Monatomic nano-enzyme drug delivery system for IBD treatment as well as preparation method and application of monatomic nano-enzyme drug delivery system

Abstract

The invention relates to the technical field of nano-materials, in particular to a monatomic nano-enzyme drug delivery system for IBD treatment and a preparation method and application of the monatomic nano-enzyme drug delivery system. The monatomic nano enzyme delivery system provided by the invention comprises an iron-doped monatomic nano enzyme Fe-SA, curcumin Cur and hyaluronic acid grafted dopamine HAD, the Fe-SA and the Cur form a Fe-SA / Cur compound through a coordination effect, and the surface of the Fe-SA / Cur compound is coated with the HAD. According to the iron-doped monatomic nano-enzyme Fe-SA, Fe atoms in the iron-doped monatomic nano-enzyme Fe-SA are distributed in a graded carbon-based matrix of the Fe-SA in a Fe-N4 co-doping form, and the iron-doped monatomic nano-enzyme Fe-SA has efficient SOD and CAT nano-enzyme activity, and can realize antioxidant self-cascade nano-enzyme reaction, effectively remove active oxygen and improve the pathological environment. According to the preparation method disclosed by the invention, a coordination compound Fe-SA / Cur is constructed by utilizing the iron-doped monatomic nano-enzyme and the curcumin, so that the anti-inflammatory effect is improved, and the characteristic of poor water solubility of the curcumin is effectively improved. According to the invention, HAD is loaded on the outer layer of a coordination compound Fe-SA / Cur to construct an oral monatomic antioxidant cascade nano-enzyme system, high acid resistance and targeted delivery capability are realized by using the remarkable stability and negative charges of HAD, and oral administration is effectively realized. In-vivo and in-vitro experiments show that the monatomic nano-enzyme delivery system provided by the invention can effectively remove intestinal free radicals, improve the intestinal environment, reduce the expression of inflammatory factors, promote the recovery of damaged tissues and effectively treat colitis. The invention provides a new thought and method for biomedical research and drug development of inflammatory bowel diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and in particular to a single-atom nanoenzyme drug delivery system for IBD treatment, and a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a disease characterized by chronic inflammation of the colonic mucosa and submucosa, primarily encompassing ulcerative colitis (UC) and Crohn's disease (CD). Common symptoms include diarrhea, abdominal pain, and rectal bleeding. Currently, clinical treatment strategies are primarily based on anti-inflammatory drugs, immunomodulators, and surgical intervention. However, these treatments have limited efficacy and are often associated with adverse side effects. A recent large population-based cohort study in Denmark demonstrated that the incidence of IBD has stabilized, but its prevalence continues to rise. Unlike Western countries, the incidence of IBD in developing countries has rapidly increased with industrialization. For example, from 1990 to 2021, both the age-standardized incidence and prevalence in China increased significantly (estimated annual percentage changes of 2.93 and 2.54, respectively). Currently, hospitalization rates in newly industrialized countries have increased significantly, placing an increasing burden on global healthcare systems.

[0003] Although the etiology of IBD is not yet fully understood, the excessive release of reactive oxygen species (ROS), leading to oxidative stress (OS), is widely considered a pathogenic factor in inflammatory diseases such as IBD. Therefore, developing strategies to eliminate ROS in the IBD microenvironment and alleviate intestinal damage is of great significance for the treatment of IBD.

[0004] The development of nanocatalytic materials has provided new insights into regulating the redox homeostasis of ROS in inflammatory diseases. Nanozymes, which combine the physical properties of nanomaterials with the catalytic activity of natural enzymes, have attracted widespread attention due to their high efficiency, safety, and ease of synthesis. Among them, self-cascading nanozymes with multiple antioxidant activities, such as superoxide dismutase (SOD) and catalase (CAT), have become an effective strategy for treating IBD by alleviating OS and improving the IBD microenvironment. Therefore, the preparation of nanozymes with self-cascading SOD-CAT activity is of great significance.

[0005] In recent years, single-atom nanozymes (SAzymes) have attracted attention due to their extremely high catalytic efficiency. These nanozymes are usually composed of a single metal atom (such as Fe, Pt) loaded on a carrier. Its main advantage lies in the single-atom structure, which can provide a high concentration of catalytic active sites and promote the exposure of these sites on the surface. In addition, single-atom nanozymes have a high specific surface area and enhanced catalytic stability. At present, researchers mainly use electronic structure to regulate the activity of single-atom nanozymes, while the macroscopic coordination strategy inside single-atom nanozymes is often overlooked. Therefore, modifying single-atom nanozymes through coordination strategies to construct cascade reaction complexes is of great significance for achieving the delivery of natural drugs and giving full play to the efficacy of nanozymes.

[0006] Among IBD treatment strategies, oral administration allows drugs to interact directly with inflammatory lesions on the intestinal mucosa, thereby improving therapeutic efficacy while reducing systemic effects on other organs. However, this approach requires drugs to have high acid resistance and specific affinity for the positively charged inflamed mucosa. Constructing an acid-resistant, negatively charged coating on the surface of single-atom nanozymes can significantly improve the efficiency of oral administration and optimize the targeted therapeutic effect. Summary of the Invention

[0007] Based on this, the present invention constructs an orally administrable single-atom antioxidant cascade nanozyme system to achieve synergistic treatment of IBD. The present invention first constructs an iron-doped single-atom nanozyme Fe-SA, in which the Fe atoms are distributed in the hierarchical carbon-based matrix of Fe-SA in the form of Fe-N4 co-doping, with efficient SOD and CAT nanozyme activity, which can realize antioxidant self-cascade nanozyme reaction, effectively remove reactive oxygen species, and improve the pathological environment. Subsequently, the present invention uses iron-doped single-atom nanozyme and curcumin to construct a coordination complex Fe-SA / Cur, which not only increases the anti-inflammatory effect, but also effectively improves the poor water solubility of curcumin. Subsequently, the present invention loads HAD on the outer layer of the coordination complex to construct an orally administrable single-atom antioxidant cascade nanozyme system Fe-SA / Cur@HAD, which utilizes the significant stability and negative charge of HAD to achieve high acid resistance and targeted delivery ability, effectively realizing oral administration. In vitro and in vivo experiments show that the system can effectively scavenge intestinal free radicals, improve the intestinal environment, reduce the expression of inflammatory factors, promote the recovery of damaged tissues, and effectively treat colitis.

[0008] In view of this, the object of the present invention is to provide a single-atom nanoenzyme drug delivery system and its preparation method and application.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] The present invention provides a single-atom nanozyme delivery system for the treatment of IBD, which includes iron-doped single-atom nanozyme Fe-SA, curcumin Cur and hyaluronic acid-grafted dopamine HAD; the Fe-SA and Cur form a Fe-SA / Cur complex through coordination, and the HAD is coated on the surface of the Fe-SA / Cur complex.

[0011] Furthermore, in the iron-doped single-atom nanozyme Fe-SA, Fe atoms are distributed in the hierarchical carbon-based matrix of Fe-SA in the form of Fe-N4 co-doping, and have superoxide dismutase (SOD)-like activity and catalase (CAT)-like activity, which can catalyze the following self-cascade reaction:

[0012] (1) SOD-like activity: converts superoxide anion O2 ·- Converted into hydrogen peroxide H2O2;

[0013] (2) CAT-like activity: decomposes hydrogen peroxide H2O2 into oxygen O2 and water H2O.

[0014] Furthermore, the preparation method of the iron-doped single-atom nanozyme Fe-SA comprises the following steps:

[0015] Zinc and iron ions coordinate with 2-methylimidazole to form Fe-ZIF-8;

[0016] The calcination reaction is carried out at 800℃ under nitrogen conditions to thermally decompose and evaporate zinc ions, while carbonizing to form a nitrogen-doped carbon structure;

[0017] Acid etching removes impurities formed during the firing process to obtain Fe-SA.

[0018] Furthermore, the preparation method of the iron-doped single-atom nanozyme Fe-SA comprises the following steps:

[0019] Zinc nitrate hexahydrate Zn(NO3)2.6H2O and iron triacetylacetonate Fe(C5H7O2)3 were ultrasonically dispersed in a methanol-tetrahydrofuran mixture, and a methanol-tetrahydrofuran mixture of 2-methylimidazole was added. After ultrasonic mixing, the coordination reaction was carried out by stirring. The precipitate was collected by centrifugation, washed with anhydrous ethanol, and dried in vacuum to obtain Fe-ZIF-8.

[0020] Fe-ZIF-8 was placed in a quartz tube, filled with N2 and calcined at 800℃ to obtain a black product;

[0021] The obtained black product was immersed in hydrochloric acid for acid etching reaction to remove impurities formed during the firing process, washed with ultrapure water and ethanol, and vacuum dried to obtain Fe-SA.

[0022] Furthermore, in the preparation of the iron-doped single-atom nanozyme Fe-SA, the weight ratio of zinc nitrate hexahydrate Zn(NO3)2.6H2O, triacetylacetonate iron Fe(C5H7O2)3, and 2-methylimidazole is: 22-23:1:21-22; the coordination reaction conditions are: stirring at 600r / min for 15min; the calcination reaction conditions are calcination reaction at 800℃ under nitrogen conditions for 2h; and the acid etching reaction conditions are soaking in 0.5M hydrochloric acid for 2h.

[0023] Furthermore, the Cur is loaded on the surface of Fe-SA through coordination, and the loading amount is 1-20% based on the mass of Fe-SA.

[0024] Furthermore, the preparation method of the Fe-SA / Cur complex comprises the following steps: dissolving Fe-SA in ethanol, adding curcumin Cur, stirring to react in the dark, removing the solvent, dialyzing to remove unreacted substances, and drying to obtain the Fe-SA / Cur complex.

[0025] Furthermore, in the preparation of the Fe-SA / Cur complex, the weight ratio of Fe-SA and Cur is 5:1, the reaction conditions are stirring and reacting in the dark for 24 hours, the solvent removal method is rotary evaporation to remove the solvent, and the dialysis time is dialysis for 3 days.

[0026] Furthermore, the HAD is formed by linking the carboxyl group of hyaluronic acid and the amino group of dopamine through an amide bond, with hyaluronic acid as the main chain and grafted dopamine as the side chain.

[0027] Furthermore, the preparation method of HAD is as follows: dissolving hyaluronic acid in ultrapure water, activating the carboxyl group with EDC and NHS, adding dopamine and stirring to react, dialyzing and freeze-drying to obtain HAD.

[0028] Furthermore, in the preparation method of HAD, the weight ratio of hyaluronic acid, EDC, NHS, and dopamine is: 1:0.5:0.3:0.5; the reaction conditions are: under nitrogen protection, hyaluronic acid is dissolved in ultrapure water, EDC and NHS are added and stirred for activation for 5 hours, dopamine is added, and the reaction is stirred for 12 hours; the dialysis conditions are: dialysis for 3 days in a 3000Da dialysis bag.

[0029] Furthermore, the coating amount of the HAD is 1-10% based on the mass of the Fe-SA / Cur complex.

[0030] Furthermore, the preparation method of the Fe-SA / Cur@HAD system includes the following steps: ultrasonically dissolving Fe-SA / Cur and HAD in ultrapure water, stirring for reaction, dialyzing, and freeze-drying to obtain the Fe-SA / Cur@HAD system.

[0031] Further, in the preparation of the Fe-SA / Cur@HAD system, the weight ratio of Fe-SA / Cur to HAD is 10:1, the reaction condition is stirring for 48 hours, and the dialysis condition is dialysis through a 3000Da dialysis bag for 3 days, with water changing every 6 hours.

[0032] Further, the Zeta potential of the single-atom nanoscale enzyme delivery system is-10 mV.

[0033] Further, the single-atom nanoscale enzyme delivery system has a curcumin Cur release rate of about 70% in 24 hours in a simulated IBD microenvironment pH = 5, and a release rate of less than 20% in simulated gastric juice pH = 1.2 and intestinal juice pH = 7.

[0034] Further, the single-atom nanoscale enzyme delivery system also has the following properties:

[0035] (1) Free radical scavenging capacity: at a concentration of 25ug / mL, the clearance rates of ABTS·, DPPH·, ·OH and O2 ·- are all ≥75%.

[0036] (2) Anti-inflammatory performance: inhibits the TLR4 / Myd88 / NF-κB related inflammatory pathway and reduces the expression of TLR4, Myd88 and NF-κB.

[0037] (3) Promote the repair of intestinal barrier function: increase the expression of E-cadherin and ZO-1 protein and promote the recovery of intestinal barrier function;

[0038] (4) Regulate intestinal flora: restore the abundance of symbiotic flora such as Proteobacteria and Firmicutes and inhibit flora imbalance.

[0039] The application also provides a preparation method of the single-atom nanoscale enzyme delivery system, comprising the following steps:

[0040] S1: Fe-SA preparation: zinc ions and iron ions are coordinated with 2-methyl imidazole to form Fe-ZIF-8; under the condition of nitrogen at 800℃, a calcination reaction is performed to pyrolyze and evaporate zinc ions, and at the same time, carbonization is performed to form a nitrogen-doped carbon structure; acid etching is performed to remove impurities formed during the calcination process, and Fe-SA is obtained.

[0041] S2: Fe-SA / Cur complex preparation: Fe-SA is dissolved in ethanol, curcumin Cur is added, and stirring reaction is performed in the dark, the solvent is removed, unreacted substances are removed by dialysis, and Fe-SA / Cur complex is obtained after drying.

[0042] S3: Fe-SA / Cur@HAD system preparation: Fe-SA / Cur and HAD are ultrasonically dissolved in ultrapure water, stirring reaction is performed, dialysis is performed, and single-atom nanoscale enzyme delivery system FCH is obtained by freeze-drying.

[0043] Furthermore, in S1, the preparation method of the Fe-SA is:

[0044] Zinc nitrate hexahydrate Zn(NO3)2.6H2O and iron triacetylacetonate Fe(C5H7O2)3 were ultrasonically dispersed in a methanol-tetrahydrofuran mixture, and a methanol-tetrahydrofuran mixture of 2-methylimidazole was added. After ultrasonic mixing, the coordination reaction was carried out by stirring. The precipitate was collected by centrifugation, washed with anhydrous ethanol, and dried in vacuum to obtain Fe-ZIF-8.

[0045] Fe-ZIF-8 was placed in a quartz tube, filled with N2 and calcined at 800℃ to obtain a black product;

[0046] The obtained black product was immersed in hydrochloric acid for acid etching reaction to remove impurities formed during the firing process, washed with ultrapure water and ethanol, and vacuum dried to obtain Fe-SA.

[0047] Furthermore, in S1, the weight ratio of Zn(NO3)2.6H2O, Fe(C5H7O2)3, and 2-methylimidazole is: 22-23:1:21-22; the coordination reaction conditions are: stirring at 600r / min for 15min; the calcination reaction conditions are calcination at 800℃ for 2h; and the acid etching reaction conditions are soaking in 0.5M hydrochloric acid for 2h.

[0048] Furthermore, in S2, the weight ratio of Fe-SA and Cur is 5:1, the reaction conditions are stirring and reacting in the dark for 24 hours, the method for removing the solvent is rotary evaporation, and the dialysis time is dialysis for 3 days.

[0049] Furthermore, in S3, the weight ratio of Fe-SA / Cur to HAD was 10:1, the reaction conditions were stirring reaction for 48 h, and the dialysis conditions were: dialysis through a 3000 Da dialysis bag for 3 days, and the water was changed every 6 hours.

[0050] The present invention also provides the use of the above-mentioned single-atom nanozyme delivery system in the preparation of IBD therapeutic drugs.

[0051] The present invention also provides an IBD therapeutic drug, which contains the above-mentioned single-atom nanozyme delivery system.

[0052] The beneficial effects of the present invention are: the present invention provides a single-atom nanozyme delivery system, which realizes the synergistic treatment of IBD. The single-atom nanozyme delivery system of the present invention comprises iron-doped single-atom nanozyme Fe-SA, curcumin Cur and hyaluronic acid-grafted dopamine HAD; the Fe-SA and Cur form a Fe-SA / Cur complex through coordination, and the HAD is coated on the surface of the Fe-SA / Cur complex. In the present invention, in the iron-doped single-atom nanozyme Fe-SA, Fe atoms are distributed in the hierarchical carbon-based matrix of Fe-SA in the form of Fe-N4 co-doping, have efficient SOD and CAT nanozyme activity, can realize antioxidant self-cascade nanozyme reaction, effectively remove reactive oxygen, and improve the pathological environment. The present invention uses iron-doped single-atom nanozyme and curcumin to construct the coordination complex Fe-SA / Cur, which not only increases the anti-inflammatory effect, but also effectively improves the poor water solubility of curcumin. This invention constructs an orally available single-atom antioxidant cascade nanozyme system by loading HAD onto the outer layer of the Fe-SA / Cur coordination complex. Leveraging HAD's remarkable stability and negative charge, it achieves high acid resistance and targeted delivery, effectively enabling oral administration. In vitro and in vivo experiments have demonstrated that this system effectively scavenges intestinal free radicals, improves the intestinal environment, reduces the expression of inflammatory factors, promotes the recovery of damaged tissue, and effectively treats colitis. This invention provides new insights and approaches for biomedical research and drug development for inflammatory bowel disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Synthesis and characterization of Fe-SA. A) Synthesis route of Fe-SA. B, C) TEM and SEM images of Fe-SA. Scale bar: 100 nm. D) HAADF-STEM image of Fe-SA (where single Fe atoms are marked with red bright spots). Scale bar: 5 nm. E) EDS spectrum of Fe-SA. F) XANES spectrum of Fe-SA. G, H) Fourier transform (FT) k of Fe-SA. 3 Weighted EXAFS spectra and fitted spectra. I, J)k 2 Weighted Fourier transform EXAFS spectra and fitted spectra. KM) Wavelet transform spectra of K-edge EXAFS oscillations of Fe foil, Fe-SA, and Fe2O3.

[0054] Figure 2Synthesis and characterization of FCH. A) Synthesis route of FCH. B) TEM images of Fe-SA, Fe-SA / Cur, and FCH. Scale bar: 100 nm. E) Fourier transform infrared (FTIR) spectroscopy. F) Ultraviolet-visible (UV–vis) spectroscopy. G) Zeta potential of Fe-SA, Fe-SA / Cur, and FCH. Data are expressed as mean ± standard deviation (SD) (n = 3), ns: p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. H) Density functional theory (DFT) calculation of the binding energy of Fe-SA / Cur at different pH values.

[0055] Figure 3 Figure 4 shows the self-cascade reaction of FCH. A-D) The scavenging ability of FCH for different free radicals. E) Schematic diagram of the self-cascade reaction. F) Dissolved oxygen at different FCH concentrations. G) Dissolved oxygen at different H2O2 concentrations. H) Michaelis-Menten kinetics of FCH-based catalase activity. I) Reaction energy diagram of Fe-SA / Cur-based superoxide dismutase (SOD) activity. J) Reaction energy diagram of Fe-SA / Cur-based catalase (CAT) activity.

[0056] Figure 4 The in vitro anti-inflammatory properties of FCH are shown. AC) Toxicity tests on NCM460 cells at different concentrations of Fe-SA, Fe-SA / Cur, and FCH. D) Intracellular reactive oxygen species (ROS) clearance under different treatments was assessed using the ROS probe DCFH-DA (Scale bar: 50 μm). E) Intracellular oxygen generation under different treatments was assessed using the oxygen-sensitive probe [Ru(dpp)₃]Cl₂ (Scale bar: 50 μm). F) Flow cytometry analysis of apoptosis in NCM460 cells under different treatments. I: Control group; II: DSS group; III: DSS + Fe-SA (25 μg / mL); IV: DSS + Fe-SA / Cur (25 μg / mL); V: DSS + FCH (25 μg / mL).

[0057] Figure 5 Evaluation of the anti-inflammatory properties of FCH in organoid models. A) Establishment and growth of human colonic organoids. B) Morphological changes in organoids following different treatments. C) Effects of different treatments on intestinal barrier proteins in organoids.

[0058] Figure 6The in vivo anti-inflammatory properties of FCH. A) Schematic diagram of the experimental process. Acute experimental colitis was induced in mice using 2.5% DSS. Mice in each group were gavaged with PBS, Fe-SA, Fe-SA / Cur, and FCH on days 1, 3, 5, and 7, respectively. Mice were sacrificed on day 8, and tissues were collected. B) Percentage change in mouse body weight. C) Change in Disease Activity Index (DAI) score. D) Images of the colon of mice in each group; E) Colon length of mice in each group. F) Representative images of H&E (hematoxylin and eosin) staining of colon tissue from each group. G) Representative images of AB-PAS (Alcian blue-periodic acid-Schiff) staining of colon tissue from each group. Data are expressed as mean ± standard deviation (SD) (n = 5), ns: p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

[0059] Figure 7 Representative images of the anti-inflammatory properties of FCH in vivo. A) Immunofluorescence staining of E-cadherin in colonic tissue; B) Representative images of immunofluorescence staining of zonula occludens 1 (ZO-1) in colonic tissue. Scale bars: 50-200 μm.

[0060] Figure 8 The in vivo anti-inflammatory properties of FCH. A) Western blot analysis of E-cadherin in colonic tissue; B) Western blot analysis of apoptosis-related proteins. C) Representative immunofluorescence (IF) images of colonic tissue stained with F4 / 80 and Ly6G. Scale bar: 100 μm.

[0061] Figure 9 RNA sequencing reveals potential mechanisms of FCH treatment for inflammatory bowel disease (IBD). A) Volcano plot of differentially expressed genes between the IBD+PBS group and the IBD+FCH group. B) GO enrichment analysis of significantly differentially expressed genes (categorized into biological process (BP), cellular component (CC), and molecular function (MF). C) KEGG pathway enrichment analysis. D) GSEA plot showing signaling pathways enriched in the FCH-treated group (including Toll-like receptor signaling, interferon signaling, and innate immune system response). E) Western blot analysis and quantification of NF-κB, TLR4, and Myd88 protein levels in the control, IBD+PBS, and IBD+FCH groups. Statistical significance is indicated as *p < 0.05, **p < 0.01, ns = not significant. RNA sequencing, n = 3 per group; Western blot, n = 4 per group.

[0062] Figure 10Fecal metabolomics analysis of the effects of FCH treatment on IBD. A) Alpha diversity (Shannon index) analysis comparing the control, DSS (DSS+PBS), and treatment (DSS+FCH) groups, n = 4. B) Principal coordinate analysis (PCoA) at the operational taxonomic unit (OTU) level. C) Microbiome dysbiosis index (MDI) analysis. D) Histogram of phylum-level abundance percentages of the gut microbiota in the three groups. E) Phylum-level species differences among multiple groups. F) Genus-level heatmap analysis of the microbial community. G) Ring plot analysis of fecal microbiota composition and distribution among different groups.

[0063] Figure 11 Fourier transform infrared spectra of hyaluronic acid (HA), dopamine and HAD.

[0064] Figure 12 Absorption spectra of hyaluronic acid (HA), dopamine and HAD.

[0065] Figure 13 Cur release curves of FCH under different pH conditions (pH 1.2, 5 and 6.8).

[0066] Figure 14 The particle size changes of FCH in simulated gastric acid.

[0067] Figure 15 The particle size changes of FCH in simulated intestinal fluid.

[0068] Figure 16 This is the ESR spectrum of FCH scavenging free radicals (DPPH·).

[0069] Figure 17 This is the ESR spectrum of FCH scavenging free radicals (ABTS·).

[0070] Figure 18 Cell apoptosis rates in different treatment groups. Data are expressed as mean ± standard deviation (SD) (n = 5), ns: p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. Ⅰ: Control group; Ⅱ: DSS group; Ⅲ: DSS + Fe-SA (25 μg / mL) group; Ⅳ: DSS + Fe-SA / Cur (25 μg / mL) group; Ⅴ: DSS + FCH (25 μg / mL) group.

[0071] Figure 19 Bioluminescence imaging of mice and colon 24 hours after treatment, showing the targeting properties of FCH.

[0072] Figure 20 Hematoxylin-eosin (H&E) staining of major organs (heart, liver, spleen, lung, kidney, and intestine) in different groups. Scale bar: 200 μm.

[0073] Figure 21 Serum biochemical analysis of mice in the control and FCH groups. Test parameters included ALT (alanine aminotransferase), AST (aspartate aminotransferase), T-Bil (total bilirubin), and CREA (creatinine). Data are expressed as mean ± standard deviation (SD) (n = 3), ns: p > 0.05 (no significant difference).

[0074] Figure 22 Routine blood tests were performed on mice in the control and FCH groups. Test parameters included white blood cell count (WBC), neutrophils (Neu), mean corpuscular hemoglobin (MCH), monocytes (Mon), lymphocytes (Lym), hemoglobin (HGB), eosinophils (Eos), red blood cell count (RBC), basophils (Bas), mean corpuscular hemoglobin concentration (MCHC), hematocrit (HCT), mean corpuscular volume (MCV), platelet packed cell (PCT), and platelet count (PLT). Data are expressed as mean ± standard deviation (SD) (n = 3). ns: p > 0.05 (no significant difference).

[0075] Figure 23 Phylogenetic trees of the control, IBD, and treatment groups. DETAILED DESCRIPTION

[0076] It should be noted that the following detailed descriptions are exemplary and are intended to provide further illustrations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the content of the present invention are encompassed within the scope that the present invention is intended to protect. Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. It should be noted that in the present invention, FCH is an abbreviation for the single-atom nanozyme delivery system Fe-SA / Cur@HAD.

[0077] Example 1 Preparation and Characterization of Fe-SA

[0078] Zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O, 1190 mg) and iron triacetylacetonate (Fe(C₅HₐO₂), 52 mg) were uniformly dispersed in a mixed solution (20 mL of methanol and 10 mL of tetrahydrofuran) using ultrasonic vibration, designated as Solution A. Simultaneously, 2-methylimidazole (2-MI, 1134 mg) was dispersed in 5 mL of a mixed solution (5 mL of methanol and 5 mL of tetrahydrofuran), designated as Solution B. Solution A and Solution B were then mixed under ultrasonic conditions and stirred at 600 rpm for 15 minutes. The resulting precipitate was collected by centrifugation, washed three times with ethanol, and dried under vacuum to obtain Fe-ZIF-8. The Fe-ZIF-8 was then placed in a quartz tube, purged with N₂, and reacted at 800°C for 2 hours. The resulting black product was immersed in 0.5 M hydrochloric acid for 2 hours, washed three times with ultrapure water and ethanol, and dried under vacuum to obtain Fe-SA.

[0079] In the present invention, first, Zn 2+ and Fe 3+ ions coordinated with 2-methylimidazole (2-MI) to form Fe-doped zeolitic imidazole framework-8 (Fe-ZIF-8). Subsequently, Fe-ZIF-8 was heat-treated at 800°C in a nitrogen (N2) atmosphere to cause the zinc ions to pyrolyze and evaporate, while carbonizing to form a nitrogen-doped carbon structure. Finally, acid etching was performed to remove impurities formed during the calcination process to obtain Fe-SA. Compared with the classic ZIF-8 structure, Fe-SA better retains the original regular dodecahedral framework. During the high-temperature pyrolysis process, its surface gradually collapsed to form a porous structure. This phenomenon can be observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) ( Figure 1 B-1C). Aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images confirm the presence of single Fe atoms ( Figure 1 D). In addition, the electron diffraction pattern shows that the four elements C, N, O and Fe are evenly distributed ( Figure 1 E).

[0080] X-ray absorption spectroscopy (including near-edge structure (XANES) and extended fine structure (EXAFS)) was used to further study the chemical coordination environment of Fe and N, K edges in Fe-SA, and the results are shown in Table 1.

[0081] Table 1

[0082]

[0083]

[0084] Compared with Fe foil and Fe2O3, the XANES curve of FeK edge in Fe-SA shows the near-edge absorption energy located between Fe foil and Fe2O3. Among them, the spatial arrangement symmetry characteristic peak at 7115eV confirms the existence of Fe-N4 structure supported by single atoms ( Figure 1 F). Fourier transform (FT)k 2 Weighted EXAFS spectra in A clear peak is shown at 2.2, which is attributed to the scattering path of Fe-N4 coordination in the first coordination shell. These signals are different from metallic Fe-Fe interactions because they correspond to the longer-range Fe-Fe ( Figure 1 G, 1H).

[0085] Using k 2 Weighted EXAFS oscillation spectroscopy is used to analyze the coordination environment of Fe in the sample. Figure 1 As shown in Figure 1, the period and frequency observed in the Fe-SA spectrum are comparable to those of the natural Fe-N4 in heme, thus distinguishing it from the oscillation spectra of Fe2O3 and Fe foil. EXAFS fitting helps to quantitatively analyze the chemical coordination environment of typical N-coordinated metal single-atom structures, such as Figure 1 As shown in J, it is precisely shown that the nitrogen coordination number around Fe is 4. Fe atoms are incorporated into the hierarchical carbon-based matrix of Fe-SA as Fe-N4 co-dopants. Finally, the resolution of k-space and R-space is investigated using the k-edge EXAFS oscillation wavelet transform spectrum ( Figure 1 K-1M), it is obvious that Fe-SA has a higher The strongest absorption is shown.

[0086] Example 2 Preparation and Characterization of HAD

[0087] 100 mg of hyaluronic acid was dissolved in 20 mL of ultrapure water under N2 protection. After it was completely dissolved, 50 mg of EDC and 30 mg of NHS were added to activate the carboxyl group. After 5 hours, 50 mg of dopamine was added and stirring was continued for 12 hours under N2 protection at a stirring rate of 500 r / min. After the reaction was completed, the mixture was placed in a 3000 Da dialysis bag and dialyzed for 3 days, and then lyophilized to collect HAD. The Fourier transform infrared spectrum and absorption spectrum of HAD are shown in Figure 2. Figure 11-12 shown.

[0088] Example 3 Preparation and Characterization of FCH

[0089] Fe-SA (50 mg) was dissolved in 10 mL of ethanol, followed by the addition of 10 mg of curcumin (Cur). The mixture was stirred for 24 h in the dark. The solvent was removed by rotary evaporation, and the residue was collected and dialyzed for 3 days to remove unreacted material. The Fe-SA / Cur was dried and collected.

[0090] Fe-SA / Cur (50 mg) and HAD (5 mg) were ultrasonically dissolved in 10 mL of ultrapure water and stirred for 48 h. The collected product was then dialyzed in a 3000 Da dialysis bag for 3 days, with the water changed every 6 hours, and then lyophilized to collect FCH.

[0091] The synthesis of FCH is as follows Figure 2 As shown in Figure A. Curcumin (Cur) is a compound derived from natural ginger with strong anti-inflammatory properties. However, its limited water solubility restricts its application range. Compared with complex delivery systems, the coordination of Fe-SA and Cur (Fe-SA / Cur) can better achieve drug delivery, greatly improving its anti-inflammatory effect. The HAD coating has significant stability and negative charge, which can ensure the oral administration of the drug and its targeted delivery.

[0092] From the TEM images, we can find that compared with Fe-SA and Fe-SA / Cur, FCH has more layers on its surface, and the original collapsed structure is gradually filled with curcumin. After being wrapped by HAD, its surface load is more layered ( Figure 2 B-2D). In addition, infrared spectroscopy and ultraviolet absorption verified the loading of curcumin and the encapsulation of HAD, proving the successful preparation of Fe-SA / cur@HAD ( Figure 2 E-2F). Zeta potential analysis showed that after HAD loading, the zeta potential was -10mV ( Figure 2 G), this large negative value sets the stage for targeted delivery to positively charged regions affected by IBD.

[0093] In vitro release experiments of Cur were conducted in simulated gastric fluid, IBD microenvironment, and intestinal fluid (pH = 1.2, 5.5, 7) to simulate different in vivo environments. Within 24 hours, approximately 70% of Cur was released in the IBD microenvironment (pH = 5.5). Less than 20% of Cur was released in simulated gastric fluid and intestinal fluid, indicating that FCH can effectively cross the gastrointestinal barrier ( Figure 13 ). Generally, the transit time of the stomach and intestine after oral administration is about 2 hours and 4 hours, respectively. FCH was incubated in simulated gastric and intestinal fluids for 4 hours to evaluate its acid resistance and drug release properties. After immersion in gastric acid or intestinal fluid for 4 hours, its structure showed almost no change, thus confirming its acid resistance and targeted drug release ability ( Figure 14 、 Figure 15 ).

[0094] To understand the interaction between curcumin and Fe-SA, density functional theory calculations were performed to analyze Fe-SA / Cur. Considering the weak acidity of the inflammatory microenvironment, the adsorption energies of Cur on Fe-N-C under acidic (pH 5.5) and physiological (pH 7) conditions were calculated (Eads). Figure 2 H). The results showed that the interaction between Cur and Fe-N-C under acidic conditions (Eads= -1.10 eV) was weaker than that under physiological conditions (Eads= -1.73 eV). This significant difference indicates that the low pH in the IBD environment can change the protonation state of the Cur molecule, thereby affecting its charge distribution and electron cloud density. This protonation change can weaken the electrostatic interaction between Fe-SA and Cur, resulting in a decrease in overall binding energy.

[0095] Example 4 Self-cascade reaction of FCH

[0096] (1) SOD activity evaluation

[0097] SOD-like activity was evaluated using an SOD assay kit (Beyotime, S0101S). According to the kit instructions, SOD-like activity was calculated by comparing the colorimetric analysis of WST-8 product.

[0098] (2) Free radical scavenging evaluation

[0099] (2.1) DPPH· scavenging experiment

[0100] DPPH· solution (100 μM, ethanol) was mixed with different concentrations of FCH (2, 5, 12.5, 25, 50 μg / mL) under light-protected conditions for 30 min. Then the mixture was centrifuged at 7000 rpm for 5 min. Then, the supernatant was collected and the absorbance was measured at 517 nm.

[0101] (2.2) ABTS· scavenging experiment

[0102] ABST solution was prepared according to the kit instructions (Beyotime, S0119), and then the ABTS· solution was mixed with FCH (2, 5, 12.5, 25, 50 μg / mL). The mixture was incubated under light-protected conditions for 10 min, the supernatant was collected and the absorbance was measured at 734 nm.

[0103] (2.3) ·OH scavenging experiment

[0104] A 0.416 mM solution of 3,3',5,5'-tetramethylbenzidine (TMB) in DMSO, a 1.0 mM solution of ferrous sulfate (FeSO4) in HAc-NaAc buffer (pH 4.5), and a 10 mM solution of hydrogen peroxide (H2O2) were mixed with FCH (2, 5, 12.5, 25, and 50 μg / mL). After standing for 5 minutes, the mixture was centrifuged at 8000 rpm for 5 minutes, and the supernatant was collected and the absorbance measured at 652 nm.

[0105] (3)CAT assessment

[0106] CAT-like activity was measured in PBS (pH 7.4) containing FCH (25 μg / mL) using H₂O₂ (1 M) as a substrate. Dissolved oxygen was measured using a dissolved oxygen meter. Kinetic assays of FCH were performed in PBS at room temperature using different concentrations of H₂O₂ (0, 0.1, 0.2, 0.3, 0.5, 1, 1.5, and 2 M). Based on equation (1), the Michaelis constant (Km) and maximum reaction rate (Vmax) values ​​were determined using Origin 2024 software.

[0107]

[0108] V: reaction rate, [S]: substrate concentration, Vmax: maximum reaction rate.

[0109] SOD is the first step in the self-cascade reaction. The SOD-like activity and free radical scavenging ability of Fe-SA were evaluated, focusing on four specific free radicals: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical (ABTS·), 1,1-diphenyl-2-trinitrophenylhydrazine radical (DPPH·), hydroxyl radical (·OH) and superoxide anion (O2· - ), these free radicals are present in large quantities in biological systems and are often associated with inflammation. As the concentration of FCH increases, its ability to scavenge these free radicals gradually increases. At a concentration of 25 μg / mL, the scavenging efficiency of all free radicals exceeds 75% ( Figure 3 A-3D).

[0110] Electron spin resonance (ESR) spectroscopy was used to visually evaluate the free radical scavenging efficiency. The electron capture agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to capture free radicals. The results showed that after FCH treatment, the free radical signal was significantly weakened, providing strong evidence for its SOD-like activity and free radical scavenging potential. Figure 16 、 Figure 17 ).

[0111] The self-cascade reaction of FCH is as follows Figure 3 E. In this process, O2·- It is converted to H2O2 through SOD-like activity. H2O2 then serves as a substrate for peroxidase and is converted to O2 and H2O through a CAT-like mechanism. The CAT-like activity of FCH is assessed using a dissolved oxygen meter. Figure 3 As shown in Figures F and 3G, the dissolved oxygen level gradually increases over time as the concentrations of H2O2 and FCH increase. This phenomenon occurs because H2O2 serves as a substrate in the CAT reaction and is a key intermediate in the self-cascade reaction. The dissolved oxygen and H2O2 concentrations produced per minute follow Michaelis-Menten kinetics, indicating that the system has significant CAT-like activity ( Figure 3 H).

[0112] The catalytic performance of Fe-NC nanozymes in two-electron SOD-like and four-electron CAT-like reactions was evaluated by DFT calculations, and the Gibbs free energy of all species (including reactants, intermediates, and products) was calculated. Figure 3 I, 3J). In the SOD-like reaction, the adsorption of the OOH reactant is exothermic, releasing 1.01 eV of energy. The formation of ·H from ·OOH is the potentially limiting step (PLS), requiring an energy input of 1.83 eV. ·H is then converted to the ·H2O2 intermediate in an exothermic step, releasing 1.96 eV. Finally, HO2 desorbs from the surface, an exothermic step (0.34 eV), completing the SOD-like reaction. In the CAT-like reaction, the adsorption and conversion of HO2 to ·O is highly exothermic, releasing 1.75 eV. The ·O intermediate then binds a second HO2 molecule in another exothermic step (0.54 eV). The subsequent conversion of ·O-HO2 to ·HO-O2 is also exothermic, releasing 0.34 eV. ·HO-O2 is then converted to ·HO and releases an O2 molecule, which is the potentially limiting step (0.36 eV). The final desorption of H2O is exothermic, releasing 0.82 eV of energy.

[0113] Through the detection of enzyme-like activity and DFT calculations, evidence was obtained that self-cascade reactions may occur, laying the foundation for further research on its anti-inflammatory biological application potential.

[0114] Example 5 Cellular anti-inflammatory efficacy of IBD

[0115] (1) Cell culture

[0116] NCM460 cells were cultured in high-glucose medium supplemented with 10% FBS and 1% PS at 37°C in a 5% CO2 atmosphere.

[0117] (2) Cell safety assessment

[0118] Biosafety was assessed by CCK-8 assay on human epithelial cells (NCM460).

[0119] (2) Intracellular ROS scavenging activity

[0120] To induce inflammatory cells in vitro, NCM460 cells were cultured at 10 5 Cells were seeded into confocal microplates and incubated with medium containing 3% DSS. Subsequently, cells were treated with Fe-SA (25 μg / mL), Fe-SA / Cur (25 μg / mL), and FCH (25 μg / mL) for 24 hours. Incubation with DFCH-DA (1 μM) and DAPI (0.5 μg / mL) was performed in the dark, and images were collected using CLASM.

[0121] (3) Intracellular O2 generation

[0122] To induce inflammatory cells in vitro, NCM460 cells were cultured at 10 5 Cells were seeded into confocal microplates and incubated with medium containing 3% DSS. Subsequently, cells were treated with Fe-SA (25 μg / mL), Fe-SA / Cur (25 μg / mL), and FCH (25 μg / mL) for 24 hours. Incubation with Ru(dpp)3Cl2 (1 μM) and DAPI (0.5 μg / mL) was performed in the dark, and images were collected using CLASM.

[0123] The biosafety was assessed by CCK-8 assay on human epithelial cells (NCM460). Figure 4 As shown in A-4C, NCM460 cells were exposed to various concentrations of Fe-SA, Fe-SA / Cur, and FCH for 24 hours. Even at the highest concentration tested (100 μg / mL), cell viability remained above 80%. These findings indicate a good safety profile with minimal toxicity.

[0124] The self-cascade reaction has been confirmed in in vitro buffer solution. To further study its intracellular self-cascade reaction, 3% dextran sulfate sodium salt (DSS) was used to induce inflammation in NCM460 cells, and then the DCFH-DA probe was used to measure the production of intracellular ROS. Figure 4 As shown in Figure D, a large amount of ROS was generated in cells after DSS induction, manifested by strong green fluorescence. However, treatment with Fe-SA, Fe-SA / Cur, or FCH significantly reduced DSS-induced intracellular ROS levels. Compared with the "DSS + Fe-SA" group, the green fluorescence in the "DSS + FCH" group was significantly weakened and almost eliminated. Notably, FCH treatment appeared to almost completely reverse DSS-induced OS. This reduction may be due to the enhanced clearance of ROS by Cur coordinated to the complex, thereby exhibiting a stronger synergistic anti-inflammatory effect.

[0125] The hypoxia probe Ru(dpp)3Cl2 was used to measure the intracellular O2 production. Figure 4 As shown in Figure E, strong red fluorescence was observed in cells after induction with 3% DSS, indicating the establishment of a hypoxic environment associated with inflammation. After treatment with FCH and other drugs, the intensity of red fluorescence in the cells was significantly reduced, indicating that the drug can increase oxygen levels in the inflammatory microenvironment, thereby alleviating the hypoxic condition. Intracellular fluorescence staining further supports the occurrence of a self-cascade reaction, indicating that the inflammatory microenvironment has been significantly improved.

[0126] In order to evaluate the effects of FCH and other factors on apoptosis after DSS induction, NCM460 cells were detected using Annexin V / PI kit and then analyzed by flow cytometry. Figure 4 F and Figure 18 As shown, the results showed that treatment with Fe-SA, Fe-SA / Cur or FCH significantly reduced DSS-induced cell apoptosis.

[0127] Example 6 Evaluation of FCH Performance in Colon Organoid Model

[0128] In 2009, Hans Clevers' laboratory in the Netherlands successfully established the first intestinal organoid culture system. Intestinal organoids retain the characteristics of their unique crypts, and their cell composition and arrangement are closely similar to those of normal intestinal epithelial structures.

[0129] Currently, intestinal organoids are widely used in the study of intestinal diseases as an important in vitro model for studying the normal physiology and pathology of intestinal epithelial cells. The present invention extracted unique colon-derived crypts from endoscopic biopsy samples of volunteers and cultured them in vitro to construct 3D organoids. Subsequently, TNF-α (50 ng / mL) and IFN-γ (50 ng / mL) were used to establish an in vitro IBD model, and FCH was administered to evaluate its therapeutic effect on IBD.

[0130] like Figure 5 As shown in Figure A, during the two-day culture period, the spherical volume of the organoids gradually increased, indicating that intestinal organoids were successfully established in vitro. In order to simulate the physiological environment, inflammatory factors (TNF-α and IFN-γ) were added to the culture medium of the organoids, resulting in significant morphological changes, impaired epithelial integrity, and the appearance of a large number of black dead cells in the lumen, thus confirming the effective induction of this in vitro IBD model ( Figure 5 B). FCH treatment significantly improved the damage of colon organoids induced by inflammatory factors ( Figure 5 B) These findings suggest that FCH has beneficial therapeutic effects in organoid IBD models.

[0131] To further visualize phenotypic changes during inflammation, organoids were observed under confocal laser microscopy. Evaluation of the effects of FCH treatment yielded similar results, as 3D reconstructions of immunofluorescence (IF)-stained organoids showed similar morphological alterations to those observed under brightfield microscopy ( Figure 5 C).

[0132] ZO-1 is essential for maintaining cell adhesion and the integrity of the epithelial barrier. It promotes calcium-dependent intercellular adhesion and promotes the formation of tight junctions, thereby preventing bacteria and harmful substances from passing through the intercellular space. Occludin is another tight junction protein that plays a key role in maintaining epithelial barrier function. Therefore, ZO-1 and Occludin are both key indicators of epithelial function and repair efficacy. In the IBD group, the expression levels of ZO-1 and Occludin stained by IF were significantly reduced compared with the control group, and the structure was discontinuous, indicating that the epithelial barrier function of the organoids was significantly impaired during inflammation ( Figure 5 C). As expected, FCH treatment significantly restored intestinal barrier function. These findings demonstrate that FCH effectively promotes the restoration of intestinal barrier function in organoids, highlighting its importance in repairing damaged intestinal architecture and providing strong support for further exploration of its in vivo applications.

[0133] Example 7 In vivo animal experiment

[0134] In the in vivo animal experiments, 6-8 week old (20-22 g) male mice were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. The acute IBD model was established by administering 2.5% DSS in drinking water.

[0135] IBD model mice were then randomly divided into four groups of six, with healthy mice serving as a control group. The groups were: control, DSS + PBS, DSS + Fe-SA (25 μg / mL), DSS + Fe-SA / Cur (25 μg / mL), and DSS + FCH (25 μg / mL). After eight days of treatment, all mice were sacrificed, and major organs and colon tissues were collected for characterization.

[0136] like Figure 6As shown in A, an acute IBD model was established by freely drinking a solution containing 2.5% w / v DSS for 7 consecutive days. At the same time, mice in the IBD+PBS, IBD+Fe-SA, IBD+Fe-SA / Cur or IBD+FCH groups were given phosphate buffered saline (PBS), Fe-SA, Fe-SA / Cur or FCH by oral gavage on days 1, 3, 5 and 7, respectively. Mice that drank normal water and were not given medication served as a control group. Body weight, macroscopic bleeding and stool consistency were monitored daily to calculate the disease activity index (DAI) score for assessing the severity of IBD. The mice were subsequently killed on day 8 to evaluate the therapeutic effect of the treatment on IBD in vivo. ICG-loaded FCH was injected into IBD mice to evaluate the targeting efficacy. Significant fluorescence intensity was observed in the IBD+FCH group, indicating that the single-atom nanozyme delivery system accumulated mainly in the abdominal area, which was further confirmed by ex vivo imaging of colon tissue ( Figure 19 ).

[0137] Mice exposed to DSS showed significant weight loss compared to the control group ( Figure 6 B). In addition, no significant difference was observed in body weight changes between the DSS+PBS group and the DSS+Fe-SA or DSS+Fe-SA / Cur groups. However, FCH treatment significantly reduced body weight loss in mice compared with PBS treatment ( Figure 6 B). Similarly, the DAI score showed that the intestinal inflammation in the IBD+FCH group was significantly milder than that in the other groups ( Figure 6 C). After the mice were sacrificed, the entire colon was carefully removed from the cecum to the anus to assess its length, which is a key indicator of the severity of inflammation. Compared with the DSS+PBS group, the DSS+FCH group showed a significant reduction in colon shortening, indicating the therapeutic effect of FCH on intestinal inflammation. The therapeutic effects of Fe-SA and Fe-SA / Cur were relatively limited, probably due to their poor targeted delivery ability. In addition, FCH was continuously administered to healthy mice for one week, followed by histological examination of major organs by HE staining, as well as routine blood tests and serum biochemical analysis. No significant toxicity of FCH was found ( Figure 20-22 ).

[0138] The distal colon tissues of each group of mice were collected for histological examination. Hematoxylin and eosin staining showed that the colon showed significant morphological damage after DSS treatment, including loss of colonic crypt structure and infiltration of inflammatory cells ( Figure 6 DE). Although Fe-SA and Fe-SA / Cur treatments showed no significant improvement, significant histopathological remission was observed after FCH treatment ( Figure 6 F).

[0139] Alcian blue-periodic acid Schiff staining (AB-PAS) was used to specifically stain mucin present in goblet cells, a key indicator of barrier function. The DSS+FCH group showed significant recovery in mucin deposition compared to the other groups ( Figure 6 G).

[0140] E-cadherin is an important intercellular adhesion molecule that plays a key role in maintaining intestinal barrier function, and its dysfunction may impair this integrity. Figure 7 As shown in A, IF staining observed a significant decrease in E-cadherin expression in the colon of the DSS group. Although there was no significant recovery of colon tissue after Fe-SA or Fe-SA / Cur treatment, FCH treatment resulted in a significant increase in E-cadherin fluorescence intensity, indicating that colon tissue was rapidly recovering and showing further structural improvement. Subsequently, ZO-1 expression in colon sections was assessed by IF. ZO-1 protein levels were significantly higher in the DSS+FCH group compared with the DSS group ( Figure 7 B). Western blotting (WB) also confirmed the effect of FCH treatment on E-cadherin expression ( Figure 8 A).

[0141] Increased intestinal epithelial cell apoptosis is not only an important pathogenic factor of IBD but also a key pathological feature. Therefore, the expression of apoptosis-related proteins in colonic tissue was evaluated by Western blotting analysis. As expected, DSS treatment led to increased levels of the pro-apoptotic proteins cleaved-Caspase 3 and Bax, while the expression of the anti-apoptotic protein BCL2 was decreased ( Figure 8 B)

[31] . This pattern was significantly reversed after FCH treatment ( Figure 8 B) These findings provide strong evidence for the efficacy of FCH in the treatment of IBD.

[0142] Macrophage infiltration is a hallmark of intestinal inflammation. Colon sections were stained with F4 / 80 (a specific marker for macrophages) and Ly6G (a specific marker for neutrophils). DSS exposure resulted in colonic epithelial damage accompanied by significant macrophage infiltration. FCH treatment resulted in a significant reduction in macrophage and neutrophil infiltration ( Figure 8 C).

[0143] Example 8 Transcriptome Analysis of FCH Therapeutic Mechanisms

[0144] To further investigate the therapeutic mechanism of FCH in DSS-induced IBD in mice, RNA sequencing analysis was performed on colon tissues to evaluate gene expression profiles. Differential expression analysis between the DSS+FCH group and the DSS+PBS group identified 595 differentially expressed genes (|log2FC| ≥ 1 and q < 0.05), of which 216 were upregulated and 289 were downregulated ( Figure 9 A). Gene ontology analysis of the differentially expressed genes revealed significant enrichment in several key biological processes, including defense responses to bacteria or viruses, regulation of innate immune responses, and cellular responses to OS ( Figure 9 B).

[0145] Gene set enrichment analysis was performed to further investigate the underlying mechanisms. Figure 9 As shown in Figure C, significant changes were found in pathways such as interferon signaling, the innate immune system, and the Toll-like receptor (TLR) signaling pathway. As classic pattern recognition receptors and key responders to invading pathogens, TLRs play a key role in the innate immune system, and the TLR signaling pathway is closely related to a variety of inflammatory diseases.

[0146] In addition, Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that terms related to NOD-like receptor signaling pathway, antigen processing and presentation, PPAR signaling pathway, Epstein-Barr virus infection, primary immunodeficiency, mucin-type O-glycan biosynthesis, and RIG-I-like receptor signaling pathway were highly enriched ( Figure 9 D). These findings provide strong evidence that FCH may improve intestinal inflammation by enhancing defense responses to microbial threats, modulating innate immune responses, and alleviating OS.

[0147] It is noteworthy that TLR4 is a highly conserved protein that is widely believed to play a key role in regulating intestinal homeostasis. To evaluate the activation of the TLR4 signaling pathway in mouse colon tissue, Western blot analysis was performed. Figure 9 As shown in Figure E, DSS administration significantly increased the expression levels of Myd88 and NF-κB. However, FCH treatment reversed this trend. A similar trend was observed for TLR4 expression, but the difference did not reach statistical significance. These results suggest that the TLR4 / Myd88 / NF-κB-related inflammatory pathway may be a key mechanism of FCH treatment in IBD.

[0148] Example 9 Effect of FCH on Intestinal Flora

[0149] The ecological balance of the intestinal commensal microbiota regulates the phenotype of the intestinal epithelium and plays a key role in the progression of IBD. Commensal microbiota and intestinal function are interdependent; dysbiosis can lead to persistent intestinal inflammation and epithelial barrier damage, and changes in the colonic microenvironment may affect microbial composition. To examine the characteristics of the intestinal microbiota in mice treated with DSS and FCH, 16S ribosomal DNA gene sequencing was used to analyze the microbial composition in fecal samples.

[0150] The alpha diversity indices of the microbial communities in all groups were analyzed, including the Shannon index, ACE index, coverage index, and Chao index. The Shannon index was significantly reduced after DSS treatment ( Figure 10 A), while no substantial changes were detected in other indices ( Figure 23 ).

[0151] For β diversity, principal coordinate analysis based on operational taxonomic units showed that there were significant differences in the bacterial community distribution among the control group, DSS (DSS+PBS) group, and treatment (DSS+FCH) group ( Figure 10 B). Subsequently, the degree of microbial imbalance was assessed using the Microbiome Dysbiosis Index (MDI). Figure 10 The DSS group showed significant dysbiosis as shown in Figure C. Although the difference did not reach statistical significance, the dysbiosis index in the treatment group was reduced compared with the DSS group, indicating that FCH has a regulatory role in maintaining microbial balance.

[0152] It has been reported that the gut microbiome of IBD patients is characterized by a decrease in the relative abundance of Firmicutes and Bacteroidetes, and an increase in Proteobacteria and Actinobacteria compared to healthy individuals. Community histogram analysis showed that the relative abundance of differential bacterial communities at the phylum level changed among the three groups ( Figure 10 D). Consistent with findings in IBD patients, the histogram shows that DSS exposure resulted in a decrease in the relative abundance of Firmicutes and an increase in Proteobacteria.

[0153] Notably, FCH treatment significantly reversed the DSS-induced imbalance in gut microbiota composition ( Figure 10 D). The differences in microbial composition among the groups were further analyzed. Interestingly, Proteobacteria and Firmicutes were identified as significantly altered microbial communities ( Figure 10 E), indicating that these changes may be closely related to the therapeutic mechanism of intestinal inflammation and FCH. In addition, the community heat map and Circos diagram intuitively depict the relative abundance and distribution of fecal microbiomes in different groups after DSS and FCH treatment ( Figure 10 Overall, these findings suggest that FCH-regulated gut microbiota may play a key role in alleviating IBD.

[0154] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A single-atom nanozyme delivery system for IBD treatment, characterized in that: The system includes an iron-doped single-atom nanozyme Fe-SA, curcumin Cur, and hyaluronic acid-grafted dopamine HAD; the Fe-SA and Cur form a Fe-SA / Cur complex through coordination, and the HAD is coated on the surface of the Fe-SA / Cur complex; in the iron-doped single-atom nanozyme Fe-SA, Fe atoms are distributed in the hierarchical carbon-based matrix of Fe-SA in the form of Fe-N4 co-doping, and have superoxide dismutase (SOD)-like activity and catalase (CAT)-like activity, and can catalyze the following self-cascade reaction: (1) SOD-like activity: converts superoxide anion O2 ·- Converted into hydrogen peroxide H2O2; (2) CAT-like activity: decomposition of hydrogen peroxide H2O2 into oxygen O2 and water H2O; The HAD is formed by linking the carboxyl group of hyaluronic acid and the amino group of dopamine through an amide bond, with hyaluronic acid as the main chain and grafted dopamine as the side chain.

2. The single-atom nanozyme delivery system according to claim 1, characterized in that The preparation method of the iron-doped single-atom nanozyme Fe-SA comprises the following steps: Zinc and iron ions coordinate with 2-methylimidazole to form Fe-ZIF-8; The calcination reaction is carried out at 800℃ under nitrogen conditions to thermally decompose and evaporate zinc ions, while carbonizing to form a nitrogen-doped carbon structure; Acid etching removes impurities formed during the firing process to obtain Fe-SA.

3. The single-atom nanozyme delivery system according to claim 2, characterized in that Preparation method of the iron-doped single-atom nanozyme Fe-SA The following steps are involved: Zinc nitrate hexahydrate and triacetylacetone were ultrasonically dispersed in a methanol-tetrahydrofuran mixture, and a methanol-tetrahydrofuran mixture of 2-methylimidazole was added. After ultrasonic mixing, the coordination reaction was carried out by stirring. The precipitate was collected by centrifugation, washed with anhydrous ethanol, and dried in vacuum to obtain Fe-ZIF-8. Fe-ZIF-8 was placed in a quartz tube, filled with N2 and calcined at 800℃ to obtain a black product; The obtained black product was immersed in hydrochloric acid for acid etching reaction to remove impurities formed during the firing process, washed with ultrapure water and ethanol, and vacuum dried to obtain Fe-SA.

4. The single-atom nanozyme delivery system according to claim 1, characterized in that The Cur is loaded on the Fe-SA surface through coordination, and the loading amount is 1-20% based on the mass of the Fe-SA.

5. The single-atom nanozyme delivery system according to claim 4, characterized in that The preparation method of the Fe-SA / Cur complex comprises the following steps: dissolving Fe-SA in ethanol, adding curcumin Cur, stirring in the dark for reaction, removing the solvent, dialyzing to remove unreacted substances, and drying to obtain the Fe-SA / Cur complex.

6. The single-atom nanozyme delivery system according to claim 1, characterized in that The coating amount of the HAD is 1-10% based on the mass of the Fe-SA / Cur complex.

7. The single-atom nanozyme delivery system according to claim 6, characterized in that The preparation method of Fe-SA / Cur@HAD includes the following steps: ultrasonically dissolving Fe-SA / Cur and HAD in ultrapure water, stirring for reaction, dialyzing, and freeze-drying to obtain the Fe-SA / Cur@HAD system.

8. The preparation method of the single-atom nanozyme delivery system according to claim 1, characterized in that: The following steps are involved: S1: Preparation of Fe-SA: Zinc and iron ions are coordinated with 2-methylimidazole to form Fe-ZIF-8; the zinc ions are calcined at 800°C in nitrogen to pyrolyze and evaporate, while carbonizing to form a nitrogen-doped carbon structure; impurities formed during the calcination process are removed by acid etching to obtain Fe-SA. S2: Preparation of Fe-SA / Cur complex: Fe-SA was dissolved in ethanol, curcumin Cur was added, and the mixture was stirred in the dark to react. The solvent was removed, unreacted substances were removed by dialysis, and the Fe-SA / Cur complex was obtained by drying. S3: Preparation of Fe-SA / Cur@HAD system: Fe-SA / Cur and HAD were ultrasonically dissolved in ultrapure water, stirred for reaction, dialyzed, and freeze-dried to obtain the single-atom nanozyme delivery system Fe-SA / Cur@HAD.

9. Use of the single-atom nanozyme delivery system according to any one of claims 1 to 7 in the preparation of IBD therapeutic drugs.

10. A drug for treating IBD, characterized in that: The drug contains the single-atom nanozyme delivery system according to any one of claims 1 to 7.

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