*3.3. Quantitative Phase Imaging*

QPI evaluated cell morphology over time, providing, at the same time, measures of several parameters such as shape, dry mass, and sphericity, as well as cellular proliferation and displacement in response to treatment [28,29].

The images (Figure 10A,B) were obtained at up to 72 h of treatment with nanospermidine, and the combination with nanofenretinide confirmed the MTT results, indicating cytotoxic effects at 200 µM with a higher activity of the combination than single nanospermidine.

**Figure 10.** QPI. Representative image at 0, 24, 48, and 72 h of BR6 cells (**A**) and NLF cells (**B**) treated with Nanospermidine (NS, 200 µM spermidine) as a single agent and in combination with Nanofenretinide (NF, 10 µM fenretinide). As a comparison, the images of the untreated cells and the cells treated with NF are shown.

Cell doubling time, displacement, dry mass, and sphericity were analyzed over 72 h at the sub-cytotoxic concentrations 50 µM and 100 µM nanospermidine to exclude the interfering effect of cell death with detachment from the substrate.

The doubling time, which is a measure of cell growth, was increased by the combination in both cell lines, with a higher effect at 100 µM than 50 µM (Figure 11A), indicating a slowing down in cell proliferation.

**Figure 11.** QPI data. (**A**) Histogram plot illustrating median cell doubling time of BR6 and NLF cells treated with nanospermidine (NS) and nanospermidine in combination with nanofenretinide (NS + NF) at the sub-cytotoxic concentrations of NS: 50 µM and 100 µM. (**B**) Displacement of BR6 and NLF cells treated with nanospermidine (NS) and nanospermidine in combination with nanofenretinide (NS + NF) at the sub-cytotoxic concentrations of NS: 50 µM and 100 µM.

Cell motility is obviously an important feature of tumor cells as it contributes to cancer invasion and metastasis [51,52]. Evaluation of cell displacement is now gaining increasing attention with improvements in time-lapse microscopy techniques [53].

In our study, we observed that cell displacement was decreased by nanospermidine, and the presence of fenretinide further limited cell displacement (Figure 11B).

In QPI, dry mass is measured from the phase delay considering that the refractive increment of biomolecules can be closely approximated by a constant [25].

A decrease in dry mass over time is indicative of a cytostatic or cytotoxic effect [54]. Sphericity quantifies the degree of cell roundness and is calculated as the ratio of the surface area of a sphere that has the same volume as the cell to the surface area of the cell. For adherent cells, such as those used in our study, sphericity provides a measure of the level of attachment of the cells to the substrate, since when the level of attachment increases the cells lose sphericity [55].

Treatment with the sub-cytotoxic concentrations of nanospermidine did not provide appreciable differences in dry mass (Figure 12A) and sphericity (Figure 12B) with respect to the controls. These results indicate that, despite the decrease in cell proliferation, the sub-cytotoxic concentrations of nanospermidine did not induce morphological variations.

**Figure 12.** QPI data. (**A**) Dry mass at 72 h of BR6 and NLF cells treated with Nanospermidine (NS) and Nanospermidine in combination with Nanofenretinide (NS + NF) at the sub-cytotoxic concentrations of NS: 50 µM and 100 µM. (**B**) Sphericity at 72 h of BR6 and NLF cells treated with nanospermidine (NS) and nanospermidine in combination with nanofenretinide (NS + NF) at the sub-cytotoxic concentrations of NS: 50 µM and 100 µM. Analysis of cell doubling time, displacement, and dry mass was mediated on six wells for each treatment.

Combination treatments, on the contrary, slightly decreased dry mass and increased sphericity in both cell lines, indicating a cytotoxic effect induced by fenretinide.
