Construction And Functional Evaluation Of A Biomimetic Gene Vector Targeting Glutathione Response In Tumors

Gene Therapy as a therapeutic approach involves the introduction of exogenous genes into the human body with the aim of repairing or replacing abnormal genes,ultimately treating diseases.In particular,gene therapy is considered a promising and safe approach to treating malignant tumors,using techniques such as gene immunotherapy,gene knockout therapy,gene modification therapy,and gene supplementation therapy.However,in both CRISPR/Cas9 gene editing and gene therapy strategies involving the delivery of exogenous gene fragments(DNA or RNA),the lack of safe and efficient gene vectors remains a major bottleneck limiting their clinical application.Compared with viral vectors,non-viral gene vectors have gradually attracted attention due to their lower immunogenicity,lower production costs,and greater gene loading capacity.Among non-viral gene vectors,cationic lipids,cationic polymers,and nanoparticles have been studied for gene delivery.Among them,polyethyleneimine(PEI)has become a research hotspot in this field due to its strong DNA condensation ability and excellent proton sponge effect.However,the stability of the "vector/gene" delivery system constructed by PEI and gene substances such as DNA and RNA is poor,and it is prone to cationic cytotoxicity when combined with other biological macromolecules during circulation in the body.Moreover,the system is a foreign substance that is easily captured and cleared by the human immune system,resulting in a short circulation time and low gene delivery efficiency.Due to its high biosafety and low immunogenicity,natural cell membranes have been widely used in the study of drug delivery systems.The various biological functions of cell membrane surface proteins can endow nanoparticles with immune evasion,long circulation time,targeted delivery,and other functions.Therefore,in this study,we combined the functional advantages of natural cell membranes and PEI to construct and evaluate a tumor-targeting gene delivery system based on cell membrane coating.The specific details are as follows.1.Construction and evaluation of gene carriers based on cell membrane-coated PEI/DNA complexes.Firstly,a series of RBCm/PEI30k/DNA,RBCm/PEI70k/DNA(referred to as RBCm-P30 Dc,RBCm-P70Dc)and RAWm/PEI30k/DNA,RAWm/PEI70k/DNA(referred to as RAWm-P30 Dc,RAWm-P70Dc)complexes with different mass ratios were obtained by squeezing red blood cell membranes(RBCm) and mouse monocyte macrophage cell membranes(RAWm)with PEI30k/DNA and PEI70k/DNA complexes.The complexes were then characterized.The results showed that all four complexes,RBCm-P30 Dc,RBCm-P70 Dc,RAWm-P30 Dc,and RAWmP70 Dc,exhibited spherical structures with particle sizes ranging from 200 nm to 300 nm.The membrane protein bands of the complexes were not significantly reduced compared to the cell membranes before squeezing,indicating successful synthesis of the complexes.Both cell membranes showed a high encapsulation efficiency of over 70% for the PEI/DNA complexes,with RBCm-P70 Dc having a maximum encapsulation efficiency of up to 80.31%.In the experiments on DNA binding protection and release,it was successfully demonstrated that the complexes can protect DNA from degradation by nucleases and can release DNA under certain conditions.The particle size and zeta potential of the complexes did not change significantly after being stored in p H 7.4 PBS buffer for 14 days,and compared to the corresponding molecular weight PEI,they showed lower protein adsorption and higher stability.Safety results showed that compared to the PEI/DNA complexes,the use of RBCmP30 Dc,RBCm-P70 Dc,RAWm-P30 Dc,and RAWm-P70 Dc complexes wrapped with cell membranes increased the survival rate of 293 T and Hela cells by around 20%.From the cell uptake experiments,it can be concluded that the complexes wrapped with cell membranes have increased uptake efficiency in both cell types,with the most significant increase observed in 293 T cells,where the maximum uptake efficiency of all four complexes increased by 10% to 15% compared to the non-wrapped complexes.In vitro transfection experiments showed that the complexes wrapped with cell membranes had significantly higher transfection efficiency in both 293 T and Hela cells,with RBCm-P70 Dc complexes showing the highest transfection efficiency.The maximum transfection efficiency of RBCm-P70 Dc complexes in 293 T and Hela cells was 64.36% and 15.91%,respectively,which were 11.21% and 6.54% higher than that of PEI70k/DNA complexes.These results demonstrate that the prepared complexes are safe,stable,and highly efficient.2.Construction and evaluation of glutathione-responsive gene vectors.Firstly,PEI was functionalized with thiol groups by conjugating with cysteine molecules containing a carboxylic group(-COOH)and a thiol group(-SH).The resulting thiolated PEI was then oxidized to form disulfide bonds with the abundant thiol groups present on the surface of cell membranes under mild conditions.The obtained product was coincubated with DNA and extruded using a liposome extruder to prepare a series of RMP30k/DNA and RMP70k/DNA complexes with mass ratio gradients,which were characterized.The results showed that RMP30k/DNA and RMP70k/DNA complexes exhibited spherical structures with particle sizes between 200 nm and 300 nm.The DNA encapsulation efficiency was over 90%,which protected the DNA from degradation by nucleases.In the presence of reduced glutathione(GSH),the release rate and efficiency of DNA were significantly improved,demonstrating strong glutathione responsiveness.The particle size and zeta potential of the complexes remained unchanged after storage in p H 7.4 PBS buffer for 14 days,indicating strong stability compared to the corresponding molecular weight PEI.Additionally,the complexes exhibited low protein adsorption,demonstrating strong stability.Based on the safety experiments,it is evident that the toxicity of RMP30 k and RMP70 k complexes to 293 T and Hela cells is significantly lower than that of the corresponding molecular weight of PEI.The results of cellular uptake experiments indicate that the maximum uptake efficiency of RMP30k/DNA and RMP70k/DNA complexes in 293 T cells is 64.49% and 73.42%,respectively,while in Hela cells,it is 51.65% and 82.74%,respectively.Compared to the previous samples,the uptake efficiency of the complexes did not show a significant change in 293 T cells,but in Hela cells,the uptake efficiency significantly increased by 8.32%.These results suggest that RMP30 k and RMP70 k complexes may be effective gene delivery systems and could be applied in gene therapy and related fields.According to the transfection experiments,compared to the previous samples,the complex with introduced disulfide bonds did not show a significant change in transfection efficiency in 293 T cells,but the maximum transfection efficiency in Hela cells increased by 4.22%.These results demonstrate that the prepared complex possesses the characteristics of safety,stability,high efficiency,and targeted drug release.This complex has great potential as a gene delivery system in various biomedical applications,including gene therapy and drug delivery.3.Construction and evaluation of a tumor-targeting gene carrier: Folic acid(FA)was used to further modify RMPs/DNA complexes to achieve targeted gene delivery.The results showed that the FA-RMP70k/DNA complex had a spherical structure with a particle size of 232.63 nm.Compared with RBCm,most membrane proteins were retained in the FA-RMP70k/DNA complex,which ensured that the original properties of the cell membrane in the FA-RMP70k/DNA complex were not significantly altered.Safety experiments showed that compared with RMP70k/DNA complexes,the toxicity of FA-RMP70k/DNA complexes to 293 T and Hela cells did not change significantly.These results demonstrate the potential of the FA-RMP70k/DNA complex as a gene delivery system in targeted biomedical applications,including gene therapy and drug delivery.According to the cell uptake experiments,the uptake efficiency of the FARMP70k/DNA complex was significantly higher than that of the RMP70k/DNA complex,with maximum uptake efficiencies of 88.36% and 96.08% in 293 T and Hela cells,respectively.Cell transfection experiments showed that the maximum transfection efficiencies of the FA-RMP70k/DNA complex in 293 T and Hela cells were 79.58% and 35.09%,respectively,representing an increase of 9.65% and 14.96% compared to the RMP70k/DNA complex.These results demonstrate that the introduction of FA molecules can further enhance the targeting ability of the complex.Based on the above,we have constructed a biomimetic gene vector that targets tumors in response to glutathione.Our research indicates that this system has good biocompatibility,stability,and targeting ability,and can significantly reduce the cytotoxicity of PEI/DNA complexes and enhance transfection efficiency.