阅读说明：本技术 巯基改性高分子化合物的水凝胶及其制备方法和用途 (Hydrogel of sulfhydryl modified macromolecular compound and preparation method and application thereof ) 是由 王文新 于 2019-11-18 设计创作，主要内容包括：本发明涉及一种巯基改性高分子化合物的水凝胶及其制备方法和应用,本发明采用了一种新颖结构的巯基改性高分子化合物,将其与丙烯酰化高分子化合物和/或丙烯酰小分子交联剂的配合而形成水凝胶,所述巯基改性高分子化合物可在生理条件下与所述丙烯酰化高分子化合物和/或丙烯酰小分子交联剂交联形成水凝胶；另外,形成的水凝胶与塑型效果及耐代谢耐降解性相关的物理性质、化学性质也具有明显优于现有技术的优势,具体的,其耐代谢耐降解性明显优于现有技术中的水凝胶；再有,由于巯基-乙烯基交联反应迅速的特点使得由所述两种化合物形成的水凝胶体系可以在注射入体内后快速原位成胶。本发明的水凝胶更加利于用于生物医药、医疗美容整形以及化妆品等领域。(The invention relates to a hydrogel of sulfhydryl modified macromolecular compound and a preparation method and application thereof, the invention adopts a sulfhydryl modified macromolecular compound with a novel structure, the sulfhydryl modified macromolecular compound is matched with an acryloyl macromolecular compound and/or an acryloyl micromolecule cross-linking agent to form the hydrogel, and the sulfhydryl modified macromolecular compound can be cross-linked with the acryloyl macromolecular compound and/or the acryloyl micromolecule cross-linking agent to form the hydrogel under physiological conditions; in addition, the formed hydrogel has the advantages of obviously superior to the prior art in physical properties and chemical properties related to the molding effect and the metabolic degradation resistance, and particularly, the metabolic degradation resistance is obviously superior to the hydrogel in the prior art; furthermore, due to the rapid reaction characteristic of the mercapto-vinyl crosslinking, the hydrogel system formed by the two compounds can be rapidly gelled in situ after being injected into a body. The hydrogel is more beneficial to the fields of biological medicine, medical cosmetology and cosmetics and the like.)

1. A hydrogel produced by gelling a system containing a mercapto group-modified polymer compound;

the sulfhydryl modified macromolecular compound is at least one of the following compounds:

a series of sulfhydryl modified macromolecular compounds, the modified macromolecular compounds structurally contain-COOH and-NH₂At least one of-OH, an acrylate group represented by the formula a, an acrylamide group represented by the formula b and an acrylamide group represented by the formula c,

the-COOH and/or-NH₂and/or-OH and/or acrylate groups and/or acrylamide groups and/or acryloyl groups are partially or completely modified to form side chains with the end groups of the following groups:

in the above groups, denotes a point of attachment; r₁Selected from hydrogen, halogen, aliphatic groups, aromatic groups, and the like; r₂And R₃Identical or different, independently of one another, from the group consisting of hydrogen, halogen, aliphatic radicals, aromatic radicals, etc.; r₄Is a fragment of a polymercapto compound;

the system further comprises at least one of the following substances:

C1. an acrylic-acylated high-molecular compound, which is a mixture of acrylic-acylated high-molecular compound,

C2. and (3) a small molecule crosslinking agent containing acryloyl.

2. The hydrogel of claim 1, wherein the-COOH and/or-NH₂and/or-OH and/or acrylate groups and/or acrylamide groups and/or acryloyl groups are partially or fully modified to form at least one of the following structures:

in the above structure, R is selected fromAlkylene groups, arylene groups, amide residues, hydrazide residues, and the like; denotes a connection point;₁denotes the point of attachment to the left radical of R;₂denotes the point of attachment to the right-hand group of R; r₁、R₂、R₃And R₄The definition of (1) is as before.

3. The hydrogel of claim 1 or 2, wherein the-COOH, -NH₂At least one of-OH, an acrylate group represented by formula a, an acrylamide group represented by formula b, and an acrylamide group represented by formula c may be directly bonded to the main chain of the polymer compound, or may be bonded to the main chain of the polymer compound through a group R ', wherein R' may be a group containing a hetero atom, an alkylene group, an arylene group, or the following linking groups:

in the above formula, R 'is alkylene or arylene, n' is an integer of 1 to 1000, and represents a connection point.

4. The hydrogel according to any one of claims 1 to 3, wherein the acrylated polymeric compound is selected from at least one of the following:

1) structurally containing-COOH, -NH₂An acrylic compound of a polymer compound of at least one of-OH, i.e., aThe polymer compound structurally contains-COOH and-NH₂An acryloylated compound formed by linking, directly or indirectly, at least one of-OH and:

R₁、R₂and R₃The definition of (1) is as before;

2) a polymer compound structurally containing at least one of an acrylate group represented by formula a, an acrylamide group represented by formula b and an acrylamide group represented by formula c.

5. The hydrogel according to claim 4, wherein in the substance of the above 1), the-COOH and/or-NH groups₂And/or some or all of the-OH groups are modified to form at least one of the following structures:

in the above structure, R is selected fromAlkylene groups, arylene groups, amide residues, hydrazide residues, and the like; denotes a connection point;₁denotes the point of attachment to the left radical of R;₂denotes the point of attachment to the right-hand group of R; r₁、R₂、R₃And R₄The definition of (1) is as before.

6. The hydrogel according to claim 4 or 5, wherein in the substance of the type 1), the group consisting of-COOH and-NH₂At least one of-OH groups may be directly bonded to the polymer compoundThe main chain may be a main chain of the polymer compound through a group R ', wherein the group R' may be a group containing a hetero atom, an alkylene group, an arylene group, or the following linking groups:

in the above formula, R 'is alkylene or arylene, n' is an integer of 1 to 1000, and represents a connection point.

7. The hydrogel of any one of claims 1-6, the substance C2. an acryloyl-containing small molecule crosslinker including, but not limited to, an acryloyl-containing small molecule compound or an acryloyl-containing oligomer; specifically, the acrylate is one or more selected from ethylene glycol diacrylate EGDA, polyethylene glycol diacrylate PEGDA, trimethylolpropane triacrylate TMPTA, pentaerythritol triacrylate PTA, pentaerythritol tetraacrylate PTTA, ditrimethylolpropane tetraacrylate DTTA, etc.

8. The hydrogel of any one of claims 1 to 7, wherein the hydrogel comprises the following characteristic structural units:

in the above unit, R₁、R₂、R₃And R₄As before, denotes the connection point.

9. A method for preparing the hydrogel of any one of claims 1 to 8, the method comprising the steps of:

gelling a system comprising:

(i) the mercapto group-modified polymer compound, and

(ii) at least one of substance C1 and substance C2;

the hydrogel is prepared.

Preferably, a solution of the thiol-modified polymer compound, a solution of the acrylated polymer compound, a solution of the small molecule cross-linking agent, and optionally a solution of at least one of other biofunctional materials, drugs, growth factors, or cell suspensions are prepared, mixed, and gelated to prepare the hydrogel.

Optionally, at least one of the other biofunctional material, the drug, the growth factor or the cell suspension may be introduced by directly adding to the solution of the thiol-modified polymer compound or the solution of the acrylated polymer compound or the solution of the small molecule cross-linking agent.

Preferably, the gel preparation process can be carried out by adding the thiol-modified polymer compound solution into the acryloyl-acylated polymer compound solution and/or the small molecule cross-linking agent solution, or can be carried out by adding the acryloyl-acylated polymer compound solution and/or the small molecule cross-linking agent solution into the thiol-modified polymer compound solution. Specifically, the two solutions can be mixed by a common syringe, a double-needle syringe, or other means.

10. Use of the hydrogel according to any one of claims 1 to 8 in the fields of biomedicine, medical cosmetic shaping, and cosmetics. In particular, it can be used for preparing drug delivery systems, dressings for soft tissue wound repair, scaffold materials for bone repair, viscoelastic agents for supporting in ophthalmic surgery, materials for preventing tissue adhesion after surgery, scaffold materials for 3D bioprinting, and the like.

Technical Field

The invention belongs to the field of biological materials, and particularly relates to a hydrogel of a sulfhydryl modified macromolecular compound, a preparation method and application thereof.

Background

Biomedical Materials, also called biological Materials for short (Biomaterials), are novel high-tech Materials for diagnosing, treating, repairing or replacing diseased tissues and organs or enhancing their functions. One of the key techniques of tissue engineering is to prepare a cell scaffold with good biocompatibility and capable of being degraded and absorbed by the body by using biological materials. The gel state is an intermediate state between solid and liquid, and the hydrogel refers to a hydrophilic crosslinked three-dimensional polymer network which can swell in water and can retain a large amount of water without dissolving, and the water content of the hydrogel can reach more than 90%. Hydrogels are an ideal class of biomaterials that, by themselves or through simple modification, can have similar, desirable physical and chemical properties as the natural extracellular matrix, while exhibiting good permeability to oxygen, nutrients, cell metabolites and water-soluble metal ions. The hydrophilic polymers for preparing the hydrogel are classified into natural polymers and synthetic polymers according to the source. Wherein the natural polymer comprises collagen, gelatin, fibrin, polysaccharide, etc., and the synthetic polymer comprises synthetic polypeptide, polyethylene glycol (PEG) and its derivatives, polymethyl methacrylate (PMMA) and its derivatives, and poly (lactic-co-glycolic acid) (PLGA) and its derivatives, etc. Injectable in situ cross-linked hydrogels have also received increased attention in recent years. Injectable in situ cross-linked hydrogels are characterized by a flowable liquid state prior to injection and, when injected into a target site, form a gel that conforms well to the shape of the target site. The injectable property not only makes the operation process simple and convenient, but also can avoid the pain of the patient caused by the implantation operation, and greatly reduces the traumatic property of the operation.

Among the natural polymers, Hyaluronic Acid (HA) HAs been receiving attention from researchers due to its excellent properties. Natural hyaluronic acid is a natural heteropolysaccharide composed of alternating units of D-glucuronic acid and N-acetylglucosamine. Decades of research have shown that hyaluronic acid is found in connective tissues of human and other vertebrates, such as the intercellular space, the tissues of the motor joints, the umbilical cord, the skin, the cartilage, the blood vessel wall, the synovial fluid, and the cockscomb. Hyaluronic acid belongs to linear high-molecular polysaccharide, and the structure of hyaluronic acid contains a disaccharide repeating unit, D-glucuronic acid in the repeating unit is connected with N-acetyl glucosamine through beta-1, 3 glycosidic bonds, and thousands of disaccharide repeating units are connected through beta-1, 4 glycosidic bonds to form a whole high-molecular straight chain and linear structure. Hyaluronic acid is usually present in the form of a sodium salt in the physiological state of the human body. The sodium hyaluronate and the gel thereof are widely applied in the fields of orthopedics, gynecology, orthopedics and the like, and can also be used as an ophthalmic preparation carrier or directly used as an ophthalmic preparation for ophthalmic surgery, namely the sodium hyaluronate products also have important application in the ophthalmic surgery. Sodium hyaluronate is also an important component of joint synovial fluid and cartilage, and can increase the viscosity and lubricating function of the joint synovial fluid by increasing the content of sodium hyaluronate in joints, and play roles of protecting cartilage, promoting joint healing and regeneration, relieving pain, increasing joint mobility and the like. And there are literature reports: a large number of animal experiments and clinical applications show that hyaluronic acid and sodium salt thereof are safe and effective ideal substances in the aspect of preventing and reducing adhesion caused by gynecological operations; among them, an aqueous solution of sodium hyaluronate is a non-newtonian fluid and has good viscoelasticity and rheological properties, and in general, a low-concentration hyaluronic acid solution mainly exhibits viscosity and a high-concentration hyaluronic acid solution mainly exhibits elasticity, so that the concentration thereof can be adjusted according to the actual use requirements.

Although natural hyaluronic acid or its sodium salt has a wide application field and has various definite application advantages, natural hyaluronic acid or its sodium salt also has definite disadvantages. First, natural hyaluronic acid or its sodium salt has a short half-life in vivo, and the degradation time in vivo is generally not more than 7 days, and the main reason for the short half-life is that natural hyaluronic acid or its sodium salt has a small average molecular weight, has good fluidity, is easily dispersed in tissues and absorbed and metabolized, and is directly expressed as low viscosity in a solution state. Secondly, natural hyaluronic acid or its sodium salt has the disadvantages of poor stability and easy degradation. Third, natural hyaluronic acid or its sodium salt has a disadvantage of being excessively hydrophilic.

Other natural high molecular compounds also have similar problems with hyaluronic acid. The hydrogel for preparing the natural macromolecular compound can solve the problems of low mechanical strength and the like to a certain extent. In the existing research, in order to obtain hydrogel of natural polymer compound with ideal physical and mechanical properties and biodegradation rate, chemical crosslinking mode is widely applied in the process of preparing hydrogel. The chemical crosslinking reaction is often applied to functional groups with high chemical activity, such as carboxyl, hydroxyl, amino, etc., and commonly used chemical crosslinking agents generally contain bifunctional groups, such as diamine, dihydrazine, dialdehyde, diol, etc., but these crosslinking agents are usually cytotoxic and, if left, will affect the biocompatibility of the hydrogel material. There is a need to develop a novel chemically crosslinked polymer hydrogel to avoid cytotoxicity caused by adding additional chemicals in the crosslinking reaction. Meanwhile, modern medicine requires that the biomaterial has certain plasticity and controllability in use, so as to realize minimally invasive treatment effect. Taking hyaluronic acid as an example, in the prior art, hydrogels prepared from hyaluronic acid as a main raw material have the following obvious disadvantages or technical prejudices: firstly, because the micromolecule crosslinking containing epoxy groups is applied to the crosslinking reaction of the hyaluronic acid, the toxic epoxy micromolecule crosslinking agent remains in the crosslinked hyaluronic acid, and after the crosslinked hyaluronic acid is prepared into hydrogel, adverse reaction or toxic action is inevitably generated, so that the application of the hydrogel of the hyaluronic acid is restricted. And secondly, the cross-linked hyaluronic acid is obtained by cross-linking reaction of the chemically modified hyaluronic acid, the hydrogel prepared by the cross-linked hyaluronic acid is high in price, and the hydrogel is improved in the aspects of viscosity, water retention, plastic effect and the like but is limited compared with the hydrogel prepared by cross-linking hyaluronic acid which is not structurally modified and reformed. In the prior art, the reaction of cross-linked hyaluronic acid generated by cross-linking reaction of hyaluronic acid requires certain reaction conditions or is harsh, in-situ cross-linking cannot be realized in a physiological state, and the product can only be realized in a pre-cross-linking and pre-filling manner, so that the application range of the product and the compliance of corresponding treatment crowds or beauty crowds are greatly influenced.

In recent years, thiol-modified polymer compounds have attracted attention of researchers because of their characteristics such as easy crosslinking to form hydrogel and oxidation resistance. The thiol modification process of the existing biocompatible macromolecule generally refers to a chemical modification process for introducing free thiol, and usually the side chain groups of polysaccharide, protein and synthetic macromolecule, such as: carboxyl, amino, hydroxyl, etc., and free thiol groups can be introduced by appropriate chemical reactions. Taking hyaluronic acid as an example, in the prior art, sulfhydrylated hyaluronic acid obtained by introducing free sulfhydryls into hyaluronic acid through chemical reaction is summarized, and although the characteristics of the sulfhydrylated hyaluronic acid are improved or enhanced in terms of physicochemical properties or biocompatibility and the like compared with natural hyaluronic acid, the following disadvantages are still not overcome enough: 1. the rate of self-crosslinking or crosslinking reactions with other substances is relatively slow, and small molecule oxidants are usually added to accelerate the crosslinking reaction. 2. After the sulfhydryl modified hyaluronic acid new compound is crosslinked to form hydrogel, compared with the existing products or products sold in the market, the key indexes of the sulfhydryl modified hyaluronic acid new compound such as physical and chemical properties, biocompatibility and the like do not have substantial advantages or sufficient distinguishing technical characteristics, and are mainly reflected in viscosity, metabolic persistence and molding effect. 3. The sulfhydrylation hyaluronic acid and the synthesis preparation method in the prior art have the disadvantages of higher toxicity or overhigh cost. The reasons are the root of influencing the industrial production and wider application of the prior thiol hyaluronic acid synthesis and preparation technology. In addition, the hydrogel prepared by crosslinking reaction of thiolated hyaluronic acid in the prior art has disadvantages or technical prejudices including: 1. in the prior art, cross-linked hyaluronic acid is obtained by cross-linking reaction of chemically modified hyaluronic acid, and the price of the hydrogel prepared by the cross-linked hyaluronic acid is higher than that of the hydrogel prepared by cross-linking natural hyaluronic acid. 2. In the prior art, crosslinked hyaluronic acid is obtained by crosslinking reaction of chemically modified hyaluronic acid, and the hydrogel prepared by the crosslinked hyaluronic acid has improved but limited improvement in the aspects of viscosity, water retention property, molding effect and the like compared with the hydrogel prepared by crosslinking natural hyaluronic acid. 3. In the prior art, chemical modification of hyaluronic acid has certain uncontrollable property, which affects the quality of cross-linked hyaluronic acid, and further causes the quality of corresponding hydrogel to fluctuate in a large range, so that the consistency of the treatment effect or the cosmetic and plastic effect of hydrogel products in different batches cannot be realized, and the hydrogel is also affected to play a greater role in the application field.

Disclosure of Invention

In order to solve the above problems, an object of the present invention is to provide a mercapto group-modified polymer compound having a novel structure, and at least one of: an acryloyl group-containing small molecule cross-linking agent, and a hydrogel having a novel structure formed by gelation of the compound or the cross-linking agent. Specifically, the invention adopts a sulfhydryl modified macromolecular compound with a novel structure, which is matched with an acryloyl macromolecular compound and/or a micromolecule cross-linking agent containing acryloyl to form hydrogel, wherein the sulfhydryl modified macromolecular compound can be cross-linked with the acryloyl macromolecular compound and/or the micromolecule cross-linking agent containing acryloyl under physiological conditions to form hydrogel; in addition, the formed hydrogel has the advantages of obviously superior to the prior art in physical properties and chemical properties related to the molding effect and the metabolic degradation resistance, and particularly, the metabolic degradation resistance is obviously superior to the hydrogel in the prior art; furthermore, due to the rapid reaction characteristic of the mercapto-vinyl crosslinking, the hydrogel system formed by the two compounds can be rapidly gelled in situ after being injected into a body. Therefore, the hydrogel is more beneficial to the fields of biological medicine, medical cosmetology and cosmetics and the like.

It is a second object of the present invention to provide a process for preparing the above hydrogel, which process has the following advantages: the crosslinking reaction does not need to add high-toxicity epoxy micromolecule crosslinking agent and catalyst, thus fundamentally avoiding the possibility of toxic substance residue in the purification process, and the like, without catalysis conditions such as illumination, heating and the like, the crosslinking reaction degree is controllable, and the cost of the crosslinking reaction is moderate and superior to that of the prior art.

The first aspect of the present invention is to provide a hydrogel, which has a completely new chemical structure and is prepared by gelation of a system containing a mercapto group-modified high-molecular compound;

the sulfhydryl modified macromolecular compound is at least one of the following compounds:

a series of sulfhydryl modified macromolecular compounds, the modified macromolecular compounds structurally contain-COOH and-NH₂-OH, an acrylate group of formula a, acryloyl of formula bAt least one of an amine group and an acryl group represented by the formula c,

the-COOH and/or-NH₂and/or-OH and/or acrylate groups and/or acrylamide groups and/or acryloyl groups are partially or completely modified to form side chains with the end groups of the following groups:

in the above groups, denotes a point of attachment; r₁Selected from hydrogen, halogen, aliphatic groups, aromatic groups, and the like; r₂And R₃Identical or different, independently of one another, from the group consisting of hydrogen, halogen, aliphatic radicals, aromatic radicals, etc.; r₄Is a fragment of a polymercapto compound;

the system further comprises at least one of the following substances:

C1. an acrylic-acylated high-molecular compound, which is a mixture of acrylic-acylated high-molecular compound,

C2. and (3) a small molecule crosslinking agent containing acryloyl.

The second aspect of the present invention provides a method for preparing the above hydrogel, which comprises the following steps:

gelling a system comprising:

(i) the mercapto group-modified polymer compound, and

(ii) at least one of substance C1 and substance C2;

the hydrogel is prepared.

The third aspect of the present invention is to provide a use of the above hydrogel, which is used in the fields of biomedicine, medical cosmetology, cosmetics, and the like.

The invention has the advantages of

The invention provides a hydrogel with an innovative structure, which is obtained by taking a sulfhydrylation modified macromolecular compound with an innovative structure as a raw material through a crosslinking reaction, and compared with the hydrogel in the prior art (for example, the hydrogel which is obtained by taking hyaluronic acid or modified hyaluronic acid as a starting material through the crosslinking reaction in the prior art), the hydrogel has unexpected technical advantages in the aspects of physical and chemical properties, modeling effect, metabolism resistance and degradation resistance and the like.

The hydrogel of the invention has the advantages that: 1. in the process of modifying and reconstructing the structure of the high molecular compound and in the subsequent crosslinking reaction process, no toxic epoxy micromolecular crosslinking agent is used, and the hydrogel product has the advantage of higher safety. 2. Compared with the hydrogel in the prior art (such as the existing cross-linked hyaluronic acid hydrogel), the hydrogel product disclosed by the invention has the technical advantages of higher viscosity, water retention, molding effect and the like. 3. The hydrogel can realize crosslinking reaction without adding any catalyst, and the reaction conditions are easier to realize, superior to the crosslinking reaction conditions of high molecular compounds in the prior art and superior to the crosslinking conditions of modified high molecular compounds in the prior art. 4. The in-situ crosslinking under the physiological condition in the true sense is realized for the first time, the end point of the crosslinking reaction is controllable, the controllability is not only reflected in the in-vitro crosslinking reaction, but also reflected in the crosslinking reaction in an animal body or a human body, and a large number of animal experiments prove that the reaction end point of the crosslinking reaction is single, stable and reproducible no matter the crosslinking reaction is in the animal body or in vitro. 5. Experimental research shows that the series of hydrogel disclosed by the invention has better stability and degradation resistance under the conditions of room temperature and accelerated stability investigation, and has better metabolic resistance in animal bodies and the like.

The invention really realizes in-situ crosslinking under physiological conditions, namely, the crosslinking reaction can be completed under the conditions of room temperature and normal pressure; or after the hydrogel is injected into tissues of an animal body or a human body, the crosslinking reaction can still be realized in the tissues, so that the degradation resistance and the metabolism resistance of the hydrogel product are obviously improved, and the use effect of the hydrogel injection product is obviously improved. And due to the unique technology of the invention, the controllability of the crosslinking degree of the hydrogel product injected into the animal body or the human body can be realized before the in vitro crosslinking or mixing stage, namely, the crosslinking reaction with controllable crosslinking reaction end point can be realized after the hydrogel product is injected into the animal body or the human body, and the safety and the treatment effect of the product are ensured.

The invention also provides a method for preparing the hydrogel, which can complete the reaction at room temperature and normal pressure, has mild reaction conditions and easy realization, and is the technical basis for realizing the in-situ crosslinking of the hydrogel under physiological conditions.

Drawings

FIG. 1 reaction equation of preparation 5;

FIG. 2 reaction equation of preparation 6;

FIG. 3 reaction equation of preparation 7;

FIG. 4 reaction equation of preparation 8;

FIG. 5 reaction equation of preparation 15;

FIG. 6 reaction equation of preparation 16;

FIG. 7 reaction equation of preparation 17;

FIG. 8 reaction equation of preparation 18;

FIG. 9 reaction equation of preparation 19;

FIG. 10 reaction equation of preparation 20;

FIG. 11 hydrogel sample cell biocompatibility experiments;

FIG. 12 in vivo modelling and support effect (height) of hydrogel samples;

FIG. 13 in vivo sculpting and support effect (basal area) of hydrogel samples;

FIG. 14 in vivo degradation assay of hydrogel samples;

FIG. 15 structural formula of HA-A1 and thereof¹H-NMR spectrum;

FIG. 16 structural formula of HA-A2 and thereof¹H-NMR spectrum;

FIG. 17 structural formula of HA-MA1 and its¹H-NMR spectrum;

FIG. 18 structural formula of HA-MA2 and its¹H-NMR spectrum;

FIG. 19 structural formulA of CHS-A and thereof¹H-NMR spectrum;

FIG. 20 structural formula of CHS-MA and thereof¹H-NMR spectrum;

FIG. 21 structural formula of Gelatin-A and the same¹H-NMR spectrum;

FIG. 22 structural formula of Gelatin-MA and the same¹H-NMR spectrum;

FIG. 23 structural formulA of CTS-A and thereof¹H-NMR spectrum;

FIG. 24 structural formula of CTS-MA and methods thereof¹H-NMR spectrum;

FIG. 25 structural formulas of HA-A1-SH1 and related compounds¹H-NMR spectrum;

FIG. 26 structural formulas of HA-A2-SH1 and related compounds¹H-NMR spectrum;

FIG. 27 structural formulas of HA-MA1-SH1 and related compounds¹H-NMR spectrum;

FIG. 28 structural formula of HA-MA2-SH1 and related compounds¹H-NMR spectrum;

FIG. 29 structural formulA of CHS-A-SH1 and methods of making the same¹H-NMR spectrum;

FIG. 30 structural formula of CHS-MA-SH1 and its¹H-NMR spectrum;

FIG. 31 structural formula of Gelatin-A-SH1 and its thereof¹H-NMR spectrum;

FIG. 32 structural formula of Gelatin-MA-SH1 and its¹H-NMR spectrum;

FIG. 33 structural formulA of CTS-A-SH1 and methods of making the same¹H-NMR spectrum;

FIG. 34 structural formula of CTS-MA-SH1 and methods for making the same¹H-NMR spectrum;

FIG. 35 structural formula of PHEMA-A and thereof¹H-NMR spectrum;

FIG. 36 structural formula of PHEMA-MA and¹H-NMR spectrum;

FIG. 37 structural formula of PVA-A and thereof¹H-NMR spectrum;

FIG. 38 structural formula of PVA-MA and the same¹H-NMR spectrum;

FIG. 39 structural formula of PHEMA-A-SH1 and its¹H-NMR spectrum;

FIG. 40 structural formula of PHEMA-MA-SH1 and its¹H-NMR spectrum;

FIG. 41 structural formula of PVA-A-SH1 and its¹H-NMR spectrum;

FIG. 42 structural formula of PVA-MA-SH1 and its thereof¹H-NMR spectrum;

FIG. 43 structural formula of HB-PEG-SH1 and its¹H-NMR spectrum;

FIG. 44 structural formulas of HA-A1-SH2 and related compounds¹H-NMR spectrum;

FIG. 45 structural formulas of HA-A1-SH3 and related compounds¹H-NMR spectrum;

FIG. 46 structural formula of HA-A2-SH2 and related compounds¹H-NMR spectrum;

FIG. 47 structural formula of HA-A2-SH3 and related compounds¹H-NMR spectrum;

FIG. 48 structural formulas of HA-A2-SH4 and related compounds¹H-NMR spectrum;

FIG. 49 structural formula of HA-A2-SH5 and related compounds¹H-NMR spectrum;

FIG. 50 structural formulas of HA-A2-SH6 and related compounds¹H-NMR spectrum;

FIG. 51 structural formulas of HA-A2-SH7 and related compounds¹H-NMR spectrum;

FIG. 52 structural formula of HA-A2-SH8 and related compounds¹H-NMR spectrum;

FIG. 53 structural formula of HA-MA1-SH5 and related compounds¹H-NMR spectrum;

FIG. 54 structural formula of HA-MA1-SH6 and related compounds¹H-NMR spectrum;

FIG. 55 structural formulas of HA-MA2-SH7 and related compounds¹H-NMR spectrum;

FIG. 56 structural formulas of HA-MA2-SH8 and related compounds¹H-NMR spectrum;

FIG. 57 reaction equation for preparation 25;

FIG. 58 reaction equation for preparation 26;

FIG. 59 reaction equation for preparation 27;

FIG. 60 reaction equation of preparation 28;

fig. 61 the reaction equation for preparation 29 (where i is 10-90%, j is 10-90%, i2+ i3 is i, j2+ j3 is j, h is j, i + j is 100%, k1 is 1-1000);

FIG. 62 reaction equation for preparation 30;

FIG. 63 reaction equation for preparation 31;

FIG. 64 the reaction equation of preparation 32;

FIG. 65 reaction equation of preparation 33;

FIG. 66 reaction equation of preparation 34;

FIG. 67 reaction equation for preparation 35;

FIG. 68 reaction equation for preparation 36;

FIG. 69 reaction equation of preparation 37;

FIG. 70 reaction equation of preparation 38;

FIG. 71 reaction equation of preparation 39;

FIG. 72 reaction equation of preparation 40;

FIG. 73 reaction equation of preparation 41;

FIG. 74 reaction equation of preparation 42.

Detailed Description

[ mercapto group-modified Polymer ]

As mentioned above, at least one of the compounds of the series shown below is required to be used in the system to be gelled according to the invention:

the thiol-modified macromolecular compound structurally contains-COOH and-NH₂At least one of-OH, an acrylate group represented by the formula a, an acrylamide group represented by the formula b and an acrylamide group represented by the formula c,

wherein the-COOH and/or-NH₂and/or-OH and/or acrylate groups and/or acrylamide groups and/or acryloyl groups are partially or completely modified to form side chains with the end groups of the following groups:

in the above groups, denotes a point of attachment;

R₁selected from hydrogen, halogen, aliphatic groups, aromatic groups, and the like; specifically, the halogen, aliphatic group and aromatic group satisfy the further definition below; preferably, R₁Selected from hydrogen, halogen, aliphatic radicals; also preferably, R₁Selected from hydrogen, halogen, C1-6 alkyl (e.g., methyl, ethyl, etc.);

R₂and R₃Identical or different, independently of one another, from the group consisting of hydrogen, halogen, aliphatic radicals, aromatic radicals, etc.; specifically, the halogen, aliphatic group and aromatic group satisfy the further definition below;

R₄is a multi-sulfhydryl compound fragment.

In a particular embodiment, the-COOH and/or-NH₂and/or-OH and/or acrylate groups and/or acrylamide groups and/or acryloyl groups are partially or fully modified to form at least one of the following structures:

in the above structure, R is selected fromAlkylene groups, arylene groups, amide residues, hydrazide residues, and the like; denotes a connection point;₁denotes the point of attachment to the left radical of R;₂denotes the point of attachment to the right-hand group of R; r₁、R₂、R₃And R₄The definition of (1) is as before.

Wherein, the-COOH, -NH₂At least one of-OH, an acrylate group represented by formula a, an acrylamide group represented by formula b, and an acrylamide group represented by formula c may be directly bonded to the main chain of the polymer compound, or may be bonded to the main chain of the polymer compound through a group R ', wherein R' may be a group containing a hetero atom, an alkylene group, an arylene group, or the following linking groups:

in the above formula, R 'is alkylene or arylene, n' is an integer of 1 to 1000, and represents a connection point.

Wherein the heteroatom containing group includes, but is not limited to: ester, amide or hydrazide residues. In particular, the ester, amide or hydrazide residue meets the further definition below.

Wherein the modified polymer compound comprises natural mucopolysaccharide polymer, such as at least one of chitosan (specifically chitosan, glycol chitosan, carboxymethyl chitosan, etc.), chondroitin sulfate, hyaluronic acid, alginate, etc.; proteins such as gelatin, fibrin, serum proteins, and the like; and/or, a synthetic polymer, such as at least one of polyvinyl alcohol, poly (meth) acrylic acid, poly (hydroxy alkyl (meth) acrylate) (e.g., poly (hydroxyethyl (meth) acrylate), etc.), hyperbranched polyethylene glycol, and the like.

Wherein the mercapto group content of the mercapto group-modified polymer compound measured by the Ellman method is 0.01 to 30mmol/g, for example, 0.1 to 10.0mmol/g, further for example, 0.3 to 5.0mmol/g, further for example, 0.5 to 3.0 mmol/g.

Wherein the molecular weight of the mercapto-modified macromolecular compound is substantially unchanged from the molecular weight of the macromolecular compound before modification.

For example, the mercapto-modified polymer compound of the present invention includes at least one of the following structures:

in the above structure, A is the structure containing at least one-COOH, -NH₂A segment of a modified polymer compound of-OH, an acrylate group represented by formula a, an acrylamide group represented by formula b, and an acryl group represented by formula c; r, R' and R₁、R₂、R₃And R₄The definition of (1) is as before; (n2+ n3)/(n1+ n2+ n3) represents the degree of acrylation; n3/(n1+ n2+ n3) represents the degree of sulfhydrylation, corresponding to the above-mentioned content of mercapto group in the mercapto-modified polymer compound measured by the Ellman method; the n1 may be 0, and if 0, the degree of acrylation is not limited, but n3/(n2+ n3) represents the degree of mercapto group, corresponding to the mercapto group content of the mercapto group-modified polymer compound measured by the Ellman method described above; the n2 may be 0, and if 0, n3/(n1+ n3) represents both the degree of acrylation and the degree of thiolation, corresponding to the mercapto group content of the mercapto-modified polymer compound measured by Ellman.

Specifically, a may be a structure as shown below:

in each of the above structures, denotes a connecting point between the main chain repeating units; represents-COOH, -NH₂-OH, -an acrylate group of formula a, -an acrylamide group of formula b, -a point of attachment between an acrylamide group of formula c and the above-mentioned fragment, or via a point of attachment between an R' group and the above-mentioned fragment.

The A can also be the residual segment or repeating unit after removing the acryloyl side chain in the following polymers Gelatin-A, Gelatin-MA, CTS-A, CTS-MA, PHEMA-A, PHEMA-MA, HB-PEG, PVA-A, PVA-MA, CHS-A or CHS-MA:

it should be noted that Gelatin-A, Gelatin-MA, CTS-A, CTS-MA, PHEMA-A, PHEMA-MA, HB-PEG, PVA-A, PVA-MA, CHS-A or CHS-MA are abbreviations for the names of polymers having the above structures, respectively, and the letters therein are not related to the letter meanings appearing in other parts of the present invention after being separated.

In the present invention, n', n1, n2, n3, n4, n5, n6, m1, m2, i, j, k1 and h are the number of repeating units in the structural formula unless otherwise specified. The value ranges fall within conventional ranges known in the art.

In one embodiment of the present invention, the series of thiol-modified polymer compounds are specifically:

the sulfhydryl modified hyaluronic acid series compound, the side chain of the repeating unit of the hyaluronic acid contains-COOH and/or-OH which is partially or totally modified to form the side chain with the end group of the following groups:

in the above groups, denotes a point of attachment;

R₁selected from hydrogen, halogen, aliphatic groups, aromatic groups, and the like; specifically, the halogen, aliphatic group and aromatic group satisfy the further definition below; preferably, R₁Selected from hydrogen, halogen, aliphatic radicals; also preferably, R₁Selected from hydrogen, halogen, C1-6 alkyl (e.g., methyl, ethyl, etc.);

R₂and R₃Identical or different, independently of one another, from the group consisting of hydrogen, halogen, aliphatic radicals, aromatic radicals, etc.; specifically, the halogen, aliphatic group and aromatic group satisfy the further definition below;

R₄is a multi-sulfhydryl compound fragment.

In a particular embodiment, the terminal group is attached to-COOH and/or-OH through an R group or directly to-COOH and/or-OH to form a side chain of at least one of the following structures:

in the structures a, b, c and d, R is selected fromAlkylene groups, arylene groups, amide residues, hydrazide residues, and the like; denotes a connection point;₁denotes the point of attachment to the left radical of R;₂denotes the point of attachment to the right-hand group of R; r₁、R₂、R₃And R₄The definition of (1) is as before.

Wherein the molecular weight of the thiol-modified hyaluronic acid ranges from five thousand to twenty million daltons. The molecular weight of the sulfhydryl modified hyaluronic acid is not changed greatly before and after modification, or the molecular weight is not changed basically.

Wherein the mercapto content of the mercapto-modified hyaluronic acid measured by the Ellman method is 0.01 to 30mmol/g, for example 0.1 to 10.0mmol/g, further for example 0.3 to 5.0mmol/g, further for example 0.5 to 3.0 mmol/g.

For example, the thiol-modified hyaluronic acid of the present invention comprises at least one of the following structures:

in the above structure, R, R₁、R₂、R₃And R₄The definition of (1) is as before; (n2+ n3)/(n1+ n2+ n3) represents the degree of acrylation; n3/(n1+ n2+ n3) represents the degree of sulfhydrylation, corresponding to the above-mentioned content of mercapto group in the mercapto-modified polymer compound measured by the Ellman method; the n1 may be 0, and if 0, the degree of acrylation is not limited, but n3/(n2+ n3) represents the degree of mercapto group, corresponding to the mercapto group content of the mercapto group-modified polymer compound measured by the Ellman method described above; the n2 may be 0, and if 0, n3/(n1+ n3) represents both the degree of acrylation and the degree of thiolation, and the mercapto content of the mercapto-modified polymer compound measured by Ellman is as followsCorrespondingly;

a is described₁The method comprises the following steps:

a is described₂Is one of the following structures:

A₁and A₂Denotes a point of attachment to COOH or OH.

Specifically, the sulfhydryl modified hyaluronic acid has at least one of the following structures, but is not limited to the following structures:

in the above structural formula, n₁、n₂And n₃The definition of (1) is as before.

As previously described, R₄Is a fragment of a polymercapto compound, e.g., -S-R₄the-SH segment may be introduced by the following, but not limited to, polythiol compounds:

wherein n4 is an integer of 2 to 30, for example, n is 2,3, 4,5 or 6; n5 is an integer of 1 to 30, for example, 1,2, 3, 4,5, etc.; n6 is an integer of 1 to 30, for example, 1,2, 3, 4,5, etc.;

4-arm-PEG-SH represents a PEG polymer containing four thiol groups; 6-arm-PEG-SH represents a PEG polymer containing six sulfhydryl groups; 8-arm-PEG-SH represents a PEG polymer containing eight thiol groups; the PEG is an abbreviation for polyethylene glycol.

[ terms and definitions ]

As previously described, R₁Selected from hydrogen, halogen, aliphatic groups, aromatic groups, and the like; r₂And R₃Identical or different, independently of one another, from the group consisting of hydrogen, halogen, aliphatic radicals, aromatic radicals, etc.

As previously mentioned, R may be selected from alkylene, arylene, amide residues, hydrazide residues and the like.

As previously mentioned, the R' may be selected from heteroatom containing groups, hydrocarbylene, arylene, and the like.

As previously mentioned, the R "may be selected from alkylene, arylene, and the like.

The halogen refers to fluorine, chlorine, bromine or iodine.

The aliphatic group is, for example, a linear or branched saturated/unsaturated aliphatic group, and specifically may be an alkyl group, an alkenyl group or an alkynyl group.

"hydrocarbyl" as used herein alone or as suffix or prefix, is for example a straight or branched chain saturated/unsaturated aliphatic radical, which may in particular be alkyl, alkenyl or alkynyl.

"alkyl" used herein alone or as suffix or prefix, is intended to include both branched and straight chain saturated aliphatic hydrocarbon groups having from 1 to 20, preferably from 1 to 6, carbon atoms. For example, "C_1-6Alkyl "denotes straight-chain and branched alkyl groups having 1,2, 3, 4,5 or 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.

"alkenyl" as used herein alone or as a suffix or prefix, is intended to include both branched and straight chain aliphatic hydrocarbon radicals containing alkenyl or alkene radicals having from 2 to 20, preferably 2-6, carbon atoms (or the particular number of carbon atoms if provided). For example, "C_2-6Alkenyl "denotes alkenyl having 2,3, 4,5 or 6 carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl, allyl, 1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methylbut-2-enyl, 3-methylbut-1-enyl, 1-pentenyl, 3-pentenyl, and 4-hexenyl.

"alkynyl" used herein alone or as a suffix or prefix is intended to include both branched and straight chain aliphatic hydrocarbon radicals containing alkynyl groups or alkynes having 2 to 20, preferably 2-6 carbon atoms (or the particular number of carbon atoms if provided). For example ethynyl, propynyl (e.g., l-propynyl, 2-propynyl), 3-butynyl, pentynyl, hexynyl and 1-methylpent-2-ynyl.

The aromatic group means an aromatic ring structure composed of 5 to 20 carbon atoms. For example: the aromatic ring structure containing 5, 6, 7 and 8 carbon atoms may be a monocyclic aromatic group such as phenyl; the ring structure containing 8, 9, 10, 11, 12, 13 or 14 carbon atoms may be polycyclic, for example naphthyl. The aromatic ring may be substituted at one or more ring positions with substituents such as alkyl, halogen, and the like, e.g., tolyl. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings"), wherein at least one of the rings is aromatic and the other cyclic rings can be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and/or heterocyclyl. Examples of polycyclic rings include, but are not limited to, 2, 3-dihydro-1, 4-benzodioxine and 2, 3-dihydro-1-benzofuran.

The "alkylene" in the present invention is a group obtained by removing one hydrogen from the above-mentioned "alkyl".

The "arylene" of the present invention is a radical obtained by removing one hydrogen from the above-mentioned "aromatic group".

The "alkylene" in the present invention is a group obtained by removing one hydrogen from the above-mentioned "alkyl".

The term "amido" as used herein alone or as suffix or prefix, refers to R^a-C (═ O) -NH-group, wherein R^aSelected from unsubstituted or optionally substituted by one or more R^bSubstituted of the following groups: alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, and the like; r^bSelected from unsubstituted or optionally substituted by one or more R^b1Substituted of the following groups: halogen, hydroxy, mercapto, nitro, cyano, alkyl, alkoxy, cycloalkyl, alkenyl, alkynyl, heterocyclyl, aryl, heteroaryl, amino, carboxy, ester, hydrazino, acyl, sulfinyl, sulfonyl, phosphoryl, and the like; each R^b1Independently of one another, from the group consisting of halogen, hydroxyl, alkyl, aryl.

The term "hydrazide" as used herein alone or as suffix or prefix, refers to R^a-C (═ O) -NH-group, wherein R^aThe definition of (1) is as before.

The "amide residue" in the present invention is a group obtained by removing one hydrogen from the "amide group".

The "hydrazide residue" in the present invention is a group obtained by removing one hydrogen from the "hydrazide group".

The term "cycloalkyl" as used herein is intended to include saturated cyclic groups having the specified number of carbon atoms. These terms may include fused or bridged polycyclic ring systems. Cycloalkyl groups have 3 to 40 carbon atoms in their ring structure. In one embodiment, the cycloalkyl group has 3, 4,5, or 6 carbon atoms in its ring structure. For example,“C_3-6cycloalkyl "denotes a group such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

The term "cycloalkenyl" as used herein is intended to include cyclic groups containing at least one alkenyl group having the specified number of carbon atoms. These terms may include fused or bridged polycyclic ring systems. Cycloalkenyl groups have 3 to 40 carbon atoms in their ring structure. In one embodiment, cycloalkenyl groups have 3, 4,5 or 6 carbon atoms in their ring structure. For example, "C_3-6Cycloalkenyl "denotes a group such as cyclopropenyl, cyclobutenyl, cyclopentenyl or cyclohexenyl.

The term "cycloalkynyl" as used herein is intended to include cyclic groups containing at least one alkynyl group having the specified number of carbon atoms. These terms may include fused or bridged polycyclic ring systems. Cycloalkynyl has 6 to 40 carbon atoms in its ring structure. In one embodiment, cycloalkynyl has 6 carbon atoms in its ring structure. For example, "C_3-6Cycloalkynyl "denotes radicals such as cyclopropynyl, cyclobutynyl, cyclopentynyl or cyclohexynyl.

As used herein, "heteroaryl" refers to a heteroaromatic heterocycle having at least one ring heteroatom (e.g., sulfur, oxygen, or nitrogen). Heteroaryl groups include monocyclic ring systems and polycyclic ring systems (e.g., having 2,3, or 4 fused rings). Examples of heteroaryl groups include, but are not limited to, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolinyl, isoquinolinyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrrolyl, oxazolyl, benzofuryl, benzothienyl, benzothiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2, 4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, benzoxazolyl, azabenzoxazolyl, imidazothiazolyl, benzo [1,4] dioxanyl, benzo [1,3] dioxolyl, and the like. In some embodiments, heteroaryl groups have from 3 to 40 carbon atoms and in other embodiments from 3 to 20 carbon atoms. In some embodiments, heteroaryl groups contain 3 to 14, 4 to 14, 3 to 7, or 5 to 6 ring-forming atoms. In some embodiments, heteroaryl has 1 to 4, 1 to 3, or 1 to 2 heteroatoms. In some embodiments, the heteroaryl group has 1 heteroatom.

The term "heterocyclyl" as used herein, refers to a saturated, unsaturated or partially saturated monocyclic, bicyclic or tricyclic ring containing from 3 to 20 atoms, wherein 1,2, 3, 4 or 5 ring atoms are selected from nitrogen, sulfur, oxygen or phosphorus, which, unless otherwise specified, may be attached through carbon or nitrogen, wherein-CH₂-the group is optionally replaced by-c (o) -; wherein unless otherwise stated to the contrary, the ring nitrogen atom or the ring sulfur atom is optionally oxidized to form an N-oxide or S-oxide or the ring nitrogen atom is optionally quaternized; wherein-NH in the ring is optionally substituted with acetyl, formyl, methyl or methanesulfonyl; and the ring is optionally substituted with one or more halogens. It is understood that when the total number of S and O atoms in the heterocyclic group exceeds 1, these heteroatoms are not adjacent to each other. If the heterocyclyl is bicyclic or tricyclic, at least one ring may optionally be a heteroaromatic ring or an aromatic ring, provided that at least one ring is non-heteroaromatic. If the heterocyclic group is monocyclic, it is not necessarily aromatic. Examples of heterocyclyl groups include, but are not limited to, piperidinyl, N-acetylpiperidinyl, N-methylpiperidinyl, N-formylpiperazinyl, N-methylsulfonylpiperazinyl, homopiperazinyl, piperazinyl, azetidinyl, oxetanyl, morpholinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, indolinyl, tetrahydropyranyl, dihydro-2H-pyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydrothiopyran-1-oxide, tetrahydrothiopyran-1, 1-dioxide, 1H-pyridin-2-one, and 2, 5-dioxoimidazolidinyl.

The term "acyl" as used herein means R^a-C (═ O) -group, wherein R^aThe definition of (1) is as before.

The term "sulfinyl" as used herein means R^a-S (═ O) -group, wherein R^aThe definition of (1) is as before.

The term "sulfonyl" as used herein means R^a-S(＝O)₂A group in which R^aThe definition of (1) is as before.

The term "phosphoryl" as used herein means R^c-P(＝O)(R^d) A group in which R^cAnd R^dIdentical or different, independently of one another, from the group consisting of unsubstituted or optionally substituted by one or more R^bSubstituted of the following groups: alkyl, cycloalkyl, alkoxy, hydroxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, and the like, R^bThe definition of (1) is as before.

The term "hydrazino" as used herein means-NHNHR^aGroup, R^aThe definition of (1) is as before.

The term "amino" as used herein refers to-NHR^aA group or-N (R)^a)₂Group, R^aThe definition of (1) is as before.

The term "amino" as used herein means-NH₂A group.

The term "carboxyl" as used herein refers to the-COOH group.

The term "ester group" as used herein means R^a-C (═ O) -O-groups or R^a-O-C (═ O) -group, wherein R^aThe definition of (1) is as before.

[ Process for producing mercapto-modified Polymer ]

As described above, the present invention provides a method for preparing the thiol-modified polymer compound, which comprises the following steps:

1) structurally containing-COOH, -NH₂A step of acrylating at least one of-OH and-NH-COOH in the structure of the polymer compound₂At least one of-OH is directly or indirectly linked to:

R₁、R₂and R₃The definition of (1) is as before; denotes a connection point;

or a polymer compound structurally containing at least one of the acrylate group represented by the formula a, the acrylamide group represented by the formula b and the acryloyl group represented by the formula c is directly used as a reaction raw material;

2) at least one of the macromolecular compounds obtained in the step 1) and a multi-sulfhydryl compound HS-R₄-SH reaction, R₄The thiol-modified macromolecular compound is prepared as defined above.

In one embodiment of the invention, the method comprises the steps of:

1) structurally containing-COOH, -NH₂A step of acrylating at least one of-OH and-NH-COOH in the structure of the polymer compound₂At least one of-OH is linked via an-R-group to or directly to:

R、R₁、R₂and R₃As before, denotes a connection point;

or the macromolecular compound structurally containing at least one of the acrylate group shown in the formula a, the acrylamide group shown in the formula b and the acryloyl group shown in the formula c is directly used as a reaction raw material;

2) at least one of the macromolecular compounds obtained in the step 1) and a multi-sulfhydryl compound HS-R₄-SH reaction, R₄The thiol-modified macromolecular compound is prepared as defined above.

In one embodiment of the present invention, there is provided a method for preparing the thiol-modified hyaluronic acid, comprising the steps of:

1) the step of acrylation of hyaluronic acid, namely, directly or indirectly connecting at least one of-COOH and-OH contained on the side chain of the repeating unit of hyaluronic acid with the following groups:

R₁、R₂and R₃Is as defined inBefore; denotes a connection point;

2) reaction of acrylated hyaluronic acid with a polymercapto compound HS-R₄-SH reaction, R₄The thiol-modified hyaluronic acid is prepared as described above.

Specifically, the step 1) is as follows: the acrylation step of hyaluronic acid is that at least one of-COOH and-OH contained in the side chain of the repeating unit of hyaluronic acid is connected with the terminal group through an R group, or is directly connected with the terminal group to form the side chain of at least one of the following structures:

r, R in the above-mentioned a structure, b structure, c structure and d structure₁、R₂、R₃And R₄The definition of (1) is as before; denotes the connection point.

In the step 1), the acrylation step may be realized by the reaction of the polymer compound to be modified and the acrylate compound, or may be realized by the reaction of the polymer compound to be modified and the acryloyl chloride compound or the acrylic anhydride compound.

The acrylate compound can be one or more of alkyl acrylate compounds, aryl acrylate compounds and glycidyl acrylate polyol compounds.

The polyhydric alcohol in the glycidyl acrylate compound is, for example, a trihydric alcohol, and specifically, glycerin, butanetriol, pentanetriol, and the like can be mentioned.

In the step 1), the acrylation step may be a conventional reaction step, and may be a reaction under conventional conditions. Typically acryloyl chloride and derivatives thereof or acrylic anhydride and derivatives thereof with a compound containing-OH, -NH₂At least one of the above-mentioned high molecular weight compounds. Or can also be usedIs glycidyl acrylate and derivatives thereof and contains-COOH, -OH and-NH₂At least one of the above-mentioned high molecular weight compounds.

In step 1), the acrylation step may be an unconventional reaction step, i.e., a high molecular compound containing a structure of formula c synthesized by a method other than the above-mentioned method is used.

In step 2), with a polymercapto compound HS-R₄the-SH reaction is carried out in a solvent. The solvent is, for example, water or an organic solvent, and further may be deionized water or dimethylformamide.

In step 2), with a polymercapto compound HS-R₄the-SH reaction is carried out under low to high temperature conditions. For example, the reaction temperature is from 0 to 80 ℃ and further from 10 to 70 ℃ and, for example, the reaction can be carried out at room temperature.

In step 2), with a polymercapto compound HS-R₄The reaction time of the-SH reaction is 0.1 to 100 hours.

In step 2), with a polymercapto compound HS-R₄The pH range of the-SH reaction is from-1 to 15. For example, the reaction pH may be 6 to 8, and further, for example, 7.

Wherein the reaction product of step 2) further comprises a post-treatment step.

Wherein the post-treatment step employs a dialysis method. Specifically, the solution after the reaction is filled into a dialysis bag (for example, a dialysis bag with a molecular weight cutoff of 2kDa or more), dialyzed with a hydrochloric acid solution (for example, pH 4) for several days (for example, 1 to 10 days, further, for example, 5 days, etc.), optionally changed with water (for example, changed with water every day or changed with water every other day) for several times (for example, two times or more, etc.), and finally the solution in the dialysis bag is collected and dried (for example, freeze-dried) to obtain a solid or viscous liquid, that is, the thiol-modified polymer compound.

The method firstly provides the Michael addition reaction of the sulfydryl of the multi-sulfydryl compound and the carbon-carbon double bond in the acryloyl group to prepare the sulfydryl modified polymer compound, the method has high sulfydryl degree, mild sulfydryl reaction conditions (which can be carried out at normal temperature in aqueous solution), no pollution, high purity of the prepared sulfydryl modified polymer compound, and is particularly suitable for further use in the fields of medicine, beauty treatment, medicine and the like.

[ Acryloylated Polymer Compound ]

As described above, the system to be gelled according to the present invention may further include a substance c1. an acrylated polymeric compound, and the acrylated polymeric compound according to the present invention may be selected from at least one of the following substances:

1) structurally containing-COOH, -NH₂And (C) at least one of-OH and-NH-COOH in the structure of the polymer compound₂An acryloylated compound formed by linking, directly or indirectly, at least one of-OH and:

R₁、R₂and R₃As before, denotes a connection point;

2) a polymer compound structurally containing at least one of an acrylate group represented by formula a, an acrylamide group represented by formula b and an acrylamide group represented by formula c.

The aforementioned substance of the 1) above, wherein the-COOH and/or-NH₂And/or some or all of the-OH groups are modified to form at least one of the following structures:

in the above structure, R is selected fromAlkylene groups, arylene groups, amide residues, hydrazide residues, and the like; denotes a connection point;₁denotes the point of attachment to the left radical of R;₂denotes the point of attachment to the right-hand group of R; r₁、R₂、R₃And R₄The definition of (1) is as before.

In the above substance 1), the group-COOH, -NH₂At least one of-OH and-OH may be directly bonded to the main chain of the polymer compound or may be bonded to the main chain of the polymer compound through a group R ', wherein R' may be a group containing a hetero atom, an alkylene group, an arylene group, or the following linking groups:

in the above formula, R 'is alkylene or arylene, n' is an integer of 1 to 1000, and represents a connection point.

Wherein the heteroatom containing group includes, but is not limited to: ester, amide or hydrazide residues. In particular, the ester, amide or hydrazide residue meets the further definitions herein.

In the above substance of the 1) above, the polymer compound to be acrylated comprises a natural mucopolysaccharide polymer, such as at least one of chitosan (specifically chitosan, glycol chitosan, carboxymethyl chitosan, etc.), chondroitin sulfate, hyaluronic acid, alginate, etc.; proteins such as gelatin, fibrin, serum proteins, and the like; and/or, a synthetic polymer, such as at least one of polyvinyl alcohol, poly (meth) acrylic acid, poly (hydroxy alkyl (meth) acrylate) (e.g., poly (hydroxyethyl (meth) acrylate), etc.), hyperbranched polyethylene glycol, and the like.

In the above substance 1), the acryloylated compound includes at least one of the following structures:

in the above structure, A is the structure containing at least one-COOH, -NH₂-OH, -a fragment of the compound to be acryloylated; r, R' and R₁、R₂、R₃And R₄The definition of (1) is as before; (m2/(m1+ m2) represents the degree of acrylation.

Specifically, a may be a structure as shown below:

in each of the above structures, denotes a connecting point between the main chain repeating units; represents-COOH, -NH₂The point of attachment between-OH and the above-mentioned fragment, or through the point of attachment between the R' group and the above-mentioned fragment.

The above 2) substance may be one of the following polymers Gelatin-A, Gelatin-MA, CTS-A, CTS-MA, PHEMA-A, PHEMA-MA, HB-PEG, PVA-A, PVA-MA, CHS-A or CHS-MA:

it should be noted that Gelatin-A, Gelatin-MA, CTS-A, CTS-MA, PHEMA-A, PHEMA-MA, HB-PEG, PVA-A, PVA-MA, CHS-A or CHS-MA are abbreviations for the names of polymers having the above structures, respectively, and the letters therein are not related to the letter meanings appearing in other parts of the present invention after being separated.

[ Small-molecule crosslinking agent ]

As mentioned above, substance C2. may also be included in the system to be gelled of the present invention as a small molecule cross-linking agent containing acryloyl groups, including but not limited to small molecule compounds containing acryloyl groups or oligomers containing acryloyl groups; specifically, the acrylate may be selected from ethylene glycol diacrylate EGDA, polyethylene glycol diacrylate PEGDA, trimethylolpropane triacrylate TMPTA, pentaerythritol triacrylate PTA, pentaerythritol tetraacrylate PTTA, ditrimethylolpropane tetraacrylate DTTA, etc.

[ hydrogel ]

As described above, the present invention provides a hydrogel produced by gelation of a system containing:

(i) the mercapto-modified polymer compound, and

(ii) at least one of substance C1 and substance C2.

In one embodiment of the present invention, the hydrogel is prepared by gelling the thiol-modified polymer compound and the acrylated polymer compound.

Wherein, the mercapto-modified macromolecular compound and the acrylic acylated macromolecular compound are fully contacted to generate a crosslinking reaction, the viscosity of the mixed system is increased immediately, and finally a uniform gel system is formed.

In one embodiment of the present invention, the hydrogel is prepared by gelling the thiol-modified polymer compound and the small-molecule crosslinking agent.

Wherein, the mercapto-modified macromolecular compound and the micromolecule cross-linking agent are fully contacted to generate cross-linking reaction, the viscosity of the mixed system is increased immediately, and finally a uniform gel system is formed.

In one embodiment of the present invention, the hydrogel is prepared by gelation of the mercapto group-modified polymer compound, the acryloyl polymer compound, and the small molecule crosslinking agent.

Wherein, the mercapto-modified macromolecular compound, the acryloyl macromolecular compound and the micromolecule cross-linking agent are fully contacted to generate cross-linking reaction, the viscosity of the mixed system is increased immediately, and finally, a uniform gel system is formed.

Wherein the hydrogel comprises the following characteristic structural units:

in the above unit, R₁、R₂、R₃And R₄As before, denotes the connection point.

Wherein the amount ratio (1 part by mass in total) of the mercapto-modified polymer compound to the acryl-acylated polymer compound is 0.01:0.99 to 0.99: 0.01. For example, it may be 0.1:0.9 to 0.9:0.1, such as 0.01:0.99, 0.1:0.9, 0.15:0.85, 0.2:0.8, 0.3:0.7, 0.4:0.6, 0.5:0.5, 0.6:0.4, 0.7:0.3, 0.8:0.2, 0.85:0.15, 0.9:0.1, 0.99:0.01 or any ratio within the interval.

Wherein the using amount ratio (1 part by mass in total) of the mercapto-modified high molecular compound to the small molecular cross-linking agent is 0.01: 0.99-0.99: 0.01. For example, it may be 0.1:0.9 to 0.9:0.1, such as 0.01:0.99, 0.1:0.9, 0.15:0.85, 0.2:0.8, 0.3:0.7, 0.4:0.6, 0.5:0.5, 0.6:0.4, 0.7:0.3, 0.8:0.2, 0.85:0.15, 0.9:0.1, 0.99:0.01 or any ratio within the interval.

Wherein the using amount ratio (1 part by mass in total) of the mercapto-modified high molecular compound, the acryloyl high molecular compound and the small molecular cross-linking agent is 0.01: 0.99-0.99: 0.01. For example, it may be 0.1:0.9 to 0.9:0.1, such as 0.01:0.99, 0.1:0.9, 0.15:0.85, 0.2:0.8, 0.3:0.7, 0.4:0.6, 0.5:0.5, 0.6:0.4, 0.7:0.3, 0.8:0.2, 0.85:0.15, 0.9:0.1, 0.99:0.01 or any ratio within the interval. Wherein, the acryloyl macromolecular compound and the micromolecule cross-linking agent can be mixed in any proportion.

The hydrogel is a stable crosslinking material formed by the addition reaction of a thiol group (-SH) of a thiol-modified high molecular compound and a carbon-carbon double bond of a substance C1 and/or a substance C2, and the crosslinking material (namely the hydrogel) has excellent mechanical properties and better physical stability and mechanical strength; in addition, the rate of metabolism in vivo is controllable. If two substances, namely C1 and C2, are introduced into the system at the same time, C2 (small molecular cross-linking agent) can participate in the cross-linking reaction of the sulfhydryl-modified macromolecular compound and C1 (acryloyl macromolecular compound), namely, the three are cross-linked together to form a stable cross-linked material. Meanwhile, the substance C1 can also be added into the gel system in a physical mixing mode, thereby achieving different application purposes. The sulfhydryl modified macromolecular compound is matched with the C1 (acrylic acylated macromolecular compound) and/or C2 (micromolecular cross-linking agent) for use, and makes up for each other, so that the three-dimensional scaffold material with excellent properties is obtained, and the application requirements of most tissue engineering can be met.

In one embodiment of the present invention, at least one of other biofunctional materials (such as hyaluronic acid, collagen, gelatin, chondroitin sulfate, chitosan, sodium alginate, etc.), drugs, growth factors, or cell suspensions, etc. may be further added to the system. The hydrogel can bring additional effect to the hydrogel by adding other biological functional materials, for example, the introduction of unmodified hyaluronic acid can increase the wound healing promotion effect of the hydrogel, the introduction of collagen or gelatin can enable the hydrogel system to be closer to the composition of organism soft tissues, the introduction of chondroitin sulfate can enhance the cartilage repair promotion effect of the hydrogel system, the introduction of positively charged biological materials such as chitosan and the like can increase the antibacterial effect of the hydrogel, and the introduction of sodium alginate can enhance the mechanical strength of the hydrogel system.

[ preparation of hydrogel ]

As described above, the present invention provides a method for preparing the above hydrogel, which comprises the steps of:

gelling a system comprising:

(i) the mercapto group-modified polymer compound, and

(ii) at least one of substance C1 and substance C2;

the hydrogel is prepared.

In a particular embodiment, the method comprises the steps of: gelling a system comprising:

(a) the thiol-modified polymer compound is a polymer compound,

(b) at least one of the following: C1. the acryloyl macromolecular compound, C2. micromolecule cross-linking agent containing acryloyl,

(c) optionally at least one of: other biofunctional materials, pharmaceuticals, growth factors and cell suspensions;

the hydrogel is prepared.

The method comprises the following steps: respectively preparing a solution of the sulfhydryl modified macromolecular compound, a solution of the acryloyl macromolecular compound, a solution of the micromolecule cross-linking agent and optionally at least one solution of other biological functional materials, medicines, growth factors or cell suspensions, mixing and gelling to prepare the hydrogel. In addition, at least one of the other biofunctional material, the drug, the growth factor, or the cell suspension may be introduced by directly adding the substance to the solution of the thiol-modified polymer compound, the solution of the acrylated polymer compound, or the solution of the small molecule cross-linking agent.

The gel preparation process can be realized by adding the sulfhydryl modified macromolecular compound solution into the acryloyl macromolecular compound solution and/or the small molecule cross-linking agent solution, or adding the acryloyl macromolecular compound solution and/or the small molecule cross-linking agent solution into the sulfhydryl modified macromolecular compound solution. Specifically, the two solutions can be mixed by a common syringe, a double-needle syringe, or other means.

The solution of the thiol-modified polymer compound may have a mass volume concentration of 0.1% to 95%, for example, 1% to 90%, and further, for example, 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%. The pH of the solution may be adjusted to 7.4 by the addition of acids, bases or buffer solutions. The buffer solution may be a phosphate buffer.

The mass volume concentration of the solution of the acryloylated high molecular compound is 0.1% to 95%, for example, 1% to 90%, and further, for example, 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%. The pH of the solution may be adjusted to 7.4 by the addition of acids, bases or buffer solutions. The buffer solution may be a phosphate buffer.

Wherein the mass volume concentration of the solution of the small molecule cross-linking agent is 0.1% to 95%, for example, 1% to 90%, and further for example, 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%. The pH of the solution may be adjusted to 7.4 by the addition of acids, bases or buffer solutions. The buffer solution may be a phosphate buffer.

The two solutions may be mixed in any ratio, for example, in equal volumes.

[ use of hydrogel ]

Hydrogels are known as a class of three-dimensional lattice of hydrophilic polymer segments that swell in water by crosslinking. The gelation process can be achieved by different reaction mechanisms including physical entanglement of polymer segments, electrostatic interaction, covalent chemical crosslinking, reversible chemical crosslinking, supramolecular chemical crosslinking, hydrophilic-hydrophobic interaction crosslinking, and the like. In recent years, with intensive research on the function of hydrogels, hydrogels have been widely used in the medical field, such as for preparing drug delivery systems, dressings for soft tissue wound repair, scaffold materials for bone repair, viscoelastic agents for supporting in ophthalmic surgery, materials for preventing tissue adhesion after surgery, and scaffold materials for 3D bioprinting, and the like, which has become a research hotspot in the fields of tissue engineering and regenerative medicine.

The hydrogel is particularly suitable for the fields of biological medicine, medical cosmetology and cosmetics and the like. In particular, it can be used for preparing drug delivery systems, dressings for soft tissue wound repair, scaffold materials for bone repair, viscoelastic agents for supporting in ophthalmic surgery, materials for preventing tissue adhesion after surgery, scaffold materials for 3D bioprinting, and the like.

The hydrogel realizes in-situ crosslinking under physiological conditions in the true sense, namely, the crosslinking reaction can be spontaneously completed under the conditions of room temperature and normal pressure; or after the hydrogel is injected into tissues of an animal body or a human body, the crosslinking reaction can still be realized in the tissues, so that the degradation resistance and the metabolism resistance of the hydrogel product are obviously improved, and the use effect of the hydrogel injection product is obviously improved. And due to the unique technology of the invention, the controllability of the crosslinking degree of the hydrogel product injected into the animal body or the human body can be realized before the in vitro crosslinking or mixing stage, namely, the crosslinking reaction with controllable crosslinking reaction end point can be realized after the hydrogel product is injected into the animal body or the human body, and the safety and the treatment effect of the product are ensured.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that various changes or modifications can be made by those skilled in the art after reading the disclosure of the present invention, and such equivalents also fall within the scope of the invention.

In the present invention, the¹The H-NMR spectrum is measured by a Varian 400MHz NMR spectrometer, the test temperature is 25 ℃, the relaxation time is 1 second, and the scanning times are 8. Specifically, 8-10 mg of the sample to be detected is taken and dissolved in 750 μ l of deuterated water, and the obtained sample solution is tested¹H-NMR spectrum.

The storage modulus is determined based on the rheological mechanical property of hydrogel, specifically, a detection instrument is a TA-DHR2 rheometer, a detection probe is a 20mm parallel plate probe, and the detection temperature is as follows: 25 ℃, shear frequency: 1Hz, shear strain: 1 percent.

The macromolecular compounds of the invention were tested for their cellular activity and biocompatibility with reference to the "GBT 16886.5-2017 medical device biological evaluation + part 5 + in vitro cytotoxicity assay" standard. Specifically, the following MTT method is a method for measuring the survival rate of cells by metabolic activity. MTT [3- (4, 5-dimethylthiozol-2-yl) -2,5-diphenyltetrazolium bromide ] in yellow aqueous solution is metabolically reduced in living cells to generate blue-violet insoluble formazan. The number of viable cells correlates with the color of formazan after it has dissolved in alcohol and is measured photometrically.

Preparation example 1 Synthesis of acrylate-modified hyaluronic acid (abbreviated as HA-A1)

In a 200 ml beaker, 1 g hyaluronic acid (purchased from Huaxi Furuida, having a weight average molecular weight of about 300kDa), 50 ml deionized water, 50 ml dimethylformamide, 12 ml triethylamine, 14 ml glycidyl acrylate were added. After stirring at room temperature until uniform and transparent, stirring was continued for 48 hours. Addition of 300 ml of acetone produced a large amount of white precipitate. The precipitate obtained by centrifugation was dissolved in 100 ml of deionized water to give a colorless transparent solution. The above solution was filled into dialysis bags (molecular weight cut-off 8kDa) and dialyzed against 5 liters of deionized water for 5 days with two water changes per day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 921 mg of white flocculent solid, i.e. HA-A1, with a yield of 92.1%.

The structural formula of HA-A1 is shown in figure 15. Fig. 15 is a schematic diagram only, showing that COOH in the hyaluronic acid partial repeating unit is esterified with glycidyl acrylate, i.e. where m2/(m1+ m2) represents the degree of acrylation, m1+ m2 ═ n, and n is the number of repeating units of unmodified hyaluronic acid. The structural formulae in the following preparation examples and examples have the same meanings as in preparation example 1, and are not repeated.

Of HA-A1¹The H-NMR spectrum is shown in FIG. 15, where a nuclear magnetic peak belonging to the acrylic function, located between 6 and 6.5ppm, is visible, confirming the successful grafting of this group into the structure of the hyaluronic acid.

Preparation example 2 Synthesis of acrylate-modified hyaluronic acid (abbreviated as HA-A2)

In a 200 ml beaker, 1 g of hyaluronic acid (purchased from Huanxifurada, having a weight average molecular weight of about 400kDa), 50 ml of deionized water, 50 ml of dimethylformamide, and 6.3 g of acrylic anhydride were added and dissolved with stirring. The solution pH was maintained at 8 ± 0.5 with 1 mol NaOH per liter and stirring was continued for 24 hours. Addition of 300 ml of acetone produced a large amount of white precipitate. The precipitate obtained by centrifugation was dissolved in 100 ml of deionized water to give a colorless transparent solution. The above solution was filled into dialysis bags (molecular weight cut-off 8kDa, Shibichun) and dialyzed against 5 liters of deionized water for 5 days with two water changes per day. Finally, collecting the solution in the dialysis bag, and freeze-drying to obtain 789 mg of white flocculent solid, namely HA-A2, with the yield of 78.9%.

The structural formula of HA-A2 is shown in figure 16.

Of HA-A2¹The H-NMR spectrum is shown in FIG. 16, where a nuclear magnetic peak belonging to the acrylic function, between 5.8 and 6.4ppm, is visible, confirming the successful grafting of this group into the structure of the hyaluronic acid.

Preparation example 3 Synthesis of methacrylate-modified hyaluronic acid (abbreviated as HA-MA1)

In a 200 ml beaker, 1 g hyaluronic acid (purchased from Huanxifurida, having a weight average molecular weight of about 400kDa), 50 ml deionized water, 50 ml dimethylformamide (Sigma), 12 ml triethylamine (Sigma), 15 ml glycidyl methacrylate were added. After stirring at room temperature until uniform and transparent, stirring was continued for 48 hours. 300 ml of acetone (Sigma) was added, resulting in a large white precipitate. The precipitate obtained by centrifugation was dissolved in 100 ml of deionized water to give a colorless solution. The above solution was filled into dialysis bags (molecular weight cut-off 8kDa, Shibichun) and dialyzed against 5 liters of deionized water for 5 days with two water changes per day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 859 mg of white flocculent solid, i.e. HA-MA1, with a yield of 85.9%.

The structural formula of HA-MA1 is shown in figure 17.

Of HA-MA1¹The H-NMR spectrum is shown in FIG. 17, where a nuclear magnetic peak belonging to the methacrylic function, located between 5.8 and 6.2ppm, is visible, confirming the successful grafting of this group into the structure of the hyaluronic acid.

Preparation example 4 Synthesis of methacrylate-modified hyaluronic acid (abbreviated as HA-MA2)

In a 200 ml beaker, 1 g of hyaluronic acid (purchased from Huaxi Furuida, having a weight average molecular weight of about 400kDa) and 100 ml of deionized water were added and dissolved with stirring at room temperature. Further, 7.7 g of methacrylic anhydride was added thereto and dissolved by stirring. The solution pH was maintained at 8 ± 0.5 with 1 mol NaOH per liter and stirring was continued for 24 hours. Addition of 200 ml of acetone (Sigma) produced a large white precipitate. The precipitate obtained by centrifugation was dissolved in 100 ml of deionized water to give a colorless transparent solution. The above solution was filled into dialysis bags (molecular weight cut-off 8kDa, Shibichun) and dialyzed against 5 liters of deionized water for 5 days with two water changes per day. Finally, the solution in the dialysis bag was collected and lyophilized to give 846 mg of white flocculent solid, HA-MA2, with a yield of 84.6%.

The structural formula of HA-MA2 is shown in figure 18.

Of HA-MA2¹The H-NMR spectrum is shown in FIG. 18, where a nuclear magnetic peak belonging to the methacrylic function, located between 5.8 and 6.2ppm, is visible, confirming that this group is presentThe clusters were successfully grafted into the structure of hyaluronic acid.

Preparation example 5 Synthesis of thiol-acrylate-modified hyaluronic acid (abbreviated as HA-A1-SH1)

In a 200 ml beaker, 1 g of HA-A1 prepared according to preparation example 1, 0.3 g of dithiothreitol (available from VWR corporation) and 100 ml of deionized water were added and dissolved with stirring at room temperature to obtain a transparent solution. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 842 mg of white flocculent solid, HA-A1-SH1 with a yield of 84.2%.

The reaction equation of HA-A1-SH1 is shown in FIG. 1, and the structural formula is shown in FIG. 1 and FIG. 25.

Of HA-A1-SH1¹The H-NMR spectrum is shown in FIG. 25, and a nuclear magnetic peak belonging to the side chain of the thiol group between 2.3 and 2.8ppm can be seen, thus proving that the thiol group is successfully grafted into the structure of the hyaluronic acid.

Preparation example 6 Synthesis of thiol-acrylate-modified hyaluronic acid (abbreviated as HA-A2-SH1)

In a 200 ml beaker, 1 g of HA-A2 prepared by the method of preparation example 2, 0.3 g of dithiothreitol (VWR), and 100 ml of deionized water were added and dissolved with stirring at room temperature to obtain a transparent solution. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 827 mg of white flocculent solid, HA-A2-SH1 with 82.7% yield.

The reaction equation of HA-A2-SH1 is shown in FIG. 2, and the structural formula is shown in FIG. 2 and FIG. 26.

Of HA-A2-SH1¹The H-NMR spectrum is shown in FIG. 26, and a nuclear magnetic peak belonging to the side chain of the thiol group between 2.6 and 2.9ppm can be seen, which proves the successful grafting of the thiol group into the structure of hyaluronic acid.

Preparation example 7 Synthesis of thiol-methacrylate-modified hyaluronic acid (abbreviated as HA-MA1-SH1)

In a 200 ml beaker, 1 g of HA-MA1 prepared by the method of preparation example 3, 0.3 g of dithiothreitol (VWR), and 100 ml of deionized water were added and dissolved with stirring at room temperature. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain about 854 mg of white flocculent solid. Thus obtaining HA-MA1-SH1 with the yield of 85.4%.

The reaction equation of HA-MA1-SH1 is shown in FIG. 3, and the structural formula is shown in FIG. 3 and FIG. 27.

Of HA-MA1-SH1¹The H-NMR spectrum is shown in FIG. 27, and a nuclear magnetic peak belonging to the side chain of the thiol group between 2.6 and 3.0ppm can be seen, thus proving the successful grafting of the thiol group into the structure of hyaluronic acid.

Preparation example 8 Synthesis of thiol-methacrylate-modified hyaluronic acid (abbreviated as HA-MA2-SH1)

In a 200 ml beaker, 1 g of HA-MA2 prepared by the method of preparation example 4, 0.3 g of dithiothreitol (VWR), and 100 ml of deionized water were added and dissolved with stirring at room temperature. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain about 833 mg of white flocculent solid. Thus, HA-MA2-SH1 was obtained with a yield of 83.3%.

The reaction equation of HA-MA2-SH1 is shown in FIG. 4, and the structural formula is shown in FIG. 4 and FIG. 28.

Of HA-MA2-SH1¹The H-NMR spectrum is shown in FIG. 28, and a nuclear magnetic peak belonging to the side chain of the thiol group between 2.6 and 3.0ppm can be seen, thus proving that the thiol group is successfully grafted into the structure of the hyaluronic acid.

Preparation example 9 Synthesis of acrylate-modified chondroitin sulfate (CHS-A for short)

A200 ml beaker was charged with 1.2 g of chondroitin sulfate (having a weight average molecular weight of about 80kDa), 50 ml of deionized water, 50 ml of dimethylformamide, and 5.4 g of acrylic anhydride, and dissolved with stirring. The solution pH was maintained at 8 ± 0.5 with 1 mol NaOH per liter and stirring was continued for 24 hours. The above solution was filled into dialysis bags (cut-off 3.5kDa, Shibichun) and dialyzed against 5 liters of deionized water for 5 days with two water changes per day. Finally, the solution in the dialysis bag was collected and lyophilized to give 781 mg of pale yellow flocculent solid, i.e., CHS-A, yield 65.1%.

The structural formulA of CHS-A is shown in figure 19.

Of CHS-A¹The H-NMR spectrum is shown in FIG. 19, where a nuclear magnetic peak of the acrylic function is visible, located between 6.0 and 6.5ppm, confirming the successful grafting of this group into the structure of chondroitin sulphate.

Preparation example 10 Synthesis of methacrylate-modified chondroitin sulfate (CHS-MA for short)

A200 ml beaker was charged with 1.2 g of chondroitin sulfate (having a weight average molecular weight of about 90kDa), 50 ml of deionized water, 50 ml of dimethylformamide, and 6.5 g of methacrylic anhydride, and dissolved by stirring. The solution pH was maintained at 8 ± 0.5 with 1 mol NaOH per liter and stirring was continued for 24 hours. The above solution was filled into dialysis bags (cut-off 3.5kDa, Shibichun) and dialyzed against 5 liters of deionized water for 5 days with two water changes per day. Finally, the solution in the dialysis bag was collected and lyophilized to give 776 mg of pale yellow flocculent solid, i.e., CHS-MA, yield 64.7%.

The structural formula of CHS-MA is shown in figure 20.

Of CHS-MA¹The H-NMR spectrum is shown in FIG. 20, where a nuclear magnetic peak belonging to the methacrylic function is visible, between 6.0 and 6.5ppm, confirming the successful grafting of this group into the structure of chondroitin sulphate.

Preparation example 11 synthetic acrylate-modified Gelatin (Gelatin-A for short)

In a 200 ml beaker, 1 g of gelatin (strength 300Blooms), 50 ml of deionized water, 50 ml of dimethylformamide and further 10 g of acrylic anhydride were added and dissolved with stirring. The solution pH was maintained at 8 ± 0.5 with 1 mol NaOH per liter and stirring was continued for 24 hours. The above solution was filled into dialysis bags (molecular weight cut-off 8kDa, Shibichun) and dialyzed against 5 liters of deionized water for 5 days with two water changes per day. Finally, the solution in the dialysis bag was collected and lyophilized to give 781 mg of a pale yellow flocculent solid, Gelatin-A, yield 78.1%.

The structural formula of Gelatin-A is shown in FIG. 21 (wherein the wavy line represents the main chain of Gelatin).

Of Gelatin-A¹The H-NMR spectrum is shown in FIG. 21, where a nuclear magnetic peak belonging to the acrylic function, located between 6.0 and 6.5ppm, is visible, confirming the successful grafting of this group into the structure of the gelatin.

Preparation example 12 Synthesis of methacrylate-modified Gelatin (Gelatin-MA)

In a 200 ml beaker, 1 g of gelatin (strength 300Blooms), 50 ml of deionized water, 50 ml of dimethylformamide and further 10 g of methacrylic anhydride were added and dissolved with stirring. The solution pH was maintained at 8 ± 0.5 with 1 mol NaOH per liter and stirring was continued for 24 hours. The above solution was filled into dialysis bags (molecular weight cut-off 8kDa, Shibichun) and dialyzed against 5 liters of deionized water for 5 days with two water changes per day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 824 mg of pale yellow flocculent solid, Gelatin-MA, yield 82.4%.

The structural outline of Gelatin-MA is shown in FIG. 22 (wherein the wavy line represents the main chain of Gelatin).

Of Gelatin-MA¹The H-NMR spectrum is shown in FIG. 22, where a nuclear magnetic peak belonging to the methacrylic function is visible, located between 5.7 and 6.2ppm, confirming the successful grafting of this group into the structure of the gelatin.

Preparation example 13 Synthesis of acrylate-modified ethylene glycol Chitosan (CTS-A for short)

In a 200 ml beaker, 1 g of ethylene glycol chitosan (with a weight average molecular weight of about 250kDa), 50 ml of deionized water, 50 ml of dimethylformamide, 8 ml of triethylamine (Sigma), 13 ml of glycidyl acrylate were added. After stirring at room temperature until uniform and transparent, stirring was continued for 48 hours. The above solution was filled into dialysis bags (cut-off 3.5kDa, Shibichun) and dialyzed against 5 liters of deionized water for 5 days with two water changes per day. Finally, the solution in the dialysis bag was collected and lyophilized to give 694 mg of light yellow flocculent solid, CTS-A, yield 69.4%.

The structure of CTS-A is shown in figure 23.

Of CTS-A¹The H-NMR spectrum is shown in FIG. 23, where a nuclear magnetic peak belonging to the acrylic function is visible between 5.8 and 6.4ppm, confirming the successful grafting of this group to ethanediolThe structure of alcohol chitosan.

Preparation example 14 Synthesis of methacrylate-modified ethylene glycol Chitosan (CTS-MA)

In a 200 ml beaker, 1 g of ethylene glycol chitosan (having a weight average molecular weight of about 200kDa), 50 ml of deionized water, 50 ml of dimethylformamide, 8 ml of triethylamine (Sigma), 13 ml of glycidyl methacrylate were added. After stirring at room temperature until uniform and transparent, stirring was continued for 48 hours. The above solution was filled into dialysis bags (cut-off 3.5kDa, Shibichun) and dialyzed against 5 liters of deionized water for 5 days with two water changes per day. Finally, the solution in the dialysis bag was collected and lyophilized to give 726 mg of light yellow flocculent solid, CTS-MA, yield 72.6%.

The structure of CTS-MA is shown in FIG. 24.

Of CTS-MA¹The H-NMR spectrum is shown in FIG. 24, where a nuclear magnetic peak belonging to the methacrylic function is visible between 5.7 and 6.2ppm, confirming the successful grafting of this group into the structure of the glycol chitosan.

Preparation example 15 Synthesis of mercapto-acrylate-modified chondroitin sulfate (abbreviated as CHS-A-SH1)

In A200 ml beaker, 1 g of CHS-A prepared by the method of preparation example 9, 0.25 g of dithiothreitol (VWR), and 100 ml of deionized water were added and dissolved with stirring at room temperature. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off 3.5kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution with pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 629 mg of pale yellow flocculent solid, thus obtaining CHS-A-SH1 with A yield of 62.9%.

The reaction equation of CHS-A-SH1 is shown in FIG. 5, and the structural formulA is shown in FIG. 5 and FIG. 29.

Of CHS-A-SH1¹The H-NMR spectrum is shown in FIG. 29, and a nuclear magnetic peak belonging to the side chain of thiol group between 2.6-3.0ppm can be seen, thus proving the successful grafting of thiol group to the structure of chondroitin sulfate.

Preparation example 16 Synthesis of mercapto-methacrylate-modified chondroitin sulfate (abbreviated as CHS-MA-SH1)

In a 200 ml beaker, 1 g of CHS-MA prepared by the method of preparation example 10, 0.25 g of dithiothreitol (VWR), and 100 ml of deionized water were added and dissolved with stirring at room temperature. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off 3.5kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution with pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag is collected and is frozen and dried to obtain 642 mg of light yellow flocculent solid, namely CHS-MA-SH1, and the yield is 64.2 percent.

The CHS-MA-SH1 reaction equation is shown in FIG. 6, and the structural formula is shown in FIG. 6 and FIG. 30.

Of CHS-MA-SH1¹The H-NMR spectrum is shown in FIG. 30, and a nuclear magnetic peak belonging to the side chain of thiol group between 2.6-3.0ppm can be seen, thus proving that the thiol group is successfully grafted to the structure of chondroitin sulfate.

Preparation example 17 Synthesis of mercapto-acrylate-modified Gelatin (Gelatin-A-SH 1 for short)

In a 200 ml beaker, 1 g of Gelatin-A prepared by the method of preparation example 11, 0.19 g of dithiothreitol (VWR), and 100 ml of deionized water were added and dissolved with stirring at room temperature. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag is collected and frozen and dried to obtain 763 mg of light yellow flocculent solid, namely Gelatin-A-SH1, with the yield of 76.3 percent.

The reaction equation of Gelatin-A-SH1 is shown in FIG. 7, and the structural formula is shown in FIG. 7 and FIG. 31.

Of Gelatin-A-SH1¹The H-NMR spectrum is shown in FIG. 31, where a nuclear magnetic peak belonging to the side chain of the thiol group is visible at a level between 2.6 and 2.8ppm, demonstrating the successful grafting of the thiol group into the structure of the gelatin.

Preparation example 18 Synthesis of mercapto-methacrylate modified Gelatin (Gelatin-MA-SH 1 for short)

In a 200 ml beaker, 1 g of Gelatin-MA prepared by the method of preparation example 12, 0.19 g of dithiothreitol (VWR), and 100 ml of deionized water were added and dissolved with stirring at room temperature. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally collecting the solution in the dialysis bag, and freeze-drying to obtain 787 mg of light yellow flocculent solid, i.e. Gelatin-MA-SH1, with the yield of 78.7%

The reaction equation of Gelatin-MA-SH1 is shown in FIG. 8, and the structural formula is shown in FIG. 8 and FIG. 32.

Of Gelatin-MA-SH1¹The H-NMR spectrum is shown in FIG. 32, and a nuclear magnetic peak belonging to the side chain of the mercapto group is seen between 2.6 and 2.7ppm, which proves the successful grafting of the mercapto group into the structure of the gelatin.

Preparation example 19 Synthesis of mercapto-acrylate-modified ethylene glycol Chitosan (CTS-A-SH 1 for short)

In A200 ml beaker, 1 g of CTS-A prepared by the method of preparation example 13, 0.25 g of dithiothreitol (VWR), and 100 ml of deionized water were added and dissolved with stirring at room temperature. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off 3.5kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution with pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag is collected and is frozen and dried to obtain light yellow flocculent solid 602 mg, namely CTS-A-SH1, with the yield of 60.2 percent.

The reaction equation of CTS-A-SH1 is shown in FIG. 9, and the structural formulA is shown in FIGS. 9 and 33.

Of CTS-A-SH1¹The H-NMR spectrum is shown in FIG. 33, and a nuclear magnetic peak belonging to a sulfydryl side chain and positioned between 2.6 and 3.0ppm can be seen, which proves that sulfydryl is successfully grafted into the structure of the glycol chitosan.

Preparation example 20 Synthesis of mercapto-methacrylate-modified Chitosan (CTS-MA-SH 1 for short)

In a 200 ml beaker, 1 g of CTS-MA prepared by the method of preparation example 14, 0.25 g of dithiothreitol (VWR), and 100 ml of deionized water were added and dissolved with stirring at room temperature. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off 3.5kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution with pH 4 for 5 days, with water changed twice a day. Finally collecting the solution in the dialysis bag, and freeze-drying to obtain 643 mg of white flocculent solid, namely CTS-MA-SH1, with the yield of 64.3 percent

The reaction equation of CTS-MA-SH1 is shown in FIG. 10, and the structural formula is shown in FIG. 10 and FIG. 34.

Of CTS-MA-SH1¹The H-NMR spectrum is shown in FIG. 34, and a nuclear magnetic peak belonging to a side chain of a thiol group between 2.5 and 2.9ppm can be seen, which proves that the thiol group is successfully grafted to the structure of chitosan.

Preparation example 21 synthetic acrylate-modified polyhydroxyethyl methacrylate (PHEMA-A for short)

In a 200 ml beaker, 2 g of polyhydroxyethyl methacrylate (Mv 20kDa, available from Sigma), 50 ml of deionized water, and 50 ml of dimethylformamide were added and dissolved by further adding 16.5 g of acrylic anhydride. The solution pH was maintained at 8 ± 0.5 with 1 mol NaOH per liter and stirring was continued for 24 hours. The above solution was filled into dialysis bags (molecular weight cut-off 2kDa, Shibichun) and dialyzed against 5 liters of deionized water for 5 days with two water changes per day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 1.42 g of a white solid, PHEMA-a, with a yield of 71.0%.

The structural formula of PHEMA-A is shown in figure 35.

Of PHEMA-A¹The H-NMR spectrum is shown in FIG. 35, where a nuclear magnetic peak belonging to the acrylic function is visible between 5.9 and 6.4ppm, confirming the successful grafting of this group into the structure of polyhydroxyethylmethacrylate.

Preparation example 22 Synthesis of methacrylate-modified polyhydroxyethyl methacrylate (PHEMA-MA)

In a 200 ml beaker, 2 g of polyhydroxyethyl methacrylate (Mv 20kDa, available from Sigma), 50 ml of deionized water, 50 ml of dimethylformamide, and 16.8 g of methacrylic anhydride were added and dissolved with stirring. The solution pH was maintained at 8 ± 0.5 with 1 mol NaOH per liter and stirring was continued for 24 hours. The above solution was filled into dialysis bags (molecular weight cut-off 2kDa, Shibichun) and dialyzed against 5 liters of deionized water for 5 days with two water changes per day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 1.48 g of a white solid, PHEMA-MA, with a yield of 74.0%.

The structural formula of PHEMA-MA is shown in figure 36.

Of PHEMA-MA¹The H-NMR spectrum is shown in FIG. 36, where a nuclear magnetic peak belonging to the methacrylic function is visible between 5.7 and 6.3ppm, demonstrating that this group is presentSuccessfully grafted to the structure of polyhydroxyethyl methacrylate.

Preparation example 23 Synthesis of acrylate-modified polyvinyl alcohol (abbreviated as PVA-A)

In a 200 ml beaker, 2 g of polyvinyl alcohol (Mw 61kDa, available from Sigma), 50 ml of deionized water, 50 ml of dimethylformamide and 13 g of acrylic anhydride were added and dissolved with stirring. The solution pH was maintained at 8 ± 0.5 with 1 mol NaOH per liter and stirring was continued for 24 hours. The above solution was filled into dialysis bags (cut-off 3.5kDa, Shibichun) and dialyzed against 5 liters of deionized water for 5 days with two water changes per day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 1.57 g of white solid, PVA-A, in 78.5% yield.

The structural formula of PVA-A is shown in figure 37.

Of PVA-A¹The H-NMR spectrum is shown in FIG. 37, where a nuclear magnetic peak belonging to the acrylic function, between 6.0 and 6.5ppm, is visible, confirming the successful grafting of this group into the structure of the polyvinyl alcohol.

Preparation example 24 Synthesis of methacrylate-modified polyvinyl alcohol (abbreviated as PVA-MA)

In a 200 ml beaker, 2 g of polyvinyl alcohol (Mw 61kDa, available from Sigma), 50 ml of deionized water, 50 ml of dimethylformamide, and 13.4 g of methacrylic anhydride were added and dissolved with stirring. The solution pH was maintained at 8 ± 0.5 with 1 mol NaOH per liter and stirring was continued for 24 hours. The above solution was filled into dialysis bags (cut-off 3.5kDa, Shibichun) and dialyzed against 5 liters of deionized water for 5 days with two water changes per day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 1.51 g of white solid, i.e., PVA-MA, with a yield of 75.5%.

The structural formula of PVA-MA is shown in figure 38.

The 1H-NMR spectrum of PVA-MA is shown in FIG. 38, where a nuclear magnetic peak belonging to the methacrylic function, located between 5.7 and 6.3ppm, is visible, confirming the successful grafting of this group into the structure of the polyvinyl alcohol.

Preparation example 25 Synthesis of mercapto-acrylate-modified polyhydroxyethyl methacrylate (PHEMA-A-SH 1 for short)

In a 200 ml beaker, 2 g of PHEMA-A prepared by the method of preparation example 21, 0.42 g of dithiothreitol (VWR), 50 ml of deionized water, and 50 ml of dimethylformamide were added and dissolved with stirring at room temperature. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (molecular weight cut-off 2kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution with pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag is collected and freeze-dried to obtain 1.67 g of white solid, namely PHEMA-A-SH1, with the yield of 83.5%.

The reaction equation of PHEMA-A-SH1 is shown in FIG. 57, and the structural formula is shown in FIGS. 57 and 39.

Of PHEMA-A-SH1¹The H-NMR spectrum is shown in FIG. 39, and a nuclear magnetic peak belonging to a sulfydryl side chain and positioned between 2.6 and 2.9ppm can be seen, so that the successful grafting of sulfydryl to the structure of the polyhydroxyethyl methacrylate is proved.

Preparation example 26 Synthesis of mercapto-methacrylate-modified polyhydroxyethyl methacrylate (PHEMA-MA-SH 1 for short)

In a 200 ml beaker, 2 g of PHEMA-MA prepared by the method of preparation 22, 0.41 g of dithiothreitol (VWR), 50 ml of deionized water, and 50 ml of dimethylformamide were added and dissolved with stirring at room temperature. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (molecular weight cut-off 2kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution with pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag is collected and freeze-dried to obtain 1.62 g of white solid, namely PHEMA-MA-SH1, with the yield of 81%.

The reaction equation of PHEMA-MA-SH1 is shown in FIG. 58, and the structural formula is shown in FIG. 58 and FIG. 40.

Of PHEMA-MA-SH1¹The H-NMR spectrum is shown in FIG. 40, and a nuclear magnetic peak belonging to a sulfydryl side chain and positioned between 2.6 and 3.0ppm can be seen, so that the successful grafting of sulfydryl to the structure of the polyhydroxyethyl methacrylate is proved.

Preparation example 27 Synthesis of mercapto-acrylate-modified polyvinyl alcohol (abbreviated as PVA-A-SH1)

In a 200 ml beaker, 1 g of PVA-A prepared by the method of preparation example 23 and 100 ml of deionized water were added, and the solution was heated with stirring until the PVA-A was completely dissolved. Subsequently, 0.47 g of dithiothreitol (VWR) was added to the solution, and the solution was dissolved with stirring at room temperature. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, collecting the solution in the dialysis bag, and freeze-drying to obtain 737 mg of white solid, namely PVA-A-SH1, with the yield of 73.7%.

The reaction equation of PVA-A-SH1 is shown in FIG. 59, and the structural formula is shown in FIGS. 59 and 41.

Of PVA-A-SH1¹The H-NMR spectrum is shown in FIG. 41, and a nuclear magnetic peak belonging to a side chain of a mercapto group between 2.6 and 3.0ppm can be seen, which proves that the mercapto group is successfully grafted to the structure of the polyvinyl alcohol.

Preparation example 28 Synthesis of mercapto-methacrylate-modified polyvinyl alcohol (abbreviated as PVA-MA-SH1)

In a 200 ml beaker, 1 g of PVA-MA prepared by the method of preparation example 24 and 100 ml of deionized water were added, and the solution was heated with stirring until the PVA-MA was completely dissolved. Subsequently, 0.47 g of dithiothreitol (VWR) was added to the solution, and the solution was dissolved with stirring at room temperature. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 718 mg of white solid, thus obtaining PVA-MA-SH1 with a yield of 71.8%.

The reaction equation of PVA-MA-SH1 is shown in FIG. 60, and the structural formula is shown in FIGS. 60 and 42.

Of PVA-MA-SH1¹The H-NMR spectrum is shown in FIG. 42, and a nuclear magnetic peak belonging to a side chain of a mercapto group can be seen between 2.5 and 3.0ppm, which proves that the mercapto group is successfully grafted into the structure of the polyvinyl alcohol.

Preparation example 29 Synthesis of thiol-modified hyperbranched PEG Polymer (abbreviation HB-PEG-SH1)

In a 200 ml beaker, 5 g of hyperbranched PEG, HB-PEG (Mw 20kDa, available from Blafar Ltd), 0.86 g of dithiothreitol (VWR), 100 ml of deionized water were added and dissolved with stirring at room temperature. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (molecular weight cut-off 2kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution with pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag is collected and is frozen and dried to obtain 3.84 g of colorless viscous liquid, thus obtaining HB-PEG-SH1 with the yield of 76.8 percent.

The reaction equation of HB-PEG-SH1 is shown in FIG. 61, and its structural formula is shown in FIGS. 61 and 43.

Of HB-PEG-SH1¹The H-NMR spectrum is shown in FIG. 43, and a nuclear magnetic peak belonging to a sulfydryl side chain and positioned between 2.5 and 2.6ppm can be seen, which proves that sulfydryl is successfully grafted into the structure of the hyperbranched PEG polymer.

Preparation example 30 Synthesis of thiol-acrylate-modified hyaluronic acid (abbreviated as HA-A1-SH2)

In a 200 ml beaker, 1 g of HA-A1 prepared according to preparation example 1, 0.42 g of 1, 4-butanedithiol (purchased from Sigma) and 100 ml of deionized water were added and dissolved with stirring at room temperature to obtain a transparent solution. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 852 mg of white flocculent solid, HA-A1-SH2 with a yield of 85.2%.

The reaction equation of HA-A1-SH2 is shown in FIG. 62, and the structural formula is shown in FIGS. 62 and 44.

Of HA-A1-SH2¹The H-NMR spectrum is shown in FIG. 44, and a nuclear magnetic peak belonging to the side chain of the thiol group between 1.6 and 1.9ppm can be seen, thus proving that the thiol group is successfully grafted into the structure of the hyaluronic acid.

Preparation example 31 Synthesis of thiol-acrylate-modified hyaluronic acid (abbreviated as HA-A1-SH3)

In a 200 ml beaker, 1 g of HA-A1 prepared according to preparation example 1, 0.43 g of 2-amino-1, 4-butanedithiol hydrochloride (purchased from Sigma) and 100 ml of deionized water were added and dissolved with stirring at room temperature to obtain a transparent solution. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 843 mg of white flocculent solid, HA-A1-SH3 with a yield of 84.3%.

The reaction equation of HA-A1-SH3 is shown in FIG. 63, and the structural formula is shown in FIGS. 63 and 45.

Of HA-A1-SH3¹The H-NMR spectrum is shown in FIG. 45, and a nuclear magnetic peak belonging to the side chain of the thiol group between 3.0 and 3.2ppm can be seen, thus proving the successful grafting of the thiol group into the structure of hyaluronic acid.

Preparation example 32 Synthesis of thiol-acrylate-modified hyaluronic acid (abbreviated as HA-A2-SH2)

In a 200 ml beaker, 1 g of HA-A2 prepared according to preparation example 2, 0.42 g of 1, 4-butanedithiol (purchased from Sigma) and 100 ml of deionized water were added and dissolved with stirring at room temperature to obtain a transparent solution. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 827 mg of white flocculent solid, HA-A2-SH2 with 82.7% yield.

The reaction equation of HA-A2-SH2 is shown in FIG. 64, and the structural formula is shown in FIGS. 64 and 46.

Of HA-A2-SH2¹The H-NMR spectrum is shown in FIG. 46, and a nuclear magnetic peak belonging to the side chain of the thiol group is seen between 1.6 and 1.9ppm, which proves the successful grafting of the thiol group into the structure of hyaluronic acid.

Preparation example 33 Synthesis of thiol-acrylate-modified hyaluronic acid (abbreviated as HA-A2-SH3)

In a 200 ml beaker, 1 g of HA-A2 prepared according to preparation example 2, 0.43 g of 2-amino-1, 4-butanedithiol hydrochloride (Sigma) and 100 ml of deionized water were added and dissolved with stirring at room temperature to obtain a transparent solution. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 833 mg of white flocculent solid, HA-A2-SH3 with a yield of 83.3%.

The reaction equation of HA-A2-SH3 is shown in FIG. 65, and the structural formula is shown in FIGS. 65 and 47.

Of HA-A2-SH3¹The H-NMR spectrum is shown in FIG. 47, and a nuclear magnetic peak belonging to a mercapto side chain between 3.0 and 3.2ppm is observed, which is evidenceThe thiol group was successfully grafted into the structure of hyaluronic acid.

Preparation example 34 Synthesis of thiol-acrylate-modified hyaluronic acid (abbreviated as HA-A2-SH4)

In a 200 ml beaker, 1 g of HA-A2 prepared according to preparation example 2, 0.38 g of 1, 3-propanedithiol (purchased from Sigma) and 100 ml of deionized water were added and dissolved with stirring at room temperature to obtain a transparent solution. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 814 mg of white flocculent solid, HA-A2-SH4 with a yield of 81.4%.

The reaction equation of HA-A2-SH4 is shown in FIG. 66, and the structural formula is shown in FIGS. 66 and 48.

Of HA-A2-SH4¹The H-NMR spectrum is shown in FIG. 48, where a nuclear magnetic peak belonging to the side chain of thiol groups between 2.5 and 2.8ppm is visible, demonstrating the successful grafting of thiol groups into the structure of hyaluronic acid.

Preparation example 35 Synthesis of thiol-acrylate-modified hyaluronic acid (abbreviated as HA-A2-SH5)

In a 200 ml beaker, 1 g of HA-A2 prepared according to preparation example 2, 0.52 g of 1, 3-benzenedithiol (purchased from Sigma) and 100 ml of deionized water were added and dissolved with stirring at room temperature to obtain a transparent solution. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 836 mg of white flocculent solid, HA-A2-SH5 with a yield of 83.6%.

The reaction equation of HA-A2-SH5 is shown in FIG. 67, and the structural formula is shown in FIGS. 67 and 49.

Of HA-A2-SH5¹The H-NMR spectrum is shown in FIG. 49, and a nuclear magnetic peak belonging to the side chain of the thiol group between 6.9 and 7.4ppm can be seen, thus proving that the thiol group is successfully grafted into the structure of the hyaluronic acid.

Preparation example 36 Synthesis of thiol-acrylate-modified hyaluronic acid (abbreviated as HA-A2-SH6)

In a 200 ml beaker, 1 g of HA-A2 prepared according to preparation example 2, 0.52 g of 1, 4-benzenedithiol (purchased from Sigma) and 100 ml of deionized water were added and dissolved with stirring at room temperature to obtain a transparent solution. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 831 mg of white flocculent solid, HA-A2-SH6 with a yield of 83.1%.

The reaction equation of HA-A2-SH6 is shown in FIG. 68, and the structural formula is shown in FIGS. 68 and 50.

Of HA-A2-SH6¹The H-NMR spectrum is shown in FIG. 50, and a nuclear magnetic peak belonging to the side chain of the thiol group and located between 6.8 and 7.0ppm can be seen, thus proving that the thiol group is successfully grafted into the structure of the hyaluronic acid.

Preparation example 37 Synthesis of thiol-acrylate-modified hyaluronic acid (abbreviated as HA-A2-SH7)

In a 200 ml beaker, 1 g of HA-A2 prepared according to preparation example 2, 0.96 g of mercaptopolyethylene glycol (purchased from Sigma) and 100 ml of deionized water were added and dissolved with stirring at room temperature to obtain a transparent solution. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 894 mg of white flocculent solid, HA-A2-SH7 with a yield of 89.4%.

The reaction equation of HA-A2-SH7 is shown in FIG. 69, and the structural formula is shown in FIGS. 69 and 51.

Of HA-A2-SH7¹The H-NMR spectrum is shown in FIG. 51, and a nuclear magnetic peak belonging to the side chain of the thiol group at 3.6ppm can be seen, which confirms successful grafting of the thiol group into the structure of hyaluronic acid.

Preparation example 38 Synthesis of thiol-acrylate-modified hyaluronic acid (abbreviated as HA-A2-SH8)

In a 200 ml beaker, 1 g of HA-A2 prepared according to preparation example 2, 0.74 g of trimethylolpropane tris (3-mercaptopropionate) (purchased from Sigma), 50 ml of deionized water and 50 ml of dimethylformamide were added and dissolved with stirring at room temperature to give a clear solution. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, collecting the solution in the dialysis bag, and freeze-drying to obtain 785 mg of white flocculent solid, namely HA-A2-SH8, with the yield of 78.5%.

The reaction equation of HA-A2-SH8 is shown in FIG. 70, and the structural formula is shown in FIGS. 70 and 52.

Of HA-A2-SH8¹The H-NMR spectrum is shown in FIG. 52, and nuclear magnetic peaks belonging to sulfhydryl side chains and located between 0.8-1.0ppm, 1.5ppm and 2.6-2.9ppm can be seen, thus proving that sulfhydryl groups are successfully grafted into the structure of hyaluronic acid.

Preparation example 39 Synthesis of thiol-methacrylate-modified hyaluronic acid (abbreviated as HA-MA1-SH5)

In a 200 ml beaker, 1 g of HA-MA1 prepared according to preparation example 3, 0.50 g of 1, 3-benzenedithiol (purchased from Sigma) and 100 ml of deionized water were added and dissolved with stirring at room temperature to obtain a transparent solution. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, collecting the solution in the dialysis bag, and freeze-drying to obtain 828 mg of white flocculent solid, namely HA-MA1-SH5, with the yield of 82.8%.

The reaction equation of HA-MA1-SH5 is shown in FIG. 71, and the structural formula is shown in FIG. 71 and FIG. 53.

Of HA-MA1-SH5¹The H-NMR spectrum is shown in FIG. 53, where a nuclear magnetic peak belonging to the side chain of the thiol group is visible at between 6.9 and 7.4ppm, demonstrating the successful grafting of the thiol group into the structure of the hyaluronic acid.

Preparation example 40 Synthesis of thiol-methacrylate-modified hyaluronic acid (abbreviated as HA-MA1-SH6)

In a 200 ml beaker, 1 g of HA-MA1 prepared according to preparation example 3, 0.50 g of 1, 4-benzenedithiol (purchased from Sigma) and 100 ml of deionized water were added and dissolved with stirring at room temperature to obtain a transparent solution. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, the solution in the dialysis bag was collected and lyophilized to obtain 833 mg of white flocculent solid, i.e., HA-MA1-SH6 with a yield of 83.3%.

The reaction equation of HA-MA1-SH6 is shown in FIG. 72, and the structural formula is shown in FIGS. 72 and 54.

Of HA-MA1-SH6¹The H-NMR spectrum is shown in FIG. 54, and a nuclear magnetic peak belonging to the side chain of the thiol group between 6.9 and 7.0ppm can be seen, thus proving that the thiol group is successfully grafted into the structure of the hyaluronic acid.

Preparation example 41 Synthesis of thiol-methacrylate-modified hyaluronic acid (abbreviated as HA-MA2-SH7)

In a 200 ml beaker, 1 g of HA-MA2 prepared according to preparation example 4, 0.92 g of mercaptopolyethylene glycol (purchased from Sigma) and 100 ml of deionized water were added and dissolved with stirring at room temperature to obtain a transparent solution. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, collecting the solution in the dialysis bag, and freeze-drying to obtain 876 mg of white flocculent solid, namely HA-MA2-SH7, with the yield of 87.6%.

The reaction equation of HA-MA2-SH7 is shown in FIG. 73, and the structural formula is shown in FIGS. 73 and 55.

Of HA-MA2-SH7¹The H-NMR spectrum is shown in FIG. 55, and a nuclear magnetic peak belonging to the side chain of the thiol group at 3.6ppm can be seen, which confirms successful grafting of the thiol group into the structure of hyaluronic acid.

Preparation example 42 Synthesis of thiol-methacrylate 2-modified hyaluronic acid (abbreviated as HA-MA2-SH8)

In a 200 ml beaker, 1 g of HA-MA2 prepared according to preparation example 4, 0.68 g of trimethylolpropane tris (3-mercaptopropionate) (purchased from Sigma), 50 ml of deionized water and 50 ml of dimethylformamide were added and dissolved with stirring at room temperature to give a transparent solution. The resulting clear solution was stirred for an additional 12 hours. The above solution was packed into dialysis bags (cut-off of 8kDa, shibixin) and dialyzed against 5 l of hydrochloric acid solution at pH 4 for 5 days, with water changed twice a day. Finally, collecting the solution in the dialysis bag, and freeze-drying to obtain 825 mg of white flocculent solid, namely HA-MA2-SH8, with the yield of 82.5%.

The reaction equation of HA-MA2-SH8 is shown in FIG. 74, and the structural formula is shown in FIG. 74 and FIG. 56.

Of HA-MA2-SH8¹The H-NMR spectrum is shown in FIG. 56, and nuclear magnetic peaks belonging to the side chain of the thiol group and located between 0.8-1.0ppm, 1.5ppm and 2.6-2.9ppm can be seen, thus proving that the thiol group is successfully grafted into the structure of the hyaluronic acid.

Example 1 preparation of hydrogel of thiol-vinyl Cross-Linked hyaluronic acid

10mg of any one of the acrylated polymers and Ethylene Glycol Diacrylate (EGDA) prepared in preparation examples 1,2, 9, 11, and 13 was dissolved in 1ml of a phosphate buffer solution (pH 7.4) to obtain a series of solutions a having a mass volume concentration of 1%.

10mg of any of the thiol-modified polymeric compounds prepared in preparation examples 5 to 8 and 15 to 20 was dissolved in 1ml of a phosphate buffer (pH 7.4) to obtain a series of solutions B having a mass volume concentration of 1%.

And uniformly mixing any one of the solution A and any one of the solution B according to the equal volume, wherein the physiological in-situ crosslinking reaction between the two macromolecular compounds occurs immediately, the solution viscosity gradually increases along with the increase of the mixing time, and finally the hydrogel is formed.

Each hydrogel in example 1 included the following characteristic structural units:

wherein R is₁、R₂And R₃The definition of (1) is as before; denotes the connection point.

The gel formation times for each set of hydrogels are listed in table 1.

TABLE 1 hydrogel gel formation time

Example 2: hydrogel storage modulus detection

Two milliliters of the hydrogel mixed solution prepared in example 1 was placed in a cylindrical mold, crosslinked at room temperature for 24 hours, and then taken out to test the storage modulus of the crosslinked samples, and each sample was tested three times. Detection instrument TA-DHR2 rheometer, detection probe 20mm parallel plate probe, detection temperature: 25 ℃, shear frequency: 1 Hz. The test results are listed in table 2.

Table 2: storage modulus comparison table:

example 3: water retention Performance test of hydrogel

The hydrogel prepared in example 1 was charged into a 20 ml glass bottle weighed in advance, and the mass of the hydrogel obtained by the mass difference subtraction method was recorded as m₀. Placing the glass bottle in a shaking table at 37 ℃, weighing the glass bottle at regular intervals to obtain the real-time mass m of the hydrogel_t. The water retention capacity of the hydrogel was calculated according to the following formula:

water retention (%) ═ m_t/m₀×100％

The water retention results are shown in table 3.

Table 3: water retention index comparison table:

example 4: in vitro degradation experiments of hydrogels

And (3) testing degradation stability: 10 ml of PBS solution were added to the hydrogel prepared in example 1 under experimental conditions of 37. + -. 0.1 ℃ and 65%. + -. 5% relative humidity. The weight of the hydrogel at the initial time point was taken as m₀The weight of the hydrogel was measured at 1,4, 8 and 16 weeks after the start of the degradation test and recorded as m_tThe degradation ratio of the hydrogel was calculated according to the following formula:

percent degradation (%) - (m)₀-m_t)/m₀×100％

The results of the in vitro degradation test of the hydrogels are shown in table 4.

Table 4 hydrogel in vitro degradation test

Example 5: hydrogel cell viability assay

The HA-SH of the present invention was tested for cellular activity and biocompatibility with reference to the "GBT 16886.5-2017 medical device biological evaluation + part 5 + in vitro cytotoxicity assay" criteria. Specifically, the following MTT method, also called MTT colorimetric method, is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-violet crystalline Formazan (Formazan) and deposit the Formazan in the cells, and dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and an enzyme linked immunosorbent assay detector is used for measuring the light absorption value of formazan at 490nm wavelength, so that the quantity of living cells can be indirectly reflected. Within a certain range of cell number, MTT crystals are formed in an amount proportional to the cell number. The specific test procedures and results are as follows:

a solution of HA-a1 prepared in preparation example 1 (10 mg/mL in phosphate buffered saline, pH 7.4) was taken and designated as solution a.

A solution of HA-A1-SH1 (10 mg/mL in phosphate buffered saline, pH 7.4) prepared in preparation example 5 was collected and designated as solution B.

Cell culture media were prepared using modified Du's eagle's medium, 10% fetal bovine serum, and 1% penicillin/streptomycin solution. L929 cells were cultured routinely, and after the cells were cultured to near confluence, the cells were digested to obtain a cell suspension.

Uniformly mixing the solution A, the solution B and the cell suspension to prepare a cell/hydrogel composite system with the volume of 50 mu L, wherein the final concentration of the cells is 1 multiplied by 10⁶cell/mL. The system was placed in a 24-well cell culture plate and 1mL of cell culture medium was added to each well for culture, where an equal number of cells on the bottom of the plate served asIs a negative control group. Samples were taken at 5% CO₂、37℃、>The cells were incubated in a cell incubator at 90% humidity for 24 hours. And (3) detecting the survival state of cells in different hydrogel samples by adopting an MTT method, and comparing the cell activity of the hydrogel group with the cell activity of a negative control group. The negative control group was 100% active. The culture was removed and 100 μ L of MTT was added to each test well and incubation continued for 4 hours. Then, the MTT solution is discarded, 200. mu.L of DMSO solution is added into each well, and after uniform shaking, the absorbance at 490nm is measured by a microplate reader. The results of the test are shown in FIG. 11. Materials with cell viability results below 70% for MTT experiments are considered potentially cytotoxic. The results show that the survival rate of cells in the hydrogel is over 70 percent, and the material has no obvious cytotoxicity and good biocompatibility.

Example 6: hydrogel modeling and support effect animal experiment

After C57BL/6 mice are anesthetized, the back is depilated, and the mice are disinfected conventionally, 120 mu L of HA-A1 and HA-A1-SH1 solution and HA-A2 and HA-A2-SH1 solution (the concentration is 10mg/mL respectively) prepared according to the method in the embodiment 1 are respectively taken, 12mg of each material is respectively dissolved in 1.2mL of PBS (phosphate buffered saline) solution, and the solution is shaken up to obtain a sample for testing for later use. The sample to be tested was each 120. mu.L aspirated by a syringe and mixed uniformly, and the resulting hydrogel precursor solution was injected into the subcutaneous part of the back of the mouse through a 24G needle. An equivalent volume of saline was injected subcutaneously into the back of mice in the same manner.

The bulge was photographed before injection, immediately after injection, at week 4, 8, and 12, measured using a vernier caliper, and recorded in detail. The molding effect of the hydrogels was evaluated by comparing the maintenance and changes in the three-dimensional morphology of the different injected samples injected into the animals. The higher the height of the injection site ridge and the smaller the base area, the better the molding and supporting effect. The results are shown in FIGS. 12 and 13. The data show that the hydrogel of the invention forms a support body capable of maintaining a certain shape after being injected into an animal body, and can better keep the shape of the injection stable.

Example 7: hydrogel plastic effect animal experiment

After C57BL/6 mice are anesthetized, the back is depilated, and the mice are disinfected conventionally, 120 mu L of HA-A1 and HA-A1-SH1 solution and HA-A2 and HA-A2-SH1 solution (the concentration is 10mg/mL respectively) prepared according to the method in the embodiment 1 are respectively taken, 12mg of each material is respectively dissolved in 1.2mL of PBS solution, and the samples for testing are obtained and are used for later use after shaking up. Respectively sucking 120 mu L of the sample for test by using a syringe, uniformly mixing, and injecting the obtained hydrogel precursor solution into the subcutaneous part of the back of the mouse through a 24G needle; one mouse is euthanized after 60 minutes of injection, and the in-situ formation condition and the form of the hydrogel in the subcutaneous tissue of the mouse are observed; the remaining animals were kept on routine rearing and the injection sites were observed photographically and the gel volume changes were recorded at 4, 8, and 12 weeks post injection. An equivalent volume of saline was injected subcutaneously into the back of mice in the same manner.

After the hydrogel precursor mixed solution was injected subcutaneously into the back of the mouse, a rounded bulge was visible at the injection site. The subcutaneous hydrogel formation in the mice was observed to be transparent intact hemispheres 60 minutes after injection. The results show that the mixed precursor solution can rapidly generate a crosslinking reaction, form hydrogel in situ during injection and maintain a certain shape. After 12 weeks of injection, a significant bulge was still observed subcutaneously in the back of the animals. The hydrogel and the local expression of the surrounding tissues are observed by cutting the skin tissues, the hydrogel has good shape, and the surrounding tissues have no inflammation, infection, necrosis and other abnormalities. The hydrogel was removed and observed and weighed, and it was found that the hydrogel remained an intact hemisphere, and that the weight was slightly reduced after weighing (see fig. 14 for results), and no significant gel fracture or disintegration, etc. was observed.

The results show that the hydrogel of the invention has superior performance in degradation resistance and maintaining colloidal stability.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.