Enabling Efficient, Fast, Biocompatible Exosome Separation via Acoustofluidics

摘要 外切体是包含生物分子(DNA、mRNA、miRNA等)的纳米大小的细胞外小泡 功能蛋白)从它们起源的细胞中分离出来。外切体几乎由所有类型的细胞分泌，因此， 它们存在于大多数生物体液中，包括血液、唾液、尿液和脑脊液。在过去的时间里 十年来，胞外体生物分子向受体细胞的转移与多种生物学行为有关 流程。因此，外切体日益成为生物医学研究的热点。 由于它们不同的分子特征，外切体被认为是一种潜在的变革性 循环生物标记物用于多种疾病的诊断和预后，包括癌症、神经退行性变 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……之外 在诊断应用中，外切体在许多治疗应用中是一种理想的药物输送系统。而当 外切体的多功能性使其成为各种生物医学应用的极佳候选者，困难 在始终如一、有效地分离外切体的情况下，极大地限制了它们的应用。当前的方法是 外显子分离涉及冗长的程序，需要训练有素的人员，重复性低， 产量低，纯度低，和/或分选后外显子完整性低。因此，这项研究存在着迫切的需求。 社区提供了一种简单、快速、高效和生物相容的方法来分离生物外切体 液体或体外细胞培养。在这个R01项目中，我们将通过开发一种声流控来解决这一未得到满足的需求 (即声学和微流体的融合)平台，用于高纯度、高产率、高生物兼容性、 自动外显子分离。拟议的声流控技术将具有以下特点：1) 自动化外切体处理，减少操作员对操作员的可变性，并实现简单、一致 通过改进的生物危害控制实现隔离；2)减少从 生物液体(例如，1毫升未稀释的血液)到分离的外切体(处理时间为5分钟，而处理时间为~8小时 使用替代技术)；3)比基准更高的外切体回收率(&gt；90%) 技术(5-25%)；4)比基准技术(~33%)更高的外切体纯度(&gt；80%)；5) 更少受到其他循环因素的污染，包括非天然血清蛋白(例如，白蛋白和 免疫球蛋白)和大小相似的颗粒，包括各种类型的脂蛋白；6)低成本和点状 护理设计；以及7)能够处理大样本和小样本(最大样本体积：~30 毫升；最小进样量：~10微米L)，这对现有方法来说是极具挑战性的。有了这些 独特的功能，建议的声流控技术有可能极大地简化和加快 与外体相关的生物医学研究的工作流程，并协助发现新的外体生物标记物。

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.