CN113234236A

CN113234236A - Viscoelastic hydrogel and preparation method and application thereof

Info

Publication number: CN113234236A
Application number: CN202110501247.1A
Authority: CN
Inventors: 刘昌胜; 袁媛; 伍自涵
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2021-05-08
Filing date: 2021-05-08
Publication date: 2021-08-10

Abstract

The invention provides a hydrogel. Specifically, the present invention provides a hydrogel having adjustable viscoelasticity formed by an injectable manner, the hydrogel being formed by a first polymer and a second polymer through a crosslinking reaction; wherein the first polymer comprises an azidoated polyhydroxy pegylated polysebacic acid glyceride; the second polymer comprises cyclic alkynyl polyhydroxy polyethylene glycol polysebacic acid glyceride. The biological safe hydrogel can be highly fit at the defect part of complex tissues, is favorable for the adhesion growth of stem cells, induces the stem cells to differentiate towards different histiocyte directions, thereby promoting the tissue repair process and improving the repair quality, and is a tissue repair material with clinical application prospect.

Description

Viscoelastic hydrogel and preparation method and application thereof

Technical Field

The invention relates to the field of material science and medicine, in particular to a viscoelastic hydrogel which is self-crosslinked in an in-vivo environment based on click chemistry and can be formed by injection, and a preparation method and application thereof. In particular for the repair of tissue defects of complex structures.

Background

Tissue defect conditions caused by congenital diseases, accidental injuries, tumor excision and the like are increased day by day, so that the tissue repair material has huge market demand, and simultaneously has higher and higher requirements on operation, mechanical properties and biological activity of the existing repair material, and particularly the tissue defects of different parts need different mechanical properties. The high-strength hydrogel can play a certain supporting role at the defect part by virtue of the mechanical property thereof, and can prevent deformation, collapse and even breakage caused by external stress.

Polysebacylic acid glyceride (PGS) is a bio-elastomer approved by the Food and Drug Administration (FDA) at present, and has good elasticity, so that the poly-sebacylic acid glyceride has potential in clinical application. However, the characteristics of in vitro molding and curing make the implantation of the material be realized by surgery, and the complicated surgical process and secondary trauma are unfavorable for doctors and patients; moreover, the excessively high modulus of the PGS material makes it difficult to release the stress in the cells growing on the surface of the material, which hinders the spreading, proliferation and differentiation of the cells. Because the polyethylene glycol (PEG) molecular chain segment has good flexibility and hydrophilicity, the polyethylene glycol (PEG) molecular chain segment is introduced into the PGS chain segment, so that the material is more hydrophilic, the mechanical property of the material can be adjusted, the material is changed from high elastomer to viscoelasticity, the cell adhesion and spreading are facilitated, and the tissue repair is promoted.

Based on the above, there is a need in the art to design a hydrogel with adjustable viscoelasticity, strong adhesion, and capable of being formed by injection, so as to minimally invasively satisfy the repair of different tissue defects in all directions.

Disclosure of Invention

The object of the present invention is to provide a hydrogel which has adjustable viscoelasticity, strong adhesion and can be formed by injection.

In a first aspect of the present invention, there is provided a viscoelastic hydrogel, characterized in that the viscoelastic hydrogel is formed by a first polymer and a second polymer through a crosslinking reaction;

wherein the first polymer comprises an azidoated polyhydroxy pegylated polysebacic acid glyceride;

the second polymer comprises cyclic alkynyl polyhydroxy polyethylene glycol polysebacic acid glyceride.

In another preferred embodiment, the viscoelastic hydrogel is formed by a crosslinking reaction of a hydrogel precursor solution a comprising a first polymer and a hydrogel precursor solution B comprising a second polymer.

In another preferred embodiment, the first polymer and the second polymer are bonded by cycloaddition reaction of azide group and cyclic alkyne group.

In another preferred embodiment, the first polymer and the second polymer form the hydrogel by click chemistry.

In another preferred embodiment, the mass ratio of the first polymer to the second polymer is 0.5-2: 0.5 to 2, preferably 0.8 to 1.2:0.8 to 1.2, more preferably 1: 1.

in another preferred embodiment, the first polymer is the polymer according to the fourth aspect of the present invention.

In another preferred embodiment, the first polymer is an azido-modified polyhydroxy pegylated polysebacic acid glyceride comprising structural units according to formula Ia or Ib:

wherein,

in the formula Ia, X₁Each independently selected from the group consisting of: H.

a₁is an integer of 1 to 20, b₁Is an integer of 5 to 60;

in the formula Ib, X₂Each independently selected from the group consisting of: H.

a₂is an integer of 1 to 20, b₂Is an integer of 5 to 60;

meaning that the azide group is linked to the first polymer backbone through a grafted chain structure.

In another preferred embodiment, the first polymer has an average of at least 1.2 structural units

Preferably at least 1.6, more preferably at least 1.8;

wherein

In another preferred embodiment, the

The degree of grafting is more than 60%, preferably more than 80%, for example 90%, 100%.

In another preferred embodiment, the first polymer is a polymer with polyhydroxy polyethylene glycol polysebacic acid glyceride (PEGS-OH) as a main chain and azide groups modified at the tail ends of side chains.

In another preferred embodiment, the first polymer is poly-hydroxyl polyethylene glycol polysebacate glyceride (PEGS-COOH) with carboxylated side chains and is obtained by grafting azidoamine through an amide bond.

In another preferred embodiment, the first polymer is obtained by taking polyhydroxy polyethylene glycol polysebacate glyceride (PEGS-OH) as a main polymer, and grafting azidoamine through an amido bond after side chain carboxylation.

In another preferred embodiment, the side chain carboxylation reagent is C4-C8 dianhydride, preferably C4-C6 dianhydride, such as succinic anhydride, glutaric anhydride, adipic anhydride.

In another preferred embodiment, the grafting ratio of the dianhydride is at least 80%, preferably at least 90%, such as 95%, 97%, 100%.

In another preferred embodiment, the reagent for grafting azidoamine is C0-C5 azidoamine, preferably 3-azidopropylamine (Az) and 4-azido-1-butylamine.

In another preferred embodiment, in the first polymer, X is₁、X₂Each independently selected from the group consisting of: H.

in another preferred embodiment, the first polymer comprises a structural unit shown as formula IV:

wherein the grafting ratio of the 3-azidopropylamine (Az) is more than 60%, preferably more than 80%, such as 90%, 100%;

a₁and b₁Is as defined above.

In another preferred embodiment, the first polymer has one or more of the following characteristics:

(a) an average of at least 1.2 azide groups per building block, preferably at least 1.6, more preferably at least 1.8;

(b) a number average molecular weight of 3.0 to 100.0kDa, preferably 6.0 to 50.0kDa, more preferably 7.0 to 20.0 kDa;

(c) the dispersion coefficient is 1.1 to 2.0, preferably 1.2 to 1.7.

In another preferred embodiment, the grafting ratio of the azide groups in the first polymer can be used to adjust the crosslinking degree of the viscoelastic hydrogel.

In another preferred embodiment, the second polymer is the second polymer according to the sixth aspect of the present invention.

In another preferred embodiment, the second polymer is a cyclic alkynylated polyhydroxy pegylated polysebacic acid glyceride comprising structural units according to formula IIa or IIb:

wherein, in the formula IIa, Y₁Each independently selected from the group consisting of: H.

cyclic alkynyl group, c₁Is an integer of 1 to 20, d₁Is an integer of 5 to 60;

in the formula IIb, Y₂Each independently selected from the group consisting of: H.

cyclic alkynyl group, c₂Is an integer of 1 to 20, d₂Is an integer of 5 to 60;

“

cyclic alkynyl "means that the cyclic alkynyl is linked to the second polymer backbone through a grafted chain structure.

In another preferred embodiment, the second polymer comprises an average of at least 1.2 structural units

Cyclic alkynyl groups, preferably at least 1.6, more preferably at least 1.8;

wherein "

In another preferred embodiment, the

The grafting degree of the cyclic alkynyl group is more than 60%, preferably more than 80%, for example 90% or 100%.

In another preferred embodiment, the

The cyclic alkynyl group includes

A cycloalkynyl group,

Heterocycloalkynyl (which includes 1-3 heteroatoms selected from N, O, S).

In another preferred embodiment, the

Cyclic alkynyl is

Dibenzocyclooctynyl group (A), (B), (C)

DBCO)。

In another preferred embodiment, the second polymer is a polymer with polyhydroxy polyethylene glycol polysebacate glyceride (PEGS-OH) as a main chain and a cyclic alkynyl group modified at the tail end of a side chain.

In another preferred embodiment, the second polymer is polyhydroxy polyethylene glycol polysebacate glyceride (PEGS-COOH) with carboxylated side chains, and the second polymer is obtained by grafting aminated cycloalkyne through amido bond.

In another preferred embodiment, the second polymer is obtained by taking polyhydroxy polyethylene glycol polysebacate glyceride as a main polymer, and grafting aminated cycloalkyne through an amide bond after side chain carboxylation.

In another preferred embodiment, the second polymer is obtained by grafting aminated dibenzocyclooctyne through an amide bond after side chain carboxylation by using polyhydroxy polyethylene glycol polysebacic acid glyceride as a main polymer.

In another preferred embodiment, the aminated dibenzocyclooctyne is selected from the group consisting of: DBCO-Amine (DBCO-Amine), DBCO-PEG (3-12) -Amine (DBCO-PEG (3-12) -Amine), sulfonic acid DBCO-Amine (Sulfo DBCO-Amine).

In another preferred embodiment, the second polymer is obtained by directly grafting a polyhydroxy polyethylene glycol glyceryl polysebacate to a carboxylated dibenzocyclooctyne.

In another preferred embodiment, the carboxylated dibenzocyclooctyne is selected from the group consisting of: DBCO-Acid (DBCO-Acid), DBCO- (C)_5-8) -acid (DBCO-C)_5-8-Acid), DBCO-PEG (3-12) -Acid (DBCO-PEG (3-12) -Acid).

In another preferred embodiment, in the second polymer, Y is₁、Y₂Each independently selected from the group consisting of: H.

in another preferred embodiment, the second polymer comprises structural units represented by formula V:

wherein the grafting ratio of the aminated Dibenzocyclooctyne (DBCO) is more than 60%, preferably more than 80%, such as 90% and 100%;

c₁and d₁Is as defined above.

In another preferred embodiment, the second polymer has one or more of the following characteristics:

(a) each structural unit comprises on average at least 1.2 cyclic alkynyl groups, preferably at least 1.6, more preferably at least 1.8;

(c) the dispersion coefficient is 1.1 to 2.0, preferably 1.2 to 1.7.

In another preferred embodiment, a is₁、a₂、c₁、c₂The same or different, each independently 1 to 20;

b is₁、b₂、d₁、d₂The same or different, each independently 5 to 60.

In another preferred embodiment, a₁＝c₁；b₁＝d₁。

In another preferred embodiment, the first polymer and the second polymer are obtained by grafting azide group and cyclic alkynyl group respectively by using the same polyhydroxy polyethylene glycol polysebacate (PEGS-COOH) with carboxylated side chains.

In another preferred embodiment, the grafting ratio of the cyclic alkyne group of the second polymer can be used to adjust the degree of crosslinking of the viscoelastic hydrogel.

In another preferred embodiment, the crosslinking degree of the viscoelastic hydrogel is 50% to 100%.

In another preferred embodiment, the viscoelastic property and the mechanical property of the viscoelastic hydrogel can be adjusted by the molecular weight length of a polyethylene glycol segment in the polyhydroxy polyethylene glycol polysebacic acid glyceride.

In another preferred embodiment, the viscoelastic hydrogel has one or more of the following characteristics:

(1) the elastic modulus is between 0.01 and 3MPa, preferably between 0.01 and 2 MPa;

(2) the creep time is between 50 and 2500s, preferably between 100 and 2000 s;

(3) the semi-relaxation time is between 10 and 2000s, preferably between 20 and 1000 s.

In another preferred example, the Young's modulus of the viscoelastic hydrogel is 1.575-0.011MPa, the creep time is 1819.63-110.34s, and the half-relaxation time is 1132-20 s.

In a second aspect of the present invention, there is provided a method for producing the viscoelastic hydrogel of the first aspect of the present invention, comprising the steps of:

(a) providing a hydrogel precursor solution A comprising a first polymer and a hydrogel precursor solution B comprising a second polymer;

(b) and mixing the hydrogel precursor solution A and the hydrogel precursor solution B, and performing rapid crosslinking to obtain the viscoelastic hydrogel.

In another preferred embodiment, the mixing is performed by injecting the hydrogel precursor solution a and the hydrogel precursor solution B into the relevant sites through a double-syringe injector, thereby mixing and forming the viscoelastic hydrogel.

In another preferred embodiment, the content of the first polymer in the hydrogel precursor solution a is 10 wt% to 100 wt%, preferably 10 wt% to 20 wt%, such as 20 wt%, 15 wt%, 10 wt% of the total mass, based on the total mass of the first polymer and the solvent.

In another preferred embodiment, the content of the second polymer in the hydrogel precursor solution B is 10 wt% to 100 wt%, preferably 10 wt% to 20 wt%, for example 20 wt%, 15 wt%, 10 wt% of the total mass, based on the total mass of the second polymer and the solvent.

In another preferred example, the solvent of the hydrogel precursor solution a and the hydrogel precursor solution B is water.

In another preferred example, the solvent of the hydrogel precursor solution a and the hydrogel precursor solution B is PBS aqueous solution.

In another preferred embodiment, in said step (b), at 10 ℃ to 40 ℃, more preferably at 25 ℃ to 37 ℃; the reaction is preferably carried out at 37 ℃.

In a third aspect of the invention, there is provided a mixture for preparing the viscoelastic hydrogel of the first aspect of the invention, the mixture comprising a hydrogel precursor solution a and a hydrogel precursor solution B.

In another preferred embodiment, the gel forming time of the mixture is 30 to 150s, preferably 30 to 90s, such as 70s, 60s, 45 s.

In another preferred embodiment, the gel forming time of the mixture can be adjusted by the molecular weight of the polyethylene glycol segment in the first polymer and/or the second polymer.

In another preferred embodiment, the mixture is gelled at 10-40 ℃, preferably 25-37 ℃.

In another preferred embodiment, the storage modulus of the mixture is 1-5Pa, preferably 1-2Pa, the loss modulus is 5-11Pa, preferably 6-10Pa, and the storage modulus is lower than the loss modulus, so that the requirement of injectability is met.

In a fourth aspect of the present invention, there is provided a polymer, wherein the polymer is an azido-esterified polyhydroxy pegylated polysebacic acid glyceride, comprising structural units represented by formula Ia or Ib:

wherein in the formula Ia, X₁Each independently selected from the group consisting of: H.

a₁is an integer of 1 to 20, b₁Is an integer of 5 to 60;

a₂is an integer of 1 to 20, b₂Is an integer of 5 to 60;

“

In another preferred embodiment, the polymer comprises at least 1.2 structural units in average

Preferably at least 1.6, more preferably at least 1.8.

In another preferred embodiment, the

In another preferred embodiment, the polymer comprises a structural unit shown as formula IV:

a₁and b₁Is as defined above.

In another preferred embodiment, the polymer has one or more of the following characteristics:

(c) the dispersion coefficient is 1.1 to 2.0, preferably 1.2 to 1.7.

In another preferred embodiment, the grafting ratio of the azide groups in the polymer can be used to adjust the degree of crosslinking of the viscoelastic hydrogel.

In another preferred embodiment, the polymer is the first polymer in the first aspect of the present invention.

In another preferred embodiment, the polymer is used to prepare a viscoelastic hydrogel according to the first aspect of the invention.

In a fifth aspect of the present invention, there is provided a process for the preparation of a polymer according to the fourth aspect of the present invention, the process comprising the steps of:

(i) mixing polyhydroxy polyethylene glycol polysebacate glyceride (PEGS-OH) with dianhydride in the presence of an inert solvent to obtain carboxylated polyhydroxy polyethylene glycol polysebacate glyceride (PEGS-COOH);

(ii) (ii) mixing PEGS-COOH obtained in the step (i) with azidoamine, and carrying out grafting reaction to obtain the polymer.

In another preferred embodiment, in the step (i), the molar ratio of the dianhydride to the hydroxyl in the PEGS-OH is 1-2: 1, preferably 1.2-1.5: 1.

In another preferred embodiment, in said step (i), the reaction is carried out at 90-120 ℃, preferably 100 ℃.

In another preferred embodiment, in said step (i), the reaction is carried out for 0.5 to 3h, preferably 1 to 2 h.

In another preferred embodiment, in step (ii), the method further includes: (ii) mixing PEGS-COOH obtained in the step (i) with Triethylamine (TEA), N' -Diisopropylcarbodiimide (DIC), N-hydroxysuccinimide (NHS) and azidoamine, and carrying out grafting reaction to obtain the polymer of claim 6.

In another preferred embodiment, in the step (ii), the azidoamine is 50 to 120 percent, preferably 60 to 100 percent, such as 60, 80, 100 percent, of the molar amount of carboxyl groups in the PEGS-COOH.

In another preferred embodiment, in the step (ii), the reaction is performed at room temperature, preferably 20 to 45 ℃, and more preferably 37 ℃.

In another preferred embodiment, in said step (ii), the reaction is carried out for 2 to 18 hours, preferably 6 to 10 hours.

In another preferred embodiment, in the step (ii), the molar ratio of N, N' -diisopropylcarbodiimide to N-hydroxysuccinimide is 0.8-1.2: 0.8-1.2, preferably 1: 1.

in another preferred embodiment, in the step (ii), the molar ratio of the N, N' -diisopropylcarbodiimide to the amount of carboxyl groups in the PEGS-COOH is 1.0 to 1.5:1, preferably 1.2 to 1.3: 1.

in another preferred example, the step (ii) further includes:

(ii-1) mixing PEGS-COOH obtained in the step (i) with N, N' -Diisopropylcarbodiimide (DIC) and N-hydroxysuccinimide (NHS) for activation;

(ii-2) adding Triethylamine (TEA) to the reaction mixture obtained in the step (ii-1), and then adding azidoamine to perform grafting reaction;

(ii-3) subjecting the reaction mixture obtained in step (ii-2) to a post-treatment to obtain the polymer of claim 6.

In another preferred embodiment, in the step (ii-1), the reaction is carried out for 1 to 5 hours, preferably 2 to 3 hours.

In another preferred embodiment, in the step (ii-2), triethylamine is added to the reaction to adjust the pH value to 8.0.

In another preferred embodiment, in the step (ii-2), the reaction is carried out for 8 to 18 hours, preferably 12 hours.

In another preferred embodiment, in the step (ii-3), the post-treatment comprises dialysis and vacuum drying.

In another preferred embodiment, the preparation method is carried out under a protective gas atmosphere.

In another preferred embodiment, the preparation method is carried out in a glove box in a protective gas atmosphere.

In a sixth aspect of the present invention, there is provided a polymer, wherein the polymer is a cyclic alkynylated polyhydroxy pegylated polysebacic acid glyceride, comprising structural units represented by formula IIa or IIb:

“

Cyclic alkynyl groups, preferably at least 1.6, more preferably at least 1.8;

wherein "

In another preferred embodiment, the

In another preferred embodiment, the

The cyclic alkynyl group includes

A substituted or unsubstituted cycloalkynyl group,

Substituted or unsubstituted heterocyclic alkynyl (which includes 1-3 heteroatoms selected from N, O, S);

wherein said substitution is by one or more groups selected from the group consisting of: halogen, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C3-C8 cycloalkyl, hydroxy, -NH₂C1-C6 amino, carboxyl, C1-C6 amido (-C (═ O) -n (rc)₂or-NH-C (═ O) (Rc), Rc being H or C1-C5 alkyl), or a substituted or unsubstituted group selected from: C1-C6 alkyl, C6-C10 aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, 5-10 membered heterocyclyl (including fused ring, spiro ring) having 1-3 heteroatoms selected from N, S and O, - (CH)₂) -C6-C10 aryl, - (CH)₂) - (5-to 10-membered heteroaryl having 1 to 3 heteroatoms selected from N, S and O), and said substitution is substituted with a substituent selected from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkoxy, oxygenAnd substituted, -CN, -OH, C6-C10 aryl.

In another preferred embodiment, the

Cyclic alkynyl is

Dibenzocyclooctynyl group (A), (B), (C)

DBCO)。

In another preferred embodiment, the polymer comprises a structural unit represented by formula V:

wherein the grafting ratio of the aminated dibenzocyclooctyne exceeds 60%, preferably exceeds 80%, such as 90% and 100%;

c₁and d₁Is as defined above.

(c) the dispersion coefficient is 1.1 to 2.0, preferably 1.2 to 1.7.

In another preferred embodiment, the grafting ratio of the cyclic alkynyl group of the polymer can be used for adjusting the crosslinking degree of the viscoelastic hydrogel.

In another preferred embodiment, the polymer is the second polymer in the first aspect of the invention.

In another preferred embodiment, the polymer according to the fourth aspect of the present invention and the polymer according to the sixth aspect of the present invention are obtained by grafting azide group and cyclic alkynyl group respectively using the same polyhydroxy pegylated polysebacic acid glyceride (PEGS-COOH) having carboxylated side chains.

In a seventh aspect of the present invention, there is provided a process for the preparation of a polymer according to the sixth aspect of the present invention, said process comprising the steps of:

(i) under the atmosphere of protective gas and in the presence of an inert solvent, mixing polyhydroxy polyethylene glycol polysebacic acid glyceride (PEGS-OH) with dianhydride to obtain carboxylated polyhydroxy polyethylene glycol polysebacic acid glyceride (PEGS-COOH);

(ii) (ii) mixing PEGS-COOH obtained in the step (i) with aminated cycloalkyne, and carrying out grafting reaction to obtain the polymer.

In another preferred embodiment, in the step (ii), the aminated cycloalkyne is an aminated dibenzocyclooctyne.

In another preferred embodiment, in step (ii), the aminated cycloalkyne is 50% to 120%, preferably 60% to 100%, such as 60%, 80%, 100% of the molar amount of carboxyl groups in the PEGS-COOH.

In another preferred embodiment, the preparation method is the same as the preparation method of the fifth aspect of the present invention, except that the azidoamine is replaced by an aminated cycloalkyne.

In an eighth aspect of the present invention, there is provided a kit for preparing the viscoelastic hydrogel of the first aspect of the present invention, the kit comprising:

(a) a first container, and a first polymer located within the container;

(b) a second container, and a second polymer located within the container.

In another preferred embodiment, the kit comprises:

(a) a first container, and a first polymer disposed in said container, said first polymer being in the form of a powder or dispersed in a solution;

(b) a second container, and a second polymer in said container, said second polymer being in the form of a powder or dispersed in a solution.

In another preferred embodiment, the kit comprises:

(a) a first container, and a precursor solution a located in the container, the precursor solution a comprising the hydrogel precursor solution a of claim 1;

(b) a second container, and a precursor solution B located in said container, said precursor solution B comprising the hydrogel precursor solution B of claim 1.

In an eighth aspect of the invention, there is provided a use of the viscoelastic hydrogel of the first aspect of the invention, (a) for the preparation of a medical material for the loading of growth factors and/or drugs, or for tissue defect filling and/or guided tissue regeneration; (b) or used for preparing a medical cosmetic material.

In a ninth aspect of the invention, there is provided a medical coating comprising a viscoelastic hydrogel according to the first aspect of the invention.

In a tenth aspect of the present invention there is provided a medical material comprising a viscoelastic hydrogel according to the first aspect of the invention.

In another preferred embodiment, the medical material is selected from the group consisting of: medical defected tissue filler and medical drug carrier.

In another preferred embodiment, the medical material is a growth factor, a drug-loaded matrix or a carrier.

In an eleventh aspect of the invention there is provided a method of filling a tissue defect and/or guiding tissue regeneration, the method comprising applying to a site in need of treatment a viscoelastic hydrogel according to the first aspect of the invention.

In a twelfth aspect of the present invention, there is provided a method for loading growth factors and/or drugs and/or cells, the method comprising mixing the viscoelastic hydrogel of the first aspect of the present invention with the growth factors and/or drugs and/or cells to be loaded.

In a thirteenth aspect of the invention, there is provided a polymer which is a polyhydroxy pegylated polyglycerol polysebacate (PEGS-OH) comprising structural units according to formula IIIa or IIIb:

wherein,

in formula IIIa, m₁Is an integer of 1 to 20, n₁Is an integer of 5 to 60;

in formula IIIb, m₂Is an integer of 1 to 20, n₂Is an integer of 5 to 60.

(a) a number average molecular weight of 3.0-100.0kDa, preferably 6.0-50.0kDa, more preferably 7.0-20.0kDa, such as 10.0kDa, 15.5kDa, 16.0 kDa;

(b) the coefficient of dispersibility is from 1.1 to 2.0, preferably from 1.2 to 1.7.

In another preferred embodiment, the number average molecular weight of the polymer is determined by the reaction time of the polymerization reaction.

(c) the glass transition temperature is-180 ℃ to-110 ℃;

(d) the crystallization temperature is-160 ℃ to-100 ℃;

(e) the melting point is-90 ℃ to-20 ℃;

(f) the thermal degradation temperature is 350-450 ℃.

In another preferred embodiment, the glass transition temperature of the polymer is-140.78 ℃, the crystallization temperature is-128.48 ℃, the melting point is-54.92 ℃, and the thermal degradation temperature is 401.04 ℃.

In another preferred embodiment, one structural unit of the polymer comprises: a sebacic acid component, a polyethylene glycol component and two glycerin components.

In another preferred example, the polymer has more hydroxyl groups and higher structural regularity relative to randomly branched PEGS obtained by direct polycondensation of polyethylene glycol, sebacic acid and glycerol.

In another preferred embodiment, the polymer is soluble in water, dimethyl sulfoxide, N' -dimethylformamide, ethanol, methanol, acetone, ethyl acetate, chloroform, tetrahydrofuran, and dichloromethane.

In another preferred embodiment, the polymer is insoluble in diethyl ether and n-hexane.

In a thirteenth aspect of the present invention, there is provided a method for producing the polymer according to the twelfth aspect of the present invention, the method comprising the steps of:

(s1) under the atmosphere of protective gas, in the presence of an inert solvent, carrying out polymerization reaction on diglycidyl sebacate (monomer 1), carboxylated polyethylene glycol (monomer 2) and bis-tetra-n-butylammonium hydroxide PEG salt (catalyst 1) to obtain polyhydroxy polyethylene glycol polysebacate shown in formula Ia;

or (s2) under the atmosphere of protective gas, in the presence of an inert solvent, carrying out polymerization reaction on sebacic acid (monomer 3), polyethylene glycol diglycidyl ester (monomer 4) and bis tetra-n-butyl ammonium hydroxide sebacate (catalyst 2) to obtain the polyhydroxy polyethylene glycol polysebacic acid glyceride shown as the formula Ib.

In another preferred embodiment, in said step (s1), monomer 2 is represented by formula A:

wherein m is₁Is an integer of 1 to 20.

In another preferred example, in the step (s1), the PEG segment of the monomer 2 has a molecular weight of 200-700 Da, preferably 250-600 Da, such as 250Da, 500Da, 600 Da.

In another preferred embodiment, in said step (s1), catalyst 1 is prepared by salifying bis-tetra-n-butylammonium hydroxide with carboxylated polyethylene glycol (COOH-PEG-COOH), and has the structure shown in formula B below:

wherein m is an integer of 1 to 20.

In another preferred embodiment, in the step (s1), the molar ratio of the monomer 1 to the monomer 2 is 0.8 to 1.2: 0.8-1.2, preferably 1: 1;

the molar ratio of the catalyst 1 to the monomer 1 is 0.004-0.008: 1, preferably 0.005-0.006: 1.

In another preferred embodiment, said step (s1) is carried out at 90-120 ℃, preferably 100 ℃.

In another preferred embodiment, said step (s1) is carried out for 12-96h, preferably 24-72h, e.g. 24h, 48h, 72 h.

In another preferred embodiment, in the step (s1), the inert solvent is N, N '-dimethylformamide, preferably anhydrous N, N' -dimethylformamide.

In another preferred example, in the step (s1), the protective gas is argon and/or high-purity nitrogen.

In another preferred embodiment, said step (s1) is performed in a glove box filled with argon atmosphere.

In another preferred embodiment, the step (s1) further comprises a post-treatment step of ether precipitation and dialysis purification.

In another preferred embodiment, in the step (s1), the monomer 2 is carboxylated polyethylene glycol with a molecular weight of 250 Da; catalyst 1 was a bis tetra-n-butyl ammonium hydroxide salt catalyst (in which the carboxylic acid moiety of the ammonium salt was a carboxylated polyethylene glycol of molecular weight 250 Da).

In another preferred embodiment, in the step (s1), the monomer 2 is carboxylated polyethylene glycol with a molecular weight of 600 Da; catalyst 1 was a bis tetra-n-butyl ammonium hydroxide salt catalyst (in which the carboxylic acid moiety of the ammonium salt was a carboxylated polyethylene glycol of molecular weight 600 Da).

In another preferred embodiment, in said step (s2), monomer 4 is represented by formula C:

wherein m is₂Is an integer of 1 to 20.

In another preferred example, in the step (s2), the molecular weight of the PEG segment of the monomer 4 is 200-700 Da, preferably 250-600 Da, such as 250Da, 500Da, 600 Da.

In another preferred embodiment, in said step (s2), catalyst 2 is bis tetra-n-butylammonium hydroxide salified with sebacic acid.

In another preferred embodiment, in the step (s2), the molar ratio of the monomer 3 to the monomer 4 is 0.8 to 1.2: 0.8-1.2, preferably 1: 1;

the molar ratio of the catalyst 2 to the monomer 3 is 0.004-0.008: 1, preferably 0.005-0.006: 1.

In another preferred embodiment, said step (s2) is carried out at 90-120 ℃, preferably 100 ℃.

In another preferred embodiment, said step (s2) is carried out for 12-96h, preferably 24-72h, e.g. 24h, 48h, 72 h.

In another preferred embodiment, in the step (s2), the inert solvent is N, N '-dimethylformamide, preferably anhydrous N, N' -dimethylformamide.

In another preferred example, in the step (s2), the protective gas is argon and/or high-purity nitrogen.

In another preferred embodiment, said step (s2) is performed in a glove box filled with argon atmosphere.

In another preferred embodiment, the step (s2) further comprises a post-treatment step of ether precipitation and dialysis purification.

In another preferred example, in the step (s2), the monomer 4 is polyethylene glycol diglycidyl ester with molecular weight of 500 Da.

In another preferred embodiment, the preparation method comprises the following steps:

(a1) under the protection of argon atmosphere, dissolving diacid and diglycidyl ester in a molar ratio of 1:1 in a solvent (wherein the diacid can be sebacic acid, carboxylated polyethylene glycol with a molecular weight of 250Da or carboxylated polyethylene glycol with a molecular weight of 600 Da; the diglycidyl ester can be diglycidyl sebacate or diglycidyl ester of polyethylene glycol with a molecular weight of 500 Da), adding a bis-tetra-n-butyl ammonium hydroxide salt catalyst in a molar ratio of 0.6 mol% (wherein the carboxylic acid part of the ammonium salt can be sebacic acid, carboxylated polyethylene glycol with a molecular weight of 250Da or carboxylated polyethylene glycol with a molecular weight of 600Da), and reacting for 1-3 days at 90-120 ℃ (preferably 100 ℃);

(b1) after the product of step (a1) is settled in ether, it is dried under vacuum at room temperature for 6-12 hours (more preferably 10-12 hours);

(c1) dialyzing and purifying the polyhydroxy PEGS polymer obtained in the step (b1) to obtain purified PEGS-OH shown in the formula a 1.

In another preferred embodiment, the preparation method of the monomer 1 comprises the following steps:

(s0) reacting epoxypropanol with sebacoyl chloride in the presence of a catalyst in a protective gas atmosphere to obtain the diglycidyl sebacate.

In another preferred embodiment, in the step (s0), the catalyst is a basic catalyst, preferably triethylamine.

In another preferred example, in the step (s0), the molar ratio of the epoxypropanol to the sebacoyl chloride is 2-3: 1, preferably 2: 1.

in another preferred embodiment, the step (s0) is performed at-20 to 0 ℃, preferably-10 to-5 ℃.

In another preferred embodiment, said step (s0) is carried out for 3 to 12h, preferably 4 to 8h, e.g. 5h, 6 h.

and dropwise adding the sebacoyl chloride solution into the mixed solution of the epoxypropane and the catalyst under the protective gas atmosphere to obtain the sebacoyl diglycidyl ester.

In another preferred embodiment, the solvent of the solution is N, N' -dimethylformamide.

In a fourteenth aspect of the invention, there is provided the use of the polymer of the twelfth aspect of the invention for the preparation of a material comprising a bioelastomer, a hydrogel, a 3D printed material, a responsive macromolecule, a conductive macromolecule.

In another preferred embodiment, the polymer is modified and/or grafted to prepare a material, and the material comprises a biological elastomer, a hydrogel, a 3D printing material, a responsive polymer and a conductive polymer.

In another preferred embodiment, the polymer is used for preparing a polymer, and the polymer is the polymer according to the fourth aspect of the invention.

In another preferred embodiment, the polymer is used for preparing a polymer, and the polymer is the polymer according to the sixth aspect of the invention.

In another preferred embodiment, the polymer is used to prepare a polymer for preparing the viscoelastic hydrogel of the first aspect of the invention.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the scheme (A) for the synthesis of polyhydroxy PEGS polymers according to the present invention; nuclear magnetic resonance hydrogen spectrogram (B) of the polyhydroxy PEGS macromolecules; fourier transform infrared spectrogram (C) of polyhydroxy PEGS polymer (PEGS-OH, lower) and branched PEGS polymer (PEGS, upper) prepared by polycondensation; the molecular weight of polyhydroxy PEGS macromolecules with different PEG block molecular weights changes with the reaction time.

FIG. 2 shows a schematic of a hydrogel of the invention with tunable viscoelasticity of the invention (A); route (B) of synthesis of the hydrogels of the invention; nuclear magnetic resonance hydrogen spectrum (C) of single-component PEGS-COOH; nuclear magnetic resonance hydrogen spectrum (D) of the mono-component PEGS-Az; nuclear magnetic resonance hydrogen spectrum (E) of mono-component PEGS-DBCO; fourier transform infrared spectrogram (F): from top to bottom: PEGS-COOH, PEGS-Az, PEGS-DBCO.

FIG. 3 shows the gel formation time as a function of temperature for the hydrogels of the invention (A): from left to right, the temperature is 37 ℃, 25 ℃ and 10 ℃; gel formation time of the hydrogels of the invention as a function of polymer concentration (B): 20 wt%, 15 wt% and 10 wt% from left to right; a photo of the hydrogel of the present invention showing a substantial sol-gel transition (C); schematic (D) of the microstructure of the hydrogel of the present invention.

FIG. 4 shows the initial modulus of the hydrogels of the present invention as a function of PEG block molecular weight and degree of crosslinking (A); the creep time varies with the PEG block molecular weight and the degree of crosslinking (B); the half relaxation time varies with the molecular weight of the PEG block and the degree of crosslinking (C).

FIG. 5 shows fluorescence plots of bone marrow mesenchymal stem cells (BMSCs) on hydrogels of the present invention of varying PEG block molecular weight and degree of cross-linking.

FIG. 6 shows a hydrogel immobilized horseradish peroxidase (HRP) release curve (A) of the present invention; HRP activity released in the first 24 hours (B); fluorescence staining pattern (C) of proliferation of 3D cultured BMSCs in hydrogel over time.

FIG. 7 shows the use of PEGS in accordance with the present invention₂₅₀-OH hydrogel repair Micro-CT (A) and Meisen trichrome section staining pattern (B) after 8 and 12 weeks of skull defect in rats.

Detailed Description

The inventor of the present invention has conducted extensive and intensive studies and, for the first time, unexpectedly found a hydrogel which has excellent rheological properties, has viscoelasticity, can realize rapid self-crosslinking in an in vivo environment, and can be formed by injection to fill a complicated defect structure.

Specifically, the hydrogel introduces a viscoelasticity-adjustable polyhydroxy polyethylene glycol sebacate glyceride (PEGS-OH) as a matrix of the hydrogel, and an injectable hydrogel system of PEGS based on click chemistry is constructed. The hydrogel has excellent injection performance, can be quickly cured and formed in an in vivo environment, does not need a catalyst, has mild crosslinking process, has no obvious toxicity to host organisms, and provides possibility for loading growth factors.

The hydrogel disclosed by the invention has excellent viscoelasticity and bioactivity, good cell compatibility and differentiation promoting performance, and ideal tissue repair effect and clinical applicability, so that the material is implanted without open surgery, the pain of a patient is reduced while the operation of a doctor is facilitated, and a new selectable material and a design thought are provided for a minimally invasive treatment tissue regeneration technology. The present invention has been completed based on this finding.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

As used herein, the terms "PEG", "polyethylene glycol" are used interchangeably.

As used herein, the terms "PGS", "polyglycerol sebacate" are used interchangeably.

As used herein, the terms "PEGS", "pegylated polysebacate", "PEGS polymer" are used interchangeably.

As used herein, the term "PGS-OH" represents a copolymer obtained by polymerizing sebacic acid (diacid monomer) and diglycidyl sebacate (diglycidyl monomer) without a PEG component.

As used herein, the term "PEG-OH" represents a copolymer derived from the polymerization of polyethylene glycol dicarboxylic acid (diacid monomer) and polyethylene glycol diglycidyl ester (diglycidyl ester monomer) without a sebacic acid component.

As used herein, the terms "PEGS-COOH", "carboxylated PEGS-OH" are used interchangeably to refer to a side chain carboxylated polyhydroxy pegylated polysebacate.

As used herein, the terms "Az", "NH", "or", or "thereof, or" a "or" a "thereof₂-Az "" 3-azidopropylamine "are used interchangeably.

As used herein, the term "PEGS-Az" refers to the pendant azido-containing hydroxypolyPEGylated polysebacic acid glyceride obtained by grafting 3-azidopropylamine onto carboxylated PEGS-OH.

As used herein, the terms "DBCO", "dibenzocyclooctyne" are used interchangeably.

As used herein, the term "NH₂-DBCO "," aminated dibenzocyclooctyne "are used interchangeably.

As used herein, the term "PEGS-DBCO" refers to hydroxy-pegylated polysebacate with a side chain containing dibenzocyclooctynyl groups, obtained by grafting an aminated dibenzocyclooctyne onto a carboxylated PEGS-OH.

As used herein, the terms "DIC", "N, N' -diisopropylcarbodiimide" are used interchangeably.

As used herein, the terms "NHS", "N-hydroxysuccinimide" are used interchangeably.

As used herein, the terms "TEA", "triethylamine" are used interchangeably.

As used herein, the terms "epoxypropanol", "glycidol" are used interchangeably.

As used herein, the terms "phosphate buffered saline", "PBS" are used interchangeably.

As used herein, the terms "mesenchymal stem cells", "BMSCs" are used interchangeably.

As used herein, the terms "horseradish peroxidase", "HRP" are used interchangeably.

As used herein, the terms "Safranin fast green tissue section staining", "Safranin-O" are used interchangeably.

Polyhydroxy Pegylated Polysebacic acid Glycerol ester (PEGS-OH)

As used herein, the terms "PEGS-OH", "polyhydroxylated PEGylated polysebacate", "polyhydroxylated PEGS" are used interchangeably to refer to the high hydroxyl content polyethylene glycol prepared in accordance with the present inventionA highly regular linear polymer of three-component block copolymerization (alternate copolymerization) of alcohol, sebacic acid and glycerol, wherein-OH represents a polyhydroxy macromolecule. PEGS_XThe subscript X in — OH refers to the molecular weight of the PEG block within one repeat unit in the PEGS. In the invention, polyhydroxy PEGS macromolecules with PEG molecular weight of 250Da, 500Da and 600Da are prepared, and the corresponding abbreviation is PEGS₂₅₀-OH”、“PEGS₅₀₀-OH”、“PEGS₆₀₀-OH”。

The polymer comprises two structures, namely PEGS-OH-1 and/or PEGS-OH-2, which are respectively shown as a formula IIIa and a formula Ib:

wherein in the formula Ia, m₁Is an integer of 1 to 20, n₁Is an integer of 5 to 60;

in the formula Ib, m₂Is an integer of 1 to 20, n₂Is an integer of 5 to 60.

A structural unit of the polymer comprises a sebacic acid component, a polyethylene glycol component and two glycerin components, and the high-content glycerin component greatly improves the hydroxyl content of the polymer and greatly enlarges the possibility of graft modification of the polymer.

Compared with randomly branched PEGS obtained by direct polycondensation of polyethylene glycol, sebacic acid and glycerol, the polymer has more hydroxyl groups, higher structural regularity and lower molecular weight distribution.

The molecular weight of the PEG block of the polymer can be adjusted, preferably 200-700 Da, more preferably 250-600 Da, such as 250Da, 500Da and 600 Da.

The polymer has one or more of the following characteristics:

(a) a number average molecular weight of 6.0-20.0kDa, preferably 7.0-16.0kDa, more preferably 10.0-15.5 kDa;

The polymer can be dissolved in water, dimethyl sulfoxide, N' -dimethylformamide, ethanol, methanol, acetone, ethyl acetate, chloroform, tetrahydrofuran and dichloromethane.

The polymer is insoluble in diethyl ether and n-hexane.

The number average molecular weight of the polymer is determined by the reaction time of the polymerization reaction.

The polymer is prepared by carboxyl monomer of sebacic acid and glycidyl ester monomer of polyethylene glycol (PEGS-OH-1) or glycidyl ester monomer of sebacic acid and carboxyl monomer of polyethylene glycol (PEGS-OH-2) under the action of a catalyst.

The polymer is subjected to ring opening polymerization of epoxy induced by acid, and under the action of an acid catalyst, carboxylate radicals react with epoxy groups in glycidyl esters, so that the reaction activity is high, and a branching reaction is rarely generated. And polymer reaction can not occur between carboxyl monomers and glycidyl ester monomers, so that regular alternating copolymerization (namely block copolymerization) linear polymer can be obtained only by directly mixing the two monomers with a catalyst.

The polymer is a block structure simultaneously provided with an elastic chain segment PGS and a flexible chain segment PEG, and the molecular weight, the molecular weight distribution, the hydroxyl content and the thermodynamic property of the polymer can be regulated, controlled and optimized through the reaction time and the length of the chain segment of the PEG. The polyhydroxy PEGS has a plurality of side hydroxyl groups, can be grafted and modified to enable the polyhydroxy PEGS to have different performances, is used in different fields, and is a high polymer material with wide application prospect.

The polymer has high elasticity of the polysebacic acid glyceride and flexibility and hydrophilicity of the polyethylene glycol, so the polymer is an excellent matrix material for preparing the hydrogel.

Preparation method of polyhydroxy polyethylene glycol polysebacic acid glyceride

The invention provides a preparation method of polyhydroxy polyethylene glycol polysebacic acid glyceride, which comprises the following steps:

(i) providing dicarboxyl and diglycidyl ester as monomers, bis tetra-N-butyl ammonium hydroxide salt as a catalyst and N, N' -dimethylformamide as a solvent;

(ii) in a glove box, dissolving a monomer and a catalyst in a solvent, and heating and stirring for a period of time under the condition of inert gas to obtain polyhydroxy PEGS (polyethylene glycol succinate) macromolecules;

in said step (i), the diglycidyl ester monomer: the molecular weight of the sebacic acid diglycidyl ester (monomer 1) is 314.37Da, and the molecular weight of the PEG diglycidyl ester (monomer 4) is 500 Da;

dicarboxylic monomers: the molecular weight of the carboxylated PEG (monomer 2) is 250Da or 600Da, and the molecular weight of the sebacic acid (monomer 3) is 202.25 Da;

in the catalyst bis tetra-n-butyl ammonium hydroxide salt, the carboxylic acid component can be carboxylated PEG (catalyst 1) with the molecular weight of 250Da or 600 Da; sebacic acid (catalyst 2), molecular weight 202.25 Da.

In the step (i) described above, the step (ii),

simultaneously adding the monomer 1, the monomer 2 and the catalyst 1 into a reaction system to obtain PEGS-OH-1 shown in a formula Ia;

and simultaneously adding the monomer 3, the monomer 4 and the catalyst 2 into a reaction system to obtain PEGS-OH-2 shown as a formula Ib.

In the step (ii) of the present invention,

the monomer feeding molar ratio is 1:1, and the catalyst dosage is 0.6 mol%;

the inert atmosphere gas can be argon or high-purity nitrogen;

the reaction temperature is 100 ℃, and the reaction time is 1-3 days.

The molecular weight of the polyhydroxy PEGS macromolecule is adjustable between 6.0kDa and 15.5kDa along with the extension of reaction time, and the PDI is 1.1 to 2.0.

The preparation method comprises the following steps:

(a1) under the protection of argon atmosphere, dissolving diacid and diglycidyl ester with a molar ratio of 1:1 in a solvent (wherein the diacid can be sebacic acid, carboxylated PEG with a molecular weight of 250Da or carboxylated PEG with a molecular weight of 600 Da; the diglycidyl ester can be diglycidyl sebacate or diglycidyl ester of polyethylene glycol with a molecular weight of 500 Da), adding a bis-tetra-n-butyl ammonium hydroxide salt catalyst with a molar ratio of 0.6 mol% (wherein the carboxylic acid part of the ammonium salt can be sebacic acid, carboxylated PEG with a molecular weight of 250Da or carboxylated PEG with a molecular weight of 600Da), and reacting for 1-3 days at 90-120 ℃ (preferably 100 ℃);

(b1) settling the product of the step (a1) in ether, and vacuum-drying at room temperature for 6-12 hours (preferably 10-12 hours) to obtain polyhydroxy polyethylene glycol polysebacate glyceride macromolecule;

(c1) dialyzing and purifying the PEGS polymer obtained in the step (b1) to obtain the purified polyethylene glycol poly-sebacic acid glyceride.

Azidoated polyhydroxy PEGylated Polysebacylic acid Glycerol ester (first Polymer)

The azido-modified polyhydroxy polyethylene glycol polysebacic acid glyceride is polyhydroxy polyethylene glycol polysebacic acid glyceride polymer with azido modified side chains.

As used herein, the terms "azidoated polyhydroxy pegylated polysebacate," "first polymer," are used interchangeably to refer to the components used to prepare the hydrogel precursor solution a of the present invention.

According to two structures of polyhydroxylated polyglycolyzed polysebacate (PEGS-OH), said first polymer may comprise structural units according to formula Ia and/or Ib:

wherein,

a₁is an integer of 1 to 20, b₁Is an integer of 5 to 60;

a₂is an integer of 1 to 20, b₂Is an integer of 5 to 60;

“

Said

Said

The grafting rate is determined by the amount of reagent for grafting azido groups.

The first polymer is a polymer which takes polyhydroxy polyethylene glycol polysebacate glyceride as a main chain and is modified with azide group at the tail end of a side chain.

The first polymer is obtained by taking polyhydroxy polyethylene glycol polysebacate glyceride (PEGS-OH) as a main polymer and grafting azido amine through amido bond after side chain carboxylation.

The side chain carboxylation reagent is C4-C8 dianhydride, C2-C8 diacid, preferably C4-C6 dianhydride, such as succinic anhydride, glutaric anhydride and adipic anhydride.

The grafting ratio of the dianhydride is at least 80%, preferably at least 90%, for example 95%, 97%, 100%.

The reagent for grafting the azido amine is C0-C6 azido amine, preferably 3-azido propylamine (Az) and 4-azido-1-butylamine.

The first polymer (PEGS-Az) obtained by using succinic anhydride as a side chain carboxylation reagent and 3-azidopropylamine (Az) as a reagent for grafting azido amine comprises a structural unit shown as a formula IV:

a₁and b₁Is as defined above.

The first polymer has one or more of the following characteristics:

(b) a number average molecular weight of 6.0-20.0kDa, preferably 7.0-16.0kDa, more preferably 10.0-15.5 kDa;

(c) the dispersion coefficient is 1.1 to 2.0, preferably 1.2 to 1.7.

The grafting ratio of the azide group in the first polymer can be used to adjust the degree of crosslinking of the hydrogel of the present invention.

Cyclic alkynylated polyhydroxy pegylated polysebacic acid glycerides (second polymers)

The cyclic alkynylated polyhydroxy pegylated polysebacic acid glyceride provided by the invention refers to a polyhydroxy pegylated polysebacic acid glyceride polymer with a side chain modifying cyclic alkynyl.

As used herein, the terms "cyclic alkynylated polyhydroxy pegylated polysebacate glycerol ester", "second polymer" are used interchangeably and refer to the components used to prepare the hydrogel precursor solution B of the present invention.

According to two structures of polyhydroxylated polyglycolysed polysebacate (PEGS-OH), said second polymer may comprise structural units of formula IIa and/or IIb:

“

Said

Said

The grafting ratio of the cyclic alkynyl group is determined by the dosage of the reagent for grafting the cyclic alkynyl group.

Said

The cyclic alkynyl group includes

A cycloalkynyl group,

Heterocycloalkynyl (which includes 1-3 heteroatoms selected from N, O, S).

Said

Cyclic alkynyl is

Dibenzocyclooctynyl group (A), (B), (C)

DBCO)。

The second polymer is a polymer with polyhydroxy polyethylene glycol polysebacic acid glyceride (PEGS-OH) as a main chain and a cyclic alkynyl group modified at the tail end of a side chain.

The second polymer is obtained by taking polyhydroxy polyethylene glycol polysebacate glyceride as a main polymer, and grafting aminated cycloalkyne through amido bond after side chain carboxylation.

The second polymer is obtained by taking polyhydroxy polyethylene glycol polysebacate glyceride as a main polymer, and grafting aminated dibenzocyclooctyne through amido bond after side chain carboxylation.

The side chain carboxylation reagent is C4-C8 dianhydride, preferably C4-C6 dianhydride, such as succinic anhydride, glutaric anhydride and adipic anhydride.

The aminated dibenzocyclooctyne is selected from the group consisting of:

DBCO-Amine (DBCO-Amine) with a chemical structural formula:

DBCO-PEG (3-12) -Amine (DBCO-PEG (3-12) -Amine) with a chemical structural formula as follows:

p is an integer of 3 to 12;

sulfonic acid DBCO-Amine (Sulfo DBCO-Amine) with a chemical structural formula as follows:

the second polymer is obtained by directly grafting polyhydroxy polyethylene glycol polysebacic acid glyceride with carboxylated dibenzocyclooctyne.

The carboxylated dibenzocyclooctyne is selected from the group consisting of:

DBCO-Acid (DBCO-Acid), the chemical structural formula of which is:

DBCO-(C_5-8) -acid (DBCO-C)_5-8-Acid) having the chemical formula:

q is an integer of 3 to 5;

DBCO-PEG (3-12) -Acid (DBCO-PEG (3-12) -Acid), the chemical structural formula of which is:

l is an integer of 3 to 12.

In the second polymer, Y₁、Y₂Each independently selected from the group consisting of: H.

the succinic anhydride is used as a side chain carboxylation reagent, and the second polymer obtained by grafting aminated dibenzocyclooctyne comprises a structural unit shown as a formula V:

c₁and d₁Is as defined above.

The second polymer has one or more of the following characteristics:

(c) the dispersion coefficient is 1.1 to 2.0, preferably 1.2 to 1.7.

The grafting ratio of the cyclic alkynyl group of the second polymer can be used for adjusting the crosslinking degree of the viscoelastic hydrogel.

A is described₁＝c₁；b₁＝d₁。

A is described₁、a₂、c₁、c₂The same or different, each independently 1 to 20;

b is₁、b₂、d₁、d₂The same or different, each independently 5 to 60.

The first polymer and the second polymer are obtained by grafting azide group and cyclic alkynyl group respectively by using the same polyhydroxy polyethylene glycol polysebacic acid glyceride (PEGS-COOH) with carboxylated side chains.

The hydrogel of the present invention

Hydrogels are a class of polymeric materials having three-dimensional crosslinked networks that are capable of absorbing and retaining large amounts of moisture. The invention provides a hydrogel which has adjustable viscoelasticity and strong adhesive force and can be formed in an injection mode.

As used herein, the terms "viscoelastic hydrogel", "hydrogel of the present invention", "PEGS hydrogel", "injectable hydrogel" are used interchangeably and refer to a hydrogel material having adjustable viscoelasticity obtained by mixing and rapidly crosslinking a gel by injecting a polyhydroxylated polyglycolized polyglycerol sebacate (PEGS-OH) as a main matrix material from a hydrogel precursor solution a and a hydrogel precursor solution B of the present invention into the relevant site by means of a double-syringe injector.

The hydrogel is formed by a crosslinking reaction of a hydrogel precursor solution A containing a first polymer and a hydrogel precursor solution B containing a second polymer;

the second polymer comprises cyclic alkynyl polyhydroxy polyethylene glycol polysebacic acid glyceride;

the mass ratio of the first polymer to the second polymer is 0.8-1.2: 0.8 to 1.2, preferably 1: 1.

the hydrogel is obtained by quickly crosslinking through a click chemical reaction between a first polymer and a second polymer.

The content of the first polymer in the hydrogel precursor solution a is 10 wt% to 100 wt%, preferably 10 wt% to 20 wt%, for example 20 wt%, 15 wt%, 10 wt% of the total mass, based on the total mass of the first polymer and the solvent.

The content of the second polymer in the hydrogel precursor solution B is 10 wt% to 100 wt%, preferably 10 wt% to 20 wt%, for example 20 wt%, 15 wt%, 10 wt% of the total mass, based on the total mass of the second polymer and the solvent.

The crosslinking degree of the hydrogel is 50-100%, preferably 60-100%.

The crosslinking degree of the hydrogel is adjusted by the grafting rate of azide group and/or cyclic alkynyl group; wherein the grafting rate of the azide group and/or the cyclic alkynyl group is adjusted by the charge ratio of the corresponding grafting reagent (the charge amount accounts for the proportion of the side chain carboxylated PEGS-OH).

The viscoelastic property and the mechanical property of the viscoelastic hydrogel can be adjusted and optimized through the molecular weight (length of a PEG block) of the PEG block in the polyhydroxy polyethylene glycol polysebacic acid glyceride.

The rheological property, the mechanical property, the gel time, the cell behavior and the tissue repair capability of the hydrogel can be optimized by regulating and controlling the molecular weight of the polyethylene glycol block in the polymer and the crosslinking degree of the hydrogel

The viscoelastic hydrogel has one or more of the following characteristics:

(2) the creep time is between 50 and 2500s, preferably between 100 and 2000 s;

The Young modulus of the viscoelastic hydrogel is 1.575-0.011MPa, the creep time is 1819.63-110.34s, and the half relaxation time is 1132-20 s.

The viscoelasticity of the hydrogel can be adjusted by the molecular weight of PEG blocks in the main chain of the polyhydroxy polyethylene glycol-based polysebacate and/or the crosslinking degree of the hydrogel. The lower the molecular weight of the PEG block, the longer the opposite PGS block, or the higher the degree of crosslinking of the hydrogel, the better the elasticity of the hydrogel; the higher the molecular weight of the PEG block, the more segmented the opposing PGS block, or the lower the degree of crosslinking of the hydrogel, the better the hydrogel's adhesion.

Method for producing hydrogel of the present invention

The invention provides a method for preparing the viscoelastic hydrogel, which comprises the following steps:

(a) adding succinic anhydride into PEGS-OH macromolecules in a glove box to carry out carboxylation, and grafting 3-azidopropylamine and aminated dibenzocyclooctyne onto carboxylated PEGS-OH by taking N, N' -diisopropylcarbodiimide and N-hydroxysuccinimide as catalysts to form a PEGS-Az and PEGS-DBCO double component;

(b) and (3) respectively filling the two components with equal mass into a double-syringe injector to form the viscoelastic solid-PEGS hydrogel after injection.

In the step (i) described above, the step (ii),

the molar weight of succinic anhydride is 1.2 times of that of hydroxyl in PEGS-OH, the solvent is anhydrous N, N' -dimethylformamide, the reaction temperature is 100 ℃, and the reaction time is 1 hour;

the feeding amount of N, N' -diisopropylcarbodiimide and N-hydroxysuccinimide is 1.2 times of the molar amount of carboxyl in PEGS-COOH;

the feeding amount of the 3-azidopropylamine and the aminated dibenzocyclooctyne is 100 percent, 80 percent and 60 percent of the molar amount of carboxyl in PEGS-COOH;

triethylamine was added to adjust the pH to 8.0, the reaction temperature was 37 ℃ and the reaction time was 6 hours.

In the step (ii), the concentration of the polymer solution is 20 wt%, 15 wt%, 10 wt%; the gel temperature was 37 deg.C, 25 deg.C, 10 deg.C.

The preparation method comprises the following steps:

(i) providing polyhydroxy PEGS, succinic anhydride, and 3-azidopropylamine (NH)₂-Az), aminated dibenzocyclooctyne (NH)₂-DBCO), N' -Diisopropylcarbodiimide (DIC), N-hydroxysuccinimide (NHS), Triethylamine (TEA) and a solvent;

(ii) in a glove box, dissolving polyhydroxy PEGS in a solvent, adding succinic anhydride, and reacting at 100 ℃ for 1 hour to obtain carboxylated PEGS-OH;

(iii) dissolving the carboxylated PEGS-OH in a solvent in a glove box, and sequentially adding TEA, DIC, NHS and NH₂-Az or NH₂Reacting at 37 ℃ for 6 hours to obtain PEGS-Az and PEGS-DBCO.

(iv) And preparing the PEGS-Az and the PEGS-DBCO into aqueous solutions, and respectively filling the aqueous solutions into a double-syringe injector to obtain the hydrogel.

The grafting rate of the carboxylated PEGS-OH is 100 percent.

The PEGS-Az grafting rate is 100%, 80% or 60%.

The PEGS-DBCO grafting rate is 100%, 80% or 60%.

The solvent is in an anhydrous state.

In the step (ii), the glove box operation comprises the following steps:

(a) and opening a vacuum pump and illumination of the glove box, and opening the high-purity nitrogen cylinder.

(b) In the control panel, a large transfer cabin is selected and inflated to ensure that the air pressure of the large transfer cabin is recovered to a normal pressure state from negative pressure;

(c) opening a large transfer cabin outside the glove box, putting needed articles into the large transfer cabin, and tightly closing the large transfer cabin;

(d) in the control panel, the large transfer chamber and the vacuum operation are selected, so that the air pressure in the large transfer chamber is reduced to a vacuum state. Then, selecting inflation operation to restore the air pressure to a normal pressure state;

(e) repeating the step d for three times, and fully cleaning the air in the large transfer cabin;

(f) opening the large transfer cabin in the glove box when the large transfer cabin is in a normal pressure state, and taking out the placed articles;

(g) weighing and adding reagents into the glove box, and sealing the reaction container after the experiment is finished;

(h) repeating the step b, and putting the articles into the large transfer cabin from the glove box;

(i) adjusting the internal pressure of the glove box to 4-1MPa, opening the large transfer cabin from the outside of the glove box and taking out the articles;

(j) and closing the large transfer chamber, selecting the large transfer chamber in the control panel, and performing vacuum operation to reduce the air pressure in the large transfer chamber to a vacuum state.

The main advantages of the invention include:

1) the viscoelastic hydrogel disclosed by the invention has excellent injectability, viscoelasticity and mechanical strength;

2) the mixture of the viscoelastic hydrogel can be injected to a required position to form gel in situ, and the gel forming time can be adjusted between 30 and 150 seconds;

3) the rheological property, the injection property, the mechanical property, the cell behavior and the tissue repair activity of the viscoelastic hydrogel can be regulated and optimized by regulating and controlling the molecular weight and the crosslinking degree of a PEG block;

4) the viscoelastic hydrogel can freely fill defect parts and can be highly fit at the defect parts;

5) the viscoelastic hydrogel disclosed by the invention has viscoelasticity which is beneficial to the adhesion and spreading of mesenchymal stem cells, promotes the tissue regeneration and improves the repair quality;

6) the viscoelastic hydrogel has a minimally invasive property, so that the viscoelastic hydrogel has a considerable prospect in clinical application.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are mole percentages and mole parts.

Example 1 Synthesis and characterization of polyhydroxy PEGS

The synthetic route is shown as A in figure 1, and specifically comprises the following steps:

(a) 4.68mL of propylene oxide and 18.9mL of triethylamine were dissolved in 100mL of toluene. Adding the mixture into a three-neck flask under the argon atmosphere, and stirring at normal temperature to uniformly mix the mixture;

(b) 6.08mL of sebacoyl chloride is dissolved in 8mL of toluene and added into a constant-pressure funnel under the atmosphere of argon;

(c) the reaction apparatus was transferred to an ethanol bath at-5 ℃ and the piston of the constant pressure funnel was opened to add dropwise the solution of sebacoyl chloride in (b) to the mixed solution in (a). The mole content of sebacoyl chloride relative to epoxypropanol in this reaction was 50%, i.e. the molar ratio of reacted acid chloride to hydroxyl was 1: 1.

(d) after 6 hours of reaction, the triethylamine hydrochloride was removed by suction filtration and the solvent was removed by rotary evaporation. And purifying by column chromatography to obtain the diglycidyl sebacate.

(e) In a glove box filled with argon, 2.39g of diglycidyl sebacate and 2.5g of carboxylated PEG were weighed, dissolved in DMF, and heated and stirred at 100 ℃ for 1 to 3 days.

(f) And purifying the prepolymer by ethanol dissolution-ultrapure water dialysis operation to remove unreacted monomers and small molecules to obtain the purified polyhydroxy PEGS polymer.

By using carboxylated PEG with different molecular weights (the number average molecular weight is respectively 250Da, 500Da and 600Da), different polyhydroxy PEGS high molecules are obtained and named as PEGS respectively₂₅₀-OH、PEGS₅₀₀-OH、PEGS₆₀₀-OH. Meanwhile, PGS-OH containing no PEG component and PEG-OH containing no sebacic acid component were prepared using the same method.

Wherein PEGS is obtained by using carboxylated PEG with number average molecular weight of 250Da₂₅₀The number average molecular weight of-OH was 12175Da, and the dispersity coefficient was 1.61.

The NMR spectrum of the obtained polyhydroxy PEGS polymer is shown as B in FIG. 1, and the peak position accords with the theoretical structure of PEGS hydrogen atom. And at a chemical shift of 3.00ppm, no peak of the epoxy functional group is observed, which indicates that all the epoxy functional groups are completely consumed by the reaction and the reaction is complete.

The Fourier transform infrared spectrum of the obtained polyhydroxy PEGS polymer is shown as C in figure 1, and is 3500cm^-1The peak intensity of hydroxyl groups is obviously enhanced compared with PEGS obtained by direct mixing and polycondensation of polyethylene glycol, sebacic acid and glycerol, which shows that the hydroxyl group content of the polyhydroxy PEGS polymer is obviously higher than that of PEGS obtained by polycondensation.

In FIG. 1, D shows the molecular weight of polyhydroxy PEGS macromolecules with different PEG block molecular weights changing with reaction time by gel permeation chromatography, and the result shows that the number average molecular weight of PEGS-OH increases with the extension of reaction time, and is not obviously influenced by the molecular weight of the PEG block, and the number average molecular weight is about 12 kDa.

Example 2 Synthesis and characterization of hydrogels of the invention

As shown in A in figure 2, polyhydroxy PEGS is chemically grafted to obtain PEGS-Az and PEGS-DBCO, and the PEGS-Az and the PEGS-DBCO are mixed in the injection process and undergo a click chemical reaction to obtain the hydrogel with a certain crosslinking degree.

The specific synthetic route is shown as B in figure 2, and comprises the following steps:

(a) in a glove box, taking 7.03g of polyhydroxy PEGS and 2.00g of succinic anhydride, fully dissolving in DMF, and mixing and stirring uniformly;

(b) sealing the device, transferring out of the glove box, and heating at 100 ℃ for 1 h;

(c) after the reaction is finished, dialyzing the product to remove impurities, and drying the product in vacuum overnight to obtain yellow liquid PEGS-COOH;

(d) taking 9.03g of PEGS-COOH, adding 252.4g of DIC and 230.18g of NHS into a glove box, dissolving in DMSO, and activating at room temperature for 3 h;

(e) after activation, adding 80 mu L of triethylamine into the reactant in the step (d), uniformly mixing, and equally dividing into two parts;

(f) 25mg of 3-azidopropylamine was added to one portion, and 50mg of aminated dibenzocyclooctyne was added to the other portion, followed by reaction at room temperature overnight;

(g) after the reaction is finished, dialyzing to remove small molecules and solvents, and then drying in vacuum to obtain PEGS-Az and PEGS-DBCO respectively;

(h) mixing the PEGS-Az and PEGS-DBCO in the step (g) to obtain the hydrogel of the invention.

The hydrogel with different crosslinking degrees can be obtained by controlling the grafting rate of functional groups of azido/cyclic alkynyl, namely controlling different proportions of charge ratios in the synthesis process. For example, in the preparation of 60% crosslinked hydrogel of the present invention, the amount of 3-azidopropylamine charged was controlled to be 60% of the molar amount of PEGS-COOH carboxyl groups, and the amount of aminated dibenzocyclooctyne was also controlled to be 60% of the molar amount of PEGS-COOH carboxyl groups.

In FIG. 2, the structures of synthesized PEGS-COOH, PEGS-Az and PEGS-DBCO are analyzed by C-E through nuclear magnetic resonance hydrogen spectroscopy, and it can be seen that no hydroxyl peak is generated in the PEGS-COOH obtained by measuring C, which indicates that the grafting rate of carboxyl reaches 100%. The PEGS-Az measured in D showed significant-C at the i, j, k (chemical shifts of 3.45, 1.91 and 1.40ppm, respectively) indices₃H₆-radical peaking. The peak also appears in the aminated dibenzocyclooctyne part in E, and PEGS-DBCO in E also appears a characteristic peak of a benzene ring at a mark k (chemical shift is 7.67-7.81ppm), which proves that the aminated dibenzocyclooctyne is successfully grafted.

In fig. 2F shows the total reflection fourier infrared spectrum. First, after the carboxylation modification, PEGS-COOH showed no significant change in the peak of functional group for PEGS-OH, since both carboxyl and hydroxyl groups were 3500cm^-1Left and right peaks. It is evident that PEGS-Az is 2140cm in length due to the introduction of azide groups and cycloalkynyl groups^-1A clear and sharp N ≡ N stretching vibration peak appears, and the PEGS-DBCO is 742cm^-1And 759cm^-1The characteristic peak of the benzene ring appears, which indicates that the azide group and the cycloalkynyl group are successfully grafted.

The results show that PEGS-Az and PEGS-DBCO are successfully prepared, and the two polymers can be directly crosslinked to obtain the hydrogel through simple mixing.

Example 3 characterization of hydrogel rheology and surface topography of the invention

The use process of the rotary rheometer comprises the following steps:

(a) turning on a rotary rheometer (Thermo Hakke, USA), setting different temperatures (37 ℃, 25 ℃, 10 ℃) and controlling the temperature, setting the frequency at 10Hz and the strain at 1.0%;

(b) weighing PEGS-Az and PEGS-DBCO macromolecules with equal mass, dissolving in PBS and preparing macromolecule solutions (20%, 15% and 10%) with different concentrations;

(c) filling the polymer solution in the step (b) into a syringe of a double-syringe injector for standby;

(d) rapidly injecting the mixture to a steel parallel plate with the diameter of 20mm by using the double-syringe injector in the step (c), and immediately starting to test;

(e) the change in storage modulus (G ') and loss modulus (G') with time is recorded.

Fig. 3, a, shows the gel formation time as a function of temperature for the hydrogels of the invention, indicating that they have formed when the storage modulus curve is higher than the loss modulus curve, and it can be seen from the figure that the loss modulus of the mixture is greater than the storage modulus at the beginning of mixing of precursor solution a and precursor solution B, i.e. the mixture is liquid after mixing, indicating that injectable requirements are met. The reaction is accelerated along with the rise of the temperature, the gelling time is obviously shortened, and the sol-gel transformation can occur within 70s at 37 ℃; almost no complete sol-gel transition was detected to occur within a test time of 10min when the temperature was as low as 10 ℃.

Fig. 3B shows the gel forming time of the hydrogel of the present invention as a function of the polymer concentration, and it can be seen that the gel forming time is shortened as the concentration is increased, the concentration is controlled to be about 15%, the sol-gel transition can occur in about 70s, and the hydrogel is suitable for the operation of surgery.

Fig. 3C shows a photograph of a hydrogel of the present invention showing a sol-gel transition in an aqueous solution at 37 ℃ for about 1min, the hydrogel was placed in an inclined vial, and the original flowing polymer solution no longer has fluidity, thus confirming that the sol-gel transition was completed. In FIG. 3, D shows the appearance of the hydrogel of the present invention, and the obvious micron-sized pores are randomly distributed on the surface of the hydrogel, which is beneficial to the migration and growth of cells, the transportation of nutrients and oxygen during the tissue repair process, and the generation and the ingrowth of blood vessels during the osteogenesis process.

Therefore, the hydrogel disclosed by the invention is short in gelling time, can be formed by injection, can be rapidly gelled in about 1min, prevents small molecules from diffusing in a body by virtue of rapid gelling, and is a good tissue repair material which can be formed by an injection mode.

Example 4 characterization of mechanical Properties of the hydrogels of the invention

In order to examine the tensile modulus and the viscoelasticity of the hydrogel in a tensile state, the hydrogel of the invention with different PEG block molecular weights (PEG block lengths) and crosslinking degrees (crosslinking rates) is selected in the research to examine the tensile property, five parallel samples are set for each group of samples to be tested, and the result is averaged. And (5) when the tensile modulus is tested, the tensile rate is 10mm/min until the tensile failure is realized, and a stress-strain curve is recorded.

In the creep test, the specimens were rapidly stretched at a rate of 50mm/min to a constant stress of 0.01MPa and held at a stress of 5000s, and the change in strain with time was recorded.

In the stress relaxation test, the bars were also rapidly stretched at a rate of 50mm/min to a constant strain of 5% and maintained at a strain of 10000s, and the change in stress over time was recorded.

As can be seen from A in FIG. 4, the hydrogel of the present invention has a somewhat lower tensile modulus with increasing molecular weight of the PEG block and decreasing degree of crosslinking. The 100% crosslinked PGS-OH group modulus reached 1.575MPa, while the 80% crosslinked PEG-OH modulus was as low as 0.011 MPa. This decrease in mechanical properties may be due to the introduction of soft segment PEG and the increase in non-crosslinking points. This indicates that the shorter the PEG block, the longer the opposite PGS block, and the higher the degree of crosslinking, the better the mechanical properties of the hydrogel of the present invention.

In the aspect of the viscoelasticity test, it can be seen from B in fig. 4 that the creep time of the hydrogel of the present invention is shortened with increase of PEG and decrease of the degree of crosslinking, i.e., the creep property is improved. The creep time of 80% cross-linked PEG-OH which is all PEG block only needs 110s, and 100% cross-linked PGS-OH which is all PGS block does not meet the requirement of creep time detection in the test time, which shows that the shorter the PEG block, the longer the opposite PGS block, and the higher the cross-linking degree, the better the elasticity of the hydrogel; the longer the PEG block, the lower the degree of crosslinking, and the better the hydrogel's viscosity. In other words, PGS moieties favor increased elasticity with the degree of crosslinking and PEG moieties favor increased viscosity with the degree of non-crosslinking.

FIG. 4C shows that another viscoelastic parameter, the half-relaxation time, and the creep time of the hydrogel of the present invention are consistent, and the increase of the length of the PEG block and the increase of the degree of crosslinking are increased from 20s of 80% crosslinked PEG-OH group to 100% crosslinked PGS-OH and PEGS₂₅₀The test time in the-OH group could not be measured.

The results show that the hydrogel of the invention can realize the conversion from elasticity to viscosity by adjusting the length of the PEG block (molecular weight of the PEG block) and the crosslinking degree. Thus, hydrogels of the present invention with different viscoelastic properties can be obtained for repair of different tissues.

Example 5 adhesion Performance of mesenchymal Stem cells on the hydrogel of the present invention

To investigate the effect of the hydrogel of the present invention on cell adhesion, the hydrogel was injected into 24-well plates and after gelation, about 10 cells were added per well⁵Individual bone marrow mesenchymal stem cells (BMSCs) and cultured for 24 h. Subsequently, it was fixed with 2.5% glutaraldehyde and cytoskeleton was stained with 5mg/mL FITC-phalloidin at 37 ℃ for 45 min. And finally, inspecting the fluorescence intensity of the sample, namely the spreading condition of the cells by using high-throughput screening.

As can be seen from FIG. 5, neither too high nor too low a viscosity is detrimental to cell adhesion, with the highest fluorescence intensity being detected in the 80% crosslinked hydrogel set of the present invention, and the best cell adhesion, i.e., a degree of viscoelasticity is beneficial for cell adhesion. And in the group of 80% crosslinking rate, the fluorescence intensity rises with the increase of PEG, which shows that the introduction of PEG is favorable for improving the hydrophilicity and flexibility of the high-elasticity PGS and is more favorable for cell adhesion on the hydrogel of the invention.

Example 6The hydrogel of the invention entraps growth factors and cells

For the investigation of the behavior of the hydrogel immobilized protein, horseradish peroxidase (HRP) is selected as a model protein to investigate the wrapping and releasing conditions of the hydrogel to the protein. To the hydrogel of the present invention, 10. mu.L of HRP solution at 50mg/mL was added and injected into a mold, and after it was solidified and molded, the protein-loaded hydrogel was soaked in 1mL of PBS and incubated at 37 ℃ for 3 days, and PBS was collected and refreshed at each time point. The released HRP activity was tested using 3,3',5,5' -tetramethylbenzidine as substrate and pure HRP as standard.

As can be seen from A in FIG. 6, HRP released somewhat abruptly, about 30% and then showed a tendency to slow-release within the first 12 hours, with the release approaching 50% at 72 hours. And as can be seen from B in fig. 6, the activity released in the first 24h is close to that of the standard, and the relative activity compared with that of the standard is about 100%, which indicates that the hydrogel of the present invention has good biocompatibility and excellent maintenance effect on the activity of the loaded protein.

For the examination of the behavior of the hydrogel-immobilized cells of the present invention, PEGS-Az and PEGS-DBCO were dissolved in α -MEM medium and then 10% was added⁶Cells were added at a density of/mL and mixed well. After injection and gelation, the culture was carried out for 1, 3 and 7 days, respectively, and the medium was changed every 2 days. Viable cells were stained green and apoptotic cells were stained red with a live-dead cell staining reagent (Dojindo, japan), and observed with a confocal fluorescence microscope.

As can be seen from C in FIG. 6, BMSCs cells proliferated over time when cultured in the hydrogel of the present invention in three dimensions, indicating that the hydrogel of the present invention has good cell compatibility and can be used as a cell carrier in the field of tissue repair.

Example 7 characterization of in situ repair of rat skull defects with hydrogels of the invention

The in vivo tissue repair effect of the hydrogel is evaluated by adopting a rat skull defect model. The experimental scheme is researched by animals in the ninth people hospital in Shanghai CityApproved by the institutional review board, meeting the relevant guidelines. Male SD rats weighing 500g at age 12 weeks were selected and each individually housed in a cage with independent temperature, light and humidity control. Before surgery, each rat was subjected to general anesthesia and intraperitoneally injected with 3.5mg/100g of sodium pentobarbital. The head of each rat was shaved and sterilized, a 1cm long incision was made along the midline of the skull from the frontal bone to the occiput, and the periosteum was pulled open to expose the entire skull. After the skin and periosteum were retracted, two symmetric, full-thickness skull defects of critical size 5mm were carefully drilled on both sides of the midline of the skull using a bone drill, using a sterile saline flush. PEGS with different degrees of crosslinking₂₅₀After injection of the-OH hydrogel into the defect, the soft tissue is sutured to achieve primary closure. Following surgery, antibiotics were administered intramuscularly twice daily for 2 consecutive days. Normal feeding after surgery, 5 rats per group were sacrificed under excessive anesthesia after 8 and 12 weeks, and after careful skull resection and cleaning, the rats were fixed in 10% neutral buffered formalin for Micro CT (a in fig. 7) and histological analysis (B in fig. 7).

The results show that for PEGS₂₅₀The group of-OH hydrogel with the crosslinking rate of 80% has the optimal bone repair effect, and the bone defect part is almost completely closed after 8 weeks, which shows that the hydrogel is beneficial to tissue repair and is an excellent tissue repair material.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A viscoelastic hydrogel formed by a crosslinking reaction of a first polymer and a second polymer;

2. The viscoelastic hydrogel of claim 1, wherein the mass ratio of the first polymer to the second polymer is from 0.5 to 2: 0.5 to 2.

3. The method of making a viscoelastic hydrogel according to claim 1 comprising the steps of:

4. A polymer which is an azido-modified polyhydroxy pegylated polysebacic acid glyceride comprising structural units according to formula Ia or Ib:

a₁is an integer of 1 to 20, b₁Is an integer of 5 to 60;

a₂is an integer of 1 to 20, b₂Is an integer of 5 to 60;

5. The method of preparing the polymer of claim 4, comprising the steps of:

6. A polymer, characterized in that the polymer is a cyclic alkynylated polyhydroxy pegylated polysebacic acid glyceride comprising structural units according to formula IIa or IIb:

“

the cyclic alkynyl group "means that the cyclic alkynyl group is linked to the second group through a grafted chain structureA dimeric polymer backbone.

7. The method of preparing the polymer of claim 6, comprising the steps of:

8. A kit for preparing the viscoelastic hydrogel of claim 1, the kit comprising:

(a) a first container, and a first polymer located within the container;

(b) a second container, and a second polymer located within the container.

9. Use of a viscoelastic hydrogel according to claim 1, (a) for the preparation of a medical material for the loading of growth factors and drugs, or for the filling of tissue defects and/or for the guided regeneration of tissue; (b) or used for preparing a medical cosmetic material.

10. A polymer, wherein the polymer is a polyhydroxy pegylated polyglycerol polysebacate (PEGS-OH) comprising structural units according to formula IIIa or IIIb:

wherein,

in formula IIIa, m₁Is an integer of 1 to 20, n₁Is an integer of 5 to 60;

in formula IIIb, m₂Is an integer of 1 to 20, n₂Is an integer of 5 to 60.

11. A method of preparing the polymer of claim 9, comprising the steps of:

(s1) under the atmosphere of protective gas, in the presence of an inert solvent, carrying out polymerization reaction on diglycidyl sebacate (monomer 1), carboxylated polyethylene glycol (monomer 2) and bis-tetra-n-butylammonium hydroxide PEG salt (catalyst 1) to obtain polyhydroxy polyethylene glycol polysebacate shown in formula IIIa;

or (s2) under the atmosphere of protective gas, in the presence of an inert solvent, carrying out polymerization reaction on sebacic acid (monomer 3), polyethylene glycol diglycidyl ester (monomer 4) and bis tetra-n-butyl ammonium hydroxide sebacate (catalyst 2) to obtain polyhydroxy polyethylene glycol polysebacate represented by formula IIIb.

12. Use of a polymer according to claim 9 for the preparation of a material comprising a bio-elastomer, a hydrogel, a 3D printed material, a responsive polymer, a conductive polymer.