Method and composition for improving skin permeability of hydrophilic substance

Abstract

The invention belongs to the technical field of daily chemical industry, and relates to a method and a composition for improving the skin permeability of a hydrotropic substance. It is found that lipopeptide can promote percutaneous absorption of macromolecular hydrophilic substances, especially hydrophilic substances with the molecular weight ranging from 800 Da to 100 kDa, meanwhile, percutaneous absorption of hydrophilic substances with the molecular weight smaller than 800 Da can be further promoted, and the lipopeptide has wide application prospects in the aspect of cosmetic preparation.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202411317839.8 filed on September 20, 2024, and the entire contents of the above patent application are hereby cited as part of this application. Technical Field

[0003] The present application belongs to the technical field of daily chemical industry, and specifically relates to a method and composition for improving the skin permeability of a hydrophilic substance. Background Art

[0004] After cosmetics are applied to the skin's surface, their active ingredients need to penetrate and accumulate in the appropriate area to take effect. For example, whitening ingredients typically need to penetrate the basal layer, the deepest layer of the epidermis, while anti-wrinkle ingredients need to penetrate even deeper into the dermis to be effective. However, due to the existence of the skin barrier, transdermal penetration of cosmetics is very difficult.

[0005] To improve the bioavailability of active ingredients, penetration enhancement technologies have become a research hotspot in the fields of pharmaceutics and cosmetics. Common penetration enhancement technologies include iontophoresis, ultrasound inhalation, microneedles, microemulsions, liposomes, nanoparticles, and enhanced permeation. However, iontophoresis, ultrasound, and microneedles require specialized equipment and operating techniques, increasing cost and complexity. Furthermore, improper use can cause skin damage or allergic reactions. Carrier systems such as microemulsions, nanoparticles, or liposomes may lead to unknown biocompatibility issues and potential toxic reactions. They may also be unstable during storage, affecting their effectiveness. Furthermore, their preparation costs are often high, potentially increasing the final price of the product. Penetration enhancers work by interacting with stratum corneum lipids to alter their structural arrangement, thereby enhancing permeability. With their low cost, flexible formulation design, and convenient processing, penetration enhancers are the best choice for increasing the transdermal absorption of active ingredients.

[0006] Lipopeptides are a highly diverse class of biosurfactants composed of hydrophilic cyclic or short linear oligopeptides and hydrophobic long-chain fatty acids. Based on their amino acid structures, they can be divided into cyclic and linear lipopeptides. Surfactin, also known as surfactant, is a natural surfactant with a unique cyclic peptide structure that imparts exceptional surface active properties, such as emulsification and dispersibility. It is widely used in cosmetics, personal care, and laundry.

[0007] WO2023032743A1 discloses the use of lipopeptides and phospholipids to improve the skin permeability of hydrophilic compounds. After testing, when lipopeptides were used alone, the dye substance was mainly retained in the stratum corneum after 6 hours. When lipopeptides and phospholipids were used together, the dye substance could not reach the dermis until 6 hours later, and the difference in permeation was not tested. Therefore, how to make lipopeptides effectively play a penetration-promoting role in a short time (e.g., about 6-16 hours) has not yet been revealed.

[0008] In addition, compared with small-molecule hydrophilic substances, large-molecule hydrophilic substances themselves are more difficult to penetrate into the skin. Although it is conceivable to use penetration enhancers as a means, this study found that the use of penetration enhancers cannot effectively increase the permeation of large-molecule hydrophilic substances, especially large-molecule hydrophilic substances with very low permeability, which are more difficult to achieve greater skin penetration.

[0009] Therefore, how to increase the skin penetration of hydrophilic substances, especially large-molecule hydrophilic substances, is still a technical problem that needs to be solved. Summary of the Invention

[0010] In response to the above problems, the present application provides a method and composition for improving the skin permeability of hydrophilic substances, which can significantly improve the permeability of hydrophilic substances, especially hydrophilic substances with a molecular weight greater than 800 Da, and improve their bioavailability.

[0011] The specific scheme of this application is as follows:

[0012] In a first aspect, the present application provides a method for increasing the skin permeability of a hydrophilic substance, comprising the step of co-applying at least one lipopeptide and at least one hydrophilic substance to the skin, wherein the hydrophilic substance comprises at least one hydrophilic substance having a molecular weight of 800 Da to 100 kDa. Preferably, the hydrophilic active substance comprises a hydrophilic active substance having a molecular weight of 800 Da to 100 kDa.

[0013] Further, in the embodiments of the present application, the molecular weight of the hydrophilic substance is, for example, 800Da, 1kDa, 2kDa, 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa, 9kDa, 10kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55kDa, 60kDa, 70kDa, 80kDa, 90kDa, 100kDa, 150kDa, 200kDa, 250kDa, 300kDa, 350kDa, 400kDa, 450kDa, 500kDa, 550kDa, 600kDa, 650kDa, 700kDa, 750kDa, 800kDa, 850kDa, 900kDa, and 1000kDa.

[0014] Furthermore, the molecular weight of the hydrophilic substance is 20 kDa-60 kDa.

[0015] Furthermore, the hydrophilic substance is a water-soluble active ingredient.

[0016] Furthermore, the hydrophilic substance includes one or more of polypeptides, proteins, polysaccharides, nucleic acids and their derivatives. Preferably, the polypeptides, proteins, polysaccharides and nucleic acid derivatives are salt derivatives.

[0017] Furthermore, the polypeptide includes one or more of insulin, angiotensin, cono peptide, antimicrobial peptide, palmitoyl pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, dodecapeptide, tridecapeptide, tetradecapeptide, pentadecapeptide, hexadecapeptide, heptadecapeptide, octadecapeptide, nonadecapeptide, and eicosapeptide.

[0018] Furthermore, the protein includes one or more of collagen, actin, fibroin, protease, hemoglobin, antibody protein, receptor protein, fibrin, ovalbumin, and casein. Preferably, the collagen is selected from natural collagen or recombinant collagen. More preferably, the recombinant collagen includes one or more of recombinant type III collagen, recombinant type XVII collagen, recombinant type I collagen, recombinant type IV collagen, recombinant type V collagen, and recombinant type XVII collagen. More preferably, the recombinant collagen includes one or more of recombinant type III collagen and recombinant type XVII collagen.

[0019] Recombinant collagen refers to collagen produced by genetic recombination techniques, in which DNA or RNA encoding the collagen is typically inserted into a suitable expression vector and transformed into host cells for expression. The DNA or RNA is inserted into the host chromosome via homologous recombination or other methods known in the art, and is then used to transform the host cells to produce the protein.

[0020] Furthermore, the recombinant type III collagen comprises an amino acid sequence as shown in SEQ ID.1, SEQ ID.2, SEQ ID.3, SEQ ID.4, SEQ ID.5 or has at least 95% homology with the amino acid sequence as shown in SEQ ID.1, SEQ ID.2, SEQ ID.3, SEQ ID.4, SEQ ID.5.

[0021] Alternatively, recombinant type III collagen is derived from, for example, Bloomcollagen TMRecombinant humanized type III collagen; Recombinant humanized type III collagen, 11917-2; Recombinant humanized type III collagen, HC1-H2.

[0022] Furthermore, the carbohydrates include one or more of glycosaminoglycans, glycogen, cellulose, and dextran. Preferably, the carbohydrates include one or more of hyaluronic acid (or hyaluronate) and its derivatives, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and heparin.

[0023] Furthermore, the nucleic acid includes one or more of a DNA molecule, an RNA molecule or a DNA-RNA chimeric nucleic acid molecule.

[0024] Furthermore, in an embodiment of the present application, the lipopeptide includes at least one or more of surfactin, lichenysin, iturin, fengycin, daptomycin, micafungin, caspofungin, and polymyxin. Furthermore, the lipopeptide includes at least surfactin.

[0025] Furthermore, the hydrophilic substance also includes a hydrophilic substance with a molecular weight less than 800 Da.

[0026] Preferably, the hydrophilic substance includes a protein with a molecular weight of 800Da-100kDa, and a hydrophilic substance with a molecular weight of less than 800Da. More preferably, the protein is selected from one or more of collagen and hydrolyzed silk fibroin. Wherein, the collagen is selected from recombinant collagen, and further, the recombinant collagen is selected from one or more of recombinant type III collagen, recombinant type XVII collagen, recombinant type I collagen, recombinant type IV collagen, recombinant type V collagen, and recombinant type XVII collagen.

[0027] Preferably, the hydrophilic substance includes a hydrophilic substance with a molecular weight of 800Da-100kDa, and a hydrophilic substance with a molecular weight less than 800Da.

[0028] Furthermore, the hydrophilic substance with a molecular weight of less than 800 Da also includes one or more of amino acids and their derivatives, small molecule peptides and their derivatives, small molecule sugars and their derivatives, mononucleotides and their derivatives, alcohols, lipids, complex polysaccharides, nut oils, vitamins and their derivatives.

[0029] Furthermore, the amino acids and their derivatives include one or more of alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, ergothioneine, icodine, 5-aminolevulinic acid, γ-aminobutyric acid, indoleacetic acid, and cystine.

[0030] Furthermore, the small molecule peptide includes one or more of dipeptides and tripeptides. Preferably, the small molecule peptide includes one or more of alanine dipeptide, carnosine, cyclic dipeptide, glycyl alanine, glycyl peptide, alanyl glutamine, glycyl-L-tyrosine, N-acetyl-L-carnosine, alanyl glutamine, BHA, glutathione, and blue copper peptide.

[0031] Furthermore, the small molecule sugar includes one or more of glucose, fructose, deoxyribose, and ribose.

[0032] Furthermore, the mononucleotide includes one or more of ribonucleotides and deoxyribonucleotides.

[0033] Furthermore, the alcohols include one or more of C16-22 alcohol, butanediol, pentanediol, octanediol, glycerol, propylene glycol, and hexanediol.

[0034] Furthermore, the lipids include one or more of cetyl olive oil, monostearate, isopropyl myristate, isopropyl palmitate, triglycerides, lecithin, ceramide, sphingomyelin, sphingolipid sugars, and sphingosine phosphate.

[0035] Preferably, the complex polysaccharide comprises one or more of glycoproteins and glycolipids.

[0036] Furthermore, the nut oil includes one or more of argan oil, macadamia nut oil, avocado oil, wheat germ oil, and olive oil.

[0037] Furthermore, the vitamins include one or more of vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, vitamin F, and vitamin K.

[0038] In a second aspect of the present application, a lipopeptide is provided for preparing a composition for improving skin permeability, wherein the composition comprises at least one lipopeptide and at least one hydrophilic substance in a physiologically acceptable medium, wherein the hydrophilic substance comprises at least a hydrophilic substance having a molecular weight of 800Da-100kDa.

[0039] Further, in the embodiments of the present application, the molecular weight of the hydrophilic substance is, for example, 800Da, 1kDa, 2kDa, 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa, 9kDa, 10kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55kDa, 60kDa, 70kDa, 80kDa, 90kDa, 100kDa, 150kDa, 200kDa, 250kDa, 300kDa, 350kDa, 400kDa, 450kDa, 500kDa, 550kDa, 600kDa, 650kDa, 700kDa, 750kDa, 800kDa, 850kDa, 900kDa, and 1000kDa.

[0040] Furthermore, the molecular weight of the hydrophilic substance is 20 kDa-60 kDa.

[0041] Furthermore, the hydrophilic substance includes one or more of polypeptides, proteins, polysaccharides, nucleic acids and their derivatives. Preferably, the polypeptides, proteins, polysaccharides and nucleic acid derivatives are salt derivatives.

[0042] Furthermore, the polypeptide includes one or more of insulin, angiotensin, cono peptide, antimicrobial peptide, palmitoyl pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, dodecapeptide, tridecapeptide, tetradecapeptide, pentadecapeptide, hexadecapeptide, heptadecapeptide, octadecapeptide, nonadecapeptide, and eicosapeptide.

[0043] Furthermore, the protein includes one or more of collagen, actin, fibroin, protease, hemoglobin, antibody protein, receptor protein, fibrin, ovalbumin, and casein.

[0044] Furthermore, the collagen comprises one or more of recombinant type III collagen, recombinant type XVII collagen, recombinant type I collagen, recombinant type IV collagen, recombinant type V collagen, and recombinant type XVII collagen. Furthermore, the collagen comprises one or more of recombinant type III collagen and recombinant type XVII collagen.

[0045] Furthermore, the carbohydrates include one or more of glycosaminoglycans, glycogen, cellulose, and dextran. Preferably, the carbohydrates include one or more of hyaluronic acid (or hyaluronate) and its derivatives, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and heparin.

[0046] Furthermore, the nucleic acid includes one or more of a DNA molecule, an RNA molecule or a DNA-RNA chimeric nucleic acid molecule.

[0047] Furthermore, in an embodiment of the present application, the lipopeptide includes at least one or more of surfactin, lichenysin, iturin, fengycin, daptomycin, micafungin, caspofungin, and polymyxin. Furthermore, the lipopeptide includes at least surfactin.

[0048] Furthermore, the hydrophilic substance also includes a hydrophilic substance with a molecular weight less than 800 Da.

[0049] Preferably, the hydrophilic substance includes a protein with a molecular weight of 800Da-100kDa, and a hydrophilic substance with a molecular weight of less than 800Da. More preferably, the protein is selected from one or more of collagen and hydrolyzed silk fibroin. Preferably, the collagen is selected from natural collagen or recombinant collagen. Further preferably, the collagen is selected from recombinant type III collagen and / or recombinant type XVII collagen, more preferably, the collagen is selected from recombinant type III collagen.

[0050] Preferably, the hydrophilic substance includes a hydrophilic substance with a molecular weight of 800Da-100kDa, and a hydrophilic substance with a molecular weight less than 800Da.

[0051] Furthermore, the hydrophilic substance with a molecular weight of less than 800Da includes one or more of amino acids and their derivatives, small molecule peptides and their derivatives, small molecule sugars and their derivatives, mononucleotides and their derivatives, alcohols, lipids, complex polysaccharides, nut oils, vitamins and their derivatives.

[0052] Furthermore, the amino acids and their derivatives include one or more of alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, ergothioneine, icodine, 5-aminolevulinic acid, γ-aminobutyric acid, indoleacetic acid, and cystine.

[0053] Furthermore, the small molecule peptide includes one or more of dipeptides and tripeptides. Preferably, the small molecule peptide includes one or more of alanine dipeptide, carnosine, cyclic dipeptide, glycyl alanine, glycyl peptide, alanyl glutamine, glycyl-L-tyrosine, N-acetyl-L-carnosine, alanyl glutamine, BHA, glutathione, and blue copper peptide.

[0054] Furthermore, the small molecule sugar includes one or more of glucose, fructose, deoxyribose, and ribose.

[0055] Furthermore, the mononucleotide includes one or more of ribonucleotides and deoxyribonucleotides.

[0056] Furthermore, the alcohols include one or more of C16-22 alcohol, butanediol, pentanediol, octanediol, glycerol, propylene glycol, and hexanediol.

[0057] Furthermore, the lipids include one or more of cetyl olive oil, monostearate, isopropyl myristate, isopropyl palmitate, triglycerides, lecithin, ceramide, sphingomyelin, sphingolipid sugars, and sphingosine phosphate.

[0058] Preferably, the complex polysaccharide comprises one or more of glycoproteins and glycolipids.

[0059] Furthermore, the nut oil includes one or more of argan oil, macadamia nut oil, avocado oil, wheat germ oil, and olive oil.

[0060] Furthermore, the vitamins include one or more of vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, vitamin F, and vitamin K.

[0061] Furthermore, the concentration of the lipopeptide in the composition is 0.01%-5% (m / v), preferably 0.05%-1% (m / v).

[0062] Furthermore, the concentration of the hydrophilic substance in the composition is 0.01%-5% (m / v), preferably 0.05%-0.1% (m / v).

[0063] Furthermore, the mass ratio of the lipopeptide and the hydrophilic substance is not limited by the ratio. Preferably, the ratio is 1:500 to 500:1. More preferably, the mass ratio of the lipopeptide and the hydrophilic substance is 1:50 to 50:1. Even more preferably, the mass ratio of the lipopeptide and the hydrophilic substance is 1:10 to 10:1.

[0064] In a third aspect, the present application provides a transdermal absorption composition, which comprises at least one lipopeptide and at least one hydrophilic substance in a physiologically acceptable medium, wherein the hydrophilic substance comprises at least a hydrophilic substance with a molecular weight of 800Da-100kDa.

[0065] Further, in the embodiments of the present application, the molecular weight of the hydrophilic substance is, for example, 800Da, 1kDa, 2kDa, 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa, 9kDa, 10kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 55kDa, 60kDa, 70kDa, 80kDa, 90kDa, 100kDa, 150kDa, 200kDa, 250kDa, 300kDa, 350kDa, 400kDa, 450kDa, 500kDa, 550kDa, 600kDa, 650kDa, 700kDa, 750kDa, 800kDa, 850kDa, 900kDa, and 1000kDa.

[0066] Furthermore, the molecular weight of the hydrophilic substance is 20 kDa-60 kDa.

[0067] Furthermore, the hydrophilic substance includes one or more of polypeptides, proteins, polysaccharides, nucleic acids and their derivatives. Preferably, the polypeptides, proteins, polysaccharides and nucleic acid derivatives are salt derivatives.

[0068] Furthermore, the polypeptide includes one or more of insulin, angiotensin, cono peptide, antimicrobial peptide, palmitoyl pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, dodecapeptide, tridecapeptide, tetradecapeptide, pentadecapeptide, hexadecapeptide, heptadecapeptide, octadecapeptide, nonadecapeptide, and eicosapeptide.

[0069] Furthermore, the protein includes one or more of collagen, actin, fibroin, protease, hemoglobin, antibody protein, receptor protein, fibrin, ovalbumin, and casein.

[0070] Furthermore, the collagen comprises one or more of recombinant type III collagen, recombinant type XVII collagen, recombinant type I collagen, recombinant type IV collagen, recombinant type V collagen, and recombinant type XVII collagen. Furthermore, the collagen comprises one or more of recombinant type III collagen and recombinant type XVII collagen.

[0071] Furthermore, the carbohydrates include one or more of glycosaminoglycans, glycogen, cellulose, and dextran. Preferably, the carbohydrates include one or more of hyaluronic acid (or hyaluronate) and its derivatives, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and heparin.

[0072] Furthermore, the nucleic acid includes one or more of a DNA molecule, an RNA molecule or a DNA-RNA chimeric nucleic acid molecule.

[0073] Furthermore, in an embodiment of the present application, the lipopeptide includes at least one or more of surfactin, lichenysin, iturin, fengycin, daptomycin, micafungin, caspofungin, and polymyxin, and further, the lipopeptide is surfactin.

[0074] Furthermore, the hydrophilic substance also includes a hydrophilic substance with a molecular weight less than 800 Da.

[0075] Preferably, the hydrophilic substance includes a protein with a molecular weight of 800Da-100kDa, and a hydrophilic substance with a molecular weight of less than 800Da. More preferably, the protein is selected from one or more of collagen and hydrolyzed silk fibroin. Wherein, the collagen is selected from recombinant collagen, and further, the recombinant collagen is selected from one or more of recombinant type III collagen, recombinant type XVII collagen, recombinant type I collagen, recombinant type IV collagen, recombinant type V collagen, and recombinant type XVII collagen.

[0076] Preferably, the hydrophilic substance includes a hydrophilic substance with a molecular weight of 800Da-100kDa, and a hydrophilic substance with a molecular weight less than 800Da.

[0077] Furthermore, the hydrophilic substance with a molecular weight of less than 800Da includes one or more of amino acids and their derivatives, small molecule peptides and their derivatives, small molecule sugars and their derivatives, mononucleotides and their derivatives, alcohols, lipids, complex polysaccharides, nut oils, vitamins and their derivatives.

[0078] Furthermore, the amino acids and their derivatives include one or more of alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, ergothioneine, icodine, 5-aminolevulinic acid, γ-aminobutyric acid, indoleacetic acid, and cystine.

[0079] Furthermore, the small molecule peptide includes one or more of dipeptides and tripeptides. Preferably, the small molecule peptide includes one or more of alanine dipeptide, carnosine, cyclic dipeptide, glycyl alanine, glycyl peptide, alanyl glutamine, glycyl-L-tyrosine, N-acetyl-L-carnosine, alanyl glutamine, BHA, glutathione, and blue copper peptide.

[0080] Furthermore, the small molecule sugar includes one or more of glucose, fructose, deoxyribose, and ribose.

[0081] Furthermore, the mononucleotide includes one or more of ribonucleotides and deoxyribonucleotides.

[0082] Furthermore, the alcohols include one or more of C16-22 alcohol, butanediol, pentanediol, octanediol, glycerol, propylene glycol, and hexanediol.

[0083] Furthermore, the lipids include one or more of cetyl olive oil, monostearate, isopropyl myristate, isopropyl palmitate, triglycerides, lecithin, ceramide, sphingomyelin, sphingolipid sugars, and sphingosine phosphate.

[0084] Preferably, the complex polysaccharide comprises one or more of glycoproteins and glycolipids.

[0085] Furthermore, the nut oil includes one or more of argan oil, macadamia nut oil, avocado oil, wheat germ oil, and olive oil.

[0086] Furthermore, the vitamins include one or more of vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, vitamin F, and vitamin K.

[0087] Furthermore, the concentration of the lipopeptide in the composition is 0.01%-5% (m / v), preferably 0.05%-1% (m / v).

[0088] Furthermore, the concentration of the hydrophilic substance in the composition is 0.01%-5% (m / v), preferably 0.05%-0.1% (m / v).

[0089] Furthermore, the mass ratio of the lipopeptide and the hydrophilic substance is not limited by the ratio. Preferably, the ratio is 1:500 to 500:1. More preferably, the mass ratio of the lipopeptide and the hydrophilic substance is 1:50 to 50:1. Even more preferably, the mass ratio of the lipopeptide and the hydrophilic substance is 1:10 to 10:1.

[0090] Typically, the active ingredients of the compositions described above are contained in a physiologically acceptable medium, particularly a cosmetically or pharmaceutically acceptable medium, which may be non-aqueous or aqueous. Thus, it may include an aqueous phase and / or an oil phase. Among the physiologically acceptable media that may be used in the present invention, their composition, amount, form of the composition, preparation method, and mode of administration may be selected by those skilled in the art based on their general knowledge, depending on the desired type of composition.

[0091] The last aspect of the present application also provides use of the composition in preparing cosmetics.

[0092] Preferably, the cosmetic comprises one or more of a cream, a lotion, a gel or an aqueous solution.

[0093] It should be noted that the present invention does not specifically limit the dosage form of the cosmetics, and all existing cosmetic dosage forms on the market are applicable.

[0094] The penetration-promoting method provided by the present invention can enable macromolecular substances to have a good penetration-promoting effect within 6 hours to 12 hours.

[0095] The penetration-enhancing method provided by the present invention can enable macromolecular substances to have a good penetration-enhancing effect at, for example, 6h, 7h, 8h, 9h, 10h, 11h, and 12h.

[0096] Beneficial effects:

[0097] The present application utilizes lipopeptides (especially sodium lipopeptide from Bacillus subtilis) as a penetration enhancer, which can significantly enhance the skin penetration of hydrophilic substances with a molecular weight of 800Da-100kDa, effectively improving their bioavailability, especially for collagen, which is known in the art to be difficult to absorb transdermally, and can achieve very significant penetration.

[0098] Furthermore, when lipopeptides (especially sodium lipopeptide from Bacillus subtilis) are used to promote the transdermal absorption of hydrophilic substances with a molecular weight of 800Da-100kDa, it can also additionally promote the skin penetration of small molecule hydrophilic substances, achieving multiple penetration-enhancing effects. DETAILED DESCRIPTION

[0099] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0100] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market. Unless otherwise specified, the percentages in this application are all weight fractions.

[0101] The raw material information of this application is shown in Table 1:

[0102] Table 1 Raw material information

[0103]

[0104] Experimental Example 1: Investigating the transdermal ability of lipopeptide sodium for macromolecular hydrophilic substances

[0105] (1) The macromolecular hydrophilic substance is sodium hyaluronate

[0106] The Franz cell diffusion pool experiment, the specific steps are as follows:

[0107] 1. Freeze the purchased suckling pig skin and thaw it naturally at room temperature before use. Wash it repeatedly with saline.

[0108] 2. Using a Franz constant temperature diffusion cell, place the ex vivo pig skin flat between the diffusion cell and the receiving cell, keeping the outer side of the stratum corneum facing the diffusion cell and the inner side facing the receiving cell, and use a metal clip to stabilize the device.

[0109] 3. Sodium hyaluronate (HA) was quantified using FITC fluorescent labeling. 0.2 g of HA was dissolved in 2 mL of 0.05 mol / L NaOH aqueous solution and sealed with a stopper. Vortex mixing was performed until completely dissolved. Subsequently, 0.04 g of fluorescent dye FITC was added and sealed with a stopper. After vortex mixing, the mixture was placed in a 95 ° C water bath for 45 min and then cooled to room temperature. 18 mL of anhydrous ethanol solution saturated with sodium chloride was added to the resulting mixture. The supernatant was removed by centrifugation. The resulting precipitate was the crude HA product labeled with FITC fluorescent labeling. 20 mL of anhydrous ethanol solution saturated with sodium chloride was added to the crude product. After vortex mixing, the labeled HA precipitate was evenly dispersed in the anhydrous ethanol solution saturated with sodium chloride. The upper alcohol solution was centrifuged and discarded. After repeated alcohol washing 6 times, the precipitate was collected and freeze-dried to obtain FITC fluorescently labeled HA powder. 3 ml of the sample solution shown in Table 2 was added to the diffusion cell as the supply solution. Three parallel experiments were set up for each group of samples to be tested. Add 7 ml of phosphate buffer solution (pH 6.8) to the receiving cell, ensure that bubbles are discharged during the addition process, and add a magnetic stirrer. The constant temperature water bath is at 37°C. The diffusion cell mouth is sealed with plastic wrap. Samples of 0.3 ml are taken at the corresponding time points and the active ingredient content is determined. At the same time, an equal volume of isothermal phosphate buffer solution (pH 6.8) is added.

[0110] Fluorescence intensity of FITC-HA was measured using a microplate reader at Ex / Em = 495 / 525 nm, with a 100 μL injection volume. A standard curve for HA was drawn using concentration and fluorescence intensity as the horizontal and vertical axes, respectively.

[0111] The experimental results are shown in Table 2.

[0112] Table 2

[0113]

[0114] (2) The macromolecular hydrophilic substance is collagen

[0115] The Franz cell diffusion pool experiment, the specific steps are as follows:

[0116] 1. Freeze the purchased suckling pig skin and thaw it naturally at room temperature before use. Wash it repeatedly with saline.

[0117] 2. Using a Franz constant temperature diffusion cell, place the ex vivo pig skin flat between the diffusion cell and the receiving cell, keeping the outer side of the stratum corneum facing the diffusion cell and the inner side facing the receiving cell, and use a metal clip to stabilize the device.

[0118] 3. Recombinant collagen was quantified using TAMRA fluorescent labeling. Weigh an appropriate amount of TAMRA and dissolve it in DMF to prepare a 10 mg / ml dye mother liquor. Weigh a certain amount of collagen and dissolve it in pH = 6.8 phosphate buffered saline to prepare a 5% collagen mother liquor. Mix the collagen mother liquor and the dye mother liquor at a dye / marker molar ratio of 10 / 1 and shake for 24 hours to fully react. Add an equal volume of water to the reaction solution and centrifuge to remove excess dye. Take the supernatant and place it in a dialysis bag to dialyze out DMF to obtain an aqueous solution of TAMRA-labeled collagen. Add 3 ml of sample solution as shown in Table 3 and Table 4 to the diffusion cell as the supply liquid, and set up three parallel experiments for each group of samples to be tested. Add 7 ml of phosphate buffer solution (pH 6.8) to the receiving cell, ensure that bubbles are discharged during the addition process, and add a magnetic stirrer. The constant temperature water bath is at 37°C. The diffusion cell mouth is sealed with plastic wrap. Samples of 0.3 ml are taken at the corresponding time points and the active ingredient content is determined. At the same time, an equal volume of isothermal phosphate buffer solution (pH 6.8) is added.

[0119] The fluorescence intensity of the labeled solution was measured using the fluorescence intensity mode of a multifunctional microplate reader, Ex / Em=543 / 572 nm, and the well plate injection volume was 100 μL.

[0120] The experimental results are shown in Tables 3 and 4.

[0121] Table 3

[0122]

[0123]

[0124] Table 4

[0125]

[0126] The results in Tables 2 to 4 show that, compared with other substances having penetration-enhancing effects, sodium subtilisin has a more prominent ability to enhance the transdermal absorption of hydrophilic substances with a molecular weight of 800Da-100kDa.

[0127] Experimental Example 2: Investigating the transdermal ability of sodium subtilisin on different collagens

[0128] The Franz cell diffusion pool experiment, the specific steps are as follows:

[0129] 1. Freeze the purchased suckling pig skin and thaw it naturally at room temperature before use. Wash it repeatedly with saline.

[0130] 2. Using a Franz constant temperature diffusion cell, place the ex vivo pig skin flat between the diffusion cell and the receiving cell, keeping the outer side of the stratum corneum facing the diffusion cell and the inner side facing the receiving cell, and use a metal clip to stabilize the device.

[0131] 3. Recombinant collagen was quantified using TAMRA fluorescent labeling. Weigh an appropriate amount of TAMRA and dissolve it in DMF to prepare a 10 mg / ml dye mother liquor. Weigh a certain amount of collagen and dissolve it in pH = 6.8 phosphate buffered saline to prepare a 5% collagen mother liquor. Mix the collagen mother liquor and the dye mother liquor at a dye / marker molar ratio of 10 / 1 and shake for 24 hours to fully react. Add an equal volume of water to the reaction solution and centrifuge to remove excess dye. Take the supernatant and place it in a dialysis bag to dialyze out DMF to obtain an aqueous solution of TAMRA-labeled collagen. Add 3 ml of sample solution as shown in Table 5 to the diffusion cell as the supply liquid, and set up three parallel experiments for each group of samples to be tested. Add 7 ml of phosphate buffer solution (pH 6.8) to the receiving cell, ensure that bubbles are discharged during the addition process, and add a magnetic stirrer. The constant temperature water bath is at 37°C. The diffusion cell mouth is sealed with plastic wrap. Samples of 0.3 ml are taken at the corresponding time points and the active ingredient content is determined. At the same time, an equal volume of isothermal phosphate buffer solution (pH 6.8) is added.

[0132] The fluorescence intensity of the labeled solution was measured using the fluorescence intensity mode of a multifunctional microplate reader, Ex / Em=543 / 572 nm, and the well plate injection volume was 100 μL.

[0133] The experimental results are shown in Table 5.

[0134] Table 5

[0135]

[0136] The results in Table 5 show that sodium subtilisin can effectively improve the transdermal absorption of collagen with different structures.

[0137] Experimental Example 3: Investigating the transdermal ability of lipopeptide sodium for small molecule hydrophilic substances

[0138] The Franz cell diffusion pool experiment, the specific steps are as follows:

[0139] 1. Freeze the purchased suckling pig skin and thaw it naturally at room temperature before use. Wash it repeatedly with saline.

[0140] 2. Using a Franz constant temperature diffusion cell, place the ex vivo pig skin flat between the diffusion cell and the receiving cell, keeping the outer side of the stratum corneum facing the diffusion cell and the inner side facing the receiving cell, and use a metal clip to stabilize the device.

[0141] 3. Add 3 ml of the sample solution shown in Table 6 to the diffusion cell as the feed solution. Set up three replicates for each sample group. Add 7 ml of phosphate buffer solution (pH 6.8) to the receiving cell, ensuring that bubbles are removed during addition. A magnetic stirrer is added. Maintain a constant temperature water bath at 37°C. Seal the diffusion cell with plastic wrap. At the appropriate time points, sample 0.3 ml and determine the active ingredient content. Simultaneously, add an equal volume of isothermal phosphate buffer solution (pH 6.8).

[0142] The concentration of β-nicotinamide mononucleotide (NMN) was determined using a microplate reader-UV spectrophotometer at an absorption wavelength of 261 nm. 100 μl of sample was added to each blank and the measurement temperature was 25°C. The NMN standard curve was plotted using concentration and absorbance as the horizontal and vertical coordinates, respectively.

[0143] The concentration of carnosine was determined by microplate reader-ultraviolet spectrophotometry at an absorption wavelength of 210 nm, with 100 μl added to each blank and a measurement temperature of 25°C. A carnosine standard curve was drawn using concentration and absorbance as the horizontal and vertical coordinates, respectively.

[0144] The concentration of vitamin C ethyl ether was determined using a microplate reader-UV spectrophotometer at an absorption wavelength of 245 nm. 100 μl of sample was added to each blank and the measurement temperature was 25°C. A standard curve of vitamin C ethyl ether was drawn using concentration and absorbance as the horizontal and vertical coordinates, respectively.

[0145] The concentration of thioneine was determined by microplate reader-ultraviolet spectrophotometry at an absorption wavelength of 254 nm, 100 μl of sample was added to each blank, and the measurement temperature was 25° C. The concentration and absorbance were used as the horizontal and vertical coordinates to draw a standard curve for thioneine.

[0146] The experimental results are shown in Table 6.

[0147] Table 6

[0148]

[0149]

[0150] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. A method for improving the skin permeability of a hydrophilic substance, characterized in that: The method comprises the steps of applying at least one lipopeptide and at least one hydrophilic substance to the skin, wherein the hydrophilic substance comprises at least a hydrophilic substance with a molecular weight of 800Da-100kDa. Preferably, the hydrophilic substance includes at least a hydrophilic substance with a molecular weight of 20 kDa-60 kDa; Preferably, the lipopeptide comprises at least one or more of surfactin, lichenysin, iturin, fengycin, daptomycin, micafungin, caspofungin, and polymyxin.

2. The method according to claim 1, characterized in that The hydrophilic substance with a molecular weight of 800Da-100kDa includes one or more of polypeptides, proteins, polysaccharides, nucleic acids and derivatives thereof.

3. The method according to claim 1 or 2, characterized in that The hydrophilic substance also includes a hydrophilic substance with a molecular weight less than 800 Da.

4. A transdermal absorption composition, characterized in that The composition comprises at least one lipopeptide and at least one hydrophilic substance in a physiologically acceptable medium, wherein the hydrophilic substance comprises at least a hydrophilic substance having a molecular weight of 800Da-100kDa. Preferably, the hydrophilic substance at least includes a hydrophilic substance with a molecular weight of 20 kDa-60 kDa.

5. The composition according to claim 4, characterized in that The concentration of the lipopeptide in the composition is 0.01%-5% (m / v), preferably 0.05%-1% (m / v).

6. The composition according to claim 4, characterized in that The concentration of the hydrophilic substance in the composition is 0.01%-5% (m / v), preferably 0.05%-0.1% (m / v).

7. Use of a lipopeptide in preparing a composition for improving skin permeability, characterized in that: The composition comprises at least one lipopeptide and at least one hydrophilic substance in a physiologically acceptable medium, wherein the hydrophilic substance comprises at least a hydrophilic substance having a molecular weight of 800Da-100kDa. Preferably, the hydrophilic substance at least includes a hydrophilic substance with a molecular weight of 20 kDa-60 kDa.

8. The composition according to any one of claims 4 to 6 or the use according to claim 7, characterized in that: The hydrophilic substance with a molecular weight of 800Da-100kDa includes one or more of polypeptides, proteins, polysaccharides, nucleic acids and derivatives thereof.

9. The composition according to any one of claims 4 to 6 or the use according to claim 7, characterized in that: The hydrophilic substance also includes a hydrophilic substance with a molecular weight less than 800 Da.

10. Use of the composition according to any one of claims 4 to 6 in the preparation of cosmetics.

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