Construction And Characterization Of Photoresponse Biomimetic Nano Gene Delivery Vector

The rapid development of gene therapy has brought hopes of cure for various human diseases,and has become the most promising treatment in the field of cancer treatment.However,how to aggregate the therapeutic gene into the target organ for specific expression so as to improve the therapeutic effect and reduce the off-target effect and other side effects has always been the most important factor restricting the clinical application of gene therapy.Therefore,it is of great significance to design and prepare efficient gene delivery carriers to achieve targeted delivery and effective expression of therapeutic genes.In the previous study,Our research group has simulated the structural characteristics of viral gene delivery vectors and constructed a biomimetic nano-vesicle gene delivery carrier based on polysaccharide.The biomimetic gene delivery vector not only can effectively prevent the removal of the reticuloendothelial system in vivo,but also can be enriched in the tumor tissue,and can be efficiently taken up by the tumor cells,thereby significantly improving the transfection efficiency of the therapeutic gene in vivo.However,how to achieve the effective expression of the therapeutic gene has not been solved.Based on this,this paper introduces a photothermal agent Au in the structure of biomimetic polysaccharide nano-vesicle gene transfer carrier,and designs and synthesizes a photoresponsive high biocompatible biomimetic nano-vesicle HTCP-Au,which is used to realize the controlled gene therapy of tumor.The research content of this paper mainly includes the following aspects:?1?Synthesis and characterization of HTCP-Au in vitro.First,Hyaluronic acid?HA?was successively connected with 12-diaminododecanoic acid and L-cysteine?L-Cys?,which was constructed by the amide bond.Then,the polyethyleneimine?PEI?was connected to the end of HA-C₁₂-Cys to get HTCP.Finally,HTCP-Au was constructed by combining Au-S bond with Au and sulfhydryl group of L-Cys.The structure of the carrier was characterized by nuclear magnetic resonance hydrogen spectrum,infrared Spectrometer,and elemental analysis.The morphological characterization was performed by transmission electron microscopy,dynamic laser light scattering and circular dichroism.The experimental results show that HTCP-Au is successfully constructed.In addition,DNA gel retardation experiments show that HTCP-Au has nucleic acid loading capacity and photothermal conversion experiment showes that HTCP-Au has the ability of photodynamic response in vitro.?2?Cell experiment in vitro.The breast cancer cell lines MCF-7 and 4T1 were used as cell models to investigate the biocompatibility,cell uptake,cell transfection and photothermal conversion performance of HTCP-Au.The Cell proliferation rate,cell cycle and apoptosis results showed that HTCP-Au and HTCP-Au/DNA had no obvious cytotoxicity.The results of flow cytometry results showed that the cells uptake rate of HTCP-Au and HTCP-Au/DNA reached more than 95%at 6 h,and the uptake of cells was related to the endocytosis mediated by clathrin.Photothermal conversion experiments showed that irradiation of HTCP-Au in breast cancer cells with 808 nm near-infrared light can significantly increase cell temperature,which further indicates that the HTCP-Au vector is photoresponsive.?3?Animal experiment in vivo.BLAB/c mice bearing S180 ascites tumor were used as models to investigate the histocompatibility,distribution and photothermal conversion activity of the photoresponse gene delivery vector in vivo.In vitro,hemolysis experiment and erythrocyte coagulation experiment showed that the hemolysis rate of htcp-au/DNA in vivo was less than 5%,and no obvious cell aggregation was observed,showing good blood compatibility.Based on the biochemical indexes of mice's blood and liver function,the changes of body weight,organ coefficient,pathological tissue section and inductively coupled plasma mass spectrometry,the experimental results showed that HTCP-Au had good biocompatibility and no obvious toxicity in vivo.Tissue distribution in vivo and in vivo imaging experiments in mice showed that HTCP-Au/DNA accumulates mainly in liver and tumor tissues,and has tumor targeting.