3.8. In Vitro Cellular Uptake

Fluorescent probe-labeled nanoparticles (C6-NPs) were prepared by replacing Asp with an equal amount of coumarin-6 (C6) under light-avoidance conditions according to Section 2.4. HepG2 cells were seeded in 6-well plates at 1 × 10⁶ cells/well and incubated for more than 12 h until 80% cell-adherent coverage was achieved, the original medium was aspirated and the wells were washed with PBS. The C6 solution and C6-NPs were diluted with a serum-free medium so that the fluorescein concentration was 1 μg/mL for both. After the cells in each group were given C6 and C6-NPs, they were incubated at a temperature of 37 °C for 4 h. At the end of the incubation, the fluorescein-containing medium was removed, and the cells were washed with PBS 2 times. Cells were digested with trypsin for 4 min and blown into cell suspension, centrifuged at 1000 rpm/min for 3 min, and then collected and washed twice with PBS. The cells were then fixed with 4% paraformaldehyde for 15 min and collected by centrifugation. Finally, the cells were resuspended with PBS, and 500 μL was placed in a flow tube, and the uptake of coumarin by HepG2 cells was detected using flow cytometry.