Polycaprolactone Based Nanofiber Scaffolds With Hirudin Composite And Its Application Studies On Endothelial Cells And Anticoagulant Effects

Ischemic diseases such as coronary artery disease(CAD)have been one of the leading causes of death from cardiovascular disease,accounting for more than 9.4 million deaths worldwide in 2016.In addition,an increasing number of patients have peripheral atherosclerosis.Patients with advanced disease often require surgical revascularization,such as coronary artery bypass grafting(CABG).The vascular grafts used in CABG are autogenous or artificial vessels,but the source of autogenous vessels is limited,so the development of artificial vessels is particularly necessary,especially the small diameter(≤6mm)artificial vessel grafts still have great limitations in clinical application.The main reasons are easy to form acute thrombus and lack of endothelialization.In this study,polycaprolactone(PCL)nanofiber scaffolds were prepared by electrospinning technology to meet the requirements of antithrombotic and endothelialization.PCL-PDA-rH composite scaffolds were further prepared by immobilization of hirudin(rH)on the surface of the scaffolds by PDA.The effects of hirudin concentration on the physical and chemical properties and anticoagulant ability of the scaffolds and the interaction with human umbilical vein endothelial cells(HUVEC)were studied.On this basis,rH-PLGA /PEI nanoparticles(rHNPs)were prepared by double emulsion solvent volatilization method to further improve the physical and chemical properties of the scaffolds,and the cytocompatibility and anticoagulant ability of the scaffolds were studied.The main contents are as follows:(1)Compared with PCL scaffolds,the hydrophilicity of PCL-PDA-RH composite scaffolds was significantly improved,which was conducive to cell adhesion.Meanwhile,the ultimate tensile strength and Young’s modulus of PCL-PDA-RH composite scaffolds were superior to those of PCL scaffolds,meeting the requirements of vascular mechanics.After soaking in PBS for 30 days,the PCL-PDA-RH composite scaffold could still keep its nanofiber structure intact,which could provide support for the formation of tissue for a long time.(2)PCL-PDA-rH nanofiber composite scaffold can support the growth and proliferation of HUVECs,and the cell spreading state is better than that of pure PCL scaffold.When hirudin concentration was 0.25mg/ m L and 0.5mg/ m L,PCL-PDA-rH composite scaffold had a slight promotion effect on the growth and proliferation of HUVECs.When hirudin solution concentration reached 1mg/m L,PCL-PDA-rH composite scaffold could significantly promote the growth and proliferation of HUVECs.All three groups of PCL-PDA-rH nanofiber composite scaffolds could promote the migration of HUVECs.APTT and TT of the three groups of PCL-PDA-rH nanofiber composite scaffolds were prolonged,while FIB decreased,indicating that the PCL-PDA-rH composite scaffolds had excellent anticoagulant ability,and the anticoagulant effect was enhanced with the increase of hirudin concentration.PCL-PDA-rH composite scaffold has the ability of anti-platelet adhesion and its hemolysis rate is far less than 5%,and the blood compatibility is good.(3)rH-PLGA /PEI nanoparticles(rHNPs)with an average particle size of 358±12nm were successfully prepared by double emulsion solvent evaporation method.rHNPs were uniformly distributed on the surface of the nanofibers by PDA.Compared with PCL scaffolds,the hydrophilicity and mechanical properties of PCL-PDA-rHNPs2.5,PCL-PDA-rHNPs5 and PCL-PDA-rHNPs10 composite scaffolds prepared with different concentrations of rHNPs were significantly improved.The results of in vitro release experiments showed that the three prepared groups of PCL-PDA-rH nanofiber scaffolds released hirudin through the degradation of rHNPs significantly improved the problem of drug release of PCL-PDA-rH nanofiber scaffolds.After degradation in PBS for 30 days,scanning electron microscope observation showed no obvious shedding phenomenon of rHNPs,and the morphology of PCL-PDA-rHNPs nanofiber scaffold did not change significantly.Compared with PCL nanofibrous scaffolds,the anticoagulant ability of the three groups of PCL-PDA-rHNPs nanofibrous scaffolds was significantly improved,APTT was extended by 8s at most,and their hemolysis rate was much lower than 5%,with good blood compatibility.PCL-PDA-rHNPs5 nanofiber scaffold promoted the growth and proliferation of HUVECs.However,PCL-PDA-rHNPs10 nanofiber scaffolds are not conducive to the growth and proliferation of HUVECs due to the high concentration of rHNPs.In this paper,PCL-PDA-rH and PCL-PDA-rHNPs nanofiber composite scaffolds have been successfully prepared.The composite scaffolds have good mechanical properties and biological activity,and are expected to become a new vascular tissue engineering materials.