A hydrogel dressing and uses thereof

Abstract

The application relates to the field of medical dressings, in particular to a hydrogel dressing and application thereof. The hydrogel dressing provided by the application is a novel double-layer hydrogel dressing, a first hydrogel loaded with Au-CeO2 nanoscale enzyme is constructed through reversible dynamic covalent cross-linking between phenylboronic acid and cis-diol, Au-CeO2 nanoscale enzyme is loaded, and a second hydrogel prepared through light cross-linking of HAMA and sodium alginate is further constructed and loaded with bFGF. The hydrogel dressing has a "time sequence step release" function, the lower layer can release Au-CeO2 in response to ROS, the upper layer is used for continuously delivering bFGF, the physical barrier of the double-layer structure and the response mechanism synergistically act, a sequential intervention strategy of "first clearing and then reconstruction" is formed: the lower layer releases Au-CeO2 in response to ROS burst to control inflammation, and the upper layer continuously supplies repair factors by relying on slow degradation to improve the healing efficiency and repair quality of a diabetic wound surface.

Description

Technical Field

[0001] This application relates to the field of medical dressings, specifically to a hydrogel dressing and its uses. Background Technology

[0002] Diabetic wounds are a typical example of refractory wounds in clinical practice. Their healing impairment primarily stems from a persistent oxidative stress microenvironment, persistent chronic inflammation, and impaired angiogenesis. In normal wound healing, the endogenous antioxidant system effectively scavenges excess reactive oxygen species (ROS), thereby promptly terminating the inflammatory response and promoting the transition of the wound from the inflammatory phase to the proliferative phase. Conversely, diabetic wounds are characterized by persistent excessive ROS production and chronic inflammation, leading to prolonged retention of M1 macrophage polarization, suppressed fibroblast function, and impaired endothelial cell migration. These pathological changes ultimately hinder granulation tissue formation and re-epithelialization.

[0003] Current clinical treatment of diabetic wounds still primarily relies on traditional methods, including surgical debridement, skin grafting, and infection control. However, these traditional methods have limited efficacy, especially when the wound is accompanied by abnormal neovascularization and persistent inflammation. On the other hand, emerging therapies such as platelet-rich plasma (PRP), hyperbaric oxygen therapy, stem cell therapy, and exosome intervention have shown promising potential in promoting the healing of diabetic wounds. Nevertheless, these emerging therapies still face significant limitations, such as rapid inactivation of active ingredients, poor delivery efficiency, and questionable long-term safety, and therefore have not yet been widely adopted in clinical practice.

[0004] In summary, developing innovative and efficient strategies for treating diabetic wounds has become an urgent clinical need.

[0005] Sustained elevation of reactive oxygen species (ROS) levels is a hallmark of impaired wound healing in diabetic patients and is closely associated with clinical manifestations such as wound chronicity, increased risk of infection, and delayed tissue repair. A chronically hyperglycemic microenvironment triggers a persistent inflammatory response in the wound site, leading to abnormal activation of numerous infiltrating immune cells and the further release of excessive ROS, disrupting redox homeostasis. Excessive ROS not only directly attack proteins, lipids, and nucleic acids, causing cellular dysfunction, but also damage vascular endothelium, inhibit angiogenesis, and degrade key components of the extracellular matrix (ECM). These effects severely hinder re-epithelialization, vascularization, and tissue remodeling. Furthermore, these changes further exacerbate inflammatory cell aggregation and the release of pro-inflammatory factors, forming a vicious cycle of "ROS-inflammation-repair impairment," leading to a chronic, difficult-to-heal wound state. Therefore, regulating redox homeostasis, promoting angiogenesis, and repairing the extracellular matrix have become key strategies for intervening in diabetic wounds and breaking this vicious cycle. Cerium dioxide nanozymes (Ce) 3+ With Ce4+ The reversible valence state transitions between these states, coupled with unique SOD and CAT dual-mimicry enzyme activities, have led to their widespread application in the treatment of various ROS-related diseases. Typically, their surface Ce... 3+ The higher the proportion, the stronger the SOD mimicry activity; while Ce 4+ Enrichment of cerium dioxide nanozymes is more conducive to the catalytic function of CAT-like nanozymes. However, the catalytic activity of cerium dioxide nanozymes is constrained by multiple factors such as surface oxygen vacancy concentration, exposed crystal facets, and size effects. Therefore, in the complex microenvironment of diabetic wounds, how to precisely control the surface valence state distribution and increase the exposure of active sites has become a core challenge to improve their catalytic efficiency. Summary of the Invention

[0006] In view of the shortcomings of the prior art, this application provides a hydrogel dressing and its use.

[0007] To achieve the above objectives, the technical solution of this application is as follows: A hydrogel dressing, comprising: a first hydrogel, which is prepared by crosslinking tetrakis(4-sulfophenyl)boric acid, polyvinyl alcohol and oxidized dextran, and Au-CeO2 nanozyme particles are loaded in the first hydrogel; a second hydrogel, which is prepared by photocrosslinking methacrylated hyaluronic acid and sodium alginate, and loaded with basic fibroblast growth factor; the first hydrogel and the second hydrogel are respectively used as the lower layer and the upper layer to form a double-layer structure hydrogel dressing.

[0008] Preferably, the Au-CeO2 nanoenzyme particles are core-shell nanostructures comprising an Au core and a CeO2 shell, wherein the Au core is encapsulated by the CeO2 shell.

[0009] Preferably, the preparation process of the Au-CeO2 nanozyme particles includes a first reaction stage involving chloroauric acid solution, cerium nitrate solution, and ammonia solution, and a second reaction stage involving the addition of cerium nitrate solution to the reaction solution in the first reaction stage for further reaction.

[0010] Preferably, in the first reaction stage, under ice bath conditions, chloroauric acid solution and cerium nitrate solution are added to deionized water, followed by rapid addition of ammonia solution, and stirring is continued for 20-30 s; in the second reaction stage, cerium nitrate solution is added to the reaction solution in the first reaction stage, and the reaction is stirred at room temperature to generate Au-CeO2 nanozyme particles. After the reaction is completed, the Au-CeO2 nanozyme particles are collected by centrifugation.

[0011] Preferably, the preparation process of the first hydrogel includes the following steps: mixing tetrakis(4-sulfophenyl)boronic acid, oxidized dextran and Au-CeO2 nanozyme particles, adding polyvinyl alcohol solution and stirring, and allowing it to stand to form a first hydrogel loaded with Au-CeO2 nanozyme particles.

[0012] Preferably, the preparation process of the second hydrogel includes the following steps: adding methacrylated hyaluronic acid, sodium alginate, basic fibroblast growth factor and photoinitiator sequentially to the surface of the first hydrogel, and completing in-situ photocrosslinking by ultraviolet light irradiation to form a second hydrogel on the surface of the first hydrogel.

[0013] The hydrogel dressings described above are used in the preparation of wound repair and treatment drugs.

[0014] Preferably, the drug is used to treat diabetic wounds.

[0015] In a first aspect of this application, a core-shell Au-CeO2 nanozyme was constructed by coating cerium dioxide onto the surface of gold nanoparticles. Utilizing the interfacial synergistic effect and strong metal-support interaction (SMSI) between the gold nanoparticles and the cerium dioxide support, the CeO2 nanozyme in the shell can be significantly promoted. 3+ / Ce 4+ The redox cycle increases oxygen vacancy density, thereby significantly enhancing the synergistic efficiency of SOD and CAT dual enzyme activities.

[0016] In a second aspect, addressing the persistent reactive oxygen species (ROS) stimulation in the early stages of diabetic wounds, this application utilizes tetratetra(4-sulfophenyl)boronic acid (TSPBA), polyvinyl alcohol (PVA), and oxidized dextran to construct a first hydrogel loaded with Au-CeO2 nanozymes via reversible dynamic covalent cross-linking between phenylboronic acid and cis-diol. TSPBA exhibits unique ROS-responsive properties: in a high-concentration H2O2 environment, the phenylboronic acid groups are oxidized to phenylboronic esters, leading to rapid dissociation of the cross-linked structure and immediate release of the Au-CeO2 nanozyme to scavenge ROS and inhibit inflammation progression.

[0017] The third aspect of this application utilizes basic fibroblast growth factor (bFGF). Basic fibroblast growth factor (bFGF) is a highly effective pro-angiogenic factor that specifically binds to receptors on the surface of endothelial cells, activating intracellular signaling pathways, significantly accelerating endothelial cell proliferation and migration, and thereby promoting angiogenesis. Furthermore, bFGF can activate fibroblast activity, accelerate the synthesis and deposition of extracellular matrix (ECM), and promote granulation tissue growth and re-epithelialization, effectively compensating for the shortcomings of relying solely on antioxidant strategies in tissue reconstruction, and providing the necessary biological scaffold and nutritional support for wound repair.

[0018] The fourth aspect of this application involves the construction of a bilayer hydrogel dressing system with a "time-sequential step-by-step release" function, aiming to break the vicious cycle of "ROS-inflammation-healing barrier". Based on the aforementioned first hydrogel designed for early-stage diabetic wounds, a second hydrogel is constructed from methacrylated hyaluronic acid (HAMA) and sodium alginate via photocrosslinking, and loaded with basic fibroblast growth factor (bFGF). The second hydrogel has high water content and a slow degradation rate, effectively protecting bFGF activity; it slowly releases bFGF after inflammation subsides, precisely matching the proliferation and remodeling stages of angiogenesis and matrix reconstruction. The physical barrier and response mechanism of the bilayer structure work synergistically to form a sequential intervention strategy of "clearing first, then rebuilding": the lower layer responds to ROS bursts by immediately releasing Au-CeO2 to control inflammation, while the upper layer relies on slow degradation to continuously supply repair factors. This precise spatiotemporal coupling design ultimately improves the healing efficiency and repair quality of diabetic wounds.

[0019] In summary, this application provides a novel bilayer hydrogel dressing (Au-CeO2-bFGF), in which the lower layer releases Au-CeO2 in response to ROS, while the upper layer continuously delivers bFGF. This hydrogel dressing, through the lower PTOD-Au-CeO2 layer, rapidly responds to the high ROS microenvironment of diabetic wounds, efficiently clearing excess ROS, thereby effectively inhibiting inflammatory responses and reducing oxidative damage. After the inflammatory microenvironment is improved, the system smoothly transitions to the upper HASA-bFGF layer, promoting extracellular matrix (ECM) remodeling and angiogenesis through the slow release of bFGF, thereby accelerating wound healing. This hydrogel dressing not only exhibits significant biocompatibility but also possesses multiple benefits, including enhanced cell migration, promotion of angiogenesis, and antioxidant and anti-inflammatory effects. In vivo animal studies have demonstrated the significant synergistic therapeutic effect of Au-CeO2 and bFGF: Au-CeO2 can block the inflammatory process, restore redox homeostasis, and inhibit the overexpression of matrix metalloproteinases (MMPs), thereby creating a stable microenvironment for bFGF to exert its optimal biological activity. This synergistic effect effectively promotes the orderly deposition of collagen and high-quality ECM remodeling, and drives angiogenesis and maturation, ultimately enabling this bilayer hydrogel dressing to significantly accelerate the closure process of diabetic wounds and greatly improve healing quality. Furthermore, its excellent biocompatibility and safety further confirm its clinical translational potential.

[0020] The bilayer hydrogel dressing provided in this application organically combines the dynamic antioxidant and anti-inflammatory functions of Au-CeO2 with the sustained repair-promoting activity of bFGF. This system can be precisely adapted to different stages of diabetic wound healing and shows great application potential in the clinical treatment of chronic refractory wounds, providing an efficient, safe, and clinically promising solution for the repair of chronic diabetic wounds. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0022] Figure 1 Schematic diagram of the preparation of Au-CeO2-bFGF and its application in the treatment of diabetic wounds in mice; Figure 2 Characterization of Au-CeO2: (A) Photograph of Au-CeO2 powder; (B) Photograph of Au-CeO2 suspension; (C) Transmission electron microscopy (TEM) image of Au-CeO2, scale bar = 50 nm; (D) EDX elemental distribution map of Au-CeO2, blue represents Au, cyan represents Ce, red represents O, scale bar = 20 nm; (E) Particle size distribution of Au-CeO2; (F) Zeta potential of Au-CeO2; (G) XRD pattern of Au-CeO2; (H) Au 4f XPS spectrum of Au-CeO2; (I) Ce 3d XPS spectrum of Au-CeO2; (J) Catalase (CAT) activity of CeO2 and Au-CeO2 (n=3); (K) Superoxide dismutase (SOD) activity of CeO2 and Au-CeO2 (n=3); (L) ABTS inhibition rates of CeO2 and Au-CeO2 (n=3); (M) RAW264.7 cells were co-incubated with DCFH-DA and treated with ROS inducer (Rosup), followed by further treatment with CeO2 and Au-CeO2, respectively. Fluorescence images were then acquired under a fluorescence microscope, scale bar = 50 μm; (N) Quantitative analysis of ROS fluorescence intensity (n=3). Figure 3Characterization of Au-CeO2-bFGF: (A) Scanning electron microscopy (SEM) image of PTOD-Au-CeO2, scale bar = 10 μm; (B) Scanning electron microscopy (SEM) image of HASA-bFGF, scale bar = 200 μm; (C) Porosity of PTOD-Au-CeO2 and HASA-bFGF (n=3); (D) Fourier transform infrared (FTIR) spectra of PTOD-Au-CeO2 and HASA-bFGF; (E) Rheological behavior of PTOD-Au-CeO2 and HASA-bFGF at frequency scans; (F) Degradation rate of PTOD-Au-CeO2 and HASA-bFGF in PBS (n=3); (G) Degradation rate of PTOD-Au-CeO2 and HASA-bFGF in H2O2 (n=3); (H) Release rates of PTOD-Au-CeO2 and HASA-bFGF in PBS (n=3); (I) Release rates of PTOD-Au-CeO2 and HASA-bFGF in H2O2 (n=3); (J) Degradation rates of PTOD-Au-CeO2 at different concentrations of H2O2; Figure 4 Antioxidant and anti-inflammatory effects of Au-CeO2-bFGF at the cellular level: (A) RAW 264.7 cells were loaded with fluorescent probes and treated with ROS inducer (Rosup), then treated with appropriate hydrogels, and the fluorescence was observed under a fluorescence microscope (scale bar = 100 μm); (B) RAW 264.7 cells were stimulated with Rosup and treated with appropriate hydrogels, and apoptosis was observed by TUNEL staining (scale bar = 100 μm); (C) Quantitative analysis of ROS fluorescence intensity (n=3); (D) Quantitative analysis of TUNEL staining fluorescence intensity (n=3); RAW 264.7 cells were stimulated with LPS and treated with appropriate hydrogels, and the levels of TNF-α (E), IL-1β (F), IL-6 (G), and IL-12 (H) in the cells were detected by ELISA (n=3); All data are expressed as mean ± standard deviation. Compared with the control group, ns ≥ 0.05, p<0.05, p<0.01, p<0.001; Figure 5Migration-promoting, tube-forming, and biocompatibility properties of Au-CeO2-bFGF: (A) Migration assay of HUVEC cells after treatment with the corresponding hydrogel, scale bar = 50 μm; (B) Wound closure rate of HUVEC cells after treatment with the corresponding hydrogel (n=3); (C) Tube-forming assay of HUVEC cells after treatment with the corresponding hydrogel, scale bar = 50 μm; (D) Quantitative analysis of tube-forming assay (n=3); (E) VEGF concentration in the supernatant of HUVEC cells after treatment with the corresponding hydrogel (n=3); (F) Viability of HUVEC cells after 24 h of treatment with the corresponding hydrogel (n=4); (G) Viability of HUVEC cells after 72 h of treatment with the corresponding hydrogel (n=4); All data are expressed as mean ± standard deviation. Compared with the control group, ns ≥ 0.05. p<0.05, p<0.01, p<0.001; Figure 6 In vivo degradation and release behavior of Au-CeO2-bFGF: (A) In vivo imaging of the degradation of Cy5-labeled PTOD-Au-CeO2 and FITC-labeled HASA-bFGF at the wound site on days 1, 3, and 7 after application; (B) In vivo imaging fluorescence quantitative analysis (n=3); (C) In vivo release of bFGF-FITC and Au-CeO2 at the wound site on days 1, 3, and 7 after application (n=3); (D) In ​​diabetic wounds... . Changes in OH activity on days 1, 3, and 7 (n=3); Figure 7 The efficacy of Au-CeO2-bFGF in treating diabetic wounds: (A) Schematic diagram of the timeline of animal experiments on Au-CeO2-bFGF treatment of diabetic wounds; (B) Representative photographs of wound healing on days 1, 3, 7, 10, and 14 after corresponding treatments in each group; (C) Wound healing rates of different treatment groups on days 7 and 14 (n=3); (D) HE staining of wound tissues in different treatment groups on days 7 and 14, scale bar = 100 μm; (E) Masson staining of wound tissues in different treatment groups on days 7 and 14, scale bar = 100 μm; (F) Quantitative analysis of inflammatory infiltration area in wound tissues of each group (n=3); (G) Quantitative analysis of collagen content in wound tissues of each group (n=3); All data are expressed as mean ± standard deviation. Compared with the control group, ns ≥ 0.05, p<0.05, p<0.01, p<0.001; Figure 8 Anti-inflammatory effect of Au-CeO2-bFGF on diabetic wounds: (A) Immunohistochemical (IHC) staining of MPO in wound tissues on days 7 and 14 of different treatment groups, scale bar = 50 μm; (B) Quantitative analysis of MPO expression in wound tissues (n=3), and levels of inflammatory factors TNF-α (C), IL-1β (D), IL-6 (E), and IL-12 (F) in wound tissues on days 7 and 14 of different treatment groups (n=4); All data are expressed as mean ± standard deviation. Compared with the control group, ns ≥ 0.05, p<0.05, p<0.01, p<0.001; Figure 9 Antioxidant effect of Au-CeO2-bFGF on diabetic wounds: (A) DHE fluorescence staining of wound tissues on days 7 and 14 of different treatment groups, scale bar = 50 μm; (B) Quantitative analysis of DHE staining fluorescence intensity (n=3); Detection of antioxidant indicators in wound tissues on days 7 and 14 after corresponding treatments: SOD (C), MDA (D), and GSH (E) (n=3); All data are expressed as mean ± standard deviation. Compared with the control group, ns ≥ 0.05, p<0.05, p<0.01, p<0.001; Figure 10 Au-CeO2-bFGF Remodeling of Extracellular Matrix (ECM) in Diabetic Wounds: (A) VG staining of wound tissues on days 7 and 14 of different treatment groups, scale bar = 50 μm; (B) Quantitative analysis of collagen fiber area in wound tissues of each group (n=3); (C) Immunofluorescence staining of type I and type III collagen in wound tissues on days 7 and 14 of different treatment groups, scale bar = 50 μm; (D) Quantitative analysis of fluorescent expression of type I collagen in wound tissues (n=3); (E) Quantitative analysis of fluorescent expression of type III collagen in wound tissues (n=3); (F) Quantitative analysis of the ratio of type I to type III collagen in wound tissues (n=3); (G) Detection of fibronectin in wound tissues on days 7 and 14 after corresponding treatments (n=3); Detection of matrix metalloproteinases (MMP2(H) and MMP9(I)) in wound tissue (n=3). All data are expressed as mean ± standard deviation. Compared with the control group, ns ≥ 0.05. p<0.05, p<0.01, p<0.001; (J) Detection of Laminin in wound tissue on day 7 and day 14 after corresponding treatment (n=3); Figure 11 The pro-angiogenic effect of Au-CeO2-bFGF on diabetic wounds: (A) Immunohistochemical (IHC) staining of CD31 in wound tissues on days 7 and 14 of different treatment groups, scale bar = 50 μm; (B) Immunohistochemical (IHC) staining of α-SMA in wound tissues on days 7 and 14 of different treatment groups, scale bar = 50 μm; (C) Quantitative analysis of CD31 expression in wound tissues (n=3); (D) Quantitative analysis of α-SMA expression in wound tissues (n=3); Detection of pro-angiogenic factors in wound tissues on days 7 and 14 after corresponding treatments: VEGF (E) and PDGF (F) (n=3); All data are expressed as mean ± standard deviation. Compared with the control group, ns ≥ 0.05, p<0.05, p<0.01, p<0.001. Detailed Implementation

[0023] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the scope of this application.

[0024] 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 limit the application. The terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0025] like Figure 1As shown, the hydrogel dressing provided in this application is a novel bilayer hydrogel dressing, wherein the lower layer can release Au-CeO2 in response to ROS, and the upper layer is used for continuous delivery of basic fibroblast growth factor (bFGF). Specifically, the lower layer is dynamically cross-linked with tetrakis(4-sulfonylurea)boronic acid (TSPBA), polyvinyl alcohol (PVA), and oxidized dextran to encapsulate Au-CeO2 (PTOD-Au-CeO2); the upper layer is formed by photocrosslinking methacrylated hyaluronic acid (HAMA) and sodium alginate to encapsulate bFGF (HASA-bFGF). The above components together constitute a bilayer hydrogel system with differentiated release characteristics.

[0026] The following are preferred embodiments of this application.

[0027] Example 1: Under ice bath conditions, 600 μL of 0.024 M chloroauric acid solution and 1.4 mL of 0.1 M cerium nitrate solution were added to 50 mL of deionized water. Then, 64 μL of ammonia solution (25-28%) dissolved in 3 mL of deionized water was rapidly added, and the mixture was stirred continuously for 25 s. Immediately afterwards, 3 mL of 0.0467 M cerium nitrate solution was added to the system, and stirring was continued at room temperature for 15 min. After the reaction was complete, the mixture was centrifuged at 4 °C and 10,000 × g for 60 min to collect the product. The resulting precipitate was washed three times with deionized water, and the centrifugation steps were repeated after each wash to separate and purify the product. Finally, the sample was dried at room temperature to obtain Au-CeO2 nanomaterials.

[0028] The following describes the physicochemical properties and related activity detection of the Au-CeO2 nanomaterials prepared in this embodiment.

[0029] 1. The dried Au-CeO2 is a blackish-purple powder. Figure 2 A), its aqueous dispersion appears purple ( Figure 2 B). Transmission electron microscopy (TEM) images show that Au-CeO2 has a core-shell nanostructure, with the Au core encapsulated by a CeO2 shell. Figure 2 C).

[0030] X-ray energy dispersive spectroscopy (EDX) elemental mapping results confirmed that Au was mainly enriched in the core region of the nanoparticles, while Ce and O were uniformly distributed. Figure 2 (D) indicates that the CeO2 shell has successfully coated the Au core.

[0031] Particle size analysis showed that the average particle size of Au-CeO2 was approximately 19.82 ± 6.31 nm, and the polydispersity index (PDI) was 0.12, indicating that it has good monodispersity. Figure 2 E). The Zeta potential of Au-CeO2 is 34.6 ± 1.03 mV ( Figure 2 F).

[0032] The Fourier transform infrared (FTIR) spectrum of Au-CeO2 is in the range of 400–600 cm⁻¹. -1 Typical Ce-O characteristic absorption peaks were observed within the range, confirming the interfacial interaction between Au and CeO2. The crystal structure of Au-CeO2 was characterized by X-ray diffraction (XRD), and the results showed characteristic diffraction peaks corresponding to Au (JCPDS No. 04-0784) and CeO2 (JCPDS No. 34-0394), respectively. Figure 2 G).

[0033] Furthermore, X-ray photoelectron spectroscopy (XPS) analysis of the elemental composition and valence state of Au-CeO2 showed that the Au 4f orbitals were at 83.9 eV (Au 0 ) and 87.6 eV (Au + Characteristic peaks appear at ( ) Figure 2 H); Ce 3d spectrum shows Ce 3+ With Ce 4+ Typical characteristic peaks of coexistence ( Figure 2 I). The above results confirm that there is a strong electronic interaction between Au and CeO2, which is beneficial to the formation of catalytic active sites.

[0034] In summary, the above results confirm that the Au-CeO2 core-shell structure has been successfully constructed.

[0035] 2. To further verify the antioxidant properties of Au-CeO2, its catalase-like (CAT) and superoxide dismutase-like (SOD) activities were investigated, and its effects on H2O2 and superoxide anion (O2) were measured, respectively. .- The scavenging ability of CeO2 was compared with that of commercial CeO2.

[0036] Experimental results showed that at a concentration of 100 μg / mL, Au-CeO2 achieved a scavenging rate of over 50% for H2O2; while commercial CeO2, even at a concentration of 200 μg / mL, did not reach 50%. Figure 2 J). Similarly, Au-CeO2 versus O2 .-The scavenging rate of Au-CeO2 was significantly better than that of CeO2, indicating that Au-CeO2 has stronger SOD-like activity. Figure 2 K).

[0037] The above results indicate that Au-CeO2 possesses multi-enzyme activity and can simultaneously scavenge O2. .- It reacts with H2O2, and its effect is significantly better than that of CeO2. Furthermore, ABTS reagent, commonly used to assess free radical scavenging ability, was employed, and the ABTS content was measured. +. The scavenging rate was further used to evaluate its antioxidant properties. The results showed that Au-CeO2 exhibited ABTS... +. The scavenging rate was significantly higher than that of CeO2, and the difference between the two widened further with increasing concentration. Figure 2 L).

[0038] The above results indicate that Au-CeO2 has excellent and stable antioxidant properties.

[0039] Simultaneously, this embodiment used RAW 264.7 cells to assess the intracellular reactive oxygen species (ROS) scavenging capacity of Au-CeO2. Cells were stimulated with ROS to simulate oxidative stress, and then detected using 2',7'-dichlorofluorescein (DCF). Results showed that the intracellular ROS level in the control group cells was significantly increased, exhibiting obvious green fluorescence; while cells treated with Au-CeO2 showed almost no green fluorescence. Figure 2 Further quantitative analysis of fluorescence intensity showed that although CeO2 could also significantly reduce green fluorescence intensity, its effect was weaker than that of Au-CeO2 (M). Figure 2 N).

[0040] In summary, the above results confirm that Au-CeO2, with its excellent enzyme-like activity, can effectively scavenge ROS and protect cells from oxidative stress damage.

[0041] Example 2: First, 200 μL of 5% tetratetra(4-sulfophenyl)boric acid (TSPBA), 50 μL of 5% oxidized dextran, and 0.25 mg of Au-CeO2 were thoroughly mixed. Then, 150 μL of 8% polyvinyl alcohol (PVA) was added and stirred rapidly to obtain a homogeneous mixture. After standing, a lower hydrogel (PTOD-Au-CeO2) was formed.

[0042] Next, 100 μL of 4% methacrylated hyaluronic acid (HAMA), 100 μL of 3% sodium alginate, 20 μL of basic fibroblast growth factor (bFGF, 100 μg / mL), and 0.6 mg of lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) were added sequentially to the surface of the lower hydrogel. After thoroughly mixing all components, the mixture was irradiated with ultraviolet light for 45 seconds to form the upper hydrogel (HASA-bFGF).

[0043] Thus, the lower and upper layers combine to ultimately construct an Au-CeO2-bFGF bilayer hydrogel.

[0044] The following describes the physicochemical properties and related activity detection of the Au-CeO2 nanomaterials prepared in this embodiment.

[0045] 1. The microstructure of Au-CeO2-bFGF was further characterized using scanning electron microscopy (SEM). Figure 3 A, 3B). The results showed that the lower hydrogel layer had a relatively uniform pore size and dense structure, while the upper layer exhibited a more loose, high-porosity three-dimensional network structure. Quantitative analysis of the porosity difference between the two layers showed that the lower layer had an average pore size of 2.68 ± 1.17 μm and a porosity of 26.29 ± 4.23%; in contrast, the upper layer had an average pore size of 53.33 ± 4.62 μm and a porosity of 56.21 ± 4.56%. Figure 3 C). This structural difference endows the bilayer hydrogel with unique functional properties: the dense lower network can effectively encapsulate Au-CeO2 and achieve ROS-responsive release; while the loose and porous upper three-dimensional network has a higher water content, which not only helps maintain the bioactivity of bFGF, but also provides a good physical environment for its long-lasting and slow release.

[0046] Fourier transform infrared (FTIR) spectroscopy analysis showed no significant difference in characteristic peaks between the lower hydrogel loaded with and unloaded Au-CeO2; similarly, the main characteristic peaks of the upper hydrogel remained essentially unchanged regardless of whether bFGF was loaded. Figure 3 D). The above results indicate that the introduction of Au-CeO2 and bFGF did not change the chemical structure of the hydrogel itself, suggesting that both exist in the hydrogel matrix in a physically embedded form.

[0047] Rheological tests were used to further elucidate the viscoelastic properties of Au-CeO2-bFGF. The storage modulus and loss modulus of PTOD-Au-CeO2 were both higher than those of HASA-bFGF. Figure 3The F indicates that PTOD-Au-CeO2 has higher rigidity. This rheological difference ensures the mechanical stability of the bilayer structure: the lower layer provides structural support and rigidity, while the upper layer maintains flexibility and conformability, thus achieving long-term in-situ adaptation in dynamic wound environments.

[0048] 2. In vitro degradation experiments further confirmed that the bilayer hydrogel exhibited gradient degradation characteristics in a simulated physiological environment (PBS solution). Within 48 h, the degradation rate of PTOD-Au-CeO2 was 14.27 ± 2.01%, while that of HASA-bFGF was only 5.99 ± 1.73% during the same period, indicating that the degradation rate of PTOD-Au-CeO2 was slightly higher than that of HASA-bFGF. Figure 3 F).

[0049] Subsequently, oxidative stress was simulated in PBS containing different concentrations of H2O2 to investigate the degradation behavior of PTOD-Au-CeO2. The results showed that the degradation rate of PTOD-Au-CeO2 significantly increased with increasing H2O2 concentration. Figure 3 To better evaluate the ROS responsiveness of PTOD-Au-CeO2, its degradation and release kinetics in PBS containing 1 mM H2O2 were further investigated. In the bilayer hydrogel degradation experiment, PTOD-Au-CeO2 was completely degraded within 48 h; although the degradation rate of HASA-bFGF was also improved, its degradation rate was only 26.05 ± 2.05% (J). Figure 3 G). This indicates that PTOD-Au-CeO2 has significant ROS responsiveness, while HASA-bFGF can maintain structural stability.

[0050] In drug release experiments using bilayer hydrogels, under simulated physiological conditions, both PTOD-Au-CeO2 and HASA-bFGF exhibited sustained-release characteristics in PBS. Figure 3 H). However, under ROS stimulation, the release of PTOD-Au-CeO2 accelerated significantly, with the cumulative release rate of bFGF reaching 92.47 ± 4.06% within 48 hours, far exceeding the 15.42 ± 1.56% in the H2O2-free group. In contrast, HASA-bFGF maintained a consistently mild and sustained release curve. Figure 3 I).

[0051] In summary, the results indicate that the PTOD-Au-CeO2 layer enables ROS-responsive release, while the HASA-bFGF layer provides long-term sustained release; their synergistic effect allows for precise adaptation to the dynamic changes in the wound microenvironment.

[0052] 3. To further verify the antioxidant and anti-inflammatory capabilities of Au-CeO2-bFGF hydrogel, RAW264.7 cells were selected as the experimental model in this embodiment.

[0053] First, an oxidative stress model was established using Rosup-induced cell culture. Then, each group was treated with the corresponding hydrogel, and intracellular reactive oxygen species (ROS) levels were detected using the DCFH-DA fluorescent probe. Results showed that, compared to the negative control group, the positive control group cells exhibited significant green fluorescence, indicating a significant increase in intracellular ROS levels and successful establishment of the oxidative stress model. Further observation revealed a significant decrease in green fluorescence intensity in the PTOD-Au-CeO2 group and the Au-CeO2-bFGF group, suggesting a substantial reduction in intracellular ROS production. Figure 4 A); while the oxidative stress levels in the PTOD group and the HASA group only decreased moderately.

[0054] Quantitative fluorescence intensity analysis showed that the Au-CeO2-bFGF group had the lowest average fluorescence intensity, followed by the PTOD-Au-CeO2 group. Figure 4 C). This result confirms that Au-CeO2-bFGF hydrogel has the strongest antioxidant effect, which originates from the release of Au-CeO2 in the bilayer hydrogel.

[0055] Meanwhile, this embodiment evaluated the effects of each treatment group on the level of apoptosis under oxidative stress. TUNEL staining results showed that the Au-CeO2-bFGF group had almost no obvious red fluorescence, while the control group showed strong red fluorescence; the red fluorescence intensity of the other groups was also lower than that of the control group. Figure 4 B). Further quantitative fluorescence analysis confirmed that the Au-CeO2-bFGF group had the lowest average fluorescence intensity ( Figure 4 (D), indicating that the hydrogel can significantly inhibit oxidative stress-induced apoptosis.

[0056] To further verify the anti-inflammatory effect of Au-CeO2-bFGF, lipopolysaccharide (LPS) was used to stimulate cells to simulate an inflammatory environment. After co-incubation with hydrogel for a period of time, the cell culture supernatant was collected, and the levels of inflammatory factors (TNF-α, IL-1β, IL-6, and IL-12) in the supernatant were measured by ELISA to evaluate its anti-inflammatory effect. Figure 4 E-4H).

[0057] The results showed that, compared with the control group, the PTOD group, PTOD-Au-CeO2 group, HASA group, HASA-bFGF group, and Au-CeO2-bFGF group all significantly reduced the concentrations of the aforementioned inflammatory factors. Among them, the Au-CeO2-bFGF group had the lowest level of inflammatory factors, followed by the PTOD-Au-CeO2 group. Furthermore, the levels of inflammatory factors in the HASA group and the HASA-bFGF group were similar.

[0058] The above results indicate that Au-CeO2-bFGF exhibits the strongest anti-inflammatory effect, and its superior performance stems from the synergistic enhancement of the anti-inflammatory activity of Au-CeO2 and the cell-repair-promoting effect of bFGF. Although PTOD and HASA also showed some anti-inflammatory effects, their effects were limited. Furthermore, the anti-inflammatory effects of HASA and HASA-bFGF were similar, indicating that adding bFGF alone without the synergistic effect of Au-CeO2 is insufficient to significantly improve the anti-inflammatory performance.

[0059] In summary, Au-CeO2-bFGF can significantly reduce ROS levels, inhibit apoptosis, and block the inflammatory process under oxidative stress and inflammation.

[0060] 4. Cell migration and tube formation assays are commonly used methods to evaluate the wound repair process at the cellular level. Human umbilical vein endothelial cells (HUVECs) can highly mimic the migration and tube formation functions of human endothelial cells, and their source is stable and reliable, making them an ideal model for studying angiogenesis during wound healing. Therefore, HUVECs were selected for subsequent experiments in this embodiment.

[0061] Cell migration assay results showed that the migration distance of the Au-CeO2-bFGF group was greater than that of the other groups. Figure 5 A). Further quantitative analysis showed that the Au-CeO2-bFGF group had the highest wound closure rate, followed closely by the HASA-bFGF group. Figure 5 B). This indicates that bFGF in Au-CeO2-bFGF plays a positive role in promoting cell migration.

[0062] Subsequently, the angiogenesis-promoting effect of Au-CeO2-bFGF was further verified through a tube formation experiment. Compared with the control group, the PTOD group, PTOD-Au-CeO2 group, HASA group, HASA-bFGF group, and Au-CeO2-bFGF group all induced the formation of more tubular structures. Figure 5 C). Among them, the Au-CeO2-bFGF group had the largest number of tubes, followed by the HASA-bFGF group ( Figure 5D). This suggests that bFGF may be the main factor promoting angiogenesis; although the hydrogel matrix itself has some angiogenesis-promoting effect, its effect is limited.

[0063] Meanwhile, we measured the VEGF levels in each group of cells. The results showed that the VEGF expression levels in the Au-CeO2-bFGF group were similar to those in the HASA-bFGF group, and significantly higher than those in the other groups. Figure 5 E), confirming the core driving role of bFGF in angiogenesis.

[0064] 5. Cell biocompatibility is a key indicator for the application of hydrogels in the biomedical field, directly determining their safety for in vivo use. To verify the cell compatibility of Au-CeO2-bFGF, this application uses the CCK-8 assay to evaluate its effect on cell viability (…). Figure 5 F-5G).

[0065] The results showed that the cell viability of the Au-CeO2-bFGF group exceeded 95% at both 24 h and 72 h of culture, with no significant difference compared to the control group. This indicates that Au-CeO2-bFGF not only possesses excellent angiogenesis-promoting ability but also exhibits high biocompatibility during long-term co-culture, laying a solid foundation for its subsequent in vivo wound repair application.

[0066] Example 3: Example 2 has preliminarily confirmed through in vitro experiments that the lower layer of the Au-CeO2-bFGF hydrogel is ROS-responsive, and the upper layer can achieve sustained release of bFGF. This example further constructs a mouse diabetic wound model to verify the efficacy of the Au-CeO2-bFGF hydrogel through animal experiments.

[0067] All animal procedures in this embodiment have been approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University and the Zhejiang Provincial Animal Experiment Ethics Committee (Approval No.: WYYY-IACUC-AEC-2026-001).

[0068] Thirty-six healthy male C57BL / 6 mice aged 6–8 weeks were selected. First, the mice were fed a high-fat diet (60% of their calories from fat) for 8 weeks. Streptozotocin (STZ) was dissolved in 1 mL of 0.1 M citrate-phosphate buffer (pH 4.5) to prepare a 1% solution. Mice were intraperitoneally injected with STZ at a dose of 25 mg / kg body weight for four consecutive days. Mice were fasted for 12 h before the first injection and resumed eating 2 h after injection. Four h after injection, they were given a 5% glucose solution, which was replaced with regular drinking water 10 h later. Subsequent STZ injections did not require fasting, and all other procedures remained the same. Fasting blood glucose was measured one week after injection. Mice with a fasting blood glucose level >16.7 mmol / L were considered successfully modeled and used for subsequent experiments.

[0069] The mice that successfully developed the model were numbered and randomly divided into 6 groups using a random number table: (1) control group; (2) PTOD group (PTOD hydrogel treatment); (3) PTOD-Au-CeO2 group (PTOD-Au-CeO2 hydrogel treatment); (4) HASA group (HASA hydrogel treatment); (5) HASA-bFGF group (HASA-bFGF hydrogel treatment); (6) Au-CeO2-bFGF group (Au-CeO2-bFGF bilayer hydrogel treatment).

[0070] 1. The lower layer of the PTOD-Au-CeO2 hydrogel was labeled with Cy5 fluorescence, and the upper layer of the HASA-bFGF hydrogel was labeled with FITC fluorescence. In short, Cy5 (10 μM) was added to the polyvinyl alcohol (PVA) hydrogel precursor solution to prepare the Cy5-labeled lower layer of the PTOD-Au-CeO2 hydrogel; similarly, FITC (50 μg / mL) was added to the HASA hydrogel precursor solution to prepare the FITC-labeled upper layer of the HASA-bFGF hydrogel.

[0071] The results showed that in the diabetic wound model, the lower layer of PTOD-Au-CeO2 degraded rapidly, and the fluorescence signal almost completely disappeared by day 7; in contrast, the upper layer of HASA-bFGF degraded slowly and maintained a strong fluorescence intensity until day 7. Figure 6 A). Further quantitative fluorescence analysis showed that by day 3, the fluorescence intensity of the lower layer of PTOD-Au-CeO2 had significantly decreased, while the fluorescence intensity of the upper layer of HASA-bFGF remained at a high level. Figure 6 B).

[0072] The release of bFGF was further investigated using the bFGF-FITC fluorescent labeling method, while the gold content was detected by ICP-MS to characterize the release of Au-CeO2. The results showed that by day 3, most of the Au-CeO2 had been released into the wound tissue; by day 7, the cumulative release of Au-CeO2 exceeded 90%. In contrast, bFGF exhibited a slow-release characteristic; even by day 7, its release rate did not exceed 30%. Figure 6 C).

[0073] To better verify the ROS responsiveness of PTOD-Au-CeO2, hydroxyl radicals in mouse wound tissue were monitored. . Changes in OH levels, because . OH activity is a key indicator reflecting ROS levels. Compared with the control group, the wound tissue in the Au-CeO2-bFGF group showed... . OH activity began to decline on day 3 and decreased significantly on day 7. Figure 6 D). This trend is highly consistent with the degradation and release patterns of PTOD-Au-CeO2, confirming that its ROS-responsive degradation mechanism is precisely controllable.

[0074] The above results demonstrate that the Au-CeO2-bFGF bilayer hydrogel achieves precise temporal regulation of "sensing-response-degradation-release" in the high ROS microenvironment of diabetic wounds: the lower layer rapidly removes ROS while simultaneously degrading, while the upper layer continuously supplies bFGF to promote repair. The two layers work synergistically to promote the healing of diabetic wounds.

[0075] 2. To comprehensively evaluate the potential of Au-CeO2-bFGF in promoting diabetic wound healing in vivo, this embodiment establishes a diabetic wound model to observe the therapeutic effect of the hydrogel. Figure 7 A). The corresponding hydrogels were applied to the diabetic wounds, and wound healing was recorded on days 1, 3, 7, 10, and 14. Compared with the control group, the wounds in the PTOD-Au-CeO2 group, HASA-bFGF group, and Au-CeO2-bFGF group all healed significantly faster. Notably, the Au-CeO2-bFGF group was nearly completely closed by day 14, demonstrating the best wound healing promotion ability. Figure 7 B). Further analysis of wound healing rates on days 7 and 14 showed that the Au-CeO2-bFGF group had the highest healing rates of 40.21 ± 4.24% and 98.25 ± 1.25%, respectively, exhibiting the fastest healing speed. The wound healing rate of the PTOD-Au-CeO2 group was slightly higher than that of the HASA-bFGF group ( Figure 7(C) This may be because Au-CeO2 in PTOD-Au-CeO2 exerts strong antioxidant and anti-inflammatory effects, creating a favorable microenvironment for the healing of diabetic wounds; in contrast, relying solely on the release of bFGF without improving the inflammatory environment resulted in poor repair effects. Furthermore, the wound closure speed in the Au-CeO2-bFGF group was the fastest among all groups, further confirming that the synergistic effect of Au-CeO2 and bFGF played the most significant role in promoting wound healing.

[0076] To further verify the therapeutic effect of Au-CeO2-bFGF, wound tissue collected on day 14 was analyzed by HE staining and Masson's trichrome staining. HE staining results showed that the control group had a large number of inflammatory cell infiltrations in the early stage of wound healing (day 7), and this phenomenon persisted until day 14. On day 7, the Au-CeO2-bFGF group had the least inflammatory cell infiltration, followed by the PTOD-Au-CeO2 group, and then the HASA-bFGF group. By day 14, the inflammatory cell infiltration in all groups had further decreased. Figure 7 D, 7F). This indicates that Au-CeO2 plays a crucial role in resisting inflammatory infiltration and significantly shortening the inflammatory period. Simultaneously, Masson trichrome staining results showed that among all groups, the Au-CeO2-bFGF group exhibited the most significant increase in collagen fibers, rising from 54.61 ± 3.31% on day 7 to 82.23 ± 1.99% on day 14, indicating its effective promotion of collagen deposition and orderly arrangement. The HASA-bFGF group followed, suggesting that bFGF makes a significant contribution to collagen deposition. Collagen deposition in the PTOD-Au-CeO2 group was also superior to the control group, which may be attributed to the antioxidant effect of Au-CeO2 mitigating the inhibition of fibroblast function by oxidative stress, thereby indirectly supporting collagen synthesis. Figure 7 E, 7G).

[0077] In summary, in Au-CeO2-bFGF, Au-CeO2 acts as the core component, exerting antioxidant and anti-inflammatory effects, while bFGF acts as a key factor, promoting collagen deposition. The synergistic effect of the two enhances the healing effect of diabetic wounds and accelerates the repair process.

[0078] 3. Persistent inflammation is a major factor hindering the healing of diabetic wounds. Myeloperoxidase (MPO), as a marker of neutrophil infiltration, can reflect the intensity of the inflammatory response. To further investigate the anti-inflammatory effect of Au-CeO2-bFGF on diabetic wounds, this example further performed MPO immunohistochemical (IHC) staining on wound tissues in each group. On day 7, compared with the control group, the number of MPO-positive cells in the Au-CeO2-bFGF group was significantly reduced, indicating a significant decrease in neutrophil infiltration; the number of MPO-positive cells in both the PTOD-Au-CeO2 group and the HASA-bFGF group was less than that in the control group, and the number in the PTOD-Au-CeO2 group was lower than that in the HASA-bFGF group (…). Figure 8 A). By day 14, the number of MPO-positive cells in all groups had further decreased, but the Au-CeO2-bFGF group maintained the lowest MPO positivity level, indicating that Au-CeO2-bFGF could effectively inhibit neutrophil infiltration, thereby alleviating local inflammation. Quantitative analysis of MPO immunohistochemical results showed that the Au-CeO2-bFGF group had the lowest MPO positivity rate on both days 7 and 14, followed closely by the PTOD-Au-CeO2 group, indicating that Au-CeO2 played a dominant role in the anti-inflammatory effect. Although the MPO positivity rate in the HASA-bFGF group showed a decreasing trend compared to the control group, the degree of decrease was weaker than that in the Au-CeO2-containing group and similar to that in the HASA group. This suggests that the inhibitory effect of bFGF alone on inflammation is limited, and the anti-inflammatory effect of the HASA-bFGF group mainly comes from the HASA hydrogel itself ( Figure 8 (B) Simultaneously, the levels of key inflammatory factors (TNF-α, IL-1β, IL-6, and IL-12) in mouse wound tissue were detected using ELISA. Results showed that on day 7, the levels of inflammatory factors in all experimental groups were significantly lower than those in the control group, with the Au-CeO2-bFGF group exhibiting the lowest levels. By day 14, the overall levels of inflammatory factors in all groups decreased, but the Au-CeO2-bFGF group remained significantly lower than the other groups.

[0079] Consistent with the above results, the PTOD-Au-CeO2 group showed significant inhibitory effects on all detected inflammatory markers, but its effect was slightly less than that of the Au-CeO2-bFGF group. The decrease in inflammatory factors in the HASA group and the HASA-bFGF group was similar, further confirming that although HASA has some anti-inflammatory ability, its effect is limited, and the addition of bFGF did not significantly improve its anti-inflammatory performance. Figure 8 C-8F).

[0080] In summary, the combined results of MPO immunohistochemistry and ELISA of inflammatory factors demonstrate that Au-CeO2-bFGF can sustainably and potently inhibit neutrophil infiltration and the expression of key pro-inflammatory factors in diabetic wounds. Its anti-inflammatory effect is significantly superior to that of a single component. This synergistic anti-inflammatory effect lays a crucial foundation for subsequent angiogenesis, collagen deposition, and reepithelialization, thereby accelerating the healing of diabetic wounds.

[0081] 4. In diabetic wounds, excessive generation of reactive oxygen species (ROS) and high levels of oxidative stress are not only markers of delayed wound healing, but also key drivers of persistent overactivation of the inflammatory response.

[0082] To evaluate the antioxidant effect of Au-CeO2-bFGF, this application used DHE staining to detect ROS levels in wound tissue on days 7 and 14, and the degree of oxidative damage was determined based on the intensity of red fluorescence. Fluorescence results showed that on days 7 and 14, the control group wound tissue exhibited strong red fluorescence, indicating severe oxidative stress and ROS accumulation. In contrast, the red fluorescence intensity of the Au-CeO2-bFGF group was significantly reduced, with the most significant difference observed on day 7, indicating that this hydrogel can effectively remove excess ROS from the wound site.

[0083] Further comparison revealed that the antioxidant effect of the PTOD-Au-CeO2 group was similar to that of the Au-CeO2-bFGF group; while the HASA-bFGF group, although showing some antioxidant capacity, was significantly weaker than the group containing Au-CeO2, which is related to the antioxidant activity of Au-CeO2 itself. Figure 9 A).

[0084] Further quantitative analysis confirmed that on days 7 and 14, the red fluorescence intensity of the Au-CeO2-bFGF group was significantly lower than that of the other groups. Figure 9 B).

[0085] The above results indicate that PTOD-Au-CeO2 can effectively reduce the oxidative stress level of diabetic wounds and promote wound healing.

[0086] 5. In the healing process of diabetic wounds, extracellular matrix (ECM) remodeling is a key step in tissue repair and regeneration. The extracellular matrix not only provides structural support for cells, but also participates in signal transduction, cell proliferation, and differentiation.

[0087] VG staining is a classic collagen-specific staining method that stains collagen fibers bright red, clearly and visually displaying their distribution, density, and arrangement within tissues. Observation of wound tissue sections from different treatment groups on days 7 and 14 showed that, compared to the control group, the Au-CeO2-bFGF group exhibited a denser, coarser, bright red collagen fiber network with a more regular arrangement, indicating that the newly formed tissue had higher structural strength and maturity. Figure 10 A).

[0088] Quantitative image analysis further confirmed that the Au-CeO2-bFGF group had the highest collagen fiber deposition in the wound area among all groups. Notably, in terms of collagen fiber regeneration, the HASA-bFGF group was superior to the PTOD-Au-CeO2 group, second only to the Au-CeO2-bFGF group; while the collagen deposition level in the HASA group was significantly lower than that in the HASA-bFGF group. Figure 10 B). This indicates that the release of bFGF can directly promote collagen synthesis.

[0089] In contrast, the advantages of the Au-CeO2-bFGF group in collagen arrangement regularity and structural maturity highlight its synergistic enhancement effect on extracellular matrix functional integration through antioxidant microenvironment regulation: the relief of oxidative stress not only reduces collagen degradation, but also optimizes the secretory rhythm of fibroblasts and the matrix cross-linking ability.

[0090] To further analyze the effects of Au-CeO2-bFGF on extracellular matrix remodeling, this application performed immunofluorescence staining analysis on type I collagen (Col I) and type III collagen (Col III) in wound tissue. The results showed that the Au-CeO2-bFGF group significantly upregulated Col III expression in the early healing stage (day 7) and significantly upregulated Col I expression in the later stage (day 14), and maintained a gradual increase in the Col III / Col I ratio.

[0091] This indicates that Au-CeO2-bFGF can effectively promote the dynamic evolution of extracellular matrix components during normal wound healing: initially, it achieves rapid filling with an immature matrix rich in Col III, followed by an orderly transition to a mature and tough matrix dominated by Col I. More importantly, the Col III / Col I ratio maintained in the Au-CeO2-bFGF group during the healing process is closer to the level of normal physiological skin.

[0092] Although the HASA-bFGF group showed a similar trend to the Au-CeO2-bFGF group, the dynamic changes in the expression intensity and ratio of Col I and Col III were slightly weaker, suggesting that although HASA-bFGF can achieve sustained release of bFGF, it still has limitations in improving the antioxidant microenvironment of diabetic wounds. Figure 10 C-10F).

[0093] 6. Fibronectin (FN) acts as a bridge for cell adhesion, migration, and tissue construction, playing a crucial role in the early stages of wound healing. The Au-CeO2-bFGF group showed the highest FN content in wound tissue on day 7; by day 14, the FN content in all groups tended to be similar. Figure 10 G). The Au-CeO2-bFGF group rapidly and efficiently increased FN content in the early stage of healing, laying a solid spatial framework and signaling basis for subsequent high-quality collagen deposition, and realizing precise temporal relay and functional synergy among extracellular matrix (ECM) components.

[0094] Laminin (LN) is a major component of the basement membrane and is crucial for epithelial regeneration and dermal-epidermal junction reconstruction. In the Au-CeO2-bFGF group, LN levels showed a similar trend to FN: on day 7, LN levels were significantly higher than in other groups, confirming that this hydrogel can effectively initiate and accelerate LN synthesis and deposition in the early healing stage, constructing a stable and functional basement "platform" for keratinocyte adhesion, migration, and differentiation. By day 14, LN levels in all groups tended to converge, which is consistent with the natural process of basement membrane reconstruction completion and stabilization. Figure 10 J). The early LN advantage established by Au-CeO2-bFGF is of decisive significance, ensuring that epidermal cells can quickly and orderly complete reepithelialization in an optimized stromal environment, thereby accelerating wound healing.

[0095] Meanwhile, the successful deposition and structural maintenance of FN and LN are highly dependent on a stable and balanced ECM degradation and remodeling environment. In diabetic wounds, persistent hyperglycemia and oxidative stress often lead to abnormally high expression of matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, resulting in excessive ECM degradation and remodeling disorder, forming a vicious cycle of "degradation greater than synthesis". Therefore, this application detected the levels of MMP-2 and MMP-9 in wound tissue. The results showed that on days 7 and 14, the levels of MMP-2 and MMP-9 in the Au-CeO2-bFGF group were significantly lower than those in the control group; the levels in the PTOD-Au-CeO2 group were also significantly lower than those in the control group. In addition, the concentrations of MMP-2 and MMP-9 in the PTOD group, HASA group, and HASA-bFGF group also decreased to varying degrees. Figure 10 H-10I).

[0096] This indicates that Au-CeO2-bFGF can effectively inhibit the overactivation of MMP-2 and MMP-9 in diabetic wounds. Its key antioxidant component (Au-CeO2) blocks the abnormal upregulation of MMPs upstream by scavenging ROS and alleviating oxidative stress, thereby stabilizing the dynamic balance between ECM degradation and remodeling and protecting the newly formed collagen, FN, and LN networks from excessive degradation. This is another core mechanism by which it promotes high-quality ECM remodeling.

[0097] In contrast, while the HASA-bFGF group showed some inhibitory effects on MMP-2 and MMP-9, its efficacy was weaker than that of the Au-CeO2-bFGF group. This again confirms the limitations of single growth factor delivery in regulating the complex pathological microenvironment of diabetes, and further highlights the synergistic therapeutic advantages of the Au-CeO2-bFGF system, which combines antioxidant microenvironment regulation with growth factor stimulation.

[0098] In summary, Au-CeO2-bFGF promotes ECM remodeling in diabetic wounds through a dual strategy of "promoting collagen synthesis" (regulating collagen dynamic transformation and enhancing early FN and LN deposition) and "inhibiting collagen degradation" (regulating MMP levels), laying a solid structural foundation for high-quality tissue regeneration.

[0099] 7. Angiogenesis is a crucial process in wound repair. The newly formed capillary network is responsible for delivering oxygen and nutrients to regenerating tissue and removing metabolic waste. Regulating the expression of key pro-angiogenic factors can effectively improve local blood supply, thereby accelerating wound healing.

[0100] CD31 and α-SMA are classic biomarkers for evaluating angiogenesis: CD31 specifically marks vascular endothelial cells and is used to directly observe microvessel density; α-SMA mainly marks pericytes and smooth muscle cells, and its presence indicates the maturity and stability of neovascular structures.

[0101] Immunohistochemical staining (IHC) of wound tissue for CD31 and α-SMA showed that, compared with the control group, the expression of CD31 and α-SMA in the Au-CeO2-bFGF group was significantly increased on days 7 and 14, and significantly higher than that in the PTOD-Au-CeO2 group and the HASA-bFGF group. The expression level in the HASA-bFGF group was closer to that in the Au-CeO2-bFGF group. Figure 11 A-11B).

[0102] Further quantitative analysis showed that on day 7, the expression levels of CD31 and α-SMA were highest in the Au-CeO2-bFGF group, followed by the HASA-bFGF group and the PTOD-Au-CeO2 group, while the control group had the lowest levels. By day 14, the Au-CeO2-bFGF group still maintained a superior expression level of CD31 and α-SMA. Figure 11 C-11D).

[0103] In addition, this application detected the content of α-SMA in wound tissue, and the trend of change was consistent with the immunohistochemical results.

[0104] The above results indicate that Au-CeO2-bFGF can effectively promote wound angiogenesis, and this ability mainly depends on the bFGF released from the upper hydrogel. The effect of the PTOD-Au-CeO2 group on wound angiogenesis alone may stem from the indirect improvement of oxidative stress by Au-CeO2, rather than a direct pro-angiogenic factor effect. Although this indirect effect contributes to some extent, it is insufficient to support sustained, efficient, and structurally mature angiogenesis.

[0105] 8. The regulation of various angiogenesis factors also plays an important role in the process of angiogenesis. Vascular endothelial growth factor (VEGF) is a core regulator of angiogenesis, and its expression level is significantly positively correlated with the density of new blood vessels.

[0106] ELISA analysis of VEGF in wound tissue showed that on days 7 and 14, the VEGF content in the Au-CeO2-bFGF group was significantly higher than that in all other groups, with the HASA-bFGF group following closely behind. Figure 11E). This result is consistent with the immunohistochemical results of CD31 and α-SMA, confirming the core mechanism by which bFGF synergistically promotes endothelial cell proliferation, migration, and angiogenesis by upregulating VEGF expression.

[0107] Furthermore, transforming growth factor-β1 (TGF-β1) also plays an important role in angiogenesis, particularly in regulating endothelial cell proliferation, migration, and lumen formation. By detecting the TGF-β1 content in wound tissue, this application found that the Au-CeO2-bFGF group showed significantly higher TGF-β1 expression on days 7 and 14 compared to other groups, with a trend consistent with that of VEGF.

[0108] As a key factor in recruiting pericytes and stabilizing vascular structure, the expression level of platelet-derived growth factor (PDGF) in the Au-CeO2-bFGF group peaked on day 7 and remained at a high level until day 14. Figure 11 The F group was significantly higher than that of the HASA-bFGF group and the PTOD-Au-CeO2 group.

[0109] The above results indicate that Au-CeO2-bFGF can synergistically upregulate the three core angiogenesis factors, VEGF, TGF-β1, and PDGF, forming a cascade regulatory network of "endothelial initiation - lumen formation - pericyte coverage," thereby driving the orderly construction of structurally complete and functionally mature new blood vessels.

[0110] It is worth noting that although both the PTOD-Au-CeO2 group and the HASA-bFGF group showed good pro-angiogenic effects, Au-CeO2-bFGF combined the advantages of both and had a more significant effect. It can not only rapidly increase the expression of angiogenic factors in the early stage, but also maintain their high levels in the later stage, thereby promoting the maturation and stability of blood vessels.

[0111] 9. The in vivo safety of Au-CeO2-bFGF is a key indicator for assessing its clinical feasibility. To comprehensively evaluate the biocompatibility of Au-CeO2-bFGF, the hydrogel was applied to wounds in healthy mice, and one week later, major organs (liver, kidney, heart, lung, and spleen) were collected for HE staining histological analysis.

[0112] Compared with healthy mice, no significant pathological changes or morphological abnormalities were observed in the major organs of mice treated with Au-CeO2-bFGF, indicating that Au-CeO2-bFGF has good biocompatibility. Simultaneously, blood cell counts and liver and kidney function indicators were detected by tail vein blood sampling. The results showed that there were no significant differences in white blood cells, red blood cells, ALT, and AST levels between the Au-CeO2-bFGF-treated group and healthy mice, further confirming that this treatment had no significant effect on the function of the major organs in mice. In conclusion, Au-CeO2-bFGF exhibits good in vivo biocompatibility, laying a solid foundation for its clinical application in the treatment of diabetic wounds.

[0113] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A hydrogel dressing, characterized in that, include: The first hydrogel was prepared by cross-linking tetra(4-sulfophenyl)boronic acid, polyvinyl alcohol and oxidized dextran, and Au-CeO2 nanozyme particles were loaded inside the first hydrogel. The second hydrogel was prepared by photocrosslinking of methacrylated hyaluronic acid and sodium alginate, and loaded with basic fibroblast growth factor. The first and second hydrogels are combined as the lower and upper layers, respectively, to form a double-layer hydrogel dressing. The Au-CeO2 nanozyme particles have a core-shell nanostructure, consisting of an Au core and a CeO2 shell, with the Au core encapsulated by the CeO2 shell. The preparation process of the first hydrogel includes: mixing 200 μL of 5% tetrakis(4-sulfophenyl)boric acid solution, 50 μL of 5% oxidized dextran solution and 0.25 mg of Au-CeO2 nanozyme particles, adding 150 μL of 8% polyvinyl alcohol solution and stirring, and allowing it to stand to form the first hydrogel; The preparation process of the second hydrogel includes: sequentially adding 100 μL of 4% methacrylated hyaluronic acid solution, 100 μL of 3% sodium alginate solution, 20 μL of 100 μg / mL basic fibroblast growth factor solution, and 0.6 mg of phenyl-2,4,6-trimethylbenzoylphosphonate lithium to the surface of the first hydrogel, and completing in-situ photocrosslinking by ultraviolet light irradiation to form the second hydrogel on the surface of the first hydrogel.

2. The hydrogel dressing according to claim 1, characterized in that, The preparation process of the Au-CeO2 nanozyme particles includes a first reaction stage involving chloroauric acid solution, cerium nitrate solution, and ammonia solution, and a second reaction stage involving the addition of cerium nitrate solution to the reaction solution in the first reaction stage for further reaction.

3. Use of the hydrogel dressing as described in any one of claims 1-2 for the preparation of wound repair and treatment drugs.

4. The use of the hydrogel dressing according to claim 3 in the preparation of wound repair and treatment drugs, characterized in that, The drug is used to treat diabetic wounds.

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