Construction Of A Novel Reactive Oxygen Species-responsive Cationic Copolymer And Its Performance In Gene Delivery

The non-viral gene vector with good biocompatibility and high transfection efficiency is the key to large-scale clinical application of gene therapy.Therefore,this study intends to design and prepare a new type of non-viral vector which is able to release DNA in response to the triggering of reactive oxygen species in the cellular microenvironment and investigates its performance in gene delivery.The specific research content and results are as follows:(1)Synthesis and structural characterization of a novel vector which can release genes in response to reactive oxygen species in the cell microenvironmentHerein,a novel reactive oxygen-responsive cationic block copolymer non-viral gene delivery vector(termed as pM-pBD)consisting of biocompatible poly(2-methacryloyloxyethyl phosphorylcholine)[p(MPC)]segment and charge reversible poly[(2-acryloyl)-ethyl-(boronic acid benzyl)-diethylammonium bromide]segment[p(BD)]was synthesized via reversible addition-fragmentation chain transfer polymerization(RAFT).The structure of the cationic copolymer was characterized by hydrogen nuclear magnetic resonance spectroscopy,high-resolution mass spectrometry and gel permeation chromatography.The results showed that the vector was successfully synthesized.The cationic copolymer pM-pBD can be broken in respond to reactive oxygen species confirmed by high-performance liquid chromatography.(2)Preparation and characterization of nanocomplex and characterization of physical and chemical propertiesThrough electrostatic interaction,the cationic copolymer pM-pBD can form nanocomplex with negatively charged DNA in a molecular self-assembly manner.The cationic p BD fragment has the characteristics of reactive oxygen species triggering charge reversal,which helps to obtain the structural dissociation of the electrostatic assembly complex triggered by reactive oxygen species,thereby realizing the function of controllable gene release.The particle size,zeta potential of the pM-pBD/p DNA nanocomplex was measure by dynamic light scatter(DLS)and the morphology of pM-pBD/p DNA nanocomplex was observed by the transmission electron microscope(TEM).The results displayed that pM-pBD and p DNA can form nanocomplex with the particle size of 88 nm?230 nm,the zeta potential of+6 m V?+30m V and the microscopic morphology was similar to spherical.The pM-pBD/p DNA nanocomplex demonstrated appreciable colloidal stability in the presence of 10%fetal bovine serum.The pM-pBD/p DNA nanocomplex at N/P ratio of 3 displayed spherical morphologies with average diameter of approximate 99.1 nm and?potential of approximate+13.8 m V.However,once upon incubation in presence of 1 mmol/L H₂O₂,the diameter of pM-pBD/p DNA nanocomplex at N/P ratio of 3 was enlarged to 330 nm and?potential was reversed to-3.91 m V due to charge reversal of BD responsive to reactive oxygen.Gel electrophoresis retardation assay demonstrated that pM-pBD can firmly bind p DNA through electrostatic interaction even in the presence of high concentration of heparin.Moreover,when the pM-pBD/p DNA nanocomplex was incubated with 1 mmol/L H₂O₂,DNA was released in response to reactive oxygen species and has little effect on the structure of DNA.(3)Intracellular transfection experiment of nanocomplexHemolysis and MTT assay demonstrated that pM-pBD/p DNA nanocomplexed showed high biocompatibility and low cytotoxicity against He La cells,even at high weight ratio of pM-pBD and p DNA(N/P)(up to 16).The results of flow cytometry showed that the uptake efficiency of pM-pBD/p DNA nanocomplex was 88.9%and the transfection efficiency was25.9%at the N/P ratio is 3.The transfection efficiency of pM-pBD/p DNA against He La cells was observed to be significantly augmented(1.5-fold)after the addition of ascorbic acid,which could stimulate the production of hydrogen peroxide.The results showed that reactive oxygen species can significantly improve the transfection efficiency of the vector.Therefore,pM-pBD represented intriguing utilities in fabrication of non-viral gene delivery systems,which entitled spatiotemporal control of gene release.