A pH-responsive anthocyanin-based hydrogel with colorimetric infection monitoring and antibacterial properties for wound healing

Abstract

Chronic and infected wounds present significant clinical challenges due to fluctuating pH levels and persistent microbial contamination, which impede healing and increase the risk of complications. Traditional wound dressings lack the capacity to actively monitor and respond to changes in the wound microenvironment. In this study, we developed a multifunctional anthocyanin-based hydrogel that integrates pH-responsive antimicrobial release with real-time visual pH indication. Synthesized via a facile one-step free radical polymerization method, the hydrogel responds to microenvironmental pH shifts with visible color changes, enabling intuitive assessment of infection status. Simultaneously, it releases the antimicrobial agent chlorhexidine gluconate in a pH-dependent manner to enhance infection control. The hydrogel's structure and physiochemical properties, including swelling behavior, mechanical strength, and tissue adhesion, were thoroughly characterized. It also exhibited notable antimicrobial and antioxidant activities. In vitro studies demonstrated excellent cytocompatibility, while in vivo evaluation using a mouse alkali burn wound model revealed significant improvement in wound healing and inflammation reduction. This work provides a promising platform for intelligent wound care by combining diagnostic and therapeutic functionalities in a single, biocompatible material.

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Rong Wang

Dr Rong Wang is a Professor at the Ningbo Institute of Materials Technology and Engineering (NIMTE), Chinese Academy of Sciences (CAS). He received his PhD degree in Chemical and Biomolecular Engineering from the National University of Singapore in 2015, and then worked as a research engineer/R&D manager at ACI Medical Pte. Ltd (Singapore) from 2015 to 2017. He joined NIMTE CAS in 2018. His research interests focus on biomedical polymeric materials, particularly in the areas of wound healing, tissue engineering, and biofunctional hydrogels.

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1. Introduction

Skin wounds, caused by various intrinsic and extrinsic factors such as hyperglycemia, bacterial infection, and mechanical injury, compromise the structural integrity and barrier function of the skin. Clinically, they represent one of the most common conditions, with etiologies including burns, surgical interventions, and chronic systemic diseases. Wound healing is a complex biological process that restores skin structure and function, typically proceeding through three sequential phases: inflammation, proliferation, and remodeling.¹ In the early inflammatory phase, immune responses initiate the release of cytokines, mediators, and enzymes, leading to the accumulation of wound exudate.² This exudate peaks during inflammation and decreases as healing progresses. Within normal range, exudate plays a beneficial role in wound recovery. However, abnormalities in its volume, composition, or regulatory mechanisms can disrupt the wound microenvironment, resulting in persistent inflammation, bacterial colonization, and impaired tissue regeneration, thereby delaying healing.³ Among various indicators of wound status, exudate pH is particularly significant. Healthy skin typically maintains a mildly acidic pH (4.2–5.6), which supports normal skin barrier function. In contrast, chronic or infected wounds often exhibit elevated exudate pH levels (7.2–10.0), a condition linked to microbial overgrowth, heightened protease activity, and localized hypoxia, all of which reflect a disrupted and unfavorable wound microenvironment.⁴

Accurate assessment and regulation of wound exudate properties, particularly pH values, are crucial for promoting healing and preventing complications. Recent advances have focused on the development of smart wound dressings capable of real-time monitoring of wound exudate by directly or indirectly detecting key parameters such as pH, temperature, oxygen concentration, and enzyme activity.^5,6 These technologies offer new opportunities for dynamic wound assessment and demonstrate significant clinical potential. However, several challenges hinder the practical application of smart wound dressings. Most current materials provide only sensing capabilities without therapeutic functions.⁷ Consequently, they offer passive feedback on complex wounds, such as those complicated by infection, but lack the capacity for active intervention or regulation. Additionally, the data generated often require external devices for reading and analysis, complicating clinical workflows and limiting timely responses and patient self-management.⁸ Furthermore, many of these materials suffer from poor flexibility, limited biocompatibility, and potential irritation to the wound bed, which restrict their suitability for long-term and stable application.⁷

An ideal smart wound dressing should integrate the functions of indication, response, and treatment. It should provide real-time detection of pH fluctuations in wound exudate, visually indicate infection status, and actively release antimicrobial agents in response to infection-associated pH changes. This integrated approach enables precise and timely intervention for infected wounds. Various pH-responsive and multifunctional smart dressings have recently been developed to facilitate early infection detection and management, thereby improving healing outcomes and reducing complications. For instance, Xu et al. reported a pH-responsive cellulose-based Janus nonwoven dressing with unidirectional fluid drainage, combining pH visualization, antibacterial and antioxidant activities, and effective exudate management. This dressing enables real-time pH monitoring in diabetic wounds, modulates the inflammatory microenvironment, and promotes tissue regeneration.⁹ Similarly, Zheng et al. developed a multifunctional carbon quantum dot-phenol red hydrogel dressing composed of polyacrylamide/chitosan. This system integrates highly sensitive colorimetric and fluorescence dual-mode pH sensing with strong antibacterial, hemostatic, and moisturizing properties.¹⁰ It also allows remote visual monitoring via smartphone, offering an intelligent platform that combines diagnostic and therapeutic functions for chronic wound care.

In this study, we developed a pH-responsive, antimicrobial-loaded anthocyanin hydrogel that simultaneously functions as an antimicrobial delivery system and a pH indicator. The hydrogel was synthesized via a one-step free radical polymerization process and enables real-time monitoring and regulation of pH fluctuations in bacteria-infected microenvironments. Its physical and chemical structure, swelling behavior, water retention capability, mechanical strength, and tissue adhesion were systematically assessed. Particular attention was given to its pH-responsive antimicrobial release profile, visible colorimetric response to pH changes, overall antimicrobial activity, and antioxidant capacity. In vitro biocompatibility was confirmed, and a mouse alkali burn wound model was employed to assess the hydrogel's wound healing efficacy and anti-inflammatory effects. Notably, the novelty of this work lies in the specific integration of anthocyanins and chlorhexidine gluconate (CHG) within the polyzwitterionic hydrogel network, simultaneously endowing the system with pH-responsive colorimetric signaling, potent antibacterial activity, and enhanced mechanical and adhesive properties. To our knowledge, such a combination of multifunctional features has rarely been reported in wound–healing hydrogels.

2. Materials and methods

2.1. Materials

Roses were purchased from Jinan Yuanshuo Economic and Trade Co., Ltd (Shandong, China). [2-(Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA, 97%), sodium hydroxide, ammonium persulfate (APS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), potassium persulfate, and zinc bis(diethyldithiocarbamate) were purchased from Aladdin Chemistry (Shanghai, China). Poly(ethylene glycol) dimethacrylate (PEGDMA, M_n 550 Da) was purchased from Sigma-Aldrich (Shanghai, China). Chlorhexidine gluconate (CHG) was purchased from Macklin (Shanghai, China). 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was purchased from Energy Chemical (Shanghai, China). Escherichia coli (E. coli) ATCC 25922 and Staphylococcus aureus (S. aureus) ATCC 25923 were purchased from American Type Culture Collection. Hematoxylin & Eosin (H&E) stain kit and Masson's trichrome stain kit were purchased from Solarbio Life Sciences (Beijing, China). Cell Counting Kit-8 (CCK-8) and Calcein/PI Cell Viability/Cytotoxicity Assay Kit were purchased from Beyotime Biotechnology (Shanghai, China). Dulbecco's Modified Eagle Medium was obtained from Thermo Fisher Scientific (Massachusetts, United States). L-929 fibroblast cells were obtained from National Collection of Authenticated Cell Cultures, Chinese Academy of Sciences.

2.2. Extraction of rose anthocyanins

Rose anthocyanins were extracted using the alcohol extraction method following a previously reported method.¹¹ Dried rose petals were ground into fine powders using a crusher, and impurities were removed by sieving. One gram of the powder was mixed with 40 mL of 60% ethanol and stirred in the dark at room temperature for 3 h to ensure complete extraction of anthocyanins. The mixture was then filtered, and the filtrate was collected. The anthocyanin solution was concentrated in a rotary evaporator at 40 °C to remove most of the ethanol. This concentrated extract was dissolved in an appropriate volume of deionized water, stirred until fully dissolved, and freeze dried. The resulting anthocyanin powder was collected and stored at 4 °C in the dark for future use.

2.3. Characterization of anthocyanins

To evaluate the color stability of anthocyanins under different environmental conditions, 1 mg of freeze-dried anthocyanin powder was dissolved in 10 mL of phosphate-buffered saline (PBS, 10 mM) with pH values ranging from 2.0 to 12.0 (adjusted with HCl or NaOH), subjected to varying temperatures (4–45 °C), natural light exposure, and prolonged UV irradiation (254 nm, 24 h). The UV-visible absorption spectra of the solutions were recorded between 400 and 800 nm using a UV-Vis spectrophotometer (Cary 300, Agilent, United States).

2.4. Preparation and characterization of anthocyanin-incorporated hydrogels

To prepare the hydrogel, 11.174 g of SBMA, 53 mg of PEGDMA, and 13.6 mg of APS were mixed with 10 mL of anthocyanin solution at predetermined concentrations to obtain a homogeneous pre-gel solution. The pre-gel solution was injected into a glass-silicone mold and placed in a water bath at 60 °C for 2 h. After polymerization, the hydrogels were removed from the mold and stored at 4 °C in the dark. The hydrogels prepared using pre-gel solution containing anthocyanin at concentrations of 0, 20, 40, 60, and 80 mg mL⁻¹ were denoted as RA0, RA2, RA4, RA6, and RA8, respectively. For CHG hydrogels, 0.2 g of CHG was added to the RA8 pre-gel formulation, resulting in RA8-CHG hydrogel. To investigate the chemical structure and composition of anthocyanin-incorporated hydrogels at varying concentrations, lyophilized samples were characterized using Fourier transform infrared (FTIR) spectroscopy (IS 50, Thermo Fisher Scientific, United States). The synthesized hydrogels were freeze-dried prior to characterization. Hydrogels’ surface elemental composition was analyzed by X-ray photoelectron spectroscopy (XPS, AXIS SUPRA+, Kratos, Japan), while the elemental distribution was examined by energy-dispersive X-ray spectroscopy (EDS) mapping using a field-emission scanning electron microscope (SEM, Sigma 300, Zeiss, Germany).

2.5. Swelling capability and water retention assay

Cylindrical hydrogel samples with a diameter of 10 mm and a thickness of 2 mm were used for the swelling assay. The initial mass of each as-prepared hydrogel was recorded as m₁. The samples were then immersed in 40 mL of PBS at different pH values (pH 5.0, 7.0, and 9.0) and allowed to swell at room temperature up to 36 h. During swelling, the samples were periodically removed, gently blotted to remove surface water, and weighed to obtain the swollen mass, recorded as m₂. The swelling ratio was calculated using eqn (1).whereas m₁ and m₂ are the initial mass of the as-prepared hydrogels and mass after swelling, respectively.

Cylindrical samples (10 mm in diameter and 2 mm in thickness) were prepared and placed at 76% relative humidity at 23 °C and 37 °C. The sample weight at different time points (M_h) was recorded over 24 h, and the water retention ratio was calculated according to eqn (2).

Weight retention (%) = M_h/M₀ × 100% (2)

whereas M₀ and M_h represent the hydrogel weight at the beginning and time h, respectively.

2.6. Mechanical tests

For the tensile test, strip-shaped hydrogel samples (4 mm in width and 2 mm in thickness) were subjected to uniaxial stretching using a universal testing machine (CMT-1104, SUST, China) at a constant rate of 50 mm min⁻¹. For the compression test, cylindrical hydrogel samples (10 mm in diameter and 5 mm in thickness) were compressed at a rate of 10% strain per minute using the universal testing machine until reaching 90% strain.

2.7. Adhesion assays

Hydrogel samples (20 mm × 20 mm) were placed between a glass slide and various substrates, including metal, glass, plastic, wood, and rubber, to assess adhesion performance. Additionally, rectangular hydrogel strips (10 mm × 50 mm) were applied to a human finger to examine adhesion under dynamic conditions. The finger was repeatedly bent, and the hydrogel's adhesion stability during movement was evaluated and recorded. The experiments were carried out with the volunteer's informed consent.

For further assessment of the hydrogel's adhesion to skin tissue, lap shear tests were conducted. Porcine skin strips (purchased from a local market, cut into sizes of 40 mm × 15 mm × 3 mm) were cleaned to remove oils and impurities and used as the adhesion substrate. Prior to testing, the skin substrates were either used directly or pre-wetted with PBS solutions at pH 5.0, 7.0, and 9.0 to simulate different wet wound environments. Hydrogel pieces (15 mm × 15 mm × 2 mm) were sandwiched between two porcine skin pieces, and a 500 g weight was placed on top for 5 min to ensure initial contact. Adhesion strength was then measured using a universal testing machine. The skin strips were fixed with clamps at both ends and pulled at 10 mm min⁻¹ until separation occurred.

2.8. pH response tests

Circular hydrogel samples (10 mm in diameter, 2 mm in thickness) were placed in a 12-well plate, with each well containing 4 mL of PBS solution at varying pH values (pH 5.0–9.0). The samples were allowed to stand for 4 h or 24 h. After immersion, color changes of the hydrogels under each pH condition were visually observed and quantitatively analyzed using a colorimeter (3nh, Shenzhen, China) to measure parameters (L*, a*, b*). The color changes (ΔE) of the hydrogels between pH 5.0 and 9.0 were calculated using eqn (3):where ΔL, Δa, and Δb represent the differences in lightness, red/green value, and yellow/blue value of the hydrogels between pH 5.0 and pH 9.0, respectively.

The ΔE values were classified into six levels based on the National Bureau of Standards (NBS) scale, calculated as NBS = ΔE × 0.92: trace (0–0.5), slight (0.5–1.5), noticeable (1.5–3.0), appreciable (3.0–6.0), much (6.0–12.0), and very much (>12.0). In addition, the RGB values of the hydrogels were measured using the Color Info Cam application (Version 2.3.1) on a smartphone, and the corresponding data were recorded.

2.9. Release behavior and antibacterial assays

To assess the release behavior of anthocyanin and CHG, 90 mg of RA8-CHG hydrogel samples were immersed in 10 mL of PBS solution at pH 5.0, 7.0, and 9.0 and incubated at 37 °C with shaking at 100 rpm for 24 h. At predetermined time intervals, aliquots of the PBS solution were withdrawn, and the concentrations of anthocyanin (measuring absorbance at 265, 272, and 320 nm at pH 5.0, 7.0, and 9.0, respectively) and CHG (measuring absorbance at 260 nm) were determined using UV-Vis spectrophotometry. Standard curve at the respective pH conditions were established in the same manner as described above.

For the pH-responsive antibacterial assay, overnight cultures of E. coli and S. aureus were centrifuged at 2700 rpm for 10 min. The bacteria were resuspended in PBS buffer (pH 5.0, 7.0, and 9.0) and adjusted to a concentration of 10⁵ CFU mL⁻¹. Hydrogel samples (10 mm in diameter, 2 mm in thickness) were added to 5 mL of the bacterial suspensions and incubated at 37 °C with shaking at 100 rpm. After 6 h, the suspensions were collected, serially diluted, and plated using the spread plate method to evaluate bacterial viability under different pH conditions. In the control group, bacteria were cultured under the same conditions without the addition of hydrogel samples.

For the zone of inhibition test, overnight cultures of E. coli and S. aureus were diluted with nutrient broth to a concentration of 10⁵ CFU mL⁻¹. The suspensions were evenly spread on solid agar plates, and hydrogel samples (10 mm in diameter, 2 mm in thickness) were placed on the inoculated surface. The plates were incubated at 37 °C for 18 h. After incubation, the inhibition zones surrounding the hydrogel samples were photographed for documentation and analysis of antibacterial activity.

2.10. Antioxidant assays

For the DPPH radical scavenging assay, 3.94 mg of DPPH was dissolved in 100 mL of anhydrous ethanol to prepare a 0.1 mmol L⁻¹ DPPH solution. Subsequently, 150 mg of hydrogel sample was added to 9 mL of the DPPH solution and incubated in the dark at room temperature for 30 min. Absorbance at 517 nm was measured before and after the reaction using a UV-Vis spectrophotometer. Radical scavenging activity was calculated using eqn (4).where A_blank is the absorbance of the DPPH solution without sample, and A_sample is the absorbance after reaction with hydrogel for 30 min.

For the ABTS radical scavenging assay, 40.6 mg of ABTS was dissolved in 10 mL of deionized water to prepare a 7.4 mmol L⁻¹ ABTS solution. Separately, 7.0 mg of potassium persulfate was dissolved in 10 mL of deionized water to obtain a 2.6 mmol L⁻¹ solution. The two solutions were mixed and incubated in the dark at room temperature for 24 h to generate the ABTS˙⁺ radical solution. Then, 150 mg of hydrogel sample was added to 9 mL of the ABTS˙⁺ solution and incubated in the dark for 30 min. Absorbance at 734 nm was measured before and after the reaction using a UV-Vis spectrophotometer. The scavenging activity was calculated using eqn (5).

where A_blank is the absorbance of the ABTS˙⁺ solution without sample, and A_sample is the absorbance after reaction with hydrogel for 30 min.

2.11. In vitro cytotoxicity and anti-inflammatory assays

The in vitro cytotoxicity assay was conducted using L-929 mouse fibroblast cells. Cells were seeded at a density of 10⁵ cells per mL in 96-well plates or confocal culture dishes. The culture medium consisted of modified Dulbecco's modified eagle's medium (DMEM), supplemented with 10% fetal bovine serum, 10⁵ U per L penicillin, and 100 mg per L streptomycin. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ for 24 h to ensure optimal growth. Hydrogel samples (50 mg) were immersed in 10 mL of complete culture medium and incubated at 37 °C for 24 h to obtain hydrogel extracts. After incubation, the original medium in the plates or dishes was replaced with either hydrogel extract, fresh culture medium (negative control), or medium containing 5 mg mL⁻¹ zinc diethyldithiocarbamate (positive control). Cells were incubated at 37 °C under 5% CO₂ for an additional 24 h. Cell viability was assessed using an enhanced CCK-8 assay kit according to the manufacturer's instructions. In parallel, cells were stained with a Calcein/PI cell viability/cytotoxicity assay kit, and their status was observed using a confocal laser scanning microscope (CLSM, TCS SP8, Leica, Germany).

Raw 264.7 macrophages in the logarithmic growth phase were harvested, and adjusted to a density of 2 × 10⁴ cells per well, then seeded into 96-well plates and cultured at 37 °C under 5% CO₂. Hydrogel samples (100 mg mL⁻¹ in culture medium) were incubated for 24 h to obtain extracts, which were subsequently diluted four-fold before use. The cells were incubated with the diluted extracts for 24 h. The culture supernatants were collected and transferred into new 96-well plates. Nitric oxide (NO) production was quantified using the NO assay kit (S0021S, Beyotime Biotechnology) in accordance with the manufacturer's instructions to assess the influence of hydrogel extracts on lipopolysaccharide (LPS)-induced NO release in Raw 264.7 cells.

2.12. Alkali burn healing experiment

All animal experiments were performed in compliance with the principles of the National Research Council's Guide for the Care and Use of Laboratory Animals, and the Laboratory animal – Guideline for ethical review of animal welfare (GB/T 35892-2018), and approved by the Institutional Animal Care and Use Committee of Zhejiang Huitong Test & Evaluation Technology Group Co., Ltd (approval no. HT-2024-LWFB-0024). Male ICR mice (25–30 g) were acclimated for one week prior to experimentation. To establish an alkali burn model, the dorsal area of the mice was shaved under anesthetization, and a 10 mm circular wound was created using a punch. A filter paper disc soaked in 1 mol L⁻¹ NaOH solution was applied to the wound for 1 min to induce an alkali burn. The control group received no treatment to the wound. The wound was then covered with a 3 M dressing. In the experimental group, RA8-CHG hydrogel was applied to the burn wound. The color change of the hydrogel was observed after 24 h. To assess the colorimetric response of the RA8-CHG hydrogel during wound healing, an additional control group was established with untreated wounds (no alkali burn). In this group, the RA8-CHG hydrogel was applied to the wounds, and the color changes were compared between the groups to evaluate the hydrogel's pH indication performance during healing of alkali burn and normal wounds. Wound healing was evaluated on days 0, 3, 7, 14, and 21 by observing wound progression. On days 7, 14, and 21, tissue samples were harvested for histological analysis. The samples were fixed in 4% paraformaldehyde for 24 h, followed by dehydration through a graded ethanol series (75%, 80%, 85%, 90%, 95%, 95%, 100%, 100%) and xylene, and then embedded in paraffin. Tissue section (4 µm) were prepared using a rotary microtome. After dewaxing with xylene and graded ethanol, the sections were stained with H&E and Masson's trichrome to evaluate morphological changes and collagen fiber reconstruction during wound healing. On day 21, healed skin tissues were collected for tensile strength testing. Samples were cut into dumbbell shapes (2 mm in narrow parallel width, 35 mm in length) and subjected to testing using a universal testing machine at a stretching speed of 5 mm min⁻¹ to assess the biomechanical properties of the healed skin and the effect of the RA8-CHG hydrogel on tissue regeneration.

2.13. Statistical analysis

Each experiment was independently performed a minimum of three times. Data are expressed as mean ± standard deviation (SD). Statistical analysis was conducted using one-way analysis of variance (ANOVA), followed by Tukey's post hoc test for multiple comparisons. A P-value of less than 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ns indicates no significant difference).

3. Results and discussion

3.1. pH responsiveness of anthocyanins

Anthocyanins were extracted from rose petals using an ethanol extraction method, as previously described.¹¹ As shown in Fig. S1, the rose anthocyanin extract exhibits distinct color changes under different pH conditions. Specifically, the solution appears red under acidic conditions (pH 2.0), purple at neutral pH (pH 7.0), and yellow under alkaline conditions (pH 10.0–12.0). These color variations arise from pH-dependent structural transformations of the anthocyanins.¹² In strongly acidic environments, anthocyanins predominantly exist as flavylium cations, which are in red color. Under mildly acidic conditions, these structures convert to colorless hemiketals. In weakly alkaline conditions, quinone-type bases form, resulting in a purple color. In strongly alkaline environments, anthocyanins convert to chalcone forms, which appear yellow (Fig. S2).^13,14 The UV-vis absorption spectra (Fig. S3) corroborate these observations. At pH 2.0, the extract exhibits a maximum absorption peak that occurs at 518 nm. As the pH increases, the maximum UV-visible absorption peak of the anthocyanin solution exhibits a pronounced red shift, shifting to 591 nm. This spectral shift is attributed to the transformation of the flavylium cation (a positively charged, highly conjugated species) to its hydrated, quinoidal base, or chalcone forms, resulting in modifications to the π–π* electronic transition energy gap.^15,16 The observed redshift is primarily attributed to the structural changes of anthocyanins under varying pH conditions.¹⁷ Furthermore, stability tests demonstrated that the anthocyanin solution remained relatively stable at pH 5–9, temperatures of 4–45 °C, and under natural light exposure, whereas prolonged UV irradiation (254 nm, 24 h) caused significant degradation (Fig. S4). This reduction is attributable to UV-induced photodegradation and structural disruption of anthocyanin molecules, which compromise their stability.

3.2. Synthesis and characterization of hydrogels

RA8-CHG hydrogel was synthesized via a straightforward one-step free radical polymerization, employing SBMA as the monomer, PEGDMA as the chemical crosslinker, and APS as the thermal initiator. Rose anthocyanin and CHG functioned as non-covalent crosslinkers, facilitating the formation of a crosslinked network (Scheme 1). During polymerization, SBMA monomers formed a three-dimensional network through free radical reactions, which PEGDMA introduced covalent crosslinking points that enhanced the structural stability of the hydrogel. Anthocyanin and CHG were incorporated into the network via non-covalent interactions with the polymer chains, reinforcing the hydrogel's integrity.

Scheme 1 Schematic illustration of the anthocyanin-incorporating hydrogel structure and its synergistic functions in infection monitoring and antibacterial activity for wound healing applications.

As shown in Fig. S5, the FTIR spectrum of RA0 exhibits an absorption peak at 1717 cm⁻¹, corresponding to the C=O stretching vibration in the SBMA units of RA0.¹⁸ In the FTIR spectrum of RA2, a peak at 1298 cm⁻¹ appears, which is likely attributed to the C–O stretching vibration of the phenolic group in anthocyanins.¹⁹ In the RA8-CHG hydrogel, this peak becomes more intense, which can be ascribed to the additional hydroxyl groups introduced by CHG. In addition, the broad absorption peak at 3437 cm⁻¹ in RA0 shifts to 3428 cm⁻¹ in RA8-CHG, suggesting the formation of additional hydrogen bonds between CHG and the hydrogel network.²⁰ The FTIR results confirm the successful synthesis of the hydrogel and indicate the presence of non-covalent intermolecular interactions within the network.

As shown in Fig. S6a, the XPS survey spectra revealed a distinct increase in the proportion of oxygen in RA8 compared with RA0, confirming the successful incorporation of anthocyanins into the hydrogel network. Fig. S6b shows two characteristic peaks at 202.0 eV (Cl 2p_1/2) and 200.3 eV (Cl 2p_3/2) in RA8-CHG,²¹ while no Cl signal is detected in RA0 or RA8, indicating the specific incorporation of CHG. EDS elemental mapping (Fig. S7 and S8) demonstrates that C, N, O, and S are uniformly distributed in RA0, RA2, RA4, RA6, and RA8, indicating a homogenous hydrogel structure. Moreover, Cl is uniformly distributed in RA8-CHG, further confirming the successful incorporation of CHG throughout the hydrogel matrix.

3.3. Mechanical properties of hydrogels

Evaluating the tensile and compressive properties of hydrogels is crucial for assessing their mechanical stability and deformability under practical conditions. These properties are key to ensuring that hydrogels maintain structural integrity and meet clinical demands when subjected to mechanical stress. Fig. 1(a) shows that the RA8-CHG hydrogel can be stretched up to 6.7 times of its own length in the tensile test. As shown in Fig. 1(b) and (c), the single-network RA0 hydrogel exhibited limited mechanical performance, with a tensile fracture stress of 40.2 kPa. Incorporation of anthocyanin markedly enhanced tensile strength, increasing from 60.9 kPa in RA2 to 77.5 kPa in RA8. This improvement is likely due to hydrogen bonding between the phenolic hydroxyl groups in anthocyanin molecules and the zwitterionic polymer chains, which increases crosslinking density and tensile performance. The RA8-CHG hydrogel demonstrated the highest tensile fracture stress of 94.1 kPa, which may be attributed to the abundant hydroxyl groups in CHG forming hydrogen bonds with the polymer network. Moreover, CHG may engage in electrostatic interactions with zwitterionic groups, introducing further physical crosslinking sites.²² Interestingly, the RA8-CHG hydrogel exhibited the lowest modulus (9.70 kPa), indicating that the incorporation of CHG not only reinforced the tensile strength through additional physical crosslinking, but also imparted greater flexibility to the network, which may be beneficial for conformal contact with irregular wound surfaces. These findings suggest that the synergistic effects of anthocyanin and CHG significantly enhance the hydrogel's tensile strength, underscoring the important role of non-covalent interactions in improving mechanical performance. The RA8-CHG hydrogel exhibited excellent elastic properties upon compression (Fig. 1(d)). Fig. 1(e) shows the representative compressive stress–strain curves for hydrogels with varying anthocyanin concentrations, along with that of the RA8-CHG hydrogel. As shown in Fig. 1(f), the RA0 hydrogel exhibited a compressive strength of 2.3 MPa. With increasing anthocyanin content, the compressive strength increased significantly, from 2.8 MPa for RA2 to 6.6 MPa for RA8, indicating that the incorporation of anthocyanins effectively enhanced the compressive performance. Following the addition of CHG, the compressive strength of RA8-CHG further increased to 8.5 MPa. Meanwhile, the RA8-CHG hydrogel showed the highest compressive modulus of 473.80 kPa, suggesting that CHG not only reinforced the network structure through additional hydrogen bonding and electrostatic interactions but also increased its resistance to deformation under load.²³ These results demonstrate that the RA8-CHG hydrogel possesses superior tensile and compressive properties, highlighting its potential for biomedical applications such as wound healing and tissue engineering.

Fig. 1 (a) Digital photographs of RA8-CHG hydrogel undergoing tensile test. (b) Representative tensile stress strain curves, and (c) tensile strength and modulus of the various hydrogels. (d) Digital photographs showing RA8-CHG hydrogel subjecting to 90% compression and then unloaded. (e) Representative compressive stress strain curves, and (f) compressive strength and modulus of the various hydrogels. (g) Swelling behaviors of various hydrogels in PBS solution at pH 5.0, 7.0, and 9.0. (h) SEM images of the cross-section of the hydrogel after swelling in PBS solution at pH 7.0 for 24 h. Scale bar: 20 µm.

3.4. Swelling capabilities and water retention ability of hydrogels

Swelling tests are critical for assessing the water absorption and expansion capacities of hydrogels in various solutions, providing key insights into their stability, antimicrobial release profiles, and biocompatibility. All hydrogels swelled in PBS solutions of various pH values (Fig. S9). As shown in Fig. 1(g), the swelling ratios of the hydrogels increased progressively, indicating strong hydration performance. After 24 h of immersion, RA0 hydrogel exhibited swelling ratios of 184.7%, 179.9%, and 172.2% at pH 5.0, 7.0, and 9.0, respectively, suggesting stable water uptake across a broad pH range. The RA8-CHG hydrogel displayed superior swelling behavior under all pH conditions tested, with swelling ratios of 213.1%, 241.1%, and 236.4% at pH 5.0, 7.0, and 9.0, respectively. Notably, the swelling ratio of the hydrogel is closely related to its internal porous structure.²⁴ As shown in Fig. 1(h), the SEM images of the swollen hydrogels revealed overall comparable porous morphologies. As shown in Fig. S10, the hydrogels exhibited excellent water retention under both conditions. At 23 °C, more than 93.05% of the initial weight was retained after 24 h, indicating minimal water loss at room temperature. At 37 °C, the water retention ratio decreased slightly but remained above 88.85% after 24 h. These results indicate that the hydrogel maintains stable water retention properties under both ambient and physiological conditions.

3.5. Adhesion properties of hydrogels

An adhesion test was conducted to evaluate the suitability of the hydrogel for wound dressing applications, ensuring firm adherence to the skin and durable protection. Investigating the adhesion properties is crucial for enhancing the hydrogel's stability and efficacy throughout the wound healing process. As shown in Fig. 2(a), RA8-CHG hydrogels exhibited strong and stable adhesion to various substrates, including metal, glass, plastic, wood, rubber, and skin. This broad substrate compatibility demonstrates the hydrogel's ability to maintain stable attachment across diverse substrates and surface morphologies, supporting its applicability in complex environments. The adhesion strength to porcine skin was quantitatively assessed using lap shear tests (Fig. 2(b)). As shown in Fig. 2(c), the RA0 hydrogel exhibited an adhesion strength of 7.0 kPa. This adhesion is attributed to hydrogen bonding between the hydroxyl groups in the stratum corneum's keratin and lipids and the zwitterionic polymer chains in the hydrogel.²⁵ Moreover, amino and carboxyl groups in skin components such as collagen and elastin can interact with the zwitterionic groups in the hydrogel via dipole–dipole interactions. Therefore, the adhesive performance can be mainly attributed to the zwitterionic groups of SBMA. Incorporation of anthocyanins into the hydrogel markedly enhanced the adhesion strength to 21.2 kPa in the RA8 hydrogel. This enhancement is due to additional hydrogen bonding from phenolic hydroxyl groups in anthocyanins, further strengthening the hydrogel–skin interface. Subsequent addition of CHG further increased the adhesion strength to 28.5 kPa. The carboxyl, hydroxyl, and amino groups in CHG interact with skin components through hydrogen bonding and electrostatic interactions, further reinforcing adhesion.²⁶ To further assess adhesion performance under wet conditions, porcine skins were pre-wetted with PBS solution at pH 5.0, 7.0, and 9.0 prior to testing. As shown in Fig. S11, the hydrogels maintained stable adhesion across the tested pH range. Fig. 2(c) shows that the adhesion strength of the hydrogels was generally unaffected by pH variation under PBS-wetted conditions. Specifically, RA6, RA8, and RA8-CHG hydrogels exhibited no significant reduction in adhesion strength under both dry and PBS-wetted conditions. In contrast, RA0, RA2, and RA4 displayed a marked decrease in adhesion strength upon PBS wetting (P < 0.05), indicating that the lack of sufficient functional groups limited the formation of stable hydrogen bonding and electrostatic interactions with the skin surface in a hydrated environment.²⁷ These findings highlight the importance of phenolic hydroxyl groups from anthocyanins and additional polar functionalities from CHG in maintaining strong and stable adhesion under physiologically relevant wet conditions, which is essential for the practical application of hydrogels as wound dressings. These results indicate that the synergistic interplay of hydrogen bonding, electrostatic interactions, and dipole–dipole interactions within the hydrogel matrix significantly enhances its adhesion to the skin (Fig. 2(d)), underscoring its potential for use in advanced wound dressing applications.

Fig. 2 (a) Photographs of RA8-CHG hydrogel adhered to metal, glass, plastic, wood, rubber, and skin. (b) Representative adhesion force displacement curves, and (c) adhesion strength of various hydrogels on porcine skin under no-wetting and pre-wetting conditions (pH 5.0, 7.0, and 9.0). Statistical significance between the no-wetting and corresponding pre-wetting groups is denoted (*P < 0.05 and **P < 0.01). (d) Possible adhesion mechanisms between RA8-CHG hydrogel and tissue.

3.6. pH responsiveness

The pH of healthy skin typically ranges from 4.2 to 5.6, whereas that of unhealed wounds often deviates significantly from this range.²⁸ For instance, in extensive wounds such as those in the early stages of diabetes, the pH of wound exudate can gradually increase to 7.0–9.0.²⁹ Burn wounds exhibit pH values ranging from 5.0 to 9.0, and in the presence of infection, the pH may rise further to approximately 6.5 to 10.0.³⁰ In alkali burn wounds, alkaline substances react with exudates to generate hydroxide ions, resulting in an alkaline exudate. As healing progresses, inflammation subsides and tissue regeneration occurs, leading to a gradual decrease in pH, eventually returning to the normal acidic range. Monitoring the pH responsiveness of hydrogels is critical for real-time tracking of wound healing, enabling timely evaluation of wound status and informed therapeutic interventions. As shown in Fig. 3(a), anthocyanins display distinct pH-responsive color changes in PBS solutions across a range of pH values. When the pH increases from 5.0 to 9.0, the RA8-CHG hydrogel exhibits a visible color transition from flesh tone to dark brown. The color differences between hydrogels at pH 5.0 and pH 9.0, quantified using a colorimeter, all exceeded the threshold for significant visual distinction (NBS = 12)³¹ (Fig. 3(b)), indicating that these changes are clearly perceptible to the naked eye. Moreover, increasing the concentration of anthocyanins enhances the observed color contrast, attributable to their strong chromatic properties. In contrast, the incorporation of CHG had no significant effect on color intensity. At a prolonged immersion time (24 h), the color difference of the RA2 hydrogel at between pH 5.0 and pH 9.0 decreased significantly (P < 0.05), which may be attributed to its lower anthocyanin content and the subsequent release of anthocyanins over time. In contrast, the RA4, RA6, RA8, and RA8-CHG hydrogels showed no significant decrease in color difference after the extended immersion (Figure S12). Analysis of RGB values extracted from RA8-CHG hydrogels at various pH levels revealed a linear correlation between the B (blue) channel and pH (Fig. 3(c)). This demonstrates the hydrogel's high sensitivity to pH changes and its capability for real-time colorimetric indication, providing a promising foundation for the development of intelligent pH monitoring systems.

Fig. 3 (a) Representative color changes of various hydrogels immersed in PBS solutions with pH values ranging from 5.0 to 9.0 for 4 h. Scale bar: 5 mm. (b) Color differences of the hydrogels in PBS at pH 5.0 and pH 9.0 for 4 h. (c) Correlation between the B (blue) component of the RGB values of the RA8-CHG hydrogel and pH. (d) Anthocyanin and (e) CHG release profiles from RA8-CHG hydrogel in PBS at pH 5.0, 7.0, and 9.0.

These hydrogels can be engineered for enhanced release of antimicrobial agents and anthocyanins in alkaline environments, thereby exerting antibacterial and antioxidant effects that promote wound healing and inhibit infection. As shown in Fig. 3(d) and (e), the release profile of the RA8-CHG hydrogel is strongly pH-dependent. Under neutral conditions (pH 7.0), the hydrogel released 21.4% of anthocyanins and 3.0% of CHG after 24 h. A slightly higher release was observed under acidic conditions (pH 5.0), with 25.7% of anthocyanins and 3.6% of CHG released. This may be attributed to the increased polarity of anthocyanins in acidic environments, facilitating their diffusion. Under alkaline conditions (pH 9.0), release was markedly enhanced, with 37.3% of anthocyanins and 4.4% of CHG released within 24 h. This increase is likely due to deprotonation of functional groups in the hydrogel matrix, which induces electrostatic repulsion and significant swelling, leading to a more porous network that facilitates diffusion of both compounds (Fig. 1(g), (h) and Fig. S9).³² In summary, the RA8-CHG hydrogel demonstrates pronounced pH-responsive release, with maximal release under alkaline conditions. This behavior supports its application as a smart, stimuli-responsive antimicrobial system, particularly advantageous for infected wounds where elevated pH is common.

3.7. Antimicrobial properties of hydrogels

The zone of inhibition test was first used to investigate the antibacterial properties of the hydrogels. As shown in Fig. 4(a), no apparent inhibition zone was observed around the RA0 hydrogel or the hydrogels only containing anthocyanin, suggesting that anthocyanins alone lack significant bactericidal activity. In contrast, a clear inhibition zone was evident around the RA8-CHG hydrogel, with diameters of 20.7 mm against E. coli and 23.3 mm against S. aureus (Fig. S13), indicating that CHG markedly enhances its antibacterial performance. The observed antibacterial activity in the RA8-CHG group is primarily attributed to CHG, which disrupts the permeability barrier of bacterial membranes, inducing leakage of intracellular contents and ultimately leading to bacterial death.³³ The antibacterial properties of the hydrogels under varying pH conditions were further evaluated. Results indicated that bacterial levels in the RA0 and RA8 hydrogel groups remained largely unchanged across pH levels. For both E. coli (Fig. 4(b)) and S. aureus (Fig. 4(c)), the RA8-CHG hydrogel exhibited the weakest antibacterial activity at pH 7.0, while the strongest effect was observed at pH 9.0, where no viable bacteria were detected in the suspension. This enhanced antibacterial activity under alkaline conditions is likely due to the increased release of CHG, as shown in Fig. 3(e). At higher pH, the hydrogel released the greatest amount of CHG, resulting in superior antibacterial efficacy. These findings indicate that the RA8-CHG hydrogel exhibits distinct pH-responsive antimicrobial behavior, with significantly enhanced efficacy under alkaline conditions. By utilizing this pH-triggered release mechanism, the RA8-CHG hydrogel enables adaptive antibacterial action in infection-prone microenvironments.

Fig. 4 (a) Representative photographs of bacterial inhibition zones for each hydrogel against E. coli and S. aureus. Scale bars: 20 mm. Antibacterial activity of various hydrogels against (b) E. coli and (c) S. aureus in suspension at pH 5.0, 7.0, and 9.0. *, **, and *** represents P < 0.05, P < 0.01, and P < 0.001, respectively.

3.8. Antioxidant properties

During wound healing, oxidative stress can cause cellular damage and impede tissue regeneration. Therefore, assessing the antioxidant properties of hydrogels is crucial to determine their potential in mitigating free radical-induced damage and promoting cell proliferation and tissue repair in localized treatments. To evaluate their antioxidant capacity, the hydrogels’ ability to scavenge DPPH and ABTS˙⁺ free radicals was investigated. As shown in Fig. 5(a), the DPPH solution treated with the RA0 hydrogel retained its dark purple color, indicating minimal radical scavenging activity. In contrast, increasing the anthocyanin content in the hydrogels led to a gradual color change from purple to yellow, with RA8 and RA8-CHG treatments showing the most pronounced effect. DPPH free radicals exhibit a strong absorbance peak at 517 nm, and a decrease at this wavelength was used to quantify scavenging activity. As shown in Fig. 5(b), RA8 and RA8-CHG hydrogels achieved scavenging rates of 76.7% and 79.9%, respectively. This activity is attributed to the phenolic hydroxyl groups in anthocyanin, which can donate hydrogen atoms to neutralize free radicals and inhibit chain oxidation reactions.³⁴ Similarly, the ABTS˙⁺ scavenging assay revealed that the RA0 hydrogel lacked radical scavenging activity, while RA8 achieved a scavenging rate of up to 98.5% (Fig. 5(a) and (c)). These findings confirm that RA8 and RA8-CHG hydrogels exhibit strong antioxidant properties and can effectively neutralize free radicals. By scavenging free radicals, anthocyanin-based hydrogels can protect cells and biomacromolecules from oxidative stress, underscoring their promise for biomedical applications.

Fig. 5 Photographs showing (a) DPPH and ABTS˙⁺ scavenging by various hydrogels. Quantitative analysis of (b) DPPH and (c) ABTS˙⁺ radical scavenging rates for each hydrogel. * and *** represents P < 0.05 and P < 0.001, respectively. (d) Cell viability of L-929 fibroblasts after incubation in hydrogel extract medium for 24 h. (e) CLSM images of L-929 fibroblasts after incubation in hydrogel extract medium for 24 h. Scale bar: 100 µm.

3.9. In vitro cytotoxicity and anti-inflammatory capability

Evaluating the cytotoxicity of hydrogels is essential to ensure their safety for biomedical applications such as wound healing. A hydrogel must demonstrate non-toxic or low-toxicity profiles to cells in order to confirm its tissue compatibility and clinical safety. In this study, the in vitro cytotoxicity of the hydrogels was evaluated following the ISO 10993-5 standard, with slight modifications to the experimental protocol. As shown in Fig. 5(d), after 24 h of incubation with L-929 fibroblasts, all hydrogel extract groups maintained cell viability above 95.8%, indicating excellent cytocompatibility of the anthocyanin-containing hydrogels. Live/dead staining further supported this finding, with most cells appearing green and only a few dead cells stained red under CLSM (Fig. 5(e)). This favorable biocompatibility is largely attributed to the intrinsic biosafety of anthocyanins, zwitterionic polymers, and CHG. Collectively, these results demonstrate that the anthocyanin-based hydrogels possess outstanding biocompatibility, highlighting their promise as safe and effective wound dressing materials.

Specifically, Raw 264.7 macrophages were treated with extracts prepared from RA0, RA8, and RA8-CHG hydrogels. To assess the anti-inflammatory activity, LPS-stimulated Raw 264.7 cells were employed, and NO, a representative pro-inflammatory mediator, was quantified as an indicator of macrophage inflammatory response. As shown in Fig. S14a, both RA8 and RA8-CHG extracts significantly suppressed LPS-induced NO production (P < 0.001). These findings demonstrated that the incorporation of anthocyanin effectively attenuates inflammatory responses, thereby highlighting their potential as anti-inflammatory biomaterials (Fig. S14b).

3.10. Monitoring and healing of alkali-burned wounds

Unlike conventional acute wound models, alkali burns represent a more severe and complex challenge due to persistent chemical damage. In this study, 10 mm diameter full-thickness wounds were created on the dorsum of ICR mice, and alkali burns were induced by applying filter paper soaked in 1 mol L⁻¹ sodium hydroxide solution to the wound sites. As demonstrated in the results above (Fig. 3), the RA8-CHG hydrogel exhibits pH-responsive color-changing properties. To further validate this functionality, the hydrogel was applied to both normal and alkali-burned wounds for 24 h, during which color changes were monitored. On normal wounds, the hydrogel appeared orange, whereas on alkali-burned wounds it turned black, indicating a clear visual distinction (Fig. S15). This shift in color reflects the pH differences between the two wound environments, confirming the hydrogel's sensitivity to environmental pH changes. Further analysis suggests that the blackening on the hydrogel on alkali-burned wounds results from its interaction with residual alkaline substances. The hydrogel likely adsorbs and neutralizes excess alkali, thereby mitigating further tissue damage. This pH modulation contributes to the stabilization of the wound microenvironment, which is beneficial for tissue regeneration.

Given that the ultimate goal of hydrogel dressing is clinical application, it is essential to validate both the efficacy and safety of RA8-CHG in promoting alkali burn healing in vivo. To this end, a mouse model was employed, with untreated animals serving as controls and the RA8-CHG hydrogel applied to the experimental group. A schematic of the experimental design is shown in Fig. 6(a). This setup enabled direct comparison of wound healing outcomes between the two groups, facilitating assessment of the hydrogel's therapeutic potential. Throughout the 21-day healing period, wound morphology was documented at various time points (Fig. 6(b) and (c)). Wounds treated with RA8-CHG hydrogel exhibited a significantly accelerated reduction in wound area compared to the control. By day 3, enhanced wound contraction and increased tissue proliferation were evident in the hydrogel-treated group (Fig. 6(d)). Scab formation was observed by day 7, forming a protective barrier over the wound. In contrast, healing in the control group was delayed, likely due to deeper tissue damage, which exacerbated muscle cell necrosis. The degradation of necrotic tissue produced lipofuscin, resulting in a yellow discoloration of the wound area.^35,36 By day 14, the scabs in the hydrogel-treated group had largely detached, and re-epithelialization was well underway. By day 21, epidermis restoration was nearly completed, although pigmentation had not yet returned to baseline. In the control group, the wounds remained dark red, possibly due to ongoing vascular remodeling or unresolved inflammation.³⁷ These findings underscore the limited regenerative capacity and extensive tissue damage associated with untreated alkali burns, highlighting the therapeutic advantage of the RA8-CHG hydrogel. These wound–healing benefits can be attributed to the hydrogel's integrated functions, including efficient absorption of wound exudate, mitigation of oxidative stress, inhibition of bacterial proliferation, and stabilization of local pH, which collectively create a favorable microenvironment for tissue repair and regeneration.

Fig. 6 (a) Schematic illustration of the alkali-burned wound healing experiment. (b) Representative images of the full-thickness dorsal skin wounds in alkali-burned mice treated with either the control (untreated) or RA8-CHG hydrogel for 21 days. (c) Wound healing process of alkali-burned mice in the control group and RA8-CHG group. (d) Quantitative analysis of wound area over time in the control and RA8-CHG groups. * represents P < 0.05. (e) Representative tensile stress–strain curves of native skin, healed skins in the control group and RA8-CHG group. (f) Comparison of tensile strength and Young's modulus among native skin, healed skin in the control group and RA8-CHG group. * (P < 0.05), ** (P < 0.01), *** (P < 0.001), and ns (not significant) were compared with the native skin group.

To evaluate the mechanical recovery of healed skin tissue, tensile testing was performed on wound sites from both groups (Fig. 6(e)). As shown in Fig. 6(f), the tensile strength of native mouse skin was 5.2 MPa. By day 21 post-injury, the control group exhibited a tensile strength of only 1.2 MPa, whereas the RA8-CHG-treated group reached 2.5 MPa. Although neither group achieved the mechanical properties of native skin, the RA8-CHG treatment significantly improved tensile strength compared to the control (P < 0.05), highlighting its beneficial effect on tissue biomechanics. Similarly, the tensile modulus of native mouse skin was 0.63 MPa. The control group's modulus was markedly reduced to 0.31 MPa (P < 0.05), while the RA8-CHG-treated group achieved a modulus of 0.57 MPa, closely approximating that of normal tissue. These findings indicate that RA8-CHG hydrogel promotes biomechanical recovery and supports functional tissue regeneration.

Although the healed tissue did not fully recover the mechanical strength of healthy skin, the RA8-CHG group exhibited significantly improved healing outcomes compared to the control group. This enhancement is attributed to the hydrogel's ability to maintain a moist microenvironment, attenuate inflammation, and promote collagen deposition. To further evaluate wound healing, histological analysis was conducted using H&E staining. As shown in Fig. 7(a), the scar width in the RA8-CHG group was significantly reduced relative to the control group, indicating more efficient tissue repair with minimized scar formation. The control group retained scab tissue in the regenerated epidermis, likely due to persistent inflammation and inadequate microenvironment regulation. In contrast, the RA8-CHG group exhibited smoother wound surfaces and minimal scab formation, underscoring the hydrogel's beneficial effect on epidermal regeneration.

Fig. 7 (a) Representative tissue sections of regenerated skin wounds from mice in the control and RA8-CHG groups were analyzed on day 21. Scar width is indicated by green arrows. Scale bar: 2 mm. (b) H&E staining and (c) Masson's trichrome staining of regenerated tissues from alkali burn wounds on days 7, 14, and 21. Red and blue arrows indicate blood vessels and neutrophils, respectively. Scale bar: 200 µm. (d) Radar chart comparing the overall performance of representative wound–healing hydrogels in terms of pH-responsiveness, antibacterial activity, antioxidant capacity, tensile strength, compressive strength, and adhesion strength.^38–44

To track tissue changes during different stages of alkali burn healing, both H&E and Masson staining were performed. On day 7 (Fig. 7(b)), the control group showed typical acute inflammatory features, including pronounced neutrophil infiltration. While neutrophils were also present in the RA8-CHG group, their infiltration was markedly reduced, suggesting an anti-inflammatory effect of the hydrogel. By day 14, inflammation had subsided in both groups, although mild signs persisted in the control group. By day 21, inflammation had largely resolved in both groups. However, enhanced vascularization was evident in the RA8-CHG group, further affirming its role in promoting tissue repair. Masson staining revealed the patterns of collagen fiber distribution and deposition (Fig. 7(c)). Collagen fibers appeared blue, while cytoplasm, muscle, and red blood cells were stained red. On day 7, the control group exhibited limited collagen accumulation near inflammatory sites. By day 21, collagen deposition had increased, with a more distinct epidermal-dermal boundary. In contrast, the RA8-CHG group demonstrated uniform collagen distribution as early as day 7. By day 14, collagen fibers were concentrated in the mid-to-deep dermal layers, and by day 21, collagen was homogeneously distributed throughout the tissue, with a well-defined epidermal–dermal interface. Collectively, these results indicate that the RA8-CHG hydrogel promotes orderly collagen deposition, enhances tissue remodeling, and contributes to more stable and functional skin regeneration. In summary, the RA8-CHG hydrogel not only accelerates wound healing but also exhibits pH-responsive colorimetric properties, modulates inflammation, and supports structural regeneration. These multifunctional characteristics highlight its potential as an intelligent wound dressing, particularly suitable for treating chronic and chemically induced wounds.

As shown in Fig. 7(d), RA8-CHG exhibited a unique combination of desirable properties, including strong antibacterial activity (>99.1%), high antioxidant activity (79.9%), excellent tensile strength (94.1 kPa), compressive strength (8.5 MPa), and adhesion strength (28.5 kPa). Such a balanced performance surpasses most previously reported systems, which typically provide only partial functionality.

4. Conclusions

In summary, a pH-responsive hydrogel (RA8-CHG) via a facile one-step free radical polymerization was developed, incorporating rose anthocyanins and CHG into a chemically crosslinked polySBMA network. The hydrogel exhibits distinct color changes under varying pH conditions, enabling real-time pH indication while simultaneously delivering antibacterial and wound–healing functions. RA8-CHG hydrogel demonstrated excellent mechanical strength, tissue adhesion, antioxidant activity, and biocompatibility. In vivo studies confirmed its capacity to accelerate healing in alkali burn wounds, reduce scar formation, and attenuate local inflammation, underscoring its clinical promise. Despite these encouraging results, several challenges, including limited sensitivity and reversibility of the pH-responsive colorimetric behavior, remain before clinical translation can be realized. Nevertheless, by integrating pH-responsive color indication with antibacterial and biocompatible features, the RA8-CHG hydrogel offers a promising platform for smart wound care, particularly in applications such as chronic wound management and next-generation intelligent dressings.

Author contributions

Jiru Miao: conceptualization, methodology, formal analysis, investigation, data curation, writing – original draft; Haiyang Chai: investigation, resources; Lei Song: resources; Xiaoxiao Liao: resources; Shuyan Chen: resources; Ying Xiao: funding acquisition; Rong Wang: conceptualization, methodology, resources, writing – review & editing, supervision, project administration, funding acquisition.

Conflicts of interest

The authors declare no conflict of interest.

Data availability

All data are available in the main text or the SI, with additional data provided upon request.

Supplementary information is available. See DOI: https://doi.org/10.1039/d5tb01386k.

Acknowledgements

This work was funded by Key Research and Development Program of Ningbo (2022Z132), and Joint Funds of the Zhejiang Provincial Natural Science Foundation of China (LZY24H150001).

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