Recent progress in polymer research to tackle infections and

Review Cite This: Biomacromolecules XXXX, XXX, XXX−XXX

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Recent Progress in Polymer Research to Tackle Infections and Antimicrobial Resistance Mohini Mohan Konai, Brinta Bhattacharjee, Sreyan Ghosh, and Jayanta Haldar* Antimicrobial Research Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru 560064, Karnataka, India ABSTRACT: Global health is increasingly being threatened by the rapid emergence of drug-resistant microbes. The ability of these microbes to form biofilms has further exacerbated the scenario leading to notorious infections that are almost impossible to treat. For addressing this clinical threat, various antimicrobial polymers, polymer-based antimicrobial hydrogels and polymer-coated antimicrobial surfaces have been developed in the recent past. This review aims to discuss such polymer-based antimicrobial strategies with a focus on their current advancement in the field. Antimicrobial polymers, whose designs are inspired from antimicrobial peptides (AMPs), are described with an emphasis on structure−activity analysis. Additionally, antibiofilm activity and in vivo efficacy are delineated to elucidate the real potential of these antimicrobial polymers as possible therapeutics. Antimicrobial hydrogels, prepared from either inherently antimicrobial polymers or biocide-loaded into polymer-derived hydrogel matrix, are elaborated followed by various strategies to engineer polymer-coated antimicrobial surfaces. In the end, the current challenges are accentuated along with future directions for further expansion of the field toward tackling infections and antimicrobial resistance.

1. INTRODUCTION Microbial resistance to conventional antibiotics is one of the biggest challenges in the field of biomedical research.1−3 Rapid emergence of drug-resistant microbes coupled with a dwindling rate of novel antibiotics entering into the clinical pipeline has created an alarming situation worldwide.4,5 On top of that, biofilm formation has further complicated the treatment of infections with conventional antibiotics.6−9 Microbes can form biofilms on abiotic surfaces (hospital walls, medical devices, implants, etc.) as well as biotic surfaces (surgical sites, wounds, lungs, urinary tract, cardiac tissues, bones, etc.) often leading to chronic infections.10−13 For combating this situation, there is a pressing need for the development of new classes of antimicrobial agents whose mechanism of action is different from most of the existing antibiotics. Additionally, novel approaches to prevent biofilm formation as well as disrupt established biofilms and cure infections are in high demand. Toward this direction, various polymer-based antimicrobial strategies have emerged, which can provide a promising solution to this global clinical problem. Inspired by antimicrobial peptides (AMPs), a plethora of polymers (new synthetic polymers and modification of existing polymers) have been developed that were shown to display potent activity against both drug-sensitive and -resistant microbes. These antimicrobial polymers primarily target the microbial membrane and display less propensity to trigger the development of resistance. Some of these polymers have also shown potent antibiofilm property and excellent in vivo efficacy even in biofilm-associated infection models (such as burn wounds, surgical wounds in mice). Some are also reported to potentiate obsolete antibiotics (such as tetracycline, rifampicin, eryth© XXXX American Chemical Society

romycin, etc.) toward multidrug-resistant Gram-negative bacteria and their biofilms. Importantly, this antibiotic resensitization has also been shown in in vivo mice model. Furthermore, polymer-based antimicrobial hydrogels have been developed as an effective strategy for treating infections. Hydrogels have widespread applications in different biomedical fields such as drug delivery, tissue engineering, wound dressing, and so forth. Various research groups have developed hydrogels based on inherently antibacterial polymer scaffolds or biocideloaded polymeric matrices, which can serve as an excellent class of wound care materials. Additional properties of these antimicrobial hydrogels include long-term antimicrobial activity and sustained release of loaded antimicrobial agents, which make them immensely useful in curing infections. Importantly, their bioadhesive nature and local antimicrobial action render them effective for treating infection in avascular regions (such as bone infections, burn-wound infections, etc.). In everyday life, we come across several surfaces that serve as potent reservoirs of deadly pathogens, allowing the formation of biofilms, which are at the core of most infections. Preventing the adherence and colonization of bacteria on biomedical devices and other relevant surfaces can limit the spread of surface-associated infections. Thus, rendering antimicrobial activity to surfaces by polymeric coating is an effective strategy. Depending on the applications, various strategies of polymeric Special Issue: Biomacromolecules Asian Special Issue Received: March 15, 2018 Revised: April 24, 2018 Published: May 2, 2018 A

DOI: 10.1021/acs.biomac.8b00458 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 1. Cationic AMPs mostly adopt amphipathic topology either in aqueous solution or in the presence of bacterial membrane. Upon adsorption of these facially segregated AMPs, the membrane integrity is disrupted through various mechanisms. Among them, most of the common modes of actions are pore formation (toroidal and barrel-stave pore model) and nonspecific membrane disintegration (carpet model). According to the conventional toroidal pore model, AMPs insert themselves across the membrane to form peptide and lipid-lined pores, whereas in the barrel-stave pore model, only peptide-lined pores are formed across the membrane. In contrast, in the carpet model, AMPs solubilize the membrane into micellar structures through nonspecific interactions with membrane lipids. Adapted with permission from ref 16. Copyright 2011, Elsevier.

the bacterial cell envelope. In contrast, the outer leaflet of the mammalian cell membrane is zwitterionic in nature, consisting of phospholipids including phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine. Thus, the cationic groups of AMP preferably interact with the negatively charged bacterial membrane over zwitterionic mammalian membrane, which makes the selective killing of bacteria possible.15 After adsorption on the bacterial membrane through electrostatic interactions, AMPs disrupt the membrane integrity by various mechanisms.16 Pore formation and nonspecific membrane disintegration are the most common modes of actions (Figure 1). According to the conventional toroidal pore model, AMPs insert themselves across the membrane to form peptide and lipid-lined pores, whereas in barrel-stave pore model, only peptide-lined pores are formed across the membrane. In contrast, in the carpet model, AMPs solubilize the membrane into micellar structures through nonspecific interactions with membrane lipids. Importantly, this unconventional membrane targeting mode of action is advantageous as the resistance development propensity is relatively slower compared to that against conventional antimicrobial drugs, which function by targeting some of the biological processes in bacteria, such as cell wall biosynthesis, protein synthesis, DNA/RNA synthesis, or folic acid synthesis.17 However, the translation of AMPs as therapeutic agents is limited due to their low in vivo stability, high manufacturing cost, and low selectivity.18 To address these limitations, various small molecular synthetic mimics of AMPs have been developed in the past.19−45 Furthermore, various research groups have dedicated attention to developing synthetic polymeric molecules as promising AMP mimics.46−53 There are many excellent reviews available that highlight various aspects of antimicrobial polymer design.46−50 In the following section, we focus our discussion on some of these novel classes of antimicrobial polymers such as polynorbornene/polyoxanorbornenes, polymethacrylates/polyacrylate, poly-β-lactams, polymalemides, and polycarbonates whose systematic advancements have ascended rapidly in recent years. 2.1.1. Polynorbornene/Polyoxanorbornene. Tew and coworkers have extensively studied norbornene- and oxanorbornene-based polymers to develop them as antimicrobial

coating, for example, noncovalent and covalent modifications are being adopted. So far, the field has been reviewed primarily with an emphasis on individual facets of antimicrobial polymer research, such as AMP-inspired antimicrobial polymers, antimicrobial hydrogels, and surfaces. Herein, for the first time we focus our interest on discuss all three aspects together to combat infections and antimicrobial resistance including the most recent developments in the field. The initial part of this review focuses on AMP-inspired antimicrobial polymers, their use in treating infections, and also their capability to resensitize obsolete antibiotics toward multidrug-resistant bacteria. The discussion will start with design principles and details of structure−activity analysis including antibiofilm properties and in vivo efficacies. This will be followed by an overview of polymer-based antimicrobial hydrogels. Additionally, polymeric coatings that can impart antimicrobial properties to surfaces will be delineated.

2. ANTIMICROBIAL POLYMERS 2.1. AMP-Inspired Antimicrobial Polymer Design. As an alternative to antibiotic therapy, various classes of polymers have been developed as promising antimicrobial agents. The basis of designing this class of molecules is inspired by the hostdefense antimicrobial peptides (AMPs). As the part of the innate immune system, AMPs are widely found in both plants as well as in the animal kingdom.14 AMPs are known to clear the microbes from the host mostly by direct killing mechanism. In some cases, they also function through modulating the host immune system.15 With a few exceptions, the peptide sequence of AMPs are rich in cationic and hydrophobic amino acid residues. Thus, either in aqueous solution or in the presence of bacterial membrane, AMPs easily adopt an amphipathic topology where the hydrophilic and hydrophobic moieties are segregated into two opposite faces (Figure 1). The presence of phosphatidylglycerol, phosphatidylserine, or cardiolipin results in an overall negative charge in the bacterial membrane. Moreover, the presence of teichoic acids (in the case of Grampositive bacteria) and lipopolysaccharides (in the case of Gramnegative bacteria) also enhances the overall negative charge of B


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Figure 2. Antimicrobial polynorbornenes and polyoxanorbornenes.

agents.54−60 A series of polymers with diverse structural variations were prepared by ring-opening metathesis polymerization (ROMP). The structure−activity relationship studies were performed to understand the effect of molecular weight (MW), charge, hydrophobicity, counterions, and polymeric architectures on antibacterial efficacy. The first series of polymers were prepared with face-segregated monomers,

where the cationic ammonium groups and hydrophobic side chains were present on the opposite sides of the polymeric backbone (Figure 2A).55 Antibacterial studies of these homopolymers indicated an initial increasing trend in minimum inhibitory concentration (MIC) values upon increasing the hydrophobicity in the polymers; however, further enhancement of hydrophobicity beyond an optimum resulted in comproC


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Figure 3. Antimicrobial polymethacrylates and polyacrylates.

cationic charges per monomer unit were found to be advantageous compared to single-charged analogue. The best selective polymer showed a huge reduction in hemolytic activity (∼1000-fold selectivity) but retained similar antibacterial activity (MIC ≈ 30 and 50 μg/mL against E. coli and S. aureus, respectively). In another report, the random copolymers were prepared by incorporating hydrophilic groups such as sugar, zwitterionic, and polyethylene glycol moieties (Figure 2D),57 which resulted in a significant reduction in hemolytic activity (HC50 = 1500 μg/mL). However, at the same time, this also resulted in decreased antimicrobial activity (MIC = 200 and 150 μg/mL against S. aureus and E. coli, respectively). This fact highlighted the importance of cationic charge in designing antibacterial polymers, which needs to be maintained to achieve higher potency. Effort was then focused on exploring the oxanorbornene-based polymers. By using various diesterfunctionalized monomers, their hydrophobicity and charges were tuned. A series of homopolymers were prepared, where the hydrophobic and cationic ester functionalities were present in the same repeating unit (Figure 2E).58 These polymers displayed molecular weight-dependent biological activities; higher activity was obtained for polymers with lower molecular

mised activity. On the contrary, hemolytic activity showed a drastic increment due to such change. The HC50 (concentration corresponding to 50% hemolysis) for the highest hydrophobic polymers reached to values of 4000 μg/mL. Investigation of the influence of molecular weight suggested that it has no significant effect on antibacterial or hemolytic activity. For further optimization, a series of random copolymers was prepared by varying the ratios of two different monomers with intermediate and high hydrophobicity (Figure 2B).54 The random copolymers bearing higher content of intermediate hydrophobic monomers were found to display better antibacterial activity (MIC values in the concentration range of 40−80 μg/mL) with retained nonhemolytic nature (HC50 value of >4000 μg/mL). For investigating the effect of cationic charge density, a set of polymers were prepared by varying the number of ammonium groups per monomer unit from one to two or three (Figure 2C).56 Interestingly, the polymers bearing a higher number of D


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hemolytic activity with HC50 values below 1 μg/mL. In contrast, the lowest MW polymers were less toxic; the best selectivity (HC50/MIC) was achieved for the polymer consisting of 17% BMA. The role of hydrophobicity was further investigated by preparing a diverse series of polymers with varied hydrophobic monomers as well as the source of cationic charges (Figure 3A).62−65 A general observation suggested that the polymers consisting of high hydrophobic content were highly antibacterial and hemolytic as well, proving it to be unsuitable for development as selective antibacterial agents. Rather, the basic design principle appeared to be an optimum balance of cationic and hydrophobic content, which needed to be maintained to achieve the most active antibacterial polymer with minimum hemolytic activity. Haeussler and co-workers have reported a set of polymethacrylates where the cationic charge was contributed either by amino or guanidino groups (Figure 3B).66 Through a reversible addition−fragmentation chain transfer (RAFT) approach, amino group-bearing copolymers of varied MW were first synthesized with low polydispersity. Guanidino groups were introduced to prepare the guanylated analogues. Antibacterial studies indicated that guanylated polymers were much more potent as antibacterial agents as compared to the amino analogues. Several guanylated polymers displayed MIC values ranging between 2 and 20 μg/mL against both S. epidermidis and C. albicans. On the other hand, the amino group bearing polymers were less effective and showed MIC values at much higher concentrations (≥128 μg/mL). Hemolytic studies revealed that guanylated polymers of both low to moderate MW and hydrophobicity were less toxic. Thus, not only the balance between charge and hydrophobicity, but also the polymer chain length, are important criteria to obtain the optimum antimicrobial polymer with the best selectivity. In another report, being inspired by naturally occurring tryptophan-rich cationic AMPs, a series of polymethacrylates were prepared (Figure 3B).67 By varying the mole percentage of indole content, both amino and guanylated polymers were synthesized. Antibacterial and hemolytic studies revealed that guanylated polymer with optimum indole content displayed the most effective antibacterial activity with MIC values of 12 μg/ mL (against S. epidermidis) and 47 μg/mL (against MRSA) with minimal toxicity toward RBCs. Yang and co-workers have demonstrated that replacing the hydrophobic unit by a hydrophilic group can be a promising strategy to improve the selectivity (Figure 3C).68 With a retained antibacterial activity (MIC ≈ 4−16 μg/mL compared to 3−8 μg/mL), a drastic increase in HC50 values (HC50 = 256−512 μg/mL compared to 2−4 μg/mL) was seen for the polymethacrylate analogue bearing a hydrophilic unit. Yang and co-workers have reported a class of random polyacrylates that were prepared by using two monomers resulting in 2-carbon spacer arm (M2 monomer) and 6-carbon spacer arm (M6 monomer) from the polymer backbone to cationic moiety, respectively. A series of polymers was synthesized by varying the mole ratio of these two monomers, M1 and M6 (Figure 3D).69 An increase in the mole percentage of M6 enhanced the antibacterial potency against E. coli. However, a less pronounced effect was seen for S. aureus due to such variation. There was no noticeable change in hemolytic activity upon increasing the mole percentage of M6 up to 90%; the HC50 remained almost constant with a value of >2000 μg/mL. However, increasing the mole percentage to 100% resulted in a sharp decrease in the HC50 value (533-fold) over mammalian cells (RBCs). However, such drastic improvement was not observed for E. coli, the best selectivity being only 10-fold. This was due to the presence of the double membrane in Gram-negative bacteria, which resulted in lesser activity and thereby lesser selectivity.59 The next set of polymers were prepared by using the monomers where both ester functionalities were cationic.60 The hydrophobicity was then tuned by using various organic counterions, such as tosylate, benzoate, hexanoate, and dodecanoate (Figure 2G). A decreased antibacterial activity was noted with increasing counterion size. Such an effect was seen to be due to lesser membrane activity of the polymers with lesser effective charge, which was originated from strong polymer−counterion complexation upon increasing the size of counterions. The effect of charge density was also investigated by preparing a class of binary random copolymers combining previously used monomers bearing mono and dicationic ester functionalities, respectively (Figure 2H).60 The hydrophobicity was also regulated by including various aliphatic ester functionalities in the monocationic monomer. A copolymer bearing the majority of monocationic monomer composition (90%) and a methyl group in the hydrophobic ester functionality showed the best selectivity toward S. aureus (650-fold). In contrast, the polymers having monomers with greater hydrophobic ester functionality in place of the methyl group was found to show good activity against E. coli. This fact therefore indicated that different mechanisms of action are possibly involved in membrane disruption of Gram-positive and -negative bacteria. 2.1.2. Polymethacrylate/Polyacrylate. Significant efforts have been directed to this class of polymers focusing on the gradual development of various structural parameters. DeGrado, Kuroda and co-workers have extensively studied these polymers by varying monomer composition, modulating the chemical nature of hydrophobic groups, and so forth. In their first report, a set of polymethacrylates was prepared by using the monomers N-(tert-butoxycarbonyl)aminoethyl methacrylate and butyl methacrylate (BMA) (Figure 3A).61 By involving free radical copolymerization with controlled concentration of chain transfer agent methyl 3-mercaptopropionate (MMP), polymers of three different molecular weight (MW) ranges were prepared. The hydrophobicity was contributed by the BMA unit, which was varied by changing its mole percentage from 0 to 60%. In contrast, the cationic charge was introduced through deprotection of Boc-groups of N-(tertbutoxycarbonyl)aminoethyl methacrylate unit. The antibacterial studies indicated a gradual decrease in MIC values upon increasing the BMA composition to ∼30%; however, further increasing the hydrophobicity did not cause any noticeable change. The polymer of lowest MW (1300−1900 Da) containing ∼30% of BMA was found to be optimum showing an MIC value of 16 μg/mL against E. coli. The hemolytic activity also showed an increasing trend with increasing hydrophobicity. The large content of hydrophobic BMA unit (30−60%) in the polymers of high MW resulted in high E


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Figure 4. Antimicrobial poly β-lactams.

which showed 208-fold selectivity toward E. coli over RBCs. Kuroda, Kamigaito, and co-workers have reported a class of self-degradable antimicrobial polyacrylates, where the cationic groups are present in the side chains and the degradable ester linkages were introduced in the backbone of the polymers (Figure 3E).70 Synthesis involved both chain and step-growth radical polymerization by using the monomers t-butyl acrylate and 3-butenyl 2-chloropropionate. The t-butyl groups of the resulting polymers were then converted into a cationic ammonium analogue by reacting with 2-Boc-ethanolamine. A series of copolymers with different structural features (such as varied molecular weight, monomer composition, and amino functionality) were prepared. The polymer bearing primary amino groups was relatively hemolytic and displayed moderate antimicrobial activity against E. coli with the best selective polymer showing an MIC value of 104 μg/mL and HC50 value >500 μg/mL. In contrast, the corresponding analogue with quaternary ammonium group was not antibacterial (MIC against E. coli was >500 μg/mL) and displayed a similar HC50 value (>500 μg/mL). Recently, Gibson, Fullam, and co-workers have reported antituberculosis activity of this class of polymers (Figure 3F).71 The poly(dimethylaminoethyl methacrylate) displayed selective antimycobacterial activity over Gramnegative P. putida and E. coli with an MIC value of 31.25 μg/ mL against M. smegmatis. Upon removal of the backbone methyl group, the resulting poly(dimethylaminoethyl acrylate) displayed decreased antimycobacterial activity. Similarly, a corresponding poly(aminoethyl methacrylate) was also found to be ineffective (MIC = 500 μg/mL). The hemolysis studies

further indicated that poly(dimethylaminoethyl methacrylate) was not toxic toward mammalian cells. Almost no hemolysis was seen even at the concentration of 5000 μg/mL. In contrast, the corresponding poly(aminoethyl methacrylate) was comparatively toxic (∼10% hemolysis was seen at 5000 μg/mL). Importantly, Kuroda and co-workers have reported a primary ammonium ethyl methacrylate homopolymer that not only showed potent anti-S. aureus activity in vitro but also displayed in vivo efficacy.72 In rat nasal infection model, the polymer was capable of reducing the S. aureus burden by a significant amount (∼103 CFU). 2.1.3. Poly β-Lactams. Gellman, Liu, and co-workers have contributed significantly in the field of antimicrobial polymers. From β-lactam monomers, they have developed nylon-3 (poly β-lactams) polymers through anionic ring-opening polymerization. This class of polymers is closely similar to natural peptides and proteins (can be considered as nylon-2 derivatives) and hence the AMPs. This nylon-3 class of polymers with sufficient backbone flexibility is capable of adopting a globally amphiphilic but conformationally irregular structure in the presence of biomembranes. Hence, this class of polymers bearing both cationic and lipophilic side chains is capable of disrupting microbial membranes preferentially to eukaryotic cell membranes. Extensive structure−activity relationship studies have been conducted by varying the chemical structures of the monomers as well as their compositions.73,74 A series of Nylon-3 polymers were prepared, where the cationic charge is contributed by MM (monomethyl) or DM (dimethyl) subunits and the hydrophobicity was F


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Figure 5. Antimicrobial polymaleimides.

sensitive toward variation in the cycloalkyl ring hydrophobicity, and the MIC values ranged more or less at a similar concentration. However, the hemolytic activity was markedly affected due to such changes. Upon increasing ring size from CHx to CHp to CO, an enhanced hemolytic activity was distinctly observed due to an overall increase in hydrophobicity of the polymer. In another report, when the cyclic subunits were replaced by incorporating corresponding acyclic hydrophobic subunits (Figure 4B),75 the antibacterial efficacy was found to be diminished by both compromised antibacterial activity as well as higher hemolytic propensity. Such changes in biological activity highlighted the role of local backbone flexibility rather than alterations in subunit distribution along the polymer chain or the changes in subunit hydrophobicity. The effect was markedly pronounced for the polymers consisting of MM as the cationic subunit. In this MM-bearing polymer when the cyclohexyl subunit was replaced by the corresponding acyclic analogue, the MIC value against E. coli was increased from 50 to >200 μg/mL. Hemolytic activity was drastically increased to 60% upon increasing the concentration to 1000 μg/mL. However, the backbone flexibility could be restricted by incorporating α,α,β,β-tetramethyl-substituted hydrophobic subunit (TM).76 Indeed, such modification resulted in a better antibacterial polymer with lesser hemolytic activity even compared to the analogous polymer bearing a cyclic hydrophobic subunit (Figure 4C). The optimized polymer consisting of a 1:1 cationic subunit (DM) and hydrophobic subunit (TM) showed activity with MIC values in the range of ≤1.6−6.3 μg/mL against various bacteria that included B. subtilis, E. coli, VREF, and MRSA. Importantly, this polymer was nontoxic and displayed a high HC10 (polymer concentration that corresponds to 10% lysis of RBC) value of 400 μg/mL. Although, the corresponding analogous polymer bearing a cyclohexyl subunit showed MIC in the similar concentration range, it was highly toxic with an HC10 value of 19 μg/mL. Furthermore, a ternary class of nylon-3 polymers were prepared by incorporating the third subunit in addition to the cationic and hydrophobic subunits (Figure 4D).77 The third subunit was rationally selected to be modestly polar but uncharged, such as serine- or glycine-like moieties. Antibacterial

provided by the subunits such as CP (cyclopentyl), CHx (cyclohexyl), CHp (cycloheptyl), and CO (cyclooctyl) (Figure 4A). An investigation of the copolymer bearing the monomers MM and CHx suggested that antibacterial activity was not strongly affected by the polymer length. There was not much variation in the MIC values when antibacterial activity was tested against four species of bacteria (such as E. coli, B. subtilis, S. aureus, and E. faecium). A slight decrease in the MIC value was observed for E. coli with increasing polymer length, whereas a slight increasing effect was observed for S. aureus. Although these variations in MIC values were small, this observation indicated that larger polymers probably face trouble in penetrating the cell wall of Gram-positive bacteria. On the contrary, the hemolytic activity was found to be strongly influenced by the length of the polymer. Polymers bearing fewer subunits (an average of 10−30) showed very weak hemolytic activity with minimum hemolytic concentration (MHC) values of ∼1000 μg/mL. However, the propensity of hemolytic activity increases dramatically for the larger polymers (≳30 subunits), where MHC values reach even lower than MIC values. Thus, the shorter polymers that inhibited the bacterial growth at lower concentrations were optimum and displayed 10- to >100-fold selectivity toward killing bacteria over RBCs. In another investigation, the effect of end-group hydrophobicity was also explored by incorporating N-terminal linear alkanoyl units with varied chain length (Figure 4A). A nonmonotonic relationship of antibacterial activity was observed upon incrementing the alkyl chain hydrophobicity. In contrast, a different trend was noted for the hemolytic activity. Upon increasing the chain length to an octanoyl group, the polymer displayed a very weak hemolytic activity (MHC values ≈ 1000 μg/mL). However, an increased propensity in hemolytic activity was shown by dodecanoyl and higher alkyl chain analogues. This increasing trend continued until the octadecanoyl analogue and the MHC value reached ∼1 μg/mL (a value that is lower compared to MIC). Furthermore, the effect of backbone hydrophobicity on antibacterial efficacy was investigated by subunit variation, which was achieved using the monomers of tunable cycloalkyl ring size (Figure 4A). A general observation indicated that the bacteria were less G


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Figure 6. Antimicrobial polycarbonates.

altering antibacterial efficacy. Both the ternary polymers and the 1:1 binary polymer displayed MIC in the same concentration range of ≤1.6−25 μg/mL. In contrast, the ternary polymers revealed HC10 values of 200−400 μg/mL, which were much higher compared to the value (HC10 = 6.25 μg/mL) displayed by the binary polymer.

studies suggested that partial replacement of hydrophobic subunits, cationic subunits, individually or both by uncharged subunit, resulted in decreased hemolytic activity with retained antibacterial efficacy. For example, partial substitution (5−20%) of 1:1 DM and CHx-bearing polymers with serine- or glycinelike subunits drastically reduced the hemolytic activity without H


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Biomacromolecules 2.1.4. Polymaleimide. Our group has focused significant attention on the polymaleimide class of polymers. Starting from commercially available polyisobutylene-alt-maleic anhydride, preparation of this class of polymers was achieved through postfunctional modification in a two-step reaction. The highly reactive anhydride ring of the precursor polymer was first functionalized with 3- aminopropyldimethylamine. The dimethylamine moieties were then quaternized using various alkylating agents with similar degrees of quaternization (∼93−98%). The first series of polymers was prepared by incorporating the alkylating agents of various hydrophobic aliphatic chains (Figure 5A).78 Antibacterial activity of this class of polymers followed a parabolic trend upon increasing the alkyl chain length. The MIC values against both E. coli and S. aureus decreased from >1000 to 3−20 μg/mL upon increasing the alkyl chain from an ethyl to octyl group followed by an increased value (100−200 μg/mL) when the chain length was increased to a decyl group. In contrast, an increasing trend was observed for hemolytic activity (HC50 value decreased from >1000 to 4 μg/mL) with increasing alkyl chain hydrophobicity. Thus, the polymer analogue having an intermediate alkyl chain (such as pentyl group) was identified as the optimum candidate showing the best selectivity (114-fold selectivity toward S. aureus over RBCs). In an attempt to further improve the antibacterial efficacy, a hydrophilic moiety (oligoethylene glycol) was incorporated in place of hydrophobic alkyl chain (Figure 5A). Although the resulting polymer showed an impressive reduction in hemolytic activity, it also displayed a drastic loss in antibacterial potency (MIC value >1000 μg/mL against E. faecium, MRSA, VRE, E. coli, and P. aeruginosa), which prevented its effectiveness as an antibacterial agent. However, the next series of polymers was prepared by introducing ester and amide functionalities in the hydrophobic alkyl chain (Figure 5B−D).79 By introducing the ester and amide functionalities, biodegradability was also achieved in the system, which is an important property for an antibacterial polymer to have systemic applications. Both classes of polymers displayed a significant reduction in hemolytic activity. Interestingly, these polymers, especially the amide-bearing polymers displayed, similar MIC values as compared to the polymers consisting of hydrophobic aliphatic chains devoid of any functional group (Figure 5A).79−81 This was an important observation, which revealed the importance of hydrogen bonding in dictating selective antibacterial activity for the first time in the field of antibacterial polymers.79 Various studies including biophysical experiments and molecular dynamics (MD) simulations have established that amide-containing polymers strongly interact with the lipid head groups of the bacterial membrane owing to the presence of a greater hydrogen bonding capable moiety (-NH-). On the contrary, the ester polymers having lower hydrogen bonding capable moiety (-O-) resulted in lesser antibacterial activity. Furthermore, the importance of hydrogen bonding was confirmed by the fact that the amide-containing polymers retained their antibacterial activity even upon decreasing the aliphatic chain length attached to the amide functionality.80 This is a unique trend that was not generally seen earlier. On the other hand, the ester series of polymers displayed an obvious trend due to such variation. The decreased antibacterial activity resulted from the decrease in aliphatic chain length. The best selective amide-containing polymer not only showed potent in vitro antibacterial activity against S. aureus and E. coli (MIC = 31 μg/ mL) but also reduced the A. baumannii burden (∼2 Log) in

vivo in burn wound infections in mice. For the effect of side chain architecture to be explored further, a series of amidecontaining polymers was prepared by employing isomerization (regio- and stereo-), cyclization, and unsaturation in hydrophobic side chains (Figure 5C and D).81 Antibacterial studies revealed that the polymers with higher cyclic side chain (such as cyclohexyl, cyclopentyl, and methylcyclopentyl) were highly antibacterial with MIC values in the range of 4−16 μg/mL (against E. coli, S. aureus, MRSA, and VRE). The lower cyclic analogue (cyclopropyl), however, displayed moderate activity with MIC in the range of 16−125 μg/mL. The toxicity studies suggested that cyclopentyl and cyclohexyl analogues were highly toxic, displaying HC50 values of 80 and 45 μg/mL, respectively. In contrast, the methylcyclopropyl was comparatively nontoxic with an HC50 value of 250 μg/mL. This optimized polymer displayed ∼50−100-fold selectivity toward bacteria over RBCs. For the regio- and stereoisomeric effects on antibacterial properties to be investigated, the polymers with isomeric side chain were prepared by keeping the number of carbon atoms constant at four (identified as the optimum (methylcyclopropyl) for the above set of polymers). The polymers bearing iso-butyl, R/S sec-butyl, and t-butyl side chains, however, displayed similar antibacterial activity (MIC in the range of 4−31 μg/mL) and toxicity profile, thus indicating that isomerization of hydrophobic side chains does not have a significant role in regulating antibacterial efficacy. However, the effect of unsaturation in the side chain hydrophobicity resulted in a significant reduction in toxicity toward mammalian cells. Both analogue polymers bearing double bond (but-3-enyl) and triple bond (but-3-ynyl) unsaturation displayed MIC of 31 μg/ mL against S. aureus as well as E. coli showing HC50 value >1000 μg/mL. Thus, cyclization and unsaturation, rather than isomerization of side chain hydrophobicity, have a profound effect toward regulating the selectivity of antimicrobial polymers. 2.1.5. Polycarbonate. Owing to their biodegradable nature, the polycarbonate class of polymers has attracted the attention of various researchers. Rapid emergence of this class of antibacterial polymers has been seen in the recent past. Yang, Hedrick, and co-workers have investigated the effects of various parameters toward antibacterial activity by preparing a series of polycarbonates.82−86 Their synthesis involved organo-catalytic ring-opening polymerization (ROP) of various functional cyclic carbonate monomers. Initially, a class of triblock polycarbonates was prepared that readily self-assembled to spherical micelles in aqueous solution (Figure 6A).82,83 These selfassembled micelles bearing cationic charges on their periphery could selectively interact with negatively charged microbial membranes through electrostatic interactions. An increased local concentration of cationic charge resulting from micelle formation leads to better interactions with negatively charged microbial membranes. The first system was a triblock copolymer, which was prepared by sequential reaction of MTC bearing monomer (3-chloropropyl 5-methyl-2-oxo-1,3dioxane-5-carboxylate) and trimethylene carbonate (TMC). The final triblock polymer consisted of two poly-TMC (PTMC) blocks and one poly-MTC (PMTC)-based cationic block (Figure 6A). In contrast, the second system was a random copolymer prepared from the monomers 5-methyl-5(3-chloropropyl)oxycarbonyl-1,3-dioxan-2-one (MTC-PrCl) and ethyloxycarbonyl-1,3-dioxan-2-one (MTC-ethyl) (Figure 6A). The first series of polymers readily self-assembled into stable nano structures, selectively disrupted microbial memI


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Biomacromolecules branes, and inhibited the growth of B. subtilis, S. aureus, MRSA, E. faecalis, and C. neoformans without showing significant hemolysis. The optimized polymer displayed MIC values in the concentration range of 5.1−16 μM against various microbes, whereas negligible hemolysis could be seen even at a concentration of 81 μM. When this polymer was injected in mice intravenously, the LD50 (the lethal dose at which half the mice were killed) was found to be 31.5 mg/kg. Importantly, the acute toxicity studies of this polymer indicated no adverse effect to the major organs (such as liver and kidney), and the balance of electrolytes in the blood remained unchanged. The second system, however appeared to reveal highly dynamic selfassembled nature and hence was capable of disassembling in the presence of microbial membrane, leading to membrane disruption.83 These polymers showed microbial growth inhibition (that included Gram-positive and -negative bacteria and fungi) with MIC values ranging between 63 and 500 μg/ mL. These values were higher compared to the critical micelle concentration (CMC) of the polymers. In the next example, a series of polycarbonate homopolymers was prepared where the hydrophobic/hydrophilic balance was controlled by varying the spacer between charged quaternary ammonium moiety and the backbone of the polymer. Some of these homopolymers displayed potent activity against various microbes (MIC values ranging between 4 and 125 μg/mL). Unlike the previous polymers, antibacterial activity did not involve self-assembly, which was clearly indicated by the higher CMC values compared to the concentration at which they showed antibacterial activity. However, these homopolymers were significantly hemolytic; the HC50 of the most active polymers were in the concentration range 125−500 μg/mL. Next, the role of the cationic group structure on the antimicrobial and hemolytic activities was extensively interrogated. A class of polycarbonates was prepared with quaternary ammonium groups of diverse nature (Figure 6B).85 In the case of polycarbonates quaternized with N,N-dimethyl alkylamines of various alkyl chain lengths, a significant improvement in antibacterial activity was seen with retention of nonhemolytic activity up to a threshold alkyl chain hydrophobicity beyond which selectivity was lost due to increased hemolytic activity. Upon increasing the chain length from methyl to butyl group, the antibacterial activity against S. aureus and E. coli was improved by 16- and 8-fold, respectively. While retaining the nonhemolytic properties (HC50 > 4000 μg/mL), the MIC value was diminished from 62.5 to 3.9 μg/mL and 125 to 15.6 μg/ mL for S. aureas and E. coli, respectively. Interestingly, when the antibacterial efficacy of hexyl- and cyclohexyl-bearing polymers were compared, the cyclic analogue was found to be less toxic (HC50 = 250 μg/mL) compared to that of the acyclic analogue (HC50 = 15.6 μg/mL), although the antibacterial activity was similar. However, the polymer bearing a benzyl group displayed a similar trend in antibacterial and hemolytic activities compared to those of the cyclohexyl analogue; the only notable improved activity was seen for P. aeruginosa (MIC value decreased from >500 to 125 μg/mL). The polycarbonate containing 1-methylimidazolinium as the cationic group displayed high selectivity toward killing E. coli and S. aureus, as it was nonhemolytic with an HC50 value >1000 μg/mL. Upon increasing the alkyl chain length to butyl group, the antibacterial activity was increased. However, the selectivity was compromised due to an undesirable increase in its hemolytic activity (HC50 = 250 μg/mL). In an effort for the antibacterial efficacy to be further optimized, another series of polycar-

bonates was prepared where two different quaternary ammonium moieties were randomly distributed in the structure (Figure 6C).85 The antibacterial and hemolytic studies indicated that 50% content of the N,N-dimethylbutylammonium group was sufficient to improve the hemolytic activity by 3-fold, and the activity was enhanced against a wide spectrum of microbes. In another report, a series of polycarbonates were prepared with propyl or hexyl side chains quaternized with various nitrogen-containing heterocycles (Figure 6D).86 These polycarbonates were found to have enhanced activity against various strains of bacteria and fungi as compared to the corresponding analogue polymers quaternized with trimethylamine. Possibly due to a greater balance between the cationic charge and hydrophobicity, this marked improvement in antimicrobial efficacy was observed. More importantly, these polymers demonstrated high HC50 values (mostly >1000 μg/ mL) and hence were highly selective toward microbial killing. Jin, Swift, and co-workers have reported a class of guanidinylated polycarbonates prepared from postsynthesis modification of alkyne-bearing polycarbonates using click chemistry (Cu(I)-catalyzed azide−alkyne cycloaddition) (Figure 6E).87 A series of polymers with various molecular weights (8, 15, and 30 kDa) and charge densities were prepared. These guanidinylated polycarbonates displayed broad-spectrum antibacterial activity with low toxicity toward mammalian cells (HC50 > 1280 μg/mL). The lowest molecular weight homopolymer bearing 100% guanidinylated repeating unit revealed the best activity with MIC values of 40 and 20 μg/mL against E. coli and S. epidermidis, respectively. Even upon diluting the charge density by reducing the guanidine content from 100 to 70 and 50%, there was no significant loss in antimicrobial activity. However, further dilution to 20% resulted in significant reduction in antibacterial potency. Recently, Cai and co-workers reported a class of antimicrobial polycarbonates consisting of primary amino groups (hydrophilic) and phenyl moieties (hydrophobic) (Figure 6F).88 A series of polymers were prepared by varying the ratio of hydrophilic and hydrophobic monomers. To study the effect of monomer sequence on antimicrobial activity, both random (randomly arranged hydrophilic and hydrophobic monomers) as well as diblock (defined hydrophobic and hydrophilic segments) copolymers were prepared. The polymer containing only hydrophilic amino groups was ineffective. In contrast, the polymers consisting of both hydrophilic and hydrophobic groups displayed potent antibacterial activity. In the case of diblock copolymers, antibacterial activity was not altered much upon varying the number of hydrophobic groups. This was possibly due to the formation of stable nanomicelle by the diblock copolymers. Thus, the hydrophobic groups are sequestered, and hence, similar antimicrobial activity resulted. This was clearly established by the fact that a random copolymer that consisted of the same number of hydrophilic and hydrophobic groups revealed much better antibacterial efficacy over diblock polymer. Further structural optimization of the random copolymers resulted in an optimum candidate containing 20 hydrophilic and 20 hydrophobic groups, which displayed excellent antibacterial activity with MIC values of 0.17, 0.55, and 0.55 μM against MRSA, MRSE, and VREF, respectively. Importantly, this polymer was not hemolytic even at a concentration of 1000 μg/mL. Yang, Hedrick, and coworkers have developed an antibacterial polycarbonate that is not only active against S. aureus and MRSA in vitro but also has potent in vivo efficacy.89 In a mouse model of systemic J


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Figure 7. (A) Chemical structures of the membrane-active polymaleimides. (B) Antibacterial activity of antibiotics and cationic polymers (Qn-prAP) against stationary phase A. baumannii. (C) Disruption of A. baumannii biofilms. Scale bars, 5 μm. (D) In vivo anti-A. baumannii activity against a mouse model of burn-wound infections. Reproduced from ref 97 under a Creative Commons Attribution 4 International License. https:// creativecommons.org/licenses/by/4.0/. Copyright 2017, Uppu et al.

infection, the polymer was found to reduce MRSA viability in the blood better than the last resort anti-MRSA antibiotic vancomycin. Additionally, no adverse effect to the liver and kidney as well as blood electrolytes could be seen after administration of the polymer. Recently, the same group reported a class of guanidinium-functionalized polycarbonates that not only displayed in vivo activity against MRSA but also showed potent efficacy against multidrug-resistant Gramnegative bacteria such as A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa.90 2.2. Antibiofilm Efficacy and Resensitization of Obsolete Antibiotics. Biofilms are well-structured surfaceadherent communities where bacteria are embedded within their self-produced extracellular matrix composed of extracellular DNA, proteins, and polysaccharides.91,92 To protect themselves from environmentally harsh conditions or any external harmful agents, bacteria are known to enter into a biofilm lifestyle that is physiologically distinct from planktonic behavior.92 Initially reported as a secret bacterial lifestyle, biofilm is now recognized as a major threat in the treatment of infections. This is the underlying cause behind chronic or persistent infections that are almost impossible to treat with conventional antibiotic therapy. The extracellular matrix poses a physical barrier for the antibiotics to penetrate into the matrix, conferring protection to the community.6 Moreover, the majority of microbial populations are present in metabolic dormancy or a molecular persistent state. This metabolically inactive population is tolerant to antibiotic treatment as they shut down the target sites of conventional antibiotics. Additionally, the increased bacterial cell density inside the biofilms favors the transfer of resistance genes, which leads to faster development of antibiotic resistance in the microbial

world. Altogether, the treatment of biofilm-associated infections remains a critical challenge that needs to be resolved urgently. Recently, antimicrobial polymers have attracted attention for resolving this challenge, and our group has taken a pioneering step in this direction. Amide side chain bearing optimized polymaleimide is capable of disrupting established biofilms of A baumannii,93 the topmost critical bacteria according to the priority pathogen list published by World Health Organization (WHO).1 Kuroda and co-workers have reported the antibiofilm property of methacrylate polymers against cariogenic bacterium S. mutans. By killing the planktonic bacteria, the polymers not only prevented biofilm formation but also removed significant biomass of preformed S. mutans biofilms.94 Gellman and coworkers also reported antibiofilm property of nylon-3 polymers.95 Interestingly, the antibiofilm efficacy was tested against C. albicans, the most notorious fungal pathogen, whose infections are often associated with biofilm formation. These nylon-3 polymers were found to display excellent biofilm inhibition property against drug-resistant C. albicans strains. When the mature biofilms were challenged with the polymers, many dead fungal cells were found inside the biofilms. For battling against a rapidly emerging AMR storm, antimicrobial polymers have also been investigated to resensitize obsolete antibiotics. Our group has reported that polymaleimide was capable of restoring the activity of the tetracycline class of antibiotics toward blaNDM‑1 containing K. pneumoniae and E. coli clinical isolates by >80−1250 fold.96 The principle behind resensitization was due to increased uptake of antibiotics resulting from membrane depolarization and permeabilization caused by the polymer. Owing to the membrane-active nature, the propensity of resistance development against the antibiotic was also reduced drastically in the combined formulation. In K


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strengthened the mechanical property, but due to the decrement in free amino groups, antimicrobial activity was decreased. This antibacterial hydrogel also displayed superior tissue adhesion property due to the presence of free dopamine functionalities. Moreover, it exhibited potent in vivo hemostatic property and accelerated wound repair capability within 7 days (Figure 8C).99 In another example, ε-poly-L-lysine-initiated polymerization of 1-vinyl-2-pyrrolidinone and N-methylol acrylamide was performed to obtain the precursor polymer for hydrogel preparation. In the presence of the enzyme plasma amine oxidase, aldehyde functionalities were generated in situ by oxidation of amino groups. These aldehyde groups reacted with the remaining amino groups and constructed the hydrogel network. The optimum formulation inhibited the growth of S. aureus (90%) and E. coli (80%). This hydrogel also displayed almost 97% wound closure within 14 days.100 Christman and co-workers have reported a class of antibacterial hydrogels, where alanine and lysine were used to prepare the peptide sequence.101 Hexamethyldisilazane-mediated ring-opening polymerization was employed to prepare this class of antibacterial polypeptides with various hydrophilicities. 6-Arm polyethylene glycol (PEG)-amide succinimidyl glutarate was used as the cross-linking agent. The three-dimensional network structure was then built by amide bond formation between carboxylic acid groups of cross-linkers and amino functionalities of polypeptides. The optimized hydrogel displayed significant antibacterial activity against both S. aureus and E. coli.101 In another example, Schneider and co-workers reported a 20residue peptide by incorporating various amino acids capable of adopting an amphiphilic β-hairpin structure due to segregation into hydrophobic and hydrophilic faces. This β-hairpin structure was capable of constructing a mechanically rigid hydrogel upon self-assembly by involving various noncovalent interactions, which when coated onto the surface inhibited the growth of MRSA (2 × 108 CFU/dm2).102 3.1.2. Chitosan-Based Hydrogels. The naturally occurring polysaccharide chitosan is well-known for its inherent antibacterial activity. Significant effort has been directed to developing antibacterial hydrogels by employing this polymer and its derivatives. Hsieh and co-workers have reported a hydrogel prepared by mixing chitosan and γ-poly(glutamic acid). The principle of gelation was based on polyelectrolyte complexation between the cationic-charged chitosan and the anionic component (γ-poly(glutamic acid)). The hydrogel displayed activity against both S. aureus and E. coli along with fibroblast cell proliferation in vitro, demonstrating that it is promising to be developed as a wound care material.103 Hanton and co-workers have reported a hydrogel prepared from polydextran aldehyde and chitosan through imine bond formation between aldehyde and amine groups.104 The hydrogel was effective against S. aureus, S. pyogenes, E. coli, and C. perf ringens, whereas no significant activity was observed against C. albicans and P. aeruginosa.104 In another strategy, researchers have come up with various hydrogels where chitosan was synthetically modified to improve the antibacterial efficacy. The most well-known synthetic strategy involves incorporation of permanent cationic charges in chitosan prior to its use for hydrogel preparation. Chan-Park and co-workers have reported a new hydrogel based on dimethyldecylammonium chitosan-graf t-poly(ethylene glycol) methacrylate and poly(ethylene glycol) (PEG) diacrylate.105 The UV irradiation leads to cross-linking of methacrylate moieties leading to hydrogel formation. This hydrogel showed excellent activity

vivo studies suggested that the combined treatment was effective as compared to individual treatment, which showed a significant reduction (∼2 log) in bacterial burden (E. coli) in a mouse model of thigh infections with good safety profile for its systemic applications. Furthermore, these polymaleimides were reported to potentiate antibiotics (erythromycin and rifampicin) to disrupt biofilms (Figure 7C), and the combined formulations were also capable of preventing the planktonic growth of dispersed cells from biofilms.97 More importantly, the combination reduced bacterial burden significantly in biofilm-associated infection models (burn and surgical wounds) caused by A. baumannii (Figure 7D) and Carbapenemaseproducing K. pneumoniae.

3. POLYMER-BASED ANTIMICROBIAL HYDROGEL A hydrogel is defined as a three-dimensional matrix that is capable of holding a large amount of water through hydrophilic interactions with its molecular network. Hydrogels have widespread applications in many biomedical fields such as drug delivery, tissue engineering, cell culture, coatings, and wound dressing, and so forth. Recently, it has also attracted enormous attention for their development to treat infections. This part of the review will focus on polymer-based antimicrobial hydrogels developed by various research groups. They can be primarily categorized into two groups: inherently antimicrobial hydrogels (where the polymers themselves are antimicrobial) and biocide-loaded hydrogels (where antimicrobial agents are loaded in the hydrogel matrix). 3.1. Inherently Antimicrobial Hydrogels. This class of hydrogels is engineered from the precursor polymers that are inherently antimicrobial (Table 1). Gelation processes involve various kinds of covalent (such as imine, amide bond, etc.) as well as noncovalent (such as electrostatic and H-bonding) interactions that ultimately help to construct the threedimensional cross-linked polymeric network. Killing of the microbes primarily takes place upon contact with the hydrogels. Antimicrobial property can be introduced in the polymers either by incorporation of various antibacterial moieties through postfunctional modification or by de novo polymer synthesis. In most of the cases, quaternary ammonium groups are incorporated to achieve the antimicrobial property. 3.1.1. Polypeptide-Based Hydrogels. Cationic amino acidbased polypeptides, such as ε-poly-L-lysine, are well-known for their inherent antimicrobial property owing to the presence of amino groups. There has been significant effort toward the development of antimicrobial hydrogels using this polypeptide.98−101 Chan-Park and co-workers have reported a hydrogel system based on ε-poly-L-lysine-graf t-methacrylamide, where the polymer cross-linking was accomplished through UV polymerization.98 These hydrogels displayed wide-spectrum antibacterial activity against various pathogens such as E. coli, P. aeruginosa, S. marcescens, and S. aureus. Additionally, it showed antifungal activity against C. albicans and F. solani. When the optimized hydrogel was challenged with microbes, it displayed ∼3 log reduction in the case of bacteria, whereas ∼1 log reduction was observed for fungi.98 Xu and co-workers have demonstrated a dopamine-modified ε-poly-L-lysine- polyethylene-glycol-based hydrogel (Figure 8).99 The dopamine moieties of the precursor polymer underwent horseradish peroxidase enzyme-induced cross-linking to build up the gel matrix (Figure 8A). The hydrogel displayed activity against S. aureus and E. coli (Figure 8B); however, increasing the amount of dopamine substitution in the polylysine backbone L


M

tetra acrylate polyethylene glycol and tetrasulfhydryl polyethylene glycol poly(L-lactide)-b-poly(ethylene glycol)-b-poly(Llactide)

quaternized ammonium moiety bearing polycarbonate

polydextran aldehyde

N-2-(hydroxyl propyl) 3-trimethyl ammonium chitosan chloride

thiol-terminated block copolymers of polyethylene glycol

benzaldehye-functionalized poly(ethylene glycol)co-poly(glycerol sebacate)

quaternized chitosan-grafted aniline

dextran aldehyde

chitosan


γ-poly(glutamic acid

chitosan

quaternized chitosan-grafted aniline

self-cross-linked

20-residue β-hairpin peptide

poly(ethylene glycol) diacrylate

electrostatic interaction

6-arm polyethylene glycol (PEG)-amide succinimidyl glutarate

polypeptide of lysine and alanine

dimethyldecyl ammonium chitosan-graf t-poly(ethylene glycol)methacrylate

noncovalent interaction

self-cross-linked

ε-poly-L-lysine-grafted poly(1-vinyl-2-pyrrolidinone-co- N-methylol acrylamide)


thiol−ene chemistry

imine bond formation



UV-polymeization


amide bond formation

enzyme-induced imine bond formation

horseradish peroxidase crosslinking of dopamine

self-cross-linked

gelation mechanism UV-polymerization

dopamine-modified ε-poly-L-lysine-polyethylene glycol

cross-linker self-cross-linked

ε-poly-L-lysine-graf t-methacrylamide

antimicrobial polymer

Table 1. Inherently Antimicrobial Hydrogels microorganism tested E. coli P. aeruginosa S. marcescens S. aureus C. albicans F. solani E. coli S. aureus E. coli S. aureus E. coli S. aureus S. aureus MRSA S. aureus E. coli S. aureus S. pyogenes E. coli C. perf ringens E. coli S. aureus F. solani E. coli S. aureus E. coli S. aureus S. aureus E. coli P. aeruginosa MRSA VRE β-lactam-resistant K. pneumoniae S. aureus E. coli C. albicans E. coli S. aureus MRSA VRE A. baumannii

111

110

109

108

107

105

104

103

102

101

100

99

98

ref

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Review 116


UV polymerization


self-cross-linked

tris(2-aminoethyl)amine, hydroxyethyl acrylate glycerol dimethacrylate

branched polyethylene imine

ABA-type triblock copolymer (A = catechol functionalized and comprised of polyethylene glycol; B = poly {[2(methacryloyloxy)-ethyl] trimethylammonium iodide} 3,4en-ionene


POEGMS (mercaptosuccinic acid + oligo (ethylene glycol) POEGDMAM (fumaryl chloride + dodecyl bis(2-hydroxyethyl) methylammonium chloride + oligo (ethylene glycol)


against both bacteria and fungi including P. aeruginosa, E. coli, S. aureus, and F. solani with 99% killing. In-depth antibacterial mechanistic investigation suggested that the polycationic hydrogel attracts anionic bacterial membrane leading to internalization of membrane components into the nanopores present in the hydrogel. This “anion sponge”-like mechanism of hydrogel results in microbial death by membrane disruption. This PEGlycated chitosan hydrogel also showed proliferation of human primary epidermal keratinocytes in vitro. The hydrogelcoated contact lens, when implanted in rabbit eyes, showed no inflammation even after 5 days.105 More recently, Ma and coworkers have reported a series of hydrogels by using synthetically modified chitosan.106−108 In one example, they prepared a hydrogel by using aniline-grafted quaternized chitosan, where polydextran aldehyde was used as the crosslinker involving imine chemistry. The optimized hydrogel demonstrated antibacterial efficacy against both S. aureus (90% killing) and E. coli (95% killing). Importantly, the activity against E. coli was retained in vivo reducing ∼91% of the bacterial burden. This degradable hydrogel was also electroactive and could enhance the proliferation of myoblasts, indicating its possibility of application in tissue engineering.107 In another report, the cross-linker was changed to benzaldehyde-functionalized poly(ethylene glycol)-co-poly(glycerol sebacate) keeping the antibacterial polymer component same. In addition to antibacterial and electro-active properties, antioxidant property was incorporated in this hydrogel to improve the wound healing.108 Recently, our group has reported an antibacterial hydrogel developed from modified chitosan and polydextran aldehyde (PDA).109 Amino groups of chitosan were partially quaternized by using glycidyltrimethylammonium chloride and the rest formed imine bonds with aldehyde groups. This system showed activity against S. aureus, E. coli, and P. aeruginosa including drug-resistant bacteria (MRSA, VRE, and β-lactam-resistant K. pneumoniae) with killing efficacies of 90−99%. This injectable hydrogel also had hemostatic and bioadhesive properties and showed potent efficacy in preventing sepsis in a mouse model of cecal ligation and puncture. Only 12.5% mice survived in control without treatment, but the survival rate increased to 62.5% when gel was applied to the punctured site. The hydrogel-treated wounds healed in 20 days and prevented bleeding from the injured liver of mice, proving its hemostatic nature. This antibacterial hydrogel was biocompatible, indicating its safe usage as an effective antibacterial sealant and wound healing material.109 3.1.3. Polycarbonate-Based Hydrogels. In the recent past, Yang and co-workers dedicated immense attention to developing polycarbonate-based antibacterial hydrogels.110−112 Initially, they prepared a quaternary moiety containing a polycarbonate polymer via an organocatalytic ring opening polymerization (ROP) at room temperature, which was then conjugated with another thiol-terminated block copolymer of polyethylene glycol.110 The resulting polymer was then used to form the hydrogel network through Michael addition reaction employing tetra-sulfhydryl polyethylene glycol. The presence of a polyethylene glycol moiety contributed to the antifouling property, and the ammonium group played an important role in its bactericidal effect. The optimum composition of hydrogel was shown to be efficacious against a wide range of pathogens (such as S. aureus, E. coli, and C. albicans). No bacterial cell (S. aureus) was allowed to adhere onto the gel-coated surfaces due to its antibacterial as well as antifouling nature.110 Similarly, poly( D -lactide)-b-cationic polycarbonate-b-poly( D -lactide)

S. aureus E. coli P. aeruginosa

115

114

113

112 hydrophobic interaction ABA′-type triblock copolymer containing vitamin E and polyethylene glycol polycarbonate backbone having hydrophobic vitamin E functionality and quaternary moiety

microorganism tested gelation mechanism cross-linker antimicrobial polymer

Table 1. continued

K. pneumoniaeC. neoformans C. albicans E. coli S. aureus C. albicans E. coli S. aureus E. coli S. aureus S. pyogenes E. coli

ref

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N


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Figure 8. (A) Schematic representation of hydrogel preparation from dopamine-grafted poly-L-lysine polymer via enzyme-induced cross-linking. (B) Bactericidal effect of hydrogel on E. coli and S. aureus. (C) Wound area contraction when treated with hydrogel for wound closure and skin repair in vivo. Reproduced with permission from ref 99. Copyright 2017, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

bacteria with low toxicity toward mammalian cells.113 Schneider and co-workers have developed a novel class of injectable hydrogels from polydextran aldehyde and branched polyethylenimine, the underlying principle of gelation being imine bond formation.114 The optimized hydrogel was capable of killing both Gram-negative and -positive bacteria without showing much toxicity toward human erythrocytes. Importantly, in a murine infection model, the hydrogel was capable of killing S. pyogenes completely in 3−5 days. This bioadhesive (adhesive stress of 2.8 kPa) hydrogel was also effective in a model of cecal ligation and puncture, preventing sepsis and improving the survival of animals (62%).114 Recently, Zeng and co-workers developed a novel hydrogel prepared from an ABAtype triblock copolymer.115 The A block was catechol functionalized and also comprised of polyethylene glycol moieties, whereas the B block consisted of poly{[2(methacryloyloxy)-ethyl] trimethylammonium iodide}. In aqueous media, this triblock copolymer undergoes selfassembly through catechol-mediated hydrogen bonding and aromatic interactions. This hydrogel inhibited the growth of E. coli and also showed antifouling properties by preventing bacterial cell adherence. Interestingly, this hydrogel also showed excellent thermosensitivity and self-healing capability.115 Tiller and co-workers have come up with a double network hydrogel.116 The antimicrobial component was prepared by using 3,4en-ionene, which was synthesized by polyaddition of trans-1,4-dibromo-2-butene and N,N,N′,N′-tetramethyl-1,3propanediamine. Reaction between the bromine end groups of 3,4en-ionene and amino groups of tris(2-aminoethyl)amine led to the formation of the antibacterial hydrogel network. This was further swollen with 2-hydroxyethyl acrylate and glycerol dimethacrylate followed by photopolymerization. The resulting double network hydrogels displayed 5 log decrease of bacterial

(PDLA-CPC-PDLA) and biodegradable poly(L-lactide)-b-poly(ethylene glycol)-b-poly(L-lactide) (PLLA-PEG-PLLA) were used together to form an antimicrobial hydrogel at 37 °C driven by noncovalent interactions.111 This polycarbonatebased hydrogel inhibited the growth of various clinically isolated bacteria (e.g., VRE, A. baumannii, K. pneumoniae, etc.) showing almost complete killing. Biofilm disruption of bacteria and fungi was also seen with ∼80% reduction in population. A vitamin E-containing polycarbonate hydrogel was synthesized in the presence of two different polymeric systems.112 The hydrogels were formed based on hydrophobic interactions between vitamin E groups and have shown both bactericidal and fungicidal activities. Additionally, this Vitamin E-containing hydrogel was also efficacious in eradicating biofilms of various microbes with ∼80% killing capability. 3.1.4. Other Polymeric Hydrogels. Various other antimicrobial polymers are also used for the rapid development of hydrogels.113−116 Zhu and co-workers have reported a class of antimicrobial hydrogels prepared based on thiol−ene chemistry from two multifunctional poly(ethylene glycol) derivatives as precursor components.113 The antibacterial component was synthesized from condensation reaction of fumaryl chloride, dodecyl bis(2-hydroxyethyl) methylammonium chloride, and oligo(ethylene glycol). The positive charge was contributed by dodecyl bis(2-hydroxyethyl) methylammonium chloride, and fumaryl chloride introduced “ene” functionality. The thiolfunctionalized polymer was synthesized by performing a condensation reaction between mercaptosuccinic acid and oligo(ethylene glycol). Mixing these multienes and multithiols bearing poly(ethylene glycol) derivatives led to a threedimentional polymeric network by covalent cross-linking via a thiol−ene reaction. The optimized hydrogel exhibited excellent antibacterial activity against both Gram-negative and -positive O


small molecular biocide

dextran methacrylate

silver nanoparticle Ag/graphene

poly(ethylene glycol) diacrylate vinylpyrrolidone acrylic acid and N,N′-methylene bisacrylamide

antimicrobial peptide (Tet213; KRWWKWWRRC)

silver nanoparticle

branched catechol-derivatized poly(ethylene glycol)

methacrylated gelatin methacryloyl-substituted tropoelastin

vancomycin

N-2-(hydroxyl propyl)3-trimethyl ammonium chitosan chloride) and polydextran aldehyde

antimicrobial peptide (HHC10; HKRWWKWIRW-NH2)

vancomycin vancomycin

poly(β-amino ester) 4-arm-PEGNH2, 4-arm-PEG-NHS, and 4-arm-PEG-CHO

poly(ethylene glycol)diacrylate pentaerythritol tetrakis(3-mercaptopropionate)

gentamicin aminoglycoside antibiotics (such as netilmicin, isepamicin, capreomycin, ribostamycin, apramycin, amikacin, paromomycin, tobramycin, neomycin)

hyaluronic acid-poly(N-isopropylacrylamide) oxidized polysaccharides (such as dextran, carboxymethyl, cellulose, alginate, chondroitin)

gold nanoparticle hydrogen peroxide

ciprofloxacin-loaded poly(lactic-co-glycolic acid) nanoparticle gentamicin

dopamine methacrylamide polyethylene glycol diacrylate carboxymethyl-chitosan

acrylamide gelatin conjugated with hydroxyl phenyl propionic acid, succinyl chitosan carboxymethyl cellulose, and hydroxyl phenyl propionic acid-modified dendritic polyglycerol

ciprofloxacin

loaded biocide

poly(2-hydroxyethyl methacrylate and poly(ethylene-glycol diacrylate) acrylic acid

hydrogel-forming component(s)

Table 2. Biocide-Loaded Antimicrobial Hydrogels

P

UV polymerization

UV polymerization


UV polymerization enzyme-induced crosslinking

covalent cross-linking by oxidation of catechol UV polymerization UV polymerization

UV polymerization amide bond and imine bond formation imine bond formation

electrochemical synthesis UV polymerization genipin-induced crosslinking temperature change imine bond formation

gelation mechanism

121 123

S. aureus S. aureus E. coli P. aeruginosa S. epidermis S. aureus S. aureus

E. coli S. aureus E. coli S. aureus S. aureus P. putida E. coli S. aureus S. epidermidis MRSA E. coli S. aureus E. coli MRSA

S. aureus MRSA S. epidermidis

119 120

E. coli S. aureus

158

157

156

139 152−154

136 138

135

126

124 125

118

ref

MRSA

microorganism tested

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Figure 9. (A) Vancomycin (Van)-loaded hydrogel formulated from precursor polymers and antibiotic release profile from the gel matrix. (B) Inhibition of bacteria (MRSA) lawn formation: (left) no Van loaded into the hydrogel, (right) 0.3 wt % Van loaded hydrogel. (C) In vivo activity against the murine model of MRSA subcutaneous infection. Reproduced with permission from ref 126. Copyright 2017, American Chemical Society.

burden against S. aureus and E. coli, whereas a 4−7 log reduction was observed against P. aeruginosa with the change of weight percentage of hydrogel components. Importantly, this class of hydrogels showed Young’s modulus in the range of 0.64−1.34 MPa with a tendency to swell by absorbing water several times more than its own weight within less than a minute.116 3.2. Hydrogels Loaded with Antimicrobial Agents. Researchers have focused enormous attention on formulating hydrogels in which various antibacterial agents are entrapped within the matrix. Conventional antibiotics, metal ions, metal nanoparticles, antimicrobial peptides, or even small molecular antibacterial agent-loaded hydrogels have been formulated

aiming for developing them as antibacterial products. A biocideloaded hydrogel is not only able to kill microbes upon contact but also releases the loaded antibacterial agents from the matrix, taking care of a wide infected region effectively. The discussion will be focused on various classes of biocide-loaded hydrogels that have emerged rapidly in recent years (Table 2). 3.2.1. Hydrogels Loaded with Antibiotics. A series of hydrogel systems have been reported where the antibiotics are entrapped either by noncovalent or covalent interactions with the hydrogel components.117−130 Kannan and co-workers have reported an amoxicillin-loaded hydrogel prepared using 4 poly(amidoamine) [G4-(NH2)64] dendrimer with peripheral thiopyridyl terminations and 8-arm thiolated polyethylene Q


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Biomacromolecules glycol as the cross-linker.117 This hydrogel system was capable of sustained delivery releasing 50−72% of amoxicillin depending on the precursor composition. In a pregnant guinea pig model, the optimized hydrogel was well tolerated with no signs of change in vaginal pH and erythema, proving it to be useful for safe treatment of genital infections in pregnant woman without any effects on the fetus.117 In another example, ciprofloxacin was loaded into the hydrogel matrix, which was formulated by using a copolymer poly(2-hydroxyethyl methacrylate). Hydrogel coating onto titanium implant showed a zone of inhibition with a diameter of 3 cm against MRSA. It was also compatible with osteoblasts, making it suitable for treating bone-associated infections.118 In another example, ciprofloxacin was first incorporated in nanoparticles prepared from poly(lactic-co-glycolic acid).119 These nanoparticles were then incorporated into a hydrogel that was constructed using dopamine methacrylamide and polyethylene glycol diacrylate. Incorporation of this dopamine derivative increased the mechanical strength of this hydrogel, making it stable under physiological conditions and thereby inhibiting the formation of bacterial film (E. coli) in in vitro flowing conditions. Importantly, the hydrogel did not show any noticeable skin reaction or toxicity.119 Various gentamicin-loaded hydrogels have been emerging in recent years. Carboxymethyl-chitosan hydrogel was prepared using genipin as the cross-linker, where gentamicin was loaded into the hydrogel matrix. This hydrogel showed biofilm inhibition capability along with mammalian cell compatibility.120 Gentamicin-loaded thermoresponsive hydrogel was also prepared using hyaluronic acid-poly(N-isopropylacrylamide). As a result of thermoresponsiveness, the hydrogel showed sol−gel transition when the temperature was increased across its lower critical solution temperature (LCST). This makes the hydrogel capable of sustained delivery of antibiotics on demand. Gentamicin showed initial burst release from the matrix within 1 h (almost 62% drug release), and the release continued up to 7 days. When rabbits were injected with this formulation under infected conditions, it reduced the infection in 7 days.121 Even soy protein isolate was also investigated as a hydrogel matrix for sustained local delivery of gentamicin.122 Cheng and co-workers have demonstrated a class of hydrogels where the aminoglycoside antibiotics such as netilmicin, isepamicin, capreomycin, ribostamycin, apramycin, amikacin, paromomycin, tobramycin, and neomycin were loaded through cross-linking with oxidized polysaccharides (such as dextran, carboxymethyl cellulose, alginate, and chondroitin) via reversible imine bond formation.123 An increment of free amino groups in antibiotic and increased dialdehyde functionality resulted in decreased gelation time of these hydrogels. These self-healing hydrogels showed potent in vitro antibacterial activity against E. coli, P. aeruginosa, S. epidermidis, and S. aureus and almost completely cleared bacteria (S. aureus) in the murine skin infection model within 3 days.123 Dziubla and co-workers reported a vancomycin-loaded hydrogel where the free amino groups of vancomycin underwent Michael-type addition with the double bonds of polyethylene glycol diacrylate to form poly(β-amino ester).124 This vancomycinincorporated precursor formed a hydrogel upon UV irradiation. Owing to the covalent linkage, vancomycin has less activity against S. aureus compared to that of the physically entrapped hydrogel. However, the antibacterial efficacy could be maintained for a longer time due to covalent incorporation of vancomycin.124 In another report by Wu and co-workers, vancomycin-incorporated hydrogels were developed from 4-

arm poly(ethylene glycol) aldehyde, 4-arm poly(ethylene glycol) succinimidyl, and 4-arm poly(ethylene glycol) amine. It was found that incorporation of imine in the hydrogel matrix improved the adhesiveness but reduced the mechanical strength. In vivo evaluation on rabbits and pigs indicated that this antibacterial and biocompatible hydrogel is capable of preventing infection and able to aid in rapid hemorrhage control. Laser confocal microscopy was used to detect bacteria on the wound, and the vancomycin-loaded gel outperformed vancomycin solution and gauze. The optimized formulation stopped bleeding within 30 s, whereas normal gauze took 5 min to stop blood flow from the wound site.125 Our group has also reported a vancomycin-loaded hydrogel system where the vancomycin was incorporated through imine bond chemistry (Figure 9A). Because of the reversible nature of the imine bond, the vancomycin release happened in a pH-dependent manner (Figure 9A). In a mouse model, the hydrogel showed excellent antibacterial efficacy with a significant reduction of bacterial burden in the site of the infected wound (∼6.1 log reduction) and surrounding tissue (∼5.8 log reduction) (Figure 9C).126 3.2.2. Hydrogels Loaded with Metal Ions and Nanoparticles. The antimicrobial property of silver has been known for a long time. Various silver-based antimicrobial products are widely being used for wound dressing applications. To date, the antimicrobial property of other metal ions has also been reported. Unfortunately, the applications of the free metal ions are limited due to their toxicity toward mammalian cells. In order to address this, significant effort has been made to develop various hydrogels embedded with these antimicrobial metal ions.131−134 Many of these hydrogel systems have immense potential as wound care materials with minimal toxicity. Furthermore, various metal nanoparticles (such as silver, gold, zinc oxide, and copper) are also well-known to have potent antimicrobial activity. The antimicrobial mechanism of action of these metal nanoparticles is primarily based on the generation of reactive oxygen species. These metal nanoparticles are also known to exert toxic effects toward mammalian cells, which limit their application to topical usages. Thus, nanoparticle-loaded hydrogels are drawing focus in the current scenario.135−142 Messersmith and co-workers have reported a silver-releasing hydrogel, where the catecholfunctionalized polyethylene glycol was used as the precursor polymer.135 The catechol present in the polymer reduced the silver ion (from silver nitrate), leading to simultaneous silver nanoparticle formation as well as covalent cross-linking. This hydrogel releases 1% of the total silver present within 14 days. It inhibited bacterial growth with negligible toxicity toward mammalian cells.135 In another example, Kong and co-workers demonstrated in situ photoreaction of an aqueous mixture of silver nitrates, poly(ethylene glycol) diacrylate, and vinylpyrrolidone, forming a silver nanoparticle-loaded hydrogel system.136 This was not only capable of inhibiting bacterial growth but also displayed antifouling property. Likewise, Kim and co-workers reported another in situ silver nanoparticleloaded hydrogel formulation.137 In basic media (pH 9), the catechol moiety of dopamine methacrylamide (monomer) was at first protected by complexation with boron (using sodium tetraborate decahydrate solution). Then, the cross-linking polymerization of this protected dopamine monomer resulted in hydrogel formation. When the silver nitrate solution was added to the hydrogel, silver nanoparticles were formed owing to the reducing property of the catechol groups. The resulting R


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Figure 10. (A) Preparation of UV-polymerized biocide-loaded dextran-based hydrogel. (B) Biocide release profile from the hydrogels for 5 days. (C) In vitro antibacterial activity of hydrogels. (D) Bacterial count in skin tissue samples of mice under treated and untreated conditions. Reproduced with permission from ref 158. Copyright 2017, American Chemical Society.

efficacy with reduced toxicity toward mammalian cells.151 Hydrogen peroxide is also well-known to have antimicrobial activity due to free radical generation. Many hydrogels have been formulated with sustained release of hydrogen peroxide that displayed effective antimicrobial efficacy along with minimal toxicity toward mammalian cells.152−154 In recent years, AMP-loaded hydrogels have attracted attention. Liskamp and co-workers have reported a system where AMP (HHC10; H-KRWWKWIRW-NH2) was covalently loaded into a hydrogel, which was prepared using poly(ethylene glycol)diacrylate and pentaerythritol tetrakis(3-mercaptopropionate) through thiol−ene chemistry. This HHC10-loaded hydrogel showed activity against various pathogenic bacteria (such as S. aureus and S. epidermis).155,156 Annabi and co-workers have demonstrated another AMP (Tet213; KRWWKWWRRC)-loaded hydrogel system where the hydrogel matrix was prepared from methacrylated gelatin (GelMA) and methacryloylsubstituted tropoelastin (MeTro) involving visible lightinduced cross-linking. The ratio of MeTro/GelMA has an effect on elastic properties of the hydrogel with elastic modulus ranging from ∼4 to 33 kPa, and the adhesive strength was between ∼500 and 1100 kPa. This sprayable hydrogel showed antibacterial efficacy against both Gram-positive (MRSA) and Gram-negative bacteria (E. coli) with biocompatibility toward mammalian cells. Approximately 80% AMP release was observed within 72 h, and reduction in bacterial growth was seen within 24 h.157 Recently, our group developed an AMP mimicking small molecular biocide-loaded hydrogels, which was prepared by UV polymerization of dextran methacrylate (Figure 10A). The resulting hydrogel killed 108 CFU/mL

hydrogels showed antibacterial as well as antifouling properties and almost 98% wound closure within 15 days of the treatment.137 Yang and co-workers prepared a hydrogel by cross-linking a Ag/graphene composite with acrylic acid and N,N′-methylene bisacrylamide.138 The optimized formulation not only showed strong inhibition of bacterial growth in vitro but also displayed excellent in vivo efficacy with promising wound healing. Significant reports are also available on gold nanoparticle-based systems. For example, a gold nanoparticleloaded silk hydrogel showed bactericidal effect by local heating upon laser exposure. A polyacrylamide hydrogel loaded with gold nanoparticle-stabilized liposomes was reported, which released the liposomes leading to bacterial cell death through membrane fusion.139 Importantly, when the hydrogel formulation was topically applied on mouse skin, it did not show any significant toxicity. Additionally, significant progress has also been seen in other nanoparticle-based hydrogels that include zinc oxide, iron oxide, copper, and so forth.143−146 3.2.3. Miscellaneous. Significant effort has also been directed toward the development of hydrogels loaded with other antibacterial agents that include salicylic acid, chlorhexidine, AMPs, synthetic small molecular biocides, and so forth.147−159 Salicylic acid is one of the most essential medications used for day-to-day life, which is known to have anti-infective ability in addition to other bioactivities. Thus, various salicylic acid-loaded hydrogel systems have been reported for their effective delivery at the site of interest.147−150 Chlorhexidine is being used as disinfectant agents for topical applications. A chlorhexidine-loaded hydrogel was also developed by Yu and co-workers that displayed antimicrobial S


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Figure 11. Various adhesion-resistant polymers used in developing an antifouling surface. PEG: poly(ethylene glycol); PCBMA: poly(carboxybetaine methacrylate); PEGMA: poly(ethylene glycol) methacrylate; PMPC: poly(2-methacryloyloxyethyl phosphorylcholine); PSBMA: poly(sulfobetainemethacrylate).

bacteria (MRSA, S. aureus, E. coli) within 2 h (Figure 10C) and also displayed antibiofilm activity. It reduced ∼99.9% MRSA colonization in a mouse model of skin infection (Figure 10D). This hydrogel was found to be biocompatibility when skin toxicity was investigated in various animal models (such as a rat model of acute dermal toxicity, guinea pig model of skin sensitization, and rabbit model of skin irritation).158

(carboxybetaine) have been shown to inhibit bacterial adhesion and biofilm formation on surfaces (Figure 11).173,174 It is postulated that the zwitterionic head can associate with a large amount of water, making the material heavily hydrophilic. This leads to reversible interactions between the incident microbes and the surface, discouraging their adhesion. These zwitterionic surfaces were shown to be promising for coating medical devices because of their biomimetic nature, which first provides biocompatibility by reducing attachment of human cells to the device and second offers protection against bacterial biofilm formation. However, these types of surfaces do not fully address the problem of microbial contamination as they have no antimicrobial functionality. 4.2. Biocide-Releasing Surfaces. The release-based approach is focused on biocide leaching in which antimicrobial compounds are released and diffuse over time from a material surface, inducing death of either nearby (but nonadhered) bacteria or adhered bacteria.175 A variety of active antimicrobials such as antibiotics, metal salts, nanoparticles, bioactive species (e.g., phage virus), and so forth have been impregnated onto surfaces to develop biocide-releasing antimicrobial surfaces.160 One of the most heavily marketed and most widespread products for suppressing microbial growth is Microban. It incorporates Triclosan (5-chloro-2-(2,4-dichlorophenoxy)-phenol), a broad spectrum phenolic antimicrobial, into a surface. The antibiotic then leaches from the surface to perform the bactericidal function. Silver has long been known to be an antimicrobial, and the metal ions (Ag+) have significant antimicrobial activity, finding use in various commercial aspects. AgION Technology’s AgION and AcryMed’s SilvaGard are two of the most well-known commercial coating products that rely on the diffusion of Ag+ ions from the substrate material and inactivate the microorganisms. However, a possible drawback of silver-based antimicrobials is the cytotoxicity of Ag+ ions toward mammalian cells. Like silver, copper has long been considered to be a hygienic material.176 Another important way of developing release-active surfaces is by immobilizing bacteriophage viruses that infect prokaryotic cells.177 The development of a phage-containing wound dressing, hydrogel-coated silicone catheter, and so forth with lytic bacteriophages has been well documented.178,179 4.3. Contact Killing. In this approach, killing of microbes is furnished upon contact.180 This can be accomplished by anchoring various classes of antimicrobials (such as, antibiotics, antimicrobial peptide, quaternary ammonium compounds, N-

4. POLYMER-COATED ANTIMICROBIAL SURFACES Among the many ways by which infectious diseases spread, the contaminated surfaces are responsible for more than 50% of all microbial infections.160 Contaminated surfaces act as reservoirs of microbes and form the basis of biofilm-associated infections of medical devices and implants.161,162 Such infections not only increase the patient sufferings and healthcare costs but may also result in death. This scenario has attracted the attention of various researchers to develop antimicrobial surfaces that can prevent infections.163,164 In this direction, three general strategies have been adopted: (i) adhesion resistance (nonfouling strategy), (ii) biocide releasing (release-based strategy), and (iii) contact killing (contact-based strategy).165 In the adhesion resistance approach, surfaces discourage the adherence of microbial cells owing to its cell-repellent nature. Release-based coatings function by leaching the loaded antimicrobials, killing both adhered as well as adjacent microbes. In the contact killing strategy, microbes are killed upon contact with the coated surfaces, where both covalent and noncovalent surface modifications have been adopted. There are many reviews in the literature that have discussed various aspects of antimicrobial surfaces.166−171 In this review, with a brief overview of nonfouling, release-active and covalently modified surfaces, we will be emphasizing noncovalent strategies for the development of antimicrobial surfaces. 4.1. Adhesion-Resistant Surfaces. An antifouling surface focuses on inhibiting microbial adhesion or reducing the capacity of microbes to adhere on the surface. Therefore, biofilm formation is prevented by averting the initial microbial attachment to surfaces. Different materials are employed as antifouling coatings. One well-established method for preventing microbial adhesion to surfaces is to covalently attach them with a layer of poly(ethylene glycol) (PEG), which involves deposition of a self-assembled monolayer (SAM) on a substrate (usually a gold surface) followed by functionalization of the SAM to contain the required PEG functionality.172 Recently, polymers with zwitterionic head groups such as poly(phosphorylcholine), poly(sulfobetaine), and polyT


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Biomacromolecules halamines, etc.) with antimicrobial polymers.180−185 Polymerbased covalent modification of a surface can be done in two ways; (i) “grafting from” technique, where polymers are grown from the surface and (ii) “grafting to” technique, in which at first polymers with suitable functionality are prepared in solution followed by tethering them to surfaces. In contrast, a noncovalent approach is based on painting of water-insoluble and organo-soluble antimicrobial polymers on different surfaces. 4.3.1. Covalently Immobilized Polymeric Coating. There are various proposed mechanisms regarding how covalently immobilized polymers kill the microbes, which has been discussed elsewhere.186 Various groups had put initial efforts in to developing nonleaching permanent antimicrobial coatings on different surfaces. Klibanov and co-workers developed a covalent coating by immobilizing poly- (4-vinyl-N-alkylpyridinium bromide) on glass.187 N-Hexyl-PVP-coated glass slides were active against various pathogens including S. aureus, S. epidermidis, E. coli, and P. aeruginosa. The polymer was further covalently attached to various surfaces, which showed activity against various airborne and water-borne bacteria.187,188 Russel and co-workers have contributed extensively in the development of antibacterial surfaces.189−195 In one of their reports, an antimicrobial polymer was synthesized directly on the surface by employing atom transfer radical polymerization (ATRP).189 Lienkamp and co-workers have engineered an antimicrobial surface coating based on polyoxonorbornenes, which were attached to various modified surfaces by UV-induced crosslinking.196 These reports involve reactions on the surfaces that are complicated and require sophisticated techniques. Researchers are also immensely focused on the development of coating procedures that do not employ reactions involving the surfaces. Recent research has resulted in the development of polymers functionalized with active linkers that can bind the polymeric structure to various surfaces. Incorporation of such linkers in the polymeric architecture paves an easier way to coat the polymers on the surfaces, which requires only one-step fabrication. Locklin and co-workers have developed a coating involving benzophenone moieties, which renders a UV-curable property to the coating material.197−199 They have developed polyethylene imine containing benzophenone and hydrophobic dodecyl chain, which impart UV-curable and antimicrobial properties, respectively.197 The polymer was spin coated on different substrates and exposed to UV light of 365 nm wavelength and were shown to be active against both Grampositive and -negative bacteria. Klibanov and co-workers have also reported a covalent coating incorporating a photoactive linker 6-(4′-azido-2-nitrophenylamino) hexanoyl into branched polyethyleneimine.200 Upon incubation of water borne pathogens E. coli and S. aureus with the photocoated cotton, no viable bacteria were detected. Dopamine has also been utilized as an anchoring agent.201−205 Various dopamineincorporated antimicrobial polymeric coatings have been reported in the recent past. Yang and co-workers developed a one-step polycarbonate-based coating that employs dopamine as a linker.201 Ring opening polymerization was at the heart of this polymerization. The brush-like polycarbonates, when coated on the surface, were active against S. aureus and E. coli. The strategy of constructing antimicrobial surfaces using purely cationic polymers is associated with deposition of dead bacterial cells. Bacteria love to adhere on cationic surfaces. Once the first layer of bacteria is attached and killed, the dead bacteria just sit and frolic on the debris, which results in fouling

of the surfaces. This fouled surface provides an environment suitable for biofilm formation. To impart nonfouling and antibiofilm properties to a surface, researchers have focused on the development of polymeric coatings that can switch between bactericidal and bacteria-repellent nature.206 Jiang and coworkers have contributed immensely toward this direction.207,208 In an earlier report, they constructed a new switchable polymer surface coating that combines the advantages of both nonfouling and bactericidal properties. When immobilized on a surface, poly(N,N-dimethyl-N(ethoxycarbonylmethyl)-N[2′(methacryloyloxy)ethyl]ammoniumbromide) on a surface killed greater than 99.9% of E. coli K12, releasing 98% of the dead bacterial cells when the cationic derivatives were hydrolyzed to nonfouling zwitterionic polymers.207 However, this suffers from the limitation that it was not reversible and the switching could occur only once. Later on, they developed a smart coating that reversibly switched between its bactericidal and antifouling properties. The reported polymeric coating was capable of switching repeatedly between its two equilibrium states, a cationic N,Ndimethyl-2-morpholinone (CB-Ring) and a zwitterionic carboxy betaine (CB-OH). This switching imparted repeated bactericidal and bacteria-repellant properties in the polymer. Under acidic conditions, CB-OH could be converted back to CB-Ring, making the surface a reversibly switchable one.208 4.3.2. Noncovalent Polymeric Coatings. Even though covalent immobilization resists leaching of the coating, it also has several challenges. The fabrication of a covalent coating requires a trained professional and involves complicated procedures. As a result, noncovalent surface modification was called into play.209 By following this principle, the preparation of antimicrobial surfaces becomes very simple, where the coating procedure is as simple as that of painting. Klibanov and co-workers have played a pioneering role in this direction.210−218 Their initial reports have demonstrated that deposition of a quaternized polyethyleneimine (PEI) derivative, N-hexyl, N-methyl-PEI on various surfaces (such as glass or polyethylene surfaces) were capable of killing airborne S. aureus. However, this antimicrobial surface developed using this shorter alkyl chain bearing PEI derivative was not permanent. The deposited antibacterial polymer was found to leach out from the surface, which partially contributed to its antibacterial activity as well.214 Later on, Klibanov and co-workers were able to overcome this issue by using PEI derivatives bearing higher hydrophobic moieties.215,217 A new antimicrobial surface was prepared by using a new PEI derivative, where the hexyl moieties were replaced with dodecyl moieties (N-dodecyl, Nmethyl-PEI). This higher hydrophobicity-bearing polymer was hypothesized to strengthen the intermolecular attractions and thus lowered the tendency to leach out from the surface. The experimental investigation indeed established that antimicrobial action was not due to leaching of the antibacterial polycation, and the mechanism of antibacterial action was primarily based on microbial contact on the coated surface. However, the resulting glass surface coated with this polymer showed potent efficacy against both bacteria (S. aureus and E. coli) as well as virus. The optimized polymer-coated glass slide showed complete killing (100%) of both bacteria and virus. The antimicrobial surface was rapidly virucidal, which was capable of killing the influenza virus within minutes (∼4 log reduction in the viral titer). Importantly, it was capable of inactivating drugresistant strains of influenza virus, poliovirus, and rotavirus with high efficacy.218 Sen and co-workers have reported a class of U


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Figure 12. (A) Hydrophobic cationic polymers (R = C18H37): (i) linear N-alkyl N-nethyl PEIs and (ii) branched N-alkyl N-methyl PEIs. (B) Antibacterial activity of a polymer-coated glass surface against E. coli. (C) Fluorescence microscopy and SEM images of bacteria (S. aureus) untreated and treated with the coated surface. (D) Mechanistic investigation of antifungal activity of polymer coatings by fluorescence microscopy (scale bar, 5 μm). Reproduced with permission from ref 224. Copyright 2014, American Chemical Society.

Figure 13. (A) Quaternary hydrophobic chitin polymers (R′ = C16H33). (B) Antibiofilm activity of the polymeric coating against S. aureus. (C) Bacterial count after harvesting catheter from the in vivo mouse model. (D) In vivo antibacterial activity of polymer-coated catheters (FESEM images). Reproduced with permission from ref 225. Copyright 2016, American Chemical Society.

anionic poly(acrylic acid).221,222 The surface coated with optimum LbL film thickness (10 nm) showed potent efficacy against both airborne and waterborne E. coli, and S. aureus including virucidal activity against influenza virus with maintained nontoxic nature toward mammalian cells. These films coating the surface were able to prevent bacterial attachment as well and thus could be developed as coating materials for the prevention of biofilm formation on various surfaces.223 Furthermore, this antimicrobial polymer was coated

antimicrobial composites by on-site precipitation of AgBr nanoparticles using a pyridinium polymer.219,220 The composites, when coated on a surface, killed bacteria as well as resisted biofilm formation. It was noteworthy that the antimicrobial action of these coated surfaces not only involved contact killing by the pyridinium polymer but also killed microbes in a releasebased manner. In another report, Hammond, Klibanov, and coworkers demonstrated a layer-by-layer (LbL) assembly of this bactericidal and virucidal cationic PEI derivative using the V


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preventing biofilm formation by both S. aureus and E. coli. Importantly, the medical-grade catheter coated with this polymer reduced MRSA burden by 3.7 log (compared to noncoated catheter) in a murine model of subcutaneous infection with no biofilm development under in vivo settings (Figure 13D). Thus, this chitin-based antibacterial polymer can be developed as a coating material for the prevention of deviceassociated infections.225

on medically relevant titanium and stainless steel surfaces. The resulting surface showed prevention of biofilm formation by S. aureus in a large-animal (sheep) trauma model, which promotes bone healing in this animal model. This indicated the potential application of this antimicrobial polymer in the development of various efficient orthopedic implants. Our group has further investigated a detailed structure−activity relationship of these polyethylenimine-based, quaternized polymer-coated surfaces. A series of quaternized polyethylenimines were prepared using precursor polymers of various molecular weights (Figure 12). PEI derivatives with various degrees of quaternization were prepared using different long chain alkyl bromides, such as 1bromododecane, 1-bromohexadecane, 1-bromooctadecane, 1bromoeicosane, and 1-bromodocosane. The resulting polymers were water insoluble and organosoluble, which were then coated on various surfaces by easily dissolving in various organic solvents. The surface coated with these polymers displayed activity against various pathogenic bacteria including drug-resistant superbugs MRSA and VRE (Figure 12). They also showed efficacy against pathogenic fungi such as Candida spp. and Cryptococcus spp. (Figure 12D). Upon contact on the polymer-coated surfaces, complete killing (∼5 log reduction in cell viability) was noted for both bacteria and fungi. This antibacterial surface also retained its efficacy in the presence of various complex mammalian fluids (such as serum, plasma, and blood) without showing significant toxicity toward mammalian cells (RBCs). Importantly, the propensity toward developing bacterial resistance was not seen even after 20 continuous passages; thus, this class of polymers could be developed as “microbicidal paint” for various biomedical and household applications.224 Recently, our group has reported another class of antimicrobial surfaces, where quaternized chitin derivatives were used.225 The water-insoluble and organosoluble antibacterial chitin derivatives were prepared by selective quaternization at the C-6 position of the sugar unit by using various N,N-dimethylalkylamines (such as N,N-dimethyltetradecylamine, N,N-dimethylhexadecyl amine) (Figure 13). By varying the molecular weights of precursor chitin polymer and the degree of quaternization, a series of antimicrobial polymers were prepared. By dissolving the polymers in methanol, they were coated on various surfaces such as polystyrene plates, glass slides, or cover glasses by employing brush, dip, or spin-coating or drop casting. Polystyrene plates coated with these polymers showed potent activity against both drug-sensitive and -resistant bacteria (MRSA and VRE) with minimal toxicity against mammalian cells (RBCs and embryo kidney cells). By increasing the alkyl chain length in the polymers with degrees of quaternization of 39 and 48%, increased antibacterial activity against S. aureus was seen. The minimum inhibitory amount (MIA) value for the polymer consisting of 48% degree of quaternization decreased from 0.48 to 0.06 μg/mm2 while the long chain was increased from -C12H25 to -C16H33. However, such drastic increment in antibacterial efficacy was not found for E. coli, where the MIA value decreased from 7.8 to 3.9 μg/ mm2. For the polymers with a higher degree of quaternization, 55%, the increasing effect on S. aureus activity was retained, but the activity against E. coli was decreased upon increasing the long chain in the polymer; the MIA value was increased from 15.6 to 31.2 μg/mm2. The surface coated with these polymers also showed potent activity against P. aeruginosa, MRSA, VRE, and β-lactam-resistant K. pneumoniae. Additionally, the surface coated with one of the best active polymers was capable of

5. CONCLUSIONS AND FUTURE PERSPECTIVES To date, the research conducted in the field of antimicrobial polymers has focused mostly on their chemical and structural aspects. As research continues to flourish in this direction, several new classes of polymers with improved antimicrobial properties are being developed. An extensive analysis of AMPinspired antimicrobial polymers has underpinned the importance of different structural parameters such as hydrophilic/ hydrophobic balance, molecular weights, charges, hydrogen bonds, polymeric architectures, and so forth in regulating their biological activity. Polymer-based antimicrobial hydrogels have been developed by Michael addition reaction, imine chemistry, amide bonding, and mussel-inspired chemistry in building the three-dimensional hydrogel network. Similarly, studies on antimicrobial surface engineering have elucidated the significance of covalent and noncovalent interactions. For evaluating the in vitro activity of antimicrobial polymers, the broth dilution procedure is a widely used method to determine the MIC values. For this purpose, measurement of the optical density is a suitable and commonly adopted technique for water-soluble antimicrobial polymers. However, for sparing soluble samples, other suitable methods need to be developed that can provide more accurate results. To date, different research groups have followed different methods to evaluate the efficacy of antimicrobial hydrogels, which demands the development of a standardized method. This will help to compare the data reported by different research groups. In the case of antimicrobial surfaces, the Japanese Industrial Standard (JIS) assay is considered as a standard method for evaluating antimicrobial activity. However, this assay does not mimic the real scenario and therefore is unable to anticipate the actual efficacy under realistic conditions. Hence, the field direly needs standardized methods for the fast and reliable evaluation of the antimicrobial activity of surfaces. Although polymers have been used in various applications, whether they can also be explored as “macromolecular drugs” for treating infectious diseases is the overarching question. The majority of studies to date are limited to the antimicrobial activity of polymeric compounds being evaluated against drugsensitive microbes in vitro. Future research should expand to drug-resistant strains including clinical isolates to establish the clinical relevance of antimicrobial polymers as drugs. There are very few reports directed toward in vivo evaluation of these macromolecular antimicrobials, and most of the existing studies focus on their use in treating topical infections. A clear challenge in the field therefore remains to explore the possibility of treating systemic infections. The detailed pharmacokinetics, pharmacodynamics, and toxicity of watersoluble antimicrobial polymers should be studied in animal models. Elaborate and in-depth studies need to be devised to determine the efficacy of antimicrobial hydrogels and surfaces in treating internal organ infections. The biocompatibility, biodegradability, and stability of polymer-based antimicrobial hydrogels and surfaces also need to be investigated in in vivo W


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Biomacromolecules models. Furthermore, the field should move forward with more focus on antibiofilm research. An inadequate effort has been made to target microbial biofilms, which are the source of a majority of infections (∼80%). Future research can target the microbial communication system (quorum sensing) to control their biofilm formation. It is well-known that quorum sensing plays an essential role in regulating the microbial biofilm lifestyle. Thus, the incorporation of strategies that inhibit microbial quorum sensing can provide a fruitful solution to this unsolved problem. Various quorum sensing inhibitors can be conjugated with antimicrobial polymers or incorporated into antimicrobial hydrogels and surface coatings, which will expectedly lead to more effective solutions for addressing the problem of biofilms.226 Last but not least, effective collaborative research involving the subject areas of polymer chemistry, microbiology, pharmacology, and toxicology is highly necessary. Sharing of knowledge by the experts of these distinct fields would be highly beneficial in leading to the discovery of innovative strategies for combating infections and antimicrobial resistance in the future.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (+91) 80-2208-2565. Fax: (+91) 80-2208-2627. Email: [email protected]. ORCID

Jayanta Haldar: 0000-0002-8068-1015 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

We thank Prof. C. N. R. Rao (JNCASR) for his constant support and encouragement. J.H. acknowledges a Sheikh Saqr Career Award Fellowship. M.M.K and B.B. thank CSIR for research fellowships.

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