*4.5. Cell Culture and Treatment with Glutathione Responsive* β*-cyclodextrin-Based Nanosponges*

Human colorectal cancer cell lines, HCT116 and HT-29 (ICLC, Interlab Cell Line Collection, Genova, Italy), and human prostatic carcinoma cell lines, DU145 and PC-3 (ICLC), were cultured in McCoy's 5A Medium and RPMI-1640 Medium, respectively. These media were supplemented with 2 mM L-glutamine, 100 UI/mL penicillin, 100 µg/mL streptomycin, and 10% (*v*/*v*) heat-inactivated fetal calf serum (Sigma, ST Louis, MO, USA) in a humidified atmosphere of 5% CO<sup>2</sup> air at 37 ◦C. At 85% confluence, cells were harvested with 0.25% trypsin and sub-cultured into 75 cm<sup>2</sup> flasks, 6-well plates or 96-well plates according to need. Cells were allowed to attach to the surface for 24 h prior to treatment. GSH-NS B and D were then suspended in a cell culture medium and diluted to the appropriate concentrations. After treatment, the cells were harvested to determine cytotoxicity, cell cycle distribution and ROS production. The cells that were not exposed to GSH-NS were used as control conditions for each experiment.
