Katherine E. S. Locock

Guanylated Polymethacrylates: A Class of Potent Antimicrobial Polymers with Low Hemolytic Activity

We have synthesized a series of copolymers containing both positively charged (amine, guanidine) and hydrophobic side chains (amphiphilic antimicrobial peptide mimics). To investigate the structure-activity relationships of these polymers, low polydispersity polymethacrylates of varying but uniform molecular weight and composition were synthesized, using a reversible addition-fragmentation chain transfer (RAFT) approach. In a facile second reaction, pendant amine groups were converted to guanidines, allowing for direct comparison of cation structure on activity and toxicity. The guanidine copolymers were much more active against Staphylococcus epidermidis and Candida albicans compared to the amine analogues. Activity against Staphylococcus epidermidis in the presence of fetal bovine serum was only maintained for guanidine copolymers. Selectivity for bacterial over mammalian cells was assessed using hemolytic and hemagglutination toxicity assays. Guanidine copolymers of low to moderate molecular weight and hydrophobicity had high antimicrobial activity with low toxicity. Optimum properties appear to be a balance between charge density, hydrophobic character, and polymer chain length. In conclusion, a suite of guanidine copolymers has been identified that represent a new class of antimicrobial polymers with high potency and low toxicity.

RAFT-derived antimicrobial polymethacrylates: elucidating the impact of end-groups on activity and cytotoxicity

Antimicrobial polymers as mimics of natural antimicrobial peptides are emerging as an alternative to classic antibiotics due to their potency, selectivity and lower susceptibility to resistance. The key chemical aspects necessary to confer high activity and selectivity to the polymer chain composition are largely known. However, little attention has been paid to how end-groups affect the overall biological activity. Here we report the use of RAFT polymerization to obtain eight well-defined cationic methacrylate polymers which bear either amine (PA1–4) or guanidine (PG1–4) pendant groups, while systematically varying the R- and Z-RAFT end-groups. These polymers were assessed in haemotoxicity assays as well as antimicrobial testing against clinically relevant pathogens; such as a vigorously biofilm forming strain of Staphylococcus epidermidis (S. epidermidis) and a vancomycin and methicillin resistant strain of Staphylococcus aureus (VISA) as well as the opportunistic fungus Candida albicans (C. albicans). The R-group was found to dominate the measured toxicity of polymers. Replacement of the anionic cyanovaleric acid R-group (PA1) with the neutral isobutyronitrile (PA3) led to over a 20 fold increase in the haemolytic activity of the polymers. The Z-group, however, was found to have more influence on the antimicrobial activity of the polymers against both VISA and C. albicans, whereby polymers with a long, lipophilic dodecylsulfanyl Z-group (PA1) were found to be more potent than those with either an ethylsulfanyl or no ZCS2-group. These results indicate that chemical control over the end-groups is a key element for achieving the desired high biological activity and selectivity, particularly when low molecular weights are required for maximum antibacterial activity.

Searching for new strategies against polymicrobial biofilm infections: guanylated polymethacrylates kill mixed fungal/bacterial biofilms

OBJECTIVES Biofilm-related human infections have high mortality rates due to drug resistance. Cohabitation of diverse microbes in polymicrobial biofilms is common and these infections present additional challenges for treatment compared with monomicrobial biofilms. Here, we address this therapeutic gap by assessing the potential of a new class of antimicrobial agents, guanylated polymethacrylates, in the treatment of polymicrobial biofilms built by two prominent human pathogens, the fungus Candida albicans and the bacterium Staphylococcus aureus. METHODS We used imaging and quantitative methods to test the antibiofilm efficacy of guanylated polymethacrylates, a new class of drugs that structurally mimic antimicrobial peptides. We further compared guanylated polymethacrylates with first-line antistaphylococcal and anti-Candida agents used as combinatorial therapy against polymicrobial biofilms. RESULTS Guanylated polymethacrylates were highly effective as a sole agent, killing both C. albicans and S. aureus when applied to established polymicrobial biofilms. Furthermore, they outperformed multiple combinations of current antimicrobial drugs, with one of the tested compounds killing 99.98% of S. aureus and 82.2% of C. albicans at a concentration of 128 mg/L. The extracellular biofilm matrix provided protection, increasing the MIC of the polymethacrylates by 2-4-fold when added to planktonic assays. Using the C. albicans bgl2ΔΔ mutant, we implicate matrix polysaccharide β-1,3 glucan in the mechanism of protection. Data for two structurally distinct polymers suggest that this mechanism could be minimized through chemical optimization of the polymer structure. Finally, we demonstrate that a potential application for these polymers is in antimicrobial lock therapy. CONCLUSIONS Guanylated polymethacrylates are a promising lead for the development of an effective monotherapy against C. albicans/S. aureus polymicrobial biofilms.

Oligomeric Cationic Polymethacrylates: A Comparison of Methods for Determining Molecular Weight

This study compares three common laboratory methods, size-exclusion chromatography (SEC), (1)H nuclear magnetic resonance (NMR), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), to determine the molecular weight of oligomeric cationic copolymers. The potential bias for each method was examined across a series of polymers that varied in molecular weight and cationic character (both choice of cation (amine versus guanidine) and relative proportion present). SEC was found to be the least accurate, overestimating Mn by an average of 140%, owing to the lack of appropriate cationic standards available, and the complexity involved in estimating the hydrodynamic volume of copolymers. MALDI-TOF approximated Mn well for the highly monodisperse (Đ < 1.1), low molecular weight (degree of polymerization (DP) <50) species but appeared unsuitable for the largest polymers in the series due to the mass bias associated with the technique. (1)H NMR was found to most accurately estimate Mn in this study, differing to theoretical values by only 5.2%. (1)H NMR end-group analysis is therefore an inexpensive and facile, primary quantitative method to estimate the molecular weight of oliogomeric cationic polymethacrylates if suitably distinct end-groups signals are present in the spectrum.

Structure–activity relationships of guanylated antimicrobial polymethacrylates

Abstract Host-defense antimicrobial peptides (AMPs) are a promising lead in the search for novel antibiotics. Many of these peptides exhibit broad-spectrum antibacterial ability, low toxicity toward human cells, and little susceptibility to induction of bacterial resistance. Our research focuses on the development of synthetic polymers that are able to mimic the amphiphilic and cation-rich characteristics of AMPs. This derives bioactive polymers that retain the activity profile of AMPs while utilizing a construct that is less expensive and easier to produce and manipulate chemically. This review details structure–activity relationships (SARs) of a new class of arginine-rich, synthetic AMP mimicking polymers (SAMPs), the guanylated polymethacrylates. These are contrasted with those of amine-based polymers that are mimics of lysine-rich AMPs. The ideal composition for candidates for practical applications was identified as those containing guanidines as a cation source, having a low molecular weight and a low level of lipophilicity. This gave polymers with high potency against Gram-positive strains of bacteria (e.g., Staphylococcus epidermidis MIC = 10 μg/mL) and low toxicity towards human red blood cells (<4% hemolysis at given MIC). This work emphasizes the need to rationalize observed biological activities based not purely on the global lipophilic and cationic character of polymers but rather to consider the profound effect that specific pendant functional groups may have on the potency, selectivity, and mechanisms behind the action of antimicrobial polymers.

Looped flow RAFT polymerization for multiblock copolymer synthesis

A looped flow process was designed for the synthesis of well-defined multiblock copolymers using reversible addition–fragmentation chain transfer (RAFT) polymerization. The reaction conditions were optimized to reach high conversions whilst maintaining a high end-group fidelity. The loop set-up proved to be a flexible, robust and time-efficient process for scaling-up multiblock copolymers.

Antimicrobial polymers : mimicking amino acid functionality, sequence control and three-dimensional structure of host-defense peptides

Peptides and proteins control and direct all aspects of cellular function and communication. Having been honed by nature for millions of years, they also typically display an unsurpassed specificity for their biological targets. This underlies the continued focus on peptides as promising drug candidates. However, the development of peptides into viable drugs is hampered by their lack of chemical and pharmacokinetic stability and the cost of large scale production. One method to overcome such hindrances is to develop polymer systems that are able to retain the important structural features of these biologically active peptides, while being cheaper and easier to produce and manipulate chemically. This review illustrates these principles using examples of polymers designed to mimic antimicrobial host-defence peptides. The host-defence peptides have been identified as some of the most important leads for the next generation of antibiotics as they typically exhibit broad spectrum antimicrobial ability, low toxicity toward human cells and little susceptibility to currently known mechanisms of bacterial resistance. Their movement from the bench to clinic is yet to be realised, however, due to the limitations of these peptides as drugs. The literature provides a number of examples of polymers that have been able to mimic these peptides through all levels of structure, starting from specific amino acid sidechains, through to more global features such as overall charge, molecular weight and threedimensional structure (e.g. α-helical). The resulting optimised polymers are able retain the activity profile of the peptides, but within a synthetic macromolecular construct that may be better suited to the development of a new generation of antimicrobial therapeutics. Such work has not only produced important new leads to combat the growing threat of antibiotic resistance, but may also open up new ways for polymers to mimic other important classes of biologically active peptides.

Sequence Control as a Powerful Tool for Improving the Selectivity of Antimicrobial Polymers

Antimicrobial polymers appear as a promising alternative to tackle the current development of bacterial resistance against conventional antibiotics as they rely on bacterial membrane disruption. This study investigates the effect of segmentation of hydrophobic and cationic functionalities on antimicrobial polymers over their selectivity between bacteria and mammalian cells. Using RAFT technology, statistical, diblock, and highly segmented multiblock copolymers were synthesized in a controlled manner. Polymers were analyzed by HPLC, and the segmentation was found to have a significant influence on their overall hydrophobicity. In addition, the amount of incorporated cationic comonomer was varied to yield a small library of bioactive macromolecules. The antimicrobial properties of these compounds were probed against pathogenic bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis), and their biocompatibility was tested using hemolysis and erythrocyte aggregation assays, as well as mammalian cell viability assays. In all cases, diblock and multiblock copolymers were found to outperform statistical copolymers, and for polymers with a low content of cationic comonomer, the multiblock showed a tremendously increased selectivity for P. aeruginosa and S. epidermidis compared to its statistical and diblock analogue. This work highlights the remarkable effect of segmentation on both the physical properties of the materials as well as their interaction with biological systems. Due to the outstanding selectivity of multiblock copolymers toward certain bacteria strains, the presented materials are a promising platform for the treatment of infections and a valuable tool to combat antimicrobial resistance.

Bio-inspired antimicrobial polymers

Abstract Traditionally, most antibiotics are relatively low molecular weight chemical compounds. Bacteria have shown the ability to acquire resistance to many antibiotics. In nature, on the other hand, there are examples of antibiotics to which resistance has not been developed. This is particularly the case for naturally occurring antimicrobial peptides. In this chapter we discuss antimicrobial peptides and their postulated mechanisms of action, followed by a review of synthetic polymers with structures inspired by biological molecules, particularly antimicrobial peptides. We also review the grafting of polymers onto biomaterials and biomedical devices, so as to generate polymeric antimicrobial coatings.