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Separation of Extracellular Vesicles by DNA-Directed Immunocapturing Followed by Enzymatic Release

preprint
submitted on 02.05.2020 and posted on 06.05.2020 by Dario Brambilla, Laura Sola, Elisa Chiodi, Natasa Zarovni, Diogo Fortunato, Mattia Criscuoli, Vincenza Dolo, Ilaria Giusti, Valentina Murdica, Riccardo Vago, Marcella Chiari
Extracellular vesicles (EVs) have attracted great interest among researchers due to their role in cell-cell communication, disease diagnosis, and drug delivery. In spite of their potential in the medical field, there is no consensus on the best method for separating microvesicles 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 is made complex by the fact that blood and cell culture media, contain a large number of 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 requires harsh conditions that hinder their use in certain types of downstream analysis. Herein, a novel capture and release approach for small extracellular vesicles (sEVs), based on DNAdirected immobilization of antiCD63 antibody is presented. The flexible DNAlinker increases the capture efficiency and allows releasing of EVs by exploiting the endonucleasic activity of DNAse I. This separation protocol works under mild conditions, enabling the release of intact vesicles that can be successfully analyzed by imaging techniques. In this article sEVs recovered from plasma were characterized by established techniques for EVs analysis including nanoparticle tracking and transmission electron microscopy.

Funding

This work was supported by European Union Horizon 2020 under Grant 766466: INDEX, Integrated Nanoparticle isolation and Detection system for complete on-chip analysis of Exosomes.

History

Email Address of Submitting Author

dario.brambilla@scitec.cnr.it

Institution

National Research Council of Italy (CNR)

Country

Italy

ORCID For Submitting Author

0000-0003-3490-4481

Declaration of Conflict of Interest

The authors declare the following competing financial interest: Marcella Chiari has filed an Italian Patent Application (102019000018239) on the bases of these data.

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