A novel anti-acne recombinant collagen-like polypeptide, and a preparation method and application thereof

Abstract

The application provides a novel anti-acne recombinant collagen-like protein polypeptide and a preparation method and application thereof. The anti-acne recombinant collagen-like protein polypeptide is obtained through artificial intelligence assisted design, has an amino acid sequence of GVRPDGFAHIRKFVSRIVRRLNIAPSKV, can play a rapid sterilization role by physically destroying the integrity of a bacterial cell membrane, has a specific and efficient inhibitory effect on propionibacterium acnes, has strong antibacterial activity, and bacteria are difficult to develop a targeted drug resistance mechanism; the polypeptide is derived from a mutant sequence of human VI collagen; the polypeptide has extremely low cytotoxicity and immunogenicity and high safety.

Description

Technical Field

[0001] This application belongs to the field of biomaterials technology, and in particular relates to novel anti-acne recombinant collagen peptides, their preparation methods, and applications. Background Technology

[0002] Acne is a chronic inflammatory skin disease affecting the hair follicles and sebaceous glands, commonly occurring in adolescents, and significantly impacting patients' quality of life and mental health. The occurrence of acne is closely related to factors such as excessive sebum secretion, abnormal keratinization of the hair follicle opening, and inflammatory responses. Among these factors, Propionibacterium acnes (…) is a contributing factor. Cutibacterium acnes The colonization and proliferation of Propionibacterium acnes are the core elements that trigger and exacerbate acne inflammation. Currently, clinical treatment for Propionibacterium acnes mainly relies on topical or oral antibiotics (such as clindamycin, erythromycin, and tetracyclines) and retinoids. However, long-term use of antibiotics can easily lead to the development of drug-resistant strains and skin microecological imbalance, while retinoids often have side effects such as dry and flaky skin, photosensitivity reactions, and teratogenic risks. Therefore, developing safe, effective, and non-resistant novel anti-acne active substances is an urgent need in the field of skin disease treatment.

[0003] Collagen is a major component of the extracellular matrix and possesses excellent biocompatibility and biodegradability. In recent years, the modification of natural proteins using genetic engineering techniques to obtain recombinant collagen peptides with novel biological functions has become a hot topic in new drug development. Functional peptides derived from the body's own proteins theoretically possess low immunogenicity and high safety, making them highly promising candidates for developing novel anti-acne agents. Summary of the Invention

[0004] This application provides a novel anti-acne recombinant collagen polypeptide, its preparation method, and its application, to solve the problems existing in related technologies. The technical solution is as follows: In a first aspect, embodiments of this application provide a novel anti-acne recombinant collagen polypeptide having the amino acid sequence shown in SEQ ID NO.1.

[0005] In one embodiment, the amino terminus and / or carboxyl terminus of the amino acid sequence shown in SEQ ID NO.1 is connected to a cell-penetrating peptide, a polyarginine sequence, or a fatty acid chain.

[0006] Secondly, embodiments of this application provide a method for preparing a novel anti-acne recombinant collagen peptide, comprising the following steps: A polypeptide with the amino acid sequence shown in SEQ ID NO.1 was synthesized by sequentially coupling amino acids from the C-terminus to the N-terminus using the solid-phase Fmoc method.

[0007] Thirdly, the embodiments of this application provide the application of the novel anti-acne recombinant collagen peptide in the preparation of Propionibacterium acnes inhibitors, wherein the effective concentration of the novel anti-acne recombinant collagen peptide in the anti-Propionibacterium acnes inhibitor is 0.1%-5wt.

[0008] Fourthly, embodiments of this application provide a Propionibacterium acnes inhibitor, wherein the Propionibacterium acnes inhibitor comprises the aforementioned novel anti-acne recombinant collagen polypeptide.

[0009] Fifthly, the embodiments of this application provide the application of novel anti-acne recombinant collagen peptides in the preparation of anti-acne products, wherein the effective concentration of the novel anti-acne recombinant collagen peptides in the anti-acne products is 0.1%-5wt.

[0010] In one embodiment, the anti-acne product is a topical medicine, cosmetic, or medical device.

[0011] Sixthly, embodiments of this application provide an anti-acne topical medicine, the topical medicine comprising the above-mentioned novel anti-acne recombinant collagen polypeptide; preferably, the topical medicine further comprises one or more pharmaceutically acceptable excipients; preferably, the dosage form of the anti-acne topical medicine includes gel, cream, solution, film, spray or patch.

[0012] In one embodiment, the effective concentration of the novel anti-acne recombinant collagen peptide in the anti-acne topical drug is 0.1%-5wt.

[0013] In a seventh aspect, embodiments of this application provide an anti-acne cosmetic, the cosmetic comprising the aforementioned novel anti-acne recombinant collagen polypeptide; preferably, the cosmetic further comprises cosmetic excipients; preferably, the cosmetic is a facial cleanser, toner, serum, lotion, cream, or mask.

[0014] In one embodiment, the effective concentration of the novel anti-acne recombinant collagen peptide in the anti-acne cosmetic is 0.1%-5wt.

[0015] Eighthly, embodiments of this application provide a medical device comprising the aforementioned novel anti-acne recombinant collagen polypeptide; preferably, the medical device is a liquid dressing, a wound repair gel, or a polypeptide-containing patch.

[0016] In one embodiment, the effective concentration of the novel anti-acne recombinant collagen peptide in the medical device is 0.1%-5wt.

[0017] In one embodiment, the liquid dressing comprises the following components in weight percentage: 0.1-0.5% of the novel anti-acne recombinant collagen polypeptide prepared in Example 1, 4-6% mannose, 1-2% trehalose, and the balance being water for injection.

[0018] The advantages or beneficial effects of the above technical solutions include at least the following: The novel anti-acne recombinant collagen peptide of this application exerts a rapid bactericidal effect by physically disrupting the integrity of bacterial cell membranes. It has a specific and highly effective inhibitory effect on Propionibacterium acnes, strong antibacterial activity, and bacteria are unlikely to develop targeted drug resistance mechanisms. The peptide of this application is derived from a mutated sequence of human type VI collagen. It has extremely low cytotoxicity and immunogenicity and high safety.

[0019] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of this application will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0020] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0021] Figure 1 The liquid chromatogram of Col6A-Pep prepared in Example 1; Figure 2 The mass spectrum of Col6A-Pep prepared in Example 1; Figure 3 The liquid chromatogram of GVR28 prepared for Comparative Example 1; Figure 4 The mass spectrum of GVR28 prepared for Comparative Example 1; Figure 5 The image shows the cytotoxicity results of Col6A-Pep prepared in Example 1; Figure 6 The cytotoxicity results of GVR28 prepared for Comparative Example 1 are shown in the figure. Figure 7 The inhibition results of Col6A-Pep (left) prepared for Example 1 and GVR28 (right) prepared for Comparative Example 1 on the standard strain ATCC 6919 of Propionibacterium acnes. Figure 8 The images show the effects of using the liquid dressings D7 and D30 of Example 4 on some subjects. Detailed Implementation

[0022] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0023] The novel anti-acne recombinant collagen polypeptide of this application has the amino acid sequence Gly-Val-Arg-Pro-Asp-Gly-Phe-Ala-His-Ile-Arg-Lys-Phe-Val-Ser-Arg-Ile-Val-Arg-Arg-Leu-Asn-Ile-Ala-Pro-Ser-Lys-Val, using the common representation method of single-letter abbreviations of 20 standard amino acids, and the corresponding amino acid sequence is shown in SEQ ID NO.1.

[0024] SEQ ID NO. 1: GVRPDGFAHIRKFVSRIVRRLNIAPSKV.

[0025] The polypeptide sequence described in this application is derived from a mutated sequence of human type VI collagen. Through assisted screening, a novel, non-natural polypeptide sequence was obtained by mutating one or more amino acids in a specific domain of type VI collagen, exhibiting extremely strong specific antibacterial activity against Propionibacterium acnes.

[0026] As one embodiment, the recombinant collagen polypeptide of this application may also be linked to functional sequences at its amino and / or carboxyl ends for promoting transdermal absorption, improving stability, prolonging half-life, or enhancing targeting, such as, but not limited to, cell-penetrating peptides (CPP), polyarginine sequences, or fatty acid chains.

[0027] The novel anti-acne recombinant collagen peptide of this application is synthesized by solid-phase Fmoc solid-phase synthesis method, in which amino acids are coupled sequentially from the C-terminus to the N-terminus to synthesize a peptide with the amino acid sequence shown in SEQ ID NO.1.

[0028] The novel anti-acne recombinant collagen polypeptide of this application can be used as an antibacterial agent, an anti-acne topical drug, an anti-acne cosmetic, or an anti-acne medical device.

[0029] The following is a further explanation using specific embodiments.

[0030] Experimental methods not specified in the examples are generally performed under standard conditions, such as those described in Molecular Cloning Laboratory Manual (Sambrook J, et al.), or as recommended by the manufacturer.

[0031] Example 1: Peptide Acquisition Solid-phase chemical synthesis of peptides: The standard Fmoc solid-phase synthesis method was used to couple amino acids sequentially from the C-terminus to the N-terminus to synthesize a peptide with the sequence SEQ ID NO.1: GVRPDGFAHIRKFVSRIVRRLNIAPSKV; the sequence was synthesized by GenScript and is abbreviated as Col6A-Pep.

[0032] The lyophilized peptides were detected by LC-MS / MS. The chromatographic column was an Inertsil ODS-SP, 4.6 mm × 250 mm. Phase A: 0.065% trifluoroacetic acid aqueous solution (v / v); Phase B: 0.05% trifluoroacetic acid in acetonitrile solution (v / v); Total flow rate: 1 mL / min; Detection wavelength: 220 nm; The LC gradient elution program is shown in Table 1. Table 1

[0033] HPLC chromatogram as shown Figure 1 As shown; mass spectrum as shown Figure 2 As shown.

[0034] After lyophilization, the molecular weight of the peptide was determined by mass spectrometry to be consistent with the theoretical value, and a pure target peptide with a purity greater than 95% was obtained.

[0035] Comparative Example 1 A polypeptide with the sequence GVRPDGFAHIRDFVSRIVRRLNIGPSKV (SEQ ID NO.2) was synthesized by sequentially coupling amino acids from the C-terminus to the N-terminus using the standard Fmoc solid-phase synthesis method. This polypeptide was synthesized by GenScript and named GVR28. Detection was performed under the LC-MS / MS detection conditions of Example 1. The HPLC chromatogram is shown below. Figure 3 As shown; mass spectrum as shown Figure 4 As shown.

[0036] After freeze-drying, mass spectrometry analysis confirmed that the molecular weight matched the theoretical value, yielding a pure target peptide with a purity greater than 95%.

[0037] Example 2 Hacat Cytotoxicity Test (1) Cell digestion and plating observation of cell state: HaCaT cells were observed under a microscope to confirm that the cell confluence reached 80%-90%, and that the cells were plump and free of contamination.

[0038] Cell digestion: Discard the old culture medium and gently wash 1-2 times with sterile PBS to remove residual serum. Add an appropriate amount of 0.25% trypsin-EDTA (enough to cover the bottom of the culture dish), and incubate at 37°C for 2-5 minutes; quickly add complete culture medium containing 10% FBS to stop the digestion.

[0039] Centrifugation and resuspension: Collect the cell suspension into a centrifuge tube and centrifuge at 1000 rpm for 3-5 min. Discard the supernatant and resuspend the cell pellet in fresh complete culture medium, mixing thoroughly by pipetting.

[0040] Cell counting: Cell concentration was determined using a cell counting chamber or an automated cell counter.

[0041] Adjusting concentration and plating: Adjust the cell suspension concentration to 5 × 10⁻⁶. 4 Cells / mL. Add 100 µL of cell suspension to each well in the central section of a 96-well plate (i.e., a seeding density of 5 × 10⁶ cells / mL). 4 (cells / mL).

[0042] To prevent edge effects: Do not add cells to the outermost ring of the 96-well plate (row 1, row 12, column A, column H). Add 100µL of sterile PBS or pure water to each well to prevent edge effects caused by liquid evaporation.

[0043] Incubation and cell adhesion: Place the 96-well plate in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere completely and resume logarithmic growth.

[0044] (2) Preparation of test samples: The test samples (Col6A-Pep, GVR28) were serially diluted with complete culture medium, and corresponding solvents were set up as blank control groups. The old culture medium in the 96-well plate was carefully aspirated and added culture medium containing different concentrations (2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 62.5 µg / mL, 31.25 µg / mL, 15.625 µg / mL, 7.8125 µg / mL) of the test sample according to the experimental design, 100 µL per well, and set up groups (each group should have at least 3-5 replicates).

[0045] Incubation: Place the culture plate back into the incubator and continue incubation for 48 hours.

[0046] (3) CCK-8 assay: Discard the drug-containing culture medium and wash gently once with PBS. Then add 100µL of fresh complete culture medium to each well.

[0047] Add CCK-8: Under light-protected conditions, add 10 µL of CCK-8 solution to each well (including the blank group). Incubation reaction: Return the culture plate to a 37°C incubator and incubate for 2 hours.

[0048] Absorbance measurement: After incubation, the 96-well plate was placed in a full-wavelength microplate reader, shaken for 10 seconds to mix, and the absorbance (OD) of each well was measured at 450 nm. Results are as follows: Figure 5 and Figure 6 As shown, Figure 5For Col6A-Pep, Figure 6 It is GVR28.

[0049] from Figure 5 The results show that the present application not only has no toxicity to Hacat, but also promotes the proliferation of Hacat cells.

[0050] Example 3: In vitro detection of anti-acne Propionibacterium acnes activity Test strain: Propionibacterium acnes standard strain ATCC 6919.

[0051] (1) Preparation of Propionibacterium acnes suspension: Propionibacterium acnes was inoculated at a ratio of 1% in thioglycolate liquid medium and cultured at 30°C for 48 hours in a constant temperature incubator to obtain Propionibacterium acnes suspension.

[0052] (2) Preparation of double-layer plates: First, pour 15 ml of thioglycolate solid medium containing 1.5% agar powder into the plate. After the medium cools and solidifies, it is used as the lower plate. Then, use a pipette to take 500 μl of activated Propionibacterium acnes and add it to a centrifuge tube containing 5 mL of thioglycolate solid medium containing 0.7% agar powder cooled to 40°C. After mixing, pour it onto the solidified lower plate and wait for it to solidify completely to become the upper plate.

[0053] (3) Add the following samples to each of the six wells: 1. Sterile water; 2. Col6A-Pep 1 mg / mL; 3. Col6A-Pep 2 mg / mL; 4. Col6A-Pep 5 mg / mL; 5. Col6A-Pep 10 mg / mL; 6. Quaternary ammonium salt-73 40 µg / mL. The following samples were added to the other 6 wells: 1. Sterile water; 2. GVR28 1 mg / mL; 3. GVR28 2 mg / mL; 4. GVR28 5 mg / mL; 5. GVR28 10 mg / mL; 6. Quaternary ammonium salt-73 40 µg / mL. (4) Place the plates in a 37℃ constant temperature incubator for 48 hours, observe the results, and take photos for recording; the inhibition diameter is shown in Table 2 and Figure 7 As shown, Figure 7 The left image shows Col6A-Pep, and the right image shows GVR28; Table 2

[0054] Example 4 The liquid dressing includes: 0.3% Col6A-Pep prepared in Example 1, 5% mannose, 1.5% trehalose, and the remainder water for injection.

[0055] The effectiveness of the liquid dressing in Example 4 was verified as follows: 1. Subjects This embodiment included a total of 17 subjects.

[0056] 2. Test method A single-group before-and-after control observation method was adopted, using the subject's pre-treatment condition as a baseline, and comparing the skin improvement at different time points after treatment.

[0057] 3. Intervention methods The procedure involves full-face microneedling with a needle length of 1.0 mm. Post-microneedling care is performed using the composition described in this application.

[0058] 4. Observation period Observations and records were taken on D0 (before treatment), D7 (7 days after treatment), and D30 (30 days after treatment). Seventeen subjects were photographed on day 7, and fourteen subjects were photographed on day 30. Facial changes in some subjects were observed as follows. Figure 8 As shown.

[0059] The evaluation was based on the VISIA numerical improvement rate, and the VISIA evaluation criteria were as follows: Improvement rate <25%: Ineffective 25% ≤ Improvement rate < 50%: General 50% ≤ Improvement rate < 75%: Effective Improvement rate ≥75%: Significantly effective.

[0060] The results are shown in Table 3: Table 3

[0061] The VISIA results indicate that the liquid dressing containing the recombinant collagen peptide Col6A-Pep described in this application significantly improves skin inflammation, sensitivity, and redness, especially with a 76.5% improvement rate in redness areas after 7 days of treatment; it also regulates sebum secretion, with a 57.1% improvement rate after 30 days of treatment; and it also has a certain improvement effect on pores and skin texture. Therefore, the liquid dressing described in this application shows significant improvement in inflammation, sensitivity, and redness in the short term, and at 30 days, it has a good objective improvement effect on acne, redness, and sebum secretion; however, its improvement effect on skin texture and pores is relatively weak.

[0062] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0063] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0064] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A novel anti-acne recombinant collagen polypeptide, characterized in that, The anti-acne recombinant collagen polypeptide has the amino acid sequence shown in SEQ ID NO.

1.

2. The novel anti-acne recombinant collagen polypeptide according to claim 1, characterized in that, The amino terminus and / or carboxyl terminus of the amino acid sequence shown in SEQ ID NO.1 is connected to a cell-penetrating peptide, a polyarginine sequence, or a fatty acid chain.

3. The method for preparing the novel anti-acne recombinant collagen polypeptide according to claim 1 or 2, characterized in that, Includes the following steps: The peptide with the amino acid sequence shown in SEQ ID NO.1 was synthesized by sequentially coupling amino acids from the C-terminus to the N-terminus using the Fmoc solid-phase synthesis method.

4. The application of the novel anti-acne recombinant collagen polypeptide according to claim 1 or 2 in the preparation of Propionibacterium acnes inhibitor, characterized in that, The effective concentration of the novel anti-acne recombinant collagen peptide in the anti-Propionibacterium acnes inhibitor is 0.1%-5wt.

5. A Propionibacterium acnes inhibitor, characterized in that, The Propionibacterium acnes inhibitor comprises the novel anti-acne recombinant collagen polypeptide as described in claim 1 or 2.

6. The application of the novel anti-acne recombinant collagen polypeptide according to claim 1 or 2 in the preparation of anti-acne products, characterized in that, The effective concentration of novel anti-acne recombinant collagen peptides in anti-acne products is 0.1%-5wt.

7. The application according to claim 6, characterized in that, The anti-acne products mentioned are topical medicines, cosmetics, or medical devices.

8. A topical anti-acne medication, characterized in that, The topical medicine includes the novel anti-acne recombinant collagen polypeptide as described in claim 1 or 2; preferably, the topical medicine further includes one or more pharmaceutically acceptable excipients; preferably, the dosage form of the anti-acne topical medicine includes gel, cream, solution, film, spray or patch.

9. An anti-acne cosmetic, characterized in that, The cosmetic product includes the novel anti-acne recombinant collagen polypeptide as described in claim 1 or 2; preferably, the cosmetic product also includes cosmetic excipients; preferably, the cosmetic product is a facial cleanser, toner, serum, lotion, cream, or mask.

10. A medical device, characterized in that, The medical device includes the novel anti-acne recombinant collagen polypeptide as described in claim 1 or 2; preferably, the medical device is a liquid dressing, a wound repair gel, or a dressing containing polypeptides.

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