Targeting Cariogenic Streptococcus mutans in Oral Biofilms with Charge-Switching Smart Antimicrobial Polymers.

Cariogenic biofilms produce strong acidic microenvironments, which is the primary cause of dental caries. Streptococcus mutans is a dominant species in cariogenic biofilms. Herein, we report a pH-responsive, charge-switching smart copolymer to selectively target and eradicate bacteria in cariogenic biofilms. To that end, the copolymer is designed to be activated in an acidic environment. The smart copolymer, Poly-1A, consists of ternary compositions of monomers with a cationic ethyl ammonium group, a carboxylic group, and a hydrophobic group in the side chains. The net charge of Poly-1A was charge neutral at neutral pH, but it switched to be cationic because the acidic carboxylate side chains were protonated and became neutral; however, the ammonium groups remained positive. Poly-1A with a net positive charge bound to the anionic surface of oral bacteria by electrostatic interactions and disrupted the bacterial membranes, causing bacterial death. Poly-1A reduced the cell viability of planktonic and biofilm S. mutans at pH 4.5, while it was not bactericidal at pH 7.4. Poly-1A did not reduce the cell viability of human gingival fibroblasts and periodontal ligament stem cells for a 1 h incubation.

Biomimetic antimicrobial polymers-Design, characterization, antimicrobial, and novel applications.

Biomimetic antimicrobial polymers have been an area of great interest as the need for novel antimicrobial compounds grows due to the development of resistance. These polymers were designed and developed to mimic naturally occurring antimicrobial peptides in both physicochemical composition and mechanism of action. These antimicrobial peptide mimetic polymers have been extensively investigated using chemical, biophysical, microbiological, and computational approaches to gain a deeper understanding of the molecular interactions that drive function. These studies have helped inform SARs, mechanism of action, and general physicochemical factors that influence the activity and properties of antimicrobial polymers. However, there are still lingering questions in this field regarding 3D structural patterning, bioavailability, and applicability to alternative targets. In this review, we present a perspective on the development and characterization of several antimicrobial polymers and discuss novel applications of these molecules emerging in the field. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease.

Molecular weight tuning optimizes poly(2-methoxyethyl acrylate) dispersion to enhance the aging resistance and anti-fouling behavior of denture base resin.

Poly(methyl methacrylate) (PMMA)-based denture base resins easily develop oral bacterial and fungal biofilms, which may constitute a significant health risk. Conventional bacterial-resistant additives and coatings often cause undesirable changes in the resin. Reduced bacterial resistance over time in the harsh oral environment is a major challenge in resin development. Poly(2-methoxyethyl acrylate) (PMEA) has anti-fouling properties; however, due to the oily/rubbery state of this polymer, and its surface aggregation tendency in a resin mixture, its direct use as a resin additive is limited. This study aimed to optimize the use of PMEA in dental resins. Acrylic resins containing a series of PMEA polymers with various molecular weights (MWs) at different concentrations were prepared, and the mechanical properties, surface gloss, direct transmittance, and cytotoxicity were evaluated, along with the distribution of PMEA in the resin. Resins with low-MW PMEA (2000 g mol-1) (PMEA-1) at low concentrations satisfied the clinical requirements for denture resins, and the PMEA was homogeneously distributed. The anti-fouling performance of the resin was evaluated for protein adsorption, bacterial and fungal attachment, and saliva-derived biofilm formation. The PMEA-1 resin most effectively inhibited biofilm formation (∼50% reduction in biofilm mass and thickness compared to those of the control). Post-aged resins maintained their mechanical properties and anti-fouling activity, and polished surfaces had the same anti-biofilm behavior. Based on wettability and tribological results, we propose that the PMEA additive creates a non-stick surface to inhibit biofilm formation. This study demonstrated that PMEA additives can provide a stable and biocompatible anti-fouling surface, without sacrificing the mechanical properties and aesthetics of denture resins.

An Antimicrobial Peptide-Mimetic Methacrylate Random Copolymer Induces Domain Formation in a Model Bacterial Membrane.

To address the emerging issue of drug-resistant bacteria, membrane-active synthetic polymers have been designed and developed to mimic host-defense antimicrobial peptides (AMPs) as antibiotic alternatives. In this study, we investigated the domain formation induced by synthetic polymer mimics of AMPs using model membranes to elucidate the biophysical principles that govern their membrane-active mechanisms. To that end, lipid vesicles mimicking Escherichia coli (E. coli) membrane were prepared using an 8:2 (molar ratio) mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), sodium salt (POPG). Our studies using differential scanning calorimetry (DSC) and fluorescence microscopy indicated that cationic amphiphilic methacrylate random copolymers induced the phase separation to form POPE- or POPG-rich domains. A rhodamine-labeled polymer also showed the binding to separated domains in the membrane. Based on these results, we propose the mechanism that the copolymers induce domain formation by clustering of anionic POPG lipids similar to natural AMPs. In addition, the time-course of polymer binding to the GUV membrane was sigmoidal, suggesting the positive feedback loop in the membrane binding. We also hypothesize that this cooperative binding of the polymer is driven by the domain formation. This study demonstrates the potential of the amphiphilic copolymers to modulate the lipid organization of cell membranes, which may provide a new strategy to design membrane-active antimicrobial agents.

Mechanistic Study of Membrane Disruption by Antimicrobial Methacrylate Random Copolymers by the Single Giant Vesicle Method.

Cationic amphiphilic polymers have been a platform to create new antimicrobial materials that act by disrupting bacterial cell membranes. While activity characterization and chemical optimization have been done in numerous studies, there remains a gap in our knowledge on the antimicrobial mechanisms of the polymers, which is needed to connect their chemical structures and biological activities. To that end, we used a single giant unilamellar vesicle (GUV) method to identify the membrane-disrupting mechanism of methacrylate random copolymers. The copolymers consist of random sequences of aminoethyl methacrylate and methyl (MMA) or butyl (BMA) methacrylate, with low molecular weights of 1600-2100 g·mol-1. GUVs consisting of an 8:2 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), sodium salt (POPG) and those with only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were prepared to mimic the bacterial (Escherichia coli) or mammalian membranes, respectively. The disruption of bacteria and mammalian cell membrane-mimetic lipid bilayers in GUVs reflected the antimicrobial and hemolytic activities of the copolymers, suggesting that the copolymers act by disrupting cell membranes. The copolymer with BMA formed pores in the lipid bilayer, while that with MMA caused GUVs to burst. Therefore, we propose that the mechanism is inherent to the chemical identity or properties of hydrophobic groups. The copolymer with MMA showed characteristic sigmoid curves of the time course of GUV burst. We propose a new kinetic model with a positive feedback loop in the insertion of the polymer chains in the lipid bilayer. The novel finding of alkyl-dependent membrane-disrupting mechanisms will provide a new insight into the role of hydrophobic groups in the optimization strategy for antimicrobial activity and selectivity.

Unexpected Enhancement of Antimicrobial Polymer Activity against Staphylococcus aureus in the Presence of Fetal Bovine Serum.

Cationic and amphiphilic polymers are known to exert broad-spectrum antibacterial activity by a putative mechanism of membrane disruption. Typically, nonspecific binding to hydrophobic components of the complex biological milieu, such as globular proteins, is considered a deterrent to the successful application of such polymers. To evaluate the extent to which serum deactivates antibacterial polymethacrylates, we compared their minimum inhibitory concentrations in the presence and absence of fetal bovine serum. Surprisingly, we discovered that the addition of fetal bovine serum (FBS) to the assay media in fact enhances the antimicrobial activity of polymers against Gram-positive bacteria S. aureus, whereas the opposite is the case for Gram-negative E. coli. Here, we present these unexpected trends and develop a hypothesis to potentially explain this unusual phenomenon.

Biomimetic tubular scaffold with heparin conjugation for rapid degradation in in situ regeneration of a small diameter neoartery.

To address the clinical need for readily available small diameter vascular grafts, biomimetic tubular scaffolds were developed for rapid in situ blood vessel regeneration. The tubular scaffolds were designed to have an inner layer that is porous, interconnected, and with a nanofibrous architecture, which provided an excellent microenvironment for host cell invasion and proliferation. Through the synthesis of poly(spirolactic-co-lactic acid) (PSLA), a highly functional polymer with a norbornene substituting a methyl group in poly(l-lactic acid) (PLLA), we were able to covalently attach biomolecules onto the polymer backbone via thiol-ene click chemistry to impart desirable functionalities to the tubular scaffolds. Specifically, heparin was conjugated on the scaffolds in order to prevent thrombosis when implanted in situ. By controlling the amount of covalently attached heparin we were able to modulate the physical properties of the tubular scaffold, resulting in tunable wettability and degradation rate while retaining the porous and nanofibrous morphology. The scaffolds were successfully tested as rat abdominal aortic replacements. Patency and viability were confirmed through dynamic ultrasound and histological analysis of the regenerated tissue. The harvested tissue showed excellent vascular cellular infiltration, proliferation, and migration with laminar cellular arrangement. Furthermore, we achieved both complete reendothelialization of the vessel lumen and native-like media extracellular matrix. No signs of aneurysm or hyperplasia were observed after 3 months of vessel replacement. Taken together, we have developed an effective vascular graft able to generate small diameter blood vessels that can function in a rat model.

Synthetic Biomimetic Polymethacrylates: Promising Platform for the Design of Anti-Cyanobacterial and Anti-Algal Agents.

Extensive, uncontrolled growth of algae and cyanobacteria is an environmental, public health, economic, and technical issue in managing natural and engineered water systems. Synthetic biomimetic polymers have been almost exclusively considered antimicrobial alternatives to conventional antibiotics to treat human bacterial infections. Very little is known about their applicability in an aquatic environment. Here, we introduce synthetic biomimetic polymethacrylates (SBPs) as a cost-effective and chemically facile, flexible platform for designing a new type of agent suitable for controlling and mitigating photosynthetic microorganisms. Since SBPs are cationic and membranolytic in heterotrophic bacteria, we hypothesized they could also interact with negatively charged cyanobacterial or algal cell walls and membranes. We demonstrated that SBPs inhibited the growth of aquatic photosynthetic organisms of concern, i.e., cyanobacteria (Microcystis aeruginosa and Synechococcus elongatus) and green algae (Chlamydomonas reinhardtii and Desmodesmus quadricauda), with 50% effective growth-inhibiting concentrations ranging between 95 nM and 6.5 µM. Additionally, SBPs exhibited algicidal effects on C. reinhardtii and cyanocidal effects on picocyanobacterium S. elongatus and microcystin-producing cyanobacterium M. aeruginosa. SBP copolymers, particularly those with moderate hydrophobic content, induced more potent cyanostatic and cyanocidal effects than homopolymers. Thus, biomimetic polymers are a promising platform for the design of anti-cyanobacterial and anti-algal agents for water treatment.

Amphiphilic polymer therapeutics: an alternative platform in the fight against antibiotic resistant bacteria.

As we are on the cusp of the "post-antibiotic" era due to rapid spread of drug resistant bacteria, there is an urgent need for new antimicrobials that are not susceptible to bacterial resistance mechanisms. In this review, we will discuss the recent development of "polymer therapeutics" with antimicrobial activity. Learning from host-defence peptides, we propose the biomimetic design of synthetic polymers to target bacterial cell membranes, which act by compromising the membrane integrity. The discussion is extended to the future challenges and opportunities of antimicrobial polymers for clinical applications.

Towards designing globular antimicrobial peptide mimics: role of polar functional groups in biomimetic ternary antimicrobial polymers.

Using atomistic molecular dynamics simulations, we study the interaction of ternary methacrylate polymers, composed of charged cationic, hydrophobic and neutral polar groups, with model bacterial membrane. Our simulation data shows that the random ternary polymers can penetrate deep into the membrane interior and partitioning of even a single polymer has a pronounced effect on the membrane structure. Lipid reorganization, on polymer binding, shows a strong affinity of the ternary polymer for anionic POPG lipids and the same is compared with the control case of binary polymers (only cationic and hydrophobic groups). While binary polymers exhibit strong propensity of acquired amphiphilic conformations upon membrane insertion, our results strongly suggest that such amphiphilic conformations are absent in the case of random ternary polymers. The ternary polymers adopt a more folded conformation, staying aligned in the direction of the membrane normal and subsequently penetrating deeper into the membrane interior suggesting a novel membrane partitioning mechanism without amphiphilic conformations. Finally, we also examine the interactions of ternary polymer aggregates with model bacterial membranes, which show that replacing some of the hydrophobic groups by polar groups leads to weakly held ternary aggregates enabling them to undergo rapid partitioning and insertion into membrane interior. Our work thus underscores the role of inclusion of polar groups into the framework of traditional binary biomimetic antimicrobial polymers and suggests different mode of partitioning into bacterial membranes, mimicking antimicrobial mechanism of globular antimicrobial peptides like Defensin.

The function of peptide-mimetic anionic groups and salt bridges in the antimicrobial activity and conformation of cationic amphiphilic copolymers.

Herein we report the synthesis of ternary statistical methacrylate copolymers comprising cationic ammonium (amino-ethyl methacrylate: AEMA), carboxylic acid (propanoic acid methacrylate: PAMA) and hydrophobic (ethyl methacrylate: EMA) side chain monomers, to study the functional role of anionic groups on their antimicrobial and hemolytic activities as well as the conformation of polymer chains. The hydrophobic monomer EMA was maintained at 40 mol% in all the polymers, with different percentages of cationic ammonium (AEMA) and anionic carboxylate (PAMA) side chains, resulting in different total net charge for the polymers. The antimicrobial and hemolytic activities of the copolymer were determined by the net charge of +3 or larger, suggesting that there was no distinct effect of the anionic carboxylate groups on the antimicrobial and hemolytic activities of the copolymers. However, the pH titration and atomic molecular dynamics simulations suggest that anionic groups may play a strong role in controlling the polymer conformation. This was achieved via formation of salt bridges between cationic and anionic groups, transiently crosslinking the polymer chain allowing dynamic switching between compact and extended conformations. These results suggest that inclusion of functional groups in general, other than the canonical hydrophobic and cationic groups in antimicrobial agents, may have broader implications in acquiring functional structures required for adequate antimicrobial activity. In order to explain the implications, we propose a molecular model in which formation of intra-chain, transient salt bridges, due to the presence of both anionic and cationic groups along the polymer, may function as "adhesives" which facilitate compact packing of the polymer chain to enable functional group interaction but without rigidly locking down the overall polymer structure, which may adversely affect their functional roles.

Aggregation of methacrylate-based ternary biomimetic antimicrobial polymers in solution.

Using detailed atomistic simulations, we explore the morphological characteristics of aggregates formed in solution phase by ternary biomimetic antimicrobial (AM) methacrylate polymers, composed of hydrophobic, charged cationic and polar functional groups and compare it with aggregate morphologies of binary methacrylate polymers, composed only of hydrophobic and charged cationic functional groups. The effect of sequence of the constituent functional groups on aggregate conformation is also studied by considering random and block sequences along the polymer backbone. Our results show that while binary polymers tend to form robust aggregates, replacing some of the hydrophobic groups with overall charge neutral polar groups weakens the aggregate considerably, leading to increased conformational fluctuations and formation of loose-packed, open aggregates, particularly in the case of random ternary polymers. Interaction energy calculations clearly suggest that the role of inclusion of polar groups in ternary polymers is two-fold: (1) to reduce possible strong local concentration of hydrophobic groups and 'smear' the overall hydrophobicity along the polymer backbone to increase the solubility of the polymers (2) to compensate the loss of attractive hydrophobic interactions by forming attractive electrostatic interactions with the charged groups and contribute to aggregation formation, albeit weak. Given that most of the naturally occurring AM peptides have contributions from all the three functional groups, this study elucidates the functionally tuneable role of inclusion of polar groups in the way AM agents interact with each other in solution phase, which can eventually dictate their partitioning behaviour into bacterial and mammalian membranes.

Understanding the Role of Shape and Composition of Star-Shaped Polymers and their Ability to Both Bind and Prevent Bacteria Attachment on Oral Relevant Surfaces.

In this study, we have prepared a series of 4- and 6-arm star-shaped polymers with varying molecular weight and hydrophobicity in order to provide insight into the role and relationship that shape and composition have on the binding and protecting of oral relevant surfaces (hydroxyapatite, HAP) from bacteria colonization. Star-shaped acrylic acid polymers were prepared by free-radical polymerization in the presence of chain transfer agents with thiol groups, and their binding to the HAP surfaces and subsequent bacteria repulsion was measured. We observed that binding was dependent on both polymer shape and hydrophobicity (star vs. linear), but their relative efficacy to reduce oral bacteria attachment from surfaces was dependent on their hydrophobicity only. We further measured the macroscopic effects of these materials to modify the mucin-coated HAP surfaces through contact angle experiments; the degree of angle change was dependent on the relative hydrophobicity of the materials suggesting future in vivo efficacy. The results from this study highlight that star-shaped polymers represent a new material platform for the development of dental applications to control bacterial adhesion which can lead to tooth decay, with various compositional and structural aspects of materials being vital to effectively design oral care products.

Author Correction: Anticancer polymers designed for killing dormant prostate cancer cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Solution-Mediated Modulation of Pseudomonas aeruginosa Biofilm Formation by a Cationic Synthetic Polymer.

Bacterial biofilms and their associated infections are a continuing problem in the healthcare community. Previous approaches utilizing anti-biofilm coatings suffer from short lifetimes, and their applications are limited to surfaces. In this research, we explored a new approach to biofilm prevention based on the hypothesis that changing planktonic bacteria behavior to result in sub-optimal biofilm formation. The behavior of planktonic Pseudomonas aeruginosa exposed to a cationic polymer was characterized for changes in growth behavior and aggregation behavior, and linked to resulting P. aeruginosa biofilm formation, biomass, viability, and metabolic activity. The incubation of P. aeruginosa planktonic bacteria with a cationic polymer resulted in the aggregation of planktonic bacteria, and a reduction in biofilm development. We propose that cationic polymers may sequester planktonic bacteria away from surfaces, thereby preventing their attachment and suppressing biofilm formation.

Anticancer polymers designed for killing dormant prostate cancer cells.

The discovery of anticancer therapeutics effective in eliminating dormant cells is a significant challenge in cancer biology. Here, we describe new synthetic polymer-based anticancer agents that mimic the mode of action of anticancer peptides. These anticancer polymers developed here are designed to capture the cationic, amphiphilic traits of anticancer peptides. The anticancer polymers are designed to target anionic lipids exposed on the cancer cell surfaces and act by disrupting the cancer cell membranes. Because the polymer mechanism is not dependent on cell proliferation, we hypothesized that the polymers were active against dormant cancer cells. The polymers exhibited cytotoxicity to proliferating prostate cancer. Importantly, the polymer killed dormant prostate cancer cells that were resistant to docetaxel. This study demonstrates a new approach to discover novel anticancer therapeutics.

Decoupling the Functional Roles of Cationic and Hydrophobic Groups in the Antimicrobial and Hemolytic Activities of Methacrylate Random Copolymers.

In this study, we report the antimicrobial and hemolytic activities of ternary statistical methacrylate copolymers consisting of cationic ammonium (amino-ethyl methacrylate: AEMA), hydrophobic alkyl (ethyl methacrylate: EMA), and neutral hydroxyl (hydroxyethyl methacrylate: HEMA) side chain monomers. The cationic and hydrophobic functionalities of copolymers mimic the cationic amphiphilicity of naturally occurring antimicrobial peptides (AMPs). The HEMA monomer units were used to separately modulate the compositions of cationic and hydrophobic monomers, and we investigated the effect of each component on the antimicrobial and hemolytic activities of copolymers. Our data indicated that increasing the number of cationic groups of the copolymers to be more than 30 mol % did not increase their antimicrobial activity against Escherichia coli. The number of cationic side chains in a polymer chain at this threshold is 5.5-7.7, which is comparable to those of natural antimicrobial peptides such as maginin (+6). The MIC values of copolymers with >30 mol % of AEMA depend on only the mol % of EMA, indicating that the hydrophobic interactions of the copolymers with E. coli cell membranes determine the antimicrobial activity of copolymers. These results suggest that the roles of cationic and hydrophobic groups can be controlled independently by design in the ternary copolymers studied here.

Branched Poly(ethylene imine)s as Anti-algal and Anti-cyanobacterial Agents with Selective Flocculation Behavior to Cyanobacteria over Algae.

Poly(ethylene imine)s (PEIs) have been widely studied for biomedical applications, including antimicrobial agents against potential human pathogens. The interactions of branched PEIs (B-PEIs) with environmentally relevant microorganisms whose uncontrolled growth in natural or engineered environments causes health, economic, and technical issues in many sectors of water management are studied. B-PEIs are shown to be potent antimicrobials effective in controlling the growth of environmentally relevant algae and cyanobacteria with dual-functionality and selectivity. Not only did they effectively inhibit growth of both algae and cyanobacteria, mostly without causing cell death (static activity), but they also selectively flocculated cyanobacteria over algae. Thus, unmodified B-PEIs provide a cost-effective and chemically facile framework for the further development of effective and selective antimicrobial agents useful for control of growth and separation of algae and cyanobacteria in natural or engineered environments.

Monitoring Antimicrobial Mechanisms of Surface-Immobilized Peptides in Situ.

Antimicrobial peptides (AMPs) in free solution can kill bacteria by disrupting bacterial cell membranes. Their modes of action have been extensively studied, and various models ranging from pore formation to carpet-like mechanisms were proposed. Surface-immobilized AMPs have been used as coatings to kill bacteria and as sensors to capture bacteria, but the interaction mechanisms of surface-immobilized AMPs and bacteria are not fully understood. In this research, an analytical platform, sum frequency generation (SFG) microscope, which is composed of an SFG vibrational spectrometer and a fluorescence microscope, was used to probe molecular interactions between surface-immobilized AMPs and bacteria in situ in real time at the solid/liquid interface. SFG probed the molecular structure of surface-immobilized AMPs while interacting with bacteria, and fluorescence images of dead bacteria were monitored as a function of time during the peptide-bacteria interaction. It was believed that upon bacteria contact, the surface-immobilized peptides changed their orientation and killed bacteria. This research demonstrated that the SFG microscope platform can examine the structure and function (bacterial killing) at the same time in the same sample environment, providing in-depth understanding on the structure-activity relationships of surface-immobilized AMPs.

Aggregation of Cationic Amphiphilic Block and Random Copoly(vinyl ether)s with Antimicrobial Activity.

In this study, we investigated the aggregation behaviors of amphiphilic poly(vinyl ether)s with antimicrobial activity. We synthesized a di-block poly(vinyl ether), B3826, composed of cationic primary amine and hydrophobic isobutyl (iBu) side chains, which previously showed antimicrobial activity against Escherichia coli. B3826 showed similar uptake behaviors as those for a hydrophobic fluorescent dye, 1,6-diphenyl-1,3,5-hexatriene, to counterpart polymers including homopolymer H44 and random copolymer R4025, indicating that the iBu block does not form strong hydrophobic domains. The cryo-TEM observations also indicated that the polymer aggregate of B3826 appears to have low-density polymer chains without any defined microscopic structures. We speculate that B3826 formed large aggregates by liquid-liquid separation due to the weak association of polymer chains. The fluorescence microscopy images showed that B3826 bonds to E. coli cell surfaces, and these bacterial cells were stained by propidium iodide, indicating that the cell membranes were significantly damaged. The results suggest that block copolymers may provide a new platform to design and develop antimicrobial materials that can utilize assembled structures and properties.