Extracellular
vesicles (EVs) secreted from probiotics, defined
as live microorganisms with beneficial effects on the host, are expected
to be new nanomaterials for EV-based therapy. To clarify the usability
of probiotic-derived EVs in terms of EV-based therapy, we systematically
evaluated their characteristics, including the yield, physicochemical
properties, the cellular uptake mechanism, and biological functions,
using three different types of probiotics: Bifidobacterium
longum, Clostridium butyricum, and Lactobacillus plantarum WCFS1. C. butyricum secreted the largest amounts of EVs,
whereas all the EVs showed comparable particle sizes and zeta potentials,
ranging from 100 to 150 nm and −8 to −10 mV, respectively.
The silkworm larvae plasma assay indicated that these EVs contain
peptidoglycan that activates the host’s immune response. Moreover,
a cellular uptake study of probiotic-derived EVs in RAW264.7 cells
(mouse macrophage-like cells) and DC2.4 cells (mouse dendritic cells)
in the presence of inhibitors (cytochalasin B, chlorpromazine, and
methyl-β-cyclodextrin) revealed that probiotic-derived EVs were
mainly taken up by these immune cells via clathrin-mediated endocytosis
and macropinocytosis. Furthermore, all the probiotic-derived EVs stimulated
the innate immune system through the production of inflammatory cytokines
(TNF-α and IL-6) from these immune cells, clarifying their utility
as a novel adjuvant formulation. These findings on probiotic-derived
EVs are valuable for understanding the biological significance of
probiotic-derived EVs and the development of EV-based immunotherapy.