A Novel Cerium Oxide Nanocomplex Comprising Lysine and Use Thereof

Abstract

The present invention relates to a cerium oxide nanocomposite having a shell layer comprising a cationic amino acid and PVP formed on a cerium oxide nanoparticle core layer; and a composition for improving skin condition comprising the same as an active ingredient. By applying lysine, which is the optimal amino acid among various amino acids capable of functioning as a multifunctional group ligand, the nanocomposite of the present invention possesses both excellent particle characteristics and a maximized ability to remove reactive oxygen species within skin tissue, thereby enabling the improvement and alleviation of various skin tissue damages caused by oxidative stress, including skin aging.

Description

Technology Field

[0001] The present invention relates to a cerium oxide nanocomposite comprising a cationic amino acid, specifically lysine, and a composition for improving skin condition comprising the same as an active ingredient. Background Technology

[0003] The skin is broadly composed of three layers: the epidermis, dermis, and hypodermis. Within the epidermis, the outermost layer, the stratum corneum, acts as a skin barrier to prevent the loss of moisture and electrolytes, while the dermis, primarily composed of connective tissue, maintains skin elasticity and supports its structure. Skin aging is broadly classified into intrinsic aging, caused by physiological factors, and extrinsic aging, caused by environmental factors. Intrinsic aging is a naturally induced phenomenon resulting from the decline of the body's physiological functions as age increases, whereas extrinsic aging refers to aging caused by ultraviolet (UV) radiation, dry air, reactive oxygen species, and other environmental stressors. When skin aging progresses due to these internal and external factors, the most representative symptoms are the weakening of the skin barrier function and the formation of wrinkles. In particular, when the skin is exposed to reactive oxygen species or free radicals, lipid peroxides are generated through oxidation, and skin constituent proteins such as collagen are deformed, accelerating the formation of wrinkles. In the dermis, fibroblasts age, reducing their ability to produce fibers and matrix, while in the epidermis, keratinocytes die, leading to a failure of the skin barrier function due to the quantitative loss of the stratum corneum. Therefore, controlling oxidative stress in skin tissue is the most important issue in inhibiting skin aging.

[0004] Meanwhile, cerium oxide exhibits thermal stability at high temperatures, and due to its lattice structure, Ce depends on the surrounding oxygen concentration. 4+ / Ce 3+ It performs redox reactions and is widely applied as an electrolyte for solid-state batteries, a material for UV filters, an oxygen sensor, an optical device, and more. Particularly in the medical field, it is gaining attention as a therapeutic composition for a wide range of diseases caused by oxidative stress and inflammation due to its excellent ability to scavenge reactive oxygen species.

[0005] However, when commonly used cerium oxide particles are manufactured as nanoparticles with fine diameters, it is very difficult to suppress aggregation and maintain uniform particle size and excellent dispersibility during mass production. Therefore, to efficiently improve skin condition using cerium oxide nanoparticles, it is important to possess optimized particle characteristics that enable the removal of skin tissue-specific oxidative stress through topical application or transdermal administration, along with excellent antioxidant effects.

[0007] Throughout this specification, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention. Prior art literature

[0009] Patent Document 1. Republic of Korea Registration No. 1864230 The problem to be solved

[0010] The inventors have made diligent research efforts to develop a nanoparticle-based antioxidant material capable of efficiently controlling oxidative damage to skin tissue, which is the main cause of skin aging. As a result, the present invention was completed by discovering that when a shell layer comprising a cationic amino acid, specifically lysine; and a polymer of Formula 1 described below, specifically polyvinylpyrrolidone (PVP), is formed on the surface of a core layer composed of cerium oxide nanoparticles, both the particle characteristics required for therapeutic nanoparticles and the efficiency of removing reactive oxygen species within skin tissue are maximized.

[0011] Therefore, the objective of the present invention is to provide a cerium oxide nanocomposite comprising lysine and PVP.

[0012] Another objective of the invention is to provide a cosmetic composition for improving skin condition and a pharmaceutical composition for preventing or treating skin damage caused by oxidative stress, comprising the above-mentioned cerium oxide nanocomposite as an active ingredient.

[0013] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings. means of solving the problem

[0015] According to one aspect of the present invention, the present invention provides a cerium oxide nanocomposite comprising the following:

[0016] (a) a cerium oxide nanoparticle core layer; and

[0017] (b)(i) one or more amino acids selected from the group consisting of lysine, arginine, and histidine and (ii) a shell layer comprising a polymer represented by the following chemical formula 1:

[0018] Chemical formula 1

[0019]

[0020] In the above chemical formula, R1 and R2 are each independently hydrogen or oxygen, and represents a single bond or a double bond, l is 1 or 2, and m is an integer from 100 to 1000.

[0021] The inventors have made diligent research efforts to develop a nanoparticle-based antioxidant material capable of efficiently controlling oxidative damage to skin tissue, which is the main cause of skin aging. As a result, it was discovered that when a shell layer comprising a cationic amino acid and a pyrrolidone polymer of Formula 1 is formed on the surface of a core layer composed of cerium oxide nanoparticles, it can be used as an excellent therapeutic composition that not only possesses excellent particle characteristics such as fine particle size, high uniformity, and dispersibility, but also maximizes the efficiency of removing reactive oxygen species within skin tissue.

[0022] In this specification, the term “cerium oxide nanoparticle” refers to trivalent (Ce 3+ ) and tetravalent (Ce 4+ It refers to nanoscale particles composed of cerium oxide containing cerium cations, specifically fine particles having a particle size of less than 500 nm.

[0023] According to a specific embodiment of the present invention, the cerium oxide nanoparticles are selected from the group consisting of cerium oxide (III) (Ce2O3) nanoparticles, cerium oxide (IV) (CeO2) nanoparticles, and mixtures thereof.

[0024] In this specification, the term “core layer” refers to the innermost layer in a multilayer composite having only one surface in contact with another layer.

[0025] In this specification, the term “shell layer” refers to a layer that surrounds a core layer within a multilayer composite and is farther from the center than the core layer.

[0026] In this specification, the term “multilayer complex” refers to a complex composed of multiple layers made of different components, and includes, without limitation, a laminated multilayer structure, a core-shell multilayer structure, and a combination thereof. Specifically, the multilayer complex of the present invention is a core-shell multilayer structure in which cerium oxide nanoparticles are present at the center and a shell layer containing a polymer of Formula 1 and amino acids surrounds it on the outside.

[0027] In this specification, the term “polymer” refers to a synthetic or natural polymer compound in which monomers of the same or different types are sequentially combined. Accordingly, polymers include homopolymers (polymers in which one type of monomer is polymerized) and copolymers prepared by the polymerization of at least two different monomers, and copolymers include copolymers (polymers prepared from two different monomers) and polymers prepared from more than two different monomers. Specifically, the polymer of Formula 1 used in the present invention is a homopolymer.

[0028] According to a specific embodiment of the present invention, in Chemical Formula 1, R1 is hydrogen, R2 is oxygen, and l is 1. According to the octet rule, when R1 is hydrogen When it is a single bond and R2 is oxygen It is obvious that is a double bond. The compound with chemical formula 1, where R1 is hydrogen, R2 is oxygen, and l is 1, is polyvinylpyrrolidone (PVP).

[0029] According to the present invention, the shell layer of the nanocomposite of the present invention comprises one or more cationic amino acids selected from the group consisting of lysine, arginine, and histidine, which act as multi-functional ligands. In this specification, the term “multi-functional ligand” refers to a molecule having two or more active functional groups and acting as a linker between said molecules by binding to two or more molecules. The cationic amino acid used in the present invention additionally comprises an amine group-containing chain capable of protonation under physiological conditions, having a carboxyl group capable of binding to the cerium oxide nanoparticles of the core layer and an amine group capable of binding to the compound of Formula 1 (e.g., PVP) of the shell layer, thereby enabling the nanocomposite of the present invention to be formed more efficiently and stably.

[0030] According to a specific embodiment of the present invention, the amino acid is lysine, and more specifically, L-lysine.

[0032] According to a specific embodiment of the present invention, the nanocomposite has an average particle size of 5 nm to 100 nm. More specifically, it has an average particle size of 10 nm to 80 nm, even more specifically, an average particle size of 15 nm to 50 nm, even more specifically, an average particle size of 15 nm to 30 nm, and most specifically, an average particle size of about 16 nm to 19 nm.

[0034] According to another aspect of the present invention, the present invention provides a cosmetic composition for improving skin condition comprising the nanocomposite of the present invention described above as an active ingredient.

[0035] In this specification, the term "improvement of skin condition" encompasses various beneficial changes, including protection of skin tissue, improvement of symptoms, improvement of lesions, and inhibition of aging, which can be achieved through the reduction or elimination of oxidative stress within skin tissue.

[0036] Specifically, the improvement of the skin condition described above is the inhibition of skin oxidation, inhibition of skin inflammation, strengthening of the skin barrier function, or prevention of skin aging.

[0037] In this specification, the term “oxidative stress” encompasses all changes within an organism caused by the disruption of the biological balance between the generation of reactive oxygen species and the detoxification of reactive intermediates or the ability to repair damage caused by reactive oxygen species. Oxidative stress is one of the major causes of skin aging, and reactive oxygen species are generated spontaneously within the human body as well as by various external factors such as ultraviolet rays, smoking, and environmental pollutants. Therefore, the cerium oxide nanocomposite of the present invention, which significantly reduces reactive oxygen species, can efficiently control skin oxidation and skin inflammation.

[0038] In this specification, the term “skin barrier function” refers to the function of skin tissue as a physical and biological barrier that blocks the permeation of harmful external molecules and inhibits internal moisture leakage. The stratum corneum of the skin epidermis, formed through a normal differentiation process, plays a key role in the barrier function of maintaining skin moisture and protecting it from external environmental stimuli, and keratinocytes, the major cells constituting the epidermis, play the most important role in the skin barrier function. As shown in the examples described below, the cerium oxide nanocomposite of the present invention removes reactive oxygen species from keratinocytes subjected to artificial oxidative shock and maintains the cell's inherent shape, ultimately significantly improving cell viability. Therefore, the composition of the present invention can alleviate damage to the stratum corneum caused by oxidative stress and efficiently restore the physical and biological barrier function of the skin epidermis.

[0039] Restoring skin barrier function can ultimately inhibit or delay skin aging by blocking skin aging caused by moisture loss and various infectious and inflammatory skin diseases caused by the penetration of pathogens.

[0040] In this specification, the term "inhibition or delay of skin aging" means preventing skin aging and restoring skin tissue to its previous state by inhibiting the aging and death of skin cells caused by oxidative stress or by strengthening skin barrier function; specifically, it encompasses the meaning of alleviating, preventing, or improving skin tissue damage, skin wrinkles, and a decrease in skin elasticity.

[0041] According to a specific embodiment of the present invention, the composition is an antioxidant composition.

[0042] The ingredients included in the cosmetic composition of the present invention include, in addition to the cerium oxide nanocomposite of the present invention as an active ingredient, ingredients commonly used in cosmetic compositions, such as, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, and carriers.

[0043] The cosmetic composition of the present invention can be prepared in any formulation conventionally manufactured in the art, and may be formulated, for example, as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto.

[0044] In the case where the formulation of the present invention is a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component.

[0045] In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, it may additionally include a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether.

[0046] When the formulation of the present invention is a solution or an emulsion, a solvent, a solubilizing agent, or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan.

[0047] In the case where the formulation of the present invention is a suspension, liquid diluents such as water, ethanol, or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used as carrier components.

[0048] In the case where the formulation of the present invention is a cleansing agent containing a surfactant, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester, etc. may be used as a carrier component.

[0049] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating skin damage caused by oxidative stress, comprising the nanocomposite of the present invention described above as an active ingredient.

[0050] In this specification, the term “skin damage due to oxidative stress” refers to skin diseases caused by oxidative stress or skin barrier dysfunction resulting therefrom, and specifically includes various pathological conditions such as skin aging due to moisture loss, various infectious diseases caused by the penetration of pathogens, and inflammatory diseases. Therefore, “skin damage due to oxidative stress” has the same meaning as “skin aging” or “skin barrier dysfunction.”

[0051] In this specification, the term “treatment” means (a) inhibition of the progression of a disease, illness, or symptom; (b) alleviation of a disease, illness, or symptom; or (c) elimination of a disease, illness, or symptom. The composition of the present invention serves to inhibit, eliminate, or alleviate skin tissue damage caused by oxidative stress by reducing reactive oxygen species and improving cell viability in skin cells subjected to oxidative stimulation. Accordingly, the composition of the present invention may serve as a therapeutic composition for skin damage on its own, or it may be applied as a therapeutic adjuvant for skin damage when administered together with other pharmacological components having antioxidant effects. Accordingly, in this specification, the terms “treatment” or “therapeutic agent” include the meaning of “therapeutic aid” or “therapeutic adjuvant.”

[0052] In this specification, the term “administration” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that an equal amount is formed within the subject’s body, and has the same meaning as “implantation,” “injection,” or “application.”

[0053] In the present invention, the term “therapeutic effective amount” refers to the content of a composition contained in an amount sufficient to provide a therapeutic or preventive effect to an individual to whom the composition of the present invention is to be administered, and includes the meaning of “preventive effective amount.”

[0054] In this specification, the term “object” includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons, or rhesus monkeys. Specifically, the object of the present invention is a human.

[0055] When the composition of the present invention is prepared as a pharmaceutical composition, it comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers used in the present invention are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0056] The pharmaceutical composition of the present invention may be administered parenterally, specifically by transdermal administration, subcutaneous administration, or topical application to the skin surface. More specifically, the pharmaceutical composition of the present invention is a transdermal agent or a topical skin application agent.

[0057] Suitable dosages of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the formulation method, mode of administration, age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity. Preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.001-10 g / kg for adults.

[0058] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-volume container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. Effects of the invention

[0060] The features and advantages of the present invention are summarized as follows:

[0061] (a) The present invention provides a cerium oxide nanocomposite having a shell layer comprising a cationic amino acid and PVP formed on a cerium oxide nanoparticle core layer; and a composition for improving skin condition comprising the same as an active ingredient.

[0062] (b) By applying lysine, which is the optimal amino acid among various amino acids capable of functioning as a multifunctional group ligand, the nanocomposite of the present invention possesses both excellent particle characteristics and maximized reactive oxygen species scavenging ability within skin tissue, thereby enabling improvement and alleviation of various skin tissue damages caused by oxidative stress, including skin aging. Brief explanation of the drawing

[0064] Figure 1 shows transmission electron microscope and visual observation images of a cerium oxide nanocomposite with lysine amino acid (Figure 1a) and a cerium oxide nanocomposite with glycine (left) and aspartic acid (right) (Figure 1b), respectively. Figure 2 shows the results of comparing the particle sizes of cerium oxide nanocomposites with various amino acids applied, where the solid line represents the result of applying lysine, the small dotted line represents the result of applying glycine, and the large dotted line represents the result of applying aspartic acid. Figure 3 shows the results of comparing the active radical scavenging ability of cerium oxide nanocomposites with various amino acids according to their concentrations. Figure 4 is a figure showing the results of comparing the active radical scavenging ability of known antioxidant substances (vitamin C and vitamin E) and the cerium oxide nanocomposite of the present invention. FIG. 5 is a figure showing the results of comparing the reactive oxygen species scavenging ability of known antioxidants (vitamin C and vitamin E) and the cerium oxide nanocomposite of the present invention, with hydrogen peroxide (H2O2) (left) and superoxide (O2 - )(intermediate) and hydroxyl radical (OH -The removal of ) was measured for each. Figure 6 is a figure showing the results of comparing the skin cell protective effects of cerium oxide nanocomposites with various amino acids. Figure 7 is the result of comparing the skin cell protective effect and whether the morphology of skin cells changed with known antioxidant substances (vitamin C and vitamin E) and the cerium oxide nanocomposite of the present invention. Figure 8 shows the results confirming the intracellular reactive oxygen species scavenging effect of the cerium oxide nanocomposite of the present invention to which lysine is applied. Figure 9 is a figure showing the results of confocal microscopy observation to compare the intracellular reactive oxygen species residue of the cerium oxide nanocomposite of the present invention to which lysine is applied with known antioxidant substances (vitamin C and vitamin E). Specific details for implementing the invention

[0065] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0067] Examples

[0068] Preparation Example 1: Synthesis of cerium oxide nanocomposites with lysine amino acid

[0069] A first solution was prepared by dissolving L-lysine hydrochloride (4.595 g, Daejeong Chemical) and sodium hydroxide (0.4 g, Deoksan Science) in deionized water (160 mL). While stirring the first solution, ethyl alcohol (125 mL) was added, and polyvinylpyrrolidone (11.55 g, Ashland) was weighed and added, after which the mixture was heated to 70°C in air to prepare a second solution. Meanwhile, a third solution was prepared by dissolving cerium(III) nitrate hexahydrate (Ce(NO3)3·6H2O, 2.7 g, Alfa Aeser, Ward Hill, MA) in ethyl alcohol (250 mL) at room temperature (approx. 20°C). Subsequently, the third solution was added to the second solution to prepare a fourth solution. Afterward, the temperature of the fourth solution was maintained at 70°C for 2 hours, and then lowered to 45°C. Through this process, ceria nanoparticles with L-lysine and polyvinylpyrrolidone bonded to their surfaces were obtained, and the nanoparticles were washed three times with acetone and deionized water to remove unreacted substances.

[0071] Preparation Example 2: Synthesis of cerium oxide nanocomposites with various amino acids

[0072] To determine whether amino acids other than lysine could be applied to the synthesis of cerium oxide nanocomposites, glycine (25 mmol, 1.877 g, Sigma-Aldrich) and aspartic acid (25 mmol, 3.328 g, Sigma-Aldrich) were added in the same molar content as the lysine applied in Experimental Example 1. The trends of the final synthesized product were observed visually, and the particle size (Z-Average, nm) of the nanocomposite was measured via dynamic light scattering analysis. The formation of cerium oxide nanocrystals was confirmed using a transmission electron microscope. As a result, only when lysine was applied were nanoparticles of uniform size (17.47 nm) obtained, and cerium oxide nanocrystals were formed, confirming a transparent final product (Fig. 1a). On the other hand, in the case of glycine, non-uniform crystals with a particle size of 271 nm were formed, resulting in an opaque final product, while in the case of aspartic acid, the particle size reached as much as 3,939 nm, and it was confirmed that precipitates were formed because crystals could not be formed (Fig. 1b). Considering the effect of nanocomposite size on dispersion, biological function, and biocompatibility, it was found that the nanocomposite with lysine had the most suitable composition.

[0074] Experimental Example 1: Comparison of Active Radical Removal Ability of Each Cerium Oxide Nanocomposite

[0075] To verify the active radical scavenging effect of the cerium oxide nanocomposites synthesized in Preparation Examples 1 and 2, an ABTS assay was performed. First, to generate ABTS radicals, a mixture of 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (7 mM) and potassium persulfate (2.45 mM) was incubated for 16 hours in the dark. Subsequently, various cerium oxide nanocomposites were mixed with the ABTS radical solution in equal proportions to achieve a concentration of 0.2 mg Ce / mL and incubated in the dark. The amount of residual ABTS radicals was confirmed by dispensing 200 μL of the incubated mixture into 96-well plates and measuring the absorbance at 734 nm. As a result, the cerium oxide nanocomposite with glycine showed a radical residue of 43%, and the cerium oxide nanocomposite with aspartic acid showed 25%, whereas when the cerium oxide nanocomposite was formed with lysine, a radical residue of 8% was observed at the same concentration (Fig. 3). Through this, it was confirmed that the formation of regular cerium oxide crystals and the uniform diameter of the nanocomposite are key factors for the efficient removal of active radicals.

[0077] Experimental Example 2: Comparison of Active Radical Scavenging Ability between Lysine-Applied Cerium Oxide Nanocomposite and Conventional Antioxidant

[0078] To verify the active radical scavenging effect between the lysine-applied cerium oxide nanocomposite of the present invention and Vitamin C and Vitamin E, which are known representative antioxidants, ABTS radicals were generated and the test substances were treated simultaneously. The same process as in Experimental Example 1 was followed, and the effects of the cerium oxide nanocomposite, Vitamin C, and Vitamin E were verified at a concentration of 50 μM, and at 10 minutes, 1 hour, 3 hours, 6 hours, and 24 hours, respectively. As a result, compared to Vitamin C and Vitamin E, the lysine-applied cerium oxide nanocomposite of the present invention exhibited significantly superior ABTS radical scavenging performance (Fig. 4), confirming that the nanocomposite of the present invention demonstrates a significantly improved active radical scavenging effect compared to existing organic compound-based antioxidants.

[0080] Experimental Example 3: Comparison of Reactive Oxygen Scavenging Ability between Lysine-Applied Cerium Oxide Nanocomposite and Conventional Antioxidant

[0081] To further evaluate the antioxidant effect of the lysine-applied nanocomposite, representative reactive oxygen species such as hydrogen peroxide (H2O2) and hydroxyl radicals ( · OH), superoxide (O2 - The removal capacity of ) was investigated. For the analysis of each reactive oxygen species, the amplex red hydrogen peroxide / peroxidase assay kit was used for hydrogen peroxide, the HORAC (hydroxyl radical antioxidant capacity) assay kit for hydroxyl radicals, and the SOD assay kit-WST for superoxide. All antioxidant substances applied in the experiment were mixed at a concentration of 10 μM, and the experiment was conducted following the representative recommended test method included in the assay kit. Through the analysis of each reactive oxygen species, it was confirmed that the ceria nanocomposite of the present invention not only surpasses Vitamin C and Vitamin E in the removal of various types of reactive oxygen species but also has the function to remove superoxide, which conventional antioxidant substances cannot remove (Fig. 5).

[0083] Experimental Example 4: Skin cell protective effect of cerium oxide nanocomposites with various amino acids

[0084] To verify the protective effect of various amino acids on skin cells using the cerium oxide nanocomposites synthesized in Preparation Examples 1 and 2, oxidative shock was applied to HaCaT keratinocytes by treating them with tBHP (Tert-Butyl Hydroperoxide), and a CCK-8 proliferation assay was performed. First, HaCaT cells were placed in a 96-well plate at a rate of 2 x 10 4Cells were dispensed per well and cultured for 24 hours. Various cerium oxide nanocomposites were treated with the same 1 μl Ce / mL of tBHP 200 μM medium and dispensed into keratinocytes at a dose of 100 μl / well. After 3 hours of treatment, the supernatant was removed, and CCK-8 reagent was diluted 1:10 in the culture medium and dispensed at a dose of 100 μl / well, followed by incubation for 2 hours. Subsequently, the viability of the keratinocytes was confirmed by measuring the absorbance at a wavelength of 450 nm. As a result, compared to cerium oxide nanocomposites with glycine and aspartic acid, the cerium oxide nanocomposite with lysine exhibited a superior protective effect by significantly preventing the death of keratinocytes caused by oxidative shock, and it was confirmed that the efficiency in improving the degree of cell death (cell death improvement rate = test group / positive control group - 1) was 20.9 at a treatment concentration of 1 μl Ce / mL, which was significantly improved compared to 14.6 for glycine and 4.6 for aspartic acid (Fig. 6).

[0086] Experimental Example 5: Comparison of Skin Cell Protective Effects of Lysine-Applied Cerium Oxide Nanocomposite and Conventional Antioxidant

[0087] To compare the skin cell protective effects of the lysine-containing cerium oxide nanocomposite with Vitamin C and Vitamin E, which are previously known representative antioxidants, the experiment was conducted following the same procedure as in Experimental Example 4, and changes in cell morphology were observed using an optical microscope. Specifically, HacaT cells were placed in a 96-well plate at a ratio of 2 x 10 4 Cells were dispensed into wells and cultured for 24 hours. Then, cerium oxide nanocomposites and positive controls, Vitamin C and Vitamin E, were diluted to 0.5 μM in tBHP 50 μM medium and dispensed at a rate of 100 μl / well to the cells. After treatment for 17 hours, the supernatant was removed, and CCK-8 reagent was diluted 1:10 in the culture medium and dispensed at a rate of 100 μl / well for 2 hours. Subsequently, the viability of keratinocytes was confirmed by measuring the absorbance at a wavelength of 450 nm. As a result, compared to existing antioxidants, the lysine-applied cerium oxide nanocomposite of the present invention showed an efficiency of 21.2 in improving the degree of cell death in keratinocytes caused by oxidative shock induced by tBHP (cell death improvement rate = test group / positive control group - 1), which was significantly improved compared to 0.95 for Vitamin C and -3.63 for Vitamin E (Fig. 7a). In addition, observation of actual cell morphological changes using an optical microscope also showed that the lysine-applied cerium oxide nanocomposite of the present invention did not induce changes in cell morphology, thereby exhibiting an excellent cytoprotective effect (Fig. 7b). Through this, it was confirmed that the unique radical scavenging performance of the lysine-applied cerium oxide nanocomposite demonstrates superior performance compared to existing organic compound-based antioxidants.

[0089] Experimental Example 6: Confirmation of the skin cell protection mechanism of lysine-applied cerium oxide nanocomposites

[0090] DCFH-DA Assay

[0091] After treating HaCaT keratinocytes with tBHP (Tert-Butyl Hydroperoxide) and lysine-coated cerium oxide nanocomposites, the antioxidant effects induced by oxidative shock were confirmed using the DCFH-DA assay, which can measure intracellular ROS. First, HaCaT cells were placed in a 96-well plate at a volume of 2.5 x 10 4Cells were dispensed at a rate of cells / well and cultured for 24 hours. The nanocomposite of the present invention was diluted to different concentrations (0.01, 0.05, 0.1 μM) in tBHP 1 mM medium and dispensed into the cells at a rate of 100 μl / well. After 6 hours of treatment, the cells were washed twice with PBS. The DCFH-DA reagent was diluted to a concentration of 10 μM in the medium, dispensed at a rate of 100 μl / well, and incubated for 30 minutes. After removing the supernatant, the cells were washed twice with PBS. 100 μl / well of PBS was dispensed, and the fluorescence intensity was measured using Excitation 470 / Emission 530. Subsequently, the supernatant was removed, the CCK-8 reagent was diluted to a 1:10 ratio in the culture medium, dispensed at a rate of 100 μl / well, and incubated for 2 hours. Subsequently, the residual reactive oxygen species in the cell were quantified by measuring the absorbance at a wavelength of 450 nm.

[0093] Confocal image

[0094] After treating HaCaT cells, which are keratinocytes, with tBHP (Tert-Butyl Hydroperoxide) and lysine-coated cerium oxide nanocomposites, confocal images were obtained using DCFH-DA, which can measure intracellular ROS. Vitamins C and E, known antioxidants, were treated as positive controls. First, HaCaT cells were placed on 8-chamber slides at a density of 4 x 10 4Cells were dispensed per chamber and cultured for 48 hours. The nanocomposite of the present invention was diluted to a concentration of 0.1 μM in tBHP 500 μM medium and dispensed into the cells at a rate of 300 μl / chamber. After treatment for 2 hours, the cells were washed once with PBS, and the DCFH-DA reagent was diluted to a concentration of 10 μM in the medium and dispensed at a rate of 300 μl / well, followed by incubation for 30 minutes. The supernatant was removed, and the cells were washed twice with PBS. Then, Hoechst was diluted in PBS at a ratio of 1:1000 and dispensed into the cells at a rate of 300 μl / chamber, followed by incubation for 5 minutes. Afterward, the cells were washed twice with PBS, all supernatant was removed, an appropriate amount of mounting medium was applied, a cover slide was placed over the cells, and images were obtained using a confocal microscope (Hoechst 405 nm, DCF-DA 488 nm, Brightfield).

[0095] Both the DCFH-DA assay (Fig. 8) and confocal imaging (Fig. 9) results confirmed that the lysine-applied cerium oxide nanocomposite of the present invention can efficiently remove intracellular reactive oxygen species compared to vitamin C and vitamin E, and through this, it was found that the skin cell protective effect shown in Experimental Example 5 was achieved through the removal of intracellular reactive oxygen species.

[0097] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 A cerium oxide nanocomposite comprising: (a) a core layer of cerium oxide nanoparticles; and (b) a shell layer comprising (i) lysine and (ii) a polymer represented by the following chemical formula 1: Chemical formula 1 In the above chemical formula, R1 is hydrogen and R2 is oxygen, and represents a single bond or a double bond, l is 1, and m is an integer from 100 to 1000. Claim 2 A nanocomposite according to claim 1, characterized in that the cerium oxide nanoparticles are selected from the group consisting of cerium oxide (III) (Ce2O3) nanoparticles, cerium oxide (IV) (CeO2) nanoparticles, and mixtures thereof. Claim 3 delete Claim 4 A nanocomposite according to claim 1, characterized in that the nanocomposite has an average particle size of 5 nm to 100 nm. Claim 5 A cosmetic composition for improving skin condition comprising a nanocomposite of any one of claims 1, 2 and 4 as an active ingredient, wherein the improvement of skin condition is selected from the group consisting of inhibition of skin oxidation, inhibition of skin inflammation, and enhancement of skin barrier function. Claim 6 delete Claim 7 A cosmetic composition according to claim 5, characterized in that the composition is an antioxidant composition. Claim 8 A pharmaceutical composition for the prevention or treatment of skin damage caused by oxidative stress, comprising a nanocomposite of any one of claims 1, 2, and 4 as an active ingredient.

Images