WO2018235976A1 - Synthesis of ceria-zirconia solid solution nanoparticles and ceria-zirconia nanocomposite, and application thereof as sepsis therapeutic agent - Google Patents

Abstract

The present invention relates to a ceria-zirconia nanocomposite, a pharmaceutical composition containing the same, and a preparation method therefor. More specifically, the present invention provides: a ceria-zirconia nanocomposite having a structure in which a surfactant encompasses ceria-zirconia solid solution nanoparticles; an antioxidant and a sepsis therapeutic agent which contain the same; and a preparation method therefor.

Description

Synthesis of ceria-zirconia solid solution nanoparticles and ceria-zirconia nanocomposites and its application as a therapeutic agent for sepsis

The present invention relates to the synthesis of ceria-zirconia nanocomposites having ceria-zirconia nanoparticles and a phospholipid-PEG layer which can easily react in vivo, and more particularly to ceria- Zirconia nanocomposite according to the present invention.

Ceria (CeO ₂ ) nanoparticles are widely used as catalysts for chemical reactions. Ceria nanoparticles have an autocatalytic function and can maintain their antioxidant properties for a long time like enzymes. However, since the efficiency is high and toxicity is strong, there is a concern about side effects and it is difficult to apply biomedical medicine.

Peritonitis is one of the diseases that surgical treatment is urgently needed. If the cause microorganisms invade the blood and proper care or surgery is not achieved in a short period of time, it may progress to sepsis. Sepsis is one of the systemic inflammatory response syndromes (SIRS) that are infected by microorganisms and cause severe inflammation in the whole body. It is important to treat the infection of organs that cause sepsis. Through physical examination, blood test, and imaging test, find the infection site of the cause of sepsis and treat with proper antibiotic. Failure to provide adequate treatment can lead to death, and in the event of impairment of body organs function or shock, it is a very high mortality. Therefore, it is necessary to prevent the inflammatory reaction and the reactive oxygen species (ROS) from occurring before the operation according to the symptoms of peritonitis in the actual treatment process. However, it is difficult to provide specific initial care for patients with peritonitis.

In 2016, the Journal of the American Medical Association (JAMA) issued a new definition of sepsis: "Life-threatening organ dysfunction caused by anomalies in the body against infection." Infection by microorganisms, resulting in excessive inflammatory reaction - increase in cytokine release, increase in vascular permeability, sympathetic nerve responsiveness, and the like. In the case of sepsis, the adverse reaction of the organism is more problematic than the infection itself, and in severe cases, it becomes a state of sepsis shock, resulting in death due to a multiple organ failure. The mortality rate of sepsis within the hospital is less than 10%, and the mortality rate due to sepsis shock. It is close to 40%. When a biopsy occurs in a patient with sepsis, the blood vessel permeability increases, metabolic acidosis (blood becomes acidic due to an increase of acidic substances other than carbon dioxide which exceeds the buffering capacity), and decrease of tissue perfusion , Even if controlled by antibiotics, long-term dysfunction continues to progress. To correct it, various medical efforts such as proper fluid supply, acidosis correction, hyperbaric administration, and hemodialysis if necessary are necessary. In other words, the most important factor in the prognosis of treatment for sepsis is how soon to start antibiotics, and the most important method is to block the infection before the outbreak of the biomarkers. Despite advances in medicine, sepsis has a golden time that is clinically not to be missed, and there is no cure to overburden the biologic reaction itself.

On the surface of the ceria nanoparticles, the conversion reaction between Ce ^{3 +} and Ce ^{4 +} occurs constantly, which enhances the catalytic action of the ceria nanoparticles. If other metal ions are added to the ceria nanoparticles, it can not only control the reaction occurring on the surface of the ceria nanoparticles, but also improve the antioxidant properties of the ceria nanoparticles. Zirconium ions are one of the metal ions that can control the reaction on the surface of ceria nanoparticles and improve their antioxidant properties.

Conventionally, it has been known that when zirconium ions are introduced into ceria nanoparticles, the stability of ceria nanoparticles at high temperatures is improved and the reactivity as a catalyst is improved. However, the application fields are mainly limited to manufacturing industries and industrial fields It was common.

International Patent Publication No. PCT / EP2012 / 063756 discloses mixed oxides comprising cerium and zirconium oxides and a process for their preparation, which mentions that the composition can be used as a catalyst in automotive engine fuels. However, the above-mentioned PCT / EP2012 / 063756 does not disclose any use of ceria-zirconia solid solution nanoparticles or the like for the treatment of antioxidants or sepsis.

Therefore, in the present invention, ceria-zirconia nanoparticles having zirconium ions introduced into ceria nanoparticles have more excellent antioxidative and anti-inflammatory effects than conventional ceria nanoparticles and can control the reactivity occurring on the surface of ceria nanoparticles, Can be minimized. Also, as the size and uniformity of the nanoparticles are smaller, the effect of the antioxidant and anti-inflammation is enhanced by increasing the surface area relative to the volume.

Furthermore, in view of the fact that the antioxidative effect of the ceria-zirconia nanoparticles of the present invention is effective in treating and preventing sepsis by applying it to an actual septicemia disease model as an in vivo experiment for the treatment and prevention of sepsis, Finished.

[Prior Art Literature]

[Patent Literature]

(Patent Document 1) International Patent Publication No. PCT / EP2012 / 063756, 'Ceria zirconia alumina composition with enhanced thermal stability'

Sepsis is an acute disease that is one of the highest rates of mortality, and substances that have an antioxidant effect in order to inhibit acute progression are needed. Therefore, the uniform nanoparticles of very small size synthesized by introducing zirconium ions into existing ceria nanoparticles are excellent in the effect of removing active oxygen (ROS) and can be used as an activator. In addition, the smaller the particle size, the higher the reactivity since the volume-to-surface area is increased. Thus, using small size ceria-zirconia nanoparticles, mortality from acute sepsis can be much lower than with conventional treatments.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the precise form disclosed. There will be.

According to an aspect of the present invention, there is provided a magnetic recording medium comprising a core layer composed of particles of ceria-zirconia, and a surfactant layer formed by bonding a surfactant to a surface of the core layer. Ceria-zirconia solid solution nanoparticles.

CE of the metal element, the greater chemical reactivity, the compound mainly or gatneunde Ce ^{3 +} oxidation state of Ce ^{4 +,} it is generally the case of a single oxide Ce ^{3 +} state are more stable, are more stable Ce ^{4 +} state. Cerium having an oxidation state of Ce ^{3 +} is referred to as cerium (III), and Ce ^{4 +} is referred to as cerium (IV). There are two kinds of cerium oxide, cerium (III) oxide (Ce ₂ O ₃ ) and cerium oxide (CeO ₂ ). At room temperature under atmospheric pressure, CeO ₂ called ceria is more stable.

* Zirconium is a silver-gray transition metal belonging to group 4 (group 4B) called titanium. Although the metal itself is highly reactive, zirconium dioxide (ZrO ₂ ), called zirconia, is a low reactivity and stable material.

The term " ceria-zirconia solid solution nanoparticle " means a monocrystalline nano-sized substance composed of a mixture of solids in which a ceria and zirconia are completely uniformly formed. do.

In an embodiment of the present invention, the core layer may be a ceria-zirconia solid solution nanoparticle characterized by a diameter of 1 to 5 nm.

In another embodiment of the present invention, the ceria-zirconia is composed of Ce _x Zr _{1 - x} O ₂ , and x is 0.1 to 1. The ceria-zirconia solid solution nanoparticles may be cerium-zirconia solid solution nanoparticles.

In another embodiment of the present invention, x may be 0.7 or less, ceria-zirconia solid solution nanoparticles.

In another embodiment of the present invention, the C ₆ -C ₂₀ alkylamine, alkyl trimethylammonium salt ((CH ₃ ) ₃ RNX, R is C ₈ -C ₂₅ and X is Br, Cl, or I), C ₁₂ Wherein the ceria-zirconia solid solution nanoparticles are selected from the group consisting of alkali metal salts of C ₁₈ fatty acids.

In order to achieve the above technical object, the oleylamine, octylamine, hexadecylamine and octadecylamine are one of the C ₆ -C ₂₀ alkylamines, other than those listed above, also the C ₆ -C ₂₀ alkylamine ≪ / RTI >

In another embodiment of the present invention, cetyltrimethylammonium bromide, octyltrimethylammonium bromide, and dodecyltrimethylammonium bromide may be included as the alkyl trimethylammonium salt, and others may be included as the alkyl trimethylammonium salt.

As according to another embodiment of the present invention, oleic acid, linoleic acid, Lau acid, pre-stick acid, lactic acid and palmitic one of stearic acid is an alkali salt of C ₁₂ -C ₁₈ fatty acid, is other than the addition, the C ₁₂ Lt; RTI ID = 0.0 > _C18 < / RTI >

In order to accomplish the above object, the present invention further provides a ceria-zirconia solid solution nanoparticle comprising the surfactant layer, wherein the ceria-zirconia solid solution nanoparticle further comprises a phospholipid-polyethylene glycol (PEG) layer adsorbed on the surface of the surfactant layer. - zirconia nanocomposites can be provided.

In another embodiment of the present invention, the ceria-zirconia nanocomposite may have a diameter of 5 to 30 nm.

In order to achieve the above object, the present invention provides an antioxidant including the ceria-zirconia nanocomposite.

In one embodiment of the present invention, in another embodiment of the present invention, the ceria-zirconia nanocomposite has a diameter of 5 to 30 nm.

In one embodiment of the present invention, the active oxygen removed by the antioxidant is any one of hydrogen peroxide (H ₂ O ₂ ), excess oxide anion (O ₂ -), and hydroxyl radical (OH) ≪ / RTI >

In order to achieve the above object, there is provided a therapeutic agent for sepsis including the ceria-zirconia nanocomposite.

In one embodiment of the present invention, the ceria-zirconia nanocomposite has a diameter of 1 to 5 nm.

As an embodiment of the present invention, there is provided a method for producing ceria-zirconia solid solution nanoparticles, comprising the steps of: (a) mixing a cerium precursor, a zirconium precursor and a surfactant to prepare a mixed solution; (b) dispersing the mixed solution; (c) heating the dispersed solution to produce a colloidal solution; (d) centrifuging the colloidal solution to precipitate only the ceria-zirconia solid solution nanoparticles, thereby providing a ceria-zirconia solid solution nanoparticle preparation method.

In one embodiment of the present invention, the cerium precursor of step (a) is selected from the group consisting of cerium (III) acetylacetonate hydrate, cerium (III) acetate hydrate, cerium (III) carbonate hydrate, cerium (III) chloride, cerium (III) chloride heptahydrate, cerium (III) bromide, cerium (III) iodide, cerium (III) nitrate hexahydrate, cerium (III) (III) sulphate hydrate and cerium (IV) sulphate. The ceria-zirconia solid solution nanoparticles of the present invention can be prepared by the following process.

In one embodiment of the present invention, the zirconium precursor of step (a) is selected from the group consisting of zirconium (IV) acetylacetonate hydrate, zirconium (IV) acetate hydrate, zirconium (IV) carbonate hydrate, zirconium (IV) Zirconium (IV) sulfate hydrate, zirconium (IV) sulfate hydrate, zirconium (IV) chloride, zirconium (IV) chloride octahydrate, zirconium (IV) Zirconium (IV) sulfate, and zirconium (IV) sulfate.

In one embodiment of the present invention, the surfactant in step (a) is selected from the group consisting of C ₆ -C ₂₀ alkylamines, alkyltrimethylammonium salts ((CH ₃ ) ₃ RNX, R is C ₈ -C ₂₅ and X is Br , Cl, or I), and an alkali salt of a C ₁₂ -C ₁₈ fatty acid. The ceria-zirconia solid solution nanoparticles may be prepared by dissolving the ceria-zirconia solid solution nanoparticles.

In one embodiment of the present invention, the step (a) of the C ₆ -C ₂₀ alkyl amine is a non oleyl amine, octyl amine, hexadecyl amine, and octadecyl amine may be, and wherein said C ₆ -C ₂₀ < / RTI > alkylamine. ≪ Desc / Clms Page number ₂ >

In another embodiment of the present invention, the alkyltrimethylammonium salt in step (a) may be cetyltrimethylammonium bromide, octyltrimethylammonium bromide, and dodecyltrimethylammonium bromide, and the other alkyltrimethylammonium salt may be included in the alkyltrimethylammonium salt The present invention provides a method for producing ceria-zirconia solid solution nanoparticles.

In one embodiment of the present invention, the alkali salt of the C ₁₂ -C ₁₈ fatty acid in step (a) may be oleic acid, linoleic acid, lauric acid, myristic acid, palmitic acid and stearic acid, The present invention provides a method for producing ceria-zirconia solid solution nanoparticles which can be included as an alkali salt of the C ₁₂ -C ₁₈ fatty acid.

In another embodiment of the present invention, the method may further include a step of cooling the colloid solution between the step (c) and the step (d), followed by washing the ceria solution with the solution of the ceria-zirconia solid solution nanoparticles have.

In one embodiment of the present invention, in the dispersion in the step (b), ceria-zirconia solid solution nanoparticles surrounded by the surfactant are dispersed using an ultrasonic disperser at room temperature for 10 to 20 minutes. And a manufacturing method thereof.

In another embodiment of the present invention, the heating conditions in the step (c) include a first step of heating to 70 to 90 캜 at a heating rate of 1 to 5 (캜 / min); And a second step of maintaining a temperature of 70 to 90 캜. The present invention also provides a method for producing ceria-zirconia solid solution nanoparticles.

According to an embodiment of the present invention, the second step is performed for 3 to 30 hours, thereby providing a method for preparing ceria-zirconia solid solution nanoparticles.

According to another aspect of the present invention, there is provided a method for producing ceria-zirconia solid solution nanoparticles, which comprises cooling the solution to 10 to 30 ° C under the cooling condition.

(A) mixing a first dispersion containing ceria-zirconia solid solution nanoparticles with a second dispersion containing a phospholipid-PEG to prepare a first mixed solution; (b) evaporating the first mixed solution to remove the solvent to obtain a powder containing the ceria-zirconia solid solution nanoparticles; (c) adding the powder to water to prepare a second mixed solution; (d) passing the second mixed solution through a filter material having a plurality of filtration holes and separating the filtrate into a filtrate. The present invention also provides a method for producing a ceria-zirconia nanocomposite.

According to an embodiment of the present invention, in the step (a), the concentration of the first dispersion containing the ceria-zirconia nanoparticles is 10 mg / ml.

In another embodiment of the present invention, there is provided a method for preparing a ceria-zirconia nanocomposite in step (a), wherein the concentration of the second dispersion containing the phospholipid-PEG is 10 mg / ml.

In order to achieve the above object, the solvent of the first dispersion and the second dispersion may be at least one selected from the group consisting of chloroform (CHCl ₃ ), dichloromethane, pentane, hexane, heptane, cyclohexane, ethyl acetate, tetrahydrofuran , Diethyl ether, and trichlorethylene. The present invention also provides a method for producing a ceria-zirconia nanocomposite.

According to an embodiment of the present invention, in the step (d), the size of the plurality of filtration holes of the filter medium is 0.1 to 1.0 micrometer (μm). .

In another embodiment of the present invention, there is provided a method for preparing a ceria-zirconia nanocomposite, further comprising the step of (e) washing the remaining phospholipid-PEG among the particles filtered on the surface of the filter material. to provide.

According to an embodiment of the present invention, ceria-zirconia solid solution nanoparticles and ceria-zirconia nanocomposite can efficiently remove active oxygen. Therefore, the ceria-zirconia nanocomposite of the present invention can exhibit an effective therapeutic effect as an antioxidant and a sepsis treatment agent.

It should be understood that the effects of the present invention are not limited to the above effects and include all effects that can be deduced from the detailed description of the present invention or the configuration of the invention described in the claims.

Fig. 1 shows the characteristics of the ceria-zirconia nanocomposite synthesized in Example 3 of the present invention.

2 shows a synthesis process of ceria-zirconia solid solution nanoparticles and ceria-zirconia nanocomposite synthesized according to Example 2 and Example 3 of the present invention.

FIG. 3 is a TEM and STEM image of a ceria-zirconia nanocomposite showing images of the surface of a ceria-zirconia nanocomposite.

FIG. 4 shows the results of XRD, XPS and DLS analysis of ceria-zirconia nanocomposites.

Figure 5 shows the results of energy dispersive spectroscopy (EDS) analysis of ceria-zirconia nanocomposites.

6 shows the cell survival rate (in vitro data (cell mortality)) of the ceria-zirconia nanocomposite.

FIG. 7 shows the results (in vivo data (mortality)) of a comparison between a ceria-zirconia experimental group and a control group for mortality by an experimental model of acute sepsis disease.

8 is a structural diagram showing a method for producing ceria-zirconia solid solution nanoparticles.

9 is a structural diagram showing a method for producing a ceria-zirconia nanocomposite.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In order to clearly illustrate the present invention, parts not related to the description are omitted, and similar parts are denoted by like reference characters throughout the specification.

Throughout the specification, when a part is referred to as being "connected" (connected, connected, coupled) with another part, it is not only the case where it is "directly connected" "Is included. Also, when an element is referred to as " comprising ", it means that it can include other elements, not excluding other elements unless specifically stated otherwise.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, the terms "comprises" or "having" and the like refer to the presence of stated features, integers, steps, operations, elements, components, or combinations thereof, But do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or combinations thereof.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Example 1. Method for producing ceria nanoparticles

(Sigma-Aldrich) and 12 mmol (3.2 g) of oleylamine ( _C18 content of about 80-90%, Acros Organics) were dissolved in 15 ml of xylene (98.5%, Sigma -Aldrich). The thus-prepared solution was dispersed at room temperature for 15 minutes using an ultrasonic disperser, and then heated to 90 ° C at a rate of 2 ° C / min. 1 ml of deionized water was poured into the solution with vigorous stirring at 90 DEG C and the solution changed from off-white to cloudy yellow, indicating that the reaction was initiated. The resulting mixture was aged at 90 < 0 > C for 3 hours to obtain a clear yellow colloid solution, which was cooled at room temperature. Thereafter, the precipitate was washed well with acetone (100 ml) using centrifugation, and the washed ceria nanoparticles were stored at a concentration of 10 mg / ml in chloroform, which may be well dispersed.

Example 2. Method for producing ceria-zirconia solid solution nanoparticles.

Cerium (III) acetylacetonate hydrate (Sigma-Aldrich) and zirconium (IV) acetylacetonate hydrate (Sigma-Aldrich) were mixed in a molar ratio of cerium (III): zirconium (IV) = 100: 0 to 20:80 Was added to 15 ml of oleylamine ( _C18 content about 80-90%, Acros Organics) (Figs. 2 and 8). The thus-prepared solution was dispersed at room temperature for 15 minutes using an ultrasonic disperser, heated to 80 ° C at a rate of 2 ° C / min, and aged for one day at 80 ° C to obtain a dark brown colloidal solution , Which was cooled at room temperature. Afterwards, the precipitate was washed well with acetone (100 ml) by centrifugation, and the washed ceria-zirconia solid solution nanoparticles were stored in chloroform at a concentration of 10 mg / ml in a well-dispersed solution.

In the present invention, in the case of the ceria-zirconia (Ce _x Zr _{1 - x} O ₂ ) solid solution nanoparticles or the ceria-zirconia nanocomposite, the sample containing the solid solution nanoparticles or the nanocomposite, , 7CZ when x is 0.7, 4CZ when x is 0.4, and 2CZ when x is 0.2. Further, 2CZ, 4CZ, and 7CZ are collectively defined as CZ NPs. For simple ceria nanoparticles, it is defined as Ce NPs.

 Energy Dispersive Spectrometry (EDS) is an optional function attached to a scanning electron microscope (SEM). When an X-ray detector detects an X-ray signal and converts it to an electronic signal, The signal is measured using a pulse processor and the energy of the detected X-ray is determined. The determined X-ray data were analyzed to confirm that the synthesis of the ceria-zirconia solid solution nanoparticles according to Example 2 was correctly performed. (Fig. 5). When referring to the EDS graph, (a) is for Ce NPs, (b) for 10CZ, (c) for 7CZ, (d) for 4CZ, .

Example 3 Preparation of Ceria-Zirconia Nanocomposite

To improve the biocompatibility of the ceria-zirconia solid solution nanoparticles, phospholipid pegylation was performed (Figs. 2 and 9). Pegylated Polyethylene glycol (PEG) is a technology that adsorbs a biocompatible, secure polymer at the boundary of a drug or other target.

Chloroform (10 mg / ml) to which 5 ml of ceria-zirconia solid solution nanoparticles had been added was added to 10 ml of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (mPEG-2000, Avanti Polar Lipids Inc) at a ratio of 1: 2 with chloroform (10 mg / ml). The solvent chloroform was evaporated using a rotary evaporator, and the remaining chloroform was removed by incubation at 70 DEG C in a vacuum drier. Then, 5 ml of water was added to the resulting powder to prepare a transparent colloidal suspension. The suspension was filtered using a filter having a size of 0.4 mu m, and then an excessive amount of mPEG-2000 was removed by ultracentrifugation. The ceria-zirconia nanocomposite encapsulated with the purified phospholipid-PEG was dispersed in distilled water.

Example 4. Analysis of SOD (superoxide dismutase) mimetic activity

  SOD is an enzyme that catalyzes a disproportionation reaction that converts excess oxygen ions into oxygen and hydrogen peroxide. It is known that almost all cells exposed to oxygen are antioxidant defense mechanisms. Excess oxidized ion scavenging activity is performed in an SOD assay kit (Sigma-Aldrich), after mixing 20 μL of each sample with 160 μL of WST-1 standard solution (0.125 mM). The reaction occurs when 20 μL of xanthine oxidase solution is added to each microplate well. When this was incubated at 37 DEG C for 20 minutes, absorbance at 450 nm was observed through a multiple plate reader (Victor X4, Perkin-Elmer) (Fig. 1 (f)). Since the inhibition rate of excess oxide ions can be calculated as the development of color decreases, the absorbance at 450 nm is a measure of quantification.

Example 5. Analysis of catalase activity

Quenching with hydrogen peroxide is carried out using Amplex ^® red hydrogen peroxide / peroxidase assay kit (molecular probes, Inc.). If horse radish peroxidase (horseradish peroxide, HRP) and Amplex ^® red reaction and the reaction with hydrogen peroxide, the red phosphor of the resonance lupine is generated (FIG. 1 (g)). At this time, the red fluorescein of resorufin (the excitation and emission maximum 571 nm, 585 nm) reflects the degree of peroxide in the solution. Hydrogen peroxide and sample are mixed to make 50 μL of hydrogen peroxide solution until final concentrations are 10 mM and 0.25 mM, respectively. When 50 μL of the Amplex ^® red / HRP standard solution is added to each microplate well, the reaction begins. The samples should be incubated at 25 ° C in the absence of light, and then the fluorescent material is measured using a multiple plate reader (Victor X4, Perkin-Elmer).

Example 6. Hydroxyl radical oxidation inhibiting ability (HORAC) analysis

Hydroxyl radical scavenging activity can be analyzed using the HORAC assay kit (Cell Biolaps, Inc. USA) (Fig. 1 (h)). Hydroxyl radicals are easily produced by mixing a hydroxyl radical-introducing agent and a Fenton reagent. By measuring the intensity of the fluorescence light (here 480 nm, emission 530 nm), the inhibition rate of the hydroxyl radical of the solution can be calculated. 20 μL of each sample (final concentration 0.125 mM) is mixed with 140 μL of the fluorescent substance (1 ×). Incubating for 30 minutes at 25 ° C, 20 μL of the hydroxyl-introducing agent (1X) and 20 μL of the Fenton's reagent are injected into each microplate well continuously. After shaking the plate for 15 seconds, incubate for 30 minutes at 25 ° C, the fluorescent material can be observed using a multiple plate reader (Victor X4).

Example 7.t - Cell viability test using BHP ( tert- butyl hydroperoxide )

20,000 RAW 264.7 cells are plated on plates with the first 96 wells and incubated for 24 hours at 37 ° C. The plate was then washed with PBS solution (phosphate buffer saline, a solution used as a suspension of organisms or tissues or organ that could not last its life with only physiological saline solution), and 10 mL of Ce NPs and 7CZ 0, 0.01, 0.02 mM). Pre-incubate the plates at 37 ° C for 24 hours by treating the plates with RAW 264.7 cells in advance. Incubating cells are washed with PBS solution, and finally 10 mL of tBHP (final concentration is 0.4 mM) is added to the cells and incubated for an additional 2 hours. After incubation, 10 mL of a solution of 3, (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (concentration 5 mg / ml) was added to each well, Incubate under the conditions. Then, the washed cells were treated with 200 mL of DMSO solution, and the cell viability was observed using a multiple plate reader (Victor X4).

Based on the above analysis (FIG. 6), it can be seen that the survival capacity of the 7CZ treated group at the concentration of 0.02 mM is the best.

Example 8. CLEC (Cecal ligation and puncture) analysis (in vivo)

In an in vivo experiment, a CLP (Cecal ligation and puncture model) model, which can actually mimic sepsis, was constructed (Fig. 7). Then, 18 mice were injected with PBS solution (control group), another 18 mice were injected with CZ NPs (experimental group), and the survival rate was observed for 14 days.

sample	Core size (nm)	HD diameter (nm)	Ce 3 + (%)	Reduction Temperature (℃)
				LT Peak	HT Peak
Ce NPs	3.55 + - 0.37	15.06 + 4.18	30.08	446.3	784.8
10CZ	2.1 ± 0.21	13.38 + - 4.05	30.42	483.3	779.1
7CZ	2.08 ± 0.22	14.21 + - 4.27	52.61	397.9	549.7
4CZ	2.19 ± 0.17	16 ± 4.54	59.81	347.8	513.2
2CZ	2.21 ± 0.18	11.01 + - 3.3	63.31	403.9	531.3

Based on the XPS analysis, we can observe the Ce ^{3 +} peak seen when the binding energies are 884.5 and 903 eV (Fig. 1 (d) and Table 1). XPS analysis means that when a sample is irradiated with X-rays having a certain energy, photoelectrons are emitted from the sample. The kinetic energy of the photoelectrons can be measured to determine the binding energy of the photoelectrons. This binding energy is a unique property of the atom that emits the photoelectron, so that the element can be analyzed.

Based on the Ce ^{3 +} peak shown in the XPS analysis, it can be seen that the more the Zr ^{4 +} ions are bonded to the ceria-zirconia nanocomposite, the more Ce ³⁺ of the ceria-zirconia nanocomposite is present . Generally, the smaller the particle size, the more Ce ^{3 +} is present in the nanoparticles. It can be seen that the greater the amount of Ce ^{3 +} , the greater the reduction effect of the excess oxide anion (O ₂ ^- ) and the hydroxyl radical (OH ·).

Furthermore, in order to compare the effect of particle size, we can analyze Ce NPs and 10CZ, which do not contain Zr ^{4 +} .

Conventional studies have shown that when the synthesis process for producing particles is carried out in aqueous solution, the particles contain more Ce ^{3 +} . For this reason, 10CZ was synthesized in the organic solution state, and 10CZ and Ce ^{3 +} / Ce ^{4 +} ratios were relatively similar, since synthesis of Ce NPs was done in aqueous solution, although 10CZ was smaller.

The difference according to the reaction state condition can be seen also in the light of the XRD analysis result (FIG. 4 (a)).

X-ray diffraction analysis is an X-ray diffraction method. When X-ray diffracts on a crystal, some of them cause diffraction. Its diffraction angle and intensity are inherent in the material structure. Using this diffraction X-ray, And information related to the quantity can be known. The analysis method for obtaining information on the structure of the crystalline material is X-ray diffraction (XRD analysis).

According to XRD analysis, 2CZ, 4CZ, and 7CZ have the same crystal structure as Ce NPs and 10CZ. Further, as the content of Zr ^{4 +} ions increases, the tetragonality is further increased because the diameter of Zr ^{4 +} ions is smaller. Therefore, it is considered that Ce ³⁺ content Judgment is deemed reasonable.

The effect of Zr ^{4 +} on the reduction of Ce ^{4 +} can be more clearly determined by analyzing the H ₂ -TPR graph (FIG. 1 (e)). Zr ^{4 +} increases the H ₂ consumption peak (LT Peak, HT Peak) at low temperature and high temperature during the reduction reaction (Table 1). Compared to Zr ^{4 +} is not included in the sample (Ce NPs, 10CZ) and Zr ^{4 +} a 2CZ, 4CZ, 7CZ contain, two peaks of a sample containing the Zr ^{4 +,} compared with Ce NPs sample , It can be seen that Zr ^{4 +} helps the overall reduction of the Ce ^{4 +} → Ce ^{3 +} reduction reaction.

As a result, high particle size is very small, Ce ^{3 +} / ^{4 +} Ce ratio, and can also easily be made when manufacturing the CZ NPs from moderate the fast playback for the removal of Ce ^{3 +} radical conditions.

(O ₂ ^- ), hydrogen peroxide (H ₂ O ₂ ) and hydroxyl radical (OH) in the active oxygen were investigated at room temperature in order to compare the active oxygen removal capacity of CZ NPs and Ce NPs.

The ability of the active oxygen removal by particle size was compared with Ce NPs and 10CZ. 10CZ of smaller size was found to be superior to active oxygen removal ability.

Compared with 7CZ and 10CZ, catalase activity of 10CZ, which has a high content of Ce ^{4 +} , was improved. This is because when hydrogen peroxide is removed, it is heavily influenced by Ce ^{4 +} . Despite the limitations of redox reactions for excess oxide anions and hydroxyl radicals, 4CZ and 2CZ, which contain less Ce ions, showed similar inhibition rates when compared to the respective analyzes of Ce NPs and 10CZ. This is supported by the fact that the ratio of Ce ^{3 +} / Ce ^{4 +} , which increases with Zr ^{4 +} , and the rate of regeneration to Ce ^{3 +} play the most important role in the reduction of excess oxide anions and hydroxyl radicals .

According to the results of all analyzes, 7CZ was found to have the most excellent ability to remove active oxygen. Furthermore, it has been confirmed that physiologically, Zr ^{4 +} is excellently added to ceria nanoparticles to remove active oxygen.

The results of the survival curve analysis using the CLP model (Example 8, FIG. 7) revealed that only 3 mice survived in the control group injected with PBS solution for 14 days, 8 mice survived and 83.3% of the control group and 55.6% of the experimental group were observed. Furthermore, in the above CLP model, it was confirmed that CZNPs are distributed around the cecum where sepsis occurs, and act on the local region. In conclusion, it can be confirmed that the ceria-zirconia solid solution nanoparticle is clearly effective as a therapeutic agent for sepsis in vivo.

In this experiment, only one dose of CZ NPs was administered to reduce the mortality rate by 27.7%. The fact that CZ NPs improved the survival rate even though they did not resolve the pathogen causing the infection indicates that the ability of CZ NPs to remove reactive oxygen has played an important role in suppressing excessive biomarkers. In addition, since CZ NPs have an effect of regeneration, they have an active oxygen (ROS) inhibitory effect continuously even once.

Furthermore, in the survival graph of the CLP model, the mortality rate during the first 2-3 days of the control group is the highest, and thereafter, the individual gradually dies. This is not much different from the 7CZ experimental group after 3 days. This suggests that it is important to treat CZ NPs rapidly in the early stages of infection, and CZ NPs react faster than Ce NPs and are more effective. As shown in the previous in vitro experiment (FIG. 6), it can be confirmed that the effect of treating CZ NPs with sepsis is superior at the stage of the initial infection reaction.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims (22)

1. A core layer composed of particles composed of ceria-zirconia;

   A surfactant layer formed by bonding a surface active agent to the surface of the core layer;

   Ceria-zirconia solid solution nanoparticles.
2. The method according to claim 1,

   Wherein the core layer has a diameter of 1 to 5 nm.
3. The method according to claim 1,

   Wherein the ceria-zirconia is represented by Ce _x Zr _{1 - x} O ₂ , and x is 0.1 to 1. The ceria-zirconia solid solution nanoparticle according to claim 1,
4. The method according to claim 1,

   The surfactant of the C ₆ -C ₂₀ alkyl amines, alkyl trimethyl ammonium salt ((CH _{3) 3} RNX, wherein R is a C ₈ -C _25, X is Br, Cl, or I), C ₁₂ -C ₁₈ fatty acids Wherein the ceria-zirconia solid solution nanoparticle is at least one selected from the group consisting of alkali metal salts and alkali salts.
5. A surfactant layer contained in the ceria-zirconia solid solution nanoparticles of any one of claims 1 to 4;

   A phospholipid-polyethylene glycol (PEG) layer adsorbed on the boundary of the surfactant layer;

   And a ceria-zirconia nanocomposite.
6. The method of claim 5,

   The ceria-zirconia nanocomposite has a diameter of 5 to 30 nm.
7. An antioxidant comprising the ceria-zirconia nanocomposite of claim 5 or claim 6.
8. The method of claim 7,

   Wherein the active oxygen removed by the antioxidant is any one of hydrogen peroxide water (H ₂ O ₂ ), excess oxide anion (O ₂ -), and hydroxyl radical (OH).
9. A therapeutic agent for sepsis comprising the ceria-zirconia nanocomposite according to claim 5 or claim 6.
10. A method for producing ceria-zirconia solid solution nanoparticles,

    (a) preparing a mixed solution by mixing a cerium precursor, a zirconium precursor, and a surfactant;

    (b) dispersing the mixed solution;

    (c) heating the dispersed solution to produce a colloidal solution;

    (d) centrifuging the colloidal solution to precipitate only ceria-zirconia solid solution nanoparticles;

    Wherein the ceria-zirconia solid solution nanoparticles are prepared by a method comprising the steps of:
11. The method of claim 10,

    The cerium precursor of step (a) is selected from the group consisting of cerium (III) acetylacetonate hydrate, cerium (III) acetate hydrate, cerium (III) carbonate hydrate, cerium (IV) hydroxide, cerium (III) ) Cerium (III) sulfate heptahydrate, cerium (III) bromide, cerium (III) iodide, cerium (III) nitrate hexahydrate, cerium (III) ) Sulfate. ≪ / RTI > < RTI ID = 0.0 > 15. < / RTI >
12. The method of claim 10,

    The zirconium precursor in step (a) is selected from the group consisting of zirconium (IV) acetylacetonate hydrate, zirconium (IV) acetate hydrate, zirconium (IV) carbonate hydrate, zirconium (IV) hydroxide, zirconium (IV) (IV) chloride, zirconium (IV) chloride octahydrate, zirconium (IV) bromide, zirconium (IV) iodide, zirconium (IV) oxynitrate hydrate, zirconium (IV) sulfate hydrate and zirconium ≪ / RTI > wherein the ceria-zirconia solid solution nanoparticles are selected from ceria-zirconia solid solution nanoparticles.
13. The method of claim 10,

    Wherein (a) the surfactant of step is C ₆ -C ₂₀ alkyl amines, alkyl trimethyl ammonium salt ((CH _{3) 3} and RNX, R is C ₈ -C _25, X is Br, Cl, or I), C ₁₂ Wherein the cerium-zirconia solid solution nanoparticles are at least one selected from the group consisting of alkali salts of C ₁₈ fatty acids.
14. The method of claim 10,

    The method of claim 1, further comprising cooling the colloid solution between (c) and (d) and then washing the ceria solution.
15. The method of claim 10,

    Wherein the dispersion in step (b) is carried out by using an ultrasonic disperser at room temperature for 10 to 20 minutes to disperse the ceria-zirconia solid solution nanoparticles.
16. The method of claim 10,

    The heating condition in the step (c) may include a first step of heating to 70 to 90 캜 at a heating rate of 1 to 5 (캜 / min);

    Maintaining a temperature of 70 to 90 캜;

    ≪ / RTI > wherein the ceria-zirconia solid solution nanoparticles are prepared from the ceria-zirconia solid solution nanoparticles.
17. 18. The method of claim 16,

    Wherein the second step maintains the ceria-zirconia solid solution nanoparticles for 3 to 30 hours.
18. 15. The method of claim 14,

    And cooling the solution to 10 to 30 占 폚 under the cooling condition.
19. A method for producing a ceria-zirconia nanocomposite,

    (a) mixing a first dispersion containing ceria-zirconia solid solution nanoparticles with a second dispersion containing a phospholipid-PEG to prepare a first mixed solution;

    (b) evaporating the first mixed solution to remove the solvent to obtain a powder containing the ceria-zirconia solid solution nanoparticles;

    (c) adding the powder to water to prepare a second mixed solution;

    (d) passing the second mixed solution through a filter medium having a plurality of filtration holes to separate the filtrate into filtrate;

    ≪ / RTI >
20. The method of claim 19,

    In the step (a)

    Wherein the concentration of the first dispersion containing the ceria-zirconia nanoparticles and the concentration of the second dispersion containing the phospholipid-PEG is 10 mg / ml.
21. The method of claim 19,

    In the step (a)

    The solvent of the first dispersion and the second dispersion is any one of chloroform (CHCl ₃ ), dichloromethane, pentane, hexane, heptane, cyclohexane, ethyl acetate, tetrahydrofuran, diethyl ether and trichlorethylene Characterized in that the ceria-zirconia nanocomposite is produced by the method.
22. The method of claim 19,

    In the step (d)

    Wherein the size of the plurality of filtration holes of the filter medium is 0.1 to 1.0 micrometer (μm).

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