*4.13. Cell Growth and Nanosponge Cellular Uptake Assays on Three-Dimensional Cell Culture*

Cell suspensions (250-cell spheroids) 40 µL were dispensed into the access hole at each cell culture site to form a hanging drop on a Perfecta3D® 96-well Hanging Drop Plate (3D Biomatrix, Ann Arbor, MI, USA). On day 8 of the HCT116 and DU145 spheroid culture, 15 µL of different GSH-NS B or GSH-NS D concentrations (0.5, 2.0, 4.0, and 6.0 mg/mL) was added to each cell hanging drop and MCS growth was analyzed at 24, 48, and 72 h after nanosponge incubation. Noteworthy is the fact that we had to use a different concentration range for 3D cell growth assay (0.5, 2.0, 4.0, and 6.0 mg/mL) to obtain the dose-response data necessary to calculate the IC<sup>50</sup> values from the one used in the 2D cell growth assay (0.5, 2.0, 4.0, and 3.0 mg/mL). Phase contrast photographs were taken by the DMI4000B microscope (Leica, Milano, Italy) and the diameter of each MCS was measured by Leica Application Suite Software (Leica) and the volume (V) was calculated using the equation V = 4/3πr 3 . Coumarin 6-loaded nanosponge uptake by MCS at the respective IC<sup>50</sup> at 24 h for GSH-NS B or GSH-NS D was analyzed by fluorescence microscopy using a DMI4000B microscope (Leica).
