Hierarchical protein assemblies with biomedical applications

Abstract

Molecular self-assembly is a key process evolved by nature, through which functional materials are generated. Among these processes, protein self-assembly into hierarchical structures provides an important avenue for the development of materials with a wide range of biomedical applications. Self-assembly exploits the formation of strong, non-covalent interactions between relatively simple building blocks to form complex morphological structures, including fibres, films, and hydrogels. When comprised of protein, these materials are often biocompatible, biodegradable, and capable of releasing cargo in a controlled manner. To this end, this thesis aims to capitalise on this unique self-assembly process to generate a class of protein-based materials with antimicrobial properties. First, an antimicrobial agent was created and subsequently combined with proteins to produce functional materials. Later, the use of an antimicrobial peptide was employed to produce a self-healing hydrogel. These innovative approaches showcase the versatility of using proteins to produce antimicrobial platforms in materials science. The rapid emergence of drug-resistant bacteria and fungi poses a critical threat to human health. As such, significant efforts have been made to synthesise therapeutic agents that act as potent antimicrobials. However, the development of novel small molecule therapeutics in this space has remained challenging. One orthogonal approach that has garnered interest is using inorganic nanoparticles as antimicrobial agents. While these nanoparticles can often overcome traditional drug resistance by targeting multiple areas of the cell, their usage in medical applications is limited due to their lack of biocompatibility. The first part of this thesis explores the synthesis and characterisation of selenium nanoparticles. Selenium, a trace element in the human body, has been found to not only be non-cytotoxic towards mammalian cells at low concentrations, but can also provide protective antioxidant properties. Selenium nanoparticles were synthesised by chemical reduction over a range of concentrations. I demonstrated that the size, stability, and dispersion of the selenium nanoparticles is concentration dependent. Moreover, through systematic exploration of assembly conditions, I optimised the antimicrobial capacity of the particles. The second part of this thesis describes the fabrication of protein-based films. Regenerated silk fibroin, a natural block copolymer with the capacity to readily re-assemble into structures with attractive physical and chemical properties that are present in native silk, was used as a protein scaffold. The regenerated silk fibroin was embedded with selenium nanoparticles and drop cast to form a hybrid organic-inorganic film. Nanoparticle aggregation is a critical issue in nanoparticle synthesis, impacting a material's structural integrity and performance. By combining the nanoparticles with silk, the nanoparticles were stabilised and remained evenly dispersed throughout the film. Furthermore, I show that this material exhibits both antibacterial and antifungal properties, while remaining highly biocompatible and noncytotoxic towards mammalian cells. By incorporating nanoparticles into the dense fibrillar network of the silk films, the protein scaffold acts not only as a material for delivery, but also protects mammalian cells from the toxic effects of bare nanoparticles. While antimicrobial films are useful for topical applications, they are limited in their ability to target internal infections. To this end, I next explored the generation of silk micron-sized hydrogels with the potential to be used for internal drug delivery. Microfluidic techniques were employed to generate droplets comprised of regenerated silk fibroin embedded with selenium nanoparticles. The thermodynamically and kinetically favourable self-assembly of silk formed uniform, spherical microcapsules, which remained stable over the course of a year. I found that the selenium-silk microcapsules had greater antimicrobial activity than that of the films. The three-dimensional structure of the capsules, created a larger surface-to-volume ratio, allowing for more forms of contact with microbes in solution, and subsequently formed a more potent antimicrobial material. The final section of this thesis explores the creation of an antimicrobial material, without the addition of nanoparticles, by using an antimicrobial peptide. I investigated a small peptide, Ac-KLVFF, derived from the Alzheimer's peptide, amyloid-β. Using Ac-KLVFF, I formed a self-healing hydrogel, capable of transitioning from a gel composition to a liquid state with the application of shear stress. During this reversible process, the hydrogel maintained similar fibril structures. The peptide also exhibited potent antimicrobial activity while remaining biocompatible at a range of concentrations. Taken together, these investigations showcase the versatility of using proteins to produce antimicrobial hierarchical structures with biomedical applications.