Synthesis And Properties Of Glucosamine-based Cationic Liposomes For Gene Delivery

The key step for gene therapy is to design an efficient and safe vector. As one of the non-viral carrier, cationic liposome combined nucleic acid molecule by electrostatic attraction. The newly formed binary complex attached to the surface of the cell membrane, which was taken up by the cell with the retraction of the cell membrane.Glycobiology played an important role in biochemistry. Carbohydrate molecules carried lots of bio-information, which made it as an integral part of living organism. These information carriers had a wide range of applications, including recognition of the bio-molecules, determination of the morphology and supplement of the energy. In result, the research of the bioactive oligosaccharides seemed particularly outstanding for the study between structure and function relationships.In this paper, two series of cationic lipids were synthesized. The four cationic lipids di-C12-GluNAc-TMA 、 di-C14-GluNAc-TMA 、di-C16-GluNAc-TMA、di-C18-GluNAc-TMA with different hydrophobic chain lengths were constructed by using glucosamine sulfate as starting material, via acetic anhydride protection, 1-O-acetic anhydride deprotection, trichloroacetimidation, glycosylation, azidation, deprotection, isopropylidenation, benzyl protection, deprotection, Williamson etherification, reductive amination and quaternization. Another four cationic lipids GluNAc-DiC12MA、GluNAc-DiC14MA、GluNAc-DiC16 MA and GluNAc-DiC18 MA with different hydrophobic chain lengths were constructed by using glucosamine sulfate as starting material, via acetic anhydride protection, 1-O-acetic anhydride deprotection, trichloroacetimidation, glycosylation, azidation, deprotection, reductive amination, tertiary amination and quaternization.The cationic liposomes and liposome/DNA complexes were characterized by Zetasizer Nano ZS, gel retardation assay, AMF and NMR. The particle size of di-C18-GluNAc-TMA was 182.4 nm and its PDI was 0.43, which indicated di-C18-GluNAc-TMA was not suitable for gene delivery. The average particle size of other seven cationic liposomes was 80-150 nm, and the size of liposome/DNA complexes were 65-140 nm. The zeta potential of cationic liposomes and its complexes were 38-60 mV and 0.5-28 mV. These data showed the seven cationic liposomes were desirable for cell transfection. Gel retardation assay was used to assess the relative binding capability of DNA to liposomes. Results revealed the increased amount of liposomes, the less light in DNA lane. When the N/P ratio added to 6, the liposome was fully integrated with plasmid DNA. The AFM suggested the glycolipids were spherical-like structures and their surfaces were distinct and smooth. The NMR displayed the chemical structure of glycolipids, which was in line with expectation.The transfection efficiency of glycolipids was evaluated by observing enhanced green fluorescent protein(EGFP) expression in HEK293 cell. It was demonstrated that GluNAc-DiC14 MA and GluNAc-DiC16 MA showed high gene transfer efficiency, other glycolipids were essentially incompetent. The data of cell viability showed almost of glycolipids exhibited lower cytotoxicity. We could summarize that GluNAc-DiC14 MA and GluNAc-DiC16 MA has higher transfection efficiency and lower cytotoxicity, which were regarded as potential and promising drug nano-carrier.GluNAc-DiC14 MA and GluNAc-DiC16 MA used as gene vector, plasmid DNA was transfected into cell line HeLa and HepG2 and it was compared with that of commercial transfection reagents Lipo2000. The transfection efficiency of the two glycolipids in HepG2 had the same results as HEK293, while both of them exhibited lower in HeLa.