Thrombolytic therapy for ischemic stroke still has several limitations, such as a narrow therapeutic time window and adverse effects. Therapeutic hypothermia is a neuroprotective strategy for stroke. In this study, we developed pH/temperature dual-responsive protein-polymer conjugates (PEG-uPA-PEG-PPG-PEG) by modifying a urokinase-type plasminogen activator (uPA) with polyethylene glycol (PEG) and poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEG-PPG-PEG, a thermosensitive polymer) via pH-sensitive imine bonds and disulfide bonds, respectively. At 37 °C and pH 7.4 (normothermia and physiological pH), PEG-uPA-PEG-PPG-PEG exhibits antiprotease hydrolysis and masked bioactivity of uPA due to the protective effect of the polymer segments wrapped around the protein surface. However, at 33 °C and pH 6.0 (hypothermia and pH at the thrombotic site), uPA loses the protective effect and recovers its bioactivity due to PEG dissociation and PEG-PPG-PEG stretching. The masked bioactivity of uPA at normothermia and physiological pH could reduce the risk of acute hemorrhage complication, and the recovery of protein activity at acidic pH and 33 °C is of great significance for thrombolytic therapy at mild hypothermia. Thus, PEG-uPA-PEG-PPG-PEG provides promising potential for therapeutic hypothermia in ischemic stroke.
Keywords: pH-responsive; protein–polymer conjugate; stroke; therapeutic hypothermia; thermoresponsive; urokinase-type plasminogen activator.