DELIVERY OF PROTEINS INTO CYTOSOL USING LLO-LIPOSOMES

描述(改编自《调查者摘要》)： 细胞是药物传递系统的重要靶点，因为许多 治疗剂的目的是干预或修饰特定的分子 发生在细胞质中的事件。许多这样的治疗方法 药物，特别是近年来发现或设计的药物，是 固有的膜-由于它们的电荷或高分子 重量。这项提议的长期目标是制定战略 以及能够将这类分子输送到胞浆中的方法 单元格空间。这项提议的主要思想是引进细胞生物学 将李斯特菌和生物化学转化为药剂学，即采取最低限度的 兼性胞内细菌李斯特氏菌的成分 单核细胞增多症，并构建基于脂质体的胞浆给药载体。 溶李素O(LLO)，这是逃逸的必要条件和充分条件 李斯特菌进入胞浆，将被提纯并并入脂质体中。 含有LLO的脂质体，它模仿李斯特菌的释放方式 进入到它们生长的细胞质中，将根据它们的 将大分子输送到细胞质中的能力。虽然建议的 战略应适用于交付其他类型的 目前的提案将重点放在大分子的交付上 蛋白质类大分子。两种类型的蛋白质在以下情况下显示效果 将被送入细胞质的将被检测：(1)一种抗原蛋白，它 诱导特异性细胞毒性T淋巴细胞(CTL)的激活；和(2)毒素 可以抑制蛋白质合成的物质。胞液递送的量将 用一种新开发的生物化学方法来测量，这种方法可以监测 细胞学递送的分子占总数的百分比 单元格关联量。体外抗原呈递分析也将是 用来测量进入胞浆的相对效率 抗原呈递途径。作为一种疫苗，这具有直接的意义 运送工具，因此将扩展到一只成熟的小鼠 淋巴细胞脉络膜脑膜炎病毒(LCMV)感染模型。诱导 针对LCMV核蛋白(NP)的CTL LIO-脂质体和随后针对病毒的保护将是 在老鼠身上进行监测。除了运送抗原蛋白外，一种 膜封闭的毒素，胶凝素，将被封装在里面 并在细胞生长抑制实验中进行检测。脂质体 循环时间较长的地层将使用LLO，以及 他们的胞浆输送效率也将得到测试。同时， 将研究LLO通过孔道形成裂解内涵体的机理。 使用生物物理和分子生物学方法。

DESCRIPTION (Adapted from Investigator's Abstract): The cytosolic space of cells is an important target for drug delivery systems since many therapeutic agents are aimed at intervening or modifying specific molecular events occurring in the cytoplasm of cells. Many of these therapeutic agents, particularly the ones discovered or designed in recent years, are intrinsically membrane-permeant due to their charge or high molecular weight. The long term objective of this proposal is to develop strategies and methods that can deliver molecules in this category into the cytosolic space of cells. The main idea of this proposal is to import cell biology and biochemistry of Listeria into pharmaceutics, i.e., to take a minimal component from a facultative intracellular bacterium, Listeria monocytogenes, and construct a liposome-based cytosolic delivery vehicle. Listeriolysin O (LLO), which is necessary and sufficient in the escape of Listeria into cytosol, will be purified and incorporated into liposomes. The LLO-containing liposomes, which mimic how Listeria deliver themselves into the cytosol where they grow, will be characterized in terms of their ability to delivery macromolecules into the cytosol. Although the proposed strategy should be applicable to the delivery of other types of macromolecules, the current proposal will focus on the delivery of proteinaceous macromolecules. Two types of proteins that show effect when delivered into the cytosol will be tested: (1) an antigenic protein that induces activation of specific cytotoxic T lymphocytes (CTL); and (2) toxins that can inhibit protein synthesis. The amount of cytosolic delivery will be measured by a newly developed biochemical assay which monitors the percent of cytosolically delivered molecules out of the total cell-associated amount. In vitro antigen presentation assays will also be employed to measure relative efficiency of delivery into the cytosolic pathway of antigen presentation. This has direct implications as a vaccine delivery vehicle, and will therefore be extended to a well-established mouse model of lymphocytic choriomeningitis virus (LCMV) infection. Induction of CTL, specific against nucleoprotein (NP) of LCMV, by NP-containing LLO-liposomes and subsequent protection from viral challenge will be monitored in mice. In addition to delivering antigenic proteins, a membrane-impermeant toxin, gelonin, will be encapsulated inside LLO-liposomes and tested in the cell growth inhibition assays. The liposome formations that have a long circulation time will be made with LLO, and their efficiency of cytosolic delivery will also be tested. Concurrently, the mechanism of endosome lysis by LLO via pore formation will be studied using biophysical and molecular biological methods.