Prussian blue nanoscale enzyme targeting inflammation and preparation method and application thereof

Abstract

The application discloses an inflammation-targeted Prussian blue nanolipase and a preparation method and application thereof, and the nanolipase can be used for preparing a drug for treating senile osteoporosis and age-related diseases, and the nanolipase comprises a macrophage membrane and citric acid modified Prussian blue nanoparticles wrapped in the macrophage membrane, the method comprises the following steps: preparing the citric acid modified Prussian blue nanoparticles, extracting the macrophage membrane, mixing a solution of the Prussian blue nanoparticles and a suspension of the macrophage membrane and carrying out ultrasonic treatment, and carrying out solid-liquid separation after stirring. The application solves the problems of short half-life and poor targeting of the existing nanolipase for treating osteoporosis, reduces off-target toxicity and enhances the stability of the curative effect, the nanolipase can more efficiently remove active oxygen, inhibit osteoclast differentiation, relieve bone loss and does not affect osteogenesis, and the nanolipase realizes long-acting, safe and efficient osteoporosis treatment as a whole, and has the application potential of being expanded to other age-related inflammatory diseases.

Description

Technical Field

[0001] This invention relates to the field of medicine, and more particularly to a nanozyme. Background Technology

[0002] Among existing treatment options for osteoporosis and other age-related diseases, a hollow, porous Prussian blue nanozyme (Li K, Hu S, Huang J, et al. Targeting ROS-induced osteoblastsenescence and RANKL production by Prussian blue nanozyme-based gene-editing platform to reverse osteoporosis. NanoToday. 2023;50:101839. doi:10.1016 / j.nantod.2023.101839) treats osteoporosis by normalizing the bone microenvironment. This study addresses the inadequacy of clinical interventions for osteoporosis by proposing a strategy of using nanozymes to regulate the disease microenvironment to inhibit osteoclast differentiation and delay osteoporosis. Hollow Prussian blue nanozymes (HPBZs) prepared by a template-free hydrothermal method were used as the research object. First, their structure and ROS scavenging properties were characterized. Then, in vitro experiments were conducted to verify their inhibitory effect on osteoclast formation and bone resorption in mouse bone marrow macrophages, while showing no effect on osteogenic formation. The molecular mechanism by which they exert their effects through inhibiting ROS generation and the NF-κB and MAPK signaling pathways was explored. Finally, in vivo experiments were conducted using an ovariectomized osteoporosis mouse model to confirm that HPBZs can alleviate bone loss and inhibit osteoclast formation, verifying their efficacy in treating osteoporosis through a normalized microenvironment. However, in the above-mentioned technical approach, Prussian blue nanozymes have poor targeting and a short half-life, resulting in a single therapeutic target and unstable efficacy, as well as increased treatment risks.

[0003] Therefore, developing nanozymes with long half-lives, good targeting, and multiple functions in the in vivo environment has become a key issue that the industry urgently needs to address. Summary of the Invention

[0004] This invention provides an inflammation-targeting Prussian blue nanozyme, its preparation method, and its application, in order to solve the technical problems of targeting and half-life of existing nanozymes mentioned in the background art.

[0005] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0006] An inflammation-targeting Prussian blue nanozyme comprises a macrophage membrane and citric acid-modified Prussian blue nanoparticles encapsulated within the macrophage membrane.

[0007] This invention focuses on the targeting effect of macrophage membranes. By encapsulating Prussian blue nanoparticles with macrophage membranes, the nanozymes are endowed with excellent targeting properties, while the half-life of the nanozymes is significantly increased, ensuring that the nanozymes can be precisely enriched at lesions of age-related diseases such as osteoporosis. Furthermore, this invention uses citric acid-modified Prussian blue nanoparticles as the encapsulation matrix, which has the advantages of small particle size and large specific surface area. Under the same mass conditions, its surface has more enzyme active sites, thus providing a larger surface area for encapsulating macrophage membranes, providing a solid foundation for fully utilizing the encapsulation effect of macrophage membranes. In addition, this invention unexpectedly discovered that the Prussian blue nanozymes prepared by encapsulation with macrophage membranes exhibited higher catalase activity in the early stages compared to Prussian blue nanoparticles alone, indicating a synergistic effect between the two.

[0008] As a further preferred embodiment of the above technical solution, the half-life of the inflammation-targeting Prussian blue nanozyme in mice is at least 1.4 times that of the half-life of citric acid-modified Prussian blue nanoparticles in mice.

[0009] Based on the same technical concept, the present invention also provides a method for preparing the above-mentioned inflammation-targeting Prussian blue nanozyme, comprising the following steps: S1. Preparation of citric acid-modified Prussian blue nanoparticles; S2. Extract macrophage cell membranes; S3. Prepare the Prussian blue nanoparticles into a solution, prepare the macrophage membrane into a suspension, mix the Prussian blue nanoparticle solution and the macrophage membrane suspension and sonicate, then separate the solid and liquid after stirring to obtain the inflammation-targeting Prussian blue nanozyme.

[0010] As a further preferred embodiment of the above technical solution, in S3, the concentration of the Prussian blue nanoparticle solution is 1~5 mg / mL, the concentration of the macrophage membrane suspension is 1~5 mg / mL, and the volume ratio of the Prussian blue nanoparticle solution to the macrophage membrane suspension is (10~40):1.

[0011] As a further preferred embodiment of the above technical solution, in S3, the ultrasonic treatment time is 10~20s.

[0012] As a further preferred embodiment of the above technical solution, in S3, the stirring temperature is 37℃, the stirring speed is 800~1200 rpm, and the stirring time is 0.5~1h.

[0013] As a further preferred embodiment of the above technical solution, in S3, solid-liquid separation is performed by centrifugation, and the solid product obtained after separation is washed.

[0014] As a further preferred embodiment of the above technical solution, in step S1, the method for preparing citric acid-modified Prussian blue nanoparticles includes the following steps: S11. Dissolve 54.1 mg of ferric chloride hexahydrate in 200 mL of 25 mM citric acid solution and heat to 60 °C to prepare solution A; S12. Dissolve 85 mg of potassium ferrocyanide in 200 mL of 25 mM citric acid solution and heat to 60 °C to prepare solution B. S13. Add solution B dropwise to solution A, stir at 60°C for 30 min, then centrifuge, wash and freeze dry to obtain the citric acid modified Prussian blue nanoparticles.

[0015] As a further preferred embodiment of the above technical solution, in S2, the method for extracting macrophage membranes includes the following steps: S21. Resuspend RAW264.7 cells in Tris-Mg buffer, lyse overnight at 4°C, add sucrose to a final concentration of 0.25M, and perform 3 freeze-thaw cycles to obtain a cell suspension. S22. Centrifuge the cell suspension and take the supernatant for further centrifugation to obtain cell membrane precipitate; S23. Wash the cell membrane precipitate twice with pre-cooled 0.25M sucrose, centrifuge again to collect the precipitate, and store it at -80℃.

[0016] Based on the same technical concept, the present invention also provides the application of the above-mentioned inflammation-targeting Prussian blue nanozyme in the preparation of a drug for treating osteoporosis and age-related diseases in the elderly.

[0017] The present invention has the following beneficial effects: The inflammation-targeting Prussian blue nanozyme provided by this invention encapsulates citric acid-modified Prussian blue nanoparticles through a macrophage membrane. This addresses the shortcomings of existing nanozymes for osteoporosis treatment, such as insufficient cytokine clearance, short half-life, and poor targeting. Furthermore, it utilizes the inflammatory chemotactic properties of the macrophage membrane to achieve precise enrichment of osteoporotic lesions, thereby reducing off-target toxicity and enhancing therapeutic stability. In addition, membrane encapsulation unexpectedly synergistically enhances the early catalase activity of the nanozyme. Combined with the advantages of small particle size and large specific surface area brought about by citric acid modification, this nanozyme can more efficiently scavenge reactive oxygen species, thereby reversing cellular aging and inhibiting inflammation. Overall, it achieves long-lasting, safe, and efficient osteoporosis treatment and has the potential for application to other age-related inflammatory diseases. Attached Figure Description

[0018] Figure 1 This is an electron microscope image of Prussian blue nanoparticles (CPB) from Example 1 of the present invention; Figure 2This is an electron microscope image of the inflammation-targeting Prussian blue nanozyme (M@CPB) of Example 1 of the present invention; Figure 3 This is the ROS scavenging ability test result of the inflammation-targeting Prussian blue nanozyme in Example 1 of the present invention; Figure 4 This is the result of the cytokine scavenging ability test of the inflammation-targeting Prussian blue nanozyme in Example 1 of the present invention; Figure 5 This is the targeting ability test result of the inflammation-targeting Prussian blue nanozyme in Example 1 of the present invention; Figure 6 This describes the biodistribution of the inflammation-targeting Prussian blue nanozyme of Example 1 of the present invention in mice. Figure 7 The results of the half-life test of the inflammation-targeting Prussian blue nanozyme of Example 1 of the present invention in mice; Figure 8 The images show a micro-CT scan and immunohistochemical examination of the mouse femur containing the inflammation-targeting Prussian blue nanozyme of Example 1 of this invention. Detailed Implementation

[0019] The present invention will be described in detail below with reference to the accompanying drawings and embodiments, but the present invention can be implemented in many different ways as defined and covered by the claims. The sintering atmosphere in all embodiments and comparative examples is air.

[0020] Example 1: The inflammation-targeting Prussian blue nanozyme of this embodiment includes a macrophage membrane and citric acid-modified Prussian blue nanoparticles encapsulated within the macrophage membrane.

[0021] The inflammation-targeting Prussian blue nanozyme in this embodiment was prepared using the following method: S1. Preparation of Prussian blue nanoparticles modified with citric acid: First, 54.1 mg of ferric chloride hexahydrate (FeCl3) was prepared. Potassium ferrocyanide (6H2O, Aladdin Biochemical Technology Co., Ltd., Shanghai, China) was dissolved in 2 mL of 25 mM citric acid solution (Shanghai Reagent Factory, Shanghai, China). The solution was then diluted to 200 mL with 25 mM citric acid solution to obtain a 1 mM solution, which was heated to 60 °C to prepare solution A. Next, 85 mg of potassium ferrocyanide was dissolved in 25 mM citric acid solution and diluted to 1 mM with 25 mM citric acid solution to 20 mL. The solution was then heated to 60 °C to prepare solution B. Finally, solution B was added dropwise to solution A, and the mixture was stirred at 60 °C for 30 min. The mixture was then centrifuged, washed, and freeze-dried to obtain citric acid-modified Prussian blue nanoparticles. The electron micrograph is shown below. Figure 1 As shown.

[0022] S2. Extraction of macrophage membranes: Macrophage membranes (MΦm) were isolated from RAW264.7 cells: Cells were resuspended in Tris-Mg buffer (pH 7.4; 10 mM Tris, 1 mM MgCl2) and lysed overnight at 4°C. Sucrose was added to a final concentration of 0.25 M, and three freeze-thaw cycles were performed (-80°C / 37°C). After centrifugation at 2000 g for 10 min, the supernatant was collected and centrifuged at 13000 rpm for 30 min to collect the cell membrane pellet. The pellet was washed twice with pre-chilled 0.25 M sucrose, centrifuged again, and stored at -80°C.

[0023] S3. Preparation of citric acid-modified Prussian blue nanoparticles coated with macrophage membranes: 1 mL of Prussian blue nanoparticle (CPBNPs) solution (1-5 mg / mL) was mixed with 0.025-0.1 mL of macrophage membrane (MΦm) suspension (1-5 mg / mL), sonicated for 20 seconds, and then stirred at 37℃ and 800-1200 rpm for 1 hour. The precipitate was collected by centrifugation and washed twice with deionized water to obtain the macrophage membrane-coated nanocomposite (M@CPB), the electron micrograph of which is shown below. Figure 2 As shown.

[0024] The ROS scavenging ability of the inflammation-targeting Prussian blue nanozyme in this embodiment was tested, and the specific method is as follows: Superoxide dismutase (SOD) activity assay: The SOD activity of CPB (0, 50, 100, 200, 400 μg / mL) was detected using a total SOD activity assay kit (WST-8 method) (Beyotime, S0101M). The SOD activity of CPB (100 μg / mL) and M@CPB (100 μg / mL) was then compared. Absorbance was measured at 650 nm, and color changes of different samples were recorded simultaneously. The SOD-like activity of the samples was expressed as the inhibition rate relative to the blank control.

[0025] Catalase (CAT) activity assay: CPB (100 μg / mL) and M@CPB (100 μg / mL) were mixed with H2O2 (5 mM) separately and incubated at 37℃ for 30 min. The change in dissolved oxygen (DO) concentration of the mixture within 10 min was detected using a portable dissolved oxygen meter (JBP-70A, China). This cycle was repeated 4 times. CPB (100 μg / mL) and M@CPB (100 μg / mL) were mixed with H2O2 (500 mM) separately at room temperature, and images were taken at 0 min and 2 h. Each sample was also mixed with 5 mmol / L H2O2 solution, and the change in dissolved oxygen (DO) within 10 min was detected and recorded using a portable dissolved oxygen meter. The hydrogen peroxide decomposition rate was calculated using the following formula: Decomposition rate (%) = (2 ΔDO / 32000) / C0×100%.

[0026] Peroxidase (POD) activity assay: 3,3′,5,5′-tetramethylbenzidine (TMB) was used as the substrate. 2 μg / mL of CPB and M@CPB were mixed with 100 mM H₂O₂ at 37°C, and the absorbance at 650 nm was continuously measured over 10 min to compare the peroxidase activity of the nanozyme. Using TMB as the substrate, 2 μg / mL of M@CPB was mixed with different concentrations of H₂O₂ (0, 0.5, 1, 1.5, 2, and 2.5 mM), and the absorbance at 650 nm was measured using a UV spectrometer after 10 min. The Michaelis-Menten equation was used to determine the catalytic parameters using the measured values: V = V max [S] / (K) m +[S]).

[0027] The results are as follows Figure 3 As shown, Figure 3 Results A showed that CPB scavenged superoxide anions (O2). - The ability of CPB to react with M@CPB is concentration-dependent. Subsequently, at a uniform concentration of 200 μg / mL, the O2 content of CPB and M@CPB was compared. - Scavenging activity: CPB against O2 - The inhibition rate was 37.70±0.06%, and M@CPB increased this inhibition rate to 42.17±0.20% (e.g., Figure 3 B and Figure 3 As shown in Figure C), the SOD-like enzyme activity of M@CPB was verified. Figure 3 The results showed that both CPB and M@CPB could rapidly degrade H2O2 within 120 min, exhibiting sustained CAT-simulated activity. Figure 3 E further intuitively reflects the CAT simulation activity of CPB and M@CPB. Figure 3 F indicates that the H2O2 decomposition rate of CPB in 10 min was 9.79±3.69%, and M@CPB increased this scavenging rate to 22.38±0.88%. Figure 3 As shown in G, under TMB color development, both CPB and M@CPB exhibit characteristic absorption peaks at 650 nm, and the absorbance increases with time. Figure 3 H). Based on the absorbance at 650 nm, the initial reaction rate under different H2O2 concentrations was calculated, and the Michaelis equation was fitted, yielding a maximum reaction rate Vmax of 18.85 μM / min and a Michaelis constant Km of 0.3008 mM. Figure 3 I).

[0028] The cytokine scavenging ability of the inflammation-targeting Prussian blue nanozyme in this embodiment was tested using the following method: MΦm and M@CPB were co-incubated with RANKL and TNF-α solutions, respectively, and their cytokine scavenging activity was verified by quantitative detection of their residual levels.

[0029] RANKL clearance quantification: RANKL (200 pg / mL) was mixed with MΦm (500 μg / mL) and M@CPB (500 μg / mL). The mixture was incubated at 37°C for 2 hours, followed by centrifugation at 14,000 rpm for 10 minutes. The RANKL concentration in the supernatant of each group was quantified using an ELISA kit. TNF-α scavenging quantification: TNF-α (500 pg / mL) was mixed with MΦm (500 μg / mL) and M@CPB (500 μg / mL). The mixture was incubated at 37°C for 2 hours, followed by centrifugation at 14,000 rpm for 10 minutes. The concentration of TNF-α in the supernatant of each group was quantified using an ELISA kit.

[0030] The results are as follows Figure 4 As shown, Figure 4 As shown in Figure A, both MΦm and M@CPB alone exhibited strong RANKL scavenging ability, with their residual concentrations decreasing to 6.47±0.06% and 5.86±0.06%, respectively; Figure 4 As shown in Figure B, both MΦm and M@CPB also exhibited significant scavenging effects on TNF-α, reducing residual levels to 38.44±0.10% and 23.89±2.35%, respectively. This indicates that M@CPB can effectively scavenge RANKL and TNF-α at the material level.

[0031] The targeting and half-life of the inflammation-targeting Prussian blue nanozyme of this embodiment were tested using the following methods: In vitro targeting study of M@CPB: First, CPB was fluorescently labeled: 25 mg / mL Ce6, 20 mg / mL EDC·HCl, and 25 mg NH2-PEG4-NH2 were dissolved in 5 mL DMSO and stirred at 1000 rpm for 6 h to synthesize NH2-Ce6. The solution was then dialyzed for 24 h in a dialysis bag. After centrifugation at 12000 rpm for 10 min, the supernatant was collected as NH2-Ce6. 10 mg / mL CPB, 20 mg / mL EDC·HCl, and 40 mg / mL N-hydroxysuccinimide were added to MES buffer and stirred at 800 rpm for 30 min. Then, 500 μg of NH2-Ce6 was added to the activated CPB, stirred for 12 h, and centrifuged at 12000 rpm to obtain the CPB Ce6 precipitate. M@CPB was obtained by coating the precipitate using the previous method. Ce6 Then, RAW264.7 macrophages, MC3T3-E1 cells, and H2O2 (400 μM)-induced senescent RAW264.7 macrophages and senescent MC3T3-E1 cells were seeded into 24-well plates containing cell spreaders. After cell adhesion, Ce6-labeled M@CPB was added to each well. Ce6 Incubate for 4 hours; discard the culture medium, wash 3 times with PBS, fix with 4% paraformaldehyde (PFA) for 30 minutes, and stain the nuclei with DAPI; observe intracellular fluorescence signals by CLSM to compare the cellular uptake efficiency of nanoparticles.

[0032] In vivo targeting study of M@CPB: Six-month-old male SAMP6 mice were injected with CPB via the tail vein. Ce6 Or M@CPB Ce6 Mice were sacrificed 36 hours after a dose of Ce6 of 2.5 mg / kg. The upper limbs, lower limbs, and lumbar vertebrae were separated. The fluorescence intensity of each tissue was measured using a small animal in vivo imaging system to analyze the distribution characteristics of the nanoparticles in vivo.

[0033] Half-life study of M@CPB: Male Kunming mice were injected with CPB via the tail vein. Ce6 Or M@CPB Ce6 (Ce6 dose 2.5 mg / kg); eyelid blood was collected at 0, 1, 2, 4, 6, 8, 12 and 24 h after injection, and the fluorescence intensity of Ce6 in plasma was measured by a small animal in vivo imaging system; the drug half-life (t1 / 2) was calculated using GraphPadPrism 8.3.0 software.

[0034] The results are as follows Figure 5-7 As shown, Figure 5 A, Figure 5As shown in Figure B, the red fluorescence signal was significantly enhanced in MC3T3-E1 and RAW264.7 cells treated with H2O2, indicating that macrophage membrane coating endows M@CPB with the ability to target inflammatory cells.

[0035] like Figure 6 A and Figure 6 As shown in B, CPB was injected. Ce6 and M@CPB Ce6 After 36 hours, fluorescence imaging was performed on the limb bones and lumbar vertebrae, and the results showed M@CPB Ce6 The group has higher fluorescence intensity ( Figure 6 A). Quantitative analysis shows that M@CPB Ce6 The fluorescence intensities of the group in the limbs, legs, and lumbar spine were CPB, respectively. Ce6 The groups were 1.67 times, 1.87 times, and 1.67 times ( Figure 6 B).

[0036] like Figure 7 As shown, CPB was administered via intravenous injection. Ce6 and M@CPB Ce6 Plasma fluorescence was detected at different time points to analyze its pharmacokinetic behavior. Figure 7 A showed that the plasma fluorescence intensity gradually decreased over time, and M@CPB Ce6 The group's descent rate was slower than that of CPB. Ce6 Group. Quantitative analysis showed that M@CPB Ce6 The half-life in Kunming mice was 1.22 h, which is shorter than that of CPB. Ce6 (0.83h) extended to 1.46 times ( Figure 7 B). It was confirmed that macrophage membrane coating can prolong the in vivo half-life of nanozymes and promote their accumulation at osteoporotic lesions.

[0037] The effects of the inflammation-targeting Prussian blue nanozyme of this embodiment on inhibiting aging and inflammation and alleviating osteoporosis were tested, and the specific methods are as follows: SMR1 mice (n=6) served as the control group and were injected with PBS via the tail vein. SAMP6 mice were randomly divided into four groups (n=6 per group): ① Model group (SAMP6): injected with PBS via the tail vein; ② CPB group (SAMP6): injected with CPB (2.5 mg / kg) via the tail vein; ③ MΦm group (SAMP6): injected with MΦm (2.5 mg / kg) via the tail vein; ④ M@CPB group (SAMP6): injected with M@CPB (2.5 mg / kg) via the tail vein. After one week of acclimatization, the mice were administered the medication every two days for eight consecutive weeks. After the last administration, the mice were sacrificed and their bones were collected.

[0038] Micro-CT examination: The right femur of a mouse was taken, fixed with 4% PFA for 24 hours, and preserved with 75% ethanol; the distal femur was scanned using a Micro-CT system to reconstruct three-dimensional images; bone microstructure parameters were analyzed: bone volume fraction (BV / TV), number of trabeculae (Tb.N), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th) and structural model index (SMI).

[0039] Histological examination: Left femurs of mice were fixed with 4% PFA for 24 h, decalcified with 10% EDTA for 30 days, and embedded in paraffin to prepare 5 μm sections. The bone sections were immunohistochemically treated with anti-TNF-α (1:200) and anti-RANKL (1:200) antibodies, dried, and sealed with neutral resin. The sections were then observed and photographed under an inverted microscope. Bone sections were incubated overnight at 4°C with DHE (1:200), p21 (1:200), CD80 (1:200), and CD206 (1:200), followed by the addition of fluorescently labeled secondary antibody for 2 hours. After DAPI staining for 10 minutes, images were taken using a Nikon (Ti-E+A1MP, Japan) confocal microscope.

[0040] The results are as follows Figure 8 As shown, the three-dimensional (3D) reconstruction results revealed that, compared with the control group, the model group exhibited significant bone loss; while the M@CPB group effectively reversed the osteoporotic phenotype (e.g., Figure 8 A). The bone volume / tissue volume ratio (BV / TV) and the number of trabecular bones (Tb.N) have also shown significant recovery (e.g. Figure 8 B. Figure 8 C). Immunohistochemical staining of bone tissue also showed that, compared with the model group, M@CPB significantly reduced the content of DHE and P21 in bone tissue (C). Figure 8 D、 Figure 8 E), inhibiting oxidative stress and cellular senescence. M@CPB also downregulated the M1 marker CD80 while upregulating the M2 marker CD206 (E). Figure 8 F, Figure 8 G), thereby promoting macrophage polarization from M1 to M2, and also significantly reducing the content of RANKL and TNF-α in bone tissue ( Figure 8 H, Figure 8 I).

[0041] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. For those skilled in the art, improvements and modifications obtained without departing from the inventive concept should also be considered within the scope of protection of the present invention.

[0042] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An inflammation-targeting Prussian blue nanozyme, characterized in that, It includes macrophage membranes and citric acid-modified Prussian blue nanoparticles encapsulated within the macrophage membranes.

2. The inflammation-targeting Prussian blue nanozyme according to claim 1, characterized in that, The inflammation-targeting Prussian blue nanozyme has a half-life in mice that is at least 1.4 times longer than that of citric acid-modified Prussian blue nanoparticles in mice.

3. A method for preparing the inflammation-targeting Prussian blue nanozyme according to claim 1 or 2, characterized in that, Includes the following steps: S1. Preparation of citric acid-modified Prussian blue nanoparticles; S2. Extract macrophage cell membranes; S3. Prepare the Prussian blue nanoparticles into a solution, prepare the macrophage membrane into a suspension, mix the Prussian blue nanoparticle solution and the macrophage membrane suspension and sonicate, then separate the solid and liquid after stirring to obtain the inflammation-targeting Prussian blue nanozyme.

4. The method for preparing the inflammation-targeting Prussian blue nanozyme according to claim 3, characterized in that, In S3, the concentration of the Prussian blue nanoparticle solution is 1~5 mg / mL, the concentration of the macrophage membrane suspension is 1~5 mg / mL, and the volume ratio of the Prussian blue nanoparticle solution to the macrophage membrane suspension is (10~40):

1.

5. The method for preparing the inflammation-targeting Prussian blue nanozyme according to claim 3, characterized in that, In S3, the ultrasonic treatment time is 10~20s.

6. The method for preparing the inflammation-targeting Prussian blue nanozyme according to claim 3, characterized in that, In S3, the stirring temperature is 37℃, the stirring speed is 800~1200rpm, and the stirring time is 0.5~1h.

7. The method for preparing the inflammation-targeting Prussian blue nanozyme according to claim 3, characterized in that, In S3, centrifugation is used for solid-liquid separation, and the resulting solid product is washed.

8. The method for preparing the inflammation-targeting Prussian blue nanozyme according to any one of claims 3-7, characterized in that, In S1, the method for preparing citric acid-modified Prussian blue nanoparticles includes the following steps: S11. Dissolve 54.1 mg of ferric chloride hexahydrate in 200 mL of 25 mM citric acid solution and heat to 60 °C to prepare solution A; S12. Dissolve 85 mg of potassium ferrocyanide in 200 mL of 25 mM citric acid solution and heat to 60 °C to prepare solution B. S13. Add solution B dropwise to solution A, stir at 60°C for 30 min, then centrifuge, wash and freeze dry to obtain the citric acid modified Prussian blue nanoparticles.

9. The method for preparing the inflammation-targeting Prussian blue nanozyme according to any one of claims 3-7, characterized in that, In S2, the method for extracting macrophage membranes includes the following steps: S21. Resuspend RAW264.7 cells in Tris-Mg buffer, lyse overnight at 4°C, add sucrose to a final concentration of 0.25M, and perform 3 freeze-thaw cycles to obtain a cell suspension. S22. Centrifuge the cell suspension and take the supernatant for further centrifugation to obtain cell membrane precipitate; S23. Wash the cell membrane precipitate twice with pre-cooled 0.25M sucrose, centrifuge again to collect the precipitate, and store it at -80℃.

10. The use of the inflammation-targeting Prussian blue nanozyme of claim 1 or 2 in the preparation of a medicament for treating osteoporosis and age-related diseases in the elderly.

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