Abstract 5580: Nanolytic Approach to Prompt Revascularization in Acute Ischemic Stroke
Background: Vascular-constrained fibrin-specific nanolytic agents could permit early revascularization in the setting of acute ischemic stroke. Development of fibrin-targeted nanolytics requires optimized surface co-presentation of homing ligands and fibrinolytic enzymes on nanoparticles without compromising bioactivity.
Objective: The study optimized human urokinase coupling and anti-fibrin homing ligand density for targeted thrombolytic efficacy and demonstrated the concept in vivo.
Methods and Results: Minimal thiolation (N-succinimidyl-S-acetylthioacetate, SATA) of urokinase (i.e., 1 to 3 SATA/molecule) diminished human clot dissolution time relative to the native molecule; greater degrees of thiolation (3 to 6 SATA/molecule) severely decreased activity. For dog clots, which digested slower than human clots, 1 to 3 SATA/urokinase did not further impair fibrinolysis rate. Concomitant coupling of thiolated urokinase and anti-fibrin antibody (50:5, 100:10, 200:20, and 400:40 ratio, respectively) to perfluorocarbon nanoparticles resulted in optimal clot dissolution for the 100:10 and 200:20 formulations. Increasing the surface urokinase-to-antibody ratio from 0:10 to 50:10, 100:10, 200:10, and 400:10 increased particle size from 273 nm (0:10) to 340 nm at the 50:10 and 100:10 enzyme levels, and to more than 400 nm with 200 urokinase molecules per particle. Acoustic microscopy (25MHz) was used to quantify binding of the fibrin-targeted PFC nanoparticles to acoustically transparent human clots. Fibrin targeting with 10 antibodies per particle provided minimal acoustic enhancement in comparison with the relatively increased (p<0.05) but equivalent signals obtained with 20 or 40 antibodies per particle. Dissolution of canine femoral clots, created by electrical injury, was monitored with low MI ultrasound under open circulation conditions following in situ targeting with the nanolytic system. Substantial subjective clot dissolution and fragmentation was observed in three animals over 60 minutes, demonstrating the nanolytic concept.
Conclusion: Nanolytic agents can target fibrinolytic enzymes to lyse nascent thrombus, which may permit early revascularization in acute ischemic stroke with minimal hemorrhagic risk.