KR102173702B1 - Peptides for targeting skin cells and use thereof - Google Patents

Abstract

The present invention provides a skin cell or skin cancer cell targeting peptide and a composition for diagnosing skin cancer, a composition for drug delivery, and a cosmetic composition comprising the peptide as an active ingredient.
The peptide can effectively recognize skin cells or skin cancer cells due to low risk of immune response and high affinity with skin cells and skin cancer cells, and the drug can be selectively delivered to skin cancer tissues or cells by the peptide. The effect of can be improved and side effects can be significantly reduced. In addition, the peptide may be used as a cosmetic composition to help improve skin such as skin regeneration or elasticity enhancement.
Through this effect, the peptide can be used in cosmetic development, skin disease or skin cancer prevention, diagnosis and treatment technology development.

Description

Peptides for targeting skin cells and their uses {PEPTIDES FOR TARGETING SKIN CELLS AND USE THEREOF}

The present invention relates to a skin cell or skin cancer cell targeting peptide and a composition for diagnosing skin cancer, a composition for drug delivery, and a cosmetic composition comprising the peptide as an active ingredient.

The skin is the largest tissue in the integumentary system, made up of a number of epithelial tissues that protect the muscles and organs in the body. When facing the external environment, the skin plays a very important role in protecting the body from pathogens. Other major functions of the skin are insulation and thermoregulation, sensory functions, and the synthesis of vitamin D and the protection of vitamin B folates.

In the skin, melanocytes are active to form pigments, or melanin is produced. These melanocytes absorb some of the potentially dangerous ultraviolet rays in the sun. It also has DNA repair enzymes that help restore UV damage, and people who lack the genes that make these enzymes are more likely to develop skin cancer.

Skin cancer is the most common cancer, accounting for 40% of all malignancies. 80% of skin cancers occur in areas exposed to sunlight such as the face, head, and wrist. Most of the people who develop skin cancer are basal cell carcinoma or squamous cell carcinoma, and melanoma accounts for 3% of skin cancer patients, and among melanoma patients, the mortality rate is 14%, which is a dangerous cancer.

Therapies performed to treat such cancer include surgery, radiation therapy, and chemotherapy, and research on the molecular mechanisms of cancer is actively progressing, but many of the currently developed therapies rely on surgical surgery. In addition, antigens specific to cancer cells and antibodies targeting them have been developed, but in the case of antibodies, there are problems such as concerns about an immune response and low efficiency of penetration into tissues. Accordingly, there is a need for the development of targeted therapeutics that are less likely to have an immune response and are easy to penetrate into tissues.

Korean Patent Registration No. 10-1585947 (registered on January 11, 2016)

In order to solve the above problems, the present invention provides a peptide for targeting skin cells.

The present invention provides a peptide for targeting skin cancer cells.

The present invention provides a skin cancer diagnostic composition and a skin cancer diagnostic kit comprising the peptide as an active ingredient.

The present invention provides a composition for drug delivery comprising the peptide as an active ingredient.

The present invention provides a cosmetic composition for skin regeneration or elasticity enhancement comprising the peptide as an active ingredient.

The present invention provides a skin cell targeting peptide consisting of an amino acid sequence represented by the following formula 1.

[Equation 1]

[abcde] _n

In Equation 1, a, b, c, d and e may each be the same or different,

Wherein a, b, c, d, and e are any one pentapeptide selected from the group consisting of XGVPG, GVPGX, VPGXG, PGXGV and GXGVP, respectively, and X of the pentapeptide is valine (V), alanine (alanine, A), may be substituted with glycine (G) or histidine (H), and n is 1 to 100.

The present invention provides a skin cancer cell targeting peptide consisting of an amino acid sequence represented by the following formula 1.

[Equation 1]

[abcde] _n

In Equation 1, a, b, c, d and e may each be the same or different,

Wherein a, b, c, d, and e are any one pentapeptide selected from the group consisting of XGVPG, GVPGX, VPGXG, PGXGV and GXGVP, respectively, and X of the pentapeptide is valine (V), alanine (alanine, A), may be substituted with glycine (G) or histidine (H), and n is 1 to 100.

The present invention provides a composition for diagnosing skin cancer comprising the peptide for targeting skin cancer cells as an active ingredient.

The present invention provides a skin cancer diagnostic kit comprising the skin cancer diagnostic composition.

The present invention provides a composition for drug delivery comprising the peptide as an active ingredient.

The present invention provides a cosmetic composition for skin regeneration or elasticity enhancement comprising the peptide for targeting skin cells as an active ingredient.

The peptide according to the present invention has a low risk of an immune response and has high affinity with skin cells and skin cancer cells, and can effectively recognize skin cells or skin cancer cells. In addition, it can be easily purified and has high stability by controlling the temperature by its temperature-sensitive properties.

The skin cell or skin cancer cell targeting peptide according to the present invention may be used as a composition for diagnosing skin cancer, and a therapeutic drug or the like may be added to the peptide to be used as a composition for drug delivery. The drug can be selectively delivered to skin cancer tissues or cells by the peptide, so that the effect of the drug can be improved and side effects can be significantly reduced. In addition, the peptide may be used as a cosmetic composition to help improve skin such as skin regeneration or elasticity enhancement.

Through this effect, the peptide can be used in cosmetic development, skin disease or skin cancer prevention, diagnosis and treatment technology development.

1 is a peptide DNA prepared according to an embodiment of the present invention.
2 is a schematic diagram of a vector used for cloning a peptide according to an embodiment of the present invention.
3 is an electrophoresis of DNA of a peptide synthesized according to an embodiment of the present invention.
4 is a schematic diagram of a vector used for expression of a peptide synthesized according to an embodiment of the present invention.
5 is an electrophoresis of a protein of a peptide according to an embodiment of the present invention.
6 shows thermal characteristics according to an experimental example of the present invention.
7 is a graph showing the results of a cytotoxicity experiment according to an experimental example of the present invention.
8 and 9 are graphs of flow cytometry results according to an experimental example of the present invention.
10 is a microscope analysis image according to an experimental example of the present invention.
11 is a microscope analysis image according to an experimental example of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention is based on the pentapeptides of XGVPG, GVPGX, VPGXG, PGXGV, and GXGVP because the types of cells and receptors that interact according to the peptide sequence type are different and amino acids of the peptide can be substituted according to the desired purpose. Thus, the guest amino acid (X) was substituted with an amino acid other than proline, among which valine (V) was substituted (V ₅ ) ₁₆ , valine (V), alanine (Alanine, A), glycine (Glycine, G) (VA ₂ G ₂ ) ₁₆ and valine (V) and histidine (H) substituted (VH ₄ ) ₁₆ were found to be able to specifically recognize skin cells. (VH ₄ ) ₁₆ This invention was completed by confirming the specific cell adhesion effect on fibroblasts, stem cells, ovarian cancer, skin epithelial cancer, keratinocytes and melanoma cells.

In the present invention, "peptide" is a polymer of amino acid residues, and may be used interchangeably with "protein" or "polypeptide".

In the present invention, the term "target" refers to specifically binding to skin cells or skin cancer cells without binding to other tissue cells.

The present invention provides a peptide for targeting skin cells.

The peptide for targeting skin cells according to the present invention may be represented by Formula 1 below.

[Equation 1]

[abcde] _n

In Equation 1, the a, b, c, d, and e may be the same or different, respectively, and the a, b, c, d, or e positions may be changed.

Wherein a, b, c, d, and e are any one pentapeptide selected from the group consisting of XGVPG, GVPGX, VPGXG, PGXGV and GXGVP, respectively, and X of the pentapeptide is valine (V), alanine (alanine, A), may be substituted with glycine (G) or histidine (H), and n may be 1 to 100.

More specifically, the peptide for targeting skin cells according to the present invention may consist of any one amino acid sequence selected from the group consisting of the following SEQ ID NO: 1 to 3, and preferably may consist of the amino acid sequence of SEQ ID NO: 3.

SEQ ID NO: 1: MGHG-[(VGVPG) ₅ ] _16- WHASNR

SEQ ID NO: 2: MGHG-[(VGVPG)(AGVPG)(GGVPG)(AGVPG)(GGVPG)] _16- WHASNR

SEQ ID NO: 3: MGHG-[(VGVPG)(HGVPG) ₄ ] ₁₆ -WHASNR

The SEQ ID NO: 1 (V ₅ ) ₁₆ , the SEQ ID NO: 2 (VA ₂ G ₂ ) ₁₆ , the SEQ ID NO: 3 can be briefly represented as (VH ₄ ) ₁₆ .

The VGVPG of SEQ ID NO: 1 is that X of the pentapeptide is substituted with valine, and is not limited thereto, and may be GVPGV, VPGVG, PGVGV, or GVGVP.

The AGVPG of SEQ ID NO: 2 is that X of the penta peptide is substituted with alanine, and may be GVPGA, VPGAG, PGAGV or GAGVP, and GGVPG is that X of the penta peptide is substituted with glycine, GVPGG, VPGGG, PGGGV or It can be GGGVP.

The HGVPG of SEQ ID NO: 3 is that X of the pentapeptide is substituted with histidine, and may be GVPGH, VPGHG, PGHGV or GHGVP.

The letters used for the amino acids refer to the following amino acids according to the standard abbreviation regulations in the field of biochemistry.

A(Ala): alanine; C(Cys): cysteine; D(Asp): aspartic acid; E(Glu): glutamic acid; F(Phe): phenylalanine; G(Gly): glycine; H(His): histidine; I(IIe): isoleucine; K(Lys): lysine; L(Leu): leucine; M(Met): methionine; N(Asn): asparagine; O(Ply)pyrrolysine; P(Pro): proline; Q(Gin): glutamine; R(Arg): arginine; S(Ser): serine; T(Thr): threonine; U(Sec): selenocysteine, V(Val): valine; W(Trp): tryptophan; Y(Tyr): Tyrosine.

According to an experimental example of the present invention, the peptide has a high affinity for skin cells and can target skin cells, and the skin cells are at least one selected from the group consisting of fibroblasts, human mesenchymal stem cells, and keratinocytes. However, it is not limited thereto.

The present invention provides a peptide for targeting skin cancer cells.

In the present invention, "tumor" refers to the physiological state of a mammal having the characteristics of unregulated cell growth or proliferation, and may be replaced with "tumor".

The peptide for targeting skin cancer cells according to the present invention may be represented by Formula 1 below.

[Equation 1]

[abcde] _n

In Equation 1, the a, b, c, d, and e may be the same or different, respectively, and the a, b, c, d, or e positions may be changed.

Wherein a, b, c, d, and e are any one pentapeptide selected from the group consisting of XGVPG, GVPGX, VPGXG, PGXGV and GXGVP, respectively, and X of the pentapeptide is valine (V), alanine (alanine, A), may be substituted with glycine (G) or histidine (H), and n may be 1 to 100.

More specifically, the peptide for targeting skin cancer cells according to the present invention may consist of any one amino acid sequence selected from the group consisting of the following SEQ ID NO: 1 to 3, and preferably may consist of the amino acid sequence of SEQ ID NO: 3.

SEQ ID NO: 1: MGHG-[(VGVPG) ₅ ] _16- WHASNR

SEQ ID NO: 2: MGHG-[(VGVPG)(AGVPG)(GGVPG)(AGVPG)(GGVPG)] _16- WHASNR

SEQ ID NO: 3: MGHG-[(VGVPG)(HGVPG) ₄ ] ₁₆ -WHASNR

The SEQ ID NO: 1 (V ₅ ) ₁₆ , the SEQ ID NO: 2 (VA ₂ G ₂ ) ₁₆ , the SEQ ID NO: 3 can be represented by (VH ₄ ) ₁₆ .

The VGVPG of SEQ ID NO: 1 is that X of the pentapeptide is substituted with valine, and is not limited thereto, and may be GVPGV, VPGVG, PGVGV, or GVGVP.

The AGVPG of SEQ ID NO: 2 is that X of the penta peptide is substituted with alanine, and may be GVPGA, VPGAG, PGAGV or GAGVP, and GGVPG is that X of the penta peptide is substituted with glycine, GVPGG, VPGGG, PGGGV or It can be GGGVP.

The HGVPG of SEQ ID NO: 3 is that X of the pentapeptide is substituted with histidine, and may be GVPGH, VPGHG, PGHGV or GHGVP.

According to an experimental example of the present invention, the peptide has a high affinity for skin cancer cells and can target skin cancer cells, and the skin cancer cells are one selected from the group consisting of skin epithelial cancer cells, ovarian cancer cells, and melanoma cells. It can be more than that. In addition, the skin cancer cells may be those in which the skin cells are tumorigenic, but the present invention is not limited thereto.

The peptide according to the present invention may be derived from nature, or may be synthesized using a known peptide synthesis method, for example, a genetic engineering method or a chemical method.

The genetic engineering method can produce a DNA sequence encoding the peptide or a functional equivalent thereof according to a conventional method. The DNA sequence can be prepared by PCR amplification using an appropriate primer. Alternatively, the DNA sequence can be synthesized by standard methods known in the art, for example, using an automatic DNA synthesizer (available from Biosearch or Applied Biosystems). The synthesized DNA sequence is operably linked thereto to be inserted into a vector containing one or more expression control sequences (eg, promoters, enhancers, etc.) that control the expression of the DNA sequence, and a recombinant expression vector formed therefrom The host cell is transformed to generate a transformant.

According to an embodiment of the present invention, the vector may be a conventional cloning vector or an expression vector, and may include a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector, etc., and thus variously produced Can be.

The "transformant" may be used interchangeably with the "host cell" and the like, and introduced into the cell by any means, for example, electric shock method, calcium phosphatase precipitation method, microinjection method, transformation method, virus infection, etc. It refers to a prokaryotic or eukaryotic cell containing heterologous DNA.

According to an embodiment of the present invention, the transformant may use all types of single-celled organisms commonly used in the field of cloning, for example, prokaryotic microorganisms such as Escherichia coli , in addition to yeast, etc. Cells derived from higher eukaryotes, including lower eukaryotic microorganisms, insect cells, plant cells, and mammals, can be used as host cells, but are not limited thereto. Since the expression level and modification of the protein differ depending on the host cell, a person skilled in the art can select and use the most suitable host cell for the purpose.

The resulting transformant can be cultured under a medium and conditions suitable for expression of the DNA sequence to recover a substantially pure peptide encoded by the DNA sequence from the culture. The recovery can be performed using a method known in the art (eg, chromatography). In the above, "substantially pure peptide" means that the peptide according to the present invention does not contain substantially any other protein derived from a host cell.

In addition, the peptides according to the present invention can be easily prepared by chemical synthesis (Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983) known in the art. Typical methods include liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Florida, 1997; A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989), but not limited thereto.

The peptide according to the present invention may include a peptide having a native amino acid sequence as well as an amino acid sequence variant thereof.

In the present invention, the term "amino acid sequence variant" means a peptide having a different sequence by deletion, insertion, non-conservative or conservative substitution, substitution of amino acid analogs, or combinations thereof in the amino acid sequence of the present invention. As a result, the activity of the molecule is not changed as a whole by the above mutation.

In addition, the peptide according to the present invention may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, and the like.

The present invention provides a composition for diagnosing skin cancer comprising the peptide as an active ingredient.

In the present invention, "diagnosis" means confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, diagnosis is to confirm the presence or characteristics of skin cancer.

The composition for diagnosing skin cancer according to the present invention may include the peptide for targeting skin cancer cells as an active ingredient. The peptide can diagnose skin cancer by reacting with a part of a skin lesion collected from a patient.

In the composition for diagnosing skin cancer according to the present invention, the skin cancer may be one or more selected from the group consisting of skin epithelial cancer, ovarian cancer, and melanoma, and other skin cancers caused by tumorigenicity of the skin cells may be included, but are limited thereto. It does not become.

In the composition for diagnosing skin cancer according to the present invention, the peptide may be labeled with any one selected from the group consisting of a color developing enzyme, a radioactive isotope, a chromopore, a luminescent material, and a fluorescent material.

As the color developing enzyme, peroxidase, alkaline phosphatase, etc. may be used, and the radioisotope may be used as ¹²⁴ I, ¹²⁵ I, ¹¹¹ In, ⁹⁹ mTc, ³² P, ³⁵ S, etc. , As the fluorescent material, FITC, RITC, rhodamine, cyanine, Texas Red, fluorescein, phycoerythrin, quantum dots, etc. can be used. have.

Further, the peptide may be labeled with an antibody epitope, a substrate, a cofactor, an inhibitor or an affinity ligand. The labeling may be performed during the process of synthesizing the peptide of the present invention, or may be performed in addition to the already synthesized peptide.

The present invention provides a skin cancer diagnostic kit comprising the skin cancer diagnostic composition.

In the kit for diagnosing skin cancer according to the present invention, the skin cancer may be one or more selected from the group consisting of skin epithelial cancer, ovarian cancer, and melanoma, and other skin cancers caused by tumorigenicity of the skin cells may be included, but are limited thereto. It does not become.

The present invention provides a composition for drug delivery comprising the peptide as an active ingredient.

The composition for drug delivery according to the present invention may include the peptide for targeting skin cells or the peptide for targeting skin cancer cells as an active ingredient. The peptide can be used as a drug delivery vehicle that selectively delivers drugs to skin cancer tissues or cells.

In the composition for drug delivery according to the present invention, the drug may be at least one selected from the group consisting of anticancer agents, antibiotics, antibacterial agents, anti-inflammatory agents, and wound healing agents, but is not limited thereto.

The drug may be an anticancer agent, and the anticancer agent may be used without limitation as long as it is used for conventional cancer treatment. The anticancer agent may be taxane or a derivative thereof, for example, docetaxel, cabazitaxel, paclitaxel, and in addition to vinblastine, vincristine, cisplain , Actinomycin-D (actinomycin-D), 5-fluouracil (5-fluouracil) and may be one or more selected from the group consisting of pembrolizumab, preferably pembrolizumab (Pembrolizumab) and It may be the same anticancer drug related to skin cancer, but is not limited thereto.

In addition, the drug may be an antibiotic, an antifungal agent, an anti-inflammatory agent, a wound treatment, and the like, and may include other treatments for skin diseases, but is not limited thereto.

The antibiotics are amicacin, aztreonam, chloramphenicol, chloramphenicol sodium succinate, ciprofloxacin, clindamycin, metronidazole, and metronidazole. gentamicin), lincomycin, tobramycin, vancomycin, polymyxin B, colistimethate sodium, or colistin may be used.

Griseofulvin, amphotericin B, nystatin or candicidin may be used as the antifungal agent.

As the anti-inflammatory agent, nonsteroidal anti-inflammatory agents such as indomethacin, naproxen, ibuprofen, piroxicam, and the like may be used, and steroidal anti-inflammatory agents such as cortisone ( cortisone), dexamethasone, prednisolone, prednisone, and the like can be used.

The wound treatment may include drugs having effects such as skin cell proliferation and regeneration, collagen synthesis, and promotion of wound healing.

In the composition for drug delivery according to the present invention, the anticancer agent or other drug may be linked or synthesized with the peptide through a method known in the art, for example, covalent bonding, crosslinking, fusion, and the like.

In addition, in the drug delivery composition according to the present invention, the peptide may be a fusion peptide formed by fusion with a protein or a peptide capable of treating cancer diseases such as TRAIL other than the anticancer agent and the other drugs.

The present invention provides a cosmetic composition for skin regeneration or elasticity enhancement, comprising the peptide for targeting skin cells as an active ingredient.

The cosmetic composition according to the present invention may further include one or more selected from the group consisting of growth factors, collagen, and skin regeneration proteins.

The "growth factor" is a generic term for a polypeptide that promotes various cell division, growth, and differentiation, and may include, for example, keratinocyte growth factor (KGF).

The skin regeneration protein may include, for example, an epidermal growth factor (EGF) called a cell regeneration factor.

In addition, in the cosmetic composition according to the present invention, the peptide may be a fusion peptide formed by fusion with skin regeneration factors such as the growth factor and collagen.

The cosmetic composition according to the present invention may further include one or more of the ingredients registered in the list of cosmetic ingredients registered with the Food and Drug Administration and the ICID (International Cosmetic Material Collection). For example, the cosmetic composition is an organic solvent, a solubilizer, a thickener, a gelling agent, a softener, an antioxidant, a suspending agent, a stabilizer, a fragrance, a surfactant, an emulsifier, a filler, a sequestering agent, a preservative, a vitamin, a blocker, It may further comprise one or more adjuvants commonly used in the cosmetic field, such as wetting agents, dyes and pigments, hydrophilic or lipophilic actives, or any other ingredients commonly used in cosmetics.

The vitamin may include a water-soluble or oil-soluble vitamin, and the water-soluble vitamin is a water-soluble vitamin that can be blended in cosmetics, preferably vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, Nicotinic acid amide, folic acid, vitamin C, vitamin H, and the like, and more preferably, vitamin C, which helps collagen synthesis, may be included. In addition, their salts (thiamine hydrochloride, sodium ascorbate, etc.) or derivatives (ascorbic acid-2-phosphate sodium salt, ascorbic acid-2-magnesium salt, etc.) may also be included, and the water-soluble vitamin is a microbial transformation method. , It can be obtained by a conventional method such as a purification method from a culture of microorganisms, an enzyme method, or a chemical synthesis method.

The cosmetic composition according to the present invention can be prepared in any formulation conventionally prepared in the technical field to which the present invention belongs. For example, skin lotion, skin softener, skin toner, gel, cream, lotion, milk lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, pack, massage cream and spray It may be formulated as, but is not limited thereto. More specifically, nourishing cream, massage cream, essence, eye cream, sun lotion, sun cream, makeup primer, makeup base, BB cream, cleansing cream, cleansing foam, cleansing water, pack, stick product, balm type It can be formulated as a product, spray or powder.

The cosmetic composition according to the present invention may be used alone or in duplicate, or may be used in duplicate with other cosmetic compositions other than the present invention. In addition, the cosmetic composition according to the present invention may be used according to a conventional method of use, and the number of times of use thereof may be varied according to a user's skin condition or taste.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Example 1> Preparation of a peptide containing valine (V), alanine (A), glycine (G) and histidine (H)

1. Peptide DNA Preparation

1 is a peptide DNA prepared according to an embodiment of the present invention.

Referring to Figure 1, the peptide is based on the pentapeptides of XGVPG, GVPGX, VPGXG, PGXGV, and GXGVP, and some of the guest amino acids corresponding to Xaa are valine (V), alanine (A) excluding prolin. , Glycine (G) and histidine (H) were substituted, and the total number of amino acids was 25. The guest amino acid may be substituted with an amino acid other than proline.

SEQ ID NO: 1: MGHG-[(VGVPG) ₅ ] _16- WHASNR

SEQ ID NO: 2: MGHG-[(VGVPG)(AGVPG)(GGVPG)(AGVPG)(GGVPG)] _16- WHASNR

SEQ ID NO: 3: MGHG-[(VGVPG)(HGVPG) ₄ ] ₁₆ -WHASNR

2 is a schematic diagram of a vector used for cloning a peptide according to an embodiment of the present invention. The peptide was cloned using the pBHA vector.

Referring to FIG. 2, Van91I and BglI restriction enzyme recognition sites were added to both ends of the peptide gene, and the molecular weight of the peptide was synthesized to increase the molecular weight of the monomer gene by repeating the restriction enzyme treatment and ligation reaction. Proceed with the method. The peptide gene can be isolated by double digestion of the cloned pBHA vector using Van91I and BglI restriction enzymes.

On the other hand, single separation using only the Van91I enzyme can generate a vector for increasing molecular weight. When ligation of the resulting insert and vector is performed, the BglI site at the 3'-end of the insert is attached to the Van91I position of the vector, so that the molecular weight of the gene can be doubled. The recognition site of Van91I is 5'-CCANNNNNTGG-3', and the recognition site of BglI is 5'-GCCNNNNNGGC-3'. 5'-CCANNNNNTGG-3' and the base moiety are specifically cleaved between the fourth and fifth. When the nucleotide sequences corresponding to N in the cut-off region are complementary to each other, the nucleotide sequences may be attached to each other by a ligation reaction. In addition, the restriction enzyme recognition site present in the original bond disappears, and a peptide gene having a molecular weight of 2 times increased can be obtained. This method was repeated several times to prepare a peptide having a desired molecular weight. In each step, DNA transfection and confirmation were performed by DNA sequencing using DH5α E. coli.

3 is an electrophoresis of the DNA of the peptide synthesized according to an embodiment of the present invention, wherein each guest amino acid is substituted with valine (V) (V ₅ ) _n , valine (V), aniline (A) And those substituted with glycine (G) are (VA ₂ G ₂ ) _n , those substituted with valine (V) and histidine (H) are named (VH ₄ ) _n , and SEQ ID NO: 1 is (V ₅ ) ₁₆ , The SEQ ID NO: 2 may be represented by (VA ₂ G ₂ ) ₁₆ , the SEQ ID NO: 3 may be represented by (VH ₄ ) ₁₆ .

3, in (A), lane 1 is DNA ladder, lane 2 is (VH ₄ ) ₄ , lane 3 is (VH ₄ ) ₈ and lane 4 is (VH ₄ ) ₁₆ , (B) In this, lane 1 is DNA ladder, lane 2 is (V ₅ ) ₁₆ , lane 3 is (VA ₂ G ₂ ) ₁₆ and lane 4 is (VH ₄ ) ₁₆ .

4 is a schematic diagram of a vector used for expression of a peptide synthesized according to an embodiment of the present invention.

Referring to FIG. 4, the peptide synthesized in the cloning step was synthesized by synthesizing a cysteine gene into an expression vector for chemical synthesis with Cy5 (Cyanine) for visualization. For expression of the peptide, it was ligated with a modified pET-32b(+) vector containing a restriction enzyme, and E. coli BL21 was used as a host vector system.

2. Expression and purification of peptides containing valine (V), alanine (A), glycine (G) and histidine (H)

E. coli BL21 transformed with the peptide-cysteine expression vector was incubated for 20 hours at 37° C. and 250 rpm in LB medium to which 100 μg/ml of ampicillin (Ampicillin, Amp) was added. The grown E. coli culture was centrifuged at 4°C and 4,000 rpm for 10 minutes to remove the culture, and the remaining E. coli precipitate was suspended in Dulbecco's Phosphate Buffered Saline (hereinafter, DPBS). After the E. coli suspension was dissolved by tip sonication, the dissolved suspension was centrifuged at 4° C. and 12,000 rpm for 15 minutes to remove cell debris.

Then, 10% polyethyleneimine (PEI) was added at a concentration of 0.5% and left on ice for 10 minutes to react with genomic DNA and residual cell debris to induce precipitation. The reaction solution was centrifuged again at 4° C. and 12,000 rpm for 15 minutes to purify only the protein supernatant. In order to selectively separate only the peptide from the protein solution, the separation process reversibly changes the hydrophilicity-hydrophobicity of the peptide according to the temperature change. First, hydrophobic folding was induced between peptide molecules under high temperature conditions, and these were separated from water molecules. To accelerate this reaction, sodium chloride (NaCl) was added to a final 2M to precipitate the peptide. Then, the precipitate obtained by centrifugation at 40° C. and 12,000 rpm for 15 minutes was resuspended in ice-cold phosphate buffer saline (hereinafter, PBS), and the peptide was dissolved again. By repeating the process of centrifuging again under high temperature conditions, only pure peptides can be purified.

The concentration of the recovered peptide-cysteine was quantitatively analyzed using the molar extinction coefficient at 280 nm. To the peptide-cysteine, tris(2-carboxyethyl)phosphin (hereinafter, TCEP) buffer was added, and the molar extinction coefficient was measured. Purity and molecular weight were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 4-20% gradient gel. The protein solution was mixed with a loading buffer containing 0.1% SDS and pretreated at 100° C. for 5 minutes. After the pretreated sample was immersed in the gel, it was operated for 150 minutes in a running buffer containing SDS at 90V. After operation, the gel was stained with Coomassie Brilliant Blue for 2 hours. Desalination was carried out with a desalting solution. The stained gel was confirmed using the Chemi Image Documentation System.

5 is an electrophoresis of a protein of a peptide according to an embodiment of the present invention. Referring to FIG. 5, lane 1 is DNA ladder, lane 2 is (V ₅ ) ₁₆ , lane 3 is (VA ₂ G ₂ ) ₁₆ and lane 4 is (VH ₄ ) ₁₆ .

<Experimental Example 1> Confirmation of the temperature-sensitive properties of the peptide

The transition temperature of the peptide-cysteine synthesized according to an embodiment of the present invention was measured based on the change in absorbance of tryptophan present in the protein at 350 nm using a UV-Vis spectrophotometer and a temperature controller.

In addition, TCEP buffer was added to the peptide-cysteine to block disulfide bonds, and the peptide was dissolved in PBS to prepare 10 μM of a peptide solution. The peptide solution was measured from 20°C to 60°C while observing the change in turbidity by increasing the temperature by 1°C/min. (VA ₂ G ₂ ) _16-C was measured from 20°C to 90°C.

6 shows thermal characteristics according to an experimental example of the present invention.

Referring to FIG. 6, (A) shows that a phase transition occurs in the peptide, and the phase transition can be visually confirmed. (B) shows the absorbance according to the temperature, and the transition temperature was defined as the temperature corresponding to the average value of the minimum and maximum absorbance (OD) before and after.

<Experimental Example 2> Peptide-Cy5 synthesis confirmation

Cysteine was introduced into the peptide to synthesize the peptide and maleimide (Mal)-Cy5. Peptide-cysteine reacted with maleimide in a pH 7.5-6.5 PBS buffer to bind the double bond of maleimide with the hydrogen of the thiol of cysteine and form a thioether bond. The absorbance of the peptide is 280 nm, the maleimide of Mal-Cy5 is 320 nm, and the Cy5 is 640 nm. The synthesis of the peptide-cysteine and Mal-Cy5 was confirmed by the disappearance of the absorbance peak of 320 nm in the peptide-Cy5, and the appearance of 280 nm of the peptide and 640 nm of Cy5.

<Experimental Example 3> Confirmation of cytotoxicity

Fibroblasts (L929), ovarian cancer cells (SKOV-3), skin epithelial cancer cells (A431), and melanoma cells (K1735) were cultured in RPMI-1640 (Hyclone). Human mesenchymal stem cells (hMSCs) are from Dulbecco's Modified Eagle Medium Low glucose (DMEM, Hyclone), and keratin cells (HaCaT) are from Dulbecco's Modified Eagle Medium Low glucose (DMEM, Hyclone). glucose). All cell lines were maintained at 37° C. and 5% CO ₂ in a medium containing 10% fetal bovine serum (FBS) and 1% antibiotic.

Cytotoxicity was measured by treating each cell with various peptide concentrations (0.1 to 20 μM). All cell lines were inoculated into a 96-well plate at a concentration of 5×10 ³ cells/well and cultured overnight to attach. Cells were treated with peptide samples diluted with serum-free medium. After 4 hours treatment with peptide-Cy5, the cells were washed with DPBS and treated with MTT solution for 2 hours. The sample plate treated with dimethyl sulfoxide (DMSO) was measured at 570 nm using a microplate reader.

7 is a graph showing the results of a cytotoxicity experiment according to an experimental example of the present invention.

Referring to FIG. 7, it was confirmed that toxicity was not observed at various concentrations of six cells and peptides, including fibroblasts, human mesenchymal stem cells, keratinocytes, ovarian cancer cells, skin epithelial cancer cells, and melanoma cells.

<Experimental Example 4> Confirmation of affinity of peptide-Cy5 to skin cells

Affinity was evaluated by comparing peptides of various sequences.

1.Flow cytometry (Fluorescence activated cell sorter, FACS)

Fibroblasts (L929), human mesenchymal stem cells (hMSCs), ovarian cancer cells (SKOV-3), cutaneous epithelial cancer cells (A431), melanoma cells (K1735) and keratinocytes (HaCaT) were each total in a 6-well plate. Incubated at 2×10 ⁵ cells/well, and 5 μM peptide-Cy5 was treated for 4 hours under maintenance conditions. After washing the cells with DPBS and centrifuging for 7 minutes at 2000 rpm, the cells were suspended with 500 μL of DPBS. Affinity was analyzed by FACS by comparing peptides of various sequences at excitation 650 nm and emission 670 nm (Cy5).

8 and 9 are graphs of flow cytometry results according to an experimental example of the present invention.

8 and 9, the synthesized (V ₅ ) ₁₆ , (VA ₂ G ₂ ) ₁₆ and (VH ₄ ) ₁₆ of the peptides containing histidine (VH ₄ ) ₁₆ are from skin cells HaCaT, A431 and K1735 It showed high affinity, and in particular, it was found to show the best affinity for A431, a skin cancer cell. As a result of additionally comparing the results of the flow cytometry with numerical values, the peptide containing histidine (VH ₄ ) ₁₆ was treated with 53 times, (V ₅ ) ₁₆ and (VA ₂ G ₂ ) ₁₆ compared to the control group not treated with the peptide. Compared to one control group, it was found to exhibit 18 times greater affinity efficiency.

2. Confocal

Fibroblasts (L929), human mesenchymal stem cells (hMSCs), keratinocytes (HaCaT), ovarian cancer cells (SKOV-3), cutaneous epithelial cancer cells (A431) and melanoma cells at a total concentration of 2×10 ⁵ cells/well (K1735) was incubated on a cover glass of a 12-well plate, and 5 μM peptide-Cy5 was treated for 4 hours under maintenance conditions. After treatment, the cells were washed twice with DPBS, fixed with 4% paraformaldehyde, and 4′,6-diamino-2-phenylindol; hereinafter, DAPI ). Images were confirmed at excitation 650 nm and emission 670 nm (Cy5) by a confocal laser scanning microscope (CLSM, LSM510 Meta, Zeiss, Germany).

10 is a microscope analysis image according to an experimental example of the present invention.

Referring to FIG. 10, it can be seen that the microscopic analysis image is consistent with the flow cytometric analysis of FIGS. 8 and 9.

<Experimental Example 5> Peptide-Cy5 affinity test for pig skin

Among the types of peptides performed in the cell experiment, an affinity experiment in pig skin was performed by selecting an experimental group with two representative (V ₅ ) ₁₆ and (VH ₄ ) ₁₆ and 1x PBS as a control.

On the back of the pig, the skin of a size of 2×2 and a thickness of 700 μm was treated with a Franz cell at a concentration of 20 μM for 4 hours, washed with DPBS and fixed with 4% paraformaldehyde to a thickness of 15 μM. The tissue was sectioned. The sectioned tissue was stained with DAPI and analyzed by confocal microscopy.

11 is a microscope analysis image according to an experimental example of the present invention.

Referring to FIG. 11, as a result of comparing the affinity in pig skin using a confocal microscope, it was confirmed that the synthesized (VH ₄ ) ₁₆ remained in the skin compared to (V ₅ ) ₁₆ .

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. Do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> PEPTIDES FOR TARGETING SKIN CELLS AND USE THEREOF <130> ADP-2019-0005 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 410 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 1 Met Gly His Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 1 5 10 15 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 20 25 30 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 35 40 45 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 50 55 60 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 65 70 75 80 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 85 90 95 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 100 105 110 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 115 120 125 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 130 135 140 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 145 150 155 160 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 165 170 175 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 180 185 190 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 195 200 205 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 210 215 220 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 225 230 235 240 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 245 250 255 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 260 265 270 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 275 280 285 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 290 295 300 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 305 310 315 320 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 325 330 335 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 340 345 350 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 355 360 365 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 370 375 380 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 385 390 395 400 Gly Val Pro Gly Trp His Ala Ser Asn Arg 405 410 <210> 2 <211> 410 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 2 Met Gly His Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly 1 5 10 15 Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val 20 25 30 Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro 35 40 45 Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly 50 55 60 Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Val 65 70 75 80 Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly 85 90 95 Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val 100 105 110 Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro 115 120 125 Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly 130 135 140 Ala Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Ala 145 150 155 160 Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly 165 170 175 Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val 180 185 190 Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro 195 200 205 Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly 210 215 220 Gly Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Gly 225 230 235 240 Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly 245 250 255 Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val 260 265 270 Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro 275 280 285 Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly 290 295 300 Val Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Ala 305 310 315 320 Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly 325 330 335 Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val 340 345 350 Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro 355 360 365 Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly 370 375 380 Ala Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly 385 390 395 400 Gly Val Pro Gly Trp His Ala Ser Asn Arg 405 410 <210> 3 <211> 410 <212> PRT <213> Artificial Sequence <220> <223> peptide <400> 3 Met Gly His Gly Val Gly Val Pro Gly His Gly Val Pro Gly His Gly 1 5 10 15 Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly Val Gly Val 20 25 30 Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro 35 40 45 Gly His Gly Val Pro Gly Val Gly Val Pro Gly His Gly Val Pro Gly 50 55 60 His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly Val 65 70 75 80 Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly 85 90 95 Val Pro Gly His Gly Val Pro Gly Val Gly Val Pro Gly His Gly Val 100 105 110 Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro 115 120 125 Gly Val Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly 130 135 140 His Gly Val Pro Gly His Gly Val Pro Gly Val Gly Val Pro Gly His 145 150 155 160 Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly 165 170 175 Val Pro Gly Val Gly Val Pro Gly His Gly Val Pro Gly His Gly Val 180 185 190 Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly Val Gly Val Pro 195 200 205 Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly 210 215 220 His Gly Val Pro Gly Val Gly Val Pro Gly His Gly Val Pro Gly His 225 230 235 240 Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly Val Gly 245 250 255 Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val 260 265 270 Pro Gly His Gly Val Pro Gly Val Gly Val Pro Gly His Gly Val Pro 275 280 285 Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly 290 295 300 Val Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His 305 310 315 320 Gly Val Pro Gly His Gly Val Pro Gly Val Gly Val Pro Gly His Gly 325 330 335 Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val 340 345 350 Pro Gly Val Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro 355 360 365 Gly His Gly Val Pro Gly His Gly Val Pro Gly Val Gly Val Pro Gly 370 375 380 His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His 385 390 395 400 Gly Val Pro Gly Trp His Ala Ser Asn Arg 405 410

Claims (15)

A peptide for targeting skin cells consisting of an amino acid sequence represented by the following formula 1:
[Equation 1]
MGHG-[abcde] _n -WHASNR
In Equation 1, a, b, c, d and e may each be the same or different,
Wherein a, b, c, d, and e are any one pentapeptide selected from the group consisting of XGVPG, GVPGX, VPGXG, PGXGV and GXGVP, respectively, and X of the pentapeptide is valine (V), alanine (alanine, A), may be substituted with glycine (G) or histidine (H),
n is from 1 to 100. The method of claim 1,
The skin cell targeting peptide,
Skin cell targeting peptide consisting of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3. The method of claim 1,
The skin cells,
Peptide for targeting skin cells, characterized in that at least one selected from the group consisting of fibroblasts, human mesenchymal stem cells and keratinocytes. A peptide for targeting skin cancer cells consisting of an amino acid sequence represented by the following formula 1:
[Equation 1]
MGHG-[abcde] _n -WHASNR
In Equation 1, a, b, c, d and e may be the same or different,
Wherein a, b, c, d, and e are any one pentapeptide selected from the group consisting of XGVPG, GVPGX, VPGXG, PGXGV and GXGVP, respectively, and X of the pentapeptide is valine (V), alanine (alanine, A), can be substituted with glycine (G) or histidine (H),
n is from 1 to 100. The method of claim 4,
The skin cancer cell targeting peptide,
A peptide for targeting skin cancer cells consisting of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3. The method of claim 4,
The skin cancer cells,
Skin cancer cell targeting peptide, characterized in that at least one selected from the group consisting of skin epithelial cancer cells, ovarian cancer cells, and melanoma cells. delete delete delete delete delete delete delete delete delete

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