Preparation And Transfection Mechanism Study Of Novel Cationic Peptide Lipid Gene Delivery System

Gene therapy is a biomedical technology which delivers the correct target gene to cells for expressing and treats diseases caused by genetic variation and gene defects.Gene therapy has great potential and good prospect in cancer and genetic disease,and has aroused extensive attention of researchers.The main research of gene therapy is to find a gene transfection vector which has high efficiency,low toxicity and can introduce gene drugs into cells and works steadily.Viral gene vector and non-viral gene vector are the main vectors used in gene therapy.Viral gene vectors include retroviruses,adenoviruses and adenoviruses,etc.,which have high transfection efficiency,but low safety and immune response.Non-viral vectors include cationic polymers,cationic polypeptides and cationic liposomes,which have attracted more and more attention due to their safety,good biocompatibility and easy access.Cationic telopeptide lipids are the most popular gene vectors among the non-viral gene vectors.Amino acids and peptides get the attention of researcher because of its immune regulation,antithrombotic function,antioxidant.Using amino acid or peptides as the basic framework of gene carrier or modifying gene carrier can promote the lysosomal escape of gene carrier and obtain high transfection efficiency.Polyamide-amine(PAMAM)is a widely used dendritic macromolecular polymer,whose terminal amino group has positively charged by protonation and can form complex with DNA.Its terminal amino group is protonated with a positive charge,which can form stable complexes with DNA,avoiding degradation by enzymes.Using PAMAM as the head of the end-state lipid can has good water solubility,high transfection rate and low cytotoxicity.There are many barriers in the gene transfer process,such as easy to be found in large amounts of proteins and enzymes in the blood,the material degradation,and the influence of the cellular structure of the biofilm cells absorb,and inside the soluble enzyme degradation,thus can not be effectively transferred to the nucleus,so the gene transfer mechanism of gene carrier need to be study.Based on the natural products,such as lysine,tartaric acid and so on,which has good biocompatibility,biodegradability,high cell targeting property,low cytotoxicity and high security,this study prepared 1generation polyamide-amine modified by lysine as the cationic end peptide lipid gene carrier of the head,and studied the gene transfer mechanism of it.Main contents of this study:(1)The synthesis and structural characterization of cationic terminal peptide lipids:The cationic end-state lipids are composed by four steps.Firstly,dodecyl chloride reacts with tartaric acid to produce dilauryl tartaric anhydride,and then lysine and 1G-PAMAM protected by ditert butyl dicarbonate react with dilauryl tartaric anhydride,the acid anhydride bond is broken,and the target product is obtained after acidification.Finally,the structure of the synthesized compound was determined by IR and~1H-NMR to confirme the cationic terminal ground state lipids to be synthesized.(2)The preparation and characterization of cationic liposomes/DNA:Cationic liposomes were synthesized by using the synthesized cationic end ground-state lipids and co-lipids DOPE.The ability of compress DNA was investigated by agarose gel block experiment.The particle size and potential of the cationic liposome were determined by Zetas izer Nano Series 90 particle size analyzer.The results showed that the synthesized cationic terminal ground state lipids could completely compress the DNA,and had the appropriate particle size and potential for transfection.(3)The cytotoxicity of cationic terminal ground lipids and their DNA complexes:The cytotoxicity of different concentrations of cationic liposomes,different cell type and different action time were investigated by MTT assay.The results showed that with the increase of cationic liposome concentration and action time,cytotoxicity also increased,without cell type dependence.The cytotoxicity of the complex increased with the increase of weight ratio.(4)The study on the gene transfer mechanism of cationic terminal ground state lipids:Cell apoptosis method was used to determine whether cationic liposomes could induce apoptosis at a certain concentration,and the relationship between the efficiency of apoptosis and the concentration and action time of liposomes..The effects of different cationic liposomes/DNA weight ratios,different cell type,serum,temperature and cell uptake inhibitors on the transfection efficiency were investigated by inverted fluorescence microscope and cell flow meter.Different cell uptake pathway inhibitors were added to study the cell entry mode of cationic terminal ground-state lipids,and the nuclei,lysosomes and DNA were stained and labeled.The distribution of DNA in cells was observed by confocal laser microscopy.It was found that the liposome could induce tumor cell apoptosis to a certain extent,but the effect was not obvious.The major endocytotic pathway of the cationic liposome into cells is the small nest protein pathway.