Bringing together engineering and entrepreneurship: understanding the role of tethered C-CHY1 in the fight against antimicrobial resistance

Alexander, Todd E

Etd

Bringing together engineering and entrepreneurship: understanding the role of tethered C-CHY1 in the fight against antimicrobial resistance

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Healthcare associated infections (HAIs) cost the US healthcare system over $45 billion to treat and cause millions of deaths annually. A large subset of HAIs are associated with medical devices that are meant to improve and save lives. Infected devices are treated using traditional antibiotics, contributing to development of antibiotic resistance. Antibiotic resistance is expected to cost $100 trillion and kill more people a year than cancer by 2050; thus, new alternative antimicrobials for the treatment of device-associated HAIs are critically needed. Antimicrobial peptides (AMPs), such as 26 amino-acid (aa), marine-derived Chrysophsin-1 (CHY1), are poised to reduce HAIs due to their broad antimicrobial activity and unique mechanisms of action that do not promote bacterial resistance. AMPs are short (12-50aa), positively charged (+2-+9) proteins found in the innate immune systems of many different species. Their high separation of hydrophilic and hydrophobic residues leads to many unique mechanisms derived from many unique secondary and tertiary structures that are not yet well understood. Despite the discovery of over 2000 natural AMPs and many more synthetically designed AMPs, none have been successfully commercialized for healthcare applications due to challenges surrounding cytotoxicity, short in vivo half-life (degradation), high costs of production and effectiveness in physiological environments (such as those with high-salt). Several strategies have been investigated to overcome these challenges, for example, truncation of cytotoxic sequences or D-amino acid substitution to improve AMP toxicity and stability; however, many of these strategies can reduce antimicrobial effectiveness. A unique strategy of increasing stability, reducing cytotoxicity, and maintaining antimicrobial activity that is relevant for medical devices is the covalent tethering (binding) of AMPs via a flexible tethering molecule to the surface. However, the effect of tethering parameters on resulting AMP mechanisms and activity is still widely debated. AMP activity can vary widely by utilizing different tethering strategies, which include additional variables such as: (1) peptide choice and properties (such as native mechanism, concentration, charge, and structure), (2) tether choice and properties (such as chemical composition, length, charge, surface density, and flexibility), and (3) testing conditions (such as temperature, solvent composition and substrate type). Some studies suggest that AMP performance may be tether-dependent, for example some AMPs require longer tethers while others do not and some need a flexible tether. Thus, models for predicting successful tethering strategies for different AMP properties, which currently do not exist, must be developed. Further, complicated and often destructive techniques, such as XPS and SEM, are typically implemented to study the relationship of all these parameters vs. antimicrobial activity, which are labor-intensive and limited in scope. Predictive models guiding tether strategy need to be constructed, but also new techniques to study tethering be developed. If these technical milestones are achieved they can serve as a predicate for commercial implementation of a host of new therapies targeted at reducing device-associated HAIs. The overall goal of this thesis was to study the relationship between antimicrobial activity of tethered C-CHY1 examining both spacer length and peptide surface density and the development of a feasible clinical business case for tethered AMPs. To achieve this goal, a traditional entrepreneurial approach was taken in which a minimally-viable product was first designed and business case analyzed, followed by studies to better optimize and understand the underlying structure-mechanism relationships. CHY1 with a C-terminus cysteine to allow for surface-binding (C-CHY1) was tethered onto a silicon dioxide surface via a flexible poly(ethylene glycol) (PEG) tether, and then both surface binding behavior and antimicrobial success of C-CHY1 were examined as a function of tether properties and reaction conditions. For these studies, quartz-crystal microbalance with dissipation (QCM-D) was the primary technique, a real-time, non-destructive flow method that was then coupled with downstream characterization techniques: fluorescent microscopy and contact angle measurements. In parallel a deep dive into domestic and international business models for commercializing AMP technologies. Specifically, tether length and surface density effects on C-CHY1 mechanisms were studied, followed by the effect of temperature, type of microbe, and salt concentration on the antimicrobial mechanisms of tethered C-CHY1. QCM-D was used to measure binding of C-CHY1 via three different length tethers, PEG molecular weight (MW) 866, 2000 and 7500, followed by microscopy to measure antimicrobial effectiveness against two model microbes Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Modeling of QCM-D data allowed for surface density and thickness to be calculated and related to C-CHY1 antimicrobial activity. PEG 7500 allowed proper C-CHY1 orientation and mobility, allowing for its native pore-forming mechanism and highest activity while PEG 866 tethers led to denser grafting and an effective, yet non-native ion displacement mechanism. The QCM-D was used to characterize the effect of salt concentration and temperature reaction conditions on the grafting density of C-CHY1 tethered via PEG 866 and PEG 7500, which was then related to antimicrobial activity. For PEG MW 866, neither temperature nor salt concentration increases significantly altered the grafting density of C-CHY1 while for PEG 7500 increasing temperature allowed for significantly increased grafting density. C-CHY1 density had no significant effect on antimicrobial activity against either microbe. Temperature of bacterial incubation did demonstrate microbe-specific changes in C-CHY1 antimicrobial activity. These results demonstrated that small changes in reaction conditions can drastically change membrane selectivity of C-CHY1. An in-depth investigation of the effects of bacterial membrane composition and temperature on soluble C-CHY1 mechanism was implemented to better understand the molecular membrane- and temperature-dependent selectivity and structure-function of C-CHY1. Supported lipid bilayers (SLBs) formed in QCM-D can be used as model membranes to elucidate AMP action mechanisms against membranes of different compositions. Two and three component SLBs representative of Gram-negative phosphatidylethanolamine (PE) and phosphatidyglycerol acid (PG) with and without charged lipopolysaccharide, LPS and Gram-positive bacteria phosphatidylcholine (PC) and PG with and without charged lipoteichoic acid, (LTA) were formed at both 23°C and 37°C. C-CHY1 at 5 µM was exposed to the different membranes and mechanistic surface action was studied. The membranes formed highly different baseline responses in QCM-D, indicative of vastly different membrane structures, thicknesses and deposition behaviors on SiO2, warranting future studies. Further, significant effects of LTA incorporation were observed in both peptide interaction and deposition. There were measurable effects of temperature on membrane formation as well as peptide interaction kinetics and even mode of interaction. Lastly, business models for the commercialization of novel medical device technologies such as surface-tethered C-CHY1 were investigated. While this technology has the potential to solve many unmet needs, there must a commercialization plan implemented in order to have an impact. There is a clear disconnect between technology development in academia and technology commercialization in industry that must be connected. Development of an entrepreneurial mindset at the graduate school level, can help bridge the gap. A thorough investigation of domestic and international business models for commercializing AMP technologies was carried out and distilled in the form of the Business Model Canvas developed by Alexander Osterwalder that can be used as a roadmap for commercialization efforts. Using the QCM-D a relationship between both spacer length and peptide surface density and the antimicrobial activity of tethered C-CHY1 was determined. A business plan was developed in order to increase the impact of this and other AMP based work. This work provides a roadmap for future researchers to quickly develop and commercial novel AMP based coating technology.

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