Abstract
With the advent of molecularly targeted agents in place of the traditionally employed chemotherapeutic cytotoxic compounds, cancer treatment has recently entered a new and exciting phase. Thus, the aim of this thesis was to explore the use of nanotechnology to deliver new-targeted cancer therapies using a carbon nanotube based interfering RNA (RNAi) gene delivery system. To consider carbon nanotubes (CNTs) as nano-bio agents for gene therapy, it is important first to understand how they behave at a cellular level. CNTs, in the form of double-walled CNTs, were oxidised and wrapped with biomolecules prior to incubation with cells. Evidence of CNT uptake and release was demonstrated through Raman spectroscopy, of single cells and cell lysates, from PCS and HeLa cancer cell lines. Results show a maximum uptake at 3 hours with 20% of CNTs being internalised by PC3 cells (5.85 mug/mL) and a consequent release within a 24-hour time frame (< 0.31 mug/mL). An increase of ID/IG ratio and loss of the outer diameter of the DWNT during incubation period suggests that CNTs are being degraded in the intracellular environment. However, the internalisation of complexes led to no significant changes in cell components, such as DNA/RNA, proteins and lipids. In addition, no significant stress, evaluated by activation of phosphorylated MAPK, was induced when cells were exposed to carbon nanotubes. Intracellular localisation and trafficking of carbon nanotubes was studied by means of antibody staining to specific cellular compartments and revealed that an endocytic pathway is involved in the internalisation of carbon nanotubes. In this endocytic pathway, carbon nanotubes were found to co-localise specifically with clathrin coated vesicles, early endosomes, lysosomes, and slow recycling endosomes. To study the feasibility of applying CNTs to deliver nucleic acids, green fluorescent protein (GFP) was employed as a reporter gene for both gene delivery and gene silencing in an in vitro model. Firstly, gene delivery protocols were optimised by transfecting GFP-encoding plasmid DNA (pDNA) to mammalian cells via CNTs. Secondly, CNTs were used to deliver siRNA to the target GFP gene integrated into the host cell genome (through Lipofectamine transfection of pDNA). Gene targeting was achieved by silencing the GFP gene through small interfering RNA-CNT complexes (siRNA-CNT) delivered to GFP expressing cells. However, results indicate that CNTs-nucleic acids (pDNA and sIRNA) complexes do not reach their target sites, resulting in poor GFP expression or silencing. Data analysed by fluorescent microscopy and flow cytometry revealed that less than 1 % of ceils were expressing the GFP gene (plasmid DNA delivery) or 2% of cells presented knockdown of GFP gene (siRNA delivery). The expression and silencing using CNTs was very low compared to delivery of constructs with transfection reagent (Lipofectamine), which led to 43% of cells expressing GFP and around 40 % of cells where the GFP gene was silenced. Nevertheless, the experience of other researchers has been that the GFP gene is very difficult to silence to a significant degree because of its high level of endogenous expression. We therefore used CNTs to deliver siRNA targeting survivin, an anti-apoptotic protein, which is known to be overexpressed in several cancer cells lines and is thought to contribute to their oncogenic character. Reduced survivin silencing effect by immunoblotting was observed when siRNA-CNTs were employed, in comparison with DharmaFECT transfection reagent. However, after extended incubation periods the silencing induced via survivin-siRNA delivered through CNT cause cells to undergo apoptosis.