Gold nanozyme containing glycol chitosan and gold particles and pharmaceutical composition containing the same for preventing or treating inflammatory bowel disease

Abstract

One aspect relates to a gold nanozyme containing glycol chitosan and gold particles, and a pharmaceutical composition containing the same for preventing or treating inflammatory bowel disease. It has been confirmed that the gold nanozyme according to one aspect and a composition containing the same suppress the expression of inflammatory factors, inhibit the production of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS), and reduce the production of intracellular nitric oxide (NO). It has also been confirmed that when the gold nanozyme further contains glycyrrhizin, it inhibits the secretion of HMGB1 (High mobility group box 1). It has also been confirmed that the gold nanozyme and a composition containing the same restore the length and weight of damaged colon, making it suitable for use in the industry / market for the prevention and / or treatment of inflammatory bowel disease.

Description

[Technical Field]

[0001] The present invention relates to a gold nanozyme containing glycol chitosan and gold particles, and a pharmaceutical composition containing the same for preventing or treating inflammatory bowel disease. [Background technology]

[0002] Inflammatory bowel disease (IBD) is a chronic disease in which environmental and genetic factors interact to cause an immune response and inflammation in the intestines. It is broadly divided into Crohn's disease, which affects the entire digestive tract, and ulcerative colitis, which occurs only in the large intestine, with ulcerative colitis having a higher incidence rate. While the exact cause of the disease is still unknown, dysregulation of the immune response is considered to be an important etiology.

[0003] Currently, the most common drugs used to treat inflammatory bowel disease are salicylates and streptosteroids. Because steroids and immunosuppressants have severe side effects and limited dosage, salicylates or biologics (anti-TNFα antibodies) are primarily prescribed. The synthetic drug mesalazine is primarily prescribed, which reduces the production of prostaglandins and leukotrienes (substances involved in inflammatory responses, found in white blood cells and immune cells) by inhibiting COX (cyclooxygenase) and LOX (lipooxygenase), and reduces the expression of intestinal inflammation-related genes by suppressing the PPAR-γ pathway. JAK inhibitors that suppress the activity of intracellular JAK substances have received clinical approval (e.g., tofacitinib) and are currently in clinical trials (e.g., TD-1473, upadacitinib, and baricitinib).

[0004] However, despite the prescription of mesalazine, a synthetic drug, the disease progresses slowly, and complications such as stenosis and fistulas occur. The overall response rate (ORR) for most patients is below 60%, with a significant number of patients not responding. In the case of biologics, the initial non-response rate is around 10-40%, and the secondary loss of response (LOR), in which efficacy declines during treatment and symptoms recur or worsen, is as high as 20-40% after one year of treatment. This is primarily due to anti-drug antibodies (ADA). Furthermore, during the clinical trial of JAK inhibitors, the US FDA recently confirmed serious side effects, including the development of heart disease, cancer, and thrombosis, putting clinical trials at risk of being discontinued. Summary of the Invention [Problem to be solved by the invention]

[0005] One aspect is to provide a gold nanozyme comprising glycol chitosan and gold particles.

[0006] Another aspect is to provide a pharmaceutical composition for preventing or treating inflammatory bowel disease, which comprises the gold nanozyme.

[0007] Yet another aspect is to provide a pharmaceutical preparation for preventing or treating inflammatory bowel disease, which comprises the gold nanozyme.

[0008] Yet another aspect is to provide a health food containing the gold nanozyme for preventing or improving inflammatory bowel disease.

[0009] Yet another embodiment involves mixing a glycol chitosan solution and a gold particle solution; oxidizing / reducing the glycol chitosan and gold particles in the mixed solution; and and a step of nanozyming the oxidized / reduced glycol chitosan and the gold particles.

[0010] Yet another aspect is to provide a method for preventing or treating inflammatory bowel disease comprising administering said gold nanozyme to an individual in need thereof.

[0011] Yet another aspect is to provide a use of said gold nanozyme for the manufacture of a medicament for the prevention or treatment of inflammatory bowel disease. [Means for solving the problem]

[0012] One embodiment provides a gold nanozyme comprising glycol chitosan and gold particles.

[0013] The term "glycol chitosan" refers to a water-soluble derivative of chitosan produced by conjugating chitosan with ethylene glycol. Glycol chitosan is non-cytotoxic, biocompatible, and can increase intestinal mucosal adsorption or drug durability.

[0014] In one embodiment, the gold particles may be gold nanoparticles.

[0015] The term "gold nanoparticles" refers to ultrafine particles made of gold, with a size of 1 to 2,500 nm, which exhibit unique and diverse properties due to their small size.

[0016] In one embodiment, the glycol chitosan may be in a form embedded in gold particles.

[0017] In one embodiment, the gold nanozyme may act as one or more enzymes selected from the group consisting of peroxidase, superoxide dismutase (SOD), and catalase.

[0018] The term "nanozyme" is a compound word of "nano" and "enzyme," and refers to a nano-substance that acts as an enzyme in the body.

[0019] The term "gold nanozyme" refers to a nanomaterial fabricated using gold particles in the nanozyme.

[0020] The term "peroxidase" refers to an enzyme that acts to reduce harmful hydrogen peroxide, which is produced during cellular metabolism, to water.

[0021] The term "superoxide dismutase (SOD)" refers to the enzyme that dismutates superoxide ions (O - It refers to an enzyme that catalyzes the disproportionation of ATP (H2O2) into hydrogen peroxide and molecular oxygen.

[0022] The term "catalase" refers to an enzyme that catalyzes the reaction of decomposing hydrogen peroxide into water and oxygen.

[0023] In one embodiment, the gold nanozyme may scavenge hydroxyl radicals.

[0024] The term "hydroxyl radical" refers to reactive oxygen species generated in living organisms.

[0025] In one embodiment, the gold nanozyme may further contain glycyrrhizin.

[0026] The term "glycyrrhizin" refers to a component extracted from licorice, and is known to be a factor that regulates glucocorticoids by acting on 11beta-HSD1 (11β-hydroxysteroid dehydrogenase type 1) hormone.

[0027] In one embodiment, the glycyrrhizin may suppress the secretion of HMGB1 (High mobility group box 1).

[0028] The term "High mobility group box 1 (HMGB1)" refers to the protein encoded by the HMGB1 gene in humans.

[0029] In one embodiment, the gold nanozyme may be stably maintained at pH 0-7.

[0030] In one specific example, the gold nanozyme may be stably maintained at pH 0 to 7, pH 0 to 6.7, pH 0 to 6.3, pH 1 to 7, pH 1 to 6.7, pH 1 to 6.3, pH 1.5 to 7, pH 1.5 to 6.7, or pH 1.5 to 6.3.

[0031] In one embodiment, we investigated whether citrate@AuNPs, alginate@AuNPs, and glycol-chitosan@AuNPs could be stably maintained in a GI tract-mimicking environment. As a result, we confirmed that glycol-chitosan@AuNPs stably maintained their properties at pH 2, pH 5, and pH 6, which are biomimetic environments of the GI tract (see Example 4).

[0032] In another embodiment, we investigated whether citrate@AuNPs, alginate@AuNPs, glycol chitosan@AuNPs, and glycol chitosan-GL@AuNPs could stably maintain their charge in a biomimetic environment (GI tract). As a result, we confirmed that chitosan@AuNPs and glycol chitosan-GL@AuNPs stably maintained their properties at pH 2 and pH 6, which are biomimetic environments (see Example 23).

[0033] The term "glycol chitosan@AuNP" refers to a gold nanozyme comprising glycol chitosan and gold nanoparticles, and "glycol chitosan-GL@AuNP" refers to a gold nanozyme comprising glycol chitosan, glycyrrhizin, and gold nanoparticles.

[0034] Another aspect provides a pharmaceutical composition for preventing or treating inflammatory bowel disease, comprising the gold nanozyme.

[0035] The "gold nanozyme" and the like may be within the ranges mentioned above.

[0036] The term "prevention" may refer to any action of suppressing inflammatory bowel disease or delaying the onset of inflammatory bowel disease in an individual by administering a pharmaceutical composition according to one embodiment.

[0037] The term "treatment" may refer to any action in which the symptoms of an individual with inflammatory bowel disease are improved or beneficially altered by administering a pharmaceutical composition according to one embodiment.

[0038] The term "administration" means introducing a given substance into an individual by an appropriate method, and "individual" means any living organism, including humans, that can have inflammatory bowel disease, such as mice, rats, livestock, etc. Specific examples include mammals, including humans.

[0039] In one embodiment, the gold nanozyme may be contained at 100 μg / ml to 150 μg / ml.

[0040] In one specific example, the gold nanozyme may be contained at 100 μg / ml to 150 μg / ml, 100 μg / ml to 140 μg / ml, 100 μg / ml to 130 μg / ml, 110 μg / ml to 150 μg / ml, 110 μg / ml to 140 μg / ml, 110 μg / ml to 130 μg / ml, 120 μg / ml to 150 μg / ml, 120 μg / ml to 140 μg / ml, or 120 μg / ml to 130 μg / ml.

[0041] In one experiment, Raw 264.7 cells and Caco-2 cells were co-cultured, and the Caco-2 cells were stimulated with hydrogen peroxide to determine the effect of the secreted HMGB1 on Raw 264.7 cells. The effects of treatment with Glycol chitosan@AuNP (125 μg / ml), Glycol chitosan-GL@AuNP (125 μg / ml), and hydrogen peroxide (HO (100 μM)) were also examined. The results showed that the activated morphology of Raw 264.7 cells was almost nonexistent in the Glycol chitosan@AuNP or Glycol chitosan-GL@AuNP treatment groups. Furthermore, the monolayer was maintained intact after treatment with chitosan@AuNP or Glycol chitosan-GL@AuNP (see Example 24).

[0042] In one embodiment, the pharmaceutical composition may suppress the expression of one or more selected from the group consisting of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), C-reactive protein (CRP), and calprotectin.

[0043] In one embodiment, we investigated whether Glycol chitosan-GL@AuNPs had an anti-inflammatory effect in a DSS-induced mouse model, and found that treatment with Glycol chitosan-GL@AuNPs reduced the expression of TNF-α, IL-6, IL-1β, iNOS, and MCP-1 (see Examples 29 and 30).

[0044] In another embodiment, we attempted to examine the level of C-reactive protein (CRP), a blood inflammatory factor. As a result, we confirmed that mice in which disease was induced with DSS had increased levels of CRP, a blood inflammatory factor, but that Glycol chitosan-GL@AuNPs restored this to a normal state (see Example 34).

[0045] In yet another embodiment, we attempted to examine the level of calprotectin, an inflammatory factor in feces, and found that the level of calprotectin was reduced by Glycol chitosan-GL@AuNP treatment (see Example 35).

[0046] In one embodiment, the pharmaceutical composition may suppress the production of one or more selected from the group consisting of intracellular reactive oxygen species (ROS), reactive nitrogen species (RNS), and nitric oxide (NO).

[0047] In one embodiment, H2O2 was treated with citrate@AuNPs, alginate@AuNPs, and glycol chitosan@AuNPs, and stained to confirm the ROS-scavenging ability of citrate@AuNPs, alginate@AuNPs, and glycol chitosan@AuNPs in intestinal epithelial cells. As a result, it was confirmed that the citrate@AuNPs, alginate@AuNPs, and glycol chitosan@AuNPs suppressed ROS production by confirming that the citrate@AuNPs, alginate@AuNPs, and glycol chitosan@AuNPs treated groups almost completely lost fluorescence (see Example 12).

[0048] In another experiment, we investigated the secretion of nitric oxide (NO) when Raw 264.7 cells were stimulated with lipopolysaccharide (LPS) and treated with citrate@AuNPs, alginate@AuNPs, and glycan chitosan@AuNPs. The results showed that citrate@AuNPs, alginate@AuNPs, and glycan chitosan@AuNPs removed approximately 50% of NO at a high concentration of 125 μg / ml when treated with a low concentration of 31.25 μg / ml. This confirmed that gold nanoparticles reduce NO production and enable the capture of RNS (see Example 13).

[0049] In one embodiment, the pharmaceutical composition may suppress cell differentiation or spleen hypertrophy.

[0050] The term "hypertrophy" refers to an abnormally large increase in the volume of an organ or tissue due to enlargement of the constituent cells.

[0051] In one embodiment, cytometry analysis was performed to confirm the M1 and M2 polarization rates in Raw 264.7 cells treated with Glycol chitosan@AuNPs and Glycol chitosan-GL@AuNPs. The results confirmed that both Glycol chitosan@AuNPs and Glycol chitosan-GL@AuNPs reduced the M1 polarization rate from 32.93% to nearly 0% after HO injury. Furthermore, while Glycol chitosan@AuNPs exhibited a higher M2 polarization rate, Glycol chitosan-GL@AuNPs maintained monocyte morphology, as evidenced by minimal changes in M1 and M2 morphology (see Example 26).

[0052] In another experiment, we investigated the enlargement of mouse spleens. As a result, we confirmed that the spleen length of the group treated with 2.5% DSS and PBS was enlarged to approximately 1.5 cm, whereas the spleen length of the groups treated with Glycol chitosan@AuNP and Glycol chitosan-GL@AuNP was shortened to a similar length as that of the normal state (see Example 27).

[0053] In one embodiment, the pharmaceutical composition may repair a damaged colon or repair tight junctions between cells.

[0054] In one embodiment, the damaged colon may be negatively altered from a normal colon.

[0055] In one embodiment, the pharmaceutical composition may restore the weight or length of a damaged colon.

[0056] In one embodiment, the weight and length of the colon of mice in the positive control group and the experimental groups (Glycol chitosan@AuNP and Glycol chitosan-GL@AuNP) were measured. As a result, it was confirmed that the groups administered with Glycol chitosan@AuNP and Glycol chitosan-GL@AuNP had a superior degree of body weight recovery, and the colon length was also restored to a similar level as the control group (see Example 27).

[0057] The term "tight junction" refers to a junction that restricts the movement of cells or substances between the two cells that it joins.

[0058] In one embodiment, we investigated the extent of tight junction (ZO-1 and occludin) restoration following treatment with Glycol chitosan@AuNPs and Glycol chitosan-GL@AuNPs. After DSS induction, feeding PBS alone disrupted tight junction proteins such as occludin and Zo-1, thereby disrupting the intestinal epithelial cell monolayer. When gold nanoparticles were added, yellow fluorescence emerged, confirming the restoration of tight junctions (see Example 28).

[0059] In one embodiment, the pharmaceutical composition may be for use in preventing damage to or regenerating the mucosa or mucus layer.

[0060] In one embodiment, we investigated the degree of mucus repair after treatment with Glycol chitosan@AuNPs and Glycol chitosan-GL@AuNPs. As a result, mucus was stained in the subepithelial goblet cells, which are responsible for mucus secretion, and we confirmed that Glycol chitosan@AuNPs and Glycol chitosan-GL@AuNPs prevented DSS-induced mucosal damage (see Example 36).

[0061] In one embodiment, the pharmaceutical composition may increase the microbiome associated with intestinal barrier protection.

[0062] The term "microbiome" refers to the microorganisms that live in the human body, including intestinal microorganisms.

[0063] In one experiment, we investigated the relative diversity of the gut microbiome. We found that Glycol chitosan-GL@AuNPs restored gut immune homeostasis without disrupting the gut microbiome in healthy mice and the DSS group treated with Glycol chitosan-GL@AuNPs. Furthermore, we confirmed that Glycol chitosan-GL@AuNP treatment significantly increased Akkermansia muciniphila, a microbiota associated with intestinal barrier protection. Further analysis at the family level confirmed that Glycol chitosan-GL@AuNP treatment significantly increased Lactobacillus intestinalis, which plays a beneficial role in intestinal barrier protection and IBD, and Muribacterium intestinale, a microbiota associated with essential anaerobic bacteria that can alleviate DSS-induced IBD (see Example 39).

[0064] In one embodiment, the inflammatory bowel disease may be one or more selected from the group consisting of Crohn's disease, ulcerative colitis, intestinal Bechet's disease, and enteritis.

[0065] The pharmaceutical composition may be provided as a pharmaceutical composition containing the active ingredient alone or containing one or more pharmaceutically acceptable carriers, excipients or diluents.

[0066] Specifically, the carrier may be, for example, a colloidal suspension, a powder, a saline solution, a lipid, a liposome, a microsphere, or a nanosphere, which may be complexed or associated with a delivery vehicle and delivered in vivo using delivery systems known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption promoters, or fatty acids.

[0067] When the pharmaceutical composition is formulated, it can be prepared using commonly used diluents or excipients such as lubricants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like. These solid formulations can be prepared by mixing the composition with at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Oral liquid formulations include suspensions, oral solutions, emulsions, syrups, and the like. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, flavorings, and preservatives, can be included. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, tween 61, cocoa butter, laurin butter, glycerol, and gelatin. When preparing eye drops, known diluents or excipients may be used.

[0068] The pharmaceutical composition may be provided in admixture with the other pharmaceutical composition for the prevention or treatment of inflammatory bowel diseases, and the other pharmaceutical composition for the prevention or treatment of inflammatory bowel diseases may be a conventionally known pharmaceutical composition for the prevention or treatment of inflammatory bowel diseases or a newly developed pharmaceutical composition for the prevention or treatment of inflammatory bowel diseases.

[0069] When the pharmaceutical composition further comprises another pharmaceutical composition for the prevention or treatment of inflammatory bowel diseases, or is provided by mixing with another pharmaceutical composition for the prevention or treatment of inflammatory bowel diseases, it is important to mix in an amount that can obtain the maximum effect with the minimum amount without causing side effects, which can be easily determined by a person skilled in the art.

[0070] The pharmaceutical composition may be administered in combination with other pharmaceutical compositions for the prevention or treatment of inflammatory bowel disease without being mixed, or may be administered simultaneously, separately, or sequentially, and may be administered in a single dose or multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by one skilled in the art.

[0071] The pharmaceutical composition can be administered orally or parenterally. When administered parenterally, the route of injection can be selected from the following: topical application to the skin, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraarterial injection, intramedullary injection, intracardiac injection, intradural injection, transdermal injection, intranasal injection, intraintestinal injection, local injection, sublingual injection, or intrathoracic injection.

[0072] The pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level can be determined by factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field.

[0073] In one embodiment, the pharmaceutical composition may be administered once a day or in divided doses. For example, it may be administered every other day or once a week. Specifically, the pharmaceutical composition may be administered at a dose of 0.001 to 1000 mg / kg / day, more specifically, at a dose of 0.1 to 100 mg / kg / day. The pharmaceutical composition may be administered once a day or in divided doses.

[0074] Another aspect provides a pharmaceutical preparation for preventing or treating inflammatory bowel disease, comprising the gold nanozyme.

[0075] The "gold nanozyme," "inflammatory bowel disease," "prevention," "treatment," etc. may be within the scope described above.

[0076] In one embodiment, the formulation may be an injection formulation, an infusion formulation, a spray formulation, or a liquid formulation.

[0077] The pharmaceutical formulation may further contain a pharmaceutically acceptable carrier that is commonly added to pharmaceutical formulations. The pharmaceutically acceptable carrier may include additives commonly used in the pharmaceutical field, such as excipients, disintegrants, binders, lubricants, emulsifiers, suspending agents, stabilizers, and pH adjusters. Sweeteners, flavors, and / or colorants may also be added, if necessary. Examples of excipients include microcrystalline cellulose, starch, silicon dioxide (SiO), sugar esters, Ludipress lactose, sucrose, maltose, fructose, sorbitol, and the like. A mixture of lactose and silicon dioxide is preferably used. The amount of the excipient used may be about 90% by weight or less based on the total weight of the pharmaceutical formulation, but is not limited thereto. Examples of disintegrants include carboxymethylcellulose calcium (CMC-Ca), carboxymethylcellulose sodium (CMC-Na), crospovidone, alginic acid, and the like. The amount of the disintegrant used may be in the range of 3 to 16% by weight based on the total weight of the pharmaceutical formulation, but is not limited thereto. The lubricant includes stearic acid, magnesium stearate, zinc stearate, glyceryl behenate, glyceryl palmitostearate, talc, etc., and may be used in an amount of about 3% by weight or less based on the total weight of the pharmaceutical formulation, but is not limited thereto.

[0078] In addition, the pharmaceutical preparations may be formulated for oral administration and may be formulated in various forms, such as tablets, films, suspensions, granules, gels, pills, tinctures, decoctions, infusions, spirits, fluid extracts, elixirs, extracts, syrups, powders, aromatic waters, and lemonades. The tablet may be formulated in various forms, such as orally disintegrating tablets, mucoadhesive tablets, dispersible tablets, sublingual tablets, buccal tablets, chewable tablets, dispensing tablets, multilayered tablets, press-coated tablets, effervescent tablets, and solution tablets. Those skilled in the art can modify these various tablets as needed. More preferably, the tablet may be a dosage form that disintegrates (dissolves) in the oral cavity (i.e., orally disintegrating or orally dissolving), such as an orally dispersible (dissolving) dosage form, such as an orally dissolving film, orally disintegrating tablet, suspension, suspension tablet, rapidly disintegrating tablet, orally disintegrating granule, orally disintegrating lozenge, sublingual tablet, powder, and / or chewable tablet.

[0079] In one embodiment, the pharmaceutical formulation can be administered at a daily dose of about 0.001 mg / kg to about 10 g / kg, with a daily dose of about 0.01 mg / kg to about 1 g / kg being preferred. However, the dosage may vary depending on the patient's condition (e.g., age, sex, weight), the severity of the condition being treated, etc. If necessary and for convenience, the total daily dosage can be divided and administered several times a day.

[0080] In still another embodiment, there is provided a health food containing the gold nanozyme for preventing or ameliorating inflammatory bowel disease.

[0081] The "gold nanozyme", "inflammatory bowel disease", "prevention", etc. may be within the scope described above.

[0082] The term "improvement" may refer to any action that at least reduces a parameter related to the condition being treated, for example, the severity of symptoms. In this regard, the health functional food can be used before or after the onset of inflammatory bowel disease, simultaneously or separately with a therapeutic drug, for the prevention or improvement of inflammatory bowel disease.

[0083] In the health functional foods, the active ingredients can be added directly to the food or used together with other foods or food ingredients, and can be used appropriately in a conventional manner. The amount of the active ingredient to be mixed can be determined appropriately depending on the intended use (prevention or improvement). Generally, when producing a food or beverage, the health functional foods can be added in an amount of about 15% by weight or less, more specifically about 10% by weight or less, based on the raw materials. However, in the case of long-term intake for health and hygiene purposes or health regulation purposes, the amount may be less than the above range.

[0084] The health functional food may further include one or more of a carrier, a diluent, an excipient, and an additive, and may be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquids. Foods to which the compound according to one embodiment can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, and health functional foods.

[0085] Specific examples of the carrier, excipient, diluent, and additive may be at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0086] In addition to the active ingredient, the health functional food may contain other essential ingredients without any particular limitation. For example, like ordinary beverages, it may contain various flavorings or natural carbohydrates as additional ingredients. Examples of the natural carbohydrates mentioned above include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as common sugars such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, and erythritol. Other flavorings that can be advantageously used include natural flavorings (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavorings (saccharin, aspartame, etc.). The proportion of the natural carbohydrates can be determined appropriately by those skilled in the art.

[0087] In addition to the above, the health functional food according to one embodiment may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers (cheese, chocolate, etc.), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. These ingredients can be used independently or in combination, and the proportions of these additives can also be appropriately selected by those skilled in the art.

[0088] The health functional food can be provided by mixing with a conventionally known health functional food for preventing or improving inflammatory bowel disease or a newly developed health functional food for preventing or improving inflammatory bowel disease.

[0089] If the health functional food further includes a health functional food for preventing or improving inflammatory bowel disease, it is important to mix the ingredients in an amount that can achieve maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art.

[0090] Yet another embodiment involves mixing a glycol chitosan solution and a gold particle solution; oxidizing / reducing the glycol chitosan and gold particles in the mixed solution; and The present invention provides a method for producing a gold nanozyme, which comprises the step of nanozyming the oxidized / reduced glycol chitosan and the gold particles.

[0091] The "glycol chitosan", "gold particles", "nanozyme", "gold nanozyme", etc. may be within the ranges described above.

[0092] In one embodiment, the glycol chitosan solution in the mixing step may further contain glycyrrhizin.

[0093] In one embodiment, the ratio of the glycol chitosan solution to the gold particle solution in the mixing step may be 1:2 to 1:10.

[0094] In one embodiment, in the nanozyme-forming step, the oxidized / reduced glycol chitosan may be embedded in the gold particles and then nanozymed.

[0095] Yet another embodiment provides a method for preventing or treating inflammatory bowel disease comprising administering said gold nanozyme to an individual in need thereof.

[0096] The terms "gold nanozyme," "individual," "administration," "inflammatory bowel disease," "prevention," "treatment," etc. may be within the scopes mentioned above.

[0097] Yet another aspect provides the use of said gold nanozyme for the manufacture of a medicament for the prevention or treatment of inflammatory bowel disease.

[0098] The "inflammatory bowel disease", "prevention", "treatment", "gold nanozyme", etc. may be within the scope described above. [Effects of the Invention]

[0099] It has been confirmed that the gold nanozyme and compositions containing the same according to one embodiment suppress the expression of inflammatory factors, inhibit the production of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS), and reduce the production of intracellular nitric oxide (NO). Furthermore, it has been confirmed that when the gold nanozyme further contains glycyrrhizin, it inhibits the secretion of HMGB1 (High mobility group box 1). Furthermore, it has been confirmed that the gold nanozyme and compositions containing the same repair damaged colon, and thus can be utilized in the industry / market for the prevention and / or treatment of inflammatory bowel disease. [Brief explanation of the drawings]

[0100] [Figure 1] FIG. 1 shows the results of measuring the absorbance of gold nanoparticles using a UV-visible spectrophotometer. [Figure 2] FIG. 2 shows the results of analyzing X-ray diffractometer (XRD) patterns of gold nanoparticles (citrate@AuNP (1:10), alginate@AuNP 0.2%, glycol chitosan@AuNP 0.32%, and glycol chitosan-GL@AuNP). [Figure 3] FIG. 3 shows transmission electron microscopy (TEM) images and TEM lattice images of gold nanoparticles (citrate@AuNP (1:10), alginate@AuNP 0.2%, and glycochitosan@AuNP 0.32%). [Figure 4] FIG. 4 shows the results of measuring the zeta potential (ZP) and dynamic light scattering (DLS) of gold nanoparticles (data shown as mean±SEM (n=3)). [Figure 5] Figure 5 shows the colloidal stability of gold nanoparticles (citrate@AuNP (1:10), alginate@AuNP 0.2%, and glycol chitosan@AuNP 0.32%) in a GI tract biomimicking environment (pH 2). [Figure 6] Figure 6 shows the colloidal stability of gold nanoparticles (Citrate@AuNP (1:10), Alginate@AuNP 0.2%, and Glycol chitosan@AuNP 0.32%) in a GI tract biomimicking environment (pH 5). [Figure 7] FIG. 7 shows the results of measuring the PDI values ​​of gold nanoparticles. [Figure 8] Figure 8 is a schematic diagram of ROS removal by Glycol chitosan@AuNP. [Figure 9] FIG. 9 shows the results of confirming the peroxidase-like activity of gold nanoparticles (data shown as Mean±SEM (n=3)). [Figure 10] FIG. 10 shows the mechanism of color change when TMB solution is added after the reaction of H2O2 with HRP. [Figure 11] FIG. 11 shows the results of confirming the catalase-like activity of gold nanoparticles (data shown as Mean±SEM (n=3)). [Figure 12] FIG. 12 shows the mechanism of hydrogen peroxide scavenging by Glycol chitosan@AuNP. [Figure 13] FIG. 13 shows the results of observing oxygen bubbles through their reaction with gold nanoparticles. [Figure 14] FIG. 14 shows the results of observing oxygen bubbles generated through the reaction between a material coated with gold nanoparticles and hydroxyl radicals. [Figure 15] FIG. 15 shows the results of TMB testing of chitosan oligosaccharide (COS) and glycol chitosan (data shown as mean±SEM (n=3)). [Figure 16] FIG. 16 is a diagram showing the reaction formula between H2O2 and catalase. [Figure 17] FIG. 17 shows peaks of products that appear upon oxidation of the amine groups of glycol chitosan and chitosan oligosaccharide. [Figure 18] FIG. 18 shows the results of evaluating the toxicity of gold nanoparticles to Caco-2 cells using the LDH assay (data shown as Mean±SEM (n=4)). [Figure 19] FIG. 19 shows the results of morphological evaluation of the toxicity of gold nanoparticles to Caco-2 cells. [Figure 20] FIG. 20 shows the results of evaluating the toxicity of gold nanoparticles to Raw 264.7 cells using the LDH assay (data shown as Mean±SEM (n=4)). [Figure 21] FIG. 21 shows the results of morphological evaluation of the toxicity of gold nanoparticles to Raw 264.7 cells. [Figure 22] FIG. 22 shows the results of confirming the total ROS scavenging ability and antioxidant properties through ABTS (2,2′-azino-bis(3-ethylbenzothiazoline 6-sulfonate)) analysis. [Figure 23] FIG. 23 shows the results of electron spin resonance measurements to confirm the SOD (superoxide dismutase)-like activity of gold nanoparticles. [Figure 24] FIG. 24 shows the results of confirming intracellular ROS scavenging activity using DCF-DA (Dichlorofluorescein-diacetate). [Figure 25] FIG. 25 shows an image of the DCF-DA results of FIG. [Figure 26] FIG. 26 shows the results of confirming the effect of gold nanoparticles on the production of NO, a type of RNS (data shown as mean±SEM (n=4); **P<0.01 and ***P<0.001). [Figure 27] FIG. 27 shows the results of confirming tight junctions using immunocytochemistry (ICC). [Figure 28] FIG. 28 shows the morphology of Caco-2 cells, confirming whether tight junctions were restored after H 2 O 2 stimulation (scale bar: 10 μm). [Figure 29] Figure 29 shows the results of FACS analysis of the M1 and M2 polarization ratios of RAW 264.7 cells (filled histograms show isotype-matched control antibody staining). Specifically, Figure 29A shows the results of analysis of the M1 and M2 polarization ratios of RAW 264.7 cells when the M1 cell surface marker contains CD80 and the M2 cell surface marker contains CD206. Figure 29B shows the results of analysis of the M1 and M2 polarization ratios of RAW 264.7 cells when the M1 cell surface marker contains iNOS and the M2 cell surface marker contains CD163. [Figure 30] Figure 30 shows the results of FACS analysis of M1- and M2-polarized populations of RAW 264.7 cells (data shown as mean ± SEM (n = 3)). Specifically, Figure 30A shows the results of FACS analysis of M1 and M2 populations of RAW 264.7 cells when the M1 cell surface marker contains CD80 and the M2 cell surface marker contains CD206. Figure 30B shows the results of FACS analysis of M1 and M2 populations of RAW 264.7 cells when the M1 cell surface marker contains iNOS and the M2 cell surface marker contains CD163. [Figure 31] FIG. 31 shows the morphology of monocytes in RAW 264.7 cells treated with LPS and gold nanoparticles together. [Figure 32] FIG. 32 shows the results of RT-PCR for pro-inflammatory cytokines TNF-α, IL-6, IL-1α, iNOS, and MCP-1 (data shown as Mean±SEM (n=3)). [Figure 33] FIG. 33 shows the results of ELISA for pro-inflammatory cytokines TNF-α, IL-6, IL-1α, iNOS, and MCP-1 (data shown as Mean±SEM (n=3)). [Figure 34] FIG. 34 shows the mechanism of action when a drug is orally administered to mice in which colitis has been induced by administration of DSS. [Figure 35] FIG. 35 shows the results of examining the degree of residue of gold nanoparticles depending on the time of day when they were administered to colon tissue, using ICP-MS (Inductively Coupled Plasma-Mass Spectrometry). [Figure 36] FIG. 36 shows the results of examining the degree of gold nanoparticles remaining in colon tissue at different times of the day using bio-transmission electron microscopy (BIO-TEM). [Figure 37] FIG. 37 shows a conjugation scheme of Glycyrrhizin and glycol chitosan. [Figure 38] FIG. 38 shows the results of H-NMR of glycol chitosan-glycyrrhizin. [Figure 39] FIG. 39 shows the results of FT-IR of glycol chitosan-glycyrrhizin. [Figure 40] FIG. 40 shows a synthesis plan for glycol chitosan and glycyrrhizin and a plan for coating them onto gold nanoparticles. [Figure 41] Figure 41 shows the DLS values ​​of gold nanoparticles (Citrate@AuNP (1:10), Alginate@AuNP 0.2%, and Glycol chitosan@AuNP 0.32%) in a GI tract biomimicking environment (pH 2 and pH 6). Data are shown as Mean ± SEM (n = 3). [Figure 42]Figure 42 shows the ZP (mV) values ​​of gold nanoparticles (Citrate@AuNP (1:10), Alginate@AuNP 0.2%, and Glycol chitosan@AuNP 0.32%) in a GI tract biomimicking environment (pH 2 and pH 6). Data are shown as Mean ± SEM (n = 3). [Figure 43] FIG. 43 shows a co-culture system of Raw 264.7 cells and Caco-2 cells. [Figure 44] FIG. 44 shows the morphology of Raw 264.7 cells in a co-culture system of Raw 264.7 cells and Caco-2 cells. [Figure 45] FIG. 45 shows the results of confirming the morphology of Caco-2 cells in a co-culture system of Raw 264.7 cells and Caco-2 cells. [Figure 46] FIG. 46 shows the results of ELISA verification of the amount of HMGB1 secretion in a co-culture system of Raw 264.7 cells and Caco-2 cells (data shown as Mean±SEM (n=4)). [Figure 47] FIG. 47 shows the results of ELISA verification of inflammatory factors secreted by Raw 264.7 cells in a co-culture system of Raw 264.7 cells and Caco-2 cells (data shown as Mean±SEM (n=4)). [Figure 48] FIG. 48 shows the results of FACS analysis of M1 and M2 polarization of Raw 264.7 cells in a co-culture system of Raw 264.7 cells and Caco-2 cells. [Figure 49] FIG. 49 shows the results of confirming populations for M1 and M2 polarization of Raw 264.7 cells in a co-culture system of Raw 264.7 cells and Caco-2 cells (data shown as Mean±SEM (n=3)). [Figure 50] FIG. 50 shows the drug treatment regimen in the mouse experiment (N=5). [Figure 51]FIG. 51 shows the results of measuring the survival rate of mice. [Figure 52] FIG. 52 shows the results of measuring the body weight of mice. [Figure 53] FIG. 53 shows the results of measuring the DAI (disease activity index) values ​​of mice. [Figure 54] FIG. 54 shows the results of measuring the length of the mouse colon. [Figure 55] FIG. 55 shows the results of confirming damage to the mouse colon. [Figure 56] FIG. 56 shows the results of measuring the weight and length of the mouse colon (data shown as Mean±SEM (n=5)). [Figure 57] Figure 57 is a photograph of a mouse anus. [Figure 58] FIG. 58 shows the results of confirming the enlargement of mouse spleens. [Figure 59] FIG. 59 shows the results of confirming the repair of tight junctions in mouse colon tissues using immunohistochemical staining. [Figure 60] FIG. 60 shows the results of ELISA validation of inflammatory factors in mouse serum (data shown as Mean±SEM (n=4)). [Figure 61] FIG. 61 shows the results of ELISA validation of inflammatory factors in mouse colon (data shown as Mean±SEM (n=4)). [Figure 62] FIG. 62 shows the results of qRT-PCR for inflammatory factors in the mouse colon (data shown as Mean±SEM (n=4)). [Figure 63] FIG. 63 shows the results of confirming the number of M1 cells in the mouse colon (data shown as Mean±SEM (n=3)). [Figure 64] FIG. 64 shows the results of flow cytometry analysis to determine the number of M1 and M2 cells in the mouse colon. [Figure 65] FIG. 65 shows the results of confirming toxicity in the major organs: heart, liver, lungs, kidneys, and spleen. [Figure 66] FIG. 66 shows the results of ELISA testing for C-reactive protein (CRP), an inflammatory factor, in mouse serum (data shown as Mean±SEM (n=4)). [Figure 67] FIG. 67 shows the results of ELISA testing of calprotectin, an inflammatory factor in mouse feces (data shown as Mean±SEM (n=4)). [Figure 68] FIG. 68 shows the results of confirming the degree of repair of the mucus layer after treatment with Glycol chitosan@AuNP and Glycol chitosan-GL@AuNP using immunohistohemistry (IHC). [Figure 69] FIG. 69 shows the results of confirming the amount of blood influx 24 hours after oral administration of Glycol chitosan-GL@AuNP (data shown as Mean±SEM (n=3)). [Figure 70] Figures 70a to 70c show the results of examining the function of major organs and hematotoxicity following oral administration of Glycol chitosan@AuNPs and Glycol chitosan-GL@AuNPs (data shown as Mean ± SEM (n = 4)). Specifically, Figure 70a shows the results of analyzing total protein, albumin, globulin, AST, ALT, BUN, total bilirubin, and creatinine. Figure 70b shows the results of analyzing RBC, HGB, HCT, RBC indices (MCV, MCH, MCHC), RDW, MPV, and PLT. Figure 70c shows the results of analyzing WBC and WBC percentages (NEU, LYM, MONO, EOS, BASO). [Figure 71]FIG. 71 shows the results of measuring the richness of OTUs (operational taxonomic units) (data shown as Mean±SEM (n=3)). [Figure 72] FIG. 72 shows the results of measuring Shannon diversity (data shown as Mean±SEM (n=3)). [Figure 73] FIG. 73 shows the results of measuring the Simpson's index for the alpha diversity of the gut microbial community (data shown as Mean±SEM (n=3)). [Figure 74] FIG. 74 shows the results of analysis of the beta diversity of gut microorganisms (data shown as Mean±SEM (n=3)). [Figure 75] FIG. 75 shows the results of a heatmap analysis of the taxonomic microbial composition at the family level. [Figure 76] FIG. 76 shows the results of confirming the relative diversity of the intestinal microbiome. [Figure 77] FIG. 77 shows the results of analyzing heat maps for taxonomic microbial composition at the phylum-species level. [Figure 78] FIG. 78 shows the results of confirming the relative abundance of Akkermansia muciniphila in the microbiota (data shown as Mean±SEM (n=3)). [Figure 79] FIG. 79 shows the results of confirming the relative abundance of Lactobacillus intestinalis in the microbiota (data shown as Mean±SEM (n=3)). [Figure 80]Figure 80 shows the results of confirming the relative abundance of Muribaculum intestinale in the microbiota (*P<0.05, **P<0.01 (PBS-treated group in 2.5% DSS group), data are shown as Mean±SEM (n=3)). DETAILED DESCRIPTION OF THE INVENTION

[0101] The present invention will be described in more detail below with reference to examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0102] Example 1. Synthesis method of gold nanoparticles (AuNPs) (1) Use of citrate Gold nanoparticles (AuNPs) were synthesized using citrate. The alloy solution (1.13 mM) and citrate solutions (5.69 mM, 7.98 mM, and 11.3 mM) were dissolved in distilled water to prepare gold-to-citrate molar ratios of 1:5, 1:7, and 1:10, respectively. The gold solution was then poured into the citrate solution, and the mixture was heated on a hot plate at 90°C while simultaneously stirring with a magnetic bar at 1,000 rpm. The mixture was centrifuged in an ultracentrifuge at 16,000 rpm for 30 minutes to obtain a precipitate.

[0103] (2) Use of Glycol Chitosan We attempted to synthesize gold nanoparticles using glycol chitosan. Glycol chitosan (0.32 w / v%) was dissolved in distilled water and heated on a hot plate at 90°C. Then, gold solution (1 mM) was slowly added dropwise to the glycol chitosan solution, and the mixture was heated on a hot plate at 90°C while simultaneously stirring with a magnetic bar at 1,000 rpm. The yellow solution gradually turned colorless and then wine-colored within 20 minutes, confirming the formation of glycol chitosan@AuNPs. The mixture was centrifuged at 16,000 rpm for 30 minutes in an ultracentrifuge to obtain a precipitate.

[0104] (3) Use of alginate We attempted to synthesize gold nanoparticles using alginate. Alginate (0.2 w / v% and 0.4 w / v%) was dissolved in distilled water and heated on a hot plate at 90 °C. Then, a gold solution (1 mM) was slowly added dropwise to the alginate solution. The mixture was heated on a hot plate at 90 °C while simultaneously stirring with a magnetic bar at 1,000 rpm. As a result, the yellow solution gradually turned colorless and then wine-colored within 20 minutes, confirming the formation of alginate-chitosan@AuNPs. The mixture was centrifuged in an ultracentrifuge at 16,000 rpm for 30 minutes to obtain a precipitate.

[0105] (4) Characteristics of gold nanoparticles The characteristics of gold nanoparticles with various ratios prepared in Example 1.(1)-(3) were investigated. Zeta potential, DLS, PDI, nanoparticle size through TEM, lattice size, and XRD were analyzed. The analytical results are shown in Table 1 below.

[0106] [Table 1]

[0107] Zeta potential analysis confirmed that Citrate@AuNP and Alginate@AuNP have a negative charge, while Glycol chitosan@AuNP has a positive charge. DLS and PDI confirmed the size and stability of gold nanoparticles in aqueous solution, while TEM and lattice parameter confirmed the size of the gold particles themselves, as well as the presence and size of a lattice structure. XRD analysis also confirmed the presence or absence of gold particles.

[0108] 2. Confirmation of Gold Nanoparticle Synthesis To confirm the synthesis of gold nanoparticles in Examples 1(1) to (3) above through SPR peaks, measurements were performed using a UV-visible spectrophotometer (NanoDrop 2000; Thermo Scientific, Wilmington, USA). Specifically, gold nanoparticles synthesized at various ratios (citrate@AuNP (1:5), citrate@AuNP (1:7), citrate@AuNP (1:10), alginate@AuNP 0.2%, alginate@AuNP 0.4%, and glycochitosan@AuNP 0.32%) were measured using the UV-visible spectrophotometer. All synthesized gold nanoparticles were confirmed to exhibit an SPR (surface plasmon peak) at approximately 530 nm (Figure 1).

[0109] 3. Characterization of Gold Nanoparticles (1) XRD (X-Ray Diffractometer) pattern analysis of gold nanoparticles To confirm the presence of elemental gold in the gold nanoparticles, the synthesized gold nanoparticles were freeze-dried to produce a powder. The gold nanoparticles were characterized using X-ray diffractometers (XRD) with D8 Advance and Bruker. The peaks at 2θ = 31.6°, 38.2°, 64.6°, and 77.6° were assigned to the (111), (200), (220), and (311) lattice planes of Au(0), respectively, confirming the presence of gold in the nanoparticles coated with various materials (Figure 2).

[0110] (2) TEM and TEM lattice analysis of gold nanoparticles To confirm the size and lattice size of gold nanoparticles, gold nanoparticles in solution were diluted with deionized water (DW) to a concentration of approximately 1 / 20. The solution was then added dropwise to cover a Cu TEM grid. A transmission electron microscope (JEM 2100F, JEOL) was used to confirm whether the nanoparticles had a particle morphology and to measure their size.

[0111] The results are shown in Figure 3. Specifically, the TEM image of the citrate@AuNP (1:10) confirmed that the nanoparticle size was approximately 27 nm, the TEM image of the alginate@AuNP 0.2% confirmed that the nanoparticle size was approximately 14.9 nm, and the TEM image of the glycochitosan@AuNP 0.32% confirmed that the nanoparticle size was approximately 8 nm (TEM (nm) shown in Figure 4). Therefore, all three nanoparticles were confirmed to be round nanoparticles. Furthermore, by measuring the lattice size inside the gold nanoparticles, it was confirmed that the lattice size was consistently 2.3 Å, 2.4 Å, and 6.6 Å for the citrate@AuNP (1:10), alginate@AuNP 0.2%, and glycochitosan@AuNP 0.32%, respectively.

[0112] (3) Measurement of ZP (zeta potential), DLS (dynamic light scattering), and PDI (poly dispersity index) values ​​of gold nanoparticles To measure the zeta potential (ZP), dynamic light scattering (DLS), and polydispersity index (PDI) values ​​of gold nanoparticles, 800 μL of gold nanoparticles in solution was placed in a cuvette and measured. The physical properties of the gold nanoparticles were measured using a Zetasizer Nano ZS (Malvern Panalytical, Worcestershire, UK).

[0113] The results are shown in Figure 4. Specifically, we confirmed that the surface charge of gold nanoparticles differed depending on the coating material. Citrate@AuNP (1:10) and Alginate@AuNP 0.2% showed negative charges at -36.2 ± 2.9 mV and -34.5 ± 11.1 mV, respectively. Glycol chitosan@AuNP 0.32% showed positive charges at 30.1 ± 1.36 mV. DLS values ​​confirmed that citrate@AuNP (1:10) had sizes of approximately 30 nm, Alginate@AuNP 0.2% had sizes of approximately 60 nm, and Glycol chitosan@AuNP 0.32% had sizes of approximately 80 nm.

[0114] 4. Confirmation of colloidal stability of gold nanoparticles by pH We investigated whether citrate@AuNPs (1:10), alginate@AuNPs (0.2%), and glycerol chitosan@AuNPs (0.32%) could be stably maintained in a biomimetic environment similar to the GI tract. Specifically, gold nanoparticles were adjusted to pH 2 and pH 6 with HCl and NaOH, respectively. Gold nanoparticles were then measured using a UV-visible spectrophotometer (NanoDrop 2000; Thermo Scientific, Wilmington, USA) after 0, 1, 3, 6, 9, and 12 hours.

[0115] As a result, we confirmed that the SPR peaks of citrate@AuNP (1:10) and alginate@AuNP began to collapse after 1 hour in a pH 2 environment, while those of glycol chitosan@AuNP 0.32% could be maintained for up to 3 hours (Figure 5). This is because the carboxylic acids of citrate and alginate undergo protonation in the acidic pH 2 environment, neutralizing the surface charge and allowing the AuNPs to aggregate. On the other hand, AuNPs coated with glycol chitosan, which has many amine groups, can avoid this resulting conjugation.

[0116] Furthermore, we confirmed that the colloidal stability of all gold nanoparticles was maintained for 12 hours in a pH 5 environment (Figure 6). Based on the results of Figures 5 and 6, we predicted that orally administered Glycol chitosan@AuNPs would be stably maintained in the stomach and intestine.

[0117] Furthermore, when gold nanoparticles were left at pH 2 and pH 6 for 12 hours, the particle size in the aqueous solution was confirmed and the PDI values ​​were measured to confirm whether they were stably and well dispersed. As a result, all PDI values ​​were below 0.5, confirming that they were stably and well dispersed (Figure 7).

[0118] 5. TMB (3,3',5,5'-Tetramethylbenzidine) Assay (1) Confirmation of peroxidase-like activity of gold nanoparticles To confirm the peroxidase properties of gold nanoparticles, AuNPs were dissolved in 80 × 10 H2O adjusted to pH 7.4, pH 5.8, and pH 2.1. -3 The mixture was incubated with 80 × 10 H2O solution (pH 7.4, pH 5.8, and pH 2.1) for 5 minutes. -3 A solution containing M, TMB, and horseradish peroxidase (HRP) (1,000 units / ml) reacted for 5 minutes was used as a positive control. The results of peroxidase-like activity (%) were normalized based on HRP activity at pH 5.8. A schematic diagram of ROS removal by glycochitosan@AuNPs is shown in Figure 8.

[0119] Figure 9 shows the results of the peroxidase-like activity of gold nanoparticles. Specifically, TMB, a substance used in colorimetric assays, reacts with ·OH generated by H2O2 and HRP to produce a blue intermediate or a yellow end product. If all ·OH is removed, the product becomes colorless. At pH 7.4, similar to the pH in the human body, all three types of gold nanoparticles were found to have almost no peroxidase activity. Furthermore, at pH 5.8, similar to the pH of colon tissue, the citrate@AuNP and glycol chitosan@AuNP gold nanoparticles had almost no peroxidase activity, at approximately 20%, while the alginate@AuNP had approximately 60% peroxidase activity, demonstrating higher peroxidase activity than the other gold nanoparticles. Furthermore, as glycol chitosan became colorless, it was confirmed that it no longer had any ability to catalyze TMB. In other words, it was confirmed that Glycol Chitosan@AuNPs lost their inherent peroxidase activity. At pH 2.1, which is similar to the pH of the stomach, it was confirmed that both gold nanoparticles and HRP had low peroxidase activity. Furthermore, the mechanism of the color change when TMB solution was added after the reaction between H2O2 and HRP is shown in Figure 10.

[0120] (2) Confirmation of catalase-like activity of gold nanoparticles We attempted to confirm the catalase properties of gold nanoparticles. Specifically, to test the catalase-like activity of citrate@AuNPs, alginate@AuNPs, and glyco-chitosan@AuNPs (125 μg / ml), we used TMB (80 × 10 mL) containing HO adjusted to pH 7.4, pH 5.8, and pH 2.1. -3M) was reacted with the gold nanoparticles for 5 minutes, and then HRP solution (1,000 units / mL) was added to detect the remaining H2O2. The catalase-like activity (%) was normalized based on the Glycol Chitosan@AuNP activity at pH 5.8. Furthermore, we confirmed that the Citrate@AuNPs and Alginate@AuNPs turned yellow before the addition of HRP, while the Glycol Chitosan turned colorless (Figure 11). At pH 5.8, similar to the pH of colonic tissue, the Citrate@AuNPs and Glycol Chitosan@AuNPs showed almost no catalase activity (approximately 20%), whereas the Alginate@AuNPs showed approximately 60% catalase activity, demonstrating their higher peroxidase activity compared to the other gold nanoparticles. Furthermore, as evidenced by the colorlessness of the Glycol Chitosan, we confirmed that it no longer had any catalytic activity for TMB. This indicates that the Glycol Chitosan@AuNPs lost their inherent catalase activity. It was confirmed that both gold nanoparticles and HRP had low peroxidase activity at pH 2.1, which is similar to the pH of the stomach.

[0121] 6. Dioxygen bubble confirmation experiment (1) Confirmation of oxygen bubbles through reaction with gold nanoparticles To confirm the oxygen generated through the reaction of gold nanoparticles with hydroxyl radicals and the catalase-like activity of Glycol chitosan@AuNPs, catalase and Glycol chitosan@AuNPs were added to H2O2 solutions and the formation of oxygen bubbles was observed. If bubbles were observed, it can be assumed that this was due to the decomposition of H2O2 into water and oxygen, which are the final products produced by catalase-like activity. The mechanism of hydrogen peroxide removal by Glycol chitosan@AuNPs is shown in Figure 12. Specifically, at 80×10 -3Citrate@AuNP (125 μg / mL), alginate@AuNP (125 μg / mL), glycochitosan@AuNP (125 μg / mL), and catalase (1,000 units / mL) were reacted with 1000 M H2O2 for 5 min to observe the generation of oxygen bubbles. As a result, glycochitosan@AuNP generated bubbles in amounts similar to those of catalase@AuNP (Figure 13).

[0122] (2) Confirmation of oxygen bubbles through the reaction of gold nanoparticle-coated materials with hydroxyl radicals The purpose of this study was to confirm the oxygen generated through the hydroxyl radical reaction of glycol chitosan and chitosan oligosaccharide (COS), which has a similar structure, as coating materials. After reacting catalase with hydrogen peroxide, the mixture was left for 5 minutes to confirm whether bubbles were generated. -3 (ii) M HO was reacted with glycol chitosan (125 μg / mL), (iii) chitosan oligosaccharide (125 μg / mL), and catalase (1,000 units / mL) for 5 minutes to observe the generation of oxygen bubbles. To confirm peroxidase-like activity, HRP (1,000 units / mL) was used as a positive control, and normalization was performed based on HRP activity. Hydroxyl radicals were generated by reacting HRP with hydrogen peroxide, and then glycol chitosan or COS was reacted to confirm the generation of bubbles (Figure 14). This indicates that amine groups are oxidized to water and oxygen.

[0123] Furthermore, a TMB assay was performed on glycol chitosan and COS. When glycol chitosan was reacted with TMB and H2O2 together, or when COS was reacted with TMB and H2O2 together, the color changed to colorless, confirming the presence of peroxidase activity (Figure 15).

[0124] 7. Confirmation of catalase-like activity of amine-rich glycol chitosan and COS The amine group was oxidized through the reaction with hydroxyl radicals, and the resulting change in functional group was confirmed by H-NMR. The reaction formula of H2O2 and catalase is shown in Figure 16. Specifically, glycol chitosan (125 μg / mL) and COS (125 μg / mL) were dissolved in deuterium oxide (DO). Then, glycol chitosan (125 μg / mL), H2O2 (80 × 10 -3 The cells were incubated with COS (125 μg / mL), H2O2 (80 × 10 -3 The catalase-like activity of amine-rich glycol chitosan and COS was measured by H-NMR (Nuclear Magnetic Resonance). The reaction of glycol chitosan with hydroxyl radicals oxidized the amine groups of glycol chitosan, and the peak of the product appeared at approximately 6.5 ppm to 7.2 ppm (Figure 17).

[0125] 8. Confirmation of cytotoxicity of gold nanoparticles to intestinal epithelial cells Various ratios of gold nanoparticles were applied to Caco-2 cells (intestinal epithelial cells), immortalized human colorectal adenocarcinoma cells (Korean Cell Line Bank, Seoul, Korea), to evaluate their cytotoxicity. Cell viability was evaluated using an LDH (lactate dehydrogenase) cytotoxicity assay kit. Specifically, Caco-2 cells (1x10 4After plating (cells / well), the cells were cultured in a CO2 incubator at 37°C for 48 hours. After washing with PBS, the cells were treated with citrate@AuNP (1:10), 0.2% alginate@AuNP, and 0.32% glycerol chitosan@AuNP (7.8 μg / ml, 15.6 μg / ml, 31.3 μg / ml, 62.5 μg / ml, and 125 μg / ml), followed by incubation for 36 hours. For blanks, 200 μl of medium alone was added to three completely empty wells (no cells), and for positive controls, 200 μl of 1% Triton X-100 was added to the cells. 100 μl of the cell supernatant was transferred to a new Eppendorf tube. In the dark, 100 μl of LDH reaction solution (MTT solution, NAD+ solution, PES solution, and lactate solution) was added to the tube, and the tube was incubated in an incubator at 37°C for up to 30 minutes. The tube was then centrifuged at 20,000 g for 15 minutes, and the supernatant of the mixture was transferred to a microplate, after which the absorbance was measured at 565 nm using a microplate reader. The blank absorbance was subtracted from the total absorbance to determine the cytotoxicity (%) (A sample -A background ) / (A maximum -A spontaneous ) Calculated by x100.

[0126] As a result, we confirmed that there was almost no toxicity at all concentrations (Figure 18).In addition, after treatment with citrate@AuNP (1:10), alginate@AuNP 0.2%, and glycol chitosan@AuNP 0.32% at concentrations (7.8 μg / ml, 15.6 μg / ml, 31.3 μg / ml, 62.5 μg / ml, and 125 μg / ml), respectively, we confirmed that Caco-2 cells formed a monolayer 36 hours later, similar to the control group (Figure 19).

[0127] 9. Confirmation of cytotoxicity of gold nanoparticles to immune cells Various ratios of gold nanoparticles were applied to Raw 264.7 cells (mouse macrophages) (Korean Cell Line Bank, Seoul, Korea), which are macrophages established in tumors in male mice induced by Abelson murine leukemia virus, to evaluate their cytotoxicity. Cell viability was evaluated using an LDH (lactate dehydrogenase) cytotoxicity assay kit. Specifically, Raw 264.7 cells (1x10) were cultured in a 96-well plate. 4 After plating (cells / well), the cells were cultured for 24 hours in a CO2-filled incubator at 37°C. After washing with PBS, the cells were treated with citrate@AuNP (1:10), 0.2% alginate@AuNP, and 0.32% glycerol chitosan@AuNP (7.8 μg / ml, 15.6 μg / ml, 31.3 μg / ml, 62.5 μg / ml, and 125 μg / ml), followed by incubation for 18 hours. For blanks, 200 μl of medium alone was added to three completely empty wells (no cells), and 200 μl of 1% Triton X-100 was added to the cells for a positive control. 100 μl of the cell supernatant was transferred to a new Eppendorf tube. In the dark, 100 μl of LDH reaction solution (MTT solution, NAD+ solution, PES solution, and lactate solution) was added to the tube, and the tube was incubated in an incubator at 37°C for up to 30 minutes. The tube was then centrifuged at 20,000 g for 15 minutes, and the supernatant of the mixture was transferred to a microplate, after which the absorbance was measured at 565 nm using a microplate reader. The blank absorbance was subtracted from the total absorbance to determine the cytotoxicity (%) (A sample -A background ) / (A maximum -A spontaneous ) Calculated by x100.

[0128] As a result, we confirmed that there was almost no toxicity at all concentrations (Figure 20). Furthermore, after 36 hours of treatment with citrate@AuNP (1:10), 0.2% alginate@AuNP, and 0.32% glycol chitosan@AuNP at concentrations (7.8 μg / ml, 15.6 μg / ml, 31.3 μg / ml, 62.5 μg / ml, and 125 μg / ml), we confirmed that Raw 264.7 cells showed toxicity and changed to a sharp morphology upon activation, but this morphology did not appear when treated with gold nanoparticles (Figure 21).

[0129] 10. Confirmation of ROS scavenging activity We attempted to confirm the ROS radical scavenging ability of gold nanoparticles. To prepare ABTS radicals (ABTS), 14 mM ABTS solution and 4.9 mM potassium persulfate were dissolved in DW. Potassium persulfate was then added to the ABTS solution to adjust the final concentration to 2.45 mM. The mixture was incubated overnight (approximately 12-16 hours) in the dark at room temperature. The ABTS solution was diluted with DW until the ABTS reading at 734 nm was 0.8. 500 μl of ABTS solution was added to a tube containing 500 μl of AuNPs and incubated at 50 rpm for 15 minutes on an orbital shaker. The mixture was then centrifuged at 20,000 g for 10 minutes in a minicentrifuge. 100 μl of the supernatant was transferred to a 96-well plate, avoiding the pellets from mixing. The reading was taken at 734 nm, and the radical scavenging activity was calculated as I% = [(Abs0-Abs1) / Abs0] × 100 (Abs0: ABTS, Abs1: sample).

[0130] As a result, we confirmed that Glycol chitosan@AuNPs showed the highest performance, and that this was concentration-dependent. We also confirmed that the capture effect was superior in the order of Citrate@AuNPs, Alginate@AuNPs, and Glycol chitosan@AuNPs (Figure 22).

[0131] 11. Electron spin resonance measurement to confirm the SOD (Superoxide Dismutase)-like activity of gold nanoparticles To confirm the superoxide dismutase (SOD) properties of gold nanoparticles, a solution of 25 μM DTPA (diethylenetriaminepentaacetic acid), 1 mM hypoxanthine, 1 unit / ml hypoxanthine oxidase, 1 M DMPO, and 1,000 unit / ml SOD (dissolved in DW) was prepared in 100 mM phosphate buffer (pH 7.4). 700 μl of DTPA solution, 200 μl of DMPO solution, and 1 ml of hypoxanthine were combined to give final concentrations of 17.5 μM, 10 mM, and 0.5 mM, respectively. The mixture was the stock solution before superoxide radical generation. 47.5 μl of the reaction mixture was transferred and 2.5 μl of xanthine oxidase was added to adjust the final xanthine oxidase concentration to 0.05 units / ml. After incubation for 30 seconds, this group served as the positive control. 50 μl of superoxide dismutase was added to the positive control solution and incubated for 30 seconds to adjust the final concentration to 500 units / ml. 50 μl of citrate@AuNP (1:5), (1:7), (1:10), 0.32% Glycol chitosan@AuNP, and 0.2% Alginate@AuNP were added to the control solution to adjust the final concentration to 125 μg / ml, and the mixture was incubated for 2 minutes. The SOD (Superoxide dismutase)-like activity of the gold nanoparticles was then measured using an electron spin resonance spectrometer (EMXplus-9.5 / 12 / P / L System (Bruker, Germany)).

[0132] SOD plays a major role in antioxidant effects by converting superoxide into H2O2 and oxygen. To confirm whether the synthesized gold nanoparticles possessed SOD-like activity, ESR experiments were performed. The higher the intensity on the y-axis, the greater the amount of superoxide produced. The strongest intensity was observed when superoxide was generated using xanthine oxidase and hypoxanthine, which served as the positive control. Subsequent treatment with superoxide dismutase resulted in a decrease in intensity. Similarly, treatment with citrate@AuNP (1:10), alginate@AuNP 0.2%, and glycol chitosan@AuNP 0.32% all decreased in intensity. Therefore, all three types of synthesized gold nanoparticles possessed SOD-like activity. Among these, glycol chitosan exhibited the greatest decrease in intensity, indicating its superior superoxide suppression effect (Figure 23).

[0133] 12. Confirmation of intracellular ROS scavenging activity To confirm the ROS-scavenging ability of gold nanoparticles in intestinal epithelial cells, a DCF-DA (Dichlorofluorescein-diacetate) assay was performed by treating H2O2 with gold nanoparticles. Caco-2 cells were cultured in a 96-well plate at 1.0x10 4 Cells were plated and cultured. Citrate@AuNPs (1:10), 0.2% alginate@AuNPs, and 0.32% glycol chitosan@AuNPs were reacted with HO (100 μM) and added to each well for 4 hours. After 4 hours, cells were stained with DCFH-DA (2',7'-dichlorofluorescin diacetate) (final concentration 2 μM) for 15 minutes and washed three times with PBS. Intracellular ROS production was measured using a microreader after irradiation with 490 nm excitation and 520 nm emission wavelengths. A group treated with HO alone served as a positive control.

[0134] As a result, it was confirmed that DCF fluorescence values ​​were reduced in most of the gold nanoparticle-treated groups compared to the positive control group (Figure 24). In addition, the positive control group emitted strong fluorescence, indicating the production of large amounts of ROS. In the gold nanoparticle-treated group, fluorescence was almost completely eliminated, indicating that gold nanoparticles suppress intracellular ROS production (Figure 25).

[0135] 13. Confirmation of RNS scavenging activity and nitric oxide (NO) production When Raw 264.7 cells were stimulated with LPS (Lipopolysaccharide) and treated with LPS and gold nanoparticles, the secreted concentration of nitric oxide (NO) was measured using Griess reagent. 5 Raw 264.7 cells seeded at 1000 cells / well were cultured in complete medium (DMEM, 10% FBS, 1% PS) for 24 hours. The cells were then cultured for 5 hours in the presence or absence of AuNPs. After washing with PBS, the cells were cultured for 20 hours in the presence or absence of LPS (1 μg / ml). Cell supernatant (100 μl) was transferred to a 96-well plate and mixed with the same volume of Griess reagent for 10 minutes. Absorbance at 546 nm was measured on the plate. NO concentrations were calculated using a NO standard curve (0 μM to 10 μM). The positive control group was treated with LPS (1 μg / ml) alone.

[0136] All gold nanoparticles were confirmed to remove approximately 50% of the high concentration of 125 μg / ml NO2 when treated with a low concentration of 31.25 μg / ml, confirming that gold nanoparticle treatment reduces NO production and enables the capture of RNS (Figure 26).

[0137] 14. Identification of tight junctions using immunocytochemistry (ICC) We investigated whether tight junctions (ZO-1, Occludin) were restored when gold nanoparticles were treated in Caco-2 cells stimulated with H2O2. Caco-2 cells (1x10 6 Caco-2 cells (1000 cells / well) were plated on the plate and cultured for 48 hours. Caco-2 cells were briefly washed with PBS and fixed in 4% paraformaldehyde. Then, the cells were washed twice for approximately 2 minutes with PBS-Tween 20. For blocking, the cells were incubated with 20% goat serum in PBS-Tween 20 at room temperature for 30 minutes. Primary antibodies (anti-mouse occludin, anti-rabbit ZO-1) diluted 100:1 in 20% goat serum in PBST-20 were incubated for 1 hour at room temperature and then washed with PBS. Secondary antibodies (goat anti-mouse-488, goat anti-rabbit-594) were diluted 1:200 in PBS and then cultured. Protected from light, the cells were washed twice for 3 minutes with PBS. Then, 1-2 drops of DAPI mounting solution (blue) were added, and the sections were covered with a cover slide and observed under a fluorescence microscope.

[0138] As a result, we confirmed that oxidative stress caused by H2O2 disrupts tight junction proteins such as occludin (green) and Zo-1 (zonula occludens-1, red), which in turn disrupts the monolayer of intestinal epithelial cells (Figure 27). We also confirmed that the degree of monolayer disruption was significantly lower when gold nanoparticles and H2O2 were treated together. This confirms that gold nanoparticles help prevent the disruption of tight junctions.

[0139] Furthermore, images of the morphology of Caco-2 cell tight junctions after 36 hours of treatment with citrate@AuNPs, alginate@AuNPs, and glycochitosan@AuNPs at respective concentrations (7.8 μg / ml, 15.6 μg / ml, 31.3 μg / ml, 62.5 μg / ml, and 125 μg / ml) are shown in Figure 28.

[0140] 15. Confirmation of M1 and M2 polarization of Raw 264.7 cells Flow cytometry was performed to confirm the M1 and M2 polarization rates in the Raw 264.7 cells treated with LPS and the LPS and gold nanoparticle (Alginate@AuNP, Glycol chitosan@AuNP) groups. Raw 264.7 cells were cultured in a 100 mm dish (2.0 × 10 6 The cells were seeded onto a medium containing 10% FBS and 1% PS and cultured for 24 hours. The cells were then washed with PBS and cultured for 5 hours with or without AuNPs (125 μg / ml). The cells were separated using a scraper and centrifuged at 12,000 rpm for 3 minutes. After removing the supernatant, the cells were redispersed in PBS and cultured at room temperature for 20 minutes with or without antibodies. The antibodies were CD206 APC-eFluor 780, CD80-FITC, and CD163-eFluor. TM 450(eBioscience TM ), iNOS-PE (eBioscience TM) were used: 1) Negative control (CD206 APC-eFluor 780, CD80-FITC co-staining), positive control (non-staining), raw cells and CD206 marker (staining), raw cells and CD80 marker (staining), raw cells stained with CD206 marker and CD80 marker (co-staining), raw cells treated with Alginate@AuNP stained with CD206 and CD80 (co-staining), raw cells treated with Glycol chitosan@AuNP stained with CD206 and CD80 (co-staining). 2) Negative control (CD163-eFluor TM 450, iNOS-PE co-staining), positive control (non-staining), Raw cell and CD163 marker (staining), Raw cell and iNOS marker (staining), Raw cell and CD163-eFluor TM 450, iNOS-PE co-staining, CD163-eFluorescence staining on raw cells treated with Alginate@AuNPs TM 450, iNOS-PE staining (co-staining), CD163-eFluorescence staining on Glycol chitosan@AuNP-treated raw cells TM Co-staining with iNOS-PE at 450. Samples were analyzed using a BD FACS (BD FACSCalibur, BD biosciences, New Jersey, USA), and data were analyzed using FlowJo V10. An unstained positive control group was used for gating.

[0141] As a result, in group 1), the M1 ratio of cells stimulated with LPS was 45.86%, an increase of approximately 7-fold compared to 6.12% in the negative control group, and in the group treated with gold nanoparticles, it further decreased to approximately 20%, confirming a decrease of approximately 50% compared to the positive control group (Figure 29A).In addition, in group 2), the M1 ratio of cells stimulated with LPS was 12.8%, an increase compared to 0.04% in the negative control group, but in the group treated with gold nanoparticles, it further decreased to approximately 7% and 4%, confirming a decrease in all compared to the positive control group (LPS treatment) (Figure 29B).

[0142] M1 and M2 polarized populations of RAW 264.7 cells were analyzed by FACS. In group 1, the percentage of M1 cells was significantly increased when stimulated with LPS, as shown in the FACS percentage graph. However, when gold nanoparticles were added to the LPS-stimulated group, the percentage of M1 cells decreased by approximately 50%. Furthermore, the increase in the percentage of M2 cells confirmed the anti-inflammatory effect (Figure 30A). The graph of total cell counts shows the average fluorescence intensity of stained cells. In group 2, the percentage of M1 cells was significantly increased when stimulated with LPS, as shown in the FACS percentage graph. However, when gold nanoparticles were added to the LPS-stimulated group, the percentage of M1 cells decreased by approximately 50% (Figure 30B).

[0143] 16. Morphological observation of Raw 264.7 cells We investigated the ability of gold nanoparticles to inhibit LPS-induced M1 polarization in Raw 264.7 cells. Raw 264.7 cells were cultured in a 24-well plate (5.0x10 4After seeding on a 1000-well plate, the cells were cultured in complete medium (DMEM, 10% FBS, 1% PS) for 24 hours. After complete culture, LPS (1 μg / ml) and gold nanoparticles (Alginate@AuNP 125 μg / ml, Glycol chitosan@AuNP 125 μg / ml) were added, and the cell morphology was observed under a microscope at 0, 1, 4, and 18 hours.

[0144] As a result, when stimulated with LPS alone, the cells reached an activated M1 state (pseudopodia) within 4 hours, but when treated with LPS and gold nanoparticles, they maintained the monocyte morphology and were not activated to M1 (Figure 31).

[0145] 17. Confirmation of pro-inflammatory cytokine secretion in Raw 264.7 cells using PCR PCR analysis was performed to confirm the extent to which gold nanoparticles inhibited the secretion of pro-inflammatory cytokines in LPS-stimulated Raw 264.7 cells. Raw 264.7 cells were cultured in a 12-well plate (1.0x10 5Cells were seeded onto a plate (100 μg / well) and cultured in complete medium for 24 hours. After washing with PBS, they were incubated with gold nanoparticles (Alginate@AuNP 125 μg / ml, Glycol chitosan@AuNP 125 μg / ml) for 5 hours. Cells were washed with PBS before being incubated with medium with or without LPS (1 μg / ml) for 20 hours. The factors analyzed were TNF-α, IL-6, IL-1 beta, MCP-1, and iNOS. Total RNA was extracted from the cells using QIAzol Lysis Reagent and an RNeasy Mini Kit (QIAGEN, Germany), followed by reverse transcription of 1 μg of RNA into cDNA using the iscript cDNA synthesis kit (BIO RAD Laboratories, USA). Then, qRT-PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems™, USA) on an Applied Biosystems Instrument (USA). GAPDH was used as an endogenous control to normalize the relative mRNA expression of the genes listed in Table 2 below.

[0146] [Table 2]

[0147] As a result, we confirmed that all of the above factors (TNF-α, IL-6, IL-1 beta, MCP-1, and iNOS) are inflammatory factors and are secreted in large amounts by cells stimulated with LPS. However, we confirmed that the secretion of all of these factors was reduced in the group treated with Glycol chitosan@AuNPs before LPS stimulation (Figure 32). Furthermore, since the secretion of inflammatory factors was further reduced by Glycol chitosan@AuNPs than by Alginate@AuNPs, we concluded that the anti-inflammatory effect was even greater. This confirmed that the inflammatory response was alleviated mainly by capturing ROS / RNS induced by LPS stimulation.

[0148] 18. Confirmation of pro-inflammatory cytokine secretion in Raw 264.7 cells using ELISA ELISA analysis was performed to confirm the extent to which gold nanoparticles inhibited the secretion of pro-inflammatory cytokines in LPS-stimulated Raw 264.7 cells. Raw 264.7 cells were cultured in a 12-well plate (1.0x10 5 The cells were seeded onto a plate (100 μg / well) and cultured in complete medium (DMEM, 10% FBS, 1% PS) for 24 hours. Afterwards, they were washed with PBS and cultured with AuNPs (125 μg / ml) for 5 hours. Before culturing the cells in medium with or without LPS (1 μg / ml) for 20 hours, the cells were washed with PBS, and the cell supernatant was obtained and stored at -20°C before analysis. ELISA validation was performed according to the protocol provided with the purchased ELISA kit. The analyzed factors were TNF-alpha (EliKine TMIL-6 (IL-6 Mouse ELISA Kit, Invitrogen), IL-1 beta (IL-1 beta Mouse ELISA Kit, Invitrogen), MCP-1 (MCP-1 Mouse ELISA Kit, Invitrogen), and iNOS (Mouse NOS2 / iNOS (Nitric Oxide Synthase 2, Inducible)).

[0149] As a result, we confirmed that all of the above factors (TNF-α, IL-6, IL-1 beta, MCP-1, and iNOS) are inflammatory factors and are secreted in large amounts by cells stimulated with LPS. However, we confirmed that the secretion of all of these factors was reduced in the group treated with Glycol chitosan@AuNPs before LPS stimulation (Figure 33). Furthermore, since the secretion of inflammatory factors was further reduced by Glycol chitosan@AuNPs than by Alginate@AuNPs, we concluded that the anti-inflammatory effect was even greater. This confirmed that the inflammatory response was alleviated mainly by capturing ROS / RNS induced by LPS stimulation.

[0150] 19. In vivo Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) We quantitatively investigated the adsorption of Glycol chitosan@AuNPs and Alginate@AuNPs to colon tissue in a mouse model of IBD induced with DSS (dextran sodium sulfate). Figure 34 shows the mechanism of drug adsorption in mice orally administered with DSS-induced colitis. The groups were as follows: Citrate@AuNPs (N = 5), Glycol chitosan@AuNPs (N = 5), and Alginate@AuNPs (N = 5). Specifically, 7-week-old male C57BL / 6 mice were housed five per cage and given drinking water supplemented with DSS for one week. They were given 60 mg / kg of Citrate@AuNPs, Glycol chitosan@AuNPs, and Alginate@AuNPs on the final day, and sacrificed 2, 6, and 24 hours later. Colon tissue was isolated and then fixed in 4% paraformaldehyde prior to ICP-MS analysis.

[0151] When gold nanoparticles were administered to colonic tissue, ICP-MS was used to examine the degree of retention over time. Drug destabilization by digestive fluids and drug clearance, such as diarrhea, reduced the bioavailability of the drug in IBD patients (Figure 35). Therefore, ICP-MS was used to examine the adsorption of citrate@AuNPs, alginate@AuNPs, and glycol chitosan@AuNPs. Glycol chitosan@AuNPs exhibited a higher degree of adsorption than other particles. This is due to the positive nature of the particles interacting with the negatively charged mucus layer. This interaction resulted in the drug remaining in the colon for approximately 24 hours. Furthermore, the retention time was approximately 3.4 times longer than that of alginate, and the elimination rate was approximately 2.7 times lower.

[0152] 20. In vivo bio-transmission electron microscopy (BIO-TEM) We aimed to qualitatively confirm the adsorption of Glycol Chitosan@AuNPs and Alginate@AuNPs to colonic tissue after oral administration in a mouse model of IBD induced with DSS (dextran sodium sulfate). The experimental materials used in this experiment were Karnofsky's fixative (initial fixation), 0.05 M sodium cacodylate buffer (for washes), 2% osmium tetroxide (1 ml) for post-fixation, 0.1 M cacodylate buffer (1 ml), 0.5% uranyl acetate for staining, ethanol (30, 50, 70, 80, and 90% concentrations) for dehydration, propylene oxide for transfer, and Spurr resin (1 ml) for infiltration. Sorensen's phosphate buffer solution, consisting of solutions A and B (A: 0.2 M NaHPO 2H 0, B: 0.2 M NaH PO 4H 0), was used for a 10-minute fixative rinse. The tissue membranes were further stained with 1% osmium tetroxide (OsO4) for 1 hour. The specimens were then washed for 10 minutes in Sorensen's phosphate buffer to remove any remaining OsO4. Dehydration was performed using different concentrations of ethanol (30%, 50%, 70%, and 90% ethanol for 10 minutes, followed by three 20-minute cycles at 100% ethanol). The Spurr kit method, which allows for the formation of epoxy resin blocks in low-viscosity embedding media, was applied to all specimens. Epoxy resin specimens were cut into 80-nm-thick sections using an ultramicrotome (EM UC7). The sections were air-dried for a minimum of 1 hour. Copper grids were mounted with uranyl acetate (2%) for 20 minutes, washed with DW, and then stained with lead citrate (0.4%) for 10 minutes.Sections were mounted on grids and viewed using an 80 kV transmission electron microscope.

[0153] As a result, it was confirmed that Alginate@AuNPs and Glycol chitosan@AuNPs remained in the mucus layer from 6 to 24 hours (Figure 36).

[0154] 21. Synthesis of Glycol Chitosan and Glycyrrhizin We attempted to further incorporate glycyrrhizin (GL), which can regulate HMGB1 activity on gold nanoparticles, into glycol chitosan. GL (2 mM and 10 ml) was dissolved in distilled water, and the pH was adjusted with 1 N NaOH until GL was completely dissolved. Then, NaIO4 (2 mM and 10 ml) was dissolved in distilled water, wrapped in foil, and kept in the dark. SP solution was slowly added dropwise to form oxidized GL (oGL). The GL solution was stirred at room temperature in the dark for 30 minutes and in the light for 30 minutes. Glycol chitosan (64 mg) was dissolved in 20 ml of DW, and the pH was adjusted with 1.0 M HCl until glycol chitosan was completely dissolved. Then, an equal volume of oGL was slowly added dropwise to the GC solution. The solution was reacted at 4°C for 2 hours, and then sodium cyanoborohydride was added in an amount equivalent to 1 / 1,000 of the total solution, followed by overnight reaction at 4°C. The resulting product was dialyzed using a 3.5 kDa MWCO dialysis bag to remove unreacted materials. The GC-GL conjugate was obtained by lyophilization for 2 days and weighed to confirm the yield. It was then dissolved in DO for H-NMR measurement. H-NMR was performed using a VNMRS 600 MHz spectrometer from Agilent Technologies, and FT-IR was performed using a Fourier transform infrared (FT-IR) spectrometer from NICOLET IS50 from Thremo Fisher Scientific.

[0155] The conjugation scheme of glycyrrhizin and glycol chitosan is shown in Figure 37. Specifically, glycyrrhizin dissolved in DW was reacted with NAIO4 to create an aldehyde group on the glucuronic acid moiety of glycyrrhizin. When oxidized glycyrrhizin was slowly added dropwise to glycol chitosan dissolved in DW, the aldehyde group and the amine group reacted via a Schiff base reaction to form an imine group, forming the Glycol Chitosan-GL intermediate. Sodium cyanoborohydride was added to this intermediate to form a stable secondary amine group.

[0156] The synthesis results of glycol chitosan-glycyrrhizin were then confirmed using H-NMR, and it was confirmed that a peak appeared at approximately 2.8 ppm when a secondary amine group was finally produced through the reaction between oxidized glycyrrhizin and the amine group of glycol chitosan (Figure 38).

[0157] The synthesis of glycol chitosan-glycyrrhizin was confirmed by FT-IR. The aromatic C=C bond of glycyrrhizin and the phenolic primary alcohol peak of glycol chitosan are characteristic peaks of each substance, and the peak of the amine group of glycol chitosan is at 3356 cm. -1 and the peak of the secondary amine group in the conjugated form appears at 3468.2 cm -1 By confirming that a broad peak appeared in this vicinity, it was confirmed that glycol chitosan and glycyrrhizin had been conjugated (FIG. 39).

[0158] 22. Synthesis of Glycol Chitosan-Glycyrrhizin Coated Gold Nanoparticles We attempted to confirm the synthesis of glycol chitosan-glycyrrhizin-coated gold nanoparticles (glycol chitosan-GL@AuNP). The synthesis scheme for glycol chitosan and glycyrrhizin, as well as the scheme for coating gold nanoparticles with glycol chitosan, are shown in Figure 40. The solution from Example 17 was added to distilled water, the pH was adjusted to 8 using 1N NaOH, and the solution was heated on a hot plate at 100°C until the GC-GL was completely dissolved. A gold solution (2 mM) was slowly added dropwise to the GC-GL solution. The yellow solution turned colorless and then wine-colored within 30 minutes, confirming the formation of GC-GL@AuNP. The AuNP solution was centrifuged at 16,000 rpm for 30 minutes. The supernatant containing excess reactants was discarded, and the AuNP pellet was redispersed in DW and analyzed using UV / vis (Nanodrop 2000).

[0159] 23. Colloidal stability of glycol chitosan-glycyrrhizin coated gold nanoparticles as a function of pH Dynamic light scattering (DLS) measurements were performed to confirm whether citrate@AuNP (1:10), alginate@AuNP (0.2%), glycol chitosan@AuNP (0.32%), and glycol chitosan-GL@AuNP remained stable in a biomimetic environment similar to the GI tract. Specifically, gold nanoparticles were adjusted to pH 2 and pH 6 with HCl and NaOH, respectively. The zeta potential of the gold nanoparticles was then monitored after 12 hours.

[0160] As a result, the value of Citrate@AuNP (1:10) was at 240 nm, which was approximately 8-fold higher than that of 30 nm, the value of Alginate@AuNP 0.2% was at 120 nm, which was approximately 2-fold higher than that of 56 nm, while the value of Glycol chitosan@AuNP 0.32% was at 60 nm, which was not significantly different from the conventional 80 nm (Figure 41).

[0161] In addition, we measured the zeta potential (mV) of citrate@AuNP (1:10), alginate@AuNP 0.2%, glycol chitosan@AuNP 0.32%, and glycol chitosan-GL@AuNP after 12 hours at pH 2 and pH 6 to confirm how stable the zeta potential was. The results are shown in Figure 42. Specifically, citrate@AuNP (1:10) had a negative charge, but after 12 hours at pH 2, its zeta potential changed to a positive charge. Furthermore, alginate@AuNP 0.2% had a zeta potential of approximately -34 mV, but after 12 hours at pH 2, its zeta potential decreased to approximately -20 mV. This indicates that the carboxylic acids of citrate and alginate were protonated, resulting in a decrease in the zeta potential. However, it was confirmed that Glycol chitosan@AuNP 0.32% maintained its zeta potential at pH 2 and pH 6. Finally, since the zeta potential value of Glycol chitosan-GL@AuNP did not change significantly, it was predicted that Glycol chitosan@AuNP and Glycol chitosan-GL@AuNP would stably maintain their properties in vivo.

[0162] Furthermore, when gold nanoparticles were left at pH 2 and pH 6 for 12 hours, the particle size in the aqueous solution was confirmed and the PDI values ​​were measured to confirm whether they were stably and well dispersed. As a result, all PDI values ​​were below 0.5, confirming that they were stably and well dispersed (Figure 7).

[0163] 24. Co-culture of Raw 264.7 and Caco-2 cells Raw 264.7 cells and Caco-2 cells were co-cultured, and the Caco-2 cells were stimulated with hydrogen peroxide to examine the effect of the secreted HMGB1 on the Raw 264.7 cells, as well as the effect of treating them with gold nanoparticles and hydrogen peroxide. The co-culture system of Raw 264.7 cells and Caco-2 cells is shown in Figure 43. To examine the secretion of pro-inflammatory cytokines, Caco-2 cells were cultured in a 6.6 mm diameter, 0.4 μm pore size (density: 2.5 × 10 4 The cells were seeded in the Transwell® insert chamber (apical side) at a density of 5.0 × 10 cells / well. Raw 264.7 cells were seeded in the basolateral chamber (density: 5.0 × 10 4 After 24 hours, the cells were divided into four groups for the experiment (Group 1: negative control, Group 2: positive control; H2O2 (100 μM), Group 3: H2O2 (100 μM) and Glycol chitosan@AuNP (125 μg / ml) were treated simultaneously, and Group 4: H2O2 (100 μM) and Glycol chitosan-GL@AuNP (125 μg / ml) were both treated in the apical chamber, and the cells were cultured for 20 hours).

[0164] The morphology of Raw 264.7 cells was examined in a co-culture system of Raw 264.7 cells and Caco-2 cells. The results confirmed that in the H2O2-treated group, Raw 264.7 cells either died or were activated by secreted HMGB1. The Glycol chitosan@AuNP or Glycol chitosan-GL@AuNP-treated groups showed almost no activated morphology of Raw 264.7 cells due to the effect of gold nanoparticles (Figure 44).

[0165] We also examined the morphology of Caco-2 cells in a co-culture system of Raw 264.7 cells and Caco-2 cells. As a result, we confirmed that the monolayer collapsed upon stimulation in the H2O2-treated group, but the monolayer remained intact after gold nanoparticle treatment (Figure 45).

[0166] 25. HMGB1, inflammatory factors (TNF-alpha, IL-6, IL-1 beta, MCP-1, and iNOS) ELISA validation of the co-culture system Analysis was performed using the cell supernatant in the basolateral chamber. ELISA validation was performed according to the protocol provided with the purchased ELISA kit. The analyzed factors were HMGB1 (EliKine TM Mouse HMGB1 ELISA Kit, Abbkine), TNF-alpha (EliKine) TM IL-6 (IL-6 Mouse ELISA Kit, Invitrogen), IL-1 beta (IL-1 beta Mouse ELISA Kit, Invitrogen), MCP-1 (MCP-1 Mouse ELISA Kit, Invitrogen), and iNOS Mouse NOS2 / iNOS (Nitric Oxide Synthase 2, Inducible).

[0167] ELISA testing of HMGB1 secretion in a co-culture system of Raw 264.7 cells and Caco-2 cells showed that when Caco-2 cells were stimulated with HO (100 μM), the highest amount of HMGB1 was secreted. However, when Glycol chitosan@AuNPs were treated with HO, the concentration of secreted HMGB1 decreased due to the destruction of the Caco-2 cells (Figure 46). Furthermore, when GL was also introduced, lower concentrations of HMGB1 were secreted than with Glycol chitosan@AuNPs. Overall, it can be seen that Glycol chitosan-GL@AuNPs inhibit HMGB1 secretion more effectively than Glycol chitosan@AuNPs.

[0168] Furthermore, ELISA testing of inflammatory factors secreted by Raw 264.7 cells in a co-culture system of Raw 264.7 and Caco-2 cells confirmed that GL, a direct antagonist of HMGB1, suppressed LPS-induced lethality and attenuated inflammation induced by M1 polarization (Figure 47). It was confirmed that the secretion of pro-inflammatory cytokines from HMGB1-activated Raw 264.7 cells was barely detectable with glycol chitosan-glycyrrhizin (Glycol chitosan-GL).

[0169] 26. FACS validation of M1 and M2 polarization of Raw 264.7 cells in a co-culture system Cytometric analysis was performed to confirm the M1 and M2 polarization rates in Raw 264.7 cells treated with gold nanoparticles (glycol chitosan@AuNP and glycol chitosan-GL@AuNP). For cytometric analysis, cells were detached using a scraper and centrifuged at 12,000 rpm for 3 minutes. After removing the supernatant, the cells were redispersed in PBS and incubated with or without antibodies for 20 minutes at room temperature. The antibodies used were CD206 APC-eFluor 780 and CD80-FITC. There were seven groups in total: negative control (staining), positive control (non-staining), raw cells with CD206 marker (staining), raw cells with CD80 marker (staining), raw cells with CD206 marker and CD80 marker (co-staining), raw cells incubated with Glycol chitosan@AuNPs and stained with CD206 and CD80 (co-staining), and raw cells incubated with Glycol chitosan-GL@AuNPs and stained with CD206 and CD80 (co-staining). Samples were analyzed using BD FACS (BD FACSCalibur, BD Biosciences, New Jersey, USA), and data were analyzed using FlowJo V10. The unstained positive control group was used for gating.

[0170] The morphology of Raw 264.7 cells was examined in a co-culture system of Raw 264.7 cells and Caco-2 cells. The results confirmed that in the H2O2-treated group, Raw 264.7 cells either died or were activated by secreted HMGB1. The Glycol chitosan@AuNP or Glycol chitosan-GL@AuNP-treated groups showed almost no activated morphology of Raw 264.7 cells due to the effect of gold nanoparticles (Figure 44).

[0171] In a co-culture system of Raw 264.7 cells and Caco-2 cells, FACS analysis of M1 and M2 polarization of Raw 264.7 cells confirmed that Glycol chitosan@AuNPs and Glycol chitosan-GL@AuNPs reduced M1 polarization from 32.93% to nearly 0% even after H2O2 injury (Figure 48). Furthermore, Glycol chitosan@AuNPs reduced M2 polarization by 53.16%, while Glycol chitosan-GL@AuNPs reduced it by approximately 0.4%, showing almost no polarization.

[0172] Analysis of the population of M1 and M2 polarization of Raw 264.7 cells in a co-culture system of Raw 264.7 cells and Caco-2 cells revealed that the Glycol chitosan@AuNPs had a higher M2 polarization rate, while the Glycol chitosan-GL@AuNPs showed almost no change in M1 and M2 morphology, suggesting that they would maintain the monocytic morphology (Figure 49). In other words, they are thought to have little stimulation on Raw 264.7 cells.

[0173] 27. Animal Experiment Preparation and Measurement Elements Seven-week-old male C57BL / 6 mice were housed five per cage and allowed to thrive for a week. The specific protocol is shown in Figure 50. Healthy mice in the negative control group were provided with normal drinking water only, while mice in the positive control group and experimental groups (glycol chitosan@AuNP and glycol chitosan-GL@AuNP) were provided with 2.5% (w / v) dextran sodium sulfate (DSS) in drinking water for 7 days, followed by normal drinking water for 7 days. The DSS-induced groups were orally administered 200 μl of 30 mg / kg Glycol chitosan@AuNPs (N=5), 200 μl of Glycol chitosan-GL@AuNPs (N=5), or 200 μl of PBS (N=5) for 2 weeks. On the final day of the experiment (Day 14), the mice were euthanized and the colon and organs (lungs, heart, kidneys, liver, and spleen) were removed. The spleen and colon weights and lengths were measured, and the extent of damage was confirmed by photographing the anus.

[0174] The results of measuring the survival rate of the mice are shown in Figure 51. The survival rates of the DSS-administered group were 80% and 60% on days 5 and 12, respectively. On the other hand, the Glycol chitosan@AuNP and GC-GL@AuNP-administered groups remained untouched, and the survival rate remained at 100%.

[0175] The results of measuring the body weight of the mice are shown in Figure 52. It was confirmed that all the groups administered with Glycol chitosan@AuNP and Glycol chitosan-GL@AuNP lost weight and recovered it by the 10th day, and that the group administered with Glycol chitosan-GL@AuNP had a superior degree of weight recovery.

[0176] The results of measuring the DAI (disease activity index) values ​​of the mice are shown in Figure 53. It was confirmed that the DAI values ​​were low in all groups administered Glycol chitosan@AuNPs and Glycol chitosan-GL@AuNPs until the end of the experiment on Day 14. The DAI index values ​​were confirmed as follows: stool consistency (hard: 0, soft: 2, diarrhea: 4), stool bleeding level measured using a Hemoccult Sensa (Beckman Coulter) (none: 0, bleeding level: 2, visible macroscopically: 4), and weight loss (weight loss less than 1%: 0, weight loss between 1-5%: 1, weight loss between 5-10%: 2, weight loss between 10-20%: 3, weight loss over 20%: 4).

[0177] The results of measuring the length of the mouse colon are shown in Figure 54. On day 14, the length of the colon tissue in the mice fed DSS and induced with IBD was approximately 5 cm, which was shorter than the control group. However, the length of the colon tissue in the mice fed gold nanoparticles was restored to a similar length to that of the control group.

[0178] The results of colon damage in mice are shown in Figure 55. The group treated with 2.5% DSS and PBS developed edema and diarrhea, and the condition of the colon deteriorated significantly, whereas the colon in the group treated with gold nanoparticles either showed a much reduced severity or was restored to a normal state.

[0179] The results of measuring the weight and length of the mouse colon are shown in FIG. 56. Both types of gold nanoparticles showed values ​​similar to those in normal conditions, confirming that they had almost no toxicity to the whole body.

[0180] Photographs of the mouse anuses are shown in Figure 57. The group treated with 2.5% DSS and PBS had poor anal condition due to bloody stools, but the group treated with gold nanoparticles showed complete recovery.

[0181] The results of confirming the enlargement of mouse spleens are shown in Figure 58. It was confirmed that the spleen length of the group treated with 2.5% DSS and PBS enlarged to approximately 1.5 cm, while the length of the group treated with gold nanoparticles became shorter, similar to the normal state.

[0182] 28. Identification of tight junctions using immunohistochemistry (IHC) We investigated the extent of tight junction (ZO-1 and occludin) repair following treatment with Glycol Chitosan@AuNPs and Glycol Chitosan-GL@AuNPs. Tissues processed onto slides were fixed in 4% paraformaldehyde. Caco-2 cells were washed twice for approximately 2 minutes with PBS-Tween 20. For blocking, the cells were incubated with 20% goat serum in PBS-Tween 20 for 30 minutes at room temperature. Primary antibodies (anti-mouse occludin, anti-rabbit ZO-1) diluted 100:1 in 20% goat serum in PBST-20 were incubated for 1 hour at room temperature and then washed with PBS. Secondary antibodies (goat anti-mouse-488, goat anti-rabbit-594) diluted 1:200 in PBS were then incubated with the cells. Protected from light, the cells were washed twice for 3 minutes with PBS. Then, 1-2 drops of DAPI (blue, nuclear stain) mounting solution were added, and the sections were covered with a cover slide and observed under a fluorescence microscope.

[0183] The results are shown in Figure 59. Specifically, after DSS induction, feeding only PBS disrupted tight junction proteins such as occludin (green) and Zo-1 (red), thereby destroying the monolayer of intestinal epithelial cells. When gold nanoparticles were added, yellow (red and green merged) fluorescence was observed, confirming that the tight junctions were restored.

[0184] 29. ELISA validation of mouse serum for HMGB1 and anti-inflammatory factors Cardiac blood was collected from mice under general anesthesia and then centrifuged at 3,000 rpm for 30 minutes at 4°C into anticoagulant-coated tubes. ELISA was performed according to the protocol provided with the kit. Specifically, experiments were performed on day 14 to confirm the anti-inflammatory effects of Glycol Chitosan-GL@AuNPs, which capture ROS / RNS and HMGB1, on serum in a DSS-induced mouse model.

[0185] The results confirmed that Glycol chitosan-GL@AuNP treatment significantly reduced HMGB1 compared to other treatment groups (PBS and Glycol chitosan@AuNP treatment). HMGB1 is actively or passively released extracellularly in necrotic tissue and stressed cells, and M1 polarization markers TNF-α, IL-6, IL-1β, iNOS, and MCP-1 were all increased by DSS administration but all decreased by gold nanoparticle treatment (Figure 60).

[0186] 30. ELISA validation for HMGB1 and anti-inflammatory factors in mouse colon The middle part of the colon tissue was cut, placed in an EP tube, and frozen in liquid nitrogen. RIPA buffer (Pierce) was used for 5 mg of tissue. TM 300 μl of HCl was added to the cells, and the cells were homogenized using a homogenizer (FastPrep-24 5G, MP Biomedicals, LLC, USA) and centrifuged at 14,800 rpm for 20 minutes at 4°C. The supernatant was then collected and stored at -20°C until analysis. Subsequently, HMGB1 (EliKine) was detected using an ELISA kit. TM Mouse HMGB1 ELISA Kit, Abbkine), TNF-alpha (EliKine) TMWe analyzed the following: Mouse TNF-α ELISA Kit (Abbkine), IL-6 (IL-6 Mouse ELISA Kit, Invitrogen), IL-1 beta (IL-1 beta Mouse ELISA Kit, Invitrogen), MCP-1 (MCP-1 Mouse ELISA Kit, Invitrogen), and iNOS Mouse NOS2 / iNOS (Nitric Oxide Synthase 2, Inducible). ELISA was performed according to the protocol provided with the kit. Specifically, experiments were performed on day 14 to confirm the anti-inflammatory effects of Glycol Chitosan-GL@AuNPs, which capture ROS / RNS and HMGB1, on the colon in a DSS-induced mouse model.

[0187] The results confirmed that Glycol chitosan-GL@AuNP treatment significantly reduced HMGB1 compared with other treatment groups (PBS and Glycol chitosan@AuNP treatment). HMGB1 was confirmed to be actively or passively released extracellularly in necrotic tissue and stressed cells. The inflammatory factors TNF-α, IL-6, IL-1β, iNOS, and MCP-1 were all increased by DSS administration but decreased by gold nanoparticle treatment (Figure 61).

[0188] 31. PCR analysis of HMGB1 and anti-inflammatory factors in mouse colon The distal portion of the colon tissue was cut, and 600 μl of RLT buffer (RNeasy Mini Kit, QIAGEN, Germany) was added per 30 mg of tissue. 10 μl of 2-mercaptoethanol (Sigma) was added per ml of RLT buffer. Homogenization was performed using a homogenizer (FastPrep-24 5G, MP Biomedicals, LLC, USA), followed by centrifugation at 14,800 rpm for 3 minutes at 4°C. The supernatant was collected. An equal volume of 70% ethanol was added and immediately pipetted. mRNA in the tissue was then extracted using the RNeasy Mini Kit (QIAGEN, Germany). 1 μg of RNA was reverse transcribed into cDNA using the iscript cDNA synthesis kit (BIO RAD Laboratories, USA), and the cDNA was then reverse transcribed using SYBR Green PCR Master Mix (Applied Biosystems Instruments, USA) on an Applied Biosystems instrument. TM qRT-PCR was performed using a qRT-PCR kit (Bio-Rad Laboratories, Inc., USA). GAPDH was used as an endogenous control to normalize the relative mRNA expression of the genes listed in Table 1.

[0189] qRT-PCR confirmed that the mRNA levels of relevant pro-inflammatory cytokines were significantly enhanced in DSS-challenged mice, while their expression was significantly decreased after treatment with glycol chitosan-coated AuNPs (Figure 62).

[0190] 32. Number of M1 and M2 polarized cells in mouse colon We aimed to determine the proportion of M1 and M2 immune cells polarized in the colonic lamina propria. After cutting the upper end of the colonic tissue, aggregated lymphoid nodules (Payer's patches) and fat were removed. The tissue was cut longitudinally and washed three times with cold PBS. Colonic tissue was dissected at 1 cm intervals and immersed in RPM I (10% FBS, 1% PS) containing 2 mM EDTA and placed in an incubator at 37.5°C, 250 rpm, for 40 minutes to separate the cells from the epithelial cell layer. The solution was filtered through a 70-micron cell strainer and centrifuged at 1,500 rpm, 4°C, for 10 minutes to obtain a pellet. Subsequently, cells were gated using CD45-APC-Cy7 (BD Pharmingen, San Diego, USA), which is consistently present in M1 and M2 immune cells, and then stained with CD206 APC-eFluor 780 and CD80-FITC. The staining method was the same as in Example 15.

[0191] After DSS induction, the PBS-treated group showed a high M1 polarization rate, whereas the Glycol chitosan@AuNP-treated group showed a lower rate, and the Glycol chitosan-GL@AuNP group showed a decrease to the control group level. In other words, gold particle treatment in an IBD model confirmed a total decrease in the M1 rate of immune cells in colon tissue (Figure 63). Furthermore, the pro-inflammatory CD80 expression level, which indicates M1-polarized macrophages, was significantly higher in colon leukocytes collected from the PBS-treated group compared to the glycol chitosan-coated AuNP-treated group. + / CD46 + The proportion of leukocytes was also found to be higher.

[0192] We also performed cytometric analysis to confirm the number of M1 and M2 cells in the mouse colon. 4After reading the immune cells, cell gating was performed based on CD45, which is expressed in all immune cells, and the M1 and M2 polarization ratios were confirmed within the gated range. As a result, Glycol chitosan-GL@AuNPs significantly reduced colonic inflammatory cytokines at both the protein and mRNA levels, and suppressed inflammatory CD80 in colonic leukocytes. + / CD45 + It was confirmed that the proportion of mononuclear cells decreased (FIG. 64).

[0193] 33.Tissue processing For histological analysis, tissues were fixed in 4% paraformaldehyde and processed automatically using a Leica TP1020 semi-enclosed benchtop tissue processor (Leica Biosystems, Wetzlar, Hesse, Germany). They were embedded in paraffin, and paraffin blocks were sectioned at 7 μm thickness using a Leica RM2145 Microtome (Leica Biosystems). Organ toxicity testing revealed no toxicity in the major organs: heart, liver, lungs, kidneys, and spleen (Figure 65).

[0194] 34. C-Reactive Protein (CRP) ELISA Validation We attempted to confirm the levels of C-reactive protein (CRP), a blood inflammatory factor. After cardiac blood collection from mice under general anesthesia, the blood was centrifuged at 3000 rpm for 30 minutes at 4°C in tubes coated with anticoagulant. ELISA was performed according to the experimental protocol included in the kit (Mouse C-Reactive Protein ELISA-ALPCO).

[0195] As a result, it was confirmed that mice in which disease was induced with DSS had increased levels of CRP, a blood inflammatory factor, but Glycol chitosan-GL@AuNP restored this to a normal state (Figure 66).

[0196] 35. Calprotectin ELISA Validation We attempted to confirm the level of calprotectin, an inflammatory factor in feces. The middle part of the colon tissue containing feces was cut, placed in an EP tube, and frozen in liquid nitrogen. RIPA buffer (Pierce) was used per 5 mg of tissue. TM After homogenization using a homogenizer (FastPrep-24 5G, MP Biomedicals, LLC, USA), the mixture was centrifuged at 14,800 rpm for 20 minutes at 4°C. After centrifugation, the supernatant was collected and stored at -20°C until analysis. ELISA was performed according to the protocol provided with the kit (Mouse Calprotectin ELISA - Cusabio).

[0197] As a result, we confirmed that calprotectin, a type of DAMP (S100A8 / 9) distinct from HMGB1, was reduced by treatment with Glycol chitosan-GL@AuNPs, thus confirming that Glycol chitosan-GL@AuNPs can regulate various types of DAMP molecules (Figure 67).

[0198] 36. Identification of mucus using immunohistochemistry (IHC) We investigated the extent of mucus layer repair following treatment with Glycol Chitosan@AuNPs and Glycol Chitosan-GL@AuNPs. Tissues processed onto slides were fixed in 4% paraformaldehyde. The cells were then washed twice for approximately 2 minutes with PBS-Tween 20. For blocking, the cells were incubated with 20% goat serum in PBS-Tween 20 for 30 minutes at room temperature. The cells were then incubated with 20% goat serum (PBST-20) and a primary antibody (anti-rabbit Muc-2, Abcam) diluted 100:1 at room temperature for 1 hour and then washed with PBS. The secondary antibody (goat anti-rabbit-488) was diluted 1:200 in PBS and then incubated with the cells. Protected from light, the cells were washed twice for 3 minutes with PBS. Then, 1-2 drops of DAPI mounting solution (blue) were added, and the sections were covered with a cover slide and observed under a fluorescence microscope.

[0199] As a result, mucus was stained in the subepithelial goblet cells, which are responsible for mucus secretion, and it was confirmed that Glycol chitosan@AuNPs and Glycol chitosan-GL@AuNPs prevented DSS-induced mucosal damage (Figure 68).

[0200] 37. Check for blood influx We attempted to confirm the blood influx of Glycol chitosan-GL@AuNPs. Seven-week-old male C57BL / 6 mice were orally administered 10 mg / kg of Glycol chitosan-GL@AuNPs, and blood samples were taken from the heart 2, 6, and 24 hours later (n = 3). The results showed that Glycol chitosan-GL@AuNPs were not detected in the blood samples taken 2, 6, and 24 hours after oral administration, confirming that Glycol chitosan-GL@AuNPs, which have the property of adsorbing to the intestinal mucosa, delayed their influx into the blood (Figure 69).

[0201] 38. Biochemical and blood tests We investigated the toxicity and blood residue of Glycol Chitosan@AuNPs and Glycol Chitosan-GL@AuNPs. Seven-week-old male Balb / c mice were orally administered PBS and Glycol Chitosan-GL@AuNPs (10 mg / kg) once daily for 14 days (n = 4). On the 14th day, serum (100 μl for biochemical testing) and plasma (200 μl for CBC testing) were collected via cardiac puncture. Samples were analyzed by Daegun Health Care (DK Korea, Seoul, Korea). Specifically, Figure 70a shows the results of analyzing total protein, albumin, globulin, AST, ALT, BUN, total bilirubin, and creatinine, Figure 70b shows the results of analyzing RBC, HGB, HCT, RBC indices (MCV, MCH, MCHC), RDW, MPV, and PLT, and Figure 70c shows the results of analyzing WBC and WBC percentages (NEU, LYM, MONO, EOS, BASO).

[0202] As shown in Figures 70a to 70c, the colored areas indicate the normal range of each index, and it was confirmed that most of the factors were within the normal range.

[0203] 39. Microbiome analysis using feces Stool analysis was used to analyze 16s ribosomal RNA gene sequencing, taxonomic classification, and diversity. Balb / c mice were orally administered PBS and Glycol chitosan-GL@AuNPs (10 mg / kg) once daily for 4 days. Stool samples were collected on day 14, stored at -80°C, and submitted to Macorgen (Seoul, Korea). Sequencing libraries for the V3 and V4 regions were constructed using the Miseq Illumina sequencing platform, and 16s RNA amplicons were amplified using V3-V4 region primers. Sequences were assigned to operational taxonomic units (OTUs) using a 0.03 cutoff and classified against the Ribosomal Database Project 16s RNA gene training set (v.9) at an 80% confidence threshold for taxonomic classification. Alpha diversity was measured using the Chao1 and Shannon indices, and the generalized UniFrac (GUniFrac) method was applied to calculate phylogenetic distances. Unweighted UniFrac measures of beta diversity were plotted along two major axes based on 10,000 reads per sample. ANOSIM analysis was performed with the vegan package (v2.5-7) in R (v4.0.5) to compare similarities between different groups, and data were processed using GraphPad Prism (version 8.3.0; GraphPad Software Inc., San Diego, CA, USA).

[0204] Measurement of OTU richness confirmed that the DSS-treated group had a change in the composition of the microbiota compared to the control group (Figure 71). Furthermore, we confirmed that imbalances in the gut microbiota, which are associated with various inflammatory and immune disorders, play an important role in the development of IBD. In this context, OTU is a unit used in analyzing microbial diversity, treating similar sequences as a single species.

[0205] By measuring the Shannon diversity value, we confirmed that the species diversity was similar within the DSS-treated groups, including the control group (Figure 72). Shannon diversity is an index that shows how diverse the data classes are within a population, and can indicate the diversity of biological species within a population.

[0206] Measurement of the Simpson index for the alpha diversity of the intestinal microbial community confirmed that there was no significant change in microbial diversity between the control and DSS groups in the Glycol chitosan@AuNP and Glycol chitosan-GL@AuNP treatment groups (Figure 73). The Simpson index can measure species diversity using the number of animal species and the distribution weight of the species; values ​​closer to 0 indicate higher diversity, and values ​​closer to 1 indicate lower diversity.

[0207] Analysis of the beta diversity of the gut microbial community showed that the beta diversity between the Glycol chitosan-GL@AuNP-treated groups in healthy mice and the DSS group was almost correlated, confirming that Glycol chitosan-GL@AuNPs could restore intestinal immune homeostasis without disrupting the gut microbiome (Figure 74).

[0208] Heat map analysis of the taxonomic microbial composition at the family level (Figure 75) confirmed that Glycol chitosan-GL@AuNPs could restore intestinal immune homeostasis without disrupting the intestinal microbiota in healthy mice and the DSS group treated with Glycol chitosan-GL@AuNPs. Furthermore, Glycol chitosan-GL@AuNP treatment significantly increased Akkermansia muciniphila, a microbiota associated with intestinal barrier protection. However, the abundance of Turicibacter sanguinis, which is highly associated with acute colitis, was reduced.

[0209] The relative diversity of the intestinal microbiome (Figure 76) confirmed that Glycol chitosan-GL@AuNPs can restore intestinal immune homeostasis without disrupting the intestinal microbiota in both healthy mice and the DSS group. Furthermore, we confirmed that Glycol chitosan-GL@AuNP treatment significantly increased Akkermansia muciniphila, a microbiota associated with intestinal barrier protection. Further analysis at the family level confirmed that Glycol chitosan-GL@AuNP treatment significantly increased Lactobacillus intestinalis, which plays a beneficial role in intestinal barrier protection and IBD, and Muribacterium intestinale, a microbiota associated with essential anaerobes that can alleviate DSS-induced IBD. However, the amount of Turicibacter sanguinis, which is highly associated with acute colitis, was confirmed to be reduced.

[0210] Heat map analysis of taxonomic microbial composition at the phylum-species level (Figure 77) confirmed that Glycol chitosan-GL@AuNPs could restore intestinal immune homeostasis without disrupting the intestinal microbiota in healthy mice and the DSS group treated with Glycol chitosan-GL@AuNPs. Furthermore, Glycol chitosan-GL@AuNP treatment significantly increased Akkermansia muciniphila, a microbiota associated with intestinal barrier protection. Further analysis at the family level confirmed that Glycol chitosan-GL@AuNP treatment significantly increased Lactobacillus intestinalis, which plays a beneficial role in intestinal barrier protection and IBD, and Muribacterium intestinale, a microbiota associated with essential anaerobes that can alleviate DSS-induced IBD. However, the abundance of Turicibacter sanguinis, which is highly correlated with acute colitis, was confirmed to be reduced.

[0211] The relative abundance of Akkermansia muciniphila in the microbiota was confirmed through family-level analysis, and it was confirmed that Glycol chitosan-GL@AuNP treatment significantly increased Akkermansia muciniphila, a microbiota related to intestinal barrier protective function (Figure 78).

[0212] The relative abundance of Lactobacillus intestinalis in the microbiota was confirmed through family-level analysis, and it was confirmed that Glycol chitosan-GL@AuNP treatment significantly increased the number of microbial species containing Lactobacillus intestinalis, which has a beneficial role in IBD (Figure 79).

[0213] The relative abundance of Muribaculum intestinale in the microbiota was confirmed through family-level analysis, and it was confirmed that Glycol chitosan-GL@AuNP treatment significantly increased Muribaculum intestinale, a microbiota associated with essential anaerobes that can alleviate DSS-induced IBD (Figure 80).

Claims

1. Gold nanozyme, comprising glycol chitosan and gold particles.

2. The gold nanozyme according to claim 1, wherein the glycol chitosan is embedded in gold particles.

3. 2. The gold nanozyme according to claim 1, which acts as one or more enzymes selected from the group consisting of peroxidase, superoxide dismutase (SOD), and catalase.

4. 2. The gold nanozyme according to claim 1, which scavenges hydroxyl radicals.

5. The gold nanozyme according to claim 1, further comprising glycyrrhizin.

6. 6. The gold nanozyme according to claim 5, wherein the glycyrrhizin inhibits the secretion of HMGB1 (High mobility group box 1).

7. The gold nanozyme according to any one of claims 1 to 6, which is stably maintained at a pH of 0 to 7.

8. A pharmaceutical composition for preventing or treating inflammatory bowel disease, comprising any one of the gold nanozymes according to claims 1 to 7.

9. 9. The pharmaceutical composition for preventing or treating inflammatory bowel disease according to claim 8, which contains the gold nanozyme at a concentration of 100 μg / ml to 150 μg / ml.

10. 9. The pharmaceutical composition for preventing or treating inflammatory bowel disease according to claim 8, wherein the pharmaceutical composition suppresses the expression of one or more proteins selected from the group consisting of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), C-reactive protein (CRP), and calprotectin.

11. 9. The pharmaceutical composition for preventing or treating inflammatory bowel disease according to claim 8, wherein the pharmaceutical composition suppresses production of one or more selected from the group consisting of intracellular reactive oxygen species (ROS: reactive oxygen species), reactive nitrogen species (RNS: reactive nitrogen species), and nitric oxide (NO: nitric oxide).

12. The pharmaceutical composition for preventing or treating inflammatory bowel disease according to claim 8, wherein the pharmaceutical composition inhibits cell differentiation or spleen hypertrophy.

13. The pharmaceutical composition for preventing or treating inflammatory bowel disease according to claim 8, wherein the pharmaceutical composition repairs damaged colon or repairs tight junctions between cells.

14. The pharmaceutical composition for preventing or treating inflammatory bowel disease according to claim 8, wherein the pharmaceutical composition is for preventing damage to or regenerating the mucosa or mucus layer.

15. The pharmaceutical composition for preventing or treating inflammatory bowel disease according to claim 8, wherein the pharmaceutical composition increases the microbiome associated with intestinal barrier protective function.

16. 9. The pharmaceutical composition for preventing or treating inflammatory bowel disease according to claim 8, wherein the inflammatory bowel disease is one or more selected from the group consisting of Crohn's disease, ulcerative colitis, intestinal Bechet's disease, and enteritis.

17. mixing a glycol chitosan solution and a gold particle solution; oxidizing / reducing the glycol chitosan and gold particles in the mixed solution; and A method for producing a gold nanozyme, comprising: forming nanozymes from the oxidized / reduced glycol chitosan and the gold particles.

18. The method for producing a gold nanozyme according to claim 17, wherein the glycol chitosan solution in the mixing step further contains glycyrrhizin.

19. 18. The method for producing a gold nanozyme according to claim 17, wherein the ratio of the glycol chitosan solution to the gold particle solution in the mixing step is 1:2 to 1:

10.

20. The method for producing a gold nanozyme according to claim 17, wherein the oxidized / reduced glycol chitosan in the nanozyme-forming step is embedded in the gold particles to form a nanozyme.

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