A novel scalable method for the production of rennet-treated milk-derived extracellular vesicles for improved curcumin oral delivery

Background: Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles secreted by all cell types and are gaining increasing attention as natural, biocompatible drug delivery nanovehicles. Among the various sources, bovine milk-derived EVs (MEVs) represent an abundant, cost-effective, and eco-friendly alternative, with demonstrated potential in delivering chemotherapeutics, bioactive natural compounds, miRNAs, and other therapeutic agents. However, the effective and scalable production of MEVs still requires protocol optimization for contaminant removal, especially casein micelles and fat globules, which may be co-isolated and compromise EV quality. This study aimed to identify an efficient and scalable strategy for MEV production suitable for oral drug delivery applications. Results: Three casein removal methods, acetic acid precipitation, ultracentrifugation, and commercial rennet, each combined with ultrafiltration/size exclusion chromatography (UF/SEC) for EV isolation were compared. Rennet treatment outperformed the other methods, yielding MEVs with superior purity, integrity, and minimal aggregation. MEVs with high quality and purity from whey derived from rennet-treated bovine milk successfully isolated by tangential flow filtration (TFF), a method more suitable for large-scale production. These vesicles were subsequently employed for the passive loading of curcumin, a hydrophobic compound with known bioactive properties but poor intrinsic bioavailability. The resulting curcumin-loaded MEVs (CurMEVs) were evaluated for their ability to traverse the intestinal barrier using in vitro and in vivo models. In addition, a prolongevity effect was observed in Caenorhabditis elegans animals supplemented with CurMEVs. Results confirmed that MEVs enhance curcumin stability and bioavailability. Conclusions: Our findings support the use of rennet combined with SEC or TFF to produce MEVs as scalable, safe, and effective delivery systems, offering a promising platform for future nutraceutical and pharmaceutical applications. In parallel, the study highlights the added value of converting dairy industry by-products, such as whey, into a sustainable source of nanocarriers.