Construction Of Biodegradable Stearylated Peptide For SiRNA Delivery And Its Evaluation In Vitro And In Vivo

Gene therapy(gene therapy) means that the exogenous gene(RNA or pDNA) is lead into specific cells, to correct for genetic abnormalities or compensate for the gene defect caused by various kinds of abnormal, to treat the disease. At present, gene therapy technology in the clinical is not mature, and can‘t be used in clinical. Exogenous genes can’t take the initiative to enter the cell, and the auxiliary carrier must be needed to help the exogenous gene enter into the cells. The biggest obstacles facing the clinical application of gene therapy is the lack of a proper gene vector(gene vector). Effectivity and safety is the two basic requirements. Therefore the development of a novel gene vector is very significant.The subject bases on the principle of cell penetrating peptide transmembrane transport, combines the relevant research reports, and designs a biodegradable polypeptide gene vector SHRss, which composed of stearic acylating arginine, histidine, cysteine polypeptide bridged by disulfide. The vitro and vivo evaluation results show that, the siRNA carrier has high delivery efficiency, while maintaining a low cytotoxicity.the first part of subject is to synthesis the Polypeptide H3CR5 C using the method of solid phase peptide synthesis, and stearyl groups is attached to the peptide with the same methods to get stearyl-H3CR5 C. HPLC is used to purify the compound, and the purity of polypeptide and stearyl peptide tested by HPLC is above 95%.HPLC-MS is used to identified polypeptide and stearic acylated peptide. Using low concentration hydrogen peroxide oxidation method to oxidize the thiol into disulfide linkage to get the SHRss, and the cysteine is used to control the bridging degree.H-NMR and GPC shows the SHRss synthesized.The second part of this topic was to evaluate biodegradable polypeptide gene vector SHRss in vitro. SHRss/siRNA nanocomplexs in the N/P=10 show the smallest particle size, in the range of 150-300 nm, potential between 25-40 mV, nanocomplexs were observed by transmission electron microscopy of the spherical approximation, the integrity of the structure. SHRss2 can be completely package siRNA in the N/P=5, and increase the stability of siRNA in serum. Luc-Hela cells have a very high cellular uptake of SHRss/siRNA nanocomplexs. SHRss/siRNA nanocomplexs showed higher siRNA delivery efficiency in Luc-Hela cells and mCherry-HEK293 cells. The research results show that the mechanisms of endocytosis, the endocytosis pathway of SHRss/si RNA nanocomplexs were mainly for clathrin-mediated endocytosis and caveolae-mediated endocytosis. SHRss/Cy3-siRNA nanocomplexs can be successful escape from endosomes in endocytosis and uniformly distributed in the cytoplasm through Microscope confocal laser. The cytotoxicity of SHRss was less than Lipofectamine2000 and PEI 25 kDa,and has a good biocompatibility, the introduction of the two disulfide bond is not significantly increased the toxicity of the carrier.The third part of this subject is to evaluate the delivery of siRNA in nude mice using SHRss2, which is the best biodegradable polypeptide gene carrier of four kinds. The results show that SHRss2/siRNA nanocomposites can be more accumulated in tumor tissues in nude mice from the tail vein injection. SHRss2/siRNA nano composite material have relatively strong interference on the expression of genes related to the tumor.