Cardoon cell cultures as a biofactory for extracellular vesicles with antisteatotic activity in an in vitro model of non-alcoholic fatty liver disease

Food-derived extracellular vesicles (EVs) hold growing interest in their applications across diverse fields, including nutraceuticals and functional foods. Plant-derived EVs present a sustainable, cost-effective alternative to other sources, addressing challenges related to safety and scalability. This research investigates the potential of cardoon (Cynara cardunculus L. var. altilis) cell suspension cultures (CSCs) as a novel biotechnological platform for EV production. EVs were isolated from cardoon CSC-conditioned media using differential ultracentrifugation and size exclusion chromatography and characterized through nanoparticle tracking analysis, interferometry, and transmission electron microscopy, showing a narrow size distribution and round-shaped morphology. Comparative proteomic and metabolomic analyses of cardoon calli and CSC EVs revealed a significant enrichment of bioactive proteins and secondary metabolites in the EV preparations. These components include key molecules associated with plant defence mechanisms and potent antioxidant and anti-inflammatory activities. In an in vitro model of non-alcoholic fatty liver disease (NAFLD) using HepG2 cells, cardoon EVs exhibited notable hepatoprotective properties. They reduced reactive oxygen species (ROS) and nitric oxide (NO) levels, enhanced cell viability, and decreased lipid accumulation, demonstrating efficacy comparable to metformin, employed as lipid-lowering drug. Mechanistically, CSC EVs activated the SIRT-1/AMPK signalling pathway, a key regulator of lipid metabolism. Under lipotoxic conditions, EV treatment enhanced AMPK phosphorylation and upregulated SIRT-1 expression. Pharmacological modulation of SIRT-1 further confirmed that CSC EVs regulate this pathway. These findings highlight the potential of cardoon EVs in tackling oxidative stress and lipid dysregulation, positioning them as a promising natural therapeutic candidate for NAFLD. By advancing the understanding of EVs isolated from plant cell cultures, this study opens avenues for their application in treating metabolic and liver-related disorders, reinforcing the potential role of EVs in nanomedicine, biotechnology, and the development of new functional food supplements.