Application of lipid nanoparticles coated with RCN2 protein in treatment of postmenopausal osteoporosis

Abstract

The invention discloses application of lipid nanoparticles coated with RCN2 protein in treatment of postmenopausal osteoporosis, and the RCN2 recombinant protein has an amino acid sequence as shown in SEQ ID NO. 1; the RCN2 recombinant protein is coated with the lipid membrane mixture, the lipid membrane mixture comprises phospholipid, and the phospholipid is coupled with a Perilipin antibody. The lipid nanoparticle provided by the invention realizes precise targeting and delivers the RCN2 protein to bone marrow fat cells, and can treat postmenopausal osteoporosis.

Description

Technical Field

[0001] The invention relates to the technical field of medicine and biochemistry, and provides an application of lipid nanoparticles coated with RCN2 protein in treating postmenopausal osteoporosis. Background Art

[0002] Osteoporosis (OP) is a chronic bone disease in the elderly characterized by bone loss, bone microstructure destruction, increased bone fragility, and easy fracture. It has the characteristics of high morbidity and high mortality of fractures, which seriously affects the quality of life of patients and brings heavy burdens to society and families. Among them, postmenopausal osteoporosis is the most common type of osteoporosis. It is a bone tissue metabolic disease characterized by rapid bone loss and increased risk of fractures caused by estrogen deficiency due to ovarian dysfunction in postmenopausal women. Studies have shown that the dynamic imbalance of bone formation and bone resorption induced by estrogen deficiency is the most important pathogenesis of postmenopausal osteoporosis. At present, the drugs used in clinical treatment of postmenopausal osteoporosis include anti-bone resorption (such as bisphosphonates and dexamethasone) and bone formation promotion (such as teriparatide). However, long-term drug treatment can lead to excessive inhibition of bone turnover in patients, and increase the risk of thrombosis and the incidence of malignant tumors. At the same time, the rebound phenomenon after treatment interruption also causes people to worry about the efficacy. Therefore, developing effective drugs to treat postmenopausal osteoporosis is of great and far-reaching significance.

[0003] RCN2 (reticulocalbin-2) as a lipolytic factor initiates bone marrow fat lipolysis by binding to the adipocyte receptor complex Neuropilin-2 / Intergrin-b1, providing energy for immune progenitor cell generation and bone formation, thereby improving the body's immunity and maintaining bone mass. However, the effect of RCN2 on osteoclast-mediated bone resorption has not been reported. At the same time, RCN2 protein intervention in vivo lacks targeting, so how to accurately target and deliver RCN2 protein to bone marrow adipocytes is also an urgent problem to be solved. Summary of the invention

[0004] The present invention provides an application of lipid nanoparticles coated with RCN2 protein in the treatment of postmenopausal osteoporosis, which achieves precise targeting and delivery of RCN2 protein to bone marrow adipocytes, and can treat postmenopausal osteoporosis.

[0005] The technical problem solved by the present invention is achieved by adopting the following technical solutions:

[0006] In a first aspect, the present application provides a lipid nanoparticle coated with RCN2 protein, wherein the lipid nanoparticle comprises:

[0007] RCN2 recombinant protein, the amino acid sequence of which is shown in SEQ ID NO.1;

[0008] A lipid membrane mixture is used to encapsulate the RCN2 recombinant protein, wherein the lipid membrane mixture includes phospholipids, and the phospholipids are coupled to the Perilipin antibody.

[0009] In some optional embodiments, the average potential of the lipid nanoparticles is 48.56±0.51 mV.

[0010] In some optional embodiments, the average particle size of the lipid nanoparticles is 133±7 nm.

[0011] In some optional embodiments, based on the total mass of the lipid nanoparticles, the lipid nanoparticles include 7.1% RCN2 recombinant protein by mass.

[0012] In some optional embodiments, the lipid nanoparticles comprise a lipid membrane mixture with a mass content of 47.3% based on the total mass of the lipid nanoparticles.

[0013] In some optional embodiments, the lipid membrane mixture further comprises PEGylated phospholipids and cholesterol.

[0014] In some optional embodiments, the molar ratio of the PEGylated phospholipid, the phospholipid and the cholesterol is 1:1:1.

[0015] In a second aspect, the present application provides a method for preparing lipid nanoparticles coated with RCN2 protein according to the first aspect, the method comprising:

[0016] Mixing the lipid membrane mixture with the RCN2 recombinant protein so that the lipid membrane mixture covers the RCN2 recombinant protein to obtain coated particles; wherein the lipid membrane mixture includes phospholipids, and the amino acid sequence of the RCN2 recombinant protein is as shown in SEQ ID NO.1;

[0017] The coated particles are mixed with a solution containing perilipin antibodies and incubated to couple the phospholipids with the perilipin antibodies, thereby obtaining the lipid nanoparticles coated with the RCN2 protein.

[0018] In a third aspect, the present application provides a phospholipid-coupled antibody solution, comprising the lipid nanoparticles of the first aspect with a mass concentration of 40-60 mg / ml or the lipid nanoparticles coated with RCN2 protein prepared by the preparation method of the second aspect, the lipid-coupled antibody solution comprising phosphate buffer solution, glycerol, gelatin, tris(hydroxymethyl)aminomethane, glycine, disodium ethylenediaminetetraacetic acid, ethanolamine, and serum albumin, and the lipid-coupled antibody solution may optionally include lipid nanoparticles with a mass concentration of 50 mg / ml.

[0019] In a fourth aspect, the present application provides a use of the lipid nanoparticles coated with RCN2 protein of the first aspect or the lipid nanoparticles coated with RCN2 protein prepared by the preparation method of the second aspect in the preparation of a drug for treating osteoporosis.

[0020] This application has at least the following beneficial effects:

[0021] Studies have found that lipid nanoparticles coated with RCN2 protein (LNP-RCN2, which is modified by Perilipin) can inhibit the expression of osteoclastogenesis-related genes CTSK and NFATc1 in vivo, and have an effective effect in inhibiting osteoclastogenesis; under the action of LNP-RCN2, the volume, thickness and number of trabeculae can be significantly reduced, while the separation of trabeculae can be significantly increased; it can improve the bone loss of OVX mice, indicating that it can treat postmenopausal osteoporosis to a certain extent. LNP-RCN2 can inhibit the formation of osteoclasts in OVX mice; it can promote the formation of osteoblasts in OVX mice. LNP-RCN2 can reduce the formation of bone marrow adipocytes in OVX mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 It is a schematic diagram of constructing lipid nanoparticles coated with RCN2 protein and a schematic diagram of the potential of lipid nanoparticles.

[0024] Figure 2 This is a schematic diagram of downregulating osteoclast differentiation indicators after lipid nanoparticles coated with RCN2 protein were injected in situ into the bone marrow cavity of OVX mice.

[0025] Figure 3 Schematic diagram of the inhibition of osteoclast differentiation after lipid nanoparticles coated with RCN2 protein were injected into the bone marrow cavity of OVX mice.

[0026] Figure 4 Schematic diagram of RCN2 protein-coated lipid nanoparticles increasing femoral bone mass in OVX mice.

[0027] Figure 5 Schematic diagram of the inhibition of osteoclast number by lipid nanoparticles coated with RCN2 protein.

[0028] Figure 6 Schematic diagram of lipid nanoparticles coated with RCN2 protein increasing the number of osteoblasts in the femur of OVX mice.

[0029] Figure 7 Schematic diagram of the reduction of femoral fat vacuoles in OVX mice by lipid nanoparticles coated with RCN2 protein. DETAILED DESCRIPTION

[0030] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0031] Example 1 Preparation of lipid nanoparticles containing RCN2 protein

[0032] ① Polycarbonate membrane was purchased from Whatman, SPC (sphingomyelin phosphorylcholine), cholesterol, DSPE-PEG and phospholipid-coupled antibodies were purchased from Xi'an Ruixi Biotechnology Co., Ltd.;

[0033] ② Dissolve phospholipids, cholesterol and polyethylene glycolated phospholipids (DSPE-PEG) in chloroform according to a predetermined molar ratio (1:1:1), and evaporate the chloroform using a rotary evaporator to form a uniform liposome film;

[0034] ③ Using a liposome extruder, extrude the liposome solution multiple times through polycarbonate membranes with different pore sizes to obtain liposomes of uniform size;

[0035] ④ Mix the RCN2 recombinant protein solution with the liposome solution, and evenly disperse the protein in the liposome by stirring or ultrasound. Incubate the mixture at room temperature for a period of time (e.g., 30 minutes to 1 hour) to ensure that the RCN2 recombinant protein is fully encapsulated in the liposome;

[0036] ⑤ Use a dialysis bag or dialysis device to dialyze the liposome solution to remove unencapsulated RCN2 recombinant protein and other impurities. After dialysis, add PBS to the required volume as needed to obtain a uniform and pure liposome-encapsulated RCN2 recombinant protein solution;

[0037] ⑥ Take 100 mg of liposomes coated with RCN2 recombinant protein and dissolve it in 1 mL of phospholipid solution, take 1 mg of Perilipin antibody and dissolve it in 1 mL of reaction buffer (PBS solution contains glycerol, Gelatin, Tris, glycine, EDTA, ethanolamine, serum protein); Perilipin antibody was purchased from abcam (ab3526).

[0038] ⑦ After 1 mL of phospholipid solution and 1 mL of Perilipin antibody are mixed evenly, the mixture is reacted at room temperature for 2 hours to achieve coupling of the "Perilipin" antibody to the phospholipid coated on the surface of the RCN2 recombinant protein;

[0039] ⑧ After the reaction, the reaction solution was transferred to an ultrafiltration tube, and all solvents were removed by centrifugal ultrafiltration at 9500 rpm and 4°C, and washed twice with pure water to remove unreacted phospholipids, i.e., Perilipin-modified lipid nanoparticles coated with RCN2 protein (LNP-RCN2, referred to as: lipid nanoparticles coated with RCN2 protein);

[0040] ⑨ The phospholipid-coupled antibody solution was stored at 4°C, and appropriate amount of samples were taken for scanning electron microscopy and transmission electron microscopy (see the structure for details). Figure 1 ).

[0041] Figure 1 Schematic diagram of constructing lipid nanoparticles (LNP-RCN2) coated with RCN2 protein; Figure A is a transmission electron microscopy schematic diagram of lipid nanoparticles, and Figures B and C are scanning electron microscopy schematic diagrams of lipid nanoparticles, indicating that the particle size is 133 ± 7nm , Figure D is a schematic diagram of the potential of lipid nanoparticles (LNP-RCN2), illustrating that the average potential of the lipid nanoparticles is 48.56±0.51 mV, that is, the particles obtained in step 8.

[0042] Meanwhile, the above steps ① to ③, and ⑥ to ⑨ were repeated without encapsulating the RCN2 recombinant protein inside the liposomes to prepare Perilipin-modified empty lipid nanoparticles (LNP-vector).

[0043] Example 2 LNP-RCN2 down-regulates osteoclast differentiation-related genes

[0044] ① After the mice were anesthetized with ether, the ovaries of both sides of the 6-week-old female mice were removed. The mice in the sham group only underwent surgery without removing the ovaries, and the sham operation group mouse model of ovariectomized mice, namely OVX mice, was constructed;

[0045] ②After 8 weeks, Perilipin-modified empty lipid nanoparticles (LNP-vector) and Perilipin-modified lipid nanoparticles coated with RCN2 protein (LNP-RCN2) were injected into the sham operation group and OVX mice through the tail vein at 20 mg / kg, once every 3 days, for a total intervention of 60 days.

[0046] ③ After 60 days, the osteoclasts of mice were isolated, cultured and intervened: the mice were anesthetized with ether, and the femurs and tibias of the sham + LNP-vector group, OVX + LNP-vector group, and OVX + LNP-RCN2 group were separated. The two ends were cut with scissors and the bone marrow was flushed out with a syringe. The cells were centrifuged at 1000 g for 3 min, the supernatant was removed, the cells were resuspended with α-MEM medium, and incubated overnight in a cell culture incubator at 37°C.

[0047] ④On the next day, collect the cells and centrifuge at 1400 rpm for 5 minutes, resuspend the cells and adjust the cell number to 1.0×10 5 cells / ml, spread 1 ml / well in a 24-well plate, add 20 μL mouse M-CSF reagent (human macrophage colony stimulating factor, Beijing Sino Biological, concentration: 2 ng / μL) to each well, and incubate in a 37°C cell culture incubator overnight.

[0048] ⑤ On the next day, 1 μL of mouse osteoclast induction reagent - RANKL (receptor activator of nuclear factor κB ligand, R&D company, concentration: 40 ng / μL) was added to each well. After incubation in a 37°C cell culture incubator for 3 days, the cells were collected by centrifugation at 1400 rpm for 5 minutes.

[0049] ⑥ Add 1 ml of RNA extraction reagent-Trizol (Hunan Aikerui Company) to each tube and let it stand at room temperature for 5-10 minutes; after 10 minutes, add 250 μL of chloroform to each well, shake vigorously on the oscillator for 15 seconds, and let it stand at room temperature for 15 minutes.

[0050] ⑦ Centrifuge at 12000g for 15 min at 4℃, transfer the upper aqueous phase to a new 1.5ml RNase free EP tube, add an equal volume of isopropanol, invert to mix, and let settle at -20℃ overnight.

[0051] ⑧ Centrifuge at 4°C, 12000g for 15 min, discard the supernatant, wash twice with 75% ethanol, dry the alcohol and add RNase free water to dissolve the RNA.

[0052] ⑨Take 1 μg of total RNA and reverse transcribe it into cDNA, add SYBR fluorescent dye and specific fluorescent quantitative PCR primers, and detect the expression of osteoclast differentiation-related genes-CTSK and NFATc1 on a fluorescent quantitative PCR instrument.

[0053] See also Figure 2 , Each value is the mean ± SEM of three tests, *p<0.05, ***p<0.001.

[0054] Table 1

[0055] Group NFATc1 relative expression level CTSK relative expression level Sham+LNP-vector 1±0.05716 1±0.04695 OVX+LNP-vector 3.476±0.1323*** 32.05±1.418*** OVX+LNP-RCN2 1.958±0.1386*** 25.95±1.071*

[0056] from Figure 2 It can be seen that compared with the Sham+LNP-vector group, the expression levels of osteoclastogenesis-related genes CTSK and NFATc1 in the OVX+LNP-vector group were significantly increased, while LNP-RCN2 intervention significantly inhibited their expression levels. This result shows that RCN2 can inhibit the expression of osteoclastogenesis-related genes CTSK and NFATc1 in vivo, and has an effective effect in inhibiting osteoclastogenesis.

[0057] Example 3 LNP-RCN2 inhibits osteoclast differentiation

[0058] After the mice were anesthetized with ether, the ovaries on both sides of the 6-week-old female mice were removed. The mice in the sham group only underwent surgery without removing the ovaries, and the ovariectomized mice and sham mice models were constructed. After 8 weeks, Perilipin-modified empty lipid nanoparticles (LNP-vector) and Perilipin-modified lipid nanoparticles coated with RCN2 protein (LNP-RCN2) were injected into the sham group and OVX mice through the tail vein at 20 mg / kg, once every 3 days, for a total of 60 days of intervention. After 60 days, the mice were anesthetized with ether, the femurs and tibias of the mice were removed, and the mouse bone marrow cells were extracted (specifically the same as steps 1 to 3 of Example 2), and 1×10 ^5 The cells / well were inoculated in a 24-well plate at a density of 1.54 × 10 cells / well, and the plates were set as follows: (sham) + LNP-vector group, OVX + LNP-vector group, and OVX + LNP-RCN2 group. Osteoclast differentiation was induced using osteoclast differentiation medium containing M-CSF reagent (human macrophage colony stimulating factor, Beijing Sino Biological, concentration: 2 ng / μL) and RANKL reagent (receptor activator of nuclear factor κB ligand, concentration: 40 ng / μL). After 6 days of culture, multinuclear osteoclasts were formed, the culture medium was discarded, osteoclasts were stained with Trap staining kit, and photos were taken.

[0059] See also Figure 3 , Each value is the mean ± SEM of three tests, *p<0.05, ***p<0.001. Figure 3It is a schematic diagram of the inhibition of osteoclast differentiation after in situ injection of perilipin-modified lipid nanoparticles coated with RCN2 protein into the bone marrow cavity of OVX mice; Figure A is a schematic diagram of tartrate-resistant acid phosphatase (Trap) staining of RCN2 inhibiting osteoclast formation, and Figure B is a statistical analysis of the number of Trap-positive (Trap+) osteoclasts in each group in Example 3.

[0060] Table 2

[0061] Group <![CDATA[Trap + Osteoclasts]]> Sham+LNP-vector 232.2±17 OVX+LNP-vector 416.9±27.71*** OVX+LNP-RCN2 298.9±15.71*

[0062] What does this set of data show? Which is the benchmark group?

[0063] Compared with OVX+LNP-vector, the number of osteoclasts in OVX+LNP-RCN2 was significantly reduced, indicating that LNP-RCN2 has the effect of inhibiting osteoclast differentiation.

[0064] Example 4 RCN2 promotes bone formation in OVX mice

[0065] After the mice were anesthetized with ether, the ovaries of both sides of the 6-week-old female mice were removed. The mice in the sham group only underwent surgery without removing the ovaries, and the ovariectomized mice and sham mice models were constructed. After 8 weeks, Perilipin-modified empty lipid nanoparticles (LNP-vector) and Perilipin-modified lipid nanoparticles coated with RCN2 protein (LNP-RCN2) were injected into the sham group and OVX mice from the tail vein at 20 mg / kg, once every 3 days, for a total of 60 days of intervention. After 60 days, the mice were anesthetized with ether, and the femur and tibia of the mice were removed. Micro-CT was used to detect and analyze the microstructure of the bone specimens, and femoral imaging scans were performed. The trabecular concentration area at the same position was selected by 3D-MED image processing software for 2D and 3D image reconstruction.

[0066] See also Figure 4 , Each value is the mean ± SEM of three tests, *p<0.05, **p<0.01, ***p<0.001. Figure 4 It is a schematic diagram of Perilipin-modified lipid nanoparticles coated with RCN2 protein increasing femoral bone mass in OVX mice; wherein, Figure A is a schematic diagram of three-dimensional reconstruction of the femur of OVX mice in which RCN2 increases bone mass, Figure B is a schematic diagram of RCN2 increasing femoral trabecular volume in OVX mice, Figure C is a schematic diagram of RCN2 increasing femoral trabecular thickness in OVX mice, Figure D is a schematic diagram of RCN2 increasing femoral trabecular number in OVX mice, and Figure E is a schematic diagram of RCN2 reducing femoral trabecular separation in OVX mice.

[0067] Table 3

[0068]

[0069]

[0070] from Figure 4 It can be seen that compared with the sham group mice, the femoral trabecular volume, trabecular thickness, and trabecular number of OVX mice were significantly reduced, while the trabecular separation was significantly increased. The above indicators were improved after LNP-RCN2 intervention. This result shows that LNP-RCN2 has the effect of improving bone loss in OVX mice.

[0071] Table 4

[0072] Group <![CDATA[Trap + Osteoclasts (N / mm 2 )]]> Sham+LNP-vector 12.280±0.847 OVX+LNP-vector 22.830±1.343** OVX+LNP-RCN2 14.210±1.107**

[0073] Figure 5 It is a schematic diagram of the inhibition of osteoclast number by lipid nanoparticles modified with Perilipin and coated with RCN2 protein; wherein, Figure A is a schematic diagram of tartrate-resistant acid phosphatase (Trap) staining, and Figure B is a statistical analysis diagram.

[0074] from Figure 5 As can be seen from Table 4, compared with the sham group mice, the number of osteoclasts in the femur of OVX mice was significantly increased, while the number of osteoclasts after LNP-RCN2 intervention was significantly reduced compared with the OVX group, indicating that LNP-RCN2 can inhibit the formation of osteoclasts in OVX mice.

[0075] Table 5

[0076] Group <![CDATA[OCN + Osteoblasts (N / mm 2 )]]> Sham+LNP-vector 10.810±0.645 OVX+LNP-vector 3.737±0.313*** OVX+LNP-RCN2 5.468±0.423*

[0077] Figure 6 It is a schematic diagram of the increase in the number of osteoblasts in the femur of OVX mice by lipid nanoparticles modified with Perilipin and coated with RCN2 protein; wherein, Figure A is a schematic diagram of osteocalcin (OCN) staining, and Figure B is a statistical analysis diagram.

[0078] from Figure 6 As can be seen from Table 5, compared with the sham group mice, the number of femoral osteoblasts in OVX mice was significantly reduced, while the number of femoral osteoblasts in mice after LNP-RCN2 intervention was significantly increased compared with the OVX group, indicating that LNP-RCN2 can promote osteoblast formation in OVX mice.

[0079] Table 6

[0080] Group <![CDATA[Number of adipocytes (N / mm 2 )]]> Sham+LNP-vector 80.520±3.256 OVX+LNP-vector 149.500±8.325*** OVX+LNP-RCN2 118.200±7.073*

[0081] Figure 7It is a schematic diagram of the reduction of the number of femoral fat vacuoles in OVX mice by Perilipin-modified lipid nanoparticles coated with RCN2 protein; wherein, Figure A is a schematic diagram of femoral HE staining, and Figure B is a statistical analysis diagram of fat vacuoles.

[0082] from Figure 7 As can be seen from Table 6, compared with the sham group mice, the number of femoral adipocytes in OVX mice was significantly increased, while the number of bone marrow adipocytes after LNP-RCN2 intervention was significantly reduced compared with the OVX group, indicating that LNP-RCN2 can reduce the formation of bone marrow adipocytes in OVX mice.

[0083] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A lipid nanoparticle coated with RCN2 protein, characterized in that: The lipid nanoparticles include: RCN2 recombinant protein, the amino acid sequence of which is shown in SEQ ID NO.1; A lipid membrane mixture is used to encapsulate the RCN2 recombinant protein, wherein the lipid membrane mixture includes phospholipids, and the phospholipids are coupled to the Perilipin antibody.

2. The lipid nanoparticle according to claim 1, characterized in that The average potential of the lipid nanoparticles was 48.56±0.51 mV.

3. The lipid nanoparticle according to claim 1, characterized in that The average particle size of the lipid nanoparticles is 133±7 nm.

4. The lipid nanoparticle according to claim 1, characterized in that Based on the total mass of the lipid nanoparticles, the lipid nanoparticles include 7.1% by mass of the RCN2 recombinant protein.

5. The lipid nanoparticle according to claim 1, characterized in that Based on the total mass of the lipid nanoparticles, the lipid nanoparticles include a lipid membrane mixture with a mass content of 47.3%.

6. The lipid nanoparticle according to claim 1, characterized in that The lipid membrane mixture also includes PEGylated phospholipids and cholesterol.

7. The lipid nanoparticle according to claim 6, characterized in that The molar ratio of the PEGylated phospholipid, the phospholipid and the cholesterol is 1:1:

1.

8. A method for preparing lipid nanoparticles coated with RCN2 protein according to any one of claims 1 to 7, characterized in that: The method comprises: Mixing the lipid membrane mixture with the RCN2 recombinant protein so that the lipid membrane mixture covers the RCN2 recombinant protein to obtain coated particles; wherein the lipid membrane mixture includes phospholipids, and the amino acid sequence of the RCN2 recombinant protein is shown in SEQ ID NO.1; The coated particles are mixed with a solution containing perilipin antibodies and incubated to couple the phospholipids with the perilipin antibodies, thereby obtaining the lipid nanoparticles coated with the RCN2 protein.

9. A phospholipid-coupled antibody solution, characterized in that: The lipid-coupled antibody solution comprises the lipid nanoparticles according to any one of claims 1 to 7 or the lipid nanoparticles prepared by the preparation method according to claim 8 at a mass concentration of 40-60 mg / ml, and the lipid-coupled antibody solution comprises phosphate buffer solution, glycerol, gelatin, tris(hydroxymethyl)aminomethane, glycine, disodium ethylenediaminetetraacetic acid, ethanolamine, and serum protein.

10. Use of the lipid nanoparticles coated with RCN2 protein according to any one of claims 1 to 7 or the lipid nanoparticles prepared by the preparation method according to claim 8 in preparing a drug for treating osteoporosis.

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