D / l amino acid alternately arranged anti-hair loss peptide and preparation method and application thereof

Abstract

The present application relates to a kind of D / L amino acid alternate arrangement anti-hair loss hair growth peptide and its preparation method and application, belong to biological medicine technical field.The amino acid sequence of the polypeptide is as shown in SEQ ID NO:1, is designed by D type and L type amino acid alternate arrangement, significantly enhance the ability of anti-protease degradation, in vitro half-life is ≥48 hours, and activate β-catenin protein expression in hair follicle stem cell.Its preparation method includes: using Rink Amide resin as carrier, using Fmoc solid-phase synthesis method sequentially coupling D / L type amino acid, after cleaving resin by trifluoroacetic acid, by high performance liquid chromatography purification, yield is ≥80%, purity is ≥96%, mass spectrum confirms molecular weight is 956.12Da.The polypeptide can be applied to the preparation of anti-hair loss hair growth cosmetics, drugs and medical devices, effectively solve the problem of low polypeptide stability, insufficient targeting in prior art.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to an innovative anti-hair-loss polypeptide with the function of regulating the Wnt / beta-catenin signal pathway and application of the polypeptide in the preparation of anti-hair-loss cosmetics, drugs or medical devices. BACKGROUND

[0002] The existing hair-growing products, such as minoxidil and plant extracts, generally have the following problems:

[0003] 1. Single target: mainly through expanding blood vessels or inhibiting androgen metabolism, the direct activation of hair follicle stem cells is limited;

[0004] 2. Insufficient stability: natural polypeptides are easily degraded by skin enzymes, resulting in low bioavailability;

[0005] 3. Low-efficiency pathway regulation: there is a lack of high-efficiency regulation molecules for the core proteins of the Wnt / beta-catenin pathway, such as beta-catenin.

[0006] The application breaks through the above technical bottlenecks by designing a polypeptide structure with alternating arrangement of D-type and L-type amino acids. SUMMARY

[0007] 1. Polypeptide structure and innovation

[0008] 1.1 Core structure formula: the amino acid sequence is shown as SEQ ID NO: 1

[0009] 1.2 Innovative design

[0010] Alternating configuration enhances stability: D-type amino acids are alternately connected with L-type, which significantly resists protease degradation, and the in-vitro half-life is greater than or equal to 48 hours;

[0011] Targeting the Wnt pathway: by activating the expression of beta-catenin protein in hair follicle stem cells HFSCs, the expression amount is increased to 1.7 times that of the control group at a concentration of 0.1%, and the hair follicle regeneration is promoted;

[0012] Optimization of helical conformation: the tryptophan Trp and histidine His in the sequence form a hydrogen bond, stabilizing the alpha-helix structure and enhancing the cell membrane penetration.

[0013] 2. Preparation method

[0014] Solid-phase synthesis method: Fmoc protection strategy is adopted, Rink Amide resin is used as a carrier, and D / L-type amino acids are coupled in sequence;

[0015] Purification and identification: HPLC purification is performed, the purity is greater than or equal to 96%, and mass spectrometry MS confirms that the molecular weight is 956.12 Da.

[0016] 3. Application field

[0017] The polypeptide can be used for preparing the following products:

[0018] Hair growth cosmetics: scalp conditioning liquid, serum, shampoo, etc.

[0019] Anti-hair loss drugs: such as external tincture, microneedle patch

[0020] Medical devices: hair follicle repair microspheres. BRIEF DESCRIPTION OF DRAWINGS

[0021] The specification is attached Figure 1 : The chemical structure diagram of the polypeptide, and the D / L type amino acid sequence is labeled in the sequence table SEQ ID NO: 1.

[0022] The specification is attached Figure 2 : Western Blot experimental result diagram.

[0023] The specification is attached Figure 3 : Characteristic analysis diagram

[0024] The specification is attached Figure 4 : HPLC result diagram

[0025] The specification is attached Figure 5 : MS result diagram

[0026] The specification is attached Figure 6 : Solubility test result diagram DETAILED DESCRIPTION

[0027] Example 1: Preparation of polypeptide

[0028] The polypeptide sequence of the application is shown in the sequence table SEQ ID NO: 1.

[0029] Rink Amide resin 0.2mmol / g was weighed, and amino acids such as Lys(Fmoc)-OH and D-Arg(Pbf)-OH were sequentially deprotected and coupled.

[0030] The resin was cleaved by TFA:H2O:TIPS=95:2.5:2.5, precipitated with ether, and freeze-dried to obtain the crude peptide.

[0031] HPLC purification was performed on a C18 column with acetonitrile / water gradient elution, and white powder was obtained after freeze-drying, with a yield of greater than or equal to 80%.

[0032] The prepared polypeptide was subjected to characteristic analysis and HPLC, MS, and solubility tests as follows:

[0033] 1. Characteristic analysis (see attached Figure 3 )

[0034] 2. HPLC results (see attached Figure 4 )

[0035] 3. MS results (see attached Figure 5 )

[0036] 4. Solubility test results (see attached Figure 6 )

[0037] Example 2: In vitro efficacy verification

[0038] 1 Materials and methods

[0039] 1.1 Test system

[0040] HFSCs human hair follicle stem cells, purchased from Punsai Biotechnology Development Co., Ltd., F5.

[0041] 1.2 Main reagents and consumables

[0042] DMEM (Gibco), fetal bovine serum (Gibco), trypsin (Gibco), β-catenin antibody, GADPH antibody;

[0043] 1.3 Main equipment

[0044] CO2 incubator, clean bench, fluorescence microscope, Western Blot membrane transfer instrument, protein electrophoresis instrument.

[0045] 1.4 Sample information

[0046]

[0047] Sample concentration: 0.1%, 0.01%

[0048] 1.5 Sample statistical analysis method

[0049] The data was calculated by using imageJ software for gray scale, and the Ctrl group was normalized.

[0050] 2 Test method

[0051] 2.1 Cell processing

[0052] (1) Experimental grouping:

[0053] a) Control group

[0054] b) 0.01% Anti-hair loss and hair growth polypeptide group

[0055] c) 0.1% Anti-hair loss and hair growth polypeptide group

[0056] (2) Select the HFSCs cells in the best growth state, routine treatment, the cell suspension density is adjusted to 2x105 per hole, with 1ml cell suspension per hole inoculated in 12-hole plate, placed in 37℃, 5% CO2 incubator. After 24h, change the liquid.

[0057] (3) After 72h of drug action, the cells are harvested, and the cell suspension is centrifuged and washed twice with pH=7.2 PBS.

[0058] (4) Add cell lysis buffer: buffer=4:1, after cell collection, ultrasonic treatment. Metal bath 100℃, 10min.

[0059] 2.2 Protein electrophoresis

[0060] (1) Correctly install the electrophoresis device, fill the inner electrophoresis tank with water to check if the device leaks, if there is leakage, reinstall it. After leak detection is completed, place it in the outer electrophoresis tank, then pour 1x electrophoresis buffer into the inner electrophoresis tank until it overflows, then add 1x electrophoresis buffer to the outer electrophoresis tank, which should not exceed the bottom of the electrophoresis frame.

[0061] (2) Take out the protein sample stored at -80℃, place it on the metal bath at 95℃ for 3min, then centrifuge it, remove the protein on the tube cap and tube wall, mix well for use.

[0062] (3) Carefully pull out the comb, add the sample in order, add 2-5uL of pre-stained protein molecular weight standard maker on both sides, start electrophoresis. First use 80V voltage, wait for the maker to appear a separated band, then change to 120V voltage until the target band is separated. After electrophoresis, cut off the upper gel and the band-free area, soak it in the transfer buffer, pay attention to keep it moist.

[0063] 2.3 Transfer and blocking

[0064] (1) Cut the PVDF membrane according to the size of the gel, make sure it is dry before use, avoid touching the membrane with your hand during the whole experiment, activate it in pure methanol for about 15s before use.

[0065] (2) The transfer is in a sandwich structure, sponge-filter paper-gel-PVDF membrane-filter paper-sponge; after the PVDF membrane is activated with methanol, soak it in the transfer buffer first, then place it on the gel, make sure the membrane and the gel are attached without air bubbles, carefully close the sandwich device, soak it in the transfer buffer, add 20% pure methanol before use, make sure the positive and negative electrodes are corresponding, put ice bags in the transfer tank, transfer at 250mA for 1-1.5h, replace the ice bags every half hour during the process.

[0066] (3) After the end of the transfer, the film is taken out and placed in an incubation box with a closed liquid, 5% skimmed milk powder and TBST buffer, placed in a 37°C shaking bed for 2 hours of blocking. According to the molecular weight of the target protein, cut the band, mark the protein name, and put it in TBST for slight wetting.

[0067] 2.4 Antibody incubation and exposure

[0068] (1) Incubate the first antibody: dilute the corresponding first antibody with 5% skimmed milk powder to 2-3 mL according to the standard ratio, immerse the band in the antibody incubation bag, make sure there is no air bubble in the bag, and incubate overnight at 4°C. Internal reference: according to the size of the target protein and the internal reference protein, cut the film and incubate separately.

[0069] (2) Incubate the second antibody: take out the band and put it into the incubation box, add TBST and wash three times, each for 5 minutes. Add the corresponding second antibody solution to each well to submerge the band, incubate at room temperature for 1-2 hours, and recover the second antibody. Wash with TBST three times, each for 5 minutes, and prepare for exposure.

[0070] (3) Prepare the luminescent solution according to the ECL luminescent solution instructions, and use it immediately and avoid light. Take out the band that needs to be exposed from the incubation box and place it on a glass plate, use filter paper to absorb the TBST on the band, then evenly drop the luminescent solution, and then place it on the sample stage of the gel imaging instrument for exposure.

[0071] 3 Test results

[0072] 0.01% of the anti-hair loss polypeptide has no obvious regulation on the β-catenin protein of hair follicle stem cells, and the gray value calculation is 1.17 times. 0.1% of the hair polypeptide can significantly up-regulate the expression of β-catenin protein, and the gray value calculation is about 1.7 times.

[0073] Sequence listing

[0074] The amino acid sequence is shown in SEQ ID NO: 1.

Claims

1. A hair loss prevention and hair growth polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1, characterized in that... D-type and L-type amino acids are alternately linked and C-terminus is amidated.

2. The anti-hair loss and hair growth polypeptide according to claim 1, characterized in that, The polypeptide regulates the Wnt / β-catenin signaling pathway by activating β-catenin protein expression in hair follicle stem cells. In vitro experiments showed that the expression level of β-catenin at a concentration of 0.1% was 1.7 times that of the control group.

3. The preparation method of the anti-hair loss and hair growth polypeptide according to claim 1 includes the following steps: a) Using Rink Amide resin as a carrier, the amino acid sequence shown in SEQ ID NO:1 was sequentially coupled using the Fmoc solid-phase synthesis method; b) The resin was pyrolyzed and then purified by high performance liquid chromatography. The mobile phase was an acetonitrile-water gradient system, and the purity was ≥95%.

4. The preparation method according to claim 3, characterized in that, In step a), the amino acid coupling reaction uses HBTU / HOBt as an activator, reacts for a certain time, and the deprotection reagent is a 20% piperidine / DMF solution.

5. A cosmetic composition comprising the anti-hair loss and hair growth polypeptide of claim 1 and a cosmetic matrix, characterized in that, The cosmetic composition is an essence, shampoo, or scalp care gel, with an added peptide content of 0.05%-0.3%.

6. A pharmaceutical composition comprising the anti-hair loss and hair growth polypeptide of claim 1 and a pharmaceutically acceptable carrier, characterized in that, The pharmaceutical composition contains a polypeptide concentration of 0.01%-0.5%, and is available in the form of a topical tincture, microneedle patch, or injection.

7. A medical device comprising the anti-hair loss and hair growth polypeptide of claim 1 and a biodegradable carrier, characterized in that, The medical device is an absorbable microsphere or a sustained-release implant with a polypeptide loading of 1-10 mg / cm³. 3 .

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