Doctor of Philosophy (PhD)
Department of Biological Sciences - Faculty of Science
Jones, Rachel A, Development of a polymer-based, pro-apoptotic cancer therapeutic, PhD thesis, Department of Biological Sciences, University of Wollongong, 2003. http://ro.uow.edu.au/theses/199
This thesis documents work directed towards the development of a novel cancer therapeutic that uses a synthetic polymer to deliver a pro-apoptotic peptide into the cytosol of cancer cells and in turn induce death by apoptosis. This was approached by (1) testing the ability of a range of synthetic polymers (poly(2-alkyl acrylic acid)s (PAAAs) and poly[1-(dimethylamino)ethyl methacrylate] (pDMAEMA)) to facilitate the release of molecules from endosomes to the cytosol, (2) conjugating a pro-apoptotic peptide (Bad(140-165)) to a suitable endosomal-disruptive polymer, and (3) testing the ability of the polymer-peptide conjugate to enter the cytosol following endocytosis and to trigger apoptosis. PAAAs were shown to be endocytosed into cells and transported to late endosomes, where acidification of the lumen by the membrane H(superscript +)-ATPase triggered polymer-mediated vesicle disruption and release of endosomal contents to the cytosol. pDMAEMA was shown to induce rapid necrosis and to be endocytosed into cells where it induced morphological changes in late endosomes/lysosomes. However, pDMAEMA did not have the ability to disrupt endosomes, and was thus not used in further experiments. In addition, the very high toxicity of pDMAEMA could also pose a significant risk of bystander cell damage in cancer therapies. A hydrazide-linker (X) was synthesised and conjugated to PAAAs. Results showed that (1) the membrane disruptive ability of PAAAs was retained following conjugation with hydrazide linker, and (2) the carboxcyclic-hydrazide bond incorporated within the linker was stable at pH 7 but not at pH 5. Finally, PAAA-X was conjugated to Bad(140-165) to form PAAA-X-Bad(140-165). Immunofluorescence assays showed that PAAA-X-Bad(140-165) partially co-localised with early endosomes and induced disruption of endosomes and release of their contents into the cytosol in a time-dependent manner. Surprisingly, cell viability was retained following uptake of PAAA-X-Bad(140-165). The reasons why apoptosis was not induced following uptake of PAAA-X-Bad(140-165) are currently unknown, however possibilities include incomplete cleavage of the linker and/or insufficient release of Bad(140-165) into the cytosol. Future experiments, such as increasing the number of linkers and/or Bad(140-165) molecules attached to molecules of PAAA, may aid in the understanding of why apoptosis was not induced. As an adjunct to studies of the effects of polymers and PAAA-X-Bad(140-165) on cells, a new technique was developed to measure mitochondrial permeability transition (MPT) in intact cells. In this method, calcein is introduced into cells by pinocytic loading. Polarised mitochondria are visualised as darkened voids within a bright green fluorescent cytosol. MPT is measured as an in-filling of calcein fluorescence into these darkened voids. This method is free of disadvantages associated with earlier developed techniques and will be valuable in studies of MPT in intact cells. However, unfortunately, technical limitations prevent the use of this method to measure MPT in cells undergoing apoptosis. Taken together, results presented in this thesis provide a firm foundation for the future development of a novel cancer therapeutic that (1) directly targets cancer cells, (2) induces apoptotic death, and (3) avoids disadvantages associated with traditional cancer therapeutics.
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