EPS-DR:Scandium (EPS-DR:Sc) Complex

Figure 5 shows the NCI plot on MNNG/HOS cells after 160 h of contact with four metal-to-ligand ratios of EPS-DR:Sc complex. For the ratios EPS-DR:Sc 1:1 and 1:2, the cell proliferation profiles are quite similar. For the metal-to-ligand ratio of 1:1, after 100 h of contact, a peak of proliferation was reached at very low NCI for all the EPS-DR concentrations considered. Then, there was a plateau that tended to zero NCI for the highest concentration of EPS-DR. For the ratios EPS-DR:Sc 1:1 and 1:2, the standard deviation of all the curves appeared very high. Likewise, even for metal-to-ligand ratio of 1:0.5 and 1:4, similar curves were obtained, that could be quite well superimposed with the control. For these two metal-to-ligand ratios, the cell proliferation started oscillating after 120 h.

The cell proliferation profiles were compared to the cell viability results. For metalto-ligand ratio of 1:0.5 and 1:4, there was a high percentage of cell viability after 160 h of contact whereas for metal-to-ligand ratio of 1:1, there was a significant decrease when increasing the concentration of EPS-DR. This behavior was very similar to the one observed on Hep:Sc complexes.

**Figure 5.** Normalized cell index plot of MNNG/HOS cells after 160 h of contact with different stoichiometric ratios L:M: (**a**) EPS-DR:Sc 1:0.5 at 50 μg mL−<sup>1</sup> (\*), 100 μg mL−<sup>1</sup> (\*), and 200 μg mL−<sup>1</sup> (\*); (**b**) EPS-DR:Sc 1:1 at 50 μg mL−1, 100 μg mL−1, and 200 μg mL−1; (**c**) EPS-DR:Sc 1:2 at 50 μg mL−<sup>1</sup> (\*), 100 μg mL−1, and 200 μg mL−<sup>1</sup> (\*\*); (**d**) EPS-DR:Sc 1:4 at 50 μg mL−<sup>1</sup> (\*), 100 μg mL−<sup>1</sup> (\*), and 200 μg mL−<sup>1</sup> (\*). \* = *p* value ≤ 0.0001, \*\* = *p* value ≤ 0.005.

#### *2.2. Human A375 Melanoma Cells*

Effect of Scandium and Polysaccharides Alone

Figure 6 shows the effect on cell proliferation of scandium, as well as the three polysaccharides alone. All the compounds reduced the cell proliferation after 95 h of contact. The effect of scandium alone was comparable to that of heparin or EPS-DR alone. Nevertheless, the EPS-DRS alone, that had the higher sulfate content, displayed a more traditional profile of detaching cells.

**Figure 6.** Normalized cell index plot of human A375 melanoma cells after 95 h of contact with (**a**) scandium at concentration of 100 μg mL−<sup>1</sup> (\*) and (**b**) polysaccharides alone at concentration of 100 μg mL−1: heparin (\*), EPS-DRS (\*), EPS-DR (\*). \* = *p* value ≤ 0.0001.

#### *2.3. Effect of Polysaccharide:Scandium Complexes*

For both EPS considered in this work, i.e., EPS-DR and -DRS, scandium-polysaccharide complexes were more efficient in reducing the cell proliferation than the EPS themselves as shown in Figure 7. For Hep:Sc (Figure 7a) and EPS-DRS:Sc (Figure 7b) complexes, at increasing metal-to-ligand ratio, a more important decrease in cell proliferation was observed. The same trend could be observed for EPS-DR:Sc complexes (Figure 7c). The best conditions for reducing the cell proliferation were reached for a metal-to-ratio of 1:2, leading to a cell proliferation reduction of approximately 55–88%.

**Figure 7.** Normalized cell index plot of human A375 melanoma cells after 95 h of contact with different scandiumpolysaccharide complexes at concentration of 100 μg mL−<sup>1</sup> and various stoichiometric ratios: (**a**) Hep:Sc 1:0.5 (\*), 1:1 (\*), 1:2 (\*), heparin alone (\*); (**b**) EPS-DRS:Sc 1:0.5 (\*), 1:1 (\*), 1:2 (\*\*), EPS-DRS alone (\*); (**c**) EPS-DR:Sc 1:0.5 (\*), 1:1 (\*), 1:2 (\*), EPS-DR alone (\*). \* = *p* value ≤ 0.0001, \*\* = *p* value ≤ 0.005.

#### *2.4. Human A549 Lung Cancer Cells*

#### 2.4.1. Effect of Scandium and Polysaccharides Alone

From Figure 8a, scandium seemed having no impact on the proliferation of lung cancer cells after 95 h of contact. By contrast, among the polysaccharides considered in this work, only EPS-DRS seemed to slow down the kinetic of the cell proliferation. By comparison to EPS-DR, containing lower sulfate content, it could be assumed so far that the sulfate content of the macromolecule played a role on the cell proliferation, at least for the cell line A549.

#### 2.4.2. Effect of Polysaccharide:Scandium Complexes

As shown in Figure 9a–c, the cell proliferation curves obtained when the cells contacted EPS-DR:Sc and Hep:Sc complexes, exhibited quite similar kinetic behaviors.

Only the metal-to-ligand ratio of 1:2 had an impact on the cell proliferation. This effect was more pronounced with EPS-DRS:Sc (Figure 9b) but for this one, there was no influence of the metal-to-ligand ratio.

**Figure 8.** Normalized cell index plot of human A549 lung cancer cells after 95 h of contact with (**a**) scandium at concentration of 100 μg mL−<sup>1</sup> (\*) and (**b**) polysaccharides alone at concentration of 100 μg mL−1: heparin (\*), EPS-DRS (\*), EPS-DR (\*). \* = *p* value ≤ 0.0001.

**Figure 9.** Normalized cell index plot of human A549 lung cancer cells after 95 h of contact with different scandiumpolysaccharide complexes at concentration of 100 μg mL−<sup>1</sup> and various stoichiometric ratios: (**a**) Hep:Sc 1:0.5 (\*), 1:1 (\*), 1:2 (\*), heparin alone(\*); (**b**) EPS-DR:Sc 1:0.5 (\*), 1:1 (\*), 1:2 (\*), EPS-DRS alone (\*); (**c**) EPS-DRS:Sc 1:0.5 (\*), 1:1 (\*), 1:2 (\*), EPS-DR alone (\*). \* = *p* value ≤ 0.0001.

#### *2.5. Human U251 Glioblastoma Cells*

Scandium alone induced a slight decrease of U251 cell proliferation (Figure 10a). Among the three polysaccharides, only heparin and EPS-DRS (Figure 10b), the most sulfated polysaccharides, slowed down the proliferation of approximately 45% compared to the untreated control group.

When considering the scandium complexes (Figure 10c–e), in particular, Hep:Sc and EPS-DR:Sc complexes showed a less efficient reduction in the proliferation of glioblastoma cells compared to the polysaccharide themselves. As observed for the other cell lines tested, there was an exception for the metal-to-ligand ratio 1:2 that influenced the kinetics. Additionally, EPS-DRS:Sc complexes were rather more efficient than DRS alone but this

activity was not correlated to the metal-to-ligand ratio as has been evidenced by the other cell lines tested above.

**Figure 10.** Normalized cell index plot of human U251 glioblastoma cells after 95 h of contact with (**a**) scandium at concentration of 100 μg mL−<sup>1</sup> (\*) and (**b**) polysaccharides alone at concentration of 100 μg mL−1: heparin (\*), EPS-DRS (\*), EPS-DR (\*). Normalized cell index plot of U251 cells after 95 h of contact with different scandium-polysaccharide complexes at concentration of 100 μg mL−<sup>1</sup> and various stoichiometric ratios: (**c**) Hep:Sc 1:0.5 (\*), 1:1 (\*), 1:2 (\*), heparin alone(\*); (**d**) EPS-DRS:Sc 1:0.5 (\*), 1:1 (\*), 1:2 (\*), EPS-DRS alone (\*); (**e**) EPS-DR:Sc 1:0.5 (\*), 1:1 (\*), 1:2 (\*), EPS-DR alone (\*). \* = *p* value ≤ 0.0001.

#### *2.6. Human MDA231 Breast Cancer Cells*

Scandium alone slightly decreased the cell proliferation of breast cancer cell, as shown in Figure 11a. Among the polysaccharides alone, only EPS-DRS demonstrated highly reducing the kinetics of proliferation (Figure 11b).

Hep:Sc and EPS-DR:Sc complexes were more efficient than the respective polysaccharides alone only at the stoichiometric ratio L:M 1:1 (Figure 11c–e), but standard deviations were very high for all the curves. EPS-DRS:Sc complexes exhibited the same effect of the polysaccharide alone (Figure 11d).

**Figure 11.** Normalized cell index plot of human MDA231 breast cancer cells after 95 h of contact with (**a**) scandium at concentration of 100 μg mL−<sup>1</sup> (\*) and (**b**) polysaccharides alone at concentration of 100 μg mL−1: heparin (\*), EPS-DRS, EPS-DR (\*). Normalized cell index plot of MDA231 cells after 95 h of contact with different scandium-polysaccharide complexes at concentration of 100 μg mL−<sup>1</sup> and various stoichiometric ratios: (**c**) Hep:Sc 1:0.5 (\*), 1:1, 1:2 (\*), heparin alone (\*); (**d**) EPS-DRS:Sc 1:0.5, 1:1, 1:2, EPS-DRS alone; (**e**) EPS-DR:Sc 1:0.5 (\*), 1:1, 1:2 (\*), EPS-DR alone (\*). \* = *p* value ≤ 0.0001.

#### *2.7. Human Caco2 Colon Cancer Cells*

For this cell line, the contact time was prolonged to 140 h to display significant effects of compounds. Scandium alone did not have any effect on cell proliferation of colon cancer cells, as shown in Figure 12a. Once again, only EPS-DRS demonstrated significantly reducing the kinetics of proliferation (Figure 12b).

**Figure 12.** Normalized cell index plot of human Caco2 colon cancer cells after 140 h of contact with (**a**) scandium at concentration of 100 μg mL−<sup>1</sup> (\*) and (**b**) polysaccharides alone at concentration of 100 μg mL−1: heparin (\*), EPS-DRS (\*), EPS-DR (\*). Normalized cell index plot of Caco2 colon cancer cells after 140 h of contact with different scandiumpolysaccharide complexes at concentration of 100 μg mL−<sup>1</sup> and various stoichiometric ratios: (**c**) Hep:Sc 1:0.5 (\*), 1:1 (\*), 1:2 (\*), heparin alone (\*); (**d**) EPS-DRS:Sc 1:0.5 (\*), 1:1 (\*), 1:2 (\*), EPS-DRS alone (\*); (**e**) EPS-DR:Sc 1:0.5 (\*), 1:1 (\*), 1:2 (\*), EPS-DR alone (\*). \* = *p* value ≤ 0.0001.

Hep:Sc and EPS-DR:Sc complexes were more efficient than the respective polysaccharides alone only at the stoichiometric ratio of 1:2 (Figure 12c,e). EPS-DRS:Sc complexes exhibited the same effect of the polysaccharide alone (Figure 12d), except at the ratio 1:1. Table 1 summarizes the effect of Hep:Sc and EPS:Sc complexes for different metal-toligand ratio and different concentrations on human osteosarcoma, melanoma, lung cancer, glioblastoma and breast cancer cell lines.

**Table 1.** Summary of the effect of Hep:Sc and EPS:Sc complexes for different metal-to-ligand ratio and different concentrations on MNNG/HOS, A375, A549, U251, MDA231 and Caco3. + = low anti-proliferative effect, ++ = medium anti-proliferative effect, +++ = high anti-proliferative effect, − = low proliferative effect, − − = medium proliferative effect, −−− = high proliferative effect, NT = not tested.

