Fig. 3: Penetration and uptake characteristics of dtEVs in PANC-1 cells.

a Enzyme-linked immunoassay (ELISA) shows both CD64^wt (K_d = 0.0589 nM) and CD64^ck (K_d = 0.0536 nM) bound to hIgG1, but not hIgG2, with high affinity (n = 3 biological independent experiments). b Flow cytometry analysis using anti-CD63 beads to capture dtEVs confirms surface CD64^ck bound to hIgG. c Internalization assay using EVs labeled with fluorescent PKH67 dye shows higher cell uptake of dtEVs loaded with humanized anti-ROR1 mAb (αROR1) than humanized anti-EGFR (αEGFR) or control IgG. Uptake of dtEVs with CK peptides (12,209 ± 1914) doubled that of Flag control (5,585 ± 755.9) (n = 3 biological independent experiments). d Schematic of transcytosis assay used to quantify the entry and exit of various nanocarriers from the top to the bottom cell monolayer separated by a Transwell® insert membrane with 5 μm pores. e CD64^ck_ αROR1 dtEVs displayed the greatest putative transcytosis (n = 5 biological independent experiments). f Pre-treating top cells with inhibitors of clathrin- mediated endocytosis (Pitstop 2, Pit2, 10 mM), caveolae- mediated endocytosis (Methyl-β-cyclodextrin, mβC, 10 mM) or exosomal secretion (neticonazole, Ntz, 10 mM) greatly prevented this putative transcytosis, but not inhibitor of macropinocytosis (Cytochalasin D, cytD, 10 µM). Con: no inhibitor (n = 5 biological independent experiments). g dtEVs penetrated tumor spheroids of PANC-1 cells (~300 µm) deeper than stEVs or LNPs after 24 h incubation (blue: DAPI). LNPs and EVs were labeled with PKH67 (green), while anti-hIgG was in red. Immunofluorescent images are representative of n = 3 biologically independent experiments. Scale bar in g: 100 μm. Data were presented as mean ± SD. For c–f, the data were analyzed by unpaired two-sided Student’s t test. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Source data are provided as a Source data file.