A novel scalable method for the production of rennet-treated milk-derived extracellular vesicles for improved curcumin oral delivery
Schifano E. Vari F. Buccini L. Karimova M. Syman K. Varnadyan D. Uccelletti D. Dinarelli S. Zuccotti M. Alfieri A. Sennato S. Mura F. Rossi M. Dini L. Tacconi S.
December 2025BioMed Central Ltd
Journal of Nanobiotechnology
2025#23Issue 1
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.
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Department of Biology and Biotechnologies “C. Darwin”, Sapienza University of Rome, Rome, Italy
Department of Physiology and Pharmacology “V. Erspamer”, Sapienza University of Rome, Rome, Italy
Department of Basic and Applied Sciences for Engineering, Sapienza University of Rome, Rome, Italy
Department of Biology Faculty of Natural Science and Geography, Abai KazakhNational Pedagogical University Almaty, Almaty, Kazakhstan
Department of Biological and Environmental Sciences and Technologies (Di.S.Te.B.A.), University of Salento, Lecce, Italy
Institute for the Structure of Matter, National Research Council, Roma, Italy
Department of Biology and Biotechnology “Lazzaro Spallanzani’, University of Pavia, Pavia, Italy
Centro Grandi Strumenti, University of Pavia, Pavia, Italy
Institute for Complex Systems (ISC)-CNR and Physics Department, Sapienza University, Rome, Italy
Research Center of Nanotechnologies for Engineering (CNIS), Sapienza University of Rome, Rome, Italy
Department of Biology and Biotechnologies “C. Darwin”
Department of Physiology and Pharmacology “V. Erspamer”
Department of Basic and Applied Sciences for Engineering
Department of Biology Faculty of Natural Science and Geography
Department of Biological and Environmental Sciences and Technologies (Di.S.Te.B.A.)
Institute for the Structure of Matter
Department of Biology and Biotechnology “Lazzaro Spallanzani’
Centro Grandi Strumenti
Institute for Complex Systems (ISC)-CNR and Physics Department
Research Center of Nanotechnologies for Engineering (CNIS)
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