Evaluation Of The Efficacy Of Exosomal Microneedle Patch Of Overexpressed TGF-β1R Mesenchymal Stem Cells In The Treatment Of Myocardial Fibrosis After Heart Attack

Part Ⅰ.Overexpression of TGF-β1R mesenchymal stem cell exosomes inhibits collagen production in rat cardiac fibroblastsObjective: To investigate the effect of overexpression of TGF-β1R mesenchymal stem cell exosomes on the induction of collagen Ⅰ and III synthesis in rat cardiac fibroblasts under hypoxic conditions.Methods: Primary cardiac fibroblasts from neonatal SD suckling rats were extracted using a mixture of trypsin and type II collagenase.Cardiac fibroblasts were identified by immunofluorescence;the hypoxic environment of cardiac fibroblasts was simulated by continuous nitrogen gas passage.TGF-β1R was transfected into rat umbilical cord mesenchymal stem cells by lentivirus to construct a stable transgenic cell line.The exosomes were extracted by ultracentrifugation,observed by transmission electron microscopy,and identified by Nanosight nanoparticle analyzer and Western blot.The effects of overexpression of TGF-β1R Uc MSCs exosomes on cardiac fibroblast value addition and collagen synthesis were evaluated by CCK-8 assay,live cell/dead cell double-staining assay,and Western blot assay for collagen Ⅰ and collagen Ⅲ expression.Results: The lentiviral plasmid overexpressing TGF-β1R was successfully constructed and efficiently transfected with Uc MSCs,whose secreted exosomes also successfully expressed the TGF-β1R protein,Uc MSCs-TGF-β1R-EXO.Uc MSCs-TGF-β1R-EXO does not affect the value-added of cardiac fibroblasts and has no toxic effect on cardiac fibroblasts,and can effectively reduce hypoxia-induced collagen Ⅰ and III synthesis by cardiac fibroblasts.Conclusion: Overexpression of TGF-β1R mesenchymal stem cell exosomes can effectively reduce the synthesis of collagen Ⅰ and III in rat cardiac fibroblasts induced by hypoxic conditions.Part Ⅱ Application of exosome microneedle patch loaded with overexpressed TGF-β1R mesenchymal stem cells in rats with myocardial infarctionObjective: To evaluate the efficacy of microneedle patches loaded with Uc MSCs exosomes overexpressing TGF-β1R in the treatment of myocardial fibrosis after myocardial infarction.Methods: In this study,HAMA microneedles were prepared by the microneedle mould method,and the morphology of the microneedles was exmlned by visual inspection,body microscopy and scanning electron microscopy;the mechanical strength was evaluated by measuring the force required to produce similar displacement of the microneedles with and without loaded exosomes;the depth of microneedle penetration was observed by stabbing the microneedles loaded with Evans blue staining and FITC into the myocardium;small animal live imaging was used to observe the effect of Cy7-labelled microneedles were distributed in vivo and the effect of HAMA on H9c2 cell value added was determined by the CCK-8 assay;whether HAMA had an effect on H9c2 cell activity was exmlned by the live/dead cell double staining assay.Rat Umbilical cord MSC exosomes overexpressing TGF-β1R were grown on microneedle patches,and SD rats(N=40)were randomly divided into control,PBS,exosome,microneedle and microneedle-loaded groups.The MI model was constructed by blocking the left anterior descending(LAD)coronary artery.And cardiac function was measured by echocardiography on postoperative day 7 and 28,and the material was taken after 28 days for testing the fibrosis area and treatment effect.Results: HAMA microneedles were found not to negatively affect H9c2 cellular value-added or their cellular activity.The microneedles loaded with overexpressed TGF-β1R exosomes were effective in reducing the area of myocardial fibrosis after myocardial infarction and improving cardiac function.It also reduced the incidence of cardiac rupture in rats due to myocardial infarction,acting to alleviate myocardial fibrosis after myocardial infarction and inhibiting the deterioration of cardiac function.Conclusion: In summary,the area of myocardial fibrosis after myocardial infarction can be effectively reduced by transplantation of microneedles loaded with high expression of TGF-β1R-EXO: in the current study,exosomes of Uc MSCs overexpressing TGF-β1R were successfully extracted;TGF-β1R-EXO significantly improved cardiac function and reduced myocardial fibrosis mina and,which may be related to TGF-β1R-EXO binding to free TGF-β1 binding,reduced activation of the Tgfβ1-Smad2/3 pathway and decreased collagen fibril production and deposition.This study will further provide a theoretical basis for genetically modified Uc MSCs in the treatment of myocardial fibrosis in the clinical setting.