Synthesis And Release In Vitro Of Responsive Nano Gene Vector Based On Tumor Microenvironment

In recent years,with the improvement of material life and medical level,the incidence rate and mortality rate of cancer also increase year by year.This is a serious threat to people's health.In modern medicine,surgery,radiotherapy and chemotherapy,as common medical means,are limited in clinical use due to the existence of toxic and side effects or tumor recurrence.The in-depth research in the field of molecular biology has led to the discovery that the essence of tumor is the over-expression of gene variation at the cellular level,which provides the theoretical basis for researchers to completely treat tumors from the root by introducing foreign normal genes to repair or inhibit mutant genes.However,a single exogenous gene can not break through the inhibition of the environment inside and outside the cell and be directly applied,so it is necessary to load,protect and transport it with a certain vector.Due to the high permeability and retention effect of tumor tissue,nanoparticles can be enriched in them,which has certain advantages in gene delivery.However,for the complex microenvironment of tumor,there are still many problems when nano carriers enter into tumor cells,such as unable to be endocytosed,difficult to escape lysosomes,unable to release effectively in cytoplasm and difficult to be transfected into nucleus.To a certain extent,it affects the application of nano carrier in gene therapy.In order to solve these problems,we designed different pH responsive nanocarriers based on the obvious pH difference between tumor tissues by external chemical modification on the basis of gold nanocarriers,and introduced nuclear targeted dexamethasone and biocompatible chitosan to improve the characteristics of the vectors,so as to overcome the problems of uptake between tissues and targeted release in cells,transfection and expression.The specific work mainly includes the following two parts(1)Based on the low pH between tumor tissues and the targeting of dexamethasone through the nuclear pore,the pH responsive gold nanoparticles gold-polyethylenimine-dichloroacrylate aminoethyl-dimethylmaleic anhydride(Au-PEI-2-ClAEA-DMMA)modified by aminoethyl dichloroacrylate and PEI-Dexa obtained by coupling PEI with dexamethasone.Ternary composite carrier DNA / PEI-Dexa / Au-PEI-2-ClAEA-DMMA was prepared by electrostatic interaction between positive PEI-Dexa loaded DNA and gold nanocarriers.Under acidic conditions between tumor tissues,the outer carrier Au-PEI-2-ClAEA-DMMA underwent charge reversal,resulting in charge exclusion,DNA / PEI-Dexa dissociated,endocytosis into cells,and pei-dexa targeted DNA into the nucleus for expression.The particle size and surface potential of the carrier were measured by potentiometric particle size analyzer to verify its stability under physiological conditions,and the best mass ratio and charge reversal ability of carrier pH response at pH = 6.0were explored.The loading capacity of the composite carrier to DNA was verified by gel electrophoresis,and the DNA loading and releasing ability of the tumor microenvironment carrier was simulated under acidic conditions.The cytotoxicity test proved that the carrier polymer must have low toxicity.In the transfection experiment,confocal microscopy showed that the vector could be well absorbed and transfected by cells.Compared with the control group,the introduction of PEI-Dexa could better promote DNA transfection,and the distribution of fluorescent protein in the nucleus was more.Therefore,the idea of PEI-Dexa /Au-PEI-2-ClAEA-DMMA vector design is verified,which has the ability of gene loading,and can intelligently respond to the complex environment to obtain the effect of DNA release in vitro,which has a certain potential in vector design and application.(2)Based on the pH difference between tumor tissues and tumor cells,Au-PEI-DMAEA and carboxylated chitosan(CCS)modified by dimethylaminoethyl acrylate were designed.Gold nanocarriers with positive charge can be combined with DNA and coated with biocompatible chitosan to obtain ternary composite carrier DNA / Au-PEI-DMAEA / CCS.When the carrier was enriched in the intercellular tissue,the outer layer of chitosan was hydrolyzed under acidic conditions,and the inner layer of carrier was exposed by endocytosis.When the carrier entered into the weak alkaline environment of pH = 7.4 in tumor cells,the hydrolysis charge repulsion of gold nano carrier occurred,and the release of DNA was achieved.The particle size analyzer and transmission electron microscope were used to explore the composite and stability of the carrier,and the charge reversal performance of the two carriers was verified under zeta potential.Gel electrophoresis experiments were carried out to investigate the loading capacity of the vector to DNA,and the release of DNA was simulated under the gradient of pH 7.4-6.5-7.4.Compared with the control group,the cytotoxicity test verified the low toxicity of chitosan inclusion,which met the requirements of cell uptake.In the transfection experiment,the fluorescent protein of the chitosan inclusion was much more than that of the control group under the confocal microscope,and the high transfection efficiency of the vector was quantitatively demonstrated under the BCA experiment.Therefore,the composite carrier can achieve the effect of gradual response release by using the pH difference inside and outside the cell,which provides a certain potential for the follow-up use of pH gradient in the treatment of tumor.