Self-assembled polypeptide derivative, hydrogel preparation, preparation method and application

Abstract

The invention provides a self-assembled polypeptide derivative, a hydrogel preparation, a preparation method and application, and particularly belongs to the technical field of biomedical materials. The structure of the self-assembled polypeptide derivative disclosed by the invention is Biotin-DFYIGSSSR, wherein Biotin is a blocked end, DF is D-configuration phenylalanine, YIG is an active sequence derived from laminin, and SSSR is an active amino acid sequence of IGF-1. The self-assembled polypeptide nano hydrogel prepared from the self-assembled polypeptide disclosed by the invention has thixotropy and self-repairing performance, and is resistant to enzymolysis and lasting in biological activity. And long-time residence in damaged tissues can be realized, so that the bioavailability is improved, and tissue repair is promoted. The self-assembled polypeptide derivative hydrogel disclosed by the invention has good biocompatibility and treatment effect, and can be safely used.

Description

A self-assembled peptide derivative, hydrogel formulation, preparation method and application Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a self-assembled polypeptide derivative, hydrogel formulation, preparation method, and application. Background Technology

[0002] The corneal epithelium is the first line of defense for the ocular surface barrier and is highly susceptible to damage from physical, chemical, and infectious factors. Under normal circumstances, the cornea can repair itself through rapid re-epithelialization. However, if persistent inflammation or metabolic abnormalities exist, epithelial defects will delay healing or fail to heal. If the epithelial defect area cannot close quickly, the risk of potential microbial infection and corneal opacity will significantly increase, ultimately leading to vision impairment and loss and increasing the need for subsequent corneal transplantation.

[0003] Current treatments include using preservative-free artificial tears, discontinuing epithelial toxic drugs, and using bandage lenses, all aimed at improving the local microenvironment of the ocular surface, but they are unlikely to fundamentally accelerate wound re-epithelialization. While autologous serum therapy can partially improve healing, it is limited by its complex procedures, significant batch-to-batch variability, and high cost. Protein therapies (such as laminin and insulin-like growth factor-1, IGF-1) can provide crucial biological signals, but their clinical application is limited by the rapid tear turnover and excessive proteolytic activity on the ocular surface. Given the shortcomings of existing treatments in terms of efficacy and practicality, there is an urgent need to develop highly effective and clinically feasible treatment strategies to promote healing. Summary of the Invention

[0004] The present invention aims to provide a self-assembled polypeptide derivative, a hydrogel formulation, a preparation method thereof, and its applications. The self-assembled polypeptide derivative of the present invention can be used to prepare a self-assembled polypeptide derivative hydrogel in which bioactive fragments are fused into a long-lasting and stable material carrier. This hydrogel product exhibits thixotropic and self-healing properties, is resistant to enzymatic hydrolysis, and has long-lasting bioactivity. It can achieve prolonged residence in damaged tissue, thereby improving bioavailability and promoting tissue repair.

[0005] This invention provides a self-assembled polypeptide derivative, wherein the structure of the self-assembled polypeptide derivative is Biotin- D FYIGSSSR, where Biotin is the end cap. D F stands for D-configuration phenylalanine, YIG is the active sequence derived from laminin, and SSSR is the active amino acid sequence of IGF-1.

[0006] The present invention also provides a product prepared based on the self-assembled polypeptide derivative described in the above technical solution, wherein the raw materials for preparation include the self-assembled polypeptide derivative and excipients described in the above technical solution.

[0007] The present invention also provides a hydrogel formulation prepared based on the self-assembled polypeptide derivative described in the above technical solution, wherein the raw materials for preparation include the self-assembled polypeptide derivative described in the above technical solution and excipients; the excipients include buffer solution and pH adjuster.

[0008] Preferably, the buffer solution comprises a PBS solution; and the pH adjuster comprises sodium carbonate.

[0009] The present invention also provides a method for preparing the hydrogel formulation described in the above technical solution, comprising the following steps: mixing the self-assembled polypeptide derivative described in the above technical solution with a buffer solution, adjusting the pH value, heating to dissolve, and cooling to obtain a self-assembled polypeptide derivative hydrogel.

[0010] Preferably, the pH value is 7-8.

[0011] The present invention also provides the application of the self-assembled polypeptide derivatives described in the above technical solutions, or the products described in the above technical solutions, or the hydrogel formulations described in the above technical solutions, or the hydrogel formulations prepared by the preparation methods described in the above technical solutions, in the preparation of tissue engineering repair products.

[0012] The present invention also provides the application of the self-assembled polypeptide derivatives described in the above technical solutions, or the products described in the above technical solutions, or the hydrogel formulations described in the above technical solutions, or the hydrogel formulations prepared by the preparation methods described in the above technical solutions, in the preparation of tissue damage repair drugs.

[0013] Preferably, the tissue damage includes corneal damage.

[0014] Preferably, the corneal injury includes corneal burns, persistent corneal epithelial defects, or keratitis.

[0015] This invention provides a self-assembled polypeptide derivative. This self-assembled polypeptide derivative can be synthesized in one step using a solid-phase synthesis method, resulting in high yield and a simple, rapid preparation process. The self-assembled polypeptide derivative of this invention can be used to prepare self-assembled polypeptide nanohydrogels with a nanoscale fiber network structure; the preparation method is simple and convenient to use. Due to the special physical properties of the self-assembled polypeptide derivative hydrogel, compared with IGF-1 protein solution, the hydrogel of this invention exhibits increased biostability and thixotropic and self-repairing behaviors (overcoming the problem of excessively rapid drug clearance caused by rapid tear turnover on the ocular surface), better resists protease degradation (avoiding excessive proteolysis), significantly prolongs the ocular surface retention time, improves bioavailability, promotes cell proliferation, and thus enhances the therapeutic effect on tissue damage. The self-assembled polypeptide derivative hydrogel of this invention has good biocompatibility and therapeutic effects and is safe to use. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 shows the Biotin- provided by the present invention. D High-resolution mass spectrum of FYIGSSSR; Figure 2 shows the Biotin-YIGSSSR and Biotin- provided by this invention. D High-resolution mass spectra of FYIGSRSS; where A is the result of Biotin-YIGSSSR; B is the result of Biotin- D Figure 3 shows the gelation process provided by the present invention; Figure 4 shows the Biotin- provided by the present invention. D Microscopic morphology of FYIGSSSR; Figure 5 shows the Biotin- provided by this invention. D Circular dichroism chromatogram of FYIGSSSR; Figure 6 shows the stability test results of the protease after treatment provided by the present invention, where A is Biotin- D Figure 7 shows the stability test results of FYIGSSSR; B is the stability test result of IGF-1 protein; Figure 7 shows the stability test results of Biotin- provided by this invention. D Rheological diagrams of FYIGSSSR; where A is the dynamic frequency scan result diagram; B is the step transition result diagram; Figure 8 is the Biotin- provided by the present invention. D Figure 9 shows the biocompatibility results of FYIGSSSR; where A represents the 24h result; B represents the 48h result; and C represents the 72h result. Figure 9 also shows the biotin- provided by this invention. D Figure 10 shows the results of in vivo imaging of the ocular surface of small animals using FYIGSSSR; where A is the in vivo imaging result; B is the relative fluorescence intensity statistics; Figure 11 shows the results of sodium fluorescein staining of corneal wounds provided by the present invention; where A is the staining result; B is the wound healing rate statistics; Figure 12 shows the results of HE staining of corneal tissue provided by the present invention. Detailed Implementation

[0018] This invention provides a self-assembled polypeptide derivative, wherein the structure of the self-assembled polypeptide derivative is Biotin- D FYIGSSSR, where Biotin is the end cap. DF represents D-configuration phenylalanine, YIG is an active sequence derived from laminin, and SSSR (SEQ ID NO. 2) is the active amino acid sequence of IGF-1. SSSR is a sequence obtained based on the C domain of insulin-like growth factor and its role in the self-assembled polypeptide derivative of this invention is to mimic the biological activity of insulin-like growth factor-1 (IGF-1). In the self-assembled polypeptide derivative of this invention, phenylalanine F is D-configuration, and all other amino acids are L-configuration. The amino acid sequence of the self-assembled polypeptide derivative of this invention is FYIGSSSR (SEQ ID NO. 1), where F is D-configuration. This invention does not specifically limit the synthesis method of the self-assembled polypeptide derivative and can use solid-phase synthesis methods for preparation. The self-assembled polypeptide derivative of this invention can self-assemble to form a β-sheet structure, forming a microstructure with a nanofiber network, and forming a hydrogel with viscoelasticity, thixotropic properties, sol-gel transition properties, and variable shear force behavior.

[0019] The present invention also provides a product prepared based on the self-assembled polypeptide derivative described in the above technical solution, wherein the raw materials for preparation include the self-assembled polypeptide derivative and excipients described in the above technical solution.

[0020] This invention also provides a hydrogel formulation prepared based on the self-assembled polypeptide derivative described in the above-described technical solution. The raw materials for preparation include the self-assembled polypeptide derivative and excipients described in the above-described technical solution; the excipients include a buffer solution and a pH adjuster. In a specific embodiment, the buffer solution includes a PBS solution; the pH adjuster includes sodium carbonate. In a specific embodiment, the self-assembled polypeptide derivative can be mixed with a 1×PBS solution to obtain a self-assembled polypeptide derivative solution. By heating and cooling the self-assembled polypeptide derivative solution, a self-assembled polypeptide nanohydrogel with a nanoscale fiber network structure can be obtained. The hydrogel formulation of this invention can supplement growth factors in vivo. Compared with IGF-1 protein, the hydrogel of this invention has good biostability, can better resist the degradation of proteases, and can prolong the retention time on the ocular surface, improve bioavailability, promote cell proliferation, inhibit cell apoptosis, and thus improve the tissue damage repair effect.

[0021] This invention also provides a method for preparing the hydrogel formulation described in the above-mentioned technical solution, comprising the following steps: mixing the self-assembled polypeptide derivative described in the above-mentioned technical solution with a buffer solution, adjusting the pH value, heating to dissolve, and cooling to obtain a self-assembled polypeptide derivative hydrogel. In a specific embodiment, the pH value is 7-8, specifically 7.4. The pH value setting of this invention can promote the dissolution of the self-assembled polypeptide derivative and avoid irritation. The method of this invention can prepare a nano-hydrogel formulation. This invention does not specifically limit the heating temperature and time; heating can be stopped once the self-assembled polypeptide derivative is dissolved. In a specific embodiment, the cooling specifically refers to cooling to room temperature. In a specific embodiment, room temperature refers to 15-30℃, specifically 20-25℃. The hydrogel formulation prepared by this invention has high stability; after vortexing the self-assembled polypeptide derivative hydrogel, a solution is formed, and after standing for 15 minutes, a hydrogel can be formed again.

[0022] The present invention also provides the application of the self-assembled polypeptide derivatives described in the above technical solutions, or the products described in the above technical solutions, or the hydrogel formulations described in the above technical solutions, or the hydrogel formulations prepared by the preparation methods described in the above technical solutions, in the preparation of tissue engineering repair products.

[0023] This invention also provides the application of the self-assembled polypeptide derivatives described in the above-described technical solutions, or the products described in the above-described technical solutions, or the hydrogel formulations prepared by the above-described technical solutions or the preparation methods described in the above-described technical solutions, in the preparation of tissue damage repair drugs. In a specific embodiment, the tissue damage includes corneal damage. In a specific embodiment, the corneal damage includes corneal burns, persistent corneal epithelial defects, or keratitis. In a specific embodiment, the corneal burns include corneal chemical burns.

[0024] In this invention, the method of using the drug (i.e., the method of delivery to the site of tissue damage) can be: dripping the hydrogel onto the site of tissue damage or injecting it into a site adjacent to the damage. When the tissue is corneal tissue, the self-assembled polypeptide derivative hydrogel of this invention can be dripped onto the ocular surface or injected into the conjunctiva.

[0025] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a self-assembled polypeptide derivative, hydrogel formulation, preparation method, and application provided by the present invention, but these should not be construed as limiting the scope of protection of the present invention.

[0026] Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0027] Example 1: Preparation and Synthesis of the Peptide The classic Fmoc-short peptide solid-phase synthesis method was used. The specific steps are as follows: 1) Weigh 0.5 mmol of 2-chlorotriphenylmethyl chloro resin (1.158 mmol / g) into a solid-phase synthesis tube, add 15 mL of anhydrous dichloromethane (DCM), and shake on a shaker for 10 min, shaking once every 2 s, to ensure 100% swelling of the 2-chlorotriphenylmethyl chloro resin; 2) Use a syringe rubber bulb to completely extrude the DCM from the solid-phase synthesis tube containing the 2-chlorotriphenylmethyl chloro resin; 3) Simultaneously, dissolve 0.75 mmol of 9-fluorenylmethoxycarbonyl protecting group (Fmoc) protected R (Arg) (1.5 times the content of 2-chlorotriphenylmethyl chloro resin) in 10 mL of DCM, and add 1.5 mmol of N... 1) Dissolve N-diisopropylethylamine (DIEPA) thoroughly, then add it to the solid-phase synthesis tube extruded from DCM in step 2). Place the tube on a shaker and react at room temperature for 2 hours. 2) Remove the reaction solution from the solid-phase synthesis tube with a syringe rubber bulb, wash three times with 10 mL of DCM for 1 minute each time, and add 16 mL of a solution containing DCM, DIEPA, and methanol (DCM, DIEPA, and methanol volume ratio of 17:1:2) to the DCM-washed solid-phase synthesis tube. Place the tube on a shaker and react at room temperature for 15 minutes to block the unreacted active groups on the 2-chlorotriphenylmethyl chloride resin, thereby improving the yield and purity of the peptide. 3) Remove the reaction solution from the solid-phase synthesis tube in step 4) with a syringe rubber bulb, wash three times with 10 mL of DCM for 1 minute each time, and then wash with 10 mL of DCM for 1 minute each time. 6) Wash three times with N,N-dimethylformamide (DMF) for 1 min each time; 7) Add 15 mL of DMF containing 20% ​​piperidine (volume ratio) to the solid-phase synthesis tube washed with DMF in step 5), place it on a shaker, and react at room temperature for 30 min. Remove the protecting group Fmoc at the N-terminus of the amino acid, and wash five times with 10 mL of DMF for 1 min each time; 8) Dissolve 1 mmol of the next Fmoc-protected amino acid (Ser) in 10 mL of DMF solution, add 2 mmol of DIEPA and 1 mmol of condensing agent HBTU, and after complete dissolution, add it to the solid-phase reaction tube extruded with DMF in step 6), place it on a shaker, and react at room temperature for 2 h. Connect the next amino acid by coupling the C-terminus of the Fmoc-amino acid to the amino acid on the resin or the N-terminus of the polypeptide chain. After the reaction is complete, wash five times with DMF for 1 min each time; 9) Repeat steps (6) and (7) (the order of amino acid connection is Ser, Ser, Gly, Ile, Tyr and DPhe), until the last amino acid is coupled, remove the Fmoc protecting group, and wash; 9) End capping: Dissolve 1 mmol of Biotin in DMF, add 2 mmol of DIEA and 1 mmol of HBTU, mix thoroughly and dissolve, then add to the solid-phase synthesis tube extruded from DMF in 8), place on a shaker, and react at room temperature for 2 h. Couple the N-terminus of the polypeptide chain on the resin through the carboxyl group at the end of Biotin. After the reaction is complete, wash with DMF 5 times, 1 min each time; 10) Prepare 10 mL of cutting solution (95% TFA (trifluoroacetic acid), 2.5% TIS (triisopropylsilane), and 2.5% H2O), place on a shaker and react at room temperature for 1 h, cut the polypeptide derivative from the resin, collect the liquid, remove the solvent with a rotary evaporator, add anhydrous diethyl ether to precipitate and obtain the crude product; 11) Purify and separate the crude polypeptide derivative product using high performance liquid chromatography (HPLC) to obtain Biotin- D FYIGSSSR polypeptide derivative.

[0028] 12) The self-assembled polypeptide derivative powder was obtained by LC-MS detection, and the results are shown in Figure 1. The structural formula of the polypeptide derivative is shown in Formula I.

[0029] , Formula I.

[0030] Comparative Example 1: Compared to Example 1, the polypeptide derivative of Comparative Example 1 does not have the D configuration F: Biotin-YIGSSSR. The amino acid sequence of this derivative is YIGSSSR (SEQ ID NO. 3). The preparation method of the polypeptide derivative of Comparative Example 1 is the same as that of Biotin-YIGSSSR in Example 1. D FYIGSSSR was used to prepare self-assembled polypeptide derivative powder by LC-MS detection, as shown in Figure 2. The structural formula is shown in Formula II. Biotin-YIGSSSR, Formula II.

[0031] Comparative Example 2: Compared to Example 1, the polypeptide derivative of Comparative Example 2 has an SSSR sequence replacement: Biotin- D FYIGSRSS, the amino acid sequence of this derivative is FYIGSRSS (SEQ ID NO.4), where F is the D configuration. The preparation method of the polypeptide derivative in Comparative Example 2 is the same as that of Biotin- in Example 1. D FYIGSSSR was used to prepare self-assembled polypeptide derivative powder by LC-MS detection, as shown in Figure 2. The structural formula is shown in Formula III. Biotin- DFYIGSRSS, Formula III; Comparative Example 3: Weigh 1.8 mg of IGF-1 protein powder (purchased from Beyotime, catalog number P5502), place it in 2 ml, add 500 μl of PBS solution (pH=7.4), and adjust the pH to 7.4 with sodium carbonate solution to obtain an IGF-1 protein solution with a concentration of 2 mg / ml.

[0032] Example 2 Preparation and performance evaluation of self-assembled peptide derivative hydrogels 1. Preparation of self-assembled peptide derivative hydrogels Self-assembled peptide derivative hydrogels were prepared by heating and cooling method 1) The synthesized self-assembled peptide Biotin- D FYIGSSSR (4 mg), Biotin-YIGSSSR (4 mg) in Comparative Example 1, and Biotin-YIGSSSR (4 mg) in Comparative Example 2. D FYIGSRSS (4 mg) was added to 1 mL of 1×PBS solution, and the pH was adjusted by adding 1 mol / L NaHCO3 solution to make the final pH value 7.4, thus obtaining three peptide solutions.

[0033] 2) Heat the obtained polypeptide solution with an alcohol lamp to completely dissolve it, and then let it cool slowly to room temperature.

[0034] Biotin- of Comparative Example 1 was prepared according to the method in Example 1. D FYIGSRSS and Biotin-YIGSSSR of Comparative Example 2 were prepared according to the method in Example 2, and Biotin-YIGSSSR was prepared at a concentration of 4 mg / ml. D Peptide solutions of FYIGSSSR and 4 mg / ml Biotin-YIGSSSR were analyzed. The gelation behavior is shown in Figure 3. Neither Biotin-YIGSSSR nor Biotin-YIGSSSR formed a stable hydrogel; instead, the gel was cloudy, appearing as a cloudy or flocculent precipitate. D FYIGSSSR can form transparent hydrogels, indicating that precise sequence structure is crucial for stable gel-forming properties, and that Biotin- D After being vortexed and dispersed, the FYIGSSSR hydrogel can still return to its colloidal state after stabilization, indicating that it possesses thixotropic and self-healing behavior, which is beneficial for biotin- D FYIGSRSS hydrogel can still spread on the corneal surface when blinking, improving bioavailability.

[0035] 2. Observation by transmission electron microscopy: The microstructure of the hydrogel was observed using a transmission electron microscope by negative staining.

[0036] 1) Use a pipette to take 10 μL of the self-assembled polypeptide derivative hydrogel Biotin- D FYIGSSSR onto a copper grid with a carbon film, let it stand for about 1 to 2 minutes, and then use filter paper to absorb the excess liquid (leaving only a thin layer of solution).

[0037] 2) After staining the hydrogel thin layer with uranyl acetate for 1 min, dry it with absorbent paper.

[0038] 3) Finally, after placing the copper mesh in a dish covered with filter paper and allowing it to dry completely at room temperature, the microstructure of the hydrogel can be observed using a transmission electron microscope.

[0039] As shown in Figure 4, transmission electron microscopy revealed that Biotin- D FYIGSSSR can form nanofibers with a diameter of approximately 9-14 nm, which interweave to form a three-dimensional network structure. Therefore, the self-assembled polypeptide derivative hydrogel provided by this invention can form nanofibers at a certain concentration, macroscopically exhibiting a stable, transparent hydrogel.

[0040] 3. Circular dichroism spectroscopy determination: The secondary structure of the sample is determined using a circular dichroism spectrometer.

[0041] 1) Add 200 μL of hydrogel sample into a 0.1 cm quartz clip, place the clip in the detector, and collect the spectrum in the wavelength range of 185 nm to 280 nm with a step size of 0.5 nm and a period of 0.5 s. The final circular dichroism chromatogram of the sample is obtained after subtracting the background value of the solvent PBS buffer.

[0042] 2) Biotin- self-assembled polypeptide derivative hydrogel D Samples of different concentrations were prepared by gradient dilution of FYIGSSSR (4 mg / ml), and their circular dichroism spectra were measured to detect secondary structures.

[0043] As shown in Figure 5, Biotin- was observed in the CD spectrum. D The FYIGSSSR hydrogel forms a positive peak at 198 nm and a negative peak at 222 nm, respectively, indicating that it adopts a β-sheet secondary structure.

[0044] 4. Biostability assay: The biostability of the self-assembled polypeptide derivative hydrogel and IGF-1 protein was tested by detecting their ability to resist proteinase K in vitro.

[0045] 1) Take a Biotin solution with a concentration of 1.2 mg / ml. DAdd 1 mL of FYIGSSSR, then add 5 μL of 0.128 mg / mL proteinase K solution, and incubate the mixture at 37°C. At different time intervals, aspirate 50 μL of sample and detect the biotin-... D The content of FYIGSSSR.

[0046] 2) Dissolve 7.7 mg of IGF-1 protein in 1 mL of PBS buffer (pH = 7.4), add 5 μL of 0.128 mg / mL proteinase K solution, and incubate at 37°C for 12 h. Take 1 μL of sample at each time point and detect the IGF-1 protein content.

[0047] As shown in Figure 6, IGF-1 protein undergoes rapid degradation within 30 minutes (B in Figure 6), while Biotin- D FYIGSSSR retained approximately 49.1% of its integrity after 6 hours of incubation (Figure 6, A). This superior resistance to enzymatic digestion stems from its D-amino acids and the β-sheet structure formed by self-assembly, which helps maintain its long-lasting activity.

[0048] 5. Rheological testing was performed using an AR2000ex system with a rheometer.

[0049] 1) A self-assembled polypeptide derivative hydrogel, Biotin-, with a concentration of 4 mg / ml was prepared by heating and cooling as described in Example 2. D FYIGSSSR.

[0050] 2) The experiment was conducted at room temperature. First, a 40 mm diameter pressure plate was installed on the instrument, and then the instrument was zeroed and balanced. After adding 1 mL of sample to the rheological tray, the temperature was set to 37℃. Time scans were performed at a constant strain of 1% and a constant frequency of 1 Hz. Then, step strain scans were performed at alternating strains of 0.1% and 100% at a constant frequency of 1 Hz.

[0051] As shown in Figure 7, the time scan (Figure 7A) indicates that with increasing scan time, the storage modulus G′ is dominant over the loss modulus G″, demonstrating that rapid and stable gelation can be achieved. The step strain results (Figure 7B) show that G′ of the hydrogel is greater than G″ at a strain of 0.1%, but significantly lower than G″ when the strain increases to 100%, indicating that the gel state transitions to a quasi-liquid state from a strain of 0.1% to 100%. When the strain returns to 0.1%, G′ immediately recovers to G″, confirming the rapid transition between the sol and gel states when the shear force changes. Biotin- DThe favorable rheological properties exhibited by FYIGSSSR facilitate effective distribution and retention on the ocular surface, indicating its potential to prolong drug residence time in the eye and improve bioavailability.

[0052] Example 3 Biocompatibility of self-assembled polypeptide derivative hydrogels cck-8 experiment 1) Place 96-well plates, pipettes, T25 cell culture flasks and other experimental consumables in a clean bench and sterilize them by UV irradiation for 30 min.

[0053] 2) Subculture corneal epithelial cells into the well plate, approximately 7 × 10⁶ cells per well. 3 A certain number of cells were collected. The 96-well plate was then placed in a cell culture incubator and cultured for 24 hours.

[0054] 3) Add different concentrations of Biotin- into the wells of a 96-well plate. D FYIGSSSR (0~400nM), repeat 6 wells for each group to prevent errors.

[0055] 4) Place the 96-well plate from step 3) back into the cell culture incubator and culture for 24h, 48h and 72h respectively. Then, transfer the 96-well plate to the clean bench, dilute the CCK-8 stock solution and culture medium at a volume ratio of 1:10, replace the culture medium in the well plate, and continue to culture in the cell culture incubator for 2h.

[0056] 5) Remove the 96-well plate from the cell culture incubator, wrap the plate with aluminum foil to protect it from light, and then place the plate in a microplate reader to measure the OD value of the cells at 450 nm. Calculate the average value of each group using 6 replicates for comparison.

[0057] As shown in Figure 8, Biotin- D FYIGSSSR polypeptide derivative hydrogel showed no significant cytotoxicity in corneal epithelial cells after 24h, 48h, and 72h at concentrations not exceeding 400 nM, indicating good cell compatibility. Furthermore, within the dosage range, the hydrogel's cell proliferation-promoting effect exhibited a bell-shaped curve, with the most significant proliferation effect observed at a concentration of 50 nM.

[0058] Example 4: Self-assembled polypeptide derivative hydrogel prolongs ocular surface retention for in vivo imaging in small animals. 1) Nine rats were randomly divided into two groups: IGF-1 protein solution group and Biotin- D In the FYIGSSSR hydrogel group, each rat was tested and observed using only its right eye.

[0059] 2) Take 10 μL of IGF-1 solution (10 ng / mL) loaded with Rhodamine B solution and 10 μL of Biotin-1 solution loaded with Rhodamine B solution. DFYIGSSSR hydrogel (4 mg / ml) was instilled into the conjunctival sac of the right eye of rats in each group.

[0060] 3) IVIS software assessment of corneal retention time: At predetermined time intervals (0 min, 1 min, 3 min, 10 min, 20 min and 30 min), in vivo animal image signals were acquired to observe the retention of ocular drops on the ocular surface.

[0061] As shown in Figure 9, this invention employs an in vivo imaging system (IVIS) with rhodamine (RhB) as a fluorescent marker for analysis. The results show that biotin- D The mean fluorescence intensity (MFI) of the FYIGSSSR hydrogel group remained at approximately 54.0% after 30 minutes, while that of the conventional IGF-1 eye drops decreased rapidly. Note: Compared to IGF-1 solution, Biotin- D FYIGSSSR hydrogel effectively resists shear stress and tear clearance caused by blinking, exhibiting prolonged ocular retention and providing continuous biostimulation in the corneal microenvironment.

[0062] Example 5: Self-assembled peptide derivative hydrogel promotes corneal injury repair. 1. Establishment of a rat model of persistent corneal epithelial defect: 1) Mark the central region of the rat cornea with a 6 mm trephine. Remove the epithelial layer using an Algerbrush-II. Cleanse the injured cornea with PBS and remove any tissue debris with a cotton swab.

[0063] 2) Rats were anesthetized by intraperitoneal injection of sodium pentobarbital solution (70 mg / kg) combined with topical anesthesia using promecaine hydrochloride eye drops. After anesthesia, the rat's cornea could be gently touched with a cotton swab. If the rat did not scratch or struggle, surgery could be performed.

[0064] 3) The model was created using only the right eye of each rat, and subsequent procedures were performed under a stereomicroscope. After marking the extent of corneal damage in the rat's eye with a 6mm trephine, all corneal epithelium within the marked area was scraped off with an AlgerBrush II.

[0065] 4) To prevent corneal infection in rats, tobramycin eye drops should be used after modeling. Analgesics can be used to relieve postoperative pain in rats and prevent them from scratching their eyes and causing secondary damage. After modeling, rats should be randomly divided into groups and treated according to their groups.

[0066] 5) Rats were randomly divided into three groups (n=3): the first group received PBS as a control; the second group received IGF-1 protein solution (10 ng / ml) as eye drops; and the third group received Biotin- D FYIGSSSR eye drops (4 mg / ml). All three groups were treated with eye drops twice daily.

[0067] 2. Slit-lamp observation of corneal wounds 1) First, anesthetize the rats using the same anesthesia method as described above.

[0068] 2) Observe and photograph the ocular surface condition of rats using diffuse illumination (inflammation, corneal condition, anterior chamber angle, and aqueous humor, etc.). Turn off the ambient light, pipette 5 μL of sodium fluorescein working solution to stain the rat cornea, and observe the epithelial healing under cobalt blue light, taking photographs as needed. Proficiency in photographing is required to ensure clear images. When taking bright-field photographs, if there are secretions or impurities on the rat corneal surface, gently moisten the ocular surface with sterile saline and use a cotton swab to help open and close the rat's eyelids to remove the foreign objects.

[0069] 3) Subsequently, ImageJ software was used to analyze and calculate the area of ​​the corneal defect region (i.e., the green-stained area) in the photograph. The results obtained represent the corneal epithelial healing speed and healing status in rats.

[0070] As shown in Figure 10, 72 hours post-injury, the IGF-1 group and the Biotin- D In the FYIGSSSR hydrogel group, the corneal wound was almost entirely covered by regenerated epithelium, while in the PBS control group, obvious defects were still visible. Quantitative results showed that twice-daily administration of IGF-1 solution and Biotin- D Both FYIGSSSR hydrogel and IGF-1 group significantly promoted healing, with healing rates of 100% and 98.23±0.64%, respectively, significantly higher than the PBS control group (91.52±2.38%), indicating that both can effectively accelerate corneal epithelial wound healing. Although there was no statistically significant difference in the final wound area between the IGF-1 group and the hydrogel group at 72 hours, the hydrogel group showed a better repair trend at 48 hours post-injury, with a faster wound closure rate than the IGF-1 group.

[0071] 3. Potential of self-assembled polypeptide derivative hydrogels to promote corneal epithelial defect repair. Hematoxylin-Eosin (HE) staining 1) Sample collection: According to the corresponding time point of observation (day 3), rats were euthanized by cervical dislocation. Immediately after euthanasia, the eyeballs were removed using sterile ophthalmic surgical instruments and the eyeballs were repeatedly rinsed with sterile physiological saline to remove blood and animal hair.

[0072] 2) Fixation: To maintain the spherical shape of the rat eyeball after fixation, use a sterile 2.5mL syringe needle to make 1-2 holes in the cleaned rat eyeball, taking care not to poke the cornea, and soak it in 10% formalin at 4℃ for 24 hours.

[0073] 3) Dehydration: Rat eyeballs were grouped and placed in disposable tissue section embedding cassettes, sealed tightly, and marked with a pencil. They were then immersed in the following solutions in sequence at room temperature for gradient dehydration: 50% (v / v) ethanol (30 min), 70% (v / v) ethanol (30 min), 80% (v / v) ethanol (30 min), 90% (v / v) ethanol (overnight), 95% (v / v) ethanol I (30 min), 95% (v / v) ethanol II (40 min), anhydrous ethanol I (10 min), anhydrous ethanol II (20 min), anhydrous ethanol + xylene (1:1 mixture, 30 min), xylene I (15 min), xylene II (15 min).

[0074] 4) Paraffin Impregnation (60℃ paraffin): The dehydrated rat eyeballs were impregnated with paraffin in the following solutions: xylene + paraffin (1:1 mixture, 30 min), paraffin I (30 min), paraffin II (30 min), paraffin III (30 min). Gloves should be worn during the operation, and the embedding cassette should be handled with tweezers to avoid burns.

[0075] 5) Embedding: After being fully impregnated with wax, the rat eyeballs are embedded to form wax blocks. Each wax block contains only one eyeball, and the rat eyeballs need to be placed in the sagittal position.

[0076] 6) Sectioning: Place the wax block in a paraffin microtome, set the coarse cut to 20μm and the section thickness to 4μm, and then section. After spreading and mounting the slides, dry them. It is important to note that during embedding, the eyeball must be properly positioned to resemble its anatomical orientation; otherwise, the sectioning direction will be affected, resulting in poor section quality.

[0077] 7) Staining: Arrange the dried tissue sections neatly on the slide holder. First, dewax the sections. The soaking order is: xylene I (10 min) — xylene II (2 min) — anhydrous ethanol + xylene (1:1, 5 min) — anhydrous ethanol (2 min) — 90% ethanol (2 min) — 80% ethanol (2 min) — 70% ethanol (2 min). Gently rinse the sections with distilled water for 2 min. The HE staining soaking order is: hematoxylin (3~8 min) — wash away excess stain with distilled water — 1% hydrochloric acid alcohol differentiation (2 min) — rinse with tap water — 0.6% lithium carbonate for blueing (2 min) — rinse with deionized water — eosin (1~3 min). Then dehydrate — 95% ethanol I (2 min) — 95% ethanol II (2 min) — anhydrous ethanol I (2 min) — anhydrous ethanol + xylene (1:1, 5 min) — xylene I (5 min) — xylene II (5 min).

[0078] 8) Mounting and microscopic observation: After HE staining, the slides can be mounted with neutral resin after the xylene has dried.

[0079] As shown in Figure 11, the corneal tissue sections of each group were analyzed by HE staining in this invention. Although the PBS control group showed some wound healing ability, its corneal epithelial cells still failed to completely cover the wound by the 3rd day after injury. In contrast, the IGF-1 group and Biotin- D All FYIGSSSR hydrogel groups achieved corneal wound closure within 72 hours post-injury. However, further histological observation revealed differences in corneal epithelial morphological reconstruction among the different treatment groups; Biotin- D The cornea treated with FYIGSSSR hydrogel not only achieved re-epithelialization, but the thickness of the regenerated corneal epithelium was also significantly greater than that of the IGF-1 group, and it exhibited a more obvious layered structure.

[0080] This invention provides a polypeptide derivative hydrogel with a structure of Biotin- D FYIGSSSR. This hydrogel, constructed by integrating laminin and insulin-like growth factor-1 (IGF-1) derived functional motifs, can spontaneously assemble into a nanofiber network with a β-sheet secondary structure. These results demonstrate that the hydrogel exhibits tunable viscoelastic and thixotropic behavior, maintaining structural stability and prolonging retention time under tear shear stress on the ocular surface, while effectively resisting enzymatic degradation. These properties significantly enhance the retention and local bioavailability of drugs or active ingredients on the ocular surface, thereby promoting corneal epithelial re-epithelialization.

[0081] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A self-assembled polypeptide derivative, characterized in that, The structure of the self-assembled polypeptide derivative is Biotin- D FYIGSSSR, where Biotin is the end cap. D F stands for D-configuration phenylalanine, YIG is the active sequence derived from laminin, and SSSR is the active amino acid sequence of IGF-1.

2. A product prepared based on the self-assembled polypeptide derivative of claim 1, characterized in that, The raw materials used in the preparation include the self-assembled polypeptide derivative and excipients as described in claim 1.

3. A hydrogel formulation prepared based on the self-assembled polypeptide derivative of claim 1, characterized in that, The raw materials used in the preparation include the self-assembled polypeptide derivative of claim 1 and excipients; the excipients include buffer solutions and pH adjusters.

4. The hydrogel formulation according to claim 3, characterized in that, The buffer solution includes a PBS solution; the pH adjuster includes sodium carbonate.

5. The method for preparing the hydrogel formulation according to claim 3 or 4, characterized in that, The process includes the following steps: mixing the self-assembled polypeptide derivative of claim 1 with a buffer solution, adjusting the pH value, heating to dissolve, and cooling to obtain a self-assembled polypeptide derivative hydrogel.

6. The preparation method according to claim 5, characterized in that, The pH value is 7-8.

7. The application of the self-assembled polypeptide derivative of claim 1, the product of claim 2, the hydrogel formulation of claim 3 or 4, or the hydrogel formulation prepared by the preparation method of claim 5 or 6 in the preparation of tissue engineering repair products.

8. The use of the self-assembled polypeptide derivative of claim 1, the product of claim 2, the hydrogel formulation of claim 3 or 4, or the hydrogel formulation prepared by the preparation method of claim 5 or 6 in the preparation of tissue damage repair drugs.

9. The application according to claim 8, characterized in that, The tissue damage includes corneal damage.

10. The application according to claim 9, characterized in that, The corneal injury includes corneal burns, persistent corneal epithelial defects, or keratitis.

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