A magnetic nano-composite preparation with multiple enzyme activities and a preparation method and application thereof

Abstract

The present application relates to a kind of magnetic nano composite preparation with multiple enzyme activities and its preparation method and application, belong to the technical field of biomedical materials.The present application is wrapped with the nano enzyme of the double-enzyme activity of superoxide dismutase and catalase on the surface of magnetic nanoparticles by continuous in-situ growth method and surface modification, and is modified nitric oxide donor material.The magnetic composite preparation prepared in the present application is uniform in size, and has high magnetic saturation strength.Under the driving of external magnetic field, nanoparticles can realize directional controllable movement.In addition, nanoparticles have multiple enzyme activities such as superoxide dismutase, catalase and nitric oxide synthase, and have significantly enhanced enzyme catalytic performance.The magnetic nano composite preparation prepared in the present application not only has good biocompatibility, but also can quickly respond to external magnetic field to realize directional controllable movement.The magnetic nano composite preparation provided in the present application can be used as tissue penetration agent for preparing wound repair drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and more specifically, relates to a magnetic nanocomposite preparation with multi-enzyme activity, its preparation method and application. Background Technology

[0002] Nanozymes are nanomaterials with intrinsic enzymatic properties, capable of effectively catalyzing substrate transformation under physiological conditions, and their kinetic mechanisms are identical to those of natural enzymes. Compared to natural enzymes, they are more functionally diverse, highly stable, easy to store, and mass-produce, making them widely applicable in analytical, sensing, and biomedical fields. They play a crucial role in decomposing harmful molecules, regulating redox balance, and alleviating inflammation by precisely modulating redox dynamics and abnormal biochemical environments. Through the rational design and optimization of the structural components, physicochemical properties, and catalytic conditions of nanozymes, precise control of their catalytic activity or the development of multi-enzyme cascade effects can be achieved, thus exhibiting precise or synergistic advantages in complex disease scenarios. However, in practical applications, the delivery efficiency of nanozymes is often unsatisfactory. Traditional nanozyme delivery methods mainly rely on passive diffusion mechanisms, resulting in poor tissue penetration and low bioavailability, significantly limiting their therapeutic efficiency in deeply damaged tissues. Summary of the Invention

[0003] To address the aforementioned deficiencies or improvement needs of existing technologies, the present invention aims to provide a method for preparing a magnetic nanocomposite formulation with multi-enzyme activity and its application. This invention utilizes a continuous in-situ growth method and surface modification to coat the surface of magnetic nanoparticles with a nanozyme possessing dual-enzyme activity (superoxide dismutase and catalase), and further modifies it with a nitric oxide donor material. The magnetic composite formulation prepared by this invention exhibits uniform size and high magnetic saturation strength, allowing the nanoparticles to achieve directional and controllable movement under an external magnetic field. Furthermore, the nanoparticles possess multi-enzyme activity resembling superoxide dismutase, catalase, and nitric oxide synthase, resulting in significantly enhanced enzyme catalytic performance. The magnetic nanocomposite formulation with multi-enzyme activity prepared by this invention not only possesses good biocompatibility but also rapidly responds to external magnetic fields, enabling deep tissue penetration and directional and controllable movement, improving the delivery efficiency of nanozymes and providing sustained release of therapeutic gases. This allows for efficient repair of wounds, particularly chronic diabetic wounds. Thus, it solves the technical problems of poor tissue penetration and low bioavailability of nanozymes in existing technologies.

[0004] According to a first aspect of the present invention, a method for preparing a magnetic nanocomposite formulation with multi-enzyme activity is provided, comprising the following steps: (1) Prepare magnetic nanozymes using method one or method two; Method 1: Magnetic nanoparticles are immersed in a solution of a nanozyme precursor that simultaneously possesses superoxide dismutase and catalase activities. The nanozyme precursor is a metal salt, and the nanozyme precursor solution also contains an oxidant, forming a complex of magnetic nanoparticles and nanozyme precursor. The complex is collected and dried, and then calcined under a non-oxidizing protective atmosphere to obtain a magnetic nanozyme with superoxide dismutase and catalase activities. Method 2: Magnetic nanoparticles are immersed in a solution of a nanozyme precursor that has both superoxide dismutase and catalase activities. The nanozyme precursor is a metal salt and an organic ligand, forming a complex of magnetic nanoparticles and nanozyme precursor. The complex is collected and dried to obtain a magnetic nanozyme with superoxide dismutase and catalase activities. (2) Mix the magnetic nanozyme obtained in step (1) with an amino-containing modifier or a hydroxyl-containing modifier evenly to modify the surface of the magnetic nanozyme and obtain the modified magnetic nanozyme. (3) Disperse the modified magnetic nanozyme obtained in step (2) in water and add nitric oxide donor material. The nitric oxide donor material is grafted onto the surface of the modified magnetic nanozyme to obtain a magnetic nanocomposite preparation with multi-enzyme activity.

[0005] Preferably, in step (1) of method one, the metal salt is a manganese salt, cerium salt, cobalt salt or copper salt, and the magnetic nanoenzyme is a manganese oxide, cerium oxide, cobalt oxide or copper oxide; In step (1) of method two, the metal salt is a manganese salt, cerium salt, cobalt salt or copper salt, and the magnetic nanozyme is a metal-organic framework formed by manganese salt, cerium salt, cobalt salt or copper salt and organic ligand. Preferably, the organic ligand is an imidazole ligand, a carboxylic acid ligand, or a nitrogen-containing heterocyclic ligand.

[0006] Preferably, the magnetic nanoparticles are iron(III) oxide, iron(II) oxide, chromium(II) oxide, rubidium-iron-boron oxide, cobalt(III) oxide, and Fe. 12 O 19 Any of the following in Sr.

[0007] Preferably, the calcination temperature is 200-500℃ and the calcination time is 2-6 h.

[0008] Preferably, the modifier is polydopamine, tannic acid, gallic acid, a coupling agent for EDC and NHS, or a silane coupling agent.

[0009] Preferably, the nitric oxide donor material is S-nitroso-N-acetylpenicillamine, S-nitrosocysteine, L-arginine, or an N-diazepine diol containing a carboxyl group.

[0010] According to another aspect of the present invention, a magnetic nanocomposite formulation with multi-enzyme activity is provided.

[0011] According to another aspect of the present invention, the application of the aforementioned magnetic nanocomposite formulation with multi-enzyme activity as a tissue penetrant in the preparation of wound repair drugs is provided.

[0012] Preferably, the application is performed under an external magnetic field with an intensity of 10-40 millitalas.

[0013] Preferably, the wound is a diabetic chronic wound.

[0014] In summary, the technical solutions conceived in this invention have the following main advantages compared with the prior art: (1) This invention constructs a novel nanoenzyme formulation with magnetic drive properties through in-situ growth and surface modification. This method not only achieves stable composite of magnetic components and enzyme-like materials, but also endows the composite formulation with the ability to respond rapidly and sensitively to external magnetic fields. Compared with the traditional passive diffusion drug delivery method, this composite formulation can achieve efficient and precise delivery of nanoenzymes and therapeutic gases to the target location under the guidance of a magnetic field, significantly improving drug utilization.

[0015] (2) The compound formulation of this invention has good biocompatibility and possesses multiple enzyme activities, including superoxide dismutase, catalase, and nitric oxide synthase. At the wound site, it can effectively remove ROS through cascade catalytic activity, releasing O2 and NO. These synergistic effects can effectively reverse oxidative damage-induced cell dysfunction, regulate inflammatory responses, and promote angiogenesis, thus making it applicable to the repair of chronic diabetic wounds.

[0016] (3) Under the control of an external magnetic field, the composite formulation of the present invention can achieve rapid enrichment in the target area, significantly increasing the local concentration of nanozymes at the lesion. At the same time, the magnetic driving effect can promote the penetration of nanozymes into the deep tissue, overcome the obstacles to drug delivery caused by the heterogeneous environment and dense matrix commonly found in chronic wounds, and thus act more effectively on deep infection or hypoxic areas, improving the therapeutic effect.

[0017] (4) Preferably, the magnetic nanoparticles can grow various dual-enzyme active materials in situ on the surface, such as manganese oxide, cerium oxide, cobalt oxide or metal-organic framework. Attached Figure Description

[0018] Figure 1 Transmission electron microscopy image of the magnetic nanocomposite formulation with multiple enzyme activities prepared in Example 1.

[0019] Figure 2The particle size distribution curve and surface Zeta potential of the magnetic nanocomposite formulation with multiple enzyme activities prepared in Example 1 are shown.

[0020] Figure 3 The hysteresis loop of the magnetic nanocomposite preparation with multiple enzyme activities prepared in Example 2.

[0021] Figure 4 The image shows a scanning electron microscope (SEM) image of the magnetic nanocomposite formulation with multiple enzyme activities prepared in Example 3.

[0022] Figure 5 Transmission electron microscopy image of the magnetic nanocomposite formulation with multiple enzyme activities prepared in Example 4.

[0023] Figure 6 The controllable trajectory change (243 s) of the magnetic nanocomposite formulation with multiple enzyme activities prepared in Example 1 under a gradient magnetic field of 15 mT.

[0024] Figure 7 The magnetic nanocomposite formulation with multiple enzyme activities prepared in Example 1 was tested with / without an external gradient magnetic field ( B) Digital photograph of penetration through the agarose gel (110 s). The white dashed line indicates the edge of the agarose gel. Scale bar: 100 μm.

[0025] Figure 8 The image shows the gas release curve of the magnetic nanocomposite formulation with multiple enzyme activities prepared in Example 1.

[0026] Figure 9 This is a biocompatibility diagram of the magnetic nanocomposite formulation with multiple enzyme activities prepared in Example 4.

[0027] Figure 10 Example 5 describes the preparation of a magnetic nanocomposite formulation with multiple enzyme activities for testing the healing performance of chronic diabetic wounds. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0029] The magnetic nanocomposite formulation with multiple enzyme activities prepared in this invention comprises the following four components: (1) A magnetic drive unit with magnetic control performance can be achieved by applying a gradient magnetic field; (2) Nanozymes; (3) Modifier; (4) Nitric oxide donor.

[0030] This invention provides a method for preparing a magnetic nanocomposite formulation with multiple enzyme activities, comprising the following steps: (1) Magnetic nanoparticles were immersed in a nanozyme precursor solution with dual enzyme activity, which simultaneously has superoxide dismutase and catalase activity. The mixture was heated and stirred, collected by an external magnet, dried, and then calcined in a tube furnace under a nitrogen atmosphere to obtain magnetic nanozymes with dual enzyme activity. (2) Mix the magnetic nanomaterials obtained in step (1) with the modifier evenly and stir for several hours to obtain surface-modified magnetic nanoenzymes, which provide abundant functional groups for subsequent grafting of nitric oxide donor materials. (3) The material obtained in step (2) is redispersed in water and mixed with the nitric oxide donor material to obtain a magnetic nanocomposite preparation with multi-enzyme activity.

[0031] In some embodiments, in step (1), the magnetic nanoparticles are iron(III) oxide, cobalt(III) oxide, rubidium iron boron, ferric oxide, chromium dioxide, or Fe. 12 O 19 At least one of Sr; In some embodiments, the magnetic nanoparticles have a particle size of 50 nm-1 μm.

[0032] In some embodiments, in step (1), the nanozyme with dual enzyme activity is manganese oxide, cerium oxide, cobalt oxide or metal-organic framework; the concentration of the nanozyme precursor solution is 10-50 mg / mL; the mass ratio of the magnetic nanoparticles to the nanozyme precursor is (0.1-0.9):1; and the mass ratio of the magnetic nanoparticles to the deionized water is 1:(100-300).

[0033] In some embodiments, in step (1), the heating temperature is 45-75℃, the stirring time is 2-8 h, the stirring speed is 100-400 rpm, the drying temperature is 20-60℃, the calcination temperature is 200-500℃, and the calcination time is 2-6 h.

[0034] In some embodiments, in step (2), the modifier introduced onto the surface of the magnetic nanoenzyme is polydopamine, tannic acid, gallic acid and its derivatives, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) coupling agent, or a silane coupling agent. The mass ratio of the magnetic nanoenzyme to deionized water is (1-10):1; the stirring speed is 100-400 rpm, and the stirring time is 6-12 h.

[0035] In some embodiments, in step (3), the outermost grafted nitric oxide donor material is: S-nitroso-N-acetylpenicillamine, S-nitrosocysteine ​​(CysNO), L-arginine, or an N-diazepinel derivative with a carboxyl group. The mass ratio of the modified magnetic nanozyme to the nitric oxide donor is 1:(10-30); the mass ratio of the modified magnetic nanozyme to the deionized water is (1-10):1, and the stirring speed is 100-400 rpm.

[0036] The magnetic nanoparticles prepared by this invention have multiple enzyme activities, with a nanoenzyme content of 20-40% and a nitric oxide donor content of 10-20%.

[0037] The magnetic nanoparticles with multiple enzyme activities prepared in this invention are used as tissue permeators in the preparation of wound repair drugs.

[0038] In some embodiments, the application is performed under an external magnetic field; the strength of the external magnetic field is 0.1-40 millitalas; and the speed can reach 20-25 μm / s when the magnetic field frequency is 30 millitalas.

[0039] In some embodiments, the motion control of the magnetically driven unit with magnetic control performance under a gradient magnetic field is generated by a permanent magnet, the direction of the magnetic field is adjusted by controlling the position of the permanent magnet, and the magnetic field strength is preferably 10-40 mT.

[0040] The following are specific examples.

[0041] Example 1 The specific steps for preparing magnetic nanocomposite formulations with multiple enzyme activities are as follows: (1) Preparation of magnetic cerium oxide nanozymes: 2.4 mmol of cerium nitrate hexahydrate and 25 mmol of sodium hydroxide were dissolved in 20 mL of ethanol and stirred at 50 °C for 20 h. Then, 50 μL of 30% H₂O₂ was added to the solution, and the mixture was stirred at room temperature for 2 h. The product was then lyophilized and collected. Next, 1 g of the precipitate was completely dissolved in 20 mL of deionized water, the pH was adjusted to 0.1–0.3, and the mixture was stirred for 2 h to obtain a Ceria precursor solution. Then, 0.1 g of Fe₃O₄, 30 mL of deionized water, and 3 mL of the Ceria precursor solution were mixed and stirred. The pH was then adjusted to neutral with ammonium hydroxide solution, and the mixture was stirred at 60 °C for 4 h. After the reaction was complete, the product was lyophilized and collected. It was then heated at 300 °C for 3 h under a nitrogen atmosphere to obtain the final product Fe₃O₄@CeO₂. In this process, the hydroxyl groups on the CeO2 surface form chemical adsorption and interfacial bonding with the hydroxyl groups on the Fe3O4, thereby generating a stable heterojunction structure. CeO2 is uniformly coated on the surface of Fe3O4 nanoparticles, forming a tight interfacial contact, which is conducive to the transfer of electrons / holes between Fe3O4 and CeO2, and improves the catalytic activity and stability of the composite material.

[0042] (2) Preparation of functionalized magnetic cerium oxide nanozymes: The product obtained in step (1) and dopamine hydrochloride were dissolved in Tris buffer solution (pH 8.4, 0.01 M) and stirred for 4 h. After the reaction was completed, the solid product was collected by magnetic separation and washed three times with deionized water to remove unreacted substances. After freeze-drying, functionalized magnetic cerium oxide nanozyme Fe3O4@CeO2 was obtained. The mass ratio of the product obtained in step (1) to dopamine hydrochloride was 1:0.5, and the mass ratio of the product to deionized water was 1:0.5.

[0043] (3) Preparation of magnetic nanoparticles with multi-enzyme activity: The product obtained in step (2) and L-arginine were dissolved in deionized water and stirred for 12 h to obtain a magnetic nanoparticle formulation Fe3O4@CeO2@PDA@L-Arg (FCPA) with multi-enzyme activity. The ratio of the product obtained in step (2) to L-arginine was 1:10, and the ratio of L-arginine to deionized water was 1:1.

[0044] Figure 1 To prepare the magnetic nanozyme, after surface functionalization and grafting with nitric oxide donor, a transmission electron microscopy image of a magnetic nanocomposite formulation with multi-enzyme activity was obtained, as shown in the image. Figure 2As shown in the figure. The results indicate that magnetic nanocomposite formulations were successfully obtained through in-situ growth and surface modification, with an average microsphere diameter of 220 nm. Analysis of the zeta potential of the magnetic nanoparticles before and after surface modification revealed a surface zeta potential of -32.5 mV before modification, 26.7 mV after modification with the modifier, and -19.2 mV after grafting with a nitric oxide donor.

[0045] Example 2 The specific steps for preparing magnetic nanocomposite formulations with multiple enzyme activities are as follows: Step (1) differs from Example 1 in that: (1) The magnetic nanoparticles used were cobalt tetroxide magnetic particles, which were purchased from Shanghai Maclean Biochemical Technology Co., Ltd. (product name: T799188). Before use, they were rinsed three times with deionized water. Then, magnetic nanozymes were prepared on the surface of magnetic cobalt tetroxide using the same method by co-precipitation. The magnetic nanoparticles were modified with dopamine on the surface using the same method (2). L-arginine was grafted onto the magnetic nanoparticles as a nitric oxide donor using the same method (3), which will not be described in detail here. Figure 3 The magnetic hysteresis loop of the prepared magnetic nanocomposite formulation with multiple enzyme activities is shown. The results show that the saturation magnetization of the magnetic nanocomposite formulation is 7.8 emu / g and the coercivity is zero, which proves the superparamagnetism of the sample. This provides support for the controllable start-up and shutdown and magnetic recovery of the magnetic nanocomposite formulation under a magnetic field.

[0046] Example 3 The specific steps for preparing magnetic nanocomposite formulations with multiple enzyme activities are as follows: Step (1) differs from Example 1 in that: (1) 12.6 mg of copper sulfate pentahydrate and 5.9 mg of sodium chloride were dissolved in 100 mL of deionized water and ultrasonically stirred until homogeneous. Then, 50 mg of Fe3O4 magnetic nanoparticles were weighed and added to the above precursor solution and ultrasonically dispersed for 15 min. 1 mL of 100 mM 4,4'-bipyridine ethanol solution was added dropwise to the resulting dispersion, and the mixture was stirred at 400 rpm for 1 h at room temperature to allow Cu-MOF to grow in situ on the Fe3O4 surface. After vacuum drying at 60 °C, Fe3O4@Cu-MOF was obtained. The same method (2) was used to perform surface dopamine functionalization modification on the magnetic nanoparticles, and the same method (3) was used to graft L-arginine as a nitric oxide donor, which will not be described in detail here.

[0047] Figure 4 Scanning electron microscope (SEM) image of the prepared multi-enzyme active magnetic nanocomposite Fe3O4@Cu-MOF@L-Arg.

[0048] Example 4 The specific steps for preparing magnetic nanocomposite formulations with multiple enzyme activities are as follows: Step (1) differs from Example 1 in that: (1) 2 g of cobalt tetroxide, 1.48 g of manganese chloride tetrahydrate and 0.5 g of polyvinylpyrrolidone were dissolved in 100 mL of deionized water and heated at 70 °C with stirring until homogeneous. Then 2.5 mL of 0.2 M potassium permanganate was added and the mixture was allowed to precipitate for 2 h. Finally, the mixture was washed three times by centrifugation with deionized water and ethanol respectively, and dried under vacuum at 60 °C to obtain Fe3O4@MnO2.

[0049] (2) The product obtained in step (1) and tannic acid were dissolved in Tris-HCl buffer solution (pH 8.4, 0.01M) and stirred for 12 h. After the reaction was completed, the solid product was collected by magnetic separation and washed three times with deionized water to remove unreacted substances. After freeze-drying, functionalized magnetic manganese oxide nanozymes were obtained. The mass ratio of the product obtained in step (1) to tannic acid was 1:0.8, and the mass ratio of the product to deionized water was 1:0.5. Subsequently, the same method was used to prepare (3) a magnetic nanocomposite preparation with multi-enzyme activity, which will not be described in detail here.

[0050] Figure 5 Transmission electron microscopy (TEM) images of Fe3O4@MnO2@L-Arg (FML), a magnetic nanocomposite formulation with multi-enzyme activity, were obtained.

[0051] Example 5 The specific steps for preparing magnetic nanocomposite formulations with multiple enzyme activities are as follows: Step (1) is the same as in Example 1, and will not be repeated here.

[0052] Steps (2) and (3) differ from those in Example 1 in that: (2) 100 mg of the magnetic nanozyme Fe3O4@CeO2 synthesized in step (1) was dispersed in 50 mL of MES buffer solution (pH 4.5 0.1 M) and stirred for 20 min. Then, EDC and NHS were added sequentially and stirred at 37 °C for 30 min for activation. The mass ratio of EDC to NHS was 1:1.

[0053] (3) Finally, S-nitroso-N-acetylpenicillamine (SNAP) was added to the activated solution. The mass ratio of S-nitroso-N-acetylpenicillamine to Fe3O4@CeO2 was 1:5. The reaction was carried out at room temperature in the dark for 8 h. After the coupling reaction was completed, glycine at a final concentration of 15 mM was added to quench the reaction. The mixture was washed 2-3 times with deionized water and then freeze-dried to obtain the magnetic nanocomposite preparation Fe3O4@CeO2@SNAP (FCS) with multiple enzyme activities.

[0054] Test Example 1 The magnetic drive performance of the magnetic nanocomposite formulation with multiple enzyme activities obtained in Example 1 was characterized by the following method: A polymethyl methacrylate (PMMA) ring with a radius R = 10 mm and a height H = 1 mm was fixed onto a microscope slide using two-component epoxy resin to construct a sample cell. 360 μL of a 0.01 mg / mL magnetic nanoparticle suspension was added to the sample cell, and after covering with a coverslip, the motion behavior of the nanomotors was observed under a gradient magnetic field. The external magnetic field was generated by a permanent magnet placed on one side of the sample cell, with a magnetic field strength of 15 mT at the center. The nanomotors were observed using a Carl Zeiss AG Axioscope 5 optical microscope, and video was recorded using an Axiocam 208 color camera to record their trajectories. The motion trajectory and velocity of the nanomotors were analyzed using image analysis software Tracker and ImageJ, as well as a self-written Python program.

[0055] Figure 6 The results of the magnetic drive performance experiment of the magnetic nanoparticles with multiple enzyme activities obtained in Example 1 show that the magnetic nanoparticles can achieve directional drive under a gradient magnetic field with a magnetic field strength of 15 mT.

[0056] Test Example 2 The magnetic agarose gelation properties of the magnetic nanocomposite formulation with multiple enzyme activities obtained in Example 1 were tested, and the specific steps are as follows: In vitro agarose gel permeation experiment: First, a 0.2% agarose gel was placed on one side of a sample cell with a radius R = 10 mm and a height H = 1 mm, filling one-third of the total sample cell volume. The remaining cavity was filled with PBS solution. FITC-labeled magnetic nanoparticles with multiple enzyme activities were pipetted into the sample cell at the end furthest from the agarose gel. An external permanent magnet was aligned with the agarose gel on the other side of the cavity. After incubation for 30 min, the sample cell was transferred to a confocal microscope (Olympus, FV1200, Japan) for quantitative analysis of the FITC-labeled magnetic nanoparticles with multiple enzyme activities permeated into the gel. Figure 7 As shown, under the induction of an external gradient magnetic field, FITC-labeled magnetic nanoparticles with various enzyme activities effectively penetrated into the agarose gel within 5 minutes.

[0057] Test Example 3 The gas-release performance of the magnetic nanocomposite formulation with multiple enzyme activities obtained in Example 2 was tested, and the specific steps are as follows: (1) Detection of nitric oxide: Prepare a series of 1 mL 5% H2O2 solutions, and then add 200 μL 1 mg / mL Co3O4@CeO2@PDA@L-Arg to them. Take samples at different time points and use a nitric oxide detection kit to detect the nitric oxide produced at different time points and plot the curve.

[0058] (2) Dissolved oxygen detection: The dissolved oxygen generated at different time points during (1) reaction was detected using a JPBJ-608 portable dissolved oxygen meter, and a curve was plotted.

[0059] Figure 8 The image shows the gas release of the magnetic nanocomposite formulation with multiple enzyme activities prepared in Example 2. The results indicate that the magnetic nanocomposite formulation can continuously release nitric oxide and oxygen within 72 h.

[0060] Test Example 4 The cytotoxicity analysis of the magnetic nanocomposite formulation with multiple enzyme activities prepared in Example 4 was performed as follows: Endothelial cells and fibroblasts were seeded into 96-well plates (3K / well) and cultured for 8–12 h. They were then co-cultured with different concentrations of the magnetic nanoparticle formulation FML (0, 50, 100, 150, 200, and 250 μg / mL). After 48 h, cytotoxicity was measured using a CCK8 assay kit (HYCEZMBIO, China) following the manufacturer's instructions. The absorbance of the solution at 450 nm was recorded using a microplate reader (TECAN, Switzerland). Cell viability was calculated using the following formula: Cell viability (%) = (Net OD value of experimental group / Net OD value of control group) × 100% like Figure 9 As shown, the multi-enzyme-active magnetic nanocomposite formulation prepared in Example 4 has good biocompatibility with endothelial cells.

[0061] Test Example 5 The magnetic nanocomposite formulation with multiple enzyme activities prepared in Example 5 was subjected to a chronic wound healing test. The specific steps are as follows: In the initial stage of the experiment, 8-week-old male C57BL / 6 mice were treated with intraperitoneal injection of streptozotocin (STZ, 50 mg / kg) for 5 days to induce diabetic mice, and a full-thickness skin wound was formed after 4 weeks. The mice were then randomly divided into 5 groups (n = 10 per group): control group (I), Fe3O4@CeO2 group (II), Fe3O4@CeO2+magnetic field group (III), FCS group (IV), and FCS+magnetic field group (V). On the day of the experiment and surgery, diabetic mice were anesthetized with sodium pentobarbital (Thermo Altair) (1%, 50 mg / kg). After shaving and disinfection, a full-thickness excision wound with a diameter of 8 mm was made on the back of all mice. Treatment was then administered after the establishment of the infected wound model and on days 0, 3, 7, and 10. Digital photographs were taken on days 0 and 14, and the wound area was measured using ImageJ software. Figure 10 As shown, FCS can efficiently repair chronic wounds under magnetic field induction, with a healing rate of over 95%.

[0062] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a magnetic nanocomposite formulation with multi-enzyme activity, characterized in that, Includes the following steps: (1) Prepare magnetic nanozymes using method one or method two; Method 1: Magnetic nanoparticles are immersed in a solution of a nanozyme precursor that simultaneously possesses superoxide dismutase and catalase activities. The nanozyme precursor is a metal salt, and the nanozyme precursor solution also contains an oxidant, forming a complex of magnetic nanoparticles and nanozyme precursor. The complex is collected and dried, and then calcined under a non-oxidizing protective atmosphere to obtain a magnetic nanozyme with superoxide dismutase and catalase activities. Method 2: Magnetic nanoparticles are immersed in a solution of a nanozyme precursor that has both superoxide dismutase and catalase activities. The nanozyme precursor is a metal salt and an organic ligand, forming a complex of magnetic nanoparticles and nanozyme precursor. The complex is collected and dried to obtain a magnetic nanozyme with superoxide dismutase and catalase activities. (2) Mix the magnetic nanozyme obtained in step (1) with an amino-containing modifier or a hydroxyl-containing modifier evenly to modify the surface of the magnetic nanozyme and obtain the modified magnetic nanozyme. (3) Disperse the modified magnetic nanozyme obtained in step (2) in water and add nitric oxide donor material. The nitric oxide donor material is grafted onto the surface of the modified magnetic nanozyme to obtain a magnetic nanocomposite preparation with multi-enzyme activity.

2. The method for preparing the magnetic nanocomposite formulation with multi-enzyme activity as described in claim 1, characterized in that, In step (1) of method one, the metal salt is a manganese salt, cerium salt, cobalt salt or copper salt, and the magnetic nanozyme is a manganese oxide, cerium oxide, cobalt oxide or copper oxide; In step (1) of method two, the metal salt is a manganese salt, cerium salt, cobalt salt or copper salt, and the magnetic nanozyme is a metal-organic framework formed by manganese salt, cerium salt, cobalt salt or copper salt and organic ligand. Preferably, the organic ligand is an imidazole ligand, a carboxylic acid ligand, or a nitrogen-containing heterocyclic ligand.

3. The method for preparing the magnetic nanocomposite formulation with multi-enzyme activity as described in claim 1 or 2, characterized in that, The magnetic nanoparticles are iron(III) oxide, iron(II) oxide, chromium(II) oxide, neodymium iron boron, cobalt(III) oxide, and Fe. 12 O 19 Any of the following in Sr.

4. The method for preparing the magnetic nanocomposite formulation with multi-enzyme activity as described in claim 1, characterized in that, The calcination temperature is 200-500℃, and the calcination time is 2-6 h.

5. The method for preparing the magnetic nanocomposite formulation with multi-enzyme activity as described in claim 1, characterized in that, The modifier is polydopamine, tannic acid, gallic acid, a coupling agent for EDC and NHS, or a silane coupling agent.

6. The method for preparing the magnetic nanocomposite formulation with multi-enzyme activity as described in claim 1, characterized in that, The nitric oxide donor material is S-nitroso-N-acetylpenicillamine, S-nitrosocysteine, L-arginine, or an N-diazepine diol containing a carboxyl group.

7. The magnetic nanocomposite preparation with multi-enzyme activity obtained by any one of claims 1-6.

8. The application of the magnetic nanocomposite formulation with multi-enzyme activity as described in claim 7 as a tissue penetrant in the preparation of wound repair drugs.

9. The application as described in claim 8, characterized in that, The application is performed under an external magnetic field with an intensity of 10-40 millitalas.

10. The application as described in claim 8 or 9, characterized in that, The wound in question is a chronic diabetic wound.

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