Fluorescent probe dil

The 293T cells were seeded in 12-well plates, which contained sterilized cover slips (WHB Biotech), incubated at 37 °C for 24 h, and transiently transfected with WT or mutant pEGFP-N2 plasmids. One day later, the cells were washed twice with PBS (phosphate buffer saline), fixed with 4% PBS-buffered formaldehyde (PFA) for 30 min at room temperature, washed twice with PBS, and then incubated with fluorescent probe Dil (Beyotime Biotech) targeted to the cell membrane for 5 min at 37°C. After washing with PBS, the nuclei were stained with DAPI (Beyotime Biotech) at room temperature for 5 min. Cover slips were mounted, and the plates were examined using a confocal microscope (Nikon A1 Microsystems).

Because Dox is intrinsically fluorescent, uptake of free Dox or NPs can be observed at 594 nm using confocal microscopy after excitation at 480 nm. Briefly, HSC-3, SCC-9, CAL-27 and HCM cells were seeded into a 6-well plate at a density of 1 × 10⁵ cells per well. All cells treated with free Dox or NPs (5 μg per well, Dox). Cells were labeled with fluorescent probe Dil (Beyotime Biotechnology, China) after 8 hours of treatment, and in vitro uptake was calculated detected by a confocal laser scanning microscopy (Carl Zeiss Microscope Systems, Jena, Germany).