Construction Of Silk Fibroin-based Coating On The Surface Of Coronary Stents And Regulation Of Its Structure And Performance

Stent intervention therapy is an effective method for clinical treatment of cardiovascular stenosis.However,the occurrence of in-stent restenosis(ISR)and late thrombosis after stent implantation is an urgent problem to be solved in clinical application.It is a feasible way to prepare novel functional cardiovascular stents by constructing functional coatings on the surface of stents.Among them,the strategy of inhibiting coagulation and smooth muscle hyperplasia and promoting endothelialization by in situ catalyzing local production of nitric oxide(NO)from endogenous donors by coating combined with metal ions is particularly attractive.Silk fibroin(SF)has good biocompatibility and can degrade slowly in vivo,which has great potential as a coating for implantation devices.However,silk fibroin as a coating for cardiovascular stents has not been studied in detail,and how to promote its functionality requires further investigation.This paper intends to use silk fibroin as the coating of coronary stent,and composite copper ions to construct NO catalytic system.By introducing chitosan to regulate its copper ion binding capacity,the effects of coating composite form,copper ion binding amount and binding form on the catalytic NO production ability were systematically studied,and their interrelationships were clarified.First,copper ions were loaded on the surface of silk fibroin films by protein-metal coordination to study the copper ion functionalization of silk fibroin films and their catalytic NO release behavior.The results showed that the concentration of copper sulfatesolution had no obvious effect on the binding of copper ions to the silk fibroin film.With the prolongation of soaking time,the more copper ions bound to silk fibroin,the copper ions on the surface of the silk fibroin film approached saturation at 60 minutes.The XPS results confirmed that copper ions may exist on the surface of the silk fibroin film in the form of copper(II)/ copper(I)coexistence.The degradation experiments showed that the copper ions bound to the surface of the silk fibroin film could be released slowly for 7 days in PBS or collagenase IA solution,and there was no significant difference in the release of copper ions between the two systems.The NO catalysis experiment showed that the loading of copper ions could significantly promote the release of NO from S-Nitroso-N-acetyl-D-penicillamine(SNAP)through catalytic action,and the content of NO released from SNAP increased by about 50% after the silk fibroin film adsorbed copper ions for 60 minutes.Secondly,in order to solve the problems of low loading rate and fast release of copper ion in silk fibroin film,chitosan(CS)with stronger ability of binding copper ion was introduced into silk fibroin film to regulate copper ion loading and catalyze NO generation capability.Different ratios of SF/CS blend films were prepared by hexafluoroisopropanol(HFIP)dissolution system,and the copper ion binding and release,catalytic NO release and cytocompatibility of the blend films were systematically studied.The results showed that with the increase of chitosan content,the loading capacity of copper ions increased significantly.With the introduction of a small amount of chitosan(5%),the copper ion loading on the surface of the blend film was about about 2.2 times that of the silk fibroin film.When the content of chitosan was increased to 15%,the copper ion loading was about 3.6 times that of the silk fibroin film.Copper ions also existed on the surface of the blend film in the form of copper(II)and copper(I)coexistence.The degradation experiments showed that the introduction of chitosan significantly improved the binding capacity of copper ions,and copper ions would not be rapidly released to cause potential cytotoxicity.And in the enzyme solution system,due to the degradation of the enzyme on the film,the release rate of copper ion was relatively higher than that in PBS,but it could still be released slowly for more than 28 days.NO catalysis experiments showed that the introduction of chitosan enhanced the copper ion loading and binding strength,and significantly promoted the release of NO from SNAP.After 28 days of degradation,the relative content of copper ions on the surface of the film with a blend ratio of silk fibroin and chitosan at 85:15 could still maintain more than 50% and had a significant catalytic release ability of NO too.The cell experiments showed that low chitosan content resulted in rapid release of copper ions and high cytotoxicity,while the excessive copper ion loading with too high chitosan content would also cause cytotoxicity.The film with a blend ratio of silk fibroin protein and chitosan at 85:15 had the best compatibility with vascular endothelial cells and the lowest toxicity and was able to support cell adhesion and rapid proliferation.Finally,the coronary metal stents were coated with three groups of SF/CS solutions with the blending ratio of 100:0,95:5 and 85:15,and combined with copper ions to study the copper ion loading,NO catalytic release and cytocompatibility.The SEM results showed that SF/CS could be firmly combined with the cobalt alloy stents to form a stable coating.The NO release experiment showed that the ability of the composite coating to catalyze the release of NO was significantly improved with the increase of chitosan content on the surface of the stent.The cell experiments showed that the coated stents could support endothelial cell growth,and the coating with a blending ratio of silk fibroin and chitosan at 85:15 had the best biocompatibility.This paper proposed the feasibility of silk fibroin/copper ions based system as a coating for metal stents and elucidated the key role of chitosan in regulating the structure and function of the coating.This paper provides new ideas and scientific basis for the development of new vascular stents.