Design And Synthesis Of Unsaturated Quaternary Ammonium Cationic Lipids For Gene Delivery

Gene therapy can regulate gene expression in specific cells to treat pathological conditions by introducing exogenous genes such as DNA,mRNA,siRNA,microRNA,or antisense oligonucleotides.In the progress,cationic liposomes are used as carriers to mediate the exogenous genes,due to their advantages of easy synthesis and low cytotoxicity.In this article,cationic lipids MU18 and GU18?C18:1?were designed and synthesized based on the previous reported MS18 and GS18?C18:0?to investigate the effects of the unsaturated hydrophobic chains on gene delivery.They consisted of quaternary ammonium headgroup,carbamate linker,and unsaturated hydrophobic domains.After purification,the chemical structures of the cationic lipids were characterized by ESI-MS,IR,¹H NMR and ¹³C NMR,and the HPLC purification was above 98%.Cationic liposomes were prepared with the cationic lipids and neutral lipid DOPE by thin-film and ultrasonic dispersing method.The physicochemical properties were characterized by transmission electron microscope and HORIBA laser particle size analyzer.The results revealed that the majority of nanoparticles appeared to be spherical.The average particle sizes of the cationic liposomes were from 130 to 190 nm,and the zeta potentials were between 50-90 mV.The abilities of the cationic liposomes to promote DNA condensation and to inhibit the migration were evaluated by electrophoretic gel retardation assay.Results demonstrated that the cationic liposomes can form lipoplexes,and DNA binding abilities of unsaturated cationic liposomes were stronger than those of saturated counterparts.Meanwhile,DNA binding capabilities of Gemini cationic liposomes were much better than those of mono-head liposomes.The transfection efficiencies of plasmid DNA mediated by the cationic liposomes were evaluated against Hela,Hep-2,and NCI-H460 cell lines in vitro.Then they were detected by an inverted fluorescence microscope qualitatively and the flow cytometry and quantitatively,respectively.The highest transfection efficiency was obtained at the N/P ratio of 3/1,and cationic liposome GU18 had higher transfection efficiency than the commercial DOTAP,and MU18 was similar to the commercial reagent.The cytotoxicity of the cationic liposomes was evaluated by MTT assay among various N/P ratios,with commercial reagent DOTAP as control.The results showed that the cationic liposomes had little cytotoxicity in the range of the N/P ratios measured.When the ratios were less than 6/1,the cell viability was all above 90%,higher than commercial reagent DOTAP.While the ratio increased to 8/1,the cell viability was decreased to about 85%,equal to DOTAP reagent.