Compositions for improving skin aging and methods of use and application thereof

Abstract

The application relates to the field of cosmetics, and specifically provides a composition for improving skin aging and application and method thereof, the composition comprising cyclin-dependent kinase 1, glutathione peroxidase 5 and cellular retinoic acid binding protein 1. The composition of the application can synergistically regulate cell proliferation, antioxidation and retinoic acid metabolic pathways, significantly improve skin photoaging, and improve bioavailability; the three protein molecules have a molecular weight less than 35 kDa, can efficiently penetrate the stratum corneum to reach the dermis, and can greatly improve the absorption rate of external use; the protein can be obtained through human recombinant expression, the risk of animal-derived protein allergy can be effectively reduced, the protein has high safety and industrialization potential, and has wide application value.

Description

Technical Field

[0001] This application relates to the field of cosmetics. Specifically, this application relates to compositions for improving skin aging, and their application and methods. Background Technology

[0002] Skin aging is a phenomenon caused by natural or unnatural factors, manifesting as dry skin, reduced elasticity, slowed epidermal renewal, and impaired barrier function. Key signs of skin aging include decreased skin elasticity, increased wrinkles, and pigmentation. Skin aging has long been a focus of attention in the fields of cosmetic and regenerative medicine. Currently, treatments for skin aging primarily include physical therapy, chemical therapy, and biological therapy, including but not limited to the use of antioxidants, UV protectants, and hormonal medications.

[0003] Existing protein-based aging treatments mainly repair damaged skin by promoting collagen synthesis, inhibiting degradation, or directly supplementing key proteins, primarily focusing on collagen and elastin. However, these treatments have the following technical problems: (1) Some animal-derived proteins are prone to causing allergic reactions; (2) Collagen plays a role in increasing skin elasticity, but for already formed wrinkles and loose skin, collagen supplementation alone is insufficient for complete repair; (3) Collagen (amino acid length: 1366AA) and elastin (786AA) have poor permeability in the skin surface due to their large molecular weight, and skin surface interventions can only produce limited effects on the epidermis, making it difficult to repair and rebuild structures such as collagen fibers in the dermis; (4) The key components in existing products usually only have a single therapeutic function, such as collagen mainly used to supplement skin collagen and elastin focused on improving skin elasticity, lacking multi-target synergistic effects; aging is a complex physiological process involving multiple aspects, such as oxidative stress, inflammatory response, and cell apoptosis, and a single protein cannot comprehensively solve the problem.

[0004] Stem cell-derived exosomes (SC-exo), as a bioactive composition containing various growth factors, cytokines, and nucleic acids, possess anti-inflammatory, antioxidant, and immunomodulatory effects, and are believed to help combat skin aging. SC-exo contains various proteins that regulate cellular functions, playing important regulatory roles in promoting skin cell growth, differentiation, oxidative damage repair, and extracellular matrix reconstruction. Previous studies have demonstrated that SC-exo, as a whole, helps improve skin wound healing and promote regeneration through various interventions such as topical application, microneedling, and patches. However, few studies have clarified which proteins play the role of the effective molecules and the mechanisms by which these molecules alleviate aging.

[0005] Meanwhile, the initial quantity of pluripotent stem cells is limited, and multiple passages may lead to decreased proliferation capacity, stem cell attenuation, and cell senescence, resulting in severe batch effects in stem cell-derived exosome products. In addition, most products currently lack a clear understanding of the composition and mechanism of action of stem cell derivatives, and large-scale production is carried out only after their effectiveness and safety are determined, and they are then added to corresponding cosmetics and skin care products.

[0006] Therefore, in the basic research and application transformation based on stem cell derivatives, it is urgent to identify the effective components or component combinations of stem cell-derived exosomes, so as to lay the foundation and focus for the application transformation of stem cell derivatives. Summary of the Invention

[0007] This application aims to at least partially address the technical problems existing in the prior art. To this end, this application proposes a composition and its application in the preparation of pharmaceuticals and cosmetics for improving skin aging, as well as a method for improving skin aging. The composition of this application significantly improves skin photoaging while enhancing bioavailability by synergistically regulating cell proliferation, antioxidant activity, and retinoic acid metabolism pathways. All three proteins have a molecular weight of less than 35 kDa, allowing them to efficiently penetrate the stratum corneum and reach the dermis, resulting in a significantly improved absorption rate when applied topically. The proteins can be obtained through human recombinant expression, effectively reducing the risk of allergic reactions to animal-derived proteins, thus possessing both high safety and industrialization potential, and has broad application value.

[0008] In one aspect of this application, a composition is provided. According to an embodiment of this application, the composition comprises: cyclin-dependent kinase 1, glutathione peroxidase 5, and retinoic acid-binding protein 1.

[0009] This application, based on the protein composition of stem cell exosomes and single-cell omics analysis of the skin after intervention, screened for potentially effective molecules and identified three proteins: cyclin-dependent kinase 1 (CDK1), glutathione peroxidase 5 (GPX5), and cellular retinoic acid-binding protein 1 (CRABP1). This application creatively discovered and verified that these three proteins are the core protein components for exosomes to exert their anti-aging effects. The combination of these three proteins can effectively alleviate cellular senescence. Combined with animal model experiments on photoaging, it was confirmed that it has a good repair effect on existing wrinkles and skin laxity, while also protecting against skin aging caused by prolonged ultraviolet radiation.

[0010] In principle, CDK1, which is related to cell proliferation, plays a key role in the control of the eukaryotic cell cycle by regulating the centrosome cycle and the initiation of mitosis. It is a key regulator of cell development and cell proliferation. The antioxidant enzyme GPX5 catalyzes the formation of the glutathione peroxidase-like protection system by catalyzing glutathione, reducing peroxides in cells and protecting cells and enzymes from oxidative damage and reactive oxygen species, including photo-oxidative stress, hydrogen peroxide, and organic hydroperoxides. The cellular retinoic acid binding protein CRABP1 can regulate the acquisition of retinoic acid by nuclear retinoic acid receptors. Retinoic acid-related metabolic processes (including retinoic acid and vitamin A-like substances) exhibit good anti-skin aging characteristics. Retinoic acid, a metabolite of retinoic acid, is also considered to be the gold standard therapy for treating photoaging.

[0011] Therefore, the composition of this application contains three proteins: CDK1, which promotes cell proliferation; GPX5, which has antioxidant properties; and CRABP1, which promotes the intracellular binding of retinoic acid. Bioinformatics results show that they are involved in processes such as oxidative stress, inflammatory responses, and apoptosis. The combination of these three proteins can exert a synergistic effect between targets, alleviating skin aging from multiple perspectives. Compared to directly using exosomes, the composition of this application uses well-defined components, which can greatly reduce the costs caused by batch-to-batch effects of exosomes and cell culture.

[0012] In addition, the above three proteins can be synthesized by humans, which reduces the likelihood of allergic reactions compared to proteins extracted from animal tissues. CDK1 has a molecular weight of 297 AA, GPX5 has a molecular weight of 162 AA, and CRABP1 has a molecular weight of 137 AA. These three proteins are only 1 / 10 to 1 / 5 the size of collagen, thus improving their skin permeability.

[0013] According to embodiments of this application, the composition may also have the following additional technical features:

[0014] According to embodiments of this application, the mass ratio of cyclin-dependent kinase 1, glutathione peroxidase 5, and retinoic acid-binding protein 1 is (0.5~5):(0.5~5):(0.5~5). In some embodiments, the mass ratio of cyclin-dependent kinase 1, glutathione peroxidase 5, and retinoic acid-binding protein 1 is (0.5~4):(0.5~4):(0.5~4), (0.6~3):(0.6~3):(0.6~3), (0.8~2):(0.8~2):(0.8~2), (0.9~1.5):(0.9~1.5):(0.9~1.5). Combining these three proteins in the above ratios can further enhance the aging-improving effect.

[0015] In another aspect of this application, a cosmetic product is proposed. According to an embodiment of this application, the cosmetic product comprises the composition described above. Thus, the cosmetic product of this application significantly improves skin aging while enhancing bioavailability by synergistically regulating cell proliferation, antioxidant activity, and retinoic acid metabolism pathways. Furthermore, it exhibits high permeability and good skin absorption; the protein can be obtained through human recombinant expression, effectively reducing the risk of allergic reactions to animal-derived proteins, possessing both high safety and industrialization potential, and has broad application value.

[0016] According to embodiments of this application, the cosmetic includes at least one of toner, lotion, face cream, eye cream, and serum; the cosmetic further includes excipients.

[0017] According to embodiments of this application, the excipients include at least one of the following: surfactants, thickeners, conditioning agents, pH adjusters, salts, preservatives, skin feel conditioning agents, emulsifiers, antioxidants, and water.

[0018] The addition of surfactants helps form a homogeneous and stable emulsion system in the aqueous and oil phases, enhances the dispersibility of components, and also helps active ingredients penetrate the skin better, thereby improving their efficacy. In some embodiments, the surfactant includes at least one selected from sodium cocoyl glycinate, sodium cocoyl glutamate, potassium cocoyl glycinate, potassium cocoyl glutamate, sodium lauroyl glycinate, sodium lauroyl glutamate, sodium methyl lauroyl taurate, cocamidopropyl hydroxysulfobetaine, disodium cocoamphodiacetate, dodecanoic acid, tetradecanoic acid, and hexadecanoic acid.

[0019] Thickeners can increase the viscosity of a product, improve its stability and feel, and make the product easy to apply and less prone to flowing. In some embodiments, the thickener includes at least one of cocamide, acrylic copolymers, acrylate copolymers, and hydroxypropyl methylcellulose.

[0020] Conditioners can improve the sensory properties of a product, such as smoothness, softness, and silkiness, providing a better user experience. In some embodiments, the conditioner includes at least one of polyquaternium salts and glycol distearate.

[0021] pH adjusters maintain the product within a specific pH range, preserving product stability, preventing microbial growth, and conforming to the physiological pH of the skin. In some embodiments, the pH adjuster includes at least one of citric acid, sodium hydroxide, and potassium hydroxide.

[0022] Salts, acting as thickeners or stabilizers, affect product viscosity and the stability of emulsion systems. In some embodiments, the salt includes at least one of sodium chloride and potassium chloride.

[0023] Preservatives protect products from microbial contamination and extend their shelf life. In some embodiments, the preservative includes at least one of potassium sorbate and phenoxyethanol.

[0024] Skin feel agents can improve how a product feels on the skin, such as providing a cooling, refreshing, moisturizing, or matte finish. In some embodiments, the skin feel agent includes at least one of cyclohexylsiloxane and polydimethylsiloxane.

[0025] Emulsifiers stabilize oil-water mixtures, prevent stratification, and form uniform emulsions or pastes. In some embodiments, the emulsifier includes at least one selected from oleyl alcohol polyether, cetyl PEG / PPG-10 / 1 polydimethylsiloxane, sodium laurylate, caprylic / capric triglyceride, polyethylene glycol, and polypropylene glycol.

[0026] Antioxidants prevent the oxidation and deterioration of oils and active ingredients in a product, maintaining its freshness and efficacy. These antioxidants include at least one of zinc stearate, tocopheryl acetate, tea polyphenols, tert-butylhydroxybenzoate, butylated hydroxytoluene, lecithin, vitamin C, and vitamin E.

[0027] According to embodiments of this application, the cosmetic further comprises at least one selected from collagen, elastin, small molecule compounds, animal extracts, microbial extracts, and plant extracts. Exemplarily, the small molecule compounds are selected from organic compound molecules with a molecular weight of less than 900 Daltons.

[0028] In another aspect, this application proposes a pharmaceutical product. According to an embodiment of this application, the pharmaceutical product comprises the aforementioned composition. Thus, the pharmaceutical product of this application significantly improves skin aging while enhancing bioavailability by synergistically regulating cell proliferation, antioxidant activity, and retinoic acid metabolism pathways. Furthermore, it exhibits high permeability and good skin absorption; the protein can be obtained through human recombinant expression, effectively reducing the risk of allergic reactions to animal-derived proteins, possessing both high safety and industrialization potential, and has broad application value.

[0029] According to embodiments of this application, the pharmaceutical product includes oral preparations, injectable preparations, or topical preparations. In some embodiments, topical preparations include topical solutions, tinctures, liniments, creams, gels, emulsions, sprays, or patches, etc.

[0030] According to embodiments of this application, the pharmaceutical product further comprises at least one of collagen, elastin, small molecule compounds, animal extracts, microbial extracts, and plant extracts.

[0031] According to embodiments of this application, the pharmaceutical product further includes pharmaceutically acceptable excipients, such as carriers, solvents, suspending agents, or excipients. Exemplary excipients may be liquids or solids, including but not limited to: pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, flow aids, sweeteners, dyes / coloring agents, flavor enhancers, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, emulsifiers, sprays, compressed air or other suitable gases, or other suitable inactive ingredients that can be used in conjunction with the pharmacodynamic compound.

[0032] In another aspect of this application, the use of the aforementioned composition in the preparation of cosmetics or pharmaceuticals is proposed. According to embodiments of this application, the cosmetics or pharmaceuticals are used to improve skin aging. The composition of this application significantly improves skin aging while enhancing bioavailability by synergistically regulating cell proliferation, antioxidant activity, and retinoic acid metabolism pathways; all three proteins have a molecular weight of less than 35 kDa, allowing them to efficiently penetrate the stratum corneum and reach the dermis, resulting in a significantly improved absorption rate for topical application; the proteins can be obtained through human recombinant expression, effectively reducing the risk of allergic reactions to animal-derived proteins, possessing both high safety and industrialization potential, and has broad application value.

[0033] According to embodiments of this application, the skin aging includes at least one of photoaging, natural aging, chemically induced aging, and inflammation-induced aging.

[0034] In another aspect, this application proposes a method for improving skin aging. According to embodiments of this application, the method includes: administering the aforementioned composition, the aforementioned cosmetic, or the aforementioned pharmaceutical product to a subject. This method significantly improves skin aging while enhancing bioavailability by synergistically regulating cell proliferation, antioxidant activity, and retinoic acid metabolism pathways; all three proteins have a molecular weight of less than 35 kDa, allowing them to efficiently penetrate the stratum corneum and reach the dermis, resulting in a significantly improved topical absorption rate; the proteins can be obtained through recombinant human expression, effectively reducing the risk of allergic reactions to animal-derived proteins, combining high safety with industrialization potential, and possessing broad application value.

[0035] According to embodiments of this application, the skin aging includes at least one of photoaging, natural aging, chemically induced aging, and inflammation-induced aging.

[0036] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0037] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0038] Figure 1 This diagram illustrates the relative changes in the SASP senescence phenotype in senescent cells after 48 hours of treatment with the three proteins, individually and in combination.

[0039] Figure 2 The image shows a photograph of the mouse's back.

[0040] Figure 3 The results of immunohistochemical staining for the mouse skin aging marker P16 are shown. + Cell percentage analysis diagram, arrow indicates P16 + cell;

[0041] Figure 4 The results of Masson staining and collagen density analysis of mouse skin are shown. In the image above, the area with higher grayscale at the top of the image represents collagen.

[0042] Figure 5 The GO analysis graph is displayed;

[0043] Figure 6 The diagram shows the analysis of mice treated with CDK1+GPX5+CRABP1 protein complex and exosomes derived from mesenchymal stem cells. Detailed Implementation

[0044] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0045] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0046] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0047] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0048] This application proposes a composition comprising: cyclin-dependent kinase 1, glutathione peroxidase 5, and retinoic acid-binding protein 1. These three proteins are core components of exosomes in their anti-aging effects. The combination of these three proteins can effectively alleviate cellular aging. Combined with animal model experiments on photoaging, it has been demonstrated that it has a good repair effect on existing wrinkles and skin laxity, while also protecting against skin aging caused by prolonged ultraviolet radiation.

[0049] The protein sequence of cyclin-dependent kinase 1 (CDK1) is as follows: Gene ID: 983, UniProtKB: P06493, and molecular weight: 34 kDa.

[0050] MEDYTKIEKIGEGTYGVVYKGRHKTTGQVVAMKKIRLESEEEGVPSTAIREISLLKELRHPNIVSLQDVLMQDSRLYLIFEFLSMDLKKYLDSIPPGQYMDSSLVKSYLYQILQGIVFCHSRRVLHRDLKPQNLLIDDKGTIKLADFGLA RAFGIPIRVYTHEVVTLWYRSPEVLLGSARYSTPVDIWSIGTIFAELATKKPLFHGDSEIDQLFRIFRALGTPNNEVWPEVESLQDYKNTFPKWKPGSLASHVKNLDENGLDLLSKMLIYDPAKRISGKMALNHPYFNDLDNQIKKM (SEQ ID NO: 1)

[0051] The protein sequence of glutathione peroxidase 5 (GPX5) is as follows: GeneID: 14780, UniProtKB ID: O75715, and molecular weight is 25 kDa.

[0052] MTTQLRVVHLLPLLLACFVQTSPKQEKMKMDCHKDEKGTIYDYEAIALNKNEYVSFKQYVGKHILFVNVATYCGLTAQYPELNALQEELKPYGLVVLGFPCNQFGKQEPGDN KEILPGLKYVRPGGGFVPSFQLFEKGDVNGEKEQKVFSFLKHSCPHPSEILGTFKSISWDPVKVHDIRWNFEKFLVGPDGIPVMRWSHRATVSSVKTDILAYLKQFKTK (SEQ ID NO:2)

[0053] The protein sequence of Cellular retinoic acid-binding protein 1 (CRABP1) is as follows: Gene ID: 1381, UniProtKB ID: P29762, and molecular weight is 15 kDa.

[0054] MPNFAGTWKMRSSENFDELLKALGVNAMLRKVAVAAASKPHVEIRQDGDQFYIKTSTTVRTTEINFKVGEGFEEETVDGRKCRSLATWENENKIHCTQTLLEGDGPKTYWTRELANDELILTFGADDVVCTRIYVRE (SEQ IDNO: 3)

[0055] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0056] Example 1

[0057] 1. Prepare the test solution

[0058] Different proteins were prepared into test solutions with DPBS (Duborough phosphate buffer), with each protein / exosome concentration at 0.05 wt%. The proteins were from MCE, MSCexo from Cyagen Biosciences (HUXUC-01001), and DPBS from Gibico.

[0059] Experimental group 1: Glutathione peroxidase 5 (GPX5);

[0060] Experimental group 2: Cyclin-dependent kinase 1 (CDK1);

[0061] Experimental group 3: Cellular retinoic acid binding protein 1 (CRABP1);

[0062] Experimental group 4: GPX5+CDK1;

[0063] Experimental group 5: GPX5 + CRABP1;

[0064] Experimental group 6: CDK1 + CRABP1;

[0065] Experimental group 7: GPX5+CDK1+CRABP1;

[0066] Experimental group 8: Exosomes derived from human umbilical cord mesenchymal stem cells (MSCexo);

[0067] Control group: PBS buffer.

[0068] 2. Cell experiments

[0069] After 13 passages, mesenchymal stem cells (MSCs) showed significant senescence and lost their rapid proliferative capacity. Senescent MSCs were seeded in 6-well plates (1 x 10^5 cells / well), and 2 mL of culture medium was added. Four replicates were performed for each treatment. The test solution prepared in step 1 was added to the MSC culture medium. Two days later, changes in the levels of senescence-associated secretory phenotype (SASP) were detected using flow cytometry-encoded microsphere array (CBA) technology to determine the cellular experimental effect of the complex protein in alleviating cellular senescence.

[0070] The results are as follows Figure 1 As shown, using CDK1 alone or in combination with CDK1+GPX5+CRABP1 significantly improves cellular senescence phenotypes. Cellular-level experimental results suggest that the combination of these three proteins can exert a better anti-aging function.

[0071] 3. Animal experiments

[0072] Thirty-two hairless nude mice were divided into four groups (mice were 3 months old, weighing 20-25g each, and were female). From day 0 to day 28, mice were anesthetized and irradiated with 9500 kDa UV light for 10 minutes each day, followed by a 3-minute cooling period before irradiation. This process was repeated six times. After day 28, noticeable wrinkles were observed on the backs of the mice. While maintaining light exposure, 100 μL of the test solution prepared in step 1 was applied to the backs of the mice every three days using a roller. The formation of wrinkles on the backs of the mice was assessed by photographs. After six interventions, the mice were euthanized, and the back skin was paraffin-embedded and sectioned. Immunohistochemical staining was used to detect the expression level of the aging marker P16, and Masson staining was used to detect collagen thickness.

[0073] like Figure 2 White light observation results showed that the CDK1+GPX5+CRABP1 protein complex solution could effectively alleviate wrinkles in mice, while CDK1 alone did not have an allergic effect.

[0074] like Figure 3 and Figure 4 As shown, the CDK1+GPX5+CRABP1 protein complex reduces the accumulation of the aging marker P16 in the skin and can promote an increase in skin collagen volume.

[0075] like Figure 5 As shown, CDK1, GPX5, and CRABP1 proteins are involved in various cell biological regulatory processes closely related to photoaging, such as oxidative stress, inflammatory response, and apoptosis.

[0076] like Figure 6 As shown, the CDK1+GPX5+CRABP1 protein complex and mesenchymal stem cell-derived exosomes (MSCexo) treated mice respectively, and the effects were comparable.

[0077] Based on the above in vivo experimental results, CDK1, GPX5 and CRABP1 proteins have the beneficial effect of delaying photoaging of mouse skin. Compared with MSCexo, the composition is more clearly defined and the properties are more stable. Under the same effect, the cost of application and conversion of compound proteins is lower and there are fewer foreseeable batch effects.

[0078] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A composition, characterized in that, include: Cyclin-dependent kinase 1, glutathione peroxidase 5, and retinoic acid-binding protein 1; The mass ratio of cyclin-dependent kinase 1, glutathione peroxidase 5, and retinoic acid-binding protein 1 is (0.5~5):(0.5~5):(0.5~5).

2. A cosmetic product, characterized in that, include: The composition of claim 1.

3. The cosmetic product according to claim 2, characterized in that, The cosmetics include at least one of toner, lotion, face cream, eye cream, and serum; The cosmetic further includes at least one of the following: collagen, elastin, small molecule compounds, animal extracts, microbial extracts, and plant extracts; The cosmetics further include excipients.

4. A medicine, characterized in that, include: The composition of claim 1.

5. The medicine according to claim 4, characterized in that, The medicine includes oral preparations, injections, or topical preparations, and the topical preparations include topical solutions, tinctures, liniments, creams, gels, emulsions, sprays, or patches; The drug further includes at least one of the following: collagen, elastin, small molecule compounds, animal extracts, microbial extracts, and plant extracts.

6. The use of the composition of claim 1 in the preparation of cosmetics or pharmaceuticals, characterized in that, The cosmetics or pharmaceuticals mentioned are used to improve skin aging.

7. The application according to claim 6, characterized in that, The skin aging mentioned includes at least one of photoaging, natural aging, chemically induced aging, and inflammation-induced aging.

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