Metal-small molecule coordination nanoparticles for treating inflammatory bowel disease as well as preparation method and application of metal-small molecule coordination nanoparticles

Abstract

The invention discloses a metal-small molecule coordination nanoparticle for treating inflammatory bowel disease and a preparation method and application thereof, and relates to the technical field of biological medicines.The preparation method of the metal-small molecule coordination nanoparticle for treating inflammatory bowel disease comprises the following steps that S1, MgCl2. 6H2O is dissolved in methyl alcohol; s2, dissolving curcumin in N, N-dimethylformamide, and stirring to promote coordination assembly; s3, the lidocaine isomeride is dissolved in DMSO (dimethyl sulfoxide); s4, dialyzing the mixed solution to remove an unreacted solvent, dispersing aggregates through ultrasonic treatment, and filtering to obtain metal-small molecule coordination nanoparticles; the prepared metal-small molecule coordination nanoparticles and inulin hydrogel serving as a carrier are prepared into a medicinal preparation; a drug delivery system is constructed by adopting the provided preparation method of the metal-small molecule coordination nanoparticles and a step-by-step self-assembly strategy, so that the retention property of the drug in the intestinal tract is enhanced while the water solubility of the drug is improved, and the drug effect is continuously exerted.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a metal-small molecule coordination nanoparticle for the treatment of inflammatory bowel disease, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic and highly relapsing inflammatory bowel disease, including ulcerative colitis and Crohn's disease. Its global prevalence continues to rise, affecting approximately 7 million people worldwide, representing a significant public health burden. The core pathological feature of IBD is severe impairment of the intestinal barrier function and imbalance of immune homeostasis. Specifically, the mechanical barrier formed by intestinal epithelial cells and their tight junctions is disrupted, leading to the infiltration of numerous immune cells and the formation of a complex inflammatory network. This results in a range of symptoms, including abdominal pain, diarrhea, and rectal bleeding, and significantly increases the risk of colon cancer. In the complex immune microenvironment of IBD, macrophage polarization is crucial in determining the inflammatory process and tissue repair. Resident macrophages in the gut are the cornerstone of maintaining immune homeostasis. Once homeostasis is lost, macrophages recruited from blood monocytes (recruited macrophages) polarize into pro-inflammatory M1 macrophages, while the transformation of repairing M2 macrophages is hindered, leading to persistent tissue damage. Therefore, reprogramming the M1 phenotype to the M2 phenotype is a highly promising drug treatment strategy for reversing the immune imbalance in IBD.

[0003] Currently, the clinical treatment of IBD still faces severe challenges. Traditional drugs such as 5-aminosalicylic acid (5-ASA), glucocorticoids, and immunosuppressants are widely used, but these drugs often fail to prevent the onset of the disease and are difficult to cure. In addition, biologics (such as TNFα immunosuppressants) are often accompanied by systemic immunosuppression, severe side effects, and drug resistance. Local administration (such as enemas), while reducing systemic exposure, suffers from short retention time and poor targeting. The emerging nano-targeting strategy has brought new hope to IBD treatment in recent years, but a considerable proportion of patients still do not respond well. Therefore, developing safe, effective, and precisely targeted novel therapeutic drugs has become an urgent need for IBD treatment. JM25-1 (2-diethylamino-N-2,5-dimethylphenylacetamide) is a lidocaine isomer with extremely weak anesthetic effects but strong anti-inflammatory effects. JM25-1 can maintain the intestinal barrier integrity in rats with irritable bowel syndrome by inhibiting mast cell activation. Network pharmacology studies have revealed the potential of JM25-1 in treating colitis; however, pharmacokinetic studies indicate that JM25-1 is rapidly metabolized orally, limiting its full efficacy in vivo. JM25-1 is rapidly absorbed and has low bioavailability regardless of whether administered by gavage or injection. This may be related to the strongly hydrophobic structure of JM25-1, directly leading to insufficient exposure of the drug at the intestinal lesion site and a short duration of action. Achieving long-term retention of JM25-1 in the intestine to ensure sustained treatment is a crucial problem that urgently needs to be solved in its current application.

[0004] In addition to anti-inflammatory effects, the drug treatment of enteritis also focuses on mucosal repair and tissue regeneration in the later stages of the disease, a crucial factor that often overlooks its importance in determining prognosis. Magnesium ions (Mg...) 2+ It has shown promising biological activity in promoting the repair of inflamed enteritis tissue. It not only neutralizes the negative charge at sites of inflamed intestinal tissue, but also... 2+ Nanoparticles coordinated with curcumin (Cur) exhibit good biocompatibility and antioxidant capacity. Based on this, this application designs a stepwise self-assembly strategy to construct smart nanomedicines: firstly, through Mg... 2+ Stable nano-units are formed through coordination with curcumin, and high-efficiency loading of JM25-1 (Mg-Cur@JM25-1) is achieved through hydrophobic interactions, thereby increasing the water solubility of JM25-1. Meanwhile, inulin hydrogel, due to its good biocompatibility, film-forming properties, and intestinal adhesion potential, allows for further mixing of the prepared nanoparticles with inulin hydrogel. The hydrogel's properties enhance drug retention in the intestine, forming a nanoparticle drug formulation that better meets clinical needs (Mg-Cur@JM25-1 Gel). Summary of the Invention

[0005] In view of this, the present invention addresses the deficiencies of the prior art, and its main objective is to provide a metal-small molecule coordination nanoparticle for the treatment of inflammatory bowel disease, its preparation method, and its application. By adopting the preparation method of the metal-small molecule coordination nanoparticle provided in this application, a stepwise self-assembly strategy is designed to construct a drug delivery system, which improves the water solubility of the drug and enhances the drug retention in the intestine, thereby continuously exerting the drug's efficacy.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing metal-small molecule coordination nanoparticles for treating inflammatory bowel disease includes the following steps: S1. Dissolve MgCl2·6H2O in methanol, disperse it by ultrasonication, and then add it dropwise to the methanol system while stirring to form a precursor solution; S2. Dissolve curcumin in N,N-dimethylformamide and add it dropwise to the precursor solution, stirring to promote coordination assembly; S3. Dissolve the lidocaine isomer in DMSO, add it to the reaction solution, and continue stirring to complete the drug loading. S4. After the reaction is complete, the mixture is dialyzed to remove unreacted small molecule reagents and organic solvents, the aggregates are dispersed by ultrasonic treatment, and then filtered to obtain metal-small molecule coordination nanoparticles.

[0007] As a preferred embodiment: the lidocaine isomer in step S3 is 2-diethylamino-N-2,5-dimethylphenylacetamide, i.e., JM25-1; the conditions for stirring to promote coordination assembly in step S2 are: continuous stirring at 37°C and 500 rpm for 60 min to promote the coordination assembly of the ligand with the magnesium ion.

[0008] As a preferred embodiment: the mass ratio of MgCl2·6H2O in step S1 to curcumin in step S2 is 300:450; the mass ratio of curcumin in step S2 to lidocaine isomer in step S3 is 450:50.

[0009] As a preferred embodiment: in step S2, magnesium ions and curcumin coordinate to form Mg-Cur nano-units; in step S3, JM25-1 is loaded onto the Mg-Cur nano-units through hydrophobic interactions to form Mg-Cur@JM25-1 nanoparticles.

[0010] A metal-small molecule coordination nanoparticle prepared by the aforementioned method for treating inflammatory bowel disease, wherein the metal-small molecule coordination nanoparticle and inulin hydrogel as a carrier are mechanically mixed to prepare a pharmaceutical formulation.

[0011] The application of the aforementioned metal-small molecule coordination nanoparticles in the preparation of a drug for treating inflammatory bowel disease.

[0012] As a preferred embodiment, the inflammatory bowel disease is ulcerative colitis.

[0013] As a preferred embodiment, the metal-small molecule coordination nanoparticles exert their drug effect by regulating macrophage polarization.

[0014] As a preferred embodiment, the regulation of macrophage polarization includes: inhibiting macrophage polarization towards the M1 phenotype, promoting macrophage transformation towards the M2 phenotype, or inhibiting the secretion of pro-inflammatory cytokines TNF-α and IL-1β.

[0015] As a preferred embodiment, the metal-small molecule coordination nanoparticles exert their drug effect by regulating communication between macrophages and epithelial cells, thereby protecting the intestinal epithelial barrier.

[0016] Compared with existing technologies, this invention has significant advantages and beneficial effects. Specifically, as can be seen from the above technical solution, the metal-small molecule coordination nanoparticles (Mg-Cur@JM25-1) and its inulin gel composite drug formulation system (Mg-Cur@JM25-1 Gel) provided by this invention exhibit significant therapeutic effects in the treatment of inflammatory bowel disease: this system utilizes Mg... 2+ The nanoparticles, coordinated with curcumin, self-assemble to form nano-modules, efficiently loading the hydrophobic drug JM25-1, significantly improving its water solubility and intestinal retention, overcoming the problems of rapid metabolism and low bioavailability of JM25-1. In vitro and in vivo experiments show that this nanomedicine can effectively regulate the polarization of macrophages from pro-inflammatory M1 to reparative M2, inhibit the secretion of inflammatory factors, restore intestinal epithelial barrier function, and exhibit excellent biocompatibility and safety. Furthermore, inulin gel further prolongs the drug's retention time in the intestine, achieving long-acting sustained release and targeted therapy, providing a novel drug treatment strategy for inflammatory bowel disease that combines anti-inflammatory, reparative, and barrier-protective functions.

[0017] To more clearly illustrate the structural features and effects of the present invention, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the screening of key pathway targets for the JM25-1 drug in colitis according to the present invention. Figure 2 This is a schematic diagram of the pharmacokinetic changes of JM25-1 under gavage and injection conditions according to the present invention; Figure 3 This is a schematic diagram illustrating the preparation and characterization of Mg-Cur@JM25-1 nanoparticles and inulin gel of the present invention; Figure 4 This is a schematic diagram illustrating the effects of Mg-Cur@JM25-1 of the present invention on macrophage proliferation, apoptosis, and polarization. Figure 5 This is a schematic diagram illustrating the effect of Mg-Cur@JM25-1 on macrophage-epithelial communication according to the present invention; Figure 6 This is a schematic diagram illustrating the therapeutic effect of the Mg-Cur@JM25-1 Gel of the present invention on mice with acute colitis. Figure 7 This is a schematic diagram illustrating the safety assessment of the Mg-Cur@JM25-1 Gel of the present invention in mice.

[0019] Explanation of reference numerals in the attached diagram: Figure 1 (A) Integration of JM25-1 with the target of colitis UC, with Venn diagram showing cross-targets; (B) GO enrichment analysis; (C) PPI network analysis; (D) Molecular docking assessment of the affinity of JM25-1 for PARP1; (E) KEGG pathway analysis; (F) Immunofluorescence detection of PARP1 protein expression in colon tissue.

[0020] Figure 2 In the image: (A) Product ion mass spectra of JM25-1 and (B) Lidocaine-d6 (internal standard); (C) Major pharmacokinetic parameters of JM25-1 after gavage or intraperitoneal administration of 15 mg / kg to rats; (D) Mean plasma concentration-time curves of JM25-1 in rats after gavage and (E) intraperitoneal injection.

[0021] Figure 3 (A) Schematic diagram of Mg-Cur@JM25-1 nanoparticle preparation. (B) TEM image of Mg-Cur@JM25-1. (C) Particle size distribution of Mg-Cur@JM25-1 and Mg-Cur. (D) 7-day particle size stability of Mg-Cur@JM25-1. (E) Zeta potential of Mg-Cur@JM25-1 and Mg-Cur. (F) UV absorption spectra of Mg-Cur@JM25-1, Mg-Cur, and JM25-1. (G) Strain scan images of Mg-Cur@JM25-1 inulin hydrogel and inulin hydrogel. (H) SEM image and surface scan images of Mg, C, and O elements in Mg-Cur@JM25-1 inulin hydrogel.

[0022] Figure 4In the middle: (A) CCK-8 assay for the proliferation of macrophages by gradient concentrations of LPS; (B) Mg-Cur, (C) Mg-Cur@JM25-1, (D) macrophages were pre-treated with JM25-1 for 1 hour, followed by LPS stimulation for 24 hours, and the proliferation rate of RAW264.7 macrophages was detected by CCK-8 assay; (E) Flow cytometry assessment of apoptosis in each group; (F) JC-1 staining image; (G) Number of CD86 positive cells in macrophages; (H) Statistical graph of CD86 positive macrophages; (I) Superoxide anion positive signal image; (J) Statistical graph of superoxide anion positive signal; (K) Immunofluorescence image (left) and statistical graph (right) of macrophage iNOS; (L) DCFH-DA fluorescence image (left) and statistical graph (right).

[0023] Figure 5 (A) Western blot analysis of the expression of iNOS, TNFα, and IL-1β in RAW264.7 macrophages by Mg-Cur@JM25-1 (left) and statistical graph (right); (B) mRNA levels of CD86, TNFα, and IL-1β in macrophages; (C) ATP levels in macrophages of each group; (D) ATP production rate of macrophages detected by the Seahorse platform; (E) ECAR values ​​of macrophages of each group; (F) Calcium ion levels of macrophages of each group; (G) RAW264.7 macrophages were divided into PBS, LPS, LPS+Mg-Cur, and LPS+Mg-Cur@JM25-1 groups, and then co-cultured with NCM460 intestinal epithelial cells. Real-time monitoring of the epithelial barrier was performed; (H) Expression of ZO-1, Claudin1, and Occludin in epithelial cells after 24 hours of incubation in the co-culture system.

[0024] Figure 6 (A) Schematic diagram of drug administration in mouse model; (B) Appearance morphology of mouse colon; (C) Pathological condition of colon tissue in each group of mice observed by H&E staining; (D) Statistical diagram of colonic villus score; (E) Statistical diagram of colonic inflammation score; (F) Statistical diagram of mouse weight; (G) Statistical diagram of colon length; (H) Statistical diagram of FD4 positive signal in peripheral blood; (I) Immunofluorescence image and transmission electron microscopy image of ZO-1 and EPAM in colon tissue; (J) Immunohistochemical image of Mucin2 and TNFα in colon tissue, and TUNEL staining apoptosis signal image.

[0025] Figure 7 (A) Pathological sections of heart, liver, spleen, lung and kidney tissues of mice in the control group and Mg-Cur@JM25-1 Gel group, H&E staining; (B) Statistical graph of changes in peripheral blood blood routine, liver function indicators and body weight of mice after one month of continuous gavage. Detailed Implementation

[0026] The present invention is as follows Figure 1 As shown in Figure 7, a method for preparing metal-small molecule coordination nanoparticles for treating inflammatory bowel disease includes the following steps: S1. Dissolve MgCl2·6H2O in methanol, disperse it by ultrasonication, and then add it dropwise to the methanol system while stirring to form a precursor solution; S2. Dissolve curcumin in N,N-dimethylformamide and add it dropwise to the precursor solution, stirring to promote coordination assembly; S3. Dissolve the lidocaine isomer in DMSO, add it to the reaction solution, and continue stirring to complete the drug loading. S4. After the reaction is complete, the mixture is dialyzed to remove unreacted small molecule reagents and organic solvents. The aggregates are dispersed by ultrasonic treatment (power 100–150 W, time 10 min), and then filtered to obtain metal-small molecule coordination nanoparticles.

[0027] In step S3, the lidocaine isomer is 2-diethylamino-N-2,5-dimethylphenylacetamide, i.e., JM25-1; the conditions for stirring to promote coordination assembly in step S2 are: continuous stirring at 37°C and 500 rpm for 60 min to promote the coordination assembly of the ligand with the magnesium ion.

[0028] In step S1, the mass ratio of MgCl2·6H2O to curcumin in step S2 is 300:450; in step S2, the mass ratio of curcumin to lidocaine isomer in step S3 is 450:50.

[0029] In step S2, magnesium ions and curcumin coordinate to form Mg-Cur nano-units; in step S3, JM25-1 is loaded onto the Mg-Cur nano-units through hydrophobic interactions to form Mg-Cur@JM25-1 nanoparticles.

[0030] A method for preparing metal-small molecule coordination nanoparticles for treating inflammatory bowel disease is used to prepare a drug formulation by mechanically mixing the metal-small molecule coordination nanoparticles and inulin hydrogel as a carrier.

[0031] Application of a metal-small molecule coordination nanoparticle in the preparation of a drug for treating inflammatory bowel disease.

[0032] This inflammatory bowel disease is ulcerative colitis.

[0033] These metal-small molecule coordination nanoparticles exert their drug effects by regulating macrophage polarization.

[0034] This regulation of macrophage polarization includes: inhibiting macrophage polarization towards the M1 phenotype, promoting macrophage transformation towards the M2 phenotype, or inhibiting the secretion of pro-inflammatory cytokines TNF-α and IL-1β.

[0035] These metal-small molecule coordination nanoparticles exert their drug effects by regulating communication between macrophages and epithelial cells, thus protecting the intestinal epithelial barrier.

[0036] Example: Metal-small molecule coordination nanoparticles for the treatment of inflammatory bowel disease, their preparation method and application Network pharmacology predicts the potential of JM25-1 in treating colitis. The chemical structure of JM25-1 was plotted and energy-optimized using ChemDraw and Chem3D software to generate its canonical SMILES representation. Subsequently, target screening was performed using multiple prediction platforms, including SwissTargetPrediction, Galaxy, and PharmMapper, limiting the prediction scope to the human species. Overlapping results from various databases were merged and deduplicated to obtain a set of non-redundant candidate targets.

[0037] Relevant disease targets were collected from public disease databases such as GeneCards, DisGeNET, and OMIM using "ulcerative colitis" as the keyword. After integrating the data and removing duplicates, intersection targets between predicted targets of JM25-1 and targets related to ulcerative colitis were identified. These intersection targets represent potential therapeutic targets for JM25-1 to exert its anti-ulcerative colitis effect.

[0038] To visualize the relationship between JM25-1, intersection targets, and ulcerative colitis, a compound-target-disease network was constructed using Cytoscape software (version 3.7.1). Key targets in the network were identified by calculating topological parameters such as degree, betweenness centrality, and proximity centrality.

[0039] To obtain functional relationships, GO annotation and KEGG enrichment analysis were performed on the intersection targets using the DAVID database. Furthermore, a protein-protein interaction (PPI) network of the intersection targets was constructed using the STRING database, with the species limited to humans and a confidence score threshold set to ≥0.4. This network was imported into Cytoscape for further topology analysis, and core genes were selected based on degree values.

[0040] JM25-1 was molecularly docked with the PARP1 protein (PDB: 7ONR) using the Surflex-Dock module in SYBYL-X. Protein pretreatment included the removal of water molecules and ligands, and Gasteiger-Huckel charge distribution was performed using an Amber7FF99 force field. JM25-1 was subjected to energy minimization using a Tripos force field. A threshold of 0.50 and an expansion value of 2 were used to generate docking sites. The docking process employed Geom mode to account for ligand flexibility.

[0041] Pharmacokinetic evaluation of JM25-1 HPLC-MS / MS conditions: The chromatographic column was a Kinetex® 2.6um C18 100 Å (50 × 2.1 mm, 2.6 μm; Phenomenex, Torrance, CA, USA); mobile phase A was water (0.1% formic acid); mobile phase B was methanol (0.1% formic acid); column temperature was 40℃; injection volume was 1 μL; gradient elution program is shown in Table 1; flow rate was 0.5 mL / min. The mass spectrometer used an ESI ion source, cation detection, and multiple reaction monitoring (MRM) scanning mode. The ion ejection voltage was 5000 eV, the ion source temperature was 550℃, the nebulizer gas was 12 psi, the curtain gas was 10 psi, and the collision gas was 8 psi. The reaction ion pairs used in the quantitative analysis were: JM25-1 m / z 235.1→86.2 and 235.1→58.1 (collision energy 30 eV, declustering voltage 50 eV, focusing voltage 200 eV, collision chamber exit voltage 15 eV), and the internal standard m / z 241.2→86.2 and 241.2→58.1 (collision energy 30 eV, declustering voltage 50 eV, focusing voltage 200 eV, collision chamber exit voltage 15 eV).

[0042] Table 1: Gradient elution procedure Time (min) A（%） B（%） 0-1 95 5 1-1.5 95→20 5→80 1.5-2.5 20 80 2.5-2.51 20→95 80→5 2.51-4 95 5 Preparation of standard curve and quality control samples: Accurately weigh a certain amount of JM25-1 and internal standard, and dissolve them in 50% methanol to prepare stock solutions of 200 μg / mL and 100 μg / mL, respectively. Further, serially dilute the JM25-1 stock solution with 50% methanol to prepare standard curve working solutions of 10, 20, 50, 100, 400, 1000, and 2000 ng / mL, and quality control working solutions of 25, 200, and 1600 ng / mL. Simultaneously, dilute the internal standard working solution to a working solution of 10 ng / mL. Add 5 μL of the JM25-1 standard curve and quality control working solutions to 50 μL of blank rat plasma, respectively, to obtain standard curve samples with concentrations ranging from 1 to 200 ng / mL and quality control plasma samples of 2.5, 20, and 160 ng / mL.

[0043] Sample preparation and determination: For rat plasma samples, add 50 μL of 50% methanol solution (to replace the JM25-1 working solution used in the standard and control samples for parallel operation), and then add 5 μL of internal standard working solution. For the standard and control samples, add 5 μL of internal standard working solution. Vortex all samples for 30 s, add 150 μL of methanol, vortex for 1 min, and centrifuge at 12000 rpm for 5 min. Transfer the supernatant to a 96-well plate and place it in the injection tray for HPLC-MS / MS analysis. The injection volume is 1 μL.

[0044] Pharmacokinetic studies: Twelve Wistar rats were used. Before the experiment, the rats were fasted for 12 hours but had free access to water. After fasting, 0.5 mL of blank blood was collected, and then JM25-1 at 15 mg / kg was administered via gavage or intraperitoneal injection. Whole blood samples of 0.4 mL were collected at 10, 20, 30, 60, 90, 120, and 180 minutes after administration. The samples were then further analyzed using 1.2 × 10⁻⁶ ppm of the drug. 4 Plasma was separated by centrifugation at rpm for 5 min and stored at -20℃. After processing, rat plasma samples were analyzed by HPLC-MS / MS to determine drug concentrations. Phoenix WinNonlin (version 6.3, Phrasight Corp, Mountain View, CA, USA) software was used to fit and analyze the measured drug concentration data, and the main pharmacokinetic parameters were calculated according to a non-compartmental model.

[0045] Preparation of Mg-Cur@JM25-1 nanoparticles 15 mL of methanol was placed in a 50 mL round-bottom flask as the reaction matrix. 300 mg of magnesium chloride hexahydrate (MgCl2·6H2O) was dissolved in 1 mL of methanol and ultrasonically dispersed (150–200 W, 5–10 min) until a homogeneous solution was obtained. This solution was then added dropwise to the methanol system using a microsyringe, and the mixture was stirred at 37°C and 500 rpm for 15 min to form a stable precursor solution. Subsequently, 450 mg of curcumin (Cur) was dissolved in 1 mL of N,N-dimethylformamide (DMF), ultrasonically dissolved until clear, and then slowly added dropwise to the reaction system. The mixture was stirred at 37°C and 500 rpm for 60 min to promote the coordination assembly of the ligand and metal ions. Finally, 50 mg of JM25-1 was dissolved in 250 μL of dimethyl sulfoxide (DMSO), ultrasonically dispersed, and then added to the reaction solution. The mixture was stirred at 37°C and 500 rpm for 3 h to ensure efficient loading of JM25-1. After the reaction, the mixture was transferred to a dialysis bag with a molecular weight cutoff of 3.5-10 kDa and dialyzed with deionized water for 72 h (changing the dialysate every 6-8 h) to remove unreacted small molecule reagents and organic solvents. The aggregates were then dispersed by ultrasonic treatment (100-150 W, 10 min) and filtered through a 0.22 μm filter membrane to obtain JM25-1-loaded nanomedicine (Mg-Cur@JM25-1). The prepared nanomedicine was mixed with inulin (300 mg / mL), heated at 70 °C for 5 min, and then cooled to room temperature to obtain inulin hydrogel Mg-Cur@JM25-1 Gel.

[0046] Characterization method of Mg-Cur@JM25-1 nanoparticles Mg-Cur@JM25-1 nanoparticles were prepared via a stepwise self-assembly method, first by magnesium ions (Mg... 2+ Mg-Cur nanoparticles were formed by coordination between Mg and curcumin (Cur), and then loaded with the drug JM25-1 through hydrophobic interactions. Subsequently, these nanoparticles were mechanically mixed with inulin hydrogel to construct a composite system. The morphology and dispersibility of the nanoparticles were observed using transmission electron microscopy (TEM), the particle size and colloidal stability during 7 days of storage were determined using dynamic light scattering (DLS), the surface potential changes of the nanoparticles were detected using Zeta potential analysis, the interaction between the nanocarrier and drug molecules was analyzed using ultraviolet-visible absorption spectroscopy (UV-Vis), and Mg was quantitatively detected using inductively coupled plasma mass spectrometry (ICP-MS). 2+ The actual load concentration of JM25-1 was determined by content and related methods to clarify the load efficiency.

[0047] CCK-8 assay for the effect of Mg-Cur@JM25-1 nanoparticles on macrophage proliferation Logarithmic growth phase mouse macrophages RAW264.7 were used at a concentration of 1*10- 4 Cells were seeded at a density of 10 cells / well in 96-well plates. After cell attachment, empty Mg-Cur, drug-loaded nanoparticles Mg-Cur@JM25-1, and the drug JM25-1 were administered at a concentration of 50 µM. 20 minutes after administration, LPS (800 ng / mL) was added. The drug preparation was performed using serum-free culture. Three replicates were set up for each sample. After 24 hours of incubation, the drug-loaded medium was discarded, and 100 μL of IMDM medium containing 10% CCK-8 was added to each well. Incubation was continued for 1 hour. The absorbance of the solution was measured at 450 nm using a microplate reader. The percentage of cell viability was calculated using the following formula: Cell inhibition rate (%) = (Drug-treated group - Control group) / Control group × 100 (%) Effects of Mg-Cur@JM25-1 nanoparticles on macrophage polarization and apoptosis Flow cytometry: Logarithmic growth phase mouse macrophages RAW264.7 were processed at a concentration of 5*10-1. 5 Cells were seeded at a density of [number] cells / well in 6-well plates. After cell adhesion, control, LPS, empty Mg-Cur, and drug-loaded nanoparticle Mg-Cur@JM25-1 groups were set up. The drug-treated groups were administered the drug at a concentration of 50 µM. 20 minutes after drug administration, LPS (800 ng / mL) was added. Drug preparation was performed using serum-free culture, and each sample was tested in triplicate. After 24 hours, cells were digested and collected using trypsin without EDTA. Intracellular ROS expression was labeled using a reactive oxygen species (ROS) assay kit, and cells were stained using an Annexin V-FITC apoptosis assay kit. The effect of the drug on cell apoptosis was detected by flow cytometry.

[0048] Immunofluorescence assay: Logarithmic growth phase mouse macrophages RAW264.7 were subjected to immunofluorescence at a concentration of 1*10-1. 5Cells were seeded at a density of 100 cells / well onto pretreated coverslips in 24-well plates. The coverslips were pre-soaked in 95% ethanol; during use, the coverslips were lifted with tweezers and a needle, the ethanol was burned off in a flame, and the cells were placed into the wells. After cooling, the cell suspension was added. Once the cells adhered, they were grouped according to the groupings (control group, LPS group, empty Mg-Cur group, drug-loaded nanoparticle Mg-Cur@JM25-1 group) and drug administration methods. After 24 hours of intervention, the supernatant was discarded, and the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. The cells were washed three times with PBS for three minutes each time, the PBS was blotted dry with absorbent paper, and normal goat serum was added to the slides. The slides were blocked for 30 minutes at room temperature. The blocking solution was blotted off with absorbent paper, and without washing, a sufficient amount of diluted primary antibody (iNOS) was added to each slide, and the slides were incubated overnight at 4°C. After washing three times with PBS, fluorescent secondary antibody was added and the slides were incubated at room temperature for 1 hour, followed by three more washes with PBS. Add DAPI and incubate in the dark for 5 min to stain the nucleus. Wash away excess DAPI by TBS for 5 min x 4 times. Wash the coverslip three times with TBS, remove and air dry upright, then use Fluor Save. TM Anti-fluorescence quenching mounting medium was used for mounting. Additionally, JC-1 is a fluorescent dye used to detect changes in mitochondrial membrane potential (ΔΨm), and a decrease or collapse of mitochondrial membrane potential is a hallmark event in the early stages of apoptosis. Therefore, JC-1 is widely used as an "indicator" for detecting early apoptosis. Cells treated with the drug were stained using the JC-1 detection kit and ROS detection kit instructions, then fixed using immunofluorescence, stained with DAPI, and mounted. Cell slides were then photographed under a confocal microscope.

[0049] Effects of Mg-Cur@JM25-1 nanoparticles on the expression of inflammatory factors in macrophages RAW264.7 macrophages were seeded into six-well plates. After cell adhesion, they were divided into control, LPS, LPS+Mg-Cur, and LPS+Mg-Cur@JM25-1 groups. After 24 hours of stimulation, cells were lysed with RIPA lysis buffer, and proteins were extracted. After SDS-PAGE electrophoresis, the proteins were transferred to PVDF membranes. The membranes were blocked in 5% skim milk, then incubated with primary antibody and HRP-labeled secondary antibody. ECL staining was used to measure the marker protein iNOS for macrophage M1 differentiation, and intracellular pro-inflammatory factors TNFα and IL-1β were also detected. For qPCR, macrophages in each group were lysed with Trizol, and total RNA was extracted using a total RNA extraction kit. RNA concentration was measured using Nano Drop, and cDNA was obtained by reverse transcription. Primers for mouse CD86, TNFα, and IL-1β were synthesized, and the target genes were amplified by real-time quantitative PCR.

[0050] Effects of Mg-Cur@JM25-1 nanoparticles on macrophage energy metabolism ATP Level Detection: To detect intracellular ATP levels, RAW264.7 macrophages were seeded in six-well plates and divided into control, LPS, LPS+Mg-Cur, and LPS+Mg-Cur@JM25-1 groups. After 24 hours of cell culture, the culture medium was removed, and cells in each group were lysed on ice using ATP detection lysis buffer. After lysis, the cells were centrifuged at 12000g for 5 minutes at 4°C, and the supernatant was used for subsequent measurements. ATP standard solutions were diluted with ATP detection lysis buffer to appropriate concentration gradients (0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM). Cell lysates and gradient concentrations of ATP standard solutions (20 μL / well) were added to the ATP detection buffer, and fluorescence was measured using a chemiluminescence analyzer. The concentration of each group was calculated based on the standard curve. To eliminate errors caused by differences in protein content during sample preparation, the protein concentration of each group was measured using a BCA kit, and the ATP concentration was then converted to nmol / mg protein.

[0051] Cellular calcium flux release: To evaluate the effect of Mg-Cur@JM25-1 on macrophage calcium flux, RAW264.7 cells were first seeded into 96-well black plates. After cell adhesion, cellular calcium flux was labeled according to the Screen Quest™ Calbryte-520 Probenecid-Free and Wash-Free Calcium Assay Kit (AAT Bioquest) instructions. First, 100 µL of Calbryte-520 AM fluorescent staining solution was added to the cells, and the cells were incubated for 30 minutes in a cell culture incubator, followed by incubation at room temperature for another 30 minutes. Baseline calcium flux was detected using a microplate reader. Then, LPS and Mg-Cur@JM25-1 were prepared to predetermined concentrations and added to the cells in the plate using the microplate reader's syringe module to monitor the effect of the drugs on cellular calcium flux.

[0052] Seahorse assays for energy metabolism: RAW264.7 macrophages were seeded into six-well plates and divided into control, LPS, LPS+Mg-Cur, and LPS+Mg-Cur@JM25-1 groups. After 24 hours of cell culture, the Seahorse XF Real-Time ATP Rate Assay Kit was used to analyze whether the main pathway for ATP production relied on oxidative phosphorylation or glycolysis. If oxidative phosphorylation was the primary pathway, the Seahorse XF Cell Mito Stress Test Kit was used to assess mitochondrial respiratory function in each group according to the instructions to observe the energy requirements of each group. If glycolysis was the primary pathway, the glycolytic capacity of each group could be assessed using the XF Glycolysis Stress Test Kit after intervention. Specifically, the day before the assay, 1 mL of hydration solution was added to the lower chamber of the XFe24 Flux Assay Kit and incubated overnight at 37°C in a CO2-free incubator. Simultaneously, XF24 cell culture plates (50 µL / well) were pre-coated with Cell-Tak™ for 20 minutes at room temperature. On the day of testing, macrophages after intervention (control group, LPS group, LPS+Mg-Cur group, and LPS+Mg-Cur@JM25-1 group) were collected and resuspended in preheated assay medium. Cells were seeded at a density of 20,000 / well into pre-coated plates, which were then centrifuged at 200 ×g for 5 minutes and incubated at 37°C for 50 minutes in a CO2-free incubator. The drugs were prepared to a final concentration using assay medium (1.5 μM oligomycin, 0.5 μM rotenone / antimycin A, 10 mM glucose, 50 mM 2-deoxyglucose), added to hydration plates, and finally the bottom layer of the XFe24 assay kit was removed and replaced with the incubated cell plate for assay on a Seahorse XF 24 instrument.

[0053] Establish a co-culture system to evaluate the role of Mg-Cur@JM25-1 nanoparticles in regulating the intestinal epithelial barrier via macrophages. Western blot observation of tight junction protein expression: Macrophages were seeded in the upper layer of Transwell and, after cell adhesion, were divided into control, LPS group, LPS+Mg-Cur@JM25-1 group, and Mg-Cur@JM25-1 group, and co-cultured with NCM460 epithelial cells pre-seeded in the lower layer. After 24 hours of culture, the expression of tight junction proteins in epithelial cells was detected by Western blot. Specific procedures: Epithelial cells were collected and thoroughly lysed on ice using RIPA lysis buffer. After centrifugation at 12000 rpm for 10 minutes, the supernatant was collected. The protein concentration in the supernatant was determined using a BCA assay kit, and the proteins were separated by SDS-PAGE gel. The proteins were then transferred to a PVDF membrane by electroporation, followed by incubation with primary antibodies (ZO-1, Occludin, Claudin1) overnight, and then incubated with secondary antibodies at room temperature for 1 hour. Finally, the membranes were developed and the band changes in each group were observed.

[0054] Real-time monitoring of epithelial barrier permeability: A real-time label-free cell analyzer (RTCA DP) was used to continuously monitor changes in transepithelial electrical resistance of intestinal epithelial cells to assess dynamic changes in barrier function. Specifically, intestinal epithelial cells were seeded in NCM460 plates on a dedicated electrode plate. After cell adhesion, the plate was replaced with a co-culture system, and the instrument automatically measured the cell index (CI) at regular intervals, with monitoring continuing for 48 hours.

[0055] An in vivo acute colitis model was established to evaluate the therapeutic effect of Mg-Cur@JM25-1 Gel. Establishment and administration of the acute colitis model: DSS powder was prepared to a final concentration (wt / vol) using sterile drinking water, and the DSS solution was replaced every two days. The acute colitis mouse model was established by directly administering drinking water containing 3% DSS to C57BL / 6 mice for seven days. Except for mice given sterile drinking water without DSS, the other groups received the same drinking water as the model group. Mice were divided into a control group, a model group, a Mg-Cur Gel group, a Mg-Cur@JM25-1 Gel group (actual drug loading concentration of 15 mg / kg), and a JM25-1 (15 mg / kg) group. Administration began on day seven and continued daily until the end of the experiment.

[0056] Disease Activity Index (DAI): From the start of the experiment, mouse body weight, fecal characteristics, and bleeding scores were continuously monitored. The DAI was measured by changes in body weight (no change = 0; 1-5% = 1; 5-10% = 2; 10-15% = 3; >15% = 4), fecal bleeding scores (normal stool = 0; brown stool = 1; red stool = 2; bloody stool = 3; massive bleeding = 4), and fecal characteristics (normal stool, good shape = 0; soft stool, soft stool sticking to the anus = 1-2; diarrhea, stool sticking to the anus = 3-4).

[0057] Observation of colitis: Mice were euthanized after the experiment, and colonic tissues from each group were collected to observe colonic length, degree of swelling, and color. The collected tissues were then thoroughly fixed in 10% formalin fixative, dehydrated, embedded in paraffin, and sectioned. The tissues were then stained with hematoxylin and eosin, and images were acquired under a biological microscope at random fields of view for pathological statistical scoring.

[0058] Immunofluorescence / immunohistochemical staining: First, immunofluorescence staining was used to observe the epithelial barrier. Colon tissue samples from each group were dehydrated and embedded, sectioned, and baked for 30 minutes. Then, paraffin sections were dewaxed in xylene, followed by a gradient hydration process: 100% ethanol for 5 min, 95%, 90%, and 80% ethanol for 2 min each, and washed three times with pure water for 2 min each. The sections were then incubated in 0.01M citrate buffer (pH 6.0) for 15 minutes on high heat. After the retrieval solution cooled to room temperature, the sections were removed and incubated at 37°C for 40 minutes with blocking buffer. Primary antibody ZO-1 and EPCAM were then added, and the sections were incubated overnight at 4°C in a humidified chamber. The next day, the sections were washed five times with PBS buffer for 5 minutes each time, followed by incubation at room temperature for one hour with secondary antibody. After five washes with PBS, DAPI staining was added for 5 minutes. Finally, after five washes, anti-fluorescence quenching mounting medium was added for mounting, and the expression of the detected proteins was observed under a microscope. In addition, the expression of tissue mucin and pro-inflammatory factors was detected by immunohistochemistry. Colon tissue sections (3 µm) from each group of mice were dewaxed with xylene, rehydrated with gradient concentrations of alcohol, and repaired with sodium citrate antigen. Non-specific binding sites were then blocked with endogenous peroxidase inhibitors and serum. The sections were subsequently incubated overnight at 4°C with primary antibodies (Mucin2, TNFα) and labeled secondary antibodies. Afterward, chromogenic substrate (DAB) was added, and color development was controlled under a microscope. Finally, hematoxylin counterstaining, dehydration and clearing, and mounting with neutral resin were performed. The localization and expression of the target antigen pairs were observed and analyzed under an optical microscope.

[0059] In vivo imaging: In vivo uptake was performed using C57BL / 6 mice. The mice were divided into the JM25-1-Cy7.5 group and the Mg-Cur@JM25-1-Cy7.5 Gel group. In vivo imaging (excitation wavelength: 745 nm; emission wavelength: 810 nm) was performed on the mice at 1, 2, 4, 6, 8 and 12 hours after gavage to observe the distribution in vivo.

[0060] Safety assessment of Mg-Cur@JM25-1 Gel Mice were continuously administered the drug Mg-Cur@JM25-1 Gel by gavage for one month, while the control group was administered physiological saline by gavage. At the end of the experiment, venous blood was collected from the mice; whole blood was used for complete blood count (CBC), and serum was used for liver and kidney function assessment. The main indicators measured included lymphocyte count (LYM), neutrophil count (NEU), mean corpuscular volume (MCV), white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), platelet count (PLT), hematocrit (HCT), alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin (ALB), and aspartate aminotransferase (AST). In addition, the collected heart, liver, spleen, lungs, and kidneys were visually observed, and abnormalities such as color, size, texture, presence of nodules, hemorrhage, and swelling were recorded. The tissues were then fixed in 10% neutral formalin, embedded in paraffin, sectioned, and stained with H&E for microscopic observation of cellular structural changes.

[0061] Discussion of Results: Network pharmacology studies have shown that JM25-1 is closely associated with PARP1 in the treatment of colitis. By integrating the drug target of JM25-1 with colitis targets, 106 potential targets for JM25-1 in the treatment of colitis were identified. Figure 1 A). GO enrichment analysis revealed that overlapping targets were significantly involved in biological processes related to inflammation and immune regulation. Top-enriched biological processes included negative regulation of apoptosis, intracellular receptor signaling pathways, positive regulation of cell population proliferation, and protein autophosphorylation. For molecular function, the targets were primarily associated with nuclear receptor activity, protein tyrosine kinase activity, and binding to the same proteins. Cellular component analysis indicated that these targets were mainly localized in the cytoplasm, nucleoplasm, and nucleus. Figure 1 B). Ten central targets were identified through PPI network analysis ( Figure 1 C), respectively INS, EGFR, ESR1, EP300, SRC, PPARG, GSK3B, PARP1, CDK2, GRB2 and MMP2 Among them, the binding affinity between JM25-1 and the central target PARP1 was evaluated by molecular docking, with a docking score of 6.433, indicating strong affinity. Figure 1D). KEGG pathway analysis showed that overlapping targets were significantly involved in multiple inflammation and immune-related signaling pathways, particularly the PI3K-Akt, PPAR, MAPK, calcium, and HIF-1 signaling pathways. Figure 1 E). Furthermore, this application observed the expression of PARP1 in the colonic tissue of colitis mice, which was significantly increased compared to the control group, while JM25-1 could restore and inhibit its expression (E). Figure 1 F).

[0062] Pharmacokinetic studies have shown that JM25-1 is rapidly metabolized and has low bioavailability. JM25-1 ( Figure 2 A) and Lidocaine-d6 (internal standard, Figure 2 B) The response is highest in cation mode, with all parent ions being protonated ions [M+H]. + Two daughter ions with the highest response were selected for monitoring. JM25-1 ion pairs and internal standard ion pairs were used for quantitative analysis. The JM25-1 ion pairs were 235.1→86.2 and 235.1→58.1, and the internal standard ion pairs were 241.2→86.2 and 241.2→58.1. The t-values ​​of JM25-1 administered by gavage were... max The peak concentration C of JM25-1 after 10 minutes of gavage was... max The area under the curve (AUC) of the drug was significantly lower than that of intraperitoneal injection, and the V of JM25-1 administered by gavage was also significantly lower. z The levels were significantly higher than those administered via intraperitoneal injection, suggesting that JM25-1 is absorbed more rapidly by gavage, but the degree of absorption is lower, resulting in low bioavailability. Figure 2 C). Gavage ( Figure 2 D) and intraperitoneal injection ( Figure 2 E) The mean drug-time curve of JM25-1 administered to mice indicates that the blood concentration of JM25-1 administered by gavage was significantly lower than that administered by intraperitoneal injection. Overall, JM25-1 exhibits relatively rapid metabolism and low bioavailability regardless of whether it is administered by injection or by gavage.

[0063] Preparation and characterization of Mg-Cur@JM25-1 Gel Figure 3 A is a flowchart of the preparation of Mg-Cur@JM25-1 Gel, which first involves magnesium ions (Mg... 2+ The Mg-Cur nanoparticles self-assemble through coordination with curcumin (Cur), forming Mg-Cur nanomodules. Subsequently, the drug JM25-1 is introduced, and drug loading is achieved through the hydrophobic interaction between the two, thus constructing the target nanoparticles. Transmission electron microscopy (TEM) observation shows that the Mg-Cur@JM25-1 nanoparticles exhibit a uniform spherical morphology. Figure 3B), with good dispersibility. Dynamic light scattering (DLS) tests showed that the particle size of Mg-Cur nanoscale units was 46.68 nm, which increased to 77.86 nm after loading JM25-1. Figure 3 (C) The significant increase in particle size provides direct evidence for successful drug loading. Stability assessment showed that the particle size distribution of Mg-Cur@JM25-1 nanoparticles did not fluctuate significantly during 7 days of storage. Figure 3 (D) indicates that it has good colloidal stability. Zeta potential testing showed that the nanoparticle potential decreased from -9.46 mV for Mg-Cur to -20.40 mV for Mg-Cur@JM25-1 (Figure 3E). The increase in the absolute value of the potential may be related to the change in the surface charge state of the nanoparticles due to JM25-1 molecular modification, further confirming the occurrence of the loading process. UV-Vis absorption spectroscopy analysis showed that Mg-Cur@JM25-1 nanoparticles had a significant absorption peak in the characteristic absorption region of JM25-1, while Mg-Cur nano-units did not have this characteristic peak, and this characteristic peak showed a significant blue shift compared to free JM25-1 ( Figure 3 F). This phenomenon stems from the strong interaction between the nanocarrier and the JM25-1 molecules, which alters the electron cloud density and energy level structure of the drug molecules, leading to changes in absorption spectral characteristics. Combined with quantitative results from inductively coupled plasma mass spectrometry (ICP-MS), Mg in the Mg-Cur@JM25-1 nanoparticle suspension... 2+ The content was 0.26 mg / L, and the actual loading concentration of JM25-1 was 5 mmol / L. The morphology, particle size, potential, spectral analysis, and quantitative characterization results corroborated each other, confirming the successful preparation of Mg-Cur@JM25-1 nanoparticles. To prolong the retention time of Mg-Cur@JM25-1 nanoparticles in the intestine, this study constructed a composite system by mechanically mixing them with inulin hydrogel. Rheological testing results showed that, compared with pure inulin hydrogel, the introduction of Mg-Cur@JM25-1 nanoparticles significantly improved the storage modulus (G') of the hydrogel in strain scanning. Figure 3 G). Figure 3 H represents the SEM image and surface scans of Mg-Cur@JM25-1 Gel, showing the presence of Mg, C, and O elements. The gel surface is porous, and the elemental overlap suggests the presence of the drug JM25-1 and Mg. 2+ Successfully packaged.

[0064] Regulation of macrophage apoptosis and differentiation by Mg-Cur@JM25-1 M1 macrophages represent the classic activated phenotype and can be induced by lipopolysaccharide (LPS). To assess its effect on cell proliferation, this application first treated cells with different concentrations of LPS. The results showed that 400 ng / mL LPS inhibited cell proliferation by 20% (…). Figure 4 (A) Therefore, this concentration was used in all subsequent experiments. Figure 4 As shown in C–D, both Mg-Cur@JM25-1 and JM25-1 significantly reversed LPS-induced proliferation inhibition, while empty Mg-Cur did not have this effect. Figure 4 B). Furthermore, LPS promoted apoptosis in RAW264.7 cells, while Mg-Cur@JM25-1 treatment attenuated this effect. Figure 4 E). Mitochondrial membrane potential depolarization (ΔΨm) is one of the hallmarks of early apoptosis. JC-1 staining results showed that LPS led to a decrease in ΔΨm, while Mg-Cur@JM25-1 intervention restored it to near-normal levels. Figure 4 F). The above results indicate that Mg-Cur@JM25-1 has proliferative and anti-apoptotic effects on macrophages. CD86 and iNOS are typical markers of the M1 macrophage phenotype. Figure 4 As shown in GH, both Mg-Cur and Mg-Cur@JM25-1 significantly inhibited CD86 expression. Simultaneously, Mg-Cur@JM25-1 treatment also significantly reduced the positive signal of iNOS (…). Figure 4 K). Mitochondrial superoxide anions promote macrophage polarization toward the M1 phenotype. Figure 4 IJ showed that Mg-Cur@JM25-1 significantly reduced ·O2 - The expression of [the substance / factor] was further examined. Intracellular reactive oxygen species (ROS) production was found to be lower in the Mg-Cur@JM25-1 group than in the LPS group. Figure 4 Therefore, these results indicate that Mg-Cur@JM25-1 can inhibit macrophage apoptosis and polarization to the M1 subtype.

[0065] Mg-Cur@JM25-1 protects the epithelial barrier by regulating macrophage glycolysis and influencing the secretion of inflammatory factors. M1 macrophages exacerbate the inflammatory response by secreting pro-inflammatory cytokines such as TNFα, IL-1β, IL-6, and IL-17A. Figure 5 A study showed that Mg-Cur@JM25-1 inhibited iNOS expression in macrophages and significantly reduced TNF-α and IL-1β secretion. Furthermore, Mg-Cur significantly inhibited LPS-induced IL-1β increase, an effect likely attributed to the anti-inflammatory properties of Mg²⁺. At the mRNA level ( Figure 5B), Mg-Cur@JM25-1 significantly reduced CD86 expression in macrophages and reversed LPS-induced upregulation of TNF-α and IL-1β. Previous studies have shown that mitochondrial reprogramming from ATP production to ROS production drives a pro-inflammatory phenotype in macrophages. To investigate whether Mg-Cur@JM25-1 regulates this mitochondrial reprogramming, this application first measured intracellular ATP levels. Compared to the LPS group, Mg-Cur@JM25-1 significantly inhibited intracellular ATP levels (B). Figure 5 C). Furthermore, mitochondrial function was further assessed using the Seahorse XF cell mitochondrial stress assay. This showed that LPS increased ATP production, while Mg-Cur@JM25-1 partially reversed the LPS-induced increase in glycolysis rate. Figure 5 (D) This change is primarily related to the glycolysis pathway. To validate this, cellular glycolysis function was subsequently assessed using the XF glycolysis stress assay kit. Consistent with the above results, it was shown that LPS enhanced extracellular acidification (ECAR) in macrophages, while Mg-Cur@JM25-1 maintained cellular homeostasis ( Figure 5 E). Simultaneously, LPS significantly increased intracellular calcium ion levels, an increase that was inhibited by Mg-Cur@JM25-1 (E). Figure 5 F). To clarify whether Mg-Cur@JM25-1 affects intestinal barrier function through macrophages, epithelial permeability was examined under co-culture conditions. Impedance-based analysis using the xCELLigence® system revealed that co-culture with LPS-polarized macrophages led to a sustained decrease in the epithelial cell index (CI), while Mg-Cur@JM25-1 pretreatment somewhat restored the CI value. Figure 5 G). Tight junction proteins are key indicators of epithelial barrier integrity, such as Figure 5 As shown in Figure H, co-culturing macrophages with LPS-polarized macrophages resulted in a significant reduction in ZO-1, Claudin1, and Occludin proteins in epithelial cells. However, macrophages pretreated with Mg-Cur@JM25-1 were able to significantly restore the expression of these tight junction proteins. These results indicate that Mg-Cur@JM25-1 helps maintain epithelial homeostasis by regulating macrophage function.

[0066] Therapeutic effects of Mg-Cur@JM25-1 on mice with colitis according to Figure 6 A schematic diagram of the experimental procedure was used for modeling and drug administration. This application established a mouse model using sodium dextran sulfate (DSS). Compared with the control group, all mice treated with DSS showed rectal bleeding and significantly shortened colon length. Figure 6B), Mg-Cur Gel and Mg-Cur@JM25-1 Gel treatments both significantly restored colon length ( Figure 6 G). Hematoxylin-eosin (H&E) staining showed severe damage to the colonic crypts in the DSS group mice. Figure 6 C, 6D), the crypt injury score of the Mg-Cur@JM25-1 Gel group was significantly lower than that of the DSS group ( Figure 6 D), while Mg-Cur@JM25-1 Gel and JM25-1 treatment significantly reduced inflammatory infiltration in the colonic tissue (D), Figure 6 E). Mouse body weight was monitored throughout the experiment. Compared to the control group, mice in the DSS group experienced a significant and sustained decrease in body weight starting from day 7. Mg-Cur Gel, Mg-Cur@JM25-1 Gel, and JM25-1 treatment alleviated this weight loss to varying degrees, with Mg-Cur@JM25-1 Gel showing the most significant effect. Figure 6 F).

[0067] In the FD4 experiment, compared with the control group, the peripheral blood fluorescein levels of mice in the DSS group were significantly increased, indicating that their intestinal barrier was significantly impaired, and treatment with Mg-Cur@JM25-1 Gel restored it to normal levels. Figure 6 H). Further assessment of the intestinal barrier using immunofluorescence detection of the colonic tight junction protein ZO-1 and the epithelial cell marker EPCAM revealed severe loss of colonic epithelial cells and reduced ZO-1 expression in the DSS group mice. Both were restored after treatment with Mg-Cur@JM25-1 Gel. These findings were corroborated by transmission electron microscopy (TEM), which showed damage to tight junctions and microvilli between colonic epithelial cells in DSS-treated mice, with partial recovery observed after administration of Mg-Cur@JM25-1Gel / JM25-1. Figure 6 I). Mucin2 is a mucin secreted by goblet cells that helps form a protective mucus layer; its expression was significantly impaired in the DSS group. Its expression was somewhat restored in the Mg-Cur@JM25-1Gel and JM25-1 groups. Figure 6 J). Furthermore, immunohistochemical analysis of TNFα in colon tissue showed an increase in the number of positive cells in the DSS group, which was restored to normal by Mg-Cur@JM25-1 Gel treatment. Finally, compared with the control group, the number of apoptotic cells in the colon tissue of mice treated with DSS was significantly increased, while it was significantly reduced under Mg-Cur@JM25-1 Gel or JM25-1 intervention. Figure 6 J, 6L).

[0068] To assess drug retention, Cy7.5-labeled JM25-1 and Mg-Cur@JM25-1 Gel were administered orally to C57BL / 6 mice (200 μL per mouse). In vivo distribution was recorded at different time points using in vivo imaging (excitation wavelength: 745 nm; emission wavelength: 810 nm). Figure 6 As shown in K, JM25-1-Cy7.5 was mostly cleared from the body within 12 hours, while Mg-Cur@JM25-1 Gel maintained a detectable signal, indicating that it has a prolonged retention time and sustained activity in vivo.

[0069] Safety assessment of Mg-Cur@JM25-1 Gel In this application, Mg-Cur@JM25-1 Gel was administered to mice via gavage for one month. Subsequently, H&E staining was used to examine the histomorphological changes of major organs, including the heart, liver, spleen, lungs, and kidneys. The results showed that Mg-Cur@JM25-1 Gel did not cause significant pathological changes in these organs. Figure 7 A). Peripheral blood was also collected for complete blood count and liver function tests, including lymphocyte count (LYM), neutrophil count (NEU), mean corpuscular volume (MCV), white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), platelet count (PLT), hematocrit (HCT), alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin (ALB), and aspartate aminotransferase (AST). All measured parameters remained within the normal range. Therefore, these results indicate that Mg-Cur@JM25-1 Gel has good biocompatibility and extremely low systemic toxicity.

[0070] In this application, (1) Mg-Cur@JM25-1 nanoparticles and inulin gel were prepared and characterized. The JM25-1 nanoparticles exhibited good dispersibility and a negative charge. A composite system was constructed by mechanically mixing the nanoparticles with inulin hydrogel, resulting in a composite system with good gel mechanical properties. Figure 3 (2) Mg-Cur@JM25-1 has the effect of protecting the intestinal barrier by regulating macrophage polarization. Specifically, Mg-Cur@JM25-1 was found to inhibit macrophage apoptosis and maintain macrophage membrane potential. At the same time, it inhibited macrophage polarization towards the pro-inflammatory M1 type (reducing the expression of CD86, iNOS, TNFα, and IL-1β) and reduced the release of superoxide anions and ROS. In terms of energy metabolism, Mg-Cur@JM25-1 can inhibit the abnormal increase of ATP in macrophages, restore their glycolytic capacity to normal levels, and reduce calcium flux release ( Figure 4In a macrophage-intestinal epithelial cell co-culture system, Mg-Cur@JM25-1 can effectively alleviate intestinal epithelial barrier damage caused by macrophage overactivation. Figure 5 (3) In vivo, an acute colitis model mouse was established to verify the efficacy of Mg-Cur@JM25-1 Gel. Mg-Cur@JM25-1 Gel can alleviate the pathological damage of colon tissue in colitis mice, restore mouse weight and colon length. The intestinal barrier was assessed by gavage detection of dextran infiltrated into peripheral blood. Colitis damage was severe, and Mg-Cur@JM25-1 Gel could significantly reduce barrier permeability, thus having a barrier protective effect. Further immunostaining of intestinal tissue revealed that Mg-Cur@JM25-1 Gel could restore the epithelial cells (EPCAM) lost in colitis mice, enhance the expression of tight junction protein ZO-1 / mucin2, reduce the secretion of cytokine TNFα, and inhibit cell apoptosis in colon tissue. We also used fluorescein Cy7.5 to label JM25-1 and Mg-Cur@JM25-1 Gel. In vivo imaging after gavage showed that the prepared smart nanomedicine could remain in the intestine for a long time. Figure 6 (4) Mg-Cur@JM25-1 Gel has high safety in vivo. After continuous gavage to mice for one month, pathological sections of the major organs (heart, liver, spleen, lung, and kidney) of the mice were found to be normal compared with the control group, and blood routine and liver function indicators were comparable to those of the control group. Figure 7 ).

[0071] The key design feature of this invention is that it provides a drug JM25-1 for treating colitis, a metal-small molecule coordination nanoparticle (Mg-Cur@JM25-1), and an inulin gel composite drug formulation system (Mg-Cur@JM25-1 Gel). This system utilizes Mg... 2+ The nanoparticles, coordinated with curcumin, self-assemble to form nano-modules, efficiently loading the hydrophobic drug JM25-1, significantly improving its water solubility and intestinal retention, overcoming the problems of rapid metabolism and low bioavailability of JM25-1. In vitro and in vivo experiments show that this nanomedicine can effectively regulate the polarization of macrophages from pro-inflammatory M1 to reparative M2, inhibit the secretion of inflammatory factors, restore intestinal epithelial barrier function, and exhibit excellent biocompatibility and safety. Furthermore, inulin gel further prolongs the drug's retention time in the intestine, achieving long-acting sustained release and targeted therapy, providing a novel drug treatment strategy for inflammatory bowel disease that combines anti-inflammatory, reparative, and barrier-protective functions.

[0072] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing metal-small molecule coordination nanoparticles for treating inflammatory bowel disease, characterized in that: Includes the following steps: S1. Dissolve MgCl2·6H2O in methanol, disperse it by ultrasonication, and then add it dropwise to the methanol system while stirring to form a precursor solution; S2. Dissolve curcumin in N,N-dimethylformamide and add it dropwise to the precursor solution, stirring to promote coordination assembly; S3. Dissolve the lidocaine isomer in DMSO, add it to the reaction solution, and continue stirring to complete the drug loading. S4. After the reaction is complete, the mixture is dialyzed to remove unreacted small molecule reagents and organic solvents, the aggregates are dispersed by ultrasonic treatment, and then filtered to obtain metal-small molecule coordination nanoparticles.

2. The method for preparing metal-small molecule coordination nanoparticles for treating inflammatory bowel disease according to claim 1, characterized in that: In step S3, the lidocaine isomer is 2-diethylamino-N-2,5-dimethylphenylacetamide, i.e., JM25-1; in step S2, the stirring conditions to promote coordination assembly are: continuous stirring at 37°C and 500 rpm for 60 min to promote the coordination assembly of the ligand with the magnesium ion.

3. The method for preparing metal-small molecule coordination nanoparticles for treating inflammatory bowel disease according to claim 1, characterized in that: In step S1, the mass ratio of MgCl2·6H2O to curcumin in step S2 is 300:450; in step S2, the mass ratio of curcumin to lidocaine isomers in step S3 is 450:

50.

4. The method for preparing metal-small molecule coordination nanoparticles for treating inflammatory bowel disease according to claim 2, characterized in that: In step S2, magnesium ions and curcumin coordinate to form Mg-Cur nano-units; in step S3, JM25-1 is loaded onto the Mg-Cur nano-units through hydrophobic interactions to form Mg-Cur@JM25-1 nanoparticles.

5. A metal-small molecule coordination nanoparticle prepared by the method for preparing metal-small molecule coordination nanoparticles for treating inflammatory bowel disease according to any one of claims 1-4, characterized in that: The metal-small molecule coordination nanoparticles and the inulin hydrogel as a carrier are mechanically mixed to prepare a pharmaceutical formulation.

6. The use of the metal-small molecule coordination nanoparticle as described in claim 5 in the preparation of a medicament for treating inflammatory bowel disease.

7. The application according to claim 6, characterized in that: The inflammatory bowel disease mentioned is ulcerative colitis.

8. The application according to claim 6, characterized in that: The metal-small molecule coordination nanoparticles exert their drug effect by regulating macrophage polarization.

9. The application according to claim 8, characterized in that: The regulation of macrophage polarization includes: inhibiting macrophage polarization towards the M1 phenotype, promoting macrophage transformation towards the M2 phenotype, or inhibiting the secretion of pro-inflammatory cytokines TNF-α and IL-1β.

10. The application according to claim 6, characterized in that: The metal-small molecule coordination nanoparticles exert their drug effect by regulating communication between macrophages and epithelial cells, thus protecting the intestinal epithelial barrier.

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