Figure 6.

Inhibition of integrins in IL-1β-ADEVs reduced the EVs uptake by primary cultured neurons in vitro. (a) The scheme of in vitro IL-1β-ADEVs uptake with integrin inhibition. Purified PKH26-labelled IL-1β-ADEVs were pre-treated with integrin inhibitor RGD peptide (0.1μM or 1μM) at 37°C for 30 min, and equal amount of pre-treated PKH26-IL-1β-ADEVs were added to primary cultured mouse cortical neurons at DIV 10, which were imaged by a live cell imaging system for three days. (b) Representative images of the indicated time. White arrow shows the fluorescent signals within the cells. Scale bar, 200 μm. (c, d) Quantification of PKH26-IL-1β-ADEVs uptake using IncuCyte software to measure (c) total integrated intensity of red fluorescence per mm² at each time point and Image J to count (d) the number of fluorescent cells at 0, 24, 48 and 72 h, across three independent experiments. Data are present as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001 with PKH26-IL-1β-ADEVs + 0.1μM RGD peptide or PKH26-IL-1β-ADEVs + 1 μM RGD peptide compared to PKH26-IL-1β-ADEVs + vehicle as determined by repeated measure two-way ANOVA with Tukey’s multiple comparisons.