Anti-inflammatory composition comprising graphene nano-structure
11786549 · 2023-10-17
Assignee
Inventors
- Kyung-sun Kang (Seoul, KR)
- Byung-Chul Lee (Seoul, KR)
- Jin Young Lee (Seoul, KR)
- Jong Bo PARK (Seoul, KR)
Cpc classification
A61P37/06
HUMAN NECESSITIES
International classification
A61K9/16
HUMAN NECESSITIES
Abstract
The present invention relates to an anti-inflammatory composition comprising a graphene nano-structure as an active ingredient. Specifically, the present invention relates to a pharmaceutical composition for preventing or treating an inflammatory disease, and a cosmetic composition or feed composition for preventing or improving an inflammatory disease, which include the anti-inflammatory composition, and a method of treating an inflammatory disease, including administering the composition to a subject in need thereof.
Claims
1. A method of treating an inflammatory bowel disease, comprising administering a composition comprising an active ingredient for treating the inflammatory bowel disease to a subject in need thereof, and thereby treating the inflammatory bowel disease, wherein the active ingredient consists of nano-sized graphene oxide (nano-GO) or a graphene quantum dot (GQD), wherein the active ingredient is plate-shaped particles, and wherein the inflammatory bowel disease is selected from the group consisting of Crohn's disease and ulcerative colitis.
2. The method of claim 1, wherein symptoms of the inflammatory disease are treated by the administering of the composition and the symptoms are a shortened colon, hair loss, reduced activity, a reduced body weight, an increased bleeding index or an increased defecation index by the administration of the composition.
3. The method of claim 1, wherein the nano-GO has a thickness of 12 nm or less and an average diameter of 15 to 50 nm.
4. The method of claim 1, wherein the GQD is a particle having an average diameter of 1 to 10 nm and a thickness of 0.5 to 3 nm.
5. The method of claim 1, wherein the composition inhibits or reduces inflammation by inhibition of the expression or secretion of a proinflammatory cytokine, inhibition of myeloperoxidase activity, inhibition of Th1 differentiation or the Th1 response, promotion of T cell activity, upregulation of an M2b macrophage, or a combination thereof.
Description
DESCRIPTION OF DRAWINGS
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MODES OF THE INVENTION
(22) Hereinafter, the configuration and effects of the present invention will be described in detail with reference to examples. These examples are merely provided to illustrate the present invention, and the scope of the present invention is not limited to these examples.
Preparation Example 1
Preparation and Biotinylation of Nano-GOs
(23) Pristine graphene oxides (GOs) were synthesized by an improved Hummer's method. To prepare nano-sized GOs, a distilled water solution of the obtained GOs (3 mg/mL) was vigorously tip-sonicated for 3 hours, and vacuum-filtered using a cellulose nitrate membrane filter (0.45 μm, GE Healthcare).
(24) In addition, nano-GOs were biotinylated by EDC coupling. First, 10 mg of N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC reagent, Sigma) was added to 10 mL of a nano-GO solution (3 mg/mL) to replace edge carboxyl groups with an EDC reagent. After 30 minutes, 20 mg of amine-PEG.sub.3-biotin (Thermo Scientific) was added to the solution to allow a reaction for 24 hours, thereby forming an amide bond between the activated terminal of nano-GO and a reactive amide group of biotin. Similar to the nano-sized GO preparation method, the final product was obtained in powder form after appropriate dialysis and filtration steps.
Preparation Example 2
Preparation and Biotinylation of GQDs
(25) GQDs were synthesized by thermo-oxidative cutting of carbon fibers (Carbon Make, South Korea) by a reaction in a 3:1 mixed solution of sulfuric acid and nitric acid (Samjeon Chemical Co., Ltd.) at 80° C. for 1 day. The solution was diluted and dialyzed using an MWCO 1 kD nitrocellulose membrane (Fisher Scientific) to remove very small fragments and the remaining acid, followed by vacuum-filtration with an inorganic membrane filter (Whatman-Anodisc 47, GE Healthcare). The final product was obtained in powder form by lyophilization.
(26) In addition, GQDs were biotinylated by the same method as used for the nano-GO of Preparation Example 1, and the final product was obtained in powder form after suitable dialysis and filtration steps.
Experimental Example 1
TEM Imaging
(27) A solution (10 μg/ml) in which each sample prepared according to the preparation examples was dispersed was adsorbed onto a 300-mesh Lacey carbon-coated copper grid (Ted Pella, Inc.) for 30 minutes. Prior to imaging, the grid was washed with several drops of distilled water and completely dried in a desiccator. The samples prepared as described above were analyzed using a high resolution-transmission electron microscope (HR-TEM, JEM-3010, JEOL Ltd.), and images were collected using a Gatan digital camera coupled with a microscope (MSC-794).
Experimental Example 2
Raman Spectroscopy
(28) For measurement of Raman spectra, a powder-type product prepared according to the preparation examples was prepared on a SiO.sub.2 base. Spectra were measured with a Renishaw micro-Raman spectrometer equipped with a 514.5 nm Ar excitation laser.
Experimental Example 3
FT-IR Spectroscopy
(29) Prior to the measurement of Fourier-transform infrared (FT-IR) spectra, the powder-type samples were completely dried in a desiccator to exclude unwanted oxygen containing peaks. Spectra were measured by a typical KBR pellet method (Nicolet 6700, Thermo Scientific).
Experimental Example 4
Experimental Animals
(30) All animal experiments were conducted according to the guidelines approved by the Institutional Animal Care and Use Committee of Seoul National University (IACUC No. SNU-170523-4). Six-week-old male C57BL/6 mice (OrientBio Inc., Sungnam, Republic of Korea) were grouped randomly, and provided 3% DSS in drinking water for 7 days (16 per group). On day 1, that is, the first day after DSS induction, mice were administered intraperitoneally 15 g/kg of nano-GOs and GQDs prepared according to the preparation examples. Body weights of the mice were measured daily, and the disease activity index (DAI) composed of body weight loss, activity, stool consistency, bleeding and a hair condition were evaluated on day 7 and day 10. After sacrificing the mice, for additional ex vivo examinations, spleen, large intestine and blood samples were collected.
Experimental Example 5
Histopathological Evaluation
(31) Collected colon samples were fixed in 10% formalin according to typical methods including dehydration with ethanol, clearing with xylene and wax infiltration with paraffin. Paraffin-embedded blocks were sectioned to a thickness of 5 μm and stained with H&E or Masson's trichrome. The loss of goblet cells, hyperemia/edema, immune cell infiltration, the presence of crypt abscesses and epithelium loss were recorded as a histopathological index, by H&E staining. The fibrotic tissue area was measured by Masson's trichrome, and quantified using ImageJ software (version 1.46r, US National Institute of Health, Bethesda, Md., USA).
Experimental Example 6
Cytokine Production
(32) To determine the secretion level of various cytokines, serum isolated from blood, the lysate of a colon and a culture supernatant of cells were prepared. To measure the extent of inflammation in vivo, a Cytometric Bead Array (CBA) kit (BD Bioscience, San Jose, Calif., USA) for mouse inflammation and ELISA kits for MPO and TGF-β1 (R&D Systems, Minneapolis, Minn., USA and Thermo Fisher Scientific, San Jose, Calif., USA, respectively) were used according to the manufacturers' protocols. To evaluate the secretion of cytokines induced from immune cells in vitro, CB kits for Th1/Th2/Th17 (BD Bioscience, San Jose, Calif., USA) and an ELISA kit for TGF-β1 (Thermo Fisher Scientific, San Jose, Calif., USA) were used. Results were detected using flow cytometry and spectrophotometry.
Experimental Example 7
Isolation and Culture of hMNCs
(33) All experimental procedures related to human umbilical cord blood (hUCB) or UCB-derived cells were conducted under approval of the Boramae Hospital Institutional Review Board (IRB) and the Seoul National University IRB. Human umbilical cord blood-mononuclear cells (hUCB-MNCs) were isolated and cultured by a known method. Specifically, UCB samples were collected immediately after birth under informed consent and parent approval. The collected UCB samples were incubated after being mixed with a HetaSep solution (Stem Cell Technologies, Vancouver, Canada) in 5:1 at room temperature for 1 hour. Afterward, the supernatant was collected with Ficoll, and mononuclear cells were isolated by centrifugation at 2,500 rpm for 20 minutes. The isolated cells were washed twice with PBS. The cells isolated as described above were subjected to experiments additionally performed in subsequent in vitro analysis.
Experimental Example 8
T Cell Isolation and Polarization
(34) Naive CD4.sup.+ T cells were isolated from freshly isolated hUCB-MNCs using a human naive CD4.sup.+ T cell isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instruction. The isolated CD4.sup.+ T cells were cultured in RPMI1640 (Gibco BRL, Grand Island, N.Y., USA) containing 10% fetal bovine serum (FBS), an anti-CD3/28 bead activator and 20 ng/ml of IL-2, which were required for proliferation of T cell subsets. For differentiation into T cell subsets, the cells were cultured in a humidified 5% CO.sub.2 atmosphere at 37° C. for 5 days in the presence/absence of nano-GOs or GQDs by adding type-specific cytokines (20 ng/ml of IFN-γ and 20 ng/ml of IL-12 for type 1 helper T cells, and 20 ng/ml of TGF-β1 for Treg cells) to a growth medium. Polarized Th1 and Treg cells were confirmed by type-specific staining and flow cytometry. For the Th1 cells, the surface was stained with CD4 antibodies, and then intracellular staining was performed with IFNγ. For Treg analysis, CD4, CD25 and IL-4 antibodies were used.
Experimental Example 9
Macrophage Isolation and Polarization
(35) Macrophages were isolated and cultured by known methods. Specifically, macrophages were isolated from freshly-isolated hUCB-MNCs using a human CD4.sup.+ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instruction. The isolated CD14.sup.+ cells were cultured in 10% FBS-containing RPMI1640. To polarize the cells to macrophage subtypes, the cells were cultured in a humidified 5% CO.sub.2 atmosphere at 37° C. for 5 days in the presence/absence of nano-GOs or GQDs by adding type-specific cytokines (20 ng/ml of IFN-γ and 1 μg/ml of LPS of M1 cells, and 20 ng/ml of IL-4 and 20 ng/ml of IL-13 for M2 cells) to a growth medium. To confirm the polarized macrophages, type-specific staining and flow cytometry were used. As a pan-macrophage marker, CD14 antibodies were used, and as specific markers for M1 and M2 subtypes, CD86 and CD206 were applied.
Experimental Example 10
Cell Proliferation Assay
(36) To measure cell proliferation, a cell proliferation ELISA kit (Roche, Indianapolis, Ind., USA) and a CCK-8 kit (Dojindo, Kumamoto, Japan) were used according to the manufacturers' instructions. For bromodeoxyuridine (BrdU) cell proliferation assay, a known method was used. Specifically, cells were incubated in a humidified 5% CO.sub.2 atmosphere at 37° C. for 2 hours with a 100 μM BrdU labeling reagent. After being fixed in a provided FixDenat solution for 30 minutes, the cells were incubated in an anti-BrdU antibody solution for 90 minutes, and in a solution of a substrate (tetramethyl-benzidine; TMB) provided at room temperature for 5 to 30 minutes. After a stop solution was poured into each well, absorbance was measured at 450 nm and 690 nm to quantify a cell proliferation level.
Experimental Example 11
Quantitative RT-PCR
(37) Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instruction. The obtained RNA was used for cDNA synthesis by a Superscript First-Strand Synthesis System (Invitrogen). A relative expression level of target mRNA was measured by an ABI 7300 detection system using a SYBR Green PCR Master Mix (Applied Biosystems, Foster City, USA). At least three independent analyses were performed for each gene.
Experimental Example 12
Cell Cycle Assay
(38) A cell cycle assay was performed according to a known protocol. Specifically, cells were fixed with ice-cold 70% ethanol at −20° C. for 30 minutes or more. The 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. The cell cycle was analyzed by flow cytometry performed on FACScalibur using Cell Quest Software (BD Bioscience, San Jose, Calif., USA).
Experimental Example 13
Apoptosis Assay
(39) An 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 by vortexing, and incubated in a room temperature dark chamber for 15 minutes. Subsequently, 400 μl of a 1×binding buffer was added to the mixture, and all samples were analyzed by flow cytometry performed on FACScalibur using Cell Quest Software.
Experimental Example 14
Immunofluorescence Assay
(40) Cells were fixed in a 4% paraformaldehyde (PFA) PBS solution at room temperature for 15 minutes, and treated with 0.25% Triton X-100 (Sigma) for 10 minutes to increase permeability. The fixed cells were incubated with a blocking solution (5% normal goat serum) for 1 hour at room temperature, and incubated with primary antibodies at 4° C. overnight. Afterward, the cells were incubated with secondary antibodies labeled with Alexa Fluor 594 (Invitrogen), and nuclei were stained by DAPI (Sigma) staining for 5 minutes.
(41) For whole tissue immunofluorescence, a paraffin slide was deparaffinized, and blocked with 5% normal goat serum-containing PBS. Sections were incubated with primary antibodies overnight, and then incubated with Alexa Fluor 594, followed by DAPI staining. Images were collected using a confocal microscope (Eclipse TE200, Nikon, Japan).
Experimental Example 15
Western Blotting
(42) A colon sample was degraded with Pro-Prep (Intron Biotechnology Co., Sungnam, Republic of Korea) to extract a protein from the tissue. The obtained protein sample was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. After the reaction was blocked with a 3% Bovine Serum Albumin (BSA) solution, the protein on the membrane was incubated with IL-10 primary antibodies at 4° C. for at least 12 hours, and then incubated with secondary antibodies. The protein-antibody complex was detected using ECL Western blotting detection reagents and an analysis system.
Experimental Example 16
Statistical Analysis
(43) The mean values of all results were expressed as the mean±standard error of mean (SEM). Statistical analyses were conducted using Student's 2-tailed t-test or one-way ANOVA followed by a Bonferroni post-hoc test for multi group comparisons using GraphPad Prism version 5.0 (GraphPad Software, San Diego, Calif., USA). Statistical significance was indicated in the description of the drawings.
Example 1
Characterization of Nano-GOs and GQDs
(44) Synthetic methods for nano-GOs and GQDs used in the present invention are schematically shown in
Example 2
DSS-Induced Colitis Improvement Effect of Graphene Nano-Structure in Mouse Models
(45) To confirm a therapeutic effect of a graphene nano-structure in DSS-induced colitis mice, on day 1 after induction of colitis, each of the nano-GOs and GQDs were intraperitoneally injected into a mouse, and a body weight, a survival rate and activity were monitored for 2 weeks. As a result, both of the nano-GOs and GQDs showed an effect of protecting a mouse from severe colitis, determined by an increased survival rate and a decreased body weight (
Example 3
Impairment of DSS-Induced Colitis Inflammatory Response by Graphene Nano-Structure
(46) The histological analysis of the colon showed that DSS induces epithelium destruction, submucosal edema, crypt abscesses and lymphocytic infiltration (
Example 4
Inhibitory Effect of Graphene Nano-Structure on CD4.SUP.+ T Cell Activity
(47) In consideration that Th1 cells play a critical role in enterocolitis, a direct effect of a graphene nano-structure in CD4.sup.+ T cells was examined. First, to determine an appropriate concentration, MNCs and primary CD4.sup.+ T cells were treated with nano-GOs and GQDs at various concentrations (
Example 5
Increase in in vivo Regulatory T Cell Infiltration by Graphene Nano-Structure
(48) The inventors had confirmed that recovery from experimental colitis is accompanied by expansion and infiltration of regulatory T cells through previous research. Accordingly, the inventors performed flow cytometry to examine whether the administration of a graphene nano-structure affects localization of Treg cells. Specifically, the administration of nano-GOs or GQDs increased colonic infiltration and splenic polarization of CD4.sup.+CD25.sup.+FoxP3.sup.+ Treg cells (
(49) Taken together, such results show that the graphene nano-structures increase the number of regulatory T cells and cytokines related thereto in vivo, which are not mediated by a direct effect on Treg polarization.
Example 6
Conversion of M1 Macrophages into M2 Type During Immune Response by Graphene Nano-Structure
Example 6-1
Conversion of M1 Macrophages into M2 Type During Immune Response by Nano-GOs
(50) Due to an imbalance between tolerogenic and protective immune responses of intestinal macrophages, colonic inflammation may occur and may be improved by alternatively activated M2-type macrophages serving to collect Treg cells and secretory cytokines thereof. Accordingly, it was examined whether treatment of nano-GOs affects the cell fate determination of macrophages. The internalization of nanoparticles was confirmed by a biotinylated carbon nano-structure and immunohistochemistry (
(51) A cell surface marker assay was conducted using flow cytometry to confirm an effect of nano-GOs on macrophage type-specific polarization. The graphene nano-structure was likely to induce naive CD14.sup.+ cells to a proinflammatory (classical) type of macrophage without other additional cytokines. With regard to M2-type macrophages, nano-GO treatment induced an increase in both of CD14.sup.+CD86.sup.+ cells and CD14.sup.+CD206.sup.+ cells. The expression of a CD206 surface marker on CD14.sup.+ cells was confirmed by immunohistochemistry (
Example 6-2
Conversion of M1 Macrophage into M2 Type During Immune Response by GQDs
(52) Whether the treatment with GQDs affects the cell fate determination of macrophages was examined. The internalization of nanoparticles was identified by a biotinylated carbon nano-structure and immunohistochemistry (
(53) A cell surface marker assay was conducted using flow cytometry to confirm an effect of GQDs on macrophage type-specific polarization. To confirm that the conversion of proinflammatory (classical) activated M1-type macrophages into anti-inflammatory M2 subtypes is caused by GQD treatment, additionally, the expression of another M2 type-specific property was examined. As a result, the expression of a hemoglobin scavenger receptor, CD163, increased in the presence of GQDs (
(54) Such results show that GQDs play a role in inflammatory resolution by downregulating an M1-like property of macrophages activated as a proinflammatory (classical) type and converting the macrophages into M2 types.
Example 7
Main Subtype M2b of Generally Activated Macrophages Induced by Nano-GOs
(55) To confirm that classically activated M1-type macrophages are converted into anti-inflammatory M2 subtypes by nano-GO treatment, additionally, the expression of another M2 type-specific property was examined. The expression of a hemoglobin scavenger receptor, CD163, increased in the presence of nano-GOs (
(56) The M2 macrophages may be classified into three or more subtypes which have distinct roles in immune systems. IL-10 secretion increased while CLEC7A and IL-1ra, which are M2a markers, decreased, and based on this, it was assumed that nano-GOs upregulate other M2-subtype cells. To prove this, M2a, M2b and M2c macrophages were induced, and the role of a graphene nano-structure was identified. As shown in the above-described result, the CD163 and CD206 expression of the M2a macrophages significantly decreased in the presence of nano-GOs (
(57) <Conclusion>
(58) In the present invention, a protective effect of a nano-sized graphene derivative, that is, a graphene nano-structure, on experimental colitis and an action mechanism thereof were analyzed.
(59) The inventors conducted a series of in vitro experiments to identify a comprehensive impact of the graphene nano-structure on innate immune cells and adaptive immune cells. A large amount of nano-GOs or GQDs was ingested by primarily isolated Th1 cells and CD14.sup.+ macrophage-like cells. It is known that proinflammatory cytokines, particularly, IL-2, IL-12 and IFN-γ and a transcription factor T-bet play a critical role in the commitment of Type 1 helper T cells. Meanwhile, representative cytokines for Th2 and Treg, such as IL-4 and IL-10, and a proinflammatory cytokine IL-6 mediated inhibition of Th1 development. The graphene nano-structure exhibited an effect of regulating an immunogenic milieu, confirmed by cytokine production. In addition, it was identified that GQDs inhibited the proliferation of CD4.sup.+T cells similarly to nano-GOs, but differently affect the development of Th1 cells.
(60) Regulatory T cells protect tissue from excessive inflammation, and help a healing process of tissue by inhibiting activated immune cells. IL-10 and TGF-β1 were involved in the development, expansion and specific role of Treg. In the present invention, it was confirmed that, although not having a direct influence on cell fate, the nano-GO treatment improves colonic and splenic infiltration of regulatory T cells by increased production of IL-10 and TGF-β1, and GQD administration improves colonic infiltration of T cells.
(61) Macrophages exhibit distinctive plasticity, and control their properties according to an environmental stimulus. Intestinal macrophages regulate homeostasis of the gut as a major regulator of immune tolerance and retain the functionality of regulatory T cells, thereby protecting tissue from an excessive immune response. In addition, alternatively-activated M2 macrophages also play a critical role in tissue recovery and a Th2 response. Therefore, it was examined how a graphene nano-structure affects macrophage polarization by treating M0, M1 and M2-type cells with nanoparticles.
(62) As a result, it was confirmed that nano-GOs and GQDs activate M0 cells to polarize the cells to M1-type cells, and suppress M2-type cells. In addition, the nano-GO or GQD treatment induces the conversion of M1 macrophages into M2 macrophages, confirming that nano-GO or GQD can be a potential immunosuppressive drug.
(63) In consideration that colonic Treg and M2-type macrophage infiltration increase during inflammation, it can be seen that the graphene nano-structure of the present invention plays a critical role in forming a regulatory loop of Treg and intestinal macrophages through IL-10 and TGF-β1 signaling.
(64) The M2 macrophages are classified into at least three subtypes having various cellular properties such as gene profiles and functions mediated by production of various cytokines. Among the M2 subtypes, M2a and M2b exhibit immunoregulatory activities and induce Th2 responses, and M2c is involved in immunosuppressive capacity and tissue-remodeling. The interaction between a subtype of these macrophages and a graphene derivative has not yet been identified. In the present invention, during M1 induction, M1-like properties were reduced, but an IL-6 level inducing M2-type cells was not changed. In addition, a representative M2-related factor such as IL-10 significantly increased in the presence of nano-GOs. M2a-related genes such as CLEC7A and IL-1Ra were downregulated by the decreased expression of cell markers, and the M2c cells showed no significant change. Rather, the proportions of M2b-subtype cells and their related products were increased by nano-GO treatment. Such results show that nano-GOs are involved in increasing TLR-mediated signaling, and thus, among M2-type cells, M2b macrophages were indirectly influenced by the graphene nano-structure of the present invention, and involved in Treg migration in the colon under inflammation.
(65) In conclusion, it was confirmed that nano-sized graphene derivatives of the present invention, specifically, nano-GOs and GQDs, have a protective effect on experimental colitis by inhibiting type 1 helper T cells and activating a regulatory loop between intestinal macrophages and regulatory T cells. Further, it was confirmed that such a therapeutic effect can be changed by the size and morphology of nanoparticles.