Cell targeted polymer self-assembly nanomicelles, and method of preparing same

Abstract

The present invention relates to a polymer self-assembly nanocarrier and a method of preparing same, the nanocarrier binding a peptide that targets a specific cell and, simultaneously, carrying an active ingredient that has been verified. The present invention can exhibit various effects such as a moisturizing effect, an increase in filaggrin production, whitening, skin barrier reinforcement and the like by selectively and efficiently delivering a drug through a targeting peptide, can be applied to skin diseases such as atopy or psoriasis, and is expected to enable the maximum effect of an active substance in the pharmaceutical and cosmetic fields to be obtained.

Description

Cell-targeting polymer self-associating nanomicelles and method for preparing the same

[0001] The present invention relates to a polymer self-associating nano-carrier that binds a peptide targeting a specific cell and simultaneously carries an effective ingredient whose effective ingredient has been verified, and a method for producing the same.

[0002] The skin, the largest organ in the human body, plays a crucial role in protecting the body, protecting against physical impact, infection, and water loss from the external environment, and maintaining body temperature. It consists of the epidermis, dermis, and subcutaneous layer. Keratinocytes, melanocytes, Langerhans cells, and fibroblasts, located in the epidermis and dermis, play a crucial role in skin tissue formation and barrier maintenance. Numerous studies have explored various topics related to skin pigmentation, wound healing, cancer treatment, and tissue regeneration using these cells.

[0003] However, demonstrating pharmacological effects in complex skin cell models is significantly more complex due to the presence of a skin barrier, including cell acquisition, endosomal and lysosomal degradation, and cell aggregation. This typically necessitates the use of modified drugs or the widespread use of nanocarriers.

[0004] Nanocarriers are a drug delivery system (DDS) technology actively being researched and commercialized in the pharmaceutical industry, enabling precise target cell attack. Recent research suggests that nanocarriers can effectively improve the solubility of insoluble active ingredients, thereby increasing the penetration rate of cosmetic ingredients and enhancing their stability. Nanocarriers have diverse potential applications in skin care, skin disease treatment, skin care, and beauty, and research on applying nanocarrier technology to functional cosmetics is rapidly advancing.

[0005] Recently, there has been a growing preference for natural plant-based active ingredients, leading to an increase in the use of plant-based active ingredients in functional cosmetics. However, effective utilization of plant-based active ingredients presents several challenges. Nanocarrier technology has been reported as a groundbreaking technology that can overcome these challenges and effectively incorporate plant-based or synthetic active ingredients into functional cosmetics. Nanocarriers enhance the stability of active ingredients and simultaneously deliver ingredients with diverse physical and chemical properties, enabling multi-effects and multi-target skincare benefits.

[0006] Types of dermal / transdermal drug delivery nanocarriers used in cosmetics include microemulsions / nanoemulsions, liposomes, lipid nanoparticles, lipid liquid crystals, nanocrystals, polymer nanocarriers, and inorganic nanocarriers.

[0007] However, when using these various nanocarriers, the amount of drug delivered to target cells is limited compared to the total dose administered, and the complexity of the skin structure and adjacent cells hinders drug absorption into target cells, raising the possibility of side effects. Therefore, the development of customized cosmetic formulation technologies is necessary to effectively deliver proven active ingredients to the target area for maximum efficacy.

[0008] Filaggrin is expressed in the uppermost epithelium, including the stratum corneum, and is an essential protein for the formation of the skin's inherent barrier function. Filaggrin genotyping and measurement of filaggrin levels have been shown to be deficient in atopic dermatitis and ichthyosis vulgaris. FLG null mutations are associated with the severity of atopic dermatitis, and the prevalence of these mutations in adults with atopic dermatitis is higher than in children. Treatments such as cyclosporine or dupilumab have been shown to normalize filaggrin expression in atopic dermatitis patients by altering the expression of skin cytokines. However, it has been reported that filaggrin genotype alone may not be sufficient to regulate filaggrin in atopic patients in the inflammatory skin environment. Studies have shown that in addition to cytokines, various other regulatory factors, such as sphingosyl-phosphorylcholine and sirtuin 1, can influence filaggrin expression. Furthermore, factors such as dry skin and bacterial imbalances can also trigger abnormal filaggrin expression, leading to atopic dermatitis. Clinical studies have shown that patients with atopic dermatitis with filaggrin gene abnormalities are more severe and persistent, and more prone to developing asthma and allergic diseases, compared to patients with normal filaggrin levels. Therefore, for patients with atopic dermatitis with filaggrin gene abnormalities, it is crucial to establish a diagnostic method to determine the presence or absence of the underlying cause, as well as a personalized treatment approach based on that underlying cause.

[0009] Melanin, a pigment found in skin, hair, and eyes, is an organic substance composed of carbohydrates, nitrogen, and amino acids, primarily derived from the amino acid tyrosine. Melanin's primary function is to protect DNA in the skin from UV exposure and prevent skin cancer. Melanin also determines skin tone and prevents sunburn.

[0010] Known methods for controlling melanin pigment formation include reducing and decolorizing large amounts of melanin pigment and inhibiting the activity of tyrosinase, the enzyme that forms melanin pigment. Chemical compounds that inhibit tyrosine primarily act by inhibiting tyrosinase activity or preventing tyrosine oxidation. These compounds include hydroquinone, arbutin, and kojic acid. Some whitening cosmetic ingredients have low stability, which can cause them to decompose or discolor, and may produce off-flavors when used. Their use is limited due to unclear in vivo efficacy and safety concerns. Furthermore, kojic acid inhibits enzyme activity by adsorbing copper ions at the active site of tyrosinase, but can cause instability and other issues when mixed into cosmetics.

[0011] Vitamin C and its derivatives are susceptible to oxidation, making them difficult to use as cosmetic ingredients. Hydroquinone, while highly effective in whitening skin, carries the risk of allergies and is irritating, toxic to melanin-producing cells and potentially causing permanent skin discoloration. Furthermore, it has recently been classified as a carcinogen and is banned for use, with some countries allowing it only in limited concentrations.

[0012] Furthermore, arbutin, a glucopyranoside derivative combined with hydroquinone, has the potential to inhibit melanin synthesis while minimizing the side effects of hydroquinone, making it a potential treatment for skin conditions characterized by increased melanin pigmentation. However, this substance has the disadvantage of being partially degraded by skin enzymes. Therefore, there is an urgent need to develop a proven, safe alternative whitening agent that can effectively reduce skin pigmentation even in small doses.

[0013] [Prior Art Literature]

[0014] (Patent Document 1) 10-2023-0061527 (filed on May 12, 2023)

[0015] (Patent Document 2) 10-2015-0175779 (filed on August 22, 2016)

[0016] (Non-patent literature 1) J. Dermatol. Sci. 2018;91:142-152.

[0017] Existing passive skin tissue drug delivery systems have limitations in that the drug is absorbed slowly and inefficiently into the desired location due to the large number of nearby cells within the skin.

[0018] To address the low absorption rate, targeting rate, and low efficiency of the passive system, a two-block copolymer consisting of mannosylerythritol lipid (MEL) linked with a maleimide linker and a hydrophobic polymer block and a hydrophilic polymer block was used to increase stability in the body and target specific cells, while containing effective ingredients with proven efficacy, such as ABT-751 and PMWC, within the micelles.

[0019] Skin protein-derived peptides have high affinity for biologically specific skin cell receptors, enabling cell-specific targeting, resulting in effective targeting of skin cells while exhibiting low immunogenicity and low cytotoxicity.

[0020] The purpose of the present invention is to establish an intelligent polymer nano micelle containing a synthetic material ABT-751 or PMWC or a natural material citron seed oil DP, which binds a specific peptide targeting fibroblasts or melanocytes and keratinocytes to the micelle and exhibits skin barrier recovery or whitening and antioxidant effects, respectively, and a manufacturing process thereof.

[0021] A cosmetic composition or pharmaceutical composition for the purpose of skin regeneration or whitening, including nano-carriers, was provided, comprising ABT-751, PMWC, and natural material citron seed oil DP.

[0022] In addition, we discovered a new mechanism of action of ABT-751, which was developed as an anticancer agent for non-small cell lung cancer, which is to strengthen the skin barrier function by increasing filaggrin expression, and we intend to utilize this in the development of functional cosmetics through this study.

[0023] In addition, PMWC, a differentiated new material with a new mechanism of action developed by mixing cysteine, a pharmaceutical material, and silicic acid, a health functional food, is intended to be utilized as an effective whitening ingredient contained within the micelle core.

[0024] In addition, we aim to utilize yuzu seed oil DP, which contains various antioxidant substances such as vitamin C, flavonoids, and carotenoids as a natural material, as an antioxidant active ingredient by loading it into micelles.

[0025] Drug delivery systems utilizing nanocarriers such as micelles and liposomes often have low drug delivery efficiency to specific areas, making it difficult for the drug to properly exert its effect.

[0026] Accordingly, in the present invention, a target-oriented polymer nano-micelle for dermal fibroblasts and melanocytes was manufactured to improve the drug delivery efficiency of ABT-751 and PMWC, and an intelligent active ingredient delivery system with excellent filaggrin regeneration and whitening efficacy was developed.

[0027] The result is a nanocarrier comprising a cell-targeting peptide and ABT-751 or PMWC in an amphiphilic self-associating diblock copolymer and an anisotropic lipid mannosylerythritol lipid (MEL)-maleimide.

[0028] The above dermal fibroblast targeting peptide provides a nanocarrier comprising a “KTTKS” peptide having a sequence of KTTKS-cysteamide.

[0029] The above melanocyte targeting peptide is provided as a nanocarrier comprising a “NAP” peptide having the sequence Ac-Nle-DHfRWGK-Cysteamide.

[0030] The above keratinocyte-targeting peptide is provided as a nanocarrier comprising a “Lam332” peptide having the sequence PPFLMLLKGSTRFC.

[0031] Also provided is a step of dissolving an amphiphilic self-associating diblock copolymer, an anisotropic lipid, and a physiologically active ingredient for dermal fibroblasts or melanocytes in a solvent and stirring the same to obtain a micelle dispersion. Also provided is a method for producing a nanocarrier, characterized by including a step of adding a dermal fibroblast-targeting peptide KTTKS or a melanin-targeting peptide NAP to the micelle dispersion and shaking the same.

[0032] Furthermore, a cosmetic composition or pharmaceutical composition for skin regeneration or whitening and an antioxidant composition comprising the nano-carrier of the present invention is provided.

[0033] The present invention utilizes a target-directed peptide to selectively and efficiently deliver drugs, thereby exhibiting various effects such as moisturizing effects, increasing filaggrin production, whitening, and strengthening the skin barrier. It is expected that it can be applied to skin diseases such as atopy and psoriasis, and that it will be possible to obtain the maximum effect of effective substances in the pharmaceutical and cosmetic fields.

[0034] Figure 1 is a scheme for the synthesis of mannosyl erythritol lipid (MEL)-maleimide of the present invention and the production of target-directed nano micelles containing the active ingredient drug.

[0035] Figure 2 shows 1H NMR spectral data of mannosylerythritol lipid (MEL)-maleimide and linker intermediate.

[0036] Figure 3 shows the results of measuring the change in nano micelle size and surface potential before and after the introduction of peptide.

[0037] Figure 4 shows the evaluation of cell efficacy of ABT-751.

[0038] Figure 5 shows the evaluation of cell efficacy of PMWC.

[0039] Figure 6 shows the skin efficacy evaluation of PMWC.

[0040] Figure 7 shows the skin efficacy evaluation of yuzu seed oil DP.

[0041] Figures 8(a to c) show the toxicity evaluation of three types of target-directed drug-loaded micelles ((a) cytotoxicity evaluation of ABT-751-loaded KTTKS micelles in dermal fibroblasts (NHDF); (b) cytotoxicity evaluation of PMWC-loaded NAP micelles in melanocytes (B16-F10); (c) cytotoxicity evaluation of citron seed oil DP-loaded Lam332 micelles in human skin keratinocytes (HaCaT).

[0042] Figure 9 shows the evaluation of cell efficacy of ABT-751-loaded KTTKS micelles.

[0043] Figure 10 shows the evaluation of cell efficacy of PMWC-supported NAP micelles.

[0044] Figure 11 shows the evaluation of cell efficacy of Lam332 micelles containing citron seed oil DP.

[0045] Hereinafter, the present invention will be described in detail.

[0046]

[0047] One aspect of the present invention is

[0048] A skin cell-targeting nano-carrier comprising a micelle formed by self-association of an amphiphilic crystalline copolymer and an anisotropic lipid, a targeting peptide bound to the anisotropic lipid, and a physiologically active ingredient inside the micelle.

[0049] Existing passive drug delivery systems for skin tissue suffer from low therapeutic efficacy due to the presence of numerous adjacent cells within the skin, hindering drug uptake into target cells. Therefore, the present invention provides active polymer nanomicelles that utilize targeting ligands to enhance drug uptake into target cells to achieve the desired effect. Furthermore, the present invention demonstrates their targeting performance for specific cells and their subsequent beneficial effects on the skin.

[0050] The above skin target cells may be, but are not necessarily limited to, dermal fibroblasts, melanocytes, or keratinocytes.

[0051] The above anisotropic lipid serves as a linker for the dermal fibroblast-targeting peptide, and mannosylerythritol lipid (MEL)-maleimide can be preferably selected. By replacing the hydroxyl group of the anisotropic lipid with a peptide linker and utilizing the maleimide-thiol reaction, which is a representative reaction of peptide conjugation, a micelle conjugated with the dermal fibroblast-targeting peptide can be prepared, but is not necessarily limited thereto, and a method widely known to those skilled in the art can be used.

[0052] The content of the anisotropic lipid may be 5 to 40 wt% based on the total weight of the entire micelle, and preferably 5 to 30 wt%. When the content of the anisotropic lipid is less than 5 wt%, there is a problem that the linker role of the dermal fibroblast-targeting peptide is insufficient, and when it exceeds 40 wt%, the content of other phases becomes relatively low, which is not preferable in terms of micelle structure formation, and when the anisotropic lipid in the micelle exceeds a certain concentration, there may be a concern that the polymer and lipid phases may separate.

[0053] The above targeting peptide is a composition that promotes migration to the desired target skin target cell, and may vary depending on the desired target skin target cell.

[0054] For example, the dermal fibroblast targeting peptide may include a "KTTKS" peptide having a sequence of KTTKS-cysteamide, the melanocyte targeting peptide may include a "NAP" peptide having a sequence of Ac-Nle-DHfRWGK-Cysteamide, and the keratinocyte targeting peptide may include a "Lam332" peptide having a sequence of PPFLMLLKGSTRFC.

[0055] In particular, the pentapeptide KTTKS (lysyl-threonyl-threonyl-lysyl-serine) is a part of collagen type I, which plays an important role in elasticity and moisturizing in the dermal layer of the skin, and is known to promote the production of growth factor TGF-β by inhibiting collagenase and maintaining extracellular matrix (ECM) and collagen type I biosynthesis and intracellular mRNA stability through its role as a 'signal peptide' within the cell, and the KTTKS forms cell-specific ligand bonds with receptors overexpressed on the surface of fibroblasts (PAR-2).

[0056] The content of the above targeting peptide can be included in the same molar ratio as the MEL-linker.

[0057] The nano-carrier of the present invention may further include a physiologically active ingredient for skin cells inside the micelle.

[0058] The above-mentioned physiologically active ingredient is not particularly limited and may be any of the conventionally known ingredients, but may also be specifically selected in consideration of the intended target skin target cells or the desired effect. For example, if dermal fibroblasts are the target cells for purposes such as skin regeneration, increasing filaggrin production, and wound healing, ABT-751 is selected; if melanocytes are the target cells for purposes such as skin, PMWC is selected; and if keratinocytes are the target cells for purposes such as strengthening the skin barrier and moisturizing effect, yuzu seed oil DP, etc. may be selected. A more detailed understanding of this can be obtained through the examples and test examples described below.

[0059] Here, ABT-751 is a substance with Cas No. 141430-65-1, also known as E7010, and is a substance known to have an effect as a microtubule synthesis inhibitor. ABT-751 is a substance that shows potential as an anticancer agent for various types of cancers, including solid tumors and blood cancers, due to its mechanism of action of interfering with the formation of the structural framework of cells, thereby arresting the cell cycle and ultimately leading to cell death (however, to the extent known to the inventor of the present invention, the effect of increasing the amount of filaggrin produced in skin cells is not known).

[0060] PMWC is methyl-2-acetylamino-3-(4-hydroxyl-3,5-dimethoxybenzoylthio)propanoate (MAHDP), and improves pigmentation in human skin pigment cells, UVB-irradiated reconstituted skin models, and clinical trials without cytotoxicity or skin irritation. PMWC inhibits the expression of melanin-producing enzymes, making MAHDP an effective treatment for hyperpigmentation.

[0061] Yuzu seed oil DP is oil obtained by steaming yuzu seed oil obtained by physically processing yuzu seeds to improve odor and remove specific substances. Yuzu seed oil DP is an ester exchange-reacted oil composition composed of 30 to 70% by weight of restructured fat in the form of monoglycerides composed of two carboxylic acids with 10 or fewer carbon atoms and one fatty acid with 10 or more carbon atoms and 30 to 70% by weight of restructured fat in the form of diglycerides composed of one carboxylic acid with 10 or fewer carbon atoms and two fatty acids with 10 or more carbon atoms. Yuzu seed oil DP can be used as a biocatalytic conversion composition in the cosmetic field for the purpose of providing a composition that provides excellent skin usability and anti-inflammatory efficacy.

[0062] Including the above amphiphilic crystalline copolymer and the amphiphilic non-crystalline copolymer, the amphiphilic crystalline copolymer may be at least one selected from the group consisting of, for example, poly(ethylene oxide)-b-poly(ε-caprolactone) copolymer (PEO-b-PCL), PE-b-PCL (poly(ethylene)-b-poly(ε-caprolactone), PHBA-b-PEO (poly(3-hydroxybutyric acid)-b-poly(ethyleneoxide), PEO-b-PAA (poly(ethylene oxide)-b-poly(acrylic acid), PEO-b-PGA (poly(ethylene oxide)-b-poly(glycolic acid), PMPC-b-PBMA (poly(2-methacryloyloxyethyl phosphorylcholine)-b-poly(butylmetacrylate), and PEO-b-PCL-b-PEO (poly(ethylene oxide)-b-poly(ε-caprolactone)-b-poly(ethylene oxide)). However, the present invention is not limited thereto. In one embodiment of the present invention, a PEO-b-PCL copolymer is used, and although this may be preferably selected, it is not necessarily limited thereto.

[0063] The average particle diameter of the micelles is not particularly limited and can be adjusted in various ways depending on the type and size of the transport target substance to be encapsulated, but an appropriate range can be set in consideration of toxicity and penetration issues. That is, when the particle diameter of the micelle is less than 2 nm, the content of the physiologically active substance is small compared to the nanocarrier, so there may be a possibility of toxicity problems due to absorption in the human body. In addition, when the particle diameter exceeds 500 nm, there may be difficulty in passing through the stratum corneum to reach the dermis layer, and there may be a possibility of particle instability problems. Accordingly, although a particle diameter within the range of 2 to 500 nm can be used, the preferable average particle diameter of the micelle may be 10 to 100 nm, more preferably 20 to 100 nm, even more preferably 25 to 75 nm, and most preferably 30 to 70 nm.

[0064]

[0065] In addition, another aspect of the present invention is

[0066] This is a method for manufacturing a skin cell-targeting nano-delivery vehicle.

[0067] In brief, it consists of (1) a process of manufacturing polymer nano micelles using an amphiphilic polymer and anisotropic lipids, and (2) a process of introducing a targeting ligand onto the surface of the manufactured polymer nano micelles.

[0068] As a result, the manufacturing method of the present invention,

[0069] A step of dissolving an amphiphilic crystalline copolymer, an anisotropic lipid, and a physiologically active ingredient for dermal fibroblasts in a solvent and stirring the same to obtain a micelle dispersion; and

[0070] The method comprises a step of adding a dermal fibroblast targeting peptide to the above micelle dispersion and shaking, and is characterized by combining the targeting peptide and the physiologically active ingredient according to the skin cell target.

[0071] That is, if the skin cell target is a dermal fibroblast, the targeting peptide is a pentapeptide KTTKS (Lys-Thr-Thr-Lys-Ser) and the physiologically active ingredient is ABT-751.

[0072] If the above skin cell target is a melanocyte, the targeting peptide is NAP and the above physiologically active ingredient is PMWC,

[0073] If the above skin cell target is a keratinocyte, the targeting peptide may be characterized as being Lam332 and the physiologically active ingredient as being citron seed oil DP, but is not necessarily limited thereto.

[0074]

[0075] Furthermore, another aspect of the present invention is

[0076] The present invention relates to a composition for external application of skin comprising the skin cell-targeting nano-carrier of the present invention. The nano-carrier of the present invention is in the form of micelles, has high stability, and, above all, has excellent direct skin penetration ability, making it highly suitable for use in compositions for external application of skin.

[0077] Additionally, the composition of the present invention may be a cosmetic composition or a pharmaceutical composition (medicinal composition).

[0078] The cosmetic composition of the present invention may be in the form of an emulsion, lotion, ointment, patch, spray, cream (oil-in-water, water-in-oil, multiphase), solution, suspension (anhydrous and aqueous), anhydrous product (oil and glycol), gel, mask, pack, or powder, and the form thereof is not particularly limited.

[0079] More specifically, the cosmetic composition of the present invention may have any one formulation selected from the group consisting of external skin ointment, cream, emollient toner, nourishing toner, pack, essence, hair tonic, shampoo, rinse, hair conditioner, hair treatment, gel, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, nourishing essence, sunscreen, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, and body cleanser, but is not limited thereto. The cosmetic composition composed of each of these formulations may contain various bases and additives necessary and appropriate for the formulation of the formulation, and the types and amounts of these ingredients can be easily selected by a person skilled in the art.

[0080] In addition, the cosmetic composition of the present invention may additionally contain excipients such as fluorescent substances, fungicides, hydrotropism inducers, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, and plant extracts.

[0081] The content of the nano-carrier of the present invention in the above cosmetic composition can be appropriately adjusted by a person skilled in the art as needed, but it is preferably included in an amount of 0.1 to 20 wt% based on the total weight of the composition. If it is included in an amount less than 0.1 wt%, the effect may be minimal, and if it is included in an amount exceeding 20 wt%, there may be problems such as ease of handling during the formulation process, storage stability, and cost issues, which may be somewhat undesirable.

[0082] A pharmaceutical composition comprising the nano-carrier of the present invention as an active ingredient can be used to improve or treat atopic skin. As described above in the "Background Art," filaggrin concentrations are very low in patients with atopic dermatitis and are significantly correlated with the severity of the disease. The nano-carrier of the present invention, loaded with ABT-751, can effectively improve or treat atopic dermatitis symptoms by increasing filaggrin production. A more detailed understanding of this can be obtained through the examples and test examples described below.

[0083] The nano-carrier of the present invention is in the form of micelles, has good penetration ability and high stability, and is therefore suitable for use in pharmaceutical compositions. In particular, it has excellent direct skin penetration ability, and is therefore suitable for use in external skin compositions.

[0084] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0085] The pharmaceutical composition of the present invention may include, in addition to the active ingredient, a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed, 1995).

[0086] The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, patient age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. Meanwhile, the dosage of the pharmaceutical composition of the present invention is preferably 0.0001-1000 mg / kg (body weight) per day.

[0087] Administration may be by topical application, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, or transdermal administration. When the pharmaceutical composition of the present invention is provided as a pharmaceutical composition specifically targeting skin cells, administration may be by topical application to the skin, but is not limited thereto. When the pharmaceutical composition of the present invention is in the form of a skin external formulation, the skin external formulation is not particularly limited, but is preferably a powder, gel, ointment, cream, liquid, or aerosol formulation.

[0088] In the present invention, as the gel base material of the external skin preparation, one or more selected from among Carbopol, Carbomer, Polyethyleneglycol, Polypropylene glycol, Polyacrylic acid, Carboxymethyl cellulose, Hydroxymethylcellulose, Polyvinylpyrrolidone, Gelatin, Alginate Salt, Chitin or Chitosan derivatives, hyaluronic acid, and collagen may be used, but is not limited thereto.

[0089]

[0090] Hereinafter, the present invention will be described in more detail with examples and test results. However, it should be made clear that the following examples are intended only to provide a detailed explanation of the invention and are not intended to limit the scope of the rights herein.

[0091]

[0092] Example

[0093] Example 1: Synthesis of an anisotropic lipid, mannosylerythritol lipid (MEL)-maleimide

[0094] Purified MEL (1 g), pyridine (367 μl), and 4-nitrophenylchloroformate (551 mg) were dissolved in anhydrous methylene chloride (32 ml), and the solution was stirred at room temperature for 1 h. The mixture was diluted with anhydrous methylene chloride and washed with water. The organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography using silica gel (ethyl acetate: n-hexane = 1:2) to obtain orthoformate compound 1 (750 mg) (colorless oil).

[0095] In the next step reaction, the orthoformate compound (100 mg) and N-(2-aminoethyl)maleimide trifluoro acetate salt (53 mg) obtained were dissolved in acetonitrile (2.8 ml), N,N-diisopropyl ethylamine (73 μl) was added, and the reaction was carried out by stirring at room temperature for 12 hours. The reactant was concentrated under reduced pressure to remove the solvent, and the mixture was diluted with anhydrous methylene chloride and washed with water. The organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography using silica gel (ethyl acetate: n-hexane = 1:2) to obtain the orthoformate compound (56 mg) (colorless oil).

[0096] Example 2: Preparation of drug-loaded polymer self-associating nanomicelles

[0097] The active ingredient raw material ABT-751 or PMWC or citron seed oil DP was weighed at 200 μM (based on 10 ml of solvent) and dissolved in 5 ml of tetrahydrofuran at 40°C. 2 mg of MEL linker and 0.1 g of PEO-b-PCL were added and dissolved. 9.9 ml of purified water was added dropwise to the reaction flask at a rate of 100 μl / min using a syringe pump device and stirred at 40°C. After the reaction was completed, tetrahydrofuran was completely removed by distillation under reduced pressure. Finally, the prepared solution was passed through a 0.2 μm syringe filter to produce drug-loaded polymer self-associating nanomicelles with a size of approximately 50 nm.

[0098] Example 3: Introduction of a targeting peptide

[0099] A peptide in a molar ratio identical to the MEL linker bound to the manufactured polymer self-associating nanomicelle solution was quantitatively added and reacted with stirring at room temperature for more than 12 hours. The resulting nanomicelles were diluted in the required ratio in DMEM (Dulbecco's Modified Egale Medium) solution and applied to cell experiments.

[0100]

[0101] Experimental Example 1: Changes in nanomicelle size before and after peptide introduction

[0102] To confirm the size and surface potential of drug-loaded polymer self-associating nanomicelles according to peptide introduction, dynamic light scattering (DLS) and zeta potential measurement were performed.

[0103] As shown in Table 1 below, it was confirmed that the size distribution and surface potential of nano micelles increased.

[0104] Particle size (nm) Surface potential (mV) ABT-751-loaded micelle 42 nm-21 KTTKS-bound ABT-752-loaded micelle 65 nm-5.48 PMWC-loaded micelle 37 nm-17 NAP-bound PMWC-loaded micelle 54 nm0.2 Yuzu seed oil-loaded micelle 40 nm-16 Lam 332-bound PMWC-loaded micelle 55 nm5.69

[0105] Test Example 2: Confirmation of drug loading within nanomicelles

[0106] To determine whether polymeric self-associating nano-micelles can support drugs, the active ingredient raw material was dissolved in an aqueous solution, passed through a 0.2 μm syringe filter, the filtrate was distilled under reduced pressure to remove water, and dissolved in methanol. A micelle filtrate with the same concentration was passed through a 0.2 μm syringe filter, the filtrate was distilled under reduced pressure to remove water, and dissolved in methanol. The sample was then subjected to UV absorbance measurement.

[0107] As shown in Table 2 below, the active ingredient raw material dissolved in water did not dissolve in water, so the absorbance intensity was low or showed almost no absorbance.

[0108]

[0109] Test Example 3: Evaluation of Cellular Efficacy of ABT-751

[0110] An efficacy evaluation was performed by treating NHEK in culture with ABT 751 to measure the amount of filaggrin expression and confirm changes in cell differentiation. ABT 751 was treated to NHEK at various concentrations (0, 0.5, 1, 2.5 μM), and an increase in filaggrin expression and cell differentiation was observed after 24 hours of treatment (Fig. 4).

[0111] Compared to the control group, which was not treated with ABT-751, the experimental group treated with ABT-751 showed increased filaggrin expression and increased cell differentiation. In particular, it was confirmed that the filaggrin expression increased in a concentration-dependent manner.

[0112] Test Example 4: Evaluation of Cellular Efficacy of PMWC

[0113] To evaluate the whitening effect on skin melanocytes, B16F10 cells were seeded in a 24-well plate at 2.0*10 4 After dispensing cells / well, the cells were cultured for 24 h. Subsequently, α-MSH (200 nM) and PMWC samples were treated simultaneously and cultured for 72 h. After completion of culture, the cells were washed with phosphate buffer (pH 7.4) and harvested into a 1.5 mL e-tube. Next, 1 N NaOH was added, and the reaction was performed at 80°C for 1 h, and the absorbance was measured at 405 nm. Arbutin (500 μg / mL) was used as a positive control.

[0114] The experimental results confirmed that melanin production increased by α-MSH stimulation and that intracellular melanin production decreased when PMWC was treated (Fig. 5).

[0115] Test Example 5: Evaluation of PMWC's Skin Efficacy

[0116] To confirm the whitening efficacy in actual tissues, 0.1% PMWC cream (DBLS-024) was applied to the UVB-induced skin of participants and observed for 8 weeks. As a result, melanin production was suppressed and whitening efficacy was observed in the UVB-irradiated eye area (Fig. 6).

[0117] Test Example 6: Evaluation of the Skin Efficacy of Yuzu Seed Oil DP

[0118] Moisture was measured on the forearm using a Corneometer (CM825, Courage and Khazaka Electronic Co., Germany), and the average value was calculated using three values. The test product, Yuzu seed oil DP, was applied at an amount of 2 ㎕ / cm 2 to the selected test area (1.5 cm * 1.5 cm) on the forearm using a micro pipette. Measurements were conducted before, immediately after, 4 hours later, and 8 hours later after application of the product. The measurement area was gently wiped with kimwipes before each measurement. The Corneometer measures the capacitance of the current delivered through a probe in contact with the skin. Moisture content and capacitance are proportional to each other, so the higher the moisture content, the higher the measured value, and the measurement coefficient is arbitrary unit (AU).

[0119] As a result of moisture measurement of Yuzu Seed Oil DP, it was confirmed that there was a change in moisture with 59.2 AU immediately after product application, 5.12 AU after 4 hours, and 4.50 AU after 8 hours. Compared to the unapplied area, Yuzu Seed Oil DP showed a statistically significant level of moisture improvement immediately after product application, 4 hours after, and 8 hours after product application, and an 8-hour moisturizing effect. (Figure 7)

[0120] Test Example 7: Cytotoxicity Evaluation of Peptide-Conjugated Drug-Loading Micelle

[0121] ABT-751-loaded micelles (065-22) containing KTTKS, PMWC-loaded micelles (LS-065-24) containing NAP, and citron seed oil DP-loaded micelles (LS-065-25) containing Lam332-loaded micelles were treated at various concentrations to NHDF cell line (fibroblasts), B16-F10 cell line (melanin-forming cells), and HaCaT cell line (keratin-forming cells) for 24 hours, and it was confirmed that there was no toxicity at each concentration (Fig. 8(a to c)).

[0122] Test Example 8: Skin elasticity evaluation of ABT-751-loaded KTTKS micelles (quantification of procollagen type I C-peptide (PIP))

[0123] Within cells, collagen is synthesized into procollagen and secreted outside the cell, and during this process, the propeptide is degraded by endopeptidase. Accordingly, the amount of human procollagen type I C-terminal peptide (PIP) produced was measured to confirm the amount of collagen produced. The experiment was conducted using NHDF cells as follows. First, the cells were cultured, washed with PBS, treated with serum-free media, and cultured for a certain period of time. After that, the supernatant of the cultured cells was obtained and the procollagen type I C-peptide was measured. As a result of the experiment, it was confirmed that the amount of PIP production increased depending on the concentration when treated with ABT-751, and when treated with "ABT-751-loaded KTTKS-bound micelles" (065-22), it was confirmed that the amount of PIP production increased by approximately 123.0 to 128.5%, when treated with "a mixture of ABT-751 material and KTTKS-bound micelles" (065-23), there was no effect, and when treated with "curcumin-loaded KTTKS micelles" (065-26), which was treated as a control, it was confirmed that the amount of PIP production increased by approximately 109.0 to 110.8% (Fig. 9).

[0124] Test Example 9: Evaluation of inhibition of melanin production in melanocytes (B16-F10) by PMWC-loaded NAP micelles

[0125] We evaluated the cellular level inhibition of melanin synthesis by treating melanocytes with a material expected to have a brightening effect. Melanocytes were cultured and treated with the drug, then centrifuged to collect the cells, and melanin was dissolved and measured. First, B16-F10 cells were cultured for 24 hours, then simultaneously treated with α-MSH (200 nM) and the sample, and incubated for 72 hours. After washing with phosphate buffer (pH 7.4), the cultured cells were harvested in a 1.5 mL e-tube, incubated with 1 N NaOH, and incubated at 60°C for 1 hour. The absorbance was measured at 405 nm. The experimental results confirmed that α-MSH stimulation increased melanin production, while PMWC treatment did not affect melanin production. Treatment with "PMWC-loaded NAP micelles" (LS-065-24) confirmed that melanin production was inhibited in a concentration-dependent manner. As a positive control, Arbutin (500 μg / ml) was treated (Fig. 10).

[0126] Test Example 10: Evaluation of the production of reactive oxygen species (H2O2) in human skin keratinocytes (HaCaT) by Lam332 micelles containing citron seed oil DP

[0127] We evaluated reactive oxygen species in keratinocytes by treating them with materials expected to reduce reactive oxygen species through intracellular DCF-DA staining. First, HaCaT cells were cultured for 24 hours, washed with PBS, and then treated with serum-free medium at various concentrations for 24 hours. After stimulation with 800 μM H2O2 for 3 hours, the cells were washed with DPBS. Next, the cells were treated with 100 μM DCF-DA and cultured for 30 minutes. Finally, the cells were washed with DPBS, harvested, and subjected to absorbance measurement or microscopic observation.

[0128] The experimental results confirmed that excessive ROS production increased by H2O2 stimulation treatment, and that citron oil DP treatment did not affect the change in ROS production amount. When "Yuja seed oil DP-loaded Lam332 micelles" (LS-065-25) was treated, it was confirmed that ROS production was suppressed by approximately 14.1 to 57.9%.

[0129] Luteolin (20 μM) was treated as a positive control (Fig. 11).

Claims

1. A skin cell-targeting nano-carrier comprising a micelle formed by self-association of an amphiphilic crystalline copolymer and an anisotropic lipid, a targeting peptide bound to the anisotropic lipid, and a physiologically active ingredient inside the micelle, wherein the above skin cell target is one of dermal fibroblasts, melanocytes, or keratinocytes, The above anisotropic lipid is mannosylerythritol lipid (MEL)-maleimide, A skin cell-targeting nano-delivery device characterized in that the targeting peptide is a pentapeptide KTTKS (Lys-Thr-Thr-Lys-Ser) whose skin cell target is a dermal fibroblast, NAP whose skin cell target is a dermal melanocyte, or Lam332 whose skin cell target is a keratinocyte.

2. A skin cell-targeting nano-delivery according to claim 1, characterized in that the physiologically active ingredient is ABT-751, PMWC, or citron seed oil DP.

3. A skin cell-targeting nano-delivery device according to claim 2, characterized in that the physiologically active ingredient is ABT-751 and the targeting peptide is pentapeptide KTTKS (Lys-Thr-Thr-Lys-Ser).

4. A skin cell-targeting nano-delivery device according to claim 2, characterized in that the physiologically active ingredient is PMWC and the targeting peptide is NAP.

5. A skin cell-targeting nano-delivery device according to claim 2, characterized in that the physiologically active ingredient is citron seed oil DP and the targeting peptide is Lam332.

6. A skin cell-targeting nano-carrier according to claim 1, characterized in that the amphiphilic crystalline copolymer is a poly(ethylene oxide)-b-poly(ε-caprolactone) copolymer (PEO-b-PCL).

7. A skin cell-targeting nano-carrier according to claim 1, characterized in that the average particle diameter of the micelle is 50 to 70 nm.

8. A step of dissolving an amphiphilic crystalline copolymer, an anisotropic lipid, and a physiologically active ingredient for targeting skin cells in a solvent and stirring the same to obtain a micelle dispersion; and A step of adding a dermal fibroblast targeting peptide to the above micelle dispersion and shaking, If the above skin cell target is a dermal fibroblast, the targeting peptide is a pentapeptide KTTKS (Lys-Thr-Thr-Lys-Ser) and the physiologically active ingredient is ABT-751. If the above skin cell target is a melanocyte, the targeting peptide is NAP and the above physiologically active ingredient is PMWC, A method for producing a skin-targeting nano-delivery device, characterized in that if the skin cell target is a keratinocyte, the targeting peptide is Lam332 and the physiologically active ingredient is citron seed oil DP.

9. A composition for external application to the skin comprising the skin cell-targeting nano-delivery agent of claim 1 or claim 2.

10. A cosmetic composition for strengthening skin barrier function, comprising the skin cell-targeting nano-delivery agent of claim 3.

11. A pharmaceutical composition for improving or treating atopic skin, comprising the skin cell-targeting nano-delivery agent of claim 3.

12. A cosmetic composition for skin whitening comprising the skin cell-targeting nano-delivery agent of claim 4.

13. A cosmetic composition for enhancing skin barrier or skin moisturizing function, comprising the skin cell-targeting nano-delivery agent of claim 5.

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