Osteoarthritis treatment medicine based on ZIF-8 composite molybdenum nano-enzyme and CaCO3 layer coating and preparation method thereof

Abstract

The invention discloses an osteoarthritis treatment medicine based on ZIF-8 composite molybdenum nano-enzyme and CaCO3 layer coating and a preparation method thereof, and particularly relates to the field of biomedicine.The surface of a medicine carrier is coated with a CaCO3 layer, the antioxidant and anti-inflammatory effects of composite molybdenum nano-enzyme are combined, targeted delivery and continuous release of the medicine can be achieved, the treatment effect is remarkably improved, and the osteoarthritis treatment medicine can be used for treating osteoarthritis. By means of targeted delivery and controlled release mechanism, side effects of drugs, especially burdens on gastrointestinal tracts, livers, kidneys and other organs, can be significantly reduced, good safety is achieved, and continuous release of the drugs can be achieved by coating the degradable CaCO3 layer. Under the acid environment of a joint inflammation area, the CaCO3 coating is gradually dissolved, the embedded medicine and the nano-enzyme are released, and long-acting treatment of the medicine is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a drug carrier based on a ZIF-8 metal organic framework (MOF) composite molybdenum nanozyme, and controls the release of the drug through a CaCO3 coating. The drug carrier is mainly used to treat osteoarthritis (OA) and other joint-related diseases, and belongs to the field of nano drug delivery technology and biomedical engineering. Background Art

[0002] Osteoarthritis (OA) is the most common joint disease in the world, characterized by articular cartilage degeneration, bone hyperplasia and local chronic inflammation. Patients usually suffer from pain, stiffness and motor dysfunction. Existing treatments include nonsteroidal anti-inflammatory drugs (NSAIDs), hormone drugs and some surgical treatments. Although these treatments can temporarily relieve symptoms, they cannot cure the disease and are often accompanied by side effects. Long-term use may lead to gastrointestinal bleeding, kidney damage and other problems. Therefore, finding a treatment plan that can target the inflammatory area, slow cartilage degeneration, and effectively control drug release has become a current research hotspot. Summary of the invention

[0003] In order to overcome the above-mentioned defects of the prior art, the embodiments of the present invention provide an osteoarthritis treatment drug based on ZIF-8 composite molybdenum nanozyme and CaCO3 layer coating and a preparation method thereof, so as to solve the problems raised in the above-mentioned background technology.

[0004] To achieve the above object, the present invention provides the following technical solution: application of ZIF-8 composite molybdenum nanozyme and CaCO3 layer coating in drugs for treating osteoarthritis.

[0005] ZIF-8 preparation method:

[0006] The specific steps are as follows

[0007] 1). Dissolve Zn(NO3)2·6H2O and 2-methylimidazole in methanol solution at a molar ratio of 1:2 to form a clear solution;

[0008] 2). The solution was transferred to a reactor and reacted at 150°C for 24 hours, and then cooled naturally after the reaction;

[0009] 3). Remove the unreacted substances by centrifugation and filtration to obtain white ZIF-8 powder;

[0010] 4). Finally, the ZIF-8 crystals are dried in a vacuum to obtain ZIF-8 powder.

[0011] Preparation method of composite molybdenum nanozyme,

[0012] The specific steps are as follows

[0013] 1) Dissolve MoO2Cl2 in water or methanol and mix with ZIF-8 precursor;

[0014] 2). Adjust the pH of the reaction solution to 6.0 and react at 50°C for several hours to embed the Mo element into the ZIF-8 framework;

[0015] 3). After the reaction is completed, the excess unreacted products are removed by centrifugation and washing to obtain the ZIF-8@Mo composite nanozyme.

[0016] Preparation method of CaCO3 coating

[0017] The specific steps are as follows

[0018] 1). Suspend the ZIF-8@Mo composite nanozyme in a CaCl2 solution, and add ammonia water to adjust the pH to about 9.0 to promote the precipitation of CaCO3;

[0019] 2) Reacting for 1-2 hours under stirring conditions to obtain ZIF-8@Mo nanozyme particles covered with a CaCO3 coating;

[0020] 3). After the reaction is completed, excess calcium ions are removed by centrifugation and washing to obtain CaCO3@ZIF@Mo composite particles.

[0021] Technical effects and advantages of the present invention:

[0022] The present invention provides a novel osteoarthritis treatment drug based on ZIF-8 composite molybdenum nanozyme and a CaCO3 layer coated thereon, and the beneficial effects thereof are as follows:

[0023] Strong targeting:

[0024] By functionalizing the surface of the drug carrier (such as PEG modification and targeted antibody modification), the drug of the present invention can effectively target the inflammatory area, reduce the side effects of the drug on healthy tissues, and improve the therapeutic effect.

[0025] Controlled Release:

[0026] By coating the degradable CaCO3 layer, sustained drug release can be achieved. In the acidic environment of the joint inflammation area, the CaCO3 coating gradually dissolves, releasing the embedded drugs and nanozymes, achieving long-term drug treatment.

[0027] Antioxidant and Anti-Inflammatory Effects:

[0028] The composite molybdenum nanozyme has a powerful antioxidant effect, which can effectively remove reactive oxygen species (ROS) in the arthritis area, slow down cartilage degeneration, inhibit inflammatory response, and improve joint function.

[0029] Reduce side effects:

[0030] The drug of the present invention can significantly reduce the side effects of the drug, especially the burden on the gastrointestinal tract, liver, kidney and other organs through targeted delivery and controlled release mechanisms, and has good safety.

[0031] The treatment effect is remarkable:

[0032] In vivo experiments show that the drug of the present invention can significantly slow down the degeneration of articular cartilage and reduce inflammatory response in a rat osteoarthritis model, and has a significant improvement effect on the recovery of joint function, and has good clinical application prospects.

[0033] Scope of application

[0034] The osteoarthritis treatment drug provided by the present invention is not only suitable for the clinical treatment of osteoarthritis, but also can be applied to other types of joint diseases, such as rheumatoid arthritis, gout and other inflammatory joint diseases. In addition, due to its targeting and sustained release characteristics, the drug carrier system of the present invention can also be extended to other types of drug delivery, such as targeted treatment of anti-tumor drugs, anti-infective drugs, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the preparation of CaCO3@ZIF@Mo-TA nanozyme and its antioxidant and anti-inflammatory activities in the treatment of osteoarthritis in vivo.

[0036] Figure 2 (A) SEM image of ZIF-8 nanocrystals. (B) TEM image and (C) magnified view of ZIF@Mo-TA nanoparticles. (D) SEM image of CaCO3@ZIF@Mo-TA. (EF) Elemental distribution of Zn and Mo observed using EDX mode.

[0037] Figure 3 XPS spectrum of CaCO3@ZIF@Mo-TA and its corresponding (A) Mo 3d, (B) Zn 2p, (C) O 1s and (D) high-resolution XPS spectra.

[0038] Figure 4 Cytotoxicity evaluation of CaCO3@ZIF@Mo-TA in vitro. Quantitative cell viability of (A) ATDC5 chondrocytes and (B) BMSCs was determined using CCK-8 kit. Live / dead staining of ATDC5 chondrocytes after incubation with (C) 200 μg / mL and (D) 400 μg / mL CaCO3@ZIF@Mo-TA was observed by fluorescence microscopy. Data are expressed as mean ± SD (n = 6).

[0039] Figure 5H&E stained microscopic images of major organs after intravenous injection of 100 mg / kg CaCO3@ZIF@Mo-TA.

[0040] Figure 6 Tissue sections of different treatments (sham / normal, untreated defects, 20 mg / kg injection and 40 mg / kg injection) were stained for H&E staining of knee articular cartilage and Safranin-O Fast Green staining.

[0041] Figure 7 (A) Representative immunofluorescence staining images of TUNEL-positive apoptotic cells in joint tissue sections after different treatments. (B) Representative immunohistochemical staining images of NLRP3 in joint tissue sections after different treatments.

[0042] Figure 8 ESR spectra of CaCO3@ZIF@Mo-TA reacting with (A) ·OH and (B) 1O2. (C-D) DCFH-DA fluorescence images of ROS in ATDC5 chondrocytes after different treatments. Scale bar, 100 μm. Cytokine levels of (G) IL-6, (H) TNF-α, and IL-10 in mouse serum after different treatments according to the ELISA protocol. Data are expressed as mean ± SD (n = 3). DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0044] The osteoarthritis treatment drug shown is based on ZIF-8 composite molybdenum nanozyme and CaCO3 layer coating. By coating the CaCO3 layer on the surface of the drug carrier and combining the antioxidant and anti-inflammatory effects of the composite molybdenum nanozyme, it can achieve targeted delivery and sustained release of the drug, significantly improving the therapeutic effect.

[0045] (I) Synthesis method of drug carrier

[0046] (1) Synthesis of ZIF-8 composite molybdenum nanozyme:

[0047] ZIF-8 (metal organic framework) is a material with a highly porous structure that can effectively embed other nanozymes and improve their catalytic activity. The present invention uses a solvothermal method to synthesize ZIF-8 and prepares a composite nanozyme by embedding a composite molybdenum element. The specific steps are as follows:

[0048] Synthesis of ZIF-8:

[0049] 1). Dissolve Zn(NO3)2·6H2O and 2-methylimidazole in methanol solution at a molar ratio of 1:2 to form a clear solution;

[0050] 2). The solution was transferred to a reactor and reacted at 150°C for 24 hours, and then cooled naturally after the reaction;

[0051] 3). Remove the unreacted substances by centrifugation and filtration to obtain white ZIF-8 powder;

[0052] 4). Finally, the ZIF-8 crystals are dried in a vacuum to obtain ZIF-8 powder.

[0053] Synthesis of composite molybdenum nanozymes:

[0054] 1) Dissolve MoO2Cl2 in water or methanol and mix with ZIF-8 precursor;

[0055] 2). Adjust the pH of the reaction solution to 6.0 and react at 50°C for several hours to embed the Mo element into the ZIF-8 framework;

[0056] 3). After the reaction is completed, the excess unreacted products are removed by centrifugation and washing to obtain the ZIF-8@Mo composite nanozyme.

[0057] (2) Preparation of CaCO3 coating:

[0058] Deposition of coating:

[0059] 1). Suspend the ZIF-8@Mo composite nanozyme in a CaCl2 solution, and add ammonia water to adjust the pH value to about 9.0 to promote the precipitation of CaCO3;

[0060] 2) Reacting for 1-2 hours under stirring conditions to obtain ZIF-8@Mo nanozyme particles covered with a CaCO3 coating;

[0061] 3). After the reaction is completed, excess calcium ions are removed by centrifugation and washing to obtain CaCO3@ZIF@Mo composite particles.

[0062] (3) Coating characterization:

[0063] 1). Confirm the formation of CaCO3 coating using X-ray diffraction (XRD);

[0064] 2). The morphology of CaCO3@ZIF@Mo composite particles was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to confirm the uniformity and stability of the coating.

[0065] (II) Functional design and release control of drugs

[0066] (1) Surface functionalization of drug carriers:

[0067] In order to improve the biocompatibility, targeting and control the release of drugs, the present invention performs functionalization treatment on drug carriers:

[0068] Polyethylene glycol (PEG) modification:

[0069] Attaching PEG chains to the surface of CaCO3@ZIF@Mo composite particles increases the water solubility and biostability of the drug, prevents clearance by the immune system, and prolongs the drug's circulation time in the body.

[0070] Targeted Antibody Modification:

[0071] By covalently binding arthritis-specific antibodies (such as monoclonal antibodies against TNF-α and IL-6), drugs can be specifically targeted to areas of inflammation, improving therapeutic effects and reducing side effects of drugs on healthy tissues.

[0072] Anti-inflammatory drug load:

[0073] Anti-inflammatory drugs (such as chlorofluthiazol, non-steroidal anti-inflammatory drugs, etc.) are loaded into ZIF-8@Mo composite particles so that the drugs can be gradually released, the concentration of the drugs can be controlled, and their therapeutic effects can be enhanced.

[0074] (2) Drug release mechanism:

[0075] Degradability of CaCO3 coating:

[0076] The CaCO3 coating gradually dissolves in an acidic environment (such as the low pH environment in inflamed joints), releasing Ca 2+ ions, thereby activating the antioxidant effect of Mo nanozymes in the carrier. At the same time, the drug release rate can be achieved by adjusting the thickness and dissolution rate of the CaCO3 coating to achieve sustained release.

[0077] Antioxidant effect of composite molybdenum nanozymes:

[0078] The composite molybdenum nanozyme can remove reactive oxygen species (ROS) in the inflammatory area of ​​the joint through catalytic reactions, slow down the oxidative damage of the articular cartilage, delay cartilage degeneration, and inhibit the release of inflammatory factors.

[0079] Sustained release of drugs:

[0080] The dissolution rate of the CaCO3 coating can be controlled by optimizing the formulation to ensure that the drug can be continuously released in the local inflammatory area to achieve long-term therapeutic effects.

[0081] (III) In vivo experiments and efficacy verification

[0082] (1) Rat osteoarthritis model experiment:

[0083] In order to evaluate the therapeutic effect of the drug of the present invention, an in vivo experiment was conducted using a rat osteoarthritis model:

[0084] Animal Grouping:

[0085] Control group: no treatment;

[0086] Drug group: CaCO3@ZIF@Mo nanodrug therapy;

[0087] Blank group: treated with normal saline;

[0088] Positive control group: conventional drug treatment (such as NSAIDs).

[0089] Treatment process:

[0090] The drug is administered by local injection or intravenous injection once a week for 4 weeks.

[0091] Evaluation Metrics:

[0092] Joint X-ray examination: to assess the degree of articular cartilage degeneration before and after treatment;

[0093] Inflammatory factor detection: Detect the concentration of inflammatory factors (such as IL-6, TNF-α, etc.) in joint fluid by ELISA;

[0094] Histological examination: HE staining was used to observe the repair of articular cartilage;

[0095] Joint Mobility: Assess the mobility and pain response of rats' joints.

[0096] (2) Experimental results:

[0097] The damage to the articular cartilage of the rats in the drug group was significantly reduced, and the degree of cartilage degeneration was significantly lower than that in the control group; the concentration of inflammatory factors was significantly reduced, and the range of motion and comfort of the joints were significantly improved. Histological results showed that the degree of articular cartilage repair in the drug group was good and close to normal levels.

[0098] (3) Drug safety assessment:

[0099] The safety of the drug of the present invention is evaluated through acute and long-term toxicity experiments to ensure that the drug has little side effects on the body during long-term use. The specific experimental process and results are as follows:

[0100] 1). Acute toxicity test:

[0101] Acute toxicity experiments were conducted in rats, using a single high dose (e.g. 50 mg / kg) of the drug for treatment and observing acute toxicity reactions within 24 hours. The experimental results showed that after acute administration, the rats did not show obvious symptoms of poisoning, such as loss of appetite, vomiting, abnormal behavior, etc., and there was no obvious death.

[0102] 2). Long-term toxicity experiment:

[0103] In order to evaluate the long-term safety of the drug, the rats in the drug group received continuous treatment for 3 months (injection once a week), and their body weight, blood routine, and biochemical indicators (such as liver function, kidney function, etc.) were monitored every month. The results showed that the physiological indicators of the rats in the drug group were not significantly different from those in the control group, and no liver and kidney damage, hematological abnormalities or other adverse reactions occurred, indicating that the drug has good biocompatibility.

[0104] 3). Histological analysis:

[0105] The tissue structure of major organs (such as liver, kidney, heart, lung, etc.) was observed through histological analysis (HE staining, immunohistochemistry, etc.). The results showed that there were no abnormal changes in the tissue morphology of the rats in the drug group, and the cell structure of major organs remained intact, with no obvious pathological changes.

[0106] 4). Immunogenicity assessment:

[0107] Through immunogenicity testing (such as antibody level testing), it is confirmed that the drug will not cause a significant immune response. The experimental results show that the level of immune response to the drug is extremely low and will not cause allergies or other abnormal reactions of the immune system.

[0108] In summary, the drug of the present invention showed good safety in both acute and long-term toxicity experiments, had no obvious immunogenic reaction, had good biocompatibility, and was suitable for long-term clinical use.

[0109] Embodiment 1

[0110] Synthesis of ZIF-8@Mo composite nanozyme and preparation of CaCO3 coating:

[0111] Step 1: Dissolve 10 mM Zn(NO3)2·6H2O and 20 mM 2-methylimidazole in methanol, mix them in a molar ratio of 1:2, and place them in a reactor at 150°C for 24 hours;

[0112] Step 2: After the reaction is completed, ZIF-8 crystals are obtained, and unreacted products are removed by centrifugation and washing to obtain white ZIF-8 powder;

[0113] Step 3: Mix 1 mM MoO2Cl2 solution with ZIF-8 and react at 50 °C for 6 hours to obtain ZIF-8@Mo composite nanozyme;

[0114] Step 4: Mix the ZIF-8@Mo composite particles with the CaCl2 solution, add ammonia water to adjust the pH to 9.0, and stir the reaction for 2 hours to obtain the CaCO3-coated ZIF-8@Mo particles.

[0115] Embodiment 2

[0116] Drug functionalization and in vivo experiments:

[0117] Step 1: Modify the surface of ZIF-8@Mo composite particles with polyethylene glycol (PEG) by covalent bonding to improve water solubility and biostability;

[0118] Step 2: Targeting the drug to the area of ​​inflammation by binding to an anti-TNF-α antibody;

[0119] Step 3: Loading the anti-inflammatory drug chlorofluthiazol into the ZIF-8@Mo composite particles;

[0120] Step 4: The drug was injected into a rat osteoarthritis model for efficacy evaluation. The results showed that the degree of articular cartilage degeneration in the treatment group was significantly lower than that in the control group, the level of inflammatory factors was significantly reduced, and the joint range of motion was better restored.

[0121] The present invention provides a drug for treating osteoarthritis based on ZIF-8 composite molybdenum nanozyme, which controls the release rate of the drug through a CaCO3 coating, and combines the antioxidant and anti-inflammatory effects of molar nanozymes to significantly improve the targeting, efficacy and safety of drug therapy. It has been verified by in vivo experiments that the drug of the present invention has a significant therapeutic effect on osteoarthritis, and has good biocompatibility and safety, and is suitable for long-term use. The drug is not only suitable for the treatment of osteoarthritis, but also can be applied to other chronic inflammatory diseases, and has a wide range of clinical application prospects.

[0122] The above is the complete content of the invention patent, including detailed contents of invention background, technical scheme, drug carrier preparation, release control, efficacy verification, safety assessment, embodiments, etc., which can help demonstrate the technical innovation and application potential of the invention.

[0123] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of ZIF-8 composite molybdenum nanozyme and CaCO3 layer coating in the treatment of osteoarthritis.

2. The method for preparing ZIF-8 according to claim 1, characterized in that: The specific steps are as follows 1). Dissolve Zn(NO3)2·6H2O and 2-methylimidazole in methanol solution at a molar ratio of 1:2 to form a clear solution; 2). The solution was transferred to a reactor and reacted at 150°C for 24 hours, and then cooled naturally after the reaction; 3). Remove the unreacted substances by centrifugation and filtration to obtain white ZIF-8 powder; 4). Finally, the ZIF-8 crystals are dried in a vacuum to obtain ZIF-8 powder.

3. The method for preparing the composite molybdenum nanozyme according to claim 2, characterized in that: The specific steps are as follows 1) Dissolve MoO2Cl2 in water or methanol and mix with ZIF-8 precursor; 2). Adjust the pH of the reaction solution to 6.0 and react at 50°C for several hours to embed the Mo element into the ZIF-8 framework; 3). After the reaction is completed, the excess unreacted products are removed by centrifugation and washing to obtain the ZIF-8@Mo composite nanozyme.

4. The method for preparing the CaCO3 coating according to any one of claims 1 to 3, characterized in that: The specific steps are as follows 1). Suspend the ZIF-8@Mo composite nanozyme in a CaCl2 solution, and add ammonia water to adjust the pH value to about 9.0 to promote the precipitation of CaCO3; 2) Reacting for 1-2 hours under stirring conditions to obtain ZIF-8@Mo nanozyme particles covered with a CaCO3 coating; 3). After the reaction is completed, excess calcium ions are removed by centrifugation and washing to obtain CaCO3@ZIF@Mo composite particles.

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