A photothermal synergistic GCP nanenzyme composite hydrogel, a preparation method thereof and application thereof in promoting healing of diabetic wounds

Abstract

The present application relates to the technical field of biological medicine, in particular to a kind of photothermal synergic GCP nanenzyme composite hydrogel and its preparation method and application in promoting diabetic wound healing.The present application successfully constructs a kind of photothermal synergic GCP nanenzyme composite hydrogel system (GCP@PG hydrogel), aims to adjust the oxidative stress microenvironment of diabetic wound by "photothermal-enzyme catalysis synergy" strategy, and combined with photothermal therapy synergistically promotes wound healing.Experiments in vitro and in vivo show that GCP@PG can effectively remove intracellular ROS and promote angiogenesis.In the full-thickness skin defect model of diabetic rats, the wound closure rate reaches 97.95% on the 14th day.The composite system is synergistically integrated by enzyme catalysis and photothermal effect, providing a new treatment strategy for diabetic wound repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a photothermal synergistic GCP nanozyme composite hydrogel, its preparation method, and its application in promoting diabetic wound healing. Background Technology

[0002] In diabetic wounds, multiple reactive oxygen species (ROS) intertwine to form a complex oxidative stress network, directly damaging cells and tissues. Currently, most materials with single enzyme mimicry activity can only target and eliminate specific ROS, failing to address the complex oxidative stress network and thus hindering wound healing. Therefore, diabetic wound healing is a pressing clinical challenge worldwide. Its pathological microenvironment is characterized by persistent inflammation, tissue hypoxia, and excessive reactive oxygen species (ROS). These factors intertwine to form a vicious cycle, hindering the wound repair process. Oxidative stress, as a core mechanism, directly damages cells, delays epithelial regeneration, and disrupts the extracellular matrix. Although various antioxidant-based treatments exist, traditional materials often suffer from poor stability and uncontrolled release, making it difficult to maintain long-term efficacy. Therefore, developing novel intelligent treatment systems that can synergistically alleviate multiple pathological abnormalities in wounds is of great significance for promoting diabetic wound healing. Summary of the Invention

[0003] The purpose of this invention is to provide a photothermal synergistic GCP nanozyme composite hydrogel, its preparation method, and its application in promoting diabetic wound healing, so as to solve the problems existing in the prior art.

[0004] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing a photothermal synergistic GCP nanozyme composite hydrogel, comprising the following steps: Cerium nitrate hexahydrate and gadolinium nitrate hexahydrate were mixed, and then acetic acid and ethylene glycol were added for a first hydrothermal treatment to obtain GC; The GC, ethylene glycol, PVP solution and chloroplatinic acid hexahydrate solution were mixed and subjected to a second hydrothermal reaction to obtain Gd / CeO2-Pt; A methacrylic acid solution and a gelatin solution were mixed, dialyzed, and freeze-dried to obtain GelMA; The GelMA, N-isopropylacrylamide, methylenebisacrylamide and I2959 were mixed to obtain PG hydrogel; The Gd / CeO2-Pt and the PG hydrogel were mixed and cross-linked to obtain a photothermal synergistic GCP nanoenzyme composite hydrogel.

[0005] Optionally, the mixture obtained by mixing cerium nitrate hexahydrate and gadolinium nitrate hexahydrate contains Ce. 3+and Gd 3+ The molar ratio is (1~4):1; The volume ratio of the mixture, the acetic acid, and the ethylene glycol is 2:2:52.

[0006] Optionally, the mass-to-volume ratio of the GC, the ethylene glycol, the PVP solution, and the chloroplatinic acid hexahydrate solution is 50 mg: 2.5 mL: 94 μL: 47 μL.

[0007] Optionally, the volume ratio of the methacrylic acid solution to the gelatin solution is 1.1:100; The methacrylic acid solution contains 1% methacrylic acid by volume; the gelatin solution contains 10% gelatin by mass.

[0008] Optionally, the mass ratio of GelMA, N-isopropylacrylamide, methylenebisacrylamide, and I2959 is 24:120:18:18.

[0009] Optionally, the mass-to-volume ratio of the Gd / CeO2-Pt to the PG hydrogel is (0.1~0.5) mg: 1 mL; The cross-linking curing conditions are: irradiation under 365 nm ultraviolet light for 3-5 minutes.

[0010] More preferably, the temperature of both the first hydrothermal reaction and the second hydrothermal reaction is 180°C, and the time is 200 minutes.

[0011] This invention provides a photothermal synergistic GCP nanozyme composite hydrogel prepared using the above-described preparation method.

[0012] This invention successfully developed a photothermal synergistic GCP nanozyme composite hydrogel system (GCP@PG hydrogel). This system combines efficient photothermal conversion with catalytic antioxidant functions to promote wound healing in diabetic patients. In vitro experiments confirmed that under high glucose and low oxygen conditions, GCP@PG significantly enhances endothelial cell viability, migration ability, and tubular structure formation. Through the photothermal-nanozyme synergistic mechanism, this system effectively scavenges intracellular reactive oxygen species, stabilizes mitochondrial membrane potential, and downregulates the expression of HIF-1α and pro-inflammatory factors. In a diabetic rat model, GCP@PG combined with near-infrared light irradiation significantly accelerated wound closure (healing rate reached 97.95% on day 14), promoted granulation tissue formation, collagen deposition, and angiogenesis, and induced macrophage polarization towards the M2 anti-inflammatory phenotype. Specific embodiments of this invention elucidate the synergistic mechanism of "photothermal therapy-nanozyme catalysis" in regulating multiple pathological abnormalities in diabetic wounds, providing new theoretical basis and experimental evidence for the development of intelligent and efficient wound dressings.

[0013] This invention provides the application of the above-mentioned photothermal synergistic GCP nanoenzyme composite hydrogel in the preparation of products that promote the healing of diabetic wounds.

[0014] More preferably, the product includes a medicine, dressing, or wound care material.

[0015] This invention provides a product that promotes wound healing in diabetic patients, the product comprising the above-mentioned photothermal synergistic GCP nanoenzyme composite hydrogel.

[0016] The present invention provides a system for promoting the healing of diabetic wounds, the system comprising the above-mentioned photothermal synergistic GCP nanoenzyme composite hydrogel and near-infrared irradiation instrument.

[0017] The present invention discloses the following technical effects: This invention successfully constructed a photothermal synergistic GCP nanozyme composite hydrogel system (GCP@PG hydrogel), aiming to regulate the oxidative stress microenvironment of diabetic wounds through a "photothermal-enzyme synergistic" strategy and synergistically promote wound healing in conjunction with photothermal therapy. First, cerium oxide nanoparticles (Gd / cerium oxide) with abundant oxygen vacancies, high photothermal conversion efficiency, and high activity were prepared by gadolinium (Gd) doping. Subsequently, platinum nanoclusters were grown on their surface to construct a Gd / CeO2-Pt composite nanozyme (GCP). This invention integrates photothermal therapy and nanozyme catalysis through the synergistic effect between platinum and Gd-doped cerium oxide (GC). Gd doping enhances the simulated activity of the cerium oxide matrix for catalase (CAT) by increasing the oxygen vacancy concentration, while the introduction of Pt nanoclusters endows the nanozyme with highly efficient photothermal conversion capabilities, achieving multi-target regulation of diabetic wounds. To achieve stable retention of nanozymes at the wound site, the prepared nanozyme (GCP) was loaded into an in-situ photocrosslinked hydrogel (within a toughened PNIPAM / hydrogel matrix) to form a composite dressing, GCP@PG. This system aims to dynamically regulate the microenvironment of diabetic wounds through the synergistic effects of multiple functions, including antioxidant and photothermal therapy, thereby providing a new strategy for efficient and intelligent wound healing. In vitro and in vivo experiments showed that GCP@PG effectively scavenges intracellular ROS and promotes angiogenesis. In a diabetic rat model of full-thickness skin defects, the wound closure rate reached 97.95% on day 14. This composite system, through the synergistic integration of enzymatic catalysis and photothermal effects, provides a novel therapeutic strategy for diabetic wound repair. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Characterization of the synthesized materials cerium oxide, GC, and GCP; where (a)~(c) are SEM images of CeO2, GC, and GCP; (d) and (e) are TEM images of GC and GCP; (f) is the elemental distribution map of GCP; (g) is the particle size distribution histogram; (h)~(j) are XRD, Raman, and XPS scan spectra; (k) is the high-resolution Ce 3d spectrum; and (l) is the O 1s XPS spectrum. Figure 2 For the evaluation of photothermal and antioxidant properties, (a) are micrographs of water, cerium oxide, GC, and GCP under NIR irradiation; (b) are the corresponding heating curves; (c) are the heating curves of GCP at different concentrations; (d) are the heating curves of GCP at different laser power densities; (e) are the temperature curves of GCP after five laser switching cycles; (f) are micrographs of O2 generation by different materials; (g) are the decomposition rate of hydrogen peroxide; (h) are the spectral absorption images of different materials for DPPH; (i) are the UV-Vis spectra; (j) are the quantitative scavenging rates; (k) are the spectral absorption images of different materials for ABTS; (l) are the UV-Vis spectra; and (m) are the quantitative scavenging rates. p<0.05, p<0.01, p < 0.001, n = 4; Figure 3 Characterization and performance analysis of the hydrogels: (a) Micrographs of PG and GCP@PG hydrogels before and after 10 minutes of NIR irradiation; (b) and (c) Scanning electron microscope (SEM) images of PG and GCP@PG; (d) Pore size distribution of the hydrogels; (e) Stress-strain curves of PG and GCP@PG; (f) Compression-rebound curves of PG and GCP@PG (5 cycles); (g) Temperature change curves of PG and GCP@PG under NIR irradiation; (h) Scavenging efficiency of different hydrogels on (ABTS)DPPH free radicals; (i) Scavenging effect of different hydrogels on ABTS free radicals. p<0.05, p<0.01, p < 0.001, n = 4; Figure 4This study evaluates the biocompatibility and angiogenesis-promoting properties of GCP@PG hydrogel. (a) Images of live / dead human umbilical vein endothelial cells (HUVECs) after co-culturing with different hydrogels for 24 and 48 hours under normal conditions; (b) corresponding cell viability data; (c) changes in the cytoskeleton morphology (F-actin / DAPI staining) of HUVECs under high glucose and hypoxia conditions after treatment with different hydrogels; (d) results of live / dead cell fluorescence staining; (e) quantitative cell viability analysis; (f) representative images from the scratch assay; (g) quantitative data on cell migration; (h) representative images from the lumen formation assay; and (i) quantitative analysis results of angiogenesis. p<0.05, p<0.01, p < 0.001, n = 4; Figure 5 The study aimed to evaluate the intracellular antioxidant and anti-inflammatory properties of GCP@PG hydrogel. (a) shows fluorescence images of intracellular reactive oxygen species (ROS) levels in HUVECs treated with different hydrogels; (b) shows the quantitative results by flow cytometry; (c) shows fluorescence images of mitochondrial membrane potential changes detected by the JC1 probe; (d) shows fluorescence images of HIF1α in HUVECs and (e) shows its relative expression level; (f)–(i) show the relative expression levels of TNF-α, IL-1β, IL-10, and Arg-1 in HUVECs. p<0.05, p<0.01, p < 0.001, n = 4; Figure 6 To evaluate the wound repair effect in a diabetic rat model; (a) is a schematic diagram of the experimental design and process; (b) shows the morphological characteristics and contours of the wound at different time points (scale bar: 20 mm); (c) shows the statistical analysis of the wound closure rate; (d) shows the H&E staining of the wound tissue on day 7 and day 14; (e) shows the histological analysis of Masson trichrome staining. p<0.05, p<0.01, p < 0.001, n = 6; Figure 7 The study investigated the anti-inflammatory and angiogenic effects of GCP@PG in vivo. (a) showed the immunohistochemical staining results for CD86 and CD206; (b) showed the quantitative analysis of CD86 expression levels; (c) showed the quantitative analysis of CD206 expression levels; (d) showed the immunofluorescence staining results for CD31 and α-SMA; (e) showed the quantitative analysis of CD31 expression levels; and (f) showed the quantitative analysis of α-SMA expression levels. p<0.05, p<0.01, p < 0.001, n = 6; Figure 8 SEM images of GC at different Ce:Gd ratios: (a) 4:1; (b) 3:1; (c) 2:1; (d) 1:1; Figure 9 XRD patterns of different materials; Figure 10 Absorption spectra of DPPH(a) and ABTS(b) free radical scavenging experiments on different materials; Figure 11 Zeta potentials for CeO2, GC, and GCP Figure 12 High-resolution transmission electron microscopy (HRTEM) images of different materials; where (a) is a TEM image of cerium oxide; (b) is a GC lattice image; and (c) is a GCP lattice image. Figure 13 High-resolution XPS spectra of platinum; Figure 14 EPR spectrum of oxygen vacancies in GC; Figure 15 The graphs show the photothermal performance evaluation of GCP nanozymes; where (a) is the heating and cooling curve of GCP; and (b) is the photothermal conversion efficiency of GCP. Figure 16 The cyclic voltammetry curves (relative to Ag / silver chloride) were recorded on the GC electrode. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1 I. Experimental Methods 1. Synthesis of Nanomaterials Synthesis of cerium oxide nanoparticles: Ce(NO3)3·6H2O (4.6 mmol) was dissolved in 2 mL of deionized water, followed by the addition of 2 mL of acetic acid and 52 mL of ethylene glycol. The mixture was placed in a polytetrafluoroethylene-lined autoclave and heated at 180 °C for 200 min. The final product was centrifuged, thoroughly washed, and dried to obtain cerium oxide nanoparticles.

[0026] Synthesis of Gd-doped cerium oxide (GC): For a sample with a nominal Ce:Gd feed ratio of 1:1, Ce(NO3)3·6H2O (2.3 mmol) and Gd(NO3)3·6H2O (2.3 mmol) were dissolved together in 2 mL of deionized water. Then, 2 mL of acetic acid and 52 mL of ethylene glycol were added. The mixture was placed in a polytetrafluoroethylene-lined autoclave and subjected to hydrothermal treatment at 180 °C for 200 minutes. The resulting solid was collected by centrifugation, washed, and dried to obtain the product named GC(1:1). By adjusting the precursor amounts according to the same scheme, samples with different Ce:Gd feed ratios can also be prepared.

[0027] For Ce:Gd (2:1) = Ce36H2O (3.07 mmol) and Gd36H2O (1.53 mmol).

[0028] For Ce:Gd (3:1) = Ce3·6H2O (3.45 mmol) and Gd3·6H2O (1.15 mmol).

[0029] For Ce:Gd (4:1) = Ce3·6H2O (3.68 mmol) and Gd3·6H2O (0.92 mmol).

[0030] The resulting products were labeled as GC (2:1), GC (3:1), and GC (4:1), respectively.

[0031] Synthesis of Gd / CeO2-Pt (GCP): First, GC nanoparticles (50 mg) were dispersed in 2.5 mL of ethylene glycol and stirred at 120 °C for 10 min. Simultaneously, aqueous solutions of PVP (125 mg dissolved in 3 mL of water) and H2PtCl6·6H2O (13 mg dissolved in 1.5 mL of water) were prepared. Then, under vigorous stirring, 94 μL of PVP solution and 47 μL of H2PtCl6 solution were added dropwise to the GC dispersion at 30-second intervals for 16 min. The reaction was maintained at 120 °C for another 10 min, and then the mixture was cooled to room temperature. The product was collected by centrifugation, washed repeatedly with deionized water and ethanol, and dried to obtain GCP.

[0032] The characterization methods for nanomaterials are as follows: morphology and elemental distribution were analyzed using a scanning electron microscope / energy dispersive spectroscopy (Zeiss GeminiSEM 360); surface zeta potential was determined using a Malvern Zetasizer Pro instrument; and PXRD was performed using a Rigaku SmartLab (Cu Kα, 5-80 2θ, scanning speed 10 min). -1 The crystal structure was analyzed, and chemical bonds were detected using a Fourier transform infrared spectrometer (Shimadzu IRTracer-100, potassium bromide particles).

[0033] 2. Evaluation of GCP photothermal performance The photothermal properties of GCP were evaluated by monitoring temperature changes under 808 nm NIR laser irradiation. A dispersion with a concentration of 200 µg / mL was typically used at 0.95 W / cm². 2 Irradiation was performed for 10 minutes at a fixed power density. To investigate the effects on key parameters, irradiation was conducted at a constant power density (0.69 W / cm²). 2 Under different power densities (0.43~1.21 W / cm²), the results were obtained. 2 Temperature rise was recorded for dispersions of 200 µg / mL and dispersions at different concentrations (50–800 µg / mL). Photothermal stability was evaluated by five on / off cycles (10 minutes of irradiation followed by cooling, concentration 200 µg / mL). Photothermal conversion efficiency was determined by measuring the 200 µg / mL dispersion at 0.69 W / cm². 2The temperature was measured by irradiating at power density for 10 minutes and monitoring the cooling curve. All temperature data and infrared images were acquired by a thermal imaging camera.

[0034] 3. Evaluation of GCP-based catalase activity This was accomplished by monitoring the decomposition of hydrogen peroxide. The material suspension (cerium oxide nanoparticles, GC, or GCP) was mixed with 80 mM hydrogen peroxide and incubated at 37°C for 30 minutes, with an 80 mM hydrogen peroxide solution serving as a control. Residual hydrogen peroxide was quantified using a commercially available detection kit. Samples in the GCP+NIR group underwent additional irradiation treatment (808 nm, 0.95 W / cm²). 2 (10 minutes). The activity of different concentrations (50–400 µg / mL) of GCP was also tested under NIR conditions. Catalytic durability was assessed by co-incubating GCP (200 µg / mL) with hydrogen peroxide: the residual hydrogen peroxide concentration was measured periodically over 30 minutes, followed by centrifugation and re-incubation with fresh hydrogen peroxide for five cycles.

[0035] 4. Evaluation of GCP's antioxidant properties 1,1-Diphenyl-2-picrylhydrazine radical (DPPH) scavenging assay: 200 µL of each sample suspension (including vitamin C (VC, as a positive control), cerium oxide nanoparticles, GC, or GCP) was mixed with 10 mM DPPH in ethanol. After incubation at 37°C for 30 minutes, the reaction mixture was centrifuged. The absorbance of the resulting supernatant was then recorded at 517 nm, with a sample-free DPPH ethanol solution serving as a control.

[0036] 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) scavenging assay: 200 µL of the corresponding material suspension (VC, cerium oxide nanoparticles, GC, or GCP) was reacted with freshly prepared ABTS working solution. After incubation at 37°C for 6 minutes, the sample was centrifuged. The absorbance of the clear supernatant was then measured at 734 nm, with the ABTS working solution without the sample serving as a control.

[0037] The formulas for calculating DPPH and ABTS clearance rates are as follows: ; OD 0、 OD 1 and OD 2 represents the absorbance values ​​of the control group, VC group, and experimental material group, respectively.

[0038] 5. Synthesis and characterization of GCP@PG hydrogel Preparation of gelatin methacrylate (GelMA): 1.1 mL of methacrylic acid (1% v / v) was added to 100 mL of 10% (w / v) gelatin solution, and the mixture was magnetically stirred at 50 °C for 3 hours. Subsequently, the mixture was dialyzed at 40 °C for 7 days using a dialysis membrane (molecular weight MWCO: 12-14 kDa) to remove unreacted reagents and byproducts. Finally, the purified product GelMA was obtained by freeze-drying at -80 to -50 °C (-80 °C in this example) for 2880 to 4320 min (i.e., 48 to 72 hours, 64 hours in this example).

[0039] Preparation of PNIPAM / GelMA hydrogel (PG): N-isopropylacrylamide (NIPAM, 120 mg), GelMA (24 mg), and methylenebisacrylamide (MBA, 18 mg) were dissolved in 1 mL of deionized water. The resulting solution was poured into a specially designed mold, and I2959 (18 mg) was added. Subsequently, a photopolymerization reaction was carried out under ultraviolet light irradiation to finally obtain the PG hydrogel.

[0040] Preparation of GCP@PG hydrogel: Gd / CeO2-Pt and PG hydrogel were mixed at a mass-volume ratio of 1 mg: 5 mL, and photocrosslinked and cured under 365 nm ultraviolet light for 4 minutes to obtain photothermal synergistic GCP nanoenzyme composite hydrogel.

[0041] Figure 3 (a) illustrates the photothermal response of PG and GCP@PG hydrogels under NIR irradiation. The specific experimental procedure involved placing equal volumes of PG and GCP@PG hydrogels on a glass slide and using a near-infrared laser (808 nm, power density typically set to 0.95 W / cm²). 2 The surface of the hydrogel was subjected to continuous irradiation. The temperature change of the hydrogel surface was monitored and recorded in real time over 10 minutes using an infrared thermal imager to evaluate the photothermal conversion efficiency and stability of the GCP nanozyme in the hydrogel matrix.

[0042] Compression-rebound cycle test: A cylindrical GCP@PG hydrogel specimen (diameter: 14.78 mm; height: 5.46 mm) was compressed on a universal testing machine at a beam speed of 2 mm / min until 80% of the initial height was reached.

[0043] The test methods for scavenging DPPH and ABTS radicals are the same as in "4. Evaluation of GCP antioxidant performance". Specifically, the PG group used PG; the GCP@PG group used GCP@PG; and the GCP@PG+ group used a combination of GCP@PG and NIR, with NIR conditions of 808 nm and 0.95 W / cm². 2 10 minutes.

[0044] 6. Biocompatibility evaluation CCK-8 assay and live / dead cell staining: Biocompatibility assessment was performed using human umbilical vein endothelial cells (HUVECs), and the experimental procedures are as follows: Cell preparation: HUVECs were seeded into 6-well plates (or 96-well plates) and cultured in a 37°C, 5% CO2 incubator until confluence reached approximately 80-90%.

[0045] High glucose treatment: Discard the original culture medium and replace it with high glucose DMEM medium (containing 10% FBS) containing 30 mM D-glucose, and pre-culture for 6-12 hours.

[0046] Hypoxia induction: The cells were then transferred to a three-gas incubator (1% O2, 5% CO2, 94% N2) and cultured for 24 hours under hypoxic conditions to construct a cell damage model of HG+Hypoxia (i.e., high glucose hypoxia model, Model group).

[0047] Control group setup: Control cells were cultured in conventional DMEM medium (low glucose / standard glucose, approximately 5.5 mM) and in a normoxic environment (21% O2, 5% CO2).

[0048] HUVECs were seeded in 96-well plates at a seeding density of 1.5 × 10⁶ cells / well. 3 ~1.5×10 4 Inoculation per well (in this example, the inoculation amount is 1.5 × 10⁻⁶) 4 (Number / well), respectively using materials (PG group, GCP@PG group or GCP@PG+NIR group (GCP@PG+ group, NIR conditions are 808 nm, 0.95 W / cm²). 2Cells were treated for 10 minutes (10 minutes) for 24 hours and 48 hours, with material concentrations of 0, 50, 100, 200, and 400 μg / mL. Conventional culture medium served as the control group (Control or Normal), while cells cultured under high glucose and hypoxic conditions served as the model group (Model). Cell viability was then measured using a CCK-8 assay kit (absorbance was detected using a microplate reader), and staining was performed using a live / dead cell staining kit (Beyotime Biotechnology Co., Ltd.). Finally, images were acquired using a fluorescence microscope (Olympus CKX53).

[0049] Cytoskeleton staining: HUVECs were incubated with hydrogel extract for 24 hours, then fixed, and the nuclei were stained with rhodamine-phalloidin for F-actin and DAPI for nuclei. Cell spreading morphology was then observed using confocal laser scanning microscopy (CLSM).

[0050] 7. Cell scratch test Human umbilical vein endothelial cells (HUVECs) were used for scratch assays. Uniform scratches were created on a monolayer of cells using a sterile pipette tip, and the wound area was imaged at 0 and 24 hours post-scraping. ImageJ software was then used for quantitative analysis of scratch closure. The specific steps are as follows: Cell seeding: HUVECs (human umbilical vein endothelial cells) were seeded into 6-well plates and cultured until confluence reached 90% or more.

[0051] Create a scratch: Use a 200 μL sterile pipette tip to make a straight scratch in the center of the cell monolayer, and wash the detached cells with PBS.

[0052] Drug treatment: Replace with serum-free culture medium containing 100~400 μg / mL GCP@PG (the final concentration of GCP@PG in this example is 200 μg / mL). Observation and recording: Observe the cell migration in the scratched area using an inverted microscope at 0, 12 and 24 hours and take pictures.

[0053] Data analysis: ImageJ software was used to measure the scratch width and calculate the cell migration rate: Migration rate = (Initial scratch area - Scratch area at measurement) / Initial scratch area × 100%.

[0054] 8. Assessment of pro-angiogenic potential To evaluate in vitro angiogenesis activity, a tubular formation assay was used. HUVECs were seeded at a density of 5,000 cells / well in 96-well plates pre-coated with 50 μL Matrigel. After treatment with GCP@PG at concentrations of 100–400 μg / mL (the final concentration of GCP@PG in this example was 200 μg / mL), the cells were incubated for 24 hours. The tubular network structure was then observed using fluorescence microscopy, and quantitative analysis was performed using ImageJ software.

[0055] 9. Intracellular reactive oxygen species scavenging experiment The intracellular reactive oxygen species (ROS) scavenging capacity of the materials was assessed using the DCFH-DA probe. HUVECs (at a concentration of 1×10⁻⁶) were used. 5 Cells were treated with different hydrogels (final concentration of hydrogel was 100-400 μg / mL, and in this example, the final concentration of hydrogel was 200 μg / mL) in the presence of 0.1 mM hydrogen peroxide for 4 hours. Cells were then stained with DCFH-DA and imaged using a fluorescence microscope. PBS was used as a negative control (Normal), and cells treated with pure 0.1 mM hydrogen peroxide served as the model group (Model). Fluorescence intensity was also quantitatively analyzed by flow cytometry.

[0056] 10. Intracellular mitochondrial membrane potential detection experiment HUVECs were 1×10 5 ~2×10 5 Density of cells / pore (2×10⁻⁶ in this embodiment) 5 The cells were seeded at a density of cells / well in 6-well plates and placed in a high glucose hypoxia model. Different forms of hydrogel material (GCP@PG, effective drug concentration of 200 μg / mL) were added and incubated together.

[0057] After incubation, the culture medium was aspirated, and the cells were washed twice with sterile PBS. Then, serum-free culture medium containing 1× JC-1 working solution was added to each well, and the cells were incubated in the dark at 37°C for 20-30 minutes.

[0058] After incubation, the cells were washed twice with 1× JC-1 staining buffer to remove free probes.

[0059] Finally, the fluorescence color change in each group of cells was observed by fluorescence microscopy, or the relative fluorescence intensity ratio of red light (J-aggregates monomer, emission wavelength of about 590 nm), which represents normal (high potential) mitochondrial membrane potential, and green light (J-monomer monomer, emission wavelength of about 525 nm), which represents decreased (low potential / damage) mitochondrial membrane potential, was quantitatively detected by flow cytometry to evaluate the repair efficacy of the hydrogel system on mitochondrial function of endothelial cells.

[0060] 11. Intracellular HIF-1α expression level and anti-inflammatory factor expression level (1) HIF-1α protein expression assay (Western Blot method): HUVECs cells from each treatment group (Control group, Model group, and different hydrogel groups) were collected, and high-potency RIPA lysis buffer containing protease and phosphatase inhibitors was added. The cells were lysed on ice for 30 minutes, and the supernatant was collected by centrifugation at 12,000 rpm for 15 minutes at 4°C. The total protein concentration was quantified using the BCA method, and denaturation was achieved by boiling with denaturing loading buffer. Equal amounts of protein samples were separated by SDS-PAGE electrophoresis and then transferred to PVDF membranes. The membranes were blocked with 5% skim milk powder at room temperature for 1-2 hours, and HIF-1α antibody and internal control antibody (GAPDH) were added and incubated overnight at 4°C. The membranes were washed three times with TBST the next day, and the corresponding horseradish peroxidase (HRP)-labeled secondary antibody was added and incubated at room temperature for 1 hour. After washing, the bands were developed using a chemiluminescence microscopy (ECL) system, and the relative expression level of HIF-1α was quantitatively analyzed by grayscale value using ImageJ software.

[0061] (2) Determination of expression levels of anti-inflammatory and pro-inflammatory factors (ELISA method): Cell culture supernatant from each group was collected, and the absolute secretion levels of pro-inflammatory factor (IL-6) and anti-inflammatory factor (IL-10) were detected using a specific enzyme-linked immunosorbent assay (ELISA) kit. Following the kit's standard instructions, the absorbance (OD value) of each well was measured at 450 nm. The expression concentration of each factor was calculated based on the standard curve to evaluate the in vitro anti-inflammatory microenvironment regulation ability of the GCP@PG hydrogel system.

[0062] 12. In vivo assessment of GCP@PG's effect on promoting diabetic wound healing All experimental procedures involving animals were conducted in accordance with the National Research Council's Guidelines for the Care and Use of Laboratory Animals. The research protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Shenzhen Baoan Traditional Chinese Medicine Hospital (Approval No.: TOP-2PZ-GM251106). Male SD rats were induced to develop a diabetic model 5 days prior to modeling by intraperitoneal injection of streptozotocin (STZ, 65 mg / kg), and a hyperglycemic state (blood glucose >300 mg / dL) was confirmed preoperatively. Under anesthesia (sodium pentobarbital, 50 mg / kg), a full-thickness circular wound with a diameter of 20 mm was created on the shaved back. Rats were randomly divided into four groups: control group, PG group, GCP@PG group, and GCP@PG+NIR group (GCP@PG+, NIR parameters: 808 nm, 0.95 W / cm²). 2Treatment was administered on days 1, 3, 5, 7, 9, and 11 post-modeling (10 minutes). The injection volume of hydrogel was 200–300 μL per animal, and in this example, it was 250 μL per animal. To record and quantify wound healing, images were taken on days 0, 3, 9, 11, and 14 to assess wound closure. ImageJ software was used for quantitative analysis of healing rate.

[0063] The formula for calculating the wound healing rate is as follows: ; S 0 represents the original wound area. S 1 represents the wound area at the measurement time point.

[0064] Wound tissue samples were collected from euthanized SD rats at designated time points (days 7 and 14 post-injury) and prepared into tissue sections. Tissue regeneration was assessed by hematoxylin and eosin (H&E) staining and Masson's trichrome staining. On day 7, the inflammatory response was detected by immunofluorescence of macrophage markers CD86 and CD206, and angiogenesis was analyzed by co-staining of endothelial marker CD31 and pericyte marker α-SMA.

[0065] 13. Statistical Analysis Unless otherwise stated, results are expressed as mean ± standard deviation of three independent replicates. Statistical significance was assessed by one-way ANOVA combined with the Tukey test (SPSS 27). p<0.05 p<0.01 p<0.001 and p < 0.0001 is considered statistically significant.

[0066] II. Experimental Results 1. Synthesis and Characterization of GCP In this embodiment, a series of cerium oxide nanomaterials with different Gd doping contents were synthesized in situ via a hydrothermal method. The morphology of samples with different doping ratios was observed by scanning electron microscopy (SEM). Figure 8 The images clearly show that as the Gd doping concentration increases, the average particle size gradually decreases, and the particle size distribution becomes more uniform. X-ray diffraction (XRD) spectra indicate that the crystal structure of samples with different doping ratios did not change significantly. Figure 9 This phenomenon may be related to Gd. 3+The doping-induced alteration of grain growth kinetics is relevant. This uniform and size-controllable nanostructure is beneficial for providing high specific surface area and optimizing mass transfer and reaction efficiency in catalytic or antioxidant applications. Furthermore, the antioxidant activity of cerium oxide materials with different Gd doping levels was preliminarily evaluated using UV-Vis absorption spectroscopy. Figure 10 As shown, the antioxidant effect of GC increases with increasing Gd doping concentration. Therefore, considering both morphological characteristics and antioxidant performance, GC with a Ce:Gd doping ratio of 1:1 was selected as the subject of further research.

[0067] Subsequently, Pt nanoclusters were grown on the GC surface via in-situ synthesis, ultimately preparing a nanoplatform GCP with an integrated synergistic photothermal catalytic mechanism. Zeta potential measurements showed that the potential of GCP was significantly lower than that of GC, which may be attributed to the introduction of platinum nanoclusters. Figure 11 The morphology of the synthesized cerium oxide nanoparticles (CeO2), GC, and GCP was observed by SEM, as shown below. Figure 1 As shown in (a)-(c), all prepared materials exhibit a typical spherical structure with a relatively uniform particle size distribution. Statistical analysis of the SEM images ( Figure 1 The (g) results show that the original cerium oxide had the largest average particle size (190.68 nm). After Gd doping, the average particle size of GC decreased significantly to 70.35 nm. The introduction of Pt nanoclusters increased the average particle size of GCP to 95.64 nm. This slight increase may be due to limited aggregation during the loading process. To further elucidate their microstructure, TEM analysis was performed on the GC and GCP samples. High-resolution TEM images showed that both had clear lattice fringes, confirming their good crystallinity. Figure 12 (b)-(c)). After Pt nanoclusters were grown on the surface, numerous fine and uniformly distributed dark spots were observed on the GCP surface. Figure 1 In (e) of the original GC, these dark spots correspond to high atomic number Pt nanoclusters. Figure 1 Compared to (d) in the previous example, the outlines of the matrix particles in GCP remained clear, indicating that Pt loading did not significantly alter the overall morphology of the cerium oxide matrix. Finally, elemental distribution analysis ( Figure 1 (f) shows that Ce, O, Gd, and Pt are uniformly distributed in GCP. This confirms that Gd was successfully doped into the cerium oxide lattice without forming an impurity phase, and also verifies that Pt exists in a highly dispersed state without agglomeration, further demonstrating the successful preparation of GCP.

[0068] Furthermore, the structure of the synthesized material was characterized in detail. XRD patterns ( Figure 1The (h) diagram shows that all samples exhibit distinct diffraction peaks at approximately 28.5, 33.1, 47.5, and 56.3 Å, corresponding to the (111), (200), (220), and (311) crystal planes of cerium oxide, respectively. No independent impurity peaks attributable to Gd or Pt were observed, indicating that Gd and Pt are highly dispersed and do not disrupt the lattice structure of cerium oxide. Raman spectroscopy ( Figure 1 In (i), all samples were at 465 cm. -1 A significant peak was observed nearby, attributed to the symmetric stretching vibration mode (F2g mode) of cerium oxide. Compared to cerium oxide, this characteristic peak broadened and decreased in intensity in GC and GCP samples. This change typically originates from Ce. 4+ Gd 3+ Substitution-induced lattice distortion and local stress. Notably, the increased low-frequency baseline and line broadening of the F2g peak in GC and GCP samples are generally attributed to enhanced defect state signals associated with oxygen vacancies, confirming that Gd doping can effectively modulate and increase oxygen vacancies in cerium oxide. Surface elemental chemical states were further analyzed by XPS. Scanning spectra (...) Figure 1 The presence of Ce, O, Gd, and Pt was clearly confirmed in (j) of the study. High-resolution analysis of the Ce 3d region (j) Figure 1 (k) shows that Ce in GC and GCP samples 3+ / Ce T (Ce) 3+ +Ce 4+ The relative proportion of Ce was significantly higher than that of cerium oxide. 3+ The presence of oxygen vacancies is direct evidence of oxygen vacancy formation to compensate for charge imbalance, and the increased proportion intuitively reflects that Gd doping induces a higher concentration of oxygen vacancies, consistent with the aforementioned Raman data. From the high-resolution O1s spectrum ( Figure 1 As can be seen from (l) in the data, compared with cerium oxide, the ratio of highly reactive adsorbed oxygen to total oxygen (O2) in GC and GCP samples is higher. Sur / O at The concentrations of Pt and Gd increased significantly. In summary, these structural characterizations confirm that Gd doping effectively introduces lattice distortion and oxygen vacancies into the cerium oxide matrix. Furthermore, the coexistence of Pt and Gd further modulates the surface chemical state, which is expected to significantly enhance the surface redox capacity and catalytic activity of the material.

[0069] 2. Evaluation of the photothermal effect and antioxidant properties of GCP The photothermal conversion capability under NIR light is a key indicator for evaluating the biomedical potential of materials. Irradiation with an 808 nm NIR laser (0.95 W / cm²) was achieved. 2 Under 10-minute conditions, the photothermal heating curves of aqueous dispersions of pure cerium oxide, GC, and GCP (all concentrations of 200 μg / mL) and pure water were tested. Figure 2 (a) and (b) in the table. The results showed that the photothermal effects of water, cerium oxide, and GC were negligible, while GCP exhibited excellent photothermal performance, with the solution temperature significantly increasing from 28.5℃ to 44.5℃ within 10 minutes of irradiation. This was attributed to the superior photothermal properties of platinum. The effects of GCP concentration and laser power on its photothermal behavior were systematically evaluated. Figure 2 As shown in (c), at a fixed laser power, the temperature increase of GCP significantly increased with increasing concentration (25-200 μg / mL), exhibiting a clear concentration dependence. Similarly, at a fixed concentration, the heating efficiency was significantly affected by the laser power density (0.5-1.5 W / cm²). 2 ) are positively correlated ( Figure 2 (d) indicates that the photothermal process is efficient and controllable. For example... Figure 2 As shown in Figure e, after five consecutive laser-switched cycles, the heating and cooling curves of the GCP sample highly overlapped with each other and showed no significant performance degradation, demonstrating its excellent photothermal cycling stability and repeatability. This lays a solid foundation for synergistic photothermal therapy in subsequent diabetic wound treatment.

[0070] To systematically evaluate the antioxidant activity of the material, this embodiment performed quantitative analysis to determine its scavenging ability against hydrogen peroxide, DPPH, and ABTS free radicals, and also investigated the synergistic enhancement effect of near-infrared (NIR) irradiation. Figure 2 As shown in (f) of section 1 and (g) of section 2, at the same concentration, the order of hydrogen peroxide decomposition ability is: GCP > GC > cerium oxide. GCP exhibits the highest scavenging efficiency, which is attributed to the abundant oxygen vacancies promoting the redox reaction. Scavenging experiments against two typical free radicals, DPPH and ABTS, further validated the broad-spectrum antioxidant properties of the material. UV-Vis absorption curves ( Figure 2 As shown in (i) of 2 and (l) of 2, the characteristic absorption peaks of DPPH and ABTS were significantly reduced after GCP treatment, which is consistent with the fading phenomenon observed in the photograph. Figure 2 (h) in 2 and (k) in 2). Quantitative statistical results ( Figure 2 (j) in 2 and (m) in 2 further confirm that GCP has the highest scavenging rate, followed by GC, while cerium oxide has the lowest activity.

[0071] The combined results of multiple free radical scavenging experiments demonstrate that the GCP composite exhibits the most superior antioxidant properties. This significant performance improvement is primarily attributed to the introduction of abundant oxygen vacancies into the cerium oxide lattice through gadolinium doping, resulting in rich catalytic active sites. This excellent antioxidant capacity lays a crucial foundation for the effective regulation of the microenvironment in diabetic wounds by the GCP composite.

[0072] 3. Characterization and performance evaluation of GCP@PG hydrogel In this embodiment, the gel was prepared by photocrosslinking of methacrylated gelatin (GelMA). ¹H NMR spectroscopy confirmed the successful synthesis of GelMA, with the proton peak corresponding to the carbon-carbon double bond appearing in the range of 5.0-5.6 ppm. Figure 13 Subsequently, a toughened polyethylene glycol (PG) hydrogel was prepared using GelMA, and a GCP@PG hydrogel was constructed by loading GCP nanoparticles. Both gels could rapidly solidify under UV irradiation. Figure 3 (a) Scanning electron microscopy (SEM) and pore size analysis showed that both PG and GCP@PG hydrogels exhibited a uniform porous network structure. Figure 3 (b) and (c) in the text), and there was no significant difference in pore size. Mechanical properties are crucial for wound dressings to resist skin deformation and maintain structural integrity. Stress-strain curves ( Figure 3 (e) indicates that the mechanical strength of GCP@PG hydrogel is higher than that of pure PG hydrogel. The hydrogel pore size distribution diagram is shown below. Figure 3 As shown in (d), the results indicate that the pore size of the GCP@PG hydrogel is 81.1 μm, and the pore size of PG is 87.5 μm. Compression-rebound cycle test ( Figure 3 (f) in Figure 14 The study confirmed that both hydrogels maintained excellent elastic resilience after five compression cycles. This superior elasticity ensures that the hydrogels can adapt to the dynamic movement of the wound site without structural damage, thus providing a stable environment for wound healing.

[0073] GCP@PG exhibits superior photothermal conversion performance, with its temperature rising to 44.8℃ after 10 minutes of NIR irradiation, significantly higher than the 24.2℃ of ordinary polyethylene glycol (PG). Figure 3 (g) in Figure 15 This confirms that GCP nanoparticles endow hydrogels with highly efficient photothermal conversion capabilities. For example... Figure 3 h in Figure 3 (i) and Figure 16 As shown, GCP@PG hydrogel exhibits significantly higher scavenging rates of DPPH and ABTS free radicals than pure PG and the control group, with scavenging rates reaching approximately 90% in both cases. The organic combination of photothermal properties and antioxidant activity makes GCP@PG hydrogel a promising candidate material for the field of diabetic wound healing.

[0074] 4. Evaluation of the biocompatibility and angiogenesis-promoting properties of GCP@PG hydrogel Biocompatibility is a core prerequisite for the clinical translation of medical wound dressings. This embodiment evaluated the cytotoxicity of PG hydrogel and its GCP@PG composite hydrogel using a human umbilical vein endothelial cell (HUVECs) system through live / dead cell staining, quantitative cell viability assays, and cytoskeleton morphology analysis, providing a safety basis for the in vivo application of the materials. Figure 4 As shown in (a) and (b), after co-culturing with PG, GCP@PG and GCP@PG+ for 24 hours and 48 hours, the cell viability remained above 90% and very few cells died, with no significant difference from the control group, indicating that the material has excellent biocompatibility. Figure 4 As shown in (c), cells treated with GCP@PG or GCP@PG+ exhibited a widely distributed F-actin network and a clear sheet-like pseudopodia structure. This orderly cytoskeleton remodeling and active sheet-like pseudopodia formation process further corroborates the material's good biocompatibility.

[0075] The core pathological feature of chronic diabetic wounds is a hyperglycemic hypoxic microenvironment. This environment significantly hinders endothelial cell proliferation, migration, and angiogenesis by inducing excessive reactive oxygen species (ROS), inhibiting cell metabolism, and disrupting signaling pathways, ultimately leading to delayed wound healing. This embodiment established an in vitro hyperglycemic hypoxic model to simulate the pathological microenvironment of diabetic wounds. Combining the photothermal properties of GCP@PG hydrogel with the antioxidant activity of GCP nanozymes, this embodiment systematically investigated the regulatory effect of this material on endothelial cell function and its pro-angiogenic mechanism. Under these conditions, the cell survival rate in the model group significantly decreased to 46.74% (…). Figure 4 (d) and (e) in the figure). GCP@PG treatment increased the survival rate to 71.46%, and further enhanced it to 87.28% (GCP@PG+) after NIR irradiation, indicating that GCP@PG composite hydrogel combined with photothermal therapy can significantly improve high glucose hypoxia-induced apoptosis.

[0076] Cell migration is a crucial step in wound healing, during which endothelial cells are recruited to the defect area to participate in tissue repair and angiogenesis. Scratch assay results ( Figure 4 (f) and (g) show that under high glucose and low oxygen conditions, the cell migration rate in the model group was only 43.82% after 24 hours. This value increased to 68.98% in the GCP@PG group and further reached 81.81% in the GCP@PG+ group, confirming that photothermal therapy can significantly promote cell migration. In vitro tubular formation experiments further confirmed (…). Figure 4 In (h) and (i) of the model group, the vascular network formed was sparse and incomplete, while the GCP@PG+ group developed a dense and complete tubular network. The total tubular structure length and number of branches in the GCP@PG+ group were more than four times that of the model group, indicating that the system can effectively promote angiogenesis.

[0077] 5. Evaluation of the intracellular antioxidant and anti-inflammatory properties of GCP@PG hydrogel ROS-scavenging biomaterials capable of combating oxidative stress damage are a key strategy for wound healing in diabetic patients. This example uses qualitative and quantitative methods to evaluate the intracellular ROS scavenging efficacy of a photothermally enhanced GCP nanozyme. Qualitative observation was performed using the DCFH-DA fluorescent probe (…). Figure 5 (a) shows that the model group exhibited strong green fluorescence, indicating high ROS accumulation; the fluorescence intensity of the PG group was slightly weakened; the GCP@PG group showed a significant decrease; and the GCP@PG+ group showed the weakest fluorescence, suggesting a gradual improvement in ROS scavenging effect. Further quantitative flow cytometry analysis ( Figure 5 As shown in (b)), the fluorescence intensity of the GCP@PG+ treatment group was only 7.70%, significantly lower than all other groups. These results clearly demonstrate that the combined strategy of photothermal therapy and GCP nanozyme can synergistically achieve efficient scavenging of intracellular ROS, thereby blocking the oxidative stress damage pathway at its source.

[0078] Mitochondria are not only a major source of intracellular reactive oxygen species (ROS), but also a key target for their damage. Their membrane potential stability directly reflects cellular energy metabolism function. Mitochondrial membrane potential detection experiments (…) Figure 5 As shown in (c) of the figure, the model group exhibited predominantly green fluorescence, indicating decreased membrane potential and mitochondrial damage. In contrast, the GCP@PG+ group showed predominantly red fluorescence, suggesting that under the synergistic effect of mild photothermal treatment, the GCP@PG composite hydrogel can alleviate mitochondrial oxidative damage by efficiently scavenging ROS, thereby maintaining mitochondrial membrane potential stability and preserving mitochondrial functional integrity. The expression level of HIF-1α directly reflects the cell's adaptation to the hypoxic microenvironment. Figure 5 As shown in (d) and (e), the model group exhibited strong HIF-1α red fluorescence, indicating that hypoxic environment induced its significant nuclear localization. The GCP@PG+ group showed the weakest red fluorescence, and its relative HIF-1α expression level was significantly reduced to 35.4% compared to the GCP@PG group. This suggests that under NIR irradiation, the photothermal effect and the synergistic effect of GCP@PG in scavenging ROS jointly inhibited the abnormal activation of HIF-1α, thereby helping to restore cellular metabolic homeostasis.

[0079] High blood sugar and low oxygen levels can induce oxidative stress and abnormal HIF-1α expression, thereby exacerbating the imbalance of inflammatory cytokines. For example... Figure 5As shown in (f) to (i), the model group exhibited a significant upregulation of pro-inflammatory factors (TNF-α, IL-1β), while the levels of anti-inflammatory mediators (IL-10, Arg-1) were significantly reduced. Administration of GCP@PG reversed these changes, downregulating the expression of pro-inflammatory factors and upregulating the levels of anti-inflammatory factors. Furthermore, near-infrared (NIR) irradiation further enhanced this regulatory effect. These combined results indicate that this material exerts a synergistic regulatory effect on the inflammatory microenvironment through the combined action of antioxidant and hypoxia regulatory mechanisms.

[0080] 6. In vivo evaluation of the efficacy of GCP@PG in promoting wound healing in diabetic patients. To verify the in vivo efficacy of the photothermal-nanozyme synergistic strategy, this embodiment uses a diabetic rat wound model for further investigation. Figure 6 As shown in (a), the experimental procedure involved creating a full-thickness skin defect model on the back of diabetic Sprague-Dawley rats, and administering treatment on days 1, 3, 5, 7, 9, and 11. Wound healing was monitored in four groups (control group, PG group, GCP@PG group, and GCP@PG+ group) over a 14-day observation period. Macroscopic wound images ( Figure 6 (b) shows that there was no significant difference in the initial wound area among the groups. Over time, the GCP@PG+ group exhibited the fastest wound contraction rate and the most complete healing effect, with the wound almost completely closed by day 14; the GCP@PG group followed, while the PG group and the control group still showed significant wound defects. Quantitative analysis of wound closure rate ( Figure 6 (c) further confirms this trend: the GCP@PG+ group achieved the highest healing rate (97.95%) on day 14, significantly better than other groups. The results indicate that NIR-irradiated GCP@PG composite hydrogels can significantly accelerate the healing process of chronic diabetic wounds, confirming the in vivo effectiveness of the "photothermal-nanozyme" synergistic treatment strategy.

[0081] To assess the histomorphological changes in healing wounds, H&E staining and Masson's trichrome staining were used to examine epidermal regeneration, inflammatory cell infiltration, and collagen deposition. Figure 6 As shown in (d), on postoperative day 7, H&E stained sections of the control group showed significant inflammatory infiltration and disordered tissue structure. In contrast, the GCP@PG+ group exhibited initial epidermal continuity, accompanied by dense granulation tissue containing newly formed capillaries and a significantly reduced inflammatory response. By day 14, the GCP@PG+ group had formed a complete epidermal layer, with neatly arranged collagen fibers in the dermis and highly mature granulation tissue. Other groups showed varying degrees of epidermal defects, scar formation, and residual inflammation. Quantitative analysis showed that the re-epithelialization rate in the GCP@PG+ treatment group reached 90.67%. Masson trichrome staining results ( Figure 6(e) further confirmed that the GCP@PG+ group showed more dense and orderly collagen deposition on postoperative days 7 and 14, with a relative collagen deposition rate of 85.31%. These results confirm that this treatment regimen can promote orderly collagen deposition and granulation tissue maturation, thereby improving the quality of wound repair.

[0082] 7. Evaluate the anti-inflammatory and angiogenesis effects of GCP@PG in vivo. One of the core pathological features of diabetic chronic wounds is the imbalance of the inflammatory microenvironment and impaired angiogenesis. Macrophage phenotypic shift (especially the polarization from the M1 pro-inflammatory phenotype to the M2 anti-inflammatory repair phenotype) plays a crucial role in regulating inflammation resolution and initiating tissue repair. In this embodiment, CD86 and CD206 were used as key biomarkers, and the inflammatory status on day 7 post-injury was assessed by immunohistochemical staining and quantitative analysis. Results ( Figure 7 Figures (a) to (c) show that the control group had the highest CD86 expression and the lowest CD206 expression; in contrast, the GCP@PG+ treatment group showed that CD86 expression decreased to 3.25%, while CD206 expression significantly increased to 69.40% (significantly higher than the 50.18% CD206 level in the GCP@PG group). These results indicate that combined photothermal therapy can effectively promote macrophage polarization towards the M2 anti-inflammatory phenotype, thereby reshaping the anti-inflammatory microenvironment.

[0083] Angiogenesis is a key indicator in the wound healing process. To verify the formation of new blood vessels, this embodiment uses a double immunofluorescence staining method for CD31 (an endothelial cell marker reflecting vascular density) and αSMA (a vascular smooth muscle cell marker reflecting vascular maturity), and performs quantitative analysis. Figure 7 (d)~(f)). On postoperative day 7, the control group showed only sparse CD31 fluorescence signal and almost undetectable α-SMA signal. In contrast, the GCP@PG+ group showed a dense and continuous CD31 fluorescence network, and α-SMA fluorescence formed a complete surrounding structure around the vascular endothelium. Quantitative results showed that the relative fluorescence intensity of CD31 and α-SMA in the GCP@PG+ group reached 24.15% and 30.48%, respectively, which were approximately 3-fold and 6-fold higher than those in the control group, and significantly higher than those in the GCP@PG group. These data indicate that the photothermal synergistic strategy can effectively improve the density and maturity of neovascularization, thereby restoring adequate blood supply to the wound.

[0084] In summary, the photothermal synergistic GCP nanozyme composite hydrogel provided by this invention, through its core mechanism of "synergistic effect of photothermal therapy and nanozyme catalysis," enables GCP@PG hydrogel to simultaneously drive macrophages to M2 polarization and promote the formation of highly mature blood vessels. This process constructs a complete regulatory cascade reaction, from optimizing the anti-inflammatory microenvironment to restoring blood supply, ultimately achieving tissue repair.

[0085] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a photothermal synergistic GCP nanozyme composite hydrogel, characterized in that, Includes the following steps: Cerium nitrate hexahydrate and gadolinium nitrate hexahydrate were mixed, and then acetic acid and ethylene glycol were added for a first hydrothermal treatment to obtain GC; The GC, ethylene glycol, PVP solution and chloroplatinic acid hexahydrate solution were mixed and subjected to a second hydrothermal reaction to obtain Gd / CeO2-Pt; A methacrylic acid solution and a gelatin solution were mixed, dialyzed, and freeze-dried to obtain GelMA; The GelMA, N-isopropylacrylamide, methylenebisacrylamide and I2959 were mixed to obtain PG hydrogel; The Gd / CeO2-Pt and the PG hydrogel were mixed and cross-linked to obtain a photothermal synergistic GCP nanoenzyme composite hydrogel.

2. The preparation method according to claim 1, characterized in that, The mixture obtained by mixing cerium nitrate hexahydrate and gadolinium nitrate hexahydrate contains Ce 3+ and Gd 3+ The molar ratio is (1~4):1; The volume ratio of the mixture, the acetic acid, and the ethylene glycol is 2:2:

52.

3. The preparation method according to claim 1, characterized in that, The mass-to-volume ratio of the GC, the ethylene glycol, the PVP solution, and the chloroplatinic acid hexahydrate solution is 50 mg: 2.5 mL: 94 μL: 47 μL.

4. The preparation method according to claim 1, characterized in that, The volume ratio of the methacrylic acid solution to the gelatin solution is 1.1:100; The methacrylic acid solution contains 1% methacrylic acid by volume; the gelatin solution contains 10% gelatin by mass.

5. The preparation method according to claim 1, characterized in that, The mass ratio of GelMA, N-isopropylacrylamide, methylenebisacrylamide, and I2959 is 24:120:18:

18.

6. The preparation method according to claim 1, characterized in that, The mass-to-volume ratio of the Gd / CeO2-Pt to the PG hydrogel is (0.1~0.5) mg: 1 mL; The cross-linking curing conditions are: irradiation under 36.5 nm ultraviolet light for 3-5 minutes.

7. A photothermal synergistic GCP nanozyme composite hydrogel prepared by the preparation method according to any one of claims 1 to 6.

8. The application of the photothermal synergistic GCP nanozyme composite hydrogel according to claim 7 in the preparation of products that promote the healing of diabetic wounds.

9. A product for promoting wound healing in diabetic patients, characterized in that, The product includes the photothermal synergistic GCP nanozyme composite hydrogel as described in claim 8.

10. A system for promoting wound healing in diabetic patients, characterized in that, The system includes the photothermal synergistic GCP nanozyme composite hydrogel and near-infrared irradiation instrument as described in claim 7.

Images