Preparation Of ROS-Responsive Exosome Coating For ICAS Treatment And Its Biological Study

ObjectiveIntracranial atherosclerotic stenosis（ICAS）is a disease dominated by chronic inflammation,and its formation is closely related to oxidative stress induced by reactive oxygen species（ROS）.In its endovascular treatment,Nitinol（Ni Ti）alloy stents play an important role,but the Ni Ti alloy stents without surface treatment have a high risk of thrombosis and in-stent restenosis after endovascular implantation.In order to improve its biocompatibility after implantation,this study intends to prepare the exosome-loaded polymer coating on the surface of Ni Ti alloy stent material,and evaluate the ROS response of the coating and its endothelialization,anti-inflammatory and anti-proliferation abilities,so as to provide some theoretical support for the development of new cerebral vascular stents in the future.Methods1.Extraction and identification of exosomes:Extractor exosomes,and the marker proteins,particle size and morphology of exosomes were detected by Western blot,nanoparticle tracking analysis（NTA）and transmission electron microscope（TEM）,respectively.2.Preparation and characterization of the coating:Through physical adhesion,Sodium alginate（SA）was first coated on the surface of Bare Nitinol（BN）alloy to form a dense activated layer.Then put it into the ROS sensitive agent M-aminpohenylboronic acid（M-ABPA）solution,use the carboxyl group of SA to chemically react with the amino group of M-ABPA,and add dihydroxyphospholipid-polyethylene glycol after the reaction is complete to form ROS response points and exosome junctions.Finally,plasma exosomes were added to form a ROS-responsive exosome coating,and the sample was denoted as SMEN.Use X-ray photoelectron spectroscopy（XPS）to characterize the grafting between coatings;atomic force microscope（AFM）and scanning electron microscope（SEM）to observe the surface morphology of Ni Ti alloy before and after coating;the contact angle measuring instrument detects the size of the water contact angle（WCA）on the Ni Ti alloy surface before and after coating.3.Hemolysis test:Soak BN and SMEN in 2%red blood cell solution,detect the absorbance with a microplate reader,and calculate the hemolysis rate.4.Platelet adhesion assay:BN and SMEN were co-incubated with platelets,and platelet adhesion was observed under SEM microscope.5.In vitro release test:To detect the ROS response point and in vitro release,SMEN were immersed in 0.5 ml PBS buffer and 0.5 ml 0.1 m M H₂O₂ solution,and the release solution was collected at 0,1,1.5,3,5 and 10 hours,respectively.6.Endothelial cell experiment:BN and SMEN were co-cultured with human umbilical vein endothelial cells（HUVECs）.The effects of samples on the proliferation and migration of HUVECs were evaluated by cell adhesion assay,CCK8 assay,scratch wound healing assay,angiogenesis assay and NO release assay.The expression of von Willebrand factor（v-WF）and vascular endothelial cadherin（VE-cadherin）in HUVECs was detected by immunofluorescence（IF）.7.Smooth muscle cell experiment:In order to verify the effect of BN and SMEN on the phenotypic switching and migration of vascular smooth muscle cells（VSMCs）,IF and Transwell assays were used for detection.8.Macrophage experiment:IF and ELISA experiments were used to detect the effect of samples on macrophage phenotype conversion and inflammatory factor secretion.9.In vivo animal experiments:BN and SMEN were implanted subcutaneously in mice,and tissue samples were obtained on the 1st and 7th day,and observed and evaluated by hematoxylin-eosin staining,Masson staining and immunohistochemical experiments.Results1.The experimental results of TEM,NTA and exosome membrane proteins Cd81 and Tsg101 showed that the exosomes were successfully extracted.2.The XPS results showed that the coating material was successfully grafted on the Ni Ti alloy surface,and the TEM and SEM results showed that the Ni Ti alloy surface changed before and after coating.At the same time,the results of the contact angle measurement instrument showed that the WCA（23.2±1.3°）of SMEN was significantly smaller than the surface WCA（87.3±1.4°）of the sample BN,forming a highly hydrophilic surface.3.The acute hemolysis test showed that the hemolysis rate of SMEN（0.001±0.0002%）is slightly higher than that of BN（0.0008±0.0003%）,but far lower than the International Organization for Standardization standard（5%）for hemolysis properties of materials,which meets clinical implantation requirements requirements.4.The results of SEM showed that the number of platelets adhered on the surface of SMEN was significantly less than that on the surface of BN.5.The results of the release experiment showed that the release rate of SMEN in H₂O₂ solution was faster than that in PBS solution.6.The results of endothelial cell adhesion assay showed that HUVECs grew well on the SMEN surface,and the SMEN showed good endothelialization on day 5.The results of CCK8 experiment showed that the cell proliferation of SMEN was 1.8,1.4 and 1.5 times that of BN at 1,3 and 5 days,respectively.The results of scratch and tube formation experiments showed that SMEN can promote endothelial cell migration and angiogenesis.In addition,within 48 hours,the NO release of SMEN was 0.49±0.1μM,which was 3 times that of BN,and the expression of VE-cadherin was higher and the expression of v-WF was lower in HUVECs on the surface of SMEN.7.The Transwell results of VSMCs showed that at 12 hours and 24 hours,the migration of VSMCs in the BN group was significantly more than that in the SMEN group.In terms of the phenotype conversion of VSMCs,the IF results showed that SMEN had the effect of inhibiting the conversion of contractile VSMCs to synthetic VSMCs（P<0.01）.8.The results of macrophages showed that SMEN could promote the secretion of anti-inflammatory cytokine IL-10（P<0.05）and the transformation of macrophages to M2 phenotype（P<0.01）,but also inhibit the secretion of pro-inflammatory cytokine IL-6（P<0.05）.9.The results of in vivo animal experiments showed that both BN and SMEN were accompanied by the infiltration of inflammatory cells on the first day of subcutaneous implantation in mice,but the expression of IL-6 was higher in the BN group.On the 7th day,it can be seen that the number of new blood vessels in the SMEN group is more than that in the BN group,showing good histocompatibility.ConclusionIn this study,ROS-responsive exosome coating with high hydrophilic properties was successfully prepared,which can effectively improve the endothelialization,anti-proliferation,and anti-thrombosis ability of Ni Ti alloy surface,which has certain application value in promoting the development of new intracranial stents and improving the therapeutic effect of ICAS.