Figure 3.

The effect of desialylation on the cellular uptake of MSC-EVs. (A) MSC-EVs were treated with different concentrations of neuraminidase for 30 min at 37 °C. The sialic acid level was determined by lectin blotting. (B) The Gal and GalNAc levels of EVs were detected by lectin blotting with 4 U neuraminidase treatment at 37 °C for 30 min. (C) The zeta potential of EVs treated with or without neuraminidase was estimated by DLS. (D) Positive markers, Alix, CD63, TSG101, and CD81, and negative marker calnexin of the MSC-EVs, E-Dox and dsE-Dox were analyzed by Western blotting. (E,F) The morphology and size of the desialylated MSC-EVs were detected by TEM and DLS, respectively. Scale bar, 100 nm. (G) MSC-EVs and desialylated MSC-EVs were labeled with ExoTracker, and their cellular uptake were analyzed by flow cytometry. (H) Uptake of free Dox, E-Dox, and dsE-Dox (loaded with 2 μmol/L Dox) were analyzed by measuring the autofluorescence of Dox using flow cytometry. The data were presented as the mean values ± SD. * p < 0.05, ** p < 0.01.