OC 53.3 - Direct Delivery of Plasmin using Clot-anchoring Thrombin-responsive Nanoparticles for Targeted Fibrinolytic Therapy

Background: Fibrin-rich clot formation in thrombo-occlusive pathologies is currently treated by systemic administration of plasminogen activators (e.g. tPA), to convert fibrin-associated plasminogen to plasmin for fibrinolytic action. However, this conversion is not restricted to clot-site only, but also occurs on circulating plasminogen, causing systemic fibrinogenolysis and bleeding risks. To address this, several past research approaches, including ours, have studied delivery of tPA packaged within clot-targeted nanoparticles for targeted action. Building on this promising strategy we aimed to explore whether a clot-targeted and clot-responsive nanoparticle platform can be designed to deliver plasmin directly, instead of tPA.

Aims: We aimed to design and evaluate a nanomedicine system that can: (1) Target clots via specific binding to activated platelets and fibrin, (2) Encapsulate plasmin instead of tPA as a direct fibrinolytic agent, and (3) Release this plasmin in a clot-responsive manner triggered by thrombin for clot-localized action.

Methods: Clot-targeted thrombin-cleavable nanoparticles (CTNPs) were manufactured using self-assembly of peptide-lipid conjugates. Plasmin-loading and its thrombin-triggered release from CTNPs were characterized by UV-Visible spectroscopy. CTNP-targeting to clots under flow was studied using microfluidics and fluorescence microscopy. Fibrinolytic effect of CTNP-delivered plasmin was studied in vitro using BioFlux microfluidic imaging and D-dimer analysis, and in vivo in a zebrafish thrombosis model.

Results: Plasmin-loaded CTNPs significantly bound to clots under shear flow and showed thrombin-triggered enhanced relase of plasmin. BioFlux studies confirmed that thrombin-triggered plasmin released from CTNPs rendered fibrinolysis similar to free plasmin, further corroborated by D-dimer analysis. In the zebrafish model, CTNP-delivered plasmin accelerated time-to-recanalization, or completely prevented occlusion when infused prior to thrombus formation.

Conclusion(s): Considering that the very short circulation half-life ( < 1 second) of plasmin prevents its systemic use but also makes it safer without off-target drug effects, clot-targeted delivery of plasmin using CTNPs can potentially enable safer and more efficacious fibrinolytic therapy.