Synthetic polymers have been used as a molecular platform to develop host-defense antimicrobial peptide (AMP) mimetics which are effective in killing drug-resistant bacteria. In this topical review, we will discuss the AMP-mimetic design and chemical optimization strategies as well as the biological and biophysical implications of AMP mimicry by synthetic polymers. Traditionally, synthetic polymers have been used as a chemical means to replicate the chemical functionalities and physicochemical properties of AMPs (e.g., cationic charge, hydrophobicity) to recapitulate their mode of action. However, we propose a new perception that AMP-mimetic polymers are an inherently bioactive platform as whole molecules, which mimic more than the side chain functionalities of AMPs. The tunable nature and chemical simplicity of synthetic random polymers facilitate the development of potent, cost-effective, broad-spectrum antimicrobials. The polymer-based approach offers the potential for many antimicrobial applications to be used directly in solution or attached to surfaces to fight against drug-resistant bacteria.

The antibacterial and antibiofilm activities of cationic amphiphilic methacrylate polymers against cariogenic bacterium S. mutans were investigated. Cationic homopolymer PE0 and copolymer PE31 containing 31 mol % of ethyl methacrylate were synthesized by reversible addition–fragmentation chain transfer polymerization. These polymers displayed bactericidal activity toward S. mutans and prevented biofilm formation by killing the planktonic bacteria. At a concentration of 1000 μg/mL when incubated for 2 h the polymers reduced >80% of biofilm biomass. When the polymer assay solution with the biofilm was vigorously mixed using a pipet for 30 s, >50% of biofilm mass was removed at a polymer concentration of 250 μg/mL. Chlorhexidine and a cationic surfactant failed to reduce the biofilm mass at the same concentration. PE0 was the most effective in removing biofilm and did not show any significant cytotoxicity to human gingival fibroblast and periodontal ligament stem cells when incubated for 10 min.

ABSTRACTThis work describes synthesis of antimicrobial methacrylate copolymers by reversible addition-fragmentation chain transfer (RAFT) polymerization and examines the versatility of this approach for improving chemical optimization to create potent, non-toxic antimicrobial polymers. Specifically, this study focuses on the radical-mediated transformation of end group of antimicrobial peptide-mimetic polymer. RAFT polymerization using 2-cyano-2-yl-dithiobenzoate provided a statistical methacrylate copolymer consisting of aminobutyl and ethyl groups in the side chains. The following radical-mediated modification using free radical initiators successfully transformed the ω-end group of parent copolymer from dithiobenzoate to a cyanoisobutyl or aminoethyl cyanopentanoate group without any significant changes to the polymer molecular weight. In general, the parent polymer and variants showed a broad spectrum of activity against a panel of bacteria, but low hemolytic activity against human red blood cells. The parent copolymer with the dithiobenzoate end-group showed highest antimicrobial and hemolytic activities as compared with other copolymers. The copolymers caused membrane depolarization in Staphylococcus aureus, while the ability of copolymers for membrane disruption is not dependent on the end-group structures. The synthetic route reported in this study will be useful for further study of the role of polymer end-groups in the antimicrobial activity of copolymers. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017, 55, 304–312

Capture and release of amphiphilic copolymers by a nano-sized polysaccharide gel (nanogel) was controlled by altering the hydrophobic binding affinity between the copolymer chains and nanogel. The antimicrobial activity of captured copolymer chains was suppressed, and regained upon release from the nanogel.

To gain an understanding of the toxicity of antimicrobial polymers to human cells, their hemolytic action was investigated using human red blood cells (RBCs). We examined the hemolysis induced by cationic amphiphilic methacrylate random copolymers, which have amino ethyl sidechains as cationic units and either butyl or methyl methacrylate as hydrophobic units. The polymer with 30 mol% butyl sidechains (B30) displayed higher hemolytic toxicity than the polymer with 59 mol% methyl sidechains (M59). B30 also induced faster release of hemoglobin from RBCs than M59. A new theoretical model is proposed based on two consecutive steps to form active polymer species on the RBC membranes, which are associated to RBC lysis. This model takes the all-or-none release of hemoglobin by the rupture of RBCs into account, providing new insight into the polymer-induced hemolysis regarding how individual or collective cells respond to the polymers.Hemolysis induced by antimicrobial methacrylate random copolymers was studied based on the new theoretical model of the all-or-none release of hemoglobin. This model provides insight into the polymer-induced hemolysis regarding how individual or collective cells response to the polymers.

This study reports a synthetic polymer functionalized with catechol groups as dental adhesives. We hypothesize that a catechol-functionalized polymer functions as a dental adhesive for wet dentin surfaces, potentially eliminating the complications associated with saliva contamination. We prepared a random copolymer containing catechol and methoxyethyl groups in the side chains. The mechanical and adhesive properties of the polymer to dentin surface in the presence of water and salivary components were determined. It was found that the new polymer combined with an Fe3+ additive improved bond strength of a commercial dental adhesive to artificial saliva contaminated dentin surface as compared to a control sample without the polymer. Histological analysis of the bonding structures showed no leakage pattern, probably due to the formation of Fe–catechol complexes, which reinforce the bonding structures. Cytotoxicity test showed that the polymers did not inhibit human gingival fibroblast cells proliferation. Results from this study suggest a potential to reduce failure of dental restorations due to saliva contamination using catechol-functionalized polymers as dental adhesives.

The function and mode of action of curcumin in modulating the formation of lipid raft domains were investigated by microscopic observation using model membranes. Curcumin induces fusion of lipid raft domains at extremely low concentrations through the alteration of the boundary between the ordered and disordered phases.

We demonstrate utilization of star-shaped polymers as high-density polymer brush coatings and their effectiveness to inhibit the adhesion of platelets and bacteria. Star polymers consisting of poly(2-hydroxyethyl methacrylate) (PHEMA) and/or poly(methyl methacrylate) (PMMA) were synthesized using living radical polymerization with a ruthenium catalyst. The polymer coatings were prepared by simple drop casting of the polymer solution onto poly(ethylene terephthalate) (PET) surfaces and then dried. Among the star polymers prepared in this study, the PHEMA star polymer (star-PHEMA) and the PHEMA/PMMA (mol. ratio of 71/29) heteroarm star polymer (star-H71M29) coatings showed the highest percentage of inhibition against platelet adhesion (78–88% relative to the non-coated PET surface) and Escherichia coli (94–97%). These coatings also showed anti-adhesion activity against platelets after incubation in Dulbecco's phosphate buffered saline or surfactant solution for 7 days. In addition, the PMMA component of the star polymers increased the scratch resistance of the coating. These results indicate that the star-polymer architecture provides high polymer chain density on PET surfaces to prevent adhesion of platelets and bacteria, as well as coating stability and physical durability to prevent exposure of bare PET surfaces. The star polymers provide a simple and effective approach to preparing anti-adhesion polymer coatings on biomedical materials against the adhesion of platelets and bacteria.

The in vitro and in vivo antimicrobial activity of primary ammonium ethyl methacrylate homopolymers (AEMPs) was investigated. AEMPs with different degrees of polymerization (DP = 7.7–12) were prepared by reversible addition–fragmentation chain-transfer (RAFT) polymerization. The AEMPs showed higher inhibitory effects against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), than Gram-negative bacteria. The AEMPs also showed potent anti-S. aureus activity in the presence of fetal bovine serum, whereas the activity of the antibiotic mupirocin was reduced under the same conditions. The AEMPs showed very little or no hemolytic activity. The cytotoxicity of AEMPs against mammalian cells HEp-2 and COS-7 was concentration-dependent, and the cell viability significantly decreased at higher polymer concentrations. The AEMPs significantly reduced the number of viable S. aureus cells in the nasal environment of cotton rats when compared to that of the control. This study demonstrates that AEMPs have potential for use in treating topical S. aureus infections.

Polymeric synthetic mimics of antimicrobial peptides (SMAMPs) have recently demonstrated similar antimicrobial activity as natural antimicrobial peptides (AMPs) from innate immunity. This is surprising, since polymeric SMAMPs are heterogeneous in terms of chemical structure (random sequence) and conformation (random coil), in contrast to defined amino acid sequence and intrinsic secondary structure. To understand this better, we compare AMPs with a “minimal” mimic, a well-characterized family of polydisperse cationic methacrylate-based random copolymer SMAMPs. Specifically, we focus on a comparison between the quantifiable membrane curvature generating capacity, charge density, and hydrophobicity of the polymeric SMAMPs and AMPs. Synchrotron small-angle X-ray scattering (SAXS) results indicate that typical AMPs and these methacrylate SMAMPs generate similar amounts of membrane negative Gaussian curvature (NGC), which is topologically necessary for a variety of membrane-destabilizing processes. Moreover, the curvature generating ability of SMAMPs is more tolerant of changes in the lipid composition than that of natural AMPs with similar chemical groups, consistent with the lower specificity of SMAMPs. We find that, although the amount of NGC generated by these SMAMPs and AMPs are similar, the SMAMPs require significantly higher levels of hydrophobicity and cationic charge to achieve the same level of membrane deformation. We propose an explanation for these differences, which has implications for new synthetic strategies aimed at improved mimesis of AMPs.

Antimicrobial and hemolytic activities of amphiphilic random copolymers were modulated by the structure of the cationic side chain spacer arms, including 2-aminoethylene, 4-aminobutylene, and 6-aminohexylene groups. Cationic amphiphilic random copolymers with ethyl methacrylate (EMA) comonomer were prepared with a range of comonomer fractions, and the library of copolymers was screened for antimicrobial and hemolytic activities. Copolymers with 4-aminobutylene cationic side chains showed an order of magnitude enhancement in their antimicrobial activity relative to those with 2-aminoethylene spacer arms, without causing adverse hemolysis. When the spacer arms were further elongated to hexylene, the copolymers displayed potent antimicrobial and hemolytic activities. The 4-aminobutylene side chain appears to be the optimal spacer arm length for maximal antimicrobial potency and minimal hemolysis, when combined with hydrophobic ethylmethacrylate in a roughly 70/30 ratio. The copolymers displayed relatively rapid bactericidal kinetics and broad-spectrum activity against a panel of Gram-positive and Gram-negative bacteria. The effect of the spacer arms on the polymer conformation in the membrane-bound state was investigated by molecular dynamics simulations. The polymer backbones adopt an extended chain conformation, parallel to the membrane surface. A facially amphiphilic conformation at the membrane surface was observed, with the primary ammonium groups localized at the lipid phoshophate region and the nonpolar side chains of EMA comonomers buried in the hydrophobic membrane environment. This study demonstrates that the antimicrobial activity and molecular conformation of amphiphilic methacrylate random copolymers can be modulated by adjustment of cationic side chain spacer arms.

Self-degradable antimicrobial copolymers bearing cationic side chains and main-chain ester linkages were synthesized using the simultaneous chain- and step-growth radical polymerization of t-butyl acrylate and 3-butenyl 2-chloropropionate, followed by the transformation of t-butyl groups into primary ammonium salts. We prepared a series of copolymers with different structural features in terms of molecular weight, monomer composition, amine functionality, and side chain structures to examine the effect of polymer properties on their antimicrobial and hemolytic activities. The acrylate copolymers containing primary amine side chains displayed moderate antimicrobial activity against E. coli but were relatively hemolytic. The acrylate copolymer with quaternary ammonium groups and the acrylamide copolymers showed low or no antimicrobial and hemolytic activities. An acrylate copolymer with primary amine side chains degraded to lower molecular weight oligomers with lower antimicrobial activity in aqueous solution. This degradation was due to amidation of the ester groups of the polymer chains by the nucleophilic addition of primary amine groups in the side chains resulting in cleavage of the polymer main chain. The degradation mechanism was studied in detail by model reactions between amine compounds and precursor copolymers.

Sum frequency generation (SFG) vibrational spectroscopy was used to analyze interactions between solid-supported lipid bilayers acting as models for cellular membranes and several membrane-active random copolymers with different lipophilic side chains, named 0R (no group), 33Me (methyl group), 11Bz (benzyl group), and 33Bu (butyl group), according to both the identity and percentage of the side chains within the polymer. Biological tests of the minimum inhibitory concentration (MIC) and hemolytic concentration were performed. The inherent surface sensitivity of SFG allowed for independent monitoring of isotopically labeled lipid bilayer leaflets as a function of concentration to study polymer−bilayer interaction mechanisms. Concentrations at which each bilayer leaflet was disrupted were quantitatively determined for each copolymer. Spectroscopic evidence of interaction with the bilayer below the critical concentrations was observed for the 11Bz polymer. The lipophilic butyl side chain of the 33Bu polymer was found to be oriented parallel to the surface normal. This research shows that SFG is a useful analytical technique which provides unique details regarding the interaction mechanisms of these membrane-active copolymers and lipid bilayers.

Cationic, amphiphilic polymers are currently being used as antimicrobial agents that disrupt biomembranes, although their mechanisms remain poorly understood. Herein, membrane association and disruption by amphiphilic polymers bearing primary, tertiary, or quaternary ammonium salt groups reveal the role of cationic group structure in the polymer−membrane interaction. The dissociation constants of polymers to liposomes of POPC were obtained by a fluorometric assay, exploiting the environmental sensitivity of dansyl moieties in the polymer end groups. Dye leakage from liposomes and solid-state NMR provided further insights into the polymer-induced membrane disruption. Interestingly, the polymers with primary amine groups induced reorganization of the bilayer structure to align lipid headgroups perpendicular to the membrane. The results showed that polymers bearing primary amines exceed the tertiary and quaternary ammonium counterparts in membrane binding and disrupting abilities. This is likely due to enhanced complexation of primary amines to the phosphate groups in the lipids, through a combination of hydrogen bonding and electrostatic interactions.

A new strategy for preparing antimicrobial surfaces by a simple dip-coating procedure is reported. Amphiphilic polycations with different mole ratios of monomers containing dodecyl quaternary ammonium, methoxyethyl, and catechol groups were synthesized by free-radical polymerization. The polymer coatings were prepared by immersing glass slides into a polymer solution and subsequent drying and heating. The quaternary ammonium side chains endow the coatings with potent antibacterial activity, the methoxyethyl side chains enable tuning the hydrophobic/hydrophilic balance, and the catachol groups promote immobilization of the polymers into films. The polymer-coated surfaces displayed bactericidal activity against Escherichia coli and Staphylococcus aureus in a dynamic contact assay and prevented the accumulation of viable E. coli, S. aureus, and Acinetobacter baumannii for up to 96 h. Atomic force microscopy (AFM) images of coating surfaces indicated that the surfaces exhibit virtually the same smoothness for all polymers except the most hydrophobic. The hydrophobic polymer without methoxyethyl side chains showed clear structuring into polymer domains, causing high surface roughness. Sum-frequency generation (SFG) vibrational spectroscopy characterization of the surface structures demonstrated that the dodecyl chains are predominantly localized at the surface−air interface of the coatings. SFG also showed that the phenyl groups of the catechols are oriented on the substrate surface. These results support our hypothesis that the adhesive or cross-linking functionality of catechol groups discourages polymer leaching, allowing the tuning of the amphiphilic balance by incorporating hydrophilic components into the polymer chains to gain potent biocidal activity.

Hemolysis induced by antimicrobial polymers was examined to gain an understanding of the mechanism of polymer toxicity to human cells. A series of cationic amphiphilic methacrylate random copolymers containing primary ammonium groups as the cationic functionality and either butyl or methyl groups as hydrophobic side chains have been prepared by radical copolymerization. Polymers with 0−47 mol % methyl groups in the side chains, relative to the total number of monomeric units, showed antimicrobial activity but no hemolysis. The polymers with 65 mol % methyl groups or 27 mol % butyl groups displayed both antimicrobial and hemolytic activity. These polymers induced leakage of the fluorescent dye calcein trapped in human red blood cells (RBCs), exhibiting the same dose−response curves as for hemoglobin leakage. The percentage of disappeared RBCs after hemolysis increased in direct proportion to the hemolysis percentage, indicating complete release of hemoglobin from fractions of RBCs (all-or-none leakage) rather than partial release from all cells (graded leakage). An osmoprotection assay using poly(ethylene glycol)s (PEGs) as osmolytes indicated that the PEGs with MW > 600 provided protection against hemolysis while low molecular weight PEGs and sucrose had no significant effect on the hemolytic activity of polymers. Accordingly, we propose the mechanism of polymer-induced hemolysis is that the polymers produce nanosized pores in the cell membranes of RBCs, causing an influx of small solutes into the cells and leading to colloid-osmotic lysis.

We examined the antibacterial and hemolytic activities in a series of amphiphilic block and random copolymers of poly(vinyl ether) derivatives prepared by base-assisting living cationic polymerization. Block and random amphiphilic copolymers with similar monomer compositions showed the same level of activity against Escherichia coli. However, the block copolymers are much less hemolytic compared to the highly hemolytic random copolymers. These results indicate that the amphiphilic copolymer structure is a key determinant of activity. Furthermore, the block copolymers induced dye leakage from lipid vesicles consisting of E. coli-type lipids, but not mammalian lipids, while the random copolymers disrupted both types of vesicles. In addition, both copolymers displayed bactericidal and hemolytic activities at concentrations 1 or 2 orders of magnitude lower than their critical (intermolecular) aggregation concentrations (CACs), as determined by light scattering measurements. This suggests that polymer aggregation or macromolecular assembly is not a requisite for the antibacterial activity and selectivity against bacteria over human red blood cells (RBCs). We speculate that different single-chain conformations between the block and random copolymers play an important role in the antibacterial action and underlying antibacterial mechanisms.

Antimicrobial polymers, designed to mimic the salient structural features of host defense peptides, are an emerging class of materials with potential for applications to combat infectious disease. Because the putative mode of action relies on physiochemical parameters of peptides such as hydrophobicity and cationic charge, rather than specific receptor-mediated interactions, the activity of the polymers can be modulated by tuning key structural parameters. While a wide diversity of chemical structures have been reported as antimicrobial polymers, a precise understanding of the structural factors which control their activity is a subject of current investigations. In this mini-review, we will outline the design principles that have been developed so far to fine tune the activity of these antimicrobial agents. The roles played by specific structural features such as cationic charge, hydrophobicity, and molecular weight will be discussed. Future directions of the field and potential challenges will be proposed.

A library of amphiphilic random copolymers containing cationic and hydrophobic side chains were prepared by copolymerization of amine-functionalized methacrylate monomers with various ratios of an alkyl methacrylate. Primary or tertiary amine groups, or quaternary ammonium groups, were utilized as the source of cationic charge in each copolymer series. The antimicrobial and hemolytic activities of these copolymers are reported, enabling a systematic assessment of the effect different amine groups exert on the biological activity of the polymers. It was shown that the copolymer composition of amphiphilic copolymers containing primary or tertiary amine groups can be tuned to achieve potent antimicrobial activity while minimizing red blood cell lysis. On the other hand, the copolymers containing quaternary ammonium groups required a greater amount of hydrophobic comonomer to express activity and showed generally lower selectivity for E. coli versus human red blood cells. Potentiometric titration data revealed the fraction of the primary or tertiary amine groups in the polymers, which are deprotonated (basic) at physiological pH. Measurements of the bactericidal and hemolytic activities in buffers of pH varying from 6 to 8 showed the impact of polymer ionization on biological activity. A decrease in the fraction of amine groups that are cationic, from α = 1.0 to 0.7, caused an enhancement of antimicrobial and hemolytic activity. As this value was decreased further to α = 0.5, loss of activity was observed. The activities of polymers containing quaternary ammonium groups were shown to be pH-independent.

Low molecular weight random copolymers bearing protonated primary amine groups and hydrophobic alkyl groups in the side chains were synthesized and their activities against E. coli, S. aureus, human red blood cells, and human epithelial carcinoma cells (HEp-2) were quantified. The mole fraction of alkyl side chains in the copolymers (falkyl) and the length of the alkyl chains were major determinants of the activities. Against E. coli cells, activity was diminished as falkyl was increased from 0 to about 0.2, but was then enhanced dramatically as falkyl was increased further. Activity against S. aureus was diminished continually with increasing falkyl. The cytotoxicity to human epithelial carcinoma cells also decreased with increasing falkyl. Conversely, hemolytic activity showed monotonic enhancement with increasing falkyl. The cationic homopolymer (falkyl = 0) completely inhibited S. aureus growth at 3 μM (10.2 μg/mL) and completely inhibited metabolic activity in HEp-2 cells at 10 μM (34 μg/mL), although it did not induce any detectable hemolysis up to 645 μM (2000 μg/mL). Polymer-induced dye leakage from liposomes provided a biophysical basis for understanding the factors which modulate the polymer−membrane interactions. Disruption of Zwitterionic POPC vesicles induced by the copolymers was enhanced as falkyl increased, following trends similar to the hemolytic activity data. The ability of the polymers to permeabilize vesicles of POPE/POPG and DOPG/Lysyl-DOPG/CL displayed trends similar to trends in their activities against E. coli and S. aureus, respectively. This was interpreted as evidence that the antimicrobial mechanism employed by the polymers involves disruption of bacterial cell membranes. An investigation of leakage kinetics revealed that the cationic homopolymer induced a gradual release of contents from POPE/POPG and DOPG/Lysyl-DOPG/CL vesicles, while the more hydrophobic copolymers induced rapid dye efflux. The results are interpreted as evidence that the cationic homopolymer and hydrophobic copolymers in this study exert their antimicrobial action by fundamentally different mechanisms of membrane disruption.

The function and mode of action of curcumin in modulating the formation of lipid raft domains were investigated by microscopic observation using model membranes. Curcumin induces fusion of lipid raft domains at extremely low concentrations through the alteration of the boundary between the ordered and disordered phases.

Capture and release of amphiphilic copolymers by a nano-sized polysaccharide gel (nanogel) was controlled by altering the hydrophobic binding affinity between the copolymer chains and nanogel. The antimicrobial activity of captured copolymer chains was suppressed, and regained upon release from the nanogel.

We demonstrate utilization of star-shaped polymers as high-density polymer brush coatings and their effectiveness to inhibit the adhesion of platelets and bacteria. Star polymers consisting of poly(2-hydroxyethyl methacrylate) (PHEMA) and/or poly(methyl methacrylate) (PMMA) were synthesized using living radical polymerization with a ruthenium catalyst. The polymer coatings were prepared by simple drop casting of the polymer solution onto poly(ethylene terephthalate) (PET) surfaces and then dried. Among the star polymers prepared in this study, the PHEMA star polymer (star-PHEMA) and the PHEMA/PMMA (mol. ratio of 71/29) heteroarm star polymer (star-H71M29) coatings showed the highest percentage of inhibition against platelet adhesion (78–88% relative to the non-coated PET surface) and Escherichia coli (94–97%). These coatings also showed anti-adhesion activity against platelets after incubation in Dulbecco's phosphate buffered saline or surfactant solution for 7 days. In addition, the PMMA component of the star polymers increased the scratch resistance of the coating. These results indicate that the star-polymer architecture provides high polymer chain density on PET surfaces to prevent adhesion of platelets and bacteria, as well as coating stability and physical durability to prevent exposure of bare PET surfaces. The star polymers provide a simple and effective approach to preparing anti-adhesion polymer coatings on biomedical materials against the adhesion of platelets and bacteria.