Construction and application of assemblable biomacromolecule gel microspheres

Abstract

The application belongs to the technical field of biological medicine, and discloses a kind of construction and application of biologic macromolecule gel microspheres that can be assembled.The application discloses a kind of gel microspheres, including gel microsphere 1 and / or gel microsphere 2.The application provides a kind of gel microspheres, which are delivered to pathological sites by the specific targeting effect of fucoidan on damaged endothelial cells.Further, gel microsphere 1 and gel microsphere 2 overcome the limitation of steric hindrance by supramolecular assembly between adamantane and cyclodextrin, provide more drug targeting anchor points, thereby improving the drug concentration at the pathological site to enhance the therapeutic efficacy of IBD.The assembly strategy of the gel microspheres provides an effective method for enhancing the drug concentration of micron-sized oral drugs at the pathological site, and provides a new idea for the delivery of micron-sized oral drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the construction and application of an assemblable biomacromolecule gel microsphere. Background Technology

[0002] Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, is a chronic, relapsing inflammatory disease of the gastrointestinal tract. The global prevalence is large and continues to grow, estimated to have exceeded 5 million cases in 2023. Its core pathological feature is oxidative stress and inflammatory damage driven by the excessive accumulation of reactive oxygen species and reactive nitrogen species (RONS) in the gut, accompanied by severe dysbiosis of the gut microbiota. Drug delivery faces significant challenges in clinical treatment. Traditional oral formulations, such as 5-aminosalicylic acid and corticosteroids, have extremely low bioavailability and are easily destroyed by gastric acid, making it difficult to achieve effective concentrations at sites of intestinal inflammation. Biologics such as monoclonal antibodies and JAK inhibitors suffer from poor stability and short half-lives, often requiring frequent intravenous administration, severely impacting patient convenience and adherence.

[0003] To overcome the limitations of traditional drugs, oral nanocarriers (such as polymer nanoparticles) have emerged. These effectively protect drugs from the acidic environment of the stomach and enhance their targeted accumulation at sites of intestinal inflammation through surface functionalization. However, nanocarriers are easily uptaken and cleared by intestinal immune cells and have weak retention capacity in the dynamic intestinal mucus layer, making long-term therapeutic effects difficult. In contrast, micron-sized carriers show unique potential: their size makes them easier to physically trap in intestinal mucus and folds, resulting in stronger initial retention and accumulation at the lesion site. However, this advantage also brings new bottlenecks—after the first dose, a large number of retained micron-sized carriers create significant steric hindrance locally, severely hindering the penetration and accumulation of new carriers in the same area during subsequent doses, leading to a decrease in therapeutic efficacy with increasing dosing frequency. Therefore, developing intelligent drug delivery systems that can achieve efficient targeting, long-term retention, and avoid the obstacles of multiple dosing has become a key direction for overcoming the treatment challenges of IBD. Summary of the Invention

[0004] The first aspect of the present invention is to provide a gel microsphere.

[0005] The second objective of this invention is to provide a method for preparing gel microspheres according to the first aspect of this invention.

[0006] The third aspect of this invention aims to provide the application of the gel microspheres of the first aspect of this invention and the preparation method of the second aspect of this invention in the preparation of medicaments for the prevention and / or treatment of inflammatory bowel disease.

[0007] The fourth aspect of this invention is to provide a drug.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a gel microsphere comprising gel microsphere 1 and / or gel microsphere 2; The gel microspheres 1 are prepared by cross-linking sodium alginate modified with adamantane, fucoidan and drugs under calcium ion conditions. The gel microspheres 2 are prepared by cross-linking sodium alginate modified with β-cyclodextrin, fucoidan, and drugs under calcium ion conditions.

[0009] In some embodiments of the present invention, the drug includes an anti-inflammatory drug.

[0010] In some embodiments of the present invention, the anti-inflammatory drug includes at least one of 5-aminosalicylic acid, acetylsalicylic acid, and itaconic acid; preferably 5-aminosalicylic acid.

[0011] In some embodiments of the present invention, the mass ratio of the adamantane-modified sodium alginate to fucoidan and the drug is 1:(0.5-2):(0.01-0.2); further, it is 1:(0.5-1.5):(0.01-0.1); and even further, it is 1:(0.8-1.2):(0.01-0.05).

[0012] In some embodiments of the present invention, the mass ratio of the β-cyclodextrin-modified sodium alginate to fucoidan and the drug is 1:(0.5-2):(0.01-0.2); further, it is 1:(0.5-1.5):(0.01-0.1); and even further, it is 1:(0.8-1.2):(0.01-0.05).

[0013] In some embodiments of the present invention, the adamantane-modified sodium alginate is prepared by a condensation reaction of sodium alginate and adamantane.

[0014] In some embodiments of the present invention, the adamantane-modified sodium alginate is prepared by a method comprising the following steps: Sodium alginate was dispersed in N,N-dimethylformamide, mixed with toluenesulfonic acid, and reacted to obtain mixture 1. Mixture 1 was mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and adamantane, and reacted to obtain adamantane-modified sodium alginate.

[0015] In some preferred embodiments of the present invention, the mass ratio of sodium alginate to toluenesulfonic acid 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and adamantane is 1:(1-2):(0.5-1.5):(0.01-0.06); further, it is 1:(1-1.5):(0.6-1):(0.01-0.03); and even further, it is 1:(1.2-1.3):(0.9-1):(0.01-0.03).

[0016] In some preferred embodiments of the present invention, the mass-to-volume ratio of sodium alginate to N,N-dimethylformamide is 1:(5-8); further, it is 1:(6-8); and even further, it is 1:(6-7).

[0017] In some preferred embodiments of the present invention, when the sodium alginate is mixed with toluenesulfonic acid, the reaction temperature is 40-70°C; further, it is 40-60°C; and even further, it is 45-55°C.

[0018] In some preferred embodiments of the present invention, when sodium alginate is mixed with toluenesulfonic acid, the reaction time is 20-50 min; further, 25-40 min; and even further, 25-35 min.

[0019] In some preferred embodiments of the present invention, when the mixture 1 is mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and adamantane, the reaction temperature is 40-70°C; further, 40-60°C; and even further, 45-55°C.

[0020] In some preferred embodiments of the present invention, when the mixture 1 is mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and adamantane, the reaction time is 20-30 h; further, 20-26 h; and even further, 23-25 ​​h.

[0021] In some preferred embodiments of the present invention, the preparation method further includes the steps of washing (e.g., repeated washing with ethanol) and drying (e.g., drying by freeze-drying).

[0022] In some embodiments of the present invention, the β-cyclodextrin-modified sodium alginate is prepared by a condensation reaction of sodium alginate and β-cyclodextrin.

[0023] In some embodiments of the present invention, the β-cyclodextrin-modified sodium alginate is prepared by a method comprising the following steps: Sodium alginate was dispersed in N,N-dimethylformamide, mixed with toluenesulfonic acid, and reacted to obtain mixture 1. Mixture 1 was mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and β-cyclodextrin, and reacted to obtain sodium alginate modified with β-cyclodextrin.

[0024] In some preferred embodiments of the present invention, the mass ratio of sodium alginate to toluenesulfonic acid 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and β-cyclodextrin is 1:(1-2):(0.5-1.5):(0.01-0.06); further, it is 1:(1-1.5):(0.6-1):(0.01-0.03); and even further, it is 1:(1.2-1.3):(0.9-1):(0.01-0.03).

[0025] In some preferred embodiments of the present invention, the mass-to-volume ratio of sodium alginate to N,N-dimethylformamide is 1:(5-8); further, it is 1:(6-8); and even further, it is 1:(6-7).

[0026] In some preferred embodiments of the present invention, when the sodium alginate is mixed with toluenesulfonic acid, the reaction temperature is 40-70°C; further, it is 40-60°C; and even further, it is 45-55°C.

[0027] In some preferred embodiments of the present invention, when sodium alginate is mixed with toluenesulfonic acid, the reaction time is 20-50 min; further, 25-40 min; and even further, 25-35 min.

[0028] In some preferred embodiments of the present invention, when the mixture 1 is mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and β-cyclodextrin, the reaction temperature is 40-70°C; further, 40-60°C; and even further, 45-55°C.

[0029] In some preferred embodiments of the present invention, when the mixture 1 is mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and β-cyclodextrin, the reaction time is 20-30 h; further, 20-26 h; and even further, 23-25 ​​h.

[0030] In some preferred embodiments of the present invention, the preparation method further includes the steps of washing (e.g., repeated washing with ethanol) and drying (e.g., drying by freeze-drying).

[0031] In some embodiments of the present invention, the particle size of the gel microspheres 1 is 1-1.5 μm; more specifically, it is 1.15 μm.

[0032] In some embodiments of the present invention, the particle size of the gel microspheres 1 is 1-1.5 μm; more specifically, it is 1.25 μm.

[0033] A second aspect of the present invention provides a method for preparing gel microspheres according to the first aspect of the present invention, comprising the following steps: dissolving adamantane-modified sodium alginate, fucoidan, and a drug in a solvent, adding calcium ion solution dropwise, and reacting to obtain gel microspheres 1 (denoted as Ad-FAMP). 5-ASA ); β-cyclodextrin-modified sodium alginate, fucoidan, and the drug were dissolved in a solvent, and calcium ion solution was added dropwise. The reaction yielded gel microspheres 2 (denoted as βCD-FAMP). 5-ASA ).

[0034] In some embodiments of the present invention, the reaction time is 20-50 min; further, 20-40 min; and even further, 25-35 min.

[0035] In some embodiments of the present invention, the solvent includes water.

[0036] In some embodiments of the present invention, the calcium ion solution is a concentrated calcium chloride solution.

[0037] In some embodiments of the present invention, the mass ratio of the adamantane-modified sodium alginate or the β-cyclodextrin-modified sodium alginate to calcium chloride is 1:(0.02-1); further, 1:(0.02-0.2); even further, 1:(0.02-0.03); and most preferably 1:0.022.

[0038] A third aspect of the invention provides the use of the gel microspheres of the first aspect of the invention or the preparation method of the second aspect of the invention in the preparation of medicaments for the prevention and / or treatment of inflammatory bowel disease.

[0039] In some embodiments of the present invention, the gel microspheres achieve the purpose of preventing and / or treating inflammatory bowel disease by enhancing intestinal drug concentration, enhancing intestinal drug retention performance, inhibiting intestinal inflammation, and improving intestinal integrity.

[0040] A fourth aspect of the present invention provides a medicament comprising the gel microspheres of the first aspect of the present invention.

[0041] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

[0042] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one selected from fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, humectants, flavoring agents, solubilizers, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, fragrances, and solvents.

[0043] In some embodiments of the present invention, the dosage form of the drug includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0044] In some embodiments of the present invention, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0045] In some embodiments of the present invention, the gastrointestinal dosage forms include, but are not limited to, enteric-coated tablets, coated tablets, film-coated tablets, sugar-coated tablets, dispersible tablets, sucking tablets, chewable tablets, effervescent tablets, scratch tablets, sustained-release and controlled-release dosage forms, sustained-release tablets, sustained-release coated tablets, controlled-release tablets, orally disintegrating tablets, lozenges, and oral patches.

[0046] In some embodiments of the present invention, the non-gastrointestinal drug delivery dosage form includes at least one of injection dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0047] In some embodiments of the present invention, the drug can be used to prevent and / or treat inflammatory bowel disease.

[0048] In some embodiments of the present invention, the drug achieves the purpose of preventing and / or treating inflammatory bowel disease by enhancing intestinal drug concentration, enhancing intestinal drug retention performance, inhibiting intestinal inflammation, and improving intestinal integrity.

[0049] In some embodiments of the present invention, the drug is administered orally, wherein the gel microspheres 2 (βCD-FAMP) are first taken orally. 5-ASA Following this, oral administration of gel microspheres 1 (Ad-FAMP) 5-ASA βCD-FAMP 5-ASA The drug is administered orally to the intestines, where it is targeted to the lesions via the specific targeting effect of fucoidan on intestinal inflammation sites in IBD. Ad-FAMP is then delivered orally as well. 5-ASA When delivered into the intestine, it transforms into Ad-FAMP through supramolecular interactions between β-cyclodextrin and adamantane. 5-ASAThe combination provides more anchor points, overcomes the spatial steric hindrance caused by micron-sized carriers, increases the drug concentration at the lesion site, enhances the efficacy of inflammatory bowel disease, and ultimately achieves significant anti-inflammatory and intestinal integrity restoration effects in mouse acute inflammatory bowel disease models and preventive inflammatory bowel disease models.

[0050] The beneficial effects of this invention are: This invention provides a gel microsphere that delivers to the lesion site via the specific targeting of fucoidan with damaged endothelial cells, achieving disease prevention and / or treatment. Furthermore, gel microspheres 1 and 2 overcome steric hindrance through supramolecular assembly between adamantane and cyclodextrin, providing more drug-targeting anchors and thus increasing drug concentration at the lesion site to enhance the therapeutic efficacy of IBD. This gel microsphere assembly strategy provides an effective method for enhancing the concentration of orally administered micron-sized drugs at the lesion site, offering a new approach to the delivery of micron-sized oral drugs. Attached Figure Description

[0051] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 Thermogravimetric characterization results for adamantane-modified sodium alginate (ALG-Ad) (b) and β-cyclodextrin-modified sodium alginate (ALG-βCD) (a).

[0052] Figure 2 Atomic force microscopy (AFM) images and particle size measurements of Ad-FAMP and βCD-FAMP are shown below; where a is the AFM image of βCD-FAMP with a scale bar of 1 μm, b is the AFM image of Ad-FAMP with a scale bar of 1 μm, c is the particle size measurement result of βCD-FAMP, and d is the particle size measurement result of Ad-FAMP.

[0053] Figure 3 To assess the stability of FAMP in the gastric acid environment (SGF).

[0054] Figure 4 The interaction between Ad-FAMP and βCD-FAMP was determined by isothermal titration calorimetry; where a represents the determination result of the interaction between Ad-FAMP5-ASA and FAMP5-ASA, b represents the determination result of the interaction between Ad-FAMP and βCD-FAMP, and c represents the determination result of the KD value of the interaction between Ad-FAMP and βCD-FAMP.

[0055] Figure 5To observe the interaction between Ad-FAMP and βCD-FAMP using confocal microscopy; where a is the confocal microscopy result of RhoB-Ad-FAMP and Cy5-FAMP, and b is the confocal microscopy result of RhoB-Ad-FAMP and Cy5-βCD-FAMP.

[0056] Figure 6 For Ad-FAMP 5-ASA and βCD-FAMP 5-ASA The therapeutic effect of the microsphere system on acute inflammatory bowel disease; where a is a diagram of the experimental process, b is a picture of the mouse tail after treatment, c is the weight change of the mouse during treatment, and d is the disease activity index score.

[0057] Figure 7 For Ad-FAMP 5-ASA and βCD-FAMP 5-ASA The therapeutic effect of the microsphere system on the prevention of inflammatory bowel disease; where a is a diagram of the experimental process, b is a picture of the mouse tail after treatment, c is the change in mouse weight during treatment, and d is the disease activity index score. Detailed Implementation

[0058] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0059] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0060] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0061] Example 1: Preparation of β-cyclodextrin-modified sodium alginate (ALG-βCD) and adamantane-modified sodium alginate (ALG-Ad) β-cyclodextrin-modified sodium alginate (ALG-βCD) was prepared by a condensation reaction between the carboxyl group on sodium alginate and the amino group on β-cyclodextrin. The specific preparation method is as follows: 3.0 g of sodium alginate (Alg) was weighed and dispersed in 20 mL of N,N-dimethylformamide (DMF). The solution was heated to 50 °C, and 3.9 g of toluenesulfonic acid (PTSA) was added. After stirring for 30 min, 2.9 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.3 g of β-cyclodextrin (βCD) were added, and the reaction was carried out under continuous stirring for 24 h. After the reaction was completed, the product was repeatedly washed with anhydrous ethanol. The washed product was then freeze-dried using a freeze dryer (Labconco, FreeZone, USA) (operating temperature ≤ -55 °C, vacuum range 0.001-1000 mbar). The freeze-dried product was β-cyclodextrin-modified sodium alginate, named ALG-βCD.

[0062] The obtained ALG-βCD was characterized by thermogravimetric analysis (TGA). The specific method of TGA was as follows: 2 mg of Alg, βCD, and ALG-βCD were weighed and added to the crucible of a thermogravimetric analyzer (NETZSCH, TG 209 F1 Libra (XX)). The mass change of each sample was then detected and analyzed under a controlled heating program (heating range 20-600°C, heating rate 0.001 K / min - 200 K / min). The results showed that the mass ratio of βCD in the synthesized ALG-βCD was 4.54% (…). Figure 1 (a) indicates the successful synthesis of ALG-βCD.

[0063] Adamantane-modified sodium alginate (ALG-Ad) was prepared by a condensation reaction between the carboxyl group on sodium alginate and the amino group on adamantane. The specific preparation method is as follows: 3.0 g of sodium alginate (Alg) was weighed and dispersed in 20 mL of N,N-dimethylformamide (DMF). The solution was heated to 50 °C, and 3.9 g of p-toluenesulfonic acid (PTSA) was added. After stirring for 30 min, 2.9 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.6 g of adamantane (Ad) were added, and the reaction was carried out under continuous stirring for 24 h. After the reaction was completed, the product was repeatedly washed with anhydrous ethanol. The washed product was then freeze-dried using a freeze dryer (Labconco, FreeZone, USA) (operating temperature ≤ -55 °C, vacuum range 0.001-1000 mbar). The freeze-dried product was β-cyclodextrin modified sodium alginate, named ALG-Ad.

[0064] The obtained ALG-Ad was characterized by thermogravimetric analysis (TGA). The specific method of TGA was as follows: 2 mg of Alg, βCD, and ALG-Ad were weighed and added to the crucible of a thermogravimetric analyzer (NETZSCH, Germany, TG 209 F1 Libra). The mass change of each sample was then detected and analyzed under a controlled heating program (heating range 20-600°C, heating rate 0.001 K / min - 200 K / min). The results showed that the mass ratio of Ad in the synthesized ALG-Ad was 10.36% (…). Figure 1 (b) indicates the successful synthesis of ALG-Ad.

[0065] Example 2 Ad-FAMP 5-ASA With βCD-FAMP 5-ASA Preparation and characterization 0.5 mg of adamantane-modified sodium alginate (ALG-Ad) or 0.5 mg of β-cyclodextrin-modified sodium alginate (ALG-βCD) prepared in Example 1 was dissolved in 1 mL of water along with 0.5 mg of fucoidan and 0.01 mg of 5-aminosalicylic acid (5-ASA). The solution was then slowly added dropwise to 10 mL of calcium chloride solution (1 mM) and stirred for 30 min to obtain Ad-FAMP gel microspheres. 5-ASA or βCD-FAMP 5-ASA .

[0066] βCD-FAMP5-ASA and Ad-FAMP5-ASA were observed using atomic force microscopy (AFM) and dynamic light scattering (DLS). The results showed that βCD-FAMP5-ASA (… Figure 2 (a and c) and Ad-FAMP5-ASA ( Figure 2 The particle sizes of b and d are 1.25 μm and 1.15 μm, respectively.

[0067] Example 3 FAMP 5-ASA Ad-AMP 5-ASA With AMP 5-ASA Preparation Dissolve 0.5 mg sodium alginate (ALG), 0.5 mg fucoidan, and 0.01 mg 5-aminosalicylic acid in 1 mL of water, and slowly add the solution dropwise to 10 mL of calcium chloride solution (1 mM). Stir for 30 min to obtain gel microspheres FAMP. 5-ASA .

[0068] 0.5 mg of adamantane-modified sodium alginate (ALG-Ad), 0.5 mg of sodium alginate (ALG), and 0.01 mg of 5-aminosalicylic acid were dissolved in 1 mL of water and slowly added dropwise to 10 mL of calcium chloride solution (1 mM). The mixture was stirred for 30 min to obtain Ad-AMP gel microspheres. 5-ASA .

[0069] Dissolve 1.0 mg sodium alginate (ALG) and 0.01 mg 5-aminosalicylic acid in 1 mL of water, and slowly add the solution dropwise to 10 mL of calcium chloride solution (1 mM). Stir for 30 min to obtain gel microspheres AMP. 5-ASA .

[0070] Example 4 βCD-FAMP 5-ASA With Ad-FAMP 5-ASA Stability in the acidic environment of the stomach βCD-FAMP5-ASA (50 μL, 10 mM) or Ad-FAMP5-ASA (50 μL, 10 mM) was added to 450 μL of simulated gastric fluid (specifically artificial gastric fluid SGF, purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number A7921) composed of dilute hydrochloric acid, pepsin, etc., and the particle size change was measured by dynamic light scattering after 0, 2, and 4 h.

[0071] The results showed that both βCD-FAMP5-ASA and Ad-FAMP5-ASA of Dunaliella salina microspheres remained stable in the gastric fluid environment. Figure 3 ).

[0072] Example 5: Isothermal calorimetric titration (ITC) detection of supramolecular interactions between βCD-FAMP and Ad on the surface of Ad-FAMP In the ITC, Ad-FAMP (50 μL, 10 mM) or FAMP (50 μL, 10 mM) was added dropwise to βCD-FAMP (500 μL, 1 mM), and after temperature equilibration, the titration data were analyzed.

[0073] The results showed that no thermal reaction was observed when Ad-FAMP was mixed with FAMP. Figure 4 In section a), a significant exothermic reaction was observed when βCD-FAMP was mixed with Ad-FAMP (an exothermic reaction caused by the supramolecular binding of βCD and Ad), with a KD value of 3.52 × 10⁻⁶. -6 M ( Figure 4 (bc).

[0074] Example 6: Observation of the aggregation of fluorescently labeled gel microspheres (Cy5-βCD-FAMP and RhoB-Ad-FAMP) using 3D confocal microscopy (3D CLSM). In this embodiment, Cy5-labeled sodium alginate (ALG-Cy5) and RhoB-labeled sodium alginate (ALG-RhoB) were prepared via a condensation reaction between the carboxyl group on sodium alginate and the amino group on Cy5 or RhoB. Cy5-labeled sodium alginate (ALG-Cy5), β-cyclodextrin-modified sodium alginate (ALG-βCD), and fucoidan were cross-linked by calcium ions to form Cy5-labeled gel microspheres Cy5-βCD-FAMP; RhoB-labeled sodium alginate (ALG-RhoB), adamantane-modified sodium alginate (ALG-Ad), and fucoidan were cross-linked by calcium ions to form RhoB-labeled gel microspheres RhoB-Ad-FAMP. The preparation method of the above gel microspheres was performed according to Example 2.

[0075] After reacting Cy5-βCD-FAMP (500 μL, 1 mM) with RhoB-Ad-FAMP (50 μL, 10 mM) for 15 min, the mixture was placed in a confocal dish and observed using a confocal microscope.

[0076] The results showed that obvious aggregates were observed after Cy5-βCD-FAMP reacted with RhoB-Ad-FAMP for 15 min, while no aggregation was observed in the control group Cy5-FAMP and RhoB-Ad-FAMP. Figure 5 ).

[0077] Example 7 βCD-FAMP 5-ASA With Ad-FAMP 5-ASA The efficacy of microsphere system in treating acute inflammatory bowel disease Seven-week-old female C57BL / 6J mice were selected, and the water was replaced with 3% sodium dextran sulfate (DSS) to establish the model. The mice were fed normally for 7 days. After the mice showed obvious bloody stools and weight loss, they were given the drug (during the drug administration period, they drank water without DSS).

[0078] Mice that successfully developed the model were randomly divided into 5 groups of 5 mice each. The 5 groups were G1: PBS; G2: 5-ASA; G3: Ad-AMP. 5-ASA +AMP 5-ASA G4: Ad-FAMP 5-ASA +FAMP 5-ASA G5: Ad-FAMP 5-ASA +βCD-FAMP 5-ASA PBS (G1) or 5-ASA (G2) (40 mg / kg) was administered orally. Additionally, groups G3, G4, and G5 were first administered Ad-AMP orally. 5-ASA or Ad-FAMP 5-ASA(5-ASA mass is 20 mg / kg), AMP is then administered orally 2 hours later. 5-ASA FAMP 5-ASA βCD-FAMP 5-ASA (5-ASA mass was 20 mg / kg). From the first day of modeling, the body weight and fecal condition of mice were monitored daily. The disease activity index (DAI) score of mice was assessed according to the criteria established by McCarthy, as shown in Table 1. Eight days after treatment, the mice were euthanized, and the intestinal tissue of the mice was removed to measure the intestinal length and weight.

[0079] Table 1 DAI Scoring Criteria

[0080] Note: In Table 1, the DAI score = (weight loss + stool characteristics + fecal occult blood / gross blood in stool) / 3. Stool characteristics scoring criteria: (1) Normal stool: formed stool; (2) Loose stool: pasty or semi-formed stool that does not adhere to the anus; (3) Loose stool: watery stool that can adhere to the anus. Mouse fecal occult blood test method: Take a small amount of stool with a cotton swab and apply it to a clean white board. Add 1-2 drops of o-toluidine glacial acetic acid solution to the surface of the stool and add 1-2 drops of 3% hydrogen peroxide to the surface of the stool. Result judgment: a. If no color is observed 2 minutes after adding the reagent, it is negative; b. If blue appears within 2 minutes after adding the reagent, it is positive. The degree of blue color is as follows: light blue gradually turns to blue after 10 seconds after adding the reagent (+), light blue-brown after adding the reagent (++), and blue-brown immediately after adding the reagent (+++).

[0081] The results are as follows Figure 6 As shown, in mice, after Ad-FAMP... 5-ASA +βCD-FAMP 5-ASA After treatment with the microsphere system, the mice showed significant improvement in rectal bleeding and body weight compared to other groups. Ad-FAMP 5-ASA +FAMP 5-ASA The treatment effect was second best, indicating that Ad-FAMP... 5-ASA The fucoidan component of FAMP5-ASA is beneficial for drug targeting of inflammatory sites, while Ad-FAMP 5-ASA and βCD-FAMP 5-ASA The combination of inflammation-targeting capabilities and surface supramolecular interactions enables more effective drug enrichment, thereby enhancing the therapeutic effect of inflammation treatment. In contrast, Ad-AMP... 5-ASA +AMP 5-ASA Due to the lack of inflammatory targeting ability and surface supramolecular interactions of fucoidan, its therapeutic effect is weaker than that of Ad-FAMP5. -ASA +FAMP 5-ASA.

[0082] Example 8 βCD-FAMP 5-ASA With Ad-FAMP 5-ASA The efficacy of microsphere systems in the treatment and prevention of inflammatory bowel disease Seven-week-old female C57BL / 6J mice were selected, and water was replaced with 3% sodium dextran sulfate (DSS) to establish the model. The mice were fed normally for 11 days, and the drug was administered on the second day (during the drug administration period, the mice continued to drink water containing 3% sodium dextran sulfate).

[0083] Mice were randomly divided into 5 groups of 5 mice each: G1: PBS; G2: 5-ASA; G3: Ad-AMP. 5-ASA +AMP 5-ASA G4: Ad-FAMP 5-ASA +FAMP 5-AS A; G5: Ad-FAMP 5-ASA +βCD-FAMP 5-ASA PBS (G1) or 5-ASA (G2) (40 mg / kg) was administered orally. Additionally, groups G3, G4, and G5 were first administered Ad-AMP orally. 5-ASA or Ad-FAMP 5-ASA (5-ASA mass is 20 mg / kg), AMP is then administered orally 2 hours later. 5-ASA FAMP 5-ASA βCD-FAMP 5-ASA (5-ASA mass was 20 mg / kg). From the first day of modeling, the body weight and fecal condition of the mice were monitored daily. The disease activity index (DAI) score of the mice was assessed according to the criteria established by McCarthy, as shown in Table 1. After 10 days of treatment, the mice were euthanized, and the intestinal tissue of the mice was removed to measure the length and weight of the intestines.

[0084] The results are as follows Figure 7 As shown, in mice, after Ad-FAMP... 5-ASA +βCD-FAMP 5-ASA After treatment with the microsphere system, mice showed significant improvement in rectal bleeding and body weight compared to other groups, indicating that Ad-FAMP... 5-ASA +βCD-FAMP 5-ASA The combination of inflammation-targeting capabilities and surface supramolecular interactions enables more effective drug enrichment, thereby enhancing the prevention and treatment of inflammation.

[0085] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A gel microsphere, comprising gel microsphere 1 and / or gel microsphere 2; in, The gel microspheres 1 are prepared by cross-linking sodium alginate modified with adamantane, fucoidan and drugs under calcium ion conditions; The gel microspheres 2 are prepared by cross-linking sodium alginate modified with β-cyclodextrin, fucoidan, and drugs under calcium ion conditions.

2. The gel microspheres according to claim 1, characterized in that, The drugs include anti-inflammatory drugs; Preferably, the anti-inflammatory drug includes at least one of 5-aminosalicylic acid, acetylsalicylic acid, and itaconic acid.

3. The gel microspheres according to claim 1 or 2, characterized in that, The adamantane-modified sodium alginate was prepared by a condensation reaction of sodium alginate and adamantane. Preferably, the adamantane-modified sodium alginate is prepared by a method comprising the following steps: Sodium alginate was dispersed in N,N-dimethylformamide, mixed with toluenesulfonic acid, and reacted to obtain mixture 1. Mixture 1 was mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and adamantane, and reacted to obtain adamantane-modified sodium alginate.

4. The gel microspheres according to claim 1 or 2, characterized in that, The β-cyclodextrin-modified sodium alginate was prepared by a condensation reaction of sodium alginate and β-cyclodextrin. Preferably, the β-cyclodextrin-modified sodium alginate is prepared by a method comprising the following steps: Sodium alginate was dispersed in N,N-dimethylformamide, mixed with toluenesulfonic acid, and reacted to obtain mixture 1. Mixture 1 was mixed with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and β-cyclodextrin, and reacted to obtain sodium alginate modified with β-cyclodextrin.

5. The gel microspheres according to claim 1 or 2, characterized in that, The mass ratio of the adamantane-modified sodium alginate to fucoidan and the drug is 1:(0.5-2):(0.01-0.2); and / or, the mass ratio of the β-cyclodextrin-modified sodium alginate to fucoidan and the drug is 1:(0.5-2):(0.01-0.2).

6. The method for preparing gel microspheres according to any one of claims 1-5, comprising the following steps: Sodium alginate modified with adamantane, fucoidan and drugs were dissolved in a solvent, calcium ion solution was added dropwise, and the reaction was carried out to obtain gel microspheres 1. β-cyclodextrin-modified sodium alginate, fucoidan, and the drug were dissolved in a solvent, and calcium ion solution was added dropwise. The reaction was carried out to obtain gel microspheres 2.

7. The gel microspheres according to claim 6, characterized in that, The reaction time is 20-50 min.

8. The use of the gel microspheres according to any one of claims 1-5 or the preparation method according to claim 6 or 7 in the preparation of medicaments for the prevention and / or treatment of inflammatory bowel disease.

9. A drug comprising the gel microspheres according to any one of claims 1-5.

10. The medicament according to claim 9, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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