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

Abstract

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.<br>

Small EVs-Associated DNA as Complementary Biomarker to Circulating Tumor DNA in Plasma of Metastatic Colorectal Cancer Patients

“…The immunoprecipitation approach we adopted in this work was recently introduced by our group [ 17 ]. It is based on the so-called DNA directed immobilization (DDI) strategy to anchor the anti-tetraspanin anti CD63 antibody on magnetic particles through a DNA-linker.…”

“…Additionally, the DNA linker can be cleaved using DNase I, enabling the release of extracellular vesicles under mild conditions. The synthesis of ssDNA-antiCD63 conjugate and the DNA sequences used for immunoprecipitation of sEVs are described in [ 17 ].…”

The Role of Surface Chemistry in the Efficacy of Protein and DNA Microarrays for Label-Free Detection: An Overview

“…Single-particle label-free imaging is widely employed for visualization of viruses [ 29 ], exosomes [ 28 , 81 ], and bacteria [ 82 ] as well as for other synthetic and biological nanoparticles [ 83 , 84 ].…”

“…The DDI method has been successfully applied to single particle capture. Particularly, improved capture efficiency has been shown, due to the added flexibility to the probes [ 81 , 90 ]. The binding of DNA-directed antibodies to the nanoparticles and of the nanoparticles to the substrate can also take place by self-assembly, where all the incubation steps are performed simultaneously [ 91 ].…”

“…Another advantage of the DDI approach is the possibility to recover the captured particles by cutting DNA strands with DNAse solutions [ 81 ]. In some cases, the biotin-streptavidin interaction is also employed as a means to ensure stable binding [ 92 ].…”