WO2021098099A1 - 巯基改性高分子化合物的水凝胶及其制备方法和用途 - Google Patents

巯基改性高分子化合物的水凝胶及其制备方法和用途 Download PDF

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WO2021098099A1
WO2021098099A1 PCT/CN2020/079823 CN2020079823W WO2021098099A1 WO 2021098099 A1 WO2021098099 A1 WO 2021098099A1 CN 2020079823 W CN2020079823 W CN 2020079823W WO 2021098099 A1 WO2021098099 A1 WO 2021098099A1
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group
solution
polymer compound
hydrogel
mercapto
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PCT/CN2020/079823
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English (en)
French (fr)
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王文新
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孛朗孚有限公司
王文新
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Priority to EP20888940.2A priority Critical patent/EP4063433B1/en
Priority to JP2022529445A priority patent/JP2023503896A/ja
Priority to AU2020386559A priority patent/AU2020386559A1/en
Priority to CA3163069A priority patent/CA3163069A1/en
Priority to US17/756,183 priority patent/US20230021037A1/en
Priority to KR1020227020479A priority patent/KR20220103756A/ko
Publication of WO2021098099A1 publication Critical patent/WO2021098099A1/zh

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Definitions

  • the invention belongs to the field of biological materials, and specifically relates to a hydrogel of sulfhydryl modified polymer compound and a preparation method and application thereof.
  • Biomedical materials namely Biomedical Materials
  • Biomaterials can also be referred to as biomaterials (Biomaterials in English). They are new high-tech materials used to diagnose, treat, repair or replace diseased tissues and organs or enhance their functions.
  • One of the key technologies of tissue engineering is to use biomaterials to prepare cell scaffolds that have good biocompatibility and can be degraded and absorbed by the body.
  • Gel state is an intermediate state between solid and liquid.
  • Hydrogel refers to a hydrophilic cross-linked three-dimensional polymer network that can swell in water and retain a large amount of water without dissolving. Its water content can reach more than 90%. Hydrogel is an ideal biological material. It can have satisfactory physical and chemical properties similar to natural extracellular matrix by itself or through simple modification.
  • hydrophilic polymers used to prepare hydrogels are classified into natural polymers and synthetic polymers according to their sources.
  • natural polymers include collagen, gelatin, fibrin, polysaccharides, etc.
  • synthetic polymers include synthetic polypeptides, polyethylene glycol (PEG) and its derivatives, polymethyl methacrylate (PMMA) and its derivatives, Polylactic acid glycolic acid copolymer (PLGA) and its derivatives, etc.
  • PEG polyethylene glycol
  • PMMA polymethyl methacrylate
  • PLGA Polylactic acid glycolic acid copolymer
  • the injectable in-situ cross-linked hydrogel is characterized by being in a flowable liquid state before injection, and after being injected to the target position, it can form a colloid that completely conforms to the shape of the target position.
  • This injectable feature not only makes the operation process simple and convenient, but also avoids the pain caused by the implantation operation to the patient, and greatly reduces the trauma of the operation.
  • hyaluronic acid is a natural heteropolysaccharide composed of alternating units of D-glucuronic acid and N-acetylglucosamine. After decades of research, it was found that it is widely present in the connective tissues of humans and other vertebrates, such as intercellular spaces, motor joint tissue, umbilical cord, skin, cartilage, blood vessel walls, synovial fluid, and combs and other tissues and organs. Both contain hyaluronic acid.
  • Hyaluronic acid is a linear polymer polysaccharide. The structure contains disaccharide repeating units.
  • the D-glucuronic acid in the repeating unit is connected to N-acetylglucosamine through ⁇ -1,3 glycosidic bonds. Thousands of two The sugar repeating units are connected by ⁇ -1,4 glycosidic bonds to form the entire polymer linear and linear structure.
  • hyaluronic acid usually exists in the form of sodium salt.
  • sodium hyaluronate and its gel are widely used in the fields of orthopedics, gynecology and plastic surgery, and can also be used as a carrier for ophthalmic preparations or directly used as ophthalmic preparations in ophthalmic surgery, that is, sodium hyaluronate products are used in ophthalmology.
  • Sodium hyaluronate is also an important component of joint synovial fluid and cartilage. By increasing the content of sodium hyaluronate in joints, it can increase the viscosity and lubricating function of joint synovial fluid, protect cartilage, promote joint healing and regeneration, and relieve Pain, increase joint mobility and other effects.
  • hyaluronic acid and its sodium salt are a safe and effective ideal substance in preventing and reducing adhesions caused by obstetrics and gynecology operations; among them, sodium hyaluronate
  • the aqueous solution is a non-Newtonian fluid with good viscoelasticity and rheology.
  • low-concentration hyaluronic acid solutions mainly show viscosity
  • high-concentration hyaluronic acid solutions mainly show elasticity, so The concentration can be adjusted according to actual needs.
  • natural hyaluronic acid or its sodium salt has a wide range of applications and has many clear application advantages
  • natural hyaluronic acid or its sodium salt also has clear disadvantages.
  • natural hyaluronic acid or its sodium salt has a short half-life in the body, and the degradation time in the body is generally no more than 7 days.
  • the main reason for the short half-life is the small average molecular weight of natural hyaluronic acid or its sodium salt. , And has good fluidity, easy to disperse in the tissues and be absorbed and metabolized, it is directly manifested as low viscosity in the solution state.
  • natural hyaluronic acid or its sodium salt has the disadvantages of poor stability and easy degradation.
  • natural hyaluronic acid or its sodium salt has the disadvantage of being too hydrophilic.
  • the cross-linking reaction of hyaluronic acid to generate cross-linked hyaluronic acid requires certain reaction conditions or the reaction conditions are relatively harsh, and the in-situ cross-linking cannot be achieved under physiological conditions.
  • the pre-cross-linking and pre-filling form realizes the product, which greatly affects the application range of the product and the compliance of the corresponding treatment population or the beauty population.
  • the existing sulfhydryl modification process of biocompatible polymers generally refers to the chemical modification process of introducing free sulfhydryl groups, usually the side chain groups of polysaccharides, proteins and synthetic polymers, such as: carboxyl, amino, hydroxyl, etc. ,
  • the free sulfhydryl group can be introduced through an appropriate chemical reaction.
  • hyaluronic acid as an example, in the prior art, the thiolated hyaluronic acid obtained by introducing free sulfhydryl groups into hyaluronic acid through a chemical reaction summarizes its characteristics. Although compared with natural hyaluronic acid, it has advantages in physical and chemical properties or biological phases. There are certain improvements or enhancements in terms of capacitance, but it is still not enough to overcome the following shortcomings: 1. The speed of self-crosslinking or crosslinking with other substances is slow, and it is usually necessary to add a small molecule oxidant to accelerate the crosslinking reaction. 2.
  • the new hyaluronic acid compound modified by sulfhydryl group, after cross-linking to form a hydrogel has no substantial advantages compared with existing commercially available products or products. Or they do not have enough distinguishing technical characteristics, which are mainly reflected in viscosity, metabolic persistence and shaping effect. 3.
  • the sulfhydryl hyaluronic acid and the synthetic preparation methods in the prior art have the disadvantages of high toxicity or high cost. The above-mentioned reasons are the root causes that affect the industrial production preparation and wider application of the existing thiolated hyaluronic acid synthesis and preparation technology.
  • hydrogels prepared from sulfhydryl hyaluronic acid through cross-linking reaction have shortcomings or technical prejudices, including: 1.
  • the chemically modified hyaluronic acid has been subjected to The cross-linking reaction obtains cross-linked hyaluronic acid, and the hydrogel prepared therefrom is more expensive than the hydrogel prepared by cross-linking natural hyaluronic acid.
  • the chemically modified hyaluronic acid is cross-linked to obtain cross-linked hyaluronic acid, and the hydrogel prepared therefrom is compared with natural hyaluronic acid through cross-linking.
  • the prepared hydrogel has improvements in viscosity, water retention, and molding effect, the improvement is limited. 3.
  • the chemical modification of hyaluronic acid in the prior art has certain uncontrollability. This uncontrollability will affect the quality of its cross-linked hyaluronic acid, which will result in a large quality of the corresponding hydrogel. Fluctuations within the range make it impossible to achieve the consistency of the therapeutic effect or cosmetic and plastic effects of hydrogel products between different batches, and it also affects the greater role of hydrogels in the application field.
  • the object of the present invention is to provide a thiol-modified polymer compound with a novel structure and at least one of the following substances: acryloyl polymer compound, small molecule crosslinking agent containing acryloyl group, which The hydrogel with a novel structure is generated by the gelation of.
  • the present invention adopts a mercapto-modified polymer compound with a novel structure, which is combined with an acryloyl polymer compound and/or a small molecule crosslinking agent containing an acryl group to form a hydrogel.
  • the sulfhydryl-modified polymer compound can be cross-linked with the acryloyl polymer compound and/or the acryl-containing small molecule cross-linking agent to form a hydrogel under physiological conditions; in addition, the formed hydrogel has a molding effect
  • the physical and chemical properties related to metabolism and degradation resistance also have obvious advantages over the prior art. Specifically, its metabolic and degradation resistance is significantly better than the hydrogel in the prior art; furthermore, due to the sulfhydryl group -The rapid vinyl cross-linking reaction allows the hydrogel system formed by the two compounds to quickly form a gel in situ after being injected into the body. Based on this, the hydrogel of the present invention is more beneficial to be used in the fields of biomedicine, medical beauty and plastic surgery, and cosmetics.
  • the second object of the present invention is to provide a method for preparing the above-mentioned hydrogel, which has the following advantages: the crosslinking reaction does not require the addition of highly toxic epoxy-based small molecule crosslinking agents and catalysts, which fundamentally avoids Poisonous substances may remain in the purification process, no catalytic conditions such as light and heating are needed, the degree of cross-linking reaction is controllable, and the cost of the cross-linking reaction is moderate and superior to the prior art.
  • the first aspect of the present invention is to provide a hydrogel, the chemical structure of the hydrogel is brand new, which is prepared by gelation of a system containing sulfhydryl-modified polymer compounds;
  • the mercapto-modified polymer compound is at least one of the following series of compounds:
  • a series of mercapto-modified polymer compounds contains -COOH, -NH 2 , -OH, acrylate groups represented by formula a, and acrylamide groups represented by formula b , At least one of the acryl group represented by formula c,
  • R 1 is selected from hydrogen, halogen, aliphatic group, aromatic group, etc.
  • R 2 and R 3 are the same or different, and are independently selected from hydrogen, halogen, aliphatic group, aromatic Groups, etc.
  • R 4 is a fragment of a polysulfhydryl compound
  • the system further contains at least one of the following substances:
  • Small molecule crosslinking agent containing acryloyl group Small molecule crosslinking agent containing acryloyl group.
  • the second aspect of the present invention is to provide a method for preparing the above-mentioned hydrogel, which includes the following steps:
  • the hydrogel is prepared.
  • the third aspect of the present invention is to provide a use of the above-mentioned hydrogel, which is used in the fields of biomedicine, medical beauty and plastic surgery, and cosmetics.
  • the present invention provides a kind of hydrogel with innovative structure obtained by cross-linking reaction using sulfhydryl modified polymer compound with innovative structure as raw material, and the hydrogel is compared with water in the prior art.
  • Gels (such as hydrogels obtained by cross-linking reactions using hyaluronic acid or modified hyaluronic acid as starting materials in the prior art) are in terms of physical and chemical properties, styling effects, and metabolic and degradation resistance. Has unexpected technical advantages.
  • the hydrogel of the present invention has the following advantages: 1. In the process of modifying and reforming the structure of the polymer compound, and in the subsequent cross-linking reaction process, no toxic epoxy-based small molecules are used for cross-linking.
  • the hydrogel product has the advantage of being safer. 2.
  • the hydrogel product of the present invention compared with the hydrogel in the prior art (for example, the existing cross-linked hyaluronic acid hydrogel), has better technologies such as viscosity, water retention, and shaping effects Advantage. 3.
  • the hydrogel of the present invention can realize the cross-linking reaction without adding any catalyst, and the reaction conditions are easier to realize, which are superior to the cross-linking reaction conditions of polymer compounds in the prior art, and are also superior to those in the prior art
  • in-situ cross-linking under real physiological conditions is realized, and the end point of the cross-linking reaction is controllable. Its controllability is not only reflected in the cross-linking reaction in vitro, but also in the body of animals or humans.
  • Cross-linking reaction A large number of animal experiments have proved that the end-point of the cross-linking reaction is single, stable and reproducible, whether in vivo or in vitro. 5.
  • Experimental studies have shown that the series of hydrogels of the present invention have better stability, resistance to degradation, and better metabolism resistance in animals under room temperature and accelerated stability inspection conditions.
  • the invention realizes in-situ cross-linking under physiological conditions in a true sense, that is, the cross-linking reaction can be completed under room temperature and normal pressure; or after being injected into the tissues of animals or humans, it can still be in the tissues.
  • the cross-linking reaction is realized, which obviously improves the degradation resistance and metabolism resistance of the hydrogel product, and obviously improves the use effect of the hydrogel injection product.
  • controllability of the degree of cross-linking of the hydrogel product injected into the animal body or human body can be realized before the cross-linking or mixing stage of the body, that is, the controllability of the degree of cross-linking of the hydrogel product injected into the animal body Or the human body can achieve a cross-linking reaction with a controllable end point of the cross-linking reaction, which ensures the safety and therapeutic effect of the product itself.
  • the present invention also proposes a method for preparing the hydrogel.
  • the method can complete the reaction at room temperature and normal pressure.
  • the reaction conditions are mild and easy to achieve, which is to realize the preparation of in-situ cross-linked water under physiological conditions.
  • the technical basis of the gel is to realize the preparation of in-situ cross-linked water under physiological conditions.
  • Figure 12 The shaping and supporting effect (height) of the hydrogel sample in vivo
  • Figure 25 The structural formula of HA-A1-SH1 and its 1 H-NMR spectrum
  • Figure 54 The structural formula of HA-MA1-SH6 and its 1 H-NMR spectrum
  • the system to be gelled in the present invention needs to use at least one of the following series of compounds:
  • the structure of the modified polymer compounds contains -COOH, -NH 2 , -OH, acrylate groups represented by formula a, acrylamide groups represented by formula b, and at least one of the acryl group represented by c,
  • part or all of the -COOH and/or -NH 2 and/or -OH and/or acrylate groups and/or acrylamide groups and/or acryl groups are modified to form end groups Is the side chain of the following groups:
  • * represents the point of attachment
  • R 1 is selected from hydrogen, halogen, aliphatic group, aromatic group, etc.; specifically, the halogen, aliphatic group, and aromatic group meet the further definition below; preferably, R 1 is selected from hydrogen, halogen, Fatty group; also preferably, R 1 is selected from hydrogen, halogen, C1-6 alkyl (such as methyl, ethyl, etc.);
  • R 2 and R 3 are the same or different, and are independently selected from hydrogen, halogen, aliphatic group, aromatic group, etc.; specifically, the halogen, aliphatic group, and aromatic group meet the further definitions below;
  • R 4 is a multimercapto compound fragment.
  • part of the -COOH and/or -NH 2 and/or -OH and/or acrylate group and/or acrylamide group and/or acryl group All are modified to form at least one of the following structures:
  • R is selected from Hydrocarbylene, arylene, amide residue, hydrazide residue, etc.; * indicates the point of attachment; 1 * indicates the point of attachment to the left group of R; 2 * indicates the point of attachment to the right group of R; R 1 , R 2 , R 3 and R 4 are as defined above.
  • At least one of -COOH, -NH 2 , -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 It is directly connected to the main chain of the polymer compound, or it can be connected to the main chain of the polymer compound through the R'group.
  • the R' can be a group containing a heteroatom, a hydrocarbylene group, an arylene group or a lower
  • R" is a hydrocarbylene group or an arylene group
  • n' is an integer from 1 to 1000
  • * represents the point of attachment.
  • heteroatom-containing groups include, but are not limited to, ester groups, amide residues or hydrazide residues.
  • ester groups include, but are not limited to, ester groups, amide residues or hydrazide residues.
  • the ester group, amide residue or hydrazide residue meets the further definition below.
  • the modified polymer compound includes natural mucopolysaccharide polymers, such as chitosan (specifically, chitosan, glycol chitosan, carboxymethyl chitosan, etc.), chondroitin sulfate , At least one of hyaluronic acid, alginate, etc.; proteins, such as gelatin, fibrin, serum protein, etc.; and/or, synthetic polymers, such as polyvinyl alcohol, poly(meth)acrylic acid, poly(meth) At least one of hydroxyalkyl acrylate (for example, polyhydroxyethyl (meth)acrylate, etc.), hyperbranched polyethylene glycol, and the like.
  • natural mucopolysaccharide polymers such as chitosan (specifically, chitosan, glycol chitosan, carboxymethyl chitosan, etc.), chondroitin sulfate , At least one of hyaluronic acid, alginate, etc.
  • the mercapto group content of the mercapto-modified polymer compound detected by the Ellman method is 0.01-30 mmol/g, for example, 0.1-10.0 mmol/g, for example, 0.3-5.0 mmol/g, and for example, 0.5-3.0 mmol/g.
  • the molecular weight of the mercapto-modified polymer compound is basically unchanged from the molecular weight of the polymer compound before modification.
  • the mercapto-modified polymer compound of the present invention includes at least one of the following structures:
  • A means that the structure contains at least one -COOH, -NH 2 , -OH, an acrylate group represented by formula a, an acrylamide group represented by formula b, and an acryl group represented by formula c Fragments of the modified polymer compound;
  • R, R', R 1 , R 2 , R 3 and R 4 have the same definitions as before; (n2+n3)/(n1+n2+n3) represents the degree of acrylation;
  • n3/(n1+n2+n3) represents the degree of sulfhydrylization, which corresponds to the sulfhydryl content of the sulfhydryl modified polymer compound detected by the Ellman method; the n1 can be 0, if it is 0, it is not used To limit the degree of acrylation, only n3/(n2+n3) represents the degree of sulfhydrylation, which corresponds to the sulfhydryl content of the sulfhydryl-modified polymer compound detected
  • the A may have the following structure:
  • * represents the connection point between the main chain repeating units; ** represents -COOH, -NH 2 , -OH, acrylate groups represented by formula a, acrylamide groups represented by formula b, The connection point between the acryl-based group represented by formula c and the aforementioned fragment, or the connection point between the R'group and the aforementioned fragment.
  • the A can also be the following polymers Gelatin-A, Gelatin-MA, CTS-A, CTS-MA, PHEMA-A, PHEMA-MA, HB-PEG, PVA-A, PVA-MA, CHS-A or The remaining fragments or repeating units after removing the acryl-containing side chain in CHS-MA:
  • Gelatin-A, Gelatin-MA, CTS-A, CTS-MA, PHEMA-A, PHEMA-MA, HB-PEG, PVA-A, PVA-MA, CHS-A or CHS-MA are respectively The abbreviation of the name of the polymer having the above structure, where the letters are separated, are not related to the meaning of the letters appearing in other parts of the present invention.
  • n, n', n1, n2, n3, n4, n5, n6, m1, m2, i, j, k1, and h all refer to the number of repeating units appearing in the structural formula. number. Its value range belongs to the conventional range known in the art.
  • the series of mercapto-modified polymer compounds are specifically:
  • * represents the point of attachment
  • R 1 is selected from hydrogen, halogen, aliphatic group, aromatic group, etc.; specifically, the halogen, aliphatic group, and aromatic group meet the further definition below; preferably, R 1 is selected from hydrogen, halogen, Fatty group; also preferably, R 1 is selected from hydrogen, halogen, C1-6 alkyl (such as methyl, ethyl, etc.);
  • R 2 and R 3 are the same or different, and are independently selected from hydrogen, halogen, aliphatic group, aromatic group, etc.; specifically, the halogen, aliphatic group, and aromatic group meet the further definitions below;
  • R 4 is a multimercapto compound fragment.
  • the end group is connected to -COOH and/or -OH through the R group or directly connected to -COOH and/or -OH to form a side chain of at least one of the following structures:
  • R is selected from Hydrocarbylene, arylene, amide residue, hydrazide residue, etc.; * indicates the point of attachment; 1 * indicates the point of attachment to the left group of R; 2 * indicates the point of attachment to the right group of R; R 1 , R 2 , R 3 and R 4 are as defined above.
  • the molecular weight of the mercapto-modified hyaluronic acid ranges from 5,000 to 20 million Daltons.
  • the molecular weight of the mercapto-modified hyaluronic acid does not change much before and after the modification, or the molecular weight is basically unchanged.
  • the mercapto group content of the mercapto-modified hyaluronic acid detected by the Ellman method is 0.01-30 mmol/g, for example, 0.1-10.0 mmol/g, for example, 0.3-5.0 mmol/g, and for example, 0.5-3.0 mmol/g.
  • the mercapto-modified hyaluronic acid of the present invention includes at least one of the following structures:
  • R, R 1 , R 2 , R 3 and R 4 are the same as before; (n2+n3)/(n1+n2+n3) represents the degree of acryl acylation; n3/(n1+n2+n3) Indicates the degree of sulfhydrylation, which corresponds to the sulfhydryl content of the sulfhydryl-modified polymer compound detected by the Ellman method; the n1 can be 0, if it is 0, the degree of acrylation is not limited, only n3 /(n2+n3) represents the degree of mercaptolation, which corresponds to the mercapto group content of the mercapto-modified polymer compound detected by the Ellman method; the n2 can be 0, if it is 0, then n3/(n1 +n3) represents both the degree of acrylation and the degree of sulfhydrylation, which corresponds to the sulfhydryl content of the sulfhydryl
  • the A 1 is:
  • the A 2 is one of the following structures:
  • a 1 and A 2 indicate the connection point with COOH or OH.
  • the mercapto-modified hyaluronic acid has at least one of the following structures, but is not limited to the following structures:
  • n 1 , n 2 and n 3 are as defined above.
  • R 4 is a polysulfhydryl compound fragment.
  • the -SR 4 -SH fragment can be introduced by, but not limited to, the following polysulfhydryl compounds:
  • n5 an integer of 1-30, such as 1, 2, 3, 4, 5, etc.
  • n6 1-30 The integer of, for example, 1, 2, 3, 4, 5, etc.
  • 4-arm-PEG-SH represents a PEG polymer containing four sulfhydryl groups
  • 6-arm-PEG-SH represents a PEG polymer containing six sulfhydryl groups
  • 8-arm-PEG-SH represents a PEG polymer containing eight sulfhydryl groups PEG polymer
  • the PEG is an abbreviation for polyethylene glycol.
  • R 1 is selected from hydrogen, halogen, aliphatic group, aromatic group, etc.
  • R 2 and R 3 are the same or different and are independently selected from hydrogen, halogen, aliphatic group, aromatic group and the like.
  • the R can be selected from alkylene groups, arylene groups, amide residues, hydrazide residues and the like.
  • the R' can be selected from heteroatom-containing groups, hydrocarbylene groups, arylene groups, and the like.
  • the R" can be selected from hydrocarbylene, arylene and the like.
  • the halogen means 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 group used alone or as a suffix or prefix in the present invention 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.
  • alkyl used alone or as a suffix or prefix in the present invention is intended to include branched and straight chain saturated aliphatic hydrocarbon groups having 1 to 20, preferably 1 to 6 carbon atoms.
  • C 1-6 alkyl means straight and branched chain alkyl groups having 1, 2, 3, 4, 5, or 6 carbon atoms.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.
  • Alkenyl used alone or as a suffix or prefix in the present invention is intended to include those having 2 to 20, preferably 2-6 carbon atoms (or if a specific number of carbon atoms is provided, then that specific number). Alkenyl or olefin branched and straight chain aliphatic hydrocarbon groups. For example, “C 2-6 alkenyl” means an alkenyl group having 2, 3, 4, 5, or 6 carbon atoms.
  • 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 group used alone or as a suffix or prefix in the present invention is intended to include those having 2 to 20, preferably 2-6 carbon atoms (or if a specific number of carbon atoms is provided, the specific number is referred to).
  • Alkynyl or branched and straight chain aliphatic hydrocarbon groups of alkynes For example, ethynyl, propynyl (e.g., 1-propynyl, 2-propynyl), 3-butynyl, pentynyl, hexynyl, and 1-methylpent-2-ynyl.
  • the aromatic group refers to an aromatic ring structure composed of 5 to 20 carbon atoms.
  • aromatic ring structures containing 5, 6, 7 and 8 carbon atoms can be monocyclic aromatic groups such as phenyl; ring structures containing 8, 9, 10, 11, 12, 13, or 14 carbon atoms It may be polycyclic such as naphthyl.
  • the aromatic ring may be substituted with substituents at one or more ring positions, and the substituents are alkyl, halogen, etc., such as tolyl.
  • aryl also includes polycyclic ring systems having two or more rings in which two or more carbons are shared by two adjacent rings (the rings are "fused rings"), in which at least One ring is aromatic and the other ring may be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and/or heterocyclyl.
  • polycyclic rings include, but are not limited to, 2,3-dihydro-1,4-benzodioxane and 2,3-dihydro-1-benzofuran.
  • the "hydrocarbylene group” in the present invention is a group obtained by removing one hydrogen from the above-mentioned "hydrocarbyl group”.
  • the "arylene group” in the present invention is a group obtained by removing one hydrogen from the above-mentioned "aromatic group”.
  • alkylene in the present invention is a group obtained by removing one hydrogen from the above "alkyl”.
  • the following groups substituted by b alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, etc.;
  • R b is selected from unsubstituted or optionally One or more of the following groups substituted by R b1 : halogen, hydroxy, mercapto, nitro, cyano, alkyl, alkoxy, cycloalkyl, alkenyl, alkynyl, heterocyclyl, aryl, heteroaryl Group, amino group, carboxyl group, ester group, hydrazine group, acyl group, sulfinyl group, sulfonyl
  • the "amide residue" in the present invention is a group obtained by removing one hydrogen from the aforementioned "amide group”.
  • the "hydrazide residue" in the present invention is a group obtained by removing one hydrogen from the above "hydrazide group”.
  • cycloalkyl as used in the present invention 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.
  • C 3-6 cycloalkyl means a group such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.
  • cycloalkenyl used in the present invention is intended to include a cyclic group containing at least one alkenyl group having the specified number of carbon atoms. These terms may include fused or bridged polycyclic ring systems.
  • the cycloalkenyl group has 3 to 40 carbon atoms in its ring structure. In one embodiment, the cycloalkenyl group has 3, 4, 5, or 6 carbon atoms in its ring structure.
  • C 3-6 cycloalkenyl means a group such as cyclopropenyl, cyclobutenyl, cyclopentenyl, or cyclohexenyl.
  • cycloalkynyl as used in the present invention 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.
  • the cycloalkynyl group has 6 to 40 carbon atoms in its ring structure. In one embodiment, the cycloalkynyl group has 6 carbon atoms in its ring structure.
  • C 3-6 cycloalkynyl means a group such as cyclopropynyl, cyclobutynyl, cyclopentynyl, or cyclohexynyl.
  • heteroaryl used in the present invention refers to a heteroaromatic heterocyclic ring having at least one ring heteroatom (for example, sulfur, oxygen, or nitrogen).
  • Heteroaryl groups include monocyclic ring systems and polycyclic ring systems (e.g., having 2, 3, or 4 fused rings).
  • heteroaryl groups include, but are not limited to, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolinyl, isoquinolinyl, thienyl, imidazolyl, thiazolyl, indole Base, pyrrolyl, oxazolyl, benzofuranyl, benzothienyl, benzothiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4 -Thiadiazole, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, benzoxazolyl, azabenzoxazolyl, imidazothiazolyl, benzo[1 ,4]dioxolyl, benzo[1,3]dioxolyl and the like
  • the heteroaryl group has 3 to 40 carbon atoms and in other embodiments has 3 to 20 carbon atoms. In some embodiments, the heteroaryl group contains 3 to 14, 4 to 14, 3 to 7, or 5 to 6 ring-forming atoms. In some embodiments, the heteroaryl group has 1 to 4, 1 to 3, or 1 to 2 heteroatoms. In some embodiments, the heteroaryl group has 1 heteroatom.
  • heterocyclic group refers to a saturated, unsaturated or partially saturated monocyclic, bicyclic or tricyclic ring containing 3 to 20 atoms, wherein 1, 2, 3, 4 or 5 ring atoms are selected from Nitrogen, sulfur, oxygen, or phosphorus, unless otherwise specified, can be connected by carbon or nitrogen, where the -CH 2 -group is optionally replaced by -C(O)-; where unless otherwise specified to the contrary, the ring nitrogen atom Or ring sulfur atoms are optionally oxidized to form N-oxides or S-oxides or ring nitrogen atoms are optionally quaternized; wherein -NH in the ring is optionally acetyl, formyl, methyl or methanesulfonate Acyl substitution; and the ring is optionally substituted with one or more halogens.
  • heterocyclic group when the total number of S atoms and O atoms in the heterocyclic group exceeds 1, these heteroatoms are not adjacent to each other.
  • the heterocyclic group 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 must not be aromatic.
  • heterocyclic groups include, but are not limited to, piperidinyl, N-acetylpiperidinyl, N-methylpiperidinyl, N-formylpiperazinyl, N-methanesulfonylpiperazinyl, homopiperazinyl , Piperazinyl, azetidinyl, oxetanyl, morpholinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, indolyl, tetrahydropyranyl, dihydro -2H-pyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydrothiopyran-1-oxide, tetrahydrothiopyran-1,1-dioxide, 1H-pyridin-2-one and 2,5 -Dioxoimidazolidinyl.
  • hydrazino used in the present invention refers to the -NHNHR a group, and the definition of Ra is the same as above.
  • amino group used in the present invention refers to a -NHR a group or a -N(R a ) 2 group, and the definition of Ra is the same as above.
  • amino refers to the -NH 2 group.
  • carboxy refers to the -COOH group.
  • the present invention provides a method for preparing the above-mentioned mercapto-modified polymer compound, which includes the following steps:
  • At least one polymer compound of acrylation step is -OH, -COOH-containing polymer compound of the structure is about, -NH 2, -OH at least one of Kind of directly or indirectly connected with the following groups:
  • R 1 , R 2 and R 3 are the same as before; * indicates the connection point;
  • a polymer compound containing at least one of the acrylate group represented by the above formula a, the acrylamide group represented by the above formula b, and the acrylamide group represented by the above formula c in structure is directly used as the reaction raw material;
  • step 2) Reacting at least one of the polymer compounds obtained in step 1) with the polymercapto compound HS-R 4 -SH, where R 4 has the same definition as before, to prepare the mercapto-modified polymer compound.
  • the method includes the following steps:
  • R, R 1 , R 2 and R 3 are the same as before, and * means the connection point;
  • a polymer compound 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 in structure is directly used as a reaction raw material;
  • step 2) Reacting at least one of the polymer compounds obtained in step 1) with the polymercapto compound HS-R 4 -SH, where R 4 has the same definition as before, to prepare the mercapto-modified polymer compound.
  • a method for preparing the above-mentioned mercapto-modified hyaluronic acid includes the following steps:
  • the acrylation step of hyaluronic acid that is, at least one of -COOH and -OH contained in the side chain of the repeating unit of hyaluronic acid is directly or indirectly connected to the following groups:
  • R 1 , R 2 and R 3 are the same as before; * indicates the connection point;
  • the step 1) is: the acrylylation step of hyaluronic acid is to connect at least one of -COOH and -OH contained in the side chain of the repeating unit of hyaluronic acid to the terminal through the R group Or directly connected to the end group to form a side chain of at least one of the following structures:
  • the acrylation step can be realized by the reaction of the polymer compound to be modified and the acrylate compound, or it can be realized by the polymer compound to be modified and the acryl chloride compound or acrylic anhydride. The reaction of the compound is achieved.
  • the acrylic compound may be one or more of an alkyl acrylate compound, an aryl acrylate compound, and a polyhydric alcohol acrylate compound.
  • the polyhydric alcohol in the glycidyl acrylate compound is, for example, a trihydric alcohol, and specifically may be glycerin, butanetriol, pentatriol, and the like.
  • the acryl acylation step can be a conventional reaction step, and the reaction can be performed under existing conventional conditions. It is usually obtained by reacting acryloyl chloride and its derivatives or acrylic anhydride and its derivatives with a polymer compound containing at least one of -OH and -NH 2. It can also be obtained by reacting glycidyl acrylate and its derivatives with a polymer compound containing at least one of -COOH, -OH, and -NH 2.
  • the acryl acylation step can be an unconventional reaction step, which is a polymer compound containing a structure of formula c synthesized by a method other than the above.
  • step 2) the reaction with the polymercapto compound HS-R 4 -SH is carried out in a solvent.
  • the solvent is, for example, water or an organic solvent, and may further be deionized water or dimethylformamide.
  • step 2) the reaction with the polymercapto compound HS-R 4 -SH is carried out under low to high temperature conditions.
  • the reaction temperature is 0-80°C, and further may be 10-70°C, for example, the reaction can be performed at room temperature.
  • step 2) the reaction time for the reaction with the polymercapto compound HS-R 4 -SH is 0.1-100 hours.
  • step 2) the pH range of the reaction with the polysulfhydryl compound HS-R 4 -SH is -1 to 15.
  • the reaction pH can be 6-8, and for example, it is 7.
  • reaction product of step 2) further includes a post-treatment step.
  • the post-processing step adopts a dialysis method.
  • a dialysis bag for example, a dialysis bag with a molecular weight cut-off of 2kDa or above
  • the water such as changing the water every day or every other day
  • dry such as freeze-drying
  • the method of the present invention proposes for the first time the Michael addition reaction of the sulfhydryl group of the polysulfhydryl compound and the carbon-carbon double bond in the acryl group to prepare the sulfhydryl modified polymer compound.
  • This method not only has a high degree of sulfhydrylization, but also a sulfhydryl group.
  • the chemical reaction conditions are mild (can be carried out in an aqueous solution at room temperature) and pollution-free, and the prepared mercapto-modified polymer compound has high purity, which is particularly suitable for further use in the fields of medicine, cosmetology, and medicine.
  • the to-be-gelled system of the present invention may also include material C1.
  • Acrylated polymer compound, the acrylated polymer compound of the present invention may be selected from at least one of the following substances:
  • An acryloyl compound of a polymer compound that contains at least one of -COOH, -NH 2 , and -OH in the structure that is, the polymer compound contains -COOH, -NH 2 , and -OH in the structure
  • R 1 , R 2 and R 3 are the same as before, and * means the connection point;
  • R is selected from Hydrocarbylene, arylene, amide residue, hydrazide residue, etc.; * indicates the point of attachment; 1 * indicates the point of attachment to the left group of R; 2 * indicates the point of attachment to the right group of R; R 1 , R 2 , R 3 and R 4 are as defined above.
  • At least one of -COOH, -NH 2 and -OH may be directly connected to the main chain of the polymer compound, or may be connected to the polymer compound through the R'group On the main chain of, the R'can be a group containing a heteroatom, a hydrocarbylene group, an arylene group or the following linking group:
  • R" is a hydrocarbylene group or an arylene group
  • n' is an integer from 1 to 1000
  • * represents the point of attachment.
  • heteroatom-containing groups include, but are not limited to, ester groups, amide residues or hydrazide residues. Specifically, the ester group, amide residue or hydrazide residue meets the further definition herein.
  • the polymer compound to be acrylylated includes natural mucopolysaccharide polymers, such as chitosan (specifically, chitosan, ethylene glycol chitosan, carboxymethyl chitosan, etc.). Sugars, etc.), at least one of chondroitin sulfate, hyaluronic acid, alginate, etc.; proteins, such as gelatin, fibrin, serum proteins, etc.; and/or, synthetic polymers, such as polyvinyl alcohol, poly(methyl) At least one of acrylic acid, polyhydroxyalkyl (meth)acrylate (for example, polyhydroxyethyl (meth)acrylate, etc.), hyperbranched polyethylene glycol, and the like.
  • chitosan specifically, chitosan, ethylene glycol chitosan, carboxymethyl chitosan, etc.
  • Sugars, etc. at least one of chondroitin sulfate, hyaluronic acid, alginate, etc
  • the acryloyl compound includes at least one of the following structures:
  • A is the fragment of the compound to be acrylylated containing at least one -COOH, -NH 2 , -OH on the structure;
  • R, R', R 1 , R 2 , R 3 and R 4 have the same definitions Before;
  • (m2/(m1+m2) represents the degree of acryloyl acylation.
  • the A may have the following structure:
  • * represents the connection point between the main chain repeating units; ** represents the connection point between -COOH, -NH 2 , -OH and the above fragments, or between the R'group and the above fragments Junction.
  • the above-mentioned second) substance can be the following polymers Gelatin-A, Gelatin-MA, CTS-A, CTS-MA, PHEMA-A, PHEMA-MA, HB-PEG, PVA-A, PVA-MA, CHS- One of A or CHS-MA:
  • Gelatin-A, Gelatin-MA, CTS-A, CTS-MA, PHEMA-A, PHEMA-MA, HB-PEG, PVA-A, PVA-MA, CHS-A or CHS-MA are respectively The abbreviation of the name of the polymer having the above structure, where the letters are separated, are not related to the meaning of the letters appearing in other parts of the present invention.
  • the system to be gelled in the present invention may also include substance C2.
  • Acrylic-containing small molecule crosslinking agent includes but not limited to acryl-containing small molecule compound or acryl-containing small molecule compound
  • the oligomer; specific can be selected from ethylene glycol diacrylate EGDA, polyethylene glycol diacrylate PEGDA, trimethylolpropane triacrylate TMPTA, pentaerythritol triacrylate PTA, pentaerythritol tetraacrylate PTTA, two (Trimethylolpropane) tetraacrylate DTTA and the like.
  • the present invention provides a hydrogel prepared by gelation of a system containing the following substances:
  • the hydrogel is prepared by gelation of the above-mentioned mercapto group-modified polymer compound and the above-mentioned acryloyl polymer compound.
  • the cross-linking reaction occurs after the mercapto-modified polymer compound and the acryloyl polymer compound are fully contacted, the viscosity of the mixed system increases immediately, and a uniform gel system is finally formed.
  • the hydrogel is prepared by gelation of the above-mentioned mercapto-modified polymer compound and the above-mentioned small molecule crosslinking agent.
  • the cross-linking reaction occurs after the mercapto-modified polymer compound and the small-molecule cross-linking agent are fully contacted, the viscosity of the mixed system increases immediately, and a uniform gel system is finally formed.
  • the hydrogel is prepared by gelation of the above-mentioned mercapto-modified polymer compound, the above-mentioned acryloyl polymer compound and the above-mentioned small molecule crosslinking agent.
  • the cross-linking reaction occurs after the mercapto-modified polymer compound is fully contacted with the acrylic polymer compound and the small molecule cross-linking agent, the viscosity of the mixed system increases immediately, and a uniform gel system is finally formed.
  • hydrogel includes the following characteristic structural units:
  • R 1 , R 2 , R 3 and R 4 have the same definitions as before, and * indicates the connection point.
  • the amount ratio of the mercapto-modified polymer compound to the acryloyl polymer compound is 0.01:0.99 to 0.99:0.01.
  • it can 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.
  • the dosage ratio of the mercapto-modified polymer compound and the small molecule crosslinking agent is 0.01:0.99 to 0.99:0.01.
  • it can 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.
  • the dosage ratio of the mercapto-modified polymer compound to the acryloyl polymer compound and the small molecule crosslinking agent is 0.01:0.99 to 0.99:0.01.
  • it can 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.
  • the acrylic polymer compound and the small molecule crosslinking agent can be mixed in any ratio.
  • the hydrogel of the present invention is formed by the addition reaction of the thiol group (-SH) of the mercapto-modified polymer compound and the carbon-carbon double bond of the substance C1 and/or the substance C2 through the addition reaction of the thiol group and the carbon-carbon double bond
  • thiol group thiol group of the mercapto-modified polymer compound
  • the carbon-carbon double bond of the substance C1 and/or the substance C2 through the addition reaction of the thiol group and the carbon-carbon double bond
  • a stable cross-linked material, the cross-linked material ie hydrogel
  • the cross-linked material ie hydrogel
  • C2 small molecule crosslinking agent
  • C1 acryloyl polymer compound
  • the substance C1 can also be added to the gel system by physical mixing, so as to achieve different application purposes.
  • the mercapto-modified polymer compound is used in combination with the C1 (acryloyl polymer compound) and/or C2 (small molecule crosslinking agent) to complement each other, thereby obtaining a three-dimensional scaffold material with excellent properties. It can meet the application requirements of most tissue engineering.
  • other biological functional materials such as hyaluronic acid, collagen, gelatin, chondroitin sulfate, chitosan, sodium alginate, etc.
  • drugs, and growth factors can be further added to the system. Or at least one of cell suspensions and the like.
  • the addition of other biological functional materials can bring additional effects to the hydrogel of the present invention.
  • the introduction of unmodified hyaluronic acid can increase the wound healing effect of the hydrogel, and the introduction of collagen or gelatin can make the hydrogel system more effective.
  • the introduction of chondroitin sulfate can enhance the cartilage repair effect of the hydrogel system
  • the introduction of positively charged biological materials such as chitosan can increase the antibacterial effect of the hydrogel
  • the introduction of sodium alginate can enhance hydration The mechanical strength of the glue system.
  • the present invention provides a method for preparing the above-mentioned hydrogel, which includes the following steps:
  • the hydrogel is prepared.
  • the method includes the following steps: gelling a system containing the following substances:
  • the hydrogel is prepared.
  • a solution of at least one of the cell suspensions is mixed and gelled to prepare the hydrogel.
  • at least one of the other biological functional materials, drugs, growth factors or cell suspensions can also be directly added to the solution of the sulfhydryl-modified polymer compound or the solution of the acryloyl polymer compound or the cell suspension.
  • the small molecule crosslinking agent is introduced into the solution.
  • the gel preparation process can be added to the acryloyl polymer compound solution and/or the small molecule crosslinking agent solution through the mercapto-modified polymer compound solution, or through the The acryloyl polymer compound solution and/or the small molecule crosslinking agent solution are added to the mercapto-modified polymer compound solution.
  • the two solutions can be mixed by a common syringe, can also be mixed by a double-needle syringe, or can be mixed by other means.
  • the mass volume concentration of the solution of the mercapto-modified polymer compound is 0.1% to 95%, for example, 1% to 90%, and further, for example, can be 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%.
  • the pH of the solution can be adjusted to 7.4 by adding acid, alkali or buffer solution.
  • the buffer solution may be a phosphate buffer solution.
  • the mass volume concentration of the solution of the acryloyl polymer compound is 0.1% to 95%, for example, 1% to 90%, and further, for example, can be 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%.
  • the pH of the solution can be adjusted to 7.4 by adding acid, alkali or buffer solution.
  • the buffer solution may be a phosphate buffer solution.
  • the mass volume concentration of the solution of the small molecule crosslinking agent is 0.1% to 95%, for example, 1% to 90%, and for example, can be 0.1%, 1%, 5%, 10%, 20%, 30%. %, 40%, 50%, 60%, 70%, 80%, 90%, 95%.
  • the pH of the solution can be adjusted to 7.4 by adding acid, alkali or buffer solution.
  • the buffer solution may be a phosphate buffer solution.
  • the two solutions can be mixed in any ratio, for example, can be mixed in equal volumes.
  • hydrogels are a three-dimensional grid formed by cross-linking hydrophilic polymer segments that can be swelled in water.
  • the gelation process can be achieved through different reaction mechanisms, including physical entanglement of polymer chain segments, electrostatic interaction, covalent chemical crosslinking, reversible chemical crosslinking, supramolecular chemical crosslinking, and hydrophilic and hydrophobic crosslinking.
  • hydrogels have been widely used in the field of medicine, such as the preparation of drug delivery systems, soft tissue wound repair dressings, scaffold materials for bone repair, and ophthalmic surgery. Viscoelastics used for support, materials used to prevent tissue adhesion after surgery, and scaffold materials used for 3D bioprinting have become research hotspots in the field of tissue engineering and regenerative medicine.
  • the hydrogel of the present invention is particularly suitable for the fields of biomedicine, medical cosmetology and plastic surgery, and cosmetics. Specifically, it can be used to prepare drug delivery systems, dressings for soft tissue wound repair, scaffold materials for bone repair, viscoelastics for support in ophthalmic surgery, materials for preventing tissue adhesion after surgery, and Scaffold materials for 3D bioprinting, etc.
  • the hydrogel of the present application realizes in-situ cross-linking under physiological conditions in a true sense, that is, the cross-linking reaction can be spontaneously completed under room temperature and normal pressure; or after being injected into the tissues of animals or humans, The cross-linking reaction can still be realized in the tissue, which obviously improves the degradation resistance and metabolism resistance of the hydrogel product, and obviously improves the use effect of the hydrogel injection product.
  • controllability of the degree of cross-linking of the hydrogel product injected into the animal body or human body can be realized before the cross-linking or mixing stage of the body, that is, the controllability of the degree of cross-linking of the hydrogel product injected into the animal body Or the human body can achieve a cross-linking reaction with a controllable end point of the cross-linking reaction, which ensures the safety and therapeutic effect of the product itself.
  • the 1 H-NMR spectrum is measured by a Varian 400MHz nuclear magnetic resonance instrument, the test temperature is 25 degrees Celsius, the relaxation time is 1 second, and the number of scans is 8 times. Specifically, 8-10 mg of the test substance is taken and dissolved in 750 microliters of deuterated water, and the 1 H-NMR spectrum of the obtained sample solution is tested.
  • the storage modulus of the present invention is based on the measurement of the rheological mechanical properties of the hydrogel.
  • the detection instrument is a TA-DHR2 rheometer
  • the detection probe is 20mm parallel plate probe
  • the detection temperature 25°C
  • the shear frequency 1Hz
  • Shear strain 1%.
  • the following MTT method refers to a detection method for measuring the survival rate of cells by metabolic activity.
  • the yellow aqueous solution MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] is metabolically reduced in living cells to produce blue-purple insoluble formazan.
  • the number of living cells is related to the color measured by a photometer after formazan is dissolved in alcohol.
  • hyaluronic acid purchased from Huaxi Freda Company, its weight average molecular weight is about 300kDa
  • 50 ml of deionized water 50 ml of dimethylformamide, 12 ml of triethylamine, 14 ml glycidyl acrylate.
  • Adding 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 obtain a colorless and transparent solution.
  • the above solution was put into a dialysis bag (molecular weight cutoff 8kDa), and dialyzed with 5 liters of deionized water for 5 days, and the water was changed twice a day. Finally, the solution in the dialysis bag was collected, and 921 mg of white flocculent solid was obtained after freeze-drying, namely HA-A1, with a yield of 92.1%.
  • m2/(m1+m2) represents the degree of acrylation
  • the above solution was put into a dialysis bag (with a molecular weight cutoff of 8kDa, Spectra), and dialyzed with 5 liters of deionized water for 5 days, and the water was changed twice a day. Finally, the solution in the dialysis bag was collected, and 789 mg of white flocculent solid was obtained after freeze-drying, namely HA-A2, and the yield was 78.9%.
  • HA-A2 The structure of HA-A2 is shown in Figure 16.
  • hyaluronic acid purchased from Huaxi Freda Company, its weight average molecular weight is about 400kDa
  • 50 ml of deionized water 50 ml of dimethylformamide (Sigma)
  • 12 ml of three Ethylamine 12 ml of three Ethylamine (Sigma)
  • 15 ml glycidyl methacrylate After stirring at room temperature until homogeneous and transparent, continue stirring for 48 hours.
  • 300 ml of acetone (Sigma) was added, resulting in a large amount of white precipitate.
  • the precipitate obtained by centrifugation was dissolved in 100 ml of deionized water to obtain a colorless solution.
  • the above solution was put into a dialysis bag (with a molecular weight cutoff of 8kDa, Spectra), and dialyzed with 5 liters of deionized water for 5 days, and the water was changed twice a day. Finally, the solution in the dialysis bag was collected, and 859 mg of white flocculent solid was obtained after freeze-drying, namely HA-MA1, with a yield of 85.9%.
  • the above solution was put into a dialysis bag (with a molecular weight cutoff of 8kDa, Spectra), and dialyzed with 5 liters of deionized water for 5 days, and the water was changed twice a day. Finally, the solution in the dialysis bag was collected, and 846 mg of white flocculent solid was obtained after freeze-drying, namely HA-MA2, with a yield of 84.6%.
  • HA-MA2 The structure of HA-MA2 is shown in Figure 18.
  • the 1 H-NMR spectrum of HA-MA2 is shown in Figure 18.
  • the nuclear magnetic peaks belonging to the methacrylic functional group between 5.8 and 6.2 ppm can be seen, which proves that the group is successfully grafted into the structure of hyaluronic acid.
  • HA-A1 prepared according to the method of Preparation Example 1, 0.3 g of dithiothreitol (purchased from VWR Company), and 100 ml of deionized water into a 200 ml beaker, and stir to dissolve at room temperature to obtain a transparent solution. The resulting clear solution was continuously stirred for 12 hours.
  • the 1 H-NMR spectrum of HA-A1-SH1 is shown in Figure 25.
  • the nuclear magnetic peak of the side chain of the sulfhydryl group located between 2.3-2.8ppm can be seen, which proves that the sulfhydryl group is successfully grafted into the structure of hyaluronic acid.
  • HA-A2 prepared according to the method of Preparation Example 2, 0.3 g of dithiothreitol (VWR), and 100 ml of deionized water into a 200 ml beaker, and stir to dissolve at room temperature to obtain a transparent solution. The resulting clear solution was continuously stirred for 12 hours.
  • HA-MA1 prepared according to the method of Preparation Example 3, 0.3 g of dithiothreitol (VWR), and 100 ml of deionized water into a 200 ml beaker, and stir to dissolve at room temperature. The resulting clear solution was continuously stirred for 12 hours.
  • the HA-MA1-SH1 was obtained with a yield of 85.4%.
  • the 1 H-NMR spectrum of HA-MA1-SH1 is shown in Figure 27.
  • the nuclear magnetic peak of the side chain of the sulfhydryl group between 2.6-3.0 ppm can be seen, which proves that the sulfhydryl group is successfully grafted into the structure of hyaluronic acid.
  • HA-MA2 prepared according to the method of Preparation Example 4, 0.3 g of dithiothreitol (VWR), and 100 ml of deionized water into a 200 ml beaker, and stir to dissolve at room temperature. The resulting clear solution was continuously stirred for 12 hours.
  • the HA-MA2-SH1 was obtained with a yield of 83.3%.
  • chondroitin sulfate (with a weight average molecular weight of about 90 kDa), 50 ml of deionized water, 50 ml of dimethylformamide were added, and 6.5 g of methacrylic anhydride was further added with stirring to dissolve.
  • the above solution was put into a dialysis bag (with a molecular weight cut-off of 3.5 kDa, Spectra Pure), and dialyzed with 5 liters of deionized water for 5 days, and the water was changed twice a day. Finally, the solution in the dialysis bag was collected, and 776 mg of light yellow flocculent solid was obtained after freeze-drying, namely CHS-MA, with a yield of 64.7%.
  • the 1 H-NMR spectrum of Gelatin-MA is shown in Fig. 22.
  • the nuclear magnetic peaks belonging to the methacrylic functional group between 5.7 and 6.2 ppm can be seen, which proves that the group is successfully grafted into the structure of gelatin.
  • CTS-A The structure of CTS-A is shown in Figure 23.
  • CTS-MA The structure of CTS-MA is shown in Figure 24.
  • PHEMA-MA prepared according to the method of Preparation Example 22, 0.41 g of dithiothreitol (VWR), 50 ml of deionized water, and 50 ml of dimethylformamide, and stir to dissolve at room temperature. The resulting clear solution was continuously stirred for 12 hours.
  • HA-A1 prepared according to the method of preparation example 1, 0.42 g of 1,4-butanedithiol (purchased from Sigma), and 100 ml of deionized water, stir and dissolve at room temperature to obtain a transparent solution .
  • the resulting clear solution was continuously stirred for 12 hours.
  • HA-A1 prepared according to the method of preparation example 1, 0.43 g of 2-amino-1,4-butanedithiol hydrochloride (purchased from Sigma), and 100 ml of deionized water, Stir and dissolve at room temperature to obtain a transparent solution. The resulting clear solution was continuously stirred for 12 hours.
  • HA-A2 prepared according to the method of preparation example 2, 0.42 g of 1,4-butanedithiol (purchased from Sigma), and 100 ml of deionized water, stir and dissolve at room temperature to obtain a transparent solution .
  • the resulting clear solution was continuously stirred for 12 hours.
  • the 1 H-NMR spectrum of HA-A2-SH2 is shown in Figure 46.
  • the nuclear magnetic peak of the side chain of the sulfhydryl group located between 1.6-1.9ppm can be seen, which proves that the sulfhydryl group is successfully grafted into the structure of hyaluronic acid.
  • HA-A2 prepared according to the method of preparation example 2, 0.43 g of 2-amino-1,4-butanedithiol hydrochloride (Sigma), 100 ml of deionized water, and stir to dissolve at room temperature. , Get a transparent solution. The resulting clear solution was continuously stirred for 12 hours.
  • HA-A2 prepared according to the method of preparation example 2, 0.38 g of 1,3-propanedithiol (purchased from Sigma), 100 ml of deionized water, and stir to dissolve at room temperature to obtain a transparent solution .
  • the resulting clear solution was continuously stirred for 12 hours.
  • HA-A2 prepared according to the method of preparation example 2, 0.52 g of 1,3-benzenedithiol (purchased from Sigma), and 100 ml of deionized water. Stir and dissolve at room temperature to obtain a transparent solution. . The resulting clear solution was continuously stirred for 12 hours.
  • HA-A2 prepared according to the method of preparation example 2, 0.52 g of 1,4-benzenedithiophenol (purchased from Sigma), and 100 ml of deionized water, stir and dissolve at room temperature to obtain a transparent solution .
  • the resulting clear solution was continuously stirred for 12 hours.
  • the 1 H-NMR spectrum of HA-A2-SH6 is shown in Figure 50.
  • the nuclear magnetic peak of the side chain of the sulfhydryl group between 6.8-7.0 ppm can be seen, which proves that the sulfhydryl group is successfully grafted into the structure of hyaluronic acid.
  • HA-A2 prepared according to the method of Preparation Example 2 into a 200 ml beaker, 0.96 g of mercaptopolyethylene glycol (purchased from Sigma), and 100 ml of deionized water, stir and dissolve at room temperature to obtain a transparent solution. The resulting clear solution was continuously stirred for 12 hours.
  • HA-A2 prepared according to the method of preparation example 2, 0.74 g of trimethylolpropane-tris(3-mercaptopropionate) (purchased from Sigma), and 50 ml of deionized water And 50 ml of dimethylformamide, stir and dissolve at room temperature to obtain a transparent solution.
  • the resulting clear solution was continuously stirred for 12 hours.
  • the 1 H-NMR spectrum of HA-A2-SH8 is shown in Figure 52.
  • the nuclear magnetic peaks belonging to the side chain of the sulfhydryl group between 0.8-1.0ppm, 1.5ppm, and 2.6-2.9ppm can be seen, which proves that the sulfhydryl group is successfully grafted to hyaluronic acid. In the structure.
  • HA-MA1 prepared according to the method of Preparation Example 3, 0.50 g of 1,3-benzenedithiol (purchased from Sigma), and 100 ml of deionized water, stir and dissolve at room temperature to obtain a transparent solution .
  • the resulting clear solution was continuously stirred for 12 hours.
  • HA-MA1 prepared according to the method of preparation example 3, 0.50 g of 1,4-benzenedithiophenol (purchased from Sigma), and 100 ml of deionized water, stir and dissolve at room temperature to obtain a transparent solution .
  • the resulting clear solution was continuously stirred for 12 hours.
  • the 1 H-NMR spectrum of HA-MA1-SH6 is shown in Fig. 54.
  • the nuclear magnetic peaks belonging to the side chain of the sulfhydryl group located between 6.9-7.0ppm can be seen, which proves that the sulfhydryl group is successfully grafted into the structure of hyaluronic acid.
  • HA-MA2 prepared according to the method of preparation example 4, 0.92 g of mercaptopolyethylene glycol (purchased from Sigma), 100 ml of deionized water were added, and the mixture was stirred and dissolved at room temperature to obtain a transparent solution. The resulting clear solution was continuously stirred for 12 hours.
  • the 1 H-NMR spectrum of HA-MA2-SH7 is shown in Fig. 55.
  • the nuclear magnetic peak of the side chain of the sulfhydryl group at 3.6 ppm can be seen, which proves that the sulfhydryl group is successfully grafted into the structure of hyaluronic acid.
  • HA-MA2 prepared according to the method of preparation example 4, 0.68 g of trimethylolpropane-tris(3-mercaptopropionate) (purchased from Sigma), and 50 ml of deionized water And 50 ml of dimethylformamide, stir and dissolve at room temperature to obtain a transparent solution.
  • the resulting clear solution was continuously stirred for 12 hours.
  • the 1 H-NMR spectrum of HA-MA2-SH8 is shown in Figure 56.
  • the nuclear magnetic peaks belonging to the side chain of the sulfhydryl group between 0.8-1.0ppm, 1.5ppm, and 2.6-2.9ppm can be seen, which proves that the sulfhydryl group is successfully grafted to hyaluronic acid. In the structure.
  • Each hydrogel in Example 1 includes the following characteristic structural units:
  • R 1 , R 2 and R 3 are the same as before; * indicates the connection point.
  • Example 2 The two milliliters of the hydrogel mixed solution prepared in Example 1 was placed in a cylindrical mold, and after cross-linking at room temperature for 24 hours, the storage modulus of the tested cross-linked samples was taken out, and each group of samples was tested three times.
  • the hydrogel prepared in Example 1 was added to a 20 ml glass bottle weighing the bottle in advance, and the mass of the hydrogel obtained by the mass difference subtraction method was recorded as m 0 .
  • the glass bottle was placed in a shaker at 37°C, and the mass was weighed at regular intervals to obtain the real-time mass of the hydrogel, which was recorded as m t .
  • the water retention capacity of hydrogel is calculated according to the following formula:
  • Degradation stability test Under the experimental conditions of a temperature of 37 ⁇ 0.1°C and a relative humidity of 65% ⁇ 5%, 10 ml of PBS solution was added to the hydrogel prepared in Example 1. The weight of the hydrogel at the initial time point is recorded as m 0 , and the weight of the hydrogel is weighed at 1, 4, 8, and 16 weeks after the start of the degradation experiment and recorded as m t , and the degradation ratio of the hydrogel is calculated according to the following formula:
  • Degradation rate (%) (m 0 -m t )/m 0 ⁇ 100%
  • the cell activity and biocompatibility of the HA-SH of the present invention are tested with reference to the standard of "GBT 16886.5-2017 Medical Device Biological Evaluation + Part 5 + In Vitro Cytotoxicity Test". Specifically, the following MTT method is used.
  • the MTT method also known as MTT colorimetric method, is a method for detecting cell survival and growth.
  • the detection principle is that the succinate dehydrogenase in the mitochondria of living cells can reduce the exogenous MTT to water-insoluble blue-purple crystal formazan (Formazan) and deposit it in the cells, while dead cells have no such function.
  • DMSO Dimethyl sulfoxide
  • an enzyme-linked immunoassay which can indirectly reflect the number of living cells.
  • the amount of MTT crystal formation is proportional to the number of cells.
  • L929 cells are cultured routinely, and after the cells are nearly confluent, the cells are digested to obtain a cell suspension.
  • the three components of solution A, solution B, and cell suspension were uniformly mixed to prepare a cell/hydrogel composite system with a volume of 50 ⁇ L, and the final concentration of cells was 1 ⁇ 10 6 cell/mL.
  • the system was placed in a 24-well cell culture plate, and 1 mL of cell culture fluid was added to each well for culture, in which the same number of cells at the bottom of the plate served as a negative control group.
  • the samples were cultured in a cell incubator for 24 hours under the conditions of 5% CO 2, 37° C., and >90% humidity.
  • the MTT method was used to detect the viability of cells in different hydrogel samples, and the cell viability of the hydrogel group was compared with the cell viability of the negative control group.
  • the negative control group is 100% active.
  • the protuberances were photographed, measured with a vernier caliper, and recorded in detail.
  • the shaping effect of the hydrogel is judged by comparing the maintenance and change of the three-dimensional shape after different injection samples are injected into the animal body. The higher the elevation of the injection site and the smaller the bottom area, the better the shaping and supporting effect.
  • the results are shown in Figure 12 and Figure 13.
  • the data shows that the hydrogel of the present invention forms a support that can maintain a certain shape after being injected into an animal, and can better maintain the stability of the shape of the injection.
  • hydrogel of the present invention has superior performance in resistance to degradation and maintaining colloidal stability.

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Abstract

一种巯基改性高分子化合物的水凝胶及其制备方法，采用巯基改性高分子化合物与丙烯酰化高分子化合物和/或丙烯酰小分子交联剂配合，巯基改性高分子化合物可在生理条件下与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。由于巯基-乙烯基交联反应迅速的特点使得形成的水凝胶体系可以在注射入体内后快速原位成胶，水凝胶适用于生物医药、医疗美容整形以及化妆品领域。

Description

巯基改性高分子化合物的水凝胶及其制备方法和用途
本申请要求2019年11月18日向中国国家知识产权局提交的专利申请号为201911130069.5，发明名称为“巯基改性高分子化合物的水凝胶及其制备方法和用途”的在先申请的优先权，该在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于生物材料领域，具体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用途。
背景技术
生物医用材料，即Biomedical Materials，亦可简称为生物材料(英文为Biomaterials)，是用于对生物体进行诊断、治疗、修复或替换病变组织、器官或增进其功能的新型高技术材料。而组织工程学的关键技术之一是用生物材料制备具有良好的生物相容性且可被肌体降解吸收的细胞支架。凝胶态是固体和液体的中间状态，水凝胶是指能够在水中发生溶胀并能保持大量水分而又不会溶解的亲水交联三维聚合物网络，其含水量可以达到90％以上。水凝胶是一类理想的生物材料，其本身或者通过简单的改性，即可具有与天然细胞外基质类似、令人满意的物理和化学性质，同时对于氧气、营养物质、细胞代谢物和水溶性金属离子表现出良好的通透性。制备水凝胶的亲水性高分子按来源分为天然高分子和合成高分子。其中，天然高分子包括胶原、明胶、纤维蛋白、多糖等，合成高分子包括合成类多肽、聚乙二醇(PEG)及其衍生物、聚甲基丙烯酸甲酯(PMMA)及其衍生物、聚乳酸乙醇酸共聚物(PLGA)及其衍生物等。近年来，可注射的原位交联水凝胶也越来越受到人们的关注。可注射原位交联水凝胶的特征在于，在注射之前呈可流动的液态，当注射到目标位置后，可形成与目标位置形状完全吻合的胶体。这种可注射的特性，不仅使得操作过程简单方便，而且可避免植入手术给患者带来的痛苦，大大降低手术的创伤性。
在所述天然高分子中，透明质酸(Hyaluronic acid，HA)因其优异的特性受到研究人员的关注。天然透明质酸是由D-葡糖醛酸和N-乙酰基葡糖胺的交替单元组成的天然杂多糖。其后经过数十年的研究，人们发现其在人和其他脊椎动物的结缔组织中广泛存在，例如细胞间隙、运动关节组织、脐带、皮肤、软骨、血管壁、滑液以及鸡冠等组织和器官中都含有透明质酸。透明质酸属于线性高分子多糖，结构中含有二糖重复单元，重复单元中的D-葡萄糖醛酸通过β-1,3糖苷键与N-乙酰基葡糖胺连接，成千上万的二糖重复单元通过β-1,4糖苷键相连，形成整个高分子直链、线性结构。在人体生理状态下，透明质酸通常以钠盐的形式存在。其中，透明质酸钠及其凝胶在骨科、妇科及整形科等领域应用广泛，还可用于作为眼用制剂载体或直接作为眼用制剂应用于眼科手术，即透明质酸钠类产品在眼科手术中也有重要应用。透明质酸钠还是关节滑液及软骨的重要组成成分，通过提高关节内透明质酸钠的含量，可以增加关节滑液的粘稠性和润滑功能，发挥保护软骨、促进关节愈合和再生、缓解疼痛、增加关节活动度等作用。且有文献报道：大量的动物实验和临床应用表明，在预防和降低妇产科手术造成的粘连方面，透明质酸及其钠盐是一种安全、有效的理想物质；其中，透明质酸钠的水溶液，是一种非牛顿型流体，有着良好的粘弹性和流变性，而且一般而言，低浓度的透明质酸溶液主要表现出粘性，高浓度的透明质酸溶液主要表现出弹性，因此可以根据实际使用的需求调整其浓度。
天然的透明质酸或其钠盐虽然应用领域广泛且具有多种明确的应用优势，但天然的透明质酸或其钠盐也有明确的缺点。首先，天然的透明质酸或其钠盐在体内的半衰期短，在生物体内降解时间一般不多于7天，造成半衰期短的主要原因为天然的透明质酸或其钠盐的平均分子量较小，且具有较好的流动性，容易分散于组织中并被吸收和代谢，其直接表现为在溶液态是低粘度的。其次，天然的透明质酸或其钠盐具有稳定性较差，易发生降解的缺点。第三，天然的透明质酸或其钠盐具有亲水性过强的缺点。
其他的天然高分子化合物也存在透明质酸的类似问题。制备天然高分子化合物的水凝胶可以一定程度上解决机械强度低等问题。现有研究中，为了获得具有理想物理机械性能和生物降解速度的天然高分子化合物的水凝胶，化学交联的方式被广泛的应用于制备水凝胶的过程中。化学交联反应中经常应用到羧基、羟基、氨基等化学活性较高的官能团，常用的化学交联剂一般含有双官能团，比如二胺、二肼、二醛、二醇等，但这些交联剂通常具有细胞毒性，如果残留将影响水凝胶材料的生物相容性。需要研究一种新型的化学交联高分子水凝胶，来避免交联反应中添加额外化学物质而带来的细胞毒性。同时，现代医学要求生物材料在使用中能够具有一定的可塑和可控性，实现微创的治疗效果。以透明质酸为例，现有技术中，以透明质酸为主要原料制成的水凝胶，具有的明显缺点或技术偏见包括以下几点：一、因含有环氧基团的小分子交联应用于透明质酸的交联反应中，而具有毒性的环氧小分子交联剂残留在交联透明质酸中，此种交联透明质酸制成水凝胶后，将不可避免地产生不良反应或毒性作用，制约了透明质酸的水凝胶的应用。二、对化学改性后的透明质酸，经交联反应而获得交联透明质酸，并由此制备得到的水凝胶，价格高昂，且相比于以未经结构修饰与改造的透明质酸经交联而制得的水凝胶，在粘度、保水性、塑型效果等方面虽有改善但改善有限。三、因现有技术中，由透明质酸经交联反应而生成交联透明质酸的反应，需要一定反应条件或反应条件较为苛刻，无法在生理状态下实现原位交联，只能以预交联并预灌充的形式实现产品，极大影响了产品的应用范围与相应治疗人群或美容人群的顺应性。
近年来，巯基改性高分子化合物因其具有易交联形成水凝胶、抗氧化等特点，引起了研究人员的关注。现有的生物相容性高分子的巯基化改性过程，一般是指引入自由巯基的化学改性过程，通常多糖、蛋白质和合成高分子的侧链基团，如：羧基、氨基、羟基等，可以通过适当的化学反应引入自由巯基。仍以透明质酸为例，现有技术中，透明质酸经化学反应引入自由巯基后获得的巯基化透明质酸，总结其特点，虽然相比天然透明质酸，在物理化学性能或生物相容性等方面具有一定改善或提高，但仍不足以克服以下缺点：1、自交联或与其他物质的交联反应的速度较慢，通常需要加入小分子氧化剂来加速交联反应。2、巯基改性后的透明质酸新化合物，在交联形成水凝胶后，其物理化学性能及生物相容性等关键指标与现有市售产品或产品相比，不具备实质性优势或不具备足够的区分技术特征，主要体现于粘度、代谢持久性和塑型效果。3、巯基化的透明质酸，现有技术中的合成制备方法，均有毒性较高或成本过高的劣势。而上述这些原因，是影响现有巯基化透明质酸合成制备技术的工业化生产制备和更广泛应用的根源。另外，现有技术中，由巯基化透明质酸经交联反应而制备的水凝胶，具有的缺点或技术偏见，包括：1、现有技术中对化学改性后的透明质酸，经交联反应获得交联透明质酸，并由此制备得到的水凝胶，相比以天然透明质酸交联而制得的水凝胶，价格更为高昂。2、现有技术中对化学改性后的透明质酸，经交联反应获得交联透明质酸，并由此制备得到的水凝胶，相比于以天然的透明质酸经交联而制得的水凝胶，在粘度、保水性、塑型效果等方面虽有改善但改善有限。3、现有技术中对透明质酸的化学改性，具有一定的不可控性，这个不可控性会对其交联透明质酸的质量产生影响，进而导致相应水凝胶的质量在很大范围内波动，无法实现不同批次间水凝胶产品治疗作用或美容整形作用的一致性，也影响了水凝胶在应用领域发挥更大作用。
发明内容
为解决上述问题，本发明的目的在于提供一种由新颖结构的巯基改性高分子化合物与下述物质的至少一种：丙烯酰化高分子化合物、含有丙烯酰基的小分子交联剂，它们的凝胶化而生成的具备新颖结构的水凝胶。具体而言，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/或含有丙烯酰基的小分子交联剂配合而形成水凝胶，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/或含有丙烯酰基的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。基于此，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。
本发明的第二个目的在于提供一种制备上述水凝胶的方法，该方法具有如下优点：交联反应不需要添加高毒性的环氧类小分子交联剂以及催化剂，从根本上避免了纯化过程的有毒物质残留可能，无需光照加热等催化条件，交联反应程度可控，交联反应的成本适中并优于现有技术。
本发明第一方面是提供一种水凝胶，该水凝胶的化学结构是全新的，其通过含有巯基改性高分子 化合物的体系的凝胶化制备得到；
所述巯基改性高分子化合物是下述系列化合物中的至少一种：
一系列巯基改性高分子化合物，其被改性的高分子化合物的结构上含有-COOH、-NH 2、-OH、式a所示丙烯酸酯类基团、式b所示丙烯酰胺类基团、式c所示丙烯酰类基团中的至少一种，
Figure PCTCN2020079823-appb-000001
所述-COOH和/或-NH 2和/或-OH和/或丙烯酸酯类基团和/或丙烯酰胺类基团和/或丙烯酰类基团的部分或全部被修饰形成端基为下述基团的侧链：
Figure PCTCN2020079823-appb-000002
上述基团中，*表示连接点；R 1选自氢，卤素，脂肪基团，芳香基团等；R 2和R 3相同或不同，彼此独立地选自氢，卤素，脂肪基团，芳香基团等；R 4为多巯基化合物片段；
所述体系中进一步含有下述物质的至少一种：
C1.丙烯酰化高分子化合物，
C2.含丙烯酰基的小分子交联剂。
本发明第二方面是提供一种上述水凝胶的制备方法，其包括以下步骤：
将含有下述物质的体系凝胶化：
(i)所述巯基改性高分子化合物，以及
(ii)物质C1和物质C2的至少一种；
制备得到所述水凝胶。
本发明的第三方面是提供一种上述水凝胶的用途，其用于生物医药，医疗美容整形，以及化妆品等领域。
本发明的有益效果
本发明提供了一种以具有创新结构的巯基化改性高分子化合物为原料，经交联反应而获得一种具有创新结构的水凝胶，并且该水凝胶相比现有技术中的水凝胶(例如现有技术中以透明质酸或改性透明质酸为起始原料并经交联反应获得的水凝胶)在物理和化学性质、塑型效果和耐代谢耐降解性等方面具有意想不到的技术优势。
本发明的水凝胶具有的优点为：1、在对高分子化合物进行化合物结构修饰与改造过程中，及在后面的交联反应过程中，均未使用具有毒性的环氧类小分子交联剂，该水凝胶产品具有更加安全的优点。2、本发明的水凝胶产品，相比于现有技术中的水凝胶(例如现有的交联透明质酸水凝胶)，具有更优秀的粘度、保水性、塑型效果等技术优势。3、本发明的水凝胶，无需添加任何催化剂即可实现交联反应，反应条件的更易于实现，优于现有技术中的高分子化合物的交联反应条件，也优于现有技术中的改性高分子化合物的交联条件。4、首次实现了真正意义上的生理条件下的原位交联，且交联反应的终点可控，其可控性不仅体现于体外的交联反应，也体现于在动物体或人体内的交联反应，经大量动物试验已证明该交联反应无论在动物体内或体外，其反应终点单一且稳定可重现。5、经实验研究表明，本发明的系列水凝胶，在室温和加速稳定性考察条件下，稳定性更好，耐降解，且在动物体内具有更优的耐代谢性等等。
本发明在真正意义上实现了生理条件下的原位交联，即在室温和常压条件下，即可完成交联反应；或在注射进入动物体或人体的组织后，仍可在组织中实现交联反应，这样就明显提高了水凝胶产品的耐降解耐代谢性能，明显提高了水凝胶注射剂产品的使用效果。且由于本发明所具备的独有技术，可以实现在体外交联或混合阶段之前，即可对注入动物体或人体的水凝胶产品的交联程度的可控性，即在注射进入动物体或人体后可实现交联反应终点可控的交联反应，保证了产品本身的安全性和治疗作用。
本发明还提出了一种制备所述水凝胶的方法，该方法是在室温和常压下即可完成反应，该反应条件的温和且易于实现，是实现制备生理条件下原位交联水凝胶的技术基础。
附图说明
图1制备例5的反应方程式；
图2制备例6的反应方程式；
图3制备例7的反应方程式；
图4制备例8的反应方程式；
图5制备例15的反应方程式；
图6制备例16的反应方程式；
图7制备例17的反应方程式；
图8制备例18的反应方程式；
图9制备例19的反应方程式；
图10制备例20的反应方程式；
图11水凝胶样品细胞生物相容性实验；
图12水凝胶样品体内塑型和支撑效果(高度)；
图13水凝胶样品体内塑型和支撑效果(底面积)；
图14水凝胶样品体内降解实验；
图15 HA-A1的结构式和其 1H-NMR谱图；
图16 HA-A2的结构式和其 1H-NMR谱图；
图17 HA-MA1的结构式和其 1H-NMR谱图；
图18 HA-MA2的结构式和其 1H-NMR谱图；
图19 CHS-A的结构式和其 1H-NMR谱图；
图20 CHS-MA的结构式和其 1H-NMR谱图；
图21 Gelatin-A的结构式和其 1H-NMR谱图；
图22 Gelatin-MA的结构式和其 1H-NMR谱图；
图23 CTS-A的结构式和其 1H-NMR谱图；
图24 CTS-MA的结构式和其 1H-NMR谱图；
图25 HA-A1-SH1的结构式和其 1H-NMR谱图；
图26 HA-A2-SH1的结构式和其 1H-NMR谱图；
图27 HA-MA1-SH1的结构式和其 1H-NMR谱图；
图28 HA-MA2-SH1的结构式和其 1H-NMR谱图；
图29 CHS-A-SH1的结构式和其 1H-NMR谱图；
图30 CHS-MA-SH1的结构式和其 1H-NMR谱图；
图31 Gelatin-A-SH1的结构式和其 1H-NMR谱图；
图32 Gelatin-MA-SH1的结构式和其 1H-NMR谱图；
图33 CTS-A-SH1的结构式和其 1H-NMR谱图；
图34 CTS-MA-SH1的结构式和其 1H-NMR谱图；
图35 PHEMA-A的结构式和其 1H-NMR谱图；
图36 PHEMA-MA的结构式和其 1H-NMR谱图；
图37 PVA-A的结构式和其 1H-NMR谱图；
图38 PVA-MA的结构式和其 1H-NMR谱图；
图39 PHEMA-A-SH1的结构式和其 1H-NMR谱图；
图40 PHEMA-MA-SH1的结构式和其 1H-NMR谱图；
图41 PVA-A-SH1的结构式和其 1H-NMR谱图；
图42 PVA-MA-SH1的结构式和其 1H-NMR谱图；
图43 HB-PEG-SH1的结构式和其 1H-NMR谱图；
图44 HA-A1-SH2的结构式和其 1H-NMR谱图；
图45 HA-A1-SH3的结构式和其 1H-NMR谱图；
图46 HA-A2-SH2的结构式和其 1H-NMR谱图；
图47 HA-A2-SH3的结构式和其 1H-NMR谱图；
图48 HA-A2-SH4的结构式和其 1H-NMR谱图；
图49 HA-A2-SH5的结构式和其 1H-NMR谱图；
图50 HA-A2-SH6的结构式和其 1H-NMR谱图；
图51 HA-A2-SH7的结构式和其 1H-NMR谱图；
图52 HA-A2-SH8的结构式和其 1H-NMR谱图；
图53 HA-MA1-SH5的结构式和其 1H-NMR谱图；
图54 HA-MA1-SH6的结构式和其 1H-NMR谱图；
图55 HA-MA2-SH7的结构式和其 1H-NMR谱图；
图56 HA-MA2-SH8的结构式和其 1H-NMR谱图；
图57制备例25的反应方程式；
图58制备例26的反应方程式；
图59制备例27的反应方程式；
图60制备例28的反应方程式；
图61制备例29的反应方程式(其中的i＝10-90％，j＝10-90％，i2+i3＝i，j2+j3＝j，h＝j，i+j＝100％，k1＝1-1000)；
图62制备例30的反应方程式；
图63制备例31的反应方程式；
图64制备例32的反应方程式；
图65制备例33的反应方程式；
图66制备例34的反应方程式；
图67制备例35的反应方程式；
图68制备例36的反应方程式；
图69制备例37的反应方程式；
图70制备例38的反应方程式；
图71制备例39的反应方程式；
图72制备例40的反应方程式；
图73制备例41的反应方程式；
图74制备例42的反应方程式。
具体实施方式
[巯基改性高分子化合物]
如前所述，本发明的待凝胶化的体系中需要采用如下所示的系列化合物中的至少一种：
系列巯基改性高分子化合物，被改性的高分子化合物的结构上含有-COOH、-NH 2、-OH、式a所示丙烯酸酯类基团、式b所示丙烯酰胺类基团、式c所示丙烯酰类基团中的至少一种，
Figure PCTCN2020079823-appb-000003
其中，所述-COOH和/或-NH 2和/或-OH和/或丙烯酸酯类基团和/或丙烯酰胺类基团和/或丙烯酰类基团的部分或全部被修饰形成端基为下述基团的侧链：
Figure PCTCN2020079823-appb-000004
上述基团中，*表示连接点；
R 1选自氢，卤素，脂肪基团，芳香基团等；具体的，所述卤素、脂肪基团、芳香基团满足下文中的进一步的定义；优选地，R 1选自氢，卤素，脂肪基团；还优选地，R 1选自氢，卤素，C1-6烷基(例如甲基、乙基等)；
R 2和R 3相同或不同，彼此独立地选自氢，卤素，脂肪基团，芳香基团等；具体的，所述卤素、脂肪基团、芳香基团满足下文中的进一步的定义；
R 4为多巯基化合物片段。
在一个具体的实施方式中，所述-COOH和/或-NH 2和/或-OH和/或丙烯酸酯类基团和/或丙烯酰胺类基团和/或丙烯酰类基团的部分或全部被修饰形成以下结构的至少一种：
Figure PCTCN2020079823-appb-000005
上述结构中，R选自
Figure PCTCN2020079823-appb-000006
亚烃基、亚芳基、酰胺残基、酰肼残基等；*表示连接点； 1*表示与R的左侧基团的连接点； 2*表示与R的右侧基团的连接点；R 1、R 2、R 3和R 4的定义同前。
其中，所述-COOH、-NH 2、-OH、式a所示丙烯酸酯类基团、式b所示丙烯酰胺类基团、式c所示丙烯酰类基团中的至少一种可以是直接连接在高分子化合物的主链上，也可以是通过R’基团连接在高分子化合物的主链上，所述R’可以是含有杂原子的基团、亚烃基、亚芳基或下述连接基团：
Figure PCTCN2020079823-appb-000007
上式中，R”是亚烃基或亚芳基，n’为1-1000的整数，*表示连接点。
其中，所述含有杂原子的基团包括但不限于：酯基、酰胺残基或酰肼残基。具体的，所述酯基、酰胺残基或酰肼残基满足下文中的进一步的定义。
其中，所述被改性的高分子化合物包含天然粘多糖聚合物，如壳聚糖类(具体可以是壳聚糖、乙二醇壳聚糖、羧甲基壳聚糖等)，硫酸软骨素，透明质酸，海藻酸盐等中的至少一种；蛋白质，如明胶、纤维蛋白、血清蛋白等；和/或，合成聚合物，如聚乙烯醇，聚(甲基)丙烯酸，聚(甲基)丙烯酸羟烷基酯(例如聚(甲基)丙烯酸羟乙酯等)，超支化聚乙二醇等中的至少一种。
其中，用Ellman法检测的所述巯基改性高分子化合物的巯基含量为0.01-30mmol/g，例如为0.1-10.0mmol/g，还例如为0.3-5.0mmol/g，再例如为0.5-3.0mmol/g。
其中，所述巯基改性高分子化合物的分子量与改性前高分子化合物的分子量基本不变。
例如，本发明的巯基改性高分子化合物包括如下结构的至少一种：
Figure PCTCN2020079823-appb-000008
Figure PCTCN2020079823-appb-000009
Figure PCTCN2020079823-appb-000010
Figure PCTCN2020079823-appb-000011
上述结构中，A为所述结构上含有至少一个-COOH、-NH 2、-OH、式a所示丙烯酸酯基团、式b所示丙烯酰胺基团、式c所示丙烯酰类基团的被改性高分子化合物的片段；R、R’、R 1、R 2、R 3和R 4的定义同前；(n2+n3)/(n1+n2+n3)表示丙烯酰化度；n3/(n1+n2+n3)表示巯基化程度，与上述的用Ellman法检测的所述巯基改性高分子化合物的巯基含量是对应的；所述n1可以为0，若为0，则不用限定丙烯酰化度，仅是n3/(n2+n3)表示巯基化程度，与上述的用Ellman法检测的所述巯基改性高分子化合物的巯基含量是对应的；所述n2可以为0，若为0，则n3/(n1+n3)既表示丙烯酰化度，又表示巯基化程度，与上述的用Ellman发检测的所述巯基改性高分子化合物的巯基含量是对应的。
具体的，所述A可以是如下所示结构：
Figure PCTCN2020079823-appb-000012
上述各结构中，*表示主链重复单元之间的连接点；**表示-COOH、-NH 2、-OH、式a所示丙烯酸酯类基团、式b所示丙烯酰胺类基团、式c所示丙烯酰类基团与上述片段之间的连接点、或者通过R’基团与上述片段之间的连接点。
所述A还可以是下述聚合物Gelatin-A、Gelatin-MA、CTS-A、CTS-MA、PHEMA-A、PHEMA-MA、HB-PEG、PVA-A、PVA-MA、CHS-A或CHS-MA中的脱除含丙烯酰侧链后的剩余的片段或重复单元：
Figure PCTCN2020079823-appb-000013
Figure PCTCN2020079823-appb-000014
需要说明的是，Gelatin-A、Gelatin-MA、CTS-A、CTS-MA、PHEMA-A、PHEMA-MA、HB-PEG、PVA-A、PVA-MA、CHS-A或CHS-MA分别是具有上述结构的聚合物名称的简写，其中的字母分开后不与本发明中其他部分出现的字母含义相关。
本发明中若没有特殊说明，其中出现的n、n’、n1、n2、n3、n4、n5、n6、m1、m2、i、j、k1、h均是指结构式中出现的重复单元的个数。其取值范围属于本领域已知的常规范围。
在本发明的一个具体实施方式中，所述系列巯基改性高分子化合物具体是：
巯基改性透明质酸系列化合物，所述透明质酸的重复单元的侧链上含有的-COOH和/或-OH部分或全部被修饰形成端基为下述基团的侧链：
Figure PCTCN2020079823-appb-000015
上述基团中，*表示连接点；
R 1选自氢，卤素，脂肪基团，芳香基团等；具体的，所述卤素、脂肪基团、芳香基团满足下文中的进一步的定义；优选地，R 1选自氢，卤素，脂肪基团；还优选地，R 1选自氢，卤素，C1-6烷基(例如甲基、乙基等)；
R 2和R 3相同或不同，彼此独立地选自氢，卤素，脂肪基团，芳香基团等；具体的，所述卤素、脂肪基团、芳香基团满足下文中的进一步的定义；
R 4为多巯基化合物片段。
在一个具体的实施方式中，所述端基通过R基团与-COOH和/或-OH相连或直接与-COOH和/或-OH相连形成以下结构的至少一种的侧链：
Figure PCTCN2020079823-appb-000016
上述a结构、b结构、c结构和d结构中，R选自
Figure PCTCN2020079823-appb-000017
亚烃基、亚芳基、酰胺残基、酰肼残基等；*表示连接点； 1*表示与R的左侧基团的连接点； 2*表示与R的右侧基 团的连接点；R 1、R 2、R 3和R 4的定义同前。
其中，所述巯基改性透明质酸的分子量范围为五千到两千万道尔顿。所述巯基改性透明质酸的分子量在改性前后变化不大，或者说分子量基本没变。
其中，用Ellman法检测的所述巯基改性透明质酸的巯基含量为0.01-30mmol/g，例如为0.1-10.0mmol/g，还例如为0.3-5.0mmol/g，再例如为0.5-3.0mmol/g。
例如，本发明的巯基改性透明质酸包括如下结构的至少一种：
Figure PCTCN2020079823-appb-000018
上述结构中，R、R 1、R 2、R 3和R 4的定义同前；(n2+n3)/(n1+n2+n3)表示丙烯酰化度；n3/(n1+n2+n3)表示巯基化程度，与上述的用Ellman法检测的所述巯基改性高分子化合物的巯基含量是对应的；所述n1可以为0，若为0，则不用限定丙烯酰化度，仅是n3/(n2+n3)表示巯基化程度，与上述的用Ellman法检测的所述巯基改性高分子化合物的巯基含量是对应的；所述n2可以为0，若为0，则n3/(n1+n3)既表示丙烯酰化度，又表示巯基化程度，与上述的用Ellman发检测的所述巯基改性高分子化合物的巯基含量是对应的；
所述A 1是：
Figure PCTCN2020079823-appb-000019
所述A 2是下述结构中的一种：
Figure PCTCN2020079823-appb-000020
A 1和A 2的结构中的*表示与COOH或OH的连接点。
具体的，所述巯基改性透明质酸具有下述结构中的至少一种但又不仅限于以下结构：
Figure PCTCN2020079823-appb-000021
Figure PCTCN2020079823-appb-000022
Figure PCTCN2020079823-appb-000023
Figure PCTCN2020079823-appb-000024
Figure PCTCN2020079823-appb-000025
Figure PCTCN2020079823-appb-000026
Figure PCTCN2020079823-appb-000027
上述结构式中，n 1、n 2和n 3的定义同前。
如前所述，R 4为多巯基化合物片段，例如，所述-S-R 4-SH片段可以由下述但不仅限于下述多巯基化合物引入：
Figure PCTCN2020079823-appb-000028
其中，n4＝2-30的整数，例如n＝2、3、4、5或6等；n5＝1-30的整数，例如为1、2、3、4、5等；n6＝1-30的整数，例如为1、2、3、4、5等；
4-arm-PEG-SH表示含有四个巯基团的PEG聚合物；6-arm-PEG-SH表示含有六个巯基基团的PEG聚合物；8-arm-PEG-SH表示含有八个巯基的PEG聚合物；所述PEG是聚乙二醇的缩写。
[术语和定义]
如前所述，R 1选自氢，卤素，脂肪基团，芳香基团等；R 2和R 3相同或不同，彼此独立地选自氢，卤素，脂肪基团，芳香基团等。
如前所述，所述R可选自亚烃基、亚芳基、酰胺残基、酰肼残基等。
如前所述，所述R’可选自含有杂原子的基团、亚烃基、亚芳基等。
如前所述，所述R”可选自亚烃基、亚芳基等。
所述卤素是指氟、氯、溴或碘。
所述脂肪基团例如为直链或支链饱和/不饱和脂肪基团，具体的可以是烷基、烯基或炔基。
本发明单独使用或用作后缀或前缀的“烃基”例如为直链或支链饱和/不饱和脂肪基团，具体的可以是烷基、烯基或炔基。
本发明单独使用或用作后缀或前缀的“烷基”意在包括具有1至20个，优选1-6个碳原子的支链和直链饱和脂族烃基。例如，“C 1-6烷基”表示具有1、2、3、4、5或6个碳原子的直链和支链烷基。烷基的实例包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、戊基和己基。
本发明单独使用或用作后缀或前缀的“烯基”意在包括具有2至20个，优选2-6个碳原子(或若提供了碳原子的具体数目，则指该具体数目)的包含烯基或烯烃的支链和直链脂族烃基。例如，“C 2-6烯基”表示具有2、3、4、5或6个碳原子的烯基。烯基的实例包括但不限于乙烯基、烯丙基、1-丙烯基、1-丁烯基、2-丁烯基、3-丁烯基、2-甲基丁-2-烯基、3-甲基丁-1-烯基、1-戊烯基、3-戊烯基和4-己烯基。
本发明单独使用或用作后缀或前缀的“炔基”意在包括具有2至20个，优选2-6个碳原子(或若提供了碳原子的具体数目，则指该具体数目)的包含炔基或炔烃的支链和直链脂族烃基。例如乙炔基、丙炔基(例如l-丙炔基、2-丙炔基)、3-丁炔基、戊炔基、己炔基和1-甲基戊-2-炔基。
所述芳香基团指由5至20个碳原子构成的芳族环结构。例如：包含5、6、7和8个碳原子的芳族环结构可以是单环芳族基团例如苯基；包含8、9、10、11、12、13或14个碳原子的环结构可以是多环的例如萘基。芳环可在一个或多个环位置取代有取代基，所述取代基为烷基、卤素等，例如甲苯基。术语“芳基”还包括具有两个或更多个环的多环环系，其中两个或更多个碳为两个相邻环所共有(所述环为“稠环”)，其中至少一个环是芳族的且其它环例如可以是环烷基、环烯基、环炔基、芳基和/或杂环基。多环的实例包括但不限于2,3-二氢-1,4-苯并二氧杂环己二烯和2,3-二氢-1-苯并呋喃。
本发明所述“亚烃基”为上述“烃基”脱除一个氢后的基团。
本发明所述“亚芳基”为上述“芳香基团”脱除一个氢后的基团。
本发明所述“亚烷基”为上述“烷基”脱除一个氢后的基团。
本发明所述单独使用或用作后缀或前缀的“酰胺基”是指R a-C(＝O)-NH-基团，其中，R a选自未取代或任选被一个或多个R b取代的下列基团：烷基、环烷基、烯基、环烯基、炔基、环炔基、杂环基、芳基、杂芳基等；R b选自未取代或任选被一个或多个R b1取代的下列基团：卤素、羟基、巯基、硝基、氰基、烷基、烷氧基、环烷基、烯基、炔基、杂环基、芳基、杂芳基、氨基、羧基、酯基、肼基、酰基、亚磺酰基、磺酰基、磷酰基等；每一个R b1彼此独立地选自卤素、羟基、烷基、芳基。
本发明所述单独使用或用作后缀或前缀的“酰肼基”是指R a-C(＝O)-NH-NH-基团，其中，R a的定义同前。
本发明所述“酰胺残基”为上述“酰胺基”脱除一个氢后的基团。
本发明所述“酰肼残基”为上述“酰肼基”脱除一个氢后的基团。
本发明使用的术语“环烷基”意在包括具有指定数目碳原子的饱和环基。这些术语可包括稠合或桥接的多环系统。环烷基在其环结构中具有3至40个碳原子。在一个实施方案中，环烷基在其环结构 中具有3、4、5或6个碳原子。例如，“C 3-6环烷基”表示例如环丙基、环丁基、环戊基或环己基的基团。
本发明使用的术语“环烯基”意在包括具有指定数目碳原子的含至少一个烯基的环基。这些术语可包括稠合或桥接的多环系统。环烯基在其环结构中具有3至40个碳原子。在一个实施方案中，环烯基在其环结构中具有3、4、5或6个碳原子。例如，“C 3-6环烯基”表示例如环丙烯基、环丁烯基、环戊烯基或环己烯基的基团。
本发明使用的术语“环炔基”意在包括具有指定数目碳原子的含至少一个炔基的环基。这些术语可包括稠合或桥接的多环系统。环炔基在其环结构中具有6至40个碳原子。在一个实施方案中，环炔基在其环结构中具有6个碳原子。例如，“C 3-6环炔基”表示例如环丙炔基、环丁炔基、环戊炔基或环己炔基的基团。
本发明使用的“杂芳基”指具有至少一个环杂原子(例如硫、氧或氮)的杂芳族杂环。杂芳基包括单环系统和多环系统(例如具有2、3或4个稠环)。杂芳基的实例包括但不限于吡啶基、嘧啶基、吡嗪基、哒嗪基、三嗪基、呋喃基、喹啉基、异喹啉基、噻吩基、咪唑基、噻唑基、吲哚基、吡咯基、噁唑基、苯并呋喃基、苯并噻吩基、苯并噻唑基、异噁唑基、吡唑基、三唑基、四唑基、吲唑基、1,2,4-噻二唑基、异噻唑基、苯并噻吩基、嘌呤基、咔唑基、苯并咪唑基、苯并噁唑基、氮杂苯并噁唑基、咪唑并噻唑基、苯并[1,4]二氧杂环己烯基、苯并[1,3]二氧杂环戊烯基等。在一些实施方案中，杂芳基具有3至40个碳原子且在其它实施方案中具有3至20个碳原子。在一些实施方案中，杂芳基包含3至14个、4至14个、3至7个或5至6个成环原子。在一些实施方案中，杂芳基具有1至4个、1至3个或1至2个杂原子。在一些实施方案中，杂芳基具有1个杂原子。
本发明使用的术语“杂环基”指包含3至20个原子的饱和、不饱和或部分饱和的单环、二环或三环，其中1、2、3、4或5个环原子选自氮、硫、氧或磷，除非另有说明，其可通过碳或氮来连接，其中-CH 2-基团任选被-C(O)-代替；其中除非另有相反说明，环氮原子或环硫原子任选被氧化以形成N-氧化物或S-氧化物或环氮原子任选被季铵化；其中环中的-NH任选被乙酰基、甲酰基、甲基或甲磺酰基取代；及环任选被一个或多个卤素取代。应该理解的是，当杂环基中S原子和O原子的总数超过1时，这些杂原子不彼此相邻。若所述杂环基为二环或三环，则至少一个环可任选为杂芳族环或芳族环，条件是至少一个环是非杂芳族的。若所述杂环基为单环，则其一定不是芳族的。杂环基的实例包括但不限于哌啶基、N-乙酰基哌啶基、N-甲基哌啶基、N-甲酰基哌嗪基、N-甲磺酰基哌嗪基、高哌嗪基、哌嗪基、氮杂环丁烷基、氧杂环丁烷基、吗啉基、四氢异喹啉基、四氢喹啉基、二氢吲哚基、四氢吡喃基、二氢-2H-吡喃基、四氢呋喃基、四氢噻喃基、四氢噻喃-1-氧化物、四氢噻喃-1,1-二氧化物、1H-吡啶-2-酮和2,5-二氧代咪唑烷基。
本发明使用的术语“酰基”是指R a-C(＝O)-基团，其中，R a的定义同前。
本发明使用的术语“亚磺酰基”是指R a-S(＝O)-基团，其中，R a的定义同前。
本发明使用的术语“磺酰基”是指R a-S(＝O) 2-基团，其中，R a的定义同前。
本发明使用的术语“磷酰基”是指R c-P(＝O)(R d)-基团，其中，R c和R d相同或不同，彼此独立地选自未取代或任选被一个或多个R b取代的下列基团：烷基、环烷基、烷氧基、羟基、烯基、环烯基、炔基、环炔基、杂环基、芳基、杂芳基等，R b的定义同前。
本发明使用的术语“肼基”是指-NHNHR a基团，R a的定义同前。
本发明使用的术语“胺基”指-NHR a基团或-N(R a) 2基团，R a的定义同前。
本发明使用的术语“氨基”指-NH 2基团。
本发明使用的术语“羧基”是指-COOH基团。
本发明使用的术语“酯基”是指R a-C(＝O)-O-基团或R a-O-C(＝O)-基团，其中，R a的定义同前。
[巯基改性高分子化合物的制备方法]
如前所述，本发明提供一种上述巯基改性高分子化合物的制备方法，其包括以下步骤：
1)结构上含有-COOH、-NH 2、-OH中的至少一种的高分子化合物的丙烯酰化步骤，即将高分子化合物的结构上含有的-COOH、-NH 2、-OH的至少一种直接或间接与如下基团连接：
Figure PCTCN2020079823-appb-000029
R 1、R 2和R 3的定义同前；*表示连接点；
或者，结构上含有上述式a所示丙烯酸酯类基团、上述式b所示丙烯酰胺类基团、上述式c所示丙烯酰类基团中至少一种的高分子化合物直接作为反应原料；
2)将步骤1)得到的高分子化合物的至少一种与多巯基化合物HS-R 4-SH反应，R 4的定义同前，制备得到所述的巯基改性高分子化合物。
在本发明的一个具体实施方式中，所述方法包括以下步骤：
1)结构上含有-COOH、-NH 2、-OH中的至少一种的高分子化合物的丙烯酰化步骤，即将高分子化合物的结构上含有的-COOH、-NH 2、-OH的至少一种通过-R-基团与如下基团连接或直接与如下基团连接：
Figure PCTCN2020079823-appb-000030
R、R 1、R 2和R 3的定义同前，*表示连接点；
或者，结构上含有式a所示丙烯酸酯类基团、式b所示丙烯酰胺类基团、式c所示丙烯酰类基团中至少一种的高分子化合物直接作为反应原料；
2)将步骤1)得到的高分子化合物的至少一种与多巯基化合物HS-R 4-SH反应，R 4的定义同前，制备得到所述的巯基改性高分子化合物。
在本发明的一个具体实施方式中，提供一种上述巯基改性透明质酸的制备方法，其包括以下步骤：
1)透明质酸的丙烯酰化步骤，即将透明质酸的重复单元的侧链上含有的-COOH、-OH的至少一种直接或间接与如下基团连接：
Figure PCTCN2020079823-appb-000031
R 1、R 2和R 3的定义同前；*表示连接点；
2)将丙烯酰化的透明质酸与多巯基化合物HS-R 4-SH反应，R 4的定义同前，制备得到所述的巯基改性透明质酸。
具体的，所述步骤1)为：透明质酸的丙烯酰化步骤，是将透明质酸的重复单元的侧链上含有的-COOH、-OH的至少一种通过R基团与所述端基相连，或者直接与所述端基相连形成以下结构的至少一种的侧链：
Figure PCTCN2020079823-appb-000032
Figure PCTCN2020079823-appb-000033
上述a结构、b结构、c结构和d结构中，R、R 1、R 2、R 3和R 4的定义同前；*表示连接点。
步骤1)中，所述丙烯酰化步骤可以是通过待改性的高分子化合物与丙烯酸酯类化合物的反应实现、也可以是通过待改性的高分子化合物与丙烯酰氯类化合物或丙烯酸酐类化合物的反应实现。
所述丙烯酸酯类化合物可以是丙烯酸烷基酯类化合物、丙烯酸芳基酯类化合物、丙烯酸缩水多元醇酯类化合物中的一种或多种。
所述丙烯酸缩水多元醇酯类化合物中的多元醇例如为三元醇，具体可以是甘油、丁三醇、戊三醇等。
步骤1)中，所述丙烯酰化步骤可以是常规的反应步骤，采用现有的常规条件反应即可。通常为丙烯酰氯及其衍生物或丙烯酸酐及其衍生物与含有-OH、-NH 2中的至少一种的高分子化合物反应获得。也可以为丙烯酸缩水甘油酯及其衍生物与含有-COOH、-OH、-NH 2中的至少一种的高分子化合物反应获得。
步骤1)中，所述丙烯酰化步骤可以是非常规的反应步骤，既采用非上述方法合成的含有式c结构的高分子化合物。
步骤2)中，与多巯基化合物HS-R 4-SH反应在溶剂中进行。所述溶剂例如为水或有机溶剂，进一步可以是去离子水或二甲基甲酰胺。
步骤2)中，与多巯基化合物HS-R 4-SH反应在低温到高温条件下进行。例如反应温度为0-80℃，进一步可以为10-70℃，例如可以在室温下反应。
步骤2)中，与多巯基化合物HS-R 4-SH反应的反应时间为0.1-100小时。
步骤2)中，与多巯基化合物HS-R 4-SH反应的pH范围为-1到15。例如反应pH可以为6-8，再例如为7。
其中，步骤2)的反应产物进一步包括后处理步骤。
其中，所述后处理步骤采用透析方法。具体的，将反应后的溶液装入透析袋(例如截留分子量2kDa或以上的透析袋)，用盐酸溶液(例如pH＝4)透析数日(例如1-10天，还例如5天等)，任选地换水(如每天换水或隔天换水)数次(例如两次或更多等)，最后收集透析袋内溶液，干燥(如冷冻干燥)后得到固体或粘稠液体、即所述的巯基改性高分子化合物。
本发明的方法中首次提出了多巯基化合物的巯基与丙烯酰类基团中的碳碳双键的迈克尔加成反应制备所述巯基改性高分子化合物，该方法不仅巯基化程度高，而且巯基化反应的条件温和(常温、水溶液中即可进行)、无污染，制备的巯基改性高分子化合物的纯度高、特别适合于进一步在医药、美容、医学等领域的使用。
[丙烯酰化高分子化合物]
如上所述，本发明的待凝胶化体系中还可以包括物质C1.丙烯酰化高分子化合物，本发明的丙烯酰化高分子化合物可选自下述物质中的至少一种：
1)结构上含有-COOH、-NH 2、-OH中的至少一种的高分子化合物的丙烯酰化化合物，即所述高分子化合物的结构上含有的-COOH、-NH 2、-OH的至少一种直接或间接与如下基团连接而形成的丙烯酰化化合物：
Figure PCTCN2020079823-appb-000034
R 1、R 2和R 3的定义同前，*表示连接点；
2)结构上含有式a所示丙烯酸酯类基团、式b所示丙烯酰胺类基团、式c所示丙烯酰类基团中至少一种的高分子化合物。
上述第1)种物质中，所述-COOH和/或-NH 2和/或-OH的部分或全部被修饰形成以下结构的至少一种：
Figure PCTCN2020079823-appb-000035
上述结构中，R选自
Figure PCTCN2020079823-appb-000036
亚烃基、亚芳基、酰胺残基、酰肼残基等；*表示连接点； 1*表示与R的左侧基团的连接点； 2*表示与R的右侧基团的连接点；R 1、R 2、R 3和R 4的定义同前。
上述第1)种物质中，所述-COOH、-NH 2、-OH中的至少一种可以是直接连接在高分子化合物的主链上，也可以是通过R’基团连接在高分子化合物的主链上，所述R’可以是含有杂原子的基团、亚烃基、亚芳基或下述连接基团：
Figure PCTCN2020079823-appb-000037
上式中，R”是亚烃基或亚芳基，n’为1-1000的整数，*表示连接点。
其中，所述含有杂原子的基团包括但不限于：酯基、酰胺残基或酰肼残基。具体的，所述酯基、酰胺残基或酰肼残基满足本文中的进一步的定义。
上述第1)种物质中，所述待丙烯酰化的高分子化合物包含天然粘多糖聚合物，如壳聚糖类(具体可以是壳聚糖、乙二醇壳聚糖、羧甲基壳聚糖等)，硫酸软骨素，透明质酸，海藻酸盐等中的至少一种；蛋白质，如明胶、纤维蛋白、血清蛋白等；和/或，合成聚合物，如聚乙烯醇，聚(甲基)丙烯酸，聚(甲基)丙烯酸羟烷基酯(例如聚(甲基)丙烯酸羟乙酯等)，超支化聚乙二醇等中的至少一种。
上述第1)种物质中，丙烯酰化化合物包括如下结构的至少一种：
Figure PCTCN2020079823-appb-000038
Figure PCTCN2020079823-appb-000039
上述结构中，A为所述结构上含有至少一个-COOH、-NH 2、-OH的待丙烯酰化化合物的片段；R、R’、R 1、R 2、R 3和R 4的定义同前；(m2/(m1+m2)表示丙烯酰化度。
具体的，所述A可以是如下所示结构：
Figure PCTCN2020079823-appb-000040
Figure PCTCN2020079823-appb-000041
上述各结构中，*表示主链重复单元之间的连接点；**表示-COOH、-NH 2、-OH与上述片段之间的连接点、或者通过R’基团与上述片段之间的连接点。
上述第2)种物质可以是下述聚合物Gelatin-A、Gelatin-MA、CTS-A、CTS-MA、PHEMA-A、PHEMA-MA、HB-PEG、PVA-A、PVA-MA、CHS-A或CHS-MA中的一种：
Figure PCTCN2020079823-appb-000042
需要说明的是，Gelatin-A、Gelatin-MA、CTS-A、CTS-MA、PHEMA-A、PHEMA-MA、HB-PEG、PVA-A、PVA-MA、CHS-A或CHS-MA分别是具有上述结构的聚合物名称的简写，其中的字母分开后不与本发明中其他部分出现的字母含义相关。
[小分子交联剂]
如上所述，本发明的待凝胶化体系中还可以包括物质C2.含丙烯酰基的小分子交联剂，该小分子交联剂包括但不限于含丙烯酰基的小分子化合物或含丙烯酰基的低聚物；具体的可以选自乙二醇二丙烯酸酯EGDA，聚乙二醇二丙烯酸酯PEGDA，三羟甲基丙烷三丙烯酸酯TMPTA，季戊四醇三丙烯酸酯PTA，季戊四醇四丙烯酸酯PTTA，二(三羟甲基丙烷)四丙烯酸酯DTTA等。
[水凝胶]
如上所述，本发明提供一种水凝胶，该水凝胶是通过含有下述物质的体系的凝胶化制备得到的：
(i)上述的巯基改性高分子化合物，以及
(ii)物质C1和物质C2的至少一种。
在本发明的一个具体实施方式中，水凝胶是通过上述的巯基改性高分子化合物与上述的丙烯酰化高分子化合物的凝胶化制备得到。
其中，巯基改性高分子化合物与丙烯酰化高分子化合物充分接触后即发生交联反应，混合体系的粘度随即增高，最终形成均一的凝胶体系。
在本发明的一个具体实施方式中，水凝胶是通过上述的巯基改性高分子化合物与上述的小分子交联剂的凝胶化制备得到。
其中，巯基改性高分子化合物与小分子交联剂充分接触后即发生交联反应，混合体系的粘度随即增高，最终形成均一的凝胶体系。
在本发明的一个具体实施方式中，水凝胶是通过上述的巯基改性高分子化合物与上述的丙烯酰化高分子化合物和上述的小分子交联剂的凝胶化制备得到。
其中，巯基改性高分子化合物与丙烯酰化高分子化合物和小分子交联剂充分接触后即发生交联反应，混合体系的粘度随即增高，最终形成均一的凝胶体系。
其中，所述水凝胶包括下述特征性结构单元：
Figure PCTCN2020079823-appb-000043
上述单元中，R 1、R 2、R 3和R 4的定义同前，*表示连接点。
其中，所述的巯基改性高分子化合物与所述的丙烯酰化高分子化合物的用量比(以质量份计，合计为1份)为0.01:0.99～0.99:0.01。例如，可以为0.1:0.9～0.9:0.1，例如为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或者区间内的任意比值。
其中，所述的巯基改性高分子化合物与所述的小分子交联剂的用量比(以质量份计，合计为1份)为0.01:0.99～0.99:0.01。例如，可以为0.1:0.9～0.9:0.1，例如为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或者区间内的任意比值。
其中，所述的巯基改性高分子化合物与所述的丙烯酰化高分子化合物和小分子交联剂的用量比(以质量份计，合计为1份)为0.01:0.99～0.99:0.01。例如，可以为0.1:0.9～0.9:0.1，例如为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或者区间内的任意比值。其中，丙烯酰化高分子化合物和小分子交联剂可以是任意比例的混合。
本发明的水凝胶，是巯基改性高分子化合物的硫醇基(-SH)与物质C1和/或物质C2的碳碳双键通过硫醇基与碳碳双键的加成反应形成的稳定的交联材料，该交联材料(即水凝胶)的力学性能优异，具有较好的物理稳定性和机械强度；另外，体内代谢速率可控。若体系中同时引入C1和C2两种物质，C2(小分子交联剂)可以参与所述巯基改性高分子化合物和C1(丙烯酰化高分子化合物)的交联反应，即三者一起交联形成稳定的交联材料。同时，物质C1也可以采用物理混合的方式加入到凝胶体系中，从而达到不同的应用目的。所述巯基改性高分子化合物，与所述C1(丙烯酰化高分子化合物)和/或C2(小分子交联剂)之间搭配使用，相互取长补短，从而获得具有优良性质的三维支架材料，能够满足多数组织工程学的应用要求。
在本发明的一个具体实施方式中，所述体系中还可以进一步加入其他生物功能材料(如透明质酸，胶原，明胶，硫酸软骨素，壳聚糖，海藻酸钠等)、药物、生长因子或者细胞悬液等中的至少一种。通过加入其它生物功能材料可以为本发明水凝胶带来额外的作用效果，如引入未改性透明质酸可增加水凝胶的促进伤口愈合作用，引入胶原或明胶可以使水凝胶体系更接近生物体软组织组成，引入硫酸软骨素可以增强水凝胶体系的促进软骨修复作用，引入壳聚糖等带正电的生物材料可以增加水凝胶的抗菌作用，引入海藻酸钠可以增强水凝胶体系的机械强度。
[水凝胶的制备]
如上所述，本发明提供了上述水凝胶的制备方法，其包括以下步骤：
将含有下述物质的体系凝胶化：
(i)所述巯基改性高分子化合物，以及
(ii)物质C1和物质C2的至少一种；
制备得到所述水凝胶。
在一个具体的实施方式中，所述方法包括以下步骤：将包含下述物质的体系凝胶化：
(a)所述巯基改性高分子化合物，
(b)下述物质的至少一种：C1.所述丙烯酰化高分子化合物，C2.含丙烯酰基的小分子交联剂，
(c)任选的下述物质的至少一种：其他生物功能材料、药物、生长因子和细胞悬液；
制备得到所述水凝胶。
其具体为：分别配制所述巯基改性高分子化合物的溶液、所述丙烯酰化高分子化合物的溶液、所述小分子交联剂的溶液和任选的其他生物功能材料、药物、生长因子或者细胞悬液中至少一种的溶液，混合，凝胶化，制备得到所述水凝胶。另外，所述其他生物功能材料、药物、生长因子或者细胞悬液中的至少一种也可以直接添加到所述巯基改性高分子化合物的溶液或所述丙烯酰化高分子化合物的溶液或所述小分子交联剂的溶液中而引入。
其中，凝胶制备过程可以通过所述的巯基改性高分子化合物溶液加入到所述的丙烯酰化高分子化合物溶液和/或所述的小分子交联剂溶液中，也可以通过所述的丙烯酰化高分子化合物溶液和/或所述的小分子交联剂溶液加入到所述的巯基改性高分子化合物溶液中。具体的，两种溶液可以通过普通注射器混合，也可以通过双针头注射器混合，也可以通过其他方式混合。
其中，所述巯基改性高分子化合物的溶液的质量体积浓度为0.1％至95％，例如为1％至90％，再例如可以是0.1％、1％、5％、10％、20％、30％、40％、50％、60％、70％、80％、90％、95％。所述溶液中可以通过加入酸、碱或缓冲溶液调节pH＝7.4。所述缓冲溶液可以为磷酸盐缓冲液。
其中，所述丙烯酰化高分子化合物的溶液的质量体积浓度为0.1％至95％，例如为1％至90％，再例如可以是0.1％、1％、5％、10％、20％、30％、40％、50％、60％、70％、80％、90％、95％。所述溶液中可以通过加入酸、碱或缓冲溶液调节pH＝7.4。所述缓冲溶液可以为磷酸盐缓冲液。
其中，所述小分子交联剂的溶液的质量体积浓度为0.1％至95％，例如为1％至90％，再例如可以是0.1％、1％、5％、10％、20％、30％、40％、50％、60％、70％、80％、90％、95％。所述溶液中可以通过加入酸、碱或缓冲溶液调节pH＝7.4。所述缓冲溶液可以为磷酸盐缓冲液。
其中，两种溶液可以以任意比例混合，例如可以按等体积混合。
[水凝胶的应用]
已知的，水凝胶是一类亲水性聚合物链段通过交联形成的可在水中溶胀的三维空间网格。凝胶化过程可以通过不同反应机理来实现，包括聚合物链段的物理缠结、静电作用、共价化学交联、可逆化学交联、超分子化学交联以及亲疏水作用交联等。近年来，随着对水凝胶功能的深入研究，水凝胶已广泛应用于医药领域，如用于制备药物传递系统、用于软组织创伤修复敷料、用于骨修复的支架材料、眼科手术中用于支撑作用的粘弹剂、用于手术后防止组织粘连的材料、以及用于3D生物打印的支架材料等，该方向已成为组织工程与再生医学领域的研究热点。
本发明的水凝胶特别适合用于生物医药，医疗美容整形，以及化妆品等领域。具体的，其可以用于制备药物传递系统、用于软组织创伤修复敷料、用于骨修复的支架材料、眼科手术中用于支撑作用的粘弹剂、用于手术后防止组织粘连的材料、以及用于3D生物打印的支架材料等。
本申请的水凝胶在真正意义上实现了生理条件下的原位交联，即在室温和常压条件下，即可自发完成交联反应；或在注射进入动物体或人体的组织后，仍可在组织中实现交联反应，这样就明显提高了水凝胶产品的耐降解耐代谢性能，明显提高了水凝胶注射剂产品的使用效果。且由于本发明所具备的独有技术，可以实现在体外交联或混合阶段之前，即可对注入动物体或人体的水凝胶产品的交联程度的可控性，即在注射进入动物体或人体后可实现交联反应终点可控的交联反应，保证了产品本身的安全性和治疗作用。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的保护范围。此外，应理解，在阅读了本发明所公开的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本发明所限定的保护范围之内。
本发明中，所述 1H-NMR谱图采用Varian 400MHz核磁共振仪测定，测试温度25摄氏度，弛豫时间1秒钟，扫描次数为8次。具体的，取待检测物8-10毫克，溶解于750微升氘代水中，所得样品溶液测试 1H-NMR谱图。
本发明的储能模量基于水凝胶的流变力学性能测定，具体的，检测仪器为TA-DHR2流变仪，检测探头20mm平行板探头，检测温度：25℃，剪切频率：1Hz，剪切应变：1％。
参照“GBT 16886.5-2017医疗器械生物学评价+第5部分+体外细胞毒性试验”标准测试本发明的高分子化合物的细胞活性和生物相容性。具体的，下述的MTT法是指通过代谢活性测定细胞的存活率的检测方法。黄色水溶液MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]在活细胞内代谢性还原，生成蓝紫色不可溶的甲臜。活细胞的数目与甲臜溶于醇类后用光度计测定的色度相关。
制备例1合成丙烯酸酯修饰的透明质酸(简称HA-A1)
在200毫升烧杯中加入1克透明质酸(购自华熙福瑞达公司，其重均分子量约为300kDa)，50毫 升去离子水，50毫升二甲基甲酰胺，12毫升三乙胺，14毫升丙烯酸缩水甘油酯。室温搅拌至均一透明后，继续搅拌48小时。加入300毫升丙酮，产生大量白色沉淀。经离心所得沉淀溶解于100毫升去离子水中，得到无色透明溶液。上述溶液装入透析袋(截留分子量8kDa)，用5升去离子水透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到921毫克白色絮状固体，即得HA-A1，收率为92.1％。
HA-A1的结构式见图15。图15仅为示意图，表示所述透明质酸部分重复单元中的COOH被丙烯酸缩水甘油酯酯化，即其中m2/(m1+m2)表示丙烯酰化程度，m1+m2＝n，n为未改性透明质酸的重复单元数。下面的制备例和实施例中的结构式的含义与制备例1的相同，就不再重复说明了。
HA-A1的 1H-NMR谱图见图15，可见位于6-6.5ppm之间的属于丙烯酸官能团的核磁峰，证明该基团成功接枝到透明质酸的结构中。
制备例2合成丙烯酸酯修饰的透明质酸(简称HA-A2)
在200毫升烧杯中加入1克透明质酸(购自华熙福瑞达公司，其重均分子量约为400kDa)，50毫升去离子水，50毫升二甲基甲酰胺，6.3克丙烯酸酐，搅拌溶解。用1摩尔每升NaOH维持溶液pH＝8±0.5，继续搅拌24小时。加入300毫升丙酮，产生大量白色沉淀。经离心所得沉淀溶解于100毫升去离子水中，得到无色透明溶液。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升去离子水透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到789毫克白色絮状固体，即得HA-A2，收率为78.9％。
HA-A2的结构式见图16。
HA-A2的 1H-NMR谱图见图16，可见位于5.8-6.4ppm之间的属于丙烯酸官能团的核磁峰，证明该基团成功接枝到透明质酸的结构中。
制备例3合成甲基丙烯酸酯修饰的透明质酸(简称HA-MA1)
在200毫升烧杯中加入1克透明质酸(购自华熙福瑞达公司，其重均分子量约为400kDa)，50毫升去离子水，50毫升二甲基甲酰胺(Sigma)，12毫升三乙胺(Sigma)，15毫升甲基丙烯酸缩水甘油酯。室温搅拌至均一透明后，继续搅拌48小时。加入300毫升丙酮(Sigma)，产生大量白色沉淀。经离心所得沉淀溶解于100毫升去离子水中，得到无色溶液。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升去离子水透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到859毫克白色絮状固体，即得HA-MA1，收率为85.9％。
HA-MA1的结构式见图17。
HA-MA1的 1H-NMR谱图见图17，可见位于5.8-6.2ppm之间的属于甲基丙烯酸官能团的核磁峰，证明该基团成功接枝到透明质酸的结构中。
制备例4合成甲基丙烯酸酯修饰的透明质酸(简称HA-MA2)
在200毫升烧杯中加入1克透明质酸(购自华熙福瑞达公司，其重均分子量约为400kDa)，100毫升去离子水，室温搅拌溶解。进一步加入7.7克甲基丙烯酸酐搅拌溶解。用1摩尔每升NaOH维持溶液pH＝8±0.5，继续搅拌24小时。加入200毫升丙酮(Sigma)，产生大量白色沉淀。经离心所得沉淀溶解于100毫升去离子水中，得到无色透明溶液。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升去离子水透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到846毫克白色絮状固体，即HA-MA2，收率为84.6％。
HA-MA2的结构式见图18。
HA-MA2的 1H-NMR谱图见图18，可见位于5.8-6.2ppm之间的属于甲基丙烯酸官能团的核磁峰，证明该基团成功接枝到透明质酸的结构中。
制备例5合成巯基-丙烯酸酯修饰的透明质酸(简称HA-A1-SH1)
在200毫升烧杯中加入1克按制备例1的方法制备的HA-A1，0.3克二硫苏糖醇(购自VWR公司)，100毫升去离子水，室温搅拌溶解，得到透明溶液。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到842毫克白色絮状固体，即得HA-A1-SH1，收率为84.2％。
HA-A1-SH1的反应方程式如图1所示，其结构式见图1和图25。
HA-A1-SH1的 1H-NMR谱图见图25，可见位于2.3-2.8ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到透明质酸的结构中。
制备例6合成巯基-丙烯酸酯修饰的透明质酸(简称HA-A2-SH1)
在200毫升烧杯中加入1克按制备例2的方法制备的HA-A2，0.3克二硫苏糖醇(VWR)，100毫升去离子水，室温搅拌溶解，得到透明溶液。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截 留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到827毫克白色絮状固体，即得HA-A2-SH1，收率为82.7％。
HA-A2-SH1的反应方程式如图2所示，其结构式见图2和图26。
HA-A2-SH1的 1H-NMR谱图见图26，可见位于2.6-2.9ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到透明质酸的结构中。
制备例7合成巯基-甲基丙烯酸酯修饰的透明质酸(简称HA-MA1-SH1)
在200毫升烧杯中加入1克按制备例3的方法制备的HA-MA1，0.3克二硫苏糖醇(VWR)，100毫升去离子水，室温搅拌溶解。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到白色絮状固体约854毫克。即得HA-MA1-SH1，收率为85.4％。
HA-MA1-SH1的反应方程式如图3所示，其结构式见图3和图27。
HA-MA1-SH1的 1H-NMR谱图见图27，可见位于2.6-3.0ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到透明质酸的结构中。
制备例8合成巯基-甲基丙烯酸酯修饰的透明质酸(简称HA-MA2-SH1)
在200毫升烧杯中加入1克按制备例4的方法制备的HA-MA2，0.3克二硫苏糖醇(VWR)，100毫升去离子水，室温搅拌溶解。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到白色絮状固体约833毫克。即得HA-MA2-SH1，收率为83.3％。
HA-MA2-SH1的反应方程式如图4所示，其结构式见图4和图28。
HA-MA2-SH1的 1H-NMR谱图见图28，可见位于2.6-3.0ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到透明质酸的结构中。
制备例9合成丙烯酸酯修饰的硫酸软骨素(简称CHS-A)
在200毫升烧杯中加入1.2克硫酸软骨素(其重均分子量约为80kDa)，50毫升去离子水，50毫升二甲基甲酰胺，5.4克丙烯酸酐，搅拌溶解。用1摩尔每升NaOH维持溶液pH＝8±0.5，继续搅拌24小时。上述溶液装入透析袋(截留分子量3.5kDa，仕必纯)，用5升去离子水透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到781毫克浅黄色絮状固体，即CHS-A，收率65.1％。
CHS-A的结构式见图19。
CHS-A的 1H-NMR谱图见图19，可见位于6.0-6.5ppm之间的丙烯酸官能团的核磁峰，证明该基团成功接枝到硫酸软骨素的结构中。
制备例10合成甲基丙烯酸酯修饰的硫酸软骨素(简称CHS-MA)
在200毫升烧杯中加入1.2克硫酸软骨素(其重均分子量约为90kDa)，50毫升去离子水，50毫升二甲基甲酰胺，进一步加入6.5克甲基丙烯酸酐搅拌溶解。用1摩尔每升NaOH维持溶液pH＝8±0.5，继续搅拌24小时。上述溶液装入透析袋(截留分子量3.5kDa，仕必纯)，用5升去离子水透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到776毫克浅黄色絮状固体，即CHS-MA，收率64.7％。
CHS-MA的结构式见图20。
CHS-MA的 1H-NMR谱图见图20，可见位于6.0-6.5ppm之间的属于甲基丙烯酸官能团的核磁峰，证明该基团成功接枝到硫酸软骨素的结构中。
制备例11合成丙烯酸酯修饰的明胶(简称Gelatin-A)
在200毫升烧杯中加入1克明胶(强度为300Blooms)，50毫升去离子水，50毫升二甲基甲酰胺，进一步加入10克丙烯酸酐搅拌溶解。用1摩尔每升NaOH维持溶液pH＝8±0.5，继续搅拌24小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升去离子水透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到781毫克浅黄色絮状固体，即Gelatin-A，收率78.1％。
Gelatin-A的结构简式见图21(其中的波浪线代表明胶的主链)。
Gelatin-A的 1H-NMR谱图见图21，可见位于6.0-6.5ppm之间的属于丙烯酸官能团的核磁峰，证明该基团成功接枝到明胶的结构中。
制备例12合成甲基丙烯酸酯修饰的明胶(简称Gelatin-MA)
在200毫升烧杯中加入1克明胶(强度为300Blooms)，50毫升去离子水，50毫升二甲基甲酰胺，进一步加入10克甲基丙烯酸酐搅拌溶解。用1摩尔每升NaOH维持溶液pH＝8±0.5，继续搅拌24小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升去离子水透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到824毫克浅黄色絮状固体，即Gelatin-MA，收率82.4％。
Gelatin-MA的结构简式见图22(其中的波浪线代表明胶的主链)。
Gelatin-MA的 1H-NMR谱图见图22，可见位于5.7-6.2ppm之间的属于甲基丙烯酸官能团的核磁峰，证明该基团成功接枝到明胶的结构中。
制备例13合成丙烯酸酯修饰的乙二醇壳聚糖(简称CTS-A)
在200毫升烧杯中加入1克乙二醇壳聚糖(其重均分子量约为250kDa)，50毫升去离子水，50毫升二甲基甲酰胺，8毫升三乙胺(Sigma)，13毫升丙烯酸缩水甘油酯。室温搅拌至均一透明后，继续搅拌48小时。上述溶液装入透析袋(截留分子量3.5kDa，仕必纯)，用5升去离子水透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到694毫克浅黄色絮状固体，即CTS-A，收率69.4％。
CTS-A结构式见图23。
CTS-A的 1H-NMR谱图见图23，可见位于5.8-6.4ppm之间的属于丙烯酸官能团的核磁峰，证明该基团成功接枝到乙二醇壳聚糖的结构中。
制备例14合成甲基丙烯酸酯修饰的乙二醇壳聚糖(简称CTS-MA)
在200毫升烧杯中加入1克乙二醇壳聚糖(其重均分子量约为200kDa)，50毫升去离子水，50毫升二甲基甲酰胺，8毫升三乙胺(Sigma)，13毫升甲基丙烯酸缩水甘油酯。室温搅拌至均一透明后，继续搅拌48小时。上述溶液装入透析袋(截留分子量3.5kDa，仕必纯)，用5升去离子水透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到726毫克浅黄色絮状固体，即CTS-MA，收率72.6％。
CTS-MA的结构式见图24。
CTS-MA的 1H-NMR谱图见图24，可见位于5.7-6.2ppm之间的属于甲基丙烯酸官能团的核磁峰，证明该基团成功接枝到乙二醇壳聚糖的结构中。
制备例15合成巯基-丙烯酸酯修饰的硫酸软骨素(简称CHS-A-SH1)
在200毫升烧杯中加入1克按制备例9的方法制备的CHS-A，0.25克二硫苏糖醇(VWR)，100毫升去离子水，室温搅拌溶解。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量3.5kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到浅黄色絮状固体629毫克，即得CHS-A-SH1，收率为62.9％。
CHS-A-SH1的反应方程式如图5所示，其结构式见图5和图29。
CHS-A-SH1的 1H-NMR谱图见图29，可见位于2.6-3.0ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到硫酸软骨素的结构中。
制备例16合成巯基-甲基丙烯酸酯修饰的硫酸软骨素(简称CHS-MA-SH1)
在200毫升烧杯中加入1克按制备例10的方法制备的CHS-MA，0.25克二硫苏糖醇(VWR)，100毫升去离子水，室温搅拌溶解。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量3.5kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到浅黄色絮状固体642毫克，即得CHS-MA-SH1，收率为64.2％。
CHS-MA-SH1反应方程式如图6所示，其结构式见图6和图30。
CHS-MA-SH1的 1H-NMR谱图见图30，可见位于2.6-3.0ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到硫酸软骨素的结构中。
制备例17合成巯基-丙烯酸酯修饰的明胶(简称Gelatin-A-SH1)
在200毫升烧杯中加入1克按制备例11的方法制备的Gelatin-A，0.19克二硫苏糖醇(VWR)，100毫升去离子水，室温搅拌溶解。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到浅黄色絮状固体763毫克，即得Gelatin-A-SH1，收率为76.3％。
Gelatin-A-SH1的反应方程式如图7所示，其结构式见图7和图31。
Gelatin-A-SH1的 1H-NMR谱图见图31，可见位于2.6-2.8ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到明胶的结构中。
制备例18合成巯基-甲基丙烯酸酯修饰的明胶(简称Gelatin-MA-SH1)
在200毫升烧杯中加入1克按制备例12的方法制备的Gelatin-MA，0.19克二硫苏糖醇(VWR)，100毫升去离子水，室温搅拌溶解。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到浅黄色絮状固体787毫克，即得Gelatin-MA-SH1，收率为78.7％
Gelatin-MA-SH1的反应方程式如图8所示，其结构式见图8和图32。
Gelatin-MA-SH1的 1H-NMR谱图见图32，可见位于2.6-2.7ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到明胶的结构中。
制备例19合成巯基-丙烯酸酯修饰的乙二醇壳聚糖(简称CTS-A-SH1)
在200毫升烧杯中加入1克按制备例13的方法制备的CTS-A，0.25克二硫苏糖醇(VWR)，100毫升去离子水，室温搅拌溶解。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量3.5kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到浅黄色絮状固体602毫克，即得CTS-A-SH1，收率为60.2％。
CTS-A-SH1的反应方程式如图9所示，其结构式见图9和图33。
CTS-A-SH1的 1H-NMR谱图见图33，可见位于2.6-3.0ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到乙二醇壳聚糖的结构中。
制备例20合成巯基-甲基丙烯酸酯修饰的壳聚糖(简称CTS-MA-SH1)
在200毫升烧杯中加入1克按制备例14的方法制备的CTS-MA，0.25克二硫苏糖醇(VWR)，100毫升去离子水，室温搅拌溶解。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量3.5kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到白色絮状固体643毫克，即得CTS-MA-SH1，收率为64.3％
CTS-MA-SH1的反应方程式如图10所示，其结构式见图10和图34。
CTS-MA-SH1的 1H-NMR谱图见图34，可见位于2.5-2.9ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到壳聚糖的结构中。
制备例21合成丙烯酸酯修饰的聚甲基丙烯酸羟乙酯(简称PHEMA-A)
在200毫升烧杯中加入2克聚甲基丙烯酸羟乙酯(Mv＝20kDa，购自Sigma公司)，50毫升去离子水，50毫升二甲基甲酰胺进一步加入16.5克丙烯酸酐搅拌溶解。用1摩尔每升NaOH维持溶液pH＝8±0.5，继续搅拌24小时。上述溶液装入透析袋(截留分子量2kDa，仕必纯)，用5升去离子水透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到1.42克白色固体，即PHEMA-A，收率71.0％。
PHEMA-A的结构式见图35。
PHEMA-A的 1H-NMR谱图见图35，可见位于5.9-6.4ppm之间的属于丙烯酸官能团的核磁峰，证明该基团成功接枝到聚甲基丙烯酸羟乙酯的结构中。
制备例22合成甲基丙烯酸酯修饰的聚甲基丙烯酸羟乙酯(简称PHEMA-MA)
在200毫升烧杯中加入2克聚甲基丙烯酸羟乙酯(Mv＝20kDa，购自Sigma公司)，50毫升去离子水，50毫升二甲基甲酰胺，进一步加入16.8克甲基丙烯酸酐搅拌溶解。用1摩尔每升NaOH维持溶液pH＝8±0.5，继续搅拌24小时。上述溶液装入透析袋(截留分子量2kDa，仕必纯)，用5升去离子水透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到1.48克白色固体，即PHEMA-MA，收率74.0％。
PHEMA-MA的结构式见图36。
PHEMA-MA的 1H-NMR谱图见图36，可见位于5.7-6.3ppm之间的属于甲基丙烯酸官能团的核磁峰，证明该基团成功接枝到聚甲基丙烯酸羟乙酯的结构中。
制备例23合成丙烯酸酯修饰的聚乙烯醇(简称PVA-A)
在200毫升烧杯中加入2克聚乙烯醇(Mw＝61kDa，购自Sigma公司)，50毫升去离子水，50毫升二甲基甲酰胺，进一步加入13克丙烯酸酐搅拌溶解。用1摩尔每升NaOH维持溶液pH＝8±0.5，继续搅拌24小时。上述溶液装入透析袋(截留分子量3.5kDa，仕必纯)，用5升去离子水透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到1.57克白色固体，即PVA-A，收率78.5％。
PVA-A的结构式见图37。
PVA-A的 1H-NMR谱图见图37，可见位于6.0-6.5ppm之间的属于丙烯酸官能团的核磁峰，证明该基团成功接枝到聚乙烯醇的结构中。
制备例24合成甲基丙烯酸酯修饰的聚乙烯醇(简称PVA-MA)
在200毫升烧杯中加入2克聚乙烯醇(Mw＝61kDa，购自Sigma公司)，50毫升去离子水，50毫升二甲基甲酰胺，进一步加入13.4克甲基丙烯酸酐搅拌溶解。用1摩尔每升NaOH维持溶液pH＝8±0.5，继续搅拌24小时。上述溶液装入透析袋(截留分子量3.5kDa，仕必纯)，用5升去离子水透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到1.51克白色固体，即PVA-MA，收率75.5％。
PVA-MA的结构式见图38。
PVA-MA的1H-NMR谱图见图38，可见位于5.7-6.3ppm之间的属于甲基丙烯酸官能团的核磁峰，证明该基团成功接枝到聚乙烯醇的结构中。
制备例25合成巯基-丙烯酸酯修饰的聚甲基丙烯酸羟乙酯(简称PHEMA-A-SH1)
在200毫升烧杯中加入2克按制备例21的方法制备的PHEMA-A，0.42克二硫苏糖醇(VWR)，50毫升去离子水，50毫升二甲基甲酰胺，室温搅拌溶解。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量2kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到白色固体1.67克，即得PHEMA-A-SH1，收率为83.5％。
PHEMA-A-SH1的反应方程式如图57所示，其结构式见图57和图39。
PHEMA-A-SH1的 1H-NMR谱图见图39，可见位于2.6-2.9ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到聚甲基丙烯酸羟乙酯的结构中。
制备例26合成巯基-甲基丙烯酸酯修饰的聚甲基丙烯酸羟乙酯(简称PHEMA-MA-SH1)
在200毫升烧杯中加入2克按制备例22的方法制备的PHEMA-MA，0.41克二硫苏糖醇(VWR)，50毫升去离子水，50毫升二甲基甲酰胺，室温搅拌溶解。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量2kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到白色固体1.62克，即得PHEMA-MA-SH1，收率为81％。
PHEMA-MA-SH1的反应方程式如图58所示，其结构式见图58和图40。
PHEMA-MA-SH1的 1H-NMR谱图见图40，可见位于2.6-3.0ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到聚甲基丙烯酸羟乙酯的结构中。
制备例27合成巯基-丙烯酸酯修饰的聚乙烯醇(简称PVA-A-SH1)
在200毫升烧杯中加入1克按制备例23的方法制备的PVA-A，100毫升去离子水，溶液加热搅拌至PVA-A完全溶解。随后溶液中加入0.47克二硫苏糖醇(VWR)，室温搅拌溶解。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到白色固体737毫克，即得PVA-A-SH1，收率为73.7％。
PVA-A-SH1的反应方程式如图59所示，其结构式见图59和图41。
PVA-A-SH1的 1H-NMR谱图见图41，可见位于2.6-3.0ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到聚乙烯醇的结构中。
制备例28合成巯基-甲基丙烯酸酯修饰的聚乙烯醇(简称PVA-MA-SH1)
在200毫升烧杯中加入1克按制备例24的方法制备的PVA-MA，100毫升去离子水，溶液加热搅拌至PVA-MA完全溶解。随后溶液中加入0.47克二硫苏糖醇(VWR)，室温搅拌溶解。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到白色固体718毫克，即得PVA-MA-SH1，收率为71.8％。
PVA-MA-SH1的反应方程式如图60所示，其结构式见图60和图42。
PVA-MA-SH1的 1H-NMR谱图见图42，可见位于2.5-3.0ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到聚乙烯醇的结构中。
制备例29合成巯基修饰的超支化PEG聚合物(简称HB-PEG-SH1)
在200毫升烧杯中加入5克超支化PEG即HB-PEG(Mw＝20kDa，购自Blafar Ltd)，0.86克二硫苏糖醇(VWR)，100毫升去离子水，室温搅拌溶解。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量2kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到无色粘稠液体3.84克，即得HB-PEG-SH1，收率为76.8％。
HB-PEG-SH1的反应方程式如图61所示，其结构式见图61和图43。
HB-PEG-SH1的 1H-NMR谱图见图43，可见位于2.5-2.6ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到超支化PEG聚合物的结构中。
制备例30合成巯基-丙烯酸酯修饰的透明质酸(简称HA-A1-SH2)
在200毫升烧杯中加入1克按制备例1的方法制备的HA-A1，0.42克1,4-丁二硫醇(购自Sigma公司)，100毫升去离子水，室温搅拌溶解，得到透明溶液。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到852毫克白色絮状固体，即得HA-A1-SH2，收率为85.2％。
HA-A1-SH2的反应方程式如图62所示，其结构式见图62和图44。
HA-A1-SH2的 1H-NMR谱图见图44，可见位于1.6-1.9ppm之间的属于巯基侧链的核磁峰，证明 巯基成功接枝到透明质酸的结构中。
制备例31合成巯基-丙烯酸酯修饰的透明质酸(简称HA-A1-SH3)
在200毫升烧杯中加入1克按制备例1的方法制备的HA-A1，0.43克2-氨基-1,4-丁二硫醇盐酸盐(购自Sigma公司)，100毫升去离子水，室温搅拌溶解，得到透明溶液。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到843毫克白色絮状固体，即得HA-A1-SH3，收率为84.3％。
HA-A1-SH3的反应方程式如图63所示，其结构式见图63和图45。
HA-A1-SH3的 1H-NMR谱图见图45，可见位于3.0-3.2ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到透明质酸的结构中。
制备例32合成巯基-丙烯酸酯修饰的透明质酸(简称HA-A2-SH2)
在200毫升烧杯中加入1克按制备例2的方法制备的HA-A2，0.42克1,4-丁二硫醇(购自Sigma公司)，100毫升去离子水，室温搅拌溶解，得到透明溶液。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到827毫克白色絮状固体，即得HA-A2-SH2，收率为82.7％。
HA-A2-SH2的反应方程式如图64所示，其结构式见图64和图46。
HA-A2-SH2的 1H-NMR谱图见图46，可见位于1.6-1.9ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到透明质酸的结构中。
制备例33合成巯基-丙烯酸酯修饰的透明质酸(简称HA-A2-SH3)
在200毫升烧杯中加入1克按制备例2的方法制备的HA-A2，0.43克2-氨基-1,4-丁二硫醇盐酸盐(Sigma)，100毫升去离子水，室温搅拌溶解，得到透明溶液。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到833毫克白色絮状固体，即得HA-A2-SH3，收率为83.3％。
HA-A2-SH3的反应方程式如图65所示，其结构式见图65和图47。
HA-A2-SH3的 1H-NMR谱图见图47，可见位于3.0-3.2ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到透明质酸的结构中。
制备例34合成巯基-丙烯酸酯修饰的透明质酸(简称HA-A2-SH4)
在200毫升烧杯中加入1克按制备例2的方法制备的HA-A2，0.38克1,3-丙二硫醇(购自Sigma公司)，100毫升去离子水，室温搅拌溶解，得到透明溶液。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到814毫克白色絮状固体，即得HA-A2-SH4，收率为81.4％。
HA-A2-SH4的反应方程式如图66所示，其结构式见图66和图48。
HA-A2-SH4的 1H-NMR谱图见图48，可见位于2.5-2.8ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到透明质酸的结构中。
制备例35合成巯基-丙烯酸酯修饰的透明质酸(简称HA-A2-SH5)
在200毫升烧杯中加入1克按制备例2的方法制备的HA-A2，0.52克1,3-苯二硫酚(购自Sigma公司)，100毫升去离子水，室温搅拌溶解，得到透明溶液。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到836毫克白色絮状固体，即得HA-A2-SH5，收率为83.6％。
HA-A2-SH5的反应方程式如图67所示，其结构式见图67和图49。
HA-A2-SH5的 1H-NMR谱图见图49，可见位于6.9-7.4ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到透明质酸的结构中。
制备例36合成巯基-丙烯酸酯修饰的透明质酸(简称HA-A2-SH6)
在200毫升烧杯中加入1克按制备例2的方法制备的HA-A2，0.52克1,4-苯二硫酚(购自Sigma公司)，100毫升去离子水，室温搅拌溶解，得到透明溶液。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到831毫克白色絮状固体，即得HA-A2-SH6，收率为83.1％。
HA-A2-SH6的反应方程式如图68所示，其结构式见图68和图50。
HA-A2-SH6的 1H-NMR谱图见图50，可见位于6.8-7.0ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到透明质酸的结构中。
制备例37合成巯基-丙烯酸酯修饰的透明质酸(简称HA-A2-SH7)
在200毫升烧杯中加入1克按制备例2的方法制备的HA-A2，0.96克巯基聚乙二醇(购自Sigma公司)，100毫升去离子水，室温搅拌溶解，得到透明溶液。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到894毫克白色絮状固体，即得HA-A2-SH7，收率为89.4％。
HA-A2-SH7的反应方程式如图69所示，其结构式见图69和图51。
HA-A2-SH7的 1H-NMR谱图见图51，可见位于3.6ppm的属于巯基侧链的核磁峰，证明巯基成功接枝到透明质酸的结构中。
制备例38合成巯基-丙烯酸酯修饰的透明质酸(简称HA-A2-SH8)
在200毫升烧杯中加入1克按制备例2的方法制备的HA-A2，0.74克三羟甲基丙烷基-三(3-巯基丙酸酯)(购自Sigma公司)，50毫升去离子水和50毫升二甲基甲酰胺，室温搅拌溶解，得到透明溶液。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到785毫克白色絮状固体，即得HA-A2-SH8，收率为78.5％。
HA-A2-SH8的反应方程式如图70所示，其结构式见图70和图52。
HA-A2-SH8的 1H-NMR谱图见图52，可见位于0.8-1.0ppm、1.5ppm、2.6-2.9ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到透明质酸的结构中。
制备例39合成巯基-甲基丙烯酸酯修饰的透明质酸(简称HA-MA1-SH5)
在200毫升烧杯中加入1克按制备例3的方法制备的HA-MA1，0.50克1,3-苯二硫酚(购自Sigma公司)，100毫升去离子水，室温搅拌溶解，得到透明溶液。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到828毫克白色絮状固体，即得HA-MA1-SH5，收率为82.8％。
HA-MA1-SH5的反应方程式如图71所示，其结构式见图71和图53。
HA-MA1-SH5的 1H-NMR谱图见图53，可见位于6.9-7.4ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到透明质酸的结构中。
制备例40合成巯基-甲基丙烯酸酯修饰的透明质酸(简称HA-MA1-SH6)
在200毫升烧杯中加入1克按制备例3的方法制备的HA-MA1，0.50克1,4-苯二硫酚(购自Sigma公司)，100毫升去离子水，室温搅拌溶解，得到透明溶液。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到833毫克白色絮状固体，即得HA-MA1-SH6，收率为83.3％。
HA-MA1-SH6的反应方程式如图72所示，其结构式见图72和图54。
HA-MA1-SH6的 1H-NMR谱图见图54，可见位于6.9-7.0ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到透明质酸的结构中。
制备例41合成巯基-甲基丙烯酸酯修饰的透明质酸(简称HA-MA2-SH7)
在200毫升烧杯中加入1克按制备例4的方法制备的HA-MA2，0.92克巯基聚乙二醇(购自Sigma公司)，100毫升去离子水，室温搅拌溶解，得到透明溶液。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到876毫克白色絮状固体，即得HA-MA2-SH7，收率为87.6％。
HA-MA2-SH7的反应方程式如图73所示，其结构式见图73和图55。
HA-MA2-SH7的 1H-NMR谱图见图55，可见位于3.6ppm的属于巯基侧链的核磁峰，证明巯基成功接枝到透明质酸的结构中。
制备例42合成巯基-甲基丙烯酸酯2修饰的透明质酸(简称HA-MA2-SH8)
在200毫升烧杯中加入1克按制备例4的方法制备的HA-MA2，0.68克三羟甲基丙烷基-三(3-巯基丙酸酯)(购自Sigma公司)，50毫升去离子水和50毫升二甲基甲酰胺，室温搅拌溶解，得到透明溶液。所得透明溶液继续搅拌12小时。上述溶液装入透析袋(截留分子量8kDa，仕必纯)，用5升pH＝4的盐酸溶液透析5天，每天换水两次。最后收集透析袋内溶液，经冷冻干燥后得到825毫克白色絮状固体，即得HA-MA2-SH8，收率为82.5％。
HA-MA2-SH8的反应方程式如图74所示，其结构式见图74和图56。
HA-MA2-SH8的 1H-NMR谱图见图56，可见位于0.8-1.0ppm、1.5ppm、2.6-2.9ppm之间的属于巯基侧链的核磁峰，证明巯基成功接枝到透明质酸的结构中。
实施例1硫醇-乙烯基交联透明质酸的水凝胶的制备
制备例1、2、9、11、13中制备任意一种的丙烯酰化高分子化合物或者乙二醇二丙烯酸酯(EGDA) 10毫克溶解于1毫升磷酸盐缓冲溶液中(pH＝7.4)，得到质量体积浓度为1％的系列溶液A。
制备例5-8，15-20中制备的任意一种巯基改性高分子化合物10毫克溶解于1毫升磷酸盐缓冲液中(pH＝7.4)，得到质量体积浓度为1％的系列溶液B。
将上述任意一种溶液A，和任意一种溶液B按等体积均匀混合，两种高分子化合物间的生理性原位交联反应随即发生，溶液粘度随混合时间增长而逐渐增加，最终形成水凝胶。
实施例1中各水凝胶包括如下特征性结构单元：
Figure PCTCN2020079823-appb-000044
其中，R 1、R 2和R 3的定义同前；*表示连接点。
各组水凝胶成胶时间列于表1中。
表1 水凝胶成胶时间
Figure PCTCN2020079823-appb-000045
实施例2：水凝胶储能模量检测
将实施例1中制备的两毫升的水凝胶混合溶液置于圆柱形模具中，室温交联24小时后取出测试交联样品储能模量，每组样品检测三次。检测仪器TA-DHR2流变仪，检测探头20mm平行板探头，检测温度：25℃，剪切频率：1Hz。测试结果列于表2中。
表2：储能模量对比表：
Figure PCTCN2020079823-appb-000046
实施例3：水凝胶的保水性能测试
将本实施例1中制备的水凝胶加入到预先称量瓶重的20毫升玻璃瓶中，由质量差减法得到水凝胶的 质量记为m 0。将玻璃瓶置于37℃摇床内，每隔一定时间称量质量得到水凝胶实时质量记为m t。水凝胶的保水能力根据下式计算：
保水率(％)＝m t/m 0×100％
保水率结果见表3。
表3：保水率指标对比表：
Figure PCTCN2020079823-appb-000047
实施例4：水凝胶的体外降解实验
降解稳定性测试：在实验条件为温度37±0.1℃，相对湿度65％±5％的条件下在实施例1中制备的水凝胶中加入10毫升PBS溶液。取初始时间点的水凝胶重量记为m 0，于降解实验开始后第1，4，8，16周称量水凝胶重量记为m t，水凝胶的降解比率根据下式计算：
降解率(％)＝(m 0-m t)/m 0×100％
水凝胶的体外降解测试结果见表4。
表4 水凝胶体外降解测试
Figure PCTCN2020079823-appb-000048
实施例5：水凝胶细胞活性实验
参照“GBT 16886.5-2017医疗器械生物学评价+第5部分+体外细胞毒性试验”标准测试本发明的HA-SH的细胞活性和生物相容性。具体的，采用下述的MTT法，MTT法又称MTT比色法，是一种检测细胞存活和生长的方法。其检测原理为活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT还原为水不溶性的蓝紫色结晶甲臜(Formazan)并沉积在细胞中，而死细胞无此功能。二甲基亚砜(DMSO)能溶 解细胞中的甲臜，用酶联免疫检测仪在490nm波长处测定其光吸收值，可间接反映活细胞数量。在一定细胞数范围内，MTT结晶形成的量与细胞数成正比。具体的测试过程和结果如下：
取制备例1制备的HA-A1的溶液(浓度10mg/mL，溶剂为磷酸盐缓冲溶液，pH＝7.4)，记为溶液A，备用。
取制备例5制备的HA-A1-SH1的溶液(浓度10mg/mL，溶剂为磷酸盐缓冲溶液，pH＝7.4)，记为溶液B，备用。
制备使用以改良杜氏依格尔培养基、10％胎牛血清和1％青霉素/链霉素溶液的细胞培养液。常规培养L929细胞，培养至细胞近汇合后，消化细胞获取细胞悬浮液。
将溶液A、溶液B、细胞悬浮液三个组分均匀混合制备体积为50μL的细胞/水凝胶复合体系，细胞的最终浓度为1×10 6cell/mL。将该体系置于24孔细胞培养板中，每孔中加入1mL细胞培养液进行培养，其中，在板底的同等数量的细胞作为阴性对照组。将样品在5％CO 2、37℃、>90％湿度条件下于细胞培养箱内培养24小时。采用MTT法检测不同水凝胶样品中细胞的存活状态，将水凝胶组细胞活性与阴性对照组的细胞活性进行比较。阴性对照组为100％活性。移除培养液，将100μL MTT加入每个试验孔中，继续孵育4小时。然后弃去MTT溶液，每孔加入200μL DMSO溶液，震荡均匀后，用酶标仪测定490nm处吸光度。测试的结果见图11。MTT实验细胞存活率结果在70％以下的材料被认为具有潜在的细胞毒性。结果显示，细胞在本发明的水凝胶中存活率均在70％以上，表明材料没有明显细胞毒性，生物相容性好。
实施例6：水凝胶塑型和支撑效果动物实验
C57BL/6小鼠麻醉后后背祛毛，常规消毒，将120μL按照实施例1中所述方法制备的HA-A1和HA-A1-SH1溶液、HA-A2和HA-A2-SH1溶液(浓度分别为10mg/mL)，即分别取各材料12mg，溶解于1.2mL的PBS(磷酸盐缓冲生理盐水)溶液中，摇匀，获得供试用样品，待用。用注射器将该供试用样品分别吸取120μL并均匀混合，将得到的水凝胶前体溶液通过24G针头注射到小鼠后背的皮下部位。同等体积的生理盐水按照同样方式注射到小鼠后背的皮下。
注射前、注射后即刻、第4周、第8周、第12周对隆起部位进行拍照、使用游标卡尺进行测量并详细记录。水凝胶的塑形效果通过对比不同注射样品注射入动物体内后立体形态的维持和变化来评判。注射部位隆起高度越高、底面积越小，说明塑形和支撑效果越好。结果见图12和图13。数据显示，本发明的水凝胶在注射入动物体内后形成能够维持一定形态的支撑体，能较好的保持注射物形态的稳定。
实施例7：水凝胶塑型效果动物实验
C57BL/6小鼠麻醉后后背祛毛，常规消毒，将120μL按照实施例1中所述方法制备的HA-A1和HA-A1-SH1溶液、HA-A2和HA-A2-SH1溶液(浓度分别为10mg/mL)，即分别取各材料12mg，溶解于1.2mL的PBS溶液中，摇匀，获得供试用样品，待用。用注射器将该供试用样品分别吸取120μL并均匀混合，将得到的水凝胶前体溶液通过24G针头注射到小鼠后背的皮下部位；注射60分钟后将一只小鼠实施安乐死，观察水凝胶在小鼠皮下原位形成情况及形态；剩余动物继续常规饲养，在注射后第4周、8周、12周对注射部位进行拍照观察并记录凝胶体积变化。同等体积的生理盐水按照同样方式注射到小鼠后背的皮下。
水凝胶前体混合溶液注射到小鼠背部皮下后，在注射部位可见圆形隆起。注射60分钟后观察到小鼠皮下形成的水凝胶为透明完整半球形。结果显示混合前体溶液能够快速发生交联反应，于注射原位形成水凝胶，并保持一定的形态。注射12周后，动物背部皮下仍能观察到明显的隆起。切开皮肤组织观察水凝胶和周围组织局部表现发现，水凝胶形态良好，周围组织未见炎症、感染、坏死等异常。将水凝胶取出后进行观察和称重，发现水凝胶保持完整的半球形，称重后发现重量稍有变小(结果见图14)，未见明显胶体破碎或解体等。
结果显示，本发明的水凝胶在耐降解和保持胶体稳定性方面性能较为优越。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种水凝胶，其特征在于，该水凝胶通过含有巯基改性高分子化合物的体系的凝胶化制备得到；
    所述巯基改性高分子化合物是下述系列化合物中的至少一种：
    一系列巯基改性高分子化合物，其被改性的高分子化合物的结构上含有-COOH、-NH 2、-OH、式a所示丙烯酸酯类基团、式b所示丙烯酰胺类基团、式c所示丙烯酰类基团中的至少一种，
    Figure PCTCN2020079823-appb-100001
    所述-COOH和/或-NH 2和/或-OH和/或丙烯酸酯类基团和/或丙烯酰胺类基团和/或丙烯酰类基团的部分或全部被修饰形成端基为下述基团的侧链：
    Figure PCTCN2020079823-appb-100002
    上述基团中，*表示连接点；R 1选自氢，卤素，脂肪基团，芳香基团等；R 2和R 3相同或不同，彼此独立地选自氢，卤素，脂肪基团，芳香基团等；R 4为多巯基化合物片段；
    所述体系中进一步含有下述物质的至少一种：
    C1.丙烯酰化高分子化合物，
    C2.含丙烯酰基的小分子交联剂。
  2. 根据权利要求1所述的水凝胶，其中，所述-COOH和/或-NH 2和/或-OH和/或丙烯酸酯类基团和/或丙烯酰胺类基团和/或丙烯酰类基团的部分或全部被修饰形成以下结构的至少一种：
    Figure PCTCN2020079823-appb-100003
    上述结构中，R选自
    Figure PCTCN2020079823-appb-100004
    亚烃基、亚芳基、酰胺残基、酰肼残基等；*表示连接点； 1*表示与R的左侧基团的连接点； 2*表示与R的右侧基团的连接点；R 1、R 2、R 3和R 4的定义同前。
  3. 根据权利要求1或2所述的水凝胶，其中，所述-COOH、-NH 2、-OH、式a所示丙烯酸酯类基团、式b所示丙烯酰胺类基团、式c所示丙烯酰类基团中的至少一种可以是直接连接在高分子化合物的主链上，也可以是通过R’基团连接在高分子化合物的主链上，所述R’可以是含有杂原子的基团、亚烃基、亚芳基或下述连接基团：
    Figure PCTCN2020079823-appb-100005
    上式中，R”是亚烃基或亚芳基，n’为1-1000的整数，*表示连接点。
  4. 根据权利要求1-3任一项所述的水凝胶，其中，所述丙烯酰化高分子化合物选自下述物质中的至少一种：
    1)结构上含有-COOH、-NH 2、-OH中的至少一种的高分子化合物的丙烯酰化化合物，即所述高 分子化合物的结构上含有的-COOH、-NH 2、-OH的至少一种直接或间接与如下基团连接而形成的丙烯酰化化合物：
    Figure PCTCN2020079823-appb-100006
    R 1、R 2和R 3的定义同前；
    2)结构上含有式a所示丙烯酸酯类基团、式b所示丙烯酰胺类基团、式c所示丙烯酰类基团中至少一种的高分子化合物。
  5. 根据权利要求4所述的水凝胶，上述第1)种物质中，所述-COOH和/或-NH 2和/或-OH的部分或全部被修饰形成以下结构的至少一种：
    Figure PCTCN2020079823-appb-100007
    上述结构中，R选自
    Figure PCTCN2020079823-appb-100008
    亚烃基、亚芳基、酰胺残基、酰肼残基等；*表示连接点； 1*表示与R的左侧基团的连接点； 2*表示与R的右侧基团的连接点；R 1、R 2、R 3和R 4的定义同前。
  6. 根据权利要求4或5所述的水凝胶，上述第1)种物质中，所述-COOH、-NH 2、-OH中的至少一种可以是直接连接在高分子化合物的主链上，也可以是通过R’基团连接在高分子化合物的主链上，所述R’可以是含有杂原子的基团、亚烃基、亚芳基或下述连接基团：
    Figure PCTCN2020079823-appb-100009
    上式中，R”是亚烃基或亚芳基，n’为1-1000的整数，*表示连接点。
  7. 根据权利要求1-6任一项所述的水凝胶，所述物质C2.含丙烯酰基的小分子交联剂包括但不限于含丙烯酰基的小分子化合物或含丙烯酰基的低聚物；具体的选自乙二醇二丙烯酸酯EGDA，聚乙二醇二丙烯酸酯PEGDA，三羟甲基丙烷三丙烯酸酯TMPTA，季戊四醇三丙烯酸酯PTA，季戊四醇四丙烯酸酯PTTA，二(三羟甲基丙烷)四丙烯酸酯DTTA等中的一种或多种。
  8. 根据权利要求1-7任一项所述的水凝胶，其中，所述水凝胶包括下述特征性结构单元：
    Figure PCTCN2020079823-appb-100010
    上述单元中，R 1、R 2、R 3和R 4的定义同前，*表示连接点。
  9. 一种权利要求1-8任一项所述水凝胶的制备方法，所述方法包括以下步骤：
    将含有下述物质的体系凝胶化：
    (i)所述巯基改性高分子化合物，以及
    (ii)物质C1和物质C2两者中的至少一种；
    制备得到所述水凝胶。
    优选地，分别配制所述巯基改性高分子化合物的溶液、所述丙烯酰化高分子化合物的溶液、所述小分子交联剂的溶液和任选的其他生物功能材料、药物、生长因子或者细胞悬液中至少一种的溶液，混合，凝胶化，制备得到所述水凝胶。
    任选地，所述其他生物功能材料、药物、生长因子或者细胞悬液中的至少一种也可以直接添加到 所述巯基改性高分子化合物的溶液或所述丙烯酰化高分子化合物的溶液或所述小分子交联剂的溶液中而引入。
    优选地，凝胶制备过程可以通过所述的巯基改性高分子化合物溶液加入到所述的丙烯酰化高分子化合物溶液和/或所述的小分子交联剂的溶液中，也可以通过所述的丙烯酰化高分子化合物溶液和/或所述的小分子交联剂的溶液加入到所述的巯基改性高分子化合物溶液中。具体的，两种溶液可以通过普通注射器混合，也可以通过双针头注射器混合，也可以通过其他方式混合。
  10. 权利要求1-8任一项所述水凝胶的用途，其用于生物医药，医疗美容整形，以及化妆品等领域。具体的，其可以用于制备药物传递系统、用于软组织创伤修复敷料、用于骨修复的支架材料、眼科手术中用于支撑作用的粘弹剂、用于手术后防止组织粘连的材料、以及用于3D生物打印的支架材料等。
PCT/CN2020/079823 2019-11-18 2020-03-18 巯基改性高分子化合物的水凝胶及其制备方法和用途 WO2021098099A1 (zh)

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