Preparation And Characterization Of Folate-coupled Chitosan Mediated Gene Survivin ShRNA Nano-carrier

Objective:Preparation of folic acid-chitosan nanoparticles,Study of the feasibility of it as a recombinant survivin shRNA plasmid vector delivery system,as well as the transfection efficiency to colorectal cancer cells(SW480),and Colorectal cancer for the study of RNAi-mediated gene therapy to lay the groundwork.Methods:Folate-coupled chitosan was prepared by the reaction of the activated folate ester with the amine group on the chitosan.The compound of folate-coupled chitosan grafted on fluorescein isothiocyanate(FITC) by chemical bond was prepared.Then FITC-labelled nanoparticles were prepared by ionically cross-linking method,and reaction with HepG2 and cells in vitro experiments.Will 20μg/mL survivin shRNA recombinant plasmid and 10μg/mL folic acidchitosan hybrid to preparation the gene complex nanocomposites.At the same time to cationic liposome gene complexes as a control.Both the above-mentioned transfected colorectal cancer cells,determination of its transfection efficiency;Western blotting detection of tumor cells after transfection of survivin protein expression.Results:The test results show that the amount of the activated folate ester used,the speed of adding agent and the reaction temperature were the main factors influencing the coupling ratio.The test results show that the amount of the activated folate ester used,the speed of adding agent and the reaction temperature were the main factors influencing the coupling ratio.Folate-coupled chitosan was stabilize coupled when the ratio of the amounts of the active folate ester to chitosan used was 1:1, the speed of adding agent was 2 mL/min,the reation temperature was 30℃and the reaction time was 12h.The average diameter of the nanoparticles was 198 nm.The Folate-coupled Chitosan Nanoparticles was form regulation under atomic force microscope,Fluorescent cells results was well.Preparation of folic acid-chitosan-mediated survivin shRNA gene recombinant plasmid nanocomposites Successfully.The effect of transfected to colorectal cancer cells,Folate-coupled Chitosan gene complex nanoparticles is stronger than cationic liposome gene complex.After transfection to colorectal cancer cells,siurvivin protein expression of Folate-coupled Chitosan gene complex nanoparticles was significantly lower than cationic liposome gene complex.Conclusion:Use Ion cross-linking method,and optimize the process conditions,preparating the small particle size,distribution of folic acid with fluorescent-coupled chitosan nanoparticles successfully.The surface of the nanoparticles with positive charge can be combined with plasmid DNA,assembled chitosan nano-coupled folate transporter system genes. Through in vitro gene transfection experiments,confirmed that folic acid -chitosan nanoparticles as a vector system capable of survivin shRNA recombinant plasmid delivery to liver cells,high-performance,low toxicity and is a highly efficient nano-gene vector,and Our research for the application of nanoparticles to achieve tumor gene therapy has laid a sound scientific basis.