Treatment of milk with various chemicals differentially affects the physicochemical and functional characteristics of extracellular vesicles

Abstract

Isolation of high-purity milk-derived extracellular vesicles (miEV) is complicated by protein impurities, such as casein. The focus of this study was to investigate whether the treatment of milk with acetic acid (AA), ethylenediaminetetraacetic acid (EDTA), and sodium citrate (SC), which are known chemicals to deplete caseins from miEV, alters their physicochemical and functional characteristics differently. For this purpose, following the treatment of milk with AA, EDTA, and SC, various subpopulations (12 K, 35 K, 70 K, and 100 K) of miEV were isolated by differential ultracentrifugation and characterized by protein quantification assay, gel electrophoresis, western blotting, dynamic light scattering, and scanning electron microscopy. In addition, the cellular uptake and immunomodulatory potential of miEV were also assessed. The results indicated that protein impurities, including caseins, were differentially depleted from miEV subpopulations by AA, EDTA, and SC. However, the acidification of milk induced partial protein aggregation. Additionally, miEV from both AA- and EDTA-treated milk exhibited a less smooth surface and higher uptake compared to those from SC-treated milk. Although all miEV significantly reduced T cell proliferation, a higher immunomodulatory potential was found in miEV from AA-treated milk. In conclusion, AA, SC, and EDTA have different efficiencies in improving the purity of miEV and can affect their physicochemical and functional characteristics differently. This issue may be critical for the efficient isolation of pure subpopulations of miEV for biomedical research.