Fig. 7

(a) Cell growth of various treatment groups determined using MTT assay. The treatment group of those treated with the combination of FL-cHy-NPs and HK-cHy-NPs did not show a significant reduction in cell growth compared to the control (p > 0.05, ns). Also, the combined formulation protects the cells from both 50 and 100 µM concentrations of CisPt. Therefore, the treatment of cells with the combination of FL-cHy-NPs and HK-cHy-NPs has significant protection efficacy against CisPt-induced cell death compared to FL-cHy-NPs or HK-cHy-NPs alone. (b) Intracellular ROS generation assay showing significant ROS generation in the CisPt only treated cells. Significantly low ROS generation was shown in the cells that were treated with FL-cHy-NPs, HK-cHy-NPs, and STS. (c) The graph showing the fluorescence intensity of MitoSOX reagent corresponded to mitochondrial superoxide generation. The assay suggested significantly low superoxide generation in FL-cHy-NPs and FL/HK-cHy-NPs treatment groups. (d) The graph showing caspase 3/7 activation in different treatment groups. The caspase 3/7 activation was significantly lowered in the cells that were treated with FL-cHy-NPs, HK-cHy-NPs, and STS. The data were compared using two-way ANOVA and each group was compared with the control group “C” by applying Dunnett’s multiple comparisons post-hoc tests. The family-wise alpha threshold confidence level was adjusted to 0.05 (95% confidence interval) during the analysis. (For 'a-d' data presented as Mean ± SD; Asterisk; ****, p < 0.0001; **, p < 0.005; *, p < 0.05; ns=not significant). (e) The western blot analysis of the samples after respective treatment with FL-cHy-NPs and HK-cHy-NPs alone or in combination. The blots are showing proteins associated with apoptotic caspase-3 pathway and control β-actin. (f) The fluorescence microscopy images (scale bar 100 μm) are showing the effect of developed FL-cHy-NPs and HK-cHy-NPs individually, and in combination on the CisPt-induced generation of ROS in HEI-OC1 cells. The first column is showing the cell nuclei stained with HOECHST-33342. The second column is showing the fluorescence of DCF (a ROS marker) in the cells. The third column is showing the cells under transmittance light. The fourth column is showing the overlay of columns 1, 2, and 3. The last column is showing a graph of the ratio of normalized intensities of overall cells and the cells that were producing ROS (getting stained with DCFH-DA). (i) Blank untreated cells: the cells that were not treated with any of the NP preparation or CisPt did not show significant ROS generation. The ratio of normalized intensities of total cells and DCF-stained cells was 1.71 ± 0.08. (ii) CisPt treated cells: The cells that were treated with CisPt only showed significant ROS generation. The ratio of normalized intensities of total cells and DCF-stained cells was 0.99 ± 0.21. (iii) FL-cHy-NPs and CisPt treated cells: the cells treated with FL-cHy-NPs and CisPt did not show significant ROS generation. The ratio of normalized intensities of total cells and DCF-stained cells was 1.71 ± 0.04. (iv) HK-cHy-NPs and CisPt treated cells: the cells treated with HK-cHy-NPs and CisPt did not show significant ROS generation. The ratio of normalized intensities of total cells and DCF-stained cells was 1.63 ± 0.35. (v) FL-cHy-NPs, HK-cHy-NPs and CisPt treated cells: the cells treated with FL-cHy-NPs, HK-cHy-NPs and CisPt did not show significant ROS generation. The ratio of normalized intensities of total cells and DCF-stained cells was 1.66 ± 0.29. (vi) STS and CisPt treated cells: the cells treated with STS and CisPt did not show significant ROS generation. The ratio of normalized intensities of total cells and DCF-stained cells was 1.53 ± 0.23. (g) The fluorescence microscopy images (scale bar 50 μm) showed the effect of developed FL-cHy-NPs and HK-cHy-NPs individually and in combination on the CisPt-induced HEI-OC1 cells cytotoxicity. The first column is showing the cell nuclei stained with HOECHST-33342. The second column is showing the fluorescence of PI (a dead cell marker) in the cells. The third column is showing the cells under transmittance light. The fourth column is showing the overlay of columns 1, 2, and 3. The last column is showing a graph of the ratio of normalized intensities of overall cells and the cells that were dead (getting PI stain). (i) Blank untreated cells: the cells that were not treated with any of the NP preparations or CisPt did not show significant cell death. The ratio of normalized intensities of total cells and PI-stained cells was 2.62 ± 0.05. (ii) CisPt treated cells: The cells that were treated with CisPt only showed significant cell death. The ratio of normalized intensities of total cells and PI-stained cells was 1.06 ± 0.08. (iii) FL-cHy-NPs and CisPt treated cells: the cells treated with FL-cHy-NPs and CisPt did not show significant cell death. The ratio of normalized intensities of total cells and PI-stained cells was 1.87 ± 0.22. (iv) HK-cHy-NPs and CisPt treated cells: the cells treated with HK-cHy-NPs and CisPt did not show significant cell death. The ratio of normalized intensities of total cells and PI-stained cells was 1.97 ± 0.15. (v) FL-cHy-NPs, HK-cHy-NPs and CisPt treated cells: the cells treated with FL-cHy-NPs, HK-cHy-NPs and CisPt did not show significant cell death. The ratio of normalized intensities of total cells and PI-stained cells was 2.41 ± 0.0.32. (vi) STS and CisPt treated cells: the cells treated with STS and CisPt did not show significant cell death. The ratio of normalized intensities of total cells and PI-stained cells was 2.03 ± 0.02. [The microscopy data were compared using one-way ANOVA and each group was compared with the control group (c; CisPt only treated) by applying Dunnett’s multiple comparisons posthoc test. The family-wise alpha threshold confidence level was adjusted to 0.05 (95% confidence interval) during the analysis and p < 0.05 was considered as a significantly different group].