Preparation and application of annexin modified FeSe diatomic nano-enzyme

Abstract

The invention belongs to the field of nano-enzyme preparation and biological application, and particularly relates to preparation and application of annexin modified FeSe diatomic nano-enzyme. A Fe-Se double-active-center interconnected porous nitrogen-doped polyhedral carbon structure is synthesized, ANXA5 protein is loaded, and the double-active-center nano enzyme Fe-Se / A5 is formed. The Fe-Se / A5 diatomic nano-enzyme makes up the deficiency of the activity of a monatomic nano-enzyme, the loaded protein enhances the targeting property and the acting effect of the nano-enzyme, significantly enhances and optimizes the mimic activity of POD, CAT, GPx and other multienzymes, has good photo-thermal performance, can efficiently generate a large amount of ROS in a tumor microenvironment, and promotes the apoptosis of tumor cells. The invention provides a new strategy for regulating and controlling the tumor microenvironment by the composite nano-enzyme, generating ROS in cooperation with photothermal therapy and promoting tumor cell apoptosis.

Description

Technical Field

[0001] The present invention belongs to the field of nanozyme preparation and biological application, and specifically relates to the preparation and application of an annexin-modified FeSe diatomic nanozyme. Background Art

[0002] Osteosarcoma is a highly malignant primary bone tumor that originates from mesenchymal cells and is characterized by the direct formation of immature bone or bone-like tissue from tumor cells. It is predominantly diagnosed in adolescents and is currently treated primarily with surgical resection supplemented with chemotherapy and radiotherapy. These treatments are associated with poor prognosis and a high risk of recurrence. Research on utilizing reactive oxygen species (ROS) generated by the tumor microenvironment to kill tumor cells has recently attracted considerable attention. Single-atom nanozymes (SAzymes), due to their unique geometric structure and electron distribution, possess excellent enzyme-like activity. They can exploit the unique environment of the tumor microenvironment to generate large amounts of ROS, promoting tumor cell apoptosis, and hold great research value. Iron single-atom nanozymes (Fe SAzymes) have been extensively studied. They possess multi-enzyme mimetic activities, such as peroxidase (POD) and catalase (CAT). They can generate ROS in the tumor microenvironment, killing tumor cells and promoting apoptosis. Fe SAzymes have been reported in the literature as peroxidase mimics or by consuming reduced glutathione for tumor catalytic therapy.

[0003] Chinese invention patent application CN117797271A (publication date: April 2, 2024) provides a tumor-targeting peptide-modified iron single-atom nanozyme preparation and its preparation method, comprising the following steps: preparing an iron single-atom nanozyme, and dissolving the iron single-atom nanozyme and cyclic peptide c (RGDfK) in deionized water, stirring for 12 to 36 hours to obtain a mixed solution; after centrifuging and washing the above mixed solution, taking the precipitate and drying it at 50 to 70°C to obtain it. The tumor-targeting peptide-modified iron single-atom nanozyme prepared by this invention has the ability to respond to the tumor microenvironment to produce efficient chemodynamic therapy, thereby achieving PDT treatment of tumors; the nanoformulation of this invention also has MRI tumor imaging performance, which plays an important role in tumor diagnosis and treatment. In addition, by combining with immune checkpoint inhibitors, it can also induce ICD to enhance tumor immunogenicity, further enhancing the effect of tumor immunotherapy. The preparation of this invention can achieve chemodynamic therapy, activation of anti-tumor immunogenicity and integrated diagnosis and treatment.

[0004] Although the existing iron single-atom nanozymes have a certain degree of targeting, they have a single active site, poor stability of the active center, easy inactivation, aggregation or redox drift of the Fe site, and lack of synergy. They have a wide range of effects in the body and are difficult to accurately target tumor cells. They may cause serious side effects and adverse reactions, and therefore have limited therapeutic effects on osteosarcoma. Compared with single metal oxides, double metal oxides have richer and more diverse properties, which makes them show great vitality in the biomedical field. The double-atom structure provides multiple active sites, which can enhance catalytic efficiency to a certain extent, and has multiple enzyme-mimicking activities, which can achieve synergistic effects of multiple therapeutic mechanisms. Summary of the Invention

[0005] In view of this, this application innovatively proposes a research plan to construct a membrane-bound protein A5 modified Fe-Se diatomic nanozyme strategy for the treatment of osteosarcoma. By synthesizing a diatomic nanozyme with a three-dimensional interconnected porous nitrogen-doped polyhedral carbon (Fe-Se-NC SA) structure with atomically dispersed Fe-Se dual active centers, the enzymatic activity of the single-atom nanozyme is improved, and then the membrane-bound protein A5 (ANXA5) is loaded on the Fe-Se-NC SA to enhance the targeting of the nanozyme. Then, its excellent POD, CAT and glutathione peroxide (GPx) enzyme activities are utilized to cooperate with photothermal therapy to promote tumor cell apoptosis, thereby achieving excellent osteosarcoma treatment effects.

[0006] The present invention provides a preparation and application of an annexin-modified FeSe diatomic nanozyme. The specific technical solution is described as follows:

[0007] A membrane-modified FeSe diatomic nanozyme, wherein the FeSe diatomic nanozyme is a three-dimensional interconnected porous nitrogen-doped polyhedral carbon (Fe-Se-NC SA) with atomically dispersed Fe-Se dual active centers, and the membrane-modified FeSe diatomic nanozyme is loaded with annexin A5;

[0008] The membrane-annexin-modified FeSe diatomic nanozyme can specifically bind to phosphatidylserine (PS) on the surface of tumor cells.

[0009] A method for preparing an annexin-modified FeSe diatomic nanozyme comprises the following steps:

[0010] Step S1: Prepare solution A and solution B respectively, wherein:

[0011] Solution A is prepared by mixing zinc nitrate hexahydrate, ferric acetylacetonate and methanol and stirring evenly;

[0012] Solution B was prepared by mixing 2-methylimidazole and methanol and stirring evenly;

[0013] Step S2: 40-80 ml of solution A prepared in step S1 was mixed with 40-80 ml of solution B, stirred, centrifuged, washed 2-5 times, and dried to obtain ZIF8@Fe(acac)3;

[0014] Step S3: The ZIF8@Fe(acac)3 prepared in step S2 is placed in an Ar atmosphere, heated to 600-1000°C for heat treatment, pickled with HNO3 in an oil bath at 25-60°C until neutral, and dried to obtain Fe-NC;

[0015] Step S4: Se powder is placed on the side of a square porcelain boat where air flows in, and Fe-NC prepared in step S3 is placed on the side where air flows out of the boat. The mixture is heated to 400-600°C at a rate of 2°C / min in an Ar atmosphere to obtain FeSe-NC.

[0016] Step S5: ANXA5 is mixed with the FeSe-NC prepared in step S4 using a liposome extruder. The ANXA5 protein is attached to the surface of the nanomaterial by repeated extrusion. The mixture is then centrifuged in a high-speed centrifuge for 20 to 30 minutes, washed 3 to 5 times, and freeze-dried in a freeze dryer to obtain Fe-Se / A5.

[0017] Furthermore, in step S1, the mass ratio of zinc nitrate hexahydrate to ferric acetylacetonate in preparing solution A is 10:1 to 50:1, and the mass-volume ratio of zinc nitrate hexahydrate to methanol is 1 g:10 to 20 ml;

[0018] Furthermore, in step S1, the mass-volume ratio of 2-methylimidazole to methanol for preparing solution B is 1 g: 5-30 mL;

[0019] Furthermore, in step S2, the mixing and stirring time of solution A and solution B is 2 to 10 minutes;

[0020] Furthermore, in step S3, the heating rate is 5°C / min;

[0021] Furthermore, in step S4, the mass ratio of Se powder to Fe-NC is 2:1 to 5:1;

[0022] Furthermore, in step S5, the high-speed centrifugal speed is 8000-10000 r / min.

[0023] This application also proposes the use of the membrane-attached protein modified FeSe diatomic nanozyme as a catalase in the treatment of osteosarcoma.

[0024] Compared with the prior art, the advantages and effects of the present invention are as follows:

[0025] 1. The present application provides a membrane-attached protein modified FeSe diatomic nanozyme. The dual active centers make up for and improve the deficiencies of the single-atom nanozyme activity. Compared with the iron single-atom nanozyme, the catalase-like activity is increased by 50% to 80%, and the peroxidase-like activity is increased by 50% to 60%; the nanozyme also has good photothermal properties and has a killing effect on tumor cells. After adding near-infrared, the killing effect on tumor cells is significantly enhanced. At a specific concentration, the killing effect of FeSe / A5 with near-infrared is 100% to 200% higher than that of FeSe / A5.

[0026] 2. The present application provides a membrane-attached protein-modified FeSe diatomic nanozyme. The loaded protein enhances the targeting of the nanozyme, enhances the accuracy of the nanozyme's action and also improves its safety.

[0027] 3. The present application provides an application of a membrane-attached protein-modified FeSe diatomic nanozyme, which enhances and optimizes the activity of enzymes such as POD, CAT and glutathione peroxide (GPx), and provides a new strategy for the composite nanozyme to regulate the tumor microenvironment, synergize with photothermal therapy to produce ROS (which can increase ROS by 70% to 200%), and promote tumor cell apoptosis; especially for inhibiting the proliferation of osteosarcoma tumor cells, under NIR irradiation, its proliferation inhibition effect is increased by 80% to 200%.

[0028] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application so that it can be implemented in accordance with the contents of the specification, and to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the following is a detailed description of the preferred embodiment of the present application in conjunction with the accompanying drawings.

[0029] Based on the detailed description of the specific embodiments of the present application in conjunction with the accompanying drawings below, those skilled in the art will become more aware of the above and other objects, advantages and features of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present application. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without inventive work. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0031] Figure 1 This is a flow chart for the preparation of the Fe-Se / A5 nanozyme prepared in the present invention;

[0032] Figure 2 This is an image of the Fe-Se / A5 nanozyme prepared in the present invention, wherein:

[0033] Figure 2 (a) is a scanning electron microscopy (SEM) image of the Fe-Se / A5 nanozyme prepared in the present invention;

[0034] Figure 2 (b) Transmission electron microscopy (TEM) image of the Fe-Se / A5 nanozyme prepared in the present invention;

[0035] Figure 2 (c) is the X-ray diffraction characterization diagram of the Fe-Se / A5 nanozyme prepared in the present invention;

[0036] Figure 2 (d) is the mapping characterization image of the Fe-Se / A5 nanozyme prepared in the present invention under transmission electron microscopy (TEM);

[0037] Figure 3 This is an X-ray energy spectrum elemental imaging (EDS-mapping) image of the Fe-Se / A5 nanozyme prepared in the present invention;

[0038] Figure 4 This is a graph evaluating the catalase-like activity of the Fe-Se / A5 nanozyme prepared in the present invention;

[0039] Figure 5 This is a graph evaluating the peroxidase activity of the Fe-Se / A5 nanozyme prepared in the present invention;

[0040] Figure 6 This is a graph evaluating the glutathione peroxidase activity of the Fe-Se / A5 nanozyme prepared in the present invention;

[0041] Figure 7 This is a diagram for evaluating the photothermal performance of the Fe-Se / A5 nanozyme prepared in the present invention;

[0042] Figure 8 This is a graph evaluating the ability of the Fe-Se / A5 nanozyme prepared in the present invention to kill 143B tumor cells;

[0043] Figure 9 This is an image of the Fe-Se / A5 nanozyme prepared by the present invention taking up targeted cells;

[0044] Figure 10 This is a graph showing the ability of the Fe-Se / A5 nanozyme prepared in the present invention to produce ROS in cells;

[0045] Figure 11 (a) is the Ki67 immunofluorescence image of the Fe-Se / A5 nanozyme prepared in the present invention after cell action:

[0046] Figure 11 (b) is a graph evaluating the Ki67 expression level of the Fe-Se / A5 nanozyme prepared in the present invention after cell action. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. In the following description, specific details such as specific configurations and components are provided only to help fully understand the embodiments of the present application. Therefore, it should be clear to those skilled in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. In addition, for clarity and brevity, the description of known functions and structures has been omitted in the embodiments.

[0048] It should be understood that references throughout this specification to "one embodiment" or "this embodiment" mean that a particular feature, structure, or characteristic associated with the embodiment is included in at least one embodiment of the present application. Therefore, the appearance of "one embodiment" or "this embodiment" throughout this specification does not necessarily refer to the same embodiment. Furthermore, these particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0049] In addition, the present application may repeat reference numerals and / or letters in different examples. This repetition is for the purpose of simplicity and clarity and does not in itself indicate the relationship between the various embodiments and / or settings discussed.

[0050] The term "and / or" in this article is only a description of the association relationship of associated objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist at the same time. The term " / and" in this article describes another type of association object relationship, indicating that two relationships can exist. For example, A / and B can mean: A exists alone, and A and B exist alone. In addition, the character " / " in this article generally indicates that the previous and subsequent associated objects are in an "or" relationship.

[0051] The term "at least one" in this article is merely a description of the association relationship between associated objects, indicating that three relationships may exist. For example, at least one of A and B can mean: A exists alone, A and B exist at the same time, and B exists alone.

[0052] It should also be noted that, in this document, relational terms such as first and second are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "include," "comprises," or any other variations thereof are intended to cover non-exclusive inclusion.

[0053] Example 1

[0054] This embodiment provides a method for preparing annexin-modified FeSe diatomic nanozyme, and the preparation process is as follows: Figure 1 As shown, the details are as follows:

[0055] Add 1-2 g zinc nitrate hexahydrate, 30-80 mg ferric acetylacetonate and 10-200 mL methanol to a beaker and stir evenly; at the same time, add 2-7 g 2-methylimidazole and 10-200 mL methanol to another beaker and stir evenly; stir the mixtures in the two beakers separately and mix them, continue stirring for 5-10 minutes; then let it stand for 12-36 hours, and then high-speed centrifuge to collect the precipitate; then add methanol to the precipitate, ultrasonicate for 10-20 minutes to fully disperse it, high-speed centrifuge for 10-30 minutes, and collect the precipitate; pour methanol again, ultrasonicate for 10-20 minutes to fully disperse it, high-speed centrifuge for 10-30 minutes, collect the precipitate, put it into an oven at 80°C, and dry it for 24-72 hours to obtain white powder ZIF8@Fe(acac)3;

[0056] Weigh the white powder ZIF8@Fe(acac)3 and put it into a tube furnace. Then, introduce argon gas and heat it to 800-1200℃ for 1-5h to obtain black powder Fe-NC.

[0057] Weigh an appropriate amount of black powder Fe-NC into a beaker, add HNO3 to dissolve it, and then centrifuge it in a high-speed centrifuge for 15 to 30 minutes. Discard the supernatant, add HNO3 to dissolve it again, repeat the above operation 2 to 3 times, collect the precipitate and dry it; weigh Se powder and Fe-NC with a mass ratio of 2:1 to 5:1, put Se powder and Fe-NC black powder into a porcelain boat and place it in a tube furnace, introduce argon into the tube furnace, heat it to 300 to 500 ° C and heat it for 2 to 8 hours to obtain FeSe-NC; then use a liposome extruder to load ANXA5 onto FeSe-NC, and finally obtain Fe-Se / A5 nanozyme. The sequence of the membrane annexin A5 is Met1-Asp320. As Figure 2As shown, scanning electron microscopy (SEM) observations of the nanomaterial's morphology, size, and structure reveal that the Fe-Se / A5 nanozyme is polyhedral with a densely porous surface, which facilitates the absorption of loaded proteins. Transmission electron microscopy (TEM) reveals that the nanozyme exhibits a hollow polyhedral structure and lacks nanoparticles. XRD analysis reveals that the synthesized diatomic nanozyme lacks the characteristic diffraction peaks of Fe and Se, indicating the absence of Fe, Se, and other metal particles. Elemental mapping reveals that C, N, Fe, and Se are uniformly distributed within the Fe-Se / A5 nanozyme.

[0058] like Figure 3 As shown in the EDS-mapping picture, the presence of C, N, Fe and Se elements can be seen, confirming the successful synthesis of the material.

[0059] The technical effect achieved by this embodiment is: This embodiment provides a method for preparing a membrane-annexin-modified FeSe diatomic nanozyme. By introducing selenium (Se) elements into Fe SAzyme, Se can regulate the electronic structure and microenvironment of Fe SAzyme through its hybrid orbital, increase the active sites of the active center of Fe SAzyme, and thus increase its enzyme-like activity.

[0060] Example 2

[0061] Based on Example 1, this example uses a dissolved oxygen meter to detect the catalase-like activity of nanozymes, such as Figure 4 As shown in the figure, among the three different nanozyme materials with the same concentration, FeSe / A5 produced the most oxygen in the same time and had the strongest catalase activity, with the enzyme activity increased by 50% to 80%, indicating that FeSe / A5 diatomic nanozyme has better catalase-like activity than iron single-atom nanozyme. Figure 5 As shown, the peroxidase activity test using TMB as a probe evaluated the nanozyme's ability to decompose hydrogen peroxide to produce ·OH. The highest absorption peak was at 652nm. Among the three different nanozyme materials with the same concentration, FeSe / A5 had the highest absorption peak at 652nm and the strongest ability to produce ·OH. The results showed that the FeSe / A5 diatomic nanozyme had superior peroxidase-like activity compared to the iron single-atom nanozyme, with enzyme activity increased by 50% to 60%.

[0062] Example 3

[0063] Based on Examples 1 and 2, this example uses DTNB as a probe to detect the residual glutathione content in the solution and verify the GPx-like enzyme biological activity of FeSe / A5. Figure 6From 0 to 8 minutes, glutathione consumption increased with the increase of FeSe / A5 reaction time, and the enzyme activity increased by 2% to 6% per minute. The results showed that FeSe / A5 has good GPx-like enzyme activity.

[0064] Example 4

[0065] Based on Examples 1 to 3, this example evaluated the photothermal performance of FeSe / A5 nanozyme.

[0066] Reference Figure 7 The nanozyme FeSe / A5 was exposed to near-infrared light at varying concentrations, and the temperature changes were recorded from 0 to 8 minutes. At the same concentration, the nanozyme's temperature gradually increased over time from 0 to 8 minutes. At the same time, the temperature of the FeSe / A5 nanozyme increased with increasing concentration at varying concentrations. These results demonstrate that the FeSe / A5 nanozyme exhibits excellent photothermal properties and is concentration-dependent.

[0067] Example 5

[0068] Based on Examples 1 to 4, this example evaluated the ability of FeSe / A5 nanozymes to kill 143B tumor cells. Figure 8 The biocompatibility of FeSe, FeSe / A5, and FeSe / A5 nanozymes was evaluated by co-culturing them with 143B cells for 24 hours under near-infrared irradiation. The same set of nanozymes exhibited toxicity to 143B cells within the experimental range of 0 to 150 μg / ml. As the concentration increased, the killing effect on tumor cells increased. As shown in the figure, at a concentration of 20 μg / ml, the killing effect of FeSe / A5 with near-infrared irradiation was 100% to 200% higher than that of FeSe / A5. These results demonstrate that the FeSe / A5 nanozyme has a tumor-killing effect, which is significantly enhanced by its targeted effect on tumor cells and the addition of near-infrared irradiation.

[0069] Example 6

[0070] Based on Examples 1 to 5, this example verifies the accuracy of Fe-Se / A5 nanozyme uptake of targeted cells. Figure 9 The 143B cytoskeleton was stained with microfilament green fluorescent dye, FeSe / A5 nanozyme was labeled with cy5 active dye, and the cell nucleus was stained with DAPI. The results showed that the 143B cytoskeleton was stained green, the cell nucleus was stained blue, and the red FeSe / A5 nanozyme was distributed in the cytoplasm, indicating that the nanozyme can target tumor cells and then be taken up and engulfed by 143B cells, which enhances the accuracy of the nanozyme action and improves safety.

[0071] Example 7

[0072] Based on Examples 1 to 6, this example evaluates the ability of nanozymes to produce ROS in cells. Figure 10 The ability of nanozyme FeSe / A5 to produce ROS in cells was determined by DCFH-DA, SOSG, and HPF fluorescent probe experiments; it was proved that nanozyme plus photothermal energy can produce more ROS, increasing ROS by 70% to 200% and enhancing oxidative stress in cells.

[0073] Example 8

[0074] Based on Examples 1 to 7, this example evaluates the ability to restrict the proliferation of osteosarcoma tumor cells. Figure 11 The proliferation of osteosarcoma cells was evaluated by studying Ki67 expression through immunofluorescence imaging; the effect of Fe-Se / A5 on cell proliferation was verified by immunofluorescence staining of proliferation-related antigen Ki67 protein. Figure 11 As shown in the figure, the weak fluorescence of Fe-Se / A5 and Fe-Se / A5+NIR groups showed that they significantly reduced the proliferation of tumor cells, especially under NIR irradiation, the ability to limit tumor cell proliferation was more obvious, and the inhibitory effect on proliferation increased by 80% to 200%.

[0075] The foregoing description is merely a preferred embodiment of the present invention and does not limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any changes, modifications, substitutions, integrations, or parameter changes to these embodiments, which fall within the spirit and principles of the present invention and achieve the same functionality through conventional substitutions, without departing from the principles and spirit of the present invention, fall within the scope of protection defined by the claims of the present invention.

Claims

1. A membrane-attached protein modified FeSe diatomic nanozyme, characterized in that: The FeSe diatomic nanozyme is a three-dimensional interconnected porous nitrogen-doped polyhedral carbon (Fe-Se-NC SA) with atomically dispersed Fe-Se dual active centers, and membrane annexin A5 is loaded on the diatomic nanozyme.

2. The method for preparing a membrane-modified FeSe diatomic nanozyme according to claim 1, characterized in that: The steps include: Step S1: Prepare solution A and solution B respectively, wherein: Solution A is prepared by mixing zinc nitrate hexahydrate, ferric acetylacetonate and methanol and stirring evenly; Solution B was prepared by mixing 2-methylimidazole and methanol and stirring evenly; Step S2: Solution A prepared in step S1 is mixed with solution B, stirred, collected by centrifugation, washed 2 to 5 times, and dried to obtain ZIF8@Fe(acac)3; Step S3: The ZIF8@Fe(acac)3 prepared in step S2 is placed in an Ar atmosphere, heated to 600-1000°C for heat treatment, pickled with HNO3 in an oil bath at 25-60°C until neutral, and dried to obtain Fe-NC; Step S4: Se powder is placed on the side of a square porcelain boat where air flows in, and Fe-NC prepared in step S3 is placed on the side where air flows out of the boat. The mixture is heated to 400-600°C at a rate of 2°C / min in an Ar atmosphere to obtain FeSe-NC. Step S5: ANXA5 is mixed with the FeSe-NC prepared in step S4 using a liposome extruder. The ANXA5 protein is attached to the surface of the nanomaterial by repeated extrusion. The mixture is then centrifuged in a high-speed centrifuge for 20 to 30 minutes, washed 3 to 5 times, and freeze-dried in a freeze dryer to obtain Fe-Se / A5.

3. The method for preparing a membrane-modified FeSe diatomic nanozyme according to claim 2, characterized in that: In the step S1, the mass ratio of zinc nitrate hexahydrate to ferric acetylacetonate in preparing solution A is 10:1 to 50:1, and the mass-volume ratio of zinc nitrate hexahydrate to methanol is 1 g:10 to 20 ml.

4. The method for preparing a membrane-modified FeSe diatomic nanozyme according to claim 3, characterized in that: In step S1, the mass-volume ratio of 2-methylimidazole to methanol in preparing solution B is 1 g: 5-30 mL.

5. The method for preparing a membrane-modified FeSe diatomic nanozyme according to claim 4, characterized in that: In step S2, the mixing and stirring time of solution A and solution B is 2 to 10 minutes.

6. The method for preparing a membrane-attached protein modified FeSe diatomic nanozyme according to claim 5, characterized in that: In step S3, the heating rate is 5°C / min.

7. The method for preparing a membrane-attached protein modified FeSe diatomic nanozyme according to claim 6, characterized in that: In the step S4, the mass ratio of Se powder to Fe-NC is 2:1 to 5:

1.

8. The method for preparing a membrane-attached protein modified FeSe diatomic nanozyme according to claim 7, characterized in that: In step S5, the high-speed centrifugal speed is 8000-10000 r / min.

9. Use of the annexin-modified FeSe diatomic nanozyme according to claim 1 in the preparation of a drug for treating osteosarcoma.

Images