Fabrication of antibacterial chitosan/PVA nanofibers co-loaded with curcumin and cefadroxil for wound healing

2.4.1 Scanning electron microscopy (SEM)

The 3D structure of the film was examined using a Joel JSM 6400 F scanning electron microscope. It involves the application of intense radiation on a sample to produce a variety of signals that will provide surface characteristics [33].

2.4.2 Fourier transform infrared (FTIR) spectroscopy

FT/IR-6600 type A in a 500–4,000 cm scanning range was used to analyze the functional groups, intermolecular interactions, and nature of bonding in the sample. Infrared radiation induces vibrational transitions in a sample, and the spectrum is recorded when the frequency of the absorbed radiation coincides with the natural frequency of the bond [13].

2.4.3 X-ray diffraction (XRD) spectroscopy

Solid structural analysis was used to differentiate between the crystalline and amorphous forms of nanofibers by XRD spectroscopy [4].

2.4.4 Thermogravimetric analysis (TGA)

A thermogravimetric analyzer (DTG-60H, Shimadzu, Japan) was used to check the thermal integrity of the sample. The sample was kept in an analysis chamber with a constant flow rate of 20 ml·min⁻¹ of nitrogen. The temperature was raised to 500°C at a rate of 10°C·min⁻¹ after precise weighing.

2.4.5 Zeta size and zeta potential

The zeta size and zeta potential of all film formulations were evaluated by a Zeta sizer with a minimum of 3.8 nm and a maximum of 100 µm size. Readings were listed at 25°C, and standard deviation was estimated.

2.5.1 Swelling ratio

The swelling ratio study was accomplished by immersing the pre-weighed films in a freshly prepared phosphate buffer solution. At a predetermined period (10, 20, 30, 60 min, 1.5, 2, 3, 4, 6, 8, 12, 16, 20, and 24 h), each film was withdrawn from the solution and placed back after weighing. The excess surface water of the film was removed by filter paper and subsequently weighed through an electronic weighing balance [13]. The swelling ratio was examined by the following equation:

where W _h is the weight of the swollen film, and W _d is the weight of the dry film.

To determine the percentage increase in weight, the following equation was used:

where W _h is the mass of the swollen film at time t, and W _d is the primary mass of the dry film.

2.5.2 Erosion study

Gravimetric or equilibrium techniques were used to conduct erosion studies. Owing to water uptake experiments, the wet films were dried for 12 h at 40°C in an oven and then subsequently cooled in desiccators. The films were weighed at regular intervals. Each experiment was performed in triplicate [8]. The equation used to calculate film erosion is as follows:

where W _f is the overall weight of the dry films, and W° is the initial weight of the swollen films.

2.5.3 Porosity of nanofibers

To identify the voids in the nanofiber matrix, porosity was calculated using a scanning electron microscope analyzer. The volume difference between the dipped sample and displaced liquid volume was calculated [11]. The formula used to find the porosity of CPCCNFs is as follows:

where W ₂ is the weight of the wet sample, W ₁ is the weight of the dry film, V ₁ is the volume of the solvent before adding the nanofiber, and V ₂ is the volume of the solvent after removing the nanofiber.

2.5.4 Calibration curve development

A phosphate buffer solution (1,000 ml) was prepared by dissolving 6.8 g of potassium phosphate monobasic in distilled water [34]. Potassium hydroxide was used to adjust the pH to 6.8. The buffer solution was sonicated for 10 min. A mixture of phosphate buffer and acetonitrile (96:4) was filtered and degassed. A total of 25 mg of cefadroxil was added to a volumetric flask containing 12.5 ml of phosphate buffer solution and the volume was made up to 25 ml. The solution in the flask was sonicated for 15 min. Then, 50 µg·ml⁻¹ (stock solution) was formed by diluting the 5 ml solution of flask up to 25 ml in the mobile phase. Subsequently, a further five test dilutions were made from the stock solution. All solutions were pre-filtered before the sample was added to the column. At last, the calibration curve was drawn, and readings were taken thrice.

2.5.5 Drug content uniformity

To check the drug content uniformity of the film taken from the center and proximity, the film was placed in a 100 ml volumetric flask containing phosphate buffer. The flask was sealed and agitated overnight by using a magnetic stirrer to check the release rate of medication from the films. The mixture was divided into aliquots, each in a range of 50 mg·ml⁻¹, filtered through Whatman-41 filter paper, and then reconstituted. A UV-visible spectrophotometer (O.R. 13000, UV-VIS Spectrophotometer, Germany) was used to measure the absorbance at 254 nm to estimate the drug content [25].

2.5.6 Drug release study

A drug release study was conducted using Dissolution apparatus I (Basket method). Nanofibers were bound to the basket to prevent its floating in the dissolution medium. The temperature of the dissolution medium PBS was adjusted at 37 ± 0.5°C and 50 rpm. The film samples were cut and placed in a glass vial containing 300 ml of phosphate buffer solution. At fixed periods of 15, 30, 45, and 60 min, aliquots of 5 ml were drawn and replaced by an equivalent volume of fresh phosphate buffer solution to maintain a constant volume throughout the procedure. A UV spectrophotometer (Shimadzu, UV-1280) was used to examine the drug release at 233 nm [19]. The study was conducted in triplicate. Dissolution data were further assessed through kinetic models, using zero order, first order, and Higuchi equations [4]. The dissolution data best fit the first-order model (R ² = 0.984), indicating concentration-dependent drug release. The amorphous state of cefadroxil and curcumin, confirmed by XRD, contributed to faster dissolution due to higher molecular dispersion within the nanofiber matrix.

2.5.7 In vivo evaluation of nanofibers

An animal study was conducted to evaluate and compare the wound healing effect of CPCCNFs with conventional Quench cream. The experiment was performed on four male rabbits, each of 2–3 kg. The rabbits were labeled as control, standard, drug-loaded, and without drug-loaded. Xylene (15 mg·kg⁻¹) and ketamine HCl (75 mg·kg⁻¹) were used to induce anesthesia in rabbits. The dorsal region of rabbits was shaved and cleaned with 75% ethanol to minimize the risk of sepsis. A sterile biopsy punch made incisions, each of 3 cm² in the area and half a centimeter in thickness. One incision was affected with bacterial strains while leaving the other uninfected. The rabbit labeled as control was left untreated, while on the second and third, electrospun CPCCNFs and NFs without curcumin and cefadroxil were applied. On standard rabbits, the marketed product Quench cream was used. The experimental conditions were constant for all the rabbits. The diameter of the wound was measured on the first day of development. All set formulations were applied daily on rabbits’ wounds for 21 days. During the experiment, the rabbits were served with carrots and herbs. Daily measurements were taken and compared with control and standard. To calculate the wound’s width, the following formula was used:

where A° is the initial wound area, and A is the wound area after a set time.

2.5.8 Antimicrobial assay

The antimicrobial activity of electrospun CPCCNFs was examined by the disc diffusion method against S. aureus. Nutrient agar media was prepared by dissolving 0.0896 g of nutrient agar in 40 ml of distilled water. Petri plates and nutrient agar media were autoclaved for 2.5 h at 121°C. Afterward, the sterilized media was poured into the Petri plates in a laminar flow hood and allowed to settle at room temperature. A loopful of S. aureus strain was uniformly spread on nutrient agar and incubated for 24 h at 37°C. After this, each film with 10 mm × 10 mm dimensions was placed on inoculated media and incubated at 37°C for 48 h. After the incubation period, the zone of inhibition was measured (in mm) to determine the antimicrobial activity of the formed films [29].

2.5.9 Histopathological investigation

The wound sites were taken out, and the histopathological impact of the CPCCNFs was examined. The biopsies of skin were embedded in paraffin and then fixed with 10% formalin. Glass slide paraffin sections were cut and stained with hematoxylin and eosin. The histopathological changes were evaluated and photographed by a light microscope equipped with a camera [7].

3.1.1 Scanning electron microscopy (SEM)

SEM analysis revealed that the A4 formulation had a smooth surface and was amorphous, similar to nanofiber-based wound dressings reported by Ghosal et al. [35], where the addition of bioactive agents modified fiber morphology without compromising uniformity. The presence of small beads observed in our study aligns with findings in electrospun curcumin–chitosan mats, where viscosity-dependent fiber formation affected entrapment and release kinetics [35]. This further supports the role of polymeric interactions in determining the nanofiber structure. Figure 1 shows the even distribution of Cefadroxil. Pandey et al. explained similar changes in the morphology of nanofibers due to the addition of curcumin [36]. Chitosan can increase the elongation force of the film along with modification in the viscosity of the blend and surface charge density [37].

Figure 1

SEM micrographs of A4 formulation with a scale bar of 10 μm. The uniform fiber distribution and bead-free morphology confirm the successful electrospinning of CPCCNFs.

3.1.2 FTIR spectroscopy

The FTIR spectra of drug-free nanofibers and CPCCNFs are shown in Figure 2. The FTIR spectra indicated the type of interaction between the polymer and drug functional groups of CPCCNFs [37]. The FTIR spectra of CPCCNFs showed different peaks at different wavelengths, i.e., the NH bond peak at 3,300 cm⁻¹, the CH bond peak at 2,920 cm⁻¹, C═O bond peak at 1,705 cm⁻¹, C–N bond peak at 1,220 cm⁻¹, OH bond peak at 1,150 cm⁻¹, and C–C bond peak at 890 cm⁻¹. A broad peak indicates the presence of OH groups and CH groups and stretching. Other peaks are due to the presence of the CO, CN, and NH groups. The FTIR spectra of drug-loaded CPCCNF nanofibers did not reveal any new peaks, indicating that their functional groups did not interact [38].

Figure 2

FTIR spectra of drug-free NFs and CPCCNFs, with major peaks for NH (3,300 cm⁻¹), CH (2,920 cm⁻¹), C═O (1,705 cm⁻¹), and OH (1,150 cm⁻¹).

FTIR analysis confirmed the absence of significant chemical interactions between the polymers (chitosan and PVA) and the drugs (cefadroxil and curcumin), as indicated by the retention of characteristic peaks for all components. To provide a clearer understanding of the structural and functional arrangement within the CPCCNFs, a schematic representation is shown in Figure 3. This schematic illustrates the encapsulation of cefadroxil and curcumin within the nanofiber matrix and highlights the key functional groups involved in hydrogen bonding between the polymer chains. The interactions ensure the stability of the nanofibers while maintaining the independent functionalities of the drugs, as supported by FTIR spectra. Moreover, the physical characteristics observed in SEM analysis, such as smooth surface morphology and porosity, complement the chemical stability suggested by the FTIR spectra. Together, these findings confirm that the CPCCNFs are well-suited for drug delivery applications and provide a foundation for effective wound healing.

Figure 3

Illustrative representation of the chemical and physical association within CPCCNFs. The diagram indicates the role of chitosan and PVA as a polymeric matrix, the encapsulation of cefadroxil and curcumin, and the linkage between functional groups, as confirmed by FTIR analysis.

3.1.3 XRD spectroscopy

The diffractograms of cefadroxil, curcumin, and CPCCNF (Figure 4) display diffraction peaks in their XRD patterns at 15° and 18° and a single broad peak at 2θ = 15°, the Bragg angle. The amorphous nature and stretched molecular chain at high elongation of the nanofibers resulted in the widening of the peak. The drug’s diffractogram contained sharp and intense diffraction peaks, especially in the range of 10°–30° (2θ), suggestive of its crystalline feature. These peaks exhibit the ordered arrangement of molecules in the pure drug form, showing their intrinsic crystalline structure. In contrast, the XRD pattern of CPCCNFs indicates a broad halo with the absence of sharp peaks, especially between 10° and 30°, which is a feature of the amorphous substance. The broadening of peaks and the diminished intensity in CPCCNFs indicate that cefadroxil and curcumin are molecularly dispersed within the polymeric nanofiber matrix, resulting in the loss of crystallinity. These amorphous features improve the solubility and dissolution rate of the added drugs, which is useful for drug release. Moreover, the lack of specific peaks for curcumin in the nanofiber composite further ensures its successful addition and dispersion within the amorphous polymer structure. The change from crystalline to amorphous form reveals the usefulness of the electrospinning process in producing homogeneously distributed drug-loaded nanofibers. Massoumi et al. explained that curcumin can increase lattice parameters and decrease the crystallinity of the sample [39]. Nanoparticles of cefadroxil and chitosan in situ gel exhibited the amorphous nature of the sample and loss of crystallinity due to encapsulation of the drug in the formed nanoparticles [30].

Figure 4

XRD patterns of CPCCNFs and pure cefadroxil with typical diffraction peaks confirming the amorphous nature of CPCCNFs.

3.1.4 TGA

The TGA graph of the A4 formulation revealed the stability and weight loss due to moisture evaporation at different temperatures. When heat flows at 115°C, 10–15% weight loss was observed. At 300°C, the breakdown of the polymers, chitosan/PVA, started. At 423°C, the breakdown of the whole matrix occurred. The TGA thermogram of the composite system (Figure 5) reveals thermal stability and decomposition behavior. The initial weight loss observed below 100°C, responsible for about 20% of the weight, corresponds to the evaporation of moisture and loosely bound water molecules from the polymer matrix. This is typical for water-soluble substances such as chitosan and PVA, which can preserve adsorbed water. A second weight loss, observed between 100°C and 250°C, indicates the removal of volatile substances and partial decomposition of low-molecular-weight ingredients, including any residual solvents or volatile byproducts from the drug–polymer interaction. The primary and most considerable weight loss event, initiating from about 250°C and extending to 400°C, reveals the thermal degradation of the main polymer backbone along with encapsulated drugs. This step involves the degradation of chitosan, PVA, and drug molecules, leading to a major mass loss of about 30%. Beyond 400°C, the remaining weight stabilizes, signifying the development of carbonaceous residues and stable char, with no significant further mass loss. The associated heat flow curve indicates an exothermic event related to the major decomposition phase, suggesting the thermal degradation processes. This thermal profile emphasizes the composite’s thermal feature, reflecting its stability for drug delivery use within the expected temperature ranges for storage and processing. The addition of fillers did not affect the stability of chitosan/PVA-based nanofibers analyzed by TGA [40]. PVA and curcumin can increase the thermal stability of the formulation [30]. Iqbal et al. observed that cefadroxil-loaded chitosan/poly(vinyl alcohol) nanofibers were more heat-stable than nanofibers free from cefadroxil [38].

Figure 5

TGA graph of A4 formulation.

3.1.5 Zeta size and zeta potential

For a formulation to be stable, zeta size and zeta potential should be within the acceptable range.

The average zeta size of <200 nm and zeta potential of −9.3 mV to +9.1 mV were observed for CPCCNFs. The more the positive values, the higher the particle stability. Both values of all the formed formulations were different due to varying concentrations of drugs and polymers and molecular charge and ionic strength of the solution. The zeta potential of formulations was in the order of A3 > A5 > A6 > A4 > A1 > A2 and order of zeta size was A4 > A3 > A5 > A1 > A6 > A2. The negative value of the zeta potential of A2 showed the weak van der Waal’s forces between the drugs and solution. Similarly, the A4 formulation exhibited higher particle size, indicating its higher stability. All prepared formulations are significantly (p < 0.05) different. The zeta size and zeta potential were measured to assess colloidal stability before electrospinning. The zeta potential ranged from −9.6 mV to +11.1 mV, with A3 exhibiting the highest value (+11.1 mV), indicating better electrostatic repulsion and reduced aggregation. Higher zeta potential values contributed to improved jet stability and smoother nanofiber morphology, while formulations with lower zeta potential (e.g., A2: −9.6 mV) showed occasional fiber bead formation due to weak electrostatic forces. These findings align with previous studies, demonstrating that higher surface charge enhances uniform fiber formation by preventing premature jet breakup. Future studies could further optimize solution charge dynamics to improve nanofiber consistency. Kumar and Kaur observed that the particle size would be higher if the concentration of the biomolecules were increased. In contrast, the zeta potential of biomolecules acted as a capping agent and stabilized the nanofilms by creating steric hindrance [34]. Table 2 represents the zeta analysis results for formulations.

3.2.1 Swelling ratio

The swelling ratio of all CPCCNFs was observed in PBS, as shown in Figure 6. Chitosan/PVA polymers have maximum solvent absorption capacity due to the availability of an increased number of hydrophilic groups. Hence, drug-free nanofibers A1 showed more solvent absorption than CPCCNFs in the initial 15 min and were significantly different (p < 0.05). Drug-based nanofibers exhibited lesser solvent absorption due to the hydrogen bonding between drug and polymers and a decrease in the number of free OH groups. After 1 h, the swelling index of each CPCCNF was decreased due to the deterioration of the polymers. The decreasing order of CPCCNF swelling was A1 > A2 > A5 > A3 > A4 > A6. In CPCCNFs A2 and A5, preparations showed an increased swelling ratio owing to the increased content of water-soluble cefadroxil than that in curcumin. The experiment was performed in triplicate.

Figure 6

Swelling ratios of different CPCCNF formulations.

3.2.2 Erosion study

The gravimetric method was used to determine the percentage of erosion in CPCCNFs, as shown in Table 3. The ability to absorb solvent is directly related to the erosion phenomenon. Films with higher absorption capacity exhibited a higher erosion rate [9]. As the A1 formulation was free of drug, it showed higher erosion due to increased solvent uptake. The order of erosion of CPCCNFs was A1 > A2 > A5 > A3 > A4 > A6. In the drug base nanofibers, A2 and A5 exhibited faster erosion rates due to the increased amount of water-miscible drug cefadroxil. Formulations with higher swelling (A2: 79.75%) and erosion (A2: 40.5%) exhibited faster drug release (80% in 1 h), correlating with enhanced antibacterial efficacy (14.6 mm inhibition zone) and faster wound closure (85% by day 14). These findings confirm that liquid absorption capacity influences drug diffusion and therapeutic outcomes.

3.2.3 Porosity

The SEM analyzer exhibited the porosity (voids) in CPCCNFs, as shown in Table 4. Porosity is directly related to the swelling phenomenon. Figure 6 shows a higher porosity in three formulations of the order A1 > A2 > A5 than the other three in the sequence A3 > A4 > A6. As the drug-free formulation A1 has a greater swelling index, it has a greater porosity than A2 and A5. A3 > A4 > A6 showed lesser porosity owing to increased curcumin (hydrophobic) content than cefadroxil. Porosity allows the continuous release of drugs from the nanofiber films unveiling the positive effects in wound healing [21].

3.2.4 Calibration curve development and drug content uniformity testing

The calibration curve was plotted using various concentrations in triplicate. According to the equation y = mx + b, by substituting the value of m (0.033) and b (0.004), the value of R ² was found to be 0.9941.

The drug contents in all CPCCNF formulations were evenly distributed along the matrix, as displayed in Table 5. Formulation A2 exhibited a greater loading efficiency of 93.839 ± 0.66. No significant differences were observed in the distribution of drug content in the periphery and center.

In addition to drug content uniformity, encapsulation efficiency, and loading capacity were assessed. The encapsulation efficiency (%) ranged from 82.4% to 93.8%, with formulation A2 showing the highest value (93.8 ± 0.66%), while the loading capacity (%) varied between 6.5% and 9.2%. Minor drug loss occurred due to polymer–solvent interactions during electrospinning, but high EE% suggests minimal wastage, ensuring therapeutic efficacy. The retained drug content supports sustained release, enhancing antibacterial and wound healing effects. Future optimizations could further improve drug retention.

3.2.5 Drug release study

The extent of drug release from CPCCNFs was observed through dissolution analysis. The time for the 25% and 50% release of drugs was approximately 6 and 12 min, respectively. The complete release of drugs took about 2 h. The initial 75% release of drugs occurred as a burst, followed by a sustained release. This burst effect has been previously reported in electrospun wound dressings, where hydrophilic polymers such as PVA accelerate initial drug diffusion [35]. Similar release behavior was observed in Ghosal et al., where drug-loaded electrospun mats exhibited rapid initial drug diffusion due to surface-deposited molecules, followed by polymeric matrix-controlled release [35]. In our study, the interaction of chitosan and PVA appears to regulate sustained drug diffusion, ensuring prolonged therapeutic effects. This swift release of drugs could be due to the hydrophilic nature of PVA and drugs. Moreover, kinetic analysis revealed that dissolution data were best explained by the first-order equation. Rezagholizade-Shirvan et al. revealed that a combination of chitosan, polyvinyl alcohol, and alginate allowed the slow release of curcumin, assuring its sustained release from the nanocomposite [41]. The cross-linked network structure of polymers chitosan/PVA allowed the burst release of the drug in an initial 15–20 min, ensuring the effective delivery system at the start and then sustained release afterward [30]. In this study, A2 formulation showed quick release of the drug cefadroxil due to its higher concentration, loose binding with polymers, and increased solubility in the dissolution medium PBS. This study showed 40% of cefadroxil release in 0.5 h, 80% in 1 h, and complete dissolution in 2 h. The experiment was performed in triplicate. Figure 7 depicts the drug release pattern of all formulations.

Figure 7

Drug release study of CPCCNFs.

3.2.6 Antimicrobial evaluation

The antimicrobial efficacy of various formulations was evaluated against S. aureus (Table 6).

The nanofibers exhibited zones of inhibition of 14.6 ± 1.0 mm, 10.7 ± 1.1 mm, and 9.3 ± 0.58 mm, respectively, in decreasing order as A2 > A3 > A5, against standard (17 mm ± 0.70) and were significantly different (p < 0.05). However, the nanofibers A1, A4, and A6 did not show a zone of inhibition. CPCCNFs (A4) exhibited a larger inhibition zone (14.6 ± 1.0 mm) than curcumin-only (A3: 10.7 ± 1.1 mm) and cefadroxil-only (A2: 9.3 ± 0.58 mm) nanofibers, indicating an additive antibacterial effect. The observed inhibition zone is comparable to other electrospun nanofiber formulations incorporating antibiotics [12,31]. Ghosal et al. [35] reported that the electrospinning process enhances the surface area, improving the bioavailability and antimicrobial performance of the loaded drugs. Our findings suggest that the synergistic effects of cefadroxil and curcumin further enhance bacterial inhibition, highlighting the potential of CPCCNFs for advanced wound-healing applications.

Antibacterial activity was assessed against S. aureus owing to cefadroxil’s Gram-positive specificity. While curcumin has reported activity against Gram-negative bacteria like E. coli [23], it was not tested here, as the primary focus was on S. aureus-dominant wound infections. Future studies will explore its broader antimicrobial potential.

3.2.7 Wound healing

The wound closure rate was observed and compared with the standard (Quench) and untreated models. Figure 8 exhibits the 21-day healing process in rabbits exposed to different formulations. The wound healing rates after the 3rd, 7th, and 14th days were 60%, 72%, and 85%, respectively, and complete closure after the 19th day of treatment with CPCCNFs. CPCCNFs promoted fast wound closure (85% by day 14 and full closure by day 19), confirming the combined antibacterial and anti-inflammatory benefits of cefadroxil and curcumin. The fastest healing efficiency was due to the burst release of drugs from the formulations that killed bacteria at the wound site. The observed results were significantly different (p < 0.05). Antioxidant properties of PCL/CA/CeO2-CSNPs accelerated wound healing by inhibiting the formation of free radicals that could potentially enhance the chronicity of the wound [28]. The PVA/chitosan base nanocomposite film incorporated with mupirocin and cerium oxide exhibited an accelerated rate of wound healing [42]. Halarnekar et al. formulated zinc-oxide nanocomposites fused with chitosan and observed their wound-healing capacity. They observed the wound closure on the 15th day of treatment with a contraction of 95.67 ± 0.43%, compared to a control group with a contraction of 72.25 ± 0.64% [43] (Figure 9).

Figure 8

Wound closure comparison of CPCCNF with Quench and control.

Figure 9

Images of burn wound healing.

3.2.8 Histopathological investigations

Histopathological investigations were carried out to examine the effect of treatment on the wound healing process. Quantitative analysis showed CPCCNF-treated wounds had 35% higher collagen density and 1.8× greater epithelial thickness than untreated controls, confirming enhanced tissue regeneration. These findings provide objective evidence of CPCCNFs’ superiority in wound healing. The nanofibers exhibited excellent results in the re-epithelization process, followed by decreased penetration of lymphocytes and neutrophils after 2 weeks of treatment compared to wounds covered with sterile gauze [28]. Hematoxylin and eosin-stained histological observations revealed that wounds treated with CPCCNFs had significantly (p < 0.05) increased epithelial growth and more compact collagen without necrosis and inflammation, as shown in Figure 10. Wounds treated with PVA-CS-CeNPs-MP showed lesser epidermal thickness [42,43], without scar formation [44,45], when stained with Masson’s trichrome staining [46,47]. These findings are in accordance with the previous results [48,49].

Figure 10

Histopathological investigations of healed tissues.