Research On SiRNA-DMAPA-CHEMS Cationic Liposomes Complex To Silence The Notch1 Gene Of SKOV3 Cells

Destination:Our research aimed at inhibiting the expression of Notchl gene in human ovarian cancer SKOV3 cells by using siRNA interference technique and evaluating the inhibitory efficiency at the cellular level. However, despite the potential of RNA interference techniques, the clinical use of siRNAs remains challenges for its fragile nature, which can be rapidly degraded by nucleases, and there are still some shortages in the delivery system of siRNA, such as low transfection efficiency and instability against the nuclease. So, the design of high efficiency, stability and security of siRNA delivery system targeting Notchl gene in SKOV3 cells is very important to enhance the resistance of ovarian cancer. In this paper, N-(cholesterylhemisuccinoyl-amino-3-propyl)-N, N-dimethylamine (DMAPA-CHEMS) was synthesized and its physicochemical properties, cytotoxicity, intracellular uptake and transfection were observed. Cationic liposomes was then prepared by depositing DMAPA-CHEMS and soy lecithin S100 as a thin film with the method of film dispersion method, and the pharmaceutical properties, stability against RNase and FBS, intracellular uptake efficiency, gene silencing efficiency, and cell proliferation of cationic liposomes were further evaluated. It gives a strong theoretical basis to build a safe and effective anti-tumor siRNA delivery system.Method:Firstly, DMAPA-CHEMS was synthesized by cholesterol, succinic anhydride DCC and NHS, and then cationic liposome was prepared with DMAPA-CHEMS and soy lecithin S100. The particle size and zeta potential of DMAPA-CHEMS cationic liposomes were measured by dynamic light scattering (DLS). The morphology was observed by transmission electron microscopy (TEM). The stability of siRNA on DMAPA-CHEMS cationic liposomes in 0. lmg/mL RNase and 25% FBS was investigated by 1.5% agarose gel electrophoresis. The cellular uptake of siRNA-DMAPA-CHEMS cationic liposomes was detected by flow cytometry and confocal laser microscopy analysis, the Notchl gene silence efficiency was evaluated by fluorescence quantitative PCR and Western Blot, and finally the cytotoxicity、proliferation and apoptosis of siRNA-DMAPA-CHEMS cationic liposomes were detected by a cell counting kit-8 (CCK-8) and an Annexin V-FITC apoptosis detection kit.ResuLt:The particle size and zeta potential of siRNA-DMAPA-CHEMS cationic liposomes were depended on N/P ratio. When the N/P ratio came to 100, the particle size was (100.94±3.72) nm and zeta potential was (46.54±4.32) mV. Flow cytometry showed an increasing percentage of fluorescence followed by increase in the N/P ratio. A highest percentage of fluorescence about 93.95% was obtained when the N/P ratio was up to 100. Confocal laser microscopy analysis showed siRNA-DMAPA-CHEMS cationic liposomes were internalized by SKOV3 rapidly in 2 h. Fluorescence quantitative PCR showed the expression levels of Notchl mRNA were significantly reduced by treatment with siRNA-DMAPA-CHEMS cationic liposomes (N/P=100). The value of 2-△△Ct was 0.34±0.04 significantly lower than 0.94±0.07 of the control group. Western Blot results showed that the Notchl-siRNA-DMAPA-CHEMS had significantly reduced the expression of Notchl protein and the protein expression is (26.47±6.23)%, significantly lower than (100.00±11.02)% of the control group at 72 h after transfection. CCK8 kit test showed that siRNA carrier itself had low cytotoxicity.When concentration of DMAPA-CHEMS cationic liposomes was less than 100 μg/mL, cell survival rate was more than 80%. But Notchl-siRNA-DMAPA-CHEMS cationic liposomes had obvious inhibitory effect on cell proliferation. When the N/P ratio was 100, at 72 h after transfection, cell survivaL rate was (60.45±2.24)%, significantly lower than (86.43 ± 0.87)% of the control group. Flow cytometry detection showed that treatment with Notchl-siRNA-DMAPA-CHEMS cationic liposomes increased the percentage of apoptotic cells.Conclusion:siRNA-DMAPA-CHEMS cationic liposome was prepared, It presented with a small particle size, positive charge, and spherical morphology, and it was very stable in 0. lmg/mL RNase and 25% FBS solution, and had superior cellular uptake and excellent gene knockdown efficiencies with high safety.