Fig. 4.
In vitro drug release monitoring and enhanced anti-tumor efficacy. a Confocal fluorescence images of KYSE-30 cells after being incubated with the RGD-f-PNPs/EPI conjugates for 5 min, 1 h, and 3 h. With a prolonged incubation time, more EPI was released. Consequently, the fluorescence intensity of both the RGD-f-PNPs and EPI gradually increased. The data suggest that it is possible to visualize the drug-releasing process using RGD-f-PNPs as a nanocarrier. Scale bar: 40 μm. b The EPI release kinetics from the RGD-f-PNPs at room temperature in PBS at pH 7.4 and 6 was measured and plotted with respect to the change of the absorbance intensity at 480 nm. The results indicated that more EPI was released in an acidic environment. Error bars represent s.d. (n = 3). c Viability of KYSE-30 cells after being treated with RGD-f-PNPs alone, EPI alone, and RGD-f-PNPs/EPI at different concentrations. Without EPI loading, the RGD-f-PNPs indicate low cytotoxicity. The cytotoxicity of RGD-f-PNPs/EPI was significantly higher than EPI alone in KYSE-30 cells, in which RGD-f-PNPs had selective targeting through RGD-integrin αvβ3 subunit binding. Especially at lower drug dose, RGD-f-PNPs/EPI indicated more significantly enhanced anti-tumor efficiency compared to EPI alone. Error bars represent s.d. (n = 3). d Confocal fluorescence images of KYSE-30 cells incubated with RGD-f-PNPs/EPI and EPI alone. A much brighter intracellular fluorescence of EPI was observed in cells incubated with RGD-f-PNPs/EPI vs. cells incubated with EPI only, indicating that the RGD-f-PNPs could facilitate the cellular uptake and intracellular accumulation of EPI into EC cells. In addition, a different internalization pattern was observed when treated with EPI alone compared to EPI-loaded RGD-f-PNPs. Scale bar: 40 μm