Material for promoting bone tissue repair as well as preparation method and application thereof

Abstract

The invention provides a material for promoting bone tissue repair as well as a preparation method and application thereof. The material for promoting bone tissue repair provided by the invention is a material which takes gold nanoparticles as a core, a zeolite imidazate skeleton grows around the core and a bioactive component, such as gallic acid, is loaded in the skeleton, namely a gold nanoparticle-zeolite imidazate skeleton synergistic gallic acid drug-loading system, and is used for promoting bone tissue repair; according to the material, bioactive components are loaded through nano-particles and a zeolite imidazate framework compound, and regeneration and repair of bone tissues are promoted by utilizing osteogenesis promoting and anti-inflammatory performance of the bioactive components.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a material for promoting bone tissue repair, a preparation method and an application thereof. Background Art

[0002] Bone defect repair has always been an important research hotspot in the field of orthopedics and dentistry. Common materials used for bone repair include autologous bone, allogeneic bone, synthetic bone and biomaterials. Among them, the application of autologous bone and allogeneic bone is limited due to surgical complexity, immune rejection, medical ethics and other issues. Although synthetic materials such as hydroxyapatite and β-tricalcium phosphate have good biocompatibility, they still have deficiencies in bone regeneration effect and anti-inflammatory properties. For patients who have insufficient bone volume for dental implants and need alveolar bone augmentation surgery, inflammation in the surgical area is a common problem with serious consequences. Therefore, how to enhance the bone regeneration ability and anti-inflammatory properties of bone repair materials is a problem that needs to be solved urgently.

[0003] At present, the existing bone repair materials lack anti-inflammatory properties: during the bone repair process, inflammatory response is an important factor affecting bone regeneration. Although some materials have introduced anti-inflammatory drugs, their effects are usually short-term and it is difficult to continuously regulate local inflammatory responses. Existing materials are usually unable to take into account the functions of anti-inflammatory and promoting bone tissue repair at the same time, resulting in limited treatment effects. In addition, these drugs often cannot act on the bone repair site in a targeted manner, and the treatment efficiency is low.

[0004] Based on the defects of current bone repair materials, it is necessary to improve them. Summary of the invention

[0005] In view of this, the present invention provides a material for promoting bone tissue repair and a preparation method and application thereof, so as to solve or at least partially solve the defects existing in the prior art.

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

[0007] The material has gold nanoparticles as the core, and the zeolite imidazolate skeleton grows around the gold nanoparticles;

[0008] The zeolite imidazolate skeleton is loaded with anti-inflammatory drugs, and the anti-inflammatory drugs include at least one of gallic acid, curcumin and resveratrol.

[0009] In a second aspect, the present invention also provides a method for preparing a material for promoting bone tissue repair, comprising the following steps:

[0010] Synthesize PVP-encapsulated gold nanoparticle solution;

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

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

[0013] 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;

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

[0015] Au NPs@ZIF-8 is added to the anti-inflammatory drug solution, stirred, and then the solid and liquid are separated, and the precipitate is collected to obtain the material for promoting bone tissue repair.

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

[0017] After heating the chloroauric acid solution, add the sodium citrate solution and continue heating until the color of the solution changes from red to yellow, thereby obtaining an Au NPs solution;

[0018] After the Au NPs solution was cooled, a polyvinyl pyrrolidone solution was added and stirred to obtain a PVP-encapsulated Au NPs solution;

[0019] The PVP-encapsulated Au NPs solution was subjected to solid-liquid separation to obtain PVP-Au NPs;

[0020] PVP-Au NPs were dissolved in methanol to obtain PVP-encapsulated gold nanoparticle solution.

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

[0022] Preferably, the first solution, the second solution, and the PVP-encapsulated gold nanoparticle solution are mixed, stirred and reacted at 20 to 25° C. for 20 to 30 hours, and the solid and liquid are separated to collect and obtain Au NPs@ZIF-8;

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

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

[0025] 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, after the chloroauric acid solution is heated to 100° C., a sodium citrate solution is added and the heating is continued, and the reaction causes the solution color to change from red to yellow, thereby obtaining an Au NPs solution;

[0027] After cooling the Au NPs solution to 20-25°C, polyvinyl pyrrolidone solution was added and stirred for 20-25h to obtain a PVP-encapsulated Au NPs solution;

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

[0029] PVP-Au NPs were dissolved in methanol to obtain a PVP-encapsulated gold nanoparticle solution;

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

[0031] 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] The polyvinyl pyrrolidone solution is a polyvinyl pyrrolidone aqueous solution, and the mass concentration of the polyvinyl pyrrolidone solution is 2.5-3%;

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

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

[0035] Preferably, Au NPs@ZIF-8 is added to the anti-inflammatory drug solution, stirred for 20 to 30 hours, and then the solid and liquid are separated. The precipitate is collected and vacuum freeze-dried to obtain a material for promoting bone tissue repair.

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

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

[0038] The material for promoting bone tissue repair of the present invention, and its preparation method and application have the following beneficial effects compared with the prior art:

[0039] 1. The material for promoting bone tissue repair of the present invention is a material with gold nanoparticles (Au NPs) as the core, a zeolitic imidazolate framework (ZIF-8) growing around it and anti-inflammatory drugs such as gallic acid (Ga) loaded inside the framework, i.e., a gold nanoparticle-zeolitic imidazolate framework enhanced gallic acid drug loading system (Ga@Au NPs@ZIF-8), which is used to promote bone tissue repair; the material loads anti-inflammatory drugs through a nanoparticle and zeolitic imidazolate framework complex, and utilizes its osteogenic and anti-inflammatory properties to promote the regeneration and repair of bone tissue;

[0040] 2. The material for promoting bone tissue repair of the present invention combines the drug delivery advantages and biological activity of gold nanoparticles and ZIF-8 by loading anti-inflammatory drugs such as gallic acid into a composite material of gold nanoparticles-zeolite imidazolate skeleton, thereby providing a multifunctional material with good bone repair effect and anti-inflammatory properties. The stable structure of the ZIF skeleton in the present invention can achieve the slow release of gallic acid, avoiding the drug resistance problem caused by the burst effect. Gallic acid has potent antioxidant and anti-inflammatory effects, and can effectively regulate the inflammatory response during bone repair. The present invention can continuously exert anti-inflammatory effects at the bone repair site by loading gallic acid into a nanocomposite material, reduce the inhibition of local inflammatory response on bone tissue regeneration, and thus accelerate the repair process of bone tissue. The present invention can not only provide sustained-release gallic acid to achieve anti-inflammatory effects, but also promote the proliferation and differentiation of osteoblasts, and accelerate the regeneration and repair of bone tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

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

[0043] Figure 2 Alkaline phosphatase staining of MC3T3-E1 cells after 14 days of osteogenic induction; Alizarin red staining of MC3T3-E1 cells after 21 days of osteogenic induction;

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

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

[0046] Figure 5 Immunofluorescence staining of inflammatory factor iNOS in RAW 264.7 cells after LPS treatment for 24 hours;

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

[0048] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described 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 creative work are within the scope of protection of the present invention.

[0049] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0050] The following are described in detail. It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments. In addition, in the description of the present application, the term "including" means "including but not limited to". Various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and simplicity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values ​​within 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 within the numbered ranges, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated in this article, it is meant to include any quoted numbers (fractions or integers) within the indicated range.

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

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

[0053] The zeolite imidazolate skeleton is loaded with anti-inflammatory drugs, and the anti-inflammatory drugs include at least one of gallic acid, curcumin and resveratrol.

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

[0055] Specifically, gold nanoparticles can promote osteogenesis and have certain immunoregulatory properties; Zeolitic Imidazolate Framework-8 (ZIF-8) is a type of metal organic framework with high porosity and stable structure. Due to its chemical stability and versatility, it has 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 drugs, thereby achieving long-term and sustained therapeutic effects. It can also have good stability in complex physiological environments 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 angiogenesis, and show good osteogenic effects in rat skull defect models. Anti-inflammatory drugs such as gallic acid (Gallic Acid, Ga, molecular formula C7H6O5) are natural polyphenol compounds widely found in plants, with strong antioxidant, anti-inflammatory and other biological activities. Gallic acid removes excess free radicals produced by tissues and can promote the proliferation and differentiation of osteoblasts to accelerate bone regeneration.

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

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

[0058] S1, synthesis of 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-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 for reaction, solid-liquid separation, and collecting to obtain Au NPs@ZIF-8;

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

[0063] S6. Add Au NPs@ZIF-8 to the anti-inflammatory drug solution, stir, separate the solid and liquid, and collect the precipitate to obtain the material for promoting bone tissue repair.

[0064] In some embodiments, the method for preparing a PVP-encapsulated gold nanoparticle solution comprises the following steps:

[0065] S11, after heating the chloroauric acid solution, adding the sodium citrate solution, and continuing to heat until the color of the solution changes from red to yellow, thereby obtaining an Au NPs solution;

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

[0067] S13, separating the PVP-encapsulated Au NPs solution into solid and liquid to obtain PVP-Au NPs;

[0068] S14. Dissolve 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, the first solution, the second solution, and the PVP-encapsulated gold nanoparticle solution are mixed, stirred and reacted at 20-25° C. for 20-30 h, and the solid and liquid are separated to collect Au NPs@ZIF-8;

[0071] Wherein, the molar concentration of the soluble zinc salt in the first solution is 25 to 35 mM;

[0072] The molar concentration of 2-methylimidazole in the second solution 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 the chloroauric acid solution is heated to 100° C., a sodium citrate solution is added and the heating is continued, and the reaction causes the color of the solution to change from red to yellow, thereby obtaining an Au NPs solution;

[0075] After cooling the Au NPs solution to 20-25°C, polyvinyl pyrrolidone solution was added and stirred for 20-25h to obtain a PVP-encapsulated Au NPs solution;

[0076] The PVP-encapsulated Au NPs solution was subjected to solid-liquid separation to obtain PVP-Au NPs;

[0077] PVP-Au NPs were dissolved in methanol to obtain a PVP-encapsulated gold nanoparticle solution;

[0078] 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%;

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

[0080] The polyvinyl pyrrolidone solution is a polyvinyl pyrrolidone aqueous solution, and the mass concentration of the polyvinyl pyrrolidone aqueous solution is 2.5-3%;

[0081] The volume ratio of chloroauric acid solution, sodium citrate solution, polyvinyl pyrrolidone 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 to separate the solid and the liquid, and the PVP-Au NPs are collected.

[0083] In some embodiments, the first solution, the second solution, and the PVP-encapsulated gold nanoparticle solution are mixed, stirred and reacted at 20-25° C. for 20-30 h, centrifuged at 8000-10000 rpm, solid-liquid separated, and Au NPs@ZIF-8 are collected.

[0084] In some embodiments, in the step of adding the anti-inflammatory drug to 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 an anti-inflammatory drug solution, stirred for 20 to 30 hours, and then the solid and liquid are separated. The precipitate is collected and vacuum freeze-dried to obtain a material for promoting bone tissue repair.

[0086] In some embodiments, in the step of adding Au NPs@ZIF-8 to the anti-inflammatory drug solution, the volume 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 invention is to combine gold nanoparticles with zeolite 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 regulate the release rate of 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 invention is to load anti-inflammatory drugs such as gallic acid into gold nanoparticles and ZIF-8 composite materials; gallic acid has strong anti-inflammatory and antioxidant activities, but its direct application is often limited by problems such as poor stability and sudden drug release. The present invention solves the stability and controllable release problems of gallic acid by adding the porous structure of ZIF-8 to the stable core of gold nanoparticles. The sustained release mechanism of the drug ensures that it can continue to play a role in the bone repair process, achieving long-term immune regulation and promoting bone regeneration. The material of the present invention is not limited to the drug delivery system, but more importantly, its multifunctional performance in bone tissue repair. The multifunctionality of the material of the present invention 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 sustained release, thereby accelerating the repair of bone tissue. This multifunctionality enables the material to cope with the complex physiological environment in bone repair, especially promoting bone regeneration while alleviating inflammatory response.

[0088] Compared with existing nanomaterials and drug delivery systems for bone repair, the materials of the present invention are multifunctional: in some complex bone injury or bone defect scenarios, single-function bone repair materials often cannot meet the treatment needs, especially when multiple functions such as anti-inflammatory and bone regeneration are required, the effects of existing materials are relatively limited. The present invention can simultaneously meet these complex bone repair needs, control inflammatory reactions, and promote bone regeneration through the design of multifunctional composite materials, significantly improving the effect of the material in actual clinical applications.

[0089] The present invention mainly uses gallic acid as a loaded drug for anti-inflammatory and bone regeneration promotion. At the same time, it can also be replaced by other drugs or bioactive molecules with similar functions according to actual needs, such as: anti-inflammatory substitutes: in addition to gallic acid, other natural active substances such as curcumin (Curcumin), resveratrol (Resveratrol), etc. can also be used as substitute drugs. These substances 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), etc. can be loaded in the composite material to accelerate the repair and regeneration of bone tissue; anti-inflammatory drugs: in certain circumstances, other anti-inflammatory drugs, such as non-steroidal anti-inflammatory drugs (NSAIDs) or corticosteroids, can also be selected to load to enhance the anti-inflammatory effect, especially in bone injuries with more severe inflammatory reactions or post-operative recovery.

[0090] The present invention can select different nanoparticles as the core. In the present invention, gold nanoparticles are used as the core to exert immune regulation and osteogenic effects. Other types of nanoparticles can also be used to change the physical and chemical properties and biological activity of the material according to different needs, such as silver nanoparticles, silicon dioxide nanoparticles, etc.

[0091] The present invention can be combined with scaffold materials for functional optimization. The present invention can be combined with existing scaffold materials or functional optimization of the scaffold structure to further expand the scope of application. By modifying the nanoparticles of the present invention on the surface of different types of tissue engineering scaffolds (such as bioceramic scaffolds, polymer scaffolds, hydrogels, electrospun fiber membranes, etc.), these scaffolds are given new functional properties to adapt to more complex tissue repair scenarios.

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

[0093] Specifically, the material of the present invention can be applied to other fields that require anti-inflammatory properties in addition to bone tissue repair 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 present invention is mainly used for bone tissue repair, it can also be applied to other types of tissue repair or regeneration by loading different effective components, such as cartilage repair, periodontal tissue repair, skin wound healing, etc.

[0094] The following further describes the material and preparation method for promoting bone tissue repair of the present application with specific examples. This section further describes the content of the present invention in conjunction with specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0095] Example 1

[0096] The present invention provides a method for preparing 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, preparing 100 mL of a 0.025% chloroauric acid aqueous solution, heating the chloroauric acid aqueous solution to 100° C. to make it boil, then adding 2 mL of a 0.1% sodium citrate aqueous solution, and continuing to heat for 20 min until the color of the solution changes from red to yellow, thereby obtaining an Au NPs solution;

[0099] S12, after cooling the Au NPs solution to room temperature (25°C), add 20 mL of a 2.5% polyvinyl pyrrolidone aqueous solution and stir for 24 h to obtain a PVP-encapsulated Au NPs (PVP-Au NPs) solution;

[0100] S13, centrifuging the PVP-encapsulated Au NPs solution at 10,000 rpm for 12 min to separate the solid and liquid, and collecting the PVP-Au NPs;

[0101] S14, dissolving the PVP-Au NPs in step S13 in 50 mL of methanol, and dispersing by ultrasonication to obtain a PVP-encapsulated gold nanoparticle solution;

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

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

[0104] S4, 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 were mixed, stirred at room temperature for 24 hours, centrifuged at 10000 rpm for 12 minutes, solid-liquid separation was performed, and Au NPs@ZIF-8 was collected;

[0105] S5, adding gallic acid 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. Add 50 mg of Au NPs@ZIF-8 prepared in step S4 to 20 mL of the anti-inflammatory drug solution, stir at room temperature for 24 hours, centrifuge at 15,000 rpm for 12 minutes, separate the solid and liquid, collect Ga@Au NPs@ZIF-8, and dry it in a vacuum freeze dryer to obtain a material for promoting bone tissue repair.

[0107] Performance Testing

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

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

[0110] Figure 2 Alkaline phosphatase staining of MC3T3-E1 cells after 14 days of osteogenic induction (i.e. Figure 2 ALP staining for14d); Alizarin red staining for 21 days of osteogenic induction in MC3T3-E1 (i.e. Figure 2 ARS staining for 21d).

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

[0112] MC3T3-E1 cells were inoculated in a 12-well plate. When the cell density increased to 90%, an osteogenic induction solution containing 25 mg / mL of Ga@Au NPs@ZIF-8 prepared in Example 1 was added to the experimental group, and the osteogenic induction solution was added to the control group. The solution was changed every 2 days. On the 14th day of induction, alkaline phosphatase staining (ALP staining) was performed using the Bio-Tian alkaline phosphatase staining kit according to the instructions; on the 21st day of induction, the generated mineralized nodules were stained with alizarin red (ARS staining) using the Saiye alizarin red staining solution according to the instructions. The results are shown in Figure 2. Figure 2 shown.

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

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

[0115] MC3T3-E1 cells were inoculated in a 6-well plate. When the cell density increased to 90%, 25 mg / mL of Ga@Au NPs@ZIF-8 (i.e. Figure 3 The osteogenic induction solution of Ga@Au NPs@ZIF-8) and Gallic Acid group (i.e. Figure 3 Gallic Acid) is an osteogenic induction solution containing 8 mg / mL gallic acid, and the control group (i.e. Figure 3 The control (in the middle) was the osteogenic induction medium, and the medium was changed every 2 days. RNA was extracted on the 7th day of induction, and reverse transcription was performed to obtain cDNA. qPCR was performed to detect the mRNA expression levels of osteogenic-related genes RUNX2 and Col-1. The results are shown in Figure 3 shown.

[0116] Figure 4 Masson staining results of paraffin sections of SD rat skull defect model 2 weeks after surgery ( Figure 4 OB in the middle is Original Bone, NB is New Bone, and the scale bar is 300 μm).

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

[0118] The skull defect model (defect diameter was 5 mm) was established in 7-week-old SPF rats under local anesthesia. Figure 4 Methacryloyl gelatin (GelMA hydrogel, catalog number EFL-GM-60) was placed in the defect area in the control group, and the experimental group (i.e. Figure 4 The Ga@Au NPs@ZIF-8 in the sample is the GelMA hydrogel containing the Ga@Au NPs@ZIF-8 prepared in Example 1. The wounds of all rats were tightly sutured. Two weeks after surgery, the rats were euthanized, and the samples were collected, fixed, decalcified, dehydrated, paraffin-embedded, sliced, and Masson-stained. The results are shown in FIG. Figure 4 shown.

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

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

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

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

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

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

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

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

[0127] Figure 4 In the study, 2 weeks after the rat skull defect model was established, Masson staining results showed that Ga@Au NPs@ZIF-8 significantly promoted the regeneration of the bone defect area.

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

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

[0130] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

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 anti-inflammatory drugs, and the anti-inflammatory drugs include at least one of gallic acid, curcumin and resveratrol.

2. The method for preparing a material for promoting bone tissue repair according to claim 1, characterized in that: 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 the anti-inflammatory drug into methanol to obtain an anti-inflammatory drug solution; Au NPs@ZIF-8 is added to the anti-inflammatory drug solution, stirred, and then the solid and liquid are separated, and the precipitate is collected to obtain the material for promoting bone tissue repair.

3. The method for preparing a material for promoting bone tissue repair according to claim 2, characterized in that: The method for preparing the PVP-encapsulated gold nanoparticle solution comprises the following steps: After heating the chloroauric acid solution, add the sodium citrate solution and continue heating until the color of the solution changes from red to yellow, thereby obtaining an Au NPs solution; After the Au NPs solution was cooled, a 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 PVP-encapsulated gold nanoparticle solution.

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

5. The method for preparing a material for promoting bone tissue repair according to claim 2, characterized in that: 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 the solid-liquid separation was performed to collect Au NPs@ZIF-8; Wherein, 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 25-35 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).

6. The method for preparing a material for promoting bone tissue repair according to claim 3, characterized in that: The chloroauric acid solution was heated to 100°C, and sodium citrate solution was added and continued to heat. 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 20-25h 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; Wherein, the chloroauric acid solution is a chloroauric acid aqueous solution, and the mass concentration of the chloroauric acid aqueous solution 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, the sodium citrate solution, the polyvinyl pyrrolidone solution and the methanol is (100-110):(2-3):(20-30):(50-60).

7. The method for preparing a material for promoting bone tissue repair according to claim 2, characterized in that: In the step of adding the anti-inflammatory drug to methanol to obtain an anti-inflammatory drug solution, the concentration of the anti-inflammatory drug in the anti-inflammatory drug solution is 3 to 5 mg / mL.

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

9. The method for preparing a material for promoting bone tissue repair according to claim 2, characterized in that: In the step of adding Au NPs@ZIF-8 to the anti-inflammatory drug solution, the volume mass ratio of the anti-inflammatory drug solution to Au NPs@ZIF-8 is (20-30) mL: (50-60) mg.

10. 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 9 in the preparation of a drug for promoting bone tissue repair.

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