*4.4. Characterization of Glutathione Responsive* β*-cyclodextrin-based Nanosponges*

The two types of GSH-NS (GSH-NS B and D), either blank or 6-coumarin loaded, were characterized in vitro to measure their physico-chemical parameters. The average diameters, polydispersity indices, and zeta potential values were determined by photon correlation spectroscopy (PCS) and electrophoretic mobility using a 90 Plus Instrument (Brookhaven, NY, USA) at a fixed angle of 90◦ and a temperature of +25 ◦C. The analyses were performed on diluted GSH-NS samples (1:30 *v*/*v*). For zeta potential determination, the samples were placed in an electrophoretic cell where an electric field of approximately 15 V/cm was applied. Three batches were analyzed for each NS type and each measured value was the average of ten repetitions. Nanosponge morphology was evaluated by transmission electron microscopy (TEM) (Philips CM10 instrument, Eindhoven, Netherlands) after the diluted aqueous nanosponge nanosuspensions were sprayed onto a Form war-coated copper grid and air-dried.
