Aa EVs can spread to systemic organs following intravenous tail vein injection.

(A) General scheme of the in vivo experimental design. Purified Aa EVs were quantified using NTS device and calculated particle numbers were used for each injection. Six-week-old male mice were intravenously administered lipid tracer, DiD-labeled Aa EVs (approximately 2.25 × 109 particles in 50 μl of PBS) for the indicated period. Created with BioRender.com. (B) The brain, heart, lung, kidney, spleen, and liver were resected, and the distributions of DiD-labeled Aa EVs were determined using an in vivo imaging system (IVIS). Although weaker fluorescence intensity was observed in the brain and lungs than in the spleen and liver, the signals became strong after 12 to 24 h of i. v. injections; IVIS signals in the brain were measured separately. Previously, we showed that Aa EVs and exRNAs specifically induce IL-6 expression in microglial cells and TNF-α expression in the brain. These proinflammatory cytokines were measured in the spleen and liver using immunofluorescence staining. Confocal image analysis was performed to detect IL-6 (green) and TNF-α (yellow) in spleen (C) and the liver (G). Red spots indicate Aa EVs. Spleen IL-6 (D), spleen TNF-α (E), liver IL-6 (H), and liver TNF-α (I) were quantified using ELISA. The levels of TNF-α and IL-6 were elevated in the spleen 12 and 24 h after injection, but not in the liver. To verify the successful Aa EV cargo delivery, one of the prominent miRNA-sized small RNAs present in Aa EV, Aa-20050, was measured by qRT-PCR in the spleen (F) and liver (J). For qRT-PCR, Cel-miR-39-3p was added (spike-in) for normalization and found to accumulate in the liver after 24 h of injection but not in the spleen; this may be attributed to rapid degradation of small RNA in these organs due to its high RNase activity. Data are presented as mean ± SD from five independent experiments. Scale bar = 100 μm. Depending on the organ, it may be possible for it to serve as both a target and an elimination organ. Most Aa EVs accumulate in the spleen and liver but induce weaker proinflammatory cytokine release, suggesting that the liver and spleen might act as elimination organs for blood-borne bEVs and their cargo, including RNAs. (TIFF)