Synthesis And Gene Transfection Efficiency Evaluation Of Amino Acids-based Cationic Liposomes

The well prospect of gene therapy as the way to tackle genetic diseases and the cancer has been widely investigated. The most important problem met in gene therapy is how to find out a high effective and low toxic gene delivery vector, which introduces genetic drugs into specific cells and can stably maintain its function. Now, gene delivery vectors are classified into viral and non-viral vectors. The cationic liposome is one of the most potential non-viral vectors.In this paper, using amino acid as starting material, eight amino acid-based cationic lipids including Glu-C12, TMA-C2-Glu-C12, TMA-C2-Glu-C8, TMA-C2-β-Ala-C12, TMA-C2-Val-C12, TMA-C2-Met-C12, TMA-C2-Gly-C12 and TMA-C2-Asp-C12 were synthesized via esterification with lauryl alcohol or n-octanol, amidation with chloroacetic chloride and quaternarization with trimethylamine.The synthetic cationic lipids were dispersed into double distilled water by ultrasonic dispersion technology to form cationic liposomes. Then liposome/EGFP complexes were prepared by combining liposomes with EGFP plasmids. The average particle size, size distribution and zeta potential of cationic liposomes and complexes were characterized by Zetasizer Nano ZS. Changing the N/P ratio could make physical structure parameters such as zeta potential and average size meet the optimal transfection conditions.Transfer assay of cationic liposomes which we prepared were investigated by transfection of EGFP plasmids into HEK293 cells in vitro. The superior commercial gene vector Lipofectamine2000 (Lipo2000) was used as control. It was shown that the cationic lipids TMA-C2-/?-Ala-C12, TMA-C2-Val-C12, TMA-C2-Met-C12 and TMA-C2-Gly-C12 with one hydrophobic chain based onβ-alanine, L-valine, L-methionine and glycine showed poor or no transfection under any N/P ratio. Those with two hydrophobic chains showed good efficiency, but L-glutamate-based TMA-C2-Glu-C12 is better than L-aspartic acid-based TMA-C2-Asp-C12. However, TMA-C2-Glu-C8 with eight carbon atoms length of hydrophobic chain and Glu-C12 unmodified with quaternary ammonium group and linking group showed no transfection.With the cationic lipid TMA-C2-Glu-C12 used as gene vector, plasmid EGFP was transfected into cell line HEK293, HBL100,293FT, A549, GC-1, Ges-1, HT1080, HUVEC, U251,3T3-L1, SW-480, NIH3T3, HeLa, MCF-7 and Hct116. And the cytotoxicity of lipid was detected by MTT assay. Results suggested that the cationic lipid TMA-C2-Glu-C12 was an extensive gene delivery vector with high efficiency and low toxicity.