A microenvironment-adaptive bilayer composite dressing for disrupting MRSA biofilms and promoting wound regeneration via ROS-mediated immune regulation

Jianan Li; Zhongwu Bei; Jian Hua; Meng Wang; Yujia Wei; Ying Qu; Bingyang Chu; Yun Yang; Dong Mo; Shiyu Liang; Xicheng Li; Qingya Liu; Meng Pan; Yutong Qian; Xiaorui Yu; Zhiyong Qian; Xiang Gao; Yongzhong Cheng

doi:10.1016/j.bioactmat.2026.05.013

. 2026 May 16;64:564–588. doi: 10.1016/j.bioactmat.2026.05.013

A microenvironment-adaptive bilayer composite dressing for disrupting MRSA biofilms and promoting wound regeneration via ROS-mediated immune regulation

Jianan Li ^a,^b,¹, Zhongwu Bei ^b,¹, Jian Hua ^c, Meng Wang ^b, Yujia Wei ^b, Ying Qu ^d, Bingyang Chu ^b, Yun Yang ^e, Dong Mo ^f, Shiyu Liang ^g, Xicheng Li ^b, Qingya Liu ^h, Meng Pan ^b, Yutong Qian ^b, Xiaorui Yu ^b, Zhiyong Qian ^b, Xiang Gao ^a,^⁎, Yongzhong Cheng ^a,^⁎⁎

PMCID: PMC13200051 PMID: 42199388

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) biofilm-infected wounds remain difficult to treat because persistent biofilm protection, oxidative stress imbalance, and unresolved inflammation jointly hinder tissue repair. Here, we developed a microenvironment-adaptive bilayer composite dressing (OQT/P) by integrating a pH-responsive copper-based nanozyme into a dynamically crosslinked hydrogel and coupling it with an outer electrospun fibrous membrane. This asymmetric structure enables stage-specific regulation of the infected wound microenvironment. In the mildly acidic infection phase, the embedded nanozyme promotes localized reactive oxygen species (ROS) generation to disrupt MRSA biofilms and enhance antibacterial efficacy. As the wound environment gradually returns toward neutrality, the system shifts toward ROS scavenging, thereby alleviating oxidative stress and suppressing inflammatory amplification. In vitro, OQT/P exhibited favorable interfacial stability, pronounced antibacterial and antibiofilm activity, good cytocompatibility, and pro-angiogenic potential. In a full-thickness MRSA biofilm-infected wound model, OQT/P markedly reduced bacterial burden and ROS accumulation, accelerated wound contraction, and achieved approximately 98.07 ± 0.90% wound closure by day 12. Histological and immunofluorescence analyses further demonstrated attenuated inflammation, enhanced collagen deposition, improved neovascularization, and more advanced tissue remodeling. Transcriptomic profiling, supported by ELISA and Western blot validation, showed that these therapeutic effects were associated with coordinated suppression of infection- and inflammation-related pathways, particularly the NF-κB, TNF, and Th17 axes, together with promotion of a repair-associated immune phenotype. Overall, this study presents a non-antibiotic strategy for MRSA biofilm-infected wounds and demonstrates the therapeutic potential of combining bilayer dressing architecture with dynamic redox regulation for infection control and regenerative repair.

Keywords: Bilayer composite dressing, pH-responsive regulation, MRSA biofilm, Microenvironment-adaptive materials, Infected wound healing

Graphical abstract

Highlights

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Bilayer dressing coordinates antibiofilm and immunomodulatory functions adaptively.
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pH-responsive nanozyme generates and then scavenges ROS to match wound phase.
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This approach suppresses inflammatory pathways to promote angiogenesis and tissue regeneration.

1. Introduction

Chronic infected wounds, particularly those complicated by methicillin-resistant Staphylococcus aureus (MRSA) biofilms, remain a substantial clinical burden [1,2]. Unlike acute wounds, MRSA biofilms form dense and stable extracellular polymeric matrices that exhibit pronounced tolerance to antibiotics, host immune defenses, and conventional wound dressings, rendering standard therapeutic strategies largely ineffective [[3], [4], [5]]. As a result, such wounds are often characterized by persistent bacterial colonization, prolonged inflammatory dysregulation, and severely delayed or arrested healing, ultimately leading to high recurrence rates and poor clinical outcomes [[6], [7], [8], [9]].

In MRSA biofilm-infected wounds, the pathological microenvironment undergoes highly dynamic evolution, in which the temporal variations of local pH and oxidative stress play pivotal roles in both infection control and tissue repair. During the early infection stage, enhanced bacterial metabolism and inflammatory responses typically drive the wound microenvironment toward mild acidity (pH 6.0–6.8), accompanied by elevated levels of reactive oxygen species (ROS). At this stage, moderate ROS generation constitutes an essential component of innate immune defense, contributing to bacterial suppression and partial disruption of biofilm structures. However, owing to the protective biofilm matrix, host-derived ROS are spatially dispersed and transient, failing to establish sustained and concentrated bactericidal activity within the biofilm. Instead, ROS preferentially accumulate in surrounding tissues, inducing bystander oxidative damage and amplifying inflammatory signaling. These observations suggest that the limited antibacterial efficacy of ROS in infected wounds arises not from their intrinsic inefficiency, but from insufficient spatial and temporal regulation [10].

As infection becomes progressively controlled, the wound microenvironment transitions toward the healing phase, accompanied by a gradual return of local pH to near-neutral values (pH 7.2–7.4). Under these conditions, persistently elevated ROS no longer confer protective benefits but instead impede tissue regeneration by damaging newly formed tissues and endothelial function, maintaining pro-inflammatory macrophage polarization, and suppressing angiogenesis and extracellular matrix remodeling. Excessive oxidative stress is therefore recognized as a key barrier preventing the orderly transition from the inflammatory to the proliferative phase of wound healing. These considerations underscore the need for microenvironment-responsive therapeutic strategies that enable stage-adaptive switching of ROS functions between antibacterial defense and tissue repair [11].

Accordingly, increasing efforts have been directed toward regulating ROS during wound healing through antioxidant biomaterials, ROS-responsive hydrogels, and catalytic nanozyme-based platforms. These approaches underscore the importance of stage-dependent redox regulation, where ROS-mediated antibacterial activity is desirable during early infection, while excessive ROS should be eliminated during the subsequent repair phase to mitigate oxidative damage and chronic inflammation. Nevertheless, most currently reported systems predominantly focus on only one side of ROS regulation, despite the fact that the pathological microenvironment of infected wounds undergoes continuous dynamic changes. This limitation highlights the need for therapeutic platforms that can sense microenvironmental evolution and adaptively switch ROS-related functions in a stage-dependent manner.

Within this context, noble metal- and transition metal–based nanozymes have emerged as promising candidates owing to their multienzyme-mimicking catalytic activities and intrinsic sensitivity to microenvironmental cues [12]. In acidic environments, proton enrichment stabilizes high-valence metal centers (e.g., Cu²⁺ or Fe³⁺) [13], facilitating electron acceptance and promoting H₂O₂/O₂ activation to generate highly oxidative ROS through Fenton-like reactions, thereby exhibiting peroxidase- or oxidase-like activities [14,15]. Conversely, under neutral or weakly alkaline conditions, metal centers shift toward lower valence states, enabling efficient ROS scavenging via dismutation or decomposition pathways and displaying superoxide dismutase- and catalase-like activities [16]. The coupling between metal valence states and local proton concentration provides a molecular basis for bidirectional ROS regulation using pH-responsive nanozymes [16]. However, effective utilization of this catalytic plasticity in infected wounds requires an appropriate carrier that can locally stabilize nanozymes while permitting dynamic interaction with the evolving wound microenvironment [17,18].

Meanwhile, bilayer or stratified wound dressings have attracted increasing attention for infected wound treatment, because they can better meet the stage-dependent functional requirements of wound healing while more closely mimicking the structural and functional asymmetry of native skin. In general, the outer layer mainly provides physical protection, barrier function, and anti-adhesion performance against external contamination, whereas the inner layer is responsible for maintaining intimate contact with the wound bed, managing exudates, and serving as a local depot for therapeutic bioactive components. Increasing evidence has shown that such layered systems can integrate antibacterial therapy, microenvironmental regulation, and tissue repair within a single platform, thus offering distinct advantages for the sequential management of infected wounds [19]. Nevertheless, studies that combine bilayer structural design with stage-adaptive bidirectional ROS regulation remain relatively limited.

Based on these considerations, we designed a microenvironment-adaptive bilayer composite dressing, in which a pH-responsive nanozyme is stably incorporated into a dynamically crosslinked hydrogel matrix and integrated with an outer electrospun fibrous layer to establish a hierarchical regulatory architecture (Scheme 1). The hydrogel is constructed from dopamine-modified oxidized dextran (ODEX-DA) and chlorogenic acid-functionalized chitosan (QCS-CGA), and further integrates a pH-responsive tannic acid–Cu–polymyxin B (TCP) nanozyme, enabling precise regulation of ROS dynamics. This design enables the dressing to respond to the evolving wound microenvironment in a stage-dependent manner. In mildly acidic biofilm niches, localized and controllable ROS generation is triggered, allowing preferential disruption of MRSA biofilms while compensating for the spatial and temporal limitations of host-derived ROS [20]. As the wound progresses into the healing phase and the local pH approaches neutrality, ROS production is adaptively suppressed and excessive oxidative stress is further scavenged, promoting immune microenvironment reprogramming from a pro-inflammatory to a pro-regenerative state that favors angiogenesis and tissue regeneration [21,22].

Scheme 1 — **Schematic illustration of the structural design of the OQT/P bilayer composite dressing and its stage-adaptive therapeutic mechanism for MRSA biofilm–infected wounds.** The outer electrospinning membrane provides a physical barrier and anti-adhesion effect to reduce bacterial invasion. The inner OQT hydrogel was tightly attached to the wound to achieve the sustained effect of functional components. In the acidic microenvironment at the early stage of infection, TCP nanoparticles showed peroxidase (POD)-/oxidase (OXD)-like activity and induced the production of ROS to achieve biofilm destruction and efficient sterilization. As the bacterial load decreases and the microenvironment tends to be neutral, TCP and polyphenol structure cooperate to remove excessive ROS and inhibit inflammatory amplification. This bidirectional ROS regulation further induces the polarization of macrophages from M1 to M2, promotes angiogenesis, collagen deposition, and tissue remodeling, and finally realizes high-quality regeneration and repair of infected wounds.

Furthermore, by integrating comprehensive in vitro and in vivo evaluations with transcriptomic analysis, we demonstrate that the therapeutic efficacy of this bilayer dressing is primarily mediated through systematic modulation of inflammation- and infection-related signaling pathways, including NF-kappa B (NF-κB), TNF, and Th17-associated axes, rather than direct activation of regenerative transcriptional programs. This microenvironment-guided, stage-adaptive regulation strategy provides a mechanistic foundation for high-quality tissue repair and offers a non-antibiotic material design paradigm for the treatment of biofilm-associated infected wounds.

2. Results and discussion

2.1. Synthesis and characterization of TCP

TCP nanoparticles were fabricated via a facile solution-based self-assembly strategy. Under alkaline conditions, the phenolic hydroxyl groups of tannic acid (TA) are deprotonated, which significantly enhances their coordination affinity toward Cu²⁺. Meanwhile, coordination interactions can also occur between Cu²⁺ and the amino functional groups of polymyxin B (PMB). In addition, partial oxidation and crosslinking of TA may be induced during this process. These cooperative interactions contribute to the formation of a stable organic–inorganic hybrid network serving as the structural “core” of TCP (Fig. 1a). The structure and composition of TCP were systematically characterized. Fourier-transform infrared (FTIR) spectra (Fig. 1b) show evident variations in characteristic absorption bands of TCP compared with those of pristine TA and PMB, indicating the establishment of strong intermolecular interactions among the components. Specifically, the absorption band around 1700 cm⁻¹, mainly attributed to aromatic C=C and carbonyl C=O vibrations of TA, exhibits noticeable attenuation and slight shifts after TCP formation, suggesting the direct involvement of carbonyl and phenolic groups in coordination with Cu²⁺. In addition, changes in the C–O stretching vibrations in the 1200–1000 cm⁻¹ region further support the occurrence of metal–phenolic coordination and/or partial oxidative crosslinking of TA molecules.

Transmission electron microscopy (TEM) images (Fig. 1c) revealed that TCP nanoparticles exhibited a quasi-spherical morphology with relatively uniform size distribution. Statistical analysis (Fig. 1d) showed that most particles were distributed in the range of 40–60 nm, with an average diameter of approximately 55 nm. Dynamic light scattering (DLS) measurements (Fig. 1e) further confirmed a relatively narrow hydrodynamic size distribution in aqueous solution. No obvious lattice fringes or well-defined crystalline planes were observed in the TEM images, indicating that the copper species were dispersed in an amorphous or low-crystallinity state within the organic framework, which is consistent with the broad diffraction features observed in the XRD pattern (Fig. 1f). Such an amorphous structure may expose more unsaturated coordination sites and structural defects, which could be beneficial for nanozyme-like catalytic activity. The XRD pattern (Fig. 1f) displayed only broad diffuse features without sharp crystalline reflections, further confirming the amorphous nature of TCP. X-ray photoelectron spectroscopy (XPS) survey spectra (Fig. 1g) revealed the presence of C, O, N, and Cu elements. In the high-resolution Cu 2p spectrum (Fig. 1h), the main peaks at approximately 934–935 eV together with the evident shake-up satellite peaks at around 940–945 eV are characteristic of Cu²⁺ species, indicating that copper predominantly exists in the divalent state. The C 1s spectrum (Fig. 1i) could be deconvoluted into C–C/C=C, C–O/C–N, and C=O components, while the O 1s spectrum (Fig. 1j) showed contributions from Cu–O, C=O, and C–O bonds, further confirming the formation of coordination interactions between Cu²⁺ ions and the polyphenolic framework [23]. In addition, SEM imaging and the corresponding EDS elemental mapping further verified the composition homogeneity of TCP nanoparticles (Fig. S1). The SEM image showed densely packed nanoparticulate morphology, while elemental mapping demonstrated that C, N, O, and Cu were uniformly distributed throughout the particles, indicating the successful and homogeneous incorporation of TA, PMB, and Cu²⁺ into the TCP nanostructure.

2.2. pH-dependent redox regulation behavior of TCP

To systematically evaluate the redox regulatory capability of TCP under different microenvironmental conditions, its ROS generation and scavenging behaviors were investigated under acidic and neutral pH, respectively. Under acidic conditions (pH 5.5), TCP exhibited pronounced ROS-generating activity, as evidenced by the dose-dependent degradation of MB in the presence of H₂O₂ (Fig. 1k). The progressive attenuation of the MB absorbance at 664 nm, accompanied by visible solution decolorization, indicates efficient catalytic activation of H₂O₂ and typical POD-like or Fenton-like activity. Further comparison of oxidation activity under different pH conditions shows that MB degradation is significantly more pronounced under acidic conditions than under neutral conditions (pH 7.2) within the same reaction system (Fig. 1l), demonstrating that the ROS generation capability of TCP is strongly pH-dependent. Specifically, the oxidative catalytic function is efficiently activated in weakly acidic environments, while it is markedly suppressed under neutral physiological conditions.

In contrast, under near-neutral conditions, TCP mainly exhibited ROS-scavenging activity. In the ox-TMB reduction assay, the absorbance of oxidized TMB gradually decreased with increasing TCP concentration, and the scavenging efficiency showed a clear concentration-dependent increase (Fig. 1m). Specifically, when the TCP concentration increased from 10 μg/mL to 200 μg/mL, the ox-TMB scavenging ratio rose from approximately 11.79 ± 2.00% to 58.14 ± 5.47%, indicating that TCP could effectively eliminate oxidative intermediates and reduce the overall oxidative level of the system [24]. Moreover, multiple free radical scavenging models, including ABTS and DPPH assays, were employed to further evaluate its antioxidant performance. Consistent results demonstrate that TCP exhibits strong scavenging ability toward different types of free radicals, with significantly enhanced efficiency at higher concentrations (Fig. 1n and Fig. S1), confirming its broad-spectrum and stable antioxidant activity [25]. TCP nanoparticles display a distinct pH-triggered redox switch: oxidative ROS generation dominates under acidic conditions characteristic of infected wounds, favoring antibacterial and antibiofilm activity, whereas ROS scavenging prevails under near-neutral conditions, contributing to redox homeostasis and protection of surrounding tissues during the healing phase. This bidirectional, microenvironment-responsive redox regulation underpins the stage-adaptive therapeutic function of TCP in infected wound management.

2.3. Preparation and characterization of QCS–CGA, ODEX–DA, and OQT hydrogels

¹H NMR spectroscopy was employed to verify the grafting of chlorogenic acid (CGA) onto the backbone of quaternized chitosan (QCS) (Fig. 2a). QCS exhibited characteristic polysaccharide proton signals at δ 3.0–4.0 ppm and a distinct resonance at δ ≈ 3.2 ppm corresponding to N⁺(CH₃)₃ groups, while CGA showed aromatic proton signals at δ 6.2–7.6 ppm. In QCS–CGA, both sets of signals were retained with slight downfield shifts and peak broadening of CGA resonances, indicating altered chemical environments and confirming covalent conjugation. FTIR spectra further supported the grafting reaction (Fig. 2b). The attenuation and slight shift of the C=O absorption of CGA (∼1720 cm⁻¹), together with the enhancement of aromatic C=C vibrations and the preservation of polysaccharide bands, suggest the involvement of carboxyl groups in the condensation reaction. UV–Vis spectroscopy showed characteristic CGA absorption bands in QCS–CGA with reduced intensity and broadened profiles (Fig. 2e), further confirming successful incorporation.

The structure of ODEX–DA was verified by ¹H NMR and FTIR analyses (Fig. 2c and d). Native dextran (DEX) exhibited typical polysaccharide proton signals at δ 3.0–4.0 ppm, while oxidized dextran (ODEX) showed new resonances at δ 5.0–5.6 ppm, corresponding to hemiacetal structures associated with introduced aldehyde groups, confirming successful oxidation. Dopamine (DA) displayed characteristic aromatic proton signals at δ 6.5–7.5 ppm and aliphatic signals at δ 2.5–3.2 ppm. In the ODEX–DA spectrum, both polysaccharide backbone signals and DA-related aromatic peaks were retained, accompanied by peak broadening and slight chemical shifts, indicating covalent conjugation via Schiff-base formation between aldehyde groups of ODEX and amino groups of DA. Consistently, FTIR spectra showed a weakened aldehyde-related C=O band at ∼1720 cm⁻¹ after DA conjugation, together with the emergence of aromatic C=C and C–O vibrations from DA, further confirming aldehyde participation in the condensation reaction. UV–Vis spectra revealed strong absorption of DA at ∼280 nm, whereas ODEX showed negligible absorption; the ODEX–DA conjugate exhibited attenuated but discernible aromatic absorption (Fig. S2), providing additional evidence for the successful incorporation of catechol moieties into the polysaccharide backbone.

A three-dimensional hydrogel network (OQ) was formed via dynamic Schiff-base crosslinking between amino groups in QCS–CGA and aldehyde groups in ODEX–DA (Fig. 2f). Stable gelation occurred only in the presence of both precursors, while incorporation of TCP nanoparticles yielded OQT hydrogels with darker coloration and enhanced structural integrity (Fig. 2g), suggesting additional metal–polyphenol coordination and secondary physical crosslinking within the network [26]. By adjusting the concentrations of QCS–CGA and ODEX–DA based on gelation time, OQ and OQT hydrogels were prepared, where OQT1, OQT2, and OQT3 correspond to hydrogels containing increasing amounts of TCP nanoparticles. SEM images revealed that both OQ and OQT hydrogels exhibited typical three-dimensional porous structures (Fig. 2i). With increasing TCP content, the pore structure became denser and the pore-size distribution shifted toward smaller and more uniform ranges, suggesting that metal–polyphenol coordination and additional physical crosslinking enhanced the network compactness. EDS elemental mapping further confirmed the uniform distribution of Cu throughout the OQT hydrogels (Fig. 2h), indicating good dispersion of TCP nanoparticles within the hydrogel matrix. To optimize the nanozyme loading within the hydrogel, formulations with increasing nanozyme concentrations (OQT1, OQT2, and OQT3) were evaluated for cytocompatibility (Fig. S3). OQT1 and OQT2 maintained cell viabilities comparable to the control and OQ groups, indicating good cytocompatibility at low to moderate nanozyme contents. In contrast, OQT3 resulted in a significant reduction in cell viability, suggesting concentration-dependent cytotoxicity at higher nanozyme loading. Based on these results, OQT2 was selected for subsequent experiments to balance biological safety and functional performance.

2.4. Injectability, self-healing, and rheological properties of OQT hydrogels

The OQT hydrogels exhibited excellent injectability and could be smoothly extruded through a syringe while retaining structural integrity after extrusion, enabling effective filling of irregular wound sites and in situ gelation (Fig. 2j) [27]. Pronounced self-healing behavior was observed for the OQT hydrogels (Fig. 2k). After being cut into separate pieces, the hydrogel fragments rapidly rejoined upon gentle contact and withstood subsequent lifting and stretching without fracture, demonstrating fast and robust autonomous healing. This efficient network reconstruction arises from the synergistic contribution of multiple dynamic interactions, including reversible Schiff-base bonds, metal–polyphenol coordination, and noncovalent interactions [28]. Compression tests further revealed that all hydrogels exhibited typical nonlinear stress–strain behavior (Fig. 2l). Compared with OQ hydrogels, OQT hydrogels exhibited markedly enhanced compressive strength, indicating that nanozyme incorporation effectively reinforces the dynamic network. The rheological results further demonstrated the favorable network stability of the hydrogels. As shown in Fig. 2m, the storage modulus (G′) of all groups remained consistently higher than the loss modulus (G″) throughout the time sweep and exhibited minimal fluctuation, indicating the formation of stable, elasticity-dominated network structures. In the strain sweep test (Fig. 2n), the hydrogels maintained relatively stable moduli within the low-strain region, whereas progressive network disruption occurred as the strain increased. Notably, OQT3 still retained a comparatively higher modulus, suggesting that incorporation of TCP reinforced the structural stability of the composite hydrogel. In the step-strain recovery test (Fig. 2o), G′ decreased markedly under high strain but rapidly recovered once the strain returned to a low level, demonstrating excellent shear-recovery capability and reversible network reconstruction. Meanwhile, the viscosity–shear rate curves (Fig. 2p) showed that all hydrogels underwent a continuous decrease in viscosity with increasing shear rate, exhibiting a typical shear-thinning behavior that is well consistent with their injectability.

The composite hydrogel system was successfully constructed at the chemical level and exhibited a combination of desirable features, including a three-dimensional porous architecture, uniform nanoparticle distribution, efficient in situ gelation, injectability, self-healing capability, and enhanced mechanical and rheological properties. In particular, the incorporation of TCP not only endowed the hydrogel with additional functional components, but also optimized the network organization and improved its mechanical performance, thereby providing a solid material basis for its subsequent application in infected wound repair.

2.5. Structural features and multifunctional performance of the asymmetric bilayer dressing

To develop a bilayer dressing with robust interfacial stability, microenvironmental responsiveness, and practical adaptability, we first characterized its degradation and ion-release behaviors. As shown in Fig. 3a, the hydrogel exhibited pronounced pH-responsive degradation. Under acidic conditions (pH 5.5), the degradation rate was markedly higher than that under near-neutral conditions (pH 7.2), and this difference became progressively more evident over time. By day 14, the degradation rate reached approximately 88.68 ± 1.50% at pH 5.5, compared with 65.10 ± 4.54% at pH 7.2. Consistent with these findings, SEM observations (Fig. S4) revealed more severe microstructural disruption in the acidic environment, where the lamellar/porous framework became looser and even partially collapsed, whereas the hydrogel maintained a relatively more intact morphology under near-neutral conditions. These results indicate that an acidic microenvironment accelerates hydrogel network disintegration and material degradation. In parallel, Cu ion release also showed a clear pH dependence (Fig. 3b). Under pH 5.5, Cu ions were released more rapidly and reached a higher cumulative release level, whereas under pH 7.4, the release profile was comparatively milder and more sustained. Notably, at 12 h, the cumulative Cu release under pH 5.5 had already reached approximately 50.97 ± 2.77%, which was substantially higher than the corresponding value of 36.90 ± 3.52% at pH 7.4. Taken together, these findings demonstrate that the system can rapidly respond to the mildly acidic microenvironment typically present in the early stage of infected wounds, enabling accelerated material degradation and enhanced release of functional Cu ions during the inflammatory/infectious phase, while maintaining a relatively moderate and sustained release behavior as the wound microenvironment gradually returns toward neutrality during healing. This stage-dependent response highlights the adaptive microenvironment-regulating capability of the bilayer system.

Fig. 3 presents the structural features and early functional performance of the hydrogel/electrospun bilayer dressing, with particular attention to its asymmetric adhesion behavior [29]. We next examined the electrospun membrane and its interfacial integration with the hydrogel layer. As shown in Fig. 3c, the PLGA@Cur electrospun membrane exhibited a uniform and continuous fibrous network, providing a suitable outer barrier for the bilayer dressing. Following plasma treatment (Fig. 3d–f), the surface wettability of the membrane was markedly improved, with the water contact angle decreasing from 133.07 ± 5.00° to 48.37 ± 1.60°, indicating a transition from a hydrophobic to a relatively hydrophilic surface. This change is expected to promote more effective wetting contact and stronger interfacial bonding between the electrospun layer and the hydrogel layer.

Notably, the macroscopic appearance of the bilayer dressing (Fig. 3e) directly confirmed the successful integration of the hydrogel layer with the electrospun membrane. The dressing maintained an intact overall shape and displayed a distinct bilayer configuration, with one side corresponding to the hydrogel layer and the other to the electrospun membrane, indicating stable integration of the two components at the macroscopic level. This observation is consistent with the subsequent cross-sectional morphology and also provides direct evidence for the practicality and structural stability of the bilayer design.

Cross-sectional SEM images further revealed a clear and compact interface between the electrospun membrane and the hydrogel, without obvious delamination or interfacial gaps (Fig. 3g), suggesting good structural integration between the two layers. This was further supported by the interfacial tensile test (Fig. S5). As the displacement increased, the interfacial adhesion strength gradually rose and reached a maximum value of approximately 5.2 kPa. Importantly, the tensile curve did not exhibit abrupt failure immediately after the peak, but instead showed a progressive fluctuating profile, indicating that interfacial failure occurred through gradual local debonding accompanied by sustained load bearing, rather than catastrophic one-step delamination. This result reflects the favorable interfacial adhesion and resistance to delamination of the bilayer dressing.

In terms of practical applicability, the finger-bending test (Fig. S6) showed that the bilayer dressing remained stably attached to the skin surface throughout the transition from the extended to the bent state, without obvious lifting, detachment, or interlayer separation, demonstrating its good flexibility and dynamic conformability. After immersion in PBS for 7 days, the bilayer structure remained intact, with a clearly distinguishable interface and no obvious debonding, delamination, or structural collapse under different placement orientations or tilted states, indicating favorable structural and interfacial stability under long-term hydrated conditions (Fig. S7). In addition, the supplementary video further showed that, when applied to the animal wound, the bilayer dressing maintained stable adhesion during normal animal movement, without noticeable lifting, slipping, or layer separation. Collectively, these results demonstrate that the bilayer dressing not only possesses a stable interface under static conditions, but also maintains good structural integrity and reliable adhesion under moist environments and dynamic mechanical disturbance.

The functional benefit of the asymmetric bilayer design is further demonstrated in an in vivo hemostasis model (Fig. 3h–j) [30]. The OQT/P dressing rapidly controls bleeding and significantly reduces blood loss compared with the control group, indicating that wound-side adhesion, fluid absorption, and physical coverage act together to enhance early hemostatic performance [31,32]. Swelling analysis (Fig. 3k and l) shows that the bilayer dressing exhibits a more moderate and stable swelling ratio than the single hydrogel system, preventing excessive expansion while maintaining effective exudate management. This behavior may be associated with the physical confinement imposed by the outer electrospun fibrous membrane and the interfacial regulation of water uptake between the two layers. Such a structure helps avoid excessive swelling, preserve dressing integrity, and maintain a suitable moist microenvironment for wound healing. Overall, the bilayer architecture combined with asymmetric adhesion improves fixation at the wound site, reduces external interference, and enhances early functional performance, providing a reliable structural basis for subsequent antibacterial and regenerative effects in infected wound healing.

2.6. Antibacterial activity and underlying mechanisms

The antibacterial activity and underlying mechanisms of the hydrogel system were systematically evaluated using Gram-positive MRSA and Gram-negative Escherichia coli (E. coli) as representative models. As illustrated in Fig. 4a, TCP nanoparticles embedded in the hydrogel matrix exhibit peroxidase POD- and Fenton-like catalytic activities, enabling localized ROS generation under infection-relevant microenvironments, thereby initiating oxidative bactericidal effects. Colony formation assays demonstrated dense bacterial growth in the control group, whereas all material-treated groups showed varying degrees of bacterial inhibition (Fig. 4b). Notably, bacterial colonies were almost completely suppressed in the OQT and OQT/P groups. Quantitative analysis of bacterial survival rates further confirmed that OQT and OQT/P exhibited the lowest survival percentages against both MRSA and E. coli, with statistically significant differences compared to OQ and OQ/P (Fig. 4c), highlighting the critical role of TCP incorporation in enhancing bactericidal efficacy. Live/dead fluorescence staining provided consistent results at the cellular level (Fig. 4d–g). Compared with the control, OQ, and OQ/P groups, bacteria exposed to OQT and OQT/P showed a pronounced increase in PI-positive (dead) cells accompanied by a marked reduction in SYTO 9-positive (live) cells, indicating effective loss of bacterial viability. It is noteworthy that OQT/P group showed stronger dead signals in both types of bacteria. The proportion of live bacteria in MRSA decreased to about 52.49 ± 2.79 %, and that in E. coli further decreased to about 34.02 ± 11.73 %, indicating that OQT/P had obvious killing effect on both types of bacteria. SEM observations further revealed pronounced morphological damage in bacteria exposed to OQT and OQT/P (Fig. 4h). While bacteria in the control and OQ groups largely maintained intact cellular morphology, MRSA and E. coli treated with OQT and OQT/P exhibited severe deformation, including membrane collapse, surface roughening, and partial envelope rupture, suggesting extensive disruption of membrane integrity.

Fig. 4 — In *Vitro* antibacterial and antibiofilm effects of OQT/P bilayer dressing. (a) Schematic illustration of the antibacterial mechanism of the OQT/P bilayer dressing. (b) *E. coli* and MRSA colonies after exposure to the control treatment, OQ, OQ/P, OQT and OQT/P bilayer dressing. (c) Quantitative analysis of MRSA and *E. coli* bacterial survival rate (n = 4). (d, e) Live/dead fluorescence staining images of MRSA and *E. coli*, respectively (SYTO 9/PI) (scale bar: 50 μm). (f, g) Live/dead ratio of MRSA and *E. coli*, respectively (n = 4). (h) SEM images showing morphological changes of MRSA and *E. coli* after different treatments (scale bar: 2 μm). (i) γ-H2AX immunofluorescence staining of MRSA after different treatments (scale bar: 50 μm). (j) Representative fluorescence images of intracellular ROS in MRSA detected by DCFH-DA staining after different treatments. (scale bar: 50 μm). (k) Quantitative analysis of γ-H2AX fluorescence intensity of MRSA. (l) Protein leakage of MRSA after different treatments. Data are presented as mean value ± SD. (∗p < 0.05; *∗∗p* < 0.01; *∗∗∗p* < 0.001; *∗∗∗∗p* < 0.0001; NS, not significant).

To elucidate the molecular mechanisms underlying bacterial inactivation, γ-H2AX immunofluorescence staining revealed markedly enhanced DNA damage signals in bacteria treated with OQT and OQT/P (Fig. 4i), indicating ROS-induced oxidative damage to genetic material. Protein leakage assays showed significantly increased extracellular protein levels following OQT and OQT/P treatment (Fig. 4l), confirming compromised membrane integrity and cytoplasmic leakage. Moreover, DCFH-DA staining demonstrated substantially elevated intracellular ROS levels in the OQT and OQT/P groups (Fig. 4j and k), providing direct evidence that TCP-mediated ROS generation plays a central role in bacterial damage [33,34]. From a synergistic structural perspective, the electrospun layer in OQ/P and OQT/P, loaded with curcumin, contributes additional antibacterial functions by physically isolating bacteria, reducing adhesion, and providing intrinsic antimicrobial activity. This effect synergizes with the cationic membrane-disrupting properties of the hydrogel matrix and the ROS-driven oxidative killing induced by TCP nanozymes. Previous studies on TA-Cu-based systems have mainly focused on direct bactericidal effects or stimulus-responsive release [35,36]. In contrast, the OQT/P system developed in this study further integrates polymyxin B, curcumin, and a bilayer structural design, enabling not only enhanced antibacterial activity but also additional functions including barrier protection, microenvironmental regulation, and repair promotion. The antibacterial performance of the system follows a progressive enhancement from moderate inhibition in OQ and OQ/P to highly efficient bactericidal activity in OQT and OQT/P, reflecting a multi-modal antibacterial mechanism involving membrane disruption, ROS-mediated molecular damage, and structural anti-adhesion effects. These results establish the hydrogel system as a potent antibacterial platform for infected wound management.

2.7. Evaluation of antibacterial ability against biofilms in vitro

The antibiofilm performance of the materials was evaluated using MRSA and E. coli as representative biofilm-forming strains. Crystal violet staining was employed to assess biofilm biomass in different treatment groups (Fig. 5a). Dense and continuous biofilm layers were observed in the control group for both strains, whereas biofilm coverage was evidently reduced in the OQ and OQ/P groups. Notably, biofilms treated with OQT and OQT/P were severely disrupted, leaving only sparse and discontinuous bacterial clusters, indicating a substantially enhanced antibiofilm effect of the composite hydrogel system [37]. Quantitative analysis further confirmed this trend (Fig. 5b and c). Compared with the control, biofilm biomass was significantly decreased in the OQ and OQ/P groups, suggesting that the hydrogel matrix itself possesses intrinsic antibiofilm activity. This effect is primarily attributed to the cationic interface introduced by quaternized chitosan, which interferes with bacterial adhesion and early-stage aggregation, thereby suppressing initial biofilm establishment. Upon incorporation of copper-based TCP nanoparticles, biofilm biomass in the OQT and OQT/P groups was further markedly reduced, demonstrating a synergistic enhancement of antibiofilm efficacy through combined physicochemical interactions.

The three-dimensional CLSM live/dead reconstruction results (Fig. 5d) further revealed the effect of the materials on biofilm viability. In the control group and the TCP-free groups, the biofilms were dominated by continuous and dense green fluorescence, indicating that live bacteria remained predominant. In contrast, in the OQT group and especially the OQT/P group, the red fluorescence signal was markedly increased, while the overall biofilm thickness and continuity were clearly reduced, suggesting extensive bacterial death accompanied by biofilm disruption. Quantitative analysis of the live/dead ratios further supported this observation. In MRSA biofilms, the proportion of live bacteria in the OQT/P group decreased to approximately 15.62 ± 1.30 %, whereas that in the control group remained around 77.04 ± 2.28 %. Similarly, in E. coli biofilms, the live bacterial ratio in the OQT/P group dropped to about 36.74 ± 1.52 %, which was substantially lower than the 92.70 ± 1.08 % observed in the control group (Fig. 5f and g). These findings indicate that the TCP-containing system not only reduces overall biofilm biomass, but also markedly impairs bacterial viability within the biofilm.

SEM observations offered direct morphological evidence of bacterial adhesion and aggregation behaviors (Fig. 5e). In the control group, both MRSA and E. coli formed dense and continuous biofilm structures, with bacteria tightly attached and embedded in abundant extracellular matrix. In contrast, after treatment with OQT and OQT/P, the biofilms became much looser and even fragmented, with reduced bacterial aggregation and a clear decrease in extracellular matrix. In addition, some bacteria exhibited obvious morphological abnormalities, including shrinkage, collapse, and incomplete cellular structures. These results suggest that the material inhibits bacterial adhesion and biofilm maturation, while effectively disrupting the integrity of established biofilms. The OQT/P bilayer dressing exhibited the strongest antibiofilm activity. It significantly reduced the biomass of MRSA and E. coli biofilms, markedly lowered the proportion of viable bacteria within the biofilm and induced loosening, fragmentation, and even disintegration of the biofilm structure. Combined with the antibacterial results described above, it can be inferred that TCP-mediated ROS generation and the accompanying damage to bacterial membrane structures may further weaken bacterial adhesion to each other and to the substrate, thereby enabling efficient elimination of mature biofilms. These results demonstrate that the bilayer system can not only suppress planktonic bacterial growth, but also effectively overcome the more challenging biofilm barrier in infected wounds, thereby providing strong support for its application in the treatment of complex infected wounds.

2.8. In vitro cyto-compatibility and hemocompatibility

The cytocompatibility and hemocompatibility of the hydrogel systems were systematically evaluated to assess their suitability for wound dressing applications. Live/dead staining showed that after 24 and 48 h of incubation, cells cultured with OQ, OQ/P, OQT, and OQT/P remained predominantly viable, with no evident cytotoxicity compared with the control group (Fig. 6a). Consistently, F-actin and nuclear staining revealed well-spread cell morphologies and intact cytoskeletal organization across all material-treated groups (Fig. 6b), indicating that the hydrogel matrices do not interfere with normal cellular behavior. CCK-8 assays further confirmed high cell viability at both time points (Fig. 6c and d). Notably, OQT and OQT/P exhibited a modest but reproducible increase in cell viability relative to the control, suggesting that incorporation of TCP nanozymes and microenvironmental regulation does not compromise cytocompatibility and may provide favorable cues for cell growth.

Hemocompatibility was evaluated by hemolysis assays to assess the safety of the bilayer dressing in blood-contacting environments (Fig. 6e). All hydrogel-treated groups exhibited hemolysis ratios well below the generally accepted safety threshold of 5% and comparable to the saline control, confirming excellent blood compatibility of the bilayer composite dressing. Cell migration was further evaluated using Transwell and scratch assays. The OQT/P group significantly enhanced HUVEC migration across the Transwell membrane compared with OQT or OQ/P alone (Fig. 6f and Fig. S9). Consistently, scratch wound assays demonstrated the most pronounced wound closure in the OQT/P group, with quantitative analysis confirming significantly accelerated migration at 12 and 24 h (Fig. 6g and h). These results indicate a synergistic pro-migratory effect arising from the integrated bilayer architecture. These findings demonstrate that the bilayer dressing combines potent antibacterial functionality with favorable cytocompatibility and pro-regenerative cellular responses, supporting its applicability for infected wound healing.

2.9. ROS scavenging–mediated anti-inflammatory and immunomodulatory effects

To elucidate the regulatory effects of the composite bilayer dressing on oxidative stress and immune responses, its ROS scavenging capacity, intracellular redox modulation, and macrophage polarization behaviors were systematically investigated. As schematically illustrated in Fig. 7a, the dressing integrates enzyme-like catalytic activity from TCP nanoparticles with the intrinsic radical-scavenging capability of polyphenol-rich hydrogel networks, enabling simultaneous elimination of multiple ROS species and restoration of local redox homeostasis, thereby suppressing inflammation-associated signal amplification [38]. The antioxidant capacity of the materials was first evaluated using representative radical systems, including •OH, DPPH, and ABTS⁺ (Fig. 7d) [28]. UV–vis spectra showed a pronounced decrease in the characteristic absorption peaks of all radicals after treatment with OQ-, OQ/P-, OQT-, and OQT/P-based systems (Fig. 7e–g). Quantitative analysis further revealed that OQT and OQT/P consistently exhibited higher scavenging efficiencies across all radical models (Fig. 7h–j). The •OH scavenging efficiency of the OQT and OQT/P groups increased to approximately 65%–67%, which was higher than that of the OQ and OQ/P groups (about 57%–59%). Meanwhile, the scavenging efficiencies toward DPPH and ABTS⁺ remained at relatively high levels across all groups, reaching approximately 85%–90% and 95%, respectively. These results indicate that incorporation of TCP further enhanced the hydroxyl radical scavenging capability of the system while preserving broad-spectrum antioxidant activity.

Fig. 7 — ROS scavenging and immunomodulatory properties of the OQT/P bilayer dressing in vitro. (a) Schematic illustration of the pH-dependent multi-enzyme-like activities of TCP nanoparticles for efficient scavenging of excessive ROS, thereby mitigating oxidative stress and inflammatory damage in infected wounds. (b) Representative fluorescence images showing intracellular ROS levels in macrophages stimulated with LPS or H₂O₂ and treated with different materials. (scale bar: 100 μm). (c) Representative fluorescence images of intracellular superoxide anion detected by DHE staining under different treatment conditions (scale bar: 100 μm). (d) Schematic illustration of chemical scavenging mechanisms for DPPH, ABTS⁺, and •OH radicals. (e–g) UV–vis absorption spectra showing the scavenging of •OH, DPPH, and ABTS⁺ radicals by different materials. (h–j) Quantitative analysis of •OH, DPPH, and ABTS⁺ radical scavenging efficiencies. (k) Flow cytometry analysis of intracellular ROS levels in RAW264.7 cells. (l) Immunofluorescence images of Arginase-1 in RAW264.7 after different treatments for 24 h. (m) Immunofluorescence images of iNOS in RAW264.7 after different treatments for 24 h (cytoskeleton: red; nucleus: blue; iNOS and Arginase-1: green). (n, o) The quantitative analysis of iNOS and Arginase-1 in RAW264.7 after different treatments for 24 h based on fluorescence microscopic images. (n = 3) (p) Flow cytometry analysis of specific marker of M1 macrophage CD86 and M2 macrophage CD206 with different experimental treatment. (q) The quantitative analysis of intracellular ROS levels in RAW264.7 cells. (n = 3) Data are presented as mean value ± SD. (∗p < 0.05; *∗∗p* < 0.01; *∗∗∗p* < 0.001; *∗∗∗∗p* < 0.0001; NS, not significant).

Intracellular redox regulation was further evaluated using LPS- and H₂O₂-induced oxidative stress models. DCFH-DA staining revealed substantial ROS accumulation after LPS or H₂O₂ stimulation, whereas treatment with OQ, OQ/P, OQT, and OQT/P markedly attenuated the fluorescence signals, with the most pronounced reduction observed in the OQT and OQT/P groups (Fig. 7b). Consistently, DHE staining showed that OQT and OQT/P effectively suppressed LPS-triggered superoxide accumulation (Fig. 7c), suggesting that these materials not only scavenge downstream ROS but may also inhibit ROS amplification at an earlier stage. This trend was further supported by flow cytometry (Fig. 7k and q). Compared with the control group, the intracellular ROS mean fluorescence intensity (MFI) in the Control + LPS group increased sharply from approximately 160 to 1700, whereas treatment with OQ, OQ/P, OQT, and OQT/P reduced the MFI to approximately 300–350 (Fig. S10). These findings demonstrate that all treatment groups effectively alleviated inflammation-induced intracellular ROS overaccumulation, with OQT and OQT/P showing the most prominent redox-regulating effect. Given the close coupling between oxidative stress and inflammatory activation, macrophage polarization was subsequently examined. Immunofluorescence staining showed that LPS stimulation induced strong expression of the M1 marker iNOS, whereas OQT and OQT/P markedly suppressed iNOS signals while enhancing the expression of the M2 marker Arg-1 (Fig. 7l–o). Flow cytometry results (Fig. 7p) further confirmed this trend. Compared with the LPS group, the proportion of M1 macrophages was markedly reduced in all treatment groups, whereas the proportion of M2 macrophages showed a gradual increase. In particular, the M1 fraction decreased from approximately 35.55% in the LPS group to about 12.47% in the OQT/P group, while the M2 fraction increased from approximately 7.81% to 26.67%. These findings indicate that the materials, especially the OQT/P bilayer dressing, can effectively suppress the activation of pro-inflammatory macrophages and promote their polarization toward a reparative phenotype.

This bilayer system exhibited pronounced ROS-scavenging and anti-inflammatory immunomodulatory activities. On the one hand, it efficiently eliminated multiple free radicals and significantly reduced intracellular ROS accumulation under inflammatory stimulation. On the other hand, by alleviating oxidative stress, it further suppressed M1 macrophage polarization while promoting the transition toward the M2 phenotype, thereby helping to shift the inflammatory microenvironment toward a state more favorable for tissue repair. Among all groups, OQT/P showed the most prominent performance in both antioxidant and immunoregulatory functions, indicating a clear synergistic effect between the bilayer dressing architecture and the functional TCP component. These findings provide important mechanistic support for its subsequent application in promoting infected wound healing [4,39].

2.10. Pro-angiogenic effects of the bilayer dressing

To systematically evaluate the regulatory effects of different dressing systems on angiogenesis-related behaviors, both pro-angiogenic signaling expression and in vitro tube formation were investigated. As illustrated in Fig. 8a, based on the previously demonstrated ROS regulation and immune microenvironment remodeling, it is reasonable to infer that the dressing system may indirectly activate VEGF-related angiogenic pathways by alleviating oxidative stress and inflammatory responses, thereby enhancing the angiogenic potential of endothelial cells. Immunofluorescence staining revealed markedly enhanced expression of VEGF-A and α-SMA in all material-treated groups compared with the control (Fig. 8d and e), with strong colocalization along the cytoskeletal structures. Quantitative analysis further confirmed significant upregulation of VEGF-A and α-SMA levels following material treatment (Fig. 8b and c), consistent with the immunofluorescence observations. These results indicate that the composite hydrogel system effectively activates molecular signals associated with angiogenesis and vascular stabilization.

Fig. 8 — Angiogenesis ability assessment OQT/P bilayer dressing. (a) Schematic diagram showing the mechanisms of angiogenesis induced by OQT/P in HUVECs. (b, c) Quantitative analysis of VEGF-A and α-SMA expression levels in HUVECs after different treatments for 24 h, based on immunofluorescence images (n = 3). (d) Immunofluorescence images of VEGF-A in HUVEC after different treatments for 24 h. (e) Immunofluorescence images of α-SMA in HUVEC after different treatments for 24 h (cytoskeleton: red; nucleus: blue; VEGF-A and α-SMA: green). (f) Representative images of angiogenesis experiments of different materials (scale bar: 800 μm). (g-k) Quantitative analysis of number of nudes, number of junctions, number of meshes, number of branches and total branches length. (n = 3). Data are presented as mean value ± SD. (∗p < 0.05; *∗∗p* < 0.01; *∗∗∗p* < 0.001; *∗∗∗∗p* < 0.0001; NS, not significant).

The functional consequences of these molecular changes were examined using HUVEC tube formation assays. Representative images show that all material-treated groups promoted the formation of more continuous and interconnected capillary-like networks relative to the control (Fig. 8f). Quantitative analysis demonstrated significant increases in total tube length, number of junctions, branching points, and meshes (Fig. 8g–k). Notably, the OQT and OQT/P groups exhibited the most pronounced enhancement across all parameters, suggesting a synergistic pro-angiogenic effect associated with the integrated bilayer design. These results demonstrate that the bilayer dressing promotes angiogenesis at both molecular and functional levels. Coupled with its ability to restore redox homeostasis and modulate inflammatory responses, the enhanced endothelial organization and vascular marker expression provide a mechanistic basis for the dressing's capacity to support neovascularization during wound repair.

2.11. In vivo evaluation of the OQT/P bilayer dressing for the treatment of MRSA biofilm–infected wounds

To evaluate therapeutic performance under clinically relevant conditions, a full-thickness MRSA biofilm–infected wound model was established in mice, and wound closure, bacterial burden, oxidative stress, and tissue regeneration were systematically assessed (Fig. 9a). This model recapitulates key pathological features of infected chronic wounds, including persistent bacterial colonization, excessive oxidative stress, and delayed healing. As shown in the representative macroscopic images (Fig. 9b), wounds in the control and commercial 3M groups displayed pronounced exudation, necrotic tissue accumulation, and limited contraction during the early healing stage. In contrast, OQ and OQ/P moderately improved wound appearance and accelerated closure, whereas OQT and especially OQT/P produced much more evident wound contraction from days 3–6 onward. This trend was further confirmed by wound outline reconstruction (Fig. 9c) and quantitative wound area analysis (Fig. 9f). By day 6, the residual wound area in the OQT/P group had decreased to approximately 7.71 ± 1.40%, markedly lower than that in the control group (69.28 ± 5.86%) and the 3M group (62.46 ± 5.96%). More importantly, during the later healing stage (days 9–12), the OQT/P group achieved nearly complete wound closure with smoother wound margins than OQT alone, leaving a residual wound area of only about 1.93 ± 0.90%. These findings indicate that the bilayer architecture confers improved dressing stability and more sustained therapeutic efficacy in the infected wound microenvironment.

To verify its in vivo antibacterial and antibiofilm efficacy, bacterial burden in the wound tissue was further evaluated. As shown in Fig. 9d, abundant MRSA colonies were observed in all groups immediately after infection establishment (day 0). After 6 days of treatment, colony numbers were dramatically reduced in the OQT group and, more notably, in the OQT/P group, approaching near-complete eradication. In contrast, substantial bacterial colonies remained in the control and 3M groups. Meanwhile, ROS staining of wound tissue (Fig. 9e) showed that the control and 3M groups still exhibited strong oxidative stress–associated fluorescence on day 6, whereas OQ and OQ/P produced only moderate reductions. By comparison, OQT and especially OQT/P caused a much more pronounced decrease in ROS signals. Quantitative analysis (Fig. 9g) further demonstrated that the relative ROS fluorescence intensity in the OQT/P group decreased to only about 0.10 ± 0.02 fold of that in the control group. Together, these findings indicate that the bilayer system not only reduces bacterial burden, but also effectively alleviates the persistently elevated ROS microenvironment in infected wounds.

Histological analyses further demonstrated the superior tissue reconstruction achieved by the bilayer dressing. At day 6, H&E staining showed persistent inflammatory cell infiltration and disorganized tissue architecture in the control and 3M groups, whereas both OQT and OQT/P markedly reduced inflammation and promoted more continuous granulation tissue formation (Fig. 9j). By day 12, wounds treated with OQT/P exhibited more complete re-epithelialization and more evident skin appendage-like structures than those treated with OQT, indicating more advanced tissue maturation (Fig. 9k). Masson trichrome staining further revealed denser and more regularly aligned collagen deposition in the OQT/P group, reflecting superior extracellular matrix remodeling and functional tissue regeneration. Quantitative analysis also supported these observations. As shown in Fig. 9h, the OQT/P group exhibited the thinnest granulation tissue, measuring approximately 55.13 ± 8.96 μm, markedly lower than that in the control group (151.13 ± 17.62 μm) and the 3M group (155.30 ± 12.62 μm), and also lower than that in the OQ and OQ/P groups. Given that excessively thick granulation tissue under inflammatory conditions often indicates persistent inflammation and abnormal tissue hyperplasia, this result suggests that inflammation was more effectively controlled in the OQT/P group, resulting in a more orderly repair process. Overall, although OQT already showed potent antibacterial and pro-healing activity, the bilayer composite dressing (OQT/P) further improved sustained bacterial suppression, ROS microenvironment regulation, and high-quality tissue reconstruction, highlighting its clear advantage for long-term treatment of complicated MRSA biofilm–infected wounds.

2.12. Histological and immunofluorescence evidence of stepwise wound microenvironment regulation in vivo

Histological and immunofluorescence analyses further demonstrate that the OQT/P bilayer dressing achieves superior in vivo therapeutic efficacy through coordinated, stage-adaptive regulation of the infected wound microenvironment. Giemsa staining reveals abundant bacterial residues in the control and 3M groups, which are only partially reduced in the OQ and OQ/P groups (Fig. 10a). In contrast, both OQT and OQT/P markedly suppress bacterial aggregation, with OQT/P exhibiting the lowest detectable bacterial burden, indicating more efficient in vivo disruption of MRSA biofilms and sustained inhibition of bacterial recolonization. Consistent with effective infection control, macrophage phenotypic analysis (Fig. 10c) shows persistent CD86⁺ M1-dominant inflammation in the control and 3M groups, whereas OQT and, more prominently, OQT/P significantly enhance CD206⁺ M2 polarization. This macrophage phenotype shift is accompanied by pronounced attenuation of IL-6 immunofluorescence (Fig. 10d) and TNF-α immunohistochemical signals (Fig. 10f), indicating effective suppression of inflammatory amplification and reprogramming of the immune microenvironment toward a pro-healing state. Following resolution of bacterial burden and inflammatory stress, vascular remodeling was evaluated. CD31/α-SMA co-staining (Fig. 10e) demonstrates enhanced neovascularization in both OQT and OQT/P groups, while OQT/P further promotes vessel stabilization and maturation, as evidenced by increased α-SMA coverage surrounding CD31⁺ endothelial structures. This observation suggests improved transition from nascent angiogenesis to functionally stabilized vasculature.

Subsequently, collagen I (Col-I) immunofluorescence (Fig. 10b) reveals more continuous and organized extracellular matrix deposition in the OQT/P group, reflecting accelerated matrix remodeling and structural maturation of regenerated tissue. Quantitative analyses across antibacterial, inflammatory, angiogenic, and remodeling-related parameters (Fig. 10g–k) consistently show that OQT/P outperforms OQT and other control groups. These results demonstrate that the bilayer OQT/P dressing orchestrates a sequential therapeutic cascade—from antibacterial and antibiofilm activity, to immune reprogramming, angiogenesis, and ultimately tissue remodeling. This stepwise regulation closely mirrors the natural biological trajectory of infected wound healing and underlies the superior in vivo regenerative efficacy of the OQT/P system.

2.13. Transcriptomic analysis reveals immune–inflammatory reprogramming induced by OQT/P

To further elucidate the molecular mechanisms by which OQT/P modulates the inflammatory microenvironment of infected wounds and promotes repair, we performed transcriptomic sequencing of wound tissues. As shown in Fig. 11a, the volcano plot revealed a pronounced reshaping of the gene expression profile after OQT/P treatment, with 487 upregulated genes and 373 downregulated genes, indicating that this bilayer dressing substantially influences transcriptional programs associated with wound healing. Hierarchical clustering analysis (Fig. 11b) further showed a clear separation between the OQT/P and control groups at the global expression level, suggesting that the treatment induced a systematic biological response rather than random local fluctuations. GO enrichment analysis (Fig. 11d) showed that the differentially expressed genes were mainly enriched in terms related to immune receptor activity, regulation of immune response, cytokine receptor activity, immune system process, and inflammatory response, indicating that the primary effect of OQT/P on infected wound repair is centered on immune and inflammatory regulation. Notably, the differentially expressed genes were also enriched in the process of detoxification of copper ion. As shown in Fig. S12, metallothionein-related genes such as Mt1, Mt2, and Mt4 displayed distinct expression patterns in the OQT/P group compared with the control group, suggesting that the host activated corresponding mechanisms for metal ion homeostasis maintenance and detoxification in response to the Cu signals released by the material. This finding indicates that functional copper ions not only participate in local microenvironmental regulation, but also induce adaptive host defense responses, thereby providing molecular-level support for both the functionality and biosafety of the material.

KEGG enrichment analysis (Fig. 11c and e) further indicated that the differentially expressed genes were mainly involved in pathways closely related to infection, inflammatory amplification, and immune reprogramming, including Staphylococcus aureus infection, cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, Th17 cell differentiation, PI3K-Akt signaling pathway, NF-κB signaling pathway, and TNF signaling pathway. In addition, Fig. S11 presents the KEGG enrichment results of all differentially expressed genes in the form of a bubble plot, further confirming that OQT/P broadly regulates infection-related pathways, cytokine networks, and inflammatory response signaling. In particular, the enrichment of the NF-κB, TNF, and Th17 pathways suggests that OQT/P may promote wound repair by suppressing inflammatory cascade amplification and alleviating pro-inflammatory immune activation [40,41]. Further GSEA results (Fig. 11h and i) showed that both the NF-κB signaling pathway and Th17 cell differentiation were negatively enriched in the OQT/P group, indicating an overall suppression of these classical pro-inflammatory pathways. Meanwhile, the heatmaps of genes related to Staphylococcus aureus infection and PI3K-Akt signaling (Fig. 11f and g) showed substantial remodeling of infection-responsive and inflammatory signaling networks after OQT/P treatment. In addition, the circular heatmap in Fig. S13 further demonstrated coordinated changes in multiple key inflammation-related genes within the TNF signaling pathway, suggesting that OQT/P does not act on a single inflammatory factor alone, but rather regulates the TNF-associated inflammatory axis at the network level. This observation is consistent with the KEGG and GSEA analyses and further supports the overall inhibitory effect of OQT/P on amplified inflammatory signaling.

To validate the sequencing findings, we further examined key inflammatory cytokines and related proteins experimentally. ELISA results (Fig. 11j–l) showed that, compared with the LPS group, OQT/P treatment significantly increased the level of the anti-inflammatory cytokine IL-10, while markedly reducing the levels of the pro-inflammatory cytokines TNF-α and IL-1β. Western blot analysis (Fig. 11m) further demonstrated that Arg-1 expression was upregulated, whereas iNOS expression was suppressed after OQT/P treatment. At the same time, the p-p65/p65 ratio was clearly reduced, indicating effective inhibition of NF-κB pathway activation. These protein-level results were highly consistent with the transcriptomic findings, demonstrating that OQT/P can significantly attenuate inflammatory responses and promote macrophage polarization toward a repair-associated phenotype.

Taken together, the transcriptomic results indicate that the regulatory effect of OQT/P on the infected wound microenvironment is not limited to a single factor, but is achieved through the coordinated modulation of multiple pathways, including infection response, cytokine networks, the NF-κB/TNF inflammatory axis, Th17 differentiation, and metal ion homeostasis regulation. On the one hand, OQT/P suppresses key signals associated with persistent infection and inflammatory amplification, thereby reducing the expression of pro-inflammatory mediators such as TNF-α, IL-1β, and iNOS. On the other hand, it enhances signals related to inflammation resolution and tissue repair, including IL-10 and Arg-1, thus driving the wound microenvironment from a persistent inflammatory state toward a repair-regenerative state. At the same time, the activation of copper ion detoxification-related genes, together with the network-level remodeling of the TNF pathway, suggests that the host possesses a certain adaptive regulatory capacity in response to the functional Cu signals released by the material. Collectively, these findings demonstrate that the OQT/P bilayer dressing exerts a coordinated therapeutic effect of infection control, inflammation suppression, and repair promotion through a multilayered and network-based molecular mechanism, thereby providing important mechanistic support for its ability to promote high-quality healing of infected wounds.

Hematological analysis and histological examination of major organs (heart, liver, spleen, lung, and kidney) revealed no significant changes between control and treated groups, indicating no systemic toxicity (Fig. S14). Blood parameters, including WBC, Neu#, Lymph#, RBC, PLT, and MPV, remained within normal physiological ranges. Histology confirmed intact tissue structures in all material-treated groups, with no evidence of copper-induced toxicity or organ damage (Fig. S15a–f).

3. Conclusion

In summary, we developed a microenvironment-adaptive bilayer dressing by integrating a pH-responsive copper-based nanozyme, a dynamically crosslinked hydrogel, and an outer electrospun fibrous membrane, enabling stage-specific treatment of MRSA biofilm-infected wounds. This system not only fulfills the sequential therapeutic demands of infection control and tissue repair, but also partially mimics the layered architecture and functional compartmentalization of native skin. Specifically, the outer fibrous membrane provides physical barrier protection and anti-adhesion capability, whereas the inner hydrogel closely adheres to the wound bed and enables local microenvironment regulation, bioactive component delivery, and maintenance of a moist healing environment. Based on this bilayer skin-mimetic design, OQT/P integrates antibacterial, antibiofilm, immunomodulatory, and pro-regenerative functions within a single platform.

Mechanistically, the key advantage of this system lies in its stage-adaptive redox regulation, namely, promoting ROS-mediated antibacterial activity during the infection stage while scavenging excessive ROS during the healing stage. As a result, OQT/P exhibited superior performance in controlling bacterial burden, alleviating oxidative stress, suppressing inflammation, promoting angiogenesis, and enhancing matrix remodeling in vivo, achieving a wound closure rate of approximately 98.07% on day 12 and a 32.8-fold increase in collagen deposition. Transcriptomic analysis further demonstrated that these therapeutic benefits were closely associated with the coordinated suppression of infection- and inflammation-related signaling pathways.

From a translational perspective, this study highlights four major advantages. First, it provides a non-antibiotic therapeutic strategy for biofilm-infected wounds. Second, the bilayer structural design enables local functional compartmentalization and synergistic action. Third, this stratified architecture partially recapitulates the organizational and interfacial features of native skin, making the material design better suited to the dual demands of barrier protection and local regulation during wound healing. Fourth, the stage-adaptive redox regulation strategy is better aligned with the dynamic evolution of the infected wound microenvironment than static antibacterial intervention. Nevertheless, several limitations should also be acknowledged. The current study was mainly validated in a murine MRSA-infected wound model, which cannot fully capture the pathological complexity of clinical chronic wounds, particularly polymicrobial wounds and diabetic refractory wounds. In addition, the long-term in vivo safety, degradation behavior, and therapeutic performance of the material in larger-animal models still require further systematic evaluation. Overall, this study suggests that effective treatment of infected wounds depends not only on bacterial eradication, but also on the precise regulation of the evolving wound microenvironment, and provides a promising strategy for the design of next-generation high-performance bioactive wound dressings.

4. Experimental section

4.1. Materials

Tannic acid (TA), polymyxin B sulfate (PMB), quaternized chitosan (QCS), sodium periodate (NaIO₄), dopamine hydrochloride (DA, 98%), copper chloride dihydrate (CuCl₂·2H₂O), 1-(3-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Aladdin Bio-Chem Technology (Shanghai, China). Dextran (DEX), chlorogenic acid (CGA), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and 2,2′-azinobis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) were obtained from Macklin (Shanghai, China). Hydrogen peroxide (H₂O₂) was purchased from ChengDu Chron Chemicals Co,. Ltd. Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich. Cell Counting Kit-8 (CCK-8), 4′,6-diamidino-2-phenylindole (DAPI, C0066), 2′,7′-Dichlorofluorescin Diacetate (Cat:D6472) and rhodamine-labelled phalloidin were supplied by Beijing Solarbio Science & Technology Co., Ltd (Beijing, China). Matrigel was provided by Xiamen Mogengel Biotechnology Co., Ltd (Xiamen, China). All reagents were of analytical grade and used as received. Deionized water was used throughout all experiments.

4.2. Synthesis and characterization of TCP NPs

TCP nanoparticles were synthesized via a coordination–polyphenol self-assembly strategy. Briefly, aqueous solutions of CuCl₂ (0.1 mg mL⁻¹), TA (1 mg mL⁻¹), and PMB (1 mg mL⁻¹) were sequentially mixed in 20 mL of deionized water under magnetic stirring for 10 min. The pH of the mixture was subsequently adjusted to 8–9 using 1 M NaOH to initiate coordination-driven polymerization. The reaction was allowed to proceed at room temperature for 2 h to complete nanoparticle formation.

To improve colloidal stability, poly (vinyl pyrrolidone) (PVP, M_n ≈ 40 000 Da) was added to the suspension for surface modification. The resulting TCP nanoparticles were collected by centrifugation and washed three times with deionized water to remove unreacted components and excess stabilizer, followed by redispersion in deionized water for subsequent use.

FTIR spectra were recorded using a Nicolet iS50 spectrometer (Thermo Scientific, USA) in the range of 4000–400 cm⁻¹ using the KBr pellet method. The morphology and internal structure of TCP nanoparticles were examined by TEM, while hydrodynamic size distribution was measured by dynamic light scattering (DLS, Malvern Instruments). Crystalline structure was analyzed by X-ray diffraction (XRD, Rigaku SmartLab SE, Japan) operated at 40 kV and 30 mA with Cu Kα radiation. Data analysis was supported by Scientific Compass www.shiyanjia.com). Elemental composition and chemical states were determined by X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha, China).

4.3. Synthesis and characterization of QCS–CGA

QCS–CGA was synthesized via an EDC/NHS-mediated amidation reaction. Briefly, 1 g of QCS was dissolved in 0.1 M MES buffer under nitrogen protection with continuous stirring until complete dissolution. Meanwhile, 1 g of CGA was dissolved in anhydrous ethanol and activated with EDC/NHS at 4 °C for 1 h. The activated CGA solution was then slowly added dropwise into the QCS solution under a nitrogen atmosphere, and the reaction was allowed to proceed at room temperature for 24 h under continuous stirring. After the reaction, the resulting mixture was transferred into a dialysis membrane (MWCO 14 kDa) and dialyzed against deionized water to remove unreacted small-molecule impurities, followed by freeze-drying to obtain QCS–CGA. The chemical structure of the product was characterized by FTIR, ¹H NMR, and UV–vis spectroscopy.

4.4. Synthesis and characterization of ODEX–DA

ODEX was prepared by sodium periodate (NaIO₄)-mediated oxidation of dextran. Briefly, 5 g of dextran was dissolved in 250 mL of deionized water, followed by the addition of 2 g NaIO₄ under dark conditions. The reaction mixture was stirred continuously at room temperature for 4 h. After the reaction, excess ethylene glycol was added to quench the reaction, and the mixture was further stirred for 2 h. The resulting solution was then transferred into a dialysis membrane (MWCO 14 kDa) and dialyzed against deionized water for 3 days to remove unreacted small molecules and byproducts, with the external water refreshed every 12 h, followed by freeze-drying to obtain ODEX.

ODEX–DA was synthesized by reacting ODEX with DA in MES buffer under a nitrogen atmosphere. Briefly, 2 g of ODEX was dissolved in 150 mL of 0.1 M MES buffer under nitrogen protection with continuous stirring until complete dissolution, followed by the addition of 0.5 g DA. The reaction was allowed to proceed at room temperature for 2 h under a nitrogen atmosphere. After the reaction, the obtained mixture was purified by dialysis (MWCO 14 kDa) against deionized water for 3 days, with the external water refreshed every 12 h, followed by freeze-drying to obtain ODEX–DA. The chemical structure of the product was characterized by FTIR, ¹H NMR, and UV–vis spectroscopy.

4.5. Preparation and characterization of OQ and OQT hydrogels

OQ and OQT hydrogels were prepared by mixing the corresponding precursor solutions at room temperature. Briefly, equal volumes of 10 wt% QCS–CGA solution and 3 wt% ODEX–DA solution were mixed, rapidly vortexed and thoroughly stirred to obtain a homogeneous mixture, and then allowed to stand at room temperature to form the OQ hydrogel. For the preparation of OQT hydrogels, TCP nanoparticle dispersion (3 mg mL⁻¹) was further added to the above precursor system, followed by gentle mixing until gelation occurred at room temperature. Gelation was evaluated by the vial inversion method, and the sample was considered gelled when no visible flow was observed after vial inversion.

The injectability of OQ and OQT hydrogels was assessed by syringe extrusion. The precursor mixtures were loaded into a 1 mL syringe and extruded at room temperature to observe their continuous extrusion behavior and gelation state. The self-healing property was evaluated by a macroscopic cut-and-heal test. Briefly, the formed hydrogels were cut into two pieces, and the separated pieces were brought back into contact to observe their reconnection and recovery of structural integrity.

The microstructures of freeze-dried hydrogels were observed using scanning electron microscopy (SEM, Hitachi S-4800). After lyophilization, the samples were sputter-coated with gold prior to SEM observation. Elemental distribution was further analyzed by energy-dispersive X-ray spectroscopy (EDS) mapping.

The rheological properties of the hydrogels were measured using a rotational rheometer (MCR 302, Anton Paar) at 25 °C. All experiments were conducted in triplicate to ensure reproducibility. Time sweep, frequency sweep, and step-strain tests were performed to evaluate the viscoelasticity and self-healing behavior of the hydrogels. Specifically, time sweep measurements were carried out at a constant strain of 0.1% and an angular frequency of 10 rad s⁻¹; frequency sweep measurements were performed over an angular frequency range of 0.1–100 rad s⁻¹ at a fixed strain of 0.1%; and step-strain measurements were alternated between low strain (1%) and high strain (800%) at a constant angular frequency of 1 rad s⁻¹ to assess the structural disruption and recovery of the hydrogel network.

The compressive properties of the hydrogels were measured using a universal testing machine. Hydrogel samples were prepared in the form of cylinders with dimensions of 8 mm × 5 mm, and the stress-strain curves were recorded to evaluate their mechanical properties.

4.6. Degradation assay of OQT hydrogel

To evaluate the degradation behavior of the OQT hydrogel under different microenvironmental conditions, lyophilized OQT hydrogel samples were weighed to obtain the initial dry weight, denoted as W₀, and then immersed in 10 mL of different buffer solutions in centrifuge tubes at 37 °C under static conditions. Acidic buffer (pH 5.5) and neutral buffer (pH 7.4) were used to simulate different wound microenvironments.

At predetermined time points, the samples were collected, gently blotted to remove excess surface liquid, and then centrifuged and lyophilized to constant weight. The remaining dry weight was recorded as W_t. The degradation experiment was continued for 14 days. To better simulate the in vivo environment, the medium was refreshed every 3 days by removing the excess old solution and adding an equal volume of fresh buffer. The degradation behavior of the OQT hydrogel was evaluated by the remaining mass ratio or degradation rate, calculated as follows:

Degradation rate (%) = (W₀ − W_t) / W₀ × 100%

where W₀ is the initial dry weight of the OQT hydrogel and W_t is the remaining dry weight at the indicated time point.

4.7. Preparation of Cur@PLGA electrospun fibrous membrane

Poly (lactic-co-glycolic acid) (PLGA, LA/GA = 50/50) and curcumin (Cur) were co-dissolved in hexafluoroisopropanol (HFIP) to prepare the electrospinning solution. Briefly, the spinning solution was prepared with a total solute concentration of 15% (w/v) and magnetically stirred at room temperature until completely dissolved to obtain a homogeneous solution. The resulting solution was then transferred into a syringe fitted with a metal needle and electrospun under the following conditions: an applied voltage of 20 kV, a flow rate of 1 mL h⁻¹, and a collecting distance of 15 cm. Electrospinning was carried out at room temperature, and the as-spun fibers were collected on the surface of the collector to form a fibrous membrane. After electrospinning, the obtained membrane was thoroughly dried under ambient or vacuum conditions to remove residual solvent, yielding the curcumin-loaded PLGA electrospun fibrous membrane, denoted as PLGA@Cur.

4.8. Bacterial culture

MRSA and E. coli were cultured in Luria–Bertani broth at 37 °C under shaking overnight to the logarithmic phase. The bacteria were harvested by centrifugation, washed twice with sterile phosphate-buffered saline (PBS, 0.01 M), and resuspended in sterile saline (0.9% NaCl). The bacterial concentration was adjusted to OD₆₀₀ = 0.1 (approximately 1 × 10⁸ CFU mL⁻¹) for subsequent experiments.

4.9. In vitro antibacterial activity

The antibacterial activity of the materials was assessed by a spread plate assay. Briefly, 200 μL of bacterial suspension (approximately 1 × 10⁸ CFU mL⁻¹) was incubated with hydrogel extracts or suspensions from different groups (OQ, OQ/P, OQT, and OQT/P) at 37 °C for 6 h under gentle shaking. The resulting mixtures were serially diluted, spread on LB agar plates, and further cultured at 37 °C for 24 h. Colony-forming units (CFUs) were counted, and the antibacterial efficiency was calculated relative to the control group.

4.10. Bacterial morphology observation by SEM

To investigate bacterial morphological changes and membrane damage after different treatments, MRSA and E. coli were treated with the five experimental groups, respectively, and then collected by centrifugation. The obtained bacterial pellets were fixed with 2.5% (v/v) glutaraldehyde at 4 °C for 4 h, followed by sequential dehydration in graded ethanol solutions (10%, 20%, 40%, 60%, 80%, and 100%, v/v). After drying and gold sputter-coating, the samples were observed by SEM to examine the morphological changes in bacterial cell structures.

4.11. Live/dead fluorescence staining of bacteria

Bacterial viability after different treatments was assessed using a SYTO 9/propidium iodide (PI) Live/Dead bacterial staining kit. Briefly, MRSA and E. coli suspensions after the indicated treatments were collected by centrifugation and gently washed with sterile PBS to remove residual culture medium and unbound material components. The bacterial pellets were then resuspended in an appropriate volume of sterile PBS, followed by incubation with SYTO 9 and PI staining solution according to the manufacturer's instructions in the dark for a certain period to allow sufficient staining. After staining, the bacterial suspension was dropped onto glass slides and observed using confocal laser scanning microscopy (CLSM). Live bacteria emitted green fluorescence, whereas dead or membrane-compromised bacteria exhibited red fluorescence. When necessary, fluorescence images from different fields were further analyzed quantitatively to evaluate bacterial viability in each group.

4.12. Intracellular ROS and protein leakage assays

Intracellular ROS levels in bacteria after different treatments were detected using the DCFH-DA probe. Briefly, bacterial suspensions from different treatment groups were collected by centrifugation and washed with sterile PBS to remove residual culture medium and unbound material components. The bacterial pellets were then resuspended in an appropriate volume of PBS and incubated with DCFH-DA at a final concentration of 20 μM at 37 °C for 30 min in the dark. After incubation, the samples were washed again with PBS to remove excess extracellular probe. The treated samples were then observed by CLSM. Intracellular ROS levels were evaluated based on the green fluorescence intensity, and the fluorescence signals of different groups were further quantified using image analysis software.

To further evaluate bacterial membrane integrity, a protein leakage assay was performed. Briefly, bacterial suspensions after different treatments were centrifuged, and the supernatants were collected for leaked protein analysis. The protein concentration in the supernatants was then determined using a bicinchoninic acid (BCA) protein assay kit according to the manufacturer's instructions. Membrane damage was assessed by comparing the protein concentrations in the supernatants among different groups. A higher protein content in the supernatant indicated greater membrane permeability and more severe membrane disruption.

4.13. In vitro antibiofilm assay

In vitro biofilm models of MRSA and E. coli were established to evaluate the antibiofilm activity of the materials. Briefly, bacterial suspensions were seeded into 12-well plates and incubated at 37 °C under static conditions for 48 h, with fresh medium replaced every 12 h to allow the formation of mature biofilms. After removing the supernatants, the wells were gently washed with sterile PBS to remove planktonic bacteria while preserving the adherent biofilms. The mature biofilms were then treated with different materials for 12 h. For morphological observation, the treated biofilms were fixed, dehydrated through a graded ethanol series, sputter-coated with gold, and observed by SEM to assess biofilm integrity and bacterial adhesion. Data analysis was supported by Ceshigo Research Service (www.ceshigo.com). For quantitative analysis, residual biofilm biomass was determined by crystal violet staining. After treatment, the wells were gently washed with PBS, stained with 0.01% (w/v) crystal violet at room temperature for 30 min, and rinsed until the unbound dye was completely removed. After air drying, the bound dye was dissolved in absolute ethanol, and the absorbance at 590 nm was measured using a microplate reader. A lower absorbance value indicated a stronger biofilm removal effect.

4.14. Live/dead staining of biofilms

Bacterial viability within biofilms was evaluated using a SYTO 9/PI Live/Dead bacterial staining kit. After material treatment, mature MRSA and E. coli biofilms were gently washed with sterile PBS to remove planktonic bacteria and residual materials, and then stained with SYTO 9/PI working solution according to the manufacturer's instructions. The stained biofilms were observed by CLSM, where live bacteria showed green fluorescence and dead or membrane-compromised bacteria showed red fluorescence. Z-stack scanning and three-dimensional reconstruction were further performed to assess biofilm integrity and bacterial viability.

4.15. Intracellular ROS detection in macrophages

RAW264.7 macrophages were stimulated with LPS for 12 h to establish an inflammatory model and then treated with different materials for 24 h. After treatment, the cells were washed with PBS and incubated with DCFH-DA working solution at 37 °C for 15 min in the dark according to the manufacturer's instructions. After removing excess probe, intracellular ROS levels were evaluated by fluorescence microscopy and flow cytometry based on green fluorescence intensity.

4.16. Antioxidant activity assay

The antioxidant activity of different materials was evaluated using DPPH, ABTS, and hydroxyl radical (•OH) scavenging assays. The experimental groups were consistent with those used in the in vivo study, including the control, OQ, OQ/P, OQT, and OQT/P groups. For the DPPH scavenging assay, hydrogel samples or the corresponding material extracts from each group were incubated with 0.1 mM DPPH ethanol solution at room temperature in the dark, and the absorbance was measured at 517 nm. For the ABTS assay, the ABTS⁺ working solution was prepared by mixing 7 mM ABTS with 2.45 mM potassium persulfate and allowing the mixture to react in the dark for 24 h. The resulting solution was then diluted to an appropriate concentration and co-incubated with hydrogel samples or the corresponding material extracts from each group, followed by absorbance measurement at 736 nm. The hydroxyl radical scavenging assay was performed based on a Fenton reaction system containing FeSO₄, H₂O₂, salicylic acid, and hydrogel samples or the corresponding material extracts from each group. After reaction, the absorbance was measured at 536 nm. The radical scavenging activity of each group was evaluated by comparison with the control group.

4.17. Macrophage polarization immunofluorescence staining

Macrophage polarization was evaluated by immunofluorescence staining of iNOS and Arg-1 in treated RAW264.7 cells. After treatment, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin. The samples were then incubated overnight at 4 °C with primary antibodies against iNOS or Arg-1, followed by incubation with the corresponding fluorophore-conjugated secondary antibodies under dark conditions. F-actin and nuclei were further stained with TRITC–phalloidin and DAPI, respectively. The stained samples were observed by CLSM to assess macrophage polarization phenotype and cytoskeletal morphology. Macrophage polarization was evaluated by immunofluorescence staining of iNOS and Arg-1 in treated RAW264.7 cells. After treatment, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin. The samples were then incubated overnight at 4 °C with primary antibodies against iNOS or Arg-1, followed by incubation with the corresponding fluorophore-conjugated secondary antibodies under dark conditions. F-actin and nuclei were further stained with TRITC–phalloidin and DAPI, respectively. The stained samples were observed by CLSM to assess macrophage polarization phenotype and cytoskeletal morphology.

4.18. In vitro tube formation assay

Tube formation assays were performed in Matrigel-coated 96-well plates to evaluate the angiogenic activity of HUVECs in response to different materials. Briefly, pre-cooled Matrigel was added to each well and allowed to polymerize at 37 °C. HUVECs suspended in material-containing medium were then seeded onto the gelled Matrigel and incubated for 6 h. Tube-like structures were imaged under an inverted microscope, and the number of junctions and total tube length were quantified using ImageJ.

4.19. Angiogenesis-related immunofluorescence staining

Angiogenesis-related protein expression in HUVECs was evaluated by immunofluorescence staining of VEGF-A and α-SMA after different material treatments. After treatment, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with BSA. The samples were then incubated overnight at 4 °C with primary antibodies against VEGF-A or α-SMA, followed by incubation with the corresponding fluorophore-conjugated secondary antibodies under dark conditions. F-actin and nuclei were further stained with TRITC–phalloidin and DAPI, respectively. The stained samples were observed by CLSM to assess angiogenesis-related protein expression and cell morphology.

4.20. Flow cytometry analysis of macrophage polarization

Macrophage polarization was analyzed by flow cytometry after different material treatments. RAW264.7 macrophages were first stimulated to establish an inflammatory model and then treated with different materials. After treatment, the cells were collected, washed with PBS, and incubated with fluorophore-conjugated antibodies against CD86 and CD206 at 4 °C in the dark. After washing, the cells were analyzed by flow cytometry, and the percentages of CD86-positive and CD206-positive cells were quantified using FlowJo software.

4.21. In vivo MRSA-infected wound model

All animal experiments were conducted in accordance with institutional guidelines and approved by the local Animal Ethics Committee. Healthy male BALB/c mice (6–8 weeks old, 18–22 g) were used in this study and acclimatized for 1 week before the experiments, with free access to food and water. Prior to surgery, the mice were anesthetized with sodium pentobarbital or isoflurane, and the dorsal hair was removed followed by routine sterilization. Under aseptic conditions, a single full-thickness excisional wound with a diameter of 10 mm was created on the dorsal surface of each mouse using a sterile biopsy punch.

Subsequently, 50 μL of MRSA suspension (1 × 10⁸ CFU mL⁻¹) was evenly applied onto the wound surface to establish the infected wound model. After inoculation, the mice were maintained for 48 h to allow bacterial colonization and infection development. The animals were then randomly divided into different treatment groups. OQ, OQ/P, OQT, OQT/P, and 3M dressings were applied to the infected wounds and fixed with medical tape to prevent displacement or detachment. The control group was treated in the same manner but without functional dressings.

Digital photographs of the wounds were taken at predetermined time points (day 0, 3, 6, 9, and 12), and the residual wound area and wound closure ratio were quantified using image analysis software to evaluate the wound-healing efficacy of different materials. At the designated endpoint, the mice were euthanized and wound tissues were harvested for subsequent microbiological, histological, and immunological analyses.

4.22. In vivo antibacterial evaluation

To quantify bacterial burden in wounds, mice were sacrificed on day 6 and wound tissues were harvested, homogenized, serially diluted, and plated on LB agar. After incubation at 37 °C for 24 h, CFUs were counted to determine bacterial loads.

4.23. Histological and immunofluorescence analysis

Wound tissues were collected on days 6 and 12 for histological and immunofluorescence analyses. For ROS detection, fresh wound tissues were prepared as frozen sections with a thickness of 7 μm and stained with dihydroethidium (DHE) to evaluate ROS levels in the wound microenvironment. For histological evaluation, wound tissues were fixed in 4% paraformaldehyde, followed by routine dehydration, paraffin embedding, and sectioning. The tissue sections were then subjected to Giemsa staining, H&E staining, and Masson's trichrome staining to assess bacterial residues, tissue morphology, and collagen deposition, respectively. After staining, the sections were observed and imaged under a microscope.

For histological evaluation, tissues were fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and subjected to Giemsa, H&E, and Masson's trichrome staining to assess bacterial residues, tissue morphology, and collagen deposition, respectively. Immunofluorescence staining was performed using antibodies against IL-6, Col-I, CD86, CD206, CD31, and α-SMA, followed by DAPI counterstaining. In addition, immunohistochemical staining of TNF-α was conducted to assess inflammatory responses in wound tissues.

4.24. Biosafety evaluation

For systemic biosafety evaluation, major organs (heart, liver, spleen, lung, and kidney) were harvested on day 12, fixed, paraffin-embedded, sectioned, and subjected to H&E staining. Blood samples were also collected for routine hematological analysis to evaluate the systemic biocompatibility of the bilayer dressings.

4.25. mRNA transcriptome sequencing analysis

To investigate the molecular mechanisms underlying the therapeutic effects of OQT/P, wound tissues from different treatment groups were collected on day 12 for transcriptomic sequencing analysis. Total RNA was extracted using TRIzol reagent according to the manufacturer's instructions. RNA quality and integrity were assessed using a NanoDrop spectrophotometer and 5300 bioanalyzer (Agilent), and qualified samples were used for subsequent library construction. mRNA libraries were prepared using an Illumina-based strategy and sequenced on a NovaSeq X Plus platform with paired-end 150 bp reads (PE150).

Raw sequencing data were subjected to quality control to obtain clean data, which were then aligned to the Mus musculus reference genome (GRCm39, Ensembl). Gene expression levels were quantified using RSEM, and differentially expressed genes (DEGs) were identified using DESeq2 with thresholds of P < 0.05 and |log2FC| ≥ 0.585. GO and KEGG enrichment analyses were subsequently performed to identify significantly regulated biological processes and signaling pathways, and GSEA was further conducted to evaluate pathway-level alterations at the transcriptomic level. The data were analyzed on the Majorbio Cloud Platform.

4.26. Statistical analysis

Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc multiple comparisons test. Data are presented as mean ± standard deviation (SD) from at least three independent experiments (n ≥ 3). Statistical significance was defined as ∗p < 0.05, with ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 indicating increasing levels of significance.

CRediT authorship contribution statement

Jianan Li: Writing – original draft, Validation, Software, Methodology, Investigation, Data curation, Conceptualization. Zhongwu Bei: Methodology, Investigation, Conceptualization. Jian Hua: Methodology, Investigation. Meng Wang: Methodology, Investigation. Yujia Wei: Methodology, Investigation. Ying Qu: Methodology. Bingyang Chu: Methodology. Yun Yang: Methodology. Dong Mo: Investigation. Shiyu Liang: Investigation. Xicheng Li: Investigation. Qingya Liu: Investigation. Meng Pan: Investigation. Yutong Qian: Investigation. Xiaorui Yu: Investigation. Zhiyong Qian: Writing – review & editing, Supervision, Funding acquisition. Xiang Gao: Writing – review & editing, Supervision, Conceptualization. Yongzhong Cheng: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

Ethics approval and consent to participate

All animal procedures were conducted in accordance with the guidelines approved by the Ethics Committee of the Animal Experimental Center, State Key Laboratory of Biotherapy, Sichuan University (Approval No. 20241127014). All experiments complied with the applicable institutional and national regulations for the care and use of laboratory animals.

Declaration of competing interest

Zhiyong Qian is an editorial board member for Bioactive Materials and was not involved in the editorial review or the decision to publish this article. All authors declare that there are no competing interests.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2024YFA1210202), the Sichuan Science and Technology Program (No. 2023NSFSC1931, China).

Footnotes

Peer review under the responsibility of editorial board of Bioactive Materials.

^{Appendix A}

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioactmat.2026.05.013.

Contributor Information

Xiang Gao, Email: xianggao@scu.edu.cn.

Yongzhong Cheng, Email: chengyz@scu.edu.cn.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Multimedia component 1

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Multimedia component 2

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Data availability

The data that support the findings of this study are available from the authors upon request.

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Data Availability Statement

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PERMALINK

A microenvironment-adaptive bilayer composite dressing for disrupting MRSA biofilms and promoting wound regeneration via ROS-mediated immune regulation

Jianan Li

Zhongwu Bei

Jian Hua

Meng Wang

Yujia Wei

Ying Qu

Bingyang Chu

Yun Yang

Dong Mo

Shiyu Liang

Xicheng Li

Qingya Liu

Meng Pan

Yutong Qian

Xiaorui Yu

Zhiyong Qian

Xiang Gao

Yongzhong Cheng

Abstract

Graphical abstract

Highlights

1. Introduction

Scheme 1.

2. Results and discussion

2.1. Synthesis and characterization of TCP

Fig. 1.

2.2. pH-dependent redox regulation behavior of TCP

2.3. Preparation and characterization of QCS–CGA, ODEX–DA, and OQT hydrogels

Fig. 2.

2.4. Injectability, self-healing, and rheological properties of OQT hydrogels

2.5. Structural features and multifunctional performance of the asymmetric bilayer dressing

Fig. 3.

2.6. Antibacterial activity and underlying mechanisms

Fig. 4.

2.7. Evaluation of antibacterial ability against biofilms in vitro

Fig. 5.

2.8. In vitro cyto-compatibility and hemocompatibility

Fig. 6.

2.9. ROS scavenging–mediated anti-inflammatory and immunomodulatory effects

Fig. 7.

2.10. Pro-angiogenic effects of the bilayer dressing

Fig. 8.

2.11. In vivo evaluation of the OQT/P bilayer dressing for the treatment of MRSA biofilm–infected wounds

Fig. 9.

2.12. Histological and immunofluorescence evidence of stepwise wound microenvironment regulation in vivo

Fig. 10.

2.13. Transcriptomic analysis reveals immune–inflammatory reprogramming induced by OQT/P

Fig. 11.

3. Conclusion

4. Experimental section

4.1. Materials

4.2. Synthesis and characterization of TCP NPs

4.3. Synthesis and characterization of QCS–CGA

4.4. Synthesis and characterization of ODEX–DA

4.5. Preparation and characterization of OQ and OQT hydrogels

4.6. Degradation assay of OQT hydrogel

4.7. Preparation of Cur@PLGA electrospun fibrous membrane

4.8. Bacterial culture

4.9. In vitro antibacterial activity

4.10. Bacterial morphology observation by SEM

4.11. Live/dead fluorescence staining of bacteria

4.12. Intracellular ROS and protein leakage assays

4.13. In vitro antibiofilm assay

4.14. Live/dead staining of biofilms

4.15. Intracellular ROS detection in macrophages

4.16. Antioxidant activity assay

4.17. Macrophage polarization immunofluorescence staining

4.18. In vitro tube formation assay

4.19. Angiogenesis-related immunofluorescence staining

4.20. Flow cytometry analysis of macrophage polarization

4.21. In vivo MRSA-infected wound model

4.22. In vivo antibacterial evaluation

4.23. Histological and immunofluorescence analysis

4.24. Biosafety evaluation

4.25. mRNA transcriptome sequencing analysis

4.26. Statistical analysis

CRediT authorship contribution statement

Ethics approval and consent to participate