Application of polarized barium titanate nanoparticles in preparation of medicine for preventing and treating inflammatory bone resorption

Abstract

The invention relates to application of polarized barium titanate nanoparticles in preparation of drugs for preventing and treating inflammatory bone resorption, and belongs to the technical field of biological medicines. The invention provides application of polarized barium titanate nanoparticles in preparation of a medicine for preventing and treating inflammatory bone resorption. The polarized barium titanate nano-particles can be endocytosed by macrophages and act on the macrophages in a targeting manner, so that a'macrophage model endocytosing the nano pBTO 'is constructed, an intracellular electric field can be provided, a macrophage model generating the electric field in the cell is constructed, osteoclast differentiation of the macrophages is inhibited in a targeting manner, and osteoclast resorption is inhibited.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of polarized barium titanate nanoparticles in the preparation of drugs for preventing and treating inflammatory bone resorption. Background Art

[0002] Inflammatory bone resorption occurs in common oral and maxillofacial diseases such as periodontitis, peri-implantitis and mandibular osteomyelitis. Macrophages, as an important component of innate immunity, play an important role in bone resorption. Studies have shown that osteoclasts can be formed from both monocyte-macrophage precursor cells in tissues and mature macrophages. Therefore, in order to inhibit bone resorption, it is necessary to find a method to inhibit macrophage osteoclast differentiation and thus inhibit osteoclast resorption. Currently commonly used anti-bone resorption drugs, such as bisphosphonates, cathepsin K inhibitors and RANKL (osteoclast differentiation factor) inhibitors, can effectively inhibit osteoclast bone resorption, but still have many side effects, including nephrotoxicity, induction of allergic reactions and jaw necrosis. In addition, these drugs cannot target the inhibition of macrophage osteoclast differentiation. Therefore, new treatment options such as targeted inhibition of macrophage osteoclast differentiation are urgently needed. Summary of the Invention

[0003] The purpose of the present invention is to overcome the shortcomings of the prior art and provide the use of polarized barium titanate nanoparticles in the preparation of drugs for preventing and treating inflammatory bone resorption.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] In a first aspect, the present invention provides the use of polarized barium titanate nanoparticles in the preparation of a drug for preventing and treating inflammatory bone resorption.

[0006] The present invention prepares biocompatible piezoelectric nanomaterial pBTO, which, after being endocytosed by macrophages, can obtain a "macrophage model of endocytosed nano-pBTO" that generates an electric field in the cell, thereby targetedly inhibiting the osteoclast differentiation of macrophages and thus inhibiting bone resorption.

[0007] Furthermore, the drug is a drug that inhibits macrophage osteoclast differentiation.

[0008] Furthermore, the drug is a drug that inhibits macrophage osteoclast differentiation by endocytosis of polarized barium titanate nanoparticles by macrophages.

[0009] Furthermore, the drug is a drug that reduces the expression level of at least one of the osteoclast differentiation-related genes Acp5, CTSK, c-FOS, DC-STAMP and Atp6v0d2.

[0010] Furthermore, the drug is a drug that inhibits osteoclastogenesis.

[0011] In a specific embodiment of the present invention, the particle size of the polarized barium titanate nanoparticles is 90 to 110 nm, and the surface potential is 1.9V to 2.1V.

[0012] Furthermore, the barium titanate nanoparticles were electrically polarized with a voltage of 2.4 to 2.6 kV for 29.5 to 30.5 minutes.

[0013] As a preferred embodiment of the present invention, the barium titanate nanoparticles are electro-polarized at a voltage of 2.5 kV for 30 minutes.

[0014] Furthermore, the preparation method of the barium titanate nanoparticles includes the following steps:

[0015] S1: uniformly mixing the titanium butoxide ethanol solution and the ammonia solution to obtain a mixture 1;

[0016] S2: Mixing the mixture 1 of step S1 and an aqueous solution of barium hydroxide octahydrate uniformly, adding diethanolamine, and mixing uniformly to obtain a mixture 2;

[0017] S3: subjecting the mixture 2 of step S2 to a hydrothermal reaction, washing and drying, to obtain barium titanate nanoparticles.

[0018] Preferably, in step S1, the volume ratio of the titanium butoxide ethanol solution to the ammonia solution is (3.9-4.1):1, the concentration of the titanium butoxide ethanol solution is 1.2-1.3 mmol / mL, and the concentration of the ammonia solution is 24.5-25.5% w / v.

[0019] Most preferably, in step S1, the volume ratio of the titanium butoxide ethanol solution and the ammonia solution is titanium butoxide ethanol solution: ammonia solution = 4:1, the concentration of the titanium butoxide ethanol solution is 1.25 mmol / mL, and the concentration of the ammonia solution is 25% w / v.

[0020] Preferably, in step S2, the volume ratio of mixture 1, barium hydroxide aqueous solution and diethanolamine is mixture 1:barium hydroxide aqueous solution:diethanolamine = (4.9-5.1):(4.9-5.1):1, and the concentration of the barium hydroxide aqueous solution is 1.35-1.45 mmol / mL.

[0021] Most preferably, in step S2, the mixture 1: barium hydroxide aqueous solution: diethanolamine = 5:5:1, and the concentration of the barium hydroxide aqueous solution is 1.4 mmol / mL.

[0022] Furthermore, in step S3, the hydrothermal reaction temperature is 199-201° C., and the reaction time is 47.5-48.5 h.

[0023] Preferably, in step S3, the hydrothermal reaction temperature is 200° C. and the time is 48 hours.

[0024] Furthermore, in step S3, the washing includes repeatedly washing the hydrothermal reaction product with water and ethanol.

[0025] Furthermore, in step S3, the drying includes drying at 59-61° C. for 23.5-24.5 hours.

[0026] Preferably, in step S3, the drying comprises drying at 60° C. for 24 hours.

[0027] Furthermore, in step S1, S2 or S3, the mixing time is 14.5 to 15.5 minutes.

[0028] Preferably, the mixing time is 15 minutes.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] Piezoelectric materials can generate electrical activity due to the asymmetric movement of ions or charges caused by slight deformation. Through coupling electricity, polarized charges are generated in the piezoelectric material, forming a piezoelectric potential and establishing a built-in electric field. The present invention prepares polarized piezoelectric nanomaterial pBTO (polarized barium titanate nanoparticles), which can be internalized by macrophages and targeted to macrophages, thereby constructing a "macrophage model that internalizes nano-pBTO", which can provide an intracellular electric field, thereby constructing a macrophage model that generates an electric field in the cell, targeting the inhibition of osteoclast differentiation of macrophages, thereby inhibiting bone resorption. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Scanning electron microscope (SEM) images of BTO and pBTO. A is BTO; B is pBTO.

[0032] Figure 2 Figure 2 shows the X-ray diffraction results and piezoelectric response of BTO and pBTO. A is the X-ray diffraction result; B is the piezoelectric response.

[0033] Figure 3 The scanning Kelvin microscope results of BTO and pBTO are shown in Figure 1. A is BTO and B is pBTO.

[0034] Figure 4 TEM images of BTO and pBTO endocytosis in RAW 264.7 macrophages. A: Control; B: BTO endocytosis; C: pBTO endocytosis. Red arrows indicate mitochondria, and white arrows indicate nanomaterials.

[0035] Figure 5Figure 2 shows the osteoclast differentiation of the macrophage model of endocytic nanomaterials displayed and counted by optical fluorescence microscopy. A shows the results of each group displayed by optical fluorescence microscopy; B shows the results of each group TRAP. + Cell number.

[0036] Figure 6 The relative expression levels of osteoclast differentiation-related genes Acp5, CTSK, c-FOS, DC-STAMP and Atp6v0d2 mRNA in the qRT-PCR "macrophage model of endocytosed nanomaterials" are shown.

[0037] Figure 7 The results of micro-computed tomography scans are shown in Figure 1. A is a 3D image, and B is the bone volume / tissue volume (BV / TV).

[0038] Figure 8 Figure 1 shows histological analysis and immunofluorescence staining of skull bones. A shows histological sections; B shows the percentage of bone erosion area.

[0039] Figure 9 Histological analysis and immunofluorescence staining of skull. A is a histological section; B is the number of TRAP-positive osteoclasts. DETAILED DESCRIPTION

[0040] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0041] The experimental animals were aged C57 / BL6 female mice (from the Experimental Animal Center of Sun Yat-sen University. All animal experiments were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University, No. SYSU-IACUC-2023).

[0042] Example 1 Preparation of BTO and pBTO

[0043] 1. Construct ferroelectric barium titanate nanoparticles by precisely adjusting the hydrothermal reaction parameters.

[0044] (1) 8.509 g (25 mmol) of titanium butoxide (99.0%, China Aladdin Co., Ltd.) was dissolved in 20 mL of anhydrous ethanol, and 5 mL of ammonia solution (25% w / v ammonia, China Aladdin Co., Ltd.) was slowly added and stirred for 15 min to obtain a titanium butoxide solution.

[0045] (2) 11.025 g (35 mmol) of barium hydroxide octahydrate (98.0%, Shanghai MacLean Biochemical Technology Co., Ltd.) was dissolved in 25 mL of deionized water and stirred in a 90° C. water bath until completely dissolved to obtain a barium hydroxide octahydrate solution.

[0046] (3) The titanium butoxide solution mixture was slowly added to the barium hydroxide octahydrate solution, stirred for 15 minutes, and 5 mL of DEA (diethanolamine, Aladdin Co., Ltd., China) was added dropwise, and stirring was continued for 15 minutes to obtain a mixture.

[0047] (4) The mixture of step (3) was transferred to a 100 mL polytetrafluoroethylene liner in a hydrothermal reactor and incubated at 200° C. for 48 h to obtain a synthesized product. The synthesized product was repeatedly washed with deionized water and ethanol and dried at 60° C. for 24 h to obtain unpolarized nano-BTO (barium titanate nanoparticles).

[0048] 2. Construct polarized BTO (pBTO).

[0049] BTO powder was placed in an insulating mold with upper and lower copper sheets serving as polarization electrodes. Polarization was then performed using a 2.5 kV electrical stimulus for 30 minutes. The BTO nanoparticles were polarized to produce pBTO. The upper and lower portions of the pBTO nanoparticles have different surface potentials, further establishing an intrinsic nanoscale electric field.

[0050] Example 2 Characterization of BTO and pBTO

[0051] 1. Scanning electron microscope (SEM) images show that the BTO and pBTO nanoparticles prepared in Example 1 are cubic in shape and uniform in size, with a size of 90 to 110 nm ( Figure 1 A and Figure 1 B).

[0052] 2. X-ray diffraction results show that the prepared barium titanate nanoparticles conform to the standard card of tetragonal barium titanate (JCPDS, PDF#75-0462), indicating that the prepared BTO and pBTO nanoparticles are tetragonal crystals with good ferroelectric properties ( Figure 2 A).

[0053] 3. The electrical properties of BTO and pBTO nanoparticles were analyzed using a piezoelectric force microscope. The results showed a typical butterfly-shaped amplitude loop and a phase change of about 180°, which demonstrated the excellent piezoelectric response of pBTO nanoparticles and the existence of ferroelectric polarization conversion under an external electric field. Figure 2 B).

[0054] 4. Scanning Kelvin microscopy results show that the surface potential of pBTO nanoparticles has increased significantly compared to before polarization, and the surface potential is 1.9~2.1V. Figure 3 A and Figure 3 B).

[0055] These results demonstrate that the present invention has successfully constructed polarized barium titanate nanoparticles with excellent piezoelectric properties. Due to the different electric potentials on the upper and lower surfaces of the nanoparticles, cells can be electrically stimulated, forming a built-in electric field at the nanoscale interface.

[0056] Example 3 Construction of a “macrophage model for endocytosing nanomaterials”

[0057] 1. Experimental Methods

[0058] RAW 264.7 macrophages were cultured in DMEM / F12 medium containing 10% fetal bovine serum, 10 mg / mL streptomycin, and 10 U / mL penicillin at 37°C in a humidified atmosphere containing 5% CO2. 100 μg / mL nanopBTO, 100 μg / mL pBTO, or PBS (phosphate buffered saline) were co-cultured with the macrophages for 2 days to allow the macrophages to internalize the nanomaterials, thereby obtaining a "macrophage model that internalizes nanomaterials" (hereinafter referred to as the cell model). The cell model was washed with PBS, pipetted, centrifuged at 3000 rpm for 2 minutes, fixed with 2.5% (v / v) glutaraldehyde solution at room temperature for 30 minutes, and stored at 4°C to obtain a fixed cell model.

[0059] The fixed cell model was refixed with 1% (w / v) OsO4 in 0.1M PB (phosphate buffer, pH 7.4) at room temperature for 2 hours and rinsed in 0.1M PB (pH 7.4) for 15 minutes three times to obtain a rinsed cell model. The rinsed cell model was dehydrated in 30% (v / v), 50% (v / v), 70% (v / v), 80% (v / v), 95% (v / v), 100% (v / v), and 100% (v / v) ethanol at room temperature for 20 minutes each, and dehydrated twice in 100% (v / v) acetone for 15 minutes each. The model was then resin-infiltrated and embedded, and then polymerized and ultrathinly sectioned to obtain cell model slices.

[0060] Cell model sections were stained with 2% (w / v) uranyl acetate saturated alcohol for 8 minutes in the dark, rinsed three times in 70% (v / v) ethanol, and then rinsed three times in ultrapure water. The sections were then stained with 2.6% (w / v) lead citrate for 8 minutes and rinsed three times in ultrapure water. The sections were then dried on filter paper, placed on a grid, and dried overnight at room temperature. Images were captured using a transmission electron microscope (TEM).

[0061] 2. Experimental Results

[0062] like Figure 4 As shown in the figure, the "macrophage model of endocytic nanomaterials" was successfully constructed, and transmission electron microscopy showed that BTO and pBTO were endocytosed by macrophages.

[0063] Example 4 Osteoclast differentiation in the macrophage model of endocytosed nanomaterials

[0064] 1. Experimental Methods

[0065] The "macrophage model of endocytic nanomaterials" constructed in Example 3 was stimulated with 50 ng / mL RANKL for 5 days to obtain a RANKL-treated cell model. The RANKL-treated cell model was fixed with 4% (w / v, g / 100 mL) paraformaldehyde for 15 minutes, permeabilized with 0.1% (v / v) Triton X-100 for 20 minutes, and incubated with TRAP (tartrate-resistant acid phosphatase) staining solution at 37°C in the dark for 1 hour to obtain a TRAP-stained cell model. The TRAP-stained cell model was then stained with DAPI (4',6-diamidino-2-phenylindole) at 37°C in the dark for 15 minutes to obtain a DAPI-stained cell model. TRAP cells with more than three nuclei were fixed with 4% (w / v, g / 100 mL) paraformaldehyde for 15 minutes, permeabilized with 0.1% (v / v) Triton X-100 for 20 minutes, and incubated with TRAP (tartrate-resistant acid phosphatase) staining solution at 37°C in the dark for 1 hour to obtain a TRAP-stained cell model. + The cells were considered osteoclasts and images were captured using an optical fluorescence microscope (Olympus, Japan).

[0066] Total RNA was extracted from RAW 264.7 cells using an RNA extraction kit (Takara, Japan, No. 9767). Total RNA was reverse transcribed using a PrimeScript™ RT kit (Takara, Japan, No. RR385S) to obtain complementary DNA (cDNA). qRT-PCR (quantitative RT-PCR) was performed on a LightCycler 480 (Roche, USA) using SYBR Green Master Mix (YEASEN, China, 11198ES08) and gene-specific primers (Table 1). The PCR system and program are shown in Tables 2 and 3, respectively. The relative expression levels of miRNAs were determined using 2 -ΔΔCT Comparison methods were standardized.

[0067] Table 1

[0068]

[0069]

[0070] Table 2

[0071]

[0072] Table 3

[0073]

[0074] 2. Experimental Results

[0075] like Figure 5 A and Figure 5 As shown in Figure B, macrophage differentiation was inhibited in the RANKL-treated "macrophage model endocytosing nanomaterials." Among them, the inhibition of differentiation was more pronounced in the "macrophage model endocytosing nano-pBTO" that endocytosed pBTO.

[0076] like Figure 6 As shown, the relative expression levels of osteoclast differentiation-related genes Acp5, CTSK, c-FOS, DC-STAMP and Atp6v0d2 mRNA were all decreased (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001).

[0077] "Macrophage model of endocytic nanomaterials" inhibits osteoclastogenesis.

[0078] Example 4 Construction of an in vivo model of skull bone resorption in mice

[0079] 1. An LPS-induced mouse calvarial osteolysis model (Beyotime, China) was established by subcutaneously injecting 25 mg / kg LPS (lipopolysaccharide) into the midsagittal suture of the skull of C57 / BL6 mice under anesthesia. Each mouse was simultaneously injected with 25 mg / kg LPS to induce calvarial osteolysis and 100 μg BTO or 100 μg pBTO to evaluate the therapeutic effects of BTO and pBTO on LPS-induced calvarial osteolysis. After 7 days, all mice were sacrificed, and their skulls were collected and fixed in 4% paraformaldehyde for 24 hours for further experiments.

[0080] 2. Micro-computed tomography

[0081] The skull of the mouse model from step 1 was scanned using a high-resolution microCT (μCT-50, SCANCO Medical AG). Scanning parameters were 70 kV, 114 μA, and 7 μm. 3D images of the skull and bone volume / tissue volume (BV / TV) were generated and analyzed using the Scanco evaluation program.

[0082] 3. Histological Analysis and Immunofluorescence Staining of Skull

[0083] The mouse model skulls from step 1 were decalcified in 10% (w / v, g / 100 mL) EDTA (pH 7.4) for 2 weeks to obtain decalcified skull samples. The decalcified skull samples were dehydrated, embedded in paraffin, and cut into 5 μm thick skull sections for hematoxylin and eosin (HE) and TRAP staining. The percentage of bone erosion area and the number of TRAP-positive osteoclasts were calculated.

[0084] 2. Experimental Results

[0085] like Figure 7 A. Figure 7 B. Figure 8 A and Figure 8 As shown in B, injection of nanoparticle pBTO into the skull can inhibit LPS-induced skull resorption, while the control group, which internalized unpolarized BTO, had no effect.

[0086] like Figure 9 A and Figure 9 As shown in B, nanoparticle pBTO injected into the skull can inhibit osteoclastogenesis.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of polarized barium titanate nanoparticles in the preparation of drugs for preventing and treating inflammatory bone resorption.

2. The use according to claim 1, characterized in that The drug is a drug that inhibits macrophage osteoclast differentiation.

3. The use according to claim 2, characterized in that The drug is a drug that inhibits macrophage osteoclast differentiation by endocytizing polarized barium titanate nanoparticles in macrophages.

4. The use according to claim 2, characterized in that The drug is a drug that reduces the expression level of at least one of the osteoclast differentiation-related genes Acp5, CTSK, c-FOS, DC-STAMP and Atp6v0d2.

5. The use according to claim 2, characterized in that The drug is a drug that inhibits osteoclastogenesis.

6. The use according to any one of claims 1 to 5, characterized in that The particle size of the polarized barium titanate nanoparticles is 90-110 nm, and the surface potential is 1.9-2.1 mV.

7. The use according to claim 6, characterized in that The method for preparing polarized barium titanate nanoparticles comprises polarizing the barium titanate nanoparticles with a voltage of 2.4 to 2.6 kV for 29.5 to 30.5 minutes.

8. The use according to claim 7, characterized in that The preparation method of the barium titanate nanoparticles comprises the following steps: S1: uniformly mixing the titanium butoxide ethanol solution and the ammonia solution to obtain a mixture 1; S2: Mixing the mixture 1 of step S1 and an aqueous solution of barium hydroxide octahydrate uniformly, adding diethanolamine, and mixing uniformly to obtain a mixture 2; S3: subjecting the mixture 2 of step S2 to a hydrothermal reaction, washing and drying, to obtain barium titanate nanoparticles.

9. The use according to claim 8, characterized in that In step S1, the volume ratio of the titanium butoxide ethanol solution to the ammonia solution is (3.9-4.1):1, the concentration of the titanium butoxide ethanol solution is 1.2-1.3 mmol / mL, and the concentration of the ammonia solution is 24.5-25.5% w / v.

10. The use according to claim 8, characterized in that In step S2, the volume ratio of mixture 1, barium hydroxide aqueous solution and diethanolamine is mixture 1:barium hydroxide aqueous solution:diethanolamine=(4.9-5.1):(4.9-5.1):1, and the concentration of barium hydroxide aqueous solution is 1.35-1.45 mmol / mL.

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