Injectable Hydrogel With Dual-Sensitive Behavior For Targeted Delivery Of Oncostatin M To Improve Cardiac Restoration After Myocardial Infarction

Objective:How to further protect cardiac function and reduce cardiac remodeling after myocardial infarction is an important clinical scientific issue.In this study,chitosan(CS),citric acid(CA)and N-isopropylacrylamide(NIPAM)were used to prepare a double sensitive injectable hydrogel with good gel-forming properties and stable biological active components.A sustained-release drug system loaded with OSM hydrogel was co-culture with cells and local multi-point injection into the surrounding area of myocardial infarction.To observe the protective effects of OSM sustained release hydrogel on cells and cardiac function after myocardial infarction at cell level and animal level,and to explore the related mechanisms.It provides scientific basis for the clinical application and transformation of OSM sustained-release hydrogel in the treatment of myocardial infarction.Methods:1.Different ratios of CS-CA-PNIPAM hydrogels were prepared by Reversible addition-fragmentation Chain Transfer Polymerization(RAFT).The physicochemical properties of hydrogels were characterized by Fourier transform infrared spectroscopy(FTIR),1H-NMR,scanning electron microscopy(SEM),rheological detection,swelling test,in vitro degradation and drug release.2.Based on the first part,the hydrogel drug sustained-release system was constructed by loading OSM and co-cultured with cells to test the cytocompatibility and biological function.The cytocompatibility of OSM sustained-release hydrogels was evaluated by CCK-8 and live/dead staining.Rhodamine B absorption test was used to observe the double sensitivity of hydrogel.The bioactivity of OSM released from hydrogel was detected by Western blot and immunofluorescence.EdU staining and flow cytometry were used to observe the effects of different materials on the proliferation and apoptosis of myocardial cells.Real-time quantitative RT-qPCR was used to detect the expression of related genes in H9c2 cells.The angiogenic ability of different materials was evaluated by endothelial cell tube formation assay.3.SD rats were used to establish a myocardial infarction model by ligation of the left anterior descending coronary artery.The therapeutic effects of different materials were observed by local multi-point microinjection into the perimyocardial infarct area of rats.After 4 weeks of treatment,cardiac function was detected by Langendorff extracorporeal perfusion system.Myocardial fibrosis and ventricular wall thickness were observed by H&E and Masson staining.TUNEL staining and immunofluorescence staining were used to observe the infiltration of macrophages,proliferation and apoptosis of cardiomyocytes and neovascularization in the perimyocardial infarction area.The expressions of inflammatory factors and angiogenesis related factors in myocardial tissue were detected by ELISA.Results:1.The results of FTIR and 1H NMR showed that CS,CA,and NIPAM were synthesized successfully to form CS-CA-PNIPAM hydrogel.The developed hydrogels have a loose porous structure with a pore size of about 10-20μm.The gel experiment show that the formation time of the hydrogel is about 30 min at 3 7℃,which indicates that the hydrogel has good temperature sensitivity.The rheological experiment show that the hydrogel has good temperature reactivity and mechanical properties.The swelling test and in vitro degradation test showed that the hydrogel had obvious pH reactivity.The simulated drug release experiments showed that the sustained drug release ability was pH dependent,and the sustained drug release time was up to 14 days.2.The results of cell experiments showed that the hydrogel loaded with OSM had good biocompatibility and bioactivity.Hydrogel loaded with OSM is more conducive to the survival and proliferation of H9c2 cardiomyocytes and can promote the transcription of cell-related genes(PI3K,AKT,mTOR,ERK).On the other hand,OSM-loaded hydrogels can enhance the tube formation ability of human umbilical vein endothelial cells(HUVECs),indicating that they can promote angiogenesis.3.H&E,and Masson results showed that hydrogel loaded with OSM had no adverse effects on important organs(liver,spleen,lung,kidney)of rats.After the injection of hydrogel,myocardial fibrosis was significantly reduced,and left ventricular wall thickness was increased.At the same time,OSM-loaded hydrogel can promote angiogenesis and neovascularization,induce macrophage polarization to M2,reduce cardiomyocyte apoptosis,promote cardiomyocyte proliferation,and retain more cardiomyocytes in the area around myocardial infarction.In addition,ELISA results showed that the hydrogel could inhibit myocardial inflammation and promote the expression of angiogenesis-related factors.Finally,the results of the cardiac function test showed that OSM-loaded hydrogel had a significant improvement effect on cardiac function after myocardial infarction.Conclusion:1.In this study,a novel CS-CA-PNIPAM hydrogel was synthesized by modifying CS with CA and NIPAM,which overcame the shortcomings of simple chitosan hydrogel.The hydrogel has good physical-chemical properties,suitable temperature and pH reactivity,and drug sustained release performance.2.The OSM-loaded hydrogel has good biocompatibility and bioactivity.The hydrogel can improve the transcription levels of Akt and ERK1/2 signaling pathways related to cell survival and proliferation and promote the survival and proliferation of cardiomyocytes.At the same time,the hydrogel can promote the tube formation of endothelial cells,indicating that it has the ability to promote angiogenesis.3.The OSM-loaded hydrogel can inhibit the inflammatory reaction of myocardial tissue,reduce myocardial fibrosis,promote angiogenesis,reduce cell apoptosis,promote the proliferation of myocardial cells,enhance the repair ability of myocardial tissue,and thus improve cardiac function.