Dispersion of cerium oxide nanoparticle-protein complex, method for eliminating active species, and method for producing dispersion of cerium oxide nanoparticle-protein complex

Abstract

The present invention addresses the problem of providing a dispersion liquid that is for a complex obtained by conjugating a protein and a cerium oxide nanoparticle, and that maintains the zeta potential and the fluid dynamic diameter of the protein. The dispersion liquid for a complex between a protein and a cerium oxide nanoparticle according to the present invention is produced by mixing a solution containing the protein with a cerium (III) salt or a solution containing cerium (III) ions, and adding an oxidizer to the resultant mixture.

Description

[Technical Field]

[0001] The present invention relates to a dispersion of a complex of cerium oxide nanoparticles and a protein, a method for producing the dispersion, and a method for scavenging active species using the dispersion. [Background technology]

[0002] Oxidative stress damages living cells and is therefore said to be related to cardiovascular diseases such as myocardial infarction and cerebral infarction, neurological diseases such as ALS and Parkinson's disease, cancer, inflammation, and aging. Because the causes of oxidative stress are reactive species (reactive oxygen species (ROS) and reactive nitrogen species (RNS)) generated in the body, antioxidant technologies that eliminate these reactive species are considered important for health management and disease prevention. Antioxidant technologies are used in various ways in daily life, such as ingesting vitamin C from food or applying astaxanthin to the skin to protect the skin and cells. These antioxidant technologies are also expected to be used in medical settings, such as by applying antioxidants to catheters to suppress inflammation or by delivering antioxidants to diseased areas through blood vessels. Therefore, it is necessary to improve biocompatibility and blood retention, and if antioxidant properties can be imparted to proteins that are abundant in the body, it is expected that they can be used as antioxidants with high biocompatibility and blood retention.

[0003] One method of imparting antioxidant properties to proteins is to combine them with antioxidants such as vitamins, but these antioxidants generally have a consumable activity and do not last long, so it is desirable to combine them with materials that have long-lasting antioxidant properties.

[0004] In contrast, cerium oxide nanoparticles (nanoceria) have antioxidant properties and are known to have catalytic activity similar to that of enzymes such as catalase and superoxide dismutase. This antioxidant property has also been reported to be effective in treating Parkinson's disease, reducing damage caused by ischemic diseases caused by cerebral infarction, and improving sperm motility. Because cerium oxide is a ceramic, it is chemically stable and has long-lasting antioxidant properties. Therefore, if cerium oxide nanoparticles can be combined with proteins, it is expected that long-lasting antioxidant properties can be imparted to proteins.

[0005] Here, Non-Patent Documents 1 and 2 disclose that when nanoceria is mixed with BSA, which is a protein, the BSA is adsorbed onto the nanoceria surface, and the amount of adsorption varies depending on the pH of the solution and the potential of the nanoceria.

[0006] Furthermore, Figure 4C of Non-Patent Document 3 discloses that the enzyme activity of nanoceria decreases when nanoceria is mixed with BSA. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Swanand Patil, Amanda Sandberg, Eric Heckert, William Self and Sudipta Seal, Biomaterials 2007, 28, 4600-4607. [Non-patent document 2] Roman Maralek, Int. J. Bioeng. and Life Sci. 2014, 8, 1346-1349. [Non-patent document 3] Biwu Liu, Zhicheng Huang, Juwen Liu, Nanoscale 2016,8, 13562-13567. Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors began investigating the possibility of obtaining a dispersion of cerium oxide nanoparticles with antioxidant properties complexed with proteins, with the goal of obtaining an antioxidant agent with high biocompatibility and blood retention. However, as shown in the comparative examples described below, when the methods described in Non-Patent Documents 1 and 2 were actually tested, aggregation of the complex occurred, resulting in a significant increase in hydrodynamic diameter. Furthermore, although aggregation could be suppressed under other conditions, the electrostatic properties of the complex, as indicated by the zeta potential, deviated significantly from those of the protein. This resulted in the problem that the complexes obtained by the methods described in Non-Patent Documents 1 and 2 lost the biocompatibility and blood retention properties that are characteristic of proteins.

[0009] Therefore, in the present invention, the objective of the present invention was to obtain a dispersion of a complex of cerium oxide nanoparticles and a protein while maintaining the hydrodynamic diameter and zeta potential of the protein. [Means for solving the problem]

[0010] To solve the above problems, the present inventors investigated methods for complexing cerium oxide nanoparticles with proteins. As a result, they discovered that by mixing a solution containing a protein with a solution containing cerium (III) ions or a cerium (III) salt, and then adding an oxidizing agent to produce a complex, it is possible to complex cerium oxide nanoparticles with proteins while maintaining the hydrodynamic diameter and zeta potential of the protein, thereby completing the present invention. The present invention is as follows.

[0011] (1) A dispersion of a complex of cerium oxide nanoparticles and protein, produced by mixing a solution containing a protein with a solution containing cerium (III) ions or a cerium (III) salt, and then adding an oxidizing agent. (2) A dispersion of a complex of cerium oxide nanoparticles and a protein according to (1), characterized in that the molecular weight of the protein is 5 kD or more and 200 kD or less. (3) A dispersion of a complex of cerium oxide nanoparticles and a protein according to (1) or (2), wherein the protein is a protein present in blood. (4) A dispersion of a complex of cerium oxide nanoparticles and a protein according to any one of (1) to (3), wherein the protein is albumin or globulin. (5) A dispersion of a complex of cerium oxide nanoparticles and a protein according to any one of (1) to (4), having a hydrodynamic diameter that is 10 times or less the hydrodynamic diameter of a solution containing the protein. (6) A dispersion of a complex of cerium oxide nanoparticles and a protein according to any one of (1) to (5), having a zeta potential at pH 7 in the range of -15 mV to +15 mV compared to the zeta potential at pH 7 of a solution containing the protein. (7) A method for eliminating active species, which comprises contacting a sample containing active species with a dispersion of a complex of cerium oxide nanoparticles and a protein according to any one of (1) to (6). (8) A method for producing a dispersion of a complex of cerium oxide nanoparticles and a protein, which comprises mixing a solution containing a protein with a solution containing cerium (III) ions or a cerium (III) salt, and then adding an oxidizing agent. [Effects of the Invention]

[0012] The dispersion of the present invention can be used as a highly biocompatible antioxidant. DETAILED DESCRIPTION OF THE INVENTION

[0013] The dispersion of the cerium oxide nanoparticle and protein complex of the present invention may be referred to herein as the dispersion of the present invention.

[0014] In the present invention, protein is a general term for a polymeric compound in which L-α-amino acids (including glycine) are linked by peptide bonds and have a molecular weight of 5 kD or more. Proteins in the present invention include natural proteins extracted and purified from natural products, recombinant proteins expressed in microorganisms such as Escherichia coli, synthetic proteins synthesized by peptide synthesis or chemical ligation, and semi-synthetic proteins in which polypeptides are added to natural proteins by ligation reactions. Proteins may also contain sugar chains, lipids, phosphate groups, etc., added during post-translational modification, or may form multimers. Furthermore, some of these may be substituted with artificial amino acids.

[0015] The protein used in the present invention is preferably a protein present in the blood, since it is required to be biocompatible and retained in the blood. Preferred examples of proteins used in the present invention include albumin and globulin. The lower limit of the molecular weight of the protein used in the present invention is preferably 5 kD or more, and more preferably 10 kD or more. The upper limit is preferably 200 kD or less, and more preferably 180 kD or less. The molecular weight range is preferably 5 kD or more and 200 kD or less, and more preferably 10 kD or more and 180 kD or less.

[0016] The dispersion of the present invention comprises a complex of cerium oxide nanoparticles composed of a mixture of Ce2O3 and CeO2 and the above-mentioned protein. The affinity for complex formation between cerium oxide and protein is presumed to involve hydrophobic interaction, hydrogen bonding, electrostatic interaction, metal chelate, or a combination thereof, but the present invention is not limited to any of these interactions.

[0017] Cerium oxide nanoparticles can be coated with polymers such as polyacrylic acid, which has carboxylic acid groups, or dextran, which has hydroxyl groups, to improve their dispersibility in solvents. Proteins contain many of the functional groups found in these polymers, either in the side chains or at the termini of amino acids, and it is believed that these functional groups form complexes with cerium oxide through the combination of these groups. Complexes of cerium oxide nanoparticles and proteins within the above molecular weight range can achieve sufficient dispersibility in solvents.

[0018] The dispersion of the present invention may contain, in addition to the above-described complex and water as a dispersion medium, other solvent components that are compatible with water. Examples of other solvent components include methanol, ethanol, propanol, isopropanol, butanol, tert-butanol, acetone, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and oligoethylene glycol. These solvent components may be contained in an amount of 50% by volume or less.

[0019] The dispersion of the present invention may contain an ionic component, such as a component that provides buffering properties, such as acetic acid, phthalic acid, succinic acid, carbonic acid, Tris(hydroxymethyl)aminomethane (Tris), 2-Morpholinoethanesulfonic acid monohydrate (MES), Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), N-(2-Acetamido)iminodiacetic acid (ADA), Piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), 2-Hydroxy-3-morpholinopropanesulfonic acid (MOPSO), N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-Morpholinopropanesulfonic acid (MOPS), N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (ADA), or N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (ADA). acid(TES), 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid(HEPES), 2-Hydroxy-N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic(TAPSO), Piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid)(POPSO), 2-Hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid(HEPSO), 3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid(HEPPS), (Tricine), N,Examples of buffering agents include N-Bis(2-hydroxyethyl)glycine(Bicine) and N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), and examples of components that do not provide buffering properties include sodium chloride and potassium chloride.

[0020] The pH of the dispersion of the present invention may be in the range of 2 to 12, preferably 4 to 10, and more preferably 5 to 8. The pH may be adjusted by adding a buffer solution, or by adding an acid such as nitric acid, sulfuric acid, or hydrochloric acid, or a base such as sodium hydroxide or potassium hydroxide.

[0021] The hydrodynamic diameter of the complex of cerium oxide nanoparticles and protein in the dispersion of the present invention is determined by measuring dynamic light scattering to derive an autocorrelation function, analyzing it using the Marquadt method, and calculating the average particle diameter from a number-transformed histogram. Dynamic light scattering is measured using an ELS-Z manufactured by Otsuka Electronics Co., Ltd. The hydrodynamic diameter of the complex in the dispersion should be no more than 10 times, and preferably no more than 5 times, the hydrodynamic diameter of the solution containing the protein.

[0022] Zeta potential is a value that represents the electrical properties of the interface of a colloid in a solvent. When a charged colloid is dispersed in a solvent, an electric double layer is formed on the colloid's surface by counterions to the surface charge of the colloid. The potential of the colloid's surface at this time is called the surface potential. Because the electric double layer is formed by the electrostatic interaction of the colloid's surface charge, ions are more strongly fixed toward the colloid. The layer of the electric double layer where counterions are more strongly fixed to the colloid's surface by electrostatic interaction is called the fixed layer, and the potential of the fixed layer is called the fixed potential. When a colloid is moved through a solvent, the fixed layer moves with the colloid. At this time, there is an interface outside the fixed layer from the colloid's perspective that moves with the colloid due to the viscosity of the solvent. This is called the slip surface or shear surface. When the potential at a point sufficiently far from the colloid is set to zero, the potential of this slip surface is defined as the zeta potential. As described above, the zeta potential varies depending on the surface charge of the colloid, and the surface charge varies due to the attachment and detachment of protons, which is dependent on the pH. Therefore, the zeta potential of the present invention is based on the value in a solution at pH 7. In addition, since the distance to the sliding surface is generally small compared to the size of the colloid, the surface of the colloid can also be approximately expressed as the sliding surface. Similarly, in the case of the dispersion used in the present invention, the surface potential of the colloid dispersed in the solvent can be considered as the zeta potential.

[0023] Zeta potential can be determined using electrokinetic phenomena such as electrophoresis, electroosmosis, backflow potential, and precipitation potential. It can be measured using a variety of methods, including microelectrophoresis, rotating grating electrophoresis, laser Doppler electrophoresis, ultrasonic vibration potential analysis, and electrodynamic acoustics. These measurements can be performed using a zeta potential measuring device. Zeta potential measuring devices are commercially available from Otsuka Electronics Co., Ltd., Malvern Instruments Ltd., Ranku Brother Ltd., PenKem Inc., and others.

[0024] Although any of the above devices can be used to measure zeta potential, laser Doppler electrophoresis is the most commonly used method, which utilizes the Doppler effect, whereby light or sound waves change frequency when they strike an object moving electrophoretically and are scattered or reflected.

[0025] When measuring the zeta potential of the dispersion of the present invention, the dispersion of the present invention can be measured as a colloidal dispersion. The dispersion may be diluted with water, or an electrolyte such as a HEPES buffer solution or a sodium chloride solution may be added before measurement. Measurement is performed by detecting scattered light or reflected light from the complex of cerium oxide nanoparticles and protein contained in the dispersion. The larger the size of the complex of cerium oxide nanoparticles and protein, the more easily it is possible to detect scattered light or reflected light at a lower concentration.

[0026] The specific conditions for measuring the zeta potential of the dispersion of the present invention by the laser Doppler method are not particularly limited, but for example, a cerium oxide nanoparticle / protein complex can be dispersed in a HEPES buffer solution (50 mM, pH 7) to a concentration of 0.1 mg / ml to 10 mg / ml, and the resulting dispersion can be placed in a measurement cell and placed in a zeta potential measurement device based on the laser Doppler electrophoresis method, at room temperature, for example, the ELS-Z from Otsuka Electronics Co., Ltd.

[0027] The zeta potential of the dispersion of the present invention may be in the range of -15 mV or more and +15 mV or less, and preferably in the range of -10 mV or more and +10 mV or less, compared to the zeta potential of the solution containing the protein at pH 7.

[0028] The dispersion of the present invention can be produced by mixing a solution containing a protein with a solution containing cerium (III) ions or a cerium (III) salt, and then adding an oxidizing agent. The method for producing the dispersion of the present invention will be described below step by step.

[0029] A protein solution can be prepared by dissolving the protein in any solvent. The solvent is preferably water or a water-compatible solvent. Specific examples of water-compatible solvents include methanol, ethanol, propanol, isopropanol, butanol, tert-butanol, acetone, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and oligoethylene glycol. These solvent components can be contained in an amount of 50% by volume or less. If the protein is difficult to dissolve, it can be dissolved by heating or ultrasonic treatment, or the salt strength can be adjusted by adding a salt such as sodium chloride or potassium chloride. In this case, the concentration of the protein solution is preferably 0.1 mg / ml or more and 10 mg / ml or less.

[0030] To mix a protein solution with a solution containing cerium(III) ions or a cerium(III) salt, you can prepare a protein solution and a solution containing cerium(III) ions separately and then mix them. Alternatively, if the protein solution is water or a water-miscible solvent, you can add the cerium(III) salt to the protein solution and then mix them. A solution containing cerium(III) ions can be prepared by dissolving a cerium(III) salt in any solvent. For example, cerium(III) nitrate hexahydrate can be used as the cerium(III) salt.

[0031] When cerium(III) nitrate hexahydrate is used as the cerium(III) salt, the mass ratio of cerium(III) nitrate hexahydrate to protein should be 0.1 or more and 100 or less, preferably 0.5 or more and 50 or less. The mixed solution is preferably mixed for 5 minutes or more until the solution becomes homogeneous.

[0032] Examples of oxidizing agents that are added after mixing a protein-containing solution with a cerium(III) ion-containing solution or a cerium(III) salt include nitric acid, potassium nitrate, hypochlorous acid, chlorous acid, chloric acid, perchloric acid, halogens, hydrogen halides, permanganates, chromic acid, dichromate, oxalic acid, hydrogen sulfide, sulfur dioxide, sodium thiosulfate, sulfuric acid, and hydrogen peroxide. Among these, hydrogen peroxide is particularly preferred. The amount of oxidizing agent added is 0.1 to 10 molar equivalents, preferably 0.5 to 2 molar equivalents, relative to the cerium(III) ion.

[0033] When a solution containing a protein is mixed with a solution containing cerium(III) ions or a cerium(III) salt and then an oxidizing agent is added, the cerium(III) ions are oxidized to cerium(IV), producing cerium oxide particles composed of a mixture of Ce2O3 and CeO2, initiating the formation of a complex between the cerium oxide nanoparticles and the protein. During this reaction, the solution turns yellow, orange, red, brown, or other colors. This coloration is due to the conversion of cerium(III) ions to cerium(IV), and the degree of coloration is determined by the ratio of cerium(III) to cerium(IV) present on the surface of the cerium oxide nanoparticles. The completion of the reaction can be determined when the color change ceases. The particle formation reaction is pH-dependent and proceeds in a weakly acidic to basic environment. As the pH becomes more acidic as the reaction progresses, the reaction solution is preferably adjusted to a pH of 5 or higher, more preferably a pH of 6 or higher, and even more preferably a pH of 7 or higher, from the time of addition of the oxidizing agent until the completion of the reaction. To adjust the pH, aqueous sodium hydroxide or aqueous ammonia can be used. The solution can be heated or cooled during the reaction, but since the protein may denature or aggregate, causing the solution to become cloudy, it is preferable to carry out the reaction within a temperature range where the solution becomes homogeneous. The reaction is usually completed within 30 minutes to 1 hour.

[0034] The dispersion of the present invention may be stored as the reaction solution after the completion of the reaction to form the complex, or may be purified after the reaction and then stored. When storing, refrigeration is preferred, and glycerol or the like may be added and then frozen. When purifying the dispersion of the present invention, the dispersion after the completion of the reaction may be filtered using an ultrafiltration membrane or dialyzed using a semipermeable membrane to remove unreacted oxidizing agent and cerium (III) ions remaining in the dispersion after the completion of the reaction. The dispersion of the present invention may be sterilized before use, for example by passing it through a sterilizing filter.

[0035] The dispersion of the present invention can be used to eliminate reactive species contained in a sample. In this specification, eliminating reactive species means that they lose their reactivity and become harmless to living organisms. Examples of reactive species that can be eliminated by the dispersion of the present invention include reactive oxygen species (ROS) and reactive nitrogen species (RNS) present in living organisms. Specifically, for example, superoxide anion (·O2 - ), hydroxyl radical (HO·), hydroperoxyl radical (HOO·), singlet oxygen (1O2), hydrogen peroxide (H2O2), nitric oxide (NO), peroxynitrite anion (ONOO·), nitrogen dioxide radical (·NO2), etc. Detoxification can be achieved, for example, by decomposing hydrogen peroxide, one of the active species, into water and oxygen, or by converting it into superoxide anion (·O2 - This refers to the conversion of oxygen radicals (H2O) to oxygen molecules and hydroxyl radicals (HO⋅) to hydroxide ions. Any sample containing these active species can be used for scavenging. This performance can be evaluated using catalase activity, which will be described later.

[0036] Catalase activity can be determined using Thermo Fisher Scientific's AmplexRed Catalase Assay Kit (A22180) according to the protocol, as described in JP-A 2018-508568. The reaction buffer included in the kit, the dispersion of the present invention, and an aqueous hydrogen peroxide solution are mixed and left to stand for 30 minutes to allow the hydrogen peroxide to decompose. The reaction solution is passed through a 30 kD ultrafiltration membrane, and the flow-through solution is mixed with the working solution included in the kit and reacted at 37°C for 30 minutes. The resorufin produced by the reaction is excited at 544 nm, and the fluorescence intensity at 590 nm is measured. The catalase activity of the dispersion of the present invention is calculated by comparing it with a calibration curve prepared using a catalase standard with known activity included in the kit. Alternatively, the EnzyChrom Catalase Assay Kit from BioAssay Systems can also be used to measure catalase activity.

[0037] When using the dispersion of the present invention to eliminate active species, the dispersion of the present invention after the completion of the above-mentioned complex formation reaction may be brought into contact with a sample containing active species, or the dispersion purified as described above may be brought into contact. As a method of contact, if the sample containing active species is liquid, the dispersion of the present invention may be added and mixed, or the dispersion of the present invention may be atomized and sprayed. If the sample is solid, the dispersion of the present invention may be added and brought into contact. The dispersion may be added during molding of fibers, tubes, beads, rubber, films, plastics, etc., or may be applied to the surface of these materials and then brought into contact with the sample containing active species.

[0038] The dispersion of the present invention can be used as an antioxidant in human or veterinary medicines for treating oxidative stress and inflammation. Specifically, the dispersion of the present invention can be administered to a subject by injection or infusion. It can also be atomized as an aerosol and administered to a subject via the respiratory tract using an inhaler. Alternatively, it can be processed into creams, lotions, or ointments using fats, fatty oils, lanolin, petrolatum, paraffin, wax, resins, plastics, glycols, higher alcohols, glycerin, water, emulsifiers, suspending agents, etc. as bases and administered to a subject via the skin. Furthermore, it can be combined with excipients and processed into gels, tablets, enteric-coated tablets, powders, suppositories, or lozenges for oral or enteral administration to a subject. Administration by the above methods can be used to prevent and treat oxidative stress-related diseases such as stroke, multiple sclerosis, amyotrophic lateral sclerosis, and ischemia-reperfusion injury.

[0039] The dispersion of the present invention can also be used as an antioxidant to coat the surfaces of medical devices such as cannulas, catheters, and stents, and artificial organs such as dialysis membranes, thereby reducing inflammation locally or systemically.

[0040] The dispersion of the present invention can be used as an antioxidant to protect skin and cells. Specifically, it can be administered to the skin by the above-mentioned methods, added to a cell culture medium, or applied to a culture vessel such as a petri dish.

[0041] The dispersion of the present invention can be used as a substitute for conventional antioxidant enzyme solutions. Specifically, as a substitute for catalase solutions, it can be used in hydrogen peroxide detection reactions and electrochemical detection reactions by coating electrodes with cerium oxide nanoparticles. It can also be used as a neutralizing solution for hydrogen peroxide used in industries such as food, semiconductor, textile, and pulp and paper manufacturing, as well as for sterilizing public baths and removing slime from pipes.

[0042] Alternatively, the dispersions of the present invention can be added to oils, detergents, foods, and animal feeds as antioxidants.

[0043] A preferred embodiment of the dispersant of the present invention is a dispersion of a complex of a protein and cerium oxide nanoparticles, wherein the hydrodynamic diameter of the complex is 10 times or less the hydrodynamic diameter of a solution containing the protein, and the zeta potential at pH 7 is in the range of -15 mV or more and +15 mV or less compared to the zeta potential at pH 7 of the solution containing the protein.

[0044] Furthermore, one preferred embodiment of the dispersant of the present invention is a dispersion of a complex of a protein and cerium oxide nanoparticles, which exhibits a catalase activity of 1.0 U / ml or more in the decomposition reaction of aqueous hydrogen peroxide using Thermo Fisher Scientific's AmplexRed Catalase Assay Kit (A22180) at a cerium oxide nanoparticle concentration of 100 μg / ml. The catalase activity of 1.0 U / ml or more in the decomposition reaction of aqueous hydrogen peroxide using the AmplexRed Catalase Assay Kit (A22180) enables the dispersant to be used as an antioxidant. A catalase activity of 1.2 U / ml or more is particularly preferred. [Example]

[0045] The present invention will be further illustrated by the following examples. Materials and Methods Bovine serum albumin (BSA), ovalbumin (OVA), human serum-derived immunoglobulin (IgG), and the commercially available nanoceria dispersion (particle size <5 nm) used in the comparative examples were obtained from Merck Ltd., and cerium (III) nitrate hexahydrate and 30% by mass hydrogen peroxide solution were obtained from Fujifilm Wako Pure Chemical Corporation. Other reagents were purchased from Fujifilm Wako Pure Chemical Industries, Ltd., Tokyo Chemical Industry Co., Ltd., and Sigma-Aldrich Japan LLC, and were used as is without further purification. The catalase activity was measured using the AmplexRed Catalase Assay Kit (A22180) from Thermo Fisher Scientific. The hydrodynamic diameter of the cerium oxide nanoparticle-protein complex was measured using the Otsuka Electronics Co., Ltd. Zeta Potential and Particle Measurement System ELS-Z.

[0046] (Reference Example 1) Measurement of hydrodynamic diameter and zeta potential of commercially available nanoceria aqueous solution, BSA aqueous solution, and OVA aqueous solution A commercially available nanoceria aqueous solution was diluted into 25 mM HEPES buffer (pH 7), and the hydrodynamic diameter and zeta potential were measured. BSA and OVA were dissolved in 25 mM HEPES buffer (pH 7), and the hydrodynamic diameter and zeta potential were measured. The results are shown in Table 1.

[0047] [Table 1]

[0048] (Comparative Examples 1 to 3) Preparation of a dispersion of a composite of cerium oxide nanoparticles and BSA To 1 ml of a 5% by mass BSA solution, 1 μl (mass ratio BSA / CeO2 = 25; Comparative Example 1), 10 μl (mass ratio BSA / CeO2 = 2.5; Comparative Example 2), or 100 μl (mass ratio BSA / CeO2 = 0.25; Comparative Example 3) of a 20% by mass commercially available nanoceria solution was added and allowed to stand at room temperature for 2 hours. After the reaction was completed, the reaction solution was concentrated and purified using a 30 kD ultrafiltration membrane, and 25 mM HEPES buffer (pH 7) was added to make the total volume 1 ml. The hydrodynamic diameter and zeta potential were measured. The results are shown in Table 2. The results showed that the hydrodynamic diameter of the dispersion prepared by simply mixing cerium oxide nanoparticles with BSA was significantly larger than that of the BSA solution, and the zeta potential of the dispersion was significantly outside the range of -15 mV to +15 mV of the zeta potential of the BSA solution at pH 7.

[0049] [Table 2]

[0050] (Examples 1 to 4) Preparation of dispersion of cerium oxide nanoparticles and BSA complex Four glass test tubes were filled with 10 ml of a 5% by weight BSA solution, and 100 μl (mass ratio BSA / Ce(NO)·6H2O = 5; Example 1), 200 μl (mass ratio BSA / Ce(NO)·6H2O = 2.5; Example 2), 400 μl (mass ratio BSA / Ce(NO)·6H2O = 1.25; Example 3), and 800 μl (mass ratio BSA / Ce(NO)·6H2O = 0.625; Example 4) of a 10% by weight commercially available cerium(III) nitrate hexahydrate solution were added. 100 μl, 200 μl, 400 μl, and 800 μl of a 1.2% by weight hydrogen peroxide solution were added to each test tube, and the mixture was stirred at room temperature for 1 hour to obtain yellow dispersions. The dispersion was concentrated and purified using a 30 kD ultrafiltration membrane, and 25 mM HEPES buffer (pH 7) was added to make the total volume 1 ml, and the hydrodynamic diameter and zeta potential were measured. The results are shown in Table 3. These results show that the hydrodynamic diameter of the dispersion of the composite of cerium oxide nanoparticles and BSA prepared above is less than 10 times that of the BSA solution (Reference Example 1, Table 1).The zeta potential of the dispersion of the composite prepared was also found to be in the range of -15 mV to +15 mV of the zeta potential of the BSA solution at pH 7.

[0051] [Table 3]

[0052] (Measurement of catalase activity of dispersions of Examples 1 to 4) The catalase activity of each of the dispersions prepared as described above was measured. Catalase activity was measured using Thermo Fisher Scientific's AmplexRed Catalase Assay Kit (A22180) according to the protocol. Briefly, 50 μl of Reaction Buffer, 25 μl of the 400 μg / ml dispersions prepared in Examples 1 to 4, and 25 μl of 40 μM aqueous hydrogen peroxide solution were mixed and allowed to stand for 30 minutes to allow the hydrogen peroxide decomposition reaction to occur. The reaction solution was passed through a 30 kD ultrafiltration membrane, and 100 μl of the flow-through solution was mixed with 50 μl of Working Solution and reacted at 37°C for 30 minutes. Resorufin produced by the reaction was excited at 544 nm, and the fluorescence intensity at 590 nm was measured. The catalase activity of each dispersion was calculated by comparing it with a calibration curve prepared using catalase preparations with known activity. The results are shown in Table 3. From these results, it was confirmed that the dispersions prepared in Examples 1 to 4 had high catalase activity.

[0053] (Examples 5 to 8) Preparation of dispersion of cerium oxide nanoparticles and OVA composite The protein BSA used in Examples 1 to 4 was changed to OVA, and the amounts of cerium (III) nitrate hexahydrate and hydrogen peroxide used were appropriately changed to prepare a dispersion of a complex with OVA. Four glass test tubes were filled with 10 ml of a 5% by weight aqueous solution of OVA, and 25 μl (mass ratio of OVA / Ce(NO)·6H2O = 20; Example 5), 50 μl (mass ratio of OVA / Ce(NO)·6H2O = 10; Example 6), 100 μl (mass ratio of OVA / Ce(NO)·6H2O = 5; Example 7), and 200 μl (mass ratio of OVA / Ce(NO)·6H2O = 2.5; Example 8) of a 10% by weight commercially available aqueous solution of cerium(III) nitrate hexahydrate was added. 25 μl, 50 μl, 100 μl, and 200 μl of a 1.2% aqueous solution of hydrogen peroxide were added to each test tube, and the mixture was stirred at room temperature for 1 hour to obtain yellow dispersions. The dispersion was concentrated and purified using a 30 kD ultrafiltration membrane, and 25 mM HEPES buffer (pH 7) was added to make the total volume 1 ml, and the hydrodynamic diameter and zeta potential were measured. The results are shown in Table 4. The results showed that the hydrodynamic diameter of the dispersion of the composite of cerium oxide nanoparticles and OVA prepared above was 10 times or less the hydrodynamic diameter of the OVA solution (Reference Example 1, Table 1).The zeta potential of the dispersion of the prepared composite was found to be in the range of -15 mV to +15 mV of the zeta potential of the OVA solution at pH 7.

[0054] [Table 4]

[0055] (Measurement of catalase activity of dispersions of Examples 5 to 8) The catalase activity of each dispersion liquid prepared as above was measured in the same manner as in Examples 1 to 4. The results are shown in Table 4. From these results, it was confirmed that the dispersions prepared in Examples 5 to 8 had high catalase activity.

[0056] (Reference Example 2) Measurement of hydrodynamic diameter and zeta potential of IgG aqueous solution The IgG was dissolved in 25 mM HEPES buffer (pH 7), and the hydrodynamic diameter and zeta potential were measured in the same manner as in Reference Example 1. The results are shown in Table 5.

[0057] [Table 5]

[0058] (Examples 9 to 12) Preparation of dispersion of cerium oxide nanoparticles and IgG complex The protein BSA used in Examples 1 to 4 was changed to IgG, and the amounts of cerium (III) nitrate hexahydrate and hydrogen peroxide used were appropriately changed to prepare a dispersion of a complex with IgG. Four glass test tubes were charged with 10 ml of a 5% by mass IgG aqueous solution, and 25 μl (mass ratio IgG / Ce(NO)·6H2O = 20; Example 9), 50 μl (mass ratio IgG / Ce(NO)·6H2O = 10; Example 10), 100 μl (mass ratio IgG / Ce(NO)·6H2O = 5; Example 11), and 200 μl (mass ratio IgG / Ce(NO)·6H2O = 2.5; Example 12) of a 10% by mass commercially available cerium(III) nitrate hexahydrate aqueous solution were added. 25 μl, 50 μl, 100 μl, and 200 μl of a 1.2% hydrogen peroxide aqueous solution were added to each test tube, and the mixture was stirred at room temperature for 1 hour to obtain yellow dispersions. The dispersion was concentrated and purified using a 30 kD ultrafiltration membrane, and 25 mM HEPES buffer (pH 7) was added to make the total volume 1 ml, and the hydrodynamic diameter and zeta potential were measured. The results are shown in Table 6. These results show that the hydrodynamic diameter of the dispersion of the cerium oxide nanoparticle / IgG complex prepared above was 10 times or less the hydrodynamic diameter of the IgG solution (Reference Example 2, Table 5).The zeta potential of the dispersion of the prepared complex was found to be in the range of -15 mV to +15 mV of the zeta potential of the IgG solution at pH 7.

[0059] [Table 6]

[0060] (Measurement of catalase activity of dispersions of Examples 9 to 12) The catalase activity of each dispersion liquid prepared as above was measured in the same manner as in Examples 1 to 4. The results are shown in Table 6. From these results, it was confirmed that the dispersions prepared in Examples 9 to 12 had high catalase activity.

Claims

1. A dispersion of a complex of cerium oxide nanoparticles and a protein, which is produced by mixing a solution containing a protein with a solution containing cerium (III) ions or a cerium (III) salt, and then adding an oxidizing agent, The oxidizing agent is hydrogen peroxide. Dispersion of cerium oxide nanoparticles and protein complexes.

2. 2. The dispersion of a complex of cerium oxide nanoparticles and a protein according to claim 1, wherein the molecular weight of the protein is 5 kD or more and 200 kD or less.

3. 3. The dispersion of a complex of cerium oxide nanoparticles and a protein according to claim 1, wherein the protein is a protein present in blood.

4. 4. The dispersion of a complex of cerium oxide nanoparticles and a protein according to claim 1, wherein the protein is albumin or globulin.

5. 5. A dispersion of a complex of cerium oxide nanoparticles and a protein according to claim 1, wherein the hydrodynamic diameter is 10 times or less the hydrodynamic diameter of a solution containing the protein.

6. 6. A dispersion of a complex of cerium oxide nanoparticles and a protein according to claim 1, wherein the zeta potential at pH 7 is in the range of -15 mV to +15 mV compared to the zeta potential at pH 7 of a solution containing the protein.

7. A method for eliminating active species, comprising contacting a sample containing active species with a dispersion of a complex of cerium oxide nanoparticles and a protein according to any one of claims 1 to 6.

8. A method for producing a dispersion of a complex of cerium oxide nanoparticles and a protein, comprising mixing a solution containing a protein with a solution containing cerium (III) ions or a cerium (III) salt, and adding hydrogen peroxide as an oxidizing agent.

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