A Nanomedicine Approach for Targeted Thrombolysis

In thrombo-occlusive vascular pathologies like myocardial infarction and stroke, rapid thrombolysis is necessary for restoring blood flow to critical organs. To accomplish this, it is advantageous to localize the delivery and action of the thrombolytic drugs specifically at the occluded (clot) site, to minimize the risks of systemic coagulopathy and hemorrhage from indiscriminate drug action. To this end, it is hypothesized that site-specific thrombolytic delivery can be achieved by engineering of nanoparticles that can specifically target the thrombus by actively binding to thrombus-associated active platelets and thereby allow controlled drug release via thrombus-relevant enzyme trigger. Accordingly, liposomal nanovehicles that can actively bind to thrombus-associated active platelets were developed in the Sen Gupta laboratory by decorating liposome surface with peptides that specifically bind integrin GPIIb-IIIa and P-selectin on active platelets. Building on this work, the current study focused on investigating the encapsulation and enzyme-triggered release of a model payload from such liposomal vehicles. For this, the model payload carboxyfluorescein (CF) was encapsulated in the aqueous core of liposomes and the encapsulation efficiency (EE) was measured by fluorescence spectrometry to be 74.70%±0.97. Subsequently the CF-loaded liposomes were subjected to incubation with the thrombus-relevant enzyme phospholipase A2 (PLA₂) at a physiologically-relevant concentration of 2.5 ng/mL, and resultant CF release at pH 7.4 in 5mM Tris-HCl buffer at 37°C was monitored over time for 5 hours by monitoring CF fluorescence. These studies showed a twofold increase in CF release from liposomes triggered by PLA₂ as compared to the absence of PLA₂. Subsequently, a relevant thrombolytic drug, streptokinase (SK), was encapsulated in these platelet-targeted liposomal constructs and PLA₂-triggred targeted thrombolysis was evaluated in vitro, using platelet-rich thrombi. Our results demonstrate the potential of platelet-targeted enzyme-triggerable vehicles as a novel nanomedicine technology for site–specific thrombolysis.