FIGURE 4.
CAR‐sEVs significantly enhanced targeting efficacy in the HepG2/C3A cell model. (A) Schematic representation of the conjugation of N₃‐sEVs with DBCO‐Cy5 (red) and DBCO‐AF488‐scFv (green) using click chemistry to generate CAR‐sEVs. (B) Flow cytometry histograms showing the fluorescence intensity of HepG2/C3A cells treated with increasing doses (10⁷, 10⁸, or 10⁹ particles) of CAR‐sEVs or N3‐sEVs for 18 h. The fluorescence was detected in the Alexa Fluor 488 and PE‐Cy5 channels. Sham treatment (no sEVs) was included as a control. (C) Quantification of targeting efficiency (%) of N3‐sEVs and CAR‐sEVs at different doses (10⁷, 10⁸, or 10⁹ particles). (D) Confocal microscopy images (630×) showing the cellular uptake of N₃‐sEVs and CAR‐sEVs in HepG2/C3A cells at various time points (0.5, 1, 2, 4, and 6 h). The images illustrate the nuclei (DAPI, blue), sEV particles with azido groups (DBCO‐Cy5, red), and DBCO‐scFv (labelled with Alexa Fluor‐488, green). The merged DIC (differential interference contrast) images to fluorescent images. Scale bar = 10 µm. (E) Flow cytometry was used to measure the mean fluorescence intensity in HepG2/C3A cells treated with N₃‐sEVs (grey) or CAR‐sEVs (red) over 6 h. All data are presented as the mean ± SEM. ***p < 0.001.