Capillary-channeled polymers fibers and films - A platform technology for exosome isolation and analytics

项目概要/摘要 希望将基础细胞生物学、疾病传播机制和临床 诊断是新技术发展的主要驱动力。在那些可能 外泌体可以沿着这些路线开发，特别有前途。外泌体很小（30-130 nm） 细胞外囊泡（EV）来源于体内的所有细胞类型，现在已被认为是体内的关键因子。 细胞内通讯它们表现出来自其起源细胞的蛋白质生物标志物，使其具有前景 用于疾病诊断的候选物。同样，许多兴趣在于外泌体的潜在用途， 药物递送载体（即，向量）。然而，在此之前，需要进行大量的基础研究。 外泌体的效用得到充分实现。基于外泌体的研究应用中的一个关键挑战， 应用在于缺乏从不同生物介质中分离囊泡的稳健和通用的方法。 虽然分离和定量方法已经发展，但没有一种方法克服了与以下相关的关键问题： 精确性和再现性，以干净、快速和经济有效地分离外来体。为了解决这一空白， 基于新型聚乙烯的极其有效的外泌体捕获和分离平台技术， 聚酯（PET）毛细管通道聚合物（C-CP）纤维和薄膜，正在开发用于 在基础研究、临床诊断和准备恢复的相关性的范围内。初步成效 显示纤维可以有效地分离细胞外囊泡，富含外泌体，具有尺寸分布， 并且产量与传统分离方法相当，时间短得多，体积小， 纯度更高。本文提出了进一步发展和验证这种外泌体分离方法 用于基础研究和临床实验室，并扩展到用于载体 应用.正如这个计划的目标所规定的那样，也正如集体“外来体”所要求的那样， 社区”，这项工作的最终目标是交付工作原型，供科学家评估。 同行和潜在的商业供应商。研究和开发计划在三个方面进行 具体目标。在第一个中，高渗透性的色谱柱由独特形状的纤维制成， 提供了一个分离和纯化外泌体的平台，适用于临床，研究， 和制备规模，上级目前的外泌体分离方法。第二，实施 自旋向下尖端形式的纤维提供了对通用和靶向外泌体的更大通用性 使用普通台式离心机收获。在第三种情况下，C-CP纤维膜可以被配置为影响纤维膜的厚度。 用于临床诊断的高效、多重侧流免疫分析。人们完全相信， 该计划的一部分将证明，新型C-CP光纤/薄膜隔离平台将被证明是一种高效、低成本、 分离外泌体用于基础生物化学研究、临床诊断的有效手段， 制备性应用，并且这些特性将导致平台的商业可用性。

Project Summary/Abstract The desire to tie aspects of fundamental cell biology, the mechanisms of disease propagation, and clinical diagnostics is a major driver for the development of new technologies. Among the biological species that may be exploited along these lines, exosomes are particularly promising. Exosomes are small (30–130 nm) extracellular vesicles (EVs) derived from all cell types within the body, which are now realized as key agents in intracellular communication. They exhibit protein biomarkers from their cells of origin, making them promising candidates for use in disease diagnostics. Likewise, much interest lies in the potential use of exosomes as drug delivery vehicles (i.e., vectors). However, a great deal of fundamental research is necessary before the utility of exosomes is fully realized. A crucial challenge in the application of exosome-based research and application lies in the lack of robust and versatile methods for vesicle isolation from diverse biological media. While isolation and quantification methods have evolved, none have overcome the key issues associated with rigor and reproducibility to cleanly, quickly, and cost-effectively isolate exosomes. To address this void, an extremely efficient platform technology for exosome capture and isolation, based on novel poly(ethylene- terephthalate) (PET) capillary-channeled polymer (C-CP) fibers and films, is being developed for applications across the scales of relevance for basic research, clinical diagnostics, and preparative recovery. Initial results show that the fibers can effectively isolate extracellular vesicles, enriched in exosomes, with size distributions and yields comparable to traditional isolation methods, in much shorter times, smaller volume scales, and higher purity. Proposed here is the further development and validation of this exosome isolation methodology for fundamental research and clinical laboratories, with extension to the preparative-scale for vector applications. As dictated by the objectives of this program, and as demanded by the collective “exosome community”, the ultimate objective of the effort is the delivery of working prototypes for evaluation by scientific peers and potential commercial providers. The Research and Development program is pursued across three Specific Aims. In the first, highly permeable chromatography columns created from unique-shaped fibers provide a platform for isolation and purification of exosomes amenable to applications on the clinical, research, and preparative scales, superior to current exosome isolation methodologies. In the second, implementation of the fibers in spin-down tip format provides greater versatility towards generic- and targeted-exosome harvesting using common, bench-top centrifuges. In the third, C-CP fiber films can be configured to affect a high-efficiency, multiplexed lateral flow immunoassay for clinical diagnostics. It is fully believed that the results of this program will demonstrate that novel C-CP fiber/film isolation platforms will prove to be an efficient, cost- effective means to isolate exosomes for use in fundamental biochemistry research, clinical diagnostics, and preparative applications, and that those characteristics will lead to commercial availability of the platfroms.