Increasing knowledge of the molecular underpinnings of disease is driving a powerful imperative to deliver agents, such as nucleic acids, to silence expression of pathologic proteins for life-saving treatment of heretofore hopeless illnesses. Although there are promising developments in strategies to deliver cell membrane impermeant nucleic acids, such as siRNA, to disease-causing cells, a safe and efficient method for targeted delivery of these agents has remained elusive. A significant hurdle for gene therapies using vascular delivery is to circumvent the endothelial barrier. We have been developing a unique technology using intravenously injected nucleic acid-loaded microbubbles (MB) which are triggered to cavitate (expand and contract) by ultrasound (US), causing transient permeabilization of the adjacent cell membrane and delivery of the therapeutic carried by the MBs. The potential of this site-specific, non-invasive delivery method is extraordinary, more so because the MBs and US transducer also provide capability for simultaneous real time image-guided navigation of therapy. Despite its promise, the mechanisms underlying the efficacy of ultrasound-triggered MB cavitation (UTMC) as a delivery platform are poorly understood. Without a sound knowledge of the fundamental mechanisms by which safe and effective biotherapeutic delivery is effected by UTMC, its ultimate bedside translation is impossible. We hypothesize that MBs cavitating in the microcirculation impart non-lethal mechanical perturbations on endothelial cells, leading to signaling events that culminate in endothelial barrier hyperpermeability. We propose in vitro studies to systematically interrogate mechanistic pathways, followed by in vivo experiments to investigate UTMC endothelial barrier effects in real time, addressing three Specific Aims: (1) Determine the mechanisms by which UTMC increases endothelial barrier permeability. We will use transwells coated with endothelial cells and manipulate candidate pathways to test the hypothesis that UTMC-induced Ca2+ influx increases endothelial permeability. We will optically measure attendant cellular events using multicolor confocal microscopy, thus correlating barrier function to cell response;; (2) Determine the relationship between in vivo MB cavitation behaviors and transendothelial transport of macromolecules (siRNA). We will use a custom ultra-high speed camera to visualize in vivo US-MB vibrations in the microcirculation to test the hypothesis that MB cavitation causes quantifiable mechanical events, then derive physical principles governing UTMC-mediated hyperpermeability;; (3): Determine extravasation pathways and cellular fate of siRNA-loaded MBs during UTMC in vivo. We will use intravital high-speed multicolor confocal microscopy in cremaste...