Polypeptide hydrogel with adjustable stress relaxation and preparation method thereof

Abstract

The invention discloses polypeptide hydrogel with adjustable stress relaxation and a preparation method thereof, and belongs to the technical field of hydrogel. The preparation method provided by the invention comprises the following steps: connecting amino acids to a polypeptide chain one by one, removing a side chain protecting group from the synthesized polypeptide through cracking, releasing the polypeptide from solid-phase resin to obtain high-purity free polypeptide, and purifying to obtain a pure polypeptide product. And finally, reacting the polypeptide pure product with maleimide functionalized star-shaped PEG to obtain a polypeptide polymer, dissolving the polypeptide polymer in water, and adjusting the pH value to obtain the polypeptide hydrogel with ultrafast relaxation time and adjustable stress relaxation. The fastest relaxation time of the hydrogel provided by the invention can reach 0.22 s, and the hydrogel utilizes the natural biodegradability and low immunogenicity of the polypeptide, so that the biocompatibility of the material is improved while the ultrafast stress relaxation characteristic is maintained, and the hydrogel has excellent biomedical prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogels, and in particular relates to a polypeptide hydrogel with adjustable stress relaxation and a preparation method thereof. Background Art

[0002] Hydrogel is a three-dimensional network material formed by hydrophilic polymers through physical or chemical cross-linking, which can absorb and lock a large amount of water while keeping the solid structure insoluble. It is soft and elastic, has excellent biocompatibility and controllability, and is widely used in biomedical fields (such as wound dressings, drug sustained release, tissue engineering), environmental protection (wastewater treatment, agricultural water conservation) and flexible electronic devices.

[0003] Relaxation time is the time required for a material to recover from a non-equilibrium state to an equilibrium state. For hydrogels, a fast (short) relaxation time means that its internal cross-linked network or molecular chains can rearrange quickly, thereby quickly releasing stress or adapting to deformation. This type of hydrogel is usually constructed by introducing dynamic reversible bonds (such as hydrogen bonds, ionic bonds, host-guest interactions, or dynamic covalent bonds), and its network can be quickly reconstructed after damage, giving the material self-healing and energy dissipation capabilities similar to biological tissues. This property makes it outstanding in dynamic biological environments, such as promoting cell migration and tissue regeneration, adapting to irregular tissue interfaces (such as nerve or muscle repair), and serving as a fatigue-resistant interface material for flexible electronic devices.

[0004] At present, hydrogels with faster relaxation times still have the following problems: (1) The relaxation time is generally more than 10 seconds. For example, the relaxation time of physically cross-linked hyaluronic acid hydrogel is about a few seconds to minutes; the relaxation time of chemically cross-linked PAAm hydrogel can reach tens of minutes to several hours.

[0005] (2) The stress relaxation rate regulation range is limited, and it is difficult to achieve in-situ control of the stress relaxation rate.

[0006] (3) Poor biocompatibility hinders its biomedical application. Summary of the invention

[0007] In view of the above-mentioned prior art, the present invention provides a polypeptide hydrogel with adjustable stress relaxation and a preparation method thereof, which solves the problems of slow relaxation time, limited range of stress relaxation rate regulation and poor biocompatibility of the prior art hydrogel.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a method for preparing a polypeptide hydrogel with adjustable stress relaxation, comprising the following steps: S1: dissolving amino acid powder in DMF to obtain an amino acid solution, and then synthesizing the amino acid solution, a deprotecting agent, a reaction condensing agent and a DMF-swollen carrier resin through a synthesis instrument to obtain a resin containing a polypeptide chain; S2: washing and drying the resin containing the polypeptide chain to obtain a resin without impurities; S3: Place the impurity-free resin in the lysate for 2-3 hours, filter the lysate, add 10 times the volume of icy ether to the filtrate, centrifuge at 8000 rpm for 5 minutes, discard the supernatant, repeat 3 times, and then air dry the precipitate for 20-30 hours to obtain the crude polypeptide product; S4: Purify the crude polypeptide product by HPLC to obtain a pure polypeptide product; S5: The pure polypeptide product and maleimide-functionalized star-shaped PEG were mixed in a molar ratio of 3-5:1, and then added to a 95% DMF aqueous solution for reaction for 24 hours, and the pH during the reaction was adjusted to 7-7.5. After the reaction, the DMF was removed by dialysis to obtain a polymer solution, and the polymer solution was lyophilized to obtain a polypeptide polymer; S6: Dissolve the polypeptide polymer in water and adjust the pH to 7-8 to obtain a polypeptide hydrogel with controllable stress relaxation.

[0009] The beneficial effects of the present invention are as follows: the present invention selects Rink Amide resin for the amidation of the C-terminus of the polypeptide, and during the synthesis, amino acids are connected to the polypeptide chain one by one by repeating the deprotection and coupling steps, and then the synthesized polypeptide is released from the solid phase resin by removing the side chain protecting groups through cleavage, thereby obtaining a high-purity free polypeptide, and then purifying to obtain a pure polypeptide product, and finally the pure polypeptide product is reacted with a maleimide-functionalized star-shaped PEG to obtain a polypeptide polymer, and the polypeptide polymer is dissolved in water, and the pH is adjusted to obtain a polypeptide hydrogel with an ultrafast relaxation time and adjustable. The method provided by the present invention utilizes the natural biodegradability and low immunogenicity of the polypeptide (the polypeptide is modified with polyethylene glycol to obtain the polypeptide polymer, and the polyethylene glycol also has excellent biocompatibility), and while maintaining the ultrafast stress relaxation characteristics, it has good biocompatibility, so that the finally obtained polypeptide hydrogel has excellent biomedical prospects.

[0010] Based on the above technical solution, the present invention can also be improved as follows.

[0011] Furthermore, the concentration of the amino acid solution is 0.15~0.25M.

[0012] Furthermore, the deprotecting agent is a piperidine solution obtained by dissolving hexahydropyridine in DMF at a volume concentration of 20%, and the reaction condensation agent is N,N-diisopropylcarbodiimide or ethyl 2-oximecyanoacetate.

[0013] The beneficial effects of adopting the further technical scheme are as follows: hexahydropyridine as an alkaline deprotecting agent can effectively neutralize the Fmoc group and make it leave the amino acid, thereby exposing the amino acid at the N-terminus, which is convenient for the subsequent condensation reaction; N, N-diisopropylcarbodiimide can effectively activate the carboxylic acid group of the amino acid to form an activated carboxylic acid derivative, making it easier for the amino group of another amino acid to react with it to form a peptide bond, and has low reactivity to the side chain of the amino acid, which means that in the condensation process, it mainly acts between the carboxylic acid and the amino group, reducing unnecessary side reactions, and ethyl 2-oxime cyanoacetate can promote the bonding reaction between amino acids under mild conditions, with a fast reaction speed, high yield, and good selectivity for amino acids.

[0014] Furthermore, the carrier resin is Rink Amide resin.

[0015] Furthermore, the washing in S2 is performed by washing with DMF three times and then washing with dichloromethane twice.

[0016] Furthermore, the cleavage solution is a mixture of trifluoroacetic acid, triisopropylsilane, water and ethanedithiol in a volume ratio of 90-95:2-3:2-3:2-3.

[0017] The beneficial effect of adopting the further technical scheme is: trifluoroacetic acid is used to break the connection bond between the polypeptide and the resin and remove the side chain protecting group, triisopropylsilane is used to capture the cationic byproducts generated during the cleavage process to prevent polypeptide modification, and ethanedithiol is used to protect easily oxidized amino acids such as Cys to prevent side reactions. The lysis solution obtained by mixing these three reagents with water in proportion can remove the side chain protecting groups of the synthesized polypeptide and release it from the solid phase resin, thereby obtaining a high-purity free polypeptide.

[0018] Furthermore, the phase A used in HPLC was 0.1% TFA aqueous solution, the phase B was 0.1% TFA acetonitrile solution, the flow rate was 10 mL / min, and the wavelengths of the dual-channel detector were 220 nm and 274 nm.

[0019] The beneficial effect of adopting the further technical solution is: reducing impurities and by-products and obtaining a high-purity polypeptide product.

[0020] Furthermore, the solid-liquid ratio of the mixture of the pure polypeptide product and the maleimide-functionalized star-shaped PEG to DMF is 10 mg: 0.5~1.5 mL.

[0021] The beneficial effect of adopting a further technical solution is that maleimide and thiol (-SH) can undergo a nucleophilic addition reaction under mild conditions to generate a stable thioether bond (-S-), and finally obtain a star-shaped polypeptide polymer.

[0022] The invention also provides a preparation method of the stress-relaxation-regulatable polypeptide hydrogel and the prepared stress-relaxation-regulatable polypeptide hydrogel.

[0023] The beneficial effects of the present invention are as follows: the polypeptide hydrogel provided by the present invention shows a gradually changing stress relaxation rate in situ, with the fastest relaxation time even being only 0.22 s; the viscoelasticity of the material changes with the pH value, from brittle quasi-elasticity to plastic viscoelasticity; and the hydrogel utilizes the natural biodegradability and low immunogenicity of the polypeptide, and has good biocompatibility while maintaining ultrafast stress relaxation properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the frequency scanning diagram of the polypeptide hydrogel in Example 1; Figure 2 It is a stress-relaxation curve diagram of Examples 2-3 and Comparative Examples 1-2; Figure 3 It is a statistical diagram of the survival rate of L929 cells; Figure 4 Live-dead staining images of L929 cells; Figure 5 Staining diagram for osteogenic differentiation of cells; Figure 6 It is a diagram of cell osteogenic differentiation activity; Figure 7 Staining diagram for chondrogenic differentiation of cells; Figure 8 This is a diagram of cell chondrogenic differentiation activity; Fig. 9 This is a self-healing experiment diagram of the polypeptide hydrogel in Comparative Example 1; Fig.10 This is a self-healing experiment diagram of the polypeptide hydrogel in Example 1. DETAILED DESCRIPTION

[0025] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0026] Example 1 A method for preparing a polypeptide hydrogel with adjustable stress relaxation comprises the following steps: S1: Dissolve the amino acid powder in DMF to prepare an amino acid solution with a concentration of 0.2M. The deprotecting agent is a piperidine solution, which is obtained by dissolving hexahydropyridine in DMF at a concentration of 20% (v / v%); the reaction condensation agent includes N, N-diisopropylcarbodiimide (DIC, dissolved in DMF at a concentration of 7.8% (v / %)) and 2-oxime cyanoacetic acid ethyl (Oxyma, 14.21g of Oyxma powder dissolved in 100mL of DMF); add the main solvent DMF to the volume required by the experiment, and finally install these solvent bottles on an automatic continuous microwave peptide synthesizer; use DMF to completely swell the Rink Amide resin, and then use the automatic continuous microwave peptide synthesizer to synthesize to obtain a resin containing a polypeptide chain; S2: Wash the resin containing the polypeptide chain with DMF for 3 times, and then with dichloromethane for 2 times. After washing, use a vacuum pump to dry the product to obtain a resin without impurities. S3: Place the impurity-free resin in a lysis solution (trifluoroacetic acid, triisopropylsilane, water and ethanedithiol mixed in a volume ratio of 92.5:2.5:2.5:2.5) for 2.5 hours (the material ratio of impurity-free resin to lysis solution is 1 g:10 mL), filter the lysis solution, add 10 times the volume of filtrate ice ether to the filtrate, mix by vortexing and ultrasonication, and centrifuge at a speed of 8000 rpm for 5 minutes. After the centrifugation, discard the supernatant, repeat the ice ether dissolution, centrifugation and discard the supernatant 3 times, and then ventilate and dry the obtained precipitate for 24 hours to obtain a crude polypeptide product; S4: The crude polypeptide product was purified by HPLC, using a reverse phase C18 column in HPLC, the system was set to 0.1% TFA aqueous solution for phase A, 0.1% TFA acetonitrile solution for phase B, the flow rate was 10 mL / min, and the wavelengths of the dual-channel detector were 220 nm and 274 nm; the crude polypeptide product was dissolved in a AB phase mixed solution (40% A, 60% B) at a concentration of 20 mg / ml, filtered with a 0.22 μm filter membrane to remove insoluble matter and then entered the chromatographic column, first using the initial mobile phase (90% A phase + 10% B phase) to run for 10 min until the baseline was stable to balance the chromatographic column, then phase B was continued at 10% volume for 2 min, and the volume was linearly increased from 10% to 90% for 30 min, and the eluent was collected when the target peak appeared, the eluent was removed of the organic solvent using a rotary evaporator and then rapidly frozen using liquid nitrogen, and finally placed in a vacuum freeze dryer to freeze-dry to obtain a pure polypeptide product; S5: The pure polypeptide product and maleimide-functionalized star-shaped PEG were mixed at a molar ratio of 4:1, and then added to a 95% DMF aqueous solution for reaction for 24 hours (the material-liquid ratio of the mixture to the DMF solution was 10 mg:1 mL), the pH during the reaction was adjusted to 7.5, and after the reaction was completed, the DMF was dialyzed to obtain a polymer solution, and the polymer solution was freeze-dried using a vacuum freeze dryer to obtain a polypeptide polymer; S6: The polypeptide polymer is dissolved in water, fully dissolved by vortexing and ultrasound, and the pH is adjusted to 8 to obtain the stress relaxation controllable polypeptide hydrogel 8-gel.

[0027] Example 2 A method for preparing a polypeptide hydrogel with adjustable stress relaxation comprises the following steps: S1: Dissolve the amino acid powder in DMF to prepare an amino acid solution with a concentration of 0.15M. The deprotecting agent is a piperidine solution, which is obtained by dissolving hexahydropyridine in DMF at a concentration of 20% (v / v%); the reaction condensation agent includes N, N-diisopropylcarbodiimide (DIC, dissolved in DMF at a concentration of 7.8% (v / %)) and 2-oxime cyanoacetic acid ethyl (Oxyma, 14.21g of Oyxma powder dissolved in 100mL of DMF); add the main solvent DMF to the volume required by the experiment, and finally install these solvent bottles on an automatic continuous microwave peptide synthesizer; use DMF to completely swell the Rink Amide resin, and then use the automatic continuous microwave peptide synthesizer to synthesize to obtain a resin containing a polypeptide chain; S2: Wash the resin containing the polypeptide chain with DMF for 3 times, and then with dichloromethane for 2 times. After washing, use a vacuum pump to dry the product to obtain a resin without impurities. S3: Place the impurity-free resin in a lysis solution (trifluoroacetic acid, triisopropylsilane, water and ethanedithiol mixed in a volume ratio of 90:3:2:2) for 2.5 hours (the material ratio of impurity-free resin to lysis solution is 1g:5mL), filter the lysis solution, add 10 times the volume of the filtrate of ice ether to the filtrate, mix by vortexing and ultrasonication, and centrifuge at a speed of 8000rpm for 5 minutes. After the centrifugation, discard the supernatant, repeat the dissolution with ice ether, centrifugation and discarding the supernatant 3 times, and then ventilate and dry the obtained precipitate for 30 hours to obtain a crude polypeptide product; S4: The crude polypeptide product was purified by HPLC, using a reverse phase C18 column in HPLC, the system was set to 0.1% TFA aqueous solution for phase A, 0.1% TFA acetonitrile solution for phase B, the flow rate was 10 mL / min, and the wavelengths of the dual-channel detector were 220 nm and 274 nm; the crude polypeptide product was dissolved in a AB phase mixed solution (40% A, 60% B) at a concentration of 20 mg / ml, filtered with a 0.22 μm filter membrane to remove insoluble matter and then entered the chromatographic column, first using the initial mobile phase (90% A phase + 10% B phase) to run for 10 min until the baseline was stable to balance the chromatographic column, then phase B was continued at 10% volume for 2 min, and the volume was linearly increased from 10% to 90% for 30 min, and the eluent was collected when the target peak appeared, the eluent was removed of the organic solvent using a rotary evaporator and then rapidly frozen using liquid nitrogen, and finally placed in a vacuum freeze dryer to freeze-dry to obtain a pure polypeptide product; S5: The pure polypeptide product and maleimide-functionalized star-shaped PEG were mixed at a molar ratio of 3:1, and then added to a 95% DMF aqueous solution for reaction for 24 hours (the material-liquid ratio of the mixture to the DMF solution was 10 mg:0.5 mL), and the pH during the reaction was adjusted to 7. After the reaction was completed, the DMF was dialyzed to obtain a polymer solution, and the polymer solution was freeze-dried using a vacuum freeze dryer to obtain a polypeptide polymer; S6: Dissolve the polypeptide polymer in water, fully dissolve it through vortex ultrasound, and adjust the pH to 7 to obtain the stress relaxation controllable polypeptide hydrogel 7-gel.

[0028] Example 3 A method for preparing a polypeptide hydrogel with adjustable stress relaxation comprises the following steps: S1: Dissolve the amino acid powder in DMF to prepare an amino acid solution with a concentration of 0.25M. The deprotecting agent is a piperidine solution, which is obtained by dissolving hexahydropyridine in DMF at a concentration of 20% (v / v%); the reaction condensation agent includes N, N-diisopropylcarbodiimide (DIC, dissolved in DMF at a concentration of 7.8% (v / %)) and 2-oxime cyanoacetic acid ethyl (Oxyma, 14.21g of Oyxma powder dissolved in 100mL of DMF); add the main solvent DMF to the volume required by the experiment, and finally install these solvent bottles on an automatic continuous microwave peptide synthesizer; use DMF to completely swell the Rink Amide resin, and then use the automatic continuous microwave peptide synthesizer to synthesize to obtain a resin containing a polypeptide chain; S2: Wash the resin containing the polypeptide chain with DMF for 3 times, and then with dichloromethane for 2 times. After washing, use a vacuum pump to dry the product to obtain a resin without impurities. S3: Place the impurity-free resin in a lysis solution (trifluoroacetic acid, triisopropylsilane, water and ethanedithiol mixed in a volume ratio of 95:3:3:2) for 2.5 hours (the material ratio of impurity-free resin to lysis solution is 1g:15mL), filter the lysis solution, add 10 times the volume of the filtrate of ice ether to the filtrate, mix by vortexing and ultrasonication, and centrifuge at a speed of 8000rpm for 5 minutes. After the centrifugation, discard the supernatant, repeat the dissolution in ice ether, centrifugation and discarding the supernatant 3 times, and then ventilate and dry the obtained precipitate for 20 hours to obtain a crude polypeptide product; S4: The crude polypeptide product was purified by HPLC, using a reverse phase C18 column in HPLC, the system was set to 0.1% TFA aqueous solution for phase A, 0.1% TFA acetonitrile solution for phase B, the flow rate was 10 mL / min, and the wavelengths of the dual-channel detector were 220 nm and 274 nm; the crude polypeptide product was dissolved in a AB phase mixed solution (40% A, 60% B) at a concentration of 20 mg / ml, filtered with a 0.22 μm filter membrane to remove insoluble matter and then entered the chromatographic column, first using the initial mobile phase (90% A phase + 10% B phase) to run for 10 min until the baseline was stable to balance the chromatographic column, then phase B was continued at 10% volume for 2 min, and the volume was linearly increased from 10% to 90% for 30 min, and the eluent was collected when the target peak appeared, the eluent was removed of the organic solvent using a rotary evaporator and then rapidly frozen using liquid nitrogen, and finally placed in a vacuum freeze dryer to freeze-dry to obtain a pure polypeptide product; S5: The pure polypeptide product and maleimide-functionalized star-shaped PEG were mixed at a molar ratio of 5:1, and then added to a 95% DMF aqueous solution for reaction for 24 hours (the material-liquid ratio of the mixture to the DMF solution was 10 mg:1.5 mL). The pH during the reaction was adjusted to 7.5. After the reaction was completed, the DMF was removed by dialysis to obtain a polymer solution. The polymer solution was freeze-dried using a vacuum freeze dryer to obtain a polypeptide polymer; S6: The polypeptide polymer is dissolved in water, fully dissolved by vortexing and ultrasound, and the pH is adjusted to 7.5 to obtain a stress relaxation controllable polypeptide hydrogel 7.5-gel.

[0029] Comparative Example 1 A method for preparing a polypeptide hydrogel comprises the following steps: S1: Dissolve the amino acid powder in DMF to prepare an amino acid solution with a concentration of 0.2M. The deprotecting agent is a piperidine solution, which is obtained by dissolving hexahydropyridine in DMF at a concentration of 20% (v / v%); the reaction condensation agent includes N, N-diisopropylcarbodiimide (DIC, dissolved in DMF at a concentration of 7.8% (v / %)) and 2-oxime cyanoacetic acid ethyl (Oxyma, 14.21g of Oyxma powder dissolved in 100mL of DMF); add the main solvent DMF to the volume required by the experiment, and finally install these solvent bottles on an automatic continuous microwave peptide synthesizer; use DMF to completely swell the Rink Amide resin, and then use the automatic continuous microwave peptide synthesizer to synthesize to obtain a resin containing a polypeptide chain; S2: Wash the resin containing the polypeptide chain with DMF for 3 times, and then with dichloromethane for 2 times. After washing, use a vacuum pump to dry the product to obtain a resin without impurities. S3: Place the impurity-free resin in a lysis solution (trifluoroacetic acid, triisopropylsilane, water and ethanedithiol mixed in a volume ratio of 92.5:2.5:2.5:2.5) for 2.5 hours (the material ratio of impurity-free resin to lysis solution is 1 g:10 mL), filter the lysis solution, add 10 times the volume of filtrate ice ether to the filtrate, mix by vortexing and ultrasonication, and centrifuge at a speed of 8000 rpm for 5 minutes. After the centrifugation, discard the supernatant, repeat the ice ether dissolution, centrifugation and discard the supernatant 3 times, and then ventilate and dry the obtained precipitate for 24 hours to obtain a crude polypeptide product; S4: The crude polypeptide product was purified by HPLC, using a reverse phase C18 column in HPLC, the system was set to 0.1% TFA aqueous solution for phase A, 0.1% TFA acetonitrile solution for phase B, the flow rate was 10 mL / min, and the wavelengths of the dual-channel detector were 220 nm and 274 nm; the crude polypeptide product was dissolved in a AB phase mixed solution (40% A, 60% B) at a concentration of 20 mg / ml, filtered with a 0.22 μm filter membrane to remove insoluble matter and then entered the chromatographic column, first using the initial mobile phase (90% A phase + 10% B phase) to run for 10 min until the baseline was stable to balance the chromatographic column, then phase B was continued at 10% volume for 2 min, and the volume was linearly increased from 10% to 90% for 30 min, and the eluent was collected when the target peak appeared, the eluent was removed of the organic solvent using a rotary evaporator and then rapidly frozen using liquid nitrogen, and finally placed in a vacuum freeze dryer to freeze-dry to obtain a pure polypeptide product; S5: The pure polypeptide product and maleimide-functionalized star-shaped PEG were mixed at a molar ratio of 4:1, and then added to a 95% DMF aqueous solution for reaction for 24 hours (the material-liquid ratio of the mixture to the DMF solution was 10 mg:1 mL), the pH during the reaction was adjusted to 7.5, and after the reaction was completed, the DMF was dialyzed to obtain a polymer solution, and the polymer solution was freeze-dried using a vacuum freeze dryer to obtain a polypeptide polymer; S6: Dissolve the polypeptide polymer in water, fully dissolve it through vortexing and ultrasound, and adjust the pH to 6 to obtain polypeptide hydrogel 6-gel.

[0030] Comparative Example 2 A method for preparing a polypeptide hydrogel comprises the following steps: S1: Dissolve the amino acid powder in DMF to prepare an amino acid solution with a concentration of 0.2M. The deprotecting agent is a piperidine solution, which is obtained by dissolving hexahydropyridine in DMF at a concentration of 20% (v / v%); the reaction condensation agent includes N, N-diisopropylcarbodiimide (DIC, dissolved in DMF at a concentration of 7.8% (v / %)) and 2-oxime cyanoacetic acid ethyl (Oxyma, 14.21g of Oyxma powder dissolved in 100mL of DMF); add the main solvent DMF to the volume required by the experiment, and finally install these solvent bottles on an automatic continuous microwave peptide synthesizer; use DMF to completely swell the Rink Amide resin, and then use the automatic continuous microwave peptide synthesizer to synthesize to obtain a resin containing a polypeptide chain; S2: Wash the resin containing the polypeptide chain with DMF for 3 times, and then with dichloromethane for 2 times. After washing, use a vacuum pump to dry the product to obtain a resin without impurities. S3: Place the impurity-free resin in a lysis solution (trifluoroacetic acid, triisopropylsilane, water and ethanedithiol mixed in a volume ratio of 92.5:2.5:2.5:2.5) for 2.5 hours (the material ratio of impurity-free resin to lysis solution is 1 g:10 mL), filter the lysis solution, add 10 times the volume of filtrate ice ether to the filtrate, mix by vortexing and ultrasonication, and centrifuge at a speed of 8000 rpm for 5 minutes. After the centrifugation, discard the supernatant, repeat the ice ether dissolution, centrifugation and discard the supernatant 3 times, and then ventilate and dry the obtained precipitate for 24 hours to obtain a crude polypeptide product; S4: The crude polypeptide product was purified by HPLC, using a reverse phase C18 column in HPLC, the system was set to 0.1% TFA aqueous solution for phase A, 0.1% TFA acetonitrile solution for phase B, the flow rate was 10 mL / min, and the wavelengths of the dual-channel detector were 220 nm and 274 nm; the crude polypeptide product was dissolved in a AB phase mixed solution (40% A, 60% B) at a concentration of 20 mg / ml, filtered with a 0.22 μm filter membrane to remove insoluble matter and then entered the chromatographic column, first using the initial mobile phase (90% A phase + 10% B phase) to run for 10 min until the baseline was stable to balance the chromatographic column, then phase B was continued at 10% volume for 2 min, and the volume was linearly increased from 10% to 90% for 30 min, and the eluent was collected when the target peak appeared, the eluent was removed of the organic solvent using a rotary evaporator and then rapidly frozen using liquid nitrogen, and finally placed in a vacuum freeze dryer to freeze-dry to obtain a pure polypeptide product; S5: The pure polypeptide product and maleimide-functionalized star-shaped PEG were mixed at a molar ratio of 4:1, and then added to a 95% DMF aqueous solution for reaction for 24 hours (the material-liquid ratio of the mixture to the DMF solution was 10 mg:1 mL), the pH during the reaction was adjusted to 7.5, and after the reaction was completed, the DMF was dialyzed to obtain a polymer solution, and the polymer solution was freeze-dried using a vacuum freeze dryer to obtain a polypeptide polymer; S6: Dissolve the polypeptide polymer in water, fully dissolve it through vortexing and ultrasound, and adjust the pH to 6.5 to obtain polypeptide hydrogel 6.5-gel.

[0031] Experimental Example 1 Rheological test: The test was conducted using a rotational rheometer MCR302 (Anton Paar, Austria), which was equipped with a PP25 (25 mm diameter) parallel plate. The polypeptide hydrogels of Examples 1 to 3 and Comparative Examples 1 to 2 were coated on the platform of the rheometer. The parallel plates were pressed down until gels overflowed on all sides. The excess gel was scraped off for testing. The test temperature was set to 25°C, and the entire test process was sealed with oil. The rheological test used a frequency sweep mode to monitor the storage modulus (G') and loss modulus (G"), and a stress-relaxation mode to calculate the relaxation time. The test results of Example 1 are shown in Figure 1. Figure 1 The test results of Examples 2 to 3 and Comparative Examples 1 to 2 are as follows: Figure 2 , Figure 1 According to the crossover frequency ωc (τ=1 / ωc) of G' and G", pH=8, the relaxation time of the hydrogel is 0.22s. Figure 2 The relaxation time was quantitatively evaluated through the stress-relaxation curve, and the experimental data were fitted to the Kohlrausch model. It was calculated that the relaxation time of the hydrogel was 1112s, 447s, 18.5s, and 1.17s when pH = 6, 6.5, 7, and 7.5, respectively.

[0032] Experimental Example 2 Biocompatibility experiment: Sterilized hydrogel (8-gel prepared in Example 1) was placed in the bottom of a 48-well plate in advance, and then L929 cells were inoculated into the wells containing the hydrogel as the experimental group, and cells without hydrogel were used as the control. The 48-well plate was cultured in a carbon dioxide cell culture incubator (37°C, 5% CO2) for 24 hours, and the cells were incubated with a CCK-8 kit for 1 hour. The absorbance at 450 nm was measured with a microplate reader. The cell survival rate in the experimental group was as follows: Figure 3 As shown (the horizontal axis is the concentration of the polypeptide polymer material), it shows that the polypeptide hydrogel of the present invention has no toxicity to L929 cells and has good biocompatibility; the morphology of L929 cells after co-culture is observed using a fluorescence microscope, and the living cells are stained green with fluorescein diacetate (FDA) and the dead cells are stained red with propidium iodide (PI) using a cell live-dead staining solution, and the cell morphology is observed again using a fluorescence microscope. The results are as follows Figure 4 There was no significant difference in cell growth between the experimental group and the control group, which also shows that the polypeptide hydrogel of the present invention has good biocompatibility.

[0033] Experimental Example 3 Cell osteogenesis experiment: mesenchymal stem cells in the logarithmic phase were dropped into cell well plates containing the polypeptide hydrogel 8-gel of Example 1 and the polypeptide hydrogel 6-gel of Comparative Example 1, respectively, and cultured in an incubator for 3 hours to allow them to adhere to the wall. The cell state was observed before staining, and then the culture medium was removed and rinsed with PBS for 3 times. 4% paraformaldehyde was added for fixation for 15 minutes, and then the formaldehyde was removed and rinsed with PBS for 3 times. The staining solution was prepared according to the instructions of the alkaline phosphatase calcium cobalt method, and then an appropriate amount of staining solution was added to cover the cell well plate, incubated in dark for 30 minutes, the staining solution was removed, and the cells were washed twice with distilled water and PBS respectively. Finally, they were observed and photographed under a microscope. The results are as follows: Figures 5 and 6 As shown, more cells were differentiated using the polypeptide hydrogel of Example 1, indicating that the polypeptide hydrogel of Example 1 can better induce cell osteogenic differentiation.

[0034] Experimental Example 4 Cell chondrogenic differentiation experiment: mesenchymal stem cells in the logarithmic phase were dropped into cell well plates containing the polypeptide hydrogel 8-gel of Example 1 and the polypeptide hydrogel 6-gel of Comparative Example 1, respectively, and cultured in an incubator for 3 hours to allow them to adhere to the wall. The cell state was observed before staining, and then the culture medium was removed and rinsed with PBS for 3 times, and 4% neutral formaldehyde solution was added to fix the cells for 45 minutes, the fixative was discarded and washed with PBS twice, and then an appropriate amount of Alcian blue staining solution was added to incubate in the dark for 30 minutes, the dye was discarded and washed with PBS twice, and finally observed and photographed under a microscope. The results are as follows: Figure 7-8 As shown, more cells were differentiated using the polypeptide hydrogel of Example 1, indicating that the polypeptide hydrogel of Example 1 can better induce osteoblastic differentiation of cells.

[0035] Experimental Example 5 Self-healing experiment: The polypeptide hydrogel 8-gel of Example 1 and the polypeptide hydrogel 6-gel of Comparative Example 1 were cut into two semicircular gels, and then they were put into contact with each other through the cut surface to observe their self-healing properties. The results are as follows: Figures 9-10 As shown, the two semicircular hydrogels of the polypeptide hydrogel of Comparative Example 1 were still in a separated state after being in contact with each other for 12 hours, while the contact surfaces of the two semicircular hydrogels of the polypeptide hydrogel of Example 1 were healed after only 2 minutes to become a complete circular hydrogel without obvious cracks.

[0036] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A method for preparing a polypeptide hydrogel with adjustable stress relaxation, characterized in that: The following steps are involved: S1: dissolving amino acid powder in DMF to obtain an amino acid solution, and then synthesizing the amino acid solution, a deprotecting agent, a reaction condensing agent and a DMF-swollen carrier resin through a synthesis instrument to obtain a resin containing a polypeptide chain; S2: washing and drying the resin containing the polypeptide chain to obtain a resin without impurities; S3: Place the impurity-free resin in the lysate for 2-3 hours, filter the lysate, add 10 times the volume of icy ether to the filtrate, centrifuge at 8000 rpm for 5 minutes, discard the supernatant, repeat 3 times, and then air dry the precipitate for 20-30 hours to obtain the crude polypeptide product; S4: Purify the crude polypeptide product by HPLC to obtain a pure polypeptide product; S5: The pure polypeptide product and maleimide-functionalized star-shaped PEG were mixed in a molar ratio of 3-5:1, and then added to a 95% DMF aqueous solution for reaction for 24 hours, and the pH during the reaction was adjusted to 7-7.

5. After the reaction, the DMF was removed by dialysis to obtain a polymer solution, and the polymer solution was lyophilized to obtain a polypeptide polymer; S6: Dissolve the polypeptide polymer in water and adjust the pH to 7-8 to obtain a polypeptide hydrogel with controllable stress relaxation.

2. The method for preparing the stress relaxation controllable polypeptide hydrogel according to claim 1, characterized in that: The concentration of the amino acid solution is 0.15-0.25M.

3. The method for preparing the stress relaxation controllable polypeptide hydrogel according to claim 1, characterized in that: The deprotecting agent is a piperidine solution obtained by dissolving hexahydropyridine in DMF at a volume concentration of 20%, and the reaction condensation agent is an N, N-diisopropylcarbodiimide solution with DMF as solvent and an ethyl 2-oximecyanoacetate solution with DMF as solvent.

4. The method for preparing the stress relaxation controllable polypeptide hydrogel according to claim 1, characterized in that: The carrier resin is Rink Amide resin.

5. The method for preparing the stress relaxation controllable polypeptide hydrogel according to claim 1, characterized in that: The washing in S2 is washing with DMF 3 times and then washing with dichloromethane 2 times.

6. The method for preparing the stress relaxation controllable polypeptide hydrogel according to claim 1, characterized in that: The lysis solution is a mixture of trifluoroacetic acid, triisopropylsilane, water and ethanedithiol in a volume ratio of 90-95:2-3:2-3:2-3.

7. The method for preparing the stress relaxation controllable polypeptide hydrogel according to claim 1, characterized in that: The HPLC used a 0.1% TFA aqueous solution as phase A, a 0.1% TFA acetonitrile solution as phase B, a flow rate of 10 mL / min, and a dual-channel detector with wavelengths of 220 nm and 274 nm.

8. The method for preparing the stress relaxation controllable polypeptide hydrogel according to claim 1, characterized in that: The solid-liquid ratio of the mixture of the pure polypeptide product and the maleimide-functionalized star-shaped PEG to DMF is 10 mg: 0.5-1.5 mL.

9. The stress relaxation controllable polypeptide hydrogel prepared by the preparation method of the stress relaxation controllable polypeptide hydrogel according to any one of claims 1 to 8.

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