A polypeptide hydrogel with controllable stress relaxation and its preparation method

Abstract

The present invention discloses a polypeptide hydrogel with adjustable stress relaxation and a preparation method thereof, belonging to the technical field of hydrogels. The preparation method provided by the present invention is to connect amino acids to the polypeptide chain one by one, and then release the synthesized polypeptide from the solid-phase resin by removing the side-chain protecting groups through cleavage, so as to obtain a free polypeptide with high purity. Then, the polypeptide pure product is obtained through purification. Finally, the polypeptide pure product reacts with maleimide-functionalized star-shaped PEG to obtain a polypeptide polymer. The polypeptide polymer is dissolved in water, and adjusting the pH can obtain a polypeptide hydrogel with an ultrafast relaxation time and adjustable stress relaxation. The fastest relaxation time of the hydrogel provided by the present invention can reach 0.22 s. Moreover, by utilizing the natural biodegradability and low immunogenicity of the polypeptide, while maintaining the ultrafast stress relaxation characteristics, the biocompatibility of the material is improved, and it has excellent prospects for biomedical applications.

Description

Technical Field

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

[0002] A hydrogel is a three-dimensional network material formed by physical or chemical cross-linking of hydrophilic polymers, which can absorb and lock a large amount of water while maintaining a solid structure without dissolution. It has a soft and elastic texture, excellent biocompatibility and controllability, and is widely used in biomedical fields (such as wound dressings, drug release, tissue engineering), environmental protection (wastewater treatment, agricultural water retention), and flexible electronic devices, etc.

[0003] The 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 the internal cross-linked network or molecular chains can quickly rearrange, thereby quickly releasing stress or adapting to deformation. Such hydrogels are usually constructed by introducing dynamic reversible bonds (such as hydrogen bonds, ionic bonds, host-guest interactions or dynamic covalent bonds), and their networks can be quickly reconstructed after being damaged, endowing the materials with self-healing and energy dissipation capabilities similar to biological tissues. This property makes them 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 fatigue-resistant interface materials for flexible electronic devices, etc.

[0004] Currently, the following problems still exist in hydrogels with relatively fast relaxation times:

[0005] (1) The relaxation time generally exceeds 10 s. For example, the relaxation time of physically cross-linked hyaluronic acid hydrogels is about several seconds to the minute level; the relaxation time of chemically cross-linked PAAm hydrogels can reach dozens of minutes to several hours.

[0006] (2) The regulation range of the stress relaxation rate is limited, and it is also difficult to achieve in-situ control of the stress relaxation rate.

[0007] (3) Poor biocompatibility hinders their biomedical applications. Summary of the Invention

[0008] 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 regulation range of stress relaxation rate, and poor biocompatibility of the existing hydrogels.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a preparation method of a polypeptide hydrogel with adjustable stress relaxation, including the following steps:

[0010] S1: Dissolve the amino acid powder in DMF to obtain an amino acid solution. Then, perform synthesis on the amino acid solution, deprotecting agent, reaction condensing agent, and DMF-swollen carrier resin through a synthesis instrument to obtain a resin containing a polypeptide chain.

[0011] S2: Wash and dry the resin containing the polypeptide chain to obtain a resin without impurities.

[0012] S3: Place the resin without impurities in the cleavage solution for 2 - 3 h, filter the cleavage solution, add ice ether with a volume 10 times that of the filtrate, centrifuge at 8000 rpm for 5 min, discard the supernatant, repeat 3 times, and then ventilate and dry the precipitate for 20 - 30 h to obtain a crude polypeptide product.

[0013] S4: Purify the crude polypeptide product using HPLC to obtain a pure polypeptide product.

[0014] S5: Mix the pure polypeptide product and maleimide-functionalized star-shaped PEG in a molar ratio of 3 - 5:1, then add them to an aqueous solution of 95% DMF and react for 24 h. Adjust the pH during the reaction to 7 - 7.5. After the reaction, perform dialysis to remove DMF to obtain a polymer solution. Freeze-dry the polymer solution to obtain a polypeptide polymer.

[0015] S6: Dissolve the polypeptide polymer in water and adjust the pH to 7 - 8 to obtain a polypeptide hydrogel with controllable stress relaxation.

[0016] The beneficial effects of the present invention are as follows: The present invention selects Rink Amide resin for C-terminal amidation of polypeptides. During synthesis, amino acids are connected to the polypeptide chain one by one through repeated deprotection and coupling steps. Then, the synthesized polypeptide is released from the solid-phase resin by removing the side-chain protecting groups through cleavage, thereby obtaining a highly pure free polypeptide. After purification, a pure polypeptide product is obtained. Finally, the pure polypeptide product reacts with maleimide-functionalized star-shaped PEG to obtain a polypeptide polymer. Dissolve the polypeptide polymer in water and adjust the pH to obtain a polypeptide hydrogel with an ultrafast relaxation time and controllability. The method provided by the present invention utilizes the natural biodegradability and low immunogenicity of polypeptides (the polypeptide is modified with polyethylene glycol to obtain a polypeptide polymer, and polyethylene glycol also has excellent biocompatibility). While maintaining the ultrafast stress relaxation characteristics, it has good biocompatibility, enabling the finally obtained polypeptide hydrogel to have excellent prospects for biomedical applications.

[0017] Based on the above technical solutions, the present invention can be further improved as follows.

[0018] Further, the concentration of the amino acid solution is 0.15 - 0.25 M.

[0019] Further, the deprotecting agent is a piperidine solution obtained by dissolving piperidine in DMF at a volume concentration of 20%, and the reaction condensing agent is N,N-diisopropylcarbodiimide or ethyl 2-cyanoacetimidate.

[0020] The beneficial effects of adopting the further technical solution are as follows: As a basic deprotecting agent, piperidine can effectively neutralize the fmoc group, causing it to leave the amino acid, thereby exposing the amino acid at the n-terminus, facilitating subsequent condensation reactions; 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 it has relatively low reactivity towards the side chains of amino acids, which means that during the condensation process, it mainly acts between the carboxylic acid and the amino group, reducing unnecessary side reactions. Ethyl 2-cyanoacetimidate can promote the bonding reaction between amino acids under mild conditions, with a fast reaction rate, high yield, and good selectivity for amino acids.

[0021] Further, the carrier resin is Rink Amide resin.

[0022] Further, the cleaning in S2 is to wash 3 times with DMF and then wash 2 times with dichloromethane.

[0023] Further, the cleavage solution is a mixed solution of trifluoroacetic acid, triisopropylsilane, water, and ethanedithiol mixed at a volume ratio of 90 - 95:2 - 3:2 - 3:2 - 3.

[0024] The beneficial effects of adopting the further technical solution are as follows: Trifluoroacetic acid is used to break the connection bond between the polypeptide and the resin and remove the side chain protecting groups. Triisopropylsilane is used to capture the cationic by-products generated during the cleavage process to prevent polypeptide modification. Ethanedithiol is used to protect easily oxidized amino acids such as Cys to prevent side reactions. The cleavage solution obtained by mixing these three reagents with water in proportion can release the synthesized polypeptide from the solid-phase resin after removing the side chain protecting groups, thereby obtaining a highly pure free polypeptide.

[0025] Further, the mobile phase A used in HPLC is an aqueous solution of 0.1% TFA, the mobile phase B is an acetonitrile solution of 0.1% TFA, the flow rate is 10 mL / min, and the wavelengths of the dual-channel detector are 220 nm and 274 nm.

[0026] The beneficial effects of adopting the further technical solution are as follows: Reduce impurities and by-products to obtain a highly pure polypeptide product.

[0027] Further, the ratio of the mixture of the pure polypeptide product and maleimide-functionalized star-shaped PEG to DMF is 10 mg:0.5 - 1.5 mL.

[0028] The beneficial effect of adopting the further technical solution is that maleimide and mercapto group (-SH) can undergo a nucleophilic addition reaction under mild conditions to form a stable thioether bond (-S-), and finally a star-shaped polypeptide polymer is obtained.

[0029] The present invention also provides a stress-relaxation adjustable polypeptide hydrogel prepared by the preparation method of the stress-relaxation adjustable polypeptide hydrogel.

[0030] The beneficial effect of the present invention is that the polypeptide hydrogel provided by the present invention shows a gradually changing stress relaxation rate in situ, and the fastest relaxation time is even only 0.22 s. The viscoelasticity of this material changes with the change of pH value, changing from brittle quasi-elasticity to plastic viscoelasticity; moreover, this hydrogel utilizes the natural biodegradability and low immunogenicity of polypeptides, and has good biocompatibility while maintaining the ultra-fast stress relaxation characteristics. Brief Description of the Drawings

[0031] Figure 1 It is the frequency sweep diagram of the polypeptide hydrogel of Example 1;

[0032] Figure 2 It is the stress-relaxation curve diagram of Examples 2-3 and Comparative Examples 1-2;

[0033] Figure 3 It is the statistical chart of the survival rate of L929 cells;

[0034] Figure 4 It is the live-dead staining diagram of L929 cells;

[0035] Figure 5 It is the staining diagram of cell osteogenic differentiation;

[0036] Figure 6 It is the activity diagram of cell osteogenic differentiation;

[0037] Figure 7 It is the staining diagram of cell chondrogenic differentiation;

[0038] Figure 8 It is the activity diagram of cell chondrogenic differentiation;

[0039] Figure 9 It is the self-healing experiment diagram of the polypeptide hydrogel of Comparative Example 1;

[0040] Figure 10 It is the self-healing experiment diagram of the polypeptide hydrogel of Example 1. Detailed Embodiments

[0041] The following detailed description of the specific embodiments of the present invention is made in conjunction with the examples.

[0042] Example 1

[0043] A method for preparing a stress-relaxation regulable polypeptide hydrogel, comprising the following steps:

[0044] S1: Place amino acid powder in DMF to dissolve and prepare an amino acid solution with a concentration of 0.2 M. The deprotecting agent is a piperidine solution obtained by dissolving hexahydropyridine in DMF at a concentration of 20% (v / v%); the reaction condensing agent includes N,N-diisopropylcarbodiimide (DIC, dissolved in DMF at a concentration of 7.8% (v / v)) and ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma, 14.21 g of Oxyma powder dissolved in 100 mL of DMF); supplement the main solvent DMF to the volume required for the experiment, and finally install these solvent bottles on an automatic continuous microwave polypeptide synthesizer; after swelling the Rink Amide resin completely with DMF, use the automatic continuous microwave polypeptide synthesizer for synthesis to obtain a resin containing a polypeptide chain;

[0045] S2: Wash the resin containing the polypeptide chain 3 times with DMF and then 2 times with dichloromethane. After the washing is completed, use a vacuum pump to evacuate and dry the product to obtain an impurity-free resin;

[0046] S3: Place the impurity-free resin in a cleavage solution (obtained by mixing trifluoroacetic acid, triisopropylsilane, water, and ethanedithiol in a volume ratio of 92.5:2.5:2.5:2.5) for 2.5 h (the material ratio of the impurity-free resin to the cleavage solution is 1 g:10 mL), filter the cleavage solution, add ice ether with a volume 10 times that of the filtrate to the filtrate, vortex and ultrasonically mix, then centrifuge at a speed of 8000 rpm for 5 min. After centrifugation, discard the supernatant, repeat the steps of dissolving with ice ether, centrifuging, and discarding the supernatant 3 times, and then ventilate and dry the obtained precipitate for 24 h to obtain a crude polypeptide product;

[0047] S4: Purify the crude polypeptide product by HPLC. In HPLC, use a reverse-phase C18 chromatographic column. Set the system A phase as 0.1% TFA aqueous solution, the B phase as 0.1% TFA acetonitrile solution, the flow rate as 10 mL / min, and the wavelengths of the dual-channel detector as 220 nm and 274 nm; dissolve the crude polypeptide product in a mixed solution of phases A and B (40% A, 60% B) at a concentration of 20 mg / ml, filter to remove insoluble substances with a 0.22 μm filter membrane and then enter the chromatographic column. First, run with the initial mobile phase (90% A phase + 10% B phase) for 10 min until the baseline is stable to equilibrate the chromatographic column. Then, the B phase continues at 10% by volume for 2 min, and then linearly gradients from 10% to 90% by volume in 30 min. Collect the eluate when the target peak appears. Remove the organic solvent from the eluate using a rotary evaporator, rapidly freeze it with liquid nitrogen, and finally freeze-dry it in a vacuum freeze dryer to obtain a pure polypeptide product;

[0048] S5: Mix the pure polypeptide product and maleimide-functionalized star-shaped PEG at a molar ratio of 4:1, and then add it to an aqueous solution of 95% DMF for reaction for 24 h (the feed ratio of the mixture to the DMF solution is 10 mg:1 mL). Adjust the pH during the reaction to 7.5. After the reaction, perform dialysis to remove DMF to obtain a polymer solution. Lyophilize the polymer solution using a vacuum freeze dryer to obtain a polypeptide polymer.

[0049] S6: Dissolve the polypeptide polymer in water, and fully dissolve it by vortex ultrasonic treatment. Adjust the pH to 8 to obtain a stress-relaxation controllable polypeptide hydrogel 8-gel.

[0050] Example 2

[0051] A preparation method of a stress-relaxation controllable polypeptide hydrogel, comprising the following steps:

[0052] S1: Place the amino acid powder in DMF to dissolve and prepare an amino acid solution with a concentration of 0.15 M. The deprotecting agent is a piperidine solution, which is obtained by dissolving hexahydropyridine in DMF at a concentration of 20% (v / v%). The reaction condensing agent includes N,N-diisopropylcarbodiimide (DIC, dissolved in DMF at a concentration of 7.8% (v / %)), and ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma, 14.21 g of Oxyma powder dissolved in 100 mL of DMF). Supplement the main solvent DMF to the volume required for the experiment. Finally, install these solvent bottles on an automatic continuous microwave polypeptide synthesizer. After swelling the Rink Amide resin completely with DMF, use the automatic continuous microwave polypeptide synthesizer for synthesis to obtain a resin containing a polypeptide chain.

[0053] S2: Wash the resin containing the polypeptide chain 3 times with DMF, and then wash it 2 times with dichloromethane. After the washing, use a vacuum pump to evacuate and dry the product to obtain an impurity-free resin.

[0054] S3: Place the impurity-free resin in a cleavage solution (obtained by mixing trifluoroacetic acid, triisopropylsilane, water, and ethanedithiol in a volume ratio of 90:3:2:2) for 2.5 h (the material ratio of the impurity-free resin to the cleavage solution is 1 g:5 mL). Filter the cleavage solution, add ice ether with a volume 10 times that of the filtrate to the filtrate, mix well by vortex ultrasonic treatment, and centrifuge at a speed of 8000 rpm for 5 min. After centrifugation, discard the supernatant. Repeat the steps of dissolving with ice ether, centrifuging, and discarding the supernatant 3 times. Then, ventilate and dry the obtained precipitate for 30 h to obtain a polypeptide crude product.

[0055] S4: Purify the crude polypeptide product using HPLC. In HPLC, use a reverse-phase C18 chromatographic column. The system is set with phase A as a 0.1% aqueous TFA solution, phase B as a 0.1% TFA acetonitrile solution, the flow rate is 10 mL / min, and the wavelengths of the dual-channel detector are 220 nm and 274 nm. Dissolve the crude polypeptide product in a mixed solution of phases A and B (40% A, 60% B) at a concentration of 20 mg / ml. After filtering through a 0.22 μm filter membrane to remove insolubles, it enters the chromatographic column. First, run with the initial mobile phase (90% phase A + 10% phase B) for 10 min until the baseline is stable to equilibrate the chromatographic column. Then, phase B continues at 10% by volume for 2 min, and then linearly gradients from 10% to 90% over 30 min. Collect the eluate when the target peak appears. Remove the organic solvent from the eluate using a rotary evaporator, then rapidly freeze it using liquid nitrogen, and finally freeze-dry it in a vacuum freeze dryer to obtain the pure polypeptide product;

[0056] S5: Mix the pure polypeptide product and maleimide-functionalized star-shaped PEG in a molar ratio of 3:1, and then add it to an aqueous solution of 95% DMF for reaction for 24 h (the feed ratio of the mixture to the DMF solution is 10 mg:0.5 mL). Adjust the pH during the reaction to 7. After the reaction, perform dialysis to remove DMF to obtain a polymer solution. Freeze-dry the polymer solution using a vacuum freeze dryer to obtain the polypeptide polymer;

[0057] S6: Dissolve the polypeptide polymer in water, and fully dissolve it by vortex ultrasonic treatment. Adjust the pH to 7 to obtain the stress relaxation controllable polypeptide hydrogel 7-gel.

[0058] Example 3

[0059] A method for preparing a stress relaxation controllable polypeptide hydrogel, comprising the following steps:

[0060] S1: Place the amino acid powder in DMF to dissolve and prepare an amino acid solution with a concentration of 0.25 M. The deprotecting agent is a piperidine solution, obtained by dissolving hexahydropyridine in DMF at a concentration of 20% (v / v%). The reaction condensing agent includes N,N-diisopropylcarbodiimide (DIC, dissolved in DMF at a concentration of 7.8% (v / v)) and ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma, 14.21 g of Oxyma powder dissolved in 100 mL of DMF). Supplement the main solvent DMF to the volume required for the experiment. Finally, install these solvent bottles on an automatic continuous microwave polypeptide synthesizer. After swelling the Rink Amide resin completely with DMF, use the automatic continuous microwave polypeptide synthesizer for synthesis to obtain the resin containing the polypeptide chain;

[0061] S2: Wash the resin containing the polypeptide chain 3 times with DMF, then wash it 2 times with dichloromethane. After the washing is completed, use a vacuum pump to evacuate and dry the product to obtain the impurity-free resin;

[0062] S3: Place the impurity-free resin in the lysis solution (obtained by mixing trifluoroacetic acid, triisopropylsilane, water, and ethanedithiol in a volume ratio of 95:3:3:2) for 2.5 h (the material ratio of the impurity-free resin to the lysis solution is 1 g:15 mL). Filter the lysis solution, add ice ether with a volume 10 times that of the filtrate to the filtrate. After vortexing and ultrasonic mixing, centrifuge at a speed of 8000 rpm for 5 min. After centrifugation, discard the supernatant. Repeat the steps of dissolving in ice ether, centrifuging, and discarding the supernatant 3 times. Then, ventilate and dry the obtained precipitate for 20 h to obtain the crude polypeptide product;

[0063] S4: Purify the crude polypeptide product using HPLC. In HPLC, use a reverse-phase C18 chromatographic column. Set the system with phase A as 0.1% aqueous TFA solution, phase B as 0.1% TFA acetonitrile solution, the flow rate as 10 mL / min, and the wavelengths of the dual-channel detector as 220 nm and 274 nm. Dissolve the crude polypeptide product in the AB-phase mixed solution (40% A, 60% B) at a concentration of 20 mg / ml. After filtering to remove insoluble substances using a 0.22-μm filter membrane, it enters the chromatographic column. First, run with the initial mobile phase (90% phase A + 10% phase B) for 10 min until the baseline is stable to equilibrate the chromatographic column. Then, phase B continues at 10% by volume for 2 min, and then linearly gradients from 10% to 90% in volume for 30 min. Collect the eluate when the target peak appears. Remove the organic solvent from the eluate using a rotary evaporator, then rapidly freeze it using liquid nitrogen, and finally freeze-dry it in a vacuum freeze dryer to obtain the pure polypeptide product;

[0064] S5: Mix the pure polypeptide product and maleimide-functionalized star-shaped PEG in a molar ratio of 5:1, and then add it to an aqueous solution of 95% DMF to react for 24 h (the material ratio of the mixture to the DMF solution is 10 mg:1.5 mL). Adjust the pH during the reaction to 7.5. After the reaction, perform dialysis to remove DMF to obtain the polymer solution. Freeze-dry the polymer solution using a vacuum freeze dryer to obtain the polypeptide polymer;

[0065] S6: Dissolve the polypeptide polymer in water, and fully dissolve it by vortexing and ultrasonic treatment. Adjust the pH to 7.5 to obtain the stress-relaxation controllable polypeptide hydrogel 7.5-gel.

[0066] Comparative Example 1

[0067] A method for preparing a polypeptide hydrogel, comprising the following steps:

[0068] S1: Place the amino acid powder in DMF to dissolve and prepare an amino acid solution with a concentration of 0.2 M. The deprotecting agent is a piperidine solution obtained by dissolving piperidine in DMF at a concentration of 20% (v / v%). The reaction condensing agent includes N,N-diisopropylcarbodiimide (DIC, dissolved in DMF at a concentration of 7.8% (v / v)) and ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma, 14.21 g of Oxyma powder dissolved in 100 mL of DMF). Make up the main solvent DMF to the required volume for the experiment. Finally, install these solvent bottles on an automatic continuous microwave polypeptide synthesizer. After swelling the Rink Amide resin completely with DMF, use the automatic continuous microwave polypeptide synthesizer for synthesis to obtain the resin containing the polypeptide chain.

[0069] S2: Wash the resin containing the polypeptide chain 3 times with DMF and then 2 times with dichloromethane. After the washing is completed, use a vacuum pump to evacuate and dry the product to obtain the resin without impurities.

[0070] S3: Place the resin without impurities in the cleavage solution (a mixture of trifluoroacetic acid, triisopropylsilane, water, and ethanedithiol in a volume ratio of 92.5:2.5:2.5:2.5) for 2.5 h (the material ratio of the resin without impurities to the cleavage solution is 1 g:10 mL). Filter the cleavage solution, add ice ether with a volume 10 times that of the filtrate to the filtrate. After vortexing and ultrasonic mixing, centrifuge at a speed of 8000 rpm for 5 min. After centrifugation, discard the supernatant. Repeat the steps of dissolving with ice ether, centrifuging, and discarding the supernatant 3 times. Then ventilate and dry the obtained precipitate for 24 h to obtain the crude polypeptide product.

[0071] S4: Purify the crude polypeptide product using HPLC. In HPLC, use a reverse-phase C18 chromatographic column. The system is set with phase A as 0.1% aqueous TFA solution, phase B as 0.1% TFA acetonitrile solution, the flow rate is 10 mL / min, and the wavelengths of the dual-channel detector are 220 nm and 274 nm. Dissolve the crude polypeptide product in the AB phase mixed solution (40% A, 60% B) at a concentration of 20 mg / ml. After filtering to remove insoluble substances with a 0.22 μm filter membrane, it enters the chromatographic column. First, run with the initial mobile phase (90% phase A + 10% phase B) for 10 min until the baseline is stable to equilibrate the chromatographic column. Then, phase B continues at 10% by volume for 2 min, and then the volume linearly gradients from 10% to 90% in 30 min. Collect the eluate when the target peak appears. Remove the organic solvent from the eluate using a rotary evaporator, then rapidly freeze it with liquid nitrogen, and finally freeze-dry it in a vacuum freeze-dryer to obtain the pure polypeptide product.

[0072] S5: Mix the pure polypeptide product and maleimide-functionalized star-shaped PEG in a molar ratio of 4:1, and then add it to an aqueous solution of 95% DMF for reaction for 24 h (the feed ratio of the mixture to the DMF solution is 10 mg:1 mL). Adjust the pH during the reaction to 7.5. After the reaction, perform dialysis to remove DMF to obtain a polymer solution. Lyophilize the polymer solution using a vacuum freeze dryer to obtain a polypeptide polymer.

[0073] S6: Dissolve the polypeptide polymer in water, and fully dissolve it by vortex ultrasonic treatment. Adjust the pH to 6 to obtain a polypeptide hydrogel 6-gel.

[0074] Comparative Example 2

[0075] A method for preparing a polypeptide hydrogel, comprising the following steps:

[0076] S1: Place the amino acid powder in DMF to dissolve and prepare an amino acid solution with a concentration of 0.2 M. The deprotecting agent is a piperidine solution, obtained by dissolving hexahydropyridine in DMF at a concentration of 20% (v / v%). The reaction condensing agent includes N,N-diisopropylcarbodiimide (DIC, dissolved in DMF at a concentration of 7.8% (v / v)) and ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma, 14.21 g of Oxyma powder dissolved in 100 mL of DMF); supplement the main solvent DMF to the volume required for the experiment. Finally, install these solvent bottles on an automatic continuous microwave polypeptide synthesizer; after swelling the Rink Amide resin completely with DMF, use the automatic continuous microwave polypeptide synthesizer for synthesis to obtain a resin containing a polypeptide chain.

[0077] S2: Wash the resin containing the polypeptide chain 3 times with DMF, and then wash it 2 times with dichloromethane. After the washing, use a vacuum pump to evacuate and dry the product to obtain an impurity-free resin.

[0078] S3: Place the impurity-free resin in a cleavage solution (obtained by mixing trifluoroacetic acid, triisopropylsilane, water, and ethanedithiol in a volume ratio of 92.5:2.5:2.5:2.5) for 2.5 h (the material ratio of the impurity-free resin to the cleavage solution is 1 g:10 mL). Filter the cleavage solution, add ice ether with a volume 10 times that of the filtrate to the filtrate, mix well by vortex ultrasonic treatment, and centrifuge at 8000 rpm for 5 min. After centrifugation, discard the supernatant. Repeat the steps of dissolving with ice ether, centrifuging, and discarding the supernatant 3 times. Then, ventilate and dry the obtained precipitate for 24 h to obtain a polypeptide crude product.

[0079] S4: Purify the crude polypeptide product using HPLC. In HPLC, use a reverse-phase C18 chromatographic column. Set the mobile phase A as 0.1% aqueous TFA solution, mobile phase B as 0.1% TFA acetonitrile solution, the flow rate is 10 mL / min, and the wavelengths of the dual-channel detector are 220 nm and 274 nm. Dissolve the crude polypeptide product in the mixed solution of mobile phases A and B (40% A, 60% B) at a concentration of 20 mg / ml. After filtering with a 0.22-μm filter membrane to remove insoluble substances, it enters the chromatographic column. First, run with the initial mobile phase (90% mobile phase A + 10% mobile phase B) for 10 min until the baseline is stable to equilibrate the chromatographic column. Then, mobile phase B continues at 10% by volume for 2 min, and then linearly gradients from 10% to 90% in volume over 30 min. Collect the eluate when the target peak appears. Remove the organic solvent from the eluate using a rotary evaporator, then rapidly freeze it with liquid nitrogen, and finally freeze-dry it in a vacuum freeze dryer to obtain the pure polypeptide product;

[0080] S5: Mix the pure polypeptide product and maleimide-functionalized star-shaped PEG in a molar ratio of 4:1, and then add it to an aqueous solution of 95% DMF for reaction for 24 h (the feed ratio of the mixture to the DMF solution is 10 mg:1 mL). Adjust the pH during the reaction to 7.5. After the reaction, perform dialysis to remove DMF to obtain the polymer solution. Freeze-dry the polymer solution using a vacuum freeze dryer to obtain the polypeptide polymer;

[0081] S6: Dissolve the polypeptide polymer in water, and fully dissolve it by vortex ultrasonic treatment. Adjust the pH to 6.5 to obtain the polypeptide hydrogel 6.5-gel.

[0082] Experimental Example 1

[0083] Rheological test: Use a rotary rheometer MCR302 (Anton Paar, Austria) for testing. The instrument is equipped with a PP25 (diameter 25 mm) parallel plate. Coat the polypeptide hydrogels of Examples 1-3 and Comparative Examples 1-2 on the platform of the rheometer. Press the parallel plate down until the gel overflows evenly around, and scrape off the excess gel for testing. Set the test temperature at 25 °C, and seal the whole test process with oil. The rheological test uses a frequency sweep mode to monitor the storage modulus (G') and loss modulus (G"), and uses a stress-relaxation mode to calculate the relaxation time. The test results of Example 1 are as Figure 1 , and the test results of Examples 2-3 and Comparative Examples 1-2 are as Figure 2 , Figure 1 Calculated according to the crossover frequency ωc of G' and G" (τ = 1 / ωc), at pH = 8, the relaxation time of the hydrogel is 0.22 s, Figure 2The relaxation time was quantitatively evaluated through the stress-relaxation curve, and the experimental data was fitted to the Kohlrausch model. The relaxation times of the hydrogels at pH = 6, 6.5, 7, and 7.5 were calculated to be 1112 s, 447 s, 18.5 s, and 1.17 s, respectively.

[0084] Experimental Example 2

[0085] Biocompatibility experiment: Sterilized hydrogel (8-gel prepared in Example 1) was pre-placed at the bottom of a 48-well plate, and then L929 cells were inoculated into the wells containing the hydrogel as the experimental group, with cells without hydrogel as the control. After the 48-well plate was further cultured in a carbon dioxide cell incubator (37 °C, 5% CO 2 ) for 24 h, after incubating the cells with a CCK-8 kit for 1 h, the absorbance at 450 nm was measured using a microplate reader. The cell survival rate in the experimental group is as Figure 3 shown (the abscissa is the concentration of the polypeptide polymer material), indicating that the polypeptide hydrogel of the present invention is non-toxic to L929 cells and has good biocompatibility; then a fluorescence microscope was used to observe the morphology of L929 cells after co-culture. After staining live cells green with fluorescein diacetate (FDA) and dead cells red with propidium iodide (PI) using a cell viability / cytotoxicity assay kit, the cell morphology was observed through a fluorescence microscope. The results are as Figure 4 shown. There is no obvious difference in cell growth between the experimental group and the control group, which also indicates that the polypeptide hydrogel of the present invention has good biocompatibility.

[0086] Experimental Example 3

[0087] Cell osteogenesis experiment: Mesenchymal stem cells in the logarithmic phase were respectively dropped into cell well plates containing the polypeptide hydrogel 8-gel of Example 1 and the polypeptide hydrogel 6-gel of Comparative Example 1, and cultured in an incubator for 3 h to allow them to adhere. The cell state was observed before staining, and then the culture medium was removed and the cells were rinsed 3 times with PBS, fixed with 4% paraformaldehyde for 15 min, and then the formaldehyde was removed and the cells were rinsed 3 times with PBS. 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, and incubated in the dark for 30 min. The staining solution was removed, and the cells were washed 2 times with distilled water and PBS respectively. Finally, the cells were observed and photographed under a microscope. The results are as Figures 5 - 6 shown. More cell differentiation was observed when using the polypeptide hydrogel of Example 1, indicating that the polypeptide hydrogel of Example 1 can better induce cell osteogenic differentiation.

[0088] Experimental Example 4

[0089] Cell chondrogenic differentiation experiment: Mesenchymal stem cells in the logarithmic phase were respectively dropped into cell culture plates containing the polypeptide hydrogel 8-gel of Example 1 and the polypeptide hydrogel 6-gel of Comparative Example 1, and cultured in an incubator for 3 h to allow them to adhere. The cell status was observed before staining, and then the culture medium was removed and the cells were rinsed 3 times with PBS. 4% neutral formaldehyde solution was added to fix the cells for 45 min, the fixing solution was discarded and the cells were washed 2 times with PBS again. Then, an appropriate amount of Alcian blue staining solution was added and incubated in the dark for 30 min. The dye was discarded and the cells were washed 2 times with PBS. Finally, the cells were observed and photographed under a microscope. The results are as Figures 7 - 8 shown. More cell differentiation occurred with the polypeptide hydrogel of Example 1, indicating that the polypeptide hydrogel of Example 1 can better induce cell chondrogenic differentiation.

[0090] Experimental Example 5

[0091] 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 semi-circular gels, and then their cut surfaces were brought into contact to observe their self-healing performance. The results are as Figures 9 - 10 shown. The two semi-circular hydrogels of the polypeptide hydrogel of Comparative Example 1 remained separated after contacting each other for 12 h. For the polypeptide hydrogel of Example 1, after only 2 minutes, the contact surface of the two semi-circular gels healed and became a complete circular hydrogel without obvious cracks.

[0092] Although the specific embodiments of the present invention have been described in detail in conjunction with the examples, it should not be construed as a limitation on the protection scope 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 efforts still fall within the protection scope 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: dissolving the polypeptide polymer in water and adjusting the pH to 7-8 to obtain a polypeptide hydrogel with adjustable stress relaxation; The carrier resin is Rink Amide resin; 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 a solvent and an ethyl 2-oxime cyanoacetate solution with DMF as a solvent; 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.

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 washing in S2 is washing with DMF 3 times and then washing with dichloromethane 2 times.

4. 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.

5. 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.

6. 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 5.

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