Polyion complex micelles as cancer therapeutic carrier for protein drugs

Anti-cancer therapeutic proteins are extensively studied as a better alternative to traditional small molecule drugs owing to their specific biological functions. Polymeric nanocarriers are widely used to deliver therapeutic agents. To overcome the disadvantages of native protein drugs, many nanocarriers have been developed to protect the protein from external enzymatic attack and to deliver them to the desired tumour sites by exploiting the enhanced permeability and retention (EPR) effect. Among the many polymeric nanocarriers for protein drugs, this thesis focuses on polyelectrolyte-protein complexes or polyion complex (PIC) micelles, which are created from charged block copolymers and counter-charged protein molecules. The interest of PIC micelles stems from the simple and non-organic preparation, as well as good protection of cargo proteins, controllable size, surface charge and response to environmental changes. Although many PIC micelles systems have been established in the literature for a wide range of therapeutic proteins, few have studied the disassembly of micelles in three-dimensional (3D) tumour spheroids due to a lack of an established technique. The aim of this thesis was to deliver anti-cancer therapeutic proteins using PIC micelles and to investigate the relationship between polymer structure to cellular uptake and release site in artificial tumour spheroids. In order to achieve this aim, Poly[poly(ethylene glycol) methyl ether acrylate] (PPEGMEA) was selected as the neutral hydrophilic block due to the superior biocompatibility and non-immunogenicity. This polymer was copolymerised with carboxylic acid bearing monomers with various lengths of carbon spacers, including acrylic acid, 2-carboxyethyl acrylate, acryloyl glycine and cryloyl aminovaleric acid. These monomers constitute the negatively charged block as a means to interact with a positively charged model protein dug, lysozyme. Another approach to increase the hydrophobicity of the core-forming part was developed by introducing hydrophobic butyl acrylate units for the purpose of generating a relatively more stable PIC micelle system. Light-sheet microscopy revealed that the disassembly of PIC micelles took place at the rim of MCF-7 human breast cancer spheroids regardless of charge density on polymers or lengths of the carbon spacers. Further studies applying confocal laser scanning microscopy (CLSM) found that the disassembly is most likely related to competing species in the cell cytosol and the cellular uptake of PIC micelles towards two-dimensional (2D) cultured MCF-7 cells is related to the hydrophobicity of the charged block. Less hydrophilicity of the charged block resulted in lower cellular uptake rate. In the final section of this thesis, three therapeutic peptide/proteins (sprouty, apoptin and melittin) were employed for comparing the cytotoxicity between PIC micelle loaded drug and their native forms towards both 2D and 3D cultured MCF-7 cancer cells. The activity of incorporated peptide/protein drugs was retained and displayed an extended activity after 7 days when compared to their free form. Melittin loaded PIC micelles were further crosslinked via click chemistry and was demonstrated an enhanced stability and different distribution of intact micelles in cell spheroids. This resulted in a slightly improved cytotoxicity towards the 3D tumour spheroid. In summary, the findings in this thesis contribute to a better understanding of the behaviour of PIC micelles in living cells. It is also expected to help in the design of PIC micelles in drug delivery area