Myerson Jacob Wheatley, He Li, Allen John Stacy, Williams Todd, Lanza Gregory, Tollefsen Douglas, Caruthers Shelton, Wickline Samuel
Washington University in Saint Louis, Department of Biomedical Engineering, USA.
Nanotechnology. 2014 Oct 3;25(39):395101. doi: 10.1088/0957-4484/25/39/395101. Epub 2014 Sep 9.
Restoring an antithrombotic surface to suppress ongoing thrombosis is an appealing strategy for treatment of acute cardiovascular disorders such as erosion of atherosclerotic plaque. An antithrombotic surface would present an alternative to systemic anticoagulation with attendant risks of bleeding. We have designed thrombin-targeted nanoparticles (NPs) that bind to sites of active clotting to extinguish local thrombin activity and inhibit platelet deposition while exhibiting only transient systemic anticoagulant effects. Perfluorocarbon nanoparticles (PFC NP) were functionalized with thrombin inhibitors (either D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone or bivalirudin) by covalent attachment of more than 15 000 inhibitors to each PFC NP. Fibrinopeptide A (FPA) ELISA demonstrated that thrombin-inhibiting NPs prevented cleavage of fibrinogen by both free and clot-bound thrombin. Magnetic resonance imaging (MRI) confirmed that a layer of thrombin-inhibiting NPs prevented growth of clots in vitro. Thrombin-inhibiting NPs were administered in vivo to C57BL6 mice subjected to laser injury of the carotid artery. NPs significantly delayed thrombotic occlusion of the artery, whereas an equivalent bolus of free inhibitor was ineffective. For thrombin-inhibiting NPs, only a short-lived (∼10 min) systemic effect on bleeding time was observed, despite prolonged clot inhibition. Imaging and quantification of in vivo antithrombotic NP layers was demonstrated by MRI of the PFC NP. (19)F MRI confirmed colocalization of particles with arterial thrombi, and quantitative (19)F spectroscopy demonstrated specific binding and retention of thrombin-inhibiting NPs in injured arteries. The ability to rapidly form and image a new antithrombotic surface in acute vascular syndromes while minimizing risks of bleeding would permit a safer method of passivating active lesions than current systemic anticoagulant regimes.
恢复抗血栓表面以抑制正在进行的血栓形成是治疗急性心血管疾病（如动脉粥样硬化斑块糜烂）的一种有吸引力的策略。抗血栓表面将为存在出血风险的全身抗凝提供一种替代方案。我们设计了靶向凝血酶的纳米颗粒（NPs），其可结合到活性凝血部位以消除局部凝血酶活性并抑制血小板沉积，同时仅表现出短暂的全身抗凝作用。通过将超过15000种抑制剂共价连接到每个全氟碳纳米颗粒（PFC NP）上，用凝血酶抑制剂（D-苯丙氨酰-L-脯氨酰-L-精氨酰氯甲基酮或比伐卢定）对全氟碳纳米颗粒进行功能化。纤维蛋白肽A（FPA）酶联免疫吸附测定表明，抑制凝血酶的纳米颗粒可防止游离和凝块结合的凝血酶对纤维蛋白原的裂解。磁共振成像（MRI）证实，一层抑制凝血酶的纳米颗粒可在体外阻止凝块生长。将抑制凝血酶的纳米颗粒体内给药至遭受颈动脉激光损伤的C57BL6小鼠。纳米颗粒显著延迟了动脉的血栓闭塞，而等量的游离抑制剂推注则无效。对于抑制凝血酶的纳米颗粒，尽管凝块抑制时间延长，但仅观察到对出血时间的短暂（约10分钟）全身作用。通过PFC NP的MRI证实了体内抗血栓纳米颗粒层的成像和定量。（19）F MRI证实颗粒与动脉血栓共定位，定量（19）F光谱表明抑制凝血酶的纳米颗粒在受伤动脉中特异性结合并保留。在急性血管综合征中快速形成并成像新抗血栓表面同时将出血风险降至最低的能力，将允许一种比当前全身抗凝方案更安全的钝化活性病变的方法。
