Probiotic-polyphenol nanoparticle colon-targeted co-delivery system as well as preparation method and application thereof

Abstract

The invention discloses a probiotic-polyphenol nanoparticle colon-targeted co-delivery system as well as a preparation method and application thereof, and belongs to the field of biological medicines. The FCPs and hyaluronic acid (HA) are combined through non-covalent interaction, so that the hydrogel has colon targeting property and mucosal adhesion at the same time, and delivery of probiotics is facilitated. The hydrogel (HF) based on polysaccharide has good biocompatibility, and can be used for effectively encapsulating the probiotics and the natural products. Then, the PDA-TH NPs and BA are incorporated into the polysaccharide chain network of HF, and finally the BA-HF-PDAT targeted co-delivery system is constructed. The BA (at) HF-PDAT targeted co-delivery system can protect BA from serious gastrointestinal stress, and is jointly assembled with PDT-TH NPs to realize dual slow release of thymol (Thy), so that the treatment effect of BA and Thy is improved, and the BA (at) HF-PDAT targeted co-delivery system contributes to treatment of clostridium difficile infection (CDI).

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a probiotic-polyphenol nanoparticle colon-targeted co-delivery system, its preparation method, and its application. Background Technology

[0002] Clostridium difficile infection ( Clostridium difficile CDI (Clostridium difficile infection) is caused by Clostridium difficile (C. difficile). difficile Clostridium difficile (CDI) is an intestinal disease caused by bacteria. Clinical manifestations include diarrhea, pseudomembranous colitis, colonic perforation, and even death, posing a significant threat to public health. The primary treatment for CDI is antibiotic therapy, including metronidazole, dapoxetine, and vancomycin. However, antibiotic treatment can further disrupt the gut microbiota balance, with approximately 15-30% of CDI patients experiencing recurrent *C. difficile* infection (rCDI). Currently, *C. difficile* is one of the most serious threats to antibiotic resistance. Despite ongoing progress in hospital management, the incidence of CDI continues to rise at a rate exceeding 4% annually. Approximately 453,000 cases of CDI are reported annually, including 83,000 recurrent cases, resulting in 29,300 deaths or prolonged hospital stays, thus creating a significant public health burden. Studies have shown that CDI disrupts the gut microbiota and impairs metabolic function. In the pathological context of CDI, *C. difficile* maintains a competitive advantage through various virulence factors such as toxins, adhesins, and biofilms, significantly inhibiting the proliferation and colonization of beneficial bacteria. During infection, the toxin (TcdB) produced by Clostridium difficile binds to the Frizzled (FZD) receptor, inhibiting the Wingless / Integrated (Wnt) signaling pathway, disrupting intestinal epithelial cell proliferation, and exacerbating the condition. Therefore, disruption of the intestinal microecology and damage to colonic tissue are closely related to the pathogenesis and progression of CDI.

[0003] Fecal microbiota transplantation (FMT) restores the gut microbiota of patients with chronic disease of the second disease (CDI) by transferring gut microbiota from healthy individuals. Despite its high efficacy, FMT has significant limitations, particularly safety concerns, including the unintended spread of drug-resistant bacteria. Furthermore, traditional probiotic therapies are often ineffective in eliminating pathogenic bacteria or achieving satisfactory therapeutic outcomes, largely due to bacterial virulence factors and the adverse environment of the digestive tract. In addition, TcdB damages the tissue repair process, exacerbating disease severity and accelerating the progression of CDI. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a probiotic-polyphenol nanoparticle colon-targeted co-delivery system, its preparation method, and its application.

[0005] Probiotic therapy can regulate the gut microbiota and alleviate CDI-induced colitis by inhibiting the growth and colonization of Clostridium difficile. Bifidobacterium adolescentis (…) Bifidobacterium adolescentis Bacillus subtilis (BA), a probiotic, can inhibit the adhesion of Clostridium difficile to intestinal epithelial cells in vitro, highlighting its potential as a promising candidate for the treatment of CDI. Furthermore, oral administration of BA can activate the Wnt / β-catenin signaling pathway, thereby enhancing intestinal epithelial cell renewal in an aging mouse model. However, oral BA activity is significantly impaired under harsh conditions of gastric acid and small intestinal fluid, hindering its colonization, increasing its accumulation in the colon, and reducing therapeutic efficacy. Therefore, innovative strategies are urgently needed to enhance the ability of oral probiotics to overcome competition from Clostridium difficile, promote its proliferation and colonization in the gut, occupy its niche, and reshape the gut microbiota and metabolism.

[0006] Several bioactive compounds have shown potential as alternative therapies for patients with chronic debilitating diseases (CDI). Thymol (Thy), a natural plant bioactive substance, effectively reduces the number of Clostridium difficile in human fecal models without adversely affecting other beneficial bacteria. Oral administration of Thy and carvacrol increases the relative abundance of Bifidobacteria in the gut microbiota, thereby alleviating DSS-induced colitis in mice. However, as a phenolic compound, Thy has low bioavailability. This limitation can be addressed by using nanoparticles, which can significantly increase the specific surface area of ​​the drug, improve its solubility, and thus enhance its bioavailability when administered orally. The use of probiotics and bioactive compounds as alternative treatments for CDI is limited by the successful delivery of these materials to specific intestinal regions, such as the colon. Appropriate delivery systems utilize biomaterials such as food-grade polysaccharides, amino acids, and proteins to encapsulate probiotics, ensuring their resistance to physical and chemical stressors during ingestion and intestinal transport, while promoting colonization and growth of probiotics in the colon. These systems primarily address challenges in the traditional gastrointestinal tract, including stomach acid, bile acids, lysozyme, and reactive oxygen species, while neglecting the critical colonization barriers caused by competition between probiotics and pathogens in the colonic microenvironment of infectious bowel diseases.

[0007] To this end, the present application develops a multifunctional probiotic-nanoparticle co-delivery platform (BA@HF-PDAT) for the treatment of CDI. Briefly, ficus carica leaf polysaccharides (FCPs) were extracted, which have anti-inflammatory and antioxidant properties. FCPs are rich in rhamnose (Rha), a biocompatible polysaccharide that remains stable in gastric and small intestinal fluids and serves as a substrate for the production of beneficial metabolites by colonic bacteria, including short-chain fatty acids and lactic acid. The present application creatively combines FCPs with hyaluronic acid (HA) through non-covalent interactions, enabling the hydrogel to have both colon targeting and mucosal adhesion, which is very beneficial for the delivery of probiotics. This polysaccharide-based hydrogel (HF) has good biocompatibility and can effectively encapsulate probiotics and natural products. Subsequently, PDA-TH NPs and BA are incorporated into the polysaccharide chain network of HF, and finally a BA@HF-PDAT co-delivery platform is constructed. The BA@HF-PDAT co-delivery platform can protect BA from severe gastrointestinal stress and co-assemble with PDT-TH NPs to double release Thy, which helps to improve the therapeutic effect of BA and Thy.

[0008] Therefore, BA and Thy treat CDI through the synergistic effect of inhibiting the growth and colonization of C. difficile and modulating intestinal cell proliferation. The present application applies BA@HF-PDAT in a mouse model. The results show that PDA-TH NPs can effectively inhibit the growth and toxin production of C. difficile, while HF can significantly enhance the resistance of BA to gastrointestinal stress. BA@HF-PDAT adapts to the specific colon conditions associated with CDI, enabling the targeted release of PDA-TH NPs and BA in inflamed colon, enhancing BA colonization, restoring the diversity and metabolic function of the intestinal microbiota in mice. Through a cell culture model, it is proved that BA@HF-PDAT can effectively scavenge reactive oxygen species (ROS), improve the survival rate of colon cells, activate the Wnt / β-catenin signaling pathway, modulate intestinal cell proliferation, and reduce CDI-induced apoptosis. These findings confirm that this multifunctional co-delivery platform is superior to single-dose probiotics or nanoparticle treatment.

[0009] To achieve the above-mentioned purpose, the technical scheme of the present application is: In a first aspect, the present application provides a probiotic-polyphenol nanoparticle colon-targeted co-delivery system (BA@HF-PDAT), comprising a probiotic polysaccharide composite gel (BA@HF) and a polyphenol nanoparticle (PDA-TH NPs), the polyphenol nanoparticle encapsulating a polyphenol to be delivered, the probiotic polysaccharide composite gel encapsulating a probiotic to be delivered, and the polyphenol nanoparticle and the probiotic being co-encapsulated in the polysaccharide composite gel; The probiotic polysaccharide composite gel is formed by combining a probiotic and a polysaccharide gel (HF); The polyphenol nanoparticles are composed of polyphenols and polydopamine nanoparticles (PDA NPs).

[0010] The PDA-TH NPs and the BA are jointly encapsulated in a network structure composed of interwoven polysaccharide chains. Most of the BA is uniformly distributed in the HF, and a small part is exposed on the outer surface of the BA@HF, and the total encapsulation rate reaches 95%.

[0011] The PDA-TH NPs interact with the polysaccharide skeleton through non-covalent interaction, which enhances the structural integrity of the co-delivery system, and the phenolic hydroxyl group acts as a molecular bridge to enhance the polysaccharide network.

[0012] The probiotic is Bifidobacterium adolescentis, which can reduce the adhesion of Clostridium difficile to the colon cell line and reduce cell damage in vitro, and other probiotics (such as Bifidobacterium pseudolongum) do not have this effect.

[0013] The polyphenol is thymol (Thy), and studies have shown that thymol has an anti-Clostridium difficile effect and does not affect the abundance of other probiotics in a human fecal culture model, and other phenolic substances (such as resveratrol) do not have this effect.

[0014] The polydopamine nanoparticles (PDA NPs) have a porous spherical morphology and are negatively charged on the surface, with an average particle size of 300-400 nm. This helps them have high dispersibility and can effectively load polyphenols.

[0015] The polyphenol nanoparticles (PDA-TH NPs) have an irregular nanospherical morphology, with an average particle size of 100-300 nm, preferably 180-220 nm, and most of the particle sizes are in the range of 20-60 nm.

[0016] In a second aspect, the present application provides a preparation method of a probiotic-polyphenol nanoparticle colon-targeted co-delivery system (BA@HF-PDAT), comprising the following steps: S1, water-alcohol precipitation method is used to extract ficus carica leaf polysaccharide (FCPs), and the ficus carica leaf polysaccharide (FCPs) and hyaluronic acid (HA) are placed in water, mixed sterilely to obtain a polysaccharide solution (HF); the BA is mixed with the HF, and sterile stirring is performed to form BA@HF; S2, PDA NPs are prepared by self-polymerization, the PDA NPs are suspended in water, Thys are added, ultrasonic stirring is performed, and PDA-TH NPs are obtained; S3, the BA@HF and the PDA-TH NPs are co-incubated, sterile stirring is performed, and BA@HF-PDAT is obtained.

[0017] The hyaluronic acid (HA) in step S1 can target the CD44 inflammatory receptor, play an inflammatory targeting role, and help the release of BA and Thy at the inflammatory site, and sodium alginate has no such effect.

[0018] In one or more embodiments, in step S1, the specific preparation process for extracting ficus carica leaf polysaccharides (FCPs) by using the water-alcohol precipitation method is as follows: (1) Soak the air-dried ficus carica leaves in alcohol, remove the alcohol solvent, and dry; (2) Crush the dried ficus carica leaves, soak them in hot water, filter the residue, and obtain a crude extract; (3) Concentrate the crude extract by rotary evaporation, add an alcohol solvent to precipitate the polysaccharides in the crude extract, alcohol precipitate the extract at low temperature, centrifuge after removing the supernatant, and then wash after removing the supernatant; (4) Remove free proteins from the crude extract by the Sevage method, separate the water phase from the denatured protein layer, repeat multiple times; swell the obtained solution in ultrapure water overnight, then place it in a semi-permeable membrane dialysis bag, seal the dialysis bag, and place it in a beaker containing ultrapure water, dialyze for 12-24 h to remove small molecules, then concentrate the liquid by rotary evaporation, and freeze-dry under vacuum to obtain ficus carica leaf polysaccharides (FCPs).

[0019] As a further technical solution, in step (1), the alcohol is ethanol, and the soaking time in ethanol is 1-3 h. The drying method is vacuum drying, and the drying temperature is 40-60°C, and the drying time is 12-48 h, preferably 24-48 h.

[0020] As a further technical solution, in step (2), the hot water temperature is 70-90°C, and the soaking time is 2-4 h, which is more conducive to obtaining a crude extract.

[0021] As a further technical solution, in step (3), the alcohol solvent is anhydrous ethanol with a final concentration of 75-85%, which is more conducive to the precipitation of polysaccharides in the crude extract. The extract is alcohol precipitated at low temperature 1-5°C for 10-14 h.

[0022] In one or more embodiments, in step S1, the ratio of ficus carica leaf polysaccharides (FCPs), HA, and water is (80-120 mg):(180-220 mg):(10-30 mL), preferably (90-110 mg):(190-210 mg):(15-25 mL). The appropriate ratio helps to exert the gastrointestinal protective effect on probiotics.

[0023] In one or more embodiments, in step S1, the sterile mixing is performed for 1-5 hours. The concentration of the polysaccharide solution (HF) is 100-200 mg / mL, such as 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, etc., preferably 140-160 mg / mL. An appropriate concentration helps to exert a protective effect while not being too viscous to be orally taken.

[0024] In one or more embodiments, in step S1, the volume ratio of the probiotic bacteria to the polysaccharide solution (HF) is (5-15):(5-15), preferably (8-12):(8-12). The concentration of the probiotic bacteria is 0.5x10 8 ~1.5x10 8 CFU / mL. An appropriate ratio of the two helps the probiotic bacteria to be uniformly dispersed in the polysaccharide gel system and the activity not to be affected by the antibacterial activity of Thy itself.

[0025] In one or more embodiments, in step S1, the sterile stirring is performed for 20-40 minutes, and the BA is wrapped in the HF to form BA@HF.

[0026] In one or more embodiments, in step S2, the specific steps for preparing the PDA NPs by the self-polymerization method include: dissolving poloxamer, dopamine hydrochloride and 1,3,5-trimethylbenzene in an alcohol solution, ultrasonically treating the mixture, then adding ammonia water, sterile stirring at room temperature, and then centrifuging, washing and drying to obtain the PDA NPs. This method realizes the preparation of PDA NPs with controllable size by inducing the self-oxidation and polymerization of dopamine in an alcohol-water system stabilized by a surfactant (poloxamer) under an alkaline environment provided by ammonia water, while 1,3,5-trimethylbenzene as an organic phase helps to form a stable interface. The synergistic effect of the components in this system ensures the stability, dispersibility and structural integrity of the nanoparticles during synthesis. The main purpose of adding ammonia water is to adjust the pH to provide an alkaline environment (pH=9.0) to induce the oxidation and self-polymerization reaction of dopamine.

[0027] As a further technical solution, the alcohol is a 50% ethanol solution, which refers to a 50wt% ethanol aqueous solution.

[0028] As a further technical solution, the ratio of poloxamer, dopamine hydrochloride, 1,3,5-trimethylbenzene, alcohol solution, and ammonia water is (0.8-1.5 g):(0.3-0.7 g):(1-5 mL):(30-50 mL):(2-8 mL), preferably (0.9-1.1 g):(0.4-0.6 g):(1-3 mL):(35-45 mL):(4-6 mL).

[0029] As a further technical solution, the ultrasonic treatment is performed for 10-30 min to achieve uniform mixing.

[0030] In one or more embodiments, in step S2, the ratio of PDA NPs, water, and polyphenol is (10-30 mg):(5-20 mL):(40-60 mg), preferably (15-25 mg):(8-12 mL):(45-55 mg). The ratio of PDA NPs to Thy is crucial. Too low a ratio of Thy can result in insufficient nanoparticle encapsulation, fast release rate, poor persistence, and possibly insufficient antibacterial or anti-inflammatory effect. Too high a ratio of Thy can result in excess PDA that may adsorb too much Thy, forming agglomerates, affecting dispersibility and bioavailability.

[0031] In one or more embodiments, in step S2, the ultrasonic treatment is performed for 10-30 min, and the stirring is performed for 1-5 h to achieve sufficient mixing reaction.

[0032] In one or more embodiments, in step S3, the sterile stirring is performed for 10-40 min, preferably 20-30 min, to promote the self-assembly of PDA-TH NPs and BA@HF and construct the polyphenol-probiotic co-delivery system BA@HF-PDAT.

[0033] In a third aspect, the present application provides a use of the above-mentioned probiotic-polyphenol nanoparticle colon-targeted co-delivery system in the preparation of a product for co-delivering polyphenol and probiotics.

[0034] In a fourth aspect, the present application provides a probiotic-polyphenol nanoparticle colon-targeted co-delivery preparation, comprising the above-mentioned probiotic-polyphenol nanoparticle colon-targeted co-delivery system.

[0035] In a fifth aspect, the present application provides a use of the above-mentioned probiotic-polyphenol nanoparticle colon-targeted co-delivery system or the above-mentioned preparation in the preparation of a medicament for treating Clostridium difficile infection.

[0036] In a sixth aspect, the present application provides a method for treating Clostridium difficile infection (CDI), the method comprising administering the above-mentioned probiotic-polyphenol nanoparticle colon-targeted co-delivery system or the above-mentioned probiotic-polyphenol nanoparticle colon-targeted co-delivery preparation to a subject.

[0037] One or some of the above technical solutions have the following advantages or beneficial effects: (1) The present application provides a probiotic-polyphenol nanoparticle colon-targeted co-delivery system (BA@HF-PDAT), which comprises a probiotic polysaccharide composite gel (BA@HF) and a polyphenol nanoparticle (PDA-TH NPs), the polyphenol nanoparticle encapsulates the polyphenol to be delivered, the probiotic polysaccharide composite gel encapsulates the probiotic to be delivered, and the polyphenol nanoparticle is encapsulated on the outer surface of the probiotic polysaccharide composite gel. Wherein, FCPs are combined with hyaluronic acid (HA) through non-covalent interaction, so that the hydrogel has colon targeting and mucosal adhesion at the same time, which is very beneficial to the delivery of probiotics. This polysaccharide-based hydrogel (HF) has good biocompatibility and can effectively encapsulate probiotics and natural products. Subsequently, PDA-TH NPs and BA are incorporated into the polysaccharide chain network of HF, and finally a BA@HF-PDAT co-delivery platform is constructed. The BA@HF-PDAT co-delivery platform can protect BA from severe gastrointestinal stress, and is assembled with PDT-TH NPs to double-release thymol (Thy), which helps to improve the therapeutic effect of BA and Thy.

[0038] (2) The present application develops a new colon-targeted hydrogel co-delivery platform (BA@HF-PDAT). The platform shows high efficacy in the precise treatment of CDI. PDA-TH NPs capable of inhibiting the virulence factors of C. difficile are designed and synthesized, and are combined with BA into a polysaccharide hydrogel for co-delivery. BA@HF-PDAT shows strong stability under harsh gastrointestinal conditions and preferentially adheres to the inflamed mucosal site for a long time. In addition, the platform inhibits the expression of C. difficile virulence factors, thereby reducing its ecological competitiveness, and enhances the colonization of BA. In a CDI mouse model, oral administration of BA@HF-PDAT can significantly alleviate the colon lesions, oxidative stress and inflammation caused by CDI. Mechanistically, intestinal cell culture models, 16S rRNA sequencing and non-targeted metabolomics show that BA@HF-PDAT effectively restores microbial diversity and metabolic function, especially amino acid and bile acid metabolism, and reshapes the intestinal microenvironment. Through the Wnt / β-catenin and Bax / Bcl-2 pathways, epithelial regeneration is activated, cell apoptosis is inhibited, and tissue repair and barrier recovery are promoted. This multifunctional strategy integrates pathogen clearance, intestinal microecological remodeling and restoration of epithelial cell proliferation capacity, providing a promising and safe alternative to traditional CDI treatment. In addition, it provides a valuable framework for developing treatment strategies for other bacterial gastrointestinal diseases. BRIEF DESCRIPTION OF DRAWINGS

[0039] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated herein by reference. The embodiments of the present application, and their

[0040] Figure 1 A schematic diagram showing the BA@HF-PDAT treatment of C. difficile infection in the embodiments of the present application; Figure 2 Characterization of PDA-TH NPs of the present application; wherein, A is a schematic diagram of PDA-TH NPs and BA@HF-PDAT, B is a TEM image of PDA NPs, C is the particle size and zeta potential of PDA NPs, D is a HRTEM image of PDA-TH NPs, E is the particle size and zeta potential of PDA-TH NPs, F is an elemental mapping of PDA-TH NPs, G is the MIC of PDA-TH against C. difficile, H is the inhibitory effect of PDA-TH on C. difficile biofilm formation, I is the MBC of PDA-TH against C. difficile, J is the effect of PDA-TH on C. difficile toxin-related genes at 12 h, K is the effect of PDA-TH on C. difficile morphology, data are expressed as mean ± SEM (n = 3); Figure 3 Characterization of BA@HF-PDAT of the present application; wherein, A is a schematic diagram of BA@HF and BA@HF-PDAT, B is a whole SEM image of BA@HF-PDAT, C is a cavity SEM image of BA@HF-PDAT, D is a surface SEM image of BA@HF-PDAT, E is the Zeta potential of BA@HF and BA@HF-PDAT, F is the infrared spectroscopy analysis of BA, BA@HF and BA@HF-PDAT, G is the XRD pattern of BA@HF and BA@HF-PDAT, H is the viscosity of BA@HF and BA@HF-PDAT, I is the modulus of BA@HF and BA@HF-PDAT, J is the thermogravimetry of BA@HF and BA@HF-PDAT, K is the T-AOC of BA@HF and BA@HF-PDAT, L is the effect of BA@HF-PDAT on BA growth, M is the survival rate data of BA, BA@HF, BA@HF-PDAT and SGF co-incubation; N is the survival rate picture comparison of BA, BA@HF, BA@HF-PDAT and SGF co-incubation; O is the survival rate of BA, BA@HF and BA@HF-PDAT and SIF co-incubation, P is the ROS scavenging ability of CON, H2O2, BA, PDA-TH, BA@HF, BA@HF-PDAT, data are expressed as mean ± SEM (n = 3); Figure 4BA@HF-PDAT alleviated TcdB-induced cell injury, wherein A is the effect of BA@HF-PDAT treatment on cell viability, B is the effect of TcdB treatment on cell viability, C is a representative image of cell morphology, the scale bar is 1 mm, and D is the effect of BA@HF-PDAT treatment on TcdB-induced reduction in cell viability; Figure 5 BA@HF-PDAT promoted the growth and colonization of probiotics in healthy and CDI mice, wherein A is a schematic diagram of an in vivo and ex vivo fluorescence imaging experimental protocol for healthy mice, B is the fluorescence signal in healthy mice at different time points, C is the ex vivo fluorescence signal of experimental animals at different time points, D is the quantification of the fluorescence signal in healthy mice in vivo for 48 h, E is the quantification of the ex vivo fluorescence signal in the cecum and colon of healthy mice, F is a schematic diagram of an in vivo and ex vivo fluorescence imaging experimental protocol for CDI mice, G is the fluorescence signal in CDI mice at different time points, H is the ex vivo fluorescence signal of CDI mice, I is the quantification of the ex vivo fluorescence signal of CDI mice, and data are represented as mean ± SEM (n = 3); Figure 6 Effect of oral BA@HF-PDAT on intestinal injury induced by CDI; wherein A is the change in body weight after mice were infected with Clostridium difficile and given different treatments, B is the DAI change curve, C is a representative image of the colon and statistical analysis, E is a representative image of HE staining (scale bar is 100 μm), Alcian Blue staining (scale bar is 100 μm), and TEM (scale bar is 200 nm), F is the concentration of TcdA and TcdB in the feces of mice, G is the level of T-AOC, MDA, and SOD in the colon, H is the mRNA expression level of IL-1β, IL-6, and TNF-α in the colon tissue, H is the mRNA expression level of IL-10 and IL-22 in the colon tissue, and data are represented as mean ± SEM (n = 6); Figure 7 Effect of oral BA@HF-PDAT on intestinal barrier integrity and tissue repair in CDI mice; wherein A is a representative immunofluorescence image of Occludin and Claudin-1 (scale bar is 100 μm), B is a representative immunofluorescence image of Ki67 (scale bar is 100 μm), C is a representative immunofluorescence image of C-caspase-3 (scale bar is 100 μm) and a representative transmission electron microscope image of colon cells (scale bar is 200 nm), D is a representative western blot image, E is the quantification and statistical analysis of the western blot image, F is the mRNA expression level of c-Myc and CyclinD1, and data are represented as mean ± SEM (n = 6); Figure 8Effects of oral BA@HF-PDAT on intestinal flora; wherein, A is intestinal flora alpha diversity index, B is intestinal flora NMDS graph, C is intestinal flora composition graph, D is the relative level of Bifidobacterium, Lactobacillus, Clostridium and Proteobacteria, E is LEfSe analysis, F is intestinal flora and index correlation analysis heat map, G is intestinal flora KEGG analysis, data is expressed by mean±SEM (n=6); Figure 9 Effects of oral BA@HF-PDAT on intestinal metabolites; wherein, A is the Venn diagram of different intestinal metabolites, B is the intestinal metabolite difference volcano plot of CDI group and CON group, C is the intestinal metabolite difference volcano plot of BA@HF-PDAT group and CDI group, D is the intestinal metabolite PCA graph, E is the intestinal metabolite KEGG analysis, F is the network analysis of intestinal flora and metabolites, G is the Mantel test analysis heat map between intestinal flora, intestinal metabolites and index, data is expressed by mean±SEM (n=6); Figure 10 PDA-TH NPs, PDA, PDAT related graphs; wherein, A is the TEM image of PDA-TH NPs; B is the HTEM image of PDA-TH NPs; C is the infrared spectrum analysis of PDA, PDAT; D is the XRD graph of PDA-TH NPs; Figure 11 Encapsulation rate and loading concentration of Thy, sterilization mechanism of PDA-TH and the influence of PDA-TH on C. difficile toxin; wherein, A is the encapsulation rate of Thy, B is the loading concentration of Thy, data of A and B is expressed by mean±SEM (n=3); C is the influence of PDA-TH on extracellular ATP level of C. difficile, D is the influence of PDA-TH on extracellular protein level of C. difficile, data of C and D is expressed by mean±SEM (n=6); E is the influence of PDA-TH on C. difficile toxin related genes at 6h, data is expressed by mean±SEM (n=6); Figure 12 Structural formula of raw material and information of ficus carica leaf polysaccharide; wherein, A is the chemical structure of dopamine, thymol and hyaluronic acid; B is the monosaccharide composition of ficus carica leaf polysaccharide; C is the molecular weight of ficus carica leaf polysaccharide, data is expressed by mean±SEM (n=3); D is the total sugar and uronic acid content of ficus carica leaf polysaccharide, data is expressed by mean±SEM (n=3); Figure 13The study aimed to assess the free radical scavenging capacity of BA, BA@HF, and BA@HF-PDAT, the quantitative in vivo fluorescence signal in CDI mice, and the levels of CAT and GSH-Px in colon tissue. Specifically, A represents the scavenging effect of different treatments on DPPH free radicals, B represents the scavenging effect of different treatments on ABTS, and data are expressed as mean ± SEM (n=3); C represents the quantitative in vivo fluorescence signal in CDI mice at different time points, and data are expressed as mean ± SEM (n=3); D represents the levels of CAT and GSH-Px in colon tissue, and data are expressed as mean ± SEM (n=6). Figure 14 For quantitative and statistical analysis of immunofluorescence images, where A is the fluorescence intensity of the closing protein, B is the fluorescence intensity of Claudin-1, C is the fluorescence intensity of Ki 67, and D is the fluorescence intensity of c-caspase-3. Data are expressed as mean ± SEM (n=6). Figure 15 A heatmap of the gut microbiota; Figure 16 A diagram of the gut microbiota composition for predicting phenotypes; Figure 17 A heatmap of sample clustering for intestinal metabolites; Figure 18 Heatmap of intestinal metabolites (FDR<0.05, VIP>1.5); Figure 19 The effect of BA@HF-PDAT on mouse body weight is shown in mean ± SEM (n=6); Figure 20 The biosafety assessment of BA@HF-PDAT is presented in the following categories: A represents the effect of oral BA@HF-PDAT on major organs of mice; B represents the effect of oral BA@HF-PDAT on blood biochemical parameters of mice; C represents the effect of oral BA@HF-PDAT on liver function of mice; and D represents the effect of oral BA@HF-PDAT on kidney function of mice. Data are expressed as mean ± SEM (n=6). Detailed Implementation

[0041] Intestinal tissue injury and disruption of intestinal microecological balance are closely related to the occurrence of Clostridium difficile infection (CDI). Traditional probiotic therapy is often ineffective in eliminating pathogenic bacteria or achieving satisfactory therapeutic effects, which is largely due to the influence of bacterial virulence factors. In addition, Clostridium difficile toxin B (TcdB) impairs the tissue repair process, thereby aggravating the severity of the disease and accelerating the progression of CDI. Against this background, the present application designs and constructs a multifunctional probiotic co-delivery platform (BA@HF-PDAT) that co-delivers Bifidobacterium adolescentis (BA) and thymol nanoparticles (PDA-TH) using polysaccharide-based hydrogel (HF), and systematically evaluates its therapeutic effect on CDI through cell culture and mouse models. BA@HF-PDAT effectively promotes probiotic colonization by enhancing intestinal adhesion, targeted delivery and inhibition of Clostridium difficile virulence factors. BA@HF-PDAT has the characteristics of gastrointestinal stability and few side effects, promotes intestinal repair by regulating intestinal cell proliferation, enhances the intestinal antioxidant and anti-inflammatory capacity, and thus relieves CDI-induced colitis. Most importantly, BA@HF-PDAT shows great potential to restore microbial metabolic function and reconstruct host-microbial interactions. Overall, this integrated co-delivery strategy provides a promising, safe and universal method for precise CDI treatment, providing valuable insights into the management of opportunistic bacterial infections associated with intestinal diseases.

[0042] In the present application, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0043] The following raw material sources are exemplary: Bifidobacterium adolescentis (CICC 6177) was purchased from the China Center of Industrial Culture Collection. Dopamine hydrochloride (CAS: 62-31-7, purity ≥98%), thymol (CAS: 89-83-8, purity ≥98%), Pluronic F-127 (CAS: 9003-11-6, purity ≥98%) were purchased from Shanghai Yuan Ye Biotechnology Co., Ltd. (Shanghai). HA (CAS: 9004-61-9, purity ≥99%) was purchased from Shanghai Maikelin Biochemical Technology Co., Ltd. Fig leaf was provided by Yangling Fig Industry Development Co., Ltd. Kanamycin, colistin, metronidazole, gentamicin, clindamycin, vancomycin and cefoxitin were purchased from Shanghai Ruizhengte Biotechnology Co., Ltd. DiR was provided by Beijing Fubaike Biotechnology Co., Ltd. LR white resin was purchased from Hydchem Biotech Co., Ltd. Clostridium difficile toxin B (TcdB) was provided by Beijing Fubaike Biotechnology Co., Ltd. Clostridium difficileThe ELISA kits for toxins (CDTs) assay were purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd., and the antioxidant capacity assay kit was purchased from Nanjing Jiancheng Technology Co., Ltd. (see Table 1 for details). Primer sequences used in the experiments were all from Sangon Biotech (Shanghai) Co., Ltd. (see Table 2 for details). Antibodies used in animal experiments were purchased from Wuhan Sanying Biotechnology Co., Ltd. and Cell Signaling Technology (CST) Biotechnology Co., Ltd. (USA) (see Table 3 for details). Antigen Unmasking Solution, skim milk powder, Dulbeco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), and Hank's balanced salt solution (HBSS) were all from Thermo Fisher Scientific (Shanghai). Other reagents were analytical grade.

[0044] Table 1 List of test kits used in the experiment

[0045] Table 2 Primer sequences and product lengths of target gene fragments

[0046] Table 3 List of antibodies used in this experiment

[0047] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0048] Example 1 1.1 Extraction of polysaccharides from fig leaves Fig leaf polysaccharides (FCPs) were extracted using a water-ethanol precipitation method. In short, after harvesting, the leaves were air-dried, soaked in ethanol for 2 hours, the ethanol was removed, and the leaves were dried in an oven at 50°C for 48 hours. The dried leaves were then crushed and soaked in hot water at 80°C for 3 hours. Residue was removed by hot filtration to obtain a crude extract. The crude extract was concentrated to 1 / 4 of its original volume using a rotary evaporator, and anhydrous ethanol (80% concentration) was added to precipitate the polysaccharides. The extract was then precipitated at 4°C for 12 hours, and most of the supernatant was removed. The remaining liquid and precipitate were centrifuged at 6000 rpm for 10 minutes, the supernatant was removed, and the extract was washed 2-3 times alternately with ethanol and acetone. Free proteins were removed from the crude extract using the Sevage method. The aqueous phase was separated from the denatured protein layer using a separation funnel, repeated 5 times. The resulting solution was swollen overnight in ultrapure water, then placed in a semi-permeable membrane dialysis bag. The dialysis bag was sealed and placed in a beaker containing ultrapure water for dialysis for 24 hours to remove small molecules. The concentrate was then rotary evaporated and freeze-dried under vacuum to obtain FCPs. The total sugar content in the FCPs was determined using the phenol-sulfuric acid method, and the uronic acid content was determined using the sulfuric acid-carbazole method. The proportion and molecular weight of monosaccharides in the FCPs were determined using high-performance liquid chromatography (HPLC, LC-20AT03030623).

[0049] 1.2 Bacterial Culture Clostridium difficile was cultured in brain heart and brain extract (BHI) medium. After reaching the optimal concentration (OD600 = 0.8), the culture was diluted 10^6 times. 7 The culture medium was inoculated onto Clostridium difficile selective medium (CCFA agar medium supplemented with 2.5% D-cycloserine, 2.5% egg yolk emulsion and 1.6 mg / L cefoxitin) to isolate single colonies of Clostridium difficile. The isolated single colonies were then re-inoculated onto BHI medium and cultured to an appropriate concentration for further analysis.

[0050] Bifidobacterium adolescentis was cultured in De Man, Rogosa, and Sharpe (MRS) media. The optimal concentration was reached. OD600 After (=0.8), dilute the culture medium by 10^ 7 The culture medium was inoculated onto Bifidobacterium adolescentis selective medium (MRS agar medium supplemented with 100 mg / L mupirocin) to isolate single colonies of Bifidobacterium adolescentis. The isolated single colonies were then re-inoculated into MRS medium and cultured to an appropriate concentration for further analysis.

[0051] 1.3 Histopathological Analysis The tissues were accurately positioned during paraffin embedding. The tissue sections with a thickness of 5 pm were deparaffinated, rehydrated, and stained with hematoxylin-eosin. Randomly selected fields of view were observed for each sample. The histomorphology was analyzed and photographed using the Panoramic MIDI system (3DHistech, Budapest, Hungary).

[0052] 1.4 Ultrastructural examination of the colon Transmission electron microscopy (TEM) was used to observe the ultrastructure of the colon. After fixation with 2.5% glutaraldehyde (% is the mass concentration percentage), the tissues were dehydrated with different gradients of ethanol (50%, 70%, 90%, 100%, % is the volume percentage). After dehydration, the tissues were embedded in LR white resin. It is worth noting that during embedding, the colon should be positioned relative to the microvilli. The tissues were cut into 80 nm thick sections using an ultramicrotome (EM UC7). The sections were stained with uranyl acetate and lead citrate. Randomly selected fields of view were observed for each sample using TEM.

[0053] 1.5 Antioxidant capacity analysis The tissue samples (100 mg) were weighed and homogenized with 0.9 mL of normal saline. A 10% homogenate was taken and centrifuged (3000 rpm, 10 min, 4°C). The supernatant was collected. The antioxidant capacity of the tissues was measured using a UV-visible spectrophotometer (UV1100, MAPADA, Shanghai). All the reagent kits were purchased from Nanjing Jiancheng Bioengineering Institute (Table 1). Each sample was measured three times, and the average value was taken.

[0054] 1.6 Immunofluorescence The paraffin block of colon tissue was spread, baked, and dried with a water bath glass slide. The section was cut at 50°C. Then the section was dehydrated and de-stoned with xylene and ethanol. Antigen extraction was performed by heat induction with antigen unmasking solution. 100 μL of sealing solution (500 mg of skim milk powder dissolved in 50 mL of PBS, pH = 7.4) was added to the section for staining. The sealing solution was removed, and the section was washed with TBST buffer. The primary antibody [occludin, claudin-1, Ki67, c-Caspase-3 (primary antibody dilution ratio: 1:2000)] was added to the section, and incubated at 4°C overnight. The primary antibody was removed, and the section was washed with TBST buffer. The section was added with fluorescent secondary antibody [goat anti-rabbit IgG (H+L), (secondary antibody dilution ratio: 1:1000)], and incubated at 26°C for 1 h. The secondary antibody was removed, and the section was washed with TBST buffer. DAPI solution was added to the section, and incubated at 26°C for 10 minutes in the dark. The DAPI solution was removed, and the section was washed with TBST buffer. Anti-fading mounting medium (G1401, Servicebio, Hubei, China) was added. Finally, the fluorescence intensity of the section was observed and analyzed by laser confocal microscope.

[0055] 1.7 Real-time quantitative PCR (qRT-PCR) Total RNA (100 mg) was isolated from each sample using a total RNA isolation kit (RC112-01, Vazyme). The concentration and purity of the total RNA were detected by a nanophotometer (840-317400). The total RNA of each sample was converted to cDNA using HiScript III RTSuperMix of a qPCR kit (R323-01, Vazyme). qRT-PCR was performed on the cDNA of each sample using an AceQ qPCR SYBR Green Master Mix kit (Q111-02, Vazyme). The gene number of mice was from NCBI, and the gene primer was purchased from Shengong Bioengineering (Shanghai) Co., Ltd. (Table 2). qRT-PCR was performed using a PCR system (ABI7500) according to the protocol. The mRNA expression of related genes was detected by the Ct method, and normalized to the expression of β-actin. -ΔΔ Ct method to detect the mRNA expression of related genes, and normalized to the expression of β-actin.

[0056] 1.8 Western blotting Proteins were extracted from supernatant with RIPA lysis and extraction buffer (89900, Thermo Scientific). Protein concentration was determined using total protein assay kit (A045-4-2, Nanjing Jiancheng Bioengineering Institute). Proteins were separated according to their molecular weight in SDS-PAGE gels under the influence of electric voltage (75 V, 30 min and 120 V, 75 min). Proteins on the gel were then transferred to polyvinylidene difluoride (PVDF) membranes for blotting. Antibodies were purchased from Wuhan USCN Life Science (Table 3). Protein blots were photographed and analyzed using Essential V6 imaging platform (Essential V6, UVITEC, St John’s Innovation Centre, England). The expression of target proteins was measured and normalized with the expression of β-actin.

[0057] 1.9 DNA extraction, 16S rDNA amplicon sequencing Total microbial genomic DNA was extracted using E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, usa). DNA concentration and purity were determined using 1.0% agarose gel electrophoresis and NanoDrop 2000 spectrophotometer (Thermo Scientific Inc., USA). Bacterial 16S rRNA hypervariable region V3-V4 was amplified with primer pair 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). PCR products were extracted from 2% agarose gel, purified using PCR clean-up kit (Shanghai Yuhua Life Science and Technology Development Co., Ltd.) according to the manufacturer's instructions, and quantified using Qubit 4.0 (Thermo Scientific, USA). Library quality was assessed on Qubit 2.0 fluorometer (Thermo Scientific, USA) and sequenced on Ion S5 XL platform (Thermo Scientific, USA). Purified amplicons were pooled in equimolar amounts for paired-end sequencing on Illumina Nextseq2000 platform (Illumina, San Diego, USA) according to the standard protocol of Shanghai Meiji Biological Medicine Science and Technology Co., Ltd. TM XL platform (Thermo Scientific, USA). Purified amplicons were pooled in equimolar amounts for paired-end sequencing on Illumina Nextseq2000 platform (Illumina, San Diego, USA) according to the standard protocol of Shanghai Meiji Biological Medicine Science and Technology Co., Ltd.

[0058] 1.10 Metabolite extraction and data matrix construction Fifty mg of fecal sample was accurately weighed and extracted with 400 pL of methanol: water (4: 1, v / v) solution. The mixture was incubated at -10 °C, then treated with a high-throughput tissue homogenizer at 50 Hz for 6 min. After vortexing for 30 s at 5 °C, the sample was stored at -20 °C for 30 min to precipitate proteins. After centrifugation at 13000 x g and 4 °C for 15 min, the supernatant was collected and transferred to a vial for LC-MS analysis. Chromatographic separation of metabolites was performed using a Thermo UHPLC system equipped with an ACQUITY UPLC HSS T3 column (100 mm x 2.1 mm id, 1.8 pm). Mass spectrometry data were collected using a Thermo UHPLC-Q Exactive HF-X mass spectrometer equipped with an electrospray ionization source, working in positive or negative ion mode. The full MS resolution was 60000, and the MS / MS resolution was 7500. Data acquisition was performed using the data-dependent acquisition (DDA) mode. Detection was performed in the mass range of 70-1050 m / z. LC / MS raw data were pre-processed using Pro-genesis QI (Waters Corporation, Milford, USA) software, and a three-dimensional data matrix in CSV format was exported. The information in this three-dimensional matrix included: sample information, metabolite name, and mass spectrometry response intensity. At the same time, metabolite identification was performed by searching databases, mainly HMDB, Metlin (https: / / metlin.scripps.edu / ) and Majorbio databases. Data were analyzed through the Majorbio cloud platform free online platform (www.cloud.majorbio.com). At least 80% of the metabolic features detected in any one group of samples were retained. To reduce errors caused by sample preparation and instrument instability, the response intensity of the sample mass spectrometry peak was normalized using a normalization method to obtain a normalized data matrix. At the same time, variables with a relative standard deviation (RSD) > 30% of the QC sample were removed, and log10 logarithmic processing was performed to obtain the final data matrix for subsequent analysis.

[0059] Example 2 2.1. Synthesis and general characterization of PDA-TH NPs PDA NPs were prepared by self-polymerization. 1 g of poloxamer (poloxamer Pluronic F127), 0.5 g of dopamine hydrochloride and 2 mL of 1,3,5-trimethylbenzene were dissolved in 40 mL of 50% ethanol solution. The mixture was ultrasonically treated for 15 min, and then mixed. Then 5 mL of ammonia water was added, and stirred at room temperature for 4 h. PDA NPs were obtained by centrifugation, washing and drying to remove the organic template. The morphology of PDA NPs was analyzed by transmission electron microscopy at an acceleration voltage of 200 kV. The Zeta potential and particle size of PDA NPs were determined by dynamic light scattering (DLS). 20 mg of PDA NPs were suspended in 10 mL of deionized water, and 50 mg of Thy was added. After stirring, ultrasonic treatment for 20 min and magnetic stirring for 4 h, PDA-TH NPs were obtained. The morphology of PDA NPs was observed by TEM at an acceleration voltage of 200 kV. The Zeta potential and particle size were determined by DLS method of Malvern Zetasizer Nano ZS. To further characterize the surface morphology and loading efficiency of PDA-TH NPs, high-resolution transmission electron microscopy was used at an acceleration voltage of 300 kV. Energy-dispersive X-ray spectroscopy was used for elemental analysis of PDA-TH. The formation mechanism of PDA-TH was confirmed by X-ray diffraction (XRD) using a D8 ADVANCE A25 system (Bruker AXS, USA). In addition, the encapsulation efficiency (EE) and loading capacity (LC) of Thy in PDA-TH NPs were determined using a 10 kDa ultrafilter (UFC501024, Millipore, USA). The calculation formulas of EE and LC are as follows:

[0060] 2.2, Evaluation of antibacterial performance of PDA-TH NPs The antibacterial performance and mechanism of PDA-TH NPs were evaluated to assess their potential for treating CDI. The inhibitory effect of PDA-TH NPs on the growth of C. difficile was evaluated by bacterial turbidity comparison method. Crystal violet staining method was used to observe the inhibitory effect of PDA-TH NPs on the formation of C. difficile biofilm. The bactericidal effect of PDA-TH NPs was further evaluated by colony counting. In addition, the influence of PDA-TH NPs on the mRNA expression of C. difficile toxins was evaluated by qRT-PCR method. The influence of PDA-TH NPs on the cell structure of C. difficile was observed by scanning electron microscopy. At the same time, the bactericidal mechanism of PDA-TH NPs was explored by determining the extracellular protein and ATP content.

[0061] 2.3, Synthesis and characterization of BA@HF@PDA T FCPs were extracted by aqueous-organic solvent precipitation method. FCPs (100 mg) and HA (200 mg) were placed in 20 mL of ultrapure water, mixed aseptically for 3 h to obtain a polysaccharide solution (HF) with a concentration of 150 mg / mL. BA (10 8 CFU / mL, 10 mL) was mixed with HF (10 mL), homogenized using a vortexer and aseptically stirred using a magnetic stirrer for 30 min. BA was encapsulated in HF to form BA@HF. The resulting BA@HF was co-incubated with PDA-TH (37 °C) and aseptically stirred for 30 min to promote the self-assembly of PDA-TH NPs with BA@HF, constructing the polyphenol-probiotic co-delivery system BA@HF-PDAT. The formation mechanism of BA@HF and BA@HF-PDAT was verified using x-ray diffraction (XRD) and energy dispersive spectrometer (EDS). The gel morphology of BA@HF and BA@HF-PDAT was observed using scanning electron microscopy. Dynamic light scattering (DLS) was used to measure the zeta potential of both formulations. The UV-vis absorbance at 600 nm and digital images were recorded to assess the turbidity of the samples.

[0062] 2.4, Performance evaluation of BA@HF@PDAT Colloidal properties, such as rheological properties, viscosity, and thermal stability, play a crucial role in determining bioavailability. Therefore, the rheological properties of BA@HF and BA@HF-PDAT were evaluated using a rheometer (FRS 1600, Anton Paar). The viscosity of BA@HF and BA@HF-PDAT was determined using a capillary viscometer (TP825, Timepower). BA@HF and BA@HF-PDAT were analyzed using a differential scanning calorimeter (HS-DSC-101A). The antioxidant potential of BA@HF and BA@HF-PDAT was evaluated using total antioxidant capacity (T-AOC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. The growth curves of BA co-incubated with HF and HF-pdat were recorded by UV- visible spectrophotometer to evaluate the effect of the carriers on BA proliferation.

[0063] 2.5, In vitro digestion analysis The protective effect of HF and HF-PDAT on BA was evaluated in simulated gastric and intestinal environments using in vitro digestion method. Briefly, 10 mL of simulated gastric fluid (SGF, containing 2000 U / mL pepsin, pH 2.0, 37°C) was prepared and incubated with BA, BA@HF and BA@HF-PDAT groups for 2 h, respectively. Then, 10 mL of simulated intestinal fluid (SIF, containing 10 mM bile salts and 100 U / mL trypsin, pH 7.4, 37°C) was added to each group and incubated for 4 h. The viable cell count was determined by plate colony counting.

[0064] 2.6, Cell culture HCoEpiC cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 2 mM L-glutamine and 1% non-essential amino acids in 6-well plates. All cells were incubated in a Midi 40 CO2incubator (Thermo Fisher Scientific, Inc.) under standard conditions (5% CO2, 95% air, 37°C). HCoEpiC cells were treated with gradient concentrations of BA@HF-PDAT for different times, and cell viability and in vitro biological safety were evaluated using CCK8 cell viability assay. TcdB is a cytotoxin and is the main factor causing intestinal cell damage by C. difficile. By treating HCoEpiC cells with different concentrations of TcdB for different times, a TcdB-induced cell damage model was established, and then evaluated using CCK8 cell viability assay. In the TcdB-induced damage model, control cells were treated with sterile vehicle, and TcdB and TcdB+BA@HF-PDAT group cells were exposed to TcdB (10 ng / mL) for 8 h. After treatment, the cells were washed with culture medium for 3 times. Control and TcdB group cells were treated with sterile vehicle for 8 h, and TcdB+BA@HF-PDAT group cells were treated with appropriate concentration of BA@HF-PDAT (20 pg / mL) for 8 h. The repair effect of BA@HF-PDAT on TcdB-induced cell damage was evaluated by observing cell morphology and CCK8 cell viability assay.

[0065] 2.7, Ethics of animal studies All mice used in this study were of C57 BL / 6 J strain, obtained from Tengxin Biotechnology Co., Ltd. (Chongqing, China). The mice were housed in an animal room with appropriate temperature and humidity conditions (ambient temperature of 22°C, relative humidity of 50%), under a 12 h light / 12 h dark cycle. The mice were allowed to eat and drink water freely. Water was sterilized by high temperature and pressure (132°C, 205.8 kPa), and sterile feed was purchased from Tengxin Biotechnology Co., Ltd. (Chongqing, China).

[0066] 2.8 In vivo biosafety evaluation Twelve 7-week-old C57BL / 6J mice were selected and administered either an appropriate concentration of BA@HF-PDAT (1 g / kg body weight / day) or an equal volume of physiological saline via gavage, with body weight recorded daily. After 7 days, the mice were anesthetized and euthanized. Major internal organs (liver, spleen, kidney) and blood samples were collected. All freshly collected specimens were fixed in 4% paraformaldehyde and embedded in paraffin.

[0067] Tissue sections with a thickness of 5 μm were dewaxed, rehydrated, and stained with hematoxylin and eosin (H&E). Blood was collected and allowed to stand at room temperature for 30 min, then centrifuged at 3000 rpm for 10 min at 4 °C to prepare serum. The obtained serum was subjected to biochemical analysis. The biosafety of BA@HF-PDAT was assessed by comparing the organ and tissue morphology and biochemical indicators (including blood biochemistry, liver function, and kidney function) of mice orally administered BA@HF-PDAT or an equal volume of physiological saline.

[0068] 2.9 Gastrointestinal transit analysis Nine 7-week-old male C57BL / 6J mice were fasted for 4 hours after 7 days of acclimatization. Figure 5 As shown in (A), mice were randomly divided into three groups: BA, BA@HF, and BA@HF-PDAT, with three mice in each group. The BA group was given BA (1×10⁻⁶). 9 CFU), labeled with DiR dye. The BA@HF group and the BA@HF-PDAT group were administered BA@HF and BA@HF-PDAT (1×10⁻⁶ CFU), respectively. 9 The mice were labeled with DiR. After gavage administration for 0.5, 2, 6, 12, 24, 48, 72, and 96 hours, mice were anesthetized with isoflurane and placed in an imaging chamber. Fluorescence signals were acquired, and images were processed using in vivo imaging software. Gastrointestinal transport was assessed based on the fluorescence distribution and intensity along the digestive tract. At the end of the experimental period, mice were euthanized, and colonic tissue was collected. Fluorescence signals were captured and analyzed using in vivo imaging software, and BA colonization efficiency was assessed based on fluorescence intensity and localization in the colonic tissue.

[0069] 2.10 Inflammation Targeting Analysis Inflammation targeting analysis, such as Figure 5(F) as described above. Six 7-week-old male C57BL / 6J mice were acclimatized for 7 days to induce intestinal dysbiosis. Briefly, mice were given an antibiotic cocktail (0.4 mg / mL kanamycin, 0.035 mg / mL gentamicin, 850 U / mL colistin, 0.215 mg / mL metronidazole, and 0.045 mg / mL vancomycin) in drinking water for 3 days, followed by intraperitoneal injection of clindamycin to mimic the microbial imbalance caused by antibiotics. After that, they were given sterile drinking water for 1 day. Then the mice were randomly divided into two groups, 3 mice in each group: Saline + BA@HF-PDAT and CDI + BA@HF-PDAT. The Saline + BA@HF-PDAT group was gavaged with 0.2 mL of saline as a baseline control, while the CDI + BA@HF-PDAT group was gavaged with 10 9 CFU / mL of C. difficile to induce an inflammatory response. The gavage was performed 7 days after C. difficile infection, after a 4-h fasting period before gavage. Both groups were gavaged with DiR-labeled BA@HF-PDAT (1 x 10 9 CFU / mL BA). After 12 h of gavage, the mice were euthanized and the colon tissue was collected. The fluorescence signal was captured and analyzed using Living Image software, and the inflammatory targeting was evaluated based on the fluorescence intensity and the localization of the colon tissue.

[0070] 2.11. Animal studies Thirty-six 6-week-old male C57BL / 6J mice were pre-fed for 1 week to acclimatize. At 7 weeks of age, the CDI mouse model was established according to the aforementioned method. Except for the CON group, the rest of the mice were given an antibiotic cocktail (0.4 mg / mL kanamycin, 0.035 mg / mL gentamicin, 850 U / mL colistin, 0.215 mg / mL metronidazole, 0.045 mg / mL vancomycin) in drinking water for 3 days. Subsequently, intraperitoneal injection of clindamycin was performed to mimic the intestinal dysbiosis caused by clinical antibiotics. Subsequently, sterile drinking water was given for 1 d, and C. difficile (10^ 8 CFU / mL, 0.2 mL) was given to induce CDI. The CON group was given the corresponding sterile vehicle by gavage. During the treatment phase, the CON and CDI groups were given sterile vehicle, while the BA, PDA-TH, BA@HF, and BA@HF-PDAT groups were given the corresponding formulations (1 g / kg body weight / d) by gavage. Body weight and disease activity index were recorded daily. Fresh fecal samples were collected by rectal sampling and stored at -80 °C for subsequent C. difficile toxin content determination, 16S sequencing, and nontargeted metabolomics analysis. At the end of the experimental period, the mice were euthanized and colon samples were collected for further analysis, including histomorphometric analysis, ultrastructural analysis, antioxidant capacity determination, qRT-PCR, western blot, and immunofluorescence analysis.

[0071] 2.12, Quantification of fecal C. difficile toxin levels TcdA and TcdB levels were quantified using ELISA kits according to the manufacturer’s instructions, as detailed in Table 1. Briefly, fecal supernatants were collected and centrifuged at 12,000 x g for 10 min at 4 °C. After 1 h incubation at 37 °C, plates were washed with buffer and horseradish peroxidase (HRP)-conjugated detection antibodies were added. After further incubation and washing, the colorimetric reaction was initiated by adding tetramethylbenzidine (TMB) substrate, followed by stopping the colorimetric reaction with sulfuric acid. The optical density (OD) values at 450 nm were recorded using a microplate reader. All samples were analyzed in duplicate to improve the reliability of the results.

[0072] 2.13, Intestinal microbiome analysis Fecal samples were collected from each mouse and stored at -80 °C until analysis (n = 6). The extraction and sequencing of sample DNA are detailed in the supplementary information. Raw sequencing reads were deposited in the NCBI Sequence Read Archive (SRA) database. Raw data were imported into the QIIME2 microbiome bioinformatics platform. Raw sequences were processed, denoised, and assembled using DADA2 to obtain ASV feature tables and species annotation tables. QIIME 2 was used to further analyze the ASV feature tables and species annotation tables of all samples. The databases used were to generate abundance tables at each taxonomic level using QIIME 2 (version 2024, https: / / qiime2.org) and to calculate beta diversity distances. Alpha diversity of the intestinal microbiome was tested and plotted using the ggplot2 package in R software (version 3.3.2). Non-metric multidimensional scaling analysis (NMDS) and microbial community structure were statistically analyzed and plotted using the ggplot2 package in R software. The microeco package in R software was used for inter-microbiome difference identification and linear discriminant analysis (LDA) effect size (LEfSe, http: / / huttenhower.sph.harvard.edu / LEfSe), focusing on the analysis of genera with an LDA score > 2, P p-values < 0.05. Microbiome data were mapped to the KEGG PATHWAY database (Kyoto Encyclopedia of Genes and Genomes, http: / / www.genome.jp / kegg / ) linking and to the corresponding KEGG compound ids and related microbiome pathways to assess their impact on biological processes. In addition, the correlation between differentially presented bacterial taxa and indicators such as inflammatory cytokines, body weight, and colon length was calculated and visualized using the ggplot2 and heatmap packages in R software through Spearman correlation analysis.

[0073] 2.14. Intestinal metabolomics analysis Fecal samples were collected from each mouse and stored at -80 °C until analysis (n = 6). LC / MS-based metabolomics was performed by Majorbio Biotech (Shanghai, China). Metabolite extraction and data matrix construction were as described in 1.9 of Example 1. The number of common metabolites and unique metabolites was quantitatively analyzed using the ggplot2 package in R software. The data were subjected to PLS-DA using the mixOmics package in R software. The volcano plot of the differential metabolites between the two groups was generated using the ggplot2 in R software. Statistical analysis of the metabolites among the three groups was performed, and the fold change of the metabolites between the groups was calculated. The differential metabolites were selected according to the criteria of VIP > 1.5 and FDR < 0.05, and the cluster heat map was generated using the mixOmics package in R software. The differential metabolite data were mapped to the KEGG PATHWAY database (Kyoto Encyclopedia of Genes and Genomes, http: / / www.genome.jp / kegg / ), and the metabolites were linked to their corresponding KEGG compound ids and related metabolic pathways to assess their impact on biological metabolic processes.

[0074] 2.15. Statistical analysis Data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism v.8 (GraphPad Software, San Diego, USA). Statistical methods used one-way ANOVA with Bonferroni correction. At the same time, the Tukey test for multiple comparisons was used to compare the significance between two groups. The statistical significance was accepted at the value of P < 0.05. P The statistical significance was accepted at the value of P < 0.05. P The statistical significance was accepted at the value of P < 0.05, and * indicates P The statistical significance was accepted at the value of P < 0.05, and ** indicates P The statistical significance was accepted at the value of P < 0.01.

[0075] Results and analysis: (1) Characterization and antibacterial properties of PDA-TH NPs The antibacterial properties of Thy were used to weaken C. difficile, while the biological activity of BA was utilized to confer a competitive advantage to jointly combat C. difficile. This synergistic mechanism constitutes a promising therapeutic strategy for CDI. Therefore, a BA-PDA NPs co-delivery system (BA@HF-PDAT) using ficollavenous polysaccharide-hyaluronic acid hydrogel (A) was developed, aiming to modulate the colonic microenvironment, enhance probiotic colonization, and repair the colonic tissue to alleviate CDI-induced colitis. Figure 2 The raw material formula of BA@HF-PDAT is as follows: Figure 12(A) as shown.

[0076] Thy was loaded into PDA NPs synthesized by EDT method, and together with poloxamer F127, the optimized PDA-TH NPs were obtained to improve the stability and bioavailability of Thy. The synthesized PDA NPs presented porous spherical morphology (Fig. 1A) with negative surface charge (Fig. 1B) and an average particle size of 316 nm (Fig. 1C), which helped them have high dispersibility and be able to effectively load polyphenols (Fig. 1D). Figure 2 Figure 2 Figure 10 HRTEM showed that the synthesized PDA-TH NPs presented irregular nanospheres (Fig. 2D) with a size of 20-60 nm (Fig. 2E) and an average particle size of 198 nm (Fig. 2F). The FT-IR of PDA-TH NPs showed the characteristic peaks of polyphenols at 1610 cm-1 and 1364 cm-1 (Fig. 2B), corresponding to the asymmetric stretching vibration of -C-O and the bending vibration of -OH (Fig. 2C) of phenolic hydroxyl groups. XRD showed the amorphous crystal state of polyphenol nanoparticles (Fig. 2D), confirming the successful synthesis of PDA-TH NPs. Single PDA-TH NPs were observed in HAADF, and elemental mapping scanning showed that N and O elements were uniformly distributed on its surface (Fig. 2F). The encapsulation efficiency was 61% (Fig. 2A), and the loading capacity could reach 275% (Fig. 2B). Notably, small particle size and high specific surface area were key determinants for improving the bioavailability of polyphenols. The particle size distribution of PDA-TH NPs (Fig. 2E) showed that the average particle size was 198 nm, and most of the particle sizes were in the range of 20-60 nm. At the same time, PDA-TH NPs played a crucial role in inhibiting the proliferation of C. difficile, inhibiting toxin production, and preventing biofilm formation. Antimicrobial evaluation showed that the minimum inhibitory concentration (MIC) was 100 mg / L, 50 mg / L could significantly inhibit the formation of C. difficile biofilm, and 200 mg / L could successfully eradicate C. difficile within 2 h (Fig. 3G-I). Further analysis of extracellular ATP and protein content confirmed that PDA-TH NPs could clear C. by destroying its cell membrane (Fig. 3C, 1D). At the sub-inhibitory concentration (50 mg / L), PDA-TH NPs had no effect on the expression of C. difficile toxin genes after 6 h of treatment (Fig. 3E). After 12 h of PDA-TH NPs treatment, PDA-TH NPs significantly down-regulated the mRNA levels of TcdA, TcdB, and TcdR, while up-regulating the expression of TcdC (Fig. 3F). Figure 2 Figure 10 -1 -1 Figure 10 Figure 10 Figure 2 Figure 11 Figure 11 Figure 3 Figure 2 Figure 11 Figure 11 Figure 2 ​​​​​​​​​​​​​​​These findings suggest that PDA-TH NPs may inhibit Clostridium difficile toxin production by suppressing the transcription of TcdA and TcdB, while simultaneously enhancing the expression of the negative regulator TcdC and interfering with the positive regulator TcdR. Finally, scanning electron microscopy (SEM) was used to investigate the morphological effects of PDA-TH NPs on Clostridium difficile. The results confirmed that PDA-TH NPs disrupted the structure of Clostridium difficile in a concentration-dependent manner, effectively inhibiting its proliferation (Figure K).

[0077] (2) Construction and characterization of BA@HF-PDAT Figure 3 (A) shows a schematic diagram of the microstructure of the BA-PDAT co-delivery system based on FCPs-hyaluronic acid hydrogel. The outer membrane of BA is rich in many modifiable functional groups, which is beneficial for its immobilization within the hydrogel. The total sugar content of FCPs is 64.2%, and the uronic acid content is 24.3%. Figure 12 D). For example Figure 12 As shown in E, FCPs contain various monosaccharides, including mannose, rhamnose, galacturonic acid, galactose, and arabinose. Among these, galacturonic acid (HG), mannose, and rhamnose were identified as the main monosaccharide components, with a total molecular weight of approximately 300 kDa. Figure 12 F). Typically, the characteristic monosaccharides of high-galacturonic acid polysaccharides (HG) and rhamnogalacturonic acid polysaccharide I (RG-I) include galacturonic acid, rhamnose, galactose, and arabinose, while hemicellulose is mainly composed of mannose. Therefore, FCPs are primarily linear, water-soluble acidic polysaccharides with a relatively high proportion of modifiable functional groups. Subsequently, PDA-TH NPs and BA were co-encapsulated in a network structure composed of interwoven polysaccharide chains. Most of the BA was uniformly distributed in the HF, while a small portion was exposed on the outer surface of BA@HF, achieving a total encapsulation efficiency of 95% ( Figure 3 B). SEM images showed that BA was mainly located within the cavity and interlayer of BA@HF-PDAT, demonstrating enhanced probiotic protection (B). Figure 3 C). Furthermore, the introduction of PDA-TH results in PDA-TH NPs being uniformly distributed on the outer surface of BA@HF-PDAT ( Figure 3 D), accompanied by a decrease in zeta potential from -35mV to -43mV at BA@HF ( Figure 3 E). An increase in the absolute value of the zeta potential contributes to the stability of the composite material, while a lower surface potential improves the interaction between the composite material and the site of gastrointestinal inflammation, thereby enhancing the local therapeutic efficacy against CDI.

[0078] FT-IR showed that 1083 cm⁻¹ in BA -1The corresponding -C-O stretching vibration peak is shifted to 1041 cm -1 and continues to shift to 1037 cm in BA@HF-PDAT -1 , indicating the successful self-assembly of the co-delivery system Figure 3 F). In addition, XRD analysis indicates that both BA@HF and BA@HF-PDAT exhibit a certain degree of structural disorder Figure 3 G). Rheological experiments show that the apparent viscosity of BA@HF and BA@HF-PDAT decreases with increasing shear rate Figure 3 H). Notably, G" (loss modulus) of BA@HF exceeds G' (storage modulus), indicating a viscosity-dominated behavior. In contrast, after the addition of PDA-TH NPs, G' of BA@HF-PDAT exceeds G", transforming it into an elasticity-dominated gel. This indicates that PDA-TH NPs enhance the structural integrity of the co-delivery system through non-covalent interactions with the polysaccharide backbone, where phenolic hydroxyl groups act as molecular bridges to enhance the polysaccharide network Figure 3 I). Differential scanning calorimetry analysis shows that the melting endothermic peak of BA@HF-PDAT shifts to 119.72 °C, indicating a slight improvement in thermodynamic stability Figure 3 J). Regarding antioxidant capacity, the addition of PDA-TH NPs significantly enhances the total antioxidant activity of the co-delivery system, as expected Figure 3 K). The results of DPPH and ABTS radical scavenging assays confirm the strong antioxidant capacity of BA@HF-PDAT, indicating its dual ability to scavenge lipophilic and hydrophilic radicals Figure 13 A, 13B). In addition, although the addition of PDA-TH NPs induces a slight delay in BA growth and slightly reduces the final colony count Figure 3 L), the rational design of the system minimizes the impact of PDA-TH NPs on BA viability during production and storage.

[0079] (3) Resistance of BA@HF-PDAT to in vitro simulated gastrointestinal environment The co-delivery platform can effectively protect probiotic bacteria and maintain bacterial viability in harsh gastrointestinal environments, which is crucial for BA to exert its therapeutic effect on CDI-induced colitis. One of the main challenges faced by oral co-delivery platforms is ensuring that BA can successfully pass through the low-pH gastric acid environment and maintain an effective active concentration. The present invention evaluated the gastric adaptation of BA, BA@HF, and BA@HF-PDAT in simulated gastric fluid (SGF) by incubating the samples in simulated gastric fluid (SGF) enriched with pepsin (pH 2.0) and measuring the number of viable bacteria at predetermined time points by plate colony counting. As Figure 3As shown in Figure M, after 0.5 h, the tolerance of BA in the BA@HF and BA@HF-PDAT groups was significantly improved compared to naked BA. However, after 1 h of exposure to SGF, the survival rate of BA in the unencapsulated group was almost undetectable. In contrast, the encapsulated BA in the BA@HF and BA@HF-PDAT groups remained viable, with the survival rate of the BA@HF-PDAT group being 21.84 times higher than that of unprotected BA. Extending the SGF incubation time to 2 h resulted in the complete death of bacteria in the naked BA group, while a large number of viable bacteria remained in the BA@HF-PDAT group, with a survival rate as high as 9.16 × 10⁻⁶. 6 CFU. Figure 3 (N) This finding is visually confirmed, as the figure shows a significant number of BAs survive in the BA@HF-PDAT system. To further assess BA survival after intestinal release, samples were incubated in simulated intestinal fluid (SIF) containing trypsin (pH 6.8). Figure 3 After 2 hours in SIF, the viability of BAs in all co-delivery systems remained relatively stable. However, after 4 hours, bacterial viability decreased slightly. Nevertheless, the survival rate of the BA@HF-PDAT group (5.66 ± 0.16 log CFU) was still significantly higher than that of the BA@HF group (4.77 ± 0.13 log CFU). These results indicate that the co-delivery system significantly improves the survival rate of BAs under gastrointestinal (GIT) conditions, which is crucial for their subsequent retention and colonization. The hydrogel-based probiotic co-delivery platform has a dual advantage: the prebiotic-like polysaccharides create a favorable microenvironment for probiotics during the preparation of the co-delivery system and form a protective film around the probiotics. The introduction of the polysaccharide network structure in PDA-TH NPs further enhances the protective effect, protecting BAs from degradation caused by the harsh gastrointestinal environment.

[0080] (4) ROS scavenging capability and mechanism of BA@HF-PDAT CDI Oxidative stress and inflammation are induced, leading to excessive ROS accumulation in damaged colonic tissue. Excessive ROS production can trigger apoptosis of intestinal epithelial cells, damage the intestinal mucosa, and disrupt certain probiotic species. Further evaluation of the antioxidant capacity of BA@HF-PDAT was conducted. HCoEpiC cells and probiotics were incubated in a medium containing H2O2. The ROS scavenging capacity of BA@HF-PDAT was then analyzed by flow cytometry. Figure 3(P) showed significantly lower percentage of ROS-positive cells compared to the H2O2 group. These findings indicated that probiotics and Thy themselves have the ability to scavenge ROS. BA@HF and BA@HF-PDAT groups showed significantly lower percentage of ROS-positive cells compared to the BA group, corresponding to 5.22-fold and 6.17-fold reduction, respectively, highlighting their effective efficiency in scavenging and eliminating intracellular ROS. Notably, the BA@HF-PDAT complex system exhibited antioxidant activity far superior to its individual components, which could be attributed to its rational design, which enhanced antioxidant capacity, effectively eliminated excess ROS, and protected intestinal cells.

[0081] (5) Protective effect of BA@HF-PDAT on TcdB-induced cell damage C. difficile mainly secretes two toxins, TcdA and TcdB. TcdA is an enterotoxin that can damage the intestinal mucosa, alter vascular permeability, cause excessive secretion of intestinal fluid, intestinal effusion, and mucosal hemorrhage and necrosis. TcdB is a cytotoxin that can induce actin depolymerization, disrupt the cytoskeleton, promote cell necrosis, and trigger inflammation and oxidative stress. Compared with TcdA, TcdB exhibits significantly higher cytotoxicity in cell culture models. Therefore, this study used TcdB to treat cells and evaluated the protective effect of BA@HF-PDAT on TcdB-induced cell damage and oxidative stress. In addition, appropriate concentration of BA@HF-PDAT (20 μg / mL) treatment for more than 8 h significantly improved cell viability ( P <0.01) Figure 4 A). In addition, exposure to 10 ng / mL TcdB for more than 8 h significantly reduced cell viability ( P <0.01) Figure 4 B). Subsequently, cells were exposed to 10 ng / mL TcdB for 8 h, washed, and then incubated with BA@HF-PDAT for another 8 h. The results showed that BA@HF-PDAT effectively reversed the TcdB-induced changes in cell morphology ( Figure 4 C) and significantly restored cell viability ( P <0.01) Figure 4 D).

[0082] (6) Mucosal adhesion ability of BA@HF-PDAT Stable colonization of probiotics in the gut is crucial for niche occupation, restoration of the gut’s colonization resistance, and effective treatment of chronic intestinal infections (CDI). BA@HF-PDAT can firmly adhere to the inflamed intestinal site by FP and HA components forming hydrogen and imine bonds with intestinal mucosal molecules, promoting probiotic colonization and providing sustained coverage and repair to the damaged tissue. To quantify the promotion of probiotic retention and colonization in vivo by HF hydrogel and HF co-mixing with PDA-TH, the fluorescent intensity of fluorescently labeled probiotics was monitored using IVIS to assess their distribution in vivo and intestinal retention over 96 h. The experimental design is shown in FIG. 5(A). After a 7-day adaptation period, healthy mice were orally administered 1 x 10 9 CFU of BA, and the fluorescent intensity was measured at predetermined time points. Compared with BA alone, BA@HF and BA@HF-PDAT exhibited rapid diffusion within 12 h, which can be attributed to the fluidic nature of the hydrogel facilitating probiotic transport. After 48 h, the fluorescent signal of unencapsulated BA was almost undetectable, while strong fluorescent signals of BA@HF and BA@HF-PDAT persisted in the colon, indicating that the co-delivery system significantly improved the survival and colonization of probiotics. Notably, the fluorescent intensity of BA@HF-PDAT remained slightly higher than that of BA@HF after 96 h, suggesting that the HF hydrogel mainly contributed to enhancing the retention of probiotics in healthy mice, while PDA-TH further strengthened the structure of the hydrogel, thereby improving its protective capacity. Moreover, Thymine can have promoted the colonization of BA by acting as a prebiotic (FIG. 5(B-C)). The fluorescent intensity quantification in FIG. 5D supports this conclusion. To further evaluate the in vivo distribution, ex vivo imaging of intestinal tissue after 96 h showed that BA@HF had strong fluorescent signals in both the cecum and colon, while the fluorescence of BA@HF-PDAT was mainly concentrated in the colon. Subsequently, to assess the adhesion capacity of BA@HF-PDAT in the inflamed colon, BA colonization in the intestinal tract of CDI mice was examined at specific time points (FIG. 6(A)). Figure 5 E). Fluorescently labeled BA was co-incubated with freshly excised intestinal tracts of CDI mice (FIG. 6(B)). Figure 5 F). After 6 h, the fluorescent intensity of BA in the CDI group was significantly higher than that in the saline group, indicating an increased retention time of BA@HF-PDAT in the inflammatory microenvironment induced by CDI. After 12 h, the fluorescent intensity was slightly higher than that at 6 h and had spread to the cecum and colon regions (FIG. 6(C)). Figure 5 (G), Figure 13C, 13D). This phenomenon could be attributed to CDI-induced diarrhea, which accelerated the transit of BA@HF-PDAT in the gastrointestinal tract. Ex vivo intestinal fluorescence imaging further confirmed these findings. Compared with the saline group, the BA fluorescence in the CDI group was significantly increased and uniformly distributed throughout the colon and cecum (Fig. 6E-F). These results suggest that BA@HF-PDAT can rapidly localize in the inflamed colon of CDI mice, thereby promoting the colonization of BA. This effect could be attributed to the inflammation-targeting property of hyaluronic acid in BA@HF-PDAT, which maximized the BA colonization before reaching the inflamed colon site. Figure 5 (H-I). These results suggest that BA@HF-PDAT can rapidly localize in the inflamed colon of CDI mice, thereby promoting the colonization of BA. This effect could be attributed to the inflammation-targeting property of hyaluronic acid in BA@HF-PDAT, which maximized the BA colonization before reaching the inflamed colon site.

[0083] (7) Intervention effect of BA@HF-PDAT on CDI-induced colitis To further elucidate the therapeutic potential of BA@HF-PDAT, its efficacy in a mouse model mimicking clinical antibiotic-induced CDI was systematically evaluated. The CDI mouse model was established using a previously described standard protocol, in which mixed antibiotics were added to the drinking water to disrupt the gut microbiota, followed by oral gavage of C. difficile to induce CDI. Subsequently, mice were randomly divided into five treatment groups and received PBS, BA, PDA-TH, BA@HF, or BA@HF-PDAT treatment for 7 days, respectively (Fig. 6A). Healthy mice served as a control group. After the treatment, mice were euthanized, and tissue and fecal samples were collected for analysis. During the entire treatment period, the body weight and disease activity index (DAI) of mice were recorded, which included parameters such as body weight change, hair roughness, and diarrhea severity. As shown in Figs. 6B-C, the body weight of CDI group mice significantly decreased, and the DAI index increased, indicating the presence of severe colitis. Compared with the CDI group, the body weight, DAI index, and colon length of mice in the BA@HF and BA@HF-PDAT groups were reduced and tended to be at the level of healthy mice. In vitro experimental analysis showed that the colon length of mice in the CDI, BA, PDA-TH, and BA@HF groups was shortened by 17.7%, 12.4%, 9.6%, and 4.5%, respectively, while the colon length of mice in the BA@HF-PDAT group was shortened by only 1.6%. In addition, the CDI-induced cecal swelling of mice in the PDA-TH, BA@HF, and BA@HF-PDAT groups was significantly alleviated (Figs. 6D-E). These results suggest that BA@HF-PDAT can effectively alleviate the CDI-induced colitis in mice. Figure 6 (B-C) as shown, the body weight of CDI group mice significantly decreased, and the DAI index increased, indicating the presence of severe colitis. Compared with the CDI group, the body weight, DAI index, and colon length of mice in the BA@HF and BA@HF-PDAT groups were reduced and tended to be at the level of healthy mice. In vitro experimental analysis showed that the colon length of mice in the CDI, BA, PDA-TH, and BA@HF groups was shortened by 17.7%, 12.4%, 9.6%, and 4.5%, respectively, while the colon length of mice in the BA@HF-PDAT group was shortened by only 1.6%. In addition, the CDI-induced cecal swelling of mice in the PDA-TH, BA@HF, and BA@HF-PDAT groups was significantly alleviated (Figs. 6D-E). These results suggest that BA@HF-PDAT can effectively alleviate the CDI-induced colitis in mice. Figure 6 D).

[0084] To assess the colonic injury, detailed histological evaluation was performed using H&E and Alcian blue staining, and a comprehensive histological scoring system was adopted, which considered loss of structural integrity, crypt and goblet cell destruction, inflammatory cell infiltration, and edema. The results showed that the colonic tissue of the CDI group exhibited severe inflammatory infiltration, epithelial damage, crypt distortion, inflammatory cell infiltration, and goblet cell depletion. Notably, the BA@HF-PDAT group showed the most effective therapeutic effect, reversing the colonic epithelial damage and inflammatory infiltration, while restoring the crypt structure and goblet cell number. Similarly, the BA@HF group also showed significant recovery of the colonic tissue morphology. The BA and PDA-TH groups showed partial restoration of the mucosal epithelial integrity, crypt structure, and goblet cell population, although there were still signs of inflammation. In addition, transmission electron microscopy (TEM) was also used to analyze the ultrastructural features of the colon. The TEM results showed that CDI caused sparse, fragmented, and disorganized microvilli in the colonic epithelium. Compared to the CDI group, the BA and PDA-TH groups showed increased microvilli length, but it was still shorter than normal, and the arrangement gradually improved, but there were still some sparse areas. In contrast, the BA@HF and BA@HF-PDAT groups had tightly arranged microvilli, and the ultrastructure of the colonic epithelium was significantly restored, approaching normal levels, indicating a better therapeutic effect. Figure 6 E). As shown in Figure 6 F, the levels of C. difficile toxins (TcdA and TcdB) in feces were quantitatively detected using an ELISA kit, and toxins were detected in all groups except the CON group. Compared to the CDI group, the TcdA and TcdB levels in the BA and BA@HF groups did not significantly decrease. However, the toxin levels in the PDA-TH and BA@HF-PDAT groups significantly decreased. These results indicate that PDA-TH plays a crucial role in reducing C. difficile toxin production, which is consistent with the in vitro experimental results.

[0085] The antioxidant capacity of colonic tissue plays a crucial role in maintaining intestinal homeostasis and preventing inflammatory damage. Enhanced antioxidant capacity has been shown to alleviate colonic injury. Therefore, the present invention evaluated the effect of BA@HF-PDAT on the antioxidant capacity of colonic tissue. The total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) levels in all treatment groups were significantly higher than in the CDI group, while the malondialdehyde (MDA) level was significantly lower. Notably, the BA@HF-PDAT group exhibited the strongest antioxidant capacity among all treatment groups, with CAT, GSH-Px, T-AOC, and SOD levels close to those of healthy mice, and the lowest MDA level in the colonic tissue. Figure 6 (G), Figure 13E, 13F). These results suggest that both BA and PDA-TH contribute to the enhancement of the antioxidant capacity of the colon tissue, which can be due to the production of antioxidant metabolites by BA and the intrinsic antioxidant properties of Thy. In addition, BA@HF and BA@HF-PDAT exhibit enhanced therapeutic effects. This enhancement can be attributed, on one hand, to the antioxidant properties of ficoll polysaccharides and, on the other hand, to the inflammation targeting and adhesion capabilities conferred by HF. These properties effectively improve the accumulation and bioavailability of BA and PDA-TH in the intestinal tract, especially in the colon, thus further enhancing their antioxidant effects. CDI induces damage to the intestinal epithelium and activates the host immune system, triggering a strong inflammatory response. The imbalance between pro- and anti-inflammatory cytokines leads to local tissue destruction and exacerbates inflammation, further aggravating intestinal damage. Compared to the CDI group, the BA, PDA-TH, BA@HF, and BA@HF-PDAT groups significantly reduced the levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) while increasing the levels of anti-inflammatory cytokines (IL-10 and IL-22). Notably, the BA@HF-PDAT group had the strongest anti-inflammatory effect, with the lowest levels of IL-1β, IL-6, and TNF-α and the highest levels of IL-10 and IL-22 in the colon tissue Figure 6 I). The anti-inflammatory effect of BA@HF-PDAT was significantly better than that of the BA group. These results suggest that BA and PDA-TH can alleviate inflammation by inhibiting toxin production, while the hyaluronic acid and ficoll polysaccharides in HF contribute to anti-inflammatory activity. The BA@HF-PDAT platform effectively integrates these components, achieving the most effective anti-inflammatory response and providing an optimized therapeutic strategy for CDI treatment.

[0086] (8) Intervention of BA@HF-PDAT on intestinal barrier damage and tissue repair dysfunction To further elucidate the repair mechanism of BA@HF-PDAT in CDI-induced colitis, the intestinal barrier integrity and tissue repair function were comprehensively evaluated using immunofluorescence staining, transmission electron microscopy (TEM), and Western blot analysis. These analyses aimed to assess the key structural and molecular changes in intestinal epithelial cells, thereby gaining insight into the protective and regenerative effects of BA@HF-PDAT on the damaged colon. The expression and distribution of tight junction proteins occludin and claudin-1, which are key markers of barrier function, were analyzed. In addition, the Wnt signaling pathway is crucial for maintaining intestinal cell self-renewal. Wnt3a, as a ligand of the Wnt signaling pathway, can activate the β-catenin-dependent transcription program, thereby promoting stem cell proliferation and maintaining stem cell properties. The pathogenesis of CDI is closely related to the inhibition of the Wnt / β-catenin signaling pathway mediated by TcdB binding to FZD receptors, leading to impaired epithelial repair. Inhibition of this pathway triggers the activation of the downstream Bax / Bcl2 signaling pathway, promoting apoptosis and further impairing intestinal healing. The expression of Ki67 was evaluated as an indicator of intestinal epithelial proliferation and repair, and transmission electron microscopy (TEM) was used for ultrastructural analysis of apoptotic cells. To elucidate the protective mechanism of BA@HF-PDAT, its effects on the Wnt / β-catenin and Bax / Bcl2 signaling pathways were detected. As Figure 7 As shown in FIG. 24(A-B), the expression of occludin and claudin-1 in the colon epithelium of the CDI group was significantly reduced, indicating severe disruption of the intestinal tight junction. Quantitative analysis of fluorescence intensity showed that BA and PDA-TH had partial protective effects on tight junction damage, while BA@HF and BA@HF-PDA-TH exhibited superior repair effects. Notably, the BA@HF-PDAT group had the highest expression levels of occludin and claudin-1, highlighting its superior intestinal barrier repair capacity (FIG. 24(A-B)). Western blot analysis consistently confirmed that BA@HF-PDAT significantly upregulated the protein levels of occludin and claudin-1, effectively repairing the intestinal mechanical barrier (FIG. 7D, Figure 7E). BA@HF-PDAT treatment significantly increased the expression of Ki67 protein compared with the CDI group, and was mainly located in the colonic crypt, further confirming its excellent ability to promote the regeneration and repair of colonic tissue. The Wnt / p-catenin signaling pathway and its downstream target genes c-Myc and cyclin D1 were evaluated. The results showed that the Wnt / p-catenin pathway was significantly inhibited in the CDI group. Compared with the CDI group, all treatment groups showed enhanced Wnt / p-catenin signaling ability Figure 7 (D-F)). Consistent with the hypothesis, CDI-induced inhibition of the Wnt / p-catenin signaling pathway led to apoptosis. C-caspase-3 immunofluorescence results showed that the expression of C-caspase-3 protein was significantly increased in the CDI group. TEM analysis showed that the cells appeared typical ultrastructural features of apoptosis, including nuclear condensation and nuclear fragmentation (Figure 7 (C), D), and the number of apoptotic cells was significantly increased in the CDI group compared with the control group (Figure 7 (E), F). Figure 17(D). All treatment groups significantly reduced C-caspase-3 protein expression. Notably, chromatin condensation was observed in the nuclei of BA and PDA-TH groups, suggesting that programmed cell death persisted, which was consistent with the results of C-caspase-3 expression. The cell morphology of BA@HF and BA@HF-PDAT groups returned to normal. Among them, the BA@HF-PDAT group had the lowest C-caspase-3 expression, suggesting the best treatment effect. The above results suggest that CDI-induced inhibition of the Wnt / β-catenin signaling pathway can activate Bax and inhibit Bcl2, leading to increased expression of P53 and C-caspase-3 proteins, inducing apoptosis and hindering colon tissue repair. BA@HF-PDAT can restore Wnt / β-catenin signaling pathway activity and effectively regulate the Bax / Bcl2 signaling pathway, thereby reducing CDI-induced apoptosis and promoting colon tissue repair. BA@HF-PDAT significantly activated the pathway, i.e., BA stimulated Wnt / β-catenin signaling by metabolites (e.g., extracellular soluble polysaccharides), while Thy also activated the pathway. In addition, PDA-TH indirectly promoted Wnt / β-catenin activation by reducing TcdB production. BA@HF-PDAT formulations showed a synergistic effect, integrating multiple components to strongly activate Wnt / β-catenin signaling and promote colon tissue repair. Consistent with hypothesis one, CDI-induced inhibition of the Wnt / β-catenin signaling pathway activated Bax and inhibited Bcl2, leading to increased expression of P53 and C-caspase-3 proteins, thereby inducing apoptosis. Similarly, BA@HF-PDAT effectively regulated the Bax / Bcl2 signaling pathway, thereby reducing CDI-induced apoptosis. These results are consistent with the observations in the cell culture model, elucidating the molecular mechanisms by which BA@HF-PDAT reduces apoptosis, promotes intestinal cell proliferation, and promotes colon repair by regulating the Wnt / β-catenin signaling pathway and the Bax / Bcl2 signaling pathway.

[0087] (9) Regulation of intestinal flora in CDI-induced colitis mice by BA@HF-PDAT Intestinal microbiota is an important indicator of intestinal health and plays a key role in the treatment of CDI and the prevention of recurrent CDI. Therefore, the purpose of the present application is to study the potential of BA@HF-PDAT in regulating the intestinal microbiota of CDI mice and ultimately preventing disease progression. Compared with the CON group, the alpha diversity index of the intestinal microbiota of the CDI group, including the Ace index, Chao1 index, Shannon index and Sobs index, was significantly reduced. This reduction is mainly due to antibiotic treatment for establishing a clinical CDI mouse model. Notably, lower alpha diversity is a feature of intestinal microbiota susceptible to CDI. The results show that the BA group and the PDA-TH group did not significantly improve alpha diversity compared with the CDI group, while the BA@HF group and the BA@HF-PDAT group significantly improved alpha diversity. Figure 8 A}.

[0088] At the same time, non-metric multidimensional scaling (NMDS) analysis was performed to evaluate the beta diversity of intestinal microbiota, in order to further understand the similarities and differences between intestinal bacterial communities. The intestinal microbiota profile of the CDI group of mice was significantly different from that of healthy mice, indicating that their microbial ecosystem was disrupted. The composition of the microbiota of the BA group and the PDA-TH group was similar to that of the CDI group, indicating that the use of BA or PDAT alone had limited effect on the recovery of the microbiota. In contrast, the microbiota profile of the BA@HF group and the BA@HF-PDAT group was closer to that of healthy mice compared with the CDI, BA and PDA-TH groups (Figure 8(B), Figure 15 ). These findings highlight the effectiveness of BA@HF and BA@HF-PDAT in alleviating intestinal microbiota dysbiosis.

[0089] Community bar plot analysis showed that Firmicutes, Bacteroidetes, Myxobacteria, and Desulfurobacteria were dominant in the CON group, and Proteobacteria and Firmicutes were dominant in the CDI group. Among them, Proteobacteria contains numerous pathogenic genera, and the increase in relative abundance of Proteobacteria is considered a potential diagnostic marker of intestinal dysbiosis and increased risk of disease. The dominant phyla in the BA and PDAT groups were similar to those in the CDI group. In contrast, the dominant phyla in the BA@HF and BA@HF-PDAT groups were Bacteroidetes, Firmicutes, and Proteobacteria, which were similar to the CON group. The Proteobacteria was significantly reduced in the BA@HF and BA@HF-PDAT groups. Bacteroidota is one of the most abundant members of the beneficial flora, responsible for polysaccharide degradation and inflammation relief, and was significantly restored in the BA@HF and BA@HF-PDAT groups. This restoration can be attributed to the large amount of nutritional substrate provided by the HF. At the genus level, the CDI group had increased abundance of pathogenic bacteria compared to the CON group, including Escherichia-Shigella (inducing severe diarrhea and intestinal inflammation), Clostridium innocua (inducing intestinal barrier damage and inflammation), Klebsiella (inducing pneumonia and liver abscess), and Enterococcus (promoting CDI and increasing the virulence of C. difficile). The microbiome composition of the BA and PDAT groups was similar to that of the CDI group. In contrast, the BA@HF and BA@HF-PDAT groups had significantly increased beneficial bacteria such as Lachnospira (reducing inflammation and oxidative stress, inhibiting the growth of harmful bacteria, and improving the intestinal mucosal barrier), Parabacteroides (regulating intestinal immunity), Akkermansia (reducing CDI, consuming mucin and producing bioactive compounds, nourishing intestinal epithelial cells and commensal probiotics), and Lactobacillus (reducing CDI, inhibiting C. difficile toxin production, and maintaining intestinal health), and their microbiome composition was more similar to that of the CON group. As shown in FIG. 8(D), the relative abundance of key microbial groups was analyzed, including Bifidobacterium, Lactobacillus, Clostridium, and Proteobacterium. The relative abundance of Bifidobacterium was significantly increased in the BA, BA@HF, and BA@HF-PDAT groups, indicating that BA successfully colonized the intestine. The relative abundance of Lactobacillus was significantly increased in the BA@HF and BA@HF-PDAT groups, indicating that the polysaccharide-based gel promoted the growth of Lactobacillus and contributed to CDI treatment. The relative abundance of Clostridium was significantly reduced in the BA@HF-PDAT group compared to the CDI group, indicating that BA@HF-PDAT showed the strongest efficacy in eliminating C. difficile from the intestinal lumen. In addition, Proteobacterium is a pathogenic bacterium associated with acute gastroenteritis and Crohn's disease. The relative abundance of Proteobacterium was significantly reduced in the BA@HF and BA@HF-PDAT groups, which can be due to the polysaccharide-based gel promoting the colonization of beneficial bacteria, thereby competitively reducing the number of Proteobacterium.

[0090] To elucidate the mechanism of BA@HF-PDAT treating CDI through host-microbe interaction, LEfSe analysis (LDA score >2, p<0.05) was performed to characterize differentially abundant bacterial taxa among groups (Fig. 8E). Then, correlation analysis was performed between the altered phenotypic indicators and microbial genera. As shown in Fig. 8F, the correlation analysis showed that the indicators of intestinal health (colon length, Claudin-1, occludin, and Ki67), antioxidant and anti-inflammatory capacity (T-AOC, SOD, GSH-Px, CAT, IL-10, and IL-22), and microbial diversity (ACE, Shannon, and Sobs index) were positively correlated with the three representative probiotics (Akumen, Lactobacillus, and Bifidobacterium) upregulated by BA@HF-PDAT and negatively correlated with the pathogenic genera (Clostridium, Proteus, and Escherichia-Shigella) inhibited by BA@HF-PDAT. In contrast, the inflammatory response (IL-1β, IL-6, and TNF-α) and oxidative stress (MDA level) were negatively correlated with the beneficial bacteria and positively correlated with the above harmful bacteria taxa. As expected, BA@HF-PDAT restored the host’s health and improved CDI by enriching the beneficial bacteria and inhibiting the harmful bacteria.

[0091] Restoration of gut microbiota composition and function is crucial for effective treatment of CDI. Therefore, the functional characteristics of the gut microbiota were assessed. BugBase analysis was used to predict gut microbial functional phenotypes (e.g., aerobicness, pathogenicity, and Gram staining). The results were consistent with the gut microbiota composition analysis, indicating that BA@HF-PDAT not only modulates the composition of the gut microbiota but also enhances its functional potential (Figure 19). Furthermore, Kyoto Encyclopedia of Genetics and Genomes (KEGG) pathway analysis was performed to determine the functional profiles of the gut microbiota in the CON, CDI, and BA@HF-PDAT groups. *Clostridium difficile* utilizes a two-component system (TCS) signaling mechanism to adapt to environmental changes, regulating the expression of virulence factors and antibiotic resistance genes, thereby promoting its proliferation and host invasion. Similarly, ATP-binding cassette (ABC) transporters in *Clostridium difficile* contribute to the expression of antibiotic resistance genes, enabling the bacteria to survive antibiotic treatment and induce disease. Compared to the CON group, the CDI group showed significantly enhanced functional activity of TCS and ABC transporters, indicating that Clostridium difficile proliferation promotes the expression of virulence and antibiotic resistance genes in the gut microbiota. Conversely, these functions were significantly inhibited in the BA@HF-PDAT group, suggesting that BA@HF-PDAT effectively eliminates Clostridium difficile, thereby reducing the expression of virulence and antibiotic resistance genes and exerting a protective effect on the host. Simultaneously, the results indicate that CDI significantly impairs the metabolic function of the gut microbiota, manifested as a significant reduction in carbon metabolism, amino acid biosynthesis, and secondary metabolite biosynthesis. Figure 8 G). BA@HF-PDAT treatment not only reconstructs the composition of the gut microbiota, but also holds promise for restoring the metabolic function of the gut microbiota.

[0092] (10) Changes in intestinal metabolites caused by BA@HF-PDAT Therefore, a non-targeted metabolomics approach was used to further investigate the effects of BA@HF-PDAT on the characteristics and function of intestinal metabolites. A total of 3192 metabolites were identified, of which 51 metabolites were specific to the CON group, 74 metabolites were specific to the CDI group, and 48 metabolites were specific to the BA@HF-PDAT group. Figure 9 A). Volcano plots showed that, compared to the CON group, the CDI group exhibited significant differences in 1215 metabolites, with 634 metabolites significantly upregulated and 581 significantly downregulated; compared to the CDI group, the BA@HF-PDAT group exhibited significant differences in 1027 metabolites, with 739 metabolites significantly upregulated and 288 significantly downregulated. Figure 9 (BC)). Principal component analysis (PCA) showed significant differences in the intestinal metabolite profiles among the three groups. Figure 9D). Cluster analysis was performed according to sample characteristics, and the top 40 differential metabolites were determined according to FDR < 0.05) and VIP value > 1.5, and then these metabolites were clustered separately. BA@HF-PDAT group showed similar metabolite abundance characteristics compared with CON group, suggesting that it recovered better metabolic homeostasis ( Figure 17 , Figure 18 ). KEGG enrichment analysis of differential metabolites showed that CDI down-regulated nucleotide metabolism, tryptophan metabolism, lipoic acid metabolism, a-linolenic acid metabolism, primary bile acid biosynthesis, and valine, leucine and isoleucine biosynthesis compared with CON group. Conversely, BA@HF-PDAT group significantly recovered nucleotide metabolism, valine, leucine and isoleucine biosynthesis, lipoic acid metabolism, primary bile acid biosynthesis, and tryptophan metabolism compared with CDI group ( Figure 9E). Nucleotides are the basic units of DNA and RNA and are important precursors for the proliferation of gut microbiota. The decrease in nucleotide metabolism reflects the decrease in metabolic flux of gut microbiota, which in turn directly impairs the intestinal barrier repair. This result indicates that BA@HF-PDAT effectively restores the gut microbiota and intestinal barrier from the perspective of metabolic intervention. Amino acid metabolism occupies the vast majority of the top 10 pathways, which is consistent with the 16S sequencing functional prediction results, indicating that amino acid metabolism plays an important role in the protective effect of BA@HF-PDAT on CDI-induced colitis. Intestinal amino acid homeostasis is crucial for maintaining intestinal health and physiological function, while amino acid metabolism disorder is closely related to disease progression and exacerbation of pathological conditions. Tryptophan metabolism is closely related to intestinal health. Inhibition of tryptophan metabolism can lead to increased mucosal damage, increased cecal bleeding, and increased IFN-γ production in CDI mice, leading to more severe pathological processes. In addition, tryptophan and its metabolites are believed to play an important role in alleviating intestinal inflammation, and BA@HF-PDAT can improve the biosynthesis and metabolism of tryptophan, which may be an important mechanism for alleviating CDI. Branched-chain amino acids (valine, leucine, and isoleucine) play a key role in regulating intestinal health and immune function. Supplementing branched-chain amino acids has been shown to promote the secretion of intestinal secretory immunoglobulin A (sIgA), enhance the defense capacity of the mucosal surface, prevent pathogenic invasion of the intestinal lamina propria, and support the host's intestinal health. Supplementing leucine can promote the proliferation of intestinal epithelial cells and promote intestinal development. In addition, branched-chain amino acids can alleviate central and muscle fatigue by regulating the levels of fatigue-related compounds (lactate, ammonia, and 5-hydroxytryptamine), energy metabolites (glucose and free fatty acids), and muscle soreness markers (lactate dehydrogenase and creatine kinase) in serum, which may partially explain the observed decrease in activity and increase in disease activity index (DAI) in CDI mice. BA@HF-PDAT significantly reversed the inhibition of tryptophan metabolism pathways and the biosynthesis of valine, leucine, and isoleucine caused by CDI, while enhancing the biosynthesis pathways of phenylalanine, tyrosine, and tryptophan, thereby highlighting its key role in alleviating CDI by regulating amino acid metabolism. The bidirectional regulatory relationship between bile acids and gut microbiota plays a crucial role in maintaining bile acid homeostasis and significantly affects gut microbiota composition, intestinal barrier integrity, and overall host health. CDI disrupts primary bile acid biosynthesis and enhances the bile secretion pathway, leading to bile acid metabolism disorder, further exacerbating gut dysbiosis. In contrast, BA@HF-PDAT treatment can effectively restore primary bile acid biosynthesis and the bile secretion pathway. Notably, as shown in Figure 6, BA@HF-PDAT treatment significantly increased the abundance of bile acid biosynthesis-related genes (FABP2, FABP7, and FABP1) and bile acid transport-related genes (ABCB11, ABCB4, and ABCB7) in the gut microbiota of CDI mice, which may be an important mechanism for alleviating CDI. In summary, BA@HF-PDAT can effectively restore the gut microbiota and intestinal barrier function in CDI-induced colitis by regulating the metabolic pathways of nucleotides, amino acids, and bile acids, which may be an important mechanism for alleviating CDI. Figure 18As shown, the restoration of bile acid-associated metabolic pathways was closely associated with the restoration of levels of intestinal deoxycholic acid (DCA) and muricholic acid (MCA). DCA is a secondary bile acid known to inhibit the growth and spore germination of C. difficile (CDI). Antibiotic-induced dysbiosis often leads to a decrease in DCA levels, thereby increasing the susceptibility to C. difficile (CDI). In addition, MCA, a mouse-specific bile acid, has also been demonstrated to inhibit the growth and spore germination of C. difficile. These findings suggest that BA@HF-PDAT can ameliorate CDI-induced colitis by modulating the bile acid levels in the mouse intestine.

[0093] To elucidate the mechanism of BA@HF-PDAT in ameliorating CDI by modulating the gut microbiota-metabolite interactions, a co-occurrence network based on Spearman correlation analysis was constructed, which contained the significantly altered bacterial taxa and metabolites ( Figure 9 F; bacteria LDA>2, metabolites VIP>1.5, FDR<0.05). The analysis showed that members of Firmicutes, Bacteroidetes, and Verrucomicrobia were strongly correlated with a series of intestinal metabolites. Most of the metabolites, including deoxycholic acid (DCA), topotecan, deoxyinosine, and deoxycytidine, were mainly enriched in pathways related to bile secretion and nucleotide metabolism. Notably, Akkermansia exhibited significant positive correlations with all these metabolites. Meanwhile, the potential beneficial metabolite DCA was positively correlated with Firmicutes and Bacteroidetes, but negatively correlated with pathogenic genera such as Escherichia-Shigella, Providencia, Proteobacteria, and Klebsiella. These findings confirmed the 16S rRNA sequencing results and highlighted the co-occurrence network formed between the key microbial taxa and metabolites altered by BA@HF-PDAT, which might synergistically contribute to its therapeutic effect on CDI.

[0094] Finally, the multi-omics analysis was integrated, and based on the Mantel test and Spearman correlation analysis, a mechanistic model of how BA@HF-PDAT alleviated CDI through the “gut microbial-metabolite” axis was constructed ( Figure 9 G). Notably, 7 metabolites—DCA, MCA, deoxyinosine, 5a,8b,9b-5,9-epoxy-3,6-olusen-8-ol, porrigenin A, e-tocopherol, and 7-dehydroxycholine-8(14)-enoic acid—were identified to be negatively correlated with the severity of CDI, and 3 metabolites—lysozyme, choline methionine, and deoxycytidine—were positively correlated with the severity of CDI. These findings suggest that the restoration of gut microbiota, metabolic reprogramming, and intestinal tissue repair might synergistically act to enable BA@HF-PDAT to effectively alleviate CDI-induced colitis.

[0095] (11) Bio-safety evaluation of BA@HF-PDAT Given the potential adverse effects of BA@HF-PDAT, it is essential to evaluate its bio-safety. Previous HCoEpiC cell co-culture experiments showed that BA@HF-PDAT of different concentrations did not produce significant cytotoxic effects, with cell viability maintained at about 100%. In addition, the in vivo bio-safety of oral BA@HF-PDAT was systematically evaluated. Oral BA@HF-PDAT did not cause significant changes in body weight of mice compared with the healthy control group (Fig. 11A). Figure 19 No obvious pathological changes were observed in the major organs (liver, spleen, and kidney) of mice compared with the healthy control group (Fig. 11B). Figure 20 Hematological analysis showed no significant differences in WBC, RBC, HGB, PLT, and MCV between groups (Fig. 11C). Figure 20 Liver function assessment showed no significant differences in AST, ALT, ALP, ALB, and TBIL between groups (Fig. 11D). Figure 15 Kidney function analysis showed no significant differences in UA, Crea, BUN, and Urea between groups (Fig. 11E). Figure 20 The above results indicate that BA@HF-PDAT has good bio-safety and smaller side effects.

[0096] In this study, a new colon-targeting hydrogel co-delivery platform (BA@HF-PDAT) was developed. This platform showed high efficacy in the precision treatment of CDI. PDA-TH NPs, which can inhibit the virulence factors of C. difficile, were designed and synthesized, and were co-delivered with BA into the polysaccharide hydrogel. BA@HF-PDAT showed strong stability under harsh gastrointestinal conditions and preferentially adhered to the inflamed mucosal site for a longer period of time. In addition, this platform inhibited the expression of C. difficile virulence factors, thereby reducing their ecological competitiveness, while enhancing the colonization of BA. In the CDI mouse model, oral BA@HF-PDAT significantly reduced the colonic lesions, oxidative stress, and inflammation caused by CDI. Mechanistically, intestinal cell culture models, 16S rRNA sequencing, and non-targeted metabolomics showed that BA@HF-PDAT effectively restored microbial diversity and metabolic function, particularly amino acid and bile acid metabolism, reshaping the intestinal microenvironment. Epithelial regeneration was activated through the Wnt / β-catenin and Bax / Bcl-2 pathways, cell apoptosis was inhibited, and tissue repair and barrier recovery were promoted. This multifunctional strategy integrated pathogen clearance, intestinal microecological remodeling, and the restoration of epithelial cell proliferation capacity, providing a promising and safe alternative to traditional CDI treatment. In addition, it provides a valuable framework for developing treatment strategies for other bacterial gastrointestinal diseases.

[0097] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. The present application can have various changes and modifications for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A probiotic-polyphenol nanoparticle colon-targeted co-delivery system, characterized in that, The probiotic-polyphenol nanoparticle colon-targeted co-delivery system comprises a probiotic polysaccharide composite gel and polyphenol nanoparticles, the polyphenol nanoparticles encapsulate polyphenols to be delivered, the probiotic polysaccharide composite gel encapsulates probiotics to be delivered, and the polyphenol nanoparticles and the probiotics are co-encapsulated in the polysaccharide composite gel; The probiotic polysaccharide composite gel is formed by combining probiotics and a polysaccharide gel; The polyphenol nanoparticles are composed of polyphenols and polydopamine nanoparticles.

2. The colon targeting co-delivery system according to claim 1, wherein, The probiotics are Bifidobacterium adolescentis; Preferably, the polyphenols are thymol; Preferably, the polydopamine nanoparticles have a porous spherical morphology, are negatively charged on the surface, and have an average particle size of 300-400 nm; Preferably, the polyphenol nanoparticles have an irregular nanospherical morphology, an average particle size of 100-300 nm, and preferably 180-220 nm.

3. A process for the preparation of the probiotic-polyphenol nanoparticle colon targeted co-delivery system as claimed in claim 1 or 2, wherein, The method comprises the following steps: S1, water-alcohol precipitation method is used to extract ficus carica leaf polysaccharide, ficus carica leaf polysaccharide and hyaluronic acid are mixed in water, and a polysaccharide solution is obtained; probiotics are mixed with the polysaccharide solution, and BA@HF is formed by sterile stirring; S2, polydopamine nanoparticles PDA NPs are prepared by self-polymerization, the PDA NPs are suspended in water, polyphenols are added, ultrasonic stirring is performed, and PDA-TH NPs are obtained; S3, BA@HF and PDA-TH NPs are co-incubated, and BA@HF-PDAT is obtained by sterile stirring.

4. The production method according to claim 3, characterized by, In step S1, the specific preparation process of the water-alcohol precipitation method for extracting ficus carica leaf polysaccharide is as follows: (1) dry ficus carica leaves are soaked in alcohol, the alcohol solvent is removed, and the leaves are dried; (2) the dried ficus carica leaves are crushed, soaked in hot water, and filtered to obtain a crude extract; (3) the crude extract is concentrated by rotary evaporation, alcohol solvent is added to precipitate the polysaccharides in the crude extract, the extract is alcohol precipitated at low temperature, the supernatant is removed after centrifugation, and the supernatant is removed and washed after centrifugation; (4) free proteins are removed from the crude extract by the Sevage method, the water phase is separated from the denatured protein layer, and the process is repeated multiple times; the obtained solution is swelled in water overnight, then placed in a semi-permeable membrane dialysis bag, dialyzed for 12-24 h, and then concentrated by rotary evaporation and vacuum freeze-dried to obtain ficus carica leaf polysaccharide.

5. The preparation method according to claim 4, characterized in that, In step (1), the alcohol is ethanol, and the soaking time in ethanol is 1-3 h; Preferably, in step (2), the hot water is at 70-90°C, and the soaking time is 2-4 h; Preferably, in step (3), the alcohol solvent is anhydrous ethanol with a final concentration of 75-85%, and the extract is alcohol precipitated at low temperature 1-5°C for 10-14 h.

6. The preparation method according to claim 3, characterized in that, In step S1, the ratio of ficus carica leaf polysaccharide, hyaluronic acid, and water is (80-120 mg):(180-220 mg):(10-30 mL), and preferably (90-110 mg):(190-210 mg):(15-25 mL); Preferably, in step S1, the sterile mixing time is 1-5 h, the concentration of the polysaccharide solution is 100-200 mg / mL, and preferably 140-160 mg / mL; Preferably, in step S1, the volume ratio of the probiotic bacteria to the polysaccharide solution is (5-15):(5-15), preferably (8-12):(8-12); the concentration of the probiotic bacteria is 0.5x10 8 ~1.5x10 8 CFU / mL. Preferably, in step S1, the sterile stirring time is 20-40 min.

7. The preparation method according to claim 3, characterized in that, In step S2, the specific steps of preparing the PDA NPs by the self-polymerization method include: dissolving poloxamer, dopamine hydrochloride and 1,3,5-trimethylbenzene in an alcohol solution, ultrasonically treating the mixture, then adding ammonia water, stirring at room temperature, and then centrifuging, washing and drying to obtain the PDA NPs; Preferably, the alcohol is a 50% ethanol solution; Preferably, the ratio of poloxamer, dopamine hydrochloride, 1,3,5-trimethylbenzene, alcohol solution and ammonia water is (0.8-1.5 g):(0.3-0.7 g):(1-5 mL):(30-50 mL):(2-8 mL), preferably (0.9-1.1 g):(0.4-0.6 g):(1-3 mL):(35-45 mL):(4-6 mL); Preferably, the ultrasonic treatment is performed for 10-30 min; Preferably, in step S2, the ratio of PDA NPs, water and polyphenol is (10-30 mg):(5-20 mL):(40-60 mg), preferably (15-25 mg):(8-12 mL):(45-55 mg); Preferably, in step S2, the ultrasonic treatment is performed for 10-30 min and the stirring is performed for 1-5 h; Preferably, in step S3, the sterile stirring is performed for 10-40 min, preferably 20-30 min.

8. Use of the probiotic-polyphenol nanoparticle colon-targeted co-delivery system of claim 1 or 2 or the probiotic-polyphenol nanoparticle colon-targeted co-delivery system prepared by the method of any one of claims 3-7 in the preparation of a product for co-delivering polyphenols and probiotics.

9. A probiotic-polyphenol nanoparticle colon-targeted co-delivery formulation, characterized in that, The probiotic-polyphenol nanoparticle colon-targeted co-delivery system of claim 1 or 2 or the probiotic-polyphenol nanoparticle colon-targeted co-delivery system prepared by the method of any one of claims 3-7.

10. Use of the probiotic-polyphenol nanoparticle colon-targeted co-delivery system of claim 1 or 2 or the probiotic-polyphenol nanoparticle colon-targeted co-delivery system prepared by the method of any one of claims 3-7 or the probiotic-polyphenol nanoparticle colon-targeted co-delivery preparation of claim 9 in the preparation of a medicament for treating Clostridium difficile infection.

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