PhD thesisTriple-negative breast cancers lack the three main breast cancer biomarkers; oestrogen-, progesterone- or HER2-receptors and chemotherapy is the only established treatment option for patients. Chemotherapies are associated with physically debilitating, off-target toxicities and tumours often develop resistance to cytotoxic compounds. Nanomedicine has the potential to improve chemotherapy efficacy and has enabled the development of small, therapeutic carriers such as microparticles (MPs). These can be made from biodegradable polymers, loaded with (sometimes poorly soluble) chemotherapeutic agents and their surfaces coated in tumour targeting proteins. CD95L has been reported to be upregulated in cancer cells identifying it a promising target. In this study, the CD95 receptor, CD95R, was fused to the Fc region of human IgG1, and baculoviral-mediated expression was optimised in Sf21 and Hi5 insect cells. The protein was purified by affinity chromatography and used to coat microparticles. CD95R-coated MPs improved the uptake into triple-negative, MDA-MB-231 cells, ostensibly via a mechanism involving CD95L. Anti-CD95L antibodies were validated following expression of CD95L-Myc-DDK in otherwise naïve HEK293T cells. The expression of supposed target ligand CD95L was then tested in MDA-MB-231 cells and found to be undetectable. Furthermore, the CD95R-coated particles were efficiently taken up by non-cancerous primary fibroblasts. The reasons for the enhanced uptake of CD95R-coated MPs were explored in MDA-MB-231 cells. Differences in the surface charge of the coated-MPs, characterised by dynamic light scattering (DLS) and electrophoresis, were ruled out as a cause. However, inhibition of the common endocytic pathways did suggest that coated-MP uptake likely occurs via macropino- or phagocytosis. The use of this system for targeted cancer treatment or for wider applications are discussed
