A single-molecule protein nanocapsule for targeted delivery of diverse cargo

用于靶向递送多种货物的单分子蛋白质纳米胶囊

• 批准号: 10374167
• 负责人: MALCOLM A LEISSRING
• 金额: $ 23.14万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10374167
• 关键词: Adopted; Affinity; Binding; Binding Sites; C-terminal; Calmodulin; Cells; Chemicals; Chimeric Proteins; Confocal Microscopy; Cysteine; Cytosol; Development; Drug Delivery Systems; Encapsulated; Endosomes; Environment; Exposure to; Extracellular Protein; Extracellular Space; Extravasation; Flow Cytometry; HIV; Immobilization; In Vitro; Insulinase; Lead; Mediating; Metalloproteases; Methods; Modification; Molecular Conformation; Mutation; Oligonucleotides; Oxidation-Reduction; Oxides; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Positioning Attribute; Proteins; Resources; Specificity; Structure; System; Therapeutic; Variant; Zinc; base; capsule; controlled release; cytotoxic; disulfide bond; experimental study; extracellular; innovative technologies; nanocapsule; novel; prevent; single molecule; targeted delivery

PROJECT SUMMARY/ABSTRACT This proposal explores the feasibility of developing a novel compound delivery system based on insulin- degrading enzyme (IDE), a zinc-metallopeptidase with a unique, nanocapsule-like structure. IDE resembles a clamshell, comprising two bowl-shaped domains connected by a “hinge” region, which allows it to adopt “open” and “closed” conformations. When closed, the protease features a large internal chamber, ~13,000-Å3 in volume, that is completely encapsulated and can accommodate cargo as large as ~8000 Da. This proposal has two principal objectives: (1) to explore the feasibility of using IDE for the encapsulation of cargo and for regulated release through extensive in vitro characterization; and (2) to develop a novel system targeting cargo exclusively to the cytosol of cells, while explicitly preventing delivery to the extracellular space. To encapsulate cargo in a reversible and controllable manner, we will generate variants of IDE containing two cysteines (S132C/E817C) positioned such that they form a disulfide bond exclusively when the protease is in the closed conformation. This double-cysteine mutation constitutes a redox-sensitive “latch” that, as shown previously, keeps IDE “locked” in the closed position in an oxidizing environment (e.g., the extracellular space) and becomes “unlocked” only when exposed to a reducing environment (e.g., cytosol). Purified nanocapsules will be subjected to an extensive battery of in vitro experiments aimed at evaluating the range of cargo that can be successfully loaded and accommodated, rates of loading and unloading and potential leakage, and the constructs will be modified as informed by these initial results. To develop a cytosol-targeting cargo delivery system based on these nanocapsules, we will incorporate a well-characterized, non-covalent cell-penetrating peptide (CPP) tag shown to efficiently translocate large proteins from the extracellular space to the cytosol and—crucially—overcome the tendency of CPP-tagged proteins to become trapped in endosomes. Together with other suitable modifications, the proposed IDE-based protein nanocapsules are expected to provide an effective, general-purpose system for encapsulating diverse cargo and delivering it exclusively to the cytosol of cells, while avoiding release into the extracellular space. If these initial, exploratory experiments are successful, this innovative technology can be conceivably be adapted for a wide variety of applications, potentially leading to powerful new methods for the targeted and regulated delivery of therapeutic compounds.

项目概要/摘要 该提案探讨了开发基于胰岛素的新型复合递送系统的可行性 降解酶（IDE），一种具有独特纳米胶囊状结构的锌金属肽酶。 IDE 类似于 翻盖式，由两个通过“铰链”区域连接的碗形区域组成，使其能够采用“开放” 和“闭合”构象。关闭时，蛋白酶具有一个大的内室，约 13,000-Å3 英寸 体积，完全封装，可容纳约 8000 Da 的货物。这个提议 有两个主要目标：（1）探索使用 IDE 封装货物和 通过广泛的体外表征进行调节释放； (2) 开发针对货物的新型系统 专门针对细胞的细胞质，同时明确阻止递送到细胞外空间。封装 以可逆可控的方式运输货物，我们将生成包含两个半胱氨酸的IDE变体 (S132C/E817C) 的位置使得它们仅在蛋白酶处于闭合状态时形成二硫键 构象。这种双半胱氨酸突变构成了一个氧化还原敏感的“锁存器”，如前所述， 在氧化环境（例如细胞外空间）中将 IDE“锁定”在关闭位置，并且 仅当暴露于还原环境（例如细胞质）时才“解锁”。纯化的纳米胶囊将 进行一系列广泛的体外实验，旨在评估可运输的货物范围 成功装载和容纳、装载和卸载率以及潜在泄漏，以及 结构将根据这些初步结果进行修改。开发细胞质靶向货物递送 基于这些纳米胶囊的系统，我们将结合一种特征良好的非共价细胞穿透 肽 (CPP) 标签可有效地将大蛋白从细胞外空间转移到胞质溶胶 至关重要的是，克服 CPP 标记蛋白被困在内体中的倾向。一起 通过其他适当的修饰，所提出的基于 IDE 的蛋白质纳米胶囊有望提供 有效的通用系统，用于封装不同的货物并将其专门输送到细胞质中 细胞，同时避免释放到细胞外空间。如果这些初步的探索性实验是 如果成功的话，这项创新技术可以适用于各种应用， 可能会产生用于靶向和调节治疗化合物递送的强大新方法。