Methodology Study Of Assembling A Stable Neutral Lipid Membrance Around Polyplex Surface

The key factor for gene therapy advancing from theory towards clinical is an appropriate delivery system. Polyplex has advantages of high gene loading density and simple preparation. Lipid gene vehicles have high cytocompatibility and good chemical modification. While assembling a neutral lipid membrane around polyplex is regarded as a unique way to take the advantages of both lipid and polymeric systems for nucleic acid delivery, to achieve this goal is not an easy task. Assembling positively charged lipids onto polyplexes may further deteriorate non-specific cell adhesion of the carrier. Using lipids with a single negative charge resulted in a thermodynamically less stable membrane. Double charged negative lipids may attach to the polyplex surface sufficiently strong, but such lipids are usually difficult to synthesize and their extra negative charges of the upper-most layer may interrupt cell adhesion. To address these issues, we report a method to assemble neutral lipid around a polyplex with sufficient stability.We synthesized an amphiphilic compound with multiple negative charges(OCAE) from oleoyl chloride and citric acid. Then we added an OCAE solution to a polyplex solution to allow the micelles formed of OCAE to adsorb on the polyplex surfaces. Finally, liposomes formed of neutral lipids were added to the system to allow the formation of a uni-lamellar membrane around the polyplex. Our hypothesis is that upon interacting with liposomes, the strongly adsorbed OCAE will spread over and function as a hydrophobic surface anchors to immobilize lipid bilayer.Various physical chemical and cellular assays were performed to characterize and confirm formation of above-mentioned design. Gel Electrophoresis result shows that lipopolyplex can package DNA plasmid successfully, and the diameter of the particle is about 280 nm, not increased obviously than the polyplex. Zeta potential decreases highly, which draws the conclusion that lipopolyplex can effectively screen the positive charge of polyplex. Transfection rusults in COS-7 Cells and HELA cells by using luciferase plasmid reveals that lipopolyplex has a high transfection efficiency. Transfection rate is closed to commercial transfection reagentsPEI25 KDa and Lipofectamine2000. Cytotoxicity of lipopolyplex is distinctly lower than both of them, especially in a high concentration of polymer at 50-200μg/mL.This design testifies that assembling a neutral lipid membrane around polyplex is viable. It’s a potential promising nucleic acid delivery vector with high transfection efficiency and low cytotoxicity.