Novel cerium oxide nanocomposite and its uses

Abstract

The present invention relates to a cerium oxide nanocomposite, a method for producing the same, and a composition containing the same as an active ingredient for preventing or treating inflammatory or autoimmune diseases. The present invention significantly improves the biomedical stability, biocompatibility, and production process efficiency of nanoparticles by applying an optimal combination of biocompatible dispersion stabilizers, while maintaining the inherent pharmacological effects of the nanoparticles, making it useful as an efficient nanoparticle therapeutic composition.

Description

[Technical field]

[0001] The present invention relates to a cerium oxide nanocomposite modified with a biocompatible dispersion stabilizer, and a composition for preventing or treating inflammatory diseases comprising the same as an active ingredient. [Background technology]

[0002] Nanoparticles, which are used for various purposes in the fields of diagnosis and treatment, show new physical / chemical properties in vivo that differ from bulk materials due to their minute size at the nano level. Currently, as much research on nanoparticles is being conducted, active development is being carried out on nanoparticles that show optimal physical properties for medical applications by adjusting the particle composition, shape, size, properties, etc. according to the purpose. On the other hand, cerium oxide has thermal stability at high temperatures and its lattice structure allows it to be converted into Ce depending on the surrounding oxygen concentration. 4+ / Ce 3+ It has a redox action and is used in a variety of applications, including electrolytes for solid-state batteries, UV filter materials, oxygen sensors, optical devices, etc. In particular, in the medical field, it has been attracting attention as a therapeutic composition for a wide range of diseases caused by oxidative stress and inflammation due to its excellent ability to remove reactive oxygen species.

[0003] Commonly used methods for producing cerium oxide particles are not only difficult to produce uniform nano-sized particles, but also difficult to produce fine particles of less than 10 nm. For example, cerium oxide nanoparticles synthesized by hydrothermal, a liquid phase method, have sizes ranging from several hundred nm to several microns, making precise particle size control difficult and poor dispersibility. As a result, methods have been proposed for producing cerium oxide nanoparticles with smaller average particle size and uniform particle size distribution, but these require inefficient processes involving high-temperature treatment at several hundred degrees Celsius, making them unsuitable for industrial mass production. Therefore, there is a need to develop a new method for efficiently producing cerium oxide nanoparticles in a mild reaction environment that have a small size of less than 50 nm, uniform distribution, excellent dispersibility, are easy to produce, and maintain their unique antioxidant effect.

[0004] Throughout this specification, a number of papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference in their entirety into this specification in order to more clearly explain the state of the art to which the present invention pertains and the contents of the present invention. Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have made extensive research efforts to develop an excellent cerium oxide nanostructure that maintains its inherent pharmacological effects while further improving the biomedical stability, biocompatibility, and production process efficiency of nanoparticles. As a result, the present inventors have found that when a polymer layer in which the pyrrolidone derivative monomer of Formula 1 is repeated and a biocompatible dispersion stabilizer layer are formed on the core of cerium oxide nanoparticles, uniform and fine particles can be stably and economically mass-produced in a sufficient reaction time without applying harsh environments such as high temperature strong base and / or strong acid conditions, thereby completing the present invention.

[0006] It is therefore an object of the present invention to provide a cerium oxide nanocomposite and a method for its preparation. Another object of the present invention is to provide a composition for preventing or treating inflammation or autoimmune diseases, which comprises the cerium oxide nanocomposite of the present invention as an active ingredient. Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims and the drawings. [Means for solving the problem]

[0007] According to one aspect, the present invention provides a cerium oxide nanocomposite comprising: (a) Core layer of cerium oxide nanoparticles; (b) an inner layer comprising a polymer represented by the following chemical formula 1: [ka] In the above formula, R1 and R2 are each independently hydrogen or oxygen; [ka] represents a single bond or a double bond, l is 1 or 2, and m is an integer of 100 to 1000; and (c) an outer layer comprising one or more biocompatible dispersion stabilizers selected from the group consisting of PGA (polyglutamic acid), PASP [poly(aspartic acid)], alginate, PAA [Poly(acrylic acid)], PMAA [Poly(methacrylic acid)], poly(methyl methacrylic acid)], PMA [poly(maleic acid)], PBMA [poly(butadiene / maleic acid)], PVPA [Poly(vinylphosphonic acid)], PSSA [Poly(styrenesulfonic acid)], PVA (polyvinyl alcohol), and dextran.

[0008] The present inventors have made extensive research efforts to develop an excellent cerium oxide nanostructure that maintains its inherent pharmacological effects while further improving the biomedical stability, biocompatibility, and production process efficiency of nanoparticles.As a result, they have found that when a polymer layer in which the pyrrolidone derivative monomer of Formula 1 is repeated and a biocompatible dispersion stabilizer layer are formed on the core of cerium oxide nanoparticles, uniform and fine particles can be stably and economically mass-produced in a sufficient reaction time without harsh environments such as high temperature strong base or strong acid conditions.

[0009] According to the conventional technology, in order to ensure in vivo stability in the process of synthesizing cerium oxide nanoparticles, PEG (polyethylene glycol) or PEG derivatives modified with succinimidyl groups are mainly used, and it is very difficult to obtain a large amount of uniform particles because of the extremely short reaction time of less than one minute and the high temperature of 95°C and strong acid / base such as HCl and NaOH. However, the nanocomposite of the present invention not only ensures stable synthesis conditions of about 2 hours of reaction time at 70°C, but also does not require a strong acid or strong base environment, efficiently overcoming the disadvantages of the conventional technology.

[0010] As used herein, the term "core layer" refers to the innermost layer in a multi-layer composite that has only one surface in contact with other layers. As used herein, the term "multilayer composite" refers to a composite consisting of multiple layers composed of different components, and includes, without limitation, a laminated multilayer structure, a core-shell multilayer structure, and combinations thereof. Specifically, the multilayer composite of the present invention has a core-shell multilayer structure in which a nanoparticle is present in the center and is surrounded by the polymer of Chemical Formula 1 and a biocompatible dispersion stabilizer at the outer corners.

[0011] In this specification, the term "inner layer" refers to a layer closer to the core than the outer layer, and the term "outer layer" refers to a layer surrounding the inner layer of the core-shell structure and farther from the core than the inner layer. The inner layer does not necessarily have to be a layer in direct contact with the core layer, and there may be an additional layer closer to the core layer than the inner layer, and the outer layer does not necessarily have to be the outermost layer, and there may be an additional outermost layer located farther from the core than the outer layer.

[0012] According to the present invention, the boundary between the core layer and the inner layer is clearly defined, but the boundary between the inner layer and the outer layer may or may not be clearly defined. When the boundary between the inner layer and the outer layer is not clearly defined, the components of the inner layer and the outer layer may be mixed in a part or the entire section of the interface.

[0013] As used herein, the term "polymer" refers to a synthetic or natural polymeric compound in which the same or different types of monomers are successively bonded. Thus, polymers include homopolymers (polymers in which one type of monomer is polymerized) and hybrid polymers made by polymerization of at least two different types of monomers, and hybrid polymers include copolymers (polymers made from two different types of monomers) and polymers made from more than two different types of monomers. Specifically, the polymer of Formula 1 used in the present invention is a homopolymer.

[0014] As used herein, the term "alkyl" refers to a linear or branched saturated hydrocarbon group, including, for example, methyl, ethyl, propyl, isopropyl, etc. C1-C3 alkyl refers to an alkyl group having an alkyl unit having 1 to 3 carbon atoms, and when C1-C3 alkyl is substituted, the carbon number of the substitute is not included. In this specification, the term "biocompatibility" means a property of not causing short-term or long-term side effects when administered into a living body and contacting cells, tissues, or body fluids of an organ, specifically including not only tissue compatibility and anticoagulant compatibility that do not cause necrosis of tissues or coagulation of blood when in contact with living tissues or blood, but also biodegradability that disappears after a certain period of time has passed after administration to the living body, and "excretion ability without accumulation after administration to the living body." Therefore, the term "biocompatible dispersion stabilizer" means a component that has the above-mentioned biocompatibility and improves the dispersibility of particles.

[0015] As used herein, the term "biodegradable" refers to the property of being naturally decomposed when exposed to a physiological solution of pH 6-8, specifically, the property of being decomposed over time in a living body by body fluids, decomposing enzymes, microorganisms, etc. According to a specific embodiment of the present invention, the cerium oxide nanoparticles used in the present invention may be cerium (IV) oxide (CeO2), cerium (III) oxide (Ce2O3), or a mixture thereof.

[0016] According to a specific embodiment of the present invention, in the above formula 1, R1 is hydrogen, R2 is oxygen, and l is 1. According to the octet rule, when R1 is hydrogen, [ka] is a single bond, and when R2 is oxygen, [ka] is a double bond. The compound of formula 1, where R1 is hydrogen, R2 is oxygen, and l is 1, is polyvinylpyrrolidone (PVP).

[0017] According to a specific embodiment of the present invention, the biocompatible dispersion stabilizer contained in the outer layer is PGA (polyglutamic acid). PGA (polyglutamic acid) may be poly-α-glutamic acid, poly-β-glutamic acid or poly-γ-glutamic acid, and is specifically poly-α-glutamic acid. Most specifically, the poly-α-glutamic acid is PLGA (poly L-glutamic acid).

[0018] According to a specific embodiment of the present invention, the nanocomposite of the present invention additionally comprises a multifunctional ligand represented by the following formula 2: [ka] In the above chemical formula, n is an integer of 3-7.

[0019] In the present specification, the term "multi-functional ligand" refers to a molecule having two or more active functional groups and binding to two or more molecules to act as a linker between the molecules. The multi-functional ligand of Formula 2 used in the present invention has a carboxyl group capable of binding to cerium oxide nanoparticles and an amine group capable of binding to PVP in the inner layer and / or PGA, PVA or dextran in the outer layer, allowing the nanocomposite of the present invention to be formed more efficiently and stably.

[0020] Specifically, in the above Chemical Formula 2, n is 5. The compound of Chemical Formula 2 where n is 5 is 6-aminohexanoic acid (6-AHA). According to a specific embodiment of the present invention, the nanocomposite of the present invention has an average particle size of 5 nm to 100 nm, more specifically 5 nm to 80 nm, most specifically 5 nm to 50 nm. According to another aspect of the present invention, there is provided a composition for preventing or treating inflammation or autoimmune diseases, comprising the nanocomplex of the present invention as an active ingredient. The cerium oxide nanocomposite used in the present invention has already been described in detail, so the description will be omitted to avoid undue duplication.

[0021] As used herein, the term "prevention" refers to inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed as having the disease or condition, but is susceptible to such disease or condition. As used herein, the term "treatment" means (a) inhibiting the development of a disease, illness, or condition; (b) alleviating a disease, illness, or condition; or (c) eliminating a disease, illness, or condition. The composition of the present invention plays a role in inhibiting, eliminating, or alleviating the development of an excessive or undesirable immune response or inflammatory symptoms by reducing reactive oxygen species and suppressing the expression of inflammatory cytokines. Thus, the composition of the present invention may be a composition for treating these diseases by itself, or may be administered together with other pharmacological ingredients having anti-inflammatory effects and applied as a therapeutic adjuvant for the above diseases. Thus, as used herein, the term "treatment" or "therapeutic agent" includes the meaning of "therapeutic adjuvant" or "therapeutic adjuvant".

[0022] As used herein, the term "administration" refers to administering a therapeutically effective amount of the composition of the present invention directly to a subject so that the same amount is formed in the subject's body, and has the same meaning as "implantation" or "injection." In the present invention, the term "therapeutically effective amount" means the content of the composition contained in an amount sufficient to provide a therapeutic or prophylactic effect to an individual to whom the composition of the present invention is to be administered, and thus includes a "prophylactically effective amount." As used herein, the term "subject" includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus monkey. Specifically, a subject of the present invention is a human.

[0023] According to a specific embodiment of the invention, the inflammatory or autoimmune disease to be prevented or treated with the composition of the invention is selected from the group consisting of rheumatoid arthritis, reactive arthritis, type 1 diabetes, systemic lupus erythematosus, multiple sclerosis, idiopathic pulmonary fibrosis, polymyositis, dermatomyositis, localized dermatosclerosis, systemic dermatosclerosis, inflammatory bowel disease, Sjogren's syndrome, Raynaud's phenomenon, Bechet's disease, Kawasaki's disease, primary biliary sclerosis, primary sclerosing cholangitis, ulcerative colitis, Crohn's disease, psoriasis, myasthenia gravis, autoimmune vasculitis, and inflammatory bowel disease. The disease is one or more diseases selected from the group consisting of acute vascular vasculitis, primary angiitis of the central nervous system, subarachnoid hemorrhage (SAH), severe cerebral infarction, intracerebral hemorrhage, hypoxic ischemic encephalopathy, traumatic brain / spinal cord injury, acute respiratory distress syndrome, cytokine storm syndrome, sepsis, and inflammatory liver disease, and more specifically, the disease is one or more diseases selected from the group consisting of subarachnoid hemorrhage, sepsis, cytokine storm syndrome, and inflammatory liver disease.

[0024] As used herein, the term "inflammatory liver disease" is intended to encompass a range of diseases involving damage to liver tissue resulting directly or indirectly from excessive or unwanted immune or inflammatory responses induced by a variety of causes. More specifically, the inflammatory liver disease is selected from the group consisting of viral hepatitis, toxoplasma hepatitis, alcoholic liver disease, toxic liver disease, acute and subacute hepatic failure, liver abscess, nonspecific reactive hepatitis, liver infarction, hepatic veno-occlusive disease, injury of liver or gallbladder, and liver transplantation-related hepatitis.

[0025] When the composition of the present invention is prepared into a pharmaceutical composition, the pharmaceutical composition of the present invention contains a pharma- ceutically acceptable carrier. The pharma- ceutically acceptable carrier contained in the pharmaceutical composition of the present invention is one that is commonly used in formulation, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the pharmaceutical composition of the present invention may additionally contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharma- ceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0026] The pharmaceutical composition of the present invention may be administered through various administration routes, specifically, parenterally, more specifically, orally, intravenously, intraarterially, subcutaneously, intraperitoneally, intradermally, intramuscularly, intraventricularly, intraspinal, inhalation, nasally, intraarticularly or topically. The suitable dosage of the pharmaceutical composition of the present invention can be variously formulated depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity of the patient, etc. The preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.0001 to 100 mg / kg for adults.

[0027] The pharmaceutical composition of the present invention may be prepared in a unit dose form or in a multi-dose container by formulating it with pharma- ceutically acceptable carriers and / or excipients in a manner that can be easily carried out by a person having ordinary skill in the art to which the present invention pertains. In this case, the dosage form may be a solution, suspension, syrup or emulsion in an oil or aqueous medium, or an extract, powder, powder, granule, tablet or capsule form, and may additionally contain a dispersant or stabilizer.

[0028] According to yet another aspect of the present invention, there is provided a method for preventing or treating inflammation or autoimmune diseases, comprising administering to a subject the above-mentioned cerium oxide nanocomposite of the present invention or a pharmaceutical composition containing the same as an active ingredient. The cerium oxide nanocomposite used in the present invention and the inflammation or autoimmune diseases that can be prevented or treated thereby have already been described in detail, so a detailed description thereof will be omitted to avoid excessive duplication.

[0029] According to yet another aspect of the present invention, there is provided a method for preparing a cerium oxide nanocomposite comprising the steps of: (a) adding a cerium precursor, a polymer represented by the following chemical formula 1, and a crosslinking compound represented by the following chemical formula 2 to a C1 to C3 alcohol solvent to prepare a mixed solution; [ka] In the above formula 1, R1 and R2 are each independently hydrogen or oxygen; [ka] represents a single bond or a double bond, l is 1 or 2, and m is an integer of 100 to 1000; [ka] In the above Chemical Formula 2, n is an integer of 3 to 7; (b) sequentially heating and cooling the mixed solution to obtain cerium oxide nanoparticles; and (c) A step of mixing the cerium oxide nanoparticles with one or more biocompatible dispersion stabilizers selected from the group consisting of PGA (polyglutamic acid), PASP [poly(aspartic acid)], alginate, PAA [Poly(acrylic acid)], PMAA [Poly(methacrylic acid)], polymethyl methacrylic acid [poly(methyl methacrylic acid)], PMA [poly(maleic acid)], PBMA [poly(butadiene / maleic acid)], PVPA [Poly(vinylphosphonic acid)], PSSA [Poly(styrenesulfonic acid)], PVA (polyvinyl alcohol) and dextran in a C1 to C3 alcohol solvent to obtain a cerium oxide nanocomposite modified with the biocompatible dispersion stabilizer.

[0030] In the present specification, the term "coating" means forming a new layer of a certain thickness by applying a specific substance onto a target surface, and the target surface and the coating substance can be modified by ionic or non-covalent bonds. The term "non-covalent bond" is a concept including not only physical bonds such as adsorption, cohesion, entanglement, and entrapment, but also bonds that occur by interactions such as hydrogen bonds and van der Waals bonds acting alone or together with the physical bonds. In the present invention, when the biocompatible dispersion stabilizer is modified, the stabilizer layer may completely surround the surface to be modified to form a sealed layer, or may form a partially sealed layer.

[0031] According to a specific embodiment of the present invention, the C1-C3 alcohol solvent is ethanol. According to a specific embodiment of the present invention, the cerium precursor is one or more precursors selected from the group consisting of cerium (III) acetate hydrate, cerium (III) acetylacetonate hydrate, cerium (III) carbonate hydrate, cerium (III) fluoride, cerium (III) chloride, cerium (III) chloride heptahydrate, cerium (III) bromide, cerium (III) iodide, cerium (III) nitrate hexahydrate, cerium (III) oxalate hydrate, cerium (III) sulfate and cerium (III) sulfate hydrate. More specifically, it is cerium (III) nitrate hexahydrate. According to a specific embodiment of the present invention, the heating is performed at 60 to 75° C. As described above, one of the advantages of the present invention is that, unlike the conventional technology in which the reaction can be carried out only for a short period of time at a high temperature of 95° C., the reaction process can be stably and sustained sufficiently at a temperature of about 70° C., making it suitable for industrial-scale mass production. Effect of the Invention

[0032] The features and advantages of the present invention can be summarized as follows: (a) The present invention provides a cerium oxide nanocomposite, a method for producing the same, and a composition for preventing or treating inflammation or autoimmune diseases containing the same as an active ingredient. (b) The present invention can be usefully used as an efficient nanoparticle therapeutic composition by applying an optimal combination of biocompatible dispersion stabilizers, which significantly improves the biomedical stability, biocompatibility and production process efficiency of nanoparticles while maintaining the inherent pharmacological effects of the nanoparticles. [Brief description of the drawings]

[0033] [Figure 1] FIG. 2 shows a transmission electron microscope image of a cerium oxide nanocomposite of the present invention. [Diagram 2] FIG. 1 shows the results of an analysis of the morphology and dispersibility of cerium oxide nanocomposites depending on the nature of the solvent. [Diagram 3] FIG. 1 shows the change in particle size of cerium oxide nanocomposite with and without the presence of compounds. [Figure 4] FIG. 1 is a diagram showing the results of an analysis of changes in particle size depending on the polyvinylpyrrolidone content. [Diagram 5] The results of analyzing the particle size of a cerium oxide nanocomposite as a function of reaction time using a dynamic light scattering device are shown below. [Figure 6A] FIG. 13 shows the results of confirming, using a surface potential analyzer, whether or not a cerium oxide nanocomposite is coated with a biocompatible dispersion stabilizer. [Figure 6B] 1 shows the results of analyzing the particle size of a cerium oxide nanocomposite using a dynamic light scattering device. [Figure 7A] FIG. 1 shows the results of evaluation of the ability of a cerium oxide nanocomposite to remove hydrogen peroxide. [Figure 7B] FIG. 1 shows the results of evaluation of the ability of a cerium oxide nanocomposite to remove hydroxyl radicals. [Figure 8] In the above examples, 10 SD rats were tested at doses of 0.001 to 0.05 mg / kg, 20 at doses of 0.1 to 0.6 mg / kg, and 20 SD rats were tested in the control group, and the survival rates were expressed as average values. [Figure 9] 1 is a graph showing the average survival rate in an experiment using 10 SD rats for the particles of the present invention, a positive control group, and a negative control group. [Figure 10] 1 is a graph showing the average survival rates in an experiment using 10 C57BL / 6 mice treated with the particles of the present invention, a positive control group, and a negative control group. [Figure 11] 1 is a graph showing the results of evaluating toxic stimulation associated with reactive oxygen species after treating hepatocytes (heap-1c1c7) in which toxic stimulation was induced by pyrogallol with particles of the present invention at different concentrations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are merely intended to more specifically illustrate the present invention, and that the scope of the present invention is not limited to these examples according to the gist of the present invention. Working Example

[0035] Example 1: Synthesis of cerium oxide nanocomposite A first solution was prepared by dissolving 6-aminohexanoic acid (6-AHA) (0.65585 g, Sigma-Aldrich, St. Louis, MO) in deionized water (30 mL). A second solution prepared by dissolving polyvinylpyrrolidone (PVP) (2.0 g, Sigma-Aldrich, St. Louis, MO) in ethyl alcohol (25 mL) was added to the first solution while stirring, and the mixture was heated to 70 °C in air to prepare a third solution. Meanwhile, a fourth solution was prepared by dissolving cerium (III) nitrate hexahydrate (Ce(NO3)3·6H2O, 0.540 g, Alfa Aeser, Ward Hill, MA) in ethyl alcohol (50 mL) at room temperature (about 20 °C). The fourth solution was then added to the third solution to prepare a fifth solution. The temperature of the fifth solution was then maintained at 70°C for 2 hours and then cooled to room temperature (approximately 20°C). Through this process, cerium oxide nanoparticles with 6-aminohexanoic acid and polyvinylpyrrolidone bound to their surfaces were obtained (Figure 1). The cerium oxide nanoparticles were then washed three times with acetone to remove unreacted materials.

[0036] Example 2: Synthesis trends of cerium oxide nanocomposite with solvent In order to understand the tendency of particles to form depending on the synthesis solvent, the formation state of the cerium oxide nanocomposite was confirmed by using a 100% aqueous solvent or a 70% ethyl alcohol solvent. #100% water-based solvent: A first solution was prepared by dissolving 6-aminohexanoic acid (0.65585 g, Sigma-Aldrich, St. Louis, MO) in deionized water (30 mL). A second solution was prepared by dissolving polyvinylpyrrolidone (2.0 g, Sigma-Aldrich, St. Louis, MO) in deionized water (25 mL) while stirring the first solution, and the mixture was heated to 70 °C in air to prepare a third solution. Meanwhile, a fourth solution was prepared by dissolving cerium (III) nitrate hexahydrate (Ce(NO3)3·6H2O, 0.540 g, Alfa Aeser, Ward Hill, MA) in deionized water (50 mL) at room temperature (about 20 °C). The fourth solution was then added to the third solution to prepare a fifth solution. The temperature of the fifth solution was then maintained at 70° C. for 2 hours and then cooled to room temperature (about 20° C.) to obtain cerium oxide nanoparticles having 6-aminohexanoic acid and polyvinylpyrrolidone bound to their surfaces. The cerium oxide nanoparticles were then washed three times with acetone to remove unreacted materials. #70% ethyl alcohol solvent Obtained in the same manner as in Example 1. Transmission electron microscope analysis showed that the cerium oxide nanocomposite synthesized in a 100% aqueous solvent showed a spider web-like tangled aggregated form, making it difficult to confirm the individual dispersibility of the particles, whereas when a 70% ethyl alcohol solvent was used, high particle dispersibility was confirmed (Figure 2).This shows that alcohol solvents are more advantageous than aqueous solvents in terms of particle reaction speed and stable dispersibility.

[0037] Example 3: Structuring of cerium oxide nanocomposites by components of synthesis reactants In order to confirm the structural change of the nanocomposite depending on the presence or absence of 6-aminohexanoic acid and polyvinylpyrrolidone during the synthesis process, the experiment was carried out by sequentially removing 6-AHA and PVP from the synthesis process of Example 1, and finally including all additives. The particle size was analyzed using a dynamic light scattering device. As a result, it was confirmed that in the absence of 6-AHA, no particles were formed, and in the absence of PVP, very large particles of over 100 nm were formed, whereas uniform particles of 10 nm were synthesized only when both 6-AHA and PVP were added to the synthesis (Figure 3).This confirmed that the two compounds, 6-AHA and PVP, participate in the synthesis in a complementary manner, and that when the two compounds are applied simultaneously, a nanocomposite with stable size and dispersity as a biomaterial can be efficiently obtained.

[0038] Example 4: Change in nanocomposite formation depending on PVP content In the present invention, in order to confirm the tendency of nanocomposite formation depending on the content of PVP, which plays the role of a dispersion stabilizer, various contents of PVP, 1.0, 1.5, 2.0 and 2.5g, were used in the synthesis process of Example 1 to examine the change. As a result of analyzing the particle size synthesized by dynamic light scattering analysis, the smallest and most uniform particles were obtained when PVP was used at a dose of 2.0g (19mg / ml). Considering the effect of the size of the nanocomposite on dispersion, function and biological stability, this can be said to be the result of searching for the most suitable synthesis conditions.

[0039] Example 5: Size formation tendency of cerium oxide nanocomposite with synthesis time In order to understand the particle size and stability depending on the synthesis maintenance time of the cerium oxide nanocomposite synthesized in Example 1, samples were taken every 30, 40, 50, 60, 70, 80, 90, and 120 minutes while maintaining the reaction of the fifth solution, and the particle size was analyzed using a dynamic light scattering device. As a result, it was confirmed that the particle size gradually decreased from 30 nm to the 5 nm level as the reaction time passed, and that the samples taken at each time had a very stable particle size, thereby confirming that the method of the present invention is a process that can produce nanoparticles with extremely excellent uniformity (FIG. 5).

[0040] Example 6: Biodispersion Stabilizer Coating of Cerium Oxide Nanocomposite A suspension was prepared by adding 3.5 mg of the cerium oxide nanoparticles prepared by the above process to 0.8 mL of sodium acetate 2.5 mM buffer. The suspension was mixed with 0.3 μmol of polyglutamic acid (PLGA) (weight average molecular weight: 9,000) dissolved in 1.2 mL of sodium acetate buffer, or the same weight of polyvinyl alcohol (PVA) (weight average molecular weight: 9,500) and dextran (weight average molecular weight: 6,000). The mixture was stirred at room temperature for 5 minutes to electrostatically bind the positive charge of 6-AHA bound to the surface of the cerium oxide nanoparticles with the surface negative charge of the polyglutamic acid, polyvinyl alcohol, dextran, etc. to obtain a cerium oxide nanocomposite.

[0041] The cerium oxide nanocomposite had a surface charge of 5 mV before coating with the biocompatible dispersion stabilizer, and after coating with PLGA, the charge was -40 mV, while PVA and dextran had charge values ​​close to -1 mV, confirming that different biocompatible dispersion stabilizers were coated onto the cerium oxide nanoparticles using this method (Figure 6A). In a saline (0.9% (w / w) sodium chloride aqueous solution) dispersion environment, which mimics the environment for actual biomedical applications, it was confirmed that the particle size was 50 to 100 nm before coating with the biocompatible dispersion stabilizer, and that after coating, the particle size improved to 10 to 50 nm in both environments in which each dispersion stabilizer was used (Figure 6B).

[0042] Example 7: Evaluation of the antioxidant effect of cerium oxide nanocomposites To determine the antioxidant effect of the cerium oxide nanocomposites coated with different biocompatible dispersion stabilizers, we investigated their ability to remove hydrogen peroxide (H2O2) and hydroxyl radical (·OH), which are representative reactive oxygen species. TMA Red Hydergen Peroxide / Peroxidase Assay Kit was used for the analysis, and a HORAC (hydroxyl radical antioxidant capacity) assay was used for the analysis of hydroxyl radicals. Through these analyses, it was confirmed that the cerium oxide nanocomposite not only has the ability to remove various types of reactive oxygen species, but that this ability is proportional to the particle concentration (Figure 7). In addition, as they were successful in removing reactive oxygen species despite being coated with different types of dispersion stabilizers, it was possible to confirm that the ability to remove reactive oxygen species originates from the core particles.

[0043] Experimental Example 1: Therapeutic effect of cerium oxide nanocomposite on non-infectious inflammatory diseases In order to confirm the therapeutic effect of the nanocomposite synthesized in the present invention on non-infectious inflammatory diseases, an experiment was carried out using an animal model of subarachnoid hemorrhage, which is a representative non-infectious inflammatory disease. Briefly, SD (Sprague-Dawley) rats (Coretech Co., Ltd.) were anesthetized with isoflurane, and the left middle cerebral artery was punctured with a 4-0 proline needle to induce subarachnoid hemorrhage (SAH). One hour after the induction of subarachnoid hemorrhage, the cerium oxide nanocomposite prepared in Example 7 was intravenously injected at 0.001, 0.01, 0.05, 0.1, 0.2, 0.4, and 0.6 mg / kg over 5 minutes, while the control SD rats were injected with the same volume of saline, and the SD rats were periodically checked for death until 14 days after the induction of subarachnoid hemorrhage. As a result, it was found that when the cerium oxide nanocomposite of the present invention was injected into SD rats, the mortality rate was significantly reduced compared to the control group. Specifically, while the mortality rate in the control group was 84.2%, when 0.6 mg / kg of the cerium oxide nanocomposite was injected, the mortality rate was reduced to 18.2% (Figure 8).

[0044] Experimental Example 2: Comparative effectiveness with existing research results for the treatment of non-infectious inflammatory diseases In order to compare and evaluate the therapeutic effects of the cerium oxide nanocomposite prepared in Example 7 and the nanocomposite previously developed by the present inventors (Korea Publication No. 10-2018-0043989) on inflammatory diseases, subarachnoid hemorrhage (SAH) was induced in SD rats (Coretech Co., Ltd.) in the same manner as in Experimental Example 1. One hour after inducing subarachnoid hemorrhage, the cerium oxide nanocomposite of the present invention was intravenously injected at 0.05 mg / kg over 5 minutes once each time, and as a positive control group, 0.25 mg / kg of the conventional cerium oxide nanocomposite was injected in the same manner into SD rats, and as a control group, the same volume of saline was injected into the SD rats. The SD rats were periodically checked for death until 14 days after inducing subarachnoid hemorrhage, and it was found that the cerium oxide nanocomposite of the present invention and the positive control group showed the same mortality rate (FIG. 9). Specifically, the mortality rate was 84.2% in the control group, 38.8% in the group injected with the nanocomposite of the present invention, and 40% in the positive control group. This indicates that the cerium oxide nanocomposite of the present invention exhibits a comparable survival rate even at 1 / 5 the dose of the positive control group, and thus exhibits a therapeutic effect about 5 times better than the positive control group. This experiment confirmed that the novel nanocomposite of the present invention has significantly improved therapeutic effect and stability compared to existing cerium oxide nanocomposites, and can therefore be used as an efficient composition for treating inflammatory diseases.

[0045] Experimental Example 3: Therapeutic effect of cerium oxide nanocomposite on infectious inflammatory disease - Sepsis To confirm the therapeutic effect of the nanocomposite of the present invention on infectious inflammatory diseases, an animal model of sepsis, a representative infectious inflammatory disease, was used. The CLP (cecal ligation and puncture) method was used to induce sepsis in 6-week-old male C57BL / 6 mice (Coretech Co., Ltd.). Specifically, the C57BL / 6 mice were anesthetized with isoflurane, disinfected with betadine solution (10% by weight povidone-iodine), and the skin was incised to expose the cecum. Then, the cecum was ligated at the distal part of the ileocecal valve using 6-0 silk, and the ligated cecum was perforated using a 26-gauge needle. Such perforation induces leakage of waste into the peritoneum, causing polymicrobial bacteremia and sepsis. Immediately after inducing sepsis in the C57BL / 6 mice, the cerium oxide nanocomposite of the present invention was intravenously injected at 0.1 mg / kg once, the conventional cerium oxide nanocomposite was injected at 0.25 mg / kg as a positive control group, and the same volume of saline was injected as a control group. The C57BL / 6 mice were then periodically checked for deaths up to 6 days after inducing sepsis, and it was confirmed that the cerium oxide nanocomposite of the present invention significantly reduced the mortality rate compared to the positive control group and the control group (FIG. 10). Specifically, the mortality rates of the control group and the positive control group were 60% and 80%, respectively, while the group injected with the nanocomposite of the present invention, which was administered at a dose only 0.4 times that of the positive control group, had a mortality rate of only 40%. From these results, it was confirmed that the nanocomposite of the present invention is a significantly improved technology that shows superior pharmacological effects even at low doses compared to conventionally developed nanocomposite.

[0046] Experimental Example 4: Therapeutic effect of cerium oxide nanocomposite on infectious inflammatory diseases - Inflammatory liver disease To investigate the therapeutic effect of the cerium oxide nanocomposite on inflammation-induced liver toxicity, the degree of liver toxicity caused by pyrogallol was measured. To evaluate liver toxicity, liver cells (heap-1c1c7) were treated with pyrogallol to induce toxic stimulation, and then treated with the cerium oxide nanocomposite at different concentrations for 1 hour to evaluate the toxic stimulation associated with reactive oxygen species. As shown in FIG. 11, it was confirmed that the cerium oxide nanocomposite of the present invention significantly reduced pyrogallol-induced liver toxicity in a concentration-dependent manner. In particular, it was shown that the liver toxicity in the group treated with 500 μM of the cerium oxide nanocomposite was reduced to a similar level as the negative control group, confirming that the cerium oxide nanocomposite has an excellent therapeutic effect on inflammation-induced liver toxicity. Although certain parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Cerium oxide nanocomposite including: (a) a core layer of cerium oxide nanoparticles; (b) an inner layer comprising a polymer represented by the following chemical formula 1: 【Chemistry 1】 In the above formula, R １ and R ２ are each independently hydrogen or oxygen; 【Chemistry 2】 represents a single bond or a double bond, l is 1 or 2, and m is an integer from 100 to 1000; and (c) PGA (polyglutamic acid), PASP (poly(aspartic acid)), alginate, PAA (poly(acrylic acid)), PMAA (poly(methacrylic acid)), polymethyl methacrylic acid (poly(methyl methacrylic acid)), PMA (poly(maleic acid)), PBMA (poly(butadiene / maleic acid)), PVPA (poly(vinylphosphonic acid)), PSSA (poly(styrenesulfonic acid) an outer layer comprising one or more biocompatible dispersion stabilizers selected from the group consisting of polyvinyl alcohol (PVA) and dextran.

2. The cerium oxide nanoparticles are cerium(III) oxide (Ce ２ O ３ ) nanoparticles, cerium(IV) oxide (CeO ２ 2. The nanocomposite of claim 1, wherein the nanocomposite is selected from the group consisting of: nanoparticles, ...

3. In the above formula 1, R １ is hydrogen, R ２ 2. The nanocomposite of claim 1, wherein: is oxygen and l is 1.

4. The nanocomposite of claim 1, wherein the biocompatible dispersion stabilizer contained in the outer layer is polyglutamic acid (PGA).

5. The nanocomposite of claim 4, wherein the PGA is poly L-glutamic acid (PLGA).

6. The nanocomposite of claim 1, further comprising a polyfunctional ligand represented by the following formula 2: 【Chemistry 3】 In the above formula, n is an integer from 3 to 7.

7. The nanocomposite of claim 6, wherein n is 5 in Formula 2.

8. The nanocomposite of claim 1, wherein the nanocomposite has an average particle size of 5 nm to 100 nm.

9. A composition for preventing or treating inflammation or autoimmune diseases, comprising the nanocomplex according to any one of claims 1 to 8 as an active ingredient.

10. The inflammatory or autoimmune disease may be rheumatoid arthritis, reactive arthritis, type 1 diabetes, systemic lupus erythematosus, multiple sclerosis, idiopathic pulmonary fibrosis, polymyositis, dermatomyositis, localized dermatosclerosis, systemic dermatosclerosis, inflammatory bowel disease, Sjogren's syndrome, Raynaud's phenomenon, Bechet's disease, Kawaki's disease, primary biliary sclerosis, primary sclerosing cholangitis, ulcerative colitis, or pulmonary edema. colis, Crohn's disease, psoriasis, myasthenia gravis, autoimmune vasculitis, primary vasculitis of the central nervous system, subarachnoid hemorrhage (SAH), severe cerebral infarction, intracerebral hemorrhage, hypoxic ischemic encephalopathy, 10. The composition according to claim 9, wherein the disease is one or more diseases selected from the group consisting of acute respiratory distress syndrome, acute encephalopathy, traumatic brain / spinal cord injury, acute respiratory distress syndrome, cytokine storm syndrome, sepsis and inflammatory liver disease.

11. The composition of claim 10, wherein the inflammatory or autoimmune disease is selected from the group consisting of subarachnoid hemorrhage, sepsis, cytokine storm syndrome and inflammatory liver disease.

12. The inflammatory liver diseases include viral hepatitis, toxoplasmosis, alcoholic liver disease, toxic liver disease, acute and subacute hepatic failure, liver abscess, nonspecific reactive hepatitis, liver infarction, hepatic veno-occlusive disease, injury to the liver or gallbladder, and hepatitis B.

12. The composition according to claim 10 or 11, wherein the anti-hepatitis agent is selected from the group consisting of hepatitis C, hepatitis B, hepatitis C of liver or gallbladder, and liver transplantation-related hepatitis.

13. A method for producing a cerium oxide nanocomposite comprising the steps of: (a) C １ ~C ３ Adding a cerium precursor, a polymer represented by the following formula 1, and a cross-linking compound represented by the following formula 2 to an alcohol solvent to prepare a mixed solution; 【Chemistry 4】 In the above formula 1, R １ and R ２ are each independently hydrogen or oxygen; 【Chemistry 5】 represents a single bond or a double bond, l is 1 or 2, and m is an integer of 100 to 1000; 【Chemistry 6】 In the above formula 2, n is an integer of 3 to 7; (b) sequentially heating and cooling the mixed solution to obtain cerium oxide nanoparticles; and (c) C １ ~C ３ In an alcohol solvent, cerium oxide nanoparticles and PGA (polyglutamic acid), PASP [poly(aspartic acid)], alginate, PAA [poly(acrylic acid)], PMAA [poly(methacrylic acid)], polymethyl methacrylic acid [poly(methyl methacrylic acid)], PMA [poly(maleic acid)], PBMA [poly(butadiene / maleic acid)], PVPA [poly(vinylphosphonic acid)], PSSA [poly(styrenesulfonic acid)] were mixed. and one or more biocompatible dispersion stabilizers selected from the group consisting of cellulose acetate (C10-C12-C6-1, ...

14. Said C １ ~C ３ 14. The method of claim 13, wherein the alcoholic solvent is ethanol.

15. 14. The method of claim 13, wherein the cerium precursor is one or more precursors selected from the group consisting of cerium(III) acetate hydrate, cerium(III) acetylacetonate hydrate, cerium(III) carbonate hydrate, cerium(III) fluoride, cerium(III) chloride, cerium(III) chloride heptahydrate, cerium(III) bromide, cerium(III) iodide, cerium(III) nitrate hexahydrate, cerium(III) oxalate hydrate, cerium(III) sulfate, and cerium(III) sulfate hydrate.

16. 14. The method of claim 13, wherein the heating is performed at 60 to 75°C.

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