Antibody-aptamer conjugate for shear-triggered tissue plasminogen activator release and thrombolysis

Pathological blood clots cause life-threatening events, including ischemic stroke and myocardial infarction. Tissue plasminogen activator (TPA), a clinically approved fibrinolytic agent, remains a frontline therapy for clot dissolution. However, its short plasma half-life and requirement for high systemic doses pose serious risks, particularly hemorrhagic complications. To address these limitations, a shear-responsive delivery platform was engineered for controlled TPA release under pathological high-shear conditions. This system, termed Single-MOlecule-based materials with structures and functions REsponsive to Shear (SMORES), consists of a shear-amplifying microparticle conjugated to a TPA-binding aptamer and functionalized with a monoclonal antibody against integrin αIIbβ3 as a platelet-targeting element. Under elevated shear conditions, the immobilized aptamer undergoes mechanical unfolding, triggering TPA release. Optical tweezers-based single-molecule force measurements probing aptamer unfolding and refolding under constant force and varied relaxation periods revealed a narrow range of threshold forces (4-10 pN) governing the transition between folded and unfolded states. Under defined flow conditions in a microfluidic channel, single-molecule flow experiments demonstrated a flow-dependent TPA release profile, with the maximum mean release observed at 50 µL/min. COMSOL-based fluid dynamics simulations estimated a corresponding tether force of 4.76 pN and a shear rate of 3,512.9 s^-1, consistent with pathological shear levels observed in stenotic vessels and aligning with optical tweezers predictions. The enzymatic activity of released TPA was confirmed by a clot dissolution assay, with the highest mean functional TPA-equivalent activity observed under the same flow condition. Together, these results demonstrate that TPA-SMORES enables mechanistically controlled, shear-triggered release of functional TPA, supporting its potential as a strategy for improving the spatial control of thrombolytic therapy.