*4.8. Nanosponge Cellular Uptake Assays*

Coumarin 6-loaded GSH-NS cellular uptake was assessed by cytofluorimetric analysis using a C6 flow cytometer (Accuri Cytometers, Ann Arbor, MI, USA) and imaging analysis using a DMI4000B fluorescence microscopy (Leica, Wetzlar, Germany). For flow cytometry analysis, 5.0 × 10<sup>4</sup> cells were seeded in a six-well culture plate. Forty-eight hours after seeding, HT-29, HCT116, DU145, and PC-3 cells were treated with the respective not-cytotoxic (IC1) and cytotoxic (IC50) concentrations of either fluorescent GSH-NS B or fluorescent GSH-NS D at 24 h. After a 24 h incubation, the cells were washed three times with phosphate-buffered saline PBS, suspended in 250 µL PBS, and run on the flow cytometer with 488 nm excitation. Intracellular fluorescence was expressed as integrated mean fluorescence intensity (iMFI), which was the product of the frequency of 6-coumarin-loaded GSH-NS positive cells and the mean fluorescence intensity.

Microscopy observation was carried out after glass coverslips were placed in 24-well plates and the cells seeded at a density of 5.0 × 10<sup>4</sup> cells/coverslip for 48 h of incubation. The coumarin 6-loaded nanosponges were then added at the respective IC<sup>50</sup> values for GSH-NS B and D, and incubated for 24 h. The cells were incubated with 1 µg/mL of 4<sup>0</sup> ,6-diamidino-2-phenylindole (DAPI) for nuclear counterstaining 30 min before the programmed stop time. After the cells were washed with PBS, the cells on the coverslip were mounted on a glass slide, observed under a fluorescence microscope, and photographed.
