Method for preparing magnesium-substituted prussian blue nanoparticles, magnesium-prussian blue nanoparticles prepared thereby, and use thereof

Abstract

The present invention relates to a method for preparing magnesium-substituted Prussian blue nanoparticles, and use thereof.

Description

Method for preparing magnesium-substituted Prussian blue nanoparticles, magnesium-Prussian blue nanoparticles prepared therefrom, and uses thereof

[0001] The present invention relates to a method for producing magnesium-substituted Prussian blue nanoparticles, magnesium-Prussian blue nanoparticles produced therefrom, and uses thereof, specifically to nanoparticles produced by magnesium substitution of Prussian blue precipitates and a method for producing such nanoparticles, and uses as pharmaceutical and / or cosmetic compositions utilizing the antioxidant effect of said nanoparticles.

[0002] Reactive Oxygen Species (ROS) refer to chemically reactive molecules containing oxygen atoms. ROS are a collective term for various types of oxygen that are primarily generated in mitochondria—small organelles within cells—and are more unstable and reactive than ordinary oxygen used in the body. While ROS are involved in signal transduction related to cell proliferation and differentiation, it is known that higher levels of ROS can promote cancer development and metastasis, DNA damage, or apoptosis. These ROS are known to play a significant role in the onset and progression of various intractable diseases, such as cancer, cardiovascular disease, neurological disorders, diabetes, and arthritis. These diseases are becoming serious social issues due to their high prevalence and mortality rates worldwide. In particular, the severity of these issues is intensifying, with cancer and cardiovascular disease ranking as the leading and second major causes of death.

[0003] Therefore, capturing these reactive oxygen species is emerging as a promising therapeutic strategy for various diseases, and consequently, there is a growing need for the development of fundamental treatments through the elimination of reactive oxygen species. However, existing treatments, such as currently used antioxidant adjuvants, are insufficient for the direct elimination of reactive oxygen species, making fundamental treatment difficult.

[0004] Therefore, there is an urgent need for the development of therapeutic agents capable of directly and effectively eliminating reactive oxygen species. However, since the development of new drugs requires ensuring long-term toxicity and stability, it is necessary to identify therapeutic agents utilizing substances with established in vivo stability.

[0005]

[0006] Accordingly, while conducting research to develop a substance that can be used as a therapeutic agent with direct and effective reactive oxygen species removal efficacy while ensuring biocompatibility, the inventors completed the present invention regarding nanoparticles fused with magnesium and Prussian blue and a method for manufacturing the same.

[0007] Accordingly, the object of the present invention is to provide a method for producing magnesium-substituted Prussian blue (Mg-PB) nanoparticles comprising: 1) a step of generating a Prussian blue precipitate through a precipitation reaction by dropping a potassium ferricyanide (K3[Fe(CN)6]) solution into a solution in which iron ions and a stabilizer are dissolved; and 2) a step of mixing a solution containing magnesium ions with the Prussian blue precipitate.

[0008] Another objective of the present invention is to provide an antioxidant comprising magnesium-Prussian blue (Mg-PB) nanoparticles.

[0009] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of diseases caused by excessive production of reactive oxygen species, comprising magnesium-Prussian blue (Mg-PB) nanoparticles.

[0010] Another objective of the present invention is to provide a quasi-drug composition for the prevention or treatment of diseases caused by excessive production of reactive oxygen species, comprising magnesium-Prussian blue (Mg-PB) nanoparticles.

[0011] Another objective of the present invention is to provide a cosmetic composition comprising magnesium-Prussian blue (Mg-PB) nanoparticles.

[0012] Another objective of the present invention is to provide a medical device comprising magnesium-Prussian blue (Mg-PB) nanoparticles.

[0013]

[0014] To achieve the above objectives, the present invention provides a method for manufacturing magnesium-substituted Prussian blue (Mg-PB) nanoparticles, comprising: 1) a step of generating a Prussian blue precipitate through a precipitation reaction by dropping a potassium ferricyanide (K3[Fe(CN)6]) solution into a solution in which iron ions and a stabilizer are dissolved; and 2) a step of mixing a solution containing magnesium ions with the Prussian blue precipitate.

[0015] To achieve another objective of the present invention, the present invention provides an antioxidant comprising magnesium-Prussian blue (Mg-PB) nanoparticles.

[0016] To achieve another objective of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of ischemic diseases comprising magnesium-Prussian blue (Mg-PB) nanoparticles.

[0017]

[0018] The present invention will be described in detail below.

[0019] In one aspect, the present invention relates to magnesium-Prussian blue (Mg-PB) nanoparticles, said particles being produced by the following method.

[0020] 1) a step of generating a Prussian blue precipitate through a precipitation reaction by dropping a potassium ferricyanide (K3[Fe(CN)6]) solution into a solution in which iron ions and a stabilizer are dissolved; and

[0021] 2) A step of mixing a solution containing magnesium ions with the above Prussian blue precipitate.

[0022] In step 1) above, Prussian blue nanoparticles (PB particles) are generated through a precipitation reaction between iron ions and potassium ferricyanide. In the reaction, the cyanide ligand of potassium ferricyanide binds to iron ions to form an Fe(III)4[Fe(II)(CN)6]3 structure. The PB particles generated as a result of the reaction have a size of 1 to 10 nm, preferably within 5 nm.

[0023] In the above process, the iron ion is Fe 2+ or Fe 3+ As such, but not limited thereto, it may be derived from one of the following substances selected from iron chloride (FeCl3, FeCl2), iron sulfate (FeSO4), iron nitrate (Fe(NO3)₃), iron citrate (FeC6H5O7), iron pyrophosphate (Fe4(P₂O7)₃), and iron acetate (Fe(C2H₃O₂)₃), and preferably may include iron chloride (FeCl3).

[0024] In the above process, a stabilizer for controlling the aggregation and size of nanoparticles may be additionally included. The stabilizer may be a substance selected from polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), citric acid, polyvinyl alcohol (PVA), polydopamine (PDA), sodium dodecyl sulfate (SDS), Tween surfactant, polymethyl methacrylate (PMMA), dextran, glutathione, cysteine, or lysine, but is not limited thereto; preferably, it may be polyvinylpyrrolidone (PVP). In one embodiment, PVP may be included as a stabilizer, i.e., a capping agent, for Prussian blue nanoparticles. PVP binds to the surface of the nanoparticles and prevents the particles generated through electrostatic interactions from aggregating with each other. As a result, the nanoparticles are evenly dispersed and can maintain a size of about 5 nm or less.

[0025] The generated Prussian blue nanoparticle precipitate is mixed with a magnesium solution, and the magnesium solution may be selected from magnesium hydroxide, magnesium chloride, magnesium oxide, magnesium nitrate, and magnesium sulfate.

[0026] In one embodiment, the PB precipitate is mixed with magnesium hydroxide (Mg(OH)2) to synthesize a magnesium-Prussian blue (Mg-PB) unit. In this case, Prussian blue acts as a catalyst to promote the decomposition reaction of magnesium hydroxide. When the materials are mixed, Mg(OH)2 decomposes to form magnesium ions (Mg 2+ ) and hydroxide ions (OH - It is converted into ), and at this time, Prussian blue lowers the energy of the reaction and accelerates the dissociation and decomposition rate of magnesium ions. The generated magnesium ions interact with the negatively charged structure of Russian blue to act as an 'ionic glue'. As magnesium ions interact with the cyanide ligands of Prussian blue, the Mg² and PB structures combine to form Mg-PB units, and the generated units react in a chain reaction to grow into Mg-PB nanoparticles. The growth of nanoparticles is due to electrostatic interactions and ionic bonding between Mg² and Prussian blue, through which Mg-PB nanoparticles exist as aggregates of individual particles. During this process, magnesium ions penetrate and substitute into the PB structure, forming 'substituted' MgPB nanoparticles that are structurally different from PB.

[0027] The above Mg-PB nanoparticles may have a size of 10 nm to 100 nm, preferably 70 nm or less, more preferably 50 nm or less.

[0028] In another aspect, the present invention relates to an antioxidant comprising magnesium-Prussian blue (Mg-PB) nanoparticles.

[0029] "Antioxidants" are substances that inhibit or delay oxidation reactions occurring within the body. Oxidation reactions are generally triggered by free radicals, which can be associated with cell damage, aging, and various diseases. Antioxidants play a role in preventing damage caused by oxidative stress by eliminating or neutralizing these free radicals.

[0030] Prussian blue has a structure in which iron(II) and iron(III) ions are bonded, and it undergoes redox reactions during the process of electron exchange between these two states. Through these reactions, peroxides (H2O₂), hydroxyl radicals (·OH), and superoxide anions (O2· - It can neutralize reactive oxygen species such as iron ions. Iron ions react with free radicals to stabilize them and prevent oxidative damage by providing or removing electrons through redox reactions.

[0031] It was confirmed that the magnesium-Prussian blue (Mg-PB) nanoparticles of the present invention have an antioxidant effect that is significantly enhanced compared to the antioxidant effect of Prussian blue (PB) due to the interaction between magnesium and Prussian blue and the structural characteristics of the nanoparticles.

[0032] In another aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of diseases caused by the excessive generation of reactive oxygen species (ROS), comprising magnesium-Prussian blue (Mg-PB) nanoparticles as an active ingredient.

[0033] If free radicals are not eliminated and accumulate excessively in the body, it leads to a state of 'oxidative stress' that damages cells and DNA. This chronic oxidative stress is known to be involved in almost all types of diseases and the aging process.

[0034] "Excessive reactive oxygen species" means that the amount of reactive oxygen species produced is greater than the antioxidant defense capacity. The above "excessive reactive oxygen species" can be identified by i) measuring the level of damage to DNA, lipids, and proteins through damage markers, which are a method of finding traces of reactive oxygen species attacking our body, or by ii) measuring whether the total antioxidant capacity in the body has decreased below a standard level (BAP test, measurement of levels of specific antioxidant enzymes such as glutathione and SOD in the body, etc.).

[0035] Diseases caused by the excessive production of the above-mentioned reactive oxygen species may include, but are not limited to, arteriosclerosis, hypertension, ischemic disease, stroke, Parkinson's disease, Alzheimer's disease, inflammatory bowel disease, rheumatoid arthritis, chronic obstructive pulmonary disease, asthma, atopic dermatitis, cancer, diabetes, chronic ulcer, burn, or wound.

[0036] "Ischemic-reperfusion injury" refers to damage that occurs when blood flow to a tissue is temporarily blocked and then restored. It is a phenomenon in which additional damage occurs to tissues during the recovery process as blood flow is restored following the blockage of oxygen and nutrient supply due to ischemia, and can occur in various organs such as the heart, brain, kidneys, and liver. The above-mentioned ischemic injury may include, but is not limited to, angina pectoris, myocardial infarction, ischemic stroke, transient ischemic attack, peripheral artery disease, renal ischemic injury, hepatic ischemic injury, or mesenteric ischemia.

[0037] In ischemic diseases, excessive reactive oxygen species (ROS) are generated as oxygen is supplied during the blood reperfusion process. These ROS damage lipids, proteins, and DNA in cell membranes, inducing apoptosis and necrosis. During this process, inflammatory cells may become activated, leading to tissue damage, while damage to intracellular mitochondria causes energy depletion, thereby accelerating cellular damage.

[0038] Among the above diseases, wounds include diabetic wounds.

[0039] It has been previously confirmed that the magnesium-Prussian blue (Mg-PB) nanoparticles of the present invention have a strong antioxidant effect, and thus can be used as a pharmaceutical composition for the prevention or treatment of diseases caused by the excessive production of reactive oxygen species.

[0040] The term "active ingredient" in the present invention refers to a component that exhibits the intended activity alone or can exhibit the intended activity together with a carrier, etc., which is inactive itself. The term "prevention" in the present invention refers to any act that can suppress or delay a disease caused by the excessive production of reactive oxygen species by administering the magnesium-Prussian blue (Mg-PB) nanoparticles according to the present invention.

[0041] In the present invention, the term "treatment" refers to any act in which the symptoms of a disease caused by excessive production of reactive oxygen species are improved, eliminated, or benefited by the administration of the magnesium-Prussian blue (Mg-PB) nanoparticles according to the present invention.

[0042] The term "improvement" in the present invention refers to any act in which a disease caused by excessive production of reactive oxygen species is improved or beneficially altered by administering a composition containing magnesium-Prussian blue (Mg-PB) nanoparticles as an active ingredient according to the present invention.

[0043]

[0044] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external formulations, suppositories, and sterile injectable solutions according to conventional methods, and may additionally include a carrier or excipient required for the formulation.

[0045] Pharmaceutically acceptable carriers, excipients, and diluents that may be additionally included in the above active ingredient include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, and mineral oil. When formulating, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0046] For example, solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as cotton, starch, calcium carbonate, sucrose or lactose, or gelatin, with the extract or compound. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and may include various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

[0047] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, witepsol, macrogol, tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used.

[0048] The pharmaceutical composition of the present invention may be administered orally or parenterally (intravenous injection, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage may vary depending on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the time, and may be selected in an appropriate form by those skilled in the art.

[0049] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceuticalally effective amount" refers to a reasonable amount applicable to medical treatment, meaning an amount sufficient to treat a disease, and the criteria may be determined based on the patient's disease, severity, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concomitant components, and other factors. The pharmaceutical composition of the present invention may be administered in combination with an individual therapeutic agent or other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of the above factors, the dosage may be determined to a level that minimizes side effects, and this can be easily determined by a person skilled in the art. Specifically, the dosage of the pharmaceutical composition may vary depending on the patient's age, weight, severity, gender, etc., and generally, an amount of 0.001 to 150 mg, more preferably 0.01 to 100 mg per kg of body weight, may be administered daily or every other day, 1 to 3 times a day. However, this is for illustrative purposes only, and the above dosage may be set differently as needed.

[0050] In another aspect, the present invention relates to a quasi-drug composition for the prevention or treatment of diseases caused by excessive production of reactive oxygen species, comprising magnesium-Prussian blue (Mg-PB) nanoparticles as an active ingredient.

[0051] In the present invention, "quasi-drug" refers to articles used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing diseases of humans or animals, among which the effect is milder than that of pharmaceuticals. For example, quasi-drugs under the Pharmaceutical Affairs Act exclude articles used for pharmaceutical purposes and include products used for the treatment or prevention of diseases in humans / animals, products that have a mild effect on the human body or do not act directly on it, etc. One embodiment may include an oral formulation, but is not limited thereto, and the formulation method, dosage, method of use, components, etc. of the quasi-drug may be appropriately selected from the ordinary technology known in the technical field.

[0052] When magnesium-Prussian blue (Mg-PB) nanoparticles according to the present invention are used as an additive to quasi-drugs, the nanoparticles may be added as they are or used together with other quasi-drugs or quasi-drug ingredients, and may be used appropriately according to conventional methods. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment).

[0053] The above-mentioned quasi-drug composition includes, but is not specifically limited to, personal hygiene products, disinfectants, or ointments. The above-mentioned personal hygiene products may specifically include, but are not specifically limited to, toothpaste, mouthwash, cleaning gel, or preservatives for prosthetics such as dentures. Additionally, the above-mentioned quasi-drug composition may specifically be manufactured and used in the form of, but is not specifically limited to, ointments, lotions, sprays, patches, creams, powders, suspensions, gels, or gels.

[0054] In another aspect, the present invention relates to a cosmetic composition for preventing or improving symptoms or diseases caused by reactive oxygen species, comprising magnesium-Prussian blue (Mg-PB) nanoparticles as an active ingredient.

[0055] The symptoms or diseases caused by the above-mentioned reactive oxygen species may include skin aging, wrinkle formation, skin pigmentation, atopy, acne, psoriasis, or eczema, but are not necessarily limited to these.

[0056] The above cosmetic product may be an ampoule, cream, lotion, toner, essence, or pack, but is not necessarily limited thereto.

[0057] The cosmetic composition according to the present invention may be composed by combining, in addition to the magnesium-Prussian blue (Mg-PB) nanoparticles, various ingredients generally used in cosmetic compositions, such as water-soluble ingredients, powder ingredients, oils, surfactants, moisturizers, viscosity modifiers, preservatives, antioxidants, fragrances, and colorants, as needed, within a range that does not reduce the effect of the present invention.

[0058] Non-limiting examples of the above-mentioned surfactants include anionic surfactants, cationic surfactants, nonionic surfactants, and amphoteric surfactants. More specifically, examples of the above-mentioned anionic surfactants include alkylbenzenesulfonates, polyoxyalkylenealkylsulfate esters, alkylsulfate esters, olefin sulfonates, alkyl phosphates, polyoxyalkylenealkylether phosphates, dialkylsulfosuccinates, fatty acid salts, etc., and examples of nonionic surfactants include polyoxyethylene alkyl ethers, polyoxyethylene fatty acid esters, polyhydric alcohol fatty acid fractional esters, polyoxyethylene polyhydric alcohol fatty acid fractional esters, polyglycerin fatty acid esters, polyoxyethylene hydrogenated castor oil derivatives, fatty acid diethanolamides, etc. In addition, cationic surfactants include tertiary aliphatic amine salts, alkyltrimethylammonium halides, dialkyldimethylammonium halides, etc., and amphoteric surfactants include amide betaine type, imidazolinium betaine type, sulfo betaine type, etc.

[0059] Examples of the above moisturizers include glycerin, propylene glycol, 1,3-butylene glycol, dipropylene glycol, sorbitol, etc. Examples of the above preservatives include benzoic acid, dehydroacetic acid, parahydroxybenzoic acid esters (methyl parahydroxybenzoate, butyl parahydroxybenzoate, etc.), phenoxyethanol, etc. In addition, examples of the above antioxidants include ascorbic acid, BHA, etc., and in addition, UV absorbers, anti-inflammatory agents, and cooling agents may be added.

[0060] The cosmetic composition of the present invention may be prepared in a formulation selected from the group consisting of solutions, topical ointments, creams, foams, nourishing lotions, softening lotions, packs, softening waters, emulsions, makeup bases, essences, soaps, liquid cleansers, bath additives, sunscreen creams, sun oils, suspensions, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing products, oils, powder foundations, emulsion foundations, wax foundations, patches, and sprays, but is not limited thereto.

[0061] The cosmetic composition of the present invention may additionally include one or more carriers acceptable in cosmetic formulations, and may appropriately incorporate, for example, oils, water, surfactants, humectants, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc., as conventional ingredients, but is not limited thereto. Here, "carrier acceptable in cosmetic formulations" refers to a compound or composition already known and used or to be developed in the future that can be included in cosmetic formulations, and which does not have toxicity, instability, or irritation beyond what the human body can tolerate when in contact with the skin. The said carrier may be included in the composition of the present invention in an amount of about 1% to about 99.99% by weight relative to its total weight, preferably about 90% to about 99.99% by weight of the composition. However, since the said ratio varies depending on the formulation in which the composition of the present invention is manufactured, its specific application site (face, neck, etc.), or its preferred application amount, the said ratio should not be understood as limiting the scope of the present invention in any aspect.

[0062] The acceptable carriers in the cosmetic formulations included in the cosmetic composition of the present invention vary depending on the formulation of the cosmetic composition.

[0063] When the formulation of the present invention is an ointment, paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. may be used as carrier components, but are not limited thereto. These may be used alone or in a mixture of two or more types.

[0064] When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component, and in particular, in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether may be additionally included, but is not limited thereto, and these may be used alone or in a mixture of two or more types.

[0065] When the formulation of the present invention is a solution or an emulsion, a solvent, a solubilizing agent, or an emulsifying agent may be 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, etc., may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan may be used, but is not limited thereto, and these may be used alone or in a mixture of two or more types.

[0066] When 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, but are not limited thereto, and these may be used alone or in a mixture of two or more.

[0067] When the formulation of the present invention is soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolyzates, isethionates, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugars, etc. may be used as carrier components, but are not limited thereto, and these may be used alone or mixed in two or more types.

[0068] The cosmetic composition of the present invention may be prepared by including the magnesium-Prussian blue (Mg-PB) nanoparticles, and the method of preparation may be selected by a person skilled in the art according to technical common sense known in the relevant technical field and is not particularly limited thereto.

[0069] In another aspect, the present invention relates to a topical skin composition for promoting wound healing and / or skin regeneration, comprising magnesium-Prussian blue (Mg-PB) nanoparticles as an active ingredient.

[0070] The above magnesium-Prussian blue (Mg-PB) nanoparticles can promote wound healing and / or skin regeneration by promoting the proliferation or migration of epidermal keratinocytes.

[0071] In the topical skin composition according to the present invention, the topical skin composition may be, for example, a pharmaceutical composition or a cosmetic composition. The description of the pharmaceutical composition or cosmetic composition is as described above.

[0072] In another aspect, the present invention relates to a medical device comprising magnesium-Prussian blue (Mg-PB) nanoparticles as an active ingredient.

[0073] The above medical device may be a wound dressing.

[0074] In the present invention, a medical device refers to a product used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing a disease, which is an instrument or material used alone or in combination on humans or animals.

[0075]

[0076] The present invention relates to magnesium-substituted Prussian blue nanoparticles and their uses. The magnesium-Prussian blue nanoparticles have strong antioxidant activity and can be used as a preventive or therapeutic agent for various diseases caused by reactive oxygen species, such as ischemic diseases, or as a medical device.

[0077]

[0078] Figure 1 is a schematic diagram showing the synthesis process of MgPB nanoparticles according to the present invention.

[0079] Figure 2 shows the results of visual observation of the MgPB nanoparticles synthesized in the present invention. In the case of MgPB, due to magnesium substitution, it exhibits a pale yellow color compared to the blue PB, and as a result of absorbance (UV-vis) measurement, the 700 nm peak, which is the characteristic peak of PB, did not appear in MgPB.

[0080] Figure 3 shows the results of confirming the magnesium ion content of MgPB synthesized in the present invention using an assay kit, where Mg is concentration-dependent. 2+ It was confirmed that the amount increased.

[0081] Figure 4 shows the results of measuring the particle size and surface potential (zeta potential) of the synthesized MgPB nanoparticles, confirming that the size of MgPB increased by approximately 10 times and the surface potential decreased.

[0082] Figure 5 is the result of TEM FFT analysis of MgPB particles, through which it was confirmed that MgPB particles also have peaks (200), (220), (400), and (420) corresponding to the crystal planes of PB, which shows that it contains particles having the same structure as PB.

[0083] In addition, as a result of confirming the constituent elements through EDS mapping, C, N, Fe, K and Mg corresponding to the existing PB were detected together, indicating that magnesium is present in the PB structure.

[0084] Figure 6 shows the results of measuring structural changes within the particles following the formation of magnesium-Prussian blue (MgPB) nanoparticles.

[0085] Figure 7 shows the results of cyclic voltammetry analysis to confirm the redox characteristics of magnesium-Prussian blue (MgPB) nanoparticles, and it was confirmed that in the case of MgPB nanoparticles, the current amount of the redox peak increased in a concentration-dependent manner.

[0086] Figure 8 shows the capture rate of peroxide measured using a hydrogen peroxide assay kit, and it was confirmed that the capture rate of MgPB nanoparticles improved in a concentration-dependent manner.

[0087] Figure 9 shows the results of measuring the change in OH radicals, confirming that MgPB nanoparticles showed a reduction rate of more than 98%.

[0088] Figure 10 shows the results of DPPH and ABTS analysis to confirm the antioxidant properties of MgPB, and it can be seen that the antioxidant efficiency of MgPB nanoparticles is improved compared to PB.

[0089] Figure 11 shows the results of comparing the cytotoxicity of MgPB and PB. PB exhibited high reactivity and toxicity at high concentrations due to its small size, but it was confirmed that MgPB nanoparticles reduced toxicity due to increased size and crystallinity caused by magnesium.

[0090] Figure 12 shows the effect on intracellular reactive oxygen species levels, confirming that the MgPB pretreatment group maintained cell viability and significantly lowered reactive oxygen species levels.

[0091] Figure 13 shows the results of verifying the effect of MgPB nanoparticles on scavenging reactive oxygen species generated within cells by establishing an ischemia-reperfusion injury (IRI) cell model. When comparing cell survival rates 2 hours after oxygen supply following 24 hours of hypoxia, the group treated with MgPB maintained a similar survival rate compared to all control groups.

[0092] Figure 14 shows the results of confirming the effects of MgPB nanoparticles on cardiac function and vascular diameter improvement using an ischemia-reperfusion injury (IRI) model.

[0093] Figure 15 is a tissue staining image to confirm the proportion of living myocardial cells in the Mg-PB nanoparticle treatment group.

[0094] Figure 16 shows the results of a blood analysis performed to evaluate the toxicity of Mg-PB nanoparticles.

[0095] Figures 17 and 18 confirm the wound healing effect (in vitro) of magnesium-Prussian blue (Mg-PB) nanoparticles.

[0096] Figures 19 and 20 confirm the wound healing effect of magnesium-Prussian blue (Mg-PB) nanoparticles in a chronic wound model (in vitro).

[0097] Figure 21 confirms the wound healing effect of magnesium-Prussian blue (Mg-PB) nanoparticles in a mouse model.

[0098] Figure 22 shows the wound healing effect of magnesium-Prussian blue (Mg-PB) nanoparticles by staining (Masson's Trichrome) a skin wound site of a mouse.

[0099] Figure 23 confirms the wound healing effect of magnesium-Prussian blue (Mg-PB) nanoparticles in a mouse model.

[0100] Figure 24 shows the results of measuring the degree of angiogenesis of Mg-PB nanoparticles.

[0101]

[0102] Hereinafter, embodiments are described in detail to specifically explain the present specification. However, the embodiments according to the present specification may be modified in various different forms, and the scope of the present specification is not to be interpreted as being limited to the embodiments described below. The embodiments of the present specification are provided to more completely explain the present specification to those with average knowledge in the art.

[0103]

[0104] Example 1. Preparation of Magnesium-Prussian Blue (Mg-PB) Nanoparticles

[0105] Mg-PB nanoparticles were synthesized via the post-synthetic modification (PSM) method. Generally, metal ion-substituted Prussian blue particles are synthesized by the precipitation of a substitution metal salt and potassium percyanide, but magnesium ions (Mg 2+ In the case of ), the size of the hydrated ions formed due to the strong hydration characteristics in aqueous solution is larger than the Prussian blue lattice channel, so substitution by direct precipitation is limited.

[0106] Accordingly, in the present invention, Prussian blue nanoparticles were first synthesized and then reacted with a magnesium hydroxide suspension to induce ion exchange between iron ions and magnesium ions within the PB lattice, thereby producing stable magnesium-substituted Prussian blue nanoparticles. As a result of EDS analysis, the Mg-PB nanoparticles synthesized by this method had an atomic ratio of magnesium to iron (Mg / Fe) of approximately 1.3, achieving an improved magnesium content compared to the conventional precipitation method.

[0107] Iron ion precursor (Fe 3+Prussian blue nanoparticles were first synthesized by reacting iron with potassium ferricyanide (K3[Fe(CN)6]). FeCl3 (10 mM), an iron ion precursor, and PVP (Polyvinylpyrrolidone, 0.5 M), a capping agent to enhance particle dispersibility, were dissolved in 8 ml of triple-distilled water. Then, 2 ml of potassium ferricyanide (K3[Fe(CN)6], 10 mM) was added dropwise to the solution while stirring to produce PB particles. Immediate PB nanoparticles were formed from the trivalent iron and the divalent iron from potassium ferricyanide, and the formed PB particles have a size of approximately 5 nm.

[0108] The formed Prussian blue nanoparticles were mixed with a magnesium hydroxide (Mg(OH)2) solution. After adding 20 mM magnesium hydroxide powder to a 1 mg / ml aqueous solution of PB particles, MgPB particles were produced through sonication. In this process, the Prussian blue nanoparticles act as a catalyst to promote the decomposition of magnesium hydroxide. During this process, ionized magnesium ions (Mg 2+ ) interacts with negatively charged Prussian blue to form multiple nanoparticles. Through this, one Mg-PB unit is formed, and these units react in a chain to undergo growth and re-assembly processes to grow into a cubic Mg-PB complex with a size of about 50 nm.

[0109] The formed Mg-PB nanoparticles have a stable structure and form a unique charge distribution through the interaction of Prussian blue and magnesium ions. The above process is illustrated in Fig. 1.

[0110]

[0111] Example 2. Characterization of Magnesium-Prussian Blue (Mg-PB) Nanoparticles

[0112] The magnesium-Prussian blue (Mg-PB) prepared in Example 1 above is a solution that exhibits a pale yellow color, unlike Prussian blue (PB). To measure the absorption characteristics of the generated nanoparticles, the UV-Vis spectrum was analyzed. PB showed a strong absorption peak near 700 nm, and it was observed that the absorption peak of Mg-PB was reduced compared to PB (Fig. 2). This indicates that the optical properties changed as magnesium bound to PB. At this time, it was confirmed that the amount of magnesium ions contained in the nanoparticles increased in a concentration-dependent manner depending on the concentration of the magnesium hydroxide solution (Fig. 3).

[0113] In addition, the nanoparticle size and surface potential were examined, and it was confirmed that the size of Mg-PB increased by approximately 10 times and the surface potential decreased, which indicates that the PB particles and magnesium ions were successfully bound (Fig. 4).

[0114] To analyze the composition of the synthesized magnesium-Prussian blue (Mg-PB) nanoparticles, TEM FFT (Fast Fourier Transform) analysis was performed. As a result, diffraction peaks corresponding to the crystal planes of Prussian blue (PB) at (200), (220), (400), and (420) were detected. Furthermore, the elemental composition of the Mg-PB particles was confirmed via EDS (Energy Dispersive Spectroscopy) mapping, revealing the detection of C, N, Fe, and K—the major constituent elements of Prussian blue—along with the additional detection of magnesium. These results demonstrate the presence of magnesium within the crystal structure of Prussian blue, confirming the formation of a new complex containing Mg. (Fig. 5)

[0115]

[0116] Example 3. Structural analysis of magnesium-Prussian blue (Mg-PB) nanoparticles

[0117] The structure of the magnesium-Prussian blue (Mg-PB) nanoparticles synthesized from Example 1 above was compared with that of PB particles using various methods. It was confirmed that the following characteristics changed during the synthesis of magnesium-Prussian blue (Mg-PB) nanoparticles. (Fig. 6)

[0118] 1) TGA (Thermogravimetric Analysis): Measurements were taken in the presence of nitrogen from RT to 800°C by increasing the temperature at a rate of 5°C per minute. A difference of 9% loss was observed in the 100-300°C range, which indicates that the water molecule content increased compared to PB as vacancies increased due to magnesium substitution in the Mg-PB structure.

[0119] 2) FT-IR (Fourier Transform Infrared Spectroscopy): ATR mode with scan rate of 4500-500 cm -1 The area was scanned. 2070 cm of PB -1 The -CN peak was separated as a new bond was formed due to magnesium substitution, which is a result confirming that a chemical bond was formed due to the substitution of magnesium ions and Prussian blue.

[0120] 3) Raman: Measured using a 785 nm wavelength laser, and the intermediate region Fe 3+ -CN-Fe 2+ Vibration range (2200-2100 cm) -1 It was confirmed that the intensity of Mg-PB nanoparticles changes depending on the change in the redox state of iron. This result confirms that new chemical bonds are formed due to magnesium substitution, similar to FT-IR.

[0121] 4) XRD (X-ray Diffraction): Measurements were performed from 10 to 60° at a scan rate of 2° / min. As a result, the peak width in Mg-PB decreased, which indicates an increase in crystallite size. In other words, it can be confirmed that the crystallinity of Prussian blue has increased due to magnesium substitution.

[0122] 5) XPS (X-ray Photoelectron Spectroscopy): Measurements were performed using a PHI 5000 versaprobe, and a decrease in Fe bonding was confirmed in the Fe 2p and N 1s spectra. This indicates that the bonding related to trivalent iron ions was reduced due to magnesium substitution.

[0123] Example 4. Confirmation of Redox Properties and Reactive Oxygen Scavenging Ability of Magnesium-Prussian Blue (Mg-PB) Nanoparticles

[0124] Cyclic voltammetry was performed to confirm the redox potential of the synthesized magnesium-Prussian blue (Mg-PB) nanoparticles and PB particles. The experiment was conducted at a scan rate of 100 mV / s. PB particles and MgPB particles were dispersed in a 0.1M KCl solution, and the solution was placed on a gold electrode to measure the current value. Through this experiment, the redox voltage values ​​of Prussian blue (PB) and Mg-PB nanoparticles were compared. As a result, as shown in Figure 7, no clear redox peak appeared in the case of PB, whereas in the case of MgPB, a clear redox peak appeared, and the corresponding current value increased in a concentration-dependent manner.

[0125] The H2O₂ scavenging ability was measured using a hydrogen peroxide assay kit. After adding 10 μM of hydrogen peroxide (H2O₂) to the sample, the amount of remaining H2O₂ was measured after a certain period of time. Through this, the hydrogen peroxide scavenging efficiency of Mg-PB nanoparticles and PB was compared. As shown in Figure 8, it was observed that the capture rate of Mg-PB nanoparticles improved in a concentration-dependent manner.

[0126] In addition, to verify the -OH radical scavenging ability of Mg-PB and PB, -OH radicals were generated using the Fenton reaction, and the reduction in radical signals by the samples was measured using an ESR (Electron Spin Resonance) instrument. As a result, as shown in Figure 9, it was confirmed that Mg-PB nanoparticles showed a reduction rate of over 98%.

[0127] The radical scavenging ability of each was measured by reacting the samples with DPPH and ABTS reagents. Radical scavenging efficiency was evaluated based on the color change phenomenon resulting from the reaction with the reagents, and through this, the DPPH and ABTS radical scavenging abilities of Mg-PB nanoparticles and PB were compared. As a result, as shown in Figure 10, it was confirmed that the antioxidant efficiency of Mg-PB nanoparticles was improved compared to PB.

[0128]

[0129] Example 5. Confirmation of Intracellular Reactive Oxygen Scavenging Ability of Magnesium-Prussian Blue (Mg-PB) Nanoparticles

[0130] 5-1) Confirmation of cytotoxicity and reactive oxygen species scavenging ability

[0131] To compare the cytotoxicity of Prussian blue (PB) and magnesium-Prussian blue (Mg-PB) nanoparticles, PB and Mg-PB nanoparticles were diluted in a medium (DMEM) at various concentrations and treated to H9c2 myocardial progenitor cells. As shown in Figure 11, PB exhibited toxicity at high concentrations due to its high reactivity caused by its small particle size. On the other hand, it was confirmed that Mg-PB nanoparticles showed relatively lower toxicity due to increased particle size and crystallinity resulting from magnesium substitution.

[0132] In addition, the reactive oxygen species scavenging ability of the two substances was compared. To induce reactive oxygen species (ROS) within the cells, 100 μM of hydrogen peroxide (H2O₂) was applied to the cells. Subsequently, the levels of reactive oxygen species in the Mg-PB nanoparticle pretreatment group and the control group (PB) were measured. As a result, as shown in Figure 12, it was confirmed that the reactive oxygen species levels in the Mg-PB nanoparticle pretreatment group were significantly lowered, and cell viability was maintained.

[0133] 5-2) Confirmation of reactive oxygen species scavenging ability in an ischemia-reperfusion injury cell model

[0134] To directly confirm the aforementioned reactive oxygen species scavenging effect within cells, an ischemia-reperfusion injury (IRI) cell model was established to verify the scavenging effect of Mg-PB nanoparticles on intracellular reactive oxygen species. Cells (HEK293) were cultured for 24 hours under hypoxic conditions (2% O2, 5% CO2), and cell viability was evaluated 2 hours after oxygen was resupplied. Cell viability was compared with the control group after treatment with various concentrations of Mg-PB nanoparticles. As a result, the Mg-PB treated groups exhibited cell viability similar to the control group at all concentrations except for 50 μg / ml. Furthermore, by measuring the intensity of the fluorescence signal caused by reactive oxygen species (ROS), it was confirmed that Mg-PB nanoparticles significantly reduced the fluorescence intensity induced by ROS. Through this, it was observed that Mg-PB nanoparticles exhibit a cytoprotective effect under hypoxic and oxygen resupplied conditions (Fig. 13).

[0135]

[0136] Example 6. Therapeutic effect of magnesium-Prussian blue (Mg-PB) nanoparticles in an IRI animal model

[0137] An ischemia-reperfusion injury (IRI) model was established in rats, and the effects of Mg-PB nanoparticles were verified through experiments using this model. After inducing an ischemic state by blocking the coronary arteries of rats for 1 hour, Mg-PB nanoparticles were intravenously injected at concentrations of 1 mg / kg, 10 mg / kg, and 30 mg / kg, respectively, 5 minutes before reperfusion. Cardiac function and vascular diameter were evaluated via ultrasound measurements on the 2nd day after surgery. As confirmed in Figure 14, the greatest improvement in cardiac function and vascular diameter compared to the control group was observed in the group administered 10 mg / kg of Mg-PB nanoparticles.

[0138] In addition, the proportion of living cardiomyocytes in the Mg-PB nanoparticle-treated group was evaluated through tissue staining. It was confirmed that the proportion of living cardiomyocytes in the group administered 10 mg / kg of Mg-PB nanoparticles increased significantly compared to the control group (Fig. 15). Furthermore, blood analysis was performed to evaluate the toxicity of Mg-PB nanoparticles. When analyzing the creatinine, AST, ALT, and BUN indices, no statistical significance was observed in any of the indices (Fig. 16). Through this, it was confirmed that Mg-PB is non-toxic.

[0139]

[0140] Example 7. Wound healing effect of magnesium-Prussian blue (Mg-PB) nanoparticles (in vitro)

[0141] 7-1) Wound healing effects through scratch assay

[0142] To verify the wound-healing efficacy of Mg-PB nanoparticles, a scratch assay was performed using human keratinocytes (HaCaT cells). A chronic wound environment was induced by adding 30 mM glucose to the cell culture medium and culturing for one week. Subsequently, cells were seeded into 6-well plates and treated with Mg-PB at various concentrations. After cell attachment, a wound was created by scraping a straight line from the center of the cell monolayer at the bottom of the culture plate using a sterile 200 μL pipette tip. Subsequent microscopic observation confirmed cell migration and proliferation, revealing significant migration and recovery characteristics in the 0.1% Mg-PB experimental group compared to the untreated control group (Figs. 17 and 18).

[0143] 7-2) Wound healing effects using a chronic wound model

[0144] To induce a chronic wound model, high concentrations of glucose (300 Um) and H2O2 were simultaneously added to HaCaT cells to induce severe oxidative stress. Subsequently, Mg-PB nanoparticles were applied to observe the wound healing effect. The untreated group of Mg-PB nanoparticles served as the control group. As a result, similar to Example 7-1, the 0.1% Mg-PB experimental group showed an excellent wound healing effect (Figs. 19 and 20).

[0145] Example 8. Wound healing effect of magnesium-Prussian blue (Mg-PB) nanoparticles (in vivo)

[0146] The wound healing effect of Mg-PB nanoparticles was confirmed using db / db mice (leptin receptor gene deletion), a type 2 diabetes model. The control group (CONT) was untreated with Mg-PB nanoparticles, and the experimental group (MW) was treated with 0.1% Mg-PB nanoparticles. After creating wounds on the backs of the mice, Mg-PB nanoparticle solution was applied every other day, and changes in wound size were observed. Significant changes in size were confirmed starting from day 13 (Figs. 21 and 23). In addition, staining (Masson's Trichrome) of the skin wounds in the db / db mice revealed that in the control group (CONT), the red epidermal layer covering the wound was very thin and incompletely formed, and regeneration was poor. On the other hand, compared to the control group, the Mg-PB nanoparticle-treated group (MW) showed that the upper red epidermal layer was regenerated more thickly and robustly, and the blue-stained collagen region densely filled the wound site in a dark and thick manner (Fig. 22). In addition, the degree of angiogenesis was measured through CD31 tissue staining, a marker present on the surface of vascular endothelial cells. The results showed that the CD31 signal in the Mg-PB nanoparticle-treated group (MW) increased by more than five times compared to the control group (Normal), indicating high vascular density (Fig. 24). Through this, the excellent angiogenic ability of Mg-PB nanoparticles was verified.

[0147] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

Claims

1. 1) a step of generating a Prussian blue (PB) precipitate through a precipitation reaction by dropping a potassium ferricyanide (K3[Fe(CN)6]) solution into a solution in which iron ions and a stabilizer are dissolved; and 2) A method for preparing magnesium-substituted Prussian blue (Mg-PB) nanoparticles comprising the step of mixing a solution containing magnesium ions with the above Prussian blue precipitate.

2. In Paragraph 1, A method for preparing magnesium-Prussian blue (Mg-PB) nanoparticles, wherein in step 2) above, the solution containing magnesium ions is selected from the group comprising magnesium hydroxide, magnesium chloride, magnesium oxide, magnesium nitrate, and magnesium sulfate.

3. In Paragraph 1, A method for preparing magnesium-Prussian blue (Mg-PB) nanoparticles, wherein the stabilizer of step 1) above is selected from the group comprising polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), citric acid, polyvinyl alcohol (PVA), polydopamine (PDA), sodium dodecyl sulfate (SDS), Tween surfactant, polymethyl methacrylate (PMMA), dextran, glutathione, cysteine, and lysine.

4. In Paragraph 1, A method for manufacturing magnesium-Prussian blue (Mg-PB) nanoparticles, wherein the magnesium-Prussian blue (Mg-PB) nanoparticles have a size of 10 to 100 nm.

5. Magnesium-Prussian blue (Mg-PB) nanoparticles produced by the manufacturing method of claim 1.

6. An antioxidant comprising magnesium-Prussian blue (Mg-PB) nanoparticles prepared by the manufacturing method of claim 1.

7. A pharmaceutical composition for the prevention or treatment of diseases caused by excessive production of reactive oxygen species, comprising magnesium-Prussian blue (Mg-PB) nanoparticles produced by the manufacturing method of claim 1 as an active ingredient.

8. In Paragraph 7, A pharmaceutical composition wherein the disease caused by the excessive generation of the above-mentioned reactive oxygen species is arteriosclerosis, hypertension, ischemic disease, stroke, Parkinson's disease, Alzheimer's disease, inflammatory bowel disease, rheumatoid arthritis, chronic obstructive pulmonary disease, asthma, atopic dermatitis, cancer, diabetes, chronic ulcer, burn, or wound.

9. In Paragraph 8, A pharmaceutical composition wherein the above-mentioned ischemic disease includes angina pectoris, myocardial infarction, ischemic stroke, transient ischemic attack, peripheral artery disease, renal ischemic injury, hepatic ischemic injury, or mesenteric ischemia.

10. A quasi-drug composition for the prevention or treatment of diseases caused by excessive production of reactive oxygen species, comprising magnesium-Prussian blue (Mg-PB) nanoparticles produced by the manufacturing method of claim 1 as an active ingredient.

11. A cosmetic composition for preventing or improving symptoms or diseases caused by reactive oxygen species, comprising magnesium-Prussian blue (Mg-PB) nanoparticles produced by the manufacturing method of claim 1 as an active ingredient.

12. In Paragraph 11, A cosmetic composition wherein the symptoms or diseases caused by the above-mentioned active oxygen are skin aging, wrinkle formation, skin pigmentation, atopy, acne, psoriasis, or eczema.

13. In Paragraph 11, The above cosmetic composition is an ampoule, cream, lotion, toner, essence, or pack.

14. A topical skin composition for promoting wound healing or skin regeneration, comprising magnesium-Prussian blue (Mg-PB) nanoparticles produced by the manufacturing method of claim 1 as an active ingredient.

15. A medical device comprising magnesium-Prussian blue (Mg-PB) nanoparticles produced by the manufacturing method of claim 1 as an active ingredient.

16. In Paragraph 15, The above medical device is a medical device that is a wound dressing.

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