Fig. 2. Biogenesis of DS-EXOs based on the MGE-mediated click chemistry of ADSCs.

(A) Confocal microscopic images of DS-modified ADSCs at different concentrations of Ac₄ManNAz. For the MGE-mediated click chemistry, ADSCs were incubated with Ac₄ManNAz for 48 hours to generate azide groups on the surface and then treated with serum-free medium containing Cy5.5-labeled DBCO-DS (10 μM) for 2 hours. (B) Quantification of the DS on the ADSCs at different concentrations of Ac₄ManNAz. Modified ADSCs were analyzed by flow cytometry to quantify the azide surface expression. (C) Intracellular tracking of DS in MDA-MB-231-CD63-GFP cells. The CD63-GFP cell line was used for MGE-mediated click chemistry to confirm the intracellular colocalization of CD63 and DS during biogenesis. Confocal microscopy images show CD63-GFP (green) and Cy5.5-labeled DS (red) in the MDA-MB-231-CD63-GFP cells. (D) Intracellular tracking of DS in the ADSCs. Time-dependent fluorescence images of the early endosomes and DS were obtained after the MGE-mediated click chemistry of ADSC. Confocal microscopy images show EEA1 (green) and Cy5.5-labeled DS (red) in the ADSCs. (E) Kinetic quantification of confocal imaging in (D). a.u., arbitrary units. (F) Secretion of the DS-decorated extracellular vesicles from the ADSCs. To visualize the extracellular secretion of the DS-decorated extracellular vesicles, the ADSCs were seeded onto a High Grid-500 glass-bottom μ-Dish. After the MGE-mediated click chemistry, images were separately obtained for the same spot using SEM and confocal microscopy. The dotted region in the top panel is magnified in the bottom panel.