*2.2. D Cell Culture Cytotoxicity of Glutathione Responsive* β*-cyclodextrin-Based Nanosponges*

The basal level of reduced glutathione was measured in each of the different cell lines, which were grouped according to cancer type. The results show that HT-29 (Figure 1A) and PC-3 cells (Figure 1B) display significantly lower GSH levels than those in HCT116 (Figure 1A) and DU145 cells (Figure 1B). Dose-response curves were then performed by exposing human colorectal cancer, HCT116 and HT-29, and human prostatic carcinoma, DU145 and PC-3, cell monolayers to various concentrations (0.5, 1.0, 2.0, and 3.0 mg/mL) of the two types of GSH-NSs with increasing disulfide bridge content (GSH-NS B and D), for 24, 48, and 72 h. Table 2 reports the IC<sup>1</sup> and IC<sup>50</sup> values of GSH-NS, which were determined at 24, 48, and 72 h of exposure.

Figure 2 reports GSH-NS IC<sup>50</sup> values according to disulfide-bond content and exposure time. These data highlight the remarkable cytotoxicity difference between the two nanosponge formulations in colorectal cancer cell lines, as lower IC<sup>50</sup> values were observed for the nanosponge formulation with the lower disulfide bridge content (GSH-NS B) at 24, 48, and 72 h (Figure 2A,B). In prostatic cancer cell lines, no significant differences in IC<sup>50</sup> values were detected for the two nanosponge formulations in DU145 cells (Figure 2C), which were characterized by the highest GSH cell content (Figure 1). Meanwhile, in PC-3 cells, a significant cytotoxicity difference was observed between the two nanosponge formulations, as the IC<sup>50</sup> values determined by the GSH-NS B formulation were lower compared with what was observed in colorectal cancer cells, even if only at 24 and 48 h (Figure 2D).

in prostatic cancer cells than in colorectal cancer cells.

GSH-NS D, in all of the cell lines, with the exception of DU145 cell line (Table 2 and Figure 2). Moreover, lower GSH-NS D IC50 values were observed on DU145 and PC-3 cells than on HCT116 and HT-29 cells at 48 and 72 h of exposure (Table 2 and Figure 2), suggesting that the nanosponge

**Figure 1.** Intracellular glutathione-responsive β-cyclodextrin (GSH) level according to cell type. The reduced glutathione content of human colorectal cancer cell lines, HCT 116 and HT-29 (**A**), and human prostatic carcinoma cell lines, DU145 and PC-3 (**B**), was measured at a basal level, that is, in untreated cells, and was expressed as nmol/µg protein. The results are mean values ± SD of three independent experiments performed in triplicate. Statistically significant difference between cell lines: \* *p* < 0.05. **Figure 1.** Intracellular glutathione-responsive β-cyclodextrin (GSH) level according to cell type. The reduced glutathione content of human colorectal cancer cell lines, HCT 116 and HT-29 (**A**), and human prostatic carcinoma cell lines, DU145 and PC-3 (**B**), was measured at a basal level, that is, in untreated cells, and was expressed as nmol/µg protein. The results are mean values ± SD of three independent experiments performed in triplicate. Statistically significant difference between cell lines: \* *p* < 0.05.


**Table 2.** Glutathione-responsive β-cyclodextrin-based nanosponge (GSH-NS) inhibition concentration (IC) values on 2D cell cultures. **Table 2.** Glutathione-responsive β-cyclodextrin-based nanosponge (GSH-NS) inhibition concentration (IC) values on 2D cell cultures.

Statistically significant difference between the two nanosponge formulations: \* *p* < 0.05. Statistically significant difference between the two nanosponge formulations: \* *p* < 0.05.

Therefore, the nanosponge formulation with the lower disulfide-bond content, GSH-NS B, was found to be more cytotoxic than the nanosponge formulation with the higher disulfide-bond content, GSH-NS D, in all of the cell lines, with the exception of DU145 cell line (Table 2 and Figure 2). Moreover, lower GSH-NS D IC<sup>50</sup> values were observed on DU145 and PC-3 cells than on HCT116 and HT-29 cells at 48 and 72 h of exposure (Table 2 and Figure 2), suggesting that the nanosponge formulation with the higher disulfide bridge concentration, GSH-NS D, has a higher cytotoxic effect in prostatic cancer cells than in colorectal cancer cells.

**Figure 2.** Cytotoxicity of glutathione responsive β-cyclodextrin-based nanosponges in 2D cell cultures. HCT116 (**A**), HT-29 (**B**), DU145 (**C**), and PC-3 (**D**) cells were incubated with GSH-NS B and GSH-NS D at different concentrations (0.5, 1.0, 2.0, 3.0 mg/mL) for 24, 48, and 72 h. Cell proliferation was evaluated by WST-1 assay and the values of the concentration required for a 50% cell growth inhibition (IC50) were determined by the proliferation curves obtained using the CalcuSyn 2.11 software (Biosoft, Cambridge, UK). The results are mean values ± St.Dev of three independent experiments, replicated eight times for each condition. Statistically significant difference between **Figure 2.** Cytotoxicity of glutathione responsive β-cyclodextrin-based nanosponges in 2D cell cultures. HCT116 (**A**), HT-29 (**B**), DU145 (**C**), and PC-3 (**D**) cells were incubated with GSH-NS B and GSH-NS D at different concentrations (0.5, 1.0, 2.0, 3.0 mg/mL) for 24, 48, and 72 h. Cell proliferation was evaluated by WST-1 assay and the values of the concentration required for a 50% cell growth inhibition (IC50) were determined by the proliferation curves obtained using the CalcuSyn 2.11 software (Biosoft, Cambridge, UK). The results are mean values ± St.Dev of three independent experiments, replicated eight times for each condition. Statistically significant difference between nanosponge formulations: \* *p* < 0.05.
