WO2013077681A1 - Transdermal delivery system of bioactive molecules of skin using intracellular molecule delivery peptides - Google Patents

Abstract

The present invention relates to a composition for transdermal delivery containing bioactive molecules of skin in which intracellular molecule delivery peptides are fused. More particularly, the composition is one in which peptides having superior in vivo molecular delivery ability are fused to bioactive molecules of skin which may not be easily permeated into the stratum corneum of skin due to molecular size and physical properties. The composition may exhibit excellent in vitro and in vivo skin permeability, and therefore, can be valuably used in delivering compounds having various advantages into skin cells and formulated into various forms. Thus, the composition can be valuably used as a material for functional cosmetics.

Description

Transdermal Delivery System of Skin Bioactive Molecules Using Intracellular Molecular Transfer Peptides

The present invention relates to a transdermal delivery composition comprising a skin bioactive molecule to which intracellular molecular transport peptides are bound, a transdermal delivery system using the skin bioactive molecule, and a method for intracellular skin delivery of the skin bioactive molecule.

The skin is a tissue that is always in contact with the outside environment. Its main function is to protect body fluids from leakage and infection, as well as act as a protective barrier against water loss. The stratum corneum of the epidermis is located on the outermost surface of the skin and prevents the loss of moisture and electrolytes outside the skin, preventing the drying of the skin and providing an environment for normal biochemical metabolism of the skin. It protects the human body from humans and plays an important role in preventing bacteria, fungi, viruses, etc. from invading the skin (Bouwstra J. A, Honeywell-Nguyen PL Gooris GS and Ponec M. Prog Lipid Res. 42: 1-36 (2003)).

There are three pathways for absorption through the skin, through the stratum corneum, through the hair follicle and sebaceous glands, and through the sweat glands (Prausnitz M. R. and Langer R. Nat Biotech. 26: 1261-1268 (2008)). The delivery of physiologically active molecules through the skin is subject to various limitations due to the structural and physical properties of the skin. To date, absorption through the stratum corneum is known as the most important absorption pathway. In particular, the stratum corneum of the skin is the natural constituent of the keratinocyte (keratinocyte) is a natural death and forms a dense structure in the outermost layer of the skin, not only evaporation of moisture but also inhibits the penetration of foreign substances, sweat and various lipid components Due to the acidity is showing around pH 5. In order to penetrate the stratum corneum barrier, the molecular weight must be as small as 1,000 or less and possess lipophilic properties (Metha R. C. and Fitzpatrick R. E. Dermatol. Ther. 20: 350-359 (2007)).

 It is known that low molecular weight synthetic compounds and natural compounds with a molecular weight of 1,000 or less that are frequently used as cosmetic ingredients can be easily transferred into cells, but macromolecules such as proteins, peptides and nucleic acids with molecular weight of 1,000 or more have a double lipid membrane structure due to their molecular weight. It is difficult to penetrate into the cell membrane. In fact, due to the intrinsic properties of the stratum corneum, which constitutes the skin barrier, they are known to have extremely low permeation efficiency of low molecular weight materials and lower permeation efficiency of high molecular weight materials. As a method for amplifying the efficiency of such small molecules and macromolecules to cross the plasma membrane of the cell, a recent interest is the method of transdermal drug delivery (TDD) (Prausnitz MR and Langer R. Nat Biotech. 26: 1261-1268 (2008). However, the biggest barrier of the method of administration through the skin is keratinocytes in the stratum corneum and keratinocyte lipids therebetween. In most skin physiologically active molecules (hereinafter, skin bioactive molecules), the percutaneous permeability is lowered due to the resistance of the stratum corneum.

Various methods for promoting the percutaneous permeability of such skin bioactive molecules have been studied, which can be roughly divided into physical, biochemical, and chemical methods. The physical method is mainly using iontophoresis, electroporation, sonophoresis, and thermal ablation. The biochemical method uses precursors to activate active ingredients during and after the skin. The chemical method is the most widely used method in the industry, and various kinds of bases, surfactants, solvents, and fatty acids are mainly in the form of complexes carrying bioactive molecules. Carrier-mediated delivery systems are being used (Prausnitz MR and Langer R. Nat Biotech. 26: 1261-1268 (2008)). Among these, much attention has recently been focused on the use of peptide-based delivery systems.

The use of peptides having cell permeability has several advantages, mainly due to the various modifications that can be made to the peptide sequence at all times. It can designate different subcellular domains and allows manipulation of carriers that can carry various forms of cargo molecules. Representative cell membrane permeable peptides are as follows.

The first transport domains discovered 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 (188)) in 1988, respectively. Separately, the transcriptional protein Tat of HIV-1, a virus that causes acquired immune deficiency syndrome (AIDS), has been found to penetrate the cell membrane and trans-activate (trans-activation) viral genes. The cell permeable Tat domain is the 48th to 57th basic amino acid sequence (GRKKRRQRRR) of the Tat protein, which has been found to play an important role in the passage of cell membranes (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 (2002) Wadia et al., Nature Medicine 10 (3), 310-315 (2004)).

Second, RQIKIYFQNRRMKWKK consisting of 16 amino acids derived from the antennapedia homeodomain of Drosophila (Joliot et al., Proc Natl Acad Sci USA 88: 1864-1868 (1991) Derossi et al., J Biol Chem 269, 10444-10450 (1994); Joliot, A. and A. Prochiantz, Nat. Cell Biol. 6 (3): 189-96 (2004).

Third, a peptide based on a membrane translocating sequence (MTS) or signal sequence, which is a receptor protein that helps to move a newly synthesized protein by RNA to the biofilm of suitable intracellular organelles in vivo. It is known that biomembrane translocation sequences, coupled with nuclear localization signals (NLS), accumulate in the cell nucleus across 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 a cargo molecule and act as an import signal when in close proximity to the cell, inducing the influx of the cargo molecule into the cell. Accordingly, attempts have been actively made to link cell membrane permeable peptides with proteins originally present in cells to be applied to biological research and disease treatment (F. Milletti, Drug Discovery Today 17: 850-860 (2012)). If cell membrane-penetrating peptides are derived from viruses such as Tat or VP22, there is a possibility of inducing an immune response in vivo, and thus, new membrane-penetrating peptides are mainly identified from secreted proteins found in humans (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 addition, in order to increase cell membrane permeability, new cell membrane-penetrating peptides have been developed by methods such as deletion, modification, and chimeric fusion on amino acid sequences (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 utility due to inefficiency or their toxicity, and delivery mechanisms are controversial. Although no exact answer has been obtained as to whether the carrier is endocytosis in the intracellular mass transfer mechanism, various mechanisms have been proposed. There is a claim that receptors do not participate and are transferred by the formation of inverted micelles. Another opinion is that proteoglycans on the cell surface play an important role in the transfer and electrostatic interactions with cell membrane-penetrating peptides. In addition, it was suggested that it does not play a big role. Some research suggests that TAT proteins do not actually undergo metastasis, but rather strongly induce electrostatic interactions with the cell surface, causing inclusions and accumulating inside the endosome, and endosomes are fused with lysosomes. It is suggested that peptides are easily degraded by proteolytic enzymes present in lysosomes. When using a substance transport carrier composed of a basic amino acid such as TAT to deliver a specific active ingredient into the cell, it is difficult to directly access to the target in the cytoplasm and high concentration treatment to deliver the active ingredient to a level that can exhibit the desired activity. There is a problem in that it is not possible to obtain the expected effect when applying it (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, in the known mediated delivery system of foreign substances, there is much controversy about the inflow mechanism of cells (F. Milletti, Drug Discovery Today 17: 850-860 (2012)). In addition, in the case of these conventionally developed peptides to date, there is no example showing that it is possible to transmit to skin cells through the stratum corneum.

Recent studies have shown that carrier-mediated delivery systems undergo metastasis through one or more mechanisms, and in the case of macromolecules, produce energy-dependent macropinocytosis, and small molecules associated with membrane permeable peptides or membrane permeable peptides may be It is assumed to have an energy independent transmission mechanism by hydrogen bonding. In any case, direct contact of the proteoglycans and cell membrane permeable peptides present in the cell membrane or cell membrane is essential for the success of intracellular delivery. Experiments such as β-cyclodextrin treatment, cytochalasin D treatment, or dominant-negative dynamine (DynK44A) expression suggest that lipid raft-mediated plenomegaly is the main mechanism (Wadia JS, Stan RV, Dowdy SF. Nat Med). 2004 Mar; 10 (3): 310-5.).

Later, in 2000, new cell membrane-penetrating peptides with improved intracellular delivery efficiency and different structural and electrostatic properties were developed. Among them, Procell Pharmaceuticals developed 'Macromolecule Intracelular Tranduction Technology: MITT) does not require the encapsulation and energy required for intracellular transfer in HIV-TAT cells, but directly interacts with the cell membrane because rigidity and integrity of the cell membrane are important factors. Direct interaction has been proposed as important (WO2008 / 093982). MTD has a higher delivery efficiency of cargo material, which can be selected from compounds, peptides, and proteins, and has the advantage of cell-to-cell delivery, compared to the conventional cell membrane permeable peptide, TAT. Its applicability is highly appreciated in the development of cosmetics.

Accordingly, the present inventors apply an improved MTD peptide having better cell permeability by modifying the amino acid sequence of the macromolecular transport domain (MTD) to the intracellular molecular transport peptide of the present invention. The present invention has been completed by demonstrating that the skin bioactive molecules which do not readily penetrate the skin are endowed with the effect of delivery in the stratum corneum or in the skin cells.

The present invention is designed to efficiently introduce skin bioactive molecules into the skin cells, which are difficult to deliver through the skin due to the size of the molecular weight or the intrinsic properties of the stratum corneum as described above, the cell improved cell permeability compared to the existing MTD peptide A transdermal delivery composition comprising a skin bioactive molecule to which an intramolecular transport peptide is bound, a transdermal delivery system using a skin bioactive molecule to which the peptide is bound, and a skin bioactive molecule to which an intracellular molecular transport peptide is bound Or to provide a method for delivery into the stratum corneum of the skin.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will 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 transdermal delivery comprising a skin bioactive molecule to which an intracellular molecular transport 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 invention, the composition is characterized in that the 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 a 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 a 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 a 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 a 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 a 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 a 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 a 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 a polynucleotide sequence of SEQ ID NO: 56, but is not limited thereto.

In another embodiment of the invention, the composition is characterized in that it penetrates the stratum corneum.

In another embodiment of the present invention, the skin bioactive molecule is characterized by binding to the N-terminus, C-terminus or sock end of the intracellular molecular transport peptide.

In another embodiment of the present invention, the skin bioactive molecule is characterized in that the binding of the amino acid of the intracellular molecule transfer peptide in the form arranged in reverse order.

In another embodiment of the present invention, the linkage is characterized by a peptide bond or a chemical bond.

In another embodiment of the invention, the chemical bonds are disulfide bonds, diamine bonds, sulfide-amine bonds, carboxyl-amine bonds, ester bonds and covalent bonds. Characterized in that selected from the group consisting of.

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

In another embodiment of the present invention, the vitamin is 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 is selected from the group consisting of.

In another embodiment of the invention, the retinoid is characterized in that it is selected from the group consisting of retinol, retinol mutated and synthetic analogues, retinal, trans, 9-cis, and 13-cis retinoic acid and etretinate.

In another embodiment of the invention, the fatty acid is characterized in that selected from the group consisting of lauric acid, stearic acid, palmitic acid, undecylenic acid, paritoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid and erucin acid .

In another embodiment of the invention, the skin bioactive molecule is characterized in that it is selected from the group consisting of anti-wrinkle agent, whitening agent, antioxidant and moisturizer.

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

In another embodiment of the present invention, the whitening agent is characterized in that it is selected from the group consisting of a whitening peptide, niacinamide, turmeric extract, arbutin, ethylascomilletel, licorice extract, ascorbyl glucoside, and magnesium ascorbyl phosphate It is done.

In another embodiment of the invention, the skin bioactive molecule is characterized in that it is selected from the group consisting of coumaric acid (coumaric acid) or acetyl pentapeptide and acetyl hexapeptide (Acetyl Hexapeptide).

In another embodiment of the invention, the composition is characterized in that it is prepared in a formulation selected from the group consisting of emulsions, creams, essences, skins, liposomes, microcapsules, composite particles, shampoos and rinses.

The present invention also provides transdermal delivery of skin bioactive molecules into skin cells, wherein the intracellular molecular transport peptides having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 35 are delivered in combination with a skin bioactive molecule. Provide a delivery system.

In one embodiment of the invention, the peptide is characterized in that encoded from a polynucleotide selected from the group consisting of SEQ ID NO: 36 to 56.

In addition, the present invention provides a method for delivering the skin bioactive molecules into the skin cells comprising the step of binding and delivering the intracellular molecular transport peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 15 to 35 with the skin bioactive molecules Provide a method.

In addition, the present invention is to deliver the skin bioactive molecules into the stratum corneum of the skin comprising the step of delivering the intracellular molecular transport peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 15 to 35 in combination with the skin bioactive molecules. Provide a method.

The composition for transdermal delivery of the present invention binds a peptide having excellent molecular transport ability in vivo to the skin bioactive molecules that are not easily permeable to the stratum corneum due to the size and physical properties of the skin stratum corneum, thereby allowing the skin bioactive molecules to be transferred to skin cells or skin Up to the stratum corneum. Accordingly, the compositions of the present invention have the advantages of antioxidant efficacy, increased angiogenesis, reduced acne symptoms, decreased line secretion, decreased aging effects, reduced wrinkles, reduced melanogenesis, lessened skin inflammation or improved skin dryness. Not only can the compound be effectively delivered to the skin tissue depth, but also can be formulated in various forms, it can be usefully used as a functional cosmetic raw material.

1 to 4 show the results of analyzing the secondary structure of the intracellular molecular transport peptide of the present invention using the PEP FOLD server program.

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 are the results of observation with confocal microscopy to measure the skin cell permeability of the intracellular molecular transport peptide of the present invention:

MR-form: improved MTD in the form A1-A2-MTD wherein A1 is methionine and A2 is arginine;

MH-form: improved MTD in the form A1-A2-MTD, wherein A1 is methionine and A2 is histidine; And

MK-form: Enhanced MTD of the form wherein A1 is methionine and A2 is lysine in the formula A1-A2-MTD.

Figure 9 is a photograph of the intracellular molecular transmission peptide of the intracellular molecular transmission peptide in the EpiOral artificial tissue model visualized by confocal microscopy.

FIG. 10 is a confocal microscope picture of the permeability in the stratum corneum and skin tissue of intracellular molecular transport peptides in an EpiDerm artificial skin model.

11 is a result of confirming the skin permeability of the intracellular molecular transport peptide in vivo by confocal microscopy.

12 is a result confirming the inhibitory effect of tyrosinase activity of the coumalic acid derivative bound to intracellular molecular transport peptides.

Figure 13 is the result confirming the inhibitory effect of the intracellular melanin production of the coumalic acid derivative coupled to the intracellular molecular transport peptides.

14 is a result confirming the inhibitory effect of collagenase activity in human normal dermal fibroblasts of intracellular molecular transport peptides and acetyl pentapeptide binding derivatives.

15 is a flow cytometry analysis for measuring skin cell permeability of intracellular molecular transport peptides and acetylhexapeptide binding derivatives.

Figure 16 shows the results of confirming the skin cell permeation position of intracellular molecular transport peptides and acetylhexapeptide binding derivatives by confocal microscopy.

Delivery of active substances through the skin has several limitations due to the structural and physical properties of the skin. In particular, the stratum corneum of the skin is keratinocyte (the major constituent cell of the skin) is a natural death and forms a dense structure in the outermost layer of the skin, inhibits the evaporation of moisture as well as the penetration of foreign substances, sweat and various lipid components Due to its acidic region near pH 5. In order to penetrate the stratum corneum, the molecular weight must be less than 1,000 and possess lipophilic properties (Metha R. C. and Fitzpatrick R. E. Dermatol. Ther. 20: 350-359 (2007)).

Low molecular synthetic compounds or natural compounds frequently used as cosmetic ingredients are known to be easily transferred into cells. Macromolecules such as proteins, peptides and nucleic acids are difficult to penetrate into cell membranes having a double lipid membrane structure due to their molecular weight. In fact, due to the intrinsic properties of the stratum corneum, which constitutes the skin barrier, they are known to have extremely low permeation efficiency of low molecular weight materials and lower permeation efficiency of high molecular weight materials. Macromolecule Intracellular Transduction Technologiy (MITT) can be used as a method for amplifying the efficiency of the small molecules and macromolecules through the plasma membrane of the cell.

Accordingly, in the present invention, a negative charge is applied to the hydrophobic domain by applying one or two hydrophilic (polar) amino acids having a positive charge to the hydrophobic macromolecular transfer domain (MTD) that has been invented (Korean Patent Publication No. 10-2009-0103957). By improving accessibility to the prominent cell membranes, we have developed an improved MTD peptide that efficiently delivers biologically active molecules into cells, and by using them, transdermal delivery compositions and transdermals that can deliver skin bioactive molecules into skin cells more efficiently. Delivery systems and methods of delivering skin bioactive molecules into skin cells or the stratum corneum of the skin.

The improved MTD peptide is a peptide described by the following Formula 1,

A 1 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);

MTD, characterized in that having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7;

[Equation 1]

A1-A2-MTD.

The peptide is a peptide capable of mediating the delivery of a biologically active molecule into cells, and is a peptide that exhibits superior cell permeability compared to the MTD peptide described in Korean Patent Application Laid-Open No. 10-2009-0103957. 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 delivery domain (MTD) peptides developed in Korean Patent Application Publication No. 10-2009-0103957, the peptide is preferably a sequence satisfying the following conditions among quantified and comparatively comparable sequences. It is not limited to:

1) the position of Proline is located in the center of the sequence;

2) the result of evaluating the possibility of extracellular secretion using the Signal P program is 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 flexibility evaluated using the Protscale (Average flexibility) program (see http://web.expasy.org/protscale/) is evaluated to be 0.36 or higher;

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

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

7) Sequence where the evaluation result of polarity using Protscale (polarity) program is evaluated to 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.

Synthesis of the peptides of the present invention and the synthesis of peptides containing cargo, which is a skin bioactive molecule, can be carried out, for example, using a device or using genetic engineering techniques.

Antioxidant efficacy, angiogenesis, decreased acne symptoms, decreased line secretion, decreased aging effects, reduced wrinkles, decreased melanin production, alleviated skin inflammation or dried skin using the intracellular molecular transport peptides It is possible to deliver compounds that have advantages such as improvement. In addition, topical application of compositions comprising them can provide a beneficial effect on the skin. The beneficial effect is to reduce or prevent damage caused by sunlight, to provide antioxidant activity, to reduce the appearance of wrinkles, including fine wrinkles on the skin, to reduce gland secretion, the aging effect It includes, but is not limited to, reducing acne, treating acne, inducing angiogenesis in hair follicles, hair growth, conducive to skin moisturizing, and inhibiting melanin production to brighten skin color.

In addition, the active ingredient deliverable into the skin cells by the intracellular molecular transfer peptide may be a compound or a protein or fragments thereof, further providing additional benefits for the skin. They are non-toxic, non-allergic and non-irritating and therefore friendly with skin tissue. Examples of suitable active cars may include, but are not limited to, vitamins and derivatives thereof, retinoids and fatty acids.

According to the invention, ascorbic acid (vitamin C), alpha-tocopherol (vitamin E) and retinoid (vitamin A) can provide beneficial properties to the skin. 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 fatty oxidation and UV exposure (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 to inhibit melanin formation and histamine secretion of cell membranes, compensates for vitamin E deficiency in the skin, is involved in preventing depigmentation of the skin, and has anti-free radical activity. Alpha-tocopherol is an antioxidant that prevents the deleterious 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 the mediators of inflammation in the skin and increase the production of procollagen, allowing for more production of type I and type III collagen.

Specifically, the cargo compound of the present invention is vitamin A, vitamin Bl, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin Dl, vitamin D2, vitamin D3, vitamin D4, Vitamins such as vitamin D5, vitamin E, and vitamin K.

Yet another cargo compound of the present invention is a retinoid. Retinoids are retinol, natural and synthetic analogues of vitamin A (retinol), vitamin A aldehydes (retinal), all trans, 9-cis, and 13-cis retinoic acid, etretinate and vitamins described in several prior documents. A acids (retinolic acid) as well as compounds having various synthetic retinoid and retinoid activities.

In addition, the cargo compounds of the invention comprise fatty acids. Fatty acids are monocarboxylic acids with saturated or unsaturated fatty tails. As defined in the International Cosmetic Ingredient Dictionary and Handbook, 7th Ed. (1997) volume 2, page 1567 (the disclosure is incorporated herein by reference), Having 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 the sebaceous gland and is used in skin care and cosmetic preparations as a moisturizer. It stabilizes the oil balance to keep the skin normal and healthy, softens the skin and reacts like an anti-keratinizing agent. Esters of palmitic acid are used to provide silky silkiness to the skin and hair. The palmitic acid acts as a carrier that can penetrate the pentapeptide into the skin, is widely used as a lubricant, and is used as an emulsifier, surfactant, and formula texturizer.

In addition, suitable fatty acids of the present invention 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, ie, the skin bioactive molecule, are bound by covalent bonds, and include, but are not limited to, ester, amide, ether and carbamide bonds. Embodiments of the invention include, but are not limited to, coumalic acid, acetylpentapeptide or acetylhexapeptide and selected intracellular molecular transport peptides.

The compounds of the present invention can 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 to improve hair growth. If a compound is used that affects hair growth, it is applied to the scalp, eyebrows or eyelashes. In addition, when applied to the skin, the intracellular molecular transport peptides and cargo compounds may reduce skin texture to reduce the appearance of fine lines and wrinkles, to improve skin elasticity, and to reduce puffiness. To soften, to provide a silky texture to the skin, to provide a moisturizing effect, to provide lubricity, to provide a bright complexion through inhibition of melanin production, to reduce the aging effect, or to other cosmetics It can be used to provide benefits.

As used herein, “skin bioactive molecules” are defined as “cosmetically or dermatologically acceptable molecules” and contact physiological tissue without excessive toxicity, incompatibility, instability, etc. And suitable compositions or compounds for use.

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

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

In terms of formulation, the compositions of the invention may be solutions, emulsions (including microemulsions), suspensions, creams, lotions, gels, powders, or other typical solids used for application to the skin and other tissues to which the compositions can be applied. Liquid compositions. Such compositions may comprise additional antimicrobial, moisturizing and hydrating agents, penetration agents, preservatives, emulsifiers, natural or synthetic oils, solvents, surfactants, detergents, gelling agents ( may include gelling agents, emollients, antioxidants, fragrances, fillers, thickeners, waxes, odor absorbers, dyestuffs, colorants, powders, viscosity-controlling agents and water And optionally, anesthetics, anti-itch actives, botanical extracts, conditioning agents, darkening or lightening agents, glitters, wetting agents ( humectant, mica, minerals, polyphenols, silicones or derivatives thereof, sunblocks, vitamins, and phytomedicinal. In some embodiments, the compositions of the present invention are formulated with the aforementioned ingredients to be stable for long periods of time, which may be useful if continuous or long term use is intended.

In some embodiments of the invention, the composition is used to treat, ameliorate or prevent skin aging. Skin damage from aging can include fine and deep wrinkles, skin lines, crevices, bumps, large holes, scalyness, loss of skin elasticity, sagging, loss of skin firmness or tautness, and discoloration. Hyperpigmented areas such as age spots and freckles, keratosis, abnormal differentiation, hyperkeratinization, elastic fibrosis, collagen breakdown, stratum corneum, dermis, epidermis, skin vascular system, And other histological changes in underlying tissues particularly in proximity to the skin. Although skin aging is aging due to external factors such as chronological aging or continuous 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, the present inventors treated the skin cells at a constant concentration using a complex in which the FITC fluorescent substance was linked to the intracellular molecular transport peptide to quantitatively determine the cell permeability of the intracellular molecular transport peptide. Afterwards, fluorescence intensity was measured using flow cytometry, and as a result, several sequences were confirmed by intracellular molecular transport peptides that delivered FITC into cells more efficiently than known protein transduction domains (PTDs). The invasion effect of the intracellular molecular transport peptides in skin cells was demonstrated.

In another specific embodiment of the present invention, the inventors have performed cell permeability and intracellular localization through confocal microscope analysis using a complex linking FITC fluorescent material to intracellular molecular transport peptides. The results can be visually confirmed. As a result, as shown in the flow cytometry results, it was confirmed that exhibited high skin cell permeability in the improved intracellular molecular transport peptide than the existing MTD peptide. In addition, it was confirmed that the intracellular molecular transport peptide has a higher cell permeability than Tat, which is known to have a relatively excellent intracellular transport efficiency among known protein transduction domains (PTDs).

In addition, the peptide having a cargo having a skin physiological activity (cargo) was confirmed to increase the physiological activity through skin permeation, thereby demonstrating that it can be used as a cosmetic raw material having a whitening or wrinkle improvement effect.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Example 1 Screening of Subject MTD Peptides for Design of Improved MTD Peptides

The inventors of the present invention, among the 193 MTD peptides developed in Korean Patent Application Publication No. 110-2009-0103957), which have been developed by the present inventors, are quantified and transmitted in a sequence that satisfies the following conditions among relative comparable sequences. Subject MTD peptide sequences were selected for ability improvement.

Conditions for the screening are as follows.

1) In the sequence of the macromolecular delivery domain, the sequence whose proline position is located in the center of the sequence is selected.

2) From the sequences of the macromolecular delivery domain, sequences having a high possibility of inducing extracellular secretion are selected.

3) Among the sequences of the macromolecular delivery domain, a sequence satisfying a specific level of the aliphatic index, which is an element that determines the physicochemical properties of the macromolecular delivery domain, is selected.

4) Among the sequences of the macromolecular delivery domain, a sequence that satisfies a certain level of flexibility, which is a factor that determines the physicochemical properties of the macromolecular delivery domain, is selected.

5) Among the sequences of the macromolecular delivery domain, a sequence that satisfies a certain level of hydropathicity, which is a factor that determines the physicochemical properties of the macromolecular delivery domain, is selected.

6) Among the sequences of the macromolecular delivery domain, a sequence that satisfies a specific level is selected for the instability index, which is a factor that determines the physicochemical properties of the macromolecular delivery domain.

7) Among the sequences of the macromolecular delivery domain, a sequence is selected in which polarity, an element that determines the physicochemical properties of the macromolecular delivery domain, satisfies a certain level.

This will be described in detail below.

To select specific representative sequences, representative sequences were determined based on the presence and location of proline, as described in step 1) above. Among the amino acids alanine, valine, proline, leucine, and isoleucine that make up the sequence of the macromolecular delivery domain, the shorter and smaller size of the proximal side affects the degree of freedom of secondary structure formation of the amino acid sequence, which is the macromolecular delivery domain. Contributes to cell membrane permeation. Among the macromolecular delivery domains (MTDs) known in the Republic of Korea Publication No. 10-2009-0103957: Novel macromolecular delivery domains and methods and uses thereof, proline is present on the sequences constituting it and the Forty-nine macromolecular delivery domains were determined, whose positions were located in the middle of the amino acid sequence of the macromolecular delivery domain and were classified as having high degree of freedom of secondary structure formation of the amino acid sequence.

Subsequently, sequences with high possibility of extracellular secretion were selected from the macromolecular delivery domains selected in step 1). Some water-soluble proteins have signals that can interact with receptors that mediate transport. In signal mediated transport, a protein has one or more signal sequences that specify the target to which it is delivered. Therefore, under the assumption that the similarity with the extracellular secretion inducing sequence can improve the transmission ability, the possibility of inducing extracellular secretion was evaluated using the Signal P program. Probability between 10% and 90% is assessed for each domain, with sequences rated at least 60% of the macromolecular delivery domains determined in step 1) selected as improved subject sequences.

Next, as described in step 3), among the macromolecular delivery domains selected in step 2), sequences satisfying a specific level of the aliphatic index, which is an element that determines the physicochemical properties of the macromolecular delivery domain, were selected. Aliphatic index is a physical feature that determines the volume of the entire molecule, which is determined by the carbon chain of the side chain of amino acids, and is evaluated as a feature that modifies the structure of the cell membrane as the macromolecular delivery domain penetrates the cell membrane. 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, and was determined using the Protparam program (see http://web.expasy.org/protparam/). The Protparam program is a useful tool that quantifies the physical properties of proteins or peptides consisting of amino acids. The aliphatic index was selected from 100 to 300 as the representative sequence for improving the transfer capacity.

Subsequently, as described in step 4), among the macromolecular delivery domains selected in step 3), a sequence was selected in which flexibility, an element that determines the physicochemical properties of the macromolecular delivery domain, satisfies a certain level. Flexibility is a physicochemical feature that represents the degree of freedom and degrees of freedom between the N- and C-terminal amino acids of the macromolecular delivery domain, and provides structural flexibility and is associated with affinity for cell membranes. Flexibility was evaluated according to the length of the amino acid sequence and the composition of the side chain sequence of the amino acid, 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 consisting of amino acids. Among the macromolecular delivery domains determined in step 3), the evaluation result of flexibility using the Protscale (Average flexibility) program was 0.36 or higher. Selected sequences were selected as representative sequences for improving transmission capacity.

Next, as described in step 5), among the macromolecular delivery domains selected in step 4), a sequence was selected in which hydropathicity, which is a factor determining the physicochemical properties of the macromolecular delivery domain, satisfies a certain level. Hydropathicity is a physical property determined by the unique properties of amino acids and is considered to be a property that determines physical properties. It is known to cause severe entanglement at 3.0 or higher. Hydropathicity was also determined by the unique value of each of the amino acid sequences of the macromolecular delivery domain and the average of the entire sequence, and evaluated using the Protparam program. Therefore, among the hydropathicity results of 1.3 to 3.8, a sequence whose hydropathicity was evaluated to 3.0 or less in the macromolecular delivery domain selected in step 4) was determined as a representative sequence for improvement.

Then, as described in step 6) among the macromolecular delivery domains selected in step 5), a sequence was determined in which the instability index of the macromolecular delivery domain satisfies a certain level. Instability index is a property that indicates the stability of the amino acid sequence is determined by the sequence of amino acids on the sequence, the higher the value has instability. This is a factor that determines the physicochemical properties of the macromolecular delivery 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, a sequence having an instability index of 30 to 60 within the macromolecular delivery domain determined in step 5) among the macromolecular delivery domains represented by instability index 0 to 130 was selected as a representative sequence for improvement.

Subsequently, among the macromolecular delivery domains selected in step 6), as described in step 7), a sequence was determined in which Polarity, an element that determines the physicochemical properties of the macromolecular delivery domain, satisfies a certain level. Polarity is a measure of the affinity for water as judged by the length of the carbon chain and the presence or absence of hydroxyl groups among the components of amino acids. This, together with hydropathicity, determines the properties of the macromolecular transport domain and is thought to affect affinity to cell membranes. Polarity is determined by the unique value of each of the amino acid sequences of the macromolecular delivery domain and the average of the entire sequence, and was evaluated using the Protscale (polarity) program. Accordingly, among the macromolecular transfer domains determined in step 6), sequences whose evaluation results were greater than 0.1 using the Protscale (polarity) program were selected as representative sequences for improvement.

Specifically, JO-103 (SEQ ID NO: 3), which is a target MTD for designing an improved MTD peptide in the macromolecular transport domain described in Korean Patent Application Publication No. 10-2009-0103957, was determined by the following procedure.

i) Of the macromolecular transfer domains (MTD) known in the Republic of Korea Patent Publication No. 10-2009-0103957: Novel macromolecular delivery domains and methods and uses thereof, JO-103 consists of the sequence of LALPVLLLA;

ii) a 90% chance of assessing the possibility of extracellular secretion using the Singal P program;

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

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

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

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

vii) is estimated to be 0.13 in the polarity evaluation using the Protscale program,

Therefore, it can be seen that JO-103 determined as a target sequence for improving transmission capacity is a target domain that satisfies all seven steps.

In the same manner, five target MTD peptides were selected for the design of the improved MTD peptide, 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).

Repositioning proline for double MTD JO-173 peptide to gain additional flexibility; The MTD 173A peptide (SEQ ID NO: 7), which is an intermediate peptide, was prepared. The MTD 173A peptide thus prepared also satisfies the selection conditions of the above seven steps, and was included in the target MTD sequence for designing an improved MTD peptide.

In addition, the intermediate peptide MTD 18m (SEQ ID NO: 6) was derived to improve the physical properties by substituting the amino acid with low hydropathicity for the JO-18 No. among the selected MTD. Satisfaction was included in the subject MTD sequence for the design of the improved MTD peptide.

Example 2 Design of Improved MTD Peptides

Based on the seven target MTDs selected in Example 1, by adding one or two hydrophilic (polar) amino acids to the hydrophobic domain to increase the access to the cell membrane, more efficiently deliver the biologically active molecules into the cell A new modified MTD peptide was intended to be made, through which the peptide described by Equation 1 was devised;

[Equation 1]

A1-A2-MTD:

A 1 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 7 amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 7 selected in Example 1.

The sequence of the improved MTD peptide designed through Equation 1 is the same as the amino acid sequence shown in SEQ ID NOs: 15 to 35, and the peptide was synthesized based on the designed amino acid sequence to confirm its cell permeability.

Example 3 Secondary Structure Analysis of Enhanced MTD Peptides

For the sequence of the improved MTD peptide designed in Example 2, the secondary structure was analyzed using the PEP FOLD server service program.

As a result, as shown in Figures 1 to 4, the sequence of binding the N-terminal sequence of the EGFP fluorescent protein to the improved MTD peptide designed in Example 2 is disclosed in Korea Patent Publication No. 10-2009-0103957 (New macromolecule) It was confirmed that the α-helix structure that can enhance the permeability of the cell membrane is maintained without impairing the structural characteristics of the large molecule transfer domain (MTD) peptide known in the delivery domain and its identification method and use.

Example 4. Synthesis of Intracellular Molecular Transfer Peptides and FITC Fusion Phosphors

The improved MTD peptide designed in Example 2 was applied to intracellular molecular transport peptides fused to the skin bioactive molecules of the present invention, and for the synthesis thereof, C-term using a general Fmoc solid phase peptide synthesis (SPPS) method. Coupling one by one from (coupling).

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

Second, all amino acid raw materials used for peptide synthesis were those protected with Tmo, N-terminus, and Trt, Boc, t-Bu, and all residues removed from acid (Fmoc-Ala-OH, Fmoc-). Val-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Thr (t-Bu) -OH, Fmoc-Ser (t-Bu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Pro-OH, Fmoc-Met-OH). For special amino acids, Fmoc-Lys (Ac) -OH and Fmoc-Lys (Dde) -OH were used.

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

Fourth, it was washed in the order of DMF, MeOH, MC, DMF.

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

In addition, in order to synthesize intracellular molecular transport peptides with fluorescence markers (FITC), intracellular molecular transport peptides were synthesized by the above synthesis method, and finally, lysine (K, Lysine) was added to proceed with peptide synthesis. FITC was then bound to the free amine residue of lysine. The synthesized intracellular molecular transfer peptide-FITC peptide was separated from the resin and purified by HPLC, and then the molecular weight of the peptide was confirmed by mass spectrometer and lyophilized to obtain intracellular molecular transfer peptide fluorescent substance. Subsequently, the synthesized intracellular molecular transport peptide phosphor was dissolved in DMSO so as to have a concentration of 1 mM in a light shielding state, and then aliquoted in a small amount in a 1.5 mL centrifuge container and stored frozen until just before use.

Example 5 Intracellular Molecular Transport Peptide Phosphors (MTD-FITC) Using Flow Cytometry in Skin Keratinocytes in vitro  Cell Permeability Check

In order to verify cell permeability, 3 μM concentration of intracellular molecular transport peptide phosphor (MTD-FITC) was treated to human keratinocyte cells (Human Keratinocyte cell line, HaCaT cell, Order No. 300493, CLS cell line service, Germany). And incubated for 1 hour. The HaCaT cells were maintained in DMED (Dubelcco'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) , Incubated at 37 ° C. under a humidified atmosphere of 5% CO ₂ . After the incubation was terminated, trypsin was treated to remove free MTD-FITC exposed to the cell membranes of the protein-treated HaCaT cells and washed three times with refrigerated PBS.

MTD-FITC peptide was applied to a flow cytometer (FACS Calibur, Beckton-Dickinson, San Diego CA, USA). For each sample, cells (1 × 10 ⁴ ) were analyzed using CellQuest Pro cytometric analysis software, and each experiment was performed three or more times. Cell permeability of each of the intracellular molecular transport peptides applied to the present invention was quantitatively analyzed for cell permeation efficiency using the existing MTD-FITC as a control.

FIG. 5 shows the results of flow cytometry analysis. The cell influx of each intracellular molecular transport peptide treated in the cell was measured using the CellQues Pro cytometric analysis software. ) Cell influx efficiency was compared by relatively evaluating the change in fluorescence.

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

Example 6 Verification of Skin Cell Permeability of Intracellular Molecular Transport Peptide Phosphor (MTD-FITC) Using Confocal Microscopy

Intracellular Molecular Transport Peptides and PTD (Tat) Proteins Known to Be Permeable to Cells were Treated at a Concentration of 3 μM in HaCaT cells, immortalized human keratinocytes, Cat No. 300493, CLS, Germany 37 After incubation for 1 hour at ℃, it was visually observed using a confocal microscope (confocal microscopy, Nikon, Germany). The day before the experiment, HaCaT cells were incubated for 24 hours in a 12-well culture plate containing glass coverslips. 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), and 5% CO Incubated at 37 ° C. under a humidified atmosphere of ₂ . HaCaT cells were treated with scrambled peptide, MTD-FITC and PTD-FITC peptide for 1 hour at a concentration of 3 μM. One hour after the treatment, cells were fixed for 20 minutes with 4% paraformaldehyde solution (paraformaldehyde, PFA) at room temperature for observation.

The cells were washed three times with PBS for direct detection of internalized FITC-peptide and counterstained with 5 mM concentration of DAPI (4 ', 6-diamidino-2-phenylindole), a nuclear fluorescence staining solution. Was performed. After 10 minutes of DAPI staining, the cells were washed three times with PBS and 20 μl of mounting media were dropped onto the slides and observed to preserve the fluorescent label of the protein. Cells treated with the intracellular molecular transport peptides were transferred to the nucleus through DAPI staining for easy identification of intracellular delivery sites of FITC-peptides and confirmed cell permeability. The confocal microscope is a Normaski filter. The prototype of the cells was observed using FITC fluorescence and DAPI fluorescence with a filter suitable for each fluorochrome. As shown in Figures 6 to 8, it was confirmed that all of the intracellular molecular transport peptides to be applied to the present invention was clearly transmitted into the cell compared to the PTD (Tat) attached to the outside of the cell membrane, and improved intracellular molecular transport It was demonstrated that the peptide has good cell permeability for skin cells.

Example 7 Confirmation of Artificial Skin Tissue Permeability of Intracellular Molecular Transfer Peptides

For analysis of ex vivo tissue distribution ability, the conventional MTD (M18m, M173) and intracellular molecular transport peptide (M1018m, M1173A), which were conjugated with FITC, were selected and processed using EpiOral skin model (MatTek, MA, USA). , Visual observation using confocal microscopy (confocal microscopy, Carl Zeisse, Germany). EpiOral artificial tissue model was incubated at 37 ° C. for 15 hours under a humidified atmosphere of 5% CO _{2 in} a 12-well plate containing 0.5 mL of the test medium provided by MatTek. The next day it was exchanged with fresh medium, 40 μl of 50 μM peptide was treated on top of EpiOral artificial tissue and incubated at 37 ° C. under a humidified atmosphere of 5% CO ₂ for 5 hours. After fixing the EpiOral artificial tissue model in 4% paraformaldehyde (PFA) for more than 15 hours, cryosections (6 μm) were prepared using a Microm HM520 cryostat (thermo). It was produced on a glass slide. The prepared slides were stained with PBS buffer for 10 minutes and stained with cell nuclei in tissues by exposure to 0.5 mM DAPI solution for 5 minutes. The stained tissue was washed with PBS buffer three times again for 10 minutes, fixed on a slide using a mounting medium, and observed under a confocal microscope. The results are shown in FIG. 9.

As shown in Figure 9, no significant level of fluorescence was observed in the negative vehicle. However, in the case of the intracellular molecular transfer peptide used in the experiment it was confirmed that permeation occurs inside the EpiOral artificial tissue model over time. In addition, it was confirmed that the intracellular molecular transport peptides (1018m, and 1173A) were more effectively delivered to the deeper 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 peptides also have excellent tissue penetrating ability to skin tissue.

Example 8. Confirmation of Artificial Skin Tissue Permeability of Intracellular Molecular Transfer Peptides

For the analysis of stratum corneum penetrating ability in ex vivo tissues, an epiderm skin model (MatTek, MA, USA) was used to select and process intracellular molecular transport peptides (M1067) and Tat peptides conjugated to FITC. confocal microscopy, Carl Zeiss, Germany). EpiDerm skin models were incubated at 37 ° C. for 15 hours under a humidified atmosphere of 5% CO _{2 in} a 12-well plate containing 0.5 ml of the test medium provided by MatTek. The next day it was exchanged with fresh medium and 40 μl of 100 μM peptide was treated over EpiDerm skin and incubated at 37 ° C. under a humidified atmosphere of 5% CO ₂ for 24 hours. After fixing the EpiDerm skin model in 4% paraformaldehyde (PFA) for at least 15 hours, cryosections (6 μm) were prepared using a Microm HM520 cryostat, Thermo. This was put on a glass slide. The prepared slides were stained with PBS buffer for 10 minutes and stained with cell nuclei in tissues by exposure to 0.5 mM DAPI solution for 5 minutes. The stained tissues were washed with PBS buffer three times for 10 minutes, and then fixed with a mounting medium, and observed with a confocal microscope. The results are shown in FIG. 10.

As shown in FIG. 10, no significant level of fluorescence was observed in the negative vehicle, and in the case of the Tat peptide, Tat-FITC treated in the stratum corneum of the uppermost layer was found to remain impermeable to tissue. In the case of intracellular molecular transport peptide (M1067) it was confirmed that the permeation of the stratum corneum of the uppermost layer to significantly penetrate into the Epiderm skin model over time. This demonstrated that intracellular molecular transport peptides (M1067) pass through the stratum corneum, known as the maximum barrier to skin uptake, and possess good skin tissue permeability.

Example 9 Intracellular Molecular Transfer Peptides Using Animal Models in vivo  Skin Tissue Permeability Check

In order to verify the in vivo skin tissue permeability of intracellular molecular transport peptides, experiments were carried out using animal models. Intracellular Molecular Transfer Peptides (M1067), Tat-FITC and FITC with fluorescent markers (FITC) attached to sterile gauze 2.5 cm x 2.5 cm per individual in 8-week-old female ICR mice (Oriental Bio, Korea). 100 μg of each alone was added and fixed to the back of the mouse using Tegarderm (3M, USA). After 1, 3, 6, 12 hours, the mouse was sacrificed by the method of cervical distal bone, the skin of the drug-coated area was extracted, and the tissue was fixed in 4% paraformaldehyde for at least 24 hours, and then frozen to a thickness of 6 μm. Sections were obtained to produce slides. The prepared slides were washed with PBS buffer for 10 minutes, and then exposed to 0.5 mM DAPI solution for 5 minutes to stain the cell nuclei in tissues. The stained tissues were washed again with PBS buffer three times for 10 minutes, and then fixed by using a mounting medium, followed by confocal microscopy, shown in FIG. 11.

As shown in FIG. 11, no fluorescence signal was detected in the negative control group, and in the FITC alone and Tat-FITC treatment groups, the fluorescence signal was specifically observed in the stratum corneum, which is the top layer of the skin, so that skin permeation did not occur. It was confirmed that no. However, in the case of the intracellular molecular transfer peptide (M1067) used in the experiment, permeation occurs to the deep tissue of the skin as the application time passes, and the permeated intracellular molecular transfer peptide (M1067) passes through the stratum corneum and the skin tissue and skin It was confirmed that the hair follicle cells also specifically present in the cytoplasm.

Example 10 Confirmation of Whitening Efficacy of Intracellular Molecular Transfer Peptides with Skin Biological Activity

10-1. Synthesis of Intracellular Molecular Transport Peptide-coumaric Acid

A 20% piperidine / N-methylpyrrolidone solution was added to the intracellular molecular transfer peptides M1067 and M2067 synthesized in Example 4 coupled to the N-terminal amino acid to remove the Fmoc group. After washing with N-methylpyrrolidone and dichloromethane (coumaric acid, Sigma, USA) commercially available was coupled. After coupling, the mixture was washed several times with N-methylpyrrolidone and dichloromethane and dried with nitrogen gas. Trifluoroacetic acid: phenol: thioanisole: water: trifluoroacetic acid (phenol: thioanisole: water: triisopropylsilane) 90: 2.5: 2.5: 2.5: 2.5 (v / v) solution of 2 to The reaction was carried out for 3 hours to remove the peptide protecting group, and the peptide-bound coumalic acid was separated from the resin, and the peptide was precipitated with diethyl ether. 10% Pd / C was added to methanol to remove the benzyl group protecting the alcohol group bonded to carbon 9 of the coumalic acid, stirred at room temperature for about 1 hour under hydrogen, and then Pd using Celite. / C was removed and the filtrate was concentrated under reduced pressure. The intracellular molecular transport peptide-coumaric acid derivative thus obtained was purified with acetonitrile containing 0.1% trifluoroacetic acid as a gradient, followed by a purified reverse phase high performance liquid chromatography column (Zobax, C8 300Å, 21.1). mm × 25 cm) to obtain intracellular molecular transport peptide-coumaric acid derivatives in which coumaric acid is bound to M1067 or M2067 intracellular molecular transport peptides having the amino acid sequence of SEQ ID NO: 16 or 23, as shown in Equation 2 below. Synthesized.

[Equation 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. Confirmation of Tyrosinase Inhibitory Activity of Intracellular Molecular Transport Peptide-coumaric Acid

The inhibitory effect of tyrosinase activity was measured using the intracellular molecule transfer peptide-coumaric acid, a compound synthesized in Example 10-1. Tyrosinase was isolated and purified from mushrooms and purchased from Sigma, USA. Tyrosine, a substrate, was dissolved in 0.05 M potassium phosphate buffer (pH6.8) 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 coumalic acid was dissolved in ethyl alcohol at a concentration of 100 mg / mL, and diluted again to an appropriate concentration. 0.5 mL of each sample tyrosine solution and buffer solution were added to the test tube, and 0.5 mL of the compound solution was added thereto, and the resultant was allowed to stand at 37 ° C. for 10 minutes. Then, 0.05 mL of 2,000 U / mL tyrosinase was added thereto and reacted at 37 ° C. for 10 minutes. I was. In this case, 0.5 mL of distilled water was added instead of each compound. The test tube containing the reaction solution was taken out of the incubator and the absorbance was measured at a wavelength of 450 nm with a spectrophotometer to determine the inhibition rate of tyrosinase activity. The inhibition rate was calculated according to Equation 1 below, and the results are shown in FIG. 12.

[Equation 1]

% Inhibition = (A-B) / A X 100

In Equation 1,

A is the absorbance at 450 nm of no inhibitor added,

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 inhibitory activity was statistically significantly increased in the case of the compound conjugated with the intracellular molecular transport peptide of the present invention, compared to the coumalic acid known to have high tyrosinase inhibitory activity.

10-3. Inhibition of Intracellular Melanin Production by Intracellular Molecular Transport Peptide-Cumaric Acid

Intracellular melanocyte production was compared using the intracellular molecular transfer peptide-coumaric acid compound synthesized in Example 10-1. Mouse-derived melanoma cells (murine melanoma, B16F1, Korea Cell Line Bank KCLB No.80007) with DMEM (Dubelcco's modified eagle medium) medium containing 10% FBS (fetal bovine serum) 1 X 10 per well in a 6-well plate It was inoculated ^{into 5} pieces and cultured in the 5% CO _2, 37 ℃ until the cell is attached to at least about 80% of the well bottom. After incubation, the medium is removed, the sample is replaced with medium diluted to an appropriate concentration (1, 10, 100 μg / mL), and then incubated for 3 days with changing medium every day under 5% CO ₂ , 37 ° C. The day after the 3 day culture, the cells from which the medium was removed are washed with PBS (phosphated buffer saline), and the cells are recovered by treating with trypsin. The recovered cells were centrifuged at 10,000 rpm for 10 minutes, and then the supernatant was removed to obtain cell pellets. The cell pellets were dried at 60 ° C., and 200 μL of 1M sodium hydroxide solution containing 10% DMSO was dissolved at 60 ° C .. Obtain intracellular melanin. With this solution, the absorbance is measured at 490 nm with a microplate reader, and some of them determine the amount of melanin per protein by specifying the amount of protein. Calibrate by blank test.

As a result, as shown in Figure 13, in the case of a compound bound to the intracellular molecular transport peptide of the present invention compared to arbutin (coutinic acid) and arbutin (coutinic acid) known to inhibit the intracellular melanin production previously, intracellular It was confirmed that the melanin production inhibitory effect significantly increased.

10-4. Skin Safety Assessment of Intracellular Molecular Transport Peptide-coumaric Acid

In order to confirm the safety as a cosmetic raw material through the primary stimulation test using the human skin of the compound intracellular molecular transfer peptide-coumaric acid and intracellular molecular transfer peptide (M1067) synthesized in Example 10-1, Note) It was performed by requesting DERMAPRO. The clinical cosmetic composition was prepared as described below.

10-4-1.Human Skin Primary Stimulation Test

More than 30 subjects were selected who met the criteria for clinical trial selection and did not meet the exclusion criteria. Subjects were removed 48 hours after application of sample material to their backs. The test site was observed 30 minutes and 24 hours after removal. Skin assessments include the Frosch & Kligman Act (Frosch PJ and Kligman AM J Am Acad Dermatol, 1 (1): 35-41 (1979)) and The Cosmetic, Toiletry, and Frangrance Association (CTFA) guideline (The Cosmetic, Toiletry and Frangrance Association, Inc. Washington, DC 20005 (1981)) was evaluated on the basis of Table 1, and the average reactivity of 48 and 72 hours was compared, and the results were determined based on the average reactivity for each composition.

Table 1 Recording of patch test reactions

Symbol	Grade	Clinical Description
+	One	Slight erythema, either spotty or diffuse
++	2	Moderate uniform erythema
+++	3	Intense erythema with edema
++++	4	Intense erythema with edema & vesicles

TABLE 2 Human skin primary irritation test result (n = 30)

number	Test substance name	No. of responder	48 hours			72 hours			Responsiveness
			1+	2+	3+	1+	2+	3+	47h	72h	Mean
One	Contains 1% intracellular molecular transport peptide	0	-	-	-	-	-	-	0.0	0.0	0.0
2	Contains 1% intracellular molecular transport peptide-coumaric acid	0	-	-	-	-	-	-	0.0	0.0	0.0
3	Contains 1% coumalic acid	0	-	-	-	-	-	-	0.0	0.0	0.0
4	Squalane	0	-	-	-	-	-	-	0.0	0.0	0.0

As shown in Table 2, the intracellular molecular transport peptide and intracellular molecular transport peptide-coumaric acid were identified as substances in the hypoallergenic category in terms of human skin primary stimulation. As a result, it was proved that the intracellular molecular transport peptides can be safely used in the human body while maintaining the functions of the skin bioactive molecules through clinical trials of specialized test institutes. Giving.

10-4-2. Preparation of intracellular molecular transport peptide-coumaric acid-containing cosmetic composition

Essence compositions containing intracellular molecular transfer peptide-coumaric acid or intracellular molecular transfer peptide were formulated as follows with the composition of Table 3 above.

1 to 6 is added to the water-dissolving tank, dissolved completely while warming up to 70 ℃, and then put into the emulsifying tank. 7 to 11 is added to the oil-dissolving tank and dissolved completely while warming up to 70 ° C. The contents were cooled to 40 ° C., and then the raw materials 12, 13, and 15 were added to an emulsification tank, mixed, and the contents were cooled to room temperature to prepare a composition for skin whitening containing intracellular molecule transfer peptide-coumaric acid.

Comparative Example 1: 1 to 6 in the water melting tank and completely dissolved while warming up to 70 ℃ and put into the emulsion tank. 7 to 11 is added to the oil-dissolving tank and dissolved completely while warming up to 70 ° C. After the contents were cooled to 40 ° C., raw materials 12 to 14 were added to an emulsification tank, mixed, and the contents were cooled to room temperature to prepare a composition containing an intracellular molecular transfer peptide.

Comparative Example 2: 1 to 6 in the water melting tank and completely dissolved while warming up to 70 ℃ and put into the emulsion tank. 7 to 11 and 16 are added to the oil-dissolving tank, dissolved completely while warming up to 70 ° C, and then mixed in an emulsifying tank. After the contents were cooled to 40 ° C., raw materials 12 and 13 were added to an emulsification tank, mixed, and the contents were cooled to room temperature to prepare a composition containing kumaric acid.

TABLE 3 Intracellular Molecular Transport Peptide-coumaric Acid-Containing Essence Composition (Unit: Weight%)

No	Raw material	Example 10-1	Comparative Example 1	Comparative Example 2
One	Purified water	84.480	84.480	64.480
2	Alcohol	0.000	0.000	20.000
3	glycerin	5.000	5.000	5.000
4	Dipropylene glycol	3.000	3.000	3.000
5	Allatoin	0.100	0.100	0.100
6	Disodium ID	0.020	0.020	0.020
7	Olive oil	2.000	2.000	2.000
8	Capric / Capric Triglycerides	2.000	2.000	2.000
9	Sodium Acre-Reid / Sodium Aricdimethyl Taurate Copolymer	0.667	0.667	0.667
10	Isohexadecane	0.667	0.667	0.667
11	Polysorbate 80	0.667	0.667	0.667
12	Chlorphenesin	0.250	0.250	0.250
13	Methylparaben	0.150	0.150	0.150
14	Intracellular molecular transport peptides	0.000	1.000	0.000
15	Intracellular Molecular Transfer Peptides-coumaric Acid	1.000	0.000	0.000
16	Coumaric acid	0.000	0.000	1.000

Example 11 Confirmation of Wrinkle Improvement Efficacy of Intracellular Molecular Transport Peptides Conjugate with Skin Bioactive Substances

11-1. Synthesis of Intracellular Molecular Transport Peptide-acetylpentapeptide

To the M1067 peptide having the amino acid sequence of SEQ ID NO: 16 synthesized in Example 4 coupled to the N-terminal amino acid sequentially synthesized acetyl penta peptide (acetylated Lys Ther Ther Lys Ser) commercially used in the cosmetic industry , 20% piperidine / N-methylpyrrolidone solution was added to remove the Fmoc group, washed several times with N-methylpyrrolidone and dichloromethane, and dried with nitrogen gas. . Trifluoroacetic acid (phenol): phenol (phenol): thioanisole (thioanisole): water (water): triisopropylsilane (triisopropylsilane) 90: 2.5: 2.5: 2.5: 2.5 (v / v) The mixture was reacted for 2 hours to 3 hours to remove the peptide protecting group, the peptide was separated from the resin, and the peptide was precipitated with diethyl ether. The MTD peptide-acetylpentapeptide derivatives thus obtained were purified by reverse phase high performance liquid chromatography column (Zobax, C8 300Å, 21.1 mm X) with acetonitrile containing 0.1% trifluoroacetic acid as a gradient. 25 cm) was used to synthesize an acetyl pentapeptide derivative in which an acetyl pentapeptide was bound to an M1067 intracellular molecular transport peptide having the amino acid sequence of SEQ ID NO.

[Equation 3]

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

*: acetylated lysine

11-2. Inhibition of Intracellular Collagenase Activity of Intracellular Molecular Transport Peptide-acetylpentapeptide

The inhibitory effect of collagenase activity in human normal skin fibroblasts was tested using the intracellular molecule transfer peptide-acetylpentapeptide, a compound synthesized in Example 11-1. Intracellular collagenase activity inhibition was measured by the method of Baeer EA et al, J Invest Dermatol, 82 (2): 162-9, 1983, and 100% of the sample was not added. . Detailed experimental method is as follows. Human normal dermal fibroblasts were dispensed into 6-well plates for cell culture and inoculated with a certain number of cells, treated with intracellular molecule transfer peptide-acetylpentapeptides at a constant concentration and incubated for 24 hours, followed by 100 μl of culture solution. The amount of collagenase in the culture was measured using the method described in Matrix metalloproteinase-1 (MMP-1) human biotrak ELISA system (GEHealthcare, USA). In the ELISA method, 100 μl of the quantitative buffer solution 2 was placed in a 96-well plate, and 100 μl of the culture solution and the standard solution diluted to 1/10 were added thereto, and the cells were incubated at room temperature for 2 hours. Remove the culture medium from the well plate, wash three times with 400 μl of wash buffer, add 100 μl of antibody solution, react at room temperature for 2 hours, and wash three times with 400 μl of wash buffer. Then, 100 μl of Peroxidase conjugate solution was added and reacted at room temperature for 2 hours. After washing three times with 400 μl of phosphate buffer (PBS), 100 μl of TMB substrate was added and reacted at room temperature for 30 minutes. 100 μl of 1M sulfuric acid solution was added and measured by ELISA Reader at 450 nm. The standard solution was dissolved in Huamn pro MMP-1 by quantitative buffer, and diluted to 0, 3.13, 6.25, 12.5, 25, 50 ng / mL, respectively. Use it. Inhibition rate was calculated according to Equation 2, the results are shown in FIG.

[Equation 2]

% Inhibition = (A-B) / A X 100

In Equation 2,

A is the absorbance at 450 nm of no sample added,

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

As a result, as shown in Figure 14, it was confirmed that in the group treated with intracellular molecule transfer peptide-acetylpentapeptide significantly inhibit the activity of intracellular collagenase in a concentration-dependent manner.

11-3. Skin Safety Verification of Intracellular Molecular Transport Peptide-Acetylpentapeptide

In order to confirm the safety as a cosmetic raw material through the first stimulation test using the human skin of the intracellular molecular transfer peptide-acetylpentapeptide, which is the compound synthesized in Example 11-1, Was performed. The clinical cosmetic composition was prepared as described below.

11-3-1. Human skin primary irritation test

More than 30 male or female subjects aged 18 to 60 who met the criteria for clinical trial selection and did not qualify for exclusion were selected. Subjects were removed 48 hours after application of sample material to their backs. The test site was observed 30 minutes and 24 hours after removal. Skin assessments include the Frosch & Kligman Act (Frosch PJ and Kligman AM J Am Acad Dermatol, 1 (1): 35-41 (1979)) and The Cosmetic, Toiletry, and Frangrance Association (CTFA) guideline (The Cosmetic, Toiletry and Frangrance Association, Inc. Washington, DC 20005 (1981)) was evaluated based on the criteria in Table 4, and the average reactivity of 48 and 72 hours was compared and the results were determined based on the average reactivity for each composition.

Table 4 Recording of patch test reactions

Symbol	Grade	Clinical Description
+	One	Slight erythema, either spotty or diffuse
++	2	Moderate uniform erythema
+++	3	Intense erythema with edema
++++	4	Intense erythema with edema & vesicles

Table 5 Human skin primary irritation test result (n = 30)

number	Test substance name	No. of responder	48 hours			72 hours			Responsiveness
			1+	2+	3+	1+	2+	3+	47h	72h	Mean
One	Contains 0.1% intracellular molecular transfer peptide	0	-	-	-	-	-	-	0.0	0.0	0.0
2	Contains 0.1% of intracellular molecular transport peptide-acetylpentapeptide	0	-	-	-	-	-	-	0.0	0.0	0.0
3	0.1% Acetylpentapeptide	0	-	-	-	-	-	-	0.0	0.0	0.0
4	Squalane	0	-	-	-	-	-	-	0.0	0.0	0.0

As shown in Table 5 above, the intracellular molecular transport peptide and the intracellular molecular transport peptide-acetylpentapeptide were shown to be substances in the hypoallergenic category in terms of human skin primary stimulation. As a result, clinical trials of specialized test institutes demonstrated that intracellular molecular transport peptides can be safely used in the human body while maintaining the function of skin bioactive molecules. It can be said that the results indicate that the value of use is remarkably superior.

11-3-2. Preparation of Intracellular Molecular Transfer Peptide-Acetylpentapeptide-Containing Cosmetic Composition

Essence compositions containing intracellular molecular transfer peptide-acetylpentapeptide or intracellular molecular transfer peptide (M1067) were formulated as follows with the composition of Table 6.

Add 1 to 5 to the water-dissolving tank, dissolve completely while warming up to 70 ℃, and put it into the emulsifying tank. 6 to 10 is added to the oil-dissolving tank, dissolved completely while warming up to 70 ° C, and then mixed in an emulsifying tank. After the contents were cooled to 40 ° C., raw materials 11, 12, and 14 were added to an emulsification tank, mixed, and the contents were cooled to room temperature to prepare a composition for improving skin wrinkles containing intracellular molecular transfer peptide-acetylpentapeptide.

Comparative Example 1: 1 to 5 in the water melting tank and completely dissolved while warming up to 70 ℃ and put into the emulsion tank. 6 to 10 is added to the oil-dissolving tank, dissolved completely while warming up to 70 ° C, and then mixed in an emulsifying tank. After the contents were cooled to 40 ° C., raw materials 11 to 13 were added to an emulsifying tank, mixed, and the contents were cooled to room temperature to prepare a composition containing intracellular molecular transfer peptides.

Comparative Example 2: 1 to 5 in the water melting tank and completely dissolved while warming up to 70 ℃ and put into the emulsion tank. 6 to 10 is added to the oil-dissolving tank, dissolved completely while warming up to 70 ° C, and then mixed in an emulsifying tank. After the contents were cooled to 40 ° C., raw materials 11, 12, and 15 were added to an emulsification tank, mixed, and the contents were cooled to room temperature to prepare a composition containing acetylpentapeptide.

Table 6 Essence Composition Containing Intracellular Molecular Transport Peptide-acetylpentapeptide (Unit: weight%)

No	Raw material	Example 11-1	Comparative Example 1	Comparative Example 2
One	Purified water	Up to 100	Up to 100	Up to 100
2	glycerin	5.000	5.000	5.000
3	Dipropylene glycol	3.000	3.000	3.000
4	Allatoin	0.100	0.100	0.100
5	Disodium ID	0.020	0.020	0.020
6	Olive oil	2.000	2.000	2.000
7	Capric / Capric Triglycerides	2.000	2.000	2.000
8	Sodium Acre-Reid / Sodium Aricdimethyl Taurate Copolymer	0.667	0.667	0.667
9	Isohexadecane	0.667	0.667	0.667
10	Polysorbate 80	0.667	0.667	0.667
11	Chlorphenesin	0.250	0.250	0.250
12	Methylparaben	0.150	0.150	0.150
13	Intracellular molecular transport peptides	0.000	0.100	0.000
14	Intracellular Molecular Transfer Peptide-Acetylpentapeptide	0.100	0.000	0.000
15	Acetyl pentapeptide	0.000	0.000	0.100

Example 12 Confirmation of Transdermal Absorption of Cosmetic Composition Combining Intracellular Molecular Transfer Peptide and Skin Bioactive Molecules

Percutaneous absorption experiments were performed in accordance with published guidelines to confirm penetration of the material obtained in the present invention into skin tissue (commonly referred to as transdermal absorption) (Test Guideline 428: Skin absroption: in vitro Method, OECD, Paris, (2004), In Vitro Skin Absorption Test Guidelines, Korea Food and Drug Administration (2010)). Percutaneous absorption experiments were used by thawing frozen stored body skin (cadaver skin, Cat No. SK11122, Hans Biomed) in PBS warmed to 32 ℃. The thawed cadaveric skin has the epidermis upward (in the direction of the donor) between the donor and the receptor of a vertical diffusion cell (Logan FDC-6, Logan instrument Corp. Somerset, NJ, USA). Faced and the dermis mounted down (receptor direction). The receptor was then filled with PBS solution (phosphate-buffered saline, pH 7.4, 32 ° C.) and left for 1 hour to allow the cadaveric skin to equilibrate with the PBS solution. Thereafter, 1 mg of 1% DMSO was added to the intracellular molecular transfer peptide, intracellular molecular transfer peptide-coumaric acid, and intracellular molecular transfer peptide-acetylpentapeptipeptides prepared in Examples 4, 10-1, and 11-1, respectively. The solution was sufficiently dissolved in 1 mL of the contained PBS solution and applied to the epidermis (1.7 cm 3 coated area), and the donor was sealed with parafilm to prevent the sample from evaporating. After 24 hours, 0.2 mL of sample was taken from the receptor and mass spectrometer was used to determine the amount of intracellular molecular transport peptide, intracellular molecular transport peptide-coumaric acid, and intracellular molecular transport peptide-acetylpentapeptipeptide. Analyze and quantify the molecular weight as shown in Table 7 below.

TABLE 7

division	Skin transmittance (%)
MTD Peptides of Example 1	11.8
MTD Peptide-Cumaric Acid of Example 10	32.7
Coumaric acid of Comparative Example 2 of Example 10	17.7
MTD Peptide-Acetylpentapeptide of Example 11	14.2
Acetylpentapeptide of Comparative Example 2 of Example 11	0.5

As shown in Table 7 above, it was found that the intracellular molecular transport peptide alone had a relatively high skin permeability of 11.8%. In the case of the intracellular molecular transport peptide combined with a skin physiologically active substance, it was able to penetrate the stratum corneum of its own skin. Compared to the skin permeation rate of lyxic acid, the intracellular molecular transport peptide-bound coumalic acid was confirmed to have doubled the permeation rate. In addition, in the case of acetyl pentapeptide, which is difficult to penetrate the stratum corneum, it was confirmed that the skin permeability increased by 28 times when binding to the intracellular molecular transport peptide. Thus, in the transdermal absorption experiments on human skin, it was again proved that the permeability of the cosmetic composition in which the intracellular molecular transport peptide and the skin bioactive molecule were combined was remarkably excellent, thereby confirming that the industrial use value of the intracellular molecular transport peptide was significant. Could.

Example 13 Verification of Skin Cell Permeability of Intracellular Molecular Transport Peptides Conjugated with Skin Bioactive Substances

13-1. Synthesis of Intracellular Molecular Transport Peptide-Acetylhexapeptide Fluorescent Compounds

Intracellular molecular transport 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, coupled to the N-terminal amino acid After sequential synthesis of acetyl hexapeptide (acetyl-Glu Glu Met Gln Arg Arg), which is commercially used in the cosmetic industry, peptides were synthesized by adding lysine (K, lysine) and then free of lysine. FITC was bound to the amine residue. The synthesized peptide-FITC was removed by adding 20% piperidine / N-methylpyrrolidone solution to remove the Fmoc group and washed several times with N-methylpyrrolidone and dichloromethane. Dry with nitrogen gas. Trifluoroacetic acid (phenol): phenol (phenol): thioanisole (thioanisole): water (water): triisopropylsilane (triisopropylsilane) 90: 2.5: 2.5: 2.5: 2.5 (v / v) The mixture was reacted for 2 hours to 3 hours to remove the peptide protecting group, the peptide was separated from the resin, and the peptide was precipitated with diethyl ether. The intracellular molecular transport peptide-acetylhexapeptide fluorescent derivative thus obtained was purified with acetonitrile containing 0.1% trifluoroacetic acid as a gradient, followed by purified reverse phase high performance liquid chromatography column (Zobax, C8 300Å). , 21.1 mm X 25 cm) to the intracellular molecular transport peptide having an amino acid sequence of SEQ ID NO: 16 or 48 as shown in the following formula 4 acetyl hexapeptide and FITC combined intracellular molecular transport peptide-acetylhexa Peptide fluorescent derivatives were synthesized. Thereafter, the synthesized peptide-FITC was dissolved in DMSO so as to have a concentration of 1 mM in a light-shielded state, and then aliquoted in small amounts in a 1.5 ml centrifuge container and stored frozen until just before use.

[Equation 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. Intracellular Molecular Transport Peptide-Acetylhexapeptide Using Flow Cytometry in vitro  Check cell permeability

In order to verify cell permeability, intracellular molecule transfer peptide-acetylhexapeptide phosphor (MTD-AH-FTIC) at a concentration of 3 μM was applied to human keratinocyte cells (Human Keratinocyte cell line, HaCaT cell, Order No. 300493, CLS cell line). service, Germany) and incubated for 1 hour. The HaCaT cells were maintained in DMED (Dubelcco'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) , Incubated at 37 ° C. under a humidified atmosphere of 5% CO ₂ . After the incubation was terminated, trypsin was treated to remove free MTD-AH-FITC exposed to the cell membrane of the protein-treated HaCaT cells, and washed three times with cold-stored PBS.

The prepared MTD-AH-FITC peptide was applied to a flow cytometer (FACS Calibur, Beckton-Dickinson, San Diego CA, USA), and for each sample, cells (1 × 10 ⁴ ) were analyzed for CellQuest Pro cytometric analysis (CellQues Pro cytometric). analysis) The software was analyzed and each experiment was performed three or more times. Cell permeability of each intracellular molecule transfer peptide applied to the present invention was quantitatively analyzed for cell permeability efficiency using acetylhexapeptide itself, a skin bioactive substance as a control.

The results were compared with the fluorescence change of each experimental group by the geometric mean in the CellQues Pro cytometric analysis software as shown in FIG. 15 to show the cellular influx of the intracellular molecular transport peptides. As a result, it was confirmed that the cell inflow efficiency of the acetylhexapeptide to which the intracellular molecular transport peptide is bound shows a high cell inflow efficiency from 200% to up to 400% compared to the efficiency of the acetylpexapeptide itself.

13-3. Confirmation of Visible Skin Cell Permeability of Intracellular Molecular Transport Peptide-Acetylhexapeptide Using Confocal Microscopy

For direct detection of internalized intracellular molecular transport peptide-acetylhexapeptide fluorescent derivatives, the cells were treated at 3 μM concentrations in HaCaT cells, immortalized human keratinocytes, Cat No. 300493, CLS, Germany. After incubation for 1 hour at 37 ℃, it was visually observed using a confocal microscope (confocal microscopy, Nikon, Germany). The day before the experiment, HaCaT cells were incubated for 24 hours in a 12-well culture plate containing glass coverslips. 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), and 5% CO Incubated at 37 ° C. under a humidified atmosphere of ² . HaCaT cells were treated with scrambled peptides, MTD-FITC and PTD-FITC peptides for 1 hour at a concentration of 5 μM. Then, the cells were fixed for 20 minutes with 4% paraformaldehyde solution (paraformaldehyde, PFA) at room temperature for observation, and washed three times with PBS, followed by DAPI (4 ', 6-diamidino-) at 5 mM concentration. Counterstain was performed with 2-phenylindole). After 10 minutes of DAPI staining, it was washed three times again with PBS, and 20 μl of mounting medium was dropped and observed on the slide to preserve the fluorescent label of the protein. Cells treated with the intracellular molecular transport peptide derivatives were identified for delivery and permeability to the nucleus through DAPI staining for easy identification of intracellular delivery sites, and confocal microscopy was performed using a normaski filter. The prototype of the cells was observed, and FITC fluorescence and DAPI fluorescence were observed with a filter suitable for each fluorochrome. As a result, as shown in Figure 16, compared to the control using the acetyl hexapeptide itself, a skin bioactive substance, the transfer peptide, the acetyl hexapeptide to which the intracellular molecular transport peptide is bound, significantly improved the transfer capacity Confirmed.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

<110> PROCELL THERAPEUTICS INC.

<120> Transdermal delivery system of dermatological active ingredients

         using cellular transduction peptides

<130> PCT01614

<150> US 61 / 563,113

<151> 2011-11-23

<160> 56

<170> KopatentIn 2.0

<210> 1

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

MTD JO-018 amino acid sequence

<400> 1

Ala Ala Leu Ile Gly Ala Val Leu Ala Pro Val Val Ala Val

  1 5 10

<210> 2

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

MTD JO-067 Amino Acid sequence

<400> 2

Ala Ala Ala Pro Ala Val Ala Ala

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<210> 3

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<212> PRT

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<220>

MTD JO-103 Amino Acid sequence

<400> 3

Leu Ala Leu Pro Val Leu Leu Leu Ala

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<210> 4

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<212> PRT

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<220>

MTD JO-159 Amino Acid sequence

<400> 4

Ile Ala Ile Ala Ala Ile Pro Ala Ile Leu Ala Leu

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<212> PRT

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MTD JO-173 Amino Acid sequence

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Ala Val Ile Pro Ile Leu Ala Val Pro

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<212> PRT

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<223> MTD 18m Amino Acid Sequence

<400> 6

Pro Ala Ala Leu Ala Ala Leu Pro Val Ala Val Val Ala Val

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<212> PRT

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MTD 173A Amino Acid Sequence

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Pro Ala Val Ile Pro Ile Leu Ala Val

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<210> 8

<211> 42

<212> DNA

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<223> MTD JO-018 polynucleotide Sequence

<400> 8

gcggcgctga ttggcgcggt gctggcgccg gtggtggcgg tg 42

<210> 9

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<212> DNA

<213> Artificial Sequence

<220>

MT223 JO-067 Polynucleotide Sequence

<400> 9

gcggcggcgc cggcggtggc ggcg 24

<210> 10

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> MTD JO-103 polynucleotide Sequence

<400> 10

ctggcgctgc cggtgctgct gctggcg 27

<210> 11

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

MTD JO-159 polynucleotide sequence

<400> 11

aatgcgaatg cggcgaatcc ggcgaatctg gcgctg 36

<210> 12

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> MTD JO-173 polynucleotide Sequence

<400> 12

gcggtgaatc cgaatctggc ggtgccg 27

<210> 13

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> MTD JO-018m polynucleotide Sequence

<400> 13

ccggcggcgc tggcggcgct gccggtggcg gtggtggcgg tg 42

<210> 14

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> MTD JO-173A polynucleotide Sequence

<400> 14

ccggcggtga atccgaatct ggcggtg 27

<210> 15

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

MTD 1018 Amino Acid Sequence

<400> 15

Met Arg Ala Ala Leu Ile Gly Ala Val Leu Ala Pro Val Val Ala Val

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<213> Artificial Sequence

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MTD 1067 Amino Acid Sequence

<400> 16

Met Arg Ala Ala Ala Pro Ala Val Ala Ala

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<220>

MTD 1103 Amino Acid Sequence

<400> 17

Met Arg Leu Ala Leu Pro Val Leu Leu Leu Ala

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<212> PRT

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<220>

MTD 1159 Amino Acid Sequence

<400> 18

Met Arg Ile Ala Ile Ala Ala Ile Pro Ala Ile Leu Ala Leu

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<212> PRT

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<400> 19

Met Arg Ala Val Ile Pro Ile Leu Ala Val Pro

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MT223 1018m Amino Acid Sequence

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Met Arg Pro Ala Ala Leu Ala Ala Leu Pro Val Ala Val Val Ala Val

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<210> 21

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<212> PRT

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<220>

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<400> 21

Met Arg Pro Ala Val Ile Pro Ile Leu Ala Val

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<223> MTD 2018 Amino Acid Sequence

<400> 22

Met His Ala Ala Leu Ile Gly Ala Val Leu Ala Pro Val Val Ala Val

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MTD 2067 Amino Acid Sequence

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Met His Ala Ala Ala Pro Ala Val Ala Ala

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MTD 2103 Amino Acid Sequence

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Met His Leu Ala Leu Pro Val Leu Leu Leu Ala

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<220>

MTD 2159 Amino Acid Sequence

<400> 25

Met His Ile Ala Ile Ala Ala Ile Pro Ala Ile Leu Ala Leu

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<220>

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Met His Ala Val Ile Pro Ile Leu Ala Val Pro

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<212> PRT

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<223> MTD 2018 m Amino Acid Sequence

<400> 27

Met His Pro Ala Ala Leu Ala Ala Leu Pro Val Ala Val Val Ala Val

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<212> PRT

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<220>

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Met His Pro Ala Val Ile Pro Ile Leu Ala Val

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MT223 3018 Amino Acid Sequence

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Met Lys Ala Ala Leu Ile Gly Ala Val Leu Ala Pro Val Val Ala Val

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MT223 3067 Amino Acid Sequence

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Met Lys Ala Ala Ala Pro Ala Val Ala Ala

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Met Lys Leu Ala Leu Pro Val Leu Leu Leu Ala

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<220>

MTD 3159 Amino Acid Sequence

<400> 32

Met Lys Ile Ala Ile Ala Ala Ile Pro Ala Ile Leu Ala Leu

  1 5 10

<210> 33

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

MTD 3173 Amino Acid Sequence

<400> 33

Met Lys Ala Val Ile Pro Ile Leu Ala Val Pro

  1 5 10

<210> 34

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> MTD 3018m Amino Acid Sequence

<400> 34

Met Lys Pro Ala Ala Leu Ala Ala Leu Pro Val Ala Val Val Ala Val

  1 5 10 15

<210> 35

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

MTD 3173A Amino Acid Sequence

<400> 35

Met Lys Pro Ala Val Ile Pro Ile Leu Ala Val

  1 5 10

<210> 36

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

MTD 1018 polynucleotide sequence

<400> 36

atgagggcgg cgctgattgg cgcggtgctg gcgccggtgg tggcggtg 48

<210> 37

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> MTD 1067 polynucleotide Sequence

<400> 37

atgagggcgg cggcgccggc ggtggcggcg 30

<210> 38

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> MTD 1103 polynucleotide Sequence

<400> 38

atgaggctgg cgctgccggt gctgctgctg gcg 33

<210> 39

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

MTD 1159 polynucleotide Sequence

<400> 39

atgaggattg cgattgcggc gattccggcg attctggcgc tg 42

<210> 40

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

MTD 1173 polynucleotide Sequence

<400> 40

atgagggcgg tgattccgat tctggcggtg ccg 33

<210> 41

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> MTD 1018m polynucleotide Sequence

<400> 41

atgaggccgg cggcgctggc ggcgctgccg gtggcggtgg tggcggtg 48

<210> 42

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

MTD 1173A polynucleotide Sequence

<400> 42

atgaggccgg cggtgattcc gattctggcg gtg 33

<210> 43

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> MTD 2018 polynucleotide sequence

<400> 43

atgcacgcgg cgctgattgg cgcggtgctg gcgccggtgg tggcggtg 48

<210> 44

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

MT223 2067 polynucleotide Sequence

<400> 44

atgcacgcgg cggcgccggc ggtggcggcg 30

<210> 45

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

MTD 2103 polynucleotide Sequence

<400> 45

atgcacctgg cgctgccggt gctgctgctg gcg 33

<210> 46

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> MTD 2159 polynucleotide Sequence

<400> 46

atgcacattg cgattgcggc gattccggcg attctggcgc tg 42

<210> 47

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

MTD 2173 polynucleotide Sequence

<400> 47

atgcacgcgg tgattccgat tctggcggtg ccg 33

<210> 48

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> MTD 2018 m polynucleotide Sequence

<400> 48

atgcacccgg cggcgctggc ggcgctgccg gtggcggtgg tggcggtg 48

<210> 49

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> MTD 2173A polynucleotide Sequence

<400> 49

atgcacccgg cggtgattcc gattctggcg gtg 33

<210> 50

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

MTD 3018 polynucleotide sequence

<400> 50

atgaaggcgg cgctgattgg cgcggtgctg gcgccggtgg tggcggtg 48

<210> 51

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

MT223 3067 Polynucleotide Sequence

<400> 51

atgaaggcgg cggcgccggc ggtggcggcg 30

<210> 52

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

MTD 3103 polynucleotide Sequence

<400> 52

atgaagctgg cgctgccggt gctgctgctg gcg 33

<210> 53

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

MT223 3159 polynucleotide Sequence

<400> 53

atgaagattg cgattgcggc gattccggcg attctggcgc tg 42

<210> 54

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

MTD 3173 polynucleotide Sequence

<400> 54

atgaaggcgg tgattccgat tctggcggtg ccg 33

<210> 55

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> MTD 3018m polynucleotide Sequence

<400> 55

atgaagccgg cggcgctggc ggcgctgccg gtggcggtgg tggcggtg 48

<210> 56

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> MTD 3173A polynucleotide Sequence

<400> 56

atgaagccgg cggtgattcc gattctggcg gtg 33

Claims (21)

1. A composition for transdermal delivery comprising a skin physiologically active molecule to which an intracellular molecule transfer peptide is bound, wherein the peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 35.
2. The composition for transdermal delivery according to claim 1, wherein the composition is a cosmetic composition.
3. The composition for transdermal delivery according to claim 1, wherein the peptide is encoded from a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 36 to 56.
4. The composition for transdermal delivery according to claim 1, wherein the composition penetrates the stratum corneum.
5. The composition for transdermal delivery according to claim 1, wherein the skin bioactive molecule binds to the N-terminus, C-terminus or sock end of the intracellular molecular transport peptide.
6.  The composition for transdermal delivery according to claim 1, wherein the skin bioactive molecule binds the amino acids of the intracellular molecule transfer peptide in a reverse order.
7. The composition for transdermal delivery according to claim 1, wherein the binding is achieved by peptide bond or chemical bond.
8. 8. The group of claim 7 wherein the chemical bond is a group consisting of disulfide bonds, diamine bonds, sulfide-amine bonds, carboxyl-amine bonds, ester bonds and covalent bonds. A composition for transdermal delivery, characterized in that selected from.
9. According to claim 1, wherein the skin bioactive molecule is a composition for transdermal delivery, characterized in that selected from the group consisting of vitamins, retinoids and fatty acids.
10. The method of claim 9, wherein the vitamin is 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 Percutaneous delivery composition, characterized in that selected from the group consisting of vitamin D5, vitamin E and vitamin K.
11. 10. The composition for transdermal delivery according to claim 9, wherein the retinoid is selected from the group consisting of retinol, retinol mutant and synthetic analogues, retinal, trans, 9-cis, and 13-cis retinolic acid and etretinate. .
12. 10. The method of claim 9, wherein the fatty acid is for transdermal delivery, characterized in that selected from the group consisting of lauric acid, stearic acid, palmitic acid, undecylenic acid, paritoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid and erucic acid. Composition.
13. According to claim 1, wherein the skin bioactive molecule is a composition for transdermal delivery, characterized in that selected from the group consisting of anti-wrinkle agent, whitening agent, antioxidant and moisturizer.
14. The composition for transdermal delivery according to claim 13, wherein the anti-wrinkle agent is selected from the group consisting of dermal action growth factor peptide or protein, retinol, retinyl palmitate, adenosine and polyethoxylatedretinamide.
15. The composition for transdermal delivery according to claim 13, wherein the whitening agent is selected from the group consisting of a whitening peptide, niacinamide, a mulberry extract, arbutin, echyl ascormyl ether, licorice extract, ascorbyl glucoside, and magnesium ascorbyl phosphate. .
16. The method of claim 1, wherein the skin bioactive molecule is selected from the group consisting of coumaric acid (coumaric acid) or acetyl pentapeptide (Acetyl pentapeptide) and acetyl hexapeptide (Acetyl Hexapeptide).
17. The composition for transdermal delivery according to claim 1, wherein the composition is prepared in a formulation selected from the group consisting of emulsions, creams, essences, skins, liposomes, microcapsules, composite particles, shampoos and rinses.
18. A transdermal delivery system for delivering a skin bioactive molecule into skin cells, wherein the system binds and delivers an intracellular molecular transfer peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-35 with a skin bioactive molecule. System.
19. The transdermal delivery system of claim 18, wherein the peptide is encoded from a polynucleotide selected from the group consisting of SEQ ID NOs: 36-56.
20. A method of delivering a skin bioactive molecule into skin cells, the method comprising binding and delivering an intracellular molecular transport peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-35 with a skin bioactive molecule Characterized in that, the method.
21. A method of delivering a skin bioactive molecule into the stratum corneum, the method comprising the step of combining and delivering an intracellular molecular transfer peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 35 with a skin bioactive molecule. Characterized in that, the method.

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