Steric modulation of thrombolytic enzymes for ischemic stroke

Abstract

Acute ischemic stroke (AIS) remains a leading cause of death and permanent disability worldwide. Despite being the only FDA-approved treatment for AIS since 1996, recombinant tissue plasminogen activator (tPA) is associated with significant risks, particularly intracranial hemorrhage (ICH). This study aimed to address these limitations through two innovative approaches: optimizing tPA and developing a nanoparticle-based delivery system for a neuroprotective agent. We successfully achieved domain-specific chemical modification of tPA, including Cys83 blocking and enzymatic oxidation of terminal galactose residues. The modified tPA was conjugated with low molecular weight heparin (LMWH), resulting in a novel tPA-LMWH complex. In vitro studies using human brain microvascular endothelial cells (HBMECs) demonstrated that while native tPA significantly compromised monolayer integrity, the sterically hindered tPA-LMWH complex markedly attenuated these detrimental effects. This was evidenced by reduced permeability and maintained trans-endothelial electrical resistance (TEER). Concurrently, we developed minocycline-loaded niosomes as a neuroprotective agent delivery system. Two niosomal compositions were prepared using the thin-film hydration technique and characterized for particle size, entrapment efficiency, and release kinetics. Both formulations exhibited favorable physicochemical properties, with mean particle sizes around 250-265 nm and high drug encapsulation efficiencies (80-87%). In vitro release studies demonstrated sustained release profiles, with composition-dependent variations in release rates. These findings lay the foundation for a combinatorial therapy that could potentially enhance the efficacy and safety of AIS treatment. The sterically hindered tPA formulation offers a promising approach to mitigate ICH risk, while minocycline-loaded niosomes present a novel strategy for targeted delivery of neuroprotective agents. Future research should focus on in vivo studies and further optimization of these systems for potential clinical application.