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的方法和台式设备，能够从处理最少的患者血清样本中高通量分离和纯化外切体。其目的是生产高纯度的材料，这些材料不含外源于外体结构的遗传、蛋白质组或脂体部分。外切体是一种天然的生物相容的纳米囊泡，由于难以分离大量均匀分散的外切体亚群用于实验，因此对其体内功能了解甚少。尽管存在这一障碍，但新的努力已经开始集中在外切体作为治疗性纳米载体的高潜力用途上。与人造纳米囊泡不同，细胞衍生的外切体表达大量复杂的靶向配体，包括天然构象中的整合素、受体甚至四氢青霉素，并含有允许变形的细胞骨架。因此，外切体正显示出作为癌症疫苗的前景，甚至已经经过改造，可以携带siRNA穿过血脑屏障。因此，有必要研究外切体在体内的功能，以了解它们在疾病发病机制中的作用，或使外切体用作治疗载体。我们建议开发一种无需任何标记的外切体分离和纯化仪器，以便这些纳米粒可以被收集、修饰并用作药物载体，而不会影响其固有的生物兼容性和功能。这项工作将使用场流分馏(FFF)和分流横向薄层传输(Splitt)技术来表征和连续分离细胞培养和鉴定的血清和/或血浆样本中的外切体。PiGale在FFF和微流体学方面拥有专业知识，他与外显体生物学专家Wickline和Hood的合作PI合作开发了一种仪器，能够根据生物物理特性表征外显体，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显体。以下是本项目的具体目标：具体目标1：利用FFF确定外切体的特征和确定最佳分离条件。具体目标2：制造和测试Splitt和Serial Splitt系统，用于连续外切体分离。具体目的3：用系列Splitt系统实现培养物与血清和/或血浆外切体的连续分离。

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.