Natural Plasma Nano-EVs for Drug Delivery

摘要 靶向递送有效药物水平和最小的非靶向影响仍然是主要的药理作用 挑战，例如蛋白质、反义寡核苷酸(ASO)和其他 五脏六腑的治疗学。允许受控药动学曲线和细胞的药物输送平台 因此，靶向，包括纳米粒、脂质体和细胞来源的细胞外小泡(EV)，正在积极地 由生物制药公司追查。然而，在设计合适的尺寸和表面方面，仍有几个关键差距 药物载体纳米颗粒的组成，这决定了它们的生物分布。尽管他们的优势 从人类干细胞或永生化细胞系产生的EV免疫原性图谱总是显示 静脉注射后网状内皮细胞(RES)在肝、脾和骨髓中的包埋(IV) 给药。相比之下，内源性、器官来源的EVS水平持续较高可能反映了 净空和更长的循环时间，这两个特性对于允许有效地将有效载荷传递到 多个目标组织。此外，表面分子特征由暴露的蛋白质和脂类组成，它们各不相同 在血浆EV亚群中，很可能决定它们对特定器官或细胞的趋向性。这项研究将 利用内源性EVS的天然工程特性开发出第一个人类血浆来源 药物递送产品。 我们开发了一种新的、灵敏的多重免疫分析方法，适用于未加工的 血浆，以基于表面暴露蛋白的特定图谱来表征血浆EV亚群。这种方法 使我们发现了一种新的内源性纳米电动汽车(NeV)亚群，其直径较小(10-40 nm) 与大多数电动汽车报告的50-200 nm直径形成鲜明对比。NEV含有蛋白质和RNA 代表几个器官的细胞标志物以及表面脂肪和蛋白质特征，以减少RES清除。 我们假设，这个新发现的EV子集是自然适应的远程间歇 器官通讯，因此非常适合药物输送。 该项目将开发一种健壮和可扩展的方法，用于从商业上分离大量NEV 获得性人体血浆。我们还将使用IN定量表征体内NEVS PK和生物分布 活体正电子发射断层扫描(PET)和荧光成像。将实现以下具体目标 在目前的提案中：目标1：纳米电动汽车的生产：放大和表征。目标2：NEV的分析 生物分布和血浆半衰期。通过证明新能源汽车改善了生物分布特性 传统的电动汽车和它们可以很容易地从人体血浆中大量分离出来，然后我们的第二阶段 SBIR将专注于优化药物加载并在疾病模型中展示疗效。

ABSTRACT Targeted delivery of efficacious drug levels with minimal off-target effects remains a major pharmacological challenge as exemplified by the poor delivery of proteins, antisense oligonucleotides (ASO), and other therapeutics to may organs. Drug delivery platforms that allow for controlled pharmacokinetic profile and cell targeting, including nanoparticles, liposomes and cell-derived extracellular vesicles (EV), are thus aggressively pursued by biopharma. However, several key gaps remain in engineering the appropriate size and surface composition of drug carrier nanoparticles, which dictates their biodistribution. Despite their favorable immunogenicity profile, EVs generated from human stem cells or immortalized cell lines invariably show entrapment by reticuloendothelial (RES) cells in liver, spleen, and bone marrow following intravenous (IV) dosing. In contrast, the persistently high plasma level of endogenous, organ derived EVs likely reflects reduced clearance and longer circulation times, both properties important for allowing for efficient payload delivery to multiple target tissues. Also, surface molecular signatures comprised of exposed proteins and lipids, which vary across plasma EV subsets, are likely to dictate their tropism to specific organs or cells. This research will capitalize on the naturally engineered properties of endogenous EVs to develop the first human plasma derived drug delivery product. We have developed a novel and sensitive multiplexed immunoassay, suitable for the use with unprocessed plasma, to characterize plasma EV subsets based on specific profiles of surface-exposed proteins. This method enabled us to discover of a novel endogenous subset of nano-EVs (nEV), whose small diameter (10-40 nm) contrasts sharply with the 50-200 nm diameter reported for most EVs. The nEVs contain protein and RNA markers of cells representing several organs as well as surface lipid and protein features to reduce RES clearance. We hypothesize that this newly discovered EV subset is naturally adapted for long-range inter- organ communications, and thus ideally suited for drug delivery. This project will develop a robust and scalable method for isolation of large amounts of nEVs from commercially acquired human plasma. We will also quantitatively characterize in vivo nEVs PK and biodistribution using in vivo positron emission tomography (PET) and fluorescence imaging. The following specific aims will be pursued in the current proposal: Aim 1: Nano-EV production: scale-up and characterization. Aim 2: Analysis of nEV biodistribution and plasma half-life. By demonstrating that nEVs have improved biodistribution properties over traditional EVs and that they can be easily isolated in large amounts from human plasma then phase 2 of our SBIR will focus on optimizing drug loading and demonstrating efficacy in disease models.