Supramolecular Self-Associating Amphiphiles Inhibit Biofilm Formation by the Critical Pathogens, Pseudomonas aeruginosa and Candida albicans.

In 2019, 4.95 million deaths were directly attributed to antimicrobial-resistant bacterial infections globally. In addition, the mortality associated with fungal infections is estimated at 1.7 million annually, with many of these deaths attributed to species that are no longer susceptible to traditional therapeutic regimes. Herein, we demonstrate the use of a novel class of supramolecular self-associating amphiphilic (SSA) salts as antimicrobial agents against the critical pathogens Pseudomonas aeruginosa and Candida albicans. We also identify preliminary structure-activity relationships for this class of compound that will aid the development of next-generation SSAs demonstrating enhanced antibiofilm activity. To gain insight into the possible mode of action for these agents, a series of microscopy studies were performed, taking advantage of the intrinsic fluorescent nature of benzothiazole-substituted SSAs. Analysis of these data showed that the SSAs interact with the cell surface and that a benzothiazole-containing SSA inhibits hyphal formation by C. albicans.

Tools to enable the study and translation of supramolecular amphiphiles.

This tutorial review focuses on providing a summary of the key techniques used for the characterisation of supramolecular amphiphiles and their self-assembled aggregates; from the understanding of low-level molecular interactions, to materials analysis, use of data to support computer-aided molecular design and finally, the translation of this class of compounds for real world application, specifically within the clinical setting. We highlight the common methodologies used for the study of traditional amphiphiles and build to provide specific examples that enable the study of specialist supramolecular systems. This includes the use of nuclear magnetic resonance spectroscopy, mass spectrometry, X-ray scattering techniques (small- and wide-angle X-ray scattering and single crystal X-ray diffraction), critical aggregation (or micelle) concentration determination methodologies, machine learning, and various microscopy techniques. Furthermore, this review provides guidance for working with supramolecular amphiphiles in in vitro and in vivo settings, as well as the use of accessible software programs, to facilitate screening and selection of druggable molecules. Each section provides: a methodology overview - information that may be derived from the use of the methodology described; a case study - examples for the application of these methodologies; and a summary section - providing methodology specific benefits, limitations and future applications.

Phospholipid headgroup composition modulates the molecular interactions and antimicrobial effects of sulfobetaine zwitterionic detergents against the "ESKAPE" pathogen Pseudomonas aeruginosa.

We determine the efficacy for three known structurally related, membrane active detergents against multidrug resistant and wild type strains of Pseudomonas aeruginosa. Accessible solution state NMR experiments are used to quantify phospholipid headgroup composition of the microbial membranes and to gain molecular level insight into antimicrobial mode of action.

Next-generation protein-based materials capture and preserve projectiles from supersonic impacts.

Extreme energy-dissipating materials are essential for a range of applications. The military and police force require ballistic armour to ensure the safety of their personnel, while the aerospace industry requires materials that enable the capture, preservation and study of hypervelocity projectiles. However, current industry standards display at least one inherent limitation, such as weight, breathability, stiffness, durability and failure to preserve captured projectiles. To resolve these limitations, we have turned to nature, using proteins that have evolved over millennia to enable effective energy dissipation. Specifically, a recombinant form of the mechanosensitive protein talin was incorporated into a monomeric unit and crosslinked, resulting in a talin shock-absorbing material (TSAM). When subjected to 1.5 km s-1 supersonic shots, TSAMs were shown to absorb the impact and capture and preserve the projectile.

Controlling the structure of supramolecular fibre formation for benzothiazole based hydrogels with antimicrobial activity against methicillin resistant Staphylococcus aureus.

Antimicrobial resistance is one of the greatest threats to human health. Gram-positive methicillin resistant Staphylococcus aureus (MRSA), in both its planktonic and biofilm form, is of particular concern. Herein we identify the hydrogelation properties for a series of intrinsically fluorescent, structurally related supramolecular self-associating amphiphiles and determine their efficacy against both planktonic and biofilm forms of MRSA. To further explore the potential translation of this hydrogel technology for real-world applications, the toxicity of the amphiphiles was determined against the eukaryotic multicellular model organism, Caenorhabditis elegans. Due to the intrinsic fluorescent nature of these supramolecular amphiphiles, material characterisation of their molecular self-associating properties included; comparative optical density plate reader assays, rheometry and widefield fluorescence microscopy. This enabled determination of amphiphile structure and hydrogel sol dependence on resultant fibre formation.

A water-soluble membrane transporter for biologically relevant cations.

Synthetic ionophores are promising therapeutic targets, yet poor water solubility limits their potential for translation into the clinic. Here we report a water-soluble, supramolecular self-associating amphiphile that functions as a cation uniporter in synthetic vesicle systems, deriving mechanistic insight through planar bilayer patch clamp experiments.

Advancements in antimicrobial nanoscale materials and self-assembling systems.

Antimicrobial resistance is directly responsible for more deaths per year than either HIV/AIDS or malaria and is predicted to incur a cumulative societal financial burden of at least $100 trillion between 2014 and 2050. Already heralded as one of the greatest threats to human health, the onset of the coronavirus pandemic has accelerated the prevalence of antimicrobial resistant bacterial infections due to factors including increased global antibiotic/antimicrobial use. Thus an urgent need for novel therapeutics to combat what some have termed the 'silent pandemic' is evident. This review acts as a repository of research and an overview of the novel therapeutic strategies being developed to overcome antimicrobial resistance, with a focus on self-assembling systems and nanoscale materials. The fundamental mechanisms of action, as well as the key advantages and disadvantages of each system are discussed, and attention is drawn to key examples within each field. As a result, this review provides a guide to the further design and development of antimicrobial systems, and outlines the interdisciplinary techniques required to translate this fundamental research towards the clinic.

Establishing the selective phospholipid membrane coordination, permeation and lysis properties for a series of 'druggable' supramolecular self-associating antimicrobial amphiphiles.

The rise of antimicrobial resistance remains one of the greatest global health threats facing humanity. Furthermore, the development of novel antibiotics has all but ground to a halt due to a collision of intersectional pressures. Herein we determine the antimicrobial efficacy for 14 structurally related supramolecular self-associating amphiphiles against clinically relevant Gram-positive methicillin resistant Staphylococcus aureus and Gram-negative Escherichia coli. We establish the ability of these agents to selectively target phospholipid membranes of differing compositions, through a combination of computational host:guest complex formation simulations, synthetic vesicle lysis, adhesion and membrane fluidity experiments, alongside our novel 1H NMR CPMG nanodisc coordination assays, to verify a potential mode of action for this class of compounds and enable the production of evermore effective next-generation antimicrobial agents. Finally, we select a 7-compound subset, showing two lead compounds to exhibit 'druggable' profiles through completion of a variety of in vivo and in vitro DMPK studies.

Supramolecular self-associating amphiphiles: determination of molecular self-association properties and calculation of critical micelle concentration using a high-throughput, optical density based methodology.

Supramolecular self-associating amphiphiles are a class of amphiphilic salt, the anionic component of which is 'frustrated' in nature, meaning multiple hydrogen bonding modes can be accessed simultaneously. Here we derive critical micelle concentration values for four supramolecular self-associating amphiphiles using the standard pendant drop approach and present a new high-throughput, optical density measurement based methodology, to enable the estimation of critical micelle concentrations over multiple temperatures. In addition, we characterise the low-level hydrogen bonded self-association events in the solid state, through single crystal X-ray diffraction, and in polar organic DMSO-d6 solutions using a combination of 1H NMR techniques. Moving into aqueous ethanol solutions (EtOH/H2O or EtOH/D2O (1 : 19 v/v)), we also show these amphiphilic compounds to form higher-order self-associated species through a combination of 1H NMR, dynamic light scattering and zeta potential studies.

The phospholipid membrane compositions of bacterial cells, cancer cell lines and biological samples from cancer patients.

While cancer now impacts the health and well-being of more of the human population than ever before, the exponential rise in antimicrobial resistant (AMR) bacterial infections means AMR is predicted to become one of the greatest future threats to human health. It is therefore vital that novel therapeutic strategies are developed that can be used in the treatment of both cancer and AMR infections. Whether the target of a therapeutic agent be inside the cell or in the cell membrane, it must either interact with or cross this phospholipid barrier to elicit the desired cellular effect. Here we summarise findings from published research into the phospholipid membrane composition of bacterial and cancer cell lines and biological samples from cancer patients. These data not only highlight key differences in the membrane composition of these biological samples, but also the methods used to elucidate and report the results of this analogous research between the microbial and cancer fields.

Towards the Application of Supramolecular Self-Associating Amphiphiles as Next-Generation Delivery Vehicles.

Herein, we present a series of supramolecular self-associating amphiphilic (SSA) salts and establish the potential for these molecular constructs to act as next-generation solution-state molecular delivery vehicles. We characterise the self-association of these SSAs, both alone and when co-formulated with a variety of drug(like) competitive guest species. Single crystal X-ray diffraction studies enable the observation of hydrogen-bonded self-association events in the solid state, whilst high resolution mass spectrometry confirms the presence of anionic SSA dimers in the gas-phase. These same anionic SSA dimeric species are also identified within a competitive organic solvent environment (DMSO-d6/0.5% H2O). However, extended self-associated aggregates are observed to form under aqueous conditions (H2O/5.0% EtOH) in both the absence and presence of these competitive guest species. Finally, through the completion of these studies, we present a framework to support others in the characterisation of such systems.

Controllable hydrogen bonded self-association for the formation of multifunctional antimicrobial materials.

SSAs are a class of supramolecular self-associating amphiphilic salt, the anionic component of which contains a covalently bound hydrogen bond donor-acceptor motif. This results in a monomeric unit which can adopt multiple hydrogen bonding modes simultaneously. Previous investigations have shown examples of SSAs to act as antimicrobial agents against clinically relevant methicillin-resistant Staphylococcus aureus (MRSA). Herein, we report an intrinsically fluorescent SSA which can self-associate producing dimers, spherical aggregates and hydrogels dependent on solvent environment, while retaining antimicrobial activity against both model Gram-positive (MRSA) and Gram-negative (Escherichia coli) bacteria. Finally, we demonstrate the SSA supramolecular hydrogel to tolerate the inclusion of the antibiotic ampicillin, leading to the enhanced inhibition of growth with both model bacteria, and derive initial molecular structure-physicochemical property-antimicrobial activity relationships.