Introduction

Currently, approximately 537 million people worldwide have diabetes [1]. By the year 2050, it is estimated that 1.31 billion people will have diabetes [2, 3]. The risk of developing diabetic foot ulcers (DFUs) is 34% [4], and over 50% of DFUs become infected [5], imposing a significant social and economic burden while severely impacting patients’ quality of life [6, 7]. Impaired blood supply and the accumulation of inflammatory mediators trigger an inflammatory response [8]. The continuous exudation of hyperglycemic fluid from diabetic chronic wounds increases bacterial colonization [9], further exacerbating the wound. Infection, persistent ROS accumulation, hyperglycemia, and vascular lesions continuously maintain a chronic inflammatory microenvironment, hindering the routine wound healing process [9,10,11].

The standardized treatment for diabetic chronic wounds typically involves necrotic tissue debridement, skin grafting, topical antibiotics, negative pressure wound therapy, hyperbaric oxygen therapy, and simple wound dressings [12, 13]. However, there is a lack of a treatment regimen that can improve the complex microenvironment of the wound. New therapies for diabetic chronic wounds are gradually emerging [9, 14,15,16]. Wound dressings offer the advantages of convenience and cost-effectiveness and have been widely used in clinical treatment studies. The latest dressings have made excellent contributions to promoting angiogenesis [17, 18], antibacterial activity [19], and reducing ROS levels [20]. However, wounds involve multiple pathological factors, and ‘single-factor’ dressings cannot comprehensively improve the wound microenvironment. As a result, they often fail to achieve the desired outcomes in complex wound microenvironment.

Disruption of the immune microenvironment is the primary factor contributing to the aforementioned pathological changes [21]. The immune microenvironment refers to the local environment within a wound where immune cells interact effectively with other cells and molecules [22]. The persistent accumulation of ROS in diabetic chronic wounds maintains the inflammatory microenvironment [23]. Bacterial infection triggers the massive recruitment of neutrophils and macrophages, which abnormally amplifies oxidative damage through excessive ROS production during pathogen clearance, potentially exacerbating tissue necrosis and progressing to life-threatening sepsis [9]. Diabetic wounds exhibit excessive accumulation of inflammatory cytokines and an imbalance in immune cell populations [16, 24]. This imbalance is a key factor hindering the healing of diabetic chronic wounds [25]. Immune cells undergo metabolic reprogramming during activation, differentiation, and the execution of their immune functions, which is crucial for their functionality [26]. For example, regulating energy metabolism enables T cells to switch between quiescent and highly proliferative states reversibly [27]. Macrophages with different phenotypes exhibit metabolic heterogeneity, with M1 macrophages primarily relying on aerobic glycolysis, while M2 macrophages depend on oxidative phosphorylation (OXPHOS) [28, 29]. Additionally, it is often overlooked that the immune cells exhibit trained Immunity, with the fundamental mechanism of non-specific enhanced responses depending on changes in epigenetic, transcriptional, and metabolic programs following transient stimulation—these changes in the program result in increased responsiveness of cells upon secondary stimulation [30]. Traditional anti-inflammatory strategies often yield only short-term effects. In recent years, an emerging trend in immunotherapy has shifted from passive suppression toward active immune remodeling. “Trained immunity” and “macrophage metabolic reprogramming” represent the forefront of this trend [31,32,33]. Specifically, by inducing macrophages to generate a persistent, beneficial pro-repair immune memory (trained immunity) and providing them with essential energy metabolic support (metabolic reprogramming), the immune microenvironment of diabetic wounds can be fundamentally and sustainably transformed, thereby accelerating the repair process. Therefore, intervening in the metabolism and trained Immunity of immune cells represents a promising immunotherapy approach.

Therefore, we designed a composite dressing CVCeCG, as shown in Fig. 1. First, we chemically bonded sodium carboxymethyl cellulose (CMC) and dopamine (DA) through carbodiimide-activated reactions to create CMC-DA. Then we added sodium iodate (NaIO₄) to the CMC-DA and gelatin (GEL) mixture under stirring conditions using a one-step method. This method rapidly oxidizes CMC-DA to CMC-PDA while crosslinking CMC-PDA with GEL via hydrogen bonds, forming CMC-PDA/GEL (abbreviated as CG) hydrogel. The oxidation of dopamine in CMC-DA and the formation of hydrogen bonds lead to the formation of the hydrogel. Additionally, we incorporated calcium peroxide(CPO)-loaded polycaprolactone microspheres (CPO@PCL), vancomycin(Van)-loaded polycaprolactone microspheres (Van@PCL), and cerium oxide nanoparticles (CeNPs) into the system during the reaction to form a hybrid hydrogel CPO@PCL/Van@PCL/CeNPs/CMC-PDA/GEL (abbreviated as CVCeCG). The primary functions of CVCeCG hydrogel dressing are: (1) The hydrogel can activate a photothermal-triggered missile-like sterilization process to eliminate infections rapidly. (2) The hydrogel can also remodel the immune microenvironment by linking epidermal/dermal cells, macrophages, and vascular dynamic regulation networks through immune training and metabolic reprogramming, and establish a mutually supportive cycle between improved immune microenvironment and angiogenesis, thereby promoting the healing of infected diabetic wounds. (3) The hydrogel improves wound hypoxia, demonstrates excellent biocompatibility, promotes cell migration, and decreases ROS levels, thereby further alleviating inflammation and promoting tissue repair. CVCeCG hydrogel possesses injectability, self-healing properties, and adhesion, making it well-suited for irregularly shaped and easily deformable wounds. After exposing CVCeCG hydrogel to near-infrared light, the rapidly elevated temperature and direct contact with polydopamine (PDA) exert a broad-spectrum bactericidal effect. The elevated temperature causes CPO@PCL to rapidly produce O₂, eliminating anaerobic bacteria and alleviating wound hypoxia, while also causing Van@PCL to release Van to eliminate bacteria rapidly. In our experiments, we found that the CVCeCG hydrogel significantly increased environmental levels of H2O2 when exposed to near-infrared (NIR) irradiation. This led to a synergistic antibacterial effect between H2O2 and the other components. Once the temperature returns to ambient conditions, the H2O2 concentration decreases again. This decrease is attributed to a gradual reduction of H2O2 by PDA and CeNPs as the temperature drops. Concurrently, the release rates of O₂ and Van decrease. We conducted evaluations using diabetic rats with full-thickness MRSA-infected wounds (Approval No. ZN2024088). This study employed MRSA-infected rats as the model system, representing a clinically challenging scenario; therefore, Van was selected as the antibiotic. However, appropriate antibiotics can be loaded in clinical practice based on specific drug susceptibility test results. Additionally, CVCeCG hydrogel establishes a dynamic regulatory network involving epidermal/dermal cells, macrophages, and blood vessels. The MCT4 transport proteins in epidermal/dermal cells increase, releasing lactic acid into the extracellular microenvironment. Extracellular lactate is taken up by macrophages through the MCT1 transporter channel, leading to metabolic reprogramming and epigenetic modifications. On the one hand, macrophages undergo metabolic reprogramming, shifting toward aerobic respiration, as evidenced by enhanced tricarboxylic acid cycle activity, which promotes M2 polarization of macrophages and their anti-inflammatory functions. On the other hand, macrophages undergo immune training to polarize toward the M2 direction. Sustained epigenetic modification promotes continuous wound healing, even after dressing removal, as it continues to promote wound healing and has long-term effects. Previous studies indicate that PDA can diminish inflammatory responses and promote macrophage polarization toward the M2 phenotype [15]. Therefore, we encourage the polarization of macrophages towards the M2 phenotype through both direct and indirect dual stimulation. CeNPs and PDA can mimic catalase (CAT) and superoxide dismutase (SOD) [34], thereby reducing ROS levels in the wound environment. The multi-component design of the CVCeCG hydrogel enables comprehensive intervention in the wound microenvironment, with synergistic effects emerging from the multifaceted engagement of its components. For instance, photothermal stimulation accelerates the rapid release of O₂, H₂O₂, and Van, thereby enhancing their antibacterial efficacy and achieving on-demand delivery of antibacterial agents. Furthermore, the multi-component formulation enhances the microenvironment, promoting not only immune enhancement and angiogenesis but also yielding long-term effects. In summary, the CVCeCG hydrogel dressing can remodel the microenvironment of infected diabetic wounds and has excellent clinical application potential.

Fig. 1
figure 1

Schematic diagram of the formation and application of CVCeCG hydrogel as a comprehensive intervention system for infected diabetic chronic wound healing

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Results and discussion

Fabrication and characterization of micro/nanoparticles

In this study, we produced sustained-release microspheres for the assembly of composite hydrogel dressings. CPO and Van’s encapsulation technology enables controlled release of O2 and Van, thereby avoiding the risk of oxygen poisoning and tissue toxicity caused by rapid release, while also eliminating the need for repeated drug use. The surface morphologies of CPO@PCL and Van@PCL were characterized by scanning electron microscopy (SEM) (Fig. 2A, C), with CPO powder and Van powder used as control groups (Fig. 2B, D). The results demonstrated that both microspheres exhibited regular spherical structures with smooth surfaces. Further energy dispersive X-ray spectrometry (EDS) (Fig. 2A-D, Figure S1A-D) revealed a uniform distribution of the oxygen (O), calcium (Ca), and chlorine (Cl) elements on the surfaces of CPO@PCL and Van@PCL, confirming successful loading of CPO and Van powders. We used a focused ion beam (FIB) to further confirm the encapsulation of CPO and Van powders in PCL microparticles. FIB-SEM cross-section observations were utilized to analyze CPO@PCL and Van@PCL(Fig. 2E, F). The results revealed that both microspheres exhibited porous structures internally, which provided favorable conditions for efficient drug loading and controlled release. Further EDS (Fig. 2E, F; Figure S1E, F) demonstrated the uniform distribution of the oxygen (O), calcium (Ca), and chlorine (Cl) elements within the microspheres, validating successful encapsulation and homogeneity of the CPO and Van powders. Next, we produced CeNPs to remove ROS from the wound. SEM imaging of CeNPs appeared spongy (Fig. 2G). The morphology of CeNPs was characterized by transmission electron microscopy (TEM) (Fig. 2H), revealing uniformly distributed nanoparticles with an average particle size of 16.74 nm (Fig. 2I). Further elemental mapping by EDS (Fig. 2H, Figure S1G) confirmed the presence of oxygen (O) and cerium (Ce) elements in CeNPs. A laser particle size analyzer systematically analyzed the particle size distribution for CPO@PCL and Van@PCL. The results demonstrated average particle sizes of 13.840 μm and 12.160 μm for CPO@PCL and Van@PCL, respectively, with uniform size distributions (Fig. 2J, K). Hydrophobic microspheres can prevent the sudden release of effective ingredients and enable the continuous release of O₂ and Van in a liquid environment. Hydrophobic microspheres enabled the sustained release of O₂ and Van in liquid environments. Systematic characterization of surface properties via water contact angle measurements revealed that CPO@PCL and Van@PCL exhibited contact angles of 70.082° ± 0.638° and 70.735° ± 0.015°, respectively, significantly higher than those of CPO (14.009° ± 0.925°) and Van (35.979° ± 0.647°). Additionally, pure PCL showed a contact angle of 77.662° ± 0.342°, slightly larger than those of CPO@PCL and Van@PCL (Fig. 2L). These results indicate that the PCL shell conferred hydrophobicity to the microspheres, enhancing their stability and drug release performance. The reduced contact angles of CPO@PCL and Van@PCL compared to pure PCL indirectly confirmed the successful encapsulation of CPO and Van powders. Drug loading capacity of CPO@PCL and Van@PCL were 9.815 ± 0.465% and 16.255 ± 0.615%, respectively, with encapsulation efficiencies of 85.047 ± 1.770% and 44.125 ± 0.361% (Figure S1H), demonstrating high drug loading capacity. Sustained release profiles over 14 days (Figure S1I, J) showed a gradual release of O2 and Van, confirming their controlled-release properties.

Fig. 2
figure 2

Fabrication and characterization of micro/nanoparticles. (A)-(D) SEM micrographs and corresponding EDS elemental mappings of CPO@PCL, CPO, Van@PCL, and Van surfaces (Scale bar = 2 μm). (E), (F) SEM micrographs and EDS elemental mappings of FIB-sectioned CPO@PCL and Van@PCL at the midsagittal plane (Scale bar = 2 μm). (G) SEM micrograph of CeNPs (Scale bar = 100 nm). (H) TEM micrograph and EDS elemental mapping of CeNPs (Scale bar = 100 nm). (I) Size distribution histogram of CeNPs based on TEM analysis. (J), (K) Particle size distributions of CPO@PCL and Van@PCL were analyzed by laser diffraction. (L) Quantitative water contact angle analysis and representative images (inset on the histogram) of hydrophobic microspheres and raw materials (****p < 0.0001)

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Fabrication and characterization of composite hydrogel

However, microspheres’ hydrophobicity may reduce their biocompatibility [35]. We address this issue by incorporating micro/nanoparticles into hydrogel dressings with excellent biocompatibility. As shown in Fig. 3A, to synthesize CG, we first formed an amide bond between CMC and DA by carbodiimide activation reaction to produce CMC-DA. Subsequently, as shown in Fig. 3B, CMC-DA and GEL have fluidity as precursors. By rapidly mixing CMC-DA with GEL and dripping it into a NaIO₄ solution, a hydrogel CG was formed. The fast oxidative polymerization of CMC-DA results in the formation of CMC-PDA. The oxidation of dopamine in CMC-DA and the formation of hydrogen bonds lead to the formation of the hydrogel. The hydrogen bonds endowed the hydrogel with stability, self-healing properties, and injectability. Micro/nanoparticles were thoroughly mixed into the reacted CG under stirring to prepare CVCeCG. 1H NMR, FTIR, and UV-vis analyses were conducted to verify CMC-DA synthesis and further validate CG formation. Figure 3C shows new characteristic peaks that emerged at 6.5–7.0. ppm in CMC-DA’s spectrum, corresponding to methylene proton signals between DA’s aromatic ring and amino. This confirms the successful chemical conjugation of DA with the CMC backbone. The degree of substitution (DS) of CMC-DA was calculated using the equation: DS = Ia/Ib, where Ia and Ib represent the peak areas at 6.5–7.0. ppm (attributed to DA) and 3.2–4.7 ppm (attributed to CMC) in 1H NMR spectrum, respectively. The DS value was determined as 0.24. The FTIR analysis (Fig. 3D) revealed that the characteristic peak of CMC-DA at 3410 cm− 1 corresponds to the stretching vibrations of O-H and N-H. The peak around 1600 cm− 1 resulted from the overlap of C = O stretching vibrations and C = C stretching vibrations of the benzene ring skeleton in dopamine. The characteristic peak at 1534 cm− 1 was attributed to the bending vibration of N-H. The peak at 1063 cm− 1 corresponded to the stretching vibration of the newly formed amide bond C-N. Compared to the characteristic peaks of CMC-DA at 3410 cm− 1 and GEL at 3423 cm− 1, the O-H and N-H stretching vibration peaks of CG decreased to 3398 cm− 1 with increased intensity, indicating the formation of numerous hydrogen bonds in the system. The characteristic peak of CG at 1555 cm− 1 was associated with the C = C stretching vibration of the benzene ring skeleton and the N-H bending vibration of the amide II band. The UV-vis analysis (Fig. 3E) showed that CMC-DA exhibited a new absorption peak at 280 nm compared to CMC, corresponding to the aromatic ring of DA. CG displayed a broad absorption band between 300–500 nm, corresponding to C = O. Next, we evaluated the mechanical properties of the hydrogel before and after loading the micro/nanoparticles. The frequency sweep results (Fig. 3F) showed that both CG and CVCeCG exhibited typical gel behaviors, where the storage modulus (G’) was consistently greater than the loss modulus (G’’), and both G’ and G’’ displayed increasing trends with rising frequency. After loading micro/nanoparticles, the G’ and G’’ of CVCeCG remained similar to those of CG, indicating the minimal impact of particle incorporation on the hydrogel’s mechanical strength. To facilitate the long-term storage of the hydrogel dressing, we investigated storage methods for freeze-dried hydrogel. Photographs of redissolved lyophilized hydrogel (Figure S2A) demonstrated that lyophilized hydrogel could reform homogeneous solutions. Frequency sweep results of redissolved lyophilized CG and CVCeCG at three solid concentrations (7.5%/15%/25%) (Fig. 3G) revealed increasing G’ and G’’ with higher solid concentrations, while the mechanical strength of redissolved hydrogels remained comparable to that of the original hydrogels. Time sweep results (Fig. 3H) showed stable G’ and G’’ values for CG (left) and CVCeCG (right) during testing, with similar moduli between CVCeCG and CG, confirming excellent structural stability. Time sweep comparisons between CVCeCG (left) and redissolved lyophilized CVCeCG with identical concentration (right) (Fig. 3I) demonstrated nearly identical G’ and G’’ profiles, suggesting negligible influence of lyophilization on rheological properties. These time/frequency sweep results collectively confirmed that lyophilized storage preserves the original mechanical strength of hydrogels. During the time sweep, G’/G’’ >1 and remains nearly constant. In the frequency sweep, G’/G’’ gradually increases with rising frequency but remains greater than 1. These results demonstrate that the CVCeCG hydrogel maintains stable gel behavior. SEM observations (Fig. 3J) revealed that CG and CVCeCG exhibited highly porous network structures with similar pore sizes, where micro/nanoparticles were uniformly dispersed within the CVCeCG network. This indicates that the hydrogels maintained appropriate pore size and distribution, and the incorporation of micro/nanoparticles had minimal impact on the hydrogel’s pore structure. The biodegradation capacity of biomaterials serves as a critical indicator for biomedical applications. CG and CVCeCG hydrogels gradually degraded over time, with all hydrogels retaining approximately 50% of their initial weight after 14 days (Figure S2B). The swelling ratios of CG and CVCeCG hydrogels followed similar trends, measuring 482.58% ± 30.60% and 519.49% ± 4.45% respectively (Figure S2C). This characteristic benefits wound repair by enabling the hydrogels to absorb biological fluids and maintain wound moisture concentrations, providing a suitable microenvironment.

Fig. 3
figure 3

Fabrication and characterization of composite hydrogel. (A) Schematic illustration of CMC-DA synthesis. (B) Photographic documentation of the hydrogel formation process. (C) 1H NMR spectra of CMC and CMC-DA. (D) FTIR spectra of hydrogels. (E) UV-Vis spectra of hydrogels. (F) Frequency sweep of CG and CVCeCG. (G) Frequency sweep of reformed CG and CVCeCG at three concentrations (7.5%/15%/25%). (H) Time sweep of CG (left) and CVCeCG (right). (I) Time sweep of CVCeCG (left) and reformed CVCeCG at identical concentrations (right). (J) SEM micrographs of CG and CVCeCG (scale bar = 10 μm)

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Self-healing properties and injectability of CVCeCG hydrogel

The hydrogen bonds between CMC-PDA and GEL endow the hydrogel with self-healing properties and injectability. The self-healing ability of the hydrogel was macroscopically studied. As shown in Fig. 4A, two hydrogel pieces injected onto fingers immediately adhered upon contact and withstood finger bending. Furthermore, two differently colored hydrogels were injected together (Fig. 4B), where the healed hydrogel exhibited uniform color mixing at the interface and could be lifted with tweezers, indicating molecular-level integration. The strain amplitude sweep test was investigated (Fig. 4C). CVCeCG hydrogel exhibited typical gel behavior with high G’ at low strain and sharp G’ decline at high strain. The critical strains for CG hydrogel and CVCeCG hydrogel were 637% and 722%, respectively, indicating network breakdown into a liquid state when exceeding these values, with CVCeCG hydrogel showing greater stability. We selected strains of 5% and 1000% to evaluate the rheological recovery behavior of CVCeCG hydrogel (Fig. 4D). At 1000% strain, G’ decreased from 180 to 64 Pa, transitioning to a fluid state. Upon reverting to 5% strain, G’ rapidly recovered to initial values. After four recovery cycles, the hydrogel restored its original state, demonstrating excellent self-healing ability. As shown in Fig. 4E, the hydrogel could be easily extruded through syringes to form continuous filaments. The hydrogel was injected into molds to produce various-shaped samples (Fig. 4F). Figure 4G further verified shear-thinning behavior with apparent viscosity decreasing as the shear rate increased. Under external forces (e.g., injection thrust), hydrogen bonds in CVCeCG hydrogel temporarily break, enhancing fluidity (reduced viscosity). The reformation of the bonds restored the solid state upon the removal of the force, enabling the hydrogel to adapt to the shape of wounds through injectability. Therefore, CVCeCG hydrogel is suitable for wounds with irregular geometries, remains stable in wounds that are subject to constant traction, and can be easily applied in clinical settings via injection.

Fig. 4
figure 4

Self-healing, injectable, adhesive, ROS-scavenging, and photothermal properties of composite hydrogels. (A), (B) Photographs demonstrating the self-healing characteristics of CVCeCG hydrogel. (C) Strain sweep results of CG and CVCeCG hydrogels. (D) Oscillatory strain sweep rheological results of the CVCeCG hydrogel. (E), (F) Photographs showing the injectable properties of CVCeCG hydrogel. (G) Shear-thinning behavior of CG and CVCeCG hydrogels in rheological tests. (H) The temperature-dependent moduli of the CVCeCG hydrogel. (I) DPPH scavenging capacity of CVCeCG with different masses (****p < 0.0001). (J)-(L) Photographic demonstrations of CVCeCG adhesive characteristics. (M) Infrared thermal images of CVCeCG under 808 nm NIR irradiation at 0.25 W/cm² and 0.5 W/cm². (N) Temperature elevation curves of CVCeCG under 808 nm NIR irradiation at 0.25 W/cm² and 0.5 W/cm² for 10 min. (O), (P) Thermal stability of the CVCeCG hydrogel under three cycles of on-off NIR irradiation (808 nm, 0.25 W/cm² or 0.5 W/cm², 10 min). (Q) O₂ and Van release profiles of CVCeCG before and after 0.5 W/cm² 808 nm NIR irradiation (n = 3). (R) Transient H2O2 increase post-NIR (n = 3)

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Adhesive, ROS-scavenging, and photothermal properties of the CVCeCG hydrogel

Dynamic temperature ramp experiments demonstrated (Fig. 4H) that CVCeCG hydrogel maintained stable gelation within the human body temperature range. We can observe that the G’ and G’’ values of the hydrogel increased as the temperature decreased, indicating that the hydrogel exhibited temperature responsiveness. Therefore, we hypothesized that reducing the temperature of the hydrogel could facilitate its easy removal. As shown in Figure S3A, we applied the CVCeCG hydrogel to the back of the hand. Direct removal resulted in some hydrogel residue remaining on the back of the hand. However, after treating the hydrogel with an ice pack for 15 s, it could be removed entirely. The shear test further evaluated this property of the CVCeCG hydrogel (Figure S3B). As shown in Figure S3C, the adhesion strength of the CVCeCG hydrogel was 0.3 kPa at 5℃ and 12.5 kPa at 37℃. We speculated that this property is due to enhanced hydrogen bonding in the CVCeCG hydrogel, which causes a significant reduction in the bonding force between the hydrogel and the interface, thereby decreasing the adhesion of the CVCeCG hydrogel [36]. The removal of commonly used wound dressings in clinical practice often leads to wound damage [37], while the CVCeCG hydrogel’s property perfectly overcomes this drawback. Furthermore, we tested adhesion by stretching CVCeCG hydrogel adhered between fingers, demonstrating persistent adhesion without detachment (Fig. 4J). When attached to the dorsal finger surface, CVCeCG hydrogel maintained adhesion while conforming to finger-bending motions (Fig. 4K). Additionally, CVCeCG hydrogel successfully adhered to rat spleen, lung, kidney, heart, and liver tissues (Fig. 4L). This photographic evidence directly confirmed the adhesive properties of CVCeCG hydrogel. Both wound sites and bacterial infections produce ROS. Excessive amounts of ROS in wounds trigger inflammation, cause cell injury, and inhibit tissue regeneration. The antiradical capacity of the hydrogel was evaluated by the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay (Fig. 4I). In DPPH solutions with identical concentration and volume, the hydrogel (1 mg, 2 mg, and 3 mg) showed DPPH residual of 30%, 25%, and 20%, respectively, confirming its dose-dependent antioxidant capacity that helps mitigate oxidative stress damage at the tissue level. Due to PDA’s excellent photothermal conversion capability, CVCeCG hydrogel generated heat under 808 nm laser irradiation. After 10-minute irradiation with 808 nm laser at intensities of 0.25 W/cm² and 0.5 W/cm², the hydrogel temperature increased to 43.13 °C and 60.43 °C, respectively (Fig. 4M, N). The photothermal responsiveness of hydrogel increases with increasing laser intensity. To better evaluate whether laser irradiation can repeatedly activate this heating behavior, the hydrogel was exposed to three consecutive on/off cycles of laser exposure (Fig. 4O, P). A stable increase in temperature was maintained after three cycles, indicating that CVCeCG hydrogel exhibits excellent photothermal stability and that its photothermal conversion can be remotely and repeatedly controlled using laser irradiation. Since 55 °C is the optimal temperature for killing bacteria without damaging cells [38], and as shown in Figs. 4N and P, the hydrogel temperature reached approximately 55 °C after 5 min of irradiation at 0.5 W/cm². Therefore, subsequent experiments employed a laser intensity of 0.5 W/cm² and an irradiation duration of 5 min. The photoresponsive heating behavior elevates hydrogel temperature, which leads to hydrogen bond dissociation in the hydrogel. Simultaneously, increased temperature enhances the kinetic energy of molecules and polymer chain segments, accelerating the diffusion of effective components from microsphere interiors to tissue. Irradiation of CVCeCG hydrogel with an 808 nm laser at 0.5 W/cm² triggered burst effective components release (Fig. 4Q). The cumulative release amounts of O2 and Van post-irradiation showed 3–7 fold increases compared to pre-irradiation levels, confirming the synergistic regulation potential of photothermal-drug release. The release of accelerated effective components further enhanced the hydrogel’s synergistic antibacterial effects. Since CPO@PCL releases H2O2 during the reaction with H2O, and PDA and CeNPs can neutralize H2O2, we evaluated the concentration of H2O2 (Fig. 4R). Considering that the wound environment is enriched with H2O2, the concentration of H2O2 was assessed not only in ddH₂O but also in 800 µM H2O2 solution. Interestingly, after the laser-irradiated hydrogel, the H2O2 concentration tended to increase during the first 20 min after the onset of the photothermal treatment, followed by a decreasing trend. In contrast, adding CVCeCG hydrogel that was not laser irradiated to the H2O2 solution resulted in a continuous decrease in the H2O2 concentration. The transient increase in the H2O2 concentration could be attributed to the massive release of H2O2 from CPO@PCL after the photothermal treatment, which was subsequently neutralized by the PDA and CeNPs. Considering H2O2’s antibacterial ability, the transient increase in H2O2 concentration was more favorable for the synergistic antimicrobial effect. In the absence of laser irradiation, the concentration of H2O2 showed a decreasing trend, which indicated that the CVCeCG hydrogel mainly acted as an H2O2 scavenger in the absence of laser irradiation.

Antibacterial properties and biocompatibility of CVCeCG hydrogel in vitro

Bacterial infections pose significant challenges to skin regeneration. In CVCeCG hydrogel, Van@PCL enables localized sustained release of Van, while components PDA and CeNPs exert direct-contact antibacterial effects. This is complemented by the sustained release of component O2, which effectively counteracts anaerobic bacterial infections. During the acute infection, the photothermal effect can directly induce thermal damage to bacteria through high temperatures while simultaneously promoting the rapid release of other effective components and facilitating the release of H2O2. This process further enhances the synergistic effect, thereby achieving the goal of rapidly eliminating bacteria in the initial stage of infection. To evaluate the synergistic antibacterial ability of CVCeCG hydrogel, the antibacterial properties were assessed by co-culturing MRSA, Gram-negative Escherichia coli (E. coli), and obligate anaerobic Bacteroides fragilis (B. fragilis) with the hydrogel at 37 °C for 24 h. The antibacterial ability of the hydrogel was assessed by colony counting on agar plates and SEM images of bacteria (Fig. 5A-C). Compared with control groups, both the CVCeCG group and the 808 nm laser-irradiated CVCeCG group (CVCeCG + NIR group) showed significantly reduced bacterial colonies on agar plates, with complete bacterial eradication observed in the CVCeCG + NIR group. The survival rates of MRSA, E. coli, and B. fragilis decreased to 27.87% ± 3.33% and 0%, 8.36% ± 0.40% and 0%, and 0% and 0%, respectively (Fig. 5E-G). SEM images revealed that bacterial morphology in the CVCeCG group and CVCeCG + NIR group underwent significant alterations compared to the smooth and intact morphology observed in the control group (Fig. 5A-C). As highlighted by white arrows, bacterial surfaces became rough with multiple depressions, indicating bacterial membrane disruption, a critical indicator for evaluating antibacterial activity. These assessments demonstrated that the CVCeCG hydrogel effectively eliminated bacteria through synergistic effects. The further reduction in colony counted on agar plates and the increased number of arrows in SEM images from the CVCeCG + NIR group confirm that the photothermal effect enhances this synergistic efficacy. Good biocompatibility is a prerequisite for the application of hydrogels in wound healing. The cytocompatibility of the hydrogel was evaluated in vitro using HUVEC cells through CCK-8 and Calcein/PI cell viability/cytotoxicity detection kits. The hydrogels were placed into the corresponding wells, and their biocompatibility was assessed on days 1, 3, and 5 with Calcein/PI detection kits. Calcein AM stained live cells (green fluorescence), while propidium iodide (PI) stained dead cells (red fluorescence). Nearly all cells in both control and experimental groups exhibited green fluorescence (live cells) (Fig. 5D), visually confirming the material’s biocompatibility. Additionally, to further investigate whether transient H₂O₂ increases induce detectable cytotoxicity, hydrogels were co-cultured with HUVECs in the same well plate. After 808 nm near-infrared irradiation of the CVCeCG hydrogel on days 1, 3, and 5, cells were stained for live/dead detection (Figure S4). Results indicated that HUVEC viability remained unaffected. Transient H₂O₂ increases do not compromise the biocompatibility of the hydrogel. Following 72 h of co-culture of the CVCeCG hydrogel with cells, CCK-8 evaluation indicated that cell viability was 103.67%±1.82%, comparable to the control group (Fig. 5H).

Fig. 5
figure 5

Antibacterial properties and biocompatibility of CVCeCG hydrogel in vitro. (A)-(C) Plate spreading cultures and SEM images of MRSA, E. coli, and B. fragilis treated with CVCeCG hydrogel (scale bar = 1 μm). (D) Live/dead staining images of CVCeCG hydrogel (scale bar = 100 μm). (E)-(G) Colony counting results from plate spreading assays of MRSA, E. coli, and B. fragilis treated with CVCeCG hydrogel (****p < 0.0001). (H) Cytotoxicity test results of CVCeCG hydrogel (ns = no significance)

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CVCeCG hydrogel demonstrates anti-inflammatory and ROS elimination capabilities in vitro

In the local immune microenvironment of diabetic wounds, excessive inflammatory responses caused by persistent macrophage infiltration are the main cause of delayed wound healing [39]. Previous studies indicated that PDA can diminish inflammatory responses and promote macrophage polarization toward the M2 phenotype [15]. RAW 264.7 macrophages were used to verify the anti-inflammatory properties of the hydrogel. As shown in Fig. 6A, LPS addition significantly increased the proportion of CD86-positive macrophages, while CVCeCG hydrogel leachate increased the proportion of CD206-positive macrophages. Compared to the Control group, the relative expression levels of CD86 and CD206 in the LPS group and LPS + CVCeCG group were 451.91%±68.47% and 99.34%±16.61%, 173.115%±42.50% and 1816.80 ± 325.29%, respectively (Fig. 6C, D). These results indicated that CVCeCG hydrogel effectively promotes the conversion of pro-inflammatory M1 to pro-reparative M2 macrophages. We performed multiplex surface marker analysis using flow cytometry (Fig. 6B). Expression of both CD86 and CD206 was detected simultaneously. Flow cytometric analysis revealed significantly elevated CD86 expression in the LPS group, whereas CD86 expression in the CVCeCG group was comparable to that in the Control group. Furthermore, CVCeCG induced upregulation of CD206 expression. This indicates that CVCeCG promotes macrophage polarization toward the M2 phenotype. We further investigated the CVCeCG hydrogel’s effects on various pro- and anti-inflammatory factors in RAW 264.7. After LPS stimulation, M1-associated pro-inflammatory mRNA levels, including interleukin 1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6), increased (Fig. 6E-G). As expected, CVCeCG hydrogel treatment significantly reduced these factors. We also examined M2-associated mRNAs, including arginase 1 (Arg-1) and interleukin-10 (IL-10), involved in anti-inflammation and tissue repair (Fig. 6H, I). CVCeCG hydrogel substantially enhanced Arg-1 and IL-10 gene expression. These results indicate that CVCeCG hydrogels inhibit the mRNA expression of pro-inflammatory biomarkers and promote the mRNA expression of anti-inflammatory biomarkers, showing their potential as an excellent anti-inflammatory material. Persistent oxidative stress caused by excessive ROS impairs wound healing and tissue regeneration [34]. To quantitatively evaluate antioxidant properties, HUVECs were cultured with 400µM H2O2 for 72 h to establish an oxidative stress model. The hydrogels were placed into the corresponding wells. Dihydroethidium (DHE) probe assessment showed significantly lower fluorescence intensity in the CVCeCG hydrogel group (Fig. 6J). The relative fluorescence intensity of the CVCeCG hydrogel group was 28.67%±10.09% (Fig. 6K), further confirming its advantage in reducing oxidative stress.

Fig. 6
figure 6

In vitro anti-inflammatory and ROS scavenging properties of hydrogel. (A) Immunofluorescence staining images of RAW264.7 (scale bar = 20 μm). (B) Flow cytometry analysis of macrophage transition toward M1/M2 phenotype. (C), (D) Quantitative analysis of RAW264.7 immunofluorescence staining (ns = no significance, ***p < 0.001, ****p < 0.0001). (E)-(G) The mRNA expression of pro-inflammatory biomarkers (IL-1β, TNF-α, and IL-6) (ns = no significance, *p < 0.05, **p < 0.01, ****p < 0.0001). (H), (I) Anti-inflammatory biomarkers (Arg-1 and IL-10) (**p < 0.01, ***p < 0.001, ****p < 0.0001). (J) DHE staining images (scale bar = 100 μm). (K) Quantitative results of DHE staining (**p < 0.01)

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Angiogenic capacity and hypoxia relief of CVCeCG hydrogel under hypoxic conditions in vitro

To evaluate the effect of hydrogel on cell migration ability, we performed scratch assays using HUVECs with photographs taken at 0, 5, 15, and 24 h for the control and hydrogel groups (Fig. 7A). Results showed significantly faster cell migration in the hydrogel group. Quantitative analysis (Fig. 7D) further confirmed a higher scratch closure rate in the hydrogel group at 24 h, indicating that the hydrogel promotes HUVEC migration. After the inflammatory phase, the wound will enter the proliferative phase. Local blood supply to the wound during the proliferative phase is a determining factor for successful healing [40]. To assess the hydrogel’s angiogenic effects under hypoxia in vitro, we conducted immunofluorescence staining and tube formation assays under hypoxic conditions (O2 < 0.1%). Cell adhesion molecule-1 (CD31)/VE-cadherin dual fluorescence staining was performed to study intercellular junctions and endothelial markers. After a 3-day hypoxia culture, immunofluorescence images and quantitative analysis (Fig. 7B, E, F) revealed significantly higher CD31 (191.2%±19.72%) and VE-cadherin (204.52%±15.48%) expression in the hydrogel group versus the control, demonstrating enhanced angiogenic capacity. A matrigel-based tube formation assay under hypoxia showed superior tubular formation of HUVECs in the hydrogel group (Fig. 7C). Quantitative analysis (Fig. 7G) indicated significantly more junctions in the hydrogel group, confirming enhanced tube formation ability. To determine whether the in vitro proangiogenic capacity of hydrogels exists under normoxic conditions, we further investigated the effects of CVCeCG hydrogel on HUVECs under normoxic conditions in vitro (Figure S5). The results indicate that under normoxic conditions, CVCeCG does not statistically significantly promote angiogenesis directly. Cell survival under hypoxia was evaluated using a Calcein/PI viability assay. Observations on days 3 and 9 (Fig. 7H) showed higher viability in the hydrogel group: On day 3, the control group had some cells with red fluorescence, while the hydrogel group maintained green fluorescence. By day 9, control cells nearly all died, whereas the hydrogel group retained dense green fluorescence, demonstrating that the hydrogel alleviates hypoxia-induced cell death.

Fig. 7
figure 7

In vitro angiogenic and hypoxia-alleviating properties of hydrogels. (A) Scratch assay results of CVCeCG hydrogel and control groups (scale bar = 100 μm). (B) Immunofluorescence staining of CD31 and VE-cadherin under hypoxic conditions for CVCeCG hydrogel and control groups (scale bar = 100 μm). (C) Tube formation assay images of control, Matrigel, and Matrigel + CVCeCG hydrogel groups under hypoxia (scale bar = 100 μm). (D) Quantitative analysis of scratch assay results (ns = no significance, *p < 0.05). (E), (F) Quantitative analysis of CD31 and VE-cadherin immunofluorescence staining in CVCeCG hydrogel and control groups under hypoxia (*p < 0.05, **p < 0.01). (G) Quantitative analysis of tube formation under hypoxic conditions (*p < 0.05). (H) Live/dead cell staining of CVCeCG hydrogel and control groups after 3-day and 9-day culture under hypoxia (scale bar = 100 μm)

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Diabetic wounds healing ability of CVCeCG hydrogel in vivo

Hydrogel dressings must first demonstrate good biocompatibility before use in clinical practice. We first evaluated its biocompatibility in Sprague Dawley (SD) rats by subcutaneously implanting the hydrogel and observing for 30 days. The results demonstrated good compatibility between the hydrogel and surrounding tissues without significant inflammation or rejection (Figure S6A). Additionally, hematoxylin and eosin (H&E) staining was performed to assess the skin, heart, liver, spleen, lungs, and kidneys of rats 30 days after subcutaneous injection of CVCeCG hydrogel, revealing no significant morphological differences compared to normal rat tissues (Figure S6B), indicating favorable in vivo biosafety. The diabetic wounds pose a risk of bleeding; thus, the ability to achieve rapid hemostasis is essential for wound healing. Subsequently, we evaluated the hemostatic capability of CVCeCG hydrogel through a rat hepatic hemostasis experiment. Both the bleeding photographs (Fig. 8B) and quantitative blood loss analysis (Fig. 8D) demonstrated that the CVCeCG hydrogel group exhibited significantly superior hemostatic effects compared to other groups, confirming its excellent hemostatic performance. The streptozotocin (STZ, 150 mg/kg)-induced diabetic SD rat model was constructed to evaluate the therapeutic effect of CVCeCG hydrogel in vivo. We successfully established diabetic rat models through 21-day continuous blood glucose and body weight monitoring of modeled diabetic rats (Figure S7). The animal experimental scheme is shown in Fig. 8A. SD rats with blood glucose levels exceeding 17 mM were selected to create full-thickness MRSA-infected wounds (diameter = 1.5 cm) on their backs. CVCeCG hydrogel with or without 808 nm laser irradiation was used as a wound dressing, 3 M dressing as a positive control, and the untreated group as a negative control. The CVCeCG + NIR group received 808 nm NIR irradiation for 5 min daily during the first three days. The SD rat wounds were evaluated for up to 14 days. Wound skin samples collected on day 1 underwent bacterial culture tests, where distinct bacterial growth was observed after 24 h (Figure S9), confirming successful MRSA infection. Macroscopic wound images, wound closure trajectories, and corresponding quantitative sizes of wound areas at different time points are shown in Fig. 8C and E. Compared with untreated or 3 M dressing-treated groups, CVCeCG hydrogel accelerated wound healing and significantly improved epidermal regeneration. The wound closure rate in the CVCeCG + NIR group reached 100% by day 14, surpassing other groups. Untreated MRSA-infected wounds showed poor healing, with partial wound reduction potentially attributable to the natural contraction of rat skin. On day 14, significantly fewer bacterial colonies were observed on culture plates from wounds in the CVCeCG and CVCeCG + NIR groups compared to other groups (Figure S9), confirming the effectiveness of the synergistic antibacterial strategy. Notably, the absence of visible bacterial colonies in the CVCeCG + NIR group further demonstrated the advantage of incorporating photothermal therapy into the synergistic antibacterial regimen. Histopathological analysis of regenerated skin during wound healing was performed through H&E staining, Masson’s trichrome staining, and Sirius red staining (Fig. 8F-J). Figure 8G and H showed that no epidermal formation was observed in any groups on day 3. Collagen deposition in wound healing is crucial for ECM formation [41]. The Untreated and 3 M dressing groups exhibited extensive and severe inflammatory cell infiltration, while the CVCeCG and CVCeCG + NIR groups showed fewer inflammatory cells. The CVCeCG and CVCeCG + NIR groups demonstrated more collagen deposition compared to Untreated and 3 M groups. Figure 8F-H revealed incomplete but extensive epidermal layers forming over wounds in CVCeCG and CVCeCG + NIR groups by day 7, whereas Untreated and 3 M groups still displayed substantial inflammatory cell infiltration. Sirius red staining results aligned with Masson staining, showing darker coloration and more collagen fibers in CVCeCG and CVCeCG + NIR groups. By day 14, Untreated and 3 M groups developed thin regenerated epidermis with persistent inflammation, while CVCeCG and CVCeCG + NIR groups formed relatively complete epidermis and dermis with minimal inflammatory cells. Notably, the CVCeCG + NIR group exhibited more intact stratum corneum and dermo-epidermal junctions than other groups. Collagen content in CVCeCG and CVCeCG + NIR groups surpassed that in controls, displaying highly organized fibrous structures closer to normal skin histology (Figure S8A, B). Quantitative analysis of Sirius Red and Masson staining (Fig. 8I, J) confirmed that the relative collagen fiber content of the CVCeCG and CVCeCG + NIR groups was significantly higher than that of the control group, with the CVCeCG + NIR group having better results than the CVCeCG group on day 14, which reflects the advantages of the synergistic strategy of photothermal involvement. Type III collagen plays a crucial role in the early stages of wound healing. Furthermore, fetal wounds heal without scarring, which is attributed to a higher proportion of type III collagen [42, 43]. Therefore, we further evaluated the relative content of type I and type III collagen, as well as their ratio, in wounds at day 7 using polarized light microscopy. As shown in Figure S10 A-C, the content of type III collagen in the CVCeCG and CVCeCG + NIR groups was higher than that in the 3 M and Control groups. Moreover, the ratio of type III collagen to type I collagen was also higher. Simultaneously, as shown in Figure S10D, there were no statistically significant differences in type I collagen content among the groups. These results indicate that wound healing was superior in the CVCeCG and CVCeCG + NIR groups, consistent with our previous analysis. Ki67 staining at day 7 (Figure S11A, B) indicated enhanced cellular proliferation in CVCeCG and CVCeCG + NIR groups, showing relative expression levels of 318.09%±36.18% (CVCeCG) and 442.82%±50.27% (CVCeCG + NIR) versus 100.27%±32.18% (Untreated) and 173.67%±46.54% (3 M). CK14 staining at day 14 (Figure S12) revealed richer hair follicles and complete epidermal regeneration in CVCeCG and CVCeCG + NIR groups, with CVCeCG + NIR exhibiting more mature epidermal structures and denser follicles than CVCeCG group. Collectively, CVCeCG hydrogel significantly eliminated infection, reduced inflammation, and promoted tissue regeneration and collagen deposition, thereby accelerating the closure and regeneration of MRSA-infected diabetic wounds.

Fig. 8
figure 8

In vivo wound healing performance of hydrogels. (A) Animal experimental procedure. (B) Corresponding images of rat liver injuries. (C) Wound photographs and closure trajectories. (D) Blood loss assessment in differently treated rat liver injury models (**p < 0.01, ***p < 0.001, ****p < 0.0001). (E) Quantitative evaluation of wound area closure (ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (F) Sirius Red staining of rat skin on days 7 and 14 (scale bar = 50 μm). (G) H&E staining of skin tissue sections collected from wound areas on days 3, 7, and 14 (scale bar = 5 mm and 100 μm). (H) Masson’s trichrome staining of skin tissue sections from wound areas on days 3, 7, and 14 (scale bar = 5 mm and 100 μm). (I) Quantitative analysis of collagen-occupied areas in Sirius Red staining (ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001). (J) Quantitative analysis of collagen-occupied areas in Masson’s trichrome staining (ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001)

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CVCeCG hydrogel promotes wound healing through anti-inflammatory, ROS-scavenging, and angiogenic effects in vivo

The immune microenvironment refers to the local environment within a wound where immune cells interact effectively with other cells and molecules [22]. Diabetic wounds exhibit excessive accumulation of inflammatory cytokines and an imbalance in immune cell populations [16, 24]. This imbalance is a key factor hindering the healing of chronic diabetic wounds [25]. Macrophages play a crucial role in skin regeneration during wound healing. M2 macrophages are related to anti-inflammatory responses and wound healing. We detected the number of M2 macrophages in skin sections and the expression levels of related inflammatory factors. On day 7, the CVCeCG and CVCeCG + NIR groups exhibited greater CD206 expression than the Untreated and 3 M groups (Fig. 9A). The relative CD206 expression levels of the 3 M group, CVCeCG group, and CVCeCG + NIR group were 127.32%±57.78%, 274.15%±36.33%, and 535.55%±65.40%, respectively (Fig. 9G). These results indicated that the interventions in the CVCeCG group and CVCeCG + NIR group had a positive regulatory effect on macrophage polarization. Common inflammatory factors such as pro-inflammatory cytokines, including IL-6 and TNF-α, and anti-inflammatory cytokines such as IL-10 and transforming growth factor-β (TGF-β) reflect the level of inflammation at the wound site [44]. The expression levels of IL-6, IL-10, TNF-α, and TGF-β in tissues were detected by immunohistochemical staining. Figure 9C-F and I-L showed that all groups had higher inflammation levels on day 3. However, the CVCeCG group and CVCeCG + NIR group expressed less IL-6 and TNF-α than the Untreated and 3 M groups. Moreover, the CVCeCG group and CVCeCG + NIR group expressed more IL-10 and TGF-β than the Untreated group and 3 M group, which might be caused by the elimination of infection, macrophage polarization, and lower local reactive oxygen. Then, we continued to observe each group’s inflammation level on day 14 (Figure S13A-H). The CVCeCG group and CVCeCG + NIR group still expressed less IL-6 and TNF-α than the Untreated group and 3 M group, which was consistent with the trend of inflammatory cell counts (Fig. 8G). Meanwhile, the CVCeCG group and CVCeCG + NIR group had an increased level of IL-10 present. Moreover, possibly because the CVCeCG group and CVCeCG + NIR group had milder inflammatory responses and complete tissue healing, the four groups showed no significant differences in TGF-β expression (Figure S13D, H). The ROS levels in the day 14 frozen sections were observed using a DHE probe (Fig. 9B). It could be found that the CVCeCG group and CVCeCG + NIR group had lower fluorescence intensity than the Untreated group and 3 M group. Quantitative analysis showed the relative fluorescence intensities of the 3 M group, CVCeCG group, and CVCeCG + NIR group were 94.845%±11.10%, 36.95%±22.97%, and 16.27%±7.03%, respectively (Fig. 9H). This indicates that the CVCeCG group and CVCeCG + NIR group had lower ROS levels in wounds, which might result from the combined effects of direct ROS scavenging and antibacterial, anti-inflammatory functions of the hydrogel. The formation of new blood vessels plays a key role in skin regeneration by supplying oxygen, nutrients, and cytokines to the wound site [45]. CD31 is a transmembrane protein expressed in early angiogenesis, indicating neovascularization. At the same time, α-smooth muscle actin (α-SMA) is a cytoplasmic protein expressed in late angiogenesis, indicating vascular smooth muscle cell maturation. Therefore, we performed double immunofluorescence staining to evaluate the angiogenic capacity of α-SMA and CD31. On day 14, more new blood vessels with higher maturity were observed in the CVCeCG group and CVCeCG + NIR group (Fig. 9M), which might be caused by the combined effects of the elimination of infection, hypoxia relief, inflammation alleviation, and reduced ROS level. Quantitative analysis showed that the relative expression levels of CD31 and α-SMA in the 3 M group, CVCeCG group, and CVCeCG + NIR group were 141.61%±21.35% and 97.69%±33.91%, 203.09%±8.50% and 262.09%±44.28%, as well as 231.70%±12.49% and 502.54%±49.84%, respectively (Fig. 9N, O). Among them, the CVCeCG + NIR group had higher α-SMA relative expression than the CVCeCG group, indicating higher vascular maturity, demonstrating the advantage of photothermal therapy.

We performed RNA-seq analysis on skin tissues at the wound from untreated and CVCeCG + NIR-treated diabetic rats to investigate how NIR-activated CVCeCG promotes healing in vivo (Fig. 10A). Differential gene expression analysis revealed 51 upregulated and 208 downregulated genes in the CVCeCG + NIR group (Fig. 10B, D). Notably, KEGG pathway analysis of these differentially expressed genes identified significant enrichment in multiple pathways associated with diabetic wound healing, such as the TNF signaling pathway, Staphylococcus aureus infection, and NOD-like receptor signaling pathway (Fig. 10C). TNF-α participates in inflammatory responses in diabetic wounds [46, 47]. TNF-α exhibits autocrine activity, inducing the secretion of IL-1β and perpetuating a cycle of chronic inflammation [48]. Activation of the NOD-like receptor protein (NLRP3) inflammasome in macrophages contributes to persistent inflammation and impaired wound healing associated with diabetes [49]. These findings align with our previous experimental data, suggesting that NIR-activated CVCeCG accelerates wound healing by modulating gene expression in diabetic wound tissue.

Fig. 9
figure 9

In vivo anti-inflammatory, ROS-scavenging, and pro-angiogenic effects of the hydrogel. (A) CD206 immunofluorescence staining images on day 7 (Scale bar = 50 μm). (B) DHE immunofluorescence staining images on day 7 (Scale bar = 50 μm). (C)-(F) IL-6, IL-10, TNF-α, and TGF-β immunohistochemical staining images on day 3 (Scale bar = 50 μm). (G) Quantitative analysis of CD206 immunofluorescence staining on day 7 (ns = no significance, *p < 0.05, **p < 0.01, ****p < 0.0001). (H) Quantitative analysis of DHE immunofluorescence staining on day 7 (ns = no significance, *p < 0.05, **p < 0.01). (I)-(L) Quantitative analysis of IL-6, IL-10, TNF-α, and TGF-β immunohistochemical staining on day 3 (ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001). (M) CD31 (green)/α-SMA (red) double immunofluorescence staining images on day 14 (Scale bar = 50 μm). (N) Quantitative analysis of CD31 immunofluorescence staining on day 14 (ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (O) Quantitative analysis of α-SMA immunofluorescence staining on day 14 (ns = no significance, *p < 0.05, **p < 0.01, ****p < 0.0001)

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Lactate modulation by CVCeCG drives metabolic reprogramming and immune training for diabetic wound healing

The tissue microenvironment plays a pivotal role in modulating immune responses and tissue repair in diabetic wounds. Building on our research, we demonstrated that NIR-activated CVCeCG hydrogel significantly enhances vascular regeneration, promotes proliferation of epidermal and dermal cells, and regulates immune homeostasis in diabetic wounds. To further elucidate the impact of CVCeCG on the tissue microenvironment, we conducted gene set enrichment analysis (GSEA). The transcriptomic analysis revealed significant enrichment of differentially expressed genes in acetylation-dependent protein binding pathways. This finding suggests that the CVCeCG hydrogel may orchestrate long-term wound healing outcomes through epigenetic modulation, specifically by regulating histone modifications within the wound microenvironment (Fig. 10E). Concurrently, results also revealed that CVCeCG-mediated transcriptional changes are predominantly enriched in inflammatory mediator regulation of TRP channels, suggesting its central role in orchestrating immunomodulatory mechanisms during wound healing (Fig. 10F). Consistent with previous studies [50, 51], lactate within the diabetic tissue microenvironment modulated immune response and wound healing. Our investigation revealed a distinct metabolic alteration following CVCeCG hydrogel treatment. Compared to untreated diabetic wounds, CVCeCG-treated wounds exhibited no significant alterations in lactate-generating enzymes (LDHA, LDHB) or the lactate influx transporter MCT1. Crucially, we observed an upregulation of the lactate efflux transporter MCT4 (Fig. 10G). This transporter-specific induction suggested enhanced cellular lactate extrusion in treated wounds. Subsequent biochemical quantification confirmed tissue lactate concentrations in the CVCeCG group increased over two-fold (p < 0.01) versus control (Fig. 10H). Importantly, the lactate is internalized by macrophages (Fig. 10I), elevating intracellular lactate levels in macrophages and consequently reprogramming the immune microenvironment to facilitate diabetic wound resolution. We further investigated how lactate levels within the tissue microenvironment drive metabolic reprogramming in macrophages. Consistent with our hypothesis, exogenous lactate supplementation significantly suppressed glycolytic function while enhancing oxidative phosphorylation in macrophages (Fig. 10J, K), indicating a metabolic shift toward aerobic respiration. Given that epidermal keratinocytes and dermal fibroblasts constitute primary lactate sources in diabetic wounds, we established a transwell co-culture model to dissect this intercellular crosstalk (Fig. 10L). Specifically, rat-derived epidermal keratinocytes were seeded in the upper chamber, separated from rat macrophages in the lower chamber. This configuration permitted lactate diffusion from keratinocytes to macrophages without direct cell contact. Crucially, both exogenous lactate supplementation and co-culture with NIR-infarred CVCeCG-treated keratinocytes elicited identical metabolic reprogramming in macrophages, with a reduction in extracellular acidification rate (Fig. 10M) coupled with increased oxygen consumption rate (Fig. 10N), confirming attenuated glycolysis and enhanced oxidative phosphorylation. These results collectively demonstrated that lactate serves as a key mediator of macrophage metabolic reprogramming in the diabetic wound niche. Trained immunity is characterized by persistent epigenetic and metabolic alterations in innate immune cells, including macrophages, monocytes, and NK cells [52]. Specifically, as supported by a clinical study from 2011 [53], elevated lactate concentrations have been observed in the wound tissue fluid of diabetic patients, indicating that lactate plays a significant role in diabetic wound healing. On the one hand, it has been reported that lactate in the skin wound area of mice can enhance lactate levels within macrophages in the local tissue microenvironment [54]. On the other hand, increased intracellular lactate promotes histone H3K18 lactylation in macrophages, which in turn upregulates the expression of genes such as Arg1 and VEGF and facilitates macrophage polarization toward the M2 phenotype [55, 56]. Building on this foundation, our study further demonstrated that the CVCeCG elevated lactate levels in tissue microenvironment, thereby enhancing histone lactylation in macrophages, promoting VEGF release, and driving M2 polarization of macrophages. Following primary CVCeCG stimulation that enhances inflammatory responses, it is critically mediated by lactate within the tissue microenvironment. Lactate drives histone lactylation to promote chromatin accessibility of inflammation-related genes, notably at the H3K18 site. Immunofluorescence confirmed increased H3K18 lactylation in F4/80 positive macrophages under NIR-activated CVCeCG hydrogel stimulation (Fig. 10O, P). Subsequent ChIP-qPCR demonstrated augmented chromatin accessibility at Arg-1 and VEGF loci concomitant with elevated H3K18 lactylation (Fig. 10Q), leading to increased mRNA expression of these genes. This epigenetic remodeling promoted M2 macrophage polarization while suppressing M1 polarization, indicating enhanced anti-inflammatory capacity and immune homeostasis in trained macrophages (Fig. 10R). Changes in histone lactylation in macrophages have epigenetic effects so that promote the enhancement of gene promoters such as Arg-1 and VEGF after wound dressing removal. ELISA results further revealed reduced secretion of IL-6, TNFα, and IL-1β, establishing sustained anti-inflammatory effects consistent with prior findings (Fig. 10S). Concurrently, heightened VEGF secretion from trained macrophages accelerated angiogenesis, aligning with observed wound healing progression. Collectively, our findings demonstrated that the CVCeCG hydrogel regulates the epidermal/dermal cell-macrophage-endothelial cell network, with the tissue microenvironment serving as the regulatory nexus (Fig. 10T). By simultaneously driving metabolic reprogramming and trained immunity in macrophages, the CVCeCG stimulated sustained VEGF secretion into the microenvironment. This cytokine-mediated crosstalk promotes robust angiogenesis, which synergizes with immune homeostasis processes to holistically accelerate diabetic wound healing.

Fig. 10
figure 10

Detected with RNA-seq, lactate modulation by CVCeCG drives metabolic reprogramming and immune training for diabetic wound healing. (A) The heatmap shows differentially expressed genes between untreated and NIR + CVCeCG groups. (B) The volcano plot depicts downregulated genes (green) and upregulated genes (red) (padj < 0.05, |Log2 FC| >1). (C) The top twenty enriched signaling pathways following KEGG enrichment analysis for differentially expressed genes. (D) PPI network of differentially expressed genes. (E) GSEA of acetylation-dependent protein binding. (F) GSEA of inflammatory mediator regulation of TRP channels. (G) Heatmap visualization of lactate-associated genes from RNA sequencing. (H) Lactate quantification in the extracellular microenvironment of diabetic rat wound healing regions. (I) Macrophage lactate detection within diabetic rat wound healing regions. (J) Glycolytic proton efflux rate (glycoPER) assessed in macrophages using Seahorse XF96 analyzer (Rotenone/Antimycin A, Rot/AA; 2-Deoxyglucose, 2-DG). (K) Mitochondrial respiration measured in macrophages via Seahorse XF96 (Oligomycin, Omy; Rotenone/Antimycin A, Rot/AA). (L) Schematic representation of the co-culture model. (M) ECAR in co-cultured macrophages by fluorometric assay (data normalized to control). (N) OCR in co-cultured macrophages by fluorometric assay (data normalized to control). (O) Representative immunofluorescence images of F4/80 positive macrophages (green) with H3K18 lactylation (H3K18la, red) at day 14 (Scale bar = 10 μm). (P) Proportion of H3K18la-positive macrophages among total F4/80-positive cells. (Q) ChIP analysis of H3K18la binding in Raw264.7 cells. (R) RT-qPCR analysis of M1/M2 polarization markers (CD86, CD206, Arg-1) and VEGF expression in lactate-treated or control Raw264.7 cells. (S) ELISA quantification of secreted factors in supernatants from lactate-post-treated or control Raw264.7 macrophages. (T) The regulatory network, including epidermal/dermal cells, macrophages, and endothelial cells within the tissue microenvironment

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Conclusion

In summary, we have synthesized a composite hydrogel dressing, CVCeCG, for the treatment of infected chronic diabetic wounds. As a comprehensive microenvironment intervention system, it can rapidly eliminate infections, reduce ROS levels, alleviate hypoxia, remodel the immune microenvironment, and promote angiogenesis. The inseparable synergistic effects of these functions promote wound healing. The CVCeCG hydrogel regulates the epidermal/dermal cell-macrophage-endothelial cell network by increasing macrophage H3K18 lactylation and mitochondrial metabolic reprogramming, thereby conferring enhanced anti-inflammatory capacity and immune homeostasis to trained macrophages. Elevated H3K18 lactylation levels are accompanied by increased expression of Arg-1 and VEGF, as well as reduced secretion of IL-6, TNFα, and IL-1β, thereby sustaining anti-inflammatory effects and promoting angiogenesis. Wound healing is still promoted after dressing removal, demonstrating long-term effects. CVCeCG hydrogel exhibits excellent self-healing properties, injectability, ROS scavenging performance, adhesion, and photothermal responsiveness. CVCeCG hydrogel can initiate a missile-like bactericidal process through photothermal activation, releasing antimicrobial components (O₂, Van, and H₂O₂) to eliminate infection rapidly. At the cellular level, CVCeCG has demonstrated strong biocompatibility, anti-inflammatory effects, the ability to scavenge intracellular ROS, promote cell migration, alleviate hypoxia, and enhance angiogenesis. Additionally, it showcases impressive hemostatic properties and tissue compatibility. In a diabetic rat model with chronic wounds infected by MRSA, CVCeCG rapidly promotes tissue healing, eliminates infection, remodels the local immune microenvironment, reduces intracellular ROS levels, promotes macrophage polarization toward the M2 phenotype, and facilitates wound vascular regeneration. Traditional immune training methods predominantly rely on the direct application of exogenous components [57]. In contrast, lactate provides a sustained, endogenous metabolic signal that more closely mimics physiological immune training processes, potentially offering superior safety and longer-lasting effects. As a metabolite already abundantly present in the wound microenvironment, our strategy essentially “turns waste into treasure” by cleverly harnessing endogenous metabolites at the lesion site to reshape immune responses. This approach may offer unique advantages in terms of biocompatibility and translational potential. This study validated the biocompatibility of the hydrogel only at the 30-day time point. Although the material had largely degraded by this stage, its long-term in vivo fate, the ultimate metabolic pathways of degradation products, and potential effects on tissue remodeling during the late healing phase remain unclear. These are critical issues that must be evaluated through longer-term animal studies before they can be translated into future clinical applications. Mechanistic investigations in this study were primarily conducted in rat models. Macrophages from different species, particularly humans, may exhibit variations in metabolic pathways and immune responses. In summary, CVCeCG provides essential insights into photothermal antibacterial effects and remodeling the immune microenvironment through trained immunity and metabolic reprogramming, offering new strategies for comprehensively improving the microenvironment of diabetic chronic wounds and demonstrating exceptional clinical translation potential.

Future directions

This study proposes a novel strategy for managing infected diabetic wounds by integrating multiple nanomaterials with photothermal hydrogels, promoting chronic wound healing through comprehensive improvement of the wound microenvironment. For further evaluation, the next step will focus on developing a scalable production process for the hydrogel and validating its efficacy and long-term safety in promoting chronic wound healing using large animal models (e.g., porcine skin injury models). Furthermore, considering that diabetic wounds often present with mixed infections in clinical settings, we plan to evaluate the efficacy of the hydrogel on such infected wounds in the future. We further propose to conduct systematic preclinical safety and toxicology assessments in compliance with regulatory requirements, thereby laying a solid foundation for the eventual application for a New Drug Clinical Trial Authorization.

Experimental section

Preparation of CPO@PCL and Van@PCL

CPO- and Van-loaded PCL microparticles were fabricated using a water-in-oil-in-water (w/o/w) double emulsion synthesis method. A 10% (w/v) PCL solution was prepared in dichloromethane (DCM) with a volume of 3 mL. Subsequently, 200 mg of CPO was dissolved in 1 mL of ethanol, while 200 mg of Van was dissolved in 0.5 mL of a 1% (w/v) aqueous polyvinyl alcohol (PVA) solution. The solutions were emulsified via ultrasonication at 30% maximum amplitude using 2-second on/off pulse cycles for 3 min each. Subsequently, 50 mL of 3% (w/v) PVA in distilled water was added to both emulsion PCL-CPO and PCL-Van, followed by additional ultrasonication at 30% amplitude with 2-second on/off pulse cycles for 5 min at room temperature. After sonication, the systems were stirred at room temperature for 24 h to evaporate the solvent, then concentrated via centrifugation at 10,000 rpm for 5 min. The microspheres were washed three times with Dulbecco’s phosphate-buffered saline (DPBS) to remove residual additives. The resulting microspheres were lyophilized and stored in a dry and cool environment until further use.

Preparation of CeNPs

According to a previous report [58], the synthesis of CeNPs proceeded as follows: Cerium nitrate (6 mmol) was dissolved in 100 mL of an aqueous ethylene glycol solution (1:1 v/v) at room temperature. The cerium nitrate solution was heated to 60 °C, followed by the addition of 20 mL NH₄OH. CeNPs were obtained after vigorous stirring for 3 h.

Preparation of CMC-DA

We synthesized CMC-DA using the same method as before [36]. 2.50 g of CMC was dissolved in 500 mL of MES solution (pH 5.0). EDC (2.40 g) and NHS (1.45 g) were added to the CMC solution and stirred for 45 min, followed by 5 g DA. The solution was stirred under a nitrogen atmosphere at 22 °C for 24 h and dialyzed against distilled water. The final product, CMC-DA powder, was obtained by lyophilization.

Fabrication of CG and CVCeCG hydrogels

CMC-DA (0.27 g) was completely dissolved in 9 mL deionized (DI) water to prepare a 3% (w/w) CMC-DA solution. Subsequently, 3 mL of 20% (w/w) GEL solution and 60 µL of 5% (w/w) oxidizing agent sodium periodate (NaIO4) solution were added. After stirring at 60℃ for 3 min, the heated solution was transferred to 37℃ to allow crosslinking and form hydrogel CG. Incorporation of micro/nanoparticles into the hydrogel system through stirring yielded CVCeCG hydrogel.

Hemostasis performance in vivo

All experimental protocols complied with the ARRIVE guidelines and institutional requirements for laboratory animal care and use, with surgical procedures approved by the Ethics Committee of Zhongnan Hospital, Wuhan University (Approval No. ZN2024088). Male Sprague-Dawley rats (180–200 g) were anesthetized using 1% sodium pentobarbital and subjected to partial hepatectomy (approximately 50% liver resection) to establish a hepatic injury model. Immediate hemostatic treatment was administered to the surgical site using commercially available gauze, gelatin sponge, or CVCeCG hydrogel, respectively. Record the amount of bleeding.

In vivo diabetic wound healing performance

Male Sprague-Dawley rats (6 weeks old, 200–220 g) were obtained from the Hubei Provincial Center for Disease Control and Prevention, Food and Drug Safety Evaluation Center (China). SD rats were fasted for 12 h. A 1% STZ (65 mg/kg, Solarbio, Beijing, China) solution in citrate-sodium citrate buffer (pH 4.4) was prepared and administered to the SD rats by intraperitoneal injection to establish a type 1 diabetes model. Blood glucose levels were measured using blood glucose test strips (Yuwell 590, China), and SD rats’ weights were measured on days 3, 7, 14, and 21 after induction. SD rats with sustained hyperglycemia (> 16.7 mmol/L) for 3 weeks were selected for subsequent experiments. After anesthesia, we applied depilatory agents to the dorsal region of the SD rats and created a full-layer wound 15 mm in diameter on their backs. Diabetic rats were randomly assigned to four groups (n = 15/group): Untreated, Tegaderm™ (3 M), CVCeCG, and CVCeCG + NIR. Wound areas were photographed and analyzed via ImageJ on days 0, 3, 7, 10, and 14. Wound exudates collected on days 1 and 14 were cultured on agar plates. Tissue samples collected on days 3, 7, and 14 were fixed in 4% paraformaldehyde for histological analysis. To assess tissue morphology, H&E, Masson’s trichrome, Sirius red, Ki67, and CK14 staining were performed. Intra-tissue levels of superoxide anions were measured using the DHE probe to analyze ROS within the tissues. Immunofluorescence staining was employed to evaluate the expression levels of mannose receptors, α-smooth muscle actin (α-SMA), and platelet endothelial CD31 to investigate macrophage polarization and angiogenesis in the tissues. Additionally, immunohistochemical staining was performed to measure the levels of IL-6, IL-10, TNF-α, and transforming growth factor-β (TGF-β) in order to assess the level of inflammation within the tissues. RNA sequencing on day 14 compared gene expression between the Untreated and CVCeCG + NIR groups. All procedures complied with institutional guidelines for laboratory animal welfare. To minimize the animals’ suffering, euthanasia was performed via cervical dislocation. The Wuhan University Animal Care and Use Committee approved the experimental protocols.

Measurement of lactate level

Intracellular and extracellular lactate levels were quantified using a Lactate Assay Kit with WST-8 (Beyotime, China). Following manufacturer’s protocol, samples were reacted with lactate dehydrogenase to catalyze lactate oxidation to pyruvate, concurrently reducing NAD + to NADH. The generated NADH subsequently reduced WST-8 to formazan via 1-methoxy-5-methylphenazinium methyl sulfate (1-mPMS) mediation, yielding formazan with peak absorbance at 450 nm. Absorbance measurements (OD450nm) were acquired using a microplate reader, with lactate concentrations determined through a standard curve. Data normalization established relative lactate levels as ratios to the mean control values.

Seahorse assay

Macrophages were seeded in Seahorse XF96 microplates and treated with designated reagents for 24 h. Prior to assay, cells were washed twice with pre-warmed Seahorse XF DMEM Base Medium supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, and 10 mM glucose (pH 7.4), followed by 60 min equilibration in a non-CO₂ incubator at 37 °C. Glycolytic function was assessed using the Glycolytic Rate Assay Kit (Agilent, USA) with sequential injections of 0.5 µM rotenone/antimycin A (Rot/AA) and 50 mM 2-deoxy-D-glucose (2-DG). Mitochondrial respiration was evaluated via the Cell Mito Stress Test Kit (Agilent, USA) using sequential injections of 1.5 µM oligomycin (Omy), 1.0 µM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and 0.5 µM Rot/AA. Real-time measurements of the glycolytic proton efflux rate (GlycoPER) and oxygen consumption rate (OCR) were performed on a Seahorse XFe96 Analyzer (Agilent). Data normalization and analysis were conducted with GlycoPER and OCR serving as quantitative proxies for glycolytic flux and oxidative phosphorylation, respectively.

Macrophages and epidermal keratinocytes co-culture system

A Transwell co-culture system (Corning, USA) was employed to investigate interactions between rat epidermal keratinocytes and bone marrow-derived macrophages without direct cell contact. Epidermal keratinocytes were isolated from Sprague-Dawley rat skin via sequential dispase/collagenase digestion as previously described [59], while BMDMs were differentiated from bone marrow monocytes using 7-day stimulation with 20 ng/mL M-CSF. Cells were co-cultured across a polyester membrane, with keratinocytes seeded in the upper chamber and macrophages in the lower chamber. All cultures were maintained at 37 °C in 5% CO₂.

OCR and ECAR assay

Oxygen consumption rate (OCR) level was assessed using the OCR fluorometric assay kit (Elabscience, China) with an oxygen-sensitive fluorescent probe. After treatment, macrophages were sealed in an airtight environment. Cellular oxygen consumption reduced ambient oxygen levels, resulting in an increase in fluorescence intensity over time. OCR was quantified by calculating the slope of the change in fluorescence intensity over time. ECAR level was assessed using the ECAR fluorometric assay kit (Elabscience, China). Concurrently, extracellular acidification rate (ECAR) was assessed using a pH-sensitive probe (Ex/Em: 490/535 nm). During glycolytic flux, proton efflux from cells acidifies the extracellular milieu, proportionally reducing probe fluorescence intensity. Data normalization established relative ECAR and OCR levels as ratios to the mean control values.

Chromatin Immunoprecipitation (ChIP)-qPCR

The ChIP assays were performed using the ChIP Assay Kit (Beyotime, China) according to the manufacturer’s specifications. Briefly, RAW264.7 cells were cross-linked with 1% formaldehyde for 10 min at room temperature, with reactions quenched using 125 mM glycine. Chromatin was fragmented to 200–500 bp fragments via ultrasonication. Sonicated lysates underwent overnight immunoprecipitation at 4 °C with anti-H3K18la antibody (1:100, PTM Bio) or normal rabbit IgG (1:250, Cell Signaling) as a negative control, alongside 2% input chromatin. Precipitated DNA and input controls were purified and quantified by real-time PCR using the comparative threshold cycle (2 − ΔΔCt) method with the following primers: Arg-1 (F: 5′-TGCTCCGTTTCGATTCTT-3′, R: 5′-TCGTGTGCCAAGTGCTATTC-3′) and VEGF (F: 5′-CGAGGGTTGGCGGCAGGAC-3′, R: 5′-CAGTGGCGGGGAGTGAGACG-3′).

ELISA detection of IL-6, IL-1β, TNF-α, and VEGF

Cytokine concentrations (IL-6, IL-1β, TNF-α, VEGF) in RAW264.7 cell supernatants were quantified using species-specific ELISA kits (Solarbio, China) following the double-antibody sandwich principle. After centrifugation (12,000 × g, 10 min), supernatants were diluted per manufacturer’s instructions. Microplates pre-coated with anti-mouse capture antibodies were incubated with serially diluted standards or pre-diluted samples (100 µL/well, 37 °C, 90 min). Biotinylated detection antibodies were added (37 °C, 60 min). Plates were rewashed before incubation with horseradish peroxidase (HRP)-conjugated streptavidin (37 °C, 30 min). After final washing, 3,3’,5,5’-tetramethylbenzidine (TMB) substrate was added (100 µL/well, 37 °C, 15 min). Absorbance was measured at 450 nm, with target cytokine concentrations calculated against standard curves using four-parameter logistic regression.

Statistical analysis

All the experimental results were expressed as the mean ± standard deviation (SD). GraphPad Prism 9 was used to analyze statistical significance through Student’s t-test. P < 0.05 was deemed statistical significance.