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Serum albumin (SA) is a versatile transport protein found at high concentrations in the plasma. The protein is known for its ability to solubilise and deliver endogenous amino acids, fatty acids and hormones, in addition to a wide array of exogenous ligands. It also demonstrates several advantages for drug delivery applications such as its biodegradability, extensive half-life, lack of toxicity and immunogenicity. In particular, albumin performs well in regards to anticancer therapeutics as it selectively accumulates in cancerous tissue due to the enhanced permeation and retention (EPR) effect. Moreover, the protein experiences increased uptake by cancerous tumours as it is rapidly metabolised as a source of glutamine and binds at high affinities to specific albumin-binding proteins that are overexpressed in cancer cells. Polymer conjugation to albumin, forming albumin-polymer bioconjugates, has demonstrated further enhancements to albumin’s properties. These benefits include reduced immunogenicity, decreased renal clearance and enhanced circulation half-life. However, the conjugation of polymer to albumin remains a significant challenge due to i) the low conjugation efficiency of attachment via the Cys34 amino acid and ii) the lack of site-specific conjugation when targeting other amino acid residues. Another pathway to form albumin-polymer bioconjugates is via supramolecular interactions, by mimicking the structures of known ligands that bind to albumin. However, due to their large size and complex structural composition, the interactions that occur between albumin and polymers are difficult to characterise. Additionally, the large bulk of the polymer chain may offset the binding effects of specific moieties that target specific binding pockets in albumin. The primary aim of this thesis is to develop an albumin-polymer bioconjugate that adopts both covalent and supramolecular modes of polymer attachment. To achieve this, a library of polymers was developed, including those bearing fatty acid moieties. The intention was for the polymers bearing the fatty acid tails to bind to the 7-9 fatty acid binding pockets in SA. Due to the lack of binding characterisation studies done for polymer and albumin systems, the overarching goal was to determine which techniques typically applied to small molecules were also applicable for polymer-albumin studies. Binding studies of small molecules and albumin typically rely on spectroscopic and calorimetric techniques which may not be feasible for analysing protein-polymer interactions. As a result, saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy was employed to observe the polymer-protein interactions from the perspective of the ligand. The techniques deemed to be applicable for albumin-polymer binding studies were carried forward to determine whether two tails of fatty acid displayed a significant change in comparison to a single fatty acid tail. The two fatty acid tails were shown to be more selective in their binding, favouring binding in the fatty acid pockets (methyl-rich regions) over areas rich in hydrophilic esters or aromatics. The aromatic moiety from the polymer also contributed significantly to the supramolecular interactions for polymers possessing two fatty acid tails. Based on these conclusions, the block copolymer bearing two fatty acid tails with the aromatic moiety, C18PM-b-PH was advanced to the final chapter of the thesis. In the final chapter, a polymer was covalently conjugated to the Cys34 residue to observe its effect on supramolecular binding with the fatty acid bearing polymers. The C18PM-b-PH block copolymer displayed a decreased affinity for the albumin-polymer bioconjugate compared to native BSA. Despite the decrease, the C18PM-b-PH was still able to demonstrate moderate to high binding affinity to the bioconjugate. This validates the feasibility and potential for an albumin-polymer bioconjugate system that incorporates both covalent and supramolecular conjugation as a vehicle for drug delivery. |
