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

Gene therapy requires safe and efficient carriers to transfer DNA or siRNA to target tissues. Cationic lipids as a important type of non-viral,cationic lipids hold promise for: their reproducibility and simplicity in preparation, their non-immunogenic nature and their efficiency in forming stable complexes even with large DNA.In this work, Using galactose as starting material, two series of galactose-based cationic lipids were synthesized. The first kind of cationic lipids, with different lengths of hydrophobic chains attached to the oxygen atom of sugar ring, such as di-C12-Gal-TMA,di-C14-Gal-TMA, di-C16-Gal-TMA, di-C18-Gal-TMA, were prepared via acetylation, selective deprotection of 1-O-acetyl, trichloroacetimidation, glucosidation, azidation, deacetylation, 3,4-O-isopropylene protection, etherification, deprotection of isopropylidene protected,reductive amination and quaternarization. The second kind of cationic lipids, with different lengths of hydrophobic chains attached to the quaternary nitrogen atom, such as Gal-DiC12 MA, Gal-DiC14 MA,Gal-DiC16 MA, Gal-DiC18 MA were prepared via acetylation, selective deprotection of 1-O-acetyl, trichloroacetimidation, glucosidation,azidation, deacetylation, reductive amination, tertiary amination and quaternarization.The zeta potential, average particle size and poly dispersition indexof cationic lipids and lipolexes were tested by Zetasizer Nano ZS. The results showed that the zeta potential of cationic lipids were 25-70 mV,and the zeta potential of two kinds of lipolexes were increased with the increasing of mole ratio, which were reached positive values when mole ratio at 4, 2 respectively. The cationic lipids were homogeneous distribution. The average partical size of cationic lipids were 130-245 nm,and the lipolexes were 100-250 nm, except di-C18-Gal-TMA/DNA,Gal-DiC18MA/DNA were reached 300-450 nm at low mole ratio.Cationic lipids and lipolexes were well dispersed and had a regular spherical surface by atom force microscopy. The DNA binding ability of the cationic lipids were assessed by gel retardation assay. As the rising of mole ratio the DNA retarding action was strengthened, DNA was completely combined by cationic lipids in mole ratio at 2, but di-C18-Gal-TMA.Using Lipo2000 as the positive control, the expression efficiency of cationic lipids carrying DNA in PC-3, HEK293, HepG2, Mat, Hela,SW480, A375, 7721 cells, and the gene silencing efficiency of cationic lipids carrying siRNA in PC-3, Mat cells were detected. The results indicated that the lipolexes di-C16-Gal-TMA/DNA had good expression efficiency in PC-3, Mat, Hela cells, while the lipolexes Gal-DiC16MA/DNA had good expression efficiency in HEK293 and Hep G2 cells. Moveover, both di-C16-Gal-TMA/siRNA andGal-DiC16MA/siRNA were have good gene silencing efficiency in PC-3and Mat cells. Meanwhile, cationic lipids di-C16-Gal-TMA and Gal-DiC16 MA had low cytotoxicity in PC-3, HEK293, HepG2, Mat,Hela cells. Thus, the cationic lipids di-C16-Gal-TMA and Gal-DiC16 MA could be used for further studies.