A controllable release nano-enzyme hydrogel for promoting chronic wound healing of diabetes and a preparation method and application thereof

Abstract

The application discloses a controllable release nano-enzyme hydrogel for promoting chronic wound healing of diabetes, and a preparation method and application thereof. The hydrogel is prepared according to the following steps: (1) preparing Pd-ZIF8@DNase I nano-enzyme particles; (2) dissolving four-arm-polyethylene glycol-o-phenylenediamine (4a-PEG-OPA) in a tris-hydroxymethyl aminomethane-hydrochloric acid (Tris-HCl) buffer solution, adding 3-aminobenzoic acid solution, and forming a hydrogel network through a Schiff base reaction; and (3) uniformly dispersing the Pd-ZIF8@DNase I nano-enzyme particles obtained in the step (1) in the hydrogel system to obtain Pd-ZIF8@DNase I hydrogel. The controllable release nano-enzyme hydrogel based on the "enzyme-induced defective metal organic framework (MOF)" structure of zeolite imidazolate framework structure material 8 (ZIF8) with high electron transfer efficiency can target neutrophil extracellular traps (NETs) and has antioxidant activity, and can promote chronic wound healing of diabetes.

Description

Technical Field

[0001] This invention relates to the field of biomedical nanomaterials technology, specifically to a controllable release nanoenzyme hydrogel that promotes the healing of chronic diabetic wounds, its preparation method, and its application. Background Technology

[0002] In a high-glucose environment, neutrophil function is significantly disrupted, exhibiting pathological features of "decreased phagocytic capacity and increased formation of neutrophil extracellular traps (NETs)." Neutrophil extracellular traps are a network structure released by activated neutrophils, composed of a DNA backbone, histones, and various antimicrobial proteins. While NETs possess a defensive function of capturing and clearing pathogens under physiological conditions, their excessive accumulation in diabetic wounds becomes a key factor hindering healing: on the one hand, histones and neutrophil elastase in NETs directly damage tissue cells and degrade the extracellular matrix; on the other hand, the DNA component of NETs maintains a persistent inflammatory state by activating the Toll-like receptor (TLR)9 signaling pathway; simultaneously, NETs can also inhibit vascular endothelial cell function, directly hindering angiogenesis. The levels of NETs markers (free DNA backbone) in the exudate of diabetic wounds are significantly higher than in normal wounds and are positively correlated with wound severity.

[0003] The high-glucose microenvironment of diabetic wounds exacerbates tissue damage through multiple mechanisms. Mitochondrial dysfunction and neutrophil respiratory bursts lead to the massive production of reactive oxygen species (ROS). ROS are not only a key inducing signal for NET formation and NETosis (neutrophil extracellular trap formation), but also form a vicious cycle with NETs—ROS promotes NET production, while myeloperoxidase (MPO) in NETs further catalyzes ROS production. This interaction significantly amplifies oxidative stress and inflammatory responses, directly damaging the function of fibroblasts and keratinocytes. Simultaneously, the high-glucose environment promotes the accumulation of advanced glycation end products (AGEs), impairs vascular endothelial cell function, and inhibits vascular endothelial growth factor (VEGF) expression. Furthermore, the protein components in NETs can directly inhibit endothelial cell migration and lumen formation, collectively leading to severe blood supply insufficiency in the wound. In terms of infection, bacteria in the wound form another pathological cycle with NETs: pathogenic bacteria such as Staphylococcus aureus and their products are strong inducers of NETosis, while excessively formed NETs become a "sanctuary" for bacteria when clearance is hindered. In particular, after NETs degrade, they may release surviving bacteria again, further aggravating the infection.

[0004] Current treatment strategies for diabetic skin wounds, which address multiple factors, have significant limitations. DNase I, which degrades NETs, ​​exhibits poor stability and limited efficacy in the wound environment, and may release trapped live bacteria due to rapid NET degradation. Topical insulin for high glucose and antioxidants for oxidative stress only address partial issues and cannot simultaneously combat NET accumulation and bacterial infection. While antibiotics can inhibit bacterial growth, their effectiveness against NET-protected biofilms is limited, and long-term use easily leads to drug resistance. Existing nanocarrier systems are mostly designed for single targets, lacking the ability to synergistically regulate multiple pathological processes including high glucose, bacteria, ROS, and NETs. Some carriers also suffer from reduced enzyme activity after immobilization and insufficient biocompatibility. Therefore, developing an integrated therapeutic platform that can simultaneously target multiple key pathological processes to achieve synergistic treatment of glucose reduction, antibacterial activity, antioxidant effects, and NET clearance is a crucial direction for overcoming the bottlenecks in the treatment of diabetic skin defects.

[0005] Therefore, there is an urgent need for a nanosystem that can achieve stable encapsulation and sustained release of DNase I, while also possessing multi-enzyme synergistic anti-inflammatory capabilities. Summary of the Invention

[0006] To address the aforementioned technical problems, the first objective of this invention is to provide a method for preparing a controllable-release nanoenzyme hydrogel that promotes the healing of chronic diabetic wounds. The second objective is to provide a controllable-release nanoenzyme hydrogel that promotes the healing of chronic diabetic wounds. The third objective is to provide its applications. This invention utilizes a controllable-release nanoenzyme hydrogel based on the high electron transfer efficiency of an enzyme-inducible defective MOF structure using zeolite imidazole ester framework material 8 (ZIF8). This nanoenzyme hydrogel can target extracellular traps (NETs) of neutrophils and also possesses antioxidant activity, thus promoting the healing of chronic diabetic wounds.

[0007] To achieve the first objective mentioned above, the present invention provides the following technical solution: a method for preparing a controllable-release nanoenzyme hydrogel that promotes the healing of chronic diabetic wounds, characterized by preparation according to the following steps:

[0008] (1) Weigh zinc nitrate and palladium nitrate and dissolve them in deionized water. Add 2-methylimidazole methanol solution dropwise and stir to mix thoroughly. Then add deoxyribonuclease I solution to encapsulate it in situ. Centrifuge and wash to obtain Pd-ZIF8@DNase I nanozyme particles.

[0009] (2) Dissolve 4a-PEG-OPA in Tris-HCl buffer (tris(hydroxymethyl)aminomethane-HCl), add 3-aminophenylboronic acid in Tris-HCl buffer solution, and form a hydrogel network through Schiff base reaction;

[0010] (3) Disperse the Pd-ZIF8@DNase I nanoenzyme particles obtained in step (1) uniformly in the hydrogel system to obtain Pd-ZIF8@DNase I hydrogel.

[0011] In the above scheme: in step (1), the molar ratio of zinc nitrate and palladium nitrate is 3:2-4:3, the molar ratio of 2-methylimidazole to metal ions is 4:1-3:1, after stirring and mixing for 10-15 hours, the concentration of deoxyribonuclease I (DNase I) solution in the mixed solution is 0.5-1 mg / mL, and the Pd-ZIF8@DNase I nanozyme particles have an icosahedral structure and a particle size of 400-500 nm.

[0012] In the above scheme: the specific operation of step (2) is to dissolve 3-aminophenylboronic acid and 4a-PEG-OPA in hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer, slowly add the 3-aminophenylboronic acid solution to the 4a-PEG-OPA solution, mix well, and then add some hydroxyethylpiperazine ethanesulfonic acid buffer. Let it stand at 37°C, invert the mixing container, and the hydrogel is obtained when the solution does not flow.

[0013] In the above scheme, the ratio of 4a-PEG-OPA to 3-aminophenylboronic acid and hydroxyethylpiperazine ethanesulfonic acid buffer is 5~6 mg:40~50 mg:1 mL. Preferably, the mass ratio of 4a-PEG-OPA to 3-aminophenylboronic acid is 8:1.

[0014] In the above scheme: In step (3), the mass concentration of Pd-ZIF8@DNase I nanozyme particles in the hydrogel is 0.5~2mg / mL.

[0015] The controlled-release nanoenzyme hydrogel for promoting the healing of chronic diabetic wounds was prepared by a method described above.

[0016] Application of controlled-release nanoenzyme hydrogels that promote the healing of chronic diabetic wounds in the preparation of biological dressings for the treatment of chronic diabetic wounds.

[0017] It can also be used to treat vasculitis ulcers, arteriosclerosis, and systemic lupus erythematosus.

[0018] This invention prepares a Pd-ZIF8@DNase I-based controlled-release nanoenzyme hydrogel to address the problems of excessive accumulation of extracellular neutrophil traps (NETs) and the easy inactivation and short in vivo residence time of conventional DNases in chronic inflammatory states such as diabetes. It constructs a composite nanosystem with multi-enzyme-mimicking activity. This system consists of two parts: first, a Pd-doped ZIF-8 metal-organic framework (MOF), during which deoxyribonuclease (DNase I) is encapsulated in situ during its synthesis to form Pd-ZIF8@DNase I nanoparticles; second, a dynamically repairable hydrogel network constructed through Schiff base bond (–C=N–) reactions, using four-armed polyethylene glycol phthalaldehyde (4a-PEG-OPA) as the cross-linking backbone and 3-aminophenylboronic acid (3-APBA) as the dynamic cross-linking unit. Pd-ZIF8@DNase I nanoparticles are uniformly dispersed within this network to form a composite hydrogel with controlled-release capability.

[0019] The Pd-ZIF8@DNase I nanoparticles are made of Zn 2+ With Pd 2+ The Pd-ZIF8 framework is formed by coordination with 2-methylimidazole, and the introduction of Pd endows the material with multi-enzyme-mimicking properties, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). During synthesis, DNase I molecules are uniformly distributed within the MOF channels, forming a Pd-ZIF8@DNase I composite structure. The presence of enzyme molecules induces some coordination defects during MOF nucleation, increasing the exposure of Pd active sites and further enhancing the enzyme-mimicking activity of the material, achieving a dual catalytic effect of "biological enzyme + nanozyme".

[0020] In the Pd-ZIF8@DNase I hydrogel, 4a-PEG-OPA provides aldehyde sites and 3-aminophenylboronic acid provides amino sites. These two groups are linked by a Schiff base reaction to form a dynamic covalent bond, constructing a flexible network. This dynamic structure endows the hydrogel with pH responsiveness and self-healing properties, enabling the gradual release of Pd-ZIF8@DNase I nanoparticles in a slightly acidic tissue environment, achieving long-term, stable enzyme release and anti-inflammatory regulation.

[0021] This composite system possesses multiple biological functions: DNase I can efficiently degrade the DNA component in NETs, ​​thus clearing NETs; Pd-ZIF8 removes excess ROS through SOD, POD, and CAT-mimicking activities, reducing inflammation and oxidative stress; and the dynamic hydrogel maintains a moist microenvironment and enables sustained-release delivery. In diabetic wounds, the entire system synergistically clears NETs, ​​reduces inflammation levels, inhibits ferroptosis, promotes fibroblast proliferation and collagen deposition, and accelerates wound repair.

[0022] The Pd-ZIF8@DNase I nanozyme hydrogel provided by this invention combines the stability of MOF-encapsulated enzymes, the synergistic effect of multi-enzyme mimicry activity, and the sustained-release properties of dynamic covalent hydrogels. It can achieve long-term and precise biological regulation in complex pathological environments and has broad application potential.

[0023] The Pd-ZIF8@DNase I nanoenzyme hydrogel provided by this invention can effectively remove excessively accumulated NETs in pathological conditions such as diabetes, restoring redox homeostasis and repair capacity of damaged tissues. In a diabetic wound model, excessive NET deposition activates endoplasmic reticulum stress, leading to fibroblast ferroptosis, impaired collagen secretion, and inducing a chronic inflammatory environment, thereby significantly delaying wound healing. This invention, through the multiple synergistic effects of the nanoenzyme system, achieves the goal of blocking NET-related pathological processes at their source.

[0024] The DNase I component in Pd-ZIF8@DNase I nanoparticles can efficiently degrade the DNA backbone in NETs, ​​reducing the toxicity of NETs to surrounding tissues. Simultaneously, the Pd-doped ZIF8 backbone exhibits multi-enzyme biomimicry, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), significantly scavenging reactive oxygen species (ROS) at wound sites and reducing oxidative stress levels. After encapsulation with DNase I, the SOD, POD, and CAT activities of Pd-ZIF8@DNase I are further enhanced compared to Pd-ZIF8. This is presumably because DNase I molecules induce local MOF structural defects during nucleation, exposing more metal active sites and thus forming an "enzyme-induced defective MOF" structure with higher electron transfer efficiency.

[0025] Through the above mechanism, the Pd-ZIF8@DNase I nanoenzyme hydrogel provided by this invention can downregulate the free DNA backbone in NETs. In addition, the dynamic covalent network of the hydrogel endows the material with pH responsiveness and sustained-release properties, which can gradually release Pd-ZIF8@DNase I in the acidic inflammatory microenvironment, achieving long-term and continuous NETs clearance and anti-inflammatory regulation.

[0026] Therefore, the Pd-ZIF8@DNase I nanoenzyme hydrogel provided by this invention can be used to prepare drugs for treating NETs-related chronic inflammatory diseases, especially suitable for pathological conditions such as diabetic chronic wounds, vasculitis ulcers, atherosclerosis, and systemic lupus erythematosus-related skin lesions. Furthermore, this material also has potential applications in diseases caused by excessive oxidative stress and impaired tissue repair, such as radiation dermatitis, difficult-to-heal surgical wounds, and infectious inflammatory models of wounds.

[0027] The application method of the Pd-ZIF8@DNase I nanoenzyme hydrogel drug prepared in this invention is not particularly limited. Representative application methods include (but are not limited to): local injection, wound application, gel patch, spray delivery, etc. Depending on the type and location of the disease, sustained-release local drug delivery or biodegradable patch treatment can also be achieved through the hydrogel carrier, which has good biocompatibility and clinical translation potential.

[0028] The present invention has the following beneficial effects:

[0029] (1) Compared with traditional free enzyme preparations and anti-inflammatory drugs, the Pd-ZIF8@DNase I nanoenzyme hydrogel preparation method provided by this invention is simple, mild, and highly controllable. By encapsulating DNase in situ during the formation of ZIF-8, enzyme activity can be effectively protected, and high encapsulation efficiency and uniform distribution can be achieved. The raw materials used are widely available, the reaction system is an aqueous system, the overall cost is low, the reproducibility is good, and it has good scalability and application prospects.

[0030] (2) The Pd-ZIF8@DNase I nanoparticles of the present invention possess synergistic catalytic activity of both bioenzymes and nanozymes. Pd doping endows ZIF-8 with multi-enzyme mimicry activities of SOD, CAT, and POD, which can efficiently scavenge reactive oxygen species (ROS), while DNase I specifically hydrolyzes the DNA backbone in the NETs structure, achieving targeted NETs removal. The synergistic effect of the two can effectively reduce the level of oxidative stress in the inflammatory microenvironment, inhibit NETs-induced endoplasmic reticulum stress, and significantly promote tissue repair.

[0031] (3) The hydrogel system provided by this invention is based on the Schiff base dynamic covalent bond between 4a-PEG-OPA and 3-aminophenylboronic acid, and has self-repair and pH-responsive properties, enabling long-term sustained release in inflammatory microacidic environments such as diabetes. This structure significantly prolongs the in vivo half-life of DNase I, avoiding the problem of rapid inactivation and clearance of free enzymes, thereby improving bioavailability and therapeutic durability.

[0032] (4) Compared with traditional single enzyme or drug treatment, the Pd-ZIF8@DNase I nanoenzyme hydrogel in this invention achieves a complex biological effect through a "multi-pathway, synergistic repair" mechanism. Experimental results show that the system can restore inflammatory markers induced by NETs, ​​and this process effectively improves the problems of excessive NETs and oxidative damage in diabetic wounds.

[0033] (5) The Pd-ZIF8@DNase I nanoenzyme hydrogel of the present invention has good biocompatibility and degradability. Its injectability and gelling properties make it suitable for various treatment scenarios such as local wound drug delivery, chronic wound dressing, and postoperative surgical repair, and have significant advantages such as local precise action, long-term sustained release, and high safety. Attached Figure Description

[0034] Figure 1 TEM observations of ZIF8, Pd-ZIF8, and Pd-ZIF8@DNase I were performed, with elemental analysis of Pd-ZIF8@DNase I using energy-dispersive X-ray spectroscopy (EDS). Particle size distribution and zeta potential were measured using dynamic light scattering (DLS).

[0035] Figure 2 Successful encapsulation of Pd-ZIF8@DNase I and crystal characterization. (A) FTIR spectra of ZIF8, Pd-ZIF8, and Pd-ZIF8@DNase I; (BC) XRD patterns of ZIF8, Pd-ZIF8, and Pd-ZIF8@DNase I; (D) BET spectra of ZIF8, Pd-ZIF8, and Pd-ZIF8@DNase I; (EH) XRD and XPS analysis of ZIF8, Pd-ZIF8, and Pd-ZIF8@DNase I, showing that DNase I introduces induced defects.

[0036] Figure 3 Comparison of SOD, POD, and CAT nanozyme activities of ZIF8, Pd-ZIF8, and Pd-ZIF8@DNase I. (AB) POD activity of CTR, ZIF8, Pd-ZIF8, Pd-ZIF8@DNase I; (C) POD activity of Pd-ZIF8@DNase I in decomposing peroxides; (D) DPPH free radical scavenging antioxidant activity of ZIF8, Pd-ZIF8, Pd-ZIF8@DNase I; (E) CAT activity of ZIF8, Pd-ZIF8, Pd-ZIF8@DNase I; (F) Macroscopic diagram of ZIF8, Pd-ZIF8, Pd-ZIF8@DNase I in decomposing hydrogen peroxide to produce oxygen; (G) Scavenging of superoxide anions by ZIF8, Pd-ZIF8, Pd-ZIF8@DNase I; (H) Scavenging of hydroxyl radicals by ZIF8, Pd-ZIF8, Pd-ZIF8@DNase I; (I) SOD activity of ZIF8, Pd-ZIF8, Pd-ZIF8@DNase I.

[0037] Figure 4Formation of Pd-ZIF8@DNase I hydrogel and its responsiveness to high sugar and acidic environments. (A) Macroscopic view of hydrogel formation; (B) SEM image of PZD-Gel hydrogel; (C) Acid and glucose responsiveness of PZD-Gel; (D) Contact angle test of PZD-Gel; (E) Adhesion of PZD-Gel; (F) Glucose responsiveness of PZD-Gel from 0h to 12h; (G) FTIR of PZD-Gel; (H) Degradation of PZD-Gel in high sugar environment; (I) Degradation of PZD-Gel in acidic environment; (J) Storage modulus and loss modulus of Gel and PZD-Gel; (K) Linear viscoelastic region of Gel and PZD-Gel; (L) Self-healing properties of PZD-Gel; (M) Swelling of PZD-Gel.

[0038] Figure 5 The healing effect of hydrogel treatment in a diabetic wound model. (A) Schematic diagram of hydrogel treatment regimen for diabetic skin wounds in mice; (B) Representative images of wounds in each treatment group on days 0, 3, 7, and 14 after induction; (C) Monitoring data on wound closure within 14 days under each treatment regimen; (D) Wound healing rate within 14 days under each treatment regimen; (E) Wound size statistics within 14 days under each treatment regimen; (F) HE and Masson staining at day 14 under each treatment regimen. Detailed Implementation

[0039] The present invention will now be further described with reference to the accompanying drawings and embodiments.

[0040] Example 1

[0041] Preparation of Pd-ZIF8@DNase I nanoparticles

[0042] Massive Zn 2+ :Pd 2+ = 3 : 2 Weigh 30 mmol zinc nitrate hexahydrate and 20 mmol palladium nitrate dihydrate and dissolve them in 10 mL deionized water. React under sonication for 15 min to form a clear solution B. Separately, dissolve 2-methylimidazole in 20 mL methanol at a molar ratio of 4:1 to metal ions and sonicate for 30 min until completely dissolved to form a clear and transparent solution A.

[0043] Solution A was slowly added dropwise to solution B under stirring to promote the formation of ZIF-8 crystal nuclei. During the reaction, after thorough mixing for 10 hours, DNase I solution (concentration of DNase I in the system was 0.5-1 mg / mL, 0.5 mg / mL was chosen in this example) was added to the mixture to in-situ encapsulate DNase I molecules within the Pd-ZIF8 structure during MOF nucleation. The system reacted at room temperature for 3 hours to form a brown suspension.

[0044] After the reaction was complete, the product was centrifuged (10,000 rpm, 10 min) and washed twice each with methanol and deionized water to remove unreacted residual ligands and free enzymes, finally yielding a light brown powder product, Pd-ZIF8@DNase I. The obtained nanoparticles were vacuum dried and stored at 4℃ for later use.

[0045] The preparation method of the control sample Pd-ZIF8 is the same as described above, but without the addition of DNase I.

[0046] Morphological characterization: The morphology of the materials was observed using transmission electron microscopy (TEM). The results showed that Pd-ZIF8 exhibited a regular dodecahedral structure with a particle size of approximately 400-500 nm; while the surface of Pd-ZIF8@DNase I was slightly rough, with irregular pores appearing in some areas, and the particle size was approximately 500 nm.

[0047] Crystal structure: X-ray diffraction (XRD) patterns show that the main diffraction peaks of both correspond to the characteristic peaks of ZIF-8, but the peak intensity of Pd-ZIF8@DNase I is slightly decreased and the full width at half maximum (FWHM) is increased, indicating that the enzyme molecule participates in the formation of crystal nuclei and induces structural defects.

[0048] Chemical composition: X-ray photoelectron spectroscopy (XPS) results showed that the Pd 3d peak in the Pd-ZIF8@DNase I sample shifted slightly to higher binding energies and a Pd–O signal appeared, indicating that a partial Pd–O–Zn coordination structure was formed during the encapsulation process, which enhanced the exposure of surface active sites.

[0049] Enzyme activity assay: The activities of SOD, POD, and CAT were detected by H2O2 decomposition assay. The results showed that Pd-ZIF8@DNase I was superior to Pd-ZIF8 in all three enzyme activity tests, indicating that the introduction of DNase I induced structural defects and enhanced electron transfer efficiency.

[0050] Example 2: Preparation of Pd-ZIF8@DNase I hydrogel

[0051] Preparation of hydrogel precursor solution:

[0052] Weigh 5 mg of 3-APBA (3-aminophenylboronic acid) and add it to centrifuge tube A. Dissolve it in 50 mM HEPES buffer (hydroxyethylpiperazine ethanesulfonic acid buffer) to a total volume of 400 µL. Weigh 40 mg of 4a-PEG-OPA and add it to centrifuge tube B. Dissolve it in 400 µL of 50 mM HEPES (pH 7.5). Take centrifuge tube C and first add 400 µL of 4a-PEG-OPA solution (from B). Slowly add 400 µL of 3-APBA solution (from A) using a pipette. Gently pipette 8–10 times to mix, then add 20 µL of 50 mM HEPES (pH 7.5) to a total volume of 1 mL. Gently invert the mixing centrifuge tube and incubate at 37°C for observation. After standing for 1–5 minutes, invert the tube. If the solution does not flow, it indicates gel formation.

[0053] Pd-ZIF8@DNase I nanoparticles (the mass concentration of Pd-ZIF8@DNase I nanoparticles in the hydrogel is 0.5~2 mg / mL, and the mass concentration of 1 mg / mL is selected in this example) were uniformly dispersed in 4a-PEG-OPA solution to form a uniform and transparent gel system, which is referred to as Pd-ZIF8@DNase I hydrogel.

[0054] The control group prepared blank hydrogels (without Pd-ZIF8@DNase I) and Pd-ZIF8 hydrogels (without enzyme).

[0055] Example 3 Performance Evaluation of Pd-ZIF8@DNase I

[0056] Enzyme activity retention: such as Figure 3 As shown, the in vitro antioxidant properties of Pd-ZIF8@DNase I hydrogel exhibited significant synergistic activity of SOD, POD, and CAT in the ROS scavenging experiment, effectively decomposing O2. - It can scavenge H2O2 and hydroxyl radicals, with a free radical scavenging rate of over 80%.

[0057] Example 4 Performance evaluation of Pd-ZIF8@DNase I hydrogel

[0058] High sugar environment and acid response: such as Figure 4As shown, Pd-ZIF8@DNase I hydrogel was placed in PBS solutions (pH 7.0–7.4 and pH 6.0–6.5) and 25 mmol of glucose, and the acid and glucose responsiveness of the hydrogel were monitored. The results showed that the hydrogel degraded slowly under neutral conditions, while the release rate increased under slightly acidic conditions, indicating good pH responsiveness. In 25 mmol of glucose, the hydrogel rapidly decomposed due to the breaking of dynamic borate ester bonds, indicating good glucose responsiveness and its ability to adapt to the inflammatory wound environment.

[0059] Example 5: Application of Pd-ZIF8@DNase I hydrogel in promoting the repair of diabetic wounds

[0060] In an STZ-induced diabetic mouse skin wound model, Pd-ZIF8@DNase I hydrogel was applied topically. Results showed that... Figure 5 As shown, the wound closure rate in the treatment group reached over 85% on day 10, while it was only about 55% in the control group. Histological examination revealed that Pd-ZIF8@DNase I hydrogel significantly reduced NET accumulation and promoted angiogenesis and collagen deposition.

[0061] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a controllable-release nanoenzyme hydrogel that promotes the healing of chronic diabetic wounds, characterized in that, Prepare according to the following steps: (1) Weigh zinc nitrate and palladium nitrate and dissolve them in deionized water. Add 2-methylimidazole methanol solution dropwise and stir to mix thoroughly. Then add deoxyribonuclease I solution to encapsulate it in situ. Centrifuge and wash to obtain Pd-ZIF8@DNaseI nanozyme particles. (2) Dissolve 4a-PEG-OPA in Tris-HCl buffer, add 3-aminophenylboronic acid in Tris-HCl buffer solution, and form a hydrogel network through Schiff base reaction; (3) Disperse the Pd-ZIF8@DNaseI nanoenzyme particles obtained in step (1) uniformly in the hydrogel system to obtain Pd-ZIF8@DNaseI hydrogel.

2. The method for preparing the controllable-release nanoenzyme hydrogel for promoting the healing of chronic diabetic wounds according to claim 1, characterized in that: In step (1), the molar ratio of zinc nitrate to palladium nitrate is 3:2-4:3, the molar ratio of 2-methylimidazole to metal ions is 4:1-3:1, and after stirring and mixing for 10-15 hours, the concentration of deoxyribonuclease I solution in the mixed solution is 0.5-1 mg / mL. The Pd-ZIF8@DNaseI nanozyme particles have an icosahedral structure and a particle size of 400-500 nm.

3. The method for preparing the controllable release nanoenzyme hydrogel for promoting the healing of chronic diabetic wounds according to claim 2, characterized in that: The specific operation of step (2) is to dissolve 3-aminophenylboronic acid and 4a-PEG-OPA in hydroxyethylpiperazine ethanesulfonic acid buffer, slowly add the 3-aminophenylboronic acid solution to the 4a-PEG-OPA solution, mix well, and then add some hydroxyethylpiperazine ethanesulfonic acid buffer. Let it stand at 37°C, invert the mixing container, and the hydrogel is obtained when the solution does not flow.

4. The method for preparing the controllable-release nanoenzyme hydrogel for promoting the healing of chronic diabetic wounds according to claim 3, characterized in that: The ratio of 4a-PEG-OPA to 3-aminophenylboronic acid and hydroxyethylpiperazine ethanesulfonic acid buffer is 5~6mg:40~50mg:1mL.

5. The method for preparing the controllable-release nanoenzyme hydrogel for promoting the healing of chronic diabetic wounds according to claim 4, characterized in that: In step (3), the mass concentration of Pd-ZIF8@DNaseI nanozyme particles in the hydrogel is 0.5~2 mg / mL.

6. A controlled-release nanoenzyme hydrogel for promoting the healing of chronic diabetic wounds prepared by the method described in any one of claims 1-5.

7. The use of the controllable release nanoenzyme hydrogel for promoting the healing of chronic diabetic wounds as described in claim 6 in the preparation of a biological dressing for the treatment of chronic diabetic wound healing.

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