Recent progress in polymer research to tackle infections and

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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 Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00458 • Publication Date (Web): 02 May 2018 Downloaded from http://pubs.acs.org on May 4, 2018

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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.

*Corresponding author: Ph. No.: (+91) 80-2208-2565 Fax: (+91) 80-2208-2627 E-mail: [email protected]

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ABSTRACT: Global health is increasingly threatened by the rapid emergence of drug-resistant microbes. The ability of these microbes to form biofilm has further exacerbated the scenario leading to notorious infections which are almost impossible to treat. To address this clinical threat, various antimicrobial polymers, polymer-based antimicrobial hydrogels and polymercoated 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 is delineated to elucidate the real potential of these antimicrobial polymers as possible therapeutics. Antimicrobial hydrogels, either prepared from inherently antimicrobial polymers or from 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 towards 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 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, 2 ACS Paragon Plus Environment

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lungs, urinary tract, cardiac tissues, bones etc.) often leading to chronic infections.10-13 To combat 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 formations as well as disrupt established biofilms and cure infections are in high demand. Towards this direction, polymer-based various 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 drugsensitive and drug-resistant microbes. These antimicrobial polymers primarily target microbial membrane and display lesser propensity to trigger resistance development. Alongside, some of these polymers have 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, erythromycin, etc.) towards multi-drug resistant Gram-negative bacteria and their biofilms. Importantly, this antibiotic re-sensitization has also been shown in in-vivo mice model. Alongside, 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 etc. Various research groups have developed hydrogels, based on inherently antibacterial polymer scaffolds or biocide loaded-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 3 ACS Paragon Plus Environment


bio-adhesive 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 of the 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 coating, for example, non-covalent 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 to discuss all three aspects together to combat infections and antimicrobial resistance including the most recent development 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 re-sensitize obsolete antibiotics towards 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 which 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 4 ACS Paragon Plus Environment

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host-defence antimicrobial peptides (AMPs). As the part of the innate immune system, AMPs are widely found in both plants as well as in 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 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

Figure 1. Cationic AMPs mostly adopt amphipathic topology either in aqueous solution or in presence of bacterial membrane. Upon adsorption of these facially segregated AMPs, the membrane integrity disrupts through various mechanisms. Among them, most of the common modes of actions are pore formation (toroidal and barrel-stave pore model) and non-specific membrane disintegration (carpet model). According to 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. Adapted with permission from ref 16. Copyright 2011 Elsevier. 5 ACS Paragon Plus Environment


presence of phosphatidylglycerol, phosphatidylserine or cardiolipin results in an overall negative charge in the bacterial membrane. Moreover, the presence of teichoic acids (in case of Grampositive bacteria) and lipopolysaccharides (in case of Gram-negative bacteria) also enhances the overall negative charge of the bacterial cell envelope. In contrast, the outer leaflet of the mammalian cell membrane is zwitterionic in nature that consists the phospholipids such as 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 bacterial membrane through electrostatic interaction, AMPs disrupt the membrane integrity by various mechanisms.16 Pore formations and non-specific membrane disintegration are the most common modes of actions (Figure 1). According to 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 Alongside, various research groups have dedicated attention in developing synthetic polymeric molecules as promising AMP mimics.46-53 There are many 6 ACS Paragon Plus Environment

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excellent reviews available that highlighted various aspects of antimicrobial polymer designs.4650

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 advancement have ascended rapidly in the recent years. 2.1.1

Polynorbornene/ Polyoxanorbornene

Tew and co-workers have extensively studied norbornene and oxanorbornene based polymers to develop as antimicrobial 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, counter ions, polymeric architectures on antibacterial efficacy. The first series of polymers were prepared with face segregated monomers, where the cationic ammonium groups and the 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 MIC (Minimum Inhibitory Concentration) values upon increasing the hydrophobicity in the polymers; however further enhancement of hydrophobicity beyond an optimum resulted in compromised activity. On contrary, hemolytic activity showed a drastic increment due to such change. The HC50 (concentration corresponds to 50% hemolysis) for the highest hydrophobic polymers reached to values of 4000 µg/mL. Investigation of the influence of molecular 7 ACS Paragon Plus Environment


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B

C

D

E

F

G

H

Figure 2. Antimicrobial polynorbornenes and polyoxanorbornenes 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 8 ACS Paragon Plus Environment

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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 non-hemolytic nature (HC50 value of >4000 µg/mL). To investigate 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 higher number of cationic charge per monomer unit were found to be advantageous compared to singlecharged analogue. The best selective polymer showed huge reduction in hemolytic activity (about 1000-fold selectivity) but retained similar antibacterial activity (MIC ∼ 30 µg/mL 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 µg/mL 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. The effort was then focused to explore the oxanorbornene-based polymers. By using various diester-functionalized 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-dependant biological activities; higher activity was obtained for polymers with lesser molecular weights. This observation was different which was not seen for norbornene-based polymers, where the hydrophobicity and charges are present on the opposite sides of the polymer backbone. This fact thus indicated that molecular 9 ACS Paragon Plus Environment


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weight dependent antibacterial efficacy possibly is linked with polymer architecture. Furthermore, various binary random copolymers were synthesized by using monomers of nonactive, non-hemolytic and active, hemolytic homopolymers (Figure 2F).58 The optimized polymers showed enhanced selectivity towards S. aureus (>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 the ester functionalities were cationic.60 The hydrophobicity was then tuned by using various organic counter ions, such as tosylate, benzoate, hexanoate and dodecanoate (Figure 2G). A decreased antibacterial activity was noticed with increasing counter ion size. Such effect was seen due to lesser membrane activity of the polymers with lesser effective charge, which was originated from strong polymercounter ion complexation upon increasing the size of counter ions. The effect of charge density was also investigated by preparing a class of binary random copolymers combining previously used monomers bearing mono and di-cationic ester functionalities respectively (Figure 2H).60 The hydrophobicity was also regulated by including various aliphatic ester functionalities in the mono-cationic monomer. A copolymer bearing majority of mono-cationic monomer composition (90%) and a methyl group in the hydrophobic ester functionality showed best selectivity towards S. aureus (650-fold). In contrast, the polymers having monomers with greater hydrophobic ester functionality in place of 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 Gram-negative bacteria. 2.1.2

Polymethacrylate/ Polyacrylate 10 ACS Paragon Plus Environment

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Significant efforts have been directed on 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 etc. In their first report, a set of polymethacrylates was prepared by using the monomers N-(tert- butoxycarbonyl)aminoethylmethacrylate and butyl methacrylate (BMA) (Figure 3A).61 By involving free radical copolymerization with controlled concentration A

B

C

F

D E

Figure 3. Antimicrobial polymethacrylates and polyacrylates. 11 ACS Paragon Plus Environment


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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-(tert-

butoxycarbonyl)aminoethylmethacrylate unit. The antibacterial studies indicated a gradual decrease in MIC values upon increasing the BMA composition to ∼30%, however further increase of 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 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 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 monomer 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 needs to be maintained to achieve most active antibacterial polymer with minimum hemolytic activity. Haeussler and coworkers 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 12 ACS Paragon Plus Environment

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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 the MIC values ranging between 2-20 µg/mL, against both S. epidermidis and C. albicans. On the other hand, the amino group bearing polymers were less effective and showed the 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 is an important criterion to meet the optimum antimicrobial polymer with 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 most effective antibacterial activity with MIC values 12 µg/mL (against S. epidermidis) and 47 µg/mL (against MRSA) with minimal toxicity towards 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), drastic increase in HC50 values (HC50 = 256-512 µg/mL compared to 2-4 µg/mL) was seen for the polymethacrylate analogue bearing 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 13 ACS Paragon Plus Environment


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 the value of > 2000 µg/mL. However, increase in the mole percentage to 100% resulted in a sharp decrease in the HC50 value (< 1.9 µg/mL). Thus, the polymer with 90 mole% of M6 is optimum which showed 208-fold selectivity towards 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 2chloropropionate. 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. Polymer bearing primary amino groups was relatively hemolytic and displayed moderate antimicrobial activity against E. coli with the best selective polymer showing MIC value of 104 µg/mL and HC50 value >500 µg/mL. In contrast, corresponding analogue with quaternary ammonium group was not antibacterial (MIC against E. coli was > 500 µg/mL) and displayed similar HC50 value (>500 µg/mL). Recently, Gibson, Fullam and coworkers have reported anti-tuberculosis activity of this class of polymers (Figure 3F).71 The poly(dimethylaminoethylmethacrylate) displayed selective anti-mycobacterial activity over Gram-negative P. putida and E. coli with the MIC value of 31.25 µg/mL against M. smegmatis. Upon removal of the backbone methyl group, the resulting poly(dimethylaminoethyl acrylate) displayed a decreased anti-mycobacterial activity. Similarly, a corresponding poly(aminoethyl methacrylate) was also found to be ineffective (MIC = 500 µg/mL). The hemolysis studies 14 ACS Paragon Plus Environment

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further indicated that poly(dimethylaminoethyl methacrylate) was not toxic towards mammalian cells. Almost no hemolysis was seen even at the concentration of 5000 µg/mL. In contrast, corresponding poly(aminoethyl methacrylate) was comparatively toxic (about 10% hemolysis was seen at 5000 µg/mL). Importantly, Kuroda and co-workers have reported a primary ammonium ethyl methacrylate homopolymer, which 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 to reduce 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

A

B

C

D

Figure 4. Antimicrobial poly β-lactams. 15 ACS Paragon Plus Environment


polymers. From β-lactam monomers, they have developed nylon-3 (poly β-lactams) polymers through anionic ring-opening polymerization. This class of polymers are closely similar to natural peptides and proteins (can be considered as nylon-2 derivatives), and hence the AMPs. These nylon-3 class of polymers with sufficient backbone flexibility are 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 are capable to disrupt microbial membranes in preference to eukaryotic cell membranes. Extensive structureactivity 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) subunit and the hydrophobicity was 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 only one order of magnitude 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 MIC value was observed for E. coli with increase in the 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 contrary, the hemolytic activity was found to be strongly influenced by the length of the polymer. Polymers bearing lesser subunits (an average of 10-30) showed very weak hemolytic activity with MHC (Minimum Hemolytic Concentration) values of ~1000 µg/mL. However, the propensity of hemolytic activity increases dramatically for the larger polymers (greater than about 30 16 ACS Paragon Plus Environment

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subunits); where MHC values reach even lower than MIC values. Thus, the shorter polymers that inhibited the bacterial growth at lesser concentrations were optimum and displayed 10-fold to >100-fold selectivity towards 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 non-monotonic relationship of antibacterial activity was observed upon increment in alkyl chain hydrophobicity. In contrast, such parabolic trend was not noticed for the hemolytic activity. Upon increasing the chain length to octanoyl group, 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 till the octadecanoyl analogue and the MHC value reached to ~ 1 µg/mL (a value which 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 sensitive towards variation in the cycloalkyl ring hydrophobicity and the MIC values were ranged more or less in the 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 change in biological activities 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 17 ACS Paragon Plus Environment


consisting of MM as the cationic subunit. In this MM bearing polymer when cyclohexyl subunit was replaced by corresponding acyclic analogue, the MIC value against E. coli was increased from 50 µg/mL 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 better antibacterial polymer with lesser hemolytic activity even compared to the analogous polymer, bearing cyclic hydrophobic subunit (Figure 4C). The optimized polymer consisted of 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 cyclohexyl subunit showed MIC in the similar concentration range, it was highly toxic with the 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-like or glycine-like moieties. Antibacterial 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-like or glycine-like subunit drastically reduced the hemolytic activity without 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,


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the ternary polymers revealed the HC10 values of 200-400 µg/mL, which was much higher compared to the value (HC10 = 6.25 µg/mL) displayed by the binary polymer. 2.1.4

Polymaleimide

Our group has focused significant attention in polymaleimide class of polymers. Starting from commercially available polyisobutylene-alt-maleic anhydride, preparation of this class of polymers was achieved through post-functional modification in a two-step reaction. The highly reactive anhydride ring of the precursor polymer was first functionalized with 3aminopropyldimethylamine. The dimethylamine moieties were then quaternized by using various alkylating agents with nearly similar degree of quaternization (~ 93-98%). The first series of polymers was prepared by incorporating the alkylating agents of varied hydrophobic aliphatic chain (Figure 5A).78 Antibacterial activity of this class of polymers followed a parabolic trend upon increasing in the alkyl chain length. The MIC value (against both E. coli and S. aureus) decreased (from >1000 μg/mL to 3-20 μg/mL) upon increasing the alkyl chain from ethyl to A

B

D

C

Figure 5. Antimicrobial polymaleimides. 19 ACS Paragon Plus Environment


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octyl group followed by an inclined value (100-200 μg/mL) when the chain length was increased to decyl group. In contrast, an increasing trend was observed for hemolytic activity (HC50 value decreased from >1000 µg/mL to 4 µg/mL) while increasing the alkyl chain hydrophobicity. Thus, the polymer analogue having intermediate alkyl chain (such as pentyl group) was identified as the optimum candidate that showed best selectivity (114-fold selectivity towards S. aureus over RBCs). In an attempt to further improve the antibacterial efficacy,

a hydrophilic

moiety (oligoethyleneglycol) 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 was >1000 µg/mL against E. faecium, MRSA, VRE, E. coli and P. aeruginosa) which prevented its effectiveness as antibacterial agent. However, the next series of polymers was prepared by introducing ester and amide functionalities in the hydrophobic alkyl chain (Figures 5B, 5C and 5D).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 huge reduction in hemolytic activity. Interestingly, these polymers, especially the amide bearing polymers displayed similar MIC values as compared to the polymers consisted 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 established that amide containing polymers strongly interacted with the lipid head groups of bacterial membrane owing to the presence of a greater hydrogen bonding capable moiety (-NH-). On contrary, the ester polymers having lower hydrogen bonding capable moiety 20 ACS Paragon Plus Environment

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(-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 on decreasing the aliphatic chain length attached to the amide functionality.80 This was a unique trend which has not generally been seen earlier. On the other hand, the ester series of polymers displayed an obvious trend due to such variation. A decreased antibacterial activity was resulted due to 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 = 31μg/mL), but also reduced the A. baumannii burden (~ 2 Log) in-vivo in burn wound infections in mice. To further explore the effect of side chain architecture, a series of amide containing polymers was prepared by employing isomerisation (regio- and stereo-), cyclization and unsaturation in hydrophobic side chains (Figures 5C and 5D).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 that displayed HC50 values of 80 μg/mL and 45 μg/mL, respectively. In contrast, the methylcyclopropyl was comparatively non-toxic with HC50 value of 250 μg/mL. This optimized polymer displayed ~ 50-100 fold selectivity towards bacteria over RBCs. To investigate the regio- and stereo-isomeric effects on antibacterial properties, the polymers with isomeric side chain were prepared by keeping number of carbon atoms constant to four (identified as the optimum (methylcyclopropyl) for above set of polymers). The polymers bearing iso-butyl, R/S sec-butyl, t-butyl side chain however displayed similar antibacterial activity (MIC in the range of 4-31 μg/mL) and toxicity 21 ACS Paragon Plus Environment


profile and thus indicated that isomerization of hydrophobic side chains does not have significant role to regulate antibacterial efficacy. However, the effect of unsaturation in the side chain hydrophobicity resulted in huge reduction in toxicity towards mammalian cells. Both the analogue polymers bearing double bond (but-3-enyl) and triple bond (but-3-ynyl) unsaturation displayed the 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 has a profound effect towards regulating selectivity of antimicrobial polymers. 2.1.5

Polycarbonate

Owing to the biodegradable nature, polycarbonate class of polymers has attracted attentions of various researchers. A 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 towards 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 which readily self-assembled to spherical micelles in aqueous solution (Figure 6A).82,83 These selfassembled micelles bearing the cationic charges on its periphery could selectively interact with negatively charged microbial membranes through electrostatic interaction. An increased local concentration of cationic charge resulting from micelle formation leads to better interactions with negatively charged microbial membrane. 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). 22 ACS Paragon Plus Environment

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Biomacromolecules

In contrast, the second system was a random copolymer which was prepared from the monomers 5-methyl-5-(3-chloropropyl)oxycarbonyl-1,3-dioxan-2-one (MTC-PrCl) and ethyloxycarbonyl1,3-dioxan-2-one (MTC-ethyl) (Figure 6A). The first series of polymers readily self-assembled into stable nano structures and selectively disrupted microbial membranes 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 self-assembled nature and hence was capable to disassemble in presence of microbial membrane leading to membrane disruption.83 These polymers showed microbial growth inhibition (that included gram-positive, gram-negative bacteria and fungi) with MIC values ranged between 63-500 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 ranged between 4-125 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 23 ACS Paragon Plus Environment


Page 24 of 99

A

B

D

C

E

F

Figure 6. Antimicrobial polycarbonates.


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Biomacromolecules

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 case of polycarbonates, which were quaternized with N,N-dimethyl alkylamines of varied alkyl chain lengths, a significant improvement in antibacterial activity was seen with retention of non-hemolytic 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-fold and 8-fold respectively. While retaining the non-hemolytic properties (HC50 > 4000 µg/mL), the MIC value was diminished from 62.5 µg/mL to 3.9 µg/mL and 125 µg/mL 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 acyclic analogue (HC50 = 15.6 µg/mL), although the antibacterial activity was similar. However, the polymer bearing benzyl group displayed similartrend in antibacterial and hemolytic activities compared to cyclohexyl analogue, only the notable improved activity was seen for P. aeruginosa (MIC value decreased from >500 µg/mL to 125 µg/mL). The polycarbonate containing 1-methylimidazolinium as the cationic group displayed high selectivity towards killing E. coli and S. aureus, as it was non-hemolytic with the 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 to further optimize the antibacterial efficacy, another series of polycarbonates were prepared where two different quaternary ammonium moieties were randomly distributed in the structure (Figure C).85 25 ACS Paragon Plus Environment


The

antibacterial

and

hemolytic

studies

indicated

Page 26 of 99

that

50%

content

of

N,N-

dimethylbutylammonium group was sufficient to improve the hemolytic activity by 3-fold, while 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 towards microbial killing. Jin, Swift and co-workers have reported a class of guanidinylated polycarbonates prepared from post-synthesis modification of alkyne bearing polycarbonates using click chemistry (Cu(I)-catalyzed azidealkyne cycloaddition) (Figure 6E).87 A series of polymers with varied molecular weight (8 kDa, 15 kDa and 30 kDa) and charge density were prepared. These guanidinylated polycarbonates displayed broad-spectrum antibacterial activity with low toxicity towards mammalian cells (HC50; >1280 µg/mL). The lowest molecular weight homopolymer bearing 100% guanidinylated repeating unit revealed the best activity with the MIC values of 40 µg/mL 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 have 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 26 ACS Paragon Plus Environment

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Biomacromolecules

monomers. To study the effect of monomer sequence on antimicrobial activity, both random (randomly arranged hydrophilic and hydrophobic monomers) as well as di-block (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 case of di-block copolymers, antibacterial activity was not much altered upon varying the number of hydrophobic groups. This was possibly due to the formation of stable nanomicelle formation by the di-block copolymers. Thus, the hydrophobic groups are sequestered and hence similar antimicrobial activity was resulted. This was clearly established by the fact that a random copolymer that consisted of same number of hydrophilic and hydrophobic groups revealed much better antibacterial efficacy. 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 the 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 co-workers have developed an antibacterial polycarbonate, which was not only active against S. aureus, MRSA in-vitro, but also had potent in-vivo efficacy.89 In a mouse model of systemic infection, the polymer was found to reduce MRSA viability in the blood better than a 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 have reported a class of

guanidinium-functionalized polycarbonates that not only displayed in-vivo activity against MRSA, but also showed potent efficacy against multi-drug-resistant Gram-negative bacteria such as A. baumannii, E. coli, K. pneumoniae and P. aeruginosa.90 27 ACS Paragon Plus Environment


2.2

Antibiofilm Efficacy and Re-sensitization of Obsolete Antibiotics

Biofilms are well-structured surface adherent communities where bacteria are embedded within their self-produced extracellular matrix composed of extracellular DNA, proteins and polysaccharides.91,92 To protect from environmental harsh conditions or any external harmful agents, bacteria are known to enter into biofilm lifestyle which is physiologically distinct from planktonic behaviour.92 Initially reported as a secret bacterial lifestyle, biofilm is now recognized as a major threat in treatment of infections. This is the underlying cause behind the chronic or persistent infections which 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 population are present in metabolic dormancy or molecular persistent state. This metabolically inactive population are tolerant to antibiotic treatment as they shut down the target sites of the conventional antibiotics. Additionally, the increased bacterial cell density inside the biofilms favours the transfer of resistance genes which leads to faster development of antibiotic resistance in microbial world. Altogether, the treatment of biofilm associated infections remains a critical challenge, which needs to be resolved urgently. Recently, antimicrobial polymers have attracted attention to resolve this challenge. 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 top most 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 the biofilm formation, but also removed significant biomass of preformed S. mutans biofilms.94 Gellman and co-workers also reported 28 ACS Paragon Plus Environment

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Biomacromolecules

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 mice model of burn-wound infection. Reproduced from ref. 97 under

a

Creative

Commons

Attribution

4

International

License

https://creativecommons.org/licenses/by/4.0/ Copyright 2017 Uppu et al. antibiofilm property of nylon-3 polymers.95 Interestingly, the antibiofilm efficacy was tested against C. albicans, the most notorious fungal pathogen, whose infections often associate 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 29 ACS Paragon Plus Environment


polymers, many dead fungal cells were found inside the biofilms. To battle against rapidly emerging AMR storm, antimicrobial polymers have also been investigated to re-sensitize the obsolete antibiotics. Our group has reported that polymaleimide was capable to restore the activity of tetracycline class of antibiotics towards blaNDM-1 containing K. pneumoniae and E. coli clinical isolates by >80-1250 fold.96 The principle behind re-sensitization 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. Invivo 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 mice 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 to prevent the planktonic growth of dispersed cells from biofilms.97 More importantly, the combination reduced bacterial burden significantly in biofilm associated infection models (burn-wound and surgicalwound) caused by A. baumannii (Figure 7D) and Carbapenemase producing K. pneumoniae. 3. POLYMER-BASED ANTIMICROBIAL HYDROGEL Hydrogel is defined as a three-dimensional matrix that is capable of holding 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 etc. Recently, it has also attracted enormous attention for their development to treat infections. This part of review will focus on polymer based antimicrobial 30 ACS Paragon Plus Environment

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Biomacromolecules

hydrogels developed by various research groups. They can be primarily categorized into two groups; inherently antimicrobial hydrogels (where the polymers itself 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, which are inherently antimicrobial. Gelation processes involve various kinds of covalent (such as imine, amide bond etc.) as well as non-covalent (such as electrostatic and H-bonding) interactions that ultimately help to construct the three-dimensional 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 post-functional modification or by de-novo polymer synthesis. In most of the cases, quaternary ammonium groups are incorporated to achieve antimicrobial property.

Table 1. Inherently antimicrobial hydrogels.

Antimicrobial

Cross-linker

Gelation mechanism

polymer

Microorganism tested


References


ε-poly-L-lysine-

self-cross-linked

UV-polymerization

Page 32 of 99

E. coli

98

graftP. aeruginosa methacrylamide S. marcescens S. aureus C. albicans F. solani dopamine-

self-cross-linked

modified ε-poly-L-

horseradish peroxidase

E. coli

99

crossS. aureus

lysine-

linking of dopamine

polyethylene glycol ε

-poly-L-lysine self-cross-linked

grafted

poly

(1-

enzyme

induced E. coli

imine

100

bond S. aureus

vinyl-2-

formation

pyrrolidinone-coN-methylol acrylamide ) polypeptide

of 6-arm

lysine and alanine

amide

polyethylene glycol

bond E. coli

formation

S. aureus

(PEG)-

amide


101

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Biomacromolecules

succinimidyl glutarate 20-residue

β- self-cross-linked

hairpin peptide

Chitosan

S. aureus

interaction

γ

Chitosan

non-covalent

MRSA

-poly(glutamic electrostatic

acid

interaction

dextran aldehyde

imine

102

S. aureus

103

E. coli bond S. aureus

formation

104

S. pyogenes E. coli C. perfringens

dimethyldecyl

poly(ethylene

ammonium

glycol) diacrylate

UV-polymeization

E. coli

105

S. aureus

chitosan-graftF. solani

poly(ethylene glycol)methaclate quaternized chitosan

polydextran grafted aldehyde

imine

bond E. coli

107

formation S. aureus

aniline quaternized chitosan

benzaldehye grafted functionalizd

imine

bond E. coli

formation


108


Page 34 of 99

poly(ethylene

aniline

S. aureus

glycol)-copoly(glycerol sebacate) N-2-(hydroxyl

polydextran

imine

propyl) 3-trimethyl aldehyde

bond S. aureus

formation

109

E. coli

ammonium P. aeruginosa

chitosan chloride)

MRSA VRE β-lactam resistant-K. pneumoniae thiol-terminated

tetra

acrylate thiol-ene chemistry

S. aureus

110

block copolymers polyethylene E. coli of

polyethylene glycol

glycol

and C. albicans

tetrasulfhydyl polyethtlene glycol

quaternized

poly(l-lactide)-b-

ammonium moiety poly(ethylene bearing

non-covalent interaction

E. coli S. aureus

glycol)-b-poly(l-


111

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Biomacromolecules

polycarbonate

lactide)

MRSA VRE A. baumannii K.

pneumoniae

C. neoformans C. albicans polycarbonate backbone

ABA’-type

hydrophobic

having triblock

hydrophobic

inter E. coli

action

112

S. aureus

copolymer

vitamin

C. albicans

E- containing

functionality

and vitamin

E

and

polyethylene

quaternary moity

glycol POEGMS

POEGDMAM

thiol-ene chemistry

(fumaryl chloride + (mercaptosuccinic dodecyl

bis(2- acid

hydroxyethyl)

oligo(ethylene

methylammonium

glycol)

chloride

E. coli S. aureus

+

+

oligo(ethylene glycol)


113


branched

poly

polyethylene imine

aldehyde

dextran imine

Page 36 of 99

bond E. coli

formation

114

S. aureus S. pyogenes

ABA type triblock self-cross-linked

non-covalent

copolymer

interaction

(A=

E. coli

115

S. aureus

116

catechol functionalized and comprised

of

polyethylene glycol,

B

=

poly{[2(methacryl oyloxy)-ethyl] trimethylammoniu m iodide} 3,4en-ionene

tris(2aminoethyl)a UV-polymerization mine,

E. coli hydroxyethyl P. aeruginosa

acrylate glycerol dimethacrylate


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Biomacromolecules

3.1.1

Polypeptide Based Hydrogels

Cationic amino acid-based polypeptides, such as ε-poly-L-lysine is well known for its inherent antimicrobial property owing to the presence of amino groups. There has been a significant effort

Figure 8. A) Schematic representation of hydrogel preparation from dopamine grafted polyL-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 WILEYVCH Verlag GmbH & Co. KGaA, Weinheim.



towards development of antimicrobial hydrogels using this polypeptide.98-101 Chan-Park and coworkers have reported a hydrogel system based on ε-poly-L-lysine-graft-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 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 amount of dopamine substitution in poly-lysine backbone strengthened the mechanical property, but due to 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 of 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 presence of the enzyme plasma amine oxidase, aldehyde functionalities were generated in-situ by oxidation of amino groups. These aldehyde groups reacted with 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 38 ACS Paragon Plus Environment

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Biomacromolecules

the peptide sequence.101 Hexamethyldisilazane mediated ring-opening polymerization was employed to prepare this class of antibacterial polypeptides with varied hydrophilicity. 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 have reported a 20-residue peptide by incorporating various amino acids, capable to adopt an amphiphilic β-hairpin structure due to segregation into hydrophobic and hydrophilic faces. This β-hairpin structure was capable to construct a mechanically rigid hydrogel upon self-assembly by involving various non-covalent interactions which when coated onto surface, inhibited the growth of MRSA (2×108 CFU/dm2).102 3.1.2

Chitosan Based Hydrogel

Naturally occurring polysaccharide, chitosan is well known for its inherent antibacterial activity. Significant effort has been directed to develop antibacterial hydrogels by employing this polymer and its derivatives. Hsieh and co-workers have reported a hydrogel, which was 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 proving it to be promising to be developed as 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. perfringens, whereas 39 ACS Paragon Plus Environment


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-graft-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 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 component into the nanopores present in the hydrogel. This ‘anion sponge’ like mechanism of hydrogel results into microbial death by membrane disruption. This PEGlycated chitosan hydrogel also showed proliferation of human primary epidermal keratinocytes in-vitro. The hydrogel coated contact lens, when implanted in rabbit eyes, showed no inflammation even after 5 days.105 More recently, Ma and co-workers have reported a series of hydrogels by using synthetically modified chitosan.106-108 In one example, they have prepared a hydrogel by using aniline grafted quaternized chitosan, where polydextran aldehyde was used as the cross-linker 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 electro-active 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 40 ACS Paragon Plus Environment

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Biomacromolecules

glycol)-co-poly(glycerol sebacate) keeping the antibacterial polymer component same. In addition to antibacterial and electro-active properties, anti-oxidant 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 bond with aldehyde groups. This system showed activity against S. aureus, E. coli, P. aeruginosa including drug-resistant bacteria (MRSA, VRE and β-lactam-resistant K. pneumoniae) with killing efficacy of 90-99%. This injectable hydrogel also had hemostatic and bioadhesive properties and showed potent efficacy in preventing sepsis in mice 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 injured liver of mice, proving its haemostatic 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 have dedicated immense attention to develop polycarbonate based antibacterial hydrogels.110-112 Initially, they have prepared quaternary moiety containing polycarbonate polymer via an organocatalytic ring opening polymerization (ROP) at room temperature which was then conjugated with thiol-terminated another 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 polyethylene-glycol moiety contributed in the antifouling property and ammonium 41 ACS Paragon Plus Environment


group played an important role in its bactericidal effect. The optimum composition of hydrogel was shown to be efficacious against wide range of pathogens (such as S. aureus, E. coli, 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) (PDLA-CPC-PDLA) and biodegradable poly(l-lactide)-bpoly(ethylene glycol)-b-poly(l-lactide) (PLLA-PEG-PLLA) were used together to form antimicrobial hydrogel at 37 °C driven by non-covalent interactions.111 This polycarbonate based 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 presence of two different polymeric systems.112 The hydrogels were formed based on hydrophobic interactions between vitamin E groups and has shown both bactericidal and fungicidal activities. Additionally, this Vitamin E containing hydrogel was also efficacious in eradicating biofilm of various microbes with around 80% killing capability. 3.1.4 Other Polymeric Hydrogels Various other antimicrobial polymers are also used for rapid development of hydrogels.113-116 Zhu and co-workers have reported a class of antimicrobial hydrogels, which was prepared based on thiol-ene chemistry from two multifunctional poly(ethylene glycol) derivatives as precursor components.113 The antibacterial component was synthesised 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 thiol functionalized polymer 42 ACS Paragon Plus Environment

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was synthesized by performing condensation reaction between mercaptosuccinic acid and oligo(ethylene glycol). Mixing these multi-enes and multi-thiols bearing poly(ethylene glycol) derivatives led to three-dimentional polymeric network by covalent crosslinking via a thiol-ene reaction. The optimized hydrogel exhibited excellent antibacterial activity against both Gramnegative and Gram-positive bacteria with low toxicity towards 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 to kill both Gram-negative and Gram-positive bacteria

without showing much toxicity towards human erythrocytes. Importantly, in a murine infection model, hydrogel was capable to kill S. pyogenes completely in 3-5 days. This bio-adhesive (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 have developed a novel hydrogel, prepared from an ABA type triblock copolymer.115 The A block was catechol functionalized and also comprised of polyethylene glycol moieties, whereas the B block was consisted of poly{[2-(methacryloyloxy)-ethyl] trimethylammonium iodide}. In aqueous media, this triblock copolymer undergoes self-assembly through catecholmediated hydrogen bonding and aromatic interactions. This hydrogel inhibited the growth of E. coli and also showed antifouling properties by preventing the bacterial cell adherence. Interestingly, this hydrogel also showed excellent thermo-sensitivity 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-1,3-propanediamine. Reaction between the bromine end groups of 3,4en-ionene and amino groups of tris(243 ACS Paragon Plus Environment


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 burden against S. aureus and E. coli while 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 to formulate hydrogels where various antibacterial agents were entrapped within the matrix. Conventional antibiotics, metal ions, metal nanoparticles, antimicrobial peptides or even small molecular antibacterial agents-loaded hydrogels have been formulated aiming for developing them as antibacterial products. Biocideloaded hydrogel is not only able to kill microbes upon contact, but also release 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 the recent years. 3.2.1 Hydrogels Loaded with Antibiotics A series of hydrogel systems have been reported where the antibiotics are entrapped either by non-covalent or covalent interactions with the hydrogel components.117-130 Kannan and coworkers have reported amoxicillin loaded hydrogel which was prepared by using 4 poly(amidoamine) [G4-(NH2)64] dendrimer with peripheral thiopyridyl terminations and 8-arm thiolated polyethylene glycol as the cross linker.117 This hydrogel system was capable of 44 ACS Paragon Plus Environment

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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 to the fetus.117 In another example, ciprofloxacin was loaded into the hydrogel matrix, which was formulated by using a copolymer poly(2hydroxyethyl methacrylate). Hydrogel coating onto titanium implant showed a zone of inhibition with 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 by using dopamine methacrylamide and polyethylene glycol diacrylate. Incorporation of this dopamine derivative increased the mechanical strength of this hydrogel making it stable in physiological conditions and thereby inhibiting the formation of bacterial film (E. coli) in in-vitro flowing condition. Importantly, the hydrogel did not show any noticeable skin reaction or toxicity.119 Gentamicin loaded various hydrogels have been emerging in recent years. Carboxymethyl-chitosan hydrogel was prepared by using genipin as the cross-linker, where gentamicin was loaded into hydrogel matrix. This hydrogel showed biofilm inhibition capability along with mammalian cell compatibility.120 Gentamicin loaded thermo-responsive hydrogel was also prepared using hyaluronic acid-poly(Nisopropylacrylamide). Table 2. Biocide-loaded antimicrobial hydrogels. Hydrogel

forming Loaded biocide

component/s poly(2-hydroxyethyl

Ciprofloxacin

Gelation

Microorganism

mechanism

tested

electrochemical

MRSA


References

118


methacrylate

and

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synthesis

poly(ethylene-glycol diacrylate) acrylic acid dopamine

ciprofloxacin-

UV-

methacrylamide

loaded

polymerization

polyethylene

E. coli

119

glycol poly(lactic-coglycolic

diacrylate

acid)

nanoparticle carboxymethyl-chitosan

genipin induced S. aureus

Gentamicin

120

crosslinking hyaluronic

acid-poly(N- gentamicin

temperature

isopropylacrylamide)

change

oxidized polysaccharides aminoglycoside

imine

(such

as

dextran, antibiotics

carboxymethyl, cellulose, as; alginate, chondroitin)

S. aureus

121

bond S. aureus

123

(such formation

E. coli

netilmicin,

isepamicin,

P. aeruginosa

capreomycin,

S. epidermis

ribostamycin, apramycin, amikacin, paromomycin, tobramycin neomycin)


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poly(β-amino ester)

Vancomycin

UV-

S. aureus

124

125

polymerization 4-arm-PEGNH2,

4-arm- Vancomycin

amide bond and S. aureus

PEG-NHS

4-arm-

imine

and

PEG-CHO

formation

N-2-(hydroxyl propyl )3- Vancomycin

imine

trimethyl

formation

chitosan

ammonium chloride)

bond

bond S. aureus

126

MRSA

and

polydextran aldehyde

branched

catechol- silver

covalent

derivatized poly(ethylene nanoparticle

crosslinking by

glycol)

oxidation

S. epidermidis

135

E. coli

136

S. aureus

138

of

catechol poly(ethylene

glycol) silver

diacrylate

UV-

nanoparticle

polymerization

vinylpyrrolidone acrylic acid and N,N′- Ag/graphene

UV-

methylene bisacrylamide

polymerization

Acrylamide

gold nanoparticle

UVpolymerization


E. coli S. aureus

139


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gelatine conjugated with hydrogen-

enzyme induced S. aureus

hydroxyl

crosslinking

phenyl peroxide

152-154

P. putida

propionic acid, succinyl chitosan

E. coli

carboxymethyl

cellulose and hydroxyl phenyl

propionic

modified

acid

dendritic

polyglycerol

poly(ethylene

antimicrobial

glycol)diacrylate

peptide (HHC10; chemistry

thiol-ene

S. aureus

156

S. epidermidis

pentaerythritol tetrakis(3- HKRWWKWIR mercaptopropionate) methacrylated

W-NH2)

gelatine antimicrobial

methacryloyl-substituted tropoelastin

peptide

UV-

(Tet213; polymerization

MRSA

157

E. coli

KRWWKWWRR C)

dextran methacrylate

small biocide

molecular UVpolymerization

S. aureus E. coli MRSA


158

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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) Invivo activity against murine model of MRSA subcutaneous infection. Reproduced with

permission from ref 126. Copyright 2017 American Chemical Society. 49 ACS Paragon Plus Environment


As a result of thermoresponsiveness, the hydrogel showed sol-gel transition when temperature was increased across its lower critical solution temperature (LCST). This makes the hydrogel capable of sustained delivery of antibiotics on demand. Gentamicinshowed initial burst release from the matrix within 1h (almost 62 % drug release) and the release continued upto 7 days. When rabbit was injected with this formulation in infected condition, 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 crosslinking 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, S. aureus and almost completely cleared bacteria (S. aureus) in murine skin infection model within three days.123 Dziubla and co-workers have reported a vancomycin-loaded hydrogel where the free amino groups of vancomycin was undergone Michael type addition with the double bonds of polyethylene glycol diacrylate to form poly(βamino ester).124 This vancomycin incorporated precursor formed hydrogel upon UV-irradiation. Owing to the covalent linkage, vancomycin has lesser activity against S. aureus compared to 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 coworkers, 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 50 ACS Paragon Plus Environment

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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 haemorrhage 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 seconds while normal gauze took 5 minutes to stop blood flow from the wound site.125 Our group have also reported a vancomycin loaded hydrogel system, where the vancomycin was incorporated through imine bond chemistry (Figure 9A). Due to reversible nature of imine bond, the vancomycin release happened in a pH dependent manner (Figure 9A). In mice model, the hydrogel showed excellent antibacterial efficacy with huge reduction of bacterial burden in the site of infected wound (~ 6.1 log reduction) and the surrounding tissue (~ 5.8 log reduction) as well (Figure 9C).126 3.2.2 Hydrogels Loaded with Metal Ions and Nanoparticles The antimicrobial property of silver is known for a long time. Silver based various antimicrobial products are widely being used for wound dressing applications. Till date, antimicrobial property of other metal ions has also been reported. Unfortunately, the applications of the free metal ions are limited due to their toxicity towards 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. Alongside, 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 generation of reactive oxygen 51 ACS Paragon Plus Environment


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species. These metal nanoparticles are also known to exert toxic effects towards mammalian cells, which limit their application for topical usages. Thus, nanoparticle loaded hydrogels are drawing focus in the current scenario.135-142 Messersmith and co-workers have reported silverreleasing hydrogel, where the catechol functionalized 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 towards mammalian cells.135 In another example, Kong and coworkers demonstrated in situ photoreaction of aqueous mixture of silver nitrates, poly(ethylene glycol) diacrylate and vinylpyrrolidone, forming silver nanoparticle loaded hydrogel system.136 This was not only capable to inhibit bacterial growth, but also displayed antifouling property. Likewise, Kim and co-workers have reported another in-situ silver nanoparticle loaded hydrogel formulation.137 In basic media (pH = 9), 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 hydrogels showed antibacterial as well as antifouling properties and almost 98% wound closure within 15 days of the treatment.137 Yang and co-workers have prepared a hydrogel by crosslinking 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 nanoparticlebased system. For example, gold 52 ACS Paragon Plus Environment

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nanoparticle loaded 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 and copper etc.143-146 3.2.3 Miscellaneous A significant effort has also been directed towards development of hydrogels loaded with other antibacterial agents that include salicylic acid, chlorhexidine, AMPs and synthetic small molecular biocides etc.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, salicylic acid loaded various hydrogel systems have been reported for its effective delivery at the site of interest.147-150 Chlorhexidine is being used as disinfectant agents for topical applications. Chlorhexidine loaded hydrogel was also developed by Yu and co-workers that displayed antimicrobial efficacy with reduced toxicity towards 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 towards mammalian cell.152-154 In the 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 hydrogel, which was prepared by using poly(ethylene glycol)diacrylate and pentaerythritol tetrakis(3mercaptopropionate) through thiol-ene chemistry. HHC10 loaded this hydrogels showed activity 53 ACS Paragon Plus Environment


against various pathogenic bacteria (such as S. aureus and S. epidermis).155-156 Annabi and coworkers have demonstrated another AMP (Tet213; KRWWKWWRRC) loaded hydrogel system, where the hydrogel matrix was prepared from methacrylated gelatine (GelMA) and methacryloyl-substituted tropoelastin (MeTro) involving visible light-induced crosslinking. The ratio of MeTro/GelMA has an effect on elastic properties of the hydrogel with elastic modulus

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


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ranging from ~ 4 to 33 kPa and the adhesive strength was in between ~ 500-1100 kPa. This sprayable hydrogel showed antibacterial efficacy against both Gram-positive (MRSA) and Gram-negative bacteria (E.coli) with biocompatibility towards mammalian cells. Around 80% AMP release was observed within 72 h and reduction in bacterial growth was seen within 24 h.157 Recently, our group has 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 bacteria (MRSA, S. aureus, E. coli) within 2 h (Figure 10C) and also displayed antibiofilm activity. It reduced ~ 99.9 % MRSA colonization in mice model of skin-infections (Figure 10D). This hydrogel was found to be biocompatibility when skin-toxicity was investigated in various animal models (such as rat model of acute dermal toxicity, guinea pig model of skin sensitization and rabbit model of skin irritation).158 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’s sufferings and healthcare costs but may also result in death. This scenario has attracted attentions 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 (non-fouling strategy), ii) biocide releasing (releasebased strategy) and iii) contact killing (contact-based strategy).165 In 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 contact killing strategy, microbes are killed upon contact with the 55 ACS Paragon Plus Environment


coated surfaces, where both covalent and non-covalent surface-modifications have been adopted. There are many reviews in literature which have discussed various aspects of antimicrobial surfaces.166-171 In this review, with a brief overview of non-fouling, release-active and covalently modified surfaces, we will be emphasizing on non-covalent strategies for development of antimicrobial surfaces. 4.1 Adhesion Resistant Surfaces 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. It involves deposition of a self-assembled monolayer (SAM) on a substrate (usually a gold surface), followed by functionalization of the

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


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SAM to contain the required PEG functionality.172 Recently, polymers with zwitterionic head groups such as poly(phosphorylcholine), poly (sulfobetaine) and poly(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 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 was shown to be promising for coating medical devices, because of their biomimetic nature which firstly provides biocompatibility by reducing attachment of human cells to the device and secondly 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 diffused over time from a material surface, inducing death either of nearby (but non-adhered) bacteria or of adhered bacteria.175 A variety of active antimicrobials such as antibiotics, metal salts or nanoparticle, bioactive species (e.g., phage virus), etc. has 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 perform bactericidal function. Silver has long been known to be an antimicrobial and the metal ions (Ag+) have a significant antimicrobial activity finding its use in various commercial aspects. AgION Technology’s AgIONTM and AcryMed’s SilvaGardTM are two of the most well-known commercial coating products which rely on the diffusion of Ag+ 57 ACS Paragon Plus Environment


ions from the substrate material and inactivate the microorganisms. But a possible drawback of silver-based antimicrobials is the cytotoxicity of Ag+ ions towards 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 Developments of a phage containing wound dressing, hydrogel-coated silicone catheter, etc. with lytic bacteriophages have 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 class of antimicrobials (such as, antibiotics, antimicrobial peptide, quaternary ammonium compounds, N-halamines, etc.) along 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, non-covalent approach is based on painting of water-insoluble and organosoluble 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 the initial efforts to develop non-leaching permanent antimicrobial coatings on different surfaces. Klibanov and coworkers 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, P. aeruginosa. The polymer was further covalently attached to various surfaces which showed activity against various airborne and water-borne 58 ACS Paragon Plus Environment

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bacteria.187,188 Russel and co-workers have contributed extensively in 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 antimicrobial surface coating based on polyoxonorbornenes, which were attached to various modified surfaces by UV induced cross-linking.196 The above mentioned reports involve reactions on the surfaces which are complicated and require sophisticated techniques. The researchers are also immensely focusing on the development of coating procedures which do not employ reactions involving the surfaces. Recent research has resulted in the development of polymers functionalized with active linkers which 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 onestep fabrication. Locklin and co-workers have developed coating involving benzophenone moieties which render 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 Gram-positive and Gram-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 dopamine-incorporated antimicrobial polymeric coatings are reported in the recent past. Yang and co-workers have developed a one-step polycarbonate-based coating which employs dopamine as a linker.201 Ring opening 59 ACS Paragon Plus Environment


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polymerization was at the heart of this polymerization. The brush-like polycarbonates when coated on surface, was 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 environment suitable for biofilm formation. To impart non-fouling and antibiofilm properties to a surface, researchers have focused on the development of polymeric coatings which can switch between its bactericidal and bacteria repellent nature.206 Jiang and co-workers have contributed immensely towards this direction.207,208 In an earlier report, they have constructed a new switchable polymer surface coating, which combines the advantages of both nonfouling and bactericidal

properties.

When

immobilized

on

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 But this suffers from the limitation that it was not reversible and the switching could occur only once. Later on, they developed a smart coating which 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,N-dimethyl-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 Non-covalent Polymeric Coatings 60 ACS Paragon Plus Environment

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Even though covalent immobilization resists leaching of the coating, it also has several challenges. The fabrication of covalent coating needs trained professional and involves complicated procedures. As a result, non-covalent surface modification was called into play.

209

By following this principle, the preparation of antimicrobial surfaces becomes very simple, where coating procedure is as simple as like 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 to kill 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 and thatpartially contributed to its antibacterial activity as well.214 Later on, Klibanov and co-workers were able to overcome this issue by using the 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, N-methyl-PEI). This higher hydrophobicity bearing polymer was hypothesised to strengthen the intermolecular attractions and thus lesser 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 mechanism of antibacterial action was primarily based on microbial contact on the coated surface. However, the resulted 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, that was capable to kill influenza virus within minutes (~ 4 log reductions in the viral titer). Importantly, the glass slide coated with this antimicrobial polymer was capable to 61 ACS Paragon Plus Environment


Figure 12. A) Hydrophobic cationic polymers ( R = C18H37) (i) Linear N-Alkyl N-Methyl PEIs, (ii) Branched N-Alkyl N-Methyl PEIs; B) Antibacterial activity of 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. inactivate drug-resistant strains of influenza virus, poliovirus and rotavirus with high efficacy.218 Sen and co-workers have reported a class of antimicrobial composite by on-site precipitation of AgBr nanoparticles using a pyridinium polymer.219,220 The composites when coated on surface, killed bacteria as well as resisted biofilm formation. It was noteworthy that the antimicrobial 62 ACS Paragon Plus Environment

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action of these coated surfaces not only involved the contact-killing by the pyridinium polymer, but also killed microbes in a release-based manner. In another report, Hammond, Klibanov and co-workers have demonstrated a layer by layer (LbL) assembly of this bactericidal and virucidal cationic PEI derivative by using the anionic poly (acrylic acid).221,222 The surface coated with optimum the LbL film thickness (10 nm) showed potent efficacy against both airborne and mentioned non-toxic nature towards mammalian cells. These films coated surface was capable to prevent bacterial attachment too, and thus could be developed as coating materials for prevention of biofilms formation on various surfaces.223 Furthermore, this antimicrobial polymer was coated on medically relevant titanium and stainless-steel surface. The resulted surface showed prevention of biofilm formation by S. aureus in large-animal (sheep) trauma model with promotes bone healing in this animal model. This indicated potential application of thisantimicrobial polymer in the development of various efficient orthopedic implants. Our group has further investigated a detailed structure-activity relationship of this polyethylenimine based quaternized polymers coated surfaces. A series of quaternized polyethylenimine were prepared by using the precursor polymers of various molecular weights (Figure 12). PEI derivatives with varied degree of quaternization was prepared using different long chain alkyl bromides, such as 1-bromododecane,

1-bromohexadecane,

1-bromooctadecane,

1-bromoeicosane,

and

1-

bromodocosane. The resulting polymers were water insoluble and organo-soluble, which were then coated on various surfaces by easily dissolving it in various organic solvent. The surface coated with these polymers displayed activity against various pathogenic bacteria including drug-resistant superbugs MRSA and VRE (Figure 12 B and 12 C). It was also capable to show efficacy against pathogenic fungi such as Candida spp. and Cryptococcus spp (Figure 12 D). Upon contact on the polymer coated surfaces, complete killing (~ 5 Log reductions in cell 63 ACS Paragon Plus Environment


viability) was noticed for both bacteria and fungi. This antibacterial surface also retained its efficacy in presence of various complex mammalian fluids (such as serum, plasma, and blood) without showing significant toxicity towards mammalian cells (RBCs). Importantly, bacterial resistance development propensity was unseen even after 20 continuous passages, thus this class of polymers could be developed as “microbicidal paint” for various biomedical and household

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 in-vivo mice model; D) In-vivo antibacterial activity of polymer-coated catheters (FESEM images) Reproduced with permission from ref. 225. Copyright 2016 American Chemical Society. 64 ACS Paragon Plus Environment

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applications.224 Recently, our group has reported another class of antimicrobial surfaces, where quaternized chitin derivatives were used. 225 The water-insoluble and organo-soluble antibacterial chitin derivatives were prepared by selective quaternization at C-6 position of the sugar unit by using various N,N-dimethylalkylamines (such as N,N-dimethyltetradecylamine, N,N-waterborne E.

coli

and

S.

aureus

including

virucidal

activity against

influenza

virus

with

dimethylhexadecylamine) (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, it was 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 drug-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 degree of quaternization 39% and 48%, an increased antibacterial activity against S. aureus was seen. The MIA (minimum inhibitory amount) value for the polymer consisted 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 noticed for E. coli, the MIA value decreased from 7.8 to 3.9 µg/mm2. For the polymers with higher degree of quaternization, 55%, the increasing effect on S. aureus activity although retained, the activity against E. coli was decreased with 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 against P. aeruginosa, MRSA, VRE and β-lactam-resistant K. pneumoniae. Additionally, the surface coated with one of the best active polymers was capable to prevent the biofilm formation by both S. aureus and E. coli. Importantly, the medical-grade catheter coated with this polymer 65 ACS Paragon Plus Environment


reduced MRSA burden by 3.7 log (compared to non-coated catheter) in a murine model of subcutaneous infection with no biofilm development under in-vivo settings (Figure 13 D). Thus, this chitin based antibacterial polymers can be developed as coating material for the prevention of device associated infections.225 5. CONCLUSIONS AND FUTURE PERSPECTIVES Till 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 having improved antimicrobial properties are being developed. An extensive analysis of AMP-inspired antimicrobial polymers has underpinned the importance of different structural parameters such as hydrophilic/hydrophobic balance, molecular weights, charges, hydrogen bonds, polymeric architectures, etc. 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 non-covalent interactions. To evaluate 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 suitable and commonly adopted technique for water-soluble antimicrobial polymers. However, for sparing soluble samples, other suitable methods need to be developed which 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. 66 ACS Paragon Plus Environment

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In case of antimicrobial surfaces, Japanese Industrial Standard (JIS) assay is considered as a standard method for evaluation of antimicrobial activity. But, this assay does not mimic the real scenario and therefore are unable to anticipate the actual efficacy under realistic conditions. Hence, the field direly needs standardized methods for fast and reliable evaluation of 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. Majority of studies till date are limited to the antimicrobial activity of polymeric compounds being evaluated against drug-sensitive microbes in-vitro. Future research should expand to drugresistant strains including clinical isolates to establish the clinical relevance of antimicrobial polymers as drugs. There are very few reports directed towards in-vivo evaluation of these macromolecular antimicrobials and most of the existing studies focus on their usage in treating topical infections. A clear challenge in the field therefore remains to explore the possibility in treating systemic infections. The detailed pharmacokinetics, pharmacodynamics and toxicity of water-soluble 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 the internal organ infections. The biocompatibility, biodegradability and stability of polymer-based antimicrobial hydrogels and surfaces also need to be investigated in in-vivo models. Furthermore, the field should move forward with more focus on antibiofilm research. An inadequate effort has been made to target microbial biofilm, which is the source of a majority of infections (about 80%). Future research can target microbial communication system (quorum sensing) to control their biofilm formation. It is well known that quorum sensing plays an essential role in regulating microbial biofilm lifestyle. Thus, incorporation of the strategies that 67 ACS Paragon Plus Environment


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 biofilm.226 Last but not the least, an 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 leading to the discovery of innovative strategies to combat infections and antimicrobial resistance in upcoming days.

AUTHOR INFORMATION Corresponding Author *(J.H.) Phone: (+91) 80-2208-2565. Fax: (+91) 80-2208-2627. E-mail: [email protected].

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

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TOC GRAPHICAL ABSTRACT


Recent progress in polymer research to tackle infections and

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