Cobalt monatomic nano-enzyme ternary composite system as well as preparation method and application thereof

Abstract

The invention provides a cobalt monatomic nano-enzyme system modified by phospholipid polyethylene glycol carboxyl. The cobalt monatomic nano-enzyme system comprises a cobalt monatomic nano-enzyme composite core and phospholipid polyethylene glycol carboxyl compounded on the surface of cobalt monatomic nano-enzyme, the cobalt monatomic nano-enzyme composite core comprises a cobalt monatomic nano-enzyme and ginsenoside loaded on the cobalt monatomic nano-enzyme. The invention also provides a bifidobacterium synergetic cobalt monatomic nano-enzyme system which is formed by combining cobalt monatomic nano-enzyme with ginsenoside Rb1 and further delivering the cobalt monatomic nano-enzyme and ginsenoside Rb1 through bifidobacterium bifidum for targeted therapy of enteritis. The combination of Co SA and Rb1 can enhance the anti-inflammatory efficacy by adjusting a key immune signal pathway, and bifidobacterium bifidum can ensure targeted delivery of microbiota and promote intestinal barrier repair. The synergistic three-component strategy integrating monatomic nano-enzyme antioxidation, natural drug immunoregulation and probiotic targeting is provided, and a promising treatment platform is provided for accurate intervention of IBD.

Description

Technical Field

[0001] This invention belongs to the field of drug preparation technology for inflammatory bowel disease, and relates to a cobalt single-atom nanozyme system modified with phospholipid polyethylene glycol carboxyl group, a cobalt single-atom nanozyme system modified with phospholipid polyethylene glycol carboxyl group grafted with 3-aminophenylboronic acid, a cobalt single-atom nanozyme system synergistic with Bifidobacterium, and their preparation methods and applications, especially a cobalt single-atom nanozyme ternary composite system and its preparation method and applications. Background Technology

[0002] Inflammatory Bowel Disease (IBD) is a chronic, relapsing, immune-mediated gastrointestinal disease, primarily including ulcerative colitis (UC) and Crohn's disease. Its typical characteristic is persistent intestinal inflammation, often accompanied by systemic symptoms such as abdominal pain, diarrhea, weight loss, rectal bleeding, and fever. With industrialization, the global incidence of IBD has increased significantly, especially in developed regions, and is closely related to lifestyle, environmental changes, and gut microbiota dysbiosis. UC, as the main subtype of IBD, can be divided into an acute exacerbation phase and a chronic persistent phase. The acute phase is characterized by sudden mucosal inflammation, massive neutrophil infiltration, and a surge of reactive oxygen species (ROS); the chronic phase is characterized by mucosal barrier damage, tissue fibrosis, and immune microenvironment dysbiosis, and conventional treatments are insufficient to achieve long-term remission. Currently, commonly used drugs for UC, such as 5-aminosalicylic acid derivatives, glucocorticoids, immunosuppressants, and biologics, can control acute symptoms, but they all have significant limitations: 5-aminosalicylic acid has poor targeting and limited efficacy in moderate to severe cases; long-term use of glucocorticoids can easily lead to metabolic abnormalities and infections; immunosuppressants may increase the risk of malignant tumors; and biologics have problems such as high immunogenicity, large differences in response, high cost, and secondary failure.

[0003] Therefore, how to develop a more suitable therapeutic agent that solves the aforementioned technical problems of existing agents and achieves a comprehensive treatment strategy that balances acute phase control and chronic phase maintenance, systemic regulation of inflammation, and barrier repair has become one of the focal points of attention for many forward-thinking researchers in the field. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a cobalt single-atom nanozyme system modified with phospholipid polyethylene glycol carboxyl groups, a cobalt single-atom nanozyme system modified with phospholipid polyethylene glycol carboxyl groups grafted with 3-aminophenylboronic acid, and a cobalt single-atom nanozyme system synergistic with Bifidobacterium, as well as their preparation methods and applications. The present invention constructs a novel ternary synergistic nanomaterial system BB@Rb1@Co SA, innovatively integrating three functional components: cobalt single-atom nanozyme (Co SA), ginsenoside Rb1, and Bifidobacterium bifidum (BB), aiming to achieve multi-dimensional and long-term intervention in ulcerative colitis (UC). In this system, Co SA, with its excellent acid-base stability and enzyme-like catalytic activity, can continuously remove excess ROS generated in the inflammatory microenvironment, alleviating oxidative stress while acting as a functional carrier to protect Rb1 to the lesion area. Moreover, the preparation process is simple, the conditions are mild, and the controllability is good, making it more suitable for industrial production and application.

[0005] This invention provides a cobalt single-atom nanozyme system modified with phospholipid polyethylene glycol carboxyl groups, comprising: a cobalt single-atom nanozyme composite core and phospholipid polyethylene glycol carboxyl groups composited on the surface of the cobalt single-atom nanozyme;

[0006] The cobalt single-atom nanozyme composite core includes a cobalt single-atom nanozyme and ginsenosides loaded on the cobalt single-atom nanozyme.

[0007] Preferably, the particle size of the cobalt single-atom nanozyme is 100~300nm;

[0008] The cobalt single-atom nanozyme has a rhombic dodecahedral morphology.

[0009] The ginsenosides include ginsenoside Rb1;

[0010] The loading of ginsenosides on cobalt single-atom nanozymes is 30%~60%;

[0011] The cobalt single-atom nanozyme contains 1% to 2% Co by mass.

[0012] The phospholipid polyethylene glycol carboxyl group is modified on the surface of the cobalt single-atom nanoenzyme composite core to form an encapsulation layer;

[0013] The thickness of the encapsulation layer is 3~10 nm.

[0014] This invention provides a 3-aminophenylboronic acid-grafted phospholipid polyethylene glycol carboxyl-modified cobalt single-atom nanozyme system, which is obtained by chemically grafting 3-aminophenylboronic acid onto the surface of the phospholipid polyethylene glycol carboxyl-modified cobalt single-atom nanozyme system described in any one of the above technical solutions.

[0015] This invention provides a cobalt single-atom nanozyme system synergistic with Bifidobacterium, comprising: Bifidobacterium and a cobalt single-atom nanozyme composite core bound to Bifidobacterium;

[0016] The cobalt single-atom nanozyme composite core includes the cobalt single-atom nanozyme composite core in the cobalt single-atom nanozyme system described in any of the above technical solutions.

[0017] Preferably, the mass ratio of the cobalt single-atom nanoenzyme composite core to the number of Bifidobacteria is (1~2) mg: (10) mg. 8 ~10 10 CFU;

[0018] The Bifidobacteria include Bifidobacterium bifidum;

[0019] When the cobalt single-atom nanozyme composite core is the cobalt single-atom nanozyme composite core in the cobalt single-atom nanozyme system described in the above technical solution, the mass ratio of the phospholipid polyethylene glycol carboxyl-modified cobalt single-atom nanozyme system to 3-aminophenylboronic acid is 1:(1~30).

[0020] When the cobalt single-atom nanozyme composite core is the cobalt single-atom nanozyme composite core in the cobalt single-atom nanozyme system described in the above technical solution, the chemical grafting specifically involves the asamide reaction of the carboxyl group at the end of the phospholipid polyethylene glycol carboxyl group with the amino group of 3-aminophenylboronic acid, and the phenylboronic acid group grafting modification on its surface.

[0021] The binding method includes specific binding;

[0022] Specifically, the combination involves the specific binding of the phenylboronic acid groups on the surface of the cobalt single-atom nanozyme system described in the above technical solution to the cis-diol structure of the glycoprotein on the surface of Bifidobacterium.

[0023] This invention provides a method for preparing a cobalt single-atom nanozyme system synergistic with Bifidobacteria, comprising the following steps:

[0024] 1) After mixing zinc salt, cobalt salt and organic solvent, 2-methylimidazole organic solution is added and reacted to obtain Co@MOF;

[0025] 2) Mix the Co@MOF obtained in the above steps with alcohol to obtain an alcohol solution, then add ammonia-alcohol solution, then add tetraethyl orthosilicate, and after reaction, obtain Co@MOF@SiO2;

[0026] 3) Under a protective atmosphere, the Co@MOF@SiO2 obtained in the above steps is pyrolyzed and then etched with an alkaline solution to obtain cobalt single-atom nanozyme CoSA;

[0027] 4) The cobalt single-atom nanozyme CoSA, ginsenosides and organic solvent obtained in the above steps are dispersed and mixed, and the separated Gs@CoSA is obtained. Then, Gs@CoSA, water-miscible organic solvent and DSPE-PEG-COOH aqueous solution are mixed again, and after ultrasonic crushing and dialysis, DSPE-PEG-COOH modified Gs@CoSA is obtained.

[0028] 5) Mix the DSPE-PEG-COOH modified Gs@Co SA, buffer, EDC and NHS obtained in the above steps, and then add 3-aminophenylboronic acid and continue mixing to obtain a single-atom nanozyme system grafted with phenylboronic acid group. Then mix the single-atom nanozyme system grafted with phenylboronic acid group with Bifidobacterium solution again to obtain a cobalt single-atom nanozyme system synergistic with Bifidobacterium.

[0029] Preferably, the zinc salt comprises zinc nitrate;

[0030] The cobalt salt includes cobalt nitrate;

[0031] In step 1), the organic solvent and the solvent in the 2-methylimidazole organic solution are each independently selected from DMF and methanol;

[0032] The molar ratio of the zinc salt to the cobalt salt is (6~9):1;

[0033] The mass ratio of the cobalt salt to 2-methylimidazole is 1:(7~10).

[0034] The mixing reaction takes 10-14 hours;

[0035] The alcohol includes ethanol;

[0036] In the ammonia-alcohol solution, the volume ratio of ammonia to alcohol is 1:(15~18).

[0037] The mass ratio of tetraethyl orthosilicate to Co@MOF is (3~4):1.

[0038] Preferably, in step 2), the reaction time is 1 to 3 hours;

[0039] In step 2), the reaction is followed by a step of letting it stand overnight.

[0040] The pyrolysis temperature is 900~1000℃;

[0041] The pyrolysis time is 3-5 hours;

[0042] The alkaline solution includes NaOH solution and / or KOH solution;

[0043] The molar concentration of the alkaline solution is 3~5M;

[0044] The etching time is 10-14 hours;

[0045] In step 4), the organic solvent includes methanol;

[0046] The mass ratio of the cobalt single-atom nanozyme CoSA to ginsenoside is 1:(1~5).

[0047] Preferably, the water-miscible organic solvent includes THF;

[0048] The dialysis is specifically performed under conditions of Mw = 8000~10000;

[0049] The buffer solution includes MES buffer;

[0050] The pH value of the buffer solution is 5-6;

[0051] The mass ratio of the DSPE-PEG-COOH modified Gs@Co SA to 3-aminophenylboronic acid is 1:(7.5~20).

[0052] The mass-to-volume ratio of the phenylboronic acid-grafted single-atom nanozyme system to the Bifidobacterium solution is 1 mg: (2~10) mL;

[0053] The Bifidobacteria include Bifidobacterium bifidum;

[0054] The concentration of the Bifidobacterium solution is 10. 8 ~10 10 CFU / mL;

[0055] The cobalt single-atom nanozyme system synergistic with Bifidobacteria is a nanozyme system used to treat inflammatory bowel disease.

[0056] The present invention also provides the application of the cobalt single-atom nanozyme system described in any one of the above technical solutions or the cobalt single-atom nanozyme system prepared by the preparation method described in any one of the above technical solutions in the preparation of drugs for treating inflammatory bowel disease.

[0057] This invention provides a cobalt single-atom nanozyme system modified with phospholipid polyethylene glycol carboxyl groups, comprising: a cobalt single-atom nanozyme composite core and phospholipid polyethylene glycol carboxyl groups composited on the surface of the cobalt single-atom nanozyme; the cobalt single-atom nanozyme composite core includes a cobalt single-atom nanozyme and ginsenosides loaded on the cobalt single-atom nanozyme. Compared with existing technologies, based on the multidimensional pathological mechanisms and treatment challenges of acute and chronic ulcerative colitis (UC), this invention suggests that nanozymes, as a class of emerging nanomaterials with intrinsic enzyme-like catalytic activity, have attracted widespread attention in the biomedical field due to their precisely tunable catalytic performance, excellent physicochemical stability, and good biocompatibility. Among them, transition metal single-atom nanozymes (SAzymes), with their monodisperse metal atoms and fully exposed catalytic sites, exhibit excellent catalytic efficiency and targeted anti-inflammatory potential, and have become a research frontier in the treatment of inflammatory diseases. In the field of UC treatment, existing studies have confirmed the application potential of SAzymes, and these breakthroughs have provided new ideas for UC treatment. However, existing SAzymes still face several key challenges: the harsh environment of the gastrointestinal tract (such as low pH and digestive enzymes) easily affects their structural stability, leading to a significant decrease in catalytic activity in the complex intestinal lumen, making it difficult to achieve sustained anti-inflammatory effects; their targeting ability to inflammatory sites is limited, their retention time is short, and their therapeutic efficacy is limited; in addition, SAzymes have insufficient inhibitory ability against pathogenic bacteria and may even disrupt the balance of gut microbiota. Most importantly, the pathological mechanism of UC involves multi-source oxidative stress, a persistent inflammatory cascade, and multiple damages to the intestinal barrier, while most current SAzymes strategies still mainly rely on the single mechanism of ROS clearance, and their in vivo experiments are mostly based on acute-phase models, lacking effective interventions for chronic-phase lesions (such as tissue fibrosis and immune microenvironment dysregulation). Furthermore, existing strategies fail to systematically address the sustained inhibition of chronic-phase inflammatory signaling pathways and the reconstruction of barrier repair capabilities, and cannot fully meet the clinical needs for treating acute-chronic alternating, complex, and protracted ulcerative colitis. Therefore, there is an urgent need to develop a novel SAzymes system with multiple synergistic effects, and to construct a multifunctional nanozyme system that can simultaneously possess antioxidant, anti-inflammatory and intestinal barrier repair functions, so as to synergistically address the multidimensional pathogenic mechanisms of UC and comprehensively improve the targeting and effectiveness of treatment.

[0058] Ginsenoside Rb1 is a natural active ingredient with significant anti-inflammatory, antioxidant, and intestinal barrier repair effects. In the treatment of ulcerative colitis (UC), it can effectively upregulate the expression of tight junction proteins such as ZO-1 and Occludin by inhibiting key inflammatory signaling pathways such as NF-κB, alleviating the inflammatory response in the acute phase and continuously promoting the recovery of mucosal barrier structure and function in the chronic phase, showing good long-term therapeutic potential. However, this ingredient suffers from low bioavailability and poor stability in the gastrointestinal environment, which seriously limits its therapeutic efficacy. A Rb1 drug delivery strategy based on a nanodelivery system can significantly improve its retention time and therapeutic efficacy in the complex intestinal microenvironment by enhancing drug stability, colonic targeting, and bioavailability, providing a new solution for the comprehensive treatment of UC.

[0059] On the other hand, probiotics, due to their intestinal targeting, gut microbiota homeostasis, and mucosal repair effects, demonstrate unique value in the intervention of UC, especially in the chronic and persistent stage. For example, Bifidobacterium bifidum (BB), as a symbiotic anaerobic bacterium naturally colonizing the hypoxic colonic lumen, possesses strong intestinal adhesion capabilities, effectively attaching to the intestinal epithelial surface to form a stable biofilm, prolonging its colonization time in the intestine. It can also secrete antibacterial substances such as short-chain fatty acids and bacteriocins, inhibiting pathogenic bacterial growth and restoring the microecological balance. More importantly, it can significantly enhance the expression of tight junction proteins and reduce intestinal permeability, thereby combating persistent barrier damage in chronic UC.

[0060] Based on this, this invention specifically designed a cobalt single-atom nanozyme system modified with phospholipid polyethylene glycol carboxyl groups, and further designed a cobalt single-atom nanozyme system synergistic with Bifidobacterium. This is a three-component synergistic nanotherapeutic composite material system of cobalt single-atom nanozyme BB@Rb1@Co SA. This invention innovatively integrates three functional components: cobalt single-atom nanozyme (Co SA), ginsenoside Rb1, and BB, aiming to achieve multi-dimensional and long-term intervention for ulcerative colitis (UC). In this system, Co SA, with its excellent acid-base stability and enzyme-like catalytic activity, can continuously remove excess ROS generated in the inflammatory microenvironment, alleviate oxidative stress, and act as a functional carrier to protect Rb1 to the lesion area. Further density functional theory (DFT) calculations were used to explore the CAT-like and SOD-like activities of CoSA, including its catalytic reaction pathway and intermediate pathway. Rb1, by inhibiting key inflammatory pathways such as NF-κB and upregulating the expression of tight junction proteins, not only alleviated acute inflammation but also significantly promoted mucosal repair and barrier reconstruction. Under the synergistic protection of CoSA, BB enhanced its colonization ability in inflamed areas of the colon and synergistically maintained intestinal microenvironment homeostasis by regulating the microbiota ecology, inhibiting the proliferation of pathogenic bacteria, and secreting beneficial metabolites. The three components construct a complementary and synergistic therapeutic loop through a synergistic mechanism of "antioxidant-anti-inflammatory repair-microbiota regulation," providing a novel strategy for the precision treatment of this type of disease.

[0061] This invention provides a novel biohybrid therapy system comprising a cobalt single-atom nanozyme (CoSA) combined with the natural saponin ginsenoside Rb1, further delivered via Bifidobacterium bifidum for targeted treatment of colitis. CoSA exhibits excellent catalase-like (CAT) and superoxide dismutase-like (SOD) activities, effectively scavenging reactive oxygen species (ROS) in inflamed colonic tissue. Binding to Rb1 enhances anti-inflammatory efficacy by modulating key immune signaling pathways, while Bifidobacterium bifidum ensures targeted delivery of the microbiota and promotes intestinal barrier repair. In a DSS-induced colitis mouse model, biohybrid therapy significantly improved clinical symptoms, restored microbial homeostasis, and reduced pro-inflammatory cytokine levels.

[0062] The BB@Rb1@CoSA system provided by this invention exhibits significant therapeutic efficacy in chronic ulcerative colitis (UC). It not only effectively remodels the gut microbiota structure and restores microbial homeostasis, but also significantly increases the levels of beneficial metabolites, synergistically strengthens epithelial barrier function, and alleviates systemic inflammation. These findings further confirm that this ternary system, through a multi-mechanism synergistic effect of "antioxidant-anti-inflammatory repair-microbiota regulation," possesses unique advantages for achieving long-term, multi-dimensional intervention therapy, providing a promising new intervention pathway for the clinical treatment of acute / chronic UC. The research results of this invention demonstrate that a synergistic three-component strategy integrating enzymatic antioxidant, phytochemical immunomodulatory, and probiotic-targeted approaches provides a promising therapeutic platform for the precise intervention of IBD. Attached Figure Description

[0063] Figure 1 SEM image of the bimetallic imidazole framework precursor Co@MOF prepared in this invention;

[0064] Figure 2 SEM image of the core-shell structured precursor Co@MOF@SiO2 prepared for this invention;

[0065] Figure 3 A simplified schematic diagram and related characterization diagrams of the preparation process of CoSA provided by the present invention;

[0066] Figure 4 Gibbs free energy curves and reaction pathway diagrams of the catalase-like catalytic cycle and the superoxide dismutase-like catalytic cycle of CoSA provided by the present invention.

[0067] Figure 5 A series of graphs representing the performance characteristics of BB@Rb1@Co SA prepared in this invention;

[0068] Figure 6 Evaluation of indicators in mice in each group during the acute UC treatment experiment provided by this invention;

[0069] Figure 7 The gut microbiota characteristics of mice in each group during the acute UC treatment experiment provided by this invention;

[0070] Figure 8 Evaluation of dominant gut microbiota and metabolite indicators in mice in each group during an acute UC treatment experiment provided by this invention;

[0071] Figure 9 Evaluation of indicators in mice in each group during the chronic UC treatment experiment provided by this invention;

[0072] Figure 10 This invention provides an assessment of the gut microbiota characteristics and metabolite indicators of mice in each group during a chronic UC treatment experiment. Detailed Implementation

[0073] To further understand the present invention, preferred embodiments are described below with reference to examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims. Those skilled in the art can refer to the content of this document to appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0074] There are no particular restrictions on the source of any raw materials used in this invention; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0075] There are no particular restrictions on the purity of any raw materials used in this invention. However, it is preferred to use analytical grade or materials that meet the relevant standards for drug purity.

[0076] All raw materials of this invention are conventional in the field, and each brand name and abbreviation is clear and distinct in its relevant application. Those skilled in the art can purchase them from the market or prepare them by conventional methods based on the brand name, abbreviation and corresponding application.

[0077] This invention provides a cobalt single-atom nanozyme system modified with phospholipid polyethylene glycol carboxyl groups, comprising: a cobalt single-atom nanozyme composite core and phospholipid polyethylene glycol carboxyl groups composited on the surface of the cobalt single-atom nanozyme;

[0078] The cobalt single-atom nanozyme composite core includes a cobalt single-atom nanozyme and ginsenosides loaded on the cobalt single-atom nanozyme.

[0079] In this invention, the particle size of the cobalt single-atom nanozyme is preferably 100~300nm, more preferably 140~260nm, and even more preferably 180~220nm.

[0080] In this invention, the cobalt single-atom nanozyme preferably has a rhombic dodecahedral morphology.

[0081] In this invention, the cobalt single-atom nanozyme is obtained by pyrolysis and etching of the precursor Co@MOF@SiO2.

[0082] In this invention, the precursor Co@MOF@SiO2 has a core-shell structure, preferably including a Co@MOF core and a SiO2 coating layer covering the surface of the Co@MOF.

[0083] In this invention, the mass content of Co in the cobalt single-atom nanozyme is preferably 1% to 2%, more preferably 1.2% to 1.8%, and even more preferably 1.4% to 1.6%.

[0084] In this invention, the particle size of the precursor Co@MOF@SiO2 is preferably 130~350nm, more preferably 180~300nm, and even more preferably 230~250nm.

[0085] In this invention, the particle size of the Co@MOF core is preferably 100~300nm, more preferably 140~260nm, and even more preferably 180~220nm.

[0086] In this invention, the thickness of the SiO2 coating layer is preferably 5~30nm, more preferably 10~25nm, and even more preferably 15~20nm.

[0087] In this invention, the mass ratio of Co@MOF to SiO2 is preferably 1:(0.4~1), more preferably 1:(0.5~0.9), and even more preferably 1:(0.6~0.8).

[0088] In this invention, the Co@MOF is preferably Co-doped ZIF-8. Zn will volatilize at high temperatures during the subsequent pyrolysis process.

[0089] In this invention, the Co@MOF preferably has a rhombic dodecahedral morphology.

[0090] In this invention, the cobalt single-atom nanozyme partially retains the morphology of the precursor Co@MOF@SiO2, and preferably has a porous and / or layered structure, more preferably a porous or layered structure.

[0091] In this invention, the pyrolysis is preferably performed under a protective atmosphere.

[0092] In this invention, the etching is preferably alkaline etching.

[0093] In this invention, in the cobalt single-atom nanozyme, Co is preferably dispersed in single-atom form and forms a local coordination structure with the N ligand.

[0094] In this invention, the N preferably includes graphitic nitrogen, pyrrole nitrogen, and pyridine nitrogen.

[0095] In this invention, the cobalt single-atom nanozyme system is preferably a nanozyme system for treating inflammatory bowel disease.

[0096] In this invention, the ginsenosides preferably include ginsenoside Rb1.

[0097] In this invention, the loading of ginsenosides on cobalt single-atom nanoenzymes is preferably ≥30%, more preferably 30%~60%, more preferably 35%~55%, and even more preferably 40%~50%.

[0098] In this invention, the phospholipid polyethylene glycol carboxyl group is modified on the surface of the cobalt single-atom nanoenzyme composite core, preferably forming an encapsulation layer.

[0099] In this invention, the thickness of the coating layer is preferably 3~10 nm, more preferably 4~9 nm, more preferably 5~8 nm, and even more preferably 6~7 nm.

[0100] This invention provides a 3-aminophenylboronic acid-grafted phospholipid polyethylene glycol carboxyl-modified cobalt single-atom nanozyme system, which is obtained by chemically grafting 3-aminophenylboronic acid onto the surface of the phospholipid polyethylene glycol carboxyl-modified cobalt single-atom nanozyme system described in any one of the above technical solutions.

[0101] This invention provides a cobalt single-atom nanozyme system synergistic with Bifidobacterium, comprising: Bifidobacterium and a cobalt single-atom nanozyme composite core bound to Bifidobacterium;

[0102] The cobalt single-atom nanozyme composite core includes the cobalt single-atom nanozyme composite core in the cobalt single-atom nanozyme system described in any of the above technical solutions.

[0103] In this invention, the preferred ratio of the mass of the cobalt single-atom nanozyme composite core to the number of Bifidobacteria is (1~2) mg: (10 mg / mg). 8 ~10 10 CFU, more preferably (1.2~1.8) mg: (30 8 ~80 10CFU, more preferably (1.4~1.6) mg: (50) 8 ~60 10 CFU.

[0104] In this invention, the bifidobacteria preferably include Bifidobacterium bifidum.

[0105] In this invention, when the cobalt single-atom nanozyme composite core is the cobalt single-atom nanozyme composite core in the cobalt single-atom nanozyme system described in the above technical solution, the mass ratio of the phospholipid polyethylene glycol carboxyl-modified cobalt single-atom nanozyme system to 3-aminophenylboronic acid is preferably 1:(1~30), more preferably 1:(6~25), and even more preferably 1:(11~20).

[0106] In this invention, when the cobalt single-atom nanozyme composite core is the cobalt single-atom nanozyme composite core in the cobalt single-atom nanozyme system described in the above technical solution, the chemical grafting is preferably performed by grafting and modifying the surface of the phenylboronic acid group by reacting the carboxyl group at the end of the phospholipid polyethylene glycol carboxyl group with the amino group of 3-aminophenylboronic acid through an amidation reaction.

[0107] In this invention, the binding method preferably includes specific binding.

[0108] In this invention, the preferred specific combination is that the phenylboronic acid groups on the surface of the cobalt single-atom nanozyme system described in the above technical solution specifically bind to the cis-diol structure of the surface glycoprotein of Bifidobacterium.

[0109] This invention provides a method for preparing a cobalt single-atom nanozyme system synergistic with Bifidobacteria, comprising the following steps:

[0110] 1) After mixing zinc salt, cobalt salt and organic solvent, 2-methylimidazole organic solution is added and reacted to obtain Co@MOF;

[0111] 2) Mix the Co@MOF obtained in the above steps with alcohol to obtain an alcohol solution, then add ammonia-alcohol solution, then add tetraethyl orthosilicate, and after reaction, obtain Co@MOF@SiO2;

[0112] 3) Under a protective atmosphere, the Co@MOF@SiO2 obtained in the above steps is pyrolyzed and then etched with an alkaline solution to obtain cobalt single-atom nanozyme CoSA;

[0113] 4) The cobalt single-atom nanozyme CoSA, ginsenosides and organic solvent obtained in the above steps are dispersed and mixed, and the separated Gs@CoSA is obtained. Then, Gs@CoSA, water-miscible organic solvent and DSPE-PEG-COOH aqueous solution are mixed again, and after ultrasonic crushing and dialysis, DSPE-PEG-COOH modified Gs@CoSA is obtained.

[0114] 5) Mix the DSPE-PEG-COOH modified Gs@Co SA, buffer, EDC and NHS obtained in the above steps, and then add 3-aminophenylboronic acid and continue mixing to obtain a single-atom nanozyme system grafted with phenylboronic acid group. Then mix the single-atom nanozyme system grafted with phenylboronic acid group with Bifidobacterium solution again to obtain a cobalt single-atom nanozyme system synergistic with Bifidobacterium.

[0115] The present invention first mixes zinc salt, cobalt salt and organic solvent, then adds 2-methylimidazole organic solution and reacts to obtain Co@MOF.

[0116] In this invention, the zinc salt preferably includes zinc nitrate.

[0117] In this invention, the cobalt salt preferably includes cobalt nitrate.

[0118] In this invention, in step 1), the organic solvent and the solvent in the 2-methylimidazole organic solution are each preferably selected independently from DMF and methanol.

[0119] In this invention, the molar ratio of the zinc salt to the cobalt salt is preferably (6~9):1, more preferably (6.5~8.5):1, and even more preferably (7~8):1.

[0120] In this invention, the mass ratio of the cobalt salt to 2-methylimidazole is preferably 1:(7~10), more preferably 1:(7.5~9.5), and even more preferably 1:(8~9).

[0121] In this invention, the mixing reaction time is preferably 10 to 14 hours, more preferably 10.5 to 13.5 hours, more preferably 11 to 13 hours, and even more preferably 11.5 to 12.5 hours.

[0122] In this invention, the Co@MOF obtained in the above steps is mixed with an alcohol to obtain an alcohol solution. Then, an ammonia-alcohol solution is added, followed by the addition of tetraethyl orthosilicate. After the reaction is carried out, Co@MOF@SiO2 is obtained.

[0123] In this invention, the alcohol preferably includes ethanol.

[0124] In this invention, the volume ratio of ammonia to alcohol in the ammonia-alcohol solution is preferably 1:(15~18), more preferably 1:(15.5~17.5), and even more preferably 1:(16~17).

[0125] In this invention, the mass ratio of tetraethyl orthosilicate to Co@MOF is preferably (3~4):1, more preferably (3.2~3.8):1, and even more preferably (3.4~3.6):1.

[0126] In this invention, in step 2), the reaction time is preferably 1 to 3 hours, more preferably 1.4 to 2.6 hours, and even more preferably 1.8 to 2.2 hours.

[0127] In this invention, step 2) preferably includes a step of letting the mixture stand overnight after the reaction.

[0128] The present invention then pyrolyzes the Co@MOF@SiO2 obtained in the above steps under a protective atmosphere, and then etches it with an alkaline solution to obtain cobalt single-atom nanozyme CoSA.

[0129] In this invention, the pyrolysis temperature is preferably 900~1000℃, more preferably 920~980℃, and even more preferably 940~960℃.

[0130] In this invention, the pyrolysis time is preferably 3 to 5 hours, more preferably 3.4 to 4.6 hours, and even more preferably 3.8 to 4.2 hours.

[0131] In this invention, the alkaline solution preferably includes NaOH solution and / or KOH solution, more preferably NaOH solution or KOH solution.

[0132] In this invention, the molar concentration of the alkaline solution is preferably 3~5M, more preferably 3.4~4.6M, and even more preferably 3.8~4.2M.

[0133] In this invention, the etching time is preferably 10 to 14 hours, more preferably 10.5 to 13.5 hours, more preferably 11 to 13 hours, and even more preferably 11.5 to 12.5 hours.

[0134] The present invention then disperses and mixes the cobalt single-atom nanozyme CoSA, ginsenosides and organic solvent obtained in the above steps, and separates the resulting Gs@CoSA. Then, Gs@CoSA, water-miscible organic solvent and DSPE-PEG-COOH aqueous solution are mixed again, and after ultrasonic crushing and dialysis, DSPE-PEG-COOH modified Gs@CoSA is obtained.

[0135] In this invention, the ginsenoside Gs includes ginsenoside Rb1.

[0136] In this invention, the organic solvent in step 4) preferably includes methanol.

[0137] In this invention, the mass ratio of the cobalt single-atom nanoenzyme CoSA to ginsenoside is preferably 1:(1~5), more preferably 1:(1~4), more preferably 1:(1~3), and even more preferably 1:(1~2).

[0138] In this invention, the water-miscible organic solvent preferably includes THF.

[0139] In this invention, the dialysis is preferably carried out under conditions of Mw=8000~10000, more preferably Mw=8400~9600, and even more preferably Mw=8800~9200.

[0140] Finally, the present invention mixes the DSPE-PEG-COOH modified Gs@Co SA, buffer, EDC and NHS obtained in the above steps, and then adds 3-aminophenylboronic acid and mixes again to obtain a single-atom nanozyme system grafted with phenylboronic acid group. Then, the single-atom nanozyme system grafted with phenylboronic acid group is mixed again with Bifidobacterium solution to obtain a cobalt single-atom nanozyme system synergistic with Bifidobacterium.

[0141] In this invention, the buffer solution preferably includes MES buffer.

[0142] In this invention, the pH value of the buffer solution is preferably 5-6, more preferably 5.2-5.8, even more preferably 5.4-5.6, and specifically 5.5.

[0143] In this invention, the mass ratio of the DSPE-PEG-COOH modified Gs@Co SA to 3-aminophenylboronic acid is preferably 1:(7.5~20), more preferably 1:(7.5~15), even more preferably 1:(7.5~10), and specifically 1:7.5.

[0144] In this invention, the preferred mass-to-volume ratio of the phenylboronic acid-grafted single-atom nanozyme system to the Bifidobacterium solution is 1 mg:(2~10) mL, more preferably 1 mg:(2~8) mL, even more preferably 1 mg:(2~6) mL, and even more preferably 1 mg:(2~4) mL. Specifically, it can be 1 mg:2 mL.

[0145] In this invention, the bifidobacteria preferably include Bifidobacterium bifidum.

[0146] In this invention, the solvent for the Bifidobacterium solution is preferably physiological saline.

[0147] In this invention, the concentration of the Bifidobacterium solution is preferably 10. 8 ~10 10 CFU / mL, more preferably 5×10⁻⁶ 8 ~80×10 8 CFU / mL, more preferably 10×10 8 ~60×10 8 CFU / mL, more preferably 30×10⁻⁶ 8 ~50×10 8 CFU / mL. Specifically, it can be 10. 8 CFU / mL.

[0148] In this invention, the cobalt single-atom nanozyme system synergistic with Bifidobacterium is preferably a nanozyme system for treating inflammatory bowel disease.

[0149] This invention employs an encapsulation-pyrolysis-etching strategy to successfully prepare cobalt single-atom materials (CoSA) using Co@MOF as a template. First, Co... 2+ Bimetallic imidazole framework precursor Co@MOF was prepared by encapsulation in a MOF template. The Co@MOF exhibited a rhombic dodecahedral morphology with uniform particle size. To prevent Co atom aggregation during pyrolysis, the Co@MOF was coated with SiO2 to obtain a core-shell structure precursor Co@MOF@SiO2, which tightly coated the Co@MOF surface, forming a complete core-shell structure. This precursor was pyrolyzed under a N2 atmosphere, followed by etching of the pyrolysis products with sodium hydroxide solution to finally obtain Co SA. Co SA retained some of its original geometry and exhibited porous and layered characteristics due to structural collapse.

[0150] Based on the potential dual-enzyme catalytic activity of CoSA, this invention constructs a novel ternary nanomaterial, BB@Rb1@CoSA, through multi-step assembly and characterizes its various properties. Utilizing the microporous structure of CoSA, highly efficient loading (loading rate greater than 30%) of the anti-inflammatory barrier repair agent ginsenoside Rb1 was achieved, forming Rb1@CoSA. Subsequently, Rb1@CoSA was encapsulated with the amphiphilic polymer DSPE-PEG-COOH (phospholipid polyethylene glycol carboxyl group). The terminal carboxyl group (-COOH) undergoes an amidation reaction with the amino group in 3-aminophenylboronic acid (3-APBA), modifying the material surface with phenylboronic acid groups. Finally, the boronic acid groups of 3-APBA specifically bind to the cis-diol structure of the BB surface glycoprotein, constructing the BB@Rb1@CoSA ternary system.

[0151] This invention provides the application of the cobalt single-atom nanozyme system described in any one of the above technical solutions or the cobalt single-atom nanozyme system prepared by the preparation method described in any one of the above technical solutions in the preparation of drugs for treating inflammatory bowel disease.

[0152] In this invention, the drug preferably comprises a cobalt single-atom nanozyme system and pharmaceutically acceptable excipients.

[0153] In this invention, the dosage form of the drug preferably includes oral preparations, injections, suppositories, inhalers, or dosage forms that can be directly applied to the intestines.

[0154] In this invention, the mass content of the polydopamine-modified cobalt single-atom nanozyme system in the pharmaceutical preparation is preferably 0.5~10 mg / mL, more preferably 1~8 mg / mL, and even more preferably 3~6 mg / mL.

[0155] In this invention, the application preferably includes the application of cobalt single-atom nanozymes in a Bifidobacterium-synergistic cobalt single-atom nanozyme system in achieving hydrogen peroxide decomposition and / or superoxide anion free radical scavenging.

[0156] In this invention, the application specifically preferably includes the application of phospholipid polyethylene glycol carboxyl groups in a cobalt single-atom nanozyme system synergistic with Bifidobacteria in relieving acute / chronic intestinal inflammation and / or promoting intestinal mucosal repair and barrier reconstruction.

[0157] In this invention, the application preferably includes the application of a cobalt single-atom nanozyme system synergistic with Bifidobacteria in enhancing the antioxidant, anti-inflammatory repair and microbial regulation of the intestinal region.

[0158] This invention provides a phospholipid polyethylene glycol carboxyl-modified cobalt single-atom nanozyme system, a 3-aminophenylboronic acid-grafted phospholipid polyethylene glycol carboxyl-modified cobalt single-atom nanozyme system, a bifidobacteria-synergistic cobalt single-atom nanozyme system, and their preparation methods and applications. The phospholipid polyethylene glycol carboxyl-modified cobalt single-atom nanozyme system and the bifidobacteria-synergistic cobalt single-atom nanozyme system designed in this invention constitute a ternary synergistic nanotherapy composite material system of cobalt single-atom nanozyme BB@Rb1@Co SA. This invention innovatively integrates three functional components: cobalt single-atom nanozyme (Co SA), ginsenoside Rb1, and BB, aiming to achieve multi-dimensional and long-term intervention for ulcerative colitis (UC). In this system, Co SA, with its excellent acid-base stability and enzyme-like catalytic activity, can continuously scavenge excess ROS generated in the inflammatory microenvironment, alleviating oxidative stress while acting as a functional carrier to protect Rb1 to the lesion area. Further density functional theory (DFT) calculations were used to explore the CAT-like and SOD-like activities of CoSA, including its catalytic reaction pathway and intermediate pathway. Rb1, by inhibiting key inflammatory pathways such as NF-κB and upregulating the expression of tight junction proteins, not only alleviated acute inflammation but also significantly promoted mucosal repair and barrier reconstruction. Under the synergistic protection of CoSA, BB enhanced its colonization ability in inflamed areas of the colon and synergistically maintained intestinal microenvironment homeostasis by regulating the microbiota ecology, inhibiting the proliferation of pathogenic bacteria, and secreting beneficial metabolites. The three components construct a complementary and synergistic therapeutic loop through a synergistic mechanism of "antioxidant-anti-inflammatory repair-microbiota regulation," providing a novel strategy for the precision treatment of this type of disease.

[0159] This invention provides a novel biohybrid therapy system comprising a cobalt single-atom nanozyme (CoSA) combined with the natural saponin ginsenoside Rb1, further delivered via Bifidobacterium bifidum for targeted treatment of colitis. CoSA exhibits excellent catalase-like (CAT) and superoxide dismutase-like (SOD) activities, effectively scavenging reactive oxygen species (ROS) in inflamed colonic tissue. Binding to Rb1 enhances anti-inflammatory efficacy by modulating key immune signaling pathways, while Bifidobacterium bifidum ensures targeted delivery of the microbiota and promotes intestinal barrier repair. In a DSS-induced colitis mouse model, biohybrid therapy significantly improved clinical symptoms, restored microbial homeostasis, and reduced pro-inflammatory cytokine levels.

[0160] The BB@Rb1@CoSA system provided by this invention exhibits significant therapeutic efficacy in chronic ulcerative colitis (UC). It not only effectively remodels the gut microbiota structure and restores microbial homeostasis, but also significantly increases the levels of beneficial metabolites, synergistically strengthens epithelial barrier function, and alleviates systemic inflammation. These findings further confirm that this ternary system, through a multi-mechanism synergistic effect of "antioxidant-anti-inflammatory repair-microbiota regulation," possesses unique advantages for achieving long-term, multi-dimensional intervention therapy, providing a promising new intervention pathway for the clinical treatment of acute / chronic UC. The research results of this invention demonstrate that a synergistic three-component strategy integrating enzymatic antioxidant, phytochemical immunomodulatory, and probiotic-targeted approaches provides a promising therapeutic platform for the precise intervention of IBD.

[0161] To further illustrate the present invention, the following embodiments provide a detailed description of a phospholipid polyethylene glycol carboxyl-modified cobalt single-atom nanozyme system, a 3-aminophenylboronic acid-grafted phospholipid polyethylene glycol carboxyl-modified cobalt single-atom nanozyme system, a Bifidobacterium-synergistic cobalt single-atom nanozyme system, their preparation methods, and applications. However, it should be understood that these embodiments are implemented under the premise of the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. They are only for further illustrating the features and advantages of the present invention, and are not intended to limit the scope of the claims of the present invention. The protection scope of the present invention is not limited to the following embodiments.

[0162] Main instruments and reagents

[0163] Zinc nitrate, cobalt nitrate, 2-methylimidazole, ammonia, tetraethyl orthosilicate, and DMPO were purchased from Sigma (Germany). Titanium sulfate, sodium hydroxide, 5-aminosalicylic acid (5-ASA), DMF, methanol, and ethanol were purchased from Aladdin Biotech (China). 3% hydrogen peroxide solution was purchased from Likang Medical Technology Co., Ltd. (China). Dopamine hydrochloride, NBT, riboflavin, L-methionine, and 4% paraformaldehyde were purchased from Yuanye Biotechnology Co., Ltd. (China). Fetal bovine serum was purchased from Ecosei Biotechnology Co., Ltd. (China). DMEM was purchased from Gibco Life Sciences (China). Trypsin and penicillin-streptomycin mixed solution were purchased from Shanghai Xiaopeng Biotechnology Co., Ltd. (China). CCK-8 was purchased from Invigentech (USA). PBS was purchased from Cytiva (UK). Enzyme-linked immunosorbent assay (ELISA) kits (IL-1β, TNF-α, and IL-6) and BCA protein assay kits were purchased from Beijing Solarbio Biotechnology Co., Ltd. (China). NO assay kit was purchased from Beyotime Biotechnology Co., Ltd. (China). MDA assay kit, GSH assay kit, TC / TG assay kit, AKP, ALT, AST, BUN, CRE, and UA assay kits were provided by Nanjing Jiancheng Biotechnology Institute (China). p65, p-p65, IκB, p-IκB, ZO-1, Occludin, Claudin-1, HO-1, and iNOS were purchased from Wuhan Sanying Biotechnology Co., Ltd. (China). NRF2 was purchased from Abmart Pharmaceutical Technology Co., Ltd. (China).

[0164] Example

[0165] 1. Synthesis of single-atom nanozymes (CoSA)

[0166] Weigh 1.07 g of zinc nitrate and 0.135 g of cobalt nitrate, add them to a mixture of 24 mL of DMF and 6 mL of methanol, and stir at room temperature for 30 min; separately dissolve 1.16 g of 2-methylimidazole in a mixture of 24 mL of DMF and 6 mL of methanol, add it to the above solution, and stir at room temperature for 12 h to obtain Co@MOF.

[0167] 108 mg of Co@MOF was dispersed in 108 mL of ethanol. After sonication, a solution of 108 mL of ethanol containing ammonia (6.48 mL) and tetraethyl orthosilicate (TEOS, 408 μL) were added dropwise while stirring. The mixture was stirred for 2 h and then aged at room temperature for 12 h. The resulting product was washed three times with deionized water and ethanol, and then dried to obtain Co@MOF@SiO2. After drying, the sample was placed in a tube furnace under nitrogen protection and calcined at 950°C for 4 h. After cooling, the resulting product was dispersed in 4 M NaOH solution and stirred for 12 h. It was then washed three times with deionized water and ethanol, and dried to obtain the final product Co SA.

[0168] 2. Construction of DSPE-PEG-COOH modified Rb1@CoSA nanoplatform

[0169] 2 mg Co SA and 2 mg ginsenoside Rb1 were dispersed in methanol, sonicated at 300 W for 15 min, stirred at 600 rpm at room temperature for 24 h, centrifuged, and the precipitate was dried to obtain Co SA@Rb1.

[0170] 1 mg of Co SA@Rb1 was added to 1 mL of THF and dissolved by sonication. Then, it was quickly added to a DSPE-PEG-COOH aqueous solution and placed in an ultrasonic cell disruptor for continuous sonication for 2 min. Subsequently, it was dialyzed for 24 h at Mw = 8000-10000 to obtain the DSPE-PEG-COOH modified Co SA@Rb1 nanoplatform.

[0171] See Figure 1 , Figure 1 SEM image of the bimetallic imidazole framework precursor Co@MOF prepared in this invention.

[0172] See Figure 2 , Figure 2 SEM image of the core-shell structured precursor Co@MOF@SiO2 prepared for this invention;

[0173] See Figure 3 , Figure 3The present invention provides a schematic diagram and related characterization diagrams of the preparation process of Co SA. (A) Schematic diagram of the Co SA synthesis process; (B) TEM (up) and HAADF-STEM (down) images of the prepared Co SA; (C) AC-STEM image and magnified view of Co SA; (D) Elemental distribution diagram of Co SA, showing the distribution of Co (yellow), C (red), and N (green); (E) XANES spectra; (F) Fourier transform EXAFS spectra; and (G) k-space of CoPc, Co SA, Co Foil, and CoO; (H) Extended X-ray absorption fine structure fitting curves of Co SA in R-space and (I) k-space; wavelet transforms for the k-space. 2 -weighted EXAFS signals of CoPc (J), Co SA (K), Co Foil (L) and CoO (M).

[0174] The preparation process and characterization of Co SA are as follows: Figure 3 As shown, Co SA (Co@MOF) was successfully constructed using an encapsulation-pyrolysis-etching method as a template. Figure 3 A). First, Co 2+ Encapsulated in a MOF template, the bimetallic imidazole framework precursor Co@MOF was obtained. Scanning electron microscopy (SEM) results showed that Co@MOF exhibited a rhombic dodecahedral structure with uniform particle size, good dispersibility, and a size of ~150 nm. Figure 1 To prevent Co atom aggregation during pyrolysis, a SiO2 coating was applied to Co@MOF, resulting in Co@MOF@SiO2. The SiO2 tightly covers the Co@MOF surface with uniform thickness, forming a complete core-shell structure with a size of ~160 nm. Figure 2 The product was pyrolyzed in an N2 atmosphere and treated with sodium hydroxide to obtain CoSA. TEM and HAADF-STEM images showed that CoSA still retained some geometric morphology and exhibited porous and layered structural features due to collapse, which provided more opportunities for ginsenoside Rb1 to bind. Figure 3B). The dispersion state of Co species was studied using aberration-corrected scanning transmission electron microscopy (AC-STEM). Figure 3 In the AC-STEM image of Co SA, relatively uniform isolated bright spots (red circles) can be observed, indicating that Co is highly dispersed at the atomic level. To further confirm the single-atom state of Co and analyze its local chemical environment, its elemental distribution and valence state characterization were further carried out. The corresponding Energy dispersive spectrometer (EDS) mapping results show that Co, C, and N elements are all uniformly distributed, consistent with the atomic-level dispersed structure. At the same time, X-ray absorption near-edge structure (XANES) indicates that the absorption edge energy of Co SA is between Co Foil and Co Pc, indicating that the valence state of Co is between 0 and +2. Figure 3 E). Meanwhile, X-ray near-edge absorption structure (XANES) spectroscopy shows that the absorption edge energy of Co SA lies between Co foil and CoPc, indicating that the valence state of Co is between 0 and +2. Figure 3 E). Further through k 2 -Weighted Fourier transform of the extended X-ray absorption fine structure (EXAFS) analysis showed that Co SA exhibited a distinct main peak at 1.44 Å, a position close to the Co-N coordination peak in CoPc (1.47 Å), which can be attributed to Co-N coordination. This peak is significantly different from the Co-O bond in CoO (1.65 Å) and the Co-Co metallic bond in Co foil (2.17 Å). Figure 3 F). This result is consistent with the atomic-level dispersion observed in AC-STEM, further confirming that Co exists in single-atom form and is mainly coordinated with N. The k-space spectra show that all sample signals are clear, the data quality is good, and the results are highly reliable. Figure 3G). To further elucidate the coordination configuration of the Co moieties in the Co SA, quantitative least-squares EXAFS curve-fitting was further performed. The fitting results in K-space and R-space clearly show that the coordination number of the central Co atom in the Co SA is approximately 4.3, and the average bond length is approximately 1.91 Å, indicating that the Co atom is in an environment coordinated with four N atoms (G). Figure 3 H~I and Table 1). Additionally, wavelet transform analysis of the k²-weighted EXAFS spectrawas was also conducted to provide the atomic configuration of the Co atom in the Co SA. The results show that the maximum intensity of Co SA occurs at approximately 4.0 Å⁻¹, which is similar to that of Co Pc (4.0 Å⁻¹), but significantly different from that of Co foil (7.0 Å⁻¹) and CoO (5.0 Å⁻¹). Figure 3 These results indicate that cobalt atoms are dispersed in single-atom form and form local coordination structures with nitrogen ligands.

[0175] See Table 1, which contains Co K-edge detection data from EXAFS.

[0176] Table 1

[0177]

[0178] See Figure 4 , Figure 4 Gibbs free energy curves and reaction pathway diagrams for the catalase-like and superoxide dismutase-like catalytic cycles of CoSA provided by this invention. (A) is a catalase-like catalytic cycle, and (B) is a superoxide dismutase-like catalytic cycle.

[0179] Depend on Figure 4 As can be seen, the free energy diagram of H2O2 decomposition at the Co SA active sites was plotted using DFT calculations. Figure 4A). The catalytic cycle begins on a clean Co SA surface (denoted as *) with a free energy of 0.00 eV. H2O2 adsorbs onto Co active sites to form *H2O2 (ΔG = -0.21 eV), followed by spontaneous O–O bond cleavage to generate *O species (ΔG = -0.94 eV). The reaction then proceeds via *OH (ΔG = -1.52 eV) to generate O2 and H2O, restoring the catalyst surface to a clean state with an energy barrier of -1.87 eV. The free energy continuously decreases throughout the reaction pathway, indicating that the process is highly thermodynamically spontaneous and suggesting that Co SA possesses potential CAT-like activity.

[0180] For the SOD-like activity of CoSA, O2 was obtained through DFT calculation. • ⁻Reaction pathways and free energy diagrams involved ( Figure 4 B). The reaction begins on a clean Co SA surface (denoted as *) with a free energy of 0.00 eV. O2•⁻ adsorbs onto the Co active sites to form *OOH, lowering the energy barrier to -1.43 eV, indicating that Co SA has a strong affinity for O2•⁻. Subsequently, spontaneous O–O bond cleavage converts to *O2 (ΔG = -0.85 eV), indicating that this conversion is thermodynamically highly spontaneous. The reaction eventually returns to the clean surface state with an energy barrier of -1.21 eV. The free energy continuously decreases throughout the catalytic pathway, indicating that Co SA can efficiently catalyze the disproportionation of O2•⁻ to O2, exhibiting potential SOD-like enzyme activity.

[0181] Comparing the two catalytic pathways, it can be seen that CoSA exhibits good thermodynamic feasibility in both CAT-like and SOD-like reactions, but significant differences still exist. Figure 4 The final-state energy barrier of the CAT-like reaction (-1.87 eV) is significantly lower than that of the SOD-like pathway (-1.21 eV), suggesting that CoSA possesses a stronger thermodynamic driving force in the catalytic decomposition of H2O2. However, the significant decreasing trend of free energy in the SOD-like pathway still indicates its outstanding catalytic potential. The above DFT calculations theoretically reveal the potential dual-enzyme catalytic mechanism of CoSA and its activity differences.

[0182] 3. Hydrogen peroxide removal capacity

[0183] H2O2 (10 mM, 100 μL), Rb1@Co SA (100 μL), and 800 μL PBS were mixed thoroughly, reacted, and then centrifuged (8000 rpm, 5 min). The supernatant was collected for later use. 24% Ti(SO2)4 (532 μL) was added to sulfuric acid solution (3.332 mL sulfuric acid solution + 16.136 mL water) to prepare a Ti(SO2)4 precursor solution. After a specified time, 100 μL of the supernatant and 100 μL of the Ti(SO2)4 precursor solution were collected. The concentration of H2O2 could be determined by the absorbance at 405 nm.

[0184] 4. Bifidobacterium bifidum armed with CoSA via DSPE-PEG-COOH linkage

[0185] 1 mg of DSPE-PEG-COOH modified Rb1@Co SA was dispersed in MES buffer (pH=5.5), followed by the addition of 32 mg EDC and 24 mg NHS, and stirred for 20 min. After stirring, 7.5 mg of 3-aminophenylboronic acid was added and stirring continued. After 24 h, the mixture was centrifuged, and 0.5 mg of the precipitate was collected and 1 mL of BB (10) was added. 8 The mixture was stirred for 15 min with CFU / mL and centrifuged to obtain BB@Rb1@Co SA. Different ratios of BB@Rb1@Co SA can be synthesized by adjusting the concentrations of BB and DSPE-PEG2000-COOH modified Rb1@Co SA.

[0186] 5. Acute colitis experiment in mice

[0187] Eighty C57BL / 6 mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. They were acclimatized for 7 days before the experiment and housed in a constant temperature environment of 22±2℃ with a 12-hour light-dark cycle and free access to water. All animal experiments were conducted in accordance with the requirements of the Experimental Animal Ethics Committee of Changchun University of Traditional Chinese Medicine. After the acclimatization period, the mice were randomly divided into 8 groups (blank group, model group, positive control group, BB group, Rb1 group, CoSA group, Rb1@CoSA group, and BB@Rb1@CoSA group), with 10 mice in each group. Except for the blank group, the remaining mice had free access to 3% DSS aqueous solution for 7 days to establish a colitis model. Subsequently, they underwent three days of oral drug treatment, with each group receiving 0.9% NaCl, 0.9% NaCl, 40 mg / kg 5-ASA, or 1×10⁻⁶ DSS, respectively. 8 CFU / kg BB, 0.6mg / kg Rb1, 2mg / kgCo SA, 2.6mg / kg Rb1@Co SA, BB@Rb1@Co SA (including 1×10 8CFU / kg BB and 2.6 mg / kg Rb1@Co SA). During the 10-day experiment, body weight changes were recorded, and feces from each group of mice were collected one day before the end of the experiment for microbiome and metabolome analysis. On the last day of the experiment, mice were euthanized, and blood, heart, liver, spleen, lungs, kidneys, and colon were collected. Serum was obtained after centrifugation of the blood and used for ELISA analysis. H&E staining was performed on the heart, liver, spleen, lungs, and kidneys, and a portion of the colon was used for H&E, PAS, Alcian Blue staining, and immunofluorescence analysis.

[0188] 6. Chronic colitis experiment in mice

[0189] Forty mice after the adaptation feeding period were randomly divided into four groups (blank group, model group, positive drug group, and BB@Rb1@Co SA group), with ten mice in each group. Except for the blank group, the remaining 30 mice were used to establish a chronic ulcerative colitis model through three cycles of 2% DSS induction (7 days of DSS induction followed by 14 days of water). After the cycle, the mice were treated with oral drugs for three days. The groups were given 0.9% NaCl, 0.9% NaCl, 40 mg / kg, and BB@Rb1@Co SA (containing 1×10⁻⁶ ppm), respectively. 8 CFU / kg BB and 2.6 mg / kg Rb1@Co SA). After the experiment, the sampling and data analysis were the same as in Experiment 5.

[0190] See Figure 5 , Figure 5 The following figures illustrate the characterization and analysis of various properties of BB@Rb1@Co SA prepared in this invention. (A) Schematic diagram of the synthesis process of BB@Rb1@Co SA; (B) UV-Vis spectra of the decomposition of H2O2 (same concentration) by Rb1@Co SA (different concentrations); (C) Time-progress curve of H2O2 decomposition by Rb1@Co SA (100 μg / mL); (D) Curve of H2O2 residual concentration in the system changing with time after multiple additions of H2O2 (10 mM); (E) Curve of dissolved oxygen concentration changing with time (Control group is the material-free system, black dot); (F) O2 concentration before and after treatment with Rb1@Co SA (100 μg / mL). •⁻ Comparison of ESR spectra of absorption intensity; (G) Schematic diagram of CAT-like and SOD-like activity mechanisms of Rb1@Co SA; (H) Bio-TEM image of BB@Rb1@Co SA system; (I) Bar chart of viable bacteria count in BB@Rb1@Co SA after 30 min in SGF; (J) Bar chart of viable bacteria count in BB@Rb1@Co SA after 5 h in SIF; H2O2 scavenging rate (K) and O2 scavenging rate of BB alone and BB@Rb1@Co SA after 30 min in SGF. • ⁻Statistical histogram of clearance rate (L); after 5 h in SIF, the clearance rates of H2O2 (M) and O2 by BB alone and BB@Rb1@Co SA. • ⁻Statistical histogram of clearance rate (N). Data are presented as mean ± SD (n=3). 30 min vs 0 min or 5 h vs 0 h, p > 0.05 and **p < 0.01, analyzed by one-way ANOVA.

[0191] This invention verifies whether the assembly strategy effectively maintains the structure and functionality of each component, focusing first on the catalytic activity of Rb1@CoSA. H2O2 is one of the main reactive oxygen species excessively generated in UC, and its accumulation significantly exacerbates intestinal oxidative stress damage. Using the Ti(SO4)2 colorimetric method, under a fixed H2O2 concentration (10 mM), the UV-Vis absorption spectra of reaction systems with different concentrations of Rb1@CoSA (0 ~ 100 μg / mL) were detected to evaluate its catalytic decomposition efficiency for H2O2. Figure 5 B). With increasing Rb1@Co SA concentration, the characteristic absorption peak of H2O2 decreased significantly, showing a concentration-dependent effect. Further time-course monitoring showed that H2O2 (10 mM) was completely decomposed within 60 min under the action of 100 µg / mL Rb1@Co SA, demonstrating highly efficient scavenging ability. Figure 5 C). To simulate the pathological microenvironment of continuous ROS generation in UC, an equal amount of H2O2 was repeatedly added to the reaction system every 60 min, and its concentration change was monitored ( Figure 5D). The results showed that Rb1@Co SA maintained stable and high catalytic efficiency in multiple rounds of reaction, with no obvious activity decay or time delay. This indicates that the material has excellent catalytic stability and can effectively cope with the persistent oxidative stress microenvironment. Based on the excellent H2O2 decomposition ability of Rb1@Co SA, it is speculated that it may catalyze the generation of O2 through catalase-like activity, thereby improving the hypoxic state in the inflamed area and benefiting intestinal barrier repair. The O2 generation in the same reaction system was monitored in real time using a dissolved oxygen meter. Figure 5 E). After adding H2O2 for 30 min, the dissolved oxygen concentration in the solution increased significantly from 6.20 mg / L to 13.15 mg / L, indicating that Rb1@CoSA can continuously release O2 while removing H2O2, showing good oxygen production capacity.

[0192] Besides H2O2, superoxide anion (O2 • (⁻) is also an important ROS involved in oxidative damage during UC pathology. Electron spin resonance (ESR) technology was used with DMPO as a spin trapping agent to investigate the effect of Rb1@CoSA on O2. • ⁻ Clearance ability ( Figure 5 F). The results showed that the ESR spectra of the Control group exhibited DMPO-O2. • - A distinct 1:1:1:1 quadruple signal peak (black line). However, after adding Rb1@Co SA (100 µg / mL), the intensity of this characteristic signal peak significantly decreased or even disappeared (red line), indicating that Rb1@Co SA can efficiently scavenge O2. • ⁻. The above results indicate that Rb1@CoSA possesses both highly efficient cascade catalytic CAT-like and SOD-like activities, and can synergistically remove excess H2O2 and O2 generated in the inflammatory microenvironment of UC. • It contains multiple reactive oxygen species, such as ⁻, which effectively alleviate oxidative stress. Figure 5 G).

[0193] Based on the excellent catalytic performance of Rb1@Co SA, the potential of the BB@Rb1@Co SA ternary system to act on the intestinal inflammatory microenvironment after oral delivery was further evaluated in an in vitro simulated environment from three aspects: bacterial conjugation behavior, gastrointestinal tolerance, and ROS scavenging capacity retention. TEM images show that typical attachment structures appear on the BB surface, which are basically consistent with the morphological characteristics of Rb1@Co SA nanoparticles. Figure 5H). The nanoparticles were uniformly distributed without aggregation, and the bacterial cell structure remained intact without significant damage. This indicates that the binding of Rb1@Co SA to BB exhibits good biocompatibility, ensuring biosafety during oral delivery. Furthermore, the nanozyme was localized only to the outer wall of BB without internalization, which facilitates its function in the colon.

[0194] Given that oral nanodelivery systems need to withstand the strong acidity of the stomach and the complex enzymatic environment of the intestine, the stability and functional retention of BB@Rb1@CoSA during gastrointestinal transport were investigated using simulated gastrointestinal fluid experiments. BB alone showed good survival in simulated physiological saline (0.9% NaCl). When incubated for 30 min, the number of viable bacteria did not decrease significantly. After extending the incubation time to 5 h, the number of viable bacteria in BB showed a certain decrease. This may be because the longer incubation time and insufficient nutrients in the simulated in vitro environment led to BB death. After incubation in simulated gastric juice (SGF) for 30 min, the number of viable bacteria in BB@Rb1@CoSA also did not decrease significantly. Figure 5 I). After incubation in simulated intestinal fluid (SIF) for 5 h, the decreasing trend of viable bacteria count in BB@Rb1@CoSA was similar to that of BB alone in simulated saline. Figure 5 J). Meanwhile, after 30 min of SGF incubation, the scavenging rate of BB@Rb1@Co SA for H2O2 and its scavenging rate for O2... •⁻ The inhibition rates remained at a high level, and were significantly higher than those of the BB group without nanozyme modification. Figure 5 K and Figure 5 Similarly, after incubation in SIF for 5 hours, the system still maintains high efficiency for H2O2 and O2. •⁻ Clearance ability ( Figure 5 M and Figure 5 N).

[0195] The results showed that BB@Rb1@Co SA could effectively resist the interference of gastric and intestinal juices, maintain efficient ROS clearance capacity and BB activity, and has the potential to be used for oral intervention of the intestinal oxidative stress microenvironment.

[0196] The therapeutic effect of BB@Rb1@Co SA on UC mice

[0197] See Figure 6 , Figure 6The following are the indicators for evaluating the mice in each group during the acute UC treatment experiment provided by this invention. (A) Survival rate chart; (B) Weight change chart; (C) Representative colon photograph, the scale bar is 1 cm; (D) Histogram of colon length; (E) Histogram of spleen index; (F) Colon tissue staining (H&E and AB-PAS) and immunofluorescence staining (Claudin-1, Occludin and ZO-1) images, the scale bar is 200 μm, respectively; (I) Immunofluorescence intensity histogram of Claudin-1 (G), Occludin (H) and ZO-1; (J) Histogram of neutral / acidic mucus area; (KN) Histogram of serum inflammatory cytokine (IL-1β, TNF-α, IL-6 and IL-10) levels. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, analyzed by one-way ANOVA.

[0198] This invention first evaluates the therapeutic effect of BB@Rb1@Co SA on dextran sulfate sodium (DSS)-induced acute UC in mice. Figure 6 An acute ulcerative colitis (UC) model was established in C57BL / 6 mice by administering drinking water containing 3% (w / v) DSS for 7 consecutive days, with a blank control group (normal drinking water, n=10). On day 8 after modeling, the UC mice were randomly divided into 7 groups (n=10): BB@Rb1@CoSA group (2.6 mg / kg Rb1@CoSA + 10... 8 CFU / kg BB); Positive group (40 mg / kg 5-Amino salicylic acid); BB group (10 8 The groups were categorized into several groups: CFU / kg, Rb1 (0.6 mg / kg), CoSA (2 mg / kg), Rb1@CoSA (2.6 mg / kg), and a model group (normal drinking water). After three consecutive days of oral administration, the intervention effect was comprehensively evaluated using multiple indicators, including survival rate, weight change, colon length, spleen index, colon tissue AB / PAS staining, H&E staining and immunofluorescence analysis, and serum inflammatory factor levels.

[0199] At the end of day 10 of the experiment, the survival rate of mice in the Model group dropped significantly to the lowest level. The survival rates of mice in other groups were all better than those in the Model group. The BB@Rb1@Co SA group had the highest survival rate of UC mice, which was slightly better than that of the Positive group. Figure 6A). Regarding weight change, the Model group experienced sustained weight loss induced by DSS, and all treatment groups were able to alleviate this trend, with the BB@Rb1@Co SA group showing the best results and a slightly better ability to maintain weight than the Positive group. Figure 6 B). Regarding colon morphology, the Model group showed shortened length and intestinal wall congestion (Model vs Control, ****p < 0.0001), while BB@Rb1@Co SA treatment significantly restored colon length and structural integrity (BB@Rb1@Co SA vs Model, **p < 0.01). Figure 6 C and Figure 6 D). Spleen index results showed that the Model group exhibited compensatory splenomegaly (elevated spleen index) due to DSS-induced inflammatory activation, while the BB@Rb1@Co SA group significantly inhibited this phenomenon (BB@Rb1@Co SA vs Model, ****p < 0.0001). Figure 6 E). Colonic tissue staining (AB, PAS and H&E) and immunofluorescence staining (Claudin-1, Occludin and ZO-1) showed that the Model group exhibited severe mucosal damage, disruption of mucus layer integrity, crypt loss, inflammatory infiltration, and low expression of tight junction proteins (Model vs Control, ****p < 0.0001). Figure 6 (FJ). Among the treatment groups, BB@Rb1@Co SA showed the most significant improvement, with better restoration of epithelial structure and barrier function than the Positive group. The mechanism may stem from the probiotic properties and targeted delivery capabilities of this ternary system, the synergistic effect of Co SA in clearing ROS and reducing inflammation, and Rb1 in repairing the intestinal barrier. The Rb1 group showed the worst effect, possibly due to the low bioavailability of single-molecule Rb1 (it may even be metabolized in the stomach). H&E staining of major organs showed that BB@Rb1@Co SA could alleviate mild splenic inflammation without causing lesions in other organs, indicating its good biocompatibility.

[0200] Regarding cytokines, the Model group showed increased levels of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) and decreased levels of the anti-inflammatory cytokine IL-10 (Model vs Control, ****p < 0.0001). BB@Rb1@Co SA was the most effective at reversing this imbalance, significantly regulating all factors (BB@Rb1@Co SA vs Model, ***p < 0.001), showing better results than the Positive group. Rb1@Co SA also performed well, followed by the BB group, with the Rb1 group showing the weakest effect. Figure 6Serum biochemistry and complete blood count results showed that AKP, ALT, and AST levels were normal in the Model group, indicating that liver function was not significantly affected. BUN, CRE, and UA were abnormally elevated, WBC, PLT, and LYM were increased, while GRAN, RBC, and HGB were decreased, suggesting inflammation and metabolic disorders in the mice (Model vs Control, ***p < 0.001). BB@Rb1@Co SA treatment significantly improved these indicators (BB@Rb1@Co SA vs Model, **p < 0.01), showing better results than other treatment groups. The Positive group was the second best, while the Rb1 group still showed the least satisfactory results.

[0201] In summary, the BB@Rb1@CoSA ternary system, through its synergistic anti-inflammatory, antioxidant, and barrier repair effects, demonstrated optimal efficacy in improving survival rate, body weight, colon morphology, cytokine balance, and physiological indicators. Its therapeutic effect is superior to that of individual components and binary systems, and also superior to positive control drugs, fully demonstrating its synergistic advantages in the treatment of acute UC.

[0202] See Figure 7 , Figure 7 The gut microbiota characteristics of mice in each group during the acute UC treatment experiment provided by this invention are shown. Among them, (A) a graph showing the fit of the neutral community model to the microbiota data; (B) a graph showing the relative abundance distribution of major microbial taxa at the phylum level in each group; (C) a heatmap showing the clustering of major microbial phyla in each group; and (D) a Circos map showing the association between phylum-level species and samples.

[0203] To investigate the impact of therapeutic intervention on the gut microbiota, this study performed 16S rRNA sequencing analysis on fecal samples from different groups. The raw data have been submitted to NCBI SRA (…). Figure 7 The fitting results of the neutral community model show that its model fit (R) is satisfactory. 2 = 0.5686) is relatively low, indicating that the microbial community construction is more of a deterministic process than a stochastic one, further validating the successful construction of the DSS-induced ulcerative colitis model. Figure 7 A).

[0204] Regarding the phylum-level microbial composition, the abundance of Bacteroidota was significantly reduced in the Model group, while Pseudomonadota and Campylobacterota, which are closely related to intestinal inflammation, were significantly enriched. After intervention with BB@Rb1@Co SA, the ratio of Bacillota / Bacteroidota tended to be balanced, and the excessive proliferation of Pseudomonadota was significantly inhibited. Figure 7B). Heatmap analysis further revealed an abnormally elevated trend of Pseudomonadota and Campylobacterota in the Model group at the phylogenetic level. BB@Rb1@Co SA treatment effectively reversed this phenomenon and increased the relative abundance of Actinomycetota (B). Figure 7 C). This further corroborates the regulatory effect of BB@Rb1@Co SA on the gut microbiota at the phylum level. Changes in these microbes may synergistically promote the relief of colitis by enhancing intestinal barrier function and regulating immune responses. The composition and distribution of microbial taxa in different samples were visualized using Circos analysis. Figure 7 D). In the Model group, the mean relative abundance of pathogenic bacteria Pseudomonadota and Campylobacterota reached 32.3% and 4.3%, respectively. After treatment with BB@Rb1@Co SA, these two types of pathogens were almost completely eliminated. At the same time, Bifidobacterium of the Actinomycetota phylum successfully colonized, and its increase helped alleviate intestinal inflammation.

[0205] See Figure 8 , Figure 8 This invention provides an assessment of the dominant gut microbiota and metabolite indicators in mice in each group during an acute UC treatment experiment. The results include: (A) LDA score distribution of significantly enriched microbial categories in each group during LEfSe analysis; (B) heatmap of differentially metabolites (short-chain fatty acids) in the gut of each group; and statistical graphs of differentially metabolites in Model vs Control (C), Positive vs Model (D), BB vs Model (E), Rb1 vs Model (F), Co SA vs Model (G), Rb1@Co SA vs Model (H), and BB@Rb1@Co SA vs Model (I).

[0206] Further validation was performed using the results of Linear Discriminant Analysis Effect Size (LEfSe) analysis. Figure 8A). Notably, only the BB@Rb1@Co SA group showed significant enrichment of beneficial bacteria, including Actinobacteria, Bifidobacteriaceae, Bifidobacteriales, and Bifidobacterium. This indicates that BB successfully achieved colon-targeted colonization after treatment with BB@Rb1@Co SA, thereby alleviating acute UC by restoring gut microbiota homeostasis. In contrast, the treatment group using BB alone did not induce a significant enrichment of the aforementioned probiotics. Furthermore, the fact that BB alone did not cause similar changes suggests that without the synergistic support of Rb1 and Co SA, BB struggles to effectively colonize and proliferate in the inflammatory microenvironment.

[0207] The improvement in the above-mentioned microbial community structure is similar to the restoration of short-chain fatty acid (SCFA) metabolism. Figure 7 and 8 The levels of SCFAs were significantly reduced in the UC model group. This decrease exacerbates intestinal inflammation and tissue damage, potentially leading to a vicious cycle of dysbiosis, metabolic disorders, and disease progression. Figure 8 B). After intervention with the BB@Rb1@Co SA ternary system, the level of SCFAs returned to normal. Figure 8 I) indicates the restoration of gut microbiota-metabolic balance. In restoring SCFA levels, regulating gut microbiota structure, and alleviating pathological manifestations of UC (such as mucosal repair and downregulation of inflammatory factors), BB@Rb1@CoSA was significantly superior to each single treatment group. Figure 6 and Figure 8 (E–H). This ternary system exhibits excellent colon-targeted release capability, effectively resisting gastric acid and bile salt environments, and precisely delivering drugs to the lesion area of ​​the colon. Subsequently, the colonized BB bacteria significantly increased the production of SCFAs such as butyrate by promoting the restoration of intestinal flora homeostasis and synergistically fermenting dietary fiber. Its overall therapeutic advantage stems from the synergistic effect of Rb1, CoSA, and BB: Rb1 regulates the immune microenvironment and inhibits excessive inflammatory response; CoSA reduces oxidative stress and accelerates epithelial repair; both together optimize the colonization microenvironment of BB. Successfully colonized BB further enhances SCFA production and promotes the restoration of flora structure. The components of BB@Rb1@CoSA are interdependent and synergistic, demonstrating the excellent comprehensive performance of this material. The rational construction of this ternary system achieves effective regulation of acute UC and precise restoration of intestinal microbiota.

[0208] See Figure 9 , Figure 9The following are the indicators for evaluating the mice in each group during the chronic UC treatment experiment provided by this invention. (A) Survival rate chart; (B) Weight change chart; (C) Representative colon photograph, the scale bar is 1 cm; (D) Histogram of colon length; (E) Histogram of spleen index; (F) Colon tissue staining (H&E and AB-PAS) and immunofluorescence staining (Claudin-1, Occludin and ZO-1) images, the scale bar is 200 μm, respectively; (G) Histogram of Claudin-1, Occludin and ZO-1 immunofluorescence intensity; (H) Histogram of neutral / acidic mucus area; (I-L) Histogram of serum inflammatory cytokine (IL-1β, TNF-α, IL-6 and IL-10) levels. *p < 0.05, **p <0.01, ***p < 0.001, ****p < 0.0001, analyzed by one-way ANOVA.

[0209] See Figure 10 , Figure 10 The following are assessments of gut microbiota characteristics and metabolite parameters in mice in the chronic UC treatment experiment provided by this invention. Among them, (A) a graph showing the fit of the neutral community model to the microbiota data; (B) a graph showing the relative abundance distribution of major microbial taxa at the phylum level in each group; (C) a heatmap of clustering at the major microbial phylum level in each group; (D) a Circos map showing the association between phylum-level species and samples; (E) a heatmap of differentially expressed metabolites (short-chain fatty acids) in the gut of each group; and statistical graphs of differentially expressed metabolites in Model vs Control (F), Positive vs Model (G), and BB@Rb1@Co SA vs Model (H).

[0210] In therapeutic studies targeting a mouse model of chronic UC, BB@Rb1@Co SA continued to demonstrate sustained and significant therapeutic effects, further highlighting its potential as a ternary synergistic therapeutic strategy in long-term UC disease intervention. Figure 9 and Figure 10 Echoing the results of acute-phase treatment, this ternary system significantly improved several core pathological indicators of chronic UC: effectively increasing long-term survival, maintaining stable weight gain, reversing colonic shortening, and significantly reducing the spleen index and alleviating splenic inflammatory infiltration, suggesting that it has a controlling effect on chronic intestinal inflammation and the accompanying systemic immune overactivation. Figure 9A–E). Histopathological results showed that the colonic mucosal crypt structure of treated mice was reconstructed, the number of goblet cells and mucus secretion function were significantly restored, and inflammatory infiltration was significantly reduced (AB / PAS and H&E staining). Immunofluorescence analysis further showed that the expression of key tight junction proteins (Claudin-1, Occludin, ZO-1) was significantly upregulated, confirming that the intestinal mucosal mechanical barrier function can still be effectively repaired by BB@Rb1@CoSA in the chronic stage. Figure 9 F–H). Regarding the regulation of the chronic inflammatory microenvironment, BB@Rb1@CoSA treatment significantly reduced the expression of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) and upregulated the level of the anti-inflammatory factor IL-10, highlighting its excellent immunomodulatory capabilities. Figure 9 In addition, this treatment system also showed comprehensive improvement in systemic metabolic and hematological abnormalities induced by chronic UC model: among serum biochemical indicators, creatinine (CRE) and uric acid (UA) levels were significantly reduced; blood routine tests showed that WBC, LYM and PLT counts tended to normalize, and the downward trend of GRAN, RBC and HGB was reversed.

[0211] The low goodness of fit (R0) of the neutral community model 2 =0.4683) indicates that under chronic UC, the assembly of the microbial community is mainly dominated by deterministic processes, further validating the successful establishment of the DSS-induced chronic UC model. Figure 10 A). Notably, mice in the Control group showed a significant increase in body weight under free-feeding conditions, along with a significant increase in the abundance of Bacillota in their gut microbiota, suggesting that this phylum may play a key role in promoting energy absorption and the obesity phenotype. In the chronic UC model group, under conditions of severe dysbiosis and metabolic disturbances, treatment with BB@Rb1@Co SA effectively reconstructed the gut microbiota composition: restoring the balance between Bacteroidota and Bacillota, inhibiting the abnormal proliferation of opportunistic pathogens such as Pseudomonadota and Campylobacterota, and promoting the colonization and enrichment of beneficial bacteria such as Bifidobacterium. Figure 10 BD). Furthermore, this treatment significantly increased SCFA levels ( Figure 10 EH), especially butyric acid, propionic acid and acetic acid.

[0212] These results demonstrate that the BB@Rb1@CoSA system exhibits significant therapeutic efficacy in chronic ulcerative colitis (UC). It not only effectively remodels the gut microbiota structure and restores microbial homeostasis, but also significantly increases the levels of beneficial metabolites, synergistically strengthens epithelial barrier function, and alleviates systemic inflammation. These findings further confirm that this ternary system, through its multi-mechanism synergistic effects of "antioxidant-anti-inflammatory repair-microbiota regulation," possesses unique advantages for achieving long-term, multi-dimensional intervention therapy, providing a promising new intervention pathway for the clinical treatment of acute / chronic UC.

[0213] The foregoing has provided a detailed description of the cobalt single-atom nanozyme ternary composite system, its preparation method, and its applications. Specific examples have been used to illustrate the principles and implementation methods of the invention. The descriptions of these embodiments are merely for the purpose of helping to understand the method and core ideas of the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including manufacturing and using any device or system, and implementing any combined method. It should be noted that for those skilled in the art, several improvements and modifications can be made to the invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims. The scope of protection of this patent is defined by the claims and may include other embodiments that can be conceived by those skilled in the art. If these other embodiments have structural elements that are not different from the wording of the claims, or if they include equivalent structural elements that are not substantially different from the wording of the claims, then these other embodiments should also be included within the scope of the claims.

Claims

1. A phospholipid polyethylene glycol carboxyl-modified cobalt monatomic nanoszyme system, characterized in that, The application relates to a phospholipid polyethylene glycol carboxyl modified cobalt monatomic nanoscale enzyme system. The cobalt monatomic nanoscale enzyme composite core comprises a cobalt monatomic nanoscale enzyme and a ginsenoside loaded on the cobalt monatomic nanoscale enzyme. The particle size of the cobalt monatomic nanoscale enzyme is 100-300 nm.

2. The Co-soloatom nanoszyme system according to claim 1, wherein, The cobalt monatomic nanoscale enzyme has a morphology of a rhombohedron. The ginsenoside comprises ginsenoside Rb1. The loading amount of the ginsenoside on the cobalt monatomic nanoscale enzyme is 30%-60%. The mass content of Co in the cobalt monatomic nanoscale enzyme is 1%-2%. The phospholipid polyethylene glycol carboxyl is modified on the surface of the cobalt monatomic nanoscale enzyme composite core to form a wrapping layer. The thickness of the wrapping layer is 3-10 nm. The phospholipid polyethylene glycol carboxyl modified cobalt monatomic nanoscale enzyme system is chemically grafted on the surface of the system by 3-aminobenzene boronic acid.

3. A 3-aminobenzoic acid grafted phospholipid polyethylene glycol carboxyl modified cobalt monatomic nanoszyme system characterized in that, The application relates to a phospholipid polyethylene glycol carboxyl modified cobalt monatomic nanoscale enzyme system.

4. A Bifidobacterium synergistic cobalt monatomic nanoszyme system, characterized in that, The cobalt monatomic nanoscale enzyme composite core comprises a cobalt monatomic nanoscale enzyme and a ginsenoside loaded on the cobalt monatomic nanoscale enzyme. The cobalt monatomic nanoscale enzyme composite core comprises a cobalt monatomic nanoscale enzyme and a ginsenoside loaded on the cobalt monatomic nanoscale enzyme. The double-bifidobacterium comprises bifidobacterium bifidum.

5. The Co-soloatom nanoszyme system according to claim 4, wherein, The ratio of the mass of the cobalt monatomic nanoscale enzyme composite core to the number of bifidobacteria is (1~2) mg:(10 8 ~10 10 ) CFU; When the cobalt monatomic nanoscale enzyme composite core is the cobalt monatomic nanoscale enzyme composite core in the cobalt monatomic nanoscale enzyme system of claim 3, the mass ratio of the phospholipid polyethylene glycol carboxyl modified cobalt monatomic nanoscale enzyme system to 3-aminobenzene boronic acid is 1: (1-30). When the cobalt monatomic nanoscale enzyme composite core is the cobalt monatomic nanoscale enzyme composite core in the cobalt monatomic nanoscale enzyme system of claim 3, the chemical grafting is that the carboxyl at the end of the phospholipid polyethylene glycol carboxyl is reacted with the amino of 3-aminobenzene boronic acid through amidation, and the benzene boronic acid group is grafted and modified on the surface. The binding mode comprises specific binding. The binding is that the benzene boronic acid group on the surface of the cobalt monatomic nanoscale enzyme system of claim 3 is specifically bound with the cis-diol structure of a glycoprotein on the surface of the double-bifidobacterium. The application relates to a phospholipid polyethylene glycol carboxyl modified cobalt monatomic nanoscale enzyme system.

6. A preparation method of a Bifidobacterium synergistic cobalt monatomic nanoszyme system, characterized in that, 1) a Co@MOF is obtained by mixing a zinc salt, a cobalt salt and an organic solvent, then adding an organic 2-methyl imidazole solution and mixing and reacting; 2) an alcohol solution is obtained by mixing the Co@MOF obtained in the above step and alcohol, then adding ammonia water-alcohol solution, and then adding tetraethyl orthosilicate and reacting; 3) a cobalt monatomic nanoscale enzyme Co SA is obtained by pyrolyzing the Co@MOF@SiO2 obtained in the above step under a protective atmosphere, and then etching with an alkali solution; 4) Gs@Co SA is obtained by dispersing and mixing the cobalt monatomic nanoscale enzyme Co SA obtained in the above step, ginsenoside and an organic solvent, and then separating; then the Gs@Co SA, a water-miscible organic solvent and a DSPE-PEG-COOH aqueous solution are mixed again, ultrasonic crushing is carried out, and dialysis is carried out to obtain the DSPE-PEG-COOH modified Gs@Co SA. ​ 5) mixing the DSPE-PEG-COOH modified Gs@Co SA, the buffer, the EDC and the NHS obtained in the above step, and then adding 3-aminobenzoic acid and continuing to mix, to obtain a 3-aminobenzoic acid group grafted single-atom nanoscale enzyme system, and then mixing the 3-aminobenzoic acid group grafted single-atom nanoscale enzyme system with a bifidobacterium solution to obtain a bifidobacterium synergistic cobalt single-atom nanoscale enzyme system.

7. The preparation method according to claim 6, characterized in that, The zinc salt comprises zinc nitrate; The cobalt salt comprises cobalt nitrate; In the step 1), the organic solvent and the solvent in the 2-methylimidazole organic solution are each independently selected from DMF and methanol; The molar ratio of the zinc salt to the cobalt salt is (6-9): 1; The mass ratio of the cobalt salt to 2-methylimidazole is 1:(7-10); The mixing reaction time is 10-14 hours; The alcohol comprises ethanol; In the ammonia water-alcohol solution, the volume ratio of ammonia water to alcohol is 1:(15-18); The mass ratio of the tetraethyl orthosilicate to Co@MOF is (3-4):

1.

8. The preparation method according to claim 6, characterized in that, In the step 2), the reaction time is 1-3 hours; In the step 2), after the reaction, the step of standing overnight is further included; The pyrolysis temperature is 900-1000℃; The pyrolysis time is 3-5 hours; The alkali solution comprises a NaOH solution and / or a KOH solution; The molar concentration of the alkali solution is 3-5M; The etching time is 10-14 hours; In the step 4), the organic solvent comprises methanol; The mass ratio of the cobalt single-atom nanoscale enzyme Co SA to ginsenosides is 1:(1-5).

9. The preparation method according to claim 6, characterized in that, The water-miscible organic solvent comprises THF; The dialysis is specifically performed under the condition of Mw=8000-10000; The buffer comprises a MES buffer; The pH value of the buffer is 5-6; The mass ratio of the DSPE-PEG-COOH modified Gs@Co SA to 3-aminobenzoic acid is 1:(7.5-20); The mass-volume ratio of the 3-aminobenzoic acid group grafted single-atom nanoscale enzyme system to the bifidobacterium solution is 1mg:(2-10)mL; The bifidobacterium comprises Bifidobacterium bifidum; The concentration of the bifidobacterium solution is 10 8 ~10 10 CFU / mL; The bifidobacterium synergistic cobalt single-atom nanoscale enzyme system is a nanoscale enzyme system for treating inflammatory bowel disease.

10. Use of the cobalt single-atom nanoscale enzyme system of any one of claims 4-5 or the bifidobacterium synergistic cobalt single-atom nanoscale enzyme system prepared by the preparation method of any one of claims 6-9 in the preparation of a drug for treating inflammatory bowel disease.

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