Caveolar Transport of Therapeutic Nanoparticles

描述(申请人提供)：治疗性纳米颗粒的腔内运输本职业发展奖申请的最终目标是为纳米技术和高分辨率显微镜领域的量化科学家王振佳博士提供机会，使他的专业知识适用于开发有效疗法这一重大问题 肺血管损伤。在这个奖项期间，王博士将被指导成功地为终身教职做好准备，成为一名独立科学家。这份申请概述了王博士将有机会发展成为一名生物医学科学家的机制，他对分子/细胞生物学和肺血管生物学的复杂性有全面的了解，并拥有活体显微镜分析健康和疾病组织所需的技能。获取这一重要的生物学信息，再加上活体显微镜的技术专长，将为王博士提供必要的工具，以确定精心设计的治疗纳米颗粒与有效传输到肺血管损伤部位所需的血管内皮细胞之间的特定关键分子相互作用。这些新获得的知识将使王博士能够开发新的治疗方法，有效地将蛋白质结合的纳米颗粒输送到血管壁上，并为利用小窝介导的细胞转运途径输送治疗性纳米颗粒奠定科学基础。了解纳米颗粒是如何通过血管壁运输的，对于优化设计与治疗分子结合的靶向纳米颗粒，以特定和有效地将生物物质输送到病理部位至关重要。我们推测，有效地将治疗性纳米颗粒输送到感染部位的一种方法是将纳米颗粒定位于内皮细胞表面的凹陷。小凹是膜内陷，直径50-100 nm，从膜内部萌发，形成细胞内小泡。根据我们的初步结果，小窝可以介导白蛋白偶联纳米颗粒的内化，我们将确定小窝介导的抗体偶联纳米颗粒的运输在多大程度上依赖于纳米颗粒的大小，纳米颗粒是否需要白蛋白涂层来靶向，以及这种运输需要什么特定的分子机制。为此，我们将解决以下目标：目标1：确定有效的空泡转运抗病毒所需的纳米颗粒的最佳性质 肿瘤坏死因子-�单抗偶联纳米粒穿过血管内皮细胞屏障。我们将确定(I)纳米颗粒成分的尺寸依赖于跨细胞作用，以及(Ii)是否需要纳米颗粒成分的白蛋白涂层来激活载货小窝的矢量(从内皮单层的顶端到底端)的运输。我们将测量抗肿瘤坏死因子-�单抗结合的纳米颗粒在Transwell小室中培养的内皮细胞单层中的运输和活细胞成像，以阐明小凹在抗体结合纳米颗粒的中介运输中的作用。目的：探讨抗肿瘤坏死因子-�单抗偶联纳米粒小窝介导细胞转运的分子机制。我们将讨论(I)腔泡颈断裂蛋白Dynamin-2，(Ii)src激活，它通过Dynamin-2和Tyr14的磷酸化诱导小窝蛋白-1(主要的腔泡膜相关蛋白)的内吞信号，以及(Iii)白蛋白结合蛋白gp60，定位于腔泡内陷，在介导抗肿瘤坏死因子-�单抗结合的纳米颗粒的跨内皮运输中的特定作用。目的：研究小窝在介导抗肿瘤坏死因子-�单抗偶联纳米粒在小鼠微血管内转运中的作用。利用活体显微镜，我们将确定小凹在野生型和小凹-1基因敲除小鼠的提睾肌小静脉中介导纳米颗粒运输的作用，这些小凹被证明缺乏小凹。我们将测试小窝介导的纳米粒转运在体内高效传递抗肿瘤坏死因子-�单抗的有效性。

DESCRIPTION (provided by applicant): 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.