Use of POCM-NP@A939572 in the preparation of a drug for treating osteoporosis

Abstract

The application belongs to the field of medicine, and discloses an application of POCM-NP@A939572 in preparation of a medicine for treating osteoporosis, wherein the POCM-NP@A939572 comprises osteoclast precursor cell membrane vesicles and NP@A939572 loaded on the osteoclast precursor cell membrane vesicles, and the NP@A939572 is PLGA nanoparticles loaded with A939572. The POCM-NP@A939572 obtained by loading the nanoparticles loaded with A939572 small molecule drugs into the osteoclast precursor cell membrane vesicles has an effect of relieving osteoporosis, can inhibit osteoclast differentiation, and can be used for prevention and relief of osteoporosis.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to the application of POCM-NP@A939572 in the preparation of drugs for treating osteoporosis. Background Technology

[0002] Skeleton is the most important supporting organ in the human body and is in a continuous process of remodeling. The dynamic balance and coupling between osteoblast-mediated bone formation and osteoclast-mediated bone resorption is a key foundation for maintaining stable bone mass and normal skeletal physiological function. Osteoclasts originate from bone marrow mononuclear cells of the hematopoietic stem cell line and fuse and differentiate into multinucleated giant cells under the influence of specific stimulating factors. They are the only cells in the body with bone resorption function and play a central role in bone remodeling and bone metabolic homeostasis. When osteoclasts are overactivated, the balance between bone formation and bone resorption is disrupted, leading to osteoporosis and other bone metabolic diseases.

[0003] Inhibiting bone resorption is one of the main strategies for treating osteoporosis. Commonly used drugs include bisphosphonates, calcitonin, RANKL (receptor activator of nuclear factor-κB ligand) monoclonal antibodies (denosumab), and strontium salts. However, these drugs still have many limitations in clinical application. For example, their long-term efficacy is unclear, long-term use may lead to atypical fractures and osteonecrosis of the mandible, and is accompanied by increased cardiovascular risk and gastrointestinal adverse reactions.

[0004] Current technologies effectively endow carriers with bone-targeting properties by extracting and purifying osteoclast precursor cell membranes and coating them onto the outer layer of nanoparticles or drug carriers. This biomimetic strategy allows nanoparticles to actively approach bone tissue during systemic circulation and achieve more efficient retention and drug release within the bone microenvironment. For example, Chinese patent CN116019779A discloses an osteoporosis treatment delivery system based on osteoclast precursor cell-targeted circBBS9 knockdown. This system uses siRNA to inhibit circRNA in osteoclast precursor cells, achieving osteoclast multinucleation and weakening bone resorption while preserving the relevant functions of osteoclast precursor cells. Simultaneously, it utilizes cell membrane microvesicles homologous to osteoclast precursor cells to deliver the siRNA in a targeted manner, achieving the therapeutic effect of osteoporosis treatment.

[0005] A939572 is an inhibitor of SCD1 (stearoyl-CoA desaturase 1). Existing research indicates that A939572 has shown therapeutic potential in metabolic diseases and cancer by regulating lipid metabolism, inhibiting inflammation (for example, Chinese patent publication number CN111973595A discloses that A939572 can alleviate acute kidney injury caused by ischemia-reperfusion injury, mainly by increasing the overall metabolic rate of tubular cells and increasing ATP energy supply, thereby promoting the survival of renal tubular cells and reducing inflammation), and tumor signaling pathways. However, there are currently no reports on its application in the prevention and treatment of osteoporosis. Summary of the Invention

[0006] The purpose of this invention is to provide the application of POCM-NP@A939572 in the preparation of drugs for treating osteoporosis. POCM-NP@A939572, obtained by loading nanoparticles loaded with the small molecule drug A939572 into osteoclast precursor cell membrane vesicles, has the effect of alleviating osteoporosis and can inhibit osteoclast differentiation, and can be used for the prevention, treatment and relief of osteoporosis.

[0007] The application of POCM-NP@A939572 in the preparation of drugs for treating osteoporosis, wherein POCM-NP@A939572 comprises osteoclast precursor cell membrane vesicles and NP@A939572 loaded onto osteoclast precursor cell membrane vesicles, wherein NP@A939572 is PLGA (polylactic acid-glycolic acid copolymer) nanoparticles encapsulating A939572.

[0008] POCM-NP@A939572 is an osteoclast precursor cell membrane vesicle loaded with A939572 (bone-targeting nanomaterial). PLGA nanoparticles loaded with A939572, coated with osteoclast precursor membranes, can target osteoclasts, thereby preventing and treating osteoporosis.

[0009] The osteoclast precursor cell membrane vesicles have a particle size of 210-230 nm, and the PLGA nanoparticles NP@A939572 loaded with A939572 have a particle size of 140-210 nm.

[0010] This invention achieves effective loading of NP@A939572 onto osteoclast precursor cell membrane vesicles by strictly controlling the particle size of the osteoclast precursor cell membrane vesicles and the particle size of NP@A939572.

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

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

[0013] This invention improves the efficacy of the drug in preventing and alleviating osteoporosis by adjusting the mass ratio of osteoclast precursor cell membrane vesicles and NP@A939572, as well as the mass ratio of PLGA to A939572 in NP@A939572.

[0014] More preferably, the mass ratio of PLGA to A939572 in NP@A939572 is 6-6.5:1, and the mass ratio of the osteoclast precursor cell membrane vesicles to the NP@A939572 loaded on the osteoclast precursor cell membrane vesicles is 1:8. The drug obtained by the present invention with the above-mentioned mass ratios has a good effect on the prevention and relief of osteoporosis caused by estrogen deficiency.

[0015] 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.

[0016] The method for preparing the osteoclast precursor cell membrane vesicles includes:

[0017] (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.

[0018] (2) Osteoclast precursor cells were resuspended and then destroyed using an ultrasonic homogenizer to obtain osteoclast precursor cell homogenate. Sucrose was added and mixed with the osteoclast precursor cell homogenate to obtain a mixture. The precipitate obtained after multiple centrifugations of the mixture is the osteoclast precursor cell membrane.

[0019] (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.

[0020] The osteoporosis mentioned is osteoporosis caused by estrogen deficiency.

[0021] The preparation method of POCM-NP@A939572 includes: ultrasonically treating the osteoclast precursor membrane and NP@A939572 using an ultrasonic disruptor, and incubating the mixture at 37°C for 1 hour to promote membrane recovery. The osteoclast precursor membrane precipitate loaded with NP@A939572 is further purified, and the precipitate is resuspended in ultrapure water to obtain POCM-NP@A939572. Optionally, the ultrasonic treatment uses an amplitude of 20-30% and a treatment time of 3 minutes; the centrifugation speed is 12000g and the time is 30 minutes.

[0022] This invention demonstrates through pharmacological experiments on the preventive and therapeutic effects of POCM-NP@A939572 on osteoporosis in ovariectomized mice that POCM-NP@A939572 can significantly prevent and therapeutically improve bone loss caused by estrogen deficiency, indicating that it has a role in preventing and treating osteoporosis and can therefore be used as a drug for the prevention and treatment of osteoporosis.

[0023] Therefore, POCM-NP@A939572 provided by this invention has the effect of alleviating osteoporosis, can inhibit osteoclast differentiation, and can be used for the prevention and relief of osteoporosis. This invention not only provides a new therapeutic use for POCM-NP@A939572, but also provides a new drug for the prevention and treatment of osteoporosis. Attached Figure Description

[0024] Figure 1 Electron micrograph of NP@A939572;

[0025] Figure 2 Electron micrograph of POCM-NP@A939572;

[0026] Figure 3 The diameter of NP@A939572;

[0027] Figure 4 The zeta potential of NP@A939572;

[0028] Figure 5 Release rates of NP@A939572 at different pH values;

[0029] Figure 6 In vitro toxicity of NP@A939572;

[0030] Figure 7 Western blot and RT-qPCR were used to inhibit osteoclast differentiation in vitro using NP@A939572.

[0031] Figure 8 TRAP and F-actin staining were used to inhibit osteoclast differentiation in NP@A939572 in vitro.

[0032] Figure 9 In vivo imaging of mouse bone tissue targeted by POCM-NP@A939572;

[0033] Figure 10 3D reconstruction image of POCM-NP@A939572 for treating osteoporosis caused by OVX;

[0034] Figure 11 For POCM-NP@A939572 treatment of osteoporosis caused by OVX, relevant bone markers were determined.

[0035] Figure 12 HE and TRAP staining images of tissues from POCM-NP@A939572 treated with OVX to induce osteoporosis. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0037] As used herein, the term "prevention" includes preventing, alleviating, suppressing, or improving symptoms or conditions of a disease; suppressing the development of complications; suppressing the development of a disease or symptom, such as controlling the progression of a disease or condition; reducing a disease or symptom; alleviating a disease or symptom; reducing complications arising from a disease or symptom; or preventing or treating signs arising from a disease or symptom. As used herein, a compound or pharmaceutical composition, when administered, can improve a disease, symptom, or condition, particularly by improving its severity, delaying onset, reducing disease progression, or shortening the duration of the condition. Whether administered regularly or intermittently, continuously or intermittently, it can be attributed to or related to the administration.

[0038] Example 1

[0039] 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, it was added to solution A to form a primary emulsion. Polyvinyl alcohol was added to water and heated to dissolve, then cooled to room temperature to obtain a 1% PVA solution. The 1% PVA solution was added to the primary emulsion and ultrasonically dissolved to form a secondary emulsion. The secondary emulsion was stirred overnight at room temperature to evaporate the dichloromethane, washed five times, and dispersed in pure water to obtain the final product, i.e., the final formulation NP@A939572.

[0040] 2. Culture of osteoclast precursor cells: Healthy 6-week-old male C57BL / 6 mice were euthanized, and the femurs and tibias / fibulae were dissected. The bone marrow cavity was flushed with phosphate-buffered saline (PFS), and the resulting cell mixture was filtered through a 0.4-micron filter and centrifuged. The cells were then seeded into 10 cm³ media in 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 to the medium, the medium was replaced with osteoclast induction medium containing 25 ng / mL MCSF and 50 ng / mL RANKL, with the medium changed every 2 days.

[0041] 3. Preparation of osteoclast precursor cell membrane vesicles: Wash osteoclast precursor cells with 1×PBS, digest with trypsin, and resuspend in TM buffer at 4°C. After resuspending, use an ultrasonic homogenizer to disrupt the osteoclast precursor cells and obtain osteoclast precursor cell homogenate. Add 1M sucrose to the osteoclast precursor cell homogenate to obtain a mixture with a final sucrose concentration of 0.25M. Centrifuge the mixture obtained in the above steps at 4°C and 2000g for 10 minutes, collect the supernatant, pour the collected supernatant into a centrifuge tube, centrifuge at 15000g at 4°C for 2 hours, carefully aspirate the supernatant, add 3 mL of 0.25M sucrose to the precipitate for resuspending, continue centrifuging at 15000g at 4°C for 2 hours, carefully aspirate the supernatant, and the precipitate is the osteoclast precursor cell membrane. After resuspending, use a liposome extruder to uniformly extrude the osteoclast precursor membrane into osteoclast precursor cell membrane vesicles of 210-230nm.

[0042] 4. Preparation of POCM-NP@A939572: Osteoclast precursor membrane vesicles (5 mg) and NP@A939572 (40 mg) were sonicated using an ultrasonic disruptor (Model 505 Sonic) with a 0.25-inch probe. The sonication parameters were: amplitude 20-30%, on 30 s, off 30 s, 3 min, repeated 6 times, with a 2 min cooling interval between each cycle. The mixture was incubated at 37 °C for 1 h to promote membrane recovery. Then, the unencapsulated nanoparticles were removed by centrifugation at 1500 g for 10 min, and the osteoclast precursor membrane precipitate loaded with NP@A939572 was further purified by centrifugation at 12000 g for 30 min. The precipitate was resuspended in ultrapure water to obtain POCM-NP@A939572 (abbreviated as POCM@NP).

[0043] Characterization of NP@A939572 in Experiment 1:

[0044] (1) DLS and Zeta potential and morphological analysis: The particle size and Zeta potential of NP@A939572 were determined using a Malvern zs-90 microscope. The morphology of NP@A939572 and POCM-NP@A939572 were observed using a transmission electron microscope (JEM-2100, JEOL).

[0045] The characterization results of NP@A939572 prepared in this embodiment are as follows: From Figure 1 (Scale bar is 0.5μm) Figure 2(Scale bar is 0.1 μm) It can be seen that the PLGA nanoparticles are spherical, with a particle size between 140-210 nm and an average particle size of 170 nm. After being coated with osteoclast precursor cell membranes, they exhibit a typical core-shell structure. The drug is not visible under TEM. The average particle size of the PLGA nanoparticles loaded with A939572, measured by DLS, is 358.9 nm, and the average potential is -21.9 mV. Figure 3 , Figure 4 ).

[0046] (2) Release under different pH conditions: 200 μL of the final preparation (containing 204 μg of A939572) was added to a dialysis bag (molecular weight cutoff 8000-14000 Da). The dialysis bag was placed in 10 mL of phosphate buffer solution with pH=6.5 and pH=7.4 respectively, and incubated at 37℃ and 100 rpm on a shaker. On days 1, 2, 3, 4, 5, 6 and 7 respectively, 1 mL of dialysate was taken and 1 mL of fresh phosphate buffer solution was added. The dialysate was filtered through a 0.22 μm filter membrane and then analyzed by HPLC.

[0047] Depend on Figure 5 The results showed that the cumulative release rate of PLGA nanoparticles was 23.8% after 7 days at pH 6.5 and 29.21% after 7 days at pH 7.4.

[0048] (3) In vitro toxicity assay: CCK-8 assay was performed using mouse BMDMs. The cytotoxicity of NPs was assessed using a commercial CCK-8 assay kit (C0005, TargetMol, Boston, USA). These cells were co-incubated with different concentrations of NP@A939572 for 24 h. Cell viability was then assessed according to the manufacturer's protocol.

[0049] Depend on Figure 6 (The number of particles on the horizontal axis is on the order of 10) 11 The results show that POCM-NP@A939572 has good safety and no cytotoxicity at high concentrations.

[0050] (4) Encapsulation efficiency determination: Pipette 100 μL of the final preparation into an EP tube, centrifuge at 12000g for 10 min, remove the supernatant, add the precipitate to a mixed solution of 5 mL dichloromethane and 1 mL anhydrous ethanol, sonicate for 2 h to obtain the demulsified solution, and use the demulsified solution to detect the encapsulation efficiency; at the same time, weigh 2 mg of A939572 into an EP tube, add 2 mL of anhydrous ethanol to prepare a 1 mg / mL stock solution, and dilute the stock solution to 1 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, and 20 μg / mL as standard solutions; perform HPLC detection on both the standard solution and the test solution;

[0051] The encapsulation efficiency of NP@A939572 prepared in Example 1 was 62.63%.

[0052] Experimental Example 2

[0053] The inhibitory effect of NP@A939572 prepared in Example 1 on osteoclast differentiation was verified in vitro. The specific method was as follows:

[0054] (1) In vitro inhibition of osteoclasts

[0055] After the extracted mouse bone marrow-derived macrophages adhered to the culture vessel, the osteoclast induction medium containing 25 ng / mL MCSF and 50 ng / mL RANKL was replaced. At the same time, different concentrations of NP@A939572 were added for treatment. After 5 days, the cells were fixed with 4% PFA for 10 min, washed 3 times with PBS, and stained with tartrate-resistant acid phosphatase (TRAP) staining solution (Sigma Aldrich).

[0056] (2) Western Blot assay

[0057] Macrophages were induced with RANKL for 3 days. Protein was extracted according to the BIO-RAD 4×Laemmli Sample Buffer (#161-0737) instructions. After lysis at room temperature for 5 min, the protein was collected and treated in a 100℃ metal bath for 10 min. SDS-PAGE preparation: A 10% separating gel and stacking gel were prepared. APS and TEMED were added for polymerization, and the gel was allowed to stand with a comb inserted. An equal volume of protein sample and marker were added for loading. Electrophoresis was performed on the separating gel at 80V, and then the voltage was adjusted to 120V to the endpoint. Transfer after electrophoresis: The gel was transferred to a methanol-activated PVDF membrane (300 mA constant current, 95 min). After transfer, the membrane was blocked with 5% skim milk for 1-2 h. Antibody incubation: Primary antibody (1:1000) was incubated overnight at 4℃, followed by 5 washes with TBST (7 min / wash); HRP-secondary antibody was incubated at room temperature for 1 h, followed by 5 washes with TBST. Target protein bands were detected.

[0058] (3) Quantitative polymerase chain reaction (qPCR)

[0059] Two days after RANKL induction of macrophages, total RNA was extracted using the Kangwei Century Ultrapure RNA Extraction Kit (CW0581). The simplified procedure was as follows: 1 mL of TRizon Reagent was added to each well for lysis for 5 min, followed by 200 μL of chloroform, mixing, and incubation for 5 min. The mixture was then centrifuged at 12000g for 10 min at 4°C. The supernatant was mixed with an equal volume of 75% ethanol and purified using a silica-based adsorption column. After washing, RNA was eluted with enzyme-free water. cDNA synthesis was then performed using the Kangwei Century Reverse Transcription Kit (CW2569). The resulting products were further detected using the Yisheng qPCR reagent (11201ES03) for real-time quantitative PCR (qPCR). Primer sequences are shown in Table 1.

[0060] Table 1 Primer sequences involved in the detection of osteoclast gene expression by real-time PCR

[0061]

[0062] In this experimental example, the characterization results of the anti-osteoclast effect of NP@A939572 prepared in Example 1 in vitro were as follows: Two days after osteoclast induction, macrophages without NP@A939572 treatment, using β-actin (ACTB) as an internal control protein, highly expressed osteoclast differentiation-related proteins, such as activated T cell nuclear factor 1 (NFATc1), proto-oncogenes, and AP-1 transcription factor subunit (c-Fos). After treatment with different concentrations of NP@A939572, the expression of osteoclast differentiation-related proteins in macrophages increased compared with the uninduced group, but decreased compared with the osteoclast induction group alone. Moreover, the higher the concentration of NP@A939572, the lower the expression of osteoclast differentiation-related proteins. Further detection of the RNA level of osteoclast-related indicators yielded consistent results, demonstrating that NP@A939572 can significantly inhibit osteoclast differentiation in vitro. Figure 7 The number of particles on the horizontal axis is on the order of 10. 11 Five days after osteoclast induction, macrophages not treated with NP@A939572 differentiated into a large number of fused, multinucleated osteoclasts, while macrophages treated with NP@A939572 differentiated into significantly fewer mature osteoclasts. Furthermore, the higher the concentration of NP@A939572, the fewer osteoclasts formed, exhibiting a concentration-dependent inhibition. Figure 8 (Scale bar is 100μm).

[0063] Experimental Example 3

[0064] The biodistribution of the nanoparticles prepared in Example 1 was verified: NPs were stained with lipophilic DiR or DiO fluorescent dyes (KGE2604-10, Jiyuan Biotechnology, Nanjing, China) and designated as CY5-POCM-NP@A939572 (POCM-CY5@NP); each mouse was injected intravenously via the tail vein with 50 μL containing 1.0 × 10⁻⁶ ppm of the nanoparticles. 10 PBS solution containing CY5-POCM-NP@A939572 particles; 6 hours post-injection, major organs of mice injected with CY5-POCM-NP@A939572 were collected and imaged using the AniView100 multimodal in vivo animal imaging system (Guangzhou Bio-Thera Solutions, Ltd.). Figure 9 As shown, CY5-POCM-NP@A939572 (POCM-CY5@NP) can effectively target mouse bone tissue (red and yellow represent different fluorescence intensities).

[0065] Test Example 4

[0066] The efficacy of POCM-NP@A939572 prepared in Example 1 in rescuing osteoporosis induced by ovariectomy was verified in vivo:

[0067] (1) Osteoporosis model and nanovesicle drug delivery

[0068] Twelve-week-old female C57BL / 6 mice were housed in specific pathogen-free cages. Mice in each group underwent surgery after intraperitoneal anesthesia with pentobarbital. The sham-operated group received a sham surgery without ovariectomy. In the ovariectomy group, the ovaries were removed via a dorsal skin approach, entering the abdominal cavity at the mid-lumbar spine level. The ovaries were ligated and removed, and the peritoneum and skin were closed layer by layer by suturing. Postoperatively, POCM-NP@A939572 was administered intravenously once weekly for 8 weeks.

[0069] (2) Tartrate-resistant acid phosphatase (TRAP) staining of bone tissue sections

[0070] Mouse femoral samples were fixed with 4% paraformaldehyde (PFA) for 48 h, and then decalcified with commercially available 10% ethylenediaminetetraacetic acid (EDTA) for 2 weeks. After decalcification, the samples were washed with PBS and then sequentially placed in a gradient of ethanol solutions for dehydration. The dehydrated samples were placed in embedding cassettes, femoral orientation adjusted, and left at room temperature until the paraffin was completely solidified. Sections of 5–10 μm thickness were then cut using a paraffin microtome. The sections were stained with TRAP using a TRAP staining kit (Solarbio, G1492, Beijing). Cell nuclei were stained with 1% methyl green staining solution (Solarbio, G1652, Beijing). The stained positive areas were analyzed using ImageJ software (NIH, USA).

[0071] (3) Microcomputed tomography (μCT) analysis

[0072] Eight weeks post-surgery, mice in all groups were euthanized, and tissue samples were harvested and fixed bilaterally in 4% paraformaldehyde at room temperature for two days. Micro-CT scans with a resolution of 9 micrometers were performed on the left femur of each group of mice after fixation, with the following parameters: voltage 70 kV, current 80 μA. 130 layers originating from the growth plate region were selected as the region of interest for data analysis and parallel 3D reconstruction. Analytical parameters included the relative bone volume of trabeculae.

[0073] (4) Statistical analysis

[0074] All statistical analyses were performed using GraphPad Prism 8.0 software. Differences between two groups were analyzed using the Student's test; comparisons of three or more groups were performed using one-way ANOVA. Experimental data are expressed as mean ± standard deviation (Mean ± SD). The criteria for statistical significance were: *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.0001.

[0075] In this experimental example, the characterization results of POCM-NP@A939572 prepared in Example 1 for in vivo rescue of osteoporosis caused by ovariectomy are as follows:

[0076] Eight weeks after ovariectomy in 12-week-old female mice, the relative bone volume of trabecular bone in the femur of the model group (OVX) was significantly lower than that in the sham-operated group; however, in the POCM-NP@A939572 intervention group, this index was significantly higher than that in the ovariectomy group, suggesting that POCM-NP@A939572 has a significant preventive and therapeutic effect on osteoporosis caused by estrogen deficiency. Figure 10 and Figure 11 Furthermore, tartrate-resistant acid phosphatase (TRAP) staining of bone tissue sections revealed a significant increase in osteoclast count after ovariectomy, while a significant decrease in osteoclast count was observed after nanovesicle treatment. Figure 12 ).

[0077] In summary, the POCM-NP@A939572 provided by this invention can inhibit the expression of osteoclast differentiation proteins in vitro, and has the effect of preventing and treating osteoporosis.

[0078] The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

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. 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. The mass ratio of PLGA to A939572 in NP@A939572 is 5-7:1; The mass ratio of the osteoclast precursor cell membrane vesicles to NP@A939572 is 1:6-10.

2. The application according to claim 1, 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.

3. 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.

4. 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.

5. The application according to any one of claims 1-4, characterized in that, The osteoporosis mentioned is osteoporosis caused by estrogen deficiency.

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