KR102031706B1 - Anti-inflammatory composition comprising nano-sized graphene oxide - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition comprising nano-sized graphene oxide as an active ingredient. Specifically, a pharmaceutical composition for preventing or treating an inflammatory disease comprising the composition, a cosmetic composition or a feed composition for preventing or improving an inflammatory disease, and administering the composition to an individual in need thereof, inflammatory A method for treating a disease.

Description

Anti-inflammatory composition comprising nano-sized graphene oxide {Anti-inflammatory composition comprising nano-sized graphene oxide}

The present invention relates to an anti-inflammatory composition comprising nano-sized graphene oxide as an active ingredient. Specifically, the present invention relates to a pharmaceutical composition for preventing or treating an inflammatory disease comprising the composition, a cosmetic composition or a feed composition for preventing or improving an inflammatory disease, and a method for preventing or treating an inflammatory disease using the composition.

Inflammation is a complex pathology caused by damage to tissues, external stimuli or defense of biological tissues against various infectious agents, due to the organic interaction of various inflammatory mediators and various immune cells in blood vessels and body fluids. For example, when an external stimulus is applied to a cell, proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) or interleukin-6 (IL-6) are increased, and the cytokine is inducible nitric (iNOS). By stimulating the expression of genes such as oxide systhase) or cyclooxygenases-2 (COX-2), NO (nitric oxide) or PGE2 (prostaglandin E2) is produced and an inflammatory reaction occurs. Cytokines, such as IL-6, IL-8, TNF-α or IFN-α, which are involved in the immune response, are synthesized and secreted by macrophages, and macrophage expression is upregulated depending on the type of cytokine. , Or downregulated (Cavaillon JM, Biomed. Pharmacother. 1994; 48 (10): 445-53).

NO has various physiological activities such as body defense, signal transduction, neurotoxicity, and vasodilation, but NO produced by iNOS in inflammatory state is vascular permeability, edema, tissue damage, genetic variation and nerve damage. Cause back. Accordingly, many studies have been conducted to develop a substance that suppresses excessive generation of NO, and phenylpropa, such as caffeic acid, chlorogenic acid, cinnamon acid, p-coumal acid, hesperidin, losmaric acid, and the like derived from various natural products. It has been reported that nooid compounds can inhibit skin inflammation (Japanese Patent Laid-Open No. 2000-319154). However, research on natural-derived components is still in its infancy, and development of more diverse components is required.

Among various inflammatory diseases, inflammatory bowel disease (IBD), represented by Chron's disease and ulcerative colitis, refers to a chronic inflammatory disease for which the cause of invading the gastrointestinal tract is not known. It is largely classified into two types of Crohn's disease and ulcerative colitis, but also includes intestinal Bechet's disease, which is relatively common in Korea. It is more common in whites and Jews than in blacks and Asians, but the number of Asians is increasing. The age of onset is between 15 and 35 years old. Symptoms of ulcerative colitis include diarrhea (blood stool and mucous stool), back loom, abdominal pain, abdominal tenderness, and weight loss. , Perianal abnormalities, abdominal tenderness and so on. Intestinal Behcet's disease is similar to these symptoms, such as diarrhea and abdominal pain and accompanied by other symptoms of Behcet's disease. Surgical treatment is the principle, but if it is difficult or complications develop, surgical treatment is performed. Although drug treatment is being performed, the therapeutic agent has not been developed as the cause is not clearly identified, and the general use of a combination of general anti-inflammatory drugs, corticosteroids, immunosuppressants, antibiotics, and other drugs is appropriate. Adjust the type and amount of medicine according to the type, severity, site, and complications of inflammation. However, this combination therapy has the disadvantage that the side effect is likely to occur. Therefore, the development of therapeutic agents for the treatment of effective inflammatory bowel disease is very important.

Accordingly, the present inventors have made diligent efforts to discover graphene derivatives having activity as biomedical therapeutic agents. As a result, graphene oxide, particularly nano-sized graphene oxide having a particle size adjusted to nanoscale, is expressed and / or expressed in inflammatory factors. The present invention was confirmed by inhibiting the secretion of inflammatory cytokines and having anti-inflammatory activity inducing differentiation of macrophages into specific subtypes.

One object of the present invention is to provide an anti-inflammatory composition comprising nano-sized graphene oxide (nano-sized graphene oxide; nano-GO) as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating an inflammatory disease in an animal, comprising administering the composition to an individual in need thereof.

As one aspect for achieving the above object, the present invention provides a composition for anti-inflammatory comprising nano-sized graphene oxide (nano-sized graphene oxide; nano-GO) as an active ingredient.

The present invention relates to the anti-oxidation of graphene oxide, especially graphene oxide whose particle size is controlled to nanoscale, inhibits the expression of inflammatory factors and / or secretion of inflammatory cytokines and induces differentiation of macrophages into certain subtypes. Inflammatory and based on findings from animal studies that can effectively prevent or treat inflammatory bowel disease.

As used herein, the term “nano-sized graphene oxide (nano-GO)” refers to a material prepared in the form of particles having a nanometer size by applying a predetermined stimulus to graphene oxide. By reference, the nano-sized graphene oxide may mean a plate-shaped particle having a predetermined thickness of 12 nm or less and an average diameter of about 15 to 50 nm. For example, the nano-sized graphene oxide may be prepared by applying ultrasonic waves to the graphene oxide (eg, by tip-sonification), but is not limited thereto. For example, the nano-sized graphene oxide may have an average diameter of 15 to 45 nm, 15 to 27 nm, 25 to 35 nm, 35 to 45 nm or 25 to 45 nm. Further, the nanoparticles may be particles having a thickness of 5 to 12 nm, 3 to 9 nm, 3 to 7 nm, 5 to 9 nm, or 5 to 7 nm, but is not limited thereto.

As used herein, the term "anti-inflammatory" refers to an action of inhibiting or reducing inflammation, and the term "inflammation" refers to a protective response occurring in the body when a living tissue is damaged, causing an inflammatory disease. to be.

In the present invention, the anti-inflammatory composition can be used for the prevention, treatment or amelioration of inflammatory diseases by showing the activity of inhibiting or reducing inflammation.

Specifically, the composition comprising the nano-sized graphene oxide of the present invention is inhibiting the expression or secretion of proinflammatory cytokines, myeloperoxidase (MPO) activity inhibition, Th1 proliferation, differentiation or reaction inhibition, inhibitory T Inflammation can be inhibited or reduced by promoting cell activity or upregulating M2b macrophages.

For example, treatment of the compositions of the present invention can reduce the secretion or expression of subsequent cytokines of the representative proinflammatory cytokines IFNγ, TNF, IL-6 and / or MCP-1. Or reduce MPO activity, indicating that it can inhibit neutrophil migration and inflammation. Furthermore, it can inhibit differentiation and / or proliferation of Th1 cells, which are known to play an important role in enteritis, and also reduce gene expression specific to them. Furthermore, inflammation can be resolved by converting M1 macrophages to M2 type during the inflammatory response. In particular, upregulation of M2b macrophages in the M2 subtype can attenuate the inflammatory response.

"Expression" includes both gene expression or protein expression.

Since the inflammatory disease refers to a disease caused by inflammation, the anti-inflammatory composition of the present invention can be used for the prevention or treatment of inflammatory diseases.

The inflammatory disease is not particularly limited as long as the symptoms can be alleviated, alleviated, ameliorated or treated by the pharmaceutical composition of the present invention. Specific examples include erythema, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, and Edison's disease. , Type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism and hypertension, scleroderma, Behcet's disease, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Meniere's syndrome, Gillian Guilian-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis, psoriasis, vitiligo, or systemic scleroderma.

Specifically, the inflammatory disease treatable by the pharmaceutical composition comprising nano-sized graphene oxide according to the present invention may be an inflammatory bowel disease. The inflammatory bowel disease refers to chronic inflammation of unknown cause occurring in the intestine, and usually refers to ulcerative colitis and Chron's disease, which are idiopathic inflammatory bowel diseases, but are relatively common in Korea. Intestinal Bechet's disease may also be included. In a broader sense, it may include both infectious enteritis such as bacterial, viral, amoeba, and tuberculosis, and inflammatory diseases occurring in all intestines such as ischemic bowel disease and radiation enteritis. The composition of the present invention is limited to the prevention or treatment of inflammatory bowel diseases, such as bacterial, viral, amoeba, tuberculous enteritis, and inflammatory diseases occurring in all intestines such as ischemic bowel disease and radiation enteritis. It can be used without, and preferably used for the prevention or treatment of Crohn's disease, ulcerative colitis and enteric Behcet's disease. As described above, the cause of inflammatory bowel disease has not yet been identified, but is genetically and environmentally affected, and is known to be mediated by immunological abnormalities. Since the cause was not clearly identified, no clear treatment was developed. Therefore, for the treatment of inflammatory bowel disease using drugs, general anti-inflammatory drugs, corticosteroids, immunosuppressants, or antibiotics are used alone or in combination to maximize therapeutic effect and minimize side effects. The method of selecting the type and the capacity is selected.

The symptoms of inflammatory bowel disease vary depending on the specific disease, but in general, abdominal pain, lower abdominal discomfort, shortening of colon, hair loss, decreased activity, weight loss, elevated bleeding index (bleeding), or increased bowel index ( Diarrhea). The same disease may have different aspects depending on the extent, location or severity of the lesion.

The prevention or treatment of inflammatory bowel disease may be performed by reducing the secretion of inflammatory cytokines IL-23 or TGF-β and / or increasing the expression of genes associated therewith.

IL-23 is a cytokine of heterodimers and plays an important role in immune response. Produced in dendritic cells, macrophage and other immune cells, it is considered a major factor affecting the balance between resistance and immunity in the intestine, and has been shown to mediate inflammation in the colon. The TGF-β synergistically works with TGF-α as a component synthesized in various tissues. It is known to play an important role in embryonic development, cell differentiation, hormone secretion and immune function. In particular, it is known to cause an inflammatory response.

As used herein, the term "prevention" means any action that inhibits or delays an inflammatory disease by administration of the composition.

As used herein, the term "treatment" means any action in which the symptoms of an inflammatory disease are improved or beneficially modified by administration of the composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" of the present invention means to exhibit a property that is not toxic to cells or humans exposed to the composition. The carrier can be used without limitation so long as it is known in the art such as buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants. Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. Typically available surfactants that facilitate migration across the membrane are those derived from steroids, but N- [1- (2,3-dioleoyl) propyl-N, N, N-trimethylammonium chloride] (DOTMA Cationic lipids, or cholesterol hemisuccinate.

In another aspect, the present invention provides a method for preventing or treating an inflammatory disease, comprising administering the pharmaceutical composition to a subject in need thereof.

In the present invention, the term "individual" means all animals including humans who may or may develop an inflammatory disease, and can effectively prevent or treat the inflammatory disease by administering the pharmaceutical composition of the present invention to an individual. The pharmaceutical composition of the present invention may be administered in parallel with existing inflammatory disease therapies.

The term "administration" of the present invention means introducing a predetermined substance into a patient in an appropriate manner, and the route of administration of the composition may be administered via any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations, in addition to the composition, include at least one excipient such as starch, calcium carbonate, sucrose, or lactose, Prepare by mixing gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups.In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, flavoring agents, preservatives, etc. Can be. However, upon oral administration, since the peptides are easy to digest, it is desirable to formulate oral compositions to coat the active agent or protect it from degradation in the stomach. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. The suppository base includes Uittepsol, Macrogol, and Twin 61. Cacao butter, laurin butter, glycerogelatin and the like can be used. Carbohydrates such as glucose, sucrose, dextran, antioxidants such as ascorbic acid, glutathione, chelating substances, small molecule proteins or other stabilizers can be used to increase the stability or absorption of the peptide.

In addition, the pharmaceutical compositions of the present invention may be administered by any device in which the active substance may migrate to target cells. Preferred modes of administration and preparations are intravenous, subcutaneous, intradermal, intramuscular, injectable and the like. Injections include non-aqueous solvents such as aqueous solvents such as physiological saline solution and ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Stabilizers (e.g. ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, to prevent microbial growth Preservatives (eg, mercury nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included.

On the other hand, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term “pharmaceutically effective amount” means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to the medical treatment and not causing side effects, wherein the effective dose level is the sex, age and weight of the patient. Well-known factors such as health status, type of disease, severity, drug activity, drug sensitivity, method of administration, time of administration, route of administration, and rate of release, duration of treatment, combination or drug used, and other medical fields. It can be easily determined by those skilled in the art according to known factors. Generally, the active substance can be administered at a dose of about 0.01 mg / kg / day to 1000 mg / kg / day. In the case of oral administration, a range of 50 to 500 mg / kg may be suitable and may be administered at least once a day.

The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

In addition, in the present invention, the anti-inflammatory composition may be a cosmetic composition for the prevention or improvement of inflammatory diseases.

Cosmetic composition of the present invention may comprise 0.0001 to 50% by weight of nano-sized graphene oxide of the present invention relative to the weight of the total composition, specifically may include 0.01 to 10% by weight, but is not limited thereto. Do not. There is an advantage in showing the excellent effect of the present invention within the above range, there is an advantage that the formulation of the composition is stabilized.

The cosmetic composition of the present invention is a solution, external ointment, cream, foam, nourishing lotion, flexible lotion, pack, flexible water, latex, makeup base, essence, soap, liquid cleanser, bath, sunscreen cream, sun oil, suspension, Emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays It is not.

The cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers formulated in general skin cosmetics, and as common components, for example, oil, water, surfactants, moisturizers, lower alcohols, thickeners , Chelating agents, pigments, preservatives, fragrances and the like may be appropriately blended, but is not limited thereto.

The cosmetically acceptable carrier included in the cosmetic composition of the present invention may vary depending on the formulation of the cosmetic composition.

When the formulation of the present invention is an ointment, paste, cream or gel, the carrier component is animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide and the like. May be used, but is not limited thereto. These may be used alone or in combination of two or more thereof.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder and the like can be used, and especially in the case of a spray, additionally chlorofluorohards. Propellants such as carboxylic, propane / butane or dimethyl ether may be included, but are not limited thereto. These may be used alone or in combination of two or more thereof.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsion may be used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Propylene glycol, 1,3-butylglycol oil, and the like can be used, and in particular, cottonseed oil, peanut oil, corn seed oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid ester May be used, but is not limited thereto. These may be used alone or in combination of two or more thereof.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, micro Crystalline cellulose, aluminum metahydroxyde, bentonite, agar or tracant may be used, but is not limited thereto. These may be used alone or in combination of two or more thereof.

When the formulation of the present invention is a soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolyzates, isethionates, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugars and the like can be used as carrier components. May be, but is not limited thereto. These may be used alone or in combination of two or more thereof.

When the formulation of the present invention is a surfactant-containing cleansing, it is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, methyltaurate, sarcositate, fatty acid as a carrier component. Amide ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like may be used, but are not limited thereto. These may be used alone or in combination of two or more thereof.

Furthermore, in the present invention, the anti-inflammatory composition may be a food additive or health functional food for preventing or improving inflammatory diseases. When the composition of the present invention is used as a food additive, the nano-sized graphene oxide may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment).

The term "health functional food" of the present invention refers to a food prepared and processed by a method of extracting, concentrating, refining, and mixing a specific ingredient as a raw material or contained in a food ingredient for the purpose of health supplement. By means of biological defense, control of the biological rhythm, prevention and recovery of diseases, such as a food designed and processed to fully exhibit the biological control function, the health food composition is for the prevention of diseases and recovery of diseases, etc. You can perform related functions.

In addition, there is no restriction on the kind of health foods in which the composition of the present invention can be used. In addition, the composition comprising the peptide of the present invention or a food-acceptable salt thereof as an active ingredient may be prepared by mixing known additives with other suitable auxiliary ingredients that may be contained in the health functional food according to the choice of those skilled in the art. Examples of foods that can be added include meat, sausages, breads, chocolates, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products, including ice cream, various soups, beverages, teas, drinks, alcoholic beverages and There is a vitamin complex, etc., the compound according to the present invention can be prepared by adding juice, tea, jelly and juice prepared with the extract as a main component.

Furthermore, in the present invention, the anti-inflammatory composition may be a feed composition for the prevention or improvement of inflammatory diseases.

The term "feed" of the present invention is any natural or artificial diet, one meal, or the like or any one of the above ingredients for the animal to eat, ingest and digest, and may refer to the nano-sized graphene oxide of the present invention. Feed comprising the active ingredient can be prepared in various forms of feed known in the art, specifically, it may include a rich feed, research feed and / or special feed.

The nano-sized graphene oxide of the present invention included in the feed composition of the present invention may vary depending on the purpose and conditions of use of the feed. For example, 0.01 to 100% by weight, more specifically, based on the total weight of the livestock feed composition. May be included in an amount of 1 to 80 wt%, but is not limited thereto.

The composition according to the invention comprises graphene oxide having a nanoscale controlled size to inhibit the secretion and / or expression of proinflammatory cytokines and to regulate the differentiation of cells involved in the inflammatory response, in particular inflammatory bowel It can be usefully used for the treatment of diseases.

1 is a diagram showing a method and characteristics of the synthesis of nano-GOs. (A) schematically shows a method for synthesizing nano-GOs according to the present invention. (B) shows a representative TEM image of the nano-GOs according to the present invention and the particle size distribution calculated therefrom. (C) shows a representative AFM image and line profile analysis of the nano-GOs according to the present invention. (D) shows a representative Raman spectrum of nano-GOs according to the present invention.
Figure 2 is a diagram showing the protective effect in the DSS-induced colitis mice by intraperitoneal injection of graphene derivatives, nano-GOs. (A to D) show experimental results for mice induced with colitis by administering 3% DSS-containing drinking water to mice for 7 days. Nano-GOs were administered intraperitoneally on day 1 after DSS administration (300 μg / head). For clinical evaluation, (A) survival rate, (B) weight loss rate, and (C) disease activity index (DAI) for colitis severity were monitored. (D) shows the shape and length of colons extracted from mice killed 10 days after colitis induction with DSS to determine bowel damage. n = 17-20 mice / group. * P <0.05, ** P <0.01, ** P <0.001. The results are expressed as mean ± SEM.
3 shows the accumulation of nano-GOs in the abdominal cavity. Mice were killed 14 days after colitis induction with DSS and nano-GOs administration. Nano-GOs were observed near the spleen and colon.
Figure 4 shows the effect of reducing the colon and systemic inflammation by nano-GOs in mice after DSS induction. After induction of colorectal cancer with DSS, mice intraperitoneally administered with nano-GOs were killed for day 10 and used for the experiment. The left side of (A) shows a representative H & E stained image of the colon cross section, and the right side shows the histopathological evaluation performed by lymphocyte infiltration and intestinal damage. Scale bar = 1 mm (top) and 500 μm (bottom). The left side of (B) shows the Masson tricolor staining result of the colon for evaluating fibrosis, and the right side shows the quantitative analysis result of the fibrotic area. Scale bar = 1 mm (top) and 500 μm (bottom). Serum was collected from colitis mice and the level of cytokine secretion indicated using a cytometric bead array (CBA) assay was measured and shown in (C). (D) represents myeloperoxidase (MPO) measured in colon tissue.
Figure 5 is a diagram showing the results of CBA analysis of colon tissue after colitis induction. CBA analysis was performed to determine the levels of cytokine secreted by colon tissue.
Figure 6 is a diagram showing the results of the experiment to determine the optimal in vitro concentration of nano-GOs. The left side of (A) shows mononuclear cells (MNCs) activated by concanavalin A in the presence of labeled nano-GOs separated from human umbilical cord blood (hUCB), The right side shows CD4 + T cells purified from hUCB and cultured in the presence of indicated concentrations of nano-GOs. (B and C) are obtained by adding 20 μg / ml of GOs to Th1 cells for 2 days, (B) proliferation by BrdU assay, (C) representative images of Th1 cells from each group Indicates.
7 is a diagram showing the inhibitory effect on the activity of Th1 differentiated cells of nano-GOs. CD4 ⁺ T cells were isolated from hUCB and induced differentiation into Th1 cells using anti-CD3 and CD28 beads in combination with recombinant IL-12 and IFN-γ in the presence of nano-GOs. (A) shows localization in Th1 differentiated cells by treatment with biotin labeled nano-GOs. Anti-GFP-biotin was used to detect biotin labeled nano-GOs. The right hand side shows a Z-stack image of Nano-GOs treated Th1 cells. Scale bar = 10 μm. (B) shows MNCs stimulated with Concanavalin A with nano-GOs separated from hUCB. MNCs were incubated for 2 days and measured by BrdU assay. (C) is the result of evaluating the proliferation of Th1 cells cultured for 2 days in the presence of nano-GOs by BrdU assay. (D) is the result of analyzing the apoptosis of Th1 cells with the Annexin V apoptosis detection kit. (E) is a cell cycle analysis result performed on Th1 cells using PI (Propidium iodide) staining. (F) is a result of analyzing the percentage of IFN-γ expressing CD4 ⁺ T cells by flow cytometry. (G) is the result of confirming mRNA expression of each group Th1 cell with respect to the displayed Th1-specific marker. (H) is the result of analyzing the displayed Th1-specific cytokines in Th1 cell supernatant by CBA analysis.
FIG. 8 shows indirect increase by nano-GOs in the ratio of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ regulatory T cells in vivo. Mice administered with nano-GOs were killed on Day 15 and colons and spleens were collected for in vitro experiments. Results were obtained from 5 to 6 mice in each group. (A) Colon invasion and (B) Spleen infiltration of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ regulatory T cells were measured by flow cytometry. (C) shows regulatory T cells observed in colon by immunostaining for FoxP3 (green), scale bar = 50 μm. FoxP3 ⁺ cells are marked with ▼. IL-10 expression of colon lysates was confirmed by (D) Western blot and (E) CBA analysis. (F) shows the results of ELISA analysis of TGF-β1 secretion levels in the colon. (G) shows the results of deflection of Treg cells by flow cytometry and analysis of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ cells. * P <0.05, ** P <0.01, *** P <0.001. The results are expressed as mean ± SEM.
9 shows the role of nano-GOs in the conversion of M1 macrophages to alternatively activated M2 type cells. CD14 ⁺ cells were isolated from hUCB and cultured in the presence of nano-GOs. (A) shows the results of localization by treating CD14 ⁺ macrophage-like cells with biotin-labeled nano-GOs. On the right is a Z-stack image of CD14 ⁺ cells treated with nano-GOs. Scale bar = 10 μm. Isolated CD14 ⁺ cells were biased into M0, M1 and M2 type cells with type-specific inducer cytokines in the presence of nano-GOs. After 7 days of culture, (B) confirmed the proliferation of cells by CCK-8 assay. Type-specific cell surface CD markers were analyzed by (C) flow cytometry and (D) immunocytochemistry. * P <0.05, ** P <0.01, *** P <0.001. The results are expressed as mean ± SEM.
10 is a diagram showing the change in cell morphology and viability as well as the differentiation capacity of macrophages by nano-GOs. The top of (A) shows a phase-contrast image of Raw264.7 cells incubated with nano-GOs. Scale bar = 100 μm. Bottom shows cell viability as determined by MTT assay. Isolated CD14 ⁺ cells were biased into M0, M1 and M2 type cells with type-specific induced cytokines in the presence of nano-GOs. (B) shows a phase difference image (scale bar = 200 µm), and (C) shows a dot plot image by flow cytometry. *** P <0.001. The results are expressed as mean ± SEM.
FIG. 11 shows that M2b macrophages are the major subtype of nano-GOs mediated M2 biased cells. Isolated CD14 ⁺ cells were incubated with GM-CSF for 2 days and incubated with IFN-γ and LPS for 5 days to differentiate into M1 type macrophages. After culturing for 7 days, the cells and the cultured medium were recovered and further analyzed. (A) shows the expression of CD163 confirmed by flow cytometry. (B) shows TNF, IFN-γ and IL-6 secretion levels measured by CBA assay. (C) is the result of analysis of the M1 and M2 type-specific gene expression by qRT-PCR. For M2 subtype biasing, CD14 ⁺ cells were incubated with M-CSF for 2 days and specific cytokine combinations (M2a, IL-4 and IL-13; M2b, Poly I: C, IL-1β and LPS) (M2c, IL-10 and TGF-β1) for 5 days. (D) shows the result of detecting the expression of M2 subtype specific CD marker by flow cytometry. (E) shows TNF and IL-6 secretion levels measured by CBA assay. (F) is a result of confirming gene expression of IL-1β and IL-10 by qRT-PCR. * P <0.05, ** P <0.005, *** P <0.001. The results are expressed as mean ± SEM.
Figure 12 shows the response of M2 subtype macrophages to nano-GOs treatment. CD14 ⁺ cells were incubated for 2 days with M-CSF and for the next 5 days with specific cytokine combinations. (A) shows a dot plot image according to flow cytometry. (B) is a result of confirming M2a subtype specific gene expression by qRT-PCR. * P <0.05, ** P <0.005, *** P <0.001. Results are expressed as mean ± SEM.

Hereinafter, the configuration and effects of the present invention through the embodiments will be described in more detail. These examples are only for illustrating the present invention, but the scope of the present invention is not limited by these examples.

Production Example  One: Nanoscale  Oxidation Of graphene (GO)  Produce

Pristine graphene oxides (GOs) were synthesized by an improved Hummer's method. To prepare nano-sized GOs, a distilled water solution of GOs (3 mg / ml) was sonicated violently for 3 hours and tip-sonicated cellulose nitrate membrane filter (0.45 μm, GE Healthcare). Vacuum-filtered.

Production Example  2: Nanoscale  Oxidation Graphene  Biotin anger

Nano-GOs were biotinylated by EDC coupling. First, 10 mg of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride, EDC reagent, Sigma) was added to 10 ml of nano-GOs. The solution was added to the solution (3 mg / ml) to replace the terminal carboxyl groups with EDC reagent. After 30 minutes, 20 mg of amine-PEG ₃ -biotin (Thermo Scientific) was added to the solution and allowed to react for 24 hours to form an amide bond between the activated end of the nano-GO and the reactive amide group of biotin. Similarly as in Preparation Example 1, the final product was obtained in the form of a powder after appropriate dialysis and filtration.

Experimental Example  One: TEM Imaging

A solution (10 μg / ml) in which a sample prepared according to the above preparation was dispersed on copper grids coated with 300 mesh lacey carbon (Ted Pella, Inc.) was adsorbed for 30 minutes. Prior to imaging the grating was washed with a few drops of distilled water and dried completely in a desiccator. The sample thus prepared was analyzed with a high resolution-transmission electron microscope (HR-TEM, JEM-3010, JEOL Ltd.), and images were collected with a Gatan digital camera (MSC-794) coupled to the microscope. .

Experimental Example  2: Raman spectroscopy measurement

The powdered product prepared according to the above preparation for Raman spectrum measurement was prepared on a SiO ₂ substrate. Spectra were measured with a Renishaw micro-Raman spectrometer equipped with a 514.5 nm Ar excitation laser.

Experimental Example  3: FT-IR spectroscopy

Prior to Fourier-transform infrared (FT-IR) spectra measurement, the powdered sample was completely dried in a desiccator to exclude unwanted oxygen containing peaks. Spectra were measured by conventional KBR pellet method (Nicolet 6700, Thermo Scientific).

Experimental Example  4: experimental animal

All animal experiments were performed in accordance with the approved guidelines of the IACUC No. SNU-170523-4. Six-week-old male C57BL / 6 mice (Orientbio, Sungnam, Republic of Korea) were randomly grouped and given 3% DSS in drinking water for 7 days (16 per group). On Day 1, ie, one day after DSS induction, mice were administered intraperitoneally with 15 g / kg dose of Nanno-GOs prepared according to the above preparation. Mice were weighed daily, and the disease activity index (DAI), which consists of weight loss, activity, stool consistency, bleeding, and hair condition, was measured on Day 7 and Day 10 (Day 10). Evaluated. After the mice were killed, spleen, large intestine and blood samples were collected for further ex vivo examinations.

Experimental Example  5: histopathological evaluation

Collected colon samples were subjected to 10% formalin according to conventional methods, including dehydration with ethanol, clearing with xylene, and wax infiltration with paraffin. Fixed. Paraffin embedded blocks were cut to 5 μm thickness and stained with H & E or Masson's trichrome. H & E staining for loss of goblet cells, hyperemia / edema, infiltration of immune cells, presence of crypt abscesses and loss of epithelium by H & E staining Recorded by histopathological index. Fibrotic tissue areas were measured by Marson tricolor staining and quantified using ImageJ software (version 1.46r, US National Institute of Health, Bethesda, MD, USA).

Experimental Example  6: cytokine generation

To determine the secretion levels of various cytokines, culture supernatants of serum, colon lysate and cells isolated from blood were prepared. To measure the extent of inflammation in vivo, Cytometric Bead Array (CBA) kits for mouse inflammation (BD Bioscience, San Jose, CA, USA) and ELISA kits for MPO and TGF-β1 (R & D, respectively) Systems, Minneapolis, MN, USA and Thermo Fisher Scientific, San Jose, Calif., USA) were used according to the manufacturer's protocol. To assess the induced cytokine secretion of in vitro immune cells, the CBA kit for Th1 / Th2 / Th17 (BD Bioscience, San Jose, CA, USA) and the ELISA kit for TGF-β1 (Thermo Fisher Scientific) , San Jose, CA, USA). The results were detected using flow cytometry and spectrophotometer.

Experimental Example  7: hMNCs  Isolation and Cultivation

All experimental procedures involving human umbilical cord blood (hUCB) or UCB-derived cells are approved by Boramae Hospital Institutional Review Board (IRB) and Seoul National University IRB. It was performed according to the instructions given. Human umbilical cord blood-mononuclear cells (hUCB-MNCs) were isolated and cultured by known methods. Specifically, UCB samples were collected immediately after childbirth with informed consent and parental approval. The collected UCB samples were mixed with HetaSep solution (Stem Cell Technologies, Vancouver, Canada) at a ratio of 5: 1 and incubated for 1 hour at room temperature. Then, the supernatant was collected with Ficoll and centrifuged at 2,500 rpm for 20 minutes to separate monocyte cells. The isolated cells were washed twice with PBS. Cells isolated as described above were further subjected to experiments performed in vitro in vitro.

Experimental Example  8: Isolation of T cells and Deflection (polarization)

Naive CD4 ⁺ T cells were isolated from freshly isolated hUCB-MNCs with human pure CD4 ⁺ T cell isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Isolated CD4 ⁺ T cells were RPMI1640 containing 10% Fetal Bovine Serum (FBS), an anti-CD3 / 28 bead activator, and 20 ng / ml IL-2, essential for T cell subsets proliferation. (Gibco BRL, Grand Island, NY, USA). To differentiate cells into T cell subtypes, type-specific cytokines (20 ng / ml of IFN-γ and 20 ng / ml of IL-12, and Treg for type 1 helper T cells) 20 ng / ml of TGF-β1) was added to the growth medium for cells and incubated at 37 ° C. in a wet 5% CO ₂ atmosphere for 5 days in the presence / absence of nano-GOs. Polarized Th1 and Treg cells were identified by type-specific staining and flow cytometry. Th1 cells were stained intracellularly with IFNγ after surface staining with CD4 antibody. Tregs were used for CD4, CD25 and IL-4 antibodies.

Experimental Example  9: Isolation of Macrophages and Deflection

Macrophages were isolated and cultured by known methods. Specifically, macrophages were isolated from hUCB-MNCs freshly isolated with human CD4 ⁺ T cell separation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Isolated CD14 + cells were cultured in RPMI1640 containing 10% FBS. To bias cells into macrophage subtypes, type-specific cytokines (20 ng / ml IFN-γ and 1 μg / ml LPS for M1 cells, and 20 ng / ml IL- for M2 cells) 4 and 20 ng / ml IL-13) were added to the growth medium and incubated for 5 days at 37 ° C. in a wet 5% CO ₂ atmosphere in the presence / absence of nano-GOs. To identify biased macrophages, type-specific staining and flow cytometry were used. CD14 antibodies were used as pan macrophage markers and CD86 and CD206 were applied as specific markers for the M1 and M2 subtypes.

Experimental Example  10: Cell Proliferation Assay

To measure cell proliferation, cell proliferation ELISA kits (Roche, Indianapolis, IN, USA) and CCK-8 kits (Dojindo, Kumamoto, Japan) were used according to the manufacturer's instructions. For bromodeoxyuridine (BrdU) cell proliferation assays, known methods were used. Specifically, cells were incubated for 2 hours at 37 ° C. in a wet 5% CO ₂ atmosphere with 100 μM BrdU labeling reagent. After 30 minutes of fixation with the provided FixDenat solution, the cells were incubated for 90 minutes in anti-BrdU antibody solution and for 5-30 minutes in the provided substrate (tetramethyl-benzidine (TMB) solution at room temperature. After pouring a stop solution into each well, absorbance was measured at 450 nm and 690 nm wavelengths to quantify cell proliferation levels.

Experimental Example  11: quantitative RT- PCR

Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. The obtained RNA was used for cDNA synthesis by the Superscript First-Strand Synthesis System (Invitrogen). Relative expression levels of target mRNA were measured with an ABI 7300 detection system using a SYBR Green PCR master mix (Applied Biosystems, Foster City, USA). At least three independent analyzes were performed for each gene.

Experimental Example  12: Cell Cycle Analysis

Cell cycle assays were performed according to known protocols. Specifically, cells were fixed with ice-cold 70% ethanol at −20 ° C. for at least 30 minutes. Fixed cells were washed with PBS and incubated with 400 μl of RNase A containing PBS (7.5 μg / ml) and propidium iodide (PI, 50 μg / ml) at 37 ° C. for 30 minutes. Cell cycles were analyzed by flow cytometry performed on FACScalibur using Cell Quest software (BD Bioscience, San Jose, Calif., USA).

Experimental Example  13: Apoptosis Assay

Apoptosis assay was performed by a known method. Specifically, cells were stained with 5 μl of FITC annexin V and 5 μl of PI in Apoptosis Detection Kits (BD Bioscience, San Jose, Calif., USA). The mixture was gently stirred with vortex and incubated for 15 minutes in the dark at room temperature. 400 μl of 1 × binding buffer was then added to the mixture, and all samples were analyzed by flow cytometry performed on a FACScalibur using Cell Quest software.

Experimental Example  14: Immunofluorescence  analysis

Cells were fixed in 4% paraformaldehyde (PFA) PBS solution for 15 minutes at room temperature and treated with 0.25% Triton X-100 (Sigma) for 10 minutes to increase permeablilized. Fixed cells were incubated with blocking solution (5% normal goat serum) for 1 hour at room temperature and incubated with primary antibody overnight at 4 ° C. Cells were then incubated with a secondary antibody labeled with Alexa Fluor 594 (Invitrogen), followed by DAPI (Sigma) staining for 5 minutes to stain nuclei.

For whole tissue immunofluorescence, paraffins on paraffin slides were deparaffinized and blocked with PBS containing 5% normal goat serum. Sections were incubated overnight with primary antibody, then incubated with Alexa Fluor 594 and DAPI stained. Images were collected with a confocal microscope (Eclipse TE200, Nikon, Japan).

Experimental Example  15: Western Blot  analysis

Colon samples were dissolved in Pro-Prep (Intron Biotechnology Co., Sungnam, Republic of Korea) to extract proteins from tissues. The obtained protein sample was separated by 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred to nitrocellulose membrane. After blocking with 3% BSA (Bovine Serum Albumin) solution, the proteins on the membrane were incubated with IL-10 primary antibody at 4 ° C. for at least 12 hours and incubated with secondary antibody. Protein and antibody complexes were detected via ECL Western blotting detection reagents and assay systems.

Experimental Example  16: statistical analysis

The mean value of all results was expressed as mean ± standard error of mean (SEM). Statistical analysis was performed after Student's 2-tailed t-test or Bonferroni for multigroup comparisons using GraphPad Prism version 5.0 (GraphPad Software, San Diego, Calif., USA). This was done using a one-way ANOVA followed by a Bonferroni post-hoc test. Statistical significance is shown in the description of the figures.

Example  1. Nano-oxidation Graphene  Characterization

Synthesis method of nano-GOs used in the present invention is schematically shown in Figure 1A. To identify the size and shape of the nano-GOs, particles were analyzed using TEM images and selected area electron diffraction (SAED) pattern analysis (FIG. 1B). And 1C). The properties of the synthesized nano-GOs were evaluated by atomic force microscopy (AFM) (Fig. 1C). In addition, Raman spectra of nano-GOs were measured (FIG. 1D). In order to place nano-GOs in immune cells, biotinylated nano-GOs were prepared. After modification of the nano-GOs to biotin, the FT-IR spectrum showed the vibration peak of the amine.

Example  2. Nano- in Mouse Models Of GOs  DSS-induced colitis improvement

To confirm the therapeutic effect of nano-GOs in DSS-induced colitis mice, the nano-GOs are injected intraperitoneally on the first day after colitis induction and body weight, survival and activity are monitored for two weeks. It was. As a result, nano-GOs showed an effect of protecting mice from severe colitis, determined by increased survival and reduced weight loss (FIGS. 2A, B). However, the protective effect on survival and body weight change was more pronounced in nano-GO-treated mice. Furthermore, on days 7 and 10, the disease activity index was measured to confirm the progress of colitis. Similar to the results from survival and body weight, nano-GO-treated mice showed significantly decreased disease activity index (FIG. 2C). On day 14 after colitis induction, crystals were collected from each group and their lengths were measured. Nano-GOs markedly blocked the reduction in colon length induced by colitis (FIG. 2D). Graphene derivatives were found in the omentum near and adjacent to visceral organs (FIG. 3). The mechanism of region specific accumulation of nano-GOs is not yet known. Overall, these results indicate that nano-GOs reduce inflammation of the colon in mice.

Example  3. DSS-Colitis-Induced Inflammation by Nano-GO Damage to the reaction

Histological analysis of the colon showed that DSS induced induced destruction of the epithelium, submucosal edema, crypt abscesses, and lymphocytic infiltration (FIG. 4A). In nano-GO-treated mice, inflammatory lesions were hardly observed compared to PBS-treated mice. Furthermore, Masson tricolor staining was performed to evaluate fibrosis in the mucosa and submucosa of the colon induced by colitis. PBS-treated mice showed collagen deposition confirmed by blue staining. Nano-GOs also significantly reduced collagen accumulation. To assess systemic inflammation, CBA assays were used to measure the levels of proinflammatory cytokines secreted from serum. Induction of IFNγ, a major cytokine accounting for Crohn's disease, was markedly inhibited in nano-GO-treated mice (FIG. 4C). Treatment of nano-GOs also reduced downstream cytokines of IFN-γ, TNF, IL-6 and MCP-1. Myeloperoxidase (MPO), a neutrophil granule constituent, is known to be proportional to the number of migrated neutrophils. Nano-GOs-treated mice showed reduced MPO activity in colon tissue compared to PBS-treated mice, indicating that nano-GOs inhibit neutrophil migration and inflammation (FIG. 4D). Thus, these results indicate that nano-GOs have immuno-suppressive properties in Crohn's disease model.

Example  4. Graphene  CD4 derivatives ⁺  T cell activity inhibitory effect

Considering that Th1 cells play an important role in enteritis, the direct effects of nano-GOs on CD4 ⁺ T cells were confirmed. First, to determine the appropriate concentration, MNCs and primary CD4 ⁺ T cells were treated with various concentrations of nano-GOs (FIG. 6A). 20 μg / ml of nano-GOs effectively inhibited proliferation of MNCs and T cells without toxicity (FIGS. 6B and 6C). In order to locate graphene derivatives in CD4 ⁺ T cells, biotin-labeled nano-GOs were treated with GFP-biotin antibody, followed by immunostaining of CD4 ⁺ Th1 cells (FIG. 7A). Biotin-labeled nano-GOs were detected in T cells. This indicates that the substance can be internalized into cells. To confirm the effect of graphene derivatives on immune cell proliferation, hUCB-derived MNCs and CD4 ⁺ T cells were treated with nano-GOs and then cultured (FIGS. 7B and C). Nano-GOs effectively inhibited the proliferation of MNCs and CD4 ⁺ T cells. Th1 inhibitory properties of nano-GOs may be the main mechanism of therapeutic effect in Crohn's disease model. Annexin V analysis showed that the nano-GOs did not affect the apoptosis pathway (FIG. 7D). However, nano-GOs slightly increased the G1 phase in the cell cycle, indicating that it inhibits cell cycle progression (FIG. 7E). To confirm the specific effects of the nano-GOs on Th1 cells, CD4 ⁺ T cells were purified from hUCB and differentiated to Th1 type in the presence of nano-GOs (FIG. 7F). As a result, nano-GOs significantly inhibited Th1 differentiation. Th1 specific gene expression was analyzed in CD4 ⁺ T cells cultured in Th1 biased conditions (FIG. 7G). Nano-GOs-treated CD4 ⁺ T cells showed markedly suppressed Th1 gene expression. To analyze functional changes in nano-GOs-treated Th1 cells, the concentration of cytokines secreted in the cell culture supernatants was measured (FIG. 7H). Secretion of IFNγ, TNF and IL-2 was significantly reduced in nano-GOs-treated cells. On the other hand, the levels of IL-6, IL-4 and IL-10 were increased in nano-GOs-treated Th1 cells. These results indicate that nano-GOs alleviate colitis by inhibiting Th1 response and promoting regulatory T cell activity.

Example  5. Nano- On the GOs  In vivo regulation of T cell infiltration

The inventors have previously confirmed that the recovery of experimental colitis is accompanied by expansion and infiltration of regulatory T cells. Thus, the present inventors confirmed by flow cytometry whether the administration of nano-GOs affect the localization of Treg cells. Administration of nano-GOs increased colonic infiltration and splenic polarization of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Tregs cells. Increased colon infiltration of Treg cells in nano-GOs treated mice was also confirmed by immunohistochemistry (FIG. 8). Next, the expression of IL-10 and TGF-β1 was determined, which are the major derivatives and products of Treg population and play an important role in Treg-mediated colitis mitigation. IL-10 expression levels were markedly increased in the colon of nano-GOs treated mice. To confirm that nano-GOs directly affect the bias of Treg cells, pure CD4 ⁺ cells were induced into regulatory T cell lineage in the presence of nano-GOs. As a result, nano-GOs did not have an increasing effect on Treg deflection, but rather interfered.

Overall, these results indicate that nano-GOs increase the number of regulatory T cells and their associated cytokines in vivo, which are not mediated by a direct effect on Treg biasing.

Example  6. Nano- On the GOs  Conversion of M1 macrophages to M2 type during immune response

Inflammation of the colon may occur due to an imbalance between the tolerogenic and protective immune responses of intestinal macrophages, which alternatively serve to recruit Treg cells and their secretory cytokines. It can be improved by alternatively activated M2 type macrophages. Therefore, it was confirmed whether treatment with nano-GOs influences the cell fate determination of macrophages. Internalization of the nanoparticles was confirmed by biotinylated graphene derivatives and immunohistochemistry (FIG. 9A). Upon treatment with nano-GOs, the proliferation of Raw264.7 cells, a mouse macrophage cell line, was increased, and enlarged cell morphology was observed (FIG. 10A). Similarly to Raw264.7 cells, primary isolated CD14 ⁺ cells also showed increased proliferation when incubated with M0 and M2 type cells with nano-GOs. In contrast, nano-GOs did not show any effective changes in M1 type cells (FIG. 9B and 10B).

The effect of nano-GOs on macrophage type-specific bias was confirmed by cell surface marker analysis using flow cytometry. All of the graphene derivatives showed a tendency to induce pure CD14 ⁺ cells into a more classical type of macrophages without other additional cytokines. In relation to M2-type macrophages, nano-GOs treatment induced an increase in both CD14 ⁺ CD86 ⁺ cells and CD14 ⁺ CD206 ⁺ cells. Expression of CD206 surface markers on CD14 ⁺ cells was confirmed by immunohistochemistry (FIG. 9D). These results indicate that nano-GOs play a role in inflammatory resolution by down-regulating M1-like properties of proinflammatory activated macrophages and by switching M2 types.

Example  7. Nano- On the GOs  Induced by As a rule  The major subtype of activated macrophages M2b

To confirm that nano-GOs treatment converts classically activated M1 type macrophages into anti-inflammatory M2 subtypes, expression of other M2 type specific properties was further confirmed. The expression of the hemoglobin scavenger receptor CD163 was increased in the presence of nano-GOs (FIGS. 11A and 11C). The levels of secretion of representative proinflammatory cytokines, TNF and IFN-γ, were markedly reduced, but no significant change was seen in IL-6 levels (FIG. 11B). Furthermore, nano-GOs treatment inhibited the mRNA expression of IL-12, IL-23, TNFα and MRF type specific transcription factors IRF5. In contrast, the level of IL-10, the most important marker of M2 macrophages, increased. However, nano-GOs treatment did not enhance, but rather inhibited, the expression of CLEC7A and IL-1ra (FIG. 11C).

M2 macrophages can be divided into three or more subtypes with distinct roles in the immune system. The secretion of IL-10 was increased while the M2a markers CLEC7A and IL-1ra were decreased, and it was assumed that the nano-GOs upregulated other M2 subtype cells. To demonstrate this, M2a, M2b and M2c macrophages were induced and the role of graphene derivatives was confirmed. As shown in previous results, CD163 and CD206 expression of M2a macrophages was significantly reduced in the presence of nano-GOs (FIG. 11D And Figure 12A). In addition, expression of the marker genes CLEC7A and IL-1ra, the transcription factor IRF4, was reduced (FIG. 12B). With respect to the M2c subtype, no noticeable change was seen. The proportion of M2b subtype macrophages was increased by nano-GOs treatment (FIGS. 11D and 12A). In addition, nano-GOs treated M2b macrophages secreted enormous amounts of TNF and IL-6 and expressed more IL-1β and IL-10 (FIGS. 11E and 11F). These results indicate that nano-GOs can attenuate inflammatory responses by upregulating M2b macrophages but not M2a or M2c.

Conclusion

Recently, carbon allotropes, including fullerenes, nanotubes and graphene derivatives, have been reported to have immunomodulatory capacity and potential for biomedical use in living organisms. In the present invention, in relation to this therapeutic potential, the protective effect of graphene derivatives, nano-GOs against experimental colitis and its mechanism of action were analyzed.

Previous studies on therapeutic uses of graphene derivatives have screened nanomaterials and established suitable conditions such as size, concentration, functional groups adsorbed on the surface and electromagnetic properties to improve biocompatibility and avoid possible risks. Has been concentrating. In one example, large GO has been reported to classically activate M1 macrophages by promoting the secretion of inflammatory cytokines and adhering to cell surfaces to activate toll-like receptor related pathways. Macrophages, on the other hand, ingested smaller GO more actively than larger GO by phagocytosis. However, all studies examining the size-dependent role of graphene oxide compare only micro- and nano-sized particles. Therefore, the present inventors attempted to evaluate the therapeutic potential and mechanism of action of nano-sized graphene derivative particles, which are easy to apply in cells and do not cause an inflammatory response. In the present invention, nano-GOs with larger side sizes more effectively ameliorate the typical symptoms of colitis. These results indicate that the properties of nano-GOs with lateral sizes of 15-50 nm are more suitable for clinical applications for colitis.

The inventors conducted a series of in vitro experiments to identify the comprehensive effects of nano-GOs on neural and adaptive immune cells. Nano-GOs were ingested in large quantities by primarily isolated Th1 cells and CD14 ⁺ macrophage like cells. Proinflammatory cytokines, particularly IL-2, IL-12, IFN-γ and the transcription factor T-bet, are known to play an important role in the commitment of type 1 helper T cells. On the other hand, representative cytokines IL-4 and IL-10 for Th2 and Treg, as well as proinflammatory cytokines IL-6, mediated Th1 development inhibition. Nano-GOs were superior in regulating the immunogenic milieu, which was confirmed by cytokine production.

Regulatory T cells protect tissues from excessive inflammation and help the tissue healing process by inhibiting activated immune cells. IL-10 and TGF-β1 were involved in the development, expansion and specific roles of Tregs. In the present invention, colonic and splenic infiltration of regulatory T cells through the increased production of IL-10 and TGF-β1, although administration of nano-GOs does not directly affect cell fate ) To improve).

Macrophages exhibit distinct plasticity and regulate their properties according to environmental stimuli. Intestinal macrophages are key regulators of immune tolerance, regulating the homeostasis of guts and protecting tissues against excessive immune responses by maintaining the functionality of regulatory T cells. In addition, 'alternatively activated M2 macrophages also play an important role in tissue repair and Th2 responses. GO-mediated determination of macrophage bias is rarely explained, and the results are now controversial. Some studies have reported that GO treatment regulates macrophage bias to M1 type in a size dependent manner, while other studies have found that GO scaffolds increase the infiltration of M2 type cells. To overcome this disagreement, we examined the effect of nano-GOs on macrophage deflection by treating nanoparticles with cells in the M0, M1 and M2 states.

As a result, it was confirmed that the nano-GOs activate the M0 cells, bias them to M1 type cells, and inhibit M2 type cells. It was also confirmed that nano-GOs-treatment could be a potential immunosuppressive drug by inducing the conversion from M1 to M2 macrophages.

Given the increased infiltration of colonic Treg and M2-type macrophages during inflammation, nano-GOs play an important role in forming regulatory loops of Tregs and intestinal macrophages through IL-10 and TGF-β1 signaling. It can be seen.

M2 macrophages are classified into at least three subtypes having various cellular characteristics such as gene profiles and distinct functions mediated by various cytokine production. Among them, M2a and M2b exhibit immunogulatory activities and induce Th2 responses, and M2c is involved in immunosuppressive capacity and tissue-remodeling. The interaction between subtypes of these macrophages and graphene derivatives is not yet known. In the present invention, during Ml induction, the Ml-like properties decrease while the IL-6 levels inducing M2 type cells do not change. In addition, representative M2-related factors such as IL-10 were significantly increased in the presence of nano-GOs. The M2a related genes CLEC7A and IL-1Ra were downregulated as the expression of cellular markers decreased, and M2c cells did not show any effective changes. Rather, the proportion of M2b subtype cells and their related products increased in response to nano-GOs treatment.

These results indicate that nano-GOs are involved in increased TLR-mediated signaling, and therefore, among M2 type cells, M2b macrophages are directly affected by nano-GOs and are involved in the migration of Tregs in the colon under inflammatory conditions. It was.

In conclusion, in the present invention, the graphene derivative, nano-sized graphene oxide (nano-GOs) inhibits type 1 helper T cells and simultaneously activates a regulatory loop between intestinal macrophages and regulatory T cells. It was confirmed to have a protective effect against colitis. In addition, these therapeutic effects were confirmed to change depending on the size and shape of the nanoparticles.

Claims (11)

A nano-sized graphene oxide (nano-sized graphene oxide; nano-GO) comprising as an active ingredient, a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease.
The method of claim 1,
The composition is to inhibit or reduce inflammation by inhibiting the expression or secretion of proinflammatory cytokines, inhibiting myeloperoxidase activity, inhibiting Th1 differentiation or response, promoting T cell activity, upregulating M2b macrophages, or a combination thereof. Pharmaceutical compositions.
delete delete The method of claim 1,
The inflammatory bowel disease is a inflammatory bowel disease selected from the group consisting of Chron's disease, ulcerative colitis, and intestinal Bechet's disease.
The method of claim 5,
The inflammatory bowel disease is characterized by accompanying symptoms of shortening of colon, hair loss, decreased activity, weight loss, increased bleeding index or increased bowel index.
Cosmetic composition for the prevention or improvement of inflammatory bowel disease comprising nano-sized graphene oxide (nano-sized graphene oxide; nano-GO) as an active ingredient.
Food composition for the prevention or improvement of inflammatory bowel disease comprising nano-sized graphene oxide (nano-sized graphene oxide; nano-GO) as an active ingredient.
Feed composition for the prevention or improvement of inflammatory bowel disease comprising nano-sized graphene oxide (nano-sized graphene oxide; nano-GO) as an active ingredient.
A method of preventing or treating inflammatory bowel disease in an animal other than a human, comprising administering the pharmaceutical composition of claim 1 to an individual in need thereof.
The method of claim 10,
Administering the pharmaceutical composition to restore shortened colon, hair loss, decreased activity, reduced body weight, elevated bleeding index or elevated defecation index.