A local blood sugar control, antioxidant, photothermal antibacterial hydrogel dressing and a preparation method and application thereof

Abstract

This invention relates to a localized hydrogel dressing with glycemic control, antioxidant, photothermal antibacterial properties, and its preparation method and application. The composition of the localized glycemic control, antioxidant, photothermal antibacterial hydrogel dressing includes: a nanoscale dumbbell structure Au-CeO2 composed of gold nanorods and cerium oxide spheres at both ends, and glucose oxidase (GO). x And a reactive oxygen species (ROS) sensitive hydrogel matrix for encapsulating the Au-CeO2 and glucose oxidase.

Description

Technical Field

[0001] This invention belongs to the field of biomedical material preparation technology, specifically relating to a localized sugar-controlling, antioxidant, photothermal antibacterial hydrogel dressing, its preparation method, and its application. Background Technology

[0002] Wounds in patients with chronic diabetes are widely recognized as a major challenge for global healthcare systems due to their high morbidity, mortality, and recurrence rates. Unlike other common wounds, chronic diabetic wounds are more susceptible to bacterial infection and excessive accumulation of reactive oxygen species (ROS) due to their complex microenvironment, thus hindering the wound healing process. Furthermore, high blood sugar levels in the local wound can lead to elevated ROS concentrations, damaging normal cells and tissues and causing persistent inflammation.

[0003] Therefore, it is of great significance to develop a multifunctional dressing that can regulate local blood glucose levels, effectively kill bacteria and remove excess ROS to reduce oxidative stress. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a hydrogel dressing with localized sugar control, antioxidant, and photothermal antibacterial properties, its preparation method, and its application in promoting diabetic wound healing.

[0005] In a first aspect, the present invention provides a hydrogel dressing for localized sugar control, anti-oxidation, and photothermal antibacterial effects. The hydrogel dressing comprises: a nanoscale dumbbell structure Au-CeO2 composed of gold nanorods and cerium oxide spheres at both ends thereends, and glucose oxidase GO. x And a reactive oxygen species (ROS) sensitive hydrogel matrix for encapsulating the Au-CeO2 and glucose oxidase.

[0006] Preferably, the length of the dumbbell-shaped Au-CeO2 structure is 80-150 nm, more preferably 100 nm; and the width is 10-30 nm, more preferably 20 nm. The reactive oxygen species (ROS) sensitive hydrogel matrix is ​​a polyvinyl alcohol (PVA)-trisodium phosphate borate (TSPBA) composite hydrogel.

[0007] Preferably, the localized sugar-controlling, antioxidant, and photothermal antibacterial hydrogel dressing has a porous structure; more preferably, the average pore size is 5-20 nm and the porosity is 5-10%.

[0008] Preferably, in the localized sugar-controlling, antioxidant, and photothermal antibacterial hydrogel dressing, the concentrations of polyvinyl alcohol and trisodium borate phosphate are both 5-10 wt%, preferably 5 wt%; the concentrations of dumbbell-structured Au-CeO2 and glucose oxidase are both 0.5-1 mg / mL, preferably 1 mg / mL.

[0009] Secondly, the present invention provides a method for preparing the above-mentioned localized sugar-controlling, antioxidant, and photothermal antibacterial hydrogel dressing, the preparation method comprising the following steps: (1) Au seed solution was added to Au growth solution to carry out Au seed growth reaction to obtain gold nanorods Au NRs, and then PtCl4 was added sequentially. 2- solutions, Ce 3+ Salt solution and deionized water were dried and purified to obtain dumbbell-shaped Au-CeO2; (2) The dumbbell-shaped Au-CeO2, glucose oxidase, polyvinyl alcohol solution, and trisodium borate solution are mixed to obtain the local sugar control, antioxidant, photothermal antibacterial hydrogel dressing.

[0010] Preferably, in step (1), the preparation process of the Au seed solution includes the following steps: injecting 5-10mM, 400-600μL NaBH4 solution into a mixture of 5-10mM, 200-250μL HAuCl4 and 0.1-0.2M, 9.75-20mL CTAB to obtain the seed solution; The preparation process of the Au growth solution includes the following steps: 5-10 mM, 1-2 mL HAuCl4 solution, 5-10 mM, 200-400 μL silver nitrate solution, and 0.5-1 M, 400-800 μL hydrochloric acid are added sequentially to 0.1-0.2 M, 400-800 μL CTAB solution and mixed. Then, 0.05-0.1 M, 160-320 mL ascorbic acid solution is added. The mixture is allowed to become colorless to obtain the Au growth solution. The Au seed growth reaction is carried out at a temperature of 25-37°C for at least 6 hours.

[0011] Preferably, in step (1), the PtCl4-containing... 2- The solution is either K₂PtCl₄ solution or H₂PtCl₄ solution, containing PtCl₄. 2- The concentration of the solution is 0.1-0.2 mM, preferably 0.1 mM; the ratio of gold nanorods (Au NRs) to K₂PtCl₄ or H₂PtCl₄ is 1-2 mol: 0.1-0.2 mol; PtCl₄ is added... 2- The reaction time for the solution is 2-5 minutes, and the reaction temperature is 25-37℃.

[0012] Preferably, in step (1), the Ce 3+ The salts are cerium nitrate, cerium chloride, and cerium acetate. 3+ The concentration of the salt solution is 5-10 mM, preferably 10 mM; gold nanorods Au NRs and Ce3+ The salt ratio is 1-2 mol: 0.5-1 mol; Ce is added. 3+ The reaction time for the salt solution is 1-2 hours, and the reaction temperature is 100-120℃.

[0013] Preferably, in step (2), the concentrations of the polyvinyl alcohol solution and the trisodium borate solution are both 5-10 wt%, preferably 5 wt%. The ratio of dumbbell-shaped Au-CeO2, glucose oxidase, polyvinyl alcohol solution, and trisodium borate solution is 2-5 mg: 2-5 mg: 2-4 mL: 2-4 mL.

[0014] Thirdly, the present invention provides an application of the above-mentioned localized sugar-controlling, antioxidant, photothermal antibacterial hydrogel dressing in the preparation of products for the treatment of diabetic wound infections.

[0015] Beneficial effects The preparation process of this invention is simple, easy to implement, pollution-free, and highly efficient. The resulting ACG multifunctional hydrogel dressing has excellent antioxidant, photothermal sterilization, and local blood glucose regulation properties, and can be used to effectively treat diabetic wound infections, showing great promise for clinical application. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating the preparation process of the dumbbell-structured Au-CeO2 and ACG hydrogel dressing, as exemplified by the present invention. Figure 2 Characterization images of the ACG hydrogel dressing prepared in Example 1: a and b are TEM images of gold nanorods and Au-CeO2 dumbbells, respectively; c and d are high-angle annular dark-field images and corresponding mapping images of the Au-CeO2 dumbbells, respectively; e and f are DLS size distribution and XRD patterns of different samples, respectively; g and h are XPS analysis of Au and Ce, respectively; i is a digital photograph of different working solutions and the gel after mixing; j is a cryo-scanning electron microscope (Cyro-SEM) image of the ACG gel; k is a representative CLSM fluorescence image of the ACG gel, in which GO x The samples were labeled with fluorescein isothiocyanate (FITC) (green), and Au-CeO2 was labeled with Cy5.5 (red). Figure 3The following are the performance evaluation results of the ACG gel prepared in Example 1 for scavenging ROS and regulating macrophages: a) ESR spectra of oxygen in different reaction systems (each solution contains 100 μm H2O2 and 0.1 mM FeCl2, a typical Fenton reaction system, followed by the addition of DMPO free radical scavenger and corresponding materials); b) oxygen generation capacity of different samples detected by oxygen probe; c) ESR spectra of ·OH in different reaction systems; d) evaluation of O2 scavenging by dumbbells of different concentrations of Au-CeO2 using WST-8 and SOD detection kits. ·- e represents the H2O2 scavenging capacity of Au-CeO2 dumbbells at different concentrations, measured using an H2O2-specific total CAT assay kit; f represents the efficiency of the Fenton reaction system in degrading methylene blue MB after adding different concentrations of Au-CeO2 dumbbells; g represents the GO content of the ACG gel under the action of H2O2. x Release curve; h represents the change in glucose content in standard glucose solution after the addition of different materials; i and j are confocal fluorescence images of ROS levels in L929 and RAW264.7 cells, respectively, with cells stained with ROS probes DCFH-DA (green fluorescence) and DAPI (blue fluorescence); k is a schematic diagram of ACG gel regulating the polarization of M1 macrophages to M2 type by clearing ROS; l represents the expression levels of M1 and M2 related genes in LPS-stimulated bone marrow (BM)-derived macrophages (BMDMs) (M1 macrophages) after different treatments; Figure 4 The photothermal antibacterial properties of the ACG gel prepared in Example 1 are evaluated as follows: a and b are 0.5-1.5 W / cm². 2 Real-time infrared thermal images and corresponding photothermal curves of Au-CeO2 dumbbells (200ppm) and ACG gel (200ppm) after 5 minutes of near-infrared laser irradiation; c and e are quantitative schematic diagrams of the relative survival rates of Staphylococcus aureus and Escherichia coli after treatment with different systems; d and f are fluorescence microscopy images of Staphylococcus aureus and Escherichia coli stained with SYTO9 / PI dye after treatment with different systems. All live bacteria are stained green by SYTO9 and dead bacteria are stained red by PI. Figure 5The following are performance evaluation results of the ACG gel prepared in Example 1 in promoting diabetic wound healing by scavenging ROS and locally controlling glucose: a) Schematic diagram of skin injury model construction and ACG gel promoting wound healing process; b) Schematic diagram of fasting blood glucose levels of healthy mice and model mice to confirm the diabetic model (n=5 mice per group); c) Representative digital images of diabetic wounds on days 3, 5 and 7 after wound healing; d) Wound closure status on days 3, 5 and 7 after wound healing; e) Wound healing rate of different groups within 7 days after wound healing; f) Quantification of wound closure in diabetic mice of each group on day 7 after wound healing (n=3); g) H&E staining results of mice in different groups on day 3 after wound healing. Figure 6 The following are the performance evaluation results of the ACG gel prepared in Example 1 in promoting the healing of diabetic wounds under Staphylococcus aureus infection: a is a schematic diagram of the skin injury model construction and the process of ACG gel promoting wound healing; b and f are real-time infrared thermal images and temperature rise curves after the infected wounds were treated with free gel, AC gel or ACG gel and then irradiated with 808 nm laser for 5 minutes (1.5 W / cm²); c is a bacterial colony photograph and corresponding bacterial quantity quantification of the infected area of ​​different groups on day 3; d is a representative digital image of the infected wounds on days 3, 5, 7 and 9 after different treatments; e is a simulated wound boundary diagram; g is the monitoring of wound closure within 9 days of each treatment; h is the glucose content in the wound on day 3 after different treatments; i is the quantification of the wound healing status of each group of diabetic mice on day 9 after wound healing; (1) PBS, (2) laser, (3) ACG gel, (4) AC gel + laser, (5) ACG gel + laser. Detailed Implementation

[0017] The present invention will be further illustrated by the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0018] First, this invention provides a hydrogel dressing for localized sugar control, anti-oxidation, and photothermal antibacterial properties. The composition of the ACG gel in this hydrogel dressing may include: a nanoscale dumbbell structure Au-CeO2 composed of gold nanorods and cerium oxide spheres at both ends, and glucose oxidase GO. x And a reactive oxygen species (ROS) sensitive hydrogel matrix for encapsulating the Au-CeO2 and glucose oxidase.

[0019] In some embodiments, the length of the dumbbell-shaped Au-CeO2 structure can be 80-150 nm, preferably 100 nm; the width can be 10-30 nm, preferably 20 nm. The Au-CeO2 dumbbell structure design used in this invention features gold nanorods in the middle with excellent photothermal conversion capabilities, while cerium oxide spheres at both ends exhibit superior antioxidant properties. The dumbbell structure separates the gold and cerium oxide, facilitating electron transfer during the reaction and thus accelerating the reaction.

[0020] In some embodiments, the reactive oxygen species (ROS)-sensitive hydrogel matrix can be a polyvinyl alcohol (PVA)-trisodium phosphate borate (TSPBA) composite hydrogel. By employing a ROS-sensitive hydrogel matrix, responsive release of components can be achieved based on the excess ROS present in diabetic wounds.

[0021] In some embodiments, the localized sugar-controlling, antioxidant, and photothermal antibacterial hydrogel dressing has a porous structure; preferably, the average pore size can be 5-20 nm, and the porosity can be 5-10%.

[0022] In some embodiments, the concentrations of polyvinyl alcohol and trisodium borate phosphate in the localized sugar-controlling, antioxidant, and photothermal antibacterial hydrogel dressing can both be 5-10 wt%, preferably 5 wt%; the concentrations of dumbbell-structured Au-CeO2 and glucose oxidase can both be 0.5-1 mg / mL, preferably 1 mg / mL. By controlling the content of the above four components within a suitable range, their respective functions can be fully utilized while avoiding damage to the overall structure of the hydrogel.

[0023] This invention is based on novel nanoscale gold cerium oxide (Au-CeO2) dumbbells and glucose oxidase (GO). x This multifunctional hydrogel dressing is used for localized blood sugar control and photothermal sterilization, protecting tissues from ROS damage and promoting the healing of infected diabetic wounds. In particular, the nano-dumbbell-structured Au-CeO2 with its elongated gold nanorods exhibits excellent photothermal antibacterial effects, while the cerium oxide at both ends possesses strong antioxidant activity, rapidly scavenging ROS and thus protecting damaged tissues from oxidative damage and alleviating photothermal-induced inflammatory responses. The released GO... x It can oxidize and consume glucose to lower local blood sugar levels.

[0024] The following, combined with Figure 1 This invention provides an exemplary method for preparing a hydrogel dressing with localized blood sugar control, antioxidant, and photothermal antibacterial properties. The preparation method may include the following steps: (1) Au seed solution was added to Au growth solution to carry out Au seed growth reaction to obtain gold nanorods Au NRs, and then PtCl4 was added sequentially.2- solutions, Ce 3+ Salt solution and deionized water were dried and purified to obtain dumbbell-shaped Au-CeO2; (2) The dumbbell structure Au-CeO2 and glucose oxidase GO x The localized sugar-controlling, antioxidant, photothermal antibacterial hydrogel dressing is obtained by mixing with polyvinyl alcohol solution and trisodium borate solution.

[0025] In some embodiments, the preparation process of the Au seed solution in step (1) may include the following steps: injecting 5-10mM, 400-600μL NaBH4 solution (e.g., 10mM, 600μL) into a mixture of 5-10mM, 200-250μL HAuCl4 (e.g., 10mM, 250μL) and 0.1-0.2M, 9.75-20mL CTAB (0.1M, 9.75mL), and then rapidly inverting for 1-3 minutes (e.g., 2 minutes) to obtain the seed solution; preferably, it is used after standing for 1-3 hours (e.g., 2 hours).

[0026] In some embodiments, the preparation process of the Au growth solution in step (1) may include the following steps: adding 5-10mM, 1-2mL HAuCl4 solution (e.g., 10mM, 2mL), 5-10mM, 200-400μL silver nitrate solution (e.g., 10mM, 40mL) and 0.5-1M, 400-800μL hydrochloric acid (e.g., 1M, 800μL) sequentially to 0.1-0.2M, 40-800mL CTAB solution (e.g., 0.1M, 320mL) and mixing them together, then adding 0.05-0.1M, 160-320mL ascorbic acid solution (e.g., 0.1M, 320mL) until the mixed solution becomes colorless to obtain the Au growth solution.

[0027] In some embodiments, in step (1), the temperature of the Au seed growth reaction can be 25-37°C and the time can be at least 6 hours.

[0028] In some implementations, in step (1), the PtCl4-containing... 2- The solution can be either K₂PtCl₄ solution or H₂PtCl₄ solution, containing PtCl₄. 2- The concentration of the solution can be 0.1-0.2 mM, preferably 0.1 mM; the ratio of gold nanorods (Au NRs) to K₂PtCl₄ or H₂PtCl₄ can be 1-2 mol: 0.1-0.2 mol; PtCl₄ is added... 2- The reaction time for the solution can be 2-5 minutes (e.g., 2 minutes), and the reaction temperature can be 25-37℃.

[0029] By adding PtCl4 2- The solution can adsorb PtCl4 onto Au NRs 2- The ions then undergo an auto-oxidation-reduction reaction with the subsequent cerium ions.

[0030] In some implementations, in step (1), the Ce 3+ The salt can be cerium nitrate, cerium chloride, or cerium acetate, with cerium nitrate being preferred; Ce 3+ The concentration of the salt solution can be 5-10 mM, preferably 10 mM; gold nanorods Au NRs and Ce 3+ The salt ratio can be 1-2 mol: 0.5-1 mol; add Ce 3+ The reaction time for the salt solution can be 1-2 hours, and the reaction temperature can be 100-120℃.

[0031] CTAB can be non-uniformly distributed around gold nanorods, with less at both ends. Therefore, the steric hindrance at the ends is smaller, and other substances are more likely to aggregate and react at the ends to form specific dumbbell structures.

[0032] In some embodiments, in step (2), the concentrations of the polyvinyl alcohol solution and the trisodium borate solution can both be 5-10 wt%, preferably 5 wt%.

[0033] In some embodiments, in step (2), the ratio of dumbbell-shaped Au-CeO2, glucose oxidase, polyvinyl alcohol solution, and trisodium borate solution can be 2-5 mg: 2-5 mg: 2-4 mL: 2-4 mL.

[0034] The ACG gel dressing prepared by the method provided in this invention, when placed on a wound, can rapidly remove excess ROS in the wound and regulate the immune microenvironment, such as inducing pro-inflammatory M1 macrophages to polarize into anti-inflammatory M2 macrophages. Furthermore, the dressing can rapidly generate heat under near-infrared laser irradiation, killing bacteria (such as Staphylococcus aureus and Escherichia coli) proliferating in the wound, preventing infection, and thus promoting the healing of diabetic wounds. More importantly, the locally released GO... x It can rapidly metabolize glucose in the wound environment, lower blood sugar levels, thereby reducing oxidative stress and depriving bacteria of their energy source, reducing the risk of infection, and thus jointly promoting wound healing. The development of the multifunctional hydrogel dressing in this invention provides an innovative platform for the treatment of diabetic wounds and has enormous clinical application potential in the future biomedical field.

[0035] The ACG multifunctional hydrogel dressing provided by this invention can be used to prepare products for the treatment of diabetic wound infections, serving as a treatment method that simultaneously controls blood sugar levels locally, eliminates reactive oxygen species (ROS), and sterilizes to promote the healing of infected diabetic wounds.

[0036] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0037] Example 1

[0038] The preparation method of the localized sugar-controlling, antioxidant, and photothermal antibacterial hydrogel dressing provided by the present invention includes the following steps: (1) Inject freshly prepared ice-cold NaBH4 solution (10mM, 600μL) into a mixture of HAuCl4 (10mM, 250μL) and CTAB (0.1M, 9.75mL), then quickly invert for 2 minutes to obtain seed solution A, and use it after standing for 2 hours; Add HAuCl4 solution (10mM, 2mL), silver nitrate solution (10mM, 400μL), and hydrochloric acid (1M, 800μL) sequentially to CTAB solution (0.1M, 40mL) and mix. Then add freshly prepared ascorbic acid solution (0.1M, 320mL) and wait for the mixed solution to turn colorless to obtain Au growth solution. Seed solution A (Au NR sample) was added to the growth solution, inverted and mixed for 2 minutes, and then the mixture was allowed to stand for at least 6 hours. The synthesized AuNRs solution (10 mL) was centrifuged and then washed with deionized water (30 mL) to remove excess surfactant. The Au NRs were then redispersed in CTAB solution (0.1 mM, 5 mL) in a 15 mL centrifuge tube. Subsequently, K2PtCl4 solution (0.1 mM) was introduced into the AuNRs solution with gentle stirring, and the resulting mixture was kept at ambient temperature for 2 minutes to promote PtCl4 growth. 2-Ions were adsorbed onto Au NRs; then, fresh Ce(AC)3 solution (10 mM) and deionized water (4.3 mL) were added sequentially to the Au NRs solution with gentle stirring; the total volume of the prepared solution was 10 mL, and the CTAB concentration was 50 μM; the solution was then placed in an oven at 100 °C for 1 hour to obtain dumbbell-shaped Au-CeO2; the obtained product was purified by centrifugation and redispersed in 10 mL of deionized water for subsequent use. (2) Dissolve polyvinyl alcohol (PVA, 1 g) in 20 mL of deionized water and gradually raise the temperature to 95 °C with continuous stirring to obtain a 5 wt% PVA aqueous solution; then, prepare a 5 wt% trisodium phosphate borate (TSPBA) aqueous solution in the same manner; dissolve 2 mg Au-CeO2 dumbbells and 2 mg glucose oxidase in 2 mL of the PVA solution prepared above; then add an equal volume of TSPBA solution to prepare the local sugar control, antioxidant, photothermal antibacterial ACG multifunctional hydrogel dressing.

[0039] Figure 2 Characterization images of the ACG hydrogel dressing prepared in Example 1: a and b are TEM images of gold nanorods and Au-CeO2 dumbbells, respectively; c and d are high-angle annular dark-field images and corresponding mapping images of the Au-CeO2 dumbbells, respectively; e and f are DLS size distribution and XRD patterns of different samples, respectively; g and h are XPS analysis of Au and Ce, respectively; i is a digital photograph of different working solutions and the gel after mixing; j is a cryo-scanning electron microscope (Cyro-SEM) image of the ACG gel; k is a representative CLSM fluorescence image of the ACG gel, in which GO x The sample was labeled with fluorescein isothiocyanate (FITC) (green), and Au-CeO2 was labeled with Cy5.5 (red).

[0040] Evaluation of the effects of Au-CeO2 dumbbells and ACG gels on superoxide anion (O2) using a superoxide dismutase (SOD) assay kit ·- The ability to clear ( ) First, the dispersed CeSACs are added to a working solution containing a water-soluble tetrazolium salt (WST). The water-soluble tetrazolium salt reacts with O2. ·-A specific reaction occurred; subsequently, xanthine oxidase was added to the mixture, and the mixture was thoroughly homogenized to obtain dumbbell-shaped Au-CeO2 and ACG gels containing an equivalent concentration of 20 μg / mL Au-CeO2; after incubation at 37°C for 20 minutes, the absorbance of different solution groups was measured at a wavelength of 450 nm; electron spin resonance (ESR) analysis: using O2 generated by the photolysis of riboflavin (50 μL, 6 mM) as a substrate, the scavenging effect of different samples was evaluated; O2 was captured using the spin trapping agent 5,5-dimethyl-1-pyrrolidone N-oxide (DMPO). ·- DMPO-O2 is formed - The adducts exhibited characteristic peaks in the ESR spectrum; changes in the intensity of these peaks were subsequently analyzed to evaluate the O2 content of the Au-CeO2 dumbbells and ACG gels. ·- Clearance ability.

[0041] The -OH scavenging activity of Au-CeO2 dumbbells and ACG gels was assessed using a microplate reader by evaluating the effective elimination of -OH generated via the Fenton reaction. A solution of FeSO4 (1 mM) and H2O2 (2 mM) was prepared in sodium acetate buffer, and various samples (Au-CeO2 dumbbells and ACG gels, each containing an equivalent concentration of Au-CeO2 at 20 μg / mL) were added to the solution. After a 5-minute reaction, 5 mM 3,3',5,5'-tetramethylbenzidine (TMB) was added, and the absorbance of the solution was measured at 650 nm. This further confirmed the ability of different samples to scavenge hydroxyl radicals generated by the Fenton reaction. ESR spectroscopy analysis was performed using the above method. Ammonium molybdate (AM) was reacted with H₂O₂ and quantified by colorimetry. H₂O₂ was reacted with Au-CeO₂ dumbbells and ACG gels (each containing an equivalent Au-CeO₂ concentration of 20 μg / mL) for 10 minutes, and then AM was added to the system. Absorbance was then measured at 405 nm. Simultaneously, the oxygen content in the solution was assessed using a dissolved oxygen analyzer. Bone marrow (BM)-derived macrophages (BMDMs) were isolated from mouse bone marrow. All cells were cultured in a humid environment at 37°C with 5% CO₂, and the culture medium was changed every 2–3 days. Macrophages subjected to different stimuli were co-incubated with ACG hydrogels, and their morphology was observed by flow cytometry and photographed to identify their phenotypic status.

[0042] Figure 3The following are the performance evaluation results of the ACG gel prepared in Example 1 for scavenging ROS and regulating macrophages: a) ESR spectra of oxygen in different reaction systems (each solution contains 100 μm H2O2 and 0.1 mM FeCl2, a typical Fenton reaction system, followed by the addition of DMPO free radical scavenger and corresponding materials); b) oxygen generation capacity of different samples detected by oxygen probe; c) ESR spectra of ·OH in different reaction systems; d) evaluation of O2 scavenging by dumbbells of different concentrations of Au-CeO2 using WST-8 and SOD detection kits. ·- e represents the H2O2 scavenging capacity of Au-CeO2 dumbbells at different concentrations, measured using an H2O2-specific total CAT assay kit; f represents the efficiency of the Fenton reaction system in degrading methylene blue MB after adding different concentrations of Au-CeO2 dumbbells; g represents the GO content of the ACG gel under the action of H2O2. x Release curve; h represents the change in glucose content in standard glucose solution after the addition of different materials; i and j are confocal fluorescence images of ROS levels in L929 and RAW264.7 cells, respectively, with cells stained with ROS probes DCFH-DA (green fluorescence) and DAPI (blue fluorescence); k is a schematic diagram of ACG gel regulating the polarization of M1 macrophages to M2 type by clearing ROS; l represents the expression levels of M1 and M2 related genes in LPS-stimulated bone marrow (BM)-derived macrophages (BMDMs) (M1 macrophages) after different treatments.

[0043] The photothermal efficacy of the dumbbell-shaped Au-CeO2 and ACG gel prepared in Example 1 was further evaluated below: 100 μL aliquots were dispensed into 96-well plates, each aliquot containing an equivalent concentration of 20 μg / mL Au-CeO2; an 808 nm laser was used at 0.5, 0.75, 1, 1.25, and 1.5 W / cm². 2 Different power densities were used to irradiate these samples for 5 minutes; temperature measurements and thermal imaging were performed at 1-minute intervals using a FLIR thermal imaging camera. Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli were cultured in antibiotic-free standard Luria Bertani (LB) medium and incubated at 37°C and 200 rpm for 12-16 hours; the optical density of the bacterial suspension at 600 nm was measured using a microplate reader; the in vitro photothermal antibacterial effects of Au-CeO2 dumbbells and ACG gel against Staphylococcus aureus and Escherichia coli were quantitatively evaluated using plate counting and live / dead bacterial staining methods; that is, Au-CeO2 dumbbells and ACG gel were added to Staphylococcus aureus (10... 6 CFU / mL) or E. coli (10 6The concentration of Au-CeO2 dumbbells was adjusted to 20 μg / mL (CFU / mL). Subsequently, Staphylococcus aureus and Escherichia coli suspensions were divided into five groups: 1) control group, 2) laser group, 3) Au-CeO2 group, 4) Au-CoO2+ laser group, and 5) ACG+ laser group. Before irradiation, the bacteria were incubated with Au-CeO2 dumbbells or ACG gel at 37°C for 6 hours; then, NIR laser (808 nm, 1.5 W / cm²) was used. 2 Groups 2, 4, and 5 of bacteria were irradiated for 5 minutes; subsequently, the bacterial suspension was diluted with PBS, and 100 μL of the resulting dilution was spread onto LB agar; after incubation, CFU were counted; bacterial viability was also measured using the standard bacterial MMT assay; for the live / dead staining assay, bacteria were stained with the SYTO9 / PI activity kit at 37°C for 30 to 60 minutes; then, the stained bacteria were placed on transparent slides and visualized using a confocal laser scanning microscope; and the morphology of the treated bacteria was detected by SEM.

[0044] Figure 4 The photothermal antibacterial properties of the ACG gel prepared in Example 1 are evaluated as follows: a and b are 0.5-1.5 W / cm². 2 Real-time infrared thermal images and corresponding photothermal curves of Au-CeO2 dumbbells (200ppm) and ACG gel (200ppm) after 5 minutes of near-infrared laser irradiation; c and e are quantitative schematic diagrams of the relative survival rates of Staphylococcus aureus and Escherichia coli after treatment with different systems; d and f are fluorescence microscopy images of Staphylococcus aureus and Escherichia coli after treatment with different systems stained with SYTO9 / PI dye. All live bacteria are stained green by SYTO9, and dead bacteria are stained red by PI.

[0045] The effect of ACG gel on wound healing in diabetic patients was evaluated using a type 1 diabetic mouse model characterized by skin wounds. To establish a streptozotocin (STZ)-induced type 1 diabetes model, 6-week-old female Babl / c mice were administered STZ via intraperitoneal injection at a dose of 50 mg / kg for 5 consecutive days. Two weeks later, blood samples were collected via the tail vein, and blood glucose levels were measured using a glucometer. Mice with blood glucose levels exceeding 15 mM were classified as diabetic and maintained under standard conditions for another four weeks before full-thickness skin wounds were created. Subsequently, full-thickness circular skin wounds with a diameter of 20 mm were induced on the back of the mice. The diabetic mice with wounds were randomly divided into five experimental groups (n=5 per group): control group, receiving 150 μL of PBS; free gel group, treated with 150 μL of free gel; G gel group, receiving 150 μL of G gel. G gel hydrogel; AC gel group, receiving 150 μL LAC gel; ACG gel group, receiving 150 μL LAC gel; hydrogel treatment was applied to the wound and changed every two days; the wound was photographed on days 0, 3, 5, and 7; the wound area was quantified and analyzed using ImageJ software; the relative wound size was calculated using the following formula: Relative wound size (%) = (Arean / Area0) × 100%, where Area0 represents the initial wound area and Arean represents the wound area at subsequent time points.

[0046] Figure 5 The following are performance evaluation results of the ACG gel prepared in Example 1 in promoting diabetic wound healing by scavenging ROS and locally controlling glucose: a) Schematic diagram of skin injury model construction and ACG gel promoting wound healing process; b) Schematic diagram of fasting blood glucose levels of healthy mice and model mice to confirm the diabetic model (n=5 mice per group); c) Representative digital images of diabetic wounds on days 3, 5 and 7 after wound healing; d) Wound closure status on days 3, 5 and 7 after wound healing; e) Wound healing rate of different groups within 7 days after wound healing; f) Quantification of wound closure in diabetic mice in each group on day 7 after wound healing (n=3); g) H&E staining results of different groups of mice on day 3 after wound healing.

[0047] Figure 6The following are the performance evaluation results of the ACG gel prepared in Example 1 in promoting the healing of diabetic wounds under Staphylococcus aureus infection: a is a schematic diagram of the skin injury model construction and the process of ACG gel promoting wound healing; b and f are real-time infrared thermal images and temperature rise curves after the infected wounds were treated with free gel, AC gel or ACG gel and then irradiated with 808 nm laser for 5 minutes (1.5 W / cm²); c is a bacterial colony photograph and corresponding bacterial quantity quantification of the infected area of ​​different groups on day 3; d is a representative digital image of the infected wounds on days 3, 5, 7 and 9 after different treatments; e is a simulated wound boundary diagram; g is the monitoring of wound closure within 9 days of each treatment; h is the glucose content in the wound on day 3 after different treatments; i is the quantification of the wound healing status of each group of diabetic mice on day 9 after wound healing; (1) PBS, (2) laser, (3) ACG gel, (4) AC gel + laser, (5) ACG gel + laser.

[0048] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A hydrogel dressing with localized blood sugar control, antioxidant, photothermal antibacterial properties, characterized in that, The composition of the localized sugar-controlling, antioxidant, and photothermal antibacterial hydrogel dressing includes: a nanoscale dumbbell structure Au-CeO2 composed of gold nanorods and cerium oxide spheres at both ends, and glucose oxidase GO. x And a reactive oxygen species (ROS) sensitive hydrogel matrix for encapsulating the Au-CeO2 and glucose oxidase.

2. The hydrogel dressing with localized blood sugar control, antioxidant, photothermal antibacterial properties according to claim 1, characterized in that, The dumbbell-shaped Au-CeO2 structure has a length of 80-150 nm, preferably 100 nm, and a width of 10-30 nm, preferably 20 nm. The reactive oxygen species (ROS) sensitive hydrogel matrix is ​​a polyvinyl alcohol (PVA)-trisodium phosphate borate (TSPBA) composite hydrogel.

3. The localized sugar-controlling, antioxidant, photothermal antibacterial hydrogel dressing according to claim 1 or 2, characterized in that, The localized sugar-controlling, antioxidant, and photothermal antibacterial hydrogel dressing has a porous structure; preferably, the average pore size is 5-20 nm and the porosity is 5-10%.

4. The topical sugar-controlling, antioxidant, and photothermal antibacterial hydrogel dressing according to any one of claims 1-3, characterized in that, In the aforementioned hydrogel dressing for localized sugar control, anti-oxidation, and photothermal antibacterial properties, the concentrations of polyvinyl alcohol and trisodium borate phosphate are both 5-10 wt%, preferably 5 wt%; the concentrations of dumbbell-structured Au-CeO2 and glucose oxidase are both 0.5-1 mg / mL, preferably 1 mg / mL.

5. A method for preparing a topical sugar-controlling, antioxidant, and photothermal antibacterial hydrogel dressing according to any one of claims 1-4, characterized in that, The preparation method includes the following steps: (1) Au seed solution was added to Au growth solution to carry out Au seed growth reaction to obtain gold nanorods Au NRs, and then PtCl4 was added sequentially. 2- solutions, Ce 3+ Salt solution and deionized water were dried and purified to obtain dumbbell-shaped Au-CeO2; (2) The dumbbell-shaped Au-CeO2, glucose oxidase, polyvinyl alcohol solution, and trisodium borate solution are mixed to obtain the local sugar control, antioxidant, photothermal antibacterial hydrogel dressing.

6. The preparation method according to claim 5, characterized in that, In step (1), the preparation process of the Au seed solution includes the following steps: injecting 5-10mM, 400-600μL NaBH4 solution into a mixture of 5-10mM, 200-250μL HAuCl4 and 0.1-0.2M, 9.75-20mL CTAB to obtain the seed solution; The preparation process of the Au growth solution includes the following steps: 5-10 mM, 1-2 mL HAuCl4 solution, 5-10 mM, 200-400 μL silver nitrate solution, and 0.5-1 M, 400-800 μL hydrochloric acid are added sequentially to 0.1-0.2 M, 400-800 μL CTAB solution and mixed. Then, 0.05-0.1 M, 160-320 mL ascorbic acid solution is added. The mixture is allowed to become colorless to obtain the Au growth solution. The Au seed growth reaction is carried out at a temperature of 25-37°C for at least 6 hours.

7. The preparation method according to claim 5 or 6, characterized in that, In step (1), the PtCl4-containing 2- The solution is either K₂PtCl₄ solution or H₂PtCl₄ solution, containing PtCl₄. 2- The concentration of the solution is 0.1-0.2 mM, preferably 0.1 mM; the ratio of gold nanorods (Au NRs) to K₂PtCl₄ or H₂PtCl₄ is 1-2 mol: 0.1-0.2 mol; PtCl₄ is added... 2- The reaction time for the solution is 2-5 minutes, and the reaction temperature is 25-37℃.

8. The preparation method according to any one of claims 5-7, characterized in that, In step (1), the Ce 3+ The salts are cerium nitrate, cerium chloride, and cerium acetate. 3+ The concentration of the salt solution is 5-10 mM, preferably 10 mM; gold nanorods Au NRs and Ce 3+ The salt ratio is 1-2 mol: 0.5-1 mol; Ce is added. 3+ The reaction time for the salt solution is 1-2 hours, and the reaction temperature is 100-120℃.

9. The preparation method according to any one of claims 5-8, characterized in that, In step (2), the concentrations of the polyvinyl alcohol solution and the trisodium borate solution are both 5-10 wt%, preferably 5 wt%. The ratio of dumbbell-shaped Au-CeO2, glucose oxidase, polyvinyl alcohol solution, and trisodium borate solution is 2-5 mg: 2-5 mg: 2-4 mL: 2-4 mL.

10. The use of any one of the localized sugar-controlling, antioxidant, photothermal antibacterial hydrogel dressings according to claims 1-4 in the preparation of products for the treatment of diabetic wound infections.

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