Michael Spelios1, Molinda Kearns, Michalakis Savva. 1. Division of Pharmaceutical Sciences, Arnold and Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, New York 11201, USA.
Abstract
The cationic lipid 1,3-dimyristoylamidopropane-2-[bis(2-dimethylaminoethane)] carbamate (1,3lb2) was applied as a delivery system for small interfering RNA (siRNA) to inhibit the production of vascular endothelial growth factor (VEGF) in vitro in human prostate carcinoma cell line PC-3. VEGF protein silencing peaked at 94% when cationic lipid-nucleic acid complexes (lipoplexes) were formulated at a nitrogen:phosphorothioate ratio (N:P) of 2 with a dose concentration of 53.7 nM, and the performance of these lipoplexes was not impeded by serum. Knockdown efficiency was maintained for at least 72 h, and an IC(50) of 12 nM lasted for 48 h. Only 20% of the total siRNA became cell-associated at this N:P, at a rate of 25 ng/h. Lipoplexes of the optimal formulation were relatively monodisperse, having an average diameter of 634 nm and a zeta potential of -21.3 mV. Formation of the 1,3lb2-siRNA complex reached 94% at an N:P of 2 and was positively cooperative; the binding constant was calculated in the range of 10(5) M(-1), and a Hill coefficient of 3 was determined. 1,3lb2 was found to be a nontoxic and potent carrier of siRNA that binds to the nucleic acid effectively and whose lipoplexes promote long-lasting inhibition, have high biological activity at low N:Ps, and are functional in the presence of serum.
The cationic pan class="Chemical">lipidn>an class="Chemical">1,3-dimyristoylamidopropane-2-[bis(2-dimethylaminoethane)] carbamate (1,3lb2) was applied as a delivery system for small interfering RNA (siRNA) to inhibit the production of vascular endothelial growth factor (VEGF) in vitro in humanprostate carcinoma cell line PC-3. VEGF protein silencing peaked at 94% when cationic lipid-nucleic acid complexes (lipoplexes) were formulated at a nitrogen:phosphorothioate ratio (N:P) of 2 with a dose concentration of 53.7 nM, and the performance of these lipoplexes was not impeded by serum. Knockdown efficiency was maintained for at least 72 h, and an IC(50) of 12 nM lasted for 48 h. Only 20% of the total siRNA became cell-associated at this N:P, at a rate of 25 ng/h. Lipoplexes of the optimal formulation were relatively monodisperse, having an average diameter of 634 nm and a zeta potential of -21.3 mV. Formation of the 1,3lb2-siRNA complex reached 94% at an N:P of 2 and was positively cooperative; the binding constant was calculated in the range of 10(5) M(-1), and a Hill coefficient of 3 was determined. 1,3lb2 was found to be a nontoxic and potent carrier of siRNA that binds to the nucleic acid effectively and whose lipoplexes promote long-lasting inhibition, have high biological activity at low N:Ps, and are functional in the presence of serum.
Authors: Regis Marty; Christophe N N'soukpoé-Kossi; David Charbonneau; Carl Maximilian Weinert; Laurent Kreplak; Heidar-Ali Tajmir-Riahi Journal: Nucleic Acids Res Date: 2008-12-22 Impact factor: 16.971