Magnesium phosphate mineralized Ce6 composite hydrogel with photodynamic therapy-mediated antibacterial, anti-inflammatory, and pro-angiogenic properties for application in infected wound healing

TL;DR

A new hydrogel dressing uses light to kill bacteria and speed up wound healing by combining a photosensitizer with magnesium phosphate nanoparticles.

Contribution

A novel mineralization approach encapsulates Ce6 in magnesium phosphate nanoparticles for sustained photodynamic therapy and wound healing.

Findings

CMP/Gel generates reactive oxygen species to effectively eradicate bacteria upon light irradiation.

Mg2+ ions released from CMP NPs reduce inflammation and promote angiogenesis and cell proliferation.

In vitro and in vivo experiments confirm CMP/Gel's efficacy in accelerating infected wound repair.

Abstract

Wound infection remains a significant clinical challenge, exacerbated by the growing prevalence of bacterial resistance due to the overuse of conventional antibiotics. Photodynamic therapy (PDT) offers a promising approach for wound sterilization that circumvents the issue of antibiotic resistance. However, conventional photosensitizers are prone to inactivation and exhibit poor retention at the wound site, limiting their clinical efficacy. To overcome these limitations, we developed a biomimetic mineralization approach to encapsulate the small-molecule photosensitizer chlorin e6 (Ce6) within magnesium phosphate nanoparticles (CMP NPs), preserving its photodynamic activity. This mineralized CMP NPs were further integrated with gelatin and natural moisturizing factor to fabricate a composite hydrogel dressing (CMP/Gel) for infected wound healing. Gelatin functions as a structural matrix that prolongs the local retention of CMP NPs, while the natural moisturizing factor enhances the hydration capacity and mechanical integrity of the dressing. Notably, magnesium ions released during the degradation of CMP NPs contribute to accelerated wound healing through multiple therapeutic effects, including antioxidant, anti-inflammatory, and pro-angiogenic activities following PDT-mediated bacterial eradication. Both in vitro and in vivo experimental results demonstrate that CMP/Gel effectively promotes infected wound repair, highlighting its potential as a novel and multifunctional therapeutic strategy for infected wound management.

Herein, we report a novel composite hydrogel dressing (CMP/Gel) for the treatment of infected wounds. Upon light irradiation, CMP/Gel generates a substantial amount of reactive oxygen species (ROS) to achieve efficient bacterial eradication. Concurrently, the sustained release of Mg2+ ions during nanoparticle degradation contributes to accelerated wound repair by mitigating residual oxidative stress, promoting cell proliferation and migration, and stimulating angiogenesis. Therefore, the CMP/Gel hydrogel dressing represents a promising and multifunctional therapeutic strategy for infected wound management, with high translational potential.Image 1

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Full text

Introduction

1

Wound healing is a highly coordinated process involving diverse cell types and intricate interactions among multiple biological pathways [[1], [2], [3]]. Disruption of skin integrity creates a vulnerable site susceptible to bacterial colonization, which can lead to substantial tissue damage and trigger a pronounced inflammatory response, resulting in delayed or non-healing wounds and potentially life-threatening systemic infections [4,5]. Although antibiotic therapy remains the standard treatment, the widespread overuse and misuse of antibiotics have accelerated the emergence of multidrug-resistant bacterial strains, increasing the risk of biofilm formation and chronic wound development [6]. Given the escalating challenge of managing complex microbial infections in clinical settings and the urgent need for effective wound care solutions, the development of novel multifunctional antibacterial dressings has become essential to improve outcomes in infected wound management.

Antimicrobial photodynamic therapy (PDT) has emerged as a promising non-antibiotic approach for preventing wound infections, owing to its distinct advantages [7]. The therapeutic mechanism involves light-mediated activation of a photosensitizer to produce cytotoxic reactive oxygen species (ROS), which induce microbial cell death through oxidative damage. This process demonstrates broad-spectrum antimicrobial activity against diverse pathogens, including drug-resistant strains, while posing a minimal risk of resistance development due to the multi-target nature of ROS action [8,9]. Furthermore, the transient lifespan and localized production of ROS restrict cytotoxic effects predominantly to the irradiated area, thereby minimizing damage to surrounding healthy tissues [10]. Despite these benefits, the clinical application of PDT is often hindered by inherent limitations of conventional photosensitizers, such as poor water solubility, low stability, and a propensity for aggregation or self-quenching, which collectively compromise their bioavailability and photodynamic efficiency [11,12]. Additionally, many photosensitizers lack specific targeting capabilities toward infection sites, resulting in suboptimal retention and potential off-target effects. Thus, overcoming these intrinsic drawbacks of conventional photosensitizers is critical for advancing PDT efficacy.

To overcome this limitation, various delivery platforms such as liposomes [13], protein nanoparticles (NPs) [14,15], polymeric micelles [[16], [17], [18]], and polymer-drug conjugates [[19], [20], [21]], have been developed. Although these systems can significantly improve biodistribution and bioavailability, their application is constrained by low drug loading capacity and the requirement for large amounts of therapeutically inert excipients. These excipients not only increase the risk of adverse effects but also elevate manufacturing costs [22,23]. To address these challenges, biomimetic mineralization strategies have been widely adopted in the development of drug delivery systems [24,25]. In situ encapsulation of photosensitizers via biomimetic mineralization enables high drug loading efficiency, while the use of biominerals as carriers effectively minimizes the toxicity and side effects associated with conventional excipients. Notably, among various biominerals, magnesium phosphate minerals demonstrate distinct advantages in promoting wound repair, as the magnesium ions (Mg^2+^) released during their degradation exert antioxidant, anti-inflammatory, and pro-angiogenic effects [26]. Therefore, we propose that embedding photosensitizers within magnesium phosphate minerals through biomimetic mineralization not only preserves photosensitizer activity and enhances bioavailability but also synergistically supports wound healing through the physiological functions of Mg^2+^ ions.

Herein, we developed a biomimetic mineralization approach (Scheme 1a) to encapsulate the photosensitizer chlorin e6 (Ce6) within magnesium phosphate nanoparticles (CMP NPs) using Ce6 as a structural template. The resulting CMP NPs were further incorporated into a composite hydrogel dressing by combining them with gelatin and a natural moisturizing factor, yielding the CMP/Gel system for infected wound healing. In this formulation, CMP NPs provide dual functionalities: red-light-responsive PDT under 630 nm irradiation and Mg^2+^-mediated therapeutic effects, including anti-inflammatory, pro-angiogenic, and tissue-repair activities (Scheme 1b). Gelatin-based hydrogels offer inherent advantages such as biocompatibility, biodegradability, and thermoreversible gelation; however, they are often limited by poor mechanical strength and rapid dehydration [27,28]. The incorporation of a natural moisturizing factor enhances the hydrogel's mechanical robustness, water retention capacity, and ionic conductivity [29,30]. Experimental results show that the CMP/Gel composite hydrogel effectively reduces bacterial burden and accelerates wound closure, demonstrating significant potential for clinical wound management and presenting an innovative, antibiotic-free strategy for treating infected wounds.Scheme 1. Schematic illustration of (a) the fabrication process and (b) the therapeutic mechanism of the CMP/Gel composite hydrogel. Schemes created with BioRender.com.Scheme 1

Experimental section

2

Materials

2.1

Magnesium chloride hexahydrate (MgCl_2_·6H_2_O), natural moisturizing factor (NMF), sodium dihydrogen phosphate anhydrous (NaH_2_PO_4_), and disodium hydrogen phosphate dodecahydrate (Na_2_HPO_4_·12H_2_O) were purchased from Aladdin Industrial Co., Ltd. Gelatin was obtained from Sigma-Aldrich. Dulbecco's modified eagle medium (DMEM) medium and fetal bovine serum (FBS) were supplied by Gibco Life Technologies. The Cell Counting Kit-8 (CCK-8) was acquired from Baoguang Biotechnology Co., Ltd. Calcein AM, propidium iodide (PI), and phosphate-buffered saline (PBS) were sourced from Solarbio Science & Technology Co., Ltd. DAPI, DCFH-DA, and the EdU assay kit were provided by Beyotime Biotechnology. CD31 and VEGF antibodies were supplied by Wuhan San Eagle Biotechnology Co., Ltd. Bacterial strains of Staphylococcus aureus (S. aureus, ATCC 25923) and Escherichia coli (E. coli, ATCC 25922) were obtained from the American Type Culture Collection (ATCC). The SYTO 9/PI bacterial live/dead double-staining kit was procured from Shanghai Maogang Biotechnology Co., Ltd. LB medium was purchased from Beijing Dingguo Changsheng Technology Co., Ltd., while agar powder and 0.1% crystal violet were obtained from Beijing Solarbio Science & Technology Co., Ltd.

Characterization

2.2

The morphology and structure of the CMP NPs were examined using a GeminiSEM 300 scanning electron microscope (SEM, Germany, ZEISS), and a Talos™ F200S transmission electron microscope (TEM, Czech Republic) operated at 200 kV, with elemental analysis performed by the attached energy-dispersive X-ray spectroscopy (EDS) system. Particle size distribution and zeta potential were measured using a NanoBrook Omni particle size analyzer (Brookhaven Instruments, UK). Crystalline phase structure was analyzed using an X'Pert Powder diffractometer (XRD, PANalytical, Netherlands), while elemental composition was determined by an ESCALAB250Xi X-ray photoelectron spectrometer (XPS, Thermo Fisher Scientific, USA). The microscopic morphology of the CMP/Gel composite hydrogel was observed using a GeminiSEM scanning electron microscope (Germany, ZEISS), and its chemical structure was analyzed via a Nicolet iS50 Fourier transform infrared spectrometer (FTIR, Thermo Fisher Scientific, USA). Swelling capacity, degradation behavior, and adhesive strength of the hydrogel were evaluated using standardized assays. Microscopic images were acquired using a Leica TCS SP8 DIVE confocal microscope (Germany) and an Olympus VS120 slide scanner (Japan). Photodynamic treatment was delivered using an LED-IB PDT instrument.

Preparation of CMP NPs

2.3

The CMP NPs were synthesized via a biomimetic mineralization strategy. In brief, 10 μL of a Ce6 solution in DMSO (40 mg/mL) was added to 970 μL of deionized water and stirred at room temperature for 5 min. Subsequently, 12.5 μL of MgCl_2_ solution (1 M) was introduced into the mixture and stirred for an additional 5 min, followed by the addition of 15 μL of phosphate solution (0.5 M) and continued stirring for 15 min. The resulting suspension was centrifuged at 11,000 rpm for 15 min. The pellet was resuspended in 900 μL of deionized water, and the washing process was repeated three times to obtain the final product, which was stored at 4 °C in the dark for subsequent use.

Preparation of CMP/Gel composite hydrogel

2.4

The CMP/Gel composite hydrogel was prepared according to a previously reported method [31]. In brief, 0.4 g of gelatin powder was first swollen in 1.76 mL of deionized water for 1 h. Subsequently, the as-prepared CMP NPs (400 μg) and NMF solution (0.24 mL) were added to the gelatin dispersion. The mixture was stirred at 50 °C for 1 h to form a homogeneous gelatin-based precursor solution, which gelled within 3∼5 min at room temperature. A pure gelatin-based hydrogel was prepared using the same protocol but without the incorporation of CMP NPs.

Photodynamic performance of CMP/Gel

2.5

The photodynamic performance of CMP/Gel composite hydrogel was evaluated in vitro using 2′,7′-dichlorodihydrofluorescein (DCFH) as a reactive oxygen species (ROS) probe. In brief, 6 μL of DCFH solution (100 μM, prepared in deoxygenated PBS, pH 7.4) was added to 6 mL of CMP/Gel dispersion. The mixture was irradiated with red light (100 mW/cm^2^), and 1 mL aliquots were collected at 0, 1, 2, 3, 4, and 5 min after irradiation. Fluorescence intensity was measured using a fluorescence spectrophotometer with excitation at 488 nm and emission at 525 nm to quantify ROS generation.

In vitro antibacterial activity of CMP/Gel

2.6

The antibacterial activity of CMP/Gel against Gram-positive S. aureus was evaluated in vitro using the filter paper disk diffusion method. Bacterial cultures stored at −80 °C were revived by streaking onto LB agar plates and incubating at 37 °C for 24 h. Single colonies were inoculated into 5 mL of fresh LB medium and cultured with shaking (180 rpm) at 37 °C for 16∼18 h until the logarithmic growth phase was reached. The optical density (OD) of the bacterial suspension was measured using a microplate reader, and the culture was diluted to concentrations ranging from 10^5^ to 10^8^ CFU/mL, followed by 15 min of static incubation. After evenly spreading each bacterial suspension onto LB agar plates, sterile filter paper disks were individually immersed in PBS, CMP (20 μg/mL), Gel, or CMP/Gel solutions, then gently blotted to remove excess liquid. Two disks were placed on each plate, with three replicates per group. The plates were incubated upside down at 37 °C for 2 h, exposed to red light (100 mW/cm^2^) for 10 min, and further incubated at 37 °C for 16∼18 h. After incubation, the diameter of the inhibition zones (clear transparent areas without bacterial growth) around the disks was measured using a vernier caliper.

To evaluate antibacterial efficacy, S. aureus suspensions (diluted to 10^8^ CFU/mL) were mixed with each test solution, with a PBS-only group serving as the control [32]. Following incubation at 37 °C for 2 h, the mixtures were exposed to red light (100 mW/cm^2^) for 10 min. Bacterial cells were collected by centrifugation (10,000 rpm, 15 min), stained in the dark with SYTO 9/PI for 10 min, washed with PBS, resuspended, and then applied onto glass slides for observation and imaging under a fluorescence microscope.

For SEM sample preparation, bacterial suspensions treated with PBS + Light, CMP + Light, Gel + Light, and CMP/Gel + Light were prepared. The bacteria were washed three times with PBS and fixed with 2.5% glutaraldehyde at 4 °C for 12 h. Subsequently, samples were dehydrated through an ethanol gradient series (30%, 50%, 70%, 80%, 90%, 95%, and 100%), with each step lasting 10 min. A 15 μL aliquot of the bacterial suspension was deposited onto a silicon wafer, sputter-coated with gold, and examined under a scanning electron microscope to assess morphological changes.

Biocompatibility evaluation

2.7

The cytotoxicity of the CMP NPs and CMP/Gel hydrogel was assessed using a CCK-8 assay. HUVECs and HaCaTs in the logarithmic growth phase were digested, counted, and seeded into 96-well plates at a density of 3 × 10^3^ cells/mL. After removal of the original medium, 90 μL of fresh high-glucose DMEM and 10 μL of the corresponding treatment solution (including PBS, CMP at 200 μg/mL, Gel, and CMP/Gel) were added to each well. Following 1 and 3 days of incubation, the solutions were aspirated and replaced with CCK-8 working solution. After incubation for 2 h, the absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated.

To visually assess cell viability, HUVECs and HaCaTs were seeded into 12-well plates at a density of 1 × 10^4^ cells/mL. After 24 h of incubation, the medium was replaced with treatment solutions and the cells were cultured for 1 or 3 days. The cells were then stained with Calcein AM and PI in PBS for 10 min in the dark. After washing with PBS, live (green) and dead (red) cells were imaged using an inverted fluorescence microscope.

Hemocompatibility was further evaluated using a hemolysis assay. Whole blood was collected from C57BL/6 mice and centrifuged at 3000 rpm for 10 min to isolate red blood cells (RBCs). The RBCs were washed with PBS (pH 7.4) until the supernatant became clear and then diluted 10-fold with PBS to prepare an RBC suspension. In Eppendorf tubes, 100 μL of the RBC suspension was mixed with 900 μL of each test sample (PBS, CMP at 20 μg/mL, Gel, and CMP/Gel), deionized water (positive control), or PBS (negative control). After incubation at 37 °C for 3 h, the mixtures were centrifuged, and 100 μL of the supernatant was transferred to a 96-well plate to measure absorbance at 540 nm. All experiments were performed with five replicates per group. The hemolysis ratio was calculated as follows:

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To assess long-term biosafety, major organs including the heart, liver, spleen, lungs, and kidneys were harvested for H&E staining to evaluate structural integrity and potential toxic responses.

HUVECs migration assay

2.8

HUVECs at 70∼80% confluence were washed twice gently with PBS and then serum-starved for 12 h in serum-free high-glucose DMEM basal medium. The cells were trypsinized, counted, and resuspended at a density of 5 × 10^3^ cells/mL. A 100 μL aliquot of the cell suspension was seeded into the upper chamber of a Transwell insert, while the lower chamber was filled with 1 mL of the corresponding solution, including PBS, CMP (20 μg/mL), Gel (hydrogel solution diluted 1:9 in medium, v/v), and CMP/Gel (hydrogel solution diluted 1:9 in medium, v/v). The plate was incubated for 24 h at 37 °C under 5% CO_2_ in the dark. After incubation, the Transwell inserts were removed, and the medium in the upper chamber was aspirated. The cells were fixed with 4% paraformaldehyde for 15 min, washed three times with PBS, and stained with 0.1% crystal violet solution for 20 min at room temperature in the dark. Non-migrated cells on the upper surface of the membrane were gently removed using a moistened sterile cotton swab. The inserts were air-dried for 10 min, and migrated cells on the lower surface of the membrane were imaged using an inverted microscope.

EdU cell proliferation assay

2.9

HUVECs at 70∼80% confluence were digested, counted, and seeded into 12-well plates at a density of 2 × 10^4^ cells/mL. After 12 h of incubation at 37 °C to allow for cell attachment, the culture medium was replaced with the respective treatment solutions, while the control group was cultured in complete medium containing 10% FBS. Following 24 h of treatment, the medium was aspirated and each well was incubated with 500 μL of 10 μM EdU working solution for 4 h at 37 °C under 5% CO_2_. After EdU labeling, the solution was removed and the cells were washed twice with PBS. The cells were fixed with 200 μL of fixation solution for 15 min at room temperature, followed by permeabilization with 200 μL of 0.5% Triton X-100 for 20 min. After permeabilization, the cells were washed twice with PBS and stained with 100 μL of DAPI solution (10 μg/mL) for 5 min at room temperature in the dark. The DAPI solution was then removed, and the cells were washed twice with PBS. The samples were imaged using an inverted fluorescence microscope. Proliferating cells labeled with EdU were detected using excitation at 488 nm (green fluorescence), and nuclei were visualized using excitation at 405 nm (blue fluorescence).

In vitro tube formation assay

2.10

HUVECs at 70∼80% confluence were washed twice gently with PBS and serum-starved in high-glucose DMEM basal medium for 12 h at 37 °C under 5% CO_2_. After trypsinization and counting, the cells were seeded into 6-well plates at a density of 1 × 10^6^ cells/mL and treated with the respective test solutions, while the control group received sterile PBS. The plates were incubated for 24 h under standard conditions. Following treatment, the cells were harvested by trypsinization and centrifugation, then resuspended at a density of 8 × 10^3^ cells/mL. A 90 μL aliquot of the cell suspension was added to each well of a 96-well plate pre-coated with Matrigel matrix and incubated at 37 °C for 4 h. Tube formation was observed and images were captured using an inverted microscope.

Immunofluorescence staining for VEGF and CD31

2.11

HUVECs at approximately 80% confluence were detached, counted, and adjusted to a density of 1 × 10^4^ cells/mL. A 500 μL aliquot of the cell suspension was seeded into 24-well plates containing pre-sterilized coverslips and gently swirled to ensure even distribution. The plates were incubated at 37 °C for 4∼6 h to allow for cell attachment. After incubation, the original medium was aspirated and replaced with the corresponding treatment solutions. Following 24 h of further culture, the cells were gently washed three times with PBS. Then, 200 μL of 4% paraformaldehyde was added to each well and incubated at room temperature for 15 min for fixation. After fixation, the cells were washed three times with PBS. After removal of residual liquid, the cells were blocked with 200 μL of 5% BSA at room temperature for 1 h. The blocking solution was discarded, and the cells were incubated with 200 μL of diluted primary antibodies against CD31 and VEGF at 4 °C overnight in the dark. The following day, the primary antibodies were removed, and the cells were washed three times with PBS. Subsequently, species-matched fluorescent secondary antibodies were added and incubated for 1 h at room temperature in the dark. After secondary antibody incubation, the cells were washed three times with PBS. Nuclei were stained with 200 μL of DAPI (10 μg/mL) for 5 min at room temperature in the dark, followed by two additional PBS washes. Coverslips were mounted cell-side up on glass slides using 10 μL of antifade mounting medium, inverted, and sealed with clear nail polish. After curing for 30 min at room temperature in the dark, images were acquired using a confocal laser scanning microscope.

Reactive oxygen species level detection

2.12

To evaluate the regulatory effects of different hydrogel materials on intracellular reactive oxygen species (ROS) levels in HUVECs under oxidative stress, we employed the DCFH-DA fluorescent probe assay combined with fluorescence microscopy and flow cytometry for quantitative analysis. An oxidative stress model was established by seeding HUVECs in high-glucose medium supplemented with 200 μmol/L H_2_O_2_ and pre-incubating for 2 h at 37 °C under 5% CO_2_. The medium was then replaced with fresh medium containing the respective treatment solutions and the cells were further incubated for 3 days. For fluorescence microscopy observation, after the culture period, the medium was removed and the cells were washed with PBS. Each well was incubated with 200 μL of DCFH-DA staining solution at 37 °C in the dark for 10∼20 min, and green fluorescence intensity within the cells was observed under a fluorescence microscope. For flow cytometric quantification, the cultured cells were trypsinized, collected by centrifugation, washed with PBS, and resuspended in 200 μL of DCFH-DA staining solution. After incubation in the dark, the fluorescence intensity of the cell population was measured by flow cytometry. This combined detection approach enables a comprehensive evaluation of the ROS-scavenging efficacy of the CMP/Gel composite hydrogel at both qualitative and quantitative levels.

In vivo antibacterial activity and wound healing evaluation

2.13

All animal experiments were conducted in accordance with institutional ethical guidelines and approved by the Animal Ethics Committee of Army Medical University (Approval No.: AMUWEC20257123). C57BL/6 mice aged 6∼8 weeks were used to establish a S. aureus-infected full-thickness skin defect model to evaluate the in vivo therapeutic efficacy of the CMP NPs and CMP/Gel composite hydrogel based on their previously demonstrated antibacterial, pro-angiogenic, and low-cytotoxicity properties in vitro.

After anesthesia with sodium pentobarbital, dorsal hair was shaved and the skin was disinfected with 75% ethanol. An 8-mm biopsy punch was used to create full-thickness skin wounds, which were inoculated with 20 μL of S. aureus suspension (10^8^ CFU/mL) and covered with a medical dressing overnight to establish infection. Infected mice were randomly assigned to eight groups: PBS, PBS + Light, CMP (20 μg/mL), CMP + Light (20 μg/mL), Gel, Gel + Light, CMP/Gel, and CMP/Gel + Light. All groups received consistent treatment conditions, with red light irradiation at 100 mW/cm^2^ for 10 min. To dynamically evaluate the wound healing process, 3 mice per group were randomly selected and humanely euthanized at each predefined time point (days 0, 3, 6, and 9) for histological analysis.

On designated days post-treatment, wound exudates were collected using sterile swabs, diluted in PBS, and plated onto LB agar plates. After 24 h of incubation at 37 °C, bacterial colonies were counted to assess in vivo antibacterial activity. Treatments were repeated every two days, and body weight and wound healing progression were monitored throughout the study. Mice were randomly euthanized on days 0, 3, 6, and 9, and wound tissues were harvested for hematoxylin and eosin (H&E) staining to evaluate inflammatory infiltration and tissue regeneration. Immunohistochemistry was performed to detect CD31 and Ki67 expression, assessing angiogenesis and cell proliferation.

Statistical analysis

2.14

All data are expressed as mean ± SD. Statistical comparisons were performed using one-way analysis of variance (ANOVA) in GraphPad Prism, with significance levels indicated as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Result and discussion

3

Synthesis and characterization of the CMP NPs

3.1

CMP NPs were synthesized through a biomimetic mineralization process using Ce6 as a template in the presence of Mg^2+^ and PO_4_^3−^ ions. Scanning electron microscopy (SEM, Fig. 1a) and transmission electron microscopy (TEM, Fig. 1b) confirmed the well-defined, spherical morphology of the CMP NPs. Dynamic light scattering (DLS) measurement further revealed an average hydrodynamic diameter of 420 nm with a polydispersity index (PDI) of 0.133 (Fig. 1c), indicating high colloidal homogeneity and excellent monodispersity. Elemental mapping confirmed the presence of C, N, O, Mg, and P, providing initial evidence for the formation of magnesium phosphate and successful incorporation of the Ce6 photosensitizer. X-ray photoelectron spectroscopy (XPS, Fig. 1d) further verified the coexistence of C, N, O, Mg, and P, with the signals from C and N reaffirming the effective loading of Ce6. The zeta potential of CMP NPs was measured to be −23.81 mV (Fig. 1e), contributing to their favorable colloidal stability. UV-Vis spectroscopy (Fig. 1f) showed that the characteristic absorption peaks of Ce6 at 395 nm and 669 nm were red-shifted to 407 nm and 673 nm in CMP NPs, respectively, suggesting π–π stacking interactions between Ce6 molecules within the nanoparticle matrix [33]. Fourier transform infrared (FTIR) spectroscopy (Fig. 1g) exhibited a marked reduction or disappearance of the C=O stretching vibration at 1702 cm^−1^, indicative of coordination between Ce6 and Mg^2+^. Additional peaks at 2964 cm^−1^ and 895 cm^−1^, assigned to C-H stretching vibrations of methyl and alkene groups, further supported the successful encapsulation of Ce6. X-ray diffraction (XRD, Fig. 1h) displayed no sharp diffraction peaks, confirming the amorphous nature of the CMP NPs. This structural feature may enhance solubility relative to crystalline counterparts, potentially promoting subsequent biodegradation. Collectively, these findings demonstrate the successful synthesis of CMP NPs and efficient encapsulation of Ce6.Fig. 1(a) SEM image showing the uniform spherical morphology of CMP NPs. (b) TEM image and corresponding elemental mapping, confirming homogeneous distribution of C, N, O, Mg, and P within the CMP NPs. (c) Hydrodynamic size distribution of CMP NPs (PDI = 0.133). (d) Full-range XPS survey spectrum of CMP NPs. (e) zeta potential of CMP NPs measured by dynamic light scattering.(f) UV-Vis absorption spectra of free Ce6 and CMP NPs. (g) FTIR spectra of Ce6 and CMP NPs. (h) XRD pattern of CMP NPs.Fig. 1

Synthesis and characterization of the CMP/Gel composite hydrogel

3.2

The CMP/Gel composite hydrogel was successfully fabricated by directly incorporating natural moisturizing factor (PCA-Na) and CMP NPs into a gelatin solution, followed by gelation at room temperature. Hydrogel formation is driven by the thermoreversible conformational transition of gelatin between random coil and triple-helix structures [34]. As a natural moisturizing factor, PCA-Na promotes ionic crosslinking between its carboxylate groups and the amino groups on gelatin chains, thereby significantly enhancing the mechanical strength of the resulting hydrogel [35].

Macroscopic gelation was visually confirmed (Fig. 2a and Fig. S1), providing initial evidence of successful formation of both Gel and CMP/Gel composite hydrogel. Furthermore, lyophilized samples of Gel (Fig. 2b) and CMP/Gel (Fig. 2c) exhibited well-defined porous morphologies under SEM. Elemental mapping revealed homogeneous distribution of P, Mg, N, O, and C throughout the CMP/Gel matrix (Fig. 2d), confirming effective incorporation of CMP NPs. FTIR spectroscopy (Fig. 2e) showed characteristic absorption peaks of gelatin at ≈1634 cm^−1^ (amide I) and ≈1544 cm^−1^ (amide II), indicating strong intermolecular interactions—primarily electrostatic attractions between protonated amide groups in gelatin and anionic groups of PCA-Na [31]. These interactions were preserved upon incorporation of CMP NPs. Rheological analysis further validated hydrogel formation. As shown in Fig. 2f (time sweep) and Fig. S2 (strain sweep), the storage modulus (G′) consistently exceeded the loss modulus (G″) across all measurements, demonstrating a dominant elastic behavior typical of a stable hydrogel network. Collectively, these results confirm the successful fabrication of the CMP/Gel composite hydrogel with efficient loading of CMP NPs.Fig. 2(a) Photographs showing the macroscopic gelation of the CMP/Gel composite hydrogel. (b) SEM images showing the internal microstructure of Gel hydrogels. (c) SEM images showing the internal microstructure of CMP/Gel hydrogels (d) SEM image and corresponding elemental mapping of the CMP/Gel composite hydrogel. (e) FTIR spectra of Gel and CMP/Gel hydrogels. (f) Rheological time-sweep analysis of the CMP/Gel composite hydrogel. (g) Swelling behavior of Gel and CMP/Gel hydrogels over 24 h. (h) In vitro degradation profiles of Gel and CMP/Gel hydrogels over 14 days. (i) Young's modulus values of Gel and CMP/Gel hydrogels measured under compression. (j) Adhesive strength of Gel and CMP/Gel hydrogels determined by lap-shear testing. Data are presented as mean ± SD, n = 3.Fig. 2

To systematically evaluate the potential of the CMP/Gel composite hydrogel as a wound dressing for infected wound healing, key functional properties including swelling capacity, degradation behavior, adhesive strength, and compressive mechanical performance were comprehensively characterized. Swelling experiments revealed that all hydrogels reached equilibrium within approximately 24 h (Fig. 2g). The CMP/Gel composite hydrogel exhibited superior swelling capacity, achieving an equilibrium swelling ratio of 49.85%, demonstrating its ability to rapidly absorb wound exudate, maintain a moist microenvironment, and facilitate nutrient exchange—features that support cell migration and tissue regeneration [36]. Degradation kinetics showed that both Gel and CMP/Gel composite hydrogel underwent a gradual slowdown in degradation after 6 days, reaching a cumulative degradation rate of 70% by day 14 (Fig. 2h), confirming their favorable biodegradability. The controlled degradation profile of CMP/Gel minimizes the risk of secondary tissue damage associated with frequent dressing changes, while sustained structural integrity ensures prolonged wound protection. Mechanically, both hydrogels displayed Young's moduli exceeding 50 kPa (Fig. 2i and Fig. S3), indicating sufficient stiffness to withstand physiological stresses and maintain structural stability [37]. Adhesion tests demonstrated comparable bonding performance between CMP/Gel and pure Gel hydrogels (Fig. 2j and Fig. S4), indicating that CMP NP incorporation does not significantly impair interfacial adhesion. Thus, the composite maintains robust adhesion while fulfilling the mechanical requirements for practical wound dressing applications. Collectively, the CMP/Gel composite hydrogel exhibits well-balanced swelling, degradation, and mechanical properties suitable for infected wound repair, highlighting its promising translational potential.

Antimicrobial activity of CMP/Gel composite hydrogel

3.3

To evaluate the photodynamic performance of the developed CMP/Gel composite hydrogel, ROS generation was assessed in vitro under red-light irradiation using 2′,7′-dichlorodihydrofluorescein (DCFH) as a fluorescent probe. Upon oxidation by ROS, the non-fluorescent DCFH is converted into a highly fluorescent product with emission at 525 nm. As shown in Fig. 3a, fluorescence intensity increased progressively with irradiation time, indicating sustained ROS production by CMP/Gel upon light activation. These results confirm the effective photodynamic activity of the CMP/Gel composite hydrogel, supporting its potential for antibacterial applications.Fig. 3(a) Time-dependent fluorescence intensity changes of the DCFH probe in CMP/Gel solution under 630 nm laser irradiation (100 mW/cm^2^). (b) Photographs of crystal violet-stained mature S. aureus biofilms after different treatments. (c) Quantitative assessment of biofilm biomass reduction based on crystal violet staining. Data are presented as mean ± SD, n = 3. (d) SEM images depicting architectural disintegration of bacterial biofilms following different treatments. (e) Representative photographs of inhibition zones against S. aureus at bacterial concentrations ranging from 10^5^ to 10^8^ CFU/mL following treatment with PBS + L, Gel + L, CMP + L, and CMP/Gel + L. (f) Quantitative analysis of inhibition zone diameters. Data are presented as mean ± SD, n = 3. (g) Fluorescence micrographs from live/dead bacterial viability staining, with green fluorescence indicating viable bacteria and red fluorescence indicating dead cells. (h) SEM images revealing morphological alterations in S. aureus after different treatments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)Fig. 3

The inhibitory and disruptive effects of the treatments on S. aureus biofilms were quantitatively assessed using crystal violet staining. As summarized in Fig. S5, in the biofilm prevention assay, both CMP + L and CMP/Gel + L showed significantly reduced staining intensity compared to PBS + L, indicating strong suppression of initial bacterial adhesion and biofilm formation. In the biofilm eradication assay (Fig. 3b and c), the CMP + L and CMP/Gel + L groups exhibited the most pronounced clearance, with the lowest residual staining, demonstrating robust degradation capacity against pre-established mature biofilms. Collectively, these results indicate that, upon red-light irradiation, both CMP NPs and CMP/Gel composite hydrogel can effectively prevent biofilm formation and eliminate existing biofilms through photodynamic mechanisms, underscoring their potential for antibiofilm applications.

To visually evaluate the eradication efficacy against mature S. aureus biofilms, biofilm architecture post-treatment was examined using SEM. As shown in Fig. 3d, the PBS + L control group displayed an intact and continuous biofilm structure. In contrast, samples treated with CMP + L and CMP/Gel + L exhibited extensive architectural disintegration, with only scattered cellular debris remaining on the surface. These findings provide visual confirmation that both CMP and CMP/Gel composite hydrogel can efficiently degrade pre-formed biofilms under red-light irradiation, demonstrating strong antibiofilm capability.

The antibacterial efficacy of different treatments (PBS + L, CMP + L, Gel + L, and CMP/Gel + L) against S. aureus, a common wound-infecting pathogen, was systematically evaluated using the disk diffusion method under red-light irradiation across bacterial concentrations ranging from 10^5^ to 10^8^ CFU/mL. As illustrated in Fig. 3e and f, both imaging and quantitative analysis of inhibition zones revealed that neither PBS + L nor Gel + L produced detectable inhibition zones at any concentration, with dense bacterial growth observed on the agar surface. In contrast, CMP + L and CMP/Gel + L exhibited marked, concentration-dependent antibacterial activity. Complete sterilization, evidenced by clear agar surfaces, was achieved at 10^5^ and 10^6^ CFU/mL. A significant reduction in bacterial colonies was observed at 10^7^ CFU/mL, while distinct inhibition zones remained visible even at 10^8^ CFU/mL. Quantitative analysis further confirmed statistically significant differences between the PBS + L group and both the CMP + L and CMP/Gel + L groups. The hydrogel also exhibited significant antibacterial activity against E. coli (Fig. S6). Noticeable inhibition zones were observed for the CMP/Gel + L group at bacterial concentrations of 10^6^ and 10^7^ CFU/mL. Even at a high bacterial load of 10^8^ CFU/mL, visible inhibition zones were still present. Quantitative analysis further confirmed that the inhibition zone diameters of the CMP/Gel + L group were significantly larger than those of the PBS + L, Gel + L, and CMP + L groups across all tested concentrations, demonstrating its sustained and potent antibacterial efficacy against Gram-negative bacteria. These findings demonstrate that CMP and CMP/Gel exert potent antibacterial effects under red-light irradiation over a broad range of bacterial loads, highlighting their promise as localized antibacterial therapies.

To assess photodynamic antibacterial efficacy, bacterial viability following various treatments was evaluated using a live/dead staining assay. As shown in Fig. 3g, distinct fluorescence patterns were observed across the four experimental groups. The PBS + L and Gel + L groups displayed only faint background fluorescence, indicating minimal bacterial killing. In contrast, the CMP + L and CMP/Gel + L groups exhibited intense and uniform red fluorescence throughout the field of view, indicating widespread bacterial death, with only sparse green signals corresponding to residual viable cells. These results further validate that both Ce6-loaded CMP and the CMP/Gel composite enable highly efficient and broad-spectrum bacterial eradication upon light activation.

To visually examine structural alterations in bacteria after treatment, bacterial cell morphology was analyzed by SEM. As demonstrated in Fig. 3h, bacteria in the PBS + L and Gel + L groups maintained intact, smooth surfaces without apparent damage. In contrast, bacteria treated with CMP + L and CMP/Gel + L showed severe morphological disruption, including membrane rupture and leakage of intracellular contents—morphological features characteristic of bacterial cell death. These observations provide direct morphological evidence that both CMP NPs and CMP/Gel composite hydrogel effectively compromise bacterial membrane integrity via ROS-mediated photodynamic action, resulting in potent bactericidal effects.

Biocompatibility of CMP/Gel composite hydrogel

3.4

Biocompatibility is a fundamental requirement for the application of biomedical materials. In this study, the cytocompatibility, hemocompatibility, and in vivo safety of CMP NPs, Gel, and the CMP/Gel composite hydrogel were systematically evaluated. For cytocompatibility assessment, HUVEC and HaCaT cell viability was determined using Calcein-AM/PI fluorescence live/dead staining. As shown in Fig. 4a, after 1 and 3 days of culture at the tested concentrations, cells in all groups exhibited high viability, with the majority of cells appearing green (live) and only rare red (dead) signals observed. The CCK-8 assay revealed that the Gel group exhibited significantly higher cell viability than the PBS group (Fig. 4b and c), confirming the intrinsic pro-proliferative property of gelatin. Moreover, both the CMP and CMP/Gel groups displayed further pronounced increases in viability, suggesting additive or synergistic contributions from gelatin and Mg^2+^ ions released from CMP. Collectively, these data substantiate the superior cytocompatibility of the CMP/Gel composite hydrogel.Fig. 4(a) Fluorescence images of cell live/dead staining of HUVECs and HaCaTs following 1- and 3-day culture under different treatment conditions. Quantitative analysis of (b) HaCaTs and (c) HUVECs viability across treatment groups. Data are presented as mean ± SD, n = 3. (d) In vitro hemolysis ratio of different samples. Data are presented as mean ± SD, n = 3. (e) H&E-stained histological sections of major organs (heart, liver, spleen, lung, and kidney) harvested from mice.Fig. 4

Regarding hemocompatibility, hemolysis assays (Fig. 4d) revealed that all experimental groups had hemolysis rates below 5%, indicating no significant hemolytic activity and compliance with international standards for biomaterial hemocompatibility. Moreover, in vivo safety evaluation in mice showed no apparent pathological lesions in H&E-stained sections of major organs following treatment with the materials combined with red light irradiation (Fig. 4e), suggesting good histocompatibility. Taken together, comprehensive evaluations at the cellular, hematological, and tissue levels consistently demonstrate that the CMP/Gel composite hydrogel exhibits robust biocompatibility, supporting its potential for clinical translation.

In vitro assessment of the CMP/Gel composite hydrogel on cell proliferation, angiogenesis, and anti-inflammatory activity

3.5

Cell migration plays a critical role in wound healing, particularly in re-epithelialization and restoration of barrier function. Building on previous CCK-8 assays that demonstrated the ability of CMP/Gel composite hydrogel to promote HUVEC and HaCaT proliferation, this study further evaluated their effects on cell migration using Transwell assays and EdU immunofluorescence staining. To specifically assess the contribution of Mg^2+^ to endothelial cell proliferation, EdU staining and quantitative analysis were performed. As shown in Fig. 5a, compared with the control group, both the CMP and CMP/Gel groups containing Mg^2+^ exhibited significant pro-proliferative effects, with a markedly increased proportion of EdU-positive cells. Notably, the Gel group exhibited a moderate yet statistically significant increase in Relative EdU positive cells compared to the PBS group (Fig. 5g), confirming its intrinsic, matrix-mediated pro-proliferative activity. Quantitative results aligned well with the distribution pattern of EdU-positive (green) cells observed in fluorescence images, indicating that Mg^2+^ plays a pivotal role in enhancing cellular proliferation.Fig. 5(a) Assessment of cell proliferation using the EdU assay. (b) Analysis of cell migration via Transwell assay. (c) Evaluation of angiogenic potential by in vitro tube formation assay. (d) Measurement of intracellular ROS levels using the DCFH-DA fluorescent probe. Immunofluorescence analysis of (e) CD31 and (f) VEGF expression. Quantitative analysis of the (g) EdU-positive cell rate, (h) cell migration and (i) branch points in the tube formation assay. Data are presented as mean ± SD, n = 3. (j-k) Relative (j) CD31 and (k) VEGF expression level analysis. Data are presented as mean ± SD, n = 3. (l) Flow cytometric detection of ROS signal intensity.Fig. 5

The migratory capacity of HUVECs was assessed using Transwell assays. Microscopic images (Fig. 5b) revealed that the number of migrated cells in the CMP and CMP/Gel groups was significantly higher than in the PBS control group, with the CMP/Gel group showing the most densely populated membrane. Quantitative analysis (Fig. 5h) showed that the number of migrated cells in the CMP/Gel and CMP NP groups was 1.9-fold and 2.0-fold higher than the control, respectively, while the Gel group exhibited a 1.5-fold increase. These consistent findings demonstrate that the CMP/Gel composite hydrogel effectively enhances both proliferation and migration of endothelial cells, outperforming its individual components.

Angiogenesis is a decisive process in wound healing, and insufficient vascularization is a major contributor to delayed tissue repair. The infected wound microenvironment can severely impair endothelial cell function and downregulate the expression of angiogenesis-related biomarkers, leading to local tissue hypoxia and compromised delivery of nutrients and oxygen [38]. These pathological conditions ultimately disrupt key regenerative processes such as cell proliferation and migration [39]. Therefore, developing novel dressings with pro-angiogenic properties has become a crucial strategy in advanced wound management. Based on this rationale, we evaluated the angiogenic potential of the CMP/Gel composite hydrogel. As illustrated in Fig. 5c and i, different treatments were assessed for their effects on HUVEC tube formation in an in vitro angiogenesis assay. Qualitatively, cells in the PBS control group appeared sparse and failed to form distinct tubular structures. The Gel group showed localized cell aggregation but only short, discontinuous segments. In contrast, both the CMP and CMP/Gel groups displayed robust pro-angiogenic activity, characterized by tightly interconnected cells forming dense, mesh-like capillary networks. The structural complexity and total tube length were substantially greater than those in the control and Gel groups. Quantitative analysis confirmed that, relative to the control, the CMP/Gel and CMP NP groups exhibited approximately 1.7-fold and 2.1-fold increases in branch point number, respectively. Collectively, these results indicate that the CMP/Gel composite hydrogel enhances endothelial tube formation and vascular network complexity via synergistic interactions between Mg^2+^ ions and the gelatin matrix, providing experimental evidence for its potential in promoting vascularization during wound healing.

VEGF and CD31 act synergistically during angiogenesis: VEGF primarily drives endothelial cell activation and neovascularization, whereas CD31 contributes to maintaining vascular integrity [40,41]. To further evaluate the pro-angiogenic potential of the CMP/Gel composite hydrogel, the expression levels of VEGF and CD31 in HUVECs were analyzed by immunofluorescence staining. Results (Fig. 5e and f) showed that, compared to the PBS control, both the CMP and CMP/Gel groups exhibited significantly stronger green fluorescence signals for CD31 and VEGF, with preserved cellular morphology and tight intercellular junctions. The CMP/Gel group displayed the most intense fluorescence among all groups. Quantitative analysis (Fig. 5j and k) confirmed that CD31 expression in the CMP/Gel group was upregulated by approximately 2.4-fold and VEGF by about 2.1-fold relative to the control group. These findings suggest that the Mg^2+^-containing CMP/Gel composite hydrogel can synergistically upregulate key angiogenic markers, potentially through Mg^2+^-mediated enhancement of endothelial function, offering molecular-level insights into its capacity to support vascularized repair in infected wounds.

Persistent overproduction of ROS due to bacterial infection can prolong the inflammatory phase of wound healing, disrupting the transition to proliferation and remodeling [42,43]. Therefore, scavenging ROS and mitigating inflammation are critical therapeutic strategies. To evaluate the ROS-scavenging capacity of the CMP/Gel composite hydrogel under oxidative stress, an in vitro model of H_2_O_2_-induced HUVECs was established and analyzed using the DCFH-DA fluorescent probe combined with flow cytometry. Fluorescence microscopy (Fig. 5d) revealed intense green fluorescence in the PBS control group, confirming successful induction of oxidative stress. In contrast, the CMP and CMP/Gel groups exhibited the weakest fluorescence signals, indicating strong ROS-scavenging activity. Flow cytometry quantification (Fig. 5l and Fig. S7) further confirmed that both CMP and CMP/Gel treatments significantly reduced intracellular ROS levels, with markedly lower fluorescence intensity compared to the PBS control and Gel groups. These results demonstrate that the Mg^2+^-containing CMP/Gel composite hydrogel effectively alleviates oxidative stress, providing experimental support for its potential to mitigate oxidative damage in infected wounds.

Antibacterial activity and wound repair effect of the CMP/Gel hydrogel in vivo

3.6

Based on the remarkable antibacterial activity and favorable biocompatibility of the CMP/Gel composite hydrogel demonstrated in vitro, a S. aureus-infected full-thickness skin defect model (inoculum concentration: 1 × 10^8^ CFU/mL) was established in C57BL/6 mice to systematically evaluate its in vivo therapeutic efficacy (Fig. 6a). Application of the CMP/Gel composite hydrogel to the wound site not only formed a physical barrier that protected the injured tissue but also helped maintain a moist wound healing environment due to its excellent water retention capacity. Furthermore, the hydrogel system enabled sustained release of CMP NPs, which, under red light irradiation, synergistically exerted antibacterial and pro-angiogenic effects.Fig. 6(a) Schematic illustration of the in vivo wound healing experimental timeline in a murine S. aureus-infected excisional wound model. (b) Representative photographs documenting the wound healing process in different treatment groups. Quantitative analysis of residual wound area percentage on days (c) 3, (d) 6, and (e) 9. (f) Bacterial colony formation assay of wound tissues. Data are presented as mean ± SD, n = 3.Fig. 6

Wound healing was monitored by digital photography every two days (Fig. 6b). On day 0, the wound areas were largely consistent across all groups. Over time, all treatment groups exhibited varying degrees of wound closure, with the CMP/Gel + L group showing the most pronounced healing response. Specifically, the PBS and PBS + L groups still displayed large unhealed wounds by day 9, indicating delayed repair. While treatment with CMP or Gel alone promoted partial healing, their combination with red light (CMP + L, Gel + L) led to accelerated wound closure. Notably, the CMP/Gel + L group achieved the most significant therapeutic outcome: the wound area was substantially reduced by day 6 and nearly completely closed with complete re-epithelialization by day 9. The newly formed tissue appeared intact and closely resembled the surrounding healthy skin in appearance. As shown in Fig. 6c–e, quantitative analysis confirmed that the CMP/Gel composite hydrogel, when combined with red light irradiation, significantly enhanced infected wound closure, demonstrating a strong synergistic effect, particularly during the mid-to-late stages of healing (days 6–9). These consistent in vivo findings indicate that the CMP/Gel + L strategy holds substantial promise for promoting infected wound repair.

The PDT antibacterial efficacy of Ce6 in CMP and CMP/Gel under red light irradiation was directly evaluated by bacterial colony culture from infected wounds. As shown in Fig. 6f, both the PBS and PBS + L groups exhibited dense bacterial growth, indicating no intrinsic antibacterial effect from the buffer or light alone. In contrast, the CMP + L and CMP/Gel + L groups showed a marked reduction in bacterial colonies upon light activation, confirming the effective PDT of Ce6. These in vivo results align well with previous in vitro antibacterial assays. In summary, the CMP/Gel composite hydrogel, when activated by red light, effectively eliminates bacteria in infected wounds through Ce6-based PDT, providing experimental evidence for its potential clinical application in infected wound management.

Fig. 7a shows a systematic evaluation of the healing efficacy of different treatments on infected wounds by H&E staining. The PBS and PBS + L groups exhibited substantial tissue defects with marked inflammatory cell infiltration, indicating that neither PBS alone nor red light irradiation effectively promotes wound repair. In the Gel and Gel + L groups, partial formation of new tissue was observed along with reduced inflammatory infiltration; however, tissue structural integrity remained poor. In contrast, the CMP/Gel + L group displayed the most pronounced reparative outcome: the wound was nearly closed, with a continuous and intact neoepithelium, well-organized collagen fibers in the dermis, and minimal inflammation. These findings demonstrate that the CMP/Gel composite hydrogel, upon red light activation, effectively eliminates bacteria through Ce6-mediated PDT and synergistically enhances tissue regeneration and structural reconstruction via the biological activity of Mg^2+^ ions, thereby significantly improving the quality of infected wound healing.Fig. 7(a) H&E staining of wound tissues following different treatments. Immunohistochemical staining of (b) CD31 and (c) Ki67 in wound tissues following different treatments.Fig. 7

Immunohistochemical staining for CD31 (Fig. 7b, an endothelial cell marker) and Ki67 (Fig. 7c, a proliferation marker) was performed on infected wound tissues to assess angiogenesis and cell proliferation under different treatments. In the PBS and PBS + L groups, CD31 and Ki67 signals were sparse and scattered, indicating limited vascularization and proliferative activity. In contrast, the CMP/Gel + L group showed densely distributed CD31-positive microvessels and abundant Ki67-positive cells, reflecting significantly enhanced angiogenesis and cellular proliferation. Quantitative analysis confirmed that both microvessel density and the cell proliferation index in the CMP/Gel + L group were significantly higher than in all other groups (Fig. S8). These results indicate that, under red light irradiation, the CMP/Gel composite hydrogel promotes angiogenesis through Mg^2+^ ions while acting synergistically with PDT to accelerate the repair of infected wounds. This study provides molecular- and cellular-level evidence that the CMP/Gel + L treatment enables high-quality wound healing through a dual mechanism combining antibacterial and pro-angiogenic effects.

Mechanistic insights into CMP/Gel-accelerated healing of infected wounds

3.7

To elucidate the molecular mechanisms by which the CMP/Gel composite hydrogel exerts Ce6-mediated PDT antibacterial and Mg^2+^-facilitated wound healing, RNA sequencing (RNA-seq) was performed on wound tissues harvested from C57BL/6 mice treated with CMP/Gel + L or PBS. Principal component analysis (PCA) revealed that samples from the PBS control group clustered primarily in the positive region of the PC1 axis, whereas those from the CMP/Gel-treated group showed a marked shift toward the negative region of PC1 (Fig. 8a). This distinct separation indicates that red light-induced PDT of CMP/Gel triggers systematic transcriptomic alterations in wound tissue, leading to significant differences in gene expression between treatment and control groups. The clear clustering pattern provides a robust foundation for subsequent in-depth analysis of differentially expressed genes (DEGs) and associated signaling pathways. Integrated analysis of volcano plot (Fig. 8b) and DEGs (Fig. 8c) results identified 1,339 significantly differentially expressed genes in CMP/Gel-treated wound tissues compared to the PBS group, including 625 upregulated and 714 downregulated genes. This transcriptome-wide reprogramming of gene expression underscores the broad regulatory impact of CMP/Gel intervention, establishing a critical molecular basis for functional enrichment and pathway validation.Fig. 8(a) PCA score plot of transcriptome data from experimental group and control group. (b) Volcano plot displaying the distribution of DEGs. (c) Bar graph showing the number of DEGs between the CMP/Gel and control groups. (d) Circular heatmap visualization of enriched gene functions. (e) Bubble plot of KEGG pathway enrichment analysis. (f) Bubble plot of GO functional enrichment analysis. (g) Schematic illustration of the proposed mechanism by which the CMP/Gel + L synergistically promotes wound healing.Fig. 8

Building upon previous in vitro and in vivo evidence demonstrating the multifaceted therapeutic effects of the CMP/Gel hydrogel including pro-angiogenic, anti-inflammatory, and pro-proliferative/migratory activities, this study further deciphered the Mg^2+^-mediated repair mechanisms at the molecular level through transcriptomic profiling of infected wounds. As shown in Fig. 8e, KEGG pathway enrichment analysis revealed that DEGs were significantly enriched in the VEGF, PI3K-Akt, and JAK-STAT signaling pathways, all of which are known to regulate angiogenesis and cell proliferation[[44], [45], [46]]. Concurrent enrichment in the NF-κB and IL-17 signaling pathways suggests a role for Mg^2+^ in attenuating excessive inflammatory responses [47]. GO enrichment analysis (Fig. 8f) further indicated that biological processes were predominantly associated with “positive regulation of angiogenesis”, “cell migration”, and “maintenance of vascular diameter”, findings consistent with in vivo observations of increased CD31-positive microvessel density and enhanced Ki67-positive cell proliferation. Circular heatmap analysis (Fig. 8d) revealed significant upregulation of key angiogenesis-related genes, including VEGF and angiopoietin. Additionally, coordinated changes were observed in the expression of genes encoding complement components (C2, C3, C4) and coagulation factors (T1, T3, T4), indicating broader modulation of immune and hemostatic systems. These results suggest that Mg^2+^ not only directly activates angiogenic programs but also contributes to remodeling the wound repair microenvironment by regulating immune responses and the coagulation-fibrinolysis balance.

Integrated mechanistic analysis supports a tripartite synergistic mechanism (Fig. 8g) by which Mg^2+^ promotes infected wound healing: (1) activation of the VEGF–PI3K/Akt signaling axis to stimulate endothelial cell proliferation and vascular maturation; (2) modulation of the JAK-STAT pathway to enhance cell migration and re-epithelialization; and (3) suppression of the NF-κB/IL-17 inflammatory axis to mitigate oxidative stress. These molecular events, together with the previously observed upregulation of CD31 and VEGF and reduction in ROS levels, form a coherent evidence chain that systematically elucidates how Mg^2+^ remodels the repair microenvironment through multi-pathway crosstalk, thereby significantly improving the quality of healing in infected wounds.

It is important to emphasize that while transcriptomic analysis revealed coordinated transcriptional modulation across multiple signaling axes, including VEGF–PI3K/Akt, JAK–STAT, and NF-κB/IL-17 pathways, the absence of direct functional validation remains a critical limitation for establishing causal mechanistic links. Consequently, the current interpretations are inherently hypothesis-generating. They reflect statistically robust correlative patterns derived from pathway enrichment, gene set variation analysis, and weighted gene co-expression network analysis, but do not constitute experimentally verified cause–effect relationships. To rigorously substantiate these molecular hypotheses, future work will implement targeted perturbation strategies in biologically relevant cell models, including pharmacological inhibition (e.g., LY294002 for PI3K; ruxolitinib for JAK), siRNA-mediated knockdown of hub genes (e.g., STAT3, RELA, IL17RA), and phenotypic rescue experiments, thereby enabling both loss-of-function confirmation and hierarchical dissection of pathway interdependencies.

Conclusion

4

In summary, we have successfully developed a magnesium phosphate mineralized Ce6 composite hydrogel (CMP/Gel) wound dressing that synergistically accelerates infected wound healing by integrating PDT with Mg^2+^-mediated pro-regenerative functions. In this system, CMP NPs serve a dual role as both a source of bioactive Mg^2+^ ions and a carrier for Ce6, while the gelatin-based hydrogel maintains a moist wound microenvironment and enables sustained release of active components. Upon red light irradiation, the CMP/Gel composite hydrogel dressing exerts potent antibacterial effects through Ce6-mediated PDT. Concurrently, the released Mg^2+^ ions effectively alleviate oxidative stress, suppress excessive inflammation, stimulate angiogenesis, and promote tissue regeneration. Both in vitro and in vivo experiments demonstrate that the combination of PDT and Mg^2+^-driven biological activities significantly enhances antibacterial efficacy and wound repair outcomes. Owing to its dual-functional capacity in combating infection and promoting regeneration, simple fabrication process, and excellent biocompatibility, the CMP/Gel composite hydrogel dressing represents a promising and clinically translatable candidate for the treatment of infected wounds.

CRediT authorship contribution statement

Yongpeng Su: Data curation, Formal analysis, Investigation. Shunying Liu: Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft. Mingdi He: Formal analysis, Methodology. Lingfei Li: Investigation, Methodology. Guihong Yang: Methodology, Visualization. Xiaohan Liu: Formal analysis, Investigation. Yiting Feng: Methodology, Visualization. Hui Tang: Methodology, Visualization. Lingbo Li: Formal analysis, Methodology, Visualization. Jianxin Wu: Formal analysis, Visualization. Zhenglin Li: Methodology, Visualization. Yi Liang: Funding acquisition, Methodology, Validation. Chao Qi: Conceptualization, Formal analysis, Funding acquisition, Project administration, Supervision, Writing – review & editing. Kaiyong Cai: Funding acquisition, Resources, Supervision. Xia Lei: Conceptualization, Funding acquisition, Resources, Supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.