# Exploring marine-based compounds as cross-linkers to improve the biocompatibility and sustainability of chitosan-based hydrogels

Lea Spitzer, Amandine Adrien, Unnimaya Thalakkale Veettil, Sheila Olza, Ana Alonso-Varona, Susana C M Fernandes

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Lea Spitzer, Amandine Adrien, Unnimaya Thalakkale Veettil, Sheila Olza, Ana Alonso-Varona, et al. Exploring marine-based compounds as cross-linkers to improve the biocompatibility and sustainability of chitosan-based hydrogels. Carbohydrate Polymer Technologies and Applications, 2024, 8, 10.1016/j.carpta.2024.100541. hal-05396369

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# Exploring marine-based compounds as cross-linkers to improve the biocompatibility and sustainability of chitosan-based hydrogels

Lea Spitzer<sup>a,b</sup>, Amandine Adrien<sup>a,b</sup>, Unnimaya Thalakkale Veettil<sup>a,b,1</sup>, Sheila Olza<sup>a,b,c</sup>, Ana Alonso-Varona<sup>c</sup>, Susana C.M. Fernandes<sup>a,b,*</sup>

## ARTICLE INFO

**Keywords:**

Natural polymers

Bioactive molecules

Algae

3D porous scaffolds

Dye-adsorber

Tissue engineering

## ABSTRACT

Chitosan is one of the most promising natural polymers, its fundamental scientific research is growing unerruptedly and has been applied in a wide and varied range of domains, including biomedical and water treatment applications. Therefore, the search and implementation of non-synthetic and non-toxic cross-linkers for chitosan-based hydrogels is crucial for the development of more sustainable and biocompatible materials. Herein, an alternative approach has been developed to explore and exploit methanolic and aqueous extracts from five red seaweed species as covalent cross-linkers for chitosan-based hydrogels. The formation of a gel could be denoted for all extracts, whereas the protein-rich methanolic extractions afforded instantaneous gel-forming ability and greater stiffness and stability. The obtained hydrogels present large porous system with high degrees of swelling up to ca. 3000 %, and were successfully applied as dye adsorbent to remove industrial dye methyl orange withing a circular process with adsorption capacities of 728.46 ± 66.17 mg/g. Furthermore, cytotoxicity and cell-adhesion studies revealed the biocompatibility of the hydrogels and their potential applicability for tissue-engineering. This work demonstrated that methanolic and aqueous extracts from different red seaweed species could replace toxic cross-linkers. Furthermore, the unexpected ability of some extracts could pave the way for the development of new formulations for additive manufacturing, in particular for 3D printing approaches.

## 1. Introduction

Hydrogels are three-dimensional (3D) polymer networks that are distinguished by high swelling capacity, high water retention and diverse physical properties. They can be classified into physical or chemical gels, depending on building up their network either via H-bonding, ionic- and hydrophobic forces, or covalent bonds, respectively (Seliktar, 2012).

Many marine derived polymers such as chitosan, collagen and alginates are widely used for the synthesis of hydrogel and 3D porous scaffolds (Claverie, McReynolds, Petitpas, Thomas, & Fernandes, 2020). Among them, chitosan is the most popular due to its biocompatibility, biodegradability (Samalens et al., 2022), antimicrobial properties and renewability (Bagheri-Khoulenjani, Taghizadeh, & Mirzadeh, 2009; Kean & Thanou, 2010). Chitosan is derived from alkaline hydrolysis of

chitin, the main structural material in the exoskeleton of arthropods (e. g. shrimps, crabs) and endoskeleton of cephalopods (e.g. cuttlefish, squid). Chitosan is a polysaccharide composed of two repeating units (N-acetyl-D-glucosamine) and (D-glucosamine) for which the acetylation degree (DA) determines its final properties (Domalik-Pyzik, Chlopek, & Pielichowska, 2019). Hydrogels based on chitosan presents a high hydrophilic surface area covered with amino (-NH₂) and hydroxyl groups (-OH), that allow the incorporation/adsorption of small (charged) molecules. Hence, they have been widely engineered as sustainable multi-functional materials either to mimic the extracellular matrix allowing their use in biomedical applications such as drug-delivery, tissue-engineering (Bhattarai, Gunn, & Zhang, 2010) or to be cast into different forms, sizes and shapes for wastewater purification for the removal of heavy metal ions and organic dyes (Crini et al., 2019).

<footnote><sup>a</sup> Université de Pau et des Pays de l'Adour, E2S UPPA, CNRS, IPREM, 64600 Anglet, France</footnote>

<footnote><sup>b</sup> MANTA-Marine Materials Research Group, E2S UPPA, Université de Pau et des Pays de l'Adour, 64600 Anglet, France</footnote>

<footnote><sup>c</sup> Department of Cell Biology and Histology, Faculty of Medicine and Nursey, University of the Basque Country (UPV/EHU), B Sarriena s/n, 48940 Leioa, Spain</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: susana.fernandes@univ-pau.fr (S.C.M. Fernandes).</footnote>

<footnote n="1">¹ Present address: Department of Materials and Environmental Chemistry, Stockholm University, SE-10691 Stockholm, Sweden.</footnote>
Chitosan-based hydrogels can be prepared by cross-linking the free amino- and hydroxyl groups along its backbone chain with other polymers or small molecules. There are numerous methods, e.g. free-radical polymerization, condensation or addition reactions, that can be considered to vary and control the cross-linking degree and porosity of the network (Pella et al., 2018). A very popular strategy is the cross-linking of chitosan using small molecules with two or more functional groups such as formaldehyde (Medellín-Castillo et al., 2021), epichlorohydrin (de Oliveira, Fernandes, & Vieira, 2006) or glutaraldehyde (Li et al., 2013). However, these kind of reagents have been reported to exhibit significant (cyto)toxicity and are not suitable for medical applications (Gao et al., 2017). As an alternative, genipin, a naturally occurring aglycone extracted from gardenia fruit, gained increasing interest as a low-toxic ($LD_{50}$ i.v. 382 mg/kg in mice) cross-linking agent (Yu, Xu, Li, & Pan, 2021), showing improved cell-adhesion(L. Gao et al., 2014) and good *in vivo* biocompatibility (Reddy, Reddy, & Jiang, 2015). Genipin reacts selectively with the primary amine groups of chitosan and leads to dark-bluish colored cross-linked hydrogels (SHYU, 2000; Zubillaga et al. 2018; Zubillaga et al. 2020). However, remaining- and recently reported toxicity (Xia et al., 2021) of genipin, as well as its high costs, (Aldrich, 2024) hampers its full use.

To avoid the incorporation of any chemical entities during the cross-linking of chitosan with other green and biocompatible molecules and/or polymers, water-soluble linking-agent N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDAC) can be considered preferable to obtain non-cytotoxic and biocompatible products (Adekogbe & Ghanem, 2005; Kuijpers et al., 2000; Lai & Li, 2010). EDAC is a common agent to covalently link amine and carboxylic acids via reactive esters (Fischer, 2010). During the past decade, many studies reported the EDAC/NHS mediated coupling of collagen (Kaczmarek, Sionkowska, Kozlowska, & Osyczka, 2018)/gelatin, (Choi et al., 1999; Huang, Onyeri, Siewe, Moshfeghian, & Madihally, 2005; Jiankang et al., 2007; Mao et al., 2003) other proteins, e.g. fibroin (Niu et al., 2022) or bioactive molecules (Fernandes et al., 2015) to chitosan. This method is very popular since EDAC is promoted as 'zero-length' cross-linker that can be easily washed out after the reaction, leading generally to biocompatible chitosan hydrogels (Huang et al., 2005).

Interestingly, red seaweeds are natural sources of carboxylic acids- and amino-rich compounds namely carbohydrates (agar-agar and carrageenan), proteins (rich in aspartic acid (Trigueros, Sanz, Filipigh, Beltrán, & Riano, 2021) and glutamic acid (Ester Trigueros et al., 2021) and smaller extents of phycobiliproteins and mycosporine-like amino acids (MAA) (Castejón, Parailloux, Izdebska, Lobinski, & Fernandes, 2021), that can be consequently considered to be cross-linked with chitosan using EDAC (Castejón, Adrien, Spitzer, & Fernandes, 2023). The hydrogels based on chitosan and red seaweeds that were reported in the literature (Feki et al., 2020; Netanel Liberman, Ochbaum, Bitton, & Arad, 2021) used the isolated carbohydrate fraction of the seaweeds as cross-linker. The exploitation of 'crude' extracts of red seaweed (considering all containing biomolecules) as cross-linker has, to the best of our knowledge, not been reported so far. Nevertheless, the latter can be of economic interest since the isolation and purification of specific molecules from red seaweed are time-consuming and expensive processes (Castejón et al., 2021).

In this context, we here report a straightforward strategy for the full exploitation of crude and non-purified methanolic- and aqueous extracts from red seaweeds as non-toxic covalent cross-linkers for chitosan-based hydrogels. The present approach was applied to five different red seaweed species: *Ahnfeltiopsis devoniensis* (AD), *Porphyra rosengurtii* (PR), *Asparagopsis armata* (AA), *Porphyra purpurea* (PP) and *Gelidium corneum* (GC). For one selected seaweed extract, the cross-linking reaction mechanism is studied using 1D & 2D NMR-, HR-MAS NMR- and IR spectroscopy to assess the covalent bonding and identify the cross-linking groups from the extract. The 3D porous morphology of the freeze-dried hydrogels is fully characterized by SEM and different

swelling experiments in aqueous solutions are done. A comprehensive study on how the amount of seaweed extract used in the process can induce changes in the porous system and impact the stability of the hydrogels is also done. Finally, the hydrogels are tested for their ability to remove toxic dyes from waste water (here: methyl orange) and tested for their biocompatibility, i.e., cytotoxicity and cell adhesion properties with the perspective of biomedical applications such as tissue engineering.

## 1.1. Hypotheses

Crude red seaweed extracts can be utilized to cross-link chitosan without prior purification and isolation of the relevant biomolecules and the resulting hydrogels are biocompatible due to their natural origin.

# 2. Experimental

## 2.1. Raw materials and chemicals

Chitosan powder with a Degree of Deacetylation (DD) of 92 % and average molecular weight (MW) of 350 kDa from squid pen was supplied by Mathani India Pvt Ltd. The red seaweed crude extracts from *Ahnfeltiopsis devoniensis* (AD), *Porphyra rosengurtii* (PR) and *Asparagopsis armata* (AA) were purchased from the Laboratory of Photobiology of the Central Research Services of the University of Malaga, Spain. These three samples were collected in the Andalusian coast between 2019 and 2020, they were received as lyophilized extracts and stored at -20 °C until use. The methanolic extraction and characterization of the three extracts were done as described previously by *De la Coba, Aguilera, Figueroa, De Gálvez, & Herrera* (2009) The red algae *Gelidium corneum* (GC) was collected in November 2021 by local fishermen at the port of Ciboure St-Jean de Luz, France. Fresh seaweed was briefly rinsed with running tap water to remove visible sand particles and epiphytes. The algae were then set to air-dry on racks in a well-ventilated room at 21 °C and kept in a cool, dark place until further use. The red algae *Porphyra purpurea* (PP) was collected and dried by Capena, Oleron island, France in Spring of 2022. The raw extracts from these last two red algae were prepared *in-house* as described below.

EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, >80 %), dimethyl sulfoxide (DMSO), anionic dye Methyl orange (≤ 100 %), NaOH, HCl and acetic acid were purchased from Sigma-Aldrich (France) and used without further purification. Dialysis was performed using a 14 kDa MWCO membrane (Sigma-Aldrich, France). Phosphate buffered saline (PBS, pH 7.2) was purchased from Sigma-Aldrich (Germany), sodium pyruvate, non-essential amino acids and fetal bovine serum (FBS) from Gibco (U.K.), penicillin-streptomycin from Lonza (Belgium), ultralow attachment cell culture plates from Corning (U.S.), sorensen buffer and glutaraldehyde from Panreac AppliChem (Spain), and hexamethyldisilazane from Electron Microscopy Science (U.S.). Murine L929 fibroblast cell line was obtained from American Type Culture Collection (ATCC, U.S.).

**Extraction of crude extracts from red algae: Gelidium corneum and Porphyra purpurea.** Before the extraction, both dried algae were ground using a ball-mill for 9 min (RETSCH PM100, Haan, Germany), alternating the direction of rotation in 3 min cycles. Powdered seaweeds were conserved in airtight containers in the dark at 4 °C until use. For the aqueous extractions, both powdered seaweeds were suspended in distilled water with a 1:40 ($w_{dry}/v$) ratio and stirred at 4 °C during 15 h. After centrifugation at 10 000 rpm, 15 min, 4 °C, to remove the algal pellet, the extracts (GC-aq and PP-aq for Gelidium corneum-aqueous and Porphyra purpurea-aqueous, respectively) were lyophilized and stored at 4 °C until further use. The methanolic extraction was carried out following the protocol of Schmid, Schürch, & Zülli (2006) with modifications. Briefly, the powdered alga was suspended in 20 % methanol with a 1:50 ($w_{dry}/v$) ratio and the extraction was carried out during 2 h at 45 °C in a water bath. After centrifugation at 10 000 rpm, 15 min, 21
<table><caption>**Table 1**

General aspect gel formation time of the hydrogels obtained using the aqueous- or methanolic-extracts of the five red seaweed species.</caption><thead><tr><th>Algae Species</th><th>Ahnfeltiopsis devoniensis<br>(AD)</th><th>Porphyra rosengurttii<br>(PR)</th><th>Asparagopsis armata<br>(AA)</th><th>Porphyra purpurea<br>(PP)</th><th>Gelidium corneum<br>(GC)</th></tr></thead><tbody><tr><td><strong>Hydrogel<br>(methanolic-extract)</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Gel formation<br>Hydrogel<br>(aqueous-extract)</strong></td><td>instantaneous<br>no extract</td><td>instantaneous<br>no extract</td><td>instantaneous<br>no extract</td><td>instantaneous</td><td>instantaneous</td></tr><tr><td><strong>Gel formation</strong></td><td>-</td><td>-</td><td>-</td><td>12 h</td><td>12 h</td></tr></tbody></table>

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°C to remove the algal pellet, methanol was eliminated using a rotary evaporator (BUCHI R-100; Flawil, Switzerland). The extracts (GC-M and PP-M for Gelidium corneum-methanolic and Porphyra purpurea-methanolic, respectively) were then lyophilized and kept at 4 °C until further use. The extracts characterization and composition, including the biochemical composition are described in detail in the Support Information (SI).

**Synthesis of the chitosan-based hydrogels.** For this study, hydrogels were prepared using the aqueous and methanolic extractions (described above) from five different seaweed species and a mass ratio of chitosan:red seaweed extract: EDAC 1:1:1. The quantity of EDAC was selected based on hydrogels with a 1:1 (w/w) ratio of chitosan:seaweed extract; while varying EDAC from 0.25 to 2 equivalents in mass. 0.25 eq and 0.5 eq were not sufficient to lead to the formation of hydrogels, they were formed from 1 eq and above.

First, chitosan was dissolved in 1 M HCl to give a 1 % (w/v) solution and the pH was adjusted between 4 and 5 using 1 M NaOH. Then, the red seaweed extract was dissolved in distilled water and EDAC was added to activate the carboxylic acid groups in the extract. The mixture was vortexed and kept at room temperature (r.t.) for 5–10 min. to assure complete reaction. Finally, the activated red seaweed extract was poured into the chitosan solution at r.t. under vigorous stirring, wherein the hydrogel was formed instantaneous or up to 12 h, depending on the extract used (see Table 1). The formed hydrogels were then purified via dialysis against distilled water for three days, frozen at -20 °C for 12 h and lyophilized at -80 °C for 24 h. The hydrogels (aerogel) were kept at r.t. for further analysis.

**Control chitosan-based hydrogel.** Physical cross-linked control hydrogel was prepared following the protocol of Nie et al. (2015). In brief, a chitosan solution (1 wt% in acetic acid) was added in a mold to a coagulation bath of 10 wt% NaOH solution until the gelation was completed (1 h). Then, the formed hydrogel was washed with distilled water until neutrality and freeze-dried at -80 °C.

## 2.2. Characterization

**Nuclear magnetic resonance (NMR).** All NMR data were acquired using a Bruker Avance NEO 400 NMR spectrometer (Billerica, U.S.) operated at 9.4 T (¹H resonance frequency 400.13 MHz), equipped with 4 mm HR-MAS probe spinning at 7 kHz. The relatively recent and advanced high-resolution magic angle spinning (HRMAS) NMR technique enables the direct application of NMR spectroscopy to semi-solid and gel-like samples. It combines the advantages of both solid- and liquid-state NMR. The experiments were recorded at 298 K by using the Bruker library zg pulse sequence and the following parameters: number of scans 64, dummy scans 4, relaxation delay 5 s, spectral width 20 ppm, transmitter offset 6.0 ppm. After exponential multiplication (line broadening of 0.3 Hz), the spectra were Fourier transformed, phased,

baseline corrected, and calibrated at 4.7 ppm on H₂O residual signal. The obtained spectral data were analyzed using TopSpin (v.4.1.4), whereas automatic phase correction and subsequent integration were applied.

Attenuated total reflectance infrared spectroscopy (ATR-IR). Fourier Transform InfraRed (FTIR) spectra were obtained on a Thermo Fisher Nicolet™ iS50 FTIR Spectrometer (Waltham, MA, U.S.) equipped with a Pike GladiATR plate (diamond crystal) for attenuated total reflectance analysis using a resolution of 4 cm⁻¹ and 128 scans. The acquisitions were fulfilled with the OMNIC Spectra software.

**Scanning Electron Microscope (SEM).** SEM was utilized to visualize the morphology of the internal cross-section of the lyophilized hydrogels using a HIROX SH-3000 (France). The samples were coated with gold (Au, 20 mA, 60 s) to enhance the conductivity for the SEM observation and the images were acquired at an operating voltage of 25 kV.

**Porosity measurements.** The porosity of the hydrogels was measured by liquid displacement of ethanol. In brief, the freeze-dried hydrogel (dry weight, W_d) was immersed in a known volume (V_1) of ethanol in a cylinder for 10 min. The resulting volume of ethanol-impregnated hydrogel in ethanol was noted as V_2, then the hydrogel was removed of the cylinder and the residual ethanol volume recorded as V_3. The total volume displaced from the hydrogel was calculated using Eq. (1):

$$V = (V_2 - V_1) + (V_1 - V_3) = V_2 - V_3 \quad (1)$$

While V_2 - V_1 presents the volume of the hydrogel and V_1-V_3 the volume of the ethanol within the hydrogel pores. The porosity of the hydrogels was then calculated using Eq. (2):

$$P(\%) = (V_1 - V_3) + (V_2 - V_3) \times 100 \quad (2)$$

**Swelling Kinetic.** The swelling behavior of the hydrogel samples was determined using a gravimetric method. A known amount of the freeze-dried samples (W_d) was immersed into distilled water at 25°C and removed at different time intervals. The swollen samples were weighted after wiping out the excess water using a filter paper (W_s) and the degree of swelling was calculated using Equation (3) (Netanel Liberman et al., 2021):

$$S(\%) = \frac{(W_s - W_d)}{W_d} \times 100 \quad (3)$$

The water uptake of the hydrogels was evaluated after immersing the samples in distilled water at 25 °C for 24 h to assure swelling-equilibria. After removal of the excess water, the wet weight of the samples (W_w) was measured via the following Eq. (4) (Zubillaga et al., 2018):

$$W(\%) = \frac{(W_w - W_d)}{W_w} \times 100 \quad (4)$$
## 2.3. Hydrogel in vitro biocompatibility

**Cytotoxicity.** Cytotoxicity test was performed according to the ISO 10993-5:2009 standard protocol by the determination of the cell viability with PrestoBlue® (Invitrogen, U.S.), a resazurin-based colorimetric assay that quantifies the mitochondrial activity of viable cells. Briefly, hydrogels were sterilized by incubation in 70 % ethanol for 2 h and washed three times with phosphate buffered saline (PBS). Then, extraction media was prepared following the ISO 10993-12:2020 standard protocol, in which each hydrogel of 54 mg was incubated in 1 mL of complete cell culture media for 24 h at 37 °C. Murine L929 fibroblasts were seeded in 96-well plates at a density of 4.10³ cells/well in complete culture medium containing Minimum Essential Medium (MEM) supplemented with 1 mM sodium pyruvate, 1 % v/v non-essential amino acids, 1 % v/v penicillin-streptomycin and 10 % v/v fetal bovine serum and allowed to adhere for 24 h at 37 °C in a humidified atmosphere containing 5 % CO₂. After incubation, the culture media was replaced with the hydrogel's extraction media, fresh complete cell culture media (negative control) or 10 % v/v dimethyl sulfoxide in complete cell culture media (positive control). Cell viability was determined at different points (0, 24, 48 and 72 h) by adding 10 % v/v PrestoBlue® reagent and incubated for 2 h at 37 °C in the dark. Finally, the absorbance at 570 nm and 600 nm was measured (Synergy HT spectrophotometer, Biotek, U. S.) and Viability (%) was calculated from Eq. (5).

$$ \text{Viability (\%)} = \frac{A_{\text{test}}}{A_{\text{control}}} \times 100 \qquad (5) $$

Where $A_{test}$ is the absorbance of the sample cells cultured with the extraction media of the material and $A_{control}$ is the absorbance of the negative control.

**Cell adhesion.** Cell adhesion and morphology of adherent cells were evaluated by SEM analysis. Previously sterilized hydrogels (0.4 × 0.4 × 0.1 cm) were placed in 24 well ultralow attachment cell culture plates and led pre-wet in complete cell culture media for 24 h at 37 °C. Then, medium was removed and drops of 15 µL of complete cell culture media containing 6.10⁴ L929 cells were seeded on the surface of each hydrogel, and allowed to adhere for 2 h at 37 °C. To maintain a hydrated environment, PBS was added into adjacent wells. After that, 500 µL of complete cell culture media was added to each well. SEM micrographs were obtained in a Hitachi S-4800 field emission scanning electron microscope (FE-SEM, Hitachi, Japan) after 1 and 3 days of L929 cells seeding on hydrogels surface. The medium was removed and all samples were rinsed three times in Sorensen buffer, fixed with 2 % v/v glutaraldehyde for 30 min at r.t. washed three times with Sorensen buffer, dehydrated using a series of increasing concentrations of ethanol solutions, and finally desiccated to the critical point with hexamethyldisilazane and then allowed to air dry. The samples were then sputtered with a gold layer under an argon atmosphere and observed with an accelerating voltage of 5 keV.

## 2.4. Hydrogel dye adsorption and recycling capacity

The adsorption of methyl orange (MO) on the red seaweed cross-linked chitosan hydrogels was studied following the intensity of the absorption band of MO at 464 nm using a Shimadzu UV-2450 UV-Visible spectrophotometer (Kyoto, Japan). A piece of 5 mg (freeze-dried) adsorbent (hydrogel) was put in 25 mL of MO solution at different concentrations (400–25 mg.L⁻¹) and stirred at 350 rpm at room temperature, while sampling out 500 µL solution for spectroscopic analysis at different time intervals. Note that for optimized adsorption results, the freeze-dried hydrogel was first swollen in acetic acid and rinsed with distilled water to remove the excess acid, before it was added to the MO solution. For the recycling step, the adsorbed dye was removed by adding the hydrogel into an aqueous solution of 0.1 M NaOH for 2 h and rinsed with distilled water until all traces of color were completely removed.

Then, the hydrogel was re-protonated in an aqueous solution of 0.1 M acetic acid for 2 h, rinsed with distilled water to remove the excess acid and finally reutilized for a following adsorption cycle. The adsorption capacity at equilibrium ($q_e$) was calculated using Eq. (6) (Yang et al., 2019):

$$ q_e = \frac{(C_0 - C_t) \times 100}{m} \qquad (6) $$

where $C_0$ and $C_t$ are the concentration of the MO solutions before and after adsorption at time t (mg.L⁻¹), respectively, and m the mass of the dry hydrogel (g). The percentage removal of the dye was calculated using Eq. (7) as follows:

$$ \text{Dye removal (\%)} = \frac{(C_0 - C_e) \times V}{m} \qquad (7) $$

where $C_0$ is the initial concentrations of the MO solution, $C_e$ the concentration at adsorption equilibrium, V the volume of the MO solution (L) and m the mass of the dry hydrogel (g).

**Statistical analysis.** The statistical analysis was performed using GraphPad Prism software v5.03 (San Diego, U.S.). For the cytotoxicity test, two-way analysis of the variance (ANOVA) followed by Bonferroni post-test was applied. The results were expressed as mean ± SD and values of p < 0.05 were considered to be statistically significant. All experiments were conducted in triplicate.

# 3. Results and discussion

## 3.1. Chitosan-based hydrogels synthesis

In this study, five methanolic and two aqueous extracts, in a total of seven extracts, from five red seaweed species *Ahnfeltiopsis devoniensis* (AD), *Porphyra rosengurttii* (PR), *Asparagopsis armata* (AA), *Porphyra purpurea* (PP) and *Gelidium corneum* (GC) were used as cross-linkers for the synthesis of chitosan-based hydrogels. As mentioned before, red seaweeds contain a complex mixture of biomolecules, bearing various COOH groups, that could each cross-link to chitosan (Castejón et al., 2021; Trigueros et al., 2021; Trigueros et al., 2021). Interestingly, the analytical characterization of seven extracts (see SI, section A: Characterization of the red seaweed extracts) showed the presence of potential cross-linker agents such as proteins, sugars, polyphenols and mycosporine-like amino-acids (Table SA1). The UV-vis, FT-IR and 1D-NMR spectra of all seven extracts are given in Fig. SA1, SA2 and SA3, respectively, and the HSQC and HMBC NMR of AD extract are displayed in Fig. SA4 and SA5, respectively.

In a first set of experiments, all extracts were tested for their capacity to be used in the cross-link reaction of chitosan under the same conditions (1:1:1 (w/w/w) ratio of chitosan:seaweed extract:EDAC). The general aspect and gel formation time of the hydrogels are depicted in Table 1.

The formation of a gel could be denoted for all seven extracts, however, resulting with a difference of stiffness, color and time of gelation needed. Interestingly, for the methanolic extracts of all five algae-species, the hydrogel formation was instantaneous, whereas for the aqueous extracts, the reaction was completed within 12 h. The instantaneous gel-formation could be exploited in further investigations for the development of 3D printing approaches. Indeed, chitosan-based hydrogels can be considered as ink material for 3D printing if their formation is rapid (Li, Wu, Chu, & Gelinsky, 2020). As chitosan-based hydrogels can mimic the extracellular matrix and modulate cell fate, the as-prepared hydrogels using the seaweed extracts present a great potential for tissue engineering and regenerative medicine due to their porous system that could facilitate cell ingrowth. For the aqueous extracts (*Porphyra purpurea* and *Gelidium corneum*) the color of the hydrogels obtained was pink and violet, suggested to arises from the phycobiliproteins, that could be washed out during the purification (not
<figure><img src="image_11.png" /><figcaption>Fig. 1. General aspect of the synthesized chitosan-based hydrogels using the methanolic extract of red algae Ahnfeltiopsis devoniensis before and after freeze-drying of: (A) CS-AD-0.5 (B) CS-AD-1, (C) CS-AD-1.5 and (D) CS-AD-2.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>Fig. 2. Red seaweed extracts. A. ¹H-NMR (400 MHz, D₂O) spectrum of the methanolic extract of red seaweed Ahnfeltiopsis devoniensis, (I) region with characteristic peaks of the proteins, (II) zoom of the latter. B. ¹³C-NMR (100.4 MHz, D₂O) spectrum of the red seaweed extract showing peaks in the characteristic region of carbonyl, proposed to arise from the proteins and the identified peaks from the sugars.</figcaption></figure>

shown). In addition, the agent EDAC could be easily removed by dialysis to assure no toxic effects from non-reacted residues. The combined results demonstrated that all algae species and their extracts have the potential to be used as cross-linker for chitosan hydrogels.

To go deeper in this work, the methanolic extract of *Ahnfeltiopsis devoniensis* (AD) was selected as a case study. To do so, different

chitosan:AD ratios (1:0.5, 1:1, 1:1.5, 1:2) with 1 mass equivalent of EDAC were tested and the obtained hydrogels were referred to as CS-AD-0.5, CS-AD-1, CS-AD-1.5 and CS-AD-2, respectively. Fig. 1 depicts the general aspect of the chitosan-based hydrogels after cross-linking reaction before and after freeze-drying for all ratios.

First, in order to get more information of the cross-linking molecules,
A

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B

<figure><img src="image_14.png" /></figure>

C

<figure><img src="image_15.png" /><figcaption>**Fig. 3.** Chitosan-based hydrogel synthesis. **A (I)** ¹H HR-MAS Spectra of chitosan-based hydrogel (CS-AD-1) from methanolic extract of Ahnfeltiopsis devoniensis, **(II)** physical cross-linked chitosan-based hydrogel (control) and **(III)** 13C HR-MAS Spectra of chitosan-based hydrogel (CS-AD-1) from seaweed cross-linkers; **B (IV)** Overlaid ATR-FTIR spectra of hydrogel CS-AD-1 (blue), methanolic extract of red seaweed Ahnfeltiopsis devoniensis and chitosan (black); **(V)** zoom of the carbonyl region between 1800 - 1400 cm⁻¹ (black curve) and red seaweed extract (red curve); and **C** Scheme representing the proposed cross-linking mechanism between chitosan and the red seaweed extract, showing the possible cross-linking units consisting of COOH groups; note that chitosan moieties show only the deacetylate unites.</figcaption></figure>

in-depth NMR investigations were performed. Fig. 2 displays the ¹H-NMR (**A**) and ¹³C-NMR (**B**) spectra of the methanolic extract of *Ahnfeltiopsis devoniensis*.

The ¹H-NMR spectra (Fig. 2A) of the extract shows characteristic peaks that were supposed to arise from carrageenan oligosaccharides residues at δ 5.19 (³J,H-1,H-2= 4 Hz) and δ 4.99 (³J,H-1,H-2= 3.4 Hz), that could be assigned to the anomeric protons, and peaks in the range of δ 4.2–3.0, that were attributed to the protons of the sugar ring (H2- H6), respectively (Rodríguez Sanchez, Matulewicz, & Ciancia, 2022) (Couplings are shown in the HSQC NMR spectrum Fig. SA4). Carrageenan are sulfated polysaccharides present in the cellular matrixes of red seaweeds (Usov, 2011). They are mainly composed of sulfated galactans, constituted by alternating 3-linked β-D-galactopyranosyl and 4 linked α-D-galactoyranosyl units, consisting of nine different families

(Rodríguez Sanchez et al., 2022). Red seaweed *Ahnfeltiopsis devoniensis* was reported to contain mainly the κ/δ-hybrid family of carrageenan (Azevedo, Torres, Sousa-Pinto, & Hilliou, 2015). In addition, the polysaccharide backbone can bear different substituents, mainly β-D-xylopyranose linked to C-6, pyruvic acid ketals and methyl ethers (Ciancia, Matulewicz, & Tuvikene, 2020). Consequently, there are free carboxylic acids groups available that could contribute to the cross-linking reaction. However, considering the carbon NMR spectra in Fig. 2B, the signals belonging to pyruvylated carrageenan (4', 6'-O-(1-carboxyethylidene)-β-D-galactopyranose; NMR signals: carboxyl at δ 175.1, acetal at δ 101.3 and methyl at δ 25.5/1.44 (Falshaw, Furneaux, & Wong, 2003; Garegg, Lindberg, & Kvarnström, 1979), could not be significantly assigned. Besides the carrageenan-oligosaccharides, numerous signals were observed in the
¹H-NMR spectra between δ 5.5–8.5 and δ 1.5–4.4, that were assigned to the peptide bonds of the proteins in the seaweed extract (NH), (C⁶H)(C⁹H) and (C¹⁰H), respectively (Raghothama, 2010). The ¹³C-NMR spectrum supported these claim with signals at δ 179.2, 178.6, 170.9 and 164.8, that are (1) in the characteristic region for carbonyl atoms (de Dios & Oldfield, 1994) and (2) showed no carbon-proton long range coupling (³J$_{Hα-CO}$) confirming peptide bonds (Fig. SA5) (Wynants, Hallenga, Van Binst, Michel, & Zanen, 1984). Other peaks belonging to bioactive compounds such as MAA were difficult to attribute due to their weak signals. Based on these results, we suggest that the cross-linking reaction proceeded mainly between the carboxylic acid groups of the proteins and the primary amino groups of chitosan, even though a reaction between the carrageenan-oligosaccharides or MAA and chitosan could not be completely excluded.

Then, ¹H and ¹³C- High Resolution Magic Angle Spinning NMR spectroscopy (HR-MAS-NMR) (Fig. 3A) was carried out to confirm the formation of covalent bonds between chitosan and the seaweed extract. HR-MAS NMR is a powerful tool that combines solution-state NMR with magic angle spinning from solid-state, enabling the analysis of intact and swollen natural samples (Farooc et al., 2013). To date, reports of HR-MAS NMR of chitosan hydrogels are scarce though they can provide further insights to the chemical composition of viscous multicomponent samples (Marzorati, Bigler, Plattner, & Vermathen, 2013).

In this work, HR-MAS spectra of hydrogel CS-AD-1 (Fig. 3A (I)) was compared to a physical cross-linked chitosan, i.e., control (Fig. 3A (II)) (obtained via gelation of solubilized CS in acidic media (Nie et al., 2015); see details in the experimental section). In the spectra (I) and (II), glucosamine unit appeared in the range of 4.50–2.50 ppm (Guaresti, Garcia-Astrain, et al., 2017). The large peak at 3.01 ppm and 2.96 ppm (yellow) from the control hydrogel (II) and CS-AD-1 hydrogel (I), respectively, were tentatively attributed to proton H-2' of deacetylated chitosan and the peak at ~ 2 ppm (green) to the H-2 of acetylated chitosan (NAc) (Capitani, De Angelis, Crescenzi, Masci, & Segre, 2001; Guaresti, García-Astrain, et al., 2017). The appearance of the additional broad peak at 3.25 ppm in the spectrum of CS-AD-1 (red) denotes the covalent cross-link between the red seaweed extract and chitosan. This claim is supported by previous reports, showing similar downfield shift due to the functionalization of NH₂ of chitosan (Guaresti, Garcia-Astrain, et al., 2017). The assignment of the covalent cross-link could be further confirmed according to the shoulder peak* in the ¹³C HR-MAS spectrum of CS-AD-1 (Fig. 3A, (III)). This phenomenon is suggested due to the 'γ-gauche effect', described as the influence of cross-link units towards the anomeric carbon of chitosan, leading to an upfield shift of its resonance (Capitani et al., 2001). Finally, a weak, broad signal could be denoted between 8.02–8.7 ppm, supposed to arise from the amide bond between seaweed extract and chitosan (Guaresti, García-Astrain, et al., 2017). Other peaks found between 54.10–79.34 ppm were assigned to the sugar-ring of chitosan as they correspond to the characteristic region reported in the literature (El Hariri El Nokab & van der Wel, 2020). The formation of a chemically cross-linked hydrogel could be further supported by ATR-FTIR spectroscopy analysis (Fig. 2B). The hydrogel spectrum (blue curve) was compared to the ATR-FTIR spectrum of non-cross-linked chitosan. The typical strong and broad band located at 3450 cm⁻¹ and 3280 cm⁻¹ could be attributed to -NH₂ and O-H stretching vibrations, respectively. The band obtained at 1381 cm⁻¹ occurred from the stretching vibration of -CN and the bands at 1149, 1067 and 1022 cm⁻¹ involved asymmetric stretching of C-O-C bridge and C-O stretching from skeletal, respectively (Kumirska et al., 2010; Peniche, Elvira, & San Roman, 1998). The formation of the amide linkage between red seaweed extract AD and chitosan was supported by the decrease and shift of the N-H bending (from NH₂) from 1516 to 1535 cm⁻¹ and the appearance of the band at 1623 cm⁻¹, corresponding to the carbonyl groups of the cross-linking. These findings were in accordance to previous studies of chemical cross-linked chitosan hydrogels, observing similar shift in the IR spectra (Bhattarai, Ramay, Gunn, Matsen, & Zhang, 2005; Guaresti, Garcia-Astrain, et al., 2017; Kurita,

<figure><img src="image_16.png" /><figcaption>Fig. 4. Swelling behaviour of hydrogel CS-AD-0.5 (brown), CS-AD-1 (blue), CS-AD-1.5 (green) and CS-AD-2 (orange) over a time period of 180 min. at r.t. Data represents average values (± SD) from 3 independent experiments.</figcaption></figure>

<caption>Table 2</caption>

Swelling degree (SD), Water-Uptake (WU) and Porosity (P) of the red seaweed cross-linked chitosan hydrogels. Data represents average values (± SD) from three independent experiments.

<table><thead><tr><th>Hydrogel</th><th>CS-AD-0.5</th><th>CS-AD-1</th><th>CS-AD-1.5</th><th>CS-AD-2</th></tr></thead><tbody><tr><td>SD (%)</td><td>3057 ± 1380</td><td>2330 ± 729</td><td>1139 ± 73</td><td>1172 ± 280</td></tr><tr><td>WU (%)</td><td>97.0 ± 2.5</td><td>96.0 ± 1.4</td><td>92.0 ± 0.4</td><td>92.0 ± 0.8</td></tr><tr><td>P (%)</td><td>93.0 ± 11.5</td><td>75.0 ± 7.1</td><td>37.0 ± 5.8</td><td>27.0 ± 4.7</td></tr></tbody></table>

Murakami, & Fujiwara, 2010; Qu, Wirsen, & Albertsson, 2000).

Due to all the results, we suggest a plausible reaction mechanism in line with previous reports from the literature (Gorgieva & Kokol, 2012; Gurer et al., 2021; Kast & Bernkop-Schnürch, 2001) (Fig. 3C). In brief, the carboxyl acid moieties that are present in the red seaweed extract (1) are activated by EDAC (2) to give O-acylsourea derivative (3) as reactive intermediate product. The latter reacts subsequently with the primary amino group of chitosan (5) under the formation of an amide bond (6). This reaction sequence is supposed to continue in a random-fashion, cross-linking the extract with chitosan to give structure 7. Remember that the seaweed extract is a complex mixture and that the bifunctional cross-linker 'red seaweed extract' (1) presents solely a simplified structure.

## 3.2. Hydrogels characterization

**Porosity, swelling behavior and water-uptake.** The four synthesized hydrogels (CS-AD-0.5 – CS-AD-2) were characterized for their swelling behavior, porosity (P) and water uptake (WU). These values are key parameters to define the ability of hydrogels to adsorb hydrophilic molecules such as drugs or dyes (Crini et al., 2019) and are relevant for their further applications. Fig. 4 shows the plot of the swelling degree (in %) over a time-period of 180 min and Table 2 the calculated values of WU and P (in %).

As can be seen, the swelling degree increased significantly from highly cross-linked hydrogel CS-AD-2 to low cross-linked hydrogel CS-AD-0.5 from 1172 % to 3057 %, respectively. All four hydrogels showed the same swelling behavior, with a rapid increase in swelling over the first 60 min followed by a moderate increase until reaching the equilibrium, which was attained between 120 and 180 min. The water uptake of the hydrogels reached 92 % for CS-AD-2, up to 97 % for CS-AD-0.5 and the porosity was calculated to 27 %, 37 %, 75 % and 93 %
<figure><img src="image_17.png" /><figcaption>**Fig. 5.** Scanning electron micrographs of: (A) CS-AD-0.5, (B) CS-AD-1, (C) CSAD-1.5 and (D) CS-AD-2 after lyophilization (50 x magnification). Scale bars= 1 mm.</figcaption></figure>

for CS-AD-2, CS-AD-1.5, CS-AD-1 and CS-AD-0.5, respectively (see Table 2). The larger pores in the less cross-linked hydrogels CS-AD-0.5 and CS-AD-1 allow the direct penetration of water and accelerate the diffusion of the aqueous fluid in the three dimensional network, which could explain the higher swelling capacities and WU (Thombare et al., 2018). Note that the swelling experiments were carried out at pH= 7, while higher swelling ratio are expected at lower pH (results not shown).

According to previous studies, natural polysaccharide-based hydrogels generally show high capabilities to hold and retain large amounts of water due to the hydrophilicity of the functional groups (hydroxyl and amino groups) in the sugar chains (Ahmad et al., 2019; Crini et al., 2019; Domalik-Pyzik et al., 2019; Huang, Liu, Huo, & Yang, 2017; Zonatto, Muniz, Tambourgi, & Paulino, 2017). Compared to physical hydrogels based on chitosan and red seaweed, (Feki et al., 2020; Netanel Liberman et al., 2021) the degree of swelling and the water uptake capacity were in the same range (lit. values between 500 and 3000 % for swelling and ca. 80–88 % for WU, respectively). However, the chitosan-based algae hydrogels in the literature used solely the polysaccharide-fraction from the microalgae extracts. Interestingly, compared to chemical cross-linked hydrogels based on chitosan and conventional cross-linking molecules such as DTBP, (Adekogbe & Ghanem, 2005) GTA (SHYU, 2000) and genipin, (Yu et al., 2021) the swelling capacity, water-uptake and porosity were significantly higher. Furthermore, the high values of swelling degree and water uptake despite the relatively low porosities in hydrogel CS-AD-2 and CS-AD-1.5 (27 and 37 %, respectively) can be related to the complex chemical structure of the seaweed cross-linker (see Table SA1).

**Morphology.** A deeper view of the microstructure of the different lyophilized hydrogels was obtained by scanning electron microscopy experiments (SEM, Fig. 5).

The SEM images depict a highly porous, irregular, cobwebbed system with an average pore size from 70 µm for CS-AD-2 to 400 µm for CS-AD-0.5. The most 'regular' structure was denoted for the lowest amount of seaweed extract (CS-AD-0.5, Fig. 5 (A)), whereas with an increasing content of the cross-linker, the surfaces became rougher and irregular. In line with its swelling-behavior, the smallest pore-sizes and 'densest' system was found for CS-AD-2 (Fig. 5 (D)), explaining the lowest

<figure><img src="image_18.png" /><figcaption>**Fig. 6.** Plot of the stability/degradation tests of hydrogel CS-AD-0.5 (brown), CS-AD-1 (blue), CS-AD-1.5 (olive) and CS-AD-2 (orange) in 1 M acetic acid solution at r.t. over a period of 8 weeks. Data represents average values (± SD) from 3 independent experiments.</figcaption></figure>

swelling degree compared to the other hydrogels. Note that the porous structure was produced during the elimination of water during the freeze-drying (-80 °C). Previous studies showed that the post-drying method has a strong correlation with the physical structure of chitosan-based hydrogels (Heimbuck, Priddy-Arrington, Sawyer, & Caldorera-Moore, 2019). Similar highly porous three-dimensional structures with interspatial voids and cobwebbed systems were found in previous studies of marine-based hydrogels consisting of red algae and chitosan (Feki et al., 2020; Netanel Liberman et al., 2021). However, the referenced studies described physical and not chemical cross-linked
<figure><img src="image_19.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_22.png" /></figure>

**Fig. 7.** Dye adsorption capacity. **A.** Plot of the dye removal (%) and adsorption capacity (mg/g) of hydrogel CS-AD-1 using different ratio hydrogel:dye. **B.** Plot of the dye removal (%) in time of hydrogel CS-AD-0.5 (brown), CS-AD-1 (blue), CS-AD-1.5 (green) and CS-AD-2 (orange) with a ratio hydrogel:dye of 6 at 25 °C. **C.** Adsorption-Desorption cycle of MO using hydrogel CS-AD-1 showing the difference steps. **D.** Plot of the dye removal (%) of four consecutive adsorption/desorption cycles using hydrogel CS-AD-1. Data represents average values (± SD) from 3 independent experiments.

hydrogels of chitosan and polysaccharides from red microalgae. For covalent cross-linked chitosan-based hydrogels using small molecules (genipin, glutaraldehyde), more compact and regular systems are generally described (Bhattarai et al., 2010; Domalik-Pyzik et al., 2019; Khapre, Pandey, & Jugade, 2021; Yu et al., 2021).

**Stability.** Considering the potential application of the hydrogels for the dye-removal tests, their stability was tested in diluted acidic medium corresponding to the pH of the dye-solution (1 M acetic acid, pH= 3-4).

Images of the stability tests are given in the Fig. 6. The results showed a significant degradation > 60 %) of hydrogel CS-AD-0.5 upon two weeks and a weight loss of 19 %, 18 % and 11 % for hydrogels CS-AD-1, CS-AD-1.5 and CS-AD-2, respectively. Upon two months, almost half of hydrogel CS-AD-1 was degraded (42 %), CS-AD-1.5 lost 31 % of its weight and CS-AD-2 denoted a weight loss of 26 %. The rapid degradability of the natural hydrogels in mild acid aqueous conditions makes them attractive compounds with respect to their end-of-life and corresponding environmental impact. On the basis of these results, hydrogel CS-AD-1 was selected for the following dye-adsorption tests (good swelling capacity (2330 %), water uptake (96 %), high porosity (75 %) and acceptable stability).

### 3.3. Dye adsorption capacity

The adsorption of textile industry dye methyl orange (MO) was studied targeting a circular removal process and recycling of the utilized hydrogel (Fig. 7). In a first study, different ratios of hydrogel CS-AD-1 and MO were tested to find the optimal adsorbent dosage. Fig. 7A shows the plot of the dye removal efficiency (%) and the adsorption capacity (μe in mg/g) against different ratios of hydrogel:dye ranging from 0.5 to 8. Note that the pH of the MO solution was 3-4 (from the azo-dye) and was not decreased in order to test the hydrogels under ‘mild’ conditions. The results showed that the effective removal of MO increased for higher amounts of hydrogel from 35 % for the ratio 0.5 to 93 % for the ratio 8 and decreased with increasing amount of hydrogel from 729 mg/g at ratio 0.5 to 115 mg/g at ratio 8, respectively. The aggregation of adsorption sites at high concentration of hydrogel could explain this behavior, leaving less surface available for the interaction with the dye (Crini & Badot, 2008). Seeking for a compromise between maximal effective dye removal and ideal adsorption capacity, the ratio 6 was chosen for the following experiments using hydrogel CS-AD-1. From Fig. 7A, the maximum adsorption capacity of 728.46 ± 66.17 mg/g showed that the hydrogel was an excellent adsorber for methyl orange
A

<figure><img src="image_23.png" /></figure>

B

<figure><img src="image_24.png" /></figure>

C

<figure><img src="image_25.png" /></figure>

**Fig. 8.** Biocompatibility studies. **A.** In vitro viability of L929 cells on CS-AD-0.5, CS-AD-1, CS-AD-1.5 and CS-AD-2 hydrogels along 72 h of incubation time. The dotted line represents the limit of acceptance established by the ISO 10993-5 (70 % of the value of the negative control); **p** < 0.01 and ***p** < 0.001. **B.** SEM micrographs of L929 seeded on CS-AD-0.5, CS-AD-1, CS-AD-1.5 and CS-AD-2 hydrogels for 24 h (I) and 72 h (II) of cultivation. The squares indicate the area that has been magnified on the right side, to show the L929 cells adhesion points to the hydrogel's surface. **C.** Appearance of the L929 cells after 72 h of culture with the corresponding extractive medium, positive or negative control. Inverted phase contrast microscope image. The scale bar represents 100 µm.

under these conditions.

In a following experiment, kinetic studies of the adsorption process were carried out to evaluate the effect of contact time for each hydrogel (Fig. 7B). The time to reach the equilibrium of adsorption for hydrogel CS-AD-0.5, CS-AD-1 and CS-AD-1.5 was 120 min with an effective dye removal of 98 %, 91 % and 87 %, respectively. Lower average adsorption values of 79 % were found for hydrogel CS-AD-2 and the time attaining equilibrium increased to more than 200 min. The decrease of the dye adsorption was assumed to be due to the smaller pore size and less available free amino groups in the highly cross-linked hydrogel CS-AD-2.

Finally, recycling studies of the adsorption/desorption of MO were carried out. The adsorption ratio of MO on hydrogel CS-AD-1 was 90 % for the first cycle, 88 %, 81 % and 76 % for the second, third and fourth cycles, respectively (Fig. 7C). The decrease of the adsorption capacity might be due to the degradation of the hydrogel during the regeneration

process (treatment with NaOH and acetic acid, see stability test Fig. 6), or the reduction of the driving force as reported by previous work (Zeng et al., 2015). Similar regeneration studies were carried out by Labidi, Salaberria, Fernandes, Labidi, & Abderrabba (2019) and could show slight decrease of the discoloration efficiency within the second and third cycle.

Chitosan-based hydrogels and their application as dye-adsorber has been extensively discussed in the past decades (Crini et al., 2019). Earlier reported studies of chitosan hydrogels (listed in Table SB1) could show that hydrogel CS-AD-1 has excellent adsorption properties and is in the range of what was previously observed for MO adsorption using natural compounds. The high adsorption of MO can be related to the numerous hydrophilic groups from chitosan and the seaweed extract, leading to electrostatic interactions and hydrogen-bonding towards the dye (Domalik-Pyzik et al., 2019). Indeed, the biosorption of chitosan hydrogels follows generally different mechanisms such as
complexation (Elwakeel, 2010), ion-exchange (Guo & Wilson, 2012); and also physical adsorption (Crini et al., 2019), explaining the efficient adsorption capacities of the seaweed hydrogels of this study. From the literature it can be seen that complex adsorbing systems, combining different biopolymers and/or oxidized graphene, obtained higher adsorption of the dye. The implication of crude red seaweed extract as complex cross-linker presents consequently an attractive alternative regarding its simple and sustainable synthesis procedure.

### 3.4. Biocompatibility studies

**Cytotoxicity.** In order to analyze the *in vitro* biocompatibility of the materials and their suitability for biomedical applications, cytotoxicity and cell adhesion tests were carried out with L929 cells (Fig. 8). The cytotoxicity tests (Fig. 8A) revealed that the hydrogels were biocompatible, as they exceeded the minimum value of 70 % viability (respect to the negative control) established by the ISO 10993-5 at 72 h. In addition, the cell viability value of all the hydrogels remained stable throughout the entire assay and the different amount of cross-linker extracts in the hydrogels did not imply a significant impact in the biocompatibility of the material, as the significant increase of cell viability for the hydrogel CS-AD-1 at 48 h was transient. These data of high *in vitro* biocompatibility of chitosan hydrogels agreed with others that have been previously reported using L929 mouse fibroblasts (L. Gao et al., 2014).

**Cell-adhesion.** The suitability of the hydrogels to allow cell adhesion was evaluated, since this property is influenced by the composition of the material and can limit its subsequent biomedical application (Todros, Todesco, & Bagno, 2021). As shown in Fig. 8B, L929 cells were well adhered and produced cytoplasmic projections with several adhesion points to the hydrogel's surface. Additional images of cell culture are given in Fig. 8C. These projections were greater with culture time and the cells had a more elongated and flatter morphology after 72 h of culture, typical of healthy adherent cells. Furthermore, there was no significant difference in cell density observed on the surface of the hydrogels. All this indicated that these chitosan-based hydrogels offer a good adhesion substrate for cells, regardless of the amount of EDAC and red seaweed.

### 4. Conclusions

In this work, we have successfully developed a simple synthesis to explore and exploit crude red seaweed extracts as covalent cross-linkers for chitosan-based hydrogels and thus confirm our hypothesis. We demonstrated that the latter could be applied to five different red seaweed species, and more importantly, use the full integrity of the extracts without separation and purification of the different bio-molecules. The in-depth NMR analysis of the extracts and hydrogels strongly suggests that the cross-linking occur mainly between chitosan and the proteins of the extracts. Due to the numerous carboxylic acid groups present in the biomolecules of the extract, various cross-linking reactions are despite possible. The physical properties of the hydrogels could be tuned by varying the amount of extract and resulted with porous systems with comparable high swelling degrees that could be related to the complex hydrophilic chemical structures of the bio-molecules in the extract.

Furthermore, the as-obtained hydrogels revealed excellent absorption capacities for the removal of toxic dyes from waste water. In an optimized protocol, the hydrogels could remove 91 % of methyl orange within 120 min and still up to 76 % after four recycling steps. Cytotoxicity and Cell-adhesion studies evidenced the good biocompatibility of the hydrogels for all different amounts of cross-linker.

In conclusion, these results open a new route to produce sustainable and biocompatible chitosan-based hydrogels from marine by-products *via* a simple synthesis step. The latter could replace toxic cross-linkers and could be envisioned to be applied in tissue-engineering purpose.

Further progress could be done to scale-up the synthesis and eventually replace EDAC by enzymatic catalysts to prevent any toxic substance during the synthesis process. Moreover, the unexpected instantaneous gel-forming ability of the methanolic extracts could pave the way for the development of new formulations for 3D printing approaches.

### CRediT authorship contribution statement

**Lea Spitzer:** Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation. **Amandine Adrien:** Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation. **Unnimaya Thalakkale Veettil:** Writing – review & editing, Validation, Methodology, Investigation. **Sheila Olza:** Writing – review & editing, Validation, Methodology, Investigation. **Ana Alonso-Varona:** Writing – review & editing, Validation, Supervision. **Susana C. M. Fernandes:** Writing – review & editing, Validation, Supervision, Methodology, Investigation, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Susana Fernandes reports financial support was provided by French National Research Agency. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Data availability

No data was used for the research described in the article.

### Acknowledgements

This work was carried under the framework of E2S UPPA Partnership Chair MANTA (Marine Materials) funded by the 'Investissements d'Avenir' French program managed by ANR (grant number #ANR-16-IDEX-0002), by the Région NouvelleAquitaine, and by the Communauté d'Agglomération du Pays Basque (France). The authors thank M. Joie and O. Lamarens for helpful experimental support on the extraction and hydrogel synthesis.

### Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.carpta.2024.100541.

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