Loading milk exosomes with urolithins boosts their delivery to the brain: Comparing the activity of encapsulated vs. free urolithins in SH-SY5Y neuroblastoma cells

The gut microbial-derived metabolites of ellagitannins and ellagic acid, urolithins (Uros) are well-established
anti-cancer metabolites according to preclinical studies. However, their efficacy is limited in systemic tissues,
including the brain, by phase-II metabolism. Exosomes (EXOs), extracellular vesicles involved in cell signaling
with the ability to cross the blood-brain barrier (BBB), could protect polyphenols from metabolism. Therefore, we
loaded milk EXOs with Uro-A, Uro-B or IsoUro-A to evaluate their brain delivery and anticancer effects compared
to non-encapsulated Uros. In Sprague-Dawley rats, perfused brain tissue analyses by UPLC-ESI-QTOF-MS showed
higher Uro levels (~3􀀀 4-fold) at 5 min following intravenous administration of EXO-Uros compared to nonencapsulated
Uros, except for Uro-B, using similar Uro concentrations (17􀀀 30 μM). Experiments on neuroblastoma
SH-SY5Y cells revealed dose-dependent antiproliferative effects for all EXO-Uros (0.3􀀀 1.2 μM), but not
with non-encapsulated Uros (10 μM). Flow cytometry analyses indicated that only EXO-Uros arrested the cell
cycle and induced apoptosis. Finally, using fluorescent-labeled EXOs and selective inhibitors, the primary
endocytic pathway was revealed to be clathrin-dependent. Overall, encapsulating Uros into EXOs is promising for
enhancing brain delivery and anticancer activity.