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. 2024 Aug 26;14(14):5413–5428. doi: 10.7150/thno.97269

Figure 3.

Figure 3

Validation of transcytosis mechanisms for mEVs intestinal absorption. (A) Fluorescence-based PK analysis results at various time points (20, 40 min, 1, 3, 6, and 24 h). (B-C) Fluorescence signal-based blood analysis at various time points (20 min, 40 min, 1, 2, 3, 4, 5, and 6 h) after oral administration of mEVs. (D) Representative histogram of flow cytometry showing a cellular uptake of mEVs in Caco-2 cells after pre-incubation with anti-FcRn antibodies (% in the histogram represents intracellular mEVs-uptaken cell populations). (E) Normalized MFI indicating intracellular mEVs-positive cells. (F) Representative fluorescence confocal images showing cellular uptake of mEVs in Caco-2 cells with FcRn pre-blocking and quantitative analysis of intracellular mEVs puncta. Cy5.5-labeled mEVs were pseudo-colored in green. (G) Schematic demonstrating the Caco-2-based in vitro intestinal transcytosis assay. (H) The NIRF images to detect Cy5.5-labeled mEVs transported to a basolateral chamber at various time points (2, 4, 6, and 12 h). (I) The cumulative quantification of fluorescence signals in (H). Ab indicates anti-FcRn antibodies. All data presented as mean ± SD, Two-tailed t-test, **P < 0.01 and ***P < 0.001, and ****P < 0.0001.