Cellular binding of the PLA-flurbiprofen nanoparticles. (A) bEnd.3 cells were treated with approximately 100 μg/cm2 PLA-flurbiprofen nanoparticles for four hours at 37°C and the cellular binding was quantified by flow cytometry. The data are shown as histograms of the FL1-H channel (red: PLA-flurbiprofen nanoparticles, black: untreated control). (B-D) bEnd.3 cells were treated with approximately 100 μg/cm2 nanoparticles. To inhibit endocytosis, the cells were treated at 4°C (B). For the cellular uptake, cells were treated for one hour (C) or four hours (D) at 37°C. After the incubation period, the cells were put on ice and washed with acidic PBS to remove the surface-bound nanoparticles. The cells were fixed with paraformaldehyde and cell nuclei were stained with DRAQ5TM. Scale bar, 10 μm. PLA, polylactide.