Study Of GSH-response Hyperbranched Poly(amido Amine)for Co-delivering Drug/gene And Its Tumor Microenvironmental Responsiveness

The high expression of glutathione(GSH)is one of the important features of tumor tissue cells that distinguish them from normal tissue cells.In recent years,tumor therapy which designed and developed for this characteristic has gradually attracted attention.The GSH-responsive polymer refers to a class of polymers that can respond and degrade in the presence of GSH in a reducing environment.Hyperbranched poly(amido amine)(HPAA),which contains a large number of disulfide bonds in the structure,is a very important one in this type of polymers.It has many advantages,such as excellent biocompatibility,easy functionalization,good water solubility and so on,so it has been widely studied in the biomedical field.On the one hand,the hyperbranched structure,a large number of functionally modifiable amino groups and abundant surface charge of HPAA provide excellent conditions for its covalent attachment with anticancer drugs and co-delivery genes,it can be rapidly degraded in tumor cells with high GSH expression to achieve efficient drugs and genes release,and thus can be used as a co-delivery carrier for the co-loading and release of drugs and genes.On the other hand,it will consume a large amount of GSH which was over-expressed in tumor cells during the degradation process,thus affecting the GSH-mediated multidrug resistance process,so it can be used alone as a potential anticancer drug,and has a strong inhibitory effect on tumor cells that have developed resistance.Based on these,the thesis aims at the characteristics of high expression of GSH in tumor cells,using HPAA as the main body to construct a novel drug/gene co-delivery vector with GSH response performance,and exploring the potential application and mechanism of action of HPAA as an anticancer drug.The specific research content is mainly divided into the following two aspects:1.GSH-responsive hyperbranched poly(amido amine)prodrugwith self-sensitizing effect for gene co-deliveryPolymeric prodrugs are favored in the field of drug carriers due to their large drug loading and effective avoidance of drug leakage,but their efficacy is often inferior to the original drugs.To solve this problem,gene therapy and sensitization therapy are often used as the supplementary means to improve efficacy.The combination of chemotherapy,gene therapy,and sensitization therapy will undoubtedly be a powerful strategy for greatly improving the therapeutic effect.However,the efficient and simple combination of the three is still an urgent problem to be solved in the current cancer treatment.In this study,a GSH-responsive cationic hyperbranched poly(amido amine)(HPAA)was prepared by Michael addition reaction,then the loading of the anticancer drug methotrexate(MTX)and functional plasmid(MMP-9 shRNA)were achieved through covalent attachment and electrostatic adsorption,respectively.Its chemical structure and the drug release were characterized by ~1H NMR and ultraviolet spectrophotometer.The redox response performance was characterized by gel permeation chromatography and dynamic light scattering.The gene loading and release were evaluated by gel electrophoresis assay,transmission electron microscopy and laser confocal microscopy.The transfection of the loaded gene in tumor cells was evaluated by transfection assay in vitro.The effect of self-sensitization chemotherapy was observed by cytotoxicity assay,cell cycle assay and intracellular GSH concentration test in vitro.At the same time,cell scratching experiments,transwell experiments,and western blotting experiments were performed to evaluate the effect of the gene loaded on inhibiting tumor cell migration.And in vitro and in vivo anti-tumor experiments were evaluated the jointly suppress tumor efficacy in vitro and in vivo.The experimental results showed that it could efficiently load drugs and genes and could respond quickly and degrade in a reducing environment.It could significantly reduce the GSH concentration in the tumor cells,and thus increased the sensitivity of tumor cells to drugs,thereby enhancing the efficacy.At the same time,in vivo and in vitro experiments have confirmed that it could effectively inhibit the tumor and had good biocompatibility.The above results indicate that HPAA-MTX as an excellent carrier with self-sensitizing effects has great potential for the co-delivery of drugs and genes.2.GSH-responsive hyperbranched polyamidoamines used to reverse multidrug resistance in tumorsThe multidrug resistance of tumors is undoubtedly one of the serious difficulties faced by current clinical cancer treatment.Studies have shown that intracellular overexpression of GSH is one of the key factors in the induction of multidrug resistance in tumor cells.Recently,as a commonly used drug/gene delivery vector,some of the inherent pharmacological activities of polymers have also been gradually discovered and studied,and they are considered as drugs for direct use in anti-tumor therapy.Based on the previous part of the work,we used the HPAA,which contains abundant disulfide bonds and can consume a large amount of GSH,to study the inhibitory effects of tumor cells that have developed resistance,and to explore the mechanism of action.The experimental results showed that the polymer had a selective effect on the inhibition of cells,which could significantly inhibit the activity of drug-resistant tumor cells,but had no effect on normal cells at the same concentration,and thus could successfully reverse the multidrug resistance of tumors.Subsequently,tests of intracellular GSH concentrations showed that GSH levels in drug-resistant tumor cells were significantly higher than those in sensitive tumor cells and normal tissue cells,and were significantly reduced after treatment with HPAA.Further tests of intracellular reactive oxygen species(ROS)concentrations showed that after treatment with HPAA,the intracellular ROS concentration was reduced as much as GSH,and the dynamic physiological balance inside cells was broken,resulting in cell damage and death.This study further expands the scope of application of synthetic polymers in the biomedical field and provides new ideas for solving the clinical problems of tumor resistance.