Construction of Antithrombotic Tissue-Engineered Blood Vessel via Reduced Graphene Oxide Based Dual-Enzyme Biomimetic Cascade

Thrombosis is one of the biggest obstacles in the clinical application of small-diameter tissue-engineered blood vessels (TEBVs). The implantation of an unmodified TEBV will lead to platelet aggregation and further activation of the coagulation cascade, in which the high concentration of adenosine diphosphate (ADP) that is released by platelets plays an important role. Inspired by the phenomenon that endothelial cells continuously generate endogenous antiplatelet substances via enzymatic reactions, we designed a reduced graphene oxide (RGO) based dual-enzyme biomimetic cascade to successively convert ADP into adenosine monophosphate (AMP) and AMP into adenosine. We used RGO as a support and bound apyrase and 5'-nucleotidase (5'-NT) on the surface of RGO through covalent bonds, and then, we modified the surface of the collagen-coated decellularized vascular matrix with the RGO-enzyme complexes, in which RGO functions as a platform with a large open surface area and minimal diffusion barriers for substrates/products to integrate two catalytic systems for cascading reactions. The experimental results demonstrate that the two enzymes can synergistically catalyze procoagulant ADP into anticoagulant AMP and adenosine successively under physiological conditions, thus reducing the concentration of ADP. AMP and adenosine can weaken or even reverse the platelet aggregation induced by ADP, thereby inhibiting thrombosis. Adenosine can also accelerate the endothelialization of TEBVs by regulating cellular energy metabolism and optimizing the microenvironment, thus ensuring the antithrombotic function and patency of TEBVs even after the RGO-enzyme complex loses its activity.