Research Of A Environment-responsive Self-assembled Dendrimer Nanocarrier For Gene Delivery

Gene therapy plays an important role in tumor therapy.It is essential to develop effective gene carriers to achieve the precise delivery and controlled release of genes,improve the therapeutic effect of gene therapy,and reduce toxicity and side effects.Dendrimers have regular molecular structure and controllable nano size.The abundant functional groups on the surface provide them with the advantages of easy modifications.Cucurbituril,a common host in supramolecular chemistry,has the advantages of low toxicity and good biocompatibility.With its hydrophobic internal cavity,it can self-assembly form a stable host-guest structure together with two specific groups through noncovalent interaction.In this thesis,we used cucurbit[8]uril(CB[8])as the supramolecular host,dendron-naphthol(DN)unit and viologen-PEG(VP)unit as the guests,and prepared a PEG-encapsulated,self-assembled dendrimeric and microenvironment-responsive nanocarrier named DNCVP(DendronNaphthol ?CB[8]?Viologen-PEG).The DNCVP can electrostatically adsorb nucleic acid to form a nanosystem and realize tumor targeted delivery of genes.First,DN unit and VP unit were obtained by multi-step reactions.The structures of all the compounds were confirmed by 1H NMR and FT-IR.The self-assemble of DN,VP and CB[8]was confirmed by UV titration,fluorescence titration and job’s experiment.The molar ratio of the three parts was proved to be 1:1:1,and the binding constants were calculated to be 7.71 × 103,4.04 × 103.Subsequently,the gene loading capacity of the carrier was evaluated by agarose gel electrophoresis.The particle size and Zeta potential of the nanosy stem were measured by DLS.The morphology of the nanosystem was determined by TEM,and the serum stability of the carrier was clarified after incubating with bovine serum albumin.Moreover,the changes of the nucleic acid bounding N/P,particle size,Zeta potential,TEM morphology and protein adsorption rate of the materials after the stimulation of acidic and reductive environments indicated the pH and redox dualresponse ability of DNCVP,and proved that PEG can effectively improve the stability of the carrier.Next,we selected two cell lines,the human embryo kidney cell HEK 293 and the mouse melanoma cell B16F10 to evaluate the cytotoxicity of the carrier by MTT.The way and the efficiency of cellular uptake of DNCVP/siRNA were evaluated by confocal laser scanning microscope and flow cytometry.The transfection efficiency of DNCVP/pEGFP in cells was evaluated by green fluorescent protein transfection experiment,and the transfection efficiency of DNCVP/pGL3 was further quantified by firefly luciferase transfection experiment,revealing the optimal N/P of 20.We used siGAPDH as model siRNA and evaluated the gene silencing efficiency of DNCVP/siGAPDH by Western Blotting(WB).We also verified that PEG could reduce the cytotoxicity of the carrier.Therefore,DNCVP is a gene carrier with low toxicity and high transfection efficiency in cellular level.Finally,we evaluated the tumor targeting and gene delevery efficiency of the carrier system in vivo.We constructed a mouse melanoma model and took live fluorescence in different time piont after tail vein injection.We evaluated the fluorescence of organs and tumor tissue 24 hours after injection,and observed the fluorescence of frozen sections of tumors to investigate the distribution of DNCVP/Cy5-siRNA in vivo and prove the tumor targeting ability.After 48 hours of tail vein injection,the expression of luciferase in various organs and tumor tissues was measured.The distribution and transfection efficiency of the system in vivo were quantified.In vivo experiments further proved that DNCVP was a gene carrier with good biocompatibility,high transfection efficiency,environmental responsive ability to achieve tumor targeting gene delivery.