Trends Sci. 2025; 22(10): 10418

Hydrogels are highly hydrated materials composed of hydrophilic polymers with networks of chemical and/or physical crosslinks between macromolecular chains [1,2]. They are used in various fields, including sensors, biomaterials, drug delivery, water treatment, tissue engineering, and soft electronics [3-7]. In wound care, hydrogels offer several advantages: they absorb exudates, maintain a moist environment, are easy to remove, and are biocompatible and safe [1,2].

Several benchmark studies have demonstrated the potential of hydrogel-based wound dressings to enhance healing rates, reduce infection, and promote tissue regeneration [8,9]. Recent advances have explored the integration of cell-derived conditioned media (CM) into hydrogel matrices, demonstrating enhanced angiogenesis and accelerated wound repair [8]. Additionally, the development of biopolymer-based smart hydrogels that respond to wound-specific stimuli has shown significant potential in overcoming limitations of traditional dressings [9]. Hydrogels can be synthesized from natural, synthetic, or hybrid polymers [10,11]. Natural polymers like alginate, gelatin, collagen, and chitosan are commonly used, while synthetic polymers include PEG, PEO, PVA, and polypeptides. However, their weak mechanical strength limits traditional hydrogel applications [12].To address this, recent developments include dual cross-linked, double network, and topological hydrogels [13,14]. Tougher hydrogels based on cellulose, chitosan, alginate, and gelatin have been explored [15]. Gelatin (G), a denatured collagen, crosslinks easily and is widely used for its biocompatibility and biodegradability. Sodium alginate (SA) is a polyanionic copolymer that forms gels via ionic interactions with divalent or trivalent cations like Ca²⁺, Cu²⁺, Ba²⁺, and Fe³⁺. Though alginate has useful properties, it lacks biological activity and is often blended with gelatin to improve hydrogel characteristics [16]. For example, Saarai et al. [16] found that a 50:50 SA:G hydrogel crosslinked with Ca²⁺ offered optimal mechanical and swelling behavior. However, monovalent salts can destabilize SA-Ca²⁺ interactions. Yari et al. [17] enhanced SA hydrogel stability using basil seed mucilage (BSM). BSM, derived from Ocimum basilicum L., contains glucomannan, xylan, and uronic acid, offering excellent water absorption [18,19]. It serves as a food stabilizer and was used here to enhance hydrogel strength and swelling [20]. Nanomaterials such as graphene oxide Jing et al. [21], silica Mohammed et al. [22], ZnO/CuO Kaptan et al. [23], and Ag Martinez-Higuera et al. [24] have also been integrated into hydrogels to boost antibacterial activity.

In this study, Cu_1.0-xAgₓO nanoparticles and BSM were incorporated into hydrogel films and evaluated for antibacterial activity, swelling, and mechanical performance. While several studies have incorporated metal oxide nanoparticles into biopolymer-based hydrogels for wound healing, most have focused on well-known polymers such as chitosan, alginate, or gelatin, and single oxide systems like CuO or Ag₂O. In contrast, this study introduces a novel strategy by utilizing basil seed mucilage (BSM)—a natural, underutilized, and highly swellable polysaccharide—as the hydrogel-forming matrix. Additionally, the
Cu_1.0-xAgₓO (0.1 ≤ x ≤ 0.7) nanoparticles represent a tunable mixed metal oxide system that leverages the synergistic antibacterial properties of CuO, Ag, and Ag₂O phases within a single nanocomposite. This approach enables composition-dependent control over antibacterial efficacy and mechanical performance, distinguishing it from previous studies using simple metal oxide blends. To the best of our knowledge, the combination of BSM-based hydrogels with compositional tuning of Cu–Ag oxide nanoparticles has not yet been reported for wound dressing applications, making this study a unique contribution to the development of multifunctional hydrogel material

Materials and methods

Materials

All chemicals were of analytical grade and used without further purification. Deionized water was used throughout. Cu_1.0-xAgₓO (x = 0.0, 0.1, 0.5, 0.9 and 1.0) was synthesized using Cu(NO₃)₂, AgNO₃, NaOH, PEG-4000, and NH₃. Hydrogel materials included sodium alginate, gelatin, glycerol, PEG-6000, NaCl, and CaCl₂. Basil seeds were sourced from a local market in Udon Thani, Thailand. Antimicrobial activity was tested against Bacillus cereus, Streptococcus thermophilus, Staphylococcus aureus, Escherichia coli, Fusarium oxysporum, and Penicillium sp. The selected bacterial strains, including Gram-positive (S. aureus, B. cereus) and Gram-negative (E. coli, S. thermophilus), are clinically relevant pathogens frequently implicated in wound infections. Their inclusion allows for evaluating the broad-spectrum antibacterial potential of the hydrogel composites. Microbial strains were maintained on nutrient agar slants.

Synthesis of Cu_1.0-xAg_xO composite

Cu_1.0-xAg_xO was synthesized using a co‑precipitation method modified from our previous work [25]. First, Cu(NO₃)₂, AgNO₃ and PEG-4000 were individually dissolved in DI water under constant stirring for 15 min. Next, the solution pH was adjusted to 12.0 0.2 using a 1.5 M NaOH solution. Subsequently, the mixed solution was stirred vigorously for 30 min until dark precipitates were obtained. After that, the precipitates were filtered using Whatman No. 93 filter paper and washed repeatedly with DI water. Then, the obtained particles were oven dried at 110 °C for 5 h and calcined at 600 °C for 4 h. For simplification, Cu_1.0-xAg_xO where x = 0.0, 0.1, 0.3, 0.5, 0.7 and 0.9 are referenced as Cu_1.0O, Cu_0.9Ag_0.1O, Cu_0.7Ag_0.3O, Cu_0.5Ag_0.5O, Cu_0.3Ag_0.7O and Cu_0.1Ag_0.9O, respectively. XRD, XPS and antimicrobial activities of the nanocomposites were further investigated. The term "CuAgO" is used solely as a general abbreviation when referring to the composite group. The highest antimicrobial activity from Cu_1.0‑xAg_xO (Cu_0.7Ag_0.3O), was used in fabrication of a Cu_0.7Ag_0.3O-loaded hydrogel film.

Preparation of BSM from basil seeds

BSM was prepared using a hydration method. First, 10 g of basil seeds were soaked in 300 mL of DI water at 40 °C for 1 h. Next, the swollen seeds were broken in a blender for 1 min and then filtered using cheese cloth. After that, the mucilage was centrifuged at 5000 rpm for 5 min to remove seed residues. Then, the BSM mucilage was oven dried at 50 °C for 24 h. Finally, brown particles of BSM were obtained and kept in a desiccator.

Preparation of BSM/HG and Cu_0.7Ag_0.3O-loaded BSM/HG

Hydrogel films with BSM concentrations of
0.0 – 0.9 %w/v were prepared following Saarai et al. [16] with slight modifications. Dried BSM was soaked in 200 mL DI water at 80 °C. Alginate (2.0% w/v), gelatin (2.0 %w/v), glycerol (2.0 %w/v), PEG-6000
(1.0 %w/v), and NaCl (0.2% w/v) were added and stirred for 30 min. The gel was cast onto a 25.4×25.4×0.2 cm aluminum plate and dried for 1 h. A 5.0 %w/v CaCl₂ solution was added for 15 min. Films were washed and labeled as HG, 0.1BSM/HG to 0.9BSM/HG. Mechanical and swelling properties were evaluated.

Cu_0.7Ag_0.3O loaded BSM/HG was prepared in a manner similar to BSM/HG. Cu_0.7Ag_0.3O particles with concentrations of 3.75, 7.50 and 15.0 mg/mL were added to the 0.5 %w/v BSM solution. Then, alginate, gelatin, glycerol, PEG-6000 and NaCl were added to the mixture. Hydrogel films with different concentrations (0.00, 3.75, 7.50 and 15.0 mg/mL) of Cu_0.7Ag_0.3O are referenced as BSM/HG, 3.75Cu_0.7Ag_0.3O/BSM/HG, 7.50Cu_0.7Ag_0.3O/BSM/HG and 15.0Cu_0.7Ag_0.3O/BSM/HG, respectively.

Characterization techniques

The crystal structure of CuAgO particles was analyzed by X-ray diffraction (XRD, Bruker D8 Advance) using Cu Kα radiation (λ = 1.54184 Å), a 2θ range of 20° – 80°, 40 mA current, and 40 kV voltage. The chemical surface composition was examined by
X-ray photoemission spectroscopy (XPS, Kratos Axis Ultra DLD) with monochromatic Al Kα radiation
(10 mA, 35 kV) under a chamber pressure of 3×10⁻⁹ Torr. The C 1s peak at 285.0 eV was used for calibration, and Cu 2p, Ag 3d, and O 1s peaks were analyzed using a Lorentzian-Gaussian function. Functional groups of Cu_0.7Ag_0.3O/BSM/HG were identified via attenuated total reflectance Fourier-transform infrared spectroscopy (ATR/FT-IR, Spectrum 2, PerkinElmer) in the 4000 – 500 cm⁻¹ range at 4 cm⁻¹ resolution. Surface morphology and elemental composition were observed using SEM-EDX (Hitachi S 3000N, 5 kV). Films were gold-coated with a K500X sputter coater (20 mA, 5 min, 9×10⁻² mbar) before SEM analysis.

Swelling tests

To determine the degree of swelling of the film samples, pre-weighed dried 1.0 1.0 cm film samples (W₁) were immersed in phosphate buffer (pH 7.4) at room temperature. The swollen films were removed after various immersion times, wiped with tissue paper to remove excess surface fluid and weighed (W₂). The percentage swelling ratio (SR, %) was computed after each time as follows:

Tensile properties

Tensile strength and elongation at break of BSM/HG films were measured using a Universal Testing Machine (GT-K01-GESTER) according to SATRA TM5 and ISO 20344 standard. Film strips (1.5×10.0 cm) were tested at a crosshead speed of 20 mm/min using a 50 mm gauge length and 5 kN load cell. All measurements were performed in 5 replicates (n = 5), and the results are reported as mean ± standard deviation.

Antimicrobial activity

The antibacterial and antifungal activities of CuAgO composites were evaluated using the agar well diffusion method. Bacterial strains (Bacillus cereus, Staphylococcus aureus, Streptococcus thermophilus, and Escherichia coli) were cultured in Mueller-Hinton broth (MHB) for 18 h at 37 °C with a 0.5 McFarland standard turbidity. Then, 100 µL of each suspension was spread on MHB agar plates. Wells (6 mm diameter) were created using a sterile cork borer, and 30 µL of CuAgO was added to each. Plates were incubated at 37 °C for 24 h. Tetracycline (50 µg) and dimethyl sulfoxide (DMSO) served as positive and negative controls. Inhibition zones were measured with a micrometer. Antifungal activity against Fusarium oxysporum and Penicillium sp. was tested similarly, using nystatin as a positive control. Antibacterial activity of BSM/HG and Cu_0.7Ag_0.3O/BSM/HG films was tested by disc diffusion. Films (6 mm) and filter paper discs were placed on agar plates and incubated at 37 °C for 24 h.

All experiments for swelling tests and antibacterial activity were conducted in triplicate (n = 3). Results are presented as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA to assess differences between groups.

Determination of minimum inhibition concentration (MIC)

The minimum inhibitory concentration (MIC) is defined as the lowest concentration of a substance that completely inhibits visible bacterial growth after incubation at 37 °C for 24 h. In this study, the MIC values of Cu_0.5Ag_0.5O and Cu_0.7Ag_0.3O against Staphylococcus aureus, Bacillus cereus, and Escherichia coli using a broth dilution method based on the protocol described by Kamonwannasit et al. [26]. Bacterial suspensions were prepared at a concentration of 1 × 10⁷ CFU/mL in Mueller-Hinton Broth (MHB). CuAgO samples were serially diluted to obtain a range of concentrations from 0.47 to 30.00 mg/L. The inoculated tubes was incubated at 37 °C for 24 h, and the MIC was recorded as the lowest concentration showing no visible bacterial growth. All experiments were conducted in triplicate to ensure reproducibility and accuracy of the results.

Results and discussion

XRD and XPS analysis

The crystal structure of CuAgO samples with different compositions calcined at 600 °C for
4 h is shown in Figure 1. The XRD patterns and their corresponding lattice planes of CuO in a Cu_1.0O sample were observed at 2 = 35.78^o ( 111), 38.89^o (111), 48.92^o ( 202), 53.69^o (020), 58.46^o (202), 61.68^o ( 113), 66.47^o (022), 68.37^o (220), 72.79^o (311) and 75.41^o (004) (JCPDS No. 45-0937). This indicates a typical CuO monoclinic structure within the C2/c space group. CuO, Ag and Ag₂O phases were detected in
Cu_1.0-xAg_xO (x = 0.1 - 0.7) samples. This indicates that a CuAgO composite was formed. An Ag₂O phase was observed at 2 = 38.40^o (011) that corresponds to the hexagonal structure of silver oxide within the P3m1 space group (JCPDS No. 72-2108). Metallic Ag was also found at 2 = 38.40^o (111), 44.63^o (200), 64.79^o (220) and 77.56^o (311), which implies the presence of the face centered cubic crystalline structure of silver within the Fm3m space group (JCPDS No. 04-0783). Detection of a CuO phase was impossible due to the very small fraction of copper in Cu_0.1Ag_0.9O samples.

XPS analysis was conducted to confirm the XRD results. This technique is normally used to identify the elements present on the surface of a specimen, as well as the valence states and interactions of each atom. Figure 2 displays the XPS spectra of O 1s, Cu 2p and Ag 3d core levels from the Cu_0.7Ag_0.3O sample. In Cu 2p spectra (Figure 2(a)), characteristic double peaks at 952.72 and 932.93 eV correspond to Cu 2p_1/2 and Cu 2p_3/2, respectively. The 19.8 eV energy gap between these levels is due to spin-orbit coupling and is in good agreement with the literature values [27-30]. Additionally, 2 bands at 942.46 and 961.02 eV can be ascribed to the shake-up satellite peaks of Cu²⁺ in CuO with d⁹ configuration in its ground state. Therefore, the appearance of the satellite peaks indicates the presence of the CuO species in the surface layer.

The XPS spectrum of Ag 3d (Figure 2(b)) reveals 2 peaks at 373.87 and 367.99 eV with an energy gap of 5.88 eV (spin-orbit splitting) indicating Ag 3d_3/2 and Ag 3d_5/2, respectively. This suggests formation of metallic silver (Ag⁰) in the composite. Moreover, the Ag 3d_3/2 and Ag 3d_5/2 core levels shift towards lower binding energies, 373.01 and 367.29 eV, respectively. This is due to the presence of silver ions in Ag₂O (Ag⁺), in agreement with the literature [31,32]. The presence of 53 % metallic Ag relative to the amount of Ag₂O is also in agreement with XRD results. The XPS spectrum of
O 1s (Figure 2(c)) shows 3 peaks at 529.23, 531.67 and 533.14 eV. The peak centered at 529.23 eV corresponds to the O 1s of Cu O or Ag O in the form of CuO and Ag₂O. The peaks at 531.67 and 533.14 eV correspond to oxygen adsorbed on the composite surface [28,32].

Figure 1 XRD patterns of CuAgO composites calcined at 600 °C for 4 h.

Figure 2 (a) Cu 2p, (b) Ag 3d and (c) O 1s XPS spectra of Cu_0.7Ag_0.3O composite calcined at 600 °C for 4 h.

Antimicrobial activity of CuAgO composites

The antibacterial activity of CuAgO at a concentration of 30 mg/mL against 4 bacterial strains was evaluated using an agar well diffusion technique. Table 1 shows the inhibition diameter around wells with CuAgO particles against bacterial strains after 24 h of incubation. A larger diameter of inhibition indicates greater antibacterial activity. As expected, the DMSO negative control was ineffective against all bacterial strains, while tetracycline (50 g/mL), a positive control, was effective with inhibition diameters of
11 29 mm. No strains were inhibited by the Cu_1.0O sample at a 30 mg/mL concentration. Inhibition zones of Cu_0.3Ag_0.7O, Cu_0.5Ag_0.5O, Cu_0.7Ag_0.3O and Cu_0.9Ag_0.1O samples developed inhibition widths ranging from
8 13 mm for all bacterial strains. Moreover, the Gram-negative bacterial strain, Escherichia coli, was only affected by the Cu_0.1Ag_0.9O sample with an inhibition diameter of 7.0 mm. These results show that Gram-positive and Gram-negative bacteria had similar susceptibility.

MIC, a parameter indicating inhibitory efficacy, refers to the lowest concentration of an antibacterial reagent that will prevent bacterial growth after 24 h of incubation. Cu_0.5Ag_0.5O and Cu_0.7Ag_0.3O samples were chosen to determine the MIC values against Staphylococcus aureus, Bacillus cereus and Escherichia coli. The results are shown in Table 2. A MIC of 3.75 mg/mL against Escherichia coli was obtained for the Cu_0.7Ag_0.3O sample, while higher MIC values were observed against Bacillus cereus (7.5 mg/mL) and Staphylococcus aureus (15.0 mg/mL). For the Cu_0.5Ag_0.5O sample, the lowest MIC value, 0.94 mg/mL, was obtained against Bacillus cereus, while a MIC value of 15 mg/mL was found against Escherichia coli and Staphylococcus aureus. The present study revealed that the Cu_0.7Ag_0.3O sample had the optimal Cu:Ag molar ratio, 0.7:0.3, for antibacterial activity. This composition was used in subsequent fabrication of Cu_0.7Ag_0.3O/BSM/HG films. Moreover, antifungal activities of all CuAgO composites were investigated against Fusarium oxysporum and Penicillium sp. These results are shown in Table 1. Nystatin (50 g/mL) was effective against Fusarium oxysporum and Penicillium sp. with inhibition zones of 2.30 and 1.50 mm, respectively. No CuAgO samples were effective against any of the fungal strains tested at 30 mg/mL.

Swelling properties of BSM/HG film

The swelling ratio of hydrogel films is used for measuring their fluid absorbing capacities, which is particularly useful for wound dressing materials. These results are shown in Figure 3. The swelling ratio of HG and 0.1BSM/HG increased gradually with time (Figure 3(A)). For 0.3BSM/HG and 0.5BSM/HG samples, the swelling ratio of the film increased sharply until 120 min of immersion in a buffer solution, and then started to decrease. The decreased swelling ratio is due to dissolution of alginate in the aqueous medium. Extension of the SA chains causes breakdown of the crosslinking bonds in the presence of phosphate anion in the buffer [33]. Thus, a portion of SA may dissolve in PBS medium, resulting in a decreased polymer mass. For 0.7BSM/HG and 0.9BSM/HG samples, the swelling ratio of the films showed similar trends where it rises quickly in the first 90 min, and then slowly increased until 240 min of immersion. After 120 min of immersion (Figure 3(B)), neat HG films exhibited a swelling ratio of 1442 %. Greater BSM filler, up to 0.5 %w/v, increased the film swelling ratio. The 0.5BSM/HG specimen exhibited a 2102 % maximal swelling ratio. Notably, this was higher than that of a commercial sodium alginate hydrogel film (50 1800 %) [34]. Among the tested films, the 0.7BSM/HG and 0.9BSM/HG films showed low swelling ratios, which was presumably due to agglomeration of BSM particles.

Tensile properties of BSM/HG films

Generally, hydrogel films used as wound dressings need better mechanical properties because the wound surface will change with body movements. The mechanical properties of HG and BSM/HG films are shown in Figure 4.

Table 1 Antimicrobial activities of synthesized CuAgO samples against 4 different bacterial and fungal strains.

Table 2 MIC of Cu_0.5Ag_0.5O and Cu_0.7Ag_0.3O against Bacillus cereus, Staphylococcus aureus and Escherichia coli.

Figure 4 Tensile properties of HG, 0.1BSM/HG, 0.3BSM/HG, 0.5BSM/HG, 0.7BSM/HG and 0.9BSM/HG.

Stress-at-break and elongation-at-break of pure HG film were 16 MPa and 37%, respectively. The tensile stress of BSM/HG films increased with BSM content up to 0.5% w/v BSM (31 MPa), and then decreased with increasing amounts of BSM. Dispersion of BSM particles in the HG is important since uniformly dispersed BSM enhances the mechanical properties of a hydrogel. However, additional increases in BSM content cause formation of excessive numbers of BSM particles and subsequent accumulation. Moreover, the elongation-at-break of BSM/HG tended to decrease relative to the amount of BSM present, related to increased tensile stress. Since the 0.5BSM/HG film sample gave better swelling and mechanical properties than other samples, it was used for incorporation with Cu_0.7Ag_0.3O particles, which showed the highest antibacterial activity.

SEM-EDX and FT-IR analysis of Cu_0.7Ag_0.3O/BSM/HG films

SEM images of BSM/HG and 7.5Cu_0.7Ag_0.3O/BSM/HG films with a 5000 magnification are presented in Figure 5. The morphology of the pure HG film was rough with a non-porous structure (Figure 5(a)). For BSM/HG films with various concentrations of BSM, film roughness increases with BSM content (Figures 5(b) - (d)), particularly in 0.7BSM/HG (Figure 5(c)) and 0.9BSM/HG (Figure 5(f)) samples. This could be due to agglomeration of BSM particles. The 0.5BSM/HG film sample seems to have the best composition with smooth and non-porous surfaces, indicating a good distribution of BSM particles in the hydrogel matrix. Moreover, SEM imagery of the Cu_0.7Ag_0.3O/BSM/HG film (Figure 5(g)) showed rough and non-porous surfaces. However, Cu_0.7Ag_0.3O particles were not observed using this technique.

Elemental distribution of Ag and Cu in the 7.5 Cu_0.7Ag_0.3O/BSM/HG film was analyzed using EDS mapping. These results are shown in Figure 6, where Cu and Ag particles in the hydrogel matrix were homogeneously distributed. Moreover, the major constituents of 7.5 Cu_0.7Ag_0.3O loaded BSM/HG films were C, O, Cl and Ca. The presence of C may be due to the carbon tape used on the SEM stub during analysis as well as substances incorporated in the hydrogel films (PEG, sodium alginate, gelatin, BSM). Trace amounts of Na in the samples are due to NaCl used in the preparation of hydrogel film. Trace amounts of Cu and Ag were also detected.

Figure 5 SEM imagery of (a) HG, (b) 0.1BSM/HG, (c) 0.5BSM/HG, (d) 0.7BSM/HG, (e) 0.9BSM/HG and (f) 7.5 Cu_0.7Ag_0.3O/BSM/HG.

Figure 6 EDS mapping displays distribution of Ag and Cu elements in 7.5 Cu_0.7Ag_0.3O/BSM/HG: (a) secondary electron image, (b) the element sensitive map of combined Cu and Ag, (c) the element sensitive map of Ag, (d) the element sensitive map of Cu, and (e) EDS spectrum of 7.5Cu_0.7Ag_0.3O/BSM/HG

Figure 7 shows the FT-IR spectra of a Cu_0.7Ag_0.3O/BSM/HG film. The peaks of all samples are less distinct. The broad peaks between 3000 and 3600 cm^-1, and 1626 cm^-1 correspond to stretching and bending vibrations of the hydroxyl (OH^-) groups of alginate, gelatin, BSM and PEG. The small peak at 2915 cm^-1 is attributed to C H stretching. Peaks at 1640 and 1539 cm^-1 can be ascribed to the C=O and C N stretching vibration of gelatin. Bands at 1640, 1435 and 1038 cm^-1 indicate C OO asymmetric stretching vibrations of free carboxylate, C OO symmetric stretching, and C O C stretching of BSM and alginate, respectively [18,35].