Brambilla Dario, Sola Laura, Ferretti Anna Maria, Chiodi Elisa, Zarovni Natasa, Fortunato Diogo, Criscuoli Mattia, Dolo Vincenza, Giusti Ilaria, Murdica Valentina, Kluszczyńska Katarzyna, Czernek Liliana, Düchler Markus, Vago Riccardo, Chiari Marcella
Institute of Chemical Science and Technology "Giulio Natta", National Research Council of Italy (CNR-SCITEC), via Mario Bianco 9, 20131 Milan, Italy.
Institute of Chemical Science and Technology "Giulio Natta", National Research Council of Italy (CNR-SCITEC), via Gaudenzio Fantoli 16/15, 20138 Milan, Italy.
Anal Chem. 2021 Apr 6;93(13):5476-5483. doi: 10.1021/acs.analchem.0c05194. Epub 2021 Mar 26.
Extracellular vesicles (EVs) have attracted considerable interest due to their role in cell-cell communication, disease diagnosis, and drug delivery. Despite their potential in the medical field, there is no consensus on the best method for separating micro- and nanovesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation of a single class of EVs, such as exosomes, is complex because blood and cell culture media contain many nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high-purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles usually requires harsh conditions that hinder their use in certain types of downstream analysis. A novel capture and release approach for small extracellular vesicles (sEVs) is presented based on DNA-directed immobilization of antiCD63 antibody. The flexible DNA linker increases the capture efficiency and allows for releasing EVs by exploiting the endonuclease activity of DNAse I. This separation protocol works under mild conditions, enabling the release of vesicles suitable for analysis by imaging techniques. In this study, sEVs recovered from plasma were characterized by established techniques for EV analysis, including nanoparticle tracking and transmission electron microscopy.
细胞外囊泡（EVs）因其在细胞间通讯、疾病诊断和药物递送中的作用而备受关注。尽管它们在医学领域具有潜力，但对于从细胞培养上清液和复杂生物流体中分离微囊泡和纳米囊泡的最佳方法尚无共识。获得良好的回收率并保持物理特性对于EVs的诊断和治疗应用至关重要。分离单一类型的EVs，如外泌体，很复杂，因为血液和细胞培养基中含有许多大小范围相同的纳米颗粒。利用免疫亲和捕获的方法可提供高纯度样品，并克服了当前使用的分离方法存在的问题。然而，捕获的纳米囊泡的释放通常需要苛刻的条件，这阻碍了它们在某些类型的下游分析中的应用。基于抗CD63抗体的DNA定向固定，提出了一种用于小细胞外囊泡（sEVs）的新型捕获和释放方法。柔性DNA接头提高了捕获效率，并允许通过利用DNA酶I的内切酶活性释放EVs。这种分离方案在温和条件下工作，能够释放适合通过成像技术进行分析的囊泡。在本研究中，通过用于EV分析的既定技术，包括纳米颗粒跟踪和透射电子显微镜，对从血浆中回收的sEVs进行了表征。
