Plant-derived extracellular vesicles possess different intrinsic therapeutic activities that can potentially help prevent or treat human diseases. These EVs can be administered as such, or ingested daily in the diet. However, food EV sources are commonly subjected to cooking processes prior to consumption. Indeed, before any biological effect can be exerted, orally-administered EVs must overcome several technological, extracellular, and cellular barriers before reaching target tissues. To explore their capacity to exert biological effects, we evaluated the stability of broccoli-derived EVs under thermal culinary conditions and gastrointestinal digestion (GI), and their ability to cross the intestinal barrier. EVs were isolated from raw broccoli, broccoli sprouts and broccoli subjected to one of five thermal culinary treatments (frying, boiling, microwaving, scalding and steaming). Nanoparticle tracking analysis (particle size distribution, concentration, and z-potential), electron microscopy, and Western blot were used to characterize the EVs. They were run through a simulated in vitro GI tract and again characterized. Potential EV transport through the intestinal barrier was assessed using transwell-cultured differentiated Caco-2 cells, and their antiproliferative effects were evaluated in intestinal and hepatic cell lines. All five thermal culinary treatments lowered EV particle concentration, protein content, and cargo, as well as influencing EV structure. Around 10 % of the broccoli-derived EVs survived GI digestion. The RNA cargo of EVs can enter intestinal cells, but only a small amount of EVs cross the intestinal barrier. Thermal treatment also reduced EV antiproliferative effects. Broccoli-derived EVs are affected by thermal culinary processing and GI digestion, and cannot cross the intestinal barrier. However, even after thermal processing they apparently maintain some potential therapeutic effects. © 2025 Elsevier B.V., All rights reserved.

Milk extracellular vesicles (EVs) represent promising drug delivery platforms, yet current isolation methods face scalability challenges. Ultracentrifugation (UC), the gold standard, is expensive and energy-intensive, limiting pharmaceutical and pharmacological implementation. This study aimed to standardize scalable EV isolation methods and evaluate cheesemaking whey as a sustainable alternative to milk for therapeutic applications. We systematically compared isolation techniques including UC, tangential flow filtration (TFF), and polyethylene glycol (PEG) co-precipitation across milk and whey from bovine, caprine, and ovine sources. Casein removal strategies using acidification and EDTA treatment were evaluated. Functional validation included in vitro simulated gastrointestinal digestion and in vivo pharmacokinetic studies of miRNA-loaded EVs in murine models. Our results demonstrate that cheesemaking whey serves as a sustainable EV source with reduced casein contamination and comparable yields to milk. Both TFF and PEG co-precipitation effectively concentrate EVs from whey, offering viable UC alternatives for pharmaceutical scale-up, though additional purification steps are required for optimal purity. Acidification effectively removes protein contaminants from milk but shows minimal benefits for whey. Cow whey provided the highest EV yields among species evaluated. Functionally, EV-encapsulated miRNAs demonstrated superior gastrointestinal stability compared to free miRNAs and exhibited enhanced bioavailability in multiple target tissues including cardiac, splenic, and skeletal muscle following oral administration. These findings establish cheesemaking whey as a sustainable, pharmaceutical-grade EV source and validate scalable isolation methods suitable for therapeutic applications, providing essential groundwork for clinical translation of milk-derived EV-based drug delivery systems. © 2025 Elsevier B.V., All rights reserved.