Novel cerium oxide nanocomposite comprising lysine and uses thereof

Abstract

The present invention relates to: a cerium oxide nanocomposite in which a shell layer comprising a cationic amino acid and PVP is formed on a core layer of cerium oxide nanoparticles; and a composition comprising same as an active ingredient for improving skin conditions. Employing lysine, which is an optimal amino acid capable of functioning as a multifunctional ligand among various amino acids, the nanocomposite of the present invention exhibits both excellent particle properties and maximized reactive oxygen species scavenging activity within skin tissue. Therefore, it can reduce and alleviate various skin tissue damages caused by oxidative stress including skin aging.

Description

Novel cerium oxide nanocomposite containing lysine and its use

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

[0002]

[0003] The skin is largely composed of three layers: the epidermis, the dermis, and the hypodermis. Within the epidermis, which is the outer layer, the stratum corneum, which constitutes the outermost layer, acts as a skin barrier, preventing the loss of moisture and electrolytes from the skin. The dermis, which is mainly composed of connective tissue, maintains the elasticity of the skin and supports its structure. Skin aging is broadly divided into intrinsic aging caused by physiological aging and extrinsic aging caused by environmental factors. Intrinsic aging is a natural aging phenomenon that occurs as the body's physiological functions decline with age, whereas extrinsic aging refers to the aging phenomenon that occurs due to ultraviolet (UV) rays, dry air, reactive oxygen species, and other environmental stressors. The most representative symptoms that appear when skin aging progresses due to these internal and external factors 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 component proteins such as collagen are modified, accelerating the formation of wrinkles. In the dermis, fibroblasts age, reducing their ability to produce fibers and matrix, and in the epidermis, keratinocytes die, leading to a quantitative loss of the stratum corneum, resulting in a breakdown of the skin barrier function. Therefore, controlling oxidative stress in skin tissue is the most important issue in preventing skin aging.

[0004] Meanwhile, cerium oxide has thermal stability at high temperatures and is oxidized to Ce depending on the surrounding oxygen concentration due to its lattice structure. 4+ / Ce 3+ It has a redox effect and is widely used as an electrolyte for solid batteries, a material for UV filters, oxygen sensors, optical devices, etc. In particular, in the medical field, it is attracting attention as a therapeutic composition for a wide range of diseases caused by oxidative stress and inflammation due to its excellent ability to remove reactive oxygen species.

[0005] However, since it is very difficult to suppress aggregation and maintain uniform particle size and excellent dispersibility during mass production when the commonly used cerium oxide particles are manufactured as nanoparticles with a fine diameter, it is important to have optimized particle characteristics that can achieve skin tissue-specific oxidative stress removal through topical application or transdermal administration along with an excellent antioxidant effect in order to effectively improve skin condition using cerium oxide nanoparticles.

[0006]

[0007] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the present invention.

[0008]

[0009] The present inventors have diligently researched and developed a nanoparticle-based antioxidant that can effectively control oxidative damage to skin tissue, a major 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 Chemical Formula 1, 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 in skin tissue are maximized.

[0010] Therefore, the purpose of the present invention is to provide a cerium oxide nanocomposite containing lysine and PVP.

[0011] Another object 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, which comprises the above cerium oxide nanocomposite as an active ingredient.

[0012] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0013]

[0014] According to one aspect of the present invention, the present invention provides a cerium oxide nanocomposite comprising:

[0015] (a) a core layer of cerium oxide nanoparticles; and

[0016] (b)(i) a shell layer comprising one or more amino acids selected from the group consisting of lysine, arginine and histidine and (ii) a polymer represented by the following chemical formula 1:

[0017] Chemical Formula 1

[0018]

[0019] In the above chemical formula, R1 and R2 are each independently hydrogen or oxygen, represents a single bond or a double bond, l is 1 or 2, and m is an integer from 100 to 1000.

[0020] The present inventors have conducted extensive research efforts to develop a nanoparticle-based antioxidant that can effectively control oxidative damage to skin tissue, a major cause of skin aging. As a result, they discovered that forming a shell layer comprising a cationic amino acid and the pyrrolidone polymer of Chemical Formula 1 on the surface of a core layer composed of cerium oxide nanoparticles not only exhibits excellent particle characteristics such as a fine particle size, high uniformity, and dispersibility, but can also be utilized as an excellent therapeutic composition that maximizes the efficiency of removing reactive oxygen species within skin tissue.

[0021] In this specification, the term “cerium oxide nanoparticle” refers to a trivalent (Ce 3+ ) and 4(Ce 4+ ) refers to nano-sized particles composed of cerium oxide containing cerium cations, specifically, fine particles having a particle size of less than 500 nm.

[0022] According to a specific embodiment of the present invention, the cerium oxide nanoparticles are selected from the group consisting of cerium(III) oxide (Ce2O3) nanoparticles, cerium(IV) oxide (CeO2) nanoparticles, and mixtures thereof.

[0023] In this specification, the term “core layer” means the innermost layer in a multilayer composite having only one surface in contact with another layer.

[0024] In this specification, the term “shell layer” means a layer that surrounds the core layer in a multilayer composite and is further from the center than the core layer.

[0025] As used herein, the term “multilayer composite” means a composite composed of multiple layers composed of different components, and includes, without limitation, a laminated multilayer structure, a core-shell multilayer structure, and combinations thereof. Specifically, the multilayer composite of the present invention is a core-shell multilayer structure in which cerium oxide nanoparticles are present at the center and a shell layer containing a polymer of chemical formula 1 and an amino acid surrounds the core.

[0026] As used herein, the term “polymer” refers to a synthetic or natural polymer compound in which monomers of the same or different types are continuously bonded. Accordingly, the polymer includes a homopolymer (a polymer formed by polymerizing one type of monomer) and a copolymer prepared by polymerizing at least two different monomers, and the copolymer includes both a copolymer (a polymer prepared from two different monomers) and a polymer prepared from more than two different monomers. Specifically, the polymer of Chemical Formula 1 used in the present invention is a homopolymer.

[0027] According to a specific embodiment of the present invention, in the chemical formula 1, R1 is hydrogen, R2 is oxygen, and l is 1. According to the octet rule, when R1 is hydrogen, is a single bond and R2 is oxygen It is obvious that it is a double bond. The compound with chemical formula 1, where R1 is hydrogen, R2 is oxygen, and l is 1, is polyvinylpyrrolidone (PVP).

[0028] According to the present invention, the shell layer of the nanocomposite of the present invention includes at least one cationic amino acid selected from the group consisting of lysine, arginine, and histidine, which acts as a multifunctional ligand. As used herein, the term “multifunctional ligand” refers to a molecule having two or more active functional groups and acting as a linker between two or more molecules by binding to them. The cationic amino acid used in the present invention has a carboxyl group capable of binding to cerium oxide nanoparticles in the core layer and an amine group capable of binding to a compound of chemical formula 1 (e.g., PVP) in the shell layer, and additionally includes an amine group-containing chain capable of being protonated under physiological conditions, thereby enabling the nanocomposite of the present invention to be formed more efficiently and stably.

[0029] According to a specific embodiment of the present invention, the amino acid is lysine, and more specifically, L-lysine.

[0030]

[0031] According to a specific embodiment of the present invention, the nanocomposite has an average particle diameter of 5 nm to 100 nm. More specifically, it has an average particle diameter of 10 nm to 80 nm, even more specifically, it has an average particle diameter of 15 nm to 50 nm, even more specifically, it has an average particle diameter of 15 nm to 30 nm, and most specifically, it has an average particle diameter of about 16 nm to 19 nm.

[0032]

[0033] 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 as an effective ingredient.

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

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

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

[0037] The term "oxidative stress" in this specification encompasses all changes in an organism caused by a disruption of the biological balance between the production of reactive oxygen species and the ability to detoxify reactive intermediates or repair damage caused by reactive oxygen species. Oxidative stress is one of the major causes of skin aging. Reactive oxygen species are generated naturally within the human body and are also generated 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 effectively control skin oxidation and inflammation.

[0038] The term "skin barrier function" as used herein refers to the function of skin tissue as a physical and biological barrier that blocks the permeation of external harmful molecules and inhibits the loss of internal moisture. The stratum corneum of the skin epidermis, which is created through a normal differentiation process, plays a key role in the barrier function that maintains skin moisture and protects it from external environmental stimuli, and keratinocytes, the main 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 that have been artificially subjected to oxidative stress, maintains the cell's inherent morphology, and ultimately significantly improves cell survival rate. 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 the 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 pathogen invasion.

[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 aging and death of skin cells caused by oxidative stress or strengthening the skin barrier function, and specifically encompasses alleviating, preventing, or improving damage to skin tissue, skin wrinkles, and decreased 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 conventional auxiliary agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, and carriers.

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

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

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

[0046] When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, and examples thereof include 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] When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as a carrier component.

[0048] When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester 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 as an active ingredient.

[0050] According to another aspect of the present invention, the present invention provides a method for preventing or treating skin damage caused by oxidative stress, comprising administering to a subject the nanocomposite of the present invention described above.

[0051] As used herein, the term "skin damage due to oxidative stress" refers to skin diseases caused by oxidative stress or skin barrier dysfunction resulting therefrom. Specifically, it encompasses various pathological conditions, such as skin aging due to moisture loss, various infectious diseases caused by pathogen invasion, and inflammatory diseases. Therefore, "skin damage due to oxidative stress" has the same meaning as "skin aging" or "skin barrier dysfunction."

[0052] As used herein, the term “treatment” means (a) inhibiting the development of a disease, condition, or symptom; (b) alleviating a disease, condition, or symptom; or (c) eliminating a disease, condition, or symptom. The composition of the present invention acts 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. Therefore, the composition of the present invention may be a therapeutic composition for skin damage on its own, or may be administered together with other pharmacological ingredients having an antioxidant effect and used as an adjuvant treatment for skin damage. Accordingly, the terms “treatment” or “therapeutic agent” as used herein include the meaning of “adjuvant treatment” or “adjuvant treatment agent.”

[0053] The term “administration” as used herein means directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the body of the subject, and has the same meaning as “transplantation,” “injection,” or “application.”

[0054] In the present invention, the term “therapeutically effective amount” means the content of the composition contained in an amount sufficient to provide a therapeutic or preventive effect to an individual to whom the composition of the present invention is to be administered, and includes “prophylactically effective amount”.

[0055] The term “subject” as used herein includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus macaque. Specifically, the subject of the present invention is a human.

[0056] When the composition of the present invention is prepared as a pharmaceutical composition, it includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier used in the present invention is one commonly used in formulations, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0057] The pharmaceutical composition of the present invention can be administered parenterally, and more specifically, can be administered transdermally, subcutaneously, or topically on the skin surface. More specifically, the pharmaceutical composition of the present invention is a transdermal agent or a topical skin agent.

[0058] The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. The preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.001-10 g / kg for adults.

[0059] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains.

[0060]

[0061] The features and advantages of the present invention are summarized as follows:

[0062] (a) The present invention provides a cerium oxide nanocomposite having a shell layer including a cationic amino acid and PVP formed on a core layer of cerium oxide nanoparticles; and a composition for improving skin condition comprising the same as an effective ingredient.

[0063] (b) The nanocomposite of the present invention has both excellent particle characteristics and maximized ability to remove reactive oxygen species within skin tissue by applying lysine, which is an optimal amino acid among various amino acids that can function as a multifunctional ligand, thereby improving and alleviating various skin tissue damages caused by oxidative stress, including skin aging.

[0064]

[0065] Figure 1 is a drawing showing transmission electron microscopy and visual observation images, respectively, of a cerium oxide nanocomposite to which lysine amino acid is applied (Figure 1a) and a cerium oxide nanocomposite to which glycine (left) and aspartic acid (right) are applied (Figure 1b).

[0066] Figure 2 shows the results of comparing the particle sizes of cerium oxide nanocomposites to which various amino acids are applied. The solid line is the result of applying lysine, the small dotted line is the result of applying glycine, and the large dotted line is the result of applying aspartic acid, respectively.

[0067] Figure 3 shows the results of comparing the active radical scavenging ability according to the concentration of cerium oxide nanocomposites to which various amino acids are applied.

[0068] Figure 4 is a drawing showing the results of comparing the active radical scavenging ability of known antioxidants (vitamin C and vitamin E) and the cerium oxide nanocomposite of the present invention.

[0069] Figure 5 is a drawing showing the results of comparing the active oxygen scavenging ability of the cerium oxide nanocomposite of the present invention with known antioxidant substances (vitamin C and vitamin E), hydrogen peroxide (H2O2) (left), superoxide (O2) - )(middle) and hydroxyl radical (OH - ) were measured respectively.

[0070] Figure 6 is a drawing showing the results of comparing the skin cell protection effects of cerium oxide nanocomposites applied with various amino acids.

[0071] Figure 7 shows the results of comparing the skin cell protection effect and morphological changes of skin cells between known antioxidants (vitamin C and vitamin E) and the cerium oxide nanocomposite of the present invention.

[0072] Figure 8 shows the results of confirming the intracellular active oxygen removal effect of the cerium oxide nanocomposite of the present invention to which lysine is applied.

[0073] Figure 9 is a drawing showing the results of confocal microscopy observation for comparing the amount of intracellular reactive oxygen species of the cerium oxide nanocomposite of the present invention to which known antioxidants (vitamin C and vitamin E) and lysine are applied.

[0074]

[0075] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0076]

[0077] Example

[0078] Manufacturing Example 1: Synthesis of cerium oxide nanocomposite with lysine amino acid applied

[0079] A first solution was prepared by dissolving L-lysine hydrochloride (4.595 g, Daejung Chemicals) and sodium hydroxide (0.4 g, Deoksan Scientific) in deionized water (160 mL). While stirring the first solution, ethyl alcohol (125 mL) was added, and polyvinylpyrrolidone (11.55 g, Ashland) was measured and added, followed by heating 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 (approximately 20°C). Thereafter, the third solution was added to the second solution to prepare a fourth solution. Thereafter, the temperature of the fourth solution was maintained at 70°C for 2 hours, and then the temperature was lowered to 45°C. Through this process, ceria nanoparticles with L-lysine and polyvinylpyrrolidone bound to the surface were obtained, and the nanoparticles were washed three times with acetone and deionized water to remove unreacted substances.

[0080]

[0081] Manufacturing Example 2: Synthesis of cerium oxide nanocomposites with various amino acids

[0082] In order 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 place of the lysine used in Experimental Example 1 in the same molar content. The final synthesized product was observed visually, and the particle size (Z-Average, nm) of the nanocomposites was measured through 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, nanoparticles of a uniform size (17.47 nm) were obtained, and cerium oxide nanocrystals were formed, confirming the formation of a transparent final product (Fig. 1a). In contrast, glycine produced non-uniform crystals with a particle size of 271 nm, resulting in an opaque final product. Aspartic acid, on the other hand, produced particles as large as 3,939 nm and failed to form crystals, resulting in a precipitate (Fig. 1b). Considering the impact of nanocomposite size on dispersion, biological function, and biocompatibility, the nanocomposite containing lysine was found to have the most suitable composition.

[0083]

[0084] Experimental Example 1: Comparison of the Active Radical Scavenging Abilities of Each Cerium Oxide Nanocomposite

[0085] To verify the scavenging effect of the cerium oxide nanocomposites synthesized through Preparation Examples 1 and 2 on active radicals, an ABTS assay was performed. First, a mixture of 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (7 mM) and potassium persulfate (2.45 mM) was incubated for 16 h in a light-shielded environment to generate ABTS radicals. Then, various cerium oxide nanocomposites were mixed with the ABTS radical solution in an equal ratio to 0.2 mg Ce / mL and incubated in a light-shielded environment. 200 μL of the incubated mixture was dispensed into a 96-well plate, and the absorbance at 734 nm was measured to confirm the remaining ABTS radicals. As a result, the radical residue was 43% for the cerium oxide nanocomposite applied with glycine, and 25% for the cerium oxide nanocomposite applied with aspartic acid, whereas 8% radical residue was observed at the same concentration when the cerium oxide nanocomposite was formed with lysine (Fig. 3). This confirmed that the formation of regular cerium oxide crystals and the uniform diameter of the nanocomposite are key factors in the efficient removal of reactive radicals.

[0086]

[0087] Experimental Example 2: Comparison of the free radical scavenging ability of lysine-applied cerium oxide nanocomposites and conventional antioxidants.

[0088] In order to verify the effect of the lysine-applied cerium oxide nanocomposite of the present invention on the scavenging of active radicals between the known representative antioxidants, vitamin C and vitamin E, ABTS radicals were generated and then the test material was treated simultaneously. The same process as Experimental Example 1 was followed, and the effects of the cerium oxide nanocomposite and vitamin C and vitamin E were verified at a concentration of 50 μM and at time intervals of 10 minutes, 1 hour, 3 hours, 6 hours, and 24 hours, respectively. As a result, the lysine-applied cerium oxide nanocomposite of the present invention exhibited significantly superior ABTS radical scavenging performance compared to vitamin C and vitamin E (Fig. 4), confirming that the nanocomposite of the present invention exhibits a significantly improved effect on the scavenging of active radicals compared to the existing organic compound-based antioxidants.

[0089] ·

[0090] Experimental Example 3: Comparison of the oxygen radical scavenging ability of cerium oxide nanocomposites containing lysine and conventional antioxidants.

[0091] To further evaluate the antioxidant effect of the lysine-applied nanocomposite, representative reactive oxygen species such as hydrogen peroxide (H2O2), hydroxyl radical (*?*OH), and superoxide (O2 -) were investigated for their scavenging ability. 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 was used for hydroxyl radicals, and the SOD assay kit-WST was used for superoxide. All antioxidants applied to the experiment were mixed at a concentration of 10 μM, and the experimental method was carried out according to the representative recommended experimental 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 of removing superoxide, which existing antioxidants cannot remove (Fig. 5).

[0092]

[0093] Experimental Example 4: Skin cell protective effect of cerium oxide nanocomposites containing various amino acids.

[0094] In order to verify the protective effect of skin cells according to the application of various amino acids using the cerium oxide nanocomposite synthesized in Manufacturing Examples 1 and 2, oxidative shock was applied to HaCaT cells, which are keratinocytes, by treating them with tBHP (Tert-Butyl hydroperoxide), and a CCK-8 assay, which is a proliferation assay, was performed. First, HacaT cells were seeded in a 96-well plate at a density of 2 x 10 4Cells were seeded per well and cultured for 24 hours, and 100 μl / well of keratinocytes were seeded with various cerium oxide nanocomposites in tBHP 200 μM medium at the same concentration of 1 μl Ce / mL. After treatment for 3 hours, the supernatant was removed, and 100 μl / well of CCK-8 reagent was diluted 1:10 in the culture medium and seeded with the same concentration. The cells were then incubated for 2 hours. The viability of keratinocytes was then confirmed by measuring the absorbance at a wavelength of 450 nm. As a result, the cerium oxide nanocomposite applied with lysine showed a superior protective effect by significantly preventing the death of keratinocytes due to oxidative shock compared to the cerium oxide nanocomposite applied with glycine and aspartic acid, and the efficiency of 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).

[0095]

[0096] Experimental Example 5: Comparison of the Skin Cell Protective Effects of Lysine-Applied Cerium Oxide Nanocomposite and Conventional Antioxidants

[0097] To compare the skin cell protection effects of cerium oxide nanocomposites with lysine and vitamin C and vitamin E, which are representative antioxidants known to exist, experiments were performed using the same process as Experimental Example 4, and cell morphological changes were observed using an optical microscope. Specifically, HacaT cells were seeded in a 96-well plate at a density of 2 x 10 4Cells were seeded per well and cultured for 24 hours. The cerium oxide nanocomposite and the positive control vitamin C and vitamin E were diluted to 0.5 μM in tBHP 50 μM medium, and 100 μl / well was seeded to the cells. After treatment for 17 hours, the supernatant was removed, and the CCK-8 reagent was diluted 1:10 in the culture medium, and 100 μl / well was seeded and incubated for 2 hours. Afterwards, the viability of keratinocytes was confirmed by measuring the absorbance at a wavelength of 450 nm. As a result, the efficiency of improving the degree of cell death of keratinocytes caused by oxidative shock induced by tBHP (cell death improvement rate = test group / positive control group-1) of 21.2 compared to existing antioxidant substances was confirmed to be significantly improved compared to 0.95 for vitamin C and -3.63 for vitamin E (Fig. 7a). In addition, the results of observing changes in the morphology of actual cells using an optical microscope showed that the lysine-applied cerium oxide nanocomposite of the present invention did not induce changes in cell morphology, thereby exhibiting an excellent cell protection effect (Fig. 7b). Through this, it was confirmed that the unique radical scavenging performance of the lysine-applied cerium oxide nanocomposite showed superior performance to existing organic compound-based antioxidants.

[0098]

[0099] Experimental Example 6: Confirmation of the Skin Cell Protection Mechanism of a Lysine-Applied Cerium Oxide Nanocomposite

[0100] DCFH-DA assay

[0101] The antioxidant effect according to oxidative shock was confirmed by treating HaCaT cells, which are keratinocytes, with tert-butyl hydroperoxide (tBHP) and lysine-applied cerium oxide nanocomposite, and then using the DCFH-DA assay, which can measure intracellular ROS. First, HaCaT cells were seeded in a 96-well plate at a density of 2.5 x 10 4Cells were seeded at 100 μl / well and cultured for 24 hours. The nanocomposite of the present invention was diluted at various concentrations (0.01, 0.05, 0.1 μM) in 1 mM tBHP medium and dispensed to the cells at 100 μl / well. After treatment for 6 hours, the cells were washed twice with PBS. The DCFH-DA reagent was diluted to a concentration of 10 μM in the medium and dispensed at 100 μl / well and incubated for 30 minutes. The supernatant was removed and the cells were washed twice with PBS. 100 μl / well of PBS was dispensed and the fluorescence intensity was measured at Excitation 470 / Emission 530. Thereafter, the supernatant was removed, and the CCK-8 reagent was diluted 1:10 in the culture medium, dispensed at 100 μl / well, and incubated for 2 hours. Afterwards, the amount of active oxygen remaining in the cells was quantified by measuring the absorbance at a wavelength of 450 nm.

[0102]

[0103] Confocal images

[0104] HaCaT cells, which are keratinocytes, were treated with tert-butyl hydroperoxide (tBHP) and lysine-modified cerium oxide nanocomposite, and confocal images were obtained using DCFH-DA, which can measure intracellular ROS. Vitamin C and vitamin E, which are known antioxidants, were treated as positive controls. First, HaCaT cells were seeded in 8-chamber slides at a density of 4 x 10 4Cells were seeded per chamber and cultured for 48 hours, and the nanocomposite of the present invention was diluted to a concentration of 0.1 μM in tBHP 500 μM medium and dispensed to the cells at 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, dispensed at 300 μl / well, and incubated for 30 minutes. The supernatant was removed and washed twice with PBS, and then Hoechst was diluted 1:1000 in PBS, dispensed to the cells at 300 μl / chamber, and incubated for 5 minutes. Thereafter, the cells were washed twice with PBS, the supernatant was completely removed, and an appropriate amount of mounting medium was applied, followed by covering with a cover slide, and images were obtained using a confocal microscope (Hoechst 405 nm, DCF-DA 488 nm, Brightfield).

[0105] 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 protection effect shown in Experimental Example 5 was achieved through the removal of intracellular reactive oxygen species.

[0106]

[0107] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Cerium oxide nanocomposite comprising: (a) a core layer of cerium oxide nanoparticles; and (b)(i) a shell layer comprising one or more amino acids selected from the group consisting of lysine, arginine and histidine and (ii) a polymer represented by the following chemical formula 1: Chemical Formula 1 In the above chemical formula, R1 and R2 are each independently hydrogen or oxygen, represents a single bond or a double bond, l is 1 or 2, and m is an integer from 100 to 1000.

2. A nanocomposite according to claim 1, characterized in that the cerium oxide nanoparticles are selected from the group consisting of cerium(III) oxide (Ce2O3) nanoparticles, cerium(IV) oxide (CeO2) nanoparticles, and mixtures thereof.

3. A nanocomposite characterized in that in the first paragraph, R1 is hydrogen, R2 is oxygen, and l is 1 in the chemical formula 1.

4. A nanocomposite according to claim 1, characterized in that the nanocomposite has an average particle diameter of 5 nm to 100 nm.

5. A cosmetic composition for improving skin condition, comprising the nanocomposite of any one of claims 1 to 4 as an active ingredient.

6. A composition according to claim 5, wherein the improvement in skin condition is selected from the group consisting of inhibition of skin oxidation, inhibition of skin inflammation, enhancement of skin barrier function, and prevention of skin aging.

7. A cosmetic composition according to claim 5, characterized in that the composition is an antioxidant composition.

8. A pharmaceutical composition for preventing or treating skin damage caused by oxidative stress, comprising the nanocomposite of any one of claims 1 to 4 as an active ingredient.

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