KR101393397B1 - Transdermal delivery system of dermatological active ingredients using cellular transduction peptides - Google Patents

Abstract

The present invention relates to a transdermal delivery composition comprising a skin biologically active molecule into which an intracellular molecule-transporting peptide is fused. Specifically, the composition is that because of the size and the physical properties of the molecular weight of the fusion peptide superior ability in vivo molecule transmitting the skin bioactive molecules are not easily transmitted through the stratum corneum, in vitro and in it exhibits excellent skin permeability in vivo . Therefore, it can be effectively used for transferring various compounds having various advantages into skin cells, and can be formulated into various forms, and thus can be usefully used as a raw material for functional cosmetic compositions .

Description

TECHNICAL FIELD The present invention relates to a transdermal delivery system for dermatological active ingredients using cellular transduction peptides,

The present invention relates to a composition for transdermal delivery comprising a skin biologically active molecule to which an intracellular molecule-transporting peptide is bound, a transdermal delivery system using the skin biologically active molecule, and a skin cell delivery method of the skin biologically active molecule.

Skin is a tissue that is always in contact with the external environment. Its main function is to prevent leakage of body fluids, prevention of infection, and protection against water loss. The stratum corneum of the epidermis is present in the outermost part of the skin and inhibits the loss of moisture and electrolytes outside the skin, thereby preventing the skin from drying out and providing an environment for normal biochemical metabolism of the skin, (Bouwstra J. A, Honeywell-Nguyen PL Gooris GS and Ponec M. Prog Lipid Res. 42: 1-36), which protects the human body from infection and prevents bacteria, fungi and viruses from invading the skin (2003)).

These pathways of absorption through the skin have three pathways: absorption through the stratum corneum, absorption through the hair follicles and sebaceous glands, and absorption through the glands (Prausnitz M. R. and Langer R. Nat Biotech. 26: 1261-1268 (2008)). The transfer of physiologically active molecules through the skin is limited by the structural and physical characteristics of the skin. As far as it is known, absorption through the stratum corneum is the most important absorption pathway. In particular, the keratinocytes of the skin are keratinocyte, which is a major constituent cell of the skin, and the keratinocyte is naturally contracted to form a dense structure in the outermost layer of the skin. It inhibits the penetration of foreign substances as well as the evaporation of water, The acidity is around pH 5. To penetrate these horny layer barriers, the molecular weight must be as small as less than 1,000, and it must be possessed of lipophilic properties (Metha R. C. and Fitzpatrick R. E. Dermatol. Ther. 20: 350-359 (2007)).

It is known that low molecular weight synthetic compounds or natural compounds having a molecular weight of 1,000 or less, which are frequently used as cosmetic raw materials, can be easily delivered into cells. However, macromolecules such as proteins, peptides and nucleic acids having a molecular weight of 1,000 or more have a double lipid membrane structure It is difficult to penetrate into the cell membrane. It is known that the permeation efficiency of low molecular weight substances is extremely low and the permeation efficiency of high molecular weight substances is even lower due to the intrinsic properties of the stratum corneum constituting the skin barrier. Transdermal drug delivery (TDD) is one of the increasingly popular methods for amplifying the efficiency of these small molecules and macromolecules passing through the plasma membrane of cells (Prausnitz MR and Langer R. Nat Biotech. 26: 1261-1268 (2008)). However, the greatest barrier to administration through the skin is keratinocytes in the stratum corneum and interstitial cells between them. Physiologically active molecules (hereinafter, skin physiologically active molecules) in most of the skin have a low transdermal permeability due to the resistance of the horny layer of the skin.

Various methods for promoting the transdermal permeability of such skin biologically active molecules have been studied and can be broadly divided into physical, biochemical, and chemical methods. The physical method is mainly a method using iontophoresis, electroporation, sonophoresis, and thermal ablation. The biochemical method uses a precursor, and the active ingredient is activated The chemical method is one of the most widely used methods in the industry. It is a form of a complex in which various kinds of bases, surfactants, solvents, fatty acids and the like mainly contain physiologically active molecules Carrier-mediated delivery systems have been used (Prausnitz MR and Langer R. Nat Biotech. 26: 1261-1268 (2008)). Of these, much attention has been focused on the use of peptide-based delivery systems.

The use of peptides with cell permeability has several advantages, mainly due to the various modifications that can be made to constant peptide sequences. This allows the designation of different subcellular domains and the manipulation of carriers capable of carrying various types of cargo molecules. A representative example of a cell membrane permeable peptide is as follows.

The first transport domains found were the Green and Loewenstein groups (Green and Loewenstein, Cell 55, 1179-1188 (188)) and the Frankel and Pabo groups (Frankel and Pabo, Cell 55, 1189-1193 Separately, we found that the transcription-related protein Tat of HIV-1, a virus that causes acquired immune deficiency syndrome (AIDS), permeates the cell membrane and cross-activates the viral gene. The transmembrane Tat domain is a 48- to 57-th basic amino acid sequence (GRKKRRQRRR) of Tat protein, which has been shown to play an important role in passing through the cell membrane (Vives et al., J. Biol. Chem. 272, 16010-16017 (1997); Futaki et al., J. Biol. Chem. 276, 5836-5840 (2001); Wadia, JS and SF Dowdy, Cum Opin. Biotechnolol. 13 (1): 52-6 ; Wadia et al., Nature Medicine 10 (3), 310-315 (2004)).

Secondly, there can be mentioned phenetylatin (RQIKIYFQNRRMKWKK) consisting of 16 amino acids derived from the Drosophila antennanapedia homeodomain (Joliot et al., Proc Natl Acad Sci USA 88: 1864-1868 (1991) Joliot, A. and A. Prochiantz, Nat. Cell Biol. 6 (3): 189-96 (2004)).

Third, peptides based on membrane translocating sequences (MTS) or signal sequences, which are responsible for the transfer of proteins newly synthesized by RNA to receptor proteins in vivo, And it is known that biotransformation sequences associated with nuclear localization signals (NLS) are accumulated in cell nuclei through cell membranes in several types of cells (JS Wadia, et al., Nat. Med. 10: 310-315 (2004); I. Nakase, et al. Biochemistry 46: 492-501 (2007); HL Amand, et al., Biochim. Biophys. Acta (2011)). These peptides bind to cargo molecules and act as import signals when they are close to cells, leading to the entry of cargo molecules into cells. Accordingly, attempts have been actively made to apply a cell membrane permeable peptide to proteins originally present in cells to apply them to biological research and disease treatment (F. Milletti, Drug Discovery Today 17: 850-860 (2012)). In the case where the cell membrane permeable peptide is derived from viruses such as Tat or VP22, there is a possibility of inducing an immune response in vivo, and thus, a secreted protein found in humans is mainly used to identify new cell membrane permeable peptides (Eguchi , A. and Dowdy, SF Trends Pharmacol. Sci. 30: 341-345 (2009); Mae, M. and Langel, U. Curr. Opin. Pharmacol. 6: 509-514 (2006); Jarver, P. et al., Trends Pharmacol. Sci. 31: 528-535 (2010)). In order to increase the permeability of the cell membrane, new cell membrane permeable peptides have been developed by deletion and modification of amino acid sequence and chimeric fusion (Taylor, BN et al., Cancer Res. 69: 537-546 (2009); Johansson, HJ et al., Mol. Ther. 16: 115-123 (2007)).

Currently known carrier-mediated delivery systems have limited efficiency due to inefficiency or self-toxicity, and delivery mechanisms are controversial. Various mechanisms have been proposed, although the exact answer has not been obtained as to whether or not endocytosis is involved in the intracellular substance transport mechanism of the carrier. It is claimed that inverted micelles are formed and not transferred to receptors. Another opinion is that proteoglycan on the cell surface plays an important role in the transfer, and that there is an electrostatic interaction with the transmembrane peptide It is also suggested that it can not play a big role in the world. Some studies have suggested that TAT proteins do not actually transfer, but they strongly induce electrostatic interactions with the cell surface, resulting in inclusion and accumulation within the endosome, and endosomes are fused with lysosomes Suggesting that peptides are easily degraded by the proteolytic enzymes present in lysosomes. In order to deliver a specific active ingredient into a cell by using a substance-transporting carrier composed of a basic amino acid such as TAT, it is difficult to directly access the target in the cytoplasm, and in order to deliver the active ingredient to a level that can exhibit the desired activity, (JS Wadia, et al., Nat. Med. 10: 310-315 (2004); I. Nakase, et al. Biochemistry 46: 492 -501 (2007); HL Amand, et al. Biochim. Biophys. Acta (2011), F. Milletti, Drug Discovery Today 17: 850-860 (2012)). As such, there is a lot of controversy about the intracellular entry mechanism of the known mediator delivery system of foreign substances (F. Milletti, Drug Discovery Today 17: 850-860 (2012)). In addition, there is no example showing that the existing developed peptides can transmit skin cells through the horny layer until now.

Recent studies have shown that carrier-mediated delivery systems can undergo metastasis via one or more mechanisms, resulting in energy-dependent macropinocytosis in the case of macromolecules, and small molecules associated with the transmembrane peptide itself or with the transmembrane peptide, It is assumed that it has an energy-independent permeation mechanism by hydrogen bonding. In any case, direct contact of the proteoglycan and the membrane permeable peptide present in the cell membrane or cell membrane is essential for the success of intracellular delivery. It has been shown that lipid raft-mediated ovarian function is the main mechanism by β-cyclodextrin treatment, cytochalasin D treatment, or dominant-negative dynamine (DynK44A) expression (Wadia JS, Stan RV, Dowdy SF. Nat Med 2004 Mar; 10 (3): 310-5.).

In 2000, new transmembrane peptides with improved cellular delivery efficiency and different structural and electrostatic properties were developed, including the Macromolecule Intracelular Tranduction Technology developed by Procel, MITT) does not require the inclusion and energy necessary for the transduction process of HIV-TAT cells in the intracellular transduction process, but since the rigidity and integrity of the cell membrane are important factors, direct interaction with the cell membrane (WO2008 / 093982). MTD has a higher efficiency of delivering a cargo material, which can be selected from among compounds, peptides, and proteins, and has advantages of cell-to-cell delivery as compared with TAT which is a conventional cell membrane permeable peptide. Its applicability to cosmetics development is highly appreciated.

Accordingly, the present inventors modified the amino acid sequence of the above-mentioned macromolecular transport domain (MTD) to apply an improved MTD peptide having better cell permeability to the intracellular molecule-transferring peptide of the present invention, The present inventors have completed the present invention by proving that the skin biologically active molecules which are not easily permeable to the skin are imparted with a delivery effect in the stratum corneum or in the skin cells.

The present invention is designed to efficiently introduce skin biologically active molecules, which are difficult to transmit through the skin due to the molecular weight or inherent characteristics of the skin horny layer, into skin cells as described above. A transdermal delivery composition comprising a skin biologically active molecule to which a molecule transfer peptide is bound, a transdermal delivery system using a skin biologically active molecule to which the peptide is bound, and a skin biologically active molecule to which an intracellular molecule transfer peptide is bound, Or into the stratum corneum of the skin.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above object,

The present invention provides a composition for percutaneous delivery comprising a skin biologically active molecule to which an intracellular molecule-transporting peptide is bound, wherein the peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 35 .

In one embodiment of the present invention, the composition is a cosmetic composition.

In another embodiment of the present invention, the peptide is characterized in that it is encoded from a polynucleotide sequence selected from the group consisting of SEQ ID NOS: 36 to 56.

In one embodiment of the present invention, the amino acid sequence of SEQ ID NO: 15 may be encoded by the polynucleotide sequence of SEQ ID NO: 36, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 16 may be encoded by the polynucleotide sequence of SEQ ID NO: 37, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 17 may be encoded by the polynucleotide sequence of SEQ ID NO: 38, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 18 may be encoded by the polynucleotide sequence of SEQ ID NO: 39, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 19 may be encoded by the polynucleotide sequence of SEQ ID NO: 40, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 20 may be encoded by the polynucleotide sequence of SEQ ID NO: 41, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 21 may be encoded by the polynucleotide sequence of SEQ ID NO: 42, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 22 may be encoded by the polynucleotide sequence of SEQ ID NO: 43, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 23 may be encoded by the polynucleotide sequence of SEQ ID NO: 44, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 24 may be encoded by the polynucleotide sequence of SEQ ID NO: 45, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 25 may be encoded by the polynucleotide sequence of SEQ ID NO: 46, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 26 may be encoded by the polynucleotide sequence of SEQ ID NO: 47, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 27 may be encoded by the polynucleotide sequence of SEQ ID NO: 48, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 28 may be encoded by the polynucleotide sequence of SEQ ID NO: 49, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 29 may be encoded by the polynucleotide sequence of SEQ ID NO: 50, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 30 may be encoded by the polynucleotide sequence of SEQ ID NO: 51, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 31 may be encoded by the polynucleotide sequence of SEQ ID NO: 52, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 32 may be encoded by the polynucleotide sequence of SEQ ID NO: 53, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 33 may be encoded by the polynucleotide sequence of SEQ ID NO: 54, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 34 may be encoded by the polynucleotide sequence of SEQ ID NO: 55, but is not limited thereto.

In another embodiment of the present invention, the amino acid sequence of SEQ ID NO: 35 may be encoded by the polynucleotide sequence of SEQ ID NO: 56, but is not limited thereto.

In another embodiment of the present invention, the composition is characterized in that it penetrates the horny layer of the skin.

In another embodiment of the present invention, the skin biologically active molecule binds to the N-terminal, C-terminal or both terminal ends of the intracellular molecule-transporting peptide.

In another embodiment of the present invention, the skin biologically active molecule is bound to the intracellular molecule transport peptide in a form in which amino acids are arranged in the reverse order.

In still another embodiment of the present invention, the binding is characterized by peptide bonding or chemical bonding.

In another embodiment of the present invention, the chemical bond may be a disulfide bond, a diamine bond, a sulfide-amine bond, a carboxyl-amine bond, an ester bond, ≪ / RTI >

In another embodiment of the present invention, it is characterized in that it is selected from the group consisting of said vitamins, retinoids and fatty acids.

In another embodiment of the present invention, the vitamin is selected from the group consisting of vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D1, vitamin D2, vitamin D3 , Vitamin D4, vitamin D5, vitamin E, and vitamin K.

In another embodiment of the present invention, the retinoid is selected from the group consisting of retinol, retinol, and synthetic analogs, retinal, trans, 9-cis, and 13-cis retinoic acid and etretinate.

In another embodiment of the present invention, the fatty acid is selected from the group consisting of lauric acid, stearic acid, palmitic acid, undecylenic acid, palmitoleic acid, oleic acid, linolic acid, linolenic acid, arachidonic acid and erucic acid .

In another embodiment of the present invention, the skin biologically active molecule is selected from the group consisting of a wrinkle-reducing agent, a whitening agent, an antioxidant, and a moisturizing agent.

In another embodiment of the present invention, the anti-wrinkle agent is selected from the group consisting of skin-acting growth factor peptide or protein, retinol, retinyl palmitate, adenosine and polyethoxylated retinamide.

In another embodiment of the present invention, the whitening agent is selected from the group consisting of whitening peptide, niacinamide, mulberry extract, arbutin, ethyl ascorbyl ether, licorice extract, ascorbyl glucoside, and magnesium ascorbyl phosphate .

In another embodiment of the present invention, the skin biologically active molecule is characterized by being selected from the group consisting of coumaric acid or acetyl pentapeptide and acetyl hexapeptide.

In another embodiment of the present invention, the composition is prepared from a formulation selected from the group consisting of an emulsion, a cream, an essence, a skin, a liposome, a microcapsule, a composite particle, a shampoo and a rinse.

The present invention also relates to a method for transdermal delivery of a skin biologically active molecule into skin cells, which comprises binding an intracellular molecule-transferring peptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 15 to 35 to a skin biologically active molecule Delivery system.

In one embodiment of the present invention, the peptide is characterized in that it is encoded from a polynucleotide selected from the group consisting of SEQ ID NOS: 36 to 56.

In addition, the present invention relates to a method for delivering a skin physiologically active molecule into skin cells, which comprises the step of binding an intracellular molecule-transferring peptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 15 to 35 to a skin physiologically active molecule, ≪ / RTI >

In addition, the present invention relates to a method for delivering a skin biologically active molecule to the skin stratum corneum, comprising the step of binding and transferring an intracellular molecular transport peptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 15 to 35 to a skin biologically active molecule ≪ / RTI >

The transdermal delivery composition of the present invention binds a peptide having excellent transcription ability in vivo to a skin biologically active molecule which is not easily permeable to the horny layer due to its molecular weight and physical properties, It can be delivered into the stratum corneum. Thus, the composition of the present invention has the advantages of antioxidant efficacy, increased angiogenesis, reduced acne symptoms, decreased secretion, reduced aging effects, reduced wrinkles, reduced melanin production, reduced skin inflammation, or improved skin dryness Compounds and the like can be effectively delivered to skin tissues in depth, and can be formulated into various forms, and thus can be usefully used as a functional cosmetic raw material.

1 to 4 are the results of analysis of the secondary structure of the intracellular molecular transport peptide of the present invention using a PEP FOLD server program.
FIG. 5 is a flow cytometry analysis result for measuring the skin cell permeability of the intracellular molecular transport peptide of the present invention.
6 to 8 show the result of observing the skin cell permeability of intracellular molecular transfer peptides of the present invention with a confocal microscope:
MR-form: an improved MTD in the formula A1-A2-MTD wherein A1 is methionine and A2 is arginine;
An improved MTD in the form of MH-form: A1-A2-MTD, wherein A1 is methionine and A2 is histidine; And
MK-form: An improved MTD in the formula A1-A2-MTD wherein A1 is methionine and A2 is lysine.
FIG. 9 is a photograph showing the intracellular permeability of an intracellular molecular transport peptide in a EpiOral artificial tissue model by a confocal microscope.
FIG. 10 is a photograph showing the penetrating ability of the intracellular molecular transfer peptide in the stratum corneum and skin tissue in a EpiDerm artificial skin model by a confocal microscope.
FIG. 11 is a graph showing the in vivo ( in vivo ) were confirmed by confocal microscopy
Fig. 12 shows the result of confirming the inhibitory effect of tyrosinase activity of a coumaric acid derivative having an intracellular molecule-transferring peptide.
FIG. 13 shows the results of confirming the inhibitory effect on intracellular melanin production of a coumaric acid derivative having an intracellular molecule-transferring peptide bound thereto.
FIG. 14 shows the results of confirming the inhibitory effect of intracellular molecular transport peptide and acetylpentapeptide-binding derivative on collagenase activity in human normal skin fibroblasts.
15 is a flow cytometry analysis result for measuring the skin cell permeability of an intracellular molecule transfer peptide and an acetylhexapeptide-binding derivative.
FIG. 16 shows the results of confocal microscopic observation of the skin cell permeation sites of the intracellular molecule transfer peptide and the acetylhexapeptide-binding derivative.

The delivery of the active substance through the skin has several limitations due to the structural and physical properties of the skin. In particular, the keratinocytes of the skin are keratinocyte, which is a major constituent cell of the skin, and the keratinocyte is naturally contracted to form a dense structure in the outermost layer of the skin. It inhibits the penetration of foreign substances as well as the evaporation of water, Which is in the acidic range near pH 5. In order to penetrate the horny layer, the molecular weight should be as small as 1,000 or less, and it must be possessed of lipophilic property (Metha R. C. and Fitzpatrick R. E. Dermatol. Ther. 20: 350-359 (2007)).

It is known that low molecular weight synthetic compounds or natural compounds frequently used as cosmetic raw materials can easily be delivered into cells. Massive molecules such as proteins, peptides and nucleic acids are difficult to penetrate into membranes having a dual lipid membrane structure due to their large molecular weight. It is known that the permeation efficiency of low molecular weight substances is extremely low and the permeation efficiency of high molecular weight substances is even lower due to the intrinsic properties of the stratum corneum constituting the skin barrier. Macromolecule Intracellular Transduction Technology (MITT) can be used as a way to amplify the efficiency of these small molecules and macromolecules passing through the plasma membrane of cells.

Accordingly, in the present invention, one or two hydrophilic (polar) amino acids positively charged in the hydrophobic macromolecular transfer domain (MTD) of Korean Patent Laid-Open No. 10-2009-0103957 have been applied to hydrophobic domains, The present invention relates to a composition for transdermal delivery capable of efficiently transferring a skin biologically active molecule into a skin cell by developing an improved MTD peptide that efficiently transfers a biologically active molecule into a cell by enhancing accessibility to an attached cell membrane, A delivery system and a method for delivering a skin biologically active molecule into a skin cell or into the stratum corneum of a skin.

The modified MTD peptide is a peptide represented by the following formula 1,

A1 is methionine (M, Met);

A2 is an amino acid selected from the group consisting of arginine (R, Arg), histidine (H, His) and lysine (K, Lys);

Wherein the MTD has an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-7.

[Formula 1]

A1-A2-MTD.

The peptide is a peptide capable of mediating the transport of a biologically active molecule into a cell, and exhibits superior cell permeability compared to the MTD peptide described in Korean Patent Laid-open No. 10-2009-0103957. In the present invention, Quot; my molecular transport peptide ".

The peptide may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 35, but is not limited thereto.

Among the 193 macromolecular transfer domain (MTD) peptides developed in Korean Patent Laid-Open No. 10-2009-0103957, the peptide is preferably a sequence which is quantitatively and comparatively comparable and satisfies the following conditions, But are not limited to:

1) the position of the proline is located at the center of the sequence;

2) The results of the extracellular secretion inducibility evaluation using the Signal P program are evaluated as 60% or more for each domain;

3) The aliphatic index evaluated using the Protparam program (see http://web.expasy.org/protparam/) is evaluated to a value between 100 and 300;

4) The evaluated flexibility is evaluated to be greater than or equal to 0.36 using the Protscale (Average flexibility) program (see http://web.expasy.org/protscale/);

5) assessed hydropathicity using the Protparam program is rated below 3.0;

6) The instability index evaluated using the Protparam program is evaluated to a value between 30 and 60;

7) Sequence in which the evaluation result of polarity using Protscale (polarity) program is evaluated as 0.1 or more.

The amino acid sequence of SEQ ID NO: 1 may be encoded by the polynucleotide sequence of SEQ ID NO: 8, but is not limited thereto.

The amino acid sequence of SEQ ID NO: 2 may be encoded by the polynucleotide sequence of SEQ ID NO: 9, but is not limited thereto.

The amino acid sequence of SEQ ID NO: 3 may be encoded by the polynucleotide sequence of SEQ ID NO: 10, but is not limited thereto.

The amino acid sequence of SEQ ID NO: 4 may be encoded by the polynucleotide sequence of SEQ ID NO: 11, but is not limited thereto.

The amino acid sequence of SEQ ID NO: 5 may be encoded by the polynucleotide sequence of SEQ ID NO: 12, but is not limited thereto.

The amino acid sequence of SEQ ID NO: 6 may be encoded by the polynucleotide sequence of SEQ ID NO: 13, but is not limited thereto.

The amino acid sequence of SEQ ID NO: 7 may be encoded by the polynucleotide sequence of SEQ ID NO: 14, but is not limited thereto.

The synthesis of the peptides of the present invention and the synthesis of peptides containing cargo as a skin biologically active molecule can be carried out, for example, by using a device or by using genetic engineering techniques.

Using the above intracellular molecular transfer peptide, the effect of antioxidant, increase of angiogenesis, decrease of acne symptom, decrease of pre-secretion, decrease of aging effect, decrease of wrinkles, decrease of melanin production, skin irritation or skin dryness Improvement can be achieved by delivering such beneficial compounds. In addition, by applying a composition containing them locally, a beneficial effect on the skin can be provided. These beneficial effects include reducing or preventing damage caused by sunlight, providing antioxidant activity, reducing the appearance of wrinkles, including fine wrinkles in the skin, reducing the secretion of the sun, , Treating acne, inducing angiogenesis in hair follicles to grow hair, helping to moisturize the skin, and inhibiting melanin production to lighten the skin color, but are not limited thereto.

In addition, the active ingredient that can be delivered into a skin cell by an intracellular molecular transport peptide can be a compound or a protein or a fragment thereof, and further provides additional skin benefits. They are non-toxic, non-allergic and non-irritating, so they have characteristics that are friendly to skin tissue. Examples of suitable active molecules include, but are not limited to, vitamins and derivatives thereof, retinoids, and fatty acids.

According to the present invention, ascorbic acid (vitamin C), alpha-tocopherol (vitamin E) and retinoid (vitamin A) can provide skin-beneficial properties. Ascorbic acid stimulates the synthesis of connective tissue and is particularly involved in the stimulation and regulation of collagen production. It helps to prevent or minimize other types of cellular damage due to persistent exposure to lipid oxidation and ultraviolet radiation (Varani, J. et al., J. Invest. Dermatol. 114: 480-486 (2000), Offord, EA et al., Free, Radical Biol. & Med. 32: 1293-1303 (2002)). Ascorbic acid helps inhibit melanin formation and histamine release in cell membranes, compensates for vitamin E deficiency in the skin, is involved in preventing skin discoloration, and has anti-free radical activity. Alpha-tocopherol is an antioxidant that prevents the detrimental effects of phospholipids and free radicals on cell membranes (J. B Chazan et al., Free Radicals and Vitamin E. Cah. Nutr. Diet. 1987 22 (l): 66-76). Retinoids block mediators of inflammation in the skin and increase the production of procollagen so that more Type I and Type III collagen are produced.

Specifically, the cargo compound of the present invention is a cargo compound of vitamin A, vitamin B, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D1, vitamin D2, Vitamins such as vitamin D5, vitamin E, and vitamin K. [

Another cargo compound of the present invention is a retinoid. Retinoids include retinol, natural and synthetic analogs of vitamin A (retinol), vitamin A aldehyde (retinal), all trans, 9-cis, and 13-cis retinoic acid, etretinate, A acids (retinoic acid) as well as compounds having various synthetic retinoids and retinoid activities.

The cargo compounds of the present invention also include fatty acids. Fatty acids are monocarboxylic acids with saturated or unsaturated fatty tail. (The disclosure of which is incorporated herein by reference) as defined in International Cosmetic Ingredient Dictionary and Handbook, 7th Ed. (1997) volume 2, page 1567), fatty acids And has at least about 7 carbon atoms. For example, palmitic acid is the most abundant natural fatty acid, a saturated fatty acid found in palm oil and other fats. Palmitic acid is also one of the major fatty acids of the skin produced by sebaceous glands and is used as a moisturizer in skin care and cosmetic preparations. It stabilizes the oil balance, keeping the skin in a normal, healthy condition, softens the skin and reacts like an anti-keratinizing agent. The esters of palmitic acid are used to provide silkiness to the skin and hair, such as silk. The palmitic acid acts as a carrier capable of penetrating the pentapeptide into the skin, is widely used as a lubricant, and is used as an emulsifier, a surfactant and a formula texturizer.

Suitable fatty acids of the present invention also include, but are not limited to, lauric acid, stearic acid, palmitic acid, undecylenic acid, palmitoleic acid, oleic acid, linoleic acid, linoleic acid, arachidonic acid and erucic acid. Additional suitable fatty acids are disclosed in the International Cosmetic Ingredient Dictionary and Handbook, 7th Ed. (1997) volume 2, page 1567. [

The intracellular molecular transport peptide and the cargo compound, i.e., the skin biologically active molecule, are linked by covalent bonds, including, but not limited to, ester, amide, ether and carbamide bonds. Embodiments of the present invention include, but are not limited to, intracellular molecular transfer peptides selected from coumaric acid, acetylpentapeptide, or acetylhexapeptide.

The compounds of the present invention may be used in many cosmetic or dermatological compositions intended for topical application. Since intracellular molecular transport peptides and cargo compounds can penetrate the skin and stimulate angiogenesis in hair follicles, these compositions can be used to prevent hair loss or improve hair growth. When using compounds that affect hair growth, apply to the scalp, eyebrows or eyelashes. When applied to the skin, the intracellular molecular delivery peptides and cargo compounds can also be used to reduce the appearance of fine lines and wrinkles, to improve skin elasticity, to reduce skin puffiness, In order to provide softness, to provide a silky texture to the skin, to provide a moisturizing effect, to provide lubrication, to provide a bright complexion through melanogenesis inhibition, to reduce the aging effect, Can be used to provide advantages.

As used herein, a " skin biologically active molecule " is defined as "a cosmetically or dermatologically acceptable molecule ", and refers to physiological tissues without undue toxicity, incompatibility, instability, Or < / RTI >

The composition of the present invention may further comprise a pharmacologically or physiologically acceptable carrier, excipient or diluent.

Examples of suitable carriers, excipients and diluents that may be included in such a composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may further include conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like in the case of pharmaceutical formulation.

In terms of formulation, the compositions of the present invention may be formulated as solutions, emulsions (including micro-emulsions), suspensions, creams, lotions, gels, powders, or other typical solids used for application to skin and other tissues Liquid compositions. Such compositions may contain additional antimicrobials, moisturizers and hydration agents, penetration agents, preservatives, emulsifiers, natural or synthetic oils, solvents, surfactants, detergents, gelling agents, emollients, antioxidants, fragrances, fillers, thickeners, waxes, odor absorbers, dyestuffs, colorants, powders, viscosity-controlling agents and water. Optionally, an anesthetic, an anti-itch active, a botanical extract, a conditioning agent, a darkening or lightening agent, a glitter, a wetting agent humectants, mica, minerals, polyphenols, silicones or derivatives thereof, sunblocks, vitamins, and phytomedicinal. In some embodiments, the compositions of the present invention are formulated with the above-described ingredients to make them stable for long periods of time, which may be useful when continuous or prolonged use is desired.

In some embodiments of the present invention, the composition is used to treat, ameliorate or prevent skin aging. Skin damage due to aging can be caused by fine and deep wrinkles, skin lines, crevices, bumps, large pores, scaliness, loss of skin elasticity, sagging, loss of skin firmness or tautness, Keratosis, abnormal differentiation, hyperkeratinization, elastic fibrosis, collagen collapse, stratum corneum, epidermis, epidermis, skin vascular system, and the like, as well as the hyperpigmented areas such as aging spots and freckles, But are not limited to, other histological changes in the underlying tissues that are particularly close to the skin. While skin aging has aging due to external factors such as chronic aging or persistent exposure to sunlight, the compositions of the present invention disclose methods for treating, ameliorating, or preventing such skin aging and other types of skin damage.

In a specific embodiment of the present invention, in order to quantitatively determine the cell permeability of an intracellular molecule-transporting peptide, the present inventors used a complex in which a FITC fluorescent substance was linked to an intracellular molecule-transporting peptide, The fluorescence intensity was measured using flow cytometry. As a result, it was confirmed that the intracellular molecule transfer peptide delivered FITC into cells intensely more efficiently than the known PTD (protein transduction domain) Of intracellular molecular transfer peptides.

In another specific embodiment of the present invention, the present inventors have conducted intracellular localization and cell permeation through a confocal microscope analysis using a complex of a FITC fluorescent substance linked to an intracellular molecule transfer peptide The results were visually verifiable. As a result, it was confirmed that the cell membrane permeability was higher in the intracellular molecule transfer peptide than in the existing MTD peptide, as in the flow cytometry results. In addition, it was confirmed that the intracellular molecular transport peptide has higher cell permeability than Tat, which is known to have a relatively high intracellular transport efficiency among PTD (protein transduction domain) known.

In addition, it has been confirmed that physiological activity through skin permeation is increased by using a peptide having a cargo having skin physiological activity, thus proving that it can be used as a cosmetic raw material having whitening or wrinkle reducing effect.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

Example  1. Improved MTD  For the design of peptides, the target MTD  Screening of peptides

Among the 193 MTD peptides developed by the present inventors, Korean Patent Laid-Open No. 110-2009-0103957, which is a technology developed by the present inventors, the present inventors have found that among the MTD peptides quantitatively and comparatively comparable, The target MTD peptide sequences for improvement of function were selected.

The conditions for the selection are as follows.

1) Sequence of the proximal position of the macromolecular transfer domain in the center of the sequence is selected.

2) Sequence of the macromolecule delivery domain with high induction of extracellular secretion is selected.

3) Among the sequences of the macromolecular transfer domain, sequences that satisfy a specific level of the aliphatic index, which determines the physicochemical properties of the macromolecular transfer domain, are selected.

4) Among the sequence of the macromolecular transfer domain, the sequence that satisfies a certain level of flexibility, which is an element that determines the physicochemical properties of the macromolecular transfer domain, is selected.

5) Among the sequences of the macromolecular delivery domain, hydropathicity, which determines the physicochemical properties of the macromolecular delivery domain, selects sequences that satisfy a certain level.

6) Among sequences of the macromolecular transfer domain, sequences whose instability index, which determines the physicochemical properties of the macromolecular transfer domain, satisfy a certain level are selected.

7) Among the sequences of the macromolecular transfer domain, sequences whose polarity, which is an element that determines the physicochemical properties of the macromolecular transfer domain, satisfy a certain level are selected.

This will be described in detail as follows.

In order to select a specific representative sequence, a representative sequence was determined based on the presence or absence of proline and its position, as described in the above step 1). Among the amino acids alanine, valine, proline, leucine and isoleucine, which constitute the sequence of the macromolecular transfer domain, proline having a short side chain and small size affects the degree of freedom of forming the secondary structure of the amino acid sequence, Lt; / RTI > cell membrane permeability. Korean Provisional Patent Publication No. 10-2009-0103957 discloses that, among the macromolecular transfer domains (MTDs) known in the novel macromolecular transfer domain and its identification method and use, proline is present on the sequence constituting it and the proline The position was located at the center of the amino acid sequence of the macromolecular transfer domain to determine the 49 macromolecular transfer domains classified as having a high degree of freedom in the formation of the secondary structure of the amino acid sequence.

Subsequently, sequences with high extracellular secretion among the macromolecular transfer domains selected in step 1) were selected. Some water-soluble proteins have a signal that can interact with receptors that mediate transport. In signal-mediated transport, proteins have one or more signal sequences that specify the target to which they are delivered. Therefore, the possibility of inducing extracellular secretion was evaluated using the Signal P program on the assumption that the similarity with the extracellular secretory induction sequence was high to improve the transcriptional activity. The probability between 10% and 90% for each domain is evaluated, and the sequences evaluated to more than 60% of the macromolecular transfer domains determined in step 1) were selected as the improved target sequences.

Sequences satisfying a certain level of the aliphatic index, which is an element that determines the physicochemical properties of the macromolecular transfer domain, were selected from the macromolecular transfer domains selected in step 2), as described in step 3). Aliphatic index is a physical feature that determines the volume of whole molecule determined by the side chain carbon chain of amino acid. It is evaluated as a feature that modifies the cell membrane structure when the macromolecular transfer domain permeates the cell membrane. The Aliphatic index is determined by the unique value of each amino acid sequence of the macromolecular delivery domain and the average of the entire sequence, determined using the Protparam program (see http://web.expasy.org/protparam/). The Protparam program was used as a useful tool for quantifying the physical properties of proteins or peptides composed of amino acids. The aliphatic index was selected as a representative sequence for the improvement of the transfection ability.

Next, among the selected macromolecular transfer domains in step 3), sequences were selected that satisfy the certain level of flexibility, an element that determines the physicochemical properties of the macromolecular transfer domain, as described in step 4). Flexibility is structurally related to affinity for cell membranes and imparts structural flexibility to the physicochemical properties of degree and degree of freedom between the N-terminal amino acid and the C-terminal amino acid of the macromolecular delivery domain. Flexibility is assessed by the length of the amino acid sequence and the configuration of the side-by-side sequence of the amino acids and evaluated using the Protscale (Average flexibility) program (see http://web.expasy.org/protscale/). The Protscale program was used as a tool to quantify the physical properties of proteins or peptides composed of amino acids. Among the macromolecular delivery domains determined in step 3), the flexibility evaluation using Protscale (Average flexibility) program was evaluated to be over 0.36 Were selected as representative sequences for improving the transfection ability.

Next, among the macromolecular delivery domains selected in step 4), the sequences that satisfy the specific level of hydropathicity, which is an element that determines the physicochemical properties of the macromolecular transfer domain, are selected, as described in step 5). Hydropathicity is a physical property determined by the unique characteristics of amino acids and is considered to be a property of determining physical properties. It is known to cause severe entanglement at a pH of 3.0 or higher. Hydropathicity is also determined by the average of the entire sequence and the unique value of each of the amino acid sequences of the macromolecular delivery domain and evaluated using the Protparam program. As a result, hydropathicity was evaluated as a representative sequence for improving hydropathicity in the macromolecular transfer domain selected in step 4) from 1.3 to 3.8.

Next, among the selected macromolecular transfer domains in step 5), the sequences whose instability index of the macromolecular delivery domain satisfies a certain level, as described in step 6), were determined. Instability index is a characteristic of stability of amino acid sequence. It is determined by sequence order of amino acids in sequence. This is a factor that determines the physicochemical properties of the macromolecular transfer domain and is considered to be a characteristic that affects the intracellular stability of the domain and was evaluated using the Protparam program. As a result, the sequences with instability index of 30 to 60 within the macromolecular transfer domain determined in step 5 of the macromolecular transfer domain showing instability index values between 0 and 130 were selected as representative sequences for improvement.

Polarity, an element that determines the physicochemical properties of the macromolecular delivery domain, as described in step 7) among the macromolecular delivery domains selected in step 6) then determined the sequence that satisfies a certain level. Polarity is a measure of affinity to water, determined by the length of the carbon chain and the presence or absence of hydroxyl groups in the amino acid constituents. It is a feature that determines the physical properties of macromolecular transfer domain with hydropathicity and also affects affinity to cell membrane. Polarity is determined by the unique values of each of the amino acid sequences of the macromolecular delivery domain and the average of the entire sequence and evaluated using the Protscale (polarity) program. Among the macromolecular transfer domains determined in step 6), a sequence having an evaluation result of polarity of 0.1 or more using the Protscale (polarity) program was selected as a representative sequence for improvement.

Specifically, the target MTD JO-103 (SEQ ID NO: 3) for designing an improved MTD peptide among the macromolecular transport domains described in Korean Patent Laid-Open No. 10-2009-0103957 was determined by the following procedure.

i) Among the macromolecular delivery domains (MTDs) known in Korean Patent Laid-Open No. 10-2009-0103957: Novel macromolecular delivery domains and their identification and use, JO-103 consists of the sequence of LALPVLLLA;

ii) 90% probability of extracellular secretion using Singal P program;

iii) the value of 271 in the aliphatic index evaluation using the Protparam program;

iv) evaluated as 0.38 in the flexibility assessment using the Protscale program;

v) determined to be 2.8 in the hydrophobicity determination using the Protparam program;

vi) determined as 52 in the instability index determination using the Protparam program;

vii) Since it is evaluated as 0.13 in the polarity evaluation using the Protscale program,

Therefore, it can be seen that the JO-103 determined as the target sequence for the improvement of the transmission capability is a target domain satisfying all of the above 7 steps.

In the same manner, five target MTD peptides were selected for the design of modified MTD peptides, and specifically, the selected target MTD peptides were MTD JO-18 (SEQ ID NO: 1), MTD JO-067 (SEQ ID NO: 2) MTD JO-103 (SEQ ID NO: 3), MTD JO-159 (SEQ ID NO: 4) and MTD JO-173 (SEQ ID NO: 5).

Changing the position of proline to the double MTD JO-173 peptide to ensure additional flexibility; The MTD 173A peptide (SEQ ID NO: 7) as an intermediate peptide was prepared. The MTD 173A peptide thus prepared was also included in the target MTD sequence for devising the improved MTD peptide since it satisfies all of the 7 selection criteria.

In addition, MTD 18m (SEQ ID NO: 6) was derived from intermediate sequence peptide MTD 18 (SEQ ID NO: 6) in order to improve the physical properties by substituting amino acids having low hydropathicity for the sequence of JO-18 among the selected MTDs. And were included in the target MTD sequence for the design of an improved MTD peptide.

Example  2. Advanced type MTD  Design of peptides

Based on the seven target MTDs selected in Example 1, one or two hydrophilic (polar) amino acids were added to the hydrophobic domain to increase accessibility to the cell membrane, thereby efficiently transferring the biologically active molecule into the cell To prepare a new improved MTD peptide, thereby designing a peptide represented by the following formula 1;

[Formula 1]

A1-A2-MTD:

A1 is methionine (M, Met);

A2 is an amino acid selected from the group consisting of positively charged arginine (R, Arg), histidine (H, His) and lysine (K, Lys);

MTD is the seven amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 7 selected in Example 1 above.

The sequence of the modified MTD peptide designed by the above formula 1 is the same as the amino acid sequence of SEQ ID NOS: 15 to 35, and a peptide was synthesized based on the designed amino acid sequence to confirm its cell permeability.

Example  3. Advanced type MTD  Peptidic Secondary structure  analysis

The sequence of the modified MTD peptide designed in Example 2 was analyzed using a PEP FOLD server service program.

As a result, as shown in FIG. 1 to FIG. 4, the sequence in which the N-terminal sequence of the EGFP fluorescent protein was combined with the modified MTD peptide designed in Example 2 was disclosed in Korean Patent Publication No. 10-2009-0103957 Helix structure capable of enhancing the permeability of the cell membrane without compromising the structural characteristics of the macromolecular transfer domain (MTD) peptide known in the art.

Example  4. Intracellular  Molecular transport peptides and FITC  Synthesis of Fused Phosphor

The modified MTD peptide designed in Example 2 was applied to intracellular molecular transfer peptides fused to the dermal biologically active molecules of the present invention. For synthesis of these peptides, a C-term One by one.

Specifically, first, the C-terminal first amino acid of the peptide was attached to a resin. The available resins can be used as needed in NH ₂ -Lys (Dde) -2-chloro-Trityl resin, NH ₂ -Met-2-chloro-Trityl resin or NH ₂ -Ser (tBu) -2-chloro- Appropriate resins were selected and used for peptide synthesis.

Second, all amino acid sources used for peptide synthesis were protected with Trt, Boc, t-Bu, etc., in which the N-terminal was protected with Fmoc and the residues were all removed from the acid (Fmoc-Ala-OH, Fmoc- -OH, Fmoc-Lys (Boc) -OH, Fmoc-Pro-OH, Val-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Thr Fmoc-Met-OH). For specific amino acids, Fmoc-Lys (Ac) -OH and Fmoc-Lys (Dde) -OH were used.

Third, Fmoc was removed by reaction with 20% piperidine in DMF at room temperature for 5 minutes twice.

Fourth, DMF, MeOH, MC and DMF were washed in order.

Fifth, TFA / EDTA / Thioanisole / TIS / H2O = 90 / 2.5 / 2.5 / 2.5 / 2.5 is used to separate the synthesized peptide from Resin and remove the protecting group of the residue (TFA = trifluoroacetic acid, EDT = 1,2-ethanedithiol , TIS = triisopropylsilane). Finally, after purification by HPLC, the molecular weight was confirmed by mass spectrometer and lyophilized to obtain a synthesis product of intracellular molecule transfer peptide.

In addition, in order to synthesize intracellular molecule transfer peptide with fluorescent indicator (FITC), intracellular molecule transfer peptide is synthesized by the above-mentioned synthesis method, and finally lysine (K, Lysine) is added to perform peptide synthesis After that, FITC was bound to the free amine residue of lysine. The synthesized intracellular molecular transport peptide-FITC peptide was separated from the resin and purified by HPLC. The molecular weight of the peptide was confirmed by a mass spectrometer and lyophilized to obtain an intracellular molecule-transferring peptide fluorescent substance. Then, the synthesized intracellular molecule-transducing peptide fluorescent substance was dissolved in DMSO to a concentration of 1 mM in the shade state, and the mixture was dispensed into a 1.5 mL centrifuge container in a small amount and kept frozen until immediately before use.

Example  5. In skin keratinocytes Flow cell  Measure ( Flow Cytometry ) In the intracellular molecular transport peptide fluorescent substance ( MTD - FITC )of in vitro Cell permeability  Confirm

(MTT-FITC) at a concentration of 3 μM was injected into a human keratinocyte cell line (HaCaT cell, Order No. 300493, CLS cell line service, Germany) to examine cell permeability. And cultured for 1 hour. The HaCaT cells were maintained in DMED (Dubelco's modified eagle medium) medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (10,000 units penicillin and 10,000 μg / mL streptomycin, invitrogen) wet (humidified) of 5% CO ₂ and incubated with 37 ℃ under air. After incubation was terminated, trypsin was treated to remove free MTD-FITC exposed to protein-treated HaCaT cell membranes and washed three times with cold PBS.

MTD-FITC peptides were applied to a flow cytometer (FACS Calibur, Beckton-Dickinson, San Diego CA, USA). For each sample, cells (1 × 10 ⁴ ) were analyzed using CellQues Pro cytometric analysis software and each experiment was performed more than 3 times. The cell permeability of each of the intracellular molecular transfer peptides used in the present invention was quantitatively compared and analyzed by using existing MTD-FITC as a control group.

FIG. 5 shows the results of flow cytometry analysis. The cell inflow efficiency of each intracellular molecular transfer peptide treated in the cells was measured by CellQues Pro cytometric analysis software, Geometric mean ) Fluorescence changes were compared relatively, and cell infusion efficiency was compared.

As a result, it was confirmed that the intracellular molecular transport peptide (MTD-FITC) exhibits excellent cell entry efficiency of existing MTD peptides and exhibits a cell entry efficiency as high as at least 140% up to 400% higher than known PTD.

Example  6. Confocal microscope  Used Intracellular  Molecular transport peptide phosphors ( MTD -FITC) skin cells Permeability  Confirm

The cells were treated with 3 μM of the intracellular molecular transfer peptide and PTD (Tat) protein, which is known to be permeable to cells, to skin-derived keratinocytes (HaCaT cell, immortalized human keratinocyte, Cat No. 300493, CLS, Germany) For 1 hour, and then observed visually using a confocal microscope (Nikon, Germany). On the day before the experiment, HaCaT cells were cultured in a 12-well culture plate containing a glass cover slip for 24 hours. HaCaT cells were maintained in DMEM medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin / streptomycin (10,000 units penicillin and 10,000 μg / mL streptomycin, Invitrogen, USA) ₂ < / RTI > under a humidified atmosphere. HaCaT cells were treated with scrambled peptide, MTD-FITC and PTD-FITC peptide at a concentration of 3 [mu] M for 1 hour. One hour after the treatment, the cells were fixed with 4% paraformaldehyde (PFA) for 20 minutes at room temperature for observation.

For direct detection of internalzed FITC-peptides, the cells were washed three times with PBS and counterstained with 5 mM concentration of DAPI (4 ', 6-diamidino-2-phenylindole) Respectively. After 10 minutes of DAPI staining, the cells were washed 3 times with PBS and 20 [mu] l of mounting media was spotted onto the slides and observed to preserve the fluorescent label of the protein. The cells treated with the intracellular molecular transfer peptide were confirmed to be transferred to the nucleus and checked for cell permeability through DAPI staining so that the intracellular delivery site of the FITC-peptide could be easily distinguished. The confocal microscope was a normaski filter, , And the FITC fluorescence and DAPI fluorescence were observed with a filter suitable for each fluorochrome. As shown in FIGS. 6 to 8, it was confirmed that all of the intracellular molecular transfer peptides applied to the present invention were clearly transferred into the cells as compared to PTD (Tat) attached to the outside of the cell membrane, Demonstrating that peptides have excellent cellular permeability to skin cells.

Example  7. Intracellular  Artificial skin tissue of molecular transfer peptides Permeability  Confirm

Ex screening vivo tissue distribution EpiOral skin model for the analysis capabilities (MatTek, MA, USA) with FITC was a conventional MTD (M18m, M173) and the intramolecular transfer peptide (M1018m, M1173A) cells joined to use the process, Confocal microscopy (Carl Zeisse, Germany) was used for visual observation. On the day before the experiment, an EpiOral artificial tissue model was cultured at 37 ° C in a humidified atmosphere of 5% CO ₂ for 15 hours in a 12-well plate containing 0.5 mL of the experimental medium provided by MatTek. The next day, 40 μl of the peptide at a concentration of 50 μM was treated above the EpiOral artificial tissue and incubated at 37 ° C. for 5 hours under a humidified atmosphere of 5% CO ₂ . After immersion for 15 hours in 4% paraformaldehyde (PFA), EpiOral artificial tissue models were fixed and frozen sections (6 μm) were prepared using Microm HM520 cryostat (Thermo) , And this was put on a glass slide. The prepared slides were washed with PBS buffer solution for 10 minutes and stained with 0.5 mM DAPI solution for 5 minutes. The stained tissue was again washed with PBS buffer three times for 10 minutes, fixed on a slide using mounting media, and observed with a confocal microscope. The results are shown in FIG.

As shown in Figure 9, no significant level of fluorescence was observed in the negative control vehicle. However, in the case of the intracellular molecule transfer peptide used in the experiment, permeation was observed to the inside of the EpiOral artificial tissue model with the lapse of time. In addition, it was confirmed that the intracellular molecule transfer peptide (1018m, and 1173A) was more efficiently transferred to the deep tissue than the conventional MTD peptides (18m and 173) used in the experiment, and the brightness of fluorescence was also increased. This demonstrates that the improved intracellular molecular transport peptide has excellent tissue permeability for skin tissue.

Example  8. Intracellular  Artificial skin tissue of molecular transfer peptides Permeability  Confirm

Ex To analyze the keratinocyte permeability in vivo tissues, the intracellular molecular transport peptide (M1067) conjugated with FITC and Tat peptide were treated with EpiDerm skin model (MatTek, MA, USA) microscopy, Carl Zeiss, Germany). On the day before the experiment, the EpiDerm skin model was incubated at 37 ° C in a humidified atmosphere of 5% CO ₂ for 15 hours in a 12-well plate containing 0.5 ml of the experimental medium provided by MatTek. The following day, 40 μl of the peptide at a concentration of 100 μM was treated at the top of the EpiDerm skin and incubated at 37 ° C. in a humidified atmosphere of 5% CO ₂ for 24 hours. After immersing EpiDerm skin model in 4% paraformaldehyde (PFA) for more than 15 hours, frozen sections (6 μm) were prepared using Microm HM520 cryostat (Thermo) This was put on a glass slide. The prepared slides were washed with PBS buffer solution for 10 minutes and stained with 0.5 mM DAPI solution for 5 minutes. The stained tissue was again rinsed with PBS buffer three times for 10 minutes, fixed with mounting media, and observed with a confocal microscope. The results are shown in FIG.

As shown in Fig. 10, no significant level of fluorescence was observed in the negative control vehicle. In the case of the Tat peptide, Tat-FITC treated on the stratum corneum of the uppermost layer did not permeate into the tissue, In the case of the intracellular molecule transfer peptide (M1067), permeation through the stratum corneum of the uppermost layer and permeation into the Epiderm skin model was observed over time. This demonstrates that intracellular molecular transport peptide (M1067) passes through the stratum corneum known as the maximum barrier for skin absorption and possesses excellent skin tissue permeability.

Example  9. Intracellular Molecular Transfer Peptides Using Animal Models in vivo  Skin tissue Permeability  Confirm

Intracellular Molecular Transfer Peptide in In order to verify the vivo skin permeability, an animal model was used. (M1067), a Tat-FITC and a FITC (FITC) conjugated with a fluorescent indicator (FITC) on sterilized gauze of 2.5 cm x 2.5 cm in size per 8-week-old female ICR mouse (Orient Bio, 100 μg each was spot-loaded and fixed to the mouse's back using Tegarderm (3M, USA). After 1, 3, 6, and 12 hours, the mice were sacrificed by cervical dislocation, skin was removed from the area to which the drug was applied, and the tissues were fixed with 4% paraformaldehyde for 24 hours or more. Slices were obtained and slides were prepared. The prepared slides were washed with PBS buffer solution for 10 minutes and exposed to 0.5 mM DAPI solution for 5 minutes to stain nuclei in the tissues. The stained tissue was again washed with PBS buffer three times for 10 minutes, fixed with mounting media, and observed with a confocal microscope, which is shown in FIG.

As shown in FIG. 11, fluorescence signals were not detected in the vehicle of the negative control, fluorescence signals were specifically observed in the stratum corneum, which is the highest layer of the skin, in FITC alone and Tat-FITC treated groups, . However, in the case of the intracellular molecular transport peptide (M1067) used in the experiment, penetration into the deep tissues of the skin occurs as the application time passes, and the permeable intracellular molecule transfer peptide (M1067) passes through the stratum corneum, It was also confirmed that the cells were specifically present in the cytoplasm of hair follicle cells.

Example  10. Skin biologically active substances Combined Intracellular  Identifying whitening efficacy of molecular transfer peptides

10-1. Intracellular  Molecular transport peptides - Kumaric  synthesis

N-methylpyrrolidone) solution was added to the intracellular molecule transfer peptides M1067 and M2067 synthesized in Example 4, which was coupled to the N-terminal amino acid, to remove the Fmoc group , Washed with N-methylpyrrolidone and dichloromethane, and coumarin acid (coumaric acid, Sigma, USA), which is commercially available, was coupled. After the coupling was completed, the solution was washed several times with N-methylpyrrolidone and dichloromethane, followed by drying with nitrogen gas. 2.5 to 2.5: 2.5 (v / v) solution of trifluoroacetic acid: phenol: thioanisole: water: triisopropylsilane (phenol: thioanisole: After 3 hours of reaction, the peptide protecting group was removed. Coumaric acid bound to the peptide was separated from the resin, and the peptide was precipitated with diethyl ether. 10% Pd / C was added to methanol, and the mixture was stirred at room temperature for about 1 hour under hydrogen to remove the benzyl group protecting the alcohol group bound to C-9 carbon of the coumaric acid. Then, Pd / C was removed and the resulting filtrate was concentrated under reduced pressure. The intracellular molecular transport peptide-coumaric acid derivative thus obtained was purified using a purified reverse phase high performance liquid chromatography column (Zobax, C8 300 Å, 21.1 袖 m) with a gradient of acetonitrile containing 0.1% trifluoroacetic acid mm X 25 cm) to obtain intracellular molecular transfer peptide-coumaric acid derivatives in which coumaric acid is bound to the M1067 or M2067 intracellular molecular transfer peptide having the amino acid sequence of SEQ ID NO: 16 or 23 as shown in the following Formula 2, Were synthesized.

[Formula 2]

(4-Hydroxycinnamoyl) Ala Ala Val Ala Pro Ala Ala Ala Arg Met

(4-Hydroxycinnamoyl) Ala Ala Val Ala Pro Ala Ala Ala His Met

10-2. Intracellular  Molecular transport peptides - Kumaric Tyrosinase  Confirm inhibitory activity

The inhibitory effect of tyrosinase activity was measured using intracellular molecule-transferring peptide-coumaric acid, which was the compound synthesized in Example 10-1. Tyrosinase was isolated and purified from mushroom and purchased from Sigma (USA). The substrate, tyrosine, was dissolved in 0.05 M potassium phosphate buffer (pH 6.8) and used as a 0.3 mg / mL solution. The compound was dissolved in distilled water at a concentration of 100 mg / mL, and the coumaric acid was dissolved in ethyl alcohol at a concentration of 100 mg / mL, and then diluted to an appropriate concentration. Add 0.5 mL of the sample solution and 0.5 mL of the buffer solution, add 0.5 mL of the compound solution, and incubate at 37 ° C for 10 minutes. Add 0.05 mL of 2,000 U / mL tyrosinase and incubate at 37 ° C for 10 minutes . The control group contained 0.5 mL of distilled water instead of each compound. The test tube containing the reaction solution was removed from the thermostat and the absorbance at 450 nm was measured with a spectrophotometer to determine the inhibition rate of tyrosinase activity. The inhibition rate was calculated according to the following equation (1), and the results are shown in FIG.

In the above equation (1)

A is the absorbance at 450 nm of the absence of the inhibitor,

B is the absorbance at 450 nm of the inhibitor added.

As a result, as shown in Fig. 12, it was confirmed that the enzyme-inhibiting activity of the compound bound to the intracellular molecule-transferring peptide of the present invention was significantly higher than that of coumaric acid, which is known to have a high tyrosinase inhibitory activity.

10-3. Intracellular  Molecular transport peptides - Kumaric Intracellular  Confirming melanin formation inhibitory activity

The intracellular molecular transport peptide-coumaric acid, a compound synthesized in Example 10-1, was used to compare the amounts of melanin production in cells during cell culture. Mouse-derived melanoma cells (B16F1, Korean Cell Line Bank KCLB No. 80007) were inoculated into a 6-well plate in DMEM (Dubelco's modified eagle medium) medium containing 10% FBS (fetal bovine serum) ⁵ , and incubate at 37 ° C in 5% CO ₂ at 37 ° C until the cells are attached to the bottom of the well by at least 80%. After the culture, the medium is removed and the sample is replaced with the medium diluted to the appropriate concentration (1, 10, 100 ㎍ / mL) and incubated for 3 days with medium exchanged every day under 5% CO ₂ at 37 ° C. 3 days incubation The next day, the cells from which the medium has been removed are washed with PBS (phosphatized buffer saline), and treated with trypsin to recover the cells. The recovered cells were centrifuged at 10,000 rpm for 10 minutes and then the supernatant was removed to obtain cell pellets. The cell pellet was dried at 60 ° C., and 200 μL of a 1 M sodium hydroxide solution containing 10% DMSO was added thereto. Intracellular melanin is obtained. Measure the absorbance at 490 nm with a microplate reader and determine the amount of melanin per protein by specifying the amount of protein. Perform a blank test and calibrate.

As a result, as shown in Fig. 13, in the case of a compound bound to intracellular molecular transfer peptide of the present invention compared to arbutin and coumaric acid, which are known to have intracellular melanin formation inhibitory effect, It was confirmed that the inhibitory effect of melanin formation was significantly increased.

10-4. Intracellular  Molecular transport peptides - Kumaric  Skin safety evaluation

The compound synthesized in Example 10-1 was subjected to a primary stimulation test using the human skin of a molecular transfer peptide-coumaric acid and an intracellular molecule transfer peptide (M1067) to confirm safety as a cosmetic raw material. Lt; / RTI > Clinical cosmetic compositions were prepared as described below.

10-4-1. Human skin primary stimulation test

We selected more than 30 subjects who met the criteria for clinical trials and did not meet the exclusion criteria. The sample was applied to the back of the subject and removed after 48 hours. The test site was observed 30 minutes and 24 hours after the removal. Skin evaluation was performed using the Frosch & Kligman method (Frosch PJ and Kligman AM J Am Acad Dermatol, 1 (1): 35-41 (1979)) and the Cosmetic, Toiletry, and Frangrance Association (CTFA) Association, Inc. Washington, DC 20005 (1981)), and the average reactivity for 48 hours and 72 hours was compared, and the results were evaluated based on the average reactivity for each composition.

As shown in Table 2, the intracellular molecular transport peptide and the intracellular molecular transport peptide-cumaric acid were identified as hypodermic categories in terms of human skin primary stimulation. This demonstrates that intracellular molecular transport peptides can be safely used for human body while maintaining the function of skin physiologically active molecules through clinical trials of professional testing laboratories, and this value is remarkably excellent as a cosmetic composition Giving.

10-4-2. Intracellular  Molecular transport peptides - Kumaric acid  contain Cosmetics  Preparation of composition

An intracellular molecule-transferring peptide-coumaric acid or an essence composition containing intracellular molecule-transferring peptide was formulated as follows in the composition of Table 3 below.

Add 1 to 6 to an aqueous dissolution tank, dissolve completely while heating to 70 ° C, and add to the emulsion tank. Add 7 to 11 to the oily melting tank, dissolve completely while heating to 70 ℃, and mix in an oil bath. After the contents were cooled to 40 캜, the raw materials 12, 13 and 15 were put into an emulsification tank and mixed. The contents were cooled to room temperature to prepare a skin whitening composition containing intracellular molecule transfer peptide-coumaric acid.

Comparative Example 1: 1 to 6 in an aqueous dissolution tank, completely dissolved while being heated to 70 DEG C, and then introduced into an emulsion tank. Add 7 to 11 to the oily melting tank, dissolve completely while heating to 70 ℃, and mix in an oil bath. After cooling the contents to 40 占 폚, the raw materials 12 to 14 were put into an emulsifying tank and mixed, and the contents were cooled to room temperature to prepare a composition containing intracellular molecule-transferring peptides.

Comparative Example 2: 1 to 6 in an aqueous dissolution tank, completely dissolved while being heated to 70 占 폚, and then introduced into an emulsification tank. Add 7 to 11 and 16 to the oily melting tank, dissolve completely while heating to 70 ℃, and mix in an emulsification tank. After the contents were cooled to 40 캜, the raw materials 12 and 13 were put into an emulsification tank and mixed. After cooling the contents to room temperature, a composition containing the coumaric acid was prepared.

Example  11. Skin biologically active substances Combined Intracellular  Confirming the wrinkle-reducing effect of the molecule transfer peptide

11-1. Intracellular  Molecular transport peptides - Acetylpentapeptide  synthesis

(Acetylated Lys Therapeutic Lys Ser) commercially available in the cosmetics industry was sequentially synthesized to M1067 peptide having the amino acid sequence of SEQ ID NO: 16 synthesized in Example 4, which was coupled to the N-terminal amino acid , 20% piperidine / N-methylpyrrolidone solution was added to the reaction mixture to remove the Fmoc group, and the resultant was washed several times with N-methylpyrrolidone and dichloromethane, followed by drying with nitrogen gas . 2.5: 2.5: 2.5 (v / v) of trifluoroacetic acid: phenol: thioanisole: water: triisopropylsilane, To react for 2 to 3 hours to remove the peptide protecting group, to separate the peptide from the resin, and to precipitate the peptide with diethyl ether. The MTD peptide-acetylpentapeptide derivative thus obtained was purified on a purified reverse phase high performance liquid chromatography column (Zobax, C8 300 Å, 21.1 mm X) using acetonitrile containing 0.1% trifluoroacetic acid as a gradient. 25 cm). As a result, an acetylpentapeptide derivative having acetylpentapeptide bound to M1067 intracellular molecular transfer peptide having the amino acid sequence of SEQ ID NO: 16 was synthesized.

[Formula 3]

Met Arg Ala Ala Pro Ala Val Ala Ala Lys * Ther Ther Lys Ser

*: acetylated lysine

11-2. Intracellular  Molecular transport peptides - Acetylpentapeptide  Intracellular Collagenase  Identification of the activity inhibition effect

The intracellular molecular transport peptide-acetylpentapeptide, which is the compound synthesized in Example 11-1, was used to test the effect of inhibiting collagenase activity in human normal skin fibroblasts. The inhibition of intracellular collagenase activity was measured by applying the method of Bauer EA et al, J Invest Dermatol, 82 (2): 162-9, 1983, and the addition of no sample was taken as 100% . The detailed experimental method is as follows. Human normal skin fibroblasts were divided into 6-well plates for cell culture and inoculated with a certain number of cells, treated with intracellular molecular transfer peptide-acetylpentapeptide at a constant concentration, cultured for 24 hours, And the amount of collagenase in the culture was measured according to the method described in Matrix metalloproteinase-1 (MMP-1) human biotrak ELISA system (GE Healthcare, USA). In the ELISA method, 100 μl of the buffer for quantitation 2 is added to a 96-well plate, and then 100 μl of each of the culture solution and the standard solution diluted 1/10 is added, followed by incubation at room temperature for 2 hours. After removing the culture solution from the well plate, wash with 400 μl of washing buffer three times, add 100 μl of antibody solution, react at room temperature for 2 hours, and wash with 400 μl of washing buffer three times. Then, 100 μl of peroxidase conjugate solution is added, reacted at room temperature for 2 hours, washed with 400 μl of phosphate buffer solution (PBS) three times, 100 μl of TMB substrate is added and reacted at room temperature for 30 minutes. Add 100 μl of 1 M sulfuric acid solution and measure with an ELISA reader at 450 nm. Dilute the standard solution with Huamn pro MMP-1 for quantitation buffer and dilute to 0, 3.13, 6.25, 12.5, 25, 50 ng / . The inhibition rate was calculated according to Equation (2), and the result is shown in Fig.

In Equation (2)

A is the absorbance at 450 nm of the sample not added,

B is the absorbance at 450 nm of the sample added.

As a result, as shown in Fig. 14, it was confirmed that inhibition of intracellular collagenase activity was significant in a concentration-dependent manner in the group treated with the intracellular molecular transport peptide-acetylpentapeptide.

11-3. Intracellular  Molecular transport peptides - Acetylpentapeptide  Confirm skin safety

In order to confirm the safety of the intracellular molecule transfer peptide-acetylpentapeptide, which is a compound synthesized in Example 11-1, as a raw material for cosmetics through a primary stimulation test using human skin, Respectively. Clinical cosmetic compositions were prepared as described below.

11-3-1. Human skin primary stimulation test

We selected more than 30 male or female subjects aged between 18 and 60 who met the criteria for clinical trials and did not meet the exclusion criteria. The sample was applied to the back of the subject and removed after 48 hours. The test site was observed 30 minutes and 24 hours after the removal. Skin evaluation was performed using the Frosch & Kligman method (Frosch PJ and Kligman AM J Am Acad Dermatol, 1 (1): 35-41 (1979)) and the Cosmetic, Toiletry, and Frangrance Association (CTFA) Association, Inc. Washington, DC 20005 (1981)), and the average reactivity for 48 hours and 72 hours was compared, and the results were evaluated based on the average reactivity for each composition.

As shown in Table 5, the intracellular molecule transfer peptide and the intracellular molecule transfer peptide-acetylpentapeptide were hypodermic in terms of primary skin stimulation. This demonstrates that intra-cellular molecular transport peptides can be safely used in the human body while maintaining the function of skin biologically active molecules through clinical trials of professional testing laboratories. This demonstrates that intracellular molecular transport peptides can be used as cosmetic compositions And the value of use is remarkably excellent.

11-3-2. Intracellular  Molecular transport peptides - Acetylpentapeptide  contain Cosmetics  Preparation of composition

An intracellular molecule transfer peptide-acetylpentapeptide or an essence composition containing intracellular molecule transfer peptide (M1067) was formulated in the composition of Table 6 as follows.

Add 1 to 5 to an aqueous dissolution tank, dissolve completely while heating to 70 ° C, and add to the emulsion tank. Add 6 to 10 to the oily melting tank and completely dissolve while heating to 70 ° C. Add to the emulsion tank and mix. After cooling the contents to 40 캜, the raw materials 11, 12 and 14 were put into an emulsification tank and mixed, and the contents were cooled to room temperature to prepare a composition for improving skin wrinkles containing the intracellular molecule transfer peptide-acetylpentapeptide.

Comparative Example 1: 1 to 5 in an aqueous dissolution tank, completely dissolved while being heated to 70 캜, and then introduced into an emulsification tank. Add 6 to 10 to the oily melting tank and completely dissolve while heating to 70 ° C. Add to the emulsion tank and mix. After cooling the contents to 40 占 폚, the raw materials 11 to 13 were put into an emulsification tank and mixed, and the contents were cooled to room temperature to prepare compositions containing intracellular molecular transfer peptides.

Comparative Example 2: 1 to 5 was added to an aqueous dissolution tank, the solution was completely dissolved while being heated to 70 DEG C, and then the solution was added to the emulsion tank. Add 6 to 10 to the oily melting tank and completely dissolve while heating to 70 ° C. Add to the emulsion tank and mix. After the contents were cooled to 40 占 폚, the raw materials 11, 12 and 15 were put into an emulsion tank and mixed. Then, the contents were cooled to room temperature to prepare a composition containing acetylpentapeptide.

Example  12. Intracellular  Molecular transport peptides and skin biologically active molecules Combined Cosmetics  Of the composition Percutaneous  Absorption confirmation

Percutaneous absorption experiments were performed according to the published guidelines to identify penetration of the material obtained in the present invention into the skin tissue (commonly referred to as percutaneous absorption) (Test Guideline 428: Skin abscession: in vitro Method, OECD, Paris, (2004), In Vitro Skin Absorption Test Guideline, Korea Food & Drug Administration (2010)). The transdermal absorption experiments were performed by thawing frozen cadaver skin (Cat No. SK11122, Hans Biomed) in PBS warmed to 32 ° C. The thawed cadaver skin was cut up (donor direction) between the donor and receptor of a vertical diffusion cell (Franz diffusion cell, Logan FDC-6, Logan instrument Corp. Somerset, NJ, USA) And the dermis was mounted down (in the direction of the receptor). The receptor was then filled with PBS solution (phosphate-buffered saline, pH 7.4, 32 ° C) and allowed to sit for 1 hour to allow cadaver skin to equilibrate with PBS solution. Then, 1 mg of intracellular molecule transfer peptide, intracellular molecule transfer peptide-coumaric acid, and intracellular molecule transfer peptide-acetylpenta peptide prepared in Examples 4, 10-1 and 11-1 were dissolved in 1% DMSO (Application area: 1.7 cm < 3 >) and the donor was sealed with a parafilm to prevent the sample from evaporating. After 24 hours, 0.2 mL of the sample was taken from the receptor, and the amount of the intracellular molecule-transferring peptide-coumaric acid and intracellular molecule-transferring peptide-acetylpentapeptide permeated through the skin was measured by a mass spectrometer ), And the results are shown in Table 7 below.

As shown in Table 7, it was also found that the skin permeability of the intracellular molecule-transporting peptide alone was also relatively high at 11.8%. In the case of intracellular molecule-transporting peptides that bind skin biologically active substances, It was confirmed that the transmittance of coumaric acid conjugated with the intracellular molecule transporting peptide was doubled as compared with the skin transmittance of ricin. In addition, in the case of acetylpentapeptide which is difficult to permeate the horny layer of skin, it was confirmed that the skin permeability was increased up to 28 times when bound to the intracellular molecule transfer peptide. As a result, the transdermal absorption test on human skin again proved that the permeability of the cosmetic composition combined with the intracellular molecule transfer peptide and the skin physiologically active molecule was remarkably excellent. Thus, the industrial use value of the intracellular molecule transfer peptide was confirmed I could.

Example  13. Skin biologically active substances Combined Intracellular  Molecular Transfer Peptide Skin cell Permeability  Confirm

13-1. Intracellular  Molecular transport peptides - Acetyl hexapeptide  Synthesis of fluorescent derivatives

The intracellular molecule transfer of M1067 having the amino acid sequence of SEQ ID NO: 16, M2067 having the amino acid sequence of SEQ ID NO: 23, and M2173A having the amino acid sequence of SEQ ID NO: 28 synthesized in Example 4, (Acetyl-Glu-Glu-Met-Gln-Arg-Arg), which is commercially used in the cosmetics industry, was synthesized sequentially, and then lysine (K, lysine) was added to the peptide to synthesize a peptide. FITC was bound to the amine residue. The synthesized peptide-FITC was obtained by removing the Fmoc group by adding 20% piperidine / N-methylpyrrolidone solution, washing it several times with N-methylpyrrolidone and dichloromethane, And dried with nitrogen gas. 2.5: 2.5: 2.5 (v / v) of trifluoroacetic acid: phenol: thioanisole: water: triisopropylsilane, To react for 2 to 3 hours to remove the peptide protecting group, to separate the peptide from the resin, and to precipitate the peptide with diethyl ether. The resulting intracellular molecular transport peptide-acetylhexapeptide fluorescent derivative was purified on a purified reverse phase high performance liquid chromatography column (Zobax, C8 300 Å) using a gradient of acetonitrile containing 0.1% trifluoroacetic acid , 21.1 mm X 25 cm) to prepare an intracellular molecule transfer peptide having the amino acid sequence of SEQ ID NO: 16 or 48 as shown in the following formula 4, and an intracellular molecular transfer peptide-acetylhexahexa with FITC coupled with acetylhexapeptide A peptide fluorescent derivative was synthesized. Then, the synthesized peptide-FITC was dissolved in DMSO to a concentration of 1 mM in a light-shielded state, and then a small amount of the peptide-FITC was dispensed into a 1.5 ml centrifuge container and kept frozen until just before use.

[Formula 4]

Met Arg Ala Ala Ala Pro Ala Val Ala Ala Glu * Glu Met Gln Arg Arg

Met His Ala Ala Ala Pro Ala Val Ala Ala Glu * Glu Met Gln Arg Arg

Met His Pro Ala Val Ile Pro Ile Leu Ala Val Glu * Glu Met Gln Arg Arg

*: acetylated glutamic acid

13-2. Flow cell  Using measurement Intracellular  Molecular transport peptides - Acetyl hexapeptide in vitro  cell Permeability  Confirm

(MTD-AH-FTIC) was incubated with 3 ÂμM of intracellular molecular transport peptide-acetylhexapeptide phosphor (MTD-AH-FTIC) on human keratinocyte cell line (HaCaT cell, Order No. 300493, CLS cell line service, Germany) and cultured for 1 hour. The HaCaT cells were maintained in DMED (Dubelco's modified eagle medium) medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (10,000 units penicillin and 10,000 μg / mL streptomycin, invitrogen) wet (humidified) of 5% CO ₂ and incubated with 37 ℃ under air. After the incubation was terminated, trypsin was treated to remove free MTD-AH-FITC exposed to the protein-treated HaCaT cell membrane and washed 3 times with chilled PBS.

For each sample, cells (1 × 10 ⁴ ) were assayed by CellQues Pro cytometric assay (Sigma-Aldrich) using a flow cytometer (FACS Calibur, Beckton-Dickinson, San Diego CA, USA) analysis software. Each experiment was performed more than 3 times. The cell permeability of each of the intracellular molecular transfer peptides applied to the present invention was quantitatively compared and analyzed by using the skin physiologically active substance acetyl hexapeptide itself as a control group.

The results are shown in FIG. 15, in which the fluorescence change of each experimental group was compared with Geometric mean in CellQues Pro cytometric analysis software, and the cell entry efficiency of the intracellular molecule transfer peptide was shown. As a result, it was confirmed that the cell entry efficiency of the acetyl hexapeptide conjugated with the intracellular molecular transfer peptide was 200% to 400% higher than that of the acetyl plexamide peptide itself.

13-3. Confocal microscope  Used Intracellular  Molecular transport peptides - Acetyl hexapeptide  Visible skin cells Permeability  Confirm

(HaCaT cell, immortalized human keratinocyte, Cat No. 300493, CLS, Germany) at a concentration of 3 [mu] M for direct detection of the internalized intracellular molecular transfer peptide-acetylhexapeptide fluorescent derivative After incubation at 37 ° C for 1 hour, it was visually observed using a confocal microscope (Nikon, Germany). On the day before the experiment, HaCaT cells were cultured in a 12-well culture plate containing a glass cover slip for 24 hours. HaCaT cells were maintained in DMEM medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin / streptomycin (10,000 units penicillin and 10,000 μg / mL streptomycin, Invitrogen, USA) ² < / RTI > under a humidified atmosphere. HaCaT cells were treated with scrambled peptides, MTD-FITC and PTD-FITC peptides at a concentration of 5 [mu] M for 1 hour. Cells were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature, washed three times with PBS, and incubated with 5 mM DAPI (4 ', 6-diamidino- 2-phenylindole). ≪ / RTI > After 10 minutes of DAPI staining, the cells were washed 3 times with PBS, and 20 μl of mounting media was dotted onto the slides to preserve the fluorescent label of the protein. The cells treated with the intracellular molecular transport peptide derivative were confirmed to be transferred to the nucleus and permeability through DAPI staining so that the intracellular delivery site could be easily distinguished. The confocal microscope was examined using a normaski filter The roundness of the cells was observed, and FITC fluorescence and DAPI fluorescence were observed with a filter corresponding to each fluorochrome. As a result, as shown in Fig. 16, in contrast to the control group using the acetyl hexapeptide itself as a skin physiologically active substance, the transfer peptide significantly improved the transmission ability of the acetyl hexapeptide bound to the intracellular molecule transfer peptide Respectively.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Attach an electronic file to a sequence list

Claims (21)

A composition for transdermal delivery comprising a skin biologically active molecule to which an intracellular molecule-transporting peptide is bound, characterized in that the peptide is composed of the amino acid sequence of SEQ ID NO: 16 or 23. The transdermal delivery composition according to claim 1, wherein the composition is a cosmetic composition. The composition according to claim 1, wherein the peptide is encoded from the polynucleotide sequence of SEQ ID NO: 37 or 44. The transdermal delivery composition according to claim 1, wherein the composition penetrates the horny layer of the skin. The transdermal delivery composition according to claim 1, wherein the skin biologically active molecule binds to the N-terminal, C-terminal, or both terminal ends of the intracellular molecule-transferring peptide.  The transdermal delivery composition according to claim 1, wherein the skin biologically active molecule is bound to the intracellular molecule transport peptide in a reverse order. The transdermal delivery composition according to claim 1, wherein the binding is carried out by peptide bonding or chemical bonding. 8. The method of claim 7, wherein the chemical bond is selected from the group consisting of a disulfide bond, a diamine bond, sulfide-amine bonds, carboxyl-amine bonds, ester bonds, ≪ / RTI > The transdermal delivery composition according to claim 1, wherein the skin biologically active molecule is selected from the group consisting of vitamins, retinoids, and fatty acids. 10. The method of claim 9, wherein the vitamin is selected from the group consisting of vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D1, vitamin D2, , Vitamin D5, vitamin E, and vitamin K. The transdermal delivery composition of claim 1, 10. The transdermal delivery composition of claim 9, wherein the retinoid is selected from the group consisting of retinol, natural and synthetic analogs of retinol, retinal, trans, 9-cis, and 13-cis retinoic acid and etretinate. . The transdermal delivery system according to claim 9, wherein the fatty acid is selected from the group consisting of lauric acid, stearic acid, palmitic acid, undecylenic acid, palytoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, Composition. The transdermal delivery composition according to claim 1, wherein the skin biologically active molecule is selected from the group consisting of a wrinkle reducing agent, a whitening agent, an antioxidant, and a moisturizing agent. 14. The transdermal delivery composition according to claim 13, wherein the anti-wrinkle agent is selected from the group consisting of skin-acting growth factor peptide or protein, retinol, retinyl palmitate, adenosine and polyethoxylated retinamide. The transdermal delivery composition according to claim 13, wherein the whitening agent is selected from the group consisting of whitening peptide, niacinamide, mulberry extract, arbutin, ethyl ascorbyl ether, licorice extract, ascorbyl glucoside, and magnesium ascorbyl phosphate . The transdermal delivery composition according to claim 1, wherein the skin biologically active molecule is selected from the group consisting of coumaric acid, acetyl pentapeptide, and acetyl hexapeptide. The composition for transdermal delivery according to claim 1, wherein the composition is prepared from an emulsion, a cream, an essence, a skin, a liposome, a microcapsule, a composite particle, a shampoo and a rinse. A system for transdermally delivering a skin biologically active molecule into a skin cell, said system comprising an intracellular molecular transport peptide comprising the amino acid sequence of SEQ ID NO: 16 or 23 in association with a dermal bioactive molecule. 19. The transdermal delivery system according to claim 18, wherein the peptide is encoded from the polynucleotide of SEQ ID NO: 37 or 44. A method for delivering a skin biologically active molecule into a skin cell in vitro, the method comprising delivering an intracellular molecular transport peptide consisting of the amino acid sequence of SEQ ID NO: 16 or 23 to a skin biologically active molecule ≪ / RTI > In vitro, a method for delivering a dermal bioactive molecule into the stratum corneum of a skin, said method comprising the step of delivering an intracellular molecular transport peptide consisting of the amino acid sequence of SEQ ID NO: 16 or 23, in association with a dermal bioactive molecule ≪ / RTI >

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