Enzyme-responsive polyamino acid, polyamino acid 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, a polyamino acid hydrogel, and a preparation method and application thereof. The enzyme-responsive polyamino acid can be used to prepare an enzyme-responsive polyamino acid hydrogel by photocrosslinking. The hydrogel has alkaline phosphatase (ALP) response activity, good biocompatibility and biodegradability, and can realize a bioorthogonal reaction under ALP catalysis to form a second crosslinked network, thereby improving mechanical properties. Based on the gradient distribution of ALP activity in osteochondral tissue, the crosslinking degree of the second network also presents a gradient distribution, forming a gradient mechanical scaffold on a spatial scale, and ultimately achieving the effect of improving cartilage repair. This solves the problem that the traditional osteochondral static gradient scaffold cannot capture and adapt to the dynamic evolution of the tissue microenvironment and endogenous cell regeneration during osteochondral repair, and has broad application prospects in the field of osteochondral tissue regeneration.

Description

Technical Field

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

[0002] The complex structure of osteochondral cartilage and the poor self-repair ability of cartilage make the repair of osteochondral defects a difficult problem in clinical practice. With the development of synthetic biomaterials with adjustable mechanical properties, the relationship between mechanical properties and cell behavior has been widely explored, including adhesion, migration, proliferation and differentiation. Therefore, using gradient mechanical cues, especially the mechanical properties of the scaffold (hardness, porosity, viscoelasticity, etc.), to regulate the chondrogenic and osteogenic differentiation of bone marrow mesenchymal stem cells at different layers and guide gradient tissue regeneration is an effective strategy for repairing osteochondral defects.

[0003] At present, mechanical scaffolds that simulate the gradient layered structure characteristics of osteochondrocytes and repair osteochondrocytes mainly include double-layer, triple-layer and gradient mechanical scaffolds. Most of the current osteochondrocyte gradient mechanical scaffolds are static scaffolds that cannot capture the dynamic changes in osteochondrocyte repair. The current dynamic mechanically enhanced hydrogels lack the ability to sense the dynamic changes in ALP concentration in the osteochondrocyte repair microenvironment and cannot regulate the mechanical strength gradient distribution to induce stem cell multidirectional differentiation. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide an enzyme-responsive polyamino acid, a polyamino acid hydrogel, and a preparation method and application thereof. The enzyme-responsive polyamino acid has ALP-responsive activity, can be prepared into an ALP-responsive polyamino acid hydrogel and form a mechanical gradient scaffold in situ in vivo under the action of the ALP activity gradient in vivo to induce multidirectional differentiation of stem cells.

[0005] The present invention provides an enzyme-responsive polyamino acid having a structure shown in Formula I:

[0006]

[0007] Formula I;

[0008] Where R1= , or ;

[0009] R2= , or ;

[0010] R3= , or ;

[0011] 1≤n≤10;

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

[0013] The present invention provides a method for preparing an enzyme-responsive polyamino acid as shown in formula I, comprising the following steps:

[0014] 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 subjected to a polymerization reaction to obtain a side-group-containing methacryloyl polyamino acid as shown in formula Ⅰ-a; when the monomer of the structure of formula (III) and / or the monomer of the structure of formula (IV) contains an amino protecting group, the polymerization reaction further includes a deprotection reaction;

[0015] The side group-containing methacryloyl polyamino acid shown in the above formula Ⅰ-a and (2-cyanobenzo[d]thiazol-6-yl)glycine shown in formula Ⅰ-b are reacted to obtain the enzyme-responsive polyamino acid shown in formula Ⅰ;

[0016] Formula (II); Formula (III); Formula (IV);

[0017]

[0018] Formula I-a; Formula I-b;

[0019]

[0020] Formula I;

[0021] Where R1= , or ;

[0022] R2= , or ;

[0023] R3= , or ;

[0024] When R2= When R4= ; When R2= or When R4=H;

[0025] R5= , or ;

[0026] R6= , or ;

[0027] 1≤n≤10;

[0028] m, x, y, z are the degrees of polymerization.

[0029] The present invention provides an enzyme-responsive polyamino acid having a structure shown in Formula II:

[0030]

[0031] Formula II;

[0032] Where R1= , or ;

[0033] R3= , or ;

[0034] 1≤n≤10;

[0035] m, x, y, z are the degrees of polymerization.

[0036] The present invention provides a method for preparing an enzyme-responsive polyamino acid as shown in formula II, comprising the following steps:

[0037] 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 (V) and a monomer of a structure of formula (IV) are subjected to a polymerization reaction to obtain a side-group-containing methacryloyl polyamino acid represented by formula II-a; when the monomer of the structure of formula (IV) contains an amino protecting group, the polymerization reaction further includes a deprotection reaction;

[0038] The side group-containing methacryloyl polyamino acid shown in the above formula II-a and N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine ​​shown in the formula II-b are reacted and deprotected to obtain the enzyme-responsive polyamino acid shown in the formula II;

[0039] Formula (II); Formula (V); Formula (IV);

[0040]

[0041] Formula II-a; Formula II-b;

[0042]

[0043] Formula II;

[0044] Where R1= , or ;

[0045] R3= , or ;

[0046] R6= , or ;

[0047] 1≤n≤10; m, x, y, z are the degree of polymerization;

[0048] Fmoc is fluorenylmethoxycarbonyl.

[0049] The invention provides an enzyme-responsive polyamino acid hydrogel, comprising an enzyme-responsive polyamino acid as shown in formula I and an enzyme-responsive polyamino acid as shown in formula II.

[0050] The present invention provides a method for preparing the enzyme-responsive polyamino acid hydrogel, comprising the following steps:

[0051] The enzyme-responsive polyamino acid shown in formula I and the enzyme-responsive polyamino acid shown in formula II undergo cross-linking reaction under light conditions to obtain an enzyme-responsive polyamino acid hydrogel.

[0052] Preferably, a photoinitiator is added in the cross-linking reaction;

[0053] The photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate;

[0054] The solvent of the cross-linking reaction is PBS buffer;

[0055] The illumination is 405nm blue light;

[0056] The cross-linking reaction time is 1 to 5 minutes;

[0057] The mass concentrations of the enzyme-responsive polyamino acid shown in formula I and the enzyme-responsive polyamino acid shown in formula II are 40-75 mg / mL, respectively.

[0058] The present invention provides the use of the enzyme-responsive polyamino acid hydrogel or the enzyme-responsive polyamino acid hydrogel prepared by the preparation method in preparing materials for inducing or promoting tissue regeneration.

[0059] Preferably, the material for inducing or promoting tissue regeneration is a hydrogel scaffold.

[0060] Compared with the prior art, the enzyme-responsive polyamino acid provided by the present invention can be used to prepare enzyme-responsive polyamino acid hydrogel, which has alkaline phosphatase (ALP) responsive activity, good biocompatibility and biodegradability, and can realize bioorthogonal reaction under ALP catalysis to form a second cross-linking network, improve mechanical properties, and based on the ALP concentration gradient distribution in osteochondral tissue, the bioorthogonal chemical cross-linking degree gradient distribution forms a gradient mechanical scaffold on a spatial scale, and finally achieves the effect of improving cartilage repair. This solves the problem that the traditional osteochondral static gradient scaffold cannot capture and adapt to the dynamic evolution of the tissue microenvironment and the endogenous regeneration ability of cells during osteochondral repair, and has broad application prospects in the field of osteochondral tissue regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 is the H NMR spectrum of methacryloyl-L-lysine-N-carboxylic anhydride monomer;

[0062] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine;

[0063] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of (2-cyanobenzo[d]thiazol-6-yl)glycine;

[0064] Figure 4 This is the H NMR spectrum of polylysine amino acid containing methacryloyl groups on the side groups;

[0065] Figure 5 This is the H NMR spectrum of polylysine amino acid containing methacryloyl groups on the side grafted with (2-cyanobenzo[d]thiazol-6-yl)glycine;

[0066] Figure 6 This is the H NMR spectrum of polylysine amino acid grafted with S-phosphocysteine ​​containing methacryloyl groups on the side groups;

[0067] Figure 7 The NMR phosphorus spectra of the side group-containing methacryloyl polylysine amino acid grafted with S-(diethoxyphosphoryl)cysteine ​​and the side group-containing methacryloyl polylysine amino acid grafted with S-phosphorylcysteine ​​(i.e., before and after deethyl protection);

[0068] Figure 8 It is the gradient bar graph of ALP enzyme activity of osteochondrocytes and tissues;

[0069] Fig. 9 It is a compression test curve diagram of enzyme-responsive polyamino acid hydrogel alkaline phosphatase catalysis at different time points from 0 to 72 hours;

[0070] Fig.10Fluorescence images of enzyme-responsive polyamino acid hydrogel co-incubated with cells;

[0071] Fig.11 This image shows the repair and regeneration effect of the full-thickness osteochondral defect 8 weeks after hydrogel implantation. DETAILED DESCRIPTION

[0072] The present invention provides an enzyme-responsive polyamino acid having a structure shown in Formula I:

[0073]

[0074] Formula I;

[0075] Where R1= , or ;

[0076] R2= , or ;

[0077] R3= , or ;

[0078] 1≤n≤10;

[0079] 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.

[0080] x is the degree of polymerization, x=1-100; preferably 5-60, more preferably 10-30. In some specific embodiments of the present invention, x is 20.

[0081] y is the degree of polymerization, x=1-100; preferably 10-60, more preferably 20-40. In some specific embodiments of the present invention, y is 30.

[0082] z is the degree of polymerization, z=1-100; preferably 10-80, more preferably 45-65. In some specific embodiments of the present invention, z is 52.

[0083] The carbonyl end of the above R1 is connected to the alkenyl group in formula I, and the alkyl end is connected to the main chain.

[0084] The carbonyl end or the near-carbonyl end of the above R2 is connected to the amino group in formula I, and the alkyl end is connected to the main chain.

[0085] The present invention provides a method for preparing the monomer represented by the above formula I, comprising the following steps:

[0086] 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 subjected to a polymerization reaction to obtain a side-group-containing methacryloyl polyamino acid as shown in formula Ⅰ-a; when the monomer of the structure of formula (III) and / or the monomer of the structure of formula (IV) contains an amino protecting group, the polymerization reaction further includes a deprotection reaction;

[0087] The side group-containing methacryloyl polyamino acid shown in the above formula Ⅰ-a and (2-cyanobenzo[d]thiazol-6-yl)glycine shown in formula Ⅰ-b are reacted to obtain the enzyme-responsive polyamino acid shown in formula Ⅰ;

[0088] Formula (II); Formula (III); Formula (IV);

[0089]

[0090] Formula I-a; Formula I-b;

[0091]

[0092] Formula I;

[0093] Where R1= , or ;

[0094] R2= , or ;

[0095] R3= , or ;

[0096] R5= , or ;

[0097] R6= , or ;

[0098] When R2= When R4= ; When R2= or When R4=H;

[0099] 1≤n≤10;

[0100] m, x, y, z are the degrees of polymerization.

[0101] The ranges of m, x, y, and z are the same as above.

[0102] Cbz is benzyloxycarbonyl.

[0103] Preferably, in the above reaction system, 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N,N-diisopropylethylamine are added to activate the condensation reaction.

[0104] The solvent for the above reaction is preferably N,N-dimethylformamide.

[0105] Preferably, a polymerization inhibitor is added in the above reaction to prevent photo-crosslinking during the reaction, and the polymerization inhibitor is preferably p-benzoquinone.

[0106] The above reaction is preferably carried out under light-shielding conditions.

[0107] The reaction temperature is preferably 20-30° C., and the reaction time is preferably 10-20 h.

[0108] Preferably, after the reaction is completed, the reaction system is precipitated and filtered in ice ether, then dissolved with dichloromethane and precipitated multiple times, and the ether on the surface of the material is drained by a vacuum drying pump to obtain a side group-containing methacryloyl polyamino acid grafted with (2-cyanobenzo[d]thiazole-6-yl)glycine.

[0109] Preferably, the side group-containing methacryloyl polyamino acid represented by the above formula Ⅰ-a is prepared according to the following method:

[0110] Using amino-terminated polyethylene glycol monomethyl ether as an initiator, methacryloyl-L-lysine / serine / threonine-N-carboxylic anhydride, Nε-benzyloxycarbonyl-L-lysine / glutamic acid / aspartic acid-N-carboxylic anhydride, Nε-acetyl-L-lysine / Nε-(2-[2-(2-methoxyethoxy)ethoxy]acetyl-L-lysine-N-carboxylic anhydride (OEG2-Lys-NCA) / Nε-benzyloxycarbonyl-L-lysine is subjected to a ring-opening polymerization reaction, and the benzyloxycarbonyl protecting group is removed by trifluoroacetic acid and an acetic acid solution of hydrogen bromide (preferably at a concentration of 33%) to obtain a side-group methacryloyl-containing polyamino acid as shown in formula I-a.

[0111] The amino-terminated polyethylene glycol monomethyl ether is preferably anhydrous amino-terminated polyethylene glycol monomethyl ether.

[0112] The solvent for the above polymerization reaction is preferably N,N-dimethylformamide.

[0113] 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: (5-60): (10-60): (10-80); more preferably 1: (10-30): (20-40): (45-65); most preferably 1: 20: 30: 52.

[0114] The reaction temperature is preferably 20-30°C, more preferably 25°C.

[0115] The reaction time is preferably 48 to 96 hours, more preferably 72 hours.

[0116] Preferably, after the reaction is completed, the reaction system is precipitated and filtered in ice ether, then dissolved with dichloromethane and precipitated multiple times, the ether on the surface of the material is drained by a vacuum drying pump, and then the amino protecting group is removed under acidic conditions, and the polyamino acid with a methacryloyl side group as shown in formula Ⅰ-a is obtained by purification by dialysis.

[0117] In the present invention, the N-carboxyanhydride structure of the above-mentioned methacryloyl-L-lysine / serine / threonine--N-carboxyanhydride, Nε-benzyloxycarbonyl-L-lysine / glutamic acid / aspartic acid-N-carboxyanhydride, Nε-acetyl-L-lysine / Nε-(2-[2-(2-methoxyethoxy)ethoxy]acetyl-L-lysine-N-carboxyanhydride (OEG2-Lys-NCA) / Nε-benzyloxycarbonyl-L-lysine is obtained by reacting amino acids with triphosgene.

[0118] In the above formula Ⅰ-b, when R4= , Ⅰ-b is That is, (2-cyanobenzo[d]thiazol-6-yl)glycine is prepared by reacting 6-amino-2-cyanobenzothiazole and glyoxylic acid under the action of sodium cyanoborohydride.

[0119] The present invention also provides an enzyme-responsive polyamino acid having a structure shown in Formula II:

[0120]

[0121] Formula II;

[0122] Where R1= , or ;

[0123] R3= , or ;

[0124] 1≤n≤10;

[0125] m, x, y, z are the degrees of polymerization.

[0126] 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.

[0127] x is the degree of polymerization, x=1-100; preferably 5-60, more preferably 10-30. In some specific embodiments of the present invention, x is 20.

[0128] y is the degree of polymerization, x=1-100; preferably 10-60, more preferably 20-40. In some specific embodiments of the present invention, y is 30.

[0129] z is the degree of polymerization, z=1-100; preferably 10-80, more preferably 45-65. In some specific embodiments of the present invention, z is 52.

[0130] The carbonyl end of the above R1 is connected to the alkenyl group in formula II, and the alkyl end is connected to the main chain.

[0131] The present invention provides a method for preparing the enzyme-responsive polyamino acid represented by the above formula II, comprising the following steps:

[0132] 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 (V) and a monomer of a structure of formula (IV) are subjected to a polymerization reaction to obtain a side-group-containing methacryloyl polyamino acid represented by formula II-a; when the monomer of the structure of formula (IV) contains an amino protecting group, the polymerization reaction further includes a deprotection reaction;

[0133] The side group-containing methacryloyl polyamino acid shown in the above formula II-a and N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine ​​shown in the formula II-b are reacted and deprotected to obtain the enzyme-responsive polyamino acid shown in the formula II;

[0134] Formula (II); Formula (V); Formula (IV);

[0135]

[0136] Formula II-a; Formula II-b;

[0137]

[0138] Formula II;

[0139] Where R1= , or ;

[0140] R3= , or ;

[0141] R6= , or ;

[0142] 1≤n≤10;

[0143] m, x, y, z are the degrees of polymerization;

[0144] Fmoc is fluorenylmethoxycarbonyl.

[0145] The ranges of m, x, y, and z are the same as above.

[0146] The conditions for the above deprotection reaction can be selected according to the type of the protecting group.

[0147] The deprotection after the reaction of the side group-containing methacryloyl polyamino acid shown in the above formula II-a and N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine ​​shown in formula II-b includes de-Fmoc and de-ethyl reactions.

[0148] Preferably, in the above reaction system, 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N,N-diisopropylethylamine are added to activate the condensation reaction.

[0149] The solvent for the above reaction is preferably N,N-dimethylformamide.

[0150] Preferably, a polymerization inhibitor is added in the above reaction to prevent photocrosslinking during the reaction, and the polymerization inhibitor is preferably p-benzoquinone.

[0151] The above reaction is preferably carried out under light-shielding conditions.

[0152] The reaction temperature is preferably 20-30° C., and the reaction time is preferably 10-20 h.

[0153] Preferably, after the reaction is completed, the reaction system is precipitated and filtered in ice ether, then dissolved with dichloromethane and precipitated multiple times, and the ether on the surface of the material is drained by a vacuum drying pump to obtain a side group-containing methacryloyl polyamino acid grafted with N-(((9H-fluorene-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine.

[0154] The product is then deprotected in N,N-dimethylformamide solvent to obtain a side-group-containing methacryloyl-containing polyamino acid grafted with S-phosphorylcysteine.

[0155] The side group-containing methacryloyl polyamino acid represented by the above formula II-a is prepared according to the following method:

[0156] Using amino-terminated polyethylene glycol monomethyl ether as an initiator, methacryloyl-L-lysine / serine / threonine-N-carboxylic anhydride, Nε-benzyloxycarbonyl-L-lysine-N-carboxylic anhydride, Nε-acetyl-L-lysine / Nε-(2-[2-(2-methoxyethoxy)ethoxy]acetyl-L-lysine-N-carboxylic anhydride (OEG2-Lys-NCA) / Nε-benzyloxycarbonyl-L-lysine-N-carboxylic anhydride are subjected to ring-opening polymerization, and the benzyloxycarbonyl protecting group is removed by trifluoroacetic acid and 33% hydrogen bromide acetic acid solution to obtain a side-group methacryloyl polyamino acid shown in formula II-a.

[0157] The amino-terminated polyethylene glycol monomethyl ether is preferably anhydrous amino-terminated polyethylene glycol monomethyl ether.

[0158] The solvent for the above polymerization reaction is preferably N,N-dimethylformamide.

[0159] 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: (5-60): (10-60): (10-80); more preferably 1: (10-30): (20-40): (45-65); most preferably 1: 20: 30: 52.

[0160] The reaction temperature is preferably 20-30°C, more preferably 25°C.

[0161] The reaction time is preferably 48 to 96 hours, more preferably 72 hours.

[0162] Preferably, after the reaction is completed, the reaction system is precipitated and filtered in ice ether, then dissolved with dichloromethane and precipitated multiple times, the ether on the surface of the material is drained by a vacuum drying pump, and then the amino protecting group is removed under acidic conditions, and the polyamino acid with a methacryloyl side group as shown in formula II-a is obtained by purification by dialysis.

[0163] The N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine ​​represented by the above formula II-b is prepared by the following method:

[0164] S1) reacting diethyl phosphite with di(2-methyl-2-propyl) N,N'-di[(9H-fluoren-9-ylmethoxy)carbonyl]-L-cysteinylstannate to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine ​​tert-butyl ester;

[0165] S2) Deprotecting the above N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine ​​tert-butyl ester in an acidic environment to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine.

[0166] Preferably, the above step S1) is carried out under the action of a catalyst, and the catalyst is preferably cesium carbonate.

[0167] The reaction solvent in step S1) is preferably acetonitrile.

[0168] The reaction temperature is preferably 20-40°C, more preferably 30°C.

[0169] The reaction time is preferably 7 to 9 hours, more preferably 8 hours.

[0170] The reaction is preferably carried out under oxygen conditions.

[0171] Preferably, after the reaction is completed, the product is purified by column chromatography. The eluents for the column chromatography are preferably ethyl acetate and n-hexane.

[0172] The acidic environment in the above step S2) is preferably provided by trifluoroacetic acid.

[0173] In some specific embodiments of the present invention, the preparation process of N-(((9H-fluorene-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine ​​shown in the above formula II-b is as follows:

[0174]

[0175] Fmoc is fluorenylmethoxycarbonyl.

[0176] On the other hand, the present invention provides an enzyme-responsive polyamino acid hydrogel, comprising the enzyme-responsive polyamino acid shown in the above formula I and the enzyme-responsive polyamino acid shown in the above formula II.

[0177] The enzyme-responsive polyamino acid shown in the above formula I and the enzyme-responsive polyamino acid shown in the above formula II form a carbon-carbon bond structure through a double bond structure under the substitution reaction of free radicals under the irradiation of a photoinitiator and blue light, so that the polymer chains are cross-linked to form a hydrogel.

[0178] The present invention prepares binary modified polyamino acids in polyamino acid gel by grafting cyanobenzothiazole and thiophosphorylcysteine ​​respectively with polyamino acids containing methacryloyl groups on the side groups. After the binary modified polyamino acids are photocrosslinked into gel, the polymer chains of the polyamino acid hydrogel containing sulfur phosphate esters on the side chains undergo phosphate hydrolysis under the action of ALP in the osteochondral microenvironment to generate 1,2-aminothiol and polymer chains containing cyanobenzothiazole on the side chains, and a bioorthogonal reaction occurs to realize a second chemical crosslinking network and improve mechanical properties. In addition, since the ALP concentration in the microenvironment is continuously increasing from articular cartilage to subchondral bone and bone tissue will continue to secrete ALP during osteochondral repair, the degree of hydrolysis of sulfur phosphate esters is increased, the degree of crosslinking of bioorthogonal chemical reactions is increased, and the mechanical properties are increased to form a spatial self-generated gradient mechanical scaffold. In the process of osteochondral repair, dynamic mechanical signals are provided to stem cells to induce them to have different proliferation and differentiation behaviors at different stages and layers, so as to adapt to the dynamic evolution of cells in the repair process and promote efficient full-layer repair of osteochondral.

[0179] The present invention selects an ALP-responsive bioorthogonal chemical reaction cross-linking network as the second cross-linking network. On the one hand, it dynamically enhances the mechanical properties on a time scale to meet the requirements of the initial cell proliferation stage, where lower hardness is beneficial to cell proliferation. In the later cell differentiation stage, it can maintain lower hardness in the cartilage layer to promote cartilage, while in the subchondral bone layer it can enhance the mechanical properties into a hard gel to regulate osteogenic differentiation, thereby accurately and efficiently promoting full-layer repair of osteochondral cartilage.

[0180] The present invention provides a method for preparing the enzyme-responsive polyamino acid hydrogel, comprising the following steps:

[0181] The enzyme-responsive polyamino acid shown in formula I and the enzyme-responsive polyamino acid shown in formula II undergo cross-linking reaction under light conditions to obtain an enzyme-responsive polyamino acid hydrogel.

[0182] Preferably, a photoinitiator is added in the cross-linking reaction.

[0183] The photoinitiator is preferably phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

[0184] The solvent for the cross-linking reaction is preferably PBS buffer.

[0185] The illumination is preferably 405 nm blue light.

[0186] The cross-linking reaction time is preferably 1 to 5 minutes, more preferably 3 minutes.

[0187] The mass concentrations of the enzyme-responsive polymer represented by Formula I and the enzyme-responsive polymer represented by Formula II are 40-75 mg / mL, more preferably 50 mg / mL.

[0188] On the other hand, the present invention provides the use of the enzyme-responsive polyamino acid hydrogel or the enzyme-responsive polyamino acid hydrogel prepared by the above preparation method in the preparation of materials for inducing or promoting tissue regeneration.

[0189] The above-mentioned material for inducing or promoting tissue regeneration is preferably a hydrogel scaffold.

[0190] Specifically, the present invention provides a hydrogel scaffold for inducing or promoting tissue regeneration, which is prepared from the above-mentioned enzyme-responsive polyamino acid hydrogel or the enzyme-responsive polyamino acid hydrogel prepared by the above-mentioned preparation method.

[0191] The enzyme-responsive polyamino acid hydrogel provided by the present invention has a dynamic ALP-responsive bioorthogonal chemical cross-linking network that senses the gradient distribution of ALP activity in the osteochondral microenvironment. The dynamic mechanical enhancement can meet the requirements of promoting cell proliferation with lower hardness in the initial cell proliferation stage, and form a mechanical gradient scaffold in the later stage to induce multidirectional differentiation of stem cells, adapt to the dynamic evolution of cells in the repair process, and promote efficient full-layer repair of osteochondrium.

[0192] In order to further illustrate the present invention, the enzyme-responsive polyamino acid composite hydrogel and its monomer, preparation method and application provided by the present invention are described in detail below in conjunction with the examples. 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.

[0193] 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.

[0194] Example 1

[0195] 1. Preparation of methacryloyl-L-lysine-N-carboxylic anhydride monomer

[0196] 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, and 1.4782 g of methacryloyl-L-lysine-N-carboxylic anhydride was obtained after separation, washing, recrystallization, and vacuum drying, with a yield of 59.6%.

[0197] Figure 1 This is the H NMR spectrum of methacryloyl-L-lysine anhydride.

[0198] 2. Preparation of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine ​​cross-linking point small molecules

[0199] The reaction flask was added with 1.3627 g cesium carbonate as catalyst, 1.1552 g diethyl phosphite, 4 g di(2-methyl-2-propyl)N,N'-di[(9H-fluoren-9-ylmethoxy)carbonyl]-L-cysteinyl stannate, and 100 mL acetonitrile, and stirred at 30 °C in an oxygen atmosphere for 8 h. After the reaction, 200 mL ethyl acetate was added, washed with water, dried and filtered, concentrated by rotary evaporation, and purified by column chromatography, with ethyl acetate and n-hexane as eluents, to obtain N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine ​​tert-butyl ester. N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine ​​tert-butyl ester was dissolved in 50 mL dichloromethane, and 50 mL trifluoroacetic acid was added. The reaction was stirred at room temperature, and the reaction was completed by spot plate tracking. After the reaction was completed, the solvent was drained to obtain 0.9723 g of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine ​​with a yield of 40.4%.

[0200] Figure 2 This is the H NMR spectrum of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine.

[0201] 3. Preparation of (2-cyanobenzo[d]thiazol-6-yl)glycine cross-linking point small molecules

[0202] 0.4 g of 6-amino-2-cyanobenzothiazole and 0.389 mg of glyoxylic acid were dissolved in acetonitrile, 0.215 sodium cyanoborohydride was added, acetic acid was added until the solution became clear, and then stirred at 25 °C for 20 min. After the reaction was completed, 50 mL of water was added, and then the solution was evaporated to half the remaining volume, extracted with ethyl acetate, and the organic layer was collected and washed with water and saturated brine three times, dried and filtered, concentrated by rotary evaporation, and purified by column chromatography to obtain 0.4091 g of (2-cyanobenzo[d]thiazol-6-yl)glycine with a yield of 76.8%.

[0203] Figure 3 This is the H NMR spectrum of (2-cyanobenzo[d]thiazol-6-yl)glycine.

[0204] 4. Preparation of polyamino acids containing methacryloyl groups on the side groups

[0205] 0.5 mmol of amino-terminated polyethylene glycol monomethyl ether (Mw = 2000) was dissolved in 100 mL of toluene, and the mixture was azeotroped in an oil bath at 130 °C for 4 h to remove excess water. The toluene was then removed by vacuum distillation to obtain anhydrous amino-terminated polyethylene glycol monomethyl ether. Anhydrous amino-terminated polyethylene glycol monomethyl ether was dissolved in 50 mL of anhydrous N,N-dimethylformamide, and then 7.5 mmol of methacryloyl-L-lysine-N-carboxylic anhydride and 42.5 mmol of L-lysine-N-carboxylic anhydride were added under nitrogen protection and stirred at 25 °C for 72 hours, during which CO2 was pumped for 15 min every day. The reaction solution was precipitated and filtered in 10 times of ice ether, then dissolved in dichloromethane and precipitated twice, and the ether on the surface of the material was drained by a vacuum drying pump. The dried product was dissolved in 30 mL of trifluoroacetic acid, and 15 mL of hydrogen bromide in acetic acid solution was added. The reaction was stirred at 25 °C for 1 h. The reaction solution was poured into 500 mL of ether and filtered. The obtained solid was dissolved in water and dialyzed in deionized water for 3 d using a dialysis bag with a molecular weight cutoff of 7000. The dialysate was changed every 4 h. The obtained solution was freeze-dried to obtain 5.9632 g of polylysine amino acid containing methacryloyl groups on the side groups, with a yield of 74.3%.

[0206] Figure 4 This is the H NMR spectrum of polylysine amino acid containing methacryloyl groups on the side groups.

[0207] 5. Preparation of polyamino acid grafted with (2-cyanobenzo[d]thiazol-6-yl)glycine containing methacryloyl side groups

[0208] 0.2534 g of 1-hydroxybenzotriazole, 0.4782 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 0.4361 g of (2-cyanobenzo[d]thiazol-6-yl)glycine were dissolved in anhydrous N,N-dimethylformamide, stirred and reacted for 1 h at 25 ° C in a nitrogen atmosphere, and 1 g of polylysine amino acid containing methacryloyl side groups, 0.3224 g of N,N-diisopropylethylamine and a catalytic amount of p-benzoquinone were added to the above reaction solution, and stirred and reacted for 15 h in the dark. After the reaction was completed, it was precipitated and filtered in 10 times of ice ether, and then dissolved in dichloromethane and precipitated twice. The ether on the surface of the material was drained by a vacuum drying pump to obtain 0.7255 g of polylysine amino acid grafted with methacryloyl side groups, with a yield of 51.7%.

[0209] Figure 5 This is the hydrogen nuclear magnetic resonance spectrum of polylysine containing methacryloyl groups on the side grafted with (2-cyanobenzo[d]thiazol-6-yl)glycine.

[0210] 6. Preparation of polyamino acid grafted with S-phosphorylcysteine ​​containing methacryloyl side groups

[0211] 0.2534 g of 1-hydroxybenzotriazole, 0.4782 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 0.89576 g of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine ​​were dissolved in anhydrous N,N-dimethylformamide, and stirred for reaction at 25 °C in a nitrogen atmosphere for 1 h. 1 g of polylysine amino acid containing methacryloyl side groups, 0.3224 g of N,N-diisopropylethylamine, and a catalytic amount of p-benzoquinone were added to the above reaction solution, and stirred for reaction for 15 h in the dark. After the reaction, the mixture was precipitated and filtered in 10 times of ice ether, then dissolved in dichloromethane and precipitated twice, and the ether on the surface of the material was drained by a vacuum drying pump to obtain a side-group containing methacryloyl polylysine amino acid grafted with N-(((9H-fluorene-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine. The dried product was dissolved in 25 mL of anhydrous N,N-dimethylformamide, 25 mL of N,N-diisopropylethylamine was added, and the reaction was stirred at room temperature for 8 h. After the reaction, the mixture was precipitated and filtered in 10 times of ice ether, then dissolved in dichloromethane and precipitated twice, and the ether on the surface of the material was drained by a vacuum drying pump to obtain 0.8102 g of side-group containing methacryloyl polylysine amino acid grafted with S-(diethoxyphosphoryl)cysteine, with a yield of 56%.

[0212] 0.8 g of S-(diethoxyphosphoryl)cysteine ​​grafted with polylysine amino acid containing methacryloyl side groups was dissolved in 100 mL of anhydrous N,N-dimethylformamide / dichloromethane mixed solvent, and 9.3 mL of trimethylsilyl bromide and 8 mL of triethylamine were added, and the mixture was heated under reflux at 65 °C for 8 h. The reaction was terminated by quenching with methanol, and the solvent was removed by vacuum pumping. 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 0.5104 g of S-phosphorylcysteine ​​grafted with polylysine amino acid containing methacryloyl side groups, with a yield of 68.8%.

[0213] Figure 6 The invention is a polylysine amino acid containing a methacryloyl group on the side group grafted with N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine.

[0214] Figure 7 Nuclear magnetic resonance phosphorus spectra of polylysine amino acid containing methacryloyl side groups grafted with S-(diethoxyphosphoryl)cysteine ​​and polylysine amino acid containing methacryloyl side groups grafted with S-phosphorylcysteine ​​(i.e. before and after deethyl protection).

[0215] Example 2. Preparation of enzyme-responsive polyamino acid hydrogel

[0216] Weigh 0.1% w / v lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) and dissolve it in PBS. Weigh an appropriate amount of 5% w / v polylysine containing methacryloyl side groups grafted with S-phosphocysteine ​​(P 18 -(Cys(PO(OH)2)), 5% w / v side-group containing methacryloyl polylysine grafted with (2-cyanobenzo[d]thiazol-6-yl)glycine solution (P 18 -CBT) was dissolved in a solution containing LAP, vortexed and ultrasonicated to fully dissolve it, and the whole process was carried out under light-proof conditions. The polymer solution was prepared as a 10% w / v pre-crosslinking solution, and 78.5 μL of the solution was respectively drawn into a polytetrafluoroethylene mold, and the enzyme-responsive polyamino acid hydrogel was prepared by 405 nm blue light crosslinking for 3 min.

[0217] Embodiment 3,

[0218] 1. Differences in the concentration levels of alkaline phosphatase activity in osteochondrocytes

[0219] Prepare osteogenic and chondrogenic culture medium according to the instructions. Digest bone marrow mesenchymal stem cells (BM-MSCs) and add 1×10 5 The cells were added to osteogenic and chondrogenic culture medium at a density of 100 / mL and cultured. The medium was changed regularly and samples were taken on the 14th day. Samples were prepared according to the alkaline phosphatase (ALP) detection kit and the ALP activity units were determined. Figure 8 As shown, it was shown that there was a unit difference in ALP activity in osteochondrocytes.

[0220] 2. Alkaline phosphatase activity concentration gradient in osteochondral tissue

[0221] The cartilage, subchondral bone and bone tissue of the knee joint of 6-month-old male healthy New Zealand white rabbits were extracted in layers, and the protein was extracted with protein extract solution. The total protein content was determined by BAC total protein kit, and then the ALP enzyme activity unit was determined by ALP detection kit. Figure 8 As shown, it shows that there is a gradient difference in ALP enzyme activity in osteocartilage.

[0222] 3. Characterization of mechanical properties of enzyme-responsive polyamino acid hydrogels before and after enzyme catalysis

[0223] The enzyme-responsive polyamino acid hydrogel material was prepared by the above-mentioned photocrosslinking method, and alkaline phosphatase was added to react at 37°C. Samples were taken at 0, 7, 12, 24, 36, 48, and 72 hours, and compression tests were performed using a universal mechanical testing machine. Fig. 9As shown, the compressive strength increases with the increase of enzyme catalysis time, which is mainly due to the increase in the degree of hydrolysis of thiophosphate, the increase in the degree of bioorthogonal chemical cross-linking, the formation of the second chemical cross-linking network, and the increase in mechanical properties, which verifies the enhancement of the mechanical properties of the gel after enzyme catalysis.

[0224] 4. Biocompatibility of enzyme-responsive polyamino acid hydrogel

[0225] The enzyme-responsive polyamino acid hydrogel was prepared by photocrosslinking and allowed to reach swelling equilibrium in complete culture medium. After sterilization by ultraviolet irradiation, the hydrogel was placed in a 24-well cell culture plate and cultured at 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. Fig.10 As shown, Fig.10 From left to right in the figure are propidium iodide (PI) staining of dead cells, calcein (AM) staining of live cells and the merged image of the two staining channels, which proves that the enzyme-responsive polyamino acid hydrogel is biocompatible.

[0226] 5. Effect of enzyme-responsive polyamino acid composite hydrogel on full-thickness repair and regeneration of osteochondral cartilage in vivo

[0227] Animal experiment of osteochondral defect in 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 right knee patella 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 femoral intercondylar articular surface. The rabbits were divided into a blank group, a photocrosslinked single network hydrogel group, i.e., a non-enzyme-responsive hydrogel group (made by crosslinking only the enzyme-responsive polyamino acid shown in formula I), and an enzyme-responsive double network hydrogel group (made by crosslinking the enzyme-responsive polyamino acid shown in formula I and the enzyme-responsive polyamino acid shown in formula II). The blank group was not implanted with hydrogel after modeling, and the remaining two groups were implanted with a photocrosslinked single network polyamino acid composite hydrogel mixed with BM-MSCs and a double network enzyme-responsive polyamino acid composite hydrogel, respectively. Eight weeks after surgery, some New Zealand white rabbits were euthanized and the femoral condyles were collected. The specimens were scanned by 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 effect of full-thickness repair and regeneration of osteochondral cartilage is as follows Fig.11 As shown in the figure, Micro-CT3D reconstruction and quantitative analysis showed that the subchondral bone repair in the photocrosslinked hydrogel group improved 8 weeks after implantation, but there were still cavities in the defect area; the new bone tissue in the enzyme-responsive 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 photocrosslinked single-network polyamino acid composite hydrogel group, the enzyme-responsive double-network hydrogel group had the best full-layer repair and regeneration effect.

[0228] 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 hydrogel, comprising an enzyme-responsive polyamino acid as shown in formula I and an enzyme-responsive polyamino acid as shown in formula II; The carbonyl end or near the carbonyl end of R2 is connected to the amino group in formula I, and the alkyl end is connected to the main chain; In Formula I and Formula II, The carbonyl end of R1 is connected to the alkenyl group in Formula I and Formula II, and the alkyl end is connected to the main chain; 1≤n≤10； m, x, y, z are the degrees of polymerization; m＝10-1000； x＝1-100； y＝1-100； z＝1-100。 2. The enzyme-responsive polyamino acid hydrogel according to claim 1, wherein the preparation method of the enzyme-responsive polyamino acid represented by formula I comprises 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 to obtain a side-group-containing methacryloyl polyamino acid as shown in formula Ⅰ-a; when the monomer of the structure of formula (III) and / or the monomer of the structure of formula (IV) contains an amino protecting group, the polymerization reaction further includes a deprotection reaction; The side group-containing methacryloyl polyamino acid shown in the above formula Ⅰ-a and (2-cyanobenzo[d]thiazol-6-yl)glycine shown in formula Ⅰ-b are reacted to obtain the enzyme-responsive polyamino acid shown in formula Ⅰ; in, when hour, when When R4=H; 1≤n≤10； Cbz is benzyloxycarbonyl; m, x, y, z are the degrees of polymerization.

3. The enzyme-responsive polyamino acid hydrogel according to claim 1, wherein the preparation method of the enzyme-responsive polyamino acid represented by formula II comprises 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 (V) and a monomer of a structure of formula (IV) are subjected to a polymerization reaction to obtain a side-group-containing methacryloyl polyamino acid as shown in formula II-a; when the monomer of the structure of formula (IV) contains an amino protecting group, a deprotection reaction is further included after the polymerization reaction; The side group-containing methacryloyl polyamino acid shown in the above formula II-a and N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(diethoxyphosphoryl)cysteine ​​shown in formula II-b are reacted and deprotected to obtain the modified polyamino acid shown in formula II; in, 1≤n≤10； m, x, y, z are the degrees of polymerization; Cbz is benzyloxycarbonyl; Fmoc is fluorenylmethoxycarbonyl.

4. The method for preparing the enzyme-responsive polyamino acid hydrogel according to claim 1, comprising the following steps: The enzyme-responsive polyamino acid shown in formula I and the enzyme-responsive polyamino acid shown in formula II undergo cross-linking reaction under light conditions to obtain an enzyme-responsive polyamino acid hydrogel.

5. The preparation method according to claim 4, characterized in that: A photoinitiator is added to the cross-linking reaction; The photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate; The solvent of the cross-linking reaction is PBS buffer; The illumination is 405nm blue light; The cross-linking reaction time is 1 to 5 minutes; The mass concentrations of the enzyme-responsive polyamino acid shown in formula I and the enzyme-responsive polyamino acid shown in formula II are 40-75 mg / mL respectively.

6. Use of the enzyme-responsive polyamino acid hydrogel according to any one of claims 1 to 3 or the enzyme-responsive polyamino acid hydrogel prepared by the preparation method according to any one of claims 4 to 5 in preparing materials for inducing or promoting tissue regeneration.

7. The use according to claim 6, characterized in that: The material for inducing or promoting tissue regeneration is a hydrogel scaffold.

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