and killed adhering S. epidermidis up to challenge numbers of 1600 CFU cm-2. Importantly, whereas the majority of papers de-scribing contact-killing of adhering bacteria neglect to dem-onstrate absence of leachables that may contribute to bacte-rial killing, killing by the above described hyperbranched coating was confirmed to be in the absence of leachables. Due to the hyperbranched nature of the coating, QACs do not only have more spatial flexibility, but also allow for mul-tiple contact points to develop between an adhering bacte-rium and the coating Accordingly, Asri et al. strengthened the current perception that electrostatic attractions, strong enough to extract anionic lipids from the bacterial cell mem-brane with subsequent leakage of the bacterial cytosol, play a major role in the working mechanism for immobilized QACs in a coating [89, 90]. Moreover, this also explains why bacte-rial strains, not susceptible to QACs in solution, are contact-killed by immobilized QACs as it provides for an entirely different working mechanism than operative in solution [91].

Wong et al. described a method of coating antibacterial thin films assembled from layer by layer application of poly-cationic N-alkylated PEIs and polyanions on silicon sub-strates [92]. Layer by layer films were built up from alternat-ing polycationic PEIs with polyanions, using three different PEI-based polycations and varying the number of bi-layers in the films. Focusing on their result with linear DMLPEI as the polycation component and PAA as the polyanion, they found that the bactericidal activity was dependent on the


number of bilayers and influenced by the pH of the PAA solution at the time of layer formation. At low pH the PAA remained relatively uncharged, which resulted in a bilayer with fewer interaction points between DMLPEI and PAA, thereby creating a bilayer with more loops and a rougher surface, displaying a higher number of cations available for interaction with bacteria (see Fig. 5). These systems proved to be more bactericidal, only requiring 1.5 bilayers for com-plete killing of airborne S. aureus, compared to 14.5 bilayers being required for the same effect when PAA with a pH of 5 was used to create bilayers. Additionally, the authors ob-served that deposition of only DMLPEI on negatively charged Si wafers lacked any antibacterial activity, confirm-ing the thought that tight binding of the positive surface charges is detrimental for the antibacterial activity of these QACs. Similar results were found in sort-like multilayer system using different QACs [93].

Fig. (5). Schematic representation of the different conformations of polymer chains resulting from PAA at different pH values. At pH 3.0, most of the PAA chains (blue) are uncharged, which results in a conformation of the DMLPEI cation (red) with most of its posi-tive charges available, leading to a high bactericidal effect. As the pH increases the PAA chains become more negatively charged, crosslinking with more of the positive charges of DMLPEI leaving less cations available for interaction with the bacterial cell mem-brane and hence less bactericidal activity. Adapted from [92] and reprinted with permission.

An alternative method to ensure availability of the posi-tive charges of QACs to keep their antimicrobial activity is by covalent immobilization on glass using a short linker molecule. Recently, Iarikov et al. functionalized glass sur-faces with epoxide groups using 3-glycidoxypropyltrimethoxysilane (GOPTS) [94]. Modified glass surfaces were then exposed to poly-allylamine (PA) so that the PA could bind via reaction of a part of its amine groups. Cova-lently bound PA showed to have more extended chains com-pared to electrostatically adsorbed PA, while chain length also increased with increased GOPTS reaction time and lower molecular weight. Accordingly, glass surfaces modi-fied with PA resulting in the most extended chains showed the highest overall killing of S. epidermidis, S.aureus and P. aeruginosa, showing reductions in adhered bacteria of 97%, 97% and 88%, respectively.

Siedenbiedel et al. developed an antimicrobial coating of a quaternized amphiphilic star block copolymer with a semi-permanent character [95]. By creating a hydrophilic antimi-

crobially active outershell, together with a hydrophobic core, micellar structures assembled in water and lead to differently structured antimicrobial coatings being developed on the surfaces after drying. The star-shaped structures made from a polystyrene core and poly(4-vinyl-N-methylpyridinium) outer shell were, when the polymers were applied in the right proportions, capable of being coated on a surface from water and showed antimicrobial activity against S. aureus. The antimicrobial activity was maintained even after rinsing, while deliberately streaming water over the surface for longer periods of time, removed the coating so that the un-modified surface was recovered, showing the non-permanent nature of their coating.

Although quaternary compounds were first used in solu-tions as disinfectants, due to their high stability they are cur-rently mainly studied for use in permanent surface coatings. The high stability contact-killing mechanism causes bacteria to disintegrate leaving the coating intact and capable of pro-tecting the surface against more bacteria. The absence of decreased efficacy by shielding of the positive charge of QACs due to adhered bacterial debris or protein adsorption is shown in in vivo studies, in which QAC coatings remained effective in preventing infection for multiple days and even induced bone healing [96, 97]. The advantages of permanent surface binding of QACs make that there are only few stud-ies on the report of QAC releasing coatings, especially since such a strategy bears the risk of QAC-induced hemolysis [98].

2.6. Polymers in Passive Coatings

PEG is often considered the gold standard for polymer brush surface modification, designed to resist fouling of the surface by many different substances [99]. By forming an osmotically driven, steric barrier to which bacteria cannot adhere, PEG modification is an example of a polymer which passively protects the surface from bacterial adhesion [100]. The low adhesion forces between bacteria and polymer brushes are believed to cause them to keep their planktonic phenotype, missing the stimulation to develop into a biofilm [101]. The passive mechanism by which PEG prevents adhe-sion of bacteria is typical for how polymer coatings protect surfaces. On their own, most polymers do not possess any antibacterial activity and hence their only way to stop bacte-rial colonization is by passive prevention of adhesion. Poly-mers can, however, be very effective in preventing bacteria to adhere, and this is why there have been many examples in which polymer surface coatings have been combined with the use of antimicrobials, combining the non-adhesiveness of the polymer brush with the killing efficacy of an antimicro-bial to improve the overall result. The possible antimicrobi-als that can be combined with polymer surface coatings have been mentioned before and include, but are not limited to, QACs, peptides and antibiotics. Surface coatings consisting of these combinations have been discussed separately in the above sections and therefore this section will mainly focus on polymer surface coatings that passively prevent bacterial adhesion.

To keep bacteria from adhering to a polymer coated sur-face, the attached polymer layer has to be in a well hydrated state, which is generally achieved by covalent immobiliza-


tion or physisorption of hydrophilic polymer chains to the surface. This strategy has been used for many years and the latest developments are driven towards the novel application of known polymers rather than the design and use of new chemicals. The application of bio-inspired attachment of polymer chains is recent, and dopamine molecules which are found to be important in the strong adhesion of marine-mussels, or derivatives of dopamine, are often applied to this end. Polydopamine, formed by coating a surface with dopa-mine, can be directly functionalized with polymer brushes, or can be used to attach an ATRP initiator for a grafting-from approach [102, 103]. Amine terminated PEO was grafted to a thin layer of polydopamine by Pop-Georgievski et al. by dip-coating of dopamine coated samples into PEO solution [103]. The resulting brush coatings were shown to be stable for multiple days, based on their ability to resist protein adsorption. ATRP formation of brushes showed anti-adhesive properties, but inclusion of a quaternized group was necessary for the desired antibacterial properties. The re-quirement for the inclusion of these antimicrobial groups, which as mentioned previously is often performed, depicts the consensus that for most applications anti-adhesiveness is not sufficient to prevent infection. This is supported by the fact that when polymer brushes are subjected to bacteria in growth media, even without firm attachment of initially ad-hering bacteria, a biofilm may still form [11].

The hydrated state of polymer brushes is vital to the anti-adhesive capacity of these coatings. To further hydrate a coating, crosslinks can be formed between the polymer chains on a surface to create a hydrogel which can hold more water than a brush-structure without collapsing and thus can increase the anti-adhesive properties of a surface. Wang et al. cross-linked PEG using an electron beam and created micro-patterned surfaces of PEG hydrogels separated at dif-ferent distances [50]. When the micron-sized hydrogel spots where separated at distances of 0.5 μm, bacteria could not adhere between the structures thus preventing bacterial adhe-sion. By slightly increasing the space between hydrogels anti-adhesive properties were still observed, while at the same time tissue cells where able to adhere to the surface due to their larger size with respect to bacteria. Alternatively, end-functionalization of PEG with dopamine molecules leads to crosslinking between PEG molecules and results in hydrogel formation. This strategy was demonstrated to de-crease bacterial adhesion by 80%; however, this was not as effective as most other PEG modifications [104]. Zhao et al. cross-linked poly(N-hydroxyethylacrylamide) and loaded the resulting hydrogel with salicylate. Using this method the authors created hydrogels exhibiting both anti-adhesive properties as a result of hydrogel formation as well as anti-bacterial properties, attributed to the release of salicylate [105]. The combination of anti-adhesive and antibacterial properties resulted in the ability to withstand bacterial adhe-sion of S. epidermidis and E. coli for over 24 h.

Polymer attachment to surfaces is effective in reducing bacterial adhesion, but due to the passive nature cannot pre-vent biofilm formation over longer periods of time. How-ever, in many temporary applications the non-adhesive na-ture of polymer surface modifications may be adequate to prolong the lifespan of a device or implant, e.g. of urinary or intra-vascular catheters. The weak adhesion forces of bacte-

ria on polymer brushes and the occurrence of fluid induced shear forces in these situations provide physical removal of bacteria not found under static fluid conditions.

2.7. Super-hydrophobicity

Hydrophobic interactions play a role in the adhesion of bacteria to surfaces, by favoring the attraction of two hydro-phobic components to remove interfacial water and lower the free energy of a particular system [106, 107]. However, ex-tremely hydrophobic surfaces have been shown to possess extraordinary anti-adhesive properties [108].

The Aizenberg group recently published a paper in which they added lubricating fluids, consisting of perfluorinated liquids, to porous polytetrafluorethylene (PTFE) to fabricate liquid-infused surfaces [109]. These surfaces were shown to be extremely resistant to bacterial adhesion and biofilm for-mation. Biofilm attachment of P. aeruginosa after 7 days was effectively zero, showing excellent anti-adhesive proper-ties and stability of the coating. In another study from the same group, it was shown that a nanostructured surface based on an epoxy-resin could be infused with the same per-fluorinated liquids to obtain a similar anti-adhesiveness and even demonstrating self-repairing behavior after physical damage [110]. Li and co-workers have also used a liquid infusion technique to create slippery, bacterial adhesion re-sistant surfaces [111]. By preparing a porous polymer sur-face of a mixture of butyl methacrylate and ethylenedi-methacrylate on glass and the subsequent addition of per-fluoropolyether, slippery surfaces were made that resisted biofilm formation of most P. aeruginosa strains included in their study. One of the used multi-resistant strains however, was still able to form a biofilm, suggesting that the results were strain dependent.

Privett et al. prepared super-hydrophobic surfaces by de-positing fluorinated silica colloids onto glass slides [112]. Briefly, (heptadecafluoro-1,1,2,2-tetrahydrodecyl)trimethoxy-silane and tetraethylorthosilicate were sonicated and added to a solution of ethanol and ammonium-hydroxide to form sil-ica colloids, which were spread-cast onto ozone/ultraviolet (UV)-treated glass slides. Static water contact angles on the coated surfaces exceeded 150 degrees and showed an over 1.75 log reduction in adhesion of S. aureus and P. aerugi-nosa. Water contact angles did not change after immersing the coating in water for over 15 days, showing good stability of the coating, although no bacterial adhesion experiments were performed after storage.

Whereas the previously mentioned studies all require fluorinated substances to achieve super-hydrophobicity, Hu et al. described an electro-spraying method to apply a super-hydrophobic biodegradable coating, without the use of such liquids [113]. In their work they present how co-electro-spraying of poly(L-lactide) and modified silica NPs onto titanium plates resulted in a coating with a water contact angle of 157°. Bacterial adhesion was reduced by 75% com-pared to poly(L-lactide) films, however, no numbers for bare titanium plates were reported. Although, like other super-hydrophobic coatings, the adhesion of mammalian cells was reduced as well, the biodegradable nature or the coating could still allow for tissue integration of an implant after degradation of the coating, thereby showing a novel feature


to make super-hydrophobic surfaces more applicable to medical implants.

The main disadvantage of super-hydrophobic coatings is that they not only restrict the adhesion of bacteria but of mammalian cells as well, which means they cannot be used in applications requiring tissue ingrowth, although the previ-ously mentioned study by Hu et al. showed that tissue inte-grating variants could be made as well, by making the coat-ing biodegradable [113]. However, even without allowing attachment of mammalian cells, there are many possible ap-plications for which super-hydrophobic surfaces are suitable and could reduce the infection rate. Especially in the pres-ence of flowing liquids, e.g. in catheter, the non-adhesiveness would promote clearance of unwanted con-taminants.

2.8. Chitosan

The use of naturally derived components is an important current theme in surface coating of materials [114-116]. Ex-amples of such materials include, hyaluronic acid, alginate, collagen, chitosan and dextran. Chitosan however, is the only one among these materials possessing an inherent antibacte-rial activity, albeit that this antibacterial activity depends on the degree of chitosan acetylation [117, 118]. Yang et al. used a biomimetic anchor and chitosan functionalized poly-mer brushes to prevent bacterial adhesion on stainless steel surfaces [117]. Barnacle cement, harvested from live barna-cles, was used to attach an ATRP initiator for formation of surface initiated PHEMA polymer brushes. Subsequently, the hydroxyl groups of the PHEMA brushes were converted into carboxyl groups that were allowed to react with the amine groups of chitosan, to achieve chitosan functionalized polymer brush surfaces. The viability of E. coli that managed to adhere on the chitosan modified polymer brush coated stainless steel was 80% lower compared to bare stainless steel, due to the bactericidal effect of chitosan. Surface com-position of the coated surfaces after 30 days showed a less than 10% loss of the barnacle cement, indicating good stabil-ity, however, the authors did not test for bacterial adhesion after this time period. Combining the anti-adhesive nature of polymers with the antibacterial properties of chitosan was achieved by Wang et al. by using a multilayer system in which the base layer consisted of a heparin/chitosan film held together by electrostatic interactions [119]. On top of the heparin/chitosan base layer a (polyvinylpyrrolidone/ poly(acrylic acid)) (PVP/PAA) layer was added by alternate deposition of PVP and PAA, after which the top layer was then cross-linked using heat treatment. The final coating was initially anti-adhesive, but demonstrated contact killing of S. aureus after the anti-adhesive top-layer had degraded after 24 h in phosphate buffered saline, exposing the bactericidal heparin/chitosan base layer. Another study demonstrated that a heparin/chitosan multilayer had antibacterial functionality and at the same time served as an osteo-inductive coating, offering a good prospective to improve the outcome of bone allograft procedures [120]. Chitosan could also be incorpo-rated into hydroxyapatite, resulting in good antibacterial properties of the coating against S. aureus, while at the same time the porous character of the hydroxyapatite enhanced osteoblast cell response, as long as chitosan concentrations remained below cytotoxic values [121].

Although chitosan already possesses antibacterial proper-ties by itself, many studies have been performed on how to improve the bactericidal effect by the use of additional anti-bacterial compounds. Since QACs have strong bactericidal capabilities, chitosan has been quaternized in several studies to increase the antibacterial properties [122-124]. Lee and co-workers studied quaternary ammonium modified chitosan brush layers [125]. Modification of chitosan by performing a Michael reaction with an acryl reagent in water, proved to be effective in introducing quaternary ammonium groups. To evaluate the effect of QAC modification, 25% and 50% QAC substitution was tested while chitosan only brushes were used as control. To immobilize the resulting chitosan and chitosan-QAC complex (CH-Q), silicon oxide surfaces were treated with GOPTS and an aqueous solution of CH (or CH-Q) was added and allowed to evaporate slowly. After evaporation, the film was left at 60°C for 12 h and the result-ing films expressed a pH dependent swelling behavior which allowed fine tuning of the film thickness. Brush layers de-creased in thickness with increasing quaternization, whereas the antimicrobial activity increased. The 50% CH-Q coating showed antibacterial activity against S. aureus, decreasing the amount of CFUs after 6 h exposure by 97% compared to uncoated control surfaces. Later studies confirmed these re-sults and showed that CH-Q coatings effectively prevented bacterial adhesion in flow conditions as well [126]. Ding et al. showed that addition of alkynyl groups to chitosan led to increased antibacterial activity of hydrogel coatings against S. aureus and E. coli [127].

Even though chitosan possesses a limited bactericidal ef-fect, its biocompatibility, along with the ease by which it can be modified, makes it a popular building block for many antibacterial surface coatings. Little is known about the pos-sible development of bacterial resistance against chitosan and it remains to be seen whether or not this occurs upon its increasing use.

3. CONCLUSIONS

Antimicrobial coatings are of ubiquitous importance, but requirements set to such coatings are most stringent in bio-medical applications, constituting the focus of this review. Prevention of bacterial adhesion and killing of bacteria, ei-ther by coatings that release antibacterial substances or through surface-associated mechanisms, are the most preva-lent approaches. A trend towards developing multi-functional coatings is becoming more apparent. Approaches based on the release of substances bear the risk of a depleted coating when needed most. Surface-associated mechanisms may suffer from attenuated efficacy due to coverage by pro-teins adsorbing from body fluids, but hitherto QACs coatings have been demonstrated to remain antimicrobially active in animal studies. From a general perspective it is impossible to tell which coating strategy will yield the best options, since this all depends on the clinical application aimed for and whether expectations are short- or long term. However, tak-ing into consideration that the era of antibiotics to control infectious biofilms will eventually come to an end, it be-comes evident that the future for biofilm control on biomate-rial implants and devices is with surface-associated modifi-cation of surfaces that are non-antibiotic related.


CONFLICT OF INTEREST

The author(s) confirm that this article content has no con-flicts of interest.

ACKNOWLEDGEMENTS

This study was funded by the UMCG, Groningen, The Netherlands. H.J. Busscher is also director of a consulting company, SASA BV (GN Schutterlaan 4, 9797 PC Thesinge, The Netherlands). Opinions and assertions contained herein are those of the authors and are not construed as necessarily representing views of the funding organization or their re-spective employers.

ABBREVIATIONS

AMP = Antimicrobial peptide

ATRP = Atom transfer radical polymerization

CaP = Calcium phosphate

CH-Q = Chitosan-QAC complex

DDAB = Didodecyldimethylammonium bromide

DMLPEI = Dodecyl,methyl-poly(ethyleneimine)

DOPA = Dopamine

DspB = Dispersin B

GOPTS = 3-Glycidoxypropyltrimethoxysilane

GS = Gentamicin sulfate

HAP = Hydroxyapatite

LDH = Layer double hydroxide

MUA = Mercaptoundecenoic acid

NP = Nanoparticle

PAA = Poly(acrylic acid)

PAH = Poly(allylamine hydrochloride)

PEG = Poly(ethylene glycol)

PEI = Poly(ethyleneimine)

PEO = poly(ethylene oxide)

pHEMA = Poly(2-hydroxyethyl methacrylate)

PLGA = Poly(lactic-co-glycolic) acid

PMAA = Poly(methacrylic acid)

PMMA = Polymethymethacrylate

PPO = Poly(propylene oxide)

PVP = Polyvinylpyrrolidone QAC = Quaternary ammonium compound

SAM = Self-assembled monolayer

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Received: August 28, 2014 Revised: August 29, 2014 Accepted: September 08, 2014

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Current Developments in Antimicrobial Surface Coatings for Biomedical Applications