RU2790539C2 - Nano-capsules covered with chitosan and their use - Google Patents

Abstract

FIELD: medicine; pharmaceutics; cosmetology; food industry.

SUBSTANCE: nano-capsule is proposed, having a size of 30-100 nm, in which a nanoparticle having a size of 5-50 nm and containing a drug and Pluronic is covered with chitosan having molecular weight of 3-20 kDa. The nano-capsule is a preparation for external application on the skin. It is obtained by a method including the first stage of obtainment of a reaction solution by dissolution of a drug and Pluronic with a value of hydrophilic-lipophilic balance (HLB) from 8 to 29 in an organic solvent and their reaction at a room temperature. At the second stage, a nanoparticle is obtained, having a size of 5-50 nm, by addition of drops of the reaction solution from the first stage to distilled water in accordance with nano-precipitation with their continuous mixing and removal of the organic solvent from the reaction solution by natural evaporation. At the third stage, the nanoparticle is covered with chitosan by addition of chitosan with molecular weight of 3-20 kDa to it in distilled water and mixing. A drug delivery system, a cosmetic composition, and a composition for a medical device are also proposed, containing the resulting nano-capsule.

EFFECT: inventions allow for obtainment of nano-capsules characterized by high stability, increase in permeability of poorly soluble drugs through the skin, providing their efficiency.

9 cl, 21 dwg, 2 tbl, 8 ex

Description

Field of invention

The present disclosure relates to a chitosan-coated nanocapsule and its use, and more specifically, to a nanocapsule in which a Pluronic-containing nanoparticle is coated with chitosan, and its use.

State of the art invention

Nanoparticles are generally defined as particles of matter 1 to 1000 nm in diameter that are larger than an atom and smaller than a cell, and have been developed as substances that can provide new applications by increasing surface area or permeability effect while decreasing their size. In particular, they are increasingly being used in the fields of electronic components using electromagnetic properties, in the medical and cosmetic industries using drug absorption properties, in the field of photocatalysts, battery production, etc.

The nanoparticle can generally be made from an inert material such as gold, tin oxide, a polymeric material such as albumin, etc. When introduced into a biological system, such particles exhibit different physical, chemical and biological properties, such as membrane permeability, optical activation, control of aggregation at the molecular level, etc., compared to larger diameter particles.

A nanocapsule is a nanometer-sized spherical shell with an empty inner space that can contain various substances. A liposome, which is a kind of typical nanocapsule, is a spherical vesicle having at least one bilayer composed of amphipathic phospholipids and other components. Because liposomes are capable of containing water-soluble drugs in their internal void space, they are used for drug delivery. However, the applications of such liposomes are limited because they are structurally unstable and have poor permeability. Thus, many tests have been carried out to improve the stability and permeability of hollow capsules, which has led to success in creating nanocapsules formed from polymers.

In the field of medicine, many studies have been carried out on the use of nanocarriers containing various drugs to deliver drugs through cell membranes for the manifestation of the therapeutic effect of drugs. Note typical drug carriers. First, the liposome is a carrier composed of phospholipids and can contain both lipophilic and hydrophilic drugs. Liposomes are formed from biocompatible materials and are non-toxic, and their surfaces can be suitably modified depending on the purpose. However, liposomes can be taken up by the reticuloendothelial system in hepatocytes or splenocytes, undergo rapid clearance from the blood, and be destroyed, so that only a small number of liposomes reach the target. To overcome this shortcoming, liposomes are subjected to surface modification with polyethylene glycol (PEG) to increase in vivo blood retention time or treatment with various antibodies or ligands to enhance targeting. The micelle serves as a carrier and is formed by copolymers having hydrophilic and hydrophobic chains. A micelle in water forms a spherical aggregate with hydrophobic regions sequestered in the center of the micelle. Research on increasing the solubility and bioavailability of insoluble drugs by incorporating insoluble drugs into the micelle core is ongoing (KWON, Ik Chan, 2010).

In addition to developing new materials for functional cosmetics for skin whitening, wrinkle reduction, antioxidant protection and anti-aging, an important task in the cosmetic industry is to develop a technology to increase the rate of transdermal absorption when applied to the skin. The skin functions as a barrier to the active ingredients so that the active ingredients, even though they are excellent in efficacy, cannot exert their effects when applied to the skin. Thus, one of the biggest challenges in the cosmetics industry is to ensure the absorption of active ingredients and maximize their effectiveness without irritating the skin. To this end, a variety of methods have been proposed to promote the absorption of the active ingredients. An active search is currently ongoing, covering effects ranging from local to systemic, demonstrating that oil-in-water (O/W) formulations of 500 nm or less are more likely to penetrate the skin (KIM, Eun Ju et al. , 2010).

The formulation approach has been predominantly studied in comparison to other methods for increasing the rate of transdermal absorption. The formulation approach can be basically divided into five forms. The first approach is a pH-sensitive polymer hydrogel. That is, a cosmetic product formulated as a pH sensitive polymer hydrogel can benefit from a system that keeps an environmentally unstable active ingredient stable and allows the active ingredient to be rapidly released and absorbed into the skin when applied to the skin. The second approach is a polymer micelle which has hydrophilic polymers and hydrophobic polymers conjugated to each other in the form of block copolymers and is thus effective for dispersing the hydrophobic active ingredient in an aqueous liquid phase. Thirdly, nanoemulsion, which is a kind of emulsion, has a particle size of 100 to 500 nm and does not undergo aggregation or fusion between particles unlike conventional emulsions, thus maintaining long-term stability even under low viscosity conditions. Fourth, a liposome is provided that consists of a lipid bilayer structurally similar to cell membranes or intercellular lipids of the stratum corneum, and thus fuses with cell membranes to effectively deliver the active ingredient to cells. Finally, ethosomes and elastic liposomes are offered. Designed to enhance skin permeability, ethosomes and elastic liposomes, unlike liposomes, have membranes that are more flexible and easier to deform (Chung, J.Y., et al., 2014).

In the food industry, nanoparticles or capsules are mainly used. They protect nutritional ingredients from external factors such as light, oxygen, humidity, temperature, etc. to reduce the loss of nutritional ingredients, and have advantages including increased value, physiological activity, stability, and target control, thus finding various applications even in high value products in the future. Being small in size and with a large surface area, compared to traditional food substances, food substances to which nanotechnology has been applied show improved permeability and retention time of particles and capsules, and thus their absorption and bioavailability in vivo will increase. In addition to improved solubility and dispersibility, the substances have the ability to pass through the difficult lipid bilayer of cells and thus can be used for efficient application of functional materials (KIM, Sae Hoon et al., 2014).

Leading to the present disclosure, the intensive and careful search carried out by the inventors of the present invention in developing a system for the delivery of physiologically effective substances, has provided a method for the manufacture of nanocapsules that have excellent stability and are characterized by a particle size of 500 nm or less, and in particular 200 nm or less. , and finding that, when manufactured by this method, nanocapsules containing insoluble drugs loaded therein exhibit high skin permeability and ensure transdermal drug delivery and drug efficacy in the body. It was also found that oral administration of the manufactured nanocapsules increased the bioavailability of the active ingredients.

As a prior art, Korean Patent No. 1698809 discloses a multilayer nanoparticle containing an insoluble drug, pluronic and chitosan with the additional inclusion of a glycol-based compound in the first core containing the drug, and a polyoxyether compound or a polyoxycastor oil compound in the second layer containing poloxamer , which differs in configuration from a nanoparticle consisting only of insoluble drug and pluronic in accordance with the present disclosure. In addition, Korean Patent No. 1748127 discloses a chitosan-coated nanoparticle containing a drug and pluronic (poloxamer), which is obtained by adding pluronic and a copolymer of lactic and glycolic acids SMHA to a drug to obtain a nanoparticle and coating the nanoparticle with chitosan. The configuration of the nanoparticle differs from the nanocapsule obtained by coating the nanoparticle consisting only of the drug and Pluronic with chitosan. Nowhere in said document is the excellent skin penetration effect of the nanocapsule according to the present disclosure described.

Non-patent document Escobar-Chavesz, J.J., et al., (2006) discloses pharmaceutical formulations, nanoparticles, temperature sensitivity, and dermal delivery with respect to Pluronic gel indicating chitosan's skin permeability. However, nowhere in said document are disclosed the nanocapsule in which the drug-Pluronic-containing nanoparticle is coated with chitosan and its skin-permeability effects.

Disclosure of invention

Technical task

One aspect of the present disclosure is to provide a chitosan-coated nanocapsule and a method for making the same.

Another aspect of the present disclosure is to provide a composition containing a nanocapsule for use in various applications.

The solution of the problem

The present disclosure relates to a nanocapsule in which a nanoparticle containing an active agent and pluronic is coated with chitosan.

The nanocapsule can be made using the first step of obtaining a reaction solution by dissolving the active agent and pluronic in an organic solvent and reacting it at room temperature; the second stage of obtaining nanoparticles by adding drops of the reaction solution from the first stage to distilled water, continuously stirring it and removing the organic solvent from the reaction solution by natural evaporation; and the third stage of coating the nanoparticles from the second stage by adding chitosan to it.

The active agent may be used in an amount of from 0 (exclusively) to 20 mass fractions (inclusive) per 100 mass fractions of Pluronic.

The active agent may be at least one selected from the group consisting of: an anti-cancer agent, an immunosuppressant, an antioxidant, an anti-inflammatory agent, an anti-wrinkle agent, an anti-hair loss agent, a wound healing agent, a skin whitening agent, a dietary supplement, an immunogen, a protein. as a therapeutic agent, revascularization agent, antifungal agent, antibiotic, antiviral agent, sedative agent, analgesic, anti-aging agent, anti-wrinkle agent, skin whitening agent, skin depigmentation agent, UV blocking agent, dye, coloring agent, deodorant products and air freshener.

The active agent may be a fat soluble or insoluble drug. The fat soluble or insoluble drug may be at least one selected from the group consisting of: fat soluble or insoluble anticancer agents including paclitaxel, docetaxel and tetradrine fat soluble or insoluble immunosuppressants including cyclosporine A and dexamethasone fat soluble or insoluble antioxidants, including tocopheryl acetate, astaxanthin, curcumin, and ascorbyl palmitate, fat-soluble or insoluble anti-inflammatory agents, including dexpanthenol and caffeic acid phenethyl ester (caffeic acid phenethyl ester), fat-soluble or insoluble anti-inflammatory agents wrinkles, including retinyl palmitate, fat-soluble or insoluble anti-hair loss preparations, including minoxidil and finasteride, fat-soluble or insoluble wound healing agents, including Centella asiatica extract and beta-sitoster ol, fat soluble or insoluble skin whitening agents including ascorbyl tetraisopalmitate, and fat soluble or insoluble nutritional supplements including tetrapeptide collagen.

The active agent may be a water-soluble drug, which may be at least one selected from the group consisting of: water-soluble anticancer agents, including doxorubicin; water-soluble anti-inflammatory agents, including phospholipase A2 (PLA2); water-soluble immunogens, including ovalbumin; water-soluble proteins as therapeutic agents, including bovine serum albumin; water-soluble wound healing agents, including the basic fibroblast growth factor (b - FGF (from the English - basic fibroblast growth factor)); and water-soluble revascularization agents, including vascular endothelial growth factor (VEGF).

The Pluronic may be at least one selected from the group consisting of Pluronic L35, Pluronic L43, Pluronic L44, Pluronic L64, Pluronic F68, Pluronic P84, Pluronic P85, Pluronic F87, Pluronic F88, Pluronic F98, Pluronic P103, Pluronic P104 , Pluronic P105, Pluronic F108, Pluronic P123 and Pluronic F127.

The nanoparticle size can vary from 5 to 80 nm and preferably from 5 to 50 nm, as measured at 32.5-37°C.

Chitosan can be characterized by a molecular weight of 3-100 kDa.

Chitosan may be contained in an amount of 0.001-200 mass fractions per 100 mass fractions of Pluronic.

The nanocapsule may have a particle size of 700 nm or less, as measured at 32.5-37°C. The particle size of the nanocapsule may be preferably 30-500 nm, more preferably 30-300 nm and most preferably 30-100 nm at 32.5-37°C.

The organic solvent in the first step may be at least one selected from the group consisting of acetone, DMSO (dimethyl sulfoxide), ethanol, acetonitrile, tetrahydrofuran, chloroform and dichloromethane.

In the second stage, distilled water can be used in a volume 4 times the volume of the organic solvent from the first stage.

The nanocapsule can enhance skin permeability by 2 times, preferably 5 times, more preferably 10 times, and even more preferably 14 times compared to the active agent alone.

In addition, the present disclosure relates to a drug delivery system, a cosmetic composition, a health functional food composition, a composition for medical instruments, and a composition for daily life items, each of which contains a nanocapsule.

The following is a detailed description of the present disclosure.

The present disclosure relates to a chitosan-coated nanocapsule, and specifically to a nanocapsule in which a nanoparticle containing an active agent and pluronic is coated with chitosan.

As used herein, the term "nanocapsule" refers to a hollow spherical capsule at the nanoscale, the empty core of which can be loaded with various substances, such as an active agent.

The nanocapsule according to the present disclosure can be obtained using a method known in the art, preferably nanoprecipitation, a membrane resuspension method, and more preferably a nanoprecipitation method.

Most preferably, a nanocapsule can be made using a method comprising the first step of obtaining a reaction solution by dissolving the active agent and pluronic in an organic solvent and reacting them at room temperature to obtain a reaction solution; the second stage of obtaining nanoparticles by adding drops of the reaction solution from the first stage to distilled water, continuously stirring it and removing the organic solvent of the reaction solution by natural evaporation; and a third step of coating the nanoparticle from the second step by adding chitosan thereto.

The organic solvent in the first step may be at least one selected from the group consisting of, but not limited to, acetone, DMSO (dimethyl sulfoxide), ethanol, acetonitrile, tetrahydrofuran, chloroform, and dichloromethane. The organic solvent is preferably at least one selected from the group consisting of acetone, tetrahydrofuran, ethanol and acetonitrile, and more preferably acetone.

In the second stage, distilled water can be used in an amount of 2-10 times, preferably 2-5 times, and more preferably 4 times the volume of the organic solvent from the first stage. If distilled water is used in a volume less than twice the volume of organic solvents, then partial precipitation may occur, and if it is 10 times higher, then the chitosan coating may be unstable, or the concentration of nanocapsules may be diluted, which is undesirable, since it requires additional method of enrichment.

The active agent is an effective substance, water-soluble or fat-soluble, and may be at least one selected from the group consisting, without limitation, of: anti-cancer agent, immunosuppressant, antioxidant, anti-inflammatory agent, anti-wrinkle agent, anti-hair loss drug, wound healing agents, skin whitening agents, nutritional supplement, immunogen, protein as a therapeutic agent, revascularization agent, antifungal agent, antibiotic, antiviral agent, sedative agent, analgesic, anti-aging agent, anti-wrinkle agent, skin whitening agent, depigmentation agent leather, UV blocking agent, dye, coloring agent, deodorant and air freshener.

The active agent may be contained in the minimum amount necessary to ensure its action and effectiveness, up to 20 mass fractions, that is, in an amount from 0 mass fractions (exclusively) to 20 mass fractions, based on 100 mass fractions of Pluronic. Preferably, the active agent may be present in an amount of 0 mass fractions (exclusively) to 10 mass fractions. If the amount of active agent exceeds 20 mass fractions, then the nanoparticles become too large in size or cannot carry all of the active agent, being unable to transport the exact effective amount of the active agent.

According to the present disclosure, "pluronic" (poloxamer) is a hydrophilic polymer characterized by temperature sensitivity, and there are derivatives with different HLB (hydrophilic-lipophilic balance) values. The Pluronic may be at least one selected from HLB 8-29 Pluronics, for example from the group consisting of: Pluronic L35, Pluronic L43, Pluronic L44, Pluronic L64, Pluronic F68, Pluronic P84, Pluronic P85, Pluronic F87, Pluronic F88, Pluronic F98, Pluronic P103, Pluronic P104, Pluronic P105, Pluronic F108, Pluronic P123 and Pluronic F127, and preferably Pluronic with HLB 15-29, for example, from the group consisting, without limitation, of: Pluronic L35, Pluronic L44, Pluronic L64, Pluronic F68, Pluronic P85, Pluronic F87, Pluronic F88, Pluronic F98, Pluronic P105, Pluronic F108 and Pluronic F127.

The nanoparticle contains an active agent and a Pluronic and is temperature sensitive so that its size varies depending on the temperature at which the measurement is made. In particular, the particle size may increase with decreasing temperature.

The nanoparticle size may be 5-80 nm and preferably 5-50 nm as measured at 32.5-37°C.

Used in this document, the term "chitosan" refers to a polysaccharide, which is obtained by partial deacetylation of chitin. Chitosan is a non-toxic, biocompatible and highly biodegradable polymer with high hydrophilicity and mucoadhesiveness. Showing a tendency to high solubility and a positive charge in an acidic environment, chitosan easily attaches to sites such as mucous membranes. In addition, chitosan has an antimicrobial effect and a hemostatic effect. Generally, chitosan is highly soluble in an acidic solution such as acetic acid, lactic acid, etc. When applied to the human body, a solution of chitosan in acid may cause skin irritation or disturbance due to pH changes in the human body.

In contrast, the chitosan according to the present disclosure, which is readily soluble in water, can overcome the problems encountered when using chitosan dissolved in an acidic solution. Chitosan may have a molecular weight of 3-100 kDa, preferably 3-20 kDa and more preferably 3-10 kDa. Chitosan with a molecular weight greater than 100 kDa is undesirable due to its poor water solubility.

Chitosan may be contained in an amount of 200 mass fractions or less, preferably in an amount of 0.001-200 mass fractions, and more preferably in an amount of 0.001-100 mass fractions per 100 mass fractions of Pluronic. Less than 0.001 mass fraction of chitosan is too small to cover the nanoparticle sufficiently, making it difficult to provide a positive charge on the surface of the nanoparticle. If chitosan is used in an amount of more than 200 mass fractions, the nanocapsule becomes too large or undergoes partial precipitation.

The nanocapsule is sensitive to temperature, and the particle size increases with decreasing temperature. The nanocapsule has a particle size of 1000 nm or less, as measured at 10°C, and decreases in size at temperatures above 10°C.

The nanocapsule preferably has a particle size of preferably 700 nm or less at 32.5-37°C, more preferably a particle size of 30-500 nm, even more preferably a particle size of 30-300 nm, and most preferably a particle size of 30-100 nm. With a particle size exceeding 700 nm, the nanocapsule does not penetrate well through the skin when applied to the skin.

The empty core of the nanocapsule can be loaded with various active agents. In addition, the nanocapsule swells at low temperatures due to its temperature sensitivity, which allows the introduction of active agents between the pluronic materials providing the composition of the nanocapsule, whereby both fat-soluble and water-soluble active agents can be retained.

Being coated with chitosan, the nanocapsules are positively charged on their surface and thus can enhance skin permeability and mucoadhesion.

The permeability through the skin of the chitosan-coated nanocapsule is significantly higher than the permeability of polymer capsules (PluNC) without chitosan, and is six times or more higher than most commercially available liposomal formulations, and can also increase two or more times, preferably five or more times, more preferably 10 times or more, and even more preferably 14 times or more compared to nanocapsules treated with the active agent alone.

The nanocapsule is produced by physical bonding between the pluronics constituting the nanoparticle and the chitosan deposited on the nanoparticle, and a separate polymer manufacturing method is not required, unlike nanoparticles made using polymers of pluronic and chitosan obtained by chemical bonds between pluronics and chitosan, and thus, the toxicity of the binder used to form the polymer can be ignored.

Moreover, the present disclosure relates to a drug delivery system containing a nanocapsule.

As used herein, the term "drug delivery system" refers to a system for delivering a therapeutically effective drug to an in vivo site in need thereof. Designed to efficiently deliver the right amount of drug to the tissue when needed, a drug delivery system can be understood as a drug composition, drug effect, formulation method, or drug formulation.

The drug delivery system may be a nanocapsule containing a therapeutically effective drug.

The drug may be at least one selected from the group consisting, without limitation, of an anti-cancer agent, an immunosuppressant, an antioxidant, an anti-inflammatory agent, an anti-wrinkle agent, an anti-hair loss agent, a wound healing agent, a skin whitening agent, a dietary supplement, an immunogen, protein as a therapeutic agent, a revascularization agent, an antifungal agent, an antibiotic, an antiviral agent, a sedative, an analgesic, an anti-aging agent, an anti-wrinkle agent, a skin whitening agent, a skin depigmentation agent, a UV blocking agent, a dye, a coloring agent, deodorant and air freshener.

Examples of the antifungal agent include, without limitation, polyenes such as amphotericin B, nystatin, fungicidin, etc., azoles, such as ketoconazole, itraconazole, etc., allylamines, such as butenafine, terbinafine, naftifine, etc. ., echinocandins such as anidulafungin, caspofungin, etc., and other antifungals such as aurones, benzoic acid, ciclopirox, flucytosine, griseofulvin, etc.

The antibiotic may be selected without limitation from penicillins, cephalosporins, polymyxins, sulfonamides, quinolines, rifampicin, aminoglycosides, macrolides, tetracyclines.

The antiviral agent may be, without limitation, an anti-influenza agent, for example, amantadine, rimantadine, oseltamivir, zanamivir, etc.; a herpes virus agent such as vidarabine, acyclovir, foscarnet, etc.; a hepatitis B virus agent such as lamivudine, entecavir, tenofovir, etc.; or an anti-human immunodeficiency virus agent, for example, zidovudine, didanosine, zalcitabine, efavirenz, rilpivirine, saquinavir, ritonavir, raltegravir, elvitegravir, dolutegravir, enfuvirtide, etc.

The sedative may be, without limitation, zolpidem, diazepam, or morphine.

The scope of analgesics includes, without limitation, acetaminophen, non-steroidal anti-inflammatory drugs, morphine, fentanyl, oxycodone, and hydromorphone.

Examples of the wound healing agent include, but are not limited to, centella asiatica, collagen, and epithelial growth factor (EGF).

The anti-inflammatory agent may be, without limitation, meloxicam, silibinin, indomethacin, propolis, caffeic acid phenethyl ester, etc.

An example of an anticancer agent can be, without limitation, paclitaxel, estrogen, doxorubicin, 5-fluorouracil, popinavir, nimusulide, progesterone, repaglinide, tetracycline, all-trans retinoic acid, luteolin, an inhibitor of the vascular endothelial growth factor receptor (VEGFR). growth factor receptor)), Wnt modulator (derived from English Wg (wingless - wingless) and Int) / β-catenin, hedgehog inhibitor and PI3K modulator (from English phosphoinositol-3-kinase - phosphoinositol-3-kinase) / Akt (from English - RAC-alpha serine / threonine-protein kinase - RAC-alpha serine / threonine protein kinase) / mTOK (from English mammalian target of rapamycin - mammalian target of rapamycin).

The immunosuppressant may be, without limitation, cyclosporin A, tacrolimus, methotrexate, rapamycin, or sirolimus.

An anti-hair loss drug that refers to a substance effective for preventing hair loss or promoting hair regrowth may include, but is not limited to, finasteride, minoxidil, cyclosporine A, a natural anti-hair loss drug such as Coicis semen extract, Rubus coreanus extract, Glycyrrhiza radix extract, Thuja orientalis extract, Angelical radix extract, Cornus officinalis extract, etc., or a peptide which is effective in promoting hair regrowth.

The active agent may be a fat soluble or insoluble drug which may be at least one selected from the group consisting of fat soluble or insoluble anticancer agents including paclitaxel, docetaxel and tetradrine, fat soluble or insoluble immunosuppressants including cyclosporin A and dexamethasone, fat-soluble or insoluble antioxidants, including tocopheryl acetate, astaxanthin, curcumin, and ascorbyl palmitate, fat-soluble or insoluble anti-inflammatory agents, including dexpanthenol and caffeic acid phenethyl ester (CARE), fat-soluble or insoluble anti-wrinkle agents, including retinyl palmitate, fat-soluble or insoluble anti-hair loss preparations, including minoxidil and finasteride, fat-soluble or insoluble wound healing agents, including Centella asiatica extract and beta-sitosterol, fat soluble or insoluble skin whitening agents including ascorbyl tetraisopalmitate; and fat soluble or insoluble nutritional supplements including tetrapeptide collagen.

The active agent may be a water-soluble drug, which may be at least one selected from the group consisting of water-soluble anticancer agents, including doxorubicin; water-soluble anti-inflammatory drugs, including phospholipase A2 (PLA2 (from the English phospholipase A2)); water-soluble immunogens, including ovalbumin; water-soluble proteins as therapeutic agents, including bovine serum albumin; water-soluble wound healing agents, including fibroblast growth factor (b-FGF); and water-soluble revascularization agents including vascular endothelial growth factor (VEGF).

The drug delivery system may contain a nanocapsule and a pharmaceutically acceptable excipient.

According to conventional methods, the drug delivery system may be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc.; means for external use; suppositories and sterile injectable solutions. Examples of the carrier, excipient and diluent to be combined in the pharmaceutical composition may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Conventionally used fillers, fillers, binders, wetting agents, disintegrants, diluents such as surfactants or auxiliaries can be used for the composition. Examples of solid formulations for oral administration may include tablets, pills, powders, granules, capsules, and the like. Such solid formulations are obtained, for example, by adding at least one excipient to the nanocapsule, for example starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition, lubricants such as magnesium stearate, talc, etc. can be used in addition to simple excipients. Examples of liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and various kinds of excipients, such as wetting agents, sweeteners, flavors, preservatives, etc., can be used in addition to simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Examples of non-aqueous suspension solvents may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injection ester such as ethylolate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cocoa butter, laurinum, glycerogelatin, and the like.

In addition, topical preparations for the skin can be prepared without limitation in the form of ointments, lotions, aerosols, patches, creams, gelling agents or gels. The external preparation on the skin may contain a penetration enhancer, for example, in particular, dimethyl sulfoxide, dimethylacetamide, dimethylformamide, a surfactant, alcohol, acetone, propylene glycol or polyethylene glycol, without limitation. The frequency of application of the topical preparation to the skin can vary greatly depending on various factors, including the age, sex and weight of the subject being treated, the particular disease or condition being treated, the severity of the disease or condition, the route of administration and the solution. prescribing physician. The external preparation on the skin is recommended to be applied ten times a day to ten times a month, preferably four times a day to four times a week, more preferably three times a day to three times a week, even more preferably once or twice per day.

The drug delivery system of the present disclosure may be administered to mammals, such as mice, pets, humans, companion animals, etc., in a variety of ways. All routes of administration may be contemplated. For example, administration can be by oral, rectal, intravenous, intramuscular, subcutaneous, dermal, dural, or intracerebroventricular routes, with transdermal administration being preferred.

The present disclosure relates to a cosmetic composition containing a nanocapsule.

The nanocapsule may contain a functional cosmetic substance that has the function of skin whitening, wrinkle reduction, anti-oxidation, anti-aging, anti-inflammatory, ultraviolet UV blocking, etc.

Examples of the skin whitening agent include, without limitation, Paper mulberry extract, arbutin, ethylascorbyl ether, licorice root oil-soluble extract, ascorbyl glycoside, niacinamide, α-bisabolol, and ascorbyltetraisopalmitate.

Examples of a wrinkle-related functional substance include, without limitation, vitamin A, vitamin A derivatives (retinyl palmitate, retinyl acetate, etc.), adenosine, and polyethoxylated retinamide.

Examples of the antioxidant functional substance can be, without limitation, vitamin A, vitamin A derivatives, vitamin E, vitamin E derivatives, carotene, lycopene, lutein, coenzyme Q10, and astaxanthin.

The cosmetic composition may include an adjuvant conventionally used in the field of cosmetics, such as a hydrophilic or lipophilic gelling agent, a hydrophilic or lipophilic active agent, a preservative, an antioxidant, a solvent, a fragrance, a bulking agent, a blocking agent, a pigment, a deodorant, a coloring agent, etc. in addition to nanocapsules.

The amount of adjuvant is at a level acceptable in the prior art. In any case, the adjuvants and their ratios can be chosen so as not to adversely affect the desired properties of the cosmetic composition in accordance with the present disclosure.

The cosmetic composition may be in at least one composition selected from the group consisting, without limitation, of lotion, skin softener, skin tonic, ampoule, astringent, cream, foundation, essence, pack, mask pack, soap, cleanser. body cleanser, foam cleanser, shampoo, rinse, hair care product, hair oil, body oil and body lotion.

The cosmetic composition can be used every day, and can also be used for an indefinite period of time. Preferably, the amount, frequency and duration of application can be adjusted according to the user's age, skin condition or skin type.

In addition, the present disclosure relates to a health functional food composition containing a nanocapsule.

The nanocapsule may include a healthy functional food material.

The health functional food can be a vitamin, mineral, probiotic, bioactive peptide, antioxidant, plant sterol, plant extract, coenzyme Q10, omega-3, astaxanthin, and the like. Preferably, it can be, but is not limited to, collagen tripeptide, red ginseng oil, astaxanthin, and omega-3.

The wellness functional food composition may include a nanocapsule and a sitologically acceptable supplement.

The health functional food composition of the present disclosure may be in the form of tablets, capsules, pills, or liquids, and examples to which the nanocapsules of the present disclosure may be added include various foods, beverages, chewing gums, teas, vitamin complexes, health-improving functional food products, etc.

According to another aspect of the present disclosure, there is provided a medical device containing a nanocapsule according to the present disclosure.

The medical device may be a filler, a wound dressing, a bone graft material, an implant coating material, an embolization agent, a diagnostic agent, or the like.

According to another aspect of the present disclosure, a composition for items necessary for everyday life is provided, containing a nanocapsule in accordance with the present disclosure.

The composition for daily life items may be, but is not limited to, a dye, a coloring agent, a deodorant, and an air freshener.

Advantageous Effects of the Present Invention

The present disclosure relates to a chitosan-coated nanocapsule and its use. According to the present disclosure, a method for manufacturing a nanocapsule having a particle size of 500 nm or less and, in particular, 200 nm or less, and excellent stability has been found, and by this method, nanocapsules containing insoluble drugs loaded therein exhibited high permeability. through the skin, guaranteed transdermal delivery of drugs and the effectiveness of drugs in the body. It was also found that oral administration of the manufactured nanocapsules increased the bioavailability of the active ingredients.

Therefore, it is expected that the chitosan-coated nanocapsules of the present disclosure will find use in the development of a delivery system that can deliver insoluble drugs or effective ingredients with markedly improved efficacy to humans and animals such as livestock, companion animals, etc. in the medical, cosmetic and food industries.

Brief disclosure of graphic materials

In FIG. 1 shows the diameters of the obtained nanoparticles in accordance with the types of Pluronic and temperatures.

In FIG. 2 shows the performance of chitosan-coated nanocapsules according to Pluronic types analyzed for (a) morphological property, (b) size, (c) polydispersity, and (d) surface charge.

In FIG. 3 shows the characteristics of chitosan-coated nanocapsules according to the molecular weights of chitosan analyzed for (a) size, (b) polydispersity, (c) surface charge, and (d) morphology.

In FIG. 4 shows the performance of chitosan-coated nanocapsules according to solvent types analyzed for (a) size, (b) polydispersity, and (c) surface charge.

In FIG. 5 shows particle sizes in accordance with methods for preparing chitosan-coated nanocapsules containing loaded drugs in accordance with the present disclosure.

In FIG. 6 shows the particle sizes of chitosan-coated nanocapsules containing drugs loaded into them, in accordance with the mixing ratios of the solvent and deionized water in their manufacture using nanoprecipitation.

In FIG. 7 shows the cytotoxicity of chitosan-coated nanocapsules according to the present disclosure.

In FIG. 8 shows the performance of chitosan-coated nanocapsules containing paclitaxel and docetaxel loaded therein according to loaded amounts of drugs analyzed for (a) size, (b) polydispersity, and (c) surface charge.

In FIG. 9 shows the performance of chitosan-coated nanocapsules loaded with (a) cyclosporine A and (b) dexamethasone according to loaded amounts of drugs analyzed for size, polydispersity, and surface charge.

In FIG. 10 shows the performance of chitosan-coated nanocapsules loaded with (a) retinyl palmitate and (b) tocopheryl acetate according to loaded amounts of drugs analyzed for size, polydispersity, and surface charge.

In FIG. 11 shows the performance of chitosan-coated nanocapsules loaded with (a) minoxidil and (b) finasteride according to loaded amounts of drugs analyzed for size, polydispersity, and surface charge.

In FIG. 12 shows the characteristics of the size and dispersion of nanocapsules according to temperatures.

In FIG. 13 shows the characteristics of chitosan-coated nanocapsules containing doxorubicin loaded therein according to the loaded amounts of drug.

In FIG. 14 shows the performance of chitosan-coated nanocapsules containing ovalbumin loaded therein according to the loaded amounts of drug.

In FIG. 15 shows the performance of chitosan-coated nanocapsules loaded with bovine serum albumin according to loaded amounts of drug.

In FIG. 16 shows whether or not chitosan-coated nanocapsules containing cyclosporin A (CsA@ChiNC), Nile red (Nile red@ChiNC) or pyrene (pyrene@ChiNC) precipitated.

In FIG. 17 shows the performance of chitosan-coated nanocapsules loaded with drugs (CsA and RP) before and after freeze-drying.

In FIG. 18 shows the skin permeability of drugs according to the types of chitosan.

In FIG. 19 shows the skin permeability of chitosan-coated nanocapsules loaded with drug (Nile red) according to the present disclosure.

In FIG. 20 shows the effects of chitosan-coated nanocapsules loaded with a drug (Nile Red) according to the present disclosure on drug delivery to the skin.

In FIG. 21 shows hair regrowth effects according to transdermal drug delivery (Cyclosporin A, CsA) with a chitosan-coated nanocapsule containing a loaded drug according to the present disclosure.

Best Mode for Carrying out the Invention

Next, the preferred embodiments of the present invention will be detailed. However, the present invention is not limited to the embodiments disclosed herein, and may be embodied in other forms. Rather, the embodiments are presented to make the present invention disclosed herein exhaustive and complete, and to sufficiently explain the idea of the present invention to those skilled in the art.

Example 1. Obtaining a nanocapsule coated with chitosan

Example 1-1. Optimization of obtaining a nanoparticle in accordance with the type of pluronic

Pluronic (poloxamer) is a non-ionic triblock copolymer consisting of a central hydrophobic chain of poly (propylene oxide) (PPO (from the English - poly (propylene oxide))), flanked by two hydrophilic chains of poly (ethylene oxide) (PEO (from the English - poly(ethylene oxide))), i.e. PEO-PPO-PEO, which is a typical temperature-sensitive polymer characterized by a temperature-dependent reversible transition of internal structures. There are Pluronic derivatives with different HLB values (hydrophilic-lipophilic balance) depending on the number of moles of PEO and PPO blocks in them. HLB in Pluronics can affect the production of nanoparticles from Pluronics. Thus, the sizes of nanoparticles were measured according to the types of pluronics.

Nanoparticles were obtained using pluronics with HLB 2-29. More specifically, 20 mg of Pluronic was dissolved in 1 ml of acetone to obtain a reaction solution. Drops of the reaction solution were slowly added to 4 ml of deionized water, which was stirred at 530 rpm. After reacting at room temperature for 12 hours or more, the acetone was removed by natural evaporation to obtain pluronic nanoparticles. The nanoparticles thus obtained were analyzed for size using a particle size analyzer (Zetasizer, Nono-Zs, Malvern) and transmission electron microscopy. The results are shown in FIG. 1.

As can be seen in FIG. 1, the measured nanoparticles composed of Pluronic F127 have a diameter of 200 nm or less at 25°C, and their diameter varies from 5 nm to 80 nm at 32.5°C to 37°C, with an average diameter of 50 nm. As for Pluronic F68, its measured diameter is 300 nm or less at 25°C and 200 nm or less at 32.5°C - 37°C.

In addition to Pluronic F127 and Pluronic F68, nanoparticles composed of Pluronics with HLB 2-29 were found to have a diameter of 200 nm at 32.5°C - 37°C.

From the disclosure above it is clear that the particle sizes can be controlled in accordance with the temperatures using pluronics. Designed nanocapsules with a diameter of 100 nm or less. To this end, the diameter of the nanoparticles was optimized to 5-80 nm and preferably 5-50 nm using pluronics with HLB 2-29.

Example 1-2. Optimization of obtaining a nanocapsule in accordance with the type of Pluronic

We studied the optimal conditions for obtaining nanocapsules in accordance with the types of pluronics. To this end, nanoparticles were prepared using Pluronics corresponding to HLB 2-29 in Table 1 below and coated with chitosan to form nanocapsules.

Briefly, 20 mg of each pluronic was dissolved in 1 ml of acetone and stirred at room temperature for 2 hours. The polymer solution was then slowly added dropwise to 4 ml of deionized water with stirring at 400 rpm. Acetone was removed from the thus obtained polymer nanoparticles for 6 hours under a fume hood. Finally, 20 mg of chitosan with a deacetylation degree of 90% and a molecular weight of 10 kDa was added to each polymer nanoparticle, followed by stirring at room temperature for 2 hours to obtain chitosan-coated nanocapsules. Chitosan-coated nanocapsules (ChiNC) designed according to Pluronic species were analyzed for size, dispersibility, and surface charge using an electrophoretic light scattering spectrophotometer (ELS-Z2, Otsuka) and the results are shown in FIG. 2.

As can be seen in FIG. 2, chitosan-coated nanocapsules (ChiNC) constructed from five Pluronics (F127, P123, P188, L35 and L81) differing in HLB (hydrophilic and lipophilic balance) by nanoprecipitation were stable without precipitation with all types of Pluronics.

When prepared from F127 and P123, the chitosan-coated nanocapsules measured for stability had a diameter of 100 nm or less, for example, a diameter ranging from 30 nm to 80 nm, with an average diameter of 60 nm at 32.5°C - 37°C. The diameter of the chitosan-coated nanocapsules obtained from P188 and L35 varied from 209 nm to 688 nm with an average diameter of 500 nm. The diameter of chitosan-coated nanocapsules obtained from L81 was up to 1.4 μm. The chitosan-coated nanocapsules prepared from F127, P123, P188, and L35 had a fineness of 0.3 or less, generally showing monodispersity. The chitosan-coated nanocapsules prepared from L81 were microsized, exhibiting somewhat greater dispersity. The measured surface charge averaged approximately +20 mV because the Pluronic capsules were heavily coated with chitosan.

It is clear from the results that Pluronics with an HLB of 8 to 29 are suitable for optimizing the compositions of chitosan-coated nanocapsules. Depending on the types of pluronics and temperatures, chitosan-coated nanocapsules with a diameter of 700 nm or less, 30-500 nm, 30-300 nm, or 30-100 nm can be formed.

Although the microcapsules are not nanosized, they can be designed even with an HLB index of 8 or less, suggesting that nano- and microsized chitosan-coated capsules can be designed as needed, for example, for application to localized areas.

Example 1-3. Optimization of obtaining a nanocapsule coated with chitosan in accordance with the molecular weight of chitosan

Based on the results of examples 1-1 and 1-2, the optimal conditions were established, taking into account the states of the pluronic, to obtain nanocapsules coated with chitosan in accordance with the molecular weights of chitosan (3 kDa - 100 kDa).

To obtain chitosan-coated nanocapsules in accordance with the present disclosure, the biocompatible material pluronic F127 (poloxamer 407) was used, which was approved by the FDA (Food and Drug Administration). Chitosan-coated nanocapsules were obtained using nanoprecipitation. Briefly, 20 mg of Pluronic F127 was dissolved in 1 ml of acetone to obtain a reaction solution, which was then slowly added dropwise to 4 ml of deionized water with stirring at 530 rpm. During the reaction at room temperature for 12 hours, acetone was removed by natural evaporation to obtain nanoparticles consisting of Pluronic (PluNC). Chitosan was added to PluNC and mixed at room temperature for one hour or longer to obtain chitosan-coated nanocapsules (ChiNC) according to the present disclosure. For this purpose, PluNC was coated with 3, 10, 20, 50, and 100 kDa chitosan. ChiNCs obtained according to the molecular weights of chitosan were analyzed for morphology, size, polydispersity index (PDI) and surface charge using a particle size analyzer (Zetasizer, Nono-Zs, Malvern) and transmission electronic microscopy. The results are shown in Fig.3.

Chitosan was mixed in an amount of 0.001-200 mass fractions with Pluronic. If the amount of chitosan was less than 0.001 mass fraction, then the nanoparticles were not sufficiently covered with chitosan, which hindered the manifestation of positive charges on the surface. More than 200 mass fractions of chitosan made the nanocapsules too large in size or led to partial precipitation. In subsequent experiments, Pluronic was mixed with chitosan in equal amounts.

As shown in FIG. 3, with respect to particle size (a), when coated with 20 kDa chitosan, nanoparticles were formed into chitosan-coated ChiNC nanocapsules (ChiNC 3K, ChiNC 10K, and ChiNC 20K) whose diameter was 100 nm or less, while the use of chitosan with molecular weight 50 kDa and 100 kDa yielded chitosan-coated ChiNC nanocapsules (ChiNC 50K and ChiNC 100K) that were approximately 200 nm in diameter. With respect to polydispersity (b), chitosan-coated nanocapsules with a molecular weight of 20 kDa or less ChiNC (ChiNC 3K, ChiNC 10K, and ChiNC 20K) were observed to have a polydispersity index of 0.2 or less, while chitosan-coated nanocapsules with molecular weights 50 kDa and 100 kDa ChiNC (ChiNC 50K and ChiNC 100K) measured a polydispersity index of approximately 0.2-0.3. With regard to surface charge (c), it was observed that PluNC itself, which was not coated with chitosan, had a negative charge of approximately -5 mV, while being coated with chitosan, the capsules had a positive charge regardless of their molecular weight. In addition, morphological examination showed spherical structures in the nanocapsules, indicating that the nanocapsule increases in size with increasing chitosan molecular weight (d).

Although not shown here, chitosan with a molecular weight greater than 100 kDa had to be dissolved in acetic acid before being applied to the particles. However, when dissolving such a large chitosan in acetic acid for coating, precipitation occurred, reducing the stability of the composition.

From the results, it becomes clear that in the chitosan-coated nanocapsules according to the present disclosure, chitosan can be stably applied to nanoparticles composed of pluronics according to the preparation method, and that the molecular weights of the chitosan to be applied affect the size and fineness of the nanocapsules. In addition, chitosan with a molecular weight of 3–20 kDa made it possible to design nanocapsules that were small and uniform in size and acquired composition stability.

Example 1-4. Optimization of obtaining a nanocapsule coated with chitosan in accordance with the solvent

Optimization was carried out with respect to the solvent used in the method for producing chitosan-coated nanocapsules.

As selected solvents, we analyzed acetone (ACE (from English - acetone)), ethanol (ETH (from English - ethanol)), tetrahydrofuran (THF), chloroform (CHL (from English - chloroform)) and dichloromethane (DCM) . When using such solvents, platforms can be created for loading various drugs (active agents) that differ in solubility in solvents.

First, 20 mg of Pluronic F127 was dissolved in 1 ml each of acetone, ethanol, and tetrahydrofuran according to nanoprecipitation, followed by a reaction for 2 hours. Then the reaction mixture was slowly added dropwise to 4 ml of deionized water with stirring at 400 rpm to obtain polymer nanocapsules, from which the solvents were then removed for 6 hours under a fume hood. Finally, 20 mg of chitosan (deacetylation 90%, molecular weight 10 kDa) was added to the nanocapsules from each solvent and stirred at room temperature for 2 hours to obtain chitosan-coated nanocapsules.

For chloroform, a single emulsion was used. A solution of 20 mg of Pluronic F127 in 1 ml of chloroform was reacted for 2 hours. After that, the reaction mixture was slowly added dropwise to 4 ml of deionized water, which was stirred at 400 rpm, to obtain nanoparticles, which were then dispersed using a homogenizer before removing the solvent for 2 hours by vacuum drying. Finally, 20 mg of chitosan (deacetylation 90%, molecular weight 10 kDa) was added to the polymer nanocapsules and stirred at room temperature for 2 hours to obtain chitosan-coated nanocapsules.

Chitosan-coated 10 kDa (ChiNC 10K) nanocapsules prepared according to nanoprecipitation and single emulsion were analyzed for size, fineness, and surface charge using an electrophoretic light scattering spectrophotometer (ELS-Z2, Otsuka).

As can be seen in Figure 4, the chitosan nanocapsules (ChiNC 10K) obtained by nanoprecipitation using organic solvents of acetone (ACE), ethanol (ETH) and tetrahydrofuran (THF), all of which are highly miscible with water, had a size of 60 nm. and a polydispersity index (PDI) of 0.3 or less, showing monodispersity.

In contrast, chitosan-coated nanocapsules (ChiNC 10K) prepared with a single emulsion using chloroform (CHL), which is an organic solvent with poor miscibility with water, were characterized by a size of approximately 750 nm (same as in dichloromethane) and showed a large index polydispersity (PDI) compared to chitosan-coated nanocapsules obtained by nanoprecipitation using acetone (ACE), ethanol (ETH), and tetrahydrofuran (THF). However, all groups of chitosan-coated nanocapsules exhibited a surface charge of approximately 20 mV, demonstrating that chitosan-coated polymer capsules, i.e., chitosan-coated nanocapsules, can be stably constructed using various solvents and methods.

Example 1-5. Development of a method for obtaining drug-loaded nanocapsules

Chitosan-coated drug-loaded nanocapsules were prepared by membrane nanoprecipitation and resuspension and characterized according to preparation methods. For this purpose, the insoluble substance cyclosporine (hereinafter referred to as "CsA") was used as a drug.

When the membrane was resuspended, 0.2 mg (2 wt%) or 0.6 mg (6 wt%) CsA was dissolved together with 10 mg of Pluronic F126 in 1 ml of acetone and stirred at room temperature for 2 hours to obtain a reaction solution. The reaction solution was kept for 2 hours under a fume hood to form a membrane upon evaporation of the acetone. 5 ml of deionized water was added to the formed membrane, stirred for 30 minutes or longer, and mixed with 10 kDa chitosan with the same mass as that of Pluronic to obtain a nanocapsule.

In another nanoprecipitation method, 0.2 mg or 0.6 mg CsA was stirred together with 10 mg Pluronic F126 in 1 ml acetone at room temperature for 2 hours to obtain a reaction solution, which was then slowly added dropwise to 4 ml deionized water with stirring at 530 rpm. The mixture was stirred for 4 hours under a fume hood so that the acetone spontaneously evaporated. To deionized water containing acetone-removed nanoparticles composed of Pluronic, 10 kDa chitosan was added in the same amount as the amount of Pluronic, followed by mixing to obtain nanocapsules. The particle size of the nanocapsules was measured using a particle size analyzer and transmission electron microscopy. The results are shown in FIG. 5.

As can be seen in FIG. 5, the CsA-free nanocapsules were about 2 times smaller in size when prepared by nanoprecipitation than when prepared by membrane resuspension. CsA-containing nanocapsules, when obtained by resuspension of the membrane, differed greatly from each other in particle size depending on the content of CsA. The nanocapsules were partially aggregated at a CsA content of 6 wt%. On the contrary, the difference in particle size depending on the content of CsA among CsA-containing nanocapsules obtained by nanoprecipitation was small compared to that among nanoparticles obtained by membrane resuspension, whereby nanocapsules could be stably designed within a certain size range without aggregation. Capsules were 100 nm or smaller at 2 wt% CsA and 200 nm or smaller at 6 wt% CsA.

The data show that nanoprecipitation is preferably used to design chitosan-coated nanocapsules containing loaded drug according to the present disclosure in order to improve formulation stability.

Example 1-6. The ratio of solvent and distilled water during nanoprecipitation

The nanocapsules obtained by nanoprecipitation were measured in size according to the ratio of acetone as the solvent of the reaction solution and deionized water used in the preparation of nanoparticles during the procedure of Example 1-5.

Chitosan-coated nanocapsules containing drug loaded therein were obtained by the same method as nanoprecipitation in Example 1-5, except that the drug was loaded at 6% by weight with acetone and deionized water mixed in a ratio of 1:5 or 1:4. The nanocapsules were analyzed for particle size and the results are shown in FIG. 6. For this purpose, the sizes of nanocapsules were measured before and after coating with chitosan.

As shown in FIG. 6, nanocapsules were formed with a size of 100 nm or less both before and after the addition of chitosan by mixing in a 1:4 ratio of acetone with deionized water, in contrast to nanocapsules formed with a 1:5 ratio of acetone and deionized water.

The result indicates that chitosan-coated nanocapsules containing the drug loaded therein according to the present disclosure can be obtained in a stable size by nanoprecipitation using solvent and deionized water at a ratio of 1:4.

Although not shown here, the solvents DMSO (dimethyl sulfoxide), ethanol, acetonitrile, tetrahydrofuran, chloroform, and dichloromethane can also provide dimensionally stable nanocapsule construction when used with deionized water at a ratio of 1:4 (see example 1-4). ).

Example 2 Cytotoxicity Analysis of a Chitosan Coated Nanocapsule

Chitosan coated nanocapsules (ChiNC) obtained in Example 1-3 were analyzed for cytotoxicity.

NIH3T3 cells were seeded at a density of 10,000 cells per well in 96-well plates and incubated for 8-12 hours. Cells were then treated with 10 μg/ml, 20 μg/ml, 50 μg/ml, or 100 μg/ml PluNC or ChiNC, both prepared in Example 1-3, for 24 hours and analyzed for cell viability using CCK8 ( counting kit 8) according to the manufacturer's instructions. The results are shown in FIG. 7. To this end, cell viability for each treatment group was expressed relative to 100% for untreated NIH3T3 cells as a control.

As shown in FIG. 7, PluNC or ChiNC provided 90% or higher cell viability at all concentrations.

The data show that the chitosan-coated nanocapsules of the present disclosure are highly biocompatible without cytotoxicity.

Example 3 Preparation Conditions for a Chitosan-Coated Nanocapsule Containing an Insoluble (Fat-Soluble) Active Agent Loaded into It

In accordance with the present disclosure, production conditions were established for chitosan-coated nanocapsules containing active agents, especially fat-soluble anti-cancer agents (paclitaxel, docetaxel), anti-inflammatory agents (dexamethasone), immunosuppressants (cyclosporine A), antioxidants (tocopheryl acetate), anti-wrinkle agents (retinyl palmitate ), anti-hair loss drugs (minoxidil, finasteride) and anti-aging agents (tocopheryl acetate, retinyl palmitate).

A solution of docetaxel, paclitaxel, dexamethasone, tocopheryl acetate, cyclosporin A or retinyl palmitate in 1 ml of acetone was mixed and reacted with 20 mg of Pluronic F127 for 2 hours. The reaction mixture was then slowly added dropwise to 4 ml of deionized water with stirring at 400 rpm to obtain drug-loaded polymer nanocapsules, from which the acetone was then removed for 6 hours under a fume hood. Finally, 20 mg of chitosan (90% deacetylation, molecular weight 10 kDa) was added to the polymer nanocapsules and stirred for 2 hours at room temperature to obtain chitosan-coated nanocapsules containing drug loaded therein. Drugs that remained unloaded were removed by ultrafiltration (Amicon Ultra-15 filter).

Separately, a solution of finasteride, minoxidil, tocopheryl acetate or retinyl palmitate in 1 ml of ethanol was mixed and reacted with 20 mg of Pluronic F127 for 2 hours. The reaction mixture was then slowly added dropwise to 4 ml of deionized water with stirring at 400 rpm to prepare drug-loaded polymer nanocapsules, from which ethanol was then removed for 6 hours under a fume hood. Finally, 20 mg of chitosan (90% deacetylation, molecular weight 10 kDa) was added to the polymer nanocapsules and stirred for 2 hours at room temperature to obtain chitosan-coated nanocapsules containing drug loaded therein. Drugs that remained unloaded were removed by ultrafiltration (Amicon Ultra-15 filter).

The drug loaded ChiNC 10K thus obtained was analyzed for size, fineness and surface charge using an electrophoretic light scattering spectrophotometer (ELS-Z2, Otsuka).

Example 3-1. Preparation of chitosan-coated nanocapsules containing loaded paclitaxel and docetaxel

As shown in FIG. 8, nanocapsules loaded with paclitaxel (PTX (from English - paclitaxel)) could be obtained, and the loading did not significantly affect the size, dispersion and surface charge until it reached 0.1 wt.%. At a loading content of 0.2 wt.%, the nanocapsules expanded to a diameter of up to 3 μm, but did not precipitate. Thus, it has been found that chitosan-coated microcapsules containing drug loaded therein can be obtained for topical application. However, partial precipitation occurred when the drug loading content reached 0.5 wt%.

It was observed that docetaxel (DOC (from English - docetaxel)) was stably loaded into chitosan-coated nanocapsules with a content of up to 2 wt. %. In addition, found out the conditions for the manufacture of the composition, which guarantee the loading of the drug with a content of up to 3 wt.% without precipitation. Thus, chitosan-coated nanocapsules and chitosan-coated microcapsules can be obtained, each containing an anticancer agent loaded therein.

Example 3-2. Obtaining nanocapsules coated with chitosan containing cyclosporine A and dexamethasone loaded into them

As can be seen in FIG. 9, cyclosporine A (CsA) was stably loaded at up to 5% by weight into chitosan-coated nanocapsules. In addition, formulating conditions were determined which guaranteed drug loading at up to 10 wt % without precipitation. Received covered with chitosan nanocapsules at a load content of 5 wt.%. Considering the applicability of slightly larger chitosan-coated nanocapsules for topical therapy, the drug could be loaded at levels up to 10% by weight. In addition, temperature sensitivity analysis showed that the chitosan-coated nanocapsules had a particle size of 700 nm or more at 10°C and 100 nm or less (30-100 nm) at 32.5°C or 37°C.

Dexamethasone (DEX (from English - dexamethasone)) could be optimally loaded at a content of up to 3 wt.%, and at a loading content of up to 5 wt.% no precipitation occurred, so that it was possible to obtain chitosan-coated nanocapsules with a size of 900 nm.

Example 3-3. Obtaining a chitosan-coated nanocapsule containing retinyl palmitate and tocopheryl acetate loaded into it

As shown in FIG. 10, it was observed that retinyl palmitate (RP (from the English - retinyl palmitate)) is stably loaded in a content of up to 5 wt.% in chitosan-coated nanocapsules. In addition, at a loading content of 10 wt.%, the nanocapsules coated with chitosan had a size of up to 100 nm, but remained unchanged in terms of dispersion and surface charge. Thus, they were stably obtained with the loading content without precipitation.

As can be understood from the data in Table 2 below, retinyl palmitate can be loaded as an active agent at up to 20 wt %, since the nanocapsules, although increased in size, did not differ much in terms of dispersity and surface charge at loading content.

With regard to tocopheryl acetate (TA (from English - tocopheryl acetate)), its optimal loading content was up to 2 wt.%, while precipitation did not occur up to 5 wt.%. Thus, it is possible to obtain chitosan-coated nanocapsules with a size of 90 nm loaded with a drug.

Example 3-4. Obtaining a chitosan-coated nanocapsule containing minoxidil and finasteride loaded into it

As can be seen in FIG. 11, minoxidil (MX (from minoxidil)) did not significantly affect the size, fineness, and charge of the surface of nanocapsules coated with chitosan until its loading content reached 5 wt. %, and thus it could optionally be loaded into chitosan-coated nanocapsules.

In addition, finasteride (FS (from English - finasteride)) could be optimally loaded at a content of up to 0.1 wt.% and did not cause precipitation at a content of up to 2 wt.%, which made it possible to obtain chitosan-coated microcapsules up to 4 μm in size. In addition, chitosan-coated nanocapsules containing 5 wt.% loaded drug were very different in terms of fineness and surface charge from chitosan-coated nanocapsules without drug loaded. Thus, the optimal production condition was up to 0.1 wt.%. For use in topical delivery via microcapsules, the drug could be loaded at levels up to 2% by weight.

As mentioned above, the conditions for obtaining chitosan-coated nanocapsules with a size of 100 nm or less, which were loaded with insoluble (fat-soluble) active agents in a content ranging from 0.1 wt.% to 20 wt.%. Therefore, they can be used to optimize the conditions for obtaining chitosan-coated nanocapsules loaded with active agents such as tetradrin, astaxanthin, curcumin, ascorbyl palmitate, caffeic acid phenethyl ester (CARE), Centella asiatica, beta-sitosterol, ascorbyltetraisopalmitate, tetrapeptide collagen. etc.

Example 4 Conditions for Obtaining a Chitosan-Coated Nanocapsule Containing a Water-Soluble Active Agent Loaded into It

In accordance with the present disclosure, conditions have been established for producing chitosan-coated nanocapsules loaded with water-soluble drugs, including anti-cancer agents (doxorubicin), immunogens (ovalbumin), proteins as therapeutic agents, and drugs (bovine serum albumin, BSA ).

A solution of 20 mg of Pluronic F127 in 1 ml of acetone was reacted for 2 hours. Then, the reaction solution was slowly added dropwise to 4 ml of deionized water with stirring at 400 rpm to obtain polymer nanoparticles, from which acetone was removed for 6 hours under a fume hood. Finally, 20 mg of chitosan (90% deacetylation, molecular weight 10 kDa) was added to the polymer nanoparticles and stirred at room temperature for 2 hours to obtain chitosan-coated nanocapsules.

A water-soluble drug (doxorubicin, BSA, or ovalbumin) was added to the thus obtained chitosan-coated nanocapsules, and then incubated at 4° C. for 2 hours. Drugs that remained unloaded were removed by ultrafiltration (Amicon Ultra-15 filter) followed by optimization of loading conditions.

Unloaded drugs were analyzed by absorbance (480 nm) for doxorubicin and by absorbance (580 nm) by Coomassie blue analysis for BSA and ovalbumin.

Chitosan-coated nanocapsules (ChiNC 10K) containing water-soluble drugs loaded therein were analyzed for size, fineness, and surface charge using an electrophoretic light scattering spectrophotometer (ELS-Z2, Otsuka).

Example 4-1. Obtaining a chitosan-coated nanocapsule containing loaded doxorubicin

In contrast to fat-soluble drugs, water-soluble drugs were loaded at low temperature (4°C) using the temperature sensitive characteristics of the chitosan-coated nanocapsules (volume expansion to 1 μm at 4°C) as shown in FIG. 12.

The chitosan-coated nanocapsules containing doxorubicin (DOX) loaded therein were similar to each other in terms of size, fineness, and surface charge, as shown in FIG. 13. In particular, the drug could be loaded stably at up to 10% by weight. Although not shown in FIG. 13, microcapsules can be formed even at a loading content of 20 mass%.

The optimal loading content was 6 wt.%, as measured by absorbance analysis.

Example 4-2. Preparation of chitosan-coated nanocapsules containing loaded ovalbumin

As shown in FIG. 14, chitosan-coated nanocapsules containing loaded ovalbumin (OVA (from English - ovalbumin)) were similar to each other in size, dispersion and surface charge without deposition up to a loading content of 5 wt.%. Absorption analysis showed that a loading content of 3% by weight was optimal.

Example 4-3. Obtaining a chitosan-coated nanocapsule containing bovine serum albumin (BSA) loaded into it

As shown in FIG. 15, chitosan-coated nanocapsules were similar in size, dispersion, and surface charge until BSA (67 kDa), which is a model drug for various proteins as therapeutics, was loaded at up to 5 wt%. Nanocapsules provide stable drug loading without precipitation. The optimal loading content was 4 wt.%, as measured by absorbance analysis.

As mentioned above, the conditions for obtaining chitosan-coated nanocapsules of 100 nm or less in size were set, into which water-soluble active agents were loaded with a content ranging from 0.1 wt.% to 20 wt.%. Accordingly, they can be used to optimize the conditions for obtaining chitosan-coated nanocapsules loaded with active agents such as phospholipase A2 (PLA2), basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF), etc. .

Example 5 Composition Stability of a Chitosan-Coated Nanocapsule Containing a Loaded Drug

Example 5-1. Stability of the Composition of a Chitosan-Coated Nanocapsule Containing a Loaded Drug

Based on the established method and condition for producing chitosan-coated nanocapsules in Example 4, chitosan-coated nanocapsules containing drugs loaded therein were obtained, while insoluble cyclosporine A (CsA) was used as an insoluble drug, and commonly available drugs Nile red and pyrene were used in as model insoluble drugs.

Together with 10 mg of Pluronic F126, 0.2 mg or 0.5 mg of CsA, Nile red or pyrene was added to 1 ml of acetone and mixed at room temperature for 2 hours to obtain a reaction mixture, which was then slowly added dropwise to 4 ml of deionized water with stirring at 530 rpm. Stirring was continued for 4 hours under a fume hood to allow for random evaporation of the acetone. After removing the acetone, nanoparticles consisting of pluronic in deionized water were mixed with the same mass of 10 kDa chitosan as the mass of pluronic to obtain chitosan-coated nanocapsules containing loaded drugs (CsA@ChiNC, Nile red@ChiNC and pyrene@ ChiNC, respectively). CsA@ChiNC, Nile red@ChiNC, and pyrene@ChiNC were analyzed for stability by monitoring precipitation upon standing at room temperature. The result is shown in Fig. 16.

As can be seen in FIG. 16, none of CsA@ChiNC, Nile Red@ChiNC, and Pyrene@ChiNC precipitated.

It is clear from the data that the chitosan-coated nanocapsules containing the drugs loaded therein according to the present disclosure are stable in terms of composition.

Example 5-2. Lyophilization Stability of a Chitosan-Coated Nanocapsule Containing a Loaded Drug

The composition of the chitosan-coated nanocapsule was analyzed for stability, especially stability in deionized water and PBS, before and after lyophilization.

In FIG. 17 shows the stability of chitosan-coated nanocapsules loaded with cyclosporin A and retinyl palmitate (RP) as measured at 37°C and 100 rpm. As can be seen, similar sizes were found in both deionized water and PBS after 4 weeks, indicating that the nanocapsules can be stably maintained.

The high stability of chitosan-coated nanocapsules was also confirmed, as it was observed that they did not undergo any size changes or settling before and after lyophilization (before freeze-drying SZ or D.Z. (before freeze-drying) and after SZ or P.Z. (after freeze drying). In particular, nanocapsules are easy to resuspend even in the absence of a cryoprotectant during lyophilization.

Example 6 Effect of transdermal drug delivery of a chitosan-coated nanocapsule containing drug loaded into it

Formulations for the transdermal delivery of drugs or active ingredients have always been subjects of increased interest in the pharmaceutical and cosmetic fields. Chitosan-coated nanocapsules loaded with drugs according to the present disclosure were analyzed for drug delivery efficiency.

Example 6-1. Optimizing the type of chitosan for skin penetration using the Franz diffusion cell system

In Example 1-3, chitosan-coated nanoparticles with a molecular weight of 20 kDa or less (ChiNC 3K, ChiNC 10K, and ChiNC 20K) were found to stably form chitosan nanoparticles of 100 nm or less. To determine which chitosan-coated nanoparticles are optimized for skin permeability, a Franz diffusion cell system was used.

In the Franz diffusion cell system, a formulation including a drug was introduced into the donor chamber while the receiving chamber was filled with saline such as PBS (phosphate buffered saline) with a permeable layer such as a permeable membrane, animal skin or a skin cell culture fixed between a donor chamber and a receiving chamber. Skin permeability can be determined by measuring the amount of drug diffusing from the donor chamber into the receiving chamber.

In the Franz diffusion cell system, 5 ml of PBS (pH 7.4 containing 0.05% polysorbate 80) was added to the receiving chamber, 1.5 × 1.5 cm human cadaver skin was fixed between the receiving chamber and the donor chamber, and each sample was placed into the donor chamber. The receiving chamber was set to 37° C. and 600 rpm and 500 μl of sample was withdrawn at sampling times of 0.5, 1, 2, 4, 8, 12, 18, and 24 hours. The amount of drug passing through the skin was determined as a function of time using high performance liquid chromatography HPLC. The results are shown in FIG. 18.

As shown in FIG. 18, CsA showed the highest skin permeability when coated with chitosan-coated nanocapsules derived from 10 kDa chitosan (ChiNC 10K). A statistically significant difference from 3 kDa chitosan was assessed as a result of the higher surface charge of ChiNC 10K than ChiNC 3K, as shown in FIG. 3.

Chitosan nanocapsules (ChiNC 10K) derived from 10 kDa chitosan significantly increased skin permeability compared to non-chitosan coated polymer capsules (PluNC) and provided skin permeability six or more times that of the most commonly used liposomal formulation.

As a result of evaluating the use of RP as a drug, RP showed 14 or more times higher skin permeability when loaded to the optimal state of ChiNC 10K than when RP alone was tested as a control, suggesting that chitosan 10 kDa can improve permeability of drugs through the skin.

Example 6-2. Measurement of skin permeability of a chitosan-coated nanocapsule using a Franz diffusion cell system

The skin permeability of the nanocapsules and chitosan-coated nanocapsules was measured using a Franz diffusion cell system in the same manner as in Example 6-1.

Chitosan-coated nanocapsules containing 2 wt % Nile Red (Nile Red@ChiNC) at 2 mg/mL were added to the donor chamber. Nile Red alone or Nile Red-containing nanocapsules without chitosan coating (Nile Red@PluNC) were used as controls. After the receiving chamber was set to 37° C. and 600 rpm, 500 µl of sample was taken from the receiving chamber at sampling times of 0.5, 1,2,3, 8, 12, and 24 hours. Fluorescence was measured in samples taken at predetermined time points. The results are shown in FIG. 19.

As shown in FIG. 19, Nile Red alone almost did not penetrate the skin, while skin permeability increased with the use of Nile Red @PluNC and Nile Red @ChiNC. In particular, @ChiNC Nile Red increased skin permeability four times or more compared to @PluNC Nile Red.

The results demonstrate excellent skin permeability of chitosan-coated nanocapsules loaded with drugs according to the present disclosure.

Example 7 Transdermal Delivery in an Animal Model

Example 7-1. Transdermal delivery of Nile Red via a chitosan-coated nanocapsule in an animal model

Chitosan-coated nanocapsules containing 2 wt% Nile Red (Nile Red@ChiNC) prepared in Example 4 were controlled to have a concentration of 2 mg/ml and applied at a dose of 300 μl for 5 days to the shaved back of each mouse . After that, back skin samples were taken and fixed for 12 hours in 10% neutral formalin. Fixed skin was immersed in cutting medium at optimal cutting temperature (OCT) and flash-frozen in liquid nitrogen at -20°C or below, followed by cutting into 20 µm thick blocks using a cutting machine. frozen sections. The blocks were attached to a glass slide to obtain skin tissue samples. They were washed with deionized water and the distribution of Nile red in the skin was observed under a fluorescent microscope. The results are shown in FIG. 20.

As shown in FIG. 20, when applied to the skin, Nile Red @ ChiNC Nile Red was found in skin tissues.

From the data, it can be understood that chitosan-coated nanocapsules loaded with drugs according to the present disclosure can deliver drugs through the skin.

Example 7-2. Drug efficacy in transdermal delivery

Cyclosporine A is used as a therapeutic agent for psoriasis and atopy and is reported to have an effect on hair regrowth. However, delivery of cyclosporine A to the skin is limited because it is insoluble in water due to its high hydrophobicity. Thus, cyclosporin A should be dissolved in an organic solvent such as acetone before being applied to the skin. However, when applied to the skin, such an organic solvent can irritate the skin and cause loss of the skin barrier.

Thus, effective penetration of cyclosporin A into the skin without irritation or damage to the skin can increase its effectiveness in hair regrowth, which provides an excellent therapeutic effect on hair loss. Thus, the chitosan-coated nanocapsules according to the present disclosure were investigated for transdermal delivery of cyclosporine A in terms of its hair regrowth effect.

Conducted experiments on the resumption of hair growth. For this purpose, female C57B/6 black mice aged 7 weeks were shaved in accordance with the recommendations of the platform of the Ministry of Food and Drug Safety. Shaved mice were divided into groups treated with saline (control (CTL)), cyclosporin A in acetone (CsA) and chitosan-coated nanocapsules containing 5 wt.% cyclosporin A obtained in example 4 (CsA @ChiNC). Each sample was applied five times a week for 4 weeks (50 mg/kg single dose for CsA). Hair regrowth effects were examined with the naked eye and the results are shown in FIG. 21.

As shown in FIG. 21, when treated with cyclosporine A in acetone (CsA), all five mice showed hair growth at week 4, at which the maximum hair regrowth effect was obtained, while hair grew naturally at a rate of less than 5% in the control (CTL). It was believed that in the group treated with CsA alone, the skin tissue and barrier were damaged by acetone in which CsA was dissolved, so that the CsA drug penetrated into the damaged skin.

Hair regrowth was observed in four out of five mice per group in the CsA@ChiNC treated group. CsA@ChiNC showed an 80% hair regrowth effect compared to CsA alone.

In addition, dorsal skin samples were taken from mice in each group and analyzed for the number and size of follicles, and the results are shown in FIG. 21(b) and (c).

As shown in FIG. 21 (b) and (c), the same number and size of hair follicles were found in the groups treated with CsA in acetone (D) and CsA@ChiNC (C).

The data show that chitosan-coated nanocapsules containing drugs loaded therein according to the present disclosure are excellent in terms of skin permeability and transdermal drug delivery efficiency, and can be used as a platform technology to deliver insoluble drugs to humans and animals.

Example 8 Enhancement of Active Ingredient Bioavailability by Oral Administration of a Nanocapsule

Nanocapsules loaded with cyclosporin A (CsA) were orally administered to male Sprague-Dawley rats (200-250 g). For this purpose, nanocapsules were introduced containing CsA loaded into them at 5 wt. % (1 ml deionized water, 50 mg/kg), using a feeding needle. Blood samples were taken at 2, 4, 6, 8, 12 and 24 hours after administration and quantitatively analyzed by HPLC.

Blood half-life and bioavailability of CsA were increased in the group treated with chitosan-coated nanocapsules compared to the group treated with nanocapsules without chitosan.

In addition, for use in the hair regrowth experiment, 7-week-old C57B/6 female black mice had their backs shaved according to the recommendations of the FDA platform as in Example 7. CsA and CsA@ChiNC solutions were orally administered three once a week for 4 weeks, as disclosed above. Higher hair regrowth effects were observed in the CsA@ChiNC treated group compared to the CsA treated group. This result was believed to be due to the improved bioavailability of the chitosan-coated nanocapsules, demonstrating that the drug delivery system using chitosan-coated nanocapsules according to the present disclosure has an excellent drug delivery effect.

Claims (14)

1. A nanocapsule having a size of 30-100 nm, in which a nanoparticle having a size of 5-50 nm and containing a drug and pluronic is coated with chitosan having a molecular weight of 3-20 kDa, wherein the drug is selected from the group consisting of paclitaxel, docetaxel, tetradrine, cyclosporin A, dexamethasone, tocopheryl acetate, astaxanthin, curcumin, ascorbyl palmitate, caffeic acid phenethyl ester (CARE), retinyl palmitate, minoxidil, finasteride, Centella asiatica, beta- sitosterol, ascorbyl tetraisopalmitate, tripeptide collagen, doxorubicin, phospholipase A2 (PLA2), ovalbumin, bovine serum albumin, basic fibroblast growth factor (b-FGF), and vascular endothelial growth factor (VEGF), and wherein the nanocapsule is a preparation for external use on the skin and obtained by a method including a first step of preparing a reaction solution by dissolving a drug and a Pluronic with a hydrophilic-lipophilic balance (HLB) value of 8 to 29 in an organic solvent and reacting them at room temperature; the second stage of obtaining a nanoparticle having a size of 5-50 nm by adding drops of the reaction solution from the first stage to distilled water in accordance with nanoprecipitation with continuous stirring, and removing the organic solvent from the reaction solution by natural evaporation; And the third stage of coating the nanoparticles from the second stage with chitosan by adding chitosan with a molecular weight of 3-20 kDa to it in distilled water and mixing. 2. Nanocapsule according to claim 1, where the drug is used in an amount of more than 0 mass fractions to 20 mass fractions per 100 mass fractions of pluronic. 3. The nanocapsule according to claim 1, wherein the Pluronic is at least one selected from the group consisting of Pluronic L35, Pluronic L43, Pluronic L44, Pluronic L64, Pluronic F68, Pluronic P84, Pluronic P85, Pluronic F87, Pluronic F88, Pluronic F98, Pluronic P103, Pluronic P104, Pluronic P105, Pluronic F108, Pluronic P123 and Pluronic F127. 4. Nanocapsule according to claim 1, where chitosan is used in an amount of 0.001-200 mass fractions per 100 mass fractions of pluronic. 5. The nanocapsule according to claim 1, wherein the organic solvent in the first step is at least one selected from the group consisting of acetone, dimethyl sulfoxide (DMSO), ethanol, acetonitrile, tetrahydrofuran, chloroform and dichloromethane. 6. Nanocapsule according to claim 1, where the volume of distilled water in the second stage is 4 times the volume of the organic solvent in the first stage. 7. A drug delivery system containing a nanocapsule according to claim 1. 8. Cosmetic composition containing a nanocapsule according to claim 1. 9. A medical device composition comprising a nanocapsule according to claim 1, wherein the medical device is selected from the group consisting of a wound covering agent and an embolization agent.

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