Protein nanoparticle for treating ulcerative colitis as well as preparation method and application thereof

Abstract

The invention relates to protein nanoparticles for treating ulcerative colitis as well as a preparation method and application thereof, and relates to the field of nano pharmaceutical preparations. 5-aminosalicylic acid is encapsulated in zein to achieve the purpose of improving the bioavailability of the 5-aminosalicylic acid by preparing protein nanoparticles of a targeted inflammation colon part, mannose grafted hyaluronic acid is used as a stabilizer to be attached to the surfaces of the zein nanoparticles through electrostatic adsorption and hydrogen-bond interaction, and the bioavailability of the 5-aminosalicylic acid is improved. Mannose can be targeted to immune cells through a mannose receptor, and can be cooperated with 5-aminosalicylic acid to treat ulcerative colitis. The hyaluronic acid has the effects of stabilizing the protein nanoparticles and targeting macrophages and intestinal epithelial cell CD44 receptors in ulcerative colitis tissues, so that aggregation of the nanoparticles in the colitis tissues is mediated, the protein nanoparticles have the functions of resisting inflammation, resisting oxidation and repairing barrier injury, and a new research direction is provided for treatment of ulcerative colitis.

Description

Technical Field

[0001] The invention belongs to the field of oral drug targeted treatment of inflammation, and specifically relates to a protein nanoparticle for treating ulcerative colitis, and a preparation method and application thereof. Background Art

[0002] Ulcerative colitis (UC) is a chronic, recurrent gastrointestinal disease with the main lesions located in the colon and rectum. Clinical symptoms such as abdominal pain and bloody diarrhea are more common. The etiology and pathogenesis have not yet been fully clarified, and may be related to environmental factors, autoimmunity, genetic susceptibility, and infectious factors. Its onset is closely related to abnormal immune function and increased inflammatory mediators. UC is an intestinal natural immune and acquired immune response that alternates between attacks and remissions. With the continuous deepening of immunological research, more and more evidence shows that immune factors have an important pathogenic role, and epithelial barrier damage plays a key role in the coordination of colitis. The incidence of ulcerative colitis is highest in Europe and the United States; in recent years, the incidence in Asia and low-income countries has also risen sharply recently. Severe ulcerative colitis gradually fibroses and even evolves into colon cancer, which seriously endangers the health of patients. Traditional treatments generally use anti-inflammatory drugs and immunosuppressants. Although they can control inflammation, they lack long-term effectiveness and have great safety risks. In recent years, targeted drug delivery preparations have been developed and applied. Targeted drug delivery is also known as localized drug release. It uses certain methods to aggregate drugs in the target area and implement targeted drug delivery to the diseased tissue. The characteristics of targeted drug preparations are drug controlled release, localized concentration, high safety, and good biocompatibility. All corresponding targeted treatments can significantly increase the drug concentration in the treatment area, improve the efficacy, reduce the drug dosage, and reduce toxic and side effects. It is a safe and efficient new preparation. In order to enhance the therapeutic effect of drugs and reduce toxic and side effects, targeted drug technology is an effective solution.

[0003] The advantages of nanoformulations are: (1) they can effectively deliver bioactive molecules to the lesion site and improve the therapeutic efficiency; (2) due to their unique nanosize, they can accumulate in the lesion site of ulcerative colitis based on the enhanced permeation and retention effect (EPR); (3) they can reduce the side effects of traditional drugs; and (4) they can improve the bioavailability of drugs. Carbohydrate macromolecules are widely used as nanocarriers because of their good biocompatibility and biodegradability.

[0004] 5-Aminosalicylic acid (5-ASA) is a drug for the treatment of mild to moderate UC. It reduces inflammation by inhibiting the release of leukotrienes from the colonic mucosa, inhibiting the migration of macrophages, and inhibiting the production of immunoglobulins by peripheral blood and intestinal lymphocytes. The disadvantage is that the effect of 5-ASA is local, not after absorption into the blood circulation. Free 5-ASA is rapidly and almost completely absorbed by the system through the small intestine, so that the amount of drug that reaches the inflamed colon is very limited, resulting in a lower success rate of treatment. Therefore, it is often introduced into the intestine through different dosage forms and routes of administration to exert pharmacological effects. Nanoparticles have unique advantages as carriers for controlled release of drugs. They can improve the solubility of drugs, change the absorption and bioavailability of drugs, change the pharmacokinetic properties of drugs, reduce their toxic side effects, and have the characteristics of targeting, sustained release, and stability.

[0005] There are many materials used to prepare nano drug carriers. Zein extracted from corn kernels is hydrophobic because its hydrophobic amino acid content exceeds 50%. Due to its insoluble properties in water, zein nanoparticles can be prepared by anti-solvent co-precipitation. Zein is widely used in oral drug delivery due to its biocompatibility, biodegradability and low toxicity. Zein is insoluble in acidic aqueous environments, which prevents drug release in the upper gastrointestinal tract and allows sustained drug release in the colon. However, zein is digested by enzymes in intestinal fluid, resulting in the destruction of the protein structure, which may lead to drug release. Limiting its degradation helps the targeted release of drugs in the colon. However, due to the structural specificity of zein, there are positive charges and hydrophobic structures, and it is often faced with problems such as low encapsulation efficiency, poor formulation stability, and lack of active targeting when used to encapsulate drugs.

[0006] Hyaluronic acid is a widely existing polysaccharide component in the extracellular matrix. It is composed of D-glucuronic acid and N-acetylglucosamine groups and has good biocompatibility. Hyaluronic acid is often used as a drug carrier and stabilizer to exert sustained release and targeting effects. At the same time, studies have shown that high molecular weight hyaluronic acid has anti-inflammatory effects. CD44 is overexpressed on colonic epithelial cells and macrophages in ulcerative colitis tissues. Hyaluronic acid binds to CD44 receptors highly expressed on the surface of colonic epithelial cells and macrophages, allowing the bound drugs to be released in a targeted manner to exert anti-inflammatory effects. The application of hyaluronic acid in nano drug release systems can enable nanoparticles to enter target cells through CD44 receptor-mediated endocytosis.

[0007] Mannose receptor is an important pattern recognition receptor and endocytosis receptor in the innate immune system. It is mainly present on the cell membrane surface of macrophages and dendritic cells recruited at the site of inflammation. Mannose can specifically bind to the mannose receptor, and studies have shown that mannose can prevent intestinal epithelial damage, repair mitochondrial dysfunction and myosin light chain kinase (MLCK)-induced tight junction damage, and can synergize with 5-aminosalicylic acid to treat ulcerative colitis.

[0008] In summary, the present invention utilizes the anti-solvent method to prepare protein nanoparticles loaded with 5-aminosalicylic acid, and uses the synthetic product of mannose grafted hyaluronic acid as a stabilizer to prepare targeted protein nanoparticles to construct a UC-targeted nano-drug delivery system. The nanoparticles can protect 5-aminosalicylic acid and target the colitis site, thereby solving the problem that the 5-aminosalicylic acid cannot reach the colitis lesion site in vivo. Under the protection and delivery of the nanocarrier, most of the drugs can pass through the stomach and small intestine intact to reach the diseased colon, and accumulate and stay in the diseased colon site for a long time, slowly releasing the drugs, thereby exerting better anti-inflammatory, antioxidant and intestinal epithelial barrier damage repair functions. Summary of the invention

[0009] The first object of the present invention is to provide a protein nanoparticle for treating ulcerative colitis. The protein nanoparticle is a protein nanoparticle that can be used for targeting macrophages and colon epithelial cells, has good biocompatibility and stability, and can be used for the treatment of ulcerative colitis.

[0010] The second object of the present invention is to provide a method for preparing the above-mentioned protein nanoparticles, which method is simple, fast and easy to promote and implement.

[0011] The third object of the present invention is to provide the use of the above protein nanoparticles in the preparation of drugs for treating ulcerative colitis.

[0012] In order to achieve the above object, the present invention adopts the following technical solution:

[0013] In the first aspect, the present invention seeks to protect a protein nanoparticle (AZ@HM) for treating ulcerative colitis, wherein the protein nanoparticle is obtained by physically cross-linking a stabilizer of mannose-grafted hyaluronic acid on the surface of zein nanoparticles (AZ) encapsulating 5-aminosalicylic acid through electrostatic adsorption.

[0014] Furthermore, in the above-mentioned protein nanoparticles: the molar ratio of mannose to hyaluronic acid is 5:1 to 20:1; the zein nanoparticles encapsulating 5-aminosalicylic acid are obtained by loading 5-aminosalicylic acid into zein nanoparticles by an anti-solvent co-precipitation method, wherein the mass ratio of 5-aminosalicylic acid to zein is 1:10 to 9:10; the mass ratio of the mannose-grafted hyaluronic acid stabilizer to the zein nanoparticles encapsulating 5-aminosalicylic acid is 1:10 to 1:1.

[0015] In the technical solution of the present invention, the drug loaded into the zein nanoparticles is 5-aminosalicylic acid, and its structure is as follows:

[0016] In the technical solution of the present invention, the structural formula of hyaluronic acid, a reactant used to prepare a stabilizer, is as follows:

[0017] Furthermore, the preparation process of the above-mentioned zein nanoparticles encapsulating 5-aminosalicylic acid is as follows: weigh zein and 5-aminosalicylic acid and dissolve them in an ethanol solution to obtain a mixed solution, slowly dropwise add the mixed solution into water under stirring to spontaneously form uniform nanoparticles, and remove ethanol by rotary evaporation to obtain zein nanoparticles encapsulating 5-aminosalicylic acid.

[0018] Furthermore, the preparation process of the above-mentioned mannose-grafted hyaluronic acid stabilizer is as follows: weigh hyaluronic acid, N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine, dissolve them in dimethyl sulfoxide, swell them under heating and stirring in a water bath at 50-60°C, weigh mannose and add it to the reaction system, react under heating and stirring in a water bath at 50-60°C, remove dimethyl sulfoxide by dialysis after the reaction, and the white powder product obtained after freeze-drying is the stabilizer.

[0019] Furthermore, the process of physically cross-linking the stabilizer of mannose-grafted hyaluronic acid on the surface of zein nanoparticles encapsulating 5-aminosalicylic acid through electrostatic adsorption is as follows: dissolving the stabilizer of mannose-grafted hyaluronic acid in water, and mixing it with the zein nanoparticles encapsulating 5-aminosalicylic acid under stirring to obtain stable and uniform protein nanoparticles for treating ulcerative colitis.

[0020] Furthermore, the concentration of the ethanol solution is 70% to 80%.

[0021] In a second aspect, the present invention claims a method for preparing the above-mentioned protein nanoparticles, the preparation method comprising the following steps:

[0022] (1) Preparing a stabilizer of mannose-grafted hyaluronic acid and zein nanoparticles encapsulating 5-aminosalicylic acid respectively:

[0023] Weigh hyaluronic acid, N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine, dissolve in dimethyl sulfoxide, swell under heating and stirring in a water bath at 50-60°C, weigh mannose and add it to the reaction system, react under heating and stirring in a water bath at 50-60°C, remove dimethyl sulfoxide by dialysis after the reaction, and the white powder product obtained after freeze-drying is the stabilizer;

[0024] Weighing zein and 5-aminosalicylic acid and dissolving them in an ethanol solution to prepare a mixed solution, slowly dropping the mixed solution into water under stirring to spontaneously form uniform nanoparticles, and removing the ethanol by rotary evaporation to obtain zein nanoparticles encapsulating 5-aminosalicylic acid;

[0025] (2) Physically cross-linking the stabilizer of mannose-grafted hyaluronic acid on the surface of zein nanoparticles encapsulating 5-aminosalicylic acid through electrostatic adsorption: The stabilizer of mannose-grafted hyaluronic acid is dissolved in water and mixed with the zein nanoparticles encapsulating 5-aminosalicylic acid under stirring to obtain stable and uniform protein nanoparticles for treating ulcerative colitis.

[0026] Furthermore, in the above-mentioned preparation method, the mass ratio of 5-aminosalicylic acid to zein in step (1) is 1:10 to 9:10; the molar ratio of mannose to hyaluronic acid is 10:1 to 20:1; and the mass ratio of the stabilizer of mannose-grafted hyaluronic acid to the zein nanoparticles encapsulating 5-aminosalicylic acid in step (2) is 1:10 to 1:1, preferably 1:10 to 1:5.

[0027] In a specific embodiment of the present invention, the mass ratio of 5-aminosalicylic acid to zein in step (1) is 1:10 to 4:5; the concentration of the ethanol solution is 70% to 80%. The stirring speed in the process of preparing zein nanoparticles encapsulating 5-aminosalicylic acid in step (1) is 3000 rpm, but is not limited thereto.

[0028] The molecular weight of the hyaluronic acid in step (1) is 5000 to 10000; the molar ratio of mannose to hyaluronic acid can be: 20:1, 15:1 or 10:1, preferably, the molar ratio of mannose to hyaluronic acid is 10:1 to 20:1, but is not limited thereto;

[0029] The molar ratio of hyaluronic acid, N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine in step (1) is 5:(5-10):4. The molar volume ratio (mmol:ml) of hyaluronic acid to dimethyl sulfoxide is 1:20-1:50.

[0030] Weigh hyaluronic acid, N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine, dissolve in dimethyl sulfoxide, swell for 4 hours under heating and stirring at 60°C water bath, stirring speed is 4500rpm; weigh mannose and add it to the reaction system, react for 48 hours under heating and stirring at 60°C water bath, stirring speed is 4500rpm. After the reaction, dialyze for 48 hours through a dialysis bag with a molecular weight of 8000 to remove dimethyl sulfoxide.

[0031] In a third aspect, the present invention seeks to protect the use of the above-mentioned protein nanoparticles in the preparation of drugs for treating ulcerative colitis.

[0032] The design principle of the technical solution of the present invention is:

[0033] 5-aminosalicylic acid is one of the main drugs commonly used in the clinic for the treatment of ulcerative colitis. 5-aminosalicylic acid exerts its anti-inflammatory effect mainly by inhibiting the production of leukotrienes, prostaglandins E and free radicals. After being directly taken orally, 5-aminosalicylic acid can be absorbed by the stomach and small intestine and then acetylated in the small intestinal epithelial cells and liver, losing its therapeutic effect. Not only is there not enough drug to reach the colon to play an anti-inflammatory role, but the drug absorbed into the body is prone to renal toxicity. Therefore, colon-targeted delivery of 5-aminosalicylic acid is particularly important. Nanoparticles have the property of aggregating at the site of colon inflammation, so 5-aminosalicylic acid is wrapped with enteric materials and prepared into nanoparticles of suitable particle size and distribution, which can achieve the purpose of targeted delivery of drugs to colon inflammation.

[0034] Zein has good self-assembly properties and can be used to prepare regular spherical nanoparticles of uniform size. It can be used for loading, transporting and controlled release of bioactive substances and drugs, and can improve the problems of low water solubility, photothermal sensitivity, acid-base sensitivity and low bioavailability of drugs. Zein also has strong biocompatibility and biodegradability. However, the nanoparticles prepared by single zein are not stable enough, and the embedding rate and loading efficiency are low, which limits its wider application. Therefore, by combining zein with proteins, polysaccharides, polyphenols, surfactants and other substances through hydrogen bonds, hydrophobic interactions, electrostatic interactions, etc., more stable zein nanoparticles can be constructed.

[0035] Active targeting can deliver therapeutic drugs directly to colonic epithelial cells and immune cells, playing a key role in ulcerative colitis. Due to the improved cellular uptake efficiency and sufficient internalization of intracellular drugs, active targeted drug delivery systems can overcome the rapid elimination and off-target effects of ulcerative colitis tissue. Macrophages are an important component of the intestinal innate immune system and maintain tissue homeostasis through immunomodulatory effects on inflammatory responses. Activated macrophages secrete a large number of proinflammatory cytokines, including interleukin-6 and tumor necrosis factor-α. In addition, various transmembrane receptors such as CD44, folate, mannose receptor, and CD98 are overexpressed not only in inflamed colitis tissues but also on the surface of activated macrophages, providing potential binding sites for active targeting systems.

[0036] Hyaluronic acid, also known as uronic acid or hyaluronan, is a non-sulfated glycosaminoglycan and an endogenous high molecular weight linear polysaccharide with repeating disaccharide units (β-1,4-d-glucuronic acid and β-1,3-N-acetyl-d-glucosamine). Hyaluronic acid is an effective material due to its excellent biocompatibility and mucoadhesion, which is superior to other natural polysaccharides for active targeting. In addition, hyaluronic acid is a natural ligand for the CD44 receptor, which is overexpressed on inflamed colonic epithelial cells and activated macrophages in patients with ulcerative colitis. Many biological functions, including cell migration and adhesion, rely on the interaction between hyaluronic acid and CD44. Hyaluronic acid is negatively charged at physiological pH and may show enhanced adhesion to inflamed tissues because ulcer tissues are composed of a large number of positively charged proteins. In addition, hyaluronic acid has been used as an active targeting ligand to modify nanocarriers to enhance the cellular uptake of drugs or nanocarriers through CD44-mediated endocytosis.

[0037] Mannose is a natural bioactive monosaccharide involved in glycoprotein metabolism and synthesis, with anti-inflammatory and antioxidant activities. Mannose treatment enhanced lysosomal integrity and limited the release of cathepsin B, preventing tight junction damage induced by mitochondrial dysfunction and myosin light chain kinase (MLCK) in the setting of intestinal epithelial damage. Mannose and 5-aminosalicylic acid have synergistic therapeutic effects on colitis, and mannose can mediate targeted endocytosis of protein nanoparticles through mannose receptors. Therefore, linking mannose to hyaluronic acid is an alternative approach for the treatment of colitis and other diseases associated with intestinal barrier dysfunction.

[0038] It can be seen from the above technical solution that compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0039] (1) The protein nanoparticles of the present invention are a colon-targeted nanoformulation that can improve the loading, transportation and controlled release of 5-aminosalicylic acid, improve its low water solubility and low bioavailability, increase the drug content and retention time in the colon, and thus better exert the anti-inflammatory effect.

[0040] (2) The hyaluronic acid in the protein nanoparticles of the present invention not only improves the stability of the nanoformulation but also increases the targeting of the nanoformulation. On the one hand, the zein nanoparticles and the stabilizer are combined by electrostatic adsorption, physical cross-linking, hydrogen bonding, etc.

[0041] (3) The mannose in the protein nanoparticles of the present invention can be targeted to immune cells via mannose receptors and can synergize with 5-ASA to treat ulcerative colitis.

[0042] (4) The protein nanoparticles of the present invention can regulate the body's immunity, effectively alleviate the symptoms of ulcerative colitis in mice and repair the barrier damage of the intestinal tract caused by inflammation. Compared with ordinary nanoparticles, the efficacy of anti-ulcerative colitis is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 These are the H NMR spectra and IR spectral scanning spectra of the reactants and synthetic products in Example 1 of the present invention, wherein Figures a, b, and c are the H NMR spectra of mannose, hyaluronic acid, and mannose-grafted hyaluronic acid stabilizer, respectively, and Figure d is the IR spectral scanning spectra of mannose, hyaluronic acid, and mannose-grafted hyaluronic acid stabilizer.

[0044] Figure 2 The particle size distribution pictures and potential diagrams of different groups of preparations in Example 4 of the present invention are shown in Figures a, b, and c, respectively, for the particle size distribution pictures of AZ, AZ@HA, and AZ@HM. Figure d is a potential diagram of different groups of preparations.

[0045] Figure 3 These are the particle size and PDI graphs of different groups of preparations in Example 5 of the present invention in artificial simulated gastric juice and artificial simulated intestinal juice, wherein Figures a, b, and c are respectively the particle size and PDI change graphs of AZ, AZ@HA, and AZ@HM in artificial simulated gastric juice at different times, and Figures d, e, and f are respectively the particle size and PDI change graphs of AZ, AZ@HA, and AZ@HM in artificial simulated intestinal juice at different times.

[0046] Figure 4 These are in vitro drug release diagrams of the three nanoparticles in Example 6 of the present invention under different conditions, wherein Figure a is an in vitro drug release diagram under artificial simulated gastric fluid conditions, and Figure b is an in vitro drug release diagram under artificial simulated intestinal fluid conditions.

[0047] Figure 5These are graphs for evaluating the cell tolerance of different cells to different concentrations of AZ@HM preparations in Example 7 of the present invention, wherein Graph a uses the HT-29 cell line as a model, and Graph b uses the RAW264.7 cell line as a model.

[0048] Figure 6 This is a graph showing the determination of the inflammatory factor content of LPS-stimulated HT-29 cells by different preparation groups in Example 8 of the present invention, wherein Figures a, b, c, and d are graphs showing the determination of the content of IL-1β, TNF-α, IL-6, and IL-10 by different preparation groups, respectively.

[0049] Figure 7 This is a graph showing the determination of inflammatory factor contents in LPS-stimulated RAW264.7 cells by different preparation groups in Example 9 of the present invention, wherein Figures a, b, c, and d are graphs showing the determination of IL-1β, TNF-α, IL-6, and IL-10 contents by different preparation groups, respectively.

[0050] Figure 8 This is a graph showing the uptake of different preparations by HT-29 cells at different times in Example 10 of the present invention.

[0051] Fig. 9 This is a graph showing the uptake of different preparations by RAW264.7 cells at different times in Example 11 of the present invention.

[0052] Fig.10 Graphs showing the effects of different preparations in Example 12 of the present invention on the level of ROS in LPS-stimulated HT-29 cells, wherein Figure a is a graph showing the level of intracellular ROS detected by flow cytometry under treatment with different preparations, and Figure b is a quantitative graph showing the fluorescence signal intensity of intracellular ROS detected by flow cytometry after treatment with different preparations.

[0053] Fig.11 Figures 14 and 15 are graphs showing the effects of different preparations on the polarization of LPS-stimulated RAW264.7 cells in Example 13 of the present invention. Figures a and b are respectively a fluorescence intensity graph and a quantitative graph of the M1 cell marker CD86 detected by flow cytometry, and Figures c and d are respectively a fluorescence intensity graph and a quantitative graph of the M2 cell marker CD206 detected by flow cytometry.

[0054] Fig.12 This is a graph showing the effects of different preparations in Example 14 of the present invention on the tight junctions of HT-29 cells stimulated by LPS.

[0055] Fig.13 Graphs showing the in vivo therapeutic effects of different preparations in Example 15 of the present invention on DSS-induced ulcerative colitis, wherein Graph a shows the weight changes of mice in each group, and Graph b shows the DAI score changes of mice in each group.

[0056] Fig.14These are the colon tissue pathology observations of different preparations in Example 16 of the present invention for treating DSS-induced ulcerative colitis, wherein Figure a is a graph showing the changes in colon length in different treatment groups, and Figure b is a H&E graph of colon tissue. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0058] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0059] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0060] Example 1 Preparation of mannose-grafted hyaluronic acid stabilizer (HM)

[0061] Weigh hyaluronic acid (0.25mM, 100.82mg) with a molecular weight of 9000, N,N'-diisopropylcarbodiimide (0.5mM, 63.1mg) and 4-dimethylaminopyridine (0.2mM, 24.434mg) and dissolve in 7mL of dimethyl sulfoxide, heat in a water bath at 60°C for 4h under stirring at 4500rpm, weigh mannose (5mM, 960mg) and add it to the reaction system, heat in a water bath at 60°C for 48h under stirring at 4500rpm, and after the reaction, dialyze for 48h through a dialysis bag with a molecular weight of 8000 to remove dimethyl sulfoxide. The white powder product obtained after freeze-drying is the stabilizer. The structure of the compound synthesized in Example 1 was determined by nuclear magnetic resonance hydrogen spectrum (1H-NMR), and the deuterated solvent was selected as D2O. The results are as follows Figure 1Figures a, b, and c are the hydrogen nuclear magnetic resonance spectra of mannose, hyaluronic acid, and mannose-grafted hyaluronic acid stabilizer, respectively. The synthetic product has the chemical shift of the characteristic groups of the reactants, and the degree of substitution is about 16%. Infrared spectroscopy (0.75-500 μm) is produced by irradiating the sample with infrared light of continuous wavelength. The molecules absorb the energy of infrared rays, causing the transition of the intramolecular vibrational energy level. The position and intensity of the absorption peaks on the infrared spectrum can be used to determine whether the compound to be tested has certain functional groups. This test uses the potassium bromide (KBr) tablet method to prepare the sample. After scanning the blank sample, the sample is scanned and measured. Figure d is the infrared spectrum scanning chart of mannose, hyaluronic acid, and mannose-grafted hyaluronic acid stabilizer. The infrared spectrum of mannose is at 2977cm -1 、2929cm -1 The peak at 1136 cm -1 、1040cm -1 It is the in-plane bending vibration peak of -OH of secondary alcohol and the stretching vibration peak of CO. The infrared spectrum of hyaluronic acid is at 3385cm -1 A broad -OH peak at 1750 cm -1 The characteristic peak of -COOH is at 1600cm -1 The NH in-plane bending vibration peak of the amide bond is at 1040 cm -1 and 946cm -1 The mannose-grafted hyaluronic acid stabilizer has the characteristic peaks of mannose and hyaluronic acid and the 1735cm -1 The characteristic peak of -COOH at 1750 cm-1 is blue-shifted due to the esterification reaction to form -COOC bond, and the carbonyl stretching vibration peak changes to 1750 cm-1. -1 .

[0062] Example 2 Preparation of Zein Nanoparticles (AZ)

[0063] Weigh 50 mg of zein and 40 mg of 5-aminosalicylic acid and dissolve them in 5 mL of 75% ethanol solution. Slowly add the solution dropwise into 10 mL of pure water at a stirring speed of 3000 rpm, and remove the ethanol by rotary evaporation at 80°C for 10 min to obtain zein nanoparticles encapsulating 5-aminosalicylic acid at a concentration of 8 mg / ml.

[0064] Example 3 Preparation of a protein nanoparticle (AZ@HA) for treating ulcerative colitis

[0065] 10 mg of hyaluronic acid was weighed and dissolved in 10 mL of purified water to obtain a stabilizer solution with a concentration of 1 mg / mL. 1 mL of zein nanoparticles (8 mg / ml) prepared in Example 2 was added to 1 mL of the stabilizer and mixed under stirring to obtain stable and uniform protein nanoparticles.

[0066] Example 4 Preparation of a protein nanoparticle (AZ@HM) for treating ulcerative colitis

[0067] Weigh 10 mg of the mannose-grafted hyaluronic acid stabilizer (HM) prepared in Example 1 and dissolve it in 10 mL of purified water to obtain a stabilizer solution with a concentration of 1 mg / mL. Take 1 mL of the zein nanoparticles (8 mg / ml) prepared in Example 2 and add it to 1 mL of the stabilizer. Mix under stirring to obtain stable and uniform protein nanoparticles. Figure 2 a, b, and c are the particle size distribution pictures of the zein nanoparticles (AZ) prepared in Example 2, the protein nanoparticles (AZ@HA) prepared in Example 3, and the protein nanoparticles (AZ@HM) prepared in Example 4, respectively. The particle size distribution of AZ is 121.23±2.49nm, the particle size distribution of AZ@HA is 135.20±1.56nm, and the particle size distribution of AZ@HM is 218.17±7.88nm. Figure d is the potential diagram of different groups of preparations, measured by Malvern laser particle size analyzer, AZ potential is 26.4±2.2mV, AZ@HA potential is -17.6±0.6mV, and AZ@HM potential is -23.3±0.9mV. Hyaluronic acid and mannose-grafted hyaluronic acid stabilizers encapsulate zein by electrostatic adsorption, physical cross-linking, hydrogen bonding, etc., so that the positively charged zein nanoparticles achieve charge reversal, which is conducive to the accumulation of protein nanoparticles in positively charged diseased areas.

[0068] Example 5 Stability of three nanoparticles

[0069] 2 mL of the three nanoparticles AZ, AZ@HA, and AZ@HM prepared in Example 2, Example 3, and Example 4 were dispersed in 10 mL of artificial simulated gastric fluid (pH = 1.2) and artificial simulated intestinal fluid (pH = 6.8), and the particle size change and polymer dispersibility index (PDI) of the nanoparticles were monitored by a Malvern laser particle size analyzer. Figure 3 a, b, c, respectively show the particle size and PDI of AZ, AZ@HA, and AZ@HM in artificial simulated gastric juice at different time points. Figures e, f, and g, respectively show the particle size and PDI of AZ, AZ@HA, and AZ@HM in artificial simulated intestinal juice at different time points. The particle size and PDI of AZ@HA and AZ@HM in artificial simulated gastric juice and artificial simulated intestinal juice are basically stable, while the particle size and PDI of AZ increase in both systems, which may be due to the digestion of zein by enzymes in the intestinal juice, resulting in the destruction of the protein structure.

[0070] Example 6 In vitro drug release of three nanoparticles under different conditions

[0071] The present invention adopts dynamic dialysis method to investigate the release behavior of drug-loaded protein nanoparticles. 2 mL of three nanoparticles AZ, AZ@HA, and AZ@HM (containing about 500 μg of 5-ASA) prepared in Example 2, Example 3, and Example 4 are accurately measured and placed in a dialysis bag (molecular weight cutoff: 3500 Da), the mouth is tightened, and 40 mL of artificial simulated intestinal fluid with a pH value of 6.8 or artificial simulated gastric fluid with a pH value of 1.2 is put into the bag, shaken at 37°C±0.5°C and 300 r / min, and 1 mL is sampled in the artificial simulated gastric fluid system at 0.5, 1, 2, 4, 6, and 8 h, and 1 mL is sampled in the artificial simulated intestinal fluid system at 0.5, 1, 2, 4, 6, 8, 10, 12, 24, and 36 h, and fresh release medium of the same volume and temperature is added at the same time. The sample was filtered through a 0.45 μm microporous membrane, the primary filtrate was discarded, 20 μL of the subsequent filtrate was taken, and the 5-amino acid salicylic acid content was determined by HPLC, and the cumulative release percentage was calculated. Figure 4 As shown in a and b, compared with the free drug 5-ASA, the cumulative release of AZ in 4 h was more than 40% in both artificial simulated intestinal fluid and artificial simulated gastric fluid, while the cumulative release of AZ@HA and AZ@HM in both solutions was less than 20%, confirming that after the zein nanoparticles were encapsulated by hyaluronic acid and synthetic stabilizers, the stability of the protein nanoparticles was improved, which helped the protein nanoparticles to target the diseased parts of the colon and achieve good retention and therapeutic effects.

[0072] Example 7 Biosafety of AZ@HM formulations on different cell lines

[0073] HT-29 cells and RAW264.7 cells in the logarithmic growth phase were seeded in a 96-well plate at a density of 5000 cells / well and cultured until the cell confluence reached 80%. 200 μL of serum-free culture medium was added to the control group, and the protein nanoparticles prepared in Example 4 were diluted with serum-free culture medium to the designed different concentrations and added to each well in the remaining groups. Three replicate wells were set up, with 200 μL in each well. After 24 hours of incubation, 20 μL of MTT solution (5 mg / mL) was added to each well, and the incubation continued at 37°C for 4 hours. The supernatant was then discarded, 200 μL of DMSO was added to dissolve it, and the absorbance was measured at 492 nm using an enzyme reader to determine whether protein nanoparticles of different concentrations were cytotoxic to HT-29 cells and RAW264.7 cells. The results are shown in Figure 5As shown in a and b, the results showed that 10, 25, 50, and 100 μg / mL protein nanoparticles had good biosafety to HT-29 cells and RAW264.7 cells, while 250 μg / mL protein nanoparticles were cytotoxic to both HT-29 cells and RAW264.7 cells, and the cell survival rates were all lower than 80%.

[0074] Example 8 Investigation of different preparation groups on inflammatory factors in HT-29 cells stimulated by LPS

[0075] Take HT-29 cells that are in good growth condition and in the logarithmic growth phase, digest them with trypsin, make single cell suspension with DMEM medium containing 10% FBS, count them with a cell counting plate, and adjust the cell concentration to 2.5x10 according to the counting results. 4 The single cell suspension of 100 μg / ml was evenly inoculated in a 96-well plate at 200 μL per well, and cultured in a 37°C, 5% CO2 incubator for 24 hours. After the cells were well attached, the cell wells were divided into a control group, a model (LPS stimulation) group, a free drug group, an AZ preparation group, an AZ@HA preparation group, and an AZ@HM preparation group. The old culture medium in each well of the control group was discarded, and a new serum-free culture medium was added. Except for the control group, LPS solution was added to all other groups, so that the final concentration of LPS solution in each well was 10 μg / mL. After 12 hours, 100 μL of serum-free culture medium was added to the control group and the model group, and free 5-ASA, three nanoparticles AZ, AZ@HA, and AZ@HM (containing about 50 μg of 5-ASA) prepared in Example 2, Example 3, and Example 4 were added to the free drug group, AZ preparation group, AZ@HA preparation group, and AZ@HM preparation group, respectively. Three duplicate wells were set up in each group. After culturing the 96-well plate in a 37°C, 5% CO2 incubator for 24 hours, the cell supernatant was collected and centrifuged at 3000 r / min for 15 minutes. The IL-1β, TNF-α, IL-6 and IL-10 kits were tested according to the instructions of the corresponding instructions. The absorbance of each group was measured with an enzyme marker, and the corresponding IL-1β, TNF-α, IL-6 and IL-10 contents were calculated. Figure 6 a, b, c, d show that after modeling, by comparing the in vitro anti-inflammatory effects of each preparation group on LPS-stimulated HT-29 cells, it was found that both AZ@HA and AZ@HM can effectively reduce the levels of pro-inflammatory inflammatory factors IL-1β, TNF-α, and IL-6 produced by LPS-stimulated HT-29 cells, and increase the level of anti-inflammatory inflammatory factor IL-10 produced by LPS-stimulated HT-29 cells. There was no significant statistical difference between the AZ@HA preparation group, the AZ@HM preparation group and the control group, indicating that after treatment in the preparation group, the levels of inflammatory factors in LPS-stimulated cells were no different from those in normal cells.

[0076] Example 9 Investigation of the effects of different preparations on inflammatory factors in LPS-stimulated RAW264.7 cells

[0077] Take RAW264.7 cells that are in good growth condition and in the logarithmic growth phase, scrape them with a cell scraper, make a single cell suspension with DMEM medium containing 10% FBS, count them with a cell counting plate, and adjust the cell concentration to 2.5x10 according to the counting results. 4 / ml single cell suspension, 200μL per well was evenly inoculated in a 96-well plate, and cultured in a 37℃, 5% CO2 incubator for 24h. After the cells were well attached, the cell wells were divided into a control group, a model (LPS stimulation) group, a free drug group, an AZ preparation group, an AZ@HA preparation group, and an AZ@HM preparation group. The old culture medium in each well of the control group was discarded, and a new serum-free culture medium was added. Except for the control group, LPS solution was added to all other groups, so that the final concentration of LPS solution in each well was 10μg / mL. After 12h, 100μL of serum-free culture medium was added to the control group and the model group, and free 5-ASA, three nanoparticles AZ, AZ@HA, and AZ@HM (containing about 50μg of 5-ASA) prepared in Example 2, Example 3, and Example 4 were added to the free drug group, AZ preparation group, AZ@HA preparation group, and AZ@HM preparation group, respectively. Three duplicate wells were set for each group. After culturing the 96-well plate in a 37°C, 5% CO2 incubator for 24 hours, the cell supernatant was collected and centrifuged at 3000 r / min for 15 minutes. The IL-1β, TNF-α, IL-6 and IL-10 kits were tested according to the instructions of the corresponding instructions. The absorbance of each group was measured with an enzyme marker, and the corresponding IL-1β, TNF-α, IL-6 and IL-10 contents were calculated. Figure 7 a, b, c, and d show that after modeling, by comparing the in vitro anti-inflammatory effects of each preparation group on LPS-stimulated RAW264.7 cells, it was found that both AZ@HA and AZ@HM can effectively reduce the levels of pro-inflammatory inflammatory factors IL-1β, TNF-α, and IL-6 produced by LPS-stimulated RAW264.7 cells, and increase the level of anti-inflammatory inflammatory factor IL-10 produced by LPS-stimulated RAW264.7 cells. There was no significant statistical difference between the AZ@HA preparation group, the AZ@HM preparation group, and the control group, indicating that after treatment in the preparation group, the levels of inflammatory factors in LPS-stimulated cells were no different from those in normal cells.

[0078] Example 10 Uptake of different preparations by HT-29 cells

[0079] The therapeutic effect of protein nanoparticles depends on rapid uptake and effective accumulation in cells. Therefore, the cellular uptake of C6-labeled protein nanoparticles (the preparation method of C6-labeled protein nanoparticles is the same as the preparation method of Example 2, except that 5-ASA is replaced by C6) was evaluated by laser scanning confocal microscopy (LSCM). As time goes by, the green fluorescence intensity gradually increases, indicating that the uptake of the preparation by HT-29 cells is time-dependent. Since the stabilizer of mannose-grafted hyaluronic acid is designed to be endocytosed by CD44 receptor-mediated endocytosis, the cells are treated with hyaluronic acid in advance and then administered. The results are as follows Figure 8 As shown, the uptake of the preparation encapsulated by hyaluronic acid and stabilizer is significantly better than that of the free drug and is time-dependent. The cells pretreated with hyaluronic acid have poor uptake of the preparation, confirming that the preparation is taken into the cell through the CD44 receptor.

[0080] Example 11 Uptake of different preparations by RAW264.7 cells

[0081] The therapeutic effect of protein nanoparticles depends on rapid uptake and effective accumulation in cells. Therefore, the cellular uptake of C6-labeled protein nanoparticles (the preparation method of C6-labeled protein nanoparticles is the same as the preparation method of Example 2, except that 5-ASA is replaced by C6) was evaluated by laser scanning confocal microscopy (LSCM). As time goes by, the green fluorescence intensity gradually increases, indicating that the uptake of the preparation by RAW264.7 cells is time-dependent. Since the stabilizer of mannose-grafted hyaluronic acid is designed to be endocytosed by CD44 receptor-mediated endocytosis, the cells are treated with hyaluronic acid in advance and then administered. The results are as follows Fig. 9 As shown, the uptake of the preparation wrapped by hyaluronic acid and stabilizer is significantly better than that of the free drug and is time-dependent. The cells pretreated with hyaluronic acid have poor uptake of the preparation, which confirms that the preparation is taken into the cell through the CD44 receptor. The expression of CD44 on the surface of different cells is different. Therefore, this example and Example 10 can confirm that the expression of CD44 on the surface of RAW264.7 cells is relatively high.

[0082] Example 12 Effects of different preparations on ROS levels in LPS-stimulated HT-29 cells

[0083] HT-29 cells in the logarithmic growth phase were cultured at 5×10 5Cells were seeded at a density of 100 μg / well in a 6-well plate and cultured until the cell confluence reached 80%. Except for the control group, 1 mL of LPS (at a concentration of 10 μg / mL) diluted with serum-free culture medium was added to each well, and three replicate wells were set up. After 12 hours, 5-ASA, and the three nanoparticles AZ, AZ@HA, and AZ@HM (containing 500 μg of 5-ASA) prepared in Example 2, Example 3, and Example 4 were added to each well except for the control group and the modeling group. The cells were incubated for 24 hours, the culture medium was removed, the cells were washed twice with PBS, and the cells were treated with trypsin EDTA, collected by centrifugation, and incubated in a serum-free culture medium containing 10 μM DCFH-DA at 37°C in the dark for 30 minutes. The cells were washed twice with PBS, and the fluorescence intensity was measured by flow cytometry. The results are as follows. Fig.10 As shown in a and b, using fluorescence intensity for quantification, the preparation group significantly reduced the ROS produced by HT-29 cells after LPS stimulation, making it close to the ROS content of normal cells, indicating that the preparation has antioxidant ability.

[0084] Example 13 Effects of different preparations on the polarization of RAW264.7 cells stimulated by LPS

[0085] RAW264.7 cells in the logarithmic growth phase were cultured at 1×10 4 The cells were seeded at a density of 100 cells / well in a 12-well plate and cultured until the cell confluence reached 80%. Except for the control group, 0.5 mL of LPS (concentration of 10 μg / mL) diluted with serum-free medium was added to each well, and three replicate wells were set. After 12 hours, 5-ASA, three nanoparticles AZ, AZ@HA, and AZ@HM (containing 500 μg of 5-ASA) prepared in Example 2, Example 3, and Example 4 were added to each well, respectively, and three replicate wells were set. After incubation for 24 hours, the medium was removed, and the cells were washed twice with PBS. The cells were scraped off with a cell scraper, and the cells were collected by centrifugation. They were fixed with a small amount of 4% paraformaldehyde for about 15 minutes, and the 4% paraformaldehyde was discarded. The cells were washed three times with PBS, and 1 mL of the primary antibody for M1 macrophage marker CD86 and the primary antibody for M2 macrophage marker CD206 were incubated at 4°C, respectively, and incubated overnight. The primary antibodies were collected, the cells were washed twice with PBS, and the secondary antibodies labeled with FITC were incubated for 4 hours. After the cells were washed twice with PBS, the fluorescence intensity was measured by flow cytometry. The results are as follows Fig.11 As shown in a, b, c, and d, under the action of the preparation, the polarization of RAW264.7 to M1 type caused by LPS modeling was effectively alleviated. From the quantitative graph, it can be seen that the content of M2 macrophages marked by CD206 was significantly upregulated under the action of the preparation group, indicating that the preparation achieved an anti-inflammatory effect.

[0086] Example 14 Effects of different preparations on tight junctions of LPS-stimulated HT-29 cells

[0087] HT-29 cells in the logarithmic growth phase were cultured at 5×10 5 The cells were inoculated at a density of 100 μg / well in a 6-well plate and cultured until the cell confluence reached 80%. Except for the control group, 1 mL of LPS (concentration of 10 μg / mL) diluted with serum-free medium was added to each well, and three replicate wells were set. After 12 hours, 5-ASA, three nanoparticles AZ, AZ@HA, and AZ@HM (containing 500 μg of 5-ASA) prepared in Example 2, Example 3, and Example 4 were added to each well, respectively, and incubated for 24 hours. The culture medium was removed, and the cells were washed twice with PBS, fixed with a small amount of 4% paraformaldehyde for about 15 minutes, and 4% paraformaldehyde was discarded. The cells were washed three times with PBS. At 4°C, HT-29 cells were incubated with a primary antibody against tight junction protein ZO-1 overnight, the primary antibody was collected, the cells were washed twice with PBS, and the secondary antibody labeled with FITC was incubated for 4 hours. 100 μL of DAPI was added to each well incubated at 37°C for 10 minutes in the dark. After the cells were washed twice with PBS, Fig.12 As shown, laser scanning confocal microscopy (LSCM) observed that after modeling, the tight junctions between HT-29 cells decreased. After treatment with the preparation group, the expression of tight junction proteins returned to the level of healthy cells, and the AZ@HM preparation group showed stronger barrier repair ability than the AZ@HA preparation group.

[0088] Example 15 In vivo therapeutic effects of different preparations on DSS-induced ulcerative colitis

[0089] Male C57BL / 6 mice were randomly divided into 6 groups, 6 mice in each group, namely normal group, 3% DSS group, 3% DSS+5-ASA treatment group, 3% DSS+AZ treatment group, 3% DSS+AZ@HA treatment group and 3% DSS+AZ@HM treatment group. After one week of adaptive feeding, the mice in the normal group drank sterilized water, and 3% DSS (w / v%, g / 100ml) was added to the drinking water of the mice in the other groups. On the 7th day of drinking 3% DSS, the normal group and the DSS group were gavaged with 200μL of normal saline, and each medication group was gavaged with 5-ASA, three nanoparticles AZ, AZ@HA, and AZ@HM (containing 1mg 5-ASA) prepared in Example 2, Example 3, and Example 4, respectively. Gavage once a day, a total of 6 times of administration, and then the mice were killed.

[0090] The diet, activity and hair condition of the mice were observed; the weight, stool characteristics and blood in stool of the mice were recorded daily, and they were scored according to the following table, and the disease activity index (DAI) of the mice was calculated. Fig.13 a, b are the body weight and DAI score graphs of mice in each group, respectively.

[0091] Table 1 DAI scoring criteria

[0092]

[0093] Example 16 Observation of colonic histopathology of DSS-induced ulcerative colitis treated with different preparations

[0094] The colon of mice (from the cecum to the anal verge) was cleaned of adhering fat tissue, flushed with saline to remove fecal residues, and the colon length was measured. Fig.14 a is a graph showing the changes in colon length in different treatment groups. The colon was cut open along the longitudinal axis of the mesentery to observe the degree of damage such as colon edema and ulcers; about 1.5 cm of the colon was cut, fixed with 10% formalin for 24 hours, embedded in paraffin, sliced, and H&E stained. The pathological changes of the colon were observed under an optical microscope at 200 times. Figure b is a H&E staining of colon tissue. DSS induction can lead to pathological changes in colon tissue, such as colon mucosal damage and inflammatory cell infiltration. The colon tissue of mice in the normal group had complete epithelial cells and crypt structures, and goblet cells were arranged tightly and neatly. The colon tissue of mice in the model group had severe lesions, with thinning of the colon mucosa, severe damage to epithelial cells and colon villi, disappearance of a large number of goblet cells and crypts, and infiltration of a large number of inflammatory cells. Compared with the 3% DSS group, the damage to the colonic mucosa and the degree of inflammatory cell infiltration in the 3% DSS+AZ@HA and 3% DSS+AZ@HM treatment groups were greatly reduced, and the crypts and goblet cells were restored, which had a good effect on protecting the colonic mucosal barrier. In addition, the degree of recovery of the mucosa, crypts and goblet cells in the colon tissues of the two groups of mice was comparable to that of the normal group of mice, and was stronger than that of the 3% DSS+5-ASA and 3% DSS+AZ treatment groups, which showed that the use of hyaluronic acid and mannose-grafted hyaluronic acid stabilizers to encapsulate zein nanoparticles can regulate the body's immunity, effectively relieve the symptoms of ulcerative colitis in mice, and repair the barrier damage of the inflammatory intestine. Compared with ordinary nanoformulations, it improves the efficacy of anti-ulcerative colitis.

Claims

1. A protein nanoparticle for treating ulcerative colitis, characterized in that: The protein nanoparticles are obtained by physically cross-linking a stabilizer of mannose grafted hyaluronic acid on the surface of zein nanoparticles encapsulating 5-aminosalicylic acid through electrostatic adsorption.

2. The protein nanoparticle according to claim 1, characterized in that: The molar ratio of mannose to hyaluronic acid is 5:1 to 20:1; the zein nanoparticles encapsulating 5-aminosalicylic acid are obtained by loading 5-aminosalicylic acid into zein nanoparticles by an anti-solvent co-precipitation method, wherein the mass ratio of 5-aminosalicylic acid to zein is 1:10 to 9:10; the mass ratio of the mannose-grafted hyaluronic acid stabilizer to the zein nanoparticles encapsulating 5-aminosalicylic acid is 1:10 to 1:

1.

3. The protein nanoparticle according to claim 1 or 2, characterized in that: The preparation process of the zein nanoparticles encapsulating 5-aminosalicylic acid is as follows: weighing zein and 5-aminosalicylic acid and dissolving them in an ethanol solution to obtain a mixed solution; slowly dropping the mixed solution into water under stirring to spontaneously form uniform nanoparticles; and removing the ethanol by rotary evaporation to obtain the zein nanoparticles encapsulating 5-aminosalicylic acid.

4. The protein nanoparticle according to claim 1 or 2, characterized in that: The preparation process of the mannose-grafted hyaluronic acid stabilizer is as follows: hyaluronic acid, N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine are weighed and dissolved in dimethyl sulfoxide, and the mixture is swelled under heating and stirring in a water bath at 50-60°C, mannose is weighed and added to the reaction system, and the mixture is reacted under heating and stirring in a water bath at 50-60°C, and the dimethyl sulfoxide is removed by dialysis after the reaction is completed. The white powder product obtained after freeze-drying is the stabilizer.

5. The protein nanoparticle according to claim 1, characterized in that: The process of physically cross-linking the stabilizer of mannose-grafted hyaluronic acid on the surface of zein nanoparticles encapsulating 5-aminosalicylic acid through electrostatic adsorption is as follows: dissolving the stabilizer of mannose-grafted hyaluronic acid in water, and mixing it with the zein nanoparticles encapsulating 5-aminosalicylic acid under stirring to obtain stable and uniform protein nanoparticles for treating ulcerative colitis.

6. The protein nanoparticle according to claim 3, characterized in that: The concentration of the ethanol solution is 70-80%.

7. The method for preparing the protein nanoparticles according to any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: (1) Preparing a stabilizer of mannose-grafted hyaluronic acid and zein nanoparticles encapsulating 5-aminosalicylic acid respectively: Weigh hyaluronic acid, N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine, dissolve in dimethyl sulfoxide, swell under heating and stirring in a water bath at 50-60°C, weigh mannose and add it to the reaction system, react under heating and stirring in a water bath at 50-60°C, remove dimethyl sulfoxide by dialysis after the reaction, and the white powder product obtained after freeze-drying is the stabilizer; Weighing zein and 5-aminosalicylic acid and dissolving them in an ethanol solution to prepare a mixed solution, slowly dropping the mixed solution into water under stirring to spontaneously form uniform nanoparticles, and removing the ethanol by rotary evaporation to obtain zein nanoparticles encapsulating 5-aminosalicylic acid; (2) Physically cross-linking the stabilizer of mannose-grafted hyaluronic acid on the surface of zein nanoparticles encapsulating 5-aminosalicylic acid through electrostatic adsorption: The stabilizer of mannose-grafted hyaluronic acid is dissolved in water and mixed with the zein nanoparticles encapsulating 5-aminosalicylic acid under stirring to obtain stable and uniform protein nanoparticles for treating ulcerative colitis.

8. The method according to claim 7, characterized in that The mass ratio of 5-aminosalicylic acid to zein in step (1) is 1:10 to 9:10; the molar ratio of mannose to hyaluronic acid is 5:1 to 20:1; The mass ratio of the stabilizer of mannose-grafted hyaluronic acid to the zein nanoparticles encapsulating 5-aminosalicylic acid in step (2) is 1:10 to 1:

1.

9. The method according to claim 7, characterized in that: The concentration of the ethanol solution in step (1) is 70-80%; the molar ratio of the hyaluronic acid, N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine is 5:(5-10):4; the molar volume ratio of the hyaluronic acid to dimethyl sulfoxide is 1:20-1:

50.

10. Use of the protein nanoparticles according to any one of claims 1 to 6 in the preparation of a drug for treating ulcerative colitis.

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