A drug-loaded intestinal adhesion composite gel microsphere, its preparation method and application

Abstract

This invention relates to an intestinal adhesion drug-loaded composite gel microsphere, its preparation method, and its application, belonging to the field of biomedical technology. The intestinal adhesion drug-loaded composite gel microsphere of this invention comprises calcium alginate gel microspheres, a protein drug encapsulated within the calcium alginate gel microspheres, and an antioxidant coated on the surface of the calcium alginate gel microspheres. Enteric-coated capsules containing these intestinal adhesion drug-loaded composite gel microspheres, when administered orally, can effectively and broadly scavenge ROS in the intestinal tract, regulate oxidative stress levels at sites of intestinal inflammation, alleviate inflammation, and reduce tissue damage, thereby achieving long-term regulation of the intestinal microenvironment to treat inflammatory bowel disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to an intestinal adhesion drug-loaded composite gel microsphere, its preparation method and application. Background Technology

[0002] Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a chronic systemic intestinal disease of unknown etiology, possibly related to a variety of factors such as genetics, environment, and autoimmunity. Symptoms of IBD include diarrhea, rectal bleeding, and weight loss, severely impacting patients' daily lives. Although certain anti-inflammatory drugs (such as aminosalicylic acid, antibiotics, glucocorticoids, and immunosuppressants) are used clinically to treat IBD, long-term use of these drugs can cause a range of side effects.

[0003] With the development and application of biopharmaceuticals, biologics such as proteins and peptides, which can selectively inhibit key mediators of the inflammatory process, have achieved significant efficacy in the treatment of IBD. For example, infliximab, which targets tumor necrosis factor (TNF)-α, was approved by the US FDA for the treatment of patients with refractory Crohn's disease, followed by the approval of adalimumab, golimumab, and sertuzumab. However, due to the molecular mechanisms of drug tolerance, not all patients benefit from these therapies. Among patients who initially respond to treatment, 30%-50% eventually lose efficacy during treatment, resulting in no durable clinical benefit while being exposed to potential side effects and toxicities. Furthermore, almost all of these protein drugs currently require injection, leading to discomfort, scarring, and local allergic reactions in patients. In contrast, oral administration offers advantages such as convenience and painlessness, improving patient compliance. Currently, a large amount of research is dedicated to oral protein delivery, mainly focusing on finding suitable protein carriers to effectively protect protein activity. Carriers such as liposomes and polyester polymers have been developed for protein delivery. However, liposomes exhibit poor stability in the gastrointestinal tract, and polyester polymers are easily degraded by gastric acid. Their degradation products may induce inflammation, limiting their application in IBD treatment. Therefore, there is an urgent need to develop new treatment methods to provide effective IBD treatment while avoiding systemic side effects, ideally through oral administration.

[0004] Reactive oxygen species (ROS) are byproducts of mitochondrial oxidative metabolism and energy production, including superoxide anions (·O2). -Chemically reactive substances such as hydrogen peroxide (H2O2) and hydroxyl radicals (HO·) are present in the colon. Excessive ROS production in the colon is a key characteristic and pathogenic factor of IBD. A sharp increase in ROS triggers high oxidative stress in cells, damages the colonic epithelial barrier, and disrupts the relationship between epithelial cells and the gut microbiota. Therefore, clearing excess ROS in the colon and modulating oxidative stress has become a feasible strategy for IBD treatment. In recent years, many studies have reported the application of metal nanozymes and inorganic reducing nanomaterials as ROS scavengers in IBD treatment. However, the potential biotoxicity of many metal nanozymes and nanomaterials limits their further application. In contrast, natural enzymes have excellent biocompatibility and can efficiently clear ROS, thus showing potential in alleviating inflammation and tissue damage. However, proteins are very fragile and unstable in the variable pH environment of the gastrointestinal tract and are easily degraded by proteases, leading to a significant reduction in the bioavailability of orally administered natural enzyme drugs, posing a challenge to oral protein delivery.

[0005] Therefore, there is an urgent need to develop oral protein delivery alternatives that can safely deliver proteins to the intestine and maintain their catalytic activity over a long period of time to provide long-term treatment for IBD and minimize systemic side effects. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an intestinal adhesion drug-loaded composite gel microsphere, its preparation method, and its application. A calcium alginate gel microsphere microreactor (protein drug@calcium alginate microspheres) loaded with protein drugs was synthesized via ion crosslinking. The biocompatibility, biodegradability, and dense hydrogel network of the calcium alginate gel microspheres encapsulate the protein drug with highly efficient catalytic activity, achieving long-term protection of the protein drug's activity in the protease-rich intestinal region. Then, to prolong the microsphere's retention time in the intestine, the protein drug@calcium alginate microspheres were modified with a bioadhesive antioxidant shell, forming an intestinal adhesion drug-loaded composite gel microsphere microreactor (protein drug@calcium alginate microspheres-antioxidant). Simultaneously, the antioxidant possesses broad-spectrum ROS scavenging ability, overcoming the limitation of protein drugs only scavenging H2O2. The synergistic effect of the protein drug and antioxidant broadly scavenges reactive oxygen species (ROS), regulating the redox balance at the inflammatory sites of inflammatory bowel disease (IBD), and achieving long-term regulation of the intestinal microenvironment for the treatment of IBD. Finally, the intestinal adhesion drug-loaded composite gel microspheres were freeze-dried and encapsulated in enteric-coated capsules for targeted delivery to the intestinal region, thereby avoiding the influence of the extreme pH environment of the stomach on protein activity and providing an oral protein delivery strategy.

[0007] The first objective of this invention is to provide an intestinal adhesion drug-loaded composite gel microsphere, comprising calcium alginate gel microspheres, a protein drug encapsulated within the calcium alginate gel microspheres, and an antioxidant coated on the surface of the calcium alginate gel microspheres.

[0008] In one embodiment of the present invention, the protein drug is selected from catalase and / or superoxide dismutase.

[0009] In one embodiment of the present invention, the antioxidant is polydopamine.

[0010] In one embodiment of the present invention, the mass ratio of the protein drug, calcium alginate gel microspheres, and antioxidant is (1-50):50:(0.5-6). This antioxidant condition ensures that the calcium alginate gel microspheres retain their high activity while being adequately modified, thereby enhancing their broad-spectrum ROS scavenging ability and bioadhesion properties.

[0011] A second objective of this invention is to provide a method for preparing the aforementioned intestinal adhesion drug-loaded composite gel microspheres, using an electrospray device as the generating device, the electrospray device comprising an injection pump, a high-voltage power supply, and a collector; the preparation method includes the following steps:

[0012] S1. The protein drug solution and calcium chloride solution are respectively loaded into the syringe pump and the collector. The high-voltage power supply is turned on to inject the protein drug solution into the calcium chloride solution to obtain a protein drug@calcium alginate microsphere solution. The protein drug solution is obtained by dissolving the protein drug in sodium alginate solution.

[0013] S2. Add an antioxidant solution to the protein drug@calcium alginate microsphere solution described in S1, adjust the pH to 7.5-8.5, and react to obtain intestinal adhesion drug-loaded composite gel microspheres.

[0014] In one embodiment of the present invention, in S1, the mass fraction of the protein drug in the protein drug solution is 0.1%-1%; the mass fraction of the sodium alginate in the sodium alginate solution is 1%-5%.

[0015] The calcium chloride solution contains 1%-5% calcium chloride by mass.

[0016] The volume ratio of the protein drug solution to the calcium chloride solution is (0.1-0.4):1.

[0017] In one embodiment of the present invention, in S1, during the injection process, the injection rate of the protein drug solution is 20 μL / min-200 μL / min, the voltage of the high-voltage power supply is 6V-14V, and the size of the needle-shaped emitter of the injection pump is selected from 28G-34G.

[0018] In one embodiment of the present invention, in S2, the final concentration of the antioxidant is 0.1 mg / mL to 1.2 mg / mL.

[0019] In one embodiment of the present invention, after S2, the step of freeze-drying the intestinal adhesion drug-loaded composite gel microsphere suspension is further included, specifically including: adding a freeze-drying protectant solution to the intestinal adhesion drug-loaded composite gel microsphere suspension for freeze-drying.

[0020] In one embodiment of the present invention, the freeze-drying protectant is selected from one or more of mannitol, sucrose and trehalose; the final concentration of the freeze-drying protectant is 2 mg / mL-10 mg / mL.

[0021] A third objective of this invention is to provide a capsule formulation prepared from the aforementioned intestinal adhesion drug-loaded composite gel microspheres. After oral administration, this capsule formulation can effectively and broadly scavenge ROS in the intestinal tract, regulate oxidative stress levels at sites of intestinal inflammation, alleviate inflammation, and reduce tissue damage, thereby achieving long-term regulation of the intestinal microenvironment for the treatment of inflammatory bowel disease.

[0022] A fourth objective of this invention is to provide the application of the aforementioned capsule formulation in the preparation of a drug for treating inflammatory bowel disease (IBD). An enteric-coated capsule formulation loaded with a protein drug@calcium alginate microspheres-antioxidant is administered orally. After the drug passes through the extreme pH environment of the stomach, the enteric-coated capsule dissolves under specific pH conditions in the colon, releasing the protein drug@calcium alginate microspheres-antioxidant. The latter achieves long-term retention in the colon due to the bioadhesiveness of the antioxidant shell. At this point, the protein drug encapsulated in the microspheres and the antioxidant shell synergistically achieve broad-spectrum scavenging of ROS, thereby regulating the oxidative stress level at the site of inflammation, alleviating intestinal inflammation, and reducing tissue damage. This achieves long-term regulation of the intestinal microenvironment to treat IBD without side effects. Therefore, the therapeutic efficacy and safety of the oral protein drug highlight its potential application in IBD treatment.

[0023] The technical solution of the present invention has the following advantages compared with the prior art:

[0024] (1) The intestinal adhesion drug-loaded composite gel microspheres described in this invention can achieve long-term protection of protein drug activity. Large-sized protein drug molecules cannot diffuse through the dense gel network structure of calcium alginate microspheres and are therefore effectively retained in sodium alginate gel microspheres, while low-molecular-weight H2O2 molecules can freely pass through the calcium alginate gel microsphere network, thereby constructing a closed H2O2 reactor. At the same time, the dense network structure of sodium alginate gel microspheres can also prevent the penetration of molecules such as proteases, protecting protein drugs from protease digestion.

[0025] (2) The intestinal adhesion drug-loaded composite gel microspheres of the present invention achieve broad-spectrum ROS scavenging through the combined action of protein drugs and antioxidants. Protein drugs can efficiently catalyze the decomposition of H2O2, but have virtually no scavenging ability for other ROS species. Antioxidants, on the other hand, can interact with a variety of ROS using their abundant reducing hydroxyl groups. To achieve a more effective ROS scavenging effect, protein drugs and antioxidants are used in combination to achieve broad-spectrum ROS scavenging.

[0026] (3) To prevent the protein from being inactivated by the variable pH environment of the gastrointestinal tract or by protease hydrolysis, the capsule formulation of this invention uses sodium alginate gel microspheres with a dense molecular network structure to encapsulate the protein drug, thereby ensuring that the protein activity remains unaffected for a longer period of time. Furthermore, to compensate for the limitation of protein drugs only being able to scavenge one type of ROS (H2O2) and to improve the bioadhesion ability of the microspheres to intestinal tissue, an antioxidant layer is coated on the surface of the protein drug@calcium alginate microspheres. Finally, to prevent the drug from being inactivated by the extreme pH of the stomach during administration, the enteric-adhesive drug-loaded composite gel microspheres are lyophilized and then encapsulated in enteric-coated capsules. The enteric-coated capsules do not degrade under the pH conditions of the stomach but degrade under the pH conditions of the intestine, thus protecting the drug from the highly acidic environment of the stomach. This effectively improves the bioavailability of oral protein administration and provides a novel and effective strategy for oral protein delivery. Attached Figure Description

[0027] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0028] Figure 1 Macroscopic and SEM images of CAT@ALG freeze-dried microspheres and CAT@ALG-PDA freeze-dried microspheres in Test Example 1 of this invention;

[0029] Figure 2 This is the encapsulation of catalase by CAT@ALG lyophilized microspheres in Test Example 2 of the present invention; where a is the encapsulation efficiency of catalase by CAT@ALG, b is the release curve of catalase in CAT@ALG within 48h, and c is the relative enzyme activity of CAT@ALG.

[0030] Figure 3 This refers to the change in relative enzyme activity of CAT@ALG-PDA during the PDA oxidative polymerization deposition process under different concentrations of dopamine in Test Example 3 of this invention.

[0031] Figure 4 The relative enzyme activities of free catalase and CAT@ALG-PDA after proteinase K digestion at different time points are shown in Test Example 3 of this invention.

[0032] Figure 5 In Test Example 4 of this invention, ALG, CAT@ALG, ALG-PDA or CAT@ALG-PDA (1 mg / mL) reacted with H2O2 (a), DPPH· free radical (b), and ABTS. +· The UV-Vis absorption spectra of the system after the reaction of free radical (c) and HO· free radical (d) and the corresponding free radical scavenging efficiency;

[0033] Figure 6 This is a graph showing the changes in cell viability of RAW 264.7 cells and HT-29 cells after incubation with different concentrations of CAT@ALG-PDA in Test Example 5 of this invention.

[0034] Figure 7 This invention demonstrates the cell-protective effect of CAT@ALG-PDA against H2O2-induced oxidative stress in Test Example 6.

[0035] Figure 8 The corresponding flow cytometry analysis of RAW 264.7 cells and HT29 cells in Test Example 7 of this invention after co-incubation with CAT@ALG, ALG-PDA or CAT@ALG-PDA (250 μg / mL) under 2mM H2O2 oxidative stress for 24 h;

[0036] Figure 9 The images show the intracellular ROS (DCF) fluorescence and relative DCF fluorescence intensity of RAW264.7 cells (a) and HT29 cells (b) in Test Example 7 of this invention after co-incubation with CAT@ALG, ALG-PDA, or CAT@ALG-PDA (250 μg / mL) under 2mM H2O2 oxidative stress for 24 h.

[0037] Figure 10 The corresponding flow cytometry analysis of RAW 264.7 cells co-incubated with different materials (250 μg / mL) and 1 μg / mL LPS for 8 h in Test Example 8 of this invention;

[0038] Figure 11 The images show the intracellular ROS (DCF) fluorescence and relative DCF fluorescence intensity of RAW 264.7 cells after co-incubation with different materials (250 μg / mL) and 1 μg / mL LPS for 8 h in Test Example 8 of this invention.

[0039] Figure 12 To test the levels of different types of ROS / RNS in RAW 264.7 cells after co-incubation with different materials (250 μg / mL) and 1 μg / mL LPS for 8 h in Example 9 of this invention, including hydrogen peroxide (H2O2) (a) and superoxide anion (O2) levels, the following tests were performed. -· Detection results of (b) and nitric oxide radicals (NO·)(c)

[0040] Figure 13 This study investigated the adhesion properties of CAT@ALG-PDA microspheres to the intestine in Test Example 10 of this invention. Specifically, (a) the distal colon of C57BL / 6 mice was incubated with free CAT, CAT@ALG, or CAT@ALG-PDA (catalase labeled with Cy7) at 37°C for 12 hours in vitro and then washed. Fluorescence signals were subsequently measured using an IVIS imaging system. (b) DSS-induced colitis SD rats were orally administered free CAT capsules, CAT@ALG capsules, or CAT@ALG-PDA capsules (catalase labeled with Cy7). The animals were sacrificed after 12 hours, and fluorescence signals in the distal colon were measured.

[0041] Figure 14 This is the CAT@ALG-PDA treatment experiment for DSS-induced IBD in Test Example 11 of the present invention; where a is the change in body weight of rats in each group during the experiment, b is the change in fecal bleeding index of rats in each group during the experiment, c is the change in fecal consistency index of rats in each group during the experiment, d is the change in disease activity index (DAI) value during the experiment, e is the colon length of colon tissue taken from rats in each group, and f is the spleen weight / body weight ratio of rats in each group.

[0042] Figure 15 The macroscopic appearance of the colon in each group in Test Example 11 of this invention;

[0043] Figure 16 These are DCF-DA stained confocal images of colon tissue sections from each group in Test Example 12 of this invention;

[0044] Figure 17 This is an ELISA analysis of inflammatory markers in the colon tissue of rats in each group during the DSS-induced IBD treatment experiment of CAT@ALG-PDA in Test Example 13 of this invention; where a is TNF-α, b is IFN-γ, c is IL-1β, d is IL-6, e is IL-12, and f is MPO.

[0045] Figure 18 These are H&E stained microscopic images of longitudinal sections of rat colon tissue from each group in the DSS-induced IBD treatment experiment using CAT@ALG-PDA in Test Example 14 of this invention.

[0046] Figure 19This is the test example 15 of the present invention, which is the treatment experiment of CAT@ALG-PDA and clinically approved drugs on DSS-induced IBD; where a is the change in body weight of rats in each group during the experiment, b is the change in fecal bleeding index of rats in each group during the experiment, c is the change in fecal consistency index of rats in each group during the experiment, d is the change in disease activity index (DAI) value during the experiment, e is the colon length of colon tissue taken from rats in each group, and f is the spleen weight / body weight ratio of rats in each group.

[0047] Figure 20 The macroscopic appearance of the colon in each group in Test Example 15 of this invention;

[0048] Figure 21 The content of hydrogen peroxide (a), superoxide anion free radical (b), and nitric oxide free radical (c) in the colon tissue of different groups of rats in Test Example 16 of this invention;

[0049] Figure 22 M1 macrophages (CD11b) in the colon of different groups of rats in Example 17 of this invention. + CD68 + CD80 + Representative flow cytometry analysis results;

[0050] Figure 23 This is an ELISA analysis of inflammatory markers in the colon tissue of rats in each group during the DSS-induced IBD treatment experiment of CAT@ALG-PDA and clinically approved drugs in Test Example 17 of this invention; where a is TNF-α, b is IL-1β, c is IL-12, d is IFN-γ, e is IL-6, and f is MPO.

[0051] Figure 24 These are H&E stained microscopic images of major organs of rats under different treatment days in Test Example 18 of this invention;

[0052] Figure 25 The results of blood biochemical analysis of rats treated with CAT@ALG-PDA in Test Example 18 of this invention are shown below; where a is alanine aminotransferase (ALT), b is urea, c is creatinine (CREA), d is alkaline phosphatase (ALP), e is aspartate aminotransferase (AST), f is red blood cell count (RBC), g is hemoglobin (HGB), h is hematocrit (HCT), i is mean corpuscular volume (MCV), j is mean corpuscular hemoglobin (MCH), k is mean corpuscular hemoglobin concentration (MCHC), and l is mean platelet volume (MPV). Detailed Implementation

[0053] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0054] In this invention, unless otherwise stated, the generating device used in the embodiments includes a high-precision injection pump, a high-voltage power supply, and a collector. The needle-shaped emitter of the injection pump is 34G in size and is placed at a constant height of 10cm above the target collector on the ground. The high-voltage power supply is connected to the emitter and the collector. The collector is a 100mm circular culture dish.

[0055] In this invention, unless otherwise stated, the preparation of the catalase solution used in the embodiments includes the following steps: dissolving catalase in a 2% sodium alginate solution to form a 1% catalase solution.

[0056] In this invention, unless otherwise stated, the calcium chloride solution used in the examples has a mass fraction of 2%.

[0057] Example 1

[0058] The CAT@ALG-PDA and its preparation method in this embodiment specifically include the following steps:

[0059] S1. 6 mL of catalase (CAT) solution and 50 mL of calcium chloride solution were respectively loaded into a high-precision syringe pump and a collector. The voltage of the high-voltage power supply was set to 12V. The catalase solution was injected into the calcium chloride solution at a flow rate of 100 μL / min using the high-precision syringe pump to obtain CAT@ALG microsphere solution.

[0060] S2. After electrospraying is completed, collect CAT@ALG microspheres and wash them twice with deionized water; add dopamine hydrochloride solution (final dopamine concentration is 0.2 mg / mL) to the supernatant, adjust the pH to ≈ 8 with sodium hydroxide solution, shake on a shaker for 6 h, and wash twice with deionized water to obtain CAT@ALG-PDA microsphere suspension.

[0061] S3. Add a 10% mannitol solution (final mannitol concentration of 6 mg / mL) to the CAT@ALG-PDA microsphere suspension, and then freeze-dry to obtain CAT@ALG-PDA lyophilized microspheres.

[0062] Test Example 1

[0063] Based on Example 1, the morphology of CAT@ALG lyophilized microspheres and CAT@ALG-PDA lyophilized microspheres was observed under a field emission scanning electron microscope (Zeiss G500). The results are as follows: Figure 1 As shown. From Figure 1 It can be seen that CAT@ALG lyophilized microspheres and CAT@ALG-PDA lyophilized microspheres have uniform morphology and low size dispersion (approximately 70 μm).

[0064] Test Example 2

[0065] (1) Investigation of CAT@ALG encapsulation and release of CAT: FITC-CAT@ALG was prepared according to the method in Example 1, except that CAT in S1 was replaced with FITC-CAT labeled with the fluorescent dye FITC. The fluorescence value of the supernatant was then measured using the standard curve method to obtain the concentration of FITC-CAT in the supernatant, which was used to calculate the encapsulation efficiency of CAT@ALG on CAT. CAT@ALG was washed with deionized water until no fluorescence signal was detected in the supernatant. Fluorescence signals were measured in the supernatant at predetermined time points (1h, 2h, 4h, 8h, 12h, 24h, 48h) to obtain the CAT release curve. The results are as follows: Figure 2 a- Figure 2 As shown in b. From Figure 2 As can be seen from this, the encapsulation efficiency of CAT in CAT@ALG is approximately 82.4%, indicating that CAT is effectively encapsulated in calcium alginate hydrogel microspheres through a rapid ionogel process. Figure 2 As can be seen from b, the amount of CAT released from CAT@ALG within 48 hours is negligible, indicating that the macromolecule CAT is effectively retained in the dense network structure of the hydrogel microspheres and cannot diffuse out.

[0066] (2) Investigation of the relative enzyme activity of CAT@ALG: 1 mL of hydrogen peroxide (50 mM) was mixed with 0.1 mL of free CAT or CAT@ALG (1 mg / mL, expressed as CAT mass) at 37 °C for 1 min, then 1 mL of ammonium molybdate (32.4 mM) was added and the mixture was cooled to 25 °C. The catalase activity was determined by measuring the absorbance of the resulting complex at 400 nm. The results are as follows: Figure 2 As shown in c. From Figure 2 c shows that the relative enzyme activity of CAT encapsulated in CAT@ALG remains at 96.7%, indicating that the low molecular weight substrate H2O2 can freely pass through the hydrogel network of the ALG hydrogel shell and react with the internally immobilized CAT, thereby constructing a closed H2O2 reactor.

[0067] Test Example 3

[0068] (1) Investigating the effect of CAT@ALG surface-modified PDA on catalase activity: CAT@ALG was co-incubated with dopamine solutions of different final concentrations (0.1 mg / mL, 0.2 mg / mL, 0.6 mg / mL, 1.2 mg / mL, 1.8 mg / mL). After incubation for 6 h, the catalase activity of CAT@ALG-PDA prepared under different conditions was measured. The results are as follows: Figure 3 As shown. From Figure 3 It can be seen that a dopamine solution concentration of 0.2 mg / mL can ensure that the microspheres maintain a high enzyme activity (91.72%).

[0069] (2) Investigating the protective effect of CAT@ALG-PDA on catalase activity: Free CAT or CAT@ALG (1 mg / mL, based on CAT mass) was incubated with proteinase K at 37℃, with a final proteinase K concentration of 0.4 mg / mL; samples were taken at predetermined time points (10 min, 20 min, 40 min, 60 min, 120 min) and catalase activity was immediately measured. The results are as follows: Figure 4 As shown. From Figure 4 It can be seen that free CAT is rapidly degraded under the action of proteinase K (0.4 mg / mL), with its activity decreasing to 14.35%, while CAT encapsulated in CAT@ALG-PDA retains approximately 95% of its initial enzyme activity after 2 hours of incubation with proteinase K. This is attributed to the dense structure of the microspheres preventing the penetration of proteinase K, thereby protecting CAT from protease degradation. This indicates that CAT encapsulated in CAT@ALG-PDA is well protected, and this structural design is crucial for the long-term catalytic activity of catalase at the site of IBD inflammation.

[0070] Test Example 4

[0071] To investigate the broad-spectrum scavenging ability of CAT@ALG-PDA against ROS, the effects of CAT@ALG-PDA on ABTS were studied. +· The abilities of the three free radicals DPPH· and OH· are detailed in the following scheme:

[0072] H2O2 scavenging assay: 0.5 mL of hydrogen peroxide (50 mM) was incubated with different concentrations of ALG, ALG-PDA, CAT@ALG, or CAT@ALG-PDA (0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL) at 37 °C for 1 min; then 0.5 mL of ammonium molybdate solution (32.4 mM) was added, and the mixture was cooled to 25 °C; the hydrogen peroxide content was quantified by measuring the absorbance of the resulting yellow complex at 400 nm, and the hydrogen peroxide scavenging rate was calculated. The results are as follows: Figure 5 As shown in a. From Figure 5As can be seen, the hydrogen peroxide content decreased significantly after incubation with CAT@ALG or CAT@ALG-PDA, while no such change was observed after incubation with ALG or ALG-PDA. This indicates that CAT effectively removes hydrogen peroxide.

[0073] ABTS +· Free radical scavenging assay: First, ABTS (7 mM) and potassium persulfate (K2S2O8, 2.45 mM) were mixed and reacted overnight at 4°C to prepare ABTS. +· Free radicals; subsequently, different concentrations (0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL) of ALG, ALG-PDA, CAT@ALG, and CAT@ALG-PDA were mixed with ABTS. +· After incubation at 0.3 mM for 10 min, the absorbance at 734 nm was measured using a UV-Vis spectrophotometer to quantify ABTS. +· ABTS can be calculated based on the free radical content. +· Free radical scavenging rate, results as follows Figure 5 As shown in b.

[0074] DPPH· free radical scavenging assay: Different concentrations of ALG, ALG-PDA, CAT@ALG, and CAT@ALG-PDA (0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL) were mixed with DPPH· (60 μM) in ethanol for 5 min. The absorbance at 517 nm was measured using a UV-Vis spectrophotometer to quantify the DPPH· free radical content and calculate the DPPH· free radical scavenging rate. The results are as follows: Figure 5 As shown in c. From Figure 5 b- Figure 5 c shows that ABTS +· The DPPH· content decreased significantly after mixing with CAT@ALG-PDA (1 mg / mL). In contrast, the free radical content in the solution after incubation with CAT@ALG did not change significantly, indicating that PDA modification significantly enhanced the ROS scavenging ability of CAT@ALG.

[0075] TMB determination: The hydroxyl radicals (HO·) generated by the Fenton reaction can oxidize colorless 3,3',5,5'-tetramethylbenzidine (TMB) to the blue compound oxTMB. Therefore, oxTMB can serve as an indicator of HO· scavenging effect of CAT@ALG-PDA. Fenton's reagent (Fe... 2+The mixture of oxTMB (10 μM; H2O2: 50 μM) and TMB (0.3 mM) was incubated for 10 min with ALG, ALG-PDA, CAT@ALG, and CAT@ALG-PDA (0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL, respectively). The absorbance of oxTMB at 654 nm was monitored using a UV-Vis spectrophotometer to quantify the HO· free radical content and calculate the HO· free radical scavenging rate. The results are as follows: Figure 5 As shown in d. From Figure 5 As can be seen from d, the content of HO· free radicals in the reaction system decreased rapidly after the addition of CAT@ALG-PDA, indicating that CAT@ALG-PDA has excellent broad-spectrum ROS scavenging ability.

[0076] Test Example 5

[0077] Investigating the biocompatibility of CAT@ALG-PDA: HT29 cells and RAW264.7 cells were seeded in 96-well plates overnight (1×10⁻⁶ cells / well). 4 Cells were incubated overnight until they adhered to the culture medium. The medium was then replaced, and different concentrations (15 μg / mL, 30 μg / mL, 60 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL) of ALG-PDA, CAT@ALG, or CAT@ALG-PDA were added to each well. After 24 hours of incubation, relative cell viability was determined using the standard MTT assay. The results are shown below. Figure 6 As shown. From Figure 6 It can be seen that after co-culturing with CAT@ALG-PDA for 24 h, HT29 and RAW 264.7 cells maintained high cell viability even at a high concentration (1000 μg / mL). This indicates that CAT@ALG-PDA has excellent biocompatibility and is suitable for oral drug delivery in therapeutic applications.

[0078] Test Example 6

[0079] Investigating the protective effect of CAT@ALG-PDA on cells under high oxidative stress (in vitro anti-inflammatory effect): HT29 cells and RAW264.7 cells were seeded into 96-well plates (1×10⁻⁶ cells / well). 4 Cells were incubated overnight. After cell attachment, they were incubated with ALG-PDA, CAT@ALG, and CAT@ALG-PDA (250 μg / mL) in an oxidizing environment (3 mM H2O2) for 24 h, respectively. The protective effects of these materials were evaluated using the standard MTT assay, and the results are as follows: Figure 7 As shown. From Figure 7It can be seen that H2O2 treatment accelerated cell death. However, CAT@ALG-PDA significantly enhanced cell viability, demonstrating superior cell protection compared to CAT@ALG and ALG-PDA under H2O2-induced oxidative stress.

[0080] Test Example 7

[0081] Further investigation into the in vitro ROS scavenging ability of CAT@ALG-PDA: HT29 and RAW264.7 cells were seeded in 12-well plates (1×10⁻⁶ cells / well). 5 Cells were incubated with ALG-PDA, CAT@ALG, or CAT@ALG-PDA (250 μg / mL) in an oxidizing environment (2 mM H2O2) at 37°C for 24 h, followed by staining with the fluorescent dye 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA, 2 μM, 30 min). Intracellular ROS levels were detected by flow cytometry, and the results are as follows: Figure 8 As shown; and cell fluorescence was imaged using a confocal microscope (Zeiss Axio-Imager LSM-800), the results are as follows. Figure 9 As shown. From Figures 8-9 As can be seen, compared with the control group, the H2O2 treatment group showed a strong green fluorescence signal due to the increased intracellular oxidative stress level. The ALG-PDA and CAT@ALG treatment groups still showed some green fluorescence signal, while the CAT@ALG-PDA treatment group significantly reduced H2O2-induced green fluorescence, indicating that CAT@ALG-PDA significantly reduced intracellular ROS levels under high oxidative stress conditions. This suggests that the synergistic effect of the PDA shell and CAT significantly alleviated oxidative stress in ROS scavenging and inhibited the inflammatory response.

[0082] Test Example 8

[0083] Investigating the intracellular ROS clearance efficiency of CAT@ALG-PDA in LPS-stimulated activated inflammatory macrophages: RAW264.7 cells were seeded into 12-well plates (1×10⁻⁶ cells per well). 5 (1 cell); after 24 h, the cells were incubated with different materials and LPS (1 μg / mL) at 37 °C for 8 h, and then stained with DCFH-DA (2 μM, 30 min); the intracellular ROS level was detected by flow cytometry, and the results are as follows. Figure 10 As shown; and cell fluorescence was imaged using a confocal microscope (Zeiss Axio-Imager LSM-800), the results are as follows. Figure 11 As shown. From Figures 10-11As can be seen, the fluorescence signal in LPS-treated RAW264.7 cells was very strong. In contrast, the fluorescence intensity of the CAT@ALG and ALG-PDA treatment groups was significantly reduced, while almost no fluorescence signal was observed in the CAT@ALG-PDA treatment group. Therefore, CAT@ALG-PDA showed the most effective ability to alleviate LPS-induced oxidative stress in RAW264.7 cells.

[0084] Test Example 9

[0085] To further evaluate the ROS scavenging effect of CAT@ALG-PDA in activated macrophages, RAW 264.7 cells were used at a concentration of 1 × 10⁶ cells per well. 6 Cells were seeded at a density in 6-well plates; after 24 h, cells were incubated at 37 °C for 8 h with ALG-PDA, CAT@ALG or CAT@ALG-PDA (250 μg / mL), and LPS (1 μg / mL), respectively; the levels of hydrogen peroxide, superoxide anion, and nitric oxide free radicals were measured using a commercially available assay kit, and the results are as follows: Figure 12 As shown. From Figure 12 It can be seen that CAT@ALG and CAT@ALG-PDA significantly reduced intracellular hydrogen peroxide ( Figure 12 a) levels, while ALG-PDA and CAT@ALG-PDA significantly reduced intracellular superoxide anion (a .... Figure 12 b) and nitric oxide ( Figure 12 c) Level. This indicates that the combination of CAT and PDA modifications gives CAT@ALG-PDA a broad-spectrum ROS scavenging capability.

[0086] Test Case 10

[0087] In vitro adhesion assay: Six-week-old male C57BL / 6 mice were sacrificed, and the distal 1.5 cm of colonic tissue (excluding the anus) was dissected; the colonic tissue was incubated with Cy7-CAT, Cy7-CAT@ALG, or Cy7@ALG-PDA solution (1 mg / mL) at 37°C for 12 h; after washing with PBS (1×), the colon was opened longitudinally, and imaging was performed using a small animal fluorescence imaging system with the lumen facing upwards. The results are as follows. Figure 13 As shown in a. From Figure 13 As can be seen, compared with Cy7-CAT@ALG or free Cy7-CAT, the colon tissue incubated with Cy7-CAT@ALG-PDA has a significantly higher fluorescence signal, indicating that PDA modification enhances the bioadhesion ability of the hydrogel microreactor to intestinal tissue.

[0088] In vivo adhesion assay: Eight-week-old male SD rats were randomly divided into Cy7-CAT, Cy7-CAT@ALG, and Cy7-CAT@ALG-PDA groups after one week of acclimatization. They were given free access to drinking water containing 4% DSS to induce acute IBD. Rats with DSS-induced acute IBD were fasted overnight. The next morning, each rat was orally administered the corresponding preparation (Cy7-CAT@ALG-PDA, Cy7-CAT@ALG, or free Cy7-CAT, 10 mg per capsule). After 24 hours, the animals were sacrificed, and the distal 3 cm of the colon was excised. Imaging was performed immediately without washing. The fluorescence signal intensity was quantitatively measured within a standard-sized region of interest (ROI) around a single colon slice using a small animal fluorescence imaging system. The background fluorescence intensity was subtracted from all specimens; the background fluorescence intensity was the average of three ROIs that did not contain any colonic tissue. The results are as follows: Figure 13 As shown in b. From Figure 13 As shown in b, the colonic fluorescence signal in IBD model rats treated with Cy7-CAT@ALG-PDA was significantly stronger than that in the Cy7-CAT@ALG or free Cy7-CAT treatment groups. This indicates that CAT@ALG-PDA has excellent bioadhesive properties, enhancing its retention in the colon and thus prolonging its duration of action at the site of inflammation.

[0089] Test Example 11

[0090] In vivo treatment study of DSS-induced IBD in rats: Eight-week-old male SD rats were randomly divided into five groups after one week of acclimatization: healthy group, DSS treatment group, CAT treatment group, CAT@ALG treatment group, and CAT@ALG-PDA treatment group. Starting from day 0, except for the healthy group, rats in the other groups had free access to drinking water containing 4% DSS for 7 consecutive days, and clinical symptoms of IBD, such as watery diarrhea, began to appear on day 1. On days 1, 3, and 5, rats in each group were administered free CAT, CAT@ALG, and CAT@ALG-PDA by gavage, respectively, with each capsule containing 10 mg of the drug. During the experiment, rat body weight, fecal consistency, and fecal occult blood were measured and recorded daily. The DAI scoring criteria were as follows: fecal consistency (hard for 0, soft for 2, diarrhea for 4), fecal occult blood (Pyramidon method, scored based on color changes, ranging from 0-4), and weight loss (less than 1% for 0, 1%-5% for 1, 5%-10% for 2, 10%-20% for 3, and more than 20% for 4). On day 6, all rats were sacrificed, colon length was measured, and the colon was gently washed with PBS. The distal colon was used for histological analysis, and the remaining tissue was stored at -80°C for future studies. Results are as follows: Figures 14-15 As shown. From Figure 14As can be seen, the healthy rats continued to gain weight, while the untreated rats lost approximately 10% of their weight by day 6, indicating that the IBD model was successfully established. The CAT-treated group showed significant weight loss, while the CAT@ALG-treated group experienced some relief from weight loss. In contrast, the CAT@ALG-PDA-treated group significantly reduced IBD-induced weight loss. Figure 14 a). The severity of inflammation, including weight loss, rectal bleeding ( Figure 14 b) and stool consistency ( Figure 14 c) Further assessment using the Disease Activity Index (DAI) score. Figure 14 d); The DAI scores of the untreated group, CAT-treated group, and CAT@ALG-treated group were significantly higher than those of the healthy group, and varying degrees of occult blood and watery diarrhea were observed. Conversely, the DAI score of the CAT@ALG-PDA-treated group was significantly lower than that of the untreated group, showing milder symptoms of occult blood and watery diarrhea. Further evaluation of the therapeutic effect of CAT@ALG-PDA showed that untreated rats exhibited colonic edema and a significant shortening of colon length ( Figure 14 e), spleen weight increases ( Figure 14 f). From Figure 15 It can be seen that the colon length of rats in the CAT@ALG-PDA treatment group was similar to that in the healthy group, colonic edema was alleviated, and spleen weight was lower. In conclusion, oral administration of CAT@ALG-PDA can enhance the therapeutic effect of DSS-induced acute IBD.

[0091] Test Example 12

[0092] Elimination of ROS in intestinal inflammatory sites by CAT@ALG-PDA: DCFH-DA staining of colon tissue sections was performed to assess ROS levels in the colon after various treatments, and the results are as follows: Figure 16 As shown. From Figure 16 It can be seen that the colon of DSS-induced acute IBD rats showed strong DCF fluorescence, while after CAT@ALG-PDA treatment, the DCF fluorescence decreased to normal levels.

[0093] Test Example 13

[0094] The preserved tissue was weighed to quantify pro-inflammatory cytokines and peroxidases in the tissue; the tissue was homogenized in PBS (1×) buffer; then centrifuged at 10,000 rpm for 10 min at 4 °C to obtain the homogenized product; a commercially available rat TNF-α, IFN-γ, IL-1β, IL-6, IL-12 and MPO ELISA kit could quantify the pro-inflammatory cytokines and peroxidases in the homogenized product, and the results are as follows. Figure 17 As shown. From Figure 17It can be seen that, compared with the untreated group, oral administration of CAT@ALG-PDA significantly reduced the levels of TNF-α, IFN-γ, IL-6, and IL-12, indicating effective relief of inflammation. However, oral administration of free CAT or CAT@ALG failed to effectively reduce the levels of pro-inflammatory cytokines in the colon, highlighting the importance of CAT@ALG-PDA's long-term catalytic activity and broad-spectrum ROS clearance capacity in IBD treatment. Furthermore, the upregulation of pro-inflammatory cytokines such as TNF-α and IL-6 promoted the recruitment of neutrophils from blood vessels and activated the NADPH oxidase pathway, leading to an imbalance in the redox environment. Neutrophil-expressed myeloperoxidase (MPO) catalyzes the production of active substances and hypochlorous acid in inflamed tissues and plays a role in the regulation of IBD. MPO can serve as a biomarker for assessing the disease status of IBD patients. The MPO levels in colonic tissues of different groups of rats were detected by ELISA. Figure 17 f). MPO levels were significantly elevated in the untreated group, while oral CAT@ALG-PDA significantly alleviated this effect, reducing MPO levels to levels comparable to the healthy group, indicating that CAT@ALG-PDA treatment effectively inhibited neutrophil infiltration in the inflamed colon. In conclusion, oral CAT@ALG-PDA demonstrates potent anti-inflammatory activity in DSS-induced acute IBD.

[0095] Test Example 14

[0096] Prior to histological analysis, distal colon tissue was gently washed with PBS and fixed in 10% formalin solution; the fixed tissue was stained with hematoxylin and eosin, and the images were observed using an optical microscope (Leica, DM4000). The results are as follows: Figure 18 As shown. From Figure 18 As can be seen, compared with the healthy group, the untreated group showed severe damage to the colonic structure, with irregular morphology, reduced villus height, damaged crypts, mucosal epithelial damage, and extensive inflammatory cell infiltration. The oral CAT@ALG treatment group restored villus height, but crypt damage and inflammatory cell infiltration remained. Notably, the oral CAT@ALG-PDA treatment group exhibited intact colonic structure, normal villus height, abundant goblet cells in the crypts, and minimal inflammatory cell infiltration, similar to the healthy group. In conclusion, oral CAT@ALG-PDA significantly enhanced the therapeutic effect in rats with acute IBD.

[0097] Test Example 15

[0098] Comparison of the therapeutic effects of CAT@ALG-PDA with clinically approved drugs: Eight-week-old male SD rats were randomly divided into five groups after one week of acclimatization: healthy group, DSS treatment group, mesalazine treatment group, adalimumab treatment group, and CAT@ALG-PDA treatment group. Starting from day 0, except for the healthy group, rats in the other groups had free access to drinking water containing 4% DSS for 7 consecutive days, and clinical symptoms of IBD, such as watery diarrhea, appeared on day 1. On days 1, 3, and 5, CAT@ALG-PDA capsules were administered orally by gavage, one capsule (each capsule containing 10 mg of drug) per dose. The dosages of mesalazine and adalimumab were based on clinical usage: mesalazine granules were administered orally by gavage, 20 mg / kg per dose; adalimumab was administered subcutaneously, 0.6 mg per dose. During the experiment, daily... Rats' body weight, fecal consistency, and fecal occult blood were measured and recorded. The DAI scoring criteria were as follows: fecal consistency (hard = 0, soft = 2, diarrhea = 4), fecal occult blood (Pyramidon method, scored based on color changes, ranging from 0-4), and weight loss (less than 1% = 0, 1%-5% = 1, 5%-10% = 2, 10%-20% = 3, more than 20% = 4). On day 6, all rats were sacrificed, colon length was measured, and the colon was gently washed with PBS. The distal colon was used for histological analysis, and the remaining tissue was stored at -80°C for future studies. Results are as follows: Figures 19-20 As shown. From Figure 19 It can be seen that oral CAT@ALG-PDA was more effective than oral mesalazine and subcutaneous adalimumab in alleviating IBD-induced weight loss. Figure 19 a). In addition, fecal occult blood tests were observed in the oral mesalazine and subcutaneous adalimumab groups ( Figure 19 b) and watery diarrhea ( Figure 19 c) Symptoms worsened over time, leading to a significant increase in the DAI score. Figure 19 d), while these symptoms improved in the CAT@ALG-PDA group. Compared with the mesalazine and adalimumab groups, both groups showed varying degrees of colonic shortening ( Figure 19 e) Colonic edema and splenomegaly ( Figure 19 f). From Figure 20 As can be seen, the CAT@ALG-PDA group significantly reduced colonic shortening and colonic edema, and had lower spleen weight. In conclusion, oral CAT@ALG-PDA provided superior therapeutic effects compared to mesalazine and adalimumab.

[0099] Test Example 16

[0100] Detection of different types of ROS in the gut: Preserved tissue was weighed for quantification of hydrogen peroxide, superoxide anion, and nitric oxide free radical levels. Analysis was performed using commercial kits for the detection of hydrogen peroxide, superoxide anion, and nitric oxide free radicals. Results are as follows: Figure 21 As shown. From Figure 21 It can be seen that the enhanced efficacy of CAT@ALG-PDA is attributed to its effective reduction of ROS and RNS concentrations, including hydrogen peroxide, superoxide anion and nitric oxide, at sites of intestinal inflammation.

[0101] Test Example 17

[0102] The collected colon tissue was placed in PBS buffer and the intestinal contents were gently washed away. The colon tissue was then homogenized in FACS buffer, digested with digestive enzymes, and filtered through nylon gauze to obtain a single-cell suspension. After staining with the corresponding fluorescently labeled antibodies (FITC-CD68, PE-CD80, PE-Cy7-CD11b), the cells were analyzed by flow cytometry. The results are as follows: Figure 22 As shown. From Figure 22 It can be seen that, compared with the healthy control group, the M1 macrophages (CD11b) in the DSS-treated group + CD68 + CD80 + The levels of M1 macrophages were elevated. Oral administration of CAT@ALG-PDA significantly reduced the levels of M1 macrophages. In contrast, mesalazine and adalimumab failed to effectively reduce M1 macrophages to the same level as CAT@ALG-PDA.

[0103] Test Example 18

[0104] The preserved tissue was weighed to quantify pro-inflammatory cytokines and peroxidases in the tissue; the tissue was homogenized in PBS (1×) buffer; and then centrifuged at 10,000 rpm for 10 min at 4 °C to obtain the homogenized product. A commercially available rat TNF-α, IFN-γ, IL-1β, IL-6, IL-12, and MPO ELISA kit can quantify the pro-inflammatory cytokines and peroxidases in the homogenized product, and the results are as follows: Figure 23 As shown. From Figure 23 It can be seen that CAT@ALG-PDA effectively reduced the levels of cytokines secreted by M1 macrophages (including TNF-α, IL-1β, and IL-12). Figure 23 a- Figure 23 c), while mesalazine and adalimumab failed to reduce the levels of these cytokines as effectively as CAT@ALG-PDA. Furthermore, CAT@ALG-PDA effectively reduced other pro-inflammatory cytokines (such as IFN-γ). Figure 23 d) IL-6 Figure 23e)) and peroxidase (MPO) Figure 23 The level of f) shows its superior efficacy in relieving inflammation.

[0105] In summary, oral CAT@ALG-PDA showed superior therapeutic effects compared to mesalazine and adalimumab.

[0106] Test Example 19

[0107] To investigate the biosafety of CAT@ALG-PDA capsules after oral administration: Eight-week-old male SD rats were randomly divided into two groups after one week of acclimatization: a healthy group and a CAT@ALG-PDA group. DSS-induced IBD rats in the CAT@ALG-PDA group were orally administered CAT@ALG-PDA at a predetermined time. These rats were sacrificed on day 6 post-treatment. Tissues from major organs were then collected, gently washed, and fixed in 10% formalin solution for histological analysis. The fixed tissues were stained with hematoxylin and eosin, and images were observed using an optical microscope (Leica DM4000). The results are as follows: Figure 24 As shown. From Figure 24 It can be seen that repeated oral administration of CAT@ALG-PDA did not cause significant tissue damage or side effects, and there was no significant difference compared with the healthy group.

[0108] In addition, after one week of acclimatization, eight-week-old male SD rats were randomly divided into two groups: a healthy group and a CAT@ALG-PDA group. Rats in the CAT@ALG-PDA group received oral CAT@ALG-PDA on day 0. Blood samples were collected on days 1, 7, and 14 for routine blood tests and biochemical blood analysis. The results are as follows: Figure 25 As shown. From Figure 25 It can be seen that CAT@ALG-PDA treatment has little effect on the concentrations of alanine aminotransferase (ALT), urea (UREA), creatinine (CREA), alkaline phosphatase (ALP), and aspartate aminotransferase (AST) in serum. Figure 25 a- Figure 25 e) indicates that the strategy did not significantly impair kidney and liver function during the monitoring period. Furthermore, these treatments were found to have an effect on parameters related to red blood cells and platelets, such as red blood cell count (RBC). Figure 25 f) Hemoglobin level (HBG, Figure 25 g), hematocrit (HCT), Figure 25 h), mean erythrocyte volume (MCV), Figure 25 i) Mean corpuscular hemoglobin (MCH) Figure 25 j) Mean corpuscular hemoglobin concentration (MCHC) Figure 25 k) and mean platelet volume (MPV), Figure 25(l) The impact is relatively small. In summary, the oral CAT@ALG-PDA treatment strategy is quite safe and has great potential for future biomedical applications.

[0109] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A drug-loaded intestinal adhesion composite gel microsphere, characterized in that, It includes calcium alginate gel microspheres, protein drugs encapsulated within the calcium alginate gel microspheres, and antioxidants coated on the surface of the calcium alginate gel microspheres.

2. The intestinal adhesion drug-loaded composite gel microspheres according to claim 1, characterized in that, The protein drug is selected from catalase and / or superoxide dismutase.

3. The intestinal adhesion drug-loaded composite gel microspheres according to claim 1, characterized in that, The antioxidant is polydopamine.

4. The intestinal adhesion drug-loaded composite gel microspheres according to claim 1, characterized in that, The mass ratio of the protein drug, calcium alginate gel microspheres, and antioxidant is (1-50):50:(0.5-6).

5. The method for preparing intestinal adhesion drug-loaded composite gel microspheres according to any one of claims 1-4, characterized in that, Using an electrospray device as the generating device, the electrospray device includes an injection pump, a high-voltage power supply, and a collector; the preparation method includes the following steps: S1. The protein drug solution and calcium chloride solution are respectively loaded into the syringe pump and the collector. The high-voltage power supply is turned on to inject the protein drug solution into the calcium chloride solution to obtain a protein drug@calcium alginate microsphere solution. The protein drug solution is obtained by dissolving the protein drug in sodium alginate solution. S2. Add an antioxidant solution to the protein drug@calcium alginate microsphere solution described in S1, adjust the pH to 7.5-8.5, and react to obtain intestinal adhesion drug-loaded composite gel microspheres.

6. The method for preparing intestinal adhesion drug-loaded composite gel microspheres according to claim 5, characterized in that, In S1, the mass fraction of the protein drug in the protein drug solution is 0.1%-1%; the mass fraction of the sodium alginate in the sodium alginate solution is 1%-5%. The calcium chloride solution contains 1%-5% calcium chloride by mass. The volume ratio of the protein drug solution to the calcium chloride solution is (0.1-0.4):

1.

7. The method for preparing intestinal adhesion drug-loaded composite gel microspheres according to claim 5, characterized in that, In S1, during the injection process, the injection rate of the protein drug solution is 20 μL / min-200 μL / min, the voltage of the high-voltage power supply is 6V-14V, and the size of the needle-shaped emitter of the injection pump is selected from 28G-34G.

8. The method for preparing intestinal adhesion drug-loaded composite gel microspheres according to claim 5, characterized in that, In S2, the final concentration of the antioxidant is 0.1 mg / mL to 1.2 mg / mL.

9. A capsule formulation, characterized in that, The capsule formulation is prepared from the intestinal adhesion drug-loaded composite gel microspheres according to any one of claims 1-4.

10. The use of the capsule formulation of claim 9 in the preparation of a medicament for treating inflammatory bowel disease.

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