Nano-gelatin Siloxane Modified With A Membrane Peptide Loading P53 Gene Inhibit The Hepatocellular Carcinoma

At present,the greatest challenge of gene therapy is still the efficient,safe gene carrier and the choice of gene therapy.Gelatin-siloxane nanomaterials(GS NPs)have the advantages of low toxicity,surface modification,and easy repeatability.In addition,it is a potential gene carrier material because its surface has a large amount of positive charge to mediate the exogenous gene into the cell and transfection.As a non-viral gene carrier,the main limiting factor is low transfection efficiency.In this project,the cell transmembrane peptide(Tat)is modified to GS NPs surface to improve transfection efficiency.Tat is a polypeptide(KYGRRRQRRKKRGC)derived from a sequence of 48 to 62 amino acids from the human immune deficiency virus 1(HIV-1)protein,containing the nucleation sequence which can promote protein and DNA into the nuclear region,and increase the efficiency of various materials.In addition,p53,as a tumor suppressor gene,is related to cell cycle,apoptosis and differentiation,which plays a monitoring role in normal conditions and participates in the response of cells to DNA damage.Therefore,the main research direction of this subject is the synthesis of GS NPs by sol-gel method,modified by Tat,and the inhibitory effect of p53 gene on liver cancer.Methods: 1)The gelatin-siloxane nanomaterials were prepared and modified by Tat.FE-SEM,TEM,DLS,Zeta potential,FTIR,TG were used to analyse physicochemical properties of the nanoparticles,such as morphology,diameter,surface potential,structure composition and so on.2)Using gel electrophoresis to detect the the encapsulation rate,release and stability of the compounds of GS and Tat-GS for p EGFP-C1-p53(p DNA)and to select the most appropriate load ratio of the cell by particle size and potential analysis.3)Evaluating the biological description of the two nanoparticles;the feasibility of the GS and Tat-GS as gene carriers were evaluated through the fluorescence observation of transfection in vitro;and discussing the entry of nanoparticles into cell and escape of lysosomes.4)MTT assay was used to detect the inhibitory effect of GS and tat-GS with p53 on liver cancer.Hoechst 33258 apoptotic cell staining and Western blot were used to study the tumor suppressive mechanism.Results: 1)The prepared gelatin-siloxane nanoparticles and modified by Tat(Tat-GS)were irregular balls,and the Tat-GS showed relatively agglomeration,with an average particle size of 289 nm compared with GS at 250 nm.The surface potential of GS and Tat-GS was 38 m V and 35 m V respectively.2)The encapsulation rate of p DNA was reduced after Tat modification,and Tat-GS/p53 can be fully wrapped at 100 ∶ 1,GS/p53 can be fully wrapped in 30 ∶ 1.However,the modification of Tat had little effect on its release and stability.Furthermore,through particle size and potential analysis,it was shown that the particle size of the complex is smaller at 200∶1.The surface shows a positive potential,which is beneficial to the cell uptake of cells,so the subsequent nanometer complex is loaded with 200∶1 ratio.3)Toxicity experiments show that GS had good biocompatibility,and decorated by Tat almost with no toxicity.The biological safety of Tat-GS was higher,and the p DNA transfection experiments showed Tat-GS had high transfection efficiency,its reason is Tat peptide as a kind of wear membrane,having the ability to enhance the carrier compounds into cells and the ability to escape the lysosome.4)Tat-GS-p53 had a stronger anticancer effect on liver cancer cells,and its tumor suppressive effect may be mediated by the expression of p53 protein,which may cause apoptosis.