**2. Results**

### *2.1. Structural Characterization of the Sulfated Fucans*

Monosaccharide anion-exchange chromatography of Fuc1 revealed a sugar composition of 97.0% ± 0.1% fucose (retention time 3.134 min) and 3.0% ± 0.1% galactose (retention time 7.910 min). Neither uronic acid residues nor glucose were detected.

Mass spectrometry of Fuc1 revealed a sulfite content (-SO3) of 29.44%, which converts into a sulfation degree (DS) of approximately 1.7 sulfate groups per sugar residue (Table 1). Both (sugar composition and DS) are in full agreemen<sup>t</sup> with the previously elucidated molecular structure [12]. An overlay of the Raman spectra from samples Fuc1, Fuc2, and Fuc3 showed no change in the relative intensities of the sulfate group vibrations (822.5 cm<sup>−</sup>1, 839.8 cm<sup>−</sup>1, 1065.3 cm<sup>−</sup>1, 1262.4 cm<sup>−</sup>1) and

methyl group vibrations (1340.9 cm<sup>−</sup>1, 1452.5 cm<sup>−</sup>1), indicating an equal ratio between the two groups and thereby showing that no desulfation occurred during the mild acid hydrolysis.

SEC-MALS (Size-exclusion chromatography-Multi-Angle Light Scattering) analysis of Fuc1 revealed a very high molecular weight average of Mw = 1548 (± 4.1) kDa [14], resulting in an average degree of polymerization (DPn) of 3512, with an approximated average monosaccharide unit weight of 290 g/mol [12]. The molar mass was broadly distributed between 300 kDa and 7 MDa, displaying coherent results throughout different injected volumes (50 μL and 100 μL, c = 1 g/L, see Figure 1). The radius of gyration as the Z-average (Rz) was found to be 83.0 nm. The overall shape of the molecule was determined through an rms conformation plot (rms radius [nm] versus M [g/mol], see Figure 2), displaying a slope (b) of 0.66 and thus indicating an overall random coil shape of the molecule (sphere b = 0.3; random coil b = 0.5; rigid rod b = 1) [15]. Considering the previously reported high degree of branching of the molecule, while having obtained the same sugar composition and degree of sulfation as shown in Kopplin et al. (2018) [12], the obtained shape of a random coil for an LH sulfated fucan molecule 3 times bigger supports the previously suggested overall structure of a large, highly flexible main chain with short side chains. Fuc2 and Fuc3 showed almost identical structural features, only differing in their degree of polymerization.

**Figure 1.** SEC-MALS (Size-exclusion chromatography-Multi-Angle Light Scattering) chromatogram of the high-molecular-weight sulfated fucan (Fuc1) giving M [g/mol] versus V [mL]; 50 μL injection (pink), 100 μL injection (green). The molar mass is plotted as a dotted line, the refractive index is displayed as an overlay.

**Table 1.** Data overview of the sulfated fucan samples used in this study. Degree of sulfation (DS), weight average molar mass (Mw), number average molar mass (Mn), degree of polymerization (DP), Z-average radius of gyration (Rz), refractive index increment (dn/dc), and slope of the RMS conformation plot (b = rms versus M).


**Figure 2.** Rms conformation plot of the high-molecular-weight sulfated fucan (Fuc1) giving the rms radius [nm] versus M [g/mol], 100 μL injection. The slope (b) = 0.66 indicates a random coil.

### *2.2. E*ff*ects on Cell Viability*

The uveal melanoma cell line OMM-1 was treated with the three LH sulfated fucans for 24 h; after that, an MTS (3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium) assay was conducted. In all three cases a dose-dependent decrease in cell viability starting with 1 μg/mL could be seen (Figure 3), but only Fuc2 and Fuc3 showed significant effects: 50 and 100 μg/mL Fuc2 lowered cell viability to 83% ± 5% (*p* < 0.01) and 76% ± 6% (*p* < 0.001), respectively. Quantities of 10, 50, and 100 μg/mL Fuc3 reduced cell viability even further to 87% ± 6%, 76% ± 5%, and 70% ± 9%, respectively. Fuc1, the sulfated fucan with the highest molecular weight, showed no significant effects on cell viability but showed a tendency at 100 μg/mL, which is not significant because of the higher variability.

**Figure 3.** Cell viability was tested in uveal melanoma cell line OMM-1 after treatment with *Laminaria hyperborea* (LH) sulfated fucans Fuc1 (**a**), Fuc2 (**b**), and Fuc3 (**c**) for 24 h. Cell viability was determined via MTS (3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and is shown as the mean and standard deviation in relation to a 100% control. Significance was evaluated with ANOVA; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 compared to control (*n* = 4).

The human RPE cell line ARPE-19 was treated with the three LH sulfated fucans for 24 h and tested with a consecutive MTS assay. Only 50 μg/mL Fuc1 increased cell viability slightly (Figure 4).

**Figure 4.** Cell viability was tested in retinal pigment epithelium (RPE) cell line ARPE-19 after treatment with LH sulfated fucans Fuc1 (**a**), Fuc2 (**b**), and Fuc3 (**c**) for 24 h. Cell viability was determined via MTS assay and is shown as the mean and standard deviation in relation to a 100% control. Significance was evaluated with ANOVA compared to the control (*n* = 4); + *p* < 0.05.

Primary porcine RPE cells were treated with the three LH sulfated fucans for 24 h and tested with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The 10 μg/mL Fuc1 lowered viability slightly, but not to a biologically relevant degree (Figure 5). This paved the way for extended incubation times to measure VEGF secretion after 72 h (see below).

**Figure 5.** Cell viability was tested in RPE cells after treatment with LH sulfated fucans Fuc1 (**a**), Fuc2 (**b**), and Fuc3 (**c**) for 24 h. Cell viability was determined via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and is shown as the mean and standard deviation in relation to an untreated control (100%). Significance was evaluated with ANOVA compared to the control (*n* = 3); \* *p* < 0.05.
