Antibacterial anti-inflammatory and osteogenesis-promoting two-dimensional bimetallic MOF and preparation and application thereof

Abstract

The application provides an antibacterial anti-inflammatory and osteogenesis-promoting two-dimensional bimetallic MOF as well as preparation and application thereof. The MOF can effectively resist bacteria and inflammation, has good biological safety, and has the ability to act for a long time. Before degradation, the two-dimensional bimetallic MOF provided by the application can effectively remove bacteria under the action of low-intensity medical US, and can remove ROS through nano-enzyme activity. After degradation, the copper and manganese ions and quercetin released by the two-dimensional bimetallic MOF can be chelated, and then the metal-ion charge transfer MLCT effect is exerted to play the SOD and CAT effects, so that the ROS adsorption effect can be continuously exerted. In addition, the copper and manganese ions and quercetin can continuously and efficiently promote bone tissue repair due to the osteogenesis and antibacterial effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to an antibacterial, anti-inflammatory and osteogenesis-promoting two-dimensional bimetallic MOF as well as a preparation and application thereof. BACKGROUND

[0002] Infective bone defect is one of the thorny problems encountered in clinical work of orthopedics. Due to local bacterial infection, bone tissue healing is slow, which will bring heavy burden to patients and society. The long-term existence of bacteria in the infection focus can induce osteoclast differentiation and activate the inflammatory signaling pathway of cells by releasing metabolites such as lipopolysaccharide (LPS), thereby inhibiting bone repair. Therefore, the efficient treatment of infective bone defect needs to solve the two problems of bacterial infection and inflammatory microenvironment. Although traditional antibiotics and surgical treatment can inhibit bacterial infection, they are poor in improving the inflammatory microenvironment of the infection focus, and the large use of antibiotics can induce bacterial drug resistance, and surgical treatment also has the risk of aggravating bone defect. Therefore, seeking an efficient method for sterilization and inhibition of local high inflammatory reaction is an urgent need for infective bone defect.

[0003] Sonodynamic therapy (SDT) is a non-invasive and efficient bacterial clearance strategy, whose basic mechanism is that under the action of ultrasound (US), the sonosensitizer is activated from the ground state to the excited state, and a series of reactions occur with the surrounding oxygen molecules to produce a large amount of reactive oxygen species (ROS). ROS can kill bacteria through multiple mechanisms, including destroying the electron transport chain, leading to the death of bacteria due to oxidative stress and energy metabolism damage. However, although ROS can effectively kill bacteria, they will also have adverse effects on surrounding normal cells and tissues. ROS can promote the polarization of macrophages M1 and thus aggravate inflammation. In addition, ROS can inhibit osteogenic differentiation by inducing mitochondrial dysfunction of stem cells. In summary, ROS is a double-edged sword. Nanocatalase is a catalytic nanomaterial with enzyme-like properties, which can simulate the structure of natural antioxidant enzymes and play the role of superoxide dismutase (SOD) and catalase (CAT), thereby catalyzing ROS to generate water and oxygen. Nanocatalase is widely used due to its cost-effectiveness, stability, adjustable catalytic activity and easy mass production. However, the development of materials that can both produce and remove ROS is a challenge for the treatment of infective bone defect. In addition, since materials that are difficult to degrade will hinder bone tissue healing and exacerbate inflammation, it is essential to choose materials with good biocompatibility for the treatment of infective bone defect.

[0004] Currently, there are many materials with good biocompatibility used for the treatment of infected bone defects. Organic photosensitizers such as porphyrin and its derivatives, 5-aminolevulinic acid, phthalocyanine, etc. have the characteristics of high SDT efficiency and fast degradation, and are widely used. In addition, degradable nanoenzymes such as MnPcBc, Prussian blue and phosphate-based glass (PBG) have also been widely used in antioxidant research. However, although good biocompatibility is conducive to preventing tissue damage and local inflammation caused by material implantation, it is a major challenge for materials with good degradation performance to exert long-term effects. Since photosensitizers or nanoenzymes rely on the inherent properties of the material to function, when the material degrades, its SDT or antioxidant effect will gradually weaken, which is not conducive to the treatment of infected bone defects. Although drug loading is a modification strategy to extend the function of degradable materials, the drug itself may still be metabolized by cells, resulting in loss of drug efficacy. Therefore, developing biomaterials with good biocompatibility and long-term efficacy is the key to the treatment of infected bone defects.

[0005] The patent with application number CN202211401896.5 discloses a high-efficiency antibacterial and osteogenesis-promoting defective MOF photosensitizer and its preparation method. The patent uses alendronate sodium-doped zirconium-based porphyrin MOF to promote sterilization by modifying the properties of the photosensitizer and to promote osteogenesis using acoustic current. However, the MOF synthesized by this method is prone to degradation and does not have long-term antibacterial and osteogenic effects. In addition, since the ROS generated by sonodynamic force can damage the activity of surrounding cells, it may exacerbate local inflammation progression, which is not conducive to bone tissue repair.

[0006] The patent with application number CN202210246313.X discloses a zinc-doped calcium phosphate ceramic microsphere with antibacterial function and a preparation method. The patent dopes zinc ions into a calcium phosphate matrix to form a composite microsphere, and the zinc ions can be released by slow degradation of the calcium phosphate, ensuring the long-term antibacterial activity of the biomaterial. However, although zinc ion release can have long-term antibacterial effects, the continued progression of inflammation at the site of bone defect can severely affect bone tissue repair, which is not conducive to the treatment of bone defects.

[0007] The patent with application number CN202410602481.7 discloses a near-infrared responsive controlled-release NO antibacterial bioceramic and its preparation method. The patent combines tetracalcium phosphate, calcium hydrogen phosphate dihydrate, Arg-ZnPc@mesoporous polydopamine microspheres, and a sodium alginate solution to prepare a biorepair material that can respond to infrared light, which can control NO release to exert long-term antibacterial effects locally. However, since the release of NO can exacerbate the inflammation of surrounding cells, it is not conducive to bone tissue repair.

[0008] The patent with application number CN202311017016.9 discloses an antibacterial and antioxidant bone-promoting multifunctional coating with environmental and photothermal responses. The coating is composed of porous titanium dioxide grown in situ on the surface of titanium or titanium alloy and ZIF-8 nanoparticles combined with curcumin. Under near-infrared light irradiation, the coating can promote sterilization by releasing zinc ions and can also promote anti-inflammatory by releasing curcumin. However, since the organic metal framework is a degradable material, curcumin is also easily metabolized by cells and naturally oxidized, so it cannot exert a long-lasting antibacterial and anti-inflammatory effect.

[0009] Summarizing the above-mentioned prior art, the problems are:

[0010] 1. The treatment of infectious bone defects needs to solve the problems of bacterial infection and inflammatory reaction. Existing treatment methods such as antibiotics or surgery can effectively solve bacterial infection, but the local presence of high inflammatory reaction will inhibit bone tissue repair. Long-term use of antibiotics will promote drug resistance, and surgical treatment also has the risk of aggravating bone defects.

[0011] 2. ROS acts as a double-edged sword, which can exert antibacterial effect by destroying bacterial biofilm. However, ROS also destroys surrounding cells, thereby aggravating inflammation of surrounding cells. Therefore, the ideal treatment strategy should be to use ROS to kill bacteria while adsorbing excess ROS to exert anti-inflammatory effect during bone tissue repair.

[0012] 3. The treatment mechanism of SDT is to excite the electron transfer in the endogenous ultrasound-sensitive agent, thereby producing a large amount of ROS. It is difficult to kill local bacteria simply by relying on SDT, and excessive ROS will also cause oxidative stress and aggravate inflammation of surrounding cells.

[0013] 4. Nanoplasma is a kind of nanomaterial with enzyme activity, which can adsorb ROS through active sites to exert anti-inflammatory effect. However, traditional nanoplasma has poor effect due to few active sites, and it is currently difficult to prepare materials with both nanoplasma activity and ultrasound-sensitive agent characteristics.

[0014] Traditional biodegradable materials have the advantage of good biocompatibility, but easy degradation also limits their action time. Since traditional antibacterial and anti-inflammatory biomaterials need to rely on drug or material itself active site to exert effect, once degraded, the effect will be greatly reduced, so it is necessary to improve the preparation method of the current biodegradable material and seek an improved strategy that can ensure that the material has good biodegradability while also continuously exerting effect. SUMMARY

[0015] (I) Technical problems solved

[0016] The present application aims at the problem that it is difficult to simultaneously resist bacteria and inflammation and the existing biological material is difficult to play a long-term role in the treatment of infectious bone defects, and proposes a preparation and application of a two-dimensional bimetallic MOF with long-acting antibacterial, anti-inflammatory and osteogenesis. The MOF can effectively resist bacteria and inflammation, and has good biological safety and long-acting ability. Before degradation, the application can effectively remove bacteria under the action of low-intensity medical US, and can remove ROS through nano-enzyme activity. After degradation, the slow-release copper and manganese ions and quercetin of the application can be chelated, and then the metal-ion charge transfer (MLCT) effect is played to play the effect of adsorbing ROS, so that the effect of adsorbing ROS can be continuously played. In addition, since the copper and manganese ions and quercetin have osteogenesis and antibacterial effects, the application can continuously and efficiently promote bone tissue repair. The present application not only makes up for the defect that the degradable material cannot continuously play a role, but also solves the key problem that it is difficult to simultaneously achieve antibacterial and anti-inflammatory in the treatment of infectious bone defects.

[0017] (ii) Technical solutions

[0018] To solve the above technical problems, the present application adopts the following technical solutions:

[0019] A two-dimensional bimetallic MOF with antibacterial, anti-inflammatory and osteogenesis, comprising 3.6mg of copper nitrate trihydrate, 40mg of polyvinylpyrrolidone, 17mg of tetra-p-terphenyl carboxylate porphyrin manganese III, 6.25mg of quercetin and 48ml of N,N-dimethylformamide. The preparation method is as follows: taking copper nitrate trihydrate and tetra-p-terphenyl carboxylate porphyrin manganese III as raw materials, using hydrothermal method to dope copper ions on tetra-p-terphenyl carboxylate porphyrin manganese III to prepare nanosheets with copper manganese double elements, and then co-incubating the nanosheets and quercetin to prepare drug-loaded two-dimensional bimetallic MOF 。

[0020] Since the copper ions will replace the manganese ions on the manganese porphyrin, the nanosheets with double active sites will be formed. The Cu2O8 and MnN4 double active sites both have the ability of SDT and adsorbing ROS, and the quercetin loaded on the two-dimensional bimetallic MOF can chelate copper and manganese ions to enhance the antioxidant capacity of quercetin after the material degrades. Therefore, the two-dimensional bimetallic MOF can continuously play the effects of antibacterial and anti-inflammatory, and efficiently promote the repair of bone tissue.

[0021] The present application also discloses a preparation method of a two-dimensional bimetallic MOF with antibacterial, anti-inflammatory and osteogenesis:

[0022] Step S1, preparation of copper MOF and two-dimensional copper-manganese doped bimetallic MOF; using copper nitrate trihydrate and manganese III tetra-p-terphenyl carboxylate porphyrin as raw materials, copper ions are doped on manganese III tetra-p-terphenyl carboxylate porphyrin by hydrothermal method to prepare nanosheets with copper-manganese double elements, and then the nanosheets are co-incubated with quercetin to prepare drug-loaded two-dimensional bimetallic MOF.

[0023] The above preparation method further comprises the following steps:

[0024] Step S2, structure and component analysis of two-dimensional bimetallic MOF;

[0025] Step S3, analysis of antioxidant performance and mechanism of two-dimensional bimetallic MOF before degradation;

[0026] Step S4, analysis of antioxidant performance and mechanism of two-dimensional bimetallic MOF after degradation;

[0027] Step S5, verify the antibacterial performance of two-dimensional bimetallic MOF before and after degradation through in vitro antibacterial experiment; Step S6, detect the biological safety of two-dimensional bimetallic MOF through BMSCs co-culture and hemolysis experiment;

[0028] Step S7, explore the osteogenesis-promoting and angiogenesis-promoting biological mechanisms of two-dimensional bimetallic MOF after co-culture with BMSCs and HUVECs through transcriptomics, single-cell transcriptomics and other omics methods, and verify the mechanisms in vitro through Western Blot and other methods;

[0029] Step S8, detect the ROS adsorption capacity of two-dimensional bimetallic MOF after co-culture with cells in vitro through flow cytometry, immunofluorescence and other methods, and verify the effect of promoting M2 differentiation of THP-1 cells;

[0030] Step S9, prepare injectable hydrogel containing two-dimensional bimetallic MOF and construct a rat model of cranial infectious defect, and verify the antibacterial, anti-inflammatory and osteogenic efficiency in vivo.

[0031] The step S1 is specifically: preparing raw materials including 3.6-14.4 mg of copper nitrate trihydrate, 40 mg of polyvinylpyrrolidone, 16 mg of tetra-carboxyphenyl porphyrin, 17 mg of manganese tetra-p-terphenyl porphyrin, 6.25 mg of quercetin and 48 ml of N,N-dimethylformamide. First, 17 mg of manganese tetra-p-terphenyl porphyrin is added to 48 ml of N,N-dimethylformamide to be stirred and dissolved until the solution is free of particles. Then, a copper nitrate trihydrate solution is prepared, and 3.6-14.4 mg of copper nitrate trihydrate is added to 16 ml of ethanol to be stirred. Next, a 1M trifluoroacetic acid solution is prepared, and 160 μl of the trifluoroacetic acid solution is added to the copper nitrate trihydrate solution to be stirred, and 40 mg of polyvinylpyrrolidone is added to the solution to be stirred uniformly. Finally, the copper nitrate trihydrate solution and the manganese tetra-p-terphenyl porphyrin solution are mixed to be stirred uniformly. The solution is placed in a muffle furnace to be heated at a rate of 2 ℃ / min to 80 ℃, and is kept at 80 ℃ for 4 h. Then, the product is washed with ethanol and centrifuged 3 times (9000 rpm, 10 min each time), and is finally dried to obtain about 25 mg of two-dimensional bimetallic MOF powder. The obtained two-dimensional bimetallic MOF powder is dissolved in 25 ml of an ethanol solution, 6.25 mg of quercetin is added, and the solution is stirred at room temperature for more than 12 h. The obtained solution is finally dried to obtain the final product.

[0032] By changing the content of the added copper nitrate trihydrate, two-dimensional bimetallic MOFs containing different copper and manganese can be prepared.

[0033] To prepare a control group, i.e., tetra-carboxyphenyl porphyrin copper not containing manganese, 16 mg of tetra-carboxyphenyl porphyrin is added to 48 ml of N,N-dimethylformamide, and is finally added to the mixed solution of copper nitrate trihydrate, trifluoroacetic acid and polyvinylpyrrolidone prepared above. The solution is placed in a muffle furnace to be heated at a rate of 2 ℃ / min to 80 ℃, and is kept at 80 ℃ for 4 h. Then, the product is washed with ethanol and centrifuged 3 times (9000 rpm, 10 min each time), and is finally dried to obtain about 25 mg of a tetra-carboxyphenyl porphyrin copper sample.

[0034] In step S2, the general morphology of the MOF is observed by field emission scanning electron microscopy (SEM), the surface structure of the MOF is observed by high-resolution transmission electron microscopy (TEM), the surface element composition and chemical bonds of the MOF are detected by X-ray photoelectron spectroscopy (XPS), the surface element composition and distribution of the MOF are observed by X-ray energy spectrum element image analysis technology (EDS), the thickness of the material is detected by atomic force microscopy (AFM), and the crystal structure of the two-dimensional bimetallic MOF is revealed by X-ray diffraction technology (XRD).

[0035] In step S3, the ability of the two-dimensional copper-manganese MOF to adsorb ROS before degradation is verified using various means such as XPS, SOD and CAT kits, and the chemical mechanism of the two-dimensional bimetallic MOF adsorbing ROS through metal active sites is revealed through theoretical calculation.

[0036] In step S4, the ability of the two-dimensional copper-manganese MOF to adsorb ROS after degradation is detected using XPS, SOD and CAT kits, and the chemical mechanism of quercetin and copper-manganese ion chelation to play an antioxidant role is revealed through theoretical calculation.

[0037] In step S5, methicillin-resistant Staphylococcus aureus is used as the research object, and the sonodynamic antibacterial performance of the two-dimensional copper-manganese bimetallic MOF is verified through in vitro antibacterial experiments, and the antibacterial effect of the MOF and bacteria after degradation is verified by releasing ions and quercetin. The difference between the two-dimensional bimetallic MOF and the traditional MOF in antibacterial performance is detected and verified by SEM and plate coating and other experimental means.

[0038] In step S7, the ability of the MOF to promote bone and blood vessels is detected and verified by various means such as immunofluorescence, PCR, and Wester Blot, and the mechanism of the MOF releasing ions to promote bone tissue repair is revealed through single-cell transcriptome.

[0039] In step S8, the ability of the MOF to protect cells from ROS damage and to regulate macrophage differentiation is detected and verified by various means such as flow cytometry and fluorescence.

[0040] In step S9, 20mg of Jelleine-1 is added to 200 microliters of DMSO, mixed and dissolved to obtain 100mg / mL Jelleine-1, which is diluted with 200ug / ml of two-dimensional bimetallic MOF solution to 8mg / ml, and incubated at 37℃ (for more than 15 minutes) to form a gel, obtaining an injectable hydrogel. The critical infective bone defect of rats is used as a model to explore the antibacterial and anti-inflammatory bone formation efficiency in vivo.

[0041] (Three) beneficial effects

[0042] (1) Excellent antibacterial effect:

[0043] The bimetallic two-dimensional MOF prepared by the present application is a high-efficiency sonosensitizer. Due to its double active sites, it can greatly enhance the electron transfer capacity compared to traditional antibacterial materials. Under the action of ultrasound, the MOF can efficiently promote electron transfer to generate ROS, thereby playing a bactericidal role. In addition, after the material degrades, the material releases copper-manganese ions and quercetin. Since copper-manganese ions and quercetin have certain bactericidal effects, the material has a persistent antibacterial effect compared to traditional MOFs.

[0044] (2) can be efficient anti-inflammatory:

[0045] The bimetallic two-dimensional MOF prepared by the application has Cu2O8 and MnN4 double active sites, and both double active sites have SOD-like and CAT-like effects. Before degradation, the material can adsorb ROS to play an antioxidant role. After degradation, the material can release quercetin and copper manganese ions, and quercetin and copper manganese ions will chelate, and the antioxidant effect of quercetin is enhanced through MLCT, so the application can continuously and efficiently play an antioxidant role, thereby promoting local inflammation treatment.

[0046] (3) can efficiently promote bone tissue repair:

[0047] The bimetallic two-dimensional MOF prepared by the application can promote osteogenesis and angiogenesis through drug and ion release. Since quercetin and copper ions have a certain effect on stimulating vascular regeneration, and copper manganese ions have a certain ability to promote osteogenesis. Therefore, the prepared copper manganese MOF can efficiently promote the regeneration of bone tissue, thereby more quickly recovering the reconstruction of bone tissue.

[0048] (4) has better biological safety:

[0049] The bimetallic two-dimensional MOF prepared by the application uses porphyrin as a raw material. Since porphyrin is an organic degradable material, it has strong biological safety, and the application uses traditional Chinese medicine quercetin as a loaded drug, which greatly improves the biocompatibility of the material itself. In addition, since the application uses hydrogel as a carrier, the biological toxicity caused by in-situ injection of MOF is avoided. Therefore, the application can more safely and effectively promote bone tissue repair.

[0050] (5) avoid the problem of bacterial drug resistance:

[0051] The bimetallic two-dimensional MOF prepared by the application can be antibacterial through ions, drugs and sonodynamic effect, thereby killing bacteria by destroying bacterial membranes and electron transport chains, and is not prone to drug resistance, thereby avoiding the problems caused by the large use of antibiotics.

[0052] (6) can treat on demand in the time dimension:

[0053] Since the bimetallic two-dimensional MOF prepared by the application performs antibacterial through sonodynamic effect, exogenous ultrasound is the key to activating the antibacterial performance of the material. In the clinical treatment process, the application of the material can develop more personalized treatment programs according to the progress stage of the patient. When facing different patients, the application of the material can control different parameters such as power and ultrasonic time, thereby killing bacteria while minimizing damage to normal tissues.

[0054] (7) can precisely position treatment in the spatial dimension:

[0055] Since the hydrogel is used as the carrier in the application, after being implanted into the lesion of a patient, the lesion can be positioned more accurately by using ultrasonic method, the influence on the surrounding tissue is minimized while the sonodynamic sterilization is performed.(8) has the potential of large-scale production:

[0056] Since the raw materials used in the application are widely sourced, the industry is mature, the preparation process is simple, safe and easy to control. In addition, the preparation process does not pollute the environment, and does not harm the operating personnel during the production process, and has the potential of large-scale production and promotion. BRIEF DESCRIPTION OF DRAWINGS

[0057] In order to more clearly illustrate the technical solutions in the embodiments of the application, the drawings needed to be used in the embodiments will be briefly introduced below.

[0058] Figure 1a TEM images of copper MOF and copper-manganese bimetallic MOF;

[0059] Figure 1b AFM images of copper MOF and copper-manganese bimetallic MOF;

[0060] Figure 1c EDS spectrum of copper-manganese bimetallic MOF;

[0061] Figure 2a ESR of ROS adsorbed by copper MOF, copper-manganese bimetallic MOF and quercetin-loaded copper-manganese bimetallic MOF;

[0062] Figure 2b ESR of ROS adsorbed by quercetin and quercetin-ion chelate;

[0063] Figure 3a ESR of ROS generated by copper MOF, copper-manganese bimetallic MOF and quercetin-loaded copper-manganese bimetallic MOF under US;

[0064] Figure 3b Antibacterial curve of copper MOF, copper-manganese bimetallic MOF and quercetin-loaded copper-manganese bimetallic MOF without adding US;

[0065] Figure 3c Antibacterial coating and SEM image of copper MOF, copper-manganese bimetallic MOF and quercetin-loaded copper-manganese bimetallic MOF without adding US;

[0066] Figure 3d Antioxidant theoretical calculation result of copper-manganese bimetallic MOF before degradation;

[0067] Figure 3e Antioxidant theoretical calculation result of copper-manganese bimetallic MOF before degradation;

[0068] Figure 4a Flow cytometry of ROS adsorption after co-culture of copper MOF, copper-manganese bimetallic MOF and quercetin-loaded copper-manganese bimetallic MOF and BMSCs;

[0069] Figure 4b Immunofluorescence image of promoting macrophage M2 polarization after co-culture of copper MOF, copper-manganese bimetallic MOF and quercetin-loaded copper-manganese bimetallic MOF and THP-1;

[0070] Figures 5a-5b ARS, ALP and immunofluorescence images of promoting stem cell osteogenesis after co-culture of copper MOF, copper-manganese bimetallic MOF and quercetin-loaded copper-manganese bimetallic MOF and BMSCs for 28 days;

[0071] Figure 5c Plate streaking image of promoting HUVEC migration after co-culture of copper MOF, copper-manganese bimetallic MOF and quercetin-loaded copper-manganese bimetallic MOF and HUVECs for 3 days;

[0072] Figure 6a SEM and appearance photograph of quercetin-loaded copper-manganese bimetallic MOF hydrogel;

[0073] Figure 6b Wound appearance image after rat skull defect modeling for 14 days;

[0074] Figure 6c Gram staining images after rat skull defect modeling for 7 and 14 days;

[0075] Figure 6d HE staining image after rat skull defect modeling for 14 days;

[0076] Figures 6e-6f CT reconstruction and BV / TV statistical image after rat skull defect modeling for 28 days. DETAILED DESCRIPTION

[0077] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific embodiments.

[0078] The experimental methods in the following examples are all conventional methods unless otherwise specified, and the experimental materials used in the following examples are all commercially available unless otherwise specified.

[0079] Example: A two-dimensional bimetallic MOF with long-acting antibacterial, anti-inflammatory and osteogenesis-promoting properties, using 3.6 mg of copper nitrate trihydrate, 40 mg of polyvinylpyrrolidone, 17 mg of manganese (III) tetra-p-terephthalate porphyrin, 6.25 mg of quercetin and 48 ml of N, N-dimethylformamide as raw materials.

[0080] Preparation and application of long-acting antibacterial anti-inflammatory and bone-forming two-dimensional bimetallic MOF, which specifically comprises the following steps:

[0081] Step one, preparation of copper MOF and two-dimensional copper manganese doped bimetallic MOF: prepare raw materials including copper nitrate trihydrate 3.6 mg, polyvinylpyrrolidone 40 mg, tetra-carboxyphenyl porphyrin 16 mg, manganese (III) tetrakis-p-benzoic acid porphyrin 17 mg, quercetin 6.25 mg and N, N-dimethylformamide 48 ml. First, 17 mg of manganese (III) tetrakis-p-benzoic acid porphyrin is added to 48 ml of N, N-dimethylformamide and stirred to dissolve until there are no particles in the solution. Then prepare a copper nitrate trihydrate solution, take 3.6 mg of copper nitrate trihydrate and add it to 16 ml of ethanol and stir. Then prepare a 1M trifluoroacetic acid solution, take 160 μl of trifluoroacetic acid solution and add it to the above copper nitrate trihydrate solution and stir, and add 40 mg of polyvinylpyrrolidone to the solution and stir until uniform. Finally, mix the copper nitrate trihydrate solution and the manganese (III) tetrakis-p-benzoic acid porphyrin solution and stir until uniform. Place the solution in a muffle furnace and heat to 80℃ at a rate of 2℃ / min and maintain for 4h. Then wash the product with ethanol and centrifuge 3 times (9000 rpm, 10 minutes each time), and finally dry to obtain ~25 mg of sample. Dissolve the obtained two-dimensional bimetallic MOF powder in 25 ml of ethanol solution, add 6.25 mg of quercetin and stir at room temperature for 12h or more, and finally dry the obtained solution to obtain the final product.

[0082] By changing the amount of copper nitrate trihydrate added, two-dimensional bimetallic MOFs containing different amounts of copper and manganese can be prepared. In this example, 7.2 mg and 14.4 mg of copper nitrate trihydrate are used as raw materials respectively, and other raw materials and steps remain unchanged to prepare two-dimensional bimetallic MOFs containing different amounts of copper and manganese.

[0083] To prepare a control group, i.e. tetra-carboxyphenyl porphyrin copper without manganese, 16 mg of tetra-carboxyphenyl porphyrin is added to 48 ml of N, N-dimethylformamide and finally added to the above prepared mixture of copper nitrate, trifluoroacetic acid, and polyvinylpyrrolidone. Place the solution in a muffle furnace and heat to 80℃ at a rate of 2℃ / min and maintain for 4h. Then wash the product with ethanol and centrifuge 3 times (9000 rpm, 10 minutes each time), and finally dry to obtain ~25 mg of tetra-carboxyphenyl porphyrin copper sample.

[0084] Step two: Structure and composition analysis of two-dimensional bimetallic MOF: The general morphology of MOF is observed by field emission scanning electron microscopy (SEM); the surface structure of MOF is observed by high-resolution transmission electron microscopy (TEM); the surface element composition and chemical bonds of MOF are detected by X-ray photoelectron spectroscopy (XPS); the surface element composition and distribution of MOF are observed by X-ray energy dispersive spectroscopy (EDS); the thickness of the material is detected by atomic force microscopy (AFM); and the crystal structure of the nanosheet is revealed by X-ray diffraction (XRD).

[0085] Step three: Analysis of antioxidant performance and mechanism of two-dimensional bimetallic MOF before degradation: The ability of two-dimensional copper-manganese MOF to adsorb ROS before degradation is verified using XPS, SOD, and CAT kits, and the chemical mechanism of nanosheet adsorbing ROS through metal active sites is revealed through theoretical calculation.

[0086] Step four: Analysis of antioxidant performance and mechanism of two-dimensional bimetallic MOF after degradation: Specifically, the ability of two-dimensional copper-manganese MOF to adsorb ROS after degradation is detected using XPS, SOD, and CAT kits, and the chemical mechanism of quercetin and copper-manganese ion chelation to play an antioxidant role is revealed through theoretical calculation.

[0087] Step five: Methicillin-resistant Staphylococcus aureus is used as the research object, and the sonodynamic antibacterial performance of two-dimensional copper-manganese bimetallic MOF is verified through in vitro antibacterial experiments, and the antibacterial effect of MOF and bacteria after degradation is verified by co-culturing. The difference between two-dimensional bimetallic MOF and traditional MOF in antibacterial performance is detected by SEM and plate coating and other experimental methods.

[0088] Step six: The biological safety of two-dimensional bimetallic MOF is detected by BMSCs co-culture and hemolysis experiment.

[0089] Step seven: The ability of MOF to promote bone formation and angiogenesis is detected and verified by immunofluorescence, PCR, and Wester Blot, and the mechanism of MOF ion release promoting bone tissue repair is revealed by single-cell transcriptome.

[0090] Step eight: Specifically, the ability of MOF to protect cells from ROS damage and regulate macrophage differentiation is detected and verified by flow cytometry and fluorescence.

[0091] Step nine: take 20 mg Jelleine-1, add 200 microliters of DMSO, mix and dissolve to obtain 100 mg / mL Jelleine-1, dilute to 8 mg / ml with a 200 μg / ml two-dimensional bimetallic MOF solution, incubate at 37°C (for more than 15 min) to form a gel, and obtain an injectable hydrogel. The critical infective bone defect model of rats is used to explore the antibacterial, anti-inflammatory and osteogenic efficiency in vivo.

[0092] The copper-manganese bimetallic MOF is synthesized by a hydrothermal method, and the material has long-acting antibacterial, anti-inflammatory and osteogenic effects. Before degradation, the material can play the roles of SDT and ROS adsorption through two active sites, and after degradation, the released copper-manganese ions and quercetin can form chelates, thereby enhancing the antioxidant effect of quercetin itself. In addition, since the copper-manganese ions and quercetin have certain antibacterial and osteogenic abilities, the material developed in the application has excellent biocompatibility and long-acting antibacterial, anti-inflammatory and osteogenic abilities.

[0093] The following takes the product prepared in the examples as an example to illustrate its performance.

[0094] 1. Material characterization

[0095] (1) Transmission electron microscopy (TEM) shows that the copper MOF and the copper-manganese MOF have an ultrathin nanosheet structure, and the sizes thereof are about 1-2 μm and 2-3 μm, respectively. Figure 1a Atomic force microscopy (AFM) shows that the average thickness of the MON(Cu) is about 2.9 nm. The thickness of the copper MOF is similar to that of the copper-manganese MOF, about 2.7 nm. Figure 1b Energy dispersive spectrometer analysis (EDS) shows that the copper-manganese MOF has a uniform surface element distribution, indicating that the nanosheet is successfully prepared. Figure 1c

[0096] 2. Antioxidant performance detection and mechanism analysis

[0097] Electron spin resonance (ESR) shows that the copper-manganese MOF loaded with quercetin has the strongest ROS adsorption capacity and can catalyze ROS to generate water and oxygen. Figure 2a In addition, it can be seen that the ions after the degradation of the copper-manganese MOF loaded with quercetin and the quercetin chelate have ROS adsorption capacity, and the effect is stronger than that of quercetin. Theoretical calculation shows that the MnN4 site of the copper-manganese bimetallic MOF before degradation has stronger activity than the traditional CuN4 site, and can play the roles of SOD and CAT through adsorption of ROS. The chelate analysis after degradation shows that quercetin can interact with copper-manganese ions to form a chelate structure, thereby promoting the distribution of electrons to form MLCT. Figure 2b 3. Antimicrobial performance detection

[0098] ​​

[0099] By the attachment Figure 3a (ESR) it can be known that the copper manganese MOF loaded with quercetin has the strongest ability to generate ROS under medical US. In addition, by the attachment Figure 3b it can be known that the copper MOF has the strongest antibacterial ability without ultrasound (i.e. after degradation), and the copper manganese MOF loaded with quercetin is second. While the coating plate experiment shows (attachment Figure 3c ) that the copper manganese MOF loaded with quercetin has the strongest antibacterial ability under the action of US.

[0100] 4. Detection of biological antioxidant, i.e. immune regulation ability

[0101] By flow cytometry (attachment Figure 4a ) it can be known that the nanosheet has strong adsorption ability of ROS, and the copper manganese MOF loaded with quercetin has the strongest adsorption ability of ROS, which can protect BMSCs from the harm of ROS. In addition, by the attachment Figure 4b i.e. immunofluorescence, it can be known that the nanosheet has strong immune regulation ability, and the copper manganese MOF loaded with quercetin has the strongest ability to promote the polarization of macrophages M2.

[0102] 5. Detection of osteogenic and angiogenic ability

[0103] By the attachment Figures 5a-5b i.e. immunofluorescence, ARS and ALP staining, it can be known that the nanosheet has certain osteogenic ability, and the copper manganese MOF loaded with quercetin has the strongest osteogenic ability, and by the attachment Figure 5c i.e. scratch experiment, it can be known that the copper manganese MOF loaded with quercetin has stronger angiogenic ability.

[0104] 6. Treatment of rat skull infectious critical bone defect

[0105] By surface electron microscopy (SEM, attachment Figure 6a ) it can be known that the hydrogel containing copper manganese MOF is successfully prepared, and the surface morphology is analyzed by SEM, and it is found that the nanomicelles can be aggregated and cross-linked. By the attachment Figures 6b-6d it can be known that the nanosheet has strong antibacterial ability in vivo, and the effect is better than that of traditional antibiotic treatment. By the attachment Figures 6e-6f it can be known that the nanosheet has the strongest osteogenic ability after 28 days, and the effect is better than that of traditional antibiotic treatment.

[0106] In summary, the two-dimensional copper-manganese bimetallic MOF loaded with quercetin has strong and persistent antibacterial, anti-inflammatory, and osteogenic ability, and can efficiently and persistently promote the repair of bone tissue. Compared with traditional biodegradable materials, the present application can exert efficient SDT effect through the bimetallic active site before the material degrades, and can exert efficient anti-inflammatory effect through the adsorption of ROS by the bimetallic active site. After the material degrades, the present application can release copper-manganese ions and quercetin to exert persistent antibacterial and osteogenic effects. In addition, since copper-manganese ions and quercetin can be chelated, they can greatly improve the antioxidant capacity of quercetin itself, thereby exerting a persistent effect. The present application is safe, efficient, simple to prepare, can provide personalized treatment for patients, and has the potential for large-scale preparation and application.

[0107] The two-dimensional bimetallic MOF loaded with quercetin prepared by the present application has the following advantages compared with the prior art for treating infected bone defects:

[0108] Compared with the patent with application number CN202211401896.5, a high-efficiency antibacterial and osteogenic defect MOF sonosensitizer and its preparation method, the two-dimensional bimetallic MOF provided by the present application has long-lasting antibacterial and antioxidant capacity, and has a long duration. Moreover, due to its ability to adsorb ROS, it has less harm to the surrounding tissue and has stronger biocompatibility.

[0109] Compared with the patent with application number CN202210246313.X, a zinc-doped calcium phosphate ceramic microsphere with antibacterial function and a preparation method, the present application can regulate local ROS to exert antioxidant effect and better promote the repair of bone tissue.

[0110] Compared with the patent with application number CN202410602481.7, near-infrared response controlled-release NO antibacterial bioceramics and a preparation method, the present application can reduce the harm of sonodynamic therapy to the surrounding tissue and can more efficiently promote the healing of bone tissue.

[0111] Compared with the patent with application number CN202311017016.9, an antibacterial and antioxidant osteogenic multifunctional coating with environmental response and photothermal response, the MOF prepared by the present application has more persistent effect and more excellent bactericidal effect. Since curcumin is easily metabolized and zinc ion slow-release has weaker bactericidal effect than SDT, the present application can more efficiently promote the repair of bone tissue.

[0112] In summary, the application provides an antibacterial, anti-inflammatory and osteogenic two-dimensional bimetallic MOF, which can effectively resist bacteria and inflammation, has good biological safety, and has the ability to act for a long time. Before degradation, the application can effectively remove bacteria under the action of low-intensity medical US, and can play the role of removing ROS through nano-enzyme activity. After degradation, the slow-release copper and manganese ions and quercetin in the application can be chelated, and then through metal-ion charge transfer (MLCT), the SOD and CAT effects can be played, so that the adsorption of ROS can be continuously played. In addition, since copper and manganese ions and quercetin have osteogenic and antibacterial effects, the application can continuously and efficiently promote bone tissue repair. The application not only makes up for the defect that degradable materials cannot continuously act, but also solves the key problem that it is difficult to achieve antibacterial and anti-inflammatory at the same time during the treatment of infected bone defects.

[0113] Finally, it should be noted that the above examples are only used to illustrate the application and not to limit the protection scope of the application. In addition, after reading the technical content of the application, those skilled in the art can make various modifications, modifications or changes to the application, and all these equivalent forms also belong to the protection scope defined by the present application.

Claims

1. A two-dimensional bimetallic MOF with antibacterial, anti-inflammatory and osteogenesis-promoting properties, characterized in that: Includes copper nitrate trihydrate 3.6mg, polyvinylpyrrolidone 40mg, tetra-p-benzoylporphyrin manganese III 17mg, quercetin 6.25mg and N,N-dimethylformamide 48ml; The preparation method comprises the following steps: S1, using copper nitrate trihydrate and tetra-p-benzoic acid porphyrin manganese III as raw materials, copper ions were doped on tetra-p-benzoic acid porphyrin manganese III by a hydrothermal method to prepare nanosheets with copper and manganese dual elements, and then the nanosheets were co-incubated with quercetin to prepare a drug-loaded two-dimensional bimetallic MOF.

2. The antibacterial, anti-inflammatory and osteogenic two-dimensional bimetallic MOF according to claim 1, characterized in that: The method further comprises the following steps: S2, structural and compositional analysis of 2D bimetallic MOFs; S3, antioxidant performance and mechanism analysis of 2D bimetallic MOF before degradation; S4, antioxidant performance and mechanism analysis of the degraded 2D bimetallic MOF; S5, the antibacterial properties of the two-dimensional bimetallic MOF before and after degradation were verified by in vitro antibacterial experiments; S6, the biosafety of the 2D bimetallic MOF was tested by BMSCs co-culture and hemolysis experiments; S7, using transcriptomics and single-cell transcriptomics methods to explore the biological mechanisms of osteogenesis and angiogenesis after co-culture of 2D bimetallic MOF with BMSCs and HUVECs, and verifying these mechanisms in vitro by Western Blot analysis; S8, flow cytometry and immunofluorescence were used to detect the ability of the two-dimensional bimetallic MOF to adsorb ROS in vitro and after cell co-culture, and to verify its effect on promoting M2 differentiation of THP-1 cells; S9, prepare an injectable hydrogel containing two-dimensional bimetallic MOF and construct a rat model of skull infection defect to verify the antibacterial and anti-inflammatory osteogenesis efficiency in vivo.

3. The antibacterial, anti-inflammatory and osteogenesis-promoting two-dimensional bimetallic MOF according to claim 1, characterized in that: Step S1 of the method is specifically: Prepare raw materials including 3.6 mg of copper nitrate trihydrate, 40 mg of polyvinylpyrrolidone, 16 mg of tetracarboxyphenylporphine, 17 mg of tetra-p-benzoic acid porphyrin manganese III, 6.25 mg of quercetin and 48 ml of N, N-dimethylformamide; first, add 17 mg of tetra-p-benzoic acid porphyrin manganese III to 48 ml of N, N-dimethylformamide and stir to dissolve the solution until there are no particles; then prepare copper nitrate trihydrate solution, take 3.6 mg of copper nitrate trihydrate and add it to 16 ml of ethanol and stir; then prepare 1 M trifluoroacetic acid solution, take 160 μl of trifluoroacetic acid solution and add it to the above copper nitrate trihydrate solution and stir. Stir, and add 40 mg of polyvinyl pyrrolidone to the solution and stir evenly; finally, the copper nitrate trihydrate solution and the tetra-benzoic acid porphyrin manganese III solution are mixed and stirred evenly, and the resulting solution is placed in a muffle furnace and heated to 80°C at 2°C / min, and kept warm for 4 hours, and then the product is washed with ethanol and centrifuged 3 times, with a centrifugal speed of 9000 rpm, each time for 10 minutes, and finally dried to obtain a two-dimensional bimetallic MOF powder sample, the obtained two-dimensional bimetallic MOF powder is dissolved in 25 ml of ethanol solution, 6.25 mg of quercetin is added and stirred at room temperature for more than 12 hours, and the obtained solution is finally dried to obtain the final product.

4. The antibacterial, anti-inflammatory and osteogenic two-dimensional bimetallic MOF according to claim 2, characterized in that: The step S2 specifically includes: observing the morphology of MOF through a field emission scanning electron microscope (SEM); observing the surface structure of MOF through a high-resolution transmission electron microscope (TEM); detecting the surface element composition and chemical bonds of MOF through X-ray photoelectron spectroscopy (XPS); observing the surface element composition and distribution of MOF through X-ray energy spectrum elemental analysis technology (EDS); detecting the thickness of the material through an atomic force microscope (AFM); and revealing the crystal structure of MOF through X-ray diffractometer technology (XRD).

5. The antibacterial, anti-inflammatory and osteogenic two-dimensional bimetallic MOF according to claim 2, characterized in that: The step S3 specifically includes: using XPS, SOD and CAT kits to verify the ability of the two-dimensional copper-manganese MOF to adsorb ROS before degradation, and revealing its chemical mechanism of adsorbing ROS through metal active sites through theoretical calculations.

6. The antibacterial, anti-inflammatory and osteogenic two-dimensional bimetallic MOF according to claim 2, characterized in that: The step S4 specifically includes: using XPS, SOD and CAT kits to detect the ability of the two-dimensional copper-manganese MOF to adsorb ROS after degradation, and revealing the chemical mechanism of the antioxidant effect of quercetin and copper-manganese ion chelation through theoretical calculation.

7. The antibacterial, anti-inflammatory and osteogenesis-promoting two-dimensional bimetallic MOF according to claim 2, characterized in that: Specifically, step S5 includes: using methicillin-resistant Staphylococcus aureus as the research object, verifying the sonodynamic antibacterial performance of the two-dimensional copper-manganese bimetallic MOF through in vitro antibacterial experiments, and co-culturing MOF and bacteria to verify the antibacterial effect of the MOF by releasing ions and quercetin after degradation, and detecting the difference in antibacterial properties between the two-dimensional bimetallic MOF and traditional MOF through SEM and plate coating experiments.

8. The antibacterial, anti-inflammatory and osteogenic two-dimensional bimetallic MOF according to claim 2, characterized in that: The step S7 specifically includes: detecting and verifying the osteogenesis and angiogenesis abilities of MOF by immunofluorescence, PCR, and Western Blot, and revealing the mechanism by which MOF promotes bone tissue repair by sustained ion release through single-cell transcriptomics.

9. The antibacterial, anti-inflammatory and osteogenesis-promoting two-dimensional bimetallic MOF according to claim 2, characterized in that: The step S8 specifically includes: detecting and verifying the ability of MOF to protect cells from ROS and regulate macrophage differentiation through flow cytometry and fluorescence.

10. The antibacterial, anti-inflammatory and osteogenesis-promoting two-dimensional bimetallic MOF according to claim 2, characterized in that: Step S9 specifically includes: taking 20 mg of Jelleine-1, adding 200 microliters of DMSO, mixing and dissolving to obtain 100 mg / mL Jelleine-1, diluting it to 8 mg / ml with 200 μg / ml of two-dimensional bimetallic MOF solution, incubating at 37°C for more than 15 minutes to form a gel, and obtaining an injectable hydrogel. The antibacterial and anti-inflammatory osteogenesis efficiency of the hydrogel was investigated in vivo using a rat critically infected bone defect model.

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