From Gene Delivery to Gene Silencing: Plasmid DNA-Transfecting Cationic Lipid 1,3-Dimyristoylamidopropane-2-[bis(2-dimethylaminoethane)] Carbamate Efficiently Promotes Small Interfering RNA-Induced RNA Interference

摘要

The cationic lipid 1,3-dimyristoylamidopropane-2-[bis(2-dimethylaminoethane)] carbamaten(1,3lb2) was applied as a delivery system for small interfering RNA (siRNA) to inhibit the production ofnvascular endothelial growth factor (VEGF) in vitro in human prostate carcinoma cell line PC-3. VEGFnprotein silencing peaked at 94%when cationic lipid-nucleic acid complexes (lipoplexes) were formulated at annitrogen:phosphorothioate ratio (N:P) of 2 with a dose concentration of 53.7 nM, and the performance ofnthese lipoplexes was not impeded by serum. Knockdown efficiency was maintained for at least 72 h, and annIC50 of 12 nM lasted for 48 h. Only 20% of the total siRNA became cell-associated at this N:P, at a rate ofn25 ng/h. Lipoplexes of the optimal formulation were relatively monodisperse, having an average diameter ofn634 nm and a ζ potential of -21.3 mV. Formation of the 1,3lb2-siRNA complex reached 94%at an N:P of 2nand was positively cooperative; the binding constant was calculated in the range of 105nM-1n, and a Hillncoefficient of 3 was determined. 1,3lb2 was found to be a nontoxic and potent carrier of siRNA that binds tonthe nucleic acid effectively and whose lipoplexes promote long-lasting inhibition, have high biological activitynat low N:Ps, and are functional in the presence of serum.