Synthesis And Gene Transfection Efficiency Evaluation Of Pentaerythritol-based And Trimethylolpropane-based Cationic Liposomes

Non-viral vectors have the potential advantages of low toxicity, low immune response, simple to use, easy preparation, easy preservation and facilitate inspection, which become a hotspot of gene medicine transfer vector. Cationic liposome as non-viral carrier and generic drugs form a binary complex by electrostatic adsorption, which can be swallowed by the reticuloendothelial system and passive targeting to various tissues and cells. Researchers improve the active targeting with structural modification.In this paper, two series of cationic lipids were syntheseized. The six pentaerythritol-based cationic lipids TMA-PER-di-C8, TMA-PER-di-C12, TMA-PER-di-C14, HEDMA-PER-di-C8, HEDMA-PER-di-C12and HEDMA-PER-di-C14which have different hydrophobic chain lengths and different cationic head were constructed by using pentaerythritol as starting material, via isopropylidenation, Williamson etherification, deprotection, allylation, iodination, tertiary amination and quaternization. TMA-TMP-di-C12, TMA-TMP-di-C14, TMA-TMP-di-C16, HEDMA-TMP-di-C12, HEDMA-TMP-di-C14and HEDMA-TMP-di-C16which possess different hydrophobic chain lengths and different cationic head were furnished from trimethylolpropane via Williamson etherification, iodination, tertiary amination and quaternization. The synthetic cationic lipids were dispersed into double distilled water by ultrasonic dispersion technology to form cationic liposomes. Then liposome/DNA complexes were prepared by combining liposomes with plasmids DNA. Formation of lipoplexes of cationic liposomes and DNA was confirmed by gel retardation assay. Results indicated that with the increase of N/P ratio, the lagging effect of DNA increased. When the N/P ratio increased to6, the liposome was fully integrated with the plasmid DNA. The average particle size, size distribution and zeta potential of cationic liposomes and liposome/DNA complexes were characterized with Zetasizer Nano ZS. The results showed that the average particle size of TMA-TMP-di-C16and its complex were726.1nm and712.5nm respectively. So, TMA-TMP-di-C16is not suitable to use as gene vector. The average particle size of other eleven kinds of cationic liposomes was between120-240nm, and the size of cationic liposome/DNA complexes was between130-270nm. Zeta potential of cationic liposomes and its complexes were about45mv and around30mv. They are suitable for cell transfection.In vitro transfection, the synthetic cationic liposomes was evaluated observing enhanced green fluorescent protein (EGFP) expression from pEGFP-C1plasmid in HEK293, using commercial transfection reagents Lipo2000as a control. The transfection efficiency of liposomes is measured by cell counting. The results indicated that the transfection efficiency of TMA-PER-di-C12, HEDMA-PER-di-C12, TMA-TMP-di-C12and HEDMA-TMP-di-C12were lower than that of Lipo2000. The transfection efficiency of TMA-PER-di-C14, HEDMA-TMP-di-C14, and HEDMA-PER-di-C14were respectively2.2,2.1and1.1times than that of Lipo2000. The transfection efficiency of TMA-TMP-di-C14was equivalent to Lipo2000. The transfection efficiency of other four liposomes was not observed. The transfection efficiency of pentaerythritol-based cationic liposomes was increased with the increase of hydrophobic chain length. The transfection efficiency of pentaerythritol-based cationic liposomes which have the same hydrophobic chain was reduced with the increase of the hydrophilicity. And the optimal length of hydrophobic chain of the trimethylolpropane-based cationic liposomes is tetradecyl. With the hydrophilicity of liposomes increases, the transfection efficiency of trimethylolpropane-based cationic liposomes which have the same hydrophobic chain increased.The transfection efficiencicy of four liposomes, TMA-PER-di-C14, HEDMA-PER-di-C14, TMA-TMP-di-C14and HEDMA-TMP-di-C14, were evalued in the cell lines HeLa and SW480, using Lipo2000as a control. The results indicated that the transfection efficiency of TMA-PER-di-C14was equivalent to Lipo2000in HeLa and SW480cell lines. Other three liposomes had low transfection efficiency, even no transfection efficiency. TMA-PER-di-C14as a gene carrier has the potential advantages of easy preparation, high transfection efficiency.