Study On Synthesis And Properties Of Glucose-based Cationic Liposomes For Gene Delivery

In the past three decades, gene therapy has shown promising potential to the treatment of human diseases such as cancer, viral infection, AIDS and cardiovascular diseases. The difficulty of gene therapy is the construction of low toxicity, high efficiency and targeting gene vector, the current research is focus on developing efficient viral and non-viral transfection vectors. Non-viral vectors have the potential advantages of low immune response, easy preservation, low cost and easy facilitate inspection. The cationic lipid is one of the most potential non-viral vectors.In this paper, two series of glucose-based cationic lipids were syntheseized. Glucose-based cationic lipids with different lengths of hydrophobic chain, di-C12-Glu-TMA, di-C14-Glu-TMA, di-C16Glu-TMA and di-C18-Glu-TMA were prepared by using glucose as starting material, via ethanoylation, deprotection of 1-O-acetyl, Schmidt reaction, glucosidation, azidation reaction, deprotection of acetyl, isopropylidination, Williamson etherification, deprotection of isopropylidene, one-point of reductive amination and quaternarization. Glucose-based cationic lipids with different lengths of hydrophobic chain, Glu-DiC12 MA, GluDiC14 MA, Glu-DiC16 MA and Glu-DiC18 MA were prepared by using glucose as starting material, via ethanoylation, deprotection of 1-O-acetyl, Schmidt reaction, glucosidation, azidation reaction, deprotection of acetyl, one-point of reductive amination, tertiary amination and quaternarization.Dissolve the lipids in double distilled water, the solution was ultrasonic until clear. Then the liposome/DNA complexes were prepared by combining liposomes with plasmids DNA. The average particle size, poly dispersition index and zeta potential of cationic lipids and complexes were characterized by Zetasizer Nano ZS. Results indicated that the average partical size of cationic liposomes were 60-250 nm except the lipid Glu-DiC18 MA, and the zeta potential of all cationic lipids were 25-70 mv;the average partical size and zeta potential of complexes were 90-180 nm and 10-40 mv at N/P ratio of their best tranfection efficiency. Cationic lipids and complexes had a regular spherical surface by atom force microscopy. The DNA binding ability of the cationic lipids were assessed by gel retardation assay. Results suggested that the binding capability of DNA to lipids increased with addition the proportion of transfection reagent in the complex. DNA delay action was strengthened with the N/P ratio increased and fully integrated with the lipids at the N/P of 3.In vitro transfection, though using commercial transfection reagents Lipo2000 as the positive control, the complexes of lipids/DNA were introduced to HEK293 cell lines, the expression of pEGFP-C3 in HEK293 cell lines was detected by cell count. Results indicated that the lipids GluDiC14 MA and Glu-DiC16 MA have commendable transfection efficiency, and di-C12-Glu-TMA, di-C14-Glu-TMA and di-C16-GluTMA have poor transfection efficiency compared with Lipo2000. However, the lipid di-C18-Glu-TMA, Glu-DiC12 MA and Glu-DiC18 MA have no transfection efficiency. The glycolipids with two hydrophobic chains in the positively charged nitrogen atoms had better transfection efficiency compared to the the hydrophobic chains linked to glucose cycle directly in ether.By using Lipo2000 as the positive control, the lipid Glu DiC14 MA and Glu-DiC16 MA which had high transfection efficiency in HEK293 cell lines were applied to transfer pEGFP-C3 into Hela and HepG2 cell lines. The result showed that the transfection efficiency of these two kinds of cationic lipid was poor than Lipo2000. MTT-based cell viability assays of lipids were performed in representative HEK293 cells. The result revealed that the lipids had lower cellular cytotoxicy than Lipo2000 expect lipid Glu-DiC12 MA.In a few words, the glucose-based cationic lipids GluDiC14 MA and Glu-DiC16 MA have higher transfection efficiency and lower cell cytotoxicy. It provided that they have the potential to vitro gene transfection and gene therapy, enriching and expanding the research of the cationic lipid.