PLGA-PEG-phosphonate polymer, bone targeting nanoparticle, and preparation method and application thereof

Abstract

The invention provides a PLGA (poly (lactic-co-glycolic acid)-PEG (polyethylene glycol)-phosphonate polymer, a bone targeting nanoparticle as well as a preparation method and application of the bone targeting nanoparticle. According to the preparation method of the PLGA-PEG-phosphonate polymer, no catalyst is needed to participate in the reaction, the reaction can be carried out at room temperature, and the preparation method is a green chemical reaction method and is more suitable for large-scale process production requirements; the bone targeting nanoparticle is formed by self-assembly of the PLGA-PEG-phosphonate polymer and PLGA-PEG, the nanoparticle can be specifically combined with hydroxyapatite in bone tissue to realize targeting of the bone tissue, and the nanoparticle can be used as a drug carrier to deliver drugs to the bone tissue, so that the bone targeting nanoparticle has a good application prospect. And bone tissue related diseases such as osteoporosis, bone cancer and osteoarthritis can be treated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a PLGA-PEG-phosphonate polymer, a bone-targeting nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Bone tissue-related diseases such as osteoporosis, bone cancer and osteoarthritis require precise drug delivery systems to improve therapeutic effect and reduce side effects. Traditional drug delivery methods often lack targeting and result in systemic side effects and low efficacy. Nanotechnology can significantly improve the stability, solubility and bioavailability of drugs by encapsulating drugs in nanoparticles. Bone-targeting nanoparticles can be obtained by modifying bone-targeting ligands such as phosphonate, peptide chain or antibody on the surface of nanoparticles, which can specifically bind to hydroxyapatite in bone tissue to achieve targeting of bone tissue.

[0003] The prior art discloses a bone-targeting nanoparticle composed of polylactic acid-glycolic acid copolymer (PLGA) and the like. However, the synthesis of the polymer of the bone-targeting nanoparticle not only requires low-temperature reaction, but also involves multiple catalysts, and the reaction conditions are relatively harsh, which is not conducive to the consistency and controllability in large-scale production.

[0004] Based on the defects of the existing bone-targeting nanoparticles, it is necessary to improve them. SUMMARY

[0005] The present application aims to at least partially solve the problems in the related art, and provides a PLGA-PEG-phosphonate polymer, a bone-targeting nanoparticle and a preparation method and application thereof.

[0006] In a first aspect, the present application provides a PLGA-PEG-phosphonate polymer, the chemical structure of which is shown as follows:

[0007]

[0008] wherein 90≤x≤130, 5≤m≤100, 5≤n≤100, m, n and x are integers;

[0009] 0≤y≤10, y is an integer;

[0010] The R is selected from any one of the following substituents:

[0011] 1≤z≤3, z is an integer.

[0012] Preferably, y=0, and R is And / or, the weight average molecular weight of the PEG segment in the PLGA-PEG-phosphonate polymer is 1000-5000 Da; the weight average molecular weight of the PLGA segment in the PLGA-PEG-phosphonate polymer is 1-150 kDa; the PLGA segment is polymerized by two monomers of lactic acid and glycolic acid, and the molar ratio of the two monomers is (50-90):(10-50).

[0013] In a second aspect, the application further provides a preparation method of the PLGA-PEG-phosphonate polymer, comprising the following steps:

[0014] The DBCO-COOH is reacted with a reactant at room temperature to obtain an intermediate product;

[0015] The intermediate product is reacted with PLGA-PEG-N3 at room temperature to obtain the PLGA-PEG-phosphonate polymer;

[0016] The chemical structural formula of the reactant is as follows:

[0017] The chemical structural formula of the intermediate product is as follows:

[0018] The chemical structural formula of the PLGA-PEG-N3 is as follows:

[0019] Preferably, the chemical structure of the intermediate product is as follows:

[0020]

[0021] The DBCO-COOH is reacted with a reactant to obtain an intermediate product, specifically comprising the following steps:

[0022] After the DBCO-COOH and HATU are mixed with the first organic solvent, an aqueous sodium alendronate solution is added, and after stirring at room temperature, the pH is adjusted to 9-10, and a white precipitate is precipitated, which is the intermediate product.

[0023] Preferably, in the step of mixing the DBCO-COOH and HATU with the first organic solvent, adding the aqueous sodium alendronate solution, and stirring at room temperature, the stirring time is 2-4 h;

[0024] And / or, the molar volume ratio of the DBCO-COOH, HATU, first organic solvent, and aqueous sodium alendronate solution is (10-14) mmol:(10-14) mmol:(8-12) mL:(8-12) mL;

[0025] The concentration of the aqueous sodium alendronate solution is 0.5-2 mol / L.

[0026] Preferably, the first organic solvent comprises at least one of methanol, ethanol, glycerol, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide.

[0027] Preferably, the intermediate product is reacted with PLGA-PEG-N3 at room temperature to obtain the PLGA-PEG-phosphonate polymer, which comprises the following steps:

[0028] The intermediate product and PLGA-PEG-N3 are added into the second organic solvent and stirred at room temperature for 2-3 hours, and then the product after the reaction is added into water, filtered, and the solid product is collected to obtain the PLGA-PEG-phosphonate polymer.

[0029] Preferably, the molar volume ratio of the intermediate product, PLGA-PEG-N3 and the second organic solvent is (1-1.2) mmol:(1-1.2) mmol:(3-7) mL;

[0030] Preferably, the second organic solvent comprises at least one of dimethyl sulfoxide, acetonitrile, N,N-dimethylformamide, methanol, ethanol, glycerol.

[0031] In a third aspect, the present application further provides a preparation method of bone-targeting nanoparticles, which comprises the following steps:

[0032] The PLGA-PEG-phosphonate polymer or the PLGA-PEG-phosphonate polymer prepared by the preparation method is dissolved in a third organic solvent, and a PVA aqueous solution is added, followed by ultrasonic treatment, stirring, separation, discarding of the precipitate, and collection of the supernatant;

[0033] The supernatant is centrifuged, and the supernatant is discarded and the precipitate is collected;

[0034] The precipitate is dissolved in water, centrifuged, and the supernatant is collected again to obtain the bone-targeting nanoparticles.

[0035] Preferably, the PLGA-PEG-phosphonate polymer and the poly(lactic-co-glycolic acid)-polyethylene glycol copolymer are dissolved in the third organic solvent, and a PVA aqueous solution with a mass concentration of 1-3% is added, followed by ultrasonic treatment for 10-20 minutes, addition of a PVA aqueous solution with a mass concentration of 0.1-0.3% again, ultrasonic treatment for 10-20 minutes again, stirring at room temperature for 10-30 hours, and then centrifugation at 700-900 g for 10-15 minutes, discarding of the precipitate, and collection of the supernatant;

[0036] The supernatant is centrifuged at 18,000-22,000 g for 25-35 minutes, and the supernatant is discarded and the precipitate is collected;

[0037] The precipitate is dissolved in water, centrifuged at 700-900g for 10-15min, and the supernatant is collected again to obtain the bone-targeting nanoparticles.

[0038] The mass / volume ratio of the PLGA-PEG-phosphonate polymer, the poly(lactic-co-glycolic acid)-polyethylene glycol copolymer, the third organic solvent, the first PVA aqueous solution, the second PVA aqueous solution, and the third PVA aqueous solution is (2-6) mg:(14-18) mg:(0.5-2) mL:(0.5-2) mL:(12-14) mL:(0.5-2) mL.

[0039] The third organic solvent comprises dichloromethane and / or chloroform.

[0040] In a fourth aspect, the application further provides a use of the PLGA-PEG-phosphonate polymer or the bone-targeting nanoparticles prepared by the preparation method in the preparation of a drug for treating bone tissue diseases.

[0041] The PLGA-PEG-phosphonate polymer, the bone-targeting nanoparticles, the preparation method and the use of the application have the following advantages over the prior art:

[0042] 1. The preparation method of the PLGA-PEG-phosphonate polymer of the application does not require any catalyst to participate in the reaction, and the reaction can occur at room temperature, which is a green chemical reaction method and is more suitable for large-scale process production requirements.

[0043] 2. The bone-targeting nanoparticles of the application are formed by self-assembly of the PLGA-PEG-phosphonate polymer and the PLGA-PEG. The nanoparticles can specifically bind to hydroxyapatite in bone tissue to achieve targeting of bone tissue. Experiments show that after injection of the bone-targeting nanoparticles (ALD-PLGA-NPs) of the application for 12h and 24h, the ALD-PLGA-NPs are almost entirely distributed in the limbs and tailbone of mice, indicating that the nanoparticles have significant targeting ability for bone, and the targeting ability is superior to that of PLGA-NPs. The bone-targeting nanoparticles of the application can be used as a drug carrier to deliver drugs to bone tissue to treat bone tissue-related diseases such as osteoporosis, bone cancer, and osteoarthritis. BRIEF DESCRIPTION OF DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0045] Figure 1 Schematic diagram of PLGA-PEG-ALD, PLGA-PEG forming ALD-PLGA-NPs by self-assembly;

[0046] Figure 2 Infrared spectra of PLGA-PEG-phosphonate polymer and PLGA-PEG-N3 synthesized in Example 1;

[0047] Figure 3 Amplification spectrum of middle partial band; Figure 2

[0048] Nuclear magnetic hydrogen spectrum of PLGA-PEG-N3 used in Example 1; (50,50,20K) 2000 Figure 4

[0049] Figure 5 Nuclear magnetic hydrogen spectrum of PLGA-PEG-ALD synthesized in Example 1; (50,50,20K) 2000

[0050] Figure 6 Zeta potential of ALD-PLGA-NPs prepared in Example 2 and PLGA-NPs prepared in Comparative Example 1;

[0051] Figure 7 Electron microscope morphology and particle size distribution of ALD-PLGA-NPs prepared in Example 2;

[0052] Figure 8 Transmission electron microscope scanning of ALD-PLGA-NPs prepared in Example 2 and PLGA-NPs prepared in Comparative Example 1 after adsorbing nano-hydroxyapatite;

[0053] Figure 9 Fluorescence distribution of ALD-PLGA-NPs prepared in Example 2 and PLGA-NPs prepared in Comparative Example 1 in mice. DETAILED DESCRIPTION

[0054] The technical solutions in the embodiments of the present application will be clearly and completely described in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0055] ​​​​​It is to be understood that the order of steps or description in the following embodiments does not imply that the preferred order of the steps or description. In addition, in the description of the present application, the term "comprising" means "including but not limited to". Various embodiments of the present application can exist in a range of forms; it should be understood that the description in a range form is merely for convenience and brevity, and should not be understood as a hard limitation on the scope of the present application; therefore, it should be considered that the described range has been specifically disclosed all possible sub-ranges and single values in the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single values in the range, such as 1, 2, 3, 4, 5 and 6, regardless of the range. In addition, whenever a numerical range is indicated in this text, it means that any cited number (fraction or integer) in the indicated range is included.

[0056] The embodiment of the present application provides a PLGA-PEG-phosphonate polymer, and a chemical structural formula is as follows:

[0057]

[0058] Wherein, 90≤x≤130, 5≤m≤100, 5≤n≤100, m, n, x are integers;

[0059] 0≤y≤10, y is an integer;

[0060] R is selected from any one of the following substituents:

[0061] 1≤z≤3, z is an integer.

[0062] In some embodiments, preferably, R is selected from any one of the following substituents:

[0063]

[0064] In some embodiments, y=0, R is At this time, the chemical structural formula of the PLGA-PEG-phosphonate polymer is:

[0065]

[0066] In some embodiments, the weight average molecular weight of the PEG segment in the PLGA-PEG-phosphonate polymer is 1000-5000 Da (Dalton); specifically, the weight average molecular weight of the PEG segment is 1000 Da, 2000 Da, 3000 Da, 4000 Da, 5000 Da, preferably the weight average molecular weight of the PEG segment is 2000 Da.

[0067] In some embodiments, the weight average molecular weight of the PLGA segment in the PLGA-PEG-phosphonate polymer is 1-150 kDa (kiloDalton); specifically, the weight average molecular weight of the PLGA segment is 1 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa; preferably, the weight average molecular weight of the PLGA segment is 20 kDa.

[0068] In some embodiments, the PLGA segment is polymerized from two monomers of lactic acid and glycolic acid, and the molar ratio of the two monomers is (50-90):(10-50); specifically, the molar ratio of the two monomers is 50:50, or 75:25, or 90:10, preferably, the molar ratio of the two monomers is 50:50.

[0069] Based on the same inventive concept, the present application also provides a preparation method of the above-mentioned PLGA-PEG-phosphonate polymer, comprising the following steps:

[0070] S1, reacting DBCO-COOH with a reactant at room temperature to obtain an intermediate product;

[0071] S2, reacting the intermediate product with PLGA-PEG-N3 at room temperature to obtain the PLGA-PEG-phosphonate polymer;

[0072] The chemical structural formula of the reactant is:

[0073] The chemical structural formula of the intermediate product is:

[0074] The chemical structural formula of PLGA-PEG-N3 is:

[0075] Specifically, the DBCO-COOH in the above embodiments is diphenyl cyclooctyne-carboxylic acid, and the chemical structural formula is: CAS:1353016-70-2; PLGA-PEG-N3 is polylactic acid-glycolic acid copolymer-polyethylene glycol-azide.

[0076] In some embodiments, the preparation method of the PLGA-PEG-phosphonate polymer is as follows:

[0077]

[0078] In some embodiments, the chemical structure of the intermediate product is as shown below:

[0079]

[0080] In some embodiments, the reaction of DBCO-COOH with the reactant to obtain the intermediate product specifically comprises the following steps:

[0081] After mixing DBCO-COOH, HATU and the first organic solvent, an aqueous sodium alendronate solution is added, and after stirring at room temperature, the pH is adjusted to 9-10, and a white precipitate is precipitated, which is the intermediate product.

[0082] Specifically, HATU is 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, with a chemical formula of C 10 H 15 OF6N6P, HATU is a commonly used polypeptide condensation reagent, which is used in the synthesis of peptide bonds between carboxyl and amino groups.

[0083] In some embodiments, in the step of mixing DBCO-COOH, HATU and the first organic solvent, and then adding an aqueous sodium alendronate solution and stirring at room temperature, the stirring time is 2-4 h.

[0084] In some embodiments, the aqueous sodium alendronate solution is prepared by adding sodium alendronate to water.

[0085] Specifically, sodium alendronate has a CAS number of 129318-43-0 and a molecular formula of C4H 12 NNaO7P2.

[0086] In some embodiments, the molar volume ratio of DBCO-COOH, HATU, the first organic solvent, and the aqueous sodium alendronate solution is (10-14) mmol:(10-14) mmol:(8-12) mL:(8-12) mL.

[0087] The concentration of the aqueous sodium alendronate solution is 0.5-2 mol / L.

[0088] In some embodiments, the first organic solvent includes at least one of methanol, ethanol, glycerol, acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide.

[0089] In some embodiments, the reaction of the intermediate product with PLGA-PEG-N3 at room temperature obtains the PLGA-PEG-phosphonate polymer, which specifically comprises the following steps:

[0090] The intermediate product and the PLGA-PEG-N3 are added into a second organic solvent, and the reaction is stirred at room temperature for 2-3 hours, and the product after the reaction is added into water, filtered, and the solid product is collected to obtain the PLGA-PEG-phosphonate polymer.

[0091] In some embodiments, the molar volume ratio of the intermediate product, the PLGA-PEG-N3 and the second organic solvent is (1-1.2) mmol:(1-1.2) mmol:(3-7) mL.

[0092] In some embodiments, the second organic solvent comprises at least one of dimethyl sulfoxide, acetonitrile, N,N-dimethylformamide, methanol, ethanol, glycerol.

[0093] In some embodiments, preferably, the synthesis route of the PLGA-PEG-phosphonate polymer is as follows:

[0094] i. Preparation of DBCO-ALD (i.e. the intermediate product mentioned above)

[0095]

[0096] ii. Preparation of PLGA-PEG-ALD (i.e. the PLGA-PEG-phosphonate polymer)

[0097]

[0098] The synthesis of the polymer PLGA-PEG-phosphonate with a novel structure designed by the present application is a green chemical reaction method, which does not require any catalyst to participate in the reaction and can occur at room temperature, and is more suitable for large-scale process production requirements.

[0099] Based on the same inventive concept, the present application also provides a preparation method of bone-targeting nanoparticles, comprising the following steps:

[0100] S1, the PLGA-PEG-phosphonate polymer, polylactic acid-glycolic acid polyethylene glycol copolymer (PLGA-PEG) mentioned above are dissolved in a third organic solvent, a PVA aqueous solution is added, and after ultrasonic treatment, stirring, separation, the precipitate is discarded, and the supernatant is collected;

[0101] S2, after centrifugation of the supernatant, the supernatant is discarded, and the precipitate is collected;

[0102] S3, the precipitate is dissolved in water, centrifuged, and the supernatant is collected again to obtain bone-targeting nanoparticles.

[0103] Specifically, the PVA in the above embodiments is polyvinyl alcohol, and the polyvinyl alcohol is added into water to obtain a PVA aqueous solution,

[0104] In some embodiments, PLGA-PEG-phosphonate polymer and polylactic acid-glycolic acid polyethylene glycol copolymer are dissolved in a third organic solvent, a PVA aqueous solution with a mass concentration of 1-3% is added, and the mixture is sonicated for 10-20 min. Then, a PVA aqueous solution with a mass concentration of 0.1-0.3% is added again, and the mixture is sonicated again for 10-20 min. The mixture is stirred at room temperature for 10-30 h, and then centrifuged at 700-900 g for 10-15 min. The precipitate is discarded, and the supernatant is collected.

[0105] Centrifuge the supernatant at 18000-22000g for 25-35 minutes, discard the supernatant, and collect the precipitate;

[0106] The precipitate was then dissolved in water and centrifuged at 700–900g for 10–15 minutes. The supernatant was collected again to obtain bone-targeting nanoparticles.

[0107] The mass-volume ratio of PLGA-PEG-phosphonate polymer, polylactic acid-glycolic acid polyethylene glycol copolymer, third organic solvent, first added PVA aqueous solution, and second added PVA aqueous solution is (2-6) mg:(14-18) mg:(0.5-2) mL:(0.5-2) mL:(12-14) mL.

[0108] The third organic solvent includes dichloromethane and / or chloroform.

[0109] Based on the same inventive concept, the present invention also provides the application of the above-mentioned PLGA-PEG-phosphonate polymer or the bone-targeting nanoparticles prepared by the above-mentioned preparation method as drug carriers in the preparation of drugs for treating bone tissue diseases.

[0110] Further reference Figure 1 As shown, it illustrates PLGA-PEG-ALD (i.e., PLGA-PEG-phosphonate polymer) and PLGA-PEG self-assembling to form ALD-PLGA-NPs (i.e., bone-targeting nanoparticles). The mass fraction of PLGA-PEG-ALD is 20%, and the mass fraction of PLGA-PEG is 80%. ALD-PLGA-NPs can serve as drug carriers to deliver drugs to bone tissue for the treatment of bone-related diseases such as osteoporosis, bone cancer, and osteoarthritis.

[0111] The following further illustrates the PLGA-PEG-phosphonate polymer, bone-targeting nanoparticle and its preparation method and application of the present application with specific examples. This part further illustrates the content of the present application in combination with specific examples, but should not be understood as a limitation of the present application. If not specifically stated, the technical means adopted in the examples are the conventional means familiar to those skilled in the art. Unless specifically stated, the reagents, methods and equipment adopted in the present application are the conventional reagents, methods and equipment in the art.

[0112] Example 1

[0113] The present example provides a PLGA-PEG-phosphonate polymer, the chemical structural formula of which is as follows:

[0114]

[0115] In the PLGA-PEG-phosphonate polymer, the weight average molecular weight of the PEG segment is 2000 Da, the weight average molecular weight of the PLGA segment in the PLGA-PEG-phosphonate polymer is 20 kDa; the PLGA segment is polymerized from two monomers of lactic acid and glycolic acid, and the molar ratio of the two monomers of lactic acid and glycolic acid is 50:50.

[0116] The preparation method of the above-mentioned PLGA-PEG-phosphonate polymer comprises the following steps:

[0117] S1, commercially available DBCO-COOH (12 mmol) and HATU (12 mmol) and 10 mL of methanol were placed in a reaction bottle and stirred at room temperature for 30 minutes, then 10 mmol of sodium alendronate dissolved in 10 mL of water was added, and stirred at room temperature for 3 h. After adjusting the pH to 10 with 0.1 M sodium hydroxide solution, white solid was precipitated, and the white solid was collected and recrystallized with ethanol to obtain pure DBCO-ALD (ESI-MS, 535.1 [M-H] + ]);

[0118] S2, 1 mmol of commercially available PLGA-PEG-N3 and 1.2 mmol of DBCO-ALD were dissolved in 5 mL of dimethyl sulfoxide into a reaction bottle, and stirred at room temperature for 2 h. The reaction liquid was poured into 500 mL of water, and the solid was collected by filtration to obtain the PLGA-PEG-phosphonate polymer;

[0119] In the PLGA-PEG-N3, PLGA (50,50,20K) -PEG 2000 -N3 is purchased from Hangzhou Xinqiao Biological Technology Co., Ltd., and the finally prepared PLGA-PEG-phosphonate polymer is denoted as PLGA (50,50,20K) -PEG 2000 -ALD; PLGA (50,50,20K)-PEG 2000 PLGA in -N3 (50,50,20K) denotes the weight average molecular weight of the PLGA segment is 20 kDa, the PLGA segment is polymerized by two monomers of lactic acid and glycolic acid, and the molar ratio of the two monomers of lactic acid and glycolic acid is 50:50; PEG 2000 denotes the weight average molecular weight of the PEG segment is 2000 Da;

[0120] The synthesis route of the above PLGA-PEG-phosphonate polymer is as follows:

[0121]

[0122] Example 2

[0123] The present embodiment provides a preparation method of bone-targeting nanoparticles, comprising the following steps:

[0124] S1, 4 mg of the PLGA-PEG-phosphonate polymer PLGA (50,50,20K) -PEG 2000 -ALD, 16 mg of PLGA (50,50,20K) -PEG 2000 (bought from Hangzhou Xinqiao Biological Technology Co., Ltd., PLGA (50,50,20K) The meanings represented are as shown above, PEG 2000 The meanings represented are as shown above) is dissolved in 1 mL of dichloromethane, 1 mL of a 2% PVA aqueous solution is added, ultrasonic treatment is performed for 15 min, 13 mL of a 0.2% PVA aqueous solution is added again, ultrasonic treatment is performed again for 15 min, stirring is performed at 500 rpm at room temperature for 15 h, then centrifugation is performed at 800 g for 10 min, the precipitate is discarded, and the supernatant is collected;

[0125] S2, the supernatant in S1 is centrifuged at 20000 g for 30 min, the supernatant is discarded, and the precipitate is collected;

[0126] S3, the precipitate in S2 is dissolved in 1 mL of water, centrifugation is performed at 800 g for 10 min, and the supernatant is collected again, i.e. bone-targeting nanoparticles (ALD-PLGA-NPs) are obtained.

[0127] Comparative Example 1

[0128] The present comparative example provides a preparation method of bone-targeting nanoparticles, comprising the following steps:

[0129] S1. Dissolve 16 mg PLGA-PEG in 1 mL of dichloromethane, add 1 mL of 2% PVA aqueous solution, sonicate for 15 min, add 13 mL of 0.2% PVA aqueous solution again, sonicate for 15 min again, stir at 500 rpm for 15 h at room temperature, then centrifuge at 800 g for 10 min, discard the precipitate and collect the supernatant.

[0130] S2. After centrifuging the supernatant in S1 at 20000g for 30min, discard the supernatant and collect the precipitate;

[0131] S3. Dissolve the precipitate from S2 in 1 mL of water, centrifuge at 800 g for 10 min, and collect the supernatant again to obtain bone-targeting nanoparticles (PLGA-NPs).

[0132] Performance testing

[0133] The infrared spectra of the PLGA-PEG-phosphonate polymer and PLGA-PEG-N3 synthesized in Example 1 are as follows: Figure 2 As shown, Figure 3 for Figure 2 Magnified spectrum of the middle band; Figures 2-3 In this context, PLGA-PEG-Ald represents the final synthesized PLGA. (50,50,20K) -PEG 2000 -ALD, PLGA-PEG-N3 indicates PLGA (50,50,20K) -PEG 2000 -N3.

[0134] from Figures 2-3 As can be seen from the infrared spectrum of PLGA-PEG-Ald, the 3000 cm⁻¹ of PLGA-PEG-N3... -1 The disappearance of the N≡N structural characteristic peak at 800-1000 cm⁻¹ indicates that a reaction has occurred; while at 800-1000 cm⁻¹... -1 The number increased by 845.27. -1 The absorption peak here is a characteristic absorption peak of the benzene ring in the structure of the reaction product.

[0135] PLGA used in Example 1 (50,50,20K) -PEG 2000 The 1H NMR spectrum of -N3 is as follows: Figure 4 As shown, the PLGA synthesized in Example 1 (50,50,20K) -PEG 2000 -ALD's 1H NMR spectrum is as follows Figure 5 As shown.

[0136] from Figures 4-5 As can be seen from this, compared to PLGA (50,50,20K) -PEG2000 - NMR spectrum of N3, in PLGA (50,50,20K) - PEG 2000 - ALD NMR hydrogen spectrum still retains PLGA (50,50,20K) - PEG 2000 - Hydrogen in the position of "1-6" in the NMR spectrum of N3, while aromatic hydrogen is increased at low field chemical shift 6.5-8.0 ppm, corresponding to Figure 5 "7" marked in the structure; the fat hydrogen is increased at high field chemical shift 2.0 ppm, corresponding to Figure 5 "8" marked in the structure; the fat hydrogen connected with active hydrogen is increased at chemical shift 4.4 ppm, corresponding to Figure 5 "9" marked in the structure; the active hydrogen is increased at chemical shift 5.8 ppm, corresponding to Figure 5 "10" marked in the structure.

[0137] The zeta potentials of the ALD-PLGA-NPs prepared in Example 2 and the PLGA-NPs prepared in Comparative Example 1 were determined using a Malvern particle size analyzer, and the results are shown in Figure 6 The zeta potential of the ALD-PLGA-NPs contains more negative charges due to the negative charges of the bisphosphonate of ALD.

[0138] The particle size and morphology of the ALD-PLGA-NPs were determined using a Malvern particle size analyzer and a transmission electron microscope, and the results are shown in Figure 7 .

[0139] As can be seen from Figure 7 , the ALD-PLGA-NPs are spherical with an average particle size of 186.3 ± 2.8 nm.

[0140] Verification of the bone targeting ability of the ALD-PLGA-NPs

[0141] In vitro experiment: the ALD-PLGA-NPs prepared in Example 2 and the PLGA-NPs prepared in Comparative Example 1 were respectively mixed with nano-hydroxyapatite under stirring at room temperature for 12 hours, then the mixture was centrifuged to remove the unbound nano-hydroxyapatite, and the supernatant was collected for transmission electron microscope scanning, and the results are shown in Figure 8 .

[0142] Figure 8 In the figure, +ALD-PLGA-NPs represents the TEM image tested after adding the ALD-PLGA-NPs, +PLGA-NPs represents the TEM image tested after adding the PLGA-NPs, and blank represents the TEM image tested after adding the nano-hydroxyapatite to water.

[0143] As can be seen from Figure 8As can be seen, compared with PLGA-NPs, ALD-PLGA-NPs can significantly adsorb nano-hydroxyapatite, which means that it has bone-targeting ability.

[0144] 100 μL of ALD-PLGA-NPs and PLGA-NPs were injected intraperitoneally into 10-week-old female C57BL / 6J mice, with PBS (phosphate-buffered saline, pH 6.8) injection serving as a control. Mice were anesthetized at 6, 12, and 24 hours post-injection, and whole-body fluorescence distribution imaging was performed using an IVIS Spectrum device. The results are as follows: Figure 9 As shown.

[0145] from Figure 9 As can be seen, 6 hours after injection, PLGA-NPs were mostly distributed in the liver of mice, while ALD-PLGA-NPs, in addition to the liver, began to show significant distribution in the limbs and tailbone of mice; 12 and 24 hours after injection, ALD-PLGA-NPs were almost entirely distributed in the limbs and tailbone of mice, indicating its significant targeting ability to bones, and its targeting ability is superior to that of PLGA-NPs.

[0146] It is understood that the technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0147] The above are merely preferred embodiments of this application, and only specifically describe the technical principles of this application. These descriptions are only for explaining the principles of this application and should not be construed as limiting the scope of protection of this application in any way. Based on this explanation, any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application, as well as other specific embodiments of this application that can be conceived by those skilled in the art without creative effort, should be included within the scope of protection of this application.

Claims

1. A PLGA-PEG-phosphonate polymer, characterized in that, Its chemical structural formula is shown below: Where 90≤x≤130, 5≤m≤100, 5≤n≤100, and m, n, and x are all integers; 0 ≤ y ≤ 10, where y is an integer; The R is selected from any of the following substituents: 1≤z≤3, where z is an integer.

2. The PLGA-PEG-phosphonate polymer as described in claim 1, characterized in that, Where y = 0, R is And / or, the weight-average molecular weight of the PEG segment in the PLGA-PEG-phosphonate polymer is 1000-5000 Da; the weight-average molecular weight of the PLGA segment in the PLGA-PEG-phosphonate polymer is 1-150 kDa; the PLGA segment is polymerized from two monomers, lactic acid and glycolic acid, wherein the molar ratio of the two monomers, lactic acid and glycolic acid, is (50-90):(10-50).

3. A method for preparing the PLGA-PEG-phosphonate polymer as described in any one of claims 1 to 2, characterized in that, Includes the following steps: DBCO-COOH was reacted with the reactants at room temperature to give an intermediate product; The intermediate product was reacted with PLGA-PEG-N3 at room temperature to obtain the PLGA-PEG-phosphonate polymer. The chemical structural formula of the reactant is as follows: The chemical structural formula of the intermediate product is as follows: The chemical structural formula of PLGA-PEG-N3 is as follows:

4. The method for preparing the PLGA-PEG-phosphonate polymer as described in claim 3, characterized in that, The chemical structure of the intermediate product is shown below: The intermediate product is obtained by reacting DBCO-COOH with the reactants, specifically through the following steps: After mixing DBCO-COOH, HATU and the first organic solvent, an aqueous solution of alendronate sodium is added. The mixture is stirred at room temperature, and then the pH is adjusted to 9-10. A white precipitate is formed, which is the intermediate product.

5. The method for preparing the PLGA-PEG-phosphonate polymer as described in claim 4, characterized in that, In the step of mixing DBCO-COOH, HATU and the first organic solvent, adding an aqueous solution of alendronate sodium, and stirring at room temperature, the stirring time is 2 to 4 hours. And / or, the molar volume ratio of DBCO-COOH, HATU, the first organic solvent, and sodium alendronate aqueous solution is (10-14) mmol:(10-14) mmol:(8-12) mL:(8-12) mL; The concentration of the sodium alendronate aqueous solution is 0.5–2 mol / L; And / or, the first organic solvent includes at least one of methanol, ethanol, glycerol, acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide.

6. The method for preparing the PLGA-PEG-phosphonate polymer as described in claim 3, characterized in that, The intermediate product is reacted with PLGA-PEG-N3 at room temperature to obtain the PLGA-PEG-phosphonate polymer, specifically including the following steps: The intermediate product and PLGA-PEG-N3 were added to a second organic solvent and stirred at room temperature for 2-3 hours. The reacted product was then added to water, filtered, and the solid product was collected to obtain the PLGA-PEG-phosphonate polymer.

7. The method for preparing the PLGA-PEG-phosphonate polymer as described in claim 6, characterized in that, The molar volume ratio of the intermediate product, PLGA-PEG-N3, and the second organic solvent was (1–1.2) mmol:(1–1.2) mmol:(3–7) mL. And / or, the second organic solvent includes at least one of dimethyl sulfoxide, acetonitrile, N,N-dimethylformamide, methanol, ethanol, and glycerol.

8. A method for preparing bone-targeting nanoparticles, characterized in that, Includes the following steps: The PLGA-PEG-phosphonate polymer or the PLGA-PEG-phosphonate polymer prepared by the preparation method according to any one of claims 1 to 2 or any one of claims 3 to 7, and the polylactic acid-glycolic acid polyethylene glycol copolymer are dissolved in a third organic solvent, PVA aqueous solution is added, ultrasonic treatment is performed, stirring is carried out, separation is performed, the precipitate is discarded, and the supernatant is collected. After centrifuging the supernatant, discard the supernatant and collect the precipitate; The precipitate was then dissolved in water, centrifuged, and the supernatant was collected again to obtain bone-targeting nanoparticles.

9. The method for preparing bone-targeting nanoparticles as described in claim 8, characterized in that, Dissolve PLGA-PEG-phosphonate polymer and polylactic acid-glycolic acid polyethylene glycol copolymer in a third organic solvent, add 1-3% PVA aqueous solution, sonicate for 10-20 min, add 0.1-0.3% PVA aqueous solution again, sonicate again for 10-20 min, stir at room temperature for 10-30 h, then centrifuge at 700-900 g for 10-15 min, discard the precipitate and collect the supernatant; Centrifuge the supernatant at 18000-22000g for 25-35 minutes, discard the supernatant, and collect the precipitate; The precipitate is then dissolved in water, centrifuged at 700-900g for 10-15 minutes, and the supernatant is collected again to obtain bone-targeting nanoparticles. The mass-volume ratio of PLGA-PEG-phosphonate polymer, polylactic acid-glycolic acid polyethylene glycol copolymer, third organic solvent, first added PVA aqueous solution, and second added PVA aqueous solution is (2-6) mg:(14-18) mg:(0.5-2) mL:(0.5-2) mL:(12-14) mL. The third organic solvent includes dichloromethane and / or chloroform.

10. The use of a PLGA-PEG-phosphonate polymer as described in any one of claims 1 to 2, or bone-targeting nanoparticles prepared by the preparation method as described in any one of claims 8 to 9, as a drug carrier in the preparation of drugs for treating bone tissue diseases.

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