Cell-penetrating peptide composition and application thereof in preparation of cosmetics

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a cell-penetrating peptide composition and application thereof in preparation of cosmetics. The cell-penetrating peptide composition is prepared from 9-polyarginine and 8-polylysine, wherein the 9-polyarginine and the 8-polylysine are used as raw materials; wherein the weight ratio of 9-polyarginine to 8-polylysine is (1-5): (1-5). The cell-penetrating peptide composition disclosed by the invention is prepared from 9-polyarginine and 8-polylysine; compared with single use of 9-polyarginine and 8-polylysine, the composite cell-penetrating peptide used in the invention has better cell-penetrating ability and synergistic cell-penetrating ability; the composition can be applied to cosmetics, so that active substances in the cosmetics can be fully absorbed and utilized; the cell-penetrating peptide can efficiently promote a plurality of active substances to enter cells in vivo and in vitro and significantly improve the transmembrane transport efficiency of the active substances, so that the concentration of the active substances in the cells is improved, and the cell-penetrating peptide has no toxic effect on the cells, does not generate immune response, and achieves the transmembrane transport effect which cannot be achieved by a single cell-penetrating peptide.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a transmembrane peptide composition and its application in the preparation of cosmetics. Background Technology

[0002] Cell-penetrating peptides (CPPs) are small amino acid sequences characterized by their ability to cross the cell membrane and transport various bioactive substances within the cell, including nucleic acids, large proteins, and other compounds. Based on their amino acid sequence, hydrophobicity, and charge, CPPs are generally classified into three categories: cationic peptides, amphiphilic peptides, and hydrophobic peptides. Cationic peptides are characterized by carrying a relatively large net positive charge at physiological pH, primarily provided by basic amino acid residues (arginine, lysine). However, different types of cell-penetrating peptides exhibit varying degrees of effectiveness in penetrating different types of active ingredients, requiring optimized selection for specific targets.

[0003] Collagen, an important structural protein, is widely found in the skin, bones, and other tissues of mammals, playing a vital role in maintaining skin elasticity and hydration. However, due to its large molecular weight, traditional forms of collagen have difficulty penetrating the skin barrier, thus limiting its effectiveness in skincare products.

[0004] In summary, this invention provides a membrane-penetrating peptide composition that exhibits good membrane-penetrating effects on collagen, and has significant application value. Summary of the Invention

[0005] In order to overcome at least one of the technical problems existing in the prior art, the present invention provides a transmembrane peptide composition and its application in the preparation of cosmetics.

[0006] The technical solution of the present invention is as follows:

[0007] The present invention first provides a transmembrane peptide composition comprising 9 polyarginine and 8 polylysine.

[0008] The amino acid sequence of the 9-polyarginine is RRRRRRRRR.

[0009] The amino acid sequence of the 8 polylysine is KKKKKKKK.

[0010] Preferably, the weight ratio of 9-polyarginine to 8-polylysine is 1-5:1-5.

[0011] Preferably, the weight ratio of 9-polyarginine to 8-polylysine is 1-3:1-3.

[0012] Preferably, the weight ratio of 9-polyarginine to 8-polylysine is 1 to 3:1.

[0013] Preferably, the weight ratio of 9-polyarginine to 8-polylysine is 1 to 2:1.

[0014] Preferably, the weight ratio of 9-polyarginine to 8-polylysine is 2:1.

[0015] Preferably, the application of the membrane-penetrating peptide composition in promoting the entry of active substances into cells.

[0016] Preferably, the active substance is collagen.

[0017] The present invention also provides an application of the above-mentioned membrane-penetrating peptide composition in the preparation of cosmetics.

[0018] Preferably, the cosmetic is a cosmetic that promotes the entry of active substances into cells.

[0019] Preferably, the active substance is collagen.

[0020] Beneficial effects: This invention provides a novel membrane-penetrating peptide composition; the membrane-penetrating peptide composition is composed of 9-polyarginine and 8-polylysine; compared with using 9-polyarginine and 8-polylysine alone, the composite membrane-penetrating peptide used in this invention has superior membrane-penetrating ability and synergistic membrane-penetrating ability; it can be applied in cosmetics to enable more complete absorption and utilization of active substances; it can efficiently promote the entry of multiple active substances into cells in vivo and in vitro, significantly improve the transmembrane transport efficiency of active substances, thereby increasing the concentration of active substances in cells, and has no toxic effect on cells, does not produce immune response, and achieves a membrane-penetrating transport effect that a single membrane-penetrating peptide cannot achieve. Attached Figure Description

[0021] Figure 1 To detect the distribution of FITC-collagen in HaCaT cells using laser confocal microscopy.

[0022] Figure 2 The average fluorescence intensity in HaCaT cells under different concentrations of transmembrane peptides is shown (*** indicates that the sample is significantly different from the Control group, p < 0.001). Detailed Implementation

[0023] The present invention will be further explained below with reference to specific embodiments, but the embodiments do not limit the present invention in any way.

[0024] The 9-polyarginine and 8-polylysine described in this invention can be purchased through conventional channels, or they can be prepared by the following method:

[0025] Preparation method of 9-polyarginine: The first step is peptide resin synthesis. Using 2Cl-Trt-Cl Resin as a carrier, Fmoc-Arg(pbf)-2Cl-Trt Resin is first synthesized, followed by deprotection, washing, and Fmoc-Arg(pbf)-OH condensation (repeated 8 times). Finally, deprotection, washing, and drying are performed to obtain the peptide resin: NH2-Arg(pbf)-Arg(pbf)-Arg(pbf)-Arg(pbf)-Arg(pbf)-Arg(pbf)-Arg(pbf)-Arg(pbf)-2Cl-Trt The second step involves cleavage (the peptide resin is acid-hydrolyzed, and then reacted with H2O, TFA, EDT, and TIS cleavage reagents), followed by concentration, precipitation with methyl tert-butyl ether, filtration, and drying to obtain crude 9-polyarginine; the third step involves purifying the crude 9-polyarginine by HPLC and lyophilizing it to obtain the finished 9-polyarginine product, which is then stored in a warehouse at 2–8°C.

[0026] 8. Preparation method of polylysine: The first step is peptide resin synthesis. Using 2Cl-Trt-Cl Resin as a carrier, Fmoc-Lys(pbf)-2Cl-Trt Resin is first synthesized, then deprotected, washed, and subjected to Fmoc-Lys(pbf)-OH condensation (repeated 7 times). Finally, deprotection, washing, and drying are performed to obtain the peptide resin: NH2-Lys(pbf)-Lys(pbf)-Lys(pbf)-Lys(pbf)-Lys(pbf)-Lys(pbf)-Lys(pbf)-2Cl-Trt The second step involves cleavage (the peptide resin is acid-hydrolyzed, and then reacted with H2O, TFA, EDT, and TIS cleavage reagents), followed by concentration, precipitation with methyl tert-butyl ether, filtration, and drying to obtain crude 8-polylysine; the third step involves purifying the crude 8-polylysine by HPLC and lyophilizing it to obtain the finished 8-polylysine product, which is then stored in a warehouse at 2–8°C.

[0027] Example 1: Preparation of the membrane-penetrating peptide composition

[0028] It is prepared by mixing 9-polyarginine and 8-polylysine in a weight ratio of 2:1.

[0029] Example 2: Preparation of the membrane-penetrating peptide composition

[0030] It is prepared by mixing 9-polyarginine and 8-polylysine in a weight ratio of 1:1.

[0031] Example 3: Preparation of membrane-penetrating peptide composition

[0032] It is prepared by mixing 9-polyarginine and 8-polylysine in a weight ratio of 1:2.

[0033] Comparative Example 1

[0034] The difference between this comparative example and Example 1 is that Comparative Example 1 uses only a single membrane-penetrating peptide, 9-polyarginine.

[0035] Comparative Example 2

[0036] The difference between this comparative example and Example 1 is that Comparative Example 2 uses only a single membrane-penetrating peptide, polylysine 8.

[0037] Experiment 1 uses an immunofluorescence cell staining model to evaluate the ability of transmembrane peptides to promote collagen transmembrane entry into cells.

[0038] 1. Solution preparation: Dissolve a certain mass of FITC-labeled collagen (0.5 mg / mL) and the membrane-penetrating peptides from Examples 1-3 and Comparative Examples 1-2 in basal culture medium to make the final concentration of the membrane-penetrating peptides 0.1%.

[0039] 2. Immunofluorescence experiment

[0040] (1) Cell culture: HaCaT cells were cultured in 10% fetal bovine serum at 37°C and 5% CO2 and then seeded into 24-well plates and cultured for 24 h.

[0041] (2) Sample addition: Discard the old culture medium and add a mixed solution of membrane-penetrating peptide-collagen or FITC-labeled collagen solution of different concentrations, and continue to culture for 24 hours.

[0042] (3) Washing: Discard the culture medium and wash with PBS 3 times, 5 min each time.

[0043] (4) Fixation: Fix cells with 4% paraformaldehyde for 30 min, then wash with PBS 3 times, 5 min each time.

[0044] (5) The staining within the cells was observed using a laser confocal microscope. The staining differences between the FITC-collagen group and the FITC-collagen-membrane-penetrating peptide mixed solution experimental group were compared. Results are shown below. Figure 1 , 2 And Table 1.

[0045] Table 1. Mean fluorescence intensity in HaCaT cells under different concentrations of transmembrane peptides

[0046]

[0047] (*** indicates that this sample is significantly different from the control group, p < 0.001)

[0048] The above results indicate that no fluorescence was detected in HaCaT cells treated with only FITC-collagen; however, FITC-collagen showed detectable fluorescence after the addition of transmembrane peptides, demonstrating that transmembrane peptides can effectively promote collagen to cross the cell membrane and enter the cell.

[0049] After HaCaT cells were treated with different ratios of transmembrane peptides for 24 hours, the average intracellular fluorescence intensity was significantly higher than that of the blank control group, and the treatment groups in Examples 1 and 2 showed extremely significant differences compared with the control group (p < 0.001). The results indicate that the transmembrane peptide composition composed of 9-polyarginine and 8-polylysine can synergistically promote collagen transmembrane entry into cells. In particular, the transmembrane peptide compositions obtained by combining 9-polyarginine and 8-polylysine in ratios of 2:1 and 1:1 in Examples 1 and 2 showed significantly higher effects in promoting collagen transmembrane entry than the single transmembrane peptides in Comparative Examples 1 and 2.

Claims

1. A membrane-penetrating peptide composition, characterized in that, It contains 9 polyarginine and 8 polylysine.

2. The membrane-penetrating peptide composition according to claim 1, characterized in that, The weight ratio of 9-polyarginine to 8-polylysine is 1-5:1-5.

3. The membrane-penetrating peptide composition according to claim 2, characterized in that, The weight ratio of 9-polyarginine to 8-polylysine is 1-3:1-3.

4. The membrane-penetrating peptide composition according to claim 2, characterized in that, The weight ratio of 9-polyarginine to 8-polylysine is 1 to 3:

1.

5. The membrane-penetrating peptide composition according to claim 2, characterized in that, The weight ratio of 9-polyarginine to 8-polylysine is 1 to 2:

1.

6. The membrane-penetrating peptide composition according to claim 2, characterized in that, The weight ratio of 9-polyarginine to 8-polylysine is 2:

1.

7. The use of the membrane-penetrating peptide composition according to claim 1 in promoting the entry of active substances into cells; Preferably, the active substance is collagen.

8. The use of the membrane-penetrating peptide composition according to any one of claims 1 to 7 in the preparation of cosmetics.

9. The application according to claim 8, characterized in that, The cosmetics mentioned are cosmetics that promote the entry of active substances into cells.

10. The application according to claim 9, characterized in that, The active substance mentioned is collagen.

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