Bioavailability and Biodistribution of Fluorophore‐labeled Exosomes from Cow's Milk after Intravenous and Oral Administration in C57Bl/6J Mice

Background MicroRNAs (miRNAs) play essential roles in gene regulation. We have reported that miRs are not only derived from endogenous synthesis but can also be obtained from cow's milk. This paradigm‐shifting discovery was corroborated by our studies suggesting that miRNAs, encapsulated in exosomes, are accumulated by endocytosis in human and rat intestinal cell cultures and subsequently secreted across the basolateral membrane. These observations have not been confirmed in a whole animal model. Hypotheses 1) Mice absorb exosomes from cow's milk; 2) exosomes accumulate in distinct tissues. Methods Exosomes were isolated from cow's milk using ultracentrifugation, and labeled using the fluorophore, 1,1‐dioctadecyl‐3,3,3,3‐tetramethylindotricarbocyanine iodide (DiR). Identity, integrity, fine dispersion, and count of exosomes were assessed using transmission electron microscopy, western blots, and nanoparticle tracker (not shown). Using a dose‐response design, exosomes were administered intravenously (i.v.) by retro‐orbital injection (1.5×10 10 , 1×10 11 , or 1 × 10 12 exosomes/g) or orally by gavage (2.5×10 10 , 1.25×10 11 , or 2.5×10 11 exosomes/g). Absorption and distribution of exosomes was monitored at timed intervals for up to 24 h using an iBox ® Small Animal Imaging system. At t=24 hours, mice were euthanized and tissues were collected for analysis in the iBox. The intestine was flushed with saline prior to analysis. Densitometry analysis was performed using VisionWorks ® LS software. Results Twenty‐four hours after i.v. administration, the vast majority of exosomes were detected in liver and spleen ( Fig. 1 ); the signal produced by the lowest dose was below detection limit. Importantly, 24 hours after oral administration the majority of exosomes localized to the small intestine with the unabsorbed fraction having moved to the colon. Of note, a fraction (<5%) of milk exosomes escaped re‐packaging in the mucosa (and retained the DiR label) and localized to the liver ( Fig. 2 ). Free DiR and unlabeled exosomes produced no detectable signal (controls). Conclusions This is the first study providing compelling evidence that 1) milk exosomes are bioavailable in mice, 2) bioavailability is <100%, 3) a fraction of orally administered exosomes escapes re‐packaging in the intestinal mucosa, and 4) milk exosomes accumulate in liver and spleen (presumably in resident macrophages). We propose that, after re‐packaging in the mucosa, the cargos of dietary exosomes are delivered to peripheral tissues. Future studies We will conduct a comprehensive analysis of milk exosome kinetics in mice to obtain an accurate assessment of bioavailability, distribution, and elimination phenomena in health and disease. Support or Funding Information Supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under award number 2015‐67017‐23181, NIH 1P20GM104320, the Gerber Foundation, the Egg Nutrition Center, the University of Nebraska Agricultural Research Division (Hatch Act), and USDA multistate group W3002.