Chitosan-Based Thermosensitive Hydrogel Loaded with Quercetin Inclusion Compound for Accelerating Infectious Wound Healing

Abstract

Background: Chitosan-based hydrogels exhibit excellent temperature-sensitive properties and are widely used as skin dressings. However, several challenges remain, such as long gelation times and difficulties releasing insoluble drugs, which limit their application in skin wound healing. In this study, we developed a novel sulfobutyl-β-cyclodextrin/quercetin@chitosan/hyaluronic acid hydrogel (Qe/SBE@CS/HA Gel). In this gel, SBE not only encapsulates Qe to form inclusion complexes, thereby enhancing the solubility of Qe, but also shortens the gelation time of thermosensitive gels through electrostatic adsorption with chitosan. Methods: Qe/SBE was prepared using the saturated solution method, while Qe/SBE@CS/HA gel was fabricated via electrostatic adsorption. The performance of the gels was evaluated using antibacterial, antioxidant, compatibility, and skin infection damage models. Results: The Qe/SBE@CS/HA Gel exhibits both thermosensitivity and acid sensitivity, releasing 91.9% of Qe in a medium with a pH of 5.0. This gel displays notable antibacterial activity and antioxidant characteristics. Furthermore, it shows excellent biocompatibility, as evidenced by hemolytic and in vivo degradation tests. The gel has the capacity to modulate chronic inflammation and facilitate angiogenesis and collagen synthesis, thereby significantly accelerating wound healing in wound and infection models. Conclusions: This multi-responsive and multifunctional gel shows potential as a therapeutic strategy for bacterial infection wounds.

1. Introduction

In recent years, chronic wounds have become increasingly challenging to treat due to the growing prevalence of chronic conditions such as age-related issues, diabetes, and biofilm-related infection [1]. Chronic wounds are characterized by persistent infections, biofilm formation, and heightened inflammation, with bacterial infections being a common cause [2]. Wound healing is a dynamic process that replaces damaged tissue and is typically divided into three consecutive stages: inflammation, proliferation, and remodeling [3]. During this process, bacterial infections often impede wound healing, leading to severe and even systemic infections [4]. Consequently, promoting the healing of infectious wounds has become a major long-term focus of clinical research. Although antibiotics can effectively control infections, prolonged use often leads to bacterial resistance and inadequate regulation of inflammation, resulting in delayed wound healing [5]. Therefore, the development of new drugs with potent antibacterial and anti-inflammatory activities is urgently required for the treatment of infectious wounds.

In recent times, Traditional Chinese Medicine (TCM) has achieved significant integration in the treatment of various diseases due to its potent bioactivity, minimal adverse effects, and robust pharmacological properties, which present lower chances of developing resistance [6,7]. Quercetin (Qe), a flavonoid compound, is widely utilized in TCM through herbs such as Rutin, Bupleurum Chinese, Mulberry Leaves, and Sophora Japonica [8]. Its diverse pharmacological properties have drawn considerable attention, as it has demonstrated the ability to combat bacterial infections, alleviate inflammation, and exhibit potent antioxidant effects [9]. Recent studies suggest that Qe can enhance the proliferation and migration of fibroblasts, and its local application has been shown to accelerate skin wound healing in mice [10,11]. However, the direct use of Qe is hindered by low availability, necessitating the development of drug delivery systems to enhance its effectiveness in skin injury repair.

Cyclodextrins are often used as carriers for poorly soluble drugs. Sulfobutyl-β-Cyclodextrin (SBE) is a polyanionic β-cyclodextrin derivative that has been approved by the FDA as a pharmaceutical excipient [12]. SBE can bind various organic, inorganic, and biological small molecules to form host-guest inclusion complexes, which improves the solubility of drugs [13]. Therefore, encapsulating Qe with SBE to form the Qe/SBE inclusion complex enhances the water solubility and bioavailability of Qe [14]. However, the Qe/SBE liquid form is difficult to stay in the damaged skin area.

Hydrogel (Gel) are increasingly used as drug carriers for wound healing due to their higher water content, biocompatibility, and biodegradability, making them suitable for a wide range of clinical applications as dressings and scaffolds [15,16]. In particular, temperature-sensitive gels can rapidly gel at body temperature, allowing them to adequately fill irregular wound sites [17]. Chitosan (CS), a natural polysaccharide, is an FDA-approved for use in biomedicine. Its numerous amino and hydroxyl groups enable the formation of self-healing gels through reactions with crosslinkers, involving dynamic imine bonds, electrostatic interactions, and hydrogen bonding [18,19,20]. Our previous research demonstrated that CS, in combination with glycerol phosphate disodium salt, could be used to prepare thermosensitive gel for vancomycin delivery to treat osteomyelitis [21]. However, the CS gel has the drawback of a long gelation time, which makes it prone to detachment when administered to the injured skin area of animals.

The characteristics of chitosan-based gels, including gelation time and gel strength, can be regulated via ionic crosslinking [22]. Hyaluronic acid (HA) and cyclodextrin often form nanomedicine delivery systems with chitosan via electrostatic adsorption and other interactions and are used for anti-tumor and anti-infection purposes [23]. However, their application in gel delivery systems is less reported.

In this study, we developed a novel sulfobutyl-β-cyclodextrin/quercetin@chitosan/hyaluronic acid gel (Qe/SBE@CS/HA Gel). In this gel, SBE serves a dual function. It not only encapsulates Qe to form inclusion complexes (Qe/SBE) but also shortens the gel’s gelation time through ion crosslinking with chitosan. Through a comprehensive and systematic evaluation of in vitro and in vivo antioxidant, antibacterial, and skin repair effects studies, we assess the feasibility of using this local delivery system for skin injury repair and provide new insights and methods for clinical treatment to promote wound healing (Scheme 1).

Scheme 1. Schematic showing application of the Qe/SBE@CS/HA Gel to heal infectious skin wounds by antibacterial and antioxidant effect (Created by the authors).

2. Results and Discussion

2.1. Construction and Characterization of a Novel Qe/SBE@CS/HA Gel

2.1.1. Construction and Formulation Optimization of Qe/SBE

The Qe/SBE inclusion complex was prepared using the saturated solution method (Figure 1A). By leveraging the hydrophobic inner cavity of SBE, the poorly soluble drug quercetin can be encapsulated. Simultaneously, by exploiting the hydrophilic outer shell of SBE, the solubility of quercetin can be enhanced.

Figure 1. Characterization of Qe/SBE (A) Preparation of Qe/SBE. (B) Phase solubility. (C) Formulation optimization (Mean ± SD, n = 3). (D) Fourier transform infrared spectroscopy analysis. (E) DSC analysis. (F) SEM analysis. note:To more clearly reflect the states of several samples within the inclusion compound, the magnification for observation was slightly different.

A series of characterizations were conducted to evaluate the properties of Qe/SBE. Solubility is an important parameter for assessing the stability of the inclusion complex formed between the host and guest [24]. As shown in Figure 1B, the solubility of quercetin in aqueous solution increases linearly with the increasing concentration of cyclodextrin. The inclusion equilibrium constant for quercetin and SBE was calculated to be 2.3 × 10³ mol⁻¹.

The Qe forms a supramolecular inclusion complex with SBE through non-covalent interactions, including hydrophobic and hydrogen bonding interactions. The formed inclusion complex exhibits good stability [25].

The formula of SBE/Qe was optimized, and it was found that the inclusion rate increases with the molar ratio of SBE/Qe. Finally, when the molar ratio of SBE/Qe reaches 5:1, the inclusion rate reaches 95.8%, and the maximum drug loading is 4.2% (Figure 1C). Further increasing the molar ratio of SBE to Qe led to no significant change in the encapsulation efficiency, while the drug loading efficiency began to decrease. This is due to the increased dosage of SBE in the formulation.

FTIR spectroscopy was used to study the successful encapsulation of quercetin into SBE (Figure 1D). The results show that the absorption peak of the carbonyl C=O stretch of Qe was detected at a wavenumber of 1654 cm⁻¹. The C=C stretch of the benzene aromatic ring was observed at 1605 cm⁻¹ and 1523 cm⁻¹.

For SBE, absorption peaks appear at 1364 cm⁻¹, 1205 cm⁻¹, and 1040 cm⁻¹, typically corresponding to the S=O asymmetric stretching vibration peaks and the symmetric stretching vibration peaks. Regarding the 1653 cm⁻¹ absorption peak generated in SBE, which is similar to the H-O-H bending vibration peak, it is speculated that this might be caused by the fact that the cavity structure of cyclodextrin is prone to adsorb water in the environment. This is similar to what has been reported in the literature.

The FTIR spectrum of the physical mixture of Qe and SBE shows some characteristic peaks of both substances, indicating that the functional groups of the two substances in the mixture largely retain their individual characteristics and there are no other interactions. The Qe/SBE physical mixture showed attenuation of the 1605 cm⁻¹ peak with no disappearance, indicating that simple hydrogen bonding between Qe and SBE only weakens the peak but cannot eliminate it. The spectrum of the Qe/SBE inclusion complex differs from that of the mixture, with some characteristic peak positions or intensities changing. The Qe/SBE inclusion complex showed the complete disappearance of the 1605 cm⁻¹ peak, which corresponds to the aromatic ring stretching vibration of Qe. This suggests that Qe may be partially embedded in the SBE cavity, causing the absorption of functional groups to disappear.

The DSC thermogram (Figure 1E) further confirmed the successful encapsulation of Qe in SBE. SBE exhibited distinct endothermic peaks at 93.3 °C, 195.5 °C, 214.0 °C, and 359.7 °C, indicating thermal changes at the corresponding temperatures. Qe showed a significant endothermic peak only at 327.1 °C. After the complexation of Qe and SBE, no obvious peaks of Qe (327.1 °C) and SBE (195.5 °C, 214.0 °C) were observed in the inclusion complex. However, there were peaks at 195.6 °C, 214.0 °C, and 323.5 °C in the Qe/SBE mixture, which were very close to the peak of Qe. This behavior was different from that of the physical mixture, where the cavity structure of SBE conceals certain functional groups of Qe, but with altered peak positions and characteristics [26]. These results indicated that Qe was encapsulated into the SBE cavity, leading to the loss of characteristic peaks.

The scanning electron microscopy (SEM) images (Figure 1F) confirmed these findings. SBE exhibits a block-like shape, while Qe presented a rod-like structure with a smooth surface. In the physical mixture, both Qe and SBE maintained their individual structures. In contrast, the morphology and shape of the Qe/SBE inclusion complex prepared via the solvent evaporation method were entirely distinct from those of the original products. It exhibited a spherical shape, and no rod-like structures resembling Qe were detected [14]. This might be because Qe is embedded in the SBE, and during the preparation process, the ethanol volatilizes and freeze drying forms a new structure. The SEM morphology of SBE is different from that reported previously, which might be related to the manufacturer.

The ¹H NMR spectra are presented in Figure S1. Compared with pure Qe and the Qe/SBE mixture, the chemical shifts of Qe at 9.30, 9.36, 9.58, 10.77, and 12.49 ppm disappeared in the inclusion complex. This suggests that these sites of Qe were enclosed in the cavity.

The successful formation of SBE/Qe inclusion complexes is the basis of the gel’s therapeutic efficacy. Its mechanism lies in the hydrophobic-hydrophilic interaction between Qe and SBE. Quercetin (Qe) is a lipophilic flavonoid with poor aqueous solubility, which limits its bioavailability in wound microenvironments. SBE, a modified cyclodextrin with a hydrophobic inner cavity and hydrophilic sulfobutyl ether side chains, can encapsulate Qe into its cavity via hydrophobic interactions [14]. We verified the formation of Qe/SBE inclusion complexes through multiple methods, such as FT-IR, DSC, SEM, and NMR. This lays the foundation for further development of the chitosan gel system.

2.1.2. Construction and Formulation Optimization of Qe/SBE@CS/HA Gel

Chitosan, due to its abundant hydroxyl and amino group structure, is often used as a gel material. Commercially available chitosan dressings lack temperature sensitivity and are difficult to completely fill irregular wounds. Meanwhile, the antioxidant and anti-inflammatory properties of single chitosan are relatively weak [27].

Furthermore, based on the CS/GP thermosensitive gel we previously constructed [21], we have developed a new thermosensitive gel composed of CS, HA, and SBE/Qe to address the problem of slow gelation speed and enable rapid gelation at the skin wound site, thus reducing waste. Qe/SBE@CS/HA Gel is a temperature sensitive gel that remains in a liquid state at low temperatures and gradually transforms into a semi-solid gel form as the temperature rises. As shown in Figure 2A, the Qe/SBE@CS/HA Gel changes from a liquid at room temperature to a solid at 37 °C, demonstrating that this gel exhibits excellent temperature sensitivity.

Figure 2. Characterizations of Qe/SBE@CS/HA Gel. (A) Photograph of Qe/SBE@CS/HA Gel aqueous solutions exhibiting sol and gel states at 15 °C and 37 °C, respectively. (B,C) Gelation time of gel (Mean ± SD, n = 3). Rheological properties exhibiting (D) viscosity (η), (E) storage modulus (G′), and (F) loss modulus (G″) of the aqueous solutions of the CS Gel, CS/HA Gel, and Qe/SBE@CS/HA Gel as a function of temperature, respectively. (G) In vitro drug release curve (Mean ± SD, n = 3). (H) In vitro degradation curve (Mean ± SD, n = 3). (I) Extracorporeal water absorption capacity curve (Mean ± SD, n = 3). (J) Fourier transform infrared spectroscopy analysis. (K) SEM image of CS Gel, CS/HA Gel, and Qe/SBE@CS/HA Gel. *** p < 0.001, ** p < 0.01, and * p < 0.05.

Gelation time is a critical parameter for medical wound dressings [28]. As illustrated in Figure 2B, the addition of HA shortens the gelation time, and an optimal gelation time of 3.92 min is achieved when the HA:CS ratio is 2:5. This is because CS contains amino groups (-NH₂), which become protonated and positively charged, while HA contains carboxyl groups (-COOH), which ionize to form negative charges. The two components form ionic bonds through electrostatic interactions, enhancing intermolecular attraction and accelerating the formation of the gel network [29].

Meanwhile, Figure 2C shows that the addition of SBE further reduces the gelation time to 2.52 min for the Qe/SBE inclusion complex. This is likely due to the formation of electrostatic bonding between the gel and the inclusion complex, significantly enhancing intermolecular interactions and providing more binding sites for crosslinking reactions [30]. Therefore, HA, SBE, and CS can all reduce the gelation time and slow down drug release through mechanisms such as electrostatic adsorption and hydrogen bonding.

2.2. Rheological Characteristics of Qe/SBE@CS/HA Gel

Temperature significantly influences the viscosity, storage modulus (G′), and loss modulus (G″) of the gel. The temperature-sensitive gel exhibits the characteristic of a sol-gel transition related to temperature. When G″ is greater than G′, it exhibits fluid properties; when G′ is greater than G″, a gel structure is formed.

As shown in Figure 2D, the three gels exhibit good fluidity in the low-temperature region (approximately 0–30 °C). As the temperature increases (30–50 °C), intermolecular interactions, such as hydrogen bonding and hydrophobic effects, are enhanced, leading to a rapid increase in viscosity.

The initial gelation temperature of the CS/HA gel is approximately 30 °C. When G′ and G″ intersect, a phase transition occurs. The intersection point represents the temperature at which the phase transition takes place. Subsequently, G′ continues to increase and becomes greater than G″ (Figure 2E,F).

The Qe/SBE inclusion complex forms more hydrogen bonds with the CS/HA gel, thereby decreasing the initial gelation temperature of the Qe/SBE@CS/HAGel and enhancing its viscoelasticity.

2.3. In Vitro Drug Release Characteristics of Qe/SBE@CS/HA Gel

CS-based Qe gels rely on the physical mixing of Qe and chitosan, resulting in low solubility, burst release, etc. [31]. Considering that the pH of the wound surface affects the healing process [32], we tested the drug release rates of Qe from Qe/SBE@CS Gel and Qe/SBE@CS/HA Gel under different pH conditions (pH 7.4 and pH 5.0).

Figure 2G shows that after 12 h in a normal physiological environment (PBS, pH = 7.4), the release amounts of Qe from Qe/SBE@CS Gel and Qe/SBE@CS/HA Gel reached approximately 41.3% and 45.5%, respectively. In an acidic environment (PBS, pH = 5.0), the release amounts of Qe from Qe/SBE@CS Gel and Qe/SBE@CS/HA Gel reached approximately 81.5% and 54.1% within 12 h, indicating a higher drug release rate in the first 12 h. After 72 h, the amounts of Qe released from Qe/SBE@CS Gel and Qe/SBE@CS/HA Gel in the acidic environment (pH = 5.0) were approximately 91.9% and 64.9%, respectively, while those in the normal physiological environment (pH = 7.4) were approximately 48.0% and 52.7%.

In the medium at pH 5.0, the cumulative release of Qe from Qe/SBE@CS/HA Gel was approximately 27% lower than that from Qe/SBE@CS Gel. This outcome cannot be ascribed to a single electrostatic adsorption mechanism, but rather to the synergistic effects of three interrelated molecular and structural alterations triggered by the incorporation of HA. CS is protonated to carry abundant positive charges (-NH₃⁺), while HA retains negatively charged carboxylate groups (-COO⁻). The introduction of HA triggers spontaneous ionic cross linking between CS, HA, and SBE via electrostatic interactions, forming a more compact three-dimensional network compared to the non-crosslinked CS Gel. Also, the zeta potentials of CS/HA Gel, Qe/SBE@CS Gel, and Qe/SBE@CS/HA Gel were −5.4 ± 0.5 mV, −16.0 ± 0.6 mV, and 19.2 ± 1.1 mV, respectively. The potential results can explain, from another perspective, that CS, HA, and SBE exert a stronger charge compression effect through spontaneous ionic crosslinking.

We tested the degradation behavior of each gel at a pH of 5.0. As shown in Figure S2, the in vitro degradation rate is: CS Gel > CS/HA Gel > Qe/SBE@CS Gel > Qe/SBE@CS/HA Gel. Under a pH 5.0 environment, the amino groups of the chitosan chains may be partially protonated, resulting in the breakage of inter-chain hydrogen bonds and swelling, thereby accelerating the gel hydrolysis and degradation. The dual introduction of HA and SBE can form a more compact network structure, slowing down the degradation rate. Therefore, the drug release of Qe/SBE@CS/HA gel is slower than that of Qe/SBE@CS Gel at pH 5.0. This may be attributed to the electrostatic adsorption of Qe/SBE within the gel matrix after gelation, which results in a slower release rate. Therefore, these results indicate that Qe/SBE@CS/HA Gel is pH-sensitive, and the addition of HA slows down the release of Qe from Qe/SBE@CS/HA Gel.

To explore the in vitro release mechanism of Qe/SBE@CS/HA Gel, various models were employed for fitting, and the results are presented in Table 1. In the realm of drug release kinetics models, the model is usually selected by comparing the correlation coefficient R² of the fitting equation. The R² values for the release curves of Qe/SBE@CS Gel and Qe/SBE@CS/HA Gel at pH 5.0 and pH 7.4, simulated using first-order release kinetics, were the highest, with values of 0.9435, 0.8882, 0.9538, and 0.9162, respectively. The fitting results indicated that the first-order model had a high R² value, suggesting that the release process followed a first-order kinetic trend, which is related to concentration-dependent drug release from the gel network.

Table 1. Release kinetics fitting of Qe/SBE@CS Gel and Qe/SBE@CS Gel in different media.

Samples	Model	Equation	R2
Qe/SBE@CS Gel pH5.0	Zero-order	Y = 50.89 + 0.77X	0.3937
	First-order	Y = 84.02 (1 − e−0.37X)	0.9435
	Higuchi	Y = 8.316X1/2 + 37.477	0.6197
	Ritger-peppas	Y = 42.573X0.208	0.8063
Qe/SBE@CS/HA Gel pH5.0	Zero-order	Y = 36.468 + 0.55X	0.5052
	First-order	Y = 57.03 (1 − e−0.348X)	0.8882
	Higuchi	Y = 5.826X1/2 + 25.775	0.7414
	Ritger-peppas	Y = 28.664X0.213	0.8895
Qe/SBE@CS Gel pH7.4	Zero-order	Y = 20.35 + 0.509X	0.5946
	First-order	Y = 43.45 (1 − e−0.433X)	0.9538
	Higuchi	Y = 5.017X1/2 + 13.054	0.8034
	Ritger-peppas	Y = 18.06X0.251	0.9043
Qe/SBE@CS/HA Gel pH7.4	Zero-order	Y = 40.216 + 0.216X	0.3840
	First-order	Y = 49.528 (1 − e−0.219X)	0.9162
	Higuchi	Y = 2.715X1/2 + 32.709	0.5702
	Ritger-peppas	Y = 29.73X0.143	0.7761

The Ritger-Peppas model is widely acknowledged for analyzing drug release mechanisms from polymeric gels. The calculated release exponent (n) values for all groups were 0.238, 0.213, 0.251, and 0.143, respectively (Table 1). The release index (n) values of each group were all less than 0.45, indicating that they conformed to the Fickian diffusion mechanism. This implies that the drug release mechanism of the Qe/SBE@CS/HA Gel exhibits both concentration-dependent release kinetics and a Fickian diffusion-dominated release mechanism.

Wang constructed the pH/temperature-sensitive chlorantraniliprole/chitosan complex, and its in vitro release kinetics follows the first-order or Ritger-Peppas model [33]. It shares similarities with the gel we constructed. Therefore, the Fickian diffusion mechanism is beneficial for our gel-based delivery system. It ensures that drug release is stable and predictable, avoiding burst release caused by gel erosion or excessive swelling. Combined with the first-order kinetic trend, the gel can maintain a sustained drug concentration at the target site, which is advantageous for improving therapeutic efficacy and reducing side effects.

2.4. Swelling, Degradation Tests and Characterization

The biodegradability of the gel is a key indicator for evaluating its biological safety [34]. As the wound heals, the applied gel gradually degrades, thereby promoting optimal wound recovery.

In the degradation test, the Qe/SBE@CS/HA Gel demonstrated a sustained degradation over a period of 14 days (Figure 2H), indicating its superior performance as a dressing in wound—healing applications.

To assess the gel’s water-absorption capacity in the presence of wound exudate, its swelling behavior was evaluated. As shown in Figure 2I, the swelling ratios of CS Gel, CS/HA Gel, Qe/SBE@CS Gel, and Qe/SBE@CS/HA Gel were 224 ± 8%, 225 ± 8%, 231 ± 6%, and 235 ± 8%, respectively. As shown in Figure S3, the swelling behavior of the gels in media with different pH values was characterized. The swelling ratios of Qe/SBE@CS Gel and Qe/SBE@CS/HA Gel in the media followed the order of pH 1.2 > pH 5.0 > pH 6.8 > pH 7.4. This phenomenon was attributed to the protonation of chitosan under low pH conditions, which enhanced the gel swelling behavior. Meanwhile, Qe/SBE@CS Gel exhibited a higher swelling ratio than Qe/SBE@CS/HA Gel in the media at pH 1.2 and pH 5.0. This result was consistent with the drug release profile, which could be explained by the more compact structure formed via electrostatic interactions among CS, HA and SBE components in Qe/SBE@CS/HA Gel, thus leading to a lower swelling ratio compared with Qe/SBE@CS Gel.

The water-absorption process of the gel is essentially a process of filling the internal pores with water. The open pores can directly come into contact with the external water, and the rapid penetration of water leads to an increase in the expansion rate. The higher the porosity, the larger the space available for water to occupy, and the higher the water absorption and swelling ratio usually is. These results indicate that Qe/SBE@CS/HA Gel possesses a denser pore structure, which facilitates the efficient absorption of accumulated wound exudate.

FT-IR spectra of Qe/SBE, CS/HA Gel, the mixture of Qe/SBE and CS/HA Gel, and Qe/SBE@CS/HA Gel are presented in Figure 2J. It is evident that both Qe/SBE and the mixture of Qe/SBE and CS/HA Gel exhibit absorption peaks corresponding to S=O at 1206 cm⁻¹ and 1040 cm⁻¹. In contrast, Qe/SBE@CS/HA Gel does not display a distinct absorption peak at 1206 cm⁻¹ and 1040 cm⁻¹. This discrepancy may be attributed to electrostatic interactions, wherein the negative charge of SO₃ is neutralized by the -NH₃⁺ moiety of chitosan. This interaction could lead to a slight alteration in the S=O bond length and a consequent modification of the S=O absorption peak.

SEM images of CS/HA Gel, SBE@CS/HA Gel, and Qe/SBE@CS/HA Gel (Figure 2K) revealed that all three gels are porous materials. Notably, Qe/SBE@CS/HA Gel exhibited a more distinct and densely interconnected porous network [35]. This porous morphology implies superior air permeability, which is beneficial for skin wound healing.

2.5. In Vitro Antibacterial Evaluation

Skin wounds are often accompanied by bacterial infections, which significantly delay the wound-healing process [36]. Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) were used to evaluate the antibacterial effects of Qe/SBE@CS/HA Gel.

As shown in Figure 3A,B, with an increasing Qe concentration, the bacterial viability of S. aureus and MRSA in the Qe/SBE@CS/HA Gel, CS/HA Gel, and Qe/SBE groups all decreased. Notably, the bacterial viability in the Qe/SBE@CS/HA Gel group was significantly lower than that in the other two groups.

Figure 3. Evaluation of the in vitro antibacterial effects of Qe/SBE@CS/HA Gel. (A,B) Growth inhibition curve of S. aureus (A) and MRSA (B) evaluated by the turbidimetric method (Mean ± SD, n = 3) note: The CS/HA Gel group does not contain the drug Qe, and the amount added is the same as the amount of chitosan used in the Qe/SBE@CS/HA Gel. (C–E) Inhibition zone diameter analysis of S. aureus and MRSA evaluated using the Oxford cup method (Mean ± SD, n = 3). (F,G) Photographs and quantification of bacterial colonies formed on an agar plate after exposure to different treatment groups, assessed via the agar diffusion assay (Mean ± SD, n = 3). *** p < 0.001.

Overall, Qe/SBE@CS/HA Gel exhibited the strongest antibacterial properties against both bacteria, indicating that the incorporation of Qe/SBE into CS/HA Gel enhanced its antibacterial performance.

The inhibition zone results (Figure 3C–E) showed that the diameter of inhibition zone is in the following order: Qe/SBE > Qe/SBE@CS/HA Gel > CS/HA Gel. The inhibition zone diameter of Qe/SBE and Qe/SBE@CS/HA Gel against S. aureus were 13.6 ± 0.4 cm and 10.6 ± 0.3 cm. The inhibition zone diameter of Qe/SBE and Qe/SBE@CS/HA Gel against MRSA were 7.8 ± 1.2 cm and 6.4 ± 0.4 cm.

Subsequently, the plate counting method was used to further evaluate the antibacterial ability of Qe/SBE@CS/HA Gel, CS/HA Gel, and Qe/SBE. As shown in Figure 3F, there was no obvious bacterial mortality in the PBS group. When CS/HA gel and Qe/SBE were co-culture with bacteria, both S. aureus and MRSA exhibited partial mortality. This effect may be due to the electrostatic binding of CS (containing amino groups) to the bacterial cell surface, which disrupts membrane permeability, combined with the ability of quercetin (with multiple phenolic hydroxyl groups) to damage the bacterial cell membrane [37]. Importantly, when Qe/SBE@CS/HA Gel was co-cultured with bacteria, nearly all bacteria colonies were eliminated, with antibacterial rates against S. aureus and MRSA reaching 94.0% ± 0.9% and 96.1% ± 0.7%, respectively.

2.6. Antioxidant Performance Evaluation

Skin wound injury and infection often induce local oxidative stress and inflammation [3]. Qe exhibits strong antioxidant activity, promoting wound healing by scavenging excessive reactive oxygen species (ROS) [38,39]. In this study, the antioxidant capacity of Qe/SBE samples was evaluated by ABTS and DPPH assays. At a quercetin Qe concentration of 50 μg/mL in Qe/SBE@CS/HA Gel, the ABTS radical scavenging rate reached 100%. Additionally, at a concentration of 100 μg/mL of Qe/SBE@CS/HA Gel, the DPPH scavenging rate also reached 100%, both demonstrating a clear dose-dependent relationship (Figure 4A,B). Compared with Qe/SBE, CS/HA Gel, and Qe/SBE@CS/HA Gel, the ABTS and DPPH scavenging capacities followed the order: Qe/SBE > Qe/SBE@CS/HA > CS/HA Gel (Figure 4C,D). To further clarify the underlying reason for the reduced antioxidant activity of Qe/SBE@CS/HA Gel compared with free Qe/SBE, we performed antioxidant activity assays on the gel extracts. As shown in Figure S4, the Qe/SBE@CS/HA Gel extracts exhibited comparable performance to free Qe/SBE in both DPPH and ABTS radical scavenging assays. This demonstrates that the antioxidant capacity of Qe/SBE remains unaffected by encapsulation within the gel. Therefore, we attribute this phenomenon to the impeded release of Qe/SBE from the gel matrix. Consistent with the in vitro release profile, the antioxidant activity of the Qe/SBE@CS/HA Gel is reduced due to the delayed release of Qe, which results from electrostatic interactions between Qe/SBE and CS within the gel matrix. Finally, to evaluate the intracellular antioxidant activity, a hydrogen peroxide-induced oxidative stress model was established in HaCat cells. As shown in Figure 4E, compared with the model group, cells treated with Qe/SBE and Qe/SBE@CS/HA Gel exhibited significantly reduced green fluorescence, indicating decreased ROS levels. Flow cytometry analysis (Figure 4F,G) quantitatively confirmed this trend, demonstrating the excellent ROS scavenging ability of both formulations.

Figure 4. Evaluation of the in vitro antioxidant effects of Qe/SBE@CS/HA Gel. (A,B) Evaluation of the in vitro antioxidant activity of Qe/SBE@CS/HA Gel using ABTS and DPPH assays at various concentrations (Mean ± SD, n = 3). (C,D) The antioxidant capacities of Qe/SBE, CS/HA Gel, and Qe/SBE@CS/HA Gel were compared using ABTS and DPPH assays at a concentration of 10 μg/mL Qe (Mean ± SD, n = 3). (E–G) Evaluation of the antioxidant capacity of the gels against hydrogen peroxide-induced cell damage, using fluorescence microscopy and flow cytometry based on the ROS method (Mean ± SD, n = 3). (H,I) Cell viability after 24 h and 72 h exposure to hydrogen peroxide, as induced by the gel (Mean ± SD, n = 5), note: Blue, red and green respectively represent CS/HA Gel, Qe/SBE and Qe/SBE@CS/HA Gel. *** p < 0.001.

Furthermore, MTT assay results (Figure 4H,I) showed that at a Qe concentration of 50 μg/mL, both Qe/SBE and Qe/SBE@CS/HA Gel significantly promoted the proliferation of H₂O₂-injured HaCat cells. This enhancement is attributed to the phenolic hydroxyl groups of Qe, which can bind to free radicals and form stable compounds, thereby reducing oxidative damage in both extracellular and intracellular environments.

Silver ion dressings, which are medical materials based on the broad-spectrum antibacterial properties of silver ions to promote wound healing, are widely used in the care of infected wounds, burns, pressure ulcers, and other types of wounds [40]. Compared with commercial silver ion dressings, our gel offers excellent antioxidant properties and lower cytotoxicity.

In summary, Qe/SBE@CS/HA Gel functions as an effective ROS scavenger and can provide antioxidant protection in both extracellular and cellular microenvironments, making it highly promising for clinical wound healing applications.

2.7. Biocompatibility

Excellent biocompatibility is essential for the clinical application of biomaterials, encompassing both cytotoxicity and hemolytic activity [41]. The effect of the gel on the proliferation of untreated HaCaT cells is used to evaluate the gel’s biocompatibility. The MTT assay demonstrated that varying concentrations of Qe did not significantly impact the viability of normal HaCat cells (Figure 5A,B), indicating that Qe/SBE@CS/HA Gel exhibits minimal cytotoxicity.

Figure 5. Evaluation of Qe/SBE@CS/HA Gel biocompatibility. (A,B) Cell viability after 24 h and 72 h of exposure to hydrogen peroxide-induced oxidative stress in the presence of the gel (Mean ± SD, n = 5). (C) Hemolysis results demonstrating blood compatibility of CS/HA Gel, Qe/SBE, and Qe/SBE@CS/HA Gel (Mean ± SD, n = 3). (D) In vivo degradation of the gel observed at 3 and 10 days post-subcutaneous implantation in mice.

The hemolysis test is crucial indicator of the biosafety of gel dressings. As shown in Figure 5C, no obvious hemolysis was observed in the CS/HA Gel, Qe/SBE, or Qe/SBE@CS/HA Gel groups compared with the positive control, and no apparent damage was noted compared with the negative control. The hemolysis rates of CS/HA Gel, Qe/SBE, and Qe/SBE@CS/HA Gel were 2.34%, 0.9%, and 0.44%, respectively, all below the 5% threshold [42], indicating excellent hemocompatibility and compliance with biological safety standards.

Furthermore, in vivo biocompatibility was assessed by observing the subcutaneous degradation of the gel. As shown in Figure 5D, after and 10 days of administration, both Qe/SBE@CS/HA Gel and CS/HA Gel gradually degraded without causing inflammation in the surrounding tissues, and neovascularization of capillaries was observed, suggesting favorable tissue integration and biodegradability.

We comprehensively evaluated the biological safety of the Qe/SBE@CS/HA Gel through a series of in vitro and in vivo experiments, including cytotoxicity tests, hemolysis tests, and in vivo degradation experiments. However, considering its subsequent clinical application, there are still deficiencies in the assessment of the material’s safety. It is necessary to investigate whether local inflammation or even systemic chronic inflammation occurs during long-term administration, as well as the systemic side effects and immunogenicity of the drug after administration.

2.8. In Vivo Wound Healing Capacity Analysis

CS is commonly used as a carrier material in tissue engineering [43]. After being loaded with Qe, the Qe/SBE@CS/HA Gel not only promotes skin tissue regeneration but also exhibits potent anti-infective properties. An MRSA-infected full-thickness skin defect mouse model was established to evaluate the gel’s wound healing capability [44], as illustrated in Figure 6A.

Figure 6. Evaluation of the in vivo wound healing effects. (A) Schematic diagram of the in vivo animal experiment schedule at different time points. note: The pink area indicates the full skin excision region, and the blue area denotes the region for drug administration and sample collection. (B,D) Photographic of wound healing in MRSA-infected full-thickness skin defects following different treatments (Mean ± SD, n = 6). (C,E) Quantitative assessment of in vivo bactericidal efficacy Qe/SBE@CS/HA Gel, demonstrating significant intergroup difference based on bacterial colonization in wound tissues at day 2 (Mean ± SD, n = 3). *** p < 0.001, ** p < 0.01, and * p < 0.05.

As shown in Figure 6B, during the treatment period, all treatment groups (CS/HA Gel, Qe/SBE, and Qe/SBE@CS/HA Gel) demonstrated superior wound healing compared to the control group (PBS). Statistical analysis of wound healing (Figure 6D) also demonstrated that the Qe/SBE@CS/HA Gel group significantly outperformed the Qe/SBE and CS/HA Gel groups. On the third day, the skin remaining rates of PBS, CS/HA Gel, Qe/SBE and Qe/SBE@CS/HA Gel group were 50.2 ± 6.4%, 43.0 ± 5.4%, 43.9 ± 7.8%, and 36.5 ± 6.3% respectively. By day 14, the wound closure rate in the Qe/SBE@CS/HA Gel group reached 99%, indicating a synergistic effect between the Qe/SBE inclusion complex and CS/HA Gel in accelerating the wound healing process. Mice are among the most widely used animal models in wound healing research. This research provides researchers with key insights into the signaling pathways involved in the healing process. Our Qe/SBE@CS/HA Gel has demonstrated favorable pro-healing efficacy in mouse models. However, wound closure in mice is predominantly achieved through wound contraction, leading to a relatively rapid healing process and an inability to fully imitate the human wound-healing process. In contrast, human wound healing mainly relies on re-epithelialization and granulation tissue formation, resulting in a much slower healing rate [45]. To achieve better clinical translation, after confirming the treatment’s efficacy in the mouse model, the pharmacological effects of the treatment are usually evaluated in rat and rabbit skin injury models. Their skin structure and physiological mechanisms are more similar to those of humans. Therefore, further evaluations in rabbit skin injury models are warranted in subsequent studies.

Moreover, the strong moisture absorption capacity of Qe/SBE@CS/HA Gel helps manage wound exudates, thereby reducing the risk of bacterial infection. This effect is further supported by bacterial culture results from wound tissue collected on day 2 (Figure 6C). Quantitative bacterial analysis (Figure 6E) revealed that only 5.1 ± 0.2% of bacteria survived on the wound surface in the Qe/SBE@CS/HA group, further validating its excellent wound healing potential. Among them, the Qe/SBE@CS/HA Gel group exhibited the most significant improvement, which can be attributed to its enhanced antibacterial and antioxidant properties.

To study wound tissue reconstruction, histological staining (H&E and Masson staining) was performed. As shown in Figure 7A,B, compared with the control group and CS/HA Gel group, the Qe/SBE and Qe/SBE@CS/HA Gel groups exhibited more complete granulation tissue formation, re-epithelialization, and collagen fiber deposition [46], which can be attributed to the strong antibacterial and antioxidant properties of Qe. In addition, epidermal thickness was significantly reduced after treatment, indicating better structural restoration. Notably, neovascularization and other skin appendages were observed in the Qe/SBE@CS/HA Gel group, further confirming its superior wound healing efficacy.

Figure 7. Wound healing evaluation by pathological tissue analysis. (A) H & E staining. (B) Masson’s trichrome staining. (C–G) Histochemical analysis following treatment with different gels on day 14. Dashed black lines indicate the epidermal boundary, and red arrows mark the newly formed blood vessels, n = 3, 100× magnification.

The degree of re-epithelialization is considered a reliable indicator for evaluating wound repair. In this study, we assessed the inflammatory response, collagen deposition, and angiogenesis. Macrophages play a pivotal role in orchestrating various phases of wound healing [47]. To evaluate the immune regulatory effects, the expression of CD86 (a marker for pro-inflammatory M1 macrophages) and CD206 (a marker for anti-inflammatory M2 macrophages) was analyzed by immunohistochemical staining [48]. Compared with other groups, the Qe/SBE@CS/HA Gel group exhibited significantly lower CD86 expression and higher CD206 expression (Figure 7C,D), suggesting that this treatment effectively promotes inflammation resolution. This effect is attributed to the antioxidant and anti-inflammatory properties of Qe, as well as the antibacterial effects of CS.

Furthermore, the Qe/SBE@CS/HA Gel group demonstrated the highest expression levels of COL-1 and COL-3 (Figure 7E,F), indicating enhanced collagen synthesis and remodeling, which supports tissue regeneration and structural repair. In addition, the number of newly formed blood vessels is a direct indicator of wound healing quality [49]. CD31 immunohistochemical staining (Figure 7G) revealed that the Qe/SBE@CS/HA Gel group had the highest density of neovascularization at the wound site among all groups, confirming that this gel formulation effectively promotes wound vascularization and accelerates healing.

3. Materials and Methods

3.1. Materials

CS (MW: 600 kDa, DD: 85%) was purchased from Zhejiang Golden-Shell Pharmaceutical Ltd. (Taizhou, China). Qe was purchased from Dalian Meilun Biotech Co., Ltd. (Dalian, China). Sulfobutyl-β-cyclodextrin sodium salt (SBE, Mw: 2163 Da, average degree of substitution: 6.5) were purchased from Cydex (San Diego, CA, USA), α-Glycerol phosphate disodium salt (α-GP)), and β-glycerol phosphate disodium salt (β-GP) were purchased from Sigma Aldrich (Saint Louis, MO, USA). Hyaluronic Acid Sodium Salt (HA, MW: 45 KDa) was purchased from Huaxi Bio-Tech Co., Ltd. (Jinan, China).

3.2. Preparation and Characterization of Qe/SBE

Phase solubility: Add an excessive amount of Qe to the SBE aqueous solution with a concentration ranging from 0 to 10 mM. After stirring at 25 °C for 48 h, remove the insoluble substances using a 0.45 µm filter membrane. Calculate the solubility of Qe. Then, according to Formula (1), calculate the inclusion equilibrium constant (K_S) of SBE with respect to Qe.

$$\mathrm{K}\mathrm{s}={\displaystyle \frac{\mathrm{K}}{{\mathrm{S}}_{\mathrm{o}}(1-\mathrm{K})}}$$

(1)

note: S₀ represents the solubility of the Qe in water at 25 °C, and K is the slope of the phase solubility curve.

The Qe/SBE inclusion complex was prepared using a saturated solution method [50]. To begin, Qe and SBE were dissolved in ethanol and water, respectively. The Qe and SBE solutions were subsequently mixed and stirred for 2 h. The ethanol solution was then removed via rotary evaporation. Following this, a 15-min centrifugation at 10,000 rpm was performed to eliminate unencapsulated drugs. The resultant mixture was freeze-dried using a freeze-drying machine (SCIENTZ-10ND, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) for 2 days to obtain the Qe/SBE inclusion compounds. We examined the inclusion complexes prepared under different molar ratios of Qe/SBE to optimize the formulation in terms of the inclusion rate and drug loading rate of Qe.

The phase solubility and inclusion rate of Qe/SBE were determined by UV spectroscopy. Qe/SBE was dissolved in ethanol to remove the unloaded Qe, and dried under vacuum. The absorbance was measured at 374 nm using an ultraviolet spectrophotometer (TU1810, Puxi, Shanghai, China). The inclusion rate and drug loading ratewere calculated as follows:

$$\mathrm{I}\mathrm{n}\mathrm{c}\mathrm{l}\mathrm{u}\mathrm{s}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}={\displaystyle \frac{{\mathrm{W}}_{\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{a}\mathrm{p}\mathrm{s}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d} \mathrm{Q}\mathrm{e}}}{{\mathrm{W}}_{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{o}\mathrm{l} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{Q}\mathrm{e}}}}\times 100\mathrm{\%}$$

(2)

$$\mathrm{D}\mathrm{r}\mathrm{u}\mathrm{g} \mathrm{l}\mathrm{o}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}={\displaystyle \frac{{\mathrm{W}}_{\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{a}\mathrm{p}\mathrm{s}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d} \mathrm{Q}\mathrm{e}}}{{\mathrm{W}}_{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{o}\mathrm{l} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{Q}\mathrm{e}}+\mathrm{W}\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{S}\mathrm{B}\mathrm{E}}}\times 100\%$$

(3)

The structure of the freeze-dried Qe/SBE inclusion complex was characterized by fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM).

FT-IR analysis: The 5 mg sample was uniformly ground and mixed with KBr before being pressed into thin sheets. The FT-IR spectrum of the sample was measured using a FT-IR spectrophotometer (FTIR-850, Tianjin Gangdong Sci. &Tech. Co., Ltd. Tianjin, China) within the range of 400–4000 cm⁻¹.

DSC analysis: The thermal behaviors were measured using DSC (DSC 3+, Mettler Toledo, Greifensee, Switzerland). Weigh 5 mg of the sample, seal it in an aluminum crucible, and use the blank aluminum crucible as a control. Set the heating rate at 10 °C/min and the heating range from 30 to 350 °C. Record the heating curve for each sample.

SEM analysis: The surface morphology of the samples was characterized by SEM (Phenom Pro, Phenom, Ambler, PA, USA). The freeze-dried samples were sputter-coated with gold and observed using SEM under an electron acceleration voltage of 5 kV.

¹H NMR analysis: ¹H NMR spectra were obtained on a Nuclear Magnetic Resonance Spectrometer (AYANCE NEO400M, Bruker, Bremen, Germany). Samples were dissolved in dimethyl sulfoxide (DMSO-d6).

3.3. Preparation and Characterization of Qe/SBE@CS/HA Gel

Dissolve CS powder in 0.1 M acetic acid at room temperature to obtain a 2.5% CS solution. The prepared solution can be stored at low temperature as a reserve solution. Slowly add HA, α-GP, β-GP and Qe/SBE solution to the CS solution while stirring in an ice bath for 15 min. The final solution was then incubated at 37 °C to form Qe/SBE@CS/HA Gel. To optimize the gelation time of the Qe/SBE@CS/HA Gel, we fixed CS/α-GP/β-GP ratio at 1:4:4 (w/w) and investigated the HA/CS ratio (0, 1:5, 2:5, 4:5, w/w), as shown in Table 2. The prepared solution can be stored at low temperature as a reserve solution. The gelation time refers to the time required for no flow to be observed for each formulation.

Table 2. Summary of prescription optimization for gels.

Sample	CS (mg)	α-GP (mg)	β-GP (mg)	HA (mg)	SBE (mg)	Qe/SBE (mg)	pH
CS/Gel	25	104	104	0	/	/	6.28 ± 0.11
CS/HA Gel-1	25	104	104	5	/	/	6.26 ± 0.11
CS/HA Gel-2	25	104	104	10	/	/	6.29 ± 0.11
CS/HA Gel-3	25	104	104	20	/	/	6.24 ± 0.11
SBE@CS/HA Gel	25	104	104	10	15	/	6.22 ± 0.15
Qe/SBE@CS/HA Gel	25	104	104	10	/	15	6.26 ± 0.12

3.4. Rheological Testing

The rheological properties of the Qe/SBE@CS/HA Gel were measured using a rheometer (Hakker Mars 40, Thermofisher Scientific, Karlsruhe, Germany) equipped with a 60 mm diameter plate and a 1 mm gap. The environmental temperature is 25 ± 0.5 °C, and the humidity is 60 ± 5%. The gel solution was applied onto rheometer plate, and measurements were conducted over a temperature range of 4–45 °C. The variation in storage modulus (G′) and loss modulus (G″) was recorded under constant strain (0.1%) and frequency (1 Hz) with a heating rate of 0.5 °C/min

3.5. Swelling Rate and In Vitro Degradation

The gel was freeze-dried to obtain a freeze-dried powder. An appropriate amount of Qe/SBE@CS/HA Gel was then added to PBS to achieve swelling equilibrium. After removal, the surface moisture was quickly absorbed with absorbent paper, and the sample was weighed to calculate the equilibrium swelling rate. In a separate experiment, an appropriate amount of Qe/SBE@CS/HA Gel was added to a PBS solution in a centrifuge tube, and the sample was weighed at different time points to determine the degradation of the gel.

3.6. In Vitro Release Effect

The Qe/SBE@CS/HA Gel was added to dialysis bag (Mw: 3500 Da) and placed in PBS with 0.5% Tween 80 (pH 7.4 and 5.0) under continuously shaking at 100 rpm. A 1 mL sample was taken at regular intervals, and fresh medium was added. The concentration of Qe was then measured using UV spectroscopy (TU-1810, PUXI, China) at 374 nm. The release rate at each time point was calculated, and the cumulative release rate in vitro was determined.

$$\mathrm{C}\mathrm{u}\mathrm{m}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{r}\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}={\displaystyle \frac{{\mathrm{C}}_{\mathrm{n}}\times \mathrm{V}+\sum {\mathrm{C}}_{\mathrm{i}}\times {\mathrm{V}}_{\mathrm{i}}}{\mathrm{M}}}\times 100\%$$

(4)

note: C_n represents the drug concentration measured at the nth sampling point, V is the total volume of the receiving medium, ΣC_i × V_i is the sum of the products of the drug concentrations of the first (n − 1) sampling points and their respective sampling volumes, and M is the total amount of drugs in the gel.

3.7. Scanning Electron Microscopy (SEM) Analysis

The surface morphology of the gel was characterized using SEM. The gel sample was frozen in liquid nitrogen and then freeze-dried to ensure complete sublimation of the water. The freeze-dried Qe/SBE@CS/HA Gel was gold-sputtered for electron microscopy analysis. SEM (SU8010, Hitachi, Tokyo, Japan) was used to observe the surface structure at an accelerating voltage of 5 kV.

3.8. In Vitro Antioxidant Activity

3.8.1. DPPH Assay

Gels with different weights and Qe content of 12.5, 25, 50, 75, 100, 150 μg/mL were weighed. Then, 3.5 mL of 100 μM DPPH solution was added to each sample, and the mixtures were incubated at room temperature for 1 h, away from light. The sample were centrifuge at 5000 rpm/min for 10 min and take the supernatant for testing. The absorbance was measured at 517 nm using UV spectroscopy.

$$\mathrm{C}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{r}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\left(\mathrm{\%}\right)={\displaystyle \frac{{\mathrm{A}}_0-\mathrm{A}}{{\mathrm{A}}_0}}\times 100\mathrm{\%}$$

(5)

note: A₀ represents the absorbance of the control group with no Qe added.

3.8.2. ABTS Assay

Gels with different weights and Qe contents of 5, 10, 20, 30, 40, 50 μg/mL, were weighed. ABTS and potassium persulfate were added, and the mixtures were incubated at room temperature for 0.5 h, Centrifuge the sample for 5 min with 5000 rpm/min to remove sediment, then collect the supernatant for subsequent measurement. The absorbance was measured at 734 nm using UV spectroscopy.

3.9. Cell Culture

HaCat cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 µg/mL streptomycin, 100 U/mL penicillin, 2.5 g/L glucose and 0.11 g/L sodium pyruvate in a humidified atmosphere containing 5% CO₂.

Gel-related materials, including carboxymethylcellulose (CMC), hyaluronic acid (HA), and Qe/SBE, are dissolved in water to create an aqueous solution. Following sterilization through a 0.22-micron filter membrane, they are ultimately prepared into gels within a sterile environment.

3.10. ROS Detection

HaCat cells were seeded into 12-well plates at a density of 10⁶ cells/well and treated with hydrogen peroxide (H₂O₂) for 4 h to induce oxidative stress damage. After treatment with Qe/SBE@CS/HA gel for 24 h, intracellular ROS levels of treated HaCat cells were quantified using the DCFH-DA method. ROS expression levels were detected using fluorescence microscopy (Leica DM I8, Wetzlar, Germany) with green fluorescence and flow cytometry (Beckman CytoFLEX S, Brea, CA, USA).

3.11. H₂O₂-Induced Cell Proliferation

HaCat cells were seeded into 96-well plates at a density of 5 × 10³ cells/well and cultured overnight. H₂O₂ was then added for 4 h to induce oxidative stress damage. The cells were treated with Qe/SBE@CS/HA gel for 24 h and 72 h. The concentration of Qe in each case is 6.25, 12.5, 25, 50, 100 μg/mL. The proliferation of HaCat cells was assessed by the MTT assay. The MTT solution was added to each well for 4 h, and DMSO was added. OD values were measured at 490 nm using a microplate reader (Thermo Multiskan, Waltham, MA, USA).

$$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\mathrm{\%})={\displaystyle \frac{{\mathrm{A}}_{\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}}}{{\mathrm{A}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}}\times 100\mathrm{\%}$$

(6)

3.12. In Vitro Biocompatibility Evaluation

3.12.1. Cell Proliferation

HaCat cells were seeded into 96-well plates at a density of 5 × 10³ cells/well and cultured overnight. The cells were then incubated with CS/HA Gel, Qe/SBE, Qe/SBE@CS/HA Gel for 24 h and 72 h. The concentration of Qe in each case is 6.25, 12.5, 25, 50, 100 μg/mL. MTT solution was added to assess cell proliferation.

3.12.2. Hemolysis Assay

Fresh blood was obtained and washed with PBS to prepare a red blood cell suspension. The red blood cell suspension was then mixed with water, CS/HA Gel, Qe/SBE, or Qe/SBE@CS/HA Gel, and incubated at 37 °C for 20 min. After incubation, the suspension was centrifuged at 1000× g for 3 min. The absorbance of the supernatant was measured at 540 nm.

$$Hemolysis rate \%={\displaystyle \frac{A_{test}-A_{negative}}{A_{positive}-A_{negative}}}\times 100\%$$

(7)

3.13. In Vitro Antibacterial Effect

3.13.1. Bacterial Culture

Staphylococcus aureus (S. aureus) and methicillin-resistant Staphylococcus aureus (MRSA, CCTCC AB 2015109) were purchased from China Typical Culture Preservation Center (Beijing, China). S. aureus and MRSA were cultured in TBST medium in a humidified atmosphere at 37 °C.

3.13.2. Turbidity Detection

CS/HA Gel, Qe/SBE, and Qe/SBE@CS/HA Gel were incubated with S. aureus or MRSA. The concentration of Qe in each case is 37.25 62.5, 125, 250, 500, 1000 μg/mL. After incubate for 48 h, turbidity was measured at a wavelength of 600 nm using microplate reader.

3.13.3. Antibiotic Disk Diffusion Test

Sterile Muller-Hinton agar was prepared, and S. aureus or MRSA was evenly spread on the surface of the agar plate. Put the medicine on the Oxford Cup and place on agar plate. The antibiotic disk was placed on the agar plate, and the plate was incubated at 37 °C for 24 h. The diameter of non-growing colonies around the disk was observed and measured by Automatic Colony Counter (Czone8, Hangzhou Xunshu Technology Co., Ltd., Hangzhou, China).

3.14. In Vivo Therapeutic Evaluation

3.14.1. Wound and Infection Model

KM male mice were purchased from Charles River Laboratory Animal Technology Co., Ltd. (Wilmington, MA, USA). All experimental procedures were approved by the Animal Experimental Ethics Committee of Zhejiang Pharmaceutical University, with ethics code 202302006. KM male mice, aged 5 weeks and weighing 20 g ± 0.2 g, were housed six per cage under air-controlled conditions (20 °C ± 1 °C and 12 h/12 h light/dark illumination cycles). After adaptive feeding, randomly group the mice according to their body weight, with 6 per group. Then, hair removal, anesthesia (50 mg/kg pentobarbital), and disinfection were performed, and full-thickness round skin defects (10.0 mm in diameter) were created on the dorsal side of mice. Subsequently, the 10⁷CFU MRSA were applied to the wound to establish an infectious full-thickness skin injury model.

3.14.2. Wound Healing Rate

PBS, CS/HA Gel, Qe/SBE, or Qe/SBE@CS/HA Gel were applied to the skin injury site of the mice in liquid form. The content of quercetin in each group was 2 mg/mL, and 100 ul was administered to each mouse. The wound area was photographed on days 0, 3, 7, 11, and 14, and the wound area was measured by Image J 1.47.

3.14.3. In Vivo Antibacterial Evaluation

To analyze the in vivo antibacterial effect, bacteria at the damaged site were collected 2 days after drug administration. The collected tissue samples were incubated with LB medium overnight and then dispersed on agar plates.

3.14.4. In Vivo Degradation Evaluation

To evaluate the degradation of the gel in vivo, the gel was injected into the subcutaneous. After anesthetizing the mice on days 3 and 10, the subcutaneous tissues were dissected to observe the remaining amount of gel and the condition of local tissues.

3.14.5. Histological and Immunohistochemistry Examination

Wound tissues were collected at the end of 14 days and fixed with 4% paraformaldehyde for histological and immunohistochemistry analysis. For each group, 3 skin tissues were randomly selected. After paraffin embedding and sectioning, the effects of the drug delivery system on scar formation and skin tissue regeneration were evaluated by HE staining and Masson’s trichrome staining. Additionally, the expression levels of collagen, CD86, CD206, and CD31 in skin tissues were assessed by immunohistochemical staining.

3.15. Statistical Analyses

All statistical analyses were performed using GraphPad Prism 8 software. Data are expressed as means ± standard deviation (SD) of measurements. The significance of differences between results was evaluated using an unpaired Student’s t-test and ANOVA. *** p < 0.001, ** p < 0.01, and * p < 0.05 were considered statistically significant.

4. Conclusions

In this study, we successfully prepared a Qe/SBE inclusion complex and incorporated it into a thermosensitive gel-based local delivery system (Qe/SBE@CS/HA Gel) composed of chitosan and hyaluronic acid. This gel exhibited excellent antibacterial, antioxidant, and wound healing-promoting properties. The introduction of SBE not only enhanced the aqueous solubility of quercetin but also interacted electrostatically with chitosan, thereby shortening the gelation time. Within the gel, Qe/SBE and chitosan form a crosslinked network gel through electrostatic adsorption, resulting in acid-responsive, sustained release of Qe. Both in vitro and in vivo experiments confirmed that the gel possesses good antibacterial activity, antioxidant capacity, and biocompatibility. In conclusion, Qe/SBE@CS/HA Gel shows potential applicability in promoting wound healing, preventing bacterial infection, and alleviating oxidative stress, though its translational value relies on further optimization of sterilization protocols, long-term stability, and scalable manufacturing processes. This work lays a foundation for further preclinical development of the gel as a potential candidate for treating infected skin wounds, pending validation in chronic wound models and comprehensive toxicity assessment.