Beinarovica, Jolanta
(2022)
New antimicrobial biomaterials based on recombinant spider silks.
PhD thesis, University of Nottingham.
Abstract
Spider silk is a protein-based material with exceptional mechanical properties together with low immunogenicity and pyrogenicity which makes it useful in biomedical applications. The cannibalistic and highly territorial nature of most spiders prevents high-density farming; therefore, the availability of their silk in a usable form is very limited. Recombinant production of silks is explored as an alternative mean of production. This study uses two miniature recombinant silk proteins (mini-spidroins) with distinct biochemical nature -NT2RepCT and 4RepCT - to create new materials for drug delivery.
A procedure for a column-free, scale up compatible purification of highly water soluble spidroin NT2RepCT has been developed. NT2RepCT was then processed into a colloidal drug delivery system that could be loaded with a model drug and exhibited a pH-dependent controlled drug release profile. It was found that NT2RepCT particles have a polydisperse size range on a micron scale, and that they are unstable in water, which makes them useful for in situ and temporary embolic applications.
Further, the 4RepCT3Aha mini-spidroin was expressed and purified. In this construct, each methionine residue is replaced with a synthetic methionine analogue L-azidohomoalanine (Aha) that carries a terminal azide in its side chain. The azide acts as a selectively chemically reactive, bioorthogonal group for bioconjugations using copper-catalysed azide-alkyne cycloaddition (CuAAC) knows as the “click reaction”. Using this methodology, a selection of antimicrobial ligands (triclosan, chloramphenicol, ciprofloxacin, erythromycin, levofloxacin, and nitroxoline) bearing a labile linker with a terminal alkyne were conjugated to 4RepCT3Aha , creating a library of antimicrobial conjugates. The resulting conjugates were processed into films that showed significant antimicrobial activity against the Gram-negative Escherichia coli (E. coli) and the Gram-positive Staphylococcus aureus (S. aureus) in a novel plate-based, high throughput-compatible assay. Further, it was found that ‘clickable’ antimicrobial ligands could also be conjugated to pre-formed Aha-bearing silk films. When the antimicrobial ligands were conjugated to pre-formed films, their biocidal activity was lower than that of films made from soluble 4RepCT3Aha -ligand conjugates but the activity was statistically significant compared to films dipped in antibiotic solution.
In addition, the 4RepCT3Aha mini-spidroin was functionalised with quaternary ammonium-based ligands via a non-labile linker prior to processing of the conjugate into a surface coating. In this approach, a cationic, contact-active antimicrobial surface was created that showed significant antimicrobial effect against E. coli in a range of conventional microbiological assays. For this material, a tailored high-throughput compatible assay to analyse the metabolic activity and biomass increase of surface-adherent bacteria was developed. In this assay, it was found that quaternary-ammonium bearing ligands have activity against E. coli, but not S. aureus or Pseudomonas aeruginosa.
In conclusion, this work describes a range of new antimicrobial materials based on miniature spider silks that can serve as a drug delivery vehicle in different biomedically relevant scenarios. By combining silk’s uniquely biocompatible nature with a tailored functionality and modifiable release kinetics, these novel biomaterials are promising candidates for drug delivery applications.
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