Fig. 3
In vitro studies of network nanocapsules. A Illustration of an in vitro BBB-crossing model. bEnd.3 cells on the transwell filter were exposed to VCNCs-Cy5.5 and CNCs-Cy5.5 for an exposure time of texp = 3 h and texp + tinc = 3 h + 24 h at a concentration of C5-HT = 25 μg/mL. Mean Cy5.5 fluorescence per cell ICy5.5 was measured by flow cytometry for bEnd.3 cells and PC-12 cells. Normalized penetration efficiencies of the nanocapsules were calculated based on these results with growth factors. B At texp + tinc = 3 h + 24 h, confocal images of VCNCs-Cy5.5 and CNCs-Cy5.5 in bEnd.3 and PC-12 cells at C5-HT = 25 μg/mL were obtained, including Cy5.5 channel (red) and DAPI channel (blue). The scale bar represents 5 μm. C Fluorescence images and D fluorescence intensities of DCFH, corresponding to the ROS level, were detected in PC-12 cells after incubation with VCNCs, CNCs, and VNCs at texp + tinc = 3 h + 24 h (C5-HT: 25 μg/mL, n = 3). The scale bar represents 100 μm. Significant differences between groups were analyzed using the one-way ANOVA method, *P < 0.05, **P < 0.01, ***P < 0.001. E The amount of liberated 5-HT was detected in PC-12 cells incubated with VCNCs, CNCs, and VNCs (C5-HT: 25 μg/mL) for 3 h in complete cell media, pH 6.4 cell media, ROSUP cell media, and pH 6.4 + ROSUP media (n = 3)