New cerium oxide nanocomposite material and its application

Abstract

The present invention relates to a cerium oxide nanocomposite and a preparation method thereof, as well as a composition containing the cerium oxide nanocomposite as an active ingredient for preventing or treating inflammation or autoimmune diseases. The present invention uses a biocompatible dispersion stabilizer composed of an optimal combination, thereby maintaining the intrinsic pharmacological effects of nanoparticles and significantly improving the biomedical stability, biocompatibility and production and processing efficiency of the nanoparticles, so that the nanoparticles can be effectively used as a high-efficiency nanoparticle therapeutic composition.

Description

Technical Field

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

[0002] Nanoparticles have been used for various purposes in the field of diagnosis and therapy, and due to their nanoscale size, they exhibit new physical / chemical properties in vivo that are different from those of bulk materials. Currently, many studies have been conducted on nanoparticles, and in this regard, great efforts have been made to develop nanoparticles that exhibit optimal properties suitable for medical use by adjusting the composition, shape, size, and properties of the particles according to the purpose.

[0003] At the same time, cerium oxide is thermally stable at high temperatures, and due to its lattice structure, Ce undergoes 4+ / Ce 3+ Due to its redox reaction, cerium oxide has various uses, such as electrolytes for solid batteries, compositions for UV filters, oxygen sensors, optical devices, etc. In particular, in the medical field, cerium oxide has become a therapeutic composition for treating a variety of diseases caused by oxidative stress and inflammation due to its excellent ability to scavenge active oxygen.

[0004] Using the commonly used synthesis methods of cerium oxide nanoparticles, it is not only difficult to evenly distribute the particles at the nanometer scale, but also difficult to synthesize particles of fine size below 10nm. For example, cerium oxide nanoparticles synthesized by hydrothermal method (which is one of the methods based on water phase) usually have a size ranging from hundreds of nanometers to several micrometers, because it is difficult to accurately control the particle size, and the particles show poor dispersibility. Therefore, methods for preparing cerium oxide nanoparticles with a small average particle size and uniform particle size distribution have been proposed, but these methods are not suitable for industrial mass production because they require inefficient processes involving high temperature treatment at hundreds of degrees Celsius.

[0005] Therefore, it is necessary to develop a new method that can effectively produce cerium oxide nanoparticles with a size of 50 nm or less, uniform size distribution and good dispersibility, and can easily produce and maintain unique antioxidant effects under mild reaction conditions.

[0006] Many papers and patent documents are cited throughout this specification, and their citations are shown. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present disclosure belongs and the content of the present disclosure. Summary of the invention

[0007] Technical issues

[0008] The present inventors have made great efforts to develop excellent cerium oxide nanostructures which maintain inherent pharmacological effects while having further improved biomedical stability, biocompatibility and efficiency in the production process of nanoparticles. As a result, the present invention was completed by the following discovery: when a polymer layer (in which the monomer of the pyrrolidone derivative of Formula 1 is repeated) and a biocompatible dispersion stabilizer layer are formed in the core of cerium oxide nanoparticles, uniform and fine particles can be stably and economically mass-produced within a sufficient reaction time without applying a harsh environment such as a strong base and / or a strong acid condition at a high temperature.

[0009] Therefore, the object of the present invention is to provide a cerium oxide nanocomposite and a preparation method thereof.

[0010] Another object of the present invention is to provide a composition for preventing or treating inflammation or autoimmune diseases, which contains the cerium dioxide nanocomposite of the present invention as an active ingredient.

[0011] Other objects and advantages of the present disclosure will become more apparent from the following detailed description of the invention, claims and drawings.

[0012] Technical Solution

[0013] In one aspect, a cerium oxide nanocomposite is provided, comprising:

[0014] (a) Core layer of cerium oxide nanoparticles;

[0015] (b) an inner layer comprising a polymer represented by the following Formula 1:

[0016] [Formula 1]

[0017]

[0018] wherein R1 and R2 are each independently hydrogen or oxygen, represents a single bond or a double bond, is 1 or 2, and m is an integer from 100 to 1000; and

[0019] (c) an outer layer comprising one or more biocompatible dispersion stabilizers selected from the group consisting of polyglutamic acid (PGA), poly(aspartic acid) (PASP), alginate, poly(acrylic acid) (PAA), poly(methacrylic acid) (PMAA), poly(methyl methacrylic acid), poly(maleic acid) (PMA), poly(butadiene / maleic acid) (PBMA), poly(vinylphosphonic acid) (PVPA), poly(styrene sulfonic acid) (PSSA), polyvinyl alcohol (PVA) and dextran.

[0020] The present inventors have made great efforts to develop excellent cerium oxide nanostructures that maintain inherent pharmacological effects while having further improved biomedical stability, biocompatibility and efficiency in the production process of nanoparticles. As a result, the present inventors have found that when a polymer layer in which a monomer of a pyrrolidone derivative of Formula 1 is repeated and a biocompatible dispersion stabilizer layer are formed at the core of cerium oxide nanoparticles, uniform fine particles can be stably and economically mass-produced within a sufficient reaction time without the harsh environment of strong alkali and strong acid conditions at high temperatures.

[0021] Because in the synthesis process of cerium oxide nanoparticles, in order to ensure in vivo stability, polyethylene glycol (PEG) or PEG derivatives modified with succinimide groups are mainly used, and the related art requires a very short reaction within 1 minute using strong acids / bases such as HCl and NaOH at a high temperature of 95°C, thus encountering great difficulties in obtaining a large number of uniform particles. However, the nanocomposite of the present invention ensures stable synthesis conditions, such as a reaction time of about 2 hours at 70°C, and does not require a strong acid or strong base environment, thereby effectively overcoming the shortcomings of the related art.

[0022] As used herein, the term "core layer" refers to the innermost layer in a multi-layer composite material having only one surface in contact with the other layers.

[0023] As used herein, the term "multilayer composite material" refers to a composite material composed of a plurality of layers composed of different components, and includes but is not limited to a laminated multilayer structure, a core-shell multilayer structure, and a combination thereof. Specifically, the multilayer composite material according to the present invention has a core-shell multilayer structure, wherein nanoparticles are present in the center, and the polymer of Formula 1 and the biocompatible dispersion stabilizer surround the nanoparticles on the outer shell.

[0024] As used herein, the term "inner layer" refers to a layer closer to the core than the outer layer, and the term "outer layer" refers to an inner layer surrounding the core-shell structure and a layer further away 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 extra layer closer to the core layer than the inner layer. The outer layer also does not necessarily have to be the outermost layer, and the outermost layer further away from the core than the outer layer may further exist.

[0025] According to the present invention, the boundary between the core layer and the inner layer is clearly distinguished, but the boundary between the inner layer and the outer layer may be clearly distinguished or may not be clearly distinguished. If the boundary between the inner layer and the outer layer is not clear, each component of the inner layer and the outer layer may be mixed near the interface or in the entire part.

[0026] As used herein, the term "polymer" refers to a synthetic or natural polymer compound in which monomers of the same or different types are continuously linked. Therefore, the polymer includes homopolymers (polymers in which one monomer is polymerized) and interpolymers prepared by polymerizing at least two different monomers, wherein the interpolymers include copolymers (polymers prepared from two different monomers) and polymers prepared from two or more different monomers. Specifically, the polymer of Formula 1 used in the present invention is a homopolymer.

[0027] As used herein, the term "alkyl" refers to a linear or branched saturated hydrocarbon group, and includes, for example, methyl, ethyl, propyl, isopropyl, etc. C1-C3 alkyl refers to an alkyl group having an alkyl unit of 1 to 3 carbon atoms, and when the C1-C3 alkyl group is substituted, the number of carbon atoms in the substituent is not included.

[0028] As used herein, the term "biocompatibility" refers to a property of not causing short-term or long-term side effects when administered in vivo and in contact with cells, tissues or body fluids of an organ, and specifically, refers not only to tissue compatibility and blood compatibility that do not cause tissue necrosis or blood coagulation in contact with biological tissues or blood, but also to biodegradability, i.e., the property of disappearing after a certain period of time after administration of the substance in vivo, and "excretibility, which is a property of being excreted from the body without accumulation after administration of the substance in vivo". Therefore, the term "biocompatible dispersion stabilizer" refers to a component that improves the dispersibility of particles while having the above-mentioned biocompatibility.

[0029] As used herein, the term "biodegradability" refers to the property of a material to decompose naturally when exposed to a physiological solution with a pH of 6 to 8, and specifically, refers to the property of a material to be decomposed over time in the body by body fluids, decomposing enzymes or microorganisms.

[0030] According to a specific embodiment of the present invention, the cerium oxide nanoparticles used in the present invention can be cerium (IV) oxide (CeO2), cerium (III) oxide (Ce 23 ) or a mixture thereof.

[0031] According to a specific embodiment of the present invention, in Formula 1, R1 is hydrogen, R2 is oxygen, is 1. According to the octet rule, it is obvious that when R1 is hydrogen, is a single bond. When R2 is oxygen, A compound of formula 1, wherein R1 is hydrogen, R2 is oxygen, and It is 1, which is polyvinylpyrrolidone (PVP).

[0032] According to a specific embodiment of the present invention, the biocompatible dispersion stabilizer contained in the outer layer is polyglutamic acid (PGA). Polyglutamic acid (PGA) can be poly-α-glutamic acid, poly-β-glutamic acid or poly-γ-glutamic acid, in particular poly-α-glutamic acid. Most specifically, poly-α-glutamic acid is poly (L-glutamic acid) (PLGA).

[0033] According to one embodiment of the present invention, the nanocomposite according to the present invention further comprises a multifunctional ligand represented by the following formula 2:

[0034] [Formula 2]

[0035]

[0036] Where n is an integer from 3 to 7.

[0037] As used herein, the term "multifunctional 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 multifunctional 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, PVP or dextran in the outer layer, so that the nanocomposite according to the present invention can be formed more effectively and stably.

[0038] Specifically, in the above Formula 2, n is 5. The compound of Formula 2 in which n is 5 is 6-aminohexanoic acid (6-AHA).

[0039] According to one 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.

[0040] According to another aspect of the present invention, the present invention provides a composition for preventing or treating inflammation or autoimmune diseases, which comprises the nanocomplex of the present invention as an active ingredient.

[0041] The cerium oxide nanocomposite used in the present invention has been described above, and thus a description thereof is omitted to avoid excessive overlap.

[0042] As used herein, the term "preventing" or "preventing" refers to inhibiting the development of a disease or condition in a subject who has not yet been diagnosed with the disease or condition but who is at risk of developing such a disease or condition.

[0043] As used herein, the term "treatment" refers to (a) inhibiting the progression of a disease, condition or symptom; (b) alleviating a disease, condition or symptom; or (c) eliminating a disease, condition or symptom. The composition according to the present invention reduces reactive oxygen and inhibits the expression of inflammatory cytokines to inhibit, eliminate or alleviate the development of symptoms caused by excessive or undesirable immune responses or inflammation. Therefore, the composition according to the present invention itself can be a composition for treating these diseases, or it can be administered in combination with other pharmacological ingredients having anti-inflammatory effects and administered as a therapeutic adjuvant for the above-mentioned diseases. Therefore, as used herein, the term "treatment" or "therapeutic agent" includes the meaning of "therapeutic adjuvant" or "therapeutic adjuvant".

[0044] As used herein, the term "administering" means directly administering a therapeutically effective amount of the composition of the present invention to a subject, thereby forming the same amount in the subject, and has the same meaning as "transplantation" or "injection".

[0045] As used herein, the term "therapeutically effective amount" refers to an amount of the contained composition sufficient to provide a therapeutic or prophylactic effect to an individual to whom the composition of the present invention is desired to be administered, and is therefore meant to include a "prophylactically effective amount."

[0046] As used herein, the term "subject" includes, but is not limited to, humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons or rhesus monkeys. Specifically, as used herein, the subject is a human.

[0047] According to a specific embodiment of the present invention, the inflammatory or autoimmune diseases prevented or treated with the composition of the present invention are one or more diseases selected from the following group: rheumatoid arthritis, reactive arthritis, type 1 diabetes, systemic lupus erythematosus, multiple sclerosis, idiopathic pulmonary fibrosis, polymyositis, dermatomyositis, localized scleroderma, systemic scleroderma, inflammatory bowel disease, Sjögren's syndrome, Raynaud's phenomenon, Behcet's disease, Kawasaki disease, primary biliary sclerosis, primary sclerosing cholangitis, ulcerative colitis, Crohn's disease, psoriasis, myasthenia gravis, autoimmune vasculitis, primary vasculitis of the central nervous system, subarachnoid hemorrhage (SAH), severe cerebral infarction, cerebral hemorrhage, hypoxic-ischemic encephalopathy, traumatic brain / spinal cord injury, acute respiratory distress syndrome, cytokine storm syndrome, sepsis and inflammatory liver disease. More specifically, the inflammatory or autoimmune disease is selected from subarachnoid hemorrhage, sepsis, cytokine storm syndrome and inflammatory liver disease.

[0048] As used herein, the term "inflammatory liver disease" refers to a series of diseases accompanied by damage to liver tissue caused by excessive or unwanted immune or inflammatory responses induced by various causes as direct or indirect causes.

[0049] More specifically, the inflammatory liver disease is selected from viral hepatitis, toxoplasmic hepatitis, alcoholic liver disease, toxic liver disease, acute and subacute liver failure, liver abscess, nonspecific reactive hepatitis, liver infarction, hepatic veno-occlusive disease, liver or gallbladder injury, and liver transplant-related hepatitis.

[0050] When being prepared as pharmaceutical composition according to the composition of the present invention, pharmaceutical composition according to the present invention comprises pharmaceutically acceptable carrier.The pharmaceutically acceptable carrier comprised in the pharmaceutical composition according to the present invention is generally used in preparation, and its example includes but is not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum, magnesium stearate, mineral oil etc. Pharmaceutical composition according to the present invention can also contain lubricant, wetting agent, sweetener, flavoring agent, emulsifier, suspending agent, preservative etc. except above-mentioned components.Suitable pharmaceutically acceptable carrier and preparation are described in detail in Leimington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences) (19th edition, 1995).

[0051] The pharmaceutical compositions according to the present disclosure can be administered by various routes of administration, specifically, parenteral, more specifically, oral, intravenous, intraarterial, subcutaneous, intraperitoneal, intradermal, intramuscular, intraventricular, intrathecal, inhalation, intranasal, intraarticular or topical.

[0052] The appropriate dosage of the pharmaceutical composition of the present invention can be specified differently according to factors such as the preparation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, reaction sensitivity, etc. For adults, the preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.0001 to 100 mg / kg.

[0053] The pharmaceutical composition of the present invention can be prepared into unit dosage form by preparing with pharmaceutically acceptable carrier and / or excipient, or can be prepared by introducing into a multi-dose container according to the method that can be easily implemented by those of ordinary skill in the art to which the present invention belongs. In this case, the preparation can be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or can also be in the form of an extract, powder, granule, tablet or capsule, and can additionally include a dispersant or stabilizer.

[0054] According to another aspect of the present invention, the present invention provides a method for preventing or treating inflammation or autoimmune diseases, the method comprising administering to a subject the cerium oxide nanocomposite of the present invention or a pharmaceutical composition containing the nanocomposite as an active ingredient. The cerium oxide nanocomposite used in the present disclosure and the inflammation or autoimmune diseases that can be prevented or treated by the cerium oxide nanocomposite have been described above, so their description will be omitted to avoid excessive overlap.

[0055] According to another aspect of the present invention, there is provided a method for preparing a cerium oxide nanocomposite, comprising:

[0056] (a) preparing a mixed solution by adding a cerium precursor, a polymer represented by the following Formula 1, and a cross-linking compound represented by the following Formula 2 to a C1-C3 alcohol solvent;

[0057] [Formula 1]

[0058]

[0059] wherein R1 and R2 are each independently hydrogen or oxygen, represents a single bond or a double bond, is 1 or 2, and m is an integer from 100 to 1000;

[0060] [Formula 2]

[0061]

[0062] Wherein, n is an integer from 3 to 7;

[0063] (b) sequentially heating and cooling the mixed solution to obtain cerium oxide nanoparticles; and

[0064] (c) mixing cerium oxide nanoparticles and one or more biocompatible dispersion stabilizers selected from the following group: polyglutamic acid (PGA), poly(aspartic acid) (PASP), alginate, poly(acrylic acid) (PAA), poly(methacrylic acid) (PMAA), poly(methyl methacrylic acid), poly(maleic acid) (PMA), poly(butadiene / maleic acid) (PBMA), poly(vinylphosphonic acid) (PVPA), poly(styrene sulfonic acid) (PSSA), polyvinyl alcohol (PVA) and dextran in a C1-C3 alcohol solvent to obtain a cerium oxide nanocomposite coated with a biocompatible dispersion stabilizer.

[0065] As used herein, the term "coating" refers to forming a new layer with a certain thickness by applying a specific material on a target surface, and the target surface and the coating material can be coated by ionic bonds or non-covalent bonds. The term "non-covalent bond" is a concept that includes not only physical bonds such as adsorption, coagulation, entanglement and embedding, but also interactive bonds such as hydrogen bonds and van der Waals bonds that occur alone or in combination with the above physical bonds. When coated with a biocompatible dispersed stabilizer in the present invention, the stabilizer layer can form a closed layer while completely surrounding the surface to be modified, or can form a partial closed layer.

[0066] According to a specific embodiment of the present invention, the C1-C3 alcohol solvent is ethanol.

[0067] 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, the cerium precursor is cerium (III) nitrate hexahydrate.

[0068] According to a specific embodiment of the present invention, the heating is performed at 60-75° C. As described above, one advantage of the present invention is that it is suitable for industrial-scale batch production, because a sufficient reaction process can be stably and continuously carried out at a temperature of about 70° C., unlike the prior art which can only react at a high temperature of 95° C. for a short time.

[0069] Beneficial Effects

[0070] The features and advantages of the present invention are summarized as follows:

[0071] (a) The present disclosure provides a cerium oxide nanocomposite and a method for preparing the same, as well as a composition for preventing or treating inflammation or autoimmune diseases, the composition containing the cerium oxide nanocomposite as an active ingredient, and

[0072] (b) By applying a biocompatible dispersion stabilizer consisting of an optimal combination, the present invention can be effectively used as an effective nanoparticle therapeutic composition to significantly improve the biomedical stability, biocompatibility and efficiency of the nanoparticle production process while maintaining the intrinsic pharmacological effects of the nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 FIG. 1 is a diagram showing a transmission electron microscope image of the cerium oxide nanocomposite of the present invention.

[0074] Figure 2 The graphs show the results of analyzing the shape and dispersibility of the cerium oxide nanocomposite according to the properties of the solvent.

[0075] Figure 3 This is a graph showing the change in particle size of the cerium oxide nanocomposite depending on the presence or absence of a compound.

[0076] Figure 4 It is a graph showing the analysis results of the change in particle size according to the amount of polyvinyl pyrrolidone.

[0077] Figure 5 This is a graph showing the results of analyzing the particle size of the cerium oxide nanocomposite at each reaction time using a dynamic light scattering device.

[0078] Figure 6a The graph shows the result of detecting whether the cerium oxide nanocomposite is coated with a biocompatible dispersion stabilizer using a surface potential analysis device. Figure 6b The results of analyzing the particle size of the cerium oxide nanocomposite using a dynamic light scattering device are shown.

[0079] FIG. 7 is a graph showing the evaluation results of the hydrogen peroxide and hydroxyl radical scavenging abilities of the cerium oxide nanocomposite.

[0080] Figure 8 It is shown that 10 SD rats at 0.001 to 0.05 mg / kg and 20 SD rats at 0.1 to 0.6 mg / kg were tested in the above-mentioned respective examples, and 20 SD rats were tested in the control group, and the survival rate thereof is expressed as the mean value.

[0081] Fig. 9 It is a graph showing that 10 SD rats were tested in the particles of the present invention, a positive control group, and a negative control group, and the average value thereof was expressed as a survival rate.

[0082] Fig.10 It is a graph in which 10 C57BL / 6 mice were tested in the particles of the present invention, a positive control group, and a negative control group, and the survival rate is expressed as the mean value thereof.

[0083] Fig.11 : is a graph showing the results of evaluating toxic stimulation associated with reactive oxygen species after treating hepatocytes (heap-1c1c7) with various concentrations of the particles of the present invention, in which the toxic stimulation was induced by pyrogallol. DETAILED DESCRIPTION

[0084] Hereinafter, the present invention is described in detail with reference to the following examples. These examples are only used to illustrate the present invention in more detail, and it is obvious to those skilled in the art that according to the spirit of the present invention, the scope of the present invention is not limited by these examples.

[0085] Example

[0086] Example 1: Synthesis of Cerium Oxide Nanocomposite

[0087] 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 (in which polyvinyl pyrrolidone (PVP) (2.0 g, Sigma-Aldrich, St. Louis, MO) was dissolved in ethanol (25 mL)) was added while stirring the first solution and the second solution was heated to 70° C. in air to prepare a third solution. At the same time, a fourth solution was prepared by dissolving cerium (III) nitrate hexahydrate (Ce(NO3)3·6H2O, 0.540 g, Alfa Aesar, Ward Hill, MA) in ethanol (50 mL) at room temperature (about 20° C.). Thereafter, a fifth solution was prepared by adding the fourth solution to the third solution. Then, the temperature of the fifth solution was maintained at 70° C. for 2 hours and then cooled to room temperature (about 20° C.). Through such treatment, cerium oxide nanoparticles with 6-aminocaproic acid and polyvinyl pyrrolidone bound to the surface were obtained (see Figure 1 ). Next, the cerium oxide nanoparticles were washed three times with acetone to remove unreacted substances.

[0088] Example 2: Synthesis Trends of Cerium Oxide Nanocomposites Depending on Solvent

[0089] In order to determine the formation tendency of particles depending on the synthesis solvent, the formation state of the cerium oxide nanocomposite was confirmed by using a 100% aqueous solvent or a 70% ethanol solvent.

[0090] #100% water-based solvent:

[0091] The first solution was prepared by dissolving 6-aminocaproic acid (0.65585 g, Sigma-Aldrich, St. Louis, MO) in deionized water (30 mL). The second solution (in which polyvinyl pyrrolidone (2.0 g, Sigma-Aldrich, St. Louis, MO) was dissolved in deionized water (25 mL)) was added during the stirring of the first solution, and then the third solution was prepared by heating to 70°C in air. At the same time, the fourth solution was prepared by dissolving cerium (III) nitrate hexahydrate (Ce(NO3)3·6H2O, 0.540 g, Alfa Aesar, Ward Hill, MA) in deionized water (50 mL) at room temperature (about 20°C). Thereafter, the fifth solution was prepared by adding the fourth solution to the third solution. Then, the temperature of the fifth solution was maintained at 70°C for 2 hours, and then cooled to room temperature (about 20°C) to obtain cerium oxide nanoparticles with 6-aminocaproic acid and polyvinyl pyrrolidone bound to the surface. Next, the cerium oxide nanoparticles were washed three times with acetone to remove unreacted substances.

[0092] #70% ethanol solvent: In the same manner as in Example 1, a solvent was obtained.

[0093] The results of analyzing the transmission electron microscopy images can confirm that the cerium oxide nanocomposite synthesized with a 100% aqueous solvent has difficult to identify individual particle dispersion and is entangled with each other and aggregated like a spider web, while the cerium oxide nanocomposite synthesized with a 70% ethanol solvent has high particle dispersion (see Figure 2 ). From the above results, it was found that alcohol solvents were more advantageous than aqueous solvents in terms of reaction rate and stable dispersion of particles.

[0094] Example 3: Structural formation of cerium oxide nanocomposites depending on the composition of the synthetic reactants

[0095] To confirm that the structural changes of the nanocomposite depend on the presence or absence of 6-aminocaproic acid and polyvinylpyrrolidone during the synthesis, 6-AHA and PVP were successively excluded from the synthesis of Example 1 and finally, the experiment was performed with all additives included. Particle size was analyzed using a dynamic light scattering device.

[0096] As a result, it was confirmed that no particles were formed in the absence of 6-AHA, and particles of 100 nm or more were formed in the absence of PVP. Only when 6-AHA and PVP were added during the synthesis, uniform particles of 10 nm in size were synthesized (see Figure 3 ). Therefore, it was confirmed that when the two compounds 6-AHA and PVP were complementary to each other in the synthesis and applied simultaneously, a nanocomposite with stable size and dispersibility could be effectively obtained as a biomaterial.

[0097] Example 4: Changes in Nanocomplex Formation Depending on the Amount of PVP

[0098] In order to confirm the tendency of nanocomposite formation depending on the amount of PVP as a dispersion stabilizer in the present invention, changes in nanocomposite formation were detected by using different amounts of PVP (1.0, 1.5, 2.0 and 2.5 g) in the synthesis process of Example 1. As a result of analyzing the size of the synthesized particles by dynamic light scattering analysis, uniform particles of the smallest size can be obtained when PVP is used at a dose of 2.0 g (19 mg / ml). Considering the influence of the size of the nanocomposite on dispersibility, function and biostability, it can be said that this is the result of finding the most suitable synthesis conditions.

[0099] Example 5: Size formation trend of cerium oxide nanocomposites depending on synthesis time

[0100] In order to identify the particle size and stability of the cerium oxide nanocomposite synthesized by Example 1 for each synthesis retention time, while maintaining the reaction of the fifth solution, samples were taken every 30, 40, 50, 60, 70, 80, 90, and 120 minutes, and the particle size was analyzed using a dynamic light scattering device. The results confirmed that as the reaction time passed, the particle size gradually decreased from 30nm to 5nm, and each sample collected at each time had a very stable particle size. Therefore, it was confirmed that the method of the present disclosure is a method for producing nanoparticles with significantly excellent uniformity (see Figure 5 ).

[0101] Example 6: Coating of Cerium Oxide Nanocomposites with Biodispersible Stabilizers

[0102] A suspension was prepared by adding 3.5 mg of cerium oxide nanoparticles prepared by the above method to 0.8 mL of sodium acetate buffer (2.5 mM). The suspension was mixed with 0.3 μmol of polyglutamic acid (PLGA) (weight average molecular weight: 9,000) or the same weight of polyvinyl alcohol (PVA) (weight average molecular weight: 9,500) and dextran (weight average molecular weight: 6,000) dissolved in 1.2 mL of sodium acetate buffer. The mixture was stirred at room temperature for 5 minutes, and the positively charged 6-AHA bound to the surface of the cerium oxide nanoparticles and the negatively charged polyglutamic acid, polyvinyl alcohol, dextran, etc. on the surface were combined by electrostatic attraction to obtain a cerium oxide nanocomposite.

[0103] It was confirmed that the cerium oxide nanocomposite had a surface potential value of 5 mV before coating with a biocompatible dispersion stabilizer, and had a charge value of -40 mV after coating with PLGA, and had a charge value close to -1 mV after coating with PVA and dextran, respectively. In addition, it was also confirmed that different biocompatible dispersion stabilizers were coated on cerium oxide nanoparticles by this method (see Figure 6a ).

[0104] It was confirmed that in a dispersion environment of physiological saline (0.9% (w / w) sodium chloride aqueous solution) simulating an actual biomedical application environment, the cerium oxide nanocomposite had a size of 50 to 100 nanometers before coating with a biocompatible dispersion stabilizer, and that the dispersion size increased from 10 nm to 50 nm (0.9% (w / w) sodium chloride aqueous solution) after coating with a biocompatible dispersion stabilizer under all conditions using each dispersion stabilizer. Figure 6b ).

[0105] Example 7: Evaluation of the Antioxidant Effect of Cerium Oxide Nanocomposites

[0106] To determine the antioxidant effect of cerium oxide nanocomposites coated with different biocompatible dispersion stabilizers, the scavenging effects of representative reactive oxygen species, hydrogen peroxide (H2O2) and hydroxyl radical (-OH), were investigated. To analyze each reactive oxygen species, Amplex TM The Red Hydergen Peroxide / Peroxidase Assay Kit was used to detect hydrogen peroxide, and the hydroxyl radical antioxidant capacity (HORAC) test was used to detect hydroxyl radicals. The various analyses confirmed that the cerium oxide nanocomposite not only has the function of removing various types of reactive oxygen species, but also that the function is proportional to the concentration of the particles (see FIG. 7 ). In addition, it can be confirmed that, although the cerium oxide nanocomposite is coated with different types of dispersion stabilizers, the ability to remove reactive oxygen species can be obtained from the core particles by successfully removing reactive oxygen species.

[0107] Experimental Example 1: Therapeutic Effects of Cerium Oxide Nanocomposites on Non-infectious Inflammatory Diseases

[0108] In order to confirm the therapeutic effect of the nanocomposite synthesized by the present invention on non-infectious inflammatory diseases, an animal model of subarachnoid hemorrhage, a representative non-infectious inflammatory disease, was used for experiments. In summary, after Sprague-Dowler (SD) rats (Koatech Co., Ltd.) were anesthetized with isoflurane, the left middle cerebral artery was punctured with a 4-0 suture needle to induce subarachnoid hemorrhage (SAH). One hour after inducing subarachnoid hemorrhage, the cerium oxide nanocomposite prepared in Example 7 was intravenously injected into the SD rats at a dose of 0.001, 0.01, 0.05, 0.1, 0.2, 0.4, and 0.6 mg / kg within 5 minutes. At the same time, the same volume of normal saline was injected into the SD rats in the control group, and the SD rats were regularly confirmed to be dead from the induction of subarachnoid hemorrhage to 14 days. The results showed that the mortality rate of SD rats injected with the cerium oxide nanocomposite according to the present invention was significantly reduced compared with that of the control group. Specifically, the mortality rate of the control group was 84.2%, while the mortality rate was reduced to 18.2% when 0.6 mg / kg of the cerium oxide nanocomposite was injected (see Figure 8 ).

[0109] Experimental Example 2: Effects compared with previous research results on the treatment of non-infectious inflammatory diseases

[0110] 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 inventors (Korean Publication No. 10-2018-0043989) on inflammatory diseases, subarachnoid hemorrhage (SAH) was induced in SD rats in the same manner as in Experimental Example 1 (Koatech Co., Ltd.). One hour after the induction of subarachnoid hemorrhage, the cerium oxide nanocomposite of the present invention was intravenously injected into the SD rats at a dose of 0.05 mg / kg for 5 minutes each time. In the same manner, conventional cerium oxide nanocomposite was injected into SD rats at a dose of 0.25 mg / kg as a positive control group, and the same volume of normal saline was injected into SD rats as a control group. From the induction of subarachnoid hemorrhage to 14 days, the SD rats were regularly checked for death, and it was found that the cerium oxide nanocomposite of the present invention and the positive control group showed the same mortality rate (see Fig. 9 ). Specifically, the mortality rate of the control group was 84.2%, the mortality rate of the nanocomposite injection group of the present invention was 38.8%, and the mortality rate of the positive control group was 40%. From the above results, it was found that the cerium oxide nanocomposite of the present invention showed a comparable survival rate even at 1 / 5 of the dose of the positive control group, thereby showing a therapeutic effect about 5 times higher than that of the positive control group. This experiment confirmed that the novel nanocomposite of the present invention has significantly improved therapeutic effects and stability compared to the previous cerium oxide nanocomposite, and can therefore be used as an effective composition for treating inflammatory diseases.

[0111] Experimental Example 3: Therapeutic Effect of Cerium Oxide Nanocomposite on Infectious Inflammatory Disease-Sepsis

[0112] In order to confirm the therapeutic effect of the nanocomposite of the present invention on infectious inflammatory diseases, an animal model of sepsis, which is a representative infectious inflammatory disease, was used. The cecal ligation and puncture (CLP) method was used to induce sepsis in 6-week-old male C57BL / 6 mice (Koatech Co., Ltd.). Specifically, C57BL / 6 mice were anesthetized with isoflurane, disinfected with betadine solution (10wt% povidone iodine), and then a skin incision was made to expose the cecum. Then, the cecum was ligated at the distal end of the ileocecal valve using 6-0 silk, and then punctured using a 26-gauge needle. This perforation causes excreta to penetrate into the peritoneum, leading to microbial bacteremia and sepsis. After inducing sepsis in C57BL / 6 mice, the cerium oxide nanocomposite of the present invention was immediately injected intravenously at 0.1 mg / kg each time, and the previous cerium oxide nanocomposite 0.25 mg / kg was injected into SD rats as a positive control group, and the same volume of saline was injected into SD rats as a control group. Thereafter, by regularly checking whether C57BL / 6 mice died from the time of sepsis induction to 6 days, it was confirmed that the cerium oxide nanocomposite according to the present invention significantly reduced the mortality rate compared with the positive control group and the control group (see Fig.10 ). Specifically, the control group and the positive control had mortality rates of 60% and 80%, respectively, whereas the group of the nanocomplex of the present invention administered at a dose only 0.4 times that of the positive control group had a mortality rate of only 40%.

[0113] These results demonstrate that the nanocomplex of the present invention is a significantly improved technology compared to previously developed nanocomplexes, showing excellent pharmacological effects even at low doses.

[0114] Experimental Example 4: Therapeutic effect of cerium oxide nanocomposite on infectious inflammatory disease-inflammatory liver disease

[0115] In order to investigate the therapeutic effect of cerium oxide nanocomplexes on inflammation-induced hepatotoxicity, the extent of hepatotoxicity induced by pyrogallol was measured. To evaluate hepatotoxicity, hepatocytes (heap-1c1c7) were treated with pyrogallol to induce toxic stimulation, and each concentration of cerium oxide nanocomplexes was treated for 1 hour to evaluate toxic stimulation associated with reactive oxygen species. Fig.11 As shown, it was confirmed that the cerium oxide nanocomposite of the present invention significantly reduced the hepatotoxicity induced by pyrogallol in a concentration-dependent manner. By showing that the hepatotoxicity of the group treated with 500 μm of the cerium oxide nanocomposite was reduced to a level similar to that of the negative control group, it was confirmed that the cerium oxide nanocomposite has an excellent therapeutic effect on hepatocellular toxicity caused by inflammation.

[0116] The specific parts of the present invention have been described in detail above, but it is obvious to those skilled in the art that such specific description is only an exemplary embodiment, and the scope of the present invention is not limited thereto. Therefore, the essential scope of the present invention will be defined by the claims and their equivalents.

Claims

1. A cerium oxide nanocomposite comprising: (a) a core layer of cerium oxide nanoparticles, wherein the cerium oxide nanoparticles are selected from the group consisting of Ce2O3 nanoparticles, CeO2 nanoparticles and mixtures thereof; (b) an inner layer comprising a polymer represented by the following formula 1 and a multifunctional ligand represented by the following formula 2: Where R1 is hydrogen and R2 is oxygen, represents a single bond or a double bond, is 1, and m is an integer from 100 to 1000; in n is the integer 5; and (c) The outer layer comprises polyglutamic acid (PGA); the nanocomposite has an average particle size of 5 nm to 100 nm.

2. The nanocomposite according to claim 1, characterized in that The PGA is poly-L-glutamic acid.

3. A composition for preventing or treating an inflammatory or autoimmune disease, comprising the nanocomplex according to any one of claims 1 to 2 as an active ingredient, wherein the inflammatory or autoimmune disease is selected from the group consisting of subarachnoid hemorrhage, sepsis and inflammatory liver disease.

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