Polymer material, drug-loaded micelle solution, drug-loaded micelle composite hydrogel as well as preparation method and application of polymer material, drug-loaded micelle solution and drug-loaded micelle composite hydrogel

Abstract

The invention discloses a polymer material, a drug-loaded micelle solution, a drug-loaded micelle composite hydrogel, and a preparation method and application of the drug-loaded micelle composite hydrogel. The polymer material is obtained by reacting oxidized dextran and oleylamine; wherein the oxidized dextran is obtained by oxidizing dextran with the molecular weight of 35000 to 45000; the oxidation degree of the oxidized dextran is 55-70%; the mass ratio of the oxidized dextran to the oleylamine is 1: (1.2-2.0). The polymer material provided by the invention can form a micelle as a carrier to entrap a hydrophobic drug, such as prednisolone. The drug-loaded micelle composite hydrogel provided by the invention can be used for treating inflammatory bowel disease IBD, can avoid damage of an acid environment to a greater extent, and prolongs the administration time.

Description

Technical Field

[0001] The invention relates to a polymer material, a drug-loaded micelle solution, a drug-loaded micelle composite hydrogel, and a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a common idiopathic inflammatory disease, primarily encompassing ulcerative colitis and Crohn's disease. Typical symptoms include abdominal pain, diarrhea, weight loss, impaired intestinal barrier integrity, and bloody stools. IBD is a complex pathological process. Intestinal microbial imbalance, impaired intestinal mucosal barrier, decreased immune function, and oxidative stress are recognized as the causes of IBD in today's medical community. The intestinal mucosa serves as the first line of defense against pathogens. Long-term inflammation of the intestinal mucosa can trigger excessive production of reactive oxygen species (ROS), further exacerbating inflammation. Therefore, eliminating inflammation and promoting mucosal repair are the primary priorities in treating IBD. Clinically, therapeutic agents include glucocorticoids, antibiotics, aminosalicylic acids, immunosuppressants, and biologics. However, long-term and frequent use of these drugs increases the risk of drug resistance, autoimmunity, hepatotoxicity, and other side effects.

[0003] Currently, nano-drug carriers are gaining increasing attention and recognition as oral colon-targeted drug delivery systems. Nano-drug carriers include various types, such as liposomes, polymer micelles, nanoparticles, and nanoclusters. Polymer micelles are core-shell nanocarriers formed by self-assembly of amphiphilic polymers. Their hydrophobic cores can be loaded with poorly soluble drugs through physical encapsulation or chemical bonding (e.g., hydrophobic interactions, π-π stacking), significantly improving drug solubility and stability. Furthermore, the nanocarrier's characteristics (typically <200 nm) and surface hydrophilic modification can reduce mucus layer adhesion, promote penetration of the mucus barrier, and accumulate at sites of inflammation, making them ideal carriers for the treatment of IBD.

[0004] CN107823178A discloses a niflumic acid colon-targeted preparation for treating irritable bowel syndrome and a preparation method thereof. The preparation method comprises: first encapsulating niflumic acid in a polymer material to form nano-micelles, and then further encapsulating the nano-micelles in a micro-pellet polymer material using a sharp hole-coagulation method; wherein the polymer material is a mixture of one or more of Pluronic F127, polylactic-co-glycolic acid copolymer (PLGA), and polyethylene glycol 1000 vitamin E succinate; and the micro-pellet polymer material is a mixture of one or more of low-ester pectin, sodium alginate, chitosan, and ethyl cellulose.

[0005] CN115850534B discloses a hyaluronic acid-cholic acid derivative, its synthesis method, and application. The method comprises: (1) synthesizing ethylenediamine hyaluronic acid; (2) using ethylenediamine hyaluronic acid and a cholic acid derivative as raw materials, subjecting the hyaluronic acid to an amidation reaction to obtain a hyaluronic acid-dehydrolithocholic acid polymer; (3) adding the hyaluronic acid-dehydrolithocholic acid polymer to water and sonicating to obtain a uniformly distributed micellar spherical particle system. The constructed micellar spherical particle system, which has a hydrophilic hyaluronic acid shell and a hydrophobic dehydrolithocholic acid core in water, is used to treat inflammatory bowel disease.

[0006] However, the application of micelles is also subject to certain challenges. For example, after oral administration, the complex gastrointestinal environment of the body can easily cause the micelle structure to rupture, leading to premature release of the drug, insufficient actual drug dose reaching the colon, and excessively short retention time. Hydrogels are natural or synthetic three-dimensional polymers with properties such as high water content, high biocompatibility, degradability, and swelling. Studies have shown that adhesive hydrogels can effectively repair damaged mucosa and tissues, and through local adhesion, they can prolong the retention time of drugs at the site of inflammation, reduce the number of drug administrations, and improve the therapeutic effect.

[0007] CN114028360A discloses a nano-drug delivery system for treating colon diseases, comprising a nanocarrier and a protective coating layer coated around the outer surface of the nanocarrier. The nanocarrier comprises porous nanoparticles and exosome active ingredients loaded onto the porous nanoparticles, and the coating layer is a hydrogel protective layer. To date, there are no reports on drug-loaded micelles formed from oxidized dextran, oleylamine, and prednisolone, nor on drug-loaded micelle composite hydrogels containing these materials, their preparation methods, or their use in the preparation of drugs for treating inflammatory bowel disease. Summary of the Invention

[0008] One object of the present invention is to provide a polymer material that can form polymer micelles as a carrier for encapsulating hydrophobic prednisolone, which is conducive to the formation of drug-loaded micelles to increase the drug loading amount and reduce the micelle particle size. Another object of the present invention is to provide a method for preparing the above-mentioned polymer material. Another object of the present invention is to provide a drug-loaded micelle solution and a preparation method thereof. Yet another object of the present invention is to provide a drug-loaded micelle composite hydrogel, which has an anti-inflammatory effect and can be used for the treatment of inflammatory bowel disease. It can prolong the administration time and avoid the sudden release of drugs to a greater extent. Yet another object of the present invention is to provide a method for preparing the above-mentioned drug-loaded micelle composite hydrogel. The last object of the present invention is to provide a use of the drug-loaded micelle composite hydrogel as described above in the preparation of drugs for treating inflammatory bowel disease.

[0009] The present invention adopts the following technical solutions to achieve the above-mentioned purpose.

[0010] On the one hand, the present invention provides a polymer material obtained by reacting oxidized dextran and oleylamine; wherein the oxidized dextran is obtained by oxidizing dextran with a molecular weight of 35,000 to 45,000; the oxidation degree of the oxidized dextran is 55 to 70%; and the mass ratio of the oxidized dextran to oleylamine is 1:1.2 to 2.0.

[0011] On the other hand, the present invention also provides a method for preparing the polymer material as described above, comprising the following steps:

[0012] 1) dissolving oxidized dextran in water to obtain an oxidized dextran solution; dissolving oleylamine in a C1-C3 alkyl alcohol to obtain an oleylamine solution;

[0013] 2) mixing the oxidized dextran solution and the oleylamine solution, then adding a boride reducing agent, reacting at 25-50° C., and after the reaction is completed, separating the reaction product into a solid and a liquid, washing the separated solid, and freeze-drying the washed solid to obtain the polymer material.

[0014] On the other hand, the present invention also provides a drug-loaded micelle solution, which is prepared from raw materials including the polymer material as described above and prednisolone, wherein the drug loading capacity of the lyophilized product of the drug-loaded micelle solution is greater than 10wt%, and the encapsulation efficiency is greater than 79wt%; the drug-loaded micelles have a nearly spherical structure and an average particle size of 70 to 85nm.

[0015] In another aspect, the present invention further provides a method for preparing the drug-loaded micelle solution as described above, comprising the following steps:

[0016] A) mixing and dissolving prednisolone, the polymer material described above, and a C1-C3 alkyl alcohol, and then concentrating under reduced pressure to obtain a film-like substance;

[0017] B) placing the film-like substance in water, and then subjecting the film-like substance to rotary evaporation to form a micelle solution, followed by centrifugation, and passing the obtained supernatant through a microporous filter membrane to obtain a drug-loaded micelle solution.

[0018] In another aspect, the present invention provides a drug-loaded micelle composite hydrogel, which is formed by an oxidized dextran solution, a thiol chitosan solution, and the drug-loaded micelle solution as described above; wherein the mass concentration ratio of the oxidized dextran solution to the thiol chitosan solution is 1:1 to 1.2, and the volume ratio of the oxidized dextran solution to the thiol chitosan solution is 1.8 to 2.2:1.

[0019] In another aspect, the present invention further provides a method for preparing the drug-loaded micelle composite hydrogel as described above, comprising the following steps:

[0020] The oxidized dextran solution and the mercapto-chitosan solution are mixed, and then the drug-loaded micelle solution as described above is added and mixed into a gel to obtain the drug-loaded micelle composite hydrogel.

[0021] According to the preparation method of the present invention, preferably, the mass concentration of the oxidized dextran solution is 20-100 mg / mL, the mass concentration of the thiol chitosan solution is 20-100 mg / mL; and the volume ratio of the oxidized dextran solution to the drug-loaded micelle solution is 2-2.5:3.

[0022] According to the preparation method of the present invention, preferably, the oxidized dextran used to prepare the polymer material and the oxidized dextran in the oxidized dextran solution are both prepared by the following steps:

[0023] (1) adding sodium periodate to the dextran solution, reacting for 3 to 5 hours in the dark, and then adding glycerol to terminate the reaction to obtain a reaction solution;

[0024] (2) The reaction solution was transferred to a dialysis bag with a molecular weight cut-off of 3400 Da for dialysis, and the dialyzate was freeze-dried to obtain the oxidized dextran.

[0025] According to the preparation method of the present invention, preferably, the mercapto chitosan is prepared by the following steps:

[0026] (a) dispersing chitosan in water to obtain a chitosan solution;

[0027] (b) adding 1-hydroxybenzotriazole to the chitosan solution and mixing to obtain a mixed solution; then adding N-acetylcysteine ​​to the mixed solution, and then dropwise adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and adjusting the pH value of the reaction system to 5.0-5.5, and reacting in the dark for 2.5-3.5 hours to obtain a reactant;

[0028] (c) dialyzing the reactant using a dialysis bag with a molecular weight cut-off of 12000 Da, and freeze-drying the dialyzate to obtain the thiol chitosan.

[0029] In the final aspect, the present invention further provides a use of the drug-loaded micelle composite hydrogel as described above in the preparation of a drug for treating inflammatory bowel disease.

[0030] The polymer material of the present invention can be used as a carrier to encapsulate hydrophobic drugs, such as prednisolone, which is conducive to the formation of drug-loaded micelles. The micelles are evenly dispersed, with an average particle size of 70 to 85 nm and a narrow particle size distribution. At the same time, the drug loading capacity of prednisolone can be increased. The drug-loaded micelle composite hydrogel of the present invention can avoid the destruction of the acidic environment to a greater extent, and fully release prednisolone (PA) after delivery to the colon environment, which can extend the administration time and avoid drug burst release to a greater extent. The drug-loaded micelle composite hydrogel of the present invention can regulate the level of inflammatory factors, inhibit the secretion of pro-inflammatory factors, increase the concentration of anti-inflammatory factors, and has a good anti-inflammatory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1(A) shows the FT-IR spectra of dextran (DEX), ODEX-H, ODEX-M, and ODEX-L.

[0032] Figure 1(B) is the hydrogen spectrum of DEX.

[0033] FIG1(C) is a hydrogen spectrum of ODEX-H obtained in Preparation Example 1.

[0034] Figure 2 This is the FT-IR graph of the polymer material ODEX-OA in Example 1.

[0035] Figure 3 This is the transmission electron microscopy result of the polymer micelle solution in Example 1.

[0036] Figure 4 This is the transmission electron microscopy result of the drug-loaded micelle solution of Example 2.

[0037] FIG5(A) is the FT-IR spectrum of the thiolated chitosan (CSSH) obtained in Preparation Example 2 and the raw material chitosan (CS).

[0038] Figure 5(B) is the hydrogen spectrum of chitosan (CS).

[0039] Figure 5(C) is the hydrogen spectrum of thiolated chitosan (CSSH).

[0040] Figure 6 These are SEM images of the drug-loaded micellar composite hydrogel obtained in Example 3. Figures A and C show the SEM images of the blank micellar hydrogel ODEX-OA-CSSH at different scales. Figures B and D show the SEM images of the drug-loaded micellar composite hydrogel ODEX-OA-PA-CSSH at different scales.

[0041] Figure 7 The drug release curves of drug-loaded micelle composite hydrogel ODEX-OA-PA-CSSH in different release media.

[0042] Figure 8The degradation curves of drug-loaded micelle composite hydrogel ODEX-OA-PA-CSSH in different pH media.

[0043] Figure 9 Shown are the changes in body weight and DAI scores of mice in different treatment groups.

[0044] FIG10(A) shows HE-stained sections of the colon of mice in different treatment groups.

[0045] FIG10(B) shows the colon histopathological scores of mice in different treatment groups.

[0046] Figure 11 The results show the levels of inflammatory cytokines in colon tissue of mice in different treatment groups. DETAILED DESCRIPTION

[0047] The present invention will be further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0048] <Polymer material, polymer micelle solution and preparation method thereof>

[0049] polymer materials

[0050] The polymer material of the present invention (which can be denoted as ODEX-OA) is obtained by reacting oxidized dextran and oleylamine. The oxidized dextran is obtained by oxidizing dextran with a molecular weight of 35,000 to 45,000; the degree of oxidation of the oxidized dextran is 55 to 70%; and the mass ratio of oxidized dextran to oleylamine is 1:1.2 to 2.0. Such a polymer material can be used as a carrier to encapsulate hydrophobic drugs, such as prednisolone. Prednisolone (prednisolone), chemical formula C 21 H 28 O5, CAS No. 50-24-8, can be referred to as PA in the present invention.

[0051] The present invention discovered that combining oxidized dextran with oleylamine can form amphiphilic polymer micelles, which not only achieves efficient loading and sustained release of poorly soluble prednisolone, but also leverages the biocompatibility and enzyme-responsive properties of oxidized dextran to further enhance colon targeting. The polymer micelle solution is uniformly dispersed and has a relatively uniform particle size. The polymer micelle solution described herein is formed from a polymer material.

[0052] In the present invention, the oxidized dextran is obtained by oxidizing dextran, wherein the molecular weight of the dextran may be 35,000 to 45,000, preferably 38,000 to 43,000, and more preferably 39,000 to 41,000. According to a specific embodiment of the present invention, the molecular weight of the dextran is 40,000 to 42,000.

[0053] In the present invention, the degree of oxidation of the oxidized dextran can be 55-70%, preferably 56-65%, more preferably 57-63%, and even more preferably 59-62%. The mass ratio of the oxidized dextran to oleylamine can be 1:1.2-2.0, preferably 1:1.4-1.9, more preferably 1:1.5-1.7, and even more preferably 1:1.6-1.7. This facilitates drug encapsulation, increases drug loading, and reduces micelle particle size. The oleylamine is 9-octadeceneamine.

[0054] Preparation method of polymer material

[0055] According to one embodiment of the present invention, a method for preparing a polymer material includes the following steps: 1) preparing an oxidized dextran solution and an oleylamine solution; and 2) forming the polymer material ODEX-OA. Optionally, the method also includes preparing the oxidized dextran. This is described in detail below.

[0056] In certain embodiments, the oxidized dextran is prepared by the following steps: (1) adding sodium periodate to a dextran solution, reacting for 3 to 5 hours in the dark, and then adding glycerol to terminate the reaction to obtain a reaction solution; (2) transferring the reaction solution to a dialysis bag for dialysis, and freeze-drying the dialyzate to obtain the oxidized dextran. The above reaction can be carried out at 40 to 65°C, preferably at 50 to 60°C. The molar ratio of sodium periodate to dextran can be 4 to 5.5:1, preferably 4.2 to 5:1, and more preferably 4.5 to 4.8:1. The reaction time is preferably 4 to 5 hours. When glycerol is added, it is preferably added dropwise. The molecular weight cutoff of the dialysis bag is greater than 3400 Da. Dialysis is performed in deionized water, and the water is changed about 3 to 4 times a day. Silver nitrate solution can be used to detect whether the dialysate has precipitated. If no precipitate is produced, the dialysis is completed. The freeze-drying temperature can be less than -50°C, for example, -80°C. The obtained oxidized dextran was stored in the dark.

[0057] In certain embodiments, oxidized dextran is dissolved in water to obtain an oxidized dextran solution; and oleylamine is dissolved in a C1-C3 alkyl alcohol to obtain an oleylamine solution. The oxidized dextran solution (oxidized dextran solution) and the oleylamine solution may be prepared in any order. The C1-C3 alkyl alcohol is selected from at least one of methanol, ethanol, and isopropanol, preferably ethanol. The present invention has found that dissolving oleylamine in ethanol is more conducive to increasing the drug loading capacity of the resulting polymer micelles or drug-loaded micelles. The mass concentration of the oxidized dextran solution may be 1-60 mg / mL, preferably 2-30 mg / mL, and more preferably 2.5-5 mg / mL. The mass concentration of the oleylamine solution may be 50-100 mg / mL, preferably 60-90 mg / mL, and more preferably 70-80 mg / mL. Controlling the concentration of the solution within the above range is more conducive to obtaining uniformly dispersed polymer micelles and drug-loaded micelles.

[0058] An oxidized dextran solution and an oleylamine solution are mixed, followed by the addition of a boride reducing agent. The reaction is carried out at 25-50°C. After the reaction is complete, the reaction product is separated into a solid and liquid state, the separated solid is washed, and the washed solid is freeze-dried to obtain the polymer material ODEX-OA. The boride reducing agent is preferably sodium cyanoborohydride. Preferably, the oxidized dextran solution and the oleylamine solution are ultrasonically mixed before the reducing agent is added. The reaction is then shaken, preferably at 35-45°C for 10-30 hours, preferably 20-25 hours. After the reaction is complete, the reaction product is transferred to ethanol and allowed to stand for 30 minutes to 1 hour before undergoing solid-liquid separation, preferably by centrifugation. After centrifugation, the supernatant is removed and the solid precipitate is collected. The separated solid is washed repeatedly with a C1-C3 alkyl alcohol and water two to three times. The C1-C3 alkyl alcohol can be methanol or ethanol, preferably methanol. The washed solid is freeze-dried below -50°C to obtain the polymer material ODEX-OA.

[0059] Preparation method of polymer micelle solution

[0060] The present invention also provides a method for preparing a polymer micelle solution, which specifically comprises the following steps: forming the polymer material ODEX-OA into a thin film-like substance; and forming the polymer micelle solution.

[0061] The above-mentioned polymer material ODEX-OA is subjected to a thin film hydration method to obtain polymer micelles. Specifically, the polymer material ODEX-OA is mixed and dissolved with a C1~C3 alkyl alcohol, and then concentrated under reduced pressure to obtain a thin film substance. Wherein, the C1~C3 alkyl alcohol is selected from methanol or ethanol, preferably methanol. The mass volume ratio of the polymer material ODEX-OA and the C1~C3 alkyl alcohol can be 10mg:5~20mL, preferably 10mg:10~15mL. After the polymer material ODEX-OA is mixed with the C1~C3 alkyl alcohol, it is preferably dissolved by ultrasound for 20~40min, for example, 30min. The temperature for reduced pressure concentration can be 55~70℃, preferably 65~70℃, and the reduced pressure concentration completely removes the organic solvent, forming a thin film at the bottom of the container to obtain a thin film substance.

[0062] The film-like substance is placed in water, and then rotary evaporated (the purpose of rotary evaporation is to dissolve the film-like substance in water to form a micellar solution), followed by centrifugation, and the resulting supernatant is passed through a microporous filter membrane to obtain a polymer micelle solution. Wherein, the amount of water used is the volume mass ratio of water to polymer material ODEX-OA, which can be 8 to 12 mL: 10 mg, preferably 9 to 11 mL: 10 mg, and more preferably 10 to 11 mL: 10 mg. Controlling the amount of water within the above range is conducive to obtaining a polymer micelle solution with a uniform particle size distribution. Then rotary evaporate, followed by centrifugation at 3000 to 4000 rpm for 8 to 15 minutes, and take the supernatant. The supernatant is passed through a microporous filter membrane of less than 1 μm, preferably a microporous filter membrane of 0.45 μm, to obtain a polymer micelle solution.

[0063] <Drug-loaded micelle solution and preparation method thereof>

[0064] Drug-loaded micelle solution

[0065] The present invention provides a drug-loaded micelle solution prepared from raw materials including the polymer material ODEX-OA and prednisolone as described above, wherein the drug-loaded micelle solution lyophilized product (i.e., the drug-loaded micelle lyophilized product) has a drug loading of 10 wt% or more and an encapsulation efficiency of 79 wt% or more; the drug-loaded micelles have a nearly spherical structure and an average particle size of 70 to 85 nm. The drug-loaded micelles are uniformly dispersed. The average particle size of the drug-loaded micelles is preferably 70 to 83 nm, more preferably 72 to 82 nm. This makes it more suitable for colon-targeted drug delivery.

[0066] The drug loading amount of the lyophilized product of the drug-loaded micelle solution (ie, the drug-loaded micelle lyophilized product) is preferably above 11 wt %, and the encapsulation efficiency is preferably above 80 wt %.

[0067] The present invention also provides a drug-loaded micelle lyophilized product, which is obtained by lyophilizing the drug-loaded micelle solution.

[0068] Preparation method of drug-loaded micelle solution

[0069] The present invention provides a method for preparing a drug-loaded micelle solution, comprising the following steps:

[0070] A) mixing and dissolving prednisolone, the polymer material ODEX-OA described above, and a C1-C3 alkyl alcohol, and then concentrating under reduced pressure to obtain a film-like substance;

[0071] B) The film-like material is placed in water, and then subjected to rotary evaporation to form a micellar solution. The solution is then centrifuged, and the resulting supernatant is filtered through a microporous filter membrane to obtain a drug-loaded micelle solution. This facilitates obtaining uniformly dispersed and uniformly distributed drug-loaded micelles.

[0072] In the present invention, the drug-loaded micelle solution is freeze-dried to obtain a drug-loaded micelle freeze-dried product, which is conducive to obtaining drug-loaded micelles with uniform dispersion and uniform particle size distribution.

[0073] The present invention adopts a thin film hydration method to prepare a drug-loaded micelle solution. In step A), the C1-C3 alkyl alcohol is selected from methanol or ethanol, preferably methanol. Preferably, prednisolone is first dissolved in the C1-C3 alkyl alcohol, and then the polymer material ODEX-OA is added for ultrasonic dissolution. The mass volume ratio of the polymer material ODEX-OA to the C1-C3 alkyl alcohol can be 10 mg:5-20 mL, preferably 10 mg:10-15 mL. The mass ratio of prednisolone to the polymer material ODEX-OA can be 2-10:10, for example, 2:10, 3:10, 4:10, 5:10, 6:10, 7:10, 8:10, 9:10, 10:10. The time for ultrasonic dissolution can be 20-40 minutes, for example, 30 minutes. The temperature for reduced pressure concentration can be 55-70°C, preferably 65-70°C. The reduced pressure concentration completely removes the organic solvent, forming a thin film at the bottom of the container to obtain a thin film-like substance.

[0074] In step B), the film-like substance is dissolved in water, and the amount of water used is that the volume mass ratio of water to the polymer material ODEX-OA can be 8-12mL:10mg, preferably 9-11mL:10mg, and more preferably 10-11mL:10mg. Controlling the amount of water within the above range is conducive to obtaining drug-loaded micelles with uniform particle size distribution and high drug loading. Then rotary evaporation (the purpose of rotary evaporation is to dissolve the film-like substance in water to form a micellar solution), followed by centrifugation at 3000-4000rpm for 8-15min, and taking the supernatant. The supernatant is passed through a microporous filter membrane below 1μm, preferably a microporous filter membrane of 0.45μm, to obtain a drug-loaded micelle solution.

[0075] In the present invention, the drug-loaded micelle solution is freeze-dried at a temperature below -50°C to obtain a drug-loaded micelle freeze-dried product.

[0076] <Drug-loaded micelle composite hydrogel and its preparation method>

[0077] Drug-loaded micelle composite hydrogel

[0078] The present invention provides a drug-loaded micelle composite hydrogel, which is formed by an oxidized dextran solution, a thiol chitosan solution and the drug-loaded micelle solution as described above; wherein the mass concentration ratio of the oxidized dextran solution and the thiol chitosan solution is 1:1 to 1.2, and the volume ratio of the two is 1.8 to 2.2:1. The drug-loaded micelle composite hydrogel of the present invention can avoid the destruction of the acidic environment to a greater extent, and fully release prednisolone (PA) after delivery to the colon environment, thereby extending the administration time and avoiding the sudden release of the drug to a greater extent. The drug-loaded micelle composite hydrogel of the present invention can regulate the level of inflammatory factors, inhibit the secretion of pro-inflammatory factors, increase the concentration of anti-inflammatory factors, and has a good anti-inflammatory effect.

[0079] In the present invention, the oxidized dextran solution is formed by dissolving oxidized dextran in phosphate buffered saline (PBS). The concentration of the phosphate buffered saline solution can be 0.01 to 0.02 M, with a pH of 7.2 to 7.4. The thiolated chitosan solution is formed by dissolving thiolated chitosan in an aqueous acetic acid solution. The concentration of the aqueous acetic acid solution can be 0.2 to 1.0 wt%, preferably 0.5 to 0.6 wt%.

[0080] The mass concentration ratio of the oxidized dextran solution to the thiolated chitosan solution can be 1:1 to 1.2, preferably 1:1 to 1.1. The volume ratio of the oxidized dextran solution to the thiolated chitosan solution can be 1.8 to 2.2:1, preferably 1.9 to 2.1:1, and more preferably 2 to 2.1:1.

[0081] The volume ratio of the oxidized dextran solution to the drug-loaded micelle solution is 2 to 2.5:3, preferably 2 to 2.3:3, and more preferably 2 to 2.1:3.

[0082] Preparation method of drug-loaded micelle composite hydrogel

[0083] The present invention provides a method for preparing a drug-loaded micellar composite hydrogel, comprising the following steps: preparing a thiol-chitosan solution; preparing an oxidized dextran solution; and a gelling reaction step. Optionally, the method further includes preparing the thiol-chitosan. This is described in detail below.

[0084] (1) Preparation steps of mercapto chitosan

[0085] In the present invention, the thiol chitosan is prepared by the following steps:

[0086] (a) dispersing chitosan in water to obtain a chitosan solution;

[0087] (b) adding 1-hydroxybenzotriazole to the chitosan solution and mixing to obtain a mixed solution; then adding N-acetylcysteine ​​to the mixed solution, and then dropwise adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and adjusting the pH value of the reaction system to 5.0-5.5, and reacting in the dark for 2.5-3.5 hours to obtain a reactant;

[0088] (c) dialyzing the reactant using a dialysis bag, and freeze-drying the dialyzate to obtain the sulfhydryl chitosan. This is conducive to obtaining sulfhydryl chitosan with higher purity.

[0089] In step (a), the concentration of chitosan in the chitosan solution may be 0.005 to 0.1 g / mL, preferably 0.01 to 0.08 g / mL, and more preferably 0.01 to 0.05 g / mL.

[0090] In step (b), the mass ratio of 1-hydroxybenzotriazole (HOBt) to chitosan can be 0.6 to 1:1, preferably 0.7 to 0.9:1, and more preferably 0.78 to 0.85:1. The mass ratio of N-acetylcysteine ​​(NAC) to chitosan can be 4 to 5:1, preferably 4.5 to 4.9:1, and more preferably 4.5 to 4.7:1. In step (b), the pH value of the reaction system is adjusted using sodium hydroxide solution or potassium hydroxide solution, preferably to 5.0. The reaction time in the dark is preferably 3 to 3.5 hours.

[0091] In step (c), the molecular weight cut-off of the dialysis bag is greater than 12,000 Da. The freeze-drying temperature is less than -50°C, for example -80°C.

[0092] (2) Preparation of thiol chitosan solution and oxidized dextran solution

[0093] According to one embodiment of the present invention, the thiolated chitosan solution is formed by dissolving thiolated chitosan in a 0.5 wt% acetic acid aqueous solution. The mass concentration of the thiolated chitosan solution can be 20-100 mg / mL, preferably 30-90 mg / mL, and more preferably 40-60 mg / mL.

[0094] According to one embodiment of the present invention, the oxidized dextran solution in this step is formed by dissolving oxidized dextran in phosphate buffer solution (PBS, 0.01M, pH = 7.2-7.4). The mass concentration of the oxidized dextran solution can be 20-100 mg / mL, preferably 30-90 mg / mL, and more preferably 40-60 mg / mL.

[0095] (3) Gelation reaction

[0096] The oxidized dextran solution and the mercapto-chitosan solution are mixed in a volume ratio, and then the drug-loaded micelle solution described above is added and mixed to form a gel to obtain the drug-loaded micelle composite hydrogel. The drug-loaded micelle composite hydrogel thus obtained has a short gelling time and good performance. For example, it can extend the drug administration time, largely avoid the sudden release of the drug, and has good anti-inflammatory properties.

[0097] In the present invention, the gel can be vortexed on a vortexer.

[0098] According to a specific embodiment of the present invention, 40 mg / mL of oxidized dextran solution and 40 mg / mL of thiol chitosan solution were mixed in a volume ratio of 2: 1 (the specific volumes were 0.2 mL and 0.1 mL, respectively), and then 0.3 mL of the drug-loaded micelle solution prepared according to the above method was added, vortexed on a vortexer, and gelled for 30 seconds to obtain a drug-loaded micelle composite hydrogel. The obtained drug-loaded micelle composite hydrogel can be recorded as ODEX-OA-PA-CSSH.

[0099] <Application>

[0100] The present invention also provides a use of the drug-loaded micelle composite hydrogel as described above in the preparation of a drug for treating inflammatory bowel disease. The drug-loaded micelle composite hydrogel of the present invention can avoid the destruction of the acidic environment to a greater extent, and fully release prednisolone (PA) after delivery to the colon environment, thereby extending the administration time and avoiding drug burst release to a greater extent. The drug-loaded micelle composite hydrogel of the present invention can regulate the level of inflammatory factors, inhibit the secretion of pro-inflammatory factors, increase the concentration of anti-inflammatory factors, and has a good anti-inflammatory effect.

[0101] <Partial test methods>

[0102] Infrared spectroscopy: Fourier transform infrared spectrophotometer (FT-IR) (IRAffinity-1 Fourier infrared spectrophotometer, Shimadzu Corporation, Japan UQS-2110007) was used with a scanning range of 4000 to 500 cm -1 , and characterized the structures of DEX, ODEX-L, ODEX-M, and ODEX-H.

[0103] 1 H NMR: The structures of DEX, ODEX, CS, and CSSH were analyzed using a 600 MHz nuclear magnetic resonance spectrometer. 4 mg of DEX and 4 mg of ODEX were dissolved in a mixture of 200 μL of deuterated chloroform and 550 μL of deuterated dimethyl sulfoxide for determination.

[0104] The degree of oxidation of oxidized dextran was determined using the hydroxylamine hydrochloride titration method, which specifically involves accurately weighing 580 mg of hydroxylamine hydrochloride, drying it to a constant mass, adding 25 mL of ultrapure water, and stirring to form a homogeneous solution. 1 mL of 0.05% (w / v) methyl orange indicator was then added to prepare a hydroxylamine hydrochloride-methyl orange solution. The solution was transferred to a volumetric flask, diluted to 100 mL, and the pH was adjusted to 4.0 with NaOH. 100 mg of the prepared ODEX sample was dissolved in 25 mL of the hydroxylamine hydrochloride-methyl orange solution and stirred at room temperature for 5 h. For comparison, 100 mg of unoxidized DEX was also weighed and dissolved in 25 mL of the hydroxylamine hydrochloride-methyl orange solution. The solution was titrated with 0.1 mol / L standard NaOH solution. When the solution color suddenly changed from red to yellow and did not fade within 30 s, and remained consistent with the control color, the NaOH consumption was recorded and recorded as ΔV. The degree of oxidation (η) was calculated as follows:

[0105] η=162ΔV×C×10 -3 / 2W;

[0106] Wherein, ΔV: volume of NaOH standard solution consumed (mL); C: concentration of NaOH standard solution (mol / L); W: mass of oxidized dextran (g); 162: molecular mass of dextran repeating unit (g / mol).

[0107] Determination of drug loading and encapsulation efficiency: The newly prepared ODEX-PA-OA drug-loaded micelle solution was pre-frozen in a -80°C refrigerator, then placed in a freeze dryer for freeze drying, a certain amount of drug-loaded micelle freeze-dried ODEX-PA-OA was weighed, and 4 times the volume of methanol solution was added for dilution, ultrasonicated for 10 minutes, and the supernatant was passed through a 0.22μm microporous filter membrane. The peak area was determined by HPLC, and the standard curve equation of PA peak area (Y) and PA concentration (X) was substituted into the equation to calculate the actual PA content in ODEX-PA-OA, and then the encapsulation efficiency (EE) and drug loading capacity (DL) of ODEX-PA-OA were calculated. Among them, the encapsulation efficiency refers to the ratio of the drug content encapsulated in the micelles to the total dosage; the drug loading capacity refers to the percentage of the drug contained in the micelles to the total amount of the preparation. The calculation formula is as follows:

[0108] Encapsulation efficiency (wt%) = actual content of PA in the lyophilized drug-loaded micelles ODEX-PA-OA / PA dosage × 100%;

[0109] Drug loading (wt%) = actual content of PA in the drug-loaded micelle lyophilized product ODEX-PA-OA / total mass of the drug-loaded micelle lyophilized product ODEX-PA-OA × 100%.

[0110] Determination of micelle particle size: The particle size of micelles in micelle solution was measured by laser particle size analyzer.

[0111] TEM determination of micelles: Take an appropriate amount of drug-loaded micelle solution or polymer micelle solution, dilute it, and add 1 to 2 drops onto a clean tin foil. Dry it at room temperature. After spraying the sample with gold, observe it under a transmission electron microscope (TEM).

[0112] SEM analysis of the hydrogel: After the gel is stabilized, the prepared hydrogel sample is placed in a freeze dryer for thorough freeze-drying. An appropriate amount of the freeze-dried preparation is adhered to the conductive adhesive on the stage. The surface of the freeze-dried preparation is gold-sprayed for 45 seconds using an ion sputtering instrument and then placed in a SEM chamber for morphology observation.

[0113] The sources of some of the raw materials in the following preparations and examples are described below: Dextran (MW 40,000), sodium cyanoborohydride (95%), and oleylamine (70%) were all purchased from Shanghai Aladdin Technology Co., Ltd. Prednisolone (99%) was purchased from Beijing Coupling Technology Co., Ltd. Chitosan (CS, degree of deacetylation 80.0%-90.0%) was purchased from Shanghai Aladdin Technology Co., Ltd.

[0114] Preparation Example 1 - Preparation of Oxidized Dextran

[0115] 50 mL of ultrapure water was added to 2.5 g of dextran (denoted as DEX, MW40000), and then stirred at 50° C. until the dextran was completely dissolved, and then cooled to room temperature to obtain a dextran solution.

[0116] 4.8 g of sodium periodate NaIO4 was added to the obtained dextran solution, and the mixture was reacted for 4 h in the dark. Then, 1 mL of glycerol was added dropwise, and the mixture was stirred for 15 min to obtain a reaction solution.

[0117] The resulting reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 3400 Da and dialyzed against deionized water, changing the water approximately three to four times daily to remove impurities such as unreacted glycerol and NaIO4. The dialysate was tested for precipitation using a 1 wt% silver nitrate solution; if no precipitation occurred, the impurities were completely removed. The resulting dialysate was freeze-dried at -80°C to obtain a white, fibrous oxidized dextran solid. The degree of oxidation was determined to be (60.75 ± 1.5)%, and the solid was stored at -20°C in the dark until further use. The resulting oxidized dextran was designated ODEX-H.

[0118] Comparative Preparation Example 1 - Preparation of Oxidized Dextran

[0119] The only difference from Preparation Example 1 was the mass of sodium periodate used. In Preparation Example 1, 3.2 g of sodium periodate was used. The degree of oxidation of the resulting oxidized dextran was (42.12 ± 0.93)%. The resulting oxidized dextran was designated ODEX-M.

[0120] Comparative Preparation Example 2 - Preparation of Oxidized Dextran

[0121] The only difference from Preparation Example 1 is the mass of sodium periodate used. Compared to Preparation Example 1, the amount of sodium periodate used was 1.6 g. The degree of oxidation of the resulting oxidized dextran was (34.83.12 ± 1.10)%. The resulting oxidized dextran was designated ODEX-L.

[0122] The FT-IR results of the oxidized dextran obtained in Preparation Example 1, Comparative Preparation Example 1, and Comparative Preparation Example 2 are shown in Figure 1(A). As shown in Figure 1(A), compared with the DEX molecular baseline, the three groups of ODEX samples with different oxidation degrees have a wave number of 1735 cm -1 Obvious characteristic peaks of aldehydes and ketones can be seen at all sites, and they become more obvious with increasing oxidation degree. At the same time, other original characteristic peaks still exist. This result indicates that the hydroxyl groups on the DEX molecules are partially oxidized to aldehyde groups, and the overall oxidation level increases from ODEX-L, ODEX-M to ODEX-H. This is consistent with the results of oxidation degree determination.

[0123] The hydrogen spectrum of DEX is shown in Figure 1(B), and the hydrogen spectrum of ODEX-H from Preparation Example 1 is shown in Figure 1(C). As shown in Figures 1(B) and 1(C), the proton peak at 3.2-4.0 ppm is derived from the chemical shift of hydrogen on the dextran ring. Compared with DEX, the new hemiacetal peak appearing in the 4.2-5.6 ppm range of ODEX indicates the presence of aldehyde groups in the polymer molecular chain. Therefore, it is preliminarily speculated that the hydroxyl groups in DEX are converted into hemiacetal or aldehyde structures within or between molecules after oxidation.

[0124] Example 1 - Preparation of polymer materials and polymer micelle solutions

[0125] 100 mg of oxidized dextran (ODEX-H) prepared in Preparation Example 1 was dissolved in 40 mL of ultrapure water to obtain an oxidized dextran solution. 0.16 g of oleylamine (OA) was dissolved in 2 mL of ethanol and stirred rapidly to dissolve it completely to obtain an oleylamine solution.

[0126] The oxidized dextran solution and oleylamine solution were mixed and sonicated for 5 minutes. Then, 0.096 g of sodium cyanoborohydride (NaCNBH3) was added and shaken at 37°C and 100 rpm for 24 hours. After the reaction, the reaction product was poured into ethanol and allowed to stand for 1 hour. The mixture was centrifuged, the supernatant removed, and the precipitate collected. The precipitate was washed twice with methanol and ultrapure water. The washed solid was freeze-dried to obtain the polymer material ODEX-OA.

[0127] 10 mg of the prepared polymer material ODEX-OA was dissolved in 10 mL of chromatographic methanol, dissolved under ultrasound for 30 min, and concentrated under reduced pressure at 68° C. for 10 min to completely evaporate the organic solvent and form a white film (i.e., a film-like substance) at the bottom of the container.

[0128] Then, 10 mL of ultrapure water at 50° C. was added to the container with the white film, followed by rotary evaporation for 20 min and centrifugation at 3500 rpm for 10 min. The supernatant was taken and filtered through a 0.45 μm microporous filter membrane to obtain a polymer micelle solution.

[0129] The FT-IR results of the polymer material ODEX-OA in Example 1 are shown in Figure 2 Compared with the FT-IR spectrum of ODEX in Figure 1(A), the FT-IR spectrum of ODEX-OA at 2854 cm -1 A very obvious absorption peak can be seen at the ODEX core, which is the symmetrical stretching vibration of the methylene (-CH2-) in the OA long chain, confirming that OA is grafted onto the ODEX skeleton.

[0130] The transmission electron microscopy results of the polymer micelle solution of Example 1 are shown in Figure 3 ,Depend on Figure 3 It can be seen that the polymer micelles are nearly spherical and have good dispersion.

[0131] Example 2 - Preparation of drug-loaded micelle solution and drug-loaded micelle lyophilizate

[0132] 100 mg of oxidized dextran (ODEX-H) prepared in Preparation Example 1 was dissolved in 40 mL of ultrapure water to obtain an oxidized dextran solution. 0.16 g of oleylamine (OA) was dissolved in 2 mL of ethanol and stirred rapidly to completely dissolve the OA to obtain an oleylamine solution.

[0133] The oxidized dextran solution and oleylamine solution were mixed and sonicated for 5 minutes. Then, 0.096 g of sodium cyanoborohydride (NaCNBH3) was added and shaken at 37°C and 100 rpm for 24 hours. After the reaction, the product was poured into ethanol and allowed to stand for 1 hour. The mixture was centrifuged, the supernatant removed, and the precipitate collected. The precipitate was washed twice with methanol and ultrapure water. The washed solid was freeze-dried to obtain the polymer material ODEX-OA.

[0134] 2 mg of prednisolone (PA) was dissolved in 10 mL of chromatographic methanol, and then 10 mg of polymer material ODEX-OA was added and dissolved by ultrasonication for 30 min. The mixture was concentrated under reduced pressure at 68°C for 10 min to completely evaporate the organic solvent and form a white film (i.e., a film-like substance) at the bottom of the container.

[0135] Then, add 10 mL of 50°C ultrapure water to the container with the white film. Rotary evaporate for 20 minutes, then centrifuge at 3500 rpm for 10 minutes. The supernatant is collected and filtered through a 0.45 μm microporous filter to obtain a drug-loaded micelle solution, which can be designated as ODEX-OA-PA.

[0136] The drug-loaded micelle solution was pre-frozen in a -80°C refrigerator, and then freeze-dried in a freeze dryer to obtain a drug-loaded micelle freeze-dried product.

[0137] The transmission electron microscopy results of the drug-loaded micelle solution of Example 2 are shown in Figure 4 .Depend on Figure 4 and Figure 3 By comparison, it can be seen that the particle size distribution of the drug-loaded micelles obtained in Example 2 is more uniform.

[0138] In vitro stability tests of drug-loaded micelles were conducted: The drug-loaded micelle solutions were stored at 4°C and 37°C for 7 days (n=3). Particle size was measured daily using a laser particle size analyzer to investigate the stability of the micelles. The results showed no significant change in particle size over the 7-day period, indicating good stability.

[0139] Comparative Examples 1 to 3

[0140] The only difference from Example 2 is that the organic solvents used in the oleylamine solutions of Comparative Examples 1, 2, and 3 are chloroform, acetone, and tetrahydrofuran, respectively. The micelle particle size and drug loading are shown in Table 1 below.

[0141] Table 1 (n=3)

[0142] serial number Organic solvent used for oleylamine solution Micellar particle size of drug-loaded micelle solution (nm) Drug loading (wt%) Example 2 ethanol 72.13±1.80 11.73±0.46 Comparative Example 1 Chloroform 110.00±2.00 4.87±0.29 Comparative Example 2 acetone 207.87±1.48 1.53±0.33 Comparative Example 3 Tetrahydrofuran 117.70±1.76 5.77±0.20

[0143] As shown in Table 1, using ethanol to dissolve OA produces smaller drug-loaded micelles and a greater drug loading capacity than the other three organic solvents. Furthermore, ethanol is less irritating and safer, making it the preferred organic solvent for dissolving OA. Although ethanol is a commonly used organic solvent, its use as a solvent for the drug-loaded micelles of the present invention to increase drug loading and reduce micelle size is unconventional.

[0144] Comparative Examples 4-5

[0145] The only difference from Example 2 is that the oxidation degree of the oxidized dextran used is different. The oxidized dextran used in Comparative Example 4 is ODEX-M (derived from Comparative Preparation Example 1). The oxidized dextran used in Comparative Example 5 is ODEX-L (derived from Comparative Preparation Example 2). The micelle particle size and drug loading are shown in Table 2 below.

[0146] Table 2 (n=3)

[0147] serial number Oxidized dextran Micellar particle size of drug-loaded micelle solution (nm) Drug loading (wt%) Example 2 From Preparation Example 1 72.13±1.80 11.73±0.46 Comparative Example 4 From Comparative Preparation Example 1 86.02±1.70 2.63±0.26 Comparative Example 5 From Comparative Preparation Example 2 200.61±2.21 3.30±0.35

[0148] As can be seen from the table, the oxidation degree of the oxidized dextran used has a significant impact on the drug loading and micelle particle size, and therefore, this is not a conventional choice in the art.

[0149] Preparation Example 2 - Preparation of mercapto chitosan

[0150] 1 g of chitosan (CS) was mixed with 92 mL of deionized water and stirred to obtain a chitosan solution.

[0151] 788 mg of 1-hydroxybenzotriazole (HOBt) was added to the chitosan solution and stirred until the solution was clear and transparent. 4.5 g of N-acetylcysteine ​​(NAC) was then added and mixed. A 100 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) solution was then added dropwise. After the addition was complete, the pH of the reaction system was adjusted to 5.0 with a 1 M sodium hydroxide solution and maintained constant. The reaction was stirred for 3 h at room temperature in the dark to obtain a reactant.

[0152] The reactants were dialyzed using a dialysis bag with a molecular weight cutoff of 12,000 Da (after boiling water treatment). The dialysis sequence was as follows: (1) dialysis against 5 mmol / L HCl and 1% NaCl for 36 h, with the dialysate replaced every 12 h; (2) dialysis against 1 mmol / L HCl for 36 h, with the dialysate replaced every 12 h. The dialyzed solution was transferred to a 500 mL bottle and freeze-dried at -80°C to obtain thiolated chitosan (CSSH).

[0153] The FT-IR of thiolated chitosan (CSSH) and raw chitosan (CS) obtained in Preparation Example 2 is shown in Figure 5(A). As shown in Figure 5(A), in the infrared absorption spectrum of CS, the wavelengths between 3200 and 3500 cm -1 The broad peak near 2800-2900 cm is the stretching vibration absorption peak of OH and NH in CS molecules. -1 The stretching vibration peaks of the methyl and methine groups on CS are 1620 cm -1 The absorption peak of amide I band (C=O) in CS molecule is 1512 cm -1 The absorption peak of amide II band (NH) is at 1527cm -1 A new absorption peak appeared near the amide bond, which is the characteristic absorption peak of the amide bond, indicating that the carboxyl group of NAC reacted with the amino group of CS. -1 A new absorption peak also appeared at , which was the stretching vibration peak of the thiol group, indicating that the thiol group on NAC was successfully connected to CS through an amide bond.

[0154] The hydrogen spectra of chitosan (CS) and thiochitosan (CSSH) are shown in Figures 5(B) and 5(C), respectively. As shown in Figures 5(B) and 5(C), the chemical shift of the characteristic peak on the sugar ring of CS is between 3.0 and 4.0 ppm. The absorption peak at 2.6 ppm corresponds to the methylene peak, which is enhanced in CSSH compared to CS. The newly appeared peak at 1.8 ppm corresponds to the methyl group in CSSH, confirming the successful synthesis of CSSH.

[0155] Example 3 - Preparation of drug-loaded micelle composite hydrogel

[0156] Dissolve 20 mg of ODEX (ODEX-H prepared in Preparation Example 1) in 0.5 mL of phosphate buffered saline (PBS) (0.01 M, pH 7.2-7.4) and stir until completely dissolved. Centrifuge at 2000 rpm for 15 minutes to remove bubbles, yielding a 40 mg / mL oxidized dextran solution. This is referred to as the ODEX solution.

[0157] 20 mg of CSSH prepared in Preparation Example 2 was dissolved in 0.5 mL of 0.5% acetic acid aqueous solution and stirred evenly. After complete dissolution, the mixture was placed in a high-speed centrifuge and centrifuged at 2000 rpm for 15 min to remove bubbles, thereby obtaining a 40 mg / mL thiol chitosan solution (denoted as CSSH solution).

[0158] The above-mentioned oxidized dextran solution and thiol chitosan solution were mixed in a ratio of 2:1 (specifically, 0.2 mL and 0.1 mL were used, respectively), and then 0.3 mL of the drug-loaded micelle solution prepared according to Example 2 was immediately added and vortexed into a gel on a vortexer. After 30 seconds, the drug-loaded micelle composite hydrogel was obtained. The drug-loaded micelle composite hydrogel had essentially no fluidity. The resulting drug-loaded micelle composite hydrogel was designated ODEX-OA-PA-CSSH.

[0159] Comparative Examples 6 to 9

[0160] The only difference from Example 3 is that the volume ratio of ODEX solution to CSSH solution is different. See Table 3 below.

[0161] Table 3

[0162]

[0163] Note: In Table 3, the concentrations of ODEX solution and CSSH solution are both 40 mg / mL.

[0164] The SEM of the drug-loaded micelle composite hydrogel obtained in Example 3 is shown in FIG. Figure 6 . Figure 6In the figure, (A) SEM results of blank micellar hydrogel ODEX-OA-CSSH (blank hydrogel here refers to blank micellar hydrogel without adding drug PA) (scale bar 500μm), (B) SEM results of drug-loaded micellar composite hydrogel ODEX-OA-PA-CSSH (scale bar 500μm), (C) SEM results of blank micellar hydrogel ODEX-OA-CSSH (scale bar 100μm), (D) SEM results of drug-loaded micellar composite hydrogel ODEX-OA-PA-CSSH (scale bar 100μm). Figure 6 Both hydrogels exhibit numerous pores, likely due to the interpenetrating three-dimensional network structure formed by the interplay of ODEX and CSSH molecules. These large pores are interwoven with numerous tiny interspersed pores. In the ODEX-OA-PA-CSSH, drug-loaded micelles can be observed embedded within the pores of these interpenetrating networks. These results demonstrate that ODEX and CSSH can form hydrogels with interpenetrating three-dimensional networks and encapsulate drugs within them.

[0165] Experimental Example - Study on the in vitro release and anti-inflammatory properties of drug-loaded micelle composite hydrogel 1. Experimental supplies and experimental animals

[0166] Dialysis bags with a molecular weight cutoff of 3400 Da were purchased from Shanghai Green Bird Technology Development Co., Ltd. Dextran sulfate sodium salt (DSS) (MW: 36,000–50,000) was purchased from Yisheng Biotechnology Co., Ltd. Sulfasalazine (SASP), batch number H31020577, was purchased from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd. TNF-α ELISA kits, IL-1β ELISA kits, and IL-10 ELISA kits, size 96T, were all purchased from Solebao Biological Reagent Co., Ltd. Experimental animals: Male Balb / c mice aged 6–8 weeks and weighing 20–22 g were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. and housed at the Experimental Animal Center of Inner Mongolia Medical University. Throughout the experiments, all animals received standardized care and were provided with standard laboratory chow and water, with regular bedding changes. All animal experiments were performed in accordance with the relevant regulations of the Medical Ethics Committee of Inner Mongolia Medical University.

[0167] The drug-loaded micelle composite hydrogels used in this experimental example were all drug-loaded micelle composite hydrogels prepared according to Example 3 (denoted as ODEX-OA-PA-CSSH).

[0168] 2. Experimental methods and results

[0169] 2.1 In vitro drug release from drug-loaded micelle composite hydrogel

[0170] 2.1.1 Experimental methods

[0171] The drug-loaded micellar composite hydrogel was placed in a dialysis bag (molecular weight cutoff of 3400 Da) and released in each of the four environments. The experimental temperature was 37°C and the rotation speed was 100 rpm. The PA concentration was measured by HPLC, and the cumulative drug release rate was calculated according to the following formula.

[0172]

[0173] In the formula, Q represents the cumulative release rate; C n and C n-1 Respectively represent the concentration of the drug in the release medium at the nth and n-1th sampling times (mg / mL); n: represents the number of sampling times; L: represents the amount of drug loaded in the hydrogel (mg).

[0174] 2.1.2 Experimental Results

[0175] The experimental results are shown in Figure 7 .from Figure 7 As can be seen, the cumulative drug release of ODEX-OA-PA-CSSH is related to the pH value of the environment and the presence of ROS. At pH 1.2 (simulated gastric fluid environment), pH 6.8 (simulated small intestine environment), pH 7.4 (simulated colon environment), and pH 7.4 + 0.5mM H2O2 (simulated inflamed colon environment), the cumulative drug release at 16 hours was 30.15%, 63.92%, 73.03%, and 79.08%, respectively. Based on these results, we can reasonably infer that the drug-loaded micelle composite hydrogel delivery system can largely avoid the damage of the acidic environment and fully release PA after delivery to the colon environment, thereby extending the drug delivery time and essentially avoiding drug burst release.

[0176] 2.2 In vitro degradation of drug-loaded micelle hydrogel composite system

[0177] 2.2.1 Experimental methods

[0178] An appropriate amount of drug-loaded micelle composite hydrogel was placed in pH 7.4 and pH 7.4 + 0.5 mM H2O2 solutions, respectively. The temperature was set at (37 ± 0.5) °C. The hydrogel blocks were removed at predetermined time points (0, 3, 6, 9, 12, 24, 36, 48, 72, and 96 h). The excess water on their surfaces was absorbed with filter paper and then weighed on an electronic balance to observe the change in gel weight over degradation time. The solution in the tube was regularly replaced with fresh buffer solution, and the degradation rate was calculated according to the formula:

[0179] Degradation rate (%) = (W0-Wi ) / W0×100%;

[0180] Where W0 is the initial mass of gel (mg), W i is the mass of the gel at the set time point (mg).

[0181] 2.2.2 Experimental results

[0182] The experimental results are shown in Figure 8 .from Figure 8 As can be seen in the figure, ODEX-OA-PA-CSSH loses nearly 80% of its mass after 96 hours of degradation in a buffer medium containing H2O2, and the degradation rate is slightly slower in a buffer medium without H2O2. This indicates that the hydrogel degrades rapidly in a simulated normal intestinal environment and a colon environment containing excessive ROS, and exhibits ROS-responsive degradation. The stimulus-responsive degradation behavior is consistent with drug release.

[0183] 2.3 Experimental results on mice with inflammatory bowel disease (IBD)

[0184] 2.3.1 Experimental methods

[0185] Establishment of an Inflammatory Bowel Disease (IBD) Mouse Model: Male Balb / c mice aged 6-8 weeks were fed an 8% (w / v) DSS solution in place of their normal drinking water for 7 days to establish the IBD model. The DSS solution was replaced every two days.

[0186] Mice were divided into the following groups: control group (Control), model group (Model), positive drug group (SASP), drug-loaded micelle composite hydrogel group (ODEX-OA-PA-CSSH), drug-loaded micelle group (ODEX-OA-PA), and blank micelle hydrogel group (ODEX-OA-CSSH). The dosing schedule for each group is shown in Table 4 below. Mice were given the appropriate treatments, and their body weight and fecal occult blood were monitored daily. After treatment, the colons of the mice were harvested for HE staining to observe colonic recovery.

[0187] Table 4

[0188]

[0189] 2.3.2 Experimental Results

[0190] (1) Changes in body weight and DAI scores Figure 9 , Figure 9In the figure, A is the DAI score, and B is the result of the change in body weight. The colon HE staining section is shown in Figure 10 (A), and the colon histopathological score is shown in Figure 10 (B). In Figure 10 (B), lowercase letters a to c represent significant differences between different groups. They are arranged in alphabetical order from large to small. P < 0.05 between adjacent letters indicates significant differences. The effect of ODEX-OA-PA-CSSH on the symptoms of IBD mice was evaluated by changes in mouse weight, stool characteristics, and blood in stool. On the 7th day after free drinking of DSS solution, the weight of mice in each group began to decrease significantly, except for the Control group, and the weight loss of mice in the Model group was the most significant ( Figure 9 A), after stopping DSS stimulation on the 9th day, the weight loss symptoms of mice in each group were alleviated. After starting treatment on the 7th day, the weight of mice in the SASP and ODEX-PA-OA-CSSH groups began to recover, and in the later stage of treatment, the weight of mice returned to normal. In addition, through the evaluation of fecal characteristics, we found that the feces of mice in the Model group were unformed and accompanied by visible blood in the stool, while the feces of mice in the ODEX-PA-OA-CSSH group were almost all formed, and the fecal occult blood test results were negative, which significantly reduced the DAI score ( Figure 9 B), improved the symptoms of IBD to a certain extent.

[0191] (2) The results of inflammatory cytokine levels in colon tissue of mice in different treatment groups are shown in Figure 11 . Figure 11 Middle, A: TNF-α; B: IL-1β; C: IL-10. Figure 11 In the figure, lowercase letters a to e indicate significant differences between different groups. They are arranged in alphabetical order from large to small. P<0.05 between adjacent letters indicates significant differences. Compared with the Control group, after DSS induction, the proinflammatory factors TNF-α and IL-1β in the colon tissue of mice were significantly increased (P<0.05). After ODEX-OA-PA-CSSH treatment, the two proinflammatory factors were significantly downregulated, and there was no significant difference compared with the Control group and the SASP group. In addition, the levels of proinflammatory factors in the ODEX-OA-PA group and the ODEX-OA-CSSH group were also increased compared with the Model group. On the contrary, DSS treatment led to a significant decrease in the level of the anti-inflammatory factor IL-10 in the colon tissue of mice. Figure 11 As shown in Figure C, IL-10 levels increased significantly after treatment with SASP and ODEX-OA-PA-CSSH. This suggests that ODEX-OA-PA-CSSH can regulate inflammatory factor levels, inhibit the secretion of pro-inflammatory factors, and increase the concentration of anti-inflammatory factors, demonstrating a favorable anti-inflammatory effect.

[0192] The present invention is not limited to the above-mentioned embodiments. Any modification, improvement, or substitution that can be conceived by those skilled in the art without departing from the essential content of the present invention shall fall within the scope of the present invention.

Claims

1. A polymer material, characterized in that The invention is obtained by reacting oxidized dextran and oleylamine; wherein the oxidized dextran is obtained by oxidizing dextran with a molecular weight of 35,000 to 45,000; the oxidation degree of the oxidized dextran is 55 to 70%; and the mass ratio of the oxidized dextran to oleylamine is 1:1.2 to 2.

0.

2. The method for preparing a polymer material according to claim 1, wherein: The following steps are involved: 1) dissolving oxidized dextran in water to obtain an oxidized dextran solution; dissolving oleylamine in a C1-C3 alkyl alcohol to obtain an oleylamine solution; 2) mixing the oxidized dextran solution and the oleylamine solution, then adding a boride reducing agent, reacting at 25-50° C., and after the reaction is completed, separating the reaction product into a solid and a liquid, washing the separated solid, and freeze-drying the washed solid to obtain the polymer material.

3. A drug-loaded micelle solution, characterized in that: The invention is prepared from raw materials including the polymer material according to claim 1 and prednisolone, wherein the drug loading amount of the lyophilized product of the drug-loaded micelle solution is above 10wt%, and the encapsulation efficiency is above 79wt%. The drug-loaded micelles have a nearly spherical structure and an average particle size of 70 to 85nm.

4. The method for preparing the drug-loaded micelle solution according to claim 3, wherein: The following steps are involved: A) mixing prednisolone, the polymer material according to claim 1 and a C1-C3 alkyl alcohol, and then concentrating under reduced pressure to obtain a film-like substance; B) placing the film-like substance in water, and then subjecting the film-like substance to rotary evaporation to form a micelle solution, followed by centrifugation, and passing the obtained supernatant through a microporous filter membrane to obtain a drug-loaded micelle solution.

5. A drug-loaded micelle composite hydrogel, characterized in that: The invention is formed by oxidized dextran solution, thiol chitosan solution and the drug-loaded micelle solution according to claim 3; wherein the mass concentration ratio of the oxidized dextran solution to the thiol chitosan solution is 1:1-1.2, and the volume ratio of the oxidized dextran solution to the thiol chitosan solution is 1.8-2.2:

1.

6. The method for preparing the drug-loaded micelle composite hydrogel according to claim 5, characterized in that: The following steps are involved: The oxidized dextran solution and the mercapto chitosan solution are mixed, and then the drug-loaded micelle solution according to claim 3 is added and mixed into a gel to obtain the drug-loaded micelle composite hydrogel.

7. The preparation method according to claim 6, characterized in that The mass concentration of the oxidized dextran solution is 20-100 mg / mL, the mass concentration of the thiol chitosan solution is 20-100 mg / mL; the volume ratio of the oxidized dextran solution to the drug-loaded micelle solution is 2-2.5:

3.

8. The preparation method according to claim 7, characterized in that The oxidized dextran used to prepare the polymer material and the oxidized dextran in the oxidized dextran solution are both prepared by the following steps: (1) adding sodium periodate to the dextran solution, reacting for 3 to 5 hours in the dark, and then adding glycerol to terminate the reaction to obtain a reaction solution; (2) The reaction solution was transferred to a dialysis bag with a molecular weight cut-off of 3400 Da for dialysis, and the dialyzate was freeze-dried to obtain oxidized dextran.

9. The preparation method according to claim 7, characterized in that The mercapto chitosan is prepared by the following steps: (a) dispersing chitosan in water to obtain a chitosan solution; (b) adding 1-hydroxybenzotriazole to the chitosan solution and mixing to obtain a mixed solution; then adding N-acetylcysteine ​​to the mixed solution, and then dropwise adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and adjusting the pH value of the reaction system to 5.0-5.5, and reacting in the dark for 2.5-3.5 hours to obtain a reactant; (c) dialyzing the reactant using a dialysis bag with a molecular weight cut-off of 12000 Da, and freeze-drying the dialyzate to obtain the thiol chitosan.

10. Use of the drug-loaded micelle composite hydrogel according to claim 5 in preparing a medicament for treating inflammatory bowel disease.

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