Thrombosis, the underlying cause of major fatal conditions like ischemic stroke and myocardial infarction, poses a challenge for treatment. Tissue plasminogen activator (tPA), the primary nonsurgical remedy, faces limitations due to high hemorrhagic risk and a low success rate in clearing thrombi. Neutrophil extracellular traps (NETs), protein-rich structures in blood clots, contribute to tPA resistance. In our microfluidic models of NET-rich clots, we discovered a core-shell structure, with the shell showing significant tPA resistance. The incorporation of DNAse, an enzyme degrading NETs, restored clot tPA susceptibility. Based on these findings, we proposed a localized co-delivery strategy using a liposomal drug delivery platform. Two types of nanoparticles were engineered: tPA-loaded nanoparticles (tPA-NP) and DNAse-loaded nanoparticles (DNAse-NP). Thrombin and neutrophil elastase-cleavable substrates ensured controlled release upon exposure to clot-specific enzymes. tPA-NP demonstrated fibrinolytic potential comparable to solvated tPA, while DNAse-NP exhibited NET-lytic activity. Alone, neither fully addressed clot lysis in functional assays. However, the combined delivery of DNAse-NP and tPA-NP achieved complete clot dissolution within 12 minutes. This approach presents a tunable, modular, and efficacious drug delivery platform for overcoming thrombosis resistant to traditional therapies.
