Preparation method and application of supramolecular blue copper peptide nanocomposite

Abstract

The present invention relates to a preparation method and application of a supramolecular blue copper peptide nanocomposite, and belongs to the technical field of cosmetics. The structure of the supramolecular blue copper peptide nanocomposite of the present invention is: Myr-RHKRHKRHK-[GHK-Cu]-X, wherein X is a negatively charged active substance. Specifically, the present invention is synthesized by Fmoc solid phase method, modified by myristoyl, to obtain myristoyl dodecapeptide: Myr-(RHK)3GHK, a complexation reaction occurs in a copper sulfate solution, to obtain a supramolecular blue copper peptide: Myr-(RHK)3-[GHK-Cu], and the supramolecular blue copper peptide solution is compounded with a negatively charged active substance to obtain a supramolecular blue copper peptide nanocomposite. Compared with commercially available blue copper peptides, the supramolecular blue copper peptide prepared by the present invention can self-assemble into nano micelles in a solution, form nanocomplexes with negatively charged active substances, can quickly penetrate the skin, and has better stability, and can better solve the current problems of limited permeability and easy inactivation of blue copper peptides; it can directly target the inside of skin cells, reduce the amount of active substances added, improve bioavailability, and exert more excellent efficacy; at the same time, the supramolecular blue copper peptide itself can be used as a carrier to form a supramolecular nanocomplex with negatively charged substances such as nucleic acids.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics, and particularly relates to a preparation method and application of a supramolecular blue copper peptide nanocomposite. Background Art

[0002] The Chinese alias of blue copper peptide is tripeptide-1 copper, and the sequence is GHK-Cu, Cu 2+ It is not the yellow color of copper metal, but appears blue in aqueous solution, so it is also called blue copper peptide. Copper uses the tripeptide GHK as a carrier to play a corresponding role by effectively complexing and transporting copper elements. Cu and GHK complement each other to make blue copper peptide have multiple biological effects: (1) Repair: It can increase blood vessel growth and antioxidant capacity, and stimulate the production of glucosamine (GAGs), helping the skin to restore self-repair. (2) Anti-inflammatory: GHK-Cu has an inhibitory effect on inflammatory response and can reduce pro-inflammatory cytokines including TNF-α, TGF-β, IL-6, etc. It effectively reduces inflammation, reduces hormone-induced redness, itching, tingling and other problems, and regenerates the skin. (3) Anti-wrinkle: It effectively blocks the nerve conduction of acetylcholine substances, thereby relaxing muscles and improving dynamic wrinkles.

[0003] However, when used, blue copper peptide is difficult to be directly absorbed by the skin due to its high hydrophilicity; and the current delivery efficiency of blue copper peptide is low; it has poor stability, is easily inactivated, and is difficult to be used with acidic substances (fruit acid, A acid, high concentration of water-soluble L-VC, salicylic acid, etc.); it cannot be used with overly acidic or overly alkaline finished products, and too low or too high pH can cause GHK-Cu to decompose, fail, discolor, and lose its function.

[0004] In view of the problem of osmotic delivery of blue copper peptide, CN 115838394 A discloses a novel method for preparing blue copper peptide, which adopts the method of modifying the side chain of lysine with fatty acids to increase the lipophilicity of blue copper peptide and improve the permeability. Its polypeptide structure is H-Gly-His-Lys(Pal)-OH, and the raw material Fmoc-Lys(Dde)-OH is expensive, and the cost of raw materials is too high; CN115089500A discloses a method for preparing supramolecular blue copper peptide microemulsion, which loads blue copper peptide into ionic liquid by preparing ionic liquid, thereby improving the permeability of blue copper peptide. However, ionic liquids generally have the problem of low purity and limited industrial application.

[0005] In response to the stability problem of blue copper peptide, CN 115501134 A discloses a compounding scheme of blue copper peptide and hyaluronic acid, and CN 115569085 A discloses a scheme of ionic liquid encapsulating blue copper peptide; the prior art generally improves the stability of blue copper peptide by compounding of complexes or encapsulating with ionic liquids, but the stability of blue copper peptide itself has not been solved.

[0006] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a supramolecular blue copper peptide nanocomposite and a preparation method thereof, so as to improve the transdermal absorption of blue copper peptide and at the same time improve the stability of raw materials, enhance the efficacy while reducing the amount of addition, and apply it to the field of cosmetic technology.

[0008] In order to achieve the above object, the technical solution adopted by the present invention comprises the following steps:

[0009] A method for preparing a supramolecular blue copper peptide nanocomposite and its application, comprising the following steps:

[0010] (1) Structural design of supramolecular blue copper peptide nanocomplex: The main structure of the blue copper peptide GHK-Cu precursor GHK tripeptide is maintained, a cell-penetrating peptide is connected, and myristoyl modification is performed. The polypeptide sequence is reconstructed and designed into a myristoyl dodecapeptide with the sequence: Myr-RHKRHKRHKGHK. The supramolecular blue copper peptide is complexed with CuSO4 solution in solution to obtain a supramolecular blue copper peptide with the sequence: Myr-RHKRHKRHK[GHK-Cu]. It is compounded with hyaluronic acid or its salt, PDRN (polydeoxyribonucleotide, ectoin) and other negatively charged active ingredients to obtain a supramolecular blue copper peptide nanocomplex with the structure: Myr-RHKRHKRHK[GHK-Cu]-X, where X is a negatively charged active ingredient such as hyaluronic acid.

[0011] (2) Preparation of myristoyl dodecapeptide: Using 2-CTC Resin as a carrier and peptide solid phase organic synthesis technology, in an N,N-dimethylformamide system, using the Fmoc synthesis strategy, myristoyl chloride is used for the final nitrogen-terminal modification to synthesize a fully protected peptide resin. The fully protected peptide resin is cleaved under the action of trifluoroacetic acid and the side chain protecting groups of the peptide are removed to obtain a crude peptide; the peptide is purified using reverse phase preparative liquid chromatography, filler: C18, phase A: 0.1% TFA / H2O, phase B: 0.1% TFA / MeOH, phase A is balanced for loading, elution procedure: 25%-85%; 60 min, preparation and purification are followed by freeze drying to obtain a purified peptide;

[0012] (3) Preparation of supramolecular blue copper peptide: The purified myristoyl dodecapeptide was dissolved in water, copper sulfate solution was added to the solution, stirred for 1-2 hours, poured into a dialysis bag, dialyzed for 24 hours, and lyophilized to obtain supramolecular blue copper peptide with the sequence Myr-RHKRHKRHK[GHK-Cu].

[0013] (4) Preparation of supramolecular blue copper peptide nanocomplex: prepare 10 mg / mL-30 mg / mL supramolecular blue copper peptide solution; prepare negatively charged active substance aqueous solution (sodium hyaluronate, PDRN, ectoine), add a certain amount of supramolecular blue copper peptide solution dropwise into the negatively charged active substance aqueous solution, and dilute the supramolecular blue copper peptide concentration to 0.07 mg / mL-1 mg / mL;

[0014] (5) Structural characterization of the supramolecular blue copper peptide nanocomplex prepared by the present invention:

[0015] Transmission electron microscopy was used for characterization, and the sample was negatively stained with 3% phosphotungstic acid. The structural morphology of the supramolecular blue copper peptide complex was nanoparticles, and DLS was used for particle size characterization.

[0016] (6) Efficacy verification of supramolecular blue copper peptide nanocomplex:

[0017] Permeability test: The distribution of fluorescent labeled substances (FITC@supramolecular copper peptide nanocomplex and FITC@copper peptide) in mouse skin was observed by fluorescence microscopy, and the changes in the fluorescence intensity of the samples were statistically analyzed to evaluate the skin penetration behavior of supramolecular copper peptide nanocomplex and copper peptide at different treatment time points. Observation by fluorescence microscopy: Compared with copper peptide, the permeability of supramolecular copper peptide nanocomplex was significantly enhanced.

[0018] Anti-aging test: Based on human skin fibroblasts, the changes in cell proliferation after the action of supramolecular blue copper peptide samples were detected; human skin fibroblasts were treated with ultraviolet light, and the changes in the content of type I collagen (Collagen I) protein were detected, indicating that the supramolecular blue copper peptide nanocomplex has significant anti-aging and anti-wrinkle effects;

[0019] Anti-inflammatory test: Based on mouse macrophages, the changes in the content of the pro-inflammatory factor TNF-α were detected after the supramolecular blue copper peptide nanocomplex and blue copper peptide acted respectively, indicating that the supramolecular blue copper peptide nanocomplex has significant anti-inflammatory and soothing effects.

[0020] (7) Stability verification of supramolecular blue copper peptide nanocomplex;

[0021] Accelerated test: The prepared supramolecular blue copper peptide nanocomplex was subjected to accelerated tests at 4°C, 25°C, 45°C, 50°C and light (UV) conditions to observe the stability of the samples. The appearance of the samples showed no abnormalities.

[0022] Anti-enzymatic test: Aminopeptidase was added to the supramolecular blue copper peptide nanocomplex and blue copper peptide solution to test the change of peptide content. The results showed that the supramolecular blue copper peptide nanocomplex can effectively inhibit the enzymatic degradation of peptides; while the blue copper peptide was quickly degraded under the action of aminopeptidase.

[0023] Test of dissociation in physiological salt environment: The supramolecular blue copper peptide nanocomplex is a nano-micelle structure that can quickly penetrate the skin barrier. After permeation, the nano-micelles need to dissociate into negatively charged active substances such as peptides and hyaluronic acid to further act on cells and exert their efficacy. The dissociation of the supramolecular blue copper peptide nanocomplex was verified by simulating the physiological salt environment of the human body: the results show that in the physiological salt environment of the human body, the supramolecular nanocomplex can dissociate, and the active substances act on cells separately to exert their efficacy.

[0024] (8) Compatibility verification of supramolecular blue copper peptide nanocomplexes;

[0025] Anion system compatibility test: Supramolecular blue copper peptide itself is positively charged and incompatible with anion system. It was prepared into a nanocomposite for compatibility test. A 0.1% macromolecular hyaluronic acid aqueous solution was prepared, and 10% supramolecular blue copper peptide nanocomposite was added to observe the compatibility of the sample. The results showed that the sample had no flocculation phenomenon and was well compatible with the anion system.

[0026] (9) Carrier function of supramolecular blue copper peptide

[0027] The sequence of the supramolecular blue copper peptide is Myr-RHKRHKRHK-[GHK-Cu]. It is amphiphilic and can be assembled into nanomicelles with a spherical structure in solution. The spherical structure contains a lipophilic core and a hydrophilic shell. The hydrophilic shell can load hydrophilic active molecules with other functions, such as ectoine.

[0028] Supramolecular blue copper peptide contains RHKRHKRHK cell-penetrating peptide, which is positively charged and can be used as a carrier to interact with negatively charged active molecules (such as HA, PDRN, etc.), encapsulate or be encapsulated to form supramolecular nanocomplexes, which can not only improve the transdermal penetration and bioavailability of polypeptide molecules, but also improve the transdermal penetration and bioavailability of macromolecules such as nucleic acids.

[0029] PDRN, also known as polydeoxyribonucleotide, has a molecular weight between 50 and 1500 kDa. It is an emerging skin anti-aging agent that is popular in the medical beauty market. However, due to its large molecular weight, strong hydrophilicity, and easy degradation by nucleases, it is not easily absorbed through the skin, so it is mostly used in injectable products. The combination of supramolecular blue copper peptide and PDRN can form a nanocomposite through positive and negative charges, promote the transdermal absorption of PDRN, and play a dual anti-aging carrier role.

[0030] In the above step (2), the degree of substitution of 2-CTC Resin is preferably 0.6-0.8 mmol / g.

[0031] In the above step (3), the concentration of the nutmeg dodecapeptide solution is preferably 10 mg / mL, and the concentration of the CuSO4.5H2O solution is preferably 4 mg / mL;

[0032] In the above step (4), the negatively charged active substance is preferably hydrolyzed sodium hyaluronate (8000Da-10000Da), and the mass ratio of supramolecular blue copper peptide to hydrolyzed sodium hyaluronate is 1:(0.1-10); the concentration of the polypeptide in the nanocomplex after dilution is (0.07mg / mL-1mg / mL), preferably 0.07mg / mL.

[0033] The beneficial effects of the present invention are:

[0034] 1. The supramolecular blue copper peptide nanocomposite of the present invention has significantly enhanced permeability compared with blue copper peptide, but it can be prepared by simply compounding the polypeptide and the negatively charged active substance aqueous solution (hyaluronic acid, PDRN, etc.) without the use of complex methods such as ionic liquids. The preparation process is simple.

[0035] 2. The supramolecular blue copper peptide nanocomplex of the present invention has a spherical nanostructure compared to the linear blue copper peptide. Enzyme hydrolysis experiments show that the supramolecular nanocomplex has better stability, can inhibit not only the degradation of polypeptides, but also the enzymatic hydrolysis of negatively charged macromolecules by hyaluronidase and nuclease, and has good compatibility with anion systems.

[0036] 3. Supramolecular blue copper peptide contains a cell-penetrating peptide structure and can directly target cells. Compared with blue copper peptide, cell uptake is enhanced. Therefore, the amount of peptide added is less, which can be as low as 70ppm, while the amount of blue copper peptide added is as high as 500ppn, which greatly reduces the cost of raw materials.

[0037] 4. The supramolecular blue copper peptide disclosed in the present invention has the function of a carrier and can be assembled into nanomicelles with a spherical structure in a solution. The spherical structure comprises a lipophilic core and a hydrophilic shell. The hydrophilic shell can carry hydrophilic active molecules with other functions. In addition, the supramolecular blue copper peptide has a cell-penetrating peptide structure, so it can carry nucleic acid molecules such as PDRN to penetrate the skin barrier, solving the application problem that nucleic acid macromolecules such as PDRN are difficult to penetrate the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The present invention is further described below in conjunction with the accompanying drawings.

[0039] Figure 1 is the HPLC spectrum of myristoyl dodecapeptide;

[0040] Figure 2 is the ESI-MS spectrum of myristoyl dodecapeptide, M=1814.2;

[0041] Figure 3 This is the transmission electron microscopy image (TEM image) of the supramolecular blue copper peptide nanocomposite;

[0042] Figure 4This is the particle size analysis diagram of the supramolecular blue copper peptide nanocomplex (DLS diagram);

[0043] Figure 5 This is a permeation experiment based on mouse skin;

[0044] A, fluorescence microscopy observation, B, fluorescence intensity;

[0045] Figure 6 The results of the cell proliferation experiment of supramolecular blue copper peptide nanocomplex based on fibroblasts;

[0046] Figure 7 The experimental results of Collage I based on the anti-wrinkle validation of UV-treated fibroblasts in vitro;

[0047] Figure 8 This is the result of soothing and anti-inflammatory experiments based on mouse macrophages;

[0048] Fig. 9 The results of the anti-enzymatic test of supramolecular blue copper peptide nanocomplex;

[0049] Fig.10 The dissociation experimental results of supramolecular blue copper peptide nanocomplex in physiological salt environment;

[0050] Fig.11 This is the DLS particle size analysis diagram of supramolecular blue copper peptide-PDRN;

[0051] Fig.12 This is the Zeta potential diagram of supramolecular blue copper peptide-PDRN. DETAILED DESCRIPTION

[0052] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0053] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0054] The following is an explanation of the abbreviations in the manual:

[0055] 2-CTC Resin (Tianjin Nankai Hecheng Technology Co., Ltd.), substitution degree 0.5 mmol / g; Trt: trityl; Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; Boc: tert-butylcarbonyl; Myristoyl-Cl: myristoyl chloride.

[0056] The analytical HPLC was Agilent 1220; the preparative HPLC was Chuangxin Hengtong LC3000, the C18 analytical column was 4.6 mm × 250 mm from Dalian Institute of Physical Chemistry, and the C18 preparative column was 10 cm × 65 cm from Chengdu Science Biology Co., Ltd.; the mass spectrometer was Waters UPLC-LC-MS;

[0057] Transmission electron microscope HITACHI HT7800, nanoparticle size and Zeta potential analyzer Malvern NanoZS90, fluorescence microscope, CO2 incubator, inverted microscope, microplate reader, ultraviolet lamp, 37℃ constant temperature incubator.

[0058] Example 1

[0059] A supramolecular blue copper peptide nanocomposite, the structure of which is: Myr-RHKRHKRHK[GHK-Cu]-HA (SEQ ID NO.1), HA is hydrolyzed sodium hyaluronate, GHK-Cu is blue copper peptide. Myr-RHKRHKRHK[GHK-Cu] is a supramolecular blue copper peptide, and the supramolecular blue copper peptide is compounded with a sodium hyaluronate aqueous solution to form a supramolecular nanocomposite.

[0060] Example 2

[0061] A method for preparing a supramolecular blue copper peptide nanocomposite comprises:

[0062] 1. Preparation of Myristoyl Dodecapeptide

[0063] (1) Synthesis of Fmoc-Lys(Boc)-2-CTC Resin

[0064] Weigh 10 g of 2-CTC Resin (SD = 0.6 mmol / g), add it to a synthesis column, swell it with 80 mL of dichloromethane for 30 min, filter and remove the solvent, add 100 mL of dichloromethane, 3.1 g (6.6 mmol) of Fmoc-Lys(Boc)-OH, and 4.2 mL (24 mmol) of N,N′-diisopropylethylamine to the synthesis column, stir and react at room temperature for 2 hours under nitrogen protection, add 15 mL of anhydrous methanol to the synthesis, wash the resin with isopropanol and N,N-dimethylformamide in turn, and obtain Fmoc-Lys(Boc)-2-CTC Resin.

[0065] (2) Synthesis of Fmoc-His(Trt)-Lys(Boc)-2-CTC Resin

[0066] Add 100 mL of 20% piperidine in N,N-dimethylformamide solution to the synthesis column, remove the Fmoc protecting group for 30 minutes, filter, and wash the filter cake twice with isopropanol and N,N-dimethylformamide, 60 mL each time. The resin is positive when tested with ninhydrin, and the resin appears black. Add 100 mL of N,N-dimethylformamide, 7.4 g (12 mmol) of Fmoc-His (Trt)-OH, 2.4 g (18 mmol) of 1-hydroxybenzotriazole, and 2.7 mL (18 mmol) of N,N-diisopropylcarbodiimide to the synthesis column, react at room temperature for 2 hours under nitrogen protection, the resin is negative when tested with ninhydrin, and the resin is colorless and transparent, filter, and wash the filter cake twice with isopropanol and N,N-dimethylformamide, 60 mL each time, to obtain Fmoc-His (Trt) -Lys (Boc) -2-CTC Resin.

[0067] (3) Synthesis of fully protected peptide resin with Fmoc protection

[0068] According to the synthesis strategy of step (2.2), 3.5 g (12 mmol) Fmoc-Gly-OH, 5.6 g (12 mmol) Fmoc-Lys (Boc) -OH, 7.4 g (12 mmol) Fmoc-His (Trt) -OH, 7.7 g (12 mmol) Fmoc-Arg (Pbf) -OH, 5.6 g (12 mmol) Fmoc-Lys (Boc) -OH, 7.4 g (12 mmol) Fmoc-His (Trt) -OH, 7.7 g (12 mmol) Fmoc-Arg (Pbf) -OH, 5.6 g (12 mmol) Fmoc-Lys (Boc) -OH, 7.4 g (12 mmol) Fmoc-His (Trt) -OH, 7.7 g (12 mmol) Fmoc c-Arg(Pbf)-OH, 5.6g(12mmol)Fmoc-Lys(Boc)-OH, 7.4g(12mmol)Fmoc-His(Trt)-OH, 7.7g(12mmol)Fmoc-Arg(Pbf)-OH, Fmoc was synthesized -Arg(Pbf)-His(Trt)-Lys(Boc)-Arg(Pbf)-His(Trt)-Lys(Boc)-Arg(Pbf)-His(Trt)-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-2-CTC Resin.

[0069] (4) Use 20% piperidine / DMF solution to remove the protecting group, and use myristoyl chloride to perform the final nitrogen end modification to obtain a fully protected peptide resin. The details are as follows:

[0070] 100 mL of 20% piperidine in N,N-dimethylformamide solution was added to the synthesis column to remove the Fmoc protecting group for 40 minutes, and the filter cake was washed twice with isopropanol and N,N-dimethylformamide, 80 mL each time. The resin was detected by ninhydrin and it was black. 100 mL of dichloromethane, 7 mL of myristoyl chloride and 15 mL of N,N-diisopropylethylamine were added to the synthesis column and reacted for 1 hour. The resin was colorless and transparent when detected by ninhydrin to obtain Myr-Arg(Pbf)-His(Trt)-Lys(Boc)-Arg(Pbf)-His(Trt)-Lys(Boc)-Arg(Pbf)-His(Trt)-Lys(Boc)-Gly-His(Trt)-Lys(Boc)-2-CTC Resin. The resin was washed with isopropanol, N,N-dimethylformamide and methanol, and then dried.

[0071] (5) The fully protected peptide resin is cleaved under the action of trifluoroacetic acid and the side chain protecting groups of the polypeptide are removed to obtain a crude polypeptide. Specifically, 132 mL of trifluoroacetic acid, 7.5 mL of triisopropylsilane, 6 mL of thioanisole, and 4.5 mL of water are added to the dried resin, reacted for 2.5 hours, filtered, the filtrate is concentrated, TFA is removed under reduced pressure, 10 times the volume of cold ether is added to the concentrate, crystallized and stirred for 1 hour, filtered, the filter cake is collected, and vacuum dried to obtain the trifluoroacetate salt of the crude myristoyl dodecapeptide;

[0072] (6) Purification:

[0073] Add 50% acetonitrile aqueous solution by volume to the crude myristoyl dodecapeptide, add a small amount of trifluoroacetic acid to assist dissolution, filter, constant volume, and prepare and purify on reverse phase chromatography (filler: C18), chromatographic procedure: A phase 0.1% TFA / H2O (v / v), B phase 0.1% TFA / ACN (v / v), A phase equilibrium loading, elution procedure: 15%-65% 60min, collect eluent. Use 50mmol / L ammonium acetate buffer to transfer salt, elution procedure: 55%-85% 60min, freeze-dry, and obtain 5.1g of pure myristoyl dodecapeptide acetate (hereinafter referred to as myristoyl dodecapeptide lyophilized powder), i.e. Myr-Arg-His-Lys-Arg-His-Lys-Arg-His-Lys-Gly-His-Lys-OH, with a yield of 46.3%.

[0074] The structure and purity of the peptide were characterized by high performance liquid chromatography and mass spectrometry. Figure 1 As shown). Electrospray mass spectrometry ESI-MS was used for structural characterization: (M+3H / 3,100) (as shown Figure 2 shown).

[0075] 2. Preparation of supramolecular blue copper peptide

[0076] 1000 mg of myristoyl dodecapeptide lyophilized powder was weighed and added to double distilled water to prepare a 10 mg / mL solution. Ultrasonic oscillation was performed for 15 min, and 4 mg / mL CuSO4.5H2O solution was added to the solution. The mixture was stirred for 1 hour, and the solution was poured into a 3500Da dialysis bag, dialyzed for 24 hours, and lyophilized to obtain a supramolecular blue copper peptide with the sequence Myr-RHKRHKRHK[GHK-Cu].

[0077] 3. Preparation of supramolecular blue copper peptide nanocomplex

[0078] Prepare and add 50 mL of 0.46 mg / mL HA (8000 Da-10000 Da) aqueous solution, drop 350 uL of 10 mg / mL supramolecular blue copper peptide solution into the hyaluronic acid aqueous solution to obtain supramolecular blue copper peptide nanocomplex, wherein the peptide concentration is diluted to 0.07 mg / mL;

[0079] 4. Structural characterization of supramolecular blue copper peptide nanocomplex

[0080] The supramolecular blue copper peptide nanocomplex was characterized by transmission electron microscopy (TEM), and the sample was negatively stained with 3% phosphotungstic acid; the particle morphology of the sample was detected: the results showed that under the conditions of peptide concentration of 0.07 mg / mL and HA of 0.46 mg / mL, the sample showed a nanoparticle morphology (such as Figure 3 shown).

[0081] At the above concentration, the particle size of the sample was detected by dynamic light scattering (DLS), and the particle size was about 180nm (such as Figure 4 shown).

[0082] Example 3

[0083] Efficacy verification of supramolecular blue copper peptide nanocomplex

[0084] 1. Permeability test of supramolecular blue copper peptide nanocomplex

[0085] Based on C57 mice, the distribution of fluorescently labeled supramolecular blue copper peptide nanocomplexes and fluorescently labeled blue copper peptides in mouse skin was observed by fluorescence microscopy, and the changes in the fluorescence intensity of the samples were statistically analyzed to evaluate the skin penetration behavior of supramolecular blue copper peptide nanocomplexes and blue copper peptides at different treatment time points. The method is as follows:

[0086] (1) Fluorescent labeling of polypeptides: 50 mg of supramolecular blue copper peptide was weighed and dissolved in aqueous solution, 200 mg of fluorescein isothiocyanate (FITC) was weighed and dissolved in methanol, the FITC solution was added to the polypeptide solution, the pH was adjusted to 8, the reaction was protected from light for 24 h, and the yellow lyophilized powder was obtained after dialysis treatment. The FITC-labeled polypeptide lyophilized powder was dissolved in water and dropped into the HA aqueous solution to prepare FITC-labeled supramolecular blue copper peptide nanocomplex, wherein the polypeptide was 0.5 mg / mL and the HA was 0.05 mg / mL, which was recorded as supramolecular blue copper peptide@FITC.

[0087] As a control group, the blue copper peptide was also treated with FITC fluorescence labeling, and yellow lyophilized powder was obtained after lyophilization. The peptide concentration was prepared to be 0.5 mg / mL solution, which was recorded as GHK-Cu@FITC.

[0088] (2) Based on C57 mice, the distribution of fluorescently labeled supramolecular blue copper peptide nanocomplexes and fluorescently labeled blue copper peptides in mouse skin was observed by fluorescence microscopy, and the changes in the fluorescence intensity of the samples were statistically analyzed to evaluate the skin penetration behavior of different samples.

[0089] (3) Experimental results: Fluorescence microscopy observation shows that the supramolecular copper peptide nanocomplex has permeation behavior. The fluorescence intensity is statistically analyzed and found that: compared with the 30min and 60min time points, the permeation amount of the sample copper peptide is not greater than that of the previous time point, while the permeation amount of the supramolecular copper peptide nanocomplex is greater than that of the previous time point, that is, as time goes by, the permeation amount of the supramolecular copper peptide nanocomplex continues to increase (e.g. Figure 5 The final results show that the permeability of the supramolecular blue copper peptide nanocomplex of the present invention is significantly higher than that of the blue copper peptide.

[0090] 2. Verification of the anti-wrinkle activity of supramolecular blue copper peptide nanocomplex

[0091] (1) Cell proliferation test based on human skin fibroblasts:

[0092] Based on human skin fibroblasts, the proliferation rate of cells after being acted upon by samples was detected; and its effect on cell proliferation was comprehensively evaluated.

[0093] Cell recovery, passage, inoculation, experimental grouping, when the cell plating rate reaches 30%-40%, take out the culture plate and mark the time periods as 0h, 24h, 48h, 72h, set up a control group and a sample group in each time period, and set up 3 replicates in each group. Prepare the test substance working solution according to Table 1.

[0094] Table 1 Preparation of cell proliferation test samples based on human skin fibroblasts

[0095]

[0096] Control group detection: discard the supernatant of all wells in the 0h time period, add 100uL PBS to each well for washing, prepare CCK-8 treatment solution in advance, the CCK-8 treatment solution is compounded by 10% CCK-8 stock solution + 90% culture medium (10% FBS), discard the PBS and add 100uCCK-8 treatment solution to each well, incubate in a light-proof incubator at 37°C and 5% CO2, aspirate 90uL in a new 96-well plate after 2h, and adjust the microplate reader to 450nm to read the OD value.

[0097] Sample loading and detection: discard the supernatant from all remaining wells, add 100uL PBS to each well for washing, discard PBS and add samples in groups, with a sample volume of 200uL per well. Add DMEM medium containing 2% FBS to the control group, and add DMEM medium containing 2% FBS of the corresponding concentration of samples to the sample group. After the sample loading is completed, place the culture plate in a 37°C, 5% CO2 incubator and continue to culture for 24h, 48h, and 72h. After the cells are cultured in the incubator for 24h, 48h, and 72h, discard the supernatant, add 100uL PBS to each well for washing, and prepare CCK-8 treatment solution in advance. The CCK-8 treatment solution is compounded with 10% CCK-8 stock solution + 90% culture medium (10% FBS).

[0098] After discarding PBS, add 100uL CCK-8 treatment solution to each well and incubate in a light-proof incubator at 37°C and 5% CO2. After 2 hours, aspirate 90uL from each well in turn and place it in a new 96-well plate. Adjust the microplate reader to 450nm to read the OD value.

[0099] Cell viability calculation: Taking 0h of each group as the control well, the relative cell viability value, SD value, and P value of each group were calculated. The results showed that compared with the blue copper peptide, the supramolecular blue copper peptide nanocomplex significantly increased the cell viability after 24h, 48h, and 72h of administration. Figure 6 shown.

[0100] (2) The anti-wrinkle efficacy of the sample was evaluated by detecting the changes in the content of type I collagen (Collagen I) after the sample was treated with ultraviolet light on a human skin fibroblast model.

[0101] Cell recovery, subculture, and inoculation were performed according to the solution preparation scheme in Table 2.

[0102] Table 2 Preparation of anti-wrinkle efficacy test samples based on UV-treated human skin fibroblasts

[0103]

[0104] UV induction: When the cell plating rate in the 12-well plate reaches 40% to 60%, the negative control group and the sample group are irradiated with UV light of appropriate intensity for a corresponding period of time according to the above detection method.

[0105] Drug administration and sample collection: After irradiation, the cells were divided into groups and the dosage was 1 mL per well. Each group had 3 replicate wells and incubated in an incubator (37°C, 5% CO2) for 48 hours. After 48 hours of incubation, the culture medium was collected and placed in an EP tube, centrifuged at 800 rpm for 5 minutes, and the supernatant was collected and stored in a -80°C refrigerator.

[0106] ELISA test: Perform the test according to the specific operating procedures in the ELISA kit instructions.

[0107] The results showed that compared with the blank group, the content of Collagen I in the negative control group decreased, indicating that the UV treatment was effective. Compared with the negative control group, the content of Collagen I in the sample blue copper peptide did not change, and the content of Collagen I in the sample supramolecular blue copper peptide nanocomplex increased. Figure 7 shown).

[0108] (3) Soothing and anti-inflammatory efficacy test based on mouse macrophages:

[0109] Raw264.7 mouse macrophages were stimulated with LPS, and the soothing and anti-inflammatory effects of supramolecular blue copper peptide nanocomplexes and blue copper peptides were evaluated by detecting the changes in the content of proinflammatory factors (TNF-α) after the action of supramolecular blue copper peptide nanocomplexes and blue copper peptides.

[0110] After cell recovery, culture and inoculation, 450 μL of the working solution containing LPS (final concentration: 5 ug / mL) was added to each well of each group and then placed in a cell culture incubator (5% CO2, 37°C) for further culture for 2 hours. After LPS stimulation, the sample working solution was prepared according to Table 3.

[0111] Table 3 Preparation of samples for soothing and anti-inflammatory efficacy test based on mouse macrophages

[0112]

[0113] Index detection: After 24 hours, collect the cell supernatant and centrifuge at 1000-1200rpm for 3 minutes. After aspirating the supernatant, the inflammatory factor indicator TNF-α can be immediately detected.

[0114] The results showed that compared with the negative control group, the TNF-α content of the supramolecular blue copper peptide nanocomplex decreased, while the TNF-α content of the blue copper peptide remained unchanged. Figure 8 (shown)

[0115] Example 4

[0116] Application of a supramolecular blue copper peptide nanocomposite

[0117] (1) Stability verification: Accelerated test: The prepared supramolecular blue copper peptide nanocomplex was subjected to accelerated tests at 4°C, 25°C, 45°C, 50°C and light (UV) conditions to observe the stability of the samples. The results are shown in Table 4;

[0118] Table 4 Accelerated test of stability of supramolecular blue copper peptide nanocomplex

[0119]

[0120] (2) Anti-enzymatic hydrolysis verification: The anti-enzymatic hydrolysis ability of the supramolecular blue copper peptide and the commercially available blue copper peptide was verified. The commercially available blue copper peptide was purchased from Chengdu Yunxi.

[0121] Prepare 10 mg / mL supramolecular blue copper peptide solution and blue copper peptide solution respectively and set aside. Determine the peptide content of the initial supramolecular blue copper peptide and blue copper peptide (Chengdu Yunxi) by high performance liquid chromatography;

[0122] 200 mg of aminopeptidase was added to the supramolecular blue copper peptide solution and the blue copper peptide solution, respectively, stirred to dissolve and disperse, and allowed to stand for 30 min, 90 min, and 120 min to detect changes in peptide content by HPLC.

[0123] The results showed that supramolecular blue copper peptide can effectively inhibit the enzymatic degradation of peptides. Under the action of aminopeptidase, the peptide content of GHK-Cu decreased by nearly 50% in 30 minutes and by nearly 90% in 120 minutes. The peptide was basically completely degraded by aminopeptidase. However, the peptide content of supramolecular blue copper peptide increased significantly in 120 minutes, and the enzymatic degradation of peptide was effectively inhibited. (e.g. Fig. 9 (shown)

[0124] (3) Physiological salt environment dissociation verification:

[0125] Prepare a 0.5 mg / mL supramolecular blue copper peptide nanocomplex solution, and use an infrared laser pen to test its micelle structure; add SBF simulated body fluid (Shangbao Bio) to the 0.5 mg / mL supramolecular blue copper peptide nanocomplex solution, and use infrared laser to test the micelle structure. SBF (Simulated body fluid) is the most commonly used solution for in vitro simulation, which is used to simulate the physiological salt environment of the human body.

[0126] According to the test, supramolecular blue copper peptide nanocomplex has a characteristic micelle structure and a significant Tyndall effect; supramolecular blue copper peptide nanocomplex in a simulated human physiological salt environment no longer has a micelle structure and the Tyndall effect disappears. Fig.10 (shown)

[0127] (4) Carrier function of supramolecular blue copper peptide:

[0128] The structural morphology of supramolecular blue copper peptide and supramolecular blue copper peptide-PDRN nanocomplex was characterized by transmission electron microscopy. The results showed that supramolecular blue copper peptide can carry PDRN to form new nanoparticles. The particle size analysis was as follows: Fig.11 As shown, it is proved that supramolecular blue copper peptide can load negatively charged active molecules. Through Zeta potential characterization, it can be seen that supramolecular blue copper peptide and PDRN form new nanoparticles (such as Fig.12 shown).

[0129] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0130] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a supramolecular blue copper peptide nanocomposite, characterized in that: The following steps are involved: (1) Structural design of supramolecular blue copper peptide nanocomplex: The structure of supramolecular blue copper peptide nanocomplex is: Myr-RHKRHKRHK-[GHK-Cu]-X; The supramolecular blue copper peptide sequence is: Myr-RHKRHKRHK-[GHK-Cu], GHK-Cu is the blue copper peptide sequence, Myr is myristoyl, and RHKRHKRHK is a cell-penetrating peptide fragment; X represents a negatively charged active substance, wherein the negatively charged active substance is one of hyaluronic acid or its salt, polydeoxyribonucleotide, and ectoine; (2) Preparation of supramolecular blue copper peptide: Myristoyl dodecapeptide was synthesized by Fmoc solid phase synthesis method, and after preparation and purification, the myristoyl dodecapeptide solution was complexed with copper sulfate solution, dialyzed and freeze-dried to obtain supramolecular blue copper peptide; the mass ratio of myristoyl dodecapeptide to copper sulfate was 1:0.04-0.1, the complexation reaction time was 1-2 hours, and the dialysis time was 24 hours; (3) Preparation of supramolecular blue copper peptide nanocomplex: prepare 10-30 mg / mL supramolecular blue copper peptide solution, add aqueous solution of negatively charged active substance for compounding and dilution, and obtain supramolecular blue copper peptide nanocomplex; The mass ratio of the supramolecular blue copper peptide to the negatively charged active component is 1:0.1-10; the final concentration of the polypeptide in the supramolecular blue copper peptide nanocomposite is 0.07-1 mg / mL.

2. The method for preparing a supramolecular blue copper peptide nanocomposite according to claim 1, characterized in that: The supramolecular blue copper peptide self-assembles to form cationic nano micelles, which are used as carriers to load hydrophilic active ingredients and nucleic acid negatively charged active ingredients.

3. Use of the supramolecular blue copper peptide nanocomposite prepared according to the method of claim 1 or 2 in cosmetics.

4. The use according to claim 3, wherein the cosmetics are cosmetics with anti-aging and soothing functions.

5. The use according to claim 3, wherein the cosmetic is an anti-wrinkle cosmetic.

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