Construction Of An Intracellular Microenvironment-responsive DNA Delivery System Based On In Situ Polymerization Technology

Gene therapy is the use of gene transfer technology to introduce foreign genes into recipient cells to treat diseases caused by gene defects.Gene therapy has developed rapidly since the concept of gene therapy was proposed in 1968 when American scientist Michael Blazer published"Changing Gene Defects:A Brighter Prospect for Medicine".The success of gene therapy is inseparable from the help of delivery vehicles to help therapeutic genes overcome various obstacles in the delivery process.Among the commonly used vectors,viral vectors have an unparalleled high transfection capacity.However,problems such as limited nucleic acid carrying capacity,lack of specificity,and unavoidable safety are the defects that viral vectors are difficult to solve.Non-viral vectors use cationic polymers,liposomes,proteins and peptides with low biotoxicity to form nanocomplexes with anionic nucleic acids based on electrostatic self-assembly and deliver them into cells,solving various safety problems of viral vectors However,non-viral vectors encapsulate nucleic acids based on self-assembly,and the assembled complexes are easily affected by poly ions in the biological environment.During the in vivo delivery process,structural dissociation is likely to occur before reaching the target,resulting in early nucleic acid leakage.The cross-linked polymer uses a cross-linking agent to connect the polymer chains as a carrier into a whole,like a net to coat the nucleic acid,which greatly enhances the stability of the non-viral vector gene delivery system.Among various methods for synthesizing cross-linked polymers,the newly emerging in-situ polymerization technology has attracted attention due to its simple steps and easy modification of functional groups.In this study,based on in situ polymerization technology,N-（3-aminopropyl）methacrylamide hydrochloride（APMA）,1-vinylimidazole（VI）,2-methacryloyloxyethylphosphorylcholinewereselected.（MPC）and N,N’-bis（acryloyl）cystamine（BACA）as functional monomers to construct a cross-linked polymer gene delivery system for delivering plasmid DNA（p DNA）.The carrier has high stability,and after being taken up by cells,the lysosome is destroyed by the proton sponge effect of the imidazole group in the acidic environment of the lysosome to realize the escape of the lysosome.After transferring to the cytoplasm,the carrier can respond to the high GSH environment in tumor cells,cleaving the reticular polymer shell,and realizing the release of p DNA.This study provides a feasible strategy for gene therapy,and the specific research contents are as follows:1.Synthetic exploration and characterization of gene delivery systemsGene delivery systems with various components and ratios were synthesized by in situ polymerization technology,and their encapsulation capacity,particle size and potential for p DNA were characterized.Finally,a synthesis strategy with N/P of 40 was selected to synthesize p DNA that could completely encapsulate p DNA.The gene delivery system is called NC（ss）₄₀.Dynamic light scattering results show that the radius of NC（ss）₄₀ is about 135 nm,and the zeta potential is about-6.5 m V.Transmission electron microscopy images show that NC（ss）₄₀ is spherical and has a uniform particle size distribution.2.Functional verification of gene delivery systemThe gene delivery system non-NC₄₀ without cross-linking agent was prepared,and the enhancement of the stability of NC（ss）₄₀ by cross-linking agent was verified by agarose gel electrophoresis experiment and heparin competition experiment.NC₄₀ has significantly improved stability;the gene delivery system NC（cc）₄₀ without GSH responsiveness was prepared,and the GSH responsiveness endowed by BACA to NC（ss）₄₀ was verified by DTT and heparin incubation experiments.The results showed that NC（ss）₄₀ could respond GSH degrades the vector,but NC（cc）₄₀ does not have this response ability;complete vector and MPC-free vector are prepared,and the enhancement of MPC to the biocompatibility of the vector is verified by hemolysis experiment;the DNase I incubation experiment is used to verify the gene delivery system.Nucleic acid protection ability,the results showed that the protection ability of NC（ss）₄₀ to p DNA in DNase I environment was significantly higher than that of non-NC₄₀.3.In vitro experiments of gene delivery systemThe cytotoxicity of NC（ss）₄₀ was detected by MTT method,and the results showed that the cell survival rate was high,and the cytotoxicity of NC（ss）₄₀ was low;the cell transfection ability of NC（ss）₄₀ was detected by luciferase transfection experiment,and the results showed that the detection intensity of luciferin was high,and the transfection ability of NC（ss）₄₀ was strong.