Green synthesis of antimicrobial alginate/hydroxyethyl cellulose hydrogels crosslinked by Ca²⁺ and dual Ca²⁺/Mg²⁺

Abstract

Eco-friendly antimicrobial hydrogels derived from carbohydrate polymers are receiving significant attention for their sustainable and cost-effective properties. This study focuses on developing antimicrobial hydrogels based on alginate and hydroxyethyl cellulose (SA/HEC) using a green chemistry approach. The impact of bivalent metal ions on cross-linking efficiency and the hydrogels’ physicochemical properties were analyzed, with hydrogels produced in various forms, such as beads, sponges, and films, characterized by SEM, XRD, TGA, FTIR, and UV–Vis spectroscopy. Calcium (Ca²⁺), magnesium (Mg²⁺), and Ca²⁺/Mg²⁺ combinations were explored, revealing that Ca²⁺ and Ca²⁺/Mg²⁺ combinations demonstrated excellent cross-linking efficiency, while Mg²⁺ alone was insufficient for cross-linking. However, adding small amounts of Ca²⁺ improved Mg²⁺ cross-linking capacity, yielding stable SA/HEC hydrogels. Beads exhibited porous structures (2–20 µm), and films with a thickness of ~ 150 µm were produced, showcasing strong mechanical and thermal stability. Additionally, in-situ synthesized silver nanoparticles (AgNPs) within SA/HEC hydrogels exhibited antimicrobial activity against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and fungal strains Candida albicans ATCC 14053 and Candida krusei ATCC 6258. This study successfully demonstrates a green chemistry method to synthesize water-insoluble hydrogels cross-linked with Ca²⁺/Mg²⁺ ions with inherent antimicrobial properties.

Introduction

Green technology to develop anti-microbial materials using hydrogels is greatly increasing because of the huge concern over environmental sustainability. Hydrogels from natural polysaccharides have unique characteristics compared to other biopolymers because of their ease of availability, non-toxicity, biocompatibility, and biodegradability, in addition to their high temperature stability and high crystallinity^1,2.

Hydrogels are three-dimensional hydrophilic polymeric networks with amine, hydroxyl, carboxyl or sulphonic groups attached to their backbone, capable of absorbing vast quantities of water³. Among the natural polysaccharides alginate and cellulose-based polymers are commanding attention due to their numerous applications in a different fields^4,5 and also both alginate and cellulose have similar structural and physicochemical properties like the natural extracellular matrix, (ECM)⁶.

Alginate is an anionic polysaccharide distributed widely in the cell walls of brown algae, where through binding with water it forms a viscous gum. It is also known as alginic acid or align. Alginic acid is a linear copolymer with homopolymeric blocks of (1–4)-linked β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G) residues, respectively, covalently linked together in different sequences or blocks. The distribution and sequence of the mannuronic and guluronic acid residues along the polymer chain are critical determinants of its physicochemical properties, including its ability to gel, viscosity, and biocompatibility. These characteristics make sodium alginate an attractive material for various applications in food, pharmaceutical, and biomedical industries^7,8.

Cellulose is one of the most abundant naturally occurring polysaccharides which has linear chains of glucose units linked by beta glycosidic linkage. Since cellulose is insoluble in water and many other organic solvents, it is desirable to work with cellulose derivatives like hydroxypropyl methylcellulose (HPMC), ethyl cellulose (EC), hydroxyethyl cellulose (HEC) and methylcellulose (MC)⁹. One of the commonly used cellulose derivatives is hydroxyethyl cellulose (HEC).

HEC is a major derivative of cellulose, where the hydroxyethyl group replaces one or more of the three hydroxyl groups present in each glucopyranoside. The properties and behavior of these polymers are highly influenced by the degree of substitution and their relative distribution within C1, C2 and C6 positions. HEC finds applications in many fields like paper, cement, paint industries because of its stabilizing, emulsifying, binding, suspending, dispersing properties^10,11.

For practical applications, sodium alginate is usually blended with other polymers to improve the mechanical, thermal and other physical properties and is used in various fields like agriculture, water treatment, tissue engineering applications, and pharmaceuticals. Alginate blended with other polymers could be cross-linked physically or chemically¹² to make them water-insoluble and improve their mechanical properties. Physical cross-linking is achieved by dipole–dipole interactions, hydrogen bonding, and hydrophobic or electrostatic forces and these hydrogels are reversible, while chemical cross-linking is permanent and involves covalent bonding making the hydrogels irreversible and more stable^13,14. The commonly used chemical cross-linkers include carbodiimide, citric acid, fumaric acid, 1,2,3,4-butane tetracarboxylic dianhydride, glutaraldehyde, and ethylenediaminetetraacetic dianhydride. Compared to chemical cross-linking, physical cross-linking is preferred in forming biocompatible materials for biomedical applications since no toxic chemicals are used here^15–22.

The carboxyl (–COOH) or hydroxyl (–OH) groups of alginate and hydroxyl (–OH) groups of hydroxyethyl cellulose are helpful in cross-linking and producing excellent materials. The green chemistry approach prefers the bivalent ions crosslinking of sodium alginate since it does not involve any toxic initiators, monomers or cross-linkers^17,18. Alginate can be cross-linked with bivalent metal ions, including Ca²⁺, Ba²⁺, Fe²⁺, Sr²⁺, Co²⁺, Ni²⁺, Zn²⁺, and Mn²⁺ and so on¹⁹. It is reported that among the bivalent cations, Mg²⁺ cannot cross-link alginate to form stable gels, under normal conditions²⁰.

It is well known that Calcium is extremely important to the human body, helping in many functions^21,22. Most of it is found in bones and very little in the extracellular fluids. Magnesium, is the fourth most abundant cation in the human body and a cofactor in more than 600 enzymatic reactions²³. Sufficient amount of magnesium has a significant effect on the precipitation of calcium carbonate²⁴.

Alginate-based antimicrobial materials have been widely studied with different cross-linking methods in which alginate was blended with different polymers and nanoparticles. To the best of our knowledge no work has been done using alginate and HEC cross-linked by a combination of Ca²⁺ and Mg²⁺ for anti-microbial studies. It is well known that HEC is a reducing as well as a stabilizing agent in the synthesis of metal nanoparticles²⁵.

In this study we aim to examine the physicochemical properties of SA/HEC hydrogels cross-linked with mono bivalent cations Ca²⁺ and Mg²⁺ and also co-crosslinked by Ca^2+/Mg²⁺ cations. The in-situ formation of silver nanoparticles and their anti-microbial efficacies in these cross-linked SA/HEC/Ca and SA/HEC/Ca/Mg hydrogels will be examined.

Materials and methods

Materials

All materials of this work are commercially available. Hydroxyethyl cellulose (HEC) was purchased from SIGMA-ALDRICH, Germany. Sodium alginate (SA) was purchased from S.D Fine-Chem (SDFCL) Limited, India. Calcium chloride (CaCl₂.H₂O) and magnesium chloride (MgCl₂.6H₂O) were purchased from Philips Harris, England. All the chemicals used in this study were of high purity and without further purification. All the solutions were prepared using millipore water.

Preparation of SA/HEC hydrogels

Sodium alginate (SA) 2.5wt% and Hydroxyethyl cellulose (HEC) 3.75wt% were used for this study. These concentrations were chosen to get the solutions of desired viscosity. After weighing the polymers, they were dissolved in water and stirred for 12 h at room temperature to get homogeneous solutions. HEC/SA blend solution was prepared by mixing HEC and SA polymer solutions prepared from the previous step in 1:1 ratio followed by stirring for 12 h at room temperature to get a homogeneous blend solution.

Cross-linking of SA/HEC hydrogels

Overall, the cross-linking was carried out using 0.1 M of CaCl₂ and MgCl₂ solutions. SA/HEC polymer was added into the CaCl₂ and MgCl₂ solutions and allowed to cross-link for 10 min. The methods used to prepare hydrogels in different forms were different. The formation of beads took place in a petri dish (10 cm diameter) which contained 25 ml of 0.1 M of the cross-linkers. Beads were formed using a pipette, by drop casting the HEC/SA blend polymers into a beaker containing the bivalent cation cross-linkers at room temperature. Thin films were produced by taking 10 ml of the HEC/SA blend polymers into a petri dish and pouring 20 ml of the bivalent cation cross-linkers over it. The Sponges were produced by freeze drying 25 ml of the HEC/SA solution in a beaker. For cross-linking, 25 ml of cross linkers were poured over the solid freeze dried polymers. For the mixture of cross-linkers 12.5 ml of each cross-linker (0.1 M CaCl₂ and 0.1 M MgCl₂) were used. After 10 min the cross-linked hydrogels were removed from the beaker and rinsed copiously with ultrapure water. The washed samples were dried at 50 °C overnight for 24 h. These dried samples were used for all the physicochemical characterization. The samples were called SA/HEC/Ca, SA/HEC-Ca/ Mg, or, SA/HEC-Mg hydrogels.

Preparation of SA/HEC/silver nanoparticles (AgNP) and cross-linking of the hydrogel

SA/HEC hydrogels (section "Preparation of SA/HEC hydrogels") were used to prepare the silver nanoparticles. To a 5 ml of the SA/HEC hydrogel 300 µl of 50 mM AgNO₃ was added and heated at 80 °C for 1 h. The color change from colorless to pale yellow and finally to brown suggested the formation of silver nanoparticles. To cross-link the SA/HEC/AgNP hydrogel with calcium 10 ml of 0.1 M CaCl₂ was taken in a petri dish and the hydrogel was dropped into the CaCl₂ using a syringe. To cross-link the hydrogel with Ca/Mg 5 ml of 0.1 M CaCl₂ and 5 ml of 0.1 M MgCl₂ were taken in a petri dish and the hydrogel was dropped into the CaCl₂/ MgCl₂ using a syringe. The immediate gelation of the hydrogel upon adding it into the cross-linker indicated the cross-linking. The cross-linked hydrogels were allowed to be in the beaker and removed after 10 min and washed with water copiously and dried at 40 °C overnight.

Characterization of SA/HEC hydrogels

Fourier transform infrared spectroscopy (FTIR)

To evaluate the structural changes of the hydrogel, the Fourier Transform Infrared (FTIR) spectra of the mixture of sodium alginate (SA) hydroxyethyl cellulose (HEC), (HEC/SA) and SA/HEC/Ca, SA/HEC-Mg, SA/HEC-Ca/Mg were recorded by FTIR IRPrestige-21 (Shimadzu) spectrometer. The analysis was carried out in a range of 400–4000 cm⁻¹ with 128 scans recorded at 4 cm⁻¹ of resolution.

Thermogravimetric analysis (TGA)

Thermo Gravimetric Analyzer (TGA) TG 209F3, NETZSCH, Germany) instrument was used to verify the thermal degeneration attributes of SA/HEC hydrogels. The starting weight of the analysis was 6 mg sample, and the analysis was carried out under N₂ and the heating rate was 10 °C/min until 700 °C.

Different scanning calorimetry (DSC)

The thermal behavior of the prepared samples was studied using SDT Q600, TA Company, by placing the samples in an aluminum crucible. The heat evolved was recorded at a 10.0 (K/min) heating rate, starting at 50 °C until 250 °C in N₂ dynamic atmosphere at a flow rate of 40.0 mL/min until 60.0 ml/min.

Morphological characterizations

Scanning electron microscopy was used on SA/HEC/Ca, SA/HEC/Ca/ Mg, and SA/HEC/Mg hydrogels to get the surface morphology and the porous structure at various magnifications. To get the cross-section images the beads, thin films, and freeze-dried hydrogels were carefully cut by using a sharp razor blade, mounted onto the sample holders, coated with gold, and then observed and imaged. The morphologies of the cross-linked SA/HEC hydrogels at room temperature were observed using a scanning electron microscope (SEM JSM-7800F) with 20 kV accelerating voltage.

X-ray diffractometer (XRD)

The XRD analysis of SA/HEC hydrogels with and without cross-linking was performed using a Philips X’pert PRO automatic diffractometer operating at 40 kV and 40 mA with a scan rate of 2°/min. The XRD spectra were recorded in the 2θ range of 10–70°.

Mechanical testing

The mechanical properties of the cross-linked SA/HEC hydrogel films were studied using a texture analyzer (TA. XT plus, Stable Micro Systems, Godalming, England) using a 5 kg load cell. Tests were done in triplicates. For this study rectangular strips with dimensions 7 mm X 70 mm were placed between two clamps at 60 mm distance. The mechanical properties like tensile strength and elongation at break were obtained directly from the software exponent connect.

UV–visible spectroscopy

UV–vis absorption spectra of the silver nanoparticles in the hydrogel were measured by using Spectrophotometer (UV-1800, Shimadzu Japan) with range of wavelength (300–700) nm. All spectra were recorded in the absorbance mode and the optical spectra were acquired, by using a quartz cuvettes (1 cm) path length.

Stability of SA/HEC hydrogels

The SA/HEC hydrogels beads were immersed in water and monitored for more than six months.

Cross-linking efficiency and stiffness of SA/HEC hydrogels

The cross-linking efficiency was tested by just immersing the cross-linked hydrogels in water. The more the cross-linking the less was the swelling effect. The stiffness was checked by just bending the films.

Antimicrobial activities of alginate and cellulosic derived compounds

The invitro antimicrobial activities for all the compounds [Sodium Alginate- Silver nanoparticles (SA/AgNPs); Hydroxyethyl cellulose-silver nanoparticles (HEC/AgNPs); Sodium Alginate- Hydroxyethyl cellulose -silver nanoparticles (SA-HEC/AgNPs); Sodium Alginate-Hydroxyethyl cellulose-Calcium/silver nanoparticles (SA/HEC/Ca/AgNPs); Sodium Alginate-Hydroxyethyl cellulose-Calcium/Magnesium-silver nanoparticles (SA/HEC/Ca/Mg/AgNPs)] were performed against the two bacterial strains, viz Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922 and two fungal strains, viz Candida albicans ATCC 14053, Candida kruzei ATCC 6258. All the cultures were resourced from the microbiology laboratory, Natural and Medical Sciences Research Center, University of Nizwa, Oman. Well-diffusion technique^26,27 on 3.8% Muller–Hinton agar (MHA) (Liofilchem, Teramo, Italy) media plates was adopted. Two -three morphologically identical colonies from each of the bacterial and fungal subculture plates were suspended in 5 mL physiological saline (0.9%) followed by the adjustment of their concentration to 0.5% McFarland standard. Following this, 200 µL of each of these solutions were spread on MHA media plate with a sterilized cotton swab. Further, the media plates were bored (bore size = 5 mm) with wells with a sterile metallic plug borer and filled with 20 µL of each of the diluted (twofold) and undiluted compounds (50%). Ciprofloxacin/Amoxycillin and amphotericin B(S) served as positive control for bacteria and fungus, respectively, while sterile distilled water was used as negative control in both the cases. Bacterial and fungal plates were incubated at 37 °C and 25 °C, respectively in the upright position until zones of inhibition were observed, which was considered an endpoint parameter for the antimicrobial activities. The diameter of the inhibition zones was measured in millimeter using the glass scale.

Results and discussion

Morphology of the SA/HEC hydrogels

The bivalent cations Ca²⁺, Mg²⁺ and Ca²⁺/Mg²⁺ were chosen for this study. The SA/HEC hydrogels were cross-linked by Ca²⁺ and also by a mixture of Ca²⁺/Mg^2+. The results showed that the Mg²⁺ failed to cross-link the SA/HEC hydrogels. As reported by Topuz et. al²⁸, Mg²⁺ ions cannot induce gelation in sodium alginate because they have less strong bonds between the ions and carboxylate groups. To produce SA/HEC hydrogels in various forms like gels, beads, sponges and films the SA/HEC hydrogels were cross-linked by Ca²⁺ ions only. Figure 1. shows the scanning electron microscope images of the SA/HEC hydrogel cross-linked by Ca²⁺ ions in various forms like beads, films, and sponges. The morphology of the surface of the beads shows that it is porous with pore size ranging from approximately 2–20 µm. SA/HEC hydrogel formed free-standing films. The thickness of the film is approximately 150 µm. The calcium crystals are seen to be uniformly spread over the surface of the film.

Fig. 1.

SEM images of the SA/HEC hydrogels cross-linked by Ca²⁺, in various forms. The inset shows the photographs of the various forms of the hydrogels.

The sponges show beautifully organized pores. The pore size is approximately 200–700 µm, showing that the pores were mesoporous, which is a very important parameter for an excellent scaffold in bone tissue engineering^29,30. The calcium crystals are seen inside and along the walls of the pores. Freeze-dried samples have a highly porous structure with an expanded state of the polymeric chains, which is reported by several researchers^31,32. All these images show that SA/HEC hydrogels cross-linked by Ca²⁺ could be used to form excellent scaffold materials for tissue engineering applications in various forms.

Physical attributes of the SA/HEC hydrogel

The transparency of the SA/HEC hydrogel films was affected by cross-linking by various metal ions. The films became less transparent with the increase in the cross-linking efficiency. SA/HEC/ Ca²⁺ showed less transparency compared to SA/HEC/ Ca²⁺/Mg²⁺, which in turn was less transparent compared to SA/HEC/Mg²⁺_. The photographs of the films of SA/HEC/X²⁺ are shown in Fig. 2. The stiffness of the films decreased in the order of SA/HEC/ Ca²⁺ > SA/HEC/ Ca²⁺/Mg²⁺ > SA/HEC/ Mg²⁺, while the flexibility of the films followed the reverse order. It can be seen from Fig. 2, that the SA/HEC/ Mg²⁺ dried films could not be peeled to get a completely free-standing film. The effect of different ions on the transparency of the films, during cross-linking can be clearly seen in Fig. 3. There is no gelation with Mg²⁺ and the hydrogel is very clear. With Ca²⁺ the cross-linking is very strong, and the lumps formed reduces the transparency whereas with Ca²⁺/Mg²⁺ the SA/HEC is cross-linked and the transparency is intermediate.

Fig. 2.

Photographs of the dried SA/HEC films cross-linked by Mg²⁺ Ca²⁺/ Mg²⁺ and Ca²⁺ ions.

Fig. 3.

Photographs of the SA/HEC hydrogels cross-linked by Mg²⁺ Ca²⁺/ Mg²⁺ and Ca²⁺ ions.

Evaluation of FTIR spectroscopy analysis data.

The FTIR spectra of the SA/HEC, SA/HEC/Ca and SA/HEC/Ca/Mg, are shown in Fig. 4. Absorption bands seen around 3300–3700 cm⁻¹ are the characteristic absorption bands of the stretching vibrations of the –OH groups of the polysaccharides of sodium alginate and hydroxyethyl cellulose^33,34. The C=O asymmetric stretching and the –COO– symmetric stretching vibrations of sodium alginate are seen at 1613 cm⁻¹ and 1415 cm⁻¹ respectively. The absorption bands seen at 2900 cm⁻¹, 1612 cm⁻¹ , and 1055 cm⁻¹ corresponds to –C–H– stretching vibration, C=O stretching vibration and C–O–C stretching vibration respectively. The strong peaks at 1012 cm⁻¹ and 952 cm⁻¹ are attributed to the stretching of C–O and C–C groups respectively^35,36.

Fig. 4.

The FTIR spectra of the SA/HEC, SA/HEC/Ca/Mg, SA/HEC/Ca.

The absorption bands after cross-linking are seen at 1061 cm⁻¹ and 611 cm⁻¹. The absorption bands of the stretching vibrations of the –OH groups of the polysaccharides are reduced after cross-linking. These hydrogels are formed by two types of interactions, hydrogen bonding (between SA and HEC) and ionic interactions (between SA and Ca²⁺ and Mg²⁺ ions). There are clear chemical shifts for the –OH stretching vibrations, C=O asymmetric stretching and the –COO– symmetric stretching vibrations after cross-linking with Ca²⁺ and Ca²⁺/ Mg²⁺. The shift is more pronounced for the hydrogels cross-linked with Ca²⁺ indicating that Ca²⁺ binds more strongly to the hydrogels compared to Ca²⁺/ Mg²⁺.

Mechanical properties of the hydrogels.

Among the various factors that affect the mechanical properties of the hydrogel films, the cross-linking agent and the polymer composition play very important roles. The tensile strength of the SA/HEC hydrogels cross-linked with different bivalent cations is shown in Table 1. The Young’s modulus for the SA/HEC/Ca film was better than SA/HEC/ Ca/Mg. With Ca²⁺ the films were harder compared to the films with Ca²⁺/Mg²⁺. The polymer chains of the SA/HEC hydrogels are organized into electronegative cavities resulting in the egg-box shape, upon cross-linking with Ca ions³⁷. More organized structure with Ca²⁺ compared to Ca²⁺/Mg²⁺ could have led to the toughening of the hydrogels resulting in the increased tensile strength of the SA/HEC/ Ca compared to the SA/HEC/ Ca /Mg.

Table 1.

Mechanical characteristics for SA/HEC/Ca, and SA/HEC/Ca/Mg films.

Hydrogel film	Modulus (MPa)	Tensile strength (MPa)	Elongation at break (%)
SA/HEC/Ca	138	0.90	6.15
SA/HEC/Ca/Mg	118	0.19	11.4

Evaluation of scanning electron microscopy and EDX analysis data.

The morphology of the cross-linked SA/HEC hydrogels is a very important parameter to be studied. Morphology is generally affected by the synthesis method, cross-linking density, and interfacial tension³⁸. Since the scaffolds were non-conducting, they were coated with gold. The micrographs of SA/HEC/Ca and SA/HEC/Ca/Mg hydrogels are shown in Fig. 4. The crystals with various morphologies were seen. As seen in the figure, plenty of crystals are there for both SA/HEC/Ca and SA/HEC/Ca/Mg hydrogels. Figure 5A shows the SEM image of SA/HEC/Ca. The length and breadth of the crystals were approximately 5–7 µm and 0.3–0.6 µm respectively. The crystal morphologies for SA/HEC/Ca/Mg is shown in Fig. 5B, in which the crystals were in the size ranging from 3 to 6 µm.

Fig. 5.

SEM images of the hydrogels (A) SA/HEC/Ca and (B) SA/HEC/Ca/Mg.

The EDX spectra of the SA/HEC/Ca/Mg and SA/HEC/Ca hydrogels films are shown in Fig. 6. The EDX spectra of SA/HEC/Ca and SA/HEC/Ca/Mg confirm the presence of Ca and Mg ions respectively. In both the spectra peaks of Au, C, O and Cl are seen. These peaks are from the SA and HEC polymers and the Au peaks have been detected due to the gold coating.

Fig. 6.

EDX analysis of (A) SA/HEC/Ca/Mg and (B) SA/HEC/Ca hydrogels.

Water stability of the SA/HEC beads crosslinked by Ca²⁺ and co-crosslinked by Ca²⁺/Mg²⁺

The water stability results of the SA/HEC beads crosslinked by Ca²⁺ and co-crosslinked by Ca²⁺/Mg²⁺ are shown in the photographs, Fig. 7. All the beads showed excellent stability except for the one cross-linked by magnesium ions. It is well known that the ion affinity for various bivalent cations towards alginate varies, in which the highest affinity is shown for Pb²⁺. Haug et. al., and Smidsrod et. al., reported the following ion affinity, Pb²⁺ > Cu²⁺ > Cd²⁺ > Ba²⁺ > Sr²⁺ > Ca²⁺ > Co²⁺, Ni²⁺, Zn²⁺ > Mn^2+39,40. Alginate consists of M-unit: β-D-mannuronic acid and G unit: α-L-guluronic acid⁴¹. A previous study¹⁹, reported the affinity of some more cations as, GG-blocks: Ba²⁺ > Sr²⁺ > Ca²⁺ > Mg²⁺ MM-blocks: Ba²⁺ > Sr²⁺ ~ Ca²⁺ MG-blocks: Ca²⁺ > Sr²⁺ ~ Ba²⁺. In general it is shown that SA has higher affinity for Ca²⁺ than Mg²⁺ ions which is also confirmed in our study.

Fig. 7.

Water Stability of the SA/HEC/Ca/Mg and SA/HEC/Ca beads.

X-ray Diffraction (XRD) analysis

Figure 8, shows the XRD diffractogram of the SA/HEC before and after cross-linking. The semi-crystalline nature of SA/HEC is shown at diffraction peaks 2θ = 13.8°, 21.16° and 42.70°, (Fig. 8.A) which is consistent with the reported data^42,43. The XRD diffractogram of the SA/HEC cross-linked with calcium and a mixture of calcium and magnesium, (Fig. 8B and C), respectively. For both the SA/HEC/Ca and SA/HEC/Ca/Mg, diffraction peaks ranging from 20 to 80° are seen. The XRD pattern of SA/HEC/ Ca shows diffraction peaks from crystal planes (100) (101) (102) (110) and (111) with a maximum peak from (101) crystal plane at 2-Theta of 33.017 degrees and SA/HEC/ Ca/Mg shows diffraction peaks with reduced intensities^44,45. It could be assumed that calcium ions were predominant in the cross linking of the SA/HEC hydrogels. These reflections confirm the in-situ growth of CaCO₃ and MgCO₃ in the SA/HEC hydrogels by mineralization process⁴⁶.

Fig. 8.

XRD spectra of (A) SA/HEC, (B) SA/HEC/Ca and (C) SA/HEC/Ca/Mg hydrogels.

Thermogravimetric analysis-TGA

The TGA analysis curves were recorded to investigate the mass loss during pyrolysis of SA/HEC, SA/HEC/Ca and SA/HEC/Ca/Mg hydrogels. The TGA curves of the SA/HEC (Fig. 9A), SA/HEC/Ca/Mg (Fig. 9B) and SA/HEC/Ca (Fig. 9C) are shown in Fig. 9. The decline in the TGA plots below 100 °C is generally attributed to the elimination of water molecules that are attached to SA and HEC. Peaks at degradation around 230 °C–250 °C are due to the destruction of glycosidic bonds and degradation of SA and HEC^47,48. After cross-linking with the Ca²⁺ and Ca²⁺/Mg²⁺ ions the moisture adsorption capacity was reduced thereby increasing the degradation temperature. After cross-linking all the temperatures were higher at the degradation phases showing that the thermal stability is increased after cross-linking. The degradation temperatures are shown in Table 2.

Fig. 9.

Normalized TGA curves (A), SA/HEC (B) SA/HEC/ Ca/Mg (C) SA/HEC/Ca.

Table 2.

TGA data before and after cross-linking SA/HEC hydrogels.

Sample	Region of decomposition	Temperature Tpeak
SA/HEC	1	50.83
	2	215
	3	625.35
SA/HEC/Ca	1	51.23
	2	225
	3	629.85
SA/HEC/ Ca/Mg	1	63.83
	2	230
	3	640.02

UV–Vis spectroscopy

In this reaction HEC as well as SA act as the reducing as well as stabilizing agent. The plenty of OH groups in HEC and SA are used to reduce the Ag ions to metallic silver particles. In this reaction the color of the solution changed from colorless to pale yellow in less than 5 min and then to dark yellow, and finally to brown color after 30 min which confirmed the formation of silver nanoparticles^49,50. The synthesis used only water as the solvent and followed a green chemistry approach of forming the silver nanoparticles. Figure 10 shows the UV–Vis spectrum of the SA/HEC/AgNPs, the inset shows the photograph of the SA/HEC/AgNPs hydrogel. The absorption peak was observed at 416 nm indicating the formation of silver nanoparticles⁵¹.

Fig. 10.

UV–Visible absorption spectrum of the SA/HEC/AgNPs hydrogel.

Evaluation of scanning electron microscopy and EDX analysis data for the SA/HEC/AgNPs hydrogel cross-linked by Ca²⁺ and a mixture of Ca²⁺ and Mg²⁺

The morphology of the SA/HEC/AgNP/Ca hydrogel beads were examined by using the SEM operating at 20 kV accelerating voltage. The beads were coated with a thin layer of gold using sputtering technique. The micrographs are shown in Fig. 11A. The presence of spherical particles as well as cubic particles were seen. The spherical particles with diameter in the range of 50–200 nm indicates the silver nanoparticles and the cubic particles in the size ranging from 1 to 5 µm indicates the calcium crystals. The EDX shown in Fig. 11B confirms the presence of Ag and Ca ions. Similar results were observed for SA/HEC/AgNP/Ca /Mg in which the size of the crystals from Ca and Mg were in the range of 1–5 µm, Fig. 11C and the EDX confirms the presence of Ca, Mg and Ag ions, Fig. 11D.

Fig. 11.

SEM images of the SA/HEC/AgNP/ hydrogels cross-linked by (A) Ca²⁺ ions, (C) Ca²⁺/Mg²⁺ ions and the EDX of SA/HEC/AgNP/Ca hydrogels (B), and SA/HEC/AgNP/Ca/Mg hydrogels (D).

Antimicrobial activities of alginate and cellulosic derived compounds

The E. coli ATCC 25922 showed susceptibility against all the diluted (except for SA/HEC/AgNPs) and undiluted compounds with the zones of inhibition ranging from 15 to 24 mm. All the compounds (except for SA/AgNPs and the undiluted sample of SA/HEC/AgNPs) exhibited antibacterial activities against S. aureus ATCC 29213 with the maximum and minimum zone of inhibition observed as 19 and 11 mm, respectively. Notably, strong antifungal activities (maximum zone of inhibition: 45 mm; minimum zone of inhibition: 25 mm) of all the tested compounds were observed against both the fungal strains of Candida. Concomitantly, all the positive controls [Ciprofloxacin and amphotericin B(S)] exhibited zones of inhibitions, while none of the negative control (sterile water) showed effects (Fig. 12; Table 3).

Fig. 12.

Media plates showing the inhibition zone across a range of compounds [Sodium Alginate–Silver nanoparticles (SA/AgNPs); Hydroxyethyl cellulose–silver nanoparticles (HEC-AgNPs); Sodium Alginate–Hydroxyethyl cellulose–silver nanoparticles (SA-HEC-AgNPs); Sodium Alginate/Hydroxyethyl cellulose–Calcium/silver nanoparticles (SA/HEC/Ca/AgNPs); Sodium Alginate–Hydroxyethyl cellulose/Calcium/Magnesium/silver nanoparticles (SA/HEC/Ca/Mg/AgNPs)] against the bacterial strains E. coli ATCC 25922 (A–E), S. aureus ATCC 29213 (F–J) and fungal strains C. albicans ATCC 14053(K) and C. kruzei ATCC 6258 (L). The red and blue arrow heads are designated for undiluted and diluted compounds, respectively, while the black indicates the Ciprofloxacin/Amoxycillin positive control for bacteria and amphotericin B(S) positive control for fungi. The yellow arrow heads specify the sterile water negative control.

Table 3.

Antimicrobial activities of all the tested compounds.

Microorganisms	E. coli ATCC 25,922		S. aureus ATCC 29,213		C. albicans ATCC 14,053	C. kruzei ATCC 6258
	Zone of inhibitions (mm)
Positive controls	29		36		40	35
Negative controls	–	–	–	–	–	–
Compounds	Undiluted	diluted	Undiluted	diluted	Undiluted	Undiluted
aSA/AgNPs	20	18	–	–	25	25
bHEC/AgNPs	22	15	16	15	40	40
cSA/HEC/AgNPs	14	–	–	11	25	30
dSA/HEC/Ca/AgNPs	23	24	19	18	45	45
eSA/HEC/Ca/Mg/AgNPs	21	18	11	19	30	35

^aSodium Alginate–Silver nanoparticles; ^bHydroxyethyl cellulose–silver nanoparticles; ^cSodium Alginate–Hydroxyethyl cellulose–silver nanoparticles; ^dSodium Alginate–Hydroxyethyl cellulose–Calcium/silver nanoparticles; ^eSodium Alginate–Hydroxyethyl cellulose–Calcium/Magnesium–silver nanoparticles.

Ca²⁺ is an excellent cross-linker for SA/HEC forming strong hydrogels it is advantageous to use Ca²⁺/Mg²⁺ in terms biomedical applications since magnesium is a very important component of bone and plays a significant role in bone metabolism and also many studies have shown the role of magnesium in stabilizing the calcium compounds^52,53.

Conclusions

This study successfully developed highly stable sodium alginate and hydroxyethyl cellulose (SA/HEC) hydrogels in various forms such as beads, sponges, and films using a green chemistry approach with water as the sole solvent. Cross-linking was achieved using bivalent cations, specifically Ca²⁺, Mg²⁺, and Ca²⁺/Mg²⁺. Notably, effective cross-linking only occurred with the combination of Ca²⁺/Mg²⁺, as Mg²⁺ alone failed to cross-link the hydrogel. Characterization using FTIR, SEM, EDX, and XRD confirmed successful crystallization and cross-linking, with enhanced thermal and mechanical properties. SA/HEC sponges displayed high porosity (200–700 µm), and beads exhibited rough surface pores (2–20 µm). In a single, efficient step, biocompatible and biodegradable scaffolds containing both calcium and magnesium were created. The synthesized SA/HEC/Ca and SA/HEC/Ca/Mg composites, along with various silver nanoparticle formulations, demonstrated effective antimicrobial properties against E. coli ATCC 25922, S. aureus ATCC 29213, C. albicans ATCC 14053, and C. krusei ATCC 6258. This straightforward, eco-friendly approach produces highly stable, antimicrobial, co-crosslinked SA/HEC hydrogels with excellent porosity, positioning them as promising scaffold materials for tissue engineering applications.

Author contributions

F.S. J. H. contributed to the concept of this article, Supervision, Data Curation, Visualization, Methodology, Writing—Original Draft, Data Curation, Visualization, Supervision; S.G. Writing—Original Draft, Data Curation, Visualization; H.S.O.S. Data Curation, Visualization, Methodology, M.S. contributed to Data Curation, Visualization, I.A contributed to Data Curation, Visualization, A.Ha. contributed to Visualization, Conceptualization, A. A: Visualization, Conceptualization, A.Hi contributed to Data Curation, Visualization, Conceptualization, Methodology, S.K. contributed to Data Curation, Visualization, Conceptualization, Methodology, Abdullah M.S. Al-Hatmi: Data Curation, Visualization, Conceptualization, Methodology.

Funding

This work was supported by the TRC research grant, (BFP/GRG/HSS/23/173). The authors acknowledge the support from the Department of Biological Sciences and Chemistry in the Faculty of Arts and Sciences at the University of Nizwa.

Data availability

Further inquiries can be directed to the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.