Functional Hydrogels Promote Chronic Infectious Wound Healing by Re-rousing Macrophage M1 and Inducing Bacterial Copper-Like Death

Functional Hydrogels Promote Chronic Infectious Wound Healing by Re-rousing Macrophage M1 and Inducing Bacterial Copper-Like Death | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Functional Hydrogels Promote Chronic Infectious Wound Healing by Re-rousing Macrophage M1 and Inducing Bacterial Copper-Like Death Chao Xiang, Chaoyu Pu, XueMei Zhong, Yong Wang, Weiyong Song, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5378421/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Traditional antibiotics are often ineffective against biofilm-associated infections, and biofilm-induced macrophage immune evasion directly halts the wound healing process. Disrupting biofilms and regulating macrophage immune functions are critical to improving wound healing. Results: In this study, we synthesized g-C 3 N 4 with peroxidase (POD) enzyme activity via thermal polymerization and copper alginate microspheres (CAM) via gas cutting. These were co-encapsulated into GelMA hydrogels to form a functionalized wound repair system (GelMA/CAM@g-C 3 N 4 ) with both anti-biofilm and local immune microenvironment remodeling capabilities. In vitro, this system exhibited excellent biocompatibility and promoted endothelial cell migration, vascular formation, and CD31 expression. It also polarized macrophages toward the M1 phenotype, restoring their pro-inflammatory functions, upregulating inflammatory cytokines (IL-1, IL-6, TNF-α), and inhibiting Staphylococcus aureus and Escherichia coli. In vivo, the system suppressed S. aureus growth, promoted angiogenesis and collagen deposition, and reshaped the pathological microenvironment to achieve wound repair and regeneration. Conclusions: This system offers a new therapeutic strategy for chronic infectious wounds. Chronic infectious wounds hydrogels microspheres immune regulation macrophages Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Chronic wounds are those that fail to restore anatomical and functional integrity within three months post-injury, affecting approximately 1–2 % of the global population[1]. Infection is considered one of the key factors contributing to chronic wounds, as it impedes the healing process, increases wound pain, places a significant economic burden on healthcare systems, and lowers patients' quality of life [2]. Over the past few decades, many new antibiotics have been developed, and antibiotics have long been considered the ultimate solution for biofilm-related bacterial infections in humans [3]. Unfortunately, the formation of biofilms is highly resistant to antibiotics, making infections increasingly challenging [4-6]. Biofilms are complex bacterial aggregates composed of bacterial cells, extracellular polymeric substances (EPS), and other microorganisms, which aid in bacterial colonization, enhance resistance to antimicrobial agents, evade host immunity, and facilitate interbacterial signaling within the community [7-9]. Staphylococcus aureus is the most common organism isolated from human chronic wounds [10]. Currently, clinical biofilm treatments involve removing infected tissue and using antibiotics in combination. However, this approach has several drawbacks, including invasive procedures, uncertain efficacy, and high treatment costs [11, 12]. Therefore, there is an urgent need to develop alternative therapeutic platforms to control pathogenic biofilm-associated infections. The normal wound healing process progresses through four overlapping cascades: hemostasis, inflammation, proliferation, and remodeling [13]. Macrophages are the first line of defense against pathogens and possess high plasticity, rapidly adapting to the complex microenvironment triggered by tissue infection or inflammation through polarization. M1 macrophages recognize, phagocytose, and kill invading bacteria, and after completing their pro-inflammatory mission, they polarize to the M2 phenotype, which promotes tissue repair. Macrophages are key regulatory factors in wound healing and tissue regeneration, ultimately leading to wound healing [14-17]. However, in chronic wounds, mature biofilms have dense EPS, which makes it difficult for macrophages to phagocytose, leading to "frustrated phagocytosis" [18]. Reactive oxygen species (ROS) have been shown to promote M1 macrophage polarization (pro-inflammatory and bactericidal) and activate macrophage oxidative bursts, acidification, and enzyme-mediated phagocytosis, including phagolysosome formation, to eliminate invading bacteria [19, 20]. Thus, reactivating the pro-inflammatory function of M1 macrophages in chronically infected wounds, reversing the immune environment, and reshaping the "physiological healing process" of the wound through ROS are considered promising therapeutic strategies for chronically infected wounds caused by biofilm-induced immune evasion [8, 21, 22]. Currently, novel nanomaterials with high peroxidase (POD)-like activity, such as noble metals, iron-based nanomaterials, transition metal dichalcogenides, and polyoxometalates, are known [23-25]. Unfortunately, these nanomaterials still have limitations, such as inherent cytotoxicity and oxidative stress under low H 2 O 2 concentrations, which can damage cellular function. Photocatalytic antibacterial therapy is an effective strategy relying on external stimuli. Due to its controllability, safety, and non-invasiveness, it has garnered increasing attention over the past few decades, with many photosensitizers developed. The metal-free polymer two-dimensional nanomaterial graphitic carbon nitride (g-C 3 N 4 ) is particularly attractive as a photosensitizer for non-antibiotic antibacterial applications due to its abundance, ease of synthesis, appropriate band structure, high stability, and low toxicity [26, 27]. More importantly, g-C 3 N 4 has been recently shown to generate micromolar concentrations of H 2 O 2 continuously, which can successfully combat established biofilms [28]. However, although the generated H 2 O 2 can fight biofilms, a single antibacterial mechanism is insufficient to completely prevent biofilm formation or provide long-term antibacterial effects at the wound site. Metal nanoparticles are known for their non-specific bacterial toxicity mechanisms, which prevent bacteria from developing resistance and broaden their antibacterial activity [29]. Copper is a classical antimicrobial metal that has been widely used to combat Gram-negative, Gram-positive, and fungal infections due to its recognized antibacterial and pro-angiogenic properties, making it essential in wound healing [30]. Studies have shown that copper ions exert antimicrobial effects through various mechanisms, including altering membrane permeability, DNA structure, metabolism, and enzyme activity [31, 32]. Thus, we aim to incorporate antimicrobial ions to achieve synergistic antibacterial effects. Alginate is a naturally occurring anionic polysaccharide extracted from brown algae, which forms stable complex structures upon contact with divalent cations (such as calcium and copper) and promotes the release of these ions in acidic conditions [33]. We developed copper alginate microspheres (CAM) for slow release, achieving long-term antibacterial effects for chronically infected wounds. To ensure the therapeutic effects of the composite system of nanomaterials and metal nanoparticles, and considering its potential as an ideal medium for wound healing, we further encapsulated the system in hydrogels, which are widely used for wound treatment due to their excellent hydrophilicity, drug-loading capacity, and sustained release properties [34]. Gelatin methacrylate (GelMA) is a gelatin derivative and denatured collagen product known for its excellent solubility and low antigenicity. The incorporation of the Arg-Gly-Asp (RGD) sequence enhances biological interactions between cells and the GelMA scaffold, making GelMA an ideal biomaterial for wound healing, morphogenesis, and tissue restoration [35]. In summary, based on the pathogenesis of biofilm formation and macrophage immune evasion in chronic infected wounds, we developed a functionalized hydrogel microsphere system. This system combats bacterial biofilms through ROS and reactivates the M1 phenotype of macrophages, while the slow release of antimicrobial metal ions provides long-lasting antibacterial effects and promotes angiogenesis, reshaping the physiological repair process of the wound. We synthesized g-C 3 N 4 with POD enzyme activity through thermal polymerization and CAM via a gas-cutting method, and finally encapsulated g-C 3 N 4 and CAM in GelMA hydrogel to construct a chronic infected wound healing system (GelMA/CAM@g-C3N4) （Scheme 1）. This system demonstrated excellent biocompatibility in vitro, promoted endothelial cell migration and vascular tube formation, reactivated M1 macrophages, and enhanced the expression of inflammatory cytokines (IL-1, IL-6, TNF-α). It significantly inhibited Staphylococcus aureus and Escherichia coli. Gene expression analysis showed that the system induced copper-like death of S. aureus by downregulating metabolic pathways such as glycolysis, oxidative phosphorylation, purine, and pyrimidine. In vivo, the system inhibited S. aureus growth, reactivated M1 macrophages by day 3, polarized macrophages to the M2 phenotype by day 7, promoted angiogenesis, and enhanced collagen deposition. This system provides a novel approach for treating biofilm-infected wounds and lays a foundation for addressing biofilm-related infectious diseases. Methods Experimental Materials Urea, sodium alginate, copper chloride, and gelatin were purchased from Aladdin Reagent (Shanghai, China); CCK-8 was obtained from Biyuntian Biotechnology (Shanghai, China); dialysis bags (8000D) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China); the bacterial live-dead staining kit was purchased from Maokang Biotechnology Co., Ltd. (Shanghai, China); Matrigel was obtained from Gelcon BioTech (Shanghai, China); photo-initiator and methacrylic anhydride were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China); DAPI was purchased from Wuhan Boster Biological Technology Ltd.; CD31 antibody, CD86 antibody, and CD206 antibody were purchased from Affinity Biosciences (Jiangsu, China); the DCFH-DA probe was purchased from Uelandy Biotechnology (Suzhou, China); FITC phalloidin was purchased from Sagon Biotech (Guangzhou, China); DMEM culture medium and fetal bovine serum were purchased from Gibco (USA). Experimental Equipment The following equipment was used: intelligent biochemical incubator (SPX-280Y, Ningbo Kesheng Laboratory Instruments Co., Ltd., China), thermostatic shaker (THZ-82, Guohua Instruments Co., Ltd., China), high-speed centrifuge (5804R, Eppendorf China Co., Ltd., China), microbiological workstation (HCB-900V, Haier Biomedical Co., Ltd., China), PCR machine (T100, Bio-Rad, USA), transmission electron microscope (HT7700, Japan), biosafety cabinet (1374, Thermo Fisher Scientific, Suzhou, China), high-speed centrifuge (5702, Eppendorf International Trading Co., Ltd., Shanghai, China), confocal microscope (Olympus, Japan), UV-Vis spectrophotometer (Shimadzu UV-2700, Japan), microplate reader (Bio-Rad, USA), fluorescence inverted microscope (CKX53, Japan), and magnetic stirrer (MS-H-Pro, Dragonlab Instrument Co., Ltd., Beijing, China). Synthesis of g-C 3 N 4 and H 2 O 2 Generation 10 g of urea was accurately weighed and dissolved completely in 30 mL of deionized water. The solution was then transferred to a crucible at 60 °C and continued to be dried for 8 hours to obtain a white crystalline solid. This solid was subsequently transferred to a muffle furnace. It was calcined at a heating rate of 5 °C/min until the temperature reached 600 °C, after which the calcination was continued for 2 hours. The resulting light yellow solid was g-C 3 N 4 . Its morphology was observed using TEM, its elemental composition and chemical bonds were confirmed by XPS, and its crystal structure was analyzed by XRD. Initially, standard H 2 O 2 solutions (0, 0.05, 0.1, 0.2, 0.4, 0.8 mmol/L) were reacted with an H 2 O 2 assay kit for 10 minutes. The absorbance was measured using a microplate reader at 405 nm to establish a standard curve. Different concentrations of g-C 3 N 4 (50, 100, 200 μg/mL) were prepared and measured at various time points (0, 0.5, 1, 1.5, 2 hours) under visible light using the H 2 O 2 assay kit. The generation of H 2 O 2 was calculated based on the standard curve. Synthesis and Characterization of Copper Alginate Microspheres (CAM) Copper alginate microspheres were synthesized by air cutting. First, 10 g of sodium alginate was weighed and dissolved in deionized water to prepare a 0.5% sodium alginate solution. Then, 1 g of CuCl 2 was weighed and dissolved in 99 mL of deionized water under ultrasonication to prepare a 1% CuCl 2 solution. The sodium alginate solution was loaded into a 10 mL syringe and pushed into a microfluidic system at a rate of 5 mL/h, where nitrogen (0.1 mpa) was used to cut the microspheres, which were then dropped into the CuCl 2 solution for cross-linking. The copper alginate microspheres were stored at 4 °C. Their morphology and structure were observed using an optical microscope, particle size was measured by ImageJ, and their microstructure and elemental composition were analyzed by SEM. Synthesis of GelMA and Combination with Copper Alginate Microspheres First, a carbonate buffer was prepared by dissolving 0.3427 g Na 2 CO 3 and 3.0915 g NaHCO 3 in 200 mL of deionized water. Then, 20 g of gelatin was dispersed in 200 mL of carbonate buffer (pH 9.0) and heated in a 50 °C oil bath until completely dissolved to make a 10 % gelatin solution. Next, 2 mL of MA (methacrylic anhydride) was slowly added to the gelatin solution using a syringe pump at a rate of 0.2 mL/min while protecting from light. After the addition, the reaction continued for 3 hours in the oil bath. Then, 100 mL of PBS was added to terminate the reaction. The mixture was centrifuged (7000 rpm, 15 min) to remove unreacted MA. The GelMA was then placed in a dialysis bag (MWCO 3500) and dialyzed at 38 °C for 2 days. Finally, the GelMA was lyophilized and stored at -20 °C. A suitable amount of GelMA was dissolved in PBS to prepare a 5 % GelMA solution, to which 0.25 % LAP (photo-initiator) was added by mass-volume ratio. Copper alginate microspheres and g-C 3 N 4 were then added to the solution, and the mixture was exposed to UV light for 30 seconds. The morphology of the GelMA hydrogel containing microspheres and g-C 3 N 4 was observed by SEM after lyophilization. Adhesion Experiment of Functionalized Hydrogels The functionalized hydrogels were prepared as described above and applied evenly to glass slides. Rat hearts, livers, spleens, lungs, and kidneys were individually placed on the hydrogel-coated slides. The adhesion was evaluated by visual observation and photographic documentation. Swelling Property Test of Functionalized Hydrogels Different systems of functionalized hydrogels were prepared (GelMA (G), GelMA/g-C 3 N 4 (G-g), GelMA/CAM (G-C), GelMA/CAM@g-C 3 N 4 (G-C-g)), lyophilized, and weighed (A 1 ). The lyophilized hydrogels were then soaked in 50 mL PBS (pH 7.4) at 37°C for 24 hours. After soaking, the hydrogels were blotted dry with filter paper and weighed again (A 2 ). The swelling ratio was calculated using the following formula: Each experiment was repeated three times. Drug Release Curve of Functionalized Hydrogels A UV standard curve for g-C 3 N 4 was established using UV spectroscopy. Specifically, different concentrations of g-C 3 N 4 (1000, 500, 250, 125, 62.25, 31.125 μg/mL) were prepared, and the UV absorption peaks were measured to create the standard curve. To obtain the drug release curve of g-C 3 N 4 , GelMA/g-C 3 N 4 hydrogels were prepared. A 10 mL hydrogel was placed in 200 mL PBS and incubated in a thermostatic shaker (37 °C; 70 rpm). At specific time points (0, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72 h), 2 mL of release medium was removed for UV analysis, and the release amount was calculated based on the standard curve. The following formula was used to calculate the release rate: where A 1 was the total amount of the drug, and A 2 was the amount of drug released at a specific time point. Similarly, the copper ion release curve was obtained. A copper ion detection kit was used to establish a standard curve based on standard copper ion concentrations (0, 10, 20, 30, 40, 50 μg/mL) measured with a microplate reader. GelMA/CAM hydrogels were prepared, and a 10 mL hydrogel was placed in 200 mL PBS. Samples were taken at specified times (0, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72 h) for copper ion analysis, and the release amount was calculated according to the standard curve. The release rate was calculated using the same formula as above. CCK-8 Assay to Evaluate the Biocompatibility of g-C 3 N 4 and Copper ions The biocompatibility of g-C 3 N 4 was evaluated using a CCK-8 assay. Human umbilical vein endothelial cells (HUVECs) at a density of 5×10³ cells were seeded into a 96-well plate and treated with DMEM (serum-free) medium containing different concentrations of g-C 3 N 4 (0, 50, 100, 150, 200, 250 μg/mL) for 24 hours. After incubation, 100 μL of CCK-8 solution was added to each well and further incubated at 37 °C for 0.5 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. This same method was used to evaluate the biocompatibility of g-C 3 N 4 in RAW264.7 cells, with the experiment repeated three times. Next, the biocompatibility of copper ions was assessed using the CCK-8 assay. HUVECs at a density of 5×10³ cells were seeded into a 96-well plate and treated with DMEM (serum-free) medium containing various concentrations of copper ions (0, 10, 20, 30, 40, 50 μg/mL) for 24 hours. After incubation, 100 μL of CCK-8 solution was added to each well and further incubated at 37°C for 0.5 hours. The absorbance at 450 nm was measured using a microplate reader to calculate cell viability. This method was similarly applied to evaluate the biocompatibility of g-C 3 N 4 in RAW264.7 cells, with the experiment repeated three times. Live/Dead Staining of Functionalized Hydrogels To assess the biocompatibility of different functionalized hydrogels, an indirect extraction method was used. Functionalized hydrogel systems, including PBS, GelMA, GelMA/g-C 3 N 4 , GelMA/CAM, and GelMA/CAM@g-C 3 N 4 , were incubated with DMEM medium at a ratio of 0.2 g/mL in accordance with national standards (GB/T 16886.5-2017) for 24 hours to obtain hydrogel extracts. HUVECs at a density of 5×10⁴ cells were seeded into a 24-well plate and treated with different hydrogel extracts for 24 hours. Live/dead staining solution was added, followed by three washes with PBS. The cells were observed and photographed using an inverted fluorescence microscope. The experiment was repeated three times. Cell Migration Assay HUVECs at a density of 1×10⁵ cells were seeded into a 6-well plate and cultured in a CO₂ incubator (37°C, 5% CO₂) until they reached confluency. A pipette tip was used to scratch the cell monolayer, creating a narrow wound gap. The cells were then incubated with extracts of PBS, GelMA, GelMA/g-C 3 N 4 , GelMA/CAM, or GelMA/CAM@g-C 3 N 4 in serum-free medium. The wound area was photographed using Cap Studio software, and Image J was used to analyze the wound area. The cell migration rate was calculated using the following formula: Where A₁ is the wound area at t = 0 h, and A₂ is the wound area at the specified time. Tube Formation Assay HUVECs at a density of 1×10⁵ cells were seeded into a 6-well plate. Once attached, the cells were incubated with serum-free DMEM extracts of PBS, GelMA, GelMA/g-C 3 N 4 , GelMA/CAM, or GelMA/CAM@g-C 3 N 4 for 24 hours. Matrigel was stored at 4°C overnight and diluted with DMEM at a 2:1 ratio. After thoroughly mixing, 75 μL of Matrigel was added to each well of a 96-well plate and allowed to solidify at 37°C for 30 minutes. HUVECs were then digested with trypsin and seeded into each well of the 96-well plate at a density of 3×10⁴ cells per well. After 12 hours, the formation of tube-like structures was observed under an inverted fluorescence microscope in Brightfield. The number of nodes and tube formation were quantified using Image J. The experiment was repeated three times. Promotion of CD31 Expression in HUVECs by Functionalized Hydrogels HUVECs at a density of 1×10⁵ cells were seeded into confocal dishes and allowed to adhere. The cells were then treated with extracts of PBS, GelMA, GelMA/g-C 3 N 4 , GelMA/CAM, or GelMA/CAM@g-C 3 N 4 for 48 hours. The extracts were aspirated, and the cells were fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and washed three times with PBS. After blocking for 20 minutes, the cells were incubated with a primary CD31 antibody at 4°C overnight, followed by a secondary antibody incubation at room temperature in the dark for 2 hours. The cells were then stained with phalloidin for 30 minutes in the dark at room temperature and counterstained with DAPI for 10 minutes. Images were captured using a confocal microscope. The fluorescence intensity of CD31 was quantified using Image J software, with the experiment repeated three times. Reactivation of RAW 264.7 Macrophage Function by Functionalized Hydrogels RAW 264.7 cells at a density of 1×10⁶ were seeded into confocal dishes and allowed to adhere. The cells were then treated with extracts of PBS, GelMA, GelMA/g-C 3 N 4 , GelMA/CAM, or GelMA/CAM@g-C 3 N 4 for 24 hours. The extracts were aspirated, and DCFH-DA working solution was added. After incubation at 37°C in the dark for 30 minutes, the cells were stained with DAPI for 10 minutes. Images were taken using a confocal microscope, and ROS expression was quantified using Image J software. The experiment was repeated three times. Similarly, RAW 264.7 cells at a density of 1×10⁶ were seeded into confocal dishes and treated with extracts of PBS, GelMA, GelMA/g-C 3 N 4 , GelMA/CAM, or GelMA/CAM@g-C 3 N 4 for 24 hours. After the extracts were aspirated, the cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked for 20 minutes. The cells were then incubated with a rabbit anti-CD86 polyclonal antibody at 4 °C overnight, followed by incubation with a secondary antibody at room temperature in the dark for 2 hours. Nuclei were stained with DAPI, and images were captured using a confocal microscope. CD86 fluorescence intensity was quantified using Image J software, with the experiment repeated three times. qPCR Analysis of RAW 264.7 Inflammatory Cytokine Expression RAW 264.7 cells at a density of 1×10⁶ were seeded into a 6-well plate and treated with extracts of PBS, GelMA, GelMA/g-C 3 N 4 , GelMA/CAM, or GelMA/CAM@g-C 3 N 4 for 24 hours. Total RNA was extracted using a rapid RNA extraction kit, and the RNA concentration was determined. The RNA was reverse-transcribed into cDNA using a reverse transcription premix kit, and qPCR was performed using primers designed for the target genes (Table 1). Gene expression was analyzed semi-quantitatively. The experiment was repeated three times. Evaluation of Antibacterial Activity of Functionalized Hydrogels by Spread Plate Method The antibacterial activity of hydrogels was evaluated using the spread plate method. Briefly, 200 μL of GelMA, GelMA/g-C 3 N 4 , GelMA/CAM, and GelMA/CAM@g-C 3 N 4 hydrogels were prepared in 48-well plates. After complete gelation, 10 μL of bacterial suspension (10 7 CFU mL −1 ) was added to the surface of the hydrogels and incubated at 37 °C for 2 hours under natural light. Subsequently, 1 mL of sterile PBS was added to each well to resuspend the bacteria. As a negative control, 10 μL of bacterial suspension (10 7 CFU mL −1 ) was added to 1 mL of PBS. Then, 100 μL of the bacterial suspension was spread evenly onto agar plates, and incubated at 37 °C in a biochemical incubator for 20 hours. The colonies were photographed and counted using Image J software, with each experiment repeated three times. Evaluation of Antibacterial Activity of Hydrogels by Live/Dead Staining The antibacterial activity of hydrogels was further assessed using a bacterial live/dead staining kit according to the manufacturer’s instructions. SYTO 9 and PI were diluted in PBS (1:1000) and mixed with the bacterial suspension at room temperature for 20 minutes. The bacterial suspension was then placed on a slide and observed under a confocal microscope, with images captured for further analysis. The experiments were repeated three times. Observation of Bacterial Morphology The bacterial morphology after treatment with different functionalized hydrogels was observed by scanning electron microscopy (SEM). Briefly, bacterial suspensions from different treatment groups were centrifuged at 4000 rpm for 10 minutes at 25°C, and the bacterial pellet was collected. The bacteria were fixed in 2.5 % glutaraldehyde at 4°C overnight. After centrifugation, the fixed samples were washed with PBS several times, followed by gradient dehydration using ethanol solutions at concentrations of 30 %, 50 %, 70 %, 85 %, 95 %, and 100 %, with each step lasting for 15 minutes. Finally, 20 μL of the dehydrated bacterial sample was dropped onto a silicon wafer and air-dried. The samples were coated with gold and observed under SEM to examine bacterial morphology. Evaluation of Anti-biofilm Activity of Functionalized Hydrogels by Crystal Violet Staining Bacterial suspensions (180 μL/well) were seeded in a 96-well plate and incubated at 37 °C in a humidified environment for 24 hours. After incubation, the planktonic bacteria were removed, and the wells were washed with PBS to remove loosely attached bacteria. PBS, GelMA, GelMA/g-C 3 N 4 , GelMA/CAM, and GelMA/CAM@g-C 3 N 4 extracts were added to the wells, and the plates were incubated at 37 °C in a humidified environment for another 24 hours. After incubation, the extracts and dead bacteria were washed away with PBS, and the remaining biofilm was fixed with 4 % formaldehyde for 15 minutes. The fixed biofilms were washed with PBS, and stained with 0.1 % crystal violet for 15 minutes at room temperature, and unbound dye was removed by washing with PBS. The plates were air-dried, and the crystal violet bound to the biofilm was solubilized using 33% acetic acid. The absorbance was measured at 590 nm, with each experiment repeated three times. Establishment of an Infected Wound Model in Animals All animal experiments were reviewed and approved by the Animal Ethics Committee of North Sichuan Medical College (Approval No: 2024094) and conducted by national animal protection guidelines. All surgical procedures were performed under anesthesia, and all efforts were made to minimize pain, suffering, and death. A total of 100 healthy male SD rats (SPF grade, 8 weeks old, weighing 220-250 g) were anesthetized with sodium pentobarbital (20 mg/kg), and their backs were shaved. Full-thickness skin wounds (10 mm in diameter) were created on the rat backs using a circular punch and surgical scalpel. The rats (n=3) were randomly divided into different groups. The wounds were infected with Staphylococcus aureus suspension (10 7 CFU/mL, 50 μL) for 24 hours to establish an infected wound model. The wounds were then treated with PBS, GelMA, GelMA/g-C 3 N 4 , GelMA/CAM, and GelMA/CAM@g-C 3 N 4 hydrogels. On days 0, 3, 7, and 14, the infected wounds were photographed, and the healing process was observed. The wound area was measured using Image J software. Additionally, on day 3, the infected wound tissues and surrounding skin were excised, homogenized in 1 mL of sterile PBS, serially diluted, and plated on solid culture media for bacterial growth. The plates were incubated in a bacterial incubator for 12 hours and photographed. During the wound healing process, skin tissue around the wound in each group was collected and fixed in 4 % paraformaldehyde. The tissue specimens were stained with hematoxylin-eosin (H&E), Masson's trichrome, Giemsa, and subjected to immunofluorescence staining for CD31, CD86, and CD206 (Chengdu Lilai Biotech Co., Ltd.). Additionally, to evaluate the in vivo toxicity of the hydrogel samples, visceral organs (heart, liver, spleen, lungs, kidneys) from the rats were collected for H&E staining. The experiments were repeated three times. Statistical Analysis All data were statistically analyzed using SPSS 24.0 and GraphPad Prism 8 software. Independent sample t-tests were used to evaluate statistical differences between the two groups, while one-way ANOVA was employed for multiple-group comparisons. Data are presented as mean ± standard deviation ( x̅±s ). "ns" indicates no significant difference; *p < 0.05, **p < 0.01, and ***p < 0.001 indicate statistically significant differences. Results Synthesis and Characterization of g-C 3 N 4 Figure 1a shows the synthesis of g-C 3 N 4 via a thermal polymerization method. Under TEM, g-C 3 N 4 exhibits a layered sheet-like structure（Figure 1b）. XPS analysis confirms that g-C 3 N 4 consists of carbon (C) and nitrogen (N) elements（Figure 1c）. The XPS valence band spectrum indicates that g-C 3 N 4 was composed of C-C, C=N-C, and N-C=N bonds（Figure 1d and 1e）. XRD results show two characteristic peaks at 13.0 ° and 27.5 °, confirming the successful synthesis of g-C 3 N 4 [36]. A hydrogen peroxide assay kit was used to detect the production of ROS and a standard curve was established, showing that the amount of ROS produced by g-C 3 N 4 was positively correlated with its concentration and light exposure time（Figure 1g and Figure S1）. Synthesis of Copper Alginate Microspheres and Construction of Functionalized Hydrogel System Figure 2a illustrates the schematic of the gas-cutting method used to synthesize copper alginate microspheres. Under a light microscope, the microspheres exhibit uniform round structures with a diameter of approximately 290.1 ± 16.3 μm（Figure 2b and 2c）. Figure 2d showed that different hydrogels formed a gel state after 30 seconds of UV light exposure. SEM analysis revealed a smooth surface for GelMA hydrogels, which became rough after incorporating g-C 3 N 4 . In the G-C-g group, microspheres were attached to the surface, and element mapping showed that the gel surface was composed of C, N, O, and Cu, confirming the successful construction of the hydrogel-microsphere system（Figure 2e and 2f）. The swelling curves of the hydrogels in different systems were similar, with no significant changed in swelling properties after the incorporation of g-C 3 N 4 and CAM. The hydrogels demonstrated excellent adhesion to the heart, liver, spleen, lungs, and kidneys（Figure 2g and Figure S2）. Using a copper ion assay kit, a standard curve for copper ions was established, and the UV spectra of different concentrations of g-C 3 N 4 were measured to create a standard curve. The release curves of functionalized hydrogel microspheres showed rapid release of g-C 3 N 4 and copper ions within 0–12 hours, followed by sustained release（Figure S3-S6）. Biocompatibility, Scratch, and Tube Formation Assays of Functionalized Hydrogel Microspheres The CCK-8 assay shows that g-C 3 N 4 concentrations exceeding 150 μg/mL significantly affect RAW264.7 cell viability, and copper ion concentrations exceeding 40 μg/mL notably reduce HUVEC cell viability. Therefore, a concentration of 150 μg/mL for g-C 3 N 4 and 40 μg/mL for copper ions was selected for subsequent experiments（Figure S7-S10）. Live-dead cell staining with extracts from different hydrogel groups showed that the functionalized hydrogel microspheres exhibited excellent biocompatibility with RAW264.7 and HUVECs（Figure 3a and Figure S11）. Scratch assay results showed that the G-g group extract slowed cell migration, while the G-C-g group significantly accelerated cell migration compared to the PBS group （Figure 3b and d）. Tube formation assays revealed that the G-g group extract reduced tube formation and total length in HUVECs, while the G-C-g group significantly promoted tube formation and total length compared to the PBS group（Figure 3c, e, and f）. After 24 hours of intervention with different hydrogel systems on HUVECs, the G-C-g group significantly promoted CD31 expression compared to the PBS group（Figure S12 and 13）. Functionalized Hydrogel Microspheres re-roused Macrophage M1 Function After treating M0 macrophages with extracts from different systems, the DCFH-DA probe results showed that both the G-g and G-C-g groups induced macrophages to exhibit a pro-inflammatory phenotype, with significantly higher ROS expression than the PBS group（Figure 4a and Figure S14）. CD86 immunofluorescence analysis showed that the G-g and G-C-g groups had higher fluorescence intensity than the PBS group（Figure 4b and Figure S15）. Further RNA extraction and PCR results indicated that the expression of pro-inflammatory factors (IL-1, IL-6, TNF-α) significantly increased in the G-g and G-C-g groups compared to the PBS group（Figure S16）. Antibiofilm Activity and Mechanism of Functionalized Hydrogel Microspheres In Vitro In infected environments, bacteria protect themselves from immune system attacks and exogenous bactericides by secreting EPS (extracellular polymeric substances) to form biofilms, which contribute to the persistence and resistance of biofilm-related infections. In this study, bacteria were seeded on the hydrogel surface, plated on agar, and observed through photographs and quantitative ImageJ analysis. Plate coating results indicated that a large number of colonies grew on the surfaces in the PBS and G groups. However, after intervention with the G-g and G-C groups, Staphylococcus aureus growth was inhibited, with the G-C-g group showing a significant reduction in bacterial colonies （Figure 5a and Figure S17）. Bacterial live-dead staining revealed a marked increase in dead bacteria in the G-C-g group compared to the PBS group （Figure 5b）. SEM analysis showed intact bacterial cell walls in the PBS and G groups, while the G-C-g group displayed disrupted bacterial walls and cytoplasmic leakage（Figure 5c and Figure S18）. Similar results were obtained when the system was tested on Escherichia coli, where the G-C-g group exhibited significant inhibitory effects on bacterial growth compared to the PBS group（Figure 5d）. Live-dead bacterial staining further confirmed that the G-C-g group had a higher proportion of dead bacteria compared to the PBS group（Figure 5e）. SEM observations revealed disrupted E. coli cell walls in the G-C-g group compared to the PBS group（Figure 5f）. Using crystal violet staining, hydrogels from different groups demonstrated that the G-C-g group exhibited significant inhibitory effects on both S. aureus and E. coli compared to the PBS group（Figure S19 and S20）. Further, after intervening with PBS and the G-C-g group on S. aureus, bacterial gene sequencing revealed similar gene distribution patterns between the G-C-g and PBS groups, which can be used for subsequent experimental validation（Figure 6a）. Compared to the PBS group, the G-C-g group exhibited upregulation of 302 metabolites and downregulation of 349 metabolites（Figure 6b）. A clustering heat map illustrated the consistency of metabolite changes after treatment, highlighting differences in metabolite expression between the two groups （Figure 6c）. KEGG enrichment analysis indicated that the functionalized hydrogel microspheres downregulated glycolysis, oxidative phosphorylation, the phosphotransferase system, amino acid metabolism (alanine, aspartate, and glutamate metabolism), purine, and pyrimidine metabolism （Figure 6d）. GO enrichment analysis revealed that the system inhibited processes such as carbohydrate derivative biosynthesis, lipoteichoic acid metabolism, and lipoteichoic acid biosynthesis（Figure 6e）. Figure 6f illustrated the antibacterial mechanism of the functionalized hydrogel microspheres. The diagram showed that the ROS generated by g-C 3 N 4 disrupts the biofilm, followed by the release of copper ions from copper alginate microspheres, which blocked bacterial ABC transporters, leading to impaired nutrient exchange. Copper ions overload within the bacteria first induced downregulation of glycolysis and oxidative phosphorylation, resulting in reduced energy metabolism. This was followed by downregulation of purine and pyrimidine metabolism, impairing DNA replication and repair systems. Additionally, triglyceride metabolism was downregulated, and copper ions induced lipid peroxidation, ultimately leading to bacterium-like copper-induced cell death. Functionalized Hydrogel Microspheres Promote Biofilm-Associated Wound Healing In Vivo Figure 7a illustrated the schematic diagram of biofilm-associated chronic wound healing and the application of functionalized hydrogel microspheres on the wound site. A circular wound model with a 10 mm diameter was created on the backs of SD rats, followed by inoculation with S. aureus. When the wound exhibited purulent exudation, successful model establishment was confirmed. Subsequently, hydrogels from different groups were applied to the wounds, and healing progress was documented. Gross images of wound healing demonstrated that interventions with the g-C 3 N 4 and CAM groups accelerated wound healing, while the G-C-g group, through the synergistic effects of g-C 3 N 4 and CAM, significantly accelerated wound healing compared to the PBS group（Figure 7b-7d）. On day 3 of wound healing, wound exudates were collected for plate analysis, revealing a reduction in bacterial numbers in the g-C3N4 and CAM groups, with the G-C-g group exhibiting the most significant reduction compared to the PBS group（Figure 7b and Figure S21）. On day 3, Giemsa staining of wound tissues showed large aggregations of S. aureus in the PBS and G groups, whereas bacterial numbers were reduced in the G-g and G-C groups. The G-C-g group showed only a few residual bacteria, significantly fewer than in other groups（Figure 7f）. H&E staining on day 7 demonstrated partial wound contraction in the G-g and G-C groups, while the G-C-g group showed significant wound contraction, leaving the shortest wound length compared to the PBS group（Figure 8a and 8c）. By day 14, wound healing had further progressed in all groups, with the G-C-g group displaying the shortest wound diameter（Figure 8a）. Masson’s staining on day 7 revealed higher collagen deposition in the G-g and G-C groups compared to the PBS group, while the G-C-g group showed the highest collagen deposition with statistical significance（Figure 8b and 8d）. By day 14, collagen deposition had further increased in all groups, with the G-C-g group exhibiting the highest collagen deposition（Figure 8b）. Immunofluorescence staining of wound tissues on day 3 revealed lower CD86 and CD206 expression in the PBS and G groups, whereas CD86 expression significantly increased in the G-g and G-C-g groups compared to the PBS group（Figure 9a）. On day 7, immunofluorescence results showed increased CD206 expression in the G-g and G-C groups, with the G-C-g group exhibiting significantly higher CD206 expression compared to the other groups（Figure 9b）. Additionally, CD31 immunofluorescence staining of wound tissues showed significantly higher CD31 expression in the G-C-g group compared to the other groups, indicating that the wound healing had progressed to the proliferation phase （Figure S22 and S23）. On day 14 of wound healing, H&E staining of the heart, liver, kidneys, lungs, and spleen of SD rats showed no significant signs of inflammation, necrosis, or other pathological changes（Figure S24）. Discussion Bacterial infectious diseases are an increasing public health challenge [37, 38]. The formation of bacterial biofilms significantly reduces the efficacy of traditional antibiotic therapies and promotes the emergence of multidrug-resistant bacteria [39, 40]. Due to the presence of EPS, biofilms operate as a self-protective structure, shielding pathogens from the innate immune system and effectively resisting antibiotic penetration, thereby posing substantial challenges to antibacterial treatment [41]. The formation of biofilms and the immune evasion they induce in macrophages both hinder the wound healing process. Therefore, disrupting bacterial biofilms and re-rousing macrophage M1 functionality are key strategies for promoting chronic wound regeneration. In this study, we developed a chronic wound repair system by synthesizing g-C 3 N 4 through thermal polymerization and CAM using gas-cutting methods. Further, we encapsulated g-C 3 N 4 and CAM into GelMA to construct a system capable of disrupting biofilms and reawakening macrophage M1 function. In recent years, with the rapid development of nanotechnology, the application of nanomaterials in the field of antibacterial therapy has achieved significant success. Among them, g-C 3 N 4 -based nanomaterials have shown remarkable progress in antibacterial applications [42]. g-C 3 N 4 is a novel inorganic non-metallic photoactive material with a two-dimensional layered aromatic polycyclic structure composed of sp2-hybridized C and N atoms, and its C-N bond length is uniform [43]. In this study, g-C 3 N 4 was successfully synthesized via thermal polymerization and characterized by TEM, XRD, and XPS analyses, including its valence band spectrum. Using an H 2 O 2 assay kit under light exposure, we demonstrated that g-C 3 N 4 exhibits peroxidase (POD) activity. Microsphere structures can effectively encapsulate active ingredients, achieving protection and sustained release [44]. Sodium alginate exchanges its Na + with divalent cations such as Ca 2+ , Cu 2+ , and Ba 2+ , forming a stable biopolymer with a unique three-dimensional structure [45]. In our constructed system, copper alginate microspheres were successfully synthesized via gas-cutting methods, and uniform spherical structures were observed under a light microscope, showing sustained release of Cu 2+ during degradation. Hydrogels are one of the most important biomaterials, attracting attention for medical applications such as wound healing, tissue engineering, and drug delivery [46]. SEM analysis revealed a uniform porous three-dimensional network structure, with microspheres and g-C 3 N 4 attached to the surface, confirming the successful construction of the functionalized hydrogel microsphere wound healing system. The RGD sequence in GelMA hydrogels promotes adhesion between the hydrogel and cells. Additionally, the water molecules in the hydrogel can form hydrogen bonds with water molecules on the wound surface, enhancing the contact between the hydrogel and the wound [47]. Furthermore, water molecules penetrate the wound tissue via physical osmosis, forming a water layer that further consolidates the connection between the hydrogel and the wound. These adhesion mechanisms play a critical role in tissue repair and regeneration. Biocompatibility aims to assess the toxicity and safety of biomaterials in cells, ensuring that therapeutic effects are achieved while minimizing potential side effects. In this study, the CCK-8 assay results indicated that when the g-C 3 N 4 concentration did not exceed 150 μg/mL and Cu 2+ concentration did not exceed 40 μg/mL, there was no significant toxicity to the cells. However, higher concentrations of nanomaterials and metal ions may cause toxicity through various mechanisms, such as disrupting the cell membrane, inducing oxidative stress, triggering intracellular metabolic disorders, and causing ionic homeostasis imbalance. Based on CCK-8 and live-dead staining assays, the functionalized hydrogel microspheres demonstrated excellent biocompatibility. Macrophages possess high plasticity and are polarized into "classically activated" pro-inflammatory M1 and "alternatively activated" anti-inflammatory M2 phenotypes during different phases of wound healing [48]. Macrophages, as a key phagocytic cell type, are part of the innate immune system, and are responsible for engulfing and processing cellular debris and pathogens [49]. The formation of biofilms enhances immune evasion and antibiotic resistance, leading to persistent local infections that hinder the wound healing process [50]. In this study, g-C 3 N 4 successfully mimicked POD enzyme activity and generated ROS. Through ROS probing, CD86 immunofluorescence, and PCR assays, the results showed that g-C 3 N 4 could promote macrophage ROS production and upregulate the expression of CD86, IL-1, IL-6, and TNF-α pro-inflammatory cytokines, successfully re-rousing macrophage M1 pro-inflammatory function. ROS, as a driving signal for M1 macrophage polarization, initiates cell cycle and metabolic reprogramming through pathways such as ATM-CHK2, NF-κB, and MAPK, regulating macrophage M1 phenotype polarization, inducing the expression of inflammatory cytokines, and enhancing their phagocytic function [51-53]. Microbial aggregation at wound sites and the development of resistance to available antibiotics are major concerns [54]. To survive under harsh conditions, including exposure to known antibiotics, microorganisms form biofilms a unique structure whose formation involves quorum sensing, environmental stress, nutrient availability, hydrodynamic conditions, intercellular communication, signaling cascades, and secondary messengers. Alternative therapies that disrupt bacterial structure and function by generating ROS and binding metal ions have gained considerable attention in recent years [55]. Wound healing involves multiple stages, including hemostasis, inflammation, proliferation, angiogenesis, and tissue remodeling, each involving interactions between different cell types [56]. Neovascularization supplies wounds with sufficient blood and oxygen to support cell survival and tissue regeneration [57]. In chronic wounds, insufficient blood perfusion exacerbates infection symptoms associated with microbes and often complicates the healing process [58]. In this study, the migration assay, tube formation assay, and CD31 immunofluorescence analysis results indicated that the functionalized hydrogel microspheres could promote endothelial cell migration, tube formation, and CD31 expression. Current research suggests that Cu 2+ can activate signaling pathways such as Wnt, PI3K/Akt, MAPK, and hypoxia-inducible factor 1α (HIF-1α), upregulating the expression of angiogenic factors, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and HIF-1α, thereby inducing angiogenesis [59, 60]. Using a chronic wound infection model with S. aureus, our system re-rousing macrophage M1 function by day 3, clearing pathogens and cellular debris, and shifting the chronic wound microenvironment towards an inflammatory phase. By day 7, macrophages had transitioned to the M2 phenotype, further promoting CD31 expression and collagen deposition, enabling the transition from the inflammatory phase to the proliferative remodeling phase, thereby greatly enhancing chronic wound healing efficiency. In summary, based on the pathological conditions of biofilm formation and macrophage immune evasion in wound sites, the functionalized hydrogel microspheres developed in this study effectively disrupted bacterial biofilms and reawakened pro-inflammatory macrophage function, thereby transforming the microenvironment of chronically infected wounds into one conducive to physiological healing, ultimately promoting wound healing. Conclusion This study addresses the critical mechanisms of biofilm formation and macrophage immune evasion, which lead to impaired wound healing, by constructing a functionalized hydrogel microsphere wound repair system (GelMA/CAM@g-C 3 N 4 ) with antibiofilm properties and the ability to re-rousing macrophage M1 function. This system effectively disrupted bacterial biofilms by generating ROS and re-roused macrophage M1 function. Additionally, the release of Cu 2+ interfered with bacterial energy metabolism, biosynthesis, and lipid metabolism processes, leading to copper-like bacterial cell death. Furthermore, this system promoted CD31 expression in endothelial cells, effectively transforming the pathological microenvironment of chronic wounds into one that facilitated regeneration. This innovative approach provides a promising solution for the treatment of chronic wounds. Declarations Funding This work was supported by Doctoral Research Launch Fund Project of North Sichuan Medical College Affiliated Hospital (CBY23-QDA23), Health Commission of Sichuan Province Medical Science and Technology Program” (24QNMP096), and Research Project Nanchong Science and Technology Bureau (23JCYJPT0036). Ethics approval and consent to participate All animal experiments in this study were approved by the Ethics Committee of the Animal Experimentation Centre of North Sichuan Medical College (approval number: 2024094). Consent for publication All authors consent for publication. Declaration of Competing Interest The authors declare no competing financial interests. Author Contribution Chao Xiang: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing-original draft. Chaoyu Pu: Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing original draft. XueMei Zhong: Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization. Yong Wang: Data curation, Project administration, Validation. Weiyong Song: Data curation, Project administration, Validation. Xingkuan Wang: Project administration, Validation, Funding. Kemiao Chen: Project administration, Validation. Kai Li: Project administration, Resources, Validation. Yue Luo: Project administration, Resources, Validation, Funding. Ke Jiang: Conceptualization, Resources. Dianming Jiang: Investigation, Methodology, Project administration, Writing – review & editing, Conceptualization. Acknowledgments We wound also like to acknowledge BioRender, which was used to create a Schematic diagram in this article. We also wish to express our deep appreciation to the North Sichuan Medical College Innovation Center for Science and Technology for providing the experimental platform that was crucial for our research. References V F, RR I, AM S, M R, D M, S K, et al. 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Oxygen tension regulating hydrogels for vascularization and osteogenesis via sequential activation of HIF-1α and ERK1/2 signaling pathways in bone regeneration. Biomaterials advances. 2024;161:213893. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Scheme1.png Scheme 1. Schematic illustration of the construction of the GelMA/CAM@g-C 3 N 4 system and its application to infected wounds. SI.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5378421","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":378436059,"identity":"64ded40c-0af5-476a-95dc-95b72adaeff9","order_by":0,"name":"Chao Xiang","email":"","orcid":"","institution":"The Third Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Xiang","suffix":""},{"id":378436061,"identity":"027f238d-6f83-4296-b0e2-40dd620733d2","order_by":1,"name":"Chaoyu Pu","email":"","orcid":"","institution":"The Affiliated Hospital of North Sichuan 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College","correspondingAuthor":false,"prefix":"","firstName":"Weiyong","middleName":"","lastName":"Song","suffix":""},{"id":378436068,"identity":"afa6b0bc-b4fb-4696-8b47-1a92876cd481","order_by":5,"name":"Xingkuan Wang","email":"","orcid":"","institution":"The Affiliated Hospital of North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xingkuan","middleName":"","lastName":"Wang","suffix":""},{"id":378436069,"identity":"5e07e936-2fe0-44db-ad58-a85eb780bd76","order_by":6,"name":"Kemiao Chen","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Kemiao","middleName":"","lastName":"Chen","suffix":""},{"id":378436070,"identity":"6d8e92f7-eb0b-47a2-8645-6f901e899f98","order_by":7,"name":"Kai Li","email":"","orcid":"","institution":"The Third Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Li","suffix":""},{"id":378436071,"identity":"5079dec5-1fd0-4cfa-bc08-c64d33108de6","order_by":8,"name":"Yue Luo","email":"","orcid":"","institution":"The Affiliated Hospital of North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Luo","suffix":""},{"id":378436072,"identity":"8f2cf2a4-3801-4607-8591-b4d0bd205a4b","order_by":9,"name":"Ke Jiang","email":"","orcid":"","institution":"The Affiliated Hospital of North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Jiang","suffix":""},{"id":378436073,"identity":"5e9d6e38-ece8-4741-ba80-25fda34d1f4c","order_by":10,"name":"Dianming Jiang","email":"data:image/png;base64,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","orcid":"","institution":"The Third Affiliated Hospital of Chongqing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Dianming","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2024-11-02 13:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5378421/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5378421/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69263967,"identity":"435a58e7-28d7-4880-8957-7ab9c9d18593","added_by":"auto","created_at":"2024-11-18 14:12:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":157314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynthesis and characterization of g-C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e.\u003c/strong\u003e (a) Schematic illustration of \u003cstrong\u003eg-C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e synthesis. (b) Morphology of \u003cstrong\u003eg-C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e under transmission electron microscopy (TEM). (c) XPS spectrum of \u003cstrong\u003eg-C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e. (d) Valence band spectrum of C 1s in \u003cstrong\u003eg-C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e. (e) Valence band spectrum of N 1s in \u003cstrong\u003eg-C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e. (f) XRD pattern of \u003cstrong\u003eg-C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e. (g) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production by \u003cstrong\u003eg-C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e at different concentrations and time points.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5378421/v1/b91d0fcaec6b09fb15a66a70.png"},{"id":69263969,"identity":"a285886f-53dc-4245-8cf6-88dafb5f5cbc","added_by":"auto","created_at":"2024-11-18 14:12:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":471623,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynthesis of copper alginate microspheres and characterization of the functionalized hydrogel system.\u003c/strong\u003e (a) Schematic illustration of copper alginate microsphere synthesis via the air-cutting method. (b) Image of copper alginate microspheres under a light microscope. (c) Microsphere size distribution under a light microscope. (d) Images of different functionalized hydrogel systems post-gelation. (e) Microstructure of different functionalized hydrogels under a scanning electron microscope (SEM). (f) Elemental mapping of GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e hydrogel. (g) Adhesion test of GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e hydrogel.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5378421/v1/2dd195456ef77e8888fa1518.png"},{"id":69263968,"identity":"bc7983ef-3516-467f-9423-b19bb7396abd","added_by":"auto","created_at":"2024-11-18 14:12:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":815592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiocompatibility, \u003c/strong\u003eCell migration assay\u003cstrong\u003e, and tube formation assay of the functionalized hydrogel microspheres.\u003c/strong\u003e (a) Live/dead staining of different functionalized hydrogel systems. (b) Cell migration assay. (c) Tube formation assay. (d) Quantitative analysis of cell migration assays. (e) Quantitative analysis of tube formation nodes. (f) Quantitative analysis of tube formation tube length. G represents GelMA; G-g represents GelMA/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e; G-C represents GelMA/CAM; G-C-g represents GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5378421/v1/13644182389dd83af84831bf.png"},{"id":69264348,"identity":"dd63179c-346f-476c-9c78-eaa87b202b7d","added_by":"auto","created_at":"2024-11-18 14:20:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":456024,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctionalized hydrogel microspheres \u003c/strong\u003eRe-rousing\u003cstrong\u003e macrophage M1 function.\u003c/strong\u003e (a) ROS probe for different functionalized hydrogel systems. (b) CD86 immunofluorescence of different functionalized hydrogel systems.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5378421/v1/c50e6b528c23d084b54e8e1a.png"},{"id":69265568,"identity":"7ab59eb1-1bb7-4528-86b2-460f4713a337","added_by":"auto","created_at":"2024-11-18 14:28:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":729817,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctionalized hydrogel microspheres exhibiting antibiofilm effects in vitro.\u003c/strong\u003e (a) Plate streaking of Staphylococcus aureus after intervention with different functionalized hydrogels. (b) Live/dead staining. (c) SEM imaging. (d) Plate streaking of \u003cem\u003eEscherichia coli \u003c/em\u003eafter intervention with different functionalized hydrogels. (e) Live/dead staining. (f) SEM imaging.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5378421/v1/f24d790f36310e48b9efb58f.png"},{"id":69264350,"identity":"b4fdc93e-f7e1-4eda-a8f4-12ad1f1e1fb6","added_by":"auto","created_at":"2024-11-18 14:20:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":284148,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibacterial mechanism analysis of functionalized hydrogel microspheres. \u003c/strong\u003e(a) Gene expression distribution across different treatment groups. (b) MA plot of gene expression in different treatment groups. (c) Clustering heatmap of different treatment groups. (d) KEGG analysis. (e) GO analysis. (f) Schematic illustration of copper-like bacterial death induced by \u003cem\u003eStaphylococcus aureus.\u003c/em\u003e G represents GelMA; G-g represents GelMA/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e; G-C represents GelMA/CAM; G-C-g represents GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5378421/v1/37fd4d850476f158f9696a6b.png"},{"id":69265756,"identity":"bf75dd92-b091-4e80-886b-003df40eb988","added_by":"auto","created_at":"2024-11-18 14:36:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":782080,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctionalized hydrogel microspheres promoting biofilm wound healing in vivo.\u003c/strong\u003e (a) Schematic illustration of biofilm model establishment and treatment of biofilm wounds with functionalized hydrogels. (b) Representative images of biofilm wounds treated with different functionalized hydrogels. (c) Tracked images of biofilm wound healing. (d) Quantitative analysis of remaining wound area during healing. (e) Plate streaking of wound exudated on day 3 post-treatment. (f) Giemsa staining of different functionalized hydrogels. G represents GelMA; G-g represents GelMA/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e; G-C represents GelMA/CAM; G-C-g represents GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5378421/v1/47d548b559e62b65c3126e75.png"},{"id":69264353,"identity":"c393e3f8-f67d-47c2-8484-cc0d6515de9f","added_by":"auto","created_at":"2024-11-18 14:20:36","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":508106,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctionalized hydrogel microspheres promoting biofilm wound healing.\u003c/strong\u003e\u003cbr\u003e\n(a) H\u0026amp;E staining after treatment with different functionalized hydrogels. (b) Masson staining after treatment with different functionalized hydrogels.\u0026nbsp;(c) Quantitative analysis of wound length after intervention with different functionalized hydrogels.\u0026nbsp;(d) Quantitative analysis of collagen deposition rate after intervention with different functionalized hydrogels. G represents GelMA; G-g represents GelMA/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e; G-C represents GelMA/CAM; G-C-g represents GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5378421/v1/87313c8eed3977fa78bd4d31.png"},{"id":69263974,"identity":"513a4d2a-9f6f-4c95-b8b5-73367bfb5127","added_by":"auto","created_at":"2024-11-18 14:12:36","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":546276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctionalized hydrogel microspheres promoting biofilm wound healing in vivo.\u003c/strong\u003e (a) Representative images of CD86 and CD206 immunofluorescence on day 3 post-treatment with different functionalized hydrogels. (b) Representative images of CD86 and CD206 immunofluorescence on day 7 post-treatment with different functionalized hydrogels.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5378421/v1/a2c12c8ac30e5bccf7363cf4.png"},{"id":70255418,"identity":"fc4090e8-ec8f-49cf-943b-c83e175f49ac","added_by":"auto","created_at":"2024-11-30 15:31:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5490189,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5378421/v1/482fe24e-1b29-461c-916b-fe697975a1b7.pdf"},{"id":69265566,"identity":"e48b265c-5048-4fc1-9747-efdc732d3f7e","added_by":"auto","created_at":"2024-11-18 14:28:36","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1167545,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1. Schematic illustration of the construction of the GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e system and its application to infected wounds.\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-5378421/v1/99e1cfec990f01d405590046.png"},{"id":69263978,"identity":"74557134-8fab-4dcb-8e9c-f8c3bea382f7","added_by":"auto","created_at":"2024-11-18 14:12:36","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":7375352,"visible":true,"origin":"","legend":"","description":"","filename":"SI.docx","url":"https://assets-eu.researchsquare.com/files/rs-5378421/v1/e1896d72a59e9b316c0a7433.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Functional Hydrogels Promote Chronic Infectious Wound Healing by Re-rousing Macrophage M1 and Inducing Bacterial Copper-Like Death","fulltext":[{"header":"Background","content":"\u003cp\u003eChronic wounds are those that fail to restore anatomical and functional integrity within three months post-injury, affecting approximately 1–2 % of the global population[1]. Infection is considered one of the key factors contributing to chronic wounds, as it impedes the healing process, increases wound pain, places a significant economic burden on healthcare systems, and lowers patients' quality of life [2]. Over the past few decades, many new antibiotics have been developed, and antibiotics have long been considered the ultimate solution for biofilm-related bacterial infections in humans [3]. Unfortunately, the formation of biofilms is highly resistant to antibiotics, making infections increasingly challenging [4-6]. Biofilms are complex bacterial aggregates composed of bacterial cells, extracellular polymeric substances (EPS), and other microorganisms, which aid in bacterial colonization, enhance resistance to antimicrobial agents, evade host immunity, and facilitate interbacterial signaling within the community [7-9]. Staphylococcus aureus is the most common organism isolated from human chronic wounds [10]. Currently, clinical biofilm treatments involve removing infected tissue and using antibiotics in combination. However, this approach has several drawbacks, including invasive procedures, uncertain efficacy, and high treatment costs [11, 12]. Therefore, there is an urgent need to develop alternative therapeutic platforms to control pathogenic biofilm-associated infections.\u003c/p\u003e\n\u003cp\u003eThe normal wound healing process progresses through four overlapping cascades: hemostasis, inflammation, proliferation, and remodeling [13]. Macrophages are the first line of defense against pathogens and possess high plasticity, rapidly adapting to the complex microenvironment triggered by tissue infection or inflammation through polarization. M1 macrophages recognize, phagocytose, and kill invading bacteria, and after completing their pro-inflammatory mission, they polarize to the M2 phenotype, which promotes tissue repair. Macrophages are key regulatory factors in wound healing and tissue regeneration, ultimately leading to wound healing [14-17]. However, in chronic wounds, mature biofilms have dense EPS, which makes it difficult for macrophages to phagocytose, leading to \"frustrated phagocytosis\" [18]. Reactive oxygen species (ROS) have been shown to promote M1 macrophage polarization (pro-inflammatory and bactericidal) and activate macrophage oxidative bursts, acidification, and enzyme-mediated phagocytosis, including phagolysosome formation, to eliminate invading bacteria [19, 20]. Thus, reactivating the pro-inflammatory function of M1 macrophages in chronically infected wounds, reversing the immune environment, and reshaping the \"physiological healing process\" of the wound through ROS are considered promising therapeutic strategies for chronically infected wounds caused by biofilm-induced immune evasion [8, 21, 22].\u003c/p\u003e\n\u003cp\u003eCurrently, novel nanomaterials with high peroxidase (POD)-like activity, such as noble metals, iron-based nanomaterials, transition metal dichalcogenides, and polyoxometalates, are known [23-25]. Unfortunately, these nanomaterials still have limitations, such as inherent cytotoxicity and oxidative stress under low H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations, which can damage cellular function. Photocatalytic antibacterial therapy is an effective strategy relying on external stimuli. Due to its controllability, safety, and non-invasiveness, it has garnered increasing attention over the past few decades, with many photosensitizers developed. The metal-free polymer two-dimensional nanomaterial graphitic carbon nitride (g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e) is particularly attractive as a photosensitizer for non-antibiotic antibacterial applications due to its abundance, ease of synthesis, appropriate band structure, high stability, and low toxicity [26, 27]. More importantly, g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e has been recently shown to generate micromolar concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e continuously, which can successfully combat established biofilms [28]. However, although the generated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e can fight biofilms, a single antibacterial mechanism is insufficient to completely prevent biofilm formation or provide long-term antibacterial effects at the wound site. Metal nanoparticles are known for their non-specific bacterial toxicity mechanisms, which prevent bacteria from developing resistance and broaden their antibacterial activity [29]. Copper is a classical antimicrobial metal that has been widely used to combat Gram-negative, Gram-positive, and fungal infections due to its recognized antibacterial and pro-angiogenic properties, making it essential in wound healing [30]. Studies have shown that copper ions exert antimicrobial effects through various mechanisms, including altering membrane permeability, DNA structure, metabolism, and enzyme activity [31, 32]. Thus, we aim to incorporate antimicrobial ions to achieve synergistic antibacterial effects. Alginate is a naturally occurring anionic polysaccharide extracted from brown algae, which forms stable complex structures upon contact with divalent cations (such as calcium and copper) and promotes the release of these ions in acidic conditions [33]. We developed copper alginate microspheres (CAM) for slow release, achieving long-term antibacterial effects for chronically infected wounds.\u003c/p\u003e\n\u003cp\u003eTo ensure the therapeutic effects of the composite system of nanomaterials and metal nanoparticles, and considering its potential as an ideal medium for wound healing, we further encapsulated the system in hydrogels, which are widely used for wound treatment due to their excellent hydrophilicity, drug-loading capacity, and sustained release properties [34]. Gelatin methacrylate (GelMA) is a gelatin derivative and denatured collagen product known for its excellent solubility and low antigenicity. The incorporation of the Arg-Gly-Asp (RGD) sequence enhances biological interactions between cells and the GelMA scaffold, making GelMA an ideal biomaterial for wound healing, morphogenesis, and tissue restoration [35].\u003c/p\u003e\n\u003cp\u003eIn summary, based on the pathogenesis of biofilm formation and macrophage immune evasion in chronic infected wounds, we developed a functionalized hydrogel microsphere system. This system combats bacterial biofilms through ROS and reactivates the M1 phenotype of macrophages, while the slow release of antimicrobial metal ions provides long-lasting antibacterial effects and promotes angiogenesis, reshaping the physiological repair process of the wound. We synthesized g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e with POD enzyme activity through thermal polymerization and CAM via a gas-cutting method, and finally encapsulated g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e and CAM in GelMA hydrogel to construct a chronic infected wound healing system (GelMA/CAM@g-C3N4) （Scheme 1）. This system demonstrated excellent biocompatibility in vitro, promoted endothelial cell migration and vascular tube formation, reactivated M1 macrophages, and enhanced the expression of inflammatory cytokines (IL-1, IL-6, TNF-α). It significantly inhibited Staphylococcus aureus and Escherichia coli. Gene expression analysis showed that the system induced copper-like death of S. aureus by downregulating metabolic pathways such as glycolysis, oxidative phosphorylation, purine, and pyrimidine. In vivo, the system inhibited S. aureus growth, reactivated M1 macrophages by day 3, polarized macrophages to the M2 phenotype by day 7, promoted angiogenesis, and enhanced collagen deposition. This system provides a novel approach for treating biofilm-infected wounds and lays a foundation for addressing biofilm-related infectious diseases.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eExperimental Materials\u003c/h2\u003e\n\u003cp\u003eUrea, sodium alginate, copper chloride, and gelatin were purchased from Aladdin Reagent (Shanghai, China); CCK-8 was obtained from Biyuntian Biotechnology (Shanghai, China); dialysis bags (8000D) were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China); the bacterial live-dead staining kit was purchased from Maokang Biotechnology Co., Ltd. (Shanghai, China); Matrigel was obtained from Gelcon BioTech (Shanghai, China); photo-initiator and methacrylic anhydride were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China); DAPI was purchased from Wuhan Boster Biological Technology Ltd.; CD31 antibody, CD86 antibody, and CD206 antibody were purchased from Affinity Biosciences (Jiangsu, China); the DCFH-DA probe was purchased from Uelandy Biotechnology (Suzhou, China); FITC phalloidin was purchased from Sagon Biotech (Guangzhou, China); DMEM culture medium and fetal bovine serum were purchased from Gibco (USA).\u003c/p\u003e\n\u003ch2\u003eExperimental Equipment\u003c/h2\u003e\n\u003cp\u003eThe following equipment was used: intelligent biochemical incubator (SPX-280Y, Ningbo Kesheng Laboratory Instruments Co., Ltd., China), thermostatic shaker (THZ-82, Guohua Instruments Co., Ltd., China), high-speed centrifuge (5804R, Eppendorf China Co., Ltd., China), microbiological workstation (HCB-900V, Haier Biomedical Co., Ltd., China), PCR machine (T100, Bio-Rad, USA), transmission electron microscope (HT7700, Japan), biosafety cabinet (1374, Thermo Fisher Scientific, Suzhou, China), high-speed centrifuge (5702, Eppendorf International Trading Co., Ltd., Shanghai, China), confocal microscope (Olympus, Japan), UV-Vis spectrophotometer (Shimadzu UV-2700, Japan), microplate reader (Bio-Rad, USA), fluorescence inverted microscope (CKX53, Japan), and magnetic stirrer (MS-H-Pro, Dragonlab Instrument Co., Ltd., Beijing, China).\u003c/p\u003e\n\u003ch3\u003eSynthesis of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e Generation\u003c/h3\u003e\n\u003cp\u003e10 g of urea was accurately weighed and dissolved completely in 30 mL of deionized water. The solution was then transferred to a crucible at 60 \u0026deg;C and continued to be dried for 8 hours to obtain a white crystalline solid. This solid was subsequently transferred to a muffle furnace. It was calcined at a heating rate of 5 \u0026deg;C/min until the temperature reached 600 \u0026deg;C, after which the calcination was continued for 2 hours. The resulting light yellow solid was g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e. Its morphology was observed using TEM, its elemental composition and chemical bonds were confirmed by XPS, and its crystal structure was analyzed by XRD.\u003c/p\u003e\n\u003cp\u003eInitially, standard H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solutions (0, 0.05, 0.1, 0.2, 0.4, 0.8 mmol/L) were reacted with an H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e assay kit for 10 minutes. The absorbance was measured using a microplate reader at 405 nm to establish a standard curve. Different concentrations of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e (50, 100, 200 \u0026mu;g/mL) were prepared and measured at various time points (0, 0.5, 1, 1.5, 2 hours) under visible light using the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e assay kit. The generation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was calculated based on the standard curve.\u003c/p\u003e\n\u003ch3\u003eSynthesis and Characterization of Copper Alginate Microspheres (CAM)\u003c/h3\u003e\n\u003cp\u003eCopper alginate microspheres were synthesized by air cutting. First, 10 g of sodium alginate was weighed and dissolved in deionized water to prepare a 0.5% sodium alginate solution. Then, 1 g of CuCl\u003csub\u003e2\u003c/sub\u003e was weighed and dissolved in 99 mL of deionized water under ultrasonication to prepare a 1% CuCl\u003csub\u003e2\u003c/sub\u003e solution. The sodium alginate solution was loaded into a 10 mL syringe and pushed into a microfluidic system at a rate of 5 mL/h, where nitrogen (0.1 mpa) was used to cut the microspheres, which were then dropped into the CuCl\u003csub\u003e2\u003c/sub\u003e solution for cross-linking. The copper alginate microspheres were stored at 4 \u0026deg;C. Their morphology and structure were observed using an optical microscope, particle size was measured by ImageJ, and their microstructure and elemental composition were analyzed by SEM.\u003c/p\u003e\n\u003ch3\u003eSynthesis of GelMA and Combination with Copper Alginate Microspheres\u003c/h3\u003e\n\u003cp\u003eFirst, a carbonate buffer was prepared by dissolving 0.3427 g Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e and 3.0915 g NaHCO\u003csub\u003e3\u003c/sub\u003e in 200 mL of deionized water. Then, 20 g of gelatin was dispersed in 200 mL of carbonate buffer (pH 9.0) and heated in a 50 \u0026deg;C oil bath until completely dissolved to make a 10 % gelatin solution. Next, 2 mL of MA (methacrylic anhydride) was slowly added to the gelatin solution using a syringe pump at a rate of 0.2 mL/min while protecting from light. After the addition, the reaction continued for 3 hours in the oil bath. Then, 100 mL of PBS was added to terminate the reaction. The mixture was centrifuged (7000 rpm, 15 min) to remove unreacted MA. The GelMA was then placed in a dialysis bag (MWCO 3500) and dialyzed at 38 \u0026deg;C for 2 days. Finally, the GelMA was lyophilized and stored at -20 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003eA suitable amount of GelMA was dissolved in PBS to prepare a 5 % GelMA solution, to which 0.25 % LAP (photo-initiator) was added by mass-volume ratio. Copper alginate microspheres and g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e were then added to the solution, and the mixture was exposed to UV light for 30 seconds. The morphology of the GelMA hydrogel containing microspheres and g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e was observed by SEM after lyophilization.\u003c/p\u003e\n\u003ch3\u003eAdhesion Experiment of Functionalized Hydrogels\u003c/h3\u003e\n\u003cp\u003eThe functionalized hydrogels were prepared as described above and applied evenly to glass slides. Rat hearts, livers, spleens, lungs, and kidneys were individually placed on the hydrogel-coated slides. The adhesion was evaluated by visual observation and photographic documentation.\u003c/p\u003e\n\u003ch3\u003eSwelling Property Test of Functionalized Hydrogels\u003c/h3\u003e\n\u003cp\u003eDifferent systems of functionalized hydrogels were prepared (GelMA (G), GelMA/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e (G-g), GelMA/CAM (G-C), GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e (G-C-g)), lyophilized, and weighed (A\u003csub\u003e1\u003c/sub\u003e). The lyophilized hydrogels were then soaked in 50 mL PBS (pH 7.4) at 37\u0026deg;C for 24 hours. After soaking, the hydrogels were blotted dry with filter paper and weighed again (A\u003csub\u003e2\u003c/sub\u003e). The swelling ratio was calculated using the following formula:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" style=\"width: 463px; height: 40.6627px;\" width=\"463\" height=\"40.6627\"\u003e\u003c/p\u003e\n\u003cp\u003eEach experiment was repeated three times.\u003c/p\u003e\n\u003ch3\u003eDrug Release Curve of Functionalized Hydrogels\u003c/h3\u003e\n\u003cp\u003eA UV standard curve for g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e was established using UV spectroscopy. Specifically, different concentrations of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e (1000, 500, 250, 125, 62.25, 31.125 \u0026mu;g/mL) were prepared, and the UV absorption peaks were measured to create the standard curve.\u003c/p\u003e\n\u003cp\u003eTo obtain the drug release curve of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, GelMA/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e hydrogels were prepared. A 10 mL hydrogel was placed in 200 mL PBS and incubated in a thermostatic shaker (37 \u0026deg;C; 70 rpm). At specific time points (0, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72 h), 2 mL of release medium was removed for UV analysis, and the release amount was calculated based on the standard curve. The following formula was used to calculate the release rate:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" height=\"43\" width=\"478\" style=\"text-align: start; color: rgb(0, 0, 0); background-color: rgb(255, 255, 255); font-size: medium; font-family: \u0026quot;\u0026quot;;\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere A\u003csub\u003e1\u003c/sub\u003e was the total amount of the drug, and A\u003csub\u003e2\u003c/sub\u003e was the amount of drug released at a specific time point.\u003c/p\u003e\n\u003cp\u003eSimilarly, the copper ion release curve was obtained. A copper ion detection kit was used to establish a standard curve based on standard copper ion concentrations (0, 10, 20, 30, 40, 50 \u0026mu;g/mL) measured with a microplate reader. GelMA/CAM hydrogels were prepared, and a 10 mL hydrogel was placed in 200 mL PBS. Samples were taken at specified times (0, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72 h) for copper ion analysis, and the release amount was calculated according to the standard curve. The release rate was calculated using the same formula as above.\u003c/p\u003e\n\u003ch3\u003eCCK-8 Assay to Evaluate the Biocompatibility of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e and Copper ions\u003c/h3\u003e\n\u003cp\u003eThe biocompatibility of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e was evaluated using a CCK-8 assay. Human umbilical vein endothelial cells (HUVECs) at a density of 5\u0026times;10\u0026sup3; cells were seeded into a 96-well plate and treated with DMEM (serum-free) medium containing different concentrations of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e (0, 50, 100, 150, 200, 250 \u0026mu;g/mL) for 24 hours. After incubation, 100 \u0026mu;L of CCK-8 solution was added to each well and further incubated at 37 \u0026deg;C for 0.5 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. This same method was used to evaluate the biocompatibility of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e in RAW264.7 cells, with the experiment repeated three times.\u003c/p\u003e\n\u003cp\u003eNext, the biocompatibility of copper ions was assessed using the CCK-8 assay. HUVECs at a density of 5\u0026times;10\u0026sup3; cells were seeded into a 96-well plate and treated with DMEM (serum-free) medium containing various concentrations of copper ions (0, 10, 20, 30, 40, 50 \u0026mu;g/mL) for 24 hours. After incubation, 100 \u0026mu;L of CCK-8 solution was added to each well and further incubated at 37\u0026deg;C for 0.5 hours. The absorbance at 450 nm was measured using a microplate reader to calculate cell viability. This method was similarly applied to evaluate the biocompatibility of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e in RAW264.7 cells, with the experiment repeated three times.\u003c/p\u003e\n\u003ch3\u003eLive/Dead Staining of Functionalized Hydrogels\u003c/h3\u003e\n\u003cp\u003eTo assess the biocompatibility of different functionalized hydrogels, an indirect extraction method was used. Functionalized hydrogel systems, including PBS, GelMA, GelMA/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, GelMA/CAM, and GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, were incubated with DMEM medium at a ratio of 0.2 g/mL in accordance with national standards (GB/T 16886.5-2017) for 24 hours to obtain hydrogel extracts. HUVECs at a density of 5\u0026times;10⁴ cells were seeded into a 24-well plate and treated with different hydrogel extracts for 24 hours. Live/dead staining solution was added, followed by three washes with PBS. The cells were observed and photographed using an inverted fluorescence microscope. The experiment was repeated three times.\u003c/p\u003e\n\u003ch3\u003eCell Migration Assay\u003c/h3\u003e\n\u003cp\u003eHUVECs at a density of 1\u0026times;10⁵ cells were seeded into a 6-well plate and cultured in a CO₂ incubator (37\u0026deg;C, 5% CO₂) until they reached confluency. A pipette tip was used to scratch the cell monolayer, creating a narrow wound gap. The cells were then incubated with extracts of PBS, GelMA, GelMA/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, GelMA/CAM, or GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e in serum-free medium. The wound area was photographed using Cap Studio software, and Image J was used to analyze the wound area. The cell migration rate was calculated using the following formula:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" style=\"text-align: start; color: rgb(0, 0, 0); background-color: rgb(255, 255, 255); font-size: medium; font-family: \u0026quot;\u0026quot;; width: 457px; height: 36.487px;\" width=\"457\" height=\"36.487\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eWhere A₁ is the wound area at t = 0 h, and A₂ is the wound area at the specified time.\u003c/p\u003e\n\u003ch3\u003eTube Formation Assay\u003c/h3\u003e\n\u003cp\u003eHUVECs at a density of 1\u0026times;10⁵ cells were seeded into a 6-well plate. Once attached, the cells were incubated with serum-free DMEM extracts of PBS, GelMA, GelMA/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, GelMA/CAM, or GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e for 24 hours. Matrigel was stored at 4\u0026deg;C overnight and diluted with DMEM at a 2:1 ratio. After thoroughly mixing, 75 \u0026mu;L of Matrigel was added to each well of a 96-well plate and allowed to solidify at 37\u0026deg;C for 30 minutes. HUVECs were then digested with trypsin and seeded into each well of the 96-well plate at a density of 3\u0026times;10⁴ cells per well. After 12 hours, the formation of tube-like structures was observed under an inverted fluorescence microscope in Brightfield. The number of nodes and tube formation were quantified using Image J. The experiment was repeated three times.\u003c/p\u003e\n\u003ch3\u003ePromotion of CD31 Expression in HUVECs by Functionalized Hydrogels\u003c/h3\u003e\n\u003cp\u003eHUVECs at a density of 1\u0026times;10⁵ cells were seeded into confocal dishes and allowed to adhere. The cells were then treated with extracts of PBS, GelMA, GelMA/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, GelMA/CAM, or GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e for 48 hours. The extracts were aspirated, and the cells were fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and washed three times with PBS. After blocking for 20 minutes, the cells were incubated with a primary CD31 antibody at 4\u0026deg;C overnight, followed by a secondary antibody incubation at room temperature in the dark for 2 hours. The cells were then stained with phalloidin for 30 minutes in the dark at room temperature and counterstained with DAPI for 10 minutes. Images were captured using a confocal microscope. The fluorescence intensity of CD31 was quantified using Image J software, with the experiment repeated three times.\u003c/p\u003e\n\u003ch3\u003eReactivation of RAW 264.7 Macrophage Function by Functionalized Hydrogels\u003c/h3\u003e\n\u003cp\u003eRAW 264.7 cells at a density of 1\u0026times;10⁶ were seeded into confocal dishes and allowed to adhere. The cells were then treated with extracts of PBS, GelMA, GelMA/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, GelMA/CAM, or GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e for 24 hours. The extracts were aspirated, and DCFH-DA working solution was added. After incubation at 37\u0026deg;C in the dark for 30 minutes, the cells were stained with DAPI for 10 minutes. Images were taken using a confocal microscope, and ROS expression was quantified using Image J software. The experiment was repeated three times.\u003c/p\u003e\n\u003cp\u003eSimilarly, RAW 264.7 cells at a density of 1\u0026times;10⁶ were seeded into confocal dishes and treated with extracts of PBS, GelMA, GelMA/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, GelMA/CAM, or GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e for 24 hours. After the extracts were aspirated, the cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked for 20 minutes. The cells were then incubated with a rabbit anti-CD86 polyclonal antibody at 4 \u0026deg;C overnight, followed by incubation with a secondary antibody at room temperature in the dark for 2 hours. Nuclei were stained with DAPI, and images were captured using a confocal microscope. CD86 fluorescence intensity was quantified using Image J software, with the experiment repeated three times.\u003c/p\u003e\n\u003ch3\u003eqPCR Analysis of RAW 264.7 Inflammatory Cytokine Expression\u003c/h3\u003e\n\u003cp\u003eRAW 264.7 cells at a density of 1\u0026times;10⁶ were seeded into a 6-well plate and treated with extracts of PBS, GelMA, GelMA/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, GelMA/CAM, or GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e for 24 hours. Total RNA was extracted using a rapid RNA extraction kit, and the RNA concentration was determined. The RNA was reverse-transcribed into cDNA using a reverse transcription premix kit, and qPCR was performed using primers designed for the target genes (Table 1). Gene expression was analyzed semi-quantitatively. The experiment was repeated three times.\u003c/p\u003e\n\u003ch3\u003eEvaluation of Antibacterial Activity of Functionalized Hydrogels by Spread Plate Method\u003c/h3\u003e\n\u003cp\u003eThe antibacterial activity of hydrogels was evaluated using the spread plate method. Briefly, 200 \u0026mu;L of GelMA, GelMA/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, GelMA/CAM, and GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e hydrogels were prepared in 48-well plates. After complete gelation, 10 \u0026mu;L of bacterial suspension (10\u003csup\u003e7\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;1\u003c/sup\u003e) was added to the surface of the hydrogels and incubated at 37 \u0026deg;C for 2 hours under natural light. Subsequently, 1 mL of sterile PBS was added to each well to resuspend the bacteria. As a negative control, 10 \u0026mu;L of bacterial suspension (10\u003csup\u003e7\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;1\u003c/sup\u003e) was added to 1 mL of PBS. Then, 100 \u0026mu;L of the bacterial suspension was spread evenly onto agar plates, and\u0026nbsp;incubated at 37 \u0026deg;C in a biochemical incubator for 20 hours. The colonies were photographed and counted using Image J software, with each experiment repeated three times.\u003c/p\u003e\n\u003ch3\u003eEvaluation of Antibacterial Activity of Hydrogels by Live/Dead Staining\u003c/h3\u003e\n\u003cp\u003eThe antibacterial activity of hydrogels was further assessed using a bacterial live/dead staining kit according to the manufacturer\u0026rsquo;s instructions. SYTO 9 and PI were diluted in PBS (1:1000) and mixed with the bacterial suspension at room temperature for 20 minutes. The bacterial suspension was then placed on a slide and observed under a confocal microscope, with images captured for further analysis. The experiments were repeated three times.\u003c/p\u003e\n\u003ch3\u003eObservation of Bacterial Morphology\u003c/h3\u003e\n\u003cp\u003eThe bacterial morphology after treatment with different functionalized hydrogels was observed by scanning electron microscopy (SEM). Briefly, bacterial suspensions from different treatment groups were centrifuged at 4000 rpm for 10 minutes at 25\u0026deg;C, and the bacterial pellet was collected. The bacteria were fixed in 2.5 % glutaraldehyde at 4\u0026deg;C overnight. After centrifugation, the fixed samples were washed with PBS several times, followed by gradient dehydration using ethanol solutions at concentrations of 30 %, 50 %, 70 %, 85 %, 95 %, and 100 %, with each step lasting for 15 minutes. Finally, 20 \u0026mu;L of the dehydrated bacterial sample was dropped onto a silicon wafer and air-dried. The samples were coated with gold and observed under SEM to examine bacterial morphology.\u003c/p\u003e\n\u003ch3\u003eEvaluation of Anti-biofilm Activity of Functionalized Hydrogels by Crystal Violet Staining\u003c/h3\u003e\n\u003cp\u003eBacterial suspensions (180 \u0026mu;L/well) were seeded in a 96-well plate and incubated at 37 \u0026deg;C in a humidified environment for 24 hours. After incubation, the planktonic bacteria were removed, and the wells were washed with PBS to remove loosely attached bacteria. PBS, GelMA, GelMA/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, GelMA/CAM, and GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e extracts were added to the wells, and the plates were incubated at 37 \u0026deg;C in a humidified environment for another 24 hours. After incubation, the extracts and dead bacteria were washed away with PBS, and the remaining biofilm was fixed with 4 % formaldehyde for 15 minutes. The fixed biofilms were washed with PBS, and stained with 0.1 % crystal violet for 15 minutes at room temperature, and unbound dye was removed by washing with PBS. The plates were air-dried, and the crystal violet bound to the biofilm was solubilized using 33% acetic acid. The absorbance was measured at 590 nm, with each experiment repeated three times.\u003c/p\u003e\n\u003ch3\u003eEstablishment of an Infected Wound Model in Animals\u003c/h3\u003e\n\u003cp\u003eAll animal experiments were reviewed and approved by the Animal Ethics Committee of North Sichuan Medical College (Approval No: 2024094) and conducted by national animal protection guidelines. All surgical procedures were performed under anesthesia, and all efforts were made to minimize pain, suffering, and death. A total of 100 healthy male SD rats (SPF grade, 8 weeks old, weighing 220-250 g) were anesthetized with sodium pentobarbital (20 mg/kg), and their backs were shaved. Full-thickness skin wounds (10 mm in diameter) were created on the rat backs using a circular punch and surgical scalpel. The rats (n=3) were randomly divided into different groups. The wounds were infected with Staphylococcus aureus suspension (10\u003csup\u003e7\u003c/sup\u003e CFU/mL, 50 \u0026mu;L) for 24 hours to establish an infected wound model. The wounds were then treated with PBS, GelMA, GelMA/g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e, GelMA/CAM, and GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e hydrogels. On days 0, 3, 7, and 14, the infected wounds were photographed, and the healing process was observed. The wound area was measured using Image J software. Additionally, on day 3, the infected wound tissues and surrounding skin were excised, homogenized in 1 mL of sterile PBS, serially diluted, and plated on solid culture media for bacterial growth. The plates were incubated in a bacterial incubator for 12 hours and photographed. During the wound healing process, skin tissue around the wound in each group was collected and fixed in 4 % paraformaldehyde. The tissue specimens were stained with hematoxylin-eosin (H\u0026amp;E), Masson\u0026apos;s trichrome, Giemsa, and subjected to immunofluorescence staining for CD31, CD86, and CD206 (Chengdu Lilai Biotech Co., Ltd.). Additionally, to evaluate the in vivo toxicity of the hydrogel samples, visceral organs (heart, liver, spleen, lungs, kidneys) from the rats were collected for H\u0026amp;E staining. The experiments were repeated three times.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n\u003cp\u003eAll data were statistically analyzed using \u003cem\u003eSPSS 24.0\u003c/em\u003e and \u003cem\u003eGraphPad Prism 8\u003c/em\u003e software. Independent sample t-tests were used to evaluate statistical differences between the two groups, while one-way ANOVA was employed for multiple-group comparisons. Data are presented as mean \u0026plusmn; standard deviation (\u003cem\u003ex̅\u0026plusmn;s\u003c/em\u003e). \u0026quot;ns\u0026quot; indicates no significant difference; *p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001 indicate statistically significant differences.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eSynthesis and Characterization of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e\u003c/h2\u003e\n\u003cp\u003eFigure 1a shows the synthesis of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e via a thermal polymerization method. Under TEM, g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e exhibits a layered sheet-like structure（Figure 1b）. XPS analysis confirms that g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e consists of carbon (C) and nitrogen (N) elements（Figure 1c）. The XPS valence band spectrum indicates that g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e was composed of C-C, C=N-C, and N-C=N bonds（Figure 1d and 1e）. XRD results show two characteristic peaks at 13.0 ° and 27.5 °, confirming the successful synthesis of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4 \u003c/sub\u003e[36]. A hydrogen peroxide assay kit was used to detect the production of ROS and a standard curve was established, showing that the amount of ROS produced by g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e was positively correlated with its concentration and light exposure time（Figure 1g and Figure S1）.\u003c/p\u003e\n\u003ch2\u003eSynthesis of Copper Alginate Microspheres and Construction of Functionalized Hydrogel System\u003c/h2\u003e\n\u003cp\u003eFigure 2a illustrates the schematic of the gas-cutting method used to synthesize copper alginate microspheres. Under a light microscope, the microspheres exhibit uniform round structures with a diameter of approximately 290.1 ± 16.3 μm（Figure 2b and 2c）. Figure 2d showed that different hydrogels formed a gel state after 30 seconds of UV light exposure. SEM analysis revealed a smooth surface for GelMA hydrogels, which became rough after incorporating g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e. In the G-C-g group, microspheres were attached to the surface, and element mapping showed that the gel surface was composed of C, N, O, and Cu, confirming the successful construction of the hydrogel-microsphere system（Figure 2e and 2f）. The swelling curves of the hydrogels in different systems were similar, with no significant changed in swelling properties after the incorporation of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e and CAM. The hydrogels demonstrated excellent adhesion to the heart, liver, spleen, lungs, and kidneys（Figure 2g and Figure S2）. Using a copper ion assay kit, a standard curve for copper ions was established, and the UV spectra of different concentrations of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e were measured to create a standard curve. The release curves of functionalized hydrogel microspheres showed rapid release of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e and copper ions within 0–12 hours, followed by sustained release（Figure S3-S6）.\u003c/p\u003e\n\u003ch2\u003eBiocompatibility, Scratch, and Tube Formation Assays of Functionalized Hydrogel Microspheres\u003c/h2\u003e\n\u003cp\u003eThe CCK-8 assay shows that g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e concentrations exceeding 150 μg/mL significantly affect RAW264.7 cell viability, and copper ion concentrations exceeding 40 μg/mL notably reduce HUVEC cell viability. Therefore, a concentration of 150 μg/mL for g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e and 40 μg/mL for copper ions was selected for subsequent experiments（Figure S7-S10）. Live-dead cell staining with extracts from different hydrogel groups showed that the functionalized hydrogel microspheres exhibited excellent biocompatibility with RAW264.7 and HUVECs（Figure 3a and Figure S11）.\u003c/p\u003e\n\u003cp\u003eScratch assay results showed that the G-g group extract slowed cell migration, while the G-C-g group significantly accelerated cell migration compared to the PBS group （Figure 3b and d）. Tube formation assays revealed that the G-g group extract reduced tube formation and total length in HUVECs, while the G-C-g group significantly promoted tube formation and total length compared to the PBS group（Figure 3c, e, and f）. After 24 hours of intervention with different hydrogel systems on HUVECs, the G-C-g group significantly promoted CD31 expression compared to the PBS group（Figure S12 and 13）.\u003c/p\u003e\n\u003ch2\u003eFunctionalized Hydrogel Microspheres re-roused Macrophage M1 Function\u003c/h2\u003e\n\u003cp\u003eAfter treating M0 macrophages with extracts from different systems, the DCFH-DA probe results showed that both the G-g and G-C-g groups induced macrophages to exhibit a pro-inflammatory phenotype, with significantly higher ROS expression than the PBS group（Figure 4a and Figure S14）. CD86 immunofluorescence analysis showed that the G-g and G-C-g groups had higher fluorescence intensity than the PBS group（Figure 4b and Figure S15）. Further RNA extraction and PCR results indicated that the expression of pro-inflammatory factors (IL-1, IL-6, TNF-α) significantly increased in the G-g and G-C-g groups compared to the PBS group（Figure S16）.\u003c/p\u003e\n\u003ch2\u003eAntibiofilm Activity and Mechanism of Functionalized Hydrogel Microspheres In Vitro\u003c/h2\u003e\n\u003cp\u003eIn infected environments, bacteria protect themselves from immune system attacks and exogenous bactericides by secreting EPS (extracellular polymeric substances) to form biofilms, which contribute to the persistence and resistance of biofilm-related infections. In this study, bacteria were seeded on the hydrogel surface, plated on agar, and observed through photographs and quantitative ImageJ analysis. Plate coating results indicated that a large number of colonies grew on the surfaces in the PBS and G groups. However, after intervention with the G-g and G-C groups, Staphylococcus aureus growth was inhibited, with the G-C-g group showing a significant reduction in bacterial colonies （Figure 5a and Figure S17）. Bacterial live-dead staining revealed a marked increase in dead bacteria in the G-C-g group compared to the PBS group （Figure 5b）. SEM analysis showed intact bacterial cell walls in the PBS and G groups, while the G-C-g group displayed disrupted bacterial walls and cytoplasmic leakage（Figure 5c and Figure S18）. Similar results were obtained when the system was tested on Escherichia coli, where the G-C-g group exhibited significant inhibitory effects on bacterial growth compared to the PBS group（Figure 5d）. Live-dead bacterial staining further confirmed that the G-C-g group had a higher proportion of dead bacteria compared to the PBS group（Figure 5e）. SEM observations revealed disrupted E. coli cell walls in the G-C-g group compared to the PBS group（Figure 5f）. Using crystal violet staining, hydrogels from different groups demonstrated that the G-C-g group exhibited significant inhibitory effects on both S. aureus and E. coli compared to the PBS group（Figure S19 and S20）.\u003c/p\u003e\n\u003cp\u003eFurther, after intervening with PBS and the G-C-g group on S. aureus, bacterial gene sequencing revealed similar gene distribution patterns between the G-C-g and PBS groups, which can be used for subsequent experimental validation（Figure 6a）. Compared to the PBS group, the G-C-g group exhibited upregulation of 302 metabolites and downregulation of 349 metabolites（Figure 6b）. A clustering heat map illustrated the consistency of metabolite changes after treatment, highlighting differences in metabolite expression between the two groups （Figure 6c）. KEGG enrichment analysis indicated that the functionalized hydrogel microspheres downregulated glycolysis, oxidative phosphorylation, the phosphotransferase system, amino acid metabolism (alanine, aspartate, and glutamate metabolism), purine, and pyrimidine metabolism （Figure 6d）. GO enrichment analysis revealed that the system inhibited processes such as carbohydrate derivative biosynthesis, lipoteichoic acid metabolism, and lipoteichoic acid biosynthesis（Figure 6e）. Figure 6f illustrated the antibacterial mechanism of the functionalized hydrogel microspheres. The diagram showed that the ROS generated by g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e disrupts the biofilm, followed by the release of copper ions from copper alginate microspheres, which blocked bacterial ABC transporters, leading to impaired nutrient exchange. Copper ions overload within the bacteria first induced downregulation of glycolysis and oxidative phosphorylation, resulting in reduced energy metabolism. This was followed by downregulation of purine and pyrimidine metabolism, impairing DNA replication and repair systems. Additionally, triglyceride metabolism was downregulated, and copper ions induced lipid peroxidation, ultimately leading to bacterium-like copper-induced cell death.\u003c/p\u003e\n\u003ch2\u003eFunctionalized Hydrogel Microspheres Promote Biofilm-Associated Wound Healing In Vivo\u003c/h2\u003e\n\u003cp\u003eFigure 7a illustrated the schematic diagram of biofilm-associated chronic wound healing and the application of functionalized hydrogel microspheres on the wound site. A circular wound model with a 10 mm diameter was created on the backs of SD rats, followed by inoculation with S. aureus. When the wound exhibited purulent exudation, successful model establishment was confirmed. Subsequently, hydrogels from different groups were applied to the wounds, and healing progress was documented. Gross images of wound healing demonstrated that interventions with the g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e and CAM groups accelerated wound healing, while the G-C-g group, through the synergistic effects of g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e and CAM, significantly accelerated wound healing compared to the PBS group（Figure 7b-7d）. On day 3 of wound healing, wound exudates were collected for plate analysis, revealing a reduction in bacterial numbers in the g-C3N4 and CAM groups, with the G-C-g group exhibiting the most significant reduction compared to the PBS group（Figure 7b and Figure S21）. On day 3, Giemsa staining of wound tissues showed large aggregations of S. aureus in the PBS and G groups, whereas bacterial numbers were reduced in the G-g and G-C groups. The G-C-g group showed only a few residual bacteria, significantly fewer than in other groups（Figure 7f）. H\u0026amp;E staining on day 7 demonstrated partial wound contraction in the G-g and G-C groups, while the G-C-g group showed significant wound contraction, leaving the shortest wound length compared to the PBS group（Figure 8a and 8c）. By day 14, wound healing had further progressed in all groups, with the G-C-g group displaying the shortest wound diameter（Figure 8a）. Masson’s staining on day 7 revealed higher collagen deposition in the G-g and G-C groups compared to the PBS group, while the G-C-g group showed the highest collagen deposition with statistical significance（Figure 8b and 8d）. By day 14, collagen deposition had further increased in all groups, with the G-C-g group exhibiting the highest collagen deposition（Figure 8b）. Immunofluorescence staining of wound tissues on day 3 revealed lower CD86 and CD206 expression in the PBS and G groups, whereas CD86 expression significantly increased in the G-g and G-C-g groups compared to the PBS group（Figure 9a）. On day 7, immunofluorescence results showed increased CD206 expression in the G-g and G-C groups, with the G-C-g group exhibiting significantly higher CD206 expression compared to the other groups（Figure 9b）. Additionally, CD31 immunofluorescence staining of wound tissues showed significantly higher CD31 expression in the G-C-g group compared to the other groups, indicating that the wound healing had progressed to the proliferation phase （Figure S22 and S23）. On day 14 of wound healing, H\u0026amp;E staining of the heart, liver, kidneys, lungs, and spleen of SD rats showed no significant signs of inflammation, necrosis, or other pathological changes（Figure S24）.\u003c/p\u003e\n"},{"header":"Discussion","content":"\u003cp\u003eBacterial infectious diseases are an increasing public health challenge [37, 38]. The formation of bacterial biofilms significantly reduces the efficacy of traditional antibiotic therapies and promotes the emergence of multidrug-resistant bacteria [39, 40]. Due to the presence of EPS, biofilms operate as a self-protective structure, shielding pathogens from the innate immune system and effectively resisting antibiotic penetration, thereby posing substantial challenges to antibacterial treatment [41]. The formation of biofilms and the immune evasion they induce in macrophages both hinder the wound healing process. Therefore, disrupting bacterial biofilms and re-rousing macrophage M1 functionality are key strategies for promoting chronic wound regeneration. In this study, we developed a chronic wound repair system by synthesizing g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e through thermal polymerization and CAM using gas-cutting methods. Further, we encapsulated g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e and CAM into GelMA to construct a system capable of disrupting biofilms and reawakening macrophage M1 function.\u003c/p\u003e\u003cp\u003eIn recent years, with the rapid development of nanotechnology, the application of nanomaterials in the field of antibacterial therapy has achieved significant success. Among them, g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e-based nanomaterials have shown remarkable progress in antibacterial applications [42]. g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e is a novel inorganic non-metallic photoactive material with a two-dimensional layered aromatic polycyclic structure composed of sp2-hybridized C and N atoms, and its C-N bond length is uniform [43]. In this study, g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e was successfully synthesized via thermal polymerization and characterized by TEM, XRD, and XPS analyses, including its valence band spectrum. Using an H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e assay kit under light exposure, we demonstrated that g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e exhibits peroxidase (POD) activity.\u003c/p\u003e\u003cp\u003eMicrosphere structures can effectively encapsulate active ingredients, achieving protection and sustained release [44]. Sodium alginate exchanges its Na\u003csup\u003e+\u003c/sup\u003e with divalent cations such as Ca\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, and Ba\u003csup\u003e2+\u003c/sup\u003e, forming a stable biopolymer with a unique three-dimensional structure [45]. In our constructed system, copper alginate microspheres were successfully synthesized via gas-cutting methods, and uniform spherical structures were observed under a light microscope, showing sustained release of Cu\u003csup\u003e2+\u003c/sup\u003e during degradation. Hydrogels are one of the most important biomaterials, attracting attention for medical applications such as wound healing, tissue engineering, and drug delivery [46]. SEM analysis revealed a uniform porous three-dimensional network structure, with microspheres and g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e attached to the surface, confirming the successful construction of the functionalized hydrogel microsphere wound healing system. The RGD sequence in GelMA hydrogels promotes adhesion between the hydrogel and cells. Additionally, the water molecules in the hydrogel can form hydrogen bonds with water molecules on the wound surface, enhancing the contact between the hydrogel and the wound [47]. Furthermore, water molecules penetrate the wound tissue via physical osmosis, forming a water layer that further consolidates the connection between the hydrogel and the wound. These adhesion mechanisms play a critical role in tissue repair and regeneration.\u003c/p\u003e\u003cp\u003eBiocompatibility aims to assess the toxicity and safety of biomaterials in cells, ensuring that therapeutic effects are achieved while minimizing potential side effects. In this study, the CCK-8 assay results indicated that when the g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e concentration did not exceed 150 μg/mL and Cu\u003csup\u003e2+\u003c/sup\u003e concentration did not exceed 40 μg/mL, there was no significant toxicity to the cells. However, higher concentrations of nanomaterials and metal ions may cause toxicity through various mechanisms, such as disrupting the cell membrane, inducing oxidative stress, triggering intracellular metabolic disorders, and causing ionic homeostasis imbalance. Based on CCK-8 and live-dead staining assays, the functionalized hydrogel microspheres demonstrated excellent biocompatibility.\u003c/p\u003e\u003cp\u003eMacrophages possess high plasticity and are polarized into \"classically activated\" pro-inflammatory M1 and \"alternatively activated\" anti-inflammatory M2 phenotypes during different phases of wound healing [48]. Macrophages, as a key phagocytic cell type, are part of the innate immune system, and are responsible for engulfing and processing cellular debris and pathogens [49]. The formation of biofilms enhances immune evasion and antibiotic resistance, leading to persistent local infections that hinder the wound healing process [50]. In this study, g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e successfully mimicked POD enzyme activity and generated ROS. Through ROS probing, CD86 immunofluorescence, and PCR assays, the results showed that g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e could promote macrophage ROS production and upregulate the expression of CD86, IL-1, IL-6, and TNF-α pro-inflammatory cytokines, successfully re-rousing macrophage M1 pro-inflammatory function. ROS, as a driving signal for M1 macrophage polarization, initiates cell cycle and metabolic reprogramming through pathways such as ATM-CHK2, NF-κB, and MAPK, regulating macrophage M1 phenotype polarization, inducing the expression of inflammatory cytokines, and enhancing their phagocytic function [51-53]. Microbial aggregation at wound sites and the development of resistance to available antibiotics are major concerns [54]. To survive under harsh conditions, including exposure to known antibiotics, microorganisms form biofilms a unique structure whose formation involves quorum sensing, environmental stress, nutrient availability, hydrodynamic conditions, intercellular communication, signaling cascades, and secondary messengers. Alternative therapies that disrupt bacterial structure and function by generating ROS and binding metal ions have gained considerable attention in recent years [55].\u003c/p\u003e\u003cp\u003eWound healing involves multiple stages, including hemostasis, inflammation, proliferation, angiogenesis, and tissue remodeling, each involving interactions between different cell types [56]. Neovascularization supplies wounds with sufficient blood and oxygen to support cell survival and tissue regeneration [57]. In chronic wounds, insufficient blood perfusion exacerbates infection symptoms associated with microbes and often complicates the healing process [58]. In this study, the migration assay, tube formation assay, and CD31 immunofluorescence analysis results indicated that the functionalized hydrogel microspheres could promote endothelial cell migration, tube formation, and CD31 expression. Current research suggests that Cu\u003csup\u003e2+\u003c/sup\u003e can activate signaling pathways such as Wnt, PI3K/Akt, MAPK, and hypoxia-inducible factor 1α (HIF-1α), upregulating the expression of angiogenic factors, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and HIF-1α, thereby inducing angiogenesis [59, 60]. Using a chronic wound infection model with S. aureus, our system re-rousing macrophage M1 function by day 3, clearing pathogens and cellular debris, and shifting the chronic wound microenvironment towards an inflammatory phase. By day 7, macrophages had transitioned to the M2 phenotype, further promoting CD31 expression and collagen deposition, enabling the transition from the inflammatory phase to the proliferative remodeling phase, thereby greatly enhancing chronic wound healing efficiency.\u003c/p\u003e\u003cp\u003eIn summary, based on the pathological conditions of biofilm formation and macrophage immune evasion in wound sites, the functionalized hydrogel microspheres developed in this study effectively disrupted bacterial biofilms and reawakened pro-inflammatory macrophage function, thereby transforming the microenvironment of chronically infected wounds into one conducive to physiological healing, ultimately promoting wound healing.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study addresses the critical mechanisms of biofilm formation and macrophage immune evasion, which lead to impaired wound healing, by constructing a functionalized hydrogel microsphere wound repair system (GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e) with antibiofilm properties and the ability to re-rousing macrophage M1 function. This system effectively disrupted bacterial biofilms by generating ROS and re-roused macrophage M1 function. Additionally, the release of Cu\u003csup\u003e2+\u003c/sup\u003e interfered with bacterial energy metabolism, biosynthesis, and lipid metabolism processes, leading to copper-like bacterial cell death. Furthermore, this system promoted CD31 expression in endothelial cells, effectively transforming the pathological microenvironment of chronic wounds into one that facilitated regeneration. This innovative approach provides a promising solution for the treatment of chronic wounds.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Doctoral Research Launch Fund Project of North Sichuan Medical College Affiliated Hospital (CBY23-QDA23), Health Commission of Sichuan Province Medical Science and Technology Program\u0026rdquo; (24QNMP096), and Research Project Nanchong Science and Technology Bureau (23JCYJPT0036).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments in this study were approved by the Ethics Committee of the Animal Experimentation Centre of North Sichuan Medical College (approval number: 2024094).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eChao Xiang: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing-original draft. Chaoyu Pu: Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing original draft. XueMei Zhong: Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization. Yong Wang: Data curation, Project administration, Validation. Weiyong Song: Data curation, Project administration, Validation. Xingkuan Wang: Project administration, Validation, Funding. Kemiao Chen: Project administration, Validation. Kai Li: Project administration, Resources, Validation. Yue Luo: Project administration, Resources, Validation, Funding. Ke Jiang: Conceptualization, Resources. Dianming Jiang: Investigation, Methodology, Project administration, Writing \u0026ndash; review \u0026amp; editing, Conceptualization.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe wound also like to acknowledge BioRender, which was used to create a Schematic diagram in this article. We also wish to express our deep appreciation to the North Sichuan Medical College Innovation Center for Science and Technology for providing the experimental platform that was crucial for our research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eV F, RR I, AM S, M R, D M, S K, et al. Chronic wounds. Nature reviews Disease primers. 2022;8(1):50.\u003c/li\u003e\n\u003cli\u003eM F, B DA, F P, A S, S S, R T, et al. Challenges in the management of chronic wound infections. Journal of global antimicrobial resistance. 2021;26:140-7.\u003c/li\u003e\n\u003cli\u003eY L, L S, L S, HC vdM, PC J, Y R, et al. Nanotechnology-based antimicrobials and delivery systems for biofilm-infection control. Chemical Society reviews. 2019;48(2):428-46.\u003c/li\u003e\n\u003cli\u003eNA K, J P, AK M, N C, S S, K V, et al. Combating bacterial biofilms and related drug resistance: Role of phyto-derived adjuvant and nanomaterials. 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Biomaterials advances. 2024;161:213893.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chronic infectious wounds, hydrogels, microspheres, immune regulation, macrophages","lastPublishedDoi":"10.21203/rs.3.rs-5378421/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5378421/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eTraditional antibiotics are often ineffective against biofilm-associated infections, and biofilm-induced macrophage immune evasion directly halts the wound healing process. Disrupting biofilms and regulating macrophage immune functions are critical to improving wound healing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e In this study, we synthesized g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e with peroxidase (POD) enzyme activity via thermal polymerization and copper alginate microspheres (CAM) via gas cutting. These were co-encapsulated into GelMA hydrogels to form a functionalized wound repair system (GelMA/CAM@g-C\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003e) with both anti-biofilm and local immune microenvironment remodeling capabilities. In vitro, this system exhibited excellent biocompatibility and promoted endothelial cell migration, vascular formation, and CD31 expression. It also polarized macrophages toward the M1 phenotype, restoring their pro-inflammatory functions, upregulating inflammatory cytokines (IL-1, IL-6, TNF-α), and inhibiting Staphylococcus aureus and Escherichia coli. In vivo, the system suppressed S. aureus growth, promoted angiogenesis and collagen deposition, and reshaped the pathological microenvironment to achieve wound repair and regeneration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e This system offers a new therapeutic strategy for chronic infectious wounds.\u003c/p\u003e","manuscriptTitle":"Functional Hydrogels Promote Chronic Infectious Wound Healing by Re-rousing Macrophage M1 and Inducing Bacterial Copper-Like Death","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-18 14:12:31","doi":"10.21203/rs.3.rs-5378421/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"57ed70d2-dc01-4837-82ea-9c71c8c7caa0","owner":[],"postedDate":"November 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-30T15:23:41+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-18 14:12:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5378421","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5378421","identity":"rs-5378421","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}