Skin penetration enhancing peptides and their applications

Abstract

The present invention belongs to the field of biotechnology and relates to a transdermal enhancement peptide and its application. The amino acid sequence of the transdermal enhancement peptide is shown in any one of SEQ ID NO: 1 to SEQ ID NO: 40. It is used to effectively deliver active ingredients below the stratum corneum and exert their efficacy. The active ingredients are not easy to penetrate the skin due to their molecular weight or the inherent characteristics of the stratum corneum. The polypeptide of the present invention is based on food sources, the source is safe and non-toxic to cells. It is a green source with good biocompatibility and high biosafety transdermal enhancement peptides, and has high application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a transdermal enhancement peptide and application thereof. Background Art

[0002] The skin is a tissue that is in constant contact with the external environment and acts as a protective barrier. Its low permeability severely limits the penetration of most active ingredients. The main permeability barrier of the skin is located in the outermost layer, the stratum corneum, which is composed of keratin-rich cells embedded in multiple lipid bilayers and has a dense structure (Peña-Juárez MC, Guadarrama-Escobar OR, Escobar-Chávez JJ. Transdermal Delivery Systems for Biomolecules. J Pharm Innov. 2022;17(2):319-332. doi: 10.1007 / s12247-020-09525-2. Epub 2021 Jan 6. PMID: 33425065; PMCID: PMC7786146.).

[0003] In order to penetrate the stratum corneum, active ingredients must pass through the tortuous lipid pathways around keratin-rich cells or be repeatedly distributed between the keratin-rich aqueous phase and the lipid phase. Therefore, only active ingredients with optimal physicochemical properties (molecular weight less than 500 Da, high hydrophobicity, and sufficient solubility in aqueous and non-aqueous solvents) can be passively transported through the stratum corneum. Compared with oral administration and injection, in order to avoid the first-pass effect in the liver, transdermal administration of active ingredients is a promising method of administration. How to further improve the efficiency of active ingredients through the skin, explore their applications in the field of skin health or various disease models, and enable large molecular active ingredients that are currently difficult to be absorbed transdermally to achieve transdermal administration are urgent issues to be solved and future research directions. In recent years, relevant studies have reported a variety of methods to promote the transdermal absorption of active ingredients, including physical methods such as iontophoresis, electroporation, ultrasound introduction, microneedles, etc.; pharmaceutical methods such as microemulsions, liposomes, nanoparticles, etc.; biochemical methods and the comprehensive application of penetration-enhancing methods, etc. Among them, penetration enhancers are still the best choice for increasing the percutaneous absorption of active ingredients due to their low cost, flexible prescription design, and convenient process. At present, widely used chemical penetration enhancers, such as sulfoxides, azones, alcohols, etc., have defects such as low biocompatibility and high irritation. In addition, newly discovered synthetic biological penetration enhancers (transdermal peptides) have gradually attracted attention. They have a wider penetration range, good biocompatibility, non-toxic irritation, rapid onset, selectivity and specificity.

[0004] At present, the main methods for screening transdermal peptides are phage display and mRNA display technology. Among them, phage display technology is widely used in screening and identifying transdermal peptides. Phage display technology is to express exogenous polypeptides on the surface of phages to obtain specific artificial phages associated with genotype and phenotype, and then interact with target protein molecules or target cells on the solid phase. Transdermal enhancement peptides are usually small peptides composed of 5 to 30 amino acids (1000 to 1500 Da). They are considered to be safe alternatives to chemical penetration enhancers. They are non-invasive, non-toxic and non-irritating. Transdermal enhancement peptides use their macromolecular substance transduction function to carry some drugs that cannot penetrate the skin through the skin. Transdermal peptides can carry and transport a wide range of drugs, from common drugs to siRNA, plasmids, DNA, proteins, etc., which can be delivered transdermally, showing strong transdermal mediation ability. Transdermal enhancement peptides are derived from phage display peptide libraries, most of which are cyclic peptides. By interacting with skin proteins (mainly keratin), it causes changes in the secondary structure of skin proteins and increases the entry of drugs into keratinocytes. The peptide that has been discovered and widely used is the SPACE peptide. When the SPACE peptide is coupled with drugs such as siRNA, it can enhance the drug's entry into the epidermis or dermis through the stratum corneum barrier. Drugs delivered by the SPACE peptide enter cells through the macropinocytosis pathway, which is not co-localized with the endolysosome, so the peptide will not be rapidly degraded by the lysosome. Chen et al. further studied that the SPACE peptide was coupled with siRNA to prepare an ethosome preparation to enhance the transdermal delivery of siRNA. This system increased the permeability of siRNA into pig skin by (6.3±1.7) times, the accumulation of siRNA in the epidermis increased by about 10 times, and the permeability of siRNA at the cellular level was also enhanced (Chen M, Zakrewsky M, Gupta V, Anselmo AC, SleeDH, Muraski JA, Mitragotri S. Topical delivery of siRNA into skin usingSPACE-peptide carriers. J Control Release. 2014 Apr 10;179:33-41. doi:10.1016 / j.jconrel.2014.01.006. Epub 2014 Jan 13. PMID: 24434423; PMCID:PMC4425738.0).

[0005] In addition, it has been reported that a short peptide TD-1 (ACSSSPSKHCG) with permeation-enhancing effect was screened by phage display technology and confirmed that TD-1 can effectively promote insulin of different conformations to pass through the skin and enter the systemic circulation. At the same time, the results suggest that TD-1 may specifically open transient channels in the skin barrier, thereby mediating insulin transport through the skin barrier (Chen Y, Shen Y, Guo X, Zhang C, Yang W, Ma M, Liu S, Zhang M, Wen LP. Transdermal protein delivery by a coadministered peptide identified via phagedisplay. Nat Biotechnol. 2006 Apr;24(4):455-60. doi: 10.1038 / nbt1193. Epub2006 Mar 26. PMID: 16565728.). Summary of the invention

[0006] First, based on the existing machine learning and artificial intelligence tools, the present invention designs a transdermal enhancement peptide library through overlapping peptide libraries, uses MLCPP 2.0 and BChemRF-CPPred prediction tools to screen transdermal enhancement peptides that can efficiently and quickly score high, and then further screens transdermal enhancement peptides with good permeability through cell experiments. The screened transdermal enhancement peptides can be used as carriers for active ingredient delivery and are widely used in the biomedical and cosmetic fields. In order to achieve the above purpose, the technical solution provided by the present invention is:

[0007] The present invention first provides a skin penetration enhancing peptide, the amino acid sequence of which is shown in any one of SEQ ID NO: 1 to SEQ ID NO: 40.

[0008] Preferably, the amino acid sequence is shown in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 19 or SEQ ID NO: 35.

[0009] Also preferably, the amino acid sequence is shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:13.

[0010] The present invention also provides a polynucleotide encoding the transdermal enhancement peptide.

[0011] The present invention also provides the use of the skin permeation enhancing peptide in the preparation of a preparation for enhancing the entry of active ingredients such as drugs into the body through the skin.

[0012] The present invention particularly provides the use of the skin permeation enhancing peptide in the preparation of skin products.

[0013] Specifically, the transdermal enhancement peptide acts as a penetration enhancer to promote the penetration of active ingredients such as drugs into the skin.

[0014] In a specific embodiment, the skin product is a medicine or a cosmetic, more specifically, the skin product is a hand cream, an eye cream, a skin moisturizing product or a skin sunscreen product.

[0015] The transdermal enhancement peptide of the present invention is derived from natural food proteins and human growth-related factors, has the advantages of safe source and non-toxicity to cells, good biocompatibility, high biosafety, and can be obtained by chemical synthesis, biosynthesis, purification and extraction, etc. The transdermal enhancement peptide of the present invention can be used to effectively deliver active ingredients below the stratum corneum and exert their efficacy, and is particularly suitable for active ingredients that are difficult to penetrate the skin due to their molecular weight or inherent characteristics of the stratum corneum. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a color diagram of the lower receiving liquid in Example 2.

[0017] Figure 2 This is the permeability effect diagram of SPP. Implementation

[0018] The present invention is further described below through specific examples in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0019] Example 1. Peptide library establishment and screening

[0020] 1) Determine the protein source

[0021] Lactoferrin is a multifunctional globular protein that is widely present in various secretions of animals and humans. Lactoferrin has multiple biological activities such as antibacterial, antioxidant, and anti-cancer. It is used in infant formula, cosmetics and other products. It is highly safe and can be used as a source of nutrition for the human body to supplement iron and amino acids. It can also be used as a drug to prevent and treat a variety of human diseases. It is used to maintain the balance of intestinal flora, prevent infection and resist viruses, inhibit tumor occurrence and metastasis, and hinder the production of free radicals in the body that damage the body.

[0022] Epidermal growth factor (EGF) is a heat-resistant single-chain low-molecular polypeptide. EGF has no sugar part, is very stable, heat-resistant and acid-resistant, and is widely present in the body fluids and various glands of animals and humans. EGF can accelerate the generation of new cells, increase the number of new cells, promote skin cell metabolism, gradually shed the dead skin layer, and maintain smooth skin texture. It has the effects of enhancing skin elasticity, reducing skin wrinkles and preventing skin aging. Therefore, EGF is often used to help tissue regeneration, repair body surface trauma and ulcers, and is used in medicines and cosmetics.

[0023] Transforming growth factor β (TGF-β) is a multifunctional cytokine belonging to the transforming growth factor family. It is a secretory protein that can regulate cell growth and differentiation by immune cells. Its key role is to regulate the inflammatory process. It is currently mainly used as a cytokine to promote fibroblast differentiation. Some studies also believe that TGF-β has potential application prospects in treating wound healing, promoting cartilage and bone repair, and treating autoimmune diseases and transplant rejection through immunosuppression.

[0024] The above proteins are mostly distributed in human and animal body fluids and have restorative effects. Lactoferrin has been used in food. A transdermal enhancing peptide library is designed based on the above green and safe proteins derived from animals and humans.

[0025] 2) AI high-throughput screening of peptide libraries

[0026] (1) The peptide library was screened using MLCPP 2.0 and BChemRF-CPPred servers. The peptide length range was set to 6-30 amino acids. The membrane penetration strength and score of the peptides were obtained through prediction and comparison.

[0027] (2) According to the predicted permeation enhancement peptide score threshold value of step (1)>0.7, 40 peptide sequences were screened and the results are shown in Table 2. The mechanisms of peptide permeation and cell membrane penetration are different, but they have something in common. Currently, there is still a lack of direct permeation effect prediction tools. The present invention uses cell membrane penetration prediction to screen permeable peptides.

[0028] 3) Peptide synthesis

[0029] The 40 transdermal enhancement peptide sequences screened out in step 1 were synthesized by solid phase chemical synthesis for subsequent experiments.

[0030] Table 1. Sequence information of 40 peptides

[0031]

[0032] Example 2: Screening and verification of transdermal enhancement peptides based on skin models

[0033] Preparation of related reagents:

[0034] 1) Skin model: Bama pig back skin, 30 days old

[0035] Transdermal receiving solution: Use sterile distilled water as solvent to prepare 0.9% sodium chloride aqueous solution.

[0036] 3) Preparation of transdermal enhancement peptide stock solution: Use sterile distilled water as solvent, prepare a transdermal enhancement peptide stock solution with a concentration of 1 mM, and then divide it into small amounts for use and store at -20°C. The present invention is selected from any one of the following amino acid sequences: SEQ ID NO:1-SEQ ID NO:40. The polypeptide is custom-made and synthesized from GenScript Biotech Co., Ltd., with green fluorescent labeled lyophilized powder, and dissolved in pure water.

[0037] Experimental protocol:

[0038] Sample #1 A blend of transdermal enhancing peptides and active ingredients

[0039] The active ingredient selected was hexapeptide (EEMQRR) with anti-wrinkle effect, SPP (SEQ ID NO: 1- SEQ ID NO: 40), TD-1, and inositol were added to the hexapeptide aqueous solution, and ultrasonicated at room temperature to fully disperse and mix. The final concentration of hexapeptide was 2 mM, the final concentration of SPP and TD-1 was 200 μM, and the final concentration of inositol was 1%.

[0040] Sample #2 In vitro skin permeation test

[0041] Franz transdermal diffusion cell was used for in vitro skin permeability test. The specific operation was as follows: Bama pigs, 30 days old, were killed and the complete back skin was cut with scissors. The dermis of the skin was wiped with a cotton ball soaked in saline to remove the adhering subcutaneous tissue, and then the skin was washed with saline, dried, wrapped with tin foil, and stored in a refrigerator at -20ºC for later use. The prepared skin was fixed between the supply chamber and the receiving chamber of the Franz diffusion cell, with the surface of the skin facing the supply chamber. The effective permeation area of ​​the diffusion cell was 1.327 cm 2 . Add 3.8 ml of 0.9% sodium chloride aqueous solution to the receiving chamber so that the liquid surface is in close contact with the skin. Add 1 ml of the mixture of transdermal enhancement peptide and active ingredient to the supply chamber. During the experiment, a water bath was used to heat the mixture to keep the temperature at 32ºC and stir at 350 r / min. The penetration effect was quantified at 8 hours.

[0042] Experimental results:

[0043] The results of the HPLC detection of the penetration enhancement of transdermal enhancing peptides on hexapeptides are shown in Table 2 below. Under the action of 200uM peptide concentration, compared with the positive control peptide TD-1 and 1% inositol, SPP-1 (SEQ ID NO: 1), SPP-7 (SEQ ID NO: 7), SPP-11 (SEQ.ID NO: 11), SPP-13 (SEQ ID NO: 13), SPP-19 (SEQ ID NO: 19) and SPP-35 (SEQID NO: 35) can penetrate the stratum corneum into the surface layer of the skin, and the penetration effect on hexapeptides is significantly better than that of the chemical penetration enhancer inositol, which is equivalent to that of the short peptide TD-1.

[0044] Among them, SPP-7 (SEQ ID NO: 7), SPP-19 (SEQ ID NO: 19) and SPP-35 (SEQ ID NO: 35) have a better penetration enhancement effect on hexapeptide than TD-1, and SPP-7 (SEQ ID NO: 7) has a penetration enhancement effect on hexapeptide that is nearly twice that of 1% inositol. In addition, the color depth of the receiving solution in the lower layer can be used to determine the penetration effect of the transdermal enhancement peptide itself. The results are as follows Figure 1 From the color of the receiving liquid, it can be seen that compared with no penetration enhancer (negative control), SPP-1 (SEQ ID NO: 1), SPP-3 (SEQ ID NO: 3), SPP-4 (EQ. ID NO: 4), SPP-13 (SEQ ID NO: 13), SPP-18 (SEQ ID NO: 18), SPP-19 (SEQ ID NO: 19), and SPP-31 (SEQ ID NO: 31) themselves have better penetration effects.

[0045] Table 2 Penetration-enhancing effect of transdermal enhancement peptides

[0046]

[0047] Example 3: SPP self-penetration ability test

[0048] Preparation of related reagents:

[0049] 1) Skin model: Bama pig back skin, 30 days old

[0050] 2) Transdermal receiving solution: Use sterile distilled water as the solvent to prepare a 0.9% sodium chloride aqueous solution.

[0051] 3) Preparation of transdermal enhancement peptide stock solution: sterile distilled water was used as solvent to prepare a transdermal enhancement peptide stock solution with a concentration of 1 mM, and then aliquoted in small amounts for use and stored at -20°C. The transdermal enhancement peptide was selected from the polypeptides shown in the amino acid sequence with good permeability found in Example 2: SPP-1 (SEQ ID NO: 1), SPP-3 (SEQ ID NO: 3), SPP-4 (EQ. ID NO: 4), SPP-13 (SEQ ID NO: 13), SPP-18 (SEQ ID NO: 18), SPP-19 (SEQ ID NO: 19), SPP-31 (SEQ ID NO: 31).

[0052] Experimental protocol:

[0053] Franz transdermal diffusion cell was used for in vitro skin permeability test. The specific operation was as follows: Bama pigs, 30 days old, were killed and the complete back skin was cut with scissors. The dermis of the skin was wiped with a cotton ball soaked in saline to remove the adhering subcutaneous tissue, and then the skin was washed with saline, dried, wrapped with tin foil, and stored in a refrigerator at -20ºC for later use. The prepared skin was fixed between the supply chamber and the receiving chamber of the Franz diffusion cell, with the surface of the skin facing the supply chamber. The effective permeation area of ​​the diffusion cell was 1.327 cm 2 . Add 3.8 ml of 0.9% sodium chloride aqueous solution to the receiving chamber so that the liquid surface is in close contact with the skin. Add 1 ml of the mixture of transdermal enhancement peptide and active ingredient to the supply chamber. During the experiment, a water bath was used to heat the mixture to keep the temperature at 32ºC and stir at 350 r / min. The penetration effect was quantified at 8 hours.

[0054] The experimental results are as follows Figure 2 As shown: at a polypeptide concentration of 200uM, compared with the positive control hexapeptide, SPP-1 (SEQ ID NO: 1), SPP-3 (SEQ ID NO: 3), SPP-4 (EQ.ID NO: 4), SPP-13 (SEQ ID NO: 13), SPP-19 (SEQ ID NO: 19), and SPP-31 (SEQ ID NO: 31) themselves have good penetration effects.

[0055] Example 4: Verification of non-toxic and non-irritating transdermal enhancement peptides based on cell models

[0056] Preparation of related reagents:

[0057] 1) Cells

[0058] Human keratinocytes (HaCaT)

[0059] 2) Preparation of transdermal enhancement peptide stock solution

[0060] Sterile distilled water was used as solvent to prepare a 3 mM transdermal enhancement peptide stock solution, which was then aliquoted in small quantities for storage at -20°C. The present invention is a polypeptide selected from any one of the following amino acid sequences: SEQ ID NO: 1-SEQ ID NO: 40.

[0061] 3) Cell lysis buffer

[0062] 1M NaOH: Weigh 4.0 g of sodium hydroxide solid and dilute to 100 mL of sodium hydroxide solution.

[0063] Experimental protocol:

[0064] Sample #1 Cytotoxicity Assay

[0065] HaCaT cells were plated in a 96-well plate at a density of 1.0×10ˆ5. After 24h of culture, different concentrations of transdermal enhancement peptides SEQ ID NO:1-SEQ ID NO:40 (5μM, 10μM, 20μM, 50μM, 100μM, 200μM) were added to the cells for 24h at 37℃ and 5%CO2. The culture medium was then discarded. 90uL of basic culture medium and 10uL CCK8 were added to each well and mixed. The cells were incubated at 37℃ for 40min. The absorbance was measured at 450nm after the incubation. Cell activity (%) = (OD of experimental group - OD of blank well) / (OD of blank control group - OD of blank well) * 100%. Cell activity <70% indicates that the transdermal enhancement peptide is cytotoxic.

[0066] Experimental results:

[0067] The cytotoxicity test results of the transdermal enhancement peptides are shown in Table 3, and the results show that the transdermal enhancement peptides (SEQ ID NO: 1-SEQ ID NO: 40) are non-toxic to HaCaT cells within the tested concentration range.

[0068] Table 3 Transdermal enhancement peptide cytotoxicity results

[0069]

[0070] +: cytotoxic; -: non-cytotoxic.

Claims

1. A skin penetration enhancing peptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO: 19 or 35.

2. The polynucleotide encoding the skin penetration enhancing peptide according to claim 1.

3. Use of a transdermal enhancement peptide in the preparation of a preparation for transdermal delivery of an active ingredient to cells, characterized in that: The amino acid sequence of the transdermal enhancement peptide is shown in any one of SEQ ID NOs: 7, 19, and 35.

4. Use of a transdermal enhancement peptide in the preparation of a skin product, characterized in that: The amino acid sequence of the transdermal enhancement peptide is shown in any one of SEQ ID NOs: 7, 19, and 35.

5. The use according to claim 4, characterized in that The skin penetration enhancing peptide acts as a penetration enhancer.

6. The use according to claim 4 or 5, characterized in that The dermatological product is a drug or cosmetic applied to the skin surface for the delivery of active ingredients.

7. The use according to claim 6, characterized in that The skin product is a hand cream, an eye cream, a skin moisturizing product, a skin anti-aging product or a skin sunscreen product.

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