**3. Discussion**

The different biological effects of sulfated fucans make them very interesting for medical research, but each sulfated fucan should be exactly characterized because of the high heterogeneity concerning these biological effects. Effects that are beneficial for the treatment of AMD were already described [2]. In this study we compared three purified sulfated fucans extracted from LH by Alginor ASA. The chemical structure was previously characterized and differs among the three samples only by the molecular weight average, which makes comparison in relation to the molecular weight possible. The aim was to prescreen these extracts as to whether they are suitable for further AMD-related research. To our knowledge, LH sulfated fucans had not been tested for this purpose before. Oxidative burden and VEGF secretion are two important factors in the risk of developing AMD and were therefore the main focus of this study. Because RPE cells are the target of the pathological mechanisms, they were chosen as the cellular models. The melanoma cell line OMM-1 acted as a model for oxidative stress protection as it is more susceptible to oxidative stress than the RPE cells.

First, the LH sulfated fucans were tested for their e ffects on the viability of primary RPE cells and the RPE cell line ARPE-19. In both cases there were no significant e ffects, which is in line with earlier studies like that by Bittkau et al. 2019, which reported that sulfated fucans in general are not antiproliferative for adherent cell lines [18]. However, this is dependent on the specific fucoidan and model system. For example Banafa et al. described antiproliferative e ffects of *Fucus vesiculosus* sulfated fucan in human breast cancer cells [19]. It was demonstrated that it arrests the cell cycle at G1, has pro-apoptotic properties, and enhances ROS formation [19]. Fuc2 and Fuc3 showed cell viability lowering e ffects in the melanoma cell line OMM-1, which could be desirable for possible use in anticancer treatments. They both have a size below 500 kDa, so the e ffects could be connected to the molecular weight and structure. Possibly, these sulfated fucans interfere with fibronectin, disturbing the adherence of the tumor cell line [20]. Of note, the attachment of OMM-1 to well plates is rather weak and the cells have no tight junctions, in contrast to the RPE and ARPE-19 cell lines. In addition, OMM-1 cells were treated at subconfluence and the sulfated fucans could slow down the proliferation of this cell line, whereas the RPE cells were already confluent.

Because oxidative burden is considered one of the main causes of the development of AMD [7], the capability of the sulfated fucans to lower oxidative stress induced by TBHP or H2O2 was investigated. In ARPE-19, no protective e ffects could be detected, and 100 μg/mL Fuc3 had a cumulative toxic effect (also for OMM-1), which means that the smallest LH sulfated fucan enhanced the toxic e ffect of TBHP and H2O2. The same outcome was achieved for the OMM-1 cell line, except for the biggest sulfated fucan Fuc1, which was protective. These results are in contrast to those for the sulfated fucans of *Sargassum angustifolium*, in which the antioxidant activity through the scavenging of radicals and reducing power increased with decreasing size of the sulfated fucan (size range from 421 to 64 kDa) [21]. Because Fuc2 and Fuc3, which were also in that range, had no e ffect at all, and due to the absence of polyphenols by virtue of the high purity, it could be assumed that Fuc1 had no scavenging e ffect but rather interacted with the antioxidant defense system of the OMM-1 cell line. The RPE cells have an already improved antioxidant defense system, controlled via Nrf-2 (transcription factor nuclear factor erythroid 2-like 2), because of the high accumulation of oxygen radicals in the photoreceptors [7]. We already showed before that several sulfated fucans have protective e ffects in the OMM-1 cell line, but rarely do they in the ARPE-19 cell line [11,16]. OMM-1 cells have decreased superoxide dismutase activity [22], which could mean that the protective e ffects of ARPE-19 are concealed by already existing antioxidant enzyme activity. That sulfated fucans can activate superoxide dismutase and Nrf-2 was shown previously [23–26].

A further important factor, mainly for the development of wet AMD, is the secretion of VEGF, which supports the angiogenesis of blood vessels in the eye. ARPE-19 cell lines and the primary RPE cells were tested for their VEGF output after sulfated fucan treatment for three days. The secreted VEGF was normalized to the cell viability. As shown before, primary RPE cells produce significantly more VEGF compared to the cell line [11] (nearly 4 times more than ARPE-19); therefore, any significant reduction of the growth factor in primary RPE cells is even more remarkable. All three LH sulfated fucans decreased VEGF significantly at 50 and 100 μg/mL, with e fficiency decreasing with smaller size in the RPE and ARPE-19 cells. This reducing e ffect could be due to the direct binding of VEGF, which inhibits the activation of the VEGF receptor. The negatively charged side chains of sulfated fucans can interact with the positively charged residues of the VEGF molecule, which could explain the interference in the binding between VEGF and its receptor [27]. This corresponds to findings in the literature [28,29]. It could be assumed that bigger size of the sulfated fucan is more e fficient in VEGF inhibition. Also, direct interaction with the VEGF receptor (VEGF-R) [28] or the suppression of the VEGF and VEGF receptor genes could be possible, as VEGF has been shown to be involved in its own regulation [3,30–33]. Additional VEGF binding assays should be performed to determine the a ffinity of the fucoidan to the growth factor, which should be considered for further studies with LH sulfated fucans. Even more striking is the e ffect in the RPE cells, in which Fuc1 and Fuc2 also decreased VEGF despite the higher secretion. For both cell types, 50 μg/mL Fuc1 had the strongest e ffect. Fuc3 showed a

tendency to stimulate VEGF secretion in RPE cells; however, this was not significant and its biological relevance is therefore uncertain. Nevertheless, it is of interest, as this small sulfated fucan may interact with VEGF di fferently to its high-molecular-weight counterpart, possibly by binding to VEGF in a VEGF-R-activating manner [34] The DS is also an important factor for the angiogenesis influencing effect [29], but the DS for all three sulfated fucans was the same.

The monosaccharide analysis by anion-exchange chromatography revealed a polysaccharide almost exclusively composed of fucose (97.0% fucose, 3.0% galactose). In addition, the sulfite content (-SO3) of 29.44% (DS = 1.7) determined by ICP-MS is in strong agreemen<sup>t</sup> with data from the previously elucidated LH sulfated fucan structure [12]. The obtained rms conformation plot slope (b) of 0.66 through SEC-MALS revealed a random coil shape for both Fuc1 and Fuc2, further supporting the previously suggested long backbone structure (1 →3 glycosidic linkages) with short side chains at 1 →2 and 1 →4 branching points. Therefore, the main di fference between Fuc1, Fuc2, and Fuc3 was the degree of polymerization, directly associating the e ffects on proliferation, oxidative stress protection, and VEGF secretion with one structural property.

It has been shown that fucoidan, due to its high degree of sulfation, is able to bind to FGF (fibroblast growth factor) receptors, similar to heparin, heparan sulfate, and sulfated alginate [35–37]. Further has it been shown that the binding and inhibition of chemo- and cytokines, such as transforming growth factor-β1 (TGF-β1), by sulfated fucans is size dependent. Even though the underlying mechanism is not ye<sup>t</sup> resolved, a similar binding preference could play a role [38].

Koyanagi et al. demonstrated that highly sulfated fucans are able to bind VEGF [29].

In addition, it has been found that polymers with conjugated inhibitors have a higher inhibitory effect on tumor necrosis factorα (TNFα) if the inhibitors are attached to higher-molecular-weight polymers; this is due to an increased di ffusion time [39]. Since the binding of VEGF to highly sulfated fucans has been proven, a similar e ffect of increased di ffusion time could play a role.

FCSP are a diverse group of macromolecules with heterogeneous molecular structures. Still, due to its almost exclusive fucose sugar composition and the full sulfation of almost every sugar unit, LH sulfated fucan is rather homogeneous in nature, even when hydrolyzed to di fferent molecular weight averages. Hence, it can be speculated that biological e ffects on cells and the metabolism are less likely caused by chemical reaction or direct receptor interactions only expressed by Fuc1 and not Fuc2 and Fuc3, but rather by physical and steric interactions such as stronger cell surface adhesion or stronger entanglement with proteins (cadherins or cytokines) due to the higher average molecular weight.

For further clarification, other FCSP with similar M w but lower DS [14] should be included in future studies, as well as high-molecular-weight hyaluronic acids, alginates, and sulfated alginates [40,41] to better elucidate and distinguish the e ffects caused by molecular size, charge density, and degree of sulfation and to determine if branching points and sugar composition play an additional role. Potential FCSP–cytokine binding needs to be investigated to exclude potential masking of the determined VEGF secretion. Finally, a chromatographic fractionation of high-molecular-weight LH sulfated fucan should be applied to narrow down the molecular weight range and the polydispersity and to ge<sup>t</sup> access to LH sulfated fucan fractions of even higher molecular weight, potentially amplifying the previously expressed e ffects and directly relating them to specific molecular weight ranges.

From the results taken together, the sulfated fucan Fuc1 from LH with a molecular weight average of 1548 kDa seems to be best candidate for further research concerning AMD-relevant mechanisms. It did not lower the cell viability of primary RPE cells, and 50 μg/mL inhibited VEGF most e fficiently. High molecular weight appears to be desirable for VEGF inhibition. We tested several sulfated fucans from di fferent algal species previously; sulfated fucans from SL were also e ffective in oxidative stress protection in the OMM-1 and ARPE-19 cell lines, and 10 μg/mL SL sulfated fucan inhibited VEGF in ARPE-19 and RPE cells [11]. The molecular weight for this sulfated fucan was 1407 kDa (unpublished data), so it is comparable to Fuc1, which also lowered VEGF significantly at 10 μg/mL in RPE and ARPE-19. This renders SL and LH sulfated fucans as promising candidates for further AMD-related

studies. Of note, the pure LH sulfated fucan in this study can be sustainably reproduced in high amounts for further experiments in perfusion cultures or in vivo studies. Furthermore, because of its protective effect in the sensitive OMM-1 cell line, it could be considered for future studies with neuronal model systems concerning oxidative stress protection. For further research, tests concerning inflammation, lipid metabolism, and complement systems would be of additional interest.
