Skatole-containing bone-targeted nanoparticles and their application in drugs for treating osteoarthritis

Abstract

The present invention belongs to the field of medicine, and specifically relates to a bone-targeted nanoparticle containing skatole and its application in a drug for treating osteoarthritis. The preparation method of the bone-targeted nanoparticle is as follows: first, dissolve skatole in acetone or ethanol to obtain a drug-containing solution; then dissolve a pyrophosphate derivative in a solvent to obtain a carrier solution; finally, drop the drug-containing solution into the carrier solution and stir, ultrasonicate for 15-20 minutes after the drop, and spray dry to obtain; the prepared bone-targeted nanoparticle containing skatole can effectively inhibit the secretion of inflammatory factors, inhibit the polarization of M1 macrophages, promote the polarization of M2 macrophages, and can also inhibit the catabolism and apoptosis of chondrocytes caused by inflammation, promote the proliferation of chondrocytes, and realize chondrocyte repair; and it has a certain inhibitory and repairing effect on cartilage damage, and can effectively treat osteoarthritis.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to bone-targeted nanoparticles containing skatole and application thereof in drugs for treating osteoarthritis. Background Art

[0002] Osteoarthritis is a common inflammatory disease worldwide, characterized by pathological changes such as cartilage damage and degeneration, subchondral bone sclerosis, and joint capsule contracture and deformation. The current clinical treatment of osteoarthritis is mainly symptomatic treatment to relieve osteoarthritis pain and inhibit inflammation. The drugs for the treatment of osteoarthritis are divided into oral non-steroidal anti-inflammatory drugs, intra-articular injection of corticosteroids, and regenerative therapies such as intra-articular injection of mesenchymal stem cells. However, these clinical treatments cannot prevent the continuous development of osteoarthritis, and the mid- and long-term efficacy is poor. In recent years, natural products have been widely used in the treatment of osteoarthritis due to their significant efficacy and high safety, and have been accepted as complementary and alternative therapies in many countries. Among them, skatole is a substance produced by intestinal flora, which has certain antibacterial and antiviral effects. Its chemical name is 3-methylindole, its molecular formula is C9H9N, and its chemical structure is:

[0003]

[0004] In addition, due to the high hardness, low blood flow, poor permeability, and special physiological and biochemical processes of bone tissue, it is difficult for drugs to effectively reach the lesion site through traditional drug administration, resulting in poor drug efficacy and many adverse reactions. In order to enable drugs to reach effective therapeutic concentrations in bone tissue to improve drug efficacy, it is necessary to improve the targeting of drugs for the treatment of osteoarthritis. Therefore, further research on the role of the natural product skatole in osteoarthritis and the search for a natural small molecule drug preparation with bone targeting and high safety is of great significance for the efficient treatment of osteoarthritis. Summary of the invention

[0005] The purpose of the present invention is to address the deficiencies of the prior art and provide a bone-targeted nanoparticle containing skatole and its use in a drug for treating osteoarthritis. The prepared bone-targeted nanoparticle containing skatole can effectively inhibit the secretion of inflammatory factors, inhibit the polarization of M1 macrophages, promote the polarization of M2 macrophages, and can also inhibit the catabolism and apoptosis of chondrocytes caused by inflammation, promote the proliferation of chondrocytes, and achieve chondrocyte repair; and it has a certain inhibitory and repairing effect on cartilage damage, and can effectively treat osteoarthritis.

[0006] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0007] A method for preparing bone-targeted nanoparticles containing skatole comprises the following steps:

[0008] A1. Dissolve skatole in acetone or ethanol to obtain a drug-containing solution;

[0009] A2, dissolving the pyrophosphate derivative in a solvent to obtain a carrier solution;

[0010] A3, drop the drug-containing solution into the carrier solution and stir, ultrasonicate for 15-20 minutes after dropwise addition, and spray dry to obtain;

[0011] Furthermore, the solid-liquid ratio of skatole in step A1 is 1 g: 10-15 mL.

[0012] Furthermore, in step A2, the solid-liquid ratio of the pyrophosphate derivative is 1 g: 50-60 mL; and the solvent is 40-50% ethanol.

[0013] Furthermore, in step A3, the mass ratio of the drug-containing solution to the carrier solution is 1:24-25; and the ultrasonic power is 100-120W.

[0014] Furthermore, the preparation method of the pyrophosphate derivative is:

[0015] (1) Compound 2 and tri(tetrabutylammonium) pyrophosphate are added to a mixed solvent, heated to 30-40°C, and reacted for 2-4 hours. After the reaction is completed, the temperature is naturally cooled to room temperature, and then sodium chloride is added and stirred for 1-2 hours to obtain modified pyrophosphoric acid. The reaction process is as follows:

[0016]

[0017] (2) Carboxymethyl chitosan and triethylamine are added to a 40-50% ethanol solution, and then modified pyrophosphoric acid is added, the temperature is raised to 70-80° C., and stirred for 5-8 hours to obtain a pyrophosphoric acid derivative.

[0018] Furthermore, the preparation method of compound 2 in step (1) is: phenylbutanol and epichlorohydrin react at 50-60° C. for 4-6 hours under the action of a catalyst to generate compound 1, and the reaction process is:

[0019] Compound 1 reacts with p-toluenesulfonyl chloride (TsCl) under the action of triethylamine and 4-dimethylaminopyridine at room temperature for 3-4 hours to generate compound 2. The reaction process is as follows:

[0020]

[0021] Furthermore, in the preparation method of the above-mentioned compound 2, the molar ratio of phenylbutanol, epichlorohydrin and catalyst is 1:1.1-1.2:0.4-0.5; the catalyst is one or a mixture of boron trifluoride, ferric chloride, aluminum chloride and zinc chloride; the molar ratio of the compound 1, p-toluenesulfonyl chloride, triethylamine and 4-dimethylaminopyridine is 1:1.1-1.2:1.5-2:0.1-0.2.

[0022] Furthermore, in step (1), the molar ratio of compound 2 to tri(tetrabutylammonium) pyrophosphate is 1:1.1-1.2; the mixed solvent is acetonitrile and tetrahydrofuran in a volume ratio of 1:0.5-1; and the molar amount of sodium chloride is the same as the molar amount of tri(tetrabutylammonium) pyrophosphate.

[0023] Furthermore, in step (2), the mass ratio of carboxymethyl chitosan, modified pyrophosphoric acid and triethylamine is 1:0.3-0.4:0.1-0.2.

[0024] The present invention provides bone-targeted nanoparticles containing skatole, which are prepared by the above-mentioned method for preparing bone-targeted nanoparticles containing skatole.

[0025] The present invention provides application of bone-targeted nanoparticles containing skatole in drugs for treating osteoarthritis.

[0026] The present invention also provides a solid preparation, comprising the above-mentioned bone-targeted nanoparticles containing skatole; the solid preparation is a tablet, a capsule, a granule or a pill.

[0027] Furthermore, the preparation method of the solid preparation is: mixing the above-mentioned bone-targeted nanoparticles containing skatole with auxiliary materials to obtain the preparation; the auxiliary materials include one or a mixture of diluents, wetting agents, adhesives, disintegrants, lubricants, fillers.

[0028] The present invention has the following beneficial effects:

[0029] The invention grafts modified pyrophosphate containing hydrophobic benzene rings and alkanes and bone-targeted pyrophosphate onto carboxymethyl chitosan molecules through chemical reactions, thereby obtaining amphiphilic pyrophosphate derivatives. The obtained pyrophosphate derivatives have no obvious toxicity to cells and can be used as pharmaceutical excipients in the biomedical field. The obtained pyrophosphate derivatives are used as drug carriers, and bone-targeted nanoparticles containing skatole are prepared through an ultrasonic method. The amphiphilic pyrophosphate derivatives have a certain surface activity, which helps to evenly disperse skatole molecules in a carrier solution, thereby promoting the pyrophosphate derivatives to form efficient encapsulation of skatole, and can also mask the irritating odor of skatole, thereby improving the drug The bone-targeted nanoparticles containing skatole prepared by the present invention have bone targeting, which is helpful to improve the efficacy of skatole in treating osteoarthritis. The in vitro release behavior of the nanoparticles is relatively slow and lasting, which can effectively inhibit the secretion of inflammatory factors, inhibit the polarization of M1 macrophages, and promote the polarization of M2 macrophages, showing anti-inflammatory and tissue repair effects, which is helpful to repair cartilage damage in osteoarthritis; it can also inhibit the catabolism and apoptosis of chondrocytes caused by inflammation, promote the proliferation of chondrocytes, and realize the repair of chondrocytes; and it has a certain inhibitory and repairing effect on cartilage damage, which can effectively treat osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the release profile of bone-targeted nanoparticles in the release medium;

[0031] Figure 2 This is a diagram showing the effect of skatole on the secretion of inflammatory factors in macrophages;

[0032] Figure 3 This is the effect diagram of skatole inhibiting chondrocyte apoptosis and promoting chondrocyte proliferation;

[0033] Figure 4 This is the semi-quantitative scoring diagram of OARSI of mouse knee joint stained with Safranin O-Fast Green;

[0034] Figure 5 This is a diagram showing the effect of skatole in treating osteoarthritis in mice. DETAILED DESCRIPTION

[0035] The following will be combined with the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0036] Skatole CAS No. 83-34-1; Carboxymethyl chitosan CAS No. 83512-85-0 (MW = 8.5 kDa, deacetylation degree = 90%, carboxymethylation degree = 85%), pharmaceutical grade; phenylbutanol CAS No. 3360-41-6; Epichlorohydrin CAS No. 106-89-8; Boron trifluoride CAS No. 7637-07-2; p-Toluenesulfonyl chloride (TsCl) CAS No. 98-59-9; Triethylamine CAS No. 121- 44-8; 4-dimethylaminopyridine CAS No. 1122-58-3; tri(tetrabutylammonium) pyrophosphate ((Bu4N)3(HO)P2O6) CAS No. 76947-02-9; sodium chloride CAS No. 7647-14-5; ethanol CAS No. 64-17-5; dichloromethane CAS No. 75-09-2; acetonitrile CAS No. 75-05-8; tetrahydrofuran CAS No. 109-99-9; all chemical reagents are commercially available.

[0037] Example 1

[0038] A method for preparing bone-targeted nanoparticles containing skatole comprises the following steps:

[0039] A1. Dissolve skatole in acetone to obtain a drug-containing solution; wherein the solid-liquid ratio of skatole is 1 g: 15 mL;

[0040] A2, dissolving the pyrophosphate derivative in a solvent to obtain a carrier solution; wherein the solid-liquid ratio of the pyrophosphate derivative is 1 g: 60 mL; and the solvent is 50% ethanol;

[0041] A3, drop the drug-containing solution into the carrier solution and stir, ultrasonicate for 20 minutes after the dropwise addition, and spray dry to obtain the product; wherein the mass ratio of the drug-containing solution to the carrier solution is 1:24; and the ultrasonic power is 120W.

[0042] The preparation method of pyrophosphate derivatives is:

[0043] (1) 15.0 g of compound 2 and 18.97 g of tri(tetrabutylammonium) pyrophosphate ((Bu4N)3(HO)P2O6) were added to 200 mL of a mixed solvent, heated to 40°C, and reacted for 2 h. After the reaction was completed, the temperature was naturally lowered to room temperature, and then 2.66 g of sodium chloride was added. The stirring was continued for 2 h, and the solvent was evaporated under reduced pressure to obtain 15.6 g of modified pyrophosphoric acid, wherein the molar ratio of compound 2 to tri(tetrabutylammonium) pyrophosphate was 1:1.2; the mixed solvent was acetonitrile and tetrahydrofuran in a volume ratio of 1:0.5; the molar amount of sodium chloride was the same as the molar amount of tri(tetrabutylammonium) pyrophosphate; the reaction process was:

[0044]

[0045] Modified pyrophosphate: ESI (m / z): 468.9 [M+H] + , 1 H-NMR (600MHz, DMSO-d6, δppm): 7.21-7.25 (m, 4H), 7.18 (t, J=7.6Hz, 1H), 4.11-4.14 ( m, 1H), 3.81-3.83 (m, 2H), 3.42-3.50 (m, 4H), 2.77 (t, J=6.0Hz, 2H), 1.58-1.60 (m, 4H).

[0046] (2) Carboxymethyl chitosan and triethylamine are added to a 40% ethanol solution, and then modified pyrophosphoric acid is added. The temperature is raised to 70°C and stirred for 8 hours. After the reaction is completed, the temperature is naturally lowered to room temperature. The solution is placed in a dialysis bag and dialyzed with distilled water for two days. The pyrophosphoric acid derivative is obtained after freeze-drying; wherein the mass ratio of carboxymethyl chitosan, modified pyrophosphoric acid and triethylamine is 1:0.4:0.1; and the degree of substitution of the modified pyrophosphoric acid in the pyrophosphoric acid derivative is determined by elemental analysis to be 13.2%.

[0047] The preparation method of compound 2 is as follows: 15.0g of phenylbutanol and 3.39g of boron trifluoride are added to 500mL of chloroform solution, 11.09g of epichlorohydrin is added dropwise under stirring, and the temperature is raised to 5°C for reaction for 6h. After the reaction is completed, the temperature is naturally lowered to room temperature, most of the chloroform is removed by rotary evaporation, and 300mL of water and 300mL of dichloromethane are added for extraction, the organic phase is collected, dried and concentrated under reduced pressure to obtain 20.5g of compound 1, wherein the molar ratio of phenylbutanol, epichlorohydrin and boron trifluoride is 1:1.2:0.5; the reaction process is:

[0048]

[0049] Compound 1: ESI (m / z): 243.1 [M+H] + , 1 H-NMR (600MHz, DMSO-d6, δppm): 7.24 (d, J=8.5Hz, 2H), 7.20-7.22 (m, 2H); 7.15 (t, J=7.6Hz, 1H), 5.37 (s , 1H), 4.10-4.13(m, 1H), 3.63-3.66(m, 2H), 3.35-3.39(m, 4H), 2.77(t, J=6.0Hz, 2H), 1.56-1.59(m, 4H).

[0050] 20.0g of compound 1, 16.72g of triethylamine and 2.02g of 4-dimethylaminopyridine were added to 400mL of anhydrous dichloromethane solution. Under ice bath condition, 18.90g of p-toluenesulfonyl chloride (TsCl) was added dropwise. After the addition was completed, stirring was continued for 1h, and then the temperature was raised to room temperature and the reaction was carried out for 4h. After the reaction was completed, 250mL of 1M hydrochloric acid solution, 400mL of saturated sodium chloride solution and 400mL of distilled water were used to wash the solution in sequence. The organic phase was collected, dried and concentrated under reduced pressure to obtain 28.6g of compound 2; wherein the molar ratio of compound 1, p-toluenesulfonyl chloride, triethylamine and 4-dimethylaminopyridine was 1:1.2:2:0.2; the reaction process was:

[0051]

[0052] Compound 2: ESI (m / z): 397.2 [M+H] + , 1 H-NMR (600MHz, DMSO-d6, δppm): 7.75 (d, J=8.6Hz, 2H), 7.45 (d, J=8.6Hz, 2H), 7.20-7.25 (m, 4H), 7.19 (t, J=7.6Hz, 1 H), 5.31-5.33 (m, 1H), 3.62-3.64 (m, 2H), 3.35-3.38 (m, 4H), 2.75 (t, J=6.0Hz, 2H), 2.43 (s, 3H), 1.57-1.59 (m, 4H).

[0053] Cytotoxicity test of the prepared pyrophosphate derivatives

[0054] The pyrophosphate derivative was dissolved in a distilled water solution, and the solution was ultrasonicated for 10 minutes at an ultrasonic power of 120W to prepare nanoparticle solutions of pyrophosphate derivatives at 0.02, 0.2, and 2 mg / mL, respectively; HepG2 cells were inoculated in DMEM culture medium containing 10% fetal bovine serum and cultured in a 37°C, 5% CO2 incubator, HepG2 cells in the logarithmic growth phase were taken, the attached cells were digested with 0.25% trypsin, and a cell suspension was prepared with DMEM culture medium containing 10% calf serum, and the HepG2 cells were inoculated into a 96-well culture plate at a density of 5000 cells / well, and after being cultured overnight at 37°C, 5% CO2 and saturated humidity, blank nanoparticle solutions of different concentrations were added. The experimental group was the nanoparticle solution of the pyrophosphate derivative, and the blank control group was added with culture medium, and each group of samples was parallel to 5 wells, and then cultured in a 37°C, 5% CO2 incubator. After 48 hours, 15uL of 5mg / mL MTT solution was added to each well. After culturing at 37°C for 4 hours, the culture was terminated, the supernatant was discarded, 150uL of DMSO solution was added to each well, and the cells were shaken in the dark for 10 minutes to fully dissolve the crystals.

[0055] The absorbance value was detected by an ELISA instrument (detection wavelength was 570 nm, reference wavelength was 630 nm), and the cell survival rate was calculated. After adding nanoparticle solutions of pyrophosphate derivatives with concentrations of 0.02, 0.2, and 2 mg / mL to the cells for 48 hours, the growth of HepG2 cells was not significantly inhibited, and the cell survival rates were all above 95%, indicating that the pyrophosphate derivatives prepared by the present invention have no obvious toxicity to cells and can be used as pharmaceutical excipients in the biomedical field.

[0056] Example 2

[0057] A method for preparing bone-targeted nanoparticles containing skatole comprises the following steps:

[0058] A1. Dissolve skatole in ethanol to obtain a drug-containing solution; wherein the solid-liquid ratio of skatole is 1 g: 10 mL;

[0059] A2, dissolving the pyrophosphate derivative in a solvent to obtain a carrier solution; wherein the solid-liquid ratio of the pyrophosphate derivative is 1 g:50 mL; and the solvent is 40% ethanol;

[0060] A3, drop the drug-containing solution into the carrier solution and stir, ultrasonicate for 15 minutes after the dropwise addition, and spray dry to obtain the product; wherein the mass ratio of the drug-containing solution to the carrier solution is 1:25; and the ultrasonic power is 100W.

[0061] The preparation method of the pyrophosphate derivative is the same as that in Example 1.

[0062] Example 3

[0063] A method for preparing bone-targeted nanoparticles containing skatole comprises the following steps:

[0064] A1. Dissolve skatole in acetone to obtain a drug-containing solution; wherein the solid-liquid ratio of skatole is 1 g: 13 mL;

[0065] A2, dissolving the pyrophosphate derivative in a solvent to obtain a carrier solution; wherein the solid-liquid ratio of the pyrophosphate derivative is 1 g:55 mL; and the solvent is 45% ethanol;

[0066] A3, drop the drug-containing solution into the carrier solution and stir, ultrasonicate for 17 minutes after the dropwise addition, and spray dry to obtain the product; wherein the mass ratio of the drug-containing solution to the carrier solution is 1:24; and the ultrasonic power is 110W.

[0067] The preparation method of the pyrophosphate derivative is the same as that in Example 1.

[0068] Comparative Example 1

[0069] Compared with Example 1, the preparation method of bone-targeted nanoparticles in this comparative example is different.

[0070] A method for preparing bone-targeted nanoparticles containing skatole comprises the following steps:

[0071] A1. Dissolve skatole in acetone to obtain a drug-containing solution; wherein the solid-liquid ratio of skatole is 1 g: 5 mL;

[0072] A2, dissolving the pyrophosphate derivative in a solvent to obtain a carrier solution; wherein the solid-liquid ratio of the pyrophosphate derivative is 1 g: 40 mL; and the solvent is 50% ethanol;

[0073] A3, add the drug-containing solution to the carrier solution and stir, ultrasonicate for 25 minutes after the addition, and spray dry to obtain the product; wherein the mass ratio of the drug-containing solution to the carrier solution is 1:20; and the ultrasonic power is 80W.

[0074] The preparation method of the pyrophosphate derivative is the same as that in Example 1.

[0075] Comparative Example 2

[0076] Compared with Example 1, the carrier solution in this comparative example is different.

[0077] A method for preparing bone-targeted nanoparticles containing skatole comprises the following steps:

[0078] A1. Dissolve skatole in acetone to obtain a drug-containing solution; wherein the solid-liquid ratio of skatole is 1 g: 15 mL;

[0079] A2, dissolving carboxymethyl chitosan in a solvent to obtain a carrier solution; wherein the solid-liquid ratio of carboxymethyl chitosan is 1g:60mL; and the solvent is 50% ethanol;

[0080] A3, drop the drug-containing solution into the carrier solution and stir, ultrasonicate for 20 minutes after the dropwise addition, and spray dry to obtain the product; wherein the mass ratio of the drug-containing solution to the carrier solution is 1:24; and the ultrasonic power is 120W.

[0081] Comparative Example 3

[0082] This comparative example provides a skatole compound.

[0083] Related experiments

[0084] 1. Determination of the morphology, particle size and drug loading of bone-targeted nanoparticles containing skatole

[0085] The morphology, particle size and drug loading of the bone-targeted nanoparticles prepared in Examples 1-3 and Comparative Examples 1-2 were measured; skatole was used as a standard, and the drug loading of the nanoparticles was determined and calculated by high performance liquid chromatography, wherein the chromatographic column was a Phenomenex C18 column (4.6 mm × 250 mm); the mobile phase A was water (containing 0.1% trifluoroacetic acid), B was acetonitrile, and the detection wavelength was 254 nm; the flow rate was 1.0 mL / min; the column temperature was 30°C; the measurement results are shown in Table 1.

[0086] Table 1 Test results of morphology, particle size and drug loading of bone-targeted nanoparticles

[0087] sample form Particle size (nm) Drug loading (%) Example 1 The particles are round in appearance and uniform in size. 162±4 18.9 Example 2 The particles are round in appearance and uniform in size. 160±3 17.6 Example 3 The particles are round in appearance and uniform in size. 161±2 18.0 Comparative Example 1 The particles are not round in appearance and are uneven in size. 260±20 11.7 Comparative Example 2 Poor formability, sticky film 380±17 8.5

[0088] It can be seen from the results in Table 1 that the preparation methods in Examples 1-3 can successfully produce nanoparticles with a round appearance, uniform size, small particle size and high drug loading; compared with Comparative Example 2 (a carrier solution containing carboxymethyl chitosan), the carrier solution used in Examples 1-3 is a pyrophosphate derivative-containing carrier solution, wherein the pyrophosphate derivative contains a hydrophobic benzene ring, an alkane structure and a hydrophilic pyrophosphate sodium salt, and a carboxymethyl chitosan structure, and the amphiphilic pyrophosphate derivative has a certain surface activity, which helps to evenly disperse the skatole molecules in the carrier solution, thereby promoting the pyrophosphate derivative to form an efficient encapsulation of skatole.

[0089] 2. Solubility and stability experiments of bone-targeted nanoparticles containing skatole

[0090] The bone-targeted nanoparticles prepared in Examples 1-3, Comparative Examples 1-2, and Comparative Example 3 were tested for solubility. For the water-solubility test, the samples were mixed with water at a ratio of 10 mg / mL. For the fat-solubility test, the samples were mixed with an organic mixed solvent (benzene, ether, carbon tetrachloride, and petroleum ether in a volume ratio of 1:1:1:1) at a ratio of 10 mg / mL. The samples were shaken for 30 seconds every 5 minutes at room temperature, and the dissolution was observed after 30 minutes. The solution stability was observed after being placed under high temperature (60°C), high humidity (RH90%), and light conditions for 10 days. The test results are shown in Table 2.

[0091] Table 2 Solubility and stability test results of bone-targeted nanoparticles

[0092]

[0093] From the results in Table 2, it can be seen that compared with Comparative Example 1 (different preparation methods), Comparative Example 2 (using carboxymethyl chitosan), and Comparative Example 3 (skatole), the bone-targeted nanoparticles prepared in Examples 1-3 have good water solubility and fat solubility, and no stratification and precipitation phenomenon occurs after being placed under high temperature, high humidity, and light conditions for 10 days. The ultrasonic method can bind skatole and the pyrophosphate derivative more tightly through intermolecular hydrogen bonds, and has good stability. This shows that the use of pyrophosphate derivatives as drug carriers to prepare skatole into amphiphilic bone-targeted nanoparticles can significantly improve the water solubility and stability of the drug, and facilitate drug storage and processing into different dosage forms.

[0094] 3. In vitro release experiment of bone-targeted nanoparticles containing skatole

[0095] The in vitro release experiment of the nanoparticles was carried out by dialysis, and the content of skatole in the release medium was measured by high performance liquid chromatography, wherein the release medium was a PBS buffer solution with a pH of 7.4 and containing 0.2% SDS.

[0096] Weigh 5 mg of the nanoparticles prepared in Examples 1-3, Comparative Examples 1-2, and Comparative Example 3, add them to 1 mL of water, place them in dialysis bags (MWCO = 3500 Da), seal them, immerse them in a conical flask containing 30 mL of release medium (PBS, pH = 7.4, containing 5% Tween 80), and put them in a 37°C constant temperature water bath oscillator at a speed of 100 r / min. Take 1 mL of sample at a preset time point, and replenish the same volume of fresh release medium at the same time. The sample is filtered through a 0.22 um microporous membrane, and the skatole content is measured by high performance liquid chromatography, and then the cumulative release amount is calculated, and a curve of cumulative release amount and time is drawn, i.e., an in vitro drug release curve, such as Figure 1 As shown, it can be seen from the release curve that compared with comparative examples 1-3, the release behavior of the nanoparticles prepared in Examples 1-3 is basically the same, all relatively slow and long-lasting, without burst release, and at 25h, the cumulative release percentage in the release medium can reach more than 80%.

[0097] 4. Effects of bone-targeted nanoparticles containing skatole on inflammatory factors and polarization of mouse macrophages

[0098] Mouse macrophage cell line RAW264.7 and chondrocyte cell line ATDC5 were co-cultured in DMEM medium containing 10% fetal bovine serum, 100U / mL penicillin and 100μg / mL streptomycin. The cells were routinely cultured and the culture medium was replaced every 2 days. When the adherence rate of RAW264.7 reached 85%, the cells were divided into five groups. The control group (CTR) was cultured in DMEM medium normally. Four groups were added with 1μg / mL lipopolysaccharide (LPS) aqueous solution to induce macrophage polarization. Then, a bone-targeted nanoparticle aqueous solution containing 5μM skatole, a bone-targeted nanoparticle aqueous solution containing 10μM skatole, and a bone-targeted nanoparticle aqueous solution containing 20μM skatole were added to the three groups, respectively. The bone-targeted nanoparticles of skatole were prepared by Example 1, and the amount added to each group was 20μL. After 24 h of culture, each group was washed three times with PBS, and total RNA was extracted with Trizol (Invitrogen). TM Kit (Invitrogen) was used to synthesize cDNA. Finally, the reaction system was prepared using SYBR Green Realtime PCR Master Mix (TOYOBO) for amplification and determination of the expression levels of inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6), M1 macrophage polarization marker nitric oxide synthase 2 (NOS2), M2 macrophage polarization marker arginase 1 (Arg1), and macrophage galactose-type lectin 1 (Mgl1). IL-1β, IL-6, NOS2, Arg1, and Mgl1 were all used as GADPH internal reference genes, and their primer sequences were synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0099] The results are as follows Figure 2 As shown, after LPS induced macrophage polarization, the expression levels of IL-1β, IL-6, and NOS2 / Arg-1 increased, and the expression level of Mgl1 decreased; after administration of an aqueous solution of bone-targeted nanoparticles containing different concentrations of skatole, the expression levels of IL-1β, IL-6, and NOS2 / Arg-1 decreased, and the expression level of Mgl1 increased, indicating that bone-targeted nanoparticles containing skatole can effectively inhibit the secretion of inflammatory factors, inhibit the polarization of M1 macrophages, and promote the polarization of M2 macrophages, showing anti-inflammatory and tissue repair effects, which is helpful for the repair of cartilage damage in osteoarthritis, thereby achieving the effect of treating osteoarthritis.

[0100] The chondrocyte ATDC5 was stained and the results were as follows Figure 3 As shown, it is shown that bone-targeted nanoparticles containing skatole can inhibit chondrocyte catabolism and apoptosis caused by inflammation, promote chondrocyte proliferation, and achieve chondrocyte repair.

[0101] 5. Experimental study on the treatment of osteoarthritis in mice with bone-targeted nanoparticles containing skatole

[0102] The osteoarthritis model was established by the meniscus instability method. The specific implementation method is as follows: After the mice were adaptively raised in the animal room, they were anesthetized with isoflurane gas, fixed in the supine position, with the left hind limb flexed at 90°, the surgical area was shaved, the surgical field was fully disinfected with type III iodine, and 75% alcohol was used for deiodination. The skin was cut to expose the patellar ligament, and the joint capsule was opened along the inner edge of the patellar ligament with a sharp knife scissors. The adipose tissue in the joint cavity was cleaned, and the medial meniscus was connected to the tibial plateau through the medial meniscus tibial ligament. The anterior horn of the medial meniscus can be pulled out after cutting with a sharp blade. After disinfection and flushing the wound, the joint cavity was sutured with 8-0 surgical sutures, and the skin was sutured with 7-0 surgical sutures to close the wound. Amoxicillin capsules (0.25 mg) were evenly applied to the wound to prevent wound infection. The sham-operated mice were sutured layer by layer after the joint capsule was opened. It was observed that the surgical incision did not exceed 1 cm, there was little bleeding in the knee joint during the operation, and the mice gradually recovered and could move freely 30 minutes to 1 hour after the operation. No overdose of anesthesia occurred, and the thread ends fell off on their own one week after the operation.

[0103] C57 / BL6 surgical mice were randomly divided into experimental group (OA+skatole), control group (OA), and sham operation group (Shame), with 8 mice in each group; 2 weeks after modeling, 6ul of nanoparticle solution containing 50μM D-tetrahydrojatrorrhizine was injected into the right knee joint of the mice in the experimental group, and 6ul of solvent (6% DMSO+10% polyoxyethylene castor oil+84% double distilled water) was injected into the control group and sham operation group respectively. The injection was performed once a week for 6 consecutive weeks. Specific injection method: After anesthetizing the mice, the skin was prepared and disinfected, and a longitudinal skin incision of about 2mm was made in the middle of the knee joint. The skin was pulled to find the knee joint, and physiological saline was drawn with a microsyringe. The midpoint of the inner edge of the patellar ligament was used as the needle point, and the injection was injected into the joint cavity obliquely from the back to the top about 1.5mm deep. After the needle was removed, the needle was slightly pressed to prevent the injection liquid from overflowing.

[0104] Eight weeks after injection, the mice were euthanized and their knee joints were fixed in paraformaldehyde for 48 h for tissue embedding, sectioning, safranin O-fast green staining, and OARSI scoring.

[0105] Semi-quantitative assessment of articular cartilage stained with Safranin O Fast Green using the OARSI scoring system:

[0106] OA changes were quantified using the histological grade score of the Osteoarthritis Research Society International (OARSI) pathology guidelines. The grading method is shown in Table 3. The scores were scored separately by two people. The semi-quantitative standard formula for determining the severity of OA was: total score = grade × grading.

[0107] Table 3 Rating criteria for OA cartilage damage tissue sections in OARSI scoring

[0108] Stage %Involvement(surface,area,volume) Stage 0 No OA activity seen Stage 1 <10％ Stage 2 10-25％ Stage 3 25-50％ Stage 4 >50％

[0109] Evaluation results such as Figure 4 As shown, the cartilage of the mice in the control group was damaged and the OARSI score was 2; in comparison, after the mice in the experimental group were treated with bone-targeted nanoparticles containing skatole, the OARSI score of the mouse joints decreased significantly, indicating that the bone-targeted nanoparticles containing skatole prepared by the present invention can effectively inhibit cartilage damage and have the effect of alleviating osteoarthritis.

[0110] Safranin O-fast green staining results are as follows Figure 5 As shown, compared with the sham operation group, the cartilage of the mice in the control group was damaged, the cartilage became thinner or even disappeared, and the arrangement of articular chondrocytes was disordered; after the mice in the experimental group were treated with bone-targeted nanoparticles containing skatole, the cartilage thickness increased and the arrangement of articular chondrocytes was more regular, which further illustrates that the bone-targeted nanoparticles containing skatole prepared by the present invention have a certain inhibitory and repairing effect on cartilage damage, and can effectively treat osteoarthritis.

[0111] It should be noted that, in this article, the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device that includes a series of elements includes not only those elements, but also includes other elements that are not explicitly listed, or also includes elements inherent to such process, method, article or device; although the embodiments of the present application have been shown and described, it can be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing bone-targeted nanoparticles containing skatole, characterized in that: The steps include: A1. Dissolve skatole in acetone or ethanol to obtain a drug-containing solution; A2, dissolving the pyrophosphate derivative in a solvent to obtain a carrier solution; A3, drop the drug-containing solution into the carrier solution and stir, ultrasonicate for 15-20 minutes after dropwise addition, and spray dry to obtain; The preparation method of the pyrophosphate derivative is: (1) Compound 2 and tri(tetrabutylammonium) pyrophosphate are added to a mixed solvent, heated to 30-40°C, and reacted for 2-4 hours. After the reaction is completed, the mixture is cooled to room temperature naturally, and then sodium chloride is added and stirred for 1-2 hours to obtain modified pyrophosphoric acid, the chemical structure of which is: (2) adding carboxymethyl chitosan and triethylamine to a 40-50% ethanol solution, then adding modified pyrophosphoric acid, heating to 70-80° C., and stirring for 5-8 hours to obtain a pyrophosphoric acid derivative; The solid-liquid ratio of skatole in step A1 is 1 g: 10-15 mL; The solid-liquid ratio of the pyrophosphate derivative in step A2 is 1 g: 50-60 mL; the solvent is 40-50% ethanol; The mass ratio of the drug-containing solution to the carrier solution in step A3 is 1:24-25; the ultrasonic power is 100-120W; The preparation method of compound 2 in step (1) is as follows: phenylbutanol and epichlorohydrin react at 50-60° C. for 4-6 hours under the action of a catalyst to generate compound 1; compound 1 reacts with p-toluenesulfonyl chloride under the action of triethylamine and 4-dimethylaminopyridine at room temperature for 3-4 hours to generate compound 2.

2. The method for preparing bone-targeted nanoparticles containing skatole according to claim 1, characterized in that: The molar ratio of the phenylbutanol, epichlorohydrin and catalyst is 1:1.1-1.2:0.4-0.5; the catalyst is one or a mixture of boron trifluoride, ferric chloride, aluminum chloride and zinc chloride; the molar ratio of the compound 1, p-toluenesulfonyl chloride, triethylamine and 4-dimethylaminopyridine is 1:1.1-1.2:1.5-2:0.1-0.

2.

3. The method for preparing bone-targeted nanoparticles containing skatole according to claim 1, characterized in that: The molar ratio of compound 2 and tri(tetrabutylammonium) pyrophosphate in step (1) is 1:1.1-1.2; the mixed solvent is acetonitrile and tetrahydrofuran in a volume ratio of 1:0.5-1; the molar amount of sodium chloride is the same as the molar amount of tri(tetrabutylammonium) pyrophosphate.

4. The method for preparing bone-targeted nanoparticles containing skatole according to claim 1, characterized in that: The mass ratio of carboxymethyl chitosan, modified pyrophosphoric acid and triethylamine in step (2) is 1:0.3-0.4:0.1-0.

2.

5. A bone-targeted nanoparticle containing skatole, characterized in that: The skatole-containing bone-targeted nanoparticles are prepared by the preparation method of any one of claims 1 to 4.

6. Use of the bone-targeted nanoparticles containing skatole according to claim 5 in the preparation of a drug for treating osteoarthritis.

Images