The in vitro cellular uptake of MPEG-PCL micelles loaded with the fluorescent probe coumarin 6 (Tokyo Chemical Industry, Japan) was assessed according to a previously described method with some modifications.32 (link),33 (link) Briefly, a sample (100 μL) of the micelles was mixed with 400 μL distilled water and 500 μL acetonitrile and vortexed for 30s before the amount of coumarin 6 loaded in the MPEG-PCL micelles was determined by HPLC under the conditions listed in Table 1 .
HUVECs were seeded in a 96-well plate at a density of 30,000 cells/well. After incubation for 24 h, the original medium was replaced with coumarin 6-loaded MPEG-PCL micelles dispersed in DMEM/F-12 solution, free coumarin 6 dissolved in DMSO-DMEM/F-12 solution at the same concentration as that of the micelles (DMSO content <0.1%) or saturated free coumarin 6 in DMEM/F-12. After incubation for 2, 8 and 24 h, the medium was discarded and the cells were washed three times with PBS to remove the un-internalized coumarin 6. Next, the cells were fixed with 4% paraformaldehyde before fluorescence images were taken and the fluorescence intensity in each group was evaluated under an inverted fluorescence microscope Axio Vert.A1 (ZEISS, Germany).
HUVECs were seeded in a 96-well plate at a density of 30,000 cells/well. After incubation for 24 h, the original medium was replaced with coumarin 6-loaded MPEG-PCL micelles dispersed in DMEM/F-12 solution, free coumarin 6 dissolved in DMSO-DMEM/F-12 solution at the same concentration as that of the micelles (DMSO content <0.1%) or saturated free coumarin 6 in DMEM/F-12. After incubation for 2, 8 and 24 h, the medium was discarded and the cells were washed three times with PBS to remove the un-internalized coumarin 6. Next, the cells were fixed with 4% paraformaldehyde before fluorescence images were taken and the fluorescence intensity in each group was evaluated under an inverted fluorescence microscope Axio Vert.A1 (ZEISS, Germany).