Delivery of Fluc-mRNA using functionalised gold nanoparticles in vitro.

Master of Science in Biochemistry. University of KwaZulu-Natal, Durban 2016.Nanomedicine, a branch of nanotechnology which includes the use of nanoparticles, is set to revolutionize the area of gene therapy. Gold nanoparticles (AuNPs) have steadily gained favour as promising gene and drug delivery agents in clinical medicine for diagnostics and the treatment of diseases such as cancer. Cancer is a chronic disease with mortality rates increasing steadily. It has been highlighted as a genetic disease with a rising need for the development of innovative gene therapy strategies and, thus, the development of safe and effective gene delivery vehicles. AuNPs feature unique properties which includes small size, high dispersity, stability and surface plasmon resonance with a capacity for ease-of-synthesis and parametric control via chemical methods. Characteristics which favour gene delivery include their biocompatibility, voluntary cellular uptake and low core toxicity. Functionalisation with cationic polymers enhances the properties of AuNPs in terms of condensation of nucleic acids, cellular internalisation, protection against nucleases and release of the therapeutic gene for expression. Over the years, gene therapy applications have routinely utilized therapeutic plasmid DNA (pDNA) for treatment. However, pDNA expression is associated with many limitations hindering cellular trafficking and nuclear entry. As a promising alternative, mRNA molecules overcome these intracellular barriers with no risk of insertional mutagenesis. In this study, the unique properties of AuNPs, cationic polymers and mRNA molecules were exploited and optimized to provide effective gene delivery vehicles for the intracellular release of mRNA for future cancer gene therapy applications, an area of research which until recently has not been investigated. AuNPs were synthesized and functionalised with chitosan (CS) and poly-l-lysine (PLL), respectively. All nanoparticles and their nanocomplexes were characterized using UV-vis spectrophotometry, ICP, FTIR, transmission electron microscopy (TEM) and Nanoparticle tracking analysis (NTA). Functionalised AuNP:Fluc-mRNA binding, compaction and nuclease protection were assessed using the gel retardation, dye displacement and nuclease protection assays, respectively. The effects of these nanocomplexes in vitro were assessed on three human cell lines, embryonic kidney (HEK293), colorectal adenocarcinoma (Caco-2) and breast adenocarcinoma (MCF-7). The cytotoxicity profile of the nanocomplexes was evaluated using the MTT assay and mRNA transfection efficiency assessed using the luciferase reporter gene assay in the three human cell lines. The AO/EB apoptosis assay was employed to confirm the mechanism of cell death as being either apoptotic or necrotic. Results show that both functionalised nanocomplexes exhibit suitable and favourable properties such as small size (CS-AuNP nanocomplex: 95.0 nm and PLL-AuNP nanocomplex: 89.4 nm), colloidal stability (zeta potential: CS-AuNP nanocomplex: 25.9 mV and PLL-AuNP nanocomplex: -97.1 mV), efficient binding and protection against nucleases, low cytotoxicity (