Thomas D. Michl

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.

Plasma polymerization of 1,1,1-trichloroethane yields a coating with robust antibacterial surface properties†

Novel, highly chlorinated surface coatings were produced via a one-step plasma polymerization (pp) of 1,1,1-trichloroethane (TCE), exhibiting excellent antimicrobial properties against the vigorously biofilm-forming bacterium Staphylococcus epidermidis.

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.

Packed Bed Bioreactor for the Isolation and Expansion of Placental-Derived Mesenchymal Stromal Cells

Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm2) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm2 format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.

Facile single-step bioconjugation of the antifungal agent caspofungin onto material surfaces via an epoxide plasma polymer interlayer

We report a facile, one-step, aqueous surface bioconjugation approach for producing an antifungal surface coating that prevents the formation of fungal biofilms. By direct reaction between surface epoxide groups and amine groups on caspofungin, it avoids the use of secondary chemicals. The coating withstands washing with detergent and reduces the growth of the fungal pathogens Candida albicans by log 6 and Candida glabrata by log 3. Importantly, we show that surface adsorption of albumin does not inhibit the activity of this antifungal coating.

Chlorine-rich plasma polymer coating for the prevention of attachment of pathogenic fungal cells onto materials surfaces

The attachment of pathogenic fungal cells onto materials surfaces, which is often followed by biofilm formation, causes adverse consequences in a wide range of areas. Here we have investigated the ability of thin film coatings from chlorinated molecules to deter fungal colonization of solid materials by contact killing of fungal cells reaching the surface of the coating. Coatings were deposited onto various substrate materials via plasma polymerization, which is a substrate-independent process widely used for industrial coating applications, using 1,1,2-trichloroethane as the process vapour. XPS surface analysis showed that the coatings were characterized by a highly chlorinated hydrocarbon polymer nature, with only a very small amount of oxygen incorporated. The activity of these coatings against human fungal pathogens was quantified using a recently developed, modified yeast assay and excellent antifungal activity was observed against Candida albicans and Candida glabrata. Plasma polymer surface coatings derived from chlorinated hydrocarbon molecules may therefore offer a promising solution to preventing yeast and mould biofilm formation on materials surfaces, for applications such as air conditioners, biomedical devices, food processing equipment, and others.

The importance of fungal pathogens and antifungal coatings in medical device infections

In recent years, increasing evidence has been collated on the contributions of fungal species, particularly Candida, to medical device infections. Fungal species can form biofilms by themselves or by participating in polymicrobial biofilms with bacteria. Thus, there is a clear need for effective preventative measures, such as thin coatings that can be applied onto medical devices to stop the attachment, proliferation, and formation of device-associated biofilms. However, fungi being eukaryotes, the challenge is greater than for bacterial infections because antifungal agents are often toxic towards eukaryotic host cells. Whilst there is extensive literature on antibacterial coatings, a far lesser body of literature exists on surfaces or coatings that prevent attachment and biofilm formation on medical devices by fungal pathogens. Here we review strategies for the design and fabrication of medical devices with antifungal surfaces. We also survey the microbiology literature on fundamental mechanisms by which fungi attach and spread on natural and synthetic surfaces. Research in this field requires close collaboration between biomaterials scientists, microbiologists and clinicians; we consider progress in the molecular understanding of fungal recognition of, and attachment to, suitable surfaces, and of ensuing metabolic changes, to be essential for designing rational approaches towards effective antifungal coatings, rather than empirical trial of coatings.

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.