NIR-responsive mild photothermal hydrogels for multimodal antibacterial therapy, immunomodulation, and regenerative repair of MRSA-infected wounds

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA)-infected wounds are difficult to heal because of persistent bacterial colonization, prolonged inflammatory responses, and restricted tissue regeneration. In this study, a near-infrared (NIR)-responsive photocurable hydrogel system (ZPPE@PLHA) was developed for localized anti-infective therapy and tissue repair by incorporating ZnO/PDA/PEG/rhEGF composite nanostructures into a PLMA/HAMA matrix. The results showed that this hydrogel enabled sustained Zn²⁺ release and exhibited a stable mild photothermal effect (mPTT) under 808 nm NIR irradiation, with the temperature rising to approximately 48.6 °C within 10 min. In vitro experiments demonstrated that the ZPPE@PLHA hydrogel possessed intrinsic antibacterial activity co-mediated by Zn²⁺ and ε-PL, and that NIR irradiation further enhanced its inhibitory effects against MRSA and E. coli. Phenotypic observations and bacterial transcriptomic analyses indicated that this antibacterial effect was associated with membrane damage, metabolic disruption, and suppression of translation-related processes. Meanwhile, ZPPE@PLHA attenuated macrophage inflammatory activation and promoted M2-associated polarization in both classically activated and lipoteichoic acid (LTA)-stimulated macrophages, and enhanced endothelial cell migration and tube formation. In an MRSA-infected rat wound model, treatment with ZPPE@PLHA hydrogel combined with NIR significantly reduced the MRSA burden, improved the macrophage-mediated inflammatory microenvironment, and accelerated wound closure and angiogenesis, accompanied by increased EGFR expression and enhanced phosphorylation of PI3K, AKT, and ERK. By integrating antibacterial, immunomodulatory, and pro-regenerative activities, this hydrogel provides a full-process therapeutic strategy for the management of acute infected wounds.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-026-04664-z.

Introduction

Skin wounds are commonly caused by various forms of trauma and are often associated with severe pain and delayed healing [1]. Disruption of the body’s primary antimicrobial barrier increases wound susceptibility to microbial invasion, which can lead to chronic infection [2, 3]. With the widespread use of antibiotics, infections caused by antibiotic-resistant bacteria have become a major global public health threat, particularly those involving methicillin-resistant Staphylococcus aureus (MRSA). MRSA frequently causes severe skin and soft tissue infections and is associated with high mortality, complications, and impaired wound healing [4, 5]. At the microscopic level, MRSA can induce persistent inflammation, immune dysregulation, and destruction of the skin barrier and extracellular matrix. Briefly, MRSA infection not only markedly promotes the excessive expression of IL-1β, IL-6, IL-8, and the NLRP3 inflammasome in keratinocytes, but also inhibits keratinocyte migration and re-epithelialization, ultimately leading to delayed wound healing [6, 7]. The infectious wound healing process involves several complex stages, including hemostasis, inflammation, cell proliferation, and tissue remodeling, which often occur alternately [8].

Although conventional wound management strategies and antibiotics can promote wound healing to some extent, they have several limitations in the treatment of infected wounds. Common dressings, such as gauze, polymer bandages, and cotton, provide physical protection, absorb exudates, and help maintain a moist wound environment; however, they have limited ability to prevent bacterial infection or actively promote tissue repair [9]. Frequent dressing changes also increase treatment and may cause secondary damage to newly regenerated tissue [10]. Moreover, the continuous emergence of resistant bacterial strains has gradually reduced the effectiveness of existing antibiotics, while prolonged antibiotic use may cause severe adverse effects or systemic toxicity [11, 12]. Importantly, successful wound healing depends not only on bacterial clearance but also on coordinated physiological processes, including angiogenesis, collagen deposition, and cell proliferation [13, 14]. However, conventional treatments primarily focus on wound cleansing or antimicrobial therapy and often neglect the critical roles of immune regulation and tissue regeneration in wound repair [15]. Therefore, there is an urgent need to develop multifunctional therapeutic strategies that can efficiently eradicate infection, attenuate inflammation, promote angiogenesis, and accelerate the healing of infected wounds.

Hydrogels have been widely investigated for the treatment of infected wounds because of their high water content, excellent biocompatibility, and three-dimensional network structure resembling the natural extracellular matrix (ECM) [16, 17]. They can absorb wound exudates, maintain a continuously moist healing microenvironment, and support cell adhesion, migration, and proliferation [18]. Hyaluronic acid methacrylate (HAMA), derived from hyaluronic acid, an important component of the skin ECM, exhibits excellent water retention capacity and biocompatibility. After crosslinking, HAMA forms a porous, stable, and soft scaffold that provides a supportive microenvironment favorable for tissue regeneration and has been widely used in drug delivery and tissue repair applications [19]. Natural ε-polylysine (ε-PL) is a cationic polypeptide composed of L-lysine units linked by amide bonds. As an FDA-approved biomaterial, ε-PL exhibits good biocompatibility and biodegradability [20]. Owing to its abundant amino groups, ε-PL can adsorb onto microbial cell surfaces through electrostatic interactions and disrupt membrane integrity, thereby exerting broad-spectrum antibacterial activity [21]. Recent studies have further demonstrated that ε-PL-based materials not only possess antibacterial properties but also promote cell adhesion and ECM deposition, highlighting their potential in tissue engineering and wound repair [22]. PLMA, obtained through methacrylation modification of ε-PL, endows hydrogels with rapid photopolymerization capability, while its cationic nature contributes to improved mechanical properties and biological functionality. Therefore, the network structure formed by combining PLMA with HAMA integrates the advantages of both components and provides an effective platform for antimicrobial therapy and wound repair.

ZnO nanoparticles (NPs) have attracted considerable attention as antimicrobial and repair-promoting functional materials because of their multiple biological effects. ZnO exhibits anti-inflammatory activity, and Zn²⁺ ions released in aqueous environments can disrupt bacterial membrane structures, inhibit metabolic activity, and interfere with genetic material synthesis, thereby producing sustained antibacterial effects [23, 24]. As an essential trace element for skin health, Zn²⁺ also regulates several wound healing-related processes, including cell proliferation, collagen deposition, and angiogenesis [25, 26]. Additionally, epidermal growth factor (EGF) is an important bioactive regulator involved in multiple stages of wound repair. By activating the EGF receptor (EGFR) signaling pathway, EGF promotes the proliferation and migration of keratinocytes and fibroblasts, thereby accelerating re-epithelialization and tissue remodeling [27]. Previous studies have demonstrated that EGF significantly promotes the repair of chronic wounds and diabetic ulcers, particularly by enhancing epidermal reconstruction and angiogenesis [28–30]. However, EGF is susceptible to degradation in vivo and has a short duration of action, which limits its clinical application. Therefore, suitable delivery systems are needed to achieve localized and sustained EGF release.

Polydopamine (PDA) is a bioinspired polymer with broadband light absorption and photothermal conversion properties similar to those of natural melanin [31]. Under near-infrared (NIR) irradiation, PDA-based nanomaterials can efficiently and controllably convert light energy into heat, enabling mild photothermal therapy (mPTT). Compared with conventional high-temperature photothermal therapy (> 60 °C), mPTT typically maintains the local temperature at a relatively low level (< 50 °C). This approach enhances local thermal stress, disrupts bacterial membrane structures, and inhibits pathogen proliferation while minimizing thermal damage to surrounding healthy tissues, thereby improving treatment safety and biocompatibility [32, 33]. Recent studies have shown that PDA-based nanomaterials can also synergistically enhance other antimicrobial strategies, such as antimicrobial drugs or functional nanomaterials, enabling efficient eradication of refractory pathogens and optimization of the wound microenvironment [34, 35]. Furthermore, PDA surfaces are rich in active functional groups, allowing covalent or non-covalent modification with various biomolecules, such as growth factors or peptides. This property further enhances cell adhesion and supports tissue regeneration in addition to the photothermal effect, thereby benefiting multiple physiological processes involved in skin wound repair [36, 37].

To address the requirements for antibacterial management, inflammation and immune regulation, and tissue remodeling at different stages of infected wound treatment, we designed and synthesized a ZnO NP-based composite photocurable hydrogel system, termed ZPPE@PLHA, for the comprehensive management of MRSA-infected wounds (Scheme 1). Briefly, PDA, carboxylated polyethylene glycol, and recombinant human EGF (rhEGF) were sequentially introduced onto the surface of ZnO NPs through self-polymerization and amidation reactions to construct multifunctional ZPPE NPs. To enable localized and sustained release, ZPPE NPs were incorporated into a PLHA matrix formed by the co-crosslinking of PLMA and HAMA, resulting in an injectable photocurable ZPPE@PLHA hydrogel system. Under NIR irradiation, this system is expected to enhance MRSA inhibition through the combined effects of mild photothermal therapy, Zn²⁺ release, and ε-PL-mediated cationic antibacterial activity. Meanwhile, released Zn²⁺ promotes macrophage-mediated immune regulation and anti-inflammatory responses, thereby establishing a favorable inflammatory and immune microenvironment for wound healing and supporting tissue remodeling after MRSA eradication. Furthermore, the sustained release of Zn²⁺ and rhEGF synergistically promotes fibroblast migration and angiogenesis, further accelerating wound repair. Additionally, the PLHA hydrogel matrix supports tissue regeneration by maintaining a moist microenvironment and promoting cell adhesion and collagen deposition. Overall, by integrating antibacterial activity, inflammatory microenvironment modulation, and tissue repair support, ZPPE@PLHA may provide a full-process therapeutic strategy for promoting the healing of MRSA-infected wounds.

Scheme 1.

Schematic illustration of the design, synthesis, and therapeutic effect of ZPPE@PLHA hydrogel for MRSA-infected wound healing. PDA, carboxyl-terminated polyethylene glycol, and recombinant human EGF (rhEGF) were sequentially introduced onto the surface of ZnO nanoparticles (NPs) through self-polymerization and amidation reactions to construct multifunctional ZPPE NPs. These ZPPE NPs were then incorporated into a photopolymerizable hydrogel system, ZPPE@PLHA, formed by the crosslinking of hyaluronic acid methacrylate (HAMA) and poly-L-lysine methacrylate (PLMA). Under near-infrared (NIR) irradiation, ZPPE@PLHA may enhance MRSA clearance through the combined effects of mild photothermal therapy, Zn²⁺ release, and ε-PL-mediated bacterial membrane disruption. Sustained Zn²⁺ release promotes macrophage polarization from the pro-inflammatory M1 phenotype toward the anti-inflammatory M2 phenotype, thereby creating a favorable inflammatory microenvironment for wound repair. In addition, rhEGF release may increase EGFR expression and enhance the phosphorylation of PI3K, AKT, and ERK, contributing to angiogenesis and tissue remodeling

Results and discussion

Synthesis and characterization of ZPPE

The preparation process of ZPPE nanocomposites is illustrated in Fig. 1A. Briefly, ZnO NPs were used as the core substrate, and PDA-coated ZnO NPs (ZnO/PDA) were readily constructed through the self-polymerization of dopamine on the ZnO surface. Subsequently, bifunctional carboxylated polyethylene glycol was used as a linker to conjugate rhEGF onto the NP surface via amide bond formation, resulting in the successful construction of ZPPE NPs. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images showed that the ZPPE NPs exhibited a spherical core–shell structure (Figs. 1B, C). The slightly irregular morphology was likely attributable to partial aggregation of ZnO NPs and PDA self-polymerization on the ZnO surface, consistent with previously reported PDA-coated nanomaterials [38, 39]. Owing to the presence of abundant uncoordinated Zn²⁺ ions on the ZnO surface, ZnO NPs exhibited a strong positive charge, with a zeta potential of 12.37 mV [40]. After PDA coating, the zeta potential markedly decreased to approximately − 7.85 mV, indicating the successful deposition of PDA [41]. Because the PDA layer contains protonatable amine groups and phenolic hydroxyl groups, these groups undergo deprotonation under alkaline conditions as the pH increases, resulting in a predominantly negative surface charge and a shift in the net surface charge from positive to negative [42]. After conjugation with bifunctional carboxylated PEG and rhEGF, the zeta potential slightly increased to approximately − 5.77 mV (Figure S1). Dynamic light scattering (DLS) analysis showed that the ZPPE NPs had an average diameter of approximately 45 nm (Figure S2). Energy-dispersive spectroscopy (EDS) analysis of the SEM images revealed the uniform distribution of Zn, O, C, N, and S within the ZPPE NPs (Fig. 1D). The elemental contents of Zn, O, C, N, and S were determined to be 67.56%, 17.51%, 13.02%, 1.19%, and 0.72%, respectively (Figure S3).

Fig. 1.

Synthesis, structural characterization, and photothermal properties of ZPPE nanoparticles (NPs). (A) Schematic illustration of the stepwise fabrication of ZPPE NPs, including PDA coating on ZnO NPs, followed by conjugation with HOOC-PEG-COOH and rhEGF. (B) SEM images of ZnO NPs and ZPPE NPs. Scale bars: 400 nm. (C) TEM images of ZPPE NPs at different magnifications. Scale bars: 100 nm (left), 50 nm (middle), and 20 nm (right). (D) SEM image and corresponding EDS elemental mapping of ZPPE NPs, showing the homogeneous distribution of Zn, O, C, N, and S elements. Scale bars: 5 μm. (E) FTIR spectra of ZnO NPs, ZnO/PDA, ZnO/PDA/PEG, and ZPPE NPs. (F) XRD patterns of ZnO NPs and ZPPE NPs. (G) XPS survey spectrum of ZPPE NPs. High-resolution XPS spectra of Zn 2p (H), C 1s (I), N 1s (J), O 1s (K), and S 2p (L), confirming the chemical states of ZnO and the successful introduction of PDA, PEG, and bioactive components onto the NP surface. (M) Infrared thermal images of ZPPE NP solutions under 808 nm NIR laser irradiation at different power densities (0.25–2.0 W/cm²). (N) Photothermal heating profiles of ZPPE NPs at different laser power densities (0.25, 0.5, 1.0, and 2.0 W/cm²). (O) Temperature elevation (ΔT) curves of water, ZP, and ZPPE under continuous NIR irradiation. (P) Concentration-dependent photothermal heating curves of ZPPE NPs (0, 1.0, 2.0, 5.0 and 10.0 mg/mL). (Q) Photothermal stability of ZPPE NPs over five repeated irradiation on/off cycles, demonstrating excellent photothermal durability and repeatability

Fourier transform infrared spectroscopy (FTIR) was performed to verify the chemical modifications at each preparation stage (Fig. 1E). ZnO NPs showed a characteristic absorption peak at approximately 390 cm⁻¹, corresponding to Zn-O stretching vibration, a typical feature of ZnO [43]. After PDA coating, a new absorption peak appeared at 1560 cm⁻¹, which was attributed to aromatic C = C stretching and N-H bending vibrations [44], indicating the successful polymerization and deposition of PDA on the ZnO surface. Furthermore, compared with pristine ZnO NPs, the FTIR peaks of ZnO/PDA were shifted, consistent with previous reports on PDA-coated nanomaterials [38]. After further modification with bifunctional carboxylated PEG, new peaks appeared at 2885 cm⁻¹ and 1100 cm⁻¹, corresponding to C-H stretching and C-O-C stretching vibrations, respectively, confirming successful PEG grafting onto the PDA surface [45]. The C = O absorption peak at 1710 cm⁻¹ was associated with amide bond formation (-CO-NH-) [46], further demonstrating the successful coupling between the carboxyl groups of PEG and the amino groups of PDA via the EDC/NHS reaction. After final conjugation with rhEGF, the ZPPE spectrum retained these characteristic peaks. The reduced absorption intensity may be attributed to hydrogen bonding and electrostatic interactions between the rhEGF molecules and PEG chains, which restricted the free vibration of functional groups [47]. The XRD patterns of pristine ZnO NPs and ZPPE nanocomposites are shown in Fig. 1F. The diffraction peaks at 2θ = 31.8°, 34.4°, 36.3°, 47.5°, 56.6°, 62.8°, 66.3°, 67.9°, 69.1°, 72.6°, and 77.0° correspond to the (100), (002), (101), (102), (110), (103), (200), (112), (201), (004), and (202) crystal planes of hexagonal wurtzite ZnO, respectively [48, 49]. The sharp and intense diffraction peaks indicated good crystallinity of ZnO NPs. After surface modification with PDA, PEG, and rhEGF, the diffraction peaks of ZPPE remained at the same positions as those of pristine ZnO, with no additional or shifted peaks, suggesting that the crystalline structure of ZnO was preserved during the functionalization. However, a slight decrease in peak intensity was observed, which may be attributed to the organic coating layers partially shielding the ZnO surface and reducing the diffraction signal [50]. These results indicate that the ZnO core retained its wurtzite crystalline phase, while surface functionalization primarily occurred through chemical modification rather than structural transformation. The surface elemental composition and chemical bonding states from the ZnO core to the final ZPPE nanocomposites were further analyzed by X-ray photoelectron spectroscopy (XPS) (Fig. 1G, Figure S4). The XPS survey spectra demonstrated the stepwise introduction of characteristic elements during each modification stage. Pristine ZnO displayed only Zn 2p and O 1s peaks. After PDA coating (ZnO/PDA), C 1s and N 1s signals appeared. Following PEG conjugation (ZnO/PDA/PEG), the O signal intensity increased. Finally, after rhEGF loading (ZPPE), a characteristic S peak appeared at approximately 164 eV, confirming the successful incorporation of rhEGF. High-resolution XPS spectra provided further evidence of chemical bonding at each modification stage (Figure S5). In pristine ZnO, the Zn 2p spectrum showed two peaks at 1021.50 eV (Zn 2p₃/₂) and 1044.55 eV (Zn 2p₁/₂) (Fig. 1H), with a binding energy separation of approximately 23.05 eV. This result is consistent with the standard Zn²⁺ state in ZnO [51], indicating that Zn existed in an oxidized state. In ZPPE, the stable Zn 2p doublet at 1021.60 eV and 1044.55 eV indicated that the chemical state of the ZnO core remained unchanged during surface modification. The C 1s spectrum of ZPPE (Fig. 1I) was deconvoluted into two main peaks at 284.80 eV and 288.80 eV, corresponding to C = C and C = O bonds, respectively. These signals were associated with the aromatic structure of PDA and the carbonyl groups in PEG/EGF, confirming the successful modification of the organic layer on the ZnO surface [52]. The N 1s spectrum (Fig. 1J) showed peaks at 399.15 eV and 401.50 eV, which were assigned to -NH₂ or amide nitrogen and protonated nitrogen (N⁺), respectively. These results further suggest that the amine groups in PDA and EGF participated in the surface coupling reaction and formed stable amide bonds. In the O 1s spectrum (Fig. 1K), the peaks at 530.30 eV and 531.85 eV correspond to lattice oxygen in Zn-O and surface Zn-OH, respectively [53], indicating that organic species covered the ZnO surface. Additionally, the S 2p spectrum (Fig. 1L) showed two peaks at 163.45 eV (S 2p₃/₂) and 164.55 eV (S 2p₁/₂), which originated from disulfide bonds in rhEGF [54], further confirming the successful loading of EGF onto the composite surface. Overall, the XPS results confirmed the presence of Zn, O, C, N, and S in ZPPE samples, with chemical states consistent with the designed structure, verifying the successful PDA coating, PEG modification, and rhEGF conjugation.

Additionally, the PDA coating endowed ZPPE NPs with mild photothermal properties under NIR stimulation. To evaluate their photothermal conversion capability, the temperature elevation of ZPPE NPs under 808 nm NIR laser irradiation was systematically investigated. At different laser power densities (Figs. 1M, N), the ΔT of ZPPE increased as the power density rose from 0.25 to 2.0 W/cm², with the maximum ΔT exceeding 40 °C, indicating a clear power-dependent photothermal response. Infrared thermal images acquired after 10 min of irradiation showed that pure water exhibited almost no obvious temperature change, remaining at approximately 23 °C, whereas ZP group displayed a marked temperature increase, reaching a maximum of approximately 43.0 ℃ (Fig. 1O, Figure S6). Compared with the ZP group and pure water control, the ZPPE group showed a further temperature increase to 45.7 °C, demonstrating its strong NIR responsiveness. The concentration-dependent photothermal performance of ZPPE was further evaluated (Fig. 1P, Figure S7). The results showed that the ΔT increased with ZPPE concentration from 1 to 10 mg/mL, reaching a maximum of 30 °C, indicating good concentration-dependent behavior. A single irradiation on/off experiment (Figure S8) showed that ZPPE rapidly heated during laser irradiation and quickly cooled after irradiation was stopped, confirming its reversible photothermal response. After five irradiation cycles (Fig. 1Q), the temperature profiles showed no noticeable attenuation, demonstrating excellent photothermal stability and repeatability. Overall, ZPPE NPs exhibited distinct power- and concentration-dependent temperature elevation under NIR irradiation, together with favorable photothermal conversion efficiency and stability. Notably, after 10 min of 808 nm NIR irradiation, the temperature of ZPPE NPs remained stable at approximately 45 °C, which falls within the typical range of mPTT. This temperature range is generally considered effective for bacterial eradication, promotion of blood circulation, and cellular repair, while avoiding thermal damage to normal tissues [55, 56]. Therefore, the photothermal properties of ZPPE suggest its potential value as an mPTT platform for infected skin wound repair.

Fabrication and characterization of ZPPE@PLHA hydrogels

To achieve the injectable delivery and local sustained release of ZPPE NPs in infected wound microenvironments, while simultaneously enabling anti-infective and tissue-repair functions, a composite photopolymerizable hydrogel system containing ZPPE NPs (ZPPE@PLHA) was fabricated. This system consists of a PLHA matrix formed by the crosslinking of PLMA and HAMA, together with uniformly dispersed ZPPE NPs. Specifically, PLMA was synthesized through the methacrylation of ε-PL with glycidyl methacrylate (GMA), which introduced double bonds into the molecular chains and endowed the polymer with photopolymerization capability. HAMA was prepared by incorporating methacrylate groups into the hyaluronic acid backbone, thereby conferring similar light-responsive properties. As shown in Fig. 2A, the ZPPE@PLHA hydrogel rapidly gelled within 20 s under 405 nm light irradiation, demonstrating excellent photopolymerization performance. The pure PLHA hydrogel appeared semitransparent, whereas the ZPPE-containing hydrogel exhibited a uniform brown color, indicating the homogeneous dispersion of ZPPE NPs within the hydrogel matrix. Figure 2B further shows the good molding integrity of different PLHA-based hydrogels. SEM results (Fig. 2C) revealed that PLHA possessed a porous, sponge-like structure with relatively uniform pore sizes. After the introduction of ZP and ZPPE, the pore walls became slightly thicker and the pore structure became denser, suggesting that ZPPE incorporation enhanced the structural support of the hydrogel. Additionally, ZPPE NPs were evenly distributed on the hydrogel surface and within the pores. SEM-assisted element mapping images (Fig. 2D) confirmed the presence and homogeneous distribution of Zn, O, C, N, and S, further verifying the uniform dispersion of ZPPE NPs throughout the hydrogel system.

Fig. 2.

Fabrication and physicochemical characterization of ZPPE@PLHA hydrogels. (A) Photographs showing the rapid in situ gelation of PLHA and ZPPE@PLHA hydrogels under 405 nm UV irradiation, demonstrating excellent photo-crosslinking capability. (B) Representative images of PLHA-, ZP@PLHA-, and ZPPE@PLHA-based hydrogels, showing uniform appearance and good structural integrity after gelation. (C) SEM images of PLHA, ZP@PLHA, and ZPPE@PLHA hydrogels. Scale bars: 100 μm. (D) SEM image and corresponding EDS elemental mapping of ZPPE@PLHA hydrogels, showing the homogeneous distribution of Zn, O, C, N, and S elements within the hydrogel matrix. Scale bars: 5 μm. (E) FTIR spectra of ZnO@PLHA, ZP@PLHA, and ZPPE@PLHA hydrogels. (F) XRD patterns of PLHA and ZPPE@PLHA hydrogels. (G) Photograph demonstrating the injectability of ZPPE@PLHA hydrogels, as evidenced by extrusion through a syringe and free-form shaping. (H) Photographs illustrating the adhesive properties of ZPPE@PLHA hydrogels at different bending angles (90°, 150°, and 180°). (I) Strain-dependent rheological properties of PLHA and ZPPE@PLHA hydrogels, showing the storage modulus (G′) and loss modulus (G″) as a function of strain. (J) Frequency-dependent rheological behavior of PLHA and ZPPE@PLHA hydrogels

FTIR and XRD analyses (Figs. 2E, F) further confirmed the successful incorporation of ZPPE into the PLHA hydrogel. In the FTIR spectrum, the characteristic C = O and C-O-C peaks of PLHA were retained after ZnO NPs incorporation, while a Zn-O absorption peak appeared, indicating the successful integration of ZnO NPs into the PLHA hydrogel. Notably, after modification with COOH-PEG-COOH and rhEGF, new characteristic peaks were observed, including a C-H stretching vibration peak at 2900 cm⁻¹, an enhanced C-O-C stretching vibration peak at 1150 cm⁻¹, and a C = O stretching vibration peak at 1638 cm⁻¹ [57, 58]. These results further confirmed the successful construction of ZPPE@PLHA. Additionally, the XRD pattern of ZPPE@PLHA retained the main diffraction peaks of ZnO, including those corresponding to the (100), (002), (101), (102), (110), and (103) planes [48], indicating that the incorporation into the hydrogel did not alter the crystalline structure of ZnO.

The injectability and adhesive properties of the ZPPE@PLHA hydrogel were further evaluated. As shown in Fig. 2G, the hydrogel could be smoothly extruded through a standard syringe and molded into various shapes under external force, demonstrating good injectability. Figure 2H shows that the hydrogel firmly adhered to the substrate at different angles, including 180°, 150°, and 90°, indicating favorable tissue adhesion, which is beneficial for wound coverage and localized fixation. Rheological strain sweep analysis (Fig. 2I) showed that, as the strain increased from 0.1% to 100%, both PLHA and ZPPE@PLHA hydrogels transitioned from a state in which the storage modulus (G′) exceeded the loss modulus (G″) to one in which G″ exceeded G′. A distinct crossover point appeared at approximately 10–20% strain, indicating network disruption under high strain and suggesting good elasticity and mechanical stability within the linear viscoelastic region. Dynamic frequency sweep analysis (Fig. 2J) further showed that G′ remained higher than G″ for both PLHA and ZPPE@PLHA hydrogels across the frequency range of 0.1–10 Hz, with relatively stable modulus changes. These results indicate that both hydrogels exhibited typical solid-like elastic behavior and possessed stable three-dimensional crosslinked network structures.

Drug release and photothermal performance of ZPPE@PLHA hydrogels

To systematically evaluate the stability and functionality of ZPPE@PLHA hydrogels under different conditions, their degradation behavior, Zn²⁺ release profile, swelling capacity, and photothermal response were comprehensively investigated. In SPF (pH 7.4), both PLHA and ZPPE@PLHA hydrogels exhibited gradual degradation over time (Fig. 3A). The ZPPE@PLHA hydrogel showed a slower overall degradation rate, with approximately 45% of its initial mass remaining after 21 days. This result suggests that ZPPE incorporation enhanced the structural stability of the hydrogel network, possibly through hydrogen bonding and electrostatic interactions between ZPPE NPs and ε-PL chains, which helped prevent rapid hydrogel degradation [59]. Under simulated mildly acidic conditions associated with infected wounds (pH 6.5) (Fig. 3B), the hydrogel degraded slightly faster than under physiological pH conditions, with less than 40% of the initial mass remaining in the ZPPE@PLHA group after 21 days. This finding indicates the pH-responsive behavior of the system, which may facilitate accelerated degradation and release in the infected skin microenvironment while minimizing interference with normal tissue growth. In addition, in the presence of hyaluronidase (Figure S9), hydrogel degradation was markedly accelerated, indicating that the hydrogel could be enzymatically metabolized under physiological conditions, which is beneficial for subsequent tissue regeneration.

Fig. 3.

Degradation behavior, Zn²⁺ release, and photothermal performance of ZPPE@PLHA hydrogels. (A) In vitro degradation profiles of PLHA and ZPPE@PLHA hydrogels in simulated physiological fluid (SPF, pH 7.4), showing gradual mass loss over time. (B) In vitro degradation behavior of PLHA and ZPPE@PLHA hydrogels in acetate buffer (pH 6.5), mimicking the mildly acidic microenvironment of infected wounds. (C) Cumulative release profiles of Zn²⁺ from ZPPE@PLHA hydrogels in SPF (pH 7.4) and acetate buffer (pH 6.5). (D) Flowchart illustrating the procedure for evaluating the photothermal performance of the hydrogel. (E) Infrared thermal images of PLHA, ZP@PLHA, and ZPPE@PLHA hydrogels under 808 nm NIR laser irradiation at different time points. (F) Temperature elevation (ΔT) curves of water, ZP@PLHA, and ZPPE@PLHA hydrogels under continuous NIR irradiation. (G) Photothermal heating profiles of ZPPE@PLHA hydrogels under different laser power densities (0.25, 0.5, 1.0, and 2.0 W/cm²). (H) Concentration-dependent photothermal heating curves of ZPPE@PLHA hydrogels with varying ZPPE contents (0, 0.25, 0.5, 1.0, and 2.0 wt%)

To evaluate the water absorption and moisture-retention capacity of the hydrogels in a wound environment, their swelling behavior was further examined (Figure S10). All three hydrogels, including PLHA, ZP@PLHA, and ZPPE@PLHA, exhibited rapid water uptake during the initial stage within 4 h and reached swelling equilibrium after approximately 12 h. Compared with PLHA, ZPPE@PLHA showed a slightly lower equilibrium swelling ratio, which may be attributed to the formation of multiple physical crosslinks by ZPPE particles within the gel network. These interactions increased the density of the internal structure and thereby limited excessive water penetration [60]. Nevertheless, ZPPE@PLHA maintained a high water content, enabling rapid exudate absorption and the maintenance of a locally moist microenvironment, which is essential for promoting cell migration and re-epithelialization [61]. ZnO NPs in this hydrogel system are expected to contribute to rapid contact-mediated antibacterial activity, immune regulation, and potential angiogenesis, thereby supporting multiple stages of infected wound repair. Therefore, understanding the Zn²⁺ release kinetics from the PLHA hydrogel is critical. The ZPPE@PLHA exhibited sustained and controllable Zn²⁺ release under both pH conditions, with a slightly higher release rate under mildly acidic conditions (pH 6.5). The released Zn²⁺ concentration reached approximately 251 µg/L after 14 days (Fig. 3C). This release pattern, characterized by a small initial burst followed by slow and stable release, may enable rapid bacterial eradication and sustained antimicrobial activity while avoiding cytotoxicity caused by excessive metal ion accumulation [62]. Thus, the system provides a favorable microenvironment for long-term wound repair.

The consistency and stability of the mPTT response were further evaluated after loading ZPPE NPs into the PLHA hydrogel (Fig. 3D). Under 808 nm NIR irradiation, the ZPPE@PLHA hydrogel exhibited a rapid temperature rise, with the central temperature reaching approximately 48.6 °C within 10 min. The corresponding thermal images showed uniform heat distribution, indicating the homogeneous dispersion of ZPPE NPs within the hydrogel matrix (Figs. 3E, F). Additional photothermal curves showed that the photothermal performance of ZPPE@PLHA was strongly dependent on both laser power density and ZPPE NP concentration. The temperature increase was enhanced as the laser power density increased from 0.25 to 2.0 W/cm² and as the ZPPE NP content increased (Figs. 3G, H, Figure S11, S12), consistent with the mild photothermal properties of ZPPE NPs. Single-cycle and repeated-cycle photothermal experiments further demonstrated that the temperature profiles of ZPPE@PLHA showed almost no attenuation after multiple irradiation on/off cycles, and the heating and cooling processes were reversible (Figure S13, S14). These results indicate excellent photothermal stability and repeatability. Overall, the ZPPE@PLHA hydrogel exhibited favorable structural stability, responsiveness to the mildly acidic infection microenvironment, controllable Zn²⁺ release, and efficient tunable mPTT performance. These properties suggest its potential for synergistic antibacterial therapy and wound healing in infected wound environments.

In vitro antibacterial activity and mechanism of ZPPE@PLHA hydrogels

To evaluate the in vitro antibacterial performance of ZPPE@PLHA hydrogel under NIR irradiation, MRSA (Gram-positive bacteria) and Escherichia coli (E. coli, Gram-negative bacteria) were used as representative bacterial models. As shown in Figs. 4A-C and Figure S15, the PLHA group exhibited moderate inhibitory effects against both bacterial strains compared with the control group, which may be attributed to the cationic ε-PL component in the PLHA matrix. Compared with PLHA, ZnO@PLHA further reduced the viability of MRSA and E. coli to 31.9% and 32.7%, respectively, indicating that ZnO incorporation and Zn²⁺ release contributed to the intrinsic antibacterial activity of the hydrogel. ZPPE@PLHA maintained strong antibacterial efficacy, suggesting that the final multifunctional modification did not compromise the antibacterial activity of ZnO or the PLHA matrix. Notably, after 808 nm NIR irradiation, the ZPPE@PLHA + NIR group almost completely inhibited colony formation of both MRSA and E. coli, indicating that PDA-mediated mild photothermal activity further enhanced the antibacterial effect of the composite hydrogel.

Fig. 4.

In vitro antibacterial activity and proposed mechanism of ZPPE@PLHA hydrogels. (A) Representative colony-forming unit (CFU) plates, SEM images, and Live/Dead fluorescence staining (SYTO9/PI) of MRSA after different treatments (Control, PLHA, ZnO@PLHA, ZPPE@PLHA, and ZPPE@PLHA + NIR). Scale bars: SEM, 500 nm; fluorescence images, 200 μm. (B) Representative CFU plates, SEM images, and Live/Dead staining of E. coli after the indicated treatments. Scale bars: SEM, 500 nm; fluorescence images, 200 μm. (C) Quantitative analysis of E. coli (left) and MRSA (right) viability following different treatments, demonstrating the markedly enhanced bactericidal efficacy of ZPPE@PLHA under NIR irradiation. (D) Volcano plot showing differentially expressed genes (DEGs) in MRSA from the ZPPE@PLHA + NIR group compared with the control group. GO and KEGG enrichment analyses of downregulated (E) and upregulated (F) DEGs in MRSA. (G) Heatmap showing the expression patterns of representative DEGs in MRSA from the control and ZPPE@PLHA + NIR groups. (H) Protein–protein interaction (PPI) network analysis of key DEGs in MRSA after ZPPE@PLHA + NIR treatment. (I) Schematic illustration of the proposed antibacterial mechanism of ZPPE@PLHA + NIR, involving PLHA-associated cationic antibacterial activity, Zn²⁺ release, and PDA-mediated mild photothermal effects. Data are presented as mean ± SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

To further investigate the antibacterial mechanism, SEM morphological observation and Live/Dead staining (SYTO9/PI) were performed to assess bacterial membrane integrity (Figs. 4A, B). SEM images showed that bacteria in the control group maintained relatively intact morphology with smooth surfaces, whereas those in the PLHA group exhibited slight shrinkage after co-culture. After treatment with ZnO@PLHA and ZPPE@PLHA, the bacteria showed surface roughening, local collapse, and other signs of structural damage. Upon NIR irradiation, both MRSA and E. coli displayed more pronounced membrane shrinkage, structural rupture, and morphological disintegration, suggesting irreversible damage to the bacterial cell membrane. Consistently, Live/Dead staining further confirmed these findings. The control group mainly exhibited SYTO9 green fluorescence, indicating intact bacterial membranes, whereas PI red fluorescence was markedly enhanced in the ZPPE@PLHA group. These results suggest that membrane disruption is an important contributor to the antibacterial activity of ZPPE@PLHA, and that NIR-triggered mild photothermal effects significantly amplify this process. Overall, the antibacterial activity of ZPPE@PLHA is mainly associated with the combined effects of the ε-PL component in the PLHA matrix, Zn²⁺ release, and NIR-triggered PDA-mediated mild photothermal therapy.

To elucidate the synergistic antibacterial eradication mechanism of ZPPE@PLHA + NIR at the molecular level, transcriptome sequencing was performed on MRSA treated with ZPPE@PLHA + NIR. The volcano plot identified 179 differentially expressed genes (DEGs), including 46 upregulated and 133 downregulated DEGs (Fig. 4D). Gene Ontology (GO) enrichment analysis showed that the downregulated DEGs were mainly associated with ribosome structure and function, translation, peptide biosynthesis, and nitrogen-containing compound biosynthesis (Fig. 4E). KEGG pathway analysis further revealed significant enrichment in ribosome, amino acid metabolism, and nitrogen metabolism-related pathways. These findings suggest that the bacterial protein synthesis system was broadly suppressed, possibly due to membrane damage-induced disruption of energy metabolism, which subsequently impaired protein synthesis [63]. In contrast, the upregulated DEGs were enriched in GO terms related to oxidative stress responses and redox activity (Fig. 4F), indicating that bacteria experienced pronounced stress and potential DNA damage under material stimulation, thereby activating repair and defense mechanisms for survival [64]. At the KEGG pathway level, upregulated genes were mainly associated with membrane transport and antimicrobial peptide tolerance, including ABC transporters, CAMP resistance, and the two-component system. These changes suggest bacteria may attempt to resist external bactericidal stress by enhancing efflux activity and regulating membrane homeostasis. However, these adaptive responses were insufficient to counteract the multimodal synergistic antibacterial effects of ZPPE@PLHA + NIR. Heatmap and protein-protein interaction (PPI) network analyses further confirmed that these transcriptomic changes were highly clustered. Genes related to transcription and translation were consistently downregulated and closely associated with energy metabolism-related genes, such as succinate dehydrogenase gene SdhA (Figs. 4G, H). These results suggest that, following membrane damage, bacteria may enter a state characterized by translational arrest and disrupted energy metabolism, ultimately leading to cell death. Notably, the downregulation of SdhA suggests the potential inhibition of succinate dehydrogenase-related metabolism, which is commonly associated with reduced respiratory activity under stress conditions [65]. Therefore, at the molecular level, ZPPE@PLHA + NIR may induce coordinated disruption of bacterial membrane integrity, metabolic activity, and translation-related processes, together with oxidative stress, collectively contributing to bacterial growth inhibition and death. Based on both phenotypic and transcriptomic evidence, we propose a possible synergistic antibacterial mechanism of ZPPE@PLHA under NIR irradiation (Fig. 4I). First, NIR-triggered mPTT moderately increases the local temperature, which may compromise bacterial membrane stability and enhance membrane permeability. Meanwhile, Zn²⁺ release may further disrupt membrane function and induce oxidative stress-related responses. In addition, the cationic PLMA/ε-PL components may interact with the negatively charged bacterial surface through electrostatic adsorption, thereby aggravating membrane perturbation and increasing bacterial susceptibility. Together, these effects are associated with bacterial membrane damage, metabolic disturbance, and suppression of translation-related processes, ultimately enhancing bacterial growth inhibition and cell death.

In vitro promotion of anti-inflammatory M2 macrophage polarization by ZPPE NPs

After efficient bacterial eradication, timely resolution of inflammation and the establishment of a pro-regenerative immune microenvironment are critical for successful wound healing. Macrophages play a central role in this process by dynamically transitioning from the pro-inflammatory M1 phenotype to the anti-inflammatory and tissue-repair-associated M2 phenotype [66, 67]. Therefore, we next investigated whether ZPPE NPs, the key functional component of the ZPPE@PLHA hydrogel, could regulate macrophage polarization in vitro. As illustrated in the schematic workflow (Fig. 5A), RAW264.7 macrophages were stimulated with LPS to induce classical M1 polarization and then treated with different NPs. As shown in Figs. 5B and C, compared with the control group, the relative fluorescence intensity of CD86 was significantly increased in the LPS-induced M1 group, confirming the successful induction of a pro-inflammatory phenotype. Treatment with ZnO, ZP, and ZPPE NPs significantly suppressed CD86 protein expression. No significant differences were observed among the three NP-treated groups, suggesting that zinc ions play a key regulatory role in macrophage polarization. Conversely, compared with the M1 group, the expression of the M2 marker CD206 was significantly upregulated in the ZnO, ZP, and ZPPE NP groups, indicating enhanced M2 macrophage polarization.

Fig. 5.

In vitro immunomodulatory effects of ZPPE NPs on macrophage polarization. (A) Schematic illustration of the experimental workflow for macrophage polarization and immunomodulation analysis. RAW264.7 macrophages were stimulated with lipopolysaccharide (LPS) to induce classical M1 polarization and subsequently treated with different NPs. (B) Representative immunofluorescence staining images of macrophages for CD86 (M1 marker, green) and CD206 (M2 marker, red) after different treatments (control, LPS, LPS + ZnO, LPS + ZP, and LPS+ZPPE NPs). Nuclei were counterstained with DAPI (blue). (C) Quantitative analysis of the relative fluorescence intensity of CD86 and CD206. (D) Representative immunofluorescence staining images of macrophages for iNOS (M1 marker, green) and CD163 (M2 marker, red) after different treatments. (E) Quantitative analysis of the relative fluorescence intensity of iNOS and CD163. (F) Relative mRNA expression levels of pro-inflammatory cytokines (Tnf-α, Il-6, and Il-1β) in macrophages after different treatments, as determined by quantitative RT-PCR. (G) Relative mRNA expression levels of anti-inflammatory markers (Il-4, Il-10, and Cd163) following treatment with different NPs. Data are presented as mean ± SD (n = 3). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

To further validate macrophage polarization at the functional protein level, macrophages were stained for inducible nitric oxide synthase (iNOS, an M1 marker) and CD163 (an M2 marker). As shown in Figs. 5D and E, LPS stimulation induced strong iNOS expression and relatively weak CD163 staining. Quantitative analysis showed that, compared with the M1 group, iNOS fluorescence intensity was significantly decreased, whereas CD163 fluorescence intensity was significantly increased in the ZnO, ZP, and ZPPE NP groups. No significant differences were detected among the three NP-treated groups. These findings suggest that ZPPE NPs attenuate M1-associated inflammatory activation and shift macrophages toward an M2-like phenotype. At the transcriptional level, the immunomodulatory effects of ZPPE NPs were further evaluated by quantifying inflammation-related cytokine expression using RT-qPCR (Fig. 5F). LPS stimulation significantly upregulated the mRNA expression of the pro-inflammatory cytokines Tnf-α, Il-6, and Il-1β. Although ZnO and ZP NPs did not significantly inhibit Il-6 expression, treatment with ZnO, ZP, and ZPPE NPs significantly downregulated Tnf-α and Il-1β expression. Conversely, compared with the M1 group, macrophages treated with ZnO, ZP, and ZPPE NPs showed consistent and significant upregulation of the M2-associated anti-inflammatory markers Il-4, Il-10, and Cd163 (Fig. 5G), indicating a marked enhancement of the anti-inflammatory and pro-regenerative phenotype. Notably, except for a significant difference in Il-4 mRNA expression between the ZnO and ZP NP groups, no significant differences were observed in the expression of the remaining inflammatory factors among the three NP-treated groups. These results further confirm the key immunomodulatory role of zinc ions in vitro.

Given that MRSA is a Gram-positive bacterium and does not contain LPS, the LPS-induced macrophage model was used only as an in vitro model of classical inflammatory macrophage activation [68]. To further evaluate the immunomodulatory effects of the nanoparticles under MRSA-related inflammatory stimulation, RAW264.7 macrophages were stimulated with lipoteichoic acid (LTA), a major cell wall component of Gram-positive bacteria [69], and then co-cultured with ZnO, ZP, or ZPPE NPs. As shown in Figure S16, compared with the LTA-stimulated group, treatment with ZnO and ZP NPs significantly reduced CD86 expression. Conversely, ZnO, ZP, and ZPPE NPs all enhanced the expression of the M2-associated marker CD206. These results further support that ZPPE NPs can regulate macrophage polarization under MRSA-related inflammatory stimulation by promoting a shift toward an M2-associated phenotype.

The observed immunomodulatory effects may be primarily attributed to the sustained release of Zn²⁺ from the ZPPE@PLHA hydrogel rather than to PDA or rhEGF. Zn²⁺ is an immunoregulatory trace element that can suppress excessive inflammatory signaling while promoting M2 macrophage polarization and the secretion of anti-inflammatory cytokines [70, 71]. Additionally, the PLHA hydrogel matrix may provide a biocompatible and low-stress microenvironment that supports M2 macrophage polarization [72]. Collectively, these results suggest that ZPPE NPs modulate macrophage polarization by attenuating M1-associated inflammatory responses and promoting M2-associated phenotypic features in vitro. This immunomodulatory capability may contribute to inflammation resolution and the establishment of a pro-healing immune microenvironment after bacterial clearance.

In vitro cytocompatibility, cell migration, and tube formation of ZPPE@PLHA hydrogels

To evaluate the safety and tissue-repair potential of the ZPPE@PLHA hydrogel for biological applications, its biocompatibility and pro-migratory effects were investigated in vitro using HUVECs. CCK-8 assay results showed that cell activity in the control, PLHA, ZnO@PLHA, ZP@PLHA and ZPPE@PLHA groups gradually increased on days 1, 3, and 5, indicating that none of the materials exhibited obvious cytotoxicity toward HUVECs (Fig. 6A). On day 5, the OD value of the ZPPE@PLHA group was comparable to those of the PLHA and control groups, suggesting that ZPPE@PLHA hydrogel exhibited good compatibility and supported endothelial cell proliferation. Consistently, Live/Dead staining showed abundant Calcein-AM-positive live cells and very few PI-positive dead cells in all hydrogel groups (Fig. 6B, Figure S17). These results indicate that the PLHA matrix, as well as the incorporation of ZnO, PDA/PEG, and rhEGF, did not induce significant cytotoxicity toward HUVECs. In addition, the hemolysis assay showed that all hydrogel groups exhibited very low hemolysis ratios, comparable to that of the PBS group and significantly lower than that of the positive control group, further confirming the good blood compatibility of the hydrogel system (Fig. 6C).

Fig. 6.

In vitro cytocompatibility and pro-migratory effects of ZPPE@PLHA hydrogels on HUVECs. (A) Viability of HUVECs cultured with control, PLHA, ZnO@PLHA, ZP@PLHA and ZPPE@PLHA groups, as evaluated by CCK-8 assay on days 1, 3, and 5. Data are presented as mean ± SD (n = 5). (B) Live/Dead staining images of HUVECs after co-culture with different hydrogels. Scale bars: 200 μm. (C) Hemolysis assay of different hydrogel groups, with PBS as the negative control and deionized water as the positive control. (D) Representative optical images of HUVEC scratch wound healing assays at 0, 12, and 24 h after treatment with control, PLHA, ZnO@PLHA, ZP@PLHA and ZPPE@PLHA groups. (E) Quantitative analysis of the scratch area at 0, 12, and 24 h. (F) Representative fluorescence images of HUVEC tube formation on Matrigel after incubation with extracts from control, PLHA, ZnO@PLHA, ZP@PLHA and ZPPE@PLHA hydrogels. Scale bars: 200 μm. (G) Quantitative analysis of total tube length. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

After confirming the favorable compatibility of the hydrogels, the effect of ZPPE@PLHA on HUVEC migration was further evaluated using a scratch assay (Fig. 6D). At 0 h, the initial scratch areas were comparable among all groups. At 12 and 24 h, PLHA showed only a limited effect on HUVEC migration compared with the control group. In contrast, the ZnO@PLHA group significantly reduced the remaining scratch area compared with PLHA, suggesting that ZnO incorporation and Zn²⁺ release promoted endothelial cell migration. The migration rates in the ZnO@PLHA, ZP@PLHA, and ZPPE@PLHA groups were significantly higher than those in the PLHA and control groups. At 12 h, the migration rate in the ZPPE@PLHA group was 2.13-fold and 2.37-fold higher than those in the PLHA and control groups, respectively (Figure S18). Notably, at 24 h, the scratch area in the ZPPE@PLHA group was almost completely covered by cells, indicating that rhEGF conjugation further enhanced endothelial cell migration. This effect may be attributed to the synergistic action of gradually released Zn²⁺ ions and bioactive rhEGF. Previous studies have shown that Zn²⁺ can regulate endothelial cell migration and proliferation, whereas EGF promotes cytoskeletal rearrangement and cell motility by activating the EGFR signaling pathway [73, 74]. Additionally, the flexible and moisture-retentive microenvironment provided by the PLHA matrix may support cell adhesion and migration [75].

Given that endothelial cell migration is closely associated with angiogenesis [76], an in vitro tube formation assay was further performed to evaluate the effect of ZPPE@PLHA on angiogenesis-related HUVEC behaviors. As shown in Fig. 6F, after 6 h of culture on Matrigel, the control and PLHA groups formed relatively scattered and incomplete tube-like networks, whereas the ZnO@PLHA and ZP@PLHA groups showed partial tubular connectivity. In contrast, HUVECs in the ZPPE@PLHA group formed clearer, more continuous, and more highly branched capillary-like networks. Quantitative analysis showed that the total tube length and number of branches in the ZnO@PLHA and ZP@PLHA groups were significantly higher than those in the PLHA group (Fig. 6G, Figure S19), indicating that ZnO NPs supported angiogenesis. Notably, ZPPE@PLHA induced the most continuous and interconnected capillary-like network and exhibited the highest total tube length among all groups. Taken together, these results demonstrate that the ZPPE@PLHA hydrogel not only exhibits good cytocompatibility in vitro but also significantly promotes endothelial cell migration and angiogenesis-related phenotypes. These findings provide important in vitro evidence supporting its potential to promote angiogenesis and tissue regeneration after infection eradication.

In vivo antibacterial evaluation of ZPPE@PLHA hydrogels in infected wound healing

To evaluate the antibacterial and wound-healing effects of the ZPPE@PLHA hydrogel in vivo, a full-thickness MRSA-infected skin wound model with a diameter of 10 mm was established. Treatment was performed with or without 808 nm NIR irradiation (Fig. 7A). Wound healing was monitored on days 0, 3, 7, 10, 14, and 21, and tissue samples were collected on days 7, 14, and 21 for further analysis. As shown in Fig. 7B, the temperature of the ZPPE@PLHA hydrogel increased to approximately 48.3 °C under 808 nm NIR irradiation, which is within the mPTT range reported in previous studies [77]. Macroscopically, distinct necrotic scabs formed on the wounds in all groups after surgery (Fig. 7C). Compared with the control and PLHA groups, the ZPPE@PLHA group showed faster wound contraction, while the ZPPE@PLHA + NIR group exhibited further accelerated wound healing by 7 days. Wound areas at different time points were delineated and marked with different colors for quantitative analysis (Fig. 7D). Relative wound area analysis showed that, on day 7, the wound area in the ZPPE@PLHA + NIR group was reduced to approximately 40% of the original area, which was significantly smaller than that in the PLHA and control groups (Fig. 7F). On days 10, 14, and 21, the wound area in the ZPPE@PLHA + NIR group remained significantly smaller than that in the ZPPE@PLHA group, suggesting that NIR-mediated mPTT may further promote wound contraction. By day 21, wounds in the ZPPE@PLHA + NIR group were almost completely healed, with smooth skin and no obvious scar formation. Notably, even without NIR irradiation, the ZPPE@PLHA group showed significantly smaller wound areas than the control group on days 3, 7, 10, 14, and 21. This accelerated healing effect may be attributed to two main factors: (i) the sustained release of Zn²⁺ and EGF, which promotes fibroblast migration and angiogenesis; and (ii) the PLHA hydrogel matrix, which maintains a moist microenvironment and supports cell adhesion and collagen deposition.

Fig. 7.

In vivo antibacterial efficacy and wound-healing performance of ZPPE@PLHA hydrogels in an MRSA-infected full-thickness skin wound model. (A) Schematic illustration of the MRSA-infected full-thickness skin wound model and the overall in vivo evaluation timeline, including antibacterial assessment, inflammatory response, immune regulation, angiogenesis, tissue remodeling, and transcriptomic analysis under NIR irradiation. (B) Infrared thermal images of MRSA-infected wounds treated with ZPPE@PLHA under 808 nm NIR laser irradiation (1.0 W cm⁻²), showing rapid and localized temperature elevation during 10 min irradiation. (C) Representative macroscopic images of infected wounds treated in the control, PLHA, ZPPE@PLHA, and ZPPE@PLHA + NIR groups at different time points (days 0, 3, 7, 10, 14, and 21). Scale bars: 2 mm. (D) Color-coded schematic diagrams of wound closure at the corresponding time points. Scale bars: 2 mm. (E) Representative photographs of bacterial colony-forming units (CFUs) cultured from wound tissues collected on day 7. (F) Quantitative analysis of relative wound area over time in the different groups. (G) Quantitative analysis of MRSA survival in wound tissues on day 7. (H) Representative hematoxylin and eosin (H&E) staining images of wound tissues collected on day 14. (I) Representative Masson’s trichrome staining images of wound tissues collected on day 14. Scale bars: 1 mm and 50 μm. Data are presented as mean ± SD (n = 5). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

The in vivo antibacterial performance was further evaluated by colony counting. The ZPPE@PLHA + NIR group showed a significant reduction in bacterial colony formation, with a bactericidal rate of approximately 95%, which was significantly higher than that of the other groups (Figs. 7E, G). This result indicates that, under mPTT, the PDA layer on the surface of ZPPE NPs rapidly converts NIR light into heat, while released Zn²⁺ and ε-PL exert synergistic antibacterial effects. Together, these effects form a triple antibacterial mechanism involving thermal inactivation, ion-mediated disruption, and antimicrobial peptide-mediated membrane damage, thereby substantially enhancing antibacterial efficacy. Histological analysis further confirmed the therapeutic effects of ZPPE@PLHA + NIR. On day 14, H&E staining showed obvious inflammation and discontinuous epithelial structures in the wounds of the control and PLHA groups. In contrast, the ZPPE@PLHA and ZPPE@PLHA + NIR groups exhibited more normalized skin architecture, intact wound tissue, and continuous epidermal regeneration, consistent with the macroscopic observations (Fig. 7H). Appropriate collagen deposition and fibrosis are essential for wound remodeling [78]. Masson staining further revealed dense and well-organized deep-blue collagen fibers in the ZPPE@PLHA + NIR group, suggesting enhanced dermal regeneration and tissue remodeling (Fig. 7I). Additionally, on day 14, new epidermis was observed in all groups. The newly formed epidermis in the ZPPE@PLHA and ZPPE@PLHA + NIR groups was significantly thinner than that in the control group, while the collagen deposition area was greater (Figure S20). This may be related to edema and epidermal cell thickening in the control group. Previous studies have suggested that a thinner regenerated epidermis may be associated with reduced scar formation [79]. On day 21, less scar tissue was observed in the ZPPE@PLHA + NIR group, indicating that early NIR intervention may facilitate rapid bacterial eradication and promote stable epidermal repair. In summary, ZPPE@PLHA hydrogel combined with NIR irradiation exhibited significant antibacterial and tissue-repair-promoting effects in an MRSA-infected wound model. Its therapeutic mechanism is mainly based on mild photothermal bacterial inactivation, sustained Zn²⁺ and ε-PL-mediated antibacterial activity, and the synergistic bioactive effects of Zn²⁺ and EGF in promoting tissue repair. Therefore, ZPPE@PLHA provides a comprehensive therapeutic strategy for the management of infected wounds.

In vivo anti-inflammatory and immunoregulatory effects of ZPPE@PLHA hydrogels

After bacterial eradication, the establishment of a favorable inflammatory and immune microenvironment is essential for effective wound repair. To further investigate the immunoregulatory effects of the ZPPE@PLHA hydrogel during infected wound healing, its in vivo anti-inflammatory activity and effects on macrophage polarization were systematically evaluated in a rat model. Immunohistochemical staining showed that, on day 14, TNF-α expression was markedly elevated in the control and PLHA groups, whereas it was significantly reduced in the ZPPE@PLHA and ZPPE@PLHA + NIR groups, indicating that the hydrogel effectively suppressed the inflammatory response. In contrast, the expression of the anti-inflammatory cytokine IL-10 was significantly increased in the ZPPE@PLHA group and was further enhanced after NIR irradiation, with the highest expression observed in the ZPPE@PLHA + NIR group (Fig. 8A, Figure S21). These results suggest that ZPPE@PLHA can downregulate pro-inflammatory responses and upregulate anti-inflammatory cytokine expression under mPTT conditions, thereby facilitating the transition from the inflammatory phase to the repair phase. ELISA results were consistent with the immunohistochemical findings (Fig. 8B). Compared with the control group, TNF-α levels in the ZPPE@PLHA and ZPPE@PLHA + NIR groups were significantly decreased on days 7 and 14, whereas IL-10 levels were significantly increased.

Fig. 8.

In vivo anti-inflammatory effects and macrophage immunomodulation of ZPPE@PLHA hydrogels in MRSA-infected wounds. (A) Representative immunohistochemical (IHC) staining images of TNF-α and IL-10 in wound tissues collected on day 14 from different treatment groups, including control, PLHA, ZPPE@PLHA, and ZPPE@PLHA + NIR. Scale bars: 1 mm. (B) ELISA quantification of TNF-α and IL-10 levels in wound tissues on day 7 (left) and day 14 (right) after different treatments. (C) Representative immunofluorescence images and quantitative analysis of the mean fluorescence intensity of macrophage polarization markers CD86 (M1, red) and CD206 (M2, green) in wound sections collected on day 7 and day 14 from different groups. Scale bars: 50 μm. Data are presented as mean ± SD (n = 5). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Immunofluorescence staining further revealed the role of ZPPE@PLHA in regulating macrophage phenotypes in vivo (Fig. 8C). On days 7 and 14, CD86 expression was markedly elevated in the control and PLHA groups but was significantly reduced in the ZPPE@PLHA group. The number of CD86-positive cells was further decreased in the ZPPE@PLHA + NIR group. Conversely, CD206 expression was significantly upregulated in the ZPPE@PLHA group and further enhanced after NIR irradiation. These findings indicate that ZPPE@PLHA treatment reduced M1-associated marker expression and enhanced M2-associated marker expression in wound tissues, thereby accelerating inflammation resolution and promoting tissue regeneration. In summary, under local mPTT, the ZPPE@PLHA hydrogel may modulate the inflammatory microenvironment through sustained Zn²⁺ release, suppression of excessive inflammation, and induction of M2 macrophage-mediated immune repair responses. This immunoregulatory effect provides a favorable inflammatory and immune microenvironment for subsequent wound regeneration and repair.

In vivo tissue repair and mechanism of ZPPE@PLHA hydrogels

Angiogenesis is a crucial step in wound repair because it supplies oxygen and nutrients to newly formed tissue and supports fibroblast migration and collagen deposition [80]. Vascular endothelial growth factor (VEGF) is a key signaling molecule that regulates neovascularization by stimulating endothelial cell proliferation, migration, and capillary formation [81]. CD31 is a specific endothelial cell marker, and its expression reflects endothelial cell abundance and the extent of new capillary formation [82]. To further investigate the tissue repair effect of ZPPE@PLHA hydrogel during the later stage of infected wound healing, immunofluorescence analysis of angiogenesis- and tissue remodeling-related markers was performed on wound tissues collected 21 days after treatment. As shown in Fig. 9A, compared with the control and pure PLHA groups, ZPPE@PLHA and ZPPE@PLHA + NIR groups showed higher expression levels of VEGF (red) and CD31 (green). Quantitative results (Figs. 9B, C) showed that the fluorescence intensities of VEGF and CD31 in the ZPPE@PLHA + NIR group were 3.5- and 3.2-fold higher, respectively, than those in the control group. These results suggest that, under mild photothermal stimulation, ZPPE@PLHA synergistically promotes endothelial cell proliferation and migration through the combined effects of rhEGF and Zn²⁺, thereby accelerating neovascularization and providing oxygen and nutrient support for tissue regeneration.

Fig. 9.

Angiogenesis promotion, tissue remodeling, and EGFR pathway activation in MRSA-infected wounds after ZPPE@PLHA treatment. (A) Representative immunofluorescence images of VEGF (red) and CD31 (green) in wound tissues collected on day 21 from the different treatment groups, including control, PLHA, ZPPE@PLHA, and ZPPE@PLHA + NIR. Nuclei were counterstained with DAPI (blue). Scale bars: 50 μm. Quantitative analysis of the mean fluorescence intensity of VEGF (B) and CD31 (C) in wound sections. (D) Representative immunofluorescence staining of α-SMA (green) in wound tissues on day 21. Scale bars: 50 μm. (E) Quantitative analysis of α-SMA fluorescence intensity in the different groups. (F) Volcano plot showing differentially expressed genes (DEGs) in wound tissues collected on day 21, comparing the PBS and ZPPE@PLHA + NIR groups. (G) GO enrichment analysis of DEGs, including biological process (BP), cellular component (CC), and molecular function (MF). (H) GSEA plots showing significant enrichment of extracellular matrix (ECM)- and collagen-related gene sets after ZPPE@PLHA + NIR treatment. (I) KEGG pathway enrichment analysis of DEGs. (J) Heatmap showing representative DEGs associated with inflammatory response and tissue remodeling. (K) Representative immunohistochemical staining of EGFR in wound tissues collected on day 21. (L) Expression levels of key downstream proteins, including EGFR, p-PI3K/PI3K, p-AKT/AKT, and p-ERK/ERK, in the ZPPE@PLHA + NIR group and the control group. (M) Schematic illustration of the proposed full-process therapeutic effects of ZPPE@PLHA hydrogels in MRSA-infected wound healing. Data are presented as mean ± SD (n = 5). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Myofibroblasts are key effector cells involved in wound contraction and collagen remodeling, and α-smooth muscle actin (α-SMA), a marker of myofibroblasts, is highly expressed during the later stages of wound repair. Upregulated α-SMA expression not only indicates myofibroblast activation but also reflects the maturation and structural stabilization of newly formed vascular walls [83, 84]. Compared with the control group, the relative fluorescence intensity of α-SMA was significantly increased in both the ZPPE@PLHA and ZPPE@PLHA + NIR groups (Figs. 9D, E). These findings suggest that ZPPE@PLHA hydrogel promotes the formation, maturation, and functional improvement of new blood vessels, thereby providing a solid physiological basis for wound tissue regeneration and orderly collagen deposition.

To further explore the potential biological mechanisms underlying the accelerated wound healing induced by ZPPE@PLHA hydrogel combined with NIR irradiation after infection eradication, RNA transcriptome analysis was performed on wound tissue collected on day 21 (n = 6 per group). The volcano plot revealed significant differences in gene expression between the ZPPE@PLHA + NIR group and control group, including 645 upregulated DEGs and 248 downregulated DEGs (Fig. 9F). These DEGs were associated with inflammation, tissue remodeling, and repair-related processes. GO enrichment analysis was then performed to classify the DEGs according to biological processes (BP), cellular components (CC), and molecular functions (MF) categories (Fig. 9G). In the BP category, the DEGs were mainly enriched in immune response- and inflammatory response-related processes. In the MF category, genes associated with zinc ion binding, calcium ion binding, and metal ion binding were significantly enriched, suggesting that ZPPE@PLHA may promote tissue repair through Zn²⁺ release and regulation of ion-related signaling pathways. In the CC category, genes associated with membranes and the extracellular matrix (ECM) were markedly enriched, indicating that ZPPE@PLHA plays an important role in angiogenesis and tissue remodeling. ECM remodeling is essential for wound healing because it supports cell adhesion, migration, and proliferation, thereby accelerating tissue repair [85]. GSEA further revealed that ZPPE@PLHA combined with NIR regulated multiple key biological processes involved in wound healing (Fig. 9H). Gene sets related to collagen-containing extracellular matrix, collagen fibril organization, extracellular matrix structural constituents, and skeletal muscle tissue development were significantly enriched in the ZPPE@PLHA + NIR group. These findings suggest that the hydrogel promotes collagen deposition and ECM remodeling and may also contribute to muscle tissue repair and regeneration.

Additionally, KEGG pathway analysis further explored the potential mechanisms by which ZPPE@PLHA hydrogel combined with NIR promoted wound healing (Fig. 9I). Several signaling pathways related to inflammation regulation and tissue remodeling were enriched, including the MAPK signaling pathway, PI3K/AKT signaling pathway, and cytokine-cytokine receptor interaction pathway. These results suggest that ZPPE@PLHA may promote angiogenesis, collagen deposition, and wound repair by regulating these pathways. To further investigate the stage-specific molecular mechanisms of ZPPE@PLHA in infected wound healing, we analyzed the expression patterns of genes associated with inflammatory responses and tissue remodeling in the ZPPE@PLHA + NIR and control groups (Fig. 9J). Among inflammation-related genes, including Il1rn, Il1r2, Il1α, Ccl27, and Ccl20, overall expression levels were lower in the ZPPE@PLHA hydrogel group than in the control group. In particular, the downregulation of pro-inflammatory genes such as Il1α and Ccl27 suggests that ZPPE@PLHA may alleviate excessive inflammation and create a favorable microenvironment for wound repair. For tissue remodeling-related genes, the ZPPE@PLHA + NIR group showed significant upregulation of collagen synthesis- and remodeling-related genes, such as Col1a1 and Col1a2, and downregulation of matrix metalloproteinase (MMP)-related genes, including Mmp3 and Mmp8. In addition, genes associated with angiogenesis and epithelial regeneration, including Esm1, Ocln, and Cldn1, were upregulated in the ZPPE@PLHA + NIR group. These results indicate that ZPPE@PLHA + NIR may promote skin tissue repair by enhancing collagen deposition and matrix remodeling while inhibiting excessive matrix degradation. Moreover, the expression levels of Egfr and Tgfa were higher in the ZPPE@PLHA + NIR group, suggesting that activation of EGFR-related signaling may accelerate wound healing by promoting cell proliferation and migration. Considering the key role of sustained rhEGF release in angiogenesis and tissue remodeling [86], EGFR expression in wound tissue was further assessed by immunohistochemistry (Fig. 9K, Figure S22). Compared with the control and PLHA groups, EGFR-positive staining was significantly enhanced in the ZPPE@PLHA group, and high EGFR expression was also maintained in the ZPPE@PLHA + NIR group. These findings indicate that the ZPPE@PLHA system enhances local EGFR-related signaling activity in wound tissues through rhEGF release. This result is consistent with the transcriptomic upregulation of repair-related genes and provides molecular support for the enhanced angiogenesis, as indicated by VEGF and CD31 expression, and tissue maturation, as indicated by α-SMA expression, observed during later-stage wound repair.

Transcriptome sequencing analysis showed that the MAPK and PI3K/AKT signaling pathways were significantly enriched after ZPPE@PLHA hydrogel treatment, and previous studies have confirmed that these pathways are closely associated with tissue remodeling [87, 88]. To further validate these findings, we examined the expression and activation status of key downstream pathway proteins (Fig. 9L, Figure S23). Compared with the control group, EGFR protein expression was upregulated in the ZPPE@PLHA + NIR group. In addition, the ratios of p-PI3K/PI3K, p-AKT/AKT, and p-ERK/ERK were significantly increased. These results indicate that ZPPE@PLHA + NIR treatment is associated with increased EGFR expression and enhanced phosphorylation of PI3K, AKT, and ERK, suggesting the potential involvement of PI3K/AKT and ERK signaling in the observed tissue repair process. Furthermore, H&E staining of major organs, including the heart, liver, spleen, lungs, and kidneys, showed no obvious tissue damage in any group (Figure S24). These findings suggest that the ZPPE@PLHA hydrogel, with or without NIR irradiation, exhibited no apparent systemic toxicity and possessed good in vivo biocompatibility. Based on the antibacterial, anti-inflammatory, immunoregulatory, angiogenic, and transcriptomic results, we summarized the proposed full-process therapeutic mechanism of the ZPPE@PLHA + NIR system in MRSA-infected wound repair (Fig. 9M). During the infection-associated phase, NIR-triggered mPTT synergizes with Zn²⁺ release and the cationic antibacterial effect of ε-PL to rapidly disrupt bacterial membrane structures and markedly reduce bacterial burden. During the inflammation-associated phase, sustained Zn²⁺ release helps suppress pro-inflammatory cytokine expression, enhances IL-10-mediated anti-inflammatory signaling, and promotes macrophage polarization from the M1 phenotype toward the M2 phenotype. These effects accelerate inflammation resolution and establish a favorable immune microenvironment for tissue repair. During the repair and remodeling phase, rhEGF and Zn²⁺ may contribute to EGFR-related signaling responses, which are potentially associated with cell migration, angiogenesis, vascular maturation, and tissue remodeling. Meanwhile, ZPPE@PLHA + NIR promotes collagen deposition and ECM structural remodeling, ultimately leading to faster, more organized wound healing with reduced scar formation. In conclusion, the multimodal antibacterial, anti-inflammatory, immunoregulatory, and pro-regenerative mechanisms of ZPPE@PLHA + NIR collectively provide a comprehensive therapeutic strategy for MRSA-infected wound management. Nevertheless, because pathway inhibition or gene-silencing experiments were not performed in this study, the involvement of EGFR/PI3K/AKT/ERK signaling should be interpreted as pathway-associated evidence rather than direct proof of causality.

Conclusion

In this study, we developed a ZnO NP-based composite photopolymerizable ZPPE@PLHA hydrogel through self-polymerization and amidation reactions for the comprehensive management of MRSA-infected wounds. By integrating mPTT, Zn²⁺- and ε-PL-mediated antibacterial activity, the ZPPE@PLHA hydrogel achieved rapid and effective MRSA eradication. Meanwhile, sustained Zn²⁺ release promoted anti-inflammatory M2 macrophage polarization, attenuated excessive inflammation, and established a favorable inflammatory microenvironment for wound repair. Additionally, rhEGF enhanced angiogenesis and tissue remodeling through EGFR-related signaling, and together with Zn²⁺ release, further accelerated wound healing. In a rat MRSA-infected wound model, ZPPE@PLHA combined with NIR irradiation effectively inhibited infection, regulated inflammation, and promoted tissue repair and regeneration, while exhibiting good in vivo biocompatibility. Overall, this hydrogel provides a promising full-process integrated therapeutic strategy and holds substantial potential for the future management of MRSA-infected wounds.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

X.S., Y.W. and Y.X. designed the research. X.S. and Z.Y. wrote the paper. X.S., C.X., Z.Y., M.W., M.L., and P.F. performed the research. M.W., X.F., and Y.W. provided professional support for animal studies. All the authors have read and approved the final manuscript.

Funding

This study was funded by Yunnan Traumatology and Orthopedics Clinical Medical Center (Grant No. 2024YNLCYXZX0187; 2024YNLCYXZX0182); Yunnan Orthopedics and Sports Rehabilitation Clinical Medicine Research Center; National Key Research and Development Program of China (Grant No. 2024YFC2418100); Basic research project of Science and Technology Department of Yunnan Province (Grant No. 202301AC070621), and Scientific Research Fund Project Department of Education of Yunnan Province (Grant No. 2024Y251).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The experiment was approved by the Animal Care and Use Committee of the Joint Logistics Support Force 920 Hospital ( Approval No. 2024-011-01).

Competing interests

The authors declare no competing interests.