Effect Of Amino Groups On The Transfection Efficiency Of Cationic Lipid Molecules Into Neuronal Cells

RNA interference(RNAi)is an RNA-dependent gene silencing process,controlled by the RNA-induced silencing complex(RISC).When endogenous or exogenous double-stranded RNA(ds RNA)is introduced into a cell,the RNAi mechanism degrades the RNA target of the ds RNA in a sequence-specific manner,there by achieving gene silencing.Among them,small interfering RNA(siRNA)and micro RNA(miRNA)have attracted much attention and have become targets for drug research.The main difference between siRNA and miRNA is that the former has a high degree of sequence specificity,while the latter has multiple targets.Due to the high efficiency of siRNA,a small amount of siRNA can degrade the target m RNA in the cell to complete the gene silencing mechanism.Therefore,siRNA is the most widely used in the development of RNAi drugs.In addition,siRNA sequence design is simple,and its immunogenicity can be significantly reduced through chemical modification.Therefore,siRNA,as a drug,has many advantages over small molecule drugs and protein drugs,and its clinical application potential is huge.However,the delivery of siRNA in vivo faces many obstacles,such as large molecules and strong negative charges,extremely poor stability,and easy degradation by nucleases in the blood.Therefore,the key to developing siRNA drugs is how to safely and effectively deliver siRNA into target cells.Efficient carrier can protect siRNA drugs from nuclease attack,and can also greatly promote the uptake of siRNA drugs by tissue cells.Among the various types of carriers currently developed,lipid nanospheres have the most potential.The hydrophilic positively charged head structure of lipid molecule is a key factor that determines the efficiency of lipid molecule to carry and deliver siRNA.This paper mainly discusses the effect of four new cationic lipid molecules prepared by adjusting the amino group arrangement on the efficiency of transfecting nerve cells.First,using Boc-L-glutamic acid and oleylamine as basic raw materials,hydrophobic tails Do Go1 and Do Go2 were synthesized through amide reaction.The hydrophilic head uses lysine as a raw material,and four cationic lipid molecules were synthesized by adjusting the arrangement of amino groups and the number of amino groups,and they were named N3,N4,N5,and N6,respectively.The molecular weight and structure of the compound were determined by the characterization methods of mass spectrometry(MS)and nuclear magnetic resonance(1HNMR).Secondly,the particle size,uniformity and stability of cationic lipid molecules combined with siRNA were measured by laser particle size and electromotive potential.Through agarose gel electrophoresis,cytotoxicity,transfection of GFP plasmid and other experiments to explore the biological activities of the four cationic lipid molecules,and the high transfection efficiency synthesized with the commercial transfection reagent Lipofectamine 2000(L2K)and our laboratory the low-toxicity Do Go4 cationic lipid molecules were compared.Experimental results show that all four lipid molecules can bind to DNA,and their cytotoxicity is greater than Do Go4 and less than L2 K.Among them,the N3 lipid molecule is the most efficient for transfecting GFP plasmid on nerve cells.Finally,the screened N3 was transfected with siRNA(FITC-siRNA,si TLP4)on microglia(BV2)to determine whether the N3 lipid molecule has the ability to deliver siRNA safely and effectively.The experimental results show that the above four cationic lipid molecules are within the range of effective gene delivery,the cytotoxicity is lower than that of L2 K,and the efficiency of plasmid transfection on nerve cells is higher than of L2 K and Do Go4.Through the analysis of experimental data,it can be found that the rule is: as the relative molecular weight of the cationic lipid molecule increases,the particle size after being combined with siRNA also increases;when the hydrophilic head has a branched structure,the increasing its electromotive force and transfection(GFP,siRNA)efficiency will decrease;when the number of modified groups is the same,the transfection(GFP,siRNA)efficiency will increase as the branch structure increases.In summary,four cationic lipid molecules were synthesized by adjusting the arrangement of amino groups in the hydrophilic head and the number of amino groups,and their structures and molecular weights were determined by 1HNMR and MS.Through the measurement of laser particle size and electromotive potential,it is determined that the four lipid molecules are within the effective delivery range of gene carriers.The biological activity of the microglial cell line(BV2)and neural cell line(N2a)was discussed through cytotoxicity and transfection experiments.This thesis provides the basis and basis for the rational design of siRNA carriers based on cationic lipid molecules.