Ultrasonication of Milk Decreases the Content of Exosomes and MicroRNAs in an Exosome-Defined Rodent Diet

Abstract

BackgroundBovine milk exosomes (BMEs) harbor regulatory proteins, lipids, and microRNAs. Consumption of an exosome- and RNA-depleted (ERD) diet elicited phenotypes compared with controls fed an exosome- and RNA-sufficient (ERS) diet in mice. All other ingredients were identical in the diets. ERD and ERS diets were prepared by substituting ultrasonicated and nonultrasonicated milk, respectively, for casein in the AIN-93G formulation. ObjectivesThe objective of this study was to assess the effect of ultrasonication of milk on exosome content and bioavailability, and cargo content. MethodsBovine milk was ultrasonicated and exosomes were isolated by ultracentrifugation [ultrasonicated exosomes (USEs)]; controls were not ultrasonicated [nonultrasonicated exosomes (NSEs)]. Exosome count, size, and morphology were assessed using a nanoparticle tracker and electron microscopy. RNAs, lipids, and proteins were analyzed by RNA sequencing and MS. Intestinal transport, bioavailability, and distribution were measured by using fluorophore-labeled USEs and NSEs in Caco-2 cells, FHs 74 Int cells, and C57BL/6J mice (n = 3; age: 6–8 wk). ResultsThe exosome count was 76% ± 22% lower in USEs than in NSEs (P < 0.05). Ultrasonication caused a degradation of ≤100% of microRNAs. USEs and NSEs contained 145 and 332 unique lipid signatures, respectively (P < 0.05). We detected a total of 525 and 484 proteins in USEs and NSEs, respectively. The uptake of USEs decreased by 46% ± 30% and 40% ± 27% compared with NSEs in Caco-2 and FHs 74 Int cells, respectively (P < 0.05). The hepatic accumulation of USEs was 48% ± 28% lower than the accumulation of NSEs in mice (P < 0.05). ConclusionsUltrasonication of milk depletes bioavailable BMEs in studies of Caco-2 cells, FHs 74 Int cells, and C57BL/6J mice and causes a near-complete degradation of microRNA cargos.