# Sucrose and Glycerol Additives: A Way to Tune the Biological and Physicochemical Properties of Agarose Hydrogels?

Victor C Igbokwe, Vincent Ball, Nour-ouda Benzaamia, Simon Gree, Sophie Hellé, Juliette Soubirou-blot, Corinne Nardin, Lydie Ploux

► To cite this version:

Victor C Igbokwe, Vincent Ball, Nour-ouda Benzaamia, Simon Gree, Sophie Hellé, et al., Sucrose and Glycerol Additives: A Way to Tune the Biological and Physicochemical Properties of Agarose Hydrogels?. Macromolecular Materials and Engineering, 2024, 10.1002/mame.202400150, hal-04647850

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# Sucrose and Glycerol Additives: A Way to Tune the Biological and Physicochemical Properties of Agarose Hydrogels?

Victor C. Igbokwe, Vincent Ball, Nour-Ouda Benzaamia, Simon Gree, Sophie Hellé, Juliette Soubirou-Blot, Corinne Nardin,* and Lydie Ploux*

Sucrose and glycerol have gained attention as additives for hydrogels, owing to their capacity to exert considerable influence over the physicochemical, mechanical, and biological characteristics of these materials. Herein, these effects on agarose hydrogels (AHs) are explored. A series of AHs are synthesized using sucrose (30% and 300% w/v) and glycerol as additives. The storage modulus (10.0–13.7 kPa) and hydrophilicity of the hydrogels (contact angle < 50°) do not vary significantly with sucrose or glycerol addition. However, sucrose enhances the hydration capacity of the hydrogels by up to 170%, whereas glycerol reduces it. Interestingly, sucrose and glycerol individually do not have bacteriostatic effects against *Staphylococcus epidermidis*, but their combination significantly (p ≤ 0.001) inhibits the growth of both *S. epidermidis* and *Pseudomonas aeruginosa* by 63% and 29%, respectively, in comparison to native agarose. Cytotoxicity testing on NIH/3T3 murine fibroblasts reveals that sucrose increases cell viability up to 98%, while glycerol reduces it below 60%. Overall, these hydrogels hold promise for antibacterial biomedical applications as wound dressing materials and surface coatings for medical devices and can also be used to formulate bioinks for 3D bioprinting.

## 1. Introduction

Hydrogels have revolutionized the design and development of innovative functional biomaterials for various biomedical

applications. These applications include tissue engineering, drug delivery, 3D bioprinting, and biosensing.[1–4] Additionally, hydrogels have been employed to coat both biotic and abiotic surfaces, such as wounds and medical devices respectively.[5,6] Hydrogels derived from natural polymers have garnered substantial interest due to their renewability, biodegradability, compatibility with living cells and tissues, and the relative ease of functional modification have rendered them indispensable.[7–9] Among the diverse spectrum of natural polymers used to synthesize hydrogels, agarose stands out prominently. This prominence can be attributed to its thermo-reversible gelation behavior, ease of modification, water solubility, and tunable mechanical properties.[10–12] Furthermore, agarose is generally regarded as biocompatible and exhibits a striking resemblance to the extracellular matrix, making it a suitable candidate for various biomedical applications, particularly in tissue engineering and regenerative medicine.[12] Notably, agarose is also biodegradable and hypoallergenic.[13]

Agarose is a neutral hydrophilic biopolymer sourced from seaweed. It comprises 400 agarobiose repeating units, composed of 1,3-linked-d-galactopyranose and 1,4-linked 3,6-anhydro-α-

V. C. Igbokwe, V. Ball, N.-O. Benzaamia, S. Hellé, L. Ploux
INSERM
UMR-S 1121 Biomaterial Bioengineering
Centre de Recherche en Biomédecine de Strasbourg
Strasbourg 67000, France
E-mail: ploux@unistra.fr

V. C. Igbokwe, V. Ball, N.-O. Benzaamia, S. Hellé, L. Ploux
Faculty of Dentistry
Université de Strasbourg
Strasbourg 67000, France

V. C. Igbokwe, V. Ball, N.-O. Benzaamia, S. Hellé, L. Ploux
CNRS
EMR 7003, Strasbourg 67000, France
S. Gree
CNRS
UMR 7361 IS2M
Université de Haute-Alsace
Mulhouse 68200, France
J. Soubirou-Blot, C. Nardin
E2S UPPA
CNRS
IPREM
Université de Pau et des Pays de l'Adour
Pau 64000, France
E-mail: corinne.nardin@univ-pau.fr

<figure><img src="image_2.png" /></figure>

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/mame.202400150

© 2024 The Author(s). Macromolecular Materials and Engineering published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

DOI: 10.1002/mame.202400150
l-galactopyranose.[14,15] This chemical structure incorporates several polar functional groups and linkages, including —OH, —O—, and C=O, conferring it with a strong hydrophilic property and the ability to form hydrogen bonds with water molecules. This property is pivotal in the context of hydrogel formation.[10] Agarose hydrogels (AHs) can be easily formulated under simple conditions without the need for catalysts, unlike hydrogels synthesized with other biopolymers such as cellulose and guar gum.[16] While native AHs have their advantages, they are inherently biologically inert and lack intrinsic antibacterial properties. This limits their applicability in antibacterial contexts, and their high water content may make them susceptible to bacterial proliferation.[17] Given that bacteriostatic or bactericidal properties are highly desirable in biomaterials for biomedical applications,[18] various studies have focused on developing antibacterial AHs through chemical modification or the incorporation of antibacterial agents, such as classical antibiotics, nanoparticles, and polyphenols.[19–23] However, the use of these antibacterial agents often presents significant challenges, including issues related to antibiotic resistance,[24–26] cytotoxicity,[27,28] environmental concerns,[29] relatively high cost of production,[30] and in some cases, limited spectrum of efficacy.[27] Considering these challenges, we explored the feasibility of using a combination of sucrose and glycerol to synthesize antibiotics-free bacteriostatic AHs.

Sucrose, glycerol, and their derivatives are widely recognized as safe additives in the fields of food and pharmaceuticals,[31–33] prompting their extensive use. In the context of biomedical applications, they have garnered substantial interest for their capacity to modify the physicochemical, mechanical, and biological properties of hydrogels. Specifically, sucrose can enhance the affinity of agarose polymer helices for solvents, thereby limiting helix aggregation tendencies and influencing the mechanical properties of agarose gels.[34] Historically, sugars have also been used in wound healing for centuries probably due to their antibacterial activity.[35] Additionally, both sucrose and its derivatives exhibit antibacterial properties at high concentrations.[36–38] However, sucrose at low concentration can be metabolized by many microorganisms and used as a carbon source,[39] thus possibly facilitating microbial growth. On the other hand, glycerol has been noted for its capacity to enhance the flexibility and softness of agarose gels by improving the mobility of polymer chains under deformation.[13] Like sucrose, glycerol and its derivatives have also shown interesting antibacterial activities.[32,40] Though specific figures on the cost and availability of sucrose and glycerol vary with factors such as location, supplier, and quantity required, the consistent use of sucrose and glycerol in various industrial and research applications strongly suggests that they are relatively inexpensive and readily accessible.

In this study, we investigated the possibility of combining sucrose and glycerol as additives to impart antibacterial properties to AHs intended for biomedical applications. To achieve this, we synthesized two series of AHs using glycerol and varied concentrations of sucrose. The concentration of glycerol used was fixed at the lower threshold of the optimal range (from 30% to 50% of the material weight) required to have a plasticizing effect, while limiting the risk of toxicity.[41] The concentration of sucrose used in this study was well below the levels (30–60% of the material weight) used in food preservation.[42] The selected concentration

<table><caption>**Table 1.** Hydrogel composition in terms of the weight of compounds used for hydrogel formulation.</caption><thead><tr><th></th><th>Sample ID</th><th>Agarose [g]</th><th>Sucrose [g]</th><th>Glycerol [g]</th></tr></thead><tbody><tr><td rowspan="5">A-series</td><td>A1</td><td>0.15</td><td>—</td><td>—</td></tr><tr><td>AG</td><td>0.15</td><td>—</td><td>6.31</td></tr><tr><td>AS<sub>30</sub></td><td>0.15</td><td>0.05</td><td>—</td></tr><tr><td>AGS<sub>30</sub></td><td>0.15</td><td>0.05</td><td>6.31</td></tr><tr><td>AS<sub>300</sub></td><td>0.15</td><td>0.45</td><td>—</td></tr><tr><td rowspan="6">B-series</td><td>AGS<sub>300</sub></td><td>0.15</td><td>0.45</td><td>6.31</td></tr><tr><td>B1</td><td>0.30</td><td>—</td><td>—</td></tr><tr><td>BG</td><td>0.30</td><td>—</td><td>6.31</td></tr><tr><td>BS<sub>30</sub></td><td>0.30</td><td>0.09</td><td>—</td></tr><tr><td>BGS<sub>30</sub></td><td>0.30</td><td>0.09</td><td>6.31</td></tr><tr><td>BS<sub>300</sub></td><td>0.30</td><td>0.90</td><td>—</td></tr><tr><td>BGS<sub>300</sub></td><td>0.30</td><td>0.90</td><td>6.31</td></tr></tbody></table>

is expected to release a minimal quantity of sucrose, thus limiting possible inflammatory drawbacks. Our experimental approach involved synthesizing two different series of hydrogels—A-series and B-series. The concentration of agarose in the B-series hydrogels was twice that of the A-series. However, it is important to note that the ratio of agarose to sucrose and agarose to glycerol remained constant within each hydrogel within each series. The primary objectives of this study were to evaluate the effect of the additives on the i) surface wettability and hydration capacity, ii) dynamic viscoelasticity, iii) bacteriostatic activity against *Staphylococcus epidermidis* and *Pseudomonas aeruginosa*, and iv) biocompatibility of AHs. The effect of the additives on the surface chemistry and the possible release of compounds from the hydrogels was evaluated as well.

## 2. Experimental Section

### 2.1. Materials

Agarose (CAS No. 9012-36-6) was purchased as a fine white powder from Sigma-Aldrich (Darmstadt, Germany) with a specific gelling temperature ($T_{gel}$) between 34.5 and 37.5 °C and a gel strength of >1200 g cm⁻² as stated by the producer. Analytical grade (99% pure) glycerol (CAS No. 56-81-5) having a boiling point of 180 °C and molecular weight of 92.09 g mol⁻¹ was purchased from Fisher Scientific GmbH (Kehl, Germany). Food grade, for human consumption sucre+ (Cestas, France) E+ organic beet sugar was purchased from Biocoop (Strasbourg, France). For the bacteriostatic activity assays, trypticasein soy broth and agar agar from Condalab (Madrid, Spain) and BD Bacto (New Jersey, USA) respectively were used. All reagents and materials were used without further purification or modification.

### 2.2. Synthesis of Hydrogels

Two sets of hydrogels (A-series and B-series) were synthesized in one-pot by varying the concentration of sucrose at 30% and 300% (w/v), while maintaining the concentration of glycerol as shown in **Table 1**. Notably, the respective concentration of agarose and sucrose in the B-series samples was consistently twice that
sample of the respective hydrogels was carefully placed into a 12-well microplate. Subsequently, the samples were inoculated with a 2 mL suspension of *P. aeruginosa* and *S. epidermidis*, respectively, preculture grown overnight in tryptic soy broth (TSB) at 37 °C in an incubator shaker. An inoculum of each test organism without any supplementary material or substance was used as a control. The samples inoculated with the test organisms were then incubated at 37 °C for 24 h in a static incubator, following which the OD₆₀₀ of the suspension was measured by a Bio-Rad SmartSpec Plus spectrophotometer, with TSB as the blank. The OD₆₀₀ values of the test organisms were converted to Log colony forming units per mL (Log CFU mL⁻¹) using an OD-CFU mL⁻¹ standard curve for the respective test organisms. The experiments were replicated a minimum of three times on at least three independent samples, thereby resulting in a total of at least nine replicates per hydrogel sample.

### 2.3.7. *In Vitro* Cytotoxicity

The cytotoxicity of the hydrogels was assessed by quantifying the viability of a NIH/3T3 mouse fibroblast cells on hydrogel extracts using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in accordance with the ISO-10993-5 protocol,[50] with some modifications. Hydrogel samples, cut into 12 mm diameter circles using a sterile biopsy punch, were carefully placed in a 24-well microplate, and sterilized with UV light for 15 min before extraction. To prepare hydrogel extracts, 1 mL of high-glucose Dulbecco's modified Eagle medium (DMEM, Dutscher France) was dispensed into the wells containing the hydrogel samples and subsequently incubated for 24 h at 37 °C. Besides, fibroblasts previously cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin), for 24 h at 37 °C in a 5% CO₂ atmosphere, were diluted with complete DMEM to achieve a final concentration of 5 × 10⁴ cells mL⁻¹. 200 µL of this cell suspension was seeded into wells of a 24-well microplate and incubated for 24 h. The medium was then replaced by the hydrogel extracts and incubated for 24 h. Complete DMEM and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. Following incubation, the supernatants were discarded while the cells attached to the wall of the microplate were washed with phosphate buffer saline (PBS). 100 µL of 0.5 mg mL⁻¹ MTT solution in DMEM was added into each well and incubated for a minimum of 2 h. After discarding the solution, 100 µL of DMSO was added to the wells to dissolve the purple formazan crystals formed. The resulting solution's absorbance was measured at 570 nm using a spectrophotometer. Cell viability was calculated using Equation 2 and reported as mean ± SD of three replicates per at least three independent samples per hydrogel.

$$ \text{Cell viability (\%)} = \frac{\text{Mean absorbance of hydrogel samples}}{\text{Mean absorbance of positive control (DMEM)}} \times 100 \quad (2) $$

### 2.4. Statistical Analysis

All data are presented as mean ± SD. To test for statistical significance of the difference between the means of the treatments

<table><caption>**Table 2.** Concentration in every compound related to the weight of the final hydrogel.</caption><thead><tr><th></th><th>Sample ID</th><th>Agarose<br/>[% w/w<sup>1</sup>]</th><th>Sucrose<br/>[% w/w<sup>1</sup>]</th><th>Glycerol<br/>[% w/w<sup>1</sup>]</th></tr></thead><tbody><tr><td rowspan="6">A-series</td><td>A1</td><td>1.3 ± 0.1</td><td>—</td><td>—</td></tr><tr><td>AG</td><td>0.8 ± 0.1</td><td>—</td><td>34.7 ± 1.6</td></tr><tr><td>AS<sub>30</sub></td><td>1.4 ± 0.2</td><td>0.5 ± 0.1</td><td>—</td></tr><tr><td>AGS<sub>30</sub></td><td>0.8 ± 0.1</td><td>0.3 ± 0.1</td><td>34.7 ± 1.6</td></tr><tr><td>AS<sub>300</sub></td><td>1.2 ± 0.1</td><td>3.6 ± 0.1</td><td>—</td></tr><tr><td>AGS<sub>300</sub></td><td>0.8 ± 0.1</td><td>2.3 ± 0.1</td><td>32.5 ± 2.1</td></tr><tr><td rowspan="6">B-series</td><td>B1</td><td>2.7 ± 0.1</td><td>—</td><td>—</td></tr><tr><td>BG</td><td>1.6 ± 0.1</td><td>—</td><td>34.7 ± 0.8</td></tr><tr><td>BS<sub>30</sub></td><td>2.8 ± 0.3</td><td>0.8 ± 0.1</td><td>—</td></tr><tr><td>BGS<sub>30</sub></td><td>1.7 ± 0.1</td><td>0.5 ± 0.1</td><td>35.5 ± 1.7</td></tr><tr><td>BS<sub>300</sub></td><td>2.6 ± 0.1</td><td>7.8 ± 0.3</td><td>—</td></tr><tr><td>BGS<sub>300</sub></td><td>1.6 ± 0.1</td><td>4.8 ± 0.3</td><td>33.9 ± 2.3</td></tr></tbody></table>

‡: referred to the weight of the material as obtained after synthesis and following stabilization of the hydration state at the room condition.

and control (A1), the normality of the data was first determined using the Shapiro-Wilk test. If the means followed a normal distribution, the Tukey post-hoc test was used to analyze the difference. However, if the means were not normally distributed, the Kruskal-Wallis's test and the Dunn's multiple comparison to analyze the difference. *, **, and *** were used to label means that were significantly different from the control at $p \le 0.05$, 0.01 and 0.001 respectively.

## 3. Results and Discussion

Two series of AHs were synthesized. The weight/weight concentration of agarose, sucrose, and glycerol relative to the final hydrogel (considered after formulation and stabilization for about 20 min under ambient conditions) are presented in **Table 2**. The concentration of agarose in the B-series of hydrogels is twice that of the A-series. The concentration of glycerol was ≈34% for all the samples, just higher than the limit allowing glycerol to play its plasticizer role.[41] The concentration of sucrose varied from 0.3 to 7.8%, thus largely lower than the limits of concentration for a preservation effect.

### 3.1. Surface Wettability

The surfaces of the hydrogels exhibited pronounced wettability (Figure 1), with CA values between 35° and 42°, which is typical of hydrophilic surfaces.[44] Also, increasing the concentration of agarose decreased the CA of all the hydrogels (Figure S2, Supporting Information), whatever the concentration in the other compounds. This suggests that agarose had a predominant influence on the wettability of the surface of the hydrogels. This surface hydrophilicity of the hydrogels was not surprising since sucrose and agarose have abundant polar chemical groups like OH in their respective chemical structures.[51]

In contrast, sucrose did not affect the CA of the hydrogels. As seen with AS30 and AS300, there was no significant difference
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Hydration capacity of the series A of hydrogels in NaCl solution (0.9% w/v): a) HC values as calculated with Equation 1 (presented as mean ± SD, n ≥ 5); b,c) Weight evolution of the hydrogels measured during (b) dehydration at 37 °C and (c) rehydration at room temperature (presented as mean ± SD, n ≥ 5).</figcaption></figure>

albeit at a sufficient concentration since AS₃₀ did not differ significantly from A1. Similarly, sucrose increased G'' as shown in Figure 3c. Notably, only hydrogels containing sucrose or its combination with glycerol differed significantly from A1. In the case of the B-series, the instability of G' and G'' prevents any conclusion about the difference between hydrogels, except that G' are significantly higher compared to the A-series (Figure 3b). This was not surprising given the expected influence of agarose (twice more concentrated in the B-series than in the A-series) on the stiffness of the hydrogel.[62]

Though not statistically significant, the reduction in G' by glycerol for the A-series was not surprising, given that glycerol is a known plasticizer that enhances the flexibility of hydrogels.[63] However, currently, there are no reports which specifically investigated the influence of glycerol on the mechanical properties of agarose hydrogels. The marginal increase in stiffness by sucrose may be attributed to enhanced hydrogen bonding with agarose polymer chains, strengthening the hydrogel network. This view was also considered by Chen et al.[64] who examined the influence of sugar concentration on the mechanical properties of a zwitterionic polymer hydrogel. Another proposition is that sucrose acts as a filler within the hydrogel matrix, increasing the packing density of polymer chains and thus stiffening the network. Furthermore, the combined effect of sucrose and glycerol on both G' and G'' looked like the effect of sucrose alone, suggesting that the binding affinity of sucrose with the polymer matrix was predominant in comparison with the plasticizing effect of glycerol.

### 3.4. Surface Chemistry

The Raman spectra of sucrose and glycerol of the hydrogel matrix of the A-series, specifically at the surface and a depth of 500 µm, are depicted in Figure 4. The spectra exhibited bands characteristics for sucrose, with notable but weak signals both within (500 µm depth) and at the hydrogel surface, probably due to the relatively low concentration of sucrose.[65] We discerned minor peaks around 1459 and 1123 cm⁻¹, associated with the scissoring vibration of CH₂ and the stretching deformation of C-OH, respectively.[47,65] Notably, these peaks were more pronounced in the AS₃₀ sample compared to AS₃₀. Interestingly, these peaks vanished within the hydrogel and the 1123 cm⁻¹ peak red shifted to 1139 cm⁻¹. This suggests probable weak intermolecular bonds due to chemical or physicochemical bonds. In addition to these observations, characteristic peaks of sucrose were observed around 1056 and 998 cm⁻¹, corresponding to the stretching of C-O in the furanoid ring and C-C in the glucose moiety, respectively.[66] Remarkably, while these peaks exhibited greater prominence at the surface of AS₃₀ compared to AS₃₀, the reverse pattern was noted within the hydrogel, possibly due to interactions with dangling polymer chains present in the bulk of the hydrogel, which were not physically crosslinked.

On the other hand, the glycerol bands exhibited higher intensity at the hydrogel surface than, potentially indicating a concentration gradient of glycerol between the surface and in the bulk of the hydrogel. An intense doublet peak was present at 1107 and
<figure><img src="image_4.png" /><figcaption>**Figure 4.** Raman spectra acquired a) at the surface and b) at 500 µm depth of the A-series hydrogels.</figcaption></figure>

latter appears to be the most plausible explanation, as increasing
the sucrose concentration, as observed in AS<sub>300</sub>, led to a slightly
more prominent band and a broad peak at 1056 cm<sup>-1</sup>, indicating
approximately 0.45% (w/v) of sucrose released. When combined
with glycerol, it was also not possible to estimate the quantity
of sucrose released from both AGS<sub>300</sub> and AGS<sub>30</sub> due to the low
intensity of the sucrose peak at 1056 cm<sup>-1</sup>.

**3.6. Bacterial Growth Inhibition**

Log CFUmL⁻¹ (Figure 6 and Figure S6a,b, Supporting Information) of the respective test bacteria, in planktonic state, was used as an index to measure their growth inhibition by the hydrogels. Similar patterns were observed for A-series and B-series. Firstly, we observed that hydrogels containing glycerol (AG) and sucrose

(AS₃₀ and AS₃₀₀) had similar effects on *S. epidermidis* than A1. While this result was expected for AS₃₀ and AS₃₀₀ due to the very low quantity of sucrose released from the hydrogels, it was unexpected in the case of AG because glycerol is commonly used as an osmotic agent in food preservation.[74,75] Osmotic agents typically reduce the water activity (Aw) of the substrate, leading to osmotic stress, plasmolysis, and impaired growth or lysis of microorganisms.[76,77] It is a general notion that bacteria require a minimum Aw of 0.91 for growth.[78] Hence lowering the Aw will consequentially impede their growth. We can thus hypothesize that the amount of glycerol released from the hydrogels may not have been sufficient to critically lower the Aw, induce osmotic stress, and finally inhibit the growth of *S. epidermidis*.

In contrast, though desirable, we found that glycerol inhibited
the growth of *P. aeruginosa* by 28%, as illustrated in Figure 6b.
The inhibitory effect of glycerol can be attributed to a reduction
<figure><img src="image_5.png" /><figcaption>**Figure 5.** ATR spectra (1200–1000 cm⁻¹ zoom) of sucrose and glycerol released from the hydrogels after immersion in water for 24 h.</figcaption></figure>

<table><caption>**Table 3.** Quantity of sucrose and glycerol released in water as estimated from the FTIR-ATR spectra.</caption><thead><tr><th>Samples</th><th>Sucrose<br>[% weight/volume]</th><th>Glycerol<br>[% weight/volume]</th></tr></thead><tbody><tr><td>AG</td><td>na</td><td>8.43</td></tr><tr><td>AS<sub>30</sub></td><td>na</td><td>Na</td></tr><tr><td>AGS<sub>30</sub></td><td>nd</td><td>7.60</td></tr><tr><td>AS<sub>300</sub></td><td>0.45</td><td>Na</td></tr><tr><td>AGS<sub>300</sub></td><td>nd</td><td>8.93</td></tr></tbody></table>

nd, not determined; na, not applicable.

in the Aw of the medium.[45] On another hand, glycerol can penetrate bacterial cells by facilitated diffusion thereby increasing intracellular osmotic pressure, weakening the membrane, and consequently inducing cellular lysis.[79] However, this finding diverges from the results reported by Scoffield et al.[80] who indicated that glycerol promotes the growth of *P. aeruginosa* by serving as a nutrient source. The discrepancy between their findings and ours may be attributed to differences in glycerol concentration and experimental conditions employed in our respective studies. Besides, the increased susceptibility of *P. aeruginosa*

<figure><img src="image_6.png" /><figcaption>**Figure 7.** Cytotoxicity of the A-series hydrogel extracts on NIH/3T3 murine fibroblasts (values presented as mean ± SD, n = 12).</figcaption></figure>

to glycerol, compared to *S. epidermidis* could be explained by the difference in the thickness of their peptidoglycan layers. Specifically, *P. aeruginosa* has a thinner peptidoglycan layer, constituting only 10% of its cell wall, while *S. epidermidis* has a significantly thicker peptidoglycan layer, composing 90% of its cell wall.[79]

Both AGS₃₀ and AGS₃₀₀ significantly inhibited the growth of *P. aeruginosa* by 29 and 26%, respectively. This level of inhibition was consistent with that observed with AG thus strongly suggesting that the inhibitory effect on *P. aeruginosa* may be principally due to the glycerol. More interestingly, although sucrose and glycerol individually had no inhibitory effect on the growth of *S. epidermidis*, the combination of both compounds in AGS₃₀ significantly (p ≤ 0.001) inhibited its growth by 63% relative to A1. This synergistic inhibitory effect of sucrose and glycerol on *S. epidermidis* was only evident with the lowest sucrose concentration, as no growth inhibition was observed with AGS₃₀₀.

### 3.7. In Vitro Cytotoxicity

The cytotoxicity of the hydrogels was assessed by measuring the metabolic activity of NIH/3T3 murine fibroblasts exposed to hydrogel extracts in DMEM. A cytotoxic effect was defined as a reduction in cell viability greater than 30%.[81] **Figure 7** shows that the viability of fibroblasts exposed to A1, AS₃₀, and AS₃₀₀ extracts

<figure><img src="image_7.png" /><figcaption>**Figure 6.** Antibacterial activity of the A-series hydrogels against a) *S. epidermidis* 35984 and b) *P. aeruginosa* ATCC 27853 (values presented as mean ± SD, n ≥ 6).</figcaption></figure>
exceeded the 70% threshold, with 81, 90, and 98% viability respectively. The increase in viability measured with the hydrogels containing sucrose suggests that sucrose positively influenced fibroblast viability in a subtle dose-dependent manner. A similar and even more pronounced trend was observed with the B-series hydrogels (Figure S7, Supporting Information), where fibroblast viability for BS30 and BS300 reached 96% and 111%, respectively. The non-toxicity of sucrose towards fibroblasts was expected, as sucrose is a preferred carbon source for both eukaryotic and prokaryotic cells.[82]

In contrast to AS30 and AS300, AG, AGS30, and AGS300 were relatively cytotoxic as the viability of fibroblasts in their respective extracts was 56%, 55%, and 62%, respectively. The cytotoxic effect was attributed to glycerol, which was the common component in these hydrogels. This finding deviates from that of Barros et al.[83] who suggested that glycerol was non-toxic to fibroblasts and keratinocytes. It was also unexpected given the widespread use of glycerol as a pharmacopeia compound in the food, pharmaceutical, and cosmetics industries for manufacturing various personal care and consumable products.[84] However, by simply doubling the concentration of sucrose, as in the case of BGS30 and BGS300 (Figure S7, Supporting Information), the viability of fibroblasts recorded 74.3% and 83% respectively. This observation gives credence to our position on the cytotoxicity of glycerol to the fibroblasts, and shows that sucrose, which promoted fibroblast viability, can counteract this toxicity.

Finally, this study explored the effects of sucrose and glycerol on the viscoelasticity, HC, surface wettability, in vitro toxicity against NIH/3T3 fibroblasts, and bacteriostatic activity of AHs. contributing to the ongoing efforts to develop hydrogels with improved or tunable mechanical, physicochemical, and antibacterial properties, and reduced cytotoxicity. Our findings revealed that, interestingly, sucrose, at the lowest sucrose concentration, in combination with glycerol synergistically inhibited the growth of *S. epidermidis*, which differs from the effect of sucrose alone and from the inhibition of the growth of *P. aeruginosa*, irrespective of the concentration of sucrose. The sucrose-glycerol combination also significantly mitigated the toxic impact of glycerol on fibroblast cells, but in a dose-dependent manner. However, it did not counteract the anti-hydration effect induced by glycerol. In terms of mechanical properties, the combination of sucrose and glycerol had a negligible effect compared to the impact of sucrose alone but differed from the effect of glycerol alone. Thus, the addition of sucrose or glycerol in AHs allows to tune hydration, an essential characteristic of hydrogels in their utility in varied biomedical applications, and even holds promise for enhancing the antibacterial properties of AHs if they are combined.

**4. Conclusions and Prospects**

In this study, we synthesized a series of AHs using glycerol and varied concentrations of sucrose as additives. The effects of these additives on the physicochemical, biological, and mechanical properties of the hydrogels were investigated. While sucrose had negligible effects on surface wettability, HC, and viscoelasticity and bacteriostatic activity of the hydrogels, it enhanced the viability of fibroblasts. On the other hand, glycerol significantly reduced both HC and viscoelasticity of the hydrogels, while mildly inhibiting the growth of *P. aeruginosa* and the viability of fibroblasts.

Finding a balance between bacteriostatic activity and biocompatibility is crucial. One strategy to achieve this balance could be to optimize the concentrations of both sucrose and glycerol in the hydrogels. Also, a supplementary additive, for instance an antibiotic, can be incorporated to augment the activities of sucrose and glycerol. Although the hydrogels synthesized in this study exhibited modest antibacterial activities, they are a viable alternative to hydrogels synthesized with high concentrations of antibiotics, which are often cytotoxic. This approach can potentially enhance the biocompatibility of the antibacterial hydrogel, making it a safer option for various biomedical applications. We envisage that our agarose-based hydrogels can potentially be used as materials for wound dressing and coatings for medical devices, and to formulate bioinks for 3D bioprinting. They can also be used as scaffolds for promoting cell growth and differentiation while concurrently minimizing the risk of infections.

**Supporting Information**

Supporting Information is available from the Wiley Online Library or from the author.

**Acknowledgements**

The authors would like to thank Payzulaeva Khava and Zülal Uğur for their contribution in the preliminary synthesis and characterization of the hydrogels.

**Conflict of Interest**

The authors declare no conflict of interest.

**Author Contributions**

V.C.I.: methodology, formal analysis (hydrogel synthesis and characterization), visualization, writing—original draft, review and editing; V.B.: investigation and formal analysis (rheology), writing—review; N.B.: investigation and formal analysis (rheology, microbiology, surface wettability, hydration capacity); S.G.: formal analysis (ATR-FTIR and Raman), writing—review; S.H.: investigation, supervision, and formal analysis (microbiology), writing—review; J.S.: conceptualization, formal analysis (hydrogel synthesis and characterization); C.N.: conceptualization, project administration, resources, supervision, validation, methodology, writing—original draft, review, and editing; L.P.: conceptualization, project administration, resources, supervision, validation, methodology, formal analysis (microbiology), visualization, writing—original draft, review, and editing. All authors have read and agreed to the published version of the manuscript.

**Data Availability Statement**

The data that support the findings of this study are available from the corresponding author upon reasonable request.

**Keywords**

agarose, bacteriostatic, glycerol, hydrogel, sucrose

Received: April 23, 2024

Revised: May 16, 2024

Published online:
[1] A. Herrmann, R. Haag, U. Schedler, *Adv. Healthcare Mater.* **2021**, *10*, 2100062.

[2] S. Jacob, A. B. Nair, J. Shah, N. Sreeharsha, S. Gupta, P. Shinu, *Pharmaceutics* **2021**, *13*, 357.

[3] W. Shi, R. He, Y. Liu, *Eur. J. Biomed. Res.* **2015**, *1*, 3.

[4] Y. Zhao, S. Song, X. Ren, J. Zhang, Q. Lin, Y. Zhao, *Chem. Rev.* **2022**, *122*, 5604.

[5] Y. Luo, L. Cui, L. Zou, Y. Zhao, L. Chen, Y. Guan, Y. Zhang, *Carbohydr. Polym.* **2022**, *294*, 119774.

[6] L. Peng, L. Chang, M. Si, J. Lin, Y. Wei, S. Wang, H. Liu, B. Han, L. Jiang, *ACS Appl. Mater. Ampmathsemicolon Interfaces* **2020**, *12*, 9718.

[7] Z. Ahmad, S. Salman, S. A. Khan, A. Amin, Z. U. Rahman, Y. O. Al-Ghamdi, K. Akhtar, E. M. Bakhsh, S. B. Khan, *Gels* **2022**, *8*, 167.

[8] D. A. Gyles, L. D. Castro, J. O. C. Silva, R. M. Ribeiro-Costa, *Eur. Polym. J.* **2017**, *88*, 373.

[9] U. S. K. Madduma-Bandarage, S. V. Madihally, *J. Appl. Polym. Sci.* **2020**, *138*, https://doi.org/10.1002/app.50376.

[10] F. Jiang, X.-W. Xu, F.-Q. Chen, H.-F. Weng, J. Chen, Y. Ru, Q. Xiao, A.-F. Xiao, *Mar Drugs* **2023**, *21*, 299.

[11] B. L. Roach, A. B. Nover, G. A. Ateshian, C. T. Hung, **2016**.

[12] P. Zarrintaj, S. Manouchehri, Z. Ahmadi, M. R. Saeb, A. M. Urbanska, D. L. Kaplan, M. Mozafari, *Carbohydr. Polym.* **2018**, *187*, 66.

[13] Q. Han, A. Wang, W. Song, M. Zhang, S. Wang, P. Ren, L. Hao, J. Yin, S. Bai, *ACS Appl Bio Mater* **2021**, *4*, 6148.

[14] J. J. Roberts, P. J. Martens, in *Biosynthetic Polym. Med. Appl.*, Elsevier, xx xx **2016**, pp. 205.

[15] V. Subramanian, D. Varade, in *Biopolym. Compos. Electron.*, Elsevier, xx xx **2017**, pp. 155.

[16] Y. Ko, J. Kim, H. Y. Jeong, G. Kwon, D. Kim, M. Ku, J. Yang, Y. Yamauchi, H.-Y. Kim, C. Lee, J. You, *Carbohydr. Polym.* **2019**, *203*, 26.

[17] Q. Bai, C. Zheng, W. Chen, N. Sun, Q. Gao, J. Liu, F. Hu, S. Pimpi, X. Yan, Y. Zhang, T. Lu, *Mater. Adv.* **2022**, *3*, 6707.

[18] J. G. Heuko, M. E. R. Duarte, A. G. Gonçalves, M. D. Noseda, F. S. Murakami, M. M. de Carvalho, D. R. B. Ducatti, *Carbohydr. Res.* **2021**, *507*, 108387.

[19] W. Gong, R. Wang, H. Huang, Y. Hou, X. Wang, W. He, X. Gong, J. Hu, *Int. J. Biol. Macromol.* **2023**, *227*, 698.

[20] J. M. Grolman, M. Singh, D. J. Mooney, E. Eriksson, K. Nuutila, *J. Burn Care Ampmathsemicolon Res.* **2019**, *40*, 900.

[21] I. P. Khosalim, Y. Y. Zhang, C. K. Y. Yiu, H. M. Wong, *Sci. Rep.* **2022**, *12*, 1971.

[22] S. Liu, X. Liu, Y. Ren, P. Wang, Y. Pu, R. Yang, X. Wang, X. Tan, Z. Ye, V. Maurizot, B. Chi, *ACS Appl. Mater. Interfaces* **2020**, *12*, 27876.

[23] N. Ninan, A. Forget, V. P. Shastri, N. H. Voelcker, A. Blencowe, *ACS Appl. Mater. Ampmathsemicolon Interfaces* **2016**, *8*, 28511.

[24] R. Carpa, A. Remizovschi, C. A. Culda, A. L. Butiuc-Keul, *Gels* **2022**, *8*, 70.

[25] B. Hu, C. Owh, P. L. Chee, W. R. Leow, X. Liu, Y.-L. Wu, P. Guo, X. J. Loh, X. Chen, *Chem. Soc. Rev.* **2018**, *47*, 6917.

[26] Y. Zhong, H. Xiao, F. Seidi, Y. Jin, *Biomacromolecules* **2020**, *21*, 2983.

[27] S.-H. Chen, Z. Li, Z.-L. Liu, L. Cheng, X.-L. Tong, F.-Y. Dai, *J. Mater. Res.* **2019**, *34*, 1911.

[28] Z. Yang, R. Huang, B. Zheng, W. Guo, C. Li, W. He, Y. Wei, Y. Du, H. Wang, D. Wu, H. Wang, *Adv. Sci.* **2021**, *8*, 2003627.

[29] S. Hamidi, F. Monajjemzadeh, M. Siahi-Shadbad, S. A. Khatibi, A. Farjami, *Polym. Eng. Ampmathsemicolon Sci.* **2022**, *63*, 5.

[30] M. A. Cook, G. D. Wright, *Sci. Transl. Med.* **2022**, *14*, eab07793.

[31] V. N. J. Lekahena, M. R. Boboleha, in *Proc. 5th Int. Conf. Food Agric. Nat. Resour. FANRes* **2019**, Atlantis Press, 2020.

[32] P. M. Schlievert, M. L. Peterson, *PLoS One* **2012**, *7*, e40350.

[33] J. R. White, *Clin. Diabetes Publ. Am. Diabetes Assoc.* **2018**, *36*, 74.

[34] V. Normand, *Carbohydr. Polym.* **2003**, *54*, 83.

[35] A. Biswas, M. Bharara, C. Hurst, R. Gruessner, D. Armstrong, H. Rilo, *J Diabetes Sci Technol* **2010**, *4*, 1139.

[36] J. A. Mora Vargas, J. O. Ortega, M. B. C. dos Santos, G. Metzker, E. Gomes, M. Boscolo, *Carbohydr. Res.* **2020**, *489*, 107957.

[37] Y. Ning, M. Ma, Y. Zhang, D. Zhang, L. Hou, K. Yang, Y. Fu, Z. Wang, Y. Jia, *Food Res Int* **2022**, *154*, 111018.

[38] S.-Y. Shao, Y.-G. Shi, Y. Wu, L.-Q. Bian, Y.-J. Zhu, X.-Y. Huang, Y. Pan, L.-Y. Zeng, R.-R. Zhang, *Molecules* **2018**, *23*, 11118.

[39] S. J. Reid, V. R. Abratt, *Appl. Microbiol. Biotechnol.* **2005**, *67*, 312.

[40] W. B. Setianto, T. Y. Wibowo, H. Yohanes, F. Illaningtyas, D. D. Anggoro, *IOP Conf. Ser. Earth Environ. Sci.* **2017**, *65*, 012046.

[41] E. Basiak, A. Lenart, F. Debeaufort, *Polymers* **2018**, *10*, 412.

[42] A. K. Yadav, S. V. Singh, *J Food Sci Technol* **2014**, *51*, 1654.

[43] A. Vigué, D. Vautier, A. Kaytoue, B. Senger, Y. Arntz, V. Ball, A. Ben Mlouka, V. Gribova, S. Hajjar-Garreau, J. Hardouin, T. Jouenne, P. Lavalle, L. Ploux, *J Funct Biomater* **2022**, *13*, 237.

[44] C. H. Kung, P. K. Sow, B. Zahiri, W. Mérida, *Adv. Mater. Interfaces* **2019**, *6*, 1900839.

[45] H. Nakagawa, T. Oyama, *Front Chem* **2019**, *7*, 731.

[46] A. Gómez-Siurana, A. Marcilla, M. Beltrán, D. Berenguer, I. Martínez-Castellanos, S. Menargues, *Thermochim. Acta* **2013**, *573*, 146.

[47] A. B. Brizuela, L. C. Bichara, E. Romano, A. Yurquina, S. Locatelli, S. A. Brandán, *Carbohydr. Res.* **2012**, *361*, 212.

[48] A. Hauswirth, R. Köhler, L. ten Bosch, G. Avramidis, C. Gerhard, *Foods* **2022**, *11*, 2786.

[49] R. Nair, S. Venkatesh, K. A. Athmaselvi, S. Thakur, *J. Food Meas. Chart. Acta* **2015**, *10*, 24.

[50] in *ANSIAMIISO 10993-52009R2014mathsemicolon Biol. Eval. Med. Devices — Part 5 Tests Vitro Cytotox*, AAMI, **2009**.

[51] G. Apte, A. Lindenbauer, J. Schemberg, H. Rothe, T.-H. Nguyen, *ACS Omega* **2021**, *6*, 10963.

[52] K. Sheng, X. Dong, Z. Chen, Z. Zhou, Y. Gu, J. Huang, *Appl. Surf. Sci.* **2022**, *591*, 153097.

[53] K. Bialik-Wąs, K. Pluta, D. Malina, M. Barczewski, K. Malarz, A. Mrozek-Wilczkiewicz, *Int. J. Mol. Sci.* **2021**, *22*, 12022.

[54] B. Xu, Y. Liu, L. Wang, X. Ge, M. Fu, P. Wang, Q. Wang, *Polymers* **2018**, *10*, 1025.

[55] P. K. GhattyVenkataKrishna, G. A. Carri, *J. Biomol. Struct. Dyn.* **2013**, *32*, 424.

[56] Y. Kataoka, N. Kitadai, O. Hisatomi, S. Nakashima, *Appl. Spectrosc.* **2011**, *65*, 436.

[57] B. S. Flowers, M. S. Mittenthal, A. H. Jenkins, D. A. Wallace, J. W. Whitley, G. P. Dennis, M. Wang, C. H. Turner, V. N. Emel'yanenko, S. P. Verevkin, J. E. Bara, *ACS Sustain. Chem. Ampmathsemicolon Eng.* **2016**, *5*, 911.

[58] Z. Y. Ben, H. Samsudin, M. F. Yhaya, *Eur. Polym. J.* **2022**, *175*, 111377.

[59] J. Tarine, S. M. Sapuan, A. Khalina, *Sci. Rep.* **2021**, *11*, 13900.

[60] L. Martikainen, K. Bertula, M. Turunen, O. Ikkala, *Macromolecules* **2020**, *53*, 9983.

[61] K. Bertula, L. Martikainen, P. Munne, S. Hietala, J. Klefström, O. Ikkala, *Nonappa*, *ACS Macro Lett.* **2019**, *8*, 670.

[62] D. Millar, M. Mennu, K. Upadhyay, C. Morley, P. Ifju, *Strain* **2021**, *57*, e12383.

[63] M. Watanabe, H. Li, M. Yamamoto, J. Horinaka, Y. Tabata, A. W. Flake, *J Biomed Mater Res B Appl Biomater* **2020**, *109*, 921.

[64] Y. Chen, W. Wang, D. Wu, M. Nagao, D. G. Hall, T. Thundat, R. Narain, *Biomacromolecules* **2018**, *19*, 596.

[65] L. S. Jr, L. M. Moreira, V. G. B. Conceição, H. L. Casalechi, I. S. Muñoz, F. F. D. Silva, M. A. S. R. Silva, R. A. D. Souza, M. T. T. Pacheco, *Spectroscopy* **2009**, *23*, 217.

[66] K. Ilaslan, I. H. Boyaci, A. Topcu, *Food Control* **2015**, *48*, 56.

[67] A. Mudalige, J. E. Pemberton, *Vib. Spectrosc.* **2007**, *45*, 27.

[68] E. Mendelovici, R. L. Frost, T. Kloprogge, *J. Raman Spectrosc.* **2000**, *31*, 1121.

[69] N. Itoh, S. E. J. Bell, *Analyst* **2017**, *142*, 994.

[70] M. Yamashina, M. Akita, T. Hasegawa, S. Hayashi, M. Yoshizawa, *Sci. Adv.* **2017**, *3*, e1701126.
[71] Y. Liu, K. Huang, Y. Zhou, D. Gou, H. Shi, *J. Phys. Chem. B* **2021**, *125*, 8099.

[72] L. F. Leopold, N. Leopold, H.-A. Diehl, C. Socaciu, *Spectroscopy* **2011**, *26*, 93.

[73] J.-J. Max, C. Chapados, *J. Phys. Chem. A* **2001**, *105*, 10681.

[74] E. K. Dermesonlouoglou, M. C. Giannakourou, *J Food Sci Technol* **2018**, *55*, 4079.

[75] K. Pattanapa, N. Therdthai, W. Chantrapornchai, W. Zhou, *Int. J. Food Sci. Ampmathsemicolon Technol.* **2010**, *45*, 1918.

[76] L. Qiu, M. Zhang, J. Tang, B. Adhikari, P. Cao, *Food Res Int* **2019**, *116*, 90.

[77] S. Zeidler, V. Müller, *Environ. Microbiol.* **2019**, *21*, 2212.

[78] L. V. Allen Jr, *Int J Pharm Compd* **2018**, *22*, 288.

[79] V. S. M. Saegeman, N. L. Ectors, D. Lismont, B. Verduyckt, J. Verhaegen, *Burns* **2008**, *34*, 205.

[80] J. Scaffield, L. Silo-Suh, *Can. J. Microbiol.* **2016**, *62*, 704.

[81] E. Jablonská, J. Kubásek, D. Vojtěch, T. Ruml, J. Lipov, *Sci. Rep.* **2021**, *11*, 6628.

[82] T. Franzino, H. Boubakri, T. Cernava, D. Abrouk, W. Achouak, S. Reverchon, W. Nasser, F. el Z. Haichar, *Plant Commun* **2022**, *3*, 100272.

[83] N. R. de Barros, R. S. dos Santos, M. C. R. Miranda, L. F. C. Bolognesi, F. A. Borges, J. V. Schiavon, R. F. C. Marques, R. D. Herculano, A. M. Q. Norberto, *Skin Res Technol* **2019**, *25*, 461.

[84] S. Goyal, N. B. Hernández, E. W. Cochran, *Polym. Int.* **2021**, *70*, 911.