A targeted peptide-modified beauty peptide composition

Abstract

The present application discloses a targeted peptide modified beauty peptide composition, which is a complex composed of a targeting peptide and a beauty peptide covalently / non-covalently. The targeting peptide has a linear or cyclic peptide composed of 3 to 30 amino acids, including an RGD polypeptide targeting the cell surface integrin αvβ3, an Otx2 polypeptide targeting the cell surface chondroitin sulfate, or a transmembrane peptide targeting the cell interior. Beauty peptides include acetylated, fatty acid acylated or non-acylated active peptides commonly seen on the market that exert effects inside and outside cells. The targeted beauty peptide composition of the present invention does not affect the activity of beauty peptides, is used for skin makeup treatment, promotes cell surface aggregation or intracellular guidance of beauty peptides, increases the chance of beauty peptides binding to intracellular and extracellular receptors, and enhances the efficacy of beauty peptides, especially anti-oxidation, anti-aging, increases the collagen content of skin tissue, and repairs the keratin structure of skin cells. It has good application potential in the field of cosmetic raw materials.

Description

Technical Field

[0001] The invention relates to the technical field of anti-oxidation and anti-aging skin care products, and in particular to a targeted peptide-modified beauty peptide composition. Background Art

[0002] Bioactive peptides are derived from proteins, usually functional peptide fragments containing 3-30 amino acids connected by amide bonds. Bioactive peptides can be signal molecules that maintain normal physiological reactions of the body, or they can be substrates that directly participate in life reactions. Beauty peptides are a type of bioactive peptides with beauty and skin care effects. Common beauty peptides can improve the survival rate and proliferation rate of aging human epidermal cells or skin fibroblasts, stimulate skin tissue activity, and increase the movement of matrix cells; they can also effectively remove the accumulation of pigments, reduce inflammation, promote the synthesis of cell components such as extracellular matrix, collagen, fibronectin, hyaluronic acid, glycosaminoglycans, repair the loss of nutrients in the skin, and maintain skin moisture and elasticity. Beauty peptides can be divided into three categories: signal peptides, carrier peptides, and inhibitory peptides. Signal peptides can bind to cell surface receptors to activate downstream cell signaling pathways, activate enzyme-linked reactions, and produce effects, such as tripeptide-1 and palmitoyl pentapeptide-4. Carrier peptides can transport trace elements Cu and Mg into cells to activate enzymatic reactions, such as blue copper peptides. Inhibitory peptides can competitively bind to cell surface receptors, inhibit the formation of neuroligin receptor complexes, reduce the release of acetylcholine, inhibit muscle contraction, and reduce wrinkles, such as acetyl hexapeptide-8. The structure of whitening nonapeptide-1 is similar to the endogenous neuropeptide melanocyte stimulating hormone (MSH-α), and can competitively bind to the melanocortin-1 receptor (MC-1R), inhibiting the stimulation of melanocyte stimulating hormone on melanocytes, reducing the activity of tyrosinase, and reducing the biosynthesis of melanin inside cells, thereby playing a role in whitening and brightening the skin. Beauty peptides have good biocompatibility, clear efficacy, high safety performance, controllable quality, selectivity and non-immunogenicity, so they are widely used in the cosmetics field. However, there are also obstacles that need to be overcome, such as medium efficiency, slow absorption, and high cost.

[0003] Integrins are a class of heterodimeric cell adhesion molecules composed of two membrane proteins, α and β. They are expressed on the cell membrane surface and in the cell matrix and are important signaling molecules for cell survival, proliferation, migration, and communication. The α chain has antigen specificity, and the β chain is related to signal transduction. 18 different α chains (α1~11, αⅡb, αD, αE, αL, αM, αV, αX) and 8 β chains (β1-8) make up the 24 known integrins, which have different substrate specificity. Integrin binding mechanisms are diverse, and a cell or extracellular matrix protein can express several different integrins, and vice versa. αvβ3 is composed of αv chain and β3, and is expressed in skin cells, endothelial cells, macrophages, neutrophils, melanoma cells, and most cancer cells. Integrin αvβ3 is a vitronectin receptor, and binding to vitronectin (VN) can maintain the integrity of capillaries and mediate platelet adhesion to VN. In addition to binding vitronectin, integrin αvβ3 can bind to fibronectin, laminin, cell adhesion factors PECAM and L1, etc. αvβ3 on vascular smooth muscle regulates arteriolar contraction and relaxation by activating intracellular L-type calcium channels, and plays a regulatory role in blood flow and vascular tension. In addition, integrins also play an important role in the occurrence and development of skin tumors. For example, integrin αvβ3 is related to the progression of melanoma cells, and the αv chain is expressed at all stages of melanoma progression. The interaction between αvβ3 and cell adhesion factors and extracellular matrix promotes the adhesion, extension and migration of tumor cells. A large number of studies have reported that the RGD peptide composed of three amino acids specifically recognizes integrin αvβ3, and can target and deliver related antibodies or anticancer drugs to the lesions for treatment. RGD has a large number of applications in tumor treatment and the development of anticancer drugs, and the therapeutic effect is significantly higher than that of drug carriers without RGD modification. As a result, a variety of short peptides based on RGD sequences have also been applied to the treatment of various cancers. At the same time, studies have shown that H2O2 induces the activation of the intracellular p38 MAPK-β3 positive feedback loop, upregulates the expression of the β3 chain, and may increase the expression of integrin αvβ3. Therefore, αvβ3 may be used as an important target for skin cells to improve skin management. RGD or RGD-derived short peptides are covalently linked to the upstream end of the beauty peptide to form a combined chimeric peptide, or the two are directly physically mixed to form a complex to verify the relevant beauty indicators of cell efficacy.

[0004] Otx2 is a homologous protein containing a highly conserved bicoid genotype homeodomain. It plays an important role in embryonic formation and postnatal development of the cerebral cortex, especially its brain plasticity. At the same time, studies have reported that Otx2 also plays an important role in maintaining the activity of the optic ganglion. Otx2 is first synthesized in the retina, then transferred and aggregated to the parvalbumin neurons (PV cells) of the visual layer, and waits until it matures, mediating the opening of the visual layer first, and then closing it during the critical period of plasticity development. Studies have reported that in the retina, Otx2 can specifically bind to ganglion neurons and bipolar neurons, and then scientists have extracted the specific recognition sequence of Otx2. Similarly, this sequence is also responsible for the targeted recognition of Otx2 to the parvalbumin neurons in the visual layer. Experimental results show that exogenously added specific recognition sequences can interact with sulfated proteoglycans around these neurons, thereby competing with endogenous Otx2 to bind to PV cells, thereby inhibiting the PV cell endocytosis of endogenous Otx2, ultimately causing PV cells to return to their pre-mature state and re-establishing visual cortical plasticity. Reports have pointed out that the key sequence of this Otx2 specific recognition of proteoglycans (PGs) is composed of 15 amino acids, and the interaction between this peptide segment and proteoglycans is the same as that of the entire Otx2 segment. Glycosaminoglycans are components that exist in large quantities on the surface of cells in the body, so the polypeptide sequence based on these 15 amino acids can be used as a targeting peptide to modify beauty peptides, increasing the concentration and efficacy of effective bioactive peptides on the cell surface and inside.

[0005] Cell-penetrating peptides are a class of polypeptide sequences originating from the homologous domain of homologous proteins that have the property of freely crossing the cell membrane, usually containing 3-50 amino acids. Therefore, the targeting peptide described in the present invention also refers to cell-penetrating peptides that can cross the cell membrane. Cell-penetrating peptides are applied to the fields of anti-tumor drugs as small molecule carriers because of their good biocompatibility, good membrane-penetrating efficiency, ability to overcome the blood-brain barrier, and low immunogenicity. With the development of peptide structure analysis and peptide synthesis technology, scientists have begun to design unique polypeptide sequences based on the relationship between the structure and function of amino acids, or to combine them using non-natural amino acid modifications and functional peptide segments, resulting in artificial synthetic polypeptides that are more efficient, less toxic, and more intelligent than natural membrane-penetrating peptides. The cell-penetrating mechanism of cell-penetrating peptides can be mainly divided into energy-dependent endocytosis mechanisms and non-energy-dependent direct transmembrane forms. A polypeptide can have both mechanisms at the same time. The endocytosis mechanism is relatively common and efficient, while the direct transmembrane mechanism transports small molecules into the cytoplasm, which can avoid the small molecule enzymatic hydrolysis of the small molecules contained due to being restricted in the endosomes to a certain extent. Most amphiphilic membrane-penetrating peptides have direct transmembrane mechanisms. Therefore, grafting the membrane-penetrating peptide onto the beauty peptide can improve the intracellular aggregation of the beauty peptide with intracellular targets to a certain extent, thereby improving the efficacy of the beauty peptide.

[0006] In daily life, oxygen free radicals (ROS) produced by normal growth and metabolism of skin cells are often in homeostatic balance in the body. However, external environmental stimulation, such as air pollution, solar ultraviolet light irradiation, or acute and chronic inflammatory diseases in the body, will produce excessive ROS, leading to protein and lipid peroxidation, destroying the defense of the body's antioxidant enzyme system, producing DNA mutations, thereby accelerating skin aging and apoptosis, and even causing diseases. Therefore, combating skin aging caused by endogenous and exogenous ROS is an important direction of anti-aging research in the cosmetics field. Inhibiting the excessive production of ROS in the body, combined with inhibiting muscle contraction caused by neurotransmitter release to produce wrinkles and supplementing skin collagen is the mainstream beauty anti-aging route. At present, there are countless antioxidant and anti-wrinkle beauty peptides on the market, mainly acetyl hexapeptide-8, tripeptide-1 copper, carnosine, glutathione, palmitoyl pentapeptide-4, nonapeptide-1, etc. However, poor transdermal permeability, high effective concentration, and unsatisfactory skin care effects have limited the application of beauty peptides. Summary of the invention

[0007] Technical issues solved

[0008] The present invention provides a targeted peptide modified beauty peptide composition, which solves the problems in the prior art that the beauty peptide has no targeting effect and cannot be enriched on the cell surface to exert its effect, so the effective dose of the beauty peptide is high, the cost is increased, and the toxicity is increased; and the molecular weight of the beauty peptide is usually large, so the ability to penetrate the skin and freely pass through the cell is weak, which reduces the efficacy of the beauty peptide acting intracellularly. The complex uses the targeted functional peptide to enrich on the cell surface or inside to increase its beauty and skin care efficacy, and more specifically increases the ability of the beauty peptide to resist oxidation, resist aging, repair the skin barrier, and replenish water; through the specific recognition function with the cell surface component or the transmembrane mechanism of efficiently penetrating the cell membrane, the aggregation concentration of the beauty peptide on the cell surface and inside the cell is enhanced, and the skin care efficacy of the targeted beauty peptide composition is enhanced.

[0009] The present invention adopts the following technical solutions:

[0010] A targeting peptide-modified cosmetic peptide composition, wherein the targeting peptide-modified cosmetic peptide composition is a complex formed by covalently or non-covalently linking a targeting peptide and a cosmetic peptide, wherein the targeting peptide is a targeting peptide targeting cell surface integrins, cell surface glycosaminoglycans (GAGs), or a cell-penetrating peptide having the characteristics of targeting the interior of a cell and freely passing through the cell membrane; the action target of the cosmetic peptide is on the cell surface or inside the cell.

[0011] Furthermore, the targeted cell surface integrin is integrin αvβ3 which is highly expressed on the surface of tumor vascular endothelial cells and on the surface of skin cells; the specific recognition polypeptide ligand sequence of integrin αvβ3 is the arginine (R)-glycine (G)-aspartic acid (D) sequence (RGD).

[0012] Furthermore, the targeting peptide and the cosmetic peptide are covalently linked via an amide bond.

[0013] Furthermore, the targeting peptide comprises 3-10 amino acids, and the amino acids are natural amino acids or non-natural amino acids, including RGD and its derivatives.

[0014] Furthermore, the RGD derivative is a linear or cyclic pentapeptide or hexapeptide containing an RGD motif, specifically a linear or cyclic RGD derivative a (RGDYK) or RGD derivative b (RGDFK) containing an RGD motif.

[0015] Further, the motif of the targeting peptide sOtx2 for cell surface glycosaminoglycans GAGs is RKQRRER; the targeting peptide is derived from the homologous protein orthodenticle homeobox protein 2 (Otx2), and is a specific binding ligand for polyanionic components of glycosaminoglycans (GAGs) chondroitin sulfate (CS) or heparan sulfate (HS) on the targeting cell membrane, and its motif is B1B2X1B3B4X2B5X3X4X5X6B6X7X8X9, wherein B1, B2, B3, B4, B5, and B6 represent arginine (R) or lysine (K); X1 and X8 are asparagine or glutamine; X2 represents aspartic acid or glutamic acid; X3, X4 and X6 represent serine or threonine; X5 represents phenylalanine, tyrosine or tryptophan; X7 represents alanine or glycine; X9 is leucine, isoleucine or valine.

[0016] Furthermore, the motif of the specific binding ligand is RKQRRERTTFTRAQL.

[0017] Furthermore, the cell-penetrating peptides with the property of targeting the interior of the cell and freely passing through the cell membrane include natural and artificially synthesized cell-penetrating peptides; the natural and artificially synthesized cell-penetrating peptides include cationic (anionic) peptides, hydrophobic peptides, and amphipathic peptides; the cationic (anionic) peptides, hydrophobic peptides, and amphipathic peptides include common classical cell-penetrating peptides Tat (GRKKRRQRRRPPQ), Penetratin (RQIKIWFQNRRMKWKK), pVEC (LLIILRRRIRKQAHAHSK), VP22 (NAATATRGRSAASRPTQRPRAPARSASRPRRPVQ), MTS (AAVALLPAVLLALLP), Pep-1 (KETWWETWWTEWSQPKKKRKVC), Transportan (GWTLNSAGYLLGKINLKALAALAKKIL), TP10 (AGYLLGKINLKALAALAKKIL), R8 (RRRRRRRR), MAP (KLALKLALKALKAALKLA) and its derivatives.

[0018] Furthermore, the beauty peptides are naturally derived and artificially synthesized bioactive peptides with beauty and skin care effects; the beauty peptides include signal peptides, carrier peptides, neuroinhibitory peptides and enzyme competitive binding peptides; the signal peptides, carrier peptides, neuroinhibitory peptides and enzyme competitive binding peptides include one or more of carnosine, snake venom peptides, acetyl hexapeptide-8 / hexapeptide-8, palmitoyl pentapeptide-4 / pentapeptide-4, palmitoyl tripeptide-5 / tripeptide-5, blue copper peptide, tripeptide-1, palmitoyl tetrapeptide-7 / tetrapeptide-7, nonapeptide-1, acetyl tetrapeptide-5 / tetrapeptide-5, myristoyl pentapeptide-4 / pentapeptide-4.

[0019] Furthermore, the non-covalent connection is achieved by incubating the targeting peptide and the beauty peptide in a physical mixing manner according to the ratio of the targeting peptide and the beauty peptide, and forming a targeting peptide beauty peptide complex by utilizing ionic interaction, hydrogen bonding, and hydrophobic interaction; the ratio of the targeting peptide to the beauty peptide is 1-5:1.

[0020] Beneficial Effects

[0021] The targeted peptide-modified cosmetic peptide composition of the present invention, using the above technical solution, has the following technical effects compared with the prior art:

[0022] 1. The cosmetic peptide of the present application has the effects of anti-oxidation, anti-aging, anti-wrinkle, repair and moisturizing, anti-aging, and promoting the synthesis of cell glycosaminoglycan and collagen; the present invention covalently links the polypeptide RGD targeting integrin αvβ3 or the Otx2 polypeptide sequence sOtx2 targeting glycosaminoglycan (GAGs) or the amphiphilic transmembrane peptide MAP with the cosmetic peptide, and evaluates the cell efficacy of the obtained chimeric targeted cosmetic peptide; the technical problems of medium efficiency, slow absorption, high cost, poor transdermal performance, and unsatisfactory skin care efficacy of cosmetic peptides are overcome;

[0023] 2. The complex uses targeted functional peptides to be enriched on the cell surface or inside to increase its beauty and skin care effects, and more specifically, to increase the ability of beauty peptides to resist oxidation, anti-aging, repair skin barrier, and replenish water;

[0024] 3. The targeted beauty peptide composition of the present invention does not affect the activity of the beauty peptide, is used for skin cosmetic treatment, promotes the cell surface aggregation or intracellular orientation of the beauty peptide, increases the chance of the beauty peptide binding to intracellular and extracellular receptors, and enhances the efficacy of the beauty peptide, especially the effects of anti-oxidation, anti-aging, increasing the collagen content of skin tissue, and repairing the keratin structure of skin cells. It has great application potential in the field of cosmetic raw materials;

[0025] 4. Use H2O2 to construct a cell oxidative damage model for skin fibroblasts HFF and immortalized keratinocytes HaCaT, and evaluate the efficacy of chimeric beauty peptides on the proliferation, apoptosis, expression of senescence protein β-galactosidase (β-gal), antioxidant enzyme system and protein gene levels of matrix metalloproteinases in oxidatively damaged cells after treatment;

[0026] 5. In the embodiment of the present invention, the amphiphilic transmembrane peptide MAP is selected as a carrier of the beauty peptide to transport the beauty peptide into the cell to exert its effect, thereby increasing the transdermal effect and cell concentration of the beauty peptide. It is worth mentioning that the amphiphilic transmembrane peptide MAP also has a certain antibacterial effect, so it can increase the stability of the beauty peptide and the synergistic effect of incorporation to a certain extent;

[0027] 6. By utilizing the principle of targeted aggregation, the concentration of beauty peptides on the cell surface and inside the cell is increased, making the efficacy of the invented chimeric beauty peptides better than the non-chimeric beauty peptides currently used on the market; compared with non-chimeric beauty peptides, targeted chimeric beauty peptides can significantly inhibit the production of intracellular ROS by 25%, increase the activity of the antioxidant enzyme SOD by about 20%, reduce the aging and apoptosis of oxidatively damaged cells, inhibit the expression levels of cellular metalloproteinase genes and proteins, and reduce the loss of cellular collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the chemical structure diagram of the RGD motif of this application.

[0029] Figure 2 This is the chemical structure diagram of the sOtx2 motif of this application.

[0030] Figure 3 This is the chemical structure diagram of the MAP motif of this application.

[0031] Figure 4 This is the chemical structure diagram of hexapeptide-8 of this application.

[0032] Figure 5 This is the chemical structure diagram of tripeptide-1 of this application.

[0033] Figure 6 This is a graph showing the relative content of ROS in HaCaT cells in Example 1 of the present application.

[0034] Figure 7 This is a diagram of the intracellular SOD activity of HaCaT in Example 1 of the present application.

[0035] Figure 8 This is a graph of Caspase-3 activity in HaCaT cells in Example 1 of the present application.

[0036] Fig. 9 This is a graph of the HYP content of HaCaT cells in Example 1 of the present application.

[0037] Fig.10 These are the micrographs of β-gal-positive HFF cells and the cell counting graphs in Example 1 of the present application, wherein a is a micrograph of β-gal-positive HFF cells, and b is a cell counting statistical graph.

[0038] Fig.11 This is a graph showing the relative content of ROS in HaCaT cells in Example 2 of the present application.

[0039] Fig.12 This is a diagram of the intracellular SOD activity of HaCaT in Example 2 of the present application.

[0040] Fig.13 This is a diagram of Caspase-3 activity in HaCaT cells in Example 2 of the present application.

[0041] Fig.14 This is a graph of the HYP content of HaCaT cells in Example 2 of this application.

[0042] Fig.15 This is a graph showing the MMP-1 content in the supernatant of HaCaT cells in Example 2 of the present application.

[0043] Fig.16 These are the micrographs of β-gal-positive HFF cells and the cell counting graphs in Example 2 of the present application, wherein a is a micrograph of β-gal-positive HFF cells, and b is a cell counting statistical graph.

[0044] Fig.17 This is a diagram of HaCaT cell proliferation in Example 3 of the present application.

[0045] Fig.18 These are the micrographs of β-gal-positive HFF cells and the cell counting graphs in Example 3 of the present application, wherein a is a micrograph of β-gal-positive HFF cells, and b is a cell counting graph.

[0046] Fig.19 This is a graph showing the elastase inhibition rate in Example 4 of the present application.

[0047] Fig. 20 These are the early cell apoptosis and Caspase-3 activity diagrams in Example 4 of the present application, wherein a is a statistical diagram of early cell apoptosis, and b is a statistical diagram of Caspase-3 activity.

[0048] Fig.21 In Example 4 of the present application, a is a statistical graph of cell HYP content, b is a statistical graph of MMP-1 protein expression content, and c is a statistical graph of MMP-1 gene level.

[0049] Fig. 22 1 is a graph showing cell apoptosis and Caspase-3 activity in Example 5 of the present application, wherein a is a statistical graph showing cell apoptosis, and b is a statistical graph showing Caspase-3 activity.

[0050] Fig.23 These are graphs of cell HYP content, MMP-1 protein and gene levels in Example 5 of the present application, wherein a is a statistical graph of cell HYP content, b is a statistical graph of MMP-1 protein expression content and c is a statistical graph of gene level. DETAILED DESCRIPTION

[0051] The following is a description of the exemplary embodiments of the present application, including various details of the embodiments of the present application to facilitate understanding, which should be considered as merely exemplary. Therefore, it should be recognized by those of ordinary skill in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for clarity and conciseness, the description of well-known functions and structures is omitted in the following description.

[0052] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0053] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified; the targeting peptides mentioned in the present invention refer to targeting peptides that target cell surface antibodies or transmembrane peptides that can pass through the cell membrane and target intracellular sites; beauty peptides as bioactive peptides can usually play a role on the cell surface and inside the cell, such as cell surface signal peptides acetyl hexapeptide-8, tripeptide-1; intracellular antioxidant peptides carnosine and glutathione.

[0054] The following will refer to the accompanying drawings to demonstrate and evaluate the efficacy of the targeted beauty peptide composition. It should be noted that only some embodiments are shown here, just to help relevant personnel understand the present invention better and more thoroughly, but it should be understood that the present invention can also be implemented in other embodiments and should not be limited by the embodiments described here.

[0055] Embodiment 1:

[0056] A targeted peptide-modified beauty peptide composition, wherein the targeted peptide-modified beauty peptide composition is integrin αvβ3 (RGD)-modified hexapeptide-8, wherein the chemical structure of the RGD motif is as follows: Figure 1 As shown, the chemical structure of hexapeptide-8 is as follows Figure 4 shown.

[0057] At present, hexapeptide-8 is one of the important members of anti-wrinkle peptides. The stability, solubility and skin permeability of hexapeptide-8 are mainly increased by modifying the acetyl group at the N-terminus of the peptide. There are data showing that acetylation can significantly improve the beauty effect of hexapeptide-8 at the cellular level. Therefore, in this example, RGD-modified hexapeptide-8 (RGD-Hexa, RGDEEMQRR) with a purity of more than 95% was obtained by F-moc peptide solid phase synthesis technology, preparative chromatography purification and mass spectrometry analysis. Hydrogen peroxide (H2O2) was used to induce oxidation of skin keratinocytes HaCaT and skin fibroblasts HFF for 2 hours to establish a cell oxidative damage model.

[0058] The H2O2-induced oxidative damage cell model was used to compare and evaluate the various cosmetic and skin care effects of RGD-Hexa and acetyl hexapeptide-8 (Wrinklend), which is widely used on the market. The intracellular ROS was quantified by fluorescent probes. Figure 6 The results showed that RGD-Hexa could significantly reduce the level of free radicals (ROS) induced by H2O2, and the effect was better than the positive control Wrinklend. At the same time, the activity of superoxide dismutase (SOD), an important member of the antioxidant enzyme system of HaCaT cells incubated with RGD-Hexa, was compared by the WST-8 method. It is known that the amount of formazan generated is negatively correlated with the activity of SOD. The absorbance of formazan was measured by a microplate reader, and then the relative activity of SOD was calculated. Figure 7The results verified that RGD-Hexa could enhance the SOD activity of oxidatively damaged keratinocytes HaCaT beyond the level of the blank control group.

[0059] After oxidatively damaged HaCaT cells were treated with RGD-Hexa, the level of ROS, a harmful substance that accelerates aging diseases, decreased, and the activity of the enzyme SOD, which has the function of clearing ROS, increased, indicating that hexapeptide-8 modified with RGD has considerable anti-aging potential.

[0060] Apoptosis is usually the final result of cell aging. Next, the activity of apoptosis-related cysteine ​​protease-3 (Caspase-3) after incubation with RGD-Hexa was compared. The fluorescent substrate of Caspase-3 and flow cytometry analysis were used to obtain the following information: Figure 8 The results showed that RGD-Hexa inhibited 30% of Caspase-3 activity caused by cellular oxidative damage, and the effect was better than the positive control Wrinklend. At the same time, the loss of collagen is also an important indicator and manifestation of aging. The important product after collagen hydrolysis is the non-essential amino acid hydroxyproline (HYP). In order to compare the collagen content of the experimental group and the control group, the intracellular HYP was quantified by colorimetry. The concentration of HYP can indirectly reflect the content of cellular collagen. Fig. 9 The results showed that RGD-Hexa could resist the loss of collagen caused by H2O2.

[0061] Based on the above results, we found that RGD-Hexa increased the antioxidant capacity of keratinocytes HaCaT, inhibited cell apoptosis and collagen loss, and then used dermal fibroblasts HFF to directly verify the anti-aging effect of RGD-Hexa. Fig.10 a Microscopic observation showed that after 72 hours of D-galactose (D-gal) induction of fibroblasts HFF, the blue crystalline precipitate in the cells, the aging-related β-galactosidase (β-gal), increased, indicating that the cell senescence of the D-gal-induced experimental group was aggravated. Co-incubation of Wrinklend or RGD-Hexa with D-gal could reduce the expression of β-gal, among which RGD-Hexa had a better anti-aging effect than the positive control Wrinklend. Fig.10 b The blue crystal cells were quantified by counting, and it can be seen that RGD-Hexa reduced β-gal to about 15%.

[0062] In summary, Example 1 demonstrated the market application potential of RGD-Hexa through the modification of RGD from the perspectives of aging-related anti-oxidation, anti-aging, anti-apoptosis, collagen content and other indicators, and also suggested the feasibility of targeted fragments to enhance the synergy of beauty peptides.

[0063] Example 2

[0064] A targeted peptide-modified beauty peptide composition, wherein the targeted peptide-modified beauty peptide composition is sOtx2-modified hexapeptide-8, and the chemical structure of the sOtx2 motif is as follows Figure 2 As shown, the chemical structure of hexapeptide-8 is as follows Figure 4 shown.

[0065] Example 2 is the same as Example 1, and is also synthesized by F-moc peptide solid phase synthesis technology, and the sOtx2-modified hexapeptide-8 (sOtx2-Hexa, RKQRREREEMQRR) with a purity of more than 95% is obtained by preparative chromatography purification and mass spectrometry analysis. The hydrogen peroxide (H2O2) oxidative damage cell evaluation model is also used to compare the efficacy of sOtx2-Hexa and the positive control peptide acetyl hexapeptide-8 (Wrinklend). Fig.11 The results showed that sOtx2-Hexa treatment reduced intracellular ROS to a greater extent than Wrinklend, indicating its antioxidant potential. Fig.12 The significant inhibitory effect of sOtx2-Hexa on the damage of antioxidant enzyme SOD in oxidatively damaged cells was further verified.

[0066] Similarly, the effects of sOtx2-Hexa on cell apoptosis and the content of HYP were also evaluated. Fig.13 This indicates that sOtx2-Hexa can improve cell apoptosis caused by H2O2 to a certain extent and reduce the activity of apoptosis-related protease Caspase-3.

[0067] Fig.14 The HaCaT cell HYP content graph proves that the targeted beauty peptide sOtx2-Hexa can improve the loss of HaCaT cell collagen caused by H2O2. At the same time, in this embodiment, the concentration of matrix metalloproteinase-1 (MMP-1) in the cell supernatant after the cells were treated with the peptide was also detected by enzyme-linked immunosorbent assay (ELISA). MMP-1 is a digestive enzyme that hydrolyzes various collagens and elastin. It is highly expressed in damaged skin and tumor lesions. Therefore, the quantitative detection of MMP-1 can further reflect the loss of tissue protein and the health of the skin. Fig.15 It showed that sOtx2-Hexa significantly alleviated the increase of MMP-1 in the supernatant caused by oxidative damage of cells. Fig.14 and Fig.15 The experimental results are not only consistent with the previous experimental results of HYP, but also reveal that one of the reasons why targeted beauty peptides maintain collagen content is to reduce the level of MMP-1 and reduce the hydrolysis of collagen.

[0068] Finally, also through Fig.16a, 16b The anti-aging effect of sOtx2-Hexa was evaluated by showing the senescence-related protein β-gal in HFF cells. The results showed that sOtx2-Hexa could significantly reduce the expression of β-gal in oxidatively damaged cells to normal cell levels.

[0069] Examples 1 and 2 successfully demonstrate that two cosmetic peptides (hexapeptide-8) modified with targeted peptide fragments targeting cell integrins or cell polysaccharides have antioxidant and anti-aging effects, and their effects are superior to the mature anti-wrinkle cosmetic peptide acetyl hexapeptide-8 on the market.

[0070] Example 3

[0071] A targeted peptide modified beauty peptide composition, wherein the targeted peptide modified beauty peptide composition is MAP modified hexapeptide-8, and the chemical structure of the MAP motif is as follows Figure 3 As shown, the chemical structure of hexapeptide-8 is as follows Figure 4 shown.

[0072] With the successful cases of hexapeptide-8 modified with different targeting fragments in Examples 1 and 2, in this example, the same synthesis and purification technology was used to synthesize hexapeptide-8 modified with the cell-penetrating peptide MAP (MAP-Hexa, KLALKLALKALKAALKLAEEMQRR). The targets of the targeting peptides shown in the previous two examples are on the cell membrane surface, while the membrane-penetrating properties of MAP are equivalent to its target being inside the cell. The cell uptake mechanism of cell-penetrating peptides mainly includes endocytosis and direct transmembrane. MAP is an amphipathic cell-penetrating peptide, and under normal circumstances, it usually enters the cell in the form of endocytosis and direct transmembrane. First, the cytotoxicity of MAP-Hexa was determined, and it was found that its cytotoxicity was 4 times that of Wrinklend, RGD-Hexa and sOtx2-Hexa, so the dosage of MAP-Hexa was reduced by 4 times (50 μg / ml) to compare its cell efficacy with 200 μg / ml Wrinklend;

[0073] Depend on Fig.17 It can be seen that 50 μg / ml MAP-Hexa can improve the proliferation of HaCaT cells after 0.4% FBS starvation, but 200 μg / ml Wrinklend has no obvious improvement effect. The positive control 50 U / ml EGF can restore the cell proliferation level after starvation to 100%. Similarly, the anti-aging effect of Wrinklend and MAP-Hexa was compared using the D-gal-induced HFF cell aging model. Fig.18It was found that MAP-Hexa can significantly reduce the positive rate of cells expressing β-gal, which is better than the positive control Wrinklend, indicating that the amphiphilicity of the membrane-penetrating fragment peptide MAP can increase the transdermal and cell penetration of the peptide compared with acetyl modification, thereby increasing the concentration of hexapeptide-8 around and inside the cells and enhancing its efficacy.

[0074] Example 4

[0075] A targeted peptide modified beauty peptide composition, wherein the targeted peptide modified beauty peptide composition is a combination of targeted peptide beauty peptides RGD-GHK (RGDGHK) with a purity of more than 95% obtained by covalently linking integrin αvβ3 (RGD) and beauty polypeptide tripeptide-1 (GHK), proving that the targeted fragment RGD can be applied to other beauty peptides. GHK, also known as dermal growth promoting factor, can promote the synthesis of extracellular matrix in the dermis of the skin, such as collagen and glycosaminoglycans, can tighten the skin, and improve and repair the protective layer of the skin. The targets of GHK are distributed inside and outside the cells, so the modification of RGD will have a target aggregation effect on GHK and enhance the efficacy of GHK. Collagen and elastin are two key proteins in the dermis of the skin, so the inhibitory activity of GHK and RGD-GHK is first compared by the in vitro elastase inhibition method. Fig.19 The RGD-GHK obtained can inhibit the activity of elastase by 25%, which is significantly higher than GHK itself. The reason is that the chemical structure configuration of RGD-GHK itself has changed, and the reaction with elastase has been enhanced, reducing the degradation of elastin by the protease; RGD-GHK binds to the active site of the enzyme and inactivates the enzyme, so the activity of the inhibitory enzyme is enhanced.

[0076] In addition to in vitro validation, similarly, the H2O2-induced cell senescence model was used to detect the apoptosis rate of skin cells HaCaT, intracellular collagen content, and matrix metalloproteinase (MMP-1) protein and gene levels to compare RGD-GHK and GHK. Fig. 20 The results showed that RGD-GHK could significantly reduce the early apoptosis of cells damaged by H2O2 and inhibit the activity of apoptosis-related protease Caspase-3. The experimental results showed that RGD-GHK had a stronger function in repairing cells than GHK. Fig.21 a shows that the concentration of hydroxyproline returned to normal levels after RGD-GHK treatment, indicating that the collagen content returned to the level before oxidative damage. Fig.21b, 21c show that RGD-GHK can significantly inhibit the expression of MMP-1 caused by cell damage at both the protein expression level and the gene level. Therefore, through the above experimental data, it is concluded that RGD-modified GHK, compared with unmodified GHK, can enhance the inhibition of protease activity to a certain extent, reduce the degradation of elastin and collagen, and inhibit the activity of apoptosis-related proteases, thereby reducing cell aging and apoptosis.

[0077] Example 5

[0078] A targeting peptide modified beauty peptide composition, wherein the targeting peptide modified beauty peptide composition is sOtx2 modified tripeptide-1, and the chemical structure of the sOtx2 motif is as follows Figure 2 As shown, the chemical structure of tripeptide-1 is as follows Figure 5 shown.

[0079] Since the efficacy mechanism of tripeptide-1 can increase the synthesis of glycosaminoglycans in the cell matrix, and sOtx2 is also a family member CS that clearly targets glycosaminoglycans, in this example, sOtx2 is grafted to tripeptide-1 to form sOtx2-GHK (RKQRRERGHK). While tripeptide-1 promotes the expression of glycosaminoglycans, it also increases the expression of sOtx2 targets, which in turn further stimulates sOtx2-GHK to be brought into the cell periphery to guide the performance of GHK. Fig. 22 It can be seen that sOtx2-GHK reduced the premature apoptosis of cells by about 10% and inhibited the activity of apoptosis-related protease Caspase-3 by about 30%. Fig.23 It was further verified that sOtx2-GHK improved the collagen content of damaged cells and reduced the expression of protease MMP-1, thus having an anti-aging effect of tightening the skin.

[0080] In summary, the five embodiments mentioned in the present invention verify that three targeting polypeptides are covalently modified to two cosmetic peptides to form new targeted cosmetic peptide molecules. This new covalent chimeric peptide can relatively enhance the multiple skin care effects of these two cosmetic peptides. Of course, although the embodiments of the present invention are limited, it is obvious that appropriate changes in various conditions can be made without departing from the spirit and scope of the present invention. It is understood that the present invention is not limited to the experimental scheme, but belongs to the scope of the claims, which includes equivalent replacements of each factor.

[0081] Although the embodiments of the present application are described above, the present application is not limited to the above specific embodiments and application fields, and the above specific embodiments are merely illustrative and instructive, rather than restrictive. A person of ordinary skill in the art can make many forms under the guidance of this specification and without departing from the scope of protection of the claims of the present application, all of which belong to the protection of the present application. Sequence Listing <110> Yunnan Yunke Specialty Plant Extraction Laboratory Co., Ltd. <120> A targeted peptide-modified beauty peptide composition <160> 20 <170> SIPOSequenceListing 1.0 <210> 1 <211> 3 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 1 Arg Gly Asp 1 <210> 2 <211> 5 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 2 Arg Gly Asp Tyr Lys 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 3 Arg Gly Asp Phe Lys 1 5 <210> 4 <211> 7 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 4 Arg Lys Gln Arg Arg Glu Arg 1 5 <210> 5 <211> 15 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 5 Arg Lys Gln Arg Arg Glu Arg Thr Thr Phe Thr Arg Ala Gln Leu 1 5 10 15 <210> 6 <211> 13 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 6 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln 1 5 10 <210> 7 <211> 16 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 7 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <210> 8 <211> 18 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 8 Leu Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His 1 5 10 15 Ser Lys <210> 9 <211> 34 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 9 Asn Ala Ala Thr Ala Thr Arg Gly Arg Ser Ala Ala Ser Arg Pro Thr 1 5 10 15 Gln Arg Pro Arg Ala Pro Ala Arg Ser Ala Ser Arg Pro Arg Arg Pro 20 25 30 Val Gln <210> 10 <211> 15 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 10 Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Pro 1 5 10 15 <210> 11 <211> 22 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 11 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val Cys 20 <210> 12 <211> 27 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 12 Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu 1 5 10 15 Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 25 <210> 13 <211> twenty one <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 13 Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu Lys Ala Leu Ala Ala Leu 1 5 10 15 Ala Lys Lys Ile Leu 20 <210> 14 <211> 8 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 14 Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <210> 15 <211> 18 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 15 Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys 1 5 10 15 Leu Ala <210> 16 <211> 9 <212> PRT <213> Synthetic sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 16 Arg Gly Asp Glu Glu Met Gln Arg Arg 1 5 <210> 17 <211> 13 <212> PRT <213> Synthetic sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 17 Arg Lys Gln Arg Arg Glu Arg Glu Glu Met Gln Arg Arg 1 5 10 <210> 18 <211> 24 <212> PRT <213> Synthetic sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 18 Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys 1 5 10 15 Leu Ala Glu Glu Met Gln Arg Arg 20 <210> 19 <211> 6 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 19 Arg Gly Asp Gly His Lys 1 5 <210> 20 <211> 10 <212> PRT <213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 20 Arg Lys Gln Arg Arg Glu Arg Gly His Lys 1 5 10

Claims

1. A targeted peptide-modified beauty peptide composition, characterized in that: The targeting peptide modified cosmetic peptide composition is a complex formed by covalently linking a targeting peptide and a cosmetic peptide, wherein the targeting peptide is a targeting peptide targeting cell surface integrin or cell surface glycosaminoglycan GAGs, and the action target of the cosmetic peptide is on the cell surface or inside the cell, and the targeting cell surface integrin is integrin αvβ3 highly expressed on the surface of tumor vascular endothelial cells and on the surface of skin cells; the specific recognition polypeptide ligand sequence of integrin αvβ3 is an arginine (R)-glycine (G)-aspartic acid (D) sequence (RGD), and the targeting peptide contains 3-10 amino acids, and the amino acids are natural amino acids or non-natural amino acids, including RGD and its derivatives; the RGD derivative is a linear or cyclic pentapeptide or hexapeptide containing an RGD motif, specifically a linear or cyclic RGD derivative a (RGDyK) and RGD derivative b (RGDFK) containing an RGD motif; The motif of the targeting peptide sOtx2 for cell surface glycosaminoglycans GAGs is RKQRRER; the targeting peptide is derived from the homologous protein orthodenticle homeobox protein 2 (Otx2), and is a specific binding ligand for the polyanionic components glycosaminoglycans (GAGs) chondroitin sulfate (CS) or heparan sulfate (HS) on the cell membrane, and the motif of the specific binding ligand is RKQRRERTTFTRAQL; the beauty peptide is hexapeptide-8 and / or tripeptide-1.

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