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pH-sensitive polyethylene glycol-conjugated urokinase nanogels (PEG-UKs) were previously reported to be a new form of UK nanogels that could release UK at certain pH values. In this study, we evaluated the effect of PEG-UK targeted to ischemic tissue with microcirculation failure in rat model of ischemic stroke and investigated the possible mechanisms of action.Surgeries were performed to induce persistent middle cerebral artery (MCA) occlusion in adult Sprague–Dawley rats. The pH distribution in the brain was mapped 1 h after ischemia using a needle-type pH micro sensor. The release curve of active UK from PEG-UK was also mapped by a continuous measurement of the peripheral blood. The thrombolytic effects of PEG-UK, when it was administrated 1 h after occlusion, including dynamic changes in the D-dimer level, neurological deficits and infarction volume, were observed. Next, the possible mechanisms underlying these effects were explored, including the BBB integrity and the extent of apoptosis and neurotoxicity. Additionally, the long-term effects of PEG-UK during the four weeks after treatment were evaluated using the dynamic changes in the body weights and clinical scores and the numbers of deaths and hemorrhagic transformations (HTs). To evaluate the systemic side effects of PEG-UK, the fluctuations of cytokines in the liver and kidney were evaluated.On average, MCA occlusion for 1 h induced an approximately 0.49 decline in the pH value (from 7.12 to 6.73), and the lowest value was 6.32 in the predominantly affected region around the cortex. PEG suspended the release of UK from PEG-UK into the circulation. When it was administrated 1 h after occlusion, PEG-UK treatment clearly reduced the severity of neurological deficits in the acute phase (P = 0.001). The relative infarct volume also decreased significantly in PEG-UK rats (P < 0.001). As to the integrity of BBB, the EB leakage in the PEG-UK group was reduced (P = 0.001). Maintenance of the expression of TIMP-1 (P = 0.032) and claudin5 (P < 0.001) and inhibition of MMP9 upregulation (P < 0.001) were observed through both immunohistochemistry and Western blot in the PEG-UK group. Moreover, the expression of both NMDAR1 (P < 0.001) and Caspase9 (P = 0.013) in PEG-UK-treated rats was reduced. As to the long-term prognosis, the rats in PEG-UK group recovered faster and better, and the numbers of deaths and HTs were not increased. No significant fluctuation in IL-1β and TNF-α was found in the PEG-UK-treated rats during the four post-treatment weeks. When PEG-UK was administrated 2.5 h after occlusion, no clearly better outcomes were observed; however, the number of HTs was not increased.Treatment with PEG-UK decreased the severity of ischemic stroke by improving ischemic brain tissue and protecting the BBB and by inhibiting apoptosis and decreasing neurotoxicity. PEG-UK could further inhibit HT through its BBB protection effect. The administration of PEG-UK also improved the long-term prognosis and had no obvious systemic side effects in rats. Our data provide new insights into the thrombolytic treatment of ischemic stroke.