An immunomodulatory hydrogel featuring antibacterial and ROS-scavenging functions for enhanced burn wound healing

Xian-Sheng Zhao; Li-Ping Zhang; Geng Wang; Qu-Yang Yang; Xiu-Jun Cheng; Chang-Yue Wu; Dan Wu; Can-Bin Dong; Yu-Tian Yang; Yi-Fan Wang; Xiu-Jun Cai; Ning-Wen Zhu

doi:10.1186/s12951-026-04126-6

. 2026 Feb 13;24:245. doi: 10.1186/s12951-026-04126-6

An immunomodulatory hydrogel featuring antibacterial and ROS-scavenging functions for enhanced burn wound healing

Xian-Sheng Zhao ^1,^3,^#, Li-Ping Zhang ^1,^#, Geng Wang ¹, Qu-Yang Yang ², Xiu-Jun Cheng ², Chang-Yue Wu ², Dan Wu ², Can-Bin Dong ², Yu-Tian Yang ¹, Yi-Fan Wang ^3,^✉, Xiu-Jun Cai ^3,^✉, Ning-Wen Zhu ^1,^2,^✉

PMCID: PMC13005498 PMID: 41689026

Abstract

Infectious wounds induce a cycle of bacterial proliferation, oxidative stress accumulation, and dysregulated macrophage polarization, collectively hindering tissue repair. Conventional wound dressings typically address these pathological factors in isolation, resulting in suboptimal therapeutic outcomes. Here, we report the design of a biocompatible multifunctional hydrogel (GAPC). This system integrates self-assembled Proanthocyanidin/Chlorhexidine nanoparticles into a dual-network GelMA/ADM scaffold. Consequently, the hydrogel exhibits simultaneous antibacterial activity, ROS scavenging, and immunomodulatory capacity. In vitro, GAPC hydrogel has superb antibacterial, antioxidant, and anti-inflammatory effects. On the other hand, GAPC hydrogel promotes M1-to-M2 macrophage transition and preserved cellular viability under oxidative stress. Furthermore, in vivo it accelerated infected burn wound closure, enhanced collagen remodeling, and stimulated neovascularization. Collectively, GAPC hydrogel interrupts the “infection-oxidative stress-inflammation” loop, offering a safe and promising option for managing infected wounds.

Graphical Abstract

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Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-026-04126-6.

Keywords: Burn wound healing, Multifunctional hydrogel, Antibacterial, ROS scavenging, Macrophage polarization, Bioactive dressing

Background

Infected wounds, especially burns, diabetic ulcers, and chronic pressure sores, represent a major clinical challenge. According to the International Society for Burn Injuries (ISBI), more than 11 million people worldwide need medical care for burns each year, and about 40% of these cases develop secondary bacterial infections [1]. These problems not only prolong hospitalization but also impair re-epithelialization and increase the risk of systemic sepsis [2]. The biology of these wounds is characterized by chronic bacterial colonization, persistent oxidative stress caused by reactive oxygen species (ROS), and sustained pro-inflammatory signaling. Together, these factors disrupt extracellular matrix (ECM) integrity, hinder the transition of macrophages from the pro-inflammatory M1 phenotype to the pro-regenerative M2 phenotype, and consequently delay wound healing [1, 3]. These chronic infections remain particularly difficult to manage due to limited drug bioavailability at the wound site and the rapid emergence of multidrug-resistant (MDR) bacteria strains [4, 5]. Therefore, developing a multifunctional biomaterial that simultaneously possesses broad-spectrum antibacterial activity, potent ROS scavenging capacity, and immunomodulatory properties is crucial for effective regeneration.

Hydrogels have emerged as one of the most promising biomaterials for wound dressings due to their high water content, injectability, and structural resemblance to the native ECM [6]. However, conventional hydrogels often exhibit intrinsic limitations, such as poor mechanical strength and uncontrolled drug release, which compromise their long-term stability in dynamic wound environments [2, 7]. Recent advances in dynamic covalent chemistry (DCC) and reversible non-covalent bonding strategies have opened new opportunities for developing self-healing hydrogels. For example, self-assembly through Schiff base or amide linkages can dissociate and reassemble at neutral pH reversibly, facilitating self-repair when damage occurs [8]. A dual-network bio-patch covalently cross-linked through C-C covalent bonds and amide bonds was reported to improve the tensile strength (200%) and self-healing effect (> 95%) [9].

Bioactive dressings, including collagen, chitosan, hyaluronic acid, and alginate, are biocompatible and biodegradable wound dressings. However, they cannot completely mimic the complexity of the natural ECM of the skin [10]. To better mimic the native ECM, we employed porcine acellular dermal matrix (ADM), which possesses a comparable structure and extracellular matrix composition to human skin [11]. ADM is decellularized to remove cellular components that could cause rejection by the immune system. In the early phase of wound healing, the ADM assists in broken skin. It also participates in skin homeostasis through interaction with cells and growth factors [12]. Furthermore, the combination of ADM with drug-embedded gelatin enhances both biomimicry and safety. Its pore size (50–200 μm) is similar to that of the natural ECM structure and facilitates fibroblast migration [13].

When it comes to designing antibacterial and antioxidant devices, the duality between efficiency and flexibility commonly suffers if all those desired properties are provided in a single agent [14]. Nanotechnology is a promising approach to improving the performance of wound dressings. Nanoparticles could be highly efficient carriers of antibacterial and antioxidant drugs, enhancing stability and local retention [15, 16]. Proanthocyanidins (PA), the class of polyphenols in grape seeds, have strong abilities to scavenge radicals, and they can also disrupt bacterial membranes by the phenolic hydroxyl groups [17]. Chlorhexidine acetate (CHX) is a biguanide general antibacterial agent that can damage the structure of the bacterial membrane in as little as 30 s, resulting in the prevention of wound infection [18–20]. The self-assembly of PA and CHX into nanoparticles, PA/CHX (named as PC), provides a theoretical combination of the two components’ orthogonal modes of action: PA for ROS scavenging and CHX for membrane disruption. These nanoparticles also stabilize drugs from degradation, extend their residency at the wound, and minimize systemic toxicities [21]. Unfortunately, there is still a shortage of carrier systems suitable to achieve the precise control of drug release with tissue adhesion and mechanical supports [22, 23].

Based on these considerations, we developed a multifunctional composite hydrogel (termed GAPC, Scheme 1A) that integrates PC nanoparticles into a GelMA/ADM-based matrix for the treatment of infected burn wounds. In this design, the PC nanoparticles serve as dual-function modules for ROS scavenging and antibacterial activity. Meanwhile, a twin dynamic crosslinking network was formed between covalent bonds from double bond radical polymerization of the gelatin skeleton and amide bonds between the -COOH group in the ADM and the -NH2 group in the gelatin. For carrier development, PC nanoparticles were incorporated into the meshwork of the hydrogel, leading to microenvironment-sensitive and sustainable drug release. Moreover, GelMA crosslinking under the 405 nm 405 nm visible light provided strong adhesion between the hydrogel and wound tissue to stay mechanically stable without joint movement.

Although various wound dressings have been developed, they often fail to address the complex ‘infection-oxidative stress-inflammation’ loop simultaneously. For instance, commercial silver-based dressings, while effective against bacteria, frequently exhibit cytotoxicity towards keratinocytes and fibroblasts, potentially delaying re-epithelialization. Similarly, traditional ADM scaffolds lack the mechanical tunability and intrinsic antibacterial activity required for infected dynamic wounds, while single-network hydrogels often suffer from poor mechanical robustness.

To overcome these limitations, we report the design of a metal-free, dual-network multifunctional hydrogel (GAPC). Unlike passive wound coverings, GAPC integrates self-assembled PC into a GelMA/ADM scaffold. This design not only ensures mechanical stability and sustained drug release but also functions as an active immunomodulator to drive the M1-to-M2 macrophage transition, offering a superior therapeutic platform over existing single function modalitie.

We systematically evaluated the physicochemical properties, cytocompatibility, hemocompatibility, and antibacterial activity of GAPC. In vitro and in vivo studies confirmed that GAPC exhibits excellent biocompatibility, tunable drug release, potent antimicrobial and antioxidant activities, and superior wound healing efficacy. Taken together, the multifunctional GAPC hydrogel provides an effective and integrated therapeutic platform for the management of infected burn wounds.

Results

Physicochemical analysis of PC

The PC nanoparticles were synthesized following previously reported methods [24], as illustrated in Scheme 1A: the polyphenolic structure of PA and the positive charge property of CHX can interact electrostatically, resulting in stable nanoparticles. Hydrogen bonding between the phenolic hydroxyl group of PA and the amino group of CHX also possibly contributes to helping stabilize the nanoparticles. The microscopic morphology of PC nanoparticles was characterized using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), (Fig. 1A). SEM images revealed a relatively smooth surface without apparent defects, and their particle size was mainly distributed between 30 and 90 nm (Fig. 1B) [25]. In addition, the elemental composition of PC was analyzed by elemental mapping. The results showed that the elements C, O, Cl, and N were uniformly distributed in its matrix (Fig. 1C) [26].

The chemical structure of the polymer was characterized by Fourier transform infrared spectroscopy (FTIR). As shown in Fig. 1D, the hydroxyl group (-OH, 3356 cm⁻¹) telescopic vibration peak of PA shifts to the low-wave number, and the amino group (-NH, 3340 cm⁻¹) of CHX widens or shifts to the low-wavelength number, which further proves that the two are involved in the formation of hydrogen bonds. Furthermore, at 229 nm, PA and PC show the same characteristic peaks, whereas at 260 nm, CHX shows characteristic peaks with PC, which strongly suggests that they are connected stably by the formation of H-bonding (Fig. 1E). PA has phenolic hydroxyl groups, which can be negatively charged. After loading with the cationic chlorhexidine (Fig. 1F), the zeta potential for the measured nanoparticles (PC) was higher.

Ultraviolet-visible (UV-vis) absorption spectrometry demonstrated that PC had the maximum absorption peak at 230 nm (Fig. S1, Supplementary Information), and then a standard curve of PAC NPs was established according to this characteristic absorption by linear regression analysis (Fig. S2). To determine its stability, we compared the PC solution (0.5 mg/mL) after placing it for different times, and all the groups showed no variation in zeta potential (Fig. 1G), indicating an unchanged surface charge and dissolution of the NP in the solution [27]. This is also corroborated by the UV-vis absorption spectra results (Fig. 1H), which show no obvious discrepancy of absorbance between groups, suggesting that PC exhibits good stability.

To test whether PC protects cells from oxidative stress, the viability of HaCaT and HUVEC cells exposed to H₂O₂ was measured using the CCK-8 assay [28]. As depicted in Fig. 1I and J, following H₂O₂ treatment, cell viability decreased to 28% and 34%, respectively. In contrast, PC treatment restored cell viability to 72% and 64%, respectively. The scavenging capabilities of PC were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), collectively referred to as reactive nitrogen species (ROS), with absorbance measurements recorded at 517 nm and 734 nm, respectively (Fig. S3). In the presence of PC at 37 °C, the DPPH solution changed from dark purple to yellow, and the ABTS solution changed from blue to gray. These colorimetric changes demonstrate that PC scavenged DPPH radicals (Fig. 1K) and ABTS radicals (Fig. 1M) in the mixed systems. This is also confirmed by the results of OD readings, which show DPPH scavenging > 90% (Fig. 1L) and a similar trend for ABTS (Fig. 1N). The absorbance of DPPH and ABTS significantly decreased following PC treatment, demonstrating the potent free radical scavenging capacity of the nanoparticles [29]. Flow cytometry analysis showed similar results, further supporting the findings of the CCK-8 results (Fig. 1O, P).

The antimicrobial activity was also evaluated for S. aureus (Gram-positive) and E. coli (Gram-negative) in the assessment of ROS scavenging capacity. Remarkably, after 4 h incubation with PC, bacterial colonies were eliminated, which suggested a longer antibiotic release (Fig. 1M-N). PC exhibited significantly potent antimicrobial activity against both S. aureus and E. coli, which was primarily ascribed to the bursting of CHX [30]. Considering the multifaceted aspects of antioxidant performance, antimicrobial potency, and biocompatibility, a dose of 1 mg/mL of PC was chosen for subsequent experiments.

Preparation and characterization of the GAPC hydrogel

Recent advances in macromolecular engineering have demonstrated that nanogel-based architectures can significantly enhance the physicochemical stability and bioactivity of encapsulated agents [31]. Inspired by these strategies, we designed the GAPC hydrogel by integrating self-assembled PC nanoparticles into a dual-network scaffold to ensure optimal structural integrity and sustained drug release. The ADM hydrogel was developed via decellularization, freeze-drying, and pepsin digestion of porcine dermis. Ultimately, the pre-gel solution was incubated for 10 min at 37 °C, to trigger gelation (Fig. 2B) [32]. The effectiveness of cell removal in ADM was examined and confirmed by H&E, Masson’s trichrome, and immunofluorescence staining. Figure 2A demonstrates that DAPI-stained nuclei (blue) were effectively removed, with collagen bundles (green) remaining. Additionally, we investigated the content of native constituents (glycosaminoglycans and hydroxyproline) and remaining DNA in ADM. As shown in Fig. 2C-E, the content of glycosaminoglycan (GAG) and hydroxyproline was similar before and after decellularization. However, the content of double-stranded DNA (dsDNA) dropped to 13 ng/mg. This suggests that decellularization successfully eliminated cellular elements and maintained the collagen bundles.

Fig. 2 — Preparation and characterization of the GAPC hydrogel. A) H&E & Masson’s trichrome stained images (scale bar:500 μm) and immunofluorescence staining images of Col I (green) and cell nucleus (blue) in the pig skin and the ADM tissue (scale bar: 200 μm). B) Schematic showing the gelation of ADM-derived hydrogel. C-E) The concentrations of glycosaminoglycans (C), dsDNA (D), and hydroxyproline (E) of the ADM. ** p < 0.001. F) Macroscopically illustration of gelation of GelMA and ADM with PC (APC) and their TEM imagery, separately. (scale bar: 500 μm). G) EDX elemental mapping for C, O, Cl, and N in GAPC (scale bar: 100 μm). H) FTIR, I) UV-vis spectra of GelMA, APC, and GAPC. J) Water absorption ratios of the hydrogels following immersion for a specified duration (n = 3). K) Compression stress-strain, L) Tensile stress-strain curves of GelMA, APC, and GAPC. M) Typical force-displacement curves of porcine skin bonded to GelMA, APC, and GAPC. N) Images showing the compressible and tensible nature of the hydrogel (scale bar: 5 mm). O) Frequency sweep tests (from 0.1 to 10 Hz) of GAPC at 37 °C. P) Rheological property of GAPC in the strain amplitude sweep (γ = 0.1–1000%) at 1 Hz and 37 °C. Q) Macroscopically illustration of the self-healing process of GAPC dyed with purple pigment and methylene blue (scale bar: 5 mm). R) Rheological property of GAPC at low (1%) and high (500%) strains with loading time of 100 s, respectively. S) Analysis of the shear-thinning characteristic in GAPC. APC = ADM+PC; GAPC = GelMA + APC; GAPC0 = GAPC without PC; GAPC1 = GAPC with PC (1 mg/mL). Data are presented as mean ± standard deviation (n = 3 independent biological replicates). Statistical significance was determined by One-way ANOVA with Tukey’s post hoc test. **** p < 0.0001 versus the Control group

PC nanoparticles were first blended with ADM to create the APC hydrogel precursor, without using toxic crosslinkers. The precursor was then mixed with GelMA. Photo-crosslinking of the methacryloyl groups formed new C-C covalent bonds, creating a 3D crosslinked network. At the same time, amide bonds formed between GelMA amino groups and ADM carboxyl groups, producing the dual-network GAPC hydrogel (Scheme 1B). SEM images showed that GAPC had a more continuous and compact microstructure (Fig. 2F). It is worth noting that the photo-crosslinking was performed using 405 nm visible light. Unlike short-wavelength UV, 405 nm light at the applied intensity shows negligible phototoxicity and thermal damage to host tissues, ensuring the biosafety of the gelation process [33]. Elemental mapping showed that N, together with C and O, was broadly distributed throughout the scaffold, consistent with the protein-based GelMA/ADM matrix. The Cl signal, attributable to CHX-associated components within the PC nanoparticles, was also detected across the scaffold (Fig. 2G), supporting the incorporation of drug-associated components within the hydrogel network. Functional groups of GAPC were also confirmed by the FTIR spectra (Fig. 2H). As clearly shown in the magnified inset (1300–1750 cm⁻¹), the GAPC hydrogel exhibited distinct spectral characteristics compared to pure GelMA and APC: The characteristic Amide I band (1600–1700 cm⁻¹) and Amide II band (~1540 cm⁻¹) in the GAPC spectrum displayed significant broadening relative to the control groups. This broadening indicates the formation of extensive intermolecular hydrogen bonds between the phenolic hydroxyl groups of PC and the amide groups of the protein matrix (GelMA/ADM), serving as the physical crosslinking nodes. Importantly, the characteristic absorption peak at 1629 cm⁻¹, corresponding to the C = C double bond of the methacrylate groups, was preserved in the GAPC pre-gel mixture. This confirms that the photo-crosslinking capability of GelMA remained intact, allowing for subsequent chemical polymerization upon blue light exposure. Additionally, UV-vis absorption spectra (Fig. 2I) displayed characteristic peaks at 263 nm and 300 nm, corresponding to the specific absorption profiles of GelMA and APC, respectively, further confirming the successful incorporation of both components.

The swelling capacity is crucial for hydrogel dressings to effectively absorb wound exudates [34]. The swelling of the hydrogels was studied in phosphate-buffered saline (PBS), as shown in Fig. 2J, Fig. S4 (Supplementary Information). At the initial stage, the hydrogels quickly absorbed liquid in the 1 st hour and then relatively slowly swelled until equilibrium was reached after 6 h [35]. The artificial moisture condition is generally believed to enhance wound healing and tissue proliferation [36]. In this study, the hydrogel is designed for treating wounds by sealing and keeping the wounded parts wet to promote healing. A water evaporation test was used to evaluate the hydrogel’s water-holding ability after wound closure [37]. As shown in Fig. S5 (Supplementary Information), the water retention of GAPC hydrogel was around 95.6% at 48 h, when compared to 100% in the sealed group and 84.2% in the unsealed group. This suggests that hydrogel may be able to reduce the loss of exudate after wound coverage to some extent and promote a moist environment for wound healing, which may facilitate the prevention of eschar formation.

Mechanical characterization was then performed to evaluate biomimetic performance. GAPC hydrogels tolerated tensile and compressive forces (Fig. 2N). As shown in Fig. 2K-L, the compressive and tensile moduli of APC were significantly lower than those of GelMA and GAPC. Lap shear tests using fresh porcine skin quantified tissue adhesion strength of the three hydrogels, showing values of 12.03 ± 1.34 kPa, 3.11 ± 1.24 kPa, and 9.17 ± 0.86 kPa, respectively (Fig. 2M, Fig. S6). These findings indicate the GAPC had better adhesion.

Self-healing capability is also a distinct property for wound healing. Macroscopic scarring was evident (Fig. 2Q), and rheologic measurements were consistent with a solid-like, elastic response [38]. Frequency sweep data demonstrated that GAPC, in the frequency range of 0.1–10 Hz, maintained its structural stability with G′ always higher than G′′ (Fig. 2O) [39], suggesting its elastomeric nature [13]. G′ and G′′ were constant until the crossover point where G′ crossed with G′′ at critical strain, over which G′′ surpassed G′, indicating network breakdown (Fig. 2P) [40]. After structural damage, all hydrogels showed effective self-healing ability (Fig. 2R) [41]. Similar results were seen in the contact test after cutting, where GAPC hydrogels showed clear shear-thinning behavior (Fig. 2S) [42]. Based on preliminary screening of mechanical properties and antibacterial efficiency, the GAPC1 group (1 mg/mL PC loading) exhibited the optimal balance between stability and bioactivity. Therefore, GAPC1 was selected as the representative formulation for subsequent comprehensive biological evaluations and in vivo studies.

Biocompatibility testing and antioxidant capacity of GAPC in vitro

To confirm the biocompatibility of GAPC, we evaluated cell viability and performed live/dead staining by co-culturing with ADSCs, HaCaT, and HUVEC cells. Cell viability and growth in the treated groups showed no significant difference compared to the control group (Fig. 3A). To further evaluate the effect on cell proliferation, the CCK-8 assay was extended to 3 days (Fig. S7). The results showed a time-dependent increase in cell viability for all groups, rising from 100% (Day 1) to nearly 300% (Day 3). The GAPC1 groups exhibited a proliferation rate comparable to the control, demonstrating that the composite hydrogel supports excellent cell growth and proliferation. And hemocompatibility, which is a crucial characteristic for hydrogel-based wound dressings [43], characterized by a hemolysis assay using mouse blood (Fig. 3B); a complementary assay using human red blood cells (hRBCs) is provided in Fig. S16. All the hydrogel-treated groups generated clear supernatants after centrifugation, and no obvious hemolysis or hemoglobin release was observed, indicating a favorable hemocompatibility.

Fig. 3 — Biocompatibility testing and Antioxidant capacity of GAPC in vitro. A) Live/Dead staining for ADSC, HaCat and HUVEC cells after leaching solutions of GAPC for 48 h (scale bar: 200 μm). B) Mouse blood hemocompatibility of GelMA, APC, and GAPC. C) The ratio of survival for HaCat cells. D) Flow cytometry results for RAW 264.7 cells. E) Images of DPPH free radicals scavenging of GAPC for 1 h (scale bar:1 cm). F) Quantitative results of the scavenging property (n = 3). G) Scratch assay images of HaCat cells after treated with H₂O₂ and leaching solutions of GAPC for different times (scale bar: 200 μm). H) Statistical analysis of migration ratio for G (n = 3). I) Assessment of tube formation in HUVECs under various treatments (scale bar: 200 μm). J) Quantifications of angular structure in each group (n = 6). K) Confocal microscopy images analysis of intracellular ROS levels in HaCat cells, with DCFH-DA marking the cytoplasm and Hoechst 33342 staining the nucleus (scale bar: 100 μm). L) Quantitative analysis of the DCFH positive rate (n = 3). M) F-actin staining of HaCat cells, with blue indicating DAPI-stained nuclei and green showing FITC-stained cytoskeleton (scale bar: 100 μm). N) Quantitative analysis of the FITC gray level (n = 3). Data are shown as mean ± standard deviation (n = 3). p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not statistically significant. Statistical significance was determined by One-way ANOVA with Tukey’s post hoc test. **** p < 0.0001 versus the Control group

Bacterial infection remains an obstacle during wound healing, causing prolonged inflammation and increased oxidative stress. This condition stimulates the excessive production of ROS, such as superoxide anion, hydrogen peroxide, hydroxyl radicals, and nitric oxide, which subsequently damage the cellular structure and prevent tissue repair [44, 45]. Thus, an antibacterial and antioxidant dual-functioned hydrogel is highly required to accelerate wound regeneration. Plant-based polyphenols such as PA and the broad-spectrum antimicrobial substance CHX acetate are known for their strong antioxidant and antibacterial effects and are frequently used as prophylaxis to prevent infections [46, 47]. Therefore, GAPC hydrogels comprising PA and CHX are an excellent candidate for an ideal multifunctional system that exhibits superior protection.

Antioxidant activity was tested using an H₂O₂-induced oxidative damage model. H₂O₂ greatly reduced HaCaT cell viability, but this effect was largely prevented when cells were co-treated with GAPC1 extracts (Fig. 3C). Similar protective effects were noted in RAW264.7 by flow cytometry of macrophages (Fig. 3D). Antioxidant activity of the materials was demonstrated through DPPH radical scavenging assays: As expected, the pure PA group (positive control) exhibited the highest scavenging efficiency due to the immediate availability of free PA. Notably, the GAPC hydrogel also demonstrated robust antioxidant capacity, scavenging over 63% of the free radicals. Although slightly lower than free PA due to the diffusion barrier of the hydrogel matrix, the activity of GAPC was significantly higher than that of the GelMA and ADM groups, confirming that the loaded PC nanoparticles retained their effective free-radical scavenging capability within the crosslinked network (Fig. 3E-F). Scratch assays showed that GAPC1 significantly promoted HaCaT cell migration with oxidative stress (Fig. 3G). In vitro tube formation test also showed that GAPC alleviated the impaired HUVEC angiogenesis induced by H₂O₂. Treatment of GAPC1 resulted in a larger branch number and total branch length, indicating the potency of GAPC1 in stimulating neovascularization (Fig. 3I) [43]. It is worth noting that under normal culture conditions (without H₂O₂), the GAPC hydrogels did not inhibit tube formation. The GAPC0 group showed similar angiogenic potential to the control, while the GAPC1 group exhibited a significantly higher number of meshes, indicating excellent basal biocompatibility and pro-angiogenic potential (Fig. S13). Moreover, DCFH-DA staining showed that the intracellular ROS levels were significantly increased in H₂O₂-incubated cells, but specifically GAPC treatment remarkably reduced this ROS production to a near-control level (Fig. 3K). Although the nuclear staining (Hoechst 33342) in the GAPC1 group appeared fainter due to live-cell staining variations, the quantitative analysis of the ROS signal (Fig. 3L) confirmed that GAPC1 significantly reduced intracellular oxidative stress compared to the H₂O₂ group. Furthermore, the cell density and morphology were cross-verified by cytoskeleton staining (Fig. 3M), where the GAPC1 group displayed a high density of spreading cells with intact filamentous actin (F-actin), comparable to the control. The quantitative analysis of FITC intensity (Fig. 3N) further demonstrated that GAPC1 effectively protected the cytoskeleton from oxidative damage.

The interplay between antioxidant activity and biocompatibility is critical for wound dressings. As highlighted in recent studies, effective ROS scavenging systems can significantly alleviate oxidative stress-induced cellular damage while maintaining high cytocompatibility [48]. Our results align with these findings, demonstrating that GAPC protects cells from H₂O₂-induced apoptosis.

Overall, GAPC1 exhibits strong protection against ROS-induced cell impairment; it provides a beneficial microenvironment for inflammatory wound healing, alleviates oxidative injury, and facilitates tissue regeneration. Therefore, GAPC1 may be a kind of multi-functional wound dressing [49].

Antibacterial capacity of CHX, PA, ADM, and GAPC in vitro

Antibacterial activity is one of the most important issues for wound dressing, because exudate and open wounds are very susceptible to bacterial colonization and proliferation. Staphylococcus aureus and Escherichia coli are the most frequent pathogens in burn wounds [50]. Four complementary approaches were used in this study to assess the antibacterial effect of GAPC hydrogels. Firstly, scanning electron microscopy (SEM) was conducted, and the SEM images showed that both strains of bacteria in the control group maintained integrity with a smooth surface, whereas the strains treated by GAPC presented membrane rupture, shrinkage, and distortion, indicating that GAPC may have killed bacteria by disturbing membrane integrity (Fig. S9) [51]. Then, the antibacterial activity was evaluated by the inhibition zone test. The CHX and GAPC samples had clear transparent zones, suggesting that antibacterial activity of the samples was observed (Fig. 4A-C). The antibacterial activity of GAPC was further confirmed by 12 h incubation of diluted S. aureus and E. coli cultures with GAPC extract or PBS added at 10% (v/v) (100 µL/mL). At 10⁶ CFU, the inhibition rate increased to 100% (Fig. 4D-G).

The live/dead bacterial staining further verified that massive dead bacteria (red fluorescence) existed in the CHX and GAPC groups (Fig. 4H). Quantitative analysis in terms of OD values also indicated that GAPC significantly inhibited the growth of the tested strains, which was mainly attributed to the CHX release (Fig. 4I-J). Overall, these results indicate that GAPC hydrogels exhibit effective and sustained antibacterial activity, which is essential for the infection prevention and management of burn wounds.

Compared to commercially available silver-based dressings, which are effective antibacterial but may exhibit cytotoxicity towards regenerating cells, the GAPC hydrogel offers a ‘pro-healing’ advantage [52]. The incorporation of PA and CHX provides a synergistic antibacterial and antioxidant effect without the cytotoxicity concerns often associated with high-concentration silver ions. Furthermore, unlike passive dressings, GAPC actively modulates the immune microenvironment by promoting the M1-to-M2 macrophage transition.

From a clinical translation perspective, the GAPC system offers distinct advantages. The lyophilized ADM and PC nanoparticles can be stored dry for extended periods, maintaining their bioactivity. The ‘mix-and-apply’ approach allows the hydrogel to be prepared immediately before use, ensuring optimal freshness. Moreover, the injectability and in situ photo-crosslinking capability allow the hydrogel to conform perfectly to irregular deep burn wounds, filling cavities that pre-formed dressings cannot effectively cover [53].

Inflammatory modulation capacity of GAPC in vitro

Macrophages have a remarkable phenotypic plasticity that enables them to rapidly adapt their phenotype in response to environmental signals in order to maintain tissue homeostasis and to promote the clearance of cellular debris and the repair of tissues [54]. Macrophages become polarized towards M1 (classically activated, pro-inflammatory) or M2 (alternatively activated, immune-regulatory, tissue remodeling) phenotypes depending on cytokine signaling [55]. Dysregulation in macrophage polarization in the inflammatory environment (pro-inflammatory M1/anti-inflammatory M2) would further increase ROS production and sustain inflammation and thereby impede wound healing [56]. The timely transformation of macrophages to relevant phenotypes can release the inflammation, promote collagen formation and new blood vessels, and establish a good environment for tissue regeneration [57]. M1 macrophages can be activated by lipopolysaccharide (LPS) and produce large amounts of inflammatory mediators, including inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α) [58]. In contrast, IL-4 or IL-10-stimulated M2 macrophages secrete anti-inflammatory signals such as arginase-1 (Arg-1), IL-10, and a number of growth factors, which suit multiple routes to support tissue repair and inflammation resolution.

The differentiation potential of macrophages plays a pivotal role in chronic wound healing, encompassing signaling pathways and mechanisms of tissue repair and regeneration. In this study, the ability of GAPC (specifically the optimized GAPC1 formulation) to induce macrophage polarization from pro-inflammatory M1 to anti-inflammatory M2 was assessed by analyzing the expression of specific markers. Undifferentiated macrophages (M0-like) were pretreated with LPS to induce M1 polarization, whereas IL-4 was used to promote M2 polarization. IL-4- or LPS-pretreated cells served as M2 and M1 reference groups, respectively (Fig. 5A). As determined by immunofluorescence staining, the fluorescence intensities of M1 markers CD86 and iNOS were significantly reduced, while those of M2 markers CD206 and Arg-1 were significantly increased in the GAPC0- and GAPC1-treated groups compared to the LPS group, indicating a trend toward M2 polarization. M2-like GAPC1-treated (GAPC1 macrophage) cells showed the same morphology, while M1-like cells displayed other morphology with elongated and multiple pseudopodia (Fig. 5B) [59]. To strictly validate this observation, quantitative analysis of the mean fluorescence intensity (MFI) was performed (Fig. S17, Supplementary Information). The results confirmed a statistically significant decrease in M1 markers and a significant increase in M2 markers for the GAPC1 group, which is consistent with the trends observed in flow cytometry.

Fig. 5 — Inflammatory modulation capacity of GAPC in vitro. A) Schematic diagram illustrating the anti-inflammatory effect of GAPC on LPS-stimulated macrophages. B) Typical immunofluorescence images showing CD86 (green), CD206 (red), iNOS (green), Arg-1(red) and cell nuclei (blue) in RAW 264.7 cells treated with IL-4, LPS, hydrogel extracts (100%, v/v) (scale bar: 50 μm). C) Flow cytometry plots depicting M1 (CD86) and M2 (CD206) cell populations after 72 h of inflammatory stimulation. D-E) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of mRNA levels for genes associated with M1-type RAW 264.7 cells (iNOS, TNF-, IL-6,) and M2-type macrophages (Arg1, IL-10, IL-4,) in RAW 264.7 cells (n = 3). F) Western blot analysis of proteins related to macrophage polarization (iNOS, CD86, CD206, and Arg1) in RAW 264.7 cells. G) Quantitative evaluation of protein levels, normalized to GAPDH, showing relative intensities. Error bars represent the mean ± standard deviation. Data are presented as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001; ns, not statistically significant. Statistical significance was determined by One-way ANOVA with Tukey’s post hoc test. **** p < 0.0001 versus the Control group

Flow cytometry further corroborated these trends. Compared with the negative control, the expression of CD86 clusters increased in the M1 group. In all hydrogel-treated groups, CD206 expression was elevated, accompanied by a relative decrease in CD86 expression (Fig. 5C). To further characterize the expression levels of these markers, the MFI was quantified (Fig. S18, Supplementary Information). Consistent with the percentage data, the GAPC1 group exhibited a significantly lower MFI for CD86 and a higher MFI for CD206 compared to the M1 group. These results also reveal that GAPC1 can effectively act as an inducer that drives pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages [60]. These results were validated by RT-qPCR (Fig. 5D, E): compared to the M1 group, mRNA levels of pro-inflammatory factors (iNOS, TNF-α, and IL-6) were significantly downregulated, and anti-inflammatory markers (Arg-1, IL-10, and IL-4) were increased in the GAPC-treated macrophages. The trends were replicated in Western blotting, and iNOS and CD86 protein levels were significantly up-regulated by LPS, whereas the opposite was true for CD206 and Arg-1, respectively. Whereas the levels of CD86 and iNOS were obviously downregulated, while CD206 and Arg-1 were upregulated in the GAPC1 treatment, revealing an evident inhibitory effect on M1-polarization.

The molecular mechanism underlying the GAPC-mediated M1-to-M2 transition is intrinsically linked to its potent ROS-scavenging capacity. ROS are not merely metabolic byproducts but also signaling mediators that influence macrophage polarization and inflammatory programs [61]. Increased intracellular ROS has been reported to facilitate NF-κB pathway activation (e.g., via IκBα phosphorylation/degradation and subsequent NF-κB activation/translocation), thereby promoting transcription of pro-inflammatory mediators, including iNOS and TNF-α [62]. In our study, GAPC exhibited strong ROS-scavenging capacity (Fig. 3K and L) and GAPC-treated macrophages showed decreased M1-associated markers (iNOS, CD86) and inflammatory cytokines (TNF-α, IL-6) together with increased M2-associated markers (Arg-1, CD206, IL-10) (Fig. 5), suggesting that an antioxidant microenvironment may contribute to macrophage reprogramming. In addition, PA has been shown to activate Nrf2 signaling in macrophages [63]. Given that Nrf2 activation is broadly associated with suppression of M1 polarization and promotion of M2-like anti-inflammatory programs [64], and that Nrf2 signaling has been implicated in regulating M2 markers such as Arg-1 and CD206 under relevant contexts [65], we propose that GAPC may act as an “immune-switch” by attenuating the ROS-linked NF-κB inflammatory axis while potentially engaging Nrf2-associated antioxidant pathways.

These results together indicate that GAPC is involved in macrophage polarization to adjust the inflammatory microenvironment, which may create a permissive environment for wound repair. This immunoregulatory potential may contribute to the anti-inflammatory effects of PA [66] and the ROS scavenging property of the hydrogel in the infected wound microenvironment that breaks the cycle of inflammation versus oxidative stress [57]. In general, GAPC hydrogels have a synergistic role in antibacterial, antioxidant, and immunomodulatory effects and could thus establish a strong theoretical basis for their use in infected burn healing. While murine models provided robust mechanistic insights, future studies incorporating primary human macrophages will be valuable to further validate the translational potential of the immunomodulatory effects observed here.

In vivo deep burn wound healing promoting efficacy of GAPC

To evaluate the effects of GAPC on burn wound healing, we established a severe burn model in BALB/c mice. Twenty-four hours after the scald injury, an 8-mm full-thickness excision wound was created on the dorsal skin. The wounds were inoculated with Staphylococcus aureus (10^⁸ CFU mL^⁻¹) for inducing the infection and then treated with different hydrogels or PBS (as the control) (Fig. 6A). Macroscopic observation of the wounds was recorded at post-treatment days 0, 3, 7, 10, and 14(Fig. 6B). Wound area was measured with ImageJ and is presented graphically (Fig. 6C). Schematic depictions of wound closure are also shown in Fig. 6C. GAPC1-treated wounds had consistently small area measurements at all time points during the experiment and had essentially completed wound closure by day 14 (Fig. 6D). Specifically, the size of the scars in the GAPC1 group was decreased by about 16% in comparison to that in the control (Fig. 6E), further indicating a significant effectiveness of the GAPC1 hydrogel in promoting wound closure and skin regeneration. Thus, the combination of CHX and PC in GAPC1 might be acting in synergy to facilitate the repair of bacteria-infected burn wounds.

Fig. 6 — In vivo deep burn wound healing promoting efficacy of GAPC. A) Experimental timeline for in vivo back skin wound (8 mm diameter) healing studies, with evaluation over 14 days. B) Pho- tographs documenting the progression of wounds on days 0, 3, 7, 10, and 14. C) Schematic illustration of skin regeneration over 14 days (scale bar: 5 mm). D) Quantitative analysis of the relative wound area percent at different time points (n = 6). E) Quantitative data of the relative scar area on day 14 (n = 6). F) Typical and Enlarged H&E and Masson-stained images of wound tissues from various groups at 7 days (scale bar: 1 mm and 100 μm, n = 6). G) Immunofluorescence staining images of the iNOS, Arg1, CD86 and CD206, in the wound tissue with different treatments on day 7 (scale bar: 100 μm, n = 3). H-K) qRT-PCR analysis of mRNA levels for genes associated with TNF-α, iNOS, Arg1 and IL-10 in the blood of mice (n = 3). Data are presented as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001; ns, not statistically significant. Statistical significance was determined by One-way ANOVA with Tukey’s post hoc test. **** p < 0.0001 versus the Control group

The architecture and presence of collagen are important to the integrity of the skin. Type I collagen is mainly responsible for providing tensile strength, and type III collagen is responsible for tensile distensibility. The network of type I and III collagens is organized in a basket-weave pattern, providing the biomechanical features specific to a normal dermis [67]. To assess the quality of the tissue, we also carried out histological tests on regenerated skin at days 7 and 14. HE and Masson tricolor staining were applied to each group of wound tissue. The results of the 7-day H&E and Masson tricolor staining analysis demonstrated a significant decrease in inflammatory cells, an increase in fibroblasts, and improved angiogenesis. This was primarily reversed by treating GAPC1, and PCs’ pro-angiogenic and anti-inflammatory properties played a major role. (Fig. 6F). Quantitative analysis of the collagen volume fraction (Fig. S19) confirmed that GAPC1 treatment significantly promoted collagen synthesis compared to the control group. On day 14, the control wounds were rich in immature granulation tissue, while the GAPC1-treated wounds displayed well-formed epidermal structures with decreased hyperplasia and almost full dermis regeneration (Fig. 7B). Masson’s staining further demonstrated that the level of collagen deposition was highest in all groups, with the degree of collagen deposition at each time point being the most significant in GAPC1. The collagen fibers of GAPC1-treated skin were denser and thicker and more regularly arranged (Fig. 7A, C).

Fig. 7 — Investigation on the tissue regeneration and angiogenesis efficacy of ADM, GAPC0 and GAPC1 in vivo. A) Typical and Enlarged H&E, Masson and Picrosirius red stained images of wound tissues from various groups at 14 days (scale bar: 1 mm and 100 μm). B) Epidermal thickness of wound tissues on day 14 (n = 3). C) Statistical analysis of collagen deposition in the entire regenerated tissues (n = 3). D-F) Statistical analysis of the collagen I and collagen III in the scar-surrounding normal regenerated skin (n = 3). G) Lacunarity analysis of collagen (n = 3). H-I) Representative images of anti-CD31(red) and anti-Ki67 (green) immunofluorescent staining in different groups on days 14 (scale bar: 100 μm, n = 3). I-J) Quantification of the capillary density and the cell proliferation rate of wound tissue in different groups on day 14 (n = 3). K) Photographic evidence of *S. aureus* colonies on TSB plates from various wound sites on day 14. L) Quantification of colony formation units from various wound sites on day 14 (n = 3). Data are presented as mean ± standard deviation (n = 3). **p < 0.01, and ***p < 0.001; ns, not statistically significant. Statistical significance was determined by One-way ANOVA with Tukey’s post hoc test. **** p < 0.0001 versus the Control group

The distribution and pattern of organization of collagen subtypes were evaluated by Picrosirius Red staining and polarized light microscopy. Type I collagen was yellow, and type III collagen was green (Fig. 7A). Wounds treated with GAPC1 demonstrated significantly increased deposition of type I collagen (Fig. 7D) and decreased expression of type III collagen (Fig. 7E), which is in line with the remodeling phase of type III collagen down-regulation and subsequent type I collagen substitution [68]. The ratio of type I to type III collagen in GAPC1-treated tissue was 4.61:1, which was significantly larger than that in the other three groups and approximately equal to that in intact skin (Fig. 7F) [67]. These findings also confirmed the capacity of GAPC hydrogel to guide skin regeneration toward normal structure.

Due to the intricate architecture of collagen, conventional measurements are not sufficient. As a result, we used fractal analysis to measure the inherent irregularity of collagen patterns. Collagen porosity (a measure of organizational complexity) was evaluated by means of the ImageJ fractal analysis plugin [69]. Porosity was greater in both the control and ADM groups, while GAPC1-treated tissue presented with lower porosity (Fig. 7G). These results imply a favorable correlation between the level of skin regeneration and collagen texture: a higher level of regeneration relates to more organized collagen content, less porosity, and a more planar arrangement, leading to less rough skin surfaces.

At the conclusion of the animal experiments, biosafety was assessed across all groups. After 14 days of treatment, major organs—including the heart, liver, spleen, lungs, and kidneys—were examined by H&E staining. Histopathological evaluation revealed no abnormalities in tissue structure (Fig. S12, Supplementary Information).

GAPC hydrogel antibacterial, anti-inflammatory, and vascular-promoting in vivo properties

Accumulating evidence suggests that modulating the oxidative microenvironment is a pivotal strategy for accelerating tissue repair. Recent pharmacological investigations have underscored that eliminating excessive ROS can reactivate suppressed regenerative pathways [70]. Consistent with this mechanism, the GAPC hydrogel interrupts the ‘infection-oxidative stress’ loop, creating a permissive environment for healing. To investigate the in vivo antibacterial activity of the hydrogel, wound tissues from each group were collected on day 14, homogenized, and plated on agar for colony culture. GAPC1-treated wounds yielded markedly fewer bacterial colonies compared with the PBS control, whereas the ADM and GAPC0 groups showed extensive bacterial growth. These findings indicate that GAPC maintains strong antibacterial activity even in the complex in vivo microenvironment, primarily attributable to the release of PC (Fig. 7K-L).

Increasing evidence suggests that macrophages play a central role in wound healing. Reducing the proportion of pro-inflammatory M1 macrophages and promoting the transition toward anti-inflammatory M2 macrophages is essential for effective repair. M2 macrophages also promote angiogenesis, collagen restructuring, and tissue reconstruction [71, 72]. Immunofluorescent staining of day 7 wound tissues showed macrophage phenotypic transitions and inflammatory marker expression (Fig. 6G). To further quantify these changes, the expression levels of M1/M2 markers were analyzed for each mouse (n = 3). As shown in Fig. S10 and Fig. S11 (Supporting Information), quantitative statistics revealed that GAPC1 significantly reduced the expression of M1 markers (CD86, iNOS) and enhanced M2 markers (CD206, Arg-1), confirming the robust immunomodulatory capacity of the hydrogel in vivo. In line with these, gene expression of pro-inflammatory (TNF‐α, iNOS) and anti‐inflammatory (Arg1, IL‐10) factors in mouse blood at day 7 was downregulated or upregulated, respectively, indicative of effective influence over the systemic inflammatory microenvironment (Fig. 6H-K) [73].

Moreover, immunofluorescent staining on day 14 wound tissues showed that the amount of CD31-positive microvascular and the percentage of Ki67-positive proliferating cells were increased in GAPC-treated wounds as compared to the other three groups (Fig. 7H-J). These in vivo data were supported by the in vitro observations, collectively suggesting that GAPC accelerates wound healing by its joint antibacterial, antioxidant, and immunomodulatory properties.

GAPC creates a superior healing microenvironment by synergistically interrupting the vicious pathogenic cycle. The antioxidant mechanism of action predominantly offered by PC in the hydrogel is probably the most critical induction point for the process. GAPC eliminates the extra ROS, heals the wound, protects the neighboring cells from oxidation, and helps cell migration and neovascularization, as well as immune regulation. The ablation of oxidative stress load is proposed to cause a macrophage shift from the M1-like pro-inflammatory phenotype to the M2-like reparative phenotype, characterized by iNOS/TNF-α/IL-6 suppression and Arg-1/IL-4/IL-10 induction, in both RAW264.7 macrophages and wound tissues. Meanwhile, sustained release of CHX by PC nanoparticles produces strong antibacterial effects, decreasing bacteria loading and inhibiting the signaling pathway to sustain chronic M1 polarization, which helps facilitate M2 transition. This leads to an immunoregulatory shift and recruitment of an M2-rich macrophage phenotype required for the next phase of healing.

The mechanistic crosstalk accounts for our in vivo findings, where the wound interface treated with GAPC1 showed improved collagen architecture, quick re-epithelialization, and effective CD31-positive vasculature development, all of which can be ascribed to the efficient control of the immune microenvironment.

A primary concern in the clinical application of chlorhexidine (CHX) is its potential dose-dependent cytotoxicity towards fibroblasts and keratinocytes, which can paradoxically delay healing if unregulated. Addressing this, the GAPC hydrogel utilizes a nanoparticle-mediated sustained release strategy. By confining CHX within self-assembled PC nanoparticles, the system prevents the initial ‘burst release’ toxicity often observed with free CHX solutions.

Our results support this safety profile: The sustained release maintains an effective bactericidal concentration while remaining below the cytotoxic threshold for mammalian cells. This is evidenced locally by the accelerated re-epithelialization and reduced scar formation observed at Day 14 (Fig. 7), and systemically by the absence of histopathological abnormalities in major organs (Heart, Liver, Spleen, Lung, Kidney) (Fig. S12). Furthermore, the reduction in chronic inflammatory markers (e.g., TNF-α, iNOS) at the wound site (Fig. 6H-K) indicates that the released CHX did not induce chronic irritation or persistent inflammation. Thus, GAPC offers a safer alternative to the repeated application of high-concentration CHX disinfectants.

Study on the mechanism of GAPC hydrogel promoting healing in vitro

Fibroblasts are the main sources of the extracellular matrix (ECM), and especially during the proliferative and remodeling stages of wound healing, collagen synthesis and deposition are essential [74]. To explore more details of the mechanism by which the GAPC hydrogel enhanced wound healing, 3T3 cells (a mouse fibroblast cell line) were used to verify the in vitro results. One such marker is Ki67, which is a well-established marker of cell cycle progression and which is up-regulated in this setting, indicating that the hydrogel may promote tissue repair through activation of cell cycle-associated pathways [75]. Immunofluorescence analysis showed that GAPC1 treatment dramatically enhanced the percentage of Ki67-positive cells (Fig. 8A, Fig. D). In addition, fibroblast behaviors were directly evaluated in vitro using 3T3 cells treated with the 100% hydrogel extract stock solution. The CCK-8 assay (OD450) showed increased cell proliferative activity in the GAPC groups (Fig. 8G). Consistently, a scratch wound assay demonstrated accelerated wound closure after GAPC extract treatment (Fig. S20). Together, these data support that GAPC promotes fibroblast viability activity and migration, which may contribute to the improved wound repair observed in vivo. Furthermore, to determine the effect on cell migration, we analyzed the expression of Vimentin [76]. As shown in Fig. 8A and Fig. B, the fluorescence intensity of Vimentin was remarkably stronger in the GAPC1 group. Since Vimentin is a cytoskeletal component critical for mesenchymal cell motility, its significant upregulation (further verified by qRT-PCR in Fig. 8F) indicates that GAPC1 promotes the migratory potential of fibroblasts, facilitating their recruitment to the wound site for ECM synthesis. In contrast, the strong α-SMA fluorescence induced by TGF-β1 was substantially suppressed by GAPC1 treatment, returning to near-baseline levels (Fig. 8C). qRT-PCR analysis further confirmed these fluorescence-based observations (Fig. 8E). Together, these results indicate active cytoskeletal remodeling. But given the accompanying α-SMA downregulation, these results imply that cellular activity is shifted to tissue repair, not fibrotic remodeling. α-SMA, as a core marker of myofibroblast differentiation [77], was downregulated, providing direct evidence that GAPC1 hydrogel effectively inhibited TGF-β1-induced fibrotic transition.

Fig. 8 — GAPC hydrogel promotes healing in vitro. A) Immunofluorescence staining images of the Vimentin, α-SMA and Ki67 in the 3T3 cells (scale bar: 50 μm). B-C) Statistics of relative fluorescence intensities of Vimentin and α-SMA (n = 3). D) Quantitative statistics of the positive rate of Ki67 (n = 3). E-F) qRT-PCR analysis of mRNA levels of α-SMA and Vimentin in the 3T3 cells (n = 3). G) CCK-8 assay (OD 450 nm) for 3T3 cells. H) Immunofluorescence staining images of the Ki67 in the HUVEC cells (scale bar: 50 μm). I) Quantitative statistics of the positive rate of Ki67 in the HUVEC cells (n = 3). Data are presented as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001, ****p < 0.0001; ns, not significant. Statistical significance was determined by One-way ANOVA with Tukey’s post hoc test

As HUVECs could serve as vascular endothelial cells, “the increased cell density is indicative of activated angiogenesis [75]. In addition, the pro-angiogenesis ability of GAPC1 in vitro was further confirmed by staining HUVECs with an immunofluorescence assay (Fig. 8H). In all groups of cells, the GAPC1-treatment group showed the greatest percentage of Ki67-positive nuclei, suggesting a strong stimulation of the endothelial cell proliferation (Fig. 8I). Such a finding indicates that, by promoting angiogenesis, GAPC1 may contribute to wound healing and tissue regeneration.

To further validate the therapeutic potential on human cells, we performed additional assays using HDF-α. As shown in Fig. S15 (Supporting Information), GAPC1 treatment effectively promoted the proliferation of HDF-α cells. Moreover, the scratch assay confirmed that GAPC1 significantly accelerated the migration of human fibroblasts even under oxidative stress conditions (Fig. S14, Supporting Information). Combined with the data on HaCaT and HUVECs, these results demonstrate the hydrogel’s broad efficacy across major human skin cell types.

The therapeutic superiority of GAPC can be attributed to its unique synergistic mechanism compared to state-of-the-art wound dressings. First, in terms of safety and mechanism, unlike commercial silver-ion dressings that rely on heavy metal toxicity to kill bacteria-often at the cost of damaging regenerating tissues-the GAPC hydrogel employs a fully organic, metal-free system. This approach provides potent broad-spectrum antibacterial activity while maintaining high biocompatibility, as evidenced by the well-preserved epidermal structures in our study. Second, regarding immunomodulation, while standard dressings merely act as passive physical barriers, GAPC actively creates a pro-regenerative microenvironment. By scavenging ROS and releasing anti-inflammatory polyphenols, it mechanically and chemically promotes the phenotype switch of macrophages from pro-inflammatory M1 to pro-healing M2. Finally, structurally, the dual-network strategy confers GAPC with mechanical properties (lap shear strength ~12 kPa) and adhesion superior to pure ADM or GelMA hydrogels, ensuring structural integrity in dynamic wound sites. Collectively, these comparative advantages highlight GAPC as a promising translational candidate for managing complex infected burn wounds. A comparative overview of GAPC and representative wound dressings is provided in Table 1.

Table 1.

Comparison of GAPC hydrogel with other representative wound dressings

Feature	GAPC Hydrogel (This Work)	Traditional ADM Scaffolds	Pure GelMA Hydrogels	Commercial Ag-based Dressings
Composition	Dual-Network (GelMA + ADM)	Single Component (Decellularized Matrix)	Single Network (Methacrylated Gelatin)	Synthetic/Cotton base + Silver ions
Mechanical Properties	Tunable & Self-healing (Double crosslinking)	Poor/Fixed mechanical strength	Brittle, lacks self-healing unless modified	Passive barrier, non-structural
Antibacterial Agent	PC Nanoparticles (Organic, Sustained Release)	None (unless loaded)	None (unless loaded)	Silver Ions (Burst release)
Biocompatibility	High (Metal-free, supports cell growth)	High (Native ECM)	High	Moderate/Low (Potential Ag+ cytotoxicity)
ROS Scavenging	High (Intrinsic PA activity)	Low/None	Low/None	Low/None
Immunomodulation	Active (Promotes M1→M2 transition)	Passive (Low immunogenicity)	Passive	Passive (Anti-infection only)
Bioactivity	Rich ECM cues (Col I, GAGs from ADM)	Rich ECM cues	Limited (Gelatin only)	None

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Conclusions

In conclusion, we have developed a multifunctional hydrogel-nanoparticle composite (GAPC) that unites antibacterial, antioxidative, and immunomodulatory activities within a single platform. The dual-network architecture endows the material with robust mechanical integrity and self-healing capability, while the embedded PC nanoparticles provide sustained ROS scavenging and bactericidal effects. Mechanistically, GAPC fosters a regenerative microenvironment by promoting M2 macrophage polarization, attenuating inflammation, and enhancing collagen remodeling and angiogenesis. Owing to its combined physicochemical stability and biological efficacy, GAPC offers a promising and translationally relevant strategy for managing complex infectious wounds.

Experimental section

Materials and chemicals

PA, CHX, hydrogen peroxide (H₂O₂), and dimethyl sulfoxide (DMSO) were purchased from Maclin (Shanghai, China). GelMA was purchased from the Engineering for Life company (Hangzhou, China). Lipopolysaccharide (LPS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2’-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) were purchased from Aladdin (Shanghai, China). CCK8 kit, Calcein-AM, FITC-phalloidin, DCFH-DA probe, propidium iodide (PI), 4’,6-diamidino-2-phenylindole (DAPI), and propidium iodide (cell viability kit) were purchased from Biosharp (Beijing, China). The cell medium and PBS were purchased from Gibco (California, USA). HaCaT, HDF-α, and HUVEC cells were purchased from Suzhou Haixing Biotechnology. Raw264.7 cells were purchased from Wuhan Procell Life Science Technology, and 3T3 cells were kindly provided by Prof. Pengyuan Liu’s group (Zhejiang University). Human red blood cells (RBCs) were purchased from Huizhi Heyuan Biotechnology (Suzhou, China). APC anti-mouse CD86 antibody and PE anti-mouse CD206/MMR antibody were purchased from Elabscience (Wuhan, China). Other antibodies were purchased from Proteintech (Wuhan, China). All other reagents were sourced from different local suppliers and used without modification.

Synthesis of PC nanoparticles

First, 100 mg CHX was dissolved in 20 mL of DDW. Then, 200 mg PA was mixed into a solution of 50 mL of ethanol and 80 mL of DDW. After sonication for 10 min at room temperature, the resulting mixture was heated to 75 °C and stirred for 12 h. The mixture was then separated by centrifugation (5000 g, 15 min). The precipitate was washed with water and ethanol. The resulting PC nanoparticles were eventually resuspended in water to a concentration of 2 mg/mL, making them suitable for future applications.

Synthesis of ADM

Porcine dermal tissue is sourced from a compliant adult Yorkshire pig (female, 10–15 months old). The dermal layer of pig skin is obtained by mechanically separating the epidermis, subcutaneous fascia, and connective tissue. Dermal fragments were decellularized through the following steps: (1) 0.25% trypsin for 6 h, followed by three washes with deionized water for 15 min each; (2) incubation in 70% ethanol for 10 h; (3) treatment with 3% hydrogen peroxide for 15 min, then washed twice with deionized water for 15 min each; and (4) incubation in a solution of 1% Triton X-100, 0.26% EDTA, and 0.69% Tris for 22 h. After decellularization, the ADM was rinsed with PBS and deionized water three times, separately. The ADM tissue was then lyophilized until completely dry.

Subsequently, the lyophilized ADM is crushed into 1–2 mm³. Then add 10 mg/mL (dry weight) and 1 mg/mL pepsin to a 0.01 M HCl solution and digest with continuous agitation at room temperature for 48 h. Undigested residues were separated by centrifugation (5,000 g, 4 °C, 10 min). This digestate is stored at 4 °C until it is used for subsequent experiments. Neutralize and dilute the digestate to 10 mg/mL with 0.1 M NaOH, 10 × PBS, and digestion on ice. The neutralizing digestive fluid at this time is called a pre-gel. The C is then incubated in an incubator at 37 °C for 10 min, after which the ADM hydrogel is formed.

Fabrication of GAPC

PC nanoparticles were initially dispersed in water and subsequently combined with a 10% (w/v) solution of ADM pre-gel and a 10% (w/v) solution of GelMA. The mixture was vortexed and supplemented with 0.04% (w/v) LAP photoinitiator, followed by thorough mixing. It was then transferred into a mold. Hydrogel formation was achieved under visible light (405 nm, 10 mW/cm² for 30 s), which initiated crosslinking. Four types of hydrogels were prepared with different concentrations of PC nanoparticles: 0, 0.25, 0.5, and 1 mg/mL. These were named GAPC0, GAPC0.25, GAPC0.5, and GAPC1, respectively.

The specific compositions and nomenclature for all hydrogel formulations prepared in this study are summarized in Table 2. Throughout the manuscript, the term ‘GAPC’ refers to the optimized GAPC1 formulation unless otherwise specified.

Table 2.

Nomenclature and composition of the hydrogel formulations

Abbreviation	Description	Components & Final Concentrations	Role in Study
PC	Nanoparticles	Self-assembled Proanthocyanidins (PA) and Chlorhexidine Acetate (CHX)	Bioactive antibacterial & antioxidant agent
APC	Precursor Mixture	ADM pre-gel (10% w/v) + PC nanoparticles	Intermediate mixture before photo-crosslinking
GAPC0	Control Hydrogel	GelMA (10% w/v) + ADM (10% w/v) + 0 mg/mL PC	Negative control (Scaffold only)
GAPC0.25	Low-dose Hydrogel	GelMA (10% w/v) + ADM (10% w/v) + 0.25 mg/mL PC	Formulation optimization
GAPC0.5	Medium-dose Hydrogel	GelMA (10% w/v) + ADM (10% w/v) + 0.5 mg/mL PC	Formulation optimization
GAPC1	Optimal Hydrogel	GelMA (10% w/v) + ADM (10% w/v) + 1.0 mg/mL PC	Key formulation used for all detailed biological and in vivo assays
GAPC	General Term	Refers to the GAPC hydrogel system in general; unless specified, implies the optimized GAPC1	General discussion of the material system

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Preparation of hydrogel extracts: To evaluate cytotoxicity and in vitro bioactivity, hydrogel extracts were prepared according to ISO 10993-12 (Biological evaluation of medical devices: Sample preparation and reference materials). Briefly, photocrosslinked hydrogels (GAPC0, GAPC1, etc.) were weighed and immersed in complete culture medium (DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin) at a mass-to-volume ratio of 0.1 g/mL (e.g., 0.5 g hydrogel in 5 mL medium). The mixtures were incubated at 37 °C for 24 h under gentle agitation. After incubation, the supernatants were collected and sterilized by filtration through a 0.22 μm syringe filter. The resulting sterile supernatant was defined as the 100% hydrogel extract stock solution. For cell-based assays, unless otherwise specified, cells were treated with the extract at 10% (v/v) (100 µL/mL), unless otherwise stated. For antibacterial assays, the 100% extract stock was added to bacterial suspensions at a final proportion of 10% (v/v) (i.e., 100 µL extract per mL bacterial suspension), unless otherwise stated. The blank control consisted of corresponding media processed under identical incubation and filtration conditions.

Physicochemical characterization

Transmission electron microscopy (TEM) images were obtained using an HT-7700 TEM (Japan). Morphology and porous structure were analyzed using a Hitachi SU-8010 scanning electron microscope (SEM, Japan) at 10 kV. Zeta potential and particle size distribution were measured with a Zetasizer Nano-ZS instrument (ZEN3600, UK). UV-vis absorption spectra were recorded on a CARY5000 micro-spectrophotometer (USA) with a 1 cm quartz cell. Fourier transform infrared spectroscopy (FTIR) was performed on a Nicolet iS50 (USA), scanning samples across 400–4000 cm⁻¹.

Rheological test of the GAPC hydrogels

Rheological properties of GAPC hydrogels were determined using a rotary rheometer, MARS 60 (HAAKE, Germany). Determination of mechanical properties of GAPC hydrogel by Zwick/Roell Z020 (Zwick, Germany). The hydrogel specimens (8 mm diameter, 8 mm height) were subjected to compression at 10 mm/min. A strain sweep test (0.1–1000% strain at 37 °C) was first performed to identify the linear viscoelastic region. Frequency sweeps (0.1–10 Hz at 37 °C) were then used to assess storage modulus (G′) and loss modulus (G″). Dynamic time sweeps were performed at 1% strain and 0.1 Hz frequency to ascertain gelation time, while self-healing capability was assessed through stepped strain amplitude cycles alternating between 1% and 500%. Finally, shear-thinning behavior was examined by measuring viscosity over shear rates from 0.1 s⁻¹ to 100 s⁻¹.

Swelling and water retention tests

The swelling ratio of GAPC hydrogels was calculated by monitoring changes in wet weight. Freeze-dried hydrogels were immersed in 10 mL PBS at 37 °C, removed at desired time points, patted dry, and weighed. The swelling ratio was calculated as Wt/W0, where Wt and W0 are the weights of the swollen and initial samples, respectively. Water uptake was determined by immersion of freeze-dried hydrogels in PBS to equilibrium swelling. The specimens were transferred to an oven at 37 °C, removed periodically, drained, and weighed. The retention was calculated by the following equation: (Ms/Md) × 100%, where Ms is the weight of the sample after partial dehydration and Md is the weight of the raw sample.

Biocompatibility evaluation of GAPC hydrogels

The cytotoxicity of GAPC hydrogels was evaluated through CCK-8. HaCaT cells (5 × 10³ cells/well) were cultivated in 96-well plates and cultured for 24 h, followed by adding the hydrogel extract (100 µL/mL, v/v). At days 1, 2, and 3, 10 µL of CCK-8 solution was added to each well and incubated for 2 h, and 100 µL of medium was then transferred to a 96-well plate, with the absorbance at 450 nm measured using a microplate reader.

Similarly, live/dead staining was analyzed for the ADSC cells, HaCaT cells, and HUVEC cells cultured on confocal dish (3 × 10⁴ cells/disc) containing hydrogel extracts. The cells were treated with Calcein-AM and PI at 37 °C in the dark for 20 min, and then the fluorescence images were acquired using a Leica STELLARIS 5 confocal microscope.

A hemolysis test was also conducted to assess hemocompatibility of GAPC hydrogels. Mouse blood and commercial hRBCs were drawn into anticoagulant-containing tubes. The RBC were separated by centrifugation at 1000 g for 10 min and washed with PBS 3 times until the supernatant became clear. Then resuspended in PBS to obtain a 2% (v/v) suspension.

Hydrogel extract (0.5 mL) and 0.5 mL suspension were added to 1.5 mL tubes and incubated at 37 °C for 60 min. The negative control was PBS, and the positive control was double-distilled water (DDW). Following incubation of samples, they were spun for 8 min at 1100 g. The supernatant (0.1 mL) was collected into a 96-well plate, and optical density was read at 540 nm. The hemolysis ratio was calculated as follows: Hemolysis = [(Ak- Ai)/(Ao- Ai)] × 100% where Ak is the absorbance of the test sample, Ao is the absorbance of the DDW control, and Ai is the absorbance of the PBS control.

Antioxidant capacity evaluation of GAPC hydrogels

Antioxidant capacity was first assessed using the DPPH assay.

Given the different physical states of the samples, two specific protocols were employed: (1) For PC Nanoparticles (Liquid Phase Assay): A concentrated 1 mM DPPH stock solution was prepared in ethanol. The reaction system (total volume: 1 mL) consisted of 900 µL of deionized water and 100 µL of the DPPH stock solution. Different masses of PC nanoparticles were dispersed into the system to achieve final concentrations of 0.25, 0.5, and 1.0 mg/mL. Pure PA (1.0 mg/mL) was used as the positive control. The mixture was incubated in the dark for 30 min. Subsequently, the samples were centrifuged to remove precipitates/nanoparticles. The supernatant was photographed, and the absorbance was measured at 519 nm. (2) For Hydrogels (Solid Phase Assay): A standard 0.1 mM DPPH solution was prepared in ethanol. Cylindrical hydrogel samples from different groups (GelMA, APC, GAPC, and PA-loaded controls) were prepared and immersed individually into 2 mL of the 0.1 mM DPPH solution. The samples were incubated in the dark for 1 h. The gross appearance of the solution was photographed to visualize the color change (from purple to yellow). The absorbance of the supernatant was measured at 519 nm to quantify the scavenging efficiency.

Scavenging (%) = [1−(A_sample/A_control)] × 100, where A_sample is the absorbance of the test group and A_control is the absorbance of the DPPH solution treated with solvent only.

Intracellular ROS assay (DCFH-DA). To assess the ability of hydrogels to reduce intracellular ROS, the DCFH-DA assay was performed. HaCaT cells (2 × 10⁴ per well) were seeded in confocal dishes and cultured for 72 h. Cells were then treated with DMEM (10% FBS) containing hydrogel extract (100 µL/mL, v/v) and H₂O₂ (650 µM) for 6 h. Untreated cells served as the negative control, and cells treated with H₂O₂ only served as the positive control. After treatment, cells were incubated with DCFH-DA (1 µL/mL in serum-free medium) for 30 min; HaCaT cells were additionally stained with Hoechst for 30 min. ROS levels were visualized with a confocal laser scanning microscope (CLSM), and fluorescence intensity was quantified using ImageJ. Parallel samples were analyzed with a Beckman flow cytometer (USA) for quantitative ROS detection.

In vitro tube formation assay. To evaluate pro-angiogenic effects an in vitro tube formation assay was conducted. A 10 µL well of Matrigel (Shanghai, China) was added to a 96-well plate that was pre-coated at 4 °C and first allowed to solidify at 37 °C, and then human umbilical vein endothelial cells (HUVECs, 4 × 10⁴ cells/well) were plated at a final concentration of 10⁵ cells/mL. Then, GAPC0 or GAPC1 hydrogel extract medium, together with a final concentration of 100 µM H₂O_2, was added. Tube formation was observed by microscopy after 12 h incubation, and the mesh structure was counted by ImageJ.

Cellular antioxidant evaluation. For additional assessment of antioxidant activity, HaCaT cells, and RAW 264.7 were seeded on 96-well plates at a density of 4 × 10³ cells/well and incubated for 24 h The culture medium was removed, and cells were treated with a 300 µM H₂O₂ solution containing the nanoparticles or hydrogels extracts. 6 h later, following incubation at 37 °C in the dark, the cell viability was detected by the CCK-8 assay.

Cell migration assessment: Epidermal cell migration was analyzed using a scratch assay. HaCaT, HDF-α, and 3T3 cells were used as experimental cells. To be brief, cells (6 × 10⁵ cells) were first inoculated into 12-well plates and cultured for 24 h until 90% confluence was reached. Subsequently, a 200 µL pipette tip was used to form a uniform scratch on the cell layer at the bottom of the plate. After washing twice with PBS, and the remaining cells were photographed. To minimize the influence of proliferation, the scratch assay was performed under low-serum conditions (1% FBS). Subsequently, GAPC0 or GAPC1 hydrogel extract medium was added, and a final concentration of 100 µM H₂O₂ was added to each well and photographed under a microscope after 24 and 48 h of incubation. The migration of cells was analyzed using ImageJ software, and the formula was calculated as scratch Healing = (S₀-S_t)/S₀ × 100%, where S₀ represents the initial scratch area, and S_t represents the scratch area after 24 and 48 h. Measurements were taken in triplicate for each group.

In vitro antibacterial assay

The antibacterial properties of GAPC hydrogels were evaluated against S. aureus and E. coli by using a disk diffusion assay. Bacterial suspensions were diluted to 1 × 10³ CFU/mL, and 100 µL aliquots were plated on Luria-Bertani (LB) agar. Sterile paper disks (10 mm diameter) were overlaid with 25 µL of different samples (PBS, CHX solution (0.1 mg/mL), PA solution (0.5 mg/mL)). For the ADM and GAPC groups, sterile solid hydrogel discs were punched into cylinders with a diameter of 10 mm (matching the paper disk size) and placed directly onto the agar surface. Following full adsorption, the disks were put on LB agar plates that had been inoculated and incubated for 12 h at 37 °C. The diameters of the inhibition zones were measured and captured on camera.

Bacterial growth assay: S. aureus and E. coli suspensions were diluted to concentrations of 10³, 10⁴, 10⁵, and 10⁶ CFU/mL, and cultured at 37 °C for 24 h in LB with 100 µL/mL GAPC extract or PBS. After incubation, 100 µL of each culture was plated onto LB agar and left at 37 °C for 48 h. CFUs were measured, and colonies were photographed.

Determination of bacterial dead and alive staining: bacterial dead and alive staining was determined by centrifuging 200 µL of bacterial solution to extract the supernatant, adding 100 µL of Calcein-AM and PI for live and dead staining, shaking the mixture for 1–2 h at 37 °C, washing it twice with PBS, resuspending it, dropping 20 µL onto the slide, and taking pictures with a fluorescence microscope.

Bacterial scanning electron microscopy detection: To assess the damage to the bacteria, the microscopic morphology of the bacteria was examined using a scanning electron microscope (SU-8010, Hitachi, Japan) set to 3 kV.

Anti-inflammation assay

Cell culture: RAW 264.7 macrophages were seeded at a density of 1 × 10⁶ cells per 6-cm dish and allowed to adhere for 24 h before treatment. The groups were defined as follows: M2 (IL-4, 50 ng/mL), M1 (LPS 500 ng/mL), GAPC0 + LPS (500 ng/mL), and GAPC1 + LPS (500 ng/mL).

Immunofluorescence: RAW 264.7 macrophages were seeded in confocal dishes and cultured for 24 h. To induce M1 polarization, cells were stimulated with LPS (500 ng/mL) for 24 h. The medium was then replaced with fresh complete medium prepared with 100% hydrogel extract stock (GAPC0 or GAPC1; prepared at 0.1 g/mL according to ISO 10993-12) with LPS maintained at 500 ng/mL, and cells were incubated for an additional 48 h (total 72 h). The M1 control group received LPS (500 ng/mL) for 72 h, while the M2 reference group was treated with IL-4 (50 ng/mL) for 72 h. Cells were then fixed and stained for CD86, CD206, iNOS, and Arg-1, which were diluted in blocking buffers at 4 °C for 12 h and subsequently conjugated with FITC-labeled secondary antibodies for 30 min. Nuclei were counterstained with DAPI. Images were captured by confocal microscopy, and the fluorescence intensity was analyzed by ImageJ software.

Analysis of RT-qPCR: Macrophages were collected for gene expression analysis, and the total RNA was isolated by TRIzol reagent. The RNA was converted to cDNA and assessed for iNOS, IL-6, TNF-α, Arg-1, IL-10, and IL-4 expression via RT-qPCR. The primer sequences are listed in Table S1 (Supplementary Information).

Western blotting: Cells were lysed with 120 µL of RIPA buffer (Beyotime Biotechnology) with protease inhibitor on day 3 after treatment. Lysates were incubated on ice and then spun for 20 min. The protein concentrations were measured by BCA assay, and the proteins were normalized and mixed with the SDS-PAGE loading buffer (Beyotime Biotechnology). The samples were denatured at 100 °C for 9 min and stored at −20 °C. After denaturation, equal amounts of protein were loaded on prefabricated gels along with 8 µL of a protein ladder (Epizyme Biomedical Technology, Shanghai, China) and separated by electrophoresis. Following electrophoresis, the proteins were transferred to PVDF membranes (BIO-RAD, USA) at 300 mA for 60 min. After 2 h blocking with 5% skim milk (Beyotime Biotechnology) at room temperature, membranes were incubated with primary antibodies at 4 °C overnight. Following washes, the membranes were incubated with HRP-coupled secondary antibodies for 1 h at 25 °C. Bands were visualized using chemiluminescent HRP substrate and imaged with a biomolecular imaging system.

Flow cytometry: Cells from the four experimental groups were filtered to obtain a uniform single-cell suspension. The cells were then co-stained with the antibodies CD86 and CD206 (BioLegend). Staining was carried out for 30 min at 4 °C, with protection from light. After staining, cells were washed with 1× PBS to remove unbound antibodies. Finally, the prepared cells were analyzed using flow cytometry.

Animal experiments

All animal experimental protocols were approved by the Ethics Committee for Animal Use of Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University (Protocol number SRRSH20250212055). BALB/c male mice, 8 weeks of age, were depilated with depilatory creams.

A post-debridement infected wound model was established to mimic clinical scenarios. (1) Burn Induction (Day −3): Third-degree burns were created on the dorsal skin of mice using a heated brass rod (90 °C, 15 s). The wounds were allowed to dry and form an eschar for 3 days. (2) Debridement and Inoculation (Day 0): On Day 0, the burn eschar was surgically excised to simulate debridement. Subsequently, 10 µL of S. aureus suspension (1 × 10⁸ CFU/mL) was inoculated onto the debrided wound bed. (3) Treatment (Day 0): The inoculated wounds were covered with Tegaderm^™ for 12 h to facilitate bacterial colonization. Afterwards, the mice were randomly divided into groups. The sterile hydrogels (GAPC0, GAPC1, etc.) were applied to cover the wound area. The dressings were secured with Tegaderm^™ and cohesive bandages, and changed every two days.

Wound contraction rate: Wound healing was observed and photographed at 0, 3, 7, 10, and 14 days. The wound area was determined using ImageJ software. Wound area rate (%) = At/A0 × 100%, where the original wound area was recorded as A0, and the wound area at the predetermined time points was recorded as At, respectively.

To study skin healing and inflammation, mice were euthanized on days 7 and 14 after treatment. The regenerated skin was collected, fixed in 4% paraformaldehyde for 60 min, and embedded in paraffin. The tissue was cut into 4 μm slices and stained with HE, Masson’s trichrome, and Picrosirius red. Inflammation and collagen around the wound were observed. For inflammation analysis in healed skin, day-7 tissue was stained with immunofluorescence for CD86, iNOS (M1 macrophages), Arg-1, and CD206 (M2 macrophages); day-1 tissue was stained with CD31and Ki67, and nuclei were stained with DAPI. The slides were examined with an inverted fluorescence microscope.

RT-qPCR Analysis: For gene expression analysis, mice’s blood from 7 days was harvested, and total RNA was extracted with TRIzol reagent. RNA was reverse-transcribed into cDNA, and expression levels of TNF-α, iNOS, Arg1, and IL-10 were quantified by RT-qPCR. Primer sequences are provided in Table S1 (Supplementary Information).

In vivo antimicrobial test: On day 14 after treatment, skin was taken from the wound site and immersed in 1 mL PBS. After 1000-fold dilution, the resulting soak solution was spread on an LB agar plate and incubated for 12 h at 37 °C to form observable colony units.

Statistical analysis

All quantitative data are presented as mean ± standard deviation (SD). Unless otherwise stated, n represents the number of independent biological replicates. For hydrogel-related assays, this implies the use of separately synthesized hydrogel batches for each replicate to account for synthesis variability. Prior to hypothesis testing, the normality of data distribution was assessed using the Shapiro-Wilk test. For normally distributed data, statistical significance was determined using One-way Analysis of Variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons between groups. Statistical analysis was performed using GraphPad Prism software (Version 9.0). Differences were considered statistically significant at p < 0.05. Significance levels are indicated as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. ‘ns’ denotes not statistically significant (p > 0.05).

Supplementary Information

Supplementary Material 1^{(85.3MB, docx)}

Acknowledgements

We thank Xiaoli Hong and Chao Bi from the Core Facilities, Zhejiang University School of Medicine, for their technical support. We also thank Prof. Pengyuan Liu’s group at Zhejiang University for kindly providing the 3T3 cells.

Abbreviations

ABTS: 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
ADM: Acellular dermal matrix
ADSCs: Adipose-derived stem cells
ANOVA: Analysis of variance
APC: ADM with PA/CHX nanoparticles
Arg-1: Arginase-1
α-SMA: Alpha-smooth muscle actin
BALB/c: BALB/c mouse strain
BCA: Bicinchoninic acid (assay)
CCK-8: Cell counting kit-8
CD31: Cluster of differentiation 31
CD86: Cluster of differentiation 86
CD206: Cluster of differentiation 206
CFU: Colony-forming unit
CHX: Chlorhexidine acetate
CLSM: Confocal laser scanning microscopy
Col I: Type I collagen
Col III: Type III collagen
DAPI: 4′,6-diamidino-2-phenylindole
DCFH-DA: 2′,7′-dichlorodihydrofluorescein diacetate
DDW: Double-distilled water
DMEM: Dulbecco’s modified eagle’s medium
DPPH: 2,2-diphenyl-1-picrylhydrazyl
dsDNA: Double-stranded DNA
ECM: Extracellular matrix
EDTA: Ethylenediaminetetraacetic acid
EDX: Energy-dispersive X-ray spectroscopy
F-actin: Filamentous actin
FBS: Fetal bovine serum
FTIR: Fourier transform infrared spectroscopy
GAG: Glycosaminoglycan(s)
GAPC: GelMA + APC (composite hydrogel)
GAPC0: GAPC without PC
GAPC1: GAPC with PC (1 mg/ml)
H&E: Hematoxylin and eosin
HaCaT: Human immortalized keratinocyte cell line
HDF-α: Human dermal fibroblasts, adult
HUVECs: Human umbilical vein endothelial cells
hRBCs: Human red blood cells
IF: Immunofluorescence
IL-4: Interleukin 4
IL-6: Interleukin 6
IL-10: Interleukin 10
iNOS: Inducible nitric oxide synthase
LB: Luria–Bertani medium
LPS: Lipopolysaccharide
MFI: Mean fluorescence intensity
NF-κB: Nuclear factor kappa B
OD: Optical density
PA: Proanthocyanidins
PBS: Phosphate-buffered saline
PC: PA/CHX nanoparticles
PI: Propidium iodide
qRT-PCR: Quantitative real-time polymerase chain reaction
ROS: Reactive oxygen species
SEM: Scanning electron microscopy
TEM: Transmission electron microscopy
TNF-α: Tumor necrosis factor alpha
UV-vis: Ultraviolet–visible (spectroscopy)

Author contributions

Authorship Contribution StatementX.-S. Zhao conceived and designed the study, performed data curation, formal analysis, and investigation, developed the methodology, managed the project, and provided resources. He also carried out software analysis, supervision, validation, visualization, and wrote the original draft of the manuscript.L.-P. Zhang contributed to investigation and methodology development, participated in project administration, provided resources, and assisted with software analysis and supervision.G. Wang was involved in methodology development, project administration, and software analysis, and contributed to validation and visualization of the data.Q.-Y. Yang, X.-J. Cheng, C.-Y. Wu, D. Wu, C.-B. Dong, and Y.-T. Yang contributed to software analysis and writing of the original draft.Y.-F. Wang, X.-J. Cai, and N.-W. Zhu provided resources, were responsible for project administration and funding acquisition, and contributed to writing—review and editing of the manuscript.All authors reviewed and approved the final version of the manuscript.

Funding

This work was supported by the Science and Technology Talent and Platform Plan Fund Project of Yunnan Province (202305AF150131), the National Key R&D Program of China(2021YFA1101100), the National Natural Science Foundation of China (82172218) and the National Key Research and Development Plan Young Scientists Program (2021YFA1102100).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All animal experimental protocols were approved by the Ethics Committee for Animal Use of Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University (Protocol number SRRSH20250212055). Animal experiments were conducted in accordance with the Guidelines for Animal Care and Use Committee of Zhejiang University.

Consent for publication

All authors of this study agreed to publish.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Xian-Sheng Zhao and Li-Ping Zhang contributed equally to this work.

Contributor Information

Yi-Fan Wang, Email: anwyf@zju.edu.cn.

Xiu-Jun Cai, Email: cxjzu@hotmail.com.

Ning-Wen Zhu, Email: zhuningwen@fudan.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1^{(85.3MB, docx)}

Data Availability Statement

No datasets were generated or analysed during the current study.

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PERMALINK

An immunomodulatory hydrogel featuring antibacterial and ROS-scavenging functions for enhanced burn wound healing

Xian-Sheng Zhao

Li-Ping Zhang

Geng Wang

Qu-Yang Yang

Xiu-Jun Cheng

Chang-Yue Wu

Dan Wu

Can-Bin Dong

Yu-Tian Yang

Yi-Fan Wang

Xiu-Jun Cai

Ning-Wen Zhu

Abstract

Graphical Abstract

Supplementary Information

Background

Scheme 1.

Results

Physicochemical analysis of PC

Fig. 1.

Preparation and characterization of the GAPC hydrogel

Fig. 2.

Biocompatibility testing and antioxidant capacity of GAPC in vitro

Fig. 3.

Antibacterial capacity of CHX, PA, ADM, and GAPC in vitro

Fig. 4.

Inflammatory modulation capacity of GAPC in vitro

Fig. 5.

In vivo deep burn wound healing promoting efficacy of GAPC

Fig. 6.

Fig. 7.

GAPC hydrogel antibacterial, anti-inflammatory, and vascular-promoting in vivo properties

Study on the mechanism of GAPC hydrogel promoting healing in vitro

Fig. 8.

Table 1.

Conclusions

Experimental section

Materials and chemicals

Synthesis of PC nanoparticles

Synthesis of ADM

Fabrication of GAPC

Table 2.

Physicochemical characterization

Rheological test of the GAPC hydrogels

Swelling and water retention tests

Biocompatibility evaluation of GAPC hydrogels

Antioxidant capacity evaluation of GAPC hydrogels

In vitro antibacterial assay

Anti-inflammation assay

Animal experiments

Statistical analysis

Supplementary Information

Acknowledgements

Abbreviations

Author contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes

Contributor Information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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