Polymeric materials, drug-loaded nanoparticle solutions, drug-loaded nanoparticle composite hydrogels, and methods of making and uses thereof

Abstract

The application discloses a polymer material, a drug-loaded nanoparticle solution, a drug-loaded nanoparticle composite hydrogel and a preparation method and application thereof. The polymer material is prepared by connecting pectin with baicalin or a derivative thereof through cystamine; two amino groups of the cystamine are respectively connected with carboxyl groups on the pectin and the baicalin or the derivative thereof through an amidation reaction to form amides; and the chemical structure of the baicalin or the derivative thereof is shown as formula (I). The polymer material can be combined with a drug for treating inflammatory bowel disease to form drug-loaded nanoparticles, and the drug-loaded nanoparticles have high drug loading and high encapsulation efficiency. The drug-loaded nanoparticle composite hydrogel prepared from the drug-loaded nanoparticles can obviously improve the symptoms of the inflammatory bowel disease.

Description

Technical Field

[0001] This invention relates to a polymer material, a drug-loaded nanoparticle solution, a drug-loaded nanoparticle composite hydrogel, and their preparation methods and uses. Background Technology

[0002] Inflammatory bowel disease (IBD) is a persistent and complex disease, primarily including Crohn's disease and ulcerative colitis. Its pathological features include persistent inflammation of the colonic mucosa and impaired intestinal barrier function, often accompanied by symptoms such as diarrhea, abdominal pain, weight loss, and even rectal bleeding. Due to its chronic nature and rising global prevalence, it places a heavy burden on healthcare systems. Currently, clinical medications for IBD include glucocorticoids, aminosalicylic acid, immunosuppressants, and biologics; however, these drugs generally suffer from instability in the gastrointestinal tract, low delivery efficiency, and a tendency to cause serious side effects in non-target tissues. Therefore, developing safe and effective novel drug delivery systems is of great value.

[0003] Oral colon-targeted drug delivery systems enable precise drug delivery to the colon. Due to their ability to increase local drug concentration, reduce systemic toxicity and dosage, and prolong retention time, they have become an important strategy for treating intestinal diseases. Based on the physiological characteristics of the colon, including pH, redox conditions, contents retention time, and gut microbiota, various colon-targeted drug delivery systems have been developed, including pH-sensitive, reactive oxygen species-responsive, time-dependent bioadhesion, and enzyme-triggered systems.

[0004] CN115806554A discloses a colon-targeted prodrug. This colon-targeted prodrug binds the drug to a targeting system with 5-aminosalicylic acid as the target, aiming to increase drug exposure in the intestine and reduce systemic absorption through this targeting system, thereby reducing drug-related side effects caused by long-term use. However, this colon-targeted prodrug only chemically links the drug to the organic compound serving as the targeting system, lacking protection for the drug. This makes the drug susceptible to structural damage during oral delivery due to the complex gastrointestinal environment, leading to premature drug leakage. Furthermore, the drug's retention time in the intestine is limited, making it difficult to maintain an effective therapeutic concentration.

[0005] Given that the targeting effect of a single drug delivery system is easily affected by fluctuations in the gastrointestinal environment, combining different drug delivery mechanisms with the same delivery system can combine the advantages of different strategies and achieve more efficient and accurate drug delivery under complex physiological conditions.

[0006] CN120535670A discloses a drug-loaded micelle composite hydrogel. This drug-loaded micelle composite hydrogel is formed from an oxidized dextran solution, a thiol-chitosan solution, and a drug-loaded micelle solution. The drug-loaded micelle solution is prepared from raw materials including a polymer material and prednisolone. The polymer material is obtained by reacting oxidized dextran with oleylamine. This drug-loaded micelle composite hydrogel can largely avoid the degradation caused by acidic environments and prolong the drug delivery time. However, the drug loading capacity of this drug-loaded micelle composite hydrogel is relatively low.

[0007] To date, there have been no reports on drug-loaded nanosols formed from pectin, cystamine, baicalin or their derivatives, or drugs, nor have there been reports on drug-loaded nanoparticle composite hydrogels containing these raw materials, their preparation methods, or their use in the preparation of drugs for treating inflammatory bowel disease. Summary of the Invention

[0008] In view of this, one object of the present invention is to provide a polymer material that can form drug-loaded nanoparticles with a high drug loading capacity with a drug. Another object of the present invention is to provide a method for preparing the above-mentioned polymer material. A further object of the present invention is to provide a drug-loaded nanoparticle solution. A further object of the present invention is to provide a method for preparing the above-mentioned drug-loaded nanoparticle solution. Yet another object of the present invention is to provide a drug-loaded nanoparticle composite hydrogel that can effectively resist the gastric acid environment, achieve drug accumulation and sustained release in the colon, and improve the symptoms of inflammatory bowel disease. Another object of the present invention is to provide a method for preparing the above-mentioned drug-loaded nanoparticle composite hydrogel. A final object of the present invention is to provide an application of the above-mentioned drug-loaded nanoparticle composite hydrogel.

[0009] The present invention achieves the above objectives using the following technical solutions.

[0010] On one hand, the present invention provides a polymer material formed by linking pectin with cystamine and baicalin or its derivatives; wherein the two amino groups of cystamine react with the carboxyl groups on pectin and baicalin or its derivatives respectively through an amidation reaction to form amides; the chemical structure of baicalin or its derivatives is shown in formula (I): (I) R is selected from hydrogen or C1 to C6 alkyl groups.

[0011] On the other hand, the present invention also provides a method for preparing the above-mentioned polymer material, which includes the following steps: 1) Pectin and cystamine dihydrochloride are subjected to an amidation reaction under a catalyst and at 20-40°C to obtain reaction solution I; wherein the weight ratio of pectin to cystamine dihydrochloride is 1:1-5; 2) Place reaction solution I in a dialysis bag, place the dialysis bag in water for the first dialysis purification, and obtain purified reaction solution I; freeze-dry purified reaction solution I to obtain polymer material intermediate; 3) The polymer material intermediate is subjected to an amidation reaction with baicalin or its derivative under a catalyst and at 20-40°C to obtain reaction solution II; wherein the weight ratio of the polymer material intermediate to baicalin or its derivative is 1:1-5. 4) Place reaction solution II in a dialysis bag, and place the dialysis bag in an aqueous solution of C1-C5 alkyl alcohols for a second dialysis purification to obtain purified reaction solution II; freeze-dry purified reaction solution II to obtain polymer material.

[0012] Another object of the present invention is to provide a drug-loaded nanoparticle solution, which is prepared from raw materials including the above-mentioned polymer material and a pharmaceutical active ingredient; wherein the above-mentioned polymer material encapsulates the pharmaceutical active ingredient to form drug-loaded nanoparticles; the weight ratio of the pharmaceutical active ingredient to the above-mentioned polymer material is 1:1.5 to 4.5; the pharmaceutical active ingredient is a pharmaceutical active ingredient for treating inflammatory bowel disease.

[0013] Another object of the present invention is to provide a method for preparing the above-mentioned drug-loaded nanoparticle solution, which includes the following steps: A) The above polymer material is mixed with C1-C5 alkyl alcohols to obtain an alcohol solution of the polymer material. Then, the active pharmaceutical ingredient is added to the alcohol solution of the polymer material to obtain an alcohol solution of the mixture. B) The alcohol solution of the mixture was rotary evaporated under reduced pressure at a speed of 85-110 rpm to obtain a thin film. C) Place the film in water, sonicate for 35-55 min, then centrifuge, take the supernatant and filter it through a microporous membrane, take the filtrate to obtain a drug-loaded nanoparticle solution; wherein, based on 1 mg of the polymer material described in claim 1, the amount of water used is 1-3 mL.

[0014] Another object of the present invention is to provide a drug-loaded nanoparticle composite hydrogel, wherein the drug-loaded nanoparticle composite hydrogel is prepared from raw materials including the above-mentioned drug-loaded nanoparticle solution, aldehyde polysaccharide solution and amino polysaccharide solution; The concentration of the aldehyde polysaccharide solution is 10-50 mg / mL; the aldehyde polysaccharide is selected from at least one of oxidized pectin, oxidized dextran, oxidized sodium alginate, oxidized sodium hyaluronate, oxidized chitosan and its derivatives, and aldehyde cellulose and its derivatives. The concentration of the aminopolysaccharide solution is 10–50 mg / mL; the aminopolysaccharide is selected from at least one of carboxymethyl chitosan, carboxyethyl chitosan, carboxypropyl chitosan, carboxybutyl chitosan, aminated chitosan, and aminated mannose. The volume ratio of the drug-loaded nanoparticle solution to the aldehyde polysaccharide solution is 1:3 to 10; the volume ratio of the aldehyde polysaccharide solution to the amino polysaccharide solution is 4 to 10:1.

[0015] Another object of the present invention is to provide a method for preparing the above-mentioned drug-loaded nanoparticle composite hydrogel, which includes the following steps: I) The above drug-loaded nanoparticle solution was mixed with the aldehyde polysaccharide solution to obtain a mixture; II) Mix the aminopolysaccharide solution with the mixture to allow the aldehyde polysaccharide and aminopolysaccharide to undergo a Schiff base reaction, thereby obtaining a drug-loaded nanoparticle composite hydrogel.

[0016] According to the preparation method of the present invention, preferably, the aldehyde polysaccharide in the aldehyde polysaccharide solution is obtained by oxidation of the precursor polysaccharide with an alkali metal perhalate.

[0017] According to the preparation method of the present invention, preferably, the alkali metal perhalate is selected from at least one of alkali metal perchlorate, perbromate, and periodate; and the alkali metal is selected from at least one of Li, Na, and K.

[0018] According to the preparation method of the present invention, preferably, the preparation method of the aldehyde polysaccharide includes the following steps: a) Add the precursor polysaccharide to water and stir to obtain a precursor polysaccharide dispersion; wherein, based on 1g of precursor polysaccharide, the amount of water used is 20-100mL. b) Add the alkali metal perhalate to the precursor polysaccharide dispersion under light-protected conditions, and react under light-protected conditions and at 20-40°C to obtain reaction solution A; then, add C2-C5 alkyldiol to reaction solution A to obtain reaction solution B; wherein, based on 1g of precursor polysaccharide, the amount of C2-C5 alkyldiol is 1-5mL. c) Place reaction solution B in a dialysis bag, then place the dialysis bag in water for dialysis purification to obtain purified reaction solution B; freeze-dry purified reaction solution B to obtain aldehyde polysaccharide.

[0019] Another object of the present invention is to provide the use of the above-mentioned drug-loaded nanoparticle composite hydrogel in the preparation of a medicament for treating inflammatory bowel disease.

[0020] The polymer material of this invention can form drug-loaded nanoparticles with drugs for treating inflammatory bowel disease, especially prednisolone. These drug-loaded nanoparticles have high drug loading and encapsulation efficiency. The drug-loaded nanoparticle composite hydrogel of this invention has good biocompatibility, can effectively resist the acidic environment of the stomach, and can achieve drug accumulation and sustained release in the colon, thus significantly improving the symptoms of inflammatory bowel disease. Attached Figure Description

[0022] Figure 1 Figure A shows the particle size distribution and zeta potential of the drug-loaded nanoparticles prepared in Example 1; where Figure A is the particle size distribution diagram and Figure B is the zeta potential diagram.

[0023] Figure 2 The image shows the zeta potential of the drug-loaded nanoparticles prepared in Example 1 under different conditions.

[0024] Figure 3 The image shows a SEM image of the drug-loaded nanoparticle composite hydrogel prepared in Example 2; the left image is magnified 100 times and the right image is magnified 500 times.

[0025] Figure 4 This is the in vitro drug release curve from Experiment Example 4.

[0026] Figure 5 The degradation curves are shown in different pH media in Experiment Example 4.

[0027] Figure 6 The DPPH scavenging rate is shown for the drug-loaded nanoparticle composite hydrogel in Experiment Example 4.

[0028] Figure 7 This is a live fluorescence image from Experiment Example 4.

[0029] Figure 8 This is the result of in vivo fluorescence quantification in Experiment Example 4.

[0030] Figure 9 The image shows the fluorescence of the isolated gastrointestinal tract in Experiment Example 4.

[0031] Figure 10 The results of fluorescence quantification of the isolated gastrointestinal tract in Experiment Example 4 are shown.

[0032] Figure 11 This is an HE-stained section of the mouse colon tissue from Experiment Example 5.

[0033] Figure 12 The figures show the levels of inflammatory factors in the colon tissue of mice in Experiment 5; where A represents the level of TNF-α, B represents the level of IL-6, and C represents the level of IL-10.

[0034] Figure 13 Figure A shows the oxidative stress level of the mouse colon tissue in Experiment 5; where Figure A represents the MPO level, Figure B represents the MDA level, and Figure C represents the SOD level.

[0035] Figure 14 This is an immunohistochemical image of the mouse colon in Experiment Example 5.

[0036] Figure 15The figures show the percentage of positive cells in the immunohistochemical images of the mouse colon in Experiment Example 5; Figure A shows the statistical results of Occludin protein, and Figure B shows the statistical results of ZO-1 protein.

[0037] Figure 16 The graph shows the weight gain curve of mice after 7 days of gavage administration of drug-loaded nanoparticle composite hydrogel in Experiment Example 5.

[0038] Figure 17 The images show HE-stained sections of various organs from the mouse in Experiment Example 5. Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0041] ​​ <Polymer Materials> The polymer material provided by this invention is formed by linking pectin with cysteine ​​and baicalin or its derivatives.

[0042] According to one embodiment of the present invention, the two amino groups of cystamine react with the carboxyl groups on pectin and baicalin or their derivatives to form amides via amidation reactions.

[0043] In this invention, the chemical structure of baicalin or its derivatives is shown in formula (I): (I) R can be selected from hydrogen or C1-C6 alkyl groups, preferably hydrogen or C1-C5 n-alkyl groups, and more preferably hydrogen or C1-C3 n-alkyl groups.

[0044] In this invention, the C1 to C6 alkyl groups may include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, methylbutyl, ethylpropyl, hexyl, methylpentyl, dimethylbutyl, and ethylbutylene.

[0045] According to a specific embodiment of the present invention, a polymer material is prepared using baicalin. Baicalin has the following structure: .

[0046] The polymer material of the present invention can form drug-loaded nanoparticles with drugs, which have high drug loading and encapsulation efficiency. According to one embodiment of the present invention, the drug loading of the drug-loaded nanoparticles can be at least 29 wt%, preferably at least 29.5 wt%, and more preferably at least 30 wt%. The encapsulation efficiency of the drug-loaded nanoparticles can be at least 90 wt%, preferably at least 90.5 wt%, and more preferably at least 91 wt%.

[0047] Theoretically, this invention can form drug-loaded nanoparticles with any active pharmaceutical ingredient, and is particularly suitable for active pharmaceutical ingredients used to treat inflammatory bowel disease, such as prednisolone.

[0048] <Preparation Methods of Polymer Materials> The present invention also provides a method for preparing the above-mentioned polymeric material, comprising a first amidation step, a first purification step, a second amidation step, and a second purification step. These are described in detail below.

[0049] First amidation step Pectin and cystamine dihydrochloride were subjected to an amidation reaction under a catalyst and at 20–40 °C to obtain reaction solution I.

[0050] According to one embodiment of the present invention, the weight ratio of pectin to cystamine dihydrochloride can be 1:1 to 5, preferably 1:1 to 4, and more preferably 1:1 to 3.

[0051] In this invention, the catalyst can be any catalyst known in the art capable of catalyzing amidation reactions. According to a preferred embodiment of the invention, the catalyst can be a combination of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide). The mass ratio of EDC to NHS can be 1–3:1, preferably 1–2.5:1, more preferably 1–2:1. The mass ratio of EDC to pectin can be 1–3:1, preferably 1–2.5:1, more preferably 1–2:1.

[0052] According to one embodiment of the present invention, the reaction temperature can be 20–40°C, preferably 22–38°C, more preferably 24–35°C. The reaction time can be 12–36 h, preferably 15–32 h, more preferably 18–28 h.

[0053] According to a preferred embodiment of the invention, the reaction can be carried out under light-protected and stirred conditions. This promotes the reaction while preventing the generation of impurities through photochemical reactions between the catalyst and intermediate products.

[0054] According to one embodiment of the present invention, pectin and a catalyst can be dissolved in water first, stirred under light-protected conditions, and then cystamine dihydrochloride can be added to the solution to carry out an amidation reaction. This can fully activate the carboxyl groups of the pectin molecular chain, which is beneficial for the amidation reaction with cystamine. According to a preferred embodiment of the present invention, based on 1g of pectin, the amount of water used can be 30-100mL, preferably 40-90mL, more preferably 45-80mL. The stirring time can be 6-24h, preferably 8-20h, more preferably 10-18h.

[0055] Limiting the reaction conditions to the above range is beneficial for the amidation reaction between cystamine and the carboxyl groups of pectin to form intermediate products, thereby increasing the yield of intermediate products and facilitating the subsequent second amidation reaction. This is also more conducive to increasing the drug loading and encapsulation efficiency of the polymer material.

[0056] In this invention, the water used in each step can be at least one of deionized water, ultrapure water, and double-distilled water; preferably at least one of deionized water and ultrapure water, more preferably deionized water or ultrapure water.

[0057] First purification step The reaction solution I was placed in a dialysis bag, and the dialysis bag was placed in water for the first dialysis purification to obtain purified reaction solution I; the purified reaction solution I was freeze-dried to obtain the polymer material intermediate.

[0058] According to one embodiment of the present invention, the molecular weight cutoff of the dialysis bag can be 7000-15000 Da, preferably 7500-14500 Da, and more preferably 8000-14000 Da. The first dialysis purification time can be 24-120 h, preferably 36-96 h, and more preferably 48-84 h. According to a preferred embodiment of the present invention, the water can be changed 2-5 times every 24 h, preferably 2-4 times, and more preferably 3-4 times. Such dialysis conditions can effectively remove unreacted cystamine dihydrochloride, EDC, NHS, and other small molecule byproducts, improving the purity of polymer material intermediates.

[0059] In this invention, freeze-drying can be achieved using any freeze-drying apparatus known in the art, without particular limitation. The freeze-drying temperature can be -120 to -18°C, preferably -100 to -20°C, and more preferably -90 to -50°C. The freeze-drying time can be 12 to 60 hours, preferably 18 to 54 hours, and more preferably 24 to 48 hours.

[0060] Second amidation step The polymer material intermediate is subjected to an amidation reaction with baicalin or its derivative under a catalyst and at 20–40 °C to obtain reaction solution II.

[0061] According to one embodiment of the present invention, the weight ratio of the polymer material intermediate to baicalin or its derivative can be 1:1 to 5, preferably 1:1 to 3.5, and more preferably 1:1 to 2.

[0062] In this invention, the catalyst can be any catalyst known in the art capable of catalyzing amidation reactions. According to a preferred embodiment of the invention, the catalyst can be a combination of EDC and NHS. The molar ratio of EDC to NHS can be 1–3:1, preferably 1–2.5:1, more preferably 1–2:1. The molar ratio of EDC to baicalin or its derivatives can be 1–5:1, preferably 1.5–4:1, more preferably 2–3:1.

[0063] According to one embodiment of the present invention, the reaction temperature can be 20–40°C, preferably 22–38°C, more preferably 24–35°C. The reaction time can be 12–36 h, preferably 15–32 h, more preferably 18–28 h. Continuous stirring can be performed during the reaction to improve reaction uniformity and reaction rate.

[0064] According to one embodiment of the present invention, a polymer material intermediate can be first added to solvent A1, followed by dilution with solvent A2 to obtain solution A. Baicalin or its derivatives and a catalyst are then added to solvent B to obtain solution B. Solution A and solution B are then mixed to carry out an amidation reaction to obtain reaction solution II. This allows the reactants to be more uniformly distributed within the reaction system, which is more conducive to the amidation reaction.

[0065] According to a preferred embodiment of the present invention, based on 1g of polymer material intermediate, the amount of solvent A1 can be 20-100mL, preferably 30-85mL, and more preferably 40-80mL. Solvent A1 can be at least one of formamide, dimethylformamide, dimethylacetamide, and N-methylpyrrolidone, preferably at least one of formamide, dimethylformamide, and dimethylacetamide, and more preferably at least one of formamide and dimethylformamide. Based on 1g of polymer material intermediate, the amount of solvent A2 can be 10-100mL, preferably 15-80mL, and more preferably 20-75mL. Solvent A2 can be at least one of dimethyl sulfoxide, tetrahydrofuran, acetone, dichloromethane, phenol, and acetonitrile, preferably at least one of dimethyl sulfoxide, tetrahydrofuran, acetone, and dichloromethane, and more preferably at least one of dimethyl sulfoxide, tetrahydrofuran, and dichloromethane.

[0066] Based on 1g of baicalin or its derivatives, the amount of solvent B can be 20-100mL, preferably 30-85mL, and more preferably 40-80mL. Solvent B can be at least one of dimethyl sulfoxide, tetrahydrofuran, acetone, dichloromethane, phenol, and acetonitrile, preferably at least one of dimethyl sulfoxide, tetrahydrofuran, acetone, and dichloromethane, and more preferably at least one of dimethyl sulfoxide, tetrahydrofuran, and dichloromethane. When preparing solution B, stirring can be performed to accelerate the dissolution of baicalin or its derivatives. The stirring time can be 15-60min, preferably 20-50min, and more preferably 25-40min.

[0067] Limiting the reaction conditions to the above range is beneficial for the amino groups in the polymer intermediate to undergo amidation reaction with the carboxyl groups in baicalin or its derivatives to form polymer materials, thereby increasing the yield of polymer materials and further improving the drug loading and encapsulation efficiency of polymer materials.

[0068] Second purification step The reaction solution II was placed in a dialysis bag, and the dialysis bag was placed in an aqueous solution of C1-C5 alkyl alcohols for a second dialysis purification to obtain purified reaction solution II; the purified reaction solution II was freeze-dried to obtain the polymer material.

[0069] In this invention, the C1-C5 alkyl alcohols can be C1-C4 alkyl alcohols, preferably C1-C3 alkyl alcohols, and more preferably at least one of methanol, ethanol, and isopropanol. According to a preferred embodiment of the invention, the volume concentration of the aqueous solution of the C1-C5 alkyl alcohols can be 1-5 vol%, preferably 1.5-4.5 vol%, and more preferably 2-4 vol%.

[0070] According to one embodiment of the present invention, the molecular weight cutoff of the dialysis bag can be 7000-15000 Da, preferably 7500-14500 Da, more preferably 8000-14000 Da. The second dialysis purification time can be 24-120 h, preferably 36-96 h, more preferably 48-84 h. According to a preferred embodiment of the present invention, the water can be changed 2-5 times every 24 h, preferably 2-4 times, and more preferably 3-4 times. Such dialysis conditions can effectively remove unreacted baicalin or its derivatives, EDC, NHS and other small molecule byproducts, improving the purity of the polymer material.

[0071] Freeze-drying is the same as the first purification step described above, and will not be repeated here.

[0072] <Drug-loaded nanoparticle solution> The present invention also provides a drug-loaded nanoparticle solution, which is prepared from raw materials including the above-mentioned polymer material and the active pharmaceutical ingredient.

[0073] According to one embodiment of the present invention, a polymer material encapsulates a drug active ingredient to form drug-loaded nanoparticles. The weight ratio of the drug active ingredient to the polymer material can be 1:1.5 to 4.5, preferably 1:1.8 to 4.2, and more preferably 1:2 to 4.

[0074] In this invention, the active pharmaceutical ingredient is preferably an active pharmaceutical ingredient for treating inflammatory bowel disease. For example, it may be selected from at least one of glucocorticoid drugs (such as prednisolone), monoclonal antibodies against inflammatory bowel disease (such as infliximab, adalimumab, vedozizumab, etc.), aminosalicylic acid preparations (such as mesalazine, etc.), and immunosuppressants (such as azathioprine, etc.), preferably at least one of glucocorticoid drugs and aminosalicylic acid preparations.

[0075] In this invention, the absolute value of the Zeta potential of the drug-loaded nanoparticles can be at least 50 mV, preferably at least 51 mV, and more preferably at least 52 mV. Such drug-loaded nanoparticles exhibit strong anti-aggregation properties, good dispersibility, and high stability; they also have high drug loading and encapsulation efficiency.

[0076] The drug loading of the drug-loaded nanoparticles can be at least 29 wt%, preferably at least 29.5 wt%, and more preferably at least 30 wt%. The encapsulation efficiency of the drug-loaded nanoparticles can be at least 90 wt%, preferably at least 90.5 wt%, and more preferably at least 91 wt%. Such drug-loaded nanoparticles have both high drug loading and encapsulation efficiency, making them very suitable for preparing drugs to treat inflammatory bowel disease.

[0077] <Preparation method of drug-loaded nanoparticle solution> This invention also provides a method for preparing the above-mentioned drug-loaded nanoparticle solution, comprising a raw material mixing step, a vacuum rotary evaporation step, and a hydration ultrasonic filtration step. This is described in detail below.

[0078] Raw material mixing steps The polymer material is mixed with C1-C5 alkyl alcohols to obtain an alcohol solution of the polymer material. Then, the active pharmaceutical ingredient is added to the alcohol solution of the polymer material to obtain an alcohol solution of the mixture.

[0079] In this invention, the C1-C5 alkyl alcohols can be C1-C4 alkyl alcohols, preferably C1-C3 alkyl alcohols, and more preferably at least one of methanol, ethanol, and isopropanol.

[0080] According to one embodiment of the present invention, based on 1 mg of polymer material, the amount of C1 to C5 alkyl alcohol can be 0.5 to 2 mL, preferably 0.75 to 1.8 mL, and more preferably 0.8 to 1.5 mL.

[0081] In this invention, after the polymer material is mixed with C1-C5 alkyl alcohols, ultrasonic treatment can be used to promote the uniform dispersion of the polymer material in the C1-C5 alkyl alcohols. The power of the ultrasonic treatment can be 100-500W, preferably 150-400W, more preferably 200-300W. The ultrasonic treatment time can be 2-20 minutes, preferably 5-18 minutes, more preferably 8-15 minutes.

[0082] In this invention, the active pharmaceutical ingredient and the dosage relationship between the active pharmaceutical ingredient and the polymer material are the same as those described in the aforementioned drug-loaded nanoparticle solution, and will not be repeated here.

[0083] Reduced pressure rotary evaporation steps The alcohol solution of the mixture was subjected to rotary evaporation under reduced pressure at a speed of 85–110 rpm to obtain a thin film.

[0084] According to one embodiment of the present invention, the rotational speed of the vacuum rotary evaporation can be 85-110 rpm, preferably 86-106 rpm, and more preferably 88-105 rpm. The temperature of the vacuum rotary evaporation can be 58-80°C, preferably 60-75°C, and more preferably 60-70°C. Such vacuum rotary evaporation conditions are beneficial for a more uniform film and for improving the drug loading and encapsulation efficiency of the subsequently prepared drug-loaded nanoparticle solution.

[0085] In this invention, vacuum rotary evaporation can be implemented using any type of vacuum rotary evaporator known in the art, without any particular limitation.

[0086] Hydration ultrasonic filtration steps The film was placed in water and sonicated for 35–55 minutes. Then it was centrifuged, and the supernatant was filtered through a microporous membrane. The filtrate was collected to obtain a drug-loaded nanoparticle solution.

[0087] According to one embodiment of the present invention, based on 1 mg of the polymer material described in claim 1, the amount of water used can be 1 to 3 mL, preferably 1.2 to 2.5 mL, and more preferably 1.3 to 2 mL. Such an amount is beneficial for more uniform hydration of the film material and for improving the drug loading and encapsulation efficiency of the drug-loaded nanoparticle solution.

[0088] According to one embodiment of the present invention, the ultrasonic treatment time can be 30-60 min, preferably 35-55 min, and more preferably 40-50 min. Such ultrasonic conditions are more conducive to improving the drug loading and encapsulation efficiency of the drug-loaded nanoparticle solution.

[0089] According to one embodiment of the present invention, the centrifugation speed can be 2000-5000 rpm, preferably 2500-4500 rpm, more preferably 3000-4000 rpm. The centrifugation time can be 2-30 min, preferably 5-25 min, more preferably 8-20 min. Such centrifugation conditions are more conducive to the enrichment of drug-loaded nanoparticles in the supernatant.

[0090] According to one embodiment of the present invention, the pore size of the microporous filter membrane can be 0.2–0.6 μm, preferably 0.22–0.55 μm, and more preferably 0.3–0.5 μm. Such a microporous filter membrane can effectively filter impurities and ensure the high purity of the drug-loaded nanoparticle solution.

[0091] <Drug-loaded nanoparticle composite hydrogel> The present invention also provides a drug-loaded nanoparticle composite hydrogel, which is prepared from raw materials including the above-mentioned drug-loaded nanoparticle solution, aldehyde polysaccharide solution and amino polysaccharide solution.

[0092] According to one embodiment of the present invention, the aldehyde polysaccharide may be selected from at least one of oxidized pectin, oxidized dextran, oxidized sodium alginate, oxidized sodium hyaluronate, oxidized chitosan and its derivatives, and aldehyde cellulose and its derivatives, preferably at least one of oxidized pectin, oxidized dextran, oxidized sodium alginate, oxidized sodium hyaluronate, and oxidized chitosan, more preferably at least one of oxidized pectin, oxidized dextran, oxidized sodium alginate, and oxidized sodium hyaluronate. The concentration of the aldehyde polysaccharide solution may be 10-50 mg / mL, preferably 12-45 mg / mL, more preferably 15-40 mg / mL.

[0093] In this invention, the solvent for the aldehyde polysaccharide solution can be at least one of PBS buffer, water, dimethyl sulfoxide, N,N-dimethylformamide, dichloromethane, and tetrahydrofuran, preferably at least one of PBS buffer, water, dimethyl sulfoxide, and N,N-dimethylformamide, more preferably at least one of PBS buffer and water, and even more preferably a PBS buffer with a concentration of 0.01-0.1M and a pH of 7.2-7.4.

[0094] According to one embodiment of the present invention, the aminopolysaccharide may be selected from at least one of carboxymethyl chitosan, carboxyethyl chitosan, carboxypropyl chitosan, carboxybutyl chitosan, aminated chitosan, and aminated mannose, preferably at least one of carboxymethyl chitosan, carboxyethyl chitosan, carboxypropyl chitosan, and carboxybutyl chitosan, and more preferably at least one of carboxymethyl chitosan and carboxyethyl chitosan. The concentration of the aminopolysaccharide solution may be 10-50 mg / mL, preferably 15-45 mg / mL, and more preferably 20-40 mg / mL.

[0095] In this invention, the solvent for the aminopolysaccharide solution can be at least one of water, dimethyl sulfoxide, N,N-dimethylformamide, dichloromethane, and tetrahydrofuran, preferably at least one of water, dimethyl sulfoxide, dichloromethane, and tetrahydrofuran, and more preferably at least one of water and dimethyl sulfoxide.

[0096] According to one embodiment of the present invention, the volume ratio of the drug-loaded nanoparticle solution to the aldehyde polysaccharide solution can be 1:3 to 10, preferably 1:3.5 to 8.5, and more preferably 1:5 to 8. Such a volume ratio is beneficial for the uniform dispersion of the drug-loaded nanoparticles in the hydrogel.

[0097] According to one embodiment of the present invention, the volume ratio of the aldehyde polysaccharide solution to the amino polysaccharide solution can be 4 to 10:1, preferably 5 to 9:1, and more preferably 6 to 8:1. Such a volume ratio is beneficial for improving the gelation time and stability of the gel.

[0098] A reasonable ratio of aldehyde polysaccharide solution, amino polysaccharide, and their dosage to drug-loaded nanoparticle solution can improve the biosafety and gastric acid resistance of drug-loaded nanoparticle composite hydrogels, and is more conducive to the enrichment and sustained release of drugs in the colon and the improvement of symptoms of inflammatory bowel disease.

[0099] <Preparation Method of Drug-Loaded Nanoparticle Composite Hydrogel> This invention also provides a method for preparing the above-mentioned drug-loaded nanoparticle composite hydrogel, including a drug-loaded nanoparticle mixing step and a gelation step. This is described in detail below.

[0100] Drug-loaded nanoparticle mixing steps The drug-loaded nanoparticle solution was mixed with the aldehyde polysaccharide solution to obtain a mixture.

[0101] According to one embodiment of the present invention, the mixing temperature can be 20-40°C, preferably 22-38°C, and more preferably 24-35°C.

[0102] In this invention, the drug-loaded nanoparticle solution can be added dropwise to the aldehyde polysaccharide solution, followed by stirring and mixing. This is more conducive to the uniform dispersion of the drug-loaded nanoparticles in the mixture. Stirring can be achieved using any stirring device or method known in the art, and is not particularly limited here. For example, a vortex mixer can be used for vortex oscillation. According to a preferred embodiment of the invention, the rotational speed of the vortex oscillation can be 500–3500 rpm, preferably 1000–3000 rpm, more preferably 1500–2500 rpm. The vortex oscillation time can be 0.2–5 min, preferably 0.5–4 min, more preferably 1–3 min.

[0103] In this invention, the aldehyde polysaccharide can be a commercially available product or obtained by oxidation of the precursor polysaccharide corresponding to the aldehyde polysaccharide. The precursor polysaccharide can be selected from at least one of pectin, dextran, sodium alginate, sodium hyaluronate, chitosan and its derivatives, cellulose and its derivatives, preferably at least one of pectin, dextran, sodium alginate, sodium hyaluronate, and chitosan, and more preferably at least one of pectin, dextran, sodium alginate, and sodium hyaluronate.

[0104] According to one embodiment of the present invention, aldehyde polysaccharides can be obtained by oxidizing precursor polysaccharides with alkali metal perhalates. The alkali metal perhalate can be selected from at least one of alkali metal perchlorates, perbromates, and periodates, preferably at least one of alkali metal perchlorates and periodates, more preferably alkali metal periodates. The alkali metal can be selected from at least one of Li (Li), Na (sodium), and K (potassium), preferably at least one of Na and K, more preferably Na or K.

[0105] According to one embodiment of the present invention, the method for preparing aldehyde polysaccharides includes a precursor polysaccharide dissolution step, an oxidation step, and a purification step. These are described in detail below.

[0106] [Precursor polysaccharide dissolution steps] Add the precursor polysaccharide to water and stir to obtain a precursor polysaccharide dispersion.

[0107] According to one embodiment of the present invention, based on 1g of precursor polysaccharide, the amount of water used can be 20-100mL, preferably 30-85mL, and more preferably 40-75mL.

[0108] In this invention, stirring can be achieved using any stirring device or method known in the art, and no particular limitation is made here. For example, it can be magnetic stirring, vortex stirring, etc. The stirring time can be 30 to 120 minutes, preferably 40 to 100 minutes, and more preferably 50 to 90 minutes. Reasonable stirring conditions are beneficial to more complete hydration and dispersion of the precursor polysaccharide.

[0109] [Oxidation Step] Alkali metal perhalate was added to the precursor polysaccharide dispersion under light-protected conditions, and an oxidation reaction was carried out under light-protected conditions and at 20-40°C to obtain reaction solution A; then, C2-C5 alkyldiol was added to reaction solution A to obtain reaction solution B.

[0110] According to one embodiment of the present invention, the oxidation reaction temperature can be 20–40°C, preferably 22–37°C, more preferably 23–35°C. The oxidation reaction time can be 6–24 h, preferably 8–20 h, more preferably 10–18 h. Such conditions are beneficial for the specific oxidation of the cis-diol structure on the precursor polysaccharide molecular chain to an aldehyde group.

[0111] According to a preferred embodiment of the present invention, stirring can also be performed during the oxidation reaction, so that the reactants are more uniformly dispersed in the system, which is conducive to a more stable reaction.

[0112] According to one embodiment of the present invention, based on 1g of precursor polysaccharide, the amount of C2-C5 alkyldiol used can be 1-5mL, preferably 1.5-4mL, and more preferably 2-3.5mL. The C2-C5 alkyldiol can be C2-C4 alkyldiol, preferably at least one of ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, and 1,4-butanediol, and more preferably at least one of ethylene glycol, 1,2-propanediol, and 1,4-butanediol.

[0113] According to a preferred embodiment of the present invention, stirring can be continued after the addition of C2-C5 alkyldiol, thereby accelerating the termination of the reaction process. The stirring time can be 5-30 min, preferably 10-25 min, and more preferably 12-20 min.

[0114] [Purification Steps] The reaction solution B was placed in a dialysis bag, and then the dialysis bag was placed in water for dialysis purification to obtain purified reaction solution B; the purified reaction solution B was freeze-dried to obtain aldehyde polysaccharide.

[0115] According to one embodiment of the present invention, the molecular weight cutoff of the dialysis bag can be 7000-15000 Da, preferably 7500-14500 Da, more preferably 8000-14000 Da. The purification time can be 24-120 h, preferably 36-96 h, more preferably 48-84 h. According to a preferred embodiment of the present invention, the water can be changed 2-5 times every 24 h, preferably 2-4 times, and more preferably 3-4 times. Such dialysis conditions are beneficial for removing unreacted raw materials and other small molecule byproducts in reaction solution B, thereby improving the purity of the aldehyde polysaccharide.

[0116] In this invention, the freeze-drying conditions are the same as those described above, and will not be repeated here.

[0117] In this invention, to avoid photoreaction of aldehyde polysaccharides, dialysis purification can be carried out under light-protected conditions.

[0118] Gel forming steps The aminopolysaccharide solution was mixed with the mixture to allow the aldehyde polysaccharide and aminopolysaccharide to undergo a Schiff base reaction, resulting in a drug-loaded nanoparticle composite hydrogel.

[0119] According to one embodiment of the present invention, the temperature of the Schiff base reaction can be 20–40°C, preferably 22–38°C, and more preferably 24–35°C. The time of the Schiff base reaction (i.e., the gelation time) can be less than or equal to 3 min, preferably less than or equal to 2 min, and more preferably less than or equal to 1 min. During the reaction, continuous stirring can be performed to improve reaction uniformity and reaction rate, thereby promoting the Schiff base reaction.

[0120] In this invention, stirring can be achieved using any stirring device or method known in the art, without particular limitation. For example, it can be oscillation, ultrasonic dispersion, magnetic stirring, etc. According to a preferred embodiment of the invention, stirring can be achieved using vortex oscillation. The rotational speed of the vortex oscillation can be 500–3500 rpm, preferably 1000–3000 rpm, more preferably 1500–2500 rpm. The duration of the vortex oscillation can be 0.2–5 min, preferably 0.4–3.5 min, more preferably 1–3 min.

[0121] Reasonable Schiff base reaction conditions are beneficial to improving the biosafety and gastric acid resistance of drug-loaded nanoparticle composite hydrogels, and are more conducive to achieving drug enrichment and sustained release in the colon and improving the symptoms of inflammatory bowel disease.

[0122] In this invention, during the cross-linking process of Schiff base reaction between aldehyde polysaccharide and amino polysaccharide, drug-loaded nanoparticles dispersed in the aldehyde polysaccharide phase can be simultaneously encapsulated and fixed in situ in a three-dimensional hydrogel network, ultimately forming a drug-loaded nanoparticle composite hydrogel.

[0123] <Applications> The present invention also provides the use of the above-mentioned drug-loaded nanoparticle composite hydrogel in the preparation of medicaments for treating inflammatory bowel disease.

[0124] According to one embodiment of the present invention, the cumulative release rate of the drug-loaded nanoparticle composite hydrogel in the gastric environment within 2 hours can be less than or equal to 23%, preferably less than or equal to 22.5%, more preferably less than or equal to 22.05%. The cumulative release rate of the drug-loaded nanoparticle composite hydrogel in the colonic region can be at least 78%, preferably at least 79%, more preferably at least 80%. Such a drug-loaded nanoparticle composite hydrogel has good acid resistance stability and sustained-release performance in the colonic region.

[0125] According to one embodiment of the present invention, the drug-loaded nanoparticle composite hydrogel can significantly enhance the expression of anti-inflammatory factors, effectively alleviate oxidative stress damage in colon tissue, and promote the expression of tight junction proteins, which helps to repair the damaged intestinal mucosal barrier.

[0126] The drug-loaded nanoparticles of this invention can effectively prolong the retention time of drugs in the gastrointestinal tract, achieve targeted distribution and enrichment in the inflamed colon, significantly improve the symptoms of inflammatory bowel disease, and are very suitable for the preparation of drugs for the treatment of inflammatory bowel disease.

[0127] <Raw Material Description> Unless otherwise specified, all raw materials used in the following examples are commercially available products.

[0128] Pectin (PEC) was purchased from Shanghai Ron Chemical Technology Co., Ltd., and its monomer chemical formula is C5H. 10 O5, CAS number 9000-69-5, galacturonic acid concentration ≥74wt%. Prednisolone, purchased from Beijing Coupling Technology Co., Ltd., chemical formula C. 21 H 28 O5, CAS number 50-24-8, purity 98wt%.

[0129] Example 1 - Preparation of polymer materials and drug-loaded nanoparticle solutions Preparation of polymer materials: 1) Dissolve 300 mg of PEC, 453 mg of EDC, and 273.3 mg of NHS in 15 mL of deionized water at room temperature (25 °C, the same below), and stir magnetically for 12 h under light-protected conditions to obtain a mixed solution. Then, add 305.4 mg of cystamine dihydrochloride (CYS) to the mixed solution and stir at room temperature for 24 h to carry out an amidation reaction to obtain reaction solution I.

[0130] 2) Place reaction solution I in a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and dialyze the bag in deionized water for 72 hours, changing the water 3 times every 24 hours to obtain purified reaction solution I. Then, freeze-dry purified reaction solution I at -80℃ for 36 hours to obtain the polymer material intermediate (denoted as PEC-SS).

[0131] 3) Dissolve 100 mg of PEC-SS in 5 mL of formamide, then dilute with 3 mL of dimethyl sulfoxide to obtain solution A. Dissolve 100 mg of baicalin (BAI), twice the molar amount of baicalin EDC, and twice the molar amount of baicalin NHS in 5 mL of dimethyl sulfoxide, and stir magnetically for 30 min to obtain solution B. Mix solution A and solution B, and stir at room temperature for 24 h to carry out the amidation reaction to obtain reaction solution II.

[0132] 4) Place reaction solution II in a dialysis bag with a molecular weight cutoff of 8000–14000 Da, and dialysis the bag in an aqueous solution of 2 vol% ethanol for 72 h for purification, changing the water 3 times every 24 h to obtain purified reaction solution II. Then, freeze-dry purified reaction solution II at -80℃ for 48 h to obtain the polymer material (denoted as PEC-SS-BAI).

[0133] Preparation of drug-loaded nanoparticle solutions: A) Mix 10 mg of PEC-SS-BAI with 10 mL of methanol and ultrasonically disperse for 10 min at a power of 250 W and an on-off-off condition (the ultrasonic device runs for 2 seconds and stops for 4 seconds) to obtain an alcoholic solution of the polymer material. Then, add prednisolone at a mass ratio of 1:2 to PEC-SS-BAI to obtain an alcoholic solution of the mixture.

[0134] B) The alcohol solution of the mixture was rotary evaporated under reduced pressure at 60°C and 90 rpm until a thin film was formed at the bottom of the container, thus obtaining the film material.

[0135] C) The membrane was placed in 15 mL of ultrapure water for hydration, then sonicated for 45 min, and then centrifuged at 3500 rpm for 10 min. The supernatant was filtered through a microporous membrane with a pore size of 0.45 μm, and the filtrate was collected to obtain the drug-loaded nanoparticle solution (the drug-loaded nanoparticles are denoted as PSL / PEC-SS-BAI).

[0136] Comparative Examples 1-2 Comparative Example 1: The only difference from Example 1 is that, when preparing the drug-loaded nanoparticle solution, the mass ratio of prednisolone to PEC-SS-BAI was 1:3; the rotary evaporation speed under reduced pressure was 80 rpm; and the film was placed in 10 mL of ultrapure water for hydration.

[0137] Comparative Example 2: The only difference from Comparative Example 2 is that the rotary evaporation speed under reduced pressure is 120 rpm.

[0138] Experimental Example 1 1. Detection of particle size and zeta potential of drug-loaded nanoparticles The drug-loaded nanoparticle solution prepared in Example 1 was added to a cuvette, and its particle size and PDI (polydispersity index) were measured using a laser particle size analyzer; the average value of three samples was taken. The drug-loaded nanoparticle solution prepared in Example 1 was injected into the potential cell of the laser particle size analyzer, and then the potential cell was placed in the sample chamber of the laser particle size analyzer. After equilibration, its Zeta potential was measured; the average value of three samples was taken.

[0139] The particle size distribution of PSL / PEC-SS-BAI nanoparticles is shown in the figure. Figure 1Figure A shows an average particle size of 235.3 ± 2.5 nm and a PDI of 0.225 ± 0.023, indicating good uniformity of the PSL / PEC-SS-BAI nanoparticles. The Zeta potential distribution of the PSL / PEC-SS-BAI nanoparticles is shown in Figure A. Figure 1 Figure B shows that the Zeta potential of the PSL / PEC-SS-BAI nanoparticles is -52.8±2.1mV. The absolute value of the Zeta potential is significantly greater than 30mV, indicating that the drug-loaded nanoparticles of the present invention have strong anti-aggregation properties and good dispersibility.

[0140] 2. Stability testing of drug-loaded nanoparticles The drug-loaded nanoparticle solutions prepared in Example 1 were placed at 4°C and room temperature for 7 days (n=3), and the Zeta potential was measured daily to investigate the stability of the drug-loaded nanoparticles.

[0141] Test results are as follows Figure 2 As shown, the Zeta potential did not change significantly within 7 days and the absolute value remained greater than 30mV, indicating that the PSL / PEC-SS-BAI nanoparticles prepared in this invention have good storage stability.

[0142] Experiment Example 2 Encapsulation efficiency and drug loading of drug-loaded nanoparticle solutions: Take 1 mL of the drug-loaded nanoparticle solutions prepared in Example 1 and Comparative Examples 1-2, respectively, dilute with 4 times the volume of methanol, then sonicate, and centrifuge at 5000 rpm for 5 min. Then, take the supernatant, filter it through a 0.22 μm filter membrane, and analyze it by high-performance liquid chromatography (HPLC) to determine the drug content in the filtrate. The encapsulation efficiency (EE) and drug loading capacity (DL) of PSL / PEC-SS-BAI are then calculated. The average value of three samples is taken. The HPLC analysis conditions are as follows: Agilent C18 column (250 mm × 4.6 mm, 5 μm), mobile phase methanol-water (65:35), detection wavelength 240 nm, column temperature 25 °C, and flow rate 1.0 mL / min. -1 The injection volume was 10 μL.

[0143] Encapsulation efficiency is defined as the ratio of the amount of drug encapsulated inside the nanoparticle to the total amount of drug added during preparation. The calculation formula is shown in equation (1): EE% = PSL / Actual PSL content in PEC-SS-BAI / PSL dosage × 100% (1).

[0144] Drug loading is defined as the percentage of the mass of the drug loaded in the nanoparticles relative to the total mass of the entire nanoparticle formulation, as shown in formula (2): DL% = PSL / actual PSL content in PEC-SS-BAI / total mass of nanoparticles × 100% (2).

[0145] The encapsulation efficiency and drug loading of the drug-loaded nanoparticle solutions are shown in Table 1.

[0146] Table 1

[0147] As shown in Table 1, the drug-loaded nanoparticle solution prepared by this invention has a high encapsulation efficiency and drug loading, and good stability.

[0148] Example 2 - Preparation of drug-loaded nanoparticle composite hydrogel Preparation of oxidized pectin: a) Add 1g of pectin to 50mL of deionized water and stir magnetically for 1h to obtain a pectin dispersion.

[0149] b) Wrap the pectin dispersion in aluminum foil to protect it from light, then add 1g of NaIO4 to the pectin dispersion and stir at room temperature for 12 hours to obtain reaction solution A. Next, add 2.25mL of ethylene glycol to reaction solution A and stir for 15 minutes to obtain reaction solution B.

[0150] c) Place reaction solution B in a dialysis bag with a molecular weight cutoff of 8000–14000 Da, and dialyze the bag in deionized water for 72 hours, changing the water three times every 24 hours to obtain purified reaction solution B. Then, freeze-dry the purified reaction solution B at -80°C to obtain oxidized pectin (denoted as OP).

[0151] Preparation of drug-loaded nanoparticle composite hydrogels: 1) Add 0.3 g of carboxymethyl chitosan (CMC) to 10 mL of deionized water and stir at room temperature for 4 h to obtain a CMC solution. Add 0.2 g of OP to 10 mL of 0.01 M PBS buffer (pH 7.2–7.4) and stir at room temperature for 4 h to obtain an OP solution.

[0152] 0.2 mL of the PSL / PEC-SS-BAI solution prepared in Example 1 was added dropwise to 1.2 mL of OP solution, and the mixture was vortexed for 1 min at room temperature and 2000 rpm to obtain a mixture. 2) Mix 0.2 mL of CMC solution with the mixture and vortex at room temperature and 2000 rpm for 1 min to obtain drug-loaded nanoparticle composite hydrogel (denoted as PSL / PEC-SS-BAI@CMC-OP).

[0153] Experimental Example 3 Morphology analysis of drug-loaded nanoparticle composite hydrogels: The PSL / PEC-SS-BAI@CMC-OP prepared in Example 2 was placed in a freeze dryer and lyophilized to constant weight. Then, the lyophilized sample was broken to expose its internal cross-section and fixed on the conductive adhesive of the sample stage. The sample was then sputtered with gold using an ion sputtering instrument. Finally, the gold-sputtered sample was placed in the chamber of a SEM (scanning electron microscope) to observe its microstructure.

[0154] SEM results are as follows Figure 3 As shown, PSL / PEC-SS-BAI@CMC-OP contains numerous pores, which are formed by the interlacing of CMC and OP molecules into a three-dimensional network structure. These pores are relatively large and interwoven with many tiny pores within. The surface roughness of the pore walls in PSL / PEC-SS-BAI@CMC-OP is significant, which can be attributed to the introduction of nanoparticles. These results demonstrate that CMC and OP can form an interconnected porous hydrogel, uniformly dispersing the drug within it.

[0155] Experiment Example 4 I. In vitro drug testing of drug-loaded nanoparticle composite hydrogels 1. In vitro drug release experiment of drug-loaded nanoparticle composite hydrogel 1.1 Experimental Methods Dialysis bags with a molecular weight cutoff of 8000–14000 Da were pretreated by boiling in water for 10 min. Then, 66.7 μg of PSL / PEC-SS-BAI@CMC-OP and 66.7 μg of free PSL were respectively loaded into the pretreated dialysis bags. The dialysis bags were then placed in a constant-temperature shaker at 37°C and 75 rpm, with a release medium volume of 20 mL. Release was performed in PBS buffer (pH 1.2) for 0–2 h, in PBS buffer (pH 6.8) for 2–6 h, and in PBS buffer (pH 7.4) for 6–24 h. At predetermined time points (0.5, 1, 2, 4, 6, 8, 10, 12, 24h), 1mL of release medium was taken, and the same volume of fresh medium was added simultaneously. After obtaining the test samples at all time points, the samples were centrifuged at 3500rpm for 10min, and the supernatant was taken. After filtration through a filter membrane with a pore size of 0.22μm, the filtrate was taken, and the PSL content was detected according to the HPLC conditions of Experiment Example 2. The cumulative drug release rate was calculated according to formula (3), and the drug release curve in the simulated gastrointestinal tract was plotted. The experiment was repeated three times (including the first time, the same below).

[0156] (3) Among them, C n The PSL concentration (μg·mL) at the nth sampling time -1 V represents the total volume of the release medium (mL); V e Sampling volume (mL); C n-1 The PSL concentration (μg·mL) at the (n-1)th sampling. -1 M0 represents the mass (mg) of PSL loaded within the drug-loaded nanoparticle composite hydrogel.

[0157] 1.2 Experimental Results Release results of PSL / PEC-SS-BAI@CMC-OP and free PSL during simulated gastrointestinal transit: Figure 4 As shown, the free PSL drug initially released approximately 71.58% in an environment with pH 1.2 (simulated gastric juice) over the first 2 hours, exhibiting a significant burst release phenomenon. The release rate then gradually slowed, reaching a plateau of over 80% by 4 hours. In contrast, the release behavior of PSL / PEC-SS-BAI@CMC-OP under the same conditions was significantly different. It released approximately 22.05% from 0 to 2 hours, and approximately 57.61% from 2 to 6 hours in an environment with pH 6.8 (simulated small intestinal juice). Upon entering the colonic environment (pH=7.4), release continued, with the curve showing a continuous upward trend, ultimately reaching over 80% release. These results indicate that the drug-loaded nanoparticle composite hydrogel of this invention can resist some gastric acid erosion, prolong the administration time, and ensure sufficient release of PSL after delivery to the colonic environment while avoiding burst release.

[0158] 2. In vitro drug degradation experiment of drug-loaded nanoparticle composite hydrogel 2.1 Experimental Methods 1.45g of PSL / PEC-SS-BAI@CMC-OP was placed in PBS buffer media with pH=1.2 and pH=6.8 respectively. The temperature was set at 37±0.5℃. PSL / PEC-SS-BAI@CMC-OP was removed at predetermined time points (0, 6, 12, 24, 36, 48, 72, 96h), excess water on the surface was absorbed with filter paper, and then weighed on an electronic balance to observe the change in weight of PSL / PEC-SS-BAI@CMC-OP with degradation time. The buffer media was changed every 12h, and the experiment was repeated in parallel three times. The sample degradation rate formula is shown in the following formula (4): (4) Where W0 is the initial weight of the hydrogel, W n The weight of the hydrogel at each set time point.

[0159] 2.2 Experimental Results The degradation curves of PSL / PEC-SS-BAI@CMC-OP measured in PBS buffer media at pH 1.2 and pH 6.8 are shown below. Figure 5 As shown, the environment with a pH of 1.2 simulates the gastric acid environment, and the environment with a pH of 6.8 simulates the slightly acidic environment of IBD (inflammatory bowel disease). PSL / PEC-SS-BAI@CMC-OP degrades slowly in the gastric acid environment, exhibiting a certain degree of acid resistance. This short-term acid resistance, to some extent, ensures the integrity of the hydrogel in gastric acid, thereby effectively preventing drug leakage. However, in the simulated IBD environment, the degradation of PSL / PEC-SS-BAI@CMC-OP accelerates, reaching almost complete degradation within 96 hours. This demonstrates that the drug-loaded nanoparticle composite hydrogel of this invention exhibits instability in the inflammatory bowel and can effectively release the drug.

[0160] 3. In vitro antioxidant experiments of drug-loaded nanoparticle composite hydrogels 3.1 Experimental Methods 5 mg of DPPH (1,1-Diphenyl-2-picrylhydrazyl radical) was mixed with 100 mL of anhydrous ethanol to prepare a solution with a concentration of 50 μg·mL⁻¹. -1 DPPH solutions were prepared using ultrapure water at concentrations of 5, 10, 15, 20, 25, and 30 mg / mL. -1 The PSL / PEC-SS-BAI@CMC-OP solution (denoted as colloidal solution) was prepared. 0.5 mL of the colloidal solution of different concentrations was mixed with 2.5 mL of DPPH solution. The mixture was incubated in the dark for 30 min, followed by centrifugation at 5000 rpm for 10 min. The supernatant was then transferred to a 96-well plate (200 µL / well). The absorbance at 517 nm was measured using a microplate reader and recorded as λ. A 1 Under the same measurement conditions, anhydrous ethanol was used instead of DPPH as a control, denoted as A 2 The absorbance was measured using ultrapure water instead of the test solution, and recorded as follows: A 0 Based on the measured absorbance, the experiment was repeated three times in parallel, and the free radical scavenging rate of DPPH was calculated using the following formula (5).

[0161] (5).

[0162] 3.2 Experimental Results The antioxidant properties of PSL / PEC-SS-BAI@CMC-OP are as follows: Figure 6As shown, PSL / PEC-SS-BAI@CMC-OP exhibits good antioxidant properties, which show an increasing trend with increasing concentration. At a concentration of 5 mg / mL... -1 At that concentration, the DPPH free radical scavenging activity was approximately 25.65%. This increased to 30 mg / mL. -1 At that time, PSL / PEC-SS-BAI@CMC-OP exhibited strong free radical scavenging ability, with a maximum scavenging rate of 71.46%. Overall results indicate that the drug-loaded nanoparticle composite hydrogel of the present invention has certain antioxidant properties and good free radical scavenging ability.

[0163] II. In vivo and in vitro distribution and targeted imaging experiments of drug-loaded nanoparticle composite hydrogels 1. Experimental Methods 1.1 Preparation of the formulation A fluorescently labeled substance (concentration 0.75 mg·kg⁻¹) was prepared according to the preparation methods of Examples 1 and 2. -1 The drug-loaded nanoparticles of DIR (1,1'-distearate-3,3,3',3'-tetramethylindole tricarbocyanine iodide) (denoted as DIR-PSL / PEC-SS-BAI) and the drug-loaded nanoparticle composite hydrogel (denoted as DIR-PSL / PEC-SS-BAI@CMC-OP).

[0164] 1.2 Establishment of the IBD Model After 7 days of acclimatization, 6-8 week old male Balb / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were given 9% (w / v) DSS (sodium dextran sulfate) solution (i.e., DSS concentration of 90 mg / mL) instead of normal drinking water via free access to water. The mice were fed DSS solution continuously for 7 days while maintaining a normal diet to establish an IBD model. The DSS solution was changed every two days to ensure solution stability.

[0165] Throughout the experiment, all animals were subject to standardized management, provided with standard laboratory feed and water, and their bedding was changed regularly. All animal-related experiments were conducted in accordance with the relevant regulations of the Medical Ethics Committee of Inner Mongolia Medical University.

[0166] 1.3 Animal Grouping IBD models were randomly divided into two groups: the M-DIR-PSL / PEC-SS-BAI group (model mice, treated with DIR-labeled drug-loaded nanoparticles) and the M-DIR-PSL / PEC-SS-BAI@CMC-OP group (model mice, treated with DIR-labeled drug-loaded nanoparticle composite hydrogel). Healthy mice were randomly divided into two groups: C-DIR-PSL / PEC-SS-BAI (control mice, treated with DIR-labeled drug-loaded nanoparticles) and C-DIR-PSL / PEC-SS-BAI@#CMC-OP (control mice, treated with DIR-labeled drug-loaded nanoparticle composite hydrogel).

[0167] 1.4 In vivo imaging Six IBD model mice and six healthy male Balb / c mice were randomly divided into two groups of three each. The mice were fasted for 12 hours but allowed free access to water. They were weighed and administered the corresponding formulations by gavage, as described in section 1.3. The mice were anesthetized at 3, 6, 12, and 24 hours after administration, and imaging analysis was performed using a small animal imaging system.

[0168] 1.5. Ex vivo imaging Six IBD model mice and six healthy male Balb / c mice were randomly divided into two groups of three each. The mice were fasted for 12 hours but allowed free access to water. They were weighed and administered the corresponding preparations by gavage according to the animal grouping method described in 1.3. Twenty-four hours after administration, the mice were sacrificed, and the gastrointestinal tissues were removed and analyzed using a small animal imaging system.

[0169] 1.6 Data Processing All fluorescence images were analyzed using Living Image software. Data from multiple groups were converted to log10 values ​​to balance the magnitude differences between different time points. Images within the same group were analyzed using the same brightness and contrast parameters. Statistical analysis and graphing were performed using Origin 2022 and GraphPad Prism 9 software. Results are expressed as mean ± standard deviation. Two-way ANOVA was used for comparisons between multiple groups, and one-way ANOVA was used for quantitative comparisons among multiple groups in a single factorial manner. Lowercase letters a through d indicate significant differences between groups, arranged alphabetically from largest to smallest. P <0.05, the difference is significant.

[0170] 2. Experimental Results 2.1 In vivo imaging results In vivo fluorescence images such as Figure 7As shown, under the same dosage, the average fluorescence intensity of DIR-PSL / PEC-SS-BAI in mice gradually decreased over time, indicating a short intestinal retention time, with almost all of it being metabolized and disappeared within 24 hours. In contrast, DIR-PSL / PEC-SS-BAI@CMC-OP still showed a significant fluorescence signal 24 hours after gavage, indicating that the hydrogel has a better adhesion and retention effect.

[0171] The results of fluorescence quantification are as follows Figure 8 As shown, comparing the model mice treated with DIR-PSL / PEC-SS-BAI@CMC-OP by gavage with the control mice, the fluorescence signal of DIR-PSL / PEC-SS-BAI@CMC-OP was stronger in the inflamed colon at different time points. This result indicates that hydrogels can enhance the targeted adhesion of drug-loaded nanoparticles in the gastrointestinal tract. The average fluorescence intensity of M-DIR-PSL / PEC-SS-BAI@CMC-OP was higher than that of the C-DIR-PSL / PEC-SS-BAI@CMC-OP treatment group. This is because in IBD model mice, inflammation leads to disruption of the intestinal mucus layer, increased vascular permeability, and changes in mucosal surface charge, significantly enhancing the mucosal adhesion of the drug-loaded nanoparticle composite hydrogel, allowing the DIR-labeled formulation to specifically accumulate at the lesion site, resulting in a strong and persistent fluorescence signal.

[0172] 2.2 Results of Ex vivo imaging Mice were administered a DIR-encapsulated drug system via gavage, and in vitro gastrointestinal imaging was observed 24 hours after administration (e.g., Figure 9 (as shown) and quantitative results (such as) Figure 10 (As shown in the image). The results showed that DIR-PSL / PEC-SS-BAI@CMC-OP enhanced drug retention, with significantly higher fluorescence intensity than DIR-PSL / PEC-SS-BAI, and the model group was stronger than the control group. In conclusion, DIR-PSL / PEC-SS-BAI@CMC-OP can effectively accumulate and continuously release drugs in inflamed colon tissue, and the results of ex vivo imaging are consistent with those of in vivo imaging.

[0173] Experimental Example 5 Pharmacodynamic experiments of drug-loaded nanoparticle composite hydrogels on IBD 1. Experimental Methods 1.1 Establishment of the IBD Model After 7 days of acclimatization, 36 male Balb / c mice were randomly divided into 6 groups of 6 mice each, as shown in Table 2. The animal experiment lasted 12 days. Except for the Control group, which had free access to water and food daily, the other 5 groups were given 9% DSS (w / v) drinking water for the first 9 days to establish an IBD model. Given that prednisolone is easily absorbed in the stomach and upper small intestine after oral administration, leading to insufficient drug concentration in the colon, a delivery system is needed to achieve colon-targeted delivery. Therefore, this experiment included drug-loaded nanoparticles and drug-loaded nanoparticle composite hydrogels to examine the delivery advantages of composite delivery systems compared to single nanoparticles. Simultaneously, a positive control group containing sulfasalazine (SASP, purchased from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd.) was used as the clinical benchmark for efficacy evaluation, while a blank hydrogel group was used to exclude interference from the biological activity of the carrier material itself. Mice were administered drugs by gavage starting on day 7 according to their groups. The Control and Model groups were administered the same volume of physiological saline by gavage at the same time, once daily for 5 consecutive days. The DSS solution should be changed every two days to ensure solution stability.

[0174] Table 2

[0175] 1.2 Collection and Processing of Animal Tissue Samples Tissue samples: 24 hours after the last administration, all mice were sacrificed and dissected. The entire colon of each mouse was removed, and after removing the mesentery and adipose tissue, it was rinsed with pre-cooled 0.9% physiological saline. The distal colon of the mouse was transversely cut and fixed with 4% paraformaldehyde. The remaining colon was used to prepare colon homogenate for the determination of inflammatory factors.

[0176] Preparation of colon homogenate: The colon tissue was minced and mixed with PBS phosphate buffer (pH=7.4) (homogenization medium) at a ratio of 1:9 (tissue weight: homogenization medium volume = 1:9). After homogenization, the mixture was centrifuged at 12000 rpm for 10 min, the supernatant was collected, aliquoted, and stored at -80℃ for later use.

[0177] 1.3. Histopathological evaluation of colon tissue Referring to the well-known HE staining experimental manual in this field, the fixed colon tissue samples were taken out, dehydrated, embedded, sectioned, stained with hematoxylin and eosin (HE), and the pathological changes of each colon sample were observed under a microscope.

[0178] 1.4. Colonic ELISA Biochemical Indicators Detection Colon tissue was processed using the same method as described in "1.2, Animal Tissue Sample Collection and Processing" for colon homogenization. The supernatant was collected and analyzed according to the instructions of the ELISA kits for tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10). All three kits were 96T and purchased from Solarbio Science & Technology Co., Ltd.

[0179] 1.5. Detection of colonic oxidative stress indicators Colon tissue was processed using the same method as colon homogenization described in "1.2. Animal Tissue Sample Collection and Processing". After taking the supernatant, the myeloperoxidase (MPO), malondialdehyde (MDA), and superoxide dismutase (SOD) were measured according to the instructions of the biochemical kits.

[0180] 1.6 Immunohistochemical analysis of the colon Referring to well-known immunohistochemical experimental manuals in this field, the fixed tissue samples were dehydrated, embedded, and sectioned. Then, they were dewaxed to water, antigen retrieval, endogenous peroxidase blocking, and serum blocking were performed in sequence. Subsequently, they were incubated with primary antibody and HRP (horseradish peroxidase) labeled secondary antibody in sequence. After DAB (diaminobenzidine) staining, hematoxylin counterstaining, dehydration, clearing, and mounting, the positive expression of peripheral membrane protein ZO-1 and closure protein Occludin was observed under a microscope.

[0181] 1.7 Safety Evaluation After 7 days of acclimatization, 12 male Balb / c mice were randomly divided into a Control group and a PSL / PEC-SS-BAI@CMC-OP group, with 6 mice in each group. The PSL / PEC-SS-BAI@CMC-OP group was administered PSL / PEC-SS-BAI@CMC-OP via gavage starting at 9:00 AM daily, while the Control group was administered an equal volume of physiological saline via gavage concurrently, for 7 consecutive days. The mice were weighed daily. 24 hours after the last administration, all mice were sacrificed and dissected. The heart, liver, spleen, lungs, and kidneys were identified and removed sequentially, weighed, fixed, and embedded. HE staining images of the tissues were collected.

[0182] 1.8 Data Processing Statistical analysis and graphing were performed using Origin 2022 and GraphPad Prism 9 software. Results are expressed as mean ± standard deviation. One-way ANOVA was used for quantitative comparisons among multiple groups. Lowercase letters a through d indicate significant differences between groups, arranged alphabetically from largest to smallest. P <0.05, the difference is significant.

[0183] 2. Experimental Results 2.1. Histopathological results of colon tissue Image of HE-stained section of mouse colon tissue as shown below Figure 11 As shown, the Control group mice had a large number of intestinal glands in the lamina propria, arranged tightly and orderly, containing a large number of goblet cells, with no inflammatory cell infiltration and no necrosis or shedding. In contrast, the Model group mice showed no necrosis of the mucosal epithelium and intestinal glands, which were replaced by proliferating connective tissue, with a significant reduction in goblet cells and a large number of inflammatory cells infiltrating into the submucosa. Compared with the Model group, the pathological damage of the colon tissue in the SASP group, PSL / PEC-SS-BAI@CMC-OP group, PSL / PEC-SS-BAI group, and PEC-SS-BAI@CMC-OP group mice was improved to varying degrees. Among them, the SASP group and PSL / PEC-SS-BAI@CMC-OP group had intact mucosal structure and clear boundaries between structural layers, contained a large number of goblet cells arranged relatively tightly, with only occasional shedding of mucosal epithelial cells and almost no inflammatory cell infiltration, indicating that PSL / PEC-SS-BAI@CMC-OP can effectively improve intestinal mucosal damage. The PSL / PEC-SS-BAI group and the PEC-SS-BAI@CMC-OP group also showed some protective effect on intestinal structure, but some inflammatory cell infiltration and goblet cell reduction were still observed.

[0184] 2.2 Results of Detection of Inflammatory Factor Levels in Colonic Tissue The levels of TNF-α, IL-6 and IL-10 in mouse colon tissue are as follows Figure 12 As shown, the expression of TNF-α and IL-6 in the colon tissue of the Model group mice was significantly higher than that in the Control group ( P <0.05. After PSL / PEC-SS-BAI@CMC-OP treatment, the levels of both pro-inflammatory factors were significantly downregulated, with no significant difference compared to the Control group and the SASP group. P＞0.05). Compared with the Model group, the levels of TNF-α and IL-6 in the PSL / PEC-SS-BAI group and the PEC-SS-BAI@CMC-OP group were decreased. After treatment with SASP and PSL / PEC-SS-BAI@CMC-OP, the level of IL-10 increased significantly to the point that there was no significant difference compared with the Control group. P ＞ 0.05). The above results indicate that PSL / PEC-SS-BAI@CMC-OP can downregulate the expression level of pro-inflammatory factors and increase the expression level of anti-inflammatory factors, thus exhibiting good anti-inflammatory activity.

[0185] 2.3 Results of oxidative stress level detection in colon tissue Oxidative stress-related indicators were detected in the colon tissue of mice in each group, and the results are as follows: Figure 13 As shown, the expression levels of MPO and MDA in the colon tissue of mice in the Control group were low, while the expression levels increased significantly after DSS induction. P <0.05. Compared with the Model group, the expression of MPO and MDA in the colon tissue of mice in all treatment groups was reduced. Among them, the PSL / PEC-SS-BAI@CMC-OP group showed no significant difference in the effect of downregulating MPO and MDA expression levels compared with the Control group and SASP group. P＞ (0.05). Conversely, DSS treatment led to a significant decrease in SOD levels in mouse colon tissue. Treatment with SASP and PSL / PEC-SS-BAI@CMC-OP significantly increased SOD levels, with better results than PSL / PEC-SS-BAI and PEC-SS-BAI@CMC-OP. This strongly suggests that PSL / PEC-SS-BAI@CMC-OP can effectively alleviate oxidative stress damage in mouse colon tissue.

[0186] 2.4 Results of barrier function test of colonic tissue The expression results of peripheral membrane protein ZO-1 and closure protein Occludin are as follows: Figure 14 and 15 As shown, compared with the control group, the protein expression of both Occludin and ZO-1 in the colonic mucosa of mice in the model group was significantly reduced. P<0.05) indicates impaired intestinal epithelial barrier structure and increased permeability. After treatment intervention in each drug-treated group, the expression of both tight junction proteins showed a significant regression (P<0.05). The ratio of ZO-1 and Occludin-positive cells in the colonic epithelium of the PSL / PEC-SS-BAI@CMC-OP group was significantly restored compared with the Model group, indicating that the drug-loaded nanoparticle composite hydrogel of the present invention can effectively promote the expression of intestinal barrier-related proteins, help maintain the integrity of the epithelial structure, and has the function of repairing the damaged intestinal mucosal barrier.

[0187] 2.5. In vivo safety test results The changes in mouse body weight after PSL / PEC-SS-BAI@CMC-OP gavage are as follows Figure 16 As shown, the weight gain trend of mice in the PSL / PEC-SS-BAI@CMC-OP group was basically the same as that in the Control group, with no significant reduction observed. The results of HE staining of major organs are shown below. Figure 17 As shown, HE staining of vital organs such as the heart, liver, spleen, lungs, and kidneys revealed no obvious pathological damage, structural abnormalities, or inflammatory cell infiltration, and no significant differences were observed compared to the Control group. These results indicate that the drug-loaded nanoparticle composite hydrogel of this invention possesses good in vivo biocompatibility.

[0188] This invention is not limited to the above-described embodiments. Any modifications, improvements, or substitutions that can be conceived by those skilled in the art without departing from the essential content of this invention fall within the scope of this invention.

Claims

1. A polymer material, characterized in that, The polymer material is formed by linking pectin with cysteine ​​and baicalin or its derivatives; wherein, the two amino groups of cysteine ​​react with the carboxyl groups on pectin and baicalin or its derivatives respectively through an amidation reaction to form amides; the chemical structure of baicalin or its derivatives is shown in formula (I): （I） R is selected from hydrogen or C1 to C6 alkyl groups.

2. A method for preparing the polymer material according to claim 1, characterized in that, Includes the following steps: 1) Pectin and cystamine dihydrochloride are subjected to an amidation reaction under a catalyst and at 20-40°C to obtain reaction solution I; wherein the weight ratio of pectin to cystamine dihydrochloride is 1:1-5; 2) Place reaction solution I in a dialysis bag, place the dialysis bag in water for the first dialysis purification, and obtain purified reaction solution I; freeze-dry purified reaction solution I to obtain polymer material intermediate; 3) The polymer material intermediate is subjected to an amidation reaction with baicalin or its derivative under a catalyst and at 20-40°C to obtain reaction solution II; wherein the weight ratio of the polymer material intermediate to baicalin or its derivative is 1:1-5. 4) Place reaction solution II in a dialysis bag, and place the dialysis bag in an aqueous solution of C1-C5 alkyl alcohols for a second dialysis purification to obtain purified reaction solution II; freeze-dry purified reaction solution II to obtain polymer material.

3. A drug-loaded nanoparticle solution, characterized in that, The drug-loaded nanoparticle solution is prepared from raw materials including the polymer material of claim 1 and the active pharmaceutical ingredient; wherein, the polymer material of claim 1 encapsulates the active pharmaceutical ingredient to form drug-loaded nanoparticles; the weight ratio of the active pharmaceutical ingredient to the polymer material of claim 1 is 1:1.5 to 4.5; and the active pharmaceutical ingredient is an active pharmaceutical ingredient for treating inflammatory bowel disease.

4. A method for preparing a drug-loaded nanoparticle solution according to claim 3, characterized in that, Includes the following steps: A) The polymer material of claim 1 is mixed with C1-C5 alkyl alcohols to obtain an alcohol solution of the polymer material. Then, the active pharmaceutical ingredient is added to the alcohol solution of the polymer material to obtain an alcohol solution of the mixture. B) The alcohol solution of the mixture was rotary evaporated under reduced pressure at a speed of 85-110 rpm to obtain a thin film. C) Place the film in water, sonicate for 35-55 min, then centrifuge, take the supernatant and filter it through a microporous membrane, take the filtrate to obtain a drug-loaded nanoparticle solution; wherein, based on 1 mg of the polymer material described in claim 1, the amount of water used is 1-3 mL.

5. A drug-loaded nanoparticle composite hydrogel, characterized in that, The drug-loaded nanoparticle composite hydrogel is prepared from raw materials including the drug-loaded nanoparticle solution, aldehyde polysaccharide solution and amino polysaccharide solution as described in claim 3; The concentration of the aldehyde polysaccharide solution is 10-50 mg / mL; the aldehyde polysaccharide is selected from at least one of oxidized pectin, oxidized dextran, oxidized sodium alginate, oxidized sodium hyaluronate, oxidized chitosan and its derivatives, and aldehyde cellulose and its derivatives. The concentration of the aminopolysaccharide solution is 10–50 mg / mL; the aminopolysaccharide is selected from at least one of carboxymethyl chitosan, carboxyethyl chitosan, carboxypropyl chitosan, carboxybutyl chitosan, aminated chitosan, and aminated mannose. The volume ratio of the drug-loaded nanoparticle solution to the aldehyde polysaccharide solution is 1:3 to 10; the volume ratio of the aldehyde polysaccharide solution to the amino polysaccharide solution is 4 to 10:

1.

6. A method for preparing the drug-loaded nanoparticle composite hydrogel according to claim 5, characterized in that, Includes the following steps: I) The drug-loaded nanoparticle solution according to claim 3 is mixed with the aldehyde polysaccharide solution to obtain a mixture; II) Mix the aminopolysaccharide solution with the mixture to allow the aldehyde polysaccharide and aminopolysaccharide to undergo a Schiff base reaction, thereby obtaining a drug-loaded nanoparticle composite hydrogel.

7. The preparation method according to claim 6, characterized in that, The aldehyde polysaccharide in the aldehyde polysaccharide solution is obtained by oxidizing the precursor polysaccharide with an alkali metal perhalate.

8. The preparation method according to claim 7, characterized in that, The alkali metal perhalate is selected from at least one of the alkali metal perchlorate, perbromate, and periodate; the alkali metal is selected from at least one of Li, Na, and K.

9. The preparation method according to claim 7 or 8, characterized in that, The preparation method of the aldehyde polysaccharide includes the following steps: a) Add the precursor polysaccharide to water and stir to obtain a precursor polysaccharide dispersion; wherein, based on 1g of precursor polysaccharide, the amount of water used is 20-100mL. b) Add the alkali metal perhalate to the precursor polysaccharide dispersion under light-protected conditions, and react under light-protected conditions and at 20-40°C to obtain reaction solution A; then, add C2-C5 alkyldiol to reaction solution A to obtain reaction solution B; wherein, based on 1g of precursor polysaccharide, the amount of C2-C5 alkyldiol is 1-5mL. c) Place reaction solution B in a dialysis bag, then place the dialysis bag in water for dialysis purification to obtain purified reaction solution B; freeze-dry purified reaction solution B to obtain aldehyde polysaccharide.

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

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