Fig. 7

Acoustic shock waves and YSA-L-ZnO NPs combination treatment in HT-29 cell spheroids. Spheroids were produced, grown for 5 days and left untreated (No NPs) or treated with nanoparticles (YSA-L-ZnO NPs or with the non-targeted L-ZnO NPs as control, both at 100 µg/mL), testing two different concentrations. After 24, 48 and 72 h, spheroids were either subjected to a multiple shock wave (SW) treatment at two different SW intensities (Energy level E6, 0.22 mJ/mm² and energy level E9, 0.35 mJ/mm²) or not (no SW). A) After 96 h from the administration of the nanoparticles, cell viability was assessed through flow cytometry, by spheroid disaggregation. Data are normalized by the control sample, which is considered to be 100% vital. *p < 0.0332, **p < 0.0021, ***p < 0.0002, ****p < 0.0001. B) Results of evaluation of apoptosis levels through Annexin V assay under flow cytometry. 100 µg/mL L-ZnO and YSA-L-ZnO were administered to spheroids and the analyses were performed after a triple SW treatment at E9 energy level C) Fluorescence microscopy images after the second SW stimulation (48 h time step) combined with L-ZnO or YSA-L-ZnO. Both slices and lateral projections from Z-stacks are presented. Spheroids were stained with Calcein AM (green) and PI (red) to mark live and dead cells, respectively; cell nuclei were also stained with Hoechst (blue)