Application of POCM-NP-coated A939572 in preparation of medicine for treating osteoporosis

Abstract

The invention belongs to the field of medicine, and discloses application of POCM-NP-coated A939572 in preparation of a medicine for treating osteoporosis, the POCM-NP-coated A939572 comprises osteoclast precursor cell membrane vesicles and NP-coated A939572 loaded on the osteoclast precursor cell membrane vesicles, and the NP-coated A939572 is PLGA (poly (lactic-co-glycolic acid)) nanoparticles entrapped with the NP-coated A939572. The POCM-NP-coated A939572 obtained by loading the nanoparticles loaded with the A939572 small molecule medicine into the osteoclast precursor cell membrane vesicles has the effect of relieving the osteoporosis, can inhibit osteoclast differentiation and can be used for preventing, treating and relieving the osteoporosis.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of medicine, and particularly relates to application of POCM-NP@A939572 in preparation of a medicine for treating osteoporosis. BACKGROUND

[0002] Bone is the most important supporting organ of the human body, and is in a continuous reconstruction process. The dynamic balance and coupling between bone formation mediated by osteoblasts and bone resorption mediated by osteoclasts are the key basis for maintaining bone mass stability and normal physiological function of the skeleton. Osteoclasts, which are derived from bone marrow mononuclear cells of the hematopoietic stem cell line, fuse and differentiate into multinucleated giant cells under the action of specific stimulating factors, and are the only cells with bone resorption function in the body, playing a core role in bone reconstruction and bone metabolic homeostasis. When osteoclasts are excessively activated, the balance between bone formation and bone resorption is broken, thereby causing osteoporosis and other bone metabolic diseases.

[0003] Inhibition of bone resorption is one of the main strategies for treating osteoporosis, and common drugs include bisphosphonates, calcitonin, RANKL (receptor activator of nuclear factor-kappa B ligand) monoclonal antibody (denosumab) and strontium salt, etc. However, these drugs still have many limitations in clinical application, such as unclear long-term efficacy, long-term use may cause atypical fractures and mandibular necrosis, accompanied by increased cardiovascular risk and digestive system adverse reactions, etc.

[0004] The existing technology extracts and purifies osteoclast precursor cell membranes, coats them on the outer layer of nanoparticles or drug carriers, which can effectively endow the carrier with bone targeting. This biomimetic strategy enables nanoparticles to actively target bone tissue in the systemic circulation and achieve more efficient retention and drug release in the bone microenvironment. For example, the Chinese patent with publication number CN116019779A discloses a circBBS9 knockdown-based osteoporosis treatment delivery system based on osteoclast precursor cells, which achieves weakening of osteoclast multinucleation and bone resorption capacity by inhibiting circRNA in osteoclast precursor cells using siRNA, while retaining the functions related to osteoclast precursor cells. At the same time, the osteoclast precursor cell-targeted delivery of siRNA is achieved by using the homologous cell membrane microvesicles of osteoclast precursor cells, thereby achieving the effect of treating osteoporosis.

[0005] A939572 is an inhibitor of SCD1 (stearoyl-CoA desaturase 1), and existing studies have shown that A939572 has therapeutic potential in the fields of metabolic diseases, cancer, etc. by regulating lipid metabolism, inhibiting inflammation (such as the Chinese patent with publication number CN111973595A discloses that A939572 can alleviate acute kidney injury caused by ischemia-reperfusion, mainly by increasing the overall metabolic rate of tubular cells, increasing ATP energy supply, thereby promoting the survival of renal tubular cells and reducing inflammation) and tumor signaling pathways. However, there is no related report on the prevention and treatment of osteoporosis. SUMMARY

[0006] The purpose of the present application is to provide the application of POCM-NP@A939572 in the preparation of drugs for treating osteoporosis. The POCM-NP@A939572 obtained by loading the A939572 small molecule drug nano-particles into the membrane vesicles of osteoclast precursor cells has the effect of relieving osteoporosis, can inhibit the differentiation of osteoclasts, and can be used for the prevention and treatment and relief of osteoporosis.

[0007] The application of POCM-NP@A939572 in the preparation of drugs for treating osteoporosis, wherein the POCM-NP@A939572 comprises membrane vesicles of osteoclast precursor cells and NP@A939572 loaded on the membrane vesicles of osteoclast precursor cells, and the NP@A939572 is PLGA (poly-lactic-co-glycolic acid) nano-particles loaded with A939572.

[0008] POCM-NP@A939572 is a kind of A939572-loaded membrane vesicles of osteoclast precursor cells (bone-targeting nanomaterials) coated with A939572-loaded PLGA nano-particles of osteoclast precursor membranes to achieve the effect of targeting osteoclasts, thereby preventing and treating osteoporosis.

[0009] The particle size of the membrane vesicles of osteoclast precursor cells is 210-230 nm, and the particle size of the A939572-loaded PLGA nano-particles NP@A939572 is 140-210 nm.

[0010] The present application strictly controls the particle size of the membrane vesicles of osteoclast precursor cells and the particle size of NP@A939572, thereby realizing the effective loading of NP@A939572 by the membrane vesicles of osteoclast precursor cells.

[0011] Preferably, the mass ratio of PLGA to A939572 in the NP@A939572 is 5-7:1.

[0012] Preferably, the mass ratio of the membrane vesicles of osteoclast precursor cells and the NP@A939572 loaded on the membrane vesicles of osteoclast precursor cells is 1:6-10.

[0013] The application has better effect on preventing and relieving osteoporosis by limiting the mass ratio of the membrane vesicle of the osteoclast precursor cell and the NP@A939572 and the mass ratio of the PLGA and the A939572 in the NP@A939572.

[0014] Further preferably, the mass ratio of the PLGA and the A939572 in the NP@A939572 is 6-6.5:1, and the mass ratio of the membrane vesicle of the osteoclast precursor cell and the NP@A939572 loaded in the membrane vesicle of the osteoclast precursor cell is 1:8. The application has better effect on preventing and relieving the osteoporosis caused by estrogen deficiency by limiting the above mass ratio.

[0015] The preparation method of the NP@A939572 comprises the following steps: dissolving the PLGA powder and then adding the A939572 solution, ultrasonic forming the primary emulsion; adding the polyvinyl alcohol solution in the primary emulsion to form the complex emulsion; stirring the complex emulsion at room temperature overnight to obtain the NP@A939572.

[0016] The preparation method of the membrane vesicle of the osteoclast precursor cell comprises: (1) extracting the bone marrow-derived macrophages of mice, adding RANKL to induce the differentiation of osteoclasts after the bone marrow-derived macrophages adhere to the wall, and collecting the osteoclast precursor cells after 3-5 days; (2) resuspending the osteoclast precursor cells, destroying the osteoclast precursor cells by using an ultrasonic disrupter after resuspension to obtain the homogenate of the osteoclast precursor cells, adding sucrose to mix with the homogenate of the osteoclast precursor cells to obtain a mixture, and obtaining the precipitate after multiple centrifugations of the mixture, which is the membrane of the osteoclast precursor cell; (3) resuspending and uniformly extruding the membrane of the osteoclast precursor cell into the membrane vesicle of the osteoclast precursor cell with a diameter of 210-230 nm by using a liposome extruder.

[0017] The osteoporosis is the osteoporosis caused by estrogen deficiency.

[0018] The preparation method of the POCM-NP@A939572 comprises: ultrasonic treatment of the membrane of the osteoclast precursor cell and the NP@A939572 by using an ultrasonic disrupter, incubation of the mixture at 37°C for 1h to promote the recovery of the membrane, and resuspension of the precipitate of the membrane of the osteoclast precursor cell loaded with the NP@A939572 in ultrapure water to obtain the POCM-NP@A939572. Optionally, the amplitude used in the ultrasonic treatment is 20-30%, the treatment time is 3min, the centrifugal treatment speed is 12000g, and the time is 30min.

[0019] The application shows that POCM-NP@A939572 can significantly prevent and treat estrogen reduction caused by bone loss, indicating that it has the effect of preventing and treating osteoporosis, and therefore can be used for preventing and treating osteoporosis drugs.

[0020] Therefore, the POCM-NP@A939572 provided by the application has the effect of relieving osteoporosis, can inhibit osteoclast differentiation, and can be used for the prevention and treatment of osteoporosis. The application not only provides a new therapeutic use of POCM-NP@A939572, but also provides a new drug for preventing and treating osteoporosis. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is an electron micrograph of NP@A939572; Figure 2 It is an electron micrograph of POCM-NP@A939572; Figure 3 It is the diameter of NP@A939572; Figure 4 It is the Zeta potential of NP@A939572; Figure 5 It is the release rate of NP@A939572 at different pH values; Figure 6 It is the in vitro toxicity of NP@A939572; Figure 7 It is the WB and RT-qPCR of NP@A939572 for inhibiting osteoclast differentiation in vitro; Figure 8 It is the TRAP and F-actin staining of NP@A939572 for inhibiting osteoclast differentiation in vitro; Figure 9 It is the in vivo imaging picture of POCM-NP@A939572 targeting mouse bone tissue; Figure 10 It is the 3D reconstruction picture of POCM-NP@A939572 treating osteoporosis caused by OVX; Figure 11 It is the related bone index of POCM-NP@A939572 treating osteoporosis caused by OVX; Figure 12 It is the HE and TRAP staining picture of POCM-NP@A939572 treating osteoporosis caused by OVX. DETAILED DESCRIPTION

[0022] The application will be further described with reference to the following specific examples. These examples are provided for illustration purposes and are not intended to limit the scope of the application. Furthermore, it is understood that various modifications can be made to the application as described herein, and such modifications are intended to be within the scope of the application. Accordingly, the application is not limited to the examples described herein.

[0023] The term "prevention" as used herein includes prophylaxis, palliation, inhibition or amelioration of the symptoms or conditions of a disease; inhibition of the development of complications; inhibition of the development of a disease or symptoms, such as control of the progression of a disease or condition; reduction of a disease or symptoms; regression of a disease or symptoms; reduction of complications resulting from a disease or symptoms, or prophylaxis or treatment of signs resulting from a disease or symptoms. As used herein, a compound or pharmaceutical composition, upon administration to a subject, can result in the amelioration of a disease, symptom or condition, especially the amelioration of the severity thereof, delay of the onset thereof, reduction of the progression thereof, or reduction of the duration thereof. The condition can be attributed to or associated with the administration, whether fixed or intermittent, continuous or discontinuous.

[0024] Example 1 1. Preparation of A939572-loaded nanoparticles: A939572 (8 mg) was dissolved in anhydrous ethanol to form solution A; PLGA powder (50 mg) was dissolved in 2 ml of dichloromethane and 0.5 ml of Tween 80, and after ultrasonic dissolution, solution A was added to form a primary emulsion, and polyvinyl alcohol was added to water and heated to dissolve, and then cooled at room temperature to obtain a 1% PVA solution. The primary emulsion was added to the 1% PVA solution and ultrasonically formed into a multiple emulsion. The multiple emulsion was stirred at room temperature overnight, and dichloromethane was volatilized, washed five times, and dispersed in pure water to obtain the final product, i.e., the final preparation NP@A939572.

[0025] 2. Culture of osteoclast precursor cells: Healthy 6-week-old male C57BL / 6 mice were euthanized, and bilateral femur and tibia were stripped, and the bone marrow cavity was flushed with phosphate buffer. The resulting cell mixture was filtered with a 0.4 micron filter and centrifuged, and then inoculated in a 10 cm dish with α-MEM medium containing 10 ng / mL macrophage colony-stimulating factor (MCSF) and 10% fetal bovine serum (FBS). After the extracted mouse bone marrow-derived macrophages adhered, the medium was replaced with an osteoclast induction solution containing 25 ng / mL MCSF and 50 ng / mL RANKL, and the medium was replaced every 2 days.

[0026] 3. Preparation of POCM-NP@A939572: The POCM-NP@A939572 was prepared by using the ultrasonic cell disrupter (Model 505 Sonic) with a 0.25 inch probe. The POCM (5 mg) and NP@A939572 (40 mg) were treated by ultrasonic at the following parameters: amplitude 20-30%, on 30 s off 30 s, 3 min, repeated 6 times, with 2 min cooling interval between each cycle. The mixture was incubated at 37 °C for 1 h to promote the recovery of the membrane. Then the un-encapsulated nanoparticles were removed by centrifugation at 1500 g for 10 min, and the POCM-NP@A939572 was further purified by centrifugation at 12000 g for 30 min. The precipitate was resuspended in ultrapure water to obtain the POCM-NP@A939572 (POCM@NP for short).

[0027] 4. Preparation of POCM-NP@A939572: The POCM-NP@A939572 was prepared by using the ultrasonic cell disrupter (Model 505 Sonic) with a 0.25 inch probe. The POCM (5 mg) and NP@A939572 (40 mg) were treated by ultrasonic at the following parameters: amplitude 20-30%, on 30 s off 30 s, 3 min, repeated 6 times, with 2 min cooling interval between each cycle. The mixture was incubated at 37 °C for 1 h to promote the recovery of the membrane. Then the un-encapsulated nanoparticles were removed by centrifugation at 1500 g for 10 min, and the POCM-NP@A939572 was further purified by centrifugation at 12000 g for 30 min. The precipitate was resuspended in ultrapure water to obtain the POCM-NP@A939572 (POCM@NP for short).

[0028] Test Example 1 Characterization of NP@A939572: (1) DLS and Zeta and morphology detection: The particle size and Zeta potential of NP@A939572 were determined by Malvern zs-90. The morphology of NP@A939572 and POCM-NP@A939572 was observed by transmission electron microscope (JEM-2100, JEOL); The characterization results of NP@A939572 prepared in this example are as follows: from Figure 1 (scale bar, 0.5 μm), Figure 2 (scale bar, 0.1 μm) It can be seen that the morphology of PLGA nanoparticles is circular, the particle size is between 140-210 nm, the average particle size is 170 nm, and after being coated with POCM, it is a typical core-shell structure. The drug is not visible under TEM. The average particle size of A939572-loaded PLGA nanoparticles measured by DLS is 358.9 nm, and the average potential is -21.9 mV (Zeta potential, 1 mg / mL, 25 °C). Figure 3、 Figure 4 ).

[0029] (2) Release under different pH conditions: 200 μL of the final preparation (containing A939572 204 μg) was added to a dialysis bag (molecular weight cut-off 8000-14000 Da), and the dialysis bag was placed in 10 mL of phosphate buffer with pH=6.5 and pH=7.4, respectively, and incubated at 37°C with 100 rpm shaking; 1 mL of dialysate was taken at 1, 2, 3, 4, 5, 6, and 7 days, respectively, and 1 mL of fresh phosphate buffer was added. After filtration through a 0.22 μm filter, the dialysate was subjected to HPLC detection; Results show that the cumulative release rate of PLGA nanoparticles under pH=6.5 conditions for 7 days is 23.8%, and the cumulative release rate under pH=7.4 conditions for 7 days is 29.21%. Figure 5 Results show that the cumulative release rate of PLGA nanoparticles under pH=6.5 conditions for 7 days is 23.8%, and the cumulative release rate under pH=7.4 conditions for 7 days is 29.21%.

[0030] (3) In vitro toxicity detection, CCK-8 detection was used: mouse BMDMs were used to evaluate the cytotoxicity of NPs by a commercial CCK-8 kit (C0005, TargetMol, Boston, USA). These cells were co-incubated with different concentrations of NP@A939572 for 24 h. Then the cell viability was detected according to the manufacturer's protocol; Results show that POCM-NP@A939572 has good safety and no cytotoxicity at high concentrations. Figure 6 The number of particles in the abscissa is of the order of 10 11 Results show that POCM-NP@A939572 has good safety and no cytotoxicity at high concentrations.

[0031] (4) Encapsulation efficiency determination: 100 μL of the final preparation was taken in an EP tube, centrifuged at 12000 g for 10 min, the supernatant was removed, and the precipitate was added to 5 mL of a mixture of dichloromethane and 1 mL of absolute ethanol. Ultrasonic was performed for 2 h to obtain a demulsified solution, which was subjected to encapsulation efficiency detection. At the same time, 2 mg of A939572 was weighed in an EP tube, 2 mL of absolute ethanol was added to prepare a 1 mg / mL stock solution, which was diluted to 1 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, and 20 μg / mL to serve as a standard solution. The standard solution and the test solution were subjected to HPLC detection together; The encapsulation efficiency of NP@A939572 prepared in Example 1 was 62.63%.

[0032] Test Example 2 The NP@A939572 prepared in Example 1 was verified for in vitro inhibition of osteoclast differentiation, and the specific method was as follows: (1) Inhibition of osteoclast differentiation in vitro After adhering the extracted mouse bone marrow-derived macrophages, replace the osteoclast induction solution containing 25 ng / mL MCSF, 50 ng / mL RANKL, and add different concentrations of NP@A939572, 5 days later, fix the cells with 4% PFA for 10 min, wash with PBS for 3 times, and use tartrate-resistant acid phosphatase (TRAP) staining solution (Sigma Aldrich) for staining.

[0033] (2) Western Blot After 3 days of RANKL induction of macrophages, extract proteins according to the instructions of BIO-RAD 4x Laemmli Sample Buffer (#161-0737), collect after 5 min of room temperature lysis, and treat with a metal bath at 100°C for 10 min. SDS-PAGE gel preparation: prepare 10% separation gel and concentrated gel, add APS and TEMED for polymerization, and insert the comb for gel formation. When loading, add an equal volume of protein sample and Marker, run to the separation gel at 80V, and then adjust to 120V for electrophoresis to the end point. After electrophoresis, transfer the membrane: transfer the gel to a methanol-activated PVDF membrane (300 mA constant current, 95 min). After membrane transfer, block with 5% skim milk for 1-2 h. Antibody incubation: incubate the primary antibody (1:1000) at 4°C overnight, wash with TBST for 5 times (7 min / time); incubate the HRP-secondary antibody at room temperature for 1 h, and wash with TBST for 5 times. Detect the target protein band.

[0034] (3) Quantitative Polymerase Chain Reaction (qPCR) After 2 days of RANKL induction of macrophages, use Kangwei Century Super Pure RNA Extraction Kit (CW0581) to extract total RNA. The brief steps are as follows: add 1 mL TRizon Reagent per well for lysis for 5 min, add 200 μL chloroform, mix well and stand for 5 min, centrifuge at 4°C, 12000g for 10 min. Mix the supernatant with an equal volume of 75% ethanol, and purify with a silica-based adsorption column. After washing, elute the RNA with enzyme-free water. Then use Kangwei Century Reverse Transcription Kit (CW2569) for cDNA synthesis. The obtained product is further detected by real-time fluorescent quantitative PCR (qPCR) using Yixing qPCR reagent (11201ES03). The primer sequences are shown in Table 1.

[0035] Table 1 Primer sequences related to the expression of osteoclast genes detected by real-time fluorescent quantitative PCR

[0036] In this test example, the results of the characterization of the anti-osteoclastogenesis effect of the NP@A939572 prepared in Example 1 in vitro are as follows: after 2 days of osteoclast induction, the macrophages not treated with NP@A939572 highly expressed osteoclast differentiation-related proteins such as activated T cell nuclear factor 1 (NFATc1), proto-oncogene, AP-1 transcription factor subunit (c-Fos) with beta-actin (ACTB) as the internal reference protein. After treatment with different concentrations of NP@A939572, the expression of osteoclast differentiation-related proteins in macrophages increased compared with the non-induced group, but the protein expression decreased compared with the simple osteoclast induction group, and the higher the concentration of NP@A939572 treatment, the less the expression of osteoclast differentiation-related proteins. Further detection of the RNA level of the osteoclast-related index obtained consistent results, proving that NP@A939572 can significantly inhibit the differentiation of osteoclasts in vitro Figure 7 , the number of particles in the abscissa is of the order of 10 11 ). After 5 days of osteoclast induction, a large number of fused and multinucleated osteoclasts were differentiated in macrophages not treated with NP@A939572, while the differentiation of mature osteoclasts in macrophages treated with NP@A939572 was significantly reduced, and the higher the concentration of NP@A939572 treatment, the fewer the number of osteoclasts formed, showing a concentration-dependent inhibition Figure 8 , the scale is 100 μm).

[0037] Test Example 3 The biodistribution of the nanoparticles prepared in Example 1 was verified: NPs were dyed with lipophilic DiR or DiO fluorescent dye (KGE2604-10, Ji Yuan Biotechnology, Nanjing, China), denoted as CY5-POCM-NP@A939572 (POCM-CY5@NP); each mouse was injected with 50 μL of PBS solution containing 1.0 × 10 10 particles of NPs through the tail vein; 6 h after injection, the main organs of the mice injected with CY5-POCM-NP@A939572 were collected and imaged by AniView100 multi-modal animal in vivo imaging system (Guangzhou Bai'ao Tai Biotechnology Co., Ltd.). As shown in Figure 9 , CY5-POCM-NP@A939572 (POCM-CY5@NP) can well target the bone tissue of mice (red and yellow represent different fluorescence intensities, respectively).

[0038] Test Example 4 The POCM-NP@A939572 prepared in Example 1 was verified for rescuing osteoporosis caused by ovary removal in vivo: (1) Osteoporosis model and nanovesicle administration Twelve-week-old female C57BL / 6 mice were raised in specific pathogen-free cages. For each group of experimental mice, after intraperitoneal injection of pentobarbital anesthesia, surgical operation was performed. The sham operation group of mice was treated with sham operation, and the bilateral ovaries were not removed. For the ovariectomy group, the abdominal cavity was accessed through the dorsal skin approach at the middle level of the bilateral lumbar vertebrae, and the bilateral ovaries were ligated and removed, and the peritoneum, skin, etc. were sutured layer by layer to close. After the operation, the POCM-NP@A939572 was injected intravenously once a week for 8 weeks.

[0039] (2) Tartrate-resistant acid phosphatase (TRAP) staining of bone tissue sections The mouse femur samples were fixed with 4% paraformaldehyde (PFA) for 48 h, and then decalcified with commercial 10% ethylenediaminetetraacetic acid (EDTA) for 2 weeks. After decalcification, the samples were washed with PBS, and then dehydrated in gradient ethanol solution. The dehydrated samples were placed in embedding boxes, the direction of the femur was adjusted, and the samples were placed at room temperature until the paraffin was completely solidified. 5-10 μm thick sections were cut using a paraffin microtome. The sections were stained with a TRAP staining kit (Solarbio, G1492, Beijing, China). The cell nuclei were stained with 1% methyl green staining solution (Solarbio, G1652, Beijing, China). The positively stained areas were analyzed by ImageJ software (NIH, USA).

[0040] (3) Micro-computed tomography (μCT) analysis The mice in each group were euthanized at 8 weeks after the operation, and the bilateral femur tissues were fixed with 4% paraformaldehyde at room temperature for 2 days. The left femur of each group of fixed mice was scanned by Micro CT with a resolution of 9 microns, and the scanning parameters were: voltage 70 kilovolts, current 80 microamperes. 130 layers starting from the growth plate region were selected as the region of interest for data analysis and three-dimensional reconstruction. The analysis indicators included: relative trabecular bone volume.

[0041] (4) Statistical analysis All statistical analyses were performed in GraphPad Prism 8.0 software. The differences between two groups were analyzed by Student's t-test; three or more groups were compared by one-way ANOVA. The experimental data were expressed as mean ± standard deviation (Mean ± SD). The criteria for determining statistical significance were: *P<0.05, **P<0.01, ***P<0.001, #P<0.0001.

[0042] In the present test example, the characterization results of POCM-NP@A939572 prepared in Example 1 in rescuing osteoporosis caused by ovariectomy in vivo are as follows: The relative bone volume of femoral trabecula of the model group mice (OVX) was significantly lower than that of the sham operation group 8 weeks after ovariectomy of 12-week-old female mice; and in the POCM-NP@A939572 intervention group, the index was significantly higher than that of the ovariectomy group, indicating that POCM-NP@A939572 has a significant prevention and treatment effect on osteoporosis caused by estrogen deficiency. Figure 10 and Figure 11 In addition, it was found by tartrate-resistant acid phosphatase (TRAP) staining of bone tissue sections that the number of osteoclasts significantly increased after ovariectomy, and the number of osteoclasts significantly decreased after treatment with nanovesicles. Figure 12

[0043] In summary, the POCM-NP@A939572 provided by the present application can inhibit the expression of differentiation protein-related osteoclasts in vitro, and has the effect of preventing and treating osteoporosis.

[0044] The above is only a preferred embodiment of the present application, and it should be noted that those skilled in the art can make several improvements and supplements without departing from the method of the present application, and these improvements and supplements should also be considered within the protection scope of the present application.​

Claims

1. The application of POCM-NP@A939572 in the preparation of drugs for treating osteoporosis, characterized in that, The POCM-NP@A939572 comprises osteoclast precursor cell membrane vesicles and NP@A939572 loaded onto osteoclast precursor cell membrane vesicles, wherein the NP@A939572 is a PLGA nanoparticle encapsulating A939572.

2. The application according to claim 1, characterized in that, The osteoclast precursor cell membrane vesicles have a particle size of 210-230 nm, and the PLGA nanoparticles loaded with A939572 have a particle size of 140-210 nm.

3. The application according to claim 1, characterized in that, The mass ratio of PLGA to A939572 in the NP@A939572 is 5-7:

1.

4. The application according to claim 3, characterized in that, The mass ratio of the osteoclast precursor cell membrane vesicles to NP@A939572 is 1:6-10.

5. The application according to claim 4, characterized in that, The mass ratio of PLGA to A939572 in NP@A939572 is 6-6.5:1, and the mass ratio of osteoclast precursor cell membrane vesicles to NP@A939572 is 1:

8.

6. The application according to claim 1, characterized in that, The preparation method of NP@A939572 includes the following steps: dissolving PLGA powder and adding A939572 solution, and ultrasonically forming a primary emulsion; adding polyvinyl alcohol solution to the primary emulsion to form a secondary emulsion; stirring the secondary emulsion at room temperature overnight to obtain NP@A939572.

7. The application according to claim 1, characterized in that, The method for preparing the osteoclast precursor cell membrane vesicles includes: (1) Extract bone marrow-derived macrophages from mice. After the bone marrow-derived macrophages adhered to the wall, RANKL was added to induce osteoclast differentiation. Osteoclast precursor cells were collected 3-5 days later. (2) Resuspend the osteoclast precursor cells, and then use an ultrasonic homogenizer to destroy the osteoclast precursor cells to obtain osteoclast precursor cell homogenate; add sucrose and mix with the osteoclast precursor cell homogenate to obtain a mixture; after centrifuging the mixture multiple times, the precipitate obtained is the osteoclast precursor cell membrane. (3) After resuspension, the osteoclast precursor cell membrane is uniformly squeezed into osteoclast precursor cell membrane vesicles of 210-230 nm using a liposome extruder.

8. The application according to any one of claims 1-7, characterized in that, The osteoporosis mentioned is osteoporosis caused by estrogen deficiency.

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