Anion Exchange Chromatography-Based Purification of Plant-derived Nanovesicles from Brassica oleracea L.: Molecular Profiling and Bioactivity in Human Cells

Clarissa Zanotti^1,2Simona Arena³Sabrina De Pascale³Valentina Ciaravolo³Rosalia Ferracane³Antonio Dario Troise³Chiara Pontecorvi¹Francesca Pacello¹Chiara Niespolo⁴Angelo Gismondi¹Andrea Scaloni³MAURO MARRA^1*

• ¹Department of Biology, University of Rome Tor Vergata, Roma, Italy
• ²PhD Program in Cellular and Molecular Biology, Department of Biology, University of Rome “Tor Vergata”, Rome, Italy., Rome, Italy
• ³Laboratory of Proteomics and Mass Spectrometry, Institute for the Animal Production System in the Mediterranean Environment, National Research Council (CNR), Napoli, Italy
• ⁴Arterra Bioscience SpA, Via Benedetto Brin 69, 80142, Naples, Italy, Naples, Italy

Plant-derived nanovesicles emerge as a promising alternative to mammalian-derived exosomes with distinct advantages, including lower immunogenicity, enhanced bioavailability, and the presence of bioactive plant metabolites. They have been shown to cross biological barriers, delivering therapeutic molecules that modulate gene expression, inflammation, oxidative stress, and cancer-related pathways. However, challenges remain that limit applicative use, including poor knowledge of their interactions with mammalian host cells and primarily the lack of a cost-effective and scalable method to obtain highly purified plant-derived nanovesicles. To address these limitations, we have developed an advanced purification platform that integrates ultrafiltration with anion exchange chromatography in a fast protein liquid chromatography system. This approach was validated using it in the isolation of plant-derived nanovesicles from Brassica oleracea L. seedlings, resulting in highly purified and concentrated preparations. Comprehensive molecular analyses, including proteomics, lipidomics, metabolomics, and miRNA profiling, characterized the nature of the purified plant-derived nanovesicles. Furthermore, their wound healing and anti-inflammatory properties were demonstrated in vitro and correlated to the potential biological activities of cargo miRNAs species by bioinformatics, highlighting the potential in nanomedicine of anion exchange-purified brassica nanovesicles. This study provides a scalable and efficient purification strategy, which might pave the way for broader applications of plant-derived nanovesicles in the clinical, nutraceutical and pharmaceutical fields.