Melanoma is the most deadly form of skin cancers, with an incidence increasing more rapidly than any other malignant cancer in the past 40 years. Metastatic melanoma is resistant to conventional treatments, such as chemotherapy and radiation therapy. Our lab has previously demonstrated that Mcl-1 is a key contributor in protecting melanoma from therapy-induced cell death. RNAi therapeutics was employed as a novel way to silence the anti-apoptotic protein by using Mcl-1 mRNA sequence-specific siRNAs in vitro. In our hands, passive non-targeted delivery of RNAi therapy into melanoma tumours has been shown to be neither effective, nor selective in vitro and in vivo. Consequently, in this study, siRNA was linked to a delivery system which expressed a ligand specifically targeting melanoma cells. Previously shown, melanoma overexpresses the cell surface ganglioside GD3, thus it is my belief that the anti-GD3 R24 monoclonal antibody can function as a targeting molecule. The antibody was linked to coated cationic liposomes (CCLs) carrying siRNA molecules. Our first step was to confirm R24 ligation to CCLs. Untargeted CCLs showed insignificant values of antibody, whereas antibody-conjugated CCLs presented approximately 30 antibodies per liposome. I also confirmed that siRNA was internalized within CCLs using spectrometry, with an encapsulation efficiency of approximately 80%. Since liposomes need to be small to be effective in vitro and in vivo, CCLs were confirmed to be less than 100nm in diameter. In vitro studies using fluorescent microscopy demonstrated greater binding to melanoma cells with antibody-conjugated CCLs as compared to untargeted CCLs. In vivo experiments showed specific localization of targeted CCLs to induced subcutaneous mouse xenograft tumours. Western blotting demonstrated greater Mcl-1 knockdown using GD3-targeted CCLs. Taken together, these results suggest that anti-GD3 antibodies can serve as targeting molecules to deliver siRNA to melanoma cells and furthermore, GD3-targeted CCLs can promote siRNA-mediated gene silencing. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2010-09-02 10:29:37.944
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/6018 |
| Date | 02 September 2010 |
| Creators | Wu, Michael Wing-Yin |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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