3.6. In Vitro Cellular Uptake Assay

The cellular uptake of HA-PLGA-NPs was assessed by laser confocal microscope (Nikon A1, Japan) and flow cytometry using Dio-labeled HA-PLGA-API-NPs in vitro. Briefly, HRT-18 and HT-29 cells were seeded in 6-well plates (2 × 10⁶ cells/well), incubated overnight in CO₂ incubator at 37 °C, then treated with 2 mL of fresh cell medium containing PLGA-DiO-NPs or HA-PLGA-DiO-NPs for 4 h in a CO₂ incubator at 37 °C. The final concentration of DiO was 2 μg/mL in all groups. The final concentration of HA-PLGA-API-NPs was 0.1 mg/mL, and the equivalent total concentration of apigenin was 3 µg/mL. To explore the role of HA in CD44-mediated cellular uptake, the cells were pre-treated with HA (5 mg/mL) for 0.5 h before being incubated with DiO-labeled HA-PLGA-NPs. The cell nuclei were stained with DAPI and observed using a fluorescence microscope. Cell samples were collected, washed, and resuspended in PBS for flow cytometry analysis.