*2.3. Glutathione Responsive* β*-cyclodextrin-Based Nanosponge Cellular Uptake on 2D Cell Cultures*

nanosponge formulations: \* *p* < 0.05.

*2.3. Glutathione Responsive β-cyclodextrin-Based Nanosponge Cellular Uptake on 2D Cell Cultures* The cellular uptake of non-cytotoxic (IC1) and cytotoxic (IC50) concentrations of fluorescent GSH-NS was analyzed by flow cytometry and fluorescence microscope imaging after 24 h exposure. HCT116, HT-29, DU145, and PC-3 cell monolayers were exposed to 6-coumarin loaded GSH-NS B or 6-coumarin loaded GSH-NS D for 24 h. Significant dose-dependent differences in nanosponge cellular uptake were observed (Figure 3A–D). Interestingly, higher nanosponge cellular uptake was observed in colorectal cancer cells (Figure 3A,B) than in prostatic cancer cells (Figure 3C,D). In particular, the highest nanosponge cellular uptake was observed in HCT116 cells (Figure 3A) and the lowest in PC-3 cells (Figure 3D). As IC50 values at 24 h of incubation in colorectal and prostatic cell line were very similar (Table 2), the lower fluorescent GSH-NS intracellular uptake in prostatic cells suggests that they are more highly sensitive to the nanosponge cytotoxic effect than the colorectal cancer cell lines. In particular, Figure 3E–G show images of untreated HCT116 cells with nuclear counterstaining (Figure 3E) and after 24 h exposure to 6-coumarin loaded GSH-NS B (Figure 3F) or 6-coumarin loaded GSH-NS D (Figure 3G). The cellular uptake of non-cytotoxic (IC1) and cytotoxic (IC50) concentrations of fluorescent GSH-NS was analyzed by flow cytometry and fluorescence microscope imaging after 24 h exposure. HCT116, HT-29, DU145, and PC-3 cell monolayers were exposed to 6-coumarin loaded GSH-NS B or 6-coumarin loaded GSH-NS D for 24 h. Significant dose-dependent differences in nanosponge cellular uptake were observed (Figure 3A–D). Interestingly, higher nanosponge cellular uptake was observed in colorectal cancer cells (Figure 3A,B) than in prostatic cancer cells (Figure 3C,D). In particular, the highest nanosponge cellular uptake was observed in HCT116 cells (Figure 3A) and the lowest in PC-3 cells (Figure 3D). As IC<sup>50</sup> values at 24 h of incubation in colorectal and prostatic cell line were very similar (Table 2), the lower fluorescent GSH-NS intracellular uptake in prostatic cells suggests that they are more highly sensitive to the nanosponge cytotoxic effect than the colorectal cancer cell lines. In particular, Figure 3E–G show images of untreated HCT116 cells with nuclear counterstaining (Figure 3E) and after 24 h exposure to 6-coumarin loaded GSH-NS B (Figure 3F) or 6-coumarin loaded GSH-NS D (Figure 3G).

**Figure 3.** Fluorescent glutathione responsive β-cyclodextrin-based nanosponge cellular uptake. HCT116 (**A**), HT-29 (**B**), DU145 (**C**), and PC-3 (**D**) cells were exposed to the respective IC1 and IC50 of 6-coumarin loaded GSH-NS B and 6-coumarin loaded GSH-NS D for 24 h and analyzed by flow cytometry. Cellular uptake was expressed as integrated mean fluorescence intensity (iMFI). Representative fluorescence images of HCT116 untreated cells (**E**), HCT116 cells exposed to 6 coumarin loaded GSH-NS B IC50 (**F**), and 6-coumarin loaded GSH-NS D IC50 (**G**) for 24 h using 4′,6- **Figure 3.** Fluorescent glutathione responsive β-cyclodextrin-based nanosponge cellular uptake. HCT116 (**A**), HT-29 (**B**), DU145 (**C**), and PC-3 (**D**) cells were exposed to the respective IC<sup>1</sup> and IC<sup>50</sup> of 6-coumarin loaded GSH-NS B and 6-coumarin loaded GSH-NS D for 24 h and analyzed by flow cytometry. Cellular uptake was expressed as integrated mean fluorescence intensity (iMFI). Representative fluorescence images of HCT116 untreated cells (**E**), HCT116 cells exposed to 6-coumarin loaded GSH-NS B IC<sup>50</sup> (**F**), and 6-coumarin loaded GSH-NS D IC<sup>50</sup> (**G**) for 24 h using 4 0 ,6-diamidino-2-phenylindole (DAPI) (blue) as nuclear counterstain (63x magnification). Statistically significant difference between IC<sup>1</sup> and IC50: \*\* *p* < 0.01; \*\*\* *p* < 0.001.

#### diamidino-2-phenylindole (DAPI) (blue) as nuclear counterstain (63x magnification). Statistically *2.4. The E*ff*ect of Glutathione Responsive* β*-cyclodextrin-Based Nanosponges on Cell Death and Cell Cycle*

significant difference between IC1 and IC50: \*\* *p* < 0.01; \*\*\* *p* < 0.001. *2.4. The Effect of Glutathione Responsive β-cyclodextrin-Based Nanosponges on Cell Death and Cell Cycle* As an increase in the number of dead or plasma membrane-damaged cells results in an increase in lactate dehydrogenase (LDH) in the culture supernatant, we measured the LDH leakage of HCT116, HT-29, DU145, and PC-3 cells after 24, 48, and 72 h of incubation with an experimental medium containing different GSH-NS B or GSH-NS D concentrations (0.5, 1.0, 2.0, and 3.0 mg/mL). No significant increase in LDH leakage percentage over untreated control cells was observed under any of the test conditions (data not shown). The lack of apparent plasma membrane-damaged cells in the LDH assay would appear to contrast significantly with the decrease in cell growth observed by WST-1 cell proliferation assay (Table 2 and Figure 2), which was performed at the same incubation As an increase in the number of dead or plasma membrane-damaged cells results in an increase in lactate dehydrogenase (LDH) in the culture supernatant, we measured the LDH leakage of HCT116, HT-29, DU145, and PC-3 cells after 24, 48, and 72 h of incubation with an experimental medium containing different GSH-NS B or GSH-NS D concentrations (0.5, 1.0, 2.0, and 3.0 mg/mL). No significant increase in LDH leakage percentage over untreated control cells was observed under any of the test conditions (data not shown). The lack of apparent plasma membrane-damaged cells in the LDH assay would appear to contrast significantly with the decrease in cell growth observed by WST-1 cell proliferation assay (Table 2 and Figure 2), which was performed at the same incubation times with the same GSH-NS concentrations. This finding prompted us to investigate the cell cycle using flow cytometry to assess any arrest of cell cycle progression.

times with the same GSH-NS concentrations. This finding prompted us to investigate the cell cycle using flow cytometry to assess any arrest of cell cycle progression. We then performed cell cycle analyses on the IC50 values of each cell line after 24 h of GSH-NS B or GSH-NS D incubation (Table 2). A significant increase in cell percentages in the G0/G1 phase and a significant decrease in S/G2/M cell percentages were observed across the entire cell population after exposure at the respective GSH-NS B or GSH-NS D IC50 values (Figure 4). Moreover, the sub-G0/G1 We then performed cell cycle analyses on the IC<sup>50</sup> values of each cell line after 24 h of GSH-NS B or GSH-NS D incubation (Table 2). A significant increase in cell percentages in the G0/G<sup>1</sup> phase and a significant decrease in S/G2/M cell percentages were observed across the entire cell population after exposure at the respective GSH-NS B or GSH-NS D IC<sup>50</sup> values (Figure 4). Moreover, the sub-G0/G<sup>1</sup> peak was absent in all cell lines. This suggests that the observed decrease in cell proliferation (Table 2 and Figure 2) was the result of alterations of cell cycle progression owing to a block in the G0/G<sup>1</sup> phase. Furthermore, the G0/G<sup>1</sup> phase cell population percentage was higher in both colorectal cancer (Figure 4A,B) and prostatic cancer (Figure 4C,D) cells after incubation with the lower disulfide-bond content nanosponge (GSH-NS B).

4C,D,G,H).

content nanosponge (GSH-NS B).

peak was absent in all cell lines. This suggests that the observed decrease in cell proliferation (Table 2 and Figure 2) was the result of alterations of cell cycle progression owing to a block in the G0/G1 phase. Furthermore, the G0/G1 phase cell population percentage was higher in both colorectal cancer (Figure 4A,B) and prostatic cancer (Figure 4C,D) cells after incubation with the lower disulfide-bond

Thus, to confirm these data, an analysis of mRNA expression of the different cyclin-dependent kinases (CDK) was performed, as they participate in cell cycle regulation, especially during the G1 to S phase transition. It was observed that *CDK1*, *CDK2*, and *CDK4* mRNA expression was downregulated in almost all cell lines, as compared with untreated cells (Figure 4B,D,F,H). Interestingly, the mRNA expression of the CDK activator, *CDC25A*, was either unaffected or down-regulated, compared with untreated cells, whereas the mRNA expression of the CDK inhibitors, *CDKN1A* and *CDKN2A*, was up-regulated compared with untreated cells (Figure 4B,D,F,H). No significant differences in the extent of cell cycle arrest were observed between the two nanosponge formulations. However, the formulation with the higher disulfide-bond content, GSH-NS D, seemed to induce a higher down-regulation in CDK mRNA and higher up-regulation in *CDKN2A* mRNA in HT-29 and DU145 cells, whereas GSH-NS B induced a higher *CDKN1A* mRNA expression in HCT116 and PC-3 cells. Furthermore, no significant differences were observed in terms of the extent of cell cycle arrest

**Figure 4.** Glutathione responsive β-cyclodextrin-based nanosponge effect on cell cycle and mRNA expression. HCT116 (**A**, **B**), HT-29 (**C**, **D**), DU145 (**E**, **F**), and PC-3 (**G**, **H**) cells were exposed to the respective GSH-NS B or GSH-NS D IC50 for 24 h. Cell cycle distribution was analyzed by flow cytometry and the data were expressed as a percentage of cells in the different phases of the cell cycle (**A**, **C**, **E**, **G**). *RRN18S* (ribosomal RNA 18S) was used for the mRNA gene expression analysis as a reference gene to normalize the data and the nanosponge-induced alterations in mRNA levels were compared with those of the controls, that is, untreated cells, fixed at 1 and shown by the dotted line. Statistically significant difference versus control: \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001. **Figure 4.** Glutathione responsive β-cyclodextrin-based nanosponge effect on cell cycle and mRNA expression. HCT116 (**A**,**B**), HT-29 (**C**,**D**), DU145 (**E**,**F**), and PC-3 (**G**,**H**) cells were exposed to the respective GSH-NS B or GSH-NS D IC<sup>50</sup> for 24 h. Cell cycle distribution was analyzed by flow cytometry and the data were expressed as a percentage of cells in the different phases of the cell cycle (**A**,**C**,**E**,**G**). *RRN18S* (ribosomal RNA 18S) was used for the mRNA gene expression analysis as a reference gene to normalize the data and the nanosponge-induced alterations in mRNA levels were compared with those of the controls, that is, untreated cells, fixed at 1 and shown by the dotted line. Statistically significant difference versus control: \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.

Thus, to confirm these data, an analysis of mRNA expression of the different cyclin-dependent kinases (CDK) was performed, as they participate in cell cycle regulation, especially during the G<sup>1</sup> to S phase transition. It was observed that *CDK1*, *CDK2*, and *CDK4* mRNA expression was down-regulated in almost all cell lines, as compared with untreated cells (Figure 4B,D,F,H). Interestingly, the mRNA expression of the CDK activator, *CDC25A*, was either unaffected or down-regulated, compared with untreated cells, whereas the mRNA expression of the CDK inhibitors, *CDKN1A* and *CDKN2A*, was up-regulated compared with untreated cells (Figure 4B,D,F,H). No significant differences in the extent of cell cycle arrest were observed between the two nanosponge formulations. However, the formulation with the higher disulfide-bond content, GSH-NS D, seemed to induce a higher down-regulation in CDK mRNA and higher up-regulation in *CDKN2A* mRNA in HT-29 and DU145 cells, whereas GSH-NS B induced a higher *CDKN1A* mRNA expression in HCT116 and PC-3 cells. Furthermore, no significant differences were observed in terms of the extent of cell cycle arrest and gene expressions between cell lines with higher (Figure 4A–F) and lower GSH content (Figure 4C,D,G,H).
