Isolation and characterization of bovine-milk derived extracellular vesicles using a modified aqueous two-phase system

Extracellular vesicles (EVs) are lipid bilayer-bound nanoparticles secreted by nearly all cells, with notable applications in intercellular communication and therapeutic delivery. However, conventional EV isolation techniques, such as ultracentrifugation and size exclusion chromatography, often face challenges like high cost, specialized equipment requirements, and low yield. In this study, we present a modified aqueous two-phase system (ATPS) as an alternative method for isolating EVs from raw bovine milk. This approach is scalable, cost-effective, and requires minimal specialized equipment, making it accessible for small laboratories. We demonstrate the efficiency of this method through the isolation and subsequent characterization of bovine milk-derived EVs, including morphological analysis, protein, and lipid quantification, and nucleic acid presence. The isolated EVs exhibited typical characteristics, morphology, specific protein markers (CD63 and TSG101), and a protein-to-lipid ratio consistent with extracellular vesicles. These findings validate the modified ATPS as a reliable and practical method for isolating milk-derived EVs, offering a promising tool for future research into their therapeutic potential and other applications.