Continuous separation of melanoma exosomes using field-flow fractionation

描述（由申请人提供）：本提案的目标是设计新方法和台式装置，以实现从最低限度处理的患者血清样品中高通量分离和纯化外泌体。目的是产生高度纯化的材料，其不含外来体结构外源的遗传、蛋白质组或脂质组部分。外泌体是天然的生物相容性纳米囊泡，鉴于分离大量均匀分散的外泌体亚群用于实验的困难，其体内功能知之甚少。尽管存在这一障碍，但新的努力已经开始关注外泌体作为治疗性纳米载体的高潜在效用。与人工纳米囊泡相反，细胞来源的外来体表达过多的复杂靶向配体，包括天然构象的整合素、受体或甚至四跨膜蛋白，并且含有允许变形性的细胞骨架。因此，外泌体显示出作为癌症疫苗的前景，甚至已经被修饰以穿过血脑屏障递送siRNA。出于这些原因，为了理解外来体在疾病发病机制中的作用或使外来体适应用作治疗递送载体的目的，研究外来体在体内功能的方法是必要的。我们建议开发一种用于分离和纯化外泌体的仪器，而无需任何标记，使得这些纳米囊泡可以被收集、修饰并用作药物载体，而不会影响其固有的生物相容性和功能性。这项工作将采用场流分离（FFF）和分流侧向薄运输（SPLITT）技术来表征和连续分离来自细胞培养物和鉴定的血清和/或血浆样品的外泌体。具有FFF和微流体专业知识的PI Gale与外泌体生物学专家Wickline和Hood合作开发了一种能够基于生物物理特性表征外泌体的仪器，并在没有任何标签或任何结构/功能损伤的情况下连续分离外泌体。以下是该项目的具体目标：具体目标1：使用FFF表征外泌体并确定最佳分离条件。具体目标2：制造和测试用于连续外泌体分离的SPLITT和串行SPLITT系统。具体目的3：证明用连续SPLITT系统连续分离培养的和血清和/或血浆外泌体。

DESCRIPTION (provided by applicant): The goal of this proposal is to design new approaches and bench-top devices that enable high throughput separation and purification of exosomes from minimally processed patient serum samples. The aim is to produce highly purified materials that are devoid of genetic, proteomic, or lipidomic moieties exogenous to exosomal structures. Exosomes are natural biocompatible nanovesicles whose in vivo function is poorly understood given the difficulty of isolating large quantities of uniformly disperse exosome subpopulations for experimentation. In spite of this hurdle, new efforts have begun to focus on the high potential utility of exosomes as therapeutic nano-carriers. In contrast to artificial nanovesicles, cell-derived exosomes express a plethora of complex targeting ligands including integrins, receptors or even tetraspanins in native conformations and contain a cytoskeleton allowing for deformability. Consequently, exosomes are showing promise as cancer vaccines and have even been modified to deliver siRNA across the blood brain barrier. For these reasons, investigating means to study exosome function in vivo for the purposes of understanding their role in disease pathogenesis or adapt exosomes for use as therapeutic delivery vehicles is warranted. We are proposing to develop an instrument for separation and purification of exosomes without any labels such that these nanovesicles can be collected, modified and used as drug carriers without affecting their inherent biocompatibility and functionality. This work will employ field-flow fractionation (FFF) and split-flow lateral thin transport (SPLITT) techniques to characterize and continuously separate exosomes from cell cultures and identified serum and/or plasma samples. PI Gale, with expertise in FFF and microfluidics, has teamed up with co-PIs Wickline and Hood who are experts in exosome biology, to develop an instrument capable of characterizing exosomes based on biophysical properties and continuously separate exosomes without any labels or any structural/functional damage. The following are the specific aims for this project: Specific Aim 1: To characterize exosomes and identify optimal separation conditions using FFF. Specific Aim 2: To fabricate and test SPLITT and serial SPLITT systems for continuous exosome separation. Specific Aim 3: To demonstrate continuous separation of cultured and serum and/or plasma exosomes with serial SPLITT system.