Enabling Efficient, Fast, Biocompatible Exosome Separation via Acoustofluidics

摘要 外泌体是含有生物分子（DNA、mRNA、miRNA和其他）的纳米尺寸的细胞外囊泡。 功能蛋白质）从它们的细胞来源。外泌体几乎从所有细胞类型分泌，因此， 它们存在于大多数生物液体中，包括血液、唾液、尿液和脑脊髓液。过去 近十年来，外泌体生物分子向受体细胞的转移已经涉及多种生物学过程， 流程.因此，外泌体越来越成为生物医学研究中许多研究的焦点。 由于其独特的分子特征，外泌体已被确定为潜在的变革性的生物学系统。 用于多种疾病的诊断和预后的循环生物标志物，包括癌症、神经退行性疾病、 疾病（即，帕金森氏症和阿尔茨海默氏症），以及肾脏、肝脏和胎盘疾病。除了 在诊断应用中，外泌体是许多治疗应用中的理想药物递送系统。而 外泌体的多功能性使它们成为各种生物医学应用的极好候选者， 外来体的一致、有效分离大大限制了它们的用途。目前的办法 外泌体分离涉及冗长的程序，需要训练有素的人员，具有低重复性，低 产率、低纯度和/或低分选后外泌体完整性。因此，迫切需要开展这方面的研究 一种简单、快速、有效和生物相容的方法，用于从生物样品中分离外来体， 液体或体外细胞培养。在这个R 01项目中，我们将通过开发一种声流控技术来解决这一未满足的需求。 (i.e.,声学和微流体的融合）平台，用于高纯度、高产量、高生物相容性， 自动化外泌体分离。所提出的声流技术将具有以下特点：1） 自动化外泌体处理，减少操作员之间的差异， 隔离结果与改进的生物危害遏制; 2）减少所需的时间量从 生物流体（例如，ImL未稀释的血液）至分离的外泌体（<5分钟处理时间vs约8小时处理时间 3）与基准相比，更高的外泌体回收率（>90%） 技术（5 - 25%）; 4）与基准技术（~33%）相比，外泌体纯度更高（>80%）; 5） 减少其他循环因子的污染，包括非天然血清蛋白（例如，白蛋白和 免疫球蛋白）和具有类似大小的颗粒，包括各种类型的脂蛋白; 6）低成本和点- 护理设计;和7）处理大和小样品体积的能力（最大样品体积：~30 mL;最小样品体积：~10 µL），这对现有方法极具挑战性。与这些 独特的功能，拟议的声流体技术有可能大大简化和加快 外泌体相关生物医学研究的工作流程，并帮助发现新的外泌体生物标志物。

Abstract Exosomes are nanosized extracellular vesicles that contain biomolecules (DNA, mRNA, miRNA, and other functional proteins) from their cell of origin. Exosomes are secreted from nearly all cell types, and as a result, they are found in most biological fluids, including blood, saliva, urine, and cerebrospinal fluid. Over the past decade, the transfer of exosomal biomolecules to recipient cells has been implicated in a variety of biological processes. Consequently, exosomes have increasingly been the focus of many studies in biomedical research. Due to their distinct molecular signatures, exosomes have been identified as a potentially transformative circulating biomarker for the diagnosis and prognosis of multiple diseases, including cancer, neurodegenerative diseases (i.e., Parkinson’s and Alzheimer’s), as well as diseases of the kidney, liver, and placenta. In addition to diagnostic applications, exosomes are an ideal drug delivery system in many therapeutic applications. While the versatility of exosomes renders them an excellent candidate for a variety of biomedical applications, difficulties in the consistent, effective isolation of exosomes have greatly limited their utility. Current approaches for exosome isolation involve lengthy procedures, require highly trained personnel, suffer from low repeatability, low yield, low purity, and/or low post-sorting exosome integrity. As a result, there exists a critical need in the research communities for a simple, rapid, efficient, and biocompatible approach for isolating exosomes form biological fluids or in vitro cell culture. In this R01 project, we will address this unmet need by developing an acoustofluidic (i.e., the fusion of acoustics and microfluidics) platform for high-purity, high-yield, high-biocompatibility, automated exosome isolation. The proposed acoustofluidic technology will have the following features: 1) Automated exosome processing which reduces operator-to-operator variability and enables simple, consistent isolation results with improved biohazard containment; 2) Reduces the amount of time necessary to go from biofluid (e.g., 1 mL undiluted blood) to isolated exosomes (<5 min processing time vs ~8 hrs processing time with alternative technologies); 3) Higher exosome recovery rate (>90%) in comparison to benchmark technologies (5‒25%); 4) Greater exosome purity (>80%) in comparison to benchmark technologies (~33%); 5) Less contamination from other circulating factors, including non-native serum proteins (e.g., albumin and immunoglobulin) and particles with similar sizes, including various types of lipoproteins; 6) Low-cost and point- of-care design; and 7) ability to handle both large and small sample volumes (maximum sample volume: ~30 mL; minimum sample volume: ~10 µL), which is extremely challenging with existing approaches. With these unique features, the proposed acoustofluidic technology has the potential to greatly simplify and expedite workflows in exosome-related biomedical research and aid in the discovery of new exosomal biomarkers.