Antibacterial Polymers: a Mechanistic Overview

Antibacterial Polymers: A Mechanistic Overview

Ao Chen,1,3 Hui Peng,1,3Idriss Blakey,1,2 Andrew K. Whittaker1,2,3*

1Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane Qld

4072

2Centre for Advanced Imaging, The University of Queensland, Brisbane Qld 4072

3ARC Centre of Excellence in Convergent Bio-Nanoscience and Technology

Bacterial fouling on surfaces is considered a major problem in modern society. Conventional methods to prevent biofilm formation often have little effect and may induce further contamination. In response to this challenge, antibacterial polymers have been designed and applied as an alternative approach to kill or inhibit bacteria and prevent the formation of biofilm. These polymers can be grouped into three broad classes, namely antibiotic-releasing polymers, polymeric antibiotics and antibiotic polymers. Antibiotic polymers are effective against bacteria both in solution and as coatings, through different mechanisms of action. In order to enhance their efficacy, antibacterial polymers have been designed with single or combined antibacterial and antibiofouling actions. The current review summarizes the mechanisms of action of antibacterial polymers, especially antibiotic polymers, both in solution and as coatings. It also discusses the antibacterial polymers with multiple functionalities.

KEYWORDS:

Antibacterial polymers, biofilms, mechanisms of action, testing

1. Introduction

Microbial fouling on the surface of materials is one of the major causes of poor hygiene. Such fouling appears in contaminated food storage, public facilities, household decorations, etc., and especially in hospitals and other medical environments. Nosocomial (hospital-based) contaminations have been considered as the major pathway of the spreads of bacterial and viral diseases1,2 and suggested to be historically responsible for several major disease outbreaks.3,4 The process of surface contamination leads to “biofilm formation”, during which a matrix of extracellular polymeric substances is excreted by the attached bacteria with the result that their colonies are protected from disinfectants. Biofilms can amplify the persistence of nosocomial pathogens,5 surpassing weeks or even months on inanimate surfaces.6 In brief, the steps in biofilm formation include initial attachment, permanent attachment and accumulation, film maturation and nourishment, and dispersion.7 It should be emphasized that the process of biofilm formation is irreversible after the initial attachment of microbes, during which large and continuous doses of disinfectants and/or antibiotics are required to eradicate the biofilm and prevent its repeated formation.8 Common disinfectants exploited include hypochlorite, reactive oxygen species (e.g. hydrogen peroxide), triclosan, silver salts, quaternary ammonium salts and alcohols.9 Advanced antibiotics have also been utilized.10 However, the presence of the biofilm significantly reduces the effects of disinfectants below lethal levels.7,11 On the other hand, continuous applications of small-molecule disinfectants or antibiotics at sub-lethal doses may induce enhanced biofilm formation12-14 and may potentially give rise to multi-resistant bacterial species.9,15,16 Hence, there is a pressing need to develop new antibacterial substances/materials to address this crucial problem.

Recent advances in the field of polymer synthesis have greatly expanded the potential reach of polymers.

The discovery of living/controlled polymerizations has enabled scientists to produce polymers with

controlled molecular weight, tailored properties and appropriate functionality for specific applications.17

Furthermore, with improvements in instrumental methods, such as NMR, fluorescence, AFM, X-ray and neutron diffraction, characterization of polymers has significantly advanced, providing clearer insight into their behavior under different situations.

The combination of polymer science and the requirement for long-lasting sterilization has given rise to antibacterial polymers, which kill or inhibit the bacteria when they approach or are in contact. The advantages of antibacterial polymers are self-evident.12,13 Compared to small-molecule disinfectants, antibacterial polymers can usually exert long-lasting resistance against bacteria with a high concentration of active components and physical/chemical stability.18 Furthermore, antibacterial polymers also possess longer shelf- life, lower toxicity toward normal tissue and lower probability of inducing drug-resistant bacterial species.19

Compared to advanced antibiotics, such as antimicrobial peptides, the cost of antibacterial polymers is significantly lower due to the use of mature methods of synthesis and processing. Thus antibacterial polymers have become recognized as a very promising class of antibacterial materials.

The past decade has witnessed rapid development of the field of antibacterial polymers, as summarized in a number of excellent reviews. In 2007, Kenawy et al. summarized the basic requirements of antibacterial polymers in a review focused on the factors that affect antibiotic activity of such materials (molecular weight, counter-ions and alkylation), the different methods of preparation, and the methods of application of antibacterial polymers.18 In the same year, Gabriel et al. surveyed the field of antibacterial polymers in which activity is determined by their amphiphilicity nature.20 Their review concentrated on host defense peptides and their synthetic mimics; finally this paper made a valuable contribution by discussing physicochemical

methods of analysis of interactions with cells. Munoz-Bonilla and Fernández-García provided in 2012 a systematic review on the different synthetic polymers with antibacterial properties, including design, synthesis and antimicrobial testing.21 More detailed discussions of the mechanism of action of antibacterial polymers were provided by Timofeeva and Kleshcheva, and Siedenbiedel and Tiller. Timofeeva and

Kleshcheva summarized the mechanisms of action of antibacterial polymers and their coatings, and the factors that influence the behavior and toxicity of the materials, including molecular weight, alkylation and structure of the amine groups.22 The review by Siedenbiedel and Tiller categorized antibacterial polymers and “contact active” coatings based on the functional mechanisms and summarized research on antibacterial polymers with multiple properties.23 Jain et al. provided an overview of the different classes of antibacterial polymers, in which the effects of molecular weight and alkyl chain length, charge density, hydrophilicity, counter-ions, and pH were briefly discussed.24 Recently, Ganewatta and Tang comprehensively surveyed the design of different types of membrane-active antibacterial polymers (antibiotic polymers) through the adjustment of structural parameters, especially the amphiphilic balance, molecular weight and the selection of cationic groups to acquire optimal antibacterial activity and biocompatibility.25 Assemblies of antibacterial polymers were also discussed. However, according to our knowledge, previous reviews, especially in early reviews, the surveys on the mechanism of action of antibacterial polymers were often incomprehensive, sometimes with obscure classification in the concept of different antibacterial polymers. In the meantime, no survey has been conducted upon the mechanisms of antibiotic polymers immobilized as coatings since

Timofeeva and Siedenbiedel, during which new results have provided deeper understanding in the mode of action of antibiotic polymer coatings.

As a result, in this review, we focus on the discussion of the mechanism of action of antibacterial polymers

and how this is related to structure elaborately and concisely. We will first clearly give out the classification of antibacterial polymers to define and differentiate the concepts, with brief introduction to respective techniques of preparation based on recent developments. Focusing on antibiotic polymers, its current understanding of the mechanism of action in solution will be comprehensively discussed in relation to the effects of different structural parameters. The properties of antibiotic polymer coatings are then presented by refining latest and representative studies into three models. Finally, the design for antibacterial polymers with multiple functionalities will be elaborated.

2. Antibacterial Polymers in Solution: Mechanisms and Key Structural Factors

2.1 Classification of Antibacterial Polymers

To date, three main concepts have been used in the design of effective antibacterial polymers: a) antibiotic- releasing polymers, b) polymeric antibiotics and c) antibiotic polymers.23 Antibiotic-releasing polymers carry antibiotics through physically- or chemically-reversible interactions, which can be released to a desired site to reach relatively high local concentrations in a controlled manner. Polymeric antibiotics are polymers that carry small-molecule antibiotics as pendant groups or repeat units which act in the same manner as their small-molecule antibacterial analogues. Antibiotic polymers usually possess the structure of a facially amphiphilic polycation and have antibacterial activities derived from the whole molecule (see Figure 1 for a schematic illustration of the three classes). However, since the mechanisms of action of antibiotic-releasing polymers and polymeric antibiotics are largely due to the properties of small molecule antibiotics, this review will concentrate on the development and properties of antibiotic polymers, and provide only a brief descriptions of the other classes of antibacterial polymers.

Figure 1: Schematic illustration of the three main classes of antibacterial polymers: a) antibiotic-releasing polymers; b) polymeric antibiotics; c) antibiotic polymers.23 Adapted from reference 23. Copyright (2012) by the authors, open access licensed by MDPI.

2.1.1 Antibiotic-releasing Polymers

Antibiotic-releasing polymers share an analogous design to certain drug carriers. Common antibacterial molecules or ions are loaded onto the polymer backbone through a range of different interactions and are then released to the target site with some degree of control. Antibiotics can be loaded into the polymers through either physical interactions or chemical bonding.

The physical interactions which are exploited for loading include simple mixing and specific physical interactions. Antibiotic metal ions or nanoparticles, such as silver,26,27 copper,28,29 gold30 and zinc,31,32 can form nanocomposites with polymers through incorporation into polymer matrices. Different loading approaches have been adopted, such as cold spraying,33 deposition from the active gas phase,34 etc. For

example, Bogdanović and colleagues prepared copper-polyaniline (Cu/PANI) nanocomposites by redox

35 2+ polymerization of aniline using CuCl2. During the reaction, the copper ions (Cu ) were reduced into copper nuclei (Cu) and encapsulated into the polyaniline matrix. The copper nanoparticles are gradually oxidized to

Cu2+ or Cu+ during application and released to exert antibacterial effects. Organic disinfectants can also be incorporated into polymers through a similar method. Muriel-Galet and colleagues prepared an antibacterial ethylene–vinyl alcohol copolymer (EVOH) through directly dissolving green tea extract (GTE) and oregano essential oil (OEO) into the polymer solution, spreading the solution and evaporating the solvent using hot air.36 Another typical approach is the formation of core-shell structures in emulsions, as reported by Pérez-

Liminana et al., who encapsulated tea tree oil into gelatine/carboxymethylcellulose microcapsules through a two-step emulsification-hardening process and applied the capsules to antibacterial textiles.37 A simple method of loading was exploited by Fisher et al., who prepared an antibacterial silicone catheter through soaking the silicone in solution of various antibacterial agents to allow it absorb the antimicrobial components.38 After drying, the catheter was endowed with long-term (over 100 days) broad spectrum antibacterial activity. The specific physical interaction mainly involved the electrostatic interactions between organic disinfectants and functional groups on the polymers, where the ionized antibiotics and polymer pendant groups become paired. An example was demonstrated by Mi et al., who loaded the conjugate base of salicylic acid by exploiting interactions with quaternary ammonium groups of poly(N,N-dimethyl-N-

(ethylcarbonylmethyl)-N-(2-(methacryloyloxy)-ethyl)ammonium salicylate) to form a non-fouling hydrogel.39

The range of chemical bonds applied for the attachment of antibacterial agents can be divided into the following: metal-ion coordination bonds,40-42 nitrogen-halogen bond (N-halamine) and hydrolyzable

functional groups such as esters43,44 and amides.45,46 Common antibiotics, including penicillin,47

Norfloxacin,48 and salicylic acid,49 have been attached to polymers through hydrolyzable functional groups and these antibiotic-releasing polymers exhibited strong antibacterial effects. Duong et al. reported the delivery of nitric oxide by poly(oligoethylene glycol methyl ether methacrylate)-b-poly(vinyl benzyl chloride)

(POEGA-b-PVBC) and poly(oligoethylene methoxy acrylate)-b-poly(2-vinyl-4,4-dimethyl-5-oxazolone monomer) (POEGA-b-PVDM) star polymer.50 Nitric oxide was attached through forming N- diazeniumdiolates (NONOates) with the amine groups on the PVBC or PVDM blocks and slowly released at pH 6.8 to effectively inhibit the formation of biofilm of P. aeruginosa. One of the most effective antibiotic- releasing polymers are polymers containing N-halamine, which released positively charged halides such as

Cl+, Br+ or I+, to kill neighboring bacteria by initiating oxidation of phospholipids in the cell membranes.

Since the discovery of N-halamine molecules by Kosugi et al.,51 the concept was further developed by several researchers such as Williams et al.,52 including attachment of the N-halamine to the polymer backbone by

Sun et al.53 The N-halamines have advantages of broad spectrum killing, long stability and can be restored after depletion by simply reacting with positive halides donors.54 Recent research has focused on the attachment of N-halamine groups onto specific surfaces and their processing into different forms, e.g. fibers

(both synthetic55 and natural56,57), mats58 and nanoparticles.59

2.1.2 Polymeric Antibiotics

As an inherent result of their release mechanism, the antibacterial activity of antibiotic-releasing polymers finally becomes depleted. The design and preparation of polymeric antibiotics aims to solve this problem through irreversible immobilization of antibacterial molecules.

Generally, polymeric antibiotics are prepared through polymerizing monomers with antibacterial agents as functional groups and are expected to behave similarly to these small-molecule counterparts. Dombrosk et al. synthesized a series of polymeric antibiotics with sulfonamides by condensation polymerization with the comonomer dimethylolurea.60 Even though the precise structures of the polymers were unclear, these polymers were shown to be stable under strong hydrolysis conditions and exhibited comparable antibacterial activity compared to the sulfonamide monomers at the same dosage.61 The authors also investigated the effects of different comonomers, dimethylolurea and formaldehyde, on the antibacterial activity of sulfonamide copolymers. It was found that the copolymers containing dimethylolurea had superior performance compared with those copolymerized with formaldehyde under identical testing conditions, indicating that the antibacterial action depends on comonomers in addition to the content of sulfonamide.62

Poly(4-vinylphenol), which carries phenol groups effective against bacterial membranes, are also potential polymeric antibiotics after being modified by sulfonation or processed through electrospinning.63 However, not all polymeric antibiotics exhibit strong antibacterial performance compared to their small molecule counterparts. One possible reason for this is that the polymerization reactions may interfere with the mechanisms of action of antibacterial functional groups, as was reported by Lawson et al., who prepared a series of polymerizable vancomycin derivatives through modifications toward different sites on vancomycin molecules.64 The authors found that the antibiotic activity of the polymers was significantly inhibited compared to that of the monomer, which was caused by the following: a) the site of modification of vancomycin molecules during monomer synthesis hinders the hydrogen bonds of the binding positions; b) the site of modification prevents the formation of intermolecular complexes (dimers) crucial to antibacterial activity; and c) the high mobility of long poly(ethylene glycol) (PEG) spacers might result in unfavorable conformations that interfere with the binding of vancomycin. Arimoto and colleagues also reported the

antibiotic behavior of vancomycin-containing polymers prepared through ring opening metathesis polymerization.65 A differentiation in the potency was observed, with the effect greatly enhanced against vancomycin-resistanct Enterococi but retarded efficacy against S. aureus and E. faecalis. It seems that the structural parameters of the polymer as well as bacterial species all play important role in the behavior of polymeric antibiotics.

A new class of polymeric antibiotics has been designed by immobilizing photo-sensitizers as the antibiotics.66

Photo-sensitizers generally possess conjugate chemical structures, which contain delocalized electrons with high density. The delocalized electrons are excited after being stimulated by light and return to ground energy level to emit energy. The radiation is able to catalyze intermolecular reactions of oxygen to form active singlet

1 molecular oxygen O2, which has a high antibiotic efficiency. Many photosensitive antibacterial polymers have been synthesized by attaching antibacterial photosensitizers to the polymer backbone, for example, porphyrin (e.g. protoporphyrin IX and zinc protoporphyrin IX),67 phenothiazine (e.g. methylene blue and toluidine blue O),68,69 and other photo-responsive dyes (e.g. gentian violet and brilliant green).66

2.1.3 Antibiotic Polymers

Due to their similarity in structure, antibiotic polymers were initially considered as a member of the class of polymeric antibiotics with immobilized quaternary ammonium salts. Such salts are widely utilized as disinfectants targeting bacterial membranes.70 However, later developments and research promoted the concept of antibiotic polymers by mimicking the physicochemical properties of natural host defense peptides,71-73 and thus underlined the fundamental property of antibiotic polymers that their antibacterial activity derives from the whole structure, instead of solely from the attached cationic groups (see Section

2.3).20 Similar to its mimicking objective, antibiotic polymers generally possess the structure of facially amphiphilic surfactants, so as to interact with the negatively charged phospholipid bilayer of the cell membrane.22,74 The hydrophilic parts are typically cations, which are able to anchor the polymer to the anionic membrane, while the hydrophobic parts are alkyl chains capable of fusing or aligning with the lipid molecules within the membrane. In most cases, the functional cationic moieties are pendant groups attached either as part of the monomer, or by post-polymerization modifications. The cationic groups can also be active in the polymer backbone if provided with sufficient hydrophobicity from the backbone or alkyl side chains.23 Examples such as linear or branched poly(ethylene imine)s (PEIs)75-77 and poly

(tetramethylenebis(diphenylphosphonio) tetramethylenedibromide)78 have been reported. An alternative structure, telechelic antibiotic polymer, has also been studied with a single antibiotic moiety at one terminal of each polymer chain. As an example, poly(2-methy-1, 3-oxazoline)-dimethyldodecylammonium bromide

(PMOx-DDA) was prepared by Waschinski et al. and Krumm et al. through ring opening polymerization.79-

The high throughput and cost effective synthesis of antibiotic polymers have greatly promoted the production compared to host defense peptides, and they can be classified according to the nature of the cationic groups.

The most commonly used moieties are quaternary ammonium groups. Therefore, these polymers are also named poly-quaternized ammonium salts (poly-QAS), including but not limited to alkylated poly(N,N- dimethylaminoethyl methacrylate) (PDMAEMA),82 PEI,75-77 alkylated polydiallylammonium

(PDADMAC),83 alkylated 4-(dimethyl-aminoemthyl)-styrene84 etc. Non-quaternized ammonium groups, such as primary and tertiary ammonium groups, are also able to kill germs once they are protonated under a certain pH range.85 Other common cationic groups (Figure 2) include: pyridinium,86-88 guanidinium73,89-91

and biguanide,92 quaternary phosphonium,78,93-95 and tertiary sulfonium.96

Figure 2: Commonly utilized cationic groups reported for antibiotic polymers.

The type of polymer backbone used in antibiotic polymers also varies widely. Polymethacrylates,91,97 polymethacrylamides98 and polystyrene derivatives99,100 are frequently adopted in the preparation of antibiotic polymers due to their wide availability and ease of reaction. Other synthetic polymers used as platforms for antibiotic polymers include polysilanes,101,102 nylon103 and polyacrylonitrile.104 Natural polymers, including cellulose,105 natural rubber106 and chitosan,105 have also been applied in this field through modification of reactive functional groups with bioactive moieties.

2.2 Mechanisms of Action of Antibiotic Polymers: The Current Understanding

The main steps involved in the killing of bacteria by antibiotic polymers were proposed by Ikeda et al.107 It was reported that only negatively charged liposomes were susceptible to the polycation in their study on the interaction between poly(hexamethylene biguanide hydrochloride) (PHMB) and a series of liposomes.107 12

Later assessment of the antibacterial activity of poly(N1-4-(2-acryloyloxyethyl)phenyl-N5-4- chlorophenylbiguanide hydrochloride) illustrated the process of killing the bacteria.92 As an example for

Gram-negative bacteria, the antibiotic polymers are initially attracted and adsorbed to the cell outer membrane through electrostatic interactions. Subsequently the membrane will be weakened and disrupted through hydrophobic interactions. The membrane selective permeability and functionality are lost. Antibiotic polymers diffuse through a thin cell wall and next the inner membranes are destroyed in a similar way. Finally, the cytoplasmic constituents leak out and the cell dies. For Gram-positive bacteria, there is no outer membrane but the polymers need to diffuse through a thicker cell wall to have action against the cytoplasmic membrane. The adsorption to and disruption of bacterial membranes (both outer and cytoplasmic) by antibiotic polymers were observed through the change of fluorescein by Rawlinson and colleagues in the study on the mechanism of PDMAEMA.108 Sovadinova and colleagues, however, reported that protonated poly(methyl 3-mercaptopropionate)-co-poly(methyl/butyl methacrylate) only penetrated the outer membrane of E. coli at a concentration lower than the minimum inhibitory concentration (MIC), indicating possible antibacterial mechanism other than significant disruption of inner membrane.19

Horvath et al. investigated the interaction of branched-PEI with a model immobilized lipid bilayer (1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC) via optical waveguide spectroscopy (Figure 3).109

From measurements of the change of optical thickness and birefringence of the layer of polymer-lipid complexes, they found the coverage of lipid was gradually replaced by PEI. Based on the observations, they proposed that the PEI chains adsorb on the membrane and insert the alkyl chains alongside the phospholipid molecules to form a complex, the polymer-lipid complex then leaves the bilayer, and finally the integrity of lipid bilayer is weakened with part of the lipid molecules exchanged by polymers.

Figure 3: Simulation of the interaction between antibiotic polymer and immobilized lipid bilayer, based on the interaction between polymer PEI and lipid bilayer POPC. (a) Adsorption of antibiotic polymer onto lipid bilayer by inserting alkyl chains; (b) PEI-lipid complex leaves the surface of lipid layer; (c) Integrity of lipid bilayer is weakened and part of the lipid is exchanged by the antibiotic polymer.109 Reprinted from reference

109. Copyright (2013) by the authors, open access licensed by MDPI.

Molecular dynamics simulations of the interaction of various antibiotic polymers with artificial membrane bilayers have also been conducted to help understand the mode of action in real systems.110,111 These studies emphasize the importance of amphiphilic structures in antibiotic polymers and illustrate the conformations adopted by polymers during interaction with membrane lipids. For example, according to the “all atom 14

molecular dynamics” computations by Ivanov et al. based on the interaction between block poly-QAS and liposome dioleoyl phosphatidylcholine (DOPC), when the interaction occurs, the cationic moieties remain outside of lipid membrane, while the hydrophobic segments insert into the lipid bilayer (Figure 4).112

Figure 4: All atom molecular dynamics illustrating the insertion of alkyl side chains of polycation into DOPC lipid bilayer. Colors of the atoms: green-carbon, red-oxygen, blue-nitrogen, silver-hydrogen, orange- phosphorus, dark blue-the lipid tail carbon atoms of DOPC.112 Reprinted with permission from reference 112.

In the work by Palermo et al., who compared the biological activity of a series of poly-QAS statistical copolymers by adjusting molecular weight, structure of ammonium group and copolymer composition, a relationship describing the polymer-membrane interactions was derived:113

⇌ ⇌ ∙ ⇌ ∙ (1)

In this model, free antibacterial polymer in solution (Pf) initially binds at specific positions on the cell surface

(S), represented as P·S. The authors proposed the formation of polymer-lipid complexes P·Sp, which causes loss of integrity and damage of the membrane. The free polymers may also interact in solution to form 15

inactive aggregates denoted as Pc. The equilibrium coefficients Kc, Kd and Kp will be affected by the hydrophobic nature of the polymers and the charge carried (see Section 2.3.2). This schematic interaction equation is valuable for researchers to better understand and design antibacterial polymers.

2.3 Key Factors Affecting Behavior of Antibiotic Polymers

According to the mechanism illustrated above, it is the interaction between the antibiotic polymers and bacterial membranes that leads to the biocidal behavior of such material. It is followed that any factor that enhances the possibility of binding with bacterial membranes can promote the antibiotic performance. The achievement of the enhancement derives from the change of a series of polymer properties through adjustments of structural parameters. In this section we discuss the effect of the structure of antibiotic polymers on interactions with the cell membrane, and subsequently on antibiotic activity.

2.3.1 Effect of Molecular Weight

The molecular weight (MW) changes the length of the polymer backbone, the overall hydrophobicity and charge density, which affect the conformation of the polymer when interacting with cell membranes. An early study on the relationship between antibacterial activity of antibiotic polymers and their MW was conducted by Ikeda et al., who found that the relation between MW and antibacterial action follows a bell-shaped curve.114 They assessed the dependence of the antibacterial activity of poly(Nl-4-(2- methacryloyloxyethyl)phenyl-N5-4-chlorophenylbiguanide hydrochloride) and poly(vinylbenzyl ammonium chloride) against S. aureus as a function of the molecular weight. Both polymers showed significantly weight- dependent properties. The polymethacrylate with biguanide pendant groups had optimal antibiotic performance within the range of MW between 50 kDa and 100 kDa, while poly(vinylbenzyl ammonium

chloride) showed monotonous increase of antibiotic action with MW to 7.7 kDa, the highest MW they were able to synthesize. The authors proposed that the adsorption, binding and membrane disruption capacities are expected to increase with MW, while the diffusion capacity is suppressed by polymers with excessive MW.

More studies on the dependence of antibacterial activity on MW have been reported. Chen et al. found a parabolic relationship between the antibiotic activity and MW, different from the bell-shaped dependence, during the study on quaternary ammonium functionalized poly(propyleneimine) dendrimers.115 It follows that an MW range of antibiotic polymers may exist for optimal antibacterial performance. A lowest average

MW is required for antibiotic polymers to maintain efficient antibacterial activity, as reported by Albert and co-authors in their study on the structural dependence of antibiotic oligoguanidines.116 The lower limit MW for oligoguanidines was shown to be 800 Da. At high MW, the reduced antibacterial action is ascribed to the

“sieving effect” by Chin et al. when investigating antibacterial polycarbonates poly(5-methyl-5-(4-

117 chloromethyl)benzylcarboxyl-1,3-dixan-2-one) (poly(MTC−OCH2BnCl)) against S. aureus. The “sieving effect” indicates that polymers with high MW might be blocked by dense peptidoglycans of the cell wall, especially for Gram-positive bacteria. This phenomenon was also observed by Lienkamp et al., whose poly(oxanorbornene)-based synthetic mimics of antimicrobial peptides, with protonated amine groups as the antibiotic groups, showed reduced antibiotic activity with increasing MW.118

In contrast to this, some researchers have observed that molecular weight had little impact on the bioactivity of the antibiotic polymer. Panarin and colleagues showed that the antibiotic activity of a series of water- soluble copolymers of N-vinylpyrrolidone and quaternized vinylamines or aminoalkyl methacrylates were independent of MW in the MW range investigated.119 Wiegand et al. compared the antibacterial action and biocompatibility of linear- and branched-PEI of different MW against E. coli and S. aureus. The result

showed that the MW did not have significant effect on antibacterial effects for either linear- or branched-PEI, but the antibiotic action depends on the structure of the polymer and even the strain of bacterial cells.77

It follows that molecular weight has a complicated effect on the antibacterial activities of antibiotic polymers, because it affects the size and net charge of the polymer. In addition, the difference in the structures of cell membranes for various bacterial species should also be taken into consideration.117,118

2.3.2 Effect of Amphiphilic Balance

Because the antibiotic polymers use the cationic group to attach to bacterial membranes and alkyl chains to insert into and disrupt the membranes, the balance between the hydrophilic (due to cationic groups and the hydrophilic non-charged groups) and hydrophobic plays an important role in the antibacterial activity of antibiotic polymers. The hydrophilicity of antibiotic polymers is majorly dictated by the mole fraction of cationic groups. These groups can be inherently charged, such as quaternary amines, or reversibly charged, such as primary and tertiary amines. On the other hand, the hydrophobicity of antibiotic polymers is derived from contributions of hydrophobic groups in the polymer, which are mainly derived from the fraction of hydrophobic comonomers and the structure of alkyl chain. (Figure 5) These two factors are interrelated. Any change in one factor affects the other oppositely and significantly influences the antibacterial activity of antibiotic polymers by changing the selectivity. In order to achieve similar efficacy and biocompatibility as host defense peptides, critical and subtle adjustments in the amphiphilic balance is required, for which detailed effects of the structural parameters mentioned above are discussed below.

Figure 5: Schematic illustration of the structural parameters of antibiotic polymers: a) Structure of ordinary antibiotic polymers; b) structure of telechelic antibiotic polymer.

(a) Different Structures of Ammonium Groups

The most exploited class of cationic groups for antibiotic polymers are quaternized ammonium salts (QASs), because of the effectiveness of small-molecule QAS disinfectants, e.g. benzalkonium chloride and centrimide.120 With four aliphatic groups attached to the nitrogen, QASs remain cationic and effective in both aqueous and dry environments. Primary and tertiary amine groups are only effective with antibiotic functions when protonated and the extent of protonation is affected by the pH of the solution. Palermo et al. compared the antibacterial behavior of antibiotic polymers with protonated primary (poly(2-aminoethyl methacrylate),

PAEMA), protonated tertiary (PDMAEMA) and quaternary ammonium groups (alkylated PDMAEMA).113

It was found that polymers with primary and tertiary amine groups exhibited a higher antibacterial effect and 19

better selectivity than the quaternized counterpart, which required higher hydrophobicity to reach similar antibiotic performance, but may also induce more cytotoxicity. Furthermore, through observing the pH dependence of antibacterial activities, they showed that the increase of pH raises the extent of deprotonation of primary/tertiary amine groups, hydrophobicity of the whole polymer, and subsequently the antibiotic and hemolytic action, but high pH caused reductions of both activities, due to the possibility of forming aggregates caused by high hydrophobicity. Timofeeva et al. reported the antibiotic activity of secondary and tertiary poly(diallylamine)-based polymers, secondary poly(diallylamine trifluoroacetate) (PDAATFA) and tertiary poly(diallylmethylamine trifluoroacetate) (PDAMATFA), and compared to the antibacterial behavior of the quaternary counterpart, poly(diallyldimethylammonium chloride) (PDADMAC).83 They found that

PDAATFA and PDAMATFA exhibited significantly higher antibiotic activity than PDADMAC until total deprotonation of the amine groups. The presence of NH2+ or NH+ groups in the PDAATFA and PDAMATFA was considered to have strong electrostatic interaction and form hydrogen bonds with oxygen atoms of phosphate, carbonyl or hydroxyl groups in the lipid, which was related to the high antibacterial efficacy.

(b) Composition of Non-antibiotic Comonomers

Most studies on the effect of the composition of non-antibiotic comonomers focused on the use of hydrophobic comonomers. Kuroda and colleagues compared the MIC and the polymer concentration

required for 50% cell lysis (HC50) of a series of protonated poly(2-aminoethyl methacrylate)-co-poly(butyl methacrylate) (PAEMA-co-PBMA) with different PBMA compositions.121 The results showed that appropriate composition of hydrophobic comonomers endows the copolymers with the most effective antibacterial behavior but the hemolysis rises with increasing hydrophobic contents. More detailed results were obtained in a subsequent study by Palermo et al., who examined the impact on the antibiotic action

113 through using methyl and butyl methacrylate as comonomers and adjusting their compositions (falkyl).

Optimal antibiotic activities of the copolymers were observed when increasing the falkyl, while the HC50/MIC

of the copolymers was found to monotonically decrease with the falkyl, indicating the loss of selectivity with increasing hydrophobicity. A subsequent study of the kinetics of liposome dye leakage induced by protonated poly(3-aminopropyl methacrylamide)-co-poly(butyl/hexyl methacrylamide) showed that polymers with high contents of hydrophobic comonomers induced fast leakage of dye, while those with high cationic compositions caused slow leakage.98 This result shows that the composition of hydrophobic comonomers determines the overall hydrophobicity of antibiotic polymers, which further modulates the permeabilization of the membranes. Different sensitivities of Gram-positive and Gram-negative species toward the composition of hydrophobic comonomers were observed by Qiao et al. when adjusting the ratio of 5-methyl-

5-ethyloxycabonyl-1,3-dioxan-2-one of poly(5-methyl-5-(3-chloropropyl) oxycabonyl-1,3-dioxan-2-one)-

co-poly(5-methyl-5-ethyloxycabonyl-1,3-dioxan-2-one) (poly(MTC-O(CH2)3Cl)-co-poly(MTC-OEt)), with quaternary ammonium groups introduced by reacting with triethylamine after polymerization.122 An optimal composition of hydrophobic comonomers (~40%) was found for Gram-positive bacteria S. aureus, while

Gram-positive cells, such as P. aeruginosa, were less responsive to charge density and require higher composition of hydrophobic groups for effective inhibition. The difference mainly derives from the distinct structures of the cell membranes of the two species, where the Gram-positive membranes contain more negatively charged lipids than the Gram-negative ones.

In addition to their hydrophobic counterparts, hydrophilic comonomers, such as PEG, have also been utilized and investigated. Allison and colleagues showed that the copolymerization with poly((ethylene glycol) methacrylate) (PEGMA) totally eliminated the hemolysis of poly(4-vinylpyridine)-co-poly((ethylene glycol)

methacrylate) (P4VP-co-PEGMA) in a certain range of the mole ratio of P4VP.87 Punia et al. reported the antibacterial activity of protonated poly(6-amino hexyl methacrylate)-co-poly( (ethylene glycol) methacrylate) (PAHMA-co-PEGMA) with different lengths and mole ratios of PEGMA.123 The hemolytic effects on red blood cells were dramatically reduced when the mole ratio of PEGMA-300 reached 33%, while the antibiotic activity was retained. Higher composition and lengths of PEGMA caused gradual reduction and abrupt loss of antibiotic behavior. It was also found that E. coli were more susceptible to the polymers compared to S. aureus, which is possibly derived from the formation of hydrogen bond between PEG chains and the polysaccharide cell wall of S. aureus. The incorporation of certain amount of hydrophilic carbohydrate-attached monomers has also been reported to remarkably increase the biocompatibility of hydrophobic antibiotic polymers.124 Hence, the composition of the hydrophilic comonomers, which manipulates the overall hydrophilicity of the polymers, can reduce the antimicrobial activity and the hemolytic action under certain mole ratio range.

During the interaction between antibiotic polymers and bacterial membranes, alkyl side chains fuse or align with the lipid molecules within the membrane so as to disrupt the integrity of the membrane. Thus the structure of alkyl side chains is critical to the antibacterial activity of the polymers. Lienkamp et al. adjusted the alkyl chain length from methyl to butyl of oxanorbornene monomers with monoamine–alkyl structures and found that the polymers with methyl/ethyl chains were inactive against E. coli but non-hemolytic while those with propyl/butyl chains behaved oppositely.118 However, longer alkyl chains cause an impairment of the antibiotic efficacy. Chin et al. studied the change of antibacterial performance and selectivity of

117 poly(MTC−OCH2BnCl). Linear alkyl chains with lengths of 1, 2, 4, 6 and 8 carbons were tested. Alkyl

chains longer than 6 were found to cause low solubility of the polymers and reduced antibiotic efficacy. The elongation of the alkyl chain can also reduce antibiotic selectivity of the polymers to general cytotoxicity. An optimal length of 6 carbons was achieved to possess the most effective antibiotic effect, and 4 carbons for best selectivity. The effects of the structures of alkyl chain were also illustrated by using linear hexyl chain, cyclohexyl group and benzyl group. The results showed that cyclohexyl and benzyl groups possessed weaker biological activity but significantly less hemolysis compared to the linear hexyl counterparts, which was related to the hydrophobicity as confirmed by water-octanol tests. Similar results were also reported by

Sambhy et al. when comparing the antibiotic and hemolytic behavior of poly(4-vinylpyridine)-co-poly(alkyl methacrylate) attached with alkyl chains of lengths from 2 to 10,125 and King et al., who studied the antibiotic difference of poly(3-((4-bromobutoxy)methyl)-3-methyloxetane)-co-poly(3-(2-(2- methoxyethoxy)ethoxymethyl)-3-methyloxetane) (PBBOx-co-PME2Ox).126 On the other hand,

Venkataraman and co-authors found a different influence of the length of alkyl chain. They prepared a series of PDMAEMA-co-PEGMA polymers and partially quaternized the tertiary amine groups with alkyl chains of different length and end groups.127 They found that the copolymers alkylated with methyl chains were of lowest MIC, and longer alkyl chains reduced the MIC. With the length of alkyl chains kept at 3 carbons, the effects of end groups of the alkyl chains were also investigated, and the antibacterial activity of the copolymers followed the trend: alkyl > alcohol >> amine ~ acid. The hemolytic effects of the above two factors were similar to other reports, i.e. more hemolysis for higher hydrophobicity. Chen et al. prepared

PDMAEMA using alkyl chains with large steric volume, rosin and eicosane (PDMAEMA-g-rosin,

PDMAEMA-g-eicosane), both groups have the same number of carbons (20 carbons).128 It was found that the partly quaternized PDMAEMA-g-rosin showed high antibacterial activity while the fully quaternized

PDMAEMA-g-rosin and PDMAEMA-g-eicosane almost lost the antibiotic efficacy. The difference was

thought to derive from the unique multi-ring structure of rosin, which has higher amphiphathicity than the eicosyl groups. However, due to the large volume, fully quaternized PDMAEMA-g-rosin shielded the cationic groups from bacterial membrane, which significantly reduced the antibacterial activity.

Of course the position of alkyl chains does not need to be necessarily connected with the cationic functional group, as was demonstrated by Ilker and colleagues in the preparation of a series of amphiphilic polynorbornene derivatives with various types of alkyl chains attached to the norbornene ring.129 Similar effect of hydrophobicity was observed as both the antibiotic and hemolytic behavior levitated when the hydrophobicity and size of the alkyl chains increased. This was explained by the stronger interaction between larger hydrophobic groups and the inner core of bacterial membranes.

It seems that the structures of alkyl side chains that have higher affinity toward the cell membranes increase the likelihood of disruption of the membranes and the antibacterial efficacy of the polymers. However, potential negative side effects of more serious hemolysis should also be considered in the design of such antibiotic polymers.

The variation of amphiphilic balance of antibiotic polymers has profound impacts on the antibacterial activity of antibiotic polymers. It has been pointed out by the group of Kuroda the possibility of optimal selectivity and biocompatibility by fine adjusting the amphiphilic balance. The kinetic equation (1) provided by them32 can be utilized to help better understand the effect of amphiphilic balance and the interaction between antibiotic polymers and membranes of bacteria:

The increase of hydrophobicity enhances hydrophobic interactions between antibiotic polymer and the cell 24

membrane, leading to lower Kd, higher Kp and more formation of polymer-lipid complex, P·Sp. However,

excessive hydrophobicity also increases Kc and shifts the reaction to the formation of more aggregates, Pc,

reducing the amount of available antibiotic polymers. On the other hand, higher charge density increases Kd

and suppresses Kc, disfavoring the inactivated formation of Pc, but the ability to disrupt cell membranes is undermined, as reflected by reduced formation of P·S. As a result, the antibacterial behavior of antibiotic polymer is a compromise of amphiphilic balance. Optimal antibacterial effect can be achieved through fine adjustment of the balance to match the specific requirements of the target bacterial membranes. For example, in the same work by Ilker et al., an optimization of biocidal and hemolytic performance was allowed by copolymerizing two types of norbornene monomers, one with high selectivity while the other with strong antibacterial function, to achieve moderate hydrophobicity in the interaction with the membranes.129

2.3.3 Effect of Copolymer Sequence

In most literature reports, linear antibiotic polymers are designed as statistical copolymers, while block copolymers or even alternating copolymers have been rarely utilized. However, copolymer sequence can have a great impact on the conformation of antibiotic polymers during the interaction with cell membranes.

Oda et al. prepared block and statistical copolymers of poly(2-aminoethyl vinyl ether) and poly(isobutyl vinyl ether) to investigate their antibacterial and cytotoxic activity.130 They found that block copolymers, with same composition and MW as statistical ones, had similar antibiotic efficacies but with significantly higher selectivity against E. coli over human red blood cells. From the hemagglutination observed for block copolymer, the authors found that the block copolymer forms single-molecule micelles with hydrophobic moieties wrapped inside and cationic groups extended toward aqueous interface. The structure of the single-

molecule micelle shields the interaction between hydrophobic chains with human red blood cells, lowering hemolysis, but it induces hemagglutination due to high charge density. On the other hand, the statistical copolymers cannot form single-molecule micelles, causing hemolysis due to the interaction between hydrophobic chain and red blood cell (Figure 6). It was also found that block copolymers were able to maintain their antibacterial activity at a concentration higher than the critical aggregation concentration

(CAC), while the efficacy reduced for statistical copolymers, which suggests that the aggregation of block copolymers may also contribute to the antibiotic performance.

Figure 6: Schematic comparison of the antibiotic and hemolytic activities of statistical (a) and block (b) antibiotic copolymers. Statistical copolymers cannot form such structures and are harmful to both bacterial and blood cells. The block copolymers form single-molecular micelles to exert antibacterial action on bacteria and avoid hemolysis, but also induce hemagglutination.130 Adapted with permission from reference

130. Copyright (2011) American Chemical Society.

Wang et al. proposed another model when investigating the antibacterial behavior of block and statistical

copolymers of PDMAEMA and PBMA.131 The same result was observed that block copolymers had similar antibacterial performance but better selectivity compared to those of statistical copolymers. The amphiphilic structure of the copolymers was considered as the main factor to change the selectivity. It was proposed by the authors that the facially amphiphilic structure of the block copolymer can only form at the junction of the blocks instead of the whole polymer, while statistical copolymers can form perfect facially amphiphilic structure along the polymer backbone through the change in polymer conformation (Figure 7). Since the two types of copolymers have similar hydrophobicity but different amphiphilic structure, it was indicated that hydrophobicity is more crucial to antibiotic activity while the amphiphilic structure is responsible for hemolysis.

Figure 7: Facially amphiphilic structures formed by block and statistical copolymers. The amphiphilic structure of statistical copolymer is along the polymer backbone (a) while that of block copolymer is at the joint of the two blocks (b) (the black dashed line serves as a note to the eye).131 Adapted with permission from reference 131. Copyright (2011) John Wiley and Sons.

The preparation of antibiotic polymers with alternating sequence was reported by Song et al. by the use of alternating ring opening polymerization.132 Through fine control over polymerization reactions, precise spacing adjustments between the adjacent monomers attached with cationic functional groups were realized.

By comparing the antibacterial performance of the alternating copolymer, as well as statistical copolymer and homopolymers prepared through the same process, it was found that precise spatial distance between the neighboring QAS groups is critical to the performance. In statistical copolymers, the irregular arrangement of cationic groups and possible shorter spacing between them resulted to locally lower hydrophobicity and antibiotic potential. Further comparison between alternating polymers and homopolymers revealed specific distance of spacing was necessary for the polymer to be antibacterial, which should be more than 4 Å threshold and at least 8 – 10 Å in range.

2.3.4 Effect of Spacers and Satellite Groups

Spacers and satellite groups are both hydrophobic chains that are not capable of killing cells, but can affect the antibacterial behavior of antibiotic polymers.

Spacers are the sections connecting the cationic group to the polymer backbone, functioning to extend the antibiotic groups into the membrane of cells, as described by the “snorkeling effect”.133 Thus it is believed that the spacers are important to the effectiveness of antibacterial polymers. Palermo et al. had a thorough investigation of the effects of spacer groups by comparing poly(N-aminoalkyl methacrylate)-co-poly(ethyl methacrylate) with different spacers.110 It was reported that longer spacers can significantly increase the antibiotic effect, antibiotic efficiency and hemolytic activity, which may be related to the rise in the hydrophobicity of the polymer. Molecular dynamics simulations confirmed the formation of facially

amphiphilic structures adopted by the antibiotic polymers and showed that elongated spacers positioned the polymer backbone closer to the lipid core of the cell membrane, consistent with the description of the

“snorkeling effect”.

Satellite groups were named by Waschinski et al., specifically referring to the opposite end group, relative to the antibacterial group, of a telechelic antibiotic polymer.79,80 These groups are non-bioactive, but affect the antibacterial behavior of telechelic antibiotic polymers profoundly. By comparing different satellite groups of polyoxazolines with N,N-dimethyldodecylammonium (DDA) end groups, including the length, structure, hydrophilicity (by protection and deprotection of tert-butyloxycarbonyl group) and whether or not cationic charge was carried, the authors illustrated that the probable mechanism for satellite groups is to assist cationic group in disrupting the membrane by inserting into the lipid bilayer of the cell membrane at the same spot as the cationic group alkyl chain. However, the effect of satellite group is limited as long satellite group brings about the excessive hydrophobicity, which can lead to aggregation of the polymer.

2.3.5 Effect of Counter-ions

Counter-ions pair with the cationic functional groups to neutralize the charge of the polymers. Strong affinity

- of counter-ions toward the polycations can reduce their solubility, such as tetrafluoroborate (BF4 ),

- - perchlorate (ClO4 ) and hexafluorophosphate (PF6 ), and further lower the antibiotic activity. This was demonstrated by the study of Kanazawa et al. studying the action of poly(tributyl(4- vinylbenzyl)phosphonium) against S. aureus.93 The results showed that the antibiotic efficacy decreased in the trend of chloride > tetraflouride > perchlorate > hexafluorophosphate as the counter-ions. This trend is

positively related to the reduced Ksps of the respective monomers, showing the relationship between Ksp and

antibacterial activity of antibiotic polymers. Lienkamp et al. detected significantly reduced antibacterial activity against E. coli and S. aureus when switching hydrophilic counter-ions trifluoroacetic ion (TFA-) to hydrophobic counter-ions in the poly(oxanorbornene), including hexanoate, dodecanoate, benzoate, and tosylate.118 It was found that polymers with hydrophobic counter-ions were not as active to disrupt bacterial membranes compared to the TFA counterparts, which possibly resulted from the formation of tight ion-pairs.

Chen et al. studied the effects of chloride and bromide anions on poly(propylene imine) dendrimers and found that the antibiotic activity of the dendrimers with bromide anions showed stronger antibacterial effects than polymers with chloride ions.115 However, Panarin et al. reported no observable influence from the counter-ions Cl-, Br- and I- on copolymers of N-vinylpyrrolidone and quaternized vinylamines or aminoalkyl methacrylates.119 It seems that the selection of counter-ions affects solubility of antibiotic polymers, which is one of the key factors related to antibacterial action. Additional effects of the counter-ions, especially on the activity of cationic groups, remain to be investigated.

3. Antibacterial Polymer Coatings

3.1 Introduction

In order to achieve more effective biofilm prevention, antibacterial polymers have also been immobilized on substrates to afford antibacterial coatings. Depending on the types of the antibacterial polymers, the antibacterial coatings can be mainly classified as antibiotic-releasing and antibiotic (contact active), while very few preparations of surface immobilized polymeric antibiotics64 have been reported. Here, only the contact active coatings are discussed.

Different immobilization methods have been adopted, which can be generally categorized into chemical and physical methods. Chemical immobilization methods include grafting from/to surfaces,134,135 crosslinking to form self-supporting films,136 in situ crosslinking polymerization,137 and being incorporated into hydrogels.138 Well-defined polymer structures and grafting density can be achieved through these methods, which are appropriate for mechanistic study. Physical methods include physical adsorption and layer-by- layer deposition. Dip coating139 and spin coating140 are simple physical adsorptions for antibacterial coating preparation. Layer-by-layer deposition needs two oppositely charged polymers to attract each other and get densely packed on substrate, which affords highly compact antibacterial coatings.5,89 These methods are simple to apply and appropriate for industrial production.

Substrates utilized for immobilization include those common in experiments, such as gold141 and glass,134 and everyday materials, e.g. nylon,142 poly(ethylene terephthalate) (PET) film,143 cellulose137,144 and poly(vinyl alcohol) (PVA) film.145

3.2 Current Understanding of the Mechanisms

Coatings of antibiotic polymers are termed “contact active” coatings, because they are able to kill the microbes when in contact. Many polycations studied in solutions have been attached to surfaces, including quaternized PEI,146,147 poly(4-vinylpyridine) (P4VP)148 and PDMAEMA.144 These coatings exhibit high efficiency and efficacy in inhibiting the growth of bacteria.

However, the modes of action of antibacterial polymer coatings may differ from those of polymers in solution.

Immobilization of antibiotic polymers disables their diffusion and further limits their capability of binding

toward the cell membrane. So far, three models have been proposed to explain the antibiotic action, which were all deduced from careful observation on the structural impacts on the behavior of antibiotic polymer immobilized.

3.2.1 Insertion Model

The mechanism of “contact active” coatings was initially considered similar to that of antibiotic polymers in solution, as reported by Tiller et al. in their study on the influence of molecular weight and alkyl chain length on the antibiotic behaviors of alkylated P4VP modified glass.148 It was found that polymers of higher MW with alkyl chain length in a certain range (3, 4, 6 and 8 carbons) were most effective, showing similar trends to antibiotic polymers in solution. The hypothesis termed the “insertion model” was proposed. This model was extended from the mode of action of antibiotic polymers in solution, which involves the insertion of polymer chains into the cell membrane to cause disruption and consequently cell death. The proposal was also indirectly supported by their observation that alkyl chains of excessive lengths (10, 12 and 16 carbons) could lead to aggregation, which prevented further interaction and insertion into bacterial membranes. This model was further strengthened by Milović et al. in their study on N-hexyl, methyl-PEI attached to glass slides.149 With the help of Live/Dead two color fluorescence tests, they were able to prove that the cell membranes ruptured when in contact with PEI chains. They also found that the coating was harmless to mammalian cells. Another antibacterial coating was prepared via non-covalent immobilization of PEI polymer.139 Even though longer alkyl chains were required to prevent leaching of the PEI, similar results were observed related to the effect of MW and hydrophobicity on the antibiotic behavior.150 Recently, Altay et al. prepared antibiotic polyoxanorborenene coatings, with quaternized pyridinium as the functional groups.

Sufficient length and flexibility, which favors the penetration of bacterial membranes, was required for the

coating to exert antibacterial activity.151 It was also found that the polymer with hexyl chains, which exhibited high but not excessive hydrophobicity, showed optimal antibiotic results. The above reports suggest that the

“insertion model” seem reasonable.

3.2.2 Ion Exchange Model

A second model was proposed by Kugler et al. in their investigations on the effect of the charge density of

P4VP grafted from small beads.152 The thresholds of charge density for high antibiotic efficiency were studied and were found to be dependent on bacterial types and bacterial metabolic states (quiescent or dividing).

More specifically, the authors considered that the “outer-layer charge density” (OLCD), the terminal area of the polycation chains, determines antibiotic activity, due to the short distance of electrostatic interaction and bulky size of the bacterial cells. Since the thickness of the P4VP monolayer (2.5 nm ± 0.1 nm) was obviously less than that of the cell membrane, the only possible antibiotic action was proposed to be via electrostatic force. Kugler and colleagues thus proposed that the polycation coating kills bacteria through exchanging the divalent cations of the cell membrane. The role of divalent cations, Ca2+ and Mg2+, is to neutralize the negative charge of phosphate group on phospholipids and lipopolysaccharides, otherwise those molecules will repeal each other and the stability of the membrane is lost. In particular, when bacteria adhere to positively charged polymer coatings, the divalent counter-ions in the cell membrane are released to reach higher system entropy, disintegrate the membrane and subsequently cause the death of cells. This hypothesis was further strengthened by Murata’s research.153 They compared the respective impacts of dry coating layer thickness and the grafting density of surface initiated PDMAEMA toward antibiotic efficiency. Different dry layer thicknesses were controlled by stopping the polymerization at different lengths of time. Measurements showed that the thickness, accessible charge density and the killing efficacy are all positively related, with

the antibiotic efficacy significantly promoted when the thickness exceeded a threshold. In another series of experiments, by continuous injection of trimethoxysilyl blocker (TMS-blocker) to inhibit surface functional groups, a surface with an immobilized initiator concentration gradient was acquired, from which the accessibility of quaternary ammonium groups of PDMAEMA layer was well controlled. The thickness of the coating, charge density and further antibacterial activity of the coatings were also found to be promoted by higher grafting density of PDMAEMA. It was again emphasized that the thickness of the coating was not sufficient to penetrate either the membrane or the cell wall of bacteria, from which they indicated that the charge may play a dominant role in antibiotic process for high density polycation coatings. Illergard and co- authors used layer-by-layer method to coat cationic polyvinylamine (PVAm) and anionic poly(acrylic acid)

(PAA) onto cellulostic fibers.154 The oxidized fibers with three-layered coatings exhibited highest antibacterial activity due to more adsorption of PVAm. Through the use of SEM and TEM, it was found that the shape of bacteria was elongated on contact with the fibers. As no nutrient was provided, the elongation was not derived from replication, but strong interaction with the polymer coating. It was emphasized that the actual contact area between the fibers and bacteria only covered a very small portion of the total bacterial surface, indicating strong electrostatic attraction caused the deformation. Moreover, the coatings were still able to exert antibiotic effects even though the surface was fully covered by dead cells. Thus the authors suggested that instead of physical interaction (insertion), the electrostatic interaction between the polymer coating and the bacterial cells be crucial to the antibacterial action of the coatings.

3.2.3 Phospholipid Sponge Model

The third mode of action was described by Bieser and Tiller as the “phospholipid sponge effect” in their study on N,N-dimethyldodecylammonium (DDA) and N,N-dimethylbutylammonium (DBA) modified

cellulose (Figure 8).155 The DDA samples all exhibited high inhibition effects, while the DBA samples showed high antibiotic ability only when the substitution rate was low, meaning more hydrophobicity was required for hydrophilic DBA. This shows that cationic density and hydrophobicity were the two main factors affecting antibacterial actions of the coating. By presuming that the negatively charged phospholipid is essential for the integrity of cell membrane, they found that anionic sodium dodecyl sulfate (SDS) solution and liposome solution with 10% phosphatidylglycerol could fully neutralize and saturate the cationic charge, which indicates strong interaction between the polycation and the phospholipids in the cell membrane. Since the polymer coating was insoluble in water, the antibiotic activity through insertion of spacer is not applicable.

Hence, a new model was proposed indicating that when bacteria are attracted toward the polycation coating, the phospholipid in the cell membrane could become destabilized by the strong positive charge of the coating, because the polymer coating acts as a “sponge” to absorb the lipid from the cell membrane. This model was supported by the work of Li et al. on quaternized dimethyldecylammonium chitosan-graft-poly(ethylene glycol) methacrylate (DMDC-Q-g-PEGMA), crosslinked with poly(ethylene glycol) (PEG) diacrylate to form a hydrogel.156 The effect of charge density and pore size of the gel were closely investigated. It was found that increasing the charge density and pore size can improve the antibiotic activity. From the fact that the dead bacteria could be easily washed off to prevent loss of effectiveness, it was proposed that the PEG hydrogel, known for its antifouling ability, and the pores, to absorb the residue of fragments of cell membrane, both contributed to the self-sterilization ability of the material. Thus the authors considered that the antibiotic activity was caused by the hydrogel absorbing the lipid in bacterial membrane. Additional proof was given by Asri et al., who synthesized shape-adaptive hyper-branched antibacterial polymer coatings.157 Branched

PEI polymers were grafted onto hyper-branched polyurethanes (PU) immobilized on glass substrates and then quaternized. Compared to antibiotic polymers in solution, where obvious damage to cell membrane can

be observed due to insertion, the immobilized hyper-branched PEI killed S. aureus by enveloping the cell, with no obvious membrane disintegration. This indicates that the membrane damage is localized to membrane-polymer interface, disproving the “insertion model”. From further experiments, which illustrated similar antibiotic activities of polycation coatings and parafilm covered clean surfaces directly charged with strong electric fields but weak DC currents, the authors disproved the ion-exchange mechanism and supported that the source of antibiotic polymer coatings comes from the strong electrostatic attraction force that can deactivate adhering cells, as described in the “phospholipid sponge model”.

On first consideration, the three models proposed to describe the interactions between polymer coatings and bacteria seem to be in conflict. However, it is likely that more than one mechanism is important, depending on the selection of structural parameters of the coating. It is also possible that the antibacterial behavior requires the synergic combination of different mechanisms. Thus further investigation of detailed polymer- cell interactions is required.

Figure 8: Illustration of the “Phospholipid Sponge Model”. Lipid molecules are strongly attracted by polycation coating and torn off from the cell membrane.155 Adapted with permission from reference 155.

4. Antibacterial Polymer Coatings with Multiple Functionalities

4.1 Necessity for Antibacterial Polymers with Multiple Functionalities

Although able to kill bacteria upon contact, antibacterial polymer coatings with single functionality, including both antibiotic-releasing and antibiotic types, are incapable of repelling the deposition of dead cells, which gradually foul the coating, exhaust the antibiotic ability and become the seed layer for biofilm formation.

This is also named “self-deactivating”.23 To address this problem, two methods have been applied: combining antibacterial polymers of different functionalities, or switching the polymer between antibiofouling and antibacterial functions on demand.

4.2 Combination of Different Antibacterial Functions

4.2.1 Antibacterial with Antibiofouling

In most studies reported to date, antibiofouling polymers have been combined with antibiotic-releasing or antibiotic polymers.

In the case of combining antibiofouling and antibiotic-releasing polymers, the antibiotic-releasing polymers can instantly inhibit the bacteria while the antibiofouling coating is able to repel the dead bacteria.

Antibiofouling coatings loaded with silver particles were synthesized by Li et al.158 The nano-hybrid coating was prepared by blending the antibiofouling polymer, poly(vinyl fluoride silicone) (BD-FT-LSR), and the

antibiotic-releasing component, mesoporous silica loaded with silver particles. The hybrid coating exhibited higher efficacy to keep the surface free of bound P. f lu ore s c en s compared to either component individually.

Fullenkamp and coauthors prepared silver nanoparticle loaded PEG hydrogel by in situ crosslinking of PEG chains with catechol groups attached by silver nitrate.159 Biological tests confirmed the antibiofouling function of PEG hydrogels and biocidal effects from the release of encapsulated silver nanoparticles. A similar coating was reported by Yin et al., which was prepared via layer-by-layer deposition of PEI and poly(acrylic acid) (PAA) and subsequent introduction of silver nanoparticles through in situ reduction of Ag+ ions.160 Chemical modification with (tridecafluoroctyl)-triethoxysilane was then conducted to endow the surface with super-hydrophobicity. The presence of fluorinated polymer at the surface mediated the release of Ag+ and prolonged the antibiotic effect longer than 14 days compared to 11 days’ burst release by the coating without the antibiofouling layer.

A second method is to combine antibiotic polymers and antibiofouling polymers. In a recent study, Yang et al. prepared such coating.161 Briefly, the surface of a stainless steel substrate was modified by being coated with barnacle cement (adhesive excreted by barnacle) or poly-dopamine, serving as the initiator anchor for surface initiated atom-transfer radical-polymerization (ATRP) of poly(2-hydroxyethyl methacrylate)

(PHEMA). After polymerization, chitosan was coupled onto PHEMA to afford the multifunctional coating.

This coating utilizes PHEMA as a highly hydrophilic antifouling coating and chitosan as the ammonium source to kill bacteria. Later, another series of coatings were synthesized by the same group to immobilize antibiofouling poly(N-hydroxyethyl acrylamide) (PHEAA) and antibiotic poly(2-(methacryloyloxy)ethyl trimethylammonium chloride) (PMETA) on the barnacle cement/polydopamine coated stainless steel through

“click” chemistry.162 Mei et al. reported a new class of antibacterial polyacrylonitrile (PAN) nanofibrous

membranes, which incorporated polyhexamethylene guanidine hydrochloride (PHGH) as the antibiotic component and heparin (HP) for antibiofouling purpose with the help of layer-by-layer technique, which exhibited high performance in both antibiotic and antibiofouling against S. aureus and E. coli. 163 This coating has been shown to efficiently keep the surface sterile without any dead bacteria attached after 24 h and was shown to be effective for as long as 2 weeks.

In addition to the combination of polymers with different functionalities, mechanical designs have also been integrated into such materials. Recently, Voo and colleagues incorporated antibiofouling PEG, substrate tethering poly(MTC-maleimide) and antibiotic poly(MTC-benzyl chloride) into a triblock copolymer and grafted the copolymer to thiol-functionalized silicone.164 Different polymerization sequences were compared.

It was found that the S-shaped coating (PEG-b-P(MTC-benzyl chloride)-b-P(MTC-maleimide)) was able to effectively prevent the formation of biofilm for at least 7 days, while the V-shaped coating (PEG-b-P(MTC- maleimide)-b-P(MTC-benzyl chloride)), cannot avoid biofilm while control surface developed biofilm especially at the 7th day.

Antibiofouling effects can also be exerted by the introduction of ordered roughness onto the surface of polymer coatings,165-167 with the antibacterial effects achieved through incorporating antibiotic/antibiotic- releasing polymers. For example, Xu et al. incorporated antibiotic function in honeycomb-patterned 2,6-di-

O-thexyldimethylsilyl cellulose (2,6-TDMS cellulose) film through the “click” chemistry to attach quaternary ammonium groups.168 Such coatings have been shown to meet the needs for both antibiofouling and antibacterial purposes.

4.2.2 Mixing Different Antibacterial Polymers

Combining two types of antibacterial polymers is a method to prolong the antibiotic effectiveness, especially after the depletion of antibiotics, when the contact-active function can further protect the surface from contamination.169,170 Sambhy et al., through partly quaternizing P4VP, achieved a dual functioning coating.171

Quaternized P4VP segments were antibiotic, while unquaternized counterparts were used as ligands to bind with AgBr particles. High antibiotic efficiencies were observed against both airborne and waterborne bacteria.

Through tuning the size of nanoparticles embedded in the polymer, a maximum length of 17 days was recorded for the sample with 1:2 AgBr/21% P4VP to remain antibiotic. The MIC of the dual-function polymer coating was 200 times less compared to that of P4VP polymer coatings and around 1/4 of AgBr solution. The duration of effectiveness (17 d) was also significantly prolonged compared to AgBr solution (1 d), while the 21% P4VP coating with twice the concentration was not able to inhibit the bacteria.

In addition, conjugate polyelectrolytes have been applied as photo-sensitive antibacterial polymers without the use of dyes, which provides the opportunity to be combined with QAS groups to enhance the overall antibacterial activity. Ji et al. reported the fabrication of a series of poly(phenylene ethynylene) (PPE) derivatives with QAS groups attached.172 These materials were confirmed to exhibit strong antibiotic activity

1 by releasing light-activated O2. The presence of QAS groups enabled the polymers to interact strongly with

1 the negatively charged bacterial membrane, especially in darkness, when no O2 is emitted to kill the bacteria.

Thus the antibiotic efficiency of the polymers was significantly enhanced. Similar antibiotic activity was also observed by Wang et al. when comparing the antibiotic action of a library of PPE polyelectrolyte and oligomer derivatives.173

4.3 Switching of Different Antibacterial Functions

Based on polymers that are responsive to certain stimuli, such as pH and temperature, several research groups have reported fabrication of antibacterial polymers with multiple functionalities.

4.3.1 Function Switching through pH-responsiveness pH-responsiveness is mainly realized when polymers contain pH-sensitive chemical groups such as esters or amides, which are hydrolysable under the control of pH. Jiang’s group has been synthesizing antibacterial polymers (coatings) which are able to switch between antibiofouling and antibacterial functions through pH adjustment. Cationic poly(N,N-dimethyl-N-(ethylcarboylmethyl)-N-(2-methacryloyloxyethyl) ammonium bromide) (PCBMA-1 C2) was synthesized through surface initiated ATRP polymerization from gold, which was highly antibiotic against E. coli.174 After the hydrolysis of terminal ester bond at pH = 10.0, the polymer changed into a zwitterionic polymer, poly(N-(carboxymethyl)-N,N-dimethyl-2-((2-methyl-1-oxo-2-propen-

1-yl)-oxy)-ethanaminium) (PCBMA-1), which was observed to release 98% of the dead bacteria. An enhancement of the coating was made by loading salicylate counter-ion on PCBMA-1 C2 to afford PCBMA-

1 C2 SA (SA refers to salicylic anion) coating.175 Upon hydrolysis or anion group exchange, controlled release of salicylate occurs to inhibit the growth of incoming bacteria. The remaining zwitterionic polymer,

PCBMA-1, is able to resist the accumulation of cells on the coating. However, such approaches of switching are nonreversible,176 so a new kind of polymer coating with a reversible function switching feature was designed by Cao et al.176,177 Poly(2-((2-hydroxy-3-(methacryloyloxy)propyl)dimethylammonio)acetate)

(polyCB-OH) was immobilized through surface initiated ATRP. After treating with TFA or acetic acid overnight, the polyCB-OH was esterified to form poly(2-(methacryloyloxymethyl)-4,4-dimethyl-6- oxomorpholin-4-ium) (polyCB-Ring). The polyCB-Ring is an antibiotic polymer bearing quaternary

ammonium groups, which can be reversed to zwitterionic polyCB-OH form through hydrolysis with weak

base (pH 7.3, Na2CO3) to repel the corpses of killed cells. This antibacterial polymer coating has achieved control over two extreme properties, hydrophobicity and hydrophilicity.

4.3.2 Function Switching through Thermal-responsiveness

The conformation of certain polymer chains collapses when the solution temperature exceeds its lower critical solution temperature (LCST) or falls below upper critical solution temperature (UCST), inducing phase separation. Poly(N-isopropylacrylamide) (PNIPAAm) is commonly exploited as thermally a responsive polymer due to its sharp LCST at 30~35 ºC178 and has been utilized in the field of antibacterial polymers. Mattheis et al. synthesized a series of thermo-switchable antibiotic PAEMA-co-PNIPAAm, with

PAEMA as the biological active component.179 The conformation of the copolymer was found to transition from coil to globule after the temperature was increased past the LCST, which was related to different antibiotic activities of the copolymers for different strains of bacteria. The antibiotic effect toward Gram positive bacteria B. subtilis remain similar before and after phase transition, while that on Gram negative bacteria E. coli was significantly enhanced after the transition. This phenomenon was explained by the shift of amphiphilicity due to the conformation change, in which the globule conformation has higher hydrophobicity and consequently higher efficiency to disrupt cell membrane. Coatings of such copolymers were prepared by crosslinking the polymer and cellulose (cotton) with poly(ethylene glycol) diglycidyl ether

(PEGDE), with similar phase transition and antibiotic behavior observed. Mi et al. prepared thermoresponsive antibacterial hydrogel dressings from PNIPAAm-b-poly(N-1-(ethoxycarbonylmethyl)-N-(3- acryloylamino-propyl)-N,N-dimethyl ammonium salicylate-b-PNIPAAm (PNIPAAm-b-PCBAA-1-C2 SA- b-PNIPAAm) triblock copolymers synthesized by RAFT polymerization.180 When applied to wounds, the

PNIPAAm block instantly transitions from water soluble to insoluble to form a physically crosslinked hydrogel due to the temperature at the wound site being higher than the LCST. Meanwhile, salicylic acid is released to prevent the wound from contamination. The cytotoxicity of such hydrogels was also evaluated and it was shown that the dressing had good biocompatibility and the initial attachment of mammalian fibroblast cells were more favored compared to polystyrene for tissue culture.

5. Future Directions

5.1 Deeper Understanding of Antibacterial Polymer-Cell Interactions

Tests on antibacterial polymers have gradually advanced to assessing their efficacy and efficiency to prevent the formation of biofilm in vitro in realistic settings.181,182 However, recent trials did not show the anticipated action against bacteria.182 This means that there is still need to improve the performance of antibacterial polymers, for which a clear vision of the mechanism of action is necessary. However, existing explanations on antibacterial polymers do not yet fully describe detailed interactions between antibacterial polymers and bacteria, while the proposed mechanisms for antibiotic polymer coatings are often conflicting.

Current investigations mainly focus on the fundamental physical/chemical properties of antibiotic polymers by investigating the effects of different structural parameters. However, new factors, such as the polymer chain conformation and dynamics, should also be considered, because they reflect the actual behavior of antibiotic polymers during the interaction and disruption of bacterial membranes. These factors can provide a more direct view of the mechanism of action. In the meantime, such valuable information has primarily been supplied by simulation and computation. This tool should be combined with experimental results in

future studies.

Of course, visualization of antibiotic processes is ideal for the solution to this problem. Fantner et al. has demonstrated the disruption fixed E. coli cell membranes by antimicrobial peptides using high-speed and high-resolution atomic force microscopy (AFM).183 The images showed that the smooth surface of cell membrane became disrupted 16 min after the introduction of antimicrobial peptide. It is believed that development in the imaging of such processes will lead to clearer insight of the interaction between polymers and bacteria.

5.2 Clinical Tests of Antibacterial Polymers In Vivo

Biofilms are a major pathway for nosocomial contamination, especially on the surface of medical devices having direct contact with our tissue. For this reason, improved biocompatibility of antibiotic polymers is essential for in vivo applications and intimate applications in vitro, such as for textiles, food packaging and coatings for water disinfection systems, etc. On the other hand, compared to the in vitro setting, the environment in vivo is more complicated, where the existence of multiple substances may hinder the mechanism of action of antibacterial polymers. A good example is the presence of serum, which is known to curtail the electrostatic interaction between the antibiotic polymer and bacterial membranes.184 Consequently, it is necessary to evaluate the behavior of candidate polymers in vivo.

Despite this recognition, most tests remain limited to in vitro assays against common bacterial species, such as S. aureus, E. coli and P. aeruginosa, with only a few tests designed for the in vivo setting.147,184 It is widely accepted that results of in vitro assays may not provide realistic information on the performance of such

materials in the human body. Henceforth, clinical trials of antibacterial polymers should be conducted185 to evaluate their potential for the intended applications, to identify clinical problems and more fundamentally to direct further improvements in antibacterial polymers.

6. Conclusions

This review summarizes the mode of action of different classes of antibacterial polymers, contact-active surfaces and some antibacterial polymers with multiple functionalities. A basic understanding of the mechanism of antibiotic polymers in solution has been recognized, but more detailed interactions between antibiotic polymers and bacterial membrane are expected, especially on the optimization of the balance of antibiotic action and cytotoxicity. Current mechanisms of immobilized antibiotic polymer have also been described, and these are not always compatible with each other. Furthermore, the design and application of antibacterial polymers with multiple functionalities are discussed, which significantly improves the antibacterial efficacy and effective life span. Future studies of antibacterial polymers should combine with interdisciplinary knowledge to achieve a deeper understanding of the mode of action. Interdisciplinary studies may be the key factor for the promising development of antibacterial polymers. Meanwhile, clinical tests of antibacterial polymers will provide more thorough information and provide new directions for further developments in this interesting and importance class of material.

Acknowledgements The authors would like to acknowledge support from the ARC Centre of Excellence in Convergent Bio-Nano Science and Technology (CE140100036).

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