Probiotic preparation and application thereof in gastrointestinal disease treatment

Abstract

The invention discloses a GastroShield composite targeting enteric-coated particle preparation as well as preparation and application thereof, and belongs to the field of biological medicines. The preparation is composed of a GastroShield four-function fusion polypeptide, an anti-TNF-alpha antibody, probiotics and a biological synergistic factor, and is prepared through three-layer treatment including sodium alginate embedding, chitosan-antibody crosslinking and Eudragit FS30D enteric coating. Wherein the GastroShield polypeptide has four functions of adhesion, pH response release, protease inhibition and antibiosis, can accurately target an inflammation site, and releases active ingredients in a colon alkaline environment. Experimental data show that the release rate of the preparation in artificial colon fluid within 4 hours reaches 95.3 + / -1.8%, the preparation can effectively repair DSS-induced ulcerative colitis mouse colon tissue, reduce the inflammatory factor level and remodel intestinal flora, and a new scheme is provided for treatment of intestinal diseases such as inflammatory bowel diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a probiotic preparation and its application in the treatment of gastrointestinal diseases. Background Art

[0002] Probiotics, as a class of active microorganisms beneficial to host health, have great potential for treating gastrointestinal diseases. They play a vital role in treating conditions such as diarrhea, constipation, and inflammatory bowel disease (IBD) by regulating the balance of the intestinal microbiome and enhancing intestinal immune function. However, current clinically used probiotic preparations still face significant technical bottlenecks, severely restricting their therapeutic efficacy and scope of application.

[0003] Inadequate gastrointestinal tolerance is the primary challenge. The highly acidic environment of the human stomach, with a pH of 1.5-3.5, results in a survival rate of conventional oral probiotics generally below 20%, with many probiotics inactivated before reaching the intestine. Although microencapsulation technologies such as sodium alginate and chitosan can improve gastric survival rates, they only provide physical protection and cannot achieve targeted release at the site of inflammation. Furthermore, the encapsulation materials can easily interfere with the metabolic activity of probiotics.

[0004] This lack of targeting further limits efficacy. Gastrointestinal diseases are often accompanied by inflammation or tissue damage in specific areas, such as the intestinal mucosal inflammation seen in patients with ulcerative colitis. However, current probiotic preparations are mostly evenly distributed in the intestine, making it difficult to enrich in lesions. While some studies have attempted to enhance mucosal adhesion by modifying anti-MUC2 monoclonal antibodies, the high cost of antibody production and poor stability have hindered industrial application.

[0005] Furthermore, existing formulations have a single mechanism of action. Single-ingredient formulations, such as Bifidobacterium animalis BLa80, can only regulate the microbiome and have limited efficacy in treating immune overactivation. Combination formulations of antibodies or antimicrobial peptides, while capable of inhibiting inflammatory factors, lack mucosal repair capabilities, and antimicrobial peptides are easily degraded by proteases. Even complex probiotic formulas (such as TT-16 complex) that incorporate multiple strains still fail to address the core issues of gastric acid damage and targeted delivery.

[0006] Existing technical attempts all have limitations: Novalac Prebiotics' acid-resistant encapsulation cannot target inflammatory sites; technologies based on engineered bacteria secreting antimicrobial peptides carry toxicity risks; and probiotic peptides produced through solid-state fermentation have low bioavailability due to their molecular weight exceeding 1000 Da. Therefore, developing innovative formulations that combine high gastrointestinal survival rates, precise targeting, and multi-mechanism synergistic intervention is crucial for breaking through technical bottlenecks in gastrointestinal disease treatment and enhancing the clinical value of probiotics. Summary of the Invention

[0007] The purpose of the present invention is to provide an innovative GastroShield composite targeted enteric-coated microparticle preparation, which achieves efficient treatment of inflammatory bowel disease through multi-component synergy and precise targeted delivery strategy.

[0008] Therefore, the present invention discloses a GastroShield tetrafunctional fusion polypeptide, the amino acid sequence of which is shown in SEQ ID NO: 1, with a total length of 85 amino acid residues and a molecular weight of approximately 9.5 kDa. Through a modular design concept, the polypeptide integrates four functional domains:

[0009] 1. Adhesion Domain: Utilizing the amino acid sequence MQKPKTGKCVPALAGC, it specifically recognizes and binds to the MUC2 mucin, which is abnormally highly expressed in inflammatory sites. ELISA assays demonstrated a binding affinity constant (Kd) of 2.34±0.17 nM for MUC2; surface plasmon resonance (SPR) analysis confirmed a Kd of 2.27 nM, significantly promoting peptide accumulation in lesions and effectively increasing local drug concentration.

[0010] 2. pH-responsive release domain: It is composed of the DDDDDK sequence. In the weakly alkaline environment of the colon (pH>7.0), the carboxyl group in this sequence dissociates, making the polypeptide negatively charged, and then self-depolymerizes, triggering the release of active fragments. Experimental results show that after incubation for 2 hours at pH 7.4, the complete polypeptide band is reduced to 26.4±1.9%, and the C-terminal active fragment accounting for 68.3±2.5% is released. The molecular weight is identified by LC-MS as 4821.9±0.7Da, which is highly matched with the antimicrobial peptide domain sequence (coverage 95%). The release half-life under this condition is 45±5min; in the pH 6.0 environment, the release rate is only 5.2±1.4% within 2 hours, thereby achieving precise colon-targeted activation.

[0011] 3. Protease inhibitory domain: The amino acid sequence CCPDACGGFLTKSIKQAF can effectively inhibit the activity of intestinal trypsin. The results of the FRET substrate assay showed that the inhibition rate of 10 μM of this peptide on trypsin reached 85.9% at 10 minutes, 84.7% at 20 minutes, and maintained at 85.6% at 30 minutes. The inhibitory activity was stable, and its half inhibitory concentration IC 50 =1.2±0.3μM, which can effectively protect the antimicrobial peptide domain from degradation by digestive enzymes and significantly prolong the stability of the peptide in the gastrointestinal tract.

[0012] 4. Antimicrobial peptide domain: It is a modified derivative of Magainin-2 with the amino acid sequence KWKSFIKKLTSAAKKVGLGALKAL. By optimizing the charge distribution and hydrophobicity, it can effectively kill pathogens such as Escherichia coli (EC 50 =7.9±0.5μM, MIC is 8μM), showing good selectivity for commensal bacteria. Experiments showed that after 48 hours of treatment with 32μM of this peptide, the survival rate of Bifidobacterium adolescentis was still as high as 84.6±2.1%.

[0013] The present invention also discloses a GastroShield composite targeted enteric-coated microparticle preparation. Calculated per gram of dry powder, the specific composition of the preparation of the present invention is as follows:

[0014] 1. Probiotics: Contains Bifidobacterium animalis BLa804×10 10 CFU / g, Lactobacillus plantarum Lp903×10 10 CFU / g, Lactobacillus casei ZM153×10 10 CFU / g, plays a role in multiple ways such as repairing the intestinal mucosal barrier, regulating the body's immune function, and competitively inhibiting the colonization of pathogens.

[0015] 2. Bioactive components: GastroShield peptide 60mg / g and anti-TNF-α antibody 15mg / g, the two work synergistically to achieve targeted antibacterial and anti-inflammatory effects, and effectively block TNF-α-mediated inflammatory signaling pathways.

[0016] 3. Biosynergistic factors: 200 mg / g of galacto-oligosaccharide as a prebiotic can synergistically promote the colonization of probiotics; 50 mg / g of magnesium oxide can locally alkalize the microenvironment and relieve acid sensitivity inhibition; 50 mg / g of baicalin can enhance the overall anti-inflammatory effect by inhibiting the NF-κB inflammatory pathway.

[0017] Preferably, the preparation of the present invention adopts a unique three-layer coating structure:

[0018] 1. First layer: 2% (w / v) sodium alginate solution is used to embed GastroShield peptides and probiotics, and 0.2M CaCl2 is used to cross-link to form a stable network structure to achieve initial protection for probiotics and peptides.

[0019] 2. The second layer: Constructing a chitosan-anti-TNF-α antibody cross-linking layer, through EDC / NHS activation technology, the antibody and chitosan are efficiently coupled, with a coupling rate of 82.3±2.5%, further stabilizing the antibody and enhancing the targeting of the preparation.

[0020] 3. The third layer uses Eudragit FS30D as the enteric coating material. According to the optimized ratio of Eudragit FS30D: triacetin: polysorbate 80 = 3:1:1, carboxyl group dissociation occurs in an environment of pH>6.8, causing the membrane structure to expand to form 20-50nm pore channels, achieving precise colon-targeted release.

[0021] The present invention also discloses a method for preparing a GastroShield composite targeted enteric-coated microparticle preparation, which comprises the following steps:

[0022] 1. Preparation of mixed bacterial suspension: Each probiotic was inoculated into MRS culture medium and cultured under appropriate conditions until the logarithmic growth phase (animal Bifidobacterium BLa80 was cultured anaerobically at 37°C for 16 h, OD 600 =1.2±0.1; Lactobacillus plantarum Lp90 was cultured aerobically at 37℃ for 12h, OD 600 =1.5±0.2; Lactobacillus casei ZM15 was cultured microaerophilically at 37℃ for 14h, OD 600 =1.3±0.1), centrifuged at 4°C and 4000 rpm for 10 min, discarded the supernatant and resuspended in sterile PBS; the three bacterial suspensions were combined to adjust the total concentration to about 1×10 11 CFU / mL, adding 5% trehalose and 3% mannitol as protective agents, and experiments have verified that this combination can make the survival rate of probiotics after freeze-drying reach 92.1±1.8%.

[0023] 2. First coating: Prepare a 2% (w / v) sodium alginate solution, sterilize it through a 0.22μm filter, add GastroShield peptide (final concentration 6mg / mL), and magnetically stir for 30 minutes. Use a syringe pump to drip the bacterial suspension-sodium alginate mixture into a 0.2M CaCl2 solution at a flow rate of 1mL / min. Cross-link for 30 minutes under magnetic stirring at 300rpm. After cross-linking, wash the microspheres three times with sterile water, centrifuge them at 4°C, 3000×g for 5 minutes, collect the microspheres, and refrigerate them for later use. Process optimization determined that the optimal parameter combination was 2% sodium alginate concentration, 6mg / mL peptide addition, 0.2M CaCl2 concentration, and 30 minutes of cross-linking time.

[0024] 3. Second layer coating: 0.5% chitosan was dissolved in 1% acetic acid solution, the pH was adjusted to 5.5 with 1 M NaOH, anti-TNF-α antibody was added to a final concentration of 1.5 mg / mL, and the mixture was stirred on ice for 10 minutes. EDC / NHS activation was performed at room temperature for 30 minutes at a molar ratio of EDC:NHS:chitosan amino group = 5:5:1. After activation, the mixture was immediately mixed with the first layer of microspheres and coupled at 37°C and 150 rpm for 30 minutes. The free antibody concentration was detected by ELISA, and the coupling efficiency was verified to be 82.3±2.5%.

[0025] 4. Third coating: Prepare an emulsion in a ratio of Eudragit FS30D: triacetin: polysorbate 80 = 3:1:1 and apply the coating using fluidized bed spray coating technology. Precisely control process parameters: inlet temperature 35°C to ensure uniform atomization of the coating solution and avoid microsphere adhesion; outlet air temperature 28°C to keep the surface moisture content of the microspheres below 2%; spray rate 2.5mL / min to achieve a uniform coating thickness of 5±0.5μm; air inlet volume 15m 3 / h to maintain good fluidity of the microspheres and eventually form an enteric coating with a dissolution threshold pH>6.8.

[0026] 5. Freeze drying: First, pre-freeze the microspheres at -20℃ for 2 hours, then transfer to -45℃ for freeze drying for 24 hours. After drying, the particle size of the microspheres is distributed between 80-150μm. Use vacuum packaging technology to store at 2-8℃ for future use.

[0027] In summary, the GastroShield composite targeted enteric-coated microparticle preparation of the present invention exhibits significant and multi-dimensional advantages in the treatment of inflammatory bowel disease.

[0028] 1. The innovatively designed GastroShield quadrifunctional fusion peptide plays a key role in targeted delivery and precision therapy. Its adhesion domain, leveraging its specific recognition of MUC2 mucin, which is highly expressed in inflammatory sites, precisely locates lesions and concentrates the drug there. The pH-responsive release domain and Eudragit FS30D enteric coating form a dual-mode responsive release system, achieving a 4-hour release rate of 95.3±1.8% in artificial colonic fluid. Compared to traditional single pH-responsive formulations, this system more precisely releases the active ingredient in the alkaline environment of the colon, significantly increasing drug concentrations at lesions while significantly reducing side effects associated with systemic drug distribution, ultimately achieving efficient and safe targeted drug delivery.

[0029] 2. The multi-component synergistic mechanism is another major breakthrough of this formulation. Probiotics, GastroShield peptides, anti-TNF-α antibodies, and bio-enhancing factors work synergistically to form a comprehensive therapeutic system. Three carefully selected probiotic strains (Bifidobacterium animalis BLa80, Lactobacillus plantarum Lp90, and Lactobacillus casei ZM15) modulate the intestinal microbiome to alleviate diarrhea, repair the intestinal barrier, and degrade purines. Their unique combination surpasses existing patented complex bacterial compounds. The antimicrobial peptide domain of the GastroShield peptide effectively kills pathogens such as Escherichia coli, while the protease inhibitory domain ensures peptide stability. The anti-TNF-α antibody blocks key inflammatory signaling pathways, while bio-enhancing factors such as baicalin further inhibit NF-κB, reducing the dosage of monoclonal antibodies by up to 40%. This multi-component synergistic effect has enabled the formulation to demonstrate excellent therapeutic efficacy in animal studies, reducing the DAI score of mice with DSS-induced ulcerative colitis to 2.8±0.3, and achieving colon length recovery and mucosal healing rates of 95%, far exceeding those of commercially available formulations and non-peptide controls.

[0030] 3. In terms of stability and practicality, the formulation, through an optimized preparation process, exhibits excellent stability and activity retention. The probiotic activity retention rate after freeze-drying exceeded 90%, and it remained well tolerated in simulated gastrointestinal fluid. A 30-day accelerated storage experiment showed that the activity retention rates of GastroShield peptide and anti-TNF-α antibody were 85.2±4.1% and 82.3±3.1%, respectively, and the viable probiotic count remained stable at 9.2×10 10 ±5.1×10 9 CFU / g, and the survival rate of live bacteria exceeds 90% after 12 months of storage at room temperature. This not only ensures the effectiveness and safety of the preparation during production, transportation, and storage, but also greatly enhances the practicality and market competitiveness of the product, providing a safe, long-term, and efficient innovative solution for the clinical treatment of inflammatory bowel disease, with extremely high development and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 SDS-PAGE detection results of GastroShield polypeptides, where 1 is GastroShield polypeptide.

[0032] Figure 2 Western blot detection results of GastroShield peptide, where 1 is GastroShield peptide.

[0033] Figure 3 LC-MS identification results.

[0034] Figure 4 Process flow chart of GastroShield composite targeted enteric-coated microparticle preparation.

[0035] Figure 5 Schematic diagram of GastroShield composite targeted enteric-coated microparticles formulation (three-layer coating).

[0036] Figure 6 DSS modeling flowchart. DETAILED DESCRIPTION

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0038] Unless otherwise noted, the reagents, methods, and equipment used in the present invention are conventional in the art. Unless otherwise noted, the reagents and materials used in the following examples were commercially available. It should be noted that the GastroShield polypeptide mentioned in the present invention is the abbreviation for the GastroShield tetrafunctional fusion polypeptide.

[0039] Example 1: Design, expression, purification and functional verification of GastroShield tetrafunctional fusion polypeptide

[0040] 1. Peptide sequence design and synthesis

[0041] GastroShield is a four-functional fusion peptide that integrates four functional domains: adhesion, pH-responsive release, protease inhibition, and antimicrobial activity:

[0042] Precise Targeting of the Adhesion Domain: The adhesion domain (MQKPKTGKCVPALAGC) ensures peptide accumulation in lesions by targeting the overexpressed MUC2 mucin at sites of inflammation. This high-affinity binding significantly increases local drug concentration and reduces potential side effects from systemic distribution.

[0043] pH-responsive depolymerization mechanism: The DDDDDK sequence becomes negatively charged and self-depolymerizes in the weakly alkaline environment of the colon (pH > 7.0), triggering the release of the active fragment. This design avoids premature degradation of the peptide in gastric acid or the small intestine, enabling colon-targeted delivery.

[0044] The barrier effect of the protease inhibitory domain: The CCPDACGGFLTKSIKQAF sequence protects the antimicrobial peptide domain from degradation by digestive enzymes by inhibiting intestinal trypsin (inhibition rate >85%). Trypsin is active in the small intestine, and its inhibition prolongs the stability of the peptide in the gastrointestinal tract, ensuring that the antimicrobial peptide remains intact before reaching the colon.

[0045] Selective Bactericidal Effects of the Antimicrobial Peptide Domain: A modified Magainin-2 derivative (KWKSFIKKLTSAAKKVGLGALKAL) demonstrates high efficacy against pathogenic bacteria such as Escherichia coli while sparing commensal bacteria. Magainin-2's α-helical structure disrupts bacterial cell membranes, while the modified sequence enhances selectivity by adjusting charge distribution or hydrophobicity.

[0046] 1.1 Polypeptide sequence: The amino acid sequence of the GastroShield polypeptide designed above (connected using a flexible linker) is shown in SEQ ID NO: 1, with a total length of 85 AA and a molecular weight of approximately 9.5 kDa.

[0047] 1.2 Gene construction: The codon-optimized gene (C segment containing 6His tag, SEQ ID NO: 2) was cloned into the pET-28a(+) vector and transformed into E. coli BL21(DE3). Positive clones were screened with 50 μg / mL kanamycin.

[0048] 1.3 Expression and purification: Pick a single clone and inoculate it into 5 mL LB-Kan medium, culture it at 37℃ for 12 h, then transfer it to 500 mL TB-Kan medium (1:100) and culture it until OD 600 =0.6, 1 mM IPTG was added, and the cells were induced at 25°C for 16 h. The cells were collected by centrifugation at 4°C, resuspended in binding buffer (20 mM Tris-HCl pH 7.9, 500 mM NaCl, 5 mM imidazole), and disrupted by ultrasonication on ice (300 W, 3 seconds on, 5 seconds off, for a total of 10 min). Centrifuged at 14,000 × g for 30 min, the supernatant was passed through a Ni-NTA column, and purified sequentially with wash buffer (containing 20 mM imidazole) and elution buffer (containing 300 mM imidazole). The eluted peak was collected and dialyzed against PBS (pH 7.4) three times at 4°C using a dialysis bag (MWCO 3.5 kDa), 2 L each time. SDS-PAGE showed ( Figure 1 ) The purified protein showed a single band at about 10 kDa with a purity of >95%. Western blot ( Figure 2 ) was verified to be correct using an anti-His antibody. The final yield was 125 ± 1.8 mg / L fermentation broth.

[0049] 2. Verification of adhesion domain binding to MUC2

[0050] The binding affinity of the peptide to recombinant human MUC2 protein was determined by ELISA. A 96-well plate was coated with MUC2 protein (ab316727, 2 μg / mL, overnight at 4°C) and blocked with 5% BSA. A gradient of peptide dilutions (0.01–100 nM) was added and incubated at room temperature for 1 hour. HRP-anti-His antibody was used for color development, and the OD was read on a microplate reader. 450 The data from three independent experiments showed that the binding curve was consistent with the Hill equation (Y = Bmax × X / (Kd + X)), with Kd = 2.34 ± 0.17 nM, Bmax = 1.632 ± 0.025, and Hill coefficient 1.02 ± 0.05. Surface plasmon resonance (SPR) validation results showed Kd = 2.27 nM (kon = 2.1 × 10 5 M-1 s -1, koff = 4.8 × 10 -4 s -1 ), indicating a single high-affinity binding site. Specific data are shown in Table 1.

[0051] Table 1 ELISA test results

[0052]

[0053] 3. pH-responsive release experiments

[0054] The peptides were dissolved in pH 6.0 (MES buffer) and pH 7.4 (PBS) and incubated at 37°C for 2 hours. SDS-PAGE silver staining showed that the intact peptide band was reduced to 26.4±1.9% at pH 7.4, and the C-terminal fragment (approximately 44AA) was released, accounting for 68.3±2.5%; while at pH 6.0, only 5.2±1.4% was released. LC-MS identified the molecular weight of the released fragment as 4821.9±0.7Da ( Figure 3 ), matching the antimicrobial peptide domain sequence (coverage 95%). Real-time monitoring showed that the release half-life at pH 7.4 was 45±5 minutes, while at pH 6.0 it was >12 hours, indicating that the peptide is rapidly activated in the alkaline environment of the intestine.

[0055] 4. Protease Inhibition Activity Assay

[0056] The FRET substrate method (Z-Gly-Pro-Arg-AMC) was used to determine the trypsin inhibitory activity. In a 96-well plate, 100nM trypsin was pre-incubated with a gradient dilution of the peptide (0.1-100μM) for 15 minutes, and then 100μM substrate was added. The fluorescence microplate reader was used to monitor the inhibitory activity for 30 minutes (λex=360nm, λem=460nm). The results showed that the inhibition rate of 10μM peptide on trypsin was stable at more than 85% (10min: 85.9%, 20min: 84.7%, 30min: 85.6%), and the IC 50 =1.2±0.3μM. Specific data are shown in Table 2.

[0057] Table 2 Luciferase RFU detection results

[0058]

[0059] 5. Antibacterial activity test

[0060] The antibacterial activity of the peptide against Escherichia coli was determined by broth dilution method. 6 CFU / mL bacterial solution was incubated with gradient concentrations of peptide (0.25-64 μM) for 18 h, and OD was measured. 600 The results showed that when the peptide concentration was ≥8μM, the inhibition rate was >88%, and the EC 50 =7.9±0.5μM, with an MIC of 8μM. Experiments on Bifidobacterium adolescentis showed that after 48 hours of treatment with 32μM peptide, the survival rate of the commensal bacteria still reached 84.6±2.1%, demonstrating the peptide's excellent selectivity. Specific data are shown in Tables 3 and 4.

[0061] Table 3 Escherichia coli inhibition rate

[0062]

[0063] Table 4 Symbiotic bacteria survival results

[0064]

[0065] This study successfully constructed and expressed a quadrifunctional fusion peptide, GastroShield, which, through modular design, achieved synergistic effects of targeted adhesion, pH-responsive release, protease inhibition, and selective antimicrobial activity. This peptide precisely enriched and released active fragments at sites of intestinal inflammation (where MUC2 is highly expressed and pH > 7.0), while simultaneously protecting the integrity of the antimicrobial peptide by inhibiting trypsin. It exhibited highly effective killing of pathogens such as Escherichia coli, while having minimal effects on commensal bacteria. These properties make it a potential candidate for the treatment of inflammatory bowel disease, intestinal infections, and other diseases, providing new insights into colon-targeted biotherapy.

[0066] Example 2: Preparation and testing of GastroShield composite targeted enteric-coated microparticles

[0067] 1. Preparation composition and ratio (calculated based on 1g dry powder)

[0068] Table 5 Preparation composition and ratio

[0069]

[0070] 2. Preparation process steps, including the following process steps: Figure 4 As shown, the schematic diagram of the three-layer coating is as follows Figure 5 shown.

[0071] Step 1. Preparation of mixed bacterial suspension

[0072] Each probiotic was cultured in MRS medium to the logarithmic phase, centrifuged (4000 rpm, 10 min), and resuspended to the target concentration. The three strains were combined to adjust the total concentration to approximately 1×10 11 CFU / mL; 5% trehalose + 3% mannitol were added as protective agents.

[0073] (1) Single bacteria culture conditions:

[0074] Bifidobacterium animalis BLa80: MRS medium, anaerobic culture at 37℃ for 16h (OD 600 =1.2±0.1);

[0075] Lactobacillus plantarum Lp90: MRS medium, aerobic culture at 37℃ for 12h (OD 600 =1.5±0.2);

[0076] Lactobacillus casei ZM15: MRS medium, microaerophilic culture at 37℃ for 14h (OD 600 =1.3±0.1);

[0077] Centrifugation: 4000 rpm (5000 g), 4°C for 10 min, discard the supernatant and resuspend in sterile PBS.

[0078] (2) Protective agent screening experiment is shown in Table 6.

[0079] Table 6 Protective agent screening test results

[0080]

[0081] Step 2. First layer of coating: sodium alginate embedding + GastroShield

[0082] Prepare 2% (w / v) sodium alginate solution, sterilize it through a 0.22 μm filter membrane, add GastroShield polypeptide (final concentration 6 mg / mL), and stir magnetically for 30 minutes. Use a syringe pump (flow rate 1 mL / min) to drop the bacterial suspension-sodium alginate mixture into 0.2 M CaCl2 solution, stir magnetically at 300 rpm for cross-linking, wash three times with sterile water after cross-linking, collect the microspheres by centrifugation at 4°C (3000×g, 5 minutes), and refrigerate for future use.

[0083] Table 7 Optimal conditions test results

[0084]

[0085] Step 3. Second coating: chitosan-anti-TNF-α antibody crosslinking

[0086] First, a chitosan solution was prepared by dissolving 0.5% chitosan in 1% acetic acid solution and adjusting the pH to 5.5 with 1M NaOH. Anti-TNF-α antibody was then added to a final concentration of 1.5 mg / mL and stirred in an ice bath for 10 minutes. EDC / NHS activation was then performed at a molar ratio of EDC:NHS:chitosan amino groups of 5:5:1 for 30 minutes at room temperature. After activation, the solution was immediately mixed with the first layer of microspheres and coupled at 37°C with shaking at 150 rpm for 30 minutes.

[0087] Finally, the free antibody concentration was detected by ELISA, and the coupling rate was calculated to be 82.3±2.5%, ensuring that this coating layer successfully achieved effective cross-linking of the antibody and chitosan.

[0088] Step 4. Third layer coating: Eudragit FS30D (enteric coating)

[0089] First, FS30D emulsion was prepared and mixed in a ratio of Eudragit FS30D: triacetin: polysorbate 80 = 3:1:1. Process optimization verified that this ratio can achieve the best membrane permeability, with a porosity of 18.7±1.2%. Subsequently, fluidized bed spray coating was used for coating. The inlet temperature was controlled at 35°C to ensure uniform atomization of the coating liquid and no adhesion. The outlet air temperature was set at 28°C to keep the surface moisture content of the microspheres below 2%. At the same time, the spray rate was controlled at 2.5mL / min to achieve a uniform coating thickness of 5±0.5μm, and the air inlet was set at 15m 3 / h, while ensuring the drying efficiency, maintaining good fluidity of the microspheres, and finally forming an enteric coating with a dissolution threshold pH>6.8.

[0090] Step 5. Freeze Drying

[0091] Prefreeze at -20℃ for 2h → freeze-dry at -45℃ for 24h; after drying, the microspheres have a particle size of 80-150μm; vacuum seal and store at 2-8℃ for later use.

[0092] 3. Preparation Inspection

[0093] 1. Determination of encapsulation efficiency and drug loading rate

[0094] First, GastroShield peptide standard solutions of different concentrations (0.1-100 μg / mL) were prepared, and the detection wavelength was 220 nm. The injection volume was 20 μL. Separation was performed on a C18 column (4.6×250 mm, 5 μm) with acetonitrile-0.1% formic acid aqueous solution (gradient elution). The standard curve (R 2 =0.9992). An appropriate amount of the prepared GastroShield composite targeted enteric-coated microparticle preparation was ultrasonically demulsified with methanol and then centrifuged. The supernatant was diluted to an appropriate concentration and sampled for analysis. The peptide content in the microparticles was calculated based on the standard curve, and the encapsulation efficiency and drug loading rate were then determined. For anti-TNF-α antibodies, an ELISA was used. Following the kit instructions, the supernatant of the microparticle preparation was processed and assayed. The content was calculated using a standard curve drawn with a standard. Each sample was assayed three times, and the intra- and inter-assay coefficients of variation (CV) were calculated.

[0095] The results showed that the encapsulation efficiency of GastroShield peptide and anti-TNF-α antibody was 84.2±1.7% and 78.5±2.1%, respectively, and the drug loading efficiency was 5.9±0.3% and 3.2±0.2%. The intra-assay coefficient of variation (CV) of the antibody determined by ELISA was <5%, and the inter-assay CV was <8%.

[0096] 2. Enteric Release Experiment

[0097] The USP paddle method was used in a dissolution apparatus with a speed set at 100 rpm and a temperature controlled at 37°C. Artificial gastric fluid (pH 1.2, composed of 0.1M HCl and 0.2% pepsin), artificial intestinal fluid (pH 6.8, pH adjusted with 0.2M Na2HPO4 and 0.1M HCl, followed by the addition of 1% pancreatin), and artificial colonic fluid (pH 7.4, composed of 0.1M PBS, 0.5% bilesalts, and 1% mucin to simulate the colonic environment) were prepared as release media. A certain amount of GastroShield composite targeted enteric-coated microparticle formulation was placed in a dissolution cup. 5 mL samples were taken at 2 and 4 hours (with an equal amount of isothermal medium added simultaneously). After filtration through a 0.45 μm filter membrane, the GastroShield polypeptide and anti-TNF-α antibody content were determined using the aforementioned HPLC-UV method and ELISA method, respectively, and the release rate at different time points was calculated.

[0098] The results (Table 8) showed that the release rate of the formulation was <5% in artificial gastric fluid (pH 1.2) over 2 hours. In artificial intestinal fluid (pH 6.8), the release rate was 52.1±3.2% over 2 hours and 78.6±2.9% over 4 hours. In artificial colonic fluid (pH 7.4), the release rate was 81.6±2.4% over 2 hours and nearly complete (95.3±1.8%) over 4 hours. The release mechanism was pH-responsive: at pH 1.2, the carboxyl group of Eudragit FS30D protonated to form a dense membrane. At pH > 6.8, the carboxyl group dissociated, causing the membrane to swell and form channels (pore size 20-50 nm).

[0099] Table 8 Enteric release test results

[0100]

[0101] 3. Probiotic Activity Retention Determination

[0102] Using the plate count method, before lyophilization, take an appropriate amount of the mixed bacterial suspension and perform a gradient dilution. Spread it on an MRS medium plate and incubate at 37°C for 24-48 hours before counting the colonies to obtain the initial viable bacterial count. After lyophilization, take an equal amount of the lyophilized preparation, reconstitute it with sterile saline, perform the same gradient dilution and plate count, calculate the viable bacterial count after lyophilization, and then calculate the activity retention rate after lyophilization. For gastric juice and intestinal fluid tolerance experiments, the lyophilized preparation was placed in artificial gastric juice (pH 1.2) and artificial intestinal juice (pH 7.4), respectively, incubated at 37°C with shaking, and samples were taken at 2 hours (gastric juice) and 4 hours (intestinal juice). After dilution, the samples were spread and counted. The viable bacterial counts were compared with the viable bacterial counts after lyophilization to calculate the survival rate at different stages.

[0103] The results showed (Table 9) that the activity retention rates of the three probiotic strains after freeze-drying were all >91% (BLa80: 91.3±1.5%, HX-LP90: 93.2±1.2%, ZM15: 92.1±1.8%). The survival rates after 2 hours of incubation in gastric fluid were >86%, and after 4 hours of incubation in intestinal fluid were >84%. The viable cell count before freeze-drying was 1.0×10 11 CFU / g, maintained at 9.1×10 10 -9.3×10 10 CFU / g.

[0104] Table 9 Probiotic activity retention test results

[0105]

[0106] 4. Preparation stability testing

[0107] Accelerated experiments were conducted at 40°C / 75% RH, with the GastroShield composite targeted enteric-coated microparticle formulation placed in a constant temperature and humidity chamber. Samples were taken at 0, 7, 14, and 30 days. The activity retention of the GastroShield peptide was determined using HPLC-UV, the activity retention of the anti-TNF-α antibody was determined using ELISA, and the viable count of the probiotic was determined using a plate count assay. Changes in the stability of the various components of the formulation over different storage times were observed.

[0108] The results are shown in Table 10. After 30 days of storage under accelerated conditions of 40°C / 75% RH, the activity of GastroShield polypeptide and anti-TNF-α antibody remained at 85.2±4.1%, 82.3±3.1%, and the viable count of probiotics was maintained at 9.2×10 10 ±5.1×10 9 CFU / g. During the period of 0-30 days, the degradation rate of peptides was 0.5% / day, that of antibodies was 0.6% / day, and the rate of decrease of viable bacteria was 0.2×10 9 CFU / g / day.

[0109] Table 10 Preparation stability test results

[0110]

[0111] 4. Summary of experimental results

[0112] This series of experiments comprehensively evaluated the key performance of the GastroShield composite targeted enteric microparticle preparation. In terms of encapsulation and drug loading, the encapsulation efficiency of GastroShield polypeptide and anti-TNF-α antibody reached 84.2±1.7% and 78.5±2.1%, respectively, and the drug loading rate was 5.9±0.3% and 3.2±0.2%. The detection method is reliable, providing a guarantee for effective drug delivery. The enteric release experiment showed that the preparation released very little in artificial gastric fluid, less than 5% within 2 hours, while in artificial intestinal fluid and colonic fluid, the release accelerated with the increase of pH, and the 4-hour release rate in colonic fluid was as high as 95.3±1.8%, which is consistent with the colon-targeted design. In terms of probiotic activity, the activity retention rate after freeze-drying exceeded 90%, and it showed good tolerance in simulated gastrointestinal fluid, ensuring the microecological regulation function. Stability experiments showed that after 30 days of accelerated storage, the activity retention rates of GastroShield peptide and anti-TNF-α antibody were 85.2±4.1% and 82.3±3.1%, respectively, and the number of viable probiotic bacteria remained at 9.2×10 10 ±5.1×10 9 CFU / g. Overall, the preparation has excellent encapsulation, targeted release, activity maintenance and stability, and has high development and application value.

[0113] Conclusion: The multi-layered enteric-coated microparticle preparation established in this example provides an innovative treatment strategy for complex intestinal diseases such as inflammatory bowel disease through the triple mechanism of "targeted delivery-intelligent release-microecological regulation" and has good drug development potential.

[0114] Example 3: GastroShield compound preparation for the treatment of DSS-induced ulcerative colitis in mice

[0115] 1. Experimental Design and Model Establishment

[0116] 1. Experimental Animals and Grouping

[0117] SPF-grade C57BL / 6 male mice, 8 weeks old, weighing 18-22 g, were selected and housed in an SPF-grade barrier facility with an ambient temperature controlled at 22 ± 2°C and a humidity maintained at 50 ± 10% under a 12-h light-dark cycle. Fifty mice were randomly divided into six groups (n = 10):

[0118] Table 11 Experimental groups and treatments

[0119]

[0120] 2.DSS Modeling Solution

[0121] Prepare 3% (w / v) DSS solution using sterile water and prepare fresh every day to ensure accurate concentration. Figure 6) are as follows: starting from day 0, mice were adaptively fed for 3 days, during which time they were weighed on an empty stomach at the same time every day (9 a.m.) and recorded accurately to 0.1 g; from day 1 to day 7, except for the normal control group, mice in other groups were given 3% DSS drinking water and corresponding interventions (gavage) were performed according to the group; on day 8, all mice were euthanized and samples were quickly collected.

[0122] 2. Detection indicators and experimental steps

[0123] 1. Disease Activity Index (DAI) Scoring Criteria and Dynamic Monitoring: Independent scoring is performed based on weight change, stool consistency, and occult / visible blood, with a total score of 12 points. Mice are scored at the same time each day (9:00 AM). The specific scoring criteria are as follows:

[0124] Table 12 Specific scoring rules

[0125] index 0 points 1 point 2 points 3 points 4 points weight changes No decline 1-5% decrease 5-10% drop 10-20% drop Decrease > 20% stool consistency Forming Soft but formed semi-formed loose stools watery stools Occult blood / visible blood Negative (occult blood-) Occult blood+ Hidden Blood++ Visible bloodstains Heavy bleeding

[0126] 2. Colon length measurement and tissue specimen preparation: After euthanasia of mice, the entire colon (from the anus to the cecum) was quickly dissected to expose the contents. The contents were carefully rinsed with PBS, dried with filter paper, and the colon length was accurately measured to 0.1 cm with a ruler. The colon was then divided into three parts for processing: the proximal 1 / 3 was fixed in 4% paraformaldehyde for 24 hours for subsequent paraffin embedding; the middle 1 / 3 was frozen in a -80°C refrigerator for inflammatory factor detection; and the distal 1 / 3 was sterilely collected for feces for intestinal flora analysis. The reference value of normal mouse colon length is 8.0±0.5 cm (n=10).

[0127] 3. Histological scoring and HE staining process: Histological scoring is based on three indicators: mucosal damage, glandular structure, and inflammatory infiltration. It uses a 0-10 score system, with lower scores indicating less severe damage. The specific scoring criteria are as follows:

[0128] Table 13 Specific scoring criteria

[0129] index 0 points 2-4 points 6-8 points 10 points Mucosal damage whole Partial erosion Extensive erosion Ulcer formation Glandular structure normal Slightly reduced Severe reduction Gland disappearance Inflammatory infiltration none A small amount of mucosal layer Mucosa + submucosa Full-thickness infiltration + abscess

[0130] The HE staining steps were as follows: first, the tissue was made into 5 μm paraffin sections and baked at 60°C for 2 h; then, the sections were dewaxed with xylene and hydrated through gradient ethanol; after staining with Harris hematoxylin for 5 min, the sections were differentiated with 1% hydrochloric acid ethanol; then, the sections were stained with eosin for 3 min, dehydrated with gradient ethanol and transparentized with xylene; finally, the sections were sealed with neutral gum and observed under a Leica DM2500 microscope at ×200 magnification.

[0131] 4. Inflammatory cytokine ELISA Assay: 100 mg of colon tissue was added to 1 mL of PBS and homogenized at 8000 rpm for 2 minutes. The sample was then centrifuged at 12000 × g for 15 minutes at 4°C and the supernatant was collected. Protein concentration was determined using the BCA assay, and the sample was uniformly adjusted to 1 mg / mL. Inflammatory cytokines such as IL-1β, TNF-α, and IL-6 were assayed strictly according to the instructions of the commercially available kits.

[0132] 5. 16S rRNA sequencing of intestinal flora: DNA was extracted from a 50mg stool sample using the QIAamp Fast DNA Stool Kit. DNA integrity was assessed by 1% agarose gel electrophoresis. DNA concentration was determined using Nanodrop to ensure the A260 / 280 ratio was within the range of 1.8-2.0. Sequencing parameters were as follows:

[0133] Table 14 Sequencing parameters

[0134]

[0135] 3. Experimental Results

[0136] 1. DAI score and colon length experiment: The inventive group performed best in terms of DAI score and colon length recovery, with a DAI score of only 2.8±0.3 on day 7 and a colon length recovery rate of 95%, significantly superior to both the commercially available group and the no-peptide control group. This demonstrates that the GastroShield compound formulation can effectively alleviate ulcerative colitis symptoms and promote colon tissue repair, with the GastroShield polypeptide playing a key synergistic role. This is shown in Table 15.

[0137] Table 15 Colon length and DAI score results

[0138] Group DAI score (Day 7) Colon length (cm) Recovery rate (%) Normal control 0.1±0.1 8.0±0.5 100 Model Blank 8.9±0.6 4.7±0.4 59 control group 8.7±0.5 4.8±0.3 60 Commercially available group 5.9±0.4 6.1±0.4 76 The present invention group 2.8±0.3 7.6±0.3 95 No peptide control group 4.5±0.4 6.8±0.3 85

[0139] The statistical method used was one-way ANOVA with Tukey's post hoc test. The results showed that the p value was < 0.01 when the group of the present invention was compared with the commercial group; p value was < 0.001 when the group of the present invention was compared with the control group; and p value was < 0.05 when the group of the present invention was compared with the control group without polypeptide.

[0140] 2. Histological Scoring and Mucosal Healing Rate Experiment: Histological observations showed that the mucosal healing rate in the group treated with the present invention was as high as 95%, with the lowest histological score. This indicates that the preparation can promote the recovery of mucosal and glandular structures and alleviate inflammatory infiltration. In contrast, the mucosal healing rate in the control group without the peptide was only 70%, confirming that the GastroShield peptide is essential for tissue repair. This is shown in Table 16.

[0141] Table 16 Histological scores and mucosal healing rates

[0142]

[0143] 3. Inflammatory Factor Detection Experiment Summary: Levels of inflammatory factors such as IL-1β, TNF-α, and IL-6 in the GastroShield group were significantly lower than those in the commercially available group and the non-peptide control group, demonstrating superior anti-inflammatory efficacy. GastroShield peptides synergize with Adalimumab to effectively block inflammatory signaling pathways and inhibit inflammatory responses, as shown in Table 17.

[0144] Table 17 Inflammatory factor levels (pg / mg protein) test results

[0145] Group IL-1β TNF-α IL-6 Normal control 5.2±1.3 8.7±2.1 3.8±0.9 Model Blank 75.6±7.8 82.3±9.1 68.5±8.3 control group 72.4±8.2 79.5±8.5 65.7±7.9 Commercially available group 35.1±4.3 38.6±5.2 32.4±4.1 The present invention group 14.3±2.1 16.8±2.5 12.7±1.8 No peptide control group 25.6±3.2 28.9±3.5 22.4±2.8

[0146] 4. Intestinal Microbiome Analysis Experimental Summary: Regarding the intestinal microbiome, the Shannon index of the inventive group approached normal levels, with a significant increase in the abundance of beneficial bacteria such as Bifidobacterium and Akkermansia, and a significant decrease in pathogenic bacteria such as Proteobacteria. The improvement in the microbiome of the no-peptide control group was less pronounced, suggesting that the GastroShield peptide helps reshape a healthy intestinal microbiome and enhance intestinal barrier function. This is shown in Tables 18 and 19.

[0147] Table 18 Diversity (Shannon Index)

[0148] Group Index value Compared with the control group Normal control 5.8±0.4 - Model Blank 2.3±0.2 -43％ control group 2.1±0.3 -47％ Commercially available group 3.5±0.4 -21％ The present invention group 5.2±0.3 -10% (close to normal) No peptide control group 4.2±0.3 -28％

[0149] Table 19 Relative abundance of key bacterial genera (%)

[0150] Genus control group Commercially available group The present invention group Normal control No peptide control group Bifidobacterium 1.2±0.3 4.6±0.8 11.6±1.5 12.3±1.8 6.5±0.8 Akkermansia 0.8±0.2 1.7±0.4 5.0±0.9 5.5±1.2 2.8±0.6 Enterococci 8.7±1.5 5.1±1.2 1.0±0.3 0.5±0.1 3.2±0.7 Proteobacteria 15.6±2.3 7.6±1.8 3.7±0.9 2.1±0.5 6.8±1.2

[0151] 4. Experimental Conclusion

[0152] This study, by adding a peptide-free control group, clarified the synergistic effects of the various components in the GastroShield compound formulation. The results showed that this formulation significantly outperformed both the commercially available formulation and the peptide-free control group in alleviating ulcerative colitis symptoms, repairing colon tissue, reducing inflammatory cytokine levels, and remodeling the intestinal flora.

[0153] GastroShield peptide plays a key role in this, with its targeted antibacterial, anti-inflammatory, and microbiome-modulating properties, forming a multi-mechanism synergistic therapeutic system with probiotics and Adalimumab. This study provides a new and effective treatment for ulcerative colitis.

[0154] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A GastroShield tetrafunctional fusion polypeptide, characterized in that: The amino acid sequence of the fusion polypeptide is shown in SEQ ID NO:

1.

2. A GastroShield composite targeted enteric-coated microparticle preparation, calculated as 1g of dry powder, characterized in that: The formulation comprises the following components: (1) Probiotics: Bifidobacterium animalis BLa80 4×10 10 CFU / g, Lactobacillus plantarum Lp903×10 10 CFU / g, Lactobacillus casei ZM15 3×10 10 CFU / g; (2) Biologically active components: 60 mg / g of the GastroShield tetrafunctional fusion polypeptide described in claim 1 and 15 mg / g of anti-TNF-α antibody; (3) Biosynergistic factors: galacto-oligosaccharide 200 mg / g, magnesium oxide 50 mg / g, baicalin 50 mg / g; The preparation adopts a three-layer coating structure, the first layer is sodium alginate embedded with GastroShield polypeptide and probiotics, the second layer is a chitosan-anti-TNF-α antibody cross-linking layer, and the third layer is Eudragit FS30D enteric coating.

3. The method for preparing the GastroShield composite targeted enteric-coated microparticle preparation according to claim 2, characterized in that: The method comprises the following steps: Step 1: Inoculate each probiotic in MRS culture medium and culture until the logarithmic growth phase. After centrifugation, resuspend to the target concentration, combine the three bacterial cultures, and add 5% trehalose and 3% mannitol as protective agents. Step 2: Prepare a 2% sodium alginate solution by mass / volume ratio, sterilize it through a 0.22 μm filter membrane, add the GastroShield tetrafunctional fusion polypeptide, and mix thoroughly. Use a syringe pump to drip the bacterial suspension-sodium alginate mixture into a 0.2 M CaCl2 solution at a flow rate of 1 mL / min. Cross-link the solution under magnetic stirring at 300 rpm. After cross-linking, wash the solution three times with sterile water, centrifuge it at 4°C and 3000 × g for 5 minutes, and collect the microspheres for later use. Step 3: Dissolve 0.5% chitosan in 1% acetic acid solution, adjust the pH to 5.5 with 1M NaOH, add anti-TNF-α antibody to a final concentration of 1.5 mg / mL, and stir on ice for 10 minutes. Activate with EDC / NHS at a molar ratio of EDC:NHS:chitosan amino groups = 5:5:1 at room temperature for 30 minutes, then immediately mix with the first layer of microspheres and couple at 37°C, 150 rpm, and shake for 30 minutes. Step 4: Prepare an emulsion in the ratio of Eudragit FS30D: triacetin: polysorbate 80 = 3:1:1, and coat with fluidized bed spray coating technology, controlling the inlet temperature at 35°C, the outlet air temperature at 28°C, the spray rate at 2.5 mL / min, and the air volume at 15 m 3 / h, forming an enteric coating with a thickness of 5±0.5μm; Step 5: Pre-freeze at -20℃ for 2h, then freeze-dry at -45℃ for 24h. The particle size of the dried microspheres is 80-150μm. After vacuum packaging, store at 2-8℃ for later use.

4. Use of the GastroShield tetrafunctional fusion polypeptide according to claim 1 in the preparation of a drug for treating inflammatory bowel disease.

5. Use of the GastroShield composite targeted enteric-coated microparticle preparation according to claim 2 in the preparation of a drug for treating inflammatory bowel disease.

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