Synthetic polypeptides and their use in promoting skin healing

Abstract

The present application provides a kind of synthetic polypeptide and its application in promoting skin healing, the amino acid sequence of the provided synthetic polypeptide is SEQ ID NO:1.The present application also provides the derivative polypeptide of the above-mentioned synthetic polypeptide.The synthetic polypeptide of the present application has obvious wound healing promoting activity, can be used to accelerate the healing of body surface skin wound, reduce scar production.Therefore, the synthetic polypeptide of the present application has excellent wound healing promoting activity, can be used as production raw material or dressing for drug, postoperative medical device, health care product, cosmetic and the like with skin repair function, has broad application prospect.

Description

Technical Field

[0001] This invention belongs to the technical field of polypeptides and their synthesis, and specifically relates to a synthetic polypeptide and its application in promoting skin healing. Background Technology

[0002] As the body's first line of defense, the skin is extremely vulnerable to trauma. Improper wound treatment can easily lead to infection or hypertrophic scarring. Therefore, effectively promoting skin wound repair is a significant challenge for researchers. Wound healing is a complex biological process and a key factor in restoring the integrity of the skin barrier.

[0003] Bioactive peptides play an indispensable role in maintaining the body's physiological activities and homeostasis. The rich functions of bioactive peptides are a popular research topic, with those isolated from plants and animals receiving particular attention. They not only play a positive role in skin wound healing but also possess excellent biological functions. Furthermore, their characteristics of being present in trace amounts, safe, and highly effective have led to their widespread application in medicine and healthcare. However, current research on the role of bioactive peptides in skin wound healing still faces some challenges. For example, the preparation of bioactive peptides often relies on traditional enzymatic hydrolysis methods, resulting in mixtures of products, and the analysis of the functional molecules of bioactive peptides is insufficient. The small molecular weight of bioactive peptides also makes effective separation and purification methods a limiting factor in their research and industrial production. When considering the effects of bioactive peptides on skin wound healing, the skin irritation potential of the resulting products should also be taken into account. Summary of the Invention

[0004] The purpose of this invention is to provide a synthetic polypeptide and its application in promoting skin healing. The synthetic polypeptide of this invention has a good therapeutic effect on wound healing, good safety, and significant efficacy.

[0005] This invention first provides a synthetic polypeptide, characterized in that the amino acid sequence of the synthetic polypeptide is as follows:

[0006] Gly-Pro-Ser-Gly-Gly-Gly-Tyr-Asp-Phe-Gly-Leu-Pro-Gly-Ala-Pro-Gly-Phe-Val-Gly (GPSGGGYDFGLPGAPGFVG) (SEQ ID NO: 1);

[0007] This invention also provides derivative polypeptides of the above-mentioned synthetic polypeptides, which comprise:

[0008] 1) A polypeptide obtained by substituting, deleting, or adding one or more amino groups to the polypeptide of SEQ ID NO:1;

[0009] 2) The polypeptide obtained by truncating the polypeptide of SEQ ID NO:1;

[0010] 3) The polypeptide obtained by tandemly combining the polypeptides in 1) or 2);

[0011] As a specific example, the amino acid sequence of the derived polypeptide is as follows:

[0012] Gly-Leu-Pro-Gly-Val-Pro-Gly-Phe-Val-Gly (GLPGVPGFVG) (SEQ ID NO: 2);

[0013] Gly-Pro-Ser-Gly-Gly-Gly-Tyr-Asp-Phe-Gly-Leu-Pro-Gly(GPSGGGYDFGLPG) (SEQ ID NO: 3);

[0014] Gly-Leu-Pro-Gly-Val-Pro-Gly-Phe(GLPGAPGF) (SEQ ID NO: 4).

[0015] In another aspect, the present invention provides a conjugated polypeptide, which is a conjugation product of the above-mentioned synthetic polypeptide and a functional molecule.

[0016] The functional molecule is a molecule that can enhance the efficacy of the synthetic peptide, such as a peptide or polysaccharide;

[0017] Another aspect of the present invention provides an use of the synthetic polypeptide in the preparation of wound healing drugs, post-skin repair medical devices, post-skin repair supplies, skin repair health products, or skin repair cosmetics.

[0018] The present invention also provides an article having the function of promoting wound healing or skin repair, wherein the article contains any one or more of the above-mentioned synthetic polypeptides or their derivative polypeptides.

[0019] The synthetic polypeptide of this invention exhibits significant wound-healing activity and can be used to accelerate the healing of skin wounds and reduce scarring. Therefore, the synthetic polypeptide of this invention possesses excellent wound-healing activity and can be used as a raw material or dressing in the production of pharmaceuticals, postoperative medical devices, health products, cosmetics, etc., with skin-repairing functions, showing broad application prospects. Attached Figure Description

[0020] Figure 1 Photographs showing the effect of peptides on wound healing in mice;

[0021] Figure 2The effect of the polypeptide with sequence SEQ ID NO:1 on the proliferation of mouse embryonic fibroblast NIH 3T3 cells is shown in the figure. Compared with the blank control, *P<0.05, **P<0.01;

[0022] Figure 3 Photographs showing the effect of the polypeptide with sequence SEQ ID NO:1 on the migration of mouse embryonic fibroblast NIH 3T3 cells, with the left image being the blank control and the right image being the polypeptide with sequence SEQ ID NO:1. Detailed Implementation

[0023] This invention uses reversed-phase liquid chromatography to separate and purify the peptides from the enzymatic hydrolysis of deer antler, which have the effect of promoting skin healing. The purification is carried out using an Eclipse XDB-C18 column, and sequencing is performed to obtain the peptides that promote skin healing. The amino acid sequence of the peptides is GPSGGGYDFGLPGAPGFVG (SEQ ID NO:1).

[0024] This invention also provides a derivative polypeptide of the synthetic polypeptide of SEQ ID NO:1, comprising:

[0025] 1) A polypeptide obtained by substituting, deleting, or adding one or more amino groups to the polypeptide of SEQ ID NO:1;

[0026] 2) The polypeptide obtained by truncating the polypeptide of SEQ ID NO:1;

[0027] 3) The polypeptide obtained by tandemly combining the polypeptides in 1) or 2);

[0028] 1) is a derivative polypeptide with high homology to SEQ ID NO:1, which also has the effect of promoting skin healing;

[0029] 2) The peptide obtained by truncating the peptide of SEQ ID NO:1 also has the effect of promoting skin healing; for example, the amino acid sequence is GLPGVPGFVG (SEQ ID NO:2), the amino acid sequence is GPSGGGYDFGLPG (SEQ ID NO:3) and the amino acid sequence is GLPGAPGF (SEQ ID NO:4).

[0030] 3) is a conjugated polypeptide obtained by tandemly selecting any two or three of the polypeptides in 1) and 2).

[0031] The conjugated polypeptide can also be a product obtained by conjugating the above-mentioned polypeptide with a functional molecule.

[0032] The functional molecule is a molecule that can enhance the efficacy of the synthetic polypeptide, such as a 3′-plus polypeptide or polysaccharide.

[0033] Another aspect of the present invention provides an use of the synthetic polypeptide in the preparation of wound healing drugs, post-skin repair medical devices, post-skin repair supplies, skin repair health products, or skin repair cosmetics.

[0034] The present invention also provides an article having the function of promoting wound healing or skin repair, wherein the article contains any one or more of the above-mentioned synthetic polypeptides or their derivative polypeptides.

[0035] The polypeptides provided by this invention can be synthesized using conventional methods, such as liquid-phase fractional synthesis, solid-phase synthesis, and biosynthesis. As a preferred embodiment of the polypeptides described in this invention, the polypeptides are synthesized using a solid-phase polypeptide synthesis process. To ensure biocompatibility, the purity of the polypeptides of this invention is ≥95%. The product can be purified using HPLC.

[0036] The polypeptides and derivatives provided by this invention can be used to prepare drugs for the treatment of skin defects and difficult-to-heal skin ulcers.

[0037] Furthermore, the skin defects and refractory skin ulcers can be large-area skin defects and refractory skin ulcers caused by various acute and chronic injuries such as inflammation, ulcers, severe burns, trauma, post-tumor surgery, and congenital malformations.

[0038] The present invention will now be described in detail with reference to the embodiments.

[0039] Example 1: Synthesis of polypeptides

[0040] The polypeptides with amino acid sequences SEQ ID NO:1-4 were synthesized according to the following steps:

[0041] Resin swelling: Place the resin in a 150 mL reaction column and add 50 mL of DCM to soak for 2 hours. Wash the resin with DMF, dry it, and repeat four times to complete the resin swelling. Deprotection: Weigh the first amino acid (protected) at the C-terminus of Fmoc, DCM, and DIEA and add them to the reactor. Then place the reactor in a shaker at 30°C and react for 2 hours. Block with methanol solution (methanol:DIEA:DCM = 1:1:2) for half an hour, then wash four times with DMF and dry it. Add 20% piperidine solution to the reactor to remove the Fmoc protecting group. After deprotection, wash four times with DMF and then dry it. Take a small amount of resin and test it using the ninhydrin method. If the resin is colored, it indicates that the deprotection was successful.

[0042] Amino acid coupling reaction: Weigh the second amino acid (protected) at the C-terminus of Fmoc, HOBT, and DIC and add them to the reactor. Then, place the reactor in a shaker at 30°C and react for 1 hour. Take a small amount of resin for testing using the ninhydrin method. If the resin is colored, it indicates incomplete condensation, and the reaction should continue. If the resin is colorless, the reaction is complete. After the reaction is complete, wash the resin four times with DMF and then dry it. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group from the resin. After deprotection, wash the resin four times with DMF and then dry it to check if the protection has been removed. Take a small amount of resin for testing using the ninhydrin method. If the resin is colored, the deprotection is successful. Connect the amino acids sequentially according to the steps.

[0043] Cleavage and separation of the synthetic peptide: The protecting groups of the peptide were completely removed using a cleavage reagent and cleaved from the resin, followed by purification. The target peptide was separated from impurities by HPLC, and the target peptide was lyophilized to obtain the product. Mass spectrometry analysis verified that the purity of the synthesized compound was greater than 97%.

[0044] Example 2: Effects of peptides on wound healing speed and quality in mice

[0045] Mice were anesthetized intravenously with 1 mL / kg of 3% sodium pentobarbital. After preparing the back of the mice, the punch was adjusted to a diameter of 5 cm and the probe was stained with gentian violet. Surgical marks were printed on the lateral third of the back of the mice, centered on the spine. The surgical area was disinfected with strong iodine and draped with sterile towels. The surgical procedure was performed entirely under sterile conditions. All skin and superficial fascia tissue of uniform size were completely removed along the imprints to the deep fascia surface, and hemostasis was achieved. A self-made sterile drug delivery tube with a sieve at the tip was embedded under sterile gauze in the back wound of the mice for easy drug administration. The blank control group was given physiological saline, while the other groups of mice were given the corresponding synthetic peptide (8 mg / mL). The incision was bandaged with sterile gauze. The healing of the wound was observed. On the 10th day, the longitudinal and transverse diameters of the unhealed wound were measured with calipers, and the healing area was calculated. Healing area = total wound area - (long diameter × short diameter × 0.785).

[0046] The results showed that topical administration significantly accelerated skin wound healing. Fourteen days after administration of the peptides in SEQ ID NO:1-4, the wound size was significantly smaller than that in the control group. Histopathological observation nine days later revealed that the control group rats had larger defects after skin injury, with some areas showing complete epithelial loss and others partially covered by epithelial tissue, but the epidermis was thinner and less continuous. After peptide administration, the epidermis repaired more completely and was thicker (Table 1).

[0047] Table 1: Comparison of Wounds and Wound Healing Speed

[0048]

[0049] Compared with the blank control, *P<0.05, **P<0.01.

[0050] Therefore, histomorphological results indicate that the peptides in SEQ ID NO:1-4 can all accelerate skin wound healing.

[0051] Example 3: Effects of peptides on the proliferation of mouse embryonic fibroblast NIH 3T3 cells

[0052] The NIH 3T3 cell concentration was diluted to 1×10⁻⁶. 4 Cells were seeded at a density of 180 μL / mL into 96-well plates and incubated at 37°C with 5% CO2 for 24 h. After complete cell adhesion, 20 μL of 10% FBS-DMEM medium containing 200 μg / mL, 100 μg / mL, 10 μg / mL, and 1 μg / mL of the peptide in SEQ ID NO:1 were added to each well. The blank control group consisted of 10% FBS-DMEM medium, with 6 replicates per group. Cells were incubated at 37°C with 5% CO2 for another 48 h. After incubation, 20 μL of 0.5 mg / mL MTT solution was added to each well, and the cells were incubated at 37°C with 5% CO2 for another 4 h. The culture medium was discarded, and 150 μL of dimethyl sulfoxide (DMSO) was added. The cells were shaken for 10 min, and the absorbance was measured at 570 nm using a microplate reader. Cell proliferation rate was calculated as follows: Cell proliferation rate = [A] 570nm (Experimental group average) - A 570nm (Blank control group) / A 570nm (Blank control group)

[0053] The results showed that the peptide of SEQ ID NO:1, at a concentration of 1-200 μg / mL, significantly promoted the proliferation of mouse embryonic fibroblasts NIH 3T3 for 24 hours. Figure 2 ).

[0054] Example 4: Effects of peptides on NIH 3T3 cell migration

[0055] Mouse embryonic fibroblast NIH 3T3 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin antibiotics. Serum-free medium or the polypeptide (10 μg / mL) of SEQ ID NO:1 was added to each well, and the cells were incubated for 24 h. After reaching the logarithmic growth phase, the cells were digested, washed once with PBS and once with serum-free medium, and then resuspended in serum-free medium. Cells were counted and the concentration adjusted to 2 × 10⁶ cells / well. 5 / mL; Add 500μL of culture medium containing 10% FBS to the lower chamber of the Transwell, and add 100-200μL of cell suspension to the upper chamber, and culture for 24h; After culture, wipe off the unmigrated cells in the upper chamber; Immerse the lower surface of the cell in 4% paraformaldehyde solution, fix for 20min, stain with crystal violet for 20min, observe the cells under a microscope and take pictures, and count the number of cells that migrated to the lower surface of the PET membrane (Table 2).

[0056] Table 2: Migrating Cell Count Table

[0057]

[0058]

[0059] Compared with the blank control, *P<0.05, **P<0.01.

[0060] The results showed that, compared with the blank control, 10 μg / mL of the peptide in SEQ ID NO:1 significantly promoted the migration of NIH 3T3 cells.

Claims

1. A synthetic polypeptide, characterized in that, The amino acid composition of the synthetic polypeptide is SEQ ID NO:

1.

2. A derived polypeptide, characterized in that, The amino acid sequence of the derived polypeptide is SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

3. The use of the polypeptide of claim 1 in the preparation of articles that promote wound healing and skin repair, wherein the articles are pharmaceuticals or cosmetics.

4. An article for wound healing or skin repair, characterized in that, The product is a pharmaceutical or cosmetic product, and the product contains the polypeptide as described in claim 1.

5. The article of claim 4, characterized in that, The product further comprises the polypeptide as described in claim 2.

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