M.A. Thesis:

Delivery of bacteria and plasmid DNA into mammalian cell with Modified polyethilenimine

Epithelial ovarian cancer (EOC) is the sixth most common cancer in women worldwide. Despite advances in detection and therapies, it still represents the most lethal gynecologic malignancy in the industrialized countries. The molecule has been considered in ovarian cancer is, CD200 molecule that relate to cancer progression. Although the immune system is capable of mounting a response against ovarian cancers, up-regulation of immune-suppressive molecule such as CD200 defeat tumor immunity. Branched polyethylenimine (PEI) is considered as the gold standard in the gene delivery by nonviral carier. In this study, we used PEI 25KDa to deliver plasmid contain shRNA against cd200 immuonoregulatory marker in epithelial ovarian cancer cell lines OVCAR-3. In order to targeting decrease cytotoxicity of nanopolymer we modified PEI with Tetrac. Derived PEI were characterized by zeta potential, Dnase and gel retardation assay. The cytotoxicity of free PEI chains and modified PEI was evaluated on OVCAR-3 cells by using the MTT test. In addition, the complex contains small shRNA plasmid exposed to the cell line for 4houres and after 72hours, the supernatant and cells were harvested. Downregulation of The surface CD200 was determined by Real Time PCR and flow cytometry using PE-conjugated anti-human CD200. DNA condensation measurement revealed that the resulted polymer could form plasmid. Particle size measurements demonstrated that modified PEI polymers were able to form nanoparticles in the size range below 190nm. MTT data showed decreased cytotoxicity of polymers. Alter PEI Deacresed the cytotoxicity in comparing to simply PEI. The resulting flow cytometry and Real Time PCR showed high efficiency in siRNA-mediated knockdown of target gene. Tetrac of branched PEI strongly reduced cytotoxicity and improved silencing efficacy. Because manipulation of the CD200-CD200R interaction affects the outcome of cancer disease, this nanoparticle may have therapeutic utility.

Keywords: polyethylenimine, nonviral carrier,shRNA, gene delivery