Elucidation of the Tissue Distribution and Host Immunostimulatory Activity of Exogenously Administered Probiotic-Derived Extracellular Vesicles for Immunoadjuvant

Extracellular vesicles (EVs) are cell-derived nanoparticles that can be used as novel biomaterials. In the development of EVs-based therapeutic systems, it is essential to understand the in vivo fate of exogenously administered EVs and subsequent biological responses mediated by EVs. Although probiotics and microorganisms that modulate the host immune system also secrete EVs, their tissue distribution and biological reactions after administration to the host have not been sufficiently elucidated. In this study, we characterized EVs released from the probiotics Bifidobacterium longum (B-EVs) and Lactobacillus plantarum WCFS1 (L-EVs) in terms of tissue distribution and immune-activating capacity after intravenous and subcutaneous administration in mice. B-EVs and L-EVs exhibited particle sizes of approximately 100-160 nm and negative zeta potentials. These EVs contained peptidoglycan, DNA, and RNA as their cargoes. Intravenously administered B-EVs and L-EVs mainly accumulated in the liver and spleen. Furthermore, liver F4/80 and splenic CD169 macrophages took up the intravenously administered EVs. Subcutaneously administered B-EVs and L-EVs accumulated in the lymph nodes and were mainly located in the B-lymphocyte zone, indicating that exogenously administered probiotic-derived EVs showed a similar biodistribution, irrespective of the EVs-secreting cell type. Evaluation of EVs-mediated immune reactions demonstrated that intravenously administered EVs showed little activation potency. In contrast, subcutaneously administered B-EVs strongly increased the expression of inflammatory cytokine (TNF-α) and co-stimulatory molecules (CD40 and CD80) than L-EVs. These findings indicate that the subcutaneous administration of B-EVs is a useful strategy for the development of novel EVs-based immunotherapies.