Evaluation Of Biological Function And Transport Mechanism Of HP1γ/pDNA/AGM-CBA Gene Delivery System Targeting Euchromatin

Objective: A guanidine-based polyaminoamine cationic polymer containing disulfide bonds has been widely studied as a novel carrier for gene delivery,but there is still a practical problem of low transfection efficiency.The HP1γ/p DNA/AGM-CBA gene delivery system constructed in this study introduced the protein HP1γ targeting euchromatin,thereby improving the transfection efficiency of cationic polymers and realizing the targeted function of the gene delivery system.In this study,the stability,transfection efficiency,intracellular transport and targeting function of this gene delivery system were further studied,which provided a theoretical basis for the design of euchromatin-targeted gene vectors with high transfection efficiency.Methods: In this study,agarose gel electrophoresis(AGE)was used to evaluate the stability of the HP1γ/p DNA/AGM-CBA gene carrier delivery system and the release ability of plasmids.Laser scanning confocal microscopy(LSCM)and flow cytometry(FCM)were used to analyze the in vitro transfection efficiency.The effect of serum on transfection efficiency was evaluated by combining confocal microscopy and flow cytometry in the case of serum intervention.The transport pathway of the complex was analyzed by confocal microscopy,flow cytometry,and LDH lactate dehydrogenase release.Immunofluorescence(IF)and laser scanning confocal microscopy were used to study the nuclear localization effect and euchromatin targeting function of this gene delivery system.Results: 1.HP1γ/p DNA/AGM-CBA could resist the acidic environment and resist the degradation of DNase and serum.2.The introduction of HP1γ improved the transfection efficiency of the gene delivery system,and the transfection efficiency reached 72.5±0.36% at the optimal N/P ratio,which was higher than that of the positive control group(p < 0.0001)and the control group without HP1γ target(p < 0.05).3.In the presence of transport inhibitors,the transfection efficiency of HP1γ/p DNA/AGM-CBA did not change significantly.The complex changes the cell membrane structure.4.After HP1γ/p DNA/AGM-CBA delivery,p DNAs were concentrated in the nucleolar region and further localized to the autochromatin region,and its nuclear localization and euchromatin targeting effect was better than that of the positive control group.Conclusion: The self-assembled HP1γ/p DNA/AGM-CBA gene delivery system has good biological stability and high transfection efficiency under the optimal N/P ratio,successfully targets foreign genes to the autochromatin region,and promotes gene expression.The experimental results show that the HP1γ/p DNA/AGM-CBA gene carrier delivery system targeting euchromatin has a good clinical application prospect.