Study On The Preparation And Properties Of Thrombin Responsive Antithrombotic Surface Coating

Foreign material surface-induced thrombosis remains the major cause of failure of implanted blood contacting medical devices.The incorporation of bioactive agents capable of inhibiting thrombosis is one of the main strategies for developing antithrombotic biomaterials.However,in most cases,antithrombotic agents are difficult to maintain their activity efficiently in the complicated blood microenvironment.In addition,the exposure of blood to the antithrombotic agent may cause disorders of hemostasis under normal conditions.Ideally,a blood contacting surface should initiate antithrombotic activity only when thrombus begins to form,while it remains ‘‘bioinert’’ in the normal blood environment.Such surface may ensure that the limited antithrombotic agents make maximal effect at the right time.At the same time,the surface should gradually covered by the endothelium layer which endow the surface with full blood compatibility.To construct such an ideal antithrombotic surface,a thrombosis-responsive fibrinolytic surface coating is developed first in this thesis.The surface keeps bio-inert in normal blood microenvironment while initiating fibrinolytic activity so as to lysis the fibrin(nascent clot)once thrombus begins to form.Subsequently,bioactive molecules capable of promoting endothelialization are further introduced to fulfill the requirement of long-term blood compatibility.The thrombosis-responsive system should be sensitive to the specific change in blood microenvironment caused by coagulation.Blood coagulation is accompanied with the generation of a series of coagulation factors.Among these factors,thrombin is of the greatest interest since it is the common product of both the intrinsic pathway and the extrinsic pathway and it is the direct enzyme that leads to the formation of fibrin.Some thrombin substrate peptides and thrombin aptamers have been used as cross-linkers to construct drug carriers capable of delivering antithrombotic agents to the target site.Such thrombin-responsive drug release concept is utilized in this thesis to construct thrombosis-responsive antithrombotic coating.In the first part of work,a thrombin-responsive fibrinolytic nanocapsule is developed.Two monomers,acrylamide(AAm)and N-(3-aminopropyl)methacrylamide hydrochloride(APM)are adsorbed on the surface of tissue plasminogen activator(t-PA)followed by in situ polymerization and crosslinking to form a thrombin-degradable hydrogel shell,named t-PA nanocapsules(t-PA NCs).The NCs prepared under optimized condition can shield t-PA completely.In the presence of thrombin,the activity of t-PA increases almost linearly with the thrombin concentration and a satisfied thrombin responsive fibrinolytic activity is proved.Such t-PA NCs can be employed not only for surface coating in the following work,but also for t-PA delivery with the potential benefit of prolonged circulation time and site-specific release at the thrombus.Subsequently,a thrombosis-responsive surface coating based on the t-PA NCs developed above is prepared.The t-PA NCs are covalently attached to various blood contacting material surfaces through a polydopamine adhesive layer.The resulting surfaces are treated with the antifouling agent glutathione(GSH)to prevent further interactions with blood/plasma components.The t-PA NCs/GSH coated surface remains inert in normal plasma microenvironment while releasing t-PA at a constant rate and promoting fibrinolysis when thrombin is present.The fibrinolytic activity increases with the increasing thrombin concentration,which demonstrates that the fibrinolytic activity can be regulated by the extent of coagulation.More importantly,the t-PA NCs/GSH modified surfaces can release sufficient t-PA for clot lysis in response to endogenous thrombin generated during the clotting process in whole blood.This work constitutes the first report of an antithrombotic coating whose function is triggered and regulated,respectively,by the appearance of thrombin and the extent of coagulation.Finally,the surface coating is further designed as to mimic the antithrombotic function of endothelium and to pro-endothelialization for long-term hemocompatibility.t-PA NCs and the heparin/poly-L-lysine nanoparticles(HPs)co-immobilized to polydopamine coated surfaces.The surface shows thrombin-responsive fibirinolytic activity,and in addition,prolongs the clotting time and promotes the adhesion and growth of endothelial cells due to the presence of heparin.Such surface coating is expected to meet different requirements at different stages of blood contacting applications.