Nanoparticle Analysis of Enveloped Virus Entry Pathways

描述（由申请人提供）：在纳米技术领域，已经对可以容纳药物和其它治疗剂的纳米尺寸颗粒的构建给予了很多关注。然而，除了使用静态配体如抗体和小肽之外，在纳米颗粒（NP）的特异性靶向方面几乎没有做过工作。这是NP设计的重要组成部分。通过选择性地将药物递送到一个细胞而不是另一个细胞，药物的功效可以大大提高，从而减少所需的剂量和潜在的副作用。病毒与理想化的NP有许多结构和物理上的相似之处。它们的直径都约为100 nm，具有亲水性外壳，循环寿命长。然而，病毒实现了很少有NP设计可以实现的壮举;它们使用病毒包膜蛋白（env）有效地靶向特定组织和细胞。envs也做一些设计的配体可能永远无法实现的事情;它们靶向特定的细胞内吞途径，进入细胞器，如高尔基体和内质网。Env也是简单的膜融合引擎，其感测周围环境以触发其货物在细胞质中的特定位点的释放。在这个提议中，我们将优化技术来构建包被在Friend鼠白血病病毒、委内瑞拉马脑炎病毒（NIH类别B剂）和埃博拉病毒（NIH类别A剂）的envs中的荧光NPs。每一个env都将病毒定位于独特的细胞受体和内吞进入途径。我们将使用最先进的共聚焦显微镜和生物化学方法研究涂层envs的细胞和细胞器靶向特性。再加上一种靶向纳米粒子的新方法，这项工作将使人们更好地了解envs在感染中的作用以及每种病毒使用的进入途径。这将为抗病毒药物开发确定新的靶点。NP还将用于快速病毒诊断测定。 项目叙述：高度纯化的病毒膜将被用于使用一种新的方法来包覆纳米颗粒，该方法保留了病毒包膜蛋白的有用特征。然后将测试纳米颗粒与细胞特异性相互作用并进入细胞的能力。这将大大有助于靶向药物治疗和疫苗设计的发展。

DESCRIPTION (provided by applicant): In the field of nanotechnology, much attention has been paid to the construction of nanometer- sized particles that can harbor drugs and other therapeutic agents. However, beyond the use of static ligands such as antibodies and small peptides, little work has been done on the specific targeting of the nanoparticles (NPs). This is an important component of NP design. By selectively delivering drugs to one cell over another, the efficacy of the drug can be drastically improved and consequently reduce the dosage needed and potential side effects. Viruses share many structural and physical similarities to an idealized NP. They are both about 100 nm in diameter, have hydrophilic shells and have long circulatory lives. However, viruses achieve a feat that few NP designs can; they efficiently target specific tissues and cells using virus envelope proteins (envs). The envs also do something that designed ligands may never achieve; they target specific cellular endocytic pathways, to access organelles such as the Golgi and the endoplasmic reticulum. Envs are also simple membrane fusion engines that sense the surrounding environment to trigger the release of their cargo in the cell cytoplasm at specific sites. In this proposal, we will optimize techniques to construct fluorescent NPs coated in the envs of Friend murine leukemia virus, Venezuelan equine encephalitis virus (NIH category B agent) and Ebola (NIH category An agent). Each env targets virus to unique cell receptors and endocytic entry pathways. We will investigate the cell and organelle targeting properties of the coated envs using state-of-the-art confocal microscopy and biochemical methods. Together with a novel way to target NPs, this work will allow a better understanding of the role of envs in infection and the entry pathways used by each virus. This will identify new targets for antiviral drug development. The NPs will also be useful in rapid virus diagnostic assays. Project Narrative: Highly purified virus membranes will be used to coat nanoparticles using a new method that preserves the useful features of the virus envelope proteins. The nanoparticles will then be tested for their ability to specifically interact with and enter cells. This will greatly aid in the development of targeted drug therapies and vaccine design.