Electrospun Chitosan–Poly(vinyl alcohol) Nanofibers Functionalized with Natural Bioactive Compounds: Design, Physicochemical Characterization and Release Profiles

TL;DR

Researchers created nanofibers from chitosan and PVA with natural compounds that could help in wound healing and tissue regeneration.

Contribution

The study introduces functionalized nanofibers with multiple bioactive compounds for wound healing applications.

Findings

Nanofibers were successfully produced with uniform morphology and influenced by the type of bioactive compounds.

The nanofibers showed high swelling capacity and pH-dependent release behavior.

Surface and wettability properties indicated compatibility with biological environments.

Abstract

This study reports the development and characterization of chitosan–poly(vinyl alcohol) (CH/PVA) nanofibers (NFs), functionalized with bioactive compounds (ACs) relevant for wound healing and tissue regeneration. CH/PVA NFs loaded with L-arginine (ARG), allantoin (ALA), royal jelly (RJ) and curcumin (CUR), either as single or co-loaded systems, were prepared by electrospinning. The polymer solutions were characterized in terms of key physicochemical properties relevant to electrospinning. The CH/PVA@ACs NFs were characterized morphologically and structurally through scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Additionally, surface-related, physical, and functional properties such as wettability, swelling behavior, and in vitro release profiles were examined. The NFs were successfully produced in a uniform and continuous manner, with the fiber diameter and morphology being influenced by the type of ACs. FTIR analysis validated the characteristic functional groups linked to both the polymeric matrix and ACs. The nanofibrous systems demonstrated a high swelling capacity and a release behavior that is dependent on pH. Analyses of surface free energy and wettability revealed favorable interfacial interactions between solid and liquid, indicating compatibility with aqueous biological environments. In summary, the developed CH/PVA@ACs NFs exhibited appropriate morphological, structural, surface, and functional properties, underscoring their potential as effective materials for wound dressings.

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1. Introduction

Nanomaterials research has expanded rapidly in recent years, driven by advances in material design and growing demand for innovative biomedical solutions. Nanotechnology enables the fabrication of materials with tailored architecture, controlled presentation of bioactive cues, and programmable drug release profiles, thereby providing advanced and effective strategies for tissue regeneration and wound healing. Representative systems include electrospun nanofibers (NFs) delivering angiogenic factors [1,2], mesoporous bioactive glass nanoparticles (NPs) promoting osteogenesis [3], and nanocomposite hydrogels providing sustained antimicrobial activity [4]. Collectively, these advances highlight the translational potential of nanostructured platforms [5] in regenerative medicine, particularly NFs that closely mimic the extracellular matrix (ECM) [6].

Tissue repair and regeneration are highly coordinated biological processes that depend on the nature of the injury and the cellular microenvironment [7]. At a cellular level, effective healing requires ECM and the spatiotemporally regulated action of growth factors and signalling molecules that govern cell proliferation, migration and differentiation [8]. Disruption of these mechanisms underlies impaired healing, a major clinical problem in chronic wounds. Chronic skin wounds affect approximately 37 million individuals worldwide and account for an estimated 3–5% of total healthcare expenditures related to wound management [9]. Their incidence continues to rise particularly in patients with diabetes mellitus (DM), vascular disease, and advanced age.

In this context, NFs have emerged as promising platforms for wound dressing, acting as temporary ECM-like substrates that support cell adhesion and modulate the wound microenvironment [10,11]. Owing to their high surface area and interconnected porosity, NFs represent effective therapeutic systems for chronic and acute wounds. Their architecture facilitates gas exchange and nutrient transport, promotes the absorption of excess exudates and allows the incorporation and release of active compounds (ACs) [12].

Chitosan (CH) is widely used in nanofibrous wound dressings due to its biocompatibility, biodegradability, low cytotoxicity, and intrinsic antimicrobial activity [13,14,15]. Owing to its polycationic character, CH readily interacts with negatively charged molecules and biological interfaces, making it a suitable matrix for the incorporation of ACs within fibrous architectures [14,15,16].

Poly(vinyl alcohol) (PVA), a synthetic, water-soluble polymer known for its excellent mechanical and film-forming properties, is often blended with CH to improve solution processability, electrospinnability, and structural stability of the resulting NFs [14,16,17]. CH/PVA-based NFs have been extensively explored as versatile platforms for the delivery of ACs, particularly natural molecules with antioxidant or antimicrobial potential, where fiber morphology, uniformity, and surface properties play a critical role in determining loading efficiency and release behaviour [14,18,19]. The architecture of NFs can be finely tuned through the control of solution properties, processing parameters, and environmental conditions, enabling the rational design of multifunctional materials with tailored physicochemical characteristics [16,20,21].

In this study, L-arginine (ARG), allantoin (ALA), royal jelly (RJ), and curcumin (CUR) were selected as representative natural ACs to functionalize the CH/PVA matrix, based on their efficiency in wound care [19]. Therefore, ARG, a precursor of nitric oxide (NO), plays a key role in promoting angiogenesis, collagen synthesis, and re-epithelialization [22]. ALA stimulates cell proliferation, thereby accelerating epidermal regeneration [5,23]. RJ, owing to its antimicrobial, anti-inflammatory, and immunomodulatory properties, provides protection against bacterial infections frequently associated with chronic wounds [24]. CUR is a powerful antioxidant that reduce oxidative stress, modulates inflammatory responses, and increases collagen production at the wound site, promoting the healing process [18,25].

Based on the beneficial effects of these ACs, several polymer-based dressing materials have been reported in the literature. For instance, RJ has been incorporated into electrospun CH/PVA NFs, resulting in uniform and hydrophilic fibers with high encapsulation efficiency, sustained release behavior, and antibacterial activity against both Gram-positive and Gram-negative bacteria [26]. Similarly, the incorporation of ARG into CH nanofibrous mats has been shown to improve wettability, mechanical properties, and long-term stability, while also promoting favorable cell–material interactions in terms of viability and proliferation [27]. CUR has also been incorporated into electrospun CH/PVA NFs, yielding uniform nanofibrous structures with preserved chemical integrity and sustained release behavior, together with pronounced antibacterial activity [28]. In addition, CH/PVA hydrogels incorporating honey and ALA have been reported to exhibit enhanced physicochemical stability, sustained release behavior, antibacterial activity, and good biocompatibility, highlighting the potential of ALA-loaded CH/PVA systems as functional biomaterials [29].

However, electrospun CH/PVA NFs co-loaded with synergistic combinations of the aforementioned ACs have not yet been reported. Therefore, the aim of the present study was to design and prepare CH/PVA NFs co-loaded with two ACs as novel synergistic systems, namely CH/PVA@ARG-ALA, CH/PVA@ARG-RJ, and CH/PVA@ALA-CUR, with the potential to enhance therapeutic performance. These nanofibrous systems were morphologically and structurally characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR) spectroscopy. In addition, their surface-related, physical, and functional properties, including wettability, swelling behavior, and in vitro release profiles, were systematically evaluated. The performance of the co-loaded CH/PVA NFs were compared with that of single-loaded systems in order to highlight the advantages of the synergistic approach.

2. Materials and Methods

2.1. Materials

CH (medium molecular weight, 75–85% degree of deacetylation), ARG (≥98%, mp 226–230 °C), ALA (≥98%, mp 230 °C), glacial acetic acid (≥99.7%), phosphate-buffered solution (PBS, 0.15 M, pH 7.4), acetate buffer (pH 5.5), sodium bicarbonate (NaHCO_3_), sodium chloride (NaCl), potassium chloride (KCl), monosodium phosphate (NaH_2_PO_4_), doubly distilled water (DDW), formamide, and α-bromonaphthalene were purchased from Sigma-Aldrich (Merck Romania SRL, București, Romania). PVA (87–89% hydrolyzed, high molecular weight, average Mw = 88–97 kDa) was purchased from Alfa Aesar (Ward Hill, MA, USA). Absolute ethanol (99.5%) was purchased from Chimreactiv (Bucharest, Romania). Dimethyl sulfoxide (DMSO; ≥95%) were purchased from MuseChem (Fairfield, NJ, USA). CUR (analytical standard, Mw = 368.38 g/mol) was purchased from Supelco (Merck KGaA, Darmstadt, Germany). Pure RJ containing 1.59 wt% 10-hydroxy-2-decenoic acid was purchased from Melidava (Crăiești, Romania).

2.2. Preparation and Characterization of CH/PVA@ACs Solutions

CH solution was prepared by dissolving CH (1% wt/v) in 2% (v/v) acetic acid at room temperature, while PVA solution (8% wt/v) was prepared in deionized water at 80 °C. The two polymeric solutions were subsequently mixed at a CH/PVA ratio of 1:3 (v/v), followed by the incorporation the ACs (ARG, ALA, RJ and CUR) as detailed in Table 1. The resulting formulations were magnetically stirred until complete homogenization was achieved.

The concentrations CH (1% w/v) and PVA (8% w/v) were selected based on preliminary trials covering a range of polymer concentrations (CH, 1–2% w/v; PVA, 3–10% w/v) aimed at balancing solution viscosity, spinnability, and fiber formation. Lower CH concentrations ensured sufficient chain mobility for electrospinning, while variations in PVA content were used to modulate fiber morphology, diameter, and uniformity. By systematically adjusting the CH:PVA ratio, formulations that minimized bead formation and yielded continuous, uniform NFs were identified at a CH/PVA ratio of 1:3 (v/v). This approach ensured that the selected concentrations provided optimal electrospinning performance while preserving the structural integrity of the nanofibrous mats.

The rheological behaviour of CH/PVA@ACs solutions was evaluated using the Ostwald model, and the viscosity coefficient, expressed in centipoise (cP), was calculated according to the corresponding equation [30]:

$$[eqn]$$

where η_1_ is the viscosity coefficient of water at 20 °C, equal to 1 cP, η_2_ is the viscosity coefficient of the samples, ρ_1_ and ρ_2_ represent the density of the reference liquid and the polymer solution.

The density of CH/PVA@ACs solutions was determined indirectly using a pycnometer (Amex, Bucharest, Romania). Surface tension was assessed with a Traube stalagmometer (Gerhardt, Bonn, Germany), and the surface tension coefficient (mN/m) was determined from the solution density and the drop count [30]. Electrical conductivity was determined using an Oakton PC 510 conductometer (Oakton Instruments, IL, USA, supplied by Metrohm Romania SRL, Bucharest, Romania) and reported in mS, while pH values were performed using a HI5522 pH-meter (Hanna Instruments, Woonsocket, RI, USA, supplied by Metrohm Romania, SRL, Bucharest, Romania) [30]. All measurements were performed in triplicate, and the results are reported as mean ± standard deviation (SD).

2.3. Preparation of CH/PVA@ACs NFs

The NFs were prepared by electrospinning using a Nanospinner Inovenso apparatus (Inovenso, Istanbul, Turkey), equipped with a high-voltage power supply, a syringe pump and a spinneret (syringe fitted with a blunt-tip needle) [31]. An aluminum foil layer was placed over a static stainless-steel plate collector (20 × 18 cm, thickness 3 mm) to facilitate the collection of the NFs. During the electrospinning process, the temperature and relative humidity were maintained at 24 ± 5 °C and 30 ± 5%, respectively. The CH/PVA@ACs solutions were loaded into a syringe, which served as the positive electrode, while the collector functioned as the counter electrode.

Based on the optimized polymer concentrations (CH, 1% w/v; PVA, 8% w/v) and the CH/PVA ratio (1:3, v/v), the loadings of the ACs were subsequently established through additional preliminary optimization studies aimed at maintaining electrospinning stability, solution homogeneity, and fiber morphological integrity. The ARG loading was fixed at 10% (w/w, relative to the total polymer content) to avoid conductivity-induced jet destabilization observed at higher loadings, whereas the RJ loading was limited to 3% (w/v) to prevent viscosity-related disturbances during fiber formation. The concentrations of ALA (4% w/v) and CUR (3% w/v) were selected according to their solubility and dispersion limits within the CH/PVA system, as higher loadings led to jet instability and morphological defects. These loadings ensured homogeneous incorporation without compromising the optimized electrospinning conditions.

Electrospinning parameters were optimized for each formulation and are summarized in Table 1. After electrospinning, the NFs were carefully removed from the aluminum foil and dried at ambient temperature overnight.

2.4. Characterization of CH/PVA@ACs NFs

2.4.1. Fourier-Transform Infrared (FT-IR) Spectroscopy

FT-IR spectra of NFs samples were recorded using an ABB MB3000 FT-IR spectrometer (ABB Measurement & Analytics, Zurich, Switzerland) equipped with an attenuated total reflectance (ATR) accessory. Spectra were recorded in the 4000–800 cm^−1^ range at a resolution of 1 cm^−1^. The NFs samples (∼1 cm^2^) were placed directly onto the ATR crystal, and gentle pressure was applied to ensure optimal contact. Each spectrum represents the average of 32 scans to improve the signal-to-noise ratio.

2.4.2. SEM Morphology and Fiber Diameter

Surface morphology was examined by SEM using a Phenom Desktop SEM (Thermo Fisher Scientific, Waltham, MA, USA). The NFs samples were sputter-coated with a thin gold layer to minimize charging effects and examined with an Inspect S scanning electron microscope operating in secondary electron model at an accelerating voltage of 30 keV. Fiber diameter distributions were determined from SEM micrographs using Phenom FiberMetric software (v. 2.9.0, Eindhoven, The Netherlands). All measurements were performed in triplicate, and the results are reported as mean ± SD.

2.4.3. Surface Free Energy and Biocompatibility-Related Surface Properties

The surface free energy properties of the CH/PVA-ACs samples were evaluated by measuring the equilibrium contact angles of three pure liquids (DDW, formamide, and α-bromonaphthalene) using a CAM PLUS microgoniometer (KSV Instrument, Helsinki, Finland) under controlled temperature conditions [30]. The CH/PVA-ACs solutions were deposited directly onto pre-cleaned and dried glass slides and allowed to dry completely at room temperature. Contact angle measurements were performed within 30 s after depositing a 1 μL droplet of each liquid onto the sample surface. The contact angle was calculated using the following equation [30]:

$$[eqn]$$

where θ is the contact angle, h represents the droplet height, and d denotes the diameter of the droplet base. The reported contact angle value corresponds to the mean ± SD calculated from at least 20 measurements performed at different locations on the sample surface.

The surface free energy components were calculated using the extended Young–Laplace approach, which describes the surface free energies of solids and liquids as the sum of Lifshitz–van der Waals (dispersive) and polar (acid–base) interaction components [30]. According to this model, the surface free energy is expressed by the following equation:

$$[eqn]$$

where γ_L_^TOT^ is the liquid total surface tension, γ^LW^ represents the Lifshitz–van der Waals (dispersive) component, γ^+^ is the electron acceptor (acidic) parameter, and γ^−^ is the electron-donor (basic) parameter. The subscripts S and L refer to the solid and liquid phases, respectively.

The derived surface free energy parameters were subsequently used to estimate surface properties related to biocompatibility. These parameters serve as indirect indicators of material–biological interactions, since surface wettability and the balance between polar and dispersive components are known to influence protein adsorption and subsequently, early biological events at the biomaterial interface [32]. Therefore, an indirect biocompatibility-related parameter was calculated according to following equation:

$$[eqn]$$

where γ^SL^ represents the solid–liquid interfacial free energy and γ^AB^ denotes polar acid–base interactions, between the biomaterial surface (S) and the reference biological liquid (L).

2.4.4. Swelling Degree

The swelling behavior of NFs samples was investigated according to the method reported by Sakthiguru & Sithique [33], with minor modifications. Square NFs specimens (2 × 2 cm) were accurately weighed to determine the initial dry mass (W_D_), and subsequently immersed in 20 mL of different aqueous media, namely DDW, PBS (pH 7.4) and pseudo-extracellular fluid (PECF, pH 8.5). All experiments were conducted at 20 °C under static conditions. The PECF solution was prepared by dissolving 2.5 g NaHCO_3_, 0.68 g NaCl, 0.22 g of KCl and 0.35 g NaH_2_PO_4_ in 100 mL of DDW. At fixed time intervals (30 min, 60 min), the NFs samples were withdrawn from the immersion medium, gently blotted with filter paper to remove excess surface liquid, and weighed to determine the swollen mass (W_S_). The swelling degree (SD) was calculated according to the following equation [33]:

$$[eqn]$$

where W_D_ and W_S_ denote the dry and swollen masses of the NFs samples, respectively. All experiments were performed in triplicate, and data are expressed as mean ± SD.

2.5. In Vitro ACs Release Profiles and Kinetic Analysis

The in vitro release of ACs was evaluated using a modified dialysis method adapted from Del Prado-Audelo et al. [34]. In short, CH/PVA@ACs NFs (150 mg) were inserted into dialysis bags (MWCO = 3.5 kDa, Thermo Scientific, Waltham, MA, USA) and submerged in 30 mL of release medium. The system was kept at 37 °C with gentle magnetic stirring (200 rpm) using a temperature-controlled water bath. Release experiments were performed in two distinct media: acetate–citrate buffer (0.01 M, pH 5.5), mimicking the physiological pH of healthy skin, and PBS (0.01 M, pH 7.4), and reflecting physiological internal environments. At predetermined time intervals, 2 mL aliquots were withdrawn from the release medium and analyzed by UV-Vis spectrophotometry (SPECORD 210 PLUS spectrophotometer) (Analytik Jena, Germany Analytik Jena GmbH+Co. KG, Jena, Germany) at the characteristic wavelength of each ACs (190 nm for ARG, 240 nm for RJ, 425 nm for CUR, and 248 nm for ALA). Following analysis, the aliquots were returned to the diffusion medium to maintain a constant volume. Quantitative determination was performed using calibration curves constructed individually for each AC under identical experimental conditions to those employed in the polymer matrix release experiments.

The percentage of in vitro release was calculated using the following equation [34,35]:

$$[eqn]$$

where C_released_ is the concentration of ACs determined at each time point from the calibration curve, and C_initial_ is the concentration of ACs incorporated into the NF matrix. All experiment was performed in triplicate, and the results are expressed as mean ± SD.

2.6. UV-Vis Calibration Curves for ACs

Calibration curves were constructed individually for each AC to enable concentration determination based on single-wavelength absorbance measurement. For this purpose, UV–Vis spectra were recorded over the 180–800 nm range. Stock solutions of every AC were initially made in the suitable solvent and subsequently serially diluted to achieve the required concentration range, maintaining absorbance values in the linear range (absorbance < 1). Due to its pronounced hydrophobicity, CUR was first dissolved in 30 μL DMSO and then diluted with DDW to the final volume. The linear regression equations corresponding to the calibration curves of each AC were as follows: y = 0.017 + 0.0009x (ARG); y = 0.0068 + 0.0067x (ALA); y = 0.0417 + 0.0085x (RJ); y = −0.11186 + 0.15086x (CUR), where y represents the absorbance and x the concentration of the respective AC.

2.7. Kinetic Modelling of ACs Release

The release kinetics of AC represents a critical parameter in the performance of controlled delivery systems, as it governs both the rate and extent of AC availability of the target site. Controlled release systems are designed to modulate the temporal release profile, typically characterized by an initial rapid release phase (“burst release”) followed by a sustained release phase, thereby enabling the maintenance of effective AC levels over an extended period. To elucidate the release mechanisms and rate-controlling steps, the experimental release data were fitted to commonly employed kinetic models, including zero-order, first-order, Higuchi, Hixon–Crowell and Korsmeyer–Peppas models.

The zero-order kinetics model is described by the following equation [36,37]:

$$[eqn]$$

where m_t_ is the amount of AC released at time t, m_b_ is the initial amount of AC in solution (assumed to be 0), and k_0_ is the zero-order release rate constant.

The first-order kinetics model is described by the following equation [36,37,38]:

$$[eqn]$$

where m_t_ is the amount of AC released at time t, m_0_ is the initial amount of AC incorporated in the delivery system and k_1_ is the first order release rate constant.

The Higuchi model, commonly applied to diffusion-controlled release systems, is given by following equation [37,38,39]:

$$[eqn]$$

where m_t_ is the amount of AC released at time t and k_H_ is the Higuchi release constant.

The Hixon–Crowell model, which accounts for changes in surface area and particle size during dissolution, is described by following equation [37,38,40]:

$$[eqn]$$

where m_0_ is the initial amount of AC incorporated in the delivery system, m_left_ is the amount of AC remaining in the system at time t, and k_H–C_ is the Hixon–Crowell rate constant.

The Korsmeyer–Peppas model is described by following equation [38]:

$$[eqn]$$

where m_t_ is the amount of AC released at time t, m_∞_ is the amount of AC released at infinitive time, k_K–P_ is the Korsmeyer–Peppas kinetic constant, and n is the release exponent indicative of the underlying release mechanism.

2.8. Statistical Analysis

Statistical analysis was performed using Student’s t-test and one-way analysis of variance (ANOVA). All experiments were conducted in triplicate, and results are expressed as mean ± SD. A p-value < 0.05 was considered statistically significant.

3. Results

3.1. Characterization of CH/PVA@ACs Solutions

The CH/PVA@ACs solutions were characterized in terms of conductivity, pH, density, and apparent viscosity, as these parameters critically influence the electrospinning process, and the corresponding results are presented in Table 2.

The electrical conductivity of polymer solutions is a key parameter in the electrospinning process, as it influences charge density within the polymer jet and, consequently, affects fibre stretching and final fibre morphology. In general, increased solution conductivity enhances jet elongation, leading to reduced NFs diameter and improved fibre uniformity, while also decreasing the tendency for bead formation [21].

All CH/PVA@ACs formulations exhibited conductivity values comparable to or higher than that of the CH/PVA matrix (4.39 ± 0.04 mS), with the exception of CH/PVA@CUR–ALA, which showed a slightly lower conductivity (4.18 ± 0.08 mS). The CH/PVA@ARG-ALA exhibited the highest conductivity (7.15 ± 0.15 mS), followed by CH/PVA@ARG-RJ (6.44 ± 0.30 mS) and CH/PVA@ARG (6.29 ± 0.14 mS). In comparison, CH/PVA@ALA and H/PVA@CUR showed moderate conductivity, 5.42 ± 0.25 mS and 5.38 ± 0.27 mS, respectively [41]. In general, formulations with ARG reliably showed higher conductivity than those lacking ARG, indicating enhanced ionic character linked to the existence of –NH_2_ group and guanidine moiety. The pH values of the polymer solutions differed based on the included ACs (Table 2). Solutions that include ARG demonstrated alkaline pH, with CH/PVA@ARG attaining a pH of 10.17 ± 0.09, in agreement with the presence of guanidine moiety. The combination of ARG with ALA led to a slightly alkaline pH (CH/PVA@ARG-ALA, pH 8.78 ± 0.14), while its association with RJ resulted in a significant reduction in pH (CH/PVA@ARG-RJ, pH 4.48 ± 0.37). This reduction may be associated with intermolecular interactions involving the free-NH_2_ groups of ALA [42], as well as with the contribution of RJ constituents with acidic character, including carbohydrates (fructose, glucose and sucrose) and organic acids such as 10-hydroxydecanoic acid (10-HDDA) and 10-hydroxy-2-decenoic acid (10-HAD), in addition to royalisin [43]. Formulations lacking ARG (CH/PVA@ALA, CH/PVA@CUR and CH/PVA@CUR-ALA) exhibited acidic to mildly acidic pH values in the range of 3–5, which is compatible with the reported stability domains of both ALA and CUR [41,42]. Density assessments revealed that all CH/PVA@AC solutions showed similar values, with ρ ≈ 1 kg m^−3^ (Table 2). This observation indicates that the inclusion of ACs does not significantly influence solution density, which seems to be mainly determined by the polymeric matrix and its molecular structure rather than the ACs.

Referring to the apparent viscosity (η), the reference CH/PVA solution exhibited a value of 157.25 ± 2.55 cP. The highest viscosity was noted for CUR-based formulations, CH/PVA@CUR (180.97 ± 0.307 cP) and CH/PVA@CUR-ALA (171.90 ± 1.25 cP), demonstrating an increase in viscosity with the adding of CUR. In comparison, formulations with ARG exhibited significantly reduced viscosity relative to the reference sample, with the lowest viscosity recorded for CH/PVA@ARG-ALA (95.09 ± 3.28 cP). The CH/PVA@ALA formulation showed a noticeable viscosity (158.66 ± 1.29 cP) similar to that of the CH/PVA matrix.

These findings suggest that the addition of CUR generally enhances the apparent viscosity of polymer solutions, while formulations with ARG show lower viscosity values, emphasizing the impact of ACs composition on the rheological properties of the electrospinning solutions.

3.2. Characterization of the CH/PVA@ACs NFs

3.2.1. Fourier Transform Infrared (FTI-R) Spectroscopy

The successful incorporation of ACs within the CH/PVA nanofibrous scaffold was confirmed by FT-IR spectroscopy, which revealed characteristic absorption bands corresponding to both the polymeric matrix and the loaded ACs (Figure 1). The FTIR spectrum of CH displayed a broad absorption band in the 3750–3000 cm^−1^ range, assigned to overlapping O–H and N–H stretching vibrations, indicative of extensive hydrogen bonding. Additional bands associated with C–H stretching vibrations (2920–2878 cm^−1^), amide I and amide II bands (1651–1543 cm^−1^), and C–O stretching modes (1149–950 cm^−1^) were consistent with the typical spectral features of CH [18,19,31,44,45,46]. PVA exhibited intense O–H stretching vibrations at 3650–3600 cm^−1^, along with characteristic C–H stretching bands in the 2939–2916 cm^−1^ range. The presence of residual acetate groups was evidenced by carbonyl stretching vibrations observed between 1713 and 1650 cm^−1^, confirming the partially hydrolyzed nature of PVA, while C–O stretching vibrations were detected around 1090 cm^−1^ [18,31,47]. In the FT-IR spectra of CH/PVA@ACs NFs, the characteristic absorption bands of both CH and PVA, together with additional signals attributable to the incorporated ACs, were observed, thereby confirming the successful embedding of ACs into nanofibrous scaffold.

ARG-loaded NFs showed bands assigned to N–H stretching vibrations (3340–3200 cm^−1^) and C=N stretching modes in the 1180–1100 cm^−1^ region [48]. The presence of ALA was supported by the absorption bands associated with N–H and C=O vibrations in the 1714–1651 cm^−1^ region, together with C–N-related vibrations between 1605 and 1327 cm^−1^ [44].

In RJ-containing NFs, peaks corresponding to amide I and amide II vibrations were detected near 1650 cm^−1^ and 1535–1560 cm^−1^, respectively, aligning with the protein components of RJ [31]. CUR-loaded NFs exhibited distinctive bands attributed to phenolic O–H stretching (3750–3550 cm^−1^), aromatic C=C/C=O vibrations (1650–1628 cm^−1^), and enolic C–O stretching modes in the 1273–1270 cm^−1^ range, aligning with the presence of CUR in the nanofibrous framework [46]. In general, the FT-IR spectra show the overlapping of distinctive bands from CH, PVA, and the incorporated ACs. No distinct new absorption bands were observed, suggesting that the ACs are incorporated predominantly through non-covalent interactions (e.g., hydrogen bonding) and physical entrapment within polymeric network.

3.2.2. SEM Morphology and Fibre Diameter

The CH/PVA formulation resulted in uniform NFs with an average diameter of 106.57 ± 0.48 nm, reflecting stable electrospinning conditions and reliable fiber formation (Figure 2A). The addition of ARG caused a minor decrease in fiber diameter (CH/PVA@ARG: 100.16 ± 0.065 nm) and produced a systematic organization of several uniform NFs, indicating enhanced jet stretching under these conditions (Figure 2B).

Formulations with ALA displayed larger fiber diameters, with CH/PVA@ALA having an average diameter of 113.10 ± 0.01 nm and fibers organized in a consistent pattern (Figure 2C). A comparable regular structure was noted for the CH/PVA@ARG–ALA formulation (103.26 ± 0.036 nm) (Figure 2D), even though the average diameter was slightly reduced, suggesting that ALA aids in stable fiber development while affecting jet viscoelasticity.

The RJ-containing formulation (CH/PVA@ARG–RJ: 112.19 ± 0.113 nm) exhibited the highest mean fiber diameter (Figure 2E) and also showed a more irregular and less uniform fiber arrangement. This morphological variability aligns with the elevated viscosity of the related polymer solution and indicates decreased jet stability during electrospinning.

For formulations containing CUR, the CH/PVA@CUR NFs showed an average diameter of 103.49 ± 0.056 nm, and SEM analysis indicated a sparse occurrence of minor particulate characteristics linked to CUR (Figure 2F). Combining CUR with ALA (CH/PVA@CUR–ALA) resulted in an increase in mean fiber diameter to 116.83 ± 0.389 nm, and the presence of nodal structures along the fibers was noted, suggesting localized heterogeneities in the nanofibrous network (Figure 2G).

Overall, these results demonstrate that changes in solution viscosity induced by different ACs are reflected not only in NFs diameter but also in fiber morphology and uniformity. Despite these variations, continuous fibrous structures were obtained for all formulations, highlighting the suitability of the CH/PVA polymeric system for electrospinning and its capacity to incorporate different ACs while maintaining structural integrity.

3.2.3. Surface Free Energy and Biocompatibility-Related Surface Properties

The high surface-to-volume ratio of NFs is associated with increased surface free energy, a physicochemical parameter that governs wettability and solid–liquid interactions at the biomaterial interface. These surface properties are particularly relevant for wound healing and tissue regeneration applications, where improved interaction with biological fluids can facilitate early biological events, such as protein adsorption and subsequent cellular attachment.

Surface free energy analysis provides valuable indirect but valuable information regarding the potential interaction of a material with biological fluids. In this context, materials exhibiting γ^SL^ values ≤ 4 mN/m are generally associated with favorable interfacial interactions and a reduced likelihood of adverse biological responses [45].

Referring to the investigated samples (Table 3), most CH/PVA@ACs formulations exhibited significantly lower γ^SL^ values compared to the reference CH/PVA NFs (14.29 ± 0.56 mN/m). γ^SL^ values within or close to the proposed favorable range were obtained for CH/PVA@ARG NFs (2.10 ± 0.23 mN/m), CH/PVA@ARG–RJ NFs (3.56 ± 0.44 mN/m), and CH/PVA@ALA NFs (4.21 ± 0.52 mN/m), indicating enhanced surface wettability and strong solid–liquid interactions. The CH/PVA@ARG-ALA NFs exhibited a higher γ^SL^ value (7.51 ± 0.15 mN/m), suggesting comparatively weaker interactions with aqueous media. This behavior may be related to intermolecular interactions between the functional groups of the incorporated ACs that partially modulate surface polarity.

In the case of CUR-containing NFs, CH/PVA@CUR displayed the highest γ^SL^ value (16.54 ± 0.23 mN/m), reflecting reduced surface wettability. However, the association of CUR with ALA resulted in a marked decrease in γ^SL^ (10.10 ± 0.25 mN/m), indicating improved interfacial interactions upon ALA incorporation, likely due to its hydrophilic character [5]. These observations are in agreement with the data reported in literature. For example, Kulkarni et al. [46] reported a water contact angle of 67.7° for CUR-loaded NFs chemically modified with thiocarbohydrazine at a comparable CUR content (3% w/v), a value closely matching the contact angle obtained for CH/PVA@CUR NFs (70.66 ± 0.39°). This similarity suggests that the intrinsic hydrophobicity of CUR tends to limit surface wettability, thereby influencing interfacial interactions with aqueous environments.

3.2.4. Swelling Degree

The SD (%) of the NFs is an important parameter that influences the release behaviour of the ACs from the polymeric matrix. Both the NFs morphology and the chemical structure of the incorporated ACs, affect membrane permeability and, consequently, the swelling capacity. High fluid absorption enhances membrane penetrability, facilitating the diffusion of ACs into the intramembranous space and potentially modulating their release profile [33].

As shown in Figure 3, the CH/PVA NFs exhibited the highest SD in DDW, reaching a maximum value of 280.15 ± 0.39% at 60 min. This behaviour indicates a pronounced affinity for aqueous media and a high absorption capacity under exudative conditions. The high swelling capacity can be primarily attributed to the hydroxyl groups present in both in CH and PVA, with facilitate interactions with polar molecules.

The swelling behavior of CUR-containing NFs was strongly dependent on the immersion medium. In PECF, CH/PVA@CUR NFs displayed a high SD, reaching 248.72 ± 0.49% at 60 min, while CH/PVA@CUR-ALA NFs showed an even higher SD of 331.58 ± 0.40% at 60 min. Notably, the SD recorded for CH/PVA@CUR-ALA NFs in PECF was comparable to that observed in DDW (Figure 3A,C). In PBS, lower values were recorded; however, CH/PVA@CUR–ALA exhibited higher values (240.29 ± 0.29% at 30 min and 278.91 ± 0.47% at 60 min) compared to CH/PVA@CUR (144.95 ± 0.65% at 30 min and 184.35 ± 0.82% at 60 min), indicating the contribution of ALA to increased fluid uptake (Figure 3B). Based on these findings, CUR appears to act predominantly as a “stiffening agent”, likely due to hydrophobic interactions and a limited capacity for hydrogen bindings [46].

NFs containing ALA generally exhibited higher SD, particularly in PBS (318.25 ± 0.65 at 30 min, 405.05 ± 0.46% at 60 min) and DDW (309.90 ± 0.20% at 30 min and 358.49 ± 0.53% at 60 min), consistent with the hydrophilic nature of ALA [33].

ARG-containing NFs exhibited high SD in DDW, reaching 557.16 ± 1.45% for CH/PVA@ARG, 602.66 ± 0.42% for CH/PVA@ARG-ALA, and 481.64 ± 1.45% for CH/PVA@ARG-RJ at 60 min. This behavior is consistent with literature reports indicating that ARG increases the overall hydrophilicity of NFs, due to the presence of amino and guanidino groups which attract water molecules and promote expansion of the polymeric network [49]. In PECF, SD of 348.78 ± 0.55%, 482.03 ± 0.27%, and 348.78 ± 0.19% were recorded at 60 min for CH/PVA@ARG, CH/PVA@ARG–ALA, and CH/PVA@ARG–RJ NFs, respectively. The synergistic effect of ARG-ALA and ARG-RJ association are evident. Specifically, ARG induces network expansion through its cationic functional groups [50], while ALA facilitates hydration due to intrinsic hydrophobicity [51]. In addition, RJ owing to its protein, sugar, and lipid content, modulates the swelling process primarily through hydrogen bonds interactions [52]. After 60 min, complete disintegration of NFs was observed.

3.3. In Vitro AC Release Profiles and Kinetic Analysis

Comparative analysis of the release profiles at pH 5.5, and 7.4 (Figure 4) demonstrates pronounced pH-dependent release behaviour of the ACs from the CH/PVA NFs, with direct relevance for wound healing applications.

At pH 5.5, ARG exhibited an approximately 21.03 ± 0.07% released within the first 30 min (Figure 4A). In contrast, ARG incorporated in combination with ALA (CH/PVA@ARG-ALA) or RJ (CH/PVA@ARG-RJ) displayed a slower and more sustained release, approximately 8.59 ± 0.5% and 14.15 ± 0.38%, respectively, within the first 30 min (Figure 4B). This behaviour is consistent with literature report [53], and can be attributed to electrostatic interactions between the positively charged groups of ARG and the negatively charged functional groups of CH/PVA nanofibrous scaffold and the co-incorporated ACs. At pH 7.4, representative of physiological conditions, ARG is released more rapidly (Figure 4C,D), approximately 81.39 ± 0.52% from CH/PVA@ARG and 73.64 ± 0.77% in combination with ALA (CH/PVA@ARG-ALA) and 62.65 ± 0.58% in combination with RJ (CH/PVA@ARG-RJ) within 24 h.

Overall, the pH-dependent release behaviour of ARG enables differential availability under distinct environmental conditions, which may be advantageous for wound healing applications by providing enhanced ARG release in neutral conditions, characteristic of the acute and subacute phase of the wound, and more sustained delivery under acidic conditions, representative for chronic, ischemic, necrotic or superinfected wounds [22].

ALA-loaded NFs (CH/PVA@ALA) exhibited a biphasic release profile at pH 5.5, characterized by an initial slow-release phase, reaching 26.53 ± 0.4% within 3 h, followed by an accelerated release up to 24 h (46.51 ± 0.18%). In contrast, at pH 7.4, ALA showed a more uniform and sustained release pattern, suggesting a pH-dependent modulation of its diffusion from the polymeric matrix.

For RJ-loaded NFs a higher release rate was observed at pH 7.4, with 74.99 ± 0.05% released within 24 h, whereas at pH 5.5 the release remained slower and more gradual (46.36 ± 0.38 within 24 h), indicating effective diffusion control exerted by the polymeric network under acidic conditions.

CUR-loaded NFs (CH/PVA@CUR) exhibited a complex, pH-sensitive release behavior. At pH 5.5, a pronounced initial burst release (25.30 ± 0.28% within 30 min) was followed by a sustained release phase, reaching a cumulative release of 89.44 ± 0.03% at 24 h. At pH 7.4, the initial burst release was attenuated (23.61 ± 0.08% within 60 min), and the cumulative release at 24 h was lower compared to the acidic medium, consistent with its reduced stability of CUR under neutral conditions. The co-association of CUR with ALA (CH/PVA@CUR-ALA) did not significantly affect the release profile of CUR at either pH value, indicating predominantly independent release kinetic of the two compounds.

Overall, single-AC-based CH/PVA NFs enabled tailored, pH-responsive release profiles, while co-loaded systems largely preserved independent release behavior, with limited interaction effects observed mainly in ARG-containing formulations.

To elucidate the release mechanisms of the ACs from CH/PVA nanofibrous systems, the experimental release data obtained at pH 5.5 and pH 7.4 were fitted to zero-order, first-order, Higuchi, Hixson–Crowell, and Korsmeyer–Peppas kinetic models.

For every formulation and pH condition, the optimal model was determined by the highest correlation coefficient (R^2^), and the Korsmeyer–Peppas release exponent (n) was applied to better understand the underlying transport mechanism (Table 4).

At pH 5.5, the release of ARG from CH/PVA@ARG NFs was most accurately characterized by the Korsmeyer–Peppas model (R^2^ = 0.971), featuring a low release exponent (n = 0.135), suggesting mainly Fickian diffusion. At pH 7.4, the release kinetics were more accurately described by the Higuchi model (R^2^ = 0.971), indicating a diffusion-controlled mechanism, whereas the elevated n value (n = 0.483) signifies a shift towards anomalous transport characteristics. A comparable release trend was noted for CH/PVA@ARG–ALA NFs, which likewise demonstrated a robust correlation with the Higuchi model (R^2^ = 0.980), indicating a diffusion-driven release mechanism with slight input from the matrix relaxation effects. The pH-sensitive release characteristics of ARG allow for quicker availability in neutral conditions and more sustained release in acidic environments, highlighting its potential significance for applications that need both rapid initiation and extended therapeutic effects.

ALA showed a notable pH-sensitive alteration in its release kinetics. At pH 5.5, the release profile of CH/PVA@ALA NFs demonstrated a strong correlation with various kinetic models, such as first-order (R^2^ = 0.994), zero-order (R^2^ = 0.985), Higuchi (R^2^ = 0.976), and Hixson–Crowell (R^2^ = 0.9916). The Korsmeyer–Peppas release exponent (n = 0.558) signifies anomalous (non-Fickian) transport, implying that ALA release in acidic environments is influenced by a combination of diffusion, matrix swelling, and erosion processes. At pH 7.4, the release mechanism transitioned to diffusion-controlled behavior, with the Korsmeyer–Peppas model demonstrating a strong fit (R^2^ = 0.995) and a low n exponent (n = 0.308), typical of Fickian diffusion. This shift indicates decreased matrix swelling and diminished polymer–drug interactions in neutral conditions.

In co-loaded systems, ALA did not meaningfully change the release kinetics of the related compounds. At pH 5.5, CH/PVA@ARG–ALA NFs showed significant correlations with first-order (R^2^ = 0.995), Higuchi (R^2^ = 0.977), and Hixson–Crowell (R^2^ > 0.994) models, with a release exponent (n = 0.308) suggesting that transport is dominated by diffusion. Likewise, CH/PVA@CUR–ALA NFs exhibited first-order kinetics (R^2^ = 0.993) alongside an unusual transport mechanism (n = 0.593). These results indicate that ALA functions as a kinetic modulator, facilitating regulated and adaptive release behavior in various pH settings.

At pH 5.5, the release of CUR from CH/PVA@CUR NFs was most accurately represented by first-order kinetics (R^2^ = 0.950), with a Korsmeyer–Peppas release exponent (n = 0.407), suggesting Fickian diffusion. A similar release mechanism was noted for CH/PVA@CUR–ALA NFs (R^2^ = 0.993, n = 0.362), indicating that ALA does not considerably affect CUR release in acidic conditions. At pH 7.4, CH/PVA@CUR NFs showed a transition to anomalous transport (n = 0.568), with the Higuchi model demonstrating the strongest correlation (R^2^ = 0.981), indicating contributions from matrix relaxation and the diminished stability of CUR in neutral settings. In comparison, CH/PVA@CUR–ALA NFs demonstrated a shift back to Fickian diffusion (n = 0.267), suggesting that ALA stabilizes the release behavior of CUR. This effect could be linked to alterations in the local microenvironment of the polymeric matrix, possibly restricting degradation processes like oxidation and hydrolysis [54]. RJ demonstrated uniform release characteristics from CH/PVA@ARG–RJ NFs at both pH levels. The release profiles were best described by the Higuchi model at pH 5.5 (R^2^ = 0.951) and pH 7.4 (R^2^ = 0.947), indicating diffusion-controlled release. The corresponding Korsmeyer–Peppas exponents (n = 0.457 at pH 5.5 and n = 0.497 at pH 7.4) place the release mechanism at the boundary between Fickian and anomalous transport, suggesting only a minor contribution of matrix relaxation.

The pH-independent kinetic behavior of RJ can be explained by its intricate composition, which includes biomolecules that have a broad range of molecular sizes and physicochemical characteristics. The combination of separate diffusion processes leads to an averaged release behavior that is largely unaffected by changes in pH.

In general, kinetic modeling reveals a distinct pH-dependent variation in release mechanisms from CH/PVA nanofibrous systems. These results emphasize the ability of CH/PVA@ACs NFs to modify their release characteristics according to environmental pH, demonstrating their appropriateness for applications that demand stage-dependent or environment-responsive delivery.

4. Discussion

The research presents the development and characterization of CH/PVA NFs loaded with natural ACs for wound healing purposes. The findings confirm the original hypothesis that the CH/PVA polymer matrix acts as a flexible platform able to adjust the release of embedded ACs based on environmental pH, thus meeting the specific needs of various wound healing phases. The physicochemical analysis of the polymeric solutions demonstrated a significant relationship between the properties of the solutions and the morphology and quality of the produced NFs. Electrical conductivity, an essential factor influencing the stability of electrospinning jets, rises with the addition of -NH_2_-containing ACs, and the CH/PVA@ARG-ALA solution displays the highest conductivity (7.15 mS).

This finding is consistent with reports indicating that increased conductivity favours uniform fibre formation and minimizes beads defects [20,55]. Importantly, the present study further demonstrates that an optimal balance between conductivity and viscosity is critical, particularly for CUR-containing formulations. Even with elevated apparent viscosities (e.g., η = 180.97 cP for CH/PVA@CUR), fibers with desirable mean diameters (103.49 ± 0.88 µm) were achieved, suggesting that conductivity plays a compensatory role in stabilizing the electrospinning process.

The pH range of polymeric solutions (3.49–10.17) indicates the inherent acid–base characteristics of the included ACs and greatly affects the stability of the complexes. Formulations containing ARG showed alkaline pH because of the guanidine moiety, but the association with ALA or RJ somewhat reduced this impact.

The ability to tune solution pH has direct implications for ACs stability; notable, CUR maintained stability in acidic environments (pH 3–5) [56], a finding corroborated by both FT-IR analysis and release behaviour.

FT-IR spectroscopy validated the effective incorporation of ACs into the CH/PVA nanofibrous matrix and indicated the presence of non-covalent interactions. Changes in band intensity and positions for -OH, -NH, C=O, and C=N groups suggest hydrogen bonding between polymers and ACs, enhancing matrix stability and enabling controlled release.

Morphological examination indicated the presence of CUR-associated crystalline domains on the NF surface, hinting at restrictions in CUR loading or complexation effectiveness. This result corresponds with literature studies indicating CUR’s propensity to crystallize because of its hydrophobic nature and conjugated π-electron framework [57], underscoring the necessity of formulation approaches that reduce phase separation.

Analysis of γ^SL^ offered significant insights into the biocompatibility of the developed systems. The CH/PVA@ARG (γ^SL^ = 2.10 ± 0.23 mN/m) and CH/PVA@ARG-RJ (γ^SL^ = 3.56 ± 0.44 mN/m) exhibited values below the widely accepted 4 mN/m threshold for favourable biomaterial–fluid interactions, indicating enhanced wettability and reduced risk of adverse biological responses. These characteristics align with the strong hydrophilicity of ARG and are expected to enhance interfacial interactions with biological fluids [58,59]. In contrast, formulations containing CUR showed higher γ^SL^ values, indicating CUR’s hydrophobic characteristics; nonetheless, co-loading with ALA markedly enhanced surface properties, affirming ALA’s effectiveness as a surface-modulating agent [51,60].

Swelling studies further supported the functional performance of the CH/PVA@ACs NFs. CH/PVA NFs exhibited maximum SD in DDW (280.15 ± 0.39% at 60 min), confirming high exudate absorption capacity. Importantly, this behaviour did not compromise the release performance of the incorporated ACs, indicating that the matrix hydration enhances functionality rather than limiting therapeutic action.

CH/PVA@ACs NFs demonstrate a release behavior that depends on pH, where co-loading typically maintains the separate release mechanisms and underscores the system’s adaptability for stage-specific wound healing applications. Release data indicate a distinct pH-dependent alteration of the transport mechanism, where acidic conditions (pH5.5), characteristic of intact skin, enhance diffusion-assisted and anomalous transport, while neutral conditions (pH7.4) primarily support diffusion-controlled release. This pH-sensitive response directly meets the clinical needs for targeted therapy at different stages of wound healing.

5. Conclusions

The research illustrates that blending natural ACs in the CH/PVA polymeric matrix significantly influences the physicochemical attributes, structural features, and functional efficacy of NFs. The adjustment of essential polymeric solution variables such as conductivity, pH, and viscosity directly affected fiber morphology, stability, and continuity, highlighting the critical role of ACs in the electrospinning process as well as the characteristics of the resulting materials. FT-IR analysis confirmed the effective incorporation of ACs and demonstrated non-covalent interactions with the polymeric network, reinforcing the development of stable systems suitable for controlled release.

Surface free energy, swelling behaviour, in vitro release and kinetics modelling results highlighted the appropriate biocompatibility as well as pH-responsive release capacity of the developed CH/PVA@ACs NFs. The identification of unique, compound-specific release mechanisms, along with maintaining pharmacokinetic independence in co-loaded formulations, signifies a crucial original contribution of this study. Significantly, ALA proved to be a potent kinetic and surface modulator, improving release control and interfacial characteristics, especially in formulations with hydrophobic substances.

It can be concluded that the co-loaded CH/PVA@ACs NFs represent a flexible and encouraging platform for applications in wound healing and tissue regeneration. By integrating structural stability, tunable release kinetics, and potential favorable biological performance, these systems provide a solid scientific basis for the future design of adaptive, stage-specific biomaterials tailored to the dynamic wound microenvironment.