Milk EVs characterization during the water buffalo lactation curve highlights the differences that affect specific cellular functions in bovine macrophages and human intestinal cells.

Extracellular Vesicles (EVs) have garnered significant attention due to their multiple functions, including intercellular communication, immune responses, tissue regeneration and their potential use as drug delivery systems in therapeutic applications. One of the most promising sources of EVs is milk (mEVs) (Mimi M.A. et al 2025; Galley J.D. et al 2020). mEVs have different advantages, such as being natural, biocompatible, abundant and can successfully pass through the harsh conditions of the gastric tract and be absorbed by intestinal cells. However, their qualitative and quantitative analysis in colostrum (colo) and milk is challenged by the presence of fat globules and casein micelles, which contribute to the complexity and heterogeneity of these biological fluids (Mecocci S. et al 2022; Wolf T et al. 2015). This study aims to characterize milk EVs during the Water Buffalo lactation curve, on both non-ultracentrifuged whey samples and EV pellets. Indeed, cell uptake experiments were contemporarily carried out to differentiate the subcellular effects on bovine macrophages and human intestinal cells. Ultracentrifuged EVs (Mecocci S. et al 2024) from colo, 15- and 60-day milk (dm) were analysed by NTA, TEM and Flow Cytometry (FC), then labelled by PKH67 and finally added to BoMac and Caco-2 cells for uptake and function studies. EV cargoes were analysed by NGS and HPLC-MS. Whey samples were analyzed post-collection. EVs characterization is based on staining with anti-CD9 and -CD63 mAbs. All FC evaluations adhered to MISEV2023 guidelines. Our experiments highlight that both colo and milk samples contain EVs with diameters ranging from ~70 nm to ~300 nm. They expressed CD9 and CD63 tetraspanins. Colo samples highlight higher EV concentration. Metabolite analysis revealed that 379 molecules were particularly abundant in coloEVs compared to milk EVs, while 619 and 764 were found to be prevalent in 15 dm and 60 dm, respectively. Uptake experiments using PKH67-labelled EVs revealed a greater internalisation of colo EVs at both 4 and 24 hours in BoMac cells, promoting cell re-adhesion and demonstrating major EV persistence over time. All the studied EVs revealed to modulate basal mitochondrial ROS production, particularly after 24h. An increase in the phagocytic activity was observed in ex vivo buffalo macrophages after pre-treatment with 60-dm EVs, assessed by pHrodo Green Zymosan uptake. In human intestinal Caco-2 cells, our data highlight that EV treatment leads to a decrease in basal hydrogen peroxide, particularly after 4h. Furthermore, ATP levels produced in Caco-2 cells in response to mitochondrial respiration (OXPHOS) and glycolysis (by Seahorse XF Real-Time ATP rate assay) increased with EV treatment, particularly with 60 dm EVs. Given that oxidative stress is associated with the release of angiogenic factors, the presence of specific cytokines was investigated, revealing, after 24h EV treatment, a significant decrease in VEGF, a key protein in the intestinal inflammatory response. EVs from colo and 15 dm appear to be the most effective in modulating VEGF levels. These findings, although still ongoing, highlight for the first time specific phenotypic, morphologic and metabolomic features of water buffalo milk EVs during the lactation curve, underscoring the role of colo and mEVs in supporting immunologic functions in bovine/bubaline cells and human gastrointestinal health. Further experiments are ongoing to confirm and extend the above-mentioned results. This research was funded by the Italian Ministry for Health (grant number RC IZSME 03/23 RC).