A PMC nanozyme and preparation method thereof

Abstract

The present invention relates to the field of medical technology, and provides a PMC nanozyme and a preparation method thereof; the PMC nanozyme comprises: a core layer, the core layer comprises oxygen atoms, phosphate ions and polyoxometalate nanoclusters of a first transition metal element; a coating layer, at least part of the coating layer is fixed on the outer surface of the core layer, and the coating layer is formed by the coordination between a second transition metal and the oxygen atoms on the core layer; wherein the first transition metal element comprises Mo; the second transition metal element comprises Mn, and the coating layer comprises a CO donor for controlling the release of CO gas. The PMC nanozyme of the present invention can utilize polyoxometalate nanoclusters in combination with CO gas therapy to synergistically treat IBD, and on the basis of endowing the nanoclusters with anti-inflammatory and intestinal epithelial barrier repair functions, the catalytic activity is enhanced, and CO is released in an environment of overexpression of reactive oxygen species, thereby alleviating oxidative stress, inhibiting inflammatory responses, restoring intestinal immune balance, and promoting the repair of mucosal damage.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a PMC nanozyme and a preparation 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). Among them, more than 90% of patients with ulcerative colitis will experience rectal bleeding, significantly reducing their quality of life. According to statistics, the number of IBD patients worldwide will be 5 million in 2023, of which the number of patients in Europe will reach 2.5-3 million, and the direct economic losses will be as high as 460-560 million euros each year. At the same time, in developing countries such as China and India, the incidence of IBD has also shown a significant upward trend. Due to its high incidence and wide impact, IBD has become a global disease, imposing a heavy burden on the public health system.

[0003] Current treatment options mainly rely on nonspecific drugs, such as the anti-inflammatory agent 5-aminosalicylic acid, glucocorticoids, and immunomodulators, but these therapies have limited effects and lack specificity. In addition, small molecule drugs may increase the risk of serious cardiovascular events and affect male reproductive function when treating IBD. Therefore, there is an urgent need to develop new drugs to avoid these side effects and effectively relieve intestinal inflammation and repair mucosal damage.

[0004] Polyoxometalates (POMs) are anionic clusters formed by the coordination of highly redox-active transition metals with oxygen atoms. 5+ 、Mo 6+ and W of W 5+ , W 6+ ) has an adjustable electronic structure. POM has been widely used in the treatment of tumors, IBD, Alzheimer's disease and abscess infections in the past few decades. Studies have shown that molybdenum-based POM exhibits excellent effects in scavenging reactive oxygen species. Similar to nanozymes such as CeO2, POM exhibits a variety of antioxidant enzyme activities, such as catalase-like, superoxide dismutase-like and glutathione peroxidase-like, which can achieve cascade reactive oxygen scavenging functions at inflammatory sites. Compared with natural enzymes, POM has the advantages of high stability, multiple catalytic activities, low cost and easy mass production. In addition, its anionic properties enable it to target positively charged IBD lesions.

[0005] Although POM has great potential as an antioxidant, its ROS scavenging effect is highly dependent on Mo. 5+ 、Mo 6+ In addition, a single POM has limited effect in regulating the complex immune microenvironment of IBD and restoring the diversity of the microbiota. Therefore, how to enhance its ability to regulate the intestinal microbiota, promote the recovery of beneficial microbiota, and reduce the proportion of pathogenic bacteria has become a key challenge in current research. Summary of the invention

[0006] The present invention provides a PMC nanozyme and a preparation method thereof, and utilizes polyoxometalate nanoclusters combined with CO gas therapy to synergistically treat IBD; that is, POM and MnBr(CO)5 are combined to construct a multifunctional delivery carrier that can target the colon, and POM can deliver MnBr(CO)5 to the inflamed colon; and CO released by MnBr(CO)5 can significantly enhance the antioxidant activity of POM, and CO also has excellent anti-inflammatory activity, and can regulate intestinal flora and restore flora homeostasis; more importantly, the excellent antioxidant and anti-inflammatory activities of POM and CO can also reshape the intestinal mucosal immune homeostasis and efficiently repair the damaged intestinal mucosal barrier, thereby achieving efficient reshaping of the colon mucosal immune homeostasis, intestinal epithelial barrier homeostasis and flora homeostasis; and ultimately achieving efficient treatment of inflammatory bowel disease.

[0007] In order to solve the above problems, the first object of the present invention is to provide a PMC nanozyme;

[0008] The second object of the present invention is to provide a method for preparing PMC nanozyme;

[0009] To achieve the first purpose of the present invention, the present invention provides a PMC nanozyme, the PMC nanozyme comprising: a core layer, the core layer comprising oxygen atoms, phosphate ions and polyoxometalate nanoclusters of a first transition metal element; a coating layer, at least a portion of the coating layer is fixed on the outer surface of the core layer, and the coating layer is formed by the coordination effect between a second transition metal and the oxygen atoms on the core layer; wherein the first transition metal element comprises Mo; the second transition metal element comprises Mn, and the coating layer comprises a CO donor for controlling the release of CO gas.

[0010] Compared with the existing technology, the technical effects achieved by adopting this technical solution are: Mo-based polyoxometalate nanoclusters have excellent reactive oxygen scavenging ability; CO gas therapy, as a new treatment method, exhibits antibacterial, antioxidant and anti-inflammatory functions, and has almost no side effects. It can activate the Nrf2 signaling pathway, synergistically upregulate the expression of HO-1, enhance the activity of antioxidant enzymes, and effectively remove excessive reactive oxygen in IBD sites; in addition, CO inhibits the Nf-KB signaling pathway, reduces the level of inflammatory factors, and promotes the polarization of macrophages to anti-inflammatory M2. In addition, CO can also regulate the flora, restore the diversity of intestinal microorganisms, and reduce pathogenic bacteria.

[0011] Therefore, the PMC nanozyme of the present application has efficient catalytic activity, CO gas sustained-release ability and good stability through the ingenious combination of polyoxometalate nanoclusters and the coating layer; thereby not only improving the function of the nanozyme, but also successfully constructing a multifunctional nanocluster platform that can be targeted and attached to the surface of damaged colon mucosa, enabling it to achieve cascade clearance of reactive oxygen species and provide sustained sustained-release CO, which is helpful for anti-inflammation and tissue repair.

[0012] In addition, the coating layer can gradually release CO gas under environmental stimulation. Low concentrations of CO have anti-inflammatory, anti-apoptotic and vasodilation effects, showing potential for application in the treatment of cardiovascular diseases and tissue damage. By controlling the coating layer, toxicity problems caused by excessive release of CO can be avoided, further improving the safety and controllability of the system. At the same time, the combination of the coating layer and the core layer enhances the structural stability of the nanozyme and reduces the risk of catalyst deactivation. The synergistic effect between the Mn and Mo elements further improves the catalytic efficiency, which not only prolongs the service life of the nanozyme, but also improves its stability in the body.

[0013] In one technical solution of the present invention, the core layer is composed of 1 phosphorus atom and 12 transition metal oxygen cluster Mo atoms.

[0014] Compared with the existing technology, the technical effects achieved by adopting this technical solution are as follows: the POM structure has excellent redox activity, can efficiently remove reactive oxygen and reduce the level of oxidative stress in the body, which is particularly important in the treatment of scenarios related to inflammation, intestinal diseases, etc., and helps to reduce cell damage caused by reactive oxygen; Mo atoms have strong oxidative ability and can catalyze a variety of reactions, thereby improving the overall catalytic efficiency of nanozymes and enabling PMC nanozymes to respond quickly at sites of inflammation; at the same time, the POM core layer of the polyoxometalate cluster has a highly stable three-dimensional structure, which can maintain good performance in the body or in complex chemical environments and is not easily degraded, thereby effectively extending the service life of the nanozyme and ensuring its continued effect during the treatment process; finally, the high customizability of the POM structure allows further modification or introduction of other functional molecules on its surface to achieve the construction of a multifunctional nanoplatform.

[0015] In one technical solution of the present invention, the chemical formula of the coating layer is MnBr(CO)5.

[0016] Compared with the existing technology, the technical effects achieved by adopting this technical scheme are as follows: MnBr(CO)5 can gradually release CO, and low concentrations of CO have anti-inflammatory, antioxidant and anti-apoptotic effects, which help to alleviate tissue damage and inhibit inflammatory response; on the one hand, CO release can reduce the production of inflammatory factors by inhibiting the Nf-kB signaling pathway, and promote the polarization of macrophages to the M2 anti-inflammatory phenotype, and can also activate the Nrf2-HO-1 signaling pathway, increase the activity of antioxidant enzymes in the body, and further alleviate the damage caused by reactive oxygen. In addition, the release of CO also has a flora regulation function, which can restore the diversity of intestinal flora, reduce the proportion of harmful bacteria, and help treat diseases such as IBD; on the other hand, the coating layer not only protects the internal POM core layer, but also improves the structural stability of the nanozyme, reduces the risk of catalyst inactivation, and at the same time, the Mn element in the coating layer and the Mo element in the core layer produce a synergistic catalytic effect, further improving the reaction efficiency of the nanozyme.

[0017] In one technical solution of the present invention, the characteristic absorption peak of PMC nanozyme in Fourier transform infrared spectrum is 500cm -1 -2500cm -1 range; and / or the absorption peak of PMC nanozyme in UV-visible spectrum is in the range of 800nm-900nm; and / or the average size of PMC nanozyme is in the range of 200nm-220nm.

[0018] Compared with the existing technology, the technical effect achieved by adopting this technical solution is: -1 -2500cm -1 The infrared absorption peak corresponds to the vibration of polyoxometalate nanoclusters and carbonyl ligands. The release process and control effect of CO ligand can be judged by detecting the intensity of carbonyl absorption peak. The absorption peak at 800nm-900nm shows that the core layer and coating layer of the nanozyme have good light absorption properties, which is related to the electronic transition between transition metal elements (Mo, Mn), indicating that PMC nanozyme is responsive under light stimulation conditions and can be used for photocontrolled release of CO or activation of catalytic reactions. The size of the nanozyme is in the range of 200nm-220nm, indicating that it is suitable for transmission through the vascular system or intercellular spaces, and has good dispersibility and stability. The smaller size helps to target penetration into damaged areas, such as inflamed intestinal tissue, and improve the accuracy of treatment. The uniformity of the nanoparticle size reduces the risk of inducing an immune response in the body and improves its biocompatibility, while ensuring high catalytic efficiency and long-term stability.

[0019] To achieve the second object of the present invention, the present invention provides a method for preparing a PMC nanozyme, comprising the following steps:

[0020] S100, preparing a core layer: dissolving a salt containing a first transition metal element and a phosphate in deionized water, adding a reducing agent, sequentially performing a precipitation process, a centrifugal process and a washing process, freeze-drying, and grinding into powder to obtain a core layer material;

[0021] S200, preparing PMC nanozyme: dispersing the compound containing the second transition metal and the core layer material in an organic solvent, stirring them in the dark, resuspending and freeze-drying them to obtain the PMC nanozyme.

[0022] Compared with the prior art, the technical effect achieved by adopting this technical solution is as follows: the salt containing the first transition metal and phosphate are dissolved in deionized water, and a reducing agent is added to form polyoxometalate nanoclusters, which are precipitated by adjusting the solution conditions, and then impurities are removed by centrifugation, unreacted components are removed by washing, the pore structure of the material is maintained by freeze-drying, and finally a powdered core layer material is obtained by grinding; the polyoxometalate nanoclusters provide abundant redox active sites, effectively remove active oxygen, and enhance antioxidant and anti-inflammatory properties; the nanostructure of the material is maintained by freeze-drying and grinding, and the activity and long-term stability of the catalyst are improved.

[0023] The compound containing the second transition metal and the core layer material are dispersed in an organic solvent and stirred to ensure uniform distribution. The process is carried out under dark conditions to prevent decomposition or degradation of the CO ligand. The final PMC nanozyme is obtained after freeze-drying to achieve its stable structure and function. The anti-inflammatory, antibacterial and intestinal flora regulating effects are achieved through the controlled release of CO. The operation is carried out under dark conditions to avoid degradation of the CO ligand and ensure the controllability of its sustained-release function.

[0024] This preparation method not only ensures the catalytic activity of the nanozyme by combining the core layer and the coating layer, but also realizes the slow release control of CO gas and avoids the risk of toxicity. Freeze-drying and resuspension treatment ensure the structural integrity of the nanozyme in complex environments, control the synergistic effect between the coating layer and the core layer, slow down the deactivation of the catalyst, and improve the application life in biological and industrial environments.

[0025] In a technical solution of the present invention, S100 specifically includes:

[0026] S110, dissolving and stirring: dissolving ammonium molybdate and sodium dihydrogen phosphate in deionized water, and stirring to obtain a first solvent;

[0027] S120, adding a reducing agent: adding ascorbic acid to the first solvent while continuously stirring to obtain a second solvent;

[0028] S130, precipitation treatment: adding anhydrous ethanol to the second solvent to perform precipitation treatment, and wait for precipitation to precipitate to obtain a first mixture;

[0029] S140, centrifugation and washing: after the first mixture is centrifuged at 10000 rpm-12000 rpm, the lower precipitate is washed with a mixed solution of anhydrous ethanol and deionized water to obtain a first precipitate;

[0030] S150, drying and grinding: freeze-drying the first precipitate, grinding it into powder, and obtaining a core layer material.

[0031] Compared with the prior art, the technical effect achieved by the technical solution is as follows: ammonium molybdate and sodium dihydrogen phosphate are dissolved in deionized water and stirred thoroughly to obtain a uniform first solvent. Ammonium molybdate provides a Mo source and sodium dihydrogen phosphate provides phosphate to form a uniform solution, which provides a basis for the assembly of polyoxometalate nanoclusters. The stirring process ensures that the solute is fully dispersed, avoids local concentration differences, and promotes the uniformity of subsequent reactions. Under continuous stirring of the first solvent, ascorbic acid is gradually added to obtain a second solvent. Ascorbic acid is used as a mild reducing agent to reduce part of the Mo 6+ Restore to Mo 5+ , adjusting the redox state of the metal oxygen clusters to enhance the catalytic activity; ascorbic acid can avoid violent reactions, so that the nanocluster structure is gradually generated, ensuring the controllability of the particle size; adding anhydrous ethanol to the second solvent to precipitate the product to obtain the first mixture; ethanol changes the polarity of the solvent, induces the precipitation of polyoxometalate nanoclusters, and improves the yield; the control of the precipitation process helps to generate nanoparticles with uniform particle size, ensuring the catalytic efficiency and stability of the material; centrifuging at 10000rpm-12000rpm, washing the lower precipitate with a mixture of anhydrous ethanol and deionized water for 3-5 times to remove impurities, Centrifugation ensures the separation of impurities in the precipitate from the supernatant, thereby improving the purity of the core layer material. Multiple washings are performed to remove unreacted precursors and by-products to avoid interference with subsequent steps. The first precipitate is freeze-dried and then ground into powder to obtain the core layer material. Freeze-drying avoids high temperature from destroying the structure of the nanomaterial, maintains a high specific surface area and porosity, and is conducive to the catalytic reaction. The powderization treatment makes the material easier to disperse in the solvent, thereby improving the coating uniformity in subsequent steps. The preparation method of the present application ensures that the core layer material has excellent catalytic performance, long-term stability and biocompatibility, laying a solid foundation for the overall performance of the PMC nanozyme.

[0032] In one technical solution of the present invention, in S110, the molar concentration ratio of ammonium molybdate to sodium dihydrogen phosphate is (1.5-2):1; and / or in S110, the stirring treatment time is between 15min-25min; and / or in S120, the molar ratio of ascorbic acid to ammonium molybdate and sodium dihydrogen phosphate in the first solvent is (10-20):1; and / or in S140, the centrifugal treatment time is between 10min-20min.

[0033] Compared with the prior art, the technical effect achieved by adopting the technical scheme is as follows: the molar concentration ratio of ammonium molybdate to sodium dihydrogen phosphate is (1.5-2):1, the ratio of ammonium molybdate to phosphate is adjusted to generate a core layer material with uniform structure and good stability, a higher Mo ratio is beneficial to increase the active sites in the Mo oxygen clusters, improve the ability to remove active oxygen, and a suitable ratio reduces the residual excess phosphate, thereby ensuring the purity of the core layer material; the stirring treatment time is 15min-25min, and sufficient stirring is performed to uniformly disperse the ammonium molybdate and sodium dihydrogen phosphate, ensure that the reactants are fully mixed, avoid local deposition, and ensure that the solution is uniform for the subsequent precipitation process. The stirring is completed in a short time, which helps to reduce the agglomeration phenomenon in the solution and improve the dispersibility of the product; the molar ratio of ascorbic acid to ammonium molybdate and sodium dihydrogen phosphate is (10-20):1, and a higher ascorbic acid ratio helps to completely reduce Mo 6+ The invention discloses a method for preparing a low-valent Mo-oxygen salt cluster and optimizing the redox properties of the polyoxometalate cluster, thereby enhancing the performance of the polyoxometalate cluster in active oxygen scavenging, and preventing the high-valent Mo oxygen cluster from being unstable and deactivated. In addition, sufficient ascorbic acid inhibits the formation of by-products, thereby ensuring that the reaction process is controllable. The centrifugation time is 10 min-20 min. Appropriate centrifugation time ensures that the precipitate is completely separated from the impurities in the supernatant, improves the purity of the core layer material, avoids particle agglomeration or incomplete precipitation due to overtime, ensures the uniform dispersion of the core layer material, reduces the possibility of material deactivation, and provides a good precursor for the subsequent coating layer preparation.

[0034] In a technical solution of the present invention, S200 specifically includes:

[0035] S210, dispersion reaction: dispersing MnBr(CO)5 and the core layer in methanol, and stirring in the dark to obtain a third solvent;

[0036] S220, removing methanol: removing methanol from the third solvent using a rotary evaporator, leaving the PMC complex;

[0037] S230, resuspension and freeze-drying: The PMC complex is resuspended with deionized water, and then freeze-dried to obtain the final PMC nanozyme.

[0038] Compared with the prior art, the technical effect achieved by adopting this technical solution is as follows: MnBr(CO)5 and the core layer material are dispersed in methanol and stirred under dark conditions. Methanol, as a good organic solvent, can promote the uniform mixing of MnBr(CO)5 and the core layer material, so that the coating layer is better attached to the surface of the core layer. The operation under dark conditions ensures the stability of the chemical structure of the coating layer, optimizes the interaction between MnBr(CO)5 and the core layer, and improves the overall structural stability and catalytic efficiency of the composite material.

[0039] A rotary evaporator was used to remove methanol, leaving behind the PMC nanozyme complex. Methanol was completely removed to prevent it from remaining in the composite material, thereby preventing its side effects in biological applications or catalytic processes. After methanol was removed, MnBr(CO)5 was tightly bound to the core layer to form a stable coating layer, which improved the mechanical and chemical stability of the material.

[0040] The PMC complex was resuspended in deionized water and then freeze-dried to obtain the final PMC nanozyme. The uniform solution formed after resuspension helps to avoid particle agglomeration and ensure good dispersibility of the final product. Freeze-drying completely removes water, keeps the nanozyme in a powder state, prolongs its storage life and prevents activity loss. The freeze-drying process is gentle and will not destroy the structure of the MnBr(CO)5 ligand and the core layer material, ensuring the catalytic activity and CO sustained-release performance of the nanozyme.

[0041] In one technical solution of the present invention, in S210, the molar concentration ratio of MnBr(CO)5 to the core layer is 1:(3-5); and / or in S210, the stirring treatment time is 20h-30h.

[0042] Compared with the existing technology, the technical effect achieved by adopting this technical scheme is: the molar concentration ratio of MnBr(CO)5 to the core layer is 1: (3-5). By controlling the ratio of MnBr(CO)5 to the core layer, the thickness of the coating layer is ensured to be appropriate, thereby regulating the release rate of CO and preventing excessive release from causing toxicity. A higher proportion of the core layer enhances the catalytic performance and reactive oxygen scavenging effect of the nanozyme; at the same time, MnBr(CO)5 ensures the effective sustained release of anti-inflammatory CO, enhances the synergistic anti-inflammatory and antioxidant effects, and a high core layer ratio helps to improve the mechanical strength and structural stability of the PMC nanozyme and reduce the risk of coating shedding.

[0043] The stirring time is 20h-30h. Long-term stirring promotes the full mixing of MnBr(CO)5 and the core layer to form a uniform coating layer, improves the stability and functional consistency of the nanozyme, and facilitates the physical adsorption and chemical interaction between MnBr(CO)5 and the core layer, thereby reducing the risk of separation and failure during subsequent use, allowing the complex to maintain good dispersion and improve its catalytic efficiency and biological performance.

[0044] After adopting the technical solution of the present invention, the following technical effects can be achieved:

[0045] (1) The polyoxometalate nanoclusters in the nuclear layer have strong antioxidant capacity and can quickly remove excess reactive oxygen species in damaged parts of the intestine, thereby alleviating oxidative stress. Inhibiting the excessive generation of reactive oxygen species helps to slow down further damage to the intestinal mucosa and reduce tissue inflammation.

[0046] (2) The coating layer can slowly release CO under controlled conditions, effectively activating the Nrf2 / HO-1 signaling pathway, increasing the activity of antioxidant enzymes, and further inhibiting oxidative stress. In addition, CO also has anti-inflammatory and anti-apoptotic effects, which can inhibit the Nf-kB signaling pathway and reduce the expression of inflammatory factors, thereby inhibiting the inflammatory response. In addition, the release of CO can also restore the diversity of intestinal flora, reduce the proportion of pathogenic bacteria, promote the proliferation of beneficial bacteria, maintain intestinal health, help alleviate the symptoms of IBD and reduce the risk of recurrence. At the same time, the vasodilatory effect of CO can improve local blood circulation, promote the healing of intestinal damaged areas, and enable PMC nanozymes to effectively adhere to the damaged intestinal mucosal surface, thereby ensuring its continued action and improving the therapeutic effect.

[0047] (3) PMC nanozymes combine multiple functions of reactive oxygen species scavenging, CO slow-release anti-inflammatory, flora regulation and tissue repair. They can reduce side effects while targeting, reduce intestinal inflammation, and relieve IBD symptoms such as abdominal pain, diarrhea and intestinal bleeding. In addition, since the slow release of CO is regulated by environmental factors, this avoids the toxicity that may be caused by excessive CO, ensuring the safety and controllability of the treatment, thereby improving the intestinal microecological system, restoring the intestinal mucosal barrier function, helping to prevent disease recurrence and prolonging the remission period. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] 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.

[0049] Figure 1 Fourier transform infrared spectra and ultraviolet spectrophotometer images of POM, MnBr(CO)5 and PMC nanozymes provided in Example 1 of the present invention;

[0050] Figure 2 The particle size and morphology of the PMC nanozyme provided in Example 1 of the present invention and the corresponding EDS elemental analysis diagram;

[0051] Figure 3XPS spectra of POM and PMC nanozymes provided in Example 1 of the present invention;

[0052] Figure 4 XPS spectra of POM-Mo 3d and PMC-Mo 3d provided in Example 1 of the present invention;

[0053] Figure 5 The XPS spectra of MnBr(CO)5-Mn 2p and PMC-Mn 2p provided in Example 1 of the present invention;

[0054] Figure 6 Zeta potential diagram of PMC nanozyme, POM and MnBr(CO)5 provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0055] 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.

[0056] The present invention provides a PMC nanozyme, which includes: a core layer, the core layer includes oxygen atoms, phosphate ions and polyoxometalate nanoclusters of a first transition metal element, and the first transition metal element includes Mo; the core layer has excellent antioxidant activity, but is easy to aggregate at the site of inflammatory bowel disease to form larger nanoparticles, which will reduce the antioxidant activity of the core layer;

[0057] A coating layer, at least part of which is fixed on the outer surface of the core layer, the coating layer is formed by the coordination between the second transition metal and the oxygen atoms on the core layer, the second transition metal element includes Mn, and the coating layer contains a CO donor for controlling the release of CO gas; MnBr(CO)5 is a CO donor molecule with the characteristic of releasing CO in response to oxidation, and can respond to the release of CO by utilizing the high concentration of ROS in the inflammatory bowel disease site; CO has excellent antioxidant and anti-inflammatory and intestinal flora regulating effects; but MnBr(CO)5 is insoluble in water and needs to be delivered to the inflammatory bowel disease site with the aid of a delivery carrier; for example, the anterior core layer has antioxidant activity, but is prone to aggregation in the inflammatory bowel disease site, which makes it an ideal targeted delivery carrier for colon drugs;

[0058] To this end, we combined POM with MnBr(CO)5 to construct a multifunctional delivery carrier that can target the colon; that is, POM can target and deliver MnBr(CO)5 to the inflamed colon; and the CO released by MnBr(CO)5 can significantly enhance the antioxidant activity of POM, and CO also has excellent anti-inflammatory activity, and can regulate intestinal flora and restore flora homeostasis; more importantly, the excellent antioxidant and anti-inflammatory activity of POM and CO can also reshape the intestinal mucosal immune homeostasis and efficiently repair the damaged intestinal mucosal barrier, thereby achieving efficient reshaping of the colon mucosal immune homeostasis, intestinal epithelial barrier homeostasis and flora homeostasis; ultimately achieving efficient treatment of inflammatory bowel disease.

[0059] The preparation method of the PMC nanozyme of the present application comprises the following steps:

[0060] S100, preparing a core layer: dissolving a salt containing a first transition metal element and a phosphate in deionized water, adding a reducing agent, sequentially performing a precipitation process, a centrifugal process and a washing process, freeze-drying, and grinding into powder to obtain a core layer material;

[0061] Preferably, ammonium molybdate and sodium dihydrogen phosphate are dissolved in deionized water and stirred sufficiently to obtain a uniform first solvent. Ammonium molybdate provides a Mo source and sodium dihydrogen phosphate provides phosphate to form a uniform solution, which provides a basis for the assembly of polyoxometalate nanoclusters. The stirring process ensures that the solute is fully dispersed, avoids local concentration differences, and promotes the uniformity of subsequent reactions.

[0062] Furthermore, the molar concentration ratio of ammonium molybdate to sodium dihydrogen phosphate is (1.5-2): 1. The ratio of ammonium molybdate to phosphate is adjusted to generate a core layer material with uniform structure and good stability. A higher Mo ratio is beneficial to increase the active sites in the Mo oxygen clusters and improve the ability to remove active oxygen. The appropriate ratio reduces the residual excess phosphate and ensures the purity of the core layer material. The stirring treatment time is 15min-25min. The ammonium molybdate and sodium dihydrogen phosphate are evenly dispersed during sufficient stirring to ensure that the reactants are fully mixed and avoid local deposition. The appropriate stirring time ensures that the solution is uniform, which lays the foundation for controlling the particle size of the core layer material in the subsequent precipitation process. Completing the stirring in a short time helps to reduce agglomeration in the solution and improve the dispersibility of the product.

[0063] Under continuous stirring of the first solvent, ascorbic acid was gradually added to obtain a second solvent. Ascorbic acid was used as a mild reducing agent to reduce part of Mo 6+ Restore to Mo5 +, adjusting the redox state of the metal oxygen clusters to improve the catalytic activity, ascorbic acid can avoid violent reactions, so that the nanocluster structure is gradually generated, ensuring the controllability of the particle size; adding anhydrous ethanol to the second solvent to precipitate the product to obtain the first mixture, ethanol changes the polarity of the solvent, induces the precipitation of polyoxometalate nanoclusters, and improves the yield. Control of the precipitation process helps to generate nanoparticles with uniform particle size, ensuring the catalytic efficiency and stability of the material;

[0064] Furthermore, the molar ratio of ascorbic acid to ammonium molybdate and sodium dihydrogen phosphate is (10-20):1. A higher ascorbic acid ratio is helpful to completely reduce Mo 6+ The low-valent state optimizes the redox properties of the polyoxometalate clusters, thereby enhancing the performance of the polyoxometalate clusters in active oxygen scavenging, while preventing the high-valent Mo oxygen clusters from being unstable and deactivated. In addition, sufficient ascorbic acid inhibits the formation of by-products, ensuring that the reaction process is controllable.

[0065] Centrifuge at 10000rpm-12000rpm, wash the lower precipitate with a mixture of anhydrous ethanol and deionized water for 3-5 times to remove impurities, centrifuge to ensure that the precipitate is separated from the impurities in the supernatant, improve the purity of the core layer material, and wash multiple times to remove unreacted precursors and by-products to avoid interference with subsequent steps;

[0066] Furthermore, the centrifugation time is 10min-20min. Appropriate centrifugation time ensures that the precipitate is completely separated from impurities in the supernatant, improves the purity of the core layer material, avoids particle agglomeration or incomplete precipitation due to overtime, ensures the uniform dispersion of the core layer material, reduces the possibility of material inactivation, and provides a good precursor for the subsequent coating layer preparation.

[0067] The first precipitate is freeze-dried and then ground into powder to obtain the core layer material. Freeze-drying avoids high temperature from destroying the structure of the nanomaterial, maintains a high specific surface area and porosity, and is conducive to the catalytic reaction. The powderization treatment makes the material easier to disperse in the solvent and improves the coating uniformity in subsequent steps. The preparation method of the present application ensures that the core layer material has excellent catalytic performance, long-term stability and biocompatibility, laying a solid foundation for the overall performance of PMC nanozymes.

[0068] S200, preparing PMC nanozyme: dispersing the compound containing the second transition metal and the core layer material in an organic solvent, stirring them in the dark, resuspending and freeze-drying them to obtain the PMC nanozyme.

[0069] Preferably, MnBr(CO)5 and the core layer material are dispersed in methanol and stirred in the dark. Methanol, as a good organic solvent, can promote the uniform mixing of MnBr(CO)5 and the core layer material, so that the coating layer is better attached to the surface of the core layer, ensuring the stability of the chemical structure of the coating layer, optimizing the interaction between MnBr(CO)5 and the core layer, and improving the overall structural stability and catalytic efficiency of the composite material;

[0070] Furthermore, the molar concentration ratio of MnBr(CO)5 to the core layer is 1:(3-5). By controlling the ratio of MnBr(CO)5 to the core layer, the thickness of the coating layer is ensured to be appropriate, thereby regulating the release rate of CO and preventing excessive release from causing toxicity. A higher proportion of the core layer enhances the catalytic performance and reactive oxygen scavenging effect of the nanozyme. At the same time, MnBr(CO)5 ensures the effective sustained release of anti-inflammatory CO and enhances the synergistic anti-inflammatory and antioxidant effects. A high core layer ratio helps to improve the mechanical strength and structural stability of the PMC nanozyme and reduce the risk of coating shedding.

[0071] The stirring time is 20h-30h. Long-term stirring promotes the full mixing of MnBr(CO)5 and the core layer to form a uniform coating layer, improves the stability and functional consistency of the nanozyme, and facilitates the physical adsorption and chemical interaction between MnBr(CO)5 and the core layer, thereby reducing the risk of separation and failure during subsequent use, allowing the complex to maintain good dispersion and improve its catalytic efficiency and biological performance.

[0072] Preferably, a rotary evaporator is used to remove methanol, leaving the PMC nanozyme complex, and the methanol is completely removed to prevent it from remaining in the composite material, thereby preventing its side effects in biological applications or catalytic processes. After the methanol is removed, MnBr(CO)5 is tightly combined with the core layer to form a stable coating layer, thereby improving the mechanical and chemical stability of the material;

[0073] The PMC complex was resuspended in deionized water and then freeze-dried to obtain the final PMC nanozyme. The uniform solution formed after resuspension helps to avoid particle agglomeration and ensure good dispersibility of the final product. Freeze-drying completely removes water, keeps the nanozyme in a powder state, prolongs its storage life and prevents activity loss. The freeze-drying process is gentle and will not destroy the structure of the MnBr(CO)5 ligand and the core layer material, ensuring the catalytic activity and CO sustained-release performance of the nanozyme.

[0074] PMC nanozymes combine the multiple functions of reactive oxygen species scavenging, CO sustained-release anti-inflammatory, flora regulation and tissue repair, providing an innovative, effective and safe solution for the treatment of inflammatory bowel disease. It can reduce side effects while targeting the disease and has broad clinical application prospects.

[0075] Embodiment 1

[0076] This embodiment provides a PMC nanozyme and a preparation method thereof, and the specific operation steps are as follows:

[0077] S110, dissolving and stirring: dissolving 5 mM ammonium molybdate and 2.92 mM sodium dihydrogen phosphate in 20 mL ddH2O, that is, the molar concentration ratio of ammonium molybdate to sodium dihydrogen phosphate is 1.71:1, and vigorously stirring for 20 minutes to obtain a first solvent;

[0078] S120, adding a reducing agent: under the condition of continuous stirring of the first solvent, adding 400 mg of ascorbic acid, that is, the molar ratio of ascorbic acid to ammonium molybdate and sodium dihydrogen phosphate in the first solvent is 14.36:1, and reacting for 90 minutes to obtain a second solvent;

[0079] S130, precipitation treatment: adding 80 mL of anhydrous ethanol to the second solvent for precipitation treatment, and waiting for precipitation to obtain a first mixture;

[0080] S140, centrifugation and washing: after the first mixture is centrifuged at 10000 rpm-12000 rpm for 15 min, the lower precipitate is washed three times with a mixed solution of anhydrous ethanol and deionized water to obtain a first precipitate;

[0081] S150, drying and grinding: freeze-drying the first precipitate and grinding it into powder to obtain a core layer material;

[0082] S210, dispersion reaction: 100 mg of MnBr(CO)5 and 300 mg of the core layer are dispersed in methanol, that is, the molar concentration ratio of MnBr(CO)5 to the core layer is 1:3, and stirred for 24 hours in the dark to obtain a third solvent;

[0083] S220, removing methanol: removing methanol from the third solvent using a rotary evaporator, leaving the PMC complex;

[0084] S230, resuspension and freeze-drying: The PMC complex is resuspended with deionized water, and then freeze-dried to obtain the final PMC nanozyme.

[0085] Embodiment 2

[0086] This embodiment provides a PMC nanozyme and a preparation method thereof. The specific operation is as in Embodiment 1, except that, in S110, dissolution and stirring: the molar concentration ratio of ammonium molybdate to sodium dihydrogen phosphate is 1.5:1, and the stirring treatment time is 15 minutes; in S120, the molar ratio of ascorbic acid to ammonium molybdate and sodium dihydrogen phosphate in the first solvent is 10:1, and in S140, the centrifugal treatment time is 10 minutes;

[0087] In S210, the molar concentration ratio of MnBr(CO)5 to the core layer is 1:5; the stirring treatment time is 20h.

[0088] Embodiment 3

[0089] This embodiment provides a PMC nanozyme and a preparation method thereof. The specific operation is as in Embodiment 1, except that, in S110, dissolution and stirring: the molar concentration ratio of ammonium molybdate to sodium dihydrogen phosphate is 2:1, and the stirring treatment time is 25 min; in S120, the molar ratio of ascorbic acid to ammonium molybdate and sodium dihydrogen phosphate in the first solvent is 20:1, and in S140, the centrifugal treatment time is 20 min;

[0090] In S210, the molar concentration ratio of MnBr(CO)5 to the core layer is 1:4.5; the stirring treatment time is 30h.

[0091] Basic physical and chemical properties and structural characterization

[0092] This study used Fourier transform infrared spectroscopy, ultraviolet spectrophotometer, transmission electron microscopy and X-ray diffraction to characterize the physical and chemical properties of Example 1, proving that the PMC nanozyme prepared by the present invention successfully adsorbed MnBr(CO)5 onto the POM surface, and proved that PMC has better antioxidant properties than POM; these research results not only provide a strong basis for a deep understanding of the structure-activity relationship of PMC nanozyme, but also lay a solid foundation for further expanding its application in biomedicine and environmental science.

[0093] Fourier transform infrared spectroscopy: The freeze-dried PMC powder of Example 1 was added to a calibrated Fourier transform infrared spectrometer to collect spectra and analyze the spectral results. Figure 1 As shown in Figure 1, by Fourier transform infrared spectroscopy analysis, we found that the -1 In the region, POM is at 1045, 926, 831, 650 and 549 cm -1 There are specific absorption peaks at 3200cm, which correspond to P-Oa bond, Mo-Od bond, Mo-Ob-Mo bond, Mo-Oc-Mo bond and δ(PO) vibration. These results prove that Keggin-type POM nanoclusters have been prepared. In addition, at 3200cm -1 There is a broad characteristic band at 400 nm, which is mainly due to the asymmetric stretching of the -OH group. The Fourier transform infrared spectrum of PMC is similar to that of POM, both retaining the complete Keggin structure and adding the unique 1986 and 617 cm -1 Characteristic peak.

[0094] UV spectrophotometer: Place 1 mL of POM, PMC and MnBr(CO)5 with the same Mo element concentration in a plastic cuvette and record the absorbance in the range of 200-1100 nm using a UV spectrophotometer. Figure 1 -B shows that the reduced activated POM has an absorption peak at 870nm, which is related to the charge transfer between metals of POM, giving POM a unique blue color. MnBr(CO)5 has a characteristic absorption peak at 380nm. These results show that we have successfully loaded MnBr(CO)5 into POM nanoclusters.

[0095] Malvern particle size analyzer: Use 1 mL of POM, PMC and MnBr(CO)5 suspension in a special dish and repeat the test three times to measure the particle size and Zeta potential of the sample. Figure 2 -A, the average size of PMC measured by the Malvern particle size analyzer is about 210nm, indicating that PMC has good dispersibility in aqueous solution, and the size of 210nm is more conducive to being phagocytosed and taken up by cells.

[0096] Energy Dispersive Spectrometer (EDS) is used to analyze the types and contents of the elements in the micro-region of the material. The formed nanoclusters were further analyzed by energy spectrum. Figure 2 -B, the P, O, and Mo elements unique to POM are distributed throughout the cluster, and the three MnBr(CO)5-specific elements Mn, Br, and C are successfully doped into POM, further proving that we have successfully prepared the PMC nanozyme loaded with CO.

[0097] X-ray Photoelectron Spectroscopy: Fix the freeze-dried powder evenly on the sample stage, evacuate the sample chamber, and use X-ray photoelectron spectrometer to detect and perform peak fitting analysis on the obtained data. Through elemental analysis of the XPS total spectrum, we further confirmed the successful preparation of PMC nanozyme, such as Figure 3 As shown. In order to gain a deeper understanding of the valence changes of Mo and Mn elements in PMC nanoclusters, we performed peak fitting analysis on the XPS results of each element. Figure 4 As shown, Mo in POM after reduction activation 5+ The proportion of Mo in PMC is 13.32%. 5+ The proportion of Mo increased to 17.06%. This indicates that in the process of preparing PMC, more 6+ Gained electrons and was reduced to Mo 5+ .Mo 5+The increase in Mo can improve the ROS scavenging ability of PMC nanozymes. 5+ It can remove ROS overexpressed in IBD sites and play an antioxidant role, thereby repairing intestinal damage. This discovery provides strong support for the application of PMC in the biomedical field. In addition, compared with MnBr(CO)5, after combining with POM, the Mn in PMC 4+ The proportion of Figure 5 As shown. 4+ The ratio of ·O is positively correlated with the catalase-like, superoxide dismutase-like, and glutathione peroxidase-like activities of the nanozymes. CAT and GSH-Px can synergistically regulate the level of H2O in the body, while SOD can convert ·O 2- Converted into O2 and H2O, they jointly remove overexpressed reactive oxygen species under oxidative stress. 5+ and Mn 4+ With the further increase of the ratio, the prepared PMC nanoclusters showed better potential ROS scavenging performance than POM.

[0098] In order to explore the interaction mechanism between POM and MnBr(CO)5, the Zeta potential of POM, MnBr(CO)5 and PMC was detected. Figure 6 As shown in the figure, the electronegativity of PMC is weakened, which indicates that there is an electrostatic interaction between POM and MnBr(CO)5. This finding is of great significance for understanding the structure-activity relationship of PMC nanozymes and optimizing their performance.

[0099] Evaluation of the therapeutic effect of PMC nanozymes on DSS-induced colitis in mice

[0100] 1. Construction of DSS-induced colitis mice

[0101] BALB / c female mice aged 6-8 weeks were selected as experimental subjects. These mice have an average weight of about 20g, are healthy and have strong adaptability. Before starting the experiment, we kept the mice in the experimental environment for three days 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 fed these mice with an aqueous solution containing 3% dextran sulfate sodium to induce acute colitis. Dextran sulfate sodium is a common chemical inducer used to simulate the pathological process of human colitis. By continuously feeding an aqueous solution containing dextran sulfate sodium, we successfully induced an acute colitis model. The choice of this model is based on its effectiveness, reliability and wide applicability. BALB / c female 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 are in a stable stage of growth and development, thereby reducing the interference of other factors on the experimental results.

[0102] 2. Treatment plan and efficacy observation

[0103] After five consecutive days of 3% DSS induction, we randomly divided the mice into five groups, each receiving a different rectal administration treatment, as follows:

[0104] 1) Normal group: The mice in this group received 200 μL of saline injection each time. As the control group, this group of mice did not receive any treatment.

[0105] 2) DSS group: This group of mice also received 200 μL of saline injection each time. This group was used to observe the natural course of DSS-induced colitis.

[0106] 3) Dex-p group: This group of mice received 200 μL of Dex-p solution (75 μg / mL) injection each time. Dex-p is a known anti-inflammatory drug used for comparative experiments.

[0107] 4) POM group: This group of mice received 200 μL of POM solution (Mo=750 μg / mL) injection each time. POM is a material with certain antioxidant effects, which is used to evaluate its therapeutic effect.

[0108] 5) PMC group: This group of mice received 200 μL of PMC solution (Mo=750 μg / mL) injection each time. PMC is the main research object of this experiment, aiming to evaluate its therapeutic effect on colitis.

[0109] Compared with the normal group, the body weight of mice in other groups began to show a significant downward trend on the third day. However, starting from the third day, the mice were given rectal administration (PBS, Dex-p, POM and PMC), and the body weight of mice between the treatment groups began to show obvious differences. On the 11th day, the body weight of mice in the DSS group after PBS treatment dropped sharply, with a body weight loss of 27.83%, while the average body weight of mice in the Dex-p treatment group, POM treatment group and PMC treatment group decreased by 18.28%, 22.24% and 9.72%, respectively, showing a certain improvement effect. In particular, the PMC treatment group showed a significant slowdown in the trend of weight loss on the 5th day of DSS induction, indicating that PMC effectively inhibited the severity of DSS-induced colitis in mice.

[0110] In addition to observing weight changes, we also closely monitored the stool viscosity and anal bleeding of each mouse. Due to the occurrence of colitis, the feces of mice also changed to a certain extent. Unlike the hard and formed normal feces of healthy mice, the feces of mice with colitis gradually became soft and moist as the number of days of induction increased, and finally bloody stools could be clearly observed with the naked eye. Mice treated with PMC had less occult blood in their stools during the treatment, and their feces were often formed.

[0111] The above tests demonstrate the excellent potential of PMC in the treatment of inflammatory bowel disease.

[0112] 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 method for preparing PMC nanozyme, characterized in that: The PMC nanozyme comprises: a core layer comprising oxygen atoms, phosphate ions and polyoxometalate nanoclusters of a first transition metal element; a coating layer, at least a portion of which is fixed to the outer surface of the core layer, the coating layer being formed by coordination between the second transition metal and oxygen atoms on the core layer; Wherein, the first transition metal element includes Mo; the core layer is composed of 1 phosphorus atom and 12 transition metal oxygen cluster Mo atoms; The second transition metal element includes Mn, and the coating layer includes a CO donor for controlling the release of CO gas, the chemical formula of the coating layer is MnBr(CO)5, and the molar concentration ratio of the MnBr(CO)5 to the core layer is 1:(3-5); The preparation method comprises the following steps: S100, preparing a core layer: dissolving a salt containing the first transition metal element and a phosphate in deionized water, adding a reducing agent, sequentially performing a precipitation process, a centrifugal process and a washing process, freeze-drying, and grinding into powder to obtain a core layer material; S200, preparing the PMC nanozyme: dispersing the compound containing the second transition metal and the core layer material in an organic solvent, stirring in the dark, resuspending and freeze-drying to obtain the PMC nanozyme; S100 specifically includes: S110, dissolving and stirring: dissolving ammonium molybdate and sodium dihydrogen phosphate in deionized water, and stirring to obtain a first solvent; S120, adding a reducing agent: adding ascorbic acid while continuously stirring the first solvent to obtain a second solvent; S130, precipitation treatment: adding anhydrous ethanol to the second solvent, performing the precipitation treatment, and waiting for precipitation to obtain a first mixture; S140, centrifugal treatment and washing treatment: after the first mixture is subjected to the centrifugal treatment at 10000 rpm-12000 rpm, the lower layer of precipitate is washed with a mixed solution of anhydrous ethanol and deionized water to obtain a first precipitate; S150, drying and grinding: subjecting the first precipitate to the freeze-drying treatment, grinding it into powder, and obtaining the core layer material.

2. The method for preparing PMC nanozyme according to claim 1, characterized in that: The characteristic absorption peak of the PMC nanozyme in Fourier transform infrared spectrum is 500 cm -1 -2500cm -1 within the scope; and / or The absorption peak of the PMC nanozyme in the UV-visible spectrum is in the range of 800nm-900nm; and / or The average size of the PMC nanozyme is in the range of 200nm-220nm.

3. The preparation method according to claim 1, characterized in that: In S110, the molar concentration ratio of the ammonium molybdate to the sodium dihydrogen phosphate is (1.5-2):1; and / or In S110, the stirring time is between 15 min and 25 min; and / or In S120, the molar ratio of the ascorbic acid to the ammonium molybdate and sodium dihydrogen phosphate in the first solvent is (10-20): 1; and / or In S140, the centrifugal treatment time is between 10 min and 20 min.

4. The preparation method according to claim 1, characterized in that: S200 specifically includes: S210, dispersion reaction: dispersing MnBr(CO)5 and the core layer in methanol, and stirring in the dark to obtain a third solvent; S220, removing methanol: removing methanol from the third solvent using a rotary evaporator to leave the PMC complex; S230, resuspension and freeze-drying: resuspend the PMC complex with deionized water, and then freeze-dry to obtain the final PMC nanozyme.

5. The preparation method according to claim 4, characterized in that: In S210, the stirring treatment time is 20h-30h.

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