CMHs nano-enzyme as well as preparation method and application method thereof

Abstract

The invention relates to the technical field of medicines, and provides a CMHs nano-enzyme as well as a preparation method and an application method thereof. The CMHs nano enzyme sequentially comprises CORM-401, a halloysite nano tube and a manganese oxide nano enzyme from inside to outside; wherein the manganese oxide nano-enzyme is generated on the surface of the halloysite nanotube through an in-situ reaction, and the carbon monoxide release molecules can release carbon monoxide. The CMHs nano-enzyme disclosed by the invention is combined with the manganese oxide nano-enzyme, the halloysite nanotube and the carbon monoxide release molecules, so that the inflammatory bowel disease is effectively treated through multiple synergistic effects; the halloysite nanotube provides a targeted delivery function, the manganese oxide nano enzyme has an anti-oxidation effect, and the carbon monoxide can further enhance the anti-oxidation effect, inhibit inflammatory reaction and relieve inflammatory related pain at the same time; the three components are matched with one another, so that the anti-oxidation and anti-inflammatory effects are enhanced, the intestinal repair can be promoted, the oxidative stress and immune disorder of a focus area are relieved, and meanwhile, the chronic pain related to the inflammatory bowel disease is effectively relieved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a CMHs nanozyme and a preparation method and an application method thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic, nonspecific intestinal inflammatory disease, mainly including ulcerative colitis (UC) and Crohn's disease (CD). The common feature of these two diseases is long-term chronic inflammation of the intestine, and patients are often at risk of serious complications such as intestinal perforation and colon cancer, so long-term treatment is required. At present, the treatment of IBD includes drug therapy and immunomodulatory therapy, but the existing therapies are not without disadvantages. For example, although traditional drugs such as 5-aminosalicylic acid (5-AminosalicylicAcid, 5-ASA) and steroids are effective, long-term use may cause side effects such as liver damage and immunosuppression. Although immunosuppressants can improve the efficacy, they may cause infection, nephrotoxicity and other complications. In addition, although biologics and targeted therapies provide new treatment directions, the side effects such as autoimmune reactions and hepatotoxicity that they may induce make these treatments also have limitations.

[0003] The pathogenesis of IBD is not yet fully understood, but oxidative stress and chronic inflammation are considered to be its core factors. Excessive reactive oxygen species (ROS) can damage the intestinal barrier, activate pro-inflammatory signaling pathways, further aggravate inflammation and tissue damage, and may also cause intestinal microbiota imbalance and nerve damage, leading to increased pain.

[0004] Therefore, there is an urgent need for a product that can clear oxidative stress, reduce inflammation, repair the intestinal barrier, regulate the intestinal microbiota, and relieve pain associated with IBD. Summary of the invention

[0005] The present invention provides a CMHs nanozyme and a preparation method and an application method thereof. The CMHs nanozyme combines manganese oxide nanozyme, halloysite nanotubes and carbon monoxide releasing molecules, and effectively improves the treatment of IBD through multiple synergistic effects; manganese oxide nanozyme has antioxidant effect, halloysite nanotubes provide targeted delivery function, and carbon monoxide can further enhance the antioxidant effect, inhibit inflammatory response, and relieve inflammatory-related pain; the three cooperate with each other, not only enhancing the antioxidant and anti-inflammatory effects, but also promoting intestinal repair, reducing oxidative stress and immune disorders in the lesion area, and effectively relieving chronic pain associated with inflammatory bowel disease.

[0006] In order to solve the above problems, the first object of the present invention is to provide a CMHs nanozyme.

[0007] The second object of the present invention is to provide a method for preparing CMHs nanozyme.

[0008] The third object of the present invention is to provide an application method of CMHs nanozyme.

[0009] To achieve the first object of the present invention, the present invention provides a CMHs nanozyme, which includes, from the inside to the outside, a carbon monoxide releasing molecule, a halloysite nanotube, and a manganese oxide nanozyme; Among them, manganese oxide nanozymes are generated on the surface of halloysite nanotubes through in situ reactions, and carbon monoxide-releasing molecules can release carbon monoxide.

[0010] In one technical solution of the present invention, the halloysite nanotube is a hollow tubular structure, and the carbon monoxide releasing molecule is arranged in the hollow tubular structure of the halloysite nanotube.

[0011] In one technical solution of the present invention, the manganese oxide nanozyme is generated by reacting with the amino groups on the surface of the halloysite nanotube.

[0012] In one embodiment of the present invention, the carbon monoxide releasing molecule includes CORM-401.

[0013] To achieve the second object of the present invention, the present invention provides a method for preparing CMHs nanozyme, comprising the following steps: S100, preparing a manganese oxide-halloysite nanotube composite material: dispersing halloysite nanotubes in an alkaline solution, and adding a potassium permanganate solution dropwise to perform an in-situ reaction to obtain a manganese oxide-halloysite nanotube composite material; S200. Synthesis of CMHs nanozyme: An alkaline solution containing carbon monoxide-releasing molecules is added dropwise to the manganese oxide-halloysite nanotube composite material to obtain CMHs nanozyme.

[0014] In a technical solution of the present invention, step S100 further includes: S110, pretreatment: dispersing the halloysite nanotube raw material in an organic solvent, and adding aminosilane dropwise into the solution to form halloysite nanotubes.

[0015] In a technical solution of the present invention, in step S110, the pretreatment is carried out under an inert atmosphere; and / or the organic solvent includes at least one of toluene, dichloromethane, and ethanol; and / or the aminosilane includes at least one of (3-aminopropyl)triethoxysilane and (3-aminopropyl)trimethoxysilane; and / or the mass ratio of the halloysite nanotube raw material, the organic solvent and the aminosilane is 1: (80-100): (20-40).

[0016] In a technical solution of the present invention, in step S100, the alkaline solution includes at least one of sodium hydroxide and potassium hydroxide; and / or the mass ratio of halloysite nanotubes, alkaline solution and potassium permanganate is 1: (0.7-1.0): (0.2-0.3).

[0017] In a technical solution of the present invention, in step S200, the mass ratio of the manganese oxide-halloysite nanotube composite material to the carbon monoxide releasing molecules is 1:(0.5-5); and / or the concentration range of the alkaline solution containing the carbon monoxide releasing molecules is between 10-20 mg / mL; and / or the pH value range of the alkaline solution containing the carbon monoxide releasing molecules is between 7-9.

[0018] To achieve the third purpose of the present invention, the present invention provides a method for using CMHs nanozyme in the treatment of inflammatory bowel disease.

[0019] After adopting the technical solution of the present invention, the following technical effects can be achieved: (1) By using halloysite nanotubes as carriers, CMHs nanozymes can accurately deliver manganese oxide nanozymes and carbon monoxide-releasing molecules to the site of inflammation, ensuring that the drugs accumulate in the lesion area, maximizing the therapeutic effect and reducing the off-target side effects of the drugs; (2) CMHs nanozymes combine the powerful antioxidant effect of manganese oxide nanozymes and the immunomodulatory effect of carbon monoxide-releasing molecules. Manganese oxide nanozymes effectively remove excess ROS in the body, thereby reducing intestinal damage caused by oxidative stress; while carbon monoxide-releasing molecules can provide multi-dimensional therapeutic effects by regulating immune responses, reducing inflammation, and relieving pain, significantly improving the condition of IBD patients; (3) The combination of CO-releasing molecules and manganese oxide nanozymes not only has significant advantages in targeted therapy, but also can accurately control the release rate of CO, ensuring that the therapeutic effect will not produce side effects due to excessive or too rapid release; at the same time, the biocompatibility and stability of halloysite nanotubes provide reliable guarantees for drug carriers, enhancing the biosafety during treatment and the feasibility of long-term application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 A flow chart of the synthesis of CMHs nanozymes provided in Example 1 of the present invention; Figure 2 This is a color change diagram of the CMHs nanozyme provided in Example 1 of the present invention; Figure 3 The Fourier transform infrared spectrum of the CMHs nanozyme provided in Example 1 of the present invention; Figure 4 This is a morphology diagram of the CMHs nanozyme provided in Example 1 of the present invention; Figure 5 The XPS peak spectrum of MHs and CMHs provided in Example 1 of the present invention; Figure 6 This is the in vitro CO release diagram of the CMHs nanozyme provided in Example 1 of the present invention; Figure 7 The cck8 cytotoxicity graph of RAW and Caco-2 cells provided in Example 1 of the present invention; Figure 8 The Caco-2 cell live-death diagram provided in Example 1 of the present invention; Fig. 9 This is a graph of intracellular ROS scavenging ability provided in Example 1 of the present invention; Fig.10 This is a graph of the intracellular RNS clearance ability provided in Example 1 of the present invention; Fig.11 This is a graph of EDU cell proliferation ability provided in Example 1 of the present invention; Fig.12 This is a graph showing the water intake and water intake trend of mice within 11 days provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0022] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] IBD is a chronic inflammatory disease of the gastrointestinal tract that mainly includes two conditions: UC and CD. Due to the risk of serious complications such as intestinal perforation, stenosis, and colorectal cancer, IBD patients usually require long-term treatment. Currently, 5-ASA and corticosteroids may cause liver damage and immunosuppression. The use of immunosuppressants increases the risk of infection and nephrotoxicity. The use of biologics and targeted therapies may suppress the immune system, leading to side effects such as autoimmune disorders and hepatotoxicity.

[0024] Although existing treatments have made some progress, the exact pathogenesis of IBD has not yet been fully elucidated. Increasing evidence shows that oxidative stress and chronic inflammation play a central role in the onset and progression of IBD. Specifically, oxidative stress refers to cell damage caused by the accumulation of excessive ROS, which not only destroys the intestinal barrier function and increases intestinal permeability, but also aggravates the inflammatory response. The occurrence of oxidative stress is due to the increase in ROS levels in the body, which exceeds the regulatory capacity of the antioxidant system, leading to worsening of tissue damage and inflammation.

[0025] At the same time, excessive accumulation of ROS will also activate pro-inflammatory signaling pathways, such as the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-KB) pathway, thereby promoting the activation of immune cells and the release of cytokines, thereby exacerbating the inflammatory response in the intestine. The vicious cycle of oxidative stress and inflammation can also lead to dysbiosis of the intestinal microbiota, further aggravating the inflammatory response. The interweaving of redox reactions and inflammatory responses makes the treatment of IBD more complex and challenging.

[0026] In addition to inflammation, redox reactions also affect the nervous system, leading to nerve damage and sensitization of pain perception. This pain caused by inflammation and oxidative stress further aggravates the suffering of IBD patients.

[0027] Given the limitations of conventional treatments, there is an urgent need for a product that can clear oxidative stress, reduce inflammation, repair the intestinal barrier, regulate the gut microbiota, and relieve pain associated with IBD.

[0028] The invention provides a CMHs nanozyme, which comprises, from the inside out, carbon monoxide releasing molecules, halloysite nanotubes and manganese oxide nanozyme.

[0029] Preferably, manganese oxide nanozymes have shown potential in anti-oxidation. Since the discovery in 2007 that ferroferric oxide nanoparticles have catalytic activity similar to that of peroxidase, the research on nanozymes has developed rapidly, especially in the acidic microenvironment of IBD, where manganese oxide nanozymes have shown strong ROS scavenging ability; however, the therapeutic potential of manganese oxide nanozymes is often limited by their targeting specificity, and non-specific distribution may cause the drug to be unable to effectively accumulate in the lesion area, thereby reducing efficacy and increasing side effects. CO has cytoprotective, immunomodulatory, antioxidant and anti-inflammatory properties. It is an endogenous signaling molecule with significant therapeutic potential. Studies have shown that CO can not only directly inhibit oxidative stress, but also reduce chronic inflammation by regulating the M1 / M2 polarization of macrophages. In addition, CO reduces nerve damage caused by oxidative stress and relieves pain by regulating neural activity. Carbon monoxide (CO) releasing molecules are compounds that can stably store carbon monoxide and can respond to elevated ROS and release carbon monoxide under pathological conditions. Since the release of CO is closely related to the severity of colon lesions, carbon monoxide can be precisely released in the lesion area, thereby achieving targeted therapy, significantly improving the treatment effect and reducing off-target side effects.

[0030] Furthermore, in order to maximize the therapeutic effect, a dedicated delivery system must be used to ensure that the carbon monoxide-releasing molecules and manganese oxide nanozymes are delivered to the inflamed colon mucosa in a targeted manner, thereby inhibiting the inflammatory response and alleviating the inflammatory response caused by excessive ROS, thereby helping to restore the immune balance of the intestine and reduce intestinal damage and pain caused by immune response disorders; Halloysite Nanotubes (Hs), as a naturally occurring aluminosilicate mineral, have good biocompatibility, stability and inherent anti-inflammatory properties, and are very suitable for gastrointestinal applications. It has become an ideal drug carrier that can accurately deliver manganese oxide and CO-releasing molecules to the site of inflammation; in addition, the surface of Hs is rich in hydroxyl and aluminum oxide groups, which is conducive to electrostatic interactions, thereby promoting the preferential accumulation of drugs in positively charged, inflamed colon tissues.

[0031] Furthermore, manganese oxide nanozymes are generated on the surface of halloysite nanotubes through an in situ reaction, and the manganese source is reduced to Mn under the action of a reducing agent. 2+ Manganese oxide adsorbs and reacts on the amino groups on the surface of Hs to form manganese oxide nanoparticles and fix them on the surface of Hs. In addition, the amino groups on the surface of Hs react with Mn 2+ The electrostatic interaction enables precise deposition in damaged tissue areas, further enhancing the therapeutic effect.

[0032] Preferably, the halloysite nanotubes are hollow tubular structures, and the CO-releasing molecules are arranged in the hollow tubular structures of the halloysite nanotubes, which can effectively improve the drug delivery and therapeutic effects. The hollow structure provides a stable storage space for the CO-releasing molecules, so that CO can be accurately released when needed; it can be activated and released in the lesion area of ​​inflammation or oxidative stress, achieving the effect of targeted treatment and enhancing its anti-inflammatory, antioxidant and immunomodulatory effects.

[0033] Furthermore, carbon monoxide releasing molecules include CORM-401. CORM-401 (Carbon Monoxide Releasing Molecule-401) has a specific chemical structure and can release CO under specific physiological or pathological conditions. CORM-401 releases CO slowly and controllably by reacting with redox substances in tissues, and under oxidative stress or other pathological conditions, CORM-401 can release carbon monoxide in response to increased ROS concentrations in the body, thereby achieving targeted therapeutic effects. The combination of CORM-401 and Hs helps to stably store and precisely release CO in the intestine or other inflammatory sites, thereby exerting antioxidant, anti-inflammatory and immunomodulatory effects.

[0034] In general, the CMHs nanozyme provided in the present application exerts a synergistic effect by combining carbon monoxide-releasing molecules, halloysite nanotubes and manganese oxide nanozymes; halloysite nanotubes, as drug carriers, effectively deliver manganese oxide nanozymes and CO-releasing molecules to the inflamed colon site by virtue of their good biocompatibility, stability and anti-inflammatory properties; in this process, manganese oxide nanozymes are generated on the surface of halloysite through in situ reactions and fixed as nanoparticles to enhance their catalytic activity; CO-releasing molecules are stably stored in the hollow tubular structure of halloysite and can be accurately released in areas of oxidative stress or inflammation, and further targeted therapy is achieved by regulating ROS, immune response and neural activity, thereby effectively improving the targeting of the drug, enhancing multiple therapeutic effects such as antioxidant, anti-inflammatory and immunomodulatory, significantly improving the therapeutic effect and reducing side effects.

[0035] The preparation method of the CMHs nanozyme of the present application comprises the following steps: S100, preparing a manganese oxide-halloysite nanotube composite material: dispersing halloysite nanotubes in an alkaline solution, and adding a potassium permanganate solution dropwise to perform an in-situ reaction to obtain a manganese oxide-halloysite nanotube composite material; S200. Synthesis of CMHs nanozyme: adding an alkaline solution containing carbon monoxide-releasing molecules dropwise into the manganese oxide-halloysite nanotube composite material to obtain the CMHs nanozyme.

[0036] Preferably, S110, pretreatment: dispersing the raw material of halloysite nanotubes in an organic solvent, and adding aminosilane dropwise to the solution to form halloysite nanotubes. Dispersing the halloysite nanotubes in an organic solvent helps to improve the dispersibility of the nanotubes, avoid nanotube aggregation, ensure uniformity in the reaction, thereby improving the surface reactivity of the nanotubes and promoting their reaction with aminosilane; the method of adding aminosilane dropwise can make the aminosilane gradually contact with the surface of halloysite and ensure the controllability of the reaction. The amino groups contained in the aminosilane molecules can react chemically with the hydroxyl groups or other functional groups on the surface of halloysite to form a covalent bond between the aminosilane and the halloysite, which not only increases the surface hydrophilicity of the halloysite, but also gives it a stronger drug carrier function, thereby enhancing its ability to bind to drug molecules or other active ingredients; in this way, the surface properties of the halloysite nanotubes are significantly improved, thereby improving its application effect in drug delivery and treatment.

[0037] Furthermore, in step S110, the pretreatment is carried out under an inert atmosphere, which can effectively prevent the surface of the halloysite nanotubes from reacting with moisture or oxygen in the air, thereby ensuring the stability and controllability of the surface modification process; the use of organic solvents including toluene, dichloromethane, ethanol, etc. can help the halloysite nanotubes to be evenly dispersed in the solution, enhance the reactivity of their surface with aminosilane, and promote the modification reaction; aminosilanes including (3-aminopropyl)triethoxysilane, (3-aminopropyl)trimethoxysilane, etc. can provide amino groups during the surface modification process, react chemically with the surface of halloysite, enhance its surface functionality, and thus improve the dispersibility, compatibility and drug carrier capacity of halloysite.

[0038] The mass ratio of organic solvent to aminosilane is set to 1: (80-100): (20-40), which can ensure the appropriate concentration of the modification reaction and effectively control the reaction rate and the stability of the reaction product; the appropriate ratio helps the aminosilane to fully react with the surface of the halloysite nanotubes, ensuring the grafting amount of the amino functional group, thereby improving the modification efficiency and surface activity of the halloysite.

[0039] In general, during the pretreatment process, after the surface of the halloysite nanotubes was treated with aminosilanization, more amino groups were introduced on the surface. These amino groups can enhance the electrostatic adsorption and chemical reactivity of the halloysite nanotubes with the manganese source in the potassium permanganate solution, which helps the manganese source to deposit on the halloysite surface and form manganese oxide nanoparticles, effectively improving the adhesion and dispersibility of manganese oxide, thereby enhancing the catalytic performance and also enhancing its stability in the biological environment; and the pretreated halloysite nanotube surface has a higher affinity, which enables it to better adsorb and carry carbon monoxide release molecules and manganese oxide nanoparticles. Through the amino treatment, the surface functional groups of the halloysite can better support the deposition and catalytic activity of manganese oxide nanoparticles, laying the foundation for the subsequent synthesis and therapeutic effect of CMHs nanozymes.

[0040] Furthermore, in step S100, the alkaline solution includes at least one of sodium hydroxide and potassium hydroxide, which helps to promote the surface modification of the halloysite nanotubes and the reaction of the manganese source. The alkaline solution can provide a suitable pH environment so that the manganese element in potassium permanganate can be effectively reduced to form manganese oxide nanoparticles and deposited on the surface of the halloysite nanotubes; sodium hydroxide, potassium hydroxide or ammonia water can provide sufficient OH - , promoting the reduction reaction of Mn element in potassium permanganate.

[0041] Preferably, the concentration range of the alkaline solution is between 0.1M-10M, and the pH value is between 12-14. By adjusting the concentration of the alkaline solution and the reaction conditions, the generation rate of manganese oxide and the morphology of the particles can be effectively controlled, further improving the deposition and fixation ability of manganese oxide on the surface of halloysite nanotubes.

[0042] The mass ratio of halloysite nanotubes, alkaline solution and potassium permanganate is 1:(0.7-1.0):(0.2-0.3), which ensures that the concentration of potassium permanganate in the reaction is sufficient to support the formation of manganese oxide while not causing over-reaction to cause precipitation or decomposition of the product, thereby maintaining the efficiency and stability of the reaction.

[0043] In general, in step S100, halloysite nanotubes are dispersed in an alkaline solution, and potassium permanganate solution is added dropwise to form manganese oxide nanoparticles through an in-situ reaction and deposited on the surface of the halloysite nanotubes; manganese oxide is directly fixed on the surface of halloysite by utilizing the amino groups on the surface of the halloysite nanotubes to react with the manganese source in the potassium permanganate, thereby preparing a manganese oxide-halloysite nanotube composite material; manganese oxide nanoparticles have strong catalytic activity and can effectively remove ROS, while the halloysite nanotubes provide a stable carrier, enhance the stability and biocompatibility of the composite material, and are suitable for application in complex biological environments.

[0044] Preferably, the mass ratio of the manganese oxide-halloysite nanotube composite material to the carbon monoxide releasing molecule is 1: (0.5-5), which can ensure the effective combination of the composite material and the CO releasing molecule, and ensure that the CO releasing molecule can play a role at an appropriate amount and rate during the treatment process; a higher CO releasing molecule ratio can increase the release of CO, enhance the anti-inflammatory, antioxidant and immunomodulatory effects, but too high a ratio may affect the catalytic performance of the manganese oxide nanozyme. The concentration range of the alkaline solution containing carbon monoxide releasing molecules is between 10-20 mg / mL, ensuring the appropriate concentration of CO releasing molecules in the solution, which can effectively respond to the oxidative stress in the lesion area and release carbon monoxide; too low a concentration may not provide sufficient CO release, and too high a concentration may cause the release rate to be too fast, resulting in side effects. The pH value of the alkaline solution containing carbon monoxide releasing molecules ranges from 7 to 9. The pH value within this range is suitable for the stability of the CO releasing molecule, and the CO releasing molecule can effectively respond to the redox reaction in the alkaline environment, thereby accurately releasing CO; too low a pH value may cause the degradation of the CO releasing molecule, and too high a pH value may affect the accuracy of its release control.

[0045] In general, in step S200, an alkaline solution containing carbon monoxide releasing molecules is added dropwise to the manganese oxide-halloysite nanotube composite material, and further reacts to generate CMHs nanozymes. The CO releasing molecules interact with the manganese oxide-halloysite nanotube composite material to form a multifunctional nanozyme, which has the catalytic ability of manganese oxide nanozymes and the carbon monoxide releasing function, so that the CMHs nanozyme can accurately release carbon monoxide in the lesion area, while exerting antioxidant, anti-inflammatory and immunomodulatory effects, thereby achieving targeted treatment and improving the treatment effect, especially showing excellent potential in controlling oxidative stress and reducing inflammatory responses.

[0046] Embodiment 1 This embodiment provides a CMHs nanozyme and a preparation method thereof, the synthesis flow chart is as follows Figure 1 The specific steps are as follows: S110, pretreatment: under a nitrogen atmosphere, dispersing a raw material of halloysite nanotubes in toluene to obtain a first solvent; adding (3-aminopropyl)triethoxysilane (APTEs) dropwise into the first solvent, reacting for 12 hours under magnetic stirring at a temperature of 90° C., collecting a precipitate by centrifugation, and washing it three times with ethanol and water respectively, and then freeze-drying the precipitate to obtain halloysite nanotubes; Among them, the mass ratio of halloysite nanotube raw material, toluene and APTEs is 1:87:30; S100, preparing a manganese oxide-halloysite nanotube composite material: dispersing halloysite nanotubes in a sodium hydroxide solution, ultrasonically treating for 10 minutes, and adding a potassium permanganate solution dropwise to obtain a mixed solution after the dropwise addition is completed; vortexing the mixed solution for 5 minutes, reacting it at 90° C. under visible light for 4 hours, centrifuging and precipitating it, and washing the precipitate with deionized water until it is neutral, and freeze-drying it to obtain a manganese oxide-halloysite nanotube composite material; The mass ratio of halloysite nanotubes, sodium hydroxide and potassium permanganate is 1:0.8:0.25; Synthesis of S200 and CMHs nanozymes: Manganese oxide-halloysite nanotube composite material was dispersed in water, and an alkaline solution containing CORM-401 was added dropwise. The mixture was stirred at room temperature in the dark for 24 h, and the precipitate was washed by centrifugation with dd water. After freeze-drying, CMHs nanozymes were obtained, and the color change was as shown in FIG. Figure 2 As shown, it changes from white to green-brown, and its morphology is as follows Figure 4 As shown; Among them, the mass ratio of the manganese oxide-halloysite nanotube composite material to CORM-401 is 1:1; the concentration of the alkaline solution containing CORM-401 is 10 mg / mL; and the pH value of the alkaline solution containing CORM-401 is 8.

[0047] Embodiment 2 This embodiment provides a CMHs nanozyme and a preparation method thereof, and the specific operation steps are as follows: S100, preparing a manganese oxide-halloysite nanotube composite material: dispersing halloysite nanotubes in a potassium hydroxide solution, ultrasonically treating for 10 minutes, and adding a potassium permanganate solution dropwise to obtain a mixed solution after the dropwise addition is completed; vortexing the mixed solution for 5 minutes, reacting it at 90° C. under visible light for 4 hours, centrifuging and precipitating it, and washing the precipitate with deionized water until it is neutral, and freeze-drying it to obtain a manganese oxide-halloysite nanotube composite material; The mass ratio of halloysite nanotubes, potassium hydroxide and potassium permanganate is 1:0.7:0.2; Synthesis of S200 and CMHs nanozymes: manganese oxide-halloysite nanotube composite material was dispersed in water, alkaline solution containing CORM-401 was added dropwise, stirred at room temperature in the dark for 24 h, precipitated by centrifugation with dd water, and freeze-dried. Figure 2 As shown, a green-brown powder is obtained, namely CMHs nanozyme; Among them, the mass ratio of the manganese oxide-halloysite nanotube composite material to CORM-401 is 1:0.5; the concentration of the alkaline solution containing CORM-401 is 15 mg / mL; and the pH value of the alkaline solution containing CORM-401 is 7.

[0048] Embodiment 3 This embodiment provides a CMHs nanozyme and a preparation method thereof, and the specific operation steps are as follows: S110, pretreatment: under a nitrogen atmosphere, dispersing a raw material of halloysite nanotubes in ethanol to obtain a first solvent; adding (3-aminopropyl)trimethoxysilane dropwise into the first solvent, reacting for 12 hours under magnetic stirring at a temperature of 90° C., collecting a precipitate by centrifugation, and washing it three times with ethanol and water respectively, and freeze-drying the precipitate after washing to obtain halloysite nanotubes; The mass ratio of the halloysite nanotube raw material, ethanol and (3-aminopropyl)trimethoxysilane is 1:100:40; S100, preparing a manganese oxide-halloysite nanotube composite material: dispersing halloysite nanotubes in a potassium hydroxide solution, ultrasonically treating for 10 minutes, and adding a potassium permanganate solution dropwise to obtain a mixed solution after the dropwise addition is completed; vortexing the mixed solution for 5 minutes, reacting it at 90° C. under visible light for 4 hours, centrifuging and precipitating it, and washing the precipitate with deionized water until it is neutral, and freeze-drying it to obtain a manganese oxide-halloysite nanotube composite material; The mass ratio of halloysite nanotubes, potassium hydroxide and potassium permanganate is 1:1:0.3; Synthesis of S200 and CMHs nanozymes: manganese oxide-halloysite nanotube composite material was dispersed in water, alkaline solution containing CORM-401 was added dropwise, stirred at room temperature in the dark for 24 h, precipitated by centrifugation with dd water, and freeze-dried. Figure 2 As shown, a green-brown powder is obtained, namely CMHs nanozyme; Among them, the mass ratio of the manganese oxide-halloysite nanotube composite material to CORM-401 is 1:5; the concentration of the alkaline solution containing CORM-401 is 20 mg / mL; and the pH value of the alkaline solution containing CORM-401 is 9.

[0049]

Basic physical and chemical properties and structural characterization

[0050] CO release performance: A sample bottle containing 2 mL of Example 1 and a CO detector (BH-90) were placed in a sealable cylindrical glass container and sealed; H2O2 at different concentrations (0.1, 0.5, 1, or 10 mM) was injected through a rubber stopper. 2 O 2 solution, triggering the release of CO, and continuously monitoring the CO concentration (ppm); the results are as follows Figure 6 As shown; at 0 mM H 2 O 2 Under these conditions, very little CO was released within 1 hour. 2 O 2 Under the same conditions, the cumulative release of CO after 1 hour reached 1.45, 2.09 and 3.02 μmol, respectively, indicating that CORM-401 in Example 1 has H 2 O 2 The responsive CO release behavior indicated that CMHs could effectively release CO at the IBD site.

[0051] Cytotoxicity of CMHs: In this study, Raw264.7 and Caco-2 cells were cultured using macrophage- and Caco-2-specific culture media provided by Wuhan Purui Biotechnology. Normal macrophages refer to RAW264.7 cells that have not been treated with LPS, and activated macrophages refer to RAW264.7 cells that have been stimulated with LPS (5 μg / mL) for 24 hours. The cytotoxicity of CMHs to Raw264.7 and Caco-2 cells was evaluated by CCK-8 assay and live / dead staining. The results are shown in Table 2. Figure 7 , Figure 8 shown.

[0052] In vitro antioxidant activity of CMHs: Activated macrophages (2×10 5Cells / culture dish) were inoculated in a culture dish and cultured for 12 hours, and then the cells were exposed to DMEM culture medium containing 100 μg / mL Example 1, and cultured for another 12 hours; after discarding the culture medium, the cells were incubated with 300 μL of PBS containing 50% Hoechst and 5 μM DCFH-DA and DAF-FM DA in DMEM culture medium for 30 minutes; then, the supernatant was collected and the fluorescence intensity of various probes in the supernatant was measured using a multifunctional microplate reader (Biotech, Epoch 2, USA); at the same time, the cells were rinsed with PBS, and the distribution of various fluorescent probes in the cells was observed using an inverted fluorescence microscope. The excitation and emission wavelengths of the DCFH-DA probe are 644 nm and 665 nm, respectively, and the excitation and emission wavelengths of DAF-FMDA are 500 nm and 515 nm, respectively. The results are shown in Figure 2. Fig. 9 , Fig.10 As shown in Figure 2, it is proved that the manganese oxide in MHs has a strong property of scavenging ROS / RNS. In addition, the CO release in CMHs helps to consume more ROS and H. 2 O 2 , thereby enhancing its cleaning effect.

[0053] CMHs promote intestinal mucosal barrier recovery in vitro: Caco-2 cells (2×10 4 Cells were seeded in 96-well plates and cultured for 24 h. 2 O 2 The cells were further exposed to fresh DMEM medium containing 100 μg / mL of Example 1 and cultured for another 24 hours. The treated Caco-2 cells were collected and analyzed using Cell Light TM EdU Apollo ® The 567 imaging kit was used to evaluate the proliferation-promoting effect of Example 1. The results were as follows: Fig.11 As shown, 500 µM H 2 O 2 After treatment, the number of EdU-positive cells decreased significantly, confirming that H 2 O 2 However, after MHs and CMHs treatment, the number of apoptotic cells decreased significantly and cell proliferation increased significantly. It is worth noting that in the CMHs treatment group, cell proliferation almost returned to normal levels, indicating that CMHs nanozymes have a significant protective effect on ROS-induced cell apoptosis.

[0054] Construction of DSS-induced colitis mice: BALB / c female mice aged 6-8 weeks were selected as experimental subjects. The average weight of these mice is about 20g, they are healthy and have strong adaptability. Before starting the experiment, we let the mice be raised in the experimental environment for a week to ensure that they adapt to the new environment and reduce the impact of environmental changes on the experimental results. After ensuring that the mice adapt to the environment, we continuously feed these mice with an aqueous solution containing 3% DSS to induce colitis. BALB / c female mice are used as experimental subjects because they are representative in terms of immune response and the occurrence of colitis. At the same time, mice aged 6-8 weeks are selected to ensure that they are in a stable stage of growth and development, thereby reducing the interference of other factors on the experimental results.

[0055] To evaluate the analgesic effect of CMHs, three mice with colitis treated with CMHs and three mice with colitis treated with PBS were selected. Each group was rectally administered with 150 μL of PBS or 1.5 mg / mL of CMHs every other day. The behavior of each mouse was monitored at a fixed time every day, and its food and water intake was accurately recorded. Fig.12 As shown, for a period of 11 days to assess their behavioral responses; As the severity of the disease increased, the amount of water and food consumed by mice gradually decreased, reflecting that the basic physiological functions of mice were severely inhibited. In contrast, mice treated with CMHs showed varying degrees of recovery in both water and food intake, indicating that CMHs treatment can alleviate pain and restore normal physiological functions in mice with DSS-induced colitis. These findings suggest that CMHs can not only modulate the underlying pathophysiology of IBD, but also positively affect the behavioral changes associated with pain in this disease.

[0056] The above tests demonstrated the excellent potential of PMC in the treatment of IBD.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A CMHs nanozyme, characterized in that: The CMHs nanozyme includes, from the inside out, carbon monoxide releasing molecules, halloysite nanotubes and manganese oxide nanozymes; Wherein, the manganese oxide nanozyme is generated on the surface of the halloysite nanotube through an in-situ reaction, and the carbon monoxide releasing molecule can release carbon monoxide.

2. The CMHs nanozyme according to claim 1, characterized in that The halloysite nanotube is a hollow tubular structure, and the carbon monoxide releasing molecule is arranged in the hollow tubular structure of the halloysite nanotube.

3. The CMHs nanozyme according to claim 1, characterized in that The manganese oxide nanozyme is generated by reacting with the amino groups on the surface of the halloysite nanotube.

4. The CMHs nanozyme according to claim 1, characterized in that The carbon monoxide releasing molecules include CORM-401.

5. A method for preparing CMHs nanozyme, characterized in that: The preparation method is used to prepare the CMHs nanozyme according to any one of claims 1 to 4, and the preparation method comprises the following steps: S100, preparing a manganese oxide-halloysite nanotube composite material: dispersing the halloysite nanotubes in an alkaline solution, and adding a potassium permanganate solution dropwise to perform an in-situ reaction to obtain the manganese oxide-halloysite nanotube composite material; S200. Synthesis of CMHs nanozyme: adding an alkaline solution containing carbon monoxide releasing molecules dropwise into the manganese oxide-halloysite nanotube composite material to obtain the CMHs nanozyme.

6. The preparation method according to claim 5, characterized in that: Step S100 also includes: S110, pretreatment: dispersing the halloysite nanotube raw material in an organic solvent to obtain a first solvent; and adding aminosilane dropwise into the first solvent to form the halloysite nanotube.

7. The preparation method according to claim 6, characterized in that: In step S110, The pretreatment is carried out under an inert atmosphere; and / or The organic solvent comprises at least one of toluene, dichloromethane and ethanol; and / or The aminosilane includes at least one of (3-aminopropyl)triethoxysilane and (3-aminopropyl)trimethoxysilane; and / or The mass ratio of the halloysite nanotube raw material, the organic solvent and the aminosilane is 1:(80-100):(20-40).

8. The preparation method according to claim 5, characterized in that: In step S100, The alkaline solution includes at least one of sodium hydroxide and potassium hydroxide; and / or The mass ratio of the halloysite nanotubes, the alkaline solution and the potassium permanganate is 1:(0.7-1):(0.2-0.3).

9. The preparation method according to claim 5, characterized in that: In step S200, The mass ratio of the manganese oxide-halloysite nanotube composite material to the carbon monoxide-releasing molecule is 1:(0.5-5); and / or The concentration of the alkaline solution containing carbon monoxide releasing molecules is in the range of 10-20 mg / mL; and / or The pH value of the alkaline solution containing carbon monoxide releasing molecules ranges from 7 to 9.

10. A method for applying CMHs nanozyme, characterized in that: Inflammatory bowel disease is treated using the CMHs nanozyme as described in any one of claims 1 to 4.

Images