FIGURE 3.

Nanotopological changes of sEVs in response to pH manipulation. (a) sEVs incubated in acidic (pH 4.00), neutral (pH 7.50), or basic (pH 10.00) buffers for 3 h before HS‐AFM imaging. sEV spherical shape is retained at pH 4.00 but deformed at pH 10.00 (scale bar, sEV_{pH 4.00}: 8 nm; sEV_{pH 7.50}: 17 nm; sEV_{pH 10.00}: 23 nm). (b) 3D structure of sEV_{pH 4.00}, sEV_{pH 7.50}, and sEV_{pH 10.00}. (c) Bar graph showing the proportion of deformed sEV after exposure to three different pH levels (black bar, normal; white bar, deformed). (d) pH level significantly alters spatial dimension parameters and circularity of sEV. Data are presented as mean ± SEM (***p < 0.001, ****p < 0.0001, n.s: not significant). (e) Bar graphs presenting the real‐time height changes (Δh) of an sEV pretreated at pH 4.00, pH 7.50, or pH 10.00. (f) sEV_{pH 10.00} is greatly stretched on PLL‐coated substrate compared with sEV_{pH 4.00} and sEV_{pH 7.50}. Values are shown as mean ± SEM (****p < 0.0001). (g) Real‐time imaging of sEV nanotopology during pH transition (neutral‐acidic‐basic). sEV shrinks at pH 4.00 and later deforms at pH 10.00 (scale bar: 30 nm). (h) Line graphs demonstrating the real‐time measurement of height, diameter, area, and circularity in response to pH change. The values are illustrated as ratio relative to the initial state (t = 0) (red: height; blue: diameter; green: area; yellow dotted: circularity; green area: pH 7.50; red area: pH 4.00; purple: pH 10.00). (i) Real‐time change in aspect ratio of sEV at different pH levels. The aspect ratio drops in the basic environment, which is consistent to the result in a premix setting (green area: pH 7.50; red area: pH 4.00; purple area: pH 10.00). (j) A schematic diagram delineating the sEV nanotopology at different pH levels. Sample size for (c), (d), and (f): sEV_{pH 4.00}, 52; sEV_{pH 7.50}, 51; sEV_{pH 10.00}, 52