An MXene@Cu-MOF heterostructure hydrogel, its preparation method and application

Abstract

This invention relates to an MXene@Cu-MOF heterostructure hydrogel, its preparation method, and its applications. The MXene@Cu-MOF heterostructure hydrogel comprises an MXene@Cu-MOF heterostructure and a biocompatible hydrogel. The MXene@Cu-MOF heterostructure consists of MXene nanosheets and Cu-MOF nanocrystals grown in situ on the MXene nanosheets. Compared with existing technologies, this invention proposes a single MXene@Cu-MOF heterostructure nanoplatform, replacing loosely coupled composite materials. It can exert both bactericidal functions and active photothermal-chemical synergistic effects, producing a cascade effect, progressing from ROS mitigation to antibacterial / biofilm eradication, and then to angiogenesis support, accelerating the closure of biofilm-infected diabetic wounds.

Description

Technical Field

[0001] This invention belongs to the technical field and relates to an MXene@Cu-MOF heterostructure hydrogel, its preparation method, and its application. Background Technology

[0002] Diabetic wound healing is characterized by persistent inflammation, recurrent or multimicrobial infection, excessive reactive oxygen species (ROS), biofilm formation, and impaired angiogenesis. These intertwined pathological processes create an adverse microenvironment resistant to wound treatment, leading to delayed healing, high amputation risk, and high socioeconomic costs. Therefore, effective treatment requires a multidimensional temporal and spatial approach to (i) eliminate acute oxidative stress to suppress the inflammatory cascade, (ii) eradicate planktonic bacteria and biofilms to reduce reinfection, and (iii) reprogram the wound bed through pro-angiogenic signaling to promote regeneration—while minimizing systemic exposure and enabling on-demand reinforcement during infection surges.

[0003] Stimulus-responsive dressings and nanoplatforms have emerged to address some of the challenges of modulating local drug delivery using pH, enzymes, or light. However, most existing systems combine multiple components through physical mixing or loose encapsulation. Such assemblies often suffer from (i) inhomogeneous distribution and aggregation (e.g., 2D nanosheet re-stacking, MOF particle aggregation), which reduces effective surface area and impairs mass / heat transfer; (ii) poor modal coupling, where one therapy (e.g., photothermal heating) cannot actively gate or amplify another (e.g., antimicrobial ion release), resulting in additive rather than synergistic effects; and (iii) limited ability to act in stages along the wound healing timeline. Importantly, wound pH is heterogeneous both spatially and temporally. While chronic wounds may be alkaline on a macroscale, the local infection / biofilm microdomain may be acidified by bacterial metabolism, providing a plausible trigger for pH-responsive carriers. Therefore, current platforms often underperform in biofilm-infected diabetic wounds because treatment must be both microenvironmentally sensitive and easily escalable.

[0004] MXenes are a class of stripped transition metal carbides / nitrides, such as Ti3C2T. x Copper-based metal-organic frameworks (Cu-MOFs; e.g., HKUST-1 or Cu-doped ZIF-8) are promising building blocks for infectious wound care due to their inherent ROS scavenging capabilities, efficient photothermal conversion via broadband near-infrared (NIR) absorption, and chemical anchoring sites provided by surface terminating groups (-O, -OH). These frameworks offer bioactive Cu with broad-spectrum antibacterial activity and pro-angiogenic signaling potential. [2+] An ion reservoir is used to replenish MXenes. Many Cu-MOFs are unstable under the typically weakly acidic conditions of infected wounds, allowing for the release of Cu triggered by pH "as needed".[2+] However, simply mixing MXene with Cu-MOF in a hydrogel rarely overcomes the fundamental problems of component segregation and uncontrolled burst release or conversely, insufficient release. Furthermore, physical mixtures typically cannot convert photothermal energy into active, programmable chemical delivery gating; therefore, the two modes remain, to some extent, independent and not truly synergistic. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems existing in the prior art, thereby providing an MXene@Cu-MOF heterostructure hydrogel, its preparation method, and its application.

[0006] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of the present invention is to provide a method for preparing MXene@Cu-MOF heterostructure hydrogel, comprising the following steps: S1. Ti3AlC2 is produced by selectively etching it using a LiF / HCl mixture, followed by layering to obtain Ti3C2Tx, which is MXene nanosheets; S2. Cu-MOF is grown in situ on MXene nanosheets to obtain MXene@Cu-MOF; S3. MXene@Cu-MOF is dispersed in a GelMA prepolymer solution and polymerized to form an MXene@Cu-MOF heterostructure hydrogel.

[0007] In some specific embodiments, in step S1, LiF is dissolved in HCl solution, Ti3AlC2 powder is slowly added under ice bath conditions for etching, centrifugation is performed, the precipitate is washed until the pH of the solution containing the precipitate is weakly acidic, and the precipitate is sonicated under an inert atmosphere and ice bath conditions to separate into layers, thereby obtaining MXene nanosheets.

[0008] In some specific embodiments, the mass ratio of LiF to Ti3AlC2 powder is (0.8~1.6):1; the concentration of the HCl solution is 9 M; The etching temperature is (30~40)℃, and the etching time is (20~30)h; Wash the precipitate until the pH of the solution containing the precipitate is (5~7). The ultrasound session lasts 30 to 60 minutes.

[0009] In some specific embodiments, in step S2, the Cu-MOF is selected from either HKUST-1 or Cu-doped ZIF-8.

[0010] In some specific embodiments, when Cu-MOF is HKUST-1, the MXene nanosheet dispersion is mixed with an aqueous solution of Cu(NO3)2-ethanol, 1,3,5-pyromellitic acid is slowly added, the reaction is carried out under acidic conditions, the product is collected by centrifugation, and the MXene@Cu-MOF composite material is obtained. When Cu-MOF is Cu-doped ZIF-8, the MXene nanosheet dispersion is mixed with Zn-containing... 2+ and Cu 2+ The solution was pre-equilibrated, and 2-methylimidazole solution was quickly added to induce Cu-doped ZIF-8 growth, thus obtaining MXene@Cu-MOF composite material.

[0011] In some specific embodiments, when Cu-MOF is HKUST-1, the ratio of MXene nanosheet dispersion, Cu(NO3)2-containing aqueous solution-ethanol, and 1,3,5-pyromellitic acid is 1 g:(5~60) mL:(2~30) g. The Cu(NO3)2 aqueous solution-ethanol was obtained by mixing Cu(NO3)2 aqueous solution and ethanol in a 1:1 volume ratio, and the Cu(NO3)2 concentration was (10~50) mM. The reaction conditions under acidic conditions are: (25~60)℃, pH (3~4) for (2~12) h.

[0012] In some specific embodiments, when the Cu-MOF is Cu-doped ZIF-8, the MXene nanosheet dispersion, containing Zn 2+ and Cu 2+ The volume ratio of the solution and the 2-methylimidazole solution was 2:1:1, wherein the solution contained Zn 2+ and Cu 2+ The total metal ion concentration in the solution is 20~100 mM, Zn 2+ With Cu 2+ The molar ratio was 95:5~80:20, the concentration of 2-methylimidazole solution was 200~800mM, and the concentration of MXene dispersion was (1.0~5.0) mg / mL.

[0013] In some specific embodiments, in step S3, GelMA and a photoinitiator are dissolved in a PBS solution to obtain a prepolymer solution, and MXene@Cu-MOF is dispersed in the prepolymer solution and photopolymerized to form an MXene@Cu-MOF heterostructure hydrogel.

[0014] In some specific embodiments, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid. The mass fraction of photoinitiator in PBS solution is 0.05–0.1 wt%, and the mass fraction of GelMA in PBS solution is 5–12 wt%. The concentration of MXene@Cu-MOF dispersed in the prepolymer solution was 0.5~1.5 mg / mL, calculated based on MXene nanosheets. The polymerization conditions were 405 nm and 10–20 mW / cm². 2 Photopolymerization for 30-60 seconds.

[0015] The second technical solution of the present invention is to provide an MXene@Cu-MOF heterostructure hydrogel, which is obtained by the preparation method described in one of the above technical solutions.

[0016] In this invention, the MXene@Cu-MOF heterostructure hydrogel comprises an MXene@Cu-MOF heterostructure and a biocompatible hydrogel. The MXene@Cu-MOF heterostructure consists of MXene nanosheets and Cu-MOF nanocrystals grown in situ on the MXene nanosheets. The MXene nanosheets are Ti3C2T. x The nanosheets, wherein the Cu-MOF is selected from either HKUST-1 or Cu-doped ZIF-8.

[0017] In this invention, MXene and Cu-MOF are integrated into a coherent 2D / 3D nanostructure, which is then transformed into a moist, shape-preserving wound hydrogel dressing. This is no longer merely a co-loaded mixture, but rather establishes true modal coupling: photothermal heating actively drives the generation of therapeutic ions, rather than simply coexisting with them. This platform provides timed, on-demand action tailored to the disease stage, enabling precise application to the wound.

[0018] In this system, MXene acts as a "framework" for conductivity, ROS quenching, and photothermal activity, while Cu-MOF forms a conformal, biodegradable "functional backpack." Surface oxide groups on MXene serve as metal ion anchoring and heterogeneous nucleation sites, guiding selective MOF crystallization at the basal plane and edges.

[0019] This heterogeneous structure integration aims to (i) prevent MXene re-stacking by inserting MOF spacers, (ii) ensure uniform dispersion and tight interfacial contact of MOFs, and (iii) establish short and efficient heat and mass transfer paths between the photothermal core and the unstable MOF domain.

[0020] The third technical solution of the present invention is to provide the application of MXene@Cu-MOF heterostructure hydrogel in the preparation of diabetic wound healing drugs.

[0021] Compared with the prior art, the present invention has the following advantages: (1) This invention proposes a single heterostructure nanoplatform of MXene@Cu-MOF. MXene acts as a "framework" for conductivity, ROS quenching, and photothermal activity, while Cu-MOF forms a conformal, biodegradable "functional backpack." The exposed MXene surface scavenge ROS to inhibit inflammatory signals ("first barrier"), while the acidic environment of infection triggers pH responsiveness and the release of baseline copper from the MOF ("self-regulating messenger"). This heterostructure not only transfers heat for bactericidal function but also accelerates the decomposition of MOF at the MXene-MOF interface, generating a Cu [2+] The eruption, in synergy with thermotherapy, eradicates resilient biomembranes through an active photothermal-chemical synergistic effect.

[0022] (2) This invention replaces the loosely coupled composite material with a single, interface-engineered heterostructure, transforming the two co-loaded reagents into a coupled system in which photothermal energy is gated and amplified to release ions. That is, in-situ growth of Cu-MOF on Ti3C2Tx produces a stable MXene@Cu-MOF heterostructure with suppressed aggregation and enhanced interfacial coupling.

[0023] (3) This invention introduces the concept of a “self-decomposing messenger”: Cu produced only when required by the microenvironment acidity (and, if necessary, controlled thermotherapy). [2+] This minimizes off-target exposure while generating negative feedback against infection. Specifically, it enables Cu to achieve both microenvironment specificity (pH responsiveness) and external programmability (NIR amplification). 2+ Release, converting photothermal input into on-demand chemical output.

[0024] (4) This invention addresses the issues of dispersion and accessible surface area by using MOF spacers to suppress MXene stacking, thereby enhancing ROS quenching and heat / mass transfer, resulting in a cascade effect—progressing from ROS mitigation to antimicrobial / biofilm eradication, and then to angiogenesis support—accelerating the closure of biofilm-infected diabetic wounds relative to physical mixtures and monomodal controls. Attached Figure Description

[0025] Figure 1Structure and morphology characterization of the synthesized MXene@Cu-MOF nanoplatform. (A) SEM micrographs of Cu-MOF, MXene(Ti3C2Tx), and MXene@Cu-MOF heterostructures. (B) TEM micrographs of Cu-MOF, MXene, and MXene@Cu-MOF. (C) AFM morphology images of Cu-MOF, MXene, and MXene@Cu-MOF: 2D height maps and corresponding 3D surface renderings. (D) EDS elemental distribution map of the synthesized MXene@Cu-MOF.

[0026] Figure 2 The in vitro antioxidant capacity of the MXene@Cu-MOF / GelMA platform. (A) MXene@Cu-MOF against ABTS• + Concentration-dependent scavenging activity of free radicals. (B) Concentration-dependent scavenging activity of MXene@Cu-MOF for DPPH• free radicals. (C) Concentration-dependent scavenging activity of MXene@Cu-MOF for superoxide anion (O2• – (D) Scavenging ability of MXene@Cu-MOF for hydroxyl radicals (OH). (E) Representative confocal images of ROS in RAW264.7 macrophages stained with DCFH-DA (red) under the following treatments: PBS (blank), H2O2 only, H2O2+GelMA, H2O2+MXene / GelMA, and H2O2+MXene@Cu-MOF / GelMA. (F) Flow cytometry quantification of ROS (DCF fluorescence) in each group of cells in (E). (G) Representative DCFH-DA staining (red) of ROS in RAW264.7 macrophages under the following treatments: PBS (blank), LPS only, LPS+GelMA, LPS+MXene / GelMA, and LPS+MXene@Cu-MOF / GelMA. (H) Flow cytometry quantification of ROS (DCF fluorescence) in each group of cells in (G). Data are expressed as mean ± standard deviation (n = 3) Figure 3 Photothermal behavior of the MXene@Cu-MOF / GelMA platform. (A) Under NIR irradiation (1.5 W / cm²). 2 (a) Infrared thermograms of control (PBS), MXene@Cu-MOF, GelMA, and MXene@Cu-MOF / GelMA (all nanosheet concentrations were 1.0 mg / mL) under NIR irradiation (1.5 W / cm²) for 10 minutes. (B) Corresponding temperature-time curves for each group in A. (C) MXene@Cu-MOF / GelMA under different nanosheet concentrations (0.5, 1.0, and 1.5 mg / mL) under NIR irradiation (1.5 W / cm²). 2(D) Temperature rise at a fixed concentration (1.0 mg / mL) under different irradiations (0.5, 1.0 and 1.5 W / cm²). 2 Temperature rise under (E) MXene@Cu-MOF / GelMA (1.0 mg / mL) after 10 minutes of NIR irradiation followed by 10 minutes of natural cooling (1.5 W / cm²). 2 (F) Photothermal stability of MXene@Cu-MOF / GelMA: at a concentration of 1.0 mg / mL, NIR irradiation (1.5 W / cm²) 2 Repeated heating-cooling cycles (10 cycles) were performed on (G)MXene@Cu-MOF in vitro with or without NIR irradiation of Cu. 2+ Release kinetics. (H)MXene@Cu-MOF / GelMA in vitro Cu with and without NIR irradiation. 2+ Release kinetics. (I) In vitro degradation curves of MXene@Cu-MOF / GelMA in PBS (pH 7.4), with and without NIR irradiation. (J) pH-dependent degradation of Cu-MOF prototype. 2+ Effects of release and NIR heating. Data are presented as mean ± standard deviation (n = 3).

[0027] Figure 4 To assess the in vitro antibacterial activity of the MXene@Cu-MOF / GelMA platform against *Escherichia coli* and *Staphylococcus aureus* under NIR. (A) Representative agar plate images of *E. coli* colonies and corresponding Live / Dead fluorescence staining after treatment with PBS (blank), GelMA, MXene@Cu-MOF, and MXene@Cu-MOF / GelMA. (B) Quantification of *E. coli* colony area (plate) and survival percentage (Live / Dead). (C) Representative agar plate images of *Staphylococcus aureus* colonies and corresponding Live / Dead staining under the same treatment as in (A). (D) Quantification of *Staphylococcus aureus* colony area and survival percentage. (E) Representative SEM micrographs of *E. coli* and *Staphylococcus aureus* after the specified treatment. Data are presented as mean ± standard deviation (n = 3); ***P < 0.001; ns, not significant.

[0028] Figure 5 For in vitro angiogenesis assessment of the MXene@Cu-MOF / GelMA platform under NIR. (A, B) EdU incorporation staining of HUVECs and EdU in each group below. +Quantification of nuclear percentage: blank (PBS), GelMA, MXene@Cu-MOF, and MXene@Cu-MOF / GelMA. (C,D) HUVEC monolayer scratch migration assay and quantification of wound closure percentage (%) at specified time points. (EG) Matrigel tube formation assay: representative images (E) and quantification of branching points (F) and total tube length (G). (HK) RT-qPCR analysis of angiogenesis-related genes (VEGF, eNOS, HIF-1a, and FGF2) in HUVECs. Data are expressed as mean ± standard deviation (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

[0029] Figure 6 The therapeutic effect of the MXene@Cu-MOF / GelMA platform in a bacterial-infected diabetic wound model. (A) Representative macroscopic images of wounds at days 5, 9, and 14 for the following groups: PBS (control), GelMA, MXene / GelMA, Cu-MOF / GelMA, and MXene@Cu-MOF / GelMA. Wound areas were plotted under the same imaging conditions. (B) Wound healing trajectories compiled from serial area measurements, showing the overlay of wound boundaries over time. (C) H&E staining of wound sections at days 9 and 14, highlighting epithelial regeneration and granulation tissue formation. (D) Quantification of epithelial thickness in each group in (C), measured at comparable distances from the wound center. (E) Masson's trichrome staining at days 9 and 14 to visualize collagen deposition and extracellular matrix remodeling. (F) Image analysis quantification of collagen area fraction (%) in sections in (E). (G) Immunohistochemistry (IHC) of type III collagen on days 9 and 14 to assess early matrix deposition. (H) Quantification of the positive area fraction (%) of type III collagen corresponding to (G). (I) IHC of type I collagen on days 9 and 14 to assess mature matrix formation. (J) Quantification of the positive area fraction (%) of type I collagen corresponding to (I). Data are presented as mean ± standard deviation (n = 6); *P < 0.05.

[0030] Figure 7In vivo assessment of antibacterial, antioxidant, macrophage polarization, and pro-angiogenic effects in an infected diabetic wound model. (A) Representative FISH images of bacterial load from wound sections from the PBS (control), GelMA, MXene / GelMA, Cu-MOF / GelMA, and MXene@Cu-MOF / GelMA groups on days 3 and 6; cell nuclei counterstained with DAPI. The same acquisition settings and regions of interest were used across groups. (B) Quantification of bacterial load per field of view from (A), obtained by automatic segmentation of FISH-positive signals (mean of multiple fields of view per animal). (C) Dihydroethidium (DHE) fluorescence staining of superoxide anions in wound tissues on days 9 and 14 of the designated groups, counterstained with DAPI nuclei. (D) Mean fluorescence intensity (MFI) of dihydroethidium (DHE) from (C), normalized according to panel instructions. (E) Immunofluorescence staining of CD206 (M2-associated macrophages) on days 9 and 14. (F) and (E) correspond to each field of view CD206 + Cell count quantification. (G) Immunofluorescence staining of CD68 (pan-macrophage marker) on days 9 and 14. (H) CD68 in each field of view corresponding to (G). + Quantification of cell counts. (I) CD31 immunofluorescence staining on days 9 and 14 to visualize microvessels. (J) Quantification of vessel count per field corresponding to (I). Data are presented as mean ± standard deviation (n = 6); *P < 0.05.

[0031] Figure 8 Whole transcriptome RNA-seq analysis of repaired skin tissue on day 14. (A) Principal component analysis (PCA) of global transcriptome profiles in the CON and EXP groups. (B) Volcano plot of differentially expressed genes (DEGs) between EXP and CON; significance was defined as |log2 fold chage| ≥ 1 (fodl change ≥ 2) and adjusted-Benjamini–Hochberg P < 0.05. (C) Heatmap of significant DEGs across samples, illustrating relative expression patterns between groups. (D) Relative expression of representative genes related to antimicrobial defense, inflammation regulation, angiogenesis, and new collagen formation in CON and EXP. (E) GO and KEGG pathway enrichment analysis of upregulated genes in EXP compared to CON (showing top enriched terms). (F) GSEA enrichment plot of selected gene sets related to antimicrobial / immune defense, inflammatory response, angiogenesis, and new collagen formation, showing positive enrichment in EXP relative to CON. Data are expressed as mean ± standard deviation (n = 3); *P < 0.05.

[0032] Figure 9 The size distribution of Cu-MOF nanocrystals. Figure 10 This is an EDS elemental distribution diagram of Cu-MOF.

[0033] Figure 11 This is an EDS elemental distribution diagram of MXene nanosheets.

[0034] Figure 12 The FTIR spectra of MXene, Cu-MOF, and MXene@Cu-MOF are shown.

[0035] Figure 13 (A) XPS broad scan spectrum of Cu-MOF, (B) High resolution XPS spectrum of MXene, (C) XRD pattern of MXene (Ti3C2Tx) nanosheets.

[0036] Figure 14 The zeta potentials are those of Cu-MOF, MXene, and MXene@Cu-MOF.

[0037] Figure 15 Anti-inflammatory effect of the MXene@Cu-MOF / GelMA platform in RAW264.7 macrophages pre-stimulated with LPS. (A, B) Representative immunofluorescence and corresponding quantification (normalized MFI or percentage of positive cells) of M1 (CD86, green) and M2 (CD206, red) markers in RAW264.7 cells treated with the following methods: LPS (positive control), IL-4 (negative / M2 control), LPS + GelMA, LPS + MXene / GelMA, and LPS + MXene@Cu-MOF / GelMA. Cell nuclei were counterstained with DAPI. (C, D) Western blot analysis of iNOS, IL-6, TNF-α, and IL-1β, and optical density quantification normalized to the loading control. Data are expressed as mean ± standard deviation (n = 3); ***P < 0.001; ns, not significant.

[0038] Figure 16 This is a schematic diagram of a cascade, on-demand treatment procedure using MXene@Cu-MOF heterostructured hydrogel for infected diabetic wounds. Detailed Implementation

[0039] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0041] Unless otherwise specified, the materials and processes used in the following embodiments or examples are conventional materials and processes employed in the art to achieve the corresponding functions.

[0042] Example 1: This embodiment provides an MXene@Cu-MOF heterostructure hydrogel, the preparation method of which includes the following steps: (1) Synthesis of MXene nanosheets: Ti3C2Tx was produced by selectively etching Ti3AlC2 using a LiF / HCl mixture, followed by stratification. Specifically, 1.0 g of LiF was dissolved in 30 mL of 9 M HCl, and 1.0 g of Ti3AlC2 powder was slowly added under stirring in an ice bath, followed by reaction at 35 °C for 24 h. The slurry was repeatedly washed by centrifugation and resuspension in water until the pH of the supernatant was approximately 6. The precipitate was sonicated under nitrogen (ice bath, 50 min) to separate into layers, and then centrifuged at low speed to remove unexfoliated particles, yielding MXene nanosheets. The supernatant containing a few layers of Ti3C2Tx was stored under nitrogen at 4 °C and used within two weeks.

[0043] (2) Cu-MOF was grown in situ on MXene nanosheets, with HKUST-1 (Cu-BTC) as a representative Cu-MOF synthesized from heterostructure.

[0044] Take 100 mL of MXene dispersion with a concentration of 1.0 mg / mL and mix it with 50 mL of a water-ethanol mixture containing Cu(NO3)2 (water-ethanol volume ratio 1:1, Cu(NO3)2 concentration approximately 25 mM). Then, slowly add approximately 2.0 g of H3BTC under stirring. The mixture is kept at approximately 40°C and pH 3.5 for approximately 8 hours to allow heterogeneous nucleation to occur on the MXene basal surface and edges. The product is collected by centrifugation, washed with ethanol and water, and redispersed in water.

[0045] For example, another Cu-MOF was grown in situ on MXene nanosheets, and another Cu-MOF was synthesized by using Cu-doped ZIF-8 as a heterostructure.

[0046] The MXene dispersion was mixed with a Zn(NO3)2·6H2O / Cu(NO3)2·3H2O mixed metal salt solution and stirred at room temperature for about 25 min to achieve pre-equilibration. Then, a 2-methylimidazole solution was rapidly added to induce Cu-doped ZIF-8 growth. Preferably, 50 mL of the mixed metal salt solution and 100 mL of 2-methylimidazole solution were added per 100 mL of MXene dispersion; the total metal ion concentration in the mixed metal salt solution was approximately 80 mM, and the Zn... 2+ With Cu 2+ The molar ratio is approximately 10:1; the concentration of the 2-methylimidazole solution is approximately 600 mM.

[0047] The composite material was washed with methanol and water and stored at 4°C. The copper loading was quantified by inductively coupled plasma-optical emission spectrometry (ICP-OES) after acid digestion in 2% HNO3.

[0048] All reported concentrations “relative to nanosheets” refer to the mass of Ti3C2Tx in the composite material.

[0049] (3) Preparation of MXene@Cu-MOF / GelMA hydrogel: GelMA was dissolved in PBS containing 0.25 wt% LAP at 37 °C to obtain a prepolymer solution; preferably, the concentration of GelMA was 5 wt%, i.e., 5 g of GelMA was added to every 100 mL of PBS. MXene@Cu-MOF was homogeneously dispersed in the GelMA prepolymer solution, and the concentration of MXene@Cu-MOF, calculated based on MXene nanosheets, was 0.5~1.5 mg / mL, preferably 1.0 mg / mL. The hydrogel was tested in a sterile mold or directly on tissue under 405 nm light (15 mW / cm²). 2 Photopolymerization takes approximately 40 seconds to form the hydrogel. The hydrogel is equilibrated in PBS before measurement.

[0050] The MXene@Cu-MOF / GelMA hydrogel (Cu-MOF is HKUST-1) prepared above was tested as follows: Test Example 1: Structural and morphological characterization of the synthesized MXene@Cu-MOF nanoplatform like Figure 9 As shown, this is the DLS intensity-weighted hydrodynamic diameter distribution of Cu-MOF nanocrystals dispersed in an aqueous medium.

[0051] like Figure 10 The image shows a representative SEM image of Cu-MOF and the corresponding EDS elemental distribution map, which shows the spatial distribution of C, N, O and Cu in Cu-MOF.

[0052] like Figure 11 As shown, it is MXene(Ti3C2T) x The representative EDS elemental distribution map shows the spatial distribution of Ti, C, O and F (surface termination groups).

[0053] like Figure 12 The image shows the FTIR spectra of MXene, Cu-MOF, and MXene@Cu-MOF. A comparison of the FTIR spectra (ATR, 4000–400 cm⁻¹) of MXene (surface -OH / -O terminating groups), Cu-MOF (ligand-related bands), and the MXene@Cu-MOF heterostructure is also shown. -1 The MXene@Cu-MOF heterostructure exhibits binding characteristics accompanied by interfacial migration / attenuation.

[0054] like Figure 13 As shown in AC, this is the XPS and XRD characterization of Cu-MOF and MXene nanosheets, and MXene(Ti3C2Tx) nanosheets.

[0055] like Figure 14 The figure shows the zeta potential of Cu-MOF, MXene, and MXene@Cu-MOF. The electrophoretic light scattering measurement zeta potential distribution (histogram) and corresponding average values ​​(bar graph, mean ± standard deviation) of Cu-MOF, MXene(Ti3C2Tx), and MXene@Cu-MOF heterostructures in aqueous electrolyte are also shown.

[0056] Depend on Figure 1 SEM and TEM images of the Cu-MOF, MXene (Ti3C2Tx), and MXene@Cu-MOF heterostructures shown in AB reveal that polyhedral Cu-MOF nanocrystals are uniformly anchored on ultrathin Ti3C2Tx (MXene) nanosheets, creating a continuous 2D / 3D contact interface rather than a loose physical mixture. In the TEM images, the MOF domains appear as discrete nanocrystals distributed across the MXene basal plane and edges, consistent with heterogeneous nucleation at oxygen termination sites. Figure 1 AFM analysis of Cu-MOF, MXene (Ti3C2Tx), and MXene@Cu-MOF heterostructures shown in Figure C reveals increased surface height and roughness in MXene@Cu-MOF compared to the original MXene, consistent with the conformal MOF capping. This MOF "spacer" domain is known to mitigate MXene recombination while maintaining accessible surface area, a recurring advantage of MOF / MXene heterostructures. Figure 1The elemental mapping of the MOF / MXene heterostructure shown in Figure D reveals the co-localization of C / N / O / Cu signals in the MOF domain, while Ti / F outlines the MXene framework, supporting close symbiosis at the nanoscale.

[0057] The above confirms the formation of the single heterostructure nanoplatform of the present invention, in which MOF nanocrystals are embedded on an MXene scaffold, rather than simply adsorbed or embedded at a distance. That is, in-situ growth of MOFs on MXene creates close contact and avoids the phase separation often observed in post-mixing methods.

[0058] Test Example 2: In vitro antioxidant test (1) Scavenging ability of ABTS free radical, DPPH free radical, superoxide anion and hydroxyl free radical ABTS• + ABTS was produced by reacting it with potassium persulfate in the dark for 12-16 hours and then diluted to an absorbance of approximately 0.70 at 734 nm. Samples at concentrations of 0-1.0 mg / mL were then reacted with ABTS. + Incubate for 6–10 minutes, and calculate the scavenging percentage based on the decrease in absorbance. DPPH radical scavenging capacity was determined by mixing the sample with an ethanol-DPPH solution (A517–0.9) in the dark for 30 minutes. Superoxide anion scavenging capacity was assessed using the xanthine-xanthine oxidase-nitroblue tetrazolium system, monitored at 560 nm. Hydroxyl radical scavenging capacity was assessed using the Fenton reaction with a salicylic acid probe, monitored at 510 nm for the chromophore. All tests included appropriate blanks and standards, and results were normalized relative to a radical control.

[0059] like Figure 2 As shown in AB, MXene@Cu-MOF exhibits concentration-dependent ABTS• within the range of 0.1–1.0 mg / mL. + The scavenging capacity of ABTS and DPPH increased monotonically relative to the free radical control, with the percentage of inhibition increasing. ABTS readings in aqueous media showed a strong quenching effect, while DPPH in alcoholic media showed a similar dose-response, consistent with known solvent and kinetic differences between the two assays. These results are consistent with the established use of ABTS and DPPH as complementary, single-electron transfer-dominated methods for ranking antioxidant capacity. Figure 2 As shown in CD, in the chemical system that generates reactive oxygen species intermediates, MXene@Cu-MOF reduces O2• in a dose-dependent manner. - The presence of •OH signals indicates that, in addition to the chromogenic ABTS / DPPH substitute, it also possesses broad-spectrum free radical mitigation capabilities.

[0060] The consistency of ABTS, DPPH, superoxide anion, and hydroxyl radical assays reinforces the conclusion that MXene@Cu-MOF possesses intrinsic antioxidant capabilities.

[0061] (2) Effects of controlling intracellular oxidative stress RAW264 macrophages were seeded in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin and cultured at 37°C and 5% CO2. RAW264 macrophages were exposed to 200–500 μM H2O2 for 1 h or 1 μg / mL LPS for 12–24 h. They were then loaded with 10 μM DCFH-DA in serum-free medium and incubated at 37°C for 30 min, followed by washing and incubation with PBS, GelMA, MXene / GelMA, or MXene@Cu-MOF / GelMA in an NIR protocol (using an 808 nm continuous-wave near-infrared laser at 1.5 W / cm²). 2 Cells were treated with a power density irradiation of 10 min. Fluorescence imaging was performed on a confocal microscope with the same exposure settings, and flow cytometry was performed on a BD FACSCelesta flow cytometer (BD Biosciences, USA), selecting viable single cells. Data were background corrected and normalized relative to controls.

[0062] like Figure 2 As shown in Figure EF, in RAW264.7 macrophages, DCFH-DA fluorescence measurements revealed that H2O2 exposure increased intracellular ROS, and the incorporation of pristine GelMA also produced a fluorescence signal, while MXene / GelMA reduced the fluorescence signal. MXene@Cu-MOF / GelMA achieved the most significant reduction in fluorescence signal, showing a significant difference compared to the H2O2 and GelMA-only control.

[0063] like Figure 2 As shown in GH, under LPS stimulation (simulating the inflammatory ROS burst in macrophages), GelMA produced less DCFH-DA fluorescence signal, MXene / GelMA reduced the fluorescence signal, while MXene@Cu-MOF / GelMA produced the largest decrease in fluorescence signal.

[0064] In the heterostructure of MXene@Cu-MOF / GelMA of this invention, the Cu-MOF domain is in close contact with the MXene surface; although copper can participate in redox chemistry, analysis of copper-based MOFs highlights their ability to modulate oxidative stress and provide antimicrobial effects when released under microenvironmental cues. Excessive ROS in diabetic wounds maintain inflammation, damage the ECM and cells, and impair angiogenesis. By reducing extracellular free radical substitutes in macrophages and intracellular DCFH-DA signaling, MXene@Cu-MOF / GelMA addresses an upstream driver leading to non-healing states. This antioxidant function complements the established antimicrobial and pro-angiogenic effects of copper-containing biomaterials and dressings in preclinical and clinical settings, demonstrating that the same platform, when appropriately adapted, can coordinate early inflammation control with later vascular support.

[0065] Test Example 3: In vitro macrophage polarization and inflammation readings RAW264.7 cells were induced to develop the M1 phenotype using LPS (1 μg / mL, 12–24 h) or as an M2 control using IL-4 (20 ng / mL, 24 h). After treatment, cells were fixed, permeabilized, and blocked with 4% paraformaldehyde. Primary antibodies against CD86 and CD206 were incubated overnight at 4°C, followed by species-specific fluorescently labeled secondary antibodies. Cell nuclei were counterstained with 4',6-diamidinyl-2-phenylindole (DAPI; Sigma, USA). Images were acquired under the same settings. For Western blotting, lysis buffer was prepared in RIPA buffer, separated by SDS-PAGE, transferred to a PVDF membrane, and used to detect iNOS, IL-6, TNF-α, and IL-1β, with β-actin or GAPDH as loading controls. Bands were visualized by chemiluminescence and quantified by optical density.

[0066] like Figure 15 As shown in Figures AB, in LPS-stimulated RAW264.7 macrophages, immunostaining revealed strong CD86 signaling and relatively weak CD206 signaling, consistent with an M1-biased state. As a control, IL-4 treatment produced the expected CD206-dominant M2-like phenotype. Incorporation of pristine GelMA produced only minor changes compared to LPS alone, while MXene / GelMA significantly reduced CD86 and increased CD206. The MXene@Cu-MOF / GelMA group showed the most significant changes, with lower CD86 and higher CD206 than the MXene / GelMA group.

[0067] like Figure 15As shown in CD, Western blots from the same treatment groups revealed elevated levels of iNOS, IL-6, TNF-α, and IL-1β in the LPS group, with density quantification normalized to the loading control. GelMA alone produced a moderate attenuation, MXene / GelMA further reduced band intensity, and MXene@Cu-MOF / GelMA produced the largest reduction relative to both LPS and GelMA controls.

[0068] Overall, these data indicate that embedding the MXene@Cu-MOF heterostructure into GelMA preserves the biocompatible matrix while, under the same assay conditions, confers stronger anti-inflammatory and remission-promoting polarization characteristics than GelMA or MXene alone.

[0069] Test Example 4: Photothermal Measurement (1) Use a device with adjustable irradiance (0.5-1.5 W / cm²). 2 Samples (PBS, GelMA, MXene@Cu-MOF dispersions, and MXene@Cu-MOF / GelMA) were irradiated for 10 min with an 808 nm continuous-wave laser. Infrared thermal images were captured using a FLIR A655sc infrared thermal imager (FLIR Systems, Inc., USA), and temperature-time curves were extracted. Concentration and irradiance dependence were observed at MXene nanosheet concentrations of 0.5–1.5 mg / mL and irradiances of 0.5–1.5 W / cm². 2 The following evaluation was conducted. Stability was tested through ten heating-cooling cycles (10 minutes of irradiation followed by 10 minutes of natural cooling). The photothermal conversion efficiency could be calculated using the Roper method based on the heating-cooling curves.

[0070] like Figure 3 As shown in Figures AB, under 808 nm NIR irradiation, the MXene@Cu-MOF dispersion exhibited a rapid and sustained temperature rise, while PBS and GelMA alone showed minimal heating. Embedding MXene@Cu-MOF into GelMA preserved robust photothermal behavior, with a slightly moderate heating rate compared to the MXene@Cu-MOF dispersion, consistent with thermal diffusion and some optical attenuation in the hydrogel matrix.

[0071] like Figure 3 As shown in CD, the temperature rise of MXene@Cu-MOF / GelMA increases with increasing MXene nanosheet concentration at a fixed irradiance, and increases with increasing irradiance at a fixed concentration, indicating that the thermal dose is controllable.

[0072] like Figure 3The heating-cooling curves shown in EF demonstrate a rapid on / off thermal response and repeatable profiles across multiple cycles, indicating the photothermal stability of the MXene@Cu-MOF heterostructure within the hydrogel network.

[0073] (2) Copper release and hydrogel degradation Place the sample in a dialysis bag (molecular weight cutoff 3.5-10 kDa), immerse it in PBS (pH 7.4) or acetate buffer (pH 6.0-6.8), and gently shake at 37°C. At specified time points, remove aliquots of the external medium and replace them with fresh buffer. For NIR-triggered release, the sample is treated at 808 nm, 1.5 W / cm² at each time point. 2 Irradiate for 10 minutes. Cu 2+ Concentration was quantified by ICP-OES using certified standards after acidification in 2% HNO3. Mass loss of the hydrogel in PBS was determined by recording the baseline wet weight and the wet weight after incubation with or without intermittent NIR irradiation; values ​​were reported as normalized residual mass.

[0074] like Figure 3 As shown by GH, in vitro copper release studies indicate the presence of baseline Cu in the dark. 2+ Release increased under NIR irradiation, whether in the free MXene@Cu-MOF heterostructure or the hydrogel-embedded form. The release magnitude and rate were higher under NIR irradiation, consistent with thermally accelerated framework degradation and faster diffusion.

[0075] like Figure 3 As shown in Figure 1, MXene@Cu-MOF / GelMA exhibits gradual mass loss in PBS (pH 7.4), with only slight additional softening or erosion under intermittent NIR exposure. This is because GelMA is largely NIR transparent, but may undergo matrix relaxation during the photothermal operation of the composite hydrogel as local temperatures rise.

[0076] like Figure 3 As shown in Figure J, the Cu-MOF prototype exhibits a larger Cu content under weakly acidic conditions than under neutral pH. 2+ Release, and NIR exposure further increases the release rate.

[0077] This invention achieves a controllable photothermal source and a thermally amplified copper reservoir in a single structure by integrating Cu-MOF onto MXene and embedding the MXene@Cu-MOF heterostructure into GelMA.

[0078] Test Example 5: In vitro antibacterial test E. coli ( E.coilATCC 25922) and Staphylococcus aureus ( S.aureus (ATCC 25923) were cultured to mid-logarithmic growth, washed, and diluted to approximately 10. 6 -10 7 CFU / mL. The bacterial suspension was incubated with PBS, GelMA, MXene@Cu-MOF, or MXene@Cu-MOF / GelMA at 37°C for 10-30 minutes, then irradiated with 808 nm NIR (1.5 W / cm²). 2 (10 minutes), unless otherwise specified. Aliquots were serially diluted and plated on LB agar plates, incubated overnight at 37°C, followed by digital imaging and image-based colony area analysis. Live / Dead staining was performed using SYTO9 and propidium iodide, according to the manufacturer's instructions; fluorescence microscopy was performed under fixed exposure. Bacteria were fixed in 2.5% glutaraldehyde, dehydrated by gradient ethanol, dried, sputter-coated, and imaged by SEM.

[0079] like Figure 4 As shown in the AD diagram, under normalized 808 nm NIR irradiation, the PBS and GelMA controls exhibited abundant colony formation and dominant green Live / Dead fluorescence on agar plates, indicating high survival rates. MXene@Cu-MOF reduced colony coverage and shifted Live / Dead readings towards decreased survival rates. The MXene@Cu-MOF / GelMA group showed the most significant effect, with sparse colonies and predominantly non-viable staining; quantitative analysis confirmed that both colony area and survival rate of *Escherichia coli* and *Staphylococcus aureus* were significantly reduced compared to the control.

[0080] like Figure 4 The SEM images shown in E indicate that the cell membranes in the control group are intact and smooth, while MXene@Cu-MOF / GelMA with NIR causes wrinkling, pitting, and partial collapse of the membranes of both bacteria, which are morphological markers of membrane damage and heat stress.

[0081] The above results are due to the local high temperature generated by the broadband NIR absorption of MXene, while the Cu-MOF component provides bioactive copper. That is, under the action of MXene@Cu-MOF / GelMA in this invention, the high temperature can destroy the membrane, denature proteins, and susceptible bacteria. Copper ions further damage the membrane, catalyze the redox cycle and ROS generation, and interact with nucleic acids and essential enzymes. This combination enhances the lethality against Escherichia coli and Staphylococcus aureus.

[0082] Test Example 6: Endothelial Cell Assay Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial cell growth medium at 37°C and 5% CO2.

[0083] EdU incorporation: Cells were treated with the specified sample, irradiated as appropriate, and incubated with EdU (10 μM) for 2 hours, followed by fixation and click chemistry detection.

[0084] Scratch migration test: The confluent monolayer was scratched with a sterile pipette tip, rinsed, treated, and imaged at baseline and specified time points; the wound area was quantified using ImageJ.

[0085] Tube formation experiment: Matrigel reduced by growth factors was polymerized in 96-well plates at a concentration of (1-2) × 10⁻⁶ per well. 4 HUVECs cells were seeded, treated, and imaged 4–8 hours later; branch points and main tube lengths were quantified using ImageJ or AngioTool.

[0086] Quantitative reverse transcription polymerase chain reaction (RT-qPCR): RNA was extracted using TRIzol, reverse transcribed, and amplified using SYBR Green on a real-time PCR system. Primers targeted vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), hypoxia-inducible factor 1-α (HIF-1α), and fibroblast growth factor 2 (FGF2); using 2 -ΔΔCt The method normalizes expression levels to GAPDH. Primer information is shown in the table below: like Figure 5 As shown in AB, under the same culture and NIR conditions, HUVECs exposed to MXene@Cu-MOF exhibited a higher EdU labeling rate than the PBS and GelMA controls. Embedding the MXene@Cu-MOF heterostructure into GelMA maintained or further enhanced the percentage of EdU++ nuclei, suggesting that the hydrogel shell does not hinder and may help maintain proliferative stimulation.

[0087] like Figure 5 As shown in CD, in the monolayer scratch test, both the PBS and GelMA groups showed baseline closure over time. MXene@Cu-MOF accelerated wound gap reduction, while MXene@Cu-MOF / GelMA produced the fastest closure under a standardized NIR protocol (using an 808 nm continuous-wave near-infrared laser, irradiated at a power density of 1.5 W / cm² for 10 min), consistent with improved migration ability.

[0088] like Figure 5 As shown in EG, on growth factor-reduced Matrigel, MXene@Cu-MOF increased the complexity of capillary-like networks compared to PBS and GelMA, manifested as more branching points and longer main tube lengths; the MXene@Cu-MOF / GelMA group produced the most complex network.

[0089] like Figure 5 RT-qPCR as shown in HK revealed upregulation of VEGF, eNOS, HIF-1α, and FGF2 in HUVECs treated with MXene@Cu-MOF, with the MXene@Cu-MOF / GelMA group showing the largest increase compared to the control. Normalization was applied to housekeeping genes and the blank control group.

[0090] Test Example 7: Animal Research Male C57BL / 6 mice (8-10 weeks old, 22-26 g) were induced to have diabetes via intraperitoneal injection of streptozotocin (50 mg / kg for 5 consecutive days). Fasting blood glucose was measured one week later; mice with blood glucose levels higher than 300 mg / dL were included. Under isoflurane anesthesia, dorsal hair was removed, and two full-thickness excision wounds were created using a 6 mm biopsy puncturist. Silicone splints were applied around each wound to minimize contraction. The wounds were inoculated with 10 7 CFU (Staphylococcus aureus) S.aureus ATCC 25923) or Staphylococcus aureus ( S.aureus ATCC 25923) and Escherichia coli ( E.coil A 1:1 mixture of PBS (ATCC 25922) (dissolved in 20-30 μL PBS) is used to establish infection. Immediate treatment is given: PBS, GelMA, MXene / GelMA, Cu-MOF / GelMA, or MXene@Cu-MOF / GelMA. Under specified conditions, NIR irradiation (808 nm, 1.0-1.5 W / cm²) is performed once daily for the first three to five days using a fixed distance and spot size. 2Wound photographs were taken with a ruler on days 0, 5, 9, and 14; area was quantified using area measurement in ImageJ. On days 9 and 14, animals were euthanized and wound tissue with healthy skin margins was collected, fixed in 4% paraformaldehyde, and embedded in paraffin. Sections (5 µm) were stained with hematoxylin and eosin (H&E) for general morphology and epithelial regeneration assessment, and Masson's trichrome staining for collagen deposition. Epidermal thickness was measured at a normalized distance from the wound center. For immunohistochemistry, antigen retrieval was performed in citrate buffer, followed by blocking, incubation with a primary antibody against type I or type III collagen, and HRP-based detection. Positive area fractions were quantified from at least five random fields of view per section using a blinded analysis. Bacterial load was assessed using a panbacterial probe by fluorescence in situ hybridization (FISH) and counterstained with DAPI. Tissue reactive oxygen species were assessed by dihydroethidium (DHE) staining and imaging under fixed exposure settings. Macrophages were labeled with antibodies against CD68 (a pan-macrophage marker) and CD206 (M2-associated), and blood vessels were observed using CD31 immunofluorescence. For each marker, at least five fields of view were analyzed for each slide and three slides for each wound. Quantification included bacterial count, mean DHE fluorescence intensity, and CD68 concentration for each field of view. + and CD206 +Cell counts and CD31[+] microvessel counts per field of view. Total RNA was extracted from wound tissues treated with MXene@Cu-MOF / GelMA (EXP) and GelMA (CON) on day 14, and RNA integrity (RIN ≥ 7) was confirmed using the RNeasy kit. Poly(A) libraries were prepared according to the manufacturer’s instructions using the NEBNextUltra II Directed RNA Library Preparation Kit (catalog number E7760S, New England Biolabs, USA). Sequencing was performed on an Illumina NovaSeq 6000, yielding 150 bp paired-end reads. Reads were subjected to quality checks (FastQC), trimming, alignment to the mouse reference genome (GRCm39) (STAR), and gene-level counts were generated using featureCounts. Differential expression analysis was performed using DESeq2 with default dispersion estimates and Benjamini–Hochberg correction. Significance was defined as adjusted P < 0.05 and absolute log2 fold change ≥ 1, unless otherwise stated. Variance-stabilized data were used for principal component analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) overcharacterization analyses were performed using ClusterProfiler; Gene Set Enrichment Analysis (GSEA) was performed using the MSigDB gene set and fgsea. Heatmaps display z-scores at the gene level. ImageJ, AngioTool, QuPath, and vendor software were used for image segmentation and quantization. Regions of interest and exposure settings were kept constant within each experiment. For infrared thermal imaging, the same emissivity settings and regions were used across groups. For flow cytometry, debris and duplexes were removed by gating before analyzing live single cells.

[0091] like Figure 6 As shown in Figure A, in a bacterially infected diabetic wound model, the PBS and GelMA control groups showed a gradual reduction in wound area over two weeks, while MXene / GelMA and Cu-MOF / GelMA accelerated closure. The MXene@Cu-MOF / GelMA group exhibited the most significant contraction on days 5, 9, and 14. Figure 6 The area measurement overlay plot shown in B reveals that the healing trajectory of MXene@Cu-MOF / GelMA is steeper compared to all comparison groups, indicating a faster progression from the inflammatory phase to the tissue-forming phase. Figure 6 As shown in CD, H&E sections on days 9 and 14 revealed that MXene@Cu-MOF / GelMA exhibited a more continuous epithelial tongue and thicker neoepithelial epidermis compared to the control group, with both MXene / GelMA and Cu-MOF / GelMA showing moderate improvement. Figure 6 Masson's trichrome staining as shown in EF revealed a larger fraction of collagen-positive areas in MXene@Cu-MOF / GelMA on days 9 and 14. Figure 6 Immunohistochemistry as shown in GJ indicates that, compared to the control group, type III collagen was higher on day 9, shifting to type I collagen by day 14.

[0092] like Figure 7 As shown in Figure AB, FISH imaging on days 3 and 6 revealed dense bacterial signals in PBS and GelMA, decreased signals in MXene / GelMA and Cu-MOF / GelMA, and the lowest bacterial load in MXene@Cu-MOF / GelMA. Figure 7 As shown in CD, dihydroethidium staining on days 9 and 14 revealed high fluorescence associated with superoxide anion in PBS and GelMA, decreased signal in MXene / GelMA and Cu-MOF / GelMA, and the lowest level in MXene@Cu-MOF / GelMA. Figure 7 As shown in EH, CD206 immunofluorescence (M2-related) increased in MXene@Cu-MOF / GelMA relative to the control group on days 9 and 14, while CD68+ cell counts (pan-macrophages) decreased or returned to normal levels compared to the inflammatory control group. This characteristic suggests a shift to a pro-remission phenotype rather than simple exhaustion. Figure 7 CD31 staining as shown in IJ revealed that on days 9 and 14, the number of microvessels in MXene@Cu-MOF / GelMA was greater than in the control group, consistent with enhanced angiogenesis.

[0093] like Figure 8 Principal component analysis (PCA) of variance-stabilized counts, as shown in Figure A, revealed significant separation between the experimental group (EXP; MXene@Cu-MOF / GelMA treatment) and the control group (CON; GelMA treatment) wounds. Volcano plots and heatmap views identified differentially expressed genes (DEGs) that met the adjusted significance threshold. Figure 8 BC). Programs upregulated in EXP include antimicrobial defense, controlled inflammation regulation, angiogenesis, and extracellular matrix tissue (BC). Figure 8 D). GO / KEGG overcharacterization analysis ( Figure 8 E) and GSEA Figure 8 F) showed that, compared to the control, the EXP pathways were positively enriched for immune defense, angiogenesis, and new collagen formation, consistent with histological and immunofluorescence readings.

[0094] The above diagram illustrates a cascade, on-demand treatment procedure using the MXene@Cu-MOF heterostructure hydrogel of this invention for infected diabetic wounds, as shown below. Figure 16 As shown: Phase I (Days 0-3): Exposed MXene surfaces scavenged ROS and attenuated inflammatory signals; the weakly acidic infection microenvironment induced pH-responsive Cu-MOF degradation, accompanied by baseline Cu degradation. 2 + Released for antibacterial action, providing a self-regulating dose that scales with acidity. Stage II (upgrade on demand): NIR irradiation activates the photothermal conversion of MXene, increasing local temperature; interfacial heating accelerates MOF decomposition and generates transient Cu. 2+ The outbreak generates a photothermal-chemical synergy, killing planktonic bacteria, disrupting biofilms, and reducing bacterial load, while the hydrogel matrix helps confine heat within the wound bed. Stage III (days 3-14): As the infection subsides, residual slow-release Cu... 2+ It supports endothelial cell migration and tubular formation, promotes angiogenesis and collagen remodeling, and facilitates macrophage polarization toward a repair phenotype.

[0095] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing an MXene@Cu-MOF heterostructure hydrogel, characterized in that, Includes the following steps: S1. Ti3AlC2 is produced by selectively etching it using a LiF / HCl mixture, followed by layering to obtain Ti3C2Tx, which is MXene nanosheets; S2. Cu-MOF is grown in situ on MXene nanosheets to obtain MXene@Cu-MOF; S3. MXene@Cu-MOF is dispersed in a GelMA prepolymer solution and polymerized to form an MXene@Cu-MOF heterostructure hydrogel.

2. The method for preparing the MXene@Cu-MOF heterostructure hydrogel according to claim 1, characterized in that, In step S1, LiF is dissolved in HCl solution, Ti3AlC2 powder is slowly added under ice bath conditions for etching, centrifugation is performed, and the precipitate is washed until the pH of the solution containing the precipitate is weakly acidic. The precipitate is then sonicated under an inert atmosphere and ice bath conditions to separate into layers, thus obtaining MXene nanosheets.

3. The preparation method according to claim 2, characterized in that, The mass ratio of LiF and Ti3AlC2 powders is (0.8~1.6):1; the concentration of the HCl solution is 9 M. The etching temperature is (30~40)℃, and the etching time is (20~30)h; Wash the precipitate until the pH of the solution containing the precipitate is (5~7). The ultrasound session lasts 30 to 60 minutes.

4. The method for preparing the MXene@Cu-MOF heterostructure hydrogel according to claim 1, characterized in that, In step S2, the Cu-MOF is selected from either HKUST-1 or Cu-doped ZIF-8.

5. The method for preparing the MXene@Cu-MOF heterostructure hydrogel according to claim 4, characterized in that, When Cu-MOF is HKUST-1, MXene nanosheet dispersion is mixed with Cu(NO3)2-containing aqueous solution-ethanol, 1,3,5-pyromellitic acid is slowly added, the reaction is carried out under acidic conditions, the product is collected by centrifugation, and MXene@Cu-MOF composite material is obtained. When Cu-MOF is Cu-doped ZIF-8, the MXene nanosheet dispersion is mixed with Zn-containing... 2+ and Cu 2+ The solution was pre-equilibrated, and 2-methylimidazole solution was quickly added to induce Cu-doped ZIF-8 growth, thus obtaining MXene@Cu-MOF composite material.

6. The method for preparing the MXene@Cu-MOF heterostructure hydrogel according to claim 5, characterized in that, When Cu-MOF is HKUST-1, the ratio of MXene nanosheet dispersion, Cu(NO3)2-containing aqueous solution-ethanol, and 1,3,5-pyromellitic acid is 1 g:(5~60) mL:(2~30) g. The Cu(NO3)2 aqueous solution-ethanol was obtained by mixing Cu(NO3)2 aqueous solution and ethanol in a 1:1 volume ratio, and the Cu(NO3)2 concentration was (10~50) mM. The reaction conditions under acidic conditions are: (25~60)℃, pH (3~4) for (2~12) h.

7. The method for preparing the MXene@Cu-MOF heterostructure hydrogel according to claim 5, characterized in that, When Cu-MOF is Cu-doped ZIF-8, MXene nanosheet dispersion, Zn-containing 2+ and Cu 2+ The volume ratio of the solution and the 2-methylimidazole solution was 2:1:1, wherein the solution contained Zn 2+ and Cu 2+ The total metal ion concentration in the solution is 20~100 mM, Zn 2+ With Cu 2+ The molar ratio was 95:5~80:20, the concentration of 2-methylimidazole solution was 200~800 mM, and the concentration of MXene dispersion was (1.0~5.0) mg / mL.

8. The method for preparing the MXene@Cu-MOF heterostructure hydrogel according to claim 1, characterized in that, In step S3, GelMA and photoinitiator are dissolved in PBS solution to obtain a prepolymer solution. MXene@Cu-MOF is dispersed in the prepolymer solution and photopolymerized to form an MXene@Cu-MOF heterostructure hydrogel.

9. The method for preparing the MXene@Cu-MOF heterostructure hydrogel according to claim 8, characterized in that, The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid. The mass fraction of photoinitiator in PBS solution is 0.05–0.1 wt%, and the mass fraction of GelMA in PBS solution is 5–12 wt%. The concentration of MXene@Cu-MOF dispersed in the prepolymer solution was 0.5~1.5 mg / mL, calculated based on MXene nanosheets. The polymerization conditions were 405 nm and 10–20 mW / cm². 2 Photopolymerization for 30-60 seconds.

10. The application of MXene@Cu-MOF heterostructure hydrogel in the preparation of diabetic wound healing drugs, characterized in that, The MXene@Cu-MOF heterostructure hydrogel is obtained by any of the preparation methods described in claims 1 to 9.

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