Preparation method and application of M2 type macrophage membrane coated FeMn diatomic nano-enzyme

Abstract

The invention belongs to the field of nano-enzyme preparation and biological application, and particularly relates to a preparation method and application of M2 type macrophage membrane coated FeMn diatomic nano-enzyme. The preparation method comprises the following steps: by taking nitrogen-doped carbon (BANT) as a carrier, loading Fe and Mn diatoms, synthesizing a novel nano material FeMnDA / BCNT diatomic nano-enzyme, extracting a macrophage membrane from natural macrophages, and coating the FeMnDA / BCNT nano material with the macrophage membrane to finally form M2 type macrophage membrane coated nano-particles, namely [MM] FeMnDA / BCNT. The [MM] FeMnDA / BCNT nano-enzyme prepared by the invention has good biological safety and simulated SOD and CAT enzyme activity, and the expression of inflammatory factors is reduced by removing excessive ROS (reactive oxygen species) in the cartilage cells induced by H2O2, so that the damage of oxidative stress to the cartilage cells is inhibited. The [MM] FeMnDA / BCNT nano-enzyme is not used for treating OA yet, so that a scientific basis is provided for further application and expansion of the diatomic nano-enzyme, and an effective strategy and a new thought are provided for treating osteoarthritis and chronic diseases related to oxidative stress in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of nanozyme preparation and biological application, and in particular to a preparation method and application of FeMn diatomic nanozyme wrapped in M2 macrophage membrane. Background Art

[0002] Osteoarthritis (OA) is a chronic degenerative cartilage disease characterized by joint pain and dysfunction and is a common, disabling condition. Its clinical symptoms primarily include joint swelling and pain, limited mobility, and even joint deformity and muscle atrophy. Currently, OA is treated primarily with medication and surgery. While medications offer only limited symptom relief, surgery is primarily reserved for patients in advanced, severe stages of the disease. Therefore, a safe and effective new pharmacological approach to treat OA is urgently needed.

[0003] It is currently believed that the most important cause of OA is an imbalance in reactive oxygen species (ROS). Reactive oxygen species (ROS) are known to be crucial to cellular processes and are essential byproducts of cellular metabolism. They include superoxide anions (O2·-), hydrogen peroxide (H2O2), and hydroxyl radicals (·OH). However, due to the oxidation of proteins, DNA, and membrane lipids, excessive ROS may be the main cause of various inflammatory diseases. The rapid development of nanomedicine has provided a new perspective for the use of multifunctional nanomaterials to eliminate ROS and treat related diseases. Nanozymes are considered to be a new type of material that is effective in treating OA due to their high catalase (CAT)- and superoxide dismutase (SOD)-like enzymatic activities, strong ability to scavenge ROS free radicals in chondrocytes, protect chondrocytes, and reduce inflammatory responses.

[0004] At the same time, biomimetic drug delivery systems, particularly cell membrane-coated nanoparticles (NPs), have attracted widespread attention. Studies have shown that macrophage-based biomimetic nanoparticles inherit the chemokine receptor proteins and adhesion proteins of the parent cells, enabling them to actively target inflammatory sites and enhance adhesion to inflammatory tissues and cells, thereby improving the efficiency of drug delivery to inflammatory sites and prolonging drug retention time. More importantly, the cytokine binding receptors on the surface of macrophage biomimetic nanoparticles can neutralize and eliminate various proinflammatory cytokines and bacterial toxins, thereby preventing these factors from activating immune cells and inhibiting downstream inflammatory cascades, effectively reducing the level of inflammatory response.

[0005] At present, there are methods to treat osteoarthritis by using the antioxidant mimicking activity of single-atom nanozymes with high atom utilization efficiency. For example, Chinese invention patent CN118403068A (publication date: July 30, 2024) discloses the preparation and application of a Fe single-atom nanozyme for targeted delivery of small interfering RNA. This patent provides a new idea for designing targeted single-atom nanozymes and loading and delivering small interfering RNA to achieve the treatment of OA. However, due to the small number of active sites of single atoms and the lack of interaction between atoms, it is difficult to meet the enzyme catalytic reaction of multiple substrates, which limits its application. Diatom nanozymes have more active sites, and the synergistic effect between heterogeneous metal atoms will further improve the catalytic performance, and have been widely concerned and recognized in the biomedical field. Based on these characteristics, the present application innovatively constructed and synthesized a diatomic nanozyme (FeMn) with nitrogen-doped carbon nanotubes (BCTN) as carriers, loaded with iron (Fe) and manganese (Mn). DA / BCNT). Then, macrophage membranes were extracted from natural macrophages and wrapped in FeMn DA / BCNT nanomaterials, M2 macrophage membrane-encapsulated nanoparticles ([MM]FeMn DA / BCNT), which can remove oxygen free radicals (ROS) by simulating the activities of superoxide dismutase (SOD) and catalase (CAT), reduce the expression of inflammatory factors, inhibit oxidative stress, and alleviate the progression of OA, and is expected to provide a newer treatment strategy for OA. Summary of the Invention

[0006] This application proposes a preparation method and application of FeMn diatomic nanozymes wrapped in M2 macrophage membranes, and its specific technical solution is described as follows.

[0007] The preparation method of FeMn diatomic nanozyme wrapped by M2 macrophage membrane comprises the following steps:

[0008] Step S1: Fe(acac)2, Mn(acac)2 and polyacrylonitrile were dissolved in N,N-dimethylformamide, and stirred continuously at 25°C for 24 to 48 hours to ensure complete dissolution to obtain a mixed solution, and the mixed solution was electrospun to obtain FeMn DA / BCNT spinning fiber;

[0009] Step S2: FeMn DA The BCNT spun fibers were immersed in the active mixture and stirred for 2 to 4 hours to promote the growth of the silica layer on the fibers. The SiO2 layer was then calcined in a H2 / Ar reducing atmosphere to decompose the SiO2 layer, and the SiO2 shell was chemically removed using a hydrofluoric acid solution.

[0010] Step S3: Rinse the product of step S2 with water and vacuum dry to obtain FeMn DA / BCNT nanozyme, and then evenly grind and store;

[0011] Step S4: Inducing IL-4 to collect RAW264.7 cells, then centrifuging the RAW264.7 cells to obtain a cell suspension, and adding a cell membrane extraction reagent and PMSF to obtain a mixture;

[0012] Step S5: After placing the mixture obtained in step S4 in an ice bath, transfer it to a cell disruptor for homogenization, and then centrifuge the homogenate to collect the cell membranes;

[0013] Step S6: using a micro-extruder to physically extrude the cell membrane obtained in step S6 through a 400 nm porous membrane to obtain a macrophage membrane;

[0014] Step S7: Containing FeMn DA The PBS of / BCNT nanozymes was mixed with macrophage membranes, and then the mixture was extruded through a 200 nm membrane using a micro extruder and sonicated to obtain M2 macrophage membrane-encapsulated FeMn DA / BCNT.

[0015] Furthermore, in step S1, the ratios of Mn(acac)2 to Fe(acac)2, polyacrylonitrile, and N,N-dimethylformamide are 1:1-1.5 g, 1:10-20 g, and 10 g:1-2 L, respectively; the electrospinning conditions are: flow rate 0.01-0.03 ml / min, voltage 15-20 kV, and the distance between the needle and the aluminum foil collector is 15-20 cm.

[0016] Furthermore, in step S2, the active mixture comprises tetraethoxysilane, ethanol, deionized water and hydrochloric acid, wherein the ratio of hydrochloric acid to tetraethoxysilane, ethanol and deionized water is 1:2-6 ml, 1:9-12 ml and 1:3-4 ml respectively; the active mixture and FeMn DA The ratio of BCNT to spinning fiber is 1 ml: 7-12 mg; the calcination condition is: calcination at 800-1000° C. for 2-3 hours.

[0017] Furthermore, in step S3, the drying time is 24 to 48 hours; in step S4, the concentration of IL-4 is 50 to 100 ng / ml, and the induction time is 45 to 50 hours; and the centrifugation condition is: centrifugation at 1000 g for 5 minutes.

[0018] Furthermore, in step S5, the ice bath time is 12 to 18 minutes, the homogenization treatment times are 30 to 35 times, and the conditions for each homogenization treatment are: 5 minutes at 30% power.

[0019] Furthermore, in step S5, centrifugation is performed twice, and the conditions are as follows: the first centrifugation condition is: centrifugation at 3000g for 10 minutes at 4°C, and the supernatant is discarded; the second centrifugation condition is: high-speed centrifugation at 14000g for 30 minutes at 4°C, and the supernatant is taken; the cell membrane is obtained from the bottom of the supernatant obtained after the second centrifugation.

[0020] Further, in step S7, the FeMn DA The concentration of / BCNT nanozyme is 0.1-0.2%.

[0021] Furthermore, in step S7, the conditions of the ultrasonic treatment are: frequency 40 kHz, power 100 W, and time 2 to 3 min.

[0022] Furthermore, the FeMn DA / BCNT nanozyme is stored in the dark. The FeMn DA / BCNT([MM]FeMn DA / BCNT) were stored in PBS at 4-5°C and used.

[0023] Application of FeMn diatomic nanozymes wrapped in M2 macrophage membranes in the preparation of drugs / devices for the treatment of osteoarthritis.

[0024] Compared with the existing technology, the advantages and effects of this application are as follows:

[0025] 1. [MM]FeMn provided by the present invention DA / BCNT nanozyme, as a new type of ROS scavenger, can effectively remove reactive oxygen species and reduce the expression of inflammatory factors, thereby alleviating the damage of oxidative stress to chondrocytes.

[0026] 2. [MM]FeMn provided by the present invention DA / BCNT nanozymes have good biocompatibility and catalytic properties, can effectively remove ROS in cells, and have significant anti-oxidative stress effects.

[0027] 3. The present invention provides a new idea for treating OA by removing ROS through diatomic nanozymes.

[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 is a brief introduction to 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 skilled 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] in:

[0032] Figure 1 [MM]FeMn prepared in this application DA / Transmission electron microscopy image of BCNT nanozyme;

[0033] Figure 2 The FeMn prepared in this application DA / Transmission electron microscopy image of BCNT nanozyme;

[0034] Figure 3 The FeMn prepared in this application DA / Scanning electron microscopy image of BCNT nanozyme;

[0035] Figure 4 The FeMn prepared in this application DA / Mapping characterization diagram of BCNT nanozyme;

[0036] Figure 5 The FeMn prepared in this application DA / EDS characterization of BCNT nanozyme;

[0037] Figure 6 The FeMn prepared in this application DA / X-ray diffraction characterization of BCNT nanozyme;

[0038] Figure 7 Western blotting analysis of FeMn DA / BCNT, [MM]FeMn DA / Results of BCNT and M2 macrophage cell membrane characteristic protein bands;

[0039] Figure 8 The FeMn prepared in this application DA / BCNT nanozyme evaluation results on CAT enzyme activity;

[0040] Figure 9 The FeMn prepared in this application DA / BCNT nanozyme SOD enzyme activity evaluation results;

[0041] Figure 10 The BCNT and FeMn prepared in this application DA / BCNT and [MM]FeMn DA / Biocompatibility evaluation results of BCNT nanozyme;

[0042] Figure 11 The BCNT and FeMn prepared in this application DA / BCNT and [MM]FeMn DA / BCNT nanozyme ROS scavenging ability results;

[0043] Figure 12 The BCNT and FeMn prepared in this application DA / BCNT and [MM]FeMn DA / Quantitative graph of the evaluation results of BCNT nanozyme's ability to scavenge ROS;

[0044] Figure 13 The BCNT and FeMn prepared in this application DA / BCNT and [MM]FeMn DA / The expression level of pro-inflammatory gene IL-6 in BCNT nanozyme;

[0045] Figure 14 The BCNT and FeMn prepared in this application DA / BCNT and [MM]FeMn DA / The expression level of the pro-inflammatory gene MMP-13 in BCNT nanozymes. DETAILED DESCRIPTION

[0046] 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.

[0047] 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.

[0048] 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.

[0049] The term "and / or" in this article is only a description of the association relationship of associated objects, indicating that there can be three relationships. 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 there can be two relationships. 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 related objects before and after are in an "or" relationship.

[0050] 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.

[0051] 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.

[0052] Example 1

[0053] This example introduces a method for preparing FeMn diatomic nanozymes wrapped in M2 macrophage membranes at a specific ratio, as follows:

[0054] 100-150 mg of Fe(acac)2 and 100 mg of Mn(acac)2, along with 1.0-2.0 g of polyacrylonitrile, were dissolved in 10-20 ml of N,N-dimethylformamide. Stirring was continued at 25°C for 24-48 hours to ensure complete dissolution. The solution was then electrospun at a flow rate of 0.02 mL / min, a voltage of 15-20 kV, and a distance of 15-20 cm between the needle and the aluminum foil collector to obtain FeMn DA / BCNT spinning fibers. Next, 200-300 mg of these fibers were immersed in an active mixture containing 4 ml of tetraethoxysilane, 15 ml of ethanol, 5 ml of deionized water and 1.5 ml of hydrochloric acid, and the mixture was vigorously stirred for 3 hours to promote the growth of the silica layer on the fiber; subsequently, it was calcined at 900 ° C for 2 hours in a 5% H2 / Ar reducing atmosphere to decompose the SiO2 layer. Finally, the SiO2 shell was chemically removed using a hydrofluoric acid solution. The fibers were thoroughly rinsed with ultrapure water and dried in a vacuum oven for 24-48 hours to obtain FeMn DA / BCNT nanozyme, grind it evenly and store it in a light-proof environment.

[0055] Subsequently, IL-4 was added to RAW264.7 cells at a concentration of 50-100 ng / mL and cultured for 24-48 hours. After the incubation period, RAW264.7 cells were harvested and centrifuged at 1000 g for 5 minutes to obtain a cell suspension, to which appropriate proportions of cell membrane extraction reagent (Beyotime, Shanghai, China) and PMSF were added. The mixture was placed in an ice bath for 15 minutes. The mixture was then transferred to a cell disruptor and homogenized 30 times with the following conditions: 5 minutes at 30% power. The homogenate was centrifuged at 3000 g for 10 minutes at 4°C, the supernatant discarded, and the supernatant was collected by high-speed centrifugation at 14000 g for 30 minutes at 4°C. The supernatant was then collected and the cell membranes at the bottom were collected. Finally, the particles were physically extruded several times through a 400 nm polycarbonate porous membrane using a microextruder (Avanti Polar Lipids, USA) to obtain macrophage membranes.

[0056] Finally, in order to DA / BCNT surface coated with macrophage membrane, 1mL containing 1mg FeMn DA The PBS containing 1% BCNT was mixed with the prepared macrophage membrane and the mixture was extruded through a 200 nm membrane at least 20 times using a microextruder. The membrane was then sonicated for 2 minutes in a bath sonicator at a frequency of 40 kHz and a power of 100 W to complete the membrane coating. DA / BCNT was stored in PBS at 4°C until use. DA / Transmission electron microscopy images of BCNT nanozymes are shown in the attached Figure 1 .

[0057] The technical effect achieved by this embodiment is: This embodiment provides a simple and novel method for preparing [MM]FeMn DA / BCNT method, through electrospinning technology, using BANT as a carrier, loading Fe and Mn diatoms, to synthesize a new type of nanomaterial FeMn DA / BCNT diatomic nanozymes, and then extracted macrophage membranes from natural macrophages and wrapped them in FeMn DA / BCNT nanomaterials, forming M2 macrophage membrane-wrapped nanoparticles, namely [MM]FeMn DA / BCNT.

[0058] Example 2

[0059] Based on Example 1, this example introduces a specific ratio of FeMn DA The preparation method of BCNT nanozyme is as follows:

[0060] 100-150 mg of Fe(acac)2 and 100 mg of Mn(acac)2, along with 1.0-2.0 g of polyacrylonitrile, were dissolved in 10-20 ml of N,N-dimethylformamide. Stirring was continued at 25°C for 24-48 hours to ensure complete dissolution. The solution was then electrospun at a flow rate of 0.02 mL / min, a voltage of 15-20 kV, and a distance of 15-20 cm between the needle and the aluminum foil collector to obtain FeMn DA / BCNT spinning fibers. Next, 200-300 mg of these fibers were immersed in an active mixture containing 4 ml of tetraethoxysilane, 15 ml of ethanol, 5 ml of deionized water and 1.5 ml of hydrochloric acid, and the mixture was vigorously stirred for 3 hours to promote the growth of the silica layer on the fiber; subsequently, it was calcined at 900 ° C for 2 hours in a 5% H2 / Ar reducing atmosphere to decompose the SiO2 layer. Finally, the SiO2 shell was chemically removed using a hydrofluoric acid solution. The fibers were thoroughly rinsed with ultrapure water and dried in a vacuum oven for 24-48 hours to obtain FeMn DA / BCNT nanozyme, grind it evenly and store it in a light-proof environment.

[0061] The FeMn DA / High-resolution transmission electron microscopy images of BCNT nanozymes are shown in the attached Figure 2 , FeMn DA / Scanning electron microscopy images of BCNT nanozymes are shown in the attached Figure 3 It can be seen that the material has a slender fibrous morphology with a fiber diameter of about 30 to 70 nm. The overall structure is interwoven to form a hollow bamboo-like structure with three-dimensional porous characteristics.

[0062] The FeMn DA / For the mapping characterization diagram of BCNT nanozyme, please see the attached Figure 4 , we can see that C, N, Fe and Mn elements are evenly dispersed, indicating that FeMn DA / BCNT nanozyme porous hollow bamboo Fe and Mn dual active sites; from FeMnDA / EDS characterization of BCNT nanozyme (attached Figure 5 ), it can be seen that C, N, Fe and Mn elements are indeed present, with the C content being 96.28%, the N content being 3.34%, the Mn content being 0.27% and the Fe content being 0.11%.

[0063] The FeMn DA / Please see the attached X-ray diffraction characterization diagram of BCNT nanozyme Figure 6 , we can see FeMn DA The two diffraction peaks of / BCNT located at 18.0° and 21.6° correspond to the 110 and 004 crystal planes of graphitic carbon, respectively. The figure does not show the diffraction peaks of Fe or Mn crystal planes, indicating the formation of atomically dispersed Fe and Mn diatoms.

[0064] Example 3

[0065] Based on Examples 1-2, this example used Western blotting to analyze FeMn DA / BCNT, [MM]FeMn DA / BCNT and M2 macrophage cell membrane characteristic protein bands, please see the attached Figure 7 , we can see [MM]FeMn DA / BCNT surface has the same key membrane antigens as macrophage surface, such as tumor necrosis factor receptor 2 (TNFR2), CD36 (oxLDL receptor) and CCR2 (monocyte chemoattractant protein-1 (MCP-1) receptor).

[0066] Example 4

[0067] Based on Examples 1-3, this example evaluates the FeMn DA / BCNT nanozyme's ability to affect CAT enzyme activity and SOD enzyme activity.

[0068] FeMn DA / Please refer to the attached figure for the evaluation results of BCNT nanozyme's ability to activate CAT enzyme Figure 8 In this embodiment, FeMn DA / BCNT was prepared into different concentrations such as 5μg / ml, 20μg / ml, 40μg / ml, 60μg / ml, and 80μg / ml, and then the CAT activity was evaluated using a CAT kit (Beyotime S0051). The experimental results showed that BCNT itself did not have CAT activity, while FeMn DAThe CAT enzyme activity of / BCNT nanozyme at concentrations of 5μg / ml, 20μg / ml, 40μg / ml, 60μg / ml and 80μg / ml was 0.2, 0.5, 0.7, 1.25 and 1.65 units / μg, respectively, showing a concentration-dependent enhancement, indicating that the nanozyme has good CAT activity.

[0069] FeMn DA / Please refer to the attached figure for the evaluation results of the SOD enzyme activity of BCNT nanozyme Figure 9 In this embodiment, FeMn DA / BCNT was prepared into different concentrations such as 5μg / ml, 20μg / ml, 40μg / ml, 60μg / ml, and 80μg / ml, and then the SOD activity was evaluated using a SOD kit (WST-8) (Beyotime S0101S). The experimental results showed that BCNT itself did not have SOD activity, while FeMn DA The SOD enzyme activities of / BCNT nanozyme at concentrations of 5μg / ml, 20μg / ml, 40μg / ml, 60μg / ml and 80μg / ml were 0.45U / mL, 0.75U / mL, 1.5U / mL, 2.4U / mL and 3U / mL, respectively, showing a concentration-dependent enhancement, indicating that the nanozyme has good SOD activity.

[0070] Example 5

[0071] Based on Examples 1-4, this example evaluates the DA / BCNT and [MM]FeMn DA / Biological activity of BCNT nanozymes.

[0072] In this example, the Cell Counting Kit-8 (CCK-8) assay system was used to detect 80 μg / ml of BCNT, FeMn DA / BCNT and [MM]FeMn DA / BCNT nanozyme toxicity to chondrocytes, the results are shown in the attached Figure 10 It can be seen that the cell activity of each group of materials is above 90%, proving that they have good biosafety.

[0073] Example 6

[0074] Based on Examples 1-5, this example evaluates the DA / BCNT and [MM]FeMn DA / BCNT nanozyme's ability to scavenge ROS.

[0075] In this example, H2O2 was used to induce chondrocytes to construct an in vitro OA cell model. In order to explore whether the material can remove ROS in cells, DCFH-DA fluorescent probe was used in combination with confocal microscopy. Green fluorescence represents ROS in cells. The results are shown in the attached figure. Figure 11 As shown in the figure, the Normal group had almost no green fluorescence, while the green fluorescence in the cells induced by H2O2 was significantly enhanced, indicating that a large amount of ROS was produced by chondrocytes under the induction of H2O2; the green fluorescence intensity of the BCNT group was basically the same as that of the H2O2 group, indicating that BCNT could not eliminate ROS in the cells; on the contrary, FeMn DA / BCNT group and [MM]FeMn DA The fluorescence intensity of the / BCNT group was significantly weakened compared with the H2O2 group, indicating that FeMn DA / BCNT and [MM]FeMn DA / BCNT can effectively remove ROS produced by cells induced by H2O2; quantitative analysis of fluorescence images using ImageJ software showed that Normal, H2O2 group, BCNT group, FeMn DA / BCNT group and [MM]FeMn DA The ROS fluorescence intensities of the BCNT group were 3, 66, 64, 32, and 33, respectively. Figure 12 ) is consistent with the trend of the microscope image. This shows that [MM]FeMn has excellent CAT and SOD-like enzyme activity. DA / BCNT can significantly remove ROS in OA chondrocytes.

[0076] Example 7

[0077] Based on Examples 1-6, this example evaluates the DA / BCNT and [MM]FeMn DA / The expression levels of pro-inflammatory genes IL-6 and MMP-13 in BCNT nanozymes.

[0078] This example uses qRT-PCR technology to analyze the expression levels of pro-inflammatory genes MMP-13 and IL-6 to study their potential in inhibiting inflammatory factors. Figure 13 and attached Figure 14 It can be seen that compared with the Normal group, the expression levels of MMP-13 in the H2O2 group and BCNT group were significantly increased, which were 0.95 and 0.96 respectively, while DA / BCNT and [MM]FeMn DAAfter treatment with FeMn / BCNT, the expression levels of MMP-13 decreased to 0.03 and 0.018, respectively. Similarly, the expression levels of IL-6 increased to 0.99 and 0.98 in the H2O2 and BCNT groups, respectively. DA / BCNT and [MM]FeMn DA / BCNT group was significantly reduced to 0.038 and 0.027. DA All factors of the / BCNT group decreased, while [MM]FeMn DA The expression levels of pro-inflammatory genes were most significantly decreased in the / NC group.

[0079] 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 variation, modification, replacement, integration, or parameter change to these embodiments, which is within the spirit and principles of the present invention and which achieves the same functionality through conventional substitutions, without departing from the principles and spirit of the present invention, falls within the scope of protection of the present invention.

Claims

1. A method for preparing FeMn diatomic nanozymes wrapped in M2 macrophage membranes, characterized in that: The following steps are involved: Step S1: Fe(acac)2, Mn(acac)2 and polyacrylonitrile are dissolved in N,N-dimethylformamide and stirred to obtain a mixed solution, and the mixed solution is electrospun to obtain FeMn DA / BCNT spinning fiber; Step S2: FeMn DA The BCNT spun fibers were immersed in the active mixture and stirred to promote the growth of a silica layer on the fibers. The SiO2 layer was then calcined in a H2 / Ar reducing atmosphere to decompose the SiO2 layer, and the SiO2 shell was chemically removed using a hydrofluoric acid solution. Step S3: Rinse the product of step S2 with water and vacuum dry to obtain FeMn DA / BCNT nanozyme, and then evenly grind and store; Step S4: Inducing IL-4 to collect RAW264.7 cells, then centrifuging the RAW264.7 cells to obtain a cell suspension, and adding a cell membrane extraction reagent and PMSF to obtain a mixture; Step S5: After placing the mixture obtained in step S4 in an ice bath, transfer it to a cell disruptor for homogenization, and then centrifuge the homogenate to collect the cell membranes; Step S6: using a micro-extruder to physically extrude the cell membrane obtained in step S6 through a 400 nm porous membrane to obtain a macrophage membrane; Step S7: Containing FeMn DA The PBS of / BCNT nanozymes was mixed with macrophage membranes, and then the mixture was extruded through a 200 nm membrane using a micro extruder and sonicated to obtain M2 macrophage membrane-encapsulated FeMn DA / BCNT.

2. The method for preparing the FeMn diatomic nanozyme wrapped by the M2 macrophage membrane according to claim 1, characterized in that: In step S1, the ratios of Mn(acac)2 to Fe(acac)2, polyacrylonitrile, and N,N-dimethylformamide are 1:1-1.5 g, 1:10-20 g, and 10 g:1-2 L, respectively; the electrospinning conditions are: flow rate 0.01-0.03 ml / min, voltage 15-20 kV, and the distance between the needle and the aluminum foil collector is 15-20 cm.

3. The method for preparing the FeMn diatomic nanozyme wrapped by the M2 macrophage membrane according to claim 2, characterized in that: In step S2, the active mixture comprises tetraethoxysilane, ethanol, deionized water and hydrochloric acid, wherein the ratio of hydrochloric acid to tetraethoxysilane, ethanol and deionized water is 1:2-6 ml, 1:9-12 ml and 1:3-4 ml respectively; the active mixture is mixed with FeMn DA The ratio of BCNT to spinning fiber is 1 ml: 7 to 12 mg.

4. The method for preparing the FeMn diatomic nanozyme wrapped by the M2 macrophage membrane according to claim 3, characterized in that: In step S3, the drying time is 24 to 48 hours; In step S4, the concentration of IL-4 is 50-100 ng / ml, the induction time is 45-50 h, and the centrifugal treatment conditions are: centrifugation at 1000 g for 5 min.

5. The method for preparing the FeMn diatomic nanozyme wrapped by the M2 macrophage membrane according to claim 4, characterized in that: In step S5, the ice bath time is 12 to 18 minutes, and the homogenization condition for each treatment is: 5 minutes at 30% power.

6. The method for preparing the FeMn diatomic nanozyme wrapped by the M2 macrophage membrane according to claim 5, characterized in that: In step S5, centrifugation is performed twice, and the conditions are: First centrifugation conditions: centrifuge at 3000 g for 10 min at 4°C and discard the supernatant; Second centrifugation conditions: high-speed centrifugation at 14,000 g for 30 min at 4°C, and the supernatant was collected; The cell membrane was obtained from the bottom of the supernatant obtained after the second centrifugation.

7. The method for preparing the FeMn diatomic nanozyme wrapped by the M2 macrophage membrane according to claim 6, characterized in that: In step S7, the FeMn DA The concentration of / BCNT nanozyme is 0.1-0.2%.

8. The method for preparing the FeMn diatomic nanozyme wrapped by the M2 macrophage membrane according to claim 7, characterized in that: In step S7, the ultrasonic treatment conditions are: frequency 40 kHz, power 100 W, and time 2 to 3 min.

9. The method for preparing the FeMn diatomic nanozyme wrapped by the M2 macrophage membrane according to claim 4 or 8, characterized in that: The FeMn DA The storage conditions of BCNT nanozyme are to keep it away from light. The FeMn wrapped by M2 macrophage membrane obtained in step S7 is DA BCNT was stored in PBS at 4-5°C until use.

10. Use of the FeMn diatomic nanozyme wrapped by the M2 macrophage membrane according to any one of claims 1 to 9 in the preparation of drugs / devices for the treatment of osteoarthritis.

Images