A kind of CMHs nanozyme, its preparation method and application method

Abstract

The present invention relates to the field of pharmaceutical technology, and provides a CMHs nanozyme, a preparation method thereof, and an application method; the CMHs nanozyme sequentially includes, from the inside out: CORM-401, halloysite nanotubes, and manganese oxide nanozyme; wherein, the manganese oxide nanozyme is generated on the surface of the halloysite nanotubes through an in-situ reaction, and the carbon monoxide-releasing molecule can release carbon monoxide. The CMHs nanozyme of the present invention combines a manganese oxide nanozyme, halloysite nanotubes, and a carbon monoxide-releasing molecule, and effectively treats inflammatory bowel disease through multiple synergistic effects; the halloysite nanotubes provide a targeted delivery function, the manganese oxide nanozyme has an antioxidant effect, and carbon monoxide can further enhance the antioxidant effect, inhibit the inflammatory response, and relieve inflammation-related pain at the same time; 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 related to inflammatory bowel disease.

Description

Technical Field

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

[0002] Inflammatory bowel disease (IBD) is a chronic non-specific intestinal inflammatory disease, mainly including ulcerative colitis (UC) and Crohn's disease (CD). The common feature of these two diseases is the long-term chronic inflammation of the intestine, and patients often face the risk of serious complications such as intestinal perforation and colon cancer, so long-term treatment is required. At present, the treatment methods of IBD include drug treatment and immunomodulatory treatment, but the existing therapies are not without drawbacks. For example, traditional drugs such as 5-aminosalicylic acid (5-ASA) and steroids are effective, but long-term use may cause side effects such as liver damage and immunosuppression. Immunosuppressants can improve the curative effect, but may cause infections, nephrotoxicity and other complications. In addition, although biological agents and targeted therapies provide new treatment directions, the side effects such as autoimmune reactions and hepatotoxicity they may cause limit these treatment methods.

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

[0004] Therefore, there is an urgent need for a product that can scavenge 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, a preparation method thereof, 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; the manganese oxide nanozyme has an antioxidant effect, the halloysite nanotubes provide a targeted delivery function, and carbon monoxide can further enhance the antioxidant effect, inhibit the inflammatory response, and relieve inflammation-related pain at the same time; 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] 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 preparation method of the CMHs nanozyme.

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

[0009] To achieve the first object of the present invention, the present invention provides a CMHs nanozyme, which successively includes, from the inside to the outside: a carbon monoxide-releasing molecule, halloysite nanotubes, and manganese oxide nanozyme; wherein, the manganese oxide nanozyme is generated on the surface of the halloysite nanotubes through an in-situ reaction, and the carbon monoxide-releasing molecule can release carbon monoxide.

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

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

[0012] In a technical solution 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 preparation method of the CMHs nanozyme, including the following steps: S100. Prepare a manganese oxide-halloysite nanotube composite material: Disperse the halloysite nanotubes in an alkaline solution, and dropwise add a potassium permanganate solution for in-situ reaction to obtain a manganese oxide-halloysite nanotube composite material; S200. Synthesis of the CMHs nanozyme: Dropwise add an alkaline solution containing a carbon monoxide-releasing molecule to the manganese oxide-halloysite nanotube composite material to obtain the CMHs nanozyme.

[0014] In a technical solution of the present invention, step S100 further includes: S110. Pretreatment: Disperse the halloysite nanotube raw material in an organic solvent, and dropwise add an amino silane to 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 amino silane 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 amino silane is 1:(80-100):(20-40).

[0016] In one 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 one technical solution of the present invention, in step S200, the mass ratio of manganese oxide-halloysite nanotube composite to carbon monoxide-releasing molecule is 1:(0.5 - 5); and / or the concentration range of the alkaline solution containing carbon monoxide-releasing molecule is between 10 - 20 mg / mL; and / or the pH value range of the alkaline solution containing carbon monoxide-releasing molecule is between 7 - 9.

[0018] To achieve the third object of the present invention, the present invention provides an application method of 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 nanozyme can accurately deliver manganese oxide nanozyme and carbon monoxide-releasing molecules to the inflammatory site, ensuring the accumulation of drugs in the lesion area, maximizing the therapeutic effect and reducing the off-target side effects of drugs; (2) CMHs nanozyme combines the strong antioxidant effect of manganese oxide nanozyme and the immunomodulatory effect of carbon monoxide-releasing molecules. Manganese oxide nanozyme effectively scavenges excessive 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 alleviating pain, significantly improving the condition of IBD patients; (3) The combination of CO-releasing molecules and manganese oxide nanozyme 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 rapid release; at the same time, the biocompatibility and stability of halloysite nanotubes provide a reliable guarantee for drug carriers, enhancing the biosafety during the treatment process and the feasibility of long-term application. Description of the Drawings

[0020] 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 in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1 It is the synthesis flow chart of CMHs nanozyme provided by Embodiment 1 of the present invention;

[0022] Figure 2 It is the color change diagram of CMHs nanozyme provided by Embodiment 1 of the present invention;

[0023] Figure 3 FTIR spectrum of CMHs nanozyme provided in the first embodiment of the present invention;

[0024] Figure 4 Morphology map of CMHs nanozyme provided in the first embodiment of the present invention;

[0025] Figure 5 XPS peak fitting spectra of MHs and CMHs provided in the first embodiment of the present invention;

[0026] Figure 6 In vitro CO release map of CMHs nanozyme provided in the first embodiment of the present invention;

[0027] Figure 7 Cck8 cytotoxicity map of RAW and Caco-2 cells provided in the first embodiment of the present invention;

[0028] Figure 8 Live / dead map of Caco-2 cells provided in the first embodiment of the present invention;

[0029] Figure 9 Intracellular ROS scavenging ability map provided in the first embodiment of the present invention;

[0030] Figure 10 Intracellular RNS scavenging ability map provided in the first embodiment of the present invention;

[0031] Figure 11 EDU cell proliferation ability map provided in the first embodiment of the present invention;

[0032] Figure 12 Water intake and water intake trend map of mice within 11 days provided in the first embodiment of the present invention. Detailed implementation manners

[0033] To make the above objects, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0034] IBD is a chronic inflammatory disease of the gastrointestinal tract, mainly including two conditions: UC and CD. Due to the risk of serious complications such as intestinal perforation, stricture, and colorectal cancer, patients with IBD usually require long-term treatment. Currently, 5-ASA and corticosteroids are used, which may cause liver damage and immunosuppression. Immunosuppressants increase the risk of infection and nephrotoxicity. Biological agents and targeted therapies may suppress the immune system, leading to side effects such as autoimmune disorders and hepatotoxicity.

[0035] Despite certain progress in existing treatment methods, the exact pathogenesis of IBD has not been fully elucidated. Increasing evidence indicates 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 disrupts the intestinal barrier function and increases intestinal permeability but also exacerbates the inflammatory response. Oxidative stress occurs due to an increase in ROS levels in the body, exceeding the regulatory capacity of the antioxidant system, leading to tissue damage and the exacerbation of inflammation.

[0036] Meanwhile, the excessive accumulation of ROS also activates pro-inflammatory signaling pathways, such as the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, thereby promoting the activation of immune cells and the release of cytokines, which further exacerbates the inflammatory response in the intestine. The vicious cycle of oxidative stress and inflammation also leads to the dysregulation of the gut microbiota, further aggravating the inflammatory response. The intertwining of redox reactions and inflammatory responses makes the treatment of IBD more complex and challenging.

[0037] In addition to inflammation, redox reactions also affect the nervous system, resulting in nerve damage and the sensitization of pain perception. This pain caused by inflammation and oxidative stress further exacerbates the suffering of IBD patients.

[0038] Given the limitations of traditional treatment methods, there is an urgent need for a product that can scavenge oxidative stress, reduce inflammation, repair the intestinal barrier, regulate the gut microbiota, and relieve pain associated with IBD.

[0039] The present invention provides a CMHs nanozyme, which sequentially includes, from the inside out: a carbon monoxide-releasing molecule, halloysite nanotubes, and manganese oxide nanozyme.

[0040] Preferably, manganese oxide nanozymes have shown potential in antioxidant applications. Since the discovery of the peroxidase-like catalytic activity of magnetite nanoparticles in 2007, the research on nanozymes has developed rapidly. Especially in the acidic microenvironment of IBD, manganese oxide nanozymes have demonstrated strong ROS scavenging ability. However, the therapeutic potential of manganese oxide nanozymes is often limited by their targeting specificity. Non-specific distribution may result in the ineffective accumulation of drugs in the lesion area, thereby reducing the therapeutic effect and increasing side effects. CO has cytoprotective, immunomodulatory, antioxidant, and anti-inflammatory properties and is an endogenous signaling molecule with significant therapeutic potential. Studies have shown that CO can not only directly inhibit oxidative stress but also alleviate chronic inflammation by regulating the M1 / M2 polarization of macrophages. In addition, CO alleviates oxidative stress-induced nerve damage and relieves pain by regulating nerve activity. Carbon monoxide (CO) releasing molecules are compounds that can stably store carbon monoxide and can release carbon monoxide in response to elevated ROS under pathological conditions. Since the release of CO is closely related to the severity of colonic lesions, precise release of carbon monoxide in the lesion area can achieve targeted therapy, significantly improve the therapeutic effect, and reduce off-target side effects.

[0041] Furthermore, to maximize the therapeutic effect, a specialized delivery system must be employed to ensure the targeted delivery of carbon monoxide releasing molecules and manganese oxide nanozymes to the inflamed colonic mucosa, which can inhibit the inflammatory response, alleviate the inflammatory response caused by excessive ROS, and thus contribute to restoring the immune balance of the intestine and reducing intestinal damage and pain caused by immune response disorders. Halloysite nanotubes (Hs), as naturally occurring aluminosilicate minerals, have good biocompatibility, stability, and inherent anti-inflammatory properties, making them very suitable for gastrointestinal applications and an ideal drug carrier that can precisely deliver manganese oxide and CO releasing molecules to the inflamed site. In addition, the surface of Hs is rich in hydroxyl and alumina groups, which is conducive to electrostatic interactions, thereby promoting the preferential accumulation of drugs in the positively charged, inflamed colonic tissue.

[0042] Furthermore, manganese oxide nanozymes are generated on the surface of halloysite nanotubes through an in-situ reaction. The manganese source is reduced to Mn 3+ 、Mn 4+ under the action of a reducing agent. Manganese oxide adsorbs and reacts on the amino groups on the surface of Hs to form manganese oxide nanoparticles and is fixed on the surface of Hs. In addition, the amino groups on the surface of Hs are precisely deposited in the damaged tissue area through electrostatic interactions with Mn 3+ 、Mn 4+ , thereby further enhancing the therapeutic effect.

[0043] Preferably, the halloysite nanotubes are of a hollow tubular structure, and the CO-releasing molecules are disposed in the hollow tubular structure of the halloysite nanotubes, which can effectively improve their drug delivery and therapeutic effects. The hollow structure provides a stable storage space for the CO-releasing molecules, so that CO can be precisely released when needed; it can be activated and released in the inflamed or oxidative stress lesion area, achieving the effect of targeted therapy and enhancing its anti-inflammatory, antioxidant and immunomodulatory effects.

[0044] Furthermore, the 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 reacts with redox substances in tissues to slowly and controllably release CO, and under oxidative stress or other pathological conditions, CORM-401 can release carbon monoxide by responding to the increased concentration of ROS in the body, achieving the effect of targeted therapy; using CORM-401 in combination with Hs helps to stably store and precisely release CO in the intestine or other inflamed sites, thereby exerting antioxidant, anti-inflammatory and immunomodulatory effects.

[0045] Generally speaking, the CMHs nanozyme provided by this application exerts a synergistic effect by combining carbon monoxide-releasing molecules, halloysite nanotubes and manganese oxide nanozyme; the halloysite nanotubes, as a drug carrier, effectively deliver the manganese oxide nanozyme and CO-releasing molecules to the inflamed colon site with its good biocompatibility, stability and anti-inflammatory properties; during this process, the manganese oxide nanozyme is generated on the surface of halloysite by in-situ reaction and fixed as nanoparticles, enhancing its catalytic activity; the CO-releasing molecules are stably stored in the hollow tubular structure of halloysite and can be precisely released in the oxidative stress or inflammation area, further realizing targeted therapy by regulating ROS, immune response and nerve activity, thereby effectively improving the drug targeting, enhancing multiple therapeutic effects such as antioxidant, anti-inflammatory and immunomodulatory effects, significantly improving the therapeutic effect and reducing side effects.

[0046] The preparation method of the CMHs nanozyme of this application includes the following steps: S100, preparing a manganese oxide-halloysite nanotube composite material: dispersing the halloysite nanotubes in an alkaline solution, and dropwise adding a potassium permanganate solution to carry out an in-situ reaction to obtain a manganese oxide-halloysite nanotube composite material; S200, synthesizing the CMHs nanozyme: dropwise adding an alkaline solution containing carbon monoxide-releasing molecules to the manganese oxide-halloysite nanotube composite material to obtain the CMHs nanozyme.

[0047] Preferably, in S110, pre-treatment: Disperse the halloysite nanotube raw material in an organic solvent, and gradually add the amino silane dropwise to the solution to form halloysite nanotubes. Dispersing the halloysite nanotubes in an organic solvent helps improve the dispersibility of the nanotubes, avoid nanotube aggregation, ensure uniformity in the reaction, thereby enhancing the surface reactivity of the nanotubes and promoting their reaction with the amino silane; the method of gradually adding the amino silane dropwise enables the amino silane to gradually contact the surface of halloysite and ensures the controllability of the reaction. The amino group contained in the amino silane molecule can undergo a chemical reaction with the hydroxyl group or other functional groups on the surface of halloysite to form a covalent bond between the amino silane and halloysite, which not only increases the surface hydrophilicity of halloysite but also endows it with a stronger drug carrier function, thus enhancing its binding ability with drug molecules or other active ingredients; in this way, the surface properties of the halloysite nanotubes are significantly improved, and further, the application effect in drug delivery and treatment is enhanced.

[0048] Further, in step S110, the pre-treatment is carried out in 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; using organic solvents including toluene, dichloromethane, ethanol, etc. can help the halloysite nanotubes to be uniformly dispersed in the solution, enhance the reactivity of their surface with the amino silane, and promote the progress of the modification reaction; the amino silane includes (3-aminopropyl)triethoxysilane, (3-aminopropyl)trimethoxysilane, etc., which 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 ability of halloysite.

[0049] The setting of the mass ratio of the organic solvent to the amino silane of 1:(80 - 100):(20 - 40) can ensure an appropriate concentration of the modification reaction, effectively control the reaction rate and the stability of the reaction product; an appropriate ratio helps the amino silane to fully react with the surface of the halloysite nanotubes, ensure the grafting amount of the amino functional groups, and thus improve the modification efficiency and surface activity of halloysite.

[0050] Generally speaking, during the pretreatment process, after the surface of halloysite nanotubes is aminosilylated, more amino groups are introduced on the surface. These amino groups can enhance the electrostatic adsorption and chemical reactivity between halloysite nanotubes and the manganese source in the potassium permanganate solution, contribute to the deposition of the manganese source on the surface of halloysite and the formation of manganese oxide nanoparticles, effectively improve the adhesion and dispersibility of manganese oxide, thereby enhancing the catalytic performance, and also enhancing its stability in the biological environment; moreover, the surface of the pretreated halloysite nanotubes has a higher affinity, enabling it to better adsorb and carry carbon monoxide-releasing molecules and manganese oxide nanoparticles. Through the amination treatment, the surface functional groups of halloysite can better support the deposition and catalytic activity of manganese oxide nanoparticles, laying the foundation for the subsequent synthesis of CMHs nanozymes and therapeutic effects.

[0051] Furthermore, in step S100, the alkaline solution includes at least one of sodium hydroxide and potassium hydroxide, which helps to promote the reaction between the surface modification of halloysite nanotubes and the manganese source. The alkaline solution can provide a suitable pH environment, enabling the manganese element in potassium permanganate to be effectively reduced to form manganese oxide nanoparticles and deposit on the surface of halloysite nanotubes; sodium hydroxide, potassium hydroxide or ammonia water can provide sufficient OH - , promoting the reduction reaction of the Mn element in potassium permanganate.

[0052] Preferably, the concentration range of the alkaline solution is between 0.1 M and 10 M, and the pH value is between 12 and 14. By adjusting the concentration of the alkaline solution and the reaction conditions, the formation 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.

[0053] The mass ratio of halloysite nanotubes, alkaline solution to potassium permanganate is 1:(0.7 - 1.0):(0.2 - 0.3), ensuring that the concentration of potassium permanganate in the reaction can sufficiently support the formation of manganese oxide, while not causing precipitation or decomposition of the product due to excessive reaction, thus maintaining the high efficiency and stability of the reaction.

[0054] Generally speaking, in step S100, halloysite nanotubes are dispersed in the alkaline solution, and potassium permanganate solution is added dropwise. Through in-situ reaction, manganese oxide nanoparticles are formed and deposited on the surface of halloysite nanotubes; by utilizing the reaction between the amino groups on the surface of halloysite nanotubes and the manganese source in potassium permanganate, manganese oxide is directly fixed on the surface of halloysite, thereby preparing a manganese oxide-halloysite nanotube composite material; manganese oxide nanoparticles have strong catalytic activity and can effectively scavenge ROS, while halloysite nanotubes provide a stable carrier, enhancing the stability and biocompatibility of the composite material, which is suitable for application in complex biological environments.

[0055] Preferably, the mass ratio of the manganese oxide-halloysite nanotube composite to the carbon monoxide-releasing molecule is 1:(0.5-5), which can ensure the effective combination of the composite and the CO-releasing molecule, and ensure that the CO-releasing molecule can play a role in an appropriate amount and rate during the treatment; a higher ratio of the CO-releasing molecule can increase the release amount of CO and 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 the carbon monoxide-releasing molecule is between 10-20 mg / mL, which ensures the appropriate concentration of the CO-releasing molecule in the solution and 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 lead to too fast a release rate and cause side effects. The pH value range of the alkaline solution containing the carbon monoxide-releasing molecule is between 7-9. 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 precisely releasing CO; too low a pH value may lead to the degradation of the CO-releasing molecule, and too high a pH value may affect the accuracy of its release control.

[0056] Generally speaking, in step S200, the alkaline solution containing the carbon monoxide-releasing molecule is added dropwise to the manganese oxide-halloysite nanotube composite, and further reacts to generate the CMHs nanozyme. The CO-releasing molecule interacts with the manganese oxide-halloysite nanotube composite to form a multifunctional nanozyme, which has the catalytic ability of the manganese oxide nanozyme and the carbon monoxide release function, enabling the CMHs nanozyme to precisely release carbon monoxide in the lesion area, while exerting antioxidant, anti-inflammatory and immunomodulatory effects, thereby achieving targeted therapy and improving the treatment effect, especially showing superior potential in controlling oxidative stress and reducing inflammatory responses.

[0057] Example 1

[0058] This example provides a CMHs nanozyme and its preparation method. The synthesis flow chart is as Figure 1As shown below, the specific operation steps are as follows: S110. Pretreatment: Under a nitrogen atmosphere, disperse the halloysite nanotube raw material in toluene to obtain a first solvent; dropwise add (3-aminopropyl)triethoxysilane (APTEs) into the first solvent, react for 12 h under magnetic stirring at a temperature of 90 °C, perform centrifugation to collect the precipitate, and wash the precipitate three times with ethanol and water respectively. After washing, the precipitate is freeze-dried to obtain halloysite nanotubes; wherein, the mass ratio of the halloysite nanotube raw material, toluene, and APTEs is 1:87:30; S100. Prepare a manganese oxide-halloysite nanotube composite material: Disperse the halloysite nanotubes in a sodium hydroxide solution, ultrasonically treat for 10 min, and dropwise add a potassium permanganate solution. After the addition is complete, obtain a mixed solution; oscillate the mixed solution with a vortex mixer for 5 min, place it under visible light and react at 90 °C for 4 h, centrifuge the precipitate, and wash the precipitate with deionized water until neutral. After freeze-drying, obtain a manganese oxide-halloysite nanotube composite material; wherein, the mass ratio of the halloysite nanotubes, sodium hydroxide, and potassium permanganate is 1:0.8:0.25; S200. Synthesis of CMHs nanozyme: Disperse the manganese oxide-halloysite nanotube composite material in water, dropwise add an alkaline solution containing CORM-401, stir in the dark at room temperature for 24 h, centrifuge and wash the precipitate with dd water, and after freeze-drying, obtain CMHs nanozyme, and its color change is as Figure 2 shown, changing from white to green-brown, and its morphology diagram is as Figure 4 shown; wherein, 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; the pH value of the alkaline solution containing CORM-401 is 8.

[0059] Example 2

[0060] This example provides a CMHs nanozyme and a preparation method thereof. The specific operation steps are as follows: S100. Prepare a manganese oxide-halloysite nanotube composite material: Disperse the halloysite nanotubes in a potassium hydroxide solution, ultrasonically treat for 10 min, and dropwise add a potassium permanganate solution. After the addition is complete, obtain a mixed solution; oscillate the mixed solution with a vortex mixer for 5 min, place it under visible light and react at 90 °C for 4 h, centrifuge the precipitate, and wash the precipitate with deionized water until neutral. After freeze-drying, obtain a manganese oxide-halloysite nanotube composite material; wherein, the mass ratio of the halloysite nanotubes, potassium hydroxide, and potassium permanganate is 1:0.7:0.2; S200. Synthesis of CMHs nanozyme: Disperse the manganese oxide-halloysite nanotube composite material in water, dropwise add an alkaline solution containing CORM-401, stir in the dark at room temperature for 24 h, centrifuge and wash the precipitate with dd water, and after freeze-drying, as Figure 2The resulting greenish-brown powder is CMHs nanozyme; wherein, the mass ratio of the manganese oxide-halloysite nanotube composite to CORM-401 is 1:0.5; the concentration of the alkaline solution containing CORM-401 is 15 mg / mL; the pH value of the alkaline solution containing CORM-401 is 7.

[0061] Example 3

[0062] This example provides a CMHs nanozyme and a preparation method thereof. The specific operation steps are as follows: S110. Pretreatment: Under a nitrogen atmosphere, disperse the halloysite nanotube raw material in ethanol to obtain a first solvent; dropwise add (3-aminopropyl)trimethoxysilane into the first solvent, react for 12 h under magnetic stirring at a temperature of 90 °C, centrifuge to collect the precipitate, and wash the precipitate three times with ethanol and water respectively. After washing, the precipitate is freeze-dried to obtain halloysite nanotubes; wherein, the mass ratio of the halloysite nanotube raw material, ethanol, and (3-aminopropyl)trimethoxysilane is 1:100:40; S100. Preparation of manganese oxide-halloysite nanotube composite: Disperse the halloysite nanotubes in a potassium hydroxide solution, ultrasonically treat for 10 min, and dropwise add a potassium permanganate solution. After the addition is complete, obtain a mixed solution; shake the mixed solution with a vortex mixer for 5 min, place it under visible light and react at 90 °C for 4 h, centrifuge the precipitate, and wash the precipitate with deionized water until neutral. After freeze-drying, obtain the manganese oxide-halloysite nanotube composite; wherein, the mass ratio of the halloysite nanotubes, potassium hydroxide, and potassium permanganate is 1:1:0.3; S200. Synthesis of CMHs nanozyme: Disperse the manganese oxide-halloysite nanotube composite in water, dropwise add an alkaline solution containing CORM-401, stir in the dark at room temperature for 24 h, centrifuge and wash the precipitate with dd water, and after freeze-drying, as Figure 2 shown, the resulting greenish-brown powder is CMHs nanozyme; wherein, the mass ratio of the manganese oxide-halloysite nanotube composite to CORM-401 is 1:5; the concentration of the alkaline solution containing CORM-401 is 20 mg / mL; the pH value of the alkaline solution containing CORM-401 is 9.

[0063]

Basic Physicochemical Properties and Structural Characterization

[0064] Fourier transform infrared spectroscopy: Add the CMHs nanozyme of Example 1 to a calibrated Fourier transform infrared spectrometer to start collecting the spectrum and analyze the spectrum results. As Figure 3 shown, through Fourier transform infrared spectroscopy analysis, we found that at 1550 cm -1 , 2930 cm -1 and 3459 cm -1New peaks were found, corresponding to the C-N, N-H, and C-H bending and stretching vibrations of APTES, further verifying the effectiveness of the modification; X-ray photoelectron spectroscopy: XPS (X-ray Photoelectron Spectroscopy), the freeze-dried powder of Example 1 was evenly fixed on the sample stage, the sample chamber was evacuated to vacuum, and detected by an X-ray photoelectron spectrometer and the obtained data was subjected to peak fitting analysis. The results are as Figure 5 shown; the analysis of XPS showed that the manganese oxide grown on the surface of Hs mainly exists in two oxidation states: Mn 3+ and Mn 4+ , with proportions of 27.14% and 72.86% respectively; this oxidation state distribution endows manganese oxide with strong oxidation ability and greatly improves its antioxidant activity.

[0065] CO release performance: The sample bottle containing 2 mL of Example 1 and a CO detector (BH-90) were placed in a sealable cylindrical glass container and sealed; different concentrations (0.1, 0.5, 1 or 10 mM) of H 2 O 2 solution was injected through the rubber stopper to trigger the release of CO, and the CO concentration (ppm) was continuously monitored; the results are as Figure 6 shown; under the condition of 0 mM of H 2 O 2 , very little CO was released within 1 hour. Under the conditions of 0.5, 1.0, and 1.5 mM of H 2 O 2 , the cumulative CO release amounts reached 1.45, 2.09, and 3.02 μmol after 1 hour respectively, thus indicating that CORM-401 in Example 1 has H 2 O 2 -responsive CO release behavior, indicating that CMHs can effectively release CO at the IBD site.

[0066] Cytotoxicity of CMHs: In this study, Raw264.7 and Caco-2 cells were cultured using macrophage and Caco-2 specific media provided by Wuhan Pure Biology. Normal macrophages refer to RAW264.7 cells untreated with LPS, and activated macrophages refer to RAW264.7 cells stimulated with LPS (5 μg / mL) for 24 hours; the cytotoxicity of CMHs to Raw264.7 and Caco-2 cells was evaluated by the CCK-8 assay and the live / dead staining method. The results are as Figure 7 , Figure 8 shown.

[0067] In vitro antioxidant activity of CMHs: The activated macrophages (2×10 5The cells / culture dishes) were inoculated into culture dishes and cultured for 12 hours. Then, the cells were exposed to the DMEM medium containing 100 μg / mL of Example 1 and cultured for another 12 hours. After discarding the medium, the cells were co-incubated with 300 μL of PBS containing 50% Hoechst and 5 μM of DCFH-DA and DAF-FM DA in DMEM medium for 30 minutes. Then, the supernatant was collected, and a multifunctional microplate reader (Biotech, Epoch 2, USA) was used to measure the fluorescence intensities of various probes in the supernatant. At the same time, the cells were rinsed with PBS, and an inverted fluorescence microscope was used to observe the distribution of various fluorescent probes in the cells. The excitation and emission wavelengths of the DCFH-DA probe were 644 nm and 665 nm, respectively, and the excitation and emission wavelengths of DAF-FM DA were 500 nm and 515 nm, respectively. The results are as Figure 9 and Figure 10 shown; thus proving 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 scavenging effect.

[0068] CMHs in vitro promoting intestinal mucosal barrier recovery performance: Caco-2 cells (2×10 4 cells / well) were inoculated into 96-well plates and cultured for 24 hours. Then, the cells were exposed to DMEM medium containing 500 μM of H 2 O 2 and cultured for another 24 hours. 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 the Cell LightTM EdU Apollo® 567 imaging kit was used to evaluate the proliferative effect of Example 1. The results are as Figure 11 shown. After treatment with 500 μM of H 2 O 2 , the number of EdU-positive cells decreased significantly, confirming the inhibitory effect of H 2 O 2 on cell proliferation. However, after treatment with MHs and CMHs, 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 recovered to the normal level, indicating that CMHs nanozymes have a significant protective effect on ROS-induced cell apoptosis.

[0069] Construction of DSS-induced colitis mice: Female BALB / c mice aged 6 - 8 weeks were selected as experimental subjects. The average weight of these mice was about 20 g, and they were in good health and highly adaptable. Before starting the experiment, we let the mice be raised in the experimental environment for one week to ensure that they adapted to the new environment and reduced the impact of environmental changes on the experimental results. After ensuring that the mice adapted to the environment, we continuously fed these mice with an aqueous solution containing 3% DSS to induce colitis. Female BALB / c mice were 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 were selected to ensure that they were in a stable stage of growth and development, thus reducing the interference of other factors on the experimental results.

[0070] To evaluate the analgesic effect of CMHs, three colitis mice treated with CMHs and three colitis mice treated with PBS were selected. Every other day, 150 μL of PBS or 1.5 mg / mL of CMHs was administered rectally to each group. The behavior of each mouse was monitored at a fixed time every day, and its food and water intake were accurately recorded. The food intake is as Figure 12 shown for 11 days to evaluate its behavioral response; as the severity of the disease increased, the water and food intake of the mice gradually decreased, which reflected that the basic physiological functions of the mice were severely inhibited. On the contrary, the mice treated with CMHs showed varying degrees of recovery in terms of water and food intake, indicating that CMHs treatment could relieve the pain of DSS-induced colitis mice and restore their normal physiological functions. These research results show that CMHs can not only regulate the underlying pathophysiology of IBD but also actively affect the pain-related behavioral changes of the disease.

[0071] The above tests demonstrated the excellent potential of PMC in the treatment of IBD.

[0072] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate 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 by an in-situ reaction, the carbon monoxide releasing molecule is CORM-401, and the carbon monoxide releasing molecule can release carbon monoxide; The preparation method of the manganese oxide nanozyme generated on the surface of the halloysite nanotube by in-situ reaction comprises the following steps: S110, pretreatment: dispersing the halloysite nanotube raw material in an organic solvent to obtain a first solvent; adding aminosilane dropwise into the first solvent to form the halloysite nanotube; 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, so that the manganese oxide nanozyme is generated on the surface of the halloysite nanotubes through an in-situ reaction, and the manganese oxide grown on the surface of the halloysite nanotubes is in two oxidation states: Mn 3+ and Mn 4+ .

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. 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 3, and the preparation method comprises the following steps: S110, pretreatment: dispersing the halloysite nanotube raw material in an organic solvent to obtain a first solvent; adding aminosilane dropwise into the first solvent to form the halloysite nanotube; 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.

5. The preparation method according to claim 4, characterized in that: 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).

6. The preparation method according to claim 4, 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).

7. The preparation method according to claim 4, characterized in that: 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 carbon monoxide-releasing molecules is between 10-20 mg / mL; and / or the pH value range of the alkaline solution containing carbon monoxide-releasing molecules is between 7-9.

8. A method for applying CMHs nanozyme, characterized in that: Use of the CMHs nanozyme as described in any one of claims 1 to 3 in the preparation and treatment of inflammatory bowel disease.

Images