122 | Tangential flow filtration enables high-yield isolation and stable characterization of grape-derived extracellular vesicles for therapeutic application

Sabino Porro^1|2, Tamer Esmail^1|2, Francesca D’Ascanio^1|2|3, Giulia Colasante^1|2, Arianna Aquilini-Mummolo,^1|2, Luana D’Onofrio^1|2, Mariagiulia Filoso^1|2, Ayesha Younas^1|2, Isabel Maria Vallejo Bermudez^4|5, Camilla Cichella^1|2, Michela Battistelli⁶, Anna Piro⁷, Serena Veschi⁷, Serena Pilato⁷, Antonella Fontana⁷, Alessandro Cama⁷, Angelo Cichelli⁸, Paola Lanuti^1|2, Federica Flamminii⁹ | ¹Department of Medicine and Aging Sciences, University “G d’Annunzio” of Chieti-Pescara, Italy; ²Center for Advanced Studies and Technology CAST; ³Molecular Immunology Group CTS208 Department of Humanities, Law and Economics, Leonardo da Vinci University, Torrevecchia Teatina, Italy; ⁴Department of Cell Biology, Physiology and Immunology, University of Cordoba, Cordoba, Spain; ⁵Immunology and Allergy Group GC01, Maimonides Biomedical Research Institute of Cordoba IMIBIC, University of Cordoba, Reina Sofia University Hospital, Cordoba, Spain; ⁶Department of Biomolecular Sciences DISB University of Urbino Carlo Bo, Italy; ⁷Department of Pharmacy, University “G d’Annunzio” of Chieti-Pescara, Italy; ⁸Department of Innovative Technologies in Medicine & Dentistry, University “G d’Annunzio” of Chieti-Pescara, Italy; ⁹Department of Promotion of Human Sciences and Quality of Life, San Raffaele Telematic University, Rome, Italy.

Grapes produce extracellular vesicles (EVs) that have gained considerable attention in recent years due to their potential applications in food, nutrition, dermatology and health. This study presents a comprehensive comparison of grape juice-derived EV isolation methods using ultracentrifugation, and tangential flow filtration. We demonstrated that tangential flow filtration yielded more grape juice-derived EVs than ultracentrifugation while preserving structural integrity. The EVs isolated were characterized using different biophysical techniques, including flow cytometry, nanoparticle tracking analysis, zeta potential, atomic force microscopy, and transmission electron microscopy. EVs isolated by ultracentrifugation displayed an average diameter of 182±90 nm, while the zeta potential was -35+ 12 mV . Atomic force microscopy and transmission electron microscopy confirmed their sizes as well as vesicular nature (i.e. their spherical shape). Flow cytometry analyses of their phenotypes showed that juice-derived EVs isolated by ultracentrifugation expressed typical plant-derived EV markers, such as penetration 1 and tetraspanin 8. Grape juice-derived EVs isolated using tangential flow filtration also showed similar results: an average diameter of 182±117 nm, a average zeta potential of -37+ 2 mV, a spherical shape, and the positivity to penetration 1 and tetraspanin 8. However, the recovery obtained by tangential flow filtration (46.3+11%) was significantly higher compared to that obtained by ultracentrifugation (14.8+ 6% ). Therefore, tangential flow filtration represents a robust and scalable approach for the isolation of grape juice–derived EVs, yielding vesicles with preserved structural and phenotypic characteristics.