WO2016182277A2

WO2016182277A2 - Transdermal delivery method for bioactive substance using nanodiamond

Info

Publication number: WO2016182277A2
Application number: PCT/KR2016/004784
Authority: WO
Inventors: 권명택; 리치제레미; 김선옥; 임선희; 지정대
Original assignee: 나노리소스 주식회사; 주식회사 에스티에스네트웍스
Priority date: 2015-05-08
Filing date: 2016-05-09
Publication date: 2016-11-17
Also published as: KR20160132296A; KR101876282B1; WO2016182277A3

Abstract

The present invention relates to a method for delivering a bioactive substance into the skin by simultaneously attaching and solubilizing a hydrophilic bioactive substance and a hydrophobic bioactive substance on a surface of nanodiamond, followed by coating on a surface of the skin. The method of the present invention enables the solubilization of the hydrophobic bioactive substance and the loading of the hydrophobic bioactive substance together with the hydrophilic bioactive substance at the same time, so that the hydrophilic bioactive substance, which is difficult to penetrate into the skin stratum corneum, together with the hydrophobic substance, is delivered into the skin, thereby improving skin penetration. In the method of the present invention, the bioactive substance can be delivered into the skin without skin irritation or toxicity. In the method of the present invention, the skin penetration of the hydrophilic active substance, which is difficult to penetrate into the skin, can be improved by using a nanodiamond composite, and, at the same time, the nanodiamond per se remains on the surface of the skin to alleviate skin dryness through a moisturizing effect.

Description

Transdermal delivery method of bioactive substance using nanodiamond

The present invention relates to a method for transdermal delivery of a physiologically active substance using nanodiamonds, and simultaneously attaches and solubilizes a hydrophilic physiologically active substance and a hydrophobic physiologically active substance to the surface of the nanodiamond, and then applies the physiologically active substance to the skin surface. It is about how to deliver.

The skin is histologically composed of epidermis, dermis, subcutaneous fat and the like, and its thickness varies according to the site, age and sex. Although various skin conditions exist, these skins each play a role of external barrier function and other physiological functions. The stratum corneum is a layer of keratinized dead cells with relatively high density of hydrophobicity insoluble protein keratin, interposed between an intercellular lipid bilayer, a thin layer of lipids. As a structure, the unique hierarchical structure of the matrix, rich in lipids and insoluble proteins, makes it impermeable to chemical factors, including various compounds from external environments. In particular, the hydrophilic bioactive material is very difficult to permeate due to this hydrophobic multiple structure of the stratum corneum.

Therefore, in general, the transport of substances through keratin, especially the hydrophilic bioactive substances in healthy skin, is not easily achieved due to various protective actions of the skin, and only a very low concentration is transmitted even if permeated. In the case of cosmetics, the physiologically active substances contained in the cosmetics must pass through the stratum corneum, the outermost layer of the skin in order to show its efficacy, and in the case of hydrophilic bioactive substances, due to the hydrophobic multiple structure of the stratum corneum and its barrier function, Bioactive substances contained in have a problem that does not sufficiently exhibit its function.

Skin penetration routes of bioactive molecules through the skin are generally divided into three types: stratum corneum, hair follicle and sebaceous gland, sweat gland. However, the direct transcellular pathway to the stratum corneum as a skin barrier is not easy and the intercellular pathway through the intercellular lipid layer is known to be relatively efficient. It uses the intercellular lipid layer pathway composed of nonpolar lipids. Polar materials can hardly penetrate the intercellular lipid layer, but hydrophobic materials are easy to permeate. However, even though penetrating the stratum corneum, absorption of bioactive molecules by the solubility and various degradation enzymes under the stratum corneum is limited, and in particular, the hydrophilic bioactive substance is very difficult to permeate due to the hydrophobic multiple structure of the stratum corneum.

Until now, the system used for the transdermal delivery of physiologically active substances contained in cosmetics is mainly to make and apply vesicles containing the active substance to materials such as surfactants, lipids, polymers, etc. In particular, the lipid component is a component of the biological membrane As it can promote transdermal penetration through the improvement of skin affinity and skin absorption of physiologically active substances, it is preferentially utilized over other materials.

In particular, liposomes, which are the most widely used vesicles in cosmetic formulations, are structures consisting of a single layer or multiple layers of lipid bilayers that are most similar to biological membranes. It is used as one of the transdermal delivery systems that facilitates skin delivery of active molecules. However, the physicochemical instability of the membrane itself, low emulsification stability, among other things, the efficiency of the drug collection and maintenance of the stability of the drug itself is very low and the penetration rate of the skin is also poor, and as a result, the efficacy is very limited.

Therefore, the carrier supporting liposome-containing bioactive molecules and improving the skin permeation should be able to maintain the stability of the carrier itself and the stabilization of the support material as much as possible, and the composition, particle size, surface charge of the particles, zeta potential It is necessary to optimize the characteristics of the carrier, such as size and pH, and ultimately, when applying skin, it is necessary to secure safety and improve efficacy by bioactive molecules.

Therefore, various studies have reported skin permeation accelerators that promote the permeation of the active material, and their permeation promotion acts mainly on the intercellular lipid bilayer and the desmosome that is involved in intercellular binding. And mechanisms that act on related proteins and directly act on the internal structure of keratinocytes. The action of a delivery enhancer on the intercellular lipid bilayer is primarily a mechanism that loosens the structure of the lipid bilayer, facilitating the delivery of the active substance. A large number of skin penetration promoters facilitate delivery through this function. It is known. Some dermal penetration promoters are known to act on desmosomes and related proteins involved in intercellular binding of keratinocytes, thereby facilitating delivery through mechanisms that separate or loosen keratinocyte intercellular bonds and increase intercellular spacing. Other skin penetration promoters are known to facilitate delivery through mechanisms that penetrate inside keratinocytes to denature keratin or affect internal structure, facilitating delivery through the transcellular route.

Skin permeation accelerators inevitably alter the structure of the stratum corneum, which is the outermost skin layer, to promote skin permeation of bioactive substances. However, due to this, most skin permeation accelerators have high cytotoxicity to skin cells and have a very high possibility of causing skin irritation such as causing skin burning and erythema upon application.

On the other hand, the production of nanodiamonds by explosion reaction is carried out in a closed metal chamber under a gas atmosphere, for example, carbon dioxide (CO2) or water (H2O) or other liquid reducing agent conditions. Obtained by explosion reaction with, 4,6-trinitrotoluene (TNT) / 1,3,5-trinitrotriazacyclohexane (also known as hexogen or RDX).

In other words, DND occurs instantaneously when an explosive substance reacts with a mixture of explosive trinitrotoluene (TNT) and white crystalline non-aqueous explosive (RDX), at a certain ratio, for example, in the tens percent by weight. In the high temperature and high pressure atmosphere, the carbon component of the composition generates nuclei of diamond crystal phase (carbon sp ³ structure), and the nucleus grows to a certain size, and on the graphite (sp ² structure) surface, C, O, H, N And a plurality of functional groups. Representative functional groups thereof are known to have a large number of COOH, C═O, NH ₂ , CHO, OH, NO ₂ , -COC- and the like.

Several studies have shown that DND has little toxicity in vivo and is biocompatible due to the stability of the structure, and has a very small particle size of several nm size and specific surface area of 250 m ² / g-450 m ² / g. It is about ten to hundred times larger than diamond and its unique electrical, chemical and optical properties, including its many hydrophilic functional groups, can be used in a wide range of industries.

The present invention provides a method for delivering a bioactive substance into the skin without skin irritation or toxicity.

The present invention provides a method for easily penetrating into the skin a hydrophilic bioactive material that is difficult to deliver into the skin.

One aspect of the present invention for achieving the above object,

A solubilization step of mixing a hydrophobic skin bioactive substance, a hydrophilic skin bioactive substance and nanodiamonds in a solvent; And a method for transdermal delivery of a bioactive material comprising applying the solubilized solution to the skin.

In another aspect, the present invention is a solubilization step of mixing hydrophobic skin bioactive material, hydrophilic skin bioactive material and nanodiamond in a solvent, separating and drying the nanodiamond complex from the solubilized solution and the nanodiamond complex It relates to a method of transdermal delivery of a bioactive material comprising the step of dispersing in a solution and applying to the skin.

The method of the present invention can carry a hydrophilic bioactive material at the same time as the solubilization of the hydrophobic bioactive material, so that the hydrophilic bioactive material, which is difficult to penetrate the stratum corneum, is delivered to the skin together with the hydrophobic material to improve skin permeability.

The method of the present invention can deliver a bioactive substance into the skin without skin irritation or toxicity.

The method of the present invention can improve the skin permeability of the hydrophilic active material that is difficult to penetrate the skin using the nanodiamond complex, and at the same time the nanodiamond itself remains on the skin surface to improve the dryness of the skin through a moisturizing effect.

The present invention can support a significant amount of bioactive materials at a molecular level corresponding to the functional groups of the nanodiamond surface, thereby providing the effects of skin bioactive materials over a long period of time.

The present invention is also applied to cosmetic compositions such as skins, lotions, essences, creams, packs for the purpose of antioxidant efficacy, wrinkle reduction, melanin reduction, skin irritation, skin UV protection, skin dryness prevention, hair loss prevention, etc. can do.

1 illustrates the surface structure of explosive nanodiamonds.

2 is a schematic diagram showing that the hydrophobic bioactive material is bound to the nanodiamond surface functional group by the solubilization step, and b of FIG. 2 is a hydrophilic bioactive material (vitamin C) and a hydrophobic bioactive material (eugenol). It is a schematic diagram showing that they are combined randomly (in any order or order).

3 is a mixture of the hydrophobic bioactive substance Eugenol and the hydrophilic bioactive substance vitamin C without using nanodiamonds in water,

4 is a solution mixed with nanodiamonds added thereto.

5 is FT-IR of ND-COOH prepared in Example 1. FIG.

6 is FT-IR of ND-Eugenol-vitamin C prepared in Example 2. FIG.

7 is an FT-IR image of ND-Glutathione-Hesperidine-Eugenol prepared in Example 3. FIG.

Figure 8 is a skin permeation experiment of Experiment 1 was taken with a confocal microscope.

9 is a quantitative value of the skin permeation experiment of Experiment 1.

The present invention relates to a method of solubilizing hydrophobic and hydrophilic bioactive materials and delivering them to the skin using nanodiamonds.

The method of transdermal delivery of a bioactive material of the present invention includes a solubilization step and a skin application step.

The solubilization step includes mixing hydrophobic skin bioactive material, hydrophilic skin bioactive material and nanodiamond in a solvent.

1 illustrates the surface structure of explosive nanodiamonds. 2 is a schematic diagram showing that the hydrophobic bioactive material is bound to the nanodiamond surface functional group by the solubilization step, and b of FIG. 2 is a hydrophilic bioactive material (vitamin C) and a hydrophobic bioactive material (eugenol). It is a schematic diagram showing that they are combined randomly (in any order or order).

Referring to FIG. ₁ , the explosive nanodiamonds have functional groups such as COOH, C═O, NH ₂ , CHO, OH, NO ₂ , and —COC—.

The surface functional groups of the nanodiamonds may be surface modified with any one or more of Carboxyl, Lactone, Hydroxy, Phenol, Thiol, and Amine.

The average size of one nanodiamond particle may be 10 nm or more and 100 nm or less, and the number of functional groups attached to one particle may be 10,000 or more and 1,000,000 or less. Thus, the nanodiamond can be combined with a very large amount of bioactive material.

In the present invention, a linker may be attached to the nanodiamond surface functional group to amplify the number of functional groups. As the linker, alkylamine, arylamine, sugar, antioxidant, protein, peptide, nucleic acid or SiCH compound may be used. The linker may be covalently bonded to a functional group.

In the present invention, the physiologically active substance can use any known substance that affects skin improvement without limitation.

The skin bioactive material may include an organic material, an inorganic material, or both thereof. To the functional group (10,000 to 1,000,000 pieces) of the surface of the nanodiamond, different kinds of bioactive substances at the molecular level may be attached.

The organic material may be vitamins, lipids, proteins, peptides, flavonoids, nucleic acids, natural substance groups.

The inorganic material may include carbon silicon-based, carbon sulfide-based, and carbon phosphide-based compounds in which Si, S, and P are bonded to carbon.

More specifically, the skin bioactive substance is eugenol, ascorbic acid, vitamin A, vitamin B, vitamin C, vitamin E, vitamin K, vitamin P, hydroquinone, hesperidin, glutathione, EGCG, retinol, adenosine, hyaluronic acid Lonic acid, tannic acid, peptides, polyphenols, flavonoids and derivatives thereof.

The molecular weight of the bioactive material may be 2 to 500,000 or less, preferably 2 to 500 or less.

The hydrophilic bioactive substance refers to a hydrophilic substance and an excellent skin improving effect, and is not particularly limited as long as it is known to those skilled in the art, for example, arbutin, adenosine, vitamin C and Hydrophilic derivatives thereof, vitamin B3, vitamin B5, vitamin H and other hydrophilic vitamins and derivatives thereof, centella asiatica extract including acetylglucosamine, madecassoside, selenium aspartate, various plant extracts and various peptide components or It may be a mixture of two or more components.

Hydrophobic physiologically active substances are predominantly hydrophobic properties, such as eugenol, Retinol, ASTAXANTHIN, CAFFEIC ACID, CARNOSIC ACID, CATECHIN, COENZYME-Q10, CURCUMIN, ELLAGIC ACID, FERULIC ACID, IDEBENONE, ISOFLAVONE, LINOLEIC ACID, LIPOC , OLEANOLIC ACID, PHLORETIN, QUERCETIN, RESVERATROL, SQUALANE, SQUALENE, TANNIC ACID, VITAMIN A, VITAMIN B VITAMIN E, VITAMIN F, CHOLESTEROL, PHYTOSPHINGOSINE, SQUALENE, GLYCOSPHINGOLIPIO

There is no restriction on the hydrophobic bioactive material and the hydrophilic bioactive material in the present invention, the hydrophobic bioactive material is a material that is easy to penetrate the stratum corneum based on the partition coefficient, and the hydrophilic bioactive material is permeable to the stratum corneum Can be a difficult substance. For example, the hydrophobic bioactive material has a distribution coefficient (log P value) of 1 to 3, and is easy to penetrate the stratum corneum, and the hydrophilic bioactive material is difficult to penetrate the skin stratum corneum (distribution coefficient (log P value). ) May be less than 1).

The hydrophobic skin bioactive material or hydrophilic skin bioactive material may be used to have antioxidant, anti-wrinkle, whitening, moisturizing or hair loss prevention activity.

The bioactive substance having the antiwrinkle activity may be selected from the group of skin cell growth factors, proteins or peptides, retinol, retinyl palmitate, adenosine and polyethoxylatedretinamide.

The bioactive substance having the whitening activity may be selected from arbutin, ascorbic acid, acetyl ascorbyl ether, ascorbyl glucoside, niacinamide, ascorbyl phosphate, bisabolol, whitening peptide group.

The solvent may be water, ethanol or a mixture of water and ethanol, or buffer solutions.

The solubilization step may control the pH according to the concentration or hydrophobicity, nanodiamond content of the hydrophobic skin bioactive material and the hydrophilic skin bioactive material.

For example, the solubilization step may be a pH of 3 to 10, preferably 5 to 8, more preferably 6 to 7.

The solubilization step may stir the solution strongly for a predetermined time. For example, the mixing time may be 24 hours.

In the mixing step, the mixed solution may be mixed (strongly) using ultrasonic waves.

The solubilization step of the present invention can be carried out by stirring the mixture at an appropriate pH condition without additives such as surfactants.

The weight ratio (or molar ratio) of the nanodiamond particles added to the solubilization step: hydrophilic bioactive material: hydrophobic bioactive material may be mixed in an appropriate range.

The method may add more than 2000ppm of the nanodiamond. More specifically, the nanodiamonds may be added in an amount of 0.001 to 20 wt% based on the solvent.

The content of the bioactive material may be 5-30% of the weight of the surface-modified nanodiamond. On the other hand, when the number of functional groups is increased by using a linker on the surface of the nanodiamond, the content of the bioactive substance may be in the range of 0.1 to 1,000% of the weight of the surface-modified nanodiamond.

Referring to Figure 2, after the solubilization step of the present invention, the hydrophilic bioactive material and the hydrophobic bioactive material are uniformly present in the solvent through non-covalent bonding with nanodiamonds.

The solubilization step is a step in which the hydrophobic skin bioactive material and the hydrophilic skin bioactive material are non-covalently bonded to the surface functional group of the nanodiamond at the same time.

The non-covalent bond between the nanodiamond and the physiologically active substance may be a hydrogen bond, an electrostatic bond, or a van der Waals bond.

The term "solubilization" used in the present invention is used as a term indicating a state in which a hydrophilic bioactive substance and a hydrophobic bioactive substance which are not dissolved in each other are uniformly mixed with a hydrophilic or hydrophobic solvent. In addition, in the present invention, when the physiologically active substance (in the case of an inorganic substance) is dispersed in a particulate state without being dissolved in a solvent, it is represented as "solubilization". Nanodiamonds of the present invention are not dissolved but dispersed in a solvent even after a solubilization step.

The present inventors have come to the present invention knowing that solubilization of hydrophilic and hydrophobic physiologically active substances that do not dissolve with each other is due to nanodiamonds.

More specifically, the inventors have determined that the bioactive materials are solubilized by the interaction between the bioactive material and the functional group present on the surface of the nanodiamond and the nanodiamond surface moisture layer.

The hydrophobic and hydrophilic bioactive materials may be present in a molecular unit by 10,000 or more and 1,000,000 or less functional groups present on the surface of the nanodiamond particles.

2B is a conceptual diagram showing that molecules of hydrophobic and hydrophilic bioactive materials are randomly bonded to the nanodiamond surface functional group randomly (in any order or order) by the mixing step.

Referring to Figure 2, there are 10,000 or more than 1,000,000 functional groups on the surface of the nanodiamond, hydrophobic bioactive material 20 is bonded to some of these functional groups, hydrophilic bioactive material to be coupled to the other part Can be. That is, a large number of hydrophilic bioactive molecules and hydrophobic bioactive molecules may be simultaneously supported on one nanodiamond particle. The molar range of the physiologically active substance bound per nanodiamond particle may be in the range of 1 × 10 ⁻⁴ mol / g −1 × 10 ⁻² mol / g.

As shown in FIG. 2, the present inventors maintain that the nanodiamond particles are stably combined with hundreds of thousands to hundreds of thousands of hydrophobic bioactive molecules and hydrophilic bioactive molecules at the same time, with 10,000 to 1,000,000 or less present on the surface of the nanodiamond. It is understood because of the functional groups and the activation energy of these functional groups.

3 is a mixture of a hydrophobic bioactive substance Eugenol and a hydrophilic bioactive substance vitamin C in water without using nanodiamonds, and FIG. 4 is a solution in which nanodiamonds are added and mixed.

3 is clearly distinguished from the drops of Eugenol, which is a hydrophobic material, and sank to the bottom of the vial, but in the case of FIG. 4, the hydrophobic bioactive material and the hydrophilic bioactive material are uniformly mixed through nanodiamonds. You can check it.

The solubilization method of the present invention can be stably supported on nanodiamonds without the molecular structure or active modification of the bioactive material.

The present invention can add a cosmetic to the solubilized solution to prepare a cosmetic composition.

The cosmetic may be a flexible lotion (skin), nourishing lotion (milk lotion), nourishing cream, massage cream or essence.

The cosmetic may include a whitening agent, a moisturizing agent, an antioxidant, an ultraviolet absorber, a surfactant, a thickener, an alcohol, a preservative, a gelling agent, a fragrance, a filler, or a dye.

The method may prepare the solubilized solution or cosmetic composition in the form of a gel or an emulsion.

The method may be prepared in powder form by drying the solubilized solution.

The present invention includes applying the solubilized solution or the cosmetic composition to the skin.

The skin coating step includes the step of releasing a hydrophilic bioactive material and a hydrophobic bioactive material which are non-covalently bound to the surface of the nanodiamond, and the hydrophilic organic material and the hydrophobic organic material together into the skin through each skin epidermal gap. Infiltration may be included.

In the release step, the hydrophilic bioactive material and the hydrophobic bioactive material non-covalently bound to the functional group of the nanodiamond can be separated from the functional group by the pH change of the skin and penetrate into the skin.

The release step may be separated from the functional group by the concentration difference between the hydrophilic bioactive material and the hydrophobic bioactive material non-covalently bonded to the functional group of the nanodiamond to penetrate into the skin. In addition, the hydrophilic bioactive material and hydrophobic bioactive material may be separated from the nanodiamond by the concentration of salts contained in body fluids such as sweat or body fluids, and may penetrate into the skin.

In the skin application step, the skin bioactive material penetrates into the skin, but the nanodiamond remains on the surface of the skin as it is.

Hereinafter, the present invention will be described in more detail with reference to examples, but these are merely to illustrate preferred embodiments of the present invention, and the examples do not limit the scope of the present invention.

Example 1

Preparation of ND-COOH

10 g of well-dispersed nanodiamonds having an average particle size of 50 nm prepared by using a nanodispersion particle dispersion process were added to 600 ml of sulfuric acid and nitric acid in a volume ratio of 3: 1 and reacted at 140 ° C. for 12 hours. After the reaction, the mixture was diluted for about 30 minutes using an ice bath to prevent volatilization and scattering of the filtrate, followed by centrifugation. Centrifugation was repeated about 10 times until the pH was neutral. The dispersed nanodiamonds thus obtained were dried. Drying was performed at 100 ° C. in an oven for 10 hours or separately using an evaporator to remove moisture. The particles obtained above were confirmed to be ND-COOH through FT-IR (FIG. 5).

ND- Eugenol -vitamin C Produce

2 g of the nanodiamond prepared in Example 1 was added to 900 mL of deionized water, dispersed for 60 minutes using ultrasonic waves, and then allowed to stand at room temperature. After titrating the pH to 7, using 0.25N NaOH solution, 100 mg of hydrophilic bioactive substance Vitamin C and 100 mg of hydrophobic bioactive substance Eugenol were added thereto, stirred for 10 minutes, and deionized water was added to 1000 mL. The solid and the liquid were separated by centrifugation at 10,000 rpm for 10 minutes and washed once to prepare ND-Eugenol-vitamin C in powder form.

The prepared ND- Eugenol-vitamin C was confirmed the presence or absence of the active material of the complex using FT-IR (Fig. 6).

Example 3

ND- Glutathione - Hesperidine - Eugenol Manufacture

2 g of the nanodiamond prepared in Example 1 was added to 900 mL of deionized water, dispersed for 60 minutes using ultrasonic waves, and then allowed to stand at room temperature. After titrating the pH to 7, using 0.25N NaOH solution, add 2mg of hydrophilic bioactive substance Glutathione, 0.2mg of hydrophobic bioactive substance, 0.2mg of Eugenol and 100mg of Eugenol for 10 minutes, and then deionized water to 1000mL. Was added. The solid and the liquid were separated by centrifugation at 10,000 rpm for 10 minutes and washed once to prepare ND-Glutathione-Hesperidine-Eugenol in powder form.

The prepared ND-Glutathione-Hesperidine-Eugenol was confirmed the presence or absence of the active material of the complex using FT-IR (Fig. 7).

Example 4 Synthesis of ND-COOH Fluorescent Derivative (Fluorescein) Using Covalent Bond

ND-COOH 1g SOCl ₂ The mixture was added to a mixture of 30 mL and 0.15 mL of anhydrous DMF, and then completely dispersed using ultrasonic waves. After the mixed solution was warmed at 70 ° C. for 24 hours, the remaining SOCl ₂ was removed by distillation under low temperature and reduced pressure.

The ND-COCl ₂ obtained in the reaction was completely mixed with anhydrous DMSO using ultrasonic waves, and 0.3 mL of pyridine and 5 g of ethylenediamine were added thereto. The mixture was stirred for 24 hours at room temperature to react, and after removing DMSO using a vacuum apparatus, the reaction mixture was washed by centrifugation (10,000 rpm, 10 minutes) with deionized water and the process was repeated five times.

After adding 1 g of ND-CONH- (CH ₂ ) ₂ -NH ₂ obtained in the reaction to a 0.1 M sodium bicarbonate solution, 5 g of fluorescent material Fluorescein was added thereto and stirred at room temperature for 24 hours. After stirring, the reaction was washed by centrifugation with deionized water (10,000 rpm, 10 minutes) and the process was repeated five times.

Skin application

The soluble solution prepared above was applied to the skin about 0.5 mL.

Comparative Example 1

In Example 1, the hydrophobic bioactive substance Eugenol and the hydrophilic bioactive substance vitamin C were added to water and stirred without adding nanodiamonds (content conditions are the same as those in Example 1).

Experiment 1; Skin non-permeation experiment of nanodiamond

Percutaneous absorption experiments were performed according to published guidelines to determine whether ND-COOH penetrated into skin tissue (transdermal absorption) (Test Guideline 428: Skin absroption: in vitro Method, OECD, Paris, (2004), In vitro skin absorption test guidelines, Korea Food and Drug Administration (2010)). For percutaneous absorption experiment, porcine skin was used, and the prepared skin was donor and receptor of a vertical diffusion cell (Logan FDC-6, Logan instrument Corp. Somerset, NJ, USA). ) The epidermis face up (in the donor direction) and the dermis down (receptor direction). The receptor was filled with PBS solution (phosphate-buffered saline, pH 7.4, 32 ° C) and allowed to stand for 1 hour so that the pig skin was in equilibrium with the PBS solution. Thereafter, the ND-COOH fluorescent derivative prepared in Example 4 was dispersed in 0.5 mL of water and applied to the skin. After 12, 24 and 48 hours, the skin tissue was recovered, washed with deionized water, and then fixed in 10% Formaldehyde for 18 hours. After fixation, cryosections (14 μm) were prepared using a Microm HM520 cryostat, Thermo, and placed on a glass slide. The prepared slides were stained with PBS buffer for 10 minutes and stained with cell nuclei in tissues by exposure to 0.2 mM DAPI solution for 7 minutes. The stained tissue was washed again with PBS buffer three times for 10 minutes, and then fixed on a slide using a mounting medium, and observed with a confocal microscope. The results are shown in FIG. 8.

In addition, by collecting a sample of the receptor at each of 6, 12, 18, 24, 30, 36, 48 hours, using a fluorescence measuring device to measure the fluorescence value of the fluorescent substance transmitted through the receptor to obtain a quantitative value and to show this in Figure 9 It was.

Referring to FIGS. 8 and 9, the fluorescent control (Fluorescein), which is a negative control group, penetrated the skin, but the fluorescence bound to ND did not penetrate the skin.

In the above, preferred embodiments of the present invention have been described in detail, but these are merely for the purpose of explanation and the scope of protection of the present invention is not limited thereto.

Claims

A solubilization step of adding hydrophobic skin bioactive substances, hydrophilic skin bioactive substances and nanodiamonds in a solvent and mixing them; And

A method for transdermal delivery of a bioactive substance comprising applying the solubilized solution to the skin.
A solubilization step of adding hydrophobic skin bioactive substances, hydrophilic skin bioactive substances and nanodiamonds in a solvent and mixing them; And

Separating and drying the nanodiamond composite from the solubilized solution; And

A method for transdermal delivery of a bioactive material comprising dispersing the nanodiamond complex in a solution and applying it to the skin.
The method of claim 1, further comprising: adding a cosmetic to the solubilized solution to prepare a cosmetic composition; And applying the cosmetic composition to the skin.
The method of transdermal delivery of a bioactive substance according to claim 2, wherein the solution comprises a cosmetic.
The method of transdermal delivery of a physiologically active substance according to claim 3 or 4, wherein the cosmetic is a flexible lotion (skin), a nourishing lotion (milk lotion), a nourishing cream, a massage cream or an essence.
The bioactive agent according to claim 3 or 4, wherein the cosmetic contains a whitening agent, a moisturizing agent, an antioxidant, an ultraviolet absorber, a surfactant, a thickener, an alcohol, a preservative, a gelling agent, a fragrance, a filler, or a dye. Method of transdermal delivery of a substance.
The method according to claim 1 or 3, wherein the method is prepared by applying the solubilized solution or composition in the form of a gel or emulsion to the skin.
The method of claim 1, wherein the solubilizing step controls the pH according to the concentration, degree of hydrophobicity, or nanodiamond content of the hydrophobic skin bioactive material and the hydrophilic skin bioactive material.
The method of claim 1, wherein the solubilizing step controls the pH to 3-10.
The method of claim 1, wherein the solubilizing step is a transdermal delivery method of a physiologically active substance, characterized in that the mixed solution is mixed using an additional ultrasonic wave.
The method of claim 1, wherein the surface functional group of the nanodiamond is any one or more of carboxyl, Lactone, Hydroxy, Phenol, Thiol, Amine.
The method of claim 1, wherein the average size of the nanodiamond particles is 10 or more and 100 nm or less, and the total number of functional groups attached to the surface is 10,000 or more and 1,000,000 or less. .
The method of claim 1, wherein the solubilizing step is characterized in that the hydrophobic skin bioactive material and the hydrophilic skin bioactive material are non-covalently bonded to and dispersed at the surface functional group of the nanodiamonds.
The method of claim 1, wherein the nanodiamond particles per one coupling is a molar range of the solubilized skin biomolecule 1 × 10 - transdermal delivery of a skin physiologically active substance, characterized in that not more than 2 mol / g ways.
The method of claim 1, wherein the skin physiologically active substance comprises at least one organic substance, inorganic substance, or both thereof.
16. The method of claim 15, wherein the organic material is a vitamin, lipid, protein, peptide, flavonoid, nucleic acid, or natural substance group.
16. The method of claim 15, wherein the inorganic material comprises carbon-silicon-based, carbon sulfide-based, and carbon phosphide-based compounds in which Si, S, and P are bonded to carbon.
According to claim 1, wherein the hydrophobic bioactive material has a partition coefficient (log P value) of 1-3 is a material that is easy to penetrate the stratum corneum, the hydrophilic bioactive material is a material that is difficult to penetrate the skin stratum corneum (distribution coefficient (log Transdermal delivery method of a physiologically active substance, characterized in that the P value) is less than 1).
The method of transdermal delivery of a physiologically active substance according to claim 1, wherein the bioactive substance has a molecular weight of 500,000 or less.
The method of claim 1, wherein the solvent is water, ethanol or a mixture of water and ethanol, or buffer solutions.
The method of claim 1, wherein the hydrophobic skin bioactive material or hydrophilic skin bioactive material has antioxidant, anti-wrinkle, whitening, moisturizing or hair loss prevention activity.
22. The method of claim 21, wherein the bioactive substance having antiwrinkle activity is selected from the group of skin cell growth factors, proteins or peptides, retinol, retinyl palmitate, adenosine and polyethoxylateddretinamide. Method of transdermal delivery of skin physiologically active substances.
The bioactive substance having whitening activity is selected from arbutin, ascorbic acid, ethylascorbyl ether, ascorbyl glucoside, niacinamide, ascorbyl phosphate, bisabolol, and whitening peptide group. Transdermal delivery method of a skin physiologically active substance.
The method of claim 1 or 2, wherein the skin coating step comprises the steps of releasing a hydrophilic bioactive material and a hydrophobic bioactive material non-covalently bonded to the nanodiamond surface; And

A method for transdermal delivery of a physiologically active substance, comprising the step of penetrating the hydrophilic organic material and hydrophobic organic material together into the skin through each skin epidermal gap.
25. The method of claim 24, wherein the release step is characterized in that the hydrophilic bioactive material and hydrophobic bioactive material non-covalently bonded to the functional group of the nanodiamond is separated from the functional group by the pH change of the skin and penetrates into the skin Method of transdermal delivery of a substance.
25. The bioactive material according to claim 24, wherein the release step is separated from the functional group by the concentration difference between the hydrophilic bioactive material and the hydrophobic bioactive material which are non-covalently bound to the functional group of the nanodiamond, and penetrates into the skin. Method of transdermal delivery.