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Membrane-Binding Peptides for Extracellular Vesicles On-Chip Analysis

preprint
revised on 01.03.2020, 08:39 and posted on 02.03.2020, 22:14 by Alessandro Gori, Alessandro Romanato, Greta Bergamaschi, Alessandro Strada, Paola Gagni, Roberto Frigerio, Dario Brambilla, Riccardo Vago, Silvia Galbiati, Silvia Picciolini, Marzia Bedoni, George G. Daaboul, Marcella Chiari, Marina Cretich
Small extracellular vesicles (EVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. EVs membrane could be then considered as a "universal" marker, alternative or complementary to traditional characteristic surface-associated proteins. Here we introduce the use of membrane sensing peptides as new, highly efficient ligands for EVs capturing onto bioanalytical chips to directly integrate EVs capturing and analysis on a microarray platform, even using serum without pre-isolation steps. EVs were analyzed by label-free, single particle counting and by fluorescence co-localization immune-staining with labelled anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general EVs baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performance are also discussed, as capturing efficiency is strictly bound to probes surface orientation and multivalency effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EVs analysis.

Funding

HYDROGEX (Regione Lombardia&Fondazione Cariplo, grant n. 2018-1720) and INDEX (European Union’s Horizon 2020 research and innovation programme under Grant Agreement N° 766466) projects are acknowledged for partial financial support to A.G., M.C. and M.C.; PRIN2015JTL4HL is also acknowledged for financial support to A.S.

History

Email Address of Submitting Author

marina.cretich@cnr.it

Institution

Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM), Milano

Country

Italy

ORCID For Submitting Author

0000-0001-8251-5275

Declaration of Conflict of Interest

Alessandro Gori, Marina Cretich and Marcella Chiari have filed a US Provisional Patent Application (62/897,042) on the base of these data.

Version Notes

Version number 2: new author's affiliation, added data on EV size distribution

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in Journal of Extracellular Vesicles

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