Combination therapeutic of rt-PA with a neuroprotective agent in the early phase of ischemic stroke is a promising therapeutic strategy. Evidences suggests that bioactive gases such as xenon have profound neuroprotective effects with advantages of ready passage through blood-brain barrier, rapid diffusion and no known side effects. However, use of these gases has been limited by lack of suitable delivery methods. Our previous research has achieved a number of milestones in developing liposomal formulations that are suitable for bioactive gas encapsulation, rt-PA association, clot targeting, thrombolysis and ultrasound-controlled bioactive gas release. The propose of this research is to develop, optimize and test a novel multifunctional echogenic liposomal (ELIP) formulation with thrombolytic agent and neuroprotective bioactive gas co-encapsulation, clot targeting, ultrasound and clot lysis modulated-release of neuroprotective gases for thrombolytic associated neuroprotective therapy. The specific aims are: (1) to optimize the encapsulation of xenon and other neuroprotective gases into ELIP for prolonged circulation time and sufficient bioactive gas release;(2) to evaluate various ultrasound parameters to trigger bioactive gases release from the proposed ELIP formulation, and (3) to optimal and identify gas and gas/cyclodextrin complexes release triggered by the interaction of rt-PA with clot during thrombolysis and once finished aims 1,2 and 3, to test the efficacy of this strategy in restoring cerebral circulation and preventing neuronal damage in an in vivo rat model of ischemic stroke. Multifunctional ELIP containing neuroprotective gases and rt-PA will be administered intravenously with carotid artery ultrasound release bioactive gas in an animal model of ischemia stroke. We anticipate that this strategy will enhance rt-PA therapeutic window and prevent neuronal damage, resulting in strengthened cerebral protection. Techniques developed will provide unique multifunctional vectors for bioactive gas encapsulation that can be easily administered, site-specific delivery and US/thrombolysis-controlled release. This may help overcome problems of incomplete thrombolysis and cerebral ischemia injury seen with current available strategies. Gas encapsulation and delivery technique developed in this research can be translated into entities in which bioactive gas delivery may be beneficial. PUBLIC HEALTH RELEVANCE: The objectives of this proposal are to develop and evaluate a recombinant tissue plasminogen activator (rt- PA) coated echogenic liposomal formulation for both ultrasound and thrombolysis controlled neuroprotective gas delivery to the cerebral circulation for the treatment of ischemic brain injury in association with thrombotic stroke. This may help overcome problems of incomplete thrombolysis and cerebral ischemia injury seen with current available strategies. Gas encapsulation and delivery technique developed in this research can be translated into entities in which bioactive gas delivery may be beneficial.