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Development of Cetuximab-conjugated lipoplexes for pulmonary metastasized cancer-directed delivery of anti-cancer siRNA

Issue Date
2013
Description
Dept. of Biomedical Laboratory Science/박사
Abstract
Pulmonary metastasis is the common result of metastatic spread to the lungs from a variety of tumors. Metastatic diseases are the leading cause of cancer-related mortality. Gene therapy using anti-cancer siRNA (small interfering RNA) has been considered to have a great potential for treatment of metastatic cancer. However, clinical application of siRNA has a very challenging task due to numerous barriers, including low stability in serum, non-specific accumulation in tissues, and poor cellular uptake. Especially, the lack of tumor specificity remains to be a major drawback for effective cancer therapy with siRNA. Hence, it is conceivable that a target-directed gene delivery system, which is more specific to tumor cells and less toxic to normal cells, would resolve this obstacle. For enhanced RNA interference in vitro and in vivo via selective accumulations in desired tissues, specific binding to target cells and facilitated intercellular trafficking are commonly attempted utilizing targeting ligands, cell- penetrating peptides, and lipidic delivery systems. Here, I have developed an immunoliposomal gene delivery system containing anti- tumoral siRNAs condensed by 9-arginine peptides (9Arg). The lipidic vesicles consist of cationic O,O''-dimyristyl-N-lysyl glutamate (DMKE) and cholesterol (Chol), grafted with polyethylene glycol (PEG). Antibodies (Cetuximab) against the epidermal growth factor receptor (EGFR) were conjugated to the PEG termini on the surface of the prepared cationic liposomes. Finally, siRNAs were pre-complexed with 9-arginine (9Arg) cell-penetrating peptides and then self-assembled with anti-EGFR liposomes into ternary complexes (anti-EGFR-9Arg-lipoplexes) in aqueous solution. The ternary complex presented a controlled size (180 nm diameter) and a zeta potential of approximately 7 mV. Luciferase siRNAs in the anti-EGFR-9Arg-lipoplexes were effectively transferred to LS174T-Luc colon cancer cells expressing EGFR on the cell surface. The transfected siRNAs were subsequently released into the cytoplasm, resulting in significant knockdown of luciferase gene expression. In cancer therapeutic experiments, Bcl-2/survivin siRNAs formulated in the anti- EGFR-9Arg-lipoplexes were able to significantly suppress transcription of the both genes in the same cancer cells, resulting in cancer cell death. In addition, the anti-EGFR-9Arg-lipoplexes were able to more selectively deliver the anti-cancer siRNAs to LS174T-Luc tumors xenografted in mice than the non-targeted 9Arg-lipoplexes. The Bcl-2 and survivin siRNA complexed in the anti-EGFR-9Arg-lipoplexes were intravenously injected to the mice carrying the pulmonary cancer of LS174T-Luc. Repeated systemic delivery of anti-EGFR-9Arg-lipoplexes significantly inhibited tumor growth whereas non-targeted 9Arg-lipoplexes were less effective. The Bcl-2 and survivin siRNAs in the anti-EGFR- 9Arg-lipoplexes resulted in simultaneous silencing of the respective genes in LS174T-Luc tumor cells. Moreover, the mice treated with the anti-EGFR-9Arg- lipoplexes exhibited prolonged survival compared to the control groups. These results suggest that theanti-EGFR-9Arg-lipoplexes may provide a promising modality for RNAi cancer therapy.
URI
http://ir.ymlib.yonsei.ac.kr/handle/22282913/134720
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2. 학위논문 > 5. Others > 기타 학위논문
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