Effects Of Peptidomimetic Amino Acids Arrangement On Gene Transfection Performance Of DoGo Lipid Nanoparticles

As a fundamental strategy,gene therapy has been proved to be an effective method for the treatment of cancer diseases,monogenetic diseases and neurological diseases.And successful gene therapy much depends on good gene delivery vehicles.The cationic lipid-based carrier is a non-viral delivery system,and it is widely used because of its good biocompatibility,low toxicity,and ease of preparation.In this paper,two cationic lipid molecules,DoGo220(branched)and DoGo310(linear),were prepared by combining two natural amino acids,e.g.glutamic acid and ornithine,and the structures were determined by ~1H nuclear magnetic resonance spectroscopy and mass spectrometry;then,DoGo220 and DoGo310 cationic lipid molecules were mixed with neutral lipid(lecithin)and PEG 2000 to obtain two lipid nanoparticles.The gene transfection efficiency of DoGo220 and DoGo310 lipid nanoparticles were compared by fluorescence quantitative and flow cytometry analysis,which proved that both DoGo220 and DoGo310 lipid nanoparticles have excellent ability to release nucleic acid in cells,while DoGo310 lipid nanoparticle showed higher transfection efficiency under the same conditions;then,the si RNA binding affinity(K_Dvalue)of the lipids were measured by fluorescence quenching experiments.The results showed that K_D of DoGo220 is 8.27 n M,K_D of DoGo310 is 3.07 n M,indicated that DoGo310 lipid nanoparticle has higher nucleic acid affinity under the same conditions.Therefore,DoGo310 had higher nucleic acid transfection efficiency.Then,the cytotoxicity,morphology and surface charge of DoGo310 lipid nanoparticle were determined by MTS method,dynamic light scattering as well as transmission electronic microscope(TEM),respectively.Combing laser scanning confocal microscope analysis and fluorescence quantitative PCR analysis,the neuronal cell targeted si RNA delivery efficiency of DoGo310 was measured.The results of cytotoxicity assay showed that more than 70%of the cells survived at a concentration of 120?g/m L.Analysis by laser particle size and transmission electronic microscope showed that DoGo310 lipid nanoparticles has a uniform nanoparticle size.The electromotive force results showed that the zeta potential of DoGo310 particles was about 30 m V,which means it would be stable in solution.The results of laser scanning confocal microscope analysis and fluorescence quantitative PCR showed that DoGo310 lipid nanoparticles can efficiently deliver si RNA to both BV2 cell line and primary mouse microglia cell,induce RNA interference,and silence the expression of endogenous gene.And no apparent cytotoxicity to neuron cell was observed.Taken together,DoGo310 lipid nanoparticles have a better ability to encapsulate and release nucleic acids and neuronal cell targeting ability,and would be a potential non-viral nanocarrier for gene delivery in vivo.This study also provides a theoretical basis for further research and development of central nervous system(CNS)targeted carrier for nucleic acids based therapeutics.