An enzyme-responsive polyamino acid, adaptive biomimetic mechanical gradient hydrogel and preparation method and application thereof

Abstract

The present invention relates to the field of tissue engineering, and in particular to an enzyme-responsive polyamino acid, an adaptive biomimetic mechanical gradient hydrogel, and a preparation method and application thereof. The enzyme-responsive polyamino acid has a structure shown in Formula I. The hydrogel prepared by the enzyme-responsive polyamino acid provides biophysical cues in a dynamic, spatiotemporal controlled manner to regulate the cell behavior of bone marrow-derived mesenchymal stem cells (BM-MSCs) loaded thereon, and by providing feedback to the ALP secreted during the repair of osteochondrocytes, self-assembly changes the secondary structure, thereby precisely regulating the mechanical microenvironment of BM-MSCs, and further regulating cell proliferation and differentiation behavior. After implantation into the body, the ALP secreted by the body itself or secreted by BM-MSCs after osteogenesis in the subsequent system diffuses to the surface of the cartilage to form an ALP gradient, and the induction system forms a self-generated gradient hydrogel scaffold through different degrees of dynamic allosteric enhancement, thereby achieving orderly repair of the multi-layer cartilage structure by BM-MSCs.

Description

Technical Field

[0001] The invention relates to the field of tissue engineering, and in particular to an enzyme-responsive polyamino acid, self-adaptive bionic mechanical gradient hydrogel, and a preparation method and application thereof. Background Art

[0002] Articular cartilage defects are difficult to repair themselves due to the lack of nutrition from blood vessels and nerves and the low proliferation capacity of chondrocytes. Cartilage defects often involve the subchondral bone and then develop into osteochondral defects, leading to osteoarthritis. Since cartilage and subchondral bone have different biological characteristics, physiological functions and tissue healing abilities, it is of great clinical significance to simultaneously reconstruct cartilage and subchondral bone at the site of osteochondral defects. Based on the characteristics of the osteochondral microenvironment and the development of cell biomechanics, using gradient-changing mechanical cues to regulate cell behavior and guide gradient tissue regeneration is an effective strategy for repairing osteochondral defects, and is also the frontier and hot spot of osteochondral defect repair research.

[0003] Existing mechanical gradient scaffolds are mainly constructed through layer-by-layer stacking, 3D printing technology, freeze-drying, electric / magnetic field driving methods, etc. to prepare mechanical gradient scaffolds with mechanical properties similar to those of natural osteocartilage structures in vitro. However, there is still a large gap between the continuity of the mechanical property gradient of the scaffold and that of the natural osteocartilage structure.

[0004] At present, most tissue engineering scaffolds can only target specific stages of the osteochondral repair process and play a positive role only for a limited time, but cannot achieve the goal of promoting the entire repair process. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide an enzyme-responsive polyamino acid, an adaptive bionic hardness gradient hydrogel and its preparation method and application, so as to provide stem cells with a specific mechanical microenvironment in different differentiation directions of bone and cartilage.

[0006] The present invention provides an enzyme-responsive polyamino acid having a structure shown in Formula I:

[0007]

[0008] Formula I;

[0009] Where R1= , or ;

[0010] R2= CH3, , , or ;

[0011] R3= , —CH2— or ;

[0012] m, x, y, z are the degrees of polymerization.

[0013] The present invention provides a method for preparing the above enzyme-responsive polyamino acid, comprising the following steps:

[0014] Using amino-terminated polyethylene glycol monomethyl ether as an initiator, a monomer of the structure of formula (II), a monomer of the structure of formula (III) and a monomer of the structure of formula (IV) are polymerized, and deprotected to obtain the enzyme-responsive polyamino acid;

[0015] Formula (II); Formula (III); Formula (IV);

[0016] Where R1= , or ;

[0017] R2= CH3, , , or ;

[0018] R3= , —CH2— or .

[0019] Preferably, the monomer of the structure of formula (II) is obtained by reacting methacryloyl amino acid and triphosgene;

[0020] The methacryloyl amino acid is selected from methacryloyl-L-lysine, methacryloyl-serine or methacryloyl-threonine.

[0021] Preferably, the monomer of formula (IV) is prepared by the following method:

[0022] S1) reacting N-(tert-butyloxycarbonyl)-amino acid tert-butyl ester with diethyl chlorophosphate to obtain diethoxyphosphoryl-N-(tert-butyloxycarbonyl)-L-tyrosine tert-butyl ester; after deprotection, diethoxyphosphoryl-amino acid is obtained;

[0023] S2) reacting the diethoxyphosphoryl-L-amino acid with triphosgene to obtain a diethoxyphosphoryl-amino acid-N-intracyclic carboxylic acid anhydride monomer;

[0024] The amino acid is selected from L-tyrosine, serine or threonine.

[0025] Preferably, the preparation method comprises the following steps:

[0026] Amino-terminated polyethylene glycol monomethyl ether is used as an initiator to initiate a first polymerization reaction of the monomer of formula (II) to obtain a first polymer intermediate;

[0027] The first polymer intermediate and the monomers of formula (III) and (IV) undergo a second polymerization reaction, followed by deprotection to obtain the enzyme-responsive polyamino acid.

[0028] The invention provides an adaptive bionic mechanical gradient hydrogel, which is the enzyme-responsive polyamino acid or a photo-crosslinked product of the enzyme-responsive polyamino acid prepared by the preparation method.

[0029] The present invention provides a method for preparing the above-mentioned adaptive bionic mechanical gradient hydrogel, comprising the following steps:

[0030] Under the action of a cross-linking agent and an initiator, the enzyme-responsive polyamino acid or the enzyme-responsive polyamino acid prepared by the preparation method is subjected to a cross-linking reaction to obtain an adaptive bionic mechanical gradient hydrogel.

[0031] Preferably, the cross-linking agent is selected from polyethylene glycol diacrylate;

[0032] The initiator is selected from phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

[0033] The present invention provides the use of the adaptive bionic mechanical gradient hydrogel or the adaptive bionic mechanical gradient hydrogel prepared by the preparation method in preparing materials for inducing or promoting tissue regeneration.

[0034] Preferably, the material for inducing or promoting tissue regeneration is a hydrogel scaffold.

[0035] Compared with the prior art, the present invention provides an enzyme-responsive polyamino acid having a structure shown in formula I.

[0036] The hydrogel prepared with the enzyme-responsive polyamino acid as a precursor provides biophysical cues in a dynamic and spatiotemporal controlled manner to regulate the cell behavior of the loaded BM-MSCs. By responding to the ALP secreted during the osteochondral repair process, the self-assembly changes the secondary structure, thereby precisely regulating the mechanical microenvironment of BM-MSCs, and then regulating cell proliferation and differentiation behavior. After implantation into the body, the ALP secreted by the body itself or in the subsequent system after BM-MSCs osteogenesis diffuses to the surface of the cartilage to form an ALP gradient, and the induction system forms a self-generated gradient hydrogel scaffold through different degrees of dynamic allosteric enhancement, thereby achieving orderly repair of the multi-layer cartilage structure by BM-MSCs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the H NMR spectrum of methacryloyl-L-lysine-N-intra-ring carboxylic anhydride monomer;

[0038] Figure 2 is the H NMR spectrum of diethoxyphosphoryl-L-tyrosine-N-intracyclic carboxylic anhydride monomer;

[0039] Figure 3 is the H NMR spectrum of polyethylene glycol monomethyl ether-b-poly(methacryloyl lysine)-b-poly(tyrosine phosphate−co−alanine);

[0040] Figure 4 The conformation percentage diagram of the secondary structure of the ALP-catalyzed polyamino acid assembly was calculated based on the CD spectrum;

[0041] Figure 5 This is the compression test diagram of enzyme-responsive polyamino acid hydrogel alkaline phosphatase catalysis at different time points from 0 to 72 hours;

[0042] Figure 6 Fluorescence images of enzyme-responsive polyamino acid hydrogel co-incubated with cells;

[0043] Figure 7 This image shows the repair and regeneration effect of the full-thickness osteochondral defect 12 weeks after hydrogel implantation. DETAILED DESCRIPTION

[0044] The present invention provides an enzyme-responsive polyamino acid having a structure shown in Formula I:

[0045]

[0046] Formula I;

[0047] Where R1= , or ;

[0048] R2= CH3, , , or ;

[0049] R3= , —CH2— or .

[0050] wherein the carbonyl end of R1 is connected to the alkenyl group in formula I.

[0051] m is the degree of polymerization, m=10-1000; preferably 10-100, more preferably 20-60. In some specific embodiments of the present invention, m is 44.

[0052] x is the degree of polymerization, x=1-100; preferably 1-60, more preferably 5-30. In some specific embodiments of the present invention, x is 10.

[0053] y is the degree of polymerization, y=1-100; preferably 10-40, more preferably 20-35. In some specific embodiments of the present invention, y is 25.

[0054] z is the degree of polymerization, z=1-100; preferably 2-10, more preferably 3-6. In some specific embodiments of the present invention, z is 4.

[0055] The present invention provides a method for preparing the above enzyme-responsive polyamino acid, comprising the following steps:

[0056] Using amino-terminated polyethylene glycol monomethyl ether as an initiator, a monomer of the structure of formula (II), a monomer of the structure of formula (III) and a monomer of the structure of formula (IV) are polymerized, and deprotected to obtain the enzyme-responsive polyamino acid;

[0057] Formula (II); Formula (III); Formula (IV);

[0058] Where R1= , or ;

[0059] R2= CH3, , , or ;

[0060] R3= , —CH2— or .

[0061] Wherein, the carbonyl end of R1 is connected to the alkenyl group in Formula II.

[0062] Among them, the monomer of the formula (II) structure is selected from methacryloyl-L-lysine-N-intracyclic carboxylic anhydride, methacryloyl-serine-N-intracyclic carboxylic anhydride monomer or methacryloyl-threonine-N-intracyclic carboxylic anhydride monomer; the monomer of the formula (III) structure is selected from alanine-intracyclic carboxylic anhydride, valine-intracyclic carboxylic anhydride, leucine-intracyclic carboxylic anhydride, isoleucine-intracyclic carboxylic anhydride or methionine-intracyclic carboxylic anhydride monomer; the monomer of the formula (IV) structure is selected from diethoxyphosphoryl-L-tyrosine-N-intracyclic carboxylic anhydride, diethoxyphosphoryl-serine-N-intracyclic carboxylic anhydride or diethoxyphosphoryl-threonine-N-intracyclic carboxylic anhydride.

[0063] Preferably, the monomer of the structure of formula (IV) is obtained by reacting methacryloyl-amino acid with triphosgene.

[0064] The methacryloyl amino acid is selected from methacryloyl-L-lysine, methacryloyl-serine or methacryloyl-threonine.

[0065] The solvent for the above reaction is preferably tetrahydrofuran.

[0066] The above reaction is preferably carried out in an inert gas atmosphere, preferably a nitrogen atmosphere.

[0067] The reaction temperature is preferably 45-65°C, more preferably 55°C; the reaction time is preferably 1-3h, more preferably 2h.

[0068] Preferably, after the reaction is completed, the reaction mixture is precipitated in excess n-hexane, separated, washed, recrystallized, and vacuum dried to obtain methacryloyl-L-lysine / serine / threonine-N-intra-ring carboxylic acid anhydride.

[0069] Preferably, the monomer of formula (IV) is prepared by the following method:

[0070] S1) reacting N-(tert-butyloxycarbonyl)-amino acid tert-butyl ester with diethyl chlorophosphate to obtain diethoxyphosphoryl-N-(tert-butyloxycarbonyl)-L-tyrosine / serine / threonine tert-butyl ester; after deprotection, diethoxyphosphoryl-amino acid is obtained;

[0071] S2) The diethoxyphosphoryl-amino acid is reacted with triphosgene to obtain a diethoxyphosphoryl-amino acid-N-intracyclic carboxylic acid anhydride monomer.

[0072] The amino acid is selected from L-tyrosine, serine or threonine.

[0073] In the reaction of step S1), the solvent is preferably dichloromethane.

[0074] The above reaction is preferably carried out in an inert gas atmosphere, preferably a nitrogen atmosphere.

[0075] The above reaction is preferably carried out in an ice bath.

[0076] Preferably, an acid binding agent is added in the above reaction, and the acid binding agent is preferably triethylamine.

[0077] The above-mentioned deprotection group is preferably carried out using trifluoroacetic acid.

[0078] In the reaction of step S2), the solvent is preferably tetrahydrofuran.

[0079] The above reaction is preferably carried out in an inert gas atmosphere, preferably a nitrogen atmosphere.

[0080] The reaction temperature is preferably 45-65°C, more preferably 55°C; the reaction time is preferably 1-3h, more preferably 2h.

[0081] Preferably, after the reaction is completed, the reaction mixture is precipitated in excess n-hexane to obtain a viscous oil, which is then vacuum dried to obtain diethoxyphosphoryl-L-tyrosine / serine / threonine-N-intra-ring carboxylic acid anhydride monomer.

[0082] Then, amino-terminated polyethylene glycol monomethyl ether is used as an initiator to polymerize methacryloyl-L-lysine / serine / threonine-N-intracyclic carboxylic anhydride monomers, diethoxyphosphoryl-L-tyrosine / serine / threonine-N-intracyclic carboxylic anhydride monomers, and alanine / valine / leucine / isoleucine / methionine-intracyclic anhydride.

[0083] The molar ratio of the amino-terminated polyethylene glycol monomethyl ether, the monomer of formula (II), the monomer of formula (III) and the monomer of formula (IV) is preferably 1: (1-60): (10-40): (2-10); more preferably 1: (5-30): (20-35): (3-6); most preferably 1: 11: 25: 4.

[0084] Firstly, an anhydrous amino-terminated polyethylene glycol monomethyl ether monomer is used as an initiator and mixed with a methacryloyl-L-lysine-N-intra-ring carboxylic anhydride monomer in an anhydrous solvent to carry out a first polymerization reaction.

[0085] Then, alanine / valine / leucine / isoleucine / methionine-intracyclic carboxylic anhydride and diethoxyphosphoryl-L-tyrosine / serine / threonine-N-intracyclic carboxylic anhydride monomers were added for a second polymerization reaction to obtain polyethylene glycol monomethyl ether-b-poly(methacryloyl lysine / serine / threonine)-b-poly(tyrosine / serine / threonine phosphate-co-alanine / valine / leucine / isoleucine / methionine).

[0086] Preferably, the anhydrous solvent is anhydrous N,N-dimethylformamide.

[0087] Preferably, the first polymerization reaction is carried out in an inert gas atmosphere, preferably a nitrogen atmosphere.

[0088] The temperature of the first polymerization reaction is preferably 20-30°C, more preferably 25°C.

[0089] The time of the first polymerization reaction is preferably 48 to 96 hours, more preferably 72 hours.

[0090] The temperature of the second polymerization reaction is preferably 20-30°C, more preferably 25°C.

[0091] The time of the second polymerization reaction is preferably 48 to 96 hours, more preferably 72 hours.

[0092] After the second polymerization reaction is completed, preferably, the reaction solution is precipitated in ice ether, and the ether on the surface of the material is drained by a vacuum drying pump. The drained product is dissolved in chloroform, and trimethylsilyl bromide and triethylamine are added to remove the ethyl protecting group. The removal of the ethyl protecting group is preferably a reflux reaction, the temperature is preferably 60° C., and the time is preferably 8 hours.

[0093] The product obtained by the above reflux was dissolved in deionized water with EDTA added, and then the pH of the solution was adjusted to near neutral, dialyzed and freeze-dried to obtain polyethylene glycol monomethyl ether-b-poly(methacryloyl lysine / serine / threonine)-b-poly(tyrosine / serine / threonine phospho-co-alanine / valine / leucine / isoleucine / methionine).

[0094] The present invention also provides an adaptive bionic mechanical gradient hydrogel, wherein the adaptive bionic mechanical gradient hydrogel is the enzyme-responsive polyamino acid or a cross-linked product of the enzyme-responsive polyamino acid prepared by the preparation method.

[0095] The above-mentioned adaptive bionic mechanical gradient hydrogel has a double network structure, including the above-mentioned enzyme-responsive polyamino acid, under the irradiation of a photoinitiator and blue light, the double bond structure forms a carbon-carbon bond structure under the substitution reaction of free radicals, so that the polymer chains are cross-linked to form a first chemical cross-linking network, and then the polyamino acid hydrogel undergoes phosphate hydrolysis of the polymer chains containing phosphate esters in the polyamino acid side chains under the action of ALP in the bone cartilage microenvironment, removes the phosphate groups, and the chain segments restore the β-folded structure under the physical interaction forces such as Π-Π stacking and hydrogen bonds, thereby forming a higher-level assembly and forming a second physical network.

[0096] The hydrogel provided by the present invention has enzyme-responsive allostery, and under the action of alkaline phosphatase (ALP), a dynamic allosteric behavior of β-folding occurs, which indicates that the hydrogel system can self-assemble into β-folding in response to alkaline phosphatase generated during bone repair, thereby enhancing the mechanical strength of the gel. ALP secreted by the body itself or secreted by bone marrow-derived mesenchymal stem cells (BM-MSCs) in the subsequent system after osteogenesis diffuses to the surface of the cartilage to form an ALP activity gradient, inducing the gel to form a mechanical strength gradually enhanced from the cartilage area to the subchondral bone area, thereby mimicking the extracellular matrix gradient mechanical properties of the natural osteochondral hierarchical structure, reducing the mechanical strength difference and stress concentration between the defect area and the surrounding tissue, strengthening the integration of the new tissue with the surrounding tissue, and providing biophysical clues to regulate cell behavior in a dynamic and spatiotemporal controlled manner, and finally realizing the functional regeneration of osteochondral tissue. This solves the problem that traditional osteochondral tissue engineering scaffolds are difficult to complete precise and orderly repair, cannot capture the dynamic evolution of the tissue mechanical microenvironment and multidirectional differentiation of stem cells in osteochondral repair, and lead to a narrow time window for the scaffold to function.

[0097] The present invention provides a method for preparing the above-mentioned adaptive bionic hardness gradient hydrogel, comprising the following steps:

[0098] Under the action of a cross-linking agent and an initiator, the enzyme-responsive polyamino acid or the enzyme-responsive polyamino acid prepared by the preparation method is subjected to a cross-linking reaction to obtain an adaptive bionic hardness gradient hydrogel.

[0099] The present invention has no particular limitation on the crosslinking agent, and it can be a water-soluble crosslinking agent containing double bonds well known to those skilled in the art. Preferably, it is polyethylene glycol diacrylate (Mw~700; EG 13 MA).

[0100] The initiator is preferably lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP).

[0101] Preferably, the above cross-linking reaction is carried out in PBS buffer.

[0102] Preferably, the cross-linking reaction is carried out under light irradiation.

[0103] The illumination is preferably 405 nm blue light.

[0104] The cross-linking reaction time is preferably 1 to 5 minutes, more preferably 3 minutes.

[0105] The present invention uses the above-mentioned enzyme-responsive dynamic allosteric hydrogel to adapt to the dynamic evolution of stem cells in the repair process in a dynamic, spatiotemporal controlled manner. The hydrogel forms a first chemical network by photocrosslinking of lysine units modified by methacrylate. At this time, the hydrogel is softer and suitable for the proliferation and chondrogenic differentiation of stem cells. When the hydrogel is subjected to the action of ALP, the phosphate ester connected to tyrosine is hydrolyzed, the hydrophobicity is enhanced, and the tyrosine unit and the alanine unit will spontaneously form a β-fold under the physical interaction force, forming a second physical network of the hydrogel, increasing the hardness of the hydrogel, and promoting the osteogenic differentiation of stem cells. Finally, the induced hydrogel system is adaptively formed to gradually increase the mechanical strength from the cartilage area to the subchondral bone area, simulating the gradient mechanical microenvironment of the natural osteocartilage structure, and achieving the purpose of precise dynamic repair of osteocartilage.

[0106] The present invention provides the use of the adaptive bionic mechanical gradient hydrogel or the adaptive bionic mechanical gradient hydrogel prepared by the preparation method in preparing materials for inducing or promoting tissue regeneration.

[0107] The material for guiding or promoting tissue regeneration is preferably a hydrogel scaffold.

[0108] Specifically, the present invention provides a hydrogel scaffold for inducing or promoting tissue regeneration, which is prepared from the above-mentioned adaptive bionic mechanical gradient hydrogel or the adaptive bionic mechanical gradient hydrogel prepared by the above-mentioned preparation method.

[0109] To further illustrate the present invention, the adaptive bionic mechanical gradient hydrogel provided by the present invention and its preparation method and application are described in detail below in conjunction with the embodiments. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the invention.

[0110] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0111] Example 1

[0112] 1. Preparation of methacryloyl-L-lysine-N-carboxylic anhydride monomer

[0113] At 25 °C and in a nitrogen atmosphere, 2.217 g of methacryloyl-L-lysine was mixed with 150 mL of anhydrous tetrahydrofuran, and triphosgene was added, and the mixture was heated to 55 °C for 2 h. After the reaction, the reaction mixture was precipitated in excess n-hexane, separated, washed, recrystallized, and vacuum dried to obtain methacryloyl-L-lysine-N-intra-ring carboxylic anhydride.

[0114] Figure 1 This is the NMR spectrum of methacryloyl-L-lysine-N-intra-ring carboxylic anhydride.

[0115] 2. Preparation of diethoxyphosphoryl-L-tyrosine-N-intracyclic carboxylic anhydride monomer

[0116] At 25 °C, in a nitrogen atmosphere, 10.0 g of N-(tert-butyloxycarbonyl)-L-tyrosine tert-butyl ester, 50 mL of dichloromethane, and 7 mL of triethylamine were mixed and placed in an ice bath. 7.0 mL of diethyl chlorophosphate was slowly added dropwise, and the reaction endpoint was determined by spotting. 80 mL of water was poured in to quench the reaction, and the reaction was extracted with dichloromethane in small amounts and multiple times. The organic layer was collected, dried over anhydrous sodium sulfate, filtered and concentrated by rotary evaporation, and then separated and purified by chromatography. The obtained diethoxyphosphoryl-N-(tert-butyloxycarbonyl)-L-tyrosine tert-butyl ester was dissolved by adding 100 mL of dichloromethane, reacted overnight with 75 mL of trifluoroacetic acid, and the reaction was tracked to the end by spotting. The solvent was then drained to obtain diethoxyphosphoryl-L-tyrosine.

[0117] At 25 °C and in a nitrogen atmosphere, 2.0 g of diethoxyphosphoryl-L-tyrosine was mixed with 150 mL of anhydrous tetrahydrofuran, and triphosgene was added, and the mixture was heated to 55 °C for 2 h. After the reaction, the reaction mixture was precipitated in excess n-hexane to obtain a viscous oily substance, which was then vacuum dried to obtain diethoxyphosphoryl-L-tyrosine-N-intra-ring carboxylic anhydride monomer.

[0118] Figure 2 This is the NMR spectrum of diethoxyphosphoryl-L-tyrosine-N-intracyclic carboxylic anhydride monomer.

[0119] 3. Preparation of polyethylene glycol monomethyl ether-b-poly(methacryloyl lysine)-b-poly(tyrosine phosphate-co-alanine)

[0120] 0.5 g of amino-terminated polyethylene glycol monomethyl ether (Mw = 2000) was dissolved in 100 mL of toluene, azeotroped in an oil bath at 130 °C for 4 hours to remove excess water, and then toluene was removed by reduced pressure distillation. Anhydrous amino-terminated polyethylene glycol monomethyl ether was dissolved in 30 mL of anhydrous N,N-dimethylformamide, and then 0.603 g of methacryloyl-L-lysine-N-carboxylic anhydride was added under nitrogen protection and stirred at 25 °C for 72 hours. Then 0.72 g of alanine-ring anhydride and 0.34 g of diethoxyphosphoryl-L-tyrosine-N-ring anhydride were added and stirred at 25 °C for 72 hours. The reaction solution was precipitated in 10 times of ice ether, and the ether on the surface of the material was drained by a vacuum drying pump. The dried product was dissolved in chloroform, trimethylsilyl bromide and triethylamine were added, and heated to reflux at 60 °C for 8 hours. Subsequently, the solvent was removed by vacuum, and the remaining solid was dissolved in deionized water with EDTA added, and then the pH of the solution was adjusted to near neutral, dialyzed and freeze-dried to obtain polyethylene glycol monomethyl ether-b-poly(methacryloyl-lysine)-b-poly(tyrosine phosphate-co-alanine) (EG 45 -K MA10 Yp4 25 ).

[0121] Figure 3 This is the NMR spectrum of polyethylene glycol monomethyl ether-b-poly(methacryloyl-lysine)-b-poly(tyrosine phosphate−co−alanine).

[0122] Example 2

[0123] Enzyme-responsive polyamino acid dynamic conformational changes:

[0124] Weigh polyethylene glycol monomethyl ether-b-poly(methacryloyl-lysine)-b-poly(tyrosine phosphate-co-alanine) (EG 45 -K MA10 Ypf 25 ) was dissolved in ddH2O at a concentration of 0.3 mg / mL and incubated with alkaline phosphatase (ALP) at 0, 0.5, 1, 2, 5, 7 and 24 h using circular dichroism spectrometer. Figure 4 As shown, under ALP catalysis, the proportion of β-folded conformation of the polyamino acid assembly increased, and dynamic conformational behavior occurred.

[0125] Example 3

[0126] 1. Preparation of enzyme-responsive dynamic allosteric hydrogel

[0127] Weigh 0.1% w / v lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) and dissolve it in PBS. Weigh an appropriate amount of 8% w / v polyethylene glycol monomethyl ether-b-poly (methacryloyl lysine)-b-poly (tyrosine phosphate-co-alanine) (EG 45 -K MA10 Ypf 25 )、5% w / vEG 13 MA was dissolved in the solution containing LAP, vortexed and ultrasonicated to fully dissolve it, and the whole process was carried out under light-proof conditions. The polymer pre-crosslinking solution was prepared, and 78.5 μL of the solution was pipetted into a polytetrafluoroethylene mold, and the enzyme-responsive dynamic allosteric hydrogel was prepared by crosslinking with 405 nm blue light for 3 min.

[0128] 2. Characterization of mechanical properties of enzyme-responsive polyamino acid hydrogels before and after enzyme catalysis

[0129] The enzyme-responsive dynamic allosteric hydrogel prepared in the above manner was added with alkaline phosphatase ALP (0.5 U mL -1 ), react at 37 ℃, take samples at 0, 7, 12, 24, 36, 48, and 72 hours, and perform compression tests using a universal mechanical testing machine to measure the breaking compression strength. Figure 5 As shown, the compressive strength increases with the increase of enzyme catalysis time, which is mainly due to the increase in the degree of phosphate hydrolysis, the increase in chain segment hydrophobicity, the formation of the second network under physical interaction, the increase in β-folding, and the increase in mechanical properties.

[0130] This indicates that the enzyme-responsive polyamino acid hydrogel prepared by the present invention forms an ALP activity gradient as ALP diffuses to the cartilage surface in vivo, forming a gradually enhanced mechanical strength from the cartilage area to the subchondral bone area, and can mimic the extracellular matrix gradient mechanical properties of the natural osteocartilage hierarchical structure.

[0131] 4. Biocompatibility of enzyme-responsive polyamino acid hydrogel

[0132] The above-mentioned photocrosslinking reaction was used to prepare the enzyme-responsive dynamic allosteric hydrogel, which was made to reach swelling equilibrium in complete culture medium. After ultraviolet irradiation sterilization, the hydrogel was placed in a 24-well cell culture plate and 1 × 10 5 The cells were inoculated at a density of 100 μg / well in a culture plate containing gel. After 48 h of culture, the cell activity was detected using a live / dead assay kit. The cells were observed under a laser scanning confocal microscope. Figure 6 As shown, Figure 6From left to right in the middle are propidium iodide (PI) staining of dead cells, calcein (AM) staining of live cells, and the merged image of the two staining channels. Figure 6 This indicates that the enzyme-responsive polyamino acid hydrogel prepared by the present invention has biocompatibility.

[0133] 5. Enzyme-responsive dynamic allosteric composite hydrogel for full-thickness repair and regeneration of osteochondral cartilage in vivo

[0134] Animal experiment on osteochondral defect of rabbit knee joint: 6-month-old male New Zealand white rabbits were anesthetized and a 2.5 cm longitudinal incision was made along the medial edge of the patella on the right knee to expose the knee joint. A 5 mm trephine was used to make a 5 mm deep osteochondral defect model in the middle of the intercondylar articular surface of the femur. The blank group was not implanted with hydrogel after modeling, and the remaining two groups were implanted with a non-enzyme-responsive single-network polyamino acid composite hydrogel group mixed with BM-MSCs (as shown in formula a) and an enzyme-responsive dynamic allosteric composite hydrogel. Twelve weeks after surgery, some New Zealand white rabbits were euthanized and the femoral condyles were collected. The specimens were scanned with micro-CT, and three-dimensional reconstruction was performed after scanning. The morphological parameters of the new bone in the ROI area were calculated using CT-Analyser software. The full-thickness repair and regeneration effect of osteochondral cartilage is shown in Figure 2. Figure 7 As shown in the figure, Micro-CT analysis showed that the subchondral bone repair in the control group improved 12 weeks after implantation, but there were still cavities in the defect area; the new bone tissue in the enzyme-responsive dynamic allosteric composite hydrogel group was obvious, and the new bone structure was similar to the surrounding tissue. There were large cavities in the bone defect area in the blank hydrogel group. It can be seen that compared with the modeling blank group without any treatment measures and the non-enzyme-responsive single network polyamino acid composite hydrogel group, the enzyme-responsive dynamic allosteric composite hydrogel group had the best full-layer repair and regeneration effect.

[0135] Formula a.

[0136] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. An enzyme-responsive polyamino acid having a structure shown in Formula I: in, The carbonyl end of R1 is connected to the alkenyl group in formula I; R2=CH3; m, x, y, z are the degrees of polymerization; m＝10-1000； x＝1-100； y＝1-100； z＝1-100。 2. The method for preparing the enzyme-responsive polyamino acid according to claim 1, comprising the following steps: Using amino-terminated polyethylene glycol monomethyl ether as an initiator, a monomer of a structure of formula (II), a monomer of a structure of formula (III), and a monomer of a structure of formula (IV) are polymerized, and deprotected to obtain the enzyme-responsive polyamino acid; in, The carbonyl end of R1 is connected to the alkenyl group in Formula II; R2=CH3; 3. The preparation method according to claim 2, characterized in that: The monomer of the formula (II) is obtained by reacting methacryloyl amino acid with triphosgene; The methacryloyl amino acid is selected from methacryloyl-L-lysine, methacryloyl-serine or methacryloyl-threonine.

4. The preparation method according to claim 2, characterized in that: The monomer of formula (IV) is prepared by the following method: S1) reacting N-(tert-butyloxycarbonyl)-amino acid tert-butyl ester with diethyl chlorophosphate to obtain diethoxyphosphoryl-N-(tert-butyloxycarbonyl)-amino acid; and removing the protecting group to obtain diethoxyphosphoryl-amino acid; S2) reacting the diethoxyphosphoryl-amino acid with triphosgene to obtain a diethoxyphosphoryl-amino acid-N-intracyclic carboxylic acid anhydride monomer; The amino acid is selected from L-tyrosine.

5. The preparation method according to claim 2, characterized in that: The preparation method comprises the following steps: Amino-terminated polyethylene glycol monomethyl ether is used as an initiator to initiate a first polymerization reaction of the monomer of formula (II) to obtain a first polymer intermediate; The first polymer intermediate and the monomers of the structure of formula (III) and the monomers of the structure of formula (IV) undergo a second polymerization reaction, and deprotection is performed to obtain the enzyme-responsive polyamino acid.

6. An adaptive bionic mechanical gradient hydrogel, characterized in that: The invention is the photo-crosslinked product of the enzyme-responsive polyamino acid according to claim 1 or the enzyme-responsive polyamino acid prepared by the preparation method according to any one of claims 2 to 5.

7. The method for preparing the adaptive biomimetic mechanical gradient hydrogel according to claim 6 comprises the following steps: Under the action of a crosslinking agent and an initiator, the enzyme-responsive polyamino acid according to claim 1 or the enzyme-responsive polyamino acid prepared by the preparation method according to any one of claims 2 to 5 is subjected to a crosslinking reaction to obtain an adaptive bionic mechanical gradient hydrogel.

8. The preparation method according to claim 7, characterized in that: The cross-linking agent is selected from polyethylene glycol diacrylate; The initiator is selected from phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt.

9. Use of the adaptive bionic mechanical gradient hydrogel according to claim 6 or the adaptive bionic mechanical gradient hydrogel prepared by the preparation method according to any one of claims 7 to 8 in preparing materials for inducing or promoting tissue regeneration.

10. The use according to claim 9, characterized in that: The material for inducing or promoting tissue regeneration is a hydrogel scaffold.

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