Cerium-based composite nano-enzyme for promoting wound healing as well as preparation method and application of cerium-based composite nano-enzyme

Abstract

The invention provides a cerium-based composite nano enzyme for promoting wound healing as well as a preparation method and application thereof, and belongs to the technical field of nano medicine. The cerium-based composite nano-enzyme is constructed by integrating cerium nanoclusters (CeNC) and amino acid into a cobalt-based MOF (Metal Organic Framework) framework, and the synthesized nano-material has a topological structure of ZIF-67. Wherein CeNC is fixed in pores and on the surface of a cobalt framework (Co-ZIF), and amino acid and 2-methylimidazole have a competitive coordination effect, replace part of ligands and participate in framework assembly. The cerium-based composite nano-enzyme has good inflammation inhibition and angiogenesis promotion capabilities, can effectively promote chronic wound healing, overcomes the problems of chronic wound treatment drugs in the prior art, establishes an example of a novel angiogenesis promotion nano-drug, and has a wide application prospect. Meanwhile, a technical support is provided for tissue repair and regeneration and ischemic disease treatment.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, and in particular to a cerium-based composite nanozyme that promotes wound healing, its preparation method, and its application. Background Technology

[0002] Delayed wound healing caused by insufficient angiogenesis is clinically defined as chronic wounds. These wounds not only significantly reduce patients' quality of life but can even be life-threatening, imposing a heavy health and economic burden. Globally, approximately 500 million people develop chronic wounds each year due to various causes such as traumatic injury, burns, and surgical incisions. The pathogenesis of chronic wounds is complex and often associated with multiple pathological conditions, including aging, obesity, inflammation, infection, diabetes, and vascular insufficiency. Current treatments primarily focus on debridement, infection control, and local wound management to promote the healing process. However, due to the complexity of the wound microenvironment, existing clinical interventions often fall short of meeting the multidimensional healing needs.

[0003] Blood vessels are crucial for tissue repair and regeneration after traumatic or pathological injuries. They ensure the efficient delivery of oxygen and nutrients to tissues and promote the removal of metabolic waste, which is essential for tissue repair and regeneration after traumatic or pathological injuries. To this end, various strategies to promote angiogenesis (such as growth factors, gene therapy, and cell therapy) have been explored to enhance chronic wound healing. However, these pro-angiogenic strategies generally still have limited efficacy and potential carcinogenic risks. Vascular endothelial growth factor (VEGF) is a core factor in promoting angiogenesis and is considered one of the most promising drug candidates in the field of therapeutic angiogenesis. However, VEGF has drawbacks such as an extremely short half-life, poor stability, a narrow dose window, and a tendency to induce severe pathological angiogenesis, which seriously hinders its clinical translation and application. Therefore, there is an urgent need for innovative methods to enhance angiogenesis to promote tissue regeneration and wound repair.

[0004] Persistent inflammation in chronic wounds induces severe oxidative stress, further hindering angiogenesis and creating a vicious cycle characterized by excessive reactive oxygen species (ROS) accumulation and insufficient angiogenesis, severely delaying wound healing. Therefore, breaking this vicious cycle is crucial for revitalizing angiogenesis. Overall, therapeutic strategies targeting the pathological microenvironment of chronic wounds, combining anti-inflammatory effects, mitigation of ROS-induced damage, and promotion of angiogenesis, hold promise as a new direction for achieving efficient wound healing.

[0005] The hypoxia-inducible factor-1α / vascular endothelial growth factor (HIF-1α / VEGF) signaling pathway plays a central role in angiogenesis and is a key regulator of angiogenesis. The complex pathological microenvironment in chronic wounds (e.g., high levels of glucose and ROS) leads to dysfunction of this signaling pathway. HIF-1α is a key regulator of cellular responses to hypoxia, activating the expression of various proteins, including VEGF, and promoting endothelial cell proliferation, migration, and angiogenesis. Given its central role in vascular remodeling, upregulating HIF-1α expression to restore angiogenic capacity has become a promising therapeutic strategy for chronic wound repair. However, HIF-1α is highly unstable in air-exposed wounds, and existing HIF-1α stabilizers have side effects such as hepatotoxicity. Therefore, there is an urgent need to develop novel HIF-1α activation methods. Recent studies have emphasized the potential activation of HIF-1α by inducing the activity of extracellular vesicles, NO, H2S, CO, iridium complexes, and Ca2+. 2+ Copper and cobalt-based nanomaterials can upregulate VEGF expression and enhance angiogenesis by stabilizing HIF-1α, thereby accelerating wound healing.

[0006] In recent years, nanozymes (a class of nanocatalysts with enzyme-like activity, including metals, metal oxides, carbon-based materials, and metal-organic frameworks (MOFs)) have attracted much attention due to their customizable biomimetic properties, high stability, ease of preparation, and cost-effectiveness. Among them, metal-organic frameworks (MOFs) have shown excellent biocatalytic potential due to their uniform pores and high specific surface area. Cobalt-based MOFs (such as ZIF-67) have become a promising class of nanozymes due to their high catalytic activity and simple synthesis. In addition, ZIF-67, as a cobalt source, can release cobalt ions, which have the functions of promoting angiogenesis and inducing anti-inflammatory macrophage polarization. However, its ROS scavenging ability is limited, which affects the therapeutic effect.

[0007] Furthermore, ideal nanomedicines for wound treatment should possess effective antioxidant activity and have Ce2 content. 3+ / Ce 4+ Cerium dioxide nanoparticles exhibit inherent antioxidant activity. However, their activity is severely limited by their tendency to aggregate and their low specific surface area, hindering their biomedical applications. Recently, ultrasmall cerium oxide nanoclusters (CeNCs) have emerged due to their large surface area and high Ce content. 3+ CeNC exhibits superior antioxidant properties due to its optimized electronic structure and proportions. These properties make CeNC particularly attractive for nanomedicine. However, this type of nanocluster suffers from thermodynamic instability and complex synthesis, making it difficult to achieve uniform and stable preparation. Therefore, the preparation of nanozymes with good antioxidant properties, high stability, and resistance to inactivation is crucial for the treatment of chronic wounds. Summary of the Invention

[0008] The purpose of this invention is to provide a cerium-based composite nanozyme that promotes wound healing, its preparation method and application. A cerium-based composite nanozyme is constructed by integrating cerium nanoclusters (CeNC) and amino acids into a cobalt-based MOF framework. It aims to accelerate chronic wound healing by breaking the vicious cycle of malignant ROS accumulation and insufficient angiogenesis. It has the characteristics of good antioxidant properties and high stability.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a cerium-based composite nanozyme that promotes wound healing. The raw materials for preparing the cerium-based composite nanozyme for promoting wound healing include the following components: Nitrates, imidazole derivatives, and amino acids.

[0010] Preferably, the molar ratio of the nitrate, imidazole derivative, and amino acid is 0.25~4:12~32:0.05~1.

[0011] Preferably, the nitrate includes one or more of cobalt nitrate and cerium ammonium nitrate.

[0012] The imidazole derivatives include 2-methylimidazole.

[0013] The amino acid includes one of tryptophan, histidine, and methionine.

[0014] This invention also provides a method for preparing the above-mentioned cerium-based composite nanozyme for promoting wound healing, characterized by comprising the following steps: (1) Dissolve the nitrate in a solvent to obtain the first solution; (2) Mix the imidazole derivatives and amino acids in a solvent to obtain a second solution; (3) Mix the first solution with the second solution, stir, let stand to react, and separate to obtain the cerium-based composite nanozyme that promotes wound healing.

[0015] Preferably, the solvents used in steps (1) and (2) are water or organic solvents.

[0016] Preferably, in step (3), the settling reaction time is 2 to 4 hours.

[0017] The present invention also provides an application of the above-mentioned cerium-based composite nanozyme for promoting wound healing in the preparation of drugs for promoting wound healing.

[0018] The present invention also provides a drug for promoting wound healing, the drug comprising the above-mentioned cerium-based composite nanoenzyme for promoting wound healing.

[0019] The beneficial effects of this invention compared to the prior art are as follows: (1) This invention constructs a cerium-based composite nanozyme by integrating cerium nanoclusters (CeNC) and amino acids into a cobalt-based MOF framework. The synthesized nanomaterial has a ZIF-67 topological structure. CeNC is immobilized in the pores and on the surface of the cobalt framework (Co-ZIF), while amino acids and 2-methylimidazole exhibit competitive coordination, replacing some ligands and participating in framework assembly. This composite nanozyme possesses excellent anti-inflammatory and angiogenesis-promoting capabilities, effectively promoting chronic wound healing. It overcomes the problems existing in current chronic wound treatment drugs, establishes a paradigm for novel angiogenesis-promoting nanomedicines, and provides technical support for tissue repair, regeneration, and the treatment of ischemic diseases.

[0020] (2) The cerium-based composite nanozyme of this invention uses Co-ZIF as the cobalt source. By releasing cobalt ions, it stabilizes HIF-1α and activates the expression of the downstream gene VEGF, thereby promoting the proliferation and migration of endothelial cells and effectively accelerating angiogenesis. Compared with directly providing exogenous VEGF protein, the nanozyme can also reduce tissue damage by regulating the wound microenvironment (such as clearing ROS and anti-inflammation). By stabilizing HIF-1α, it can continuously stimulate the body's own cells to synthesize and secrete VEGF. Moreover, endogenous expression is usually regulated by in vivo feedback mechanisms, which is more in line with local needs, thereby significantly reducing the risk of pathological angiogenesis.

[0021] (3) Co-ZIF has been reported to have a size greater than 1 micrometer, which severely limits its potential in most cutting-edge biomedical applications, especially those requiring systemic drug delivery and nuclear action. Therefore, this invention uses amino acids as size modifiers to reduce the size of Co-ZIF from the micrometer level to about 200 nm, which greatly improves its catalytic activity and bioavailability.

[0022] (4) In this invention, the spatial confinement effect of Co-ZIF effectively fixes CeNC and prevents its aggregation. The internal electron transfer from Co to Ce increases the Ce of CeNC. 3+ These structural features significantly enhance the antioxidant efficacy of TCC, giving it superior ROS scavenging capabilities. Furthermore, tryptophan reduces the size of TCC from the micrometer scale of Co-ZIF to approximately 200 nanometers, significantly enhancing its antioxidant activity and bioavailability for wound healing. Both in vitro and in vivo experiments demonstrate that TCC enhances wound healing by scavenging ROS, inhibiting inflammation, and promoting angiogenesis through upregulation of HIF-1α / VEGF levels, elucidating its therapeutic advantages in chronic wound healing. In conclusion, this TCC-based therapeutic strategy, which reverses the vicious cycle of ROS accumulation and insufficient angiogenesis, establishes a promising paradigm for chronic wound management.

[0023] (5) The composite nanozyme of the present invention can also stabilize HIF-1α by releasing cobalt ions to activate the HIF-1α / VEGF pathway, and promote angiogenesis by inducing macrophages to polarize to the anti-inflammatory M2 phenotype. In vitro and in vivo studies have shown that TCC reverses the cascade of insufficient angiogenesis induced by ROS accumulation by synergistically clearing ROS, inhibiting inflammation and promoting angiogenesis, thereby significantly accelerating the healing of chronic wounds. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 These are morphology images of different products in Experimental Example 1 of the present invention, where a is the morphology image of Co-ZIF, b is the morphology image of Ce / Co-ZIF, and c is the morphology image of TCC. Figure 2 The XRD spectra of Co-ZIF, Ce / Co-ZIF, and TCC in Experimental Example 1 of this invention are shown below. Figure 3 The UV absorption spectra of Co-ZIF, Ce / Co-ZIF, and TCC in Experimental Example 1 of this invention are shown below. Figure 4 The images shown are HRTEM images of the TCC in Experimental Example 1 of this invention, where a is the HRTEM image of the TCC, CeNC is within the white box, and the inset shows the corresponding lattice fringes; b is the HRTEM image of the TCC, CeNC is within the yellow circle. Figure 5 The pore size distribution diagrams of Co-ZIF, Ce / Co-ZIF, and TCC in Experimental Example 1 of this invention are shown. Figure 6 The Ce 3d XPS spectra of Ce / Co-ZIF and TCC in Experimental Example 1 of this invention are shown.

[0026] Figure 7 The Co 2p XPS spectra of Co-ZIF, Ce / Co-ZIF, and TCC in Experimental Example 1 of this invention are shown below. Figure 8 The image shows the antioxidant activity of Co-ZIF, Ce / Co-ZIF and TCC in Experimental Example 2 of this invention, where a represents SOD-like activity and b represents CAT-like enzyme activity. Figure 9The images show the in vitro angiogenesis effects of Co-ZIF, Ce / Co-ZIF, and TCC in Experimental Example 2 of this invention, where a is the angiogenesis experiment, b is the total blood vessel length statistics, and c is the branch point number statistics. Figure 10 The angiogenesis mechanism of Co-ZIF, Ce / Co-ZIF and TCC in Experimental Example 2 of this invention is shown, where a is the relative expression of HIF-1α and b is the relative expression of VEGF. Figure 11 The figure shows the in vitro antioxidant activity detection results of Co-ZIF, Ce / Co-ZIF and TCC in Experiment Example 2 of the present invention, where a is the fluorescence microscopy image of the ROS probe (DCFH-DA) and b is the ROS fluorescence intensity statistics. Figure 12 The in vitro anti-inflammatory properties of Co-ZIF, Ce / Co-ZIF and TCC in Experimental Example 2 of this invention are shown in Figure a, where a is a representative immunofluorescence image of CD86 and CD206 in Raw264.7 cells, b is the statistical data of CD86 expression, and c is the statistical data of CD206 expression. Figure 13 This is an example of the in vivo wound healing effects of Co-ZIF, Ce / Co-ZIF and TCC in Experiment 2 of the present invention. Among them, a is a photograph of the wound healing process after different nanozyme treatments on days 0, 3, 7 and 14, b is a superimposed image of the wound area after various treatments on days 0, 3, 7 and 14, and c is a quantitative analysis of the wound closure rate. Figure 14 The histological evaluation results of Co-ZIF, Ce / Co-ZIF and TCC in Experimental Example 2 of this invention are shown in Figure a. Masson staining is shown, with control, Co-ZIF, Ce / Co-ZIF and TCC from left to right; and the quantitative statistics of collagen volume fraction are shown in Figure b. Figure 15 The in vivo angiogenesis effects of Co-ZIF, Ce / Co-ZIF and TCC in Experimental Example 2 of this invention are shown, where a is Doppler flow imaging (DBFI) and b is a statistical map of blood flow regions. Figure 16 This is an H&E staining image of the main organs in Experiment Example 2 of this invention; Figure 17 The antioxidant activities of commercial CeO2, ZIF-67, Ce / Co-ZIF and TCC in Experimental Example 3 of this invention are shown in Figure A, where A is a bar chart of SOD values, b is a bar chart of CAT values, and C is a bar chart of SOD values ​​and CAT values. Figure 18 This is the Ce 3d XPS plot of commercial CeO2 in Experimental Example 3 of this invention. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] Example 1 Example 1 of this invention provides a method for preparing composite nanozymes (TCC), the specific steps of which are as follows: 0.87 g of cobalt nitrate hexahydrate and 86.6 mg of cerium ammonium nitrate were added to 150 mL of deionized water and stirred until completely dissolved. Then, 14.78 g of 2-methylimidazole and 0.3 g of tryptophan were dissolved in 150 mL of water. The two solutions were mixed and stirred for 5 minutes, and then allowed to stand for 3 hours. The purple precipitate obtained by centrifugation was TCC. The resulting solid was washed three times with ultrapure water, centrifuged, and freeze-dried for later use.

[0033] Example 2 Example 1 of this invention provides a method for preparing composite nanozymes (TCC) using organic phases. The difference from Example 1 is that deionized water is replaced with methanol. The specific steps are as follows: 0.87 g of cobalt nitrate hexahydrate and 86.6 mg of cerium ammonium nitrate were added to 150 mL of methanol and stirred until completely dissolved. Then, 14.78 g of 2-methylimidazole and 0.3 g of tryptophan were dissolved in 150 mL of methanol. The two solutions were mixed and stirred for 5 minutes, and then allowed to stand for 3 hours. The purple precipitate obtained by centrifugation was TCC. The resulting solid was washed three times with ultrapure water, centrifuged, and freeze-dried for later use.

[0034] Example 3 Example 3 of this invention provides a method for preparing the composite nanozyme Trp@Ce / Co-ZIF(90), the specific steps of which are as follows: 0.87 g of cobalt nitrate hexahydrate and 86.6 mg of cerium ammonium nitrate were added to 150 mL of methanol and stirred until completely dissolved. Then, 17.31 g of imidazole-2-methyl (ICA) and 0.3 g of tryptophan were dissolved in 150 mL of methanol. The two solutions were mixed and stirred for 5 minutes, and then allowed to stand for 3 hours. The purple precipitate obtained by centrifugation was Trp@Ce / Co-ZIF(90). The resulting solid was washed three times with ultrapure water, centrifuged, and freeze-dried for later use.

[0035] Example 4 Example 4 of this invention provides a method for preparing the composite nanozyme Trp@Ce / Co-ZIF(7), the specific steps of which are as follows: 0.87 g of cobalt nitrate hexahydrate and 86.6 mg of cerium ammonium nitrate were added to 150 mL of methanol and stirred until completely dissolved. Then, 21.29 g of benzimidazole (BIM) and 0.3 g of tryptophan were dissolved in 150 mL of methanol. The two solutions were mixed and stirred for 5 minutes and allowed to stand for 3 hours. The purple precipitate obtained by centrifugation was Trp@Ce / Co-ZIF(7). The obtained solid was washed three times with ultrapure water, centrifuged, and freeze-dried for later use.

[0036] Example 5 Example 5 of this invention provides a method for preparing the composite nanozyme His@Ce / Co-ZIF, the specific steps of which are as follows: 0.87 g of cobalt nitrate hexahydrate and 86.6 mg of cerium ammonium nitrate were added to 150 mL of deionized water and stirred until completely dissolved. Then, 14.78 g of 2-methylimidazole and 0.3 g of histidine (His) were dissolved in 150 mL of water. The two solutions were mixed and stirred for 5 minutes, and then allowed to stand for 3 hours. The purple precipitate obtained by centrifugation was His@Ce / Co-ZIF. The resulting solid was washed three times with ultrapure water, centrifuged, and freeze-dried for later use.

[0037] Example 6 Example 6 of this invention provides a method for preparing the composite nanozyme Met@Ce / Co-ZIF, the specific steps of which are as follows: 0.87 g of cobalt nitrate hexahydrate and 86.6 mg of cerium ammonium nitrate were added to 150 mL of deionized water and stirred until completely dissolved. Then, 14.78 g of 2-methylimidazole and 0.3 g of methionine (Met) were dissolved in 150 mL of water. The two solutions were mixed and stirred for 5 minutes, and then allowed to stand for 3 hours. The purple precipitate obtained by centrifugation was Met@Ce / Co-ZIF. The resulting solid was washed three times with ultrapure water and then freeze-dried for later use.

[0038] Example 7 Example 7 of this invention provides a method for preparing the composite nanozyme Trp@Ce-ZIF, the specific steps of which are as follows: 0.87 g of cobalt nitrate hexahydrate and 86.6 mg of cerium ammonium nitrate were added to 150 mL of deionized water and stirred at room temperature until completely dissolved. Then, 14.78 g of 2-methylimidazole and 0.3 g of tryptophan were dissolved in 150 mL of water. The two solutions were mixed and stirred for 5 minutes, and then allowed to stand for 3 hours. The pale yellow precipitate obtained by centrifugation was Trp@Ce-ZIF. The resulting solid was washed three times with ultrapure water, centrifuged, and freeze-dried for later use.

[0039] Example 8 Example 8 of this invention provides a method for preparing the composite nanozyme Trp@10%Ce / Co-ZIF, the specific steps of which are as follows: 0.82 g of cobalt nitrate hexahydrate and 173 mg of cerium ammonium nitrate were added to 150 mL of deionized water and stirred until completely dissolved. Then, 14.78 g of 2-methylimidazole and 0.3 g of tryptophan were dissolved in 150 mL of water. The two solutions were mixed and stirred for 5 minutes, and then allowed to stand for 3 hours. The purple precipitate obtained by centrifugation was Trp@10%Ce / Co-ZIF. The resulting solid was washed three times with ultrapure water, centrifuged, and freeze-dried for later use.

[0040] Example 9 Example 9 of this invention provides a method for preparing the composite nanozyme Trp@Ce / Co-ZIF, the specific steps of which are as follows: 0.87 g of cobalt nitrate hexahydrate and 86.6 mg of cerium ammonium nitrate were added to 150 mL of deionized water and stirred until completely dissolved. Then, 14.78 g of 2-methylimidazole and 0.1 g of tryptophan were dissolved in 150 mL of water. The two solutions were mixed and stirred for 5 minutes, and then allowed to stand for 3 hours. The purple precipitate obtained by centrifugation was Trp@Ce / Co-ZIF. The resulting solid was washed three times with ultrapure water, centrifuged, and freeze-dried for later use.

[0041] Comparative Example 1 Comparative Example 1 of this invention provides a method for synthesizing Co-ZIF, the specific steps of which are as follows: 0.92 g of cobalt nitrate hexahydrate was dissolved in 150 mL of deionized water by stirring until completely dissolved. Then, 14.78 g of 2-methylimidazole was dissolved in 150 mL of water. The two solutions were mixed and stirred for 5 minutes, and then allowed to stand for 3 hours. The purple precipitate obtained by centrifugation was Co-ZIF. The resulting solid was washed three times with ultrapure water, centrifuged, and freeze-dried for later use.

[0042] Comparative Example 2 Comparative Example 2 of this invention provides a method for synthesizing Ce / Co-ZIF, the specific steps of which are as follows: 0.87 g of cobalt nitrate hexahydrate and 86.6 mg of cerium ammonium nitrate were added to 150 mL of deionized water and stirred until completely dissolved. Then, 14.78 g of 2-methylimidazole was dissolved in 150 mL of water. The two solutions were mixed and stirred for 5 minutes, and then allowed to stand for 3 hours. The purple precipitate obtained by centrifugation was Ce / Co-ZIF. The resulting solid was washed three times with ultrapure water, centrifuged, and freeze-dried for later use.

[0043] Experimental Example 1 Experimental Example 1 of this invention characterized the TCC, Co-ZIF, and Ce / Co-ZIF prepared in Example 1, Comparative Example 1, and Comparative Example 2. The specific steps are as follows: (1) The morphology of the composite nanozyme (TCC), Co-ZIF, and Ce / Co-ZIF was detected, and the results are as follows: Figure 1 As shown.

[0044] Figure 1The results show that Ce / Co-ZIF is a composite nanozyme material that integrates CeNC into a cobalt-based MOF framework. Compared with the micron-sized Co-ZIF and Co / Ce-ZIF, the size of TCC particles is around 200 nm, which is beneficial to enhance their antioxidant activity and bioavailability.

[0045] (2) The composite nanozyme (TCC), Co-ZIF, and Ce / Co-ZIF were characterized, and the results are as follows: Figures 2-7 As shown.

[0046] Figure 2 The XRD spectrum shows that TCC has the same XRD characteristic peaks as the ZIF-67 material reported in the literature, which indicates that TCC retains the topology of ZIF-67.

[0047] Figure 3 The UV absorption spectrum showed that TCC exhibited a characteristic UV-Vis absorption peak at 280 nm, corresponding to tryptophan, proving that tryptophan was successfully introduced into the crystal framework.

[0048] Figure 4 HRTEM images of TCC confirmed the uniform distribution of CeNC (approximately 1.5 nm) on the TCC surface and within the mesoporous structure. The measured cluster lattice spacing of 0.31 nm, corresponding to the CeO2(111) crystal plane, further confirmed the spontaneous formation of CeNC during the synthesis process.

[0049] Figure 5 The pore size distribution map shows that the specific surface areas of Co-ZIF, Ce / Co-ZIF, and TCC range from 1477 m². 2 g -1 (Co-ZIF) dropped to 1348 m 2 g -1 (Ce / Co-ZIF), finally decreasing to 1023 m. 2 g -1 (TCC), which is related to CeNC and tryptophan occupying the pores.

[0050] Figure 6 Quantitative analysis of the Ce 3d XPS spectrum identified 10 analytical peaks, corresponding to mixed Ce... 3+ (Pink) and Ce 4+ (Blue) Oxidation state. Ce in Ce / Co-ZIF and TCC. 3+ / Ce 4+ The ratios were 1.1 and 1.3, respectively.

[0051] Figure 7 The Co 2p XPS spectrum shows that, compared to Co-ZIF, the Co 2p of Ce / Co-ZIF is significantly different.3 / 2 and 2p 1 / 2 The XPS peaks shifted significantly to higher binding energies by 0.5 and 0.6 eV. A similar shift was observed in TCC. Meanwhile, peak area analysis indicated that, compared to Co-ZIF, Co in Ce / Co-ZIF and TCC... 2+ / Co 3+ The ratios were all reduced. These observations clearly demonstrate that charge transfer from cobalt to cerium leads to an enhanced electron density at Ce sites within the CeNC confined within the ZIF framework. Comparative analyses show that Ce / Co-ZIF and TCC exhibit higher antioxidant activity compared to Co-ZIF. This spontaneous incorporation of small-sized CeNCs and Co-Ce electron transfer will promote Ce... 3+ / Ce 4+ The dynamic redox cycle enhances the catalytic antioxidant performance of TCC.

[0052] Experimental Example 2 Experimental Example 2 of this invention evaluated the effects of TCC, Co-ZIF, and Ce / Co-ZIF prepared in Example 1, Comparative Example 1, and Comparative Example 2. The specific steps are as follows: (1) Antioxidant activity detection: (1.1) SOD-like activity: The SOD-like activities of TCC, Co-ZIF, and Ce / Co-ZIF were quantified using a total SOD activity assay kit (WST-8 method). The results are as follows: Figure 8 As shown.

[0053] (1.2) CAT-like activity: 25 μg mL -1 TCC, Co-ZIF, and Ce / Co-ZIF were incubated with 30 mM H2O2 at 37°C for 30 minutes. After the reaction, 100 μL of the supernatant was centrifuged and added to a 96-well plate, followed by the addition of 100 μL of ammonium molybdate (20 mg / mL). -1 The mixture was reacted with residual H2O2 and allowed to stand for 10 minutes to form a yellow complex. The absorbance of the characteristic absorption peak of the yellow complex at 405 nm was then analyzed using a microplate reader. The results are as follows: Figure 8 As shown.

[0054] Figure 8 The results showed that TCC exhibited superior SOD-like activity, with a higher scavenging capacity for ·O compared to Co-ZIF and Ce / Co-ZIF. 2- The abilities are 10.2 times and 1.5 times higher, respectively. For CAT-like activity, under the same conditions, TCC's H2O2 scavenging rate is greater than 80%, far exceeding Ce / Co-ZIF (66.6%) and Co-ZIF (7.6%).

[0055] (2) Evaluation of in vitro angiogenesis-promoting effects: HUVECs were inoculated onto the polymer matrix and treated with TCC, Co-ZIF, and Ce / Co-ZIF (25 μg / mL), respectively. -1 After incubation at 37°C for 6 hours, the endothelial cell network was imaged using a bright-field microscope. The total tube length and branch point density in ≥5 random fields per well were quantified using ImageJ software with an angiogenesis analysis plugin. The relative expression of HIF-1α and VEGF was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The results are shown below. Figure 9 , 10 As shown.

[0056] Figure 9 Mid-field imaging revealed that the Co-ZIF, Ce / Co-ZIF, and TCC groups exhibited stronger vessel-forming capabilities, while the control group showed sparse and immature structures. Quantitative analysis of total vessel length showed that, compared with the control group, the total vessel length of the Co-ZIF, Ce / Co-ZIF, and TCC groups increased by 71%, 110%, and 164%, respectively. Simultaneously, the branch point density of Co-ZIF, Ce / Co-ZIF, and TCC increased by 101%, 140%, and 188%, respectively, with TCC demonstrating superior pro-angiogenic capacity.

[0057] Figure 10 Real-time quantitative polymerase chain reaction (RT-qPCR) results showed that, compared with the control group, the expression levels of angiogenesis genes (HIF-1α and VEGF) in the H2O2-treated group were significantly reduced. In contrast, Ce / Co-ZIF and TCC treatments effectively reversed the H2O2-induced decrease in the expression levels of angiogenesis-related genes. In particular, the expression levels of angiogenesis genes after TCC treatment were close to those in the control group, indicating that TCC has a strong angiogenesis capacity. Co-ZIF treatment had little effect on the expression of angiogenesis-related genes, a phenomenon attributed to its insufficient antioxidant capacity.

[0058] (3) Evaluation of in vitro antioxidant activity Excessive ROS accumulation can induce endothelial dysfunction by disrupting the HIF-1α / VEGF signaling pathway, ultimately leading to delayed wound healing. To assess the ROS scavenging capacity of the material against H2O2-induced oxidative stress damage, HUVECs were cultured in confocal culture dishes for 24 hours and then co-treated with Co-ZIF, Ce / Co-ZIF, and TCC (25 μg mL⁻¹) and H2O2 (100 μM) for 24 hours. Intracellular ROS levels were detected using the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe, and fluorescence intensity was measured using ImageJ software. Results are as follows: Figure 11As shown.

[0059] Figure 11 The results showed that H2O2 treatment induced a 5.2-fold increase in fluorescence intensity compared to the control group. Treatment with Co-ZIF, Ce / Co-ZIF, and TCC eliminated excess ROS, reducing ROS levels by 30% (Co-ZIF), 66% (Ce / Co-ZIF), and 83% (TCC), respectively. Notably, the ROS levels in the TCC-treated group were close to those in the control group. Simultaneously, the fluorescence intensities of the Ce / Co-ZIF and TCC groups were significantly lower than those in the Co-ZIF group, indicating that encapsulated CeNCs exhibited stronger ROS scavenging activity.

[0060] The results showed that TCC could effectively alleviate ROS and reverse the functional limitation of the ROS-driven HIF-1α / VEGF signaling pathway, thereby breaking the vicious cycle of insufficient ROS-angiogenesis and accelerating wound healing. TCC has a dual mechanism to promote angiogenesis: (i) the released cobalt ions can stabilize HIF-1α; (ii) the high antioxidant capacity can reduce the inhibition of HIF-1α / VEGF signaling by ROS, thereby creating a favorable microenvironment for wound healing.

[0061] (4) In vitro anti-inflammatory properties To further evaluate the nanozyme-mediated immune microenvironment, this invention performed macrophage polarization analysis. Using the monocyte-macrophage cell line (Raw 264.7) as an inflammatory cell model, Raw 264.7 cells were seeded in confocal culture dishes and treated with TCC, Co-ZIF, and Ce / Co-ZIF (25 μg / mL), respectively. -1 Treat with culture for 24 hours, then use LPS (100 ng / mL) -1 Cells were stimulated for 24 hours. Subsequently, cells were fixed with 4% paraformaldehyde for 15 minutes, permeated with 0.1% Triton X-100 for 10 minutes, and arrested with 5% BSA at 25°C for 1 hour. Raw 264.7 cells were then incubated with primary antibodies (rabbit anti-CD86 antibody and rabbit anti-mannose receptor antibody) at 4°C for 12 hours. After washing three times with PBS (pH=7.4), cells were incubated with fluorescent secondary antibody at 37°C in the dark for 1 hour. Finally, cell nuclei were reverse stained with DAPI at 37°C for 15 minutes. Samples were imaged using a laser confocal scanning microscope (CLSM), and the average fluorescence intensity was quantified using ImageJ software. The results are shown below. Figure 12 As shown.

[0062] Figure 12The results showed that, compared with the control group, LPS stimulation induced a significant shift from the M1 to the M2 phenotype, characterized by a significant increase in the M1-associated marker (CD86) and a substantial decrease in the M2-associated marker (CD206). In contrast, Ce / Co-ZIF and TCC treatments reversed this trend, with CD86 expression significantly inhibited by 32% and 48%, respectively, compared with the LPS group. Conversely, CD206 expression increased by 1.8-fold and 1.9-fold, respectively, in the Ce / Co-ZIF and TCC treatment groups. Therefore, Ce / Co-ZIF and TCC treatments significantly increased the polarization rate of M2 / M1 macrophages. Among them, TCC treatment showed the most significant anti-inflammatory activity, effectively inhibiting M1 macrophage polarization and promoting M2 macrophage polarization.

[0063] (5) Assessment of chronic wound treatment in the body: A full-thickness skin defect model was established using eight-week-old C57BL / 6 mice (20±2 g). All animal experiments were approved by the Animal Experiment Management Committee of Air Force Medical University to ensure that the animals received ethical and humane treatment (KY20243341). After anesthetizing the mice with isoflurane, the hair on their backs was removed with depilatory cream, and the skin was disinfected with 75% ethanol. Then, a circular wound with a diameter of 10 mm was created on the back of each mouse. Subsequently, the mice were randomly divided into four experimental groups and administered PBS (control group), Co-ZIF, Ce / Co-ZIF, and TCC (0.5 mg / mL) every two days, respectively. -1 The wound was covered with 50 μL of a novel coronavirus. Photographs were taken on days 0, 3, 7, and 14 post-surgery to visually record wound healing progress. The residual wound area (%) was determined by measuring the wound area at the wound boundary. The residual wound area percentage was calculated using the following formula: Residual wound area percentage (%) = (Actual wound area / Original wound area) × 100%. Simultaneously, Doppler flow imaging (DBFI) was used to assess blood flow recovery on days 7 and 14. On day 14 post-surgery, mice were euthanized, and skin tissue samples were collected from the wound. Collagen composition was analyzed to evaluate wound healing effectiveness. The results are as follows: Figures 13-16 As shown.

[0064] Figure 13 The results showed that, compared with the control group, the wound area in all three material groups was significantly reduced on day 3. On day 7, the wound closure rate in the TCC group reached 72%, with the wound almost completely healed and covered by new epithelial tissue. By day 14, the wound closure rates in the Co-ZIF group, Ce / Co-ZIF group, and TCC group reached 91%, 95%, and 99%, respectively, significantly exceeding the control (81%). Importantly, after 14 days of treatment, the healing rate in the TCC group approached 100%, indicating almost complete wound healing.

[0065] Figure 14 The results showed that, compared with the control group, Masson staining revealed greater collagen fiber deposition and more ordered and denser collagen arrangement in the Ce / Co-ZIF and TCC groups. Quantitative analysis showed that the TCC group had the highest collagen deposition density among all groups, which was 1.9 times and 1.1 times that of the control group and Ce / Co-ZIF group, respectively, at day 14, indicating that TCC had the strongest wound repair ability.

[0066] Figure 15 Doppler flow imaging (DBFI) was used to monitor skin microvascular blood flow. Results showed that both the Ce / Co-ZIF and TCC groups had more mature vascular networks in the early and late stages of treatment (7 and 14 days post-surgery). The blood flow area in the TCC group at 7 and 14 days post-surgery was 2.7 times and 1.6 times that of the control group, respectively. Quantitative analysis further validated the superior angiogenesis-promoting ability of TCC in vivo, with its blood flow area significantly larger than other groups. These findings are highly consistent with in vitro tube formation capacity analysis, in vivo wound progression observation, and histological analysis of skin wound tissue. These data from in vitro and in vivo experiments collectively demonstrate that TCC is an effective angiogenesis promoter that can accelerate wound healing by enhancing angiogenesis.

[0067] Figure 16 Histological examination using H&E staining showed that, compared with the control group, the tissue structures of the major organs (heart, liver, spleen, lungs, and kidneys) in all treatment groups showed no obvious signs of inflammation or toxicity, confirming that the evaluated nanozymes have good biocompatibility and systemic safety, supporting their potential for clinical translation.

[0068] Experimental Example 3 In Experiment 3 of this invention, commercial CeO2 was used as a control, and the antioxidant activity of commercial CeO2 was tested using the method described in Experiment 2. The results are as follows: Figure 17 , 18 As shown.

[0069] Figure 17 , 18 The results show that, compared to commercial CeO2 and the pure cobalt framework (Co-ZIF), TCC and Ce / Co-ZIF exhibit significantly enhanced antioxidant activity. This improved activity is mainly attributed to the successful encapsulation of ultrasmall cerium nanoclusters, a structure that effectively overcomes the material limitations of commercial CeO2. Commercial CeO2 typically has a large particle size and nearly complete Ce content. 4+ The chemical state results in a lower specific surface area, poor accessibility of active sites, and a lack of Ce. 3+ / Ce 4+The oxidation state equilibrium necessary for redox cycles limits the antioxidant enzyme activity of certain enzymes. In contrast, CeNC in TCC and Ce / Co-ZIF possesses an ultra-small size, which not only greatly increases the specific surface area and exposes more active sites, but also significantly enhances Ce's antioxidant capacity. 3+ The proportion of Ce. 3+ Sites are key active centers for scavenging reactive oxygen species (ROS), and an increase in their content directly enhances the activity of the material.

[0070] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cerium-based composite nanoscale enzyme for promoting wound healing, characterized in that, The preparation raw material of the cerium-based composite nanoscale enzyme for promoting wound healing comprises the following components: nitrate, imidazole derivative, amino acid.

2. The cerium-based composite nanoscale enzyme for promoting wound healing according to claim 1, wherein, The molar ratio of the nitrate, imidazole derivative and amino acid is 0.25-4:12-32:0.05-1.

3. The cerium-based composite nanoscale enzyme for promoting wound healing according to claim 1, wherein, The nitrate comprises one or more of cobalt nitrate and cerium ammonium nitrate; The amino acid comprises one of tryptophan, histidine and methionine; The imidazole derivative comprises 2-methyl imidazole.

4. A method for preparing the cerium-based composite nanoscale enzyme for promoting wound healing according to any one of claims 1-3, characterized in that, The method comprises the following steps: (1) dissolving the nitrate in a solvent to obtain a first solution; (2) mixing the imidazole derivative and amino acid in a solvent to obtain a second solution; (3) mixing the first solution and the second solution, stirring, standing for reaction, and separating to obtain the cerium-based composite nanoscale enzyme for promoting wound healing.

5. The method of claim 4, wherein the cerium-based composite nanoszyme for promoting wound healing is prepared by the steps of: (a) preparing a cerium-based composite nanoszyme; (b) adding a wound healing agent to the cerium-based composite nanoszyme; and (c) drying the cerium-based composite nanoszyme. In steps (1) and (2), the solvent is water or an organic solvent.

6. The method of claim 4, wherein the cerium-based composite nanoszyme for promoting wound healing is prepared by the steps of: In step (3), the standing time is 2-4 hours.

7. Use of the cerium-based composite nanoscale enzyme for promoting wound healing according to any one of claims 1-3 or prepared by the method according to any one of claims 5-6 in the preparation of a drug for promoting wound healing.

8. A medicament for promoting wound healing, characterized by, The drug comprises the cerium-based composite nanoscale enzyme for promoting wound healing according to any one of claims 1-3.

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