Construction And Evaluation Of Multilevel Microenvironment "Adaptive" Gene Delivery System

After the central principle was put forward,it is recognized that the root cause of the disease is the harmful defect or mutation of the gene,and gene therapy came into being.However,due to the defect that the gene itself is easily degraded,a vector is needed to carry the gene for treatment.Although viral vectors have extremely high gene transfection efficiency,their immunogenicity is fatal,and foreign genes may be permanently integrated into the host cell chromosomes,and there is a potential risk of cancer induction.In recent years,with the development of nano medical technology,since non viral vectors are convenient for functional modification to achieve ideal results in the process of gene delivery,they have become a hot spot in current research.In this study,aiming at the multi-level physiological barriers faced by non-viral vectors in the process of gene delivery,a gene delivery system with multi-level responsiveness was designed and synthesized.Within the new gene delivery system,polylysine(PLys)is as the core,disulfide bond(S-S)cross-links and bind genes,temperature-sensitive intermediate layer polypropylacrylamide(PNIPAM)protects genes,polyethylene glycol(PEG)shell prolongs blood circulation time,active targeting(RGD)increases tumor enrichment,and matrix metalloproteinase 2(MMP-2)cleave short peptide(GPLGVRG)de PEGylation to increase endocytosis and intracellular transport efficiency.This study provides a feasible strategy for gene therapy,the research content is as follows:1.Synthesis and characterization of cationic carrierMultiple gene delivery vectors PEG-PLys-SH,PEG-GPLGVRG-PLys-SH,RGD-PEG-GPLGVRG-PLys-SH,PNIPAM-PLys-SH were synthesized by synthetic methods such as amide reaction,click reaction and ring-opening polymerization reaction.2.Preparation and physical and chemical properties of vector/gene complexThe agarose gel electrophoresis experiment and heparin competition experiment verified the ability and stability of the vector to bind genes;the particle size of each vector/gene complex was between 80-90 nm determined by dynamic light scattering(DLS),and the potential of the complexes was between 3-5 m V;MTT cytotoxicity experiments have confirmed that the cell survival rate is high,demonstrating that the carrier has good biocompatibility.3.Microenvironmental responsiveness of vectors and vector/gene complexes and gene transfection of vector/gene complexes at all levelsThe agarose gel electrophoresis experiment and heparin competition experiment verified the responsiveness of the vector/gene complexes to intracellular reduction environment;nuclease protection,DLS,transmission electron microscopy(TEM),proton nuclear magnetic resonance spectroscopy(~1H NMR)verified the temperature response of the temperature-sensitive complex.With the rise of temperature from room temperature to physiological temperature,the intermediate barrier changes from hydrophilic to hydrophobic;laser confocal microscopy(CLSM)and date from flow cytometry(FCM)demonstrated that the introduction of RGD,an active targeting group,-can increased the uptake efficiency of vector/gene complexes in cells by about 3 times;gel permeation chromatography(GPC),DLS,CLSM,FCM,lactate dehydrogenase(LDH)activity experiments displayed the performance of MMP in response to de PEGylation.After de PEGylation,the molecular weight of the carrier changes significantly,the potential of vector/gene complexes increases,the uptake capacity in cells was increased by about 6 times,and the lysosomal escape speed was accelerated;the gene transfection ability of the vector/gene complexes was observed by the inverted fluorescence microscope,and the introduction of active targeting RGD and de PEGylation strategies all improved the gene transfection ability of vector/gene complexes.4.Tumor suppression ability of temperature-sensitive vector/gene complex combined with sorafenib in vivoIn vivo tumor suppression experiments of tumor-bearing mice,it was proved that compared with the only gene or drug treatment groups,the tumor volume in the combined treatment group increased the most slowly and the tumor suppression effect was the most obvious.Hematoxylin-eosin(HE)staining showed that all the treatment groups had no obvious toxic and side effects on the main organs of mice.Immunohistochemistry of VEGF confirmed the antitumor effect of the therapeutic gene by inhibiting expression ofvascular endothelial growth factor(VEGF).