Preparation method and application of diagnosis and treatment integrated photoactive composite hydrogel

Abstract

The invention relates to the field of biomedicine, and discloses a preparation method and application of diagnosis and treatment integrated photoactive composite hydrogel. The triphenylamine derivative TPAB with an A-pi-D-pi-A conjugated structure is synthesized by adopting TPA-CHO and MN as monomers and adopting a simple and efficient Schiff base reaction, Cu < 2 + > is further introduced into a TPAB molecule, and a stable metal complex is formed through a coordinate bond. Further, through a Schiff base reaction, TPAB-Cu < 2 + > and modified hyaluronic acid (mHA) are covalently linked to form a cross-linked network, and meanwhile, emodin is doped through hydrogen bond coupling, so that the space-time responsive hybrid hydrogel dressing EM (at) mHA-TPAB-Cu < 2 + > with the dynamic cross-linked network is finally obtained. The hydrogel is an ideal candidate material for comprehensive diagnosis and treatment of drug-resistant bacteria wound infection.

Description

Technical Field

[0001] This application relates to the biomedical field, and more specifically, to a method for preparing and using a photoactive composite hydrogel that integrates diagnosis and treatment. Background Technology

[0002] Drug-resistant bacterial infections pose a significant challenge to clinical treatment, and the increasing antibiotic resistance of pathogens exacerbates this problem, severely weakening treatment effectiveness and patient prognosis. To address the need for effective interventions against multidrug-resistant (MDR) bacterial infections, there is an urgent need to develop next-generation treatment platforms that integrate infection control, immune modulation, tissue regeneration, and real-time treatment monitoring.

[0003] Photodynamic therapy (PDT) based on small organic molecule photosensitizers offers a promising treatment strategy for MDR bacterial wound infections, with advantages including biocompatibility, non-invasiveness, and spatiotemporal selectivity. In traditional PDT, photosensitizers convert ambient oxygen molecules into reactive oxygen species (ROS) under light irradiation, directly killing MDR bacteria by oxidizing their lipids, proteins, and DNA. Photosensitizers are classified into type I and type II. Type I photosensitizers primarily generate ROS (O2-· and ·OH) through electron transfer or hydrogen extraction processes, while type II photosensitizers primarily convert ground-state oxygen into singlet oxygen (1O2) through energy transfer processes. Recent studies have also shown that through precise molecular design, photosensitizers can possess microenvironment-responsive properties, enabling non-invasive monitoring of treatment progress. However, despite the significant advantages of PDT in treating MDR bacterial wound infections, several challenges remain to be addressed. On the one hand, most existing small-molecule photosensitizers are oxygen-dependent (type II photosensitizers), while the infection microenvironment is usually in a hypoxic state. Furthermore, biofilm formation further exacerbates this hypoxia, significantly reducing the therapeutic efficacy of oxygen-dependent photosensitizers. On the other hand, due to the complexity of the infection microenvironment, synthesizing small-molecule photosensitizers with both high selectivity and high sensitivity remains a significant challenge, and the synthesis of microenvironment-responsive photosensitizers often requires complex and stringent reaction conditions. Moreover, wound repair is a highly complex and dynamically evolving biological process, typically involving four overlapping and sequentially progressive stages: hemostasis, inflammation, proliferation, and remodeling. In an infection environment, excessive ROS accumulation and the excessive inflammatory response induced by bacterial death often severely hinder the normal wound healing process. Timely regulation of macrophage transformation from the pro-inflammatory M1 phenotype to the anti-inflammatory and reparative M2 phenotype is crucial for suppressing inflammatory responses and promoting cell proliferation and tissue regeneration.

[0004] Emodin (6-methyl-1,3,8-trihydroxyanthraquinone) is a phenolic anthraquinone compound with multiple pharmacological activities, obtained from natural plants such as rhubarb, knotweed, and coptis. This compound exhibits excellent antioxidant and anti-inflammatory properties by effectively scavenging reactive oxygen species (ROS) by providing hydrogen atoms, electrons, or protons. Recent studies have shown that emodin can promote the polarization of macrophages from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype and significantly inhibit inflammation-related signaling pathways. However, the low solubility, poor bioavailability, and limited transdermal absorption of emodin hinder its application in wound treatment. Developing suitable drug delivery systems is crucial to fully utilizing its anti-inflammatory properties to promote the healing of infected wounds. Hydrogels, as hydrophilic soft materials with a hydrophilic three-dimensional porous network structure, can absorb wound exudate, isolate the wound from the external environment, and maintain a moist wound environment, thus becoming ideal wound dressings and functional drug carriers. Hyaluronic acid, as a major component of the extracellular matrix, possesses excellent biocompatibility, biodegradability, and low immunogenicity, while also promoting collagen deposition and angiogenesis to some extent. Therefore, if functional photosensitizers and emodin can be integrated into the hyaluronic acid biomolecular network through physicochemical structural design, it is possible to promote the clearance of MDR bacteria, remodel the inflammatory microenvironment, promote tissue regeneration, and achieve in-situ monitoring of MDR bacterial-infected wounds. Summary of the Invention

[0005] The purpose of this invention is to address the technical problems in existing wound treatment technologies for drug-resistant bacterial infections, such as insufficient photodynamic performance of photosensitizers in hypoxic environments, complex and insufficiently specific synthesis processes of microenvironment-responsive molecules, limited application of emodin, and the single function of traditional treatment platforms. To this end, this application utilizes TPAB-Cu, which possesses significant type I photodynamic activity and microenvironment-responsive properties. 2+ The complex was reacted with modified hyaluronic acid (mHA) to synthesize a covalent macromolecular network, and emodin (EM) was loaded into the network via hydrogen bonds. Ultimately, a spatiotemporally responsive hybrid hydrogel (EM@mHA-TPAB-Cu) with a dynamically cross-linked network was obtained. 2+ This enables efficient and collaborative diagnosis and treatment of wounds infected with MDR bacteria.

[0006] To achieve the above-mentioned objectives, this application adopts the following technical solution:

[0007] In the first aspect, this application provides a photoactive composite hydrogel EM@mHA-TPAB-Cu that integrates diagnosis and treatment. 2+ The preparation method includes the following steps: Step 1: Using TPA-CHO and MN as monomers, TPAB is synthesized via a simple and efficient Schiff base reaction, and Cu is further introduced. 2+ TPAB-Cu is formed through coordinate bonds.2+ ; Step 2: Dissolve emodin in DMSO and mHA in deionized water, then mix the two and add the TPAB-Cu obtained in Step 1. 2+ The solution was ultrasonically treated to form a homogeneous solution, stirred at room temperature, and then dialyzed to remove DMSO, yielding EM@mHA-TPAB-Cu. 2+ Composite hydrogel.

[0008] Further, step 1 includes: Step 1-1: Dissolve TPA-CHO and MN in a solvent, reflux under an inert atmosphere, filter, recrystallize to obtain TPAB powder; Step 1-2: Dissolve the TPAB obtained in Step 1-1 in a solvent, then add Cu(NO3)2, stir at room temperature, dry, purify, and dry again to obtain the composite photosensitizer TPAB-Cu. 2+ .

[0009] Furthermore, in step 2, emodin is dissolved in DMSO to obtain a 15wt% emodin solution; and mHA is dissolved in deionized water to obtain a 10wt% mHA solution.

[0010] Furthermore, in step 2, the emodin solution, mHA solution, and TPAB-Cu 2+ The dosage ratio is 5mL:5mL:10mg.

[0011] Further, the specific steps of step 2 are as follows: dissolving emodin in dimethyl sulfoxide to obtain an emodin solution, dissolving mHA in deionized water to obtain an mHA solution, then mixing the emodin solution and the mHA solution, and adding TPAB-Cu. 2+ The mixture was sonicated for 10 minutes to form a homogeneous solution, stirred at room temperature for 6 hours, and after dialysis to remove DMSO, EM@mHA-TPAB-Cu was obtained. 2+ Composite hydrogel.

[0012] Further, the preparation method of mHA is as follows: sodium hyaluronate is dissolved in deionized water, then sodium periodate solution is slowly added, and the mixture is stirred continuously at room temperature for 4 hours. Then, ethylene glycol is added to terminate the reaction, and the mixture is stirred for another hour. The resulting mixture is dialyzed against 12 kDa MWCO for 3 days, and then freeze-dried for 24 hours to obtain mHA.

[0013] Secondly, this application provides a therapeutically integrated photoactive composite hydrogel EM@mHA-TPAB-Cu 2+ It is prepared by the preparation method described above.

[0014] Thirdly, this application provides the photoactive composite hydrogel EM@mHA-TPAB-Cu. 2+ Use in the preparation of medicaments for treating wound infections caused by drug-resistant bacteria.

[0015] Fourthly, this application provides the photoactive composite hydrogel EM@mHA-TPAB-Cu. 2+ Use in the preparation of products for diagnosing wound infections caused by drug-resistant bacteria.

[0016] In summary, this application has the following beneficial effects: This invention synthesizes a novel triphenylamine derivative (TPAB) with an A-π-D-π-A conjugated structure using TPA-CHO and MN as monomers via a simple and efficient Schiff base reaction, and further incorporates Cu... 2+ The TPAB molecule is introduced, forming a stable metal complex through coordinate bonds. Furthermore, via a Schiff base reaction, TPAB-Cu... 2+ By covalently coupling with mHA and doping with emodin via hydrogen bonding, a spatiotemporally responsive hybrid hydrogel dressing with a dynamic cross-linked network, EM@mHA-TPAB-Cu, was ultimately obtained. 2+ .

[0017] For the treatment of wounds infected with MDR bacteria, this hybrid hydrogel dressing can be activated by 400 nm laser irradiation to generate ROS that kill MDR bacteria. Subsequently, the gradual release of emodin in the hydrogel cross-linked network clears excess ROS, inhibits the expression of inflammatory cytokines, and polarizes macrophages to the M2 anti-inflammatory phenotype. Furthermore, the hybrid hydrogel dressing promotes cell proliferation, cell migration, angiogenesis, and epithelial regeneration. Additionally, this hybrid hydrogel dressing exhibits changes in fluorescence intensity when interacting with endogenous hydrogen sulfide in the microenvironment of methicillin-resistant Staphylococcus aureus (MRSA) infection, enabling in-situ monitoring of the treatment process. These combined properties make this hybrid hydrogel an ideal candidate material for treating wounds infected with MDR bacteria. Attached Figure Description

[0018] Figure 1 TPAB-Cu 2+ Molecular structure and photophysical characterization. Among them, a) 1 The 1H NMR spectrum illustrates the synthesis process of the TPAB molecule. (b) Cu 2+ Molecular structural evolution during complexation with TPAB at different molar ratios. c) Job diagram showing Cu 2+ The 2:1 stoichiometric ratio of TPAB to TPAB. d) DFT calculations of TPAB and TPAB-Cu 2+ HOMO and LUMO. e) ESR signal display TPAB-Cu2+ ROS generation under 400 nm laser irradiation. f) TPAB-Cu 2+ Fluorescence intensity changes in response to H2S. g) Linear relationship between relative fluorescence intensity (F / F0) and H2S concentration. h) Calculation of TPAB-Cu based on the Benesi-Hildebrand equation. 2+ Linear fluorescence titration curve with H2S complexation constant. i) TPAB-Cu 2+ Reversibility of the fluorescent probe's response to H2S. j) TPAB-Cu under the response of competing ions to H2S. 2+ The effect of fluorescence intensity.

[0019] Figure 2 FTIR spectra of MN, TPA-CHO, and TPAB. The FTIR spectrum of MN is shown at 3351 cm⁻¹. -1 An absorption peak for the stretching vibration of -NH appears at 3303 cm⁻¹, and it is observed at 3303 cm⁻¹. -1 and 3240cm -1 An absorption peak for the stretching vibration of -NH2 appears at 1687 cm⁻¹. For TPA-CHO, the absorption peak for the stretching vibration of -CHO appears at 1687 cm⁻¹. -1 At the site. After MN reacts with TPA-CHO, 1687 cm -1 The absorption peak of the -CHO stretching vibration at 3309 cm⁻¹ was significantly weakened, while the absorption peak of the -NH stretching vibration shifted to 3309 cm⁻¹. -1 (Blue shift). Additionally, at 1593cm... -1 A new characteristic peak corresponding to the stretching vibration of the Schiff base bond (-N=C) appeared, confirming the successful synthesis of the target product TPAB.

[0020] Figure 3 a) UV-Vis absorption spectrum of TPAB. b) Fluorescence spectrum of TPAB, with a maximum excitation wavelength of 400 nm and a maximum emission wavelength of 480 nm.

[0021] Figure 4 a) TPAB (10 μM) and Cu at different concentrations 2+ b) Fluorescence titration curves of different Cu 2+ Fluorescence intensity of the complex at 480 nm at the specified concentration.

[0022] Figure 5 a) TPAB-Cu 2+ Fluorescence titration curves of the probe (10 μM) with different concentrations of H2S. b) TPAB-Cu 2+ Fluorescence intensity at 480 nm under different H2S concentrations.

[0023] Figure 6 a) Preparation of mHA. b) The reaction of HA and mHA. 1 H NMR spectrum.

[0024] Figure 7 mHA, EM and TPAB-Cu in hydrogel 2+ A schematic diagram of intermolecular interactions between molecules.

[0025] Figure 8 :EM@mHA-TPAB-Cu 2+ Structural characterization of the hydrogel. a) EM@mHA-TPAB-Cu 2+ Macroscopic observation of hydrogel formation. b) EM@mHA-TPAB-Cu 2+ Representative photographs of the hydrogel self-healing process. c) EM@mHA-TPAB-Cu 2+ Adhesion and deformability. d) mHA, EM@mHA and EM@mHA-TPAB-Cu 2+ Rheological properties at strain rates from 0.01 to 10. e) EM, mHA, EM@mHA and EM@mHA-TPAB-Cu 2+ FTIR spectrum. f) SEM image showing EM@mHA-TPAB-Cu 2+ High-porosity microstructure. g) EDS elemental distribution of the hybrid hydrogel, showing carbon (C), oxygen (O), nitrogen (N), and copper (Cu). h) Curves of continuous EM release from the hybrid hydrogel under different pH conditions (accelerated experimental conditions). i, j) mHA, EM@mHA, and EM@mHA-TPAB-Cu 2+ ROS scavenging time-dependent curves for ABTS and DPPH radicals.

[0026] Figure 9 a) EM@mHA-TPAB-Cu 2+ XPS full spectrum scan. b) EM@mHA-TPAB-Cu 2+ Cu 2p XPS energy spectrum.

[0027] Figure 10 SEM images of mHA and EM@mHA: a) mHA and b) EM@mHA.

[0028] Figure 11 The ROS scavenging rate of the hybrid hydrogel against (a) ABTS and (b) DPPH radicals changed with prolonged incubation time. c) The hybrid hydrogel was evaluated from 0 to 32 mg / mL by the UV-Vis spectral changes of ABTS radicals. -1ROS scavenging curves within the concentration range. d) Evaluation of the hybrid hydrogel at concentrations from 0 to 7.5 mg / mL by UV-Vis spectral changes of DPPH radicals. -1 ROS scavenging curves within the concentration range.

[0029] Figure 12 :EM@mHA-TPAB-Cu 2+ Biocompatibility study of hydrogels. a, b) L929 cells and HUVECs in PBS (control group), mHA, EM@mHA, and EM@mHA-TPAB-Cu 2+ c, d) Staining results of live / dead cells on days 1, 3, and 5 under co-culture conditions. c) Quantitative analysis of cell viability of L929 cells and HUVECs under the same culture conditions using the CCK8 assay. e) Microscopic images of L929 cells after scratch assay, showing cell migration. f) Quantitative histogram of scratch assay results. g) Immunofluorescence images of L929 cells, showing staining results of type I collagen (green), F-actin (red), and cell nucleus (blue). (h) Quantitative fluorescence analysis of type I collagen expression. i) Hemolysis assay for blood compatibility assessment of mixed hydrogels. Statistical significance markers are as follows: p<0.05; p<0.01; p < 0.001; , p<0.0001.

[0030] Figure 13 :EM@mHA-TPAB-Cu 2+ In vitro antibacterial properties of the hydrogel. a, b) Representative images of bacterial colonies, live / dead staining detection, and assays with PBS, mHA, EM@mHA, and EM@mHA-TPAB-Cu. 2+ and EM@mHA-TPAB-Cu 2+ Scanning electron microscopy images of bacteria after laser treatment. Statistical analysis of colony counts and live / dead staining of (c, d) MRSA and (e, f) ESBL-E. coli. g) Reactive oxygen species (ROS) levels in MRSA (with / without laser irradiation) as detected by DCFH-DA in different treatment groups. h) Quantitative fluorescence analysis of ROS levels in each laser-treated group. i) Schematic diagram of the photodynamic antibacterial mechanism of hybrid hydrogel. ① Control group; ② mHA group; ③ EM@mHA group; ④ EM@mHA-TPAB-Cu 2+ Group; ⑤EM@mHA-TPAB-Cu 2+ Laser group.

[0031] Figure 14 :EM@mHA-TPAB-Cu 2+ In vitro anti-biofilm activity of the composite hydrogel. a) Three-dimensional CLSM images and (b, c) corresponding quantitative fluorescence results, showing the effects of PBS, mHA, EM@mHA, and EM@mHA-TPAB-Cu. 2+ and EM@mHA-TPAB-Cu 2+ Changes in fluorescence intensity of mature MRSA biofilms (stained with Syto-9 and PI) after laser treatment. (d, e) Viable cell counts and agar plate photographs of immature MRSA biofilms in each group. ① Control group; ② mHA group; ③ EM@mHA group; ④ EM@mHA-TPAB-Cu 2+ Group; ⑤EM@mHA-TPAB-Cu 2+ Laser group.

[0032] Figure 15 Z-axis superimposed three-dimensional images of MRSA biofilms after different treatment groups.

[0033] Figure 16 :EM@mHA-TPAB-Cu 2+ In vitro polarization assay of RAW264.7 macrophages induced by composite hydrogel. a, b) Immunofluorescence staining results of CD86 (M1 marker, red) and CD206 (M2 marker, green) in RAW264.7 macrophages after different treatments. c) Western blot analysis showing the expression levels of CD86 and CD206 in RAW264.7 macrophages. d) RT-qPCR detection of CD86 expression in RAW264.7 macrophages. e) RT-qPCR detection of CD206 expression in RAW264.7 macrophages. ELISA analysis f) Secretion of TNF-α and g) IL-6 cytokines in the supernatant of RAW264.7 macrophages. h) Flow cytometry detection of CD86 and CD206 expression in RAW264.7 macrophages. ① Control group; ② LPS group; ③ LPS+mHA group; ④ LPS+EM@mHA group; ⑤ LPS+EM@mHA-TPAB-Cu 2+ Group.

[0034] Figure 17 Statistical analysis of Western blot results showed the protein expression levels of a) CD86 and b) CD206 in RAW264.7 macrophages after treatment with different groups. ① Control group; ② LPS group; ③ LPS+mHA group; ④ LPS+EM@mHA group; ⑤ LPS+EM@mHA-TPAB-Cu 2+Group.

[0035] Figure 18 :EM@mHA-TPAB-Cu 2+ Transcriptome analysis of the mechanism by which composite hydrogels regulate macrophage reprogramming. a) LPS group and LPS+EM@mHA-TPAB-Cu 2+ Volcano plots of the groups. Red indicates upregulated genes, green indicates downregulated genes. b) Heatmaps show the overall expression levels of the two groups of genes. c) Bioprocess (BP) analysis and d) KEGG analysis revealed the potential pathways of differentially expressed genes. GSEA showed that LPS+EM@mHA-TPAB-Cu 2+ The number of genes enriched in the e) TNF, f) JAK-STAT and g) IL-17 pathways was significantly reduced in the group.

[0036] Figure 19 :LPS+EM@mHA-TPAB-Cu 2+ Gene network diagram of IL-17, JAK-STAT and TNF pathways in the treatment group.

[0037] Figure 20 :EM@mHA-TPAB-Cu 2+ In vivo treatment and monitoring of MRSA-infected wounds using hybrid hydrogels. a) Schematic diagram depicting the treatment and monitoring process. b) Representative photographs of wound healing in different treatment groups on days 7, 14, and 21, and corresponding wound trajectories for each group. c) Wound closure rates in different groups on days 7, 14, and 21. d) Representative images of CD68 co-stained with iNOS or Arg1 on days 3 and 7, and corresponding statistical analysis. e) Quantitative fluorescence analysis of M1 and f) M2 macrophages in tissue sections. g) Images of MRSA colonies in wound tissue cultured on agar plates on day 7. h) In vivo H2S fluorescence imaging of infected and uninfected wounds treated with hybrid hydrogels at different time points, providing a promising strategy for real-time monitoring of treatment progress. i) Quantitative fluorescence analysis evaluating the treatment process.

[0038] Figure 21 Histological analysis showed that EM@mHA-TPAB-Cu 2+The effect of composite hydrogel on accelerating tissue regeneration in MRSA-infected wounds. a) HE staining images of wound samples. b) CK14 immunofluorescence staining. c) α-SMA immunofluorescence staining at different time points in different treatment groups. d) Representative Masson staining images showing collagen deposition. e, f) Immunofluorescence staining images showing the distribution of type I and type III collagen. g) Quantitative analysis of CK14 positive areas. h) Quantitative analysis of α-SMA density among groups. i) Number of newly formed hair follicles in different treatment groups obtained by Masson staining.

[0039] Figure 22 H&E stained tissue sections of key organs such as the heart, liver, spleen, lungs and kidneys.

[0040] Figure 23 TPAB-Cu 2+ Fluorescence imaging capability of endogenous and exogenous H2S in cells. a) TPAB-Cu 2+ Bright-field and fluorescence images of L929 cells treated with different concentrations of H2S were detected. (b) TPAB-Cu 2+ Fluorescent image of stained MRSA.

[0041] Figure 24 Using a commercial sulfide detection kit, following the manufacturer's instructions, the H2S content in wound exudate collected from uninfected and infected mice was quantitatively determined.

[0042] Figure 25 :EM@mHA-TPAB-Cu 2+ Design principles and synthesis methods of composite hydrogels. Detailed Implementation

[0043] The technical solutions and effects of this application will be further described in detail below with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the invention and are not intended to limit the invention.

[0044] Example 1: Composite hydrogel EM@mHA-TPAB-Cu 2+ Preparation Step 1, TPAB-Cu 2+ Synthesis 1-1, Synthesis of TPA-CHO Triphenylamine (5.0 g, 20 mmol) was dissolved in DMF (10.0 mL), and POCl3 (9.4 mL, 102.0 mmol) was added dropwise at 0°C. The reaction mixture was stirred at room temperature until the color turned red. The solution was then heated to 60°C and reacted for 2 hours. After the reaction was complete, the mixture was cooled in ice water and neutralized with sodium bicarbonate. The precipitate was filtered off, and the solution was dried over P2O5. The product was purified by column chromatography using CHCl2 as eluent to give a yellow powder containing 90.12% N,N-bis(4-formylphenyl)aniline (TPA-CHO). 1 H NMR ( Figure 1 a, 600 MHz, DMSO-d6): δ (ppm) 9.88 (s, 2H), 7.84 (d, J=8.7 Hz, 4H), 7.47 (t, J=7.9 Hz, 2H), 7.31 (t, J=7.4 Hz, 1H), 7.24-7.20 (m, 2H), 7.17 (d, J=8.6 Hz, 4H).

[0045] 1-2, Synthesis of MN Methyl 4-aminobenzoate (6.0 g, 40.0 mmol) was added to a hydrazine hydrate solution (30.0 mL), and refluxed at 80°C for 24 hours under a nitrogen atmosphere. Then, 10.0 mL of ethanol was added and the mixture was stirred for 1 hour. After cooling to room temperature, the precipitate was filtered, recrystallized from ethanol, and dried under vacuum to give a white crystalline product. The yield of 4-aminophenylhydrazine (MN) was 89.21%. 1 HNMR ( Figure 1 a, 600 MHz, DMSO-d6): δ (ppm) 9.26 (s, 1H), 7.54 (d, J=8.8 Hz, 2H), 6.53 (d, J=7.8 Hz, 2H), 5.56 (d, J=7.4 Hz, 2H), 4.28 (s, 1H).

[0046] 1-3, Synthesis of TPAB The TPA-CHO (0.3013 g, 1.0 mmol) obtained in step 1 and the MN (0.3345 g, 2.0 mmol) obtained in step 2 were dissolved in ethanol (20.0 mL), refluxed at 80°C for 8 hours under a nitrogen atmosphere, the precipitate was filtered, and recrystallized with anhydrous ethanol to obtain a yellow powder. The yield of TPAB was 59.89%. 1 H NMR ( Figure 1a, 600 MHz, DMSO-d6): δ (ppm) 10.03 (s, 2H), 8.10 (s, 2H), 7.83 (d, J=8.7 Hz, 4H), 7.56 (d, J=7.7 Hz, 4H), 7.37 (d, J=7.6Hz, 4H), 7.35 (s, 1H), 7.19 (d, J=8.2 Hz, 2H), 7.13 (d, J=8.5 Hz, 2H), 7.03 (d, J=8.7 Hz, 4H).

[0047] 1-4, TPAB-Cu 2+ Synthesis The TPAB (56.76 mg, 0.1 mmol) obtained in step 3 was dissolved in ethanol, followed by the addition of Cu(NO3)2 powder (M(Cu 2+ (M(TPAB) = 1:1 or 2:1 or 3:1), stirred at room temperature for 24 hours, dried by rotary evaporation, the resulting product was dissolved in CHCl2, and then extracted three times with CHCl2 and water (1:1, v / v). The purified solution was dried by rotary evaporation and further dried under vacuum to obtain TPAB-Cu. 2+ . 1 H NMR ( Figure 1 b, 600 MHz, DMSO-d6): δ (ppm) 9.83 (s, 2H), 8.00 (s, 2H), 7.8 (d, J=8.7 Hz, 4H), 7.52 (d, J=7.7 Hz, 4H), 7.31 (s, 1H), 7.03 (d, J=8.7 Hz, 4H).

[0048] Step 2: Synthesis of mHA 0.4 g of sodium hyaluronate was dissolved in 400 mL of deionized water, followed by slow addition of sodium periodate solution (3 mL, 0.5 M). The mixture was stirred continuously at room temperature for 4 hours, then 3 mL of ethylene glycol was added to terminate the reaction, and stirring was continued for 1 hour. The resulting mixture was dialyzed against 12 kDa MWCO for 3 days, followed by freeze-drying for 24 hours to obtain mHA. The 1H NMR results are as follows: Figure 6 As shown.

[0049] Step 3: EM@mHA-TPAB-Cu 2+ Preparation of composite hydrogels Emodin was dissolved in dimethyl sulfoxide to obtain an emodin solution, and mHA was dissolved in deionized water to obtain an mHA solution. Then, the emodin solution (5 mL, 15 wt%) and the mHA solution (5 mL, 10 wt%) were mixed, and TPAB-Cu was added. 2+(10 mg), the mixture was sonicated for 10 minutes to form a homogeneous solution, stirred at room temperature for 6 hours, and after dialysis to remove DMSO, EM@mHA-TPAB-Cu was obtained. 2+ Composite hydrogels. The hydrogel structure is as follows: Figure 7 As shown.

[0050] Example 2: Performance Testing Experimental methods: (1) EM from EM@mHA-TPAB-Cu 2+ Release in hybrid hydrogels: The release behavior of EM in hybrid hydrogels was evaluated using UV-Vis spectrophotometry based on standard absorption curves. Hydrogels (30 mg) were encapsulated in dialysis bags (MWCO 2 kDa) and immersed in PBS solutions at pH 6, 7, and 8, respectively. At predetermined time intervals, 0.5 mL of PBS solution was taken for UV-Vis absorption analysis, and an equal volume of fresh medium was added.

[0051] (2) ROS removal capability assessment: ABTS+· and DPPH· were used as indicators to evaluate the ROS scavenging activity of the mixed hydrogel. For the ABTS assay, the ABTS+· working solution was prepared by mixing ABTS (1 mL, 7.4 mM) aqueous solution with K2S2O8 (1 mL, 2.6 mM aqueous solution) and storing in the dark for 12 hours. Subsequently, the mixed hydrogel was added sequentially to 1200 μL of PBS (pH=7.4) with 20 μL of the ABTS+· working solution and incubated at 37°C for different times. For the DPPH assay, the mixed hydrogel was mixed with DPPH ethanol solution (1 mL, 50 μg / mL)... -1 Mix the solutions and add anhydrous ethanol to a total volume of 1.5 mL. Incubate the solution in the dark for different times. The reduction degree of ABTS+· and DPPH· is quantified by measuring the absorbance changes at 734 nm and 517 nm, respectively. The scavenging effect of the hydrogel on ABTS free radicals and DPPH free radicals is calculated using the following formula: Clearance rate (%) = (A0 - A) / A0 × 100%; Where A0 represents the initial absorbance of ABTS+ at 734 nm or DPPH+ at 517 nm, and A represents the absorbance after incubation of the working solution with the hydrogel for different times. ROS scavenging experiments for other groups were performed using the same method as for the hybrid hydrogels. For experiments with different concentrations of hybrid hydrogels, their ROS scavenging activity was evaluated by incubating the hydrogel with the working solution for 1 hour.

[0052] (3) EM@mHA-TPAB-Cu 2+Cytotoxicity studies with HUVECs: The experiment used a high-glucose medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin) to culture cells. mHA, EM@mHA, and EM@mHA-TPAB-Cu were used as the inoculants. 2+ The solution was filtered through a 0.22 μm filter membrane and then diluted to 20 mg / mL with the corresponding cell culture medium. The toxicity of the two hydrogels to L929 and HUVEC cells was assessed using the CCK-8 assay. Cells were cultured at 6 × 10⁶ cells / mL. 3 Seeds were planted at a density of cells / well in 96-well plates. After 24 hours of incubation, the medium was replaced with a medium containing 100 μL of hydrogel (control group, mHA group, EM@mHA group, EM@mHA-TPAB-Cu). 2+ The samples were cultured in groups for 1, 3, and 5 days, respectively. After the culture was completed, the culture medium was removed, 10 μL of CCK-8 reagent was added to each well, and the samples were incubated at 37°C for 1 hour. The absorbance was then measured at a wavelength of 450 nm.

[0053] (4) Cell staining and cell migration experiments: Cell viability was further assessed using live / dead cell staining and cell migration assays. Cells were cultured in drug-containing media (control group, mHA, EM@mHA, EM@mHA-TPAB-Cu). 2+ After treatment for 1, 3 and 5 days, L929 and HUVEC cells were stained with calcein AM and propidium iodide (PI) for 30 minutes and observed under a fluorescence microscope (Olympus CKX53).

[0054] In the migration experiment, L929 cells were loaded at a rate of 2 × 10⁻⁶. 4 Cells were seeded at a density of [number] cells / well in 6-well plates and incubated for 24 hours. Vertical scratches were made in each well using a 200 μL pipette tip, followed by three washes with PBS to remove non-adherent cells. Cells were then cultured in a hydrogel-containing medium. Cell migration was monitored at 0, 6, 12, and 24 hours using fluorescence microscopy, and the migration area was quantified using ImageJ software. The migration rate was calculated using the following formula: Migration rate (%) = [(A1) A2) / A1] × 100%; Where A1 represents the initial scratch area and A2 represents the remaining area after cultivation.

[0055] L929 cell type I collagen immunofluorescence staining experiment: L929 cells were injected with 1×10⁻⁶ cells... 4 Cells were seeded at a density of 1:1 in 96-well plates and incubated for 24 hours. Subsequently, different experimental groups (control group, mHA group, EM@mHA group, EM@mHA-TPAB-Cu) were used. 2+Cells were treated with [group A] and incubated for another 24 hours. After removing the culture medium, the cells were washed three times with PBS. Cells were fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes, and blocked with QuickBlock™ blocking buffer at room temperature for 1 hour. Primary antibody against type I collagen was added, and the cells were incubated at 4°C for 24 hours, followed by incubation with secondary antibody at room temperature in the dark for 2 hours. Cells were then stained with TRITC phalloidin at room temperature for 1 hour, washed three times with PBS, and finally stained with DAPI for 30 minutes. After a final PBS wash, the cells were observed under a fluorescence microscope.

[0056] (5) Hemolysis test: Blood (1 mL) was collected from the rat tail vein and centrifuged at 1000 rpm. The supernatant was discarded, and the red blood cells were washed three times with PBS. 0.5 mL of the red blood cell suspension was then mixed with 0.5 mL of pure water, PBS, mHA, EM@mHA, or EM@mHA-TPAB-Cu, respectively. 2+ Mix. Incubate the mixture at 37°C for 1 hour. After incubation, centrifuge at 1000 rpm and measure the absorbance of the supernatant at 542 nm using a UV-Vis spectrophotometer.

[0057] (6) In vitro antibacterial activity assessment: MRSA was cultured overnight in LB medium at 37°C and 220 rpm with shaking until the logarithmic growth phase. Then, 1×10⁻⁶ cells / mL were added. 8 CFU / mL bacterial suspension was mixed with control group (PBS), mHA, EM@mHA, and EM@mHA-TPAB-Cu, respectively. 2+ and EM@mHA-TPAB-Cu 2+ Laser (400 nm, 0.05 W cm⁻¹) -2 The samples were incubated for 1 hour (5 minutes). Collected MRSA cells were stained using a live / dead bacterial viability assay kit (SYTO 9 and PI) according to the manufacturer's instructions. Bacterial viability was observed using fluorescence microscopy, and MRSA morphology was detected using scanning electron microscopy (SEM). After treatment, the MRSA suspension was centrifuged, washed with PBS, and fixed with 2.5% glutaraldehyde solution at 4°C for 12 hours. Samples were dehydrated by a gradient of ethanol, replaced with tert-butanol, freeze-dried, and labeled with gold before SEM observation. For bacterial counting, the MRSA suspension was serially diluted, inoculated onto agar plates, and incubated for 24 hours. The number of viable bacteria was quantified according to the Chinese national standard GB / T 4789.2. The reactive oxygen species (ROS) generation capacity of the hydrogel was assessed using the DCFH-DA probe: DCFH-DA was added to each sample after 1 hour of incubation, and the samples were incubated at 37°C in the dark for 30 minutes. The amount of ROS generated was detected by fluorescence microscopy.

[0058] (7) In vitro antibacterial biofilm activity experiment: To establish a mature bacterial biofilm, 1% glucose (500 μL / mL) will be added. -1 LB medium was added to a confocal compatible culture plate, followed by inoculation with a bacterial suspension derived from a single colony (approximately 1 × 10⁻⁶). 7 CFU mL -1 The culture was incubated at 37 °C for 2 days, with fresh medium replaced daily, eventually forming a mature biofilm. In the biofilm disruption experiment, different treatments (500 μg / mL) were applied to the formed biofilm. -1 ), including control group (PBS), mHA, EM@mHA, EM@mHA-TPAB-Cu 2+ and EM@mHA-TPAB-Cu 2+ Laser (400 nm, 0.05 W cm⁻¹) -2 (5 minutes). After treatment, the culture plates were incubated at 37 °C for 24 hours. The plates were gently washed twice with PBS to remove unbound cells and residual reagents. The biofilms were then stained using a live / dead bacteria viability assay kit (SYTO and PI) according to the manufacturer's instructions. Fluorescence images were acquired using a confocal laser scanning microscope (CLSM), and the three-dimensional structure of the biofilms was reconstructed for analysis.

[0059] (8) In vitro immunomodulation experiments: RAW264.7 macrophages (5 × 10⁻⁶) 5 Cells were seeded in 6-well plates and cultured overnight. Cells were stimulated with LPS (500 ng / mL) for 1 hour, with untreated RAW264.7 cells serving as a control. Subsequently, cells were treated with PBS, mHA, EM@mHA, and EM@mHA-TPAB-Cu, respectively. 2+ Macrophages were treated for 24 hours. After centrifugation, cells were washed and lysed using RIPA lysis buffer. The levels of inflammatory cytokines in the supernatant were detected using an ELISA kit. The remaining cells were assessed for macrophage polarization using flow cytometry. After washing, cells were resuspended in 500 μL PBS containing FITC-labeled anti-mouse CD86 antibody and PE-labeled anti-mouse CD206 antibody. Samples were incubated at 4°C in the dark for 30 minutes, and surface marker expression was analyzed using a BD FACSCanto II flow cytometer.

[0060] (9) Transcriptomics analysis and RT-qPCR: To investigate EM@mHA-TPAB-Cu 2+To investigate the effect of treatment on macrophage gene expression, mRNA sequencing technology (mRNA-seq) was used in this study. Macrophages were seeded in 6-well plates and pre-stimulated with lipopolysaccharide (500 ng / mL) for 1 hour to induce immune imbalance, and then treated with PBS (control group) or EM@mHA-TPAB-Cu 2+ (experimental group) for 24 hours. Total mRNA was extracted using chloroform lysis method, and three biological replicates were set for each group. Sequencing and bioinformatics analysis were completed by Shanghai Bioanalysis Co., Ltd. To verify the transcriptomics results, the mRNA expression levels of markers (CD86 and CD206) related to M1 macrophage polarization before and after treatment were quantitatively detected by RT-qPCR. All experiments were set with three replicates to ensure the reproducibility of the results.

[0061] (10)Evaluation of the expression of genes related to in vitro angiogenesis: Human umbilical vein endothelial cells (HUVECs) were co-cultured with each experimental group. Cell proteins were extracted using a BCA protein assay kit, separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% bovine serum albumin (BSA) for 1 hour, and then incubated with primary antibodies against VEGF, VEGFR, phosphorylated Akt and actin. After incubation with the secondary antibody for 30 minutes, the protein signals were developed using an enhanced chemiluminescence detection kit. Finally, the band optical density was quantitatively analyzed using ImageJ software.

[0062] (11)In vivo evaluation of infectious wound healing: Full-thickness skin defects (about 10 mm in diameter) were created on the backs of 8-week-old male BALB / c mice. The wounds were infected with MRSA (1×10 8 CFU·mL -1 , 50 μL) for 48 hours. On day 0 and day 5, the infected wounds were treated with the control group (PBS), mHA, EM@mHA and EM@mHA-TPAB-Cu 2+ laser (400 nm, 0.05 W·cm -2 , for 5 minutes). On day 7, wound tissues of each experimental group were collected and ground, and the bacterial suspension was spread on LB agar plates after centrifugation. The wound healing was monitored using a digital camera on days 7, 14 and 21, and the data were analyzed using Image J software. The wound healing rate was calculated as: (A0 - A t ) / A0 × 100%, where A0 and A t represent the initial wound area and the remaining wound area at each time point, respectively.

[0063] ((12) Histological and immunohistochemical analysis: Mice were sacrificed at a preset time point, tissues were collected and fixed with 4% paraformaldehyde (PFA) for 24 hours. Subsequently, histological and immunohistochemical evaluations were performed. Hematoxylin-eosin (H&E) staining and Masson's trichrome staining were used to evaluate the pathological status and wound healing. Collagen deposition (type I and type III collagen), epithelialization (CK-14), and neovascularization (α-SMA) during wound healing were detected by immunofluorescence staining. To further explore the macrophage immune response at different healing stages, immunofluorescence staining of inducible nitric oxide synthase (iNOS), arginase-1 (Arg1), and CD68 was also performed. All procedures were carried out according to the manufacturer's guidelines. Fluorescent images were acquired using a fluorescence microscope.

[0064] (13)Fluorescent monitoring of hydrogen sulfide 1) Fluorescent titration experiment: Sodium hydrosulfide (a donor of hydrogen sulfide) was used for in vitro detection. In an aqueous solution, sodium hydrosulfide dissociates into Na + and HS - and then reacts with H + to generate hydrogen sulfide. First, a 10 mM DMSO stock solution of TPAB-Cu 2+ was prepared. 3 μL of this solution was mixed with 100 μL of phosphate buffer saline (PBS). Subsequently, different concentrations of sodium hydrosulfide solutions were added, and the final volume was adjusted to 3 mL with DMSO. After the addition of different concentrations of sodium hydrosulfide, the fluorescence emission spectra of TPAB-Cu 2+ were recorded at an excitation wavelength of 400 nm. A linear detection curve was obtained through the Stern-Volmer equation, and the limit of detection (LOD) was calculated using the IUPAC formula.

[0065] LOD=3σ / K where σ represents the standard deviation of the fluorescence intensity measurements of 20 TPAB -Cu 2+ samples (10 µM), and K is the slope of the linear equation.

[0066] The complexation constant (Ka) of TPAB-Cu 2+ with hydrogen sulfide was calculated using the Benesi-Hildebrand equation:

[0067] where F0 represents the fluorescence intensity of TPAB-Cu 2+ at 10 µM, F represents the fluorescence intensity of TPAB-Cu 2+ after the addition of different concentrations of hydrogen sulfide, and Fmax represents the fluorescence intensity of TPAB-Cu 2+The saturated fluorescence intensity bound to hydrogen sulfide, where [M] represents the hydrogen sulfide concentration. This is illustrated by plotting 1 / [H₂S] on the x-axis and 1 / [F-F₀] on the y-axis. Figure 1 The complexation constant (K) was determined. a ).

[0068] To evaluate TPAB-Cu 2+ The selectivity of the fluorescent probe was demonstrated by using 10 µM TPAB-Cu. 2+ The solution contained sodium hydrosulfide (10 µM) and several competing molecules / ions (L-cysteine, DL-homocysteine, glutathione, S2O3). - HSO3 - S2O8 2- Cr2O7 - PO4 3- HPO4 - NO 2- The mixture was prepared at a concentration of 100 µM. Fluorescence spectra were recorded at room temperature.

[0069] 2) Imaging of hydrogen sulfide in MRSA: A single MRSA colony was picked up using an inoculation loop and added to 5 mL of LB broth. The culture was incubated with shaking at 37 °C for 4 hours, with a final bacterial concentration of approximately 1.0 × 10⁻⁶. 8 CFU mL -1 After centrifuging the bacterial suspension, wash three times with PBS buffer. Then, transfer 200 µL of the suspension to a 96-well plate and add 10 µL of TPAB-Cu. 2+ Incubate at 10 µM for different time periods. Add 10 µL of bacterial suspension to a glass slide and gently cover with a coverslip to avoid air bubbles. Finally, image the bacteria using an inverted fluorescence microscope.

[0070] 3) Imaging of hydrogen sulfide in living cells: L929 cells were imaged at 1.0 × 10⁻⁶. 4 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 hours. The plates were then replaced with TPAB-Cu. 2+ Fresh culture medium (10 µM) was added, with a total volume of 100 µL. Cells were incubated at 37 °C and 5% CO2 for 30 minutes, followed by washing three times with PBS. Different concentrations of sodium hydrosulfide (5 µM, 10 µM, and 20 µM) were then added, and the cells were incubated at 37 °C for another 30 minutes. After washing three more times with PBS, fluorescence images were acquired using a fluorescence microscope.

[0071] Statistical analysis: Quantitative results were based on at least five samples per group, with each experiment independently repeated at least three times. Results are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8.0 software, employing either one-way or two-way ANOVA. A p-value <0.05 was considered statistically significant. Statistical significance is indicated in the legend as follows: P<0.05, P<0.01, P<0.001, P<0.0001.

[0072] Experimental results: (1) EM@mHA-TPAB-Cu 2+ Synthesis and Characterization of Hydrogels In this embodiment, methyl hyaluronic acid (mHA) was successfully prepared by oxidizing HA with sodium periodate. Figure 6 a) of mHA 1 The presence of a new proton peak near 5.0 ppm in the H NMR spectrum confirms the presence of the aldehyde group, indicating the successful synthesis of aldehyde-functionalized HA. Figure 6 (b) Then, the natural herbal emodin was added to the mHA to obtain a uniform yellow solution. Figure 8 (a) Finally, add TPAB-Cu to the mixture. 2+ After stirring continuously for 1 hour, EM@mHA-TPAB-Cu was formed. 2+ Hybrid hydrogel. The gel network consists of TPAB-Cu. 2+ The Schiff base reaction between mHA and ( ) forms ( Figure 7 Thanks to the reversible nature of dynamic Schiff base bonds, the resulting hydrogel exhibits significant self-healing capabilities. Figure 8 (b) Even when cut into multiple pieces, it can quickly return to its original shape. Furthermore, this hybrid hydrogel exhibits excellent adhesion properties, which is of significant value for wound dressing applications. Figure 8 (c) Rheological analysis showed that, compared with liquid mHA and EM@mHA, EM@mHA-TPAB-Cu 2+ The hydrogel exhibits solid-like behavior under low strain (<2%), with its storage modulus exceeding its loss modulus, indicating the formation of a stable cross-linked network. Figure 8 (d) This is attributed to the combination of methhydroxyacrylate (mHA) and TPAB-Cu. 2+The covalent crosslinking between them enhances the structural stability of the hydrogel. In addition, this hybrid hydrogel can be injected through a syringe, enabling it to adapt to irregular and deep wound defects. It can act as a microbial protection barrier while maintaining an optimal moist environment, thus promoting wound healing.

[0073] The molecular structure of the hybrid hydrogel was characterized by Fourier transform infrared spectroscopy (FTIR) ( Figure 8 in e). In the spectrum of EM@mHA-TPAB-Cu 2+ , the characteristic C=O stretching vibration peak of mHA (1643 cm -1 ) disappeared, and at the same time, a new absorption band appeared at about 1604 cm -1 , corresponding to the C=N bond. This spectral change confirmed the formation of Schiff base bonds between mHA and TPAB-Cu 2+ . X-ray photoelectron spectroscopy (XPS) further confirmed the successful construction of EM@mHA-TPAB-Cu 2+ ( Figure 9 ). The morphology of EM@mHA-TPAB-Cu 2+ was observed by scanning electron microscopy (SEM). As shown in f in Figure 8 , this hybrid hydrogel presented an interconnected honeycomb-like three-dimensional network structure. Due to a higher crosslinking density, compared with mHA and EM@mHA, this hybrid hydrogel exhibited a more stable structure and smaller pore sizes ( Figure 10 ). The high porosity of EM@mHA-TPAB-Cu 2+ can enhance the reactive oxygen species (ROS) response ability and promote the subsequent delivery of EM. Related energy spectrum analysis (EDS) showed that the porous framework of the hybrid hydrogel contained carbon, nitrogen, oxygen, and copper elements, indicating a strong interaction between modified hyaluronic acid (mHA) and TPAB-Cu 2+ ( Figure 8 in g).

[0074] Due to the accumulation of acidic by-products generated by bacterial metabolism and pus, the microenvironment of infected wounds is usually acidic in the early stage. As shown in h in Figure 8 , the hybrid hydrogel gradually released EM over time under different pH conditions. Notably, compared with the alkaline condition, the EM release rate was significantly faster in acidic and neutral environments, which helped to effectively scavenge excessive reactive oxygen species (ROS) in the inflammatory microenvironment. Studies have shown that timely scavenging of ROS in the inflammatory microenvironment can promote the polarization of macrophages to the M2 phenotype, thus accelerating the wound healing process. The ROS scavenging ability of this hybrid hydrogel was evaluated by measuring the consumption of ABTS and DPPH free radicals. As shown in Figure 8As shown in i, compared with the mHA group, EM@mHA and EM@mHA-TPAB-Cu 2+ ABTS in the group +· The absorbance at 734 nm gradually decreased over time. Figure 11 (a, c) Similarly, within 8 hours, EM@mHA and EM@mHA-TPAB-Cu2+ hydrogel effectively removed DPPH. · ( Figure 8 j in Figure 11 (b, d in the original text). These results indicate that the EM component significantly enhances the reactive oxygen species scavenging capacity of the spatiotemporally responsive hybrid hydrogel, thereby promoting macrophage polarization to the M2 phenotype and facilitating tissue regeneration.

[0075] (2) EM@mHA-TPAB-Cu 2+ Biocompatibility of hydrogels Excellent biocompatibility of hydrogel dressings is crucial for effective wound management. Fibroblasts (L929) play a vital role in wound healing, and their migration and functional alterations influence tissue regeneration. Endothelial cells (HUVECs) participate in angiogenesis and make significant contributions to the healing process. The biocompatibility of spatiotemporally responsive hybrid hydrogels was assessed using live / dead cell staining and CCK-8 assays. L929 and HUVEC cells were respectively treated with PBS (control group), mHA, EM@mHA, and EM@mHA-TPAB-Cu. 2+ The culture was carried out for 1, 3, and 5 days in total. (Example) Figure 12 As shown in figures a and b, the cell count in all groups increased over time, indicating that the hydrogel has excellent biocompatibility. CCK-8 assays further confirmed the proliferative activity of cells in all groups, with EM@mHA-TPAB-Cu showing the highest cell count. 2+ Hybrid hydrogels showed the most favorable effect on cell proliferation. Figure 12 (c, d in the original text). To further investigate the biological behavior of fibroblasts in response to hybrid hydrogels, scratch assays and collagen secretion assays were performed on L929 cells. Figure 12 As shown in e, after co-culturing for 0, 6, 12, and 24 hours, the cell scratch area gradually decreased in all groups. EM@mHA-TPAB-Cu 2+ The cell migration rates of the groups were 3.9 times, 1.7 times, and 1.2 times that of the control group, mHA group, and EM@mHA group, respectively, indicating that the designed components in the hybrid hydrogel are beneficial for promoting cell migration. Figure 12 (f) Fibroblasts are the main cells that synthesize and secrete type I collagen during wound healing. Early collagen synthesis is crucial for tissue remodeling and scar reduction. Immunofluorescence staining showed mHA, EM@mHA, and EM@mHA-TPAB-Cu2+ The L929 cells in the group secreted more type I collagen than those in the control group. Figure 12 (g in the text). Quantitative fluorescence intensity analysis further confirmed that the mixed hydrogel can effectively promote collagen secretion by fibroblasts (g in the text). Figure 12 (h in the original text). The blood compatibility of this hybrid hydrogel was further evaluated using a hemolysis test. mHA, EM@mHA, and EM@mHA-TPAB-Cu 2+ The hemolysis rate in all groups was less than 3%, indicating that it has excellent blood compatibility. Figure 12 (i) These results indicate that EM@mHA-TPAB-Cu 2+ Hybrid hydrogels exhibit excellent biocompatibility and are potential candidate materials for wound healing management.

[0076] (3) EM@mHA-TPAB-Cu 2+ In vitro antibacterial properties of hydrogels Effective antibacterial treatment is crucial for preventing the worsening of infected wounds. The newly developed photosensitizer TPAB-Cu2+ exhibits excellent photodynamic activity. To evaluate the antibacterial efficacy of the spatiotemporally responsive hybrid hydrogel, we conducted in vitro experiments against Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative extended-spectrum β-lactamase-producing Escherichia coli (ESBL-E. coli). MRSA was reacted with PBS, mHA, EM@mHA, and EM@mHA-TPAB-Cu2+, respectively. 2+ (No laser) and EM@mHA-TPAB-Cu 2+ Laser (400 nm, 0.05 W cm⁻¹) -2 Incubate for a total of 1 hour (5 minutes), followed by bacterial counting, live / dead staining, and scanning electron microscopy imaging. Figure 13 (af) in the text. Figure 4 As shown in a, EM@mHA and EM@mHA-TPAB-Cu 2+ (Without laser) No significant antibacterial activity was shown. However, after photoactivation, EM@mHA-TPAB-Cu 2+ The hydrogel almost completely killed MRSA, demonstrating a strong photodynamic bactericidal effect. Colony forming unit (CFU) analysis confirmed a significant reduction in the number of viable bacteria. Figure 13 c). Live / dead staining further revealed the effects of laser irradiation on EM@mHA-TPAB-Cu. 2+ The bacterial membrane structure in the group was severely damaged, with yellow fluorescence (a superposition of green and red signals) being the dominant color. Figure 13 (a) Quantitative analysis showed that 93% of the bacteria in this group were PI-positive (dead) ( Figure 13 (d in the text)

[0077] The study also observed that the material exhibited sustained antibacterial effects against ESBL-E. coli, highlighting the broad-spectrum antibacterial properties of the mixed hydrogel. Figure 13 (b, e, f in the image). Scanning electron microscopy images confirmed that photodynamic therapy can induce structural damage in MRSA and ESBL-E. coli, including cell deformation, shrinkage, and membrane rupture. Figure 13 (a, b) These morphological changes stem from ROS disrupting bacterial membrane integrity, ultimately leading to bacterial death. To elucidate the antibacterial mechanism, researchers used DCFH as an indicator of ROS generation—this substance is oxidized to the green fluorescent substance DCF under the action of ROS. Figure 13 As shown in g, no obvious green fluorescence was detected in the unirradiated group, PBS, mHA, and EM@mHA under light illumination. However, the laser-irradiated EM@mHA-TPAB-Cu group... 2+ The hydrogel exhibited strong DCF fluorescence, confirming its ROS generation efficiency ( Figure 13 (h in the text). This photodynamic activity originates from TPAB-Cu. 2+ Photosensitizers promote the generation of various reactive oxygen species (ROS) through type I and type II photoreactions, thereby effectively killing bacteria under both normoxic and hypoxic conditions. Figure 13 (i in the text).

[0078] In chronically infected wounds, drug-resistant bacteria often form biofilms composed of extracellular polymeric substances. These substances act as barriers to antibiotic penetration, leading to persistent infection and delayed healing. Therefore, this study evaluated the anti-biofilm ability of spatiotemporally responsive hydrogels. Biofilm structure and bacterial activity were analyzed using live / dead cell staining and confocal laser scanning microscopy (CLSM). Figure 14 (a) The reconstructed three-dimensional biofilm model shows that PBS, mHA, EM@mHA, and EM@mHA-TPAB-Cu 2+ The (untreated) group retained complete biomembrane structures. In contrast, the laser-irradiated EM@mHA-TPAB-Cu group... 2+ The hydrogel significantly disrupted the biofilm structure and cleared embedded methicillin-resistant Staphylococcus aureus (MRSA). Figure 14 (b and c in the text). It is noteworthy that the biofilm thickness was significantly reduced after treatment. Figure 15 This is attributed to reactive oxygen species (ROS)-mediated degradation of the biofilm matrix. To verify the bacterial clearance effect in the biofilm, MRSA ( ) was extracted and quantified by CFU detection. Figure 14 (d) Laser-irradiated EM@mHA-TPAB-Cu 2+ The group showed a significant reduction in bacterial load compared to all other groups. Figure 14(e). In summary, these findings highlight EM@mHA-TPAB-Cu 2+ The superior antibacterial and antibiofilm properties of hybrid hydrogels suggest their potential application in treating infected wounds, especially those involving multidrug-resistant pathogens.

[0079] (4) EM@mHA-TPAB-Cu 2+ In vitro immunomodulatory properties of hydrogels Macrophages, as innate immune cells, play a central role in regulating the inflammatory response during wound healing. Classically activated M1 macrophages exhibit pro-inflammatory activity, while alternatively activated M2 macrophages participate in anti-inflammatory responses and tissue repair. In the early stages of chronic wound healing, macrophages primarily polarize towards the M1 phenotype, secreting various pro-inflammatory cytokines. Persistent infiltration of M1 macrophages exacerbates local inflammation and hinders tissue regeneration. Therefore, promoting macrophage polarization from the M1 to the M2 phenotype is considered a promising strategy for suppressing excessive inflammation and accelerating wound healing. Given the inherent anti-inflammatory properties of the natural compound emodin, we hypothesize EM@mHA-TPAB-Cu 2+ Hydrogels can promote macrophage polarization from the M1 to the M2 phenotype. To verify the immunomodulatory capacity of this spatiotemporally responsive hydrogel, RAW264.7 macrophages were first polarized to the M1 phenotype by lipopolysaccharide (LPS) stimulation, and then co-cultured with the hydrogel. Immunofluorescence staining of macrophage markers CD86 (M1) and CD206 (M2) showed that the hydrogel containing EM significantly downregulated CD86 expression and upregulated CD206 levels, indicating successful polarization to the M2 phenotype. Figure 16 (a, b) Western blot analysis further validated the expression levels of CD86 and CD206, and the results were consistent with the trends of immunofluorescence detection data, indicating that emodin plays a central role in macrophage polarization. Figure 16 c in the middle; Figure 17 Furthermore, reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis showed that in macrophages treated with emodin-loaded hydrogels, CD86 mRNA was significantly downregulated while MRC1 mRNA encoding CD206 was upregulated, confirming a phenotypic shift at the transcriptional level. Figure 16 (d, e). Enzyme-linked immunosorbent assay (ELISA) results further confirmed that the hydrogel containing EM significantly inhibited the secretion of M1-related pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Figure 16 (f, g in the original text). Finally, the immunomodulatory effects of this hybrid hydrogel were quantitatively assessed using flow cytometry. Figure 16 As shown in h, after EM@mHA-TPAB-Cu 2+After hydrogel treatment, CD86 + The proportion of M1 macrophages decreased from 40.6% to 18.9%, while the proportion of CD206+ M2 macrophages increased from 6.9% to 22.3%. These results indicate that the hybrid hydrogel can effectively inhibit the expression of pro-inflammatory cytokines and promote macrophage polarization towards an anti-inflammatory phenotype, highlighting its therapeutic potential in regulating inflammation and promoting wound healing.

[0080] To investigate the immunomodulatory and anti-inflammatory mechanisms of the hybrid hydrogel, we studied LPS-induced M1 polarized macrophages treated with PBS and EM@mHA-TPAB-Cu 2+ Transcriptome sequencing (RNA-seq) was performed on the hydrogel-treated groups. Differentially expressed genes (DEGs) were identified based on a statistical threshold of p < 0.05 and |log2 (fold change)| ≥ 1. Compared with the PBS control group, the mixed hydrogel treatment group resulted in the upregulation of 621 genes and the downregulation of 1375 genes. Figure 18 (a, b) Gene ontology (GO) analysis showed that DEGs were significantly enriched in biological processes such as immune regulation, inflammation suppression, reactive oxygen species (ROS) regulation, and tissue regeneration, suggesting that this hybrid hydrogel has multifaceted regulatory capabilities. Figure 18 c). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes (DEGs) revealed significant enrichment in multiple metabolic and signaling pathways, primarily involving the IL-17, JAK-STAT, and TNF-α pathways. These pathways play crucial roles in inflammation regulation, immune response, cell proliferation, and tissue regeneration throughout the wound healing process. Figure 18 (d in the text). The gene interaction network map comprehensively demonstrates the functional connections between these key pathways. Figure 19 Notably, genes associated with the IL-17, JAK-STAT, and TNF-α signaling pathways were observed in EM@mHA-TPAB-Cu 2+ Significantly downregulated in the group ( Figure 18 (eg), indicating that hydrogels can inhibit pro-inflammatory signaling and promote macrophage polarization toward the pro-regenerative M2 phenotype.

[0081] (5) EM@mHA-TPAB-Cu 2+ In vivo evaluation of hydrogels for the treatment of infected wounds To investigate the therapeutic effects of spatiotemporally responsive hybrid hydrogels in vivo, we established a full-thickness MRSA-infected wound model. The wound model construction, processing, and evaluation process are as follows: Figure 20 As shown in a, MRSA-infected wounds were treated with PBS (control group), mHA, EM@mHA, and EM@mHA-TPAB-Cu on days 0 and 5, respectively.2+ Treatment. All treatment groups were exposed to light (400 nm, 0.05 W cm⁻², 5 minutes). The wound healing process was observed, and tissue samples were collected at different time points for histological analysis. Figure 20 As shown in b, the healing trajectories of each group are similar but their rates differ. It is noteworthy that EM@mHA-TPAB-Cu 2+ The group showed significantly accelerated healing, with the wound area reduced to only 7.1% of its original size by day 21. Figure 20 (c) This highlights the remarkable therapeutic potential of this multifunctional hydrogel against MDR bacterial infections.

[0082] To further evaluate the inflammatory modulatory properties of this hybrid hydrogel, we investigated the biological behavior of macrophages in wound tissue across all treatment groups. To assess this process, M1 and M2 macrophages in wound tissue were stained with CD68 / inducible nitric oxide synthase (iNOS) and CD68 / arginase-1 (Arg1) fluorescently labeled antibodies on days 3 and 7, respectively. In contrast, on day 7, EM@mHA-TPAB-Cu 2+ The expression level of Arg1, a marker of M2, in the hybrid hydrogel group was significantly higher than that in the PBS group, mHA group, and EM@mHA group, indicating that the hybrid hydrogel can effectively promote the polarization of macrophages from the M1 phenotype to the M2 phenotype in wound tissue. Figure 20 f). To assess the antibacterial effect, MRSA strains were collected from wound tissue on day 7. Figure 20 g in EM@mHA-TPAB-Cu 2+ The group achieved near-complete MRSA removal at the wound site, significantly outperforming all other groups, fully demonstrating its powerful photodynamic antibacterial effect.

[0083] Given TPAB-Cu 2+ Fluorescence imaging capability for exogenous and endogenous hydrogen sulfide in cells ( Figure 23 We used an in vivo fluorescence imaging system to monitor the spatiotemporal dynamics of hydrogen sulfide levels at infected wound sites. By combining in vivo fluorescence imaging with quantitative biochemical analysis, we conducted a systematic and time-resolved study of changes in endogenous H2S levels during treatment. Related monitoring experiments were performed on days 0, 3, 5, and 7 in MRSA-infected and uninfected mouse wound models. In the uninfected group, only hybrid hydrogel was applied without laser irradiation. Figure 20As shown in h, in vivo IVIS fluorescence imaging results indicated that infected wounds exhibited significant and dynamically increasing fluorescence signals during treatment, while uninfected wounds maintained extremely low fluorescence levels under the same treatment and imaging conditions. This suggests that the fluorescence activation of the hydrogel mainly originates from stimulation by the infection microenvironment, rather than the background signal of the material itself. To further achieve direct quantification of endogenous H2S, we collected wound exudate from infected and uninfected mice at the above time points and analyzed it using a commercial sulfide detection kit according to the kit instructions. Figure 24 Quantitative results showed that the H2S content in infected wounds was significantly higher than that in the uninfected group, and gradually decreased during treatment, consistent with the trend of infection control and inflammation relief. Subsequently, we performed quantitative region of interest (ROI) analysis on the corresponding IVIS images. Figure 20 As shown in i, the relative fluorescence intensity obtained exhibits a good linear correlation with the independently measured H2S concentration in the exudate, further demonstrating that the fluorescence response of this hybrid hydrogel in vivo can accurately reflect changes in H2S levels in infected wounds (see Figure 1). Figure 20 (d and g in the text). In summary, these results fully demonstrate that the constructed spatiotemporally responsive hybrid hydrogel can not only achieve effective treatment of drug-resistant bacterial infections, but also serve as a reliable platform for real-time, dynamic, and visual monitoring of the infection treatment process.

[0084] Subsequently, we systematically evaluated the tissue regeneration effects in each treatment group. Histological changes in infected wounds were observed using hematoxylin and eosin (H&E) staining. Figure 21 As shown in a, the EM@mHA group and the EM@mHA-TPAB-Cu 2+ All groups showed significant granulation tissue formation and epithelial regeneration, while wound healing was significantly delayed in the PBS and mHA groups. Notably, EM@mHA-TPAB-Cu 2+ The group showed significantly thickened granulation tissue at all detection time points, and by day 21, fully integrated epidermal structures and skin appendages were observed. To further assess epidermal regeneration, researchers performed immunofluorescence staining on cytokeratin 14 (CK14), a key marker of basal keratinocyte and epithelial regeneration. Figure 21 As shown in b, the EM@mHA group and EM@mHA-TPAB-Cu 2+ CK14 expression was significantly elevated in all groups, with the latter showing the largest CK14-positive area, consistent with H&E staining results, indicating enhanced epidermal remodeling. Figure 21 g in (the middle part).

[0085] Effective skin regeneration also relies on a strong angiogenic capacity to deliver nutrients and oxygen to healing tissue. Immunofluorescence staining of α-smooth muscle actin (α-SMA), a marker used to identify mature vascular systems, showed that compared to the PBS group, the mHA group, EM@mHA group, and EM@mHA-TPAB-Cu group... 2+ The density of new blood vessels in the group showed a progressive increase. Figure 21 c). Wherein EM@mHA-TPAB-Cu 2+ The group exhibited the most significant angiogenesis, and quantitative vascular density analysis further confirmed this phenomenon. Figure 21 (h in the original text). This enhancement effect can be attributed to the synergistic effect of the functional components of the mixed hydrogel. To explore its molecular mechanism, researchers performed Western blot analysis. Vascular endothelial growth factor (VEGF), as a key factor in angiogenesis, promotes endothelial cell proliferation, migration, and angiogenesis. After binding to its receptor (such as VEGFR-2), VEGF can activate multiple intracellular signaling pathways, including the Akt pathway.

[0086] To investigate collagen remodeling, a hallmark of mature wound healing, we used Masson staining and immunofluorescence assays for type I (Col I) and type III (Col III) collagen. On day 7, the PBS group showed disordered and sparse collagen fibers, while the mHA and EM@mHA groups exhibited moderate collagen regeneration. Figure 21 In contrast, EM@mHA-TPAB-Cu 2+ This group exhibited the densest and most well-structured collagen deposits. By day 21, this group not only formed thicker, oriented collagen bundles but also showed the highest number of skin appendages, including hair follicles. Figure 21 The "i" in the figure indicates that tissue remodeling and appendage regeneration have entered an advanced stage. Immunofluorescence analysis ( Figure 21 Further, in sections e and f), it was shown that the levels of Col I and Col III in all treatment groups increased over time, with EM@mHA-TPAB-Cu showing the highest levels. 2+ The group consistently maintained the highest expression levels of both collagen types. These findings collectively confirm the superior performance of this hydrogel in promoting collagen synthesis, matrix remodeling, and overall skin tissue reconstruction.

[0087] EM@mHA-TPAB-Cu 2+The accelerated wound closure observed in the group, characterized by enhanced epithelial regeneration, promoted angiogenesis, and accelerated collagen remodeling, highlights the significant therapeutic effect of this hydrogel against drug-resistant bacterial infections. This effect is attributed to its integrated photodynamic antibacterial activity and immunomodulatory potential. Furthermore, H&E staining of major organs (heart, liver, spleen, lungs, and kidneys) on day 21 showed no histopathological abnormalities in any group. Figure 22 The study confirmed the biocompatibility of the hydrogel. These findings establish the potential of this spatiotemporally responsive hybrid hydrogel as a therapeutic and diagnostic candidate material for treating drug-resistant bacterial infections.

[0088] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. Therapeutic photoactive composite hydrogel EM@mHA-TPAB-Cu 2+ The preparation method of the [method] is characterized by, Includes the following steps: Step 1: Using TPA-CHO and MN as monomers, TPAB is synthesized via a simple and efficient Schiff base reaction, and Cu is further introduced. 2+ TPAB-Cu is formed through coordinate bonds. 2+ ; Step 2: Dissolve emodin in DMSO and mHA in deionized water, then mix the two and add the TPAB-Cu obtained in Step 1. 2+ The solution was ultrasonically treated to form a homogeneous solution, stirred at room temperature, and then dialyzed to remove DMSO, yielding EM@mHA-TPAB-Cu. 2 + Composite hydrogel.

2. The preparation method according to claim 1, characterized in that, Step 1 includes: Step 1-1: Dissolve TPA-CHO and MN in a solvent, reflux under an inert atmosphere, filter, recrystallize to obtain TPAB powder; Step 1-2: Dissolve the TPAB obtained in Step 1-1 in a solvent, then add Cu(NO3)2, stir at room temperature, dry, purify, and dry again to obtain the composite photosensitizer TPAB-Cu. 2+ .

3. The preparation method according to claim 1, characterized in that, In step 2, emodin is dissolved in DMSO to obtain a 15wt% emodin solution; mHA is dissolved in deionized water to obtain a 10wt% mHA solution.

4. The preparation method according to claim 3, characterized in that, In step 2, the emodin solution, mHA solution, and TPAB-Cu 2+ The dosage ratio is 5mL:5mL:10mg.

5. The preparation method according to claim 4, characterized in that, The specific steps of step 2 are as follows: emodin is dissolved in dimethyl sulfoxide to obtain an emodin solution, and mHA is dissolved in deionized water to obtain an mHA solution. Then, the emodin solution and mHA solution are mixed, and TPAB-Cu is added. 2+ The mixture was sonicated for 10 minutes to form a homogeneous solution, stirred at room temperature for 6 hours, and after dialysis to remove DMSO, EM@mHA-TPAB-Cu was obtained. 2+ Composite hydrogel.

6. Therapeutic photoactive composite hydrogel EM@mHA-TPAB-Cu 2+ Its characteristics are, It is prepared by the preparation method according to any one of claims 1 to 5.

7. The photoactive composite hydrogel EM@mHA-TPAB-Cu according to claim 6 2+ Use in the preparation of medicaments for treating wound infections caused by drug-resistant bacteria.

8. The photoactive composite hydrogel EM@mHA-TPAB-Cu according to claim 6 2+ Use in the preparation of products for diagnosing wound infections caused by drug-resistant bacteria.

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