Design and Synthesis of Antimicrobial Polypeptide Polymers
AuthorRasines Mazo, Alicia
AffiliationChemical and Biomolecular Engineering
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2023-05-26.
© 2021 Alicia Rasines Mazo
The goals of this thesis were to explore the impact of architectural design on the potency of antimicrobial polypeptide polymers. To this aim, we firstly investigated the synthesis and kinetics of flexible polymeric macroinitiators obtained via photo reversible addition-fragmentation chain-transfer polymerization (RAFT) for the ring opening polymerization of N-carboxyanhydrides. This study revealed the bottlebrush polymers benefit from a cooperative folding of alpha helices in chlorinated solvent, resulting in fast kinetic rates. Furthermore, free primary amine and trimethylsilylated macroinitiators were shown to offer similar initiation efficiency, kinetics, and polypeptide control, which allowed the selection of appropriate initiators and polymerization conditions for future applications. Based on this synthetic knowledge, amphipathic bottlebrush polypeptide polymers, termed brush Structurally Nanoengineered Antimicrobial Peptide Polymers (brush SNAPPs) were synthesized. Their direct antibacterial activity was evaluated against Gram-positive and Gram-negative pathogens by means of in vitro assays. The outcome of the structure activity investigation revealed the bottlebrush morphology is indeed antibacterial, with short bottlebrushes of backbone degree of polymerization of 16 behaving similarly to star shaped SNAPPs and displaying preference for Gram-positive bacteria. In contrast, a brush SNAPP with longer backbone length of 190 displayed greater efficacy when challenged with Gram-negative Acinetobacter baumannii. These results, combined with the reduced cytotoxicity of the brush SNAPP architecture provide guidance for the treatment of Gram-negative infections. Aiming to further reduce the observed mammalian toxicity of polypeptides, we next focused on PEGylation as a means to improve SNAPP biocompatibility. Three different avenues for the polymerization of brush poly(ethylene glycol) methyl ether acrylate (PEGA) with high chain end fidelity were investigated. Blue LED-activated RAFT polymerization yielded remarkable alpha- and omega- group retention compared to thermal and UV activated RAFT methods. This technique was applied toward the synthesis of discrete polypeptide nanogels comprised of a comb-brush PEGA and bioactive polypeptide corona, aiming to selectively target Gram-positive pathogens. Antibacterial evaluation of the SNAPP nanogel hybrid against Escherichia coli and Staphylococcus aureus demonstrated this architectural design furnishes bacteriostatic and bactericidal properties, with selective targeting of S. aureus whilst also achieving an improved therapeutic profile and antifouling protection. Together, these results have generated important considerations for the future design of antimicrobial polypeptide therapeutic agents of varied architecture.
KeywordsNCA ROP; antibacterial polypeptide; SNAPP; architecture of polypeptides; structure-activity study of polypeptides
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