| Objective Based on the specific pathological characteristics of continuous hypoxia and elevated reactive oxygen species(ROS)in the cardiac microenvironment after acute myocardial infarction,a nanoparticle nucleic acid delivery system that responds to both hypoxia and ROS was constructed,and after loading with injectable hyaluronic acid hydrogel,to study its therapeutic effect on myocardial infarction.Methods The first part: Preparation and physicochemical characterization of B-PDEA/DNA nanocomposites:(1)Preparation of non-viral carrier cationic polymer P-boric acid benzyl quaternized polyacrylic acid N,N-diethylaminoethyl(B-PDEA),to characterize the structure of B-PDEA and the characteristics of charge reversal in response to ROS.(2)Amplify the plasmids used in the research and verify by sequencing.(3)Prepare B-PDEA/DNA nanocomposites,and characterize its morphology,size,potential,and DNA compression package ability.(4)Use H2O2 to simulate a high ROS environment.After co-incubation of B-PDEA/DNA with H2O2,perform agarose gel electrophoresis experiments and DLS potentiometric measurement to verify its ROS responsiveness.(5)In the four types of cells,293 T,HT22,hMSC and primary cardiomyocytes,the classic cationic polymer carrier polyethyleneimine(PEI)was used as a positive control to study the cytotoxicity of B-PDEA.The second part: The functional characterization of B-PDEA/DNA:(1)In the four types of cells,293 T,HT22,hMSC and primary cardiomyocytes,PEI was used as a positive control to compare the transfection ability of B-PDEA.Determine the optimal nitrogen to phosphorus ratio for B-PDEA transfection and the optimal DNA content for transfection in a 24-well plate.(2)Observe the cellular uptake and dissociation of B-PDEA/DNA nanocomposites in cardiomyocytes.(3)Take B-PDEA/pSV40 as the control group and B-PDEA/pEPO-SV40 as the experimental group.After transfection,continue to culture in a hypoxic incubator for 48 hours,and then measure the expression level of the target gene to verify the ability of the EPO enhancer to induce high expression of the target gene under hypoxia.(4)After B-PDEA/DNA transfection,incubate with different concentrations of H2O2,measure the transfection efficiency,and verify its ROS response ability.The third part: Experimental study of B-PDEA/DNA nanocomposites for the treatment of acute myocardial infarction in SD rats:(1)The SD rat myocardial infarction model was constructed.The small animal in vivo imager verified the effect of injectable hyaluronic acid hydrogel on increasing the retention rate of B-PDEA/DNA nanocomposites.(2)The SD rats were randomly divided into 4 groups: PBS group,hyaluronic acid(HA)group,B-PDEA/DNA nanocomposites group,injectable hyaluronic acid hydrogel equipped with B-PDEA/DNA nanocomposites system(B-PDEA/DNA/HA)group.After MI,the treatments of each group were injected.One month later,the heart function of each group was evaluated by ultrasound.The paraffin sections of the heart infarction area were subjected to Masson staining to study the therapeutic effect of B-PDEA/DNA/HA on acute myocardial infarction.Results The first part:Preparation and physicochemical characterization of B-PDEA/DNA nanocomposites:(1)The result of proton NMR showed that the prepared B-PDEA was 100% quaternized and the chemical structure was correct.The charge reversal of B-PDEA’s ROS response is time-dependent and ROS concentration-dependent.(2)The results of plasmid sequencing showed that the recombinant plasmid used for the treatment of myocardial infarction successfully inserted the EPO enhancer and the therapeutic gene VEGF.(3)The size of the prepared B-PDEA/DNA nanocomposites is about 50-60 nm,and the potential is about +30mv.Nucleic acid gel results show that B-PDEA/DNA can wrap DNA well in the range of N/P=10-50 to prevent DNA leakage and migration.(4)After B-PDEA/DNA was incubated with a final concentration of 1m M H2O2 for 24 h,the results of nucleic acid gel showed the release of DNA and the DLS potential measurement revealed that the potential of B-PDEA/DNA was reversed to about-20 mv,which proved the ROS oxidation responsiveness of B-PDEA.(5)It was found in293 T,HT22,hMSC and primary cardiomyocytes that the toxicity of B-PDEA was significantly less than that of PEI.The second part:The functional characterization of B-PDEA/DNA:(1)Taking the N/P ratio PEI/DNA nanocomposites at the highest transfection efficiency in the corresponding cells as a positive control,B-PDEA/DNA nanocomposites with different N/P was transfected into 293 T,HT22,hMSC and primary cardiomyocytes.The results showed that B-PDEA/DNA was transferred in the above four cells,and the transfection efficiency increases with the increase of N/P.The optimal N/P ratio for transfection of B-PDEA/DNA nanocomposites is N/P=30,and the optimal DNA content for transfection in a 24-well plate is 1μg per well.(2)Observe the B-PDEA/DNA transfected primary cardiomyocytes under laser confocal.DNA has been transferred into the cardiomyocytes at 5 minutes,and a large amount of DNA has been transferred into the cells at 4 hours.Most of the B-PDEA/DNA nanocomposites did not dissociate after5 h of transfection,but at 16 h,the B-PDEA/DNA nanocomposites basically dissociated and released a large amount of DNA.(3)After transfection,the transfection efficiency was measured after cultured in a hypoxic incubator.The results showed that the transfection efficiency of the B-PDEA/pEPO-SV40 group was higher than that of the B-PDEA/pSV40 group,indicating that the expression level of the target gene in the B-PDEA/pEPO-SV40 group is significantly higher.(4)After transfection,the transfection efficiency was measured after co-incubation with different concentrations of H2O2.The results showed that the transfection efficiency of the co-incubation group with 50μm or100μm H2O2 was higher than that of the non-H2O2 co-incubation group.The third part: Experimental study of B-PDEA/DNA nanocomposites for the treatment of acute myocardial infarction in SD rats:(1)Ligation of the left anterior descending coronary artery in SD rats to construct an acute myocardial infarction model,after injecting the B-PDEA/DNA nanocomposites with 5wt% injectable HA hydrogel,the results of the small animal in vivo imager showed that compared with the direct use of the B-PDEA/DNA nanocomposites group,there are still more B-PDEA/DNA nanocomposites remaining in the myocardial infarct area on the 7th day after B-PDEA/DNA nanocomposites combined application 5wt% injectable HA hydrogel group.(2)One month after the treatment of myocardial infarction,echocardiographic function testing showed that the B-PDEA/DNA nanocomposites and B-PDEA/DNA/HA group can improve heart function,and the effect of B-PDEA/DNA/HA group is more significant.Masson staining of the infarct area showed that compared with the PBS group,the thickness of the ventricular wall in the B-PDEA/DNA/HA group was significantly thicker and the degree of fibrosis was reduced.Conclusion The B-PDEA/DNA nanocomposites constructed in this study has dual responsiveness to hypoxia and ROS.Compared with the traditional cationic polymer PEI,B-PDEA has less cytotoxicity and exhibits higher cytotoxicity in a variety of cells.Transfection efficiency.In vivo experiments on myocardial infarction treatment showed that 5wt% injectable HA hydrogel equipped with B-PDEA/DNA nanocomposites can increase the retention rate of B-PDEA/DNA nanocomposites in myocardial infarction area and improve the therapeutic effect of B-PDEA/DNA nanocomposites on myocardial infarction.The results of Masson staining showed that the fibrosis of the myocardial infarction area in the B-PDEA/DNA/HA group was reduced,and the thickness of the ventricular wall increased.B-PDEA/DNA/HA is expected to achieve good gene therapy effects in acute myocardial infarction. |
