*3.5. PGS-NPs Morphological Characterization*

Most promising unloaded and loaded PGS-NPs were characterized through transmission electron microscopy (TEM). Samples for TEM imaging were prepared by dropping PGS-NPs suspensions on single-side-polished copper grids coated with a formvar film and dried at room temperature. Samples were observed using a Talos L120C transmission electron microscope (Thermo Fisher Scientific, Milan, Italy) equipped with a 4K digital camera Ceta CMOS (Thermo Fisher Scientific). Obtained images were elaborated using Nanoscope software and the particle size distribution was calculated. A minimum of 150 nanoparticles were selected in each acquired TEM image.

Dynamic light scattering (DLS) analysis was carried out by using a Nanozetasizer (Malvern Instruments) equipped with a 4.0 mW He–Ne laser operating at 633 nm and an avalanche photodiode detector in order to determine the average size of the obtained PGS-NPs. All the measurements were repeated at least 3 times. Colloidal stability of PGS-NPs at different concentrations (0.2 and 0.1 mg/mL) was investigated by means of DLS at 37 ◦C. PGS-NP size was monitored at different times from sample preparation (time 0, 24, 48, 72 h). The same experiment was carried out using the Dulbecco's Modified Eagle Medium (DMEM, Euroclone, Milano, Italy) supplemented as described below and stored at 37 ◦C in a humidified 5% CO2 atmosphere.
