Fig. 4: Trafficking of dtEVs in recipient cells.

a Nanocarriers labeled with PKH26 (red) were taken up by endocytosis and underwent vesicular trafficking in the recipient cells, from the endosome to the lysosome. b PANC-1 cells were initially treated with fluorescent nanoparticles (red) for 2 h and then fixed for co-staining with anti-Rab5 to visualize the colocalization in early endosome (green). Colocalization in recipient cells showed a similar level for LNPs, non-targeted IgG-EVs and dtEVs (colocalization % (Col%): LNP: 89.0 ± 3.5; non-targeted IgG-EV: 88.17 ± 5.2; dtEV: 81.50 ± 3.7, n = 3 biological independent experiments). c When comparing the colocalization (yellow) of LNPs, dtEVs, and non-targeted IgG-EVs, we observed less colocalization of dtEVs in lysosomes (green) after 8 h incubation (Col%: LNP: 26.17 ± 4.0; non-targeted IgG-EV: 13.53 ± 4.87; dtEV: 5.73 ± 1.45, n = 3 biological independent experiments). d A fluobody recognition system was used to monitor the interaction of dtEV lumens within vesicular trafficking. Recipient cells were pre-transfected with an anti-GFP fluobody tagged with mCherry (red), which is expressed in the cytosol. Upon release of the dtEV lumen, the cytosolic anti-GFP fluobody could recognize CD63^GFP on the dtEV lumens, leading to temporary local accumulation. e PANC-1 cells expressing anti-GFP_mCherry fluobody were incubated with dtEVs containing CD63^GFP for 2, 4, and 8 h. The induced accumulation of anti-GFP fluobody was observed to increase from 2 to 4 and 8 h, suggesting the potential of lumen releasing of dtEV. Fluorescent images are representative of n = 3 biologically independent experiments. Scale bar in b, c, and e are 10 μm. Data were presented as mean ± SD. For b and c, the data were analyzed by unpaired two-sided Student’s t test. (ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Source data are provided as a Source data file.