The goal of the present work was to design and test an acute-use nanoparticle-based antithrombotic agent that exhibits sustained local inhibition of thrombin without requiring a systemic anticoagulant effect to function against acute arterial thrombosis. To demonstrate proof of concept, we functionalized the surface of liposomes with multiple copies of the direct thrombin inhibitor, d-phenylalanyl-l-prolyl-l-arginyl-chloromethyl ketone (PPACK), which exhibits high affinity for thrombin as a free agent but manifests too rapid clearance in vivo to be effective alone. The PPACK-liposomes were formulated as single unilamellar vesicles, with a diameter of 170.78 ± 10.59 nm and a near neutral charge. In vitro models confirmed the inhibitory activity of PPACK-liposomes, demonstrating a KI′ of 172.6 nM. In experimental clots in vitro, treatment of formed clots completely abrogated any further clotting upon exposure to human plasma. The liposomes were evaluated in vivo in a model of photochemical-induced carotid artery injury, resulting in significantly prolonged arterial occlusion time over that of controls (69.06 ± 5.65 min for saline treatment, N = 6, 71.33 ± 9.46 min for free PPACK treated; N = 4, 85.75 ± 18.24 min for precursor liposomes; N = 4, 139.75 ± 20.46 min for PPACK-liposomes; P = 0.0049, N = 6). Systemic anticoagulant profiles revealed a rapid return to control levels within 50 min, while still maintaining antithrombin activity at the injury site. The establishment of a potent and long-acting anticoagulant surface over a newly forming clot with the use of thrombin targeted nanoparticles that do not require systemic anticoagulation to be effective offers an alternative site-targeted approach to the management of acute thrombosis.
- drug delivery