WO2016006744A1

WO2016006744A1 - Peptide-sirna complex for transdermal delivery using intracellular molecular transport peptide, and use thereof

Info

Publication number: WO2016006744A1
Application number: PCT/KR2014/006278
Authority: WO
Inventors: 이병규; 신기덕; 김한수; 윤진아; 라포조그래커; 들르브아시드릭; 유어뱅일세; 덴자데니얼
Original assignee: 주식회사 프로셀테라퓨틱스; 인스티튜트 큐리
Priority date: 2014-07-11
Filing date: 2014-07-11
Publication date: 2016-01-14

Abstract

The present invention relates to a method for effectively delivering an siRNA, which encodes a material regulating a biofunction, into an organism by using an intracellular molecular transport peptide. Specifically, the composition in which an siRNA, which cannot readily permeate into a cell due to the molecular weight size thereof and a characteristic of having an excessive negative charge, and a peptide having remarkable intracellular molecular transport ability are chemically fused, does not exhibit toxicity to skin cells, can be effectively used for delivering an siRNA, which can effectively inhibit a Kif13A gene playing an important role in melanin formation, into skin cells, and can be formulated into various forms, and thus can be useful as a differentiated material for a functional whitening cosmetic material.

Description

Peptide-SIRNA Complexes for Transdermal Delivery Using Intracellular Molecular Transfer Peptides and Uses thereof

The present invention relates to a peptide-siRNA complex for transdermal delivery using an intracellular molecular transfer peptide and its use, and more particularly, by covalent bonding of intracellular molecular transfer peptide to Kif13A-siRNA that inhibits melanogenesis. It relates to a composition for transdermal delivery comprising the formed complex and its use.

Generally, the skin is composed of epidermis, dermis and subcutaneous fat, and is a very important organ that plays various roles such as protective function, barrier function, temperature control function, excretory function and respiratory function (Proksch E, Brandner JM, Jensen JM. Exp Dermatol. 17 (12): 1063-72. 2008). The stratum corneum of the epidermis is the outermost part of the skin and plays an important role in preventing moisture loss, mechanical stress and infections outside the skin (Sotiropoulou PA, Blanpain C. Cold Spring Harb Perspect Biol. 4: a008383 2012).

The color of this skin is fundamentally determined by melanin (Nesterov A, Zhao J, Minter D, Hertel C, Ma W, Abeysinghe P, Hong M, Jia Q. Chem Pharm Bull. 56 (9): 1292-96. 2008). In particular, the difference in skin color by race is indicated by the ability of melanocytes in melanocytes and the number, maturity, and presence of melanosomes to the epidermis. In other words, melanosomes are generally surrounded by a membrane to form agglomerative melanosomal complexes in white epidermal cells. However, in melanosomes, melanosomes are scattered and appear dark. In black, genetically more melanin is produced than in white, and the type of melanin produced is higher in eumelanin, and more melanin is spread in keratinocytes. Looks black (Riley PA. Int J Biochem Cell Biol. 29: 1235-39. 1997).

Melanin is produced in melanocytes in the basal layer of epidermal tissue, which accounts for about 5% of the cells in the epidermis, and some are mixed in the middle of keratinocytes. Structurally, it has a dendritic protrusion, which extends to the periphery to transport the melanosomes.

Melanosomes are melanin-filled membranous granules that move melanin to the end of dendritic processes. According to the morphological criteria, it develops in four stages (maturation). If there is a lot of eumelanin, it is oval dark brown, and if there is a lot of peomelanin, it becomes yellowish red. In the first stage (Stage 1), a pre-melanosome in the form of nonpigmented vacuole, which is derived from an endosomal system, is formed, and in the second stage (Stage 2), it is free. In the third stage (Stage3), melanin begins to accumulate in the melanoma, and in the final stage (Stage 4), it develops into fully mature melanosomes (Van Den Bossche). K, Naeyaert JM, Lambert J. Traffic. 7. 769-78. 2006). In order to form mature melanosomes in stages 3 to 4, an effector for synthesizing tyrosinase and tyrp1 (tyrosinase-related protein1) from the Golgi and moving them to pre-melanosomes is required. (Raposo G, Marks MS. Nat Rev Mol Cell Biol. 8: 786-707, 2007), although little is known about the mechanism of this migration, adhesions that serve to support enzymes in transport vesicles Protein complex (AP) is required, and adhesion protein complexes are known to be classified into four types (AP-1, AP-2, AP-3, AP-4) (Bonifacino JS, Glick BS.Cell. 116: 153-166. 2004). Adhesion protein complex works with Kif13A (kinesin superfamily protein 13A) to allow melanin to be produced in melanosomes through a three-step process. In the first process, AP-1 reacts with a signal of the cytoplasmic region of the four kinds of adhesion protein complexes, causing Tyrp1 in the melanosome to move to the endosome. In the second step, AP-1 reacts with Kif13A, forming a tube in the endosome so that Kif13A can move the endosomes along the microtubule to the periphery of the cell and make contact with the melanosome. In the third step, the tube-formed endosome is conjugated with the melanosome, and Tyrp1, which is required for melanin formation, is transferred to the melanosome (Delevoye C, Hurbain I, Tenza D, Sibarita J, Uzan-Gafsou S, Ohno). H, Geert W, Verkleij AJ, Salamero J, Marks MA, Raposo G. J Cell Biol. 187: 247-264. 2009).

Melanin is a type of amino acid tyrosine that undergoes oxidation and is a synthetic pigment. It is produced by the addition of tyrosine or by the condensation of tyrosine and cysteine. Mainly involved are tyrosinase and tyrosinase-related protein-1 (Tyrp-1), which converts tyrosine into dopa (DOPA, dihydroxyphenylalanine) and dopaquinone (dopaquinone). To promote this. The resulting dopaquinone passes through two biosynthetic pathways that are synthesized as eumelanin and pheomelanin (US20120321575A1), eumelanin appears as a brown to black pigment, and pheomelanin (pheomelanin) It appears as a yellow to red pigment. This melanin protects the skin from UV rays and neutralizes free radicals produced by byproducts of various inflammatory conditions.However, when melanin is overproduced as a defense against UV rays, it is continuously released out of the skin. The spots form on the skin, or the skin becomes black.

Since it is not cosmetically good, various whitening methods for preventing excessive deposition of melanin on the skin and reducing the pigment content in the skin have attracted attention. Its main methods include UV protection, rapid bleaching of melanin pigment, blocking the transfer of biosynthetic melanin to keratinocytes, and inhibiting tyrosinase activity, a rate determining step of melanin biosynthesis. How to block the signal transduction that induces tyrosinase activity and biosynthesis promotion from the cell membrane of melanocytes, and administers a substance that is specifically toxic to melanocytes (Son KH, Heo MY.Int). J Cosmeti Sci. 35: 9-18. 2013).

However, the stratum corneum of the skin is the natural constituent of the keratinocytes (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 of pH around 5, the delivery of physiologically active substances through the skin, in particular the stratum corneum is subject to various restrictions due to the structural and physical properties of the skin. That is, in order for the skin bioactive substance to penetrate the stratum corneum barrier, the molecular weight must be less than 1,000 and possess lipophilic properties (Metha RC, Fitzpatric RE. Dermatol Ther. 20: 350-359. 2007).

Therefore, various methods for promoting the percutaneous permeability of such skin bioactive substances have been studied, and these methods can be largely divided into physical, biochemical, and chemical methods. The physical method is mainly a method of permeating the material using electroporation, sonophoresis, and thermal ablation. The biochemical method uses a precursor to activate and activate the active ingredient during or after the skin. It is converted into a form of a component that can be expressed to show activity after skin penetration. Chemical methods are the most widely used methods in the industry, and carrier-mediated delivery systems in which a variety of bases, surfactants, solvents, and fatty acids are formed in a complex containing mainly bioactive molecules. systems are 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 peptide-based transport domains discovered were HIV-Tat, which was established in 1988 by 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), respectively, indicate that the transcriptional protein Tat of HIV-1, a virus that causes acquired immune deficiency syndrome (AIDS), penetrates the cell membrane and trans-activates viral genes. Found. 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)).

Next, RQIKIYFQNRRMKWKK consisting of 16 amino acids 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)), biofilm translocation sequence Peptides based on (membrane translocating sequence, MTS) or signal sequences are also known as representative peptide domains. These sequences are recognized by receptor proteins that help transfer the newly synthesized protein by RNA to the biofilm of the appropriate intracellular organelles in vivo, and the biofilm potential sequence coupled with the nuclear localization signal (NLS) Several types of cells are known to accumulate in the nucleus through the cell membrane (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)). However, when cell membrane-penetrating peptides are derived from viruses such as Tat or VP22, there is a possibility of inducing an immune response in vivo. Therefore, studies are underway to minimize immunogenicity mainly by developing new membrane-penetrating peptides 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. The exact answer to whether the carrier's intracellular transport mechanism is endocytosis has not been answered, but 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 studies have reported that TAT-proteins do not directly penetrate cell membranes, but accumulate inside endosomes, which are formed by strongly inducing electrostatic interactions with the cell surface and causing inclusions. It has been suggested that Tat-proteins are easily degraded by proteolytic enzymes present in lysosomes by fusion with lysosomes present in (Trabulo S, Cardoso AL, Mano M, De Lima MC. 961-993. 2010). 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 deliver the TAT-effective substance to the target in the cytoplasm and deliver the active ingredient to a level that can exhibit the required activity. To this end, high concentrations of treatment are required, and it has been reported that it is not possible to sufficiently obtain the expected effects of transport sequences such as TAT and the transport of bioactive substance complexes (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)).

In recent studies, carrier-mediated delivery systems have metastases through one or more mechanisms, and if the transporter is a macromolecule, causes energy-dependent macropinocytosis, and small molecules associated with the cell membrane permeable peptide or the cell membrane permeable peptide are electrostatic. It is known to have an energy-independent transmission mechanism by attraction or hydrogen bonding. This suggests that direct contact of proteoglycans and cell membrane permeable peptides on the cell membrane or cell membranes is essential for the success of intracellular delivery, such as β-cyclodextrin treatment, cytokalcin-D treatment, or Dybk44A (dominant-negative dynamine) expression. It can be seen that lipid raft-mediated macrophage action is the main mechanism (Wadia JS, Stan RV, Dowdy SF. Nat Med. 2004 Mar; 10 (3): 310-5.). As such, known peptide-mediated delivery systems with a high content of basic amino acids remain controversial about the cellular influx mechanism (F. Milletti, Drug Discovery Today 17: 850-860 (2012)). In the case of existing developed peptides, there have been no examples showing that it is possible to transmit to skin cells through the stratum corneum.

However, in 2000, a new cell membrane permeable peptide, the Macromolecule Transduction Domain (MTD), which has improved intracellular delivery efficiency and differs in structure and electrostatic properties, was developed. Technology (Macromolecule Intracelular Tranduction Technology (MITT) 'and its macromolecular transduction domain can provide a solution to the transmission limits of these peptides. The macromolecular transport domain (MTD) exhibits a completely different mechanism from those of transport peptide carriers with high levels of TAT, phenyratin, and other basic amino acids, and is characterized by little incorporation and energy required for intracellular transport. . In addition, since the rigidity and integrity of the cell membrane act as an important factor of cell membrane permeation, direct interaction with the cell membrane is important, and compared with the conventional cell membrane permeable peptides TAT and phenitratin. Cargo materials such as low-molecular weight compounds, peptides and proteins have high delivery efficiency and cell-to-cell delivery.

Accordingly, the present inventors have optimized the macromolecular transport domain (MTD), selected a transport domain having better cell permeability, and applied it to the intracellular molecular transport peptide of the present invention. By covalently binding a skin bioactive molecule having a molecular weight of 1,000 or more, such as Kif13A-siRNA, which induces a whitening effect to such intracellular molecular transport peptides, it can be effectively delivered into the stratum corneum or into skin cells. On the basis of this, the present invention has been completed.

The present invention is designed to efficiently introduce Kif13A-siRNA into melanocytes, which are difficult to deliver through the skin due to the molecular weight and the intrinsic properties of the stratum corneum. As described above, the covalent linkage between intracellular transport peptides and Kif13A-siRNA To provide a composition for transdermal delivery comprising a complex made through, a transdermal delivery system using a siRNA covalently bonded to the peptide, and a method for delivering an intracellular molecular transfer peptide to the skin cells or the stratum corneum of the covalently bonded siRNA The purpose.

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 complex in which intracellular molecular transport peptides and siRNAs are covalently linked, wherein the peptides comprise an amino acid sequence as set forth in SEQ ID NO: 5.

In one embodiment of the invention, the peptides are characterized in that they are encoded from the polynucleotide sequence set forth in SEQ ID NO: 6.

In another embodiment of the present invention, the siRNA is characterized by inhibiting melanin formation by inhibiting the expression of Kinesin superfamily protein 13A (Kif13A).

In another embodiment of the present invention, the siRNA is characterized by consisting of a sense RNA strand consisting of SEQ ID NO: 1 and an antisense RNA strand consisting of SEQ ID NO: 2.

In another embodiment of the present invention, the covalent bond is characterized in that the non-degradable bond or degradable bond.

In another embodiment of the invention, the non-degradable bond is characterized in that the amide bond or phosphate bond.

In another embodiment of the present invention, the degradable bond is characterized in that selected from the group consisting of disulfide bonds, acid decomposable bonds, ester bonds, anhydride bonds, biodegradable bonds and enzyme degradable bonds.

In another embodiment of the invention, the covalent bond is Thioether linker, S-HyNic (6-hydrazino-nicotinic acid) / 4-FB (4-formylbenzoate) linker and 4FB (N-succinimidyl-4-formylbenzamide) / hydroxylamine And a linker selected from the group consisting of linkers.

In another embodiment of the present invention, the covalent bond is characterized in that made by 4FB (N-succinimidyl-4-formylbenzamide) / hydroxylamine linker.

In another embodiment of the present invention, the composition is characterized in that the cosmetic composition.

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, serums, liposomes, microcapsules, composite particles, shampoos and rinses.

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

The present invention also provides a system for transdermal delivery of siRNA into skin cells, wherein the system covalently transfers intracellular molecular transfer peptides consisting of the amino acid sequence of SEQ ID NO: 5 with siRNA.

In one embodiment of the present invention, the siRNA is characterized by consisting of a sense RNA strand consisting of SEQ ID NO: 1 and an antisense RNA strand consisting of SEQ ID NO: 2.

Furthermore, the present invention provides a method for delivering siRNA into skin cells, the method comprising covalently delivering an intracellular molecular transfer peptide consisting of the amino acid sequence of SEQ ID NO: 5 with siRNA. do.

In addition, the present invention provides a method for delivering siRNA into the stratum corneum, the method comprising the step of covalently delivering the intracellular molecular transport peptide consisting of the amino acid sequence of SEQ ID NO: 5 and siRNA to provide.

In addition, the present invention provides a cosmetic method comprising the step of whitening the skin by topically applying to the epidermis by covalently bonding the intracellular molecular transport peptide consisting of the amino acid sequence of SEQ ID NO: 5 and siRNA.

The composition for transdermal delivery of the present invention binds peptides with excellent molecular transport ability in vivo to Kif13A, which is not easily permeable to the skin stratum corneum due to the size of the molecular weight and high level of negative charge, thereby allowing Kif13A in the stratum corneum and melanocytes. Can be delivered effectively. In addition, it does not cause cytotoxicity in comparison with the conventional liposome transmission method. Therefore, it can be safely and effectively reduce the production of melanin and can be formulated in various forms can be usefully used as a whitening functional cosmetic raw material.

1 is a photograph showing that the MTD2173A peptide, Kif13A and con siRNA are covalently bonded.

Figure 2 shows the results of measuring the cell proliferation of MTD2173A-siRNA.

Figure 3 shows the results of measuring the cytotoxicity to siRNA transporter using MTD2173A-siRNA and liposomes.

FIG. 4 shows the results of transfecting siRNA into cells using liposomes (A) and intracellular delivery using MTD2173A-siRNA in real-time PCR experiments.

5 shows the results of (A) transfecting siRNA into cells using liposomes and (B) Western blot of intracellular delivery using MTD2173A-siRNA to inhibit Kif13A protein expression. It is shown.

FIG. 6 shows the results of transfecting siRNA into cells using (A) liposomes and (B) inhibiting melanin production in cells using MTD2173A-siRNA.

Figure 7 shows the inhibition of beta galactosidase activity of mouse organ sections administered with M1173A-LacZ-siRNA-Cy3, M2173A-LacZ-siRNA-Cy3, M3173A-LacZ-siRNA-Cy3 and M1018m-LacZ-siRNA-Cy3. The results observed with a focus microscope are shown.

Figure 8 shows the reaction of mouse organ sections administered with M1173A-LacZ-siRNA-Cy3, M2173A-LacZ-siRNA-Cy3, M3173A-LacZ-siRNA-Cy3 and M1018m-LacZ-siRNA-Cy3 overnight in X-gal staining solution. After staining the tissue by hematoxylene eosin staining and showing the results of beta galactosidase activity observed.

It is known that low molecular synthetic compounds or natural compounds can be easily delivered into cells, and macromolecules such as proteins, peptides and nucleic acids have a limitation in that they are difficult to penetrate into cell membranes having a double lipid membrane structure because of their molecular weight. Moreover, the 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 such a stratum corneum, the molecular weight must be as small as 1,000 or less, and it must be possible to possess lipophilic properties. Therefore, various effective materials used as cosmetic raw materials are known to have extremely low permeation efficiency of low molecular weight materials and lower permeation efficiency of high molecular weight materials due to the intrinsic properties of the stratum corneum, which actually constitutes the skin barrier. Such small molecules and macromolecules may use the 'Macromolecule Intracellular Transduction Technologiy (MITT)' as a method for enhancing skin permeation efficiency.

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 improved MTD peptides that efficiently deliver biologically active molecules into cells, and by using them, transdermal delivery compositions, transdermal delivery systems, which can more efficiently deliver siRNA into skin cells. Provided are methods and cosmetic methods for delivering siRNA into skin cells or stratum corneum.

Hereinafter, the present invention will be described in detail.

The present invention provides a transdermal delivery system composition comprising a complex in which intracellular molecular transport peptides and siRNAs are covalently linked, wherein the peptides comprise an amino acid sequence as set forth in SEQ ID NO: 5.

In the present invention, the peptide is a peptide capable of mediating the delivery of a biologically active molecule into cells, and is a peptide that exhibits excellent cell permeability compared to the MTD peptide described in Korean Patent Application Laid-Open No. 10-2009-0103957. Referred to it as "intracellular molecular transport peptide."

The peptide may have an amino acid sequence of SEQ ID NO: 5, but is not limited thereto. In addition, the amino acid sequence of SEQ ID NO: 5 may be encoded by the polynucleotide sequence of SEQ ID NO: 6, but is not limited thereto.

In the present invention, siRNA has an effect of inhibiting the expression of Kif13A (kinesin superfamily protein 13A) to inhibit melanin formation, more specifically, consisting of a sense RNA strand consisting of SEQ ID NO: 1 and an antisense RNA strand consisting of SEQ ID NO: 2 It may be, but is not limited thereto.

Kif13A-siRNA, which exhibits whitening activity, inhibits Kif13A expression, and thus the endosome does not migrate to the periphery of the cell and thus does not form an endothelial tube capable of contact with the melanosome. Thus, Tyrp1, which is required for melanin formation, may not migrate to melanosomes and may inhibit melanin formation (Delevoye C, Hurbain I, Tenza D, Sibarita J, Uzan-Gafsou S, Ohno H, Geert W, Verkleij AJ, Salamero J, Marks MA, Raposo G. J Cell Biol. 187: 247-264. 2009).

Based on this mechanism, the treatment of Kif13A-siRNA on highly pigmented human melanoma cells (MNT-1) cells significantly reduced the number of mature melanosomes and decreased melanin content by 40% compared to the control group. Delevoye C, Hurbain I, Tenza D, Sibarita J, Uzan-Gafsou S, Ohno H, Geert W, Verkleij AJ, Salamero J, Marks MA, Raposo G. J Cell Biol. 187: 247-264. 2009).

In the present invention, the intracellular molecular transport peptides and siRNAs are preferably bound by covalent bonds. The binding between the peptide and the siRNA may be made by covalent or non-covalent bond, since the non-covalent bond between the negatively-charged siRNA and the positively-charged peptide has a problem in that a precipitate is formed due to neutralization of the conjugate, thereby inactivating the function of the peptide. to be. The covalent bonds may be either non-degradable bonds or degradable bonds. At this time, the non-degradable bond is an amide bond or phosphate bond, and the degradable bond is a disulfide bond, an acid decomposable bond, an ester bond, anhydride bond, a biodegradable bond, or Enzyme-degradable bonds, and the like, but are not limited thereto. Moreover, the covalent bonds are Thioether linker, S-HyNic (6-hydrazino-nicotinic acid) / 4-FB (4-formylbenzoate) linker and 4FB (N-succinimidyl-4-formylbenzamide) /

It is preferably made by a linker selected from the group consisting of hydroxylamine linkers, more preferably 4FB (N-succinimidyl-4-formylbenzamide) / hydroxylamine linkers.

The compositions of the present invention can be usefully used to improve, prevent, prevent or treat whitening of many cosmetic or dermatological compositions, in particular skin intended for topical application.

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 one embodiment of the present invention, in order to deliver the intracellular non-permeable whitening functional Kif13A siRNA into the cell, a complex was prepared in which the KD1373 siRNA was linked to the MTD2173A peptide, and treated with MTD2173A-Kif13A siRNA to the cell, followed by Real time PCR. Western blot was used to observe the gene expression and protein expression of the target gene Kif13A. As a result, MTD2173A-Kif13A siRNA prepared by using MTD2173A was observed by observing the effect of reducing the mRNA expression and protein expression of the target gene to a level similar to the method using liposomes, which is a method of delivering siRNA into cells. It proved to be an intracellular delivery method.

In another embodiment of the present invention, MTD2173A-Kif13A siRNA was used to observe cell safety through cell proliferation assay and cell cytotocicity experiment. As a result, MTD2173A-Kif13A siRNA did not inhibit the proliferation of the cells even at the treatment concentration of up to 1.5 μM, it was found that no cytotoxicity.

In addition, in another embodiment of the present invention, as a result of treatment of MTD2173A-Kif13A siRNA to human-derived melanocytes (MNT1), the intracellular delivery of Kif13A siRNA using Lipofectamine, which has previously been used to inhibit melanin production By confirming the efficacy is superior to the method, it was confirmed that the MTD is a carrier of a new siRNA. Melanin inhibitory effect of MTD2173A-Kif13A siRNA in the cell demonstrated that the foreign material can be used as a cosmetic raw material with whitening efficacy.

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.

[실시예]EXAMPLE

실시예 1. 세포 내 분자 전송 펩티드 합성Example 1. Intracellular Molecular Transport Peptide Synthesis

Synthesis of intracellular molecular transport peptides linked amino acids one by one from the C-terminus using the conventional Fmoc solid phase peptide synthesis (SPPS) method.

More specifically, first, the C-terminal first amino acid of the peptide is Fmoc-Lys using HCTU (5-Chloro-1- [bis (dimethylamino) methylene] -1 H -benzotriazolium 3-oxide hexafluorophosphate) concentrate on a solid support. (e-ivDde) -OH was added.

Next, all amino acid raw materials used for peptide synthesis were those protected by Trt, Boc, t-Bu, etc., in which N-term was protected by Fmoc, and all residues were 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 (e-ivDde) -OH were used.

Next, Fmoc was removed by reacting twice at room temperature for 5 minutes using piperidine dissolved in DMF (Dimethylformamide) at a concentration of 20%.

Next, after washing with DMF, methyl alcohol (MeOH), methyl chloride (MC), DMF in order, O- (1H-6-Chlorobenzotriazole-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) The hydroxylamine linker was attached using a concentrated material.

Next, the peptide synthesized using TFA / EDT / Thioanisole / TIS / H2O = 90 / 2.5 / 2.5 / 2.5 / 2.5 (TFA = trifluoroacetic acid, EDT = 1,2-ethanedithiol, TIS = triisopropylsilane) was isolated from the resin. And residue protecting groups.

Finally, after purification by high performance liquid chromatography (HPLC) system, MS confirmed and freeze-dried to finally obtain the intracellular molecular transport peptide (hereinafter referred to as 'MTD2173A').

As a result, specific information of the MTD2173A obtained in Example 1 is as follows:

MTD _2173A

Met His Pro Ala Val Ile Pro Ile Leu Ala Val-hydroxylamine (SEQ ID NO: 5)

That is, the MTD2173A has a hydroxylamine linker attached to the intracellular molecular transport peptide having the amino acid sequence of SEQ ID NO: 5.

실시예 2. MTD2173A와 siRNA 공유결합Example 2. Coupling siRNA with MTD2173A

In order to covalently bond the MTD2173A peptide obtained in Example 1 with Kif13A siRNA and con siRNA, the experiment was carried out as follows. That is, the covalent bond between the MTD2173A peptide and siRNA obtained in Example 1 was covalently bonded using 4FB-siRNA and hydroxylamine-peptide according to the Oligonucleotide / peptide conjugation method of Innopep.

More specifically, first, S-4FB (N-succinimidyl-4-formylbenzamide) is dissolved in DMF solution, and the S-4FB is conjugated to 20 times the siRNA and siRNA concentrations (conjugation buffer, 100 mM sodium phosphate, 150 mM sodium). chloride, pH 6.0) and allowed to react at room temperature for 2 hours. Thereafter, desalting purification was performed for exchanging buffer solution with excess S-4FB present in the reaction mixture.

Next, the prepared 4FB-modified siRNA and hydroxylamine peptide having a molar ratio of 5 times thereof were mixed in a TurboLink Catalyst Buffer (10 mM Phosphate, 15 mM Sodium Chloride, 10 mM aniline, pH 6.0) solution. And reacted at room temperature for 2 hours.

Finally, the excess peptide was removed from the reactants using ultrafiltration (diafiltration, Sartorius Vivaspin), and the reaction result was confirmed with a 2% NuSieve GTG agarose gel as shown in FIG. Drying completed the preparation of the peptide-siRNA complex.

On the other hand, specific information of the siRNA used in the above embodiment is shown in Table 1 below.

Table 1

division	Sequence information
sense Kif13A siRNA	S-4FB-GGCGGGUAGCGAAAGAGUA dTdT (SEQ ID NO: 1)
Antisense Kif13A siRNA	UACUCUUUCGCUACCCGCC dAdG (SEQ ID NO: 2)
sense con siRNA	S-4FB-CCUACGCCACCAAUUUCGU dTdT (SEQ ID NO: 3)
Antisense con siRNA	ACGAAAUUGGUGGCGUAGG dTdT (SEQ ID NO: 4)

실시예 3. 세포 내 분자 전송 펩티드-siRNA 복합체의 세포 안전성 확인Example 3. Confirmation of Cell Safety of Intracellular Molecular Transport Peptide-siRNA Complexes

3-1. 세포 증식능 실험3-1. Cell proliferation test

In order to confirm the cellular safety of the intracellular molecular transport peptide-siRNA complex prepared by Example 2, the experiment was carried out as follows.

Human-derived melanocytes (Human melanocyte, MNT1) containing 10% FBS (fetal bovine serum) Modified DMEM (Dubelcco's modified eagle medium, 5% AIM V medium, 1% Pyruvate Sodium, 1% non-essential amino acids, 1% penicillin / streptomycin) medium was inoculated at 1 X 10 ⁴ per well in a 96-well plate and incubated for 24 hours at 5% CO ₂ , 37 ℃. After completion of the culture, the medium was removed and the sample obtained in Example 2 was used as a sample, and the sample was replaced with a medium diluted to a suitable concentration (37.5, 75, 150, 750, 1500 nmol / L), and then 5% CO. ₂ , incubated for 2 days at 37 ℃. In order to confirm the cell proliferation ability, 10 μl of WST-1 solution was treated to the cultured cells, and then cultured at 37 ° C. for 1 hour, and the value was measured at 450 nm with an ELISA reader. % Growth rate was calculated, and the result is shown in FIG.

[Equation 1]

% Growth = (B / A) X 100

In Equation 1,

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

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

As shown in FIG. 2, it could be confirmed that even if the treatment concentration of MTD2173A-siRNA was increased, it did not affect the proliferation ability of the cells.

3-2. 세포 독성 확인3-2. Cytotoxicity Check

To test for cytotoxicity, the cell cytotoxicity assay kit provided by Promega, USA was used to measure the degree of leakage of lactate dehydrogenase (LDH) inside cells. Observed. More specifically, human melanocytes (Human melanocyte, MNT1) containing 10% FBS (fetal bovine serum) modified DMEM (Dubelcco's modified eagle medium, 5% AIM V medium, 1% Pyruvate Sodium, 1% non Inoculated at 1 X 10 ⁴ per well in a 96-well plate with -essential amino acids, 1% penicillin / streptomycin) medium and incubated at 5% CO ₂ , 37 ° C for 24 hours. After incubation, the medium was removed, and the sample obtained in Example 2 was used as a sample, and the sample was replaced with a medium diluted to an appropriate concentration (150, 300 nmol / L), followed by 5% CO ₂ at 37 ° C for 2 days. Incubated for In order to measure the maximum amount of LDH effluent from the cells, 10 μl of 10X cell lysis buffer was treated to 96-well plate cells and incubated at 37 ° C. for 1 hour. To confirm cytotoxicity, 50 μl of culture was taken from each well and transferred to a new 96-well plate. 50 μl of the mixed solution was treated in each 96-well plate and incubated at room temperature for 30 minutes in dark conditions. After 50 µl of the reaction stop solution was treated in each well, its value was measured at a wavelength of 490 nm with an ELISA reader, and then cytotoxicity (%) was calculated by Equation 2 below. Indicated.

[Equation 2]

Cytotoxicity (%) = (B / A) X 100

In Equation 2,

A is the absorbance at 490 nm in which LDH is secreted extracellularly,

B is the absorbance at 490 nm of the sample added.

As shown in FIG. 3, siRNA delivery into cells using liposomes showed up to 6.5% toxicity, whereas MTD2173A-siRNA showed a relatively low toxicity level of 0.5%, indicating that it is safer than liposomes. From this, it can be seen that the intracellular molecular transport peptide of the present invention has a remarkably good value as an intracellular transporter.

실시예 4. 세포 내 분자 전송 펩티드-siRNA 복합체의 목표유전자 발현 억제능 확인Example 4. Confirmation of target gene expression inhibitory activity of intracellular molecular transport peptide-siRNA complex

Human melanocytes (MNT1) are derived from DMEM (Dubelcco's modified eagle medium, 5% AIM V medium, 1% Pyruvate Sodium, 1% non-essential amino acids containing 10% FBS (fetal bovine serum)) Inoculated at 1 X 10 ⁵ per well in a 6-well plate with 1% penicillin / streptomycin medium, 5% CO ₂ , and incubated at 37 ° C for 24 hours, followed by 1% penicillin / streptomycin (penicillin / Medium without streptomycin) was pretreated and incubated. In solution A, 1.5 μl of 100 μM siRNA was added to 98.5 μl of Opti-MEM medium, and 6 μl of RNAi MAX (Invitrogen) was prepared in 94 μl of Opti-MEM medium, respectively, and then solution A and B were mixed. After incubation at room temperature for 5 minutes, another sample, MTD2173A-siRNA, was added to 194 μl of Opti-MEM medium and 6 μl of 100 μM MTD2173A-siRNA was incubated for 5 minutes, and then treated evenly to the cells. After incubation for 24 hours, the cells were collected and inoculated with 1.2 × 10 ⁵ cells in 6-well and 6 × 10 ⁴ cells in 12-well. The next day, the sample was processed again as above.

4-1. Real time PCR(RT-PCR)을 이용한 목표 유전자의 mRNA 상대적 정량 분석4-1. MRNA Relative Quantitative Analysis of Target Genes Using Real Time PCR (RT-PCR)

After RNA interference the media was removed and treated with Trizol (Invitrogen, USA) to extract and quantify total RNA. The same amount of RNA corresponding to 2 μg was used as a template, and cDNA was synthesized using a cDNA synthesis kit (RevertAid first strand cDNA Synthesis kit, Thermo scientific). More specifically, 2 μg of RNA was added to a 250 μl Eppendorf tube, 1 μl of oligo dT, 4 μl of 5X Reaction buffer, 1 μl of RiboLock RNase Inhibitor, 2 μl of 10 mM dNTP Mix, and 1 μl of RevertAid M-MuLV reverse Transcriptase. Diethyl pyrocarbonate (DEPC) -treated distilled water was added so that the total volume was 20 μl. This was synthesized cDNA for 1 hour at 42 ℃ by a gene amplifier (Mastercycler pro, Eppendorf, Germany), the enzyme activity was terminated for 5 minutes at 70 ℃. The synthesized cDNA was diluted 1/10 with distilled water, and 10 μl of SYBR Green PCR master mix 2X (Applied Biosystems, USA), 1 μl qPCR primer, and cDNA in each well of a 96-well plate to analyze the expression level of Kif13A. 1 μl and distilled water 8 μl were added to form a mixed solution. Meanwhile, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), which is a housekeeping gene, was used as a standard gene to correct mRNA expression, and qPCR primers were produced by Bionia (Kif13A: Cat # P141985, GAPDH: Cat). # P267613, Bioneer, Korea).

The mixed solution contained in a 96-well plate was subjected to real-time PCR using a 7500 Fast Real Time PCR System (Applied Biosystems, USA). The difference in ΔCt values was determined using Con siRNA as a control to the Ct value of Kif13A corrected by the GAPDH gene. MRNA expression rate of Kif13A was compared using Gene Expression Formula 2 ^(-ΔCt) , and the results are shown in FIG. 4.

As shown in FIG. 4, the MNT1 cell line in which Kif13A siRNA was introduced into a liposome, which is a conventional method, was introduced into the MNT1 cell line when the mRNA amount of the Kif13A gene and MTD2173A-Kif13A siRNA were introduced into the MNT1 cell line. When compared, the reduction effect was confirmed to be similar to 75%, 80%, and from the above results it can be seen that the MTD2173A-Kif13A siRNA of the present invention is excellent in the intracellular penetration effect.

4-2. Western Blot을 이용한 목표유전자의 발현분석4-2. Expression Analysis of Target Gene Using Western Blot

After RNA interference, cells were washed twice with PBS and then treated with 150 μl of lysis buffer (50 mM Tris, 150 mM NaCl, 10 mM EDTA, 1% Triton, 1% protease inhibitor, pH 8) per well and 30 on ice. It was left for a minute. Cell lysate was collected using a scraper and centrifuged (13,000 rpm, 4 ° C) to collect only the supernatant. The amount of protein in the supernatant was measured by BCA protein assay kit (Pierce), and a certain amount of protein was separated by electrophoresis on SDS-polyacrylamide gel, followed by nitrocellulose membrane (NC). Western blotting was performed by transferring, and the result is shown in FIG. 5. In this case, an anti-Kif13A polyvalent antibody (polyclonal Ab, Cat # A301-077A) purchased from Bethyl Laboratories, Inc. was used as the primary antibody. To analyze the results, Chemiluminescence Luminol Reagent (Chemiluminescence luminol reagent) and image analysis equipment LAS 4000 (GE healthcare) was used.

As shown in Figure 5, the con siRNA and MTD2173A-con siRNA used as a control did not inhibit the protein expression of the Kif13A gene, but in the experimental group treated with Kif13A siRNA and MTD2173A-Kif13A siRNA was confirmed that the expression of Kif13A is completely suppressed. Could. From the above results, similar to the effect of inhibiting the expression of target protein by intracellular delivery of Kif13A siRNA using liposomes, MTD2173A-Kif13A siRNA was also delivered to the cell by MTD2173A-Kif13A siRNA to suppress expression of target gene protein. Could be observed.

실시예 5. 세포내 분자 전송 펩티드-siRNA의 멜라닌 생성 저해 활성 확인Example 5 Identification of Melanin Inhibitory Activity of Intracellular Molecular Transport Peptide-siRNA

As in Example 4, cells in which the expression of the target gene was suppressed were washed with PBS (phosphated buffer saline), and the cells were recovered by treating with trypsin. The recovered cells were centrifuged at 10,000 rpm for 10 minutes and the supernatant was removed to obtain cell pellets. Some of the recovered cells were observed using a TC10 ^™ Automated Cell Counter (Bio-Rad, USA). The cell pellet was dried at 60 ° C., and 200 μL of 1M sodium hydroxide solution containing 10% DMSO was sufficiently dissolved at 60 ° C. to obtain intracellular melanin. Dilute with Melanin solution step by step to make a standard solution. Transfer the standard solution and the solution to a 96-well plate, measure the absorbance at 490 nm with a microplate reader, quantify using a standard curve, and use the cell number per cell. The amount of melanin produced was determined and the results are shown in FIG. 6.

As shown in FIG. 6, the amount of melanin synthesized was reduced by 9.5% when the melanin-forming cells were compared with the transfected Kif13A siRNA using liposomes and the amount of melanin produced in the control-transfected cells. I could confirm that. On the contrary, in the cells treated with MTD2173A-Kif13A siRNA, the synthesized melanin reduction effect was increased to 21.6% when the amount of melanin produced in the cells treated with MTD2173A-con siRNA, a control group, was compared.

From the above results, the MTD2173A-siRNA complex of the present invention not only effectively delivers siRNA-like substances into cells in a manner different from the existing intracellular transfection methods, but also the delivered substances can effectively exert their effects in cells. I could see that.

In addition, MTD2173A-siRNA specific to kinesin Kif13A, which plays an important role in melanin formation, can effectively inhibit melanin production and could be effectively used as a whitening substance.

실시예 6. 베타갈락토시다제 활성 억제능 확인Example 6. Confirmation of inhibitory activity of beta galactosidase activity

B6 ROSA26 mice continuously expressing beta galactosidase were treated with 200 μg / head of M1173A-LacZ-siRNA-Cy3, M2173A-LacZ-siRNA-Cy3, M3173A-LacZ-siRNA-Cy3 and M1018m-LacZ-siRNA-Cy3 for 3 days. Two days after intravenous administration, mice were sacrificed to sacrifice the lungs. The extracted lungs were fixed in 4% paraformaldehyde for 15 hours or longer, and frozen sections were prepared using a micron cryo-extruder, and then placed on a glass slide to produce slides for microscopic observation. The prepared slides were washed with phosphate buffer solution for 10 minutes and stained with cell nuclei in tissue for 5 minutes with 0.5 mM DAPI solution. The stained tissue was washed again three times with a buffer solution for 10 minutes, and then fixed with an overlapping medium and observed under a confocal microscope. The results are shown in FIG. 7.

On the other hand, specific information of the MTD1173A, MTD3173A, and MTD1018m used in the experiment is as follows:

MTD _{1018 m}

Met Arg Pro Ala Ala Leu Ala Ala Leu Pro Val Ala Val Val Ala Val (SEQ ID NO: 7)

MTD _1173A

Met Arg Pro Ala Val Ile Pro Ile Leu Ala Val (SEQ ID NO: 8)

MTD _3173A

Met Lys Pro Ala Val Ile Pro Ile Leu Ala Val (SEQ ID NO: 9)

As shown in FIG. 7, no significant level of fluorescence was observed in the negative control, while M1173A-LacZ-siRNA-Cy3, M2173A-LacZ-siRNA-Cy3, M3173A-LacZ-siRNA-Cy3 and M1018m- were used in the experiment. In the case of LacZ-siRNA-Cy3, it was observed that permeation of cells of lung tissue occurred.

In addition, after the slides prepared as described above were reacted with X-gal dye solution overnight, the tissues were stained with hematoxyleneeocin staining, and then beta galactosidase activity was observed. The results are shown in FIG. 8. It was. As shown in FIG. 8, beta galactosidase activity was observed throughout most organ sections in the negative control group, but with M1173A-LacZ-siRNA-Cy3, M2173A-LacZ-siRNA-Cy3, M3173A-LacZ-siRNA-Cy3 and It was confirmed that beta galactosidase activity was reduced in the organ section of the mouse administered with M1018m-LacZ-siRNA-Cy3. However, in the case of M1173A, M3173A, and M1018m, it was observed by confocal microscopy that the permeation of cells in the lung tissue was less efficient than M2173A, which showed that the efficacy of M2173A was excellent even in the result of beta galactosidase activity. Could confirm. Through this, it can be seen that M2173A can effectively deliver siRNA, which is difficult to be delivered intracellularly, into organ tissues, and effectively exert its efficacy in the tissues to which the delivered substance or drug is delivered.

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.

Claims

A composition for transdermal delivery comprising a complex covalently linked to an intracellular molecular transport peptide and an siRNA,

The peptide is a transdermal delivery composition, characterized in that consisting of the amino acid sequence of SEQ ID NO: 5.
The composition for transdermal delivery according to claim 1, wherein the peptide is encoded from a polynucleotide sequence set forth in SEQ ID NO: 6.
The composition of claim 1, wherein the siRNA inhibits the expression of Kif13A (kinesin superfamily protein 13A) to inhibit melanin formation.
The composition for transdermal delivery according to claim 3, wherein the siRNA is composed of a sense RNA strand consisting of SEQ ID NO: 1 and an antisense RNA strand consisting of SEQ ID NO: 2.
According to claim 1, wherein the covalent bonds, characterized in that the non-degradable bonds or degradable bonds, compositions for transdermal delivery.
The composition for transdermal delivery according to claim 5, wherein the non-degradable bond is an amide bond or a phosphate bond.
The composition for transdermal delivery according to claim 5, wherein the degradable bond is selected from the group consisting of disulfide bonds, acid decomposable bonds, ester bonds, anhydride bonds, biodegradable bonds, and enzyme degradable bonds.
The covalent bond of claim 1, wherein the covalent bond is composed of a thioether linker, S-HyNic (6-hydrazino-nicotinic acid) / 4-FB (4-formylbenzoate) linker, and 4FB (N-succinimidyl-4-formylbenzamide) / hydroxylamine linker. A composition for transdermal delivery, characterized by comprising a linker selected from the group.
According to claim 1, The covalent bond is characterized in that the transdermal delivery composition, characterized in that made by 4FB (N-succinimidyl-4-formylbenzamide) / hydroxylamine linker.
The composition for transdermal delivery of claim 1, wherein the composition is a cosmetic composition.
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, serums, liposomes, microcapsules, composite particles, shampoos and rinses.
The composition for transdermal delivery according to claim 1, wherein the composition penetrates the stratum corneum.
A transdermal delivery system for delivering siRNA into skin cells, wherein the system is characterized in that the intracellular molecular transport peptide consisting of the amino acid sequence of SEQ ID NO: 5 is covalently linked to the siRNA for delivery.
The system according to claim 13, wherein the siRNA is composed of a sense RNA strand consisting of SEQ ID NO: 1 and an antisense RNA strand consisting of SEQ ID NO: 2.
A method of delivering siRNA into skin cells, the method comprising covalently delivering an intracellular molecular transfer peptide consisting of the amino acid sequence of SEQ ID NO: 5 with siRNA.
The method according to claim 15, wherein the siRNA is composed of a sense RNA strand consisting of SEQ ID NO: 1 and an antisense RNA strand consisting of SEQ ID NO: 2.
A method of delivering an siRNA into the stratum corneum, wherein the method comprises covalently delivering an intracellular molecular transfer peptide consisting of the amino acid sequence of SEQ ID NO: 5 with the siRNA.
18. The method of claim 17, wherein the siRNA is comprised of a sense RNA strand consisting of SEQ ID NO: 1 and an antisense RNA strand consisting of SEQ ID NO: 2.
An intracellular molecular transfer peptide consisting of an amino acid sequence set forth in SEQ ID NO: 5 and a siRNA are covalently bonded to the topical application to the epidermis, thereby whitening the skin.
The method of claim 19, wherein the siRNA is composed of a sense RNA strand consisting of SEQ ID NO: 1 and an antisense RNA strand consisting of SEQ ID NO: 2.