ABCG2 in bovine and ovine milk extracellular vesicles: Evidence of substrate accumulation in a cellular model.

Milk-derived extracellular vesicles (EVs) are emerging as key mediators of molecular transfer within the mammary gland and may contribute to the secretion of bioactive compounds and xenobiotics into milk. The ATP-binding cassette transporter ABCG2 plays a central role in drug and metabolite secretion into milk; however, its presence and functionality in milk-derived EVs from livestock species remain poorly understood. In this study, EVs from bovine and ovine milk were isolated by ultracentrifugation and size exclusion chromatography and characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blot. ABCG2 was consistently detected in EV-enriched fractions from both species. While EV size distributions were similar (∼160-170 nm), significant differences in zeta potential may suggest species-specific vesicle properties. To investigate the association between ABCG2 expression and substrate accumulation, EVs derived from parental and ovine ABCG2-transduced MDCK-II cells were evaluated using topotecan as a model substrate. EV preparations derived from ABCG2-expressing cells contained significantly more topotecan than those obtained from parental cells. These findings demonstrate the presence of ABCG2 in EV-enriched fractions isolated from bovine and ovine milk and provide supportive in vitro evidence that ABCG2 expression in donor cells is associated with increased topotecan recovery in cell-derived EV preparations. The results identify a potential EV-associated mechanism with possible implications for xenobiotic transfer, dairy safety, and the development of EV-based delivery systems.