An elastin biomimetic polypeptide with high bioactivity of beta helix structure, preparation method and application

Abstract

This invention discloses a highly bioactive β-helix elastin biomimetic polypeptide, its preparation method, and its applications. The polypeptide has the amino acid sequence shown in SEQ ID NO.1, is soluble in water or buffer solution to form a clear solution, and spontaneously forms a stable β-helix conformation in solution. The polypeptide solution has a defined phase transition temperature (Tt) of 70-80℃, ensuring stable dissolution at room temperature and skin application temperatures. The preparation method includes solid-phase synthesis, purification and identification, dissolution preparation, and necessary temperature treatment steps. This polypeptide solution can be used to prepare skincare products for anti-wrinkle and UV damage repair, such as serums, sprays, and complex solutions. This invention has advantages such as a well-defined sequence, simple preparation, no cross-linking required, and good stability, and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of elastin biomimetic technology, specifically relating to a highly bioactive β-helix elastin biomimetic polypeptide, its preparation method and application, and particularly to a soluble elastin polypeptide solution with a β-helix conformation, its preparation method and its application in anti-wrinkle and UV irradiation damage repair. Background Technology

[0002] Elastin is a key structural protein in the extracellular matrix (ECM) of vertebrates, mainly distributed in tissues requiring high elasticity and reversible deformation capacity, such as skin, blood vessel walls, lung tissue, and ligaments. The formation of skin wrinkles is closely related to the decline of skin elasticity. Skin elasticity primarily originates from the supporting network formed by collagen and elastin in the extracellular matrix. With age and the accumulation of environmental stimuli, especially ultraviolet (UVA / UVB) radiation, elastin in the skin breaks down or becomes structurally disordered, collagen degradation accelerates, and oxidative stress is enhanced, inflammatory factors are upregulated, and matrix metalloproteinases (MMPs) expression increases, leading to photoaging manifestations such as increased fine lines, sagging, roughness, and decreased elasticity. Therefore, developing active ingredients that can improve skin elasticity and promote repair after UV irradiation is an important direction in the field of anti-wrinkle and skin repair.

[0003] In skincare products, peptide active ingredients have attracted widespread attention due to their well-defined sequences, good biocompatibility, and direct application in solution form. Among them, elastin-like peptides (ELPs) are typically designed based on repetitive motifs, can undergo conformational changes in solution, and exhibit temperature-responsive properties. However, current solutions of ELP active ingredients still have shortcomings: on the one hand, conformational formation and stability are often influenced by multiple factors, lacking a clearly defined and easily verifiable temperature characteristic window; on the other hand, some systems have low phase transition temperatures, which may lead to phase separation, turbidity, or insufficient stability at room temperature or operating temperature, thus limiting their formulation development, storage, and transportation as solution-based skincare active ingredients; furthermore, current research focuses more on ELP gels or materialization, and there is still a lack of targeted solutions for systems that "do not require crosslinking, maintain a solution state, have high phase transition temperatures, and can be used for anti-wrinkle and UV repair."

[0004] Therefore, there is an urgent need to provide an elastin polypeptide solution with a well-defined sequence, easy preparation, the ability to form a β-helical conformation in solution and a well-defined phase transition temperature window, so as to achieve a stable, reproducible and industrially suitable solution-type active polypeptide product for anti-wrinkle and UV-induced damage repair. Summary of the Invention

[0005] To address the shortcomings of existing elastin-like peptides, such as low phase transition temperature, easy phase separation at room temperature, and the need for cross-linking before use, this invention aims to provide an elastin-inspired biomimetic peptide, its solution, preparation method, and applications. The peptide described in this invention possesses the technical advantages of well-defined conformation, structural stability, high phase transition temperature, easy solubility, convenient use, and convenient preparation.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an elastin-inspired polypeptide with a β-helix structure, wherein the elastin-inspired polypeptide is a single-chain polypeptide sequence, preferably an elastin-like sequence, capable of forming a β-helix conformation based on β-turn in aqueous solution or buffer solution, and exhibiting reversible elastic behavior and / or temperature-responsive behavior.

[0007] In some embodiments, the elastin-inspired polypeptide comprises the following amino acid sequence (SEQ ID NO. 1): GLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGI.

[0008] In some embodiments, the elastin-inspired polypeptide comprises elastin-like repeating structural units, the repeating structural units comprising VPGXG or XGXXPG or equivalent structures thereof, wherein X is any amino acid residue; preferably, X is selected from one or more of A, V, L, I, and G.

[0009] In some embodiments, the elastin-inspired polypeptide allows for conservative or equivalent substitutions without compromising its β-helix conformational ability and reversible elastic behavior; preferably, the substitution number is 1–3 amino acid residues.

[0010] Secondly, the present invention further provides a soluble elastin polypeptide solution, the solution comprising the polypeptide described in the first aspect and a solvent, wherein the solvent is water or a buffer solution; the solution is a clear solution and can be used as a solution-type active ingredient in skin care and repair products.

[0011] In some embodiments, the soluble elastin polypeptide solution contains a polypeptide concentration of 0.001-100 mg / mL, preferably 0.01-50 mg / mL, and more preferably 0.1-20 mg / mL.

[0012] In some embodiments, the solution does not contain a crosslinking agent and is not subjected to crosslinking treatment.

[0013] In some embodiments, the polypeptide solution has a defined phase transition temperature of 70-80 °C.

[0014] Preferably, the phase transition temperature is determined by measuring a turbidity curve. More preferably, the solution remains clear and stable within the range of room temperature and skin application temperature, without macroscopic phase separation or precipitation.

[0015] In some embodiments, the elastin-inspired polypeptide is capable of forming a β-helical conformation in aqueous or buffer solutions, and this conformation can be confirmed or characterized by at least one of the following methods: (1) Circular dichroism (CD) test; (2) Fourier transform infrared spectroscopy (FTIR) test; (3) Nuclear magnetic resonance (NMR) test; (4) Turbidity curves were used to determine the temperature-response phase transition behavior.

[0016] Thirdly, in some embodiments, the formation conditions of the β-helical conformation include, but are not limited to, any combination of the following factors: pH 5.0–9.0; Ionic strength 0–200 mM; Temperature 4–80℃; Peptide concentration: 0.1–300 mg / mL.

[0017] Fourthly, the present invention further provides a method for preparing the β-helix elastin biomimetic polypeptide, the method comprising: (a) Solid-phase synthesis route (SPPS) In some embodiments, the preparation method includes: (1) Resin-bound peptides were obtained by stepwise coupling of amino acids according to the target sequence using a solid-phase peptide synthesis method; (2) Cleavage and deprotection to obtain crude polypeptide; (3) Purification was performed using reversed-phase high-performance liquid chromatography; (4) The target polypeptide is obtained by freeze drying.

[0018] Preferably, the solid-phase synthesis method is the Fmoc solid-phase synthesis method, wherein the coupling system of the above method is selected from one or more of HBTU, HATU, DIC / Oxyma, and PyBOP, the base is diisopropylethylamine (DIPEA), and the pyrolysis system is a TFA (trifluoroacetic acid) system containing a trapping agent (e.g., triisopropylsilane (TIS), water, and ethylenedithiol (EDT)).

[0019] (ii) Reorganization of expression routes In some embodiments, the preparation method includes: (1) Construct the nucleic acid sequence encoding the elastin biomimetic polypeptide and ligate it into an expression vector; (2) Introduce the expression vector into host cells to induce expression; (3) The expression product is purified to obtain the elastin biomimetic polypeptide; (4) The structure and purity of the polypeptide were identified.

[0020] Preferably, the host cell is Escherichia coli; purification can be performed by affinity chromatography or by utilizing the temperature-responsive phase transition of ELP.

[0021] Fifthly, the present invention further provides a method for preparing the polypeptide solution, comprising: polypeptide preparation (solid-phase synthesis / purification / identification) → dissolution and preparation → filtration and sterilization → dispensing. Preferably, the polypeptide is purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and its molecular weight is confirmed by MS, and its purity is confirmed by HPLC analysis.

[0022] In a sixth aspect, the present invention further provides the use of the polypeptide solution in the preparation of anti-wrinkle and UV-damage repair products, wherein the products are selected from one or more of serums, sprays, and compound solutions.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects: The final product of this invention is a soluble polypeptide solution, which does not require cross-linking and materialization steps. The preparation process is simple and suitable for the development of skin care and repair products.

[0024] (1) The sequence is well-defined and easy to prepare: The elastin biomimetic polypeptide provided by the present invention is a single sequence with a simple and well-defined structure. It can be prepared by solid-phase synthesis or by recombinant expression, thereby improving the feasibility of preparation and batch consistency.

[0025] (2) Better conformation controllability: The polypeptide can form a β-helix structure under solution conditions. This structure can be characterized and confirmed by CD, NMR, FTIR and other means, thereby achieving traceability of "sequence-structure-performance" and improving the stability of material performance.

[0026] (3) The phase change temperature is between 70-80℃, which keeps the solution in a stable dissolution state within the range of room temperature and skin use temperature, reduces the risk of phase separation, and is conducive to storage, transportation and compatibility with the formulation system.

[0027] (4) Diverse material processing forms: The elastin biomimetic polypeptide can be processed into various forms such as hydrogel, film, coating, fiber, microsphere, etc., which makes it easy to select the appropriate processing method according to different applications.

[0028] (5) The solution can be directly used as an active polypeptide solution for anti-wrinkle and UV irradiation damage repair products, with a clear application path.

[0029] (6) The solution is compatible with common skin care solution systems, making it easy to form various dosage forms (serum, spray, freeze-dried reconstituted solution, etc.). Attached Figure Description

[0030] Figure 1 This is a predicted structural diagram of the polypeptide described in Example 1 of the present invention.

[0031] Figure 2 This is a comparison curve of the root mean square deviation (RMSD) of the main chain of the polypeptide under different dielectric constants as described in Example 2 of the present invention, which changes over time.

[0032] Figure 3 This is a molecular dynamics simulation conformation diagram of the polypeptide described in Example 2 of the present invention under different dielectric constants (ε=40.0 (Figure ac) and ε=80.0 (Figure df)).

[0033] Figure 4 This is the mass spectrometry characterization diagram of the polypeptide described in Example 3 of the present invention.

[0034] Figure 5 This is the Fourier transform infrared spectrum characterization of the polypeptide described in Example 3 of the present invention.

[0035] Figure 6 This is the CD spectrum described in Embodiment 6 of the present invention.

[0036] Figure 7 This is a phase transition temperature fitting diagram of the polypeptide described in Example 5 of the present invention.

[0037] Figure 8 This is a CCK-8 cell activity assay of the polypeptide described in Example 7 of this invention.

[0038] Figure 9 This is a cell adhesion detection diagram of the polypeptide described in Example 7 of the present invention.

[0039] Figure 10 This is a live / dead cell detection image of the polypeptide described in Example 7 of the present invention.

[0040] Figure 11 This is a cell proliferation detection diagram of the polypeptide described in Example 7 of the present invention.

[0041] Figure 12 This is a cell migration diagram of the polypeptide described in Example 7 of the present invention.

[0042] Figure 13 This is a diagram showing the morphological and quantitative results of UVB-induced damage to the caudal fin of zebrafish induced by the polypeptide described in Example 8 of this invention.

[0043] Figure 14 This is a diagram showing the ROS fluorescence imaging and quantification results of the polypeptide described in Example 8 of this invention in zebrafish larvae. Detailed Implementation

[0044] The technical solution of the present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following description.

[0045] This invention provides a soluble elastin polypeptide solution, its preparation method, and its uses. The polypeptide solution maintains its solution state without cross-linking or material processing, and has a clear β-helix conformation and phase transition temperature, thereby being used for anti-wrinkle and UV irradiation damage repair.

[0046] (1) Preparation of polypeptide sequences: The amino acid sequence of the target polypeptide used in this embodiment is shown in SEQ ID NO. 1.

[0047] (2) Structural prediction: Molecular conformation prediction was performed using AlphaFold Server (https: / / alphafoldserver.com / , with AlphaFold3 as the base model), a deep learning-based protein 3D structure prediction platform. The aforementioned amino acid sequence was input into the server system, and deep learning conformation prediction computation was initiated using the system's default parameters without any additional ligand or template intervention.

[0048] (3) Structure acquisition: After the computation is complete, the set of predicted models output by the system is extracted. Using model confidence (e.g., pLDDT score, i.e., predicted local distance difference test score) as the evaluation criterion, the three-dimensional structural model with the highest confidence score is selected and downloaded. This model is exported as a PDB (Protein Data Bank) file, representing one of the stable spatial folds (β-spiral conformation) of the peptide in a near-native state, such as... Figure 1 As shown, it is used as the initial conformational model of the polypeptide.

[0049] The PDB structure file obtained in this embodiment can be directly used in subsequent molecular simulation steps.

[0050] This embodiment uses the protein three-dimensional structure obtained in Example 1 as the initial conformation, and performs simulation calculations using a self-constructed OpenMM implicit solvent molecular dynamics simulation script. Stability and mechanical analysis are then performed on the simulation data. The specific steps are as follows: (1) Simulation preparation Based on the OpenMM molecular simulation framework, a self-written implicit solvent molecular simulation script was developed. This script employs a pure protein force field (amber14 / protein.ff14SB) combined with a GBSA (Generalized Born-Surface Area) implicit solvent model. The GB (Generalized Born) term handles electrostatic and dielectric shielding effects under different dielectric conditions, while the SA term (using the ACE model) calculates the nonpolar surface area to accurately preserve hydrophobic interactions within the structure (such as the hydrophobic stacking of Val / Pro residues). The script systematically sets multiple sets of different dielectric constants (ε=4.0, 10.0, 20.0, 40.0, 80.0) while maintaining the internal dielectric constant of the solute at a fixed value of 1.0.

[0051] (2) Construction of the equilibrium system The PDB structure file downloaded in Example 1 is imported into the script described above. The script first calls PDBFixer to automatically repair the structure and adds hydrogen atoms at a specific pH (e.g., pH=7.0). Subsequently, the system is built under aperiodic boundary conditions, and high-precision energy minimization and conformational equilibrium are performed sequentially. After equilibrium is achieved, a productive molecular dynamics simulation is performed using the Langevin kinetic integrator (friction coefficient set to 1.0 / ps, integration step size 2 fs, simulation temperature 300 K, simulation duration 20 ns), outputting high-compression ratio atomic motion trajectory files (DCD format) and log files containing energy states (CSV format) for each dielectric constant. (3) Trajectory dynamics feature extraction The atomic motion trajectory files were post-processed and analyzed using the MDAnalysis trajectory analysis tool to extract and calculate several core conformational dynamic parameters, including: root mean square deviation (RMSD) time series of the main chain, radius of gyration (Rg), evolution of the number of intramolecular hydrogen bonds, and occupancy rate. At the same time, high-precision dihedral analysis (sequence-dihedral diagram) was performed on the protein backbone and side chains, and the degree of angular fluctuation of each residue in the main chain (ϕ, ψ) and side chain (χ1) was systematically quantified to evaluate the local flexibility and overall structural compactness of the polypeptide chain from multiple dimensions. (4) Analysis of simulation results Based on the above multi-dimensional conformational dynamics simulation data, such as Figure 2 and Figure 3As shown, the conclusion is that the stability of the specific polypeptide structure (such as the β-spiral conformation) is significantly dependent on the solvent dielectric environment. At specific low dielectric constants (ε=4.0 and ε=40.0), the electrostatic shielding effect within the system weakens, and the polypeptide can maintain a high-density key internal hydrogen bond network. Its RMSD, cyclotron radius, and overall flexibility index remain at extremely low levels, exhibiting short-term conformational stability (9 ns). However, with changes in the environmental dielectric constant, the hydrogen bond network exhibits significant instability compared to ε=4.0 and ε=40.0, and the structure no longer maintains its initial state but collapses into other conformations. These calculation results quantitatively confirm at the atomic scale that the synergistic effect of the internal dielectric environment and hydrogen bonds is the core driving force for maintaining the specific folding conformation of the polypeptide.

[0052] The target elastin polypeptide sequence is as follows: GLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGI (SEQ ID NO.1) (1) Raw materials and reagents Resin: Rinkamide resin; Protected amino acids: Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Ile-OH; Coupling reagent: HBTU; Alkali: DIPEA; Deprotection solution: 20% piperidine / DMF; Lysis buffer: TFA / water / TIS; Washing solvents: DMF, DCM; Purification: C18 reversed-phase HPLC system; Identification: ESI-MS, HPLC analysis.

[0053] (2) Solid-phase synthesis steps a) Resin swelling: Weigh the resin and add it to the reaction column, and use DCM to swell for more than 4 hours; b) The N-terminal Fmoc protecting group was removed by a 20% piperidine / N,N-dimethylformamide (DMF) solution, and the complete removal of the protecting group was detected by a colorimetric reaction; c) The N-terminus protected by Fmoc (4 eq) amino acids, HOBT (4 eq) and HBTU (4 eq) were dissolved in DMF, activated at low temperature for 20 min, and then DIEA (6 eq) was added dropwise to the solution. The solution was mixed and added to the reactor, and the reaction was carried out for 3 hours.

[0054] d) After the reaction was complete, the reaction solution was withdrawn from the reactor, and the resin was washed three times with DMF and DCM, respectively. A colorimetric reaction confirmed complete amino acid condensation. The resin was then treated three times with a 20% piperidine / DMF solution. The resin was washed three times with DMF and DCM, and a colorimetric reaction confirmed complete removal of the protecting groups. e) Repeat the "deprotection-coupling-washing" cycle until the target sequence is obtained; the target sequence is: GLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGI; f) Finally, deprotection and washing are performed to obtain resin-bound linear peptides.

[0055] (3) Pyrolysis and crude product precipitation Place the resin in a lysis flask, add TFA / water / TIS (e.g., 95:2.5:2.5, volume ratio), and lyse for 3-4 hours; filter to remove the resin and collect the lysis buffer; drop the lysis buffer into ice-cold ether to precipitate the crude peptide; centrifuge to collect the precipitate and wash it 3 times with ice-cold ether; obtain the crude product and dry it for later use.

[0056] (4) Purification and identification The crude product was dissolved in water or a water / acetonitrile mixed solvent containing 0.1% TFA and purified by C18 reversed-phase HPLC with gradient elution in an acetonitrile (A) / water (B) system. The main peak was collected and lyophilized. After purification, the molecular weight was determined by ESI-MS to confirm the correct sequence. The purity was determined by analytical HPLC. The product with a purity ≥90% was taken as the target elastin biomimetic peptide.

[0057] Molecular weight identification: Mass spectrometry characterization diagram as shown Figure 4 As shown, the measured molecular weight is 3934.8 Da, which is highly consistent with the theoretical value of 3932.6 Da within the error range, proving that the target sequence was successfully synthesized.

[0058] Functional group characterization: Fourier transform infrared spectrum as shown Figure 6 As shown, the position of the characteristic peak of the amide band proves that there is a typical β-helix structure within the polypeptide molecule.

[0059] 1) Solvent and buffer system The peptide obtained in Example 1 was dissolved in deionized water or a buffer solution, which may be PBS, phosphate buffer, or Tris buffer. PBS buffer (pH 7.2–7.4) is preferred to simulate skin contact and improve solution stability.

[0060] 2) Dissolution and concentration preparation (1) Weigh the target polypeptide powder, add solvent and gently shake or vortex at low speed to mix; (2) Prepare peptide concentrations of 0.1–20 mg / mL. In some embodiments, this can be extended to concentrations of 0.01–100 mg / mL in water, PBS, phosphate buffer, or Tris buffer. (3) If necessary, the solution can be left to stand at 4°C for several hours to promote complete dissolution and obtain a clear solution.

[0061] 3) Sterilization filtration and preservation (1) The solution was sterilized by filtration through a 0.22 μm filter membrane; (2) Dispense into sterile containers and store at 4°C for later use; (3) If used in a freeze-drying reconstitution system, freeze-drying can be performed after this step.

[0062] Note: The solution system of this invention does not contain cross-linking agents, does not undergo cross-linking reactions, and remains in solution state as the final product form.

[0063] 1) Sample preparation Take the polypeptide solution obtained in Example 4 and set a concentration point of 5 mg / mL.

[0064] 2) Turbidity curve determination (1) Use a UV-Vis spectrophotometer to monitor the turbidity of the solution at a wavelength of 350 nm; (2) Set a temperature gradient heating program, for example, from 20℃ to 90℃, with a heating rate of 5℃ / min; (3) Record the absorbance curve as a function of temperature.

[0065] 3) Phase transition temperature determination The phase transition temperature Tt is defined as the inflection point temperature at which the turbidity curve rises significantly or the temperature corresponding to the peak value of the first derivative.

[0066] 4) Experimental Results Experimental results show that the turbidity of the polypeptide solution increases significantly within a specific temperature range during heating, indicating that the solution undergoes a temperature-induced phase transition. Further analysis of the inflection point of the turbidity curve reveals that the phase transition temperature Tt of the polypeptide solution of this invention is located in the range of 70–80℃.

[0067] Furthermore, the peptide solution remained clear and stable within the range of room temperature and skin application temperature (e.g., 25–45°C), with no obvious turbidity or precipitation observed. This indicates that the peptide solution has a high phase transition temperature window, which is beneficial for its formulation development, storage and transportation, and topical skin care applications as a solution-type active ingredient.

[0068] 1) CD spectrum scanning (1) Take the polypeptide solution obtained in Example 4 and dilute it to a concentration of 0.2 mg / mL suitable for CD determination if necessary; (2) Circular dichroism spectroscopy was used to scan in the range of 190–260 nm to record the secondary structure spectrum.

[0069] 2) Temperature scanning and cyclic testing (1) Set the temperature scan range, for example, 10–90℃ or a wider range; (2) Record the characteristic CD signal as a function of temperature; (3) Perform heating-cooling cycles (e.g., 20℃→90℃→20℃) to evaluate the formation and reversibility of the β-helical conformation.

[0070] 3) Result determination The trend of peptides forming β-helical conformations in solution can be characterized by changes in characteristic peaks in CD spectra and temperature scan curves, and their conformational stability and reversibility can be evaluated.

[0071] Experimental results: Secondary structure: CD spectrum as shown Figure 6 As shown, a distinct β-helix characteristic signal was observed in the range of 190–230 nm, indicating that the polypeptide self-assembled into a stable ordered conformation in aqueous solution. After a heating-cooling cycle, it could return to the formed β-helix structure, demonstrating its reversible change capability.

[0072] Cell line: Mouse fibroblast L929; Culture medium: MEM + 10% fetal bovine serum (FBS); Peptide solution: The soluble elastin biomimetic peptide solution obtained in Example 4 was used; Reagents: CCK-8 kit, crystal violet, Live / Dead staining reagent (Calcein-AM / PI); Concentration gradient: The peptide solution was diluted with culture medium to 0 (control), 10, 50, 100, 200, 400, and 800 μg / mL; Sterilization by filtration: All peptide solutions were filtered through a 0.22 μm filter membrane before use.

[0073] Note: The 0 mg / mL group served as a blank control group (culture medium only).

[0074] L929 cells were seeded at 3 × 10³ cells / well in 96-well plates and cultured at 37°C and 5% CO2 for 24 h to allow cell adhesion. The original culture medium was discarded, and different concentrations of peptide solution were added to each treatment group, with 5 replicates per group. The cells were cultured for 24 h, 48 h, and 72 h, respectively. At each time point, 10 μL of CCK-8 reagent was added and incubated for 1–2 h. The absorbance (OD value) was read at 450 nm, and the background was corrected using blank culture medium wells. Calculate relative cell activity (%): Relative activity = (OD) 样品 -OD 空白 ) / (OD 对照 -OD空白 () × 100%, results are shown in Figure 5 .

[0075] First, add 5 mg / mL peptide solution to the bottom of the 6-well plate and soak for at least 24 hours. Then, aspirate the peptide solution and wash once with PBS before seeding L929 cells (1×10⁻⁶). 6 Add 1 mg / mL of culture medium containing peptide to each well; incubate at 37°C for 1 h, 2 h, and 4 h; after reaching the time points, gently wash 1–2 times with PBS to remove non-adhered cells; Crystal violet staining was used to quantify the number of adherent cells; crystal violet was dissolved in SDS, and 100 μL was added to a 96-well plate, with absorbance measured at 570 nm using a microplate reader. The number of adherent cells and their spreading morphology were recorded by microscopy.

[0076] like Figure 9 As shown, the peptide solution can increase the number of early-adhering cells and promote cell spreading, indicating that the peptide solution of the present invention has the effect of promoting cell adhesion. Compared with the control group, the cell adhesion rate of the peptide solution treatment group is increased, indicating that it can provide a favorable cellular microenvironment for skin repair.

[0077] L929 cells were seeded in 1×10⁶ cells per 35 mm confocal dish. 6 Cells per well were cultured for 24 hours. After adhesion, 1 mg / mL of peptide solution was added for treatment. After 1, 3 and 5 days of culture, the cells were washed with PBS and then added with Live / Dead staining solution and incubated in the dark for 30 minutes. The cells were observed using a confocal microscope. Green cells were live cells and red cells were dead cells. The proportion of live cells was counted and photographed.

[0078] L929 cells were seeded at 3 × 10³ cells / well in 96-well plates and cultured at 37°C and 5% CO2 for 24 h to allow cell adhesion. The original culture medium was discarded, and different concentrations of peptide solution were added to each treatment group, with 5 replicates per group. The cells were cultured for 1, 3, and 5 days. At each time point, 10 μL of CCK-8 reagent was added and incubated for 1–2 h. The absorbance (OD value) was read at 450 nm, and the background was corrected using blank culture medium wells. Calculate relative cell activity (%): Relative activity = (OD) 样品 -OD 空白 ) / (OD 对照 -OD 空白 ) × 100%.

[0079] L929 cells were fed at a rate of 1×10⁻⁶ 6Inoculate each cell / plate into a 35mm plate. After 24 hours of adhesion, remove and discard the culture medium. Make a scratch in the center using a pipette tip, wash 2-3 times with PBS, then add 1mg / mL peptide solution for microscopic photography for 0 hours. After 24 hours of incubation, take microscopic photos again for comparison, and use ImageJ software for measurement and quantitative analysis.

[0080] Experimental results: The CCK-8 results showed that, within the concentration range of 10–800 μg / mL, the cell viability of the polypeptide solution treatment groups was not lower than that of the control group, and the cell viability showed an increasing trend, indicating that the polypeptide solution of the present invention has good cell compatibility. This indicates that the soluble polypeptide solution has good cell compatibility and can significantly improve the viability of L929 cells, suggesting that it has the effect of promoting cell proliferation.

[0081] Live / Dead staining is shown in the attached image. Figure 10 The results show that the polypeptide solution treatment group exhibits predominantly green fluorescence with very little red dead cell signal, and shows no significant cytotoxicity compared to the control group, further demonstrating that the polypeptide solution of this invention has good biocompatibility.

[0082] Safety and basic activity: CCK-8 cell viability assay results (see...) Figure 8 The results indicate that the peptides are non-toxic within the test range of 0-800 μg / mL; Figure 11 The results showed that the peptide significantly promoted cell proliferation in a concentration-dependent manner.

[0083] Repairability: Scratch test results are as follows Figure 12 As shown, after 24 hours of treatment, the scratch healing rate of the polypeptide group was significantly higher than that of the control group, proving that it has the ability to induce cell migration and accelerate wound healing.

[0084] Cell viability and quality: staining results of live and dead cells are as follows Figure 10 As shown, the polypeptide group cells exhibited dense green fluorescence (live cells) and very little red fluorescence (dead cells), demonstrating excellent biocompatibility.

[0085] Adhesion promotion: Results of cell adhesion experiments are as follows Figure 9 As shown, the cell adhesion speed and spreading area on the peptide-treated matrix were significantly improved, proving that peptides can effectively enhance the interaction between cells and the extracellular matrix.

[0086] (1) Laboratory animals and grouping Healthy wild-type zebrafish were selected and domesticated under standard conditions before the experiment, with a light / dark cycle of 14h / 10h and a water temperature maintained at 28±1℃. The zebrafish were randomly divided into the following experimental groups: Blank control group: no damage treatment was performed, only cultured in medium; Model group: damage model established, no peptide treatment added; Positive control group (PC): damage was treated with a positive control repair agent; Peptide treatment group: damage was treated with the peptide solution of this invention. The solution used in the peptide treatment group was the soluble elastic peptide solution prepared in Example 2.

[0087] (2) Experiment on UVB-induced caudal fin damage and repair in zebrafish Zebrafish larvae, 2 days post-fertilization, were selected, with 15 larvae per well, and randomly divided into a blank control group, a UVB model group, a positive control group, and a polypeptide treatment group. The positive control group was treated with a tea polyphenol solution with a mass concentration of 10 mg / mL. The concentration was determined according to the Chinese industry standard TZHCA014-2022 and related literature on the application of tea polyphenols in UV-induced zebrafish sunburn models. The aforementioned studies have shown that tea polyphenols have a good repair effect on UV-induced zebrafish tail fin damage. The polypeptide treatment group was treated with the polypeptide solution of the present invention.

[0088] Before the experiment, the E3 culture medium was discarded, and 3 mL of the corresponding experimental solution was added to each group. The mixture was incubated at 28.5℃ for 2 hours, with the blank control group receiving no treatment. Except for the blank control group, all zebrafish larvae in the other groups received UVB irradiation. UVB irradiation was performed twice, 15 minutes each time, with a 30-minute interval between the two irradiations, for a total irradiation dose of 1.87 J / cm². After irradiation, the zebrafish larvae were placed in fresh E3 culture medium and cultured at 28.5℃ for another 22 hours. Images of the caudal fins of the zebrafish larvae were acquired at the set culture time points. The larvae were fixed in 3% low-melting-point agarose, and the caudal fin area was imaged using an inverted fluorescence microscope. The caudal fin area was measured and quantitatively analyzed using ImageJ software.

[0089] The fin contraction inhibition rate Q (%) is calculated according to the following formula: Q (%) = (A1−A2) / (A3−A2)×100%, where A1 is the average caudal fin area of ​​the treatment group, A2 is the average caudal fin area of ​​the model group, and A3 is the average caudal fin area of ​​the blank control group.

[0090] like Figure 13As shown, in the UVB irradiation-induced caudal fin injury model, the caudal fins of zebrafish larvae in the model group showed obvious contraction and structural damage, and the caudal fin outline was incomplete. Compared with the model group, after treatment with the polypeptide solution of the present invention, the damaged area of ​​the zebrafish caudal fin was significantly reduced, the repaired area was increased, and the caudal fin outline was more continuous and complete. Its overall morphology was close to that of the blank control group and the positive control group.

[0091] The above results indicate that the polypeptide solution of the present invention can effectively alleviate the damage to the caudal fin of zebrafish caused by UVB irradiation, promote the repair and structural restoration of damaged tissues, and show a good anti-ultraviolet damage repair effect.

[0092] (3) Evaluation of antioxidant activity in zebrafish Zebrafish larvae, 3 days post-fertilization, were randomly divided into four groups: a blank control group, a model group, a positive control group, and a peptide treatment group. The positive control group was treated with a 0.1 mg / mL dipotassium glycyrrhizate solution, the concentration of which was determined based on prior literature on the use of dipotassium glycyrrhizate as a plant-derived antioxidant in a zebrafish ROS oxidative stress model. The peptide treatment group received the peptide solution of this invention. Each group of zebrafish larvae was treated with 3 mL of the corresponding experimental solution at 28.5℃ for 1 h. Subsequently, the model group, positive control group, and peptide treatment group were treated with 10 μM copper sulfate (CuSO4) solution for 20 min to induce oxidative stress in the zebrafish larvae. After treatment, the larvae were rinsed three times in fresh E3 culture medium. Then, the reactive oxygen species (ROS) levels in the zebrafish larvae were detected by incubating with a 20 μg / mL DCFH-DA fluorescent probe at 28.5℃ in the dark for 1 h. After incubation, the larvae were washed with culture medium and fixed in 3% low-melting-point agarose gel. The zebrafish larvae were imaged using an Olympus SZX16 fluorescence stereomicroscope, and the fluorescence signal intensity was quantitatively analyzed using ImageJ software.

[0093] like Figure 14 The fluorescence imaging results showed that the fluorescence signal in zebrafish larvae of the blank control group was weak and uniformly distributed; after CuSO4-induced oxidative stress, the ROS fluorescence signal in the model group was significantly enhanced, indicating a significant increase in oxidative stress level. Compared with the model group, the ROS fluorescence intensity in zebrafish larvae of the positive control group (dipotassium glycyrrhizate) was significantly reduced; the ROS fluorescence signal in zebrafish larvae of the peptide treatment group was also significantly weakened, and its fluorescence intensity was close to that of the positive control group.

[0094] Quantitative analysis of fluorescence intensity showed that the ROS fluorescence intensity in zebrafish larvae treated with the peptide was significantly lower than that in the model group, and the difference was statistically significant. This indicates that the peptide solution of the present invention can effectively scavenge or inhibit the generation of reactive oxygen species induced by oxidative stress and has good in vivo antioxidant activity.

[0095] The above description is merely a few exemplary embodiments of the present invention. For those skilled in the art, the present invention can be modified and varied in practice depending on specific preparation conditions, and is not intended to limit the present invention. Everything within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A biomimetic polypeptide with a highly bioactive β-helix structure, characterized in that, The elastin-inspired polypeptide is a single-chain polypeptide with the amino acid sequence SEQ ID NO.1: GLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGI.

2. An elastin-inspired polypeptide solution, characterized in that, The solution comprises the elastin biomimetic polypeptide of claim 1 and a solvent, wherein the solvent is water or a buffer solution, and the solution is a clear solution.

3. The elastin-inspired polypeptide solution as described in claim 2, characterized in that, The buffer solution is selected from one or more of PBS, phosphate buffer, and Tris buffer.

4. The elastin biomimetic polypeptide solution as described in claim 2 or 3, characterized in that, The concentration of the polypeptide is 0.001-100 mg / mL.

5. The elastin biomimetic polypeptide solution according to any one of claims 2-3, characterized in that, The phase transition temperature Tt of the solution is 70-80℃.

6. The elastin biomimetic polypeptide solution according to any one of claims 2-3, characterized in that, The solution remains clear and stable within the temperature range of 25-45°C, without macroscopic phase separation or precipitation.

7. The elastin biomimetic polypeptide solution according to any one of claims 2-3, characterized in that, The solution does not contain cross-linking agents and is not subjected to cross-linking treatment.

8. The elastin biomimetic polypeptide solution according to any one of claims 2-3, characterized in that, The polypeptide forms a β-helix conformation in solution.

9. A method for preparing the elastin biomimetic polypeptide solution according to any one of claims 2-8, characterized in that, The process includes the following steps: (1) preparing the elastin biomimetic polypeptide of claim 1; (2) dissolving the polypeptide in water or buffer solution to prepare a clear solution; (3) sterilizing and dispensing the solution to obtain the final product.

10. The method as described in claim 9, characterized in that, Step (1) The peptide was prepared by solid-phase peptide synthesis and purified by reversed-phase high-performance liquid chromatography.

11. The method according to any one of claims 9-10, characterized in that, Purification was performed using reversed-phase high-performance liquid chromatography (RP-HPLC) with a water / acetonitrile elution system containing 0.05–0.1% TFA or formic acid. After purification, the molecular weight was confirmed by mass spectrometry, and the purity was confirmed by HPLC to be ≥90%.

12. The use of the elastin biomimetic polypeptide solution according to any one of claims 2-8 in the preparation of anti-wrinkle products and / or UV-induced damage repair products, characterized in that, The product is a topical solution formulation.

13. The application of claim 12, characterized in that... The product is intended for use in repair products, which are serums, sprays, or lyophilized reconstituted solutions.

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