A GI-M nanoparticle-loaded hydrogel for diabetic wound repair and application

Abstract

The application discloses a kind of GI-M nanoparticle hydrogel for the repair of diabetic wound and application, belong to the technical field of biomedical materials;The application first encapsulates GOx, INS in Zn-Fe MOF to obtain GI-M nanoparticles, then adds GI-M nanoparticles in the process of preparing ROS-responsive hydrogel with TSPBA, PVA, to obtain GI-M nanoparticle hydrogel;Experimental demonstration shows that GI-M nanoparticles in GI-M nanoparticle hydrogel can quickly respond to glucose in the microenvironment of diabetic wound and trigger cascade catalytic reaction, GOx catalytic decomposition of glucose leads to microenvironment acidification, produces H2O2, excites Zn-Fe MOF peroxidase-like activity catalysis H2O2 and produces •OH, plays antibacterial activity while MOF skeleton degrades and orderly releases INS, and glucose content can control the strength of cascade catalytic reaction and accurate release of INS.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a GI-M nanoparticle-loaded hydrogel for the repair of diabetic wounds and its application. Background Technology

[0002] The typical chronic hyperglycemia in diabetes severely disrupts the normal wound healing process and significantly increases the risk of amputation and death in patients. The microenvironment of diabetic wounds is highly complex, characterized by a complex interplay of multiple pathological features, including persistent hyperglycemia, susceptibility to bacterial infection, abnormal oxidative stress levels, and long-term inflammatory responses, forming a vicious cycle that is difficult to break and poses a serious challenge to existing treatment methods. In this complex pathological context, the interaction between the imbalance of reactive oxygen species (ROS) homeostasis and blood glucose fluctuations has become the core obstacle to effective wound healing.

[0003] In the process of diabetic wound repair, reactive oxygen species (ROS) play a dual role: at physiological levels, ROS help with antibacterial activity and promote angiogenesis, while excessive accumulation leads to oxidative damage and increased inflammation, hindering the healing process. Therefore, precise regulation of ROS is key to improving the effectiveness of diabetic wound repair. This means generating sufficient ROS during the infection stage to exert a highly effective antibacterial effect, and promptly removing excess ROS in the later stages of healing to reduce tissue damage. In addition, local glucose level regulation is also crucial for improving the effectiveness of diabetic wound repair. Common strategies include using glucose oxidase (GOx) to catalyze blood glucose reduction or constructing a pH-responsive insulin controlled-release system. However, these methods have significant limitations, such as poor stability of natural enzymes and short-lived blood glucose-lowering effects. Furthermore, most insulin carriers are not sensitive enough to changes in the physiological environment, which can easily lead to dysregulation of insulin release and the risk of hypoglycemia.

[0004] Therefore, developing an intelligent treatment system that can autonomously respond to glucose levels, precisely regulate ROS, and release insulin on demand is crucial for improving the healing effect of diabetic wounds. Summary of the Invention

[0005] The first objective of this invention is to provide a GI-M nanoparticle-loaded hydrogel for the repair of diabetic wounds, and the second objective is to provide applications of the GI-M nanoparticle-loaded hydrogel.

[0006] The first objective of this invention is achieved as follows: a GI-M nanoparticle-loaded hydrogel for diabetic wound repair is prepared by fabricating a ROS-responsive hydrogel from GI-M nanoparticles co-encapsulated with glucose oxidase and insulin. The preparation method is as follows: 1) Dissolve zinc acetate dihydrate and ferric chloride hexahydrate together in deionized water, and stir continuously at 750-850 rpm for 10-20 minutes at room temperature to form a mixed metal salt aqueous solution. The Zn in the mixed metal salt aqueous solution... 2+ Fe 3+ The molar ratio is 7.5:1, and when the content of ferric chloride hexahydrate is 1 mmol, the content of deionized water is 90~110 mL; 2) Add 10 mg of glucose oxidase and 10 mg of insulin to 1 mmol of ferric chloride hexahydrate. Add glucose oxidase and insulin to the mixed metal salt aqueous solution and stir until fully dispersed to obtain mixed solution A; 3) Prepare an aqueous solution of 2-methylimidazole by dissolving 1 mmol of 2-methylimidazole in 2-3 mL of deionized water. Add the aqueous solution of 2-methylimidazole to the mixed solution A at a molar ratio of ferric chloride hexahydrate to 2-methylimidazole of 1:40. Stir the mixture continuously at room temperature for 25-35 min at a speed of 750-850 rpm. After the reaction is completed, let the mixture stand at room temperature for 20-28 h to age. 4) After aging, centrifuge at 7500~8500 rpm for 18~22 min. Wash the separated precipitate with deionized water at 7500~8500 rpm 2~4 times. Freeze-dry the washed solid product at -180℃ to obtain GI-M nanoparticles that co-encapsulate glucose oxidase and insulin. 5) Dissolve 10 mg of GI-M nanoparticles obtained in step 4) in 1 mL of 2.5~3.5 wt% TSPBA aqueous solution to obtain mixed solution B; mix mixed solution B with 8~10 wt% PVA aqueous solution at a volume ratio of 1:0.8~1.2, let stand at room temperature for 5 min, and crosslink to form GI-M nanoparticle-loaded hydrogel.

[0007] The second objective of this invention is achieved by the application of the GI-M nanoparticle hydrogel for wound repair in diabetic patients in the preparation of diabetic wound repair dressings.

[0008] In the prior art, metal-organic frameworks (MOFs) exhibit significant advantages in drug delivery and biocatalysis due to their high specific surface area, tunable pore structure, abundant metal active sites, and good biocompatibility. This invention selects a Zn-Fe bimetallic organic framework MOF (i.e., Zn-Fe MOF) as a drug carrier, encapsulating glucose oxidase (Gox) and insulin (INS) within it to obtain GI-M nanoparticles. Then, during the preparation of ROS-responsive hydrogels using N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine (TSPBA) and polyvinyl alcohol (PVA), GI-M nanoparticles are added as an additive to obtain a GI-M nanoparticle-loaded hydrogel.

[0009] Experiments have demonstrated that GI-M nanoparticles in GI-M nanoparticle-loaded hydrogels can rapidly respond to glucose in the microenvironment of diabetic infected wounds, triggering a cascade catalytic reaction mechanism. Gox catalyzes the decomposition of glucose, leading to microenvironment acidification, which in turn stimulates the peroxidase-like activity of Zn-Fe MOF, catalyzing the generation of hydroxyl radicals (•OH) from H2O2, thereby exerting antibacterial activity and promoting the degradation of the MOF skeleton and the orderly release of INS. The gradually released INS and Gox accelerate the rate of glucose reduction. The cascade catalytic reaction gradually weakens as the catalytic substrate decreases, effectively controlling the amount of •OH generated and INS released, and matching it with the degree of repair of the infected wound, avoiding excessive glucose reduction and the continuous generation of ROS in the microenvironment, and reducing potential damage.

[0010] The present invention has the following advantages over the prior art: (1) When the GI-M nanoparticle hydrogel described in this invention is applied, the high glucose content in the wound microenvironment is the basis for triggering the cascade catalytic reaction and orderly releasing INS. The glucose content reduces and weakens the cascade catalytic reaction and controls the amount of INS released, forming a closed-loop regulated mechanism for repairing diabetic infected wounds, thereby improving the wound repair effect. (2) The GI-M nanoparticle hydrogel described in this invention uses glucose as a single signal to simultaneously trigger two core therapeutic functions: namely, generating •OH through cascade catalytic reaction to achieve efficient antibacterial effect, and achieving orderly and precise release of INS through the influence of the strength of cascade catalytic reaction on the degradation of MOF skeleton, thus forming a synergistic linkage mechanism of hypoglycemia and antibacterial effect. (3) The GI-M nanoparticle hydrogel described in this invention generates antibacterial components and lowers blood sugar by triggering a cascade catalytic reaction with glucose. At the same time, it regulates the strength of the cascade catalytic reaction by changing the glucose content, avoiding excessive blood sugar reduction and continuous generation of ROS microenvironment, thus forming a protective mechanism in the repair process. Therefore, the GI-M nanoparticle-loaded hydrogel described in this invention can overcome the shortcomings of existing diabetic wound treatment technologies. It can achieve antibacterial, repair, and precise regulation of ROS and on-demand insulin release by autonomously responding to glucose levels, and has broad application prospects. Attached Figure Description

[0011] Figure 1 This is a transmission electron microscope image of Zn-Fe(MOF) in Example 1; Figure 2 This is the XRD pattern of Zn-Fe(MOF) in Example 1; Figure 3 This is the FT-IR image of the GI-M nanoparticles in Example 2; Figure 4 The following is an in vitro cascade catalytic reaction in Experiment Example 1, where: A is a schematic diagram of the cascade catalytic reaction process, B is the glucose consumption curve of GI-M nanoparticle-loaded hydrogels at different concentrations, C is the pH change curve of GI-M nanoparticle-loaded hydrogels at different glucose concentrations, and D is the generation of hydroxyl radicals (•OH). Figure 5 The in vitro antibacterial effect of GI-M in Experiment Example 2 is shown in Figure 2. In Figure 2, A represents the colony status on the plates of different treatment groups, and B represents the bacterial survival rate of different treatment groups. Figure 6 The treatment effects on infected wounds in diabetic mice under different treatment groups in Experiment Example 3 are shown in Figure A, where A represents the changes in wound area in different treatment groups during the treatment period, and B represents the percentage of remaining wound area in different treatment groups. Detailed Implementation

[0012] The present invention will now be described in more detail with reference to the accompanying drawings, embodiments, and test examples. However, the present invention is not limited thereto. Any modifications or improvements based on the present invention should be covered within the scope of protection of the present invention. Unless otherwise specified, the reagents, culture media, buffer solutions and other raw materials used in the present invention can be purchased from the market or prepared in the laboratory by known or conventional methods.

[0013] The present invention discloses a GI-M nanoparticle-loaded hydrogel for diabetic wound repair, which is a ROS-responsive hydrogel made by co-encapsulating GI-M nanoparticles containing glucose oxidase and insulin. The preparation method is as follows: 1) Dissolve zinc acetate dihydrate and ferric chloride hexahydrate together in deionized water, and stir continuously at 750-850 rpm for 10-20 minutes at room temperature to form a mixed metal salt aqueous solution. The Zn in the mixed metal salt aqueous solution... 2+ Fe 3+ The molar ratio is 7.5:1, and when the content of ferric chloride hexahydrate is 1 mmol, the content of deionized water is 90~110 mL; 2) Add 10 mg of glucose oxidase and 10 mg of insulin to 1 mmol of ferric chloride hexahydrate. Add glucose oxidase and insulin to the mixed metal salt aqueous solution and stir until fully dispersed to obtain mixed solution A; 3) Prepare an aqueous solution of 2-methylimidazole by dissolving 1 mmol of 2-methylimidazole in 2-3 mL of deionized water. Add the aqueous solution of 2-methylimidazole to the mixed solution A at a molar ratio of ferric chloride hexahydrate to 2-methylimidazole of 1:40. Stir the mixture continuously at room temperature for 25-35 min at a speed of 750-850 rpm. After the reaction is completed, let the mixture stand at room temperature for 20-28 h to age. 4) After aging, centrifuge at 7500~8500 rpm for 18~22 min. Wash the separated precipitate with deionized water at 7500~8500 rpm 2~4 times. Freeze-dry the washed solid product at -180℃ to obtain GI-M nanoparticles that co-encapsulate glucose oxidase and insulin. 5) Dissolve 10 mg of GI-M nanoparticles obtained in step 4) in 1 mL of 2.5~3.5 wt% TSPBA aqueous solution to obtain mixed solution B; mix mixed solution B with 8~10 wt% PVA aqueous solution at a volume ratio of 1:0.8~1.2, let stand at room temperature for 5 min, and crosslink to form GI-M nanoparticle-loaded hydrogel.

[0014] Preferably, step 1) involves continuously stirring at 800 rpm for 15 minutes at room temperature to form a mixed metal salt aqueous solution.

[0015] Preferably, in step 1), the content of deionized water in the mixed metal salt aqueous solution is 100 mL when the content of ferric chloride hexahydrate is 1 mmol.

[0016] Preferably, in step 3), 1 mmol of 2-methylimidazole is dissolved in 2.5 mL of deionized water to prepare an aqueous solution of 2-methylimidazole. After adding the aqueous solution of 2-methylimidazole to the mixed solution A, the mixture is stirred continuously at 800 rpm for 30 min at room temperature. After the reaction is completed, the mixed system is allowed to stand at room temperature for 24 h for aging.

[0017] Preferably, after aging in step 4), the precipitate is centrifuged at 8000 rpm for 20 min, and the separated precipitate is washed three times with deionized water at 8000 rpm.

[0018] Preferably, in step 5), 10 mg of GI-M nanoparticles are dissolved in 1 mL of 3 wt% TSPBA aqueous solution.

[0019] Preferably, in step 5), the mixed solution B is mixed with 9wt% PVA aqueous solution at a volume ratio of 1:1.

[0020] The present invention relates to the application of GI-M nanoparticle hydrogel for wound repair in diabetic patients in the preparation of diabetic wound repair dressings.

[0021] Preferably, the diabetic wound is a diabetic infected wound.

[0022] Example 1: Preparation and characterization of Zn-Fe bimetallic organic framework nanoparticles 1) Weigh 27.03 mg of ferric chloride hexahydrate (FeCl3·6H2O, 0.1 mmol) and 165.13 mg of zinc acetate dihydrate (Zn(CH3COO)2·2H2O, 0.75 mmol) and dissolve them together in 10 mL of deionized water. Stir continuously at 800 rpm for 15 min at room temperature to form a mixed metal salt aqueous solution. 2) Weigh 328.44 mg (4 mmol) of 2-methylimidazole and dissolve it in 10 mL of deionized water. Add the 2-methylimidazole aqueous solution to the mixed metal salt aqueous solution and stir continuously at 800 rpm for 30 min at room temperature. After the reaction is completed, let the mixture stand at room temperature for 24 h to age. 3) Then, centrifuge at 8000 rpm for 20 min. The separated precipitate is washed three times with deionized water at 8000 rpm. The washed solid product is freeze-dried at -180℃ to obtain Zn-Fe bimetallic organic framework nanoparticles, namely Zn-Fe MOF.

[0023] Characterization results: such as Figure 1 As shown, transmission electron microscopy (TEM) characterization revealed that the Zn-Fe MOF prepared in Example 1 exhibited a smooth, spindle-shaped structure; energy-dispersive X-ray spectroscopy (EDS) surface scanning analysis confirmed that iron (Fe) and zinc (Zn) elements were uniformly distributed throughout the material. Figure 2 As shown, the X-ray diffraction (XRD) was used for characterization. The XRD pattern showed a broadened diffraction peak at 13.7°, which belongs to the typical crystal structure of MOF(Zn). At the same time, a sharp diffraction peak was observed at 11.87°, corresponding to the reflection of the crystal plane of graphite carbon. The above results indicate that the prepared Zn-Fe MOF has a semi-crystalline structure consistent with the reported features.

[0024] Example 2 Preparation of GI-M nanoparticles 1) Weigh 27.03 mg of ferric chloride hexahydrate (FeCl3·6H2O, 0.1 mmol) and 165.13 mg of zinc acetate dihydrate (Zn(CH3COO)2·2H2O, 0.75 mmol) and dissolve them together in 10 mL of deionized water. Stir continuously at 800 rpm for 15 min at room temperature to form a mixed metal salt aqueous solution. 2) Add 1 mg of glucose oxidase (GOx) and 1 mg of insulin (INS) to the mixed metal salt aqueous solution, and stir until fully dispersed to obtain mixed solution A; 3) Weigh 328.44 mg (4 mmol) of 2-methylimidazole and dissolve it in 10 mL of deionized water. Add the 2-methylimidazole aqueous solution to mixed solution A and stir continuously at 800 rpm for 30 min at room temperature. After the reaction is complete, let the mixture stand at room temperature for 24 h to age. 4) After aging, centrifuge at 8000 rpm for 20 min. The separated precipitate is washed three times with deionized water at 8000 rpm. The washed solid product is freeze-dried at -180℃ to obtain GI-M nanoparticles co-encapsulated with GOx and INS.

[0025] To confirm the successful encapsulation of GOx and INS, Fourier transform infrared spectroscopy (FT-IR) analysis was performed on the GI-M nanoparticles prepared in Example 2: [Example data would be inserted here] Figure 3 As shown, in the infrared spectrum of GI-M nanoparticles, at 2909 cm⁻¹... -1 A new absorption peak appeared, which is attributed to the stretching vibration of the methyl group (-CH3) in insulin and glucose oxidase molecules; at the same time, the intensity of the characteristic absorption peaks (amide I band and amide II band) representing protein amide bonds was significantly enhanced; the above spectral changes together confirm that GOx and INS were successfully encapsulated in the Zn-Fe MOF backbone.

[0026] Example 3 Preparation of GI-M nanoparticles 1) Weigh 27.03 mg of ferric chloride hexahydrate (FeCl3·6H2O, 0.1 mmol) and 165.13 mg of zinc acetate dihydrate (Zn(CH3COO)2·2H2O, 0.75 mmol) and dissolve them together in 9 mL of deionized water. Stir continuously at 750 rpm for 20 min at room temperature to form a mixed metal salt aqueous solution. 2) Add 1 mg of glucose oxidase (GOx) and 1 mg of insulin (INS) to the mixed metal salt aqueous solution, and stir until fully dispersed to obtain mixed solution A; 3) Weigh 328.44 mg (4 mmol) of 2-methylimidazole and dissolve it in 8 mL of deionized water. Add the 2-methylimidazole aqueous solution to mixed solution A and stir continuously at 750 rpm for 35 min at room temperature. After the reaction is complete, let the mixture stand at room temperature for 20 h to age. 4) After aging, the product was centrifuged at 7500 rpm for 22 min. The separated precipitate was washed four times with deionized water at 7500 rpm. The washed solid product was freeze-dried at -180℃ to obtain GI-M nanoparticles encapsulated with GOx and INS. Referring to Example 2, Fourier transform infrared spectroscopy (FT-IR) analysis was performed on the GI-M nanoparticles prepared in Example 3, confirming that GOx and INS were successfully encapsulated in the Zn-Fe MOF framework.

[0027] Example 4 Preparation of GI-M nanoparticles 1) Weigh 27.03 mg of ferric chloride hexahydrate (FeCl3·6H2O, 0.1 mmol) and 165.13 mg of zinc acetate dihydrate (Zn(CH3COO)2·2H2O, 0.75 mmol) and dissolve them together in 11 mL of deionized water. Stir continuously at 850 rpm for 10 min at room temperature to form a mixed metal salt aqueous solution. 2) Add 1 mg of glucose oxidase (GOx) and 1 mg of insulin (INS) to the mixed metal salt aqueous solution, and stir until fully dispersed to obtain mixed solution A; 3) Weigh 328.44 mg (4 mmol) of 2-methylimidazole and dissolve it in 12 mL of deionized water. Add the 2-methylimidazole aqueous solution to mixed solution A and stir continuously at 850 rpm for 25 min at room temperature. After the reaction is complete, let the mixture stand at room temperature for 28 h to age. 4) After aging, the product was centrifuged at 8500 rpm for 18 min. The separated precipitate was washed twice with deionized water at 8500 rpm. The washed solid product was freeze-dried at -180℃ to obtain GI-M nanoparticles encapsulated with GOx and INS. Referring to Example 2, Fourier transform infrared spectroscopy (FT-IR) analysis was performed on the GI-M nanoparticles prepared in Example 4, confirming that GOx and INS were successfully encapsulated in the Zn-Fe MOF framework.

[0028] Example 5 Preparation of IM Nanoparticles In the preparation of the IM nanoparticles prepared in Example 2, step 2) only 1 mg of insulin (INS) was added to the mixed metal salt aqueous solution and stirred until fully dispersed to obtain mixed solution A. The other steps were the same as in Example 2 to obtain IM nanoparticles encapsulated with INS. In accordance with Example 2, Fourier transform infrared spectroscopy (FT-IR) analysis was performed on the IM nanoparticles prepared in Example 5 to confirm that INS was successfully encapsulated in the Zn-Fe MOF framework.

[0029] Example 6 Preparation of GM Nanoparticles In the preparation of the mixture according to Example 2, step 2) only 1 mg of glucose oxidase (GOx) was added to the mixed metal salt aqueous solution and stirred until fully dispersed to obtain mixed solution A. The other steps were the same as in Example 2 to obtain GM nanoparticles encapsulated with Gox. According to Example 2, Fourier transform infrared spectroscopy (FT-IR) analysis was performed on the GM nanoparticles prepared in Example 6 to confirm that GOx was successfully encapsulated in the Zn-Fe MOF framework.

[0030] Example 7 Preparation of GI-M nanoparticle-loaded hydrogel 10 mg of the GI-M nanoparticles prepared in Example 2 was dissolved in 1 mL of 3 wt% TSPBA (N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine, the same below) aqueous solution to obtain mixed solution B; mixed solution B was mixed with 9 wt% PVA (polyvinyl alcohol, the same below) aqueous solution at a volume ratio of 1:1 and allowed to stand at room temperature for 5 min to crosslink and form a GI-M nanoparticle-loaded hydrogel.

[0031] Example 8 Preparation of GI-M nanoparticle-loaded hydrogel 10 mg of the GI-M nanoparticles prepared in Example 3 was dissolved in 1 mL of 2.5 wt% TSPBA aqueous solution to obtain mixed solution B; mixed solution B was mixed with 10 wt% PVA aqueous solution at a volume ratio of 1:0.8 and allowed to stand at room temperature for 5 min to crosslink and form a GI-M nanoparticle-loaded hydrogel.

[0032] Example 9 Preparation of GI-M nanoparticle-loaded hydrogel 10 mg of the GI-M nanoparticles prepared in Example 4 was dissolved in 1 mL of 3.5 wt% TSPBA aqueous solution to obtain mixed solution B; mixed solution B was mixed with 8 wt% PVA aqueous solution at a volume ratio of 1:1.2 and allowed to stand at room temperature for 5 min to crosslink and form a GI-M nanoparticle-loaded hydrogel.

[0033] Example 10 Preparation of Zn-Fe MOF-loaded hydrogel 10 mg of Zn-Fe MOF prepared in Example 1 was dissolved in 1 mL of 3 wt% TSPBA aqueous solution to obtain mixed solution B; mixed solution B was mixed with 9 wt% PVA aqueous solution at a volume ratio of 1:1 and allowed to stand at room temperature for 5 min to crosslink and form Zn-FeMOF-loaded hydrogel.

[0034] Example 11 Preparation of IM Nanoparticle-Loaded Hydrogel 10 mg of the IM nanoparticles prepared in Example 5 was dissolved in 1 mL of 3 wt% TSPBA aqueous solution to obtain mixed solution B; mixed solution B was mixed with 9 wt% PVA aqueous solution at a volume ratio of 1:1 and allowed to stand at room temperature for 5 min to crosslink and form a hydrogel loaded with IM nanoparticles.

[0035] Example 12 Preparation of GM nanoparticle-loaded hydrogel 10 mg of the GM nanoparticles prepared in Example 6 was dissolved in 1 mL of 3 wt% TSPBA aqueous solution to obtain mixed solution B; mixed solution B was mixed with 9 wt% PVA aqueous solution at a volume ratio of 1:1 and allowed to stand at room temperature for 5 min to crosslink and form a GM nanoparticle-loaded hydrogel.

[0036] Experimental Example 1: In Vitro Catalytic Cascade Reaction Experiment of GI-M Nanoparticles 1. Test method: The 3,5-dinitrosalicylic acid method (DNS) was used to quantitatively analyze glucose concentration. The absorbance of glucose standards at different concentrations was measured at a wavelength of 534 nm using ultraviolet-visible spectroscopy (UV-Vis) to construct a standard curve for quantitative analysis. 1-1. Take 15 mL of each of three groups of 10 mg / mL glucose aqueous solutions, add GI-M nanoparticles prepared in Example 2 to each solution, and adjust the pH to 7.4. Incubate the mixtures together at 37°C for 45 h. Collect 100 µL of the reaction mixture at 10 h, 15 h, 20 h, 25 h, 30 h, 35 h, 40 h, and 45 h. Immediately add DNS reagent to the collected reaction mixtures to terminate the reaction and develop color. Then calculate the real-time concentration of free glucose at each sampling time point according to the standard curve prepared in step 1-1. 1-2. The GI-M nanoparticles prepared in Example 2 were mixed with deionized water to obtain three sets of test solutions. The concentration of GI-M nanoparticles in each set of test solutions was 2 mg / mL. Glucose was added to two of the test solutions, and the glucose concentrations were controlled to be 5 mg / mL and 10 mg / mL, respectively. The pH value of each set was adjusted to 7.4, and the solutions were incubated together at 37°C for 5 h. The pH value of the reaction system was measured and recorded using a precision pH meter at 0 h, 1 h, 2 h, 3 h, 4 h, and 5 h. 1-3. Take 5 mL of PBS buffer with pH values ​​of 5.0, 6.0, and 7.4 respectively, and add GI-M nanoparticles prepared in Example 2 to each. Control the concentration of GI-M nanoparticles to 2 mg / mL. After reacting at 37℃ for 45 min, centrifuge each group and take 0.5 mL of the supernatant. Add 0.12 mg TMB (3,3',5,5'-tetramethylbenzidine). After reacting for 45 min, perform UV-Vis spectroscopy analysis. Hydroxyl radicals (•OH) can oxidize colorless TMB to generate a blue product with a characteristic absorption peak at 652 nm. This experiment is used to evaluate the generation of •OH.

[0037] 2. Test Results: 2-1, such as Figure 4 As shown in Figure B, as the concentration of GI-M nanoparticles changed to 0.5 mg / mL, 1 mg / mL, and 2 mg / mL, the glucose concentration at each measurement time point decreased sequentially. When the concentration of GI-M nanoparticles was 2 mg / mL, after 45 h of reaction, the glucose concentration decreased from the initial concentration of 10 mg / mL to 1.9 mg / mL. Therefore, the effective consumption of glucose showed a characteristic of dependence on the concentration of GI-M nanoparticles. Combined with the trend of the curve, it can be seen that GI-M nanoparticles have the ability to continuously and efficiently consume glucose. 2-2, such as Figure 4 As shown in Figure C, when the glucose concentration is 0, the pH value remains almost unchanged over 5 hours; when the glucose concentration is 5 mg / mL, the pH value continuously decreases from 7.8 to around 6.7 over 5 hours; when the glucose concentration increases to 10 mg / mL, the pH value continuously decreases from 7.8 to below 6.3 over 5 hours. Therefore, the pH regulation ability of GI-M nanoparticles exhibits a glucose concentration-dependent characteristic. This phenomenon is consistent with the reaction mechanism of glucose oxidase (GOx) catalyzing the oxidation of glucose to gluconic acid, confirming that GI-M nanoparticles have the ability to respond to a hyperglycemic microenvironment and transform into an acidic environment. 2-3, such as Figure 4 As shown in Figure D, when pH=5.0, the amount of •OH generated is significantly higher than that at pH values ​​of 6.0 and 7.4, proving that GI-M nanoparticles can still exhibit the inherent peroxidase-like activity of Zn-Fe MOF, and can catalyze the production of highly reactive •OH from hydrogen peroxide (H2O2).

[0038] Experimental Example 2: In vitro antibacterial experiment of GI-M nanoparticles Using Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) as pathogenic bacteria, the in vitro antibacterial properties of GI-M nanoparticles were evaluated. The specific procedure is as follows: 1. Experimental Groups: Control group: Phosphate-buffered saline (PBS) was used as the test reagent, and PBS was purchased from the market; MOF(Zn-Fe) group: Zn-Fe MOF prepared in Example 1 was used as the test reagent; MOF(Zn-Fe) / INS group: IM nanoparticles prepared in Example 5 were used as test reagents; MOF(Zn-Fe) / GOx group: GM nanoparticles prepared in Example 6 were used as test reagents; GI-M group: GI-M nanoparticles prepared in Example 2 were used as test reagents.

[0039] 2. Test methods: 2-1. Escherichia coli (E. coli ATCC 25922) and Staphylococcus aureus (S. aureus ATCC 25923) were inoculated into LB liquid medium separately using standard methods and cultured at 37°C for 16 h. Then, the bacterial concentration was diluted with sterile PBS to 1 × 10⁻⁶. 8 CFU / mL bacterial suspension; LB liquid medium formula: per 1L of medium, containing 10g tryptone, 5g yeast extract, 10g NaCl, with the remainder being sterile water; 2-2. Glucose was added to different Escherichia coli and Staphylococcus aureus bacterial cultures, respectively, and the glucose concentration was controlled at 10 mg / mL (to ensure sufficient reaction substrate). Then, PBS (control group), Zn-Fe MOF, IM nanoparticles, GM nanoparticles, and GI-M nanoparticles were added according to the groups in step 1, until the concentration of each group was 400 μg / mL. Each group was placed in a constant temperature shaker at 37℃ and cultured for 2 h (simulating the reaction process under physiological conditions). 2-3. After the reaction, take 10 μL of bacterial solution from each group, dilute it serially, and spread it evenly on the surface of LB agar plates. Invert the agar plates and incubate them in a 37℃ constant temperature incubator for 18 h. Then count the colony forming units (CFU) on the agar plates and calculate the bacterial killing rate of each experimental group relative to the control group.

[0040] 3. Test Results: like Figure 5 As shown in Figures A and B, a large number of colonies were observed to grow in the Control group, the MOF(Zn-Fe) group, and the MOF(Zn-Fe) / INS group, indicating that Zn-Fe MOF alone or insulin-loaded did not show significant antibacterial activity under the experimental conditions. In contrast, the MOF(Zn-Fe) / Gox group and the GI-M group showed a kill rate of over 95% against Escherichia coli and Staphylococcus aureus, demonstrating excellent antibacterial activity. This indicates that the encapsulated Gox effectively responds to the high glucose environment, thereby reducing the environmental pH, activating the peroxidase-like activity of Zn-Fe MOF, and increasing •OH in the environment to fully exert its antibacterial activity.

[0041] Experimental Example 3: Diabetic Wound Repair Experiment Using GI-M Nanoparticle Hydrogel 1. Experimental Groups: Control group: PBS with phosphate buffered saline was used as the test reagent; MOF(Zn-Fe)@Gel group: Zn-Fe MOF hydrogel prepared in Example 10 was used as the test reagent; MOF(Zn-Fe) / INS@Gel group: The IM nanoparticle-loaded hydrogel prepared in Example 11 was used as the test reagent; MOF(Zn-Fe) / Gox@Gel group: The GM nanoparticle-loaded hydrogel prepared in Example 12 was used as the test reagent; GI-M@Gel group: The GI-M nanoparticle-loaded hydrogel prepared in Example 7 was used as the test reagent.

[0042] 2. Test methods: 2-1. ICR male mice were fed a high-fat / high-sugar diet for two weeks. A diabetic model was then induced by intraperitoneal injection of streptozotocin (STZ) 55 mg / kg. Fasting blood glucose (FBG) levels were monitored weekly by collecting tail vein blood. A diabetic state was defined as FBG > 16.7 mmol / L. The mice were anesthetized with isoflurane and their back hair was shaved. The back skin was first cleaned with 75% medical alcohol, and then a 10 mm diameter full-thickness skin excision wound was created on the back of the mice using a skin sampler. 100 µL of mixed bacterial solution was inoculated into the wound, and the mice were fed normally for 24 hours to establish an infected wound. The mixed bacterial solution consisted of 50 µL each of Escherichia coli and Staphylococcus aureus from step 2-1 of Experiment Example 2. 2-2. On days 1, 4, and 7, 100 µL of the test drug for each group was dripped onto the infected wound according to the group, and then the animals were fed normally until day 14. 2-3. Take photos on days 1, 4, 7 and 14 to observe the wound healing of each group, and use ImageJ software to calculate the wound area of ​​each group.

[0043] 3. Test Results: like Figure 6 As shown, no signs of wound healing were observed in any group on day 1, with almost no difference in efficacy. On day 4, all groups showed wound repair effects. Although the MOF(Zn-Fe)@Gel, MOF(Zn-Fe) / INS@Gel, and MOF(Zn-Fe) / GOx@Gel groups showed better repair effects than the Control group, they were significantly inferior to the GI-M@Gel group. On days 7 and 14, the repair effects of all groups further improved over time, especially the repair effect of the GI-M@Gel group. The gap between his experimental groups was significant; by day 14, the infected wounds in the GI-M@Gel group were close to healing, while other groups still had obvious abscesses or unhealed wounds; throughout the trial, the repair effect of the MOF(Zn-Fe) / INS@Gel group was significantly worse than that of the MOF(Zn-Fe) / GOx@Gel group, and the MOF(Zn-Fe)@Gel group was worse than the MOF(Zn-Fe) / INS@Gel and MOF(Zn-Fe) / GOx@Gel groups, only slightly better than the Control group.

[0044] Based on the above experimental results, it can be seen that when the GI-M nanoparticle-loaded hydrogel comes into contact with the hyperglycemic microenvironment of a diabetic wound, a cascade catalytic reaction mechanism is formed: GOx catalyzes glucose in the wound microenvironment, generating gluconic acid and H2O2, leading to local microenvironment acidification; the acidic microenvironment activates Zn-Fe The peroxidase-like activity of MOF catalyzes the generation of hydroxyl radicals (•OH) from H2O2, thus exhibiting excellent antibacterial activity and promoting the degradation of the MOF skeleton. As the MOF skeleton degrades, INS is released in an orderly manner, which, together with Gox, accelerates the reduction of local blood glucose levels. When the blood glucose level in the wound returns to normal, the GOx catalytic substrate decreases, the cascade reaction mechanism automatically weakens, and the release of INS is also controlled. Thus, the GI-M nanoparticles in the GI-M nanoparticle-loaded hydrogel of this invention have a rapid response capability to glucose in the microenvironment of diabetic infected wounds. Based on the GOx catalytic decomposition of glucose triggering a cascade catalytic reaction mechanism, •OH is rapidly generated and INS is released. The cascade catalytic reaction and INS release are effectively controlled according to changes in blood glucose levels, thereby achieving timely regulation of blood glucose and ROS levels in the microenvironment as the degree of infection gradually decreases, effectively preventing potential damage caused by excessive blood glucose reduction and the continuous generation of ROS in the microenvironment.

[0045] In contrast, the IM nanoparticle-loaded hydrogel, lacking Gox encapsulation, cannot rapidly respond to glucose in the microenvironment of diabetic infected wounds. Because it cannot alter the pH and H2O2 levels in the microenvironment, the peroxidase-like activity of the Zn-Fe MOF is limited, affecting MOF backbone degradation and INS release. This primarily relies on the release of Zn from the Zn-Fe MOF... 2+ It activates the vascular endothelial growth factor pathway, promoting angiogenesis and epithelial regeneration. Although the repair effect of the GM nanoparticle-loaded hydrogel is better than that of the IM nanoparticle-loaded hydrogel, since it only encapsulates Gox, the cascade catalytic reaction effect is not as good as that of the GI-M nanoparticle-loaded hydrogel because it does not carry INS. This leads to a significant difference in repair effect between the two.

[0046] Therefore, the GI-M nanoparticle-loaded hydrogel described in this invention can be used as a dressing for the repair of diabetic infected wounds, and has broad application value.

Claims

1. A GI-M nanoparticle-loaded hydrogel for diabetic wound repair, characterized in that, The GI-M nanoparticle-loaded hydrogel is a ROS-responsive hydrogel made by co-encapsulating GI-M nanoparticles containing glucose oxidase and insulin. The preparation method is as follows: 1) Dissolve zinc acetate dihydrate and ferric chloride hexahydrate together in deionized water, and stir continuously at 750-850 rpm for 10-20 minutes at room temperature to form a mixed metal salt aqueous solution. The Zn in the mixed metal salt aqueous solution... 2+ Fe 3+ The molar ratio is 7.5:1, and when the content of ferric chloride hexahydrate is 1 mmol, the content of deionized water is 90~110 mL; 2) Add 10 mg of glucose oxidase and 10 mg of insulin to 1 mmol of ferric chloride hexahydrate. Add glucose oxidase and insulin to the mixed metal salt aqueous solution and stir until fully dispersed to obtain mixed solution A; 3) Prepare an aqueous solution of 2-methylimidazole by dissolving 1 mmol of 2-methylimidazole in 2-3 mL of deionized water. Add the aqueous solution of 2-methylimidazole to the mixed solution A at a molar ratio of ferric chloride hexahydrate to 2-methylimidazole of 1:

40. Stir the mixture continuously at room temperature for 25-35 min at a speed of 750-850 rpm. After the reaction is completed, let the mixture stand at room temperature for 20-28 h to age. 4) After aging, centrifuge at 7500~8500 rpm for 18~22 min. Wash the separated precipitate with deionized water at 7500~8500 rpm 2~4 times. Freeze-dry the washed solid product at -180℃ to obtain GI-M nanoparticles that co-encapsulate glucose oxidase and insulin. 5) Dissolve 10 mg of GI-M nanoparticles obtained in step 4) in 1 mL of 2.5~3.5 wt% TSPBA aqueous solution to obtain mixed solution B; mix mixed solution B with 8~10 wt% PVA aqueous solution at a volume ratio of 1:0.8~1.2, let stand at room temperature for 5 min, and crosslink to form GI-M nanoparticle-loaded hydrogel.

2. The GI-M nanoparticle-loaded hydrogel according to claim 1, characterized in that, Step 1) involves continuously stirring at 800 rpm for 15 minutes at room temperature to form a mixed metal salt aqueous solution.

3. The GI-M nanoparticle-loaded hydrogel according to claim 1, characterized in that, When the concentration of ferric chloride hexahydrate in the mixed metal salt aqueous solution in step 1) is 1 mmol, the deionized water content is 100 mL.

4. The GI-M nanoparticle-loaded hydrogel according to claim 1, characterized in that, Step 3) Prepare a 2-methylimidazole aqueous solution by dissolving 1 mmol of 2-methylimidazole in 2.5 mL of deionized water. After adding the 2-methylimidazole aqueous solution to the mixed solution A, stir continuously at 800 rpm for 30 min at room temperature. After the reaction is completed, let the mixed system stand at room temperature for 24 h to age.

5. The GI-M nanoparticle-loaded hydrogel according to claim 1, characterized in that, After aging in step 4), centrifuge at 8000 rpm for 20 min. Wash the separated precipitate three times with deionized water at 8000 rpm.

6. The GI-M nanoparticle-loaded hydrogel according to claim 1, characterized in that, In step 5), 10 mg of GI-M nanoparticles are dissolved in 1 mL of 3 wt% TSPBA aqueous solution.

7. The GI-M nanoparticle-loaded hydrogel according to claim 1, characterized in that, In step 5), mixed solution B is mixed with 9wt% PVA aqueous solution at a volume ratio of 1:

1.

8. The use of the GI-M nanoparticle hydrogel of any one of claims 1 to 7 for wound repair in diabetic patients in the preparation of diabetic wound repair dressings.

9. The application according to claim 8, characterized in that, The diabetic wound is an infected diabetic wound.

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