Anti-inflammatory and tissue regeneration effects of a chlorogenic acid/hyaluronic acid hydrogel on methicillin-resistant Staphylococcus aureus-infected diabetic wounds

Abstract

Infections triggered by bacteria in diabetic wounds continue to pose a significant challenge, primarily due to the inflammatory microenvironment induced by high glucose levels, which favor bacterial growth. Hence, developing dressings tailored for diabetic wound treatment has become particularly crucial. Here, we prepared a composite hydrogel derived from natural polymers as a wound dressing. This composite hydrogel was fabricated by the cross-linking of hyaluronic acid (HA) grafted with chlorogenic acid (CA) and phenylboronic acid (PBA) and the incorporation of copper sulfide nanoparticles (CuS NPs). The hydrogels exhibited adequate adhesive properties and self-healing capabilities. By releasing the natural polyphenol CA, the hydrogel showed promising antioxidant performance, excellent promotion of cell proliferation, and angiogenesis properties, thereby effectively promoting tissue repair. The treatment on an in vivo diabetes wound model indicated that the dressing contributed to wound closure, re-epithelialization, collagen deposition, and the downregulation of inflammatory factors. This multifunctional hydrogel presented a potent strategy for managing infected diabetic wounds and showed significant promise for clinical translation.

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1. Introduction

Diabetes, as a chronic disease, is increasing in incidence rate year by year and is expected to increase to 12.2% by 2045.¹ For diabetics, the long-term instability in blood sugar levels can lead to serious complications, including diabetic ketosis and retinal vascular disease,² as well as large numbers of wound infections and ulcer problems.³ The wounds in patients with diabetes typically heal slowly due to the vasoconstriction and rapid bacterial proliferation induced by hyperglycemia. The long-term exposed skin wounds further provide a breeding ground for bacteria.^4,5 If the wound is not treated promptly, it can become very difficult to heal, which might lead to ulceration or even amputation. Inspired by the characteristics of diabetic wounds, more and more wound dressing systems are focused on the field of diabetic wounds.^6,7 Hu et al. loaded berberine into natural living microalgae to form a bioactive hydrogel. Under laser irradiation, the hydrogel can continuously release berberine and produce reactive oxygen species (ROS),⁸ thus producing antibacterial ability against methicillin-resistant Staphylococcus aureus (MRSA).⁹ In addition, tissue repair at the wound site is crucially important. Liu et al. developed a self-assembled antibacterial quaternary ammonium salt polymer hydrogel without loading drugs and cytokines, which was simple and effective in promoting tissue regeneration and wound repair.¹⁰ Therefore, it was important to promote tissue repair in conjunction with treatment.¹¹ Current diabetic wound dressings (e.g., hydrocolloids and alginate) often fail to address the triad of bacterial resistance, chronic inflammation, and impaired angiogenesis. Moreover, frequent dressing changes disrupt healing and increase infection risk. There is an urgent need for multifunctional dressings that integrate antimicrobial, antioxidant, and pro-regenerative properties. There is a strong need to develop a non-toxic hydrogel dressing that has excellent antimicrobial and antioxidant capabilities and promotes tissue regeneration and angiogenesis.^12–14

HA, as a naturally occurring linear polysaccharide polymer, is a major component of the extracellular matrix.¹⁵ Therefore, it is highly biocompatible and non-immunogenic.¹⁶ In addition, it has been shown that HA can enhance the migration and differentiation of mesenchymal and epithelial cells, as well as improve angiogenesis and collagen deposition to alter wound healing ability.^17,18 CA is a natural active ingredient extracted from plants such as Lonicerae Japonicae Flos and coffee beans.¹⁹ It exists as one of the most abundant and powerful polyphenolic compounds in the human diet, and is endowed with strong antioxidant activity.^20,21 In addition, CA can disrupt the structure of bacterial cell membranes and interfere with bacterial metabolic processes, thereby exerting antibacterial effects.^22,23 At the same time, CA can inhibit inflammation damage by regulating the expression of related proteins in the inflammatory response,^19,20 such as downregulation of the expression level of tumor necrosis factor-α (TNF-α) and reducing the cytokine levels of interleukin-1β (IL-1β) and interleukin-6 (IL-6).²⁴ Recent advances in photothermal therapy (PTT) leverage nanomaterials like CuS NPs to convert NIR light into localized heat,^25,26 enabling precise bacterial eradication. Unlike antibiotics, PTT avoids resistance and synergizes with tissue-regenerative agents, making it ideal for chronic wounds. CuS NPs is an inorganic photothermal agent with a low-cost, simple preparation, and strong absorption ability in the NIR-II region.^27,28 Meanwhile, Cu²⁺ can act as an essential element released by CuS NPs to promote wound healing,²⁹ and stimulate angiogenesis by upregulating the expression of vascular endothelial growth factor.^30,31 Copper ions are also important catalysts for many enzymes, proteins, and transcription factors, such as vascular endothelial growth factor (VEGF) and nerve growth factor.³² They play an important role in many wound healing processes, and are used as preservatives and antibacterial agents.³³ Therefore, we hypothesized that hydrogels developed by mixing the biopolymer HA with the natural active ingredient CA rich in hydroxyl groups in an aqueous solution would exhibit significant antimicrobial activity and therapeutic effects on diabetic wounds through their combined antioxidant, antibacterial, and pro-regenerative properties. To prove our hypothesis, as shown in Scheme 1, we developed a novel multifunctional hydrogel, CuS@HPCA, for diabetic wound healing. This work distinguishes itself from existing systems through several key conceptual advances.

Scheme 1 Schematic of the preparation of the composite hydrogel CuS@HPCA and its application in infected wound healing.

Unlike conventional hydrogels where drugs are merely encapsulated, CA was employed as a dynamic crosslinker via pH-sensitive phenylboronate ester bonds. This design enables intelligent, sustained release of CA specifically in the acidic wound microenvironment, maximizing its therapeutic efficacy while minimizing premature release. At the same time, a sophisticated feedback mechanism was operated: the acidic infection triggered CA release to combat bacteria and inflammation, while the NIR-triggered photothermal effect not only directly eradicated bacteria but also enhanced the release of CA, creating a powerful synergistic antibacterial action.

In brief, we have developed an antimicrobial hydrogel that not only promotes tissue regeneration but also offers a new blueprint for designing intelligent, antibiotic-free drug delivery systems. The hydrogel, which named as CuS@HPCA, was characterized with acid sensitivity and sustained release profile of CA in the inflammatory environment. The antibacterial ability against MRSA, the biocompatibility and the ability to promote the cell migration and angiogenesis of the hydrogel were studied. Finally, the MRSA model of a wound infection in diabetic mice was established in vivo to investigate the effect of the composite hydrogel on tissue regeneration and anti-inflammatory performance. In brief, we have successfully prepared a new antimicrobial hydrogel that significantly promotes tissue regeneration for chronic diabetic wounds, and at the same time offers new solutions for antibiotic-free drug delivery systems.

2. Materials and methods

2.1 Materials

HUVECs, L929 cells were purchased from ATCC. MRSA ATCC 43300 was kindly provided by the Department of Chemistry, Nankai University (Tianjin, China). Hyaluronic acid (30 kDa), 3-aminophenylboronic acid (98%), chlorogenic acid (98%), carbodiimide hydrochloride (EDC, 99%), and N-hydroxy succinimide (NHS, 98%) were purchased from Beijing InnoChem Science & Technology Co., Ltd. CuCl₂ (98%), sodium citrate (98%) and Na₂S (98%) were sourced from Shanghai Macklin Biochemical Co., Ltd.

2.2. Synthesis and characterization of HAPBA

According to the literature, HA (1.00 g, 2.5 mmol) and APBA (0.68 g, 5.0 mmol) were dissolved in 50 mL of deionized (DI) water and 20 mL of DMSO in the flask, then a solution of EDC (0.48 g, 2.5 mmol) and NHS (0.28 g, 2.5 mmol) in 30 mL DMSO was added to the flask with vigorous stirring for 24 h. The obtained reaction solution was placed in 1 mM NaOH solution for dialysis (MWCO 3000) for 2 days, and then subjected to dialysis in DI water for 2 days, and finally freeze-dried to obtain HAPBA. ¹H NMR spectroscopy (Ascend-600, Bruker, Germany) was used to characterize the structure of HAPBA.

2.3. Synthesis and characterization of CuS NPs

CuS NPs was synthesized using the previous method with modifications.³⁴ CuCl₂ (13.5 mg, 0.1 mmol) and sodium citrate (20 mg, 0.06 mmol) were dissolved in 100 mL of DI water under continuous stirring. Na₂S·9H₂O (24 mg, dissolved in 10 mL DI water) and the solution were added dropwise to the above solution at room temperature with continuous stirring for 5 min, and then the reaction temperature was increased to 90 °C. After stirring for another 30 min, CuS NPs was obtained. The particle size and polydispersity index (PDI) were analyzed by dynamic light scattering (DLS) (Nano ZS90, Malvern Instruments Ltd, UK) and the morphology was observed by transmission electron microscopy (TEM, FEI tecnaif-20, the United States). Then, the UV visible absorption spectra of 100 μg mL⁻¹, 200 μg mL⁻¹, and 400 μg mL⁻¹ CuS NPs solution were collected by a UV spectrophotometer (PurkinjeTU1901, China) within a wavelength range from 400 to 1100 nm.

2.4. Preparation and characterization of hydrogels

80 mg HAPBA was dissolved in 1 mL CuS NPs dispersion with CuS concentrations of 100, 200, and 400 μg mL⁻¹, respectively. After that, 5 mg CA was dissolved in 200 μL DI water and added to the above solution, and then the reaction system was rested for 1 hour to obtain the cross-linked hydrogel. The hydrogels loaded with 100, 200, or 400 μg mL⁻¹ CuS NPs were named CuS1@HPCA, CuS2@HPCA and CuS4@HPCA, respectively. Similarly, 5 mg CA was also dissolved in 200 μL DI water and added to 80 mg HAPBA in 1 mL DI water to prepare a blank hydrogel, which was named as HPCA. The gelation state of the hydrogel was detected by the tube inversion method. HAPBA and CA solution were mixed, and the test tube was inverted every 10 seconds at room temperature. Once the mixture could not flow within 60 seconds after inversion, the crosslinking was considered to be complete. Fourier transform infrared (FTIR) spectroscopy (Scientific Nicolet 870, the United States) was used to assess the structure of the hydrogels. Scanning electron microscopy (SEM, HITACHI SU8020, Japan) was used to examine the surface features of the hydrogels and the distribution of copper and sulfur in the hydrogel through energy dispersive spectrometer (EDS).

2.5. Rheological property tests

The rheological analysis of the fabricated hydrogels was performed by using a rheometer (Anton Paar MCR 702e, Austria) with a 25 mm parallel plate and a 1 mm gap setting. The storage (G′) and loss (G″) modulus of hydrogels were measured at 25 °C. The frequency sweep was measured with an angular frequency of 1 to 100 rad s⁻¹ at a constant strain of 1% in the linear viscoelastic region. Strain alternating sweeps were performed at a fixed frequency (1.00 Hz) from light strain (γ = 1%) to large strain (γ = 300%).

2.6. Swelling ratio tests

The swelling properties of HPCA and CuS@HPCA hydrogels were tested in DI water at 25 °C. The swollen hydrogel was removed at a specific time, weighed after the surface water was wiped off, the initial weight of the lyophilized hydrogel was called W_o and the weight of the hydrogel was recorded as W_t (n = 3).

2.7. Adhesion and self-healing tests

For characterization of the adhesiveness of the hydrogel, the substrates of glass, plastic, metal, were selected. The hydrogel was cut into two parts at room temperature to investigate the self-healing behavior of the hydrogel, and the two separate parts were allowed to re-contact for 5 minutes. The recovered hydrogel was stretched to observe the damaged degrees.

2.8. Photothermal performance

To evaluate the photothermal performance of CuS NPs, 1 mL of CuS NPs at different concentrations (50, 100, 200, and 400 μg mL⁻¹) were irradiated with NIR laser (1064 nm Changchun Optoelectronics, MDL-III) with a power density of 1.0 W cm⁻² for 6 min, and the temperature change of the solution was monitored every 1 minute. In addition, the photothermal stability of CuS NPs at a concentration of 400 μg mL⁻¹ was examined by four cycles (1.0 W cm⁻²) with or without laser irradiation. Similarly, the photothermal properties of the CuS NPs-loaded hydrogels were tested by the same method.

2.9. CA release study

The cumulative release of CA was investigated in PBS (pH 7.4 and pH 6.0 at 37 °C). At a predetermined time point, the supernatant was taken out and an aliquot amount of fresh PBS was supplemented. The cumulative released amount of CA was quantified by UV-vis spectrometer. The photothermal controlled release of CA was alternated between turning on and off the laser every five minutes, and the testing method was the same as above.

2.10. Hemolysis assessment

Briefly, 500 μL of rabbit blood was centrifuged at 3000 rpm for 5 min and then washed 3 times with PBS to a final concentration of 5%. The 500 μL HPCA hydrogel or CuS@HPCA hydrogel impregnation solution was then mixed with 500 μL of erythrocytes in a 1.5 mL EP tube, and incubated for 4 h at 37 °C. Samples were centrifuged at 3000 rpm for 5 min, and the absorbance of the supernatant was measured at 545 nm using a UV-vis spectrophotometer. PBS and Triton X-100 (1%) were used as negative and positive controls, respectively.

The hemolysis rate was calculated by using the following formula:

where OD_n represents the absorbance of the negative group, OD_e represents the absorbance of the experimental group, and OD_p represents the absorbance of the positive group, respectively.

2.11. Cytocompatibility assessment

The cytocompatibility properties of HPCA and CuS@HPCA hydrogels against L929 cells and HUVECs were determined by 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay and Live/Dead cell staining. L929 cells and HUVECs (1.0 × 10⁵ cells per well) were seeded on 96-well plates and incubated for 24 h, extracts of hydrogels at different concentrations were added and then incubated for 24 and 72 h. After that, 10.0 μL MTT (5.0 μg mL⁻¹ in PBS) was added to each well and incubated for 4 h. The OD value of formazan dissolving in 100.0 μL DMSO of each well was recorded at 570 nm using a microplate reader (Bio-T Instruments, Inc., United States). The cell viability ratio was calculated by the following formula:
where OD_t is the OD value of the 24 or 72 hour culture of the added sample, and OD_c is the 24 or 72 hour OD value of the DMEM culture without FBS.

2.12. Antioxidation experiment

100 μM DPPH and different samples in 2 mL of ethanol were mixed and reacted at 37 °C for 30 min in the dark. PBS was used as the control group. The absorbance of the mixture was measured at 517 nm using a UV-Vis spectrophotometer. The DPPH scavenging activity was calculated using the following formula:
where A_b is the absorbance of the control and A_h is the absorbance of the sample.

2.13. ROS fluorescent staining

In order to study the ROS scavenging effect of hydrogels, the ROS level of cells treated with LPS (1 μg mL⁻¹) was detected by fluorescence probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). L929 cells were inoculated into each well of a 24 well plate (1 × 10⁵ cells per well) and attached to the plate overnight. Hydrogel extract was added to each well, and the cells were cultured in each well (with or without hydrogel) in LPS containing growth medium for 4 hours. After removal of the culture medium, the cells were incubated in the probe solution at 37 °C for 20 minutes, followed by microplate reading and fluorescence microscopy (Echo Revolve Generation 2, the United States) imaging.

2.14. Photothermal and antibacterial performance

In order to investigate the antibacterial efficiency of the hydrogel, MRSA (10⁸ CFU per mL) and hydrogels were incubated for 24 h, and then irradiated for five minutes by near-infrared laser. The bacterial growth rate was measured by plate counting method. The bactericidal effect was determined as the decrease of CFU per mL after 24 hours, and photographs of the (lysogeny broth) LB tablets were taken.

2.15. Cell migration assay

Cell migration was determined by wound scratching assay. The L929 cell suspension with a density of 5.0 × 10⁵ cells per well was seeded in 6-well plates and cultured for 24 hours. Scratches on each well at the bottom of the plate were made and washed 3 times with PBS. The medium was replaced with different hydrogel leachates, and then were incubated for 24 hours. The scratching pictures were taken by optical microscope (Olympus CKKX41, Japan).

2.16. Tube formation evaluation

Matrigel were added to the pre-chilled 96-well plates and leaved at 37 °C for 1 h. HUVEC-GFP cells were seeded into plates in a density of 4 × 10³ cells per well. The cells with 100 μL of different samples were incubated. After 6 hours incubation, tube forming images were obtained by fluorescence microscope. The total length and branches number were analyzed by ImageJ in 3 random regions per well.

2.17. In vivo diabetes wound healing assay

All animal experiments complied with the National Institutes of Health guide and were approved by the Animal Welfare Ethics Committee, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (approval no.: 2024B187), and were carried out in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals. Kunming mice (50 males, 20–30 g) were purchased from SPF (Beijing) Biotechnology Co., Ltd, which were raised on a high-fat diet at room temperature, in a clean and ventilated environment. The mice were administered by intraperitoneal injection of streptozotocin for 3 consecutive days. When glucose levels in mice consistently exceeded 16.8 mmol L⁻¹, the diabetes model was regarded successfully established.³⁵ A full-thickness wound with a diameter of 8 mm was produced on the back of each mouse after being anaesthetized by isoflurane. All the mice were divided into four groups, which were named as model group, HPCA group, CuS4@HPCA group, and CuS4@HPCA + NIR group, respectively. The wounds were covered with HPCA and CuS4@HPCA hydrogel, respectively. The CuS4@HPCA + NIR group was irradiated by near-infrared light for five minutes. The wound area was observed and photographed on days 0, 3, 7 and 14, respectively. After 7 days of treatment, half of the mice in each group were sacrificed for hematoxylin and eosin (H&E) staining and Masson staining. The rest of the mice were sacrificed for the immunohistochemistry assay of inflammatory factors TNF-α, IL-6, IL-1β, and immunofluorescence staining to observe the expression of CD31 and VEGF, and H&E and Masson staining were also performed after 14 days treatment.

2.18. Statistical analysis

All data were shown as mean ± standard deviation (SD) and each experiment was performed for at least three times. The statistical analysis was examined by T-test and one-way ANOVA. All statistical tests were denoted as significant when p values * < 0.05, highly significant when ** < 0.01, and extremely significant when *** < 0.001.

3. Results and discussion

3.1. Synthesis and characterization of HAPBA

As shown in Fig. 1A, PBA was grafted onto hyaluronic acid through EDC/NHS chemistry. Fig. 1B shows the ¹HNMR spectra of the three samples, where the peak at δ = 7.0–8.0 ppm in HAPBA was considered the successful grafting of PBA to HA. The degree of substitution was calculated as 28.3% based on the ratio of the aromatic proton integral (7.0–8.0 ppm, 4H, –C₆H₄) to methyl proton peak integral (peak b, 2.0 ppm, 3H, –CH₃) of HAPBA.

Fig. 1 Characterization of hydrogels. (A) The synthesis of HAPBA. (B) ¹H NMR of PBA, HA, and HAPBA. (C) FT-IR spectra of CA, HAPBA and HPCA. (D) UV-vis spectra of the CuS NPs. (E) Particle size distribution and TEM of the CuS NPs. (F) SEM micrograph of the HPCA and CuS@HPCA hydrogels and the distribution of the Cu element and S element in the CuS@HPCA hydrogel. (G) Gelation of HPCA.

3.2. Characterization of CuS NPs

As shown in Fig. 1D, CuS NPs with different concentrations exhibit broad absorption at 900–1100 nm, which might endow CuS NPs with near-infrared laser-induced photothermal conversion capability. The CuS NPs were synthesized by hydrothermal assay, and TEM images of CuS NPs are shown in Fig. 1E, exhibiting a uniform spherical shape with sizes at approximately 10 nm. In addition, the size was confirmed by dynamic light scattering (DLS). The hydrated particle size of CuS NPs was 42.00 ± 15.29 nm with a PDI of 0.171, indicating that CuS NPs had good dispersiblity in water.

3.3. Preparation and characterization of the hydrogel

The peak detected at 851 cm⁻¹ in the FTTR spectrum (Fig. 1C) represents the =CH stretching vibration contributed by the benzene ring. The C=C bending vibration of the phenyl group was also observed at 1621 cm⁻¹. The cross-linking reaction was demonstrated using HAPBA and CA solutions by the tube inversion method (Fig. 1G). Then, SEM was utilized to evaluate the porous structure of the hydrogels (Fig. 1F), which was favorable in facilitating the transport of nutrients and oxygen. Each group of hydrogels showed a porous structure, and the copper and sulfur elements were evenly distributed in the hydrogels according to EDS analysis.

3.4. Rheological property tests

The rheological properties of the dynamic hydrogels are shown in Fig. 2A. The frequency sweep tests of both hydrogels showed that the modulus increased slightly, but G″ continued to be lower than G′ in the angular frequency range from 1 to 100 rad s⁻¹, indicating that the elastic properties of these dynamic hydrogels were well maintained. At the same time, a continuous step strain test was carried out to study the rheological recovery behavior (Fig. 2B). When the strain reached the critical value of 300%, the value of the elastic modulus of each group of hydrogels immediately decreased from 1152 to 340 Pa, which was lower than the viscous modulus (485 Pa). As soon as the low strain (1%) was applied, the elastic modulus and viscous modulus were immediately restored. This showed that the collapse and recovery of all hydrogel networks was reversible, with rapid and efficient self-healing capability.

Fig. 2 (A) Rheological performance of the hydrogels at 1% strain in the frequency range of 1–100 rad s⁻¹. (B) G′ and G″ at alternate step strains of 1% and 300% in each cycle (3 cycles). (C) The swelling ratios of the hydrogels. (D) The self-healing performance of the hydrogels. (E) The adhesive performance of the hydrogels to glass, plastics and metal.

3.5. Swelling tests

As an ideal wound dressing, hydrogels should be able to absorb exudate while providing a moist healing environment. As shown in Fig. 2C, all four hydrogels had a significant swelling rate of up to 800% in pH7.4 PBS at room temperature. At the same time, this hydrogel had the characteristics of rapid water absorption and swelling within five minutes, which could be used for the rapid absorption of wound exudate.

3.6. Self-healing and adhesion tests

As shown in Fig. 2D, the half portion of the hydrogel stained with methyl red could combine with another one to form a new hydrogel, which exhibited good tensile properties. It was speculated that the self-healing properties of hydrogels was attributed to the hydrogen bond formed in the hydrogel networks. It was crucial to cover the wound site with a dressing that exhibited excellent media adhesion and self-healing ability to provide a good healing environment, so as to promote long-term tissue regeneration. As shown in Fig. 2E, the hydrogel demonstrated excellent adhesion capabilities to different surfaces (plastic, metal, and glass), even in lifting a 200 g balancing weight.

3.7. Photothermal performances of CuS NPs and hydrogels

CuS NPs have been reported in many literature studies to have photothermal properties.³⁶ The temperatures of the CuS NPs (100 μg mL⁻¹, 200 μg mL⁻¹, 400 μg mL⁻¹) suspensions were increased significantly within 6 minutes laser irradiation. At the same time, CuS NPs exhibited a stable photothermal heating effect and a similar warming pattern during the four heating–cooling cycles (Fig. 3A and B). Similarly, the hydrogels loaded with CuS NPs at a concentration of 400 μg mL⁻¹ reached 50 °C after five minutes of irradiation, which was faster than free CuS NPs (Fig. 3C and D). This might be ascribed to the better distribution of CuS NPs in the hydrogel. In addition, considering the photothermal heating competence, the concentrations of 200 μg mL⁻¹ and 400 μg mL⁻¹ were selected for subsequent experiments and defined as CuS2@HPCA and CuS4@HPCA, respectively.

Fig. 3 Photothermal and release behaviors of the hydrogels. (A) Temperature and NIR irradiation time curves of the CuS NPs under different concentrations. (B) Photothermal heating and cooling cycle curves of 400 μg mL⁻¹ CuS NPs. (C) Temperature and NIR irradiation time curves of the CuS@HPCA hydrogels under different concentrations. (D) Photothermal heating and cooling cycle curves of CuS4@HPCA. (E) CA release curves of the composite hydrogels at pH 7.4 and 5.5, respectively. (F) CA release curves of the composite hydrogels in four heating and cooling cycles.

3.8. CA release study

The hydrogel was acid-responsive since the phenylborate bond could be broken under acidic conditions. As shown in Fig. 3E, CA showed a higher release at pH 5.5 with a cumulative release of 67.6% in 60 h. However, only 17.6% of CA was released at pH 7.4 in the same period. The hydrogel demonstrated good release behavior under acidic conditions, providing a prerequisite for responsive inflammatory microenvironmental therapy. In addition, the release behavior of CA from hydrogels loaded with CuS NPs was determined by repeating the laser irradiation for four times to verify whether the heat generated from the photothermal reagent could affect the release behavior of CA. As shown in Fig. 3F, it was suggested that the increased heat contributed to the release of CA by comparison of the release curves of CuS2@HPCA and CuS4@HPCA. Nearly 45% of CA could be released from the CuS4@HPCA hydrogel in 50 minutes, hinting that the heat generated by the CuS NPs could effectively promote the release of CA from the hydrogels.

3.9. Hemolysis assessment

The good hemocompatibility of the hydrogels is shown in Fig. 4F. The hemolysis rate of the hydrogel extracts was less than 2%, even at a concentration of 20 mg mL⁻¹. Therefore, it was assumed that all hydrogels fabricated in this study have high potential to be used as wound dressings.

Fig. 4 The biocompatibility and antibacterial properties of the composite hydrogels. The cell viability of L929 cells after incubation with the HPCA, CuS2@HPCA and CuS4@HPCA extracts for 1 day (A) and 3 days (B), respectively. The cell viability of HUVECs after incubation with the HPCA, CuS2@HPCA and CuS4@HPCA extracts for 1 day (C) and 3 days (D). (E) The live/dead staining of L929 cells and HUVECs after incubation with HPCA, CuS2@HPCA and CuS4@HPCA extracts for 3 days (scale bar = 200 μm). (F) The hemolysis rate curve of composite hydrogel. (G and H) Images of the photothermal effect-assisted bactericidal performance of the hydrogels for MRSA eradication and the quantitative statistics of their respective antibacterial activity.

3.10. Cytocompatibility assessment

L929 and HUVECs were employed as model cells to investigate the biocompatibility of the hydrogels. The cytotoxicity of the hydrogels was assessed by MTT assay. All of the prepared hydrogel extracts were incubated with cells for 24 h and 72 h, respectively. As shown in Fig. 4A–D, no significant variation in the cell viability between different groups of hydrogels was observed after 24 h and 72 h incubation, respectively. The cell viability was kept at more than 80% even at a concentration as high as 20 mg mL⁻¹, indicating that the hydrogels had good biocompatibility to L929 cells and HUVECs. The biocompatibility of each hydrogel was also tested by Live/Dead staining. The results are shown in Fig. 4E, where the red fluorescence was virtually invisible relative to the intensity of green fluorescence. The results of the Live/Dead assay demonstrated that most of the L929 cells and HUVECs remained viable after 3 days of incubation with the hydrogel extracts. Such results indicated that the current hydrogels possessed good cytocompatibility and were suitable to be employed as a wound dressing.

3.11. Photothermal and antibacterial performance

Once a diabetes wound was infected with bacteria, it would cause severe wound infection and was difficult to heal. Therefore, the antibacterial performance of wound dressings was particularly important. In this work, MRSA was used to evaluate the antibacterial effect of hydrogels in vitro. After incubation with hydrogels for 24 hours, as shown in Fig. 4G and H, the antibacterial rate of the HPCA hydrogel was only 58.6% after incubation with hydrogels for 24 hours. However, its antimicrobial effect was enhanced by laser irradiation after loading CuS NPs into the hydrogel. The antibacterial rate reached 97.9% in the CuS4@HPCA + NIR group. Therefore, it could be derived that the good antibacterial properties of the hydrogel composites were contributed by the dual mode action of the bactericidal CA and the photothermal agent CuS NPs.

3.12. Antioxidation experiment

As reported in the literature, the polyphenol structure of CA could eliminate free radicals and had good antioxidant properties to protect the wound environment.^37,38 Therefore, the antioxidant property of hydrogels was investigated by the DPPH˙ clearance experiment and ROS scavenging assay. As shown in Fig. 5A, with the increase of the hydrogel concentration from 0.31 to 10 mg mL⁻¹, the scavenging efficiency of DPPH˙ increased from 0% to more than 83%, and no significant difference between each group was observed. Such result suggested that the antioxidant properties of the hydrogels were mainly contributed by the CA performance.

Fig. 5 (A) DPPH˙ clearance of hydrogel extracts. (B and C) Quantitative statistics and photographs of ROS fluorescence staining. (D and E) Photographs and quantitative statistics of the HUVEC migration at 0 and 24 h. (F) Images of the HUVEC tube formation on the Matrigel. (G) Total length after incubating with the hydrogel extracts. (H) Number of vascular branches after incubating with the hydrogel extracts.

3.13. ROS removal assay

The excessive accumulation of ROS in the wound bed was a key factor leading to the development of chronic wounds. LPS was used to induce an increase of intracellular ROS levels to simulate the wound environment (Fig. 5B and C). A DCFH-DA probe was used for comparison of the intracellular ROS. The fluorescence intensity of each hydrogel group was decreased as compared with the control group, which confirmed the key role of CA in ROS clearance. The results showed that the intracellular ROS level was lower in the CuS2@HPCA and CuS4@HPCA groups than that in the HPCA group, hinting that the composite hydrogel could achieve ROS removal capability and better protection for the wound environment.

3.14. Cell scratching experiment

The effect of hydrogels on the cell migration of HUVECs was evaluated by cell scratching experiment. As shown in Fig. 5D and E, the 24 h migration rate of the CuS2@HPCA group reached 58.1%. As a comparison, the migration rate of the HPCA group was only 22.5%. Moreover, the best cell migration ratio was observed in the CuS4@HPCA group, with a 24 h migration rate of 71.2%. These results indicated that CuS NPs played an important role in promoting cell migration.

3.15. Tube formation evaluation

The angiogenesis effect of hydrogels on HUVEC-GFP cells were assessed in vitro by tube-forming method (Fig. 5F). The control group was incapable of forming branching junctions and agglomerated on the Matrigel. Quantitative analysis of the number of vascular branches (Fig. 5G) and total vascular length (Fig. 5H) showed that the CuS4@HPCA group had the longest vascular length and best number of branches, and exhibited better pro-angiogenic effect as compared with other groups.

3.16. In vivo studies

We then further investigated the in vivo efficacy of CuS4@HPCA in chronic wound healing using a full-thickness skin wound model of diabetic mice infected with MRSA (Fig. 6A). The wound photographs were taken at days 0, 3, 7, and 14 (Fig. 6B) and the wound healing area was quantified using ImageJ (Fig. 6C).

Fig. 6 The wound healing effect of various hydrogel groups. (A) Illustration of the timeline of the diabetic animal experiments. (B and C) Representative photographs and schematic of the diabetic wound on days 0, 3, 7 and 14, respectively. (D) Quantitative analysis of the wound closure rate.

The wound healing was observed in all groups on days 0, 3, 7 and 14. In general, the CuS4@HPCA + NIR group showed the fastest healing compared to other groups during the 14 day treatment period. The quantification of the wound area (Fig. 6D) revealed that the wound on day 14 in the CuS4@HPCA group was essentially closed, whereas as much as 15.5% of the wound area remained unclosed in the model group. These findings suggested that the CuS4@HPCA hydrogels had good intrinsic antimicrobial activity and wound healing promotion effect. In addition, the antimicrobial activity of the CuS4@HPCA hydrogels was further enhanced by near-infrared irradiation, resulting in the rapid elimination of bacteria and significantly accelerated wound healing.

3.17. Histological immunofluorescence evaluation

As shown in Fig. 7A, the H&E staining of tissue sections on days 7 and 14 post-treatment exhibited maximal skin tissue damage, local epidermal cell necrosis and sloughing, and a large number of inflammatory cells (neutrophils), and diffused infiltrates were manifested in the model group. In contrast, only a trivial amount of a diffused infiltration of inflammatory cells was observed in the dermis, hair follicles and sweat gland structures in the CuS4@HPCA + NIR group. In the process of wound repair, the content of collagen was an important indicator to evaluate the repair extent (Fig. 7B). As shown in Fig. 7D, the collagen was observed by Masson staining, which was quantitatively analyzed by Image J. The CuS4@HPCA + NIR group demonstrated the highest amount of collagen deposition, which could reach 42.7% in 7 days, while the model group exhibited only 4.5%. On day 14, the collagen deposition from the CuS4@HPCA + NIR group increased to 55.8%, with a comparison of only 12.2% of that observed in the model group, hinting the better wound healing efficacy of the CuS4@HPCA hydrogel with the assistance of NIR light irradiation.

Fig. 7 (A and B) The H&E and Masson staining on days 7 and 14 (scale bar = 100 μm). (D) Statistical analysis of the relative area covered by collagen in the regenerated tissue on the basis of Masson staining for each group. (C) Images of CD31 and VEGF immunofluorescence staining on day 14 (scale bar = 50 nm). (E and F) The relative coverage areas of CD31 and VEGF in the immunofluorescence staining on day 14.

At the same time, it has been shown that the production of blood vessels and fibrous tissue was positively correlated with wound healing, therefore platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial growth factor (VEGF) were used to investigate the angiogenesis effect of the respective modalities. As shown in Fig. 7C, significant green (CD31) and red (VEGF) fluorescence were observed in all treatment groups. However, only weak fluorescence was observed in the model group, suggesting that the composited hydrogel could stimulate the formation of microvasculature, which could also be illustrated from the quantitative analysis (Fig. 7E and F). Particularly, the red fluorescence of the CuS4@HPCA + NIR group was significantly enhanced, suggesting that Cu²⁺ could stimulate endothelial cell proliferation and angiogenesis.

Cytokines such as TNF-α, IL-1β, and IL-6 in skin tissues were analyzed by immunohistochemical staining to identify the inflammatory responses. As shown in Fig. 8A, it could be observed that the expression of the three inflammatory factors had significant differences between the control group and treatment groups, respectively. Significant downregulation of TNF-α, IL-1β and IL-6 was observed in the CuS4@HPCA + NIR group, indicating the best anti-inflammatory effect realized by this group. Moreover, it was demonstrated that CuS4@HPCA + NIR could help to increase the amount of neovascularization by upregulating the expression of CD31 and VEGF, while inhibiting the expression of TNF-α, IL-1β, and IL-6 for reducing the inflammation responses at the same time, both of which contributed to the promotion of wound healing.

Fig. 8 Inflammatory levels in the healed skin tissue. (A) Representative photographs of the expression of TNF-α, IL-6 and IL-1β in immunohistochemical staining on day 14 after wound establishment (scale bar = 100 μm). (B–D) Quantitative analysis of TNF-α, IL-6, and IL-1β.

4. Conclusions

In this work, an acid-sensitive natural hyaluronic acid hydrogel with antibacterial and photothermal effect was successfully prepared as a candidate wound dressing to promote wound healing and tissue regeneration for diabetes. The hydrogel was cross-linked by HAPBA and CA. Due to the chemical and physical double crosslinking, the hydrogels was exhibited with excellent adhesion and self-healing ability, as well as good biocompatibility, antioxidant and antibacterial capabilities. It should be noted that the hydrogels had good potency in the promotion of angiogenesis and cell migration. The results from the in vivo animal wound healing experiments indicated that the CuS4@HPCA hydrogel reduced pro-inflammatory cytokines and enhanced angiogenesis, demonstrating multifunctionality critical for diabetic wound repair. Combined with its 97.9% antibacterial efficacy, this system shows promise as an adjunct therapy to standard care. Further preclinical studies in large-animal models and safety profiling are required to validate clinical translatability. In summary, the CuS4@HPCA hydrogel had great potential in applications such as a dressing material for wound management in diabetes.

Author contributions

Yiqun Wang: data curation, formal analysis, investigation, validation and writing original draft. Lingyu Jia: investigation, methodology, data curation, validation. Sihan Shen: data curation, validation, review & editing. Zhangran Zhu: data curation, formal analysis, methodology. Weiyan Cai: methodology, investigation, data curation, validation. Gejing De: formal analysis, resources, data curation. Miyi Yang: methodology, validation. Shuiming Xiao: data curation, resources, project administration. Yanjun Chen: supervision, resources, project administration. Yu Zhao: funding acquisition, methodology, resources, supervision, project administration, validation. Shuang Liu: funding acquisition, formal analysis, methodology. Qinghe Zhao: conceptualization, supervision, resources, writing – review & editing, funding acquisition.

Conflicts of interest

The authors declare that they have no conflict of interest.

Data availability

The data are available from the corresponding author on reasonable request.

Acknowledgements

This study was financially supported by the National Key R&D Program of China (2024YFA1209900 and 2024YFA1209901), the National Natural Science Foundation of China (81974461 and 22005344), the Fundamental Research Funds for the Central Public Welfare Research Institutes (No. ZXKT23023 and ZXKT23020), the Science and Technology Innovation Teams of Shanxi Province (202204051001030), and Traditional Chinese Medicine (TCM) Science and Technology Innovation Project of Shanxi Province (2025KJZY008).

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