Selenium Bio-Nanocomposite Based on Alteromonas macleodii Mo169 Exopolysaccharide: Synthesis, Characterization, and In Vitro Antioxidant Activity

In this study, the novel exopolysaccharide (EPS) produced by the marine bacterium Alteromonas macleodii Mo 169 was used as a stabilizer and capping agent in the preparation of selenium nanoparticles (SeNPs). The synthesized nanoparticles were well dispersed and spherical with an average particle size of 32 nm. The cytotoxicity of the EPS and the EPS/SeNPs bio-nanocomposite was investigated on human keratinocyte (HaCaT) and fibroblast (CCD-1079Sk) cell lines. No cytotoxicity was found for the EPS alone for concentrations up to 1 g L−1. A cytotoxic effect was only noticed for the bio-nanocomposite at the highest concentrations tested (0.5 and 1 g L−1). In vitro experiments demonstrated that non-cytotoxic concentrations of the EPS/SeNPs bio-nanocomposite had a significant cellular antioxidant effect on the HaCaT cell line by reducing ROS levels up to 33.8%. These findings demonstrated that the A. macleodii Mo 169 EPS can be efficiently used as a stabilizer and surface coating to produce a SeNP-based bio-nanocomposite with improved antioxidant activity.


Introduction
Selenium (Se) is an essential trace mineral for biological functions, including the synthesis of selenoenzymes capable of regulating important physiological functions of the human body [1]. Se is present in the selenocysteine residue (SEC) of various antioxidant enzymes (e.g., glutathione peroxidase (GPX), thioredoxin reductase (TXNRD), and selenoprotein P(SELENOP)), and acts as their redox center, being essential for their activity [2]. These antioxidant enzymes can effectively protect the membrane structures of organisms from oxidative damage [3]. Apart from its antioxidant activity, Se intake has several health benefits, including the regulation of the thyroid hormone metabolism, an anticancer effect (mainly chemopreventive), and the modulation of the immune system response in inflammatory disorders (e.g., diabetes, bone toxicity, colitis, and drug-induced toxicity) [1,2]. Nonetheless, Se's biological activity strongly depends on its chemical form and the absorbed dose [4]. With most European agricultural soils being classified as having low Se concentrations [5], sodium selenite (Na 2 SeO 3 ), sodium selenate (Na 2 SeO 4 ), or selenomethionine (SeMet) are commonly found as Se sources in food supplements [4]. However, and antioxidant ability of the synthesized bio-nanocomposite were investigated through in vitro assays.

Preparation of the Bio-Nanocomposites
The SeNPs were synthesized according to the procedure described by Yan et al. [17], with minor modifications. A 2 g L −1 EPS solution was prepared (5 mL), and the pH was adjusted to~8 with NaOH (0.1 M). Control samples were prepared using deionized water instead of the EPS solution. A quantity of 500 µL of a 100 mM Na 2 SeO 3 stock solution was added to the EPS or control solutions, to achieve a Se 4+ concentration of 10 mM. After stirring, 1 mL of an ascorbic acid solution (200 mM) was added to the tubes, followed by incubation at room temperature, for 1 h, protected from the light. SeNPs' formation was noticed by the appearance of a red color, which was confirmed by UV-visible spectra measurements (CamSpec M509T spectrophotometer) in the wavelength range of 200-800 nm. After NPs' synthesis, the EPS/NPs bio-nanocomposites were dialyzed using a 12 kDa MWCO (molecular weight cut-off) membrane (ZelluTrans/Roth) against deionized water, for 48 h. The purified bio-nanocomposite was maintained at 4 • C or lyophilized for further analysis. The reactions were performed in triplicates.

Characterization of the Bio-Nanocomposites
The selenium content in the bio-nanocomposite was determined by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) (Ultima, Horiba Jobin-Yvon, France, equipped with a 40.68 MHz RF generator, Czerny-Turner monochromator with 1.00 m (sequential) and autosampler AS500). The zeta potential measurements were performed in a Zetasiser Nano ZS, model ZEN (Malvern Panalytical), adopting electrophoretic cells (disposable folded hair cells; reference DTS1070) at 25 • C. Zeta potential was calculated by adopting the Smoluchowski equation [18]. The particle size of the EPS/SeNPs bionanocomposite (diluted 10 times) was determined by dynamic light scattering (DLS) using Photocor equipment (helium-neon laser, 633 nm, 20 mW, 90 • C) and a Brookhaven BI9000 autocorrelator. All measurements were performed at least three times and the raw data were analyzed with Dynals Software (SoftScientific, Tallinn, Estonia). Transmission electron microscopy (TEM) (JEM 1400; JEOL Europe, Zaventem, Belgium) was employed for the determination of the SeNPs' morphology and size distribution, as described by Concórdio-Reis et al. [18]. Fourier transform infrared (FT-IR) spectra of the samples were recorded on a Perkin-Elmer Spectrum II spectrometer (Waltham, MA, USA) over 500-4500 cm −1 after 10 scans. X-ray diffraction (XRD) analysis was performed using a benchtop MiniFlex II X-ray diffractometer from Rigaku (Tokyo, Japan) with a Cu X-ray tube (30 KV/15 mA). The 2θ scans were performed from 10 • to 60 • , with a step size of 0.01 • . Thermogravimetric (TG) analysis was conducted using a Thermogravimetric Analyzer Labsys EVO (Setaram, France), with a heating rate of 10 • C min −1 , from 25 to 500 • C. (DMEM) supplemented with 10% (v/v) of heat-inactivated fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (PS). CCD-1079Sk cells were cultured in DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) non-essential amino acids (NEAA). Cells were maintained at 37 • C with 5% CO 2 , as described by Concórdio-Reis et al. [18].

Cytotoxicity Assays
The HaCaT and CCD-1079Sk cells were seeded into 96-well plates at a density of 4.5 × 10 5 cells mL −1 and 1.5 × 10 5 cells mL −1 and allowed to grow for 72 and 24 h, respectively. Then, the cells were incubated with the EPS and the EPS/SeNPs bio-nanocomposite diluted in the respective culture medium with 0.5% FBS (125-1000 mg L −1 ). Control experiments were performed by incubating the cells with only a culture medium (0.5% FBS). After 24 h, the cells were washed once with PBS (Sigma-Aldrich, St. Louis, MO, USA) and the cell viability was assessed through the CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA), which contained MTS reagent. The optical density was measured at 490 nm using a BioTek EPOCH2 Microplate Reader (BioTek Instruments, Winooski, VT, USA) and cell viability was expressed in terms of the percentage of living cells relatively to the control.

Cellular Antioxidant Activity
Cellular antioxidant assays were performed following previously described methods [19,20], with some modifications. HaCaT cells were seeded at a density of 1.4 × 10 5 cells cm −2 in 96 well plates and the formation of intracellular reactive oxygen species (ROS) was monitored using 2 ,7 -dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe. Then, 72 h after seeding, cells were washed with PBS and treated with selected non-toxic concentrations (62.5 µg mL −1 ; 125 µg mL −1 ; 250 µg mL −1 ) of the samples and 25 µM DCFH-DA in PBS for 1 h. Subsequently, cells were washed again with PBS and incubated with the stress inducer (600 µM AAPH in PBS) for 1 h. After that, fluorescence was measured in a FL800 microplate fluorescence reader (Bio-Tek Instruments, Winooski, VT, USA) (Ex/Em 485 ± 20/528 ± 20 nm). The results were expressed as ROS percentage relative to the untreated control (cells treated with DCFH-DA and AAPH). Three independent experiments were performed, each sample was measured in triplicate.

Data Analysis
Three independent experiments were performed in at least duplicate and the results were expressed in terms of mean ± SD (standard deviation). When homogeneous variance and a normal distribution of the data were verified, the results were analyzed by one-way analysis of variance (ANOVA), followed by the Tukey or Bonferroni tests for multiple comparisons. If the data were not normally distributed or in the case of heterogeneous variances, the statistical analysis was performed by Student's t-test. Differences resulting in p < 0.05 were considered statistically significant. Adopting Lucia G Software, version 4.80 (Laboratory Imaging s.r.o-Nicon, Praha, Czech Republic), the equivalent diameter of at least 1000 particles was analyzed in view to determine particle size distribution, mean and corresponding percentiles: 10, 50, 90, and 99.

NPs Synthesis
For SeNPs' synthesis, the EPS was investigated for the stabilization of the SeNPs, and ascorbic acid was added to the mixture as a reducing agent. Ascorbic acid was previously used as a reducing agent in the synthesis of SeNPs stabilized by C. sinensis fungus Cs-HK1 EPS [8], carboxylated curdlans [17], β-D-glucan [21], chitosan [22], arabinogalactans [9], lectinan [23], gum Arabic [24], pectin [3], and by the polysaccharides of G. lemaneiformis [4], G. Livida [10], Oudemansiella raphanipies [6], Lycium barbarum [25], and Catathelasma ventricosum [26]. Within 1 h of incubation at room temperature with 10 mM of Se 4+ , the solution's color changed from colorless to light yellow and, finally, to red ( Figure 1, insert), indicating the formation of amorphous or monoclinic SeNPs [8,9,17,25]. This alteration in color may be ascribed to the excitation of surface plasmon resonance (SPR) of SeNP, resulting in the SPR band observed in the UV-vis spectra of SeNPs (235 nm) ( Figure 1). The SeNPs synthesized by other polysaccharides presented a SPR band at a slightly higher wavelength (260-273 nm) [3,4,17,21,25]. Nonetheless, differences in the absorption peak were reported when the synthesis conditions were altered [8,9] and might be related to differences in SeNPs' crystallinity and particle size [9]. For instance, in the study that reported the use of arabinogalactan as a stabilizer for SeNPs' synthesis, an increase in SeO 3 2− concentration from 30 to 120 mM resulted in particles with higher dimensions, as well as an increase in the intensity of the absorption band, and a shift in its position from 260 nm to higher wavelengths was noticed [9]. Thus, it is expected that lower selenium concentrations, such as the 10 mM used in this study, would result in SPR bands located at lower wavelengths. Additionally, the study performed by Xiao et al. [8] using C. sinensis Cs-HK1 EPS for the stabilization of the SeNPs showed that the ratio of selenium to EPS can also affect the characteristics of the SPR band, including its intensity and width. An increase in the Se: EPS ratio from 1:1 to 1:3 led to the formation of unstable large-sized SeNPs, characterized by a broader SPR band with a peak at a higher wavelength (306 nm) [8]. [4], G. Livida [10], Oudemansiella raphanipies [6], Lycium barbarum [25], and Catathelasma ventricosum [26]. Within 1 h of incubation at room temperature with 10 mM of Se 4+ , the solution's color changed from colorless to light yellow and, finally, to red (Figure 1, insert) indicating the formation of amorphous or monoclinic SeNPs [8,9,17,25]. This alteration in color may be ascribed to the excitation of surface plasmon resonance (SPR) of SeNP resulting in the SPR band observed in the UV-vis spectra of SeNPs (235 nm) ( Figure 1) The SeNPs synthesized by other polysaccharides presented a SPR band at a slightly higher wavelength (260-273 nm) [3,4,17,21,25]. Nonetheless, differences in the absorption peak were reported when the synthesis conditions were altered [8,9] and might be related to differences in SeNPs' crystallinity and particle size [9]. For instance, in the study that reported the use of arabinogalactan as a stabilizer for SeNPs' synthesis, an increase in SeO3 2− concentration from 30 to 120 mM resulted in particles with higher dimensions, as well as an increase in the intensity of the absorption band, and a shift in its position from 260 nm to higher wavelengths was noticed [9]. Thus, it is expected that lower selenium concentrations, such as the 10 mM used in this study, would result in SPR bands located at lower wavelengths. Additionally, the study performed by Xiao et al. [8] using C. sinensis Cs-HK1 EPS for the stabilization of the SeNPs showed that the ratio of selenium to EPS can also affect the characteristics of the SPR band, including its intensity and width. An increase in the Se: EPS ratio from 1:1 to 1:3 led to the formation of unstable large-sized SeNPs, characterized by a broader SPR band with a peak at a higher wavelength (306 nm) [8]. Although the control experiment without the addition of EPS also changed from colorless to red, the presence of precipitates was noticed (Figure 1, insert). Similarly previous studies reported the formation of brown/black aggregates that precipitated after Se 4+ incubation with ascorbic acid in the absence of the stabilizer agent [8,9,17,25]. Bare SeNPs are known to be highly unstable in aqueous solutions. Due to their high surface energy, these NPs tend to form strong interparticle bonds that result in their aggregation Although the control experiment without the addition of EPS also changed from colorless to red, the presence of precipitates was noticed (Figure 1, insert). Similarly, previous studies reported the formation of brown/black aggregates that precipitated after Se 4+ incubation with ascorbic acid in the absence of the stabilizer agent [8,9,17,25]. Bare SeNPs are known to be highly unstable in aqueous solutions. Due to their high surface energy, these NPs tend to form strong interparticle bonds that result in their aggregation and precipitation [8,17,25]. Thus, adding natural polysaccharides, such as A. macleodii Mo 169 EPS, is essential for the synthesis of stable and well-dispersed SeNPs. In the presence of the EPS, NPs' synthesis occurred in the polysaccharide's molecular microenvironment and the EPS acts as a soft template to stabilize the SeNPs by controlling the crystal nucleation and growth processes [1]. Moreover, the functional groups of the polysaccharide would facilitate the interaction of the SeNPs with the target cells and their lipidic barriers [1].

Composition
After purification by dialysis, the EPS/SeNPs bio-nanocomposite contained 183.8 mg L −1 of selenium. Nonetheless, the diffractogram of the bio-nanocomposite did not present the typical Bragg peaks of crystalline selenium ( Figure 2B). Only one large band at about 20 • was detected in both diffractograms (Figure 2), which can be attributed to the polysaccharide's amorphous nature. Similar results were observed in previous reports [3,4,6,10,17,21,23]. Although the typical peaks of crystalline Se appear in the XRD spectrum of the purified SeNPs, these disappeared in the presence of the polysaccharides C. sinensis Cs-HK1 EPS [8] and carboxylated curdlan [17], and the bio-nanocomposites showed the same pattern as the amorphous polysaccharide, suggesting that the SeNPs were bound by the polysaccharide matrix, leading to the occurrence of amorphous SeNPs [8,17].
nucleation and growth processes [1]. Moreover, the functional groups of the polysaccharide would facilitate the interaction of the SeNPs with the target cells and their lipidic barriers [1].

Composition
After purification by dialysis, the EPS/SeNPs bio-nanocomposite contained 183.8 mg L −1 of selenium. Nonetheless, the diffractogram of the bio-nanocomposite did not present the typical Bragg peaks of crystalline selenium ( Figure 2B). Only one large band at about 20° was detected in both diffractograms (Figure 2), which can be attributed to the polysaccharide's amorphous nature. Similar results were observed in previous reports [3,4,6,10,17,21,23]. Although the typical peaks of crystalline Se appear in the XRD spectrum of the purified SeNPs, these disappeared in the presence of the polysaccharides C. sinensis Cs-HK1 EPS [8] and carboxylated curdlan [17], and the bio-nanocomposites showed the same pattern as the amorphous polysaccharide, suggesting that the SeNPs were bound by the polysaccharide matrix, leading to the occurrence of amorphous SeNPs [8,17].

Colloidal Stability
The colloidal stability of the synthesized EPS/SeNPs bio-nanocomposite was evaluated through the measurement of its zeta potential. The EPS/SeNPs bionanocomposite presented a high negative zeta potential of −46.43 ± 1.36 mV. This high electrokinetic potential can easily explain this enhancement of the physicochemical stability of the colloidal suspension through ionic, or electrostatic, repulsion afforded by the anionic charges of EPS which has migrated on their surface [18,27]. This value was consistent with the values found in the literature for polysaccharide-stabilized SeNP, namely the polysaccharides from G. lemaneiformis (−47.1 mV) [4] and G. Livida (−46.77 mV) [10]. Smaller negative potential values were reported for SeNPs prepared with carboxylated curdlans (−17 to −28 mV) [17], O. raphanipies polysaccharide (−14.1 mV) [6], and L. barbarum polysaccharides (−37 mV) [25], which might be related to differences in composition (e.g., content in uronic acids) and molecular weight between polysaccharides [17,25].

SeNPs Morphology
TEM micrographs of the purified EPS/SeNPs bio-nanocomposite showed that the synthesized SeNPs were monodispersed and homogenous spherical particles ( Figure 3).

Colloidal Stability
The colloidal stability of the synthesized EPS/SeNPs bio-nanocomposite was evaluated through the measurement of its zeta potential. The EPS/SeNPs bio-nanocomposite presented a high negative zeta potential of −46.43 ± 1.36 mV. This high electrokinetic potential can easily explain this enhancement of the physicochemical stability of the colloidal suspension through ionic, or electrostatic, repulsion afforded by the anionic charges of EPS which has migrated on their surface [18,27]. This value was consistent with the values found in the literature for polysaccharide-stabilized SeNP, namely the polysaccharides from G. lemaneiformis (−47.1 mV) [4] and G. Livida (−46.77 mV) [10]. Smaller negative potential values were reported for SeNPs prepared with carboxylated curdlans (−17 to −28 mV) [17], O. raphanipies polysaccharide (−14.1 mV) [6], and L. barbarum polysaccharides (−37 mV) [25], which might be related to differences in composition (e.g., content in uronic acids) and molecular weight between polysaccharides [17,25].

SeNPs Morphology
TEM micrographs of the purified EPS/SeNPs bio-nanocomposite showed that the synthesized SeNPs were monodispersed and homogenous spherical particles ( Figure 3). Interestingly, a fainted sphere-like layer around the SeNPs was observed (Figure 3, insert). This coating layer can be assigned to the EPS, suggesting that the high molecular weight polysaccharide had an important role in the dispersion and stabilization of the SeNPs. No SeNPs' aggregates were noticed in the TEM images (Figure 3), which occurred in the previous study where carboxyl curdlan Cur-4 was used as a stabilizer and capping agent [17]. In that study, the authors concluded that the compact random coils that Cur-4 exhibited might have limited the interaction between the polymer's chains and the SeNPs, envisaging the importance of chain conformation on SeNPs' stability, size, and morphology [17]. [17]. In that study, the authors concluded that the compact exhibited might have limited the interaction between the polym envisaging the importance of chain conformation on SeN morphology [17].

SeNPs and Bio-Nanocomposite's Size
The particle size distribution of the SeNPs was homogeno nm for percentile 50 and 90, respectively ( Figure 4). With an a nm, the SeNPs' size is within the range of values reported in the with sizes ranging from 5 to 200 nm have shown improved pro and in vivo against the oxidative effects of free radicals [28 potential antioxidant activity of the EPS/SeNPs bio-nanocompo

SeNPs and Bio-Nanocomposite's Size
The particle size distribution of the SeNPs was homogenous, ranging from 22 to 76 nm for percentile 50 and 90, respectively ( Figure 4). With an average particle size of 32 nm, the SeNPs' size is within the range of values reported in the literature (Table 1). SeNPs with sizes ranging from 5 to 200 nm have shown improved protective properties in vitro and in vivo against the oxidative effects of free radicals [28,29], which indicated the potential antioxidant activity of the EPS/SeNPs bio-nanocomposite.
Interestingly, a fainted sphere-like layer around the SeNPs was observed (Figure 3, in This coating layer can be assigned to the EPS, suggesting that the high molecular w polysaccharide had an important role in the dispersion and stabilization of the SeNPs SeNPs' aggregates were noticed in the TEM images (Figure 3), which occurred in previous study where carboxyl curdlan Cur-4 was used as a stabilizer and capping a [17]. In that study, the authors concluded that the compact random coils that C exhibited might have limited the interaction between the polymer's chains and the Se envisaging the importance of chain conformation on SeNPs' stability, size, morphology [17].

SeNPs and Bio-Nanocomposite's Size
The particle size distribution of the SeNPs was homogenous, ranging from 22 nm for percentile 50 and 90, respectively ( Figure 4). With an average particle size nm, the SeNPs' size is within the range of values reported in the literature (Table 1). Se with sizes ranging from 5 to 200 nm have shown improved protective properties in and in vivo against the oxidative effects of free radicals [28,29], which indicated potential antioxidant activity of the EPS/SeNPs bio-nanocomposite.  The mean hydrodynamic diameter of the colloidal SeNPs coated by the EPS was determined by DLS (Table 1). The EPS/SeNPs bio-nanocomposite had a diameter of 297 ± 4 nm, a value significantly higher in comparison with that obtained in TEM (22-76 nm). Nonetheless, this difference in particle size was also previously reported for other EPS/NPs bionanocomposites [4,8,10,[30][31][32][33][34], and can be attributed to the differences in both techniques (e.g., detection method or sample conditioning) [30]. Additionally, the hydrodynamic diameter includes the hydration layer and polymer coating, thus resulting in larger values [10,17,34]. Nonetheless, in this study, the SeNPs stabilized by A. macleodii Mo 169 EPS presented a higher value (297 ± 4 nm) compared with those reported in the literature ( Table 1). As examples, those prepared with the galactose-rich polysaccharides of G. lemaneiformis (92.5-137.7 nm) [4] and G. Livida (115.4 nm) [10], Larix principis-rupprechtii arabinogalactans (94.24-173.2 nm) [9], lectinan (100 nm) [23], gum Arabic (145-170 nm) [24], and the L. barbarum polysaccharide composed of arabinose, xylose, glucose, and galactose (105.4 nm) [25].
Interestingly, all these polysaccharides presented a lower Mw (0.092-18.4 kDa) than that found for A. macleodii Mo 169 EPS (1.6 and 4.6 MDa) [15]. The closest value was found for SeNPs prepared with carboxylated curdlan Cur-4 with a Mw of 0.57 MDa (243.4 nm) [17], suggesting that the large Mw of A. macleodii Mo 169 EPS could cause the larger particle size of the EPS/SeNPs bio-nanocomposite.

EPS-NPs Interaction
FTIR analysis was carried out to investigate the surface functional groups of the A. macleodii Mo169 EPS involved in the stabilization of the SeNPs. An alteration in the shape and intensity of the band found between 3000 and 3500 cm −1 , which corresponds to the stretching frequencies of hydroxyl groups (O-H) [9,15,32], was found in the bionanocomposite in comparison with the EPS alone ( Figure 5). A shift to a lower wavenumber was noticed in the peak of the stretching vibration of C-H that appeared at 2925 cm −1 in the EPS ( Figure 5A) [9,17]. Significant alterations were found in the adsorption region characteristic of the C=O asymmetric (1596 cm −1 ) and symmetric (1300-1450 cm −1 ) stretching vibrations of the carboxylates from the uronic acids [15,35], which suggests that these groups played an important role in the stabilization of the SeNPs. Additionally, alterations were noticed in the region of 1722 cm −1 assigned to the C=O stretching of the acyl substituents [18]. These results revealed the importance of hydroxyl and carboxylate groups in the stabilization of the SeNPs. In previous studies, SeNPs' aggregation seemed to be avoided through the interactions between the NPs and the hydroxyl groups of different polysaccharides, including lectinan [23], arabinogalactan [9], pectin [3], gum Arabic [24], curdlan [17], and C. sinensis EPS [8], and L. barbarum [25], and Lignosus rhinocerotis [21] polysaccharides. In addition to the hydroxyl groups, the imino groups of G. Livida and G. lemaneiformis polysaccharides also seemed to be involved in SeNPs' stabilization [4,10].

Assessment of Cytotoxicity
The EPS and EPS/SeNPs bio-nanocomposite biocompatibility was investigated on human skin cell lines, namely CCD-1079Sk fibroblasts ( Figure 6A) and HaCaT keratinocytes ( Figure 6B). As described in ISO 10993-5, a cytotoxic effect was considered when cell viability decreased below 70%. As presented in Figure 6, the EPS alone did not show any cytotoxic effect on either cell line for concentrations up to 1000 mg L −1 , indicating its biocompatibility. Regarding the cytotoxicity of the EPS/SeNPs bio-nanocomposite concentrations of 500 mg L −1 (containing 47 mgSe L −1 ) and 1000 mg L −1 (containing 92 mgSe L −1 ) were cytotoxic, causing a reduction in CCD-1079Sk and HaCaT cell viability superior to 55% ( Figure 6A) and 89% ( Figure 6B), respectively. Nonetheless, in the presence of 125 mg L −1 and 250 mg L −1 of EPS/SeNPs bio-nanocomposite (11 mgSe L −1 and 23 mgSe L −1 respectively), no cytotoxic effect was considered since cell viability of both cell lines exceeded 87 ± 15% ( Figure 6). Similar results were obtained by other authors for SeNPs (83.6 nm) coated with G. lemaneiformis polysaccharides, where RAW 264.7 cells' viability was maintained above 77% for concentrations up to 20 mgSe L −1 [4]. Moreover, in another study, RWPE-1 cells maintained their viability in the presence of 400 mg L −1 of pectincoated SeNPs [3], a concentration superior to that found in this study (250 mg L −1 ). In addition to the differences in cell lines' sensitivity towards metals [18], the size of those SeNPs was superior (41 nm, compared with 32 nm for A. macleodii Mo 169 EPS), and the content in Se of the bio-nanocomposite might also be different, resulting in the differences observed.

Assessment of Cytotoxicity
The EPS and EPS/SeNPs bio-nanocomposite biocompatibility was investigated on human skin cell lines, namely CCD-1079Sk fibroblasts ( Figure 6A) and HaCaT keratinocytes ( Figure 6B). As described in ISO 10993-5, a cytotoxic effect was considered when cell viability decreased below 70%. As presented in Figure 6, the EPS alone did not show any cytotoxic effect on either cell line for concentrations up to 1000 mg L −1 , indicating its biocompatibility. Regarding the cytotoxicity of the EPS/SeNPs bio-nanocomposite, concentrations of 500 mg L −1 (containing 47 mg Se L −1 ) and 1000 mg L −1 (containing 92 mg Se L −1 ) were cytotoxic, causing a reduction in CCD-1079Sk and HaCaT cell viability superior to 55% ( Figure 6A) and 89% ( Figure 6B), respectively. Nonetheless, in the presence of 125 mg L −1 and 250 mg L −1 of EPS/SeNPs bio-nanocomposite (11 mg Se L −1 and 23 mg Se L −1 , respectively), no cytotoxic effect was considered since cell viability of both cell lines exceeded 87 ± 15% ( Figure 6). Similar results were obtained by other authors for SeNPs (83.6 nm) coated with G. lemaneiformis polysaccharides, where RAW 264.7 cells' viability was maintained above 77% for concentrations up to 20 mg Se L −1 [4]. Moreover, in another study, RWPE-1 cells maintained their viability in the presence of 400 mg L −1 of pectin-coated SeNPs [3], a concentration superior to that found in this study (250 mg L −1 ). In addition to the differences in cell lines' sensitivity towards metals [18], the size of those SeNPs was superior (41 nm, compared with 32 nm for A. macleodii Mo 169 EPS), and the content in Se of the bio-nanocomposite might also be different, resulting in the differences observed. Bioengineering 2023, 10, x FOR PEER REVIEW 10 of 13

Evaluation of Cellular Antioxidant Activity
Free radicals such as reactive oxygen species (ROS) can cause oxidative stress in essential cellular structures of living organisms, leading to altered functionality [19,36]. Many oxidative stress-related diseases, such as cardiovascular and immunity diseases, type-II diabetes, cancer, or aging, are related to the accumulation of excessive ROS [7]. Antioxidants, by reducing the level of ROS, can be effective in the protection of the cells against oxidative stress [7]. The capacity of the A. macleodii Mo 169 EPS and its SeNPs bionanocomposite to reduce AAPH-induced ROS production in HaCaT cells was evaluated ( Figure 7). The EPS alone did not reduce ROS generation at a cellular level. Nonetheless, EPS/SeNPs bio-nanocomposite concentrations of 125 mg L −1 (11 mgSe L −1 ) and 250 mg L −1 (23 mgSe L −1 ) significantly reduced ROS production (p ≤ 0.001) by 25.9% and 33.8% (Figure  7), suggesting a dose-dependent cellular antioxidant capacity. Comparable results were reported in the literature for other SeNPs-containing bio-nanocomposites. For instance, ROS production in H2O2-induced HepG2 cells was also significantly decreased in the presence of SeNPs coated with C. sinensis EPS [7]. In that study, the inhibition of ROS production seemed to occur in a dose-dependent matter and was more intense as the SeNPs' size decreased from 150 to 50 nm [7]. In addition, SeNPs capped with Bacillus paralicheniformis SR14 EPS were more efficient in reducing the H2O2-induced ROS production by IPEC-J2 cells than chemically synthesized SeNPs [37], suggesting that the polysaccharide coating might be beneficial for the antioxidant activity.

Evaluation of Cellular Antioxidant Activity
Free radicals such as reactive oxygen species (ROS) can cause oxidative stress in essential cellular structures of living organisms, leading to altered functionality [19,36]. Many oxidative stress-related diseases, such as cardiovascular and immunity diseases, type-II diabetes, cancer, or aging, are related to the accumulation of excessive ROS [7]. Antioxidants, by reducing the level of ROS, can be effective in the protection of the cells against oxidative stress [7]. The capacity of the A. macleodii Mo 169 EPS and its SeNPs bionanocomposite to reduce AAPH-induced ROS production in HaCaT cells was evaluated ( Figure 7). The EPS alone did not reduce ROS generation at a cellular level. Nonetheless, EPS/SeNPs bio-nanocomposite concentrations of 125 mg L −1 (11 mgSe L −1 ) and 250 mg L −1 (23 mgSe L −1 ) significantly reduced ROS production (p ≤ 0.001) by 25.9% and 33.8% (Figure  7), suggesting a dose-dependent cellular antioxidant capacity. Comparable results were reported in the literature for other SeNPs-containing bio-nanocomposites. For instance, ROS production in H2O2-induced HepG2 cells was also significantly decreased in the presence of SeNPs coated with C. sinensis EPS [7]. In that study, the inhibition of ROS production seemed to occur in a dose-dependent matter and was more intense as the SeNPs' size decreased from 150 to 50 nm [7]. In addition, SeNPs capped with Bacillus paralicheniformis SR14 EPS were more efficient in reducing the H2O2-induced ROS production by IPEC-J2 cells than chemically synthesized SeNPs [37], suggesting that the polysaccharide coating might be beneficial for the antioxidant activity.

Evaluation of Cellular Antioxidant Activity
Free radicals such as reactive oxygen species (ROS) can cause oxidative stress in essential cellular structures of living organisms, leading to altered functionality [19,36]. Many oxidative stress-related diseases, such as cardiovascular and immunity diseases, type-II diabetes, cancer, or aging, are related to the accumulation of excessive ROS [7]. Antioxidants, by reducing the level of ROS, can be effective in the protection of the cells against oxidative stress [7]. The capacity of the A. macleodii Mo 169 EPS and its SeNPs bionanocomposite to reduce AAPH-induced ROS production in HaCaT cells was evaluated ( Figure 7). The EPS alone did not reduce ROS generation at a cellular level. Nonetheless, EPS/SeNPs bio-nanocomposite concentrations of 125 mg L −1 (11 mgSe L −1 ) and 250 mg L −1 (23 mgSe L −1 ) significantly reduced ROS production (p ≤ 0.001) by 25.9% and 33.8% (Figure  7), suggesting a dose-dependent cellular antioxidant capacity. Comparable results were reported in the literature for other SeNPs-containing bio-nanocomposites. For instance, ROS production in H2O2-induced HepG2 cells was also significantly decreased in the presence of SeNPs coated with C. sinensis EPS [7]. In that study, the inhibition of ROS production seemed to occur in a dose-dependent matter and was more intense as the SeNPs' size decreased from 150 to 50 nm [7]. In addition, SeNPs capped with Bacillus paralicheniformis SR14 EPS were more efficient in reducing the H2O2-induced ROS production by IPEC-J2 cells than chemically synthesized SeNPs [37], suggesting that the polysaccharide coating might be beneficial for the antioxidant activity.

Evaluation of Cellular Antioxidant Activity
Free radicals such as reactive oxygen species (ROS) can cause oxidative stress in essential cellular structures of living organisms, leading to altered functionality [19,36]. Many oxidative stress-related diseases, such as cardiovascular and immunity diseases, type-II diabetes, cancer, or aging, are related to the accumulation of excessive ROS [7]. Antioxidants, by reducing the level of ROS, can be effective in the protection of the cells against oxidative stress [7]. The capacity of the A. macleodii Mo 169 EPS and its SeNPs bionanocomposite to reduce AAPH-induced ROS production in HaCaT cells was evaluated ( Figure 7). The EPS alone did not reduce ROS generation at a cellular level. Nonetheless, EPS/SeNPs bio-nanocomposite concentrations of 125 mg L −1 (11 mgSe L −1 ) and 250 mg L −1 (23 mgSe L −1 ) significantly reduced ROS production (p ≤ 0.001) by 25.9% and 33.8% (Figure  7), suggesting a dose-dependent cellular antioxidant capacity. Comparable results were reported in the literature for other SeNPs-containing bio-nanocomposites. For instance, ROS production in H2O2-induced HepG2 cells was also significantly decreased in the presence of SeNPs coated with C. sinensis EPS [7]. In that study, the inhibition of ROS production seemed to occur in a dose-dependent matter and was more intense as the SeNPs' size decreased from 150 to 50 nm [7]. In addition, SeNPs capped with Bacillus paralicheniformis SR14 EPS were more efficient in reducing the H2O2-induced ROS production by IPEC-J2 cells than chemically synthesized SeNPs [37], suggesting that the polysaccharide coating might be beneficial for the antioxidant activity.
) were performed by incubating the cells with only culture medium. Statistically significant differences comparing samples with the control were calculated according to the t-test (***, p ≤ 0.001, **** p ≤ 0.0001).

Evaluation of Cellular Antioxidant Activity
Free radicals such as reactive oxygen species (ROS) can cause oxidative stress in essential cellular structures of living organisms, leading to altered functionality [19,36]. Many oxidative stress-related diseases, such as cardiovascular and immunity diseases, type-II diabetes, cancer, or aging, are related to the accumulation of excessive ROS [7]. Antioxidants, by reducing the level of ROS, can be effective in the protection of the cells against oxidative stress [7]. The capacity of the A. macleodii Mo 169 EPS and its SeNPs bio-nanocomposite to reduce AAPH-induced ROS production in HaCaT cells was evaluated (Figure 7). The EPS alone did not reduce ROS generation at a cellular level. Nonetheless, EPS/SeNPs bionanocomposite concentrations of 125 mg L −1 (11 mg Se L −1 ) and 250 mg L −1 (23 mg Se L −1 ) significantly reduced ROS production (p ≤ 0.001) by 25.9% and 33.8% (Figure 7), suggesting a dose-dependent cellular antioxidant capacity. Comparable results were reported in the literature for other SeNPs-containing bio-nanocomposites. For instance, ROS production in H 2 O 2 -induced HepG2 cells was also significantly decreased in the presence of SeNPs coated with C. sinensis EPS [7]. In that study, the inhibition of ROS production seemed to occur in a dose-dependent matter and was more intense as the SeNPs' size decreased from 150 to 50 nm [7]. In addition, SeNPs capped with Bacillus paralicheniformis SR14 EPS were more efficient in reducing the H 2 O 2 -induced ROS production by IPEC-J2 cells than chemically synthesized SeNPs [37], suggesting that the polysaccharide coating might be beneficial for the antioxidant activity.

Conclusions
The present study showed that the exopolysaccharide produced by the marine bacterium A. macleodii Mo169 can be used as a stabilizer and dispersing agent in the formation of SeNPs via the redox system of selenite and ascorbic acid. The synthesized SeNPs had an average particle size of 32 nm and were coated with the polysaccharide, forming a bio-nanocomposite with 297 nm. Thanks to the EPS's high content in negatively charged functional groups (i.e., carboxylic) and substituents (i.e., pyruvate, sulfate, and lactate), the synthesized SeNPs were stable and well dispersed in the solution. Additionally, the biocompatibility and cellular antioxidant potential of the synthesized EPS/SeNPs bio-nanocomposite was investigated. The SeNPs coated with the EPS had a low cytotoxicity towards CCD-1079Sk fibroblasts and HaCaT keratinocytes, since with concentrations up to 250 mg L −1 (containing 23 mgSe L −1 ), high cell viability was maintained. Moreover, the bio-nanocomposite exhibited cellular antioxidant capacity in vitro for concentrations above 125 mg L −1 (11 mgSe L −1 ). These results suggest that the EPS was suitable for the stabilization of SeNPs and can have a future in the development of novel biomaterials or formulations with therapeutic applications. In this study, the potential of marine biopolymers in nanotechnology and biomedical applications is also evidenced.

Evaluation of Cellular Antioxidant Activity
Free radicals such as reactive oxygen species (ROS) can cause oxidative stress essential cellular structures of living organisms, leading to altered functionality [19,3 Many oxidative stress-related diseases, such as cardiovascular and immunity diseas type-II diabetes, cancer, or aging, are related to the accumulation of excessive ROS [ Antioxidants, by reducing the level of ROS, can be effective in the protection of the ce against oxidative stress [7]. The capacity of the A. macleodii Mo 169 EPS and its SeNPs b nanocomposite to reduce AAPH-induced ROS production in HaCaT cells was evaluat (Figure 7). The EPS alone did not reduce ROS generation at a cellular level. Nonethele EPS/SeNPs bio-nanocomposite concentrations of 125 mg L −1 (11 mgSe L −1 ) and 250 mg L (23 mgSe L −1 ) significantly reduced ROS production (p ≤ 0.001) by 25.9% and 33.8% (Figu 7), suggesting a dose-dependent cellular antioxidant capacity. Comparable results we reported in the literature for other SeNPs-containing bio-nanocomposites. For instan ROS production in H2O2-induced HepG2 cells was also significantly decreased in the pre ence of SeNPs coated with C. sinensis EPS [7]. In that study, the inhibition of ROS produ tion seemed to occur in a dose-dependent matter and was more intense as the SeNPs' si decreased from 150 to 50 nm [7]. In addition, SeNPs capped with Bacillus paralicheniform SR14 EPS were more efficient in reducing the H2O2-induced ROS production by IPEC cells than chemically synthesized SeNPs [37], suggesting that the polysaccharide coati might be beneficial for the antioxidant activity.

Evaluation of Cellular Antioxidant Activity
Free radicals such as reactive oxygen species (ROS) can cause oxidative str essential cellular structures of living organisms, leading to altered functionality [1 Many oxidative stress-related diseases, such as cardiovascular and immunity dis type-II diabetes, cancer, or aging, are related to the accumulation of excessive RO Antioxidants, by reducing the level of ROS, can be effective in the protection of th against oxidative stress [7]. The capacity of the A. macleodii Mo 169 EPS and its SeNP nanocomposite to reduce AAPH-induced ROS production in HaCaT cells was eva (Figure 7). The EPS alone did not reduce ROS generation at a cellular level. Noneth EPS/SeNPs bio-nanocomposite concentrations of 125 mg L −1 (11 mgSe L −1 ) and 250 m (23 mgSe L −1 ) significantly reduced ROS production (p ≤ 0.001) by 25.9% and 33.8% (F 7), suggesting a dose-dependent cellular antioxidant capacity. Comparable results reported in the literature for other SeNPs-containing bio-nanocomposites. For ins ROS production in H2O2-induced HepG2 cells was also significantly decreased in the ence of SeNPs coated with C. sinensis EPS [7]. In that study, the inhibition of ROS pr tion seemed to occur in a dose-dependent matter and was more intense as the SeNP decreased from 150 to 50 nm [7]. In addition, SeNPs capped with Bacillus paralicheni SR14 EPS were more efficient in reducing the H2O2-induced ROS production by IP cells than chemically synthesized SeNPs [37], suggesting that the polysaccharide c might be beneficial for the antioxidant activity. xygen species (ROS) can cause oxidative stress in organisms, leading to altered functionality [19,36]. es, such as cardiovascular and immunity diseases, e related to the accumulation of excessive ROS [7]. f ROS, can be effective in the protection of the cells city of the A. macleodii Mo 169 EPS and its SeNPs biouced ROS production in HaCaT cells was evaluated uce ROS generation at a cellular level. Nonetheless, ntrations of 125 mg L −1 (11 mgSe L −1 ) and 250 mg L −1 S production (p ≤ 0.001) by 25.9% and 33.8% (Figure ular antioxidant capacity. Comparable results were eNPs-containing bio-nanocomposites. For instance, epG2 cells was also significantly decreased in the nensis EPS [7]. In that study, the inhibition of ROS se-dependent matter and was more intense as the 50 nm [7]. In addition, SeNPs capped with Bacillus efficient in reducing the H2O2-induced ROS producsynthesized SeNPs [37], suggesting that the polysacr the antioxidant activity. ) were performed by incubating the cells only with AAPH. Statistically significant differences comparing samples with the negative control were calculated according to the t-test (***, p ≤ 0.001, **** p ≤ 0.0001).

Conclusions
The present study showed that the exopolysaccharide produced by the marine bacterium A. macleodii Mo169 can be used as a stabilizer and dispersing agent in the formation of SeNPs via the redox system of selenite and ascorbic acid. The synthesized SeNPs had an average particle size of 32 nm and were coated with the polysaccharide, forming a bio-nanocomposite with 297 nm. Thanks to the EPS's high content in negatively charged functional groups (i.e., carboxylic) and substituents (i.e., pyruvate, sulfate, and lactate), the synthesized SeNPs were stable and well dispersed in the solution. Additionally, the biocompatibility and cellular antioxidant potential of the synthesized EPS/SeNPs bionanocomposite was investigated. The SeNPs coated with the EPS had a low cytotoxicity towards CCD-1079Sk fibroblasts and HaCaT keratinocytes, since with concentrations up to 250 mg L −1 (containing 23 mg Se L −1 ), high cell viability was maintained. Moreover, the bio-nanocomposite exhibited cellular antioxidant capacity in vitro for concentrations above 125 mg L −1 (11 mg Se L −1 ). These results suggest that the EPS was suitable for the stabilization of SeNPs and can have a future in the development of novel biomaterials or formulations with therapeutic applications. In this study, the potential of marine biopolymers in nanotechnology and biomedical applications is also evidenced.