Synthetic polypeptides for biomedical and bioactive applications

© 2016 Dr. Steven Josef ShirbinSynthetic polypeptides are bioinspired mimics of natural polypeptides, readily prepared through controlled synthetic polymerization processes. Their use has offered chemists and biologists around the world the ability to precisely control the synthesis, scale-up, modification and engineering of polypeptides with properties similar to those seen in the natural world. The plethora of possible functionality gives synthetic polypeptides an array of properties shown to be highly useful in the biomedical and antimicrobial (bioactive) fields. Despite this, significant deficiencies surrounding the application of synthetic polypeptide materials in these fields remain. This thesis reports on the fabrication and testing of novel synthetic polypeptide and synthetic polypeptide-based materials to address deficiencies related to the application of synthetic polypeptides in nanoparticle drug delivery, cellular scaffolds for tissue engineering and antimicrobial materials for water treatment. For drug delivery, investigations into the use of synthetic polypeptide-based nanoparticles for cisplatin delivery have traditionally focused on micelle assemblies. Different synthetic polypeptide-based self-assemblies such as vesicles offers the prospect of introducing a new biocompatible and biodegradable architecture for cisplatin delivery. In this study, the preparation of novel cancer-targeting synthetic polypeptide-based vesicles for cisplatin drug delivery is described. The vesicles were prepared through a novel drug-induced self-assembly process. Folic acid was conjugated to the vesicle corona to form an active targeting drug delivery system. In vitro studies on these targeted vesicles showed significantly higher cellular binding/uptake and dose-dependent cytotoxicity toward cancerous cells (HeLa) compared to non-cancerous cells (NIH-3T3). Next, preliminary studies into the preparation of aptamer (advanced targeting ligands composed of single strand nucleotides) targeted synthetic poly(L-glutamic acid)-based drug delivery systems was investigated. Poly(L-glutamic acid) or PLG, has been utilized in a range of synthetic polypeptide-based drug delivery systems owing to its biocompatibility and favorable enzymatic biodegradability profiles. Whilst aptamers have been used as advanced targeting ligands in a wide range of polymeric nanoparticle drug delivery systems, they have yet to be investigated in delivery systems composed of PLG. Conjugation of a model single stranded DNA (ssDNA) aptamer to poly(ethylene glycol)-b-poly(L-glutamic acid) (PEG-b-PLG) block copolymers, common synthetic PLG-based nanoparticle precursors, was achieved through thiol-maleimide coupling chemistries and the conjugates successfully isolated through preparative gel electrophoresis. The DNA-polymer conjugation and isolation protocols established in this work offer potential use in future studies employing aptamer-targeting of PLG-based delivery systems. For tissue engineering, the biocompatible, biodegradable and cell adhesive properties of synthetic polypeptides makes them useful materials for the fabrication of 3D polymeric hydrogels with macroporous morphologies ideally suited for cell in-growth. Traditionally, synthetic polypeptides have been used as partial components of these gel networks, and often require side-chain modifications to allow for cross-linking to take place, thus hindering the effective study of these materials as cellular scaffolds. In this study, macroporous hydrogels composed entirely of synthetic polypeptides were prepared through direct cross-linking of a single poly(L-glutamic acid)-b-poly(L-lysine) (PLG-b-PLL) polypeptide component under cryogelation conditions. Tuning the relative ratios of the amino acid constituents could result in cryogels with very different pore structures, swelling, and mechanical properties, suitable for a range of soft tissue engineering applications. These cryogels were shown to be enzymatically biodegradable and demonstrated excellent biocompatibility, cell attachment and cell proliferation profiles with mammalian fibroblast (NIH-3T3) cells. The inherent antimicrobial (bioactive) properties of peptides are utilized in this study through the preparation of synthetic polypeptide-based cryogels with inherent antimicrobial (bioactive) properties, for potential water purification applications. Traditionally, the effective bioactive properties of antimicrobial cryogels come from the incorporation of known antimicrobial agents to the gel structure rather than from the polymer itself. The leaching of these toxic agents is commonly reported in these systems, leading to potential toxicity issues. Cryogels composed of a polycationic poly(L-lysine) and hydrophobic poly(D,L-valine) copolymer were prepared with the gels displaying high swelling, and inherent antimicrobial activity against E. coli after brief 1 h exposure, with no toxic leaching. Compared to a conventional ‘nanoporous’ hydrogel, the cryogel macropores and their integrity were found to be crucial for bactericidal activity where they allow for effective uptake of bacteria into the gels, and provide a confined environment and increased surface area for contact of the bacteria with the antimicrobial polymer walls. The materials prepared in this thesis and the study of their properties, demonstrate an advancement in the scientific understanding and applicability of synthetic polypeptides in the relevant biomedical and bioactive fields