Microsphere bone repair material loaded with small-molecular targeted drug and preparation method of microsphere bone repair material

Abstract

The invention belongs to the technical field of bone repair materials, and discloses a microsphere bone repair material loaded with a micromolecular targeted drug and a preparation method thereof. The preparation method comprises the following steps: uniformly mixing hydroxyapatite nano-particles or selenium-doped hydroxyapatite nano-particles prepared by adopting an LSS method with a gelatin solution of polyvinyl alcohol to serve as a water phase; uniformly mixing the PLGA, the fat-soluble micromolecular targeted drug and the organic solvent to serve as an oil phase; uniformly mixing the oil phase and the water phase by adopting an equal-density emulsion method to obtain an equal-density emulsion; and forming uniform emulsion liquid drops from the equidensity emulsion by adopting a micro-fluidic method, solidifying the emulsion liquid drops in a collection phase under an ice bath to form microspheres, and carrying out post-treatment to obtain the microsphere bone repair material loaded with the small molecular targeted drug. The preparation method disclosed by the invention is simple to operate and high in controllability, and the prepared microsphere bone repair material has a porous microsphere structure with uniform size and pore diameter, is excellent in biological safety, osteogenic activity and anti-inflammatory activity, and can be effectively applied to alveolar bone defects.

Description

Technical Field

[0001] The present invention relates to the technical field of bone repair materials, and in particular to a microsphere bone repair material loaded with small molecule targeted drugs and a preparation method thereof. Background Art

[0002] Periodontitis is one of the most common oral diseases. It can be difficult to detect in its early stages, incurable in its later stages, can trigger systemic diseases, and affects a wide range of people. According to the Third National Oral Epidemiology Survey, its prevalence in my country is as high as 45%. Therefore, its treatment represents a significant societal need. Periodontitis can cause alveolar bone defects, and severe alveolar bone resorption requires bone grafting to repair and regenerate the alveolar bone. However, due to the complexity of periodontal tissue structure, the influence of oral microorganisms, and inflammatory factors, periodontal regeneration has long been a clinically challenging medical problem that needs to be addressed.

[0003] Currently, the efficacy of bone grafting for periodontitis is not ideal, which is related to the existing bone grafting materials. The existing bone grafting materials mainly include autologous bone, allogeneic bone and artificial bone. Among them, autologous bone requires a second operation, which increases the pain of patients; allogeneic bone has limited donors and may have the risk of infectious diseases, as well as the risk of rejection. Therefore, artificial bone has become the most commonly used bone repair material in clinical practice.

[0004] However, current artificial bone powder uses hydroxyapatite as its primary raw material. While its preparation method is relatively mature, its controllability is poor, resulting in uneven size and particle size. This in turn leads to unstable pore structures, hindering cell ingrowth, and limited surface area, hindering biomineralization. Furthermore, artificial bone powder prepared by these methods often has weak osteogenic activity, limited biological functions, and lacks anti-inflammatory activity. Summary of the Invention

[0005] In order to address the technical problems of the above-mentioned existing bone repair materials, such as weak osteogenic activity, single biological function, lack of anti-inflammatory activity, and uneven size and particle size, which result in unstable pore structure provided by the material that is not conducive to cell ingrowth and limited surface area that is not conducive to biomineralization, the purpose of the present invention is to provide a microsphere bone repair material loaded with small molecule targeted drugs and a preparation method thereof. The present invention adopts isopycnic emulsion technology and microfluidics technology to introduce fat-soluble small molecule targeted drugs in the process of constructing porous microspheres based on hydroxyapatite nanoparticles, thereby obtaining a microsphere bone repair material with uniform size and pore size, thereby achieving improvements in its osteogenic effect and anti-inflammatory activity, enabling it to be effectively used for the repair of alveolar bone defects.

[0006] The microsphere bone repair material loaded with small molecule targeted drugs and the preparation method thereof of the present invention are achieved through the following technical solutions:

[0007] The first purpose of the present invention is to provide a method for preparing a microsphere bone repair material loaded with small molecule targeted drugs, so as to introduce fat-soluble small molecule targeted drugs into the preparation process of the porous microspheres to obtain a microsphere bone repair material loaded with small molecule targeted drugs.

[0008] It should be noted that the present invention takes into account that periodontal immunity has significant circadian oscillations. Studies have shown that circadian rhythm disorders can aggravate the progression of macrophage dysfunction and alveolar bone loss in patients with periodontitis, and aggravate periodontitis by regulating the rhythmic core gene BMAL1. Regulating macrophage phenotype and cytokine secretion helps to reduce the level of periodontitis inflammation, improve the periodontal microenvironment, thereby reducing tissue damage or promoting periodontal tissue regeneration. Among the core circadian clock genes, REV-ERBs is a link closely related to periodontal immunity and bone regeneration. REV-ERBa is a member of the REV-ERBs family. As a nuclear receptor, it inhibits the transcription and expression of the core gene BMAL1, and forms a feedback loop of gene expression with core links of the circadian clock such as BMAL1 and CLOCK. Studies have shown that REV-ERBs are closely related to inflammation and are a key link between the circadian clock and immune function. Therefore, REV-ERBs may be a unique therapeutic target for human inflammatory diseases. Nuclear receptor REV-ERBs can inhibit inflammasome activation under pharmacological activation by small molecule agonists, or reduce IL-6 production by inhibiting Tlr4 in macrophages, blocking the inflammatory response, or inhibit LPS-induced macrophage M1 polarization through the phosphatidylinositol-3-kinase (PI3K) signaling pathway. Due to the extensive connection between REV-ERBs and periodontal inflammation, targeting REV-ERBs to regulate periodontal inflammation and correct its abnormal fluctuations, thereby providing a favorable microenvironment for bone regeneration, has become a promising strategy for promoting alveolar bone regeneration. Therefore, in some preferred embodiments of the present invention, the lipid-soluble small molecule targeted drug used is a targeted agonist of Rev-ERBs to enhance the anti-inflammatory activity of microsphere bone repair materials. In some more preferred embodiments of the present invention, considering that compound STL1267 is one of the most effective and safest small molecule compounds currently targeting REV-ERBs, compound STL1267 is preferably used as a lipid-soluble small molecule targeted drug to enhance the anti-inflammatory activity of microsphere bone repair materials.

[0009] The present invention takes into account that if the carrier is prepared first, and then the drug is loaded on the carrier to achieve the composite of the drug and the carrier, there will be uneven loading effect, and the loaded drug will affect the structure of the carrier, resulting in an impact on its osteogenic activity. Therefore, in order to ensure that the fat-soluble small molecule targeted drug can be introduced during the preparation of the porous microspheres, and microspheres with stable morphology and uniform pores are obtained, the present invention preferably first adopts an isopycnic emulsion method to homogenize the fat-soluble small molecule targeted drug and the above-mentioned nanoparticle matrix into a stable isopycnic emulsion, which is used as a liquid raw material for further forming porous microspheres. The isopycnic emulsion is then formed into uniform emulsion droplets by a microfluidic method, solidified in the collection phase, and then obtained by post-processing. A microsphere bone repair material loaded with small molecule targeted drugs is obtained. And the preparation method of a microsphere bone repair material loaded with small molecule targeted drugs of the present invention specifically includes the following steps:

[0010] Step 1, prepare the aqueous phase:

[0011] Polyvinyl alcohol is dispersed in a gelatin solution to obtain a polyvinyl alcohol gelatin solution; and a nanoparticle matrix is added to the polyvinyl alcohol gelatin solution and mixed to obtain an aqueous phase.

[0012] Among them, the present invention uses gelatin as a template and adopts hydroxyapatite nanoparticles prepared by the LSS method or selenium-doped hydroxyapatite nanoparticles as the nanoparticle matrix for constructing porous microspheres, so that the used nanoparticle matrix has a natural bone hydroxyapatite hierarchical structure, providing an excellent osteogenic activity basis for the obtained porous microspheres.

[0013] In some preferred embodiments of the present invention, polyvinyl alcohol is dispersed in a gelatin solution to obtain a polyvinyl alcohol-gelatin solution. The polyvinyl alcohol-gelatin solution serves as a dispersing solvent and porogen for dispersing the nanoparticle matrix. The PVA is used to provide surface tension to stabilize the emulsion later. In some preferred embodiments of the present invention, the mass concentration of the polyvinyl alcohol in the polyvinyl alcohol solution is 1.5% to 2.5%, and the mass concentration of the gelatin solution is 10% to 20%.

[0014] In some preferred embodiments of the present invention, the mass ratio of the nanoparticle matrix in the aqueous phase to the polyvinyl alcohol in the polyvinyl alcohol gelatin solution is 1.2-8:1.

[0015] It should be noted that in order to ensure that the nanoparticle matrix has a biomimetic hierarchical mineralized hydroxyapatite structure and excellent osteogenic activity, in some preferred embodiments of the present invention, it is prepared by the following preparation method:

[0016] The LSS method is adopted, with gelatin as a template and a solvent containing octadecylamine and linoleic acid as a reaction solvent. After being mixed with a reactive ion source in the reaction solvent, an incubation treatment is performed, in which octadecylamine and linoleic acid respectively regulate the orderly assembly of ions provided by the reactive ion source on the active groups of the gelatin to form a nanoparticle matrix with a natural bone hydroxyapatite hierarchical structure, thereby promoting osteogenic differentiation at the nano level, thereby providing osteogenic activity for the microsphere material.

[0017] Wherein, when preparing the hydroxyapatite nanoparticles, the reaction ion source is Ca 2+ Source and PO4 3+ When preparing selenium-doped hydroxyapatite nanoparticles, the reaction ion source is Ca 2+ Source, PO4 3+ Source and SeO3 2- source.

[0018] In some preferred embodiments of the present invention, when preparing the hydroxyapatite nanoparticles, gelatin and Ca 2+ Source and PO4 3+ The dosage ratio of the source is 35mg-45mg:1mmol:0.58mmol. In other preferred embodiments of the present invention, when preparing selenium-doped hydroxyapatite nanoparticles, gelatin and Ca 2+ Source, PO4 3+ Source and SeO3 2- The usage ratio of the source is 35mg~45mg:1mmol:0.58mmol:0.0561mmol.

[0019] It should also be noted that, in some preferred embodiments of the present invention, when gelatin, a solvent containing octadecylamine and linoleic acid, and a reactive ion source are mixed, Ca 2+ The source is dispersed in water to form Ca 2+ source solution; dispersing gelatin in water to form a gelatin aqueous solution; 2+ The source solution and the gelatin aqueous solution were mixed in a 37°C water bath to obtain a mixed solution A. Octadecylamine, linoleic acid and anhydrous ethanol were mixed in a volume ratio of 1:8:32 to obtain a reaction solvent. The mixed solution A and the reaction solvent were mixed to obtain a mixed solution B. 3+ source, or PO4 3+ Source and SeO3 2-The source is dispersed in water to obtain a mixed solution C, and the mixed solution C is added dropwise to the mixed solution B and stirred for 1 to 3 hours to obtain a suspension. The obtained suspension is then incubated. In some preferred embodiments of the present invention, the suspension is incubated at 20°C to 45°C for 24 to 168 hours, the final precipitate is collected by centrifugation, and washed alternately with anhydrous ethanol and deionized water for at least 6 times to obtain hydroxyapatite nanoparticles or selenium-doped hydroxyapatite nanoparticles, which are then stored in anhydrous ethanol at a storage temperature of 2°C to 8°C.

[0020] In some preferred embodiments of the present invention, in order to avoid the introduction of other impurities, before dispersing the nanoparticle matrix in the gelatin solution of polyvinyl alcohol, the nanoparticle matrix stored in anhydrous ethanol is first centrifuged, washed with water at least 3 times, and then dispersed in the gelatin solution of polyvinyl alcohol.

[0021] In some preferred embodiments of the present invention, the gelatin is macromolecular porcine gelatin.

[0022] In some preferred embodiments of the present invention, the Ca 2+ The source is Ca(NO3)2·4H2O.

[0023] In some preferred embodiments of the present invention, the PO4 3+ The source is trisodium phosphate.

[0024] In some preferred embodiments of the present invention, the SeO3 2- The source is sodium selenite.

[0025] Step 2, prepare the oil phase:

[0026] PLGA and fat-soluble small molecule targeted drugs are dispersed in an organic solvent to obtain an oil phase.

[0027] In some preferred embodiments of the present invention, the molecular weight of the PLGA used is 45000Da. In some preferred embodiments of the present invention, the organic solvent used is a mixed solvent of ethyl acetate and dichloromethane, wherein the mass ratio of ethyl acetate and dichloromethane is not limited, because the relative amounts of ethyl acetate and dichloromethane will be adjusted later to adjust the density of the oil phase to the same as that of the aqueous phase. In some preferred embodiments of the present invention, a mixed solvent with a mass ratio of ethyl acetate and dichloromethane of 7:5 to 8:6 can be used to dissolve or disperse PLGA and fat-soluble small molecule targeted drugs, so that the density of the oil phase can be adjusted to the same as that of the aqueous phase after subsequent processing.

[0028] In some preferred embodiments of the present invention, the fat-soluble small molecule targeted drug is uniformly dispersed in the organic solvent by ultrasonic dispersion, and the ultrasonic frequency is 20kHz to 100kHz, and the drug is ultrasonicated until dissolved.

[0029] In some preferred embodiments of the present invention, when preparing the oil phase, the mass ratio of the fat-soluble small molecule targeted drug to the PLGA is 0.1 mg to 20 mg: 0.1 g to 0.3 g.

[0030] Step 3, prepare an equal density emulsion:

[0031] The oil phase and the water phase are mixed uniformly by adopting an isodensity emulsion method to obtain a stable isodensity emulsion.

[0032] It should be noted that, in some preferred embodiments of the present invention, the oil phase and the aqueous phase are mixed and then emulsified at a high speed to ensure that the oil phase and the aqueous phase are fully mixed to form a stable isodensity emulsion, and the mixing rate of the high-speed homogenization emulsification is 15000 r / min to 20000 r / min, and the mixing time is 20s to 30s.

[0033] In some preferred embodiments of the present invention, when the aqueous phase and the oil phase are mixed, the mass ratio of the fat-soluble small molecule targeted drug in the oil phase to the nanoparticle matrix in the aqueous phase is 0.1 mg to 20 mg: 0.06 g to 0.12 g.

[0034] Step 4, preparation of microspheres:

[0035] The isodensity emulsion is formed into uniform emulsion droplets by adopting a microfluidics method; the emulsion droplets are placed in a collection phase under ice bath conditions to solidify at low temperature to form microspheres.

[0036] It should be noted that, in some preferred embodiments of the present invention, the collection phase used is a polyvinyl alcohol solution, so that uniform emulsion droplets fall into the collection phase one by one, and due to the presence of surface tension mainly provided by PVA, the liquid remains stable and gradually solidifies at low temperature, thereby forming microspheres.

[0037] In other preferred embodiments of the present invention, the collection phase is a polyvinyl alcohol solution with a mass concentration of 1.5% to 2.5%.

[0038] In some preferred embodiments of the present invention, the temperature of the low-temperature coagulation is 2°C to 16°C.

[0039] Step 5, post-processing of microspheres:

[0040] The microspheres are stirred to remove the organic solvent; after heating, they are washed to remove the collection phase, and freeze-dried to obtain a microsphere bone repair material loaded with small molecule targeted drugs.

[0041] It should be noted that, in some preferred embodiments of the present invention, after the uniform emulsion droplets solidify at low temperature to form microspheres, the mixture is stirred at room temperature for 10 to 15 hours to completely evaporate the dichloromethane and ethyl acetate in the oil phase.

[0042] The stirred product is treated in a water bath at 30°C to 50°C for 2h to 4h to melt the gelatin, thereby releasing the microspheres from the gelatin, and washed with water at least 3 times to remove the gelatin on the surface of the microspheres. The product is then freeze-dried to remove excess water while retaining the porous microsphere structure, thereby obtaining a microsphere bone repair material with a porous structure loaded with small molecule targeted drugs.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] This invention uses gelatin as a template and hydroxyapatite nanoparticles or selenium-doped hydroxyapatite nanoparticles prepared by the LSS method as the nanoparticle matrix for constructing porous microspheres. This nanoparticle matrix has the hierarchical structure of natural bone hydroxyapatite, providing a foundation for the resulting porous microspheres' excellent osteogenic activity. A gelatin solution of polyvinyl alcohol is used as a porogen, mixed with the nanoparticle matrix, and then used as the aqueous phase. A mixture of PLGA, a lipid-soluble small molecule targeted drug, and an organic solvent serves as the oil phase. Then, by combining isopycnic emulsion technology and microfluidic technology, the oil phase and the water phase are mixed to obtain an isopycnic emulsion with excellent uniformity and stability. The isopycnic emulsion is then subjected to a microfluidic method, and the collection phase as the continuous phase and the isopycnic emulsion as the discontinuous phase are allowed to intersect in a microfluidic device to form uniform droplets. The collection phase is then removed by post-treatment and freeze-dried, thereby achieving the preparation of a large number of uniform solid microspheres, and obtaining a microsphere bone repair material with specific pores and loaded with small molecule targeted drugs. The present invention introduces fat-soluble small molecule targeted drugs in the process of preparing a microsphere bone repair material with a porous structure, not only combining the two steps of carrier preparation and load loading into one step, simplifying the preparation process, but also structurally improving the pores and appearance of the microsphere bone repair material, solving the current problem of uneven morphology and size of porous microspheres, forming a uniform size and uniform pore diameter structure that is conducive to biomineralization and cell ingrowth, and providing a bone repair-promoting microenvironment.

[0045] The preparation method of the present invention is simple to operate and highly controllable. The microsphere bone repair material prepared in the present invention has a porous microsphere structure with uniform size and pore size. Its large surface area and Gel-SeHA component facilitate the provision of a large number of biomineralization sites, and the uniform pore size promotes cell ingrowth, thereby facilitating osteogenesis. The simultaneous introduction of poly(lactic-co-glycolic acid) (PLGA) and a fat-soluble small molecule targeted drug further enhances the material's biosafety, provides osteogenesis and anti-inflammatory activity, and can be effectively applied in applications such as alveolar bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a schematic diagram of the preparation process of the microsphere bone repair material of the present invention.

[0047] Figure 2 FSEM images of the microspheres of Example 1, wherein (a) is a low-magnification view of Example 1, and (b) is a high-magnification view of Example 1.

[0048] Figure 3 FSEM images of the microspheres of Example 2, wherein (a) is a low-magnification view of Example 2, and (b) is a high-magnification view of Example 2.

[0049] Figure 4 FSEM images of the microspheres of Example 3, wherein (a) is a low-magnification view of Example 3, and (b) is a high-magnification view of Example 3.

[0050] Figure 5 FSEM images of the microspheres of Example 4, wherein (a) is a low-magnification view of Example 4, and (b) is a high-magnification view of Example 4.

[0051] Figure 6 FSEM images of the microspheres of Example 5, wherein (a) is a low-magnification view of Example 5, and (b) is a high-magnification view of Example 5.

[0052] Figure 7 FSEM images of the microspheres of Example 6, wherein (a) is a low-magnification view of Example 6, and (b) is a high-magnification view of Example 6.

[0053] Figure 8 FSEM images of the microspheres of Example 7, wherein (a) is a low-magnification view of Example 7, and (b) is a high-magnification view of Example 7.

[0054] Figure 9 1 is the pH change curve and mass degradation curve of the microspheres after the in vitro microsphere degradation experiment of Example 1.

[0055] Figure 10The particle size distribution of the microspheres in Example 1, wherein (a) shows the size of some microspheres under a light microscope, and (b) shows the particle size distribution of the microspheres under a light microscope using Image J statistics.

[0056] Figure 11 This is an SEM image of the hydroxyapatite structure in the microspheres of Example 1.

[0057] Figure 12 TEM image of the hydroxyapatite structure in the microspheres of Example 1, Figure 12 In the figure, (a) is a TEM image at a scale of 0.5 μm, and (b) is an enlarged view of (a).

[0058] Figure 13 is the SEM image of the hydroxyapatite structure in the microspheres of Gel-SeHA and Example 1, Figure 13 In the figure, (a) is a SEM image of Gel-SeHA, and (b) is a SEM image of the hydroxyapatite structure in the microspheres of Example 1.

[0059] Figure 14 The live-death staining diagram of cells after co-culture with microspheres at different concentrations. Figure 14 In the figure, (a) is a live and dead staining image of RAW264.7 cells after co-culture with microspheres at a concentration of 0 mg / mL, (b) is a live and dead staining image of RAW264.7 cells after co-culture with microspheres at a concentration of 0.25 mg / mL, (c) is a live and dead staining image of RAW264.7 cells after co-culture with microspheres at a concentration of 0.5 mg / mL, (d) is a live and dead staining image of RAW264.7 cells after co-culture with microspheres at a concentration of 1 mg / mL, (e) is a live and dead staining image of BMSC cells after co-culture with microspheres at a concentration of 0 mg / mL, (f) is a live and dead staining image of BMSC cells after co-culture with microspheres at a concentration of 0.25 mg / mL, (g) is a live and dead staining image of BMSC cells after co-culture with microspheres at a concentration of 0.5 mg / mL, and (h) is a live and dead staining image of BMSC cells after co-culture with microspheres at a concentration of 1 mg / mL.

[0060] Figure 15 Figure 2 is the proliferation curve of cells after co-culture with microspheres of different concentrations within 4 days, wherein (a) is the proliferation curve of BMSCs after co-culture with microspheres of different concentrations within 4 days, and (b) is the proliferation curve of RAW264.7 after co-culture with microspheres of different concentrations within 4 days.

[0061] Figure 16Figure 1 is the immunofluorescence staining result of the cell adhesion experiment after co-culture, wherein (a) is the immunofluorescence staining result of the BMSC cell adhesion experiment after 3 days of co-culture, (b) is the immunofluorescence staining result of the RAW264.7 cell adhesion experiment after 3 days of co-culture, (c) is the immunofluorescence staining result of the BMSC cell adhesion experiment after 7 days of co-culture, and (d) is the immunofluorescence staining result of the RAW264.7 cell adhesion experiment after 7 days of co-culture.

[0062] Figure 17 Figure 1 is an SEM image of RAW264.7 cells after co-culture, wherein (a) is an SEM image of RAW264.7 cells at a scale of 200 μm after 3 days of co-culture, (b) is an SEM image of RAW264.7 cells at a scale of 30 μm after 3 days of co-culture, (c) is an SEM image of RAW264.7 cells at a scale of 200 μm after 7 days of co-culture, and (d) is an SEM image of RAW264.7 cells at a scale of 50 μm after 7 days of co-culture. Figure 17 It can be seen that the microspheres prepared in Example 1 are conducive to adhesion, and RAW264.7 cells have normal morphology on the microspheres and can continue to proliferate.

[0063] Figure 18 Figure 1 is an SEM image of BMSC cells after co-culture, wherein (a) is an SEM image of BMSC cells at a scale of 200 μm after 3 days of co-culture, (b) is an SEM image of BMSC cells at a scale of 50 μm after 3 days of co-culture, (c) is an SEM image of BMSC cells at a scale of 200 μm after 7 days of co-culture, and (d) is an SEM image of BMSC cells at a scale of 50 μm after 7 days of co-culture. Figure 18 It can be seen that the microspheres prepared in Example 1 are conducive to adhesion, and the BMSC cells have normal morphology on the microspheres and can continue to proliferate.

[0064] Figure 19 These are SEM images of the microspheres of Comparative Example 1 and the microspheres of Example 1 after biomineralization experiments, wherein (a) is an SEM image of the microspheres of Comparative Example 1 at a scale of 200 μm, (b) is an SEM image of the microspheres of Example 1 at a scale of 200 μm, (c) is an SEM image of the microspheres of Comparative Example 1 at a scale of 20 μm, and (d) is an SEM image of the microspheres of Example 1 at a scale of 20 μm.

[0065] Figure 20 for Figure 19 EDX element analysis results of Figure (d), where (a) is the distribution diagram of the C element, (b) is the distribution diagram of the O element, (c) is the distribution diagram of the Ca element, and (d) is the distribution diagram of the P element. DETAILED DESCRIPTION

[0066] The technical solutions in the embodiments of the present invention are described clearly and completely below. It should be noted that in the following embodiments of the present invention, the PLGA used has a specification of Mw=45000Da. The gelatin used is pigskin gelatin purchased from Sigma.

[0067] Example 1

[0068] See also Figure 1 This embodiment provides a microsphere bone repair material loaded with small molecule targeted drugs, which is prepared by the following steps:

[0069] Step 1, prepare the aqueous phase:

[0070] 1) Dissolve 0.236 g of Ca(NO3)2·4H2O in 2 mL of deionized water to obtain Ca 2+ Source solution. Disperse 40 mg of pig skin gelatin in 3 mL of ionized water to obtain a gelatin aqueous solution. 2+ The source solution and the gelatin aqueous solution were mixed in a 37° C. water bath to obtain a mixed solution A.

[0071] 2) Octadecylamine, linoleic acid, and anhydrous ethanol were mixed in a volume ratio of 1:8:32 to obtain a reaction solvent.

[0072] 3) The obtained mixed solution A was mixed with 12.3 mL of the reaction solvent to obtain a mixed solution B.

[0073] 4) Disperse 0.0952 g of trisodium phosphate and 0.0097 g of sodium selenite in 3 mL of deionized water to obtain a mixed solution C.

[0074] 5) The obtained mixed solution C was added dropwise to the mixed solution B, and stirred at 37° C. for 2 h to obtain a suspension.

[0075] 6) The suspension was incubated in a processor at 37°C for 72 hours. The final precipitate was collected by centrifugation and washed six times alternately with anhydrous ethanol and deionized water to obtain selenium-doped hydroxyapatite nanoparticles, labeled as Gel-SeHA. The Gel-SeHA was then stored in anhydrous ethanol at 4°C.

[0076] 7) Dispersing polyvinyl alcohol in the gelatin solution to prepare a polyvinyl alcohol gelatin solution with a mass concentration of 2% of polyvinyl alcohol and a mass concentration of 10% of gelatin.

[0077] 8) The Gel-SeHA stored in anhydrous ethanol in step 6) was centrifuged and washed three times with ultrapure water. Then, 0.08 g of the washed Gel-SeHA was placed in 1 mL of the above polyvinyl alcohol gelatin solution and dispersed evenly with a Pasteur pipette to obtain an aqueous phase.

[0078] Step 2, prepare the oil phase:

[0079] 1) Ethyl acetate and dichloromethane were mixed in a mass ratio of 8:5 to obtain 3.6 g of organic solvent.

[0080] 2) 0.18 g of PLGA and 10 mg of the lipid-soluble small molecule targeted drug STL1267 were placed in the above organic solvent and dispersed evenly by ultrasonication for 1 minute to obtain an oil phase. That is, in this example, the concentration of PLGA in the oil phase was 5%.

[0081] Step 3, prepare an equal density emulsion:

[0082] 1) After mixing the oil phase and aqueous phase obtained above, dichloromethane or ethyl acetate is added dropwise to suspend the water droplets by observing whether the water droplets float or sink in the oil phase, thereby obtaining an oil phase with the same density as the aqueous phase. High-speed homogenization is performed at a speed of 20,000 r / min and a time of 25 seconds to emulsify the isopycnic suspension into a stable, uniform, isopycnic emulsion.

[0083] Step 4, preparation of microspheres:

[0084] 1) Place an equal density emulsion in a glass syringe to prevent the organic solvent from dissolving the wall of an ordinary syringe, and place the glass syringe on a syringe pump for standby use.

[0085] 2) A polyvinyl alcohol solution with a mass concentration of 2% is used as a collection phase, and the solution is placed in a medical syringe, and the medical syringe is placed on another syringe pump for standby use.

[0086] 3) Install an "L"-shaped needle at the output end of the glass syringe, connect a silicone hose to the output end of the medical syringe, insert the "L"-shaped needle into the other end of the silicone hose 2 cm away from the output tube, and insert the capillary glass tube filled with silicone hose 1 mm below the needle. The connection diagram is shown in the figure. Figure 1 As shown in step 4.

[0087] 4) After turning on both syringe pumps and adjusting them to a steady flow rate, the emulsion flowing from the glass syringe is sheared into uniformly sized droplets by the flowing collection phase in the surrounding silicone hose and squeezed into the capillary glass tube. The end of the capillary glass tube is immersed in a beaker filled with the collection phase, which is then placed in an ice bath and slowly stirred. The uniformly sized emulsion droplets fall one by one into the collection phase. Due to surface tension, primarily provided by the PVA, the liquid remains stable and gradually solidifies in the low-temperature ice bath, forming microspheres. This device can also be directly replaced with a glass cross-flow focusing microfluidic chip.

[0088] Step 5, post-processing of microspheres:

[0089] After the microspheres solidify, the dichloromethane and ethyl acetate in the oil phase are evaporated by stirring overnight. The microspheres are released from the gelatin by placing them in a warm water bath at 45°C for 4 hours, and then washed three times with ultrapure water to remove the gelatin. Finally, the microspheres are freeze-dried to obtain a microsphere bone repair material loaded with small molecule targeted drugs, which is then placed in a desiccator for long-term storage.

[0090] Example 2

[0091] See also Figure 1 This embodiment provides a microsphere bone repair material loaded with small molecule targeted drugs, and the difference between this embodiment and embodiment 1 is only that:

[0092] In this example, when preparing the aqueous phase, the amount of washed Gel-SeHA used was 0.1 g.

[0093] Example 3

[0094] See also Figure 1 This embodiment provides a microsphere bone repair material loaded with small molecule targeted drugs, and the difference between this embodiment and embodiment 1 is only that:

[0095] In this embodiment, the concentration of PLGA in the oil phase is 3%.

[0096] Example 4

[0097] See also Figure 1 This embodiment provides a microsphere bone repair material loaded with small molecule targeted drugs, and the difference between this embodiment and embodiment 1 is only that:

[0098] In this example, when preparing the aqueous phase, the amount of washed Gel-SeHA used was 0.12 g.

[0099] Example 5

[0100] See also Figure 1 This embodiment provides a microsphere bone repair material loaded with small molecule targeted drugs, and the difference between this embodiment and embodiment 1 is only that:

[0101] In this embodiment, when preparing the aqueous phase, the amount of the polyvinyl alcohol gelatin solution used is 0.5 mL.

[0102] Example 6

[0103] See also Figure 1 This embodiment provides a microsphere bone repair material loaded with small molecule targeted drugs, and the difference between this embodiment and embodiment 1 is only that:

[0104] In this example, when preparing the aqueous phase, the amount of the polyvinyl alcohol gelatin solution used was 1.5 mL.

[0105] Example 7

[0106] See also Figure 1 This embodiment provides a microsphere bone repair material loaded with small molecule targeted drugs, and the difference between this embodiment and embodiment 1 is only that:

[0107] In this example, when preparing the aqueous phase, the amount of the polyvinyl alcohol gelatin solution used was 2.0 mL.

[0108] Comparative Example 1

[0109] This comparative example provides a microsphere bone repair material loaded with small molecule targeted drugs, and the difference between this comparative example and Example 1 is only that:

[0110] In this comparative example, no polyvinyl alcohol gelatin solution was added when preparing the aqueous phase.

[0111] Experimental part

[0112] It should be noted that, for the sake of convenience, the following experimental section will refer to the microsphere bone repair material as microsphere.

[0113] (1) Morphology test

[0114] The present invention takes the microspheres of Example 1 to Example 6 as examples, and performs electron scanning electron microscopy tests on them respectively, and the test results are as follows: Figure 2-Figure 8 shown.

[0115] Figure 2 FSEM images of the microspheres of Example 1, wherein (a) is a low-magnification view of Example 1 and (b) is a high-magnification view of Example 1. Figure 2 It can be seen that the microspheres of Example 1 have a through-pore structure, and the small pores within the spheres and the large pores between the spheres form a multi-level pore structure.

[0116] Figure 3 The FSEM images of the microspheres of Example 2 are shown in Figure 2, wherein (a) is a low-magnification view of Example 2 and (b) is a high-magnification view of Example 2. Figure 3 and Figure 2 It can be seen that increasing the Gel-SeHA loading amount will result in excessive filling of inorganic matter in the microspheres and reduced pores.

[0117] Figure 4 The FSEM images of the microspheres of Example 3, wherein (a) is a low-magnification view of Example 3, and (b) is a high-magnification view of Example 3. Figure 4 and Figure 2 It can be seen that reducing the PLGA content leads to insufficient microsphere scaffold components, making it difficult to form a pore structure, which will also affect the pore formation of the microspheres.

[0118] Figure 5 The FSEM images of the microspheres of Example 4 are shown in Figure 4, wherein (a) is a low-magnification view of Example 4 and (b) is a high-magnification view of Example 4. Figure 5 and Figure 2 It can be seen that further increasing the Gel-SeHA content will result in the PLGA scaffold of the microspheres being unable to accommodate a large amount of inorganic matter, resulting in the precipitation of inorganic matter in the microspheres and shrinkage of the morphology.

[0119] Figure 6 The FSEM images of the microspheres of Example 5 are shown in Figure 5, wherein (a) is a low-magnification view of Example 5 and (b) is a high-magnification view of Example 5. Figure 6 and Figure 2 It can be seen that reducing the amount of polyvinyl alcohol gelatin solution from 1 mL to 0.5 mL, that is, reducing the porogen, will lead to a decrease in the pores of the microspheres.

[0120] Figure 7 The FSEM images of the microspheres of Example 6, wherein (a) is a low-magnification view of Example 6, and (b) is a high-magnification view of Example 6. Figure 5 and Figure 2 It can be seen that when the porogen is increased to 1.5 mL, the pores of the microspheres increase significantly, but the excessively high porosity makes the microsphere structure looser and more fragile.

[0121] Figure 8 FSEM images of the microspheres of Example 7, wherein (a) is a low-magnification view of Example 7, and (b) is a high-magnification view of Example 7. Figure 8 and Figure 2 It can be seen that when the porogen is further increased to 2 mL, the microsphere structure becomes more fragile and collapses under the SEM voltage.

[0122] In summary, increasing the Gel-SeHA loading amount will reduce the porosity of the microspheres, increasing the scaffold component PLGA will help form a pore structure, and increasing the content of the porogen will increase the porosity of the microspheres, but excessive pores will cause the microsphere structure to collapse.

[0123] (2) In vitro microsphere degradation experiment

[0124] In order to explore the stability of microspheres and simulate the in vivo degradation performance, the present invention took the microspheres of Example 1 as an example and conducted an in vitro microsphere degradation experiment. The test method of the in vitro microsphere degradation experiment is as follows:

[0125] Weigh 0.03g of freeze-dried microspheres and place them in a 5mL centrifuge tube, number and record the actual mass. Add 3mL of PBS, and the resulting microsphere concentration is 0.01g / mL. Incubate in a constant temperature shaker at 37°C and a shaking speed of 70r / min to simulate the in vivo environment. After a certain period of time, aspirate the supernatant to measure the pH and draw a pH change curve. Wash five times with ddH2O, measure the mass after freeze-drying, subtract it from the original recorded actual mass, obtain the degraded mass, draw a mass degradation curve, and organize the pH change curve and mass degradation curve as shown below. Figure 9 shown.

[0126] And by Figure 9 The test results show that the mass fraction and pH of the microspheres change slowly and the acid production is slow. Therefore, the degradation rate of the spheres of this component is moderate and can fit the degradation time window required for bone formation.

[0127] (3) Particle size distribution

[0128] In order to explore whether the particle size of the microspheres obtained by the microfluidic method is uniform, the microspheres of Example 1 were taken as an example, and the particle size distribution was statistically analyzed using ImageJ. The test results are as follows: Figure 10 shown. Figure 10 In the figure, (a) shows the size of some microspheres under light microscopy, and (b) shows the particle size distribution of microspheres under light microscopy using ImageJ statistics.

[0129] And by Figure 10 The test results show that the microspheres prepared by the microfluidic method are uniform in size and have an ideal particle size distribution.

[0130] Based on the above research, the present invention takes the microspheres of Example 1 as an example and further conducts the following tests on the hydroxyapatite structure in the microspheres.

[0131] Figure 11 This is an SEM image of the hydroxyapatite structure in the microspheres of Example 1. The purple pseudo-color portion in the image is the hydroxyapatite structure in the microspheres.

[0132] Figure 12TEM image of the hydroxyapatite structure in the microspheres of Example 1, Figure 12 In the figure, (a) is a TEM image at a scale of 0.5 μm, and (b) is an enlarged view of (a). It can be seen that the hydroxyapatite structure has an excellent biomimetic mineralization hierarchical structure, which is beneficial to promoting osteogenic differentiation at the nano level.

[0133] Figure 13 is the SEM image of the hydroxyapatite structure in the microspheres of Gel-SeHA and Example 1, Figure 13 In the figure, (a) is the SEM image of Gel-SeHA, and (b) is the SEM image of the hydroxyapatite structure in the microspheres of Example 1. Figure 13 As can be seen from Figures (a) and (b), the original morphology of Gel-SeHA in the left figure is still maintained after being loaded into the microspheres.

[0134] (IV) Cytotoxicity and adhesion tests

[0135] In order to explore the biosafety of the microspheres of the present invention, the microspheres prepared in Example 1 were used as an example to co-culture the microspheres with cells to detect their cytotoxicity and adhesion. The test results are as follows: Figure 14-Figure 13 shown.

[0136] The culture conditions for co-culture are:

[0137] The microspheres prepared in Example 1 were irradiated under UV light for 8–10 hours, then washed three times with sterile PBS. The microspheres were then dissolved in complete culture medium and cultured directly with cells. Live / dead staining, cell adhesion, and proliferation on the microsphere surfaces were observed. BMSCs and RAW264.7 cells were used for co-culture. Both the RAW264.7 and BMDM cells used in the present experiments were obtained from the Cell Bank of the Chinese Academy of Sciences in Shanghai, China.

[0138] Complete culture medium composition: α medium for BMSC, high-glucose DMEM medium for RAW264.7, 10% FBS, 1% penicillin-streptomycin (P / S, double-antibody). Cell culture environment at 37°C, 5% CO2. 1. Effects of different microsphere concentrations on cells after co-culture

[0139] The concentrations of the microspheres prepared in Example 1 in complete culture medium were 0 mg / mL, 0.25 mg / mL, 0.5 mg / mL and 1 mg / mL, respectively.

[0140] I. Staining Experiment

[0141] The present invention takes the microspheres of Example 1 as an example, and stains the cells co-cultured with microspheres at different concentrations according to the method of the following live and dead staining instructions. The method of the live and dead staining instructions is as follows:

[0142] 1) Approximately 1×10 5 Seed cells per well into 24-well plates.

[0143] 2) After 12 to 24 hours of adherence, when the cells have grown to 90% to 100%, the cells are treated with the culture medium suspension containing the microspheres at different concentrations.

[0144] 3) After 2 days, remove the cells from the corresponding time groups and prepare them for staining in the dark.

[0145] 4) Prepare the working solution according to the instructions and place it on ice away from light.

[0146] 5) Washing: For adherent cells, remove the culture medium and wash the cells once with PBS.

[0147] 6) Staining: Add 250 μL of Calcein AM / PI working solution to each well of a 24-well plate and incubate at 37°C in the dark for 30 min.

[0148] 7) Slide preparation: After the staining incubation is completed, wash the cells with PBS and add anti-fluorescence quenching sealing solution directly to each well of the plate. The cell samples can then be observed under a fluorescence microscope.

[0149] 8) Detection: Analyze cells by fluorescence microscopy within 2 hours, observe the staining effect under the fluorescence microscope, and organize the observed cell staining images as follows: Figure 14 shown.

[0150] Figure 14 The live-death staining diagram of cells after co-culture with microspheres at different concentrations. Figure 14 In the figure, (a) is a live and dead staining image of RAW264.7 cells after co-culture with microspheres at a concentration of 0 mg / mL, (b) is a live and dead staining image of RAW264.7 cells after co-culture with microspheres at a concentration of 0.25 mg / mL, (c) is a live and dead staining image of RAW264.7 cells after co-culture with microspheres at a concentration of 0.5 mg / mL, (d) is a live and dead staining image of RAW264.7 cells after co-culture with microspheres at a concentration of 1 mg / mL, (e) is a live and dead staining image of BMSC cells after co-culture with microspheres at a concentration of 0 mg / mL, (f) is a live and dead staining image of BMSC cells after co-culture with microspheres at a concentration of 0.25 mg / mL, (g) is a live and dead staining image of BMSC cells after co-culture with microspheres at a concentration of 0.5 mg / mL, and (h) is a live and dead staining image of BMSC cells after co-culture with microspheres at a concentration of 1 mg / mL.

[0151] It can be seen that microspheres with different concentration gradients have no obvious effect on the activity of the two cells, which preliminarily indicates that the microspheres have good biosafety.

[0152] II. CCK8 test

[0153] The present invention takes the microspheres of Example 1 as an example, and performs CCK8 testing on cells co-cultured with different concentrations of microspheres according to the method in the CCK-8 instruction manual. The method in the CCK-8 instruction manual is as follows:

[0154] 100 μL of cell suspension was added to each well of a 96-well plate. After the wells were pre-cultured in an incubator for 24 hours to adhere to the wall, the cells were treated with the above-mentioned microspheres at different concentrations for 1, 2, 3, and 4 days. Subsequently, the old culture medium was removed and the cells were washed twice with new complete culture medium. Then, 100 μL of new culture medium and 10 μL of CCK-8 solution were added to each well. After incubation at 37°C in the dark for 30 minutes, the OD value at 450 nm was measured with a microplate reader and plotted as shown below. Figure 15 Cell proliferation curves are shown.

[0155] Figure 15 In the figure, (a) shows the proliferation curve of BMSCs after co-culture with microspheres of different concentrations within 4 days, and (b) shows the proliferation curve of RAW264.7 after co-culture with microspheres of different concentrations within 4 days. It can be seen that there is no difference in the proliferation curve of microspheres compared with the control group, indicating that the microspheres have no obvious toxicity and are suitable for cell proliferation.

[0156] 2. Effects of different culture times on cells after co-culture

[0157] I. Cell Adhesion Assay

[0158] The present invention uses the microspheres of Example 1 as an example, and conducts a cell adhesion experiment on the co-cultured cells according to the following method, and the cell adhesion experiment method is as follows:

[0159] 1) After co-culturing cells with 0.5 mg / mL microspheres for 3 or 7 days, the microspheres were collected and placed in a centrifuge tube.

[0160] 2) The culture medium was aspirated and the microspheres were washed twice with 1×PBS (pH=7.4) preheated at 37°C.

[0161] 3) Fix the cells with 4% formaldehyde solution in PBS at room temperature for 10 minutes.

[0162] 4) After fixation, wash the cells 2-3 times with PBS at room temperature for 10 minutes each time.

[0163] 5) PBS containing 0.1% Triton X-100 was added and incubated at room temperature for 10 minutes to enhance cell permeability.

[0164] 6) Aspirate the above liquid and wash the cells with PBS 2 to 3 times, each time for 10 minutes.

[0165] 7) Take 200 μL of the prepared 100 nM TRITC-labeled phalloidin working solution, cover the microspheres, and incubate at room temperature in the dark for 30 minutes.

[0166] 8) Wash with PBS three times, 5 min each time.

[0167] 9) Add anti-fluorescence quenching mounting medium containing DAPI.

[0168] The present invention performs fluorescence observation under a fluorescence microscope or confocal microscope, selects TRITC excitation / emission filter, Ex / Em=540 / 570nm; and DAPI excitation / emission filter, Ex / Em=364 / 454nm, and the immunofluorescence staining results of the cell adhesion experiment after co-culture are as follows Figure 16 shown.

[0169] Figure 16 Figure 2 shows the immunofluorescence staining results of BMSC cell adhesion experiment after 3 days of co-culture, Figure 2 shows the immunofluorescence staining results of RAW264.7 cell adhesion experiment after 3 days of co-culture, Figure 2 shows the immunofluorescence staining results of BMSC cell adhesion experiment after 7 days of co-culture, and Figure 2 shows the immunofluorescence staining results of RAW264.7 cell adhesion experiment after 7 days of co-culture.

[0170] And by Figure 16 It can be seen that the microspheres prepared in Example 1 are conducive to adhesion, and the cells on the microspheres have normal morphology and can continue to proliferate.

[0171] II. SEM scanning test

[0172] The present invention also conducted SEM scanning tests on RAW264.7 cells and BMSC cells after co-culture of 0.5 mg / mL microspheres with cells for 3 days or 7 days. The sample preparation before SEM scanning test was as follows: after co-culture, the microspheres were gently collected and washed once with PBS, and the microspheres were fixed in 2.5% glutaraldehyde for electron microscopy at 4°C for 4 hours, and then washed 3 times with ddH2O. After the microspheres were freeze-dried, scanning electron microscopy imaging was performed, and the test results were as follows: Figure 17 and Figure 18 shown.

[0173] Figure 17Figure 1 is an SEM image of RAW264.7 cells after co-culture, wherein (a) is an SEM image of RAW264.7 cells at a scale of 200 μm after 3 days of co-culture, (b) is an SEM image of RAW264.7 cells at a scale of 30 μm after 3 days of co-culture, (c) is an SEM image of RAW264.7 cells at a scale of 200 μm after 7 days of co-culture, and (d) is an SEM image of RAW264.7 cells at a scale of 50 μm after 7 days of co-culture. Figure 17 It can be seen that the microspheres prepared in Example 1 are conducive to adhesion, and RAW264.7 cells have normal morphology on the microspheres and can continue to proliferate.

[0174] Figure 18 Figure 1 is an SEM image of BMSC cells after co-culture, wherein (a) is an SEM image of BMSC cells at a scale of 200 μm after 3 days of co-culture, (b) is an SEM image of BMSC cells at a scale of 50 μm after 3 days of co-culture, (c) is an SEM image of BMSC cells at a scale of 200 μm after 7 days of co-culture, and (d) is an SEM image of BMSC cells at a scale of 50 μm after 7 days of co-culture. Figure 18 It can be seen that the microspheres prepared in Example 1 are conducive to adhesion, and the BMSC cells have normal morphology on the microspheres and can continue to proliferate.

[0175] (5) Biomineralization experiment

[0176] The present invention takes the microspheres of Comparative Example 1 and the microspheres of Example 1 as examples, and conducts a microsphere mineralization experiment according to the following method, and a biomineralization test is conducted by the following method:

[0177] 2 mg of anhydrous calcium chloride and 6 mg of MgCl2·6H2O were added to 20 mL of Dulbecco's phosphate buffered saline as simulated body fluid, recorded as SBF. The microsphere sample was immersed in SBF and incubated on a shaker at 37°C and 70 rpm for 3 days to induce the formation of calcium phosphate compounds. After the sample was rinsed with deionized water and freeze-dried three times, the product after the mineralization experiment was obtained. The product after the mineralization experiment was subjected to SEM scanning test and EDX elemental analysis test, and the test results were as follows: Figure 19 and Figure 20 shown.

[0178] Figure 19The SEM images of the microspheres of Comparative Example 1 and Example 1 after biomineralization experiments, wherein (a) is the SEM image of the microspheres of Comparative Example 1 at a scale of 200 μm, (b) is the SEM image of the microspheres of Example 1 at a scale of 200 μm, (c) is the SEM image of the microspheres of Comparative Example 1 at a scale of 20 μm, and (d) is the SEM image of the microspheres of Example 1 at a scale of 20 μm. Figure 19 It can be seen that compared with the non-porous microspheres in Comparative Example 1, the porous microspheres prepared in Example 1 can provide a larger surface area and more mineralization sites for biomineralization, and exhibit better biomineralization performance.

[0179] Figure 20 for Figure 19 The EDX element analysis results of Figure (d) in the figure, where (a) is the distribution diagram of C element, (b) is the distribution diagram of O element, (c) is the distribution diagram of Ca element, and (d) is the distribution diagram of P element. Figure 20 It can be seen that there is a large amount of calcium and phosphorus enrichment in the mineralization site, indicating successful biomineralization.

[0180] In summary, the microsphere bone repair material prepared by the preparation method of the present invention has a porous microsphere structure with uniform size and pore size. Its large surface area and Gel-SeHA component facilitate the provision of a large number of biomineralization sites, and the uniform pore size facilitates cell ingrowth, thereby promoting osteogenesis. The simultaneous introduction of polylactic-co-glycolic acid (PLGA) and fat-soluble small molecule targeted drugs further enhances the biosafety of the material, providing osteogenesis and anti-inflammatory activity, making the microsphere bone repair material prepared by the present invention effectively applicable to applications such as alveolar bone defects.

[0181] Obviously, the above embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A method for preparing a microsphere bone repair material loaded with small molecule targeted drugs, characterized in that: The following steps are involved: dispersing polyvinyl alcohol in a gelatin solution to obtain a polyvinyl alcohol gelatin solution; The polyvinyl alcohol gelatin solution is used as a porogen and mixed with a nanoparticle matrix to obtain an aqueous phase; wherein the nanoparticle matrix has a natural bone hydroxyapatite hierarchical structure and is hydroxyapatite nanoparticles or selenium-doped hydroxyapatite nanoparticles prepared by an LSS method using gelatin as a template; Dispersing PLGA and fat-soluble small molecule targeted drugs in an organic solvent to obtain an oil phase; The oil phase and the water phase are mixed uniformly by an isopycnic emulsion method to obtain a stable isopycnic emulsion; The isodensity emulsion is formed into uniform emulsion droplets by a microfluidic method; the emulsion droplets are placed in a collection phase under ice bath conditions and solidified at low temperature to form microspheres; wherein the collection phase is a polyvinyl alcohol solution; The microspheres are stirred to remove the organic solvent; after heating, they are washed to remove the collection phase, and freeze-dried to obtain a microsphere bone repair material loaded with small molecule targeted drugs.

2. The preparation method according to claim 1, wherein The fat-soluble small molecule is a targeted agonist of Rev-Erba.

3. The preparation method according to claim 2, wherein The targeted agonist of Rev-Erba is compound STL1267.

4. The preparation method according to claim 1, wherein The mass ratio of the fat-soluble small molecule targeted drug in the oil phase to the nanoparticle matrix in the water phase is 0.1 mg to 20 mg: 0.06 g to 0.12 g.

5. The preparation method according to claim 1, wherein The mass ratio of the fat-soluble small molecule targeted drug to the PLGA is 0.1 mg to 20 mg: 0.1 g to 0.3 g.

6. The preparation method according to claim 1, wherein The mass ratio of the nanoparticle matrix in the aqueous phase to the polyvinyl alcohol in the polyvinyl alcohol gelatin solution is 1.2 to 8:1; The mass concentration of polyvinyl alcohol in the polyvinyl alcohol gelatin solution is 1.5% to 2.5%.

7. The preparation method according to claim 1, wherein The nanoparticle matrix is prepared by the following steps: The LSS method is adopted, with gelatin as a template and a solvent containing octadecylamine and linoleic acid as a reaction solvent. After the gelatin is mixed with a reactive ion source in the reaction solvent, an incubation treatment is performed, so that the ions provided by the reactive ion source are respectively regulated by octadecylamine and linoleic acid to assemble in an orderly manner on the active groups of the gelatin, thereby forming a nanoparticle matrix with a natural bone hydroxyapatite hierarchical structure; Wherein, when preparing the hydroxyapatite nanoparticles, the reaction ion source is Ca 2+ Source and PO4 3+ ; When preparing selenium-doped hydroxyapatite nanoparticles, the reactive ion source is Ca 2+ Source, PO4 3+ Source and SeO3 2- source.

8. The preparation method according to claim 7, wherein When preparing the hydroxyapatite nanoparticles, gelatin and Ca 2+ Source and PO4 3+ The dosage ratio of the source is 35mg~45mg:1mmol:0.58mmol; When preparing Se-doped hydroxyapatite nanoparticles, gelatin and Ca 2+ Source, PO4 3+ Source and SeO3 2- The usage ratio of the source is 35mg~45mg:1mmol:0.58mmol:0.0561mmol.

9. The preparation method according to claim 7, wherein The incubation temperature is 20° C. to 45° C., and the incubation time is 24 hours to 168 hours.

10. A microsphere bone repair material loaded with small molecule targeted drugs prepared by the preparation method according to any one of claims 1 to 9.

Images