Preparation and characterization of anionic oligopeptide-modified tissue plasminogen activator for triggered delivery: An approach for localized thrombolysis

Purpose: The study sought to synthesize anionic peptide-conjugated tissue plasminogen activator (tPA) for its targeted/triggered delivery, where tPA's activity would be masked in the circulation and regenerated at the thrombus site by a commonly used anticoagulant, heparin, to minimize tPA associated bleeding complications. Methods: tPA was conjugated to Polyglutamate, and the activity of oligoanion-modified tPA was tested by fibrinolytic assay. Separately human serum albumin (HSA) was conjugated to protamine and the formation of its electrostatic complex with anionic peptide was monitored by Forster Resonance Energy Transfer (FRET). The masking of tPA-activity via steric hindrance created by albumin, and subsequent regeneration with therapeutic dose of heparin was tested by enzymatic assay. Stability of 'camouflaged-tPA' was determined in human plasma. Using fluorescence microscope, binding of camouflaged-tPA with activated platelets was monitored. Heparin modulated clot-lysis was evaluated in human blood clot. Results: The anionic tPA retained similar to 97% activity of the unmodified-tPA. FRET experiments confirmed the electrostatic interaction between polyglutamate and protamine which was subsequently reversed by heparin. Complexation with HSA-protamine masked similar to 60% of tPA activity which was fully regenerated by heparin. The complex retained its prodrug character in human plasma after incubation at 37 degrees C. Fluorescence microscopic study confirmed binding of the construct with activated platelets. In lysing human clot, the camouflage could mask tPA-activity until it was triggered at a heparin level of 0.4 U/mL. Conclusion: Oligoanion-modified tPA could be used for targeted/triggered delivery where its enzymatic activity could be masked by HSA-protamine conjugate and successfully regenerated by therapeutic dose of heparin. (C) 2012 Elsevier Ltd. All rights reserved.