Synthesis, Characterization, Applications and Computational Studies of CA4 Hydrogels Incorporated with ZnO Nanoparticles [CA4-ZnO]

2.1.1. Stage I: Synthesis of Pre-Polymers

Citric acid (CA) was used as the monomer. A quantity of 0.025 mol (4.803 g) of citric acid (CA) was dissolved in 5 mL of ethanol and then transferred into a round-bottom flask fitted with a mechanical stirrer until the monomer was completely dissolved in the ethanol.

Triethanolamine (TEA) [0.025 mol (3.3006 g)], dissolved in 5 mL of ethanol, was added dropwise to the citric acid solution using a dropping funnel. The reaction mixture was stirred at 140 °C for 1 h. The formation of a sticky white gel indicated the formation of the citric acid–triethanolamine (CT) prepolymer).

2.1.2. Stage II: Synthesis of Bio-Polymeric Hydrogels

2-Furoic acid (FA) [0.025 mol (2.802 g)] was dissolved in 5 mL of ethanol, added to the CT prepolymer, and stirred continuously with a mechanical stirrer at 140 °C for 2 h. The formation of a glassy brown gel of citric acid–triethanolamine–2-furoic acid (CTF) confirmed the synthesis of the parent hydrogel. The resultant gel was immersed in pure ethanol for 24 h to remove unreacted monomers and subsequently dried in a vacuum oven at 35 °C for 24 h. A similar procedure was followed by altering the chemical composition of the monomers. Table 1 lists the experimental details of the synthesized biopolymeric hydrogels. Among the different hydrogel series, CA₄ was selected for further investigation based on its superior swelling behavior compared with the other synthesized hydrogels.

2.1.3. Incorporation of Zinc oxide (ZnO) Nanoparticles in CA4 Biopolymeric Hydrogels

A total of 0.5 wt% zinc oxide (ZnO) nanoparticles with a particle size of less than 100 nm were incorporated into the CA₄ biopolymeric hydrogel matrix and stirred continuously at 35 °C for 1 h, resulting in the formation of biopolymeric nanocomposite hydrogels [CA₄-ZnO]. Similarly, 1.0 wt% and 2.0 wt% ZnO nanoparticles were separately incorporated into the CA₄ biopolymeric hydrogels using the same procedure, as shown in Table 2. The ZnO concentrations (0.5, 1.0, and 2.0 wt%) were calculated based on the total weight of the synthesized polymer hydrogel matrix. The samples were named CA₄Z₁, CA₄Z₂, and CA₄Z₃ based on the composition of the CA₄ hydrogel matrix and the ZnO nanoparticles. Here, CA₄ represents the base hydrogel formulation (0.040 + 0.025 + 0.025 mol), Z indicates ZnO nanoparticles, whereas the numbers 1, 2, and 3 correspond to 0.5 wt%, 1.0 wt%, and 2.0 wt% ZnO nanoparticles, respectively.

2.2.1. Fourier Transform Infrared (FTIR) Spectroscopic Analysis

The molecular structure of the CA₄-ZnO nanocomposite was characterized using a Shimadzu 8400S FTIR spectrophotometer. Samples were prepared using the standard KBr pellet method, and the spectra were recorded over the range of 4000–500cm⁻¹.

2.2.2. UV–Vis Spectrophotometry

An ultraviolet–visible double-beam (UV–Vis) spectrophotometer was used to characterize the CA₄-ZnO nanocomposite. Samples of the compound were collected periodically to monitor the completion of the reaction. The sample spectra were recorded over a wavelength range of 190–1100 nm.

2.2.3. Scanning Electron Microscopy–Energy-Dispersive X-ray Spectroscopy (SEM -EDX)

To evaluate the effects of polymer and cross-linker concentrations on the surface morphology, the CA₄-ZnO hydrogel was characterized using scanning electron microscopy (SEM). SEM coupled with energy-dispersive X-ray spectroscopy (EDX) enabled rapid and accurate qualitative and quantitative analysis of the elemental composition, providing valuable insights into the hydrogel structure.

2.2.4. High-Resolution Transmission Electron Microscopy (HR-TEM)

High-resolution transmission electron microscopy (HR-TEM), operated at 200–300 kV, was used to obtain high-resolution images and structural information at the nanoscale.

2.2.5. Thermogravimetric Analysis (TGA)

The thermal properties of the polymeric hydrogels were investigated using simultaneous thermogravimetric analysis (TGA) with an SDT Q600 (TA Instruments). The TGA curves were recorded over a temperature range from room temperature to 500 °C.

2.2.6. Swelling Behavior

A 0.200 g sample of dried hydrogel was immersed in buffer solutions with pH values ranging from 2.0 to 11.0 at room temperature. At regular time intervals, the swollen hydrogels were carefully removed from the swelling medium, gently blotted with filter paper to remove excess surface water, weighed, and then returned to the original swelling medium. This procedure ensured the accurate determination of the water absorption capacity. Finally, excess surface water was removed from the hydrogels with filter paper. The swelling percentage was calculated using Equation (1):

where, W_I is the initial weight of the dried hydrogel and W_Sq is the weight of the swollen sample at equilibrium.

2.3.1. Antibacterial Studies

The antibacterial activity was evaluated using the conventional agar well diffusion method. Each bacterial isolate was cultured in brain heart infusion (BHI) broth and diluted to approximately 10⁵ colony-forming units (CFU)/mL. The bacterial suspensions were flood-inoculated onto the surface of Mueller–Hinton agar and allowed to dry. Sterile cork borers were used to create wells with a diameter of 5 mm in the agar medium. Subsequently, 30 µL of the sample solution (50 µg compound dissolved in 500 µL DMSO) was placed in each well. The plates were incubated at 37 °C for 18 h. Antimicrobial activity was evaluated by measuring the diameter of the inhibition zone. Dimethyl sulfoxide (DMSO) was used as the solvent control, while ciprofloxacin was used as the standard antibacterial agent. The tests were performed in triplicate. The hydrogels were tested against Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae, and Escherichia coli.

2.3.2. Antifungal Studies

The antifungal activity was evaluated against C. albicans and A. niger. Clotrimazole (20 mg/well) was used as the standard. The diameter of the inhibition zone (mm) was used as an indicator of activity against the test pathogens.

2.3.3. Cytotoxic Studies

The mouse fibroblast cell line (L929) was seeded in a 96-well plate at a concentration of 1 × 10⁴ cells/well in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1× antibiotic–antimycotic solution. The cells were incubated in a CO₂ incubator maintained at 37 °C with 5% CO₂. After washing with 200 μL of 1× phosphate-buffered saline (PBS), the cells were cultured for 24 h in serum-free medium containing different concentrations of the test sample, with 25% DMSO used as the positive control. At the end of the treatment period, the culture medium was aspirated from the cells. MTT solution [0.5 mg/mL of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] prepared in 1X PBS was added, and the cells were incubated for 4 h at 37 °C in a CO₂ incubator. After the incubation period, the medium containing MTT was discarded, and the cells were rinsed with 200 μL of PBS. The formed crystals were dissolved in 100 μL of DMSO and mixed thoroughly. The formation of purple-blue formazan dye was quantified by measuring the absorbance at 570 nm using a microplate reader.

2.3.4. Antioxidant Studies

Blois’s (1958) method was used to measure the sample’s DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity. A 0.5 mL aliquot of the sample solution in methanol was mixed with 2.5 mL of a 0.5 mM methanolic DPPH solution. The reaction mixture was shaken well and kept in the dark at room temperature for 30 min. The absorbance was measured at 517 nm using a UV spectrophotometer. Ascorbic acid was used as the positive control. The percentage of DPPH free radical scavenging activity was determined using the standard formula.

2.3.5. Computational Studies

A preliminary computational study was conducted to investigate the antibacterial activity and protein-binding properties of CA₄ hydrogels and ZnO nanoparticle-incorporated hydrogels. Therefore, the CA₄-ZnO nanocomposite hydrogels were subjected to molecular docking studies.

3.1.1. FTIR Spectroscopy of CA4-ZnO Nanocomposite Hydrogels

A stretching frequency observed at 3360 cm⁻¹ corresponds to the hydrogen-bonded O-H stretching vibration [19,20]. The decrease in the stretching frequency from 3414 cm⁻¹ to 3360 cm⁻¹ may be attributed to the incorporation of ZnO nanoparticles into the polymer network. The peak at 2982 cm⁻¹ represents the C-H stretching vibration. A distinct sharp peak at 1732 cm⁻¹ is ascribed to C=O stretching [21]. New absorption peaks at 1610 cm⁻¹ and 1474 cm⁻¹ were attributed to COO⁻ stretching vibrations. The prominent spectral peak at 1294 cm⁻¹ is due to the presence of C-N stretching vibrations found in triethanolamine. The C-O stretching vibration was observed at 1176 cm⁻¹ in the polyester network. The peaks at 1073 cm⁻¹ and 1041 cm⁻¹ were attributed to C-O-C stretching vibrations. The spectral peak appeared at 670 cm^−1, confirming the incorporation of zinc oxide nanoparticles into the main chain of hydrogels, as shown in Figure 1 [22].

3.1.2. UV Spectroscopy of [CA4-ZnO] Nanocomposite Hydrogels

UV–Vis spectroscopy is a key analytical technique used to characterize citric acid-, 2-furoic acid-, and triethanolamine-based hydrogels incorporated with zinc oxide (ZnO) nanoparticles. This method helps confirm the incorporation of ZnO nanoparticles into the hydrogel matrix. Figure 2 shows the UV–Vis spectral findings of CA₄-ZnO nanocomposite hydrogels. ZnO nanoparticles absorb UV–Vis light over the wavelength range of 250–800 nm [23]. The UV-Visible spectrum of the CA₄-ZnO nanocomposite revealed a broad absorption peak at 300 nm, corresponding to an absorbance value of 0.1. The absorbance of a nanocomposite hydrogel is caused by n→π* transitions. The λmax primarily corresponds to chromophores containing carbonyl groups. This transition involves the promotion of an electron from a nonbonding (n) orbital, specifically a lone pair on a heteroatom such as oxygen, to an antibonding (π*) orbital.

3.1.3. SEM and EDX Analysis of CA4-ZnO Nanocomposite Hydrogels

The surface morphology of the synthesized CA₄-ZnO nanocomposite hydrogel was examined using the CA₄Z₃ sample by scanning electron microscopy (SEM). The SEM image (Figure 3) reveals a heterogeneous, nonporous surface morphology with a rough structure attributed to the hydrogel matrix containing ZnO nanoparticles, which are irregularly distributed throughout the network. The interaction between the hydrogel polymer chains and ZnO nanoparticles enhances the swelling ability and functional properties of the hydrogel.

To validate the synthesis of the CA₄-ZnO nanocomposite hydrogel CA₄Z₃, EDX analysis was performed, and the resulting peaks are shown in Figure 4. The EDX analysis confirmed the presence of ZnO nanoparticles in the synthesized nanocomposite. The elemental composition of the nanocomposite, expressed in weight percentage, revealed the presence of carbon (C), oxygen (O), nitrogen (N), and zinc (Zn) at 56.16%, 30.69%, 3.29%, and 7.51%, respectively. The plot of CA4-ZnO nanocomposite hydrogels shows the presence of carbon, nitrogen, oxygen, and zinc (Table 3).

3.1.4. HR-TEM Analysis of CA4-ZnO Nanocomposite Hydrogels

The size and structure of nanoparticles within the hydrogel network were examined using transmission electron microscopy (TEM). The TEM images showed that the spherical ZnO nanoparticles were uniformly distributed within the CA₄-ZnO hydrogel matrix. The average particle size of the CA₄-ZnO nanocomposite was approximately 38 nm (Figure 5). The well-defined spherical morphology of the ZnO nanoparticles indicates strong interactions between the hydrogel matrix and ZnO nanoparticles. Furthermore, the presence of lower concentrations of spherical ZnO nanoparticles enhanced the biological activity, as evidenced by the antibacterial studies [24].

3.1.5. TGA of [CA4-ZnO] Nanocomposite Hydrogels

Thermogravimetric analysis (TGA) was performed to investigate the thermal stability of the CA₄-ZnO nanocomposite hydrogel, as shown in Figure 6. The TGA curve shows three stages of degradation. The initial stage of degradation occurred between 50 °C and 100 °C, resulting in a 9% weight loss due to the removal of residual water. The second stage of degradation occurred between 150 °C and 250 °C, resulting in a 23% weight loss due to the decomposition of 2-furoic acid substituents from the hydrogel matrix (Figure 6). The third stage of degradation was observed at 300 °C, resulting in a 16% weight loss due to the decomposition of the polymer network containing embedded ZnO nanoparticles [25]. Overall, the TGA study revealed that the ZnO nanocomposite hydrogel exhibited considerable thermal stability, with several degradation stages involving water loss and polymer decomposition. In general, ZnO nanoparticles exhibit high thermal stability under normal conditions. However, their stability can be affected by factors such as impurities, interactions with other materials, and exposure to high temperatures, which may lead to structural degradation. In the present study, the interaction between the biopolymeric hydrogel matrix and ZnO nanoparticles resulted in reduced thermal stability, likely due to polymeric filler interactions [26].

3.1.6. Swelling Equilibrium (%) of CA4-ZnO Nanocomposite Hydrogels

ZnO nanoparticles at concentrations of 0.5%, 1.0%, and 2.0%, designated as CA₄Z₁, CA₄Z₂, and CA₄Z₃, respectively, were incorporated into CA₄ hydrogels. The swelling behavior of the CA₄-ZnO nanocomposite hydrogels at various pH values (2.0, 4.0, 7.0, 9.0, and 11.0) was evaluated, and the results are presented in Table 4. The hydrogels exhibited pH-dependent swelling, with maximal swelling observed under neutral to slightly alkaline conditions (pH 7–9), owing to the ionization of functional groups such as -COOH and -OH in the polymeric matrix, which increases electrostatic repulsion and favors higher water uptake. CA₄Z₃ had the largest swelling percentage (127% at pH 7), followed by CA₄Z₂ (121%) and CA₄Z₁ (117%), as shown in Figure 7. Increasing the concentration of ZnO nanoparticles appears to enhance the swelling capacity, which may be attributed to their interactions with the hydrophilic functional groups within the hydrogel network. At acidic pH (2–4), the swelling ratios decreased due to protonation of functional groups, which promoted hydrogen bonding and network contraction. At a higher alkaline pH of 11, swelling was partially reduced due to charge shielding and partial degradation of the hydrogel network [27]. Overall, the swelling investigation demonstrates that the incorporation of ZnO nanoparticles into the hydrogel is advantageous for applications in drug delivery, wound dressing, and biomedical systems requiring controlled release under physiological pH conditions [28]. The biological activities in the present study were evaluated using CA₄Z₃ nanocomposite hydrogels due to their favorable swelling behavior.

3.2.1. Antibacterial Activity

CA₄Z₃ outperformed all other [CA₄-ZnO] hydrogel compositions in terms of antibacterial performance, as shown in Figure 8. The antibacterial activity was evaluated against Gram-positive bacterial strains (S. aureus and B. subtilis) and Gram-negative bacterial strains (E. coli and K. pneumoniae), using ciprofloxacin as the standard reference drug. The standard drug ciprofloxacin produced inhibition zones of 25 mm against S. aureus, 18 mm against B. subtilis, 20 mm against K. pneumoniae, and 30 mm against E. coli. The CA₄Z₃ hydrogels exhibited inhibition zones of 0 mm against E. coli, 8 mm against S. aureus, 10 mm against B. subtilis, and 6 mm against K. pneumoniae (Figure 8). These findings indicate that the hydrogel exhibited moderate inhibitory activity against S. aureus, B. subtilis, and K. pneumoniae, whereas E. coli showed no antibacterial activity, which may be attributed to variations in cell membrane permeability toward the antibacterial agent [29]. The obtained results demonstrate that CA₄Z₃ hydrogels exhibited moderate inhibitory effects, indicating their potential for biological applications.

3.2.2. Antifungal Activity

The hydrogels were evaluated for antifungal activity against fungal strains, including Candida albicans and Aspergillus niger, using the agar well diffusion method. The standard antifungal drug clotrimazole exhibited inhibition zones of 16 mm against Candida albicans and 26 mm against Aspergillus niger. In comparison, the CA₄Z₃ hydrogel at a concentration of 300 mg/well produced a 4 mm inhibition zone against Candida albicans, while no inhibition (0 mm) was observed against Aspergillus niger (Figure 9). These findings revealed that the antifungal activity of the hydrogel was significantly lower than that of the standard drug. The limited inhibition may be attributed to the robust chitinous cell walls and melanin pigments present in these fungi, which enhance resistance to oxidative stress and reduce the susceptibility of the cells to ZnO nanoparticle attack [30]. Nevertheless, the incorporation of ZnO nanoparticles into polymeric hydrogel matrices may provide a synergistic antifungal platform with potential applications in biomedical and agricultural fields.

3.2.3. Cytotoxicity Activity

Several studies have investigated the cytotoxicity of ZnO nanoparticles using the MTT assay, demonstrating their effects on a wide range of animal cell types [31]. Figure 10 shows the cell viability% of CA₄Z₃ nanocomposite hydrogels. The concentrations of 25, 50, 100, 250, and 500 µg/mL yielded percentages of 95, 90, 90, 85, and 82%, respectively. CA₄Z₃ was classified as non-toxic according to the criteria defined by ISO 10993-5 [32]. The average cell viability was greater than 80% at all concentrations. As the result, among the CA₄-ZnO nanocomposite hydrogels, CA₄Z₃ exhibited a higher percentage of cell viability, particularly at lower concentrations (25, 50, and 100 µg/mL), indicating its suitability and safety for biomedical applications (Figure 11).

3.2.4. Antioxidant Activity

The antioxidant activity of CA₄Z₃ nanocomposite hydrogels was evaluated using the DPPH radical-scavenging assay, with ascorbic acid as the reference standard. The inhibition rates ranged from 32% to 94% at different concentrations (25, 50, 100, 250, and 500 µg/mL), with corresponding values of 32%, 42%, 67%, 90%, and 94%, respectively. CA₄Z₃ hydrogels exhibited antioxidant activity of 42%, 53%, 61%, 68%, and 79% at concentrations of 25, 50, 100, 250, and 500 µg/mL, respectively, as shown in Figure 12.

At lower concentrations (25 and 50 μg/mL), CA₄Z₃ exhibited moderate inhibition, whereas ascorbic acid showed comparatively lower inhibition. At medium concentrations (100–250 µg/mL), the antioxidant activity of CA₄Z₃ gradually increased, followed by a further increase in activity at higher concentrations. At the maximum concentration (500 µg/mL), CA₄Z₃ exhibited good inhibition compared with ascorbic acid. These results suggest that the ZnO nanocomposite hydrogel possesses significant antioxidant properties, which may be attributed to the interaction of ZnO nanoparticles with free radicals. This may promote electron donation and hydrogen transfer to DPPH radicals through the hydrophilic functional groups (-OH, -NH, and -COOH) present in the hydrogel matrix. The interaction between ZnO nanoparticles and the polymeric network facilitates enhanced radical-scavenging efficacy. The IC₅₀ of CA₄Z₃ was 149 µg/mL for the DPPH method. The scavenging activity and IC₅₀ value exhibit an inverse relationship [33]. Similar findings have also been reported by Patel et al. (2011) [34].

3.3.1. Density Functional Theory (DFT)

Since the CA₄-ZnO nanocomposite hydrogels exhibited biological activity, DFT and molecular docking studies were conducted to explore their mechanism of action. Initially, an intrinsic reaction coordinate (IRC) analysis was performed using the three basic monomers, namely citric acid, triethanolamine, and 2-furoic acid, which were utilized in the synthesis of the hydrogels. The IRC study revealed the structure selected for optimization at the DFT level. The monomers were further optimized at the 6-31G(d) level using the B3LYP (Becke, 3-parameter, Lee–Yang–Parr) functional, and the FMOs (Frontier Molecular Orbitals) were visualized to evaluate the molecular orbital aspects of reactivity. After geometry optimization, the molecular electrostatic potential (MESP) was generated to identify probable reactive regions of the molecule. The optimized molecules were subsequently used for docking studies. The DFT-derived values for the CA₄-ZnO nanocomposite hydrogels are presented in Table 5.

The FMO analysis revealed that, although the three monomers combine to form the hydrogel network, the IRC results indicated that the FMOs were unevenly distributed across the individual monomers. After optimization, it was observed that the HOMO (Highest Occupied Molecular Orbital) was primarily localized on the triethanolamine moiety of the hydrogel, whereas the LUMO (Lowest Unoccupied Molecular Orbital) was distributed slightly over the citric acid moiety, in close proximity to the ZnO nanoparticles within the hydrogel network (Figure 13). The 2-furoic acid moiety exhibited no lobes over its structure, indicating the absence of significant reactive regions within this segment. The denser lobes observed near the ZnO nanoparticle region suggest the presence of prominent reactive regions around its center. The MESP illustrates the distribution of electrostatic potential across the molecular surface, including the presence of positive potential regions, as shown in Figure 14 and Figure 15. These findings suggest that the corresponding moiety contributes to the desired biological activity through electronic effects arising from the coexistence of negative and positive potential regions. A higher-potential region was observed near the ZnO nanoparticle region, and the biological activity was enhanced through the incorporation of ZnO nanoparticles into the hydrogel matrix.

3.3.2. Molecular Docking (PDB ID: 1N67 and 4DXD)

In view of their biomedical properties, the nanocomposite hydrogels were subjected to docking studies against important receptors associated with antibacterial activity and wound healing, including the FtsZ (filamenting temperature-sensitive mutant Z) protein complex [35] and the Staphylococcus aureus protein target Clumping Factor A [36]. Infections caused by drug-resistant strains of Klebsiella pneumoniae represent a major challenge to the efficacy of conventional antibiotics. Therefore, there is an increasing need to investigate alternative antimicrobial therapies, particularly hydrogels synthesized from simple monomeric compounds [37]. Considering the biological importance of hydrogel derivatives, the present study aimed to synthesize, characterize, and investigate the biological potential of the hydrogel using a simple, convenient, and eco-friendly synthetic methodology.

The molecular docking study was conducted to evaluate the interactions between CA₄-ZnO nanocomposite hydrogels and the FtsZ protein complex (PDB ID: 4DXD), given their strong antibacterial properties. Additionally, CA₄-ZnO nanocomposite hydrogels were evaluated against the Staphylococcus aureus protein Clumping Factor A (PDB ID: 1N67), which demonstrated significant antibacterial activity. Both nanocomposite hydrogels exhibited favorable docking scores. However, the CA₄-ZnO nanocomposite hydrogel demonstrated stronger and more favorable binding interactions with the Clumping Factor A protein, as shown in Table 6. The CA₄-ZnO nanocomposite hydrogel also demonstrated superior ligand efficiency across all evaluated parameters, including standard ligand efficiency (−0.188), surface area-adjusted ligand efficiency (−0.633), and log-normalized ligand efficiency (−1.543).

In the CA₄-ZnO–Clumping Factor A complex, the ligand is tightly anchored via hydrogen bonds with polar residues (ASN267, SER268, and GLN235) and stabilized by hydrophobic contacts (VAL270, VAL323, ALA269, and ILE232) and potential electrostatic interactions with ASP273. Water-mediated interactions and minimal solvent exposure further enhance binding stability. These findings suggest that CA₄-ZnO nanocomposite hydrogels may serve as effective modulators of S. aureus proteins.

3.3.3. ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity)

The water solubility of a compound (LogS) represents the solubility of the compound at 25 °C. Drugs with higher water solubility are absorbed more efficiently than lipid-soluble compounds, and the obtained value of −2.99 indicates favorable aqueous solubility. Similarly, intestinal absorption predicts the percentage of an orally administered drug that is absorbed. The low absorption value observed in this study suggests that the compound may be more effectively administered through alternative routes rather than oral delivery.

The blood-brain barrier (BBB) indicates the ability of a drug to penetrate the brain and represents an important factor in minimizing side effects and toxicity while enhancing therapeutic efficacy. The evaluated nanocomposite hydrogels were predicted to cross the BBB, as presented in Table 7. The obtained value suggests favorable BBB permeability for the investigated drug delivery system.