Preparation And Characterization Of Calcitonin Gene-related Peptide Modified Silk Fibroin Material

Cardiovascular disease is a serious threat to human health.In order to solve cardiovascular disease,researchers have conducted several researches on vascular grafts,but small-diameter(internal diameter<6mm)vascular grafts cannot achieve ideal patency.This is mainly due to thrombosis and intimal hyperplasia after transplantation.Silk fibroin(SF)has excellent biocompatibility,good physical and chemical properties,while calcitonin gene-related peptide can promote endothelial cell growth and inhibit smooth muscle cell proliferation.Therefore,calcitonin gene-related peptide modified silk fibrion material applied to small diameter artificial blood vessels,will help promote endothelialization,inhibit intimal hyperplasiaand prevent thrombosis,which is expected to achieve the clinical application of small diameter artificial blood vessels.Silk fibroin was prepared by mixed adipic acid with silk fibroin and using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide(EDC)as cross-linker or treating with ethanol after mixing.The film was then loaded with a calcitonin gene-related peptide using electrostatic adsorption.The effect of adipic acid(AA)-modified silk fibroin(SF)material on the calcitonin gene-related peptide's loading was investigated.It was hoped that the calcitonin gene-related peptide could be stably loaded on silk fibroin without using chemical reaction principle.Ethanol-treated SF/AA film had good hot water stability.When the mass ratio SF:AA=100:1.5,the protein loss rate of the sample was minimized stable.With the increase of SF:AA ratio,the zeta potential of the surface on mixed film gradually decreased from SF:AA=100:0 to 100:2.5,and the surface Zeta potential decreased from-19.5 mV to-27.7 mV,indicating that the material surface after mixing had more negative charge.The composition and structure of ethanol-treated SF/AA films were analyzed byFourier transform infrared spectroscopy(FTIR),X-ray diffraction(XRD)and X-ray photoelectron spectroscopy(XPS).The results of FTIR and XRD indicated that adipic acid induced silk fibroin molecules to form more ?-sheet structures.XPS results showed that silk fibroin material introduced new C=O group.EDC was used to cross-linked adipic acid and silk fibroin,and the silk fibroin filmsl also had good hot water stability.When the SF:AA=100:1.5,silk fibroin molecular reacted fully.The zeta potential of the silk fibroin aqueous solution after the adipic acid cross-linking reaction gradually decreased,from SF:AA=100:0 to 100:2.5,the Zeta potential changed from-2..7 mV to-5.4 mV;the surface Zeta potential of silk fibroin films also gradually decreased,SF:AA=100:0?100:2.5,and the surface Zeta potential decreased from-14.3 mV to-32.6 mV,indicating that the surface of the adipic acid cross-linked and modified silk fibroin films had more negative charges.FTIR and XRD results showed that tadipic acid cross-linked and modified silk fibroin material induced the formation of a more stable secondary structure,mainly by significantly increasing the ?-sheet structure of silk fibroin.With a small amount of alpha helix and beta turn.XPS results showed that the modified silk fibroin material introduced a new C=O,-(CH2)4-group and formed a new amide bond.Comprehensive thermal dissolubility,Zeta potential,FTIR,XRD and XPS results,EDC cross-linked adipic acid modified silk fibroin samples SF:AA=100:0,SF:AA=100:1 and SF:AA=100:2.5 were selected to load calcitonin gene-related peptide.The calcitonin gene-related peptide was loaded on the adipic acid-crosslinked and modified silk fibroin films,which increased with the increase of adipic acid content.The maximum amount of calcitonin gene-related peptide loaded on the modified silk fibroin film with SF:AA=100:2.5 was 500 ng/cm2;which was consistent with the result of Zeta-potential of surface modification of silk fibroin cross-linked with adipic acid.Calcitonin gene-related peptide which was loaded on the adipic acid cross-linked and modified silk fibroin films(SF:AA=100:2.5)was released slowly first at different pH(5,7.4,and 9).Calcitonin gene-related peptide was always released slowly but was released slowly at 12h and quickly and massively after 12h at pH 7.4 and 9.The release of calcitonin gene-related peptide at pH 7.4 which was loaded on the different proportions of adipic acid cross-linked and modified silk fibroin films(SF;AA=100:1,and 100:2.5)were always slowly and the calcitonin gene-related peptide on material(SF:AA=100:0)was released quick during 12h at pH 7.4.