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

项目摘要/摘要 该方案探索了开发基于胰岛素的新型复合给药系统的可行性。 降解酶(IDE)，一种具有独特的纳米胶囊状结构的锌金属肽酶。IDE类似于 翻盖，由两个碗形域组成，由一个“铰链”区域连接，这使得它可以采用“开放”。 和“封闭”构象。当关闭时，该酶具有一个大的内部腔，约13,000-ä3英寸 体积，它是完全封装的，可以容纳多达8000 Da的货物。这项建议 有两个主要目标：(1)探索使用集成开发环境对货物和货物进行封装的可行性 通过广泛的体外特性调节释放；以及(2)开发一种新的靶向货物的系统 排他地进入细胞胞浆，同时明确阻止输送到细胞外空间。封装，封装 货物以可逆和可控的方式，我们将产生含有两个半胱氨酸的IDE变体 (S132C/E817C)，定位为当蛋白酶处于闭合状态时，它们只形成二硫键 构象。如前所述，这种双半胱氨酸突变构成了一个氧化还原敏感的“闩锁”， 使IDE在氧化环境(例如，细胞外空间)中处于关闭状态，并且 只有当暴露在还原环境(如胞浆)中时，才会变得“解锁”。纯化的纳米胶囊将 接受广泛的体外实验，目的是评估可以 成功装载和容纳、装卸速度和可能的泄漏，以及 构造将根据这些初始结果进行修改。开发一种细胞溶胶靶向货物递送 基于这些纳米胶囊的系统，我们将结合一种特征良好的、非共价的细胞穿透 多肽(CPP)标签被证明能有效地将大蛋白从细胞外空间转移到胞浆 而且--至关重要的是--克服了CPP标记的蛋白质被困在内吞体内的倾向。同舟共济 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一种 高效、通用的系统，用于封装不同的货物并将其专门发送到 细胞，同时避免释放到细胞外空间。如果这些最初的探索性实验 成功后，这项创新技术可以被改编成广泛的应用， 潜在地为靶向和规范的治疗化合物的递送带来了强大的新方法。

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.