An injectable polypeptide hydrogel that rapidly forms in situ in the body and a preparation method thereof

Abstract

The invention discloses an injectable polypeptide hydrogel that is rapidly formed in situ in vivo and a preparation method thereof, and relates to the technical field of polypeptide hydrogel preparation. The preparation method comprises the following steps: (1) modifying the click reaction active groups of sulfhydryl and maleimide at both ends of the polypeptide to obtain a hybrid polypeptide molecule; configuring into an aqueous solution to obtain an α-helical parallel dimer aqueous solution; (2) adding tris(2-carboxyethyl)phosphine hydrochloride and mixing to obtain a mixed solution; then polymerizing at room temperature to obtain a rigid polymer solution; (3) adjusting the pH value, standing or injecting into the body at room temperature to obtain a hydrogel. The hydrogel of the present invention has the characteristics of pH response, adjustable mechanical strength, injection in liquid form, and rapid in situ formation of a hydrogel in a physiological environment, effectively solving the problems of low mechanical strength, poor biosafety, and poor adhesion of the hydrogel to damaged tissue in the existing injectable hydrogel technology.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide hydrogel preparation, and in particular to an injectable polypeptide hydrogel that is rapidly formed in situ in vivo and a preparation method thereof. Background Art

[0002] Hydrogels are highly hydrated three-dimensional networks formed by cross-linking polymer chains. They have a wide range of applications in biomedicine, tissue engineering, drug delivery, soft robotics, and sensing, and have garnered significant attention. Injectable hydrogels are hydrogels that can be delivered to a target site via injection. Due to their viscoelasticity and diffusivity, injectable hydrogels can promote tissue regeneration in a variety of ways, from simple mechanical support and spatiotemporally controlled cell or therapeutic agent delivery to the local recruitment and regulation of host cells to promote tissue regeneration. Currently, injectable hydrogels have been widely used for local drug storage, sustained drug release, avoiding first-pass effects, and improving patient compliance, and have achieved encouraging results in the treatment of various diseases, including trauma, cardiovascular disease, bone repair, and tumors.

[0003] However, because reported injectable hydrogels must be implanted in the form of shaped hydrogels, their injectability requires a high level of fluidity in the hydrogel material itself. Consequently, developed injectable hydrogels generally exhibit poor adhesion to surrounding damaged tissues, and most injectable hydrogels possess only a single biological activity, limiting their potential for multiple therapeutic benefits at the treatment site. Furthermore, low strength is the most obvious and unavoidable drawback of injectable hydrogels. Physical damage within the body can lead to infection and further induce inflammatory responses.

[0004] Therefore, it is very necessary to develop a biosafe injectable hydrogel that can be implanted into animals in liquid form and can quickly form in situ at the implantation site, while also being able to adjust its strength according to therapeutic requirements. Summary of the Invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide an injectable polypeptide hydrogel that can be rapidly formed in situ in vivo and a preparation method thereof. The hydrogel has the characteristics of being responsive to pH, having adjustable strength, being injectable in liquid form, and being able to rapidly form a hydrogel in situ in a physiological environment. This effectively solves the problems of low mechanical strength, poor biosafety, and poor adhesion of the hydrogel to damaged tissue in existing injectable hydrogel technology.

[0006] The present invention solves the above-mentioned technical problem with the following technical solution: providing a method for preparing an injectable polypeptide hydrogel that is rapidly formed in situ in vivo, comprising the following steps:

[0007] (1) Modifying the two ends of the polypeptide with thiol and maleimide click reaction active groups to obtain a hybrid polypeptide molecule; then preparing it into a 50-70 mg / mL aqueous solution, at which time the hybrid polypeptide molecule is in the state of α-helical parallel dimers, to obtain an α-helical parallel dimer aqueous solution;

[0008] (2) adding 0.6 equivalents of tris(2-carboxyethyl)phosphine hydrochloride to the aqueous solution of the α-helical parallel dimer obtained in step (1), and mixing to obtain a mixed solution; then, polymerizing the mixture at room temperature for 24 hours, and then adding maleimide to terminate the reaction to obtain a rigid polymer solution;

[0009] (3) The pH value of the rigid polymer solution obtained in step (2) is adjusted to 7.5, and the solution is allowed to stand at room temperature or injected into the body in the form of a solution to obtain an injectable polypeptide hydrogel that is rapidly formed in situ in the body.

[0010] Furthermore, in step (1), the polypeptide is a SwitCCh polypeptide or a P9SN polypeptide.

[0011] Furthermore, the amino acid sequence of the SwitCCh polypeptide is shown in SEQ ID NO. 1, which is ENQSLEQENSQLKQEISQLEQEIQQLHYG; the amino acid sequence of the P9SN polypeptide is shown in SEQ ID NO. 2, which is ENQSLEQKNSQLKQEISQLEQEIQQLEYG.

[0012] Furthermore, in step (1), the hybrid polypeptide molecule is Mal-SwitCCh-Cys or Mal-P9SN-Cys.

[0013] Furthermore, the hybrid polypeptide molecule Mal-SwitCCh-Cys, whose chemical formula is C 157 H 244 N 44 O 60 S, specifically, the click reaction active groups maleimide and sulfhydryl (introduced through cysteine) are modified at both ends of the SwitCCh polypeptide sequence, and its structure is as follows Figure 1 shown.

[0014] Furthermore, the Mal-SwitCCh-Cys molecule was prepared by the following method:

[0015] S1. The synthesis was performed using the American CEM Liberty Blue fully automatic peptide analyzer with a synthesis equivalent of 0.25 mmol. The amount of various amino acids was calculated according to the preset sequence. 2.51 g of asparagine was dissolved in 21 mL of N'N-dimethylformamide, 1.88 g of cysteine was dissolved in 16 mL of N'N-dimethylformamide, 8.43 g of glutamine was dissolved in 69 mL of N'N-dimethylformamide, 4.52 g of glutamic acid was dissolved in 53 mL of N'N-dimethylformamide, 0.36 g of glycine was dissolved in 6 mL of N'N-dimethylformamide, 0.58 g of histidine was dissolved in 6 mL of N'N-dimethylformamide, 1.14 g of isoleucine was dissolved in 16 mL of N'N-dimethylformamide, 2.62 g of leucine was dissolved in 37 mL of N'N-dimethylformamide, and 1.04 g of lysine was dissolved in 11 mL In N'N' dimethylformamide, weigh 2.46g of serine and dissolve it in 32mL of N'N' dimethylformamide, weigh 0.56g of tyrosine and dissolve it in 6mL of N'N' dimethylformamide; dissolve the polypeptide under ultrasound, and after dissolution, install each reagent tube on the instrument. Weigh 14.21g of ethyl 2-oxime cyanoacetate solid and dissolve it in 100mL of N'N' dimethylformamide to prepare ethyl 2-oxime cyanoacetate solution; take 7.8mL of N,N-diisopropylcarbodiimide solution and mix it in 92.2mL of N'N' dimethylformamide to prepare DIC solution; measure 168mL of 20wt% piperidine solution as deprotection solvent; finally, weigh 0.367mg of Rink amide AM resin and place it in the reaction vessel; CEM Liberty The steps for peptide synthesis on the Blue fully automatic peptide analyzer are as follows: swelling the resin, adding the deprotection solvent, microwave heating to 70°C, removing the deprotection solvent, washing three times, adding the corresponding amino acid, adding the coupling agent, microwave heating to 70°C for 110 seconds or 230 seconds, and washing three times. This process is repeated 30 times for a total of 30 amino acids in the sequence, and the solid-phase synthesis is completed.

[0016] S2. Add 0.45 g of maleimidobutyric acid, 0.38 g of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 0.435 mL of N,N-diisopropylethylamine, and 8 mL of N'N-dimethylformamide to the resin after solid-phase synthesis in S1, and rotate the mixture at room temperature for 1 h. Repeat this process twice.

[0017] S3. Add 10 mL of lysis buffer to the resin obtained in S2, then place on a rotator for 2 h. Then add 30 mL of anhydrous ether and mix thoroughly. Centrifuge at 8000 rpm for 5 min. Repeat the centrifugation three times to obtain a crude white hybrid polypeptide product.

[0018] S4. Dissolve the crude white hybrid polypeptide obtained in S3 in a 10 mg / mL mixed solvent, and then purify it by liquid phase purification, wherein mobile phase A is acetonitrile, mobile phase B is water containing 0.3vt% trifluoroacetic acid, and the elution gradient is 80%-20% water, 20%-80% acetonitrile, elution time is 30 minutes, and the pure product is obtained after 17 minutes;

[0019] S5. The pure product obtained in S4 was evaporated to remove acetonitrile and water, and 20 mL of water was retained. The product was placed in liquid nitrogen for cooling, and lyophilized at -80°C and 1.0 Pa vacuum for 72 h to obtain the pure product powder of Mal-SwitCCh-Cys.

[0020] Furthermore, in step S3, the lysis solution contains 9.25 mL of trifluoroacetic acid, 0.25 mL of water, 0.25 mL of 1,2-ethanedithiol and 0.25 mL of triisopropylsilane.

[0021] Furthermore, in step S4, the mixed solvent is prepared by mixing water and acetonitrile in a volume ratio of 6:4.

[0022] Furthermore, the hybrid polypeptide molecule Mal-P9SN-Cys, whose chemical formula is C 157 H 249 N 43 O 60 S, specifically, the two ends of the P9SN polypeptide sequence are modified with click reaction active groups maleimide and sulfhydryl (introduced through cysteine), and its structure is as follows Figure 2 shown.

[0023] Furthermore, the hybrid polypeptide molecule Mal-P9SN-Cys was prepared by the following method:

[0024] S1. The synthesis was performed using the American CEM Liberty Blue fully automatic peptide analyzer with a synthesis equivalent of 0.25 mmol. The amount of various amino acids was calculated according to the preset sequence. 2.51 g of asparagine was dissolved in 21 mL of N'N-dimethylformamide, 1.88 g of cysteine was dissolved in 16 mL of N'N-dimethylformamide, 8.43 g of glutamine was dissolved in 69 mL of N'N-dimethylformamide, 4.09 g of glutamic acid was dissolved in 48 mL of N'N-dimethylformamide, 0.36 g of glycine was dissolved in 6 mL of N'N-dimethylformamide, 0.58 g of histidine was dissolved in 6 mL of N'N-dimethylformamide, 1.14 g of isoleucine was dissolved in 16 mL of N'N-dimethylformamide, 2.62 g of leucine was dissolved in 37 mL of N'N-dimethylformamide, and 1.97 g of lysine was dissolved in 20 mL In N'N' dimethylformamide, weigh 2.46g of serine and dissolve it in 32mL of N'N' dimethylformamide, weigh 0.56g of tyrosine and dissolve it in 6mL of N'N' dimethylformamide; dissolve the polypeptide under ultrasound, and after dissolution, install each reagent tube on the instrument. Weigh 14.21g of ethyl 2-oxime cyanoacetate solid and dissolve it in 100mL of N'N' dimethylformamide to prepare ethyl 2-oxime cyanoacetate solution; take 7.8mL of N,N-diisopropylcarbodiimide solution and mix it in 92.2mL of N'N' dimethylformamide to prepare DIC solution; measure 168mL of 20wt% piperidine solution as deprotection solvent; finally, weigh 0.367mg of Rink amide AM resin and place it in the reaction vessel; CEM Liberty The steps for peptide synthesis on the Blue fully automatic peptide analyzer are as follows: swelling the resin, adding the deprotection solvent, microwave heating to 70°C, removing the deprotection solvent, washing three times, adding the corresponding amino acid, adding the coupling agent, microwave heating to 70°C for 110 seconds or 230 seconds, and washing three times. This process is repeated 30 times for a total of 30 amino acids in the sequence, and the solid-phase synthesis is completed.

[0025] S2. Add 0.45 g of maleimidobutyric acid, 0.38 g of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 0.435 mL of N,N-diisopropylethylamine, and 8 mL of N'N-dimethylformamide to the resin after solid-phase synthesis in S1, and rotate the mixture at room temperature for 1 h. Repeat this process twice.

[0026] S3. Add 10 mL of lysis buffer to the resin obtained in S2, then place on a rotator for 2 h. Then add 30 mL of anhydrous ether and mix thoroughly. Centrifuge at 8000 rpm for 5 min. Repeat the centrifugation three times to obtain a crude white hybrid polypeptide product.

[0027] S4. Dissolve the crude white hybrid polypeptide obtained in S3 in a 10 mg / mL mixed solvent, and then purify it by liquid phase purification, wherein mobile phase A is acetonitrile, mobile phase B is water containing 0.3vt% trifluoroacetic acid, and the elution gradient is 80%-20% water, 20%-80% acetonitrile, elution time is 30 minutes, and the pure product is obtained after 17 minutes;

[0028] S5. The pure product obtained in S4 was evaporated to remove acetonitrile and water, and 20 mL of water was retained. The product was placed in liquid nitrogen for cooling, and lyophilized at -80°C and 1.0 Pa vacuum for 72 h to obtain the pure product powder of Mal-P9SN-Cys.

[0029] Furthermore, in step S3, the lysis solution contains 9.25 mL of trifluoroacetic acid, 0.25 mL of water, 0.25 mL of 1,2-ethanedithiol and 0.25 mL of triisopropylsilane.

[0030] Furthermore, in step (1), a 60 mg / mL aqueous solution is prepared.

[0031] Furthermore, the rigid polymer solution obtained in step (2) was dialyzed in deionized water for 24 hours and freeze-dried to obtain white rigid polymer powder.

[0032] The injectable polypeptide hydrogel that is rapidly formed in situ in vivo is prepared by the method for preparing the injectable polypeptide hydrogel that is rapidly formed in situ in vivo.

[0033] The present invention has the following beneficial effects:

[0034] 1. The present invention first grafts maleimide and cysteine onto a polypeptide to form a hybrid polypeptide. After being prepared into an aqueous solution, the hybrid polypeptide is in the state of an α-helical parallel dimer. Under acidic conditions (pH 4), it forms a rigid polymer through head-to-tail covalent polymerization. By adjusting the environmental pH to neutral, the rigid polymer can be split into two to form flexible polypeptide chains that can be entangled with each other, thereby forming a hydrogel network. Figure 3 As shown. And the polypeptide molecule can be replaced by other responsive α-helical dimer polypeptides, not just limited to SwitCCh and P9SN; the polypeptide is achieved by the click reaction between maleimide and the sulfhydryl group on cysteine to achieve head-to-tail covalent polymerization of the polypeptide. This process can be replaced by other forms of intermolecular covalent condensation to achieve head-to-tail connection between dimers, thereby forming a rigid polymer, such as Figure 4 At the same time, the hydrogel has the characteristics of being pH-responsive, having adjustable strength, being injectable in liquid form, and being able to rapidly form a hydrogel in situ in a physiological environment. This effectively solves the problems of low strength, poor biosafety, and poor adhesion of the hydrogel to damaged tissue in existing technologies.

[0035] 2. The present invention has developed a hydrogel that can be injected into the body in liquid form. The hydrogel slowly forms at room temperature, and heating can significantly accelerate its solution-hydrogel conversion process. Therefore, after the hydrogel solution is implanted in the body, it can be heated in situ within only 5 minutes under the action of body temperature.

[0036] 3. The injectable polypeptide hydrogel of the present invention, which rapidly forms in situ in vivo, not only exhibits excellent injectability but also adheres to contacting tissues through in situ gelation. Furthermore, the hydrogel's strength can be adjusted over a wide range by adjusting its concentration to suit the requirements of different tissues or organs, thus overcoming the shortcomings of conventional injectable hydrogels in terms of strength. The rigid polymer solution, the precursor of the polypeptide hydrogel, remains liquid at high concentrations and can be injected in situ to form a high-strength hydrogel. It also exhibits high biosafety and good tissue compatibility.

[0037] 4. The precursor of the injectable polypeptide hydrogel that is rapidly formed in situ in vivo is a rigid polymer freeze-dried powder, which is convenient for long-term storage and long-distance transportation. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the structure of the hybrid polypeptide molecule Mal-SwitCCh-Cys;

[0039] Figure 2 Schematic diagram of the structure of the hybrid polypeptide molecule Mal-P9SN-Cys;

[0040] Figure 3 Schematic diagram of the flexible chain entanglement of polypeptide hydrogel;

[0041] Figure 4 Schematic diagram of the formation of rigid polymer;

[0042] Figure 5 Schematic diagram of the Mal-SwitCCh-Cys mass spectrometry results of hybrid polypeptide molecules;

[0043] Figure 6 Schematic diagram of the hybrid polypeptide molecule Mal-SwitCCh-Cys in the state of α-helical parallel dimer;

[0044] Figure 7 The pH of the rigid polymer solution was adjusted to 7.5 to form a hydrogel and the rheological results of the hydrogel;

[0045] Figure 8 Schematic diagram of the rapid formation of hydrogel in mice after adjusting the pH to neutral. DETAILED DESCRIPTION

[0046] The principles and features of the present invention are described below. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, conventional conditions or manufacturer-recommended conditions were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0047] Example 1

[0048] An injectable polypeptide hydrogel that is rapidly formed in situ in vivo, the preparation method of which comprises the following steps:

[0049] (1) The two ends of the SwitCCh polypeptide were modified with thiol and maleimide click reaction active groups to obtain the hybrid polypeptide molecule Mal-SwitCCh-Cys. The mass spectrometry results are as follows: Figure 5 As shown; then it is prepared into a 60 mg / mL aqueous solution, at which time the hybrid polypeptide molecule is in the state of α-helical parallel dimers, as shown Figure 6 As shown, an aqueous solution of α-helical parallel dimers was obtained;

[0050] (2) adding 0.6 equivalents of tris(2-carboxyethyl)phosphine hydrochloride to the aqueous solution of the Mal-SwitCCh-Cys hybrid polypeptide parallel dimer obtained in step (1) and mixing to obtain a mixed solution; then, polymerizing the solution at room temperature for 24 hours, adding maleimide to terminate the reaction, thereby obtaining a rigid polymer solution;

[0051] (3) The pH value of the rigid polymer solution obtained in step (2) was adjusted to 7.5, and the solution was allowed to stand at room temperature for 2 hours to obtain an injectable polypeptide hydrogel that was rapidly formed in situ in vivo. The rheological results of this process are shown in FIG. Figure 7 shown.

[0052] Among them, the hybrid polypeptide molecule Mal-SwitCCh-Cys has a chemical formula of C 157 H 244 N 44 O 60 S, specifically, the click reaction active groups maleimide and sulfhydryl (introduced through cysteine) are modified at both ends of the SwitCCh polypeptide sequence, and its structure is as follows Figure 1 shown.

[0053] In step (1), the hybrid polypeptide molecule Mal-SwitCCh-Cys is prepared by the following method:

[0054] S1. The synthesis was performed using the American CEM Liberty Blue fully automatic peptide analyzer with a synthesis equivalent of 0.25 mmol. The amount of various amino acids was calculated according to the preset sequence. 2.51 g of asparagine was dissolved in 21 mL of N'N-dimethylformamide, 1.88 g of cysteine was dissolved in 16 mL of N'N-dimethylformamide, 8.43 g of glutamine was dissolved in 69 mL of N'N-dimethylformamide, 4.52 g of glutamic acid was dissolved in 53 mL of N'N-dimethylformamide, 0.36 g of glycine was dissolved in 6 mL of N'N-dimethylformamide, 0.58 g of histidine was dissolved in 6 mL of N'N-dimethylformamide, 1.14 g of isoleucine was dissolved in 16 mL of N'N-dimethylformamide, 2.62 g of leucine was dissolved in 37 mL of N'N-dimethylformamide, and 1.04 g of lysine was dissolved in 11 mL In N'N' dimethylformamide, weigh 2.46g of serine and dissolve it in 32mL of N'N' dimethylformamide, weigh 0.56g of tyrosine and dissolve it in 6mL of N'N' dimethylformamide; dissolve the polypeptide under ultrasound, and after dissolution, install each reagent tube on the instrument. Weigh 14.21g of ethyl 2-oxime cyanoacetate solid and dissolve it in 100mL of N'N' dimethylformamide to prepare ethyl 2-oxime cyanoacetate solution; take 7.8mL of N,N-diisopropylcarbodiimide solution and mix it in 92.2mL of N'N' dimethylformamide to prepare DIC solution; measure 168mL of 20wt% piperidine solution as deprotection solvent; finally, weigh 0.367mg of Rink amide AM resin and place it in the reaction vessel; CEM Liberty The steps for peptide synthesis on the Blue fully automatic peptide analyzer are as follows: swelling the resin, adding the deprotection solvent, microwave heating to 70°C, removing the deprotection solvent, washing three times, adding the corresponding amino acid, adding the coupling agent, microwave heating to 70°C for 110 seconds or 230 seconds, and washing three times. This process is repeated 30 times for a total of 30 amino acids in the sequence, and the solid-phase synthesis is completed.

[0055] S2. Add 0.45 g of maleimidobutyric acid, 0.38 g of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 0.435 mL of N,N-diisopropylethylamine, and 8 mL of N'N-dimethylformamide to the resin after solid-phase synthesis in S1, and rotate the mixture at room temperature for 1 h. Repeat this process twice.

[0056] S3. Add 10 mL of lysis buffer to the resin obtained in S2, then place on a rotator for lysis for 2 h, then add 30 mL of anhydrous ether and mix thoroughly, centrifuge at 8000 rpm for 5 min, and repeat the centrifugation three times to obtain a crude white hybrid polypeptide product; the lysis buffer contains 9.25 mL of trifluoroacetic acid, 0.25 mL of water, 0.25 mL of 1,2-ethanedithiol, and 0.25 mL of triisopropylsilane;

[0057] S4. Dissolve the crude white hybrid polypeptide obtained in S3 in a 10 mg / mL mixed solvent, and then purify by liquid phase purification, wherein mobile phase A is acetonitrile, mobile phase B is water containing 0.3vt% trifluoroacetic acid, and the elution gradient is 80%-20% water, 20%-80% acetonitrile, elution time is 30 minutes, and the pure product is obtained after 17 minutes; the mixed solvent is a mixture of water and acetonitrile in a volume ratio of 6:4;

[0058] S5. The pure product obtained in S4 was evaporated to remove acetonitrile and water, and 20 mL of water was retained. The product was placed in liquid nitrogen for cooling, and lyophilized at -80°C and 1.0 Pa vacuum for 72 h to obtain the pure product powder of Mal-SwitCCh-Cys.

[0059] The rigid polymer solution obtained in step (2) was dialyzed in deionized water for 24 hours and freeze-dried to obtain a white rigid polymer powder. The powder was dissolved in deionized water and the pH was adjusted to 7.5 before being injected subcutaneously into mice. A hydrogel was formed within 2 minutes and a stable colloid was obtained within 5 minutes. Figure 8 shown.

[0060] Example 2

[0061] An injectable polypeptide hydrogel that is rapidly formed in situ in vivo, the preparation method of which comprises the following steps:

[0062] (1) The two ends of the P9SN polypeptide were modified with thiol and maleimide click reaction active groups, respectively, to obtain a hybrid polypeptide molecule Mal-P9SN-Cys; then, a 60 mg / mL aqueous solution was prepared, in which the hybrid polypeptide molecule was in the state of an α-helical parallel dimer, to obtain an α-helical parallel dimer aqueous solution;

[0063] (2) adding 0.6 equivalents of tris(2-carboxyethyl)phosphine hydrochloride to the aqueous solution of the hybrid polypeptide parallel dimer obtained in step (1), and mixing to obtain a mixed solution; then, polymerizing the mixture at room temperature for 24 hours, adding maleimide to terminate the reaction, and obtaining a rigid polymer solution;

[0064] (3) The pH value of the rigid polymer solution obtained in step (2) is adjusted to 7.5, and the solution is allowed to stand at room temperature for 2 hours to obtain an injectable polypeptide hydrogel that is rapidly formed in situ in vivo.

[0065] Wherein, in step (1), the hybrid polypeptide molecule Mal-P9SN-Cys is prepared by the following method:

[0066] S1. The synthesis was performed using the American CEM Liberty Blue fully automatic peptide analyzer with a synthesis equivalent of 0.25 mmol. The amount of various amino acids was calculated according to the preset sequence. 2.51 g of asparagine was dissolved in 21 mL of N'N-dimethylformamide, 1.88 g of cysteine was dissolved in 16 mL of N'N-dimethylformamide, 8.43 g of glutamine was dissolved in 69 mL of N'N-dimethylformamide, 4.09 g of glutamic acid was dissolved in 48 mL of N'N-dimethylformamide, 0.36 g of glycine was dissolved in 6 mL of N'N-dimethylformamide, 0.58 g of histidine was dissolved in 6 mL of N'N-dimethylformamide, 1.14 g of isoleucine was dissolved in 16 mL of N'N-dimethylformamide, 2.62 g of leucine was dissolved in 37 mL of N'N-dimethylformamide, and 1.97 g of lysine was dissolved in 20 mL In N'N' dimethylformamide, weigh 2.46g of serine and dissolve it in 32mL of N'N' dimethylformamide, weigh 0.56g of tyrosine and dissolve it in 6mL of N'N' dimethylformamide; dissolve the polypeptide under ultrasound, and after dissolution, install each reagent tube on the instrument. Weigh 14.21g of ethyl 2-oxime cyanoacetate solid and dissolve it in 100mL of N'N' dimethylformamide to prepare ethyl 2-oxime cyanoacetate solution; take 7.8mL of N,N-diisopropylcarbodiimide solution and mix it in 92.2mL of N'N' dimethylformamide to prepare DIC solution; measure 168mL of 20wt% piperidine solution as deprotection solvent; finally, weigh 0.367mg of Rink amide AM resin and place it in the reaction vessel; CEM Liberty The steps for peptide synthesis on the Blue fully automatic peptide analyzer are as follows: swelling the resin, adding the deprotection solvent, microwave heating to 70°C, removing the deprotection solvent, washing three times, adding the corresponding amino acid, adding the coupling agent, microwave heating to 70°C for 110 seconds or 230 seconds, and washing three times. This process is repeated 30 times for a total of 30 amino acids in the sequence, and the solid-phase synthesis is completed.

[0067] S2. Add 0.45 g of maleimidobutyric acid, 0.38 g of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 0.435 mL of N,N-diisopropylethylamine, and 8 mL of N'N-dimethylformamide to the resin after solid-phase synthesis in S1, and rotate the mixture at room temperature for 1 h. Repeat this process twice.

[0068] S3. Add 10 mL of lysis buffer to the resin obtained in S2, then place on a rotator for lysis for 2 h, then add 30 mL of anhydrous ether and mix thoroughly, centrifuge at 8000 rpm for 5 min, and repeat the centrifugation three times to obtain a crude white hybrid polypeptide product; the lysis buffer contains 9.25 mL of trifluoroacetic acid, 0.25 mL of water, 0.25 mL of 1,2-ethanedithiol, and 0.25 mL of triisopropylsilane;

[0069] S4. Dissolve the crude white hybrid polypeptide obtained in S3 in a 10 mg / mL mixed solvent, and then purify it by liquid phase purification, wherein mobile phase A is acetonitrile, mobile phase B is water containing 0.3vt% trifluoroacetic acid, and the elution gradient is 80%-20% water, 20%-80% acetonitrile, elution time is 30 minutes, and the pure product is obtained after 17 minutes;

[0070] S5. The pure product obtained in S4 was evaporated to remove acetonitrile and water, and 20 mL of water was retained. The product was placed in liquid nitrogen for cooling, and lyophilized at -80°C and 1.0 Pa vacuum for 72 h to obtain the pure product powder of Mal-SwitCCh-Cys.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing an injectable polypeptide hydrogel that is rapidly formed in situ in vivo, characterized in that: The following steps are involved: (1) Modify the two ends of the polypeptide with thiol and maleimide click reaction active groups to obtain a hybrid polypeptide molecule; then prepare it into a 50-70 mg / mL aqueous solution, at which time the hybrid polypeptide molecule is in the state of α-helical parallel dimer, and obtain an α-helical parallel dimer aqueous solution; (2) adding 0.6 equivalents of tris(2-carboxyethyl)phosphine hydrochloride to the aqueous solution of the α-helical parallel dimer obtained in step (1) and mixing them to obtain a mixed solution; then, polymerizing the mixture at room temperature for 24 hours, adding maleimide to terminate the reaction, thereby obtaining a rigid polymer solution; (3) adjusting the pH value of the rigid polymer solution obtained in step (2) to 7.5, allowing it to stand at room temperature or injecting it into the body in the form of a solution to obtain an injectable polypeptide hydrogel that is rapidly formed in situ in the body; In step (1), the polypeptide is a SwitCCh polypeptide or a P9SN polypeptide; the amino acid sequence of the SwitCCh polypeptide is shown in SEQ ID NO.1, which is: ENQSLEQENSQLKQEISQLEQEIQQLHYG; the amino acid sequence of the P9SN polypeptide is shown in SEQ ID NO.2, which is: ENQSLEQKNSQLKQEISQLEQEIQQLEYG.

2. The method for preparing an injectable polypeptide hydrogel that is rapidly formed in situ in vivo according to claim 1, wherein: In step (1), the hybrid polypeptide molecule is Mal-SwitCCh-Cys or Mal-P9SN-Cys.

3. The method for preparing an injectable polypeptide hydrogel that is rapidly formed in situ in vivo according to claim 1, wherein: In step (1), a 60 mg / mL aqueous solution is prepared.

4. The method for preparing an injectable polypeptide hydrogel that is rapidly formed in situ in vivo according to claim 1, wherein: The rigid polymer solution obtained in step (2) was dialyzed in deionized water for 24 h and freeze-dried to obtain white rigid polymer powder.

5. The injectable polypeptide hydrogel that is rapidly formed in situ in vivo is prepared by the method for preparing the injectable polypeptide hydrogel that is rapidly formed in situ in vivo according to any one of claims 1 to 4.

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