Investigating the selectivity of sequence-controlled antimicrobial polymers synthesised by RAFT polymerisation

Bacterial resistance has become a pressing clinical issue for which the World Health Organisation has raised awareness due to the low levels of infection control reached recently. This global issue has been considered as a failure in antibiotic drug discovery and new types of antibiotics have recently been investigated to tackle it. For this reason, new types of antibiotics have recently been investigated. Among these, antimicrobial peptides (AMPs) have attracted an increased interest and a few of them are currently undergoing clinical trials. AMPs are peptides comprised of 10-50 amino-acids with cationic and hydrophobic residues, present in the innate immune system of multicellular organisms, hence the terminology host-defence peptides. The majority of these peptides, adopt an amphipathic helical conformation with the cationic functionalities on one side of the coil and the hydrophobic ones on the other. AMPs have demonstrated a broad-range antimicrobial activity, which is associated to their mechanism of action based on bacterial membrane disruption. Indeed, the positively charged residues of AMPs have been shown to interact with the negatively charged phospholipids of bacterial membranes. Following the binding of AMPs to bacterial membranes, the hydrophobic residues of AMPs insert into the membrane, inducing the formation of pores. Once the integrity of the bacterial membrane is compromised, there is leakage of the intracellular material, which leads to bacterial cell death. Remarkably, as AMPs seem to target bacterial membrane instead of a specific ligand, they do not seem to evoke bacterial resistance against these peptides. Indeed, only minor structural changes are required for bacteria to exhibit a reduced susceptibility towards conventional antibiotics, whereas more significant changes in the structure of the bacterial membrane would be necessary to prevent the antimicrobial action of AMPs. This membrane interaction based on electrostatic interactions allows AMPs to be selective towards bacteria over mammalian cells to a certain extent

Investigating cell uptake of guanidinium-rich RAFT polymers : impact of comonomer and monomer distribution

A range of well-defined guanidinium-rich linear polymers with demonstrable efficiency for cellular internalization were developed. A protected guanidinium-functional acrylamide monomer (di-Boc-guanidinium ethyl acrylamide, GEAdiBoc) was synthesized and then polymerized via RAFT polymerization to yield well-defined homopolymers, which were then deprotected and functionalized with a fluorescein dye to observe and quantify their cellular uptake. The cellular uptake of these homopolymers was first compared to analogous polyarginines, which are commonly used in modern drug delivery. Following this, a range of well-defined guanidinium-rich copolymers were prepared in which the monomer distribution was varied using a convenient one-pot sequential RAFT polymerization approach. Systematic quantification of the cell uptake of these compounds, supported by fluorescent confocal microscopy data, revealed that while the overall hydrophobicity of the resulting copolymers has a direct impact on the amount of copolymer taken up by cells, the distribution of monomers has an influence on both the extent of uptake and the relative extent to which each route of internalization (endocytosis vs direct translocation) is exploited

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 three-dimensional 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

Targeting intracellular, multi-drug resistant Staphylococcus aureus with guanidinium polymers by elucidating the structure-activity relationship

Intracellular persistence of bacteria represents a clinical challenge as bacteria can thrive in an environment protected from antibiotics and immune responses. Novel targeting strategies are critical in tackling antibiotic resistant infections. Synthetic antimicrobial peptides (SAMPs) are interesting candidates as they exhibit a very high antimicrobial activity. We first compared the activity of a library of ammonium and guanidinium polymers with different sequences (statistical, tetrablock and diblock) synthesized by RAFT polymerization against methicillin-resistant S. aureus (MRSA) and methicillin-sensitive strains (MSSA). As the guanidinium SAMPs were the most potent, they were used to treat intracellular S. aureus in keratinocytes. The diblock structure was the most active, reducing the amount of intracellular MSSA and MRSA by two-fold. We present here a potential treatment for intracellular, multi-drug resistant bacteria, using a simple and scalable strategy

The type VII secretion system protects Staphylococcus aureus against antimicrobial host fatty acids

The Staphylococcus aureus type VII secretion system (T7SS) exports several proteins that are pivotal for bacterial virulence. The mechanisms underlying T7SS-mediated staphylococcal survival during infection nevertheless remain unclear. Here we report that S. aureus lacking T7SS components are more susceptible to host-derived antimicrobial fatty acids. Unsaturated fatty acids such as linoleic acid (LA) elicited an increased inhibition of S. aureus mutants lacking T7SS effectors EsxC, EsxA and EsxB, or the membrane-bound ATPase EssC, compared to the wild-type (WT). T7SS mutants generated in different S. aureus strain backgrounds also displayed an increased sensitivity to LA. Analysis of bacterial membrane lipid profiles revealed that the esxC mutant was less able to incorporate LA into its membrane phospholipids. Although the ability to bind labelled LA did not differ between the WT and mutant strains, LA induced more cell membrane damage in the T7SS mutants compared to the WT. Furthermore, proteomic analyses of WT and mutant cell fractions revealed that, in addition to compromising membranes, T7SS defects induce oxidative stress and hamper their response to LA challenge. Thus, our findings indicate that T7SS contribute to maintaining S. aureus membrane integrity and homeostasis when bacteria encounter antimicrobial fatty acids

Der „neue“ Münzfundkatalog Mittelalter/Neuzeit der Numismatischen Kommission der Länder in der Bundesrepublik Deutschland (Ein Zwischenbericht)

Im Jahre 1941 erschien in den Blättern für deutsche Landesgeschichte ein programmatischer Aufsatz über „Die deutschen Münzfunde“. Autor war der Braunschweiger Museumsdirektor Prof. Dr. Wilhelm Jesse (1887–1972). Er gehörte zu den ersten Mitgliedern der 1943 gegründeten Braunschweigischen Wissenschaftlichen Gesellschaft. Seine Ideen zur Fundnumismatik inspirierten bald Walter Hävernick (1905–1983) zu seiner Konzeption einer flächendeckenden Funddokumentation für Thüringen (1943/1955) und Deutschland (1950). Aus dem maschinenschriftlichen Fundkatalog Mittelalter/Neuzeit "seiner" Numismatischen Kommission der Länder in der Bundesrepublik Deutschland ist ein halbes Jahrhundert später der EDV-Münzfundkatalog hervorgegangen

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

Reverse-phase high performance liquid chromatography (RP-HPLC) as a powerful tool to characterise complex water-soluble copolymer architectures

Recent progress in modern polymer synthesis techniques has led to the design of complex functional materials, which can be difficult to analyse accurately. While size-exclusion chromatography (SEC) or mass spectrometry (MS) is typically used to gain information about molecular weight distribution, chemical structure and molecular architecture, there is a lack of available methods for characterising compositional heterogeneity (i.e. monomer distribution). In contrast to SEC in which separation occurs by hydrodynamic volume, interaction-based chromatography (IC) separates compounds according to their affinity for a stationary phase, which has proven useful in gaining information about the general chemical structure of copolymers in the past. Here, we explore the potential of reverse-phase high performance liquid chromatography (RP-HPLC) as a tool for the characterisation of monomer segmentation in charged water-soluble copolymers. A library of acrylamide copolymeric systems, prepared via reversible addition–fragmentation chain transfer (RAFT) polymerisation, is used to demonstrate the influence of monomer distribution (diblock, multiblock and statistical) on the elution time. The robustness of the method is tested by studying a range of copolymers with varying charges, charge contents and hydrophobicities, as well as by using various solvent systems or column lengths. Results highlight the efficiency of RP-HPLC to separate copolymers with varying segmentations, with a limitation observed for branched architectures