Caveolar Transport of Therapeutic Nanoparticles The ultimate objective of this Career Development Award application is to provide Dr. Zhenjia Wang, a quantitative scientist in the fields of nanotechnology and high resolution microscopy, the opportunity to adapt his expertise to the significant problem of developing effective therapies for lung vascular injury. During this award Dr. Wang will be mentored to be successfully prepared for an tenure- track faculty position as an independent scientist. This application outlines the mechanisms through which Dr. Wang will be provided the opportunity to develop into a biomedical scientist with a comprehensive understanding of the complexities of molecular/cellular biology and lung vascular biology, and together with the skills needed for intravital microscopy in analyzing both health and disease tissue. The acquisition of this important body of biological information together with the technical expertise of intravital microscopy will provide Dr. Wang with the necessary tools to identify specific critical molecular interactions between carefully designed therapeutic nanoparticles and the vascular endothelium required for effective transit to sites of vascular injury in lung. These newly acquired bodies of knowledge will allow Dr. Wang to develop novel therapeutic approaches for delivering protein-conjugated nanoparticles efficiently and effectively across the vessel wall and to establish the scientific underpinnings for exploiting the caveolae-mediated pathway of transcytosis for the delivery of therapeutic nanoparticles. Understanding how nanoparticles are transported across the vessel wall is critical for the optimal design of targeted nanoparticles conjugated to therapeutic molecules to specifically and efficiently get biologics to sites of pathology. We postulate that one way to efficiently deliver therapeutic nanoparticles to sites of infection, is b targeting nanoparticles to caveolar invaginations on the surface of endothelial cells. Caveolae are membrane invaginations, 50-100 nm in diameter that bud internally from the membrane to form intracellular vesicles. Based on our preliminary results that caveolae can mediate the internalization albumin-conjugated nanoparticles, we will determine to what degree caveolae- mediated transport of antibody-conjugated nanoparticles is dependent on nanoparticle size, whether nanoparticles require albumin-coating for targeting, and what specific molecular machinery this transport requires. To this end we will address the following aims: Aim 1: To determine the optimal properties of nanoparticles required for efficient caveolar transport of anti TNF- mAb-conjugated nanoparticles across the vascular endothelial barrier. We will determine (i) the size-dependence of the nanoparticle component on transcytosis and (ii) if albumin coating of the nanoparticle component is required to activate vectoral (from apical to basal side of the endothelial monolayer) trafficking of the cargo laden caveolae. We will measure anti-TNF- mAb- conjugated nanoparticle transport across a monolayer of endothelial cells grown in a Transwell chamber and live cell imaging, to elucidate the role of caveolae in mediating transport of antibody- conjugated nanoparticles. Aim 2: To identify molecular mechanisms of caveolae-mediated transcytosis of anti-TNF- mAb-conjugated nanoparticles. We will address the specific roles of (i) the caveolar neck scission protein dynamin-2, (ii) Src activation, which induces a endocytosis signal by the phosphorylation of dynamin-2 and Tyr14 on caveolin-1 (the primary caveolar membrane-associated protein), and (iii) the albumin-binding protein, gp60, localized to caveolar invaginations, in mediating transendothelial transport of anti-TNF- mAb-conjugated nanoparticles. Aim 3: To determine the role of caveolae in mediating transport of anti-TNF- mAb- conjugated nanoparticles in microvessels of mice. Using intravital microscopy we will determine the role of caveolae in mediating nanoparticle transport in cremaster muscle venules of wild type and caveolin-1 knockout mice, shown to lack caveolae. We will test the usefulness of caveolae-mediated transcytosis of nanoparticles for efficient and effective delivery of anti-TNF- mAb in vivo.