Use of a co-atom nanoscale enzyme in preventing radiation-induced intestinal injury

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the application of a Co single-atom nanozyme in the prevention of radiation-induced intestinal injury. The invention first prepares a ZnCo-MOF material by coordinating zinc and cobalt metal ions with a 2-methylimidazole organic ligand via a solvothermal synthesis method. Subsequently, this MOF precursor is pyrolyzed at high temperature to convert it into a Co single-atom nanozyme, ultimately constructing a nanozyme material that combines multi-enzyme mimicry characteristics with oral pH responsiveness. This material achieves a dual core effect in the treatment of abdominal / pelvic tumors: enhancing the efficacy of radiotherapy and protecting the intestine from radiation damage. Its intestinal radiation protection mechanism is achieved by downregulating the PI3K / AKT pathway, reducing the formation of the extracellular neutrophil network (NETs), and scavenging reactive oxygen species in the intestine, thereby protecting the intestine from radiation damage. This invention provides a novel technical approach and implementation path for the clinical prevention and treatment of acute intestinal radiation injury.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to the application of a Co single-atom nanozyme in the prevention of radiation-induced intestinal injury. Background Technology

[0002] Currently, radiotherapy (RT) has become a major treatment for malignant tumors. Its mechanisms of action include directly damaging biomolecules and inducing indirect oxidative damage through the production of excessive reactive oxygen species (ROS), ultimately leading to cell death. However, in clinical applications, the existence of tumor heterogeneity (such as hypoxia) significantly reduces the radiation sensitivity of tumors compared to normal tissues. Therefore, high-energy ionizing radiation (IR) inevitably causes irreversible radiation damage to normal tissues, thus limiting the efficacy of radiotherapy for tumors. Among these, the intestines are particularly sensitive to ionizing radiation, and intestinal damage after radiotherapy has a significant impact on the prognosis of patients with abdominopelvic cancer. Therefore, achieving radiation protection of normal tissues and improving the efficiency of radiotherapy for abdominopelvic tumors is both an urgent clinical need and a major challenge that needs to be overcome.

[0003] Currently, the clinically recognized radioprotective agent amifostine, while able to prevent radiation-induced intestinal injury by improving crypt cell survival, requires intravenous injection and has high toxicity and significant side effects. Although some progress has been made in small-molecule radioprotective agents such as vitamin E and astaxanthin, which can prevent radiation-induced intestinal injury by scavenging reactive oxygen species (ROS), these organic molecules have drawbacks such as poor water solubility, short blood circulation time, and rapid metabolic rate, hindering the development of oral formulations. Furthermore, protecting the intestine from radiation damage by scavenging ROS may weaken the therapeutic effect of radiotherapy on tumors, a problem that has previously received little attention. Therefore, the development of novel and stable oral radioprotective agents is of paramount importance.

[0004] Single-atom nanozymes (SANs), with their precisely defined atomic structures mimicking the active sites of natural enzymes, combine the advantages of both natural enzymes and nanozymes, possessing unique characteristics such as high activity, excellent stability, and low cost. In recent years, the field of single-atom nanozymes has developed rapidly, successfully uncovering multi-enzyme-like activities such as peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT). In biomedical applications that mimic natural enzymes, nanozymes have shown great potential and achieved significant results. However, the preparation of bifunctional nanozymes that can respond to dynamic biological microenvironments and possess different enzyme activities to meet diverse biomedical needs still faces significant challenges. Furthermore, research on the related biological mechanisms of nanozymes is still in its early stages, which also introduces uncertainty into their application in the field of radiation protection. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, this invention constructs an orally administered pH-responsive Co single-atom nanozyme (Co-SAN) with multi-enzyme mimicry properties. This nanozyme can achieve dual effects in the treatment of abdominal / pelvic tumors: enhancing the efficacy of radiotherapy and protecting the intestine from radiation damage.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides the application of Co-Single Atom Nanozyme Co-SAN in the preparation of drugs to prevent radiation-induced intestinal injury. The Co-Single Atom Nanozyme Co-SAN is obtained by solvothermal synthesis of zinc and cobalt metal ions with 2-methylimidazolium organic ligands to form ZnCo-MOF, followed by high-temperature calcination and pyrolysis.

[0007] The nanozyme Co-SAN constructed in this invention can respond to pH changes in the biological microenvironment to simulate different enzyme activities, thus overcoming the challenges of radiotherapy and radiation protection for abdominal / pelvic tumors. Co-SAN exhibits excellent catalytic activities of CAT, SOD, and GPx in the intestinal environment, significantly alleviating oxidative stress and protecting the normal intestine from radiation damage through its superior ROS scavenging ability. Simultaneously, this composite nanocarrier can exert CAT, OXD, NOX, and GSHOx activities in the tumor microenvironment, both alleviating tumor hypoxia and generating reactive oxygen species to enhance the efficacy of radiotherapy for abdominal and pelvic tumors. In vitro experiments have demonstrated that this composite nanocarrier possesses good biocompatibility and excellent radiation protection capabilities for normal tissues. Subsequent in vivo experiments strongly confirmed the significant potential of Co-SAN in preventing intestinal side effects of radiotherapy and enhancing the efficacy of tumor radiotherapy. More importantly, flow cytometry and RNA sequencing revealed a novel mechanism for protecting against radiation-induced intestinal injury: Co-SAN exerts its protective effect against radiation-induced intestinal injury by downregulating the PI3K / AKT pathway to reduce the formation of the extracellular neutrophil network (NETs). This also elucidates the crucial role of NETs in radiation damage. These findings provide a feasible approach for constructing single-atom nanomedicines to mitigate the side effects of clinical radiotherapy.

[0008] Preferably, the preparation method of the Co single-atom nanozyme Co-SAN includes the following steps: S1. Disperse zinc salt and cobalt salt in methanol solution, mix well, then add methanol solution containing 2-methylimidazole, mix again, and carry out solvothermal reaction. After reaction, wash and dry to obtain ZnCo-MOF. S2. ZnCo-MOF is placed in an inert atmosphere and calcined at high temperature to obtain Co-SAN.

[0009] More preferably, in S1, the molar ratio of zinc ions, cobalt ions and 2-methylimidazole is 2:1:8-10.

[0010] More preferably, in S1, the temperature of the solvothermal reaction is 35-45°C and the time is 5-8 h.

[0011] More preferably, in S1, the zinc salt includes zinc nitrate, zinc chloride, zinc acetate, or zinc sulfate, and the cobalt salt includes cobalt nitrate, cobalt chloride, cobalt acetate, or cobalt sulfate.

[0012] More preferably, in S1, the mixing is performed by room temperature ultrasonic treatment.

[0013] More preferably, in S2, the high-temperature calcination is to raise the temperature to 800-1000°C at a heating rate of 3-7°C / min and maintain it at that temperature for 2-5 hours.

[0014] Preferably, the Co-SAN single-atom nanozyme protects the intestine from radiation damage by downregulating the PI3K / AKT pathway, reducing the formation of neutrophil extracellular network (NETs), and clearing intestinal reactive oxygen species (ROS).

[0015] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0016] More preferably, the excipients include at least one of the following: excipients, solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, coating materials, pH adjusters, absorbents, diluents, and antioxidants.

[0017] More preferably, the dosage form of the drug includes injections, tablets, capsules, granules, powders, syrups, or oral liquids.

[0018] Preferably, the administration methods of the Co single-atom nanozyme Co-SAN include oral administration, injection administration, subcutaneous administration, inhalation administration, and rectal administration.

[0019] Compared with the prior art, the beneficial effects of the present invention are: This invention first prepares ZnCo-MOF materials by coordinating zinc and cobalt metal ions with 2-methylimidazole organic ligands via a solvothermal synthesis method. Subsequently, the MOF precursor is pyrolyzed at high temperature to transform it into Co-SAN single-atom nanozymes, ultimately constructing a nanozyme material that combines multi-enzyme mimicry properties with oral pH responsiveness. This material achieves dual core effects in the treatment of abdominal / pelvic tumors: significantly enhancing the efficacy of radiotherapy on the one hand, and effectively protecting the intestine from radiation damage on the other. Its intestinal radiation protection mechanism is achieved by downregulating the PI3K / AKT pathway, reducing the formation of the extracellular neutrophil network (NETs), and scavenging intestinal reactive oxygen species (ROS), thereby protecting the intestine from radiation damage. This invention provides a novel technical approach and implementation path for the clinical prevention and treatment of acute intestinal radiation injury. Attached Figure Description

[0020] Figure 1 Physicochemical properties of Co-SAN-mimicked enzyme activity; Figures show: (a) Schematic diagram of pH-responsive multi-enzyme simulated activity of Co-SAN. (b) Bar chart of ABTS radical scavenging efficiency of Co-SAN under different pH conditions. (c) Curves showing the change in O2 generation efficiency over time when 10 mM H2O2 is mixed with PBS, N / C, Co-NPs, and Co-SAN. (d) Bar chart of H2O2 scavenging efficiency of Co-SAN under different pH conditions. (e) UV-Vis absorption spectra and glutathione peroxidase (GPx) activity assessment of the reaction of GSH with DTNB under different conditions. (f) Bar chart of glutathione (GSH) consumption of Co-SAN under different pH conditions. (g) Effects of different concentrations of Co-SAN on ABTS· + DPPH·, O2· - (h) Three-dimensional bar chart of H2O2 removal efficiency of Co-SAN. (i) UV-Vis spectrum (time gradient) of Co-SAN catalyzing NADH oxidation reaction. (j) UV-Vis spectrum (time gradient) of Co-SAN catalyzing NADPH oxidation reaction. (k) Bar chart of NAD(P)H consumption by Co-SAN under different pH conditions. (l) Detection of O2 by DMPO scavenger. •- Electron spin resonance (ESR) spectra of ·OH. (m) Electron spin resonance spectra of ·OH detected by ESR spectroscopy. (n) Three-dimensional bar charts of simulated activities of different concentrations of Co-SAN on NOX, OXD, POD, and GSHOx.

[0021] Figure 2Oral administration of Co-SAN provides comprehensive protection against radiation-induced intestinal damage; Figure: (a) Gastrointestinal fluorescence images of mice 1, 2, 4, 8, and 12 hours after oral administration of Co-SAN@Cy7 (containing an equal amount of Cy7); Cy7 channel: excitation wavelength 740 nm; emission wavelength 770 nm; white dashed circles indicate the area for quantitative analysis of fluorescence intensity. (b) Schematic diagram of the experimental procedure. (c) Changes in mouse body weight (n=3 biologically independent animals), data are expressed as mean ± standard deviation; the p-value between the 12 Gy abdominal X-ray irradiation group (IR) and the IR-Co-SAN group was calculated using a two-tailed t-test. (d) Survival curves of mice irradiated with a lethal dose of 16 Gy abdominal X-ray (n=5 biologically independent animals), median survival: PBS group >50 days; IR+PBS group 21 days; IR+N / C group 21 days; IR+Co-NPs group 28 days; IR+Co-SAN group >50 days; p-values ​​were calculated using a Log-rank (Mantel-Cox) test. (e) Colon length of mice after irradiation in each group (n=5 biologically independent animals). (f) Flow cytometry analysis of ROS content in intestinal tissues of each group (n=3 biologically independent animals). Data are expressed as mean ± standard deviation. P-values ​​between the IR group and the IR+Co-SAN group were calculated using a two-tailed t-test. (gh) HE staining images (g) and villus length (h) of small intestine (duodenum, jejunum, ileum) after treatment with IR+PBS, IR+N / C, IR+Co-NPs, and IR+Co-SAN (n=6 biologically independent animals). Scale bar = 100 µm. The experiment was independently repeated three times, and the results were consistent. (i) Representative immunofluorescence images of tight junction protein-3, occlusion protein, ZO-1, F4 / 80, γ-H2AX, and Tunnel expression in the intestines of each group after irradiation (n=5 biologically independent animals). Data are expressed as mean ± standard deviation. P-values ​​were calculated using a two-tailed t-test. Compared with the IR+PBS group ( <0.05, <0.01, <0.001, ns., no statistical significance).

[0022] Figure 3 Co-SAN mitigates radiation-induced intestinal damage by influencing the PI3K / AKT pathway; Figure: (a) Mice were orally administered Co-SAN and then irradiated with 12 Gy abdominal X-rays (IR), followed by RNA sequencing analysis. KEGG analysis showed the top 20 significantly enriched pathways. (b) GSEA analysis of genes altered after combined IR and Co-SAN treatment, showing normalized enrichment fractions (NES) and p-values. (c) Neutrophils were irradiated with 8 Gy X-rays and then treated with Co-SAN (80 μg / mL) or LY294002 (10 μM) for 24 hours, followed by Western blot analysis. (d) Schematic diagram of the experimental procedure. (e) Mouse body weight (n=3 biologically independent animals). Data are expressed as mean ± standard deviation. The p-value between the 12 Gy abdominal X-ray (IR) + Co-SAN group and the IR + Co-SAN + LY294002 group was calculated using a two-tailed t-test. (f) Survival curves of mice irradiated with a lethal dose of 16 Gy abdominal X-rays (n = 5 biologically independent animals), median survival: PBS group > 50 days; IR+PBS group 15 days; IR+LY294002 group 21 days; IR+Co-SAN group > 50 days; IR+Co-SAN+LY294002 group 35 days. (gh) HE-stained images (g) and villus length (h) of the small intestine (duodenum, jejunum, and ileum) after treatment with PBS, IR+PBS, IR+LY294002, IR+Co-SAN, and IR+Co-SAN+LY294002 (n = 6 biologically independent animals), scale bar = 100 µm. This experiment was independently repeated three times, with similar results. Detailed Implementation

[0023] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0024] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0025] In the radiotherapy of abdominopelvic tumors, achieving radiation protection of normal tissues while improving treatment efficiency is both an urgent clinical need and a major challenge that needs to be overcome. While the clinically recognized protectant amifostine can prevent radiation-induced intestinal injury by improving crypt cell survival, it requires intravenous injection and has significant toxicity and side effects. Although small-molecule protectants such as vitamin E and astaxanthin have made progress in scavenging reactive oxygen species (ROS), their poor water solubility, short blood circulation, and rapid metabolism hinder the development of oral formulations.

[0026] To this end, this invention constructs an orally administered pH-responsive Co-14 nanozyme (Co-SAN) with multi-enzyme mimicry properties. This nanozyme can achieve dual effects in the treatment of abdominal / pelvic tumors: enhancing the efficacy of radiotherapy while protecting the intestine from radiation damage. Preliminary studies show that orally administered Co-SAN has excellent stability in the gastric environment, ensuring its smooth arrival in the intestine and prolonging its residence time. Notably, in the intestinal microenvironment (pH=8.0), Co-SAN exhibits antioxidant properties of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx), effectively scavenging excess reactive oxygen species (ROS) and powerfully and rapidly reducing radiotherapy toxicity in the alkaline intestinal environment, protecting the intestine from radiation damage. More importantly, in the slightly acidic tumor microenvironment (pH=6.5), Co-SAN exhibits oxidase (OXD), NADPH oxidase (NOX), glutathione peroxidase (GSHOx), and catalase (CAT) activities, significantly enhancing the efficacy of tumor radiotherapy and accelerating tumor cell death. This study reveals a novel mechanism by which Co-SAN prevents radiation-induced intestinal injury. Neutrophils are well-known as among the first cells recruited to acutely injured tissues after radiation exposure, triggering a series of inflammatory responses. The extracellular reticular structures (NETs) formed by neutrophils play a dual role in this inflammation. NETs assist the body in defending against invading substances, but overexpression can lead to apoptosis of normal cells. Flow cytometry and RNA sequencing analysis confirmed that Co-SAN protects the intestine from acute radiation damage by downregulating the PI3K / AKT pathway, inhibiting NET formation, and simultaneously scavenging reactive oxygen species (ROS). These findings not only deepen our understanding of the mechanisms of radiation-induced intestinal injury but also provide new insights into the treatment of abdominal / pelvic tumors.

[0027] To fully and clearly present the technical solution and significant advantages of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0028] Example 1: Synthesis and Characterization of Nanozyme Co-SAN (1) Synthesis of ZnCo-MOF: Zn(NO3)2·6H2O (1.116 g) and Co(NO3)2·6H2O (0.5 g) were dispersed in a methanol solution (100 mL) and sonicated at room temperature for 20 min (approximately 200 W). Simultaneously, a methanol solution (100 mL) containing 2-methylimidazole (1.232 g) was injected, and sonication was continued at room temperature for another 20 min. The reaction was then carried out at 37 °C for 6 hours. After the reaction, the precipitate was washed three times with methanol and then dried under vacuum at 65 °C to obtain the final product.

[0029] (2) Synthesis of Co-SAN: ZnCo-MOF powder was placed in a tube furnace and heated to 900℃ at a rate of 5℃ / min under the protection of flowing nitrogen (0.5m / s), and held at this temperature for 3 hours. After the reaction, the sample was cooled to room temperature and Co-SAN was collected.

[0030] Comparative Example 1: Synthesis and Characterization of N / C Nanozymes (1) Synthesis of ZIF-8: First, 2-methylimidazole (1.232 g) was dispersed in a methanol solution (100 mL), followed by the addition of a methanol solution (100 mL) containing Zn(NO3)2·6H2O (1.116 g). The mixture was sonicated at room temperature (around 200 W) for 5 minutes and then placed at 37°C overnight. After the reaction, the precipitate was washed three times with methanol and then dried under vacuum at 65°C to obtain the final product.

[0031] (2) Synthesis of N / C: ZIF-8 powder was placed in a tube furnace and heated to 900°C at a rate of 5°C / min under the protection of flowing nitrogen (0.5 m / s), and held at this temperature for 3 hours. After the reaction, the N / C was collected after the sample cooled to room temperature.

[0032] Comparative Example 2: Synthesis and Characterization of Nanozymes Co-NPs (1) Synthesis of ZIF-67: First, 2-methylimidazole (1.232 g) was dispersed in a methanol solution (100 mL), followed by the addition of a methanol solution (100 mL) containing Co(NO3)2·6H2O (1.0 g). The mixture was sonicated at room temperature (around 200 W) for 5 minutes and then placed at 37 °C overnight. After the reaction, the precipitate was washed three times with methanol and then dried under vacuum at 65 °C to obtain the final product.

[0033] (2) Synthesis of Co-NPs: ZIF-67 powder was placed in a tube furnace and heated to 900℃ at a rate of 5℃ / min under the protection of flowing nitrogen (0.5m / s), and held at this temperature for 3 hours. After the reaction, the sample was cooled to room temperature and Co-NPs were collected.

[0034] Experimental Example 1: Enzyme Activity Characterization of Nanozymes 1. Experimental Methods (1) The antioxidant capacity of Co-SAN, N / C and Co-NPs was detected by 2,2-diphenyl-1-picrylhydrazine (DPPH). 80 μL of Co-SAN or N / C or Co-NPs (1 mg / mL) was added to 920 μL of DPPH solution (50 μg / mL), incubated for 30 min, and then the absorbance was measured at 519 nm.

[0035] (2) The antioxidant properties of Co-SAN, N / C, and Co-NPs at different pH values ​​were determined by the 2,2'-azobis(3-ethylbenzothiazole-6-sulfonic acid) (ABTS) method. 80 μL of Co-SAN, N / C, or Co-NPs (1 mg / mL) was added to 920 μL of ABTS at different pH values ​​(7.4, 7.8, 8.4, and 9.0). •+ After incubation in a buffer solution for 5 minutes, the absorbance at 734 nm was measured.

[0036] (3) To test the stability of Co-SAN, N / C and Co-NPs, they were dispersed in pH 1.2 buffer and incubated for 3 hours. After washing three times with deionized water, their antioxidant properties were further tested by the ABTS method.

[0037] (4) Detection of catalase mimicry activities of Co-SAN, N / C, and Co-NPs. The catalase mimicry activities of Co-SAN, N / C, or Co-NPs were explored by detecting the ability of samples to catalyze the decomposition of H2O2 to produce oxygen. After adding Co-SAN, N / C, or Co-NPs (2.5 μg / mL) to a buffer solution containing hydrogen peroxide (50 mM) (20 mM, pH 7.8), the amount of oxygen generated was immediately monitored using a dissolved oxygen analyzer (JPSJ-606L, Laixi, China).

[0038] (5) The inhibition rate of hydrogen peroxide by different concentrations of Co-SAN, N / C or Co-NPs was determined by a hydrogen peroxide detection kit (Bristol Biotech, China), and the pH dependence of its CAT-simulated activity was investigated.

[0039] (6) The superoxide dismutase mimic activities of Co-SAN, N / C, and Co-NPs were determined. The effects of different concentrations of Co-SAN, N / C, or Co-NPs on superoxide anion (O2) activity were further detected using a total SOD activity assay kit (Bio-Thera Solutions, China). •- The inhibition rate of ).

[0040] (7) Determination of glutathione peroxidase and oxidase mimicry activities of Co-SAN, N / C, and Co-NPs. The GPx mimicry activities of Co-SAN, N / C, or Co-NPs were determined using the 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) probe method. The reaction system contained glutathione (2 mM), H2O2 (0.1 mM, 0.5 mM, and 1 mM), and Co-SAN, N / C, or Co-NPs (80 μg / mL), prepared in buffer (20 mM, pH 7.8). After incubation in the dark for 30 minutes, DTNB was added, and the absorbance at 420 nm was measured.

[0041] (8) The pH dependence of the glutathione peroxidase (GPx) mimic activities of Co-SAN, N / C and Co-NPs was further investigated by adjusting the pH of the buffer solution (7.4, 7.8, 8.4 and 9.0). At the same time, the GSHOx mimic activity was determined by a similar method, but without the addition of H2O2 solution.

[0042] (9) Detection of NADPH oxidase mimic activity. The NADPH oxidase mimic activities of Co-SAN, N / C, and Co-NPs were detected by the change in absorbance of NADPH at 340 nm. Co-SAN, N / C, or Co-NPs (80 μg / mL) were added to a buffer solution containing NADH (200 μg / mL) and reacted under different pH conditions. The absorbance of the reactants at 260 nm and 340 nm was measured. Subsequently, the H2O2 generated during the oxidation reaction of different concentrations of NADH was detected using a hydrogen peroxide detection kit. The NADPH oxidation experiment was also measured using the same method as for NADH.

[0043] (10) Peroxidase mimicry and oxidase mimicry activities of Co-SAN, N / C and Co-NPs. The POD mimicry activities of Co-SAN, N / C or Co-NPs were detected by the 3,5,3',5'-tetramethylbenzidine (TMB) colorimetric method. The reaction solution containing Co-SAN or N / C or Co-NPs (80 μg / mL), H2O2 (0.1 mM) and TMB (0.5 mM) was prepared in PBS buffer (20 mM, pH 5.0), and the absorbance at 652 nm was measured.

[0044] (11) The OXD-simulated activities of Co-SAN, N / C, and Co-NPs were detected by 1,2-diaminobenzene (OPD) colorimetric method. Co-SAN, N / C, or Co-NPs (80 μg / mL) were added to PBS buffer (20 mM, pH 6.0) containing OPD (1 mM) and reacted, and the absorbance at 417 nm was measured. Simultaneously, the O2 generated at different oxidation times was detected by 1,3-diphenylisobenzofuran (DPBF). •- .

[0045] (12) To investigate the concentration- and pH-dependent peroxidase (POD) mimicry and oxidase (OXD) mimicry activities of Co-SAN, N / C, and Co-NPs, and to simultaneously conduct ·OH and O2 studies. •-The detection of ) was carried out by changing the concentration of the material in the reaction system (five gradients: 5, 10, 20, 40, and 80 μg / mL) and the pH value of the buffer solution (five gradients: 4.0, 5.0, 6.0, 6.8, and 7.4). Among them, •OH and O2... •- The generation of •OH was detected by electron paramagnetic resonance (ESR). For the detection of •OH, a reaction solution containing Co-SAN or N / C or Co-NPs (80 μg / mL), H₂O₂ (1 mM), and DMPO (0.1 mM) was prepared in a buffer solution (20 mM, pH 6.0). To prevent O₂... •- Interference was introduced by adding superoxide dismutase to the reaction solution. After sonication, ESR analysis was performed using EMXplus (Bruker). This was to detect O2. •- To generate the reaction solution, Co-SAN, N / C, or Co-NPs (80 μg / mL) were added to a PBS buffer (20 mM, pH 6.0) containing NADPH (200 μg / mL) and DMPO (0.1 mM). DMSO was added during the experiment to eliminate •OH interference before measuring the ESR spectrum of the reaction solution.

[0046] 2. Experimental Results Inspired by its structural similarity to natural enzymes, this invention explores the multiple enzyme mimicry activities of Co-SAN. The mechanism by which Co-SAN possesses both functional enzyme catalytic properties and pH-responsive characteristics is demonstrated. First, the reactive oxygen species (ROS) scavenging capacity of Co-SAN was evaluated using ABTS and DPPH free radical scavenging experiments. Compared to Co-NPs and N / C, Co-SAN exhibits superior ABTS• + and DPPH • Scavenging ability ( Figure 1 (a) Notably, Co-SAN exhibited low ABTS radical scavenging rate at pH 7.4, and its activity gradually increased with increasing pH, indicating a significant pH-dependent antioxidant capacity. To investigate the chemical stability of Co-SAN, it was dispersed in a buffer solution at pH 1.2 and incubated for 3 hours. Remarkably, its ABTS• scavenging activity was […]. + The ability to generate O2 remained excellent and was positively correlated with the concentration of Co-SAN, indicating that the compound has excellent stability under strongly acidic conditions. Encouraged by this phenomenon, the enzyme activity of Co-SAN under different conditions was further evaluated. Given that the release of O2 in the presence of H2O2 can be used to assess CAT-like enzyme activity, the experiment revealed that Co-SAN has a stronger O2 generation capacity than Co-NPs and N / C under the same conditions. Figure 1 (b) It is worth noting that Co-SAN exhibits highly efficient hydrogen peroxide decomposition capabilities over a wide pH range, indicating its superior CAT-like characteristics in a broad range of applications.Figure 1 (c) Furthermore, Co-SAN effectively scavenges hydrogen peroxide in a pH-dependent manner in the presence of glutathione (GSH), indicating that it possesses good glutathione peroxidase (GPx) mimicry activity. Figure 1 (d) It is worth noting that, in order to determine the O2 of Co-SAN... •- Scavenging efficiency (i.e., simulated SOD activity) was tested at different concentrations of Co-SAN using an SOD activity assay kit. The results confirmed that Co-SAN exhibited excellent SOD-simulating activity and effectively scavenged O2 under alkaline pH conditions. •- However, its activity is significantly reduced under acidic pH conditions. Figure 1 (e). This demonstrates that Co-SAN, by mimicking the pH dependence of multiple enzymes such as SOD, CAT, and GPx, can synergistically scavenge H2O2 and O2. - It exhibits unparalleled activity in scavenging reactive oxygen species under alkaline conditions.

[0047] The oxidation of 1,2-diaminobenzene (OPD) was used as a typical catalytic reaction to investigate the oxidase (OXD)-like activity of Co-SAN. The yellow reaction solution exhibited a characteristic absorption peak at 417 nm, indicating that this material exhibits superior oxidase-like activity compared to Co-NPs and N / C. Furthermore, the simulated OXD activity of Co-SAN increased with decreasing pH, exhibiting almost no enzyme activity under neutral pH conditions. Figure 1 (f) It is well known that the mimicry ability of NAD(P)H oxidase (NOX) is also related to tumor metabolism. Therefore, the mimicry activity of Co-SAN for NAD(P)H oxidase was investigated by UV-Vis absorption spectroscopy. Notably, the typical absorption peak of NADH at 340 nm decreased significantly with time, while that of NAD... + The absorption peak at 260 nm is significantly enhanced. Figure 1 The g in the figure indicates that Co-SAN oxidizes NADH to NAD through a stepwise process. + It exhibits highly efficient NOx mimicry activity. Simultaneously, in the NADH oxidation reaction involving Co-SAN, H₂O₂ generation is clearly detected with increasing NADH concentration, which is beneficial for the generation of •OH during the POD mimicry activity. Furthermore, due to its similarity to NADH, Co-SAN also exhibits significant oxidase-like activity towards NADPH. Figure 1(h) To investigate the effect of pH, NOX activity was measured under different pH conditions. The results showed that NAD(P)H consumption decreased significantly with increasing pH, indicating that an acidic environment is the optimal condition for enhancing the NADH oxidase-like activity of Co-SAN. To further elucidate the reactive oxygen species, electron spin resonance (ESR) technology was used with 5,5-dimethyl-1-pyrrolidone N-oxide (DMPO) as a scavenger. The characteristic signal of the DMPO-OOH adduct verified the presence of O2 in the reaction system. •- The generation ( Figure 1 (i in the text).

[0048] As expected, due to Co-SAN's NOx-mimicking catalytic properties, its O2 content decreases after NADPH treatment. •- The signal intensity was slightly increased compared to Co-SAN alone, indicating its excellent NOx and OXD mimicry activity. A significant peak with an intensity ratio of 1:2:2:1 was detected in the presence of a mixture of Co-SAN and H2O2, attributed to the formation of •OH, suggesting that Co-SAN can serve as a potential alternative to natural POD nanozymes. Figure 1 (j) Furthermore, compared to the control group N / C and Co-NPs, Co-SAN effectively consumed glutathione (GSH) in a concentration-dependent manner, confirming its superior glutathione oxidase (GSHOx) activity. Figure 1 (k in the text). Given the acidic nature of the tumor microenvironment, Co-SAN can enhance reactive oxygen species generation and alleviate tumor hypoxia by leveraging its oxidase properties, highlighting its potential to significantly improve the efficacy of radiotherapy.

[0049] Experimental Example 2: Study on the protective effect of nanozyme Co-SAN against intestinal radiation damage 1. Experimental Methods (see experimental procedure) Figure 2 (b) The synthesized Co-SAN (5 mg) was dispersed in 5 mL of PBS solution containing 5 mg Cy7. The solution was then gently shaken in the dark at 4 °C for 24 hours. Finally, Co-SAN@Cy7 was separated from the solution by centrifugation (3200 g, 10 min). The separated solution was administered orally to mice via gavage, and the gastrointestinal retention of Co-SAN was assessed at different time points.

[0050] Five-week-old male C57BL / 6J mice were randomly divided into five groups: PBS group (normal control), IR+PBS group (radiation injury group), IR+N / C group, IR+Co-NPs group, and IR+Co-SAN group. After fasting for 12 hours, mice in the IR+Co-SAN group were orally administered 100 μL of PBS solution containing 5 mg / kg Co-SAN, while the control group was given equal volumes of PBS, N / C, and Co-NPs solutions, respectively. Four hours later, mice in the radiation group underwent peritoneal X-ray irradiation (from the diaphragm to the pelvis) at a dose of 12 Gy and a dose rate of 500 cGy / min using a Vital Beam X-ray machine from X-RAD (USA). The interval between Co-SAN administration and radiation was determined by detecting the degree of intestinal damage in mice. The specific method was as follows: after measuring the intestinal villi of mice, the mice were irradiated with X-rays at a dose of 12 Gy at four time points: 1, 2, 4, and 6 hours. The optimal interval was then selected by comparing the intestinal damage after irradiation at different time points.

[0051] Five days after mouse modeling, peripheral blood and intestinal tissue were collected, homogenized, and analyzed using an ELISA kit (Wuhan ELK Biotechnology Co., Ltd.) to quantitatively detect representative pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β) in radiation injury. Simultaneously, body weight changes in a subset of mice were recorded over one month. In addition, the intestines of mice were removed on the 5th day after radiotherapy, and the length of the colon and rectum was compared. The degree of intestinal radiation damage was analyzed by HE staining and PAS staining, and intestinal radiation damage-related indicators were detected by immunofluorescence staining. The antibodies used and their sources are as follows: tight junction protein 1 (ZO-1, rabbit polyclonal antibody, catalog number 61-7300, Thermo Fisher Scientific, Guangzhou, China), tight junction protein 3 (Claudin3, rabbit polyclonal antibody, catalog number 34-1700, Thermo Fisher Scientific, Guangzhou, China), occlusion protein (Ms polyclonal IgG, catalog number 33-1500, Thermo Fisher Scientific, Guangzhou, China), γ-H2AX (rabbit polyclonal IgG, Cell Signaling Technology, USA), TUNEL, CD206 (rabbit polyclonal IgG, catalog number 91992S, Cell Signaling Technology, USA), and F4 / 80 (rat monoclonal antibody, catalog number ab6640, abcam, USA).

[0052] Simultaneously, intestinal segments from different treatment groups were collected in EP tubes. The intestinal tissue from the EP tubes was removed, placed on crushed ice, and washed twice with an appropriate amount of 1640 medium. The tissue was weighed, and 5 ml of each aliquot was mixed with 50 μl of 1×HBSS + 0.5 M EDTA + 1 mM DTT. The tissue was cut into 5 mm pieces, transferred to 50 ml centrifuge tubes, and pre-digestion solution was added. The mixture was incubated at 37°C and 145 rpm for 30 minutes, followed by filtration through a 70 μm filter. After washing the tissue with 1640 buffer, DNase and collagenase were added to digest the tissue. After thorough mixing, the mixture was incubated at 37°C for 1 hour. After filtration through a 70 μm filter, the tissue was washed again. The filtrate was centrifuged at 400 g for 10 minutes, the supernatant was discarded, and the precipitate was retained. This process was repeated. The tissue was washed twice, the supernatant was discarded, and the precipitate was retained. Some tissue samples were tested using a ROS detection kit, while the remaining tissue samples were incubated at 4°C in the dark for 30 minutes with antibodies CD45 (FITC, 147710, Biolegend, China), CD11b (PE, 101207, Biolegend, China), and Ly6g (APC, 164506, Biolegend, China), followed by washing and resuspending in HBSS buffer for flow cytometry analysis.

[0053] 2. Experimental Results Gastrointestinal fluorescence imaging showed ( Figure 2 (a) Co-SAN can be effectively enriched in the intestine (especially the colon region), and the fluorescence intensity reaches a high level after about 2 hours. Weight changes show ( Figure 2 In mice receiving 12 Gy abdominal radiation (c), the weight recovery trend in the IR+Co-SAN group was significantly better than that in the IR group, IR+PBS group, and IR+N / C group, approaching the level of the normal PBS group, indicating that Co-SAN can alleviate radiation-induced weight loss. Survival curves show ( Figure 2 (d) Under a lethal dose of 16 Gy, the median survival of mice in the IR+Co-SAN group was >50 days, comparable to the unirradiated PBS group; while the median survival of the IR+PBS group and the IR+N / C group was only 21 days, and the IR+Co-NPs group was 28 days, indicating that Co-SAN can significantly improve the survival rate of irradiated mice. Colon length showed ( Figure 2 (e) After radiation, the colon length in the IR+Co-SAN group was significantly longer than that in the IR+PBS and IR+N / C groups, and approached that of the normal PBS group, indicating that Co-SAN can inhibit radiation-induced colonic atrophy. Flow cytometry analysis of reactive oxygen species (ROS) content in intestinal tissue showed ( Figure 2(f) After radiation, the ROS level in the IR+PBS group was significantly increased; while the ROS level in the IR+Co-SAN group was significantly decreased, approaching that of the normal PBS group, indicating that Co-SAN can effectively clear radiation-induced intestinal ROS and inhibit oxidative stress damage. HE staining showed ( Figure 2 In the IR+PBS and IR+N / C groups, significant damage (structural disorder and atrophy) was observed in the villi of the small intestine (duodenum, jejunum, ileum); while the villi structure of the IR+Co-SAN group was close to that of the normal PBS group. Quantitative analysis of villi length (g) Figure 1 The results (h) also confirmed that it was significantly superior to other radiation treatment groups, indicating that Co-SAN can effectively protect the morphology and structure of the small intestinal villi. Immunofluorescence results showed ( Figure 2 In the IR+PBS group after radiation exposure, the expression of apoptosis markers (TUNEL, γ-H2AX) was significantly increased; the expression of tight junction proteins (occlusion protein, Claudin3, ZO-1) was significantly decreased (suggesting intestinal barrier disruption); and the expression of inflammatory markers (F4 / 80) was increased (suggesting inflammatory activation). In the IR+Co-SAN group, all of the above indicators were significantly improved, with reduced apoptosis, restored tight junction protein expression, and inhibited inflammatory activation. These results demonstrate the protective effect of Co-SAN on intestinal cells and the barrier at the molecular level, including apoptosis, barrier function, and inflammatory response. These results indicate that oral Co-SAN has a comprehensive and potent protective effect against radiation-induced intestinal damage.

[0054] Experimental Example 3: Study on the Mechanism of Co-SAN Nanozyme in Protecting Against Radiation-Induced Intestinal Injury 1. Experimental Methods (see experimental procedure) Figure 3 d) C57BL / 6J mice were first orally administered Co-SAN, and a radiation injury model was established 4 hours later. Intestinal tissue was collected from the mice on the 5th day after the model was established. In addition, 5-week-old male C57BL / 6J mice were irradiated with abdominal X-rays at a dose of 16 Gy (lethal dose) and a dose rate of 500 cGy / min. The survival time of the mice was then monitored and recorded. All the above doses were based on literature data (Zhang D, Zhong D, Ouyang J, et al. Microalgae-based oral microcarriers for gut microbiota homeostasis and intestinal protection in cancer radiotherapy[J]. Nature communications, 2022, 13(1): 1413.).

[0055] Total RNA was extracted from intestinal tissues of mice in different treatment groups using the Qiagen RNeasy Mini Kit and sequenced on the Illumina Novaseq platform. After sequencing, the sequencing quality of the fastq files was assessed using FastQC (v 0.11.9) to ensure all samples met high-quality standards. Simultaneously, Illumina adapter sequences were trimmed using Trim galore (v 0.6.7), and 150 bp paired end reads were aligned to the mouse reference genome (GRCm39) using hisat2 (v2.2.1). Differentially expressed gene comparison analysis was performed using DEseq2 (v1.40.2), GSEA and KEGG pathway enrichment analysis was conducted using the "clusterProfiler" R package (v4.8.3), and data visualization was achieved using the ggplot2 package (v3.4.4).

[0056] Intestinal tissue sections from mice in different treatment groups were selected for immunofluorescence staining to detect extracellular trap markers of neutrophils (Mpo and CitH3). The sections were scanned and analyzed using Pannoramic SCAN (3DHISTECH, Hungary).

[0057] Five-week-old male C57BL / 6J mice were randomly divided into five groups: PBS group (saline control), IR+PBS group (radiation damage group), IR+LY294002 group (LY294002: CAS No. 154447-36-6; a specific phosphatidylinositol 3-kinase PI3K small molecule inhibitor, which blocks the PI3K / Akt signaling pathway), IR+Co-SAN group, and IR+Co-SAN+LY294002 group. Mice were fasted for 12 hours, and then orally administered 100 μL of PBS containing 5 mg / kg Co-SAN to the IR+Co-SAN+LY294002 group. The control group was given an equal volume of PBS and Co-SAN solution, respectively. Mice were then irradiated with 12 Gy X-rays at a dose rate of 500 cGy / min (X-RAD, VitalBeam, USA). For five consecutive days after radiotherapy, mice were intraperitoneally injected with LY294002 (MCE HY-10108, 10 mg / kg) to inhibit the PI3K / AKT pathway. Body weight changes in some mice were recorded over one month. On day 5 after radiotherapy, the mice's intestines were harvested, and the degree of damage was analyzed by HE and PAS staining. Immunofluorescence staining was used to analyze intestinal radiation damage-related indicators, including ZO-1, tight junction protein 3, occlusion protein, γ-H2AX, TUNEL, CD206, and F4 / 80.

[0058] To verify the inhibitory effects of the inhibitors LY294002 and Co-SAN on the PI3K / Akt pathway, Western blotting analysis was used to detect changes in the expression of related proteins. The antibodies used were as follows: PI3K (rabbit polyclonal IgG, ab191606, abcam, USA), p-PI3K (rabbit polyclonal antibody, 4228S, Cell Signaling Technology, USA), AKT (rabbit polyclonal antibody, 9272S, Cell Signaling Technology, USA), and p-AKT (rabbit polyclonal antibody, 9271S, Cell Signaling Technology, USA). The sample processing procedure was as follows: intestinal tissue samples and neutrophils from mice in different treatment groups were lysed with RIPA lysis buffer in an ice bath for 30 minutes, centrifuged at 12,000 g for 10 minutes, and the supernatant was collected. Protein concentration was determined using the bovine serum albumin (BSA) method before Western blotting analysis.

[0059] 2. Experimental Results KEGG enrichment results from RNA sequencing showed that ( Figure 3 In the study (a), the PI3K / AKT signaling pathway was significantly enriched in differentially expressed genes (P-value very low), suggesting that this pathway is one of the core mechanisms by which Co-SAN exerts its intestinal radiation protection effect. Enrichment analysis was performed on the gene set after radiation combined with Co-SAN treatment (…). Figure 3 In section b), the normalized enrichment fraction (NES) of the PI3K-AKT signaling pathway was -1.37, with a P-value < 0.001, further clarifying that Co-SAN can significantly regulate gene expression in this pathway. Immunoblotting results showed ( Figure 3 In irradiated neutrophils (c), the phosphorylation levels of PI3K and AKT (p-PI3K, p-AKT) were significantly increased; however, the phosphorylation level was inhibited after the addition of the PI3K inhibitor LY294002; Co-SAN treatment maintained or enhanced PI3K / AKT activation. Simultaneously, the expression of neutrophil traps (Mpo, Cith3) was also regulated by this pathway, indicating that Co-SAN can affect the function of irradiated neutrophils by activating the PI3K / AKT pathway. Changes in body weight ( Figure 3 (e) shows that after receiving 12 Gy radiation, the weight recovery trend of mice in the IR+Co-SAN group was significantly better than that in the IR+PBS group; however, when combined with LY294002, the weight recovery effect was significantly inhibited, suggesting that the PI3K / AKT pathway is a key mechanism for Co-SAN to maintain body weight. Survival curves ( Figure 3f) showed that at a lethal dose of 16 Gy, the median survival of mice in the IR+Co-SAN group was >50 days (comparable to the unirradiated PBS group); the median survival of the IR+Co-SAN+LY294002 group decreased to 35 days, indicating that blocking the PI3K / AKT pathway weakens the survival protection effect of Co-SAN. HE staining showed ( Figure 3 In the IR+PBS group, the villi of the small intestine (duodenum, jejunum, ileum) were severely damaged (atrophic, structurally disordered); the villi structure of the IR+Co-SAN group was close to normal; while the degree of villi damage in the IR+Co-SAN+LY294002 group was significantly more severe than that in the IR+Co-SAN group. Quantitative analysis of villi length (g) Figure 3 The trend was also confirmed by h). This indicates that Co-SAN protects the morphology of intestinal villi through the PI3K / AKT pathway. The above results show that Co-SAN effectively alleviates radiation-induced intestinal damage by activating the PI3K / AKT signaling pathway; while the PI3K inhibitor LY294002 can block this protective effect.

[0060] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. The application of Co-SAN single-atom nanozyme in the preparation of drugs for preventing radiation-induced intestinal injury, characterized in that, The Co-Single Atom Nanozyme Co-SAN is obtained by solvothermal synthesis of ZnCo-MOF by coordinating zinc and cobalt metal ions with 2-methylimidazolium organic ligands, followed by high-temperature calcination and pyrolysis.

2. The application according to claim 1, characterized in that, The preparation method of the Co single-atom nanozyme Co-SAN includes the following steps: S1. Disperse zinc salt and cobalt salt in methanol solution, mix well, then add methanol solution containing 2-methylimidazole, mix again, and carry out solvothermal reaction. After reaction, wash and dry to obtain ZnCo-MOF. S2. ZnCo-MOF is placed in an inert atmosphere and calcined at high temperature to obtain Co-SAN.

3. The application according to claim 2, characterized in that, In S1, the molar ratio of zinc ions, cobalt ions and 2-methylimidazole is 2:1:8-10.

4. The application according to claim 2, characterized in that, In S1, the temperature of the solvothermal reaction is 35-45℃ and the time is 5-8 h.

5. The application according to claim 2, characterized in that, In S1, the zinc salt includes zinc nitrate, zinc chloride, zinc acetate, or zinc sulfate, and the cobalt salt includes cobalt nitrate, cobalt chloride, cobalt acetate, or cobalt sulfate.

6. The application according to claim 2, characterized in that, In S2, the high-temperature calcination is to raise the temperature to 800-1000℃ at a heating rate of 3-7℃ / min and maintain it at that temperature for 2-5 hours.

7. The application according to claim 1, characterized in that, The Co-SAN single-atom nanozyme protects the intestine from radiation damage by downregulating the PI3K / AKT pathway, reducing the formation of the extracellular network of neutrophils, and clearing reactive oxygen species in the intestine.

8. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The excipients include at least one of the following: excipients, solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, coating materials, pH adjusters, absorbents, diluents, and antioxidants.

10. The application according to claim 8, characterized in that, The dosage forms of the drug include injections, tablets, capsules, granules, powders, syrups, or oral liquids.

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