Antibacterial, anti-inflammatory and osteogenesis-promoting two-dimensional bimetallic MOF (Metal Organic Framework) as well as preparation and application thereof

Abstract

The invention provides an antibacterial, anti-inflammatory and osteogenesis-promoting two-dimensional bimetallic MOF (Metal Organic Framework) as well as preparation and application thereof. The two-dimensional bimetal MOF can effectively resist bacteria and inflammation, has good biological safety and long-acting function exerting capacity, can efficiently remove bacteria under the action of low-strength medical US before degradation, and can exert the function of removing ROS through nano-enzyme activity. After degradation, copper and manganese ions slowly released by the two-dimensional bimetal MOF and quercetin can be chelated, SOD and CAT-like effects are achieved through metal-ion charge transfer MLCT, the ROS adsorption effect can be continuously achieved, and in addition, due to the fact that the copper and manganese ions and quercetin have osteogenesis and antibacterial effects, bone tissue repair can be continuously and efficiently promoted.

Description

Technical field:

[0001] The present invention relates to the field of biomedicine, and in particular to an antibacterial, anti-inflammatory and osteogenesis-promoting two-dimensional bimetallic MOF and its preparation and application. Background technology:

[0002] Infected bone defects are one of the thorny problems encountered in orthopedic clinical work. Due to local bacterial infection, bone tissue heals slowly, which will bring a heavy burden to patients and society. The long-term presence 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 infected bone defects requires solving both bacterial infection and inflammatory microenvironment. Although traditional antibiotics and surgical treatments can inhibit bacterial infection, they are not effective in improving the inflammatory microenvironment of the infection focus, and the large-scale use of antibiotics will induce bacterial resistance, and surgical treatment also has the risk of aggravating bone defects. Based on this, seeking a method of efficient sterilization and inhibition of local high inflammatory response is an urgent need for infected bone defects.

[0003] Sonodynamic therapy (SDT) is a non-invasive and highly effective bacterial removal strategy. Its 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 a variety of mechanisms, including disrupting the electron transport chain, causing bacteria to die due to oxidative stress and impaired energy metabolism. However, although ROS can effectively kill bacteria, they also have adverse effects on surrounding normal cells and tissues. ROS can aggravate inflammation by promoting macrophage M1 polarization. In addition, ROS can also inhibit osteogenic differentiation by inducing mitochondrial dysfunction in stem cells. In short, ROS is a double-edged sword. Nanozymes are catalytic nanomaterials with enzyme-like properties. They can mimic the structure of natural antioxidant enzymes, play the role of superoxide dismutase (SOD) and catalase (CAT), and then catalyze ROS to generate water and oxygen. Nanozymes are widely used due to their cost-effectiveness, stability, adjustable catalytic activity and ease of large-scale production. However, developing materials that can both generate and scavenge ROS is a challenge for the treatment of infected bone defects. In addition, since difficult-to-degrade materials can hinder bone tissue healing and aggravate inflammation, selecting materials with good biocompatibility is crucial for the treatment of infected bone defects.

[0004] At present, many materials with good biocompatibility are used to treat infected bone defects. Organic sonosensitizers such as porphyrin and its derivatives, 5-aminolevulinic acid, phthalocyanine, etc. have the characteristics of high SDT efficiency and fast degradation and are therefore widely used. In addition, degradable nanozymes, such as MnPcBc, Prussian blue and phosphate-based glass (PBG), have also been widely used in antioxidant research. However, although good biocompatibility is beneficial to prevent tissue damage and local inflammation caused by material implantation, it is a major challenge for materials with good degradation properties to exert long-term effects. Since sonosensitizers or nanozymes 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 prolong the function of degradable materials, the drug itself may still be metabolized by cells, resulting in loss of efficacy. Therefore, the development of 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 defective MOF sonosensitizer with high efficiency in antibacterial and osteogenesis and its preparation method. The patent uses sodium alendronate doped with zirconium-based porphyrin MOF, which can promote sterilization by modifying the properties of the sonosensitizer and promote osteogenesis by using acoustic current. However, the MOF synthesized by this method is easily degraded and does not have long-term antibacterial and osteogenesis effects. In addition, since the ROS generated by acoustic power will damage the activity of surrounding cells, it may aggravate the progression of local inflammation and is not conducive to the repair of bone tissue.

[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 the calcium phosphate matrix to form a composite microsphere. The zinc ions can be slowly degraded and released by calcium phosphate, ensuring the long-term antibacterial activity of the biomaterial. However, although the sustained release of zinc ions can play a long-term antibacterial role, the continued progression of inflammation in the local bone defect will seriously affect the repair of bone tissue, which is not conducive to the treatment of bone defects.

[0007] The patent with application number CN202410602481.7 discloses near-infrared responsive controlled release of NO antibacterial bioceramics and preparation methods. The patent combines tetracalcium phosphate, calcium hydrogen phosphate dihydrate, Arg-ZnPc@mesoporous polydopamine microspheres and sodium alginate solution to prepare a bioremediation material that can respond to infrared light, which can exert a long-term antibacterial effect locally by controlling the release of NO. However, the release of NO will aggravate the inflammation of surrounding cells, which is not conducive to bone tissue repair.

[0008] The patent with application number CN202311017016.9 discloses an antibacterial, antioxidant and osteogenic multifunctional coating with environmental response and photothermal response. The patent consists of porous titanium dioxide grown in situ on the surface of titanium or titanium alloy and organic metal framework ZIF-8 nanoparticles with composite curcumin, which can promote bactericidal effect by releasing zinc ions under near-infrared light irradiation, and can also promote anti-inflammation 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 lasting antibacterial and anti-inflammatory effect.

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

[0010] 1. Treatment of infected bone defects requires solving two problems: bacterial infection and inflammatory response. Existing treatments such as antibiotics or surgery can effectively solve bacterial infections, but the local high inflammatory response will inhibit the repair of bone tissue. Long-term use of antibiotics will promote drug resistance, and surgical treatment also has the risk of aggravating bone defects.

[0011] 2. ROS is a double-edged sword that can play an antibacterial role by destroying bacterial biofilms. However, ROS can also damage surrounding cells, thereby aggravating the inflammation of surrounding cells. Therefore, the ideal treatment strategy should be to use ROS to kill bacteria while exerting an anti-inflammatory effect by absorbing excess ROS during the bone tissue repair stage.

[0012] 3. The mechanism of SDT treatment is to stimulate electron transfer in sonosensitizers through exogenous ultrasound, thereby generating a large amount of ROS. It is difficult to kill local bacteria by relying solely on SDT, and excessive ROS will also cause oxidative stress and aggravate the inflammation of surrounding cells.

[0013] 4. Nanozymes are nanomaterials with enzyme activity, which can exert anti-inflammatory effects by adsorbing ROS through active sites. However, traditional nanozymes have poor effects due to their small number of active sites, and it is currently difficult to prepare materials that have both nanozyme activity and sonosensitizer properties.

[0014] Although traditional biodegradable materials have the advantage of good biocompatibility, they are easy to degrade, which limits their duration of action. Since traditional antibacterial and anti-inflammatory biomaterials rely on drugs or the active sites of the materials themselves to work, their effects will be greatly reduced once they are degraded. Therefore, it is necessary to improve the current preparation methods of biodegradable materials and seek an improvement strategy that can ensure that the materials have good biodegradability while also being able to continue to work. Summary of the invention:

[0015] 1. Technical issues to be solved

[0016] The present invention aims at the problem that it is impossible to simultaneously fight against bacteria and inflammation during the treatment of infected bone defects and that existing biomaterials are difficult to play a long-term role, and proposes the preparation and application of a two-dimensional bimetallic MOF with long-term antibacterial, anti-inflammatory and osteogenesis. The MOF can effectively fight against bacteria and inflammation, and in addition has good biosafety and the ability to play a long-term role. Before degradation, the invention can efficiently remove bacteria under the action of low-intensity medical US, and can play a role in removing ROS through nanozyme activity. After degradation, the copper-manganese ions and quercetin released by the invention can be chelated, and then play a SOD and CAT-like effect through metal-ion charge transfer (MLCT), so that the role of adsorbing ROS can be continuously played. In addition, since copper-manganese ions and quercetin have osteogenesis and antibacterial effects, the invention can continuously and efficiently promote bone tissue repair. The present invention not only makes up for the defect that degradable materials cannot play a role continuously, but also solves the key problem that it is difficult to achieve antibacterial and anti-inflammatory effects simultaneously during the treatment of infected bone defects.

[0017] (II) Technical solution

[0018] In order to solve the above technical problems, the present invention adopts the following technical solutions:

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

[0020] Since copper ions replace manganese ions on manganese porphyrin, nanosheets with dual active sites are formed. Both Cu2O8 and MnN4 dual active sites have the ability to perform SDT and adsorb 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 is degraded. Therefore, the two-dimensional bimetallic MOF can continuously exert antibacterial and anti-inflammatory effects and efficiently promote the repair of bone tissue.

[0021] The present invention also discloses a method for preparing a two-dimensional bimetallic MOF with antibacterial, anti-inflammatory and bone-promoting properties:

[0022] Step S1, preparing copper MOF and two-dimensional copper-manganese-doped bimetallic MOF; using copper nitrate trihydrate and tetra-p-benzoic acid porphyrin manganese III as raw materials, using a hydrothermal method to dope copper ions on tetra-p-benzoic acid porphyrin manganese III to prepare nanosheets with copper and manganese dual elements, and then co-incubating the nanosheets with quercetin to prepare a drug-loaded two-dimensional bimetallic MOF.

[0023] The above preparation method also includes the following steps:

[0024] Step S2, structural and composition analysis of two-dimensional bimetallic MOF;

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

[0026] Step S4, analyzing the antioxidant properties and mechanism of the two-dimensional bimetallic MOF after degradation;

[0027] Step S5, verifying the antibacterial properties of the two-dimensional bimetallic MOF before and after degradation through in vitro antibacterial experiments; Step S6, detecting the biosafety of the two-dimensional bimetallic MOF through BMSCs co-culture and hemolysis experiments;

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

[0029] Step S8, detecting the ability of the two-dimensional bimetallic MOF to adsorb ROS after in vitro co-culture with cells by various means such as flow cytometry and immunofluorescence, and verifying its effect of promoting M2 differentiation of THP-1 cells;

[0030] Step S9, prepare an injectable hydrogel containing a two-dimensional bimetallic MOF and construct a rat model of infected skull defect to verify the antibacterial and anti-inflammatory osteogenesis efficiency in vivo.

[0031] In the above step S1, specifically: prepare raw materials including 3.6-14.4 mg of copper nitrate trihydrate, 40 mg of polyvinyl pyrrolidone, 16 mg of tetracarboxyl phenyl porphine, 17 mg of tetraphenyl porphyrin manganese III, 6.25 mg of quercetin and 48 ml of N, N-dimethylformamide. First, add 17 mg of tetraphenyl porphyrin manganese III to 48 ml of N, N-dimethylformamide and stir to dissolve until the solution has no particles. Then prepare copper nitrate trihydrate solution, take 3.6-14.4 mg of copper nitrate trihydrate and add it to 16 ml of ethanol and stir. Secondly, after preparing 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 polyvinyl pyrrolidone to the solution and stir evenly. Finally, mix the copper nitrate trihydrate solution and the tetraphenyl porphyrin manganese III solution and stir evenly. The solution was placed in a muffle furnace and heated to 80°C at 2°C / min and kept warm for 4 hours. The product was then washed and centrifuged 3 times with ethanol (9000rpm, 10 minutes each time), and finally dried to obtain ~25mg of two-dimensional bimetallic MOF powder. The obtained two-dimensional bimetallic MOF powder was dissolved in 25ml of ethanol solution, 6.25mg of quercetin was added and stirred at room temperature for more than 12 hours, and the obtained solution was finally dried to obtain the final product.

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

[0033] To prepare a control group, i.e., copper tetracarboxyphenylporphyrin without manganese, 16 mg of copper tetracarboxyphenylporphyrin was added to 48 ml of N, N-dimethylformamide, and finally added to the above-prepared copper nitrate trihydrate, trifluoroacetic acid, and polyvinyl pyrrolidone mixture. The solution was placed in a muffle furnace and heated to 80°C at 2°C / min and kept warm for 4 hours. The product was then washed with ethanol and centrifuged 3 times (9000 rpm, 10 minutes each time), and finally dried to obtain a 25 mg copper tetracarboxyphenylporphyrin sample.

[0034] In step S2, specifically, 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 spectrum elemental imaging 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 diffractometer technology (XRD).

[0035] In step S3, the ability of the two-dimensional copper-manganese MOF to adsorb ROS before degradation is verified by 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 calculations.

[0036] In step S4, XPS, SOD and CAT kits are used to detect the ability of the two-dimensional copper-manganese MOF to adsorb ROS after degradation, and the chemical mechanism of the antioxidant effect of quercetin and copper-manganese ion chelation is revealed through theoretical calculations.

[0037] In step S5, methicillin-resistant Staphylococcus aureus was used as the research object, and the sonodynamic antibacterial performance of the two-dimensional copper-manganese bimetallic MOF was verified by in vitro antibacterial experiments, and the MOF and bacteria were co-cultured to verify the antibacterial effect of the degradation by releasing ions and quercetin. The difference between the two-dimensional bimetallic MOF and the traditional MOF in antibacterial properties was detected by various experimental methods such as SEM and plate coating.

[0038] In step S7, the ability of MOF to promote osteogenesis and angiogenesis is detected and verified by immunofluorescence, PCR, Western Blot and other methods, and the mechanism of MOF slow-release ions promoting bone tissue repair is revealed by single-cell transcriptome.

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

[0040] In step S9, specifically, 20 mg of Jelleine-1 was taken, 200 microliters of DMSO was added, and 100 mg / mL of Jelleine-1 was obtained by mixing and dissolving, and then diluted to 8 mg / ml with 200 μg / ml of two-dimensional bimetallic MOF solution, and incubated at 37°C (more than 15 min) to obtain an injectable hydrogel. The antibacterial and anti-inflammatory osteogenesis efficiency was explored in vivo using a rat critically infected bone defect model.

[0041] (III) Beneficial effects

[0042] (1) Excellent antibacterial effect:

[0043] The bimetallic two-dimensional MOF prepared by the present invention is a highly efficient sonosensitizer. Due to its dual active sites, it can greatly enhance the electron transfer ability compared with traditional antibacterial materials. Under the action of ultrasound, the MOF will efficiently promote electron transfer to generate ROS, thereby exerting a bactericidal effect. In addition, after the material is degraded, the material will release copper manganese ions and quercetin. Since copper manganese ions and quercetin both have certain bactericidal effects, this material has a lasting antibacterial effect compared with traditional MOF.

[0044] (2) Highly effective anti-inflammatory:

[0045] The bimetallic two-dimensional MOF prepared by the present invention has dual active sites of Cu2O8 and MnN4, and both active sites have SOD-like and CAT-like effects. Before degradation, the material can adsorb ROS to exert an antioxidant effect. 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 will be enhanced through MLCT. Therefore, the present invention can continuously and efficiently exert an antioxidant effect, thereby promoting the treatment of local inflammation.

[0046] (3) Can effectively promote bone tissue repair:

[0047] The bimetallic two-dimensional MOF prepared by the present invention can promote osteogenesis and angiogenesis through drug and ion sustained release. Since quercetin and copper ions have certain effects of stimulating angiogenesis, and copper-manganese ions have certain osteogenesis ability, the prepared copper-manganese MOF can effectively promote the regeneration of bone tissue, thereby restoring the reconstruction of bone tissue more quickly.

[0048] (4) Better biosafety:

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

[0050] (5) Avoid bacterial resistance problems:

[0051] The bimetallic two-dimensional MOF prepared by the present invention can be antibacterial through ions, drugs and sonic power, and can kill bacteria by destroying bacterial membranes and electron transport chains, etc. It is not easy to produce drug resistance, thus avoiding the problems caused by the large-scale use of antibiotics.

[0052] (6) Treatment can be provided on demand in the time dimension:

[0053] Since the bimetallic two-dimensional MOF prepared by the present invention is antibacterial through sonodynamics, exogenous ultrasound is the key to activating the antibacterial properties of the material. In the clinical treatment process, the application of this material can formulate a more personalized treatment plan according to the patient's stage of progression. When facing different patients, the application of this material can adjust different parameters such as power, ultrasound time, etc., thereby sterilizing while minimizing damage to normal tissues.

[0054] (7) Treatment can be precisely positioned in the spatial dimension:

[0055] Since the present invention uses hydrogel as a carrier, after implantation into the patient's lesion, the ultrasound method can be used to more accurately locate the lesion, while minimizing the impact on surrounding tissues during sonodynamic sterilization. (8) It has the potential for large-scale production:

[0056] Since the raw materials used in the present invention are widely available and 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 cause harm to operators during the production process, and has the potential for large-scale production and promotion. Description of the drawings:

[0057] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments are 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 This is the EDS spectrum of the copper-manganese bimetallic MOF;

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

[0062] Figure 2b The ESR of quercetin and quercetin-ion chelate adsorbing ROS;

[0063] Figure 3a ESR of ROS produced by Cu MOF, Cu-Mn bimetallic MOF, and Cu-Mn bimetallic MOF loaded with quercetin under US;

[0064] Figure 3b The antibacterial curves of copper MOF, copper-manganese bimetallic MOF and copper-manganese bimetallic MOF loaded with quercetin without adding US;

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

[0066] Figure 3d The theoretical calculation results of the anti-oxidation of copper-manganese bimetallic MOF before degradation;

[0067] Figure 3e The theoretical calculation results of the anti-oxidation of copper-manganese bimetallic MOF before degradation;

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

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

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

[0071] Figure 5c Plate streaking diagram showing that copper MOF, copper-manganese bimetallic MOF, and copper-manganese bimetallic MOF loaded with quercetin promoted HUVECs migration after co-culture with HUVECs for 3 days;

[0072] Figure 6a The SEM and appearance photos of the copper-manganese bimetallic MOF hydrogel loaded with quercetin;

[0073] Figure 6b This is the appearance of the wound 14 days after the rat skull defect model was created;

[0074] Figure 6c The Gram staining images of rats after 7 and 14 days of skull defect modeling;

[0075] Figure 6d HE staining of rat skull defect model 14 days later;

[0076] Figure 6e-6f CT reconstruction and BV / TV statistics of rat skull defect model 28 days later. Specific implementation method:

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

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

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

[0080] The preparation and application of a two-dimensional bimetallic MOF with long-lasting antibacterial, anti-inflammatory and osteogenesis-promoting properties specifically include the following steps:

[0081] Step 1, preparation of copper MOF and two-dimensional copper-manganese doped bimetallic MOF: prepare raw materials including 3.6 mg of copper nitrate trihydrate, 40 mg of polyvinyl pyrrolidone, 16 mg of tetracarboxyphenylporphine, 17 mg of tetraphenylporphyrin manganese (III), 6.25 mg of quercetin and 48 ml of N, N-dimethylformamide. First, add 17 mg of tetraphenylporphyrin manganese (III) to 48 ml of N, N-dimethylformamide and stir to dissolve until the solution has 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. Secondly, after preparing 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 polyvinyl pyrrolidone to the solution and stir evenly. Finally, mix the copper nitrate trihydrate solution and the tetraphenylporphyrin manganese (III) solution and stir evenly. The solution was placed in a muffle furnace and heated to 80°C at 2°C / min and kept warm for 4 hours. The product was then washed and centrifuged 3 times with ethanol (9000rpm, 10 minutes each time), and finally dried to obtain ~25 mg of sample. The obtained two-dimensional bimetallic MOF powder was dissolved in 25 ml of ethanol solution, 6.25 mg of quercetin was added and stirred at room temperature for more than 12 hours, and the obtained solution was finally dried to obtain the final product.

[0082] By changing the content of copper nitrate trihydrate added, a two-dimensional bimetallic MOF containing different copper and manganese can be prepared. In this example, 7.2 mg and 14.4 mg of copper nitrate trihydrate were used as raw materials, and other raw materials and steps remained unchanged to prepare a two-dimensional bimetallic MOF containing different copper and manganese.

[0083] To prepare a control group, i.e., copper tetracarboxyphenylporphyrin without manganese, 16 mg of copper tetracarboxyphenylporphyrin was added to 48 ml of N,N-dimethylformamide, and finally added to the above-prepared copper nitrate, trifluoroacetic acid, and polyvinyl pyrrolidone mixture. The solution was placed in a muffle furnace and heated to 80°C at 2°C / min and kept warm for 4 hours. The product was then washed with ethanol and centrifuged 3 times (9000 rpm, 10 minutes each time), and finally dried to obtain a 25 mg copper tetracarboxyphenylporphyrin sample.

[0084] Step 2, structural and composition analysis of two-dimensional bimetallic MOF: observe the general morphology of MOF by field emission scanning electron microscopy (SEM); observe the surface structure of MOF by high-resolution transmission electron microscopy (TEM); detect the surface element composition and chemical bonds of MOF by X-ray photoelectron spectroscopy analysis (XPS); observe the surface element composition and distribution of MOF by X-ray energy spectrum elemental imaging analysis technology (EDS); detect the thickness of the material by atomic force microscopy (AFM); and reveal the crystal structure of the nanosheets by X-ray diffractometer technology (XRD).

[0085] Step 3. Analysis of the antioxidant properties and mechanism of the two-dimensional bimetallic MOF before degradation: XPS, SOD, CAT kits and other methods were used to verify the ability of the two-dimensional copper-manganese MOF to adsorb ROS before degradation, and theoretical calculations were used to reveal the chemical mechanism of the nanosheets adsorbing ROS through metal active sites.

[0086] Step 4: Analysis of the antioxidant properties and mechanisms of the two-dimensional bimetallic MOF after degradation: Specifically, XPS, SOD and CAT kits were used to detect the ability of the two-dimensional copper-manganese MOF to adsorb ROS after degradation, and theoretical calculations were used to reveal the chemical mechanism by which quercetin and copper-manganese ion chelation exert antioxidant effects.

[0087] Step 5: Using methicillin-resistant Staphylococcus aureus as the research object, the sonodynamic antibacterial performance of the two-dimensional copper-manganese bimetallic MOF was verified through in vitro antibacterial experiments, and MOF and bacteria were co-cultured to verify the antibacterial effect of ions and quercetin released after degradation. The difference between the antibacterial properties of the two-dimensional bimetallic MOF and traditional MOF was detected through various experimental methods such as SEM and plate coating.

[0088] Step 6: Detect the biosafety of the two-dimensional bimetallic MOF through BMSCs co-culture and hemolysis experiments.

[0089] Step 7: Detect and verify the osteogenesis and angiogenesis capabilities of MOF through immunofluorescence, PCR, Western Blot and other methods, and reveal the mechanism by which MOF slow-release ions promote bone tissue repair through single-cell transcriptomics.

[0090] Step 8: Specifically, the ability of MOF to protect cells from ROS and regulate macrophage differentiation is detected and verified through flow cytometry, fluorescence and other methods.

[0091] Step 9: Take 20 mg of Jelleine-1, add 200 microliters of DMSO, mix and dissolve to obtain 100 mg / mL Jelleine-1, dilute to 8 mg / ml with 200 μg / ml two-dimensional bimetallic MOF solution, incubate at 37°C (more than 15 minutes) to gel, and obtain injectable hydrogel. Using the critical infected bone defect model of rats, the antibacterial and anti-inflammatory osteogenesis efficiency was explored in vivo.

[0092] The present invention adopts a hydrothermal method to synthesize a copper-manganese bimetallic MOF, and the material has long-lasting antibacterial, anti-inflammatory, and osteogenesis effects. Before degradation, it can play the role of SDT and adsorb ROS through dual active sites. After degradation, the released copper-manganese ions and quercetin can be chelated, thereby enhancing the antioxidant effect of quercetin itself. In addition, since copper-manganese ions and quercetin have certain antibacterial and anti-inflammatory abilities, the material developed by the present invention has excellent biocompatibility and long-lasting antibacterial and anti-inflammatory abilities.

[0093] The performance of the product prepared in the embodiment is described below by taking it as an example.

[0094] 1. Material Characterization

[0095] (1) By attaching Figure 1a Transmission electron microscopy (TEM) shows that copper MOF and copper manganese MOF have ultra-thin nanosheet structures, with sizes of approximately 1-2 μm and 2-3 μm, respectively. Figure 1b The average thickness of MON(Cu) measured by atomic force microscopy (AFM) is about 2.9nm). The thickness of copper MOF is comparable to that of copper manganese MOF, which is about 2.7nm. Figure 1c Energy dispersive spectroscopy (EDS) analysis showed that the elements on the surface of CuMn MOF were uniformly distributed, indicating that the nanosheets were successfully prepared.

[0096] 2. Antioxidant performance testing and mechanism analysis

[0097] By attaching Figure 2a Electron paramagnetic resonance (ESR) showed that the copper-manganese MOF loaded with quercetin had the strongest ROS adsorption capacity and could catalyze ROS to generate water and oxygen. Figure 2b It can be seen that the degraded copper-manganese MOF loaded with quercetin has the ability to adsorb ROS, and the effect is stronger than quercetin. Theoretical calculations show that the MnN4 site of the copper-manganese bimetallic MOF before degradation has stronger activity than the traditional CuN4 site, and can play the role of SOD and CAT by adsorbing ROS. The chelation 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.

[0098] 3. Antibacterial performance testing

[0099] By attaching Figure 3a (ESR) showed that the copper-manganese MOF loaded with quercetin had the strongest ability to generate ROS under medical US. Figure 3b It can be seen that the copper MOF has the strongest antibacterial ability without ultrasound (i.e. after degradation), while the copper-manganese MOF loaded with quercetin is second. The coating experiment shows that (attached Figure 3c ) The copper-manganese MOF loaded with quercetin has the strongest antibacterial ability under the action of US.

[0100] 4. Biological antioxidant and immune regulation ability detection

[0101] By flow cytometry ( Figure 4a ) It can be seen that the nanosheets have a strong ability to adsorb ROS, and the copper-manganese MOF loaded with quercetin has the strongest ability to adsorb ROS, which can protect BMSCs from the harm of ROS. Figure 4b That is, immunofluorescence showed that the nanosheets had strong immune regulatory ability, and the copper-manganese MOF loaded with quercetin had the strongest ability to promote macrophage M2 polarization.

[0102] 5. Osteogenesis and angiogenesis ability testing

[0103] By attaching Figure 5a-5b That is, immunofluorescence, ARS and ALP staining showed that the nanosheets had a certain osteogenic ability, and the copper-manganese MOF loaded with quercetin had the strongest osteogenic ability. Figure 5c That is, the scratch experiment shows that the copper-manganese MOF loaded with quercetin has stronger angiogenic ability.

[0104] 6. Treatment of infected critical bone defects in rat skull

[0105] By surface electron microscopy (SEM, attached Figure 6a ) It can be seen that the hydrogel containing copper-manganese MOF was successfully prepared, and the surface morphology was analyzed by SEM, and it was found that the nanomicelles could aggregate and cross-link. Figure 6b-6d It can be seen that the nanosheets have strong antibacterial ability in the body, and the effect is better than traditional antibiotic treatment. Figure 6e-6f It can be seen that the nanosheets have the strongest bone-forming ability after 28 days, and the effect is better than traditional antibiotic treatment.

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

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

[0108] Compared with the patent with application number CN202211401896.5, a defective MOF sonosensitizer with high efficiency antibacterial and osteogenesis and its preparation method, the two-dimensional bimetallic MOF provided by the present invention has long-lasting antibacterial and antioxidant capabilities, and due to its ability to adsorb ROS, it has little harm to surrounding tissues 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 invention can regulate local ROS, thereby exerting an antioxidant effect and better promoting the repair of bone tissue.

[0110] Compared with the patented near-infrared responsive controlled-release NO antibacterial bioceramics and preparation method with application number CN202410602481.7, the present invention can reduce the harm of acoustic dynamics to surrounding tissues, and can promote the healing of bone tissue more efficiently.

[0111] Compared with the environmentally responsive and photothermally responsive antibacterial, antioxidant and osteopromoting multifunctional coating of the patent application number CN202311017016.9, the MOF prepared by the present invention has a more lasting effect and a better bactericidal effect. Since curcumin is easily metabolized and the sustained release of zinc ions is weaker than the bactericidal effect of SDT, the present invention can promote bone tissue repair more efficiently.

[0112] In summary, the present invention provides a two-dimensional bimetallic MOF that is antibacterial, anti-inflammatory and promotes osteogenesis, which can effectively resist bacteria and inflammation, and has the ability to exert its effect for a long time while having good biosafety. Before degradation, the present invention can efficiently remove bacteria under the action of low-intensity medical US, and can play a role in removing ROS through nanozyme activity. After degradation, the sustained-release copper manganese ions and quercetin of the present invention can be chelated, and then exert SOD and CAT effects through metal-ion charge transfer (MLCT), so that the effect of adsorbing ROS can be continuously exerted. In addition, since copper manganese ions and quercetin have osteogenesis and antibacterial effects, the present invention can continuously and efficiently promote bone tissue repair. The present invention not only makes up for the defect that degradable materials cannot exert their effects continuously, but also solves the key problem that it is difficult to achieve antibacterial and anti-inflammatory effects at the same time during the treatment of infectious bone defects.

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

Claims

1. A two-dimensional bimetallic MOF with antibacterial, anti-inflammatory and bone-promoting properties, characterized in that: It includes copper nitrate trihydrate 3.6 mg, polyvinyl pyrrolidone 40 mg, tetra-p-benzoic acid porphyrin manganese III 17 mg, quercetin 6.25 mg and N, N-dimethylformamide 48 ml; 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 are 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 are co-incubated with quercetin to prepare a drug-loaded two-dimensional bimetallic MOF.

2. The antibacterial, anti-inflammatory and bone-promoting 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 properties and mechanism analysis of 2D bimetallic MOF before degradation; S4, Antioxidant properties and mechanism analysis of 2D bimetallic MOF after degradation; 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 2D bimetallic MOF was tested by BMSCs co-culture and hemolysis experiments; S7, explore the biological mechanism of osteogenesis and angiogenesis after co-culture of 2D bimetallic MOF with BMSCs and HUVECs by transcriptomics and single-cell transcriptomics methods, and verify its mechanism by Western Blot method in vitro; S8, flow cytometry and immunofluorescence methods 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 in promoting M2 differentiation of THP-1 cells; S9, prepare injectable hydrogels containing two-dimensional bimetallic MOFs and construct a rat model of infected skull defect to verify the antibacterial and anti-inflammatory osteogenesis efficiency in vivo.

3. The antibacterial, anti-inflammatory and bone-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 polyvinyl pyrrolidone, 16 mg of tetracarboxyphenylporphine, 17 mg of tetraphenylporphyrin manganese III, 6.25 mg of quercetin and 48 ml of N, N-dimethylformamide; first, add 17 mg of tetraphenylporphyrin manganese III to 48 ml of N, N-dimethylformamide and stir to dissolve until the solution has 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, mix the copper nitrate trihydrate solution and the tetraphenylporphyrin manganese III benzoate solution and stir evenly, put the resulting solution into a muffle furnace and heat it to 80°C at 2°C / min, and keep it warm for 4 hours, then wash the product with ethanol and centrifuge it 3 times, the centrifugal speed is 9000rpm, each time for 10 minutes, and finally dry it to obtain a two-dimensional bimetallic MOF powder 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 more than 12 hours, and finally dry the obtained solution to obtain the final product.

4. The antibacterial, anti-inflammatory and bone-promoting 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 bone-promoting 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 bone-promoting 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 bone-promoting two-dimensional bimetallic MOF according to claim 2, characterized in that: The step S5 is specifically as follows: taking 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 bone-promoting 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 of MOF slow-release ions promoting bone tissue repair by single-cell transcriptome.

9. The antibacterial, anti-inflammatory and bone-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 by flow cytometry and fluorescence.

10. The antibacterial, anti-inflammatory and bone-promoting two-dimensional bimetallic MOF according to claim 2, characterized in that: The step S9 is specifically as follows: take 20 mg of Jelleine-1, add 200 microliters of DMSO, mix and dissolve to obtain 100 mg / mL Jelleine-1, dilute to 8 mg / ml with 200 μg / ml two-dimensional bimetallic MOF solution, incubate at 37°C for more than 15 minutes to form a gel, and obtain an injectable hydrogel. Using the critical infected bone defect of rats as a model, the antibacterial and anti-inflammatory osteogenesis efficiency is explored in vivo.

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