An oral composite microsphere for treating ulcerative colitis based on intestinal ROS storm pathological axis regulation, and a preparation method and application thereof

Abstract

This invention discloses an oral composite microsphere for treating ulcerative colitis based on the regulation of the intestinal ROS storm pathological axis, its preparation method, and its application. It aims to address the technical problems of existing ulcerative colitis treatments, such as the difficulty in reversing the core pathological cycle, the easy inactivation of oral antioxidant drugs and nanozymes by gastric acid, poor targeting, and insufficient antioxidant capacity of probiotics used alone. The oral composite microsphere uses Clostridium butyricum spores as a biological carrier, first loading Ir with peroxidase, superoxide dismutase, and catalase-like activities. 10 -KB nanozymes are then encapsulated using sodium alginate-calcium ion crosslinking. This invention utilizes the pH-responsive gel properties of sodium alginate to achieve anti-acid protection and colon-targeted release, synergistically leveraging the flora regulation, mucosal repair, and Ir... 10 The highly efficient ROS scavenging, mitochondrial protection, and PANoptosis inhibition functions of KB nanozymes achieve a synergistic therapeutic goal of "precise delivery, highly efficient antioxidation, and microecological regulation".

Description

Technical Field

[0001] This invention belongs to the field of ulcerative colitis treatment and oral targeted drug delivery technology, specifically an oral composite microsphere for treating ulcerative colitis based on the regulation of the intestinal ROS storm pathological axis, its preparation method and application. Background Technology

[0002] Ulcerative colitis (UC) is a chronic inflammatory bowel disease with complex etiologies and difficult to cure. Its core pathological features include intestinal mucosal barrier disruption, excessive inflammatory response, and gut microbiota imbalance. Currently, its pathogenesis is believed to be closely related to a multi-cascade reaction network consisting of oxidative stress, mitochondrial damage, programmed cell death, and gut microbiota imbalance. Numerous studies have shown that the "ROS storm" pathological axis, formed by the excessive accumulation of reactive oxygen species (ROS) in the gut, plays a key driving role in the development and progression of UC. Excessive ROS not only directly damages the integrity of the intestinal epithelium but may also induce mitochondrial membrane potential collapse and promote abnormal opening of the mPTP, thereby triggering PANoptosis, a complex cell death process including apoptosis and pyroptosis, ultimately forming a vicious cycle of "ROS storm—mitochondrial damage—barrier disruption—intensified inflammation." Therefore, effectively blocking the ROS storm and restoring intestinal homeostasis is an important scientific problem that needs to be solved in the treatment of UC.

[0003] Current clinical treatments for ulcerative colitis (UC) (such as aminosalicylic acids, glucocorticoids, and biologics) can provide short-term relief from inflammation, but they are unable to effectively reverse the core pathological process of "ROS storm—mitochondrial damage—barrier disruption," resulting in limited therapeutic efficacy and a high relapse rate. Oral antioxidants are one direction of research aimed at blocking ROS storms, but these drugs often face challenges such as gastric acid inactivation, difficulty in lesion localization, and low bioavailability, significantly limiting their clinical application value.

[0004] Against this backdrop, nanozymes have gradually become a focus of research in related therapies. Nanozymes have advantages such as ultrastability and multi-enzyme-like activity, which can efficiently remove excess ROS and overcome some of the shortcomings of traditional antioxidant drugs to a certain extent. However, they still have significant shortcomings in practical applications: they are easily destroyed by gastric acid when taken orally and are difficult to precisely target intestinal lesions; at the same time, their function is relatively singular, only able to remove ROS and unable to regulate intestinal flora homeostasis, thus making it difficult to break the vicious cycle of "ROS storm - increased inflammation" and limiting their application effects.

[0005] Meanwhile, probiotic therapy has demonstrated unique advantages in the treatment of UC, with the application potential of Clostridium butyricum attracting attention. Clostridium butyricum spores possess certain acid and heat resistance properties, allowing them to be delivered from the gastrointestinal tract to the colon and germinate to produce butyric acid, thus aiding in mucosal barrier repair and gut microbiota regulation. However, the stability of conventional Clostridium butyricum preparations in highly acidic environments still needs improvement. More importantly, its antioxidant capacity is relatively weak, making it difficult to combat the severe oxidative damage caused by ROS storms, and its use alone is unlikely to achieve the desired therapeutic effect for UC.

[0006] In summary, current clinical treatments struggle to break through the core pathological cycle driven by ROS storms, and both nanozymes and Clostridium butyricum, when used alone, have significant limitations, failing to achieve the synergistic therapeutic effects of "precise delivery—highly effective antioxidation—microecological regulation." Therefore, developing an oral drug that can simultaneously achieve anti-gastric acid and colon-targeted delivery, while also possessing ROS clearance, mitochondrial protection, PANoptosis inhibition, microbial homeostasis restoration, and mucosal repair functions, is of great significance for overcoming the bottleneck in UC treatment. Summary of the Invention

[0007] The purpose of this invention is to provide an oral composite microsphere for the treatment of ulcerative colitis based on the regulation of the intestinal ROS storm pathological axis, as well as its preparation method and application. It achieves the effects of resisting gastric acid, targeted release in the colon, simultaneous removal of excess ROS in the intestine, protection of mitochondria in intestinal epithelial cells, inhibition of PANoptosis in intestinal epithelial cells, and restoration of gut microbiota homeostasis by germinating and producing butyric acid from Clostridium butyricum, ultimately promoting the repair of colitis.

[0008] The objective of this invention is achieved through the following technical solution: An oral composite microsphere for treating ulcerative colitis based on the regulation of the intestinal ROS storm pathological axis uses Clostridium butyricum spores as a biological carrier. First, Ir10-KB nanozyme with ROS scavenging activity is loaded, and then the composite microspheres are formed by cross-linking and embedding with sodium alginate-calcium ions. The Ir 10 -KB nanozymes are complexes formed by loading Ir nanoparticles onto Ketjen black oxide (KBO) as a carrier, and possess peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT)-like activities. As one of the possible implementations of this application, the method for preparing Clostridium butyricum spores is as follows: Clostridium butyricum is inoculated into enhanced Clostridium medium (RCM), anaerobic culture is carried out, the vegetative cells are inactivated by heat treatment, the spore precipitate is collected by centrifugation, washed with PBS, purified by sucrose solution density gradient centrifugation, and freeze-dried for later use.

[0009] As some possible implementations of this application, the parameters for preparing Clostridium butyricum spores are as follows: anaerobic culture temperature is 35~39℃, culture time is 40~52h; heat treatment temperature is 75~85℃, heat treatment time is 8~12min; PBS washing times are 2~4 times; the sucrose solution density gradient consists of a lower layer of sucrose solution of 35%~45% and an upper layer of sucrose solution of 55%~65%.

[0010] Furthermore, to achieve the above objectives, this application also provides a method for preparing oral composite microspheres for treating ulcerative colitis, comprising the following steps: (1) Ir 10 -KB nanozyme was dissolved in PBS buffer, Clostridium butyricum spore powder was added, and the mixture was gently stirred and incubated at room temperature to allow Ir to develop. 10 -KB nanozymes were loaded onto the spore surface, the precipitate was collected by centrifugation, and washed with PBS to obtain Spore@Ir 10 -KB complex; (2) Dissolve sodium alginate in deionized water to prepare sodium alginate aqueous solution, and add Spore@Ir 10 -KB complex was ultrasonically dispersed to form a homogeneous suspension; (3) The suspension was injected into an aqueous calcium chloride solution and crosslinked at room temperature. After the reaction was completed, the product was collected by filtration, washed, and freeze-dried to obtain oral composite microspheres.

[0011] As some possible implementations of this application, in step (1), Clostridium butyricum spore powder and Ir 10 -KB nanozyme mass ratio is (5–20):1. Preferably, Clostridium butyricum spore powder and Ir 10 The mass ratio of -KB nanozymes is 10:1.

[0012] As one possible implementation of this application, in step (2), the spore concentration in the suspension is 1–5 mg / mL.

[0013] Furthermore, in order to achieve the above objectives, this application also provides the use of oral composite microspheres for treating ulcerative colitis in the preparation of a medicament for treating ulcerative colitis based on the regulation of the intestinal ROS storm pathological axis. Compared with the prior art, the beneficial effects of the present invention are: 1. The advantages of the oral compound microspheres combined with microecological therapy and antioxidant nanoenzyme therapy provided by this invention include the fact that butyric acid produced after Clostridium butyricum spores germinate can help repair the mucosal barrier, regulate intestinal flora homeostasis, and promote M2 macrophage polarization; Ir 10-KB nanozymes possess peroxidase, superoxide dismutase, and catalase-like activities, enabling them to efficiently remove excess ROS in the intestine, intervene in the vicious cycle of "ROS storm-mitochondrial damage-barrier disruption," and inhibit PANoptosis of intestinal epithelial cells, forming a multi-target synergistic regulatory effect, which helps to overcome the limitations of using nanozymes or Clostridium butyricum alone.

[0014] 2. The oral composite microspheres provided by this invention are formed by cross-linking and encapsulating sodium alginate-calcium ions to form pH-responsive gel microspheres, which can achieve colon-targeted release, laying the foundation for subsequent efficient therapeutic effects. At the same time, it improves the problems of poor targeting and low bioavailability of traditional oral antioxidant drugs and nanozymes. In addition, the acid and bile salt resistance of Clostridium butyricum spores further enhances the stability of the carrier during intestinal delivery.

[0015] 3. The oral composite microspheres provided by this invention have good biocompatibility, and the components of the system have good biocompatibility. In vivo experiments in mice show that oral administration did not cause significant damage to liver and kidney function or tissues, and the hemolytic toxicity was low, providing safety support for subsequent clinical applications. Attached Figure Description

[0016] Figure 1 : Butyric acid bacillus spore formation was verified by TEM.

[0017] Figure 2 Spore@Ir 10 Characterization diagram of the physicochemical properties of KB and sodium alginate composite microspheres. Among them, a. Ir 10 -KB、Spore@Ir 10 -KB TEM detection. b. Spore@Ir 10 -SEM and mapping of KB sodium alginate microspheres. c. Spore and Ir 10 -KB combined confocal focal length. d. Ir 10 -KB particle size detection. e. Ir 10 -KB, Spore, Spore@Ir 10 -KB Zeta potential.

[0018] Figure 3 : Measurement of Spore and Spore@Ir at different time points 10 -KB solution's OD600.

[0019] Figure 4 Spore@Ir 10 -KB in vitro experimental study on the treatment of UC. Among them, a.Control, H2O2, Spore, and Ir... 10-KB and Spore@Ir 10 - The effect of KB groups on ROS clearance in NCM460 cells. b. Statistical analysis of ROS fluorescence intensity in NCM460 cells after different treatments. c. JC-1 fluorescence imaging in NCM460 cells after different treatments (scale bar: 50 μm). d. Statistical analysis of JC-1 fluorescence imaging. e. MitoSOX red fluorescence imaging in NCM460 cells after different treatments (scale bar: 50 μm). f. Statistical graph of MitoSOX red fluorescence intensity. g. Phenotypic expression of M2 macrophages in different groups (scale bar: 50 μm). h. Statistical analysis of M2 macrophage expression under different treatments. ij. Statistical analysis of M1 macrophage expression under different treatments. k. Ir 10 - Schematic diagram of how KB regulates the inflammatory microenvironment by acting on macrophages.

[0020] Figure 5 : Detection of intracellular reactive oxygen species (ROS) and statistical analysis of fluorescence intensity in cells of different treatment groups using the DCFH-DA probe. a. Detection of intracellular ROS in different treatment groups using the DCFH-DA probe. b. Statistical analysis of intracellular fluorescence intensity in different treatment groups.

[0021] Figure 6 : ROS indexes and statistical analysis graphs for each group detected by flow cytometry. Wherein, a. Flow cytometry results graph; b. Statistical analysis graph.

[0022] Figure 7 Flow cytometry analysis of NCM460 cell apoptosis and statistical data in each group. a. Flow cytometry results; b. Statistical data.

[0023] Figure 8 Different concentrations of Ir 10 -KB's CCK8 experiment on NCM460 cells.

[0024] Figure 9 Spore@Ir 10 - The therapeutic effect of KB / Alg on a DSS-induced mouse model of ulcerative colitis. The study included: a. a schematic diagram of the UC model procedure and treatment; b. changes in mouse body weight; c. disease activity index in each group of mice; and d. Ir at different time points. 10 -KB / Alg and Spore@Ir 10 e. In vitro fluorescence imaging of the gastrointestinal tract of mice with KB / Alg protein. f. Anatomical photographs of the colon of mice in different groups on day 15.

[0025] Figure 10 Different concentrations of Ir 10 -KB hemolysis rate and images.

[0026] Figure 11 HE images of mouse heart, liver, spleen, lungs and kidneys.

[0027] Figure 12 Statistical data on mouse serum CK, UREA, AST, and CREA.

[0028] Figure 13 Spore@Ir 10 -KB / Alg's therapeutic effect and regulatory effect on the gut microbiota in a DSS-induced mouse model of ulcerative colitis. Specifically: a. Relative abundance of gut microbiota at the phylum level in different treatments. b. LEfSe plot showing the differences between the DSS group and Spore@Ir. 10 -KB / Alg gut microbiota composition. Among them, cf. shows an α diversity block diagram of microbial richness (Chao and OTUS) and diversity (Shannon and Simpson). Detailed Implementation

[0029] Example 1: Preparation and microstructure characterization of core raw materials, complexes, and oral composite microspheres (e.g.) Figures 1-2 ).

[0030] 1.1 Preparation of Clostridium butyricum spore powder: Clostridium butyricum was inoculated onto reinforced Clostridium medium (RCM) and anaerobically cultured at 37℃ for 48 h. The vegetative cells were inactivated by heat treatment (80℃, 10 min). The spore precipitate was collected by centrifugation, washed three times with PBS, and purified by density gradient centrifugation (40% / 60% sucrose solution). After freeze-drying, Clostridium butyricum spore powder was obtained. The successful preparation of the spores was verified by SEM. Figure 1 ).

[0031] 1.2 Ir 10 Preparation of -KB: (1) Preparation of Ketjen black oxide (KBO): Take 2 g of raw Ketjen black (KB) powder and add it to a 500 mL round bottom flask. Add 100 mL of nitric acid (boiling point 83℃) and place it under reflux at 70℃ for 2 h. After the reaction is completed, immediately dilute the reaction solution with warm water at about 50℃. Then wash it with warm water by centrifugation (total water volume is about 3 L) until the pH value of the washing solution becomes neutral. Finally, place the washed solid under vacuum drying at 60-80℃ to obtain Ketjen black oxide (KBO). (This experiment needs to be carried out in a fume hood to avoid the spread of odor.)

[0032] (2) Loading and reducing Ir metal: Weigh 50 mg of the KBO prepared above, add it to 100 mL of deionized water and ultrasonically disperse for 30-60 min to ensure that the KBO is uniformly dispersed without obvious agglomeration; add 5 mL of IrCl3·xH2O aqueous solution with a concentration of 10 mg / mL, and place the mixture on a magnetic stirrer and stir continuously for 11 h. Then quickly add NaBH4 aqueous solution (1.0 M, 10 mL), stir for 15 s and let stand for 4-6 h. After the reaction is complete, centrifuge the system to collect the precipitate, wash it 3 times with deionized water, and finally place the washed precipitate under vacuum drying at 60℃ to obtain the final Ir10-KB nanozyme.

[0033] 1.3 Spore@Ir 10 Specific preparation method of the -KB complex: (1) Spore pre-dispersion: Take Clostridium butyricum spore powder, add it to PBS buffer, gently blow or vortex at low speed to disperse it fully, and obtain a spore suspension with a concentration of 3 mg / mL.

[0034] (2) Ir 10 -KB Nanozyme Loading: Ir10-KB nanozyme was dissolved in PBS buffer, and Clostridium butyricum spores were loaded with Ir... 10 Ir10-KB nanozyme was slowly added to the above spore suspension at a mass ratio of 10:1. The mixture was then incubated at room temperature with magnetic stirring (200 rpm) for 3 hours to load the Ir10-KB nanozyme onto the spore surface.

[0035] (3) Separation and washing: After incubation, the mixture was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the mixture was resuspended with PBS buffer and washed 2–3 times to remove unloaded Ir10-KB nanozymes.

[0036] (4) Product collection: The obtained precipitate was resuspended in a small amount of PBS buffer to obtain Spore@Ir 10 -KB complex suspension, or Spore@Ir obtained by freeze-drying. 10 -KB complex powder.

[0037] 1.4 Spore@Ir 10 Specific preparation method of -KB / Alg oral composite microspheres: (1) Preparation of sodium alginate solution: Weigh sodium alginate and dissolve it in deionized water. Stir magnetically until completely dissolved. Prepare a sodium alginate aqueous solution with a mass fraction of 2.5 wt%. Let it stand to remove bubbles before use.

[0038] (2) Spore@Ir 10-KB dispersion: Take the above Spore@Ir 10 -KB complex suspension (5% of sodium alginate solution volume) was added to sodium alginate solution, gently stirred and dispersed by low-power ultrasonication (100 W, mode: ultrasonication for 30 s, intermittent for 30 s) for 15 min to form a homogeneous suspension.

[0039] (3) Ca 2+ Cross-linking to form spheres: The suspension was added dropwise to 100 mM CaCl2 aqueous solution at a constant rate of 0.5 mL / min using a syringe. The cross-linking reaction was carried out at room temperature for 30 min to form Spore@Ir. 10 -KB / Alg composite microspheres.

[0040] (4) Microsphere collection and processing: After cross-linking, the microspheres are collected by filtering through a 200-mesh filter and washed three times with deionized water to remove excess Ca. 2+ Subsequently, it was freeze-dried to obtain Spore@Ir 10 -KB / Alg oral compound microspheres.

[0041] Ir was examined using transmission electron microscopy (TEM). 10 -KB and Spore@Ir 10 The microstructure of -KB was characterized. For example... Figure 2 As shown in a, Ir 10 -KB nanomaterials exhibit a uniform nanoscale structure; after binding with Clostridium butyricum spores, Ir 10 -KB is evenly distributed and successfully loaded onto the spore surface, forming a stable Spore@Ir 10 The -KB composite system showed no obvious aggregation, indicating that the construction strategy has good controllability and stability.

[0042] Subsequently, regarding Spore@Ir 10 - The microsphere structure formed by encapsulating KB with sodium alginate was analyzed by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). For example... Figure 2 As shown in b, Spore@Ir 10 -KB / Alg microspheres exhibit a regular spherical structure with a relatively dense and uniform surface; elemental mapping results further confirm the uniform distribution of Ir elements within the microspheres, indicating that Ir... 10 -KB was successfully encapsulated inside sodium alginate microspheres, providing a structural basis for its oral delivery and colon-targeted release.

[0043] To further verify Ir 10The binding between -KB and spores was analyzed using confocal laser scanning microscopy to examine the interaction between spores and Ir. 10 -KB spatial colocalization was observed. For example... Figure 2 As shown in c, Ir 10 The fluorescence signal of -KB highly overlaps with the spore outline, indicating that Ir 10 -KB can be stably adsorbed or loaded onto the spore surface, further confirming that Spore@Ir 10 The successful construction of the -KB composite structure. Regarding physicochemical properties, dynamic light scattering (DLS) results show that Ir... 10 -KB has a relatively uniform particle size distribution. Figure 2 d) Suitable for surface assembly with microbial carriers. Zeta potential analysis results show that it is compatible with free Ir 10 Compared to Spore, Spore@Ir 10 The surface potential of -KB changes significantly ( Figure 2 (e) This indicates the existence of effective interfacial interactions between the two, providing a charge basis for the stability of the composite system. Furthermore, measurements of Spore and Spore@Ir at different time points... 10 - OD600 values ​​of KB solution ( Figure 3 The results showed that neither the OD600 value nor the OD600 value decreased significantly during the experimental period, indicating that Spore@Ir 10 The -KB complex system exhibits excellent time stability, ensuring its effectiveness in subsequent oral delivery and functional performance.

[0044] Spore@Ir 10 -Study on the in vitro effects and mechanisms of KB in treating UC ( Figure 4 ).

[0045] To evaluate Spore@Ir 10 The regulatory effect of KB on oxidative stress damage was investigated by constructing an H2O2-induced oxidative stress system using normal human colonic epithelial cells NCM460 as a model. Figure 4 As shown in figure a, compared with the control group, H2O2 treatment significantly enhanced intracellular ROS fluorescence signal, while Spore or Ir alone... 10 -KB processing can reduce ROS levels to some extent. It's worth noting that Spore@Ir 10 The -KB treatment group showed the most significant ROS scavenging effect, almost restoring ROS to near-normal levels. Further quantitative fluorescence analysis results ( Figure 4 b) Consistent with imaging results, indicating that Spore@Ir 10-KB exhibits superior synergistic ROS scavenging capabilities compared to single-component formulations. Fluorescence inverted microscopy and flow cytometry further validated the ROS scavenging effect, such as... Figure 5 (For specific reference Figure 5 a and Figure 5 b) and Figure 6 (For specific reference Figure 6 a and Figure 6 b).

[0046] Given that excessive ROS is a significant cause of mitochondrial dysfunction, the JC-1 probe was further used to assess changes in mitochondrial membrane potential in NCM460 cells under different treatment conditions. Figure 4 As shown in c, after H2O2 treatment, the red fluorescence of JC-1 cells was significantly weakened while the green fluorescence was enhanced, indicating a significant decrease in mitochondrial membrane potential; while Spore or Ir 10 -KB processing can partially reverse this change. In contrast, Spore@Ir 10 The -KB treatment group exhibited a stronger red fluorescence signal, suggesting that it could significantly maintain mitochondrial membrane potential stability. The corresponding red / green fluorescence ratio statistics are as follows (…). Figure 4 d) This further validated the above conclusions. Simultaneously, mitochondrial ROS levels were detected using the MitoSOX probe. For example... Figure 4 As shown in Figure e, H2O2 treatment significantly induced excessive ROS production in mitochondria, while Spore@Ir10-KB treatment significantly reduced the red fluorescence intensity of MitoSOX. Quantitative analysis results ( Figure 4 f) indicates that Spore@Ir 10 -KB showed a significant advantage in inhibiting mitochondrial ROS production, suggesting that it can effectively block the positive feedback amplification process of "ROS-mitochondrial damage". Flow cytometry was used to detect apoptosis and statistical data of NCM460 cells in each group. Figure 7 , please refer to Figure 7 a) and 7b) indicate that Spore@Ir 10 -KB exhibits superior synergistic anti-apoptotic ability compared to single-component formulations.

[0047] Considering the close relationship between oxidative stress and the immune microenvironment, further investigation of Ir... 10 -KB's regulatory role in macrophage phenotypic polarization. For example... Figure 4 As shown in g, compared with the control group, Ir 10 -KB treatment significantly enhanced the fluorescence expression of macrophage M2-related markers, and its statistical results ( Figure 4 h) showed a significantly increased proportion of M2 macrophages. Conversely, Ir 10-KB treatment significantly inhibited the expression of M1 macrophage-related markers ( Figure 4 i), corresponding quantitative analysis results ( Figure 4 j) This further confirms its inhibitory effect on pro-inflammatory polarization. 10 -Schematic diagram of how KB regulates the inflammatory microenvironment by acting on macrophages ( Figure 4 k).

[0048] The above results indicate that Ir 10 -KB can improve the inflammatory microenvironment by regulating macrophage polarization and promoting the formation of anti-inflammatory phenotypes. This was verified using a CCK8 assay at different concentrations of Ir. 10 - Effect of KB on NCM460 cell proliferation ( Figure 8 ).

[0049] Spore@Ir 10 -KB / Alg in vivo experiment to alleviate DSS-induced ulcerative colitis in mice ( Figure 9 ).

[0050] To further verify Spore@Ir 10 The therapeutic potential of the -KB / Alg oral delivery system for ulcerative colitis in vivo was investigated by constructing a mouse UC model induced by dextran sulfate sodium (DSS), and administration and evaluation were performed according to the established experimental protocol. Figure 9 a) The therapeutic effect was systematically evaluated by comprehensively analyzing the drug's delivery behavior in the gastrointestinal tract, overall disease phenotype, and colonic histological changes. First, the body weight changes in mice were continuously monitored. For example... Figure 9 As shown in b, the body weight of mice in the model group decreased significantly after DSS treatment, indicating a gradual worsening of colitis symptoms; in contrast, Spore@Ir 10 The -KB / Alg treatment group showed a significantly reduced rate of weight loss, with a recovery trend observed in the later stages of treatment, demonstrating superior improvement compared to the Spore / Alg alone group. Consistent with this, the Disease Activity Index (DAI) results indicated that Spore@Ir... 10 -KB / Alg treatment significantly reduced the combined symptoms of DSS-induced diarrhea, bloody stools, and weight loss, effectively alleviating disease severity. Figure 9 c). To verify the gastrointestinal transport and colonic accumulation capabilities of the composite delivery system in vivo, Ir10-KB was labeled with the fluorescent dye IR-820, and Ir was further analyzed. 10 -KB / Alg and Spore@Ir 10 -KB / Alg was used to perform in vitro fluorescence imaging analysis of the gastrointestinal tract of mice at different time points. For example... Figure 9 As shown in d, Ir 10The fluorescence signal of -KB / Alg rapidly weakened over time; In contrast, Spore@Ir 10 -KB / Alg exhibited a stronger and more persistent fluorescence signal in the colon, indicating that the introduction of the spore carrier significantly enhanced the targeted accumulation and retention capacity of the oral system in the colitis lesion area. Hemolysis assay ( Figure 10 ) To validate and evaluate the biosafety (hemolytic toxicity) of different concentrations of Ir10-KB. HE images of mouse heart, liver, spleen, lungs, and kidneys ( Figure 11 This indicates the safety of the experiment, and at the same time, the statistical data of creatine kinase (CK), urea (UREA), aspartate aminotransferase (AST), and creatinine (CREA) in mouse serum are also included. Figure 12 The results showed that none of the indicators were significantly different from those in the normal control group, further confirming the effectiveness of Spore@Ir. 10 -KB / Alg, after oral administration, did not cause significant damage to liver and kidney function or tissues in mice, demonstrating good in vivo biocompatibility. At the organ level, mice were sacrificed on day 15, and their colons were harvested for anatomical observation. Figure 9 As shown in e, the DSS model group mice showed a significant shortening of the colon accompanied by inflammatory manifestations such as congestion and edema; while Spore@Ir 10 The -KB / Alg treatment group showed significant improvement in colon appearance. Quantitative results of colon length further confirmed the effectiveness of Spore@Ir. 10 -KB / Alg can significantly alleviate DSS-induced colonic shortening ( Figure 9 f), suggesting that it has a good protective effect against colonic inflammation damage.

[0051] Spore@Ir 10 -KB / Alg remodels DSS-induced intestinal immune microenvironment and gut microbiota homeostasis in UC mice ( Figure 13 ).

[0052] To further evaluate Spore@Ir 10 The immunomodulatory effects of KB / Alg on ulcerative colitis and its intestinal microecological remodeling in vivo were systematically analyzed in a DSS-induced UC mouse model. Given the central role of gut microbiota imbalance in the pathogenesis of UC, 16S rRNA sequencing analysis was performed on the gut microbiota composition of different mouse groups. Figure 13 As shown in figure a, at the phylum level, DSS treatment significantly disrupted the gut microbiota structure, while Spore@Ir 10 -KB / Alg treatment significantly restored the gut microbiota composition, showing a trend reversion towards the healthy control group. LEfSe analysis further revealed ( Figure 13 b), Spore@Ir 10-KB / Alg treatment significantly enriched various beneficial bacteria associated with anti-inflammation and gut homeostasis, while the DSS group was dominated by pro-inflammatory bacteria. α-Diversity analysis results ( Figure 13 c– Figure 13 f) showed that DSS significantly reduced the richness (Chao and OTUs) and diversity (Shannon and Simpson indices) of the gut microbiota, while Spore@Ir 10 -KB / Alg treatment significantly improved the above indicators, indicating that this system can effectively restore DSS-induced damage to gut microbiota diversity. Spore@Ir 10 -KB / Alg demonstrates significant comprehensive therapeutic advantages in a DSS-induced ulcerative colitis model by synergistically regulating intestinal immune inflammatory responses, repairing intestinal epithelial barrier function, and reshaping intestinal microbial homeostasis.

Claims

1. An oral composite microsphere for treating ulcerative colitis based on the regulation of the intestinal ROS storm pathological axis, characterized in that, Using Clostridium butyricum spores as a biological carrier, Ir was first loaded with Ir, which has ROS scavenging activity. 10 -KB nanozyme, which is then cross-linked and encapsulated with sodium alginate-calcium ions to form composite microspheres; The Ir 10 -KB nanozymes are complexes formed by loading Ir nanoparticles with Ketjen black oxide as a carrier, and they have peroxidase, superoxide dismutase and catalase-like activities. The method for preparing the oral composite microspheres includes the following steps: Ir 10 -KB nanozyme was dissolved in PBS buffer, Clostridium butyricum spore powder was added, and the mixture was gently stirred and incubated at room temperature to allow Ir to develop. 10 -KB nanozymes were loaded onto the spore surface, the precipitate was collected by centrifugation, and washed with PBS to obtain Spore@Ir 10 -KB complex; Dissolve sodium alginate in deionized water to prepare a sodium alginate aqueous solution, then add Spore@Ir 10 -KB complex was ultrasonically dispersed to form a homogeneous suspension; The suspension was injected into an aqueous calcium chloride solution and crosslinked at room temperature. After the reaction was completed, the product was collected by filtration, washed, and freeze-dried to obtain oral composite microspheres.

2. The oral composite microspheres for treating ulcerative colitis according to claim 1, characterized in that, The method for preparing Clostridium butyricum spores is as follows: Clostridium butyricum is inoculated into a Clostridium-enriched culture medium, anaerobic culture is carried out, the vegetative cells are inactivated by heat treatment, the spore precipitate is collected by centrifugation, washed with PBS, purified by sucrose solution density gradient centrifugation, and then freeze-dried for later use.

3. The oral composite microspheres for treating ulcerative colitis according to claim 2, characterized in that, The parameters for preparing Clostridium butyricum spores are as follows: anaerobic culture temperature is 35~39℃, culture time is 40~52h; heat treatment temperature is 75~85℃, heat treatment time is 8~12min; PBS washing times are 2~4 times; the sucrose solution density gradient consists of a lower layer of sucrose solution of 35%~45% and an upper layer of sucrose solution of 55%~65%.

4. A method for preparing oral composite microspheres for treating ulcerative colitis according to any one of claims 1-3, characterized in that, Includes the following steps: Ir 10 -KB nanozyme was dissolved in PBS buffer, Clostridium butyricum spore powder was added, and the mixture was gently stirred and incubated at room temperature to allow Ir to develop. 10 -KB nanozymes were loaded onto the spore surface, the precipitate was collected by centrifugation, and washed with PBS to obtain Spore@Ir 10 -KB complex; Dissolve sodium alginate in deionized water to prepare a sodium alginate aqueous solution, then add Spore@Ir 10 -KB complex was ultrasonically dispersed to form a homogeneous suspension; The suspension was injected into an aqueous calcium chloride solution and crosslinked at room temperature. After the reaction was completed, the product was collected by filtration, washed, and freeze-dried to obtain oral composite microspheres.

5. The method for preparing oral composite microspheres according to claim 4, characterized in that, Clostridium butyricum spore powder and Ir 10 The mass ratio of -KB nanozymes is (5–20):

1.

6. The use of the oral composite microspheres for treating ulcerative colitis according to any one of claims 1-3 in the preparation of a medicament for treating ulcerative colitis based on the regulation of the intestinal ROS storm pathological axis.

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