A polyamino acid hydrogel, its preparation method and application

Abstract

The present invention relates to a polyamino acid hydrogel and its preparation method and application. The method comprises the following steps: mixing glutamic acid-N-carboxyl anhydride or side-chain-modified glutamic acid-N-carboxyl anhydride with N-carboxyl anhydride containing a sulfhydryl group with a solvent, adding an initiator to react, and obtaining polyamino acid I; dissolving sarcosine-N-carboxyl anhydride with a solvent, then adding a multifunctional initiator to react, and obtaining a polymeric intermediate; then dissolving the polymeric intermediate with a solvent, and adding 3-maleimidopropionic acid hydroxysuccinimide ester to react, and obtaining polyamino acid II; preparing a polyamino acid I solution and a polyamino acid II solution, and then mixing the solution with a short peptide solution, and gelling to obtain a polyamino acid hydrogel. The polyamino acid hydrogel obtained by the present invention has excellent biocompatibility and biodegradability, can well support stem cell growth and cartilage differentiation, and has good immunoregulatory properties.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a polyamino acid hydrogel and a preparation method and application thereof. Background Art

[0002] Articular cartilage damage is an extremely common joint lesion. Mature articular cartilage lacks a blood supply and has limited self-repair capabilities. Left untreated, osteochondral defects increase the risk of future osteoarthritis by sixfold. Furthermore, osteoarticular diseases are a leading cause of healthcare expenditure, making early treatment and intervention crucial.

[0003] There are many ways to treat cartilage defects. Currently, the most commonly used method in clinical practice is microfracture repair. However, the human articular cartilage is hyaline cartilage, and the cartilage repaired by microfracture is fibrocartilage. The mechanical strength of fibrocartilage is not as high as that of hyaline cartilage, so it is more easily broken under the pressure of repetitive daily activities. Therefore, microfracture cannot repair the actual damaged cartilage itself, but only provides a substitute with similar properties. Currently, tissue engineering technology provides a very promising treatment strategy for cartilage damage. Suitable tissue engineering scaffolds can play a local supporting role and enable the damaged tissue to be reconstructed into suitable living tissue. The structure of hydrogel is similar to the extracellular matrix of natural cartilage, so it is a very important material for cartilage repair and regeneration.

[0004] Hydrogels are three-dimensional mesh materials formed by cross-linking hydrophilic polymer chains, typically with a water content exceeding 70%. Due to their high water content, large porosity, and excellent biocompatibility, they are currently being developed for applications such as controlled drug release and cell culture scaffolds.

[0005] Compared to non-covalently cross-linked hydrogels, covalently cross-linked hydrogels typically exhibit higher mechanical properties and a moderate degradation rate, making them more suitable for cartilage tissue repair. However, the polymers used in traditional covalently cross-linked hydrogel design often have poor chemical modifiability and are difficult to be compatible with biological organisms.

[0006] Polyethylene glycol-based hydrogels are currently widely used in research and clinical applications. Polyethylene glycol has good water solubility and excellent biocompatibility. However, its widespread use in food, pharmaceuticals, cosmetics, and other fields has led to the development of antibodies against it in most people, making it potentially immunogenic. Furthermore, polyethylene glycol is difficult to degrade in the body, posing certain safety risks.

[0007] Polyamino acids are a type of polymer material with broad biological application prospects. The chemical diversity of their side chains allows for easy modification or cross-linking. At the same time, their main chain structure, similar to that of natural polypeptides, enables them to form secondary structures similar to proteins, such as α-helices and β-sheets, thereby improving the mechanical properties of the material. The material also possesses excellent biocompatibility and biodegradability. Polyamino acid hydrogels have been widely used in drug delivery, stem cell culture, artificial tissue, and other fields. However, at present, the application range of polyamino acid hydrogels in cartilage repair is relatively narrow, and the types of monomers used are relatively limited. In addition, to achieve both water solubility and low swelling properties, polyamino acid precursors are often prepared by blocking with polyethylene glycol segments, but this also introduces issues with the immunogenicity and degradability of polyethylene glycol segments.

[0008] Therefore, it is necessary to design a new type of polymer with low immunogenicity, high biocompatibility and excellent degradability as a hydrogel substrate for cartilage repair. Summary of the Invention

[0009] In order to solve one or more technical problems existing in the prior art, the present invention provides a polyamino acid hydrogel and a preparation method and application thereof.

[0010] In a first aspect, the present invention provides a method for preparing a polyamino acid hydrogel, the method comprising the following steps:

[0011] (1) uniformly mixing glutamic acid-N-carboxylic anhydride or side-chain-modified glutamic acid-N-carboxylic anhydride with N-carboxylic anhydride containing a thiol group using a first solvent, and then adding an initiator to carry out a polymerization reaction to obtain polyamino acid I;

[0012] (2) dissolving sarcosine-N-carboxylic anhydride in a second solvent, then adding a multifunctional initiator to carry out a polymerization reaction to obtain a polymer intermediate, then dissolving the polymer intermediate in a third solvent and adding 3-maleimidopropionic acid hydroxysuccinimide ester to carry out a polymer end group modification reaction to obtain polyamino acid II;

[0013] (3) Polyamino acid I and polyamino acid II are prepared into polyamino acid I solution and polyamino acid II solution respectively using phosphate buffer, and then the polyamino acid I solution, polyamino acid II solution and short peptide solution are mixed evenly and gelled to obtain polyamino acid hydrogel.

[0014] Preferably, the side chain modification group of the glutamic acid-N-carboxylic anhydride is selected from one or more of ethylene glycol, diethylene glycol, diethylene glycol monomethyl ether, triethylene glycol monomethyl ether, tetraethylene glycol monomethyl ether, ethanolamine, 3-hydroxypropionic acid and lactic acid; the N-carboxylic anhydride containing a thiol group is selected from one or more of cysteine-N-carboxylic anhydride, homocysteine-N-carboxylic anhydride and penicillamine-N-carboxylic anhydride; the initiator in step (1) is a reagent containing an amino group or a thiol group with a certain nucleophilicity, preferably, the initiator includes but is not limited to one or more of n-hexylamine, benzylamine, mercaptoethanol, methyl glutamate, diethylamine, isopropylamine and cyclohexylamine, more preferably, the initiator is benzylamine; and / or the short peptide contained in the short peptide solution is cysteine-arginine-glycine-aspartic acid.

[0015] Preferably, the side chain modification group of the glutamic acid-N-carboxyl anhydride is triethylene glycol monomethyl ether, and the structural formula of the triethylene glycol monomethyl ether-modified glutamic acid-N-carboxyl anhydride is shown in the following formula a:

[0016]

[0017] The N-carboxyl anhydride containing a thiol group is cysteine-N-carboxyl anhydride, and the structural formula of the cysteine-N-carboxyl anhydride is shown in the following formula b:

[0018]

[0019] The polyamino acid I is polyamino acid P (EG3Glu-co-Cys), and the structural formula is shown in the following formula I:

[0020]

[0021] The polyamino acid II is polyamino acid PSar-Mal4, and its structural formula is shown in the following formula II:

[0022]

[0023] Preferably, in step (1), the molar ratio of the side chain-modified glutamic acid-N-carboxyl anhydride, the thiol-containing N-carboxyl anhydride and the initiator is (200-400): (10-100): 1, preferably 250:50: 1; the concentration of the initiator is 0.3-0.6 mol / L, preferably 0.5 mol / L; the amount of the first solvent is 2-8 mL, preferably 3 mL; the temperature of the polymerization reaction is room temperature, and the polymerization reaction time is 3-6 h, preferably 4 h; and / or the polymerization reaction uses unprotected thiol-containing N-carboxyl anhydride directly as a polymerization monomer to participate in the reaction, so that the obtained polyamino acid I has a branched structure.

[0024] Preferably, in step (2): the multifunctional initiator is a compound having multiple nucleophilic amino groups and / or thiol groups, preferably, the multifunctional initiator includes but is not limited to one or more of ethylenediamine, triaminoethylamine, pentaerythritol, lysine, and lysine methyl ester, more preferably, the multifunctional initiator is pentaerythritol; the molar ratio of the sarcosine-N-carboxylic anhydride, the multifunctional initiator and the 3-maleimidopropionic acid hydroxysuccinimide ester is 1:(0.004-0.006):(0.05-0.15), preferably 1:0.005:0.1; the temperature of the polymerization reaction is room temperature, and the time of the polymerization reaction is 18-30 hours, preferably 24 hours; and / or the temperature of the polymer end group modification reaction is room temperature, and the time of the polymer end group modification reaction is 18-30 hours, preferably 24 hours.

[0025] Preferably, in step (3): the concentration of the polyamino acid I solution is 80-120 mg / mL, preferably 100 mg / mL; and / or the concentration of the polyamino acid II solution is 120-180 mg / mL, preferably 150 mg / mL.

[0026] Preferably, in step (3): the short peptide solution is prepared using phosphate buffer; and / or the molar ratio of the total amount of thiol groups contained in the polyamino acid I solution and the short peptide solution to the maleimide groups contained in the polyamino acid II solution is (0.9-1.1):1, preferably 1:1.

[0027] Preferably, in step (3): the mass fraction of the prepared polyamino acid hydrogel is 5% to 20%, preferably 10%.

[0028] In a second aspect, the present invention provides a polyamino acid hydrogel prepared by the preparation method described in the first aspect of the present invention.

[0029] In a third aspect, the present invention provides the use of the polyamino acid hydrogel prepared by the preparation method described in the first aspect of the present invention in the field of biomedicine; preferably, it is used in stem cell culture and tissue engineering, and more preferably, it is used as a cartilage repair material in stem cell culture and tissue engineering.

[0030] Compared with the prior art, the present invention has at least the following beneficial effects:

[0031] (1) The present invention addresses the problems of poor biocompatibility and difficult degradation of traditional carbon skeleton cartilage repair hydrogels. By adopting a full polyamino acid skeleton and cross-linking agent design, the obtained polyamino acid hydrogel has excellent biocompatibility and biodegradability.

[0032] (2) The main chain of the polyamino acid hydrogel in the present invention contains a thioester bond, which has higher chemical activity than an amide bond, making the polyamino acid hydrogel have better degradation performance.

[0033] (3) In some preferred embodiments of the present invention, a polyglutamic acid derivative with a side chain modified with triethylene glycol monomethyl ether is selected as the polyamino acid gel skeleton. The polyamino acid monomer itself is electrically neutral and has good water solubility, thereby solving the problem of high swelling ratio caused by the need to introduce ionic amino acids in traditional polyamino acid gels, and also avoiding the potential immunogenicity and degradation risks caused by the introduction of long-chain polyethylene glycol.

[0034] (4) In some preferred embodiments of the present invention, cysteine-N-carboxylic anhydride (CysNCA) is used as a comonomer to prepare polyamino acids containing a certain amount of thiol groups. This method is prepared by a one-step synthesis method. The synthesis method is simple and convenient. Compared with the protection-deprotection strategy and post-modification strategy commonly used in traditional methods, the operation is simpler and the modification efficiency is more controllable.

[0035] (5) The CysNCA copolymerization strategy selected in the present invention adds NCA containing active thiol groups into the copolymerization system, so that the obtained polyamino acid has a branched structure.

[0036] (6) In some preferred embodiments of the present invention, the polyamino acid backbone P( L -EG3Glu-co- L -Cys) presents an α-helical secondary structure in aqueous solution. At the same time, the presence of thiol groups during the polymerization process gives it certain branching characteristics. The presence of helical structure and branching gives the hydrogel stronger and more adjustable mechanical properties compared to traditional polyamino acid hydrogels, making it suitable for cartilage repair.

[0037] (7) The crosslinker used in the present invention is a four-arm polysarcosine functionalized with maleimide end groups, which forms a chemically crosslinked hydrogel through a maleimide-thiol reaction. This reaction uses an in situ crosslinking strategy, enabling rapid gelation at room temperature after mixing the two precursors, facilitating mixing with cells and other functional substances, while also being simple to operate.

[0038] (8) The polyamino acid hydrogel prepared by the present invention has excellent biocompatibility and can well support stem cell growth and cartilage differentiation. Its degradation process is consistent with the cartilage repair process, and it has an immunomodulatory function, promoting the differentiation of macrophages to the M2 phenotype and showing immunomodulatory properties during long-term implantation.

[0039] (9) Compared with traditional cartilage repair hydrogel materials, the polyamino acid hydrogel obtained in the present invention has better immunomodulatory properties and can well induce the polarization of M2 macrophages, ultimately achieving the effect of promoting tissue repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The polyamino acid P (EG3Glu-co-Cys) in Example 1 of the present invention is 1 H NMR spectrum (400M, D2O).

[0041] Figure 2 This is a size exclusion chromatography curve (DMF) of the polyamino acid P (EG3Glu-co-Cys) in Example 1 of the present invention.

[0042] Figure 3 This is a circular dichroism spectrum curve of the polyamino acid P (EG3Glu-co-Cys) in Example 1 of the present invention.

[0043] Figure 4 The polyamino acid PSar-Mal4 in Example 1 of the present invention is 1 H NMR spectrum (400M, D2O).

[0044] Figure 5 Schematic diagram of the synthesis of the polyamino acid hydrogel PAA-RGD obtained in Example 1 of the present invention.

[0045] Figure 6 This is the rheological oscillation frequency sweep curve of the polyamino acid hydrogel PAA-RGD obtained in Example 1 of the present invention. This curve was obtained through oscillation frequency sweep testing using a rotational rheometer. The test temperature was 25°C. Before the test, the gel was first subjected to an oscillation amplitude sweep at a frequency of 1 Hz to determine its linear viscoelastic region. Subsequently, an oscillation frequency sweep was performed within the linear viscoelastic region over a sweep range of 0.01-100 rad / s. The results demonstrate that the obtained polyamino acid hydrogel is a relatively stable, self-supporting gel.

[0046] Figure 7 This is the in vitro swelling curve of the polyamino acid hydrogel PAA-RGD obtained in Example 1 of the present invention. The polyamino acid hydrogel (50 μL) prepared in the present invention was immersed in phosphate buffer at 37°C for a specified time, and the mass of the gel after swelling was recorded. The ratio of the mass after swelling to the initial mass before swelling is the swelling ratio.

[0047] Figure 8 This is the compression test curve of the polyamino acid hydrogel PAA-RGD obtained in Example 1 of the present invention.

[0048] Figure 9This is a scanning electron microscope image of the polyamino acid hydrogel PAA-RGD obtained in Example 1 of the present invention.

[0049] Figure 10 Graphs showing cell death and viability staining results of the polyamino acid hydrogel PAA-RGD obtained in Example 1 of the present invention after culturing PB-MSCs for 3, 7, and 14 days.

[0050] Figure 11 This is a diagram showing the expression of type II collagen in the polyamino acid hydrogel PAA-RGD obtained in Example 1 of the present invention after culturing PB-MSCs for 14 days.

[0051] Figure 12 This is a schematic diagram of cartilage repair using the polyamino acid hydrogel PAA-RGD obtained in the present invention.

[0052] Figure 13 This is a general view of the polyamino acid hydrogel PAA-RGD obtained in Example 1 of the present invention repairing rabbit osteochondral defects after 6 weeks and 12 weeks.

[0053] Figure 14 These are MRI images of the polyamino acid hydrogel PAA-RGD obtained in Example 1 of the present invention repairing rabbit osteochondral defects after 6 and 12 weeks.

[0054] Figure 15 These are micro-CT images of the polyamino acid hydrogel PAA-RGD obtained in Example 1 of the present invention repairing rabbit osteochondral defects after 6 and 12 weeks.

[0055] Figure 16 This is a SEM image of the polyamino acid hydrogel PAA-RGD obtained in Example 1 of the present invention repairing rabbit osteochondral defects 12 weeks later.

[0056] Figure 17 Graphs showing the toluidine blue staining results of the polyamino acid hydrogel PAA-RGD obtained in Example 1 of the present invention repairing rabbit osteochondral defects after 6 and 12 weeks.

[0057] Figure 18 This is a diagram showing the immunohistochemical results of polarized macrophages infiltrating around the polyamino acid hydrogel PAA-RGD obtained in Example 1 of the present invention.

[0058] Figure 19 This is a graph showing the international cartilage repair scoring results of PAA-RGD, PEG-RGD, and GelMA hydrogels in Example 1 of the present invention.

[0059] Figure 20 This is a graph showing the polarization area of M2 infiltrating macrophages around PAA-RGD, PEG-RGD, and GelMA hydrogels in Example 1 of the present invention.

[0060] Figure 21 The structure of PEG-RGD hydrogel and the schematic diagram of the hydrogel.

[0061] Figure 22 Schematic diagram of the structure of GelMA hydrogel.

[0062] In the figure: Regarding the results of the rabbit osteochondral defect repair experiment, Blank corresponds to the experimental results of the blank group in which the poly amino acid hydrogel PAA-RGD in Example 1 was not implanted after the rabbit osteochondral defect, and PAA-RGD corresponds to the experimental results of the poly amino acid hydrogel PAA-RGD in Example 1 was implanted after the rabbit osteochondral defect. DETAILED DESCRIPTION

[0063] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0064] In a first aspect, the present invention provides a method for preparing a polyamino acid hydrogel, the method comprising the following steps:

[0065] (1) using a first solvent to uniformly mix glutamic acid-N-carboxylic anhydride or side chain modified glutamic acid-N-carboxylic anhydride with N-carboxylic anhydride containing a thiol group, and then adding an initiator to carry out a polymerization reaction to obtain a polyamino acid I. Preferably, the polyamino acid I is a polyamino acid P (EG3Glu-co-Cys); in a specific embodiment of the present invention, preferably, before performing the following step (2), the step of purifying the polyamino acid I is further included; in some preferred embodiments of the present invention, the side chain modified glutamic acid-N-carboxylic anhydride is triethylene glycol monomethyl ether modified L -EG3GluNCA, the N-carboxylic anhydride containing a thiol group is cysteine-N-carboxylic anhydride L -CysNCA, the initiator is benzylamine initiator BnNH2, and the reaction formula for preparing the polyamino acid P(EG3Glu-co-Cys) in step (1) of the present invention is as follows:

[0066]

[0067] (2) using a second solvent to dissolve sarcosine-N-carboxylic anhydride SarNCA, then adding a multifunctional initiator to carry out a polymerization reaction to obtain a polymer intermediate, then using a third solvent to dissolve the polymer intermediate and adding 3-maleimidopropionic acid hydroxysuccinimide ester NHS-MAL to carry out a polymer end group modification reaction to obtain polyamino acid II, i.e., polyamino acid PSar-Mal4; in some specific embodiments of the present invention, before the polymer intermediate is subjected to the polymer end group modification reaction, the step of purifying the polymer intermediate is further included; before carrying out the following step (3), the step of purifying the polyamino acid II is further included;

[0068] (3) Polyamino acid I and polyamino acid II are prepared into polyamino acid I solution and polyamino acid II solution respectively using phosphate buffer solution (PBS), and then the polyamino acid I solution, polyamino acid II solution and short peptide solution (such as CRGD solution) are mixed evenly, and gelation is performed (for example, gelation occurs within 10 minutes of standing at room temperature) to obtain a polyamino acid hydrogel, such as PAA-RGD hydrogel.

[0069] The present invention preferably uses NCA ring-opening polymerization to synthesize L -EG3Glu unit of the polyamino acid, the obtained polymer is uncharged, and has good water solubility, suitable for use as a gel skeleton; the present invention preferably uses the cysteine NCA monomer copolymerization method to synthesize the polyamino acid containing cysteine units, avoiding the tedious protection and deprotection steps, introducing sulfhydryl cross-linking points, and is used for the preparation of cartilage repair gel; at the same time, the cysteine NCA monomer gives the material a certain branching feature, so that it has good mechanical properties; the present invention synthesizes a polyamino acid with a specific secondary structure; compared with the polyamino acid with a secondary structure such as a random coil, the obtained polyamino acid with an α-helix The gel material formed by amino acids has higher mechanical properties; the present invention uses maleimide-modified polysarcosine as a cross-linker, and the cross-linker is also completely degradable; the thiol-maleimide reaction is fast, efficient, and easy to operate; the polyamino acid hydrogel obtained by the present invention is covalently linked to the short peptide CRGD and has excellent cell adhesion; of course, the present invention can also use polyamino acid skeletons with different secondary structures such as polyproline to adjust the mechanical properties of the gel to meet the needs of different cell differentiation; and the present invention can introduce more functional small molecules with thiol functional groups and covalently link them to the gel to give the gel more functions.

[0070] According to some preferred embodiments, the side chain modification group of the glutamic acid-N-carboxylic anhydride is selected from one or more of ethylene glycol, diethylene glycol, diethylene glycol monomethyl ether, triethylene glycol monomethyl ether, tetraethylene glycol monomethyl ether, ethanolamine, 3-hydroxypropionic acid and lactic acid; the N-carboxylic anhydride containing a sulfhydryl group is selected from one or more of cysteine-N-carboxylic anhydride, homocysteine-N-carboxylic anhydride and penicillamine-N-carboxylic anhydride; the present invention has no particular restriction on the sources of these side chain modified glutamic acid-N-carboxylic anhydride and the N-carboxylic anhydride containing a sulfhydryl group, for example, they can be products directly purchased on the market or products synthesized by existing methods; the N-carboxylic anhydride in step (1) The initiator is an amino- or thiol-containing reagent with a certain nucleophilicity. Preferably, the initiator includes but is not limited to one or more of n-hexylamine, benzylamine, mercaptoethanol, methyl glutamate, diethylamine, isopropylamine, and cyclohexylamine. More preferably, the initiator is benzylamine. The first solvent, the second solvent, and / or the third solvent are selected from at least one of N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, N-ethylpyrrolidone, and dimethyl sulfoxide. Preferably, the first solvent, the second solvent, and the third solvent are N,N-dimethylformamide. And / or the short peptide contained in the short peptide solution is cysteine-arginine-glycine-aspartic acid.

[0071] According to some preferred embodiments, the side chain modification group of the glutamic acid-N-carboxyl anhydride is triethylene glycol monomethyl ether, and the structural formula of the triethylene glycol monomethyl ether-modified glutamic acid-N-carboxyl anhydride is shown in Formula a below:

[0072]

[0073] The N-carboxyl anhydride containing a thiol group is cysteine-N-carboxyl anhydride, and the structural formula of the cysteine-N-carboxyl anhydride is shown in the following formula b:

[0074]

[0075] The polyamino acid I is polyamino acid P (EG3Glu-co-Cys), and the structural formula is shown in the following formula I:

[0076]

[0077] In formula I, m, n, o, and p are the numbers of repeating units of a copolymer obtained by copolymerizing a certain amount of glutamic acid-N-carboxylic anhydride modified with triethylene glycol monomethyl ether and cysteine-N-carboxylic anhydride in a polyamino acid I, wherein o and p are the numbers of branched chain-containing repeating units and branched segment repeating units in the polymer. The present invention can obtain different values of m, n, o, and p according to different feed ratios. Preferably, according to different feed methods, the optional ratio of m:n:o in the polyamino acid I is (200-400):(4-40):(10-60), and more preferably, m:n:o=120:7.5:16.3.

[0078] According to some preferred embodiments, the polyamino acid II is polyamino acid PSar-Mal4, and the structural formula is shown in the following formula II:

[0079]

[0080] In formula II, x is the number of repeating units on each arm of polyamino acid II. The present invention has no particular limitation on the value of x. Preferably, the range of x is 30 to 70, and more preferably, x=60.

[0081] According to some preferred embodiments, in step (1): the molar ratio of the side chain modified glutamic acid-N-carboxyl anhydride, the N-carboxyl anhydride containing a thiol group and the initiator is (200-400): (10-100): 1, preferably 250:50: 1; the present invention has found that the molar ratio of the side chain modified glutamic acid-N-carboxyl anhydride, the N-carboxyl anhydride containing a thiol group and the initiator is (200-400): (10-100): 1, and the selection of this molar ratio makes the proportion of cysteine containing a thiol group in the polyamino acid I obtained under the above conditions moderate, meeting the requirements of cross-linking; at the same time, a certain branched structure is formed, which is conducive to improving the degradation performance of the gel.

[0082] According to some preferred embodiments, in step (1), the concentration of the initiator is 0.3 to 0.6 mol / L, preferably 0.5 mol / L; the amount of the first solvent is 2 to 8 mL (e.g., 2, 3, 4, 5, 6, 7 or 8 mL), preferably 3 mL; the temperature of the polymerization reaction is room temperature (e.g., room temperature 15 to 35° C.), the time of the polymerization reaction is 3 to 6 h (e.g., 3, 4, 5 or 6 h), preferably 4 h; and / or the polymerization reaction uses unprotected thiol-containing N-carboxylic anhydride directly as a polymerization monomer to participate in the reaction, so that the obtained polyamino acid I has a branched structure.

[0083] According to some preferred embodiments, in step (2): the multifunctional initiator is a compound having multiple nucleophilic amino groups and / or thiol groups, preferably, the multifunctional initiator includes but is not limited to one or more of ethylenediamine, triaminoethylamine, pentaerythritol, lysine, and lysine methyl ester, more preferably, the multifunctional initiator is pentaerythritol; the molar ratio of the sarcosine-N-carboxylic anhydride, the multifunctional initiator and the 3-maleimidopropionic acid hydroxysuccinimide ester is 1:(0.004-0.1%). 0.006): (0.05-0.15) (e.g. 1:0.004:0.05, 1:0.004:0.08, 1:0.004:0.1, 1:0.004:0.12, 1:0.004:0.15, 1:0.005:0.05, 1:0.005:0.08, 1:0.005:0.1, 1:0.005:0.12, 1:0.005:0.15, 1:0.006:0.05, 1:0.006:0.08, 1:0.006 :0.1, 1:0.006:0.12 or 1:0.006:0.15), preferably 1:0.005:0.1; The present invention found that the molar ratio of the sarcosine-N-carboxylic anhydride, the multifunctional initiator and the 3-maleimidopropionic acid hydroxysuccinimide ester is 1:(0.004-0.006):(0.05-0.15), at which the sarcosine-N-carboxylic anhydride can be completely converted into a polyamino acid, and its molecular weight matches that of the polyamino acid I; at the same time, at this molar ratio, the polyamino acid The end group of amino acid II can be completely converted into maleimide, and can react with thiol to form a gel quickly; the temperature of the polymerization reaction is room temperature (e.g., room temperature 15-35°C), and the time of the polymerization reaction is 18-30 hours (e.g., 18, 20, 24, 28 or 30 hours), preferably 24 hours; and / or the temperature of the polymer end group modification reaction is room temperature (e.g., room temperature 15-35°C), and the time of the polymer end group modification reaction is 18-30 hours (e.g., 18, 20, 24, 28 or 30 hours), preferably 24 hours.

[0084] According to some preferred embodiments, in step (3), the concentration of the polyamino acid I solution is 80-120 mg / mL (e.g., 80, 85, 90, 95, 100, 105, 110, 115 or 120 mg / mL), preferably 100 mg / mL; and / or the concentration of the polyamino acid II solution is 120-180 mg / mL (e.g., 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175 or 180 mg / mL), preferably 150 mg / mL. The present invention has found that when mixed within this concentration range, both polyamino acid precursors can be well dissolved, and the gelation rate of the obtained polyamino acid hydrogel is faster.

[0085] According to some preferred embodiments, in step (3): the short peptide solution is prepared using phosphate buffer, preferably, the concentration of the short peptide solution is 20 mg / mL; and / or the molar ratio of the total amount of thiol groups contained in the polyamino acid I solution and the short peptide solution to the maleimide groups contained in the polyamino acid II solution is (0.9-1.1):1, preferably 1:1.

[0086] According to some more preferred embodiments, in step (1), the molar ratio of the side chain modified glutamic acid-N-carboxyl anhydride, the thiol-containing N-carboxyl anhydride and the initiator is (200-400): (10-100): 1; in step (2), the molar ratio of the sarcosine-N-carboxyl anhydride, the multifunctional initiator and the 3-maleimidopropionic acid hydroxysuccinimide ester is 1: (0.004-0.006): (0.05-0.15); in step (3), the concentration of the polyamino acid I solution is 80-120 mg / mL, and the concentration of the polyamino acid II solution is 120-180 mg / mL; the present invention has obtained the optimal experimental conditions for preparing the polyamino acid hydrogel of the present invention after a large number of creative experiments, which can ensure that the polyamino hydrogel of the present invention with the best performance is obtained.

[0087] According to some preferred embodiments, in step (3): the gelation is performed at room temperature for 8 to 15 minutes (e.g., 8, 9, 10, 12 or 15 minutes), preferably 10 minutes;

[0088] The gelation is performed at room temperature for 10 minutes. Preferably, the mass fraction of the obtained polyamino acid hydrogel is 5 to 20%, preferably 10%. In the present invention, the mass fraction of the polyamino acid hydrogel refers to the mass percentage of the polyamino acid contained in the polyamino acid hydrogel.

[0089] According to some specific embodiments, the preparation of the polyamino acid hydrogel PAA-RGD comprises the following steps:

[0090] ①Synthetic polyamino acid P (EG3Glu-co-Cys)

[0091] At room temperature, L -EG3GluNCA (9.3 mmol) and L-CysNCA (1.8 mmol) was dissolved in DMF (3.0 mL), and benzylamine initiator (75 μL x 0.5 M) was added. The reaction was stirred at room temperature for 4 h. FT-IR was used to detect the completion of polymerization. After polymerization, the mixture was precipitated with diethyl ether and centrifuged at 4000 rpm for 10 min. The supernatant was discarded. The polymer was further purified by dialysis to obtain a white powdery solid (80% yield), which was polyamino acid P(EG3Glu-co-Cys).

[0092] ②Synthetic polyamino acid PSar-Mal4

[0093] SarNCA (8.70 mmol) was added to a 20 mL sample vial and dissolved in LC-MS-grade DMF (9 mL). Freshly prepared pentaerythritol initiator (570 μL x 0.075 M) was added and a magnetic separator was added to initiate the reaction. After 24 hours of reaction, polymerization was confirmed by FT-IR. Upon completion of the polymerization, the vial was removed from the glove box, precipitated with diethyl ether, and centrifuged at 4000 rpm for 10 minutes. The supernatant was discarded, and the resulting polymer intermediate was dried in a vacuum oven.

[0094] To obtain a terminally maleimide-modified polymer, the resulting polymer intermediate was dissolved in DMF, and 3-hydroxysuccinimide maleimidopropionate (NHS-MAL) (0.91 mmol) was added. The reaction was allowed to react overnight at room temperature. The product was precipitated with diethyl ether and centrifuged at 4000 rpm for 10 minutes. The supernatant was discarded, air-dried, and the resulting polymer was reconstituted with water. The polymer was dialyzed to remove small molecule impurities and lyophilized to obtain a white solid (81% yield), the polyamino acid PSar-Mal4.

[0095] ③Synthesis of polyamino acid hydrogel PAA-RGD

[0096] PBS was used to prepare polymer P(EG3Glu-co-Cys) and polymer PSar-Mal4 into a stock solution of 100 mg / mL and 150 mg / mL, respectively. PBS was used to prepare a short peptide cysteine-arginine-glycine-aspartic acid (CRGD) into a CRGD solution. The molar ratio of the total amount of thiol groups in the short peptide and polymer P(EG3Glu-co-Cys) to the maleimide in PSar-Mal4 was 1:1. A specific volume of the stock solution and the CRGD solution were aspirated and mixed, and gelation occurred within 10 minutes of standing at room temperature to prepare the preset polyamino acid hydrogel PAA-RGD.

[0097] The present invention has no particular limitation on the sources of the raw materials used. For example, the raw materials can be directly purchased from the market or synthesized by existing methods.

[0098] In a second aspect, the present invention provides a polyamino acid hydrogel prepared by the preparation method described in the first aspect of the present invention.

[0099] In a third aspect, the present invention provides the use of the polyamino acid hydrogel prepared by the preparation method described in the first aspect of the present invention in the field of biomedicine; preferably, it is used in stem cell culture and tissue engineering, and more preferably, it is used as a cartilage repair material in stem cell culture and tissue engineering.

[0100] The present invention will be further described below by way of examples, but the protection scope of the present invention is not limited to these examples.

[0101] Example 1

[0102] ①Synthetic polyamino acid P (EG3Glu-co-Cys)

[0103] At room temperature, L -EG3GluNCA (9.3 mmol) and L -CysNCA (1.8 mmol) was dissolved in DMF (3.0 mL), and benzylamine initiator (75 μL x 0.5 M) was added. The reaction was stirred at room temperature at 25°C for 4 h. FT-IR was used to verify the completion of polymerization. After polymerization, the mixture was precipitated with diethyl ether and centrifuged at 4000 rpm for 10 min. The supernatant was discarded. The polymer was further purified by dialysis to obtain a white powdery solid (80% yield), which was polyamino acid P(EG3Glu-co-Cys). The obtained polyamino acid P (EG3Glu-co-Cys) has a branched structure, which is specifically characterized as follows: the thiol content in the polymer is characterized by Ellman's reagent, and the result obtained shows that each chain segment contains 7.5 thiol groups; the total amino group / thiol content in the polymer is characterized by TNBS reagent, and the result obtained shows that each chain segment contains 17.5 amino groups (number of terminal units); according to the above analytical chemistry results, polymer nuclear magnetic resonance hydrogen spectrum and elemental analysis results, the number of thioesters in each chain segment in the polymer is 16.5; in the branched polymer, the degree of branching = (branching unit + terminal unit) / total number of repeating units, that is, the degree of branching in the obtained polymer polyamino acid P (EG3Glu-co-Cys) is (16.5 + 17.5) / 147 = 0.23.

[0104] ②Synthetic polyamino acid PSar-Mal4

[0105] SarNCA (8.70 mmol) was added to a 20 mL sample vial and dissolved in LC-MS-grade DMF (9 mL). Freshly prepared pentaerythritol initiator (570 μL x 0.075 M) was added and a magnetic separator was added to initiate the reaction. After 24 hours at room temperature (25°C), polymerization completion was confirmed using FT-IR. After polymerization, the vial was removed from the glove box, precipitated with diethyl ether, and centrifuged at 4000 rpm for 10 minutes. The supernatant was discarded, and the resulting polymer intermediate was dried in a vacuum oven.

[0106] To obtain a terminally maleimide-modified polymer, the resulting polymer intermediate was dissolved in DMF (9 mL) and 3-hydroxysuccinimide maleimidopropionate (NHS-MAL) (0.91 mmol) was added. The reaction was allowed to react overnight at 25°C. The product was precipitated with diethyl ether and centrifuged at 4000 rpm for 10 minutes. The supernatant was discarded, air-dried, and the resulting polymer was reconstituted with water. The polymer was dialyzed to remove small molecule impurities and lyophilized to obtain a white solid (81% yield), the polyamino acid PSar-Mal4.

[0107] ③Synthesis of polyamino acid hydrogel PAA-RGD

[0108] PBS was used to prepare polymer P(EG3Glu-co-Cys) and polymer PSar-Mal4 into a stock solution of 100 mg / mL and 150 mg / mL, respectively. PBS was used to prepare a short peptide cysteine-arginine-glycine-aspartic acid (CRGD) into a CRGD solution. The concentration of the CRGD solution was 20 mg / mL. The molar ratio of the total amount of thiol groups in the short peptide and polymer P(EG3Glu-co-Cys) to the maleimide in PSar-Mal4 was 1:1. A specific volume of the stock solution and CRGD solution was aspirated and mixed, and gelation occurred after standing at room temperature at 25°C for 10 minutes to prepare the preset polyamino acid hydrogel PAA-RGD.

[0109] In this example, the performance of the prepared polyamino acid hydrogel PAA-RGD was tested: the rheological oscillation frequency scanning curve of the polyamino acid hydrogel PAA-RGD was as follows: Figure 6 As shown; the in vitro swelling curve of polyamino acid hydrogel PAA-RGD is as shown Figure 7 As shown; the compression test curve of polyamino acid hydrogel PAA-RGD is as follows Figure 8 As shown; the scanning electron microscopy image of polyamino acid hydrogel PAA-RGD is shown Figure 9 shown.

[0110] The cell death and viability staining results of the polyamino acid hydrogel PAA-RGD prepared in this example after culturing PB-MSCs cells for 3 days, 7 days, and 14 days are shown in the figure. Figure 10As shown in the figure, the culture method of PB-MSCs refers to Chinese patent ZL201110187286.5; the expression of type II collagen in polyamino acid hydrogel PAA-RGD after culturing PB-MSCs for 14 days is shown in the figure. Figure 11 shown; from Figure 11 It can be seen that type II collagen is highly expressed in chondrocytes, but not in undifferentiated mesenchymal stem cells. After 14 days of culture, the cell nuclei were stained with DAPI, while the type II collagen content on the cell surface was expressed with COLII. Figure 11 The high COLII fluorescence intensity showed that PAA-RGD hydrogel had an excellent effect in promoting the chondrogenic differentiation of PB-MSCs.

[0111] The schematic diagram of cartilage repair of the polyamino acid hydrogel PAA-RGD prepared in this example is shown in FIG. Figure 12 As shown: Osteochondral defects were created in the femoral trochlear groove of the knee joint of New Zealand white rabbits. A 5mm diameter corneal trephine was used to create an osteochondral defect with a diameter of 5mm and a depth of 3mm. The PAA-RGD hydrogel containing PB-MSCs of the same diameter and depth prepared in advance was then filled into the defect. The gross images of the rabbit osteochondral defects repaired by the polyamino acid hydrogel PAA-RGD at 6 and 12 weeks are shown in the figure below. Figure 13 As shown; MRI imaging of polyamino acid hydrogel PAA-RGD repairing rabbit osteochondral defects after 6 and 12 weeks Figure 14 As shown in the figure: Compared with the simple defect group, the subchondral bone edema after PAA-RGD hydrogel repair was significantly reduced, and the continuity of the cartilage was better, indicating that it has good cartilage repair ability; the micro-CT imaging of polyamino acid hydrogel PAA-RGD on rabbit osteochondral defects after 6 weeks and 12 weeks is shown in the figure Figure 15 As shown: Compared with the simple defect group, PAA-RGD hydrogel has the ability to significantly promote subchondral bone repair; SEM images of polyamino acid hydrogel PAA-RGD repairing rabbit cartilage defects after 12 weeks are shown as follows: Figure 16 As shown, in Figure 16 In the figure, Normal represents the SEM image of the normal state before the rabbit cartilage defect, and Blank represents the SEM image of the newly filled tissue 12 weeks after the rabbit simple osteochondral defect modeling (without any hydrogel filling and repair). The results show that the smoothness of the newly filled cartilage surface after PAA-RGD hydrogel repair is closer to that of normal cartilage, while the newly filled tissue of the blank group is very rough.

[0112] Figure 17 shows the results of toluidine blue staining of the polyamino acid hydrogel PAA-RGD prepared in this example for repairing rabbit cartilage defects after 6 and 12 weeks: Toluidine blue can dye cartilage into blue-purple, while bone tissue and fibrous tissue cannot be colored. Therefore, the dark-stained part in the figure indicates the cartilage part. Figure 17 N refers to normal cartilage, R refers to repair cartilage, and the arrow indicates the junction of normal cartilage and repair cartilage. Figure 17 It was found that the newly formed cartilage of PAA-RGD hydrogel was similar to normal cartilage, while the blank control group failed to repair and produce cartilage tissue, and the osteochondral defects were filled with connective tissue or fibrous tissue; this indicates that PAA-RGD has very good cartilage repair ability.

[0113] The immunohistochemical results of the polarized macrophages infiltrating around the polyamino acid hydrogel PAA-RGD prepared in this example for 14 days are as follows: Figure 18 As shown: Macrophages are crucial to the body's immune regulation. Macrophages are activated into M1 type, i.e., classically activated macrophages (CD86+), through the classical activation pathway. They have a pro-inflammatory effect, promote the activation of the body's early inflammatory response, and are not conducive to tissue repair. Macrophages can also be activated into M2 type, i.e., alternatively activated macrophages (CD206+), which exert an anti-inflammatory effect and are beneficial to tissue repair. The present invention conducted immunohistochemical analysis on macrophages infiltrating around PAA-RGD and found that the cells around it were mainly M2 macrophages (the arrows indicate representative CD206-positive cells), and there were basically no M1 macrophages. The research results of the present invention show that PAA-RGD hydrogel has the effect of promoting the polarization of macrophages to M2.

[0114] Example 2

[0115] ①Synthesis of polyglutamic acid-polycysteine copolymer P(Glu-co-Cys)

[0116] At room temperature, glutamic acid-N-carboxylic anhydride ( L -GluNCA) (5 mmol) and L-CysNCA (1 mmol) was dissolved in DMF (10.0 mL), and benzylamine initiator (100 μL × 0.5 M) was added, and the reaction was stirred at 10°C for 18 hours. FT-IR was used to detect whether the polymerization was complete. After the polymerization was completed, it was precipitated with ether and centrifuged at 4000 rpm for 10 minutes. The supernatant was discarded. The polymer was further purified by dialysis, and a white powder solid was finally obtained (yield 50%), which was the polyamino acid P (Glu-co-Cys). The obtained polyamino acid P (Glu-co-Cys) had a pH-dependent dissolution behavior, dissolving well in phosphate buffer at pH = 7.4 and insoluble in a weakly acidic environment. This property can be used to design smart gels with responsive pores / adjustable mechanical properties; the reaction formula for the synthesis of polyglutamic acid-polycysteine copolymer P (Glu-co-Cys) is shown below.

[0117]

[0118] ② is the same as step ② in Example 1.

[0119] ③ is basically the same as step ③ in Example 1, except that; this example uses polyglutamic acid-polycysteine copolymer P(Glu-co-Cys) to replace P(EG3Glu-co-Cys) in Example 1 to perform step ③.

[0120] Example 3

[0121] ①Synthesis of a copolymer of glutamic acid and cysteine with a side chain modified with diethylene glycol monomethyl ether (P(EG2Glu-co-Cys))

[0122] At room temperature, glutamic acid-N-carboxyl anhydride ( L -EG2GluNCA) (9.3 mmol) and L -CysNCA (1.8 mmol) was dissolved in DMF (3.0 mL), and benzylamine initiator (75 μL×0.5 M) was added, and the reaction was stirred at room temperature of 25°C for 4 hours. FT-IR was used to detect whether the polymerization was complete. After the polymerization was completed, it was precipitated with ether and centrifuged at 4000 rpm for 10 minutes. The supernatant was discarded. The polymer was further purified by dialysis to obtain a white powdery solid (yield 76%), which was polyamino acid P (EG2Glu-co-Cys). The obtained polyamino acid P (EG2Glu-co-Cys) also has a branched structure and can react with PSar-Mal4 to prepare a hydrogel. At the same time, due to LThe -EG2Glu side chain has a critical solution temperature (LCST) close to human body temperature, and its LCST performance can be used to regulate the permeability of the hydrogel; the reaction formula for synthesizing a glutamic acid and cysteine copolymer P(EG2Glu-co-Cys) with a side chain modified with diethylene glycol monomethyl ether is shown below.

[0123]

[0124] ② is the same as step ② in Example 1.

[0125] ③ is basically the same as step ③ in Example 1, except that; this example uses P(EG2Glu-co-Cys) to replace P(EG3Glu-co-Cys) in Example 1 to perform step ③.

[0126] Example 4

[0127] ①Synthesis of a copolymer of glutamic acid and polypenicillamine with triethylene glycol monomethyl ether as the side chain

[0128] At room temperature, L -EG3GluNCA (9.3mmol) and penicillamine-N-carboxylic anhydride ( L -PenNCA) (1.8mmol) was dissolved in DMF (3.0mL), benzylamine initiator (75μL×0.5M) was added, and the reaction was stirred at room temperature of 25°C for 4h. FT-IR was used to detect whether the polymerization was complete. After the polymerization was completed, it was precipitated with ether and centrifuged at 4000rpm for 10min. The supernatant was discarded. The polymer was further purified by dialysis to finally obtain a white powdery solid (yield 67%), which was polyamino acid P (EG3Glu-co-Pen). Due to L The side chain of -PenNCA is sterically hindered, resulting in a polyamino acid P(EG3Glu-co-Pen) lacking a branched structure and capable of being characterized by SEC. However, its thiol groups remain reactive and can react with PSar-Mal4 to form a hydrogel. Furthermore, penicillamine possesses certain biological functions, enabling the hydrogel to achieve sustained release of penicillamine in vivo. The reaction equation for synthesizing a copolymer of glutamic acid and polypenicillamine, P(EG3Glu-co-Pen), with a triethylene glycol monomethyl ether side chain, is shown below.

[0129]

[0130] ② is the same as step ② in Example 1.

[0131] ③ is basically the same as step ③ in Example 1, except that; this example uses P(EG3Glu-co-Pen) to replace P(EG3Glu-co-Cys) in Example 1 to perform step ③.

[0132] The present invention compared the performance of the polyamino acid hydrogel PAA-RGD prepared in Example 1 with PEG-RGD and GelMA hydrogels. The results are shown in Tables 1 and 2.

[0133] Table 1: Comparison of properties of polyamino acid hydrogels.

[0134]

[0135] The international cartilage repair score results of three hydrogels, PAA-RGD, PEG-RGD and GelMA, are shown in the figure. Figure 19 As shown in Table 1; the symbol "-" in Table 1 indicates that the performance indicator does not exist. The results in Table 1 show that the international cartilage repair scores corresponding to GelMA and PEG-RGD hydrogels at 6 weeks were lower than those of the blank control group. This is because the GelMA and PEG-RGD groups failed to degrade in vivo at 6 weeks, hindering the ingrowth of new cartilage, while the blank control group was filled with fibrous tissue or scar tissue at 6 weeks, resulting in a higher international cartilage repair score.

[0136] Table 2 Results of M2 polarization area of infiltrating macrophages around hydrogels.

[0137] M2 polarization area% PAA-RGD PEG-RGD GelMA 14 days 13.27 2.39 3.8

[0138] The M2 polarization area results of the infiltrating macrophages around the three hydrogels of PAA-RGD, PEG-RGD and GelMA for 14 days, as shown in the figure. Figure 20 shown.

[0139] In Tables 1 and 2, PEG-RGD is a commonly used hydrogel for scientific research and commercial use. The structural diagram of PEG-RGD hydrogel is shown in FIG. Figure 21 As shown; GelMA is a commonly used hydrogel for scientific research and commercial use, and the structural diagram of GelMA hydrogel is as shown Figure 22 In Tables 1 and 2, the results of PEG-RGD and GelMA hydrogels were obtained using the same method as that used for PAA-RGD hydrogel.

[0140] Parts of the present invention that are not described in detail are well known to those skilled in the art.

[0141] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for preparing a polyamino acid hydrogel, characterized in that: The method comprises the following steps: (1) using a first solvent to uniformly mix glutamic acid-N-carboxylic anhydride or side chain-modified glutamic acid-N-carboxylic anhydride and N-carboxylic anhydride containing a thiol group, and then adding an initiator to carry out a polymerization reaction to obtain polyamino acid I; (2) dissolving sarcosine-N-carboxylic anhydride in a second solvent, then adding a multifunctional initiator to carry out a polymerization reaction to obtain a polymer intermediate, then dissolving the polymer intermediate in a third solvent and adding 3-maleimidopropionic acid hydroxysuccinimide ester to carry out a polymer end group modification reaction to obtain polyamino acid II; (3) Polyamino acid I and polyamino acid II are prepared into polyamino acid I solution and polyamino acid II solution respectively using phosphate buffer, and then the polyamino acid I solution, polyamino acid II solution and short peptide solution are mixed evenly and gelated to obtain polyamino acid hydrogel.

2. The preparation method according to claim 1, wherein: The side chain modification group of the glutamic acid-N-carboxylic anhydride is selected from one or more of ethylene glycol, diethylene glycol, diethylene glycol monomethyl ether, triethylene glycol monomethyl ether, tetraethylene glycol monomethyl ether, ethanolamine, 3-hydroxypropionic acid and lactic acid; The sulfhydryl-containing N-carboxyl anhydride is selected from one or more of cysteine-N-carboxyl anhydride, homocysteine-N-carboxyl anhydride, and penicillamine-N-carboxyl anhydride; The initiator in step (1) is a reagent containing an amino group or a thiol group with a certain nucleophilicity, and / or The short peptide contained in the short peptide solution is cysteine-arginine-glycine-aspartic acid.

3. The preparation method according to claim 2, wherein: The initiator includes but is not limited to one or more of n-hexylamine, benzylamine, mercaptoethanol, methyl glutamate, diethylamine, isopropylamine, and cyclohexylamine.

4. The preparation method according to claim 2, wherein: The initiator is benzylamine.

5. The preparation method according to claim 2, wherein: The side chain modification group of the glutamic acid-N-carboxyl anhydride is triethylene glycol monomethyl ether, and the structural formula of the glutamic acid-N-carboxyl anhydride modified with triethylene glycol monomethyl ether is shown in the following formula a: Formula a; The N-carboxyl anhydride containing a thiol group is cysteine-N-carboxyl anhydride, and the structural formula of the cysteine-N-carboxyl anhydride is shown in the following formula b: Formula b; The polyamino acid I is polyamino acid P (EG3Glu- co -Cys), the structural formula is shown in the following formula I: Formula I; The polyamino acid II is polyamino acid PSar-Mal4, and its structural formula is shown in the following formula II: Formula II.

6. The preparation method according to any one of claims 1 to 5, characterized in that In step (1): The molar ratio of the side chain modified glutamic acid-N-carboxyl internal anhydride, the thiol-containing N-carboxyl internal anhydride and the initiator is (200-400): (10-100): 1; The concentration of the initiator is 0.3-0.6 mol / L; The amount of the first solvent is 2-8 mL; The polymerization reaction temperature is room temperature, and the polymerization reaction time is 3 to 6 hours; and / or In the polymerization reaction, unprotected thiol-containing N-carboxyl lactic anhydride is directly used as a polymerization monomer to participate in the reaction, so that the obtained polyamino acid I has a branched structure.

7. The preparation method according to claim 6, characterized in that In step (1): The molar ratio of the side chain modified glutamic acid-N-carboxyl anhydride, the thiol-containing N-carboxyl anhydride and the initiator is 250:50:

1.

8. The preparation method according to claim 6, characterized in that In step (1): The concentration of the initiator is 0.5 mol / L.

9. The preparation method according to claim 6, characterized in that In step (1): The amount of the first solvent used is 3 mL.

10. The preparation method according to claim 6, characterized in that In step (1): The polymerization reaction temperature is room temperature, and the polymerization reaction time is 4 hours.

11. The preparation method according to any one of claims 1 to 5, characterized in that In step (2): The multifunctional initiator is a compound having multiple amino groups and / or thiol groups with nucleophilicity; The molar ratio of the sarcosine-N-carboxylic anhydride, the multifunctional initiator and the 3-maleimidopropionic acid hydroxysuccinimide ester is 1: (0.004-0.006): (0.05-0.15); The polymerization reaction temperature is room temperature, and the polymerization reaction time is 18 to 30 hours; and / or The temperature of the polymer end group modification reaction is room temperature, and the time of the polymer end group modification reaction is 18 to 30 hours.

12. The preparation method according to claim 11, characterized in that In step (2): The multifunctional initiator includes but is not limited to one or more of ethylenediamine, triaminoethylamine, pentaerythritol, lysine, and lysine methyl ester.

13. The preparation method according to claim 11, characterized in that In step (2): The multifunctional initiator is pentaerythritol.

14. The preparation method according to claim 11, characterized in that In step (2): The molar ratio of the sarcosine-N-carboxylic anhydride, the multifunctional initiator and the 3-maleimidopropionic acid hydroxysuccinimide ester is 1:0.005:0.

1.

15. The preparation method according to claim 11, characterized in that In step (2): The polymerization reaction time is 24 hours.

16. The preparation method according to claim 11, characterized in that In step (2): The polymer end group modification reaction time is 24 hours.

17. The preparation method according to any one of claims 1 to 5, characterized in that In step (3): The concentration of the polyamino acid I solution is 80-120 mg / mL; and / or The concentration of the polyamino acid II solution is 120-180 mg / mL.

18. The preparation method according to claim 17, characterized in that: In step (3): The concentration of the polyamino acid I solution is 100 mg / mL.

19. The preparation method according to claim 17, characterized in that In step (3): The concentration of the polyamino acid II solution is 150 mg / mL.

20. The preparation method according to any one of claims 1 to 5, characterized in that In step (3): The short peptide solution is prepared using phosphate buffer; and / or The molar ratio of the total amount of thiol groups contained in the polyamino acid I solution and the short peptide solution to the maleimide groups contained in the polyamino acid II solution is (0.9-1.1):

1.

21. The preparation method according to claim 20, characterized in that In step (3): The molar ratio of the total amount of thiol groups contained in the polyamino acid I solution and the short peptide solution to the maleimide groups contained in the polyamino acid II solution is 1:

1.

22. The preparation method according to any one of claims 1 to 5, characterized in that In step (3): The mass fraction of the prepared polyamino acid hydrogel is 5%~20%.

23. The preparation method according to claim 22, characterized in that In step (3): The mass fraction of the prepared polyamino acid hydrogel is 10%.

24. The polyamino acid hydrogel prepared by the preparation method according to any one of claims 1 to 23.

25. Use of the polyamino acid hydrogel prepared by the preparation method according to any one of claims 1 to 23 in preparing cartilage repair materials.

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