Growth factor FGF, SCF-delivering cell penetrating peptide, and wound healing composition thereof

Abstract

The present invention relates to a cell penetrating peptide for delivering growth factors FGF and SCF, and a cosmetic composition thereof for wound healing, dermatitis prevention and treatment, and skin barrier formation. According to the present invention, a cell-penetrating peptide in which a carrier-transmitting peptide (CDP) is bonded to FGF or SCF according to the present invention enables a growth factor to effectively penetrate the skin or cells, thereby ensuring further convenience while improving the intrinsic efficacy of the growth factor in vivo as much as possible. The composition not only has excellent effects on wound healing, skin barrier formation, dermatitis improvement and transdermal delivery of growth factors, but also is very suitable for related industries, i.e., pharmacy, especially in the field of cosmetics.

Description

Technical Field

[0001] This relates to a growth factor FGF and a cell-penetrating peptide that delivers SCF, and compositions thereof for wound healing, prevention and treatment of dermatitis, formation of skin barrier, and cosmetic use. Background Technology

[0002] The skin covers the entire body and is generally composed of three layers: the epidermis, dermis, and subcutaneous fat. The epidermis, the outermost layer, contains the stratum corneum, which functions as a skin barrier, protecting the body from various external irritants (Li Hengyu, 2008). The stratum corneum is composed of multiple layers of flattened keratinocytes, filled with hydrophobic, insoluble fibrous proteins called keratin. Between each keratinocyte are intercellular lipids composed of ceramides, free fatty acids, cholesterol, etc., forming a "layered structure." Due to this structural feature, it can inhibit moisture evaporation from the skin and effectively protect the body from external pressure or harmful stimuli. However, the active ingredients in cosmetics cannot be easily absorbed (Honeywell-Nguyen and Bouwstra, 2005).

[0003] Technologies that improve transdermal absorption are crucial. An exceptionally strong skin barrier prevents the absorption of active ingredients; therefore, even highly effective ingredients cannot exert their full potential when applied to the skin. Thus, it is essential to promote skin absorption of active ingredients and maximize their efficacy.

[0004] Cell penetrating peptides (CPPs) are short peptides of approximately 10-30 units that penetrate cell membranes, most of which are derived from protein-transduction domains (PTDs) or membrane-translocating sequences (MTS). Therefore, CPPs are also referred to as PTDs or MTSs. Unlike conventional pathways for external substances to enter the cell, CPPs play a crucial role, translocating into the cell without damaging the cell membrane. Even DNA or large proteins that cannot cross the cell membrane can be delivered into the cell, and numerous studies have been conducted on them since the 1990s.

[0005] It is understood that there are currently reports of CPPs derived from about 50 different proteins, but the precise mechanism by which they enter cells has not yet been clearly revealed. However, in some studies that use CPPs, when ordinary external substances enter the cell, they are locked by the endosomes within the cell, then broken down by lysosomes and absorbed by the cytoplasm. In contrast, CPPs enter the cell through endocytosis, where they detach from the endosomes, avoid being broken down by lysosomes, and enter the cell.

[0006] Firstly, it is understood that well-known cell-penetrating peptides (CPPs), such as HIV-Tat and antennapedia, are mainly short peptides with fewer than 30 positively charged amino acids. They are not only proteins, but can also deliver DNA, RNA, fats, carbohydrates, compounds, or viruses into cells. They are reported to be receptor-independent and penetrate the cell membrane through mechanisms such as endocytosis, phagocytosis, and direct penetration.

[0007] HIV-Tat is the most widely used peptide, and its safety has been questioned because its peptides are derived from the virus.

[0008] In view of the above needs, a novel cell-penetrating peptide with higher delivery efficiency for carriers such as compounds, peptides and proteins than traditional CPP was developed using the in silico method. This cell-penetrating peptide is called a carrier delivery peptide (CDP).

[0009] FGF (fibroblast growth factor) is a growth factor that is effective against fibroblasts in the human body. It is an oligopeptide composed of 155 amino acids with a molecular weight of 17.3 kDa. It specifically binds to the fibroblast growth factor receptor (FGFR) on the cell surface, working on various cells in the body, including blood vessel, muscle, bone, and nerve cells, to promote cell proliferation and differentiation. Furthermore, it has strong angiogenesis and neuroprotective effects. When injuries occur in the body, it is an essential growth factor for healing, showing remarkable efficacy in treating burns, bedsores, and skin wounds. In the physiological activity of the skin, it is effective in enhancing the activity of skin cells reduced by aging, promoting regeneration after surgery and injury, and promoting collagen synthesis. In the hair, it helps increase the number and growth of hair follicle cells, contributing to hair formation and regeneration, and maintaining healthy black hair.

[0010] SCF (stem cell factor) is a stem cell growth factor, existing as a kit-ligan, KL, or steel factor, and is a cytokine bound to the c-Kit receptor (CD117). SCF is produced by fibroblasts and endothelial cells, existing in both membrane-bound and water-soluble protein forms, and participates in hematopoiesis, spermatogenesis, and melanin formation. Soluble SCF has a molecular weight of 18.5 kDa and forms a dimer. As the most important growth factor for embryonic development, it participates in adult hematopoiesis, spermatogenesis, and melanin formation. Stem cells exist in almost all cells, including the skin, and SCF, as an essential factor for activating stem cells, can prevent skin aging and promote the formation of new hair follicles.

[0011] To overcome the drawbacks of the aforementioned traditional technologies, the inventors have recombined cell-penetrating peptides that can more effectively deliver growth factors (FGF, SCF) through cell or skin penetration. It has been confirmed that, when used, these peptides can be applied to moisturizing and anti-inflammatory effects in inflammatory response-induced environments, as well as wound healing in pruritus-induced conditions, by utilizing the morphology of the recombinant cell-penetrating growth factor protein. Furthermore, it has been confirmed that they can prevent dermatitis while forming a skin barrier, thus also making them suitable for cosmetics. This invention was completed based on the above. Summary of the Invention

[0012] The problem to be solved

[0013] The purpose of this invention is to provide a cell-penetrating peptide (CPP) for which a carrier delivery peptide (CDP) is bonded to an FGF (stem cell factor) peptide.

[0014] The purpose of this invention is to provide a recombinant expression vector that expresses a cell-penetrating peptide of a carrier delivery peptide (CDP) bonded to an FGF (fibroblast growth factor) or SCF (stem cell factor) peptide.

[0015] Furthermore, another object of the present invention is to provide a pharmaceutical composition for wound treatment that includes cell-penetrating peptides as an active ingredient.

[0016] Furthermore, another object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of dermatitis, which includes cell-penetrating peptides as an active ingredient.

[0017] Furthermore, another object of the present invention is to provide a pharmaceutical composition that includes a cell-penetrating peptide as an active ingredient for treating skin barrier formation caused by dermatitis.

[0018] Furthermore, another objective of the present invention is to provide a cosmetic composition for improving skin barrier and skin moisturizing by including cell-penetrating peptides as an active ingredient.

[0019] Furthermore, another object of the present invention is to provide a composition for transdermal delivery of growth factors.

[0020] Problem Solution

[0021] This invention provides a cell-penetrating peptide (CPP) for which a carrier delivery peptide (CDP) is bonded to an FGF (fibroblast growth factor) or SCF (stem cell factor) peptide.

[0022] The present invention provides a recombinant expression vector that expresses a cell-penetrating peptide of FGF (fibroblast growth factor) or SCF (stem cell factor) peptide bonded to a carrier delivery peptide (CDP).

[0023] Furthermore, the present invention provides a pharmaceutical composition for wound treatment that includes a cell-penetrating peptide as an active ingredient.

[0024] Furthermore, the present invention provides a pharmaceutical composition for the prevention and treatment of dermatitis, which includes cell-penetrating peptides as an active ingredient.

[0025] Furthermore, the present invention provides a pharmaceutical composition for treating skin barrier formation caused by dermatitis, including cell-penetrating peptides as an active ingredient.

[0026] Furthermore, the present invention provides a cosmetic composition for improving skin barrier and skin moisturizing by including cell-penetrating peptides as an active ingredient.

[0027] Furthermore, the present invention provides a composition for transdermal delivery of growth factors.

[0028] Invention Effects

[0029] Cell-penetrating peptides are FGF or SCF with carrier delivery peptides (CDPs) bonded to them, as described in this invention. They enable growth factors to effectively penetrate the skin or cells, thereby maximizing the inherent efficacy of the growth factors in vivo while ensuring further convenience. They not only have excellent effects on wound healing, skin barrier formation, dermatitis improvement, and transdermal delivery of growth factors, but are also very suitable for related industries, namely, pharmaceuticals, especially the cosmetics field. Attached Figure Description

[0030] Figure 1 This invention illustrates a view of a recombinant expression vector for the production of SCF-CDP (peptide-bonded vector delivery peptide) or FGF-CDP recombinant protein.

[0031] A: A view of the carrier produced by SCF-CDP.

[0032] B: A view of the carrier produced by FGF-CDP.

[0033] Figure 2 This is a view of the expression and purification of SCF-CDP or FGF-CDP recombinant proteins in this invention.

[0034] A: Electrophoresis results of SCF-CDP recombinant protein

[0035] B: Electrophoresis results of FGF-CDP recombinant protein

[0036] Figure 3 This is a view of the skin cell penetration ability of the SCF-CDP or FGF-CDP recombinant protein in this invention.

[0037] A: Negative control group (BSA)

[0038] B: Penetration ability of SCF-CDP recombinant protein

[0039] C: Penetration ability of FGF-CDP recombinant protein

[0040] Figure 4 This is a cytotoxic view of different concentrations of SCF-CDP, FGF-CDP, and SCF+FGF-CDP recombinant proteins in human fibroblasts with cytokine-induced dermatitis (TNF-α, IL-4, IL-13, IL-22).

[0041] A: Cytotoxicity of FGF-CDP recombinant protein

[0042] B: Cytotoxicity of SCF-CDP recombinant protein

[0043] C: Cytotoxicity of SCF+FGF-CDP recombinant protein

[0044] Figure 5 This is a graph evaluating the wound healing efficacy of SCF-CDP, FGF-CDP, and SCF+FGF-CDP recombinant proteins in a dermatitis model.

[0045] Figure 6 In a dermatitis model, the phosphorylation of the signal transduction protein ERK of SCF-CDP, FGF-CDP, and SCF+FGF-CDP recombinant proteins was confirmed, and the diagrams of cell translocation and differentiation were verified.

[0046] Figure 7In a dermatitis model, the ratios of the signal transduction proteins p-ERK1 / 2 / ERK1 / 2 and p-Egr1 / ERK1 / 2 of the SCF-CDP, FGF-CDP, and SCF+FGF-CDP recombinant proteins were confirmed to verify the views of cell translocation and differentiation.

[0047] A: The ratio of p-ERK1 / 2 / ERK1 / 2

[0048] B: The ratio of p-Egr1 / ERK1 / 2

[0049] Figure 8 In a dermatitis model, the ratios of the signal transduction proteins p-Rac1 / GAPDH and p-Akt / GAPDH of the recombinant SCF-CDP, FGF-CDP, and SCF+FGF-CDP proteins were confirmed to verify the view of cell translocation and differentiation.

[0050] A: The ratio of p-Rac1 / GAPDH

[0051] B: The ratio of p-Akt / GAPDH

[0052] Figure 9 This is a view of the expression of moisturizing (HAS2) mRNA after treating skin keratinocytes (HaCaT) with dermatitis-inducing substances at different time points (24H, 48H, 72H) and then treating them with SCF-CDP, FGF-CDP and SCF+FGF-CDP recombinant proteins.

[0053] Figure 10 This is a view of the skin barrier improvement efficacy of SCF-CDP, FGF-CDP, and SCF+FGF-CDP recombinant proteins in a dermatitis model.

[0054] A: Expressions of CLDN1 and CLDN4

[0055] B: Formulaic charts for CLDN1 expression

[0056] C: Formulaic charts for CLDN4 expression

[0057] Figure 11 This is a view of skin keratinocytes (HaCaT) treated with dermatitis-inducing substances and expressed anti-inflammatory factor mRNA through treatment with SCF-CDP, FGF-CDP and SCF+FGF-CDP recombinant proteins.

[0058] A: mRNA expression of inflammatory cytokine (IL-1α)

[0059] B: mRNA expression of inflammatory cytokines (IL-1β) Detailed Implementation

[0060] The present invention will now be described in detail with reference to the accompanying drawings through implementation examples. However, the following implementation examples are illustrative of the invention, and detailed descriptions of well-known technologies or structures may be omitted if they unnecessarily confuse those skilled in the art with the essential points of the invention, without limiting the invention. Various modifications and applications are possible within the scope of the following claims and their equivalents.

[0061] Furthermore, the terminology used in this specification is for the reasonable expression of preferred embodiments of the invention and may vary depending on the user's, the user's, or the conventions of the technical field to which this invention pertains. Therefore, the definition of these terms should be based on the entire contents of this specification. Throughout this specification, when a part "includes" a constituent element, unless specifically stated to the contrary, it means that other constituent elements are not excluded, but rather further included.

[0062] Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art related to this invention. Furthermore, while this specification describes preferred methods or specimens, similar or equivalent methods are also included within the scope of this invention. All publications referenced in this specification are incorporated herein by reference.

[0063] This invention provides a cell-penetrating peptide (CPP) for which a carrier delivery peptide (CDP) is bonded to an FGF (fibroblast growth factor) or SCF (stem cell factor) peptide.

[0064] The cell-penetrating peptide may be an FGF or SCF with a CDP bonded to its C-terminus, but is not limited to this.

[0065] The CDP may include the amino acid of sequence number 1, the FGF may include the amino acid of sequence number 2, and the SCF may include the amino acid of sequence number 3.

[0066] The FGF and CDP bonded recombinant peptide (FGF-CDP) may include the amino acid of sequence number 4, and the SCF and CDP bonded recombinant peptide (SCF-CDP) may include the amino acid of sequence number 5.

[0067] In this invention, the term "carrier-delivered peptide (CDP)" refers to a novel cell-penetrating peptide developed by the inventors using the in silico method, which has a higher delivery efficiency for carriers such as compounds, peptides, and proteins compared to existing CPPs.

[0068] The peptide or variant is bound to a biologically active substance, delivering the substance into cells or living organisms, exhibiting activity related to physiological phenomena or therapeutic purposes. Cell-penetrating peptides or variants, possessing the substance delivery function of this invention, are extremely small peptides, thus enabling biological interference against potentially generated active substances.

[0069] The variant can be modified to have the following structure: the amino acids of the peptide are substituted or deleted in whole or in part, or the stability of the local amino acid sequence is increased in vivo.

[0070] or

[0071] To improve hydrophilicity, local amino acid sequences are modified, or some or all amino acids are replaced with L- or D-amino acids, or local amino acids are several modified monolayers. The modified variant is given cell-penetrating and intracellular delivery capabilities.

[0072] The present invention provides a recombinant expression vector that expresses a cell-penetrating peptide of FGF (fibroblast growth factor) or SCF (stem cell factor) peptide bonded to a carrier delivery peptide (CDP).

[0073] The recombinant expression vector may include a peptide sequence having amino acid sequences of sequence numbers 1 to 5 and a tag sequence for easy purification of the peptide, such as consecutive histidine codons, maltose-binding protein codons, and Myc codons, and may further include a fusion partner for increasing the solubility of the fusion compound. Furthermore, for the overall structural and functional stability of the peptide or the flexibility of the proteins encoded by individual genes, it may further include a spacer amino acid or base sequence containing one or more glycine, proline, or amino acid AAY. Moreover, to remove unnecessary portions of the peptide, it may include sequences that can be specifically cleaved by enzymes, expression emptying sequences, and intracellular delivery confirmation markers or reporter gene sequences, but is not limited thereto. The expression emptying sequence used in the recombinant expression vector of the present invention consists of a regulatory domain, which includes a specific promoter on cells, tissues, and internal organs where target DNA and / or RNA are selectively delivered or expressed.

[0074] Furthermore, the present invention provides a pharmaceutical composition for wound treatment that includes a cell-penetrating peptide as an active ingredient.

[0075] The compositions described in this invention can be used as pharmaceutical compositions for prevention and treatment, and may further include pharmaceutically permissible carriers. "Pharmaceutically permissible" means physiologically permissible and generally does not cause gastrointestinal disturbances, dizziness, or other allergic reactions or similar reactions when taken by humans. For example, pharmaceutically permissible carriers include oral carriers such as lactose, starch, cellulose derivatives, magnesium stearate, and stearic acid, as well as non-oral carriers such as water, suitable essential oils, physiological saline, aqueous glucose, and ethylene glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-p-hydroxybenzoate, and chlorobutanol. In addition, pharmaceutically permissible carriers can be found in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995). The pharmaceutical composition described in this invention, together with the aforementioned pharmaceutically permissible carrier, can be formulated into dosage forms in a suitable manner according to methods known in the art. That is, the pharmaceutical composition described in this invention can be formulated into various non-oral or oral dosage forms according to known methods. Representative non-oral dosage forms include injections, preferably isotonic aqueous solutions or suspensions. Injections can be prepared using suitable dispersants, wetting agents, and suspending agents according to techniques known in the art. For example, various components can be dissolved in physiological saline or buffer solutions to prepare injectable formulations. Furthermore, oral dosage forms include powders, granules, tablets, pills, and capsules, etc., and are not limited thereto.

[0076] The pharmaceutical composition formulated using the above method can be administered at an effective dose via multiple routes, including oral, transdermal, subcutaneous, intravenous, or intramuscular routes. Here, "effective dose" refers to the amount at which a patient exhibits a preventive or therapeutic effect. The dosage of the pharmaceutical composition described in this invention can be appropriately selected based on the route of administration, the recipient, age, gender, weight, individual differences, and disease condition. Preferably, the pharmaceutical composition described in this invention can be formulated with different active ingredient contents according to the severity of the disease; however, preferably, it can be administered repeatedly throughout the day at an effective dose of 0.1–1000 mg / kg / day. Furthermore, the composition described in this invention can also be administered concurrently with known chemicals that have preventive, ameliorative, or therapeutic effects.

[0077] Furthermore, the present invention provides a pharmaceutical composition for the prevention and treatment of dermatitis, which includes cell-penetrating peptides as an active ingredient.

[0078] The cause of the wound may be dermatitis, but the dermatitis is caused by the expression of inflammatory cytokines and may be selected from the group consisting of atopic dermatitis, seborrheic dermatitis, xerotic dermatitis, contact dermatitis and nummular dermatitis, but is not limited to this here.

[0079] Furthermore, the present invention provides a pharmaceutical composition for treating skin barrier formation caused by dermatitis, including cell-penetrating peptides as an active ingredient.

[0080] In this invention, the term "skin barrier" is formed on the outermost layer of the skin, namely the stratum corneum. As a protective barrier for the skin, it prevents the body from losing the water and electrolytes it needs, and conversely, it prevents harmful pathogens from invading the skin.

[0081] Furthermore, the present invention provides a cosmetic composition for improving skin barrier and skin moisturizing by including cell-penetrating peptides as an active ingredient.

[0082] The cosmetic compositions of this invention can be formulated into dosage forms comprising the group consisting of: solutions, topical ointments, creams, foams, nourishing lotions, softening lotions, masks, toners, lotions, sunscreens, serums, soaps, liquid cleansers, bath additives, sunscreens, sunscreen oils, suspensions, emulsions, pastes, gels, skin lotions, powders, soaps, surfactant-oil-based cleansers, skin oils, foundation creams, foundation lotions, skin wax foundations, repair solutions, and sprays, but are not limited thereto. Furthermore, the cosmetic compositions of this invention may further include one or more of the cosmetically permissible carriers typically formulated in skincare and cosmetic products. For example, oils, water, surfactants, moisturizers, low-alcohol content, thickeners, chelating agents, pigments, preservatives, fragrances, etc., may be appropriately incorporated as common ingredients, but are not limited thereto.

[0083] Furthermore, the present invention provides a composition for transdermal delivery of growth factors.

[0084] The growth factor, as a biologically active substance, exhibits activity related to physiological phenomena or therapeutic purposes, manifested as delivery into cells or the body. Furthermore, the biologically active substance can be DNA, RNA, protein, fat, carbohydrate, and chemical compounds. The chemical compounds can be anticancer agents, immunotherapies, antiviral agents, antibiotics, and growth, evolution, or differentiation factors of organisms. In this invention, the cell-penetrating peptide with substance delivery function is an extremely short peptide, thus minimizing potential biological interference from the active substance. The peptide or variant and the fusion of the biologically active substance are delivered to the living organism via intravenous, intraperitoneal, intramuscular, subcutaneous, intradermal, intranasal, mucosal, inhalation, and oral administration. To deliver the fusion to specific cells, tissues, or visceral organs, the biologically active substance can be bonded to the extracellular portion of a ligand that selectively binds to a receptor specifically expressed in that cell, tissue, or visceral organ, or to a monoclonal antibody (mAb) that specifically binds to such a receptor or ligand, and to a modified morphology. The binding of the peptide to the biologically active substance can be based on indirect binding at the nucleotide level using cloning methods with an expression vector, or direct binding between the peptide and the biologically active substance through chemical or physical covalent or non-covalent bonds.

[0085] The present invention is further described in detail through the following embodiments. However, the following embodiments are only used to illustrate the content of the present invention and are not intended to limit the present invention.

[0086] [Experimental Example 1] Preparation of recombinant expression vectors for producing SCF-CDP (Cargo Delivery Peptide) or FGF-CDP recombinant proteins

[0087] CDP’s growth factor delivery efficacy is significant in the self-developed cell-penetrating peptide. To verify its significant effect, the cell-penetrating peptide was bound to SCF or FGF to prepare SCF-CDP and FGF-CDP recombinant proteins for use as experimental groups.

[0088] First, SCF or FGF moieties were prepared via gene synthesis. Cell-penetrating peptides were attached to the synthesized gene C-terminal via PCR, followed by cleavage with NdeI / XhoI, including stopping codon expression, without expressing the c-terminal histogram. Ligation was performed on the pET 28a(+) vector, and then transformation was performed on BL21(DE3) to prepare a recombinant expression vector. This was performed by Cosmogenetech. The prepared vector is shown below. Figure 1 As shown.

[0089] Table 1

[0090]

[0091] [Experimental Example 2] Expression and purification of SCF-CDP (Cargo Delivery Peptide) or FGF-CDP recombinant protein

[0092] The recombinant protein was cultured as follows: Plasmids expressing the recombinant protein were transformed into *E. coli* BL21(DE3) using a heat shock method (42°C, 90 seconds) to prepare gene-modified organisms. This included... The transformed strain of kanamycin was cultured on LB medium at 37°C for 24 hours. LB medium Add culture medium once With kanamycin The cells were cultured a second time at 37°C until the OD600 value of the cells reached 0.8 to 1.0.

[0093] Recombinant protein expression was induced by adding 1 mM IPTG (Isopropylβ-d-1-thiogalactopyranoside) and cultured at 37°C and 200 rpm for 20 hours. Cells were collected by centrifugation and lysed. Then, the cells were centrifuged at 10,000 rpm for 1 hour at 4°C, yielding only the supernatant. The recombinant protein was purified using a Nickel column, followed by dialysis with sodium phosphate buffer (pH 7.4) for 20 hours. To verify the purification of the recombinant protein, electrophoresis was performed on a 10% SDS-PAGE gel.

[0094] like Figure 2As shown, recombinant protein was observed after Elution on the SDS-PAGE gel. This confirms that the recombinant protein was successfully purified according to the experimental protocol.

[0095] [Experimental Example 3] Skin tissue penetration ability of SCF-CDP (Cargo Delivery Peptide) or FGF-CDP recombinant protein

[0096] Using Alexa 488-NHS (Invitrogen), the process of labeling SCF-CDP or FGF-CDP recombinant proteins with fluorescent labeling (FITC) was carried out at room temperature for 1 hour.

[0097] Alexa 488-NHS (Invitrogen) was dissolved in DMSO and added to a solution containing recombinant protein, allowing it to react at room temperature for 1 hour. To remove residual staining, dye was removed using a Pierce dye removal column (ThermoScientific).

[0098] Fluorescently labeled recombinant protein was applied to piglet skin and allowed to react at room temperature for 1 hour. After removing residual solution from the tissue with paper towels, fixation was performed using 4% paraformaldehyde at 4°C for 12 hours. Residual 4% paraformaldehyde was then removed from the tissue with paper towels, followed by dehydration with 15% and 30% sucrose solutions. The dehydrated tissue was prepared into cryosections (20 μm), placed on slides, and FITC fluorescence was observed using confocal microscopy. The results of the FITC fluorescence observations are as follows: Figure 3 As shown.

[0099] from Figure 3 It has been confirmed that, compared with the negative control group (BSA), the recombinant protein (SCF-CDP or FGF-CDP) described in this invention has a higher skin penetration ability.

[0100] [Experimental Example 4] In vitro skin conditioning to induce dermatitis

[0101] A common symptom in patients with psoriasis and atopic dermatitis is elevated expression of TNF-α (Tumor necrosis factor) and the interleukin family compared to normal skin. This can trigger inflammatory responses, decreased hydration, and damage to the skin barrier. In particular, increased keratin expression in these patients induces hyperkeratosis (increased stratum corneum thickness), resulting in slower keratin shedding compared to normal skin. However, the skin's moisturizing effect is significantly lower than normal skin, which can quickly lead to pruritus and skin wounds and barrier damage caused by scratching.

[0102] The inventors constructed an in vitro skin inflammation model by inducing an increase in IL-1 and a decrease in HAS2 (hyaluronic acid synthesis 2) that are prevalent in dermatitis patients. This starvation process was performed for 72 hours. On a 24-well plate, samples were collected at 5x10... 4 Seeding of HaCaT (keratinocytes) cells was performed in cells / wells and then cultured for 24 hours. Next, starvation was performed by treating the cells with FBS-free DMEM. Cytokine-derived media were then treated on FBS-free DMEM, and the recovered cells were lysed using Trizol to obtain RNA only. The resulting RNA concentration was brought to 1.5 μg, and the optimal time for reverse transcription (RT) via cDNA was determined, thus constructing a skin inflammation model.

[0103] [Experimental Example 5] Cytotoxicity experiments of SCF-CDP or FGF-CDP and SCF+FGF-CDP recombinant proteins were conducted in a dermatitis model.

[0104] To confirm the cytotoxicity of the raw material cells, determine the maximum treatment concentration, and test the cytotoxicity of purified SCF-CDP, FGF-CDP, and SCF+FGF-CDP recombinant proteins, cytotoxicity was assessed using the MTT assay after treating cells with the recombinant proteins at different concentrations.

[0105] In human fibroblast cells, treatment with cytokines (TNF-α, IL-4, IL-13, IL-22) induced dermatitis. Specifically, 5x10⁶ cells were seeded on 96-well plates. 5Cells were cultured for 24 hours. After media removal, cells were treated with 100 μL of recombinant protein solutions at different concentrations on serum-free media and cultured for 24 hours. MTT reagent was added to each well (10 μL per well), and cells were further cultured for 4 hours. The plates were shaken for 1 minute, and absorbance was measured at OD 420–480 nm.

[0106] like Figure 4 As shown, control and comparative studies confirmed that SCF-CDP, FGF-CDP, and SCF+FGF-CDP recombinant proteins were non-toxic at a maximum of 10 pm.

[0107] [Experimental Example 6] Evaluation of the wound healing efficacy of SCF-CDP or FGF-CDP and SCF+FGF-CDP recombinant proteins in a dermatitis model.

[0108] Patients with dermatitis often develop wounds due to scratching caused by itching. This study investigated the efficacy of recombinant proteins SCF-CDP, FGF-CDP, and SCF+FGF-CDP in wound healing methods. ERK1 / 2 and the PI3K / Akt pathway are representative transcriptional regulators involved in cell proliferation and translocation; phosphorylation of ERK1 / 2 triggers the synthesis of Egr1 in the cell nucleus. Migration assays confirmed whether the recombinant proteins described in this invention affect cell proliferation and translocation. Specifically, HaCaT cells were seeded at 8 x 10⁵ cells per well plate and cultured for 24 hours. Subsequently, starvation was performed by treating with DMEM without FBS.

[0109] Using a pipette tip, a wound was artificially created in the center of the test plate. Cytokine derivatives (TNF-α, IL-4, IL-13, IL-22) and SCF-CDP or FGF-CDP and SCF+FGF-CDP proteins at a concentration of 10 pm were treated on DMEM without FBS. Cell translocation was then observed using images at time intervals (24H, 48H, 72H).

[0110] from Figure 5 It has been confirmed that SCF-CDP or FGF-CDP and SCF+FGF-CDP recombinant proteins affect cell proliferation and translocation, and have a wound healing effect.

[0111] Furthermore, to confirm whether the signal transduction protein ERK involved in cell translocation and differentiation was phosphorylated, the cells after the experiment were scraped off using a scraper and kept for later use.

[0112] Specifically, particulate matter was obtained by centrifugation at 14,000 rpm for 20 minutes, and then quantified using a bradford assay. Proteins were separated by size using SDS-PAGE electrophoresis, and then transferred to a membrane using a transfer. Subsequently, different treatments with ERK, p-ERK, and GAPDH antibodies were performed to confirm the analysis results of wound healing-promoting transcription factors and the increased gene expression rate.

[0113] like Figure 6 As shown, the analysis results of the increase in the expression rate of wound healing-promoting transcription factors and genes confirmed that SCF-CDP or FGF-CDP and SCF+FGF-CDP recombinant proteins affect cell migration and differentiation and have a wound healing effect.

[0114] And, as Figure 7 As shown, the SCF-CDP treatment group increased ERK1 / 2 phosphorylation by 1.3 times compared to the inflammation treatment group, and the FGF-CDP treatment group increased ERK1 / 2 phosphorylation by 1.2 times compared to the inflammation treatment group.

[0115] And, as Figure 8 As shown, the SCF-CDP treatment group increased p-Akt expression by 1.2-fold compared to the inflammation treatment group, while the FGF-CDP treatment group increased it by 2.1-fold compared to the inflammation treatment group. It was confirmed that the FGF-CDP treatment group increased Rac1 expression by 1.5-fold compared to the inflammation treatment group.

[0116] Based on the above results, SCF-CDP promoted cell translocation by increasing Egr1 and p-Akt expression, while FGF-CDP induced cell translocation by increasing p-Akt and Rac1 expression.

[0117] [Experimental Example 7] Evaluation of the moisturizing efficacy of SCF-CDP or FGF-CDP and SCF+FGF-CDP recombinant proteins in a dermatitis model.

[0118] Hyaluronan synthase 2 (HAS2) protein plays a crucial role in hyaluronic acid synthesis. Patients with allergic dermatitis commonly exhibit downregulated HAS2 expression, leading to impaired moisturizing function. Among HAS families, human cells show the highest HAS2 expression. Aquaporin 3 (AQP3), one of the moisturizing-related genes found in human cell membranes, functions as a cellular drainage system, promoting the diffusion of synthesized water. It has been confirmed that in dermatitis models, the recombinant proteomes of SCF-CDP, FGF-CDP, and SCF+FGF-CDP affect the expression of HAS2 and AQP3.

[0119] Specifically, on the 6well plate, 5x10 5 HaCaT cells were seeded and cultured for 24 hours. Then, they were starved by treating with DMEM without FBS. Next, on the FBS-free DMEM, cells were treated with Cytokine derivatives and recombinant proteins SCF-CDP, FGF-CDP, and SCF+FGF-CDP. Cells were then lysed and recovered using Trizol to obtain only RNA. The resulting RNA concentration was brought to 1.5 μg, and the increased HAS2 mRNA expression was confirmed by reverse transcription (RT) of cDNA. The positive control group used retinoic acid (RTN), a substance previously reported to increase HAS2 expression.

[0120] like Figure 9 As shown, compared to the Cytokine-treated group, the positive control group (RTN) showed approximately 9.48-fold increased HAS2 expression, 33.1-fold increased SCF-CDP, and 20.21-fold increased FGF-CDP, while the mixed treatment group showed a 23.56-fold increase. This confirms that, compared to the positive control group (RTN), the increase in HAS2 expression was equivalent to 349.16% (SCF-CDP), 213.19% (FGF-CDP), and 248.52% (mixed treatment group), respectively, indicating a higher expression of the moisturizing gene. This suggests that upregulating HAS2 expression increases hyaluronic acid synthesis, resulting in higher moisturizing efficacy for patients with allergic dermatitis.

[0121] [Experimental Example 8] Evaluation of the skin barrier function of SCF-CDP or FGF-CDP and SCF+FGF-CDP recombinant proteins in a dermatitis model.

[0122] Tight junction protein-1 (CLDN1) is a transmembrane protein known as Tight Junction (TJ), a representative protein that tightly binds cells together, strengthening the skin barrier. Tight junction protein-4 (CLDN4) participates in the formation of the skin barrier by targeting ions and molecules in keratinocytes. In allergic dermatitis, the expression of TJ is significantly reduced, leading to a weakened skin barrier and triggering itching. Therefore, a dermatitis model confirmed that two cell-penetrating growth factors (SCF-CDP and FGF-CDP) alter the expression levels of CLDN1 and CLDN4.

[0123] like Figure 10 As shown, the Cytokine-treated group reduced CLDN1 expression by approximately 50% compared to the untreated group, confirming that the skin barrier is weakened by Cytokine treatment. SCF-CDP increased CLDN1 expression by approximately 20% and CLDN4 expression by 40% compared to the Cytokine-treated group. FGF-CDP increased CLDN4 expression by 20% compared to the Cytokine-treated group, and the combined treatment group increased CLDN4 expression by 60% compared to the Cytokine-treated group. Inflammatory keratinocytes are expected to contribute to the formation of the skin barrier with ions and water molecules.

[0124] [Experimental Example 9] Anti-inflammatory effects of SCF-CDP or FGF-CDP and SCF+FGF-CDP recombinant proteins in a dermatitis model.

[0125] In the pathogenesis of allergic dermatitis, in the initial stage, keratinocytes produce IL-1α, IL-1β, TNF-α, IL-6, and IL-8, which in turn triggers T cells and monocytes to infiltrate and induce an inflammatory response, thereby activating various immune cells and releasing multiple cytokines. The anti-inflammatory effects of SCF-CDP and FGF-CDP have been confirmed in a dermatitis model that guides the release of multiple cytokines.

[0126] The positive control group used brassinolide, whose anti-inflammatory effects have been verified. As a result, all experimental groups (IL-1α, IL-1β) showed anti-inflammatory effects, thus confirming that the experiment was completed normally.

[0127] like Figure 11As shown, FGF-CDP inhibited IL-1α expression by approximately 17.8% compared to the cytokine treatment group, and the mixed treatment group inhibited it by 20% compared to the cytokine treatment group. SCF-CDP inhibited IL-1β by approximately 7.4% compared to the cytokine treatment group, FGF-CDP inhibited it by 22.3%, and the mixed treatment group inhibited it by 25.5%. This indicates that SCF-CDP and FGF-CDP exhibit anti-inflammatory efficacy in IL-1 family-targeted skin diseases, including atopic dermatitis.

Claims

1. A cell-penetrating peptide of which a carrier delivery peptide is bonded to a fibroblast growth factor or stem cell factor peptide.

2. The cell-penetrating peptide according to claim 1, characterized in that: The cell-penetrating peptide has fibroblast growth factor bonded to its C-terminus or stem cell factor.

3. The cell-penetrating peptide according to claim 1, characterized in that: The fibroblast growth factor includes amino acids of sequence number 1, the fibroblast growth factor includes amino acids of sequence number 2, and the stem cell factor includes amino acids of sequence number 3.

4. The cell-penetrating peptide according to claim 1, characterized in that: The fibroblast growth factor and the fibroblast growth factor-bonded recombinant peptide fibroblast growth factor include the amino acid of sequence number 4.

5. The cell-penetrating peptide according to claim 1, characterized in that: The recombinant peptide stem cell factor-fibroblast growth factor (SCM-fibroblast growth factor) includes amino acid sequence number 5.

6. A recombinant expression vector that expresses a vector delivery peptide (a cell-penetrating peptide of fibroblast growth factor) bonded to a fibroblast growth factor or stem cell factor peptide.

7. A pharmaceutical composition for wound treatment that includes the cell-penetrating peptide of claim 1 as an active ingredient.

8. The composition according to claim 7, characterized in that: The wound was caused by dermatitis.

9. A pharmaceutical composition for the prevention and treatment of dermatitis, comprising the cell-penetrating peptide of claim 1 as an active ingredient.

10. The pharmaceutical composition of claim 9, characterized in that: The cell-penetrating peptide forms a skin barrier and inhibits verification.

11. A pharmaceutical composition for treating skin barrier formation caused by dermatitis, including the cell-penetrating peptide of claim 1 as an active ingredient.

12. A cosmetic composition for improving skin barrier and skin moisturizing, including the cell-penetrating peptide of claim 1 as an active ingredient.

13. A composition for transdermal delivery of growth factors, including the cell-penetrating peptide of claim 1 as an active ingredient.

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