Macrophage-Targeting Gene Silencing Orchestrates Myocardial Microenvironment Remodeling Tow Ard The Anti-Inflammatory Treatment Of Ischemia-Reperfusion(IR) Injury

Part Ⅰ Preparation and characterization of redox-responsive branched poly(β-amino ester)nanocompositesObjective:To design and synthesize the redox-responsive branched poly(β-amino ester)(BPAE-SS)containing disulfide bond,and form nanocomposite BSs NCs with siMOF,then modify the surface with carboxylated mannan(Man-COOH),and finally characterize the properties of the nanocomposite MBSs NCs.Methods:The yellow viscous oil-like BPAE-SS was synthesized by chemical method.The molecular weight(MWs)and reduction sensitivity of the polymer were determined by gel permeation chromatography(GPC),and the structure of the polymer was reflected by the conformational curve between the radius of gyration(Rg)and the molecular weight.BPAE-SS was dissolved in acetic acid buffer solution,then BSs NCs were formed with siMOF by electrostatic adsorption,and then MBSs NCs were formed by modifying a layer of Man-COOH on the surface of the composite.Agarose gel electrophoresis(AGE)and dynamic light scattering(DLS)were used to determine the optimum ratio of MBSs NCs by measuring the different mass ratios of BPAE-SS to siMOF and Man-COOH to siMOF.DLS and AGE were used to evaluate the size change and siRNA release of BSs NCs nanocomposite before and after glutathione(GSH)treatment,and siRNA release in different concentrations of heparin sodium.The serum stability of MBSs NCs was determined by AGE and DLS.Results:The molecular weight of BPAE-SS was 21200 and the polydispersity index(PDI)was 1.58.The molecular weight of BPAE-SS treated with dithiothreitol(DTT)decreased significantly,which confirmed the reduction sensitivity of BPAE-SS.The slope of BPAE-SS was about 0.2,which indicated its branching structure.The results of AGE and DLS showed that:(1)When the mass ratio of Man-COOH/BPAE-SS/siMOF was 20/30/1,the optimum particle size of MBSs NCs was～170 nm and the potential was～6 mV.The results were consistent with those observed by transmission electron microscope.(2)After GSH treatment,the particle size of BSs NCs increased obviously and the condensation ability of siMOF decreased.In addition,heparin sodium replacement test showed that the concentration of heparin sodium required for siMOF release decreased significantly.(3)MBSs NCs maintained good stability in serum.Conclusion:MBSs NCs have good branching structure and GSH responsiveness,and maintain stability in serum,which is beneficial to the release of siRNA in cells as needed.Part Ⅱ Mechanism of MOF/miR21 Gene Silencing Induced by MBSsm Nanocomposite in MacrophagesObjective:To study the mechanism of MOF/miR21 gene silencing induced by MBSsm nano-composites in macrophages.Methods:Flow cytometry was used to detect the ability of nanocomposite MBSs NCs to deliver siRNA into macrophages,and confocal laser scanning microscope(CLSM)was used to evaluate the intracellular release ability and lysosomal escape ability of nanocomposite MBSs NCs.Mannose targeting assay was used to study the mechanism of MBSs NCs targeted endocytosis mediated by Man-COOH and 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium bromide(MTT)was used to evaluate the toxicity of MBSs NCs.The expression of MOF mRNA and KBTBD7 mRNA in macrophages treated with nano-composites was detected by real-time PCR,and then the gene silencing effect of MBSs NCs and MBSm NCs was evaluated.CLSM was used to detect ROS scavenging ability of MBSsm NCs in macrophages.Finally,CLSM,real-time PCR and Western blot techniques were used to study the mechanism of MBSsm NCs in coordinating myocardial microenvironment remodeling.Results:Flow cytometry showed that MBSs NCs had strong intracellular siRNA delivery ability,and CLSM showed that MBSs NCs could release siRNA well and escape from lysosomes.The results of mannose targeting experiment showed that the higher the concentration of mannose was,the lower the uptake level of MBSs NCs cells was.MTT results showed that MBSs NCs had no obvious cytotoxicity.The results of Real-time PCR showed that when the mass ratio of BPAE-SS/siMOF was 30,the gene silencing efficiency was the best.Compared with other nano-composites,the expression of MOF mRNA and KBTBD7 mRNA in macrophages treated with MBSs NCs was the lowest,which indicated that MBSs NCs had high gene silencing efficiency.Macrophages induced by lipopolysaccharide(LPS)could release a large amount of reactive oxygen species(ROS).Through CLSM observation,it was found that ROS in macrophages treated with MBSsm NCs had the highest clearance,and little green fluorescence was seen,which indicated that MBSsm NCs combined with siMOF and miR21 could effectively clear ROS produced in macrophages and alleviate oxidative stress.The results of CLSM,real-time PCR and Western blot showed that under the simulated myocardial reperfusion oxidation and inflammation environment,MBSsm NCs efficiently delivered siMOF and miR21 into macrophages,cleared ROS and alleviated inflammatory reaction,and promoted the phenotype of macrophages to change from M1(inflammatory type)to M2(anti-inflammatory type),and further inhibited autophagy of myocardial cells through crosstalk between macrophages and myocardial cells.Conclusion:MBSsm NCs have high transfection efficiency and can escape from lysosomes.Targeted macrophages can efficiently deliver siMOF and miR21 into macrophages to clear ROS,reduce inflammatory reaction,promote macrophage phenotype from M1 to M2,and reduce autophagy of myocardial cells,thus coordinating the remodeling of myocardial microenvironment.Part Ⅲ Research on the effect of MBSsm Nanocomposite in the Treatment of Myocardial Ischemia-reperfusion InjuryObjective:To study the effect of MBSsm nanocomposite in the treatment of myocardial ischemia-reperfusion injury.Methods:The mouse model with myocardial ischemia-reperfusion was established.There were 6 groups,such as PBS group,MBSc group,BSsm group,MBSsm group,MBSs group and MBSm group.The ability of MBSs NCs to deliver Cy5-siRNA into ischemic tissue and the ability of MBSs NCs to target myocardial macrophages were detected by IVIS optical imaging system.The expressions of MOF mRNA,Kbtbd7 mRNA,TNF-α mRNA,IL-6 mRNA,IL-10 mRNA and Argl mRNA were detected by real-time PCR after siMOF and miR21 were delivered to myocardial ischemia by MBSsm NCs.The expressions of MOF and KBTBD7 protein were detected by Western blot.At 3 days after operation,Cardiac ejection fraction(EF)and left ventricular fractional shortening(FS)were measured by echocardiography to evaluate cardiac function.After 7 days,the heart tissue sections were analyzed histologically.The myocardial infarction area was measured by 2,3,5-triphenyltetrazolium chloride(TTC)staining,the myocardial injury degree was evaluated by HE staining,the myocardial apoptosis degree was detected by TUNEL method,and the myocardial fibrosis degree was evaluated by Masson’s trichrome(MT)staining.Results:The results of IVIS optical imaging system showed that MBSs NCs had the highest distribution level in myocardial ischemic tissues,and flow cytometry showed that MBSs NCs had strong ability to target myocardial macrophages.The results of real-time PCR analysis showed that compared with the other five groups,MBSsm group had the best effect of gene silencing,and Western blot showed that MOF and KBTBD7 protein expression levels were the lowest.At 3 days after operation,TTC staining showed that the myocardial infarction area in MBSsm group was the smallest,while cardiac ultrasound showed that the cardiac function of MBSsm group basically recovered to normal level.At 7 days after operation,histological analysis showed that the myocardial tissue of MBSsm group had no significant difference with normal myocardial tissue.Conclusion:MBSsm nanocomposite can deliver siMOF and miR21 to myocardial ischemia tissue,which can alleviate myocardial ischemia-reperfusion injury and promote the recovery of cardiac function by alleviating oxidative stress and inhibiting inflammatory reaction,coordinating myocardial microenvironment remodeling.