A manganese-doped ceria nanoscale enzyme-loaded grifola frondosa polysaccharide hydrogel, a preparation method and application thereof

Abstract

The application discloses a kind of loaded manganese doped ceria nanoscale enzyme's Pachyman hydrogel and its preparation method and application, it is related to biomedical technical field.The preparation method is first prepared by high-temperature thermal decomposition method fat-soluble manganese doped ceria nanoscale enzyme, then it is surface-modified using distearoyl phosphatidyl ethanolamine to obtain water-soluble nanoscale enzyme;Polyvinyl alcohol solution is mixed uniformly with Pachyman powder, water-soluble nanoscale enzyme, borax solution is added to crosslink and form hydrogel, after freeze-room temperature processing alternately, it is obtained.The energy storage modulus of the prepared hydrogel is 120-350 Pa at pH 6.5-7.2, 37 ℃, and the adhesion is greater than or equal to 8.5 kPa.The hydrogel prepared by the application has good biocompatibility, can efficiently remove active oxygen, relieve intestinal oxidative stress, and has suitable adhesion and degradation characteristics, can be administered by enema, and be used for preparing drugs for treating irritable bowel syndrome.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozymes, its preparation method, and its application. Background Technology

[0002] Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder characterized by recurrent abdominal pain, bloating, and changes in bowel habits. Based on stool characteristics, it can be divided into subtypes such as diarrheal IBS (IBS-D) and constipation IBS (IBS-C), with diarrheal IBS being the most common clinically. The disease has a high global prevalence, primarily affecting young and middle-aged adults. Its prolonged and recurrent course not only severely impacts patients' quality of life but also imposes a heavy medical burden on society. The etiology and pathogenesis of IBS are not yet fully understood, but it is generally believed to be a brain-gut axis dysfunction resulting from the interaction of multiple factors, including visceral hypersensitivity, abnormal gastrointestinal motility, impaired intestinal mucosal barrier, low-grade inflammation, intestinal flora imbalance, and psychological factors. Existing conventional treatments (such as antispasmodics and antidiarrheals) primarily aim to relieve symptoms, but have limitations such as slow onset of action, significant individual variability in efficacy, and difficulty in precisely addressing core pathological aspects.

[0003] Numerous studies have shown that persistent intestinal dysfunction can lead to decreased antioxidant capacity in IBS patients, triggering reactive oxygen species (ROS)-mediated oxidative stress. Excessive ROS can directly damage intestinal epithelial cells, disrupt tight junctions, increase intestinal permeability, and promote the release of pro-inflammatory cytokines, further affecting enteric nerve function and causing visceral hypersensitivity and abdominal pain. Furthermore, abnormalities in the serotonin (5-HT) signaling pathway are also a key pathological mechanism of IBS. Elevated 5-HT levels in the intestinal mucosa of patients can activate primary afferent neurons, inducing visceral hypersensitivity and accelerating intestinal transit. Targeting these mechanisms, artificial nanozymes possessing both superoxide dismutase (SOD) and catalase (CAT) activities can efficiently catalyze the ROS cascade reaction, converting it into water and oxygen, thus achieving local antioxidant effects. Among these, cerium oxide nanoparticles (CeNPs) exhibit unique Ce³⁺ activity. + / Ce 4+ Its valence-state switching ability shows promising application prospects in scavenging ROS; the incorporation of manganese ions (Mn) can further enhance its catalytic activity. Poria cocos polysaccharide (PCP), as the active ingredient of the traditional Chinese medicine Poria cocos, possesses bioactivities such as immunomodulation, anti-inflammation, and intestinal barrier repair. Based on this, this study designed a Poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozymes. Through enema local administration, the drug is directly delivered to the core reaction site of IBS symptoms—the colorectal region—aiming to provide a novel local drug delivery strategy for IBS treatment through multiple mechanisms, including scavenging excess ROS, reducing oxidative stress, and regulating the intestinal immune microenvironment.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] Based on the pathophysiological characteristics of irritable bowel syndrome (IBS) and the limitations of existing drug treatments, the present invention aims to provide a poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozyme, its preparation method, and its application for the treatment of IBS.

[0006] This invention is implemented as follows: In a first aspect, the present invention provides a method for preparing a Poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozyme, comprising the following steps: Manganese acetate and cerium acetate were dissolved in a mixed solvent of oleylamine and xylene. After stirring and sonication, the mixture was heated under inert gas protection and water was added to carry out the reaction. After the reaction was completed, the mixture was centrifuged, washed and redispersed to obtain a lipid-soluble manganese-doped cerium dioxide nanozyme. The lipid-soluble manganese-doped cerium dioxide nanozyme was surface-modified with distearate phosphatidylethanolamine, and after removing the organic solvent, water was added for ultrasonic dispersion. Then, it was purified by membrane filtration and dialysis to obtain water-soluble manganese-doped cerium dioxide nanozyme. Take a polyvinyl alcohol solution, add poria cocos polysaccharide powder and the water-soluble manganese-doped cerium dioxide nanozyme, stir evenly, then add borax solution for cross-linking to form a hydrogel; the obtained hydrogel is subjected to alternating freezing and room temperature treatment to obtain poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozyme.

[0007] In an optional embodiment, the molar ratio of manganese acetate to cerium acetate is (2-4):(3-6); the volume ratio of oleylamine to xylene is 1:1 to 3:1; the reaction temperature is 90℃-110℃, and the reaction time is 1-4 hours.

[0008] In an optional embodiment, the mass ratio of the lipid-soluble manganese-doped cerium dioxide nanozyme to distearate phosphatidylethanolamine is 1:2 to 1:5; the surface modification reaction is carried out in chloroform or dichloromethane solvent under stirring at room temperature for 2-6 hours.

[0009] In an optional embodiment, the polyvinyl alcohol solution has a mass-volume fraction of 5%-15%, and the borax solution has a mass-volume fraction of 1%-5%; the volume ratio of the polyvinyl alcohol solution to the borax solution is 5:1 to 10:1.

[0010] In an optional embodiment, the amount of Poria cocos polysaccharide added is 2%-15% of the mass of polyvinyl alcohol; the amount of water-soluble manganese-doped cerium dioxide nanozyme added is 0.1%-5% of the mass of polyvinyl alcohol, based on the mass of the manganese-doped cerium dioxide nanozyme.

[0011] In an optional embodiment, the freeze-room temperature alternation process includes the following steps: freezing at -20°C to -10°C for 2-8 hours, then thawing at room temperature for 1-2 hours, and repeating the alternation process 1-3 times.

[0012] Secondly, the present invention provides a Poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozyme, wherein the hydrogel has a storage modulus of 120-350 Pa and an adhesion force ≥8.5 kPa at pH 6.5-7.2 and 37℃.

[0013] Thirdly, the present invention provides the application of a poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozyme in the preparation of a drug for the prevention or treatment of irritable bowel syndrome.

[0014] In an optional implementation, the hydrogel is used in the preparation of drugs for efficiently scavenging reactive oxygen species, alleviating oxidative stress, regulating the intestinal immune microenvironment, reducing chronic inflammation, repairing intestinal barrier function, or regulating the 5-HT signaling pathway.

[0015] In an optional implementation, the drug is administered via enema.

[0016] The present invention has the following beneficial effects: This invention provides a hydrogel of Poria cocos polysaccharide loaded with manganese-doped cerium dioxide nanozyme (CeMn@PCPGel). This formulation uses polyethylene glycol-modified cerium-manganese nanozyme and Poria cocos polysaccharide as active ingredients, administered locally in the rectum to achieve synergistic treatment through multiple pathways. This hydrogel formulation can efficiently scavenge reactive oxygen species in the lesion area, alleviate oxidative stress, regulate the intestinal immune microenvironment, reduce chronic inflammation, repair intestinal barrier function, and modulate the 5-HT signaling pathway, thereby significantly improving IBS-D-related diarrhea symptoms and visceral hypersensitivity. This dosage form not only increases the concentration and retention time of the drug at the intestinal lesion site but also reduces adverse reactions caused by systemic exposure, providing a novel local drug delivery strategy with good translational potential for the clinical treatment of IBS-D. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The particle size, PDI, and Zeta potential of the CeMn NP-PEG nanozyme in the embodiments of the present invention are shown below. Figure 2These are representative TEM images of CeMn NP and CeMn NP-PEG nanozymes from embodiments of the present invention; Figure 3 This is an EDS elemental diagram of the nanozyme CeMn NP in the embodiments of the present invention; Figure 4 The SEAD and XRD patterns of the nanozyme in the embodiments of the present invention reveal the crystal structure of CeMn NP; Figure 5 This is a schematic diagram illustrating the preparation of CeMn@PCP Gel in an embodiment of the present invention; Figure 6 These are scanning electron microscope (SEM) images of hydrogel PCP Gel and CeMn@PCP Gel in embodiments of the present invention; Figure 7 The following figures illustrate the performance testing of hydrogel PCP Gel and CeMn@PCP Gel in this embodiment of the invention: Figures (A) and (B) show the swelling test of the gel's rheological properties, Figure (C) shows the viscosity test of the gel, Figure (D) shows the swelling experiment of the gel, and Figure (E) shows the degradation experiment of the gel. Figure 8 This is an example of the detection of the enzyme-like activity of CeMn@PCP Gel in this invention. Figure (A) shows the ability to scavenge ABTS, Figure (B) shows glutathione activity, Figure (C) shows POD activity, Figure (D) shows CAT-like activity, Figure (E) shows superoxide dismutase-like enzyme activity, and Figure (F) shows the ability to scavenge hydroxyl radicals. Figure 9 The results of the cytotoxicity test of CeMn NP-PEG nanozyme against bone marrow-derived macrophages (BMDM) cells in this embodiment of the invention are shown. Figure 10 This invention illustrates the uptake of CeMn NP-PEG nanozyme by BMDM cells in an embodiment of the invention. Figure (A) shows an inverted fluorescence microscope image of CeMn NP-PEG uptake by BMDM cells, and Figure (B) shows the flow cytometry statistics of CeMn NP-PEG uptake by BMDM cells. Figure 11 This invention illustrates the removal of excess ROS from cells by CeMn@PCP Gel extract in an embodiment of the invention. Figure (A) shows the analysis of intracellular ROS fluorescence intensity using an inverted fluorescence microscope, and Figure (B) shows the analysis of intracellular ROS fluorescence intensity using flow cytometry. Figure 12This invention provides an evaluation of the in vivo therapeutic effect of CeMn@PCP Gel in this embodiment. Figure (A) illustrates the animal experiment process, Figure (B) shows the changes in body weight of rats in each treatment group, Figure (C) shows the food intake of rats in each treatment group, Figure (D) shows the sucrose preference test of rats in each treatment group, Figure (E) shows the fecal water content of rats in each treatment group, and Figure (F) shows the abdominal wall withdrawal reflex AWR score of rats in each treatment group. Figure 13 This is a schematic diagram of H&E staining, AB staining, and PAS staining of colon tissue from rats in each treatment group in this embodiment of the invention. Figure 14 In this embodiment of the invention, CeMn@PCP Gel treatment can improve the intestinal barrier in IBS rats. Figure (A) shows the immunohistochemical staining results of ZO-1 and Claudin-1, and Figures (B) and (C) show the expression levels of ZO-1 and Claudin-1 mRNA in the colon of rats in each treatment group by RT-qPCR. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0020] The following provides a detailed description of the Poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozymes, its preparation method, and its application.

[0021] The Poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozymes integrates multiple pharmacological activities and possesses a synergistic therapeutic mechanism. On one hand, manganese doping significantly enhances the catalytic performance of cerium dioxide nanozymes (CeNPs): Mn incorporation not only optimizes the nanozyme particle size but also increases the surface Ce³⁺ content. + / Ce 4+ The proportions and valence cycle rates of these enzymes endow the material with stronger superoxide dismutase (SOD) and catalase (CAT) activities. This dual-enzyme activity can mimic the body's natural antioxidant enzyme system, catalyzing the conversion of excess reactive oxygen species (ROS) generated at the lesion site into water and oxygen, thereby efficiently scavenging ROS, blocking oxidative stress responses, inhibiting the pro-inflammatory function of macrophages, and reshaping the intestinal inflammatory microenvironment. On the other hand, the Poria cocos polysaccharide (PCP) itself, as a carrier, has clear anti-inflammatory and immunomodulatory effects. It can further consolidate the anti-inflammatory effect and reduce visceral hypersensitivity by regulating the intestinal flora, repairing the intestinal barrier, and inhibiting the TLR4 / NF-κB signaling pathway.

[0022] In a first aspect, the present invention provides a method for preparing a Poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozyme, comprising the following steps: S1. Preparation of lipid-soluble manganese-doped cerium dioxide nanozyme (CeMn NP): Manganese acetate and cerium acetate were dissolved in a mixed solvent of oleylamine and xylene. After stirring and sonication, the mixture was heated under inert gas protection and water was added to carry out the reaction. After the reaction was completed, the mixture was centrifuged, washed and redispersed to obtain lipid-soluble manganese-doped cerium dioxide nanozyme.

[0023] In some embodiments, the molar ratio of manganese acetate to cerium acetate is (2-4):(3-6); the volume ratio of oleylamine to xylene is 1:1 to 3:1; the reaction temperature is 90℃-110℃, and the reaction time is 1-4 hours.

[0024] S2. Preparation of water-soluble manganese-doped cerium dioxide nanozyme (CeMn NP-PEG): The lipid-soluble manganese-doped cerium dioxide nanozyme was subjected to a surface modification reaction with distearate phosphatidylethanolamine (DSPE). After removing the organic solvent, water was added for ultrasonic dispersion, followed by membrane filtration and dialysis purification to obtain the water-soluble manganese-doped cerium dioxide nanozyme.

[0025] In some embodiments, the mass ratio of the lipid-soluble manganese-doped cerium dioxide nanozyme to distearate phosphatidylethanolamine is 1:2 to 1:5; the surface modification reaction is carried out in chloroform or dichloromethane solvent with stirring at room temperature for 2-6 hours.

[0026] In this application, the distearate phosphatidylethanolamine is selected as DSPE-mPEG. 2k By adopting DSPE-mPEG 2k Surface modification of lipid-soluble manganese-doped cerium dioxide nanozymes offers advantages due to the unique structure of this amphiphilic molecule. The hydrophobic DSPE phospholipid chains act as "anchors," firmly inserting into the oil-phase coating of the nanozyme through hydrophobic interactions, achieving stable encapsulation. Meanwhile, the hydrophilic mPEG long chains form a dense hydration layer on the nanozyme surface, acting as a "barrier" for steric stabilization; PEG with a molecular weight of 2000 is preferred. This modification not only endows the nanozyme with excellent water solubility and dispersibility, enabling uniform loading in hydrogel systems, but more importantly, it endows the nanozyme with "stealth" properties, effectively avoiding recognition and clearance by the reticuloendothelial system. This significantly increases the retention and accumulation of the nanozyme at intestinal lesions, thereby enhancing its efficacy in scavenging reactive oxygen species and regulating the immune microenvironment. This provides a crucial pharmaceutical basis for subsequent enema administration in the treatment of irritable bowel syndrome.

[0027] S3. Preparation of Poria cocos polysaccharide hydrogel loaded with nanozyme (CeMn@PCP Gel): Take polyvinyl alcohol solution, add Poria cocos polysaccharide powder and the water-soluble manganese-doped cerium dioxide nanozyme, stir evenly, add borax solution for mixing and cross-linking to form hydrogel; the obtained hydrogel is subjected to alternating freezing-room temperature treatment to obtain Poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozyme.

[0028] In some preferred embodiments, the preparation of the polyvinyl alcohol (PVA) solution and the borax solution includes the following steps: 98.0 g of deionized water and 2.0 g of PVA are added to a 100 mL Erlenmeyer flask; 96.0 g of deionized water and 4.0 g of borax are added to another 100 mL Erlenmeyer flask. The two flasks are then placed on a heating device and heated to 80°C. The mixture is stirred continuously at an appropriate speed for 30 min at this temperature until the solution is completely dissolved and becomes clear and transparent. Finally, the resulting solution is cooled to 37°C for later use.

[0029] In some embodiments, the polyvinyl alcohol solution has a mass-volume fraction of 5%-15%, and the borax solution has a mass-volume fraction of 1%-5%; the volume ratio of the polyvinyl alcohol solution to the borax solution is 5:1 to 10:1.

[0030] In some embodiments, the amount of Poria cocos polysaccharide added is 2%-15% of the mass of polyvinyl alcohol; the amount of water-soluble manganese-doped cerium dioxide nanozyme added is 0.1%-5% of the mass of polyvinyl alcohol, based on the mass of the manganese-doped cerium dioxide nanozyme.

[0031] In an optional embodiment, the freeze-room temperature alternation process includes the following steps: freezing at -20°C to -10°C for 2-8 hours, then thawing at room temperature for 1-2 hours, and repeating the alternation process 1-3 times.

[0032] Preferably, the freezing temperature for the alternating freezing-room temperature treatment is -20°C.

[0033] This application employs a freeze-thaw cycle to stabilize the structure of hydrogels. Its core advantage lies in significantly improving the mechanical properties and structural stability of the hydrogels through the dynamic reconstruction of the physical cross-linking network. During the freezing stage, water forms ice crystals within the system, forcing polyvinyl alcohol (PVA) molecular chains to locally concentrate and arrange themselves in an orderly manner within the ice crystal gaps. This promotes the recombination of hydrogen bonds between PVA chains and the uniform distribution of borate ester bonds. Subsequent thawing at room temperature melts the ice crystals, releasing the previously occupied space and forming a denser and more uniform three-dimensional network pore structure within the gel. After multiple freeze-thaw cycles, PVA molecular chains accumulate in microcrystalline regions, further enhancing the mechanical strength and elasticity of the gel.

[0034] In addition, after completing the freeze-to-room temperature treatment, the sample can be placed in a laminar flow hood and sterilized by continuous irradiation with a 254 nm ultraviolet lamp for 12 hours to ensure the sterility and biosafety necessary for subsequent cell experiments.

[0035] It should be noted that all room temperatures mentioned in this application refer to 15-30°C, which can be obtained by those skilled in the art based on publicly available information.

[0036] Secondly, the present invention provides a Poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozyme, wherein the hydrogel has a storage modulus of 120-350 Pa and an adhesion force ≥8.5 kPa at pH 6.5-7.2 and 37℃.

[0037] Thirdly, the present invention provides the application of a poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozyme in the preparation of a drug for the prevention or treatment of irritable bowel syndrome.

[0038] In some embodiments, the hydrogel is used in the preparation of drugs for efficiently scavenging reactive oxygen species, relieving oxidative stress, regulating the intestinal immune microenvironment, reducing chronic inflammation, repairing intestinal barrier function, or regulating the 5-HT signaling pathway.

[0039] Specifically, CeMn@PCP Gel integrates the highly efficient ROS scavenging ability of nanozymes with the immunomodulatory properties of polysaccharides. Through multi-target synergistic effects—scavenging excess ROS, reducing oxidative stress, reshaping immune homeostasis, enhancing intestinal barrier function, and regulating visceral sensitivity—it provides an innovative approach to IBS treatment that combines high efficiency, safety, and precision.

[0040] In some embodiments, the drug is administered via enema. In this application, CeMn@PCP Gel achieves highly efficient and safe local treatment by precisely targeting the lesion. Enema administration delivers the hydrogel directly to the core area of ​​IBS symptoms—the colorectal region—creating a high local drug concentration for rapid onset of action. Simultaneously, it effectively avoids the risks of upper gastrointestinal degradation and systemic exposure associated with oral administration, significantly reducing systemic side effects.

[0041] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0042] Example 1 This embodiment provides a method for preparing a Poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozymes, such as... Figure 5 As shown, it includes the following steps: S1. Preparation of lipid-soluble manganese-doped cerium dioxide nanozyme (CeMn NP): 0.4 mM manganese acetate tetrahydrate, 0.6 mM cerium acetate, and 15 mL oleylamine were co-dispersed in 15 mL xylene. The mixture was stirred at room temperature for 30 min, followed by sonication for 2 h, and then allowed to stand overnight with stirring. Under a nitrogen atmosphere, the reaction system was heated to 90 °C, and 1 mL of distilled water was quickly added. The temperature was then increased to 110 °C and the reaction was carried out for 3 h. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 10 min, the precipitate was collected, and redissolved in chloroform to obtain the lipid-soluble nanozyme CeMn NP.

[0043] S2. Preparation of water-soluble manganese-doped cerium dioxide nanozyme (CeMn NP-PEG): In preparing the water-soluble nanozyme, 10 mg of lipophilic nanozyme was reacted with 50 mg of DSPE-mPEG2k. After the reaction, the mixture was placed at 60℃ and the solvent was completely removed using a rotary evaporator. Then, 5 mL of distilled water was added, and the mixture was thoroughly dispersed under ultrasonic assistance to obtain the water-soluble nanozyme CeMn NP-PEG. The obtained product was filtered through a 0.22 μm filter membrane and then dialyzed in deionized water for 48 h using a dialysis bag with a molecular weight cutoff of 14 kDa for further purification. The purified CeMn NP-PEG sample was stored at 4℃ and its concentration was determined for later use.

[0044] S3. Preparation of Poria cocos polysaccharide hydrogel-supported nanozyme (CeMn@PCP Gel): 98.0 g of deionized water and 2.0 g of PVA were added to one 100 mL Erlenmeyer flask; 96.0 g of deionized water and 4.0 g of borax were added to another 100 mL Erlenmeyer flask. The two flasks were then placed on a heating device and heated to 80 °C. The mixture was stirred continuously at an appropriate speed for 30 min at this temperature until the solution was completely dissolved and became clear and transparent. Finally, the resulting solution was cooled to 37 °C for later use.

[0045] 900 μL of 2.0% PVA solution was transferred into a 2 mL EP tube, followed by the addition of 20.0 mg PCP powder and CeMn NP-PEG (Ce concentration 0.5 μg / mL), and the mixture was stirred thoroughly until homogeneous. Then, 100 μL of 4.0% Borax solution was added, and the mixture was gently stirred until fully homogeneous, thus forming CeMn@PCP Gel in situ.

[0046] The prepared hydrogel sample was first in The sample was frozen and allowed to stand at 20 °C for 12 h to complete the pre-stabilization treatment. Then, it was transferred to room temperature for 4 h to equilibrate, allowing for phase transition and structural relaxation. Finally, the sample was placed in a laminar flow hood and sterilized by continuous irradiation with a 254 nm UV lamp for 12 h.

[0047] Example 2 This embodiment characterizes and detects the CeMn NP and CeMn NP-PEG prepared in Example 1, such as... Figure 1 , Figure 2 , Figure 3 , Figure 4 and Figure 6 As shown.

[0048] Depend on Figure 1 The particle size, PDI, and Zeta potential of CeMn NP-PEG are shown. The particle size of CeMn NP-PEG is approximately 11.92 ± 1.94 nm, and the Zeta potential is -11.91 ± 1.84 mV. The polydispersity index (PI) is used to characterize the particle size distribution of nanoparticles. The smaller the value, the lower the degree of dispersion and the more concentrated the particle size distribution. The PI of CeMn NP-PEG is less than 0.3, indicating that the nanoparticles have a uniform particle size distribution, meet the standards for nano-formulations, and are suitable for subsequent experiments.

[0049] Depend on Figure 2 High-resolution transmission electron microscopy (HRTEM) revealed that CeMn NP exhibited good crystallinity and a polyhedral quasi-spherical morphology with an average core diameter of approximately 1.7 nm. Surface modification with DPSE-mPEG2k increased the particle size of CeMn NP-PEG to 2.2 nm, while the lattice fringes showed significant attenuation, indicating successful polyethylene glycol treatment of the nanoparticle surface.

[0050] Depend on Figure 3 The EDS elemental diagram of the nanozyme CeMn NP shown indicates that Ce and Mn elements are uniformly distributed and enriched on the surface of CeMn NP.

[0051] Depend on Figure 4Selected area electron diffraction (SAED) and X-ray diffraction (XRD) analyses jointly revealed the crystal structure characteristics of CeMn-NP. The SAED pattern showed clear and continuous diffraction rings, confirming the definite crystalline properties of the nanoparticles. The XRD pattern observed distinct characteristic diffraction peaks at 2θ = 29°, 33°, 48°, 56°, and 77°, whose positions matched the (111), (200), (220), (311), and (331) crystal planes of the cubic fluorite structure, further clarifying that the nanomaterial possesses a cubic fluorite structure. These results indicate that the prepared CeMn-NP exhibits good crystallinity and structural consistency.

[0052] Figure 6 Scanning electron microscopy (SEM) images further revealed the microstructure of the hydrogel. As shown in the figure, the hydrogel prepared with 2% PVA and 4% borax exhibits a uniform and highly interconnected porous network structure with a relatively uniform pore size distribution and a dense and orderly overall morphology, demonstrating ideal three-dimensional skeletal features of the hydrogel.

[0053] Example 3 This embodiment characterizes and detects the PCP Gel and CeMn@PCP Gel prepared in Example 1, such as... Figure 7 As shown.

[0054] Figure 7 Figure (A) shows the rheological tests of hydrogel PCP Gel and CeMn@PCP Gel. Frequency scanning results under 1% constant strain conditions indicate that both PCP Gel and CeMn@PCP Gel exhibit typical elastic-dominant behavior within the test frequency range (10¹-10² Hz). Their storage modulus (G′) is consistently higher than their loss modulus (G″), demonstrating stable solid-like elasticity and structural recovery properties under these conditions. The difference in viscosity characteristics between the gel and PCP Gel was assessed using a rotational viscometer. A B25 shaft was used, and 8 mL samples were continuously subjected to a fixed torque for 70 minutes at a constant temperature of 25 ℃. The viscosity of the hydrogel was measured, and before each test, the sample was ensured to be evenly covered after the shaft was immersed to ensure data reliability. The viscosity value during the stable phase was recorded. By comparing the average viscosity of the two and analyzing their change trend over time, the differences in rheological properties between the two were revealed. The final result, as shown in Figure (B), is that the incorporation of Poria cocos polysaccharide PCP significantly increased the viscosity of the hydrogel system, thereby enhancing its adhesive properties. Figure (C) shows the swelling experiment of the gel. The specific experimental procedure is to directly immerse an accurately weighed (W0) freeze-dried PCP gel hydrogel sample into an excess of swelling medium, place it in a constant temperature environment, carefully remove the sample with tweezers at a preset time point, place it on filter paper and let it stand for a moment to absorb excess surface moisture, and then quickly weigh it (W0).t When the mass change does not exceed 2% in three consecutive measurements, swelling equilibrium is considered to have been reached. The final results show that although the original gel gel exhibits a faster initial swelling rate, the PCP gel shows a slower and more persistent water absorption curve, indicating prolonged drug release. The hydrogel degradation experiment is shown in Figure (D). Pre-weighed (W0) block hydrogels were placed in PBS at 37 ℃ and pH 7.4 for degradation. Samples were periodically removed, washed, lyophilized, and weighed (W0). t The mass residual rate was calculated to assess the degradation behavior. Degradation studies showed that both Gel and PCP Gel were completely degraded within 3 hours, while PCP Gel had a slower mass loss rate and more stable degradation behavior.

[0055] Example 4 In this embodiment, the CeMn@PCP Gel prepared in Example 1 was subjected to enzyme-like activity detection, such as... Figure 8 As shown.

[0056] Figure 8 Figure (A) shows the ABTS activity assay: 100 μL of hydrogel extract CeMn@PCP Gel (Ce: 10 μg / mL, PCP: 1 mg / mL), 100 μL of CeMn NP-PEG (Ce: 10 μg / mL), and PCPGel (PCP: 1 mg / mL) were mixed with 400 μL of ABTS working solution (7.4 mmol / L ABTS stock solution + 2.6 mmol / L K2S2O8 stock solution) in a 2 mL centrifuge tube and allowed to react for 10 min. After the reaction, 100 μL of the mixture was transferred to a 96-well plate, and the absorbance at 734 nm was measured. Each measurement was repeated three times. The control (A0) consisted of 400 μL of ABTS solution mixed with 100 μL of ethanol. The results showed that CeMn NP-PEG alone exhibited moderate ABTS radical scavenging efficiency, with a scavenging rate of 17% at a concentration of 10 μg / mL. In contrast, PCP Gel (PCP: 1 mg / mL) exhibited a significantly higher ABTS free radical scavenging rate of 80.8%, indicating its intrinsic antioxidant activity. Notably, incorporating CeMn-NP-PEG into the PCP Gel matrix (Ce: 10 μg / mL; PCP Gel: 1 mg / mL) did not impair the free radical scavenging performance, as CeMn@PCP Gel maintained a high scavenging rate of 81.6%.

[0057] Figure (B) shows the glutathione activity assay. Following the manufacturer's instructions, the GSH reducing power of the samples was determined using a reduced glutathione (GSH) assay kit (Jiancheng Biotechnology Institute, Nanjing, China). The samples tested included: 100 μL of hydrogel extract CeMn@PCP Gel (Ce: 10 μg / mL, PCP: 2.0 mg / mL), 100 μL of PCP Gel (PCP: 2.0 mg / mL), and 100 μL of CeMn NP-PEG solution (Ce: 10 μg / mL). The results showed that the GSH-like activity of CeMn@PCP Gel reached 26.5 μmol / L, significantly higher than that of CeMn NP-PEG (12.3 μmol / L) and PCPGel (13.6 μmol / L).

[0058] Figure (C) shows the POD activity assay. In a 96-well plate, 40 μL of hydrogel extract CeMn@PCP Gel (Ce: 4 μg / mL, PCP: 1 mg / mL), 40 μL of CeMn NP-PEG (Ce: 4 μg / mL), and PCP Gel (PCP: 1 mg / mL) were mixed with 3,3′,5,5′-tetramethylbenzidine (TMB, 20 μL), double-distilled water (ddH2O, 120 μL), and hydrogen peroxide (H2O2, 10 mM, 20 μL), and allowed to react for 5 min. After the reaction, a full wavelength scan was performed in the range of 300–999 nm. The results showed that CeMn NP-PEG, PCP Gel, and CeMn@PCP Gel all exhibited POD-like activity, with CeMn@PCP Gel showing the strongest enzyme mimicry performance.

[0059] Figure (D) shows the superoxide dismutase (SOD) mimicry activity assay. The samples were analyzed using a total SOD activity assay kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. The samples tested included: 20 μL of hydrogel extract CeMn@PCP Gel (Ce: 10 μg / mL, PCP: 2.0 mg / mL), 20 μL of PCP Gel (PCP: 2.0 mg / mL), and 20 μL of CeMn NP-PEG solution (Ce: 10 μg / mL). The results showed that the SOD-like activities of CeMnNP-PEG and PCP Gel were 0.28 U / μg and 0.21 U / μg, respectively. In contrast, CeMn@PCP Gel exhibited the strongest SOD-like activity, reaching 0.61 U / μg.

[0060] Figure (E) shows the detection of hydroxyl radical scavenging ability. The samples were tested using a hydroxyl radical detection kit (Bestbio, Shanghai, China) according to the manufacturer's instructions. Specific samples tested included: 200 μL of hydrogel extract CeMn@PCP Gel (Ce: 2.3 μg / mL, PCP: 2.0 mg / mL), 200 μL of PCP Gel (PCP: 2.0 mg / mL), and 200 μL of CeMn NP-PEG solution (Ce: 2.3 μg / mL). The results showed that the concentrations of CeMn NP-PEG, PCP Gel, and CeMn@PCP Gel were 4.57 U / μg, 42.4 U / μg, and 46.7 U / μg, respectively, indicating that CeMn@PCP Gel exhibited the strongest antioxidant activity against ·OH.

[0061] Example 5 The CeMn NP-PEG prepared in Example 1 was used to test the cytotoxicity of bone marrow-derived macrophages (BMDM). The steps included: 6-8 week old C57BL / 6 mice were sacrificed, and after systemic disinfection with 75% ethanol, the femur and tibia were aseptically separated, and the attached soft tissue was removed. The ends of the bones were cut off, and the bone marrow cavity was flushed with pre-cooled DMEM medium to obtain a bone marrow cell suspension. This suspension was filtered through a 70 μm cell sieve, centrifuged, and resuspended in DMEM complete medium containing 40 ng / mL M-CSF and 20% FBS. The cells were then induced and cultured at 37 ℃ in a 5% CO2 incubator for 4 days to obtain mature BMDM cells for subsequent experiments. Four days later, BMDMs cells seeded in 96-well plates were incubated with different concentrations of CeMn NP-PEG (Ce: 0.125, 0.25, 0.5, 1, 2, 4, 8 μg / mL) in a 37 ℃, 5% CO2 incubator for 24 h. After incubation, 10 μL of MTT solution (5 mg / mL, prepared with PBS) was added to each well, gently mixed, and cultured for another 4 h. After 4 h, the culture medium was aspirated from the wells to avoid damaging the cells, and 150 μL of dimethyl sulfoxide was added to each well. The plates were gently shaken on a shaker for 15 min to fully dissolve the formazan crystals. The absorbance (OD value) of each well was then measured at 570 nm. The results are shown below. Figure 9 As shown. Figure 9 The results showed that the CeMn NP-PEG nanozyme at a concentration of 8 μg / mL was not toxic to cells.

[0062] Example 6: This embodiment detects the uptake of CeMn NP-PEG by BMDM cells, including the following steps: isolated bone marrow-derived macrophages (BMDM) are packaged at 2 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 cells / well in 12-well plates and cultured for 4 days to promote differentiation into BMDM. Cells were then co-cultured with Nile Red-labeled CeMn NP-PEG (Ce: 0.5 μg / mL) for 2, 5, 10, 15, 30, 60, and 120 min, respectively. After incubation, cells were washed three times with PBS to remove excess nanoparticles. Cell uptake of CeMn NP-PEG was then assessed using flow cytometry (NovoCyte Quanteon VBYR, Agilent Technologies, USA) and fluorescence microscopy (BZ-X800LE; KEYENCE, Japan). Figure 10 The results showed that both quantitative and qualitative analyses confirmed that CeMn NP-PEG could be rapidly absorbed by BMDM cells in a time-dependent manner.

[0063] Example 7 This embodiment examines the ability of CeMn@PCP Gel extract to remove excess ROS from cells, including the following steps: BMDM is seeded into 12-well plates at a seeding density of 2.0 × 10⁶ cells per well. 5 Cells were cultured in DMEM for 24 h to form a monolayer. Except for the control group, which used normal DMEM medium, the other groups were treated simultaneously with 400 μM H2O2 and one of the following percolates: free CeMn NP-PEG (Ce: 0.5 μg / mL), PCP Gel (PCP: 200 μg / mL), or CeMn@PCP Gel (Ce: 0.5 μg / mL, PCP: 200 μg / mL) for 4 h. After treatment, the cells were washed three times with PBS and then incubated with the ROS probe DCFH-DA (10 μM) for 30 min. The ROS fluorescence intensity in BMDM cells was quantified by flow cytometry (NovoCyte Quanteon VBYR, Agilent, USA) and observed under an inverted fluorescence microscope (BZ-X800LE, KEYENCE, Japan). Figure 11 This indicates that the gel bound to PCP Gel and CeMn NP-PEG maintained its antioxidant capacity. When comparing the three, CeMn@PCP Gel showed the strongest ability to reduce intracellular ROS levels.

[0064] Example 8 This embodiment evaluates the in vivo therapeutic effect of CeMn@PCP Gel.

[0065] 1. IBS-D model replication: A rat IBS-D model was established using a two-factor approach combining acetic acid stimulation and restraint stress. Rats were anesthetized by isoflurane inhalation to ensure they were unconscious and minimize physical stress during the procedure. A 4% acetic acid solution was slowly injected into the rat colon (6 cm from the anus) using a lubricated catheter. The initial acetic acid dose was 0.2 mL, increasing by 0.1 mL daily until the fourth day, when the dose was maintained at 0.5 mL. The rats underwent acetic acid rectal stimulation once daily, while the control group received PBS enemas. The rats were kept in a head-down, tail-up position for 30 seconds to allow sufficient contact of the acetic acid with the colonic mucosa, inducing local low-grade inflammation and epithelial barrier damage. Seven days after acetic acid infusion, the rats were placed in a fixed cage with ligatures for 1 hour of restraint stress. While the rats' movement was not restricted, scratching of the face with the upper limbs was limited. This procedure activated the abnormal gut-brain axis response, ultimately leading to stable IBS-D symptoms. The restraint stress lasted for one week. The control group received no other treatment. After the experiment ended on day 14, in order to verify whether the IBS-D rat model was successfully established, the rats' physical condition was evaluated by changes in body weight gain, and the severity of visceral hypersensitivity and diarrhea symptoms was evaluated by abdominal withdrawal reflex (AWR) score, changes in body temperature near the anus in the rat's abdomen, food intake, sugar water preference, and fecal water content (FWC).

[0066] 2. Grouping and administration: In addition to the normal control group, the successfully established IBS-D model rats were randomly divided into four groups (n=6): model group, free CeMnNP-PEG group (Ce: 0.75 μg / g), PCP Gel group (PCP: 30 μg / g), and CeMn@PCP Gel group (Ce: 0.75 μg / g, PCP: 30 μg / g). In the normal control group and the model group, 0.5 mL of PBS was administered rectally every other day. The other treatment groups were also administered 0.5 mL of the drug rectally every other day for two consecutive weeks after the modeling was completed.

[0067] 3. Experimental Results: Figure 12 Figure (B) shows the changes in body weight of rats in each treatment group. Body weight was measured and recorded daily before, after, and during treatment. The initial body weight before modeling was used as a baseline, and the percentage change in body weight was calculated to assess the trend of body weight change in each group. According to rat body weight monitoring, the rate of increase in body weight in the model group was slower compared to the healthy control group. Treatment with free CeMn NP-PEG, PCP Gel, and CeMn@PCP Gel increased the rate of increase in body weight, particularly in the CeMn@PCP Gel group.

[0068] Figure (C) shows the food intake measurements of rats in each treatment group. The food intake experiment assessed visceral sensitivity. Rats were housed in pairs in one cage, fasted and deprived of water for 12 hours, and then given weighed feed. Food intake was calculated based on feed consumption over 12 hours. The results showed that, compared with the healthy control group, the food intake in the model group increased most significantly after treatment with CeMn@PCP Gel.

[0069] Figure (D) shows the sucrose preference test in rats of each treatment group. The sucrose intake test evaluated the treatment of depression. Two rats were housed per cage, fasted and deprived of water for 12 hours, and then given one bottle each of 1% sucrose solution and distilled water. The sucrose preference index was calculated based on the consumption of the two bottles over 12 hours. Depressive behavior was assessed by comparing the sucrose preference indices of each group. The results showed that compared with the healthy control group, the sucrose preference value was decreased in the model group, and increased after treatment with CeMn@PCP Gel.

[0070] Figure (E) shows the determination of fecal water content in rats of each treatment group. The experimental procedure involved collecting feces from each rat for 1 hour every other day and weighing it using an electronic balance; this was the wet weight of the feces. The feces were then placed in an oven at 60 °C for 3 hours until the weight no longer changed; this weight was the dry weight. The results showed that the fecal water content of the rats after modeling was relatively high. After treatment with CeMn@PCPGel, the fecal water content decreased, and the fecal morphology gradually returned to normal.

[0071] Figure (F) shows the abdominal withdrawal reflex (AWR) in rats of each treatment group. To assess visceral hypersensitivity in a rat model of irritable bowel syndrome, the abdominal withdrawal reflex test was performed using a colonic balloon dilation method. Before the end of the experiment, rats were fasted for 12 h, then anesthetized with isoflurane. A catheter with a balloon was lubricated with medical coupling agent and gently inserted into the rat's colon and rectum (approximately 6 cm from the anus). The catheter and tail were fixed with medical tape to secure the catheter's position. After the rats awoke, visceral sensitivity was assessed within 5 min by rapidly inflating the balloon to achieve dilation pressures of 0.2 mL, 0.2 mL, and 0.4 mL within short periods, each lasting 20 s. The experiment was repeated three times, and the response of the rat's abdominal wall to rectal balloon dilation stimulation was observed. The final results showed that the abdominal stretch reflex (AWR) scores of rats in the CeMn NP-PEG group, PCP Gel group, and CeMn@PCP Gel group were all lower than those in the IBS-D group, with the most significant difference observed in the CeMn@PCP Gel group. This suggests that CeMn@PCP Gel can effectively alleviate the depressive state in IBS-D rats.

[0072] Example 9 In this embodiment, to evaluate the effect of CeMn@PCP Gel on improving colonic histopathology, H&E staining, AB staining, and PAS staining were performed on the colonic tissues of rats in each treatment group in Example 9. The results are as follows: Figure 13 , Figure 14 As shown.

[0073] like Figure 13 As shown, the colonic tissue structure of the normal group rats was intact, with no obvious edema or inflammatory cell infiltration. In contrast, the colonic tissue of the IBS-D model group rats showed obvious local inflammatory cell infiltration (indicated by black arrows). After treatment with CeMn NP-PEG, PCP Gel, and CeMn@PCP Gel, the degree of inflammatory cell infiltration was alleviated to varying degrees, with the CeMn@PCP Gel group showing the most significant improvement.

[0074] like Figure 14 Immunohistochemical staining results for ZO-1 and Claudin-1 showed that, compared with the normal group, the expression of Claudin-1 and ZO-1 in the colon tissue of rats in the model group was significantly reduced. After treatment with CeMn@PCP Gel, the expression levels of Claudin-1 and ZO-1 in the rat colon were significantly increased. Similarly, qRT-PCR results showed that all drug treatments upregulated the expression of these genes to varying degrees. Among them, the CeMn@PCP Gel group showed the most significant reversal effect.

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a hydrogel of Poria cocos polysaccharide loaded with manganese-doped cerium dioxide nanozyme, characterized in that, It includes the following steps: Manganese acetate and cerium acetate were dissolved in a mixed solvent of oleylamine and xylene. After stirring and sonication, the mixture was heated under inert gas protection and water was added to carry out the reaction. After the reaction was completed, the mixture was centrifuged, washed and redispersed to obtain a lipid-soluble manganese-doped cerium dioxide nanozyme. The lipid-soluble manganese-doped cerium dioxide nanozyme was surface-modified with distearate phosphatidylethanolamine, and after removing the organic solvent, water was added for ultrasonic dispersion. Then, it was purified by membrane filtration and dialysis to obtain water-soluble manganese-doped cerium dioxide nanozyme. Take a polyvinyl alcohol solution, add poria cocos polysaccharide powder and the water-soluble manganese-doped cerium dioxide nanozyme, stir evenly, then add borax solution for cross-linking to form a hydrogel; the obtained hydrogel is subjected to alternating freezing and room temperature treatment to obtain poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozyme.

2. The method for preparing a Poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozyme according to claim 1, characterized in that, The molar ratio of manganese acetate to cerium acetate is (2-4):(3-6); the volume ratio of oleylamine to xylene is 1:1 to 3:1; the reaction temperature is 90℃-110℃, and the reaction time is 1-4 hours.

3. The method for preparing a Poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozyme according to claim 1, characterized in that, The mass ratio of the lipid-soluble manganese-doped cerium dioxide nanozyme to distearate phosphatidylethanolamine is 1:2 to 1:5; the surface modification reaction is carried out in chloroform or dichloromethane solvent with stirring at room temperature for 2-6 hours.

4. The method for preparing a Poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozyme according to claim 1, characterized in that, The polyvinyl alcohol solution has a mass-volume fraction of 5%-15%, and the borax solution has a mass-volume fraction of 1%-5%; the volume ratio of the polyvinyl alcohol solution to the borax solution is 5:1 to 10:

1.

5. The method for preparing a Poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozyme according to claim 1, characterized in that, The amount of Poria cocos polysaccharide added is 2%-15% of the mass of polyvinyl alcohol; the amount of water-soluble manganese-doped cerium dioxide nanozyme added is 0.1%-5% of the mass of polyvinyl alcohol, based on the mass of the manganese-doped cerium dioxide nanozyme.

6. The method for preparing a Poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozyme according to claim 1, characterized in that, The freeze-room temperature alternation process includes the following steps: freezing at -20°C to -10°C for 2-8 hours, then thawing at room temperature for 1-2 hours, and repeating the alternation process 1-3 times.

7. A Poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozymes, prepared by the method according to any one of claims 1-6, characterized in that, The hydrogel has a storage modulus of 120-350 Pa and an adhesion force of ≥8.5 kPa at pH 6.5-7.2 and 37℃.

8. The use of the Poria cocos polysaccharide hydrogel loaded with manganese-doped cerium dioxide nanozyme as described in claim 7 in the preparation of a medicament for the prevention or treatment of irritable bowel syndrome.

9. The application according to claim 8, characterized in that, The hydrogel is used in the preparation of drugs for efficiently scavenging reactive oxygen species, relieving oxidative stress, regulating the intestinal immune microenvironment, reducing chronic inflammation, repairing intestinal barrier function, or regulating the 5-HT signaling pathway.

10. The application according to claim 8, characterized in that, The drug is administered via enema.

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