A material for promoting bone tissue repair and its preparation method and application

Abstract

The application provides a material for promoting bone tissue repair and a preparation method and application thereof. The material for promoting bone tissue repair is a gold nanoparticle-zoelitic imidazolate framework synergistic gallic acid drug delivery system, wherein the gold nanoparticle is taken as a core, a zoelitic imidazolate framework is grown around the core, and a bioactive component, such as gallic acid, is loaded in the framework. The material is used for promoting bone tissue repair. The material loads the bioactive component through a nanoparticle and a zoelitic imidazolate framework compound, utilizes the osteogenesis and anti-inflammatory performance of the material, and promotes the regeneration and repair of bone tissue.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, and particularly relates to a material for promoting bone tissue repair and a preparation method and application thereof. BACKGROUND

[0002] Bone defect repair has been an important research focus in the field of orthopedics and the field of dentistry. Common materials for bone repair include autologous bone, allogeneic bone, synthetic bone and biological materials. The application of autologous bone and allogeneic bone is limited due to surgical complexity, immune rejection, medical ethics and other problems. Synthetic materials such as hydroxyapatite and beta-tricalcium phosphate have good biocompatibility, but they still have deficiencies in bone regeneration effect and anti-inflammatory performance. For patients with insufficient bone volume for dental implantation, surgical bone augmentation of alveolar bone is required. Inflammation in the surgical area is a common and serious problem. Therefore, how to enhance the bone regeneration ability and anti-inflammatory performance of bone repair materials is a problem to be solved at present.

[0003] At present, the existing bone repair materials have the problem of lacking anti-inflammatory performance: during the bone repair process, inflammatory reaction is an important factor affecting bone regeneration. Although some materials introduce anti-inflammatory drugs, their effect is usually short-term and difficult to continuously regulate local inflammatory reaction. The existing materials usually cannot simultaneously consider the functions of anti-inflammation and promotion of bone tissue repair, resulting in limited treatment effect. In addition, these drugs often cannot target the bone repair site, and the treatment efficiency is low.

[0004] Based on the defects of the existing bone repair materials, it is necessary to improve them. SUMMARY

[0005] Therefore, the present application provides a material for promoting bone tissue repair and a preparation method and application thereof to solve or at least partially solve the defects in the prior art.

[0006] In a first aspect, the present application provides a material for promoting bone tissue repair, comprising: gold nanoparticles and a zeolitic imidazolate framework;

[0007] The material takes gold nanoparticles as the core, and the zeolitic imidazolate framework grows around the gold nanoparticles.

[0008] The zeolitic imidazolate framework internally loads an anti-inflammatory drug, and the anti-inflammatory drug comprises at least one of gallic acid, curcumin and resveratrol.

[0009] In a second aspect, the present application further provides a preparation method of the material for promoting bone tissue repair, comprising the following steps:

[0010] Synthesizing a PVP-encapsulated gold nanoparticle solution;

[0011] dissolving the soluble zinc salt in methanol to obtain a first solution;

[0012] dissolving 2-methylimidazole in methanol to obtain a second solution;

[0013] mixing the first solution, the second solution and the PVP-encapsulated gold nanoparticle solution, stirring and reacting, solid-liquid separation and collection to obtain Au NPs@ZIF-8;

[0014] adding an anti-inflammatory drug into methanol to obtain an anti-inflammatory drug solution;

[0015] adding Au NPs@ZIF-8 into the anti-inflammatory drug solution, stirring, solid-liquid separation and collection to obtain a precipitate, i.e. a material for promoting bone tissue repair.

[0016] Preferably, the method for preparing the PVP-encapsulated gold nanoparticle solution comprises the following steps:

[0017] heating the chloroauric acid solution, adding sodium citrate solution and continuing to heat until the color of the solution changes from red to yellow to obtain an Au NPs solution;

[0018] cooling the Au NPs solution, adding polyvinylpyrrolidone solution and stirring to obtain a PVP-encapsulated Au NPs solution;

[0019] solid-liquid separation of the PVP-encapsulated Au NPs solution to obtain PVP-Au NPs;

[0020] dissolving the PVP-Au NPs in methanol to obtain a PVP-encapsulated gold nanoparticle solution.

[0021] Preferably, the soluble zinc salt comprises at least one of zinc nitrate, zinc sulfate or zinc acetate.

[0022] Preferably, after mixing the first solution, the second solution and the PVP-encapsulated gold nanoparticle solution, stirring and reacting at 20-25℃ for 20-30h, solid-liquid separation and collection to obtain Au NPs@ZIF-8;

[0023] Preferably, the molar concentration of the soluble zinc salt in the first solution is 25-35mM;

[0024] Preferably, the molar concentration of 2-methylimidazole in the second solution is 25-35mM;

[0025] Preferably, the volume ratio of the first solution, the second solution and the PVP-encapsulated gold nanoparticle solution is (50-60):(50-60):(10-20).

[0026] Preferably, the chloroauric acid solution is heated to 100℃, then the sodium citrate solution is added, and the reaction is continued under heating until the solution color changes from red to yellow, and the Au NPs solution is obtained;

[0027] The Au NPs solution is cooled to 20-25℃, then the polyvinylpyrrolidone solution is added, and stirred for 20-25h to obtain the PVP-encapsulated Au NPs solution;

[0028] The PVP-encapsulated Au NPs solution is subjected to solid-liquid separation to obtain the PVP-Au NPs;

[0029] The PVP-Au NPs is dissolved in methanol to obtain the PVP-encapsulated gold nanoparticle solution;

[0030] Preferably, the chloroauric acid solution is an aqueous chloroauric acid solution, and the mass concentration of the aqueous chloroauric acid solution is 0.025-0.03%;

[0031] Preferably, the sodium citrate solution is an aqueous sodium citrate solution, and the mass concentration of the aqueous sodium citrate solution is 0.1-0.2%;

[0032] Preferably, the polyvinylpyrrolidone solution is an aqueous polyvinylpyrrolidone solution, and the mass concentration of the aqueous polyvinylpyrrolidone solution is 2.5-3%;

[0033] Preferably, the volume ratio of the chloroauric acid solution, the sodium citrate solution, the polyvinylpyrrolidone solution, and the methanol is (100-110):(2-3):(20-30):(50-60).

[0034] Preferably, in the step of adding the anti-inflammatory drug into the methanol to obtain an anti-inflammatory drug solution, the concentration of the anti-inflammatory drug in the anti-inflammatory drug solution is 3-5mg / mL.

[0035] Preferably, in the step of adding the Au NPs@ZIF-8 into the anti-inflammatory drug solution, the volume-to-mass ratio of the anti-inflammatory drug solution to the Au NPs@ZIF-8 is (20-30)mL:(50-60)mg.

[0036] Preferably, in the step of adding the Au NPs@ZIF-8 into the anti-inflammatory drug solution, the volume-to-mass ratio of the anti-inflammatory drug solution to the Au NPs@ZIF-8 is (20-30)mL:(50-60)mg.

[0037] In a third aspect, the present application also provides a use of the material for promoting bone tissue repair or the material prepared by the preparation method in the preparation of a drug for promoting bone tissue repair.

[0038] The material for promoting bone tissue repair, the preparation method and application thereof have the following beneficial effects relative to the prior art:

[0039] 1. The material for promoting bone tissue repair is a gold nanoparticle (Au NPs) as a core, a zeolitic imidazolate framework-8 (ZIF-8) growing around the core and an anti-inflammatory drug such as gallic acid (Ga) loaded in the framework, namely a Ga@Au NPs@ZIF-8 drug delivery system, for promoting bone tissue repair.

[0040] 2. The material for promoting bone tissue repair has the advantages of the gold nanoparticle and the ZIF-8 drug delivery system and the biological activity thereof by loading the anti-inflammatory drug such as gallic acid in the composite material of the gold nanoparticle and the ZIF-8, and provides a multifunctional material with good bone repair effect and anti-inflammatory performance. BRIEF DESCRIPTION OF DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0042] Figure 1 TEM image of the material for promoting bone tissue repair Ga@Au NPs@ZIF-8 prepared in Example 1;

[0043] Figure 2 Alizarin red staining of MC3T3-E1 osteogenic induction for 21 days;

[0044] Figure 3 qPCR results for MC3T3-E1 cells after 7 days of osteogenic induction;

[0045] Figure 4 Masson staining results for paraffin sections of SD rat skull defect model 2 weeks after operation;

[0046] Figure 5 Inflammatory factor iNOS immunofluorescence staining of RAW 264.7 cells after 24 hours of LPS treatment;

[0047] Figure 6 qPCR results of RAW 264.7 cells after 24 hours of LPS treatment. DETAILED DESCRIPTION

[0048] 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. 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.

[0049] It should be noted that similar reference numerals and letters represent similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0050] The following will be described in detail respectively. It should be noted that the sequence of the following embodiments is not limited as the preferred sequence of the embodiments. 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 type; it should be understood that the description in a range type is only for the 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., as well as single numbers 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.

[0051] The present application provides a material for promoting bone tissue repair, comprising: gold nanoparticles and zeolite imidazolate framework;

[0052] The material takes gold nanoparticles as the core, and the zeolite imidazolate framework grows around the gold nanoparticles;

[0053] The anti-inflammatory drug loaded inside the zeolitic imidazolate framework includes at least one of gallic acid, curcumin, and resveratrol.

[0054] The material for promoting bone tissue repair of the present application is a kind of material with gold nanoparticles (Au nanoparticle, Au NPs) as the core, zeolitic imidazolate framework (Zeolitic Imidazolate Framework-8, ZIF-8) growing around and loading anti-inflammatory drugs such as gallic acid (Gallic acid, Ga) inside the framework, namely gold nanoparticle-zeolitic imidazolate framework synergistic gallic acid drug delivery system (Ga@Au NPs@ZIF-8), which is used for promoting bone tissue repair. The material loads anti-inflammatory drugs such as gallic acid through the nanoparticle and zeolitic imidazolate framework complex, utilizes its osteogenic and anti-inflammatory properties, and promotes the regeneration and repair of bone tissue.

[0055] Specifically, gold nanoparticles can promote osteogenesis and have certain immunoregulatory properties; zeolitic imidazolate framework (Zeolitic Imidazolate Framework-8, ZIF-8) is a kind of metal-organic framework with high porosity and stable structure, and due to its chemical stability and multifunctionality, it shows unique advantages in drug delivery and tissue engineering. The porous structure of ZIF-8 allows it to be used as a drug carrier to control the release rate of the drug, thereby achieving long-acting and sustained therapeutic effect. And it can have good stability in complex physiological environment and is not easy to degrade or fail. At the same time, ZIF-8 has multiple biological activities, ZIF-8 can promote osteoblast differentiation and promote angiogenesis, and shows good osteogenic effect in rat calvarial defect model. Anti-inflammatory drugs such as gallic acid (Gallic Acid, Ga, molecular formula C7H6O5) are a kind of natural polyphenolic compounds widely existing in plants, which have strong antioxidant and anti-inflammatory biological activities. Gallic acid can scavenge excess free radicals generated in tissues, and can promote the proliferation and differentiation of osteoblasts to accelerate bone regeneration.

[0056] The present application combines the drug delivery advantages and biological activities of gold nanoparticles and ZIF-8 by loading anti-inflammatory drugs such as gallic acid into the composite material of gold nanoparticles-ZIF-8, and provides a multifunctional material with good bone repair effect. The stable structure of the ZIF skeleton in the present application can achieve slow release of gallic acid, avoiding the problem of drug resistance caused by burst effect. Gallic acid has strong antioxidant and anti-inflammatory effects, and can effectively regulate the inflammatory response in the bone repair process. By loading gallic acid into the nanocomposite material, the present application can continuously exert anti-inflammatory effect at the bone repair site, reduce the inhibition of local inflammatory response on bone tissue regeneration, and thus accelerate the bone tissue repair process. The present application not only provides slow-release gallic acid to achieve long-term anti-inflammatory effect, but also promotes the proliferation and differentiation of osteoblasts, and accelerates the regeneration and repair of bone tissue.

[0057] Based on the same inventive concept, the present application also provides a preparation method of a material for promoting bone tissue repair, comprising the following steps:

[0058] S1, synthesizing a PVP-encapsulated gold nanoparticle solution;

[0059] S2, dissolving a soluble zinc salt in methanol to obtain a first solution;

[0060] S3, dissolving 2-methylimidazole (2-Methylimidazole, 2-ME) in methanol to obtain a second solution;

[0061] S4, mixing the first solution, the second solution and the PVP-encapsulated gold nanoparticle solution, stirring and reacting, solid-liquid separation, and collecting Au NPs@ZIF-8;

[0062] S5, adding an anti-inflammatory drug to methanol to obtain an anti-inflammatory drug solution;

[0063] S6, adding Au NPs@ZIF-8 to the anti-inflammatory drug solution, stirring, and then solid-liquid separation to collect the precipitate, i.e. the material for promoting bone tissue repair.

[0064] In some embodiments, the preparation method of the PVP-encapsulated gold nanoparticle solution comprises the following steps:

[0065] S11, heating the chloroauric acid solution, adding sodium citrate solution, continuing to heat, and changing the color of the solution from red to yellow to obtain an Au NPs solution;

[0066] S12, cooling the Au NPs solution, adding a polyvinyl pyrrolidone (PVP) solution, and stirring to obtain a PVP-encapsulated Au NPs solution;

[0067] S13, solid-liquid separation of the PVP-encapsulated Au NPs solution to obtain PVP-Au NPs;

[0068] S14, dissolving the PVP-Au NPs in methanol to obtain a PVP-encapsulated gold nanoparticle solution.

[0069] In some embodiments, the soluble zinc salt includes at least one of zinc nitrate, zinc sulfate or zinc acetate.

[0070] In some embodiments, after mixing the first solution, the second solution and the PVP-encapsulated gold nanoparticle solution, the mixture is stirred at 20-25°C for 20-30 hours, and then solid-liquid separation is performed to obtain Au NPs@ZIF-8.

[0071] In the first solution, the molar concentration of the soluble zinc salt is 25-35 mM.

[0072] In the second solution, the molar concentration of 2-methylimidazole is 25-35 mM.

[0073] The volume ratio of the first solution, the second solution and the PVP-encapsulated gold nanoparticle solution is (50-60):(50-60):(10-20).

[0074] In some embodiments, after heating the chloroauric acid solution to 100°C, the sodium citrate solution is added, and the heating is continued until the color of the solution changes from red to yellow, thereby obtaining an Au NPs solution.

[0075] After cooling the Au NPs solution to 20-25°C, the polyvinylpyrrolidone solution is added, and the mixture is stirred for 20-25 hours to obtain a PVP-encapsulated Au NPs solution.

[0076] Solid-liquid separation of the PVP-encapsulated Au NPs solution to obtain PVP-Au NPs.

[0077] Dissolving the PVP-Au NPs in methanol to obtain a PVP-encapsulated gold nanoparticle solution.

[0078] The chloroauric acid solution is an aqueous chloroauric acid solution, and the mass concentration of the aqueous chloroauric acid solution is 0.025-0.03%.

[0079] The sodium citrate solution is an aqueous sodium citrate solution, and the mass concentration of the aqueous sodium citrate solution is 0.1-0.2%.

[0080] The polyvinylpyrrolidone solution is an aqueous polyvinylpyrrolidone solution, and the mass concentration of the aqueous polyvinylpyrrolidone solution is 2.5-3%.

[0081] The volume ratio of chloroauric acid solution, sodium citrate solution, polyvinylpyrrolidone solution and methanol is (100-110):(2-3):(20-30):(50-60).

[0082] In some embodiments, the PVP-encapsulated Au NPs solution is centrifuged at 8000-10000 rpm, solid-liquid separation is performed, and PVP-Au NPs are collected.

[0083] In some embodiments, after the first solution, the second solution and the PVP-encapsulated gold nanoparticle solution are mixed, stirring reaction is performed at 20-25℃ for 20-30 h, centrifugation is performed at 8000-10000 rpm, solid-liquid separation is performed, and Au NPs@ZIF-8 are collected.

[0084] In some embodiments, in the step of adding the anti-inflammatory drug into methanol to obtain an anti-inflammatory drug solution, the concentration of the anti-inflammatory drug in the anti-inflammatory drug solution is 3-5 mg / mL.

[0085] In some embodiments, Au NPs@ZIF-8 is added to the anti-inflammatory drug solution, stirring is performed for 20-30 h, solid-liquid separation is performed, a precipitate is collected, vacuum freeze-drying is performed, and a material for promoting bone tissue repair is obtained.

[0086] In some embodiments, in the step of adding Au NPs@ZIF-8 to the anti-inflammatory drug solution, the volume-to-mass ratio of the anti-inflammatory drug solution to Au NPs@ZIF-8 is (20-30) mL:(50-60) mg.

[0087] The primary innovation of the present application is the combination of gold nanoparticles and zeolitic imidazolate framework (ZIF-8) to form a multifunctional composite material; ZIF-8 has a highly ordered pore structure and good chemical stability, and can load drugs and control the release rate of the drugs. The uniqueness of this structure is that the stable combination of the two is achieved through a specific synthesis process, forming a composite material with high porosity, good drug loading capacity and stability; another key point of the present application is to load anti-inflammatory drugs such as gallic acid into the gold nanoparticle and ZIF-8 composite material; gallic acid has strong anti-inflammatory and antioxidant activity, but its direct application is often limited by poor stability and drug burst release. The present application solves the stability and controlled release problems of gallic acid by using the stable core of gold nanoparticles and the porous structure of ZIF-8. The sustained release mechanism of the drug ensures that it can continuously play a role in the bone repair process, achieving long-acting immune regulation and promoting bone regeneration. The material of the present application is not limited to a drug delivery system, but more importantly, it has multifunctional performance in bone tissue repair. The multifunctionality of the material of the present application is one of its core innovations. The composite material not only has anti-inflammatory function, but also promotes the proliferation and differentiation of osteoblasts through drug release, thereby accelerating the repair of bone tissue. This multifunctionality enables the material to cope with the complex physiological environment in bone repair, especially in relieving inflammation while promoting bone regeneration.

[0088] Compared with existing nanomaterials and drug delivery systems for bone repair, the material of the present application has multifunctionality: in some complex bone injury or bone defect scenarios, a single-function bone repair material often cannot meet the treatment needs, especially in cases where multiple functions such as anti-inflammatory and bone regeneration are required, the effect of existing materials is limited. The present application can simultaneously address these complex bone repair needs through the design of a multifunctional composite material, control inflammation, promote bone regeneration, and significantly improve the effect of the material in actual clinical applications.

[0089] The present application mainly uses gallic acid as a loaded drug for anti-inflammatory and bone regeneration promotion, and according to actual needs, other drugs or bioactive molecules with similar functions can be used as substitutes, such as: anti-inflammatory substitutes: in addition to gallic acid, other natural active substances such as curcumin and resveratrol can also be used as substitute drugs, which also have good anti-inflammatory and antioxidant properties; bone regeneration promoting drugs / bioactive molecules: other bioactive factors that promote bone tissue repair such as bone morphogenetic protein (BMP) can be loaded in the composite material to accelerate the repair and regeneration of bone tissue; anti-inflammatory drugs: in specific cases, other anti-inflammatory drugs such as non-steroidal anti-inflammatory drugs (NSAIDs) or corticosteroids can also be loaded to enhance the anti-inflammatory effect, especially in severe inflammatory reactions after bone injury or surgery.

[0090] The application can select different nanoparticles as the core. Gold nanoparticles are used as the core in the application to play the role of immune regulation and bone formation promotion. According to different needs, other types of nanoparticles can also be used to change the physical and chemical properties and biological activity of the material, such as silver nanoparticles, silicon dioxide nanoparticles, etc.

[0091] The application can be combined with a stent material for functional optimization. The application can be combined with an existing stent material or optimized in terms of structure and function to further expand the application range. By modifying the nanoparticles of the application on the surface of different types of tissue engineering scaffolds (such as bioceramic scaffolds, polymer scaffolds, hydrogels, electrospun fiber membranes, etc.), new functional properties are given to these scaffolds to adapt to more complex tissue repair scenarios.

[0092] Based on the same inventive concept, the application also provides a use of the above-mentioned material for promoting bone tissue repair or the material prepared by the above-mentioned preparation method in the preparation of a drug for promoting bone tissue repair.

[0093] Specifically, the material of the application can also be applied to other fields requiring anti-inflammatory properties due to its significant anti-inflammatory properties. By adjusting the type of loaded drugs, the material can be widely used in wound dressings, dental materials, etc. to inhibit inflammation and promote tissue healing. Although the application is mainly applied to the repair of bone tissue, it can also be applied to other types of tissue repair or regeneration by loading different effective components. For example, cartilage repair, periodontal tissue repair, skin wound healing, etc.

[0094] The material for promoting bone tissue repair and the preparation method thereof of the application are further illustrated in the following specific examples. This part further illustrates the content of the application in combination with specific examples, but should not be understood as a limitation of the application. If not specifically stated, the technical means used in the examples is a conventional means known to those skilled in the art. Unless specifically stated, the reagents, methods and equipment used in the application are conventional reagents, methods and equipment in the art.

[0095] Example 1

[0096] The application provides a preparation method of a material for promoting bone tissue repair, comprising the following steps:

[0097] S1, synthesizing a PVP-encapsulated gold nanoparticle solution, specifically comprising the following steps:

[0098] S11, prepare 100 mL of 0.025% chloroauric acid aqueous solution, heat the chloroauric acid aqueous solution to 100°C to boil, then add 2 mL of 0.1% sodium citrate aqueous solution, continue to heat for 20 min, and the color of the solution changes from red to yellow to obtain an Au NPs solution;

[0099] S12, after the Au NPs solution is cooled to room temperature (25°C), 20 mL of 2.5% polyvinylpyrrolidone aqueous solution is added, and stirring is performed for 24 h to obtain a PVP-encapsulated Au NPs (PVP-Au NPs) solution;

[0100] S13, the PVP-encapsulated Au NPs solution is centrifuged at 10000 rpm for 12 min to separate the solid and liquid, and the PVP-Au NPs are collected;

[0101] S14, the PVP-Au NPs in step S13 are dissolved in 50 mL of methanol, and ultrasonic dispersion is performed to obtain a PVP-encapsulated gold nanoparticle solution;

[0102] S2, Zn(NO3)2·6H2O is dissolved in methanol to obtain a first solution; the molar concentration of Zn(NO3)2·6H2O in the first solution is 25 mM;

[0103] S3, 2-methylimidazole (2-Methylimidazole, 2-ME) is dissolved in methanol to obtain a second solution; the molar concentration of 2-methylimidazole in the second solution is 25 mM;

[0104] S4, after 50 mL of the first solution, 50 mL of the second solution, and 10 mL of the PVP-encapsulated gold nanoparticle solution in step S14 are mixed, stirring is performed at room temperature for 24 h, and centrifugation is performed at 10000 rpm for 12 min to separate the solid and liquid, and the Au NPs@ZIF-8 is collected;

[0105] S5, gallic acid is added to methanol to obtain an anti-inflammatory drug solution; the concentration of gallic acid in the anti-inflammatory drug solution is 3 mg / mL;

[0106] S6, 50 mg of the Au NPs@ZIF-8 in step S4 is added to 20 mL of the anti-inflammatory drug solution, stirring is performed at room temperature for 24 h, and centrifugation is performed at 15000 rpm for 12 min to separate the solid and liquid, and the Ga@Au NPs@ZIF-8 is collected, and drying is performed by a vacuum freeze dryer to obtain the material for promoting bone tissue repair.

[0107] Performance test

[0108] Figure 1TEM image of Ga@Au NPs@ZIF-8 prepared in Example 1 for promoting bone tissue repair.

[0109] As can be seen from Figure 1 , Ga@Au NPs@ZIF-8 presents a porous hollow structure due to the loading of gallic acid.

[0110] Figure 2 ALP staining for 14d) and Alizarin Red S (ARS) staining for 21d) of MC3T3-E1 osteogenic induction. Figure 2 ALP staining for 14d) and Alizarin Red S (ARS) staining for 21d) of MC3T3-E1 osteogenic induction. Figure 2 ALP staining for 14d) and Alizarin Red S (ARS) staining for 21d) of MC3T3-E1 osteogenic induction.

[0111] Figure 2 The specific experimental process is as follows:

[0112] MC3T3-E1 cells were inoculated in 12-well plates, and when the cell density grew to 90%, 25 mg / mL Ga@Au NPs@ZIF-8 prepared in Example 1 was added to the experimental group, and the control group was osteogenic induction liquid, and the liquid was changed every 2 days. According to the instructions, alkaline phosphatase staining (ALP staining) was performed using the Bicun alkaline phosphatase staining kit when induced to the 14th day; when induced to the 21st day, the produced mineralized nodule was stained with Alizarin Red S (ARS) according to the instructions, and the results are shown in Figure 2 .

[0113] Figure 3 qPCR results of MC3T3-E1 osteogenic induction for 7d (*, p<0.05).

[0114] Figure 3 The specific experimental process is as follows:

[0115] MC3T3-E1 cells were inoculated in 6-well plates, and when the cell density grew to 90%, 25 mg / mL Ga@Au NPs@ZIF-8 prepared in Example 1 (i.e. Figure 3 Ga@Au NPs@ZIF-8) was added to the experimental group, and the Gallic Acid group (i.e. Figure 3 Gallic Acid) was an osteogenic induction liquid containing 8 mg / mL gallic acid, and the control group (i.e. Figure 3 Control) was an osteogenic induction liquid, and the liquid was changed every 2 days. RNA was extracted when induced to the 7th day, reverse transcription was performed to obtain cDNA, and qPCR was performed to detect the mRNA expression level of osteogenesis-related genes RUNX2 and Col-1, and the results are shown inFigure 3 as shown.

[0116] Figure 4 Masson staining results of paraffin sections of SD rat skull defect model 2 weeks after operation Figure 4 Original Bone, NB for New Bone, scale bar for 300 pm).

[0117] Figure 4 The specific experimental process is as follows:

[0118] 7-week-old SPF rats were anesthetized and modeled for skull defect model (defect diameter of 5 mm), the control group (i.e. Figure 4 Control) placed methylacrylated gelatin (GelMA hydrogel, item number EFL-GM-60) in the defect area, and the experimental group (i.e. Figure 4 Ga@Au NPs@ZIF-8) was GelMA hydrogel containing Ga@Au NPs@ZIF-8 prepared in Example 1, and the wounds of all rats were tightly sutured, and the rats were euthanized 2 weeks after operation, and then fixed, decalcified, dehydrated, paraffin-embedded, sectioned, and Masson stained, and the results are as shown. Figure 4

[0119] Figure 5 Inflammatory factor iNOS immunofluorescence staining of RAW 264.7 cells after 24 hours of LPS treatment (red for cytoskeleton, blue for nucleus, and green for iNOS).

[0120] Figure 5 The specific experimental process is as follows:

[0121] RAW 264.7 cells were inoculated in a confocal dish, and when the cell density grew to 90%, the control group (i.e. Figure 5 Control) was complete culture medium, the LPS group (i.e. Figure 5 LPS) was a culture medium containing LPS (lipopolysaccharide, 1 pg / mL), and the Ga@AuNPs@ZIF-8 group (i.e. Figure 5 Ga@Au NPs@ZIF-8) was a culture medium containing LPS (1 pg / mL) and Ga@AuNPs@ZIF-8 (25 mg / mL) prepared in Example 1. After 24 hours, fixation was performed for immunofluorescence staining, and the results are as shown. Figure 5

[0122] Figure 6 qPCR results of RAW 264.7 cells after 24 hours of LPS treatment.

[0123] Figure 6 ​​The specific experimental process is as follows:

[0124] RAW 264.7 cells were inoculated in a 6-well plate, and after the cell density grew to 90%, the control group (i.e. Figure 6 Control) was complete culture medium, the LPS group (i.e. Figure 6 LPS) was culture medium containing LPS (1 μg / mL), and the Ga@Au NPs@ZIF-8 group (i.e. Figure 6 Ga@Au NPs@ZIF-8) was culture medium containing LPS (1 μg / mL) and Ga@Au NPs@ZIF-8 (25 mg / mL) prepared in Example 1. After 24 hours, mRNA was extracted, and the mRNA expression level of the inflammation-related gene iNOS was detected by qPCR, and the results are shown in Figure 6 .

[0125] Figure 2 The ALP staining results show that Ga@Au NPs@ZIF-8 promotes the ALP expression of osteoblasts; the alizarin red staining results show that Ga@Au NPs@ZIF-8 promotes the mineralization of osteoblasts.

[0126] Figure 3 The qRT-PCR results show that Ga@Au NPs@ZIF-8 significantly increases the Runx2 and Col-1 expression levels of osteoblasts.

[0127] Figure 4 After the rat skull defect model was established for 2 weeks, the Masson staining results show that Ga@Au NPs@ZIF-8 significantly promotes the regeneration of the bone defect area.

[0128] Figures 5-6 The qRT-PCR results and iNOS immunofluorescence staining results show that Ga@Au NPs@ZIF-8 significantly reduces the iNOS expression level of RAW 264.7 cells under LPS stimulation.

[0129] The present application has been verified by in vivo and in vitro experiments, and the Ga@Au NPs@ZIF-8 prepared by the present application can effectively promote bone regeneration and has good anti-inflammatory effect.

[0130] The above only describes the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A material for promoting bone tissue repair, characterized in that: including gold nanoparticles and zeolite imidazolate framework; The material has gold nanoparticles as the core, and the zeolite imidazolate skeleton grows around the gold nanoparticles; The zeolite imidazolate skeleton is loaded with an anti-inflammatory drug, and the anti-inflammatory drug is gallic acid; The method for preparing the material for promoting bone tissue repair comprises the following steps: Synthesize PVP-encapsulated gold nanoparticle solution; dissolving a soluble zinc salt in methanol to obtain a first solution; dissolving 2-methylimidazole in methanol to obtain a second solution; The first solution, the second solution, and the PVP-encapsulated gold nanoparticle solution were mixed, stirred for reaction, and solid-liquid separation was performed to collect Au NPs@ZIF-8; adding an anti-inflammatory drug to methanol to obtain an anti-inflammatory drug solution; Adding Au NPs@ZIF-8 to an anti-inflammatory drug solution, stirring, and performing solid-liquid separation, the precipitate is collected to obtain a material for promoting bone tissue repair. The method for preparing the PVP-encapsulated gold nanoparticle solution comprises the following steps: After heating the chloroauric acid solution, sodium citrate solution was added and continued to heat until the color of the solution changed from red to yellow to obtain an Au NPs solution; After cooling the Au NPs solution, polyvinyl pyrrolidone solution was added and stirred to obtain a PVP-encapsulated Au NPs solution; The PVP-encapsulated Au NPs solution was subjected to solid-liquid separation to obtain PVP-Au NPs; PVP-Au NPs were dissolved in methanol to obtain a PVP-encapsulated gold nanoparticle solution; The molar concentration of the soluble zinc salt in the first solution is 25 to 35 mM; The molar concentration of 2-methylimidazole in the second solution is 25 to 35 mM; The volume ratio of the first solution, the second solution, and the PVP-encapsulated gold nanoparticle solution is (50-60):(50-60):(10-20); The chloroauric acid solution is an aqueous solution of chloroauric acid, and the mass concentration of the aqueous solution of chloroauric acid is 0.025-0.03%; The sodium citrate solution is an aqueous sodium citrate solution, and the mass concentration of the aqueous sodium citrate solution is 0.1-0.2%; The polyvinyl pyrrolidone solution is a polyvinyl pyrrolidone aqueous solution, and the mass concentration of the polyvinyl pyrrolidone solution is 2.5-3%; The volume ratio of the chloroauric acid solution, sodium citrate solution, polyvinyl pyrrolidone solution, and methanol is (100-110):(2-3):(20-30):(50-60); The concentration of the anti-inflammatory drug in the anti-inflammatory drug solution is 3 to 5 mg / mL; The volume mass ratio of anti-inflammatory drug solution to Au NPs@ZIF-8 is (20~30) mL: (50~60) mg.

2. The method for preparing a material for promoting bone tissue repair according to claim 1, wherein: The following steps are involved: Synthesize PVP-encapsulated gold nanoparticle solution; dissolving a soluble zinc salt in methanol to obtain a first solution; dissolving 2-methylimidazole in methanol to obtain a second solution; The first solution, the second solution, and the PVP-encapsulated gold nanoparticle solution were mixed, stirred for reaction, and solid-liquid separation was performed to collect Au NPs@ZIF-8; adding an anti-inflammatory drug to methanol to obtain an anti-inflammatory drug solution; Adding Au NPs@ZIF-8 to an anti-inflammatory drug solution, stirring, and performing solid-liquid separation, the precipitate is collected to obtain a material for promoting bone tissue repair. The method for preparing the PVP-encapsulated gold nanoparticle solution comprises the following steps: After heating the chloroauric acid solution, sodium citrate solution was added and continued to heat until the color of the solution changed from red to yellow to obtain an Au NPs solution; After cooling the Au NPs solution, polyvinyl pyrrolidone solution was added and stirred to obtain a PVP-encapsulated Au NPs solution; The PVP-encapsulated Au NPs solution was subjected to solid-liquid separation to obtain PVP-Au NPs; PVP-Au NPs were dissolved in methanol to obtain a PVP-encapsulated gold nanoparticle solution; The molar concentration of the soluble zinc salt in the first solution is 25 to 35 mM; The molar concentration of 2-methylimidazole in the second solution is 25 to 35 mM; The volume ratio of the first solution, the second solution, and the PVP-encapsulated gold nanoparticle solution is (50-60):(50-60):(10-20); The chloroauric acid solution is an aqueous solution of chloroauric acid, and the mass concentration of the aqueous solution of chloroauric acid is 0.025-0.03%; The sodium citrate solution is an aqueous sodium citrate solution, and the mass concentration of the aqueous sodium citrate solution is 0.1-0.2%; The polyvinyl pyrrolidone solution is a polyvinyl pyrrolidone aqueous solution, and the mass concentration of the polyvinyl pyrrolidone solution is 2.5-3%; The volume ratio of the chloroauric acid solution, sodium citrate solution, polyvinyl pyrrolidone solution, and methanol is (100-110):(2-3):(20-30):(50-60); The concentration of the anti-inflammatory drug in the anti-inflammatory drug solution is 3 to 5 mg / mL; The volume mass ratio of anti-inflammatory drug solution to Au NPs@ZIF-8 is (20~30) mL: (50~60) mg.

3. The method for preparing a material for promoting bone tissue repair according to claim 2, wherein: The soluble zinc salt includes at least one of zinc nitrate, zinc sulfate or zinc acetate.

4. The method for preparing a material for promoting bone tissue repair according to claim 2, wherein: The first solution, the second solution, and the PVP-encapsulated gold nanoparticle solution were mixed, stirred and reacted at 20-25° C. for 20-30 h, and solid-liquid separation was performed to collect Au NPs@ZIF-8.

5. The method for preparing a material for promoting bone tissue repair according to claim 2, wherein: The chloroauric acid solution was heated to 100°C, sodium citrate solution was added, and heating was continued. The reaction caused the solution color to change from red to yellow, and an AuNPs solution was obtained. After cooling the Au NPs solution to 20-25°C, polyvinyl pyrrolidone solution was added and stirred for 20-25 h to obtain a PVP-encapsulated Au NPs solution; The PVP-encapsulated Au NPs solution was subjected to solid-liquid separation to obtain PVP-Au NPs; PVP-Au NPs were dissolved in methanol to obtain PVP-encapsulated gold nanoparticle solution.

6. The method for preparing a material for promoting bone tissue repair according to claim 2, wherein: Au NPs@ZIF-8 was added to the anti-inflammatory drug solution, stirred for 20 to 30 hours, and then the solid and liquid were separated. The precipitate was collected and freeze-dried in vacuum to obtain a material for promoting bone tissue repair.

7. Use of the material for promoting bone tissue repair according to claim 1 or the material for promoting bone tissue repair prepared by the preparation method according to any one of claims 2 to 6 in the preparation of a drug for promoting bone tissue repair.

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