Sulfhydrylation biocompatible polymer derivative, preparation method thereof, extracellular matrix-imitating injectable in-situ cross-linking hydrogel and application of extracellular matrix-imitating injectable in-situ cross-linking hydrogel

Abstract

The invention relates to the technical field of biomedical material modification, in particular to a sulfhydrylation biocompatible polymer derivative, a preparation method thereof, extracellular matrix imitating injectable in-situ cross-linked hydrogel and application thereof. According to the invention, a pre-acylation strategy is firstly carried out on the biocompatible polymer containing both amino and carboxyl, so that a three-dimensional cross-linked byproduct formed by coupling of self amido bonds is avoided, meanwhile, the number of side chain carboxyl for sulfhydrylation modification is increased, and the accurate and adjustable pre-acylation rate can optimize the physical and chemical properties of the derivative; meanwhile, a disulfide bond is used as a protective precursor, active sulfydryl is introduced through amido bond coupling, self-oxidation of the active sulfydryl in the reaction process is avoided, sulfydryl activity is guaranteed, hydrazine-containing reagents are avoided, safety is further improved, virus residues and immunological rejection risks are avoided, universality is high, and the application prospect is wide; and in combination with a high-concentration preparation method, the method has the advantage of large-scale production, and further widens the industrial application potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomaterial modification, and particularly relates to a thiolated biocompatible polymer derivative, a preparation method thereof, and an injectable in-situ crosslinking hydrogel simulating extracellular matrix and application thereof. BACKGROUND

[0002] Biocompatible polymers such as gelatin, which is a partial hydrolysis product of collagen, including type A gelatin prepared by acid hydrolysis and type B gelatin prepared by alkaline hydrolysis, are a kind of protein containing 18 kinds of amino acids, and retain a large number of collagen bioactive sequences. Biocompatible polymers such as recombinant human collagen are obtained by gene engineering means, i.e., expressing human collagen genes in host cells (such as E. coli, yeast, mammalian cells) with high efficiency. The recombinant human collagen has the advantages of precise sequence design, high batch consistency, good solubility, no risk of viral residue, low immunogenicity, and excellent biological safety. The recombinant expression technology can also be used to prepare gelatin analogs, i.e., recombinant gelatin, which has a clear amino acid sequence, a concentrated molecular weight distribution, and excellent biocompatibility and processability, and has a wide potential application prospect in the field of biological medicine. However, both gelatin and recombinant gelatin have important performance defects, such as good water solubility but low viscosity, easy flow and diffusion, insufficient mechanical properties, too fast in vivo degradation and absorption, and inability to provide long-term stable support, which makes it difficult to meet the clinical application requirements of tissue regeneration scaffolds, wound healing dressings, and the like. Chemical modification and crosslinking are an effective way to solve the above-mentioned performance defects, and thiolation modification is an important part of chemical modification of (recombinant) gelatin and recombinant human collagen. The introduction of thiol groups into biocompatible polymers can obtain derivatives with good biocompatibility and no toxic by-products, and also avoids the toxic and adverse reactions of traditional chemical crosslinking agents, which can well meet the needs of many clinical applications.

[0003] Biocompatible polymers such as gelatin and collagen have similar molecular structures composed of multiple amino acids, and the side chains contain both amino groups (-NH2) and carboxyl groups (-COOH), of which the carboxyl group is an important target group for chemical modification, and the number is usually large. For example, type B gelatin contains 28 side chain amines and 118 side chain carboxyl groups per 1000 amino acid residues; the number of side chain carboxyl groups of type A gelatin is about 54 / 1000 amino acid residues, and the number of side chain amines is similar to that of type B gelatin. The amphoteric biocompatible polymer is usually dissolved only in aqueous solution, and the selection of chemical modification methods is very limited.

[0004] The existing technology (BS Jacobson, et al., Analytical Biochemistry, 106(1), 1980, 114-117; Journal of Molecular Biology, 104, 1, 1976: 243-261) describes the coupling reaction principle of biocompatible polymers containing carboxyl groups (-COOH) with acylhydrazine or amino reagents, such as... Figure 1 As shown. By Figure 1 It is known that in a weakly acidic aqueous environment (typically with a pH of approximately 4-6), the carboxyl groups of biocompatible polymers can be activated by water-soluble carbodiimides such as EDCI to form O-acetylisourea intermediates (e.g., Figure 1 The active intermediate (a) formed as shown in the figure can then undergo nucleophilic addition with an unprotonated amino reagent to form an amide bond (-NH(CO)-) coupling (e.g. Figure 1 The compound formed as shown in (b) has a high amino (-NH2) pKa (typically pH ≈ 8-11), while under these weakly acidic conditions (pH ≈ 4-6), it is predominantly protonated (-NH3) without nucleophilic addition activity. + It exists in the form of ) with very few non-protonated active amino groups (usually <0.1%), making it difficult to react with O-acetylisourea intermediates ( Figure 1 (a) The active intermediate rapidly reacts to form an amide bond coupling, while the half-life of the O-acetylisourea intermediate is only a few seconds to 2 minutes. It rapidly reacts with water to rearrange and form a stable and inactive N-acetylisourea byproduct (such as...). Figure 1 (c) Inactive compound formed as shown in the diagram. Therefore, to improve reaction efficiency, the nucleophilic addition reaction time needs to be extended. Thus, reagents such as N-hydroxy(sulfonated)succinimide (Sulfo-NHS) are usually added during the reaction to generate a more stable, hydrolysis-resistant active intermediate, whose half-life can be significantly extended to approximately 4-5 hours (e.g., Figure 1 (d) The hydrolysis-resistant intermediate and (e) compound formed as shown in the diagram.

[0005] Therefore, for the biocompatible polymer compounds such as gelatin and collagen which have both amino and carboxyl groups in the side chain, after activating the side chain carboxyl group, EDCI / (sulfo)-NHS can be used to effectively form amide bond coupling with the amino group in the side chain of the same molecule, which has become the most important chemical method for preparing gelatin and collagen cross-linked materials (Z Ghassemi et al., Biopolymers 2018 109(9) e23232; J-W Luo et al., Materials Science Engineering C, 98, 2019, 628-634; D-V Bax et al., Acta Biomaterialia, 49, 2017, 218-234; L Gu et al., Trends in Biotechnology, 37(5), 2019, 464-491). However, the inevitable coupling cross-linking side reaction of the amino group in the side chain of the same molecule seriously restricts the chemical modification of the carboxyl group in the side chain of gelatin and collagen by introducing thiol groups through amide bond coupling. The poor water solubility of the thiolated derivatives obtained limits their application in the field of biomedicine.

[0006] The current technology solves the above problem (side reaction of coupling cross-linking of amino group in the side chain of the same molecule) by using a highly reactive hydrazine reagent (-(CO)NHNH2) to couple with the activated biocompatible polymer such as gelatin. The pKa of the hydrazine reagent and the conventional amino reagent is different. The amino reagent has a higher pKa (pH≈8-11), while the pKa of the hydrazine reagent is significantly reduced due to the adjacent electron-withdrawing group (pH≈2.5-4.5), so the amino group of the hydrazine reagent usually exists in the form of non-protonation (-(CO)NHNH2) under weakly acidic conditions (pH=4-6), which can efficiently nucleophilic addition with O-acetyl isourea intermediate to form hydrazine bond coupling (-(CO)NHNH(CO)-, as shown in the route of forming compound (b) in Figure 1

[0007] ​Based on the above reaction principle, existing research (X-Z Shu. et al, Biomaterials, 2003, 24(21):3825-3834; CN101220090A) uses dihydrazide containing disulfide bond as hydrazine reagent to react with activated gelatin, nucleophilic addition coupling reaction occurs to form hydrazine bond, and dihydrazide modified gelatin is obtained, and then a reducing agent is used to reduce the disulfide bond into a derivative with free thiol group. However, one of the raw materials of dihydrazide containing disulfide bond is hydrazine, and the coupling of hydrazine bond has the potential to hydrolyze into hydrazine in vivo, and hydrazine belongs to 2B potential carcinogens. In the current bio-pharmaceutical field, regulations are becoming more and more strict, and the long-term safety of the cross-linked biomaterials in the body has attracted the attention of clinical experts, and should be avoided as much as possible (Robin A Cox. et al, Canadian Journal of Chemistry, 1984, 62:1613-1617; L-B Witkin, AMA Archives of Industrial Health, 1956, 13(1):34-36; S-H Kenyon. et al, Biochemical Pharmacology, 1999, 57(11):1311-1319; L-A Torres-de la Roche et al., Journal of Clinical Medicine, 11, 2022, 931). SUMMARY

[0008] For the safety of medical field, to solve the technical problems of potential risks brought by hydrazine reagent in the prior art and the technical problem that amino reagent cannot be used to obtain thiol-modified derivative in the conventional path, the present application provides a thiol-modified biocompatible polymer derivative and a preparation method thereof, and an injectable in-situ cross-linked hydrogel simulating extracellular matrix and its use. In the process of preparing the thiol-modified derivative of biocompatible polymer, the biocompatible polymer containing amino and carboxyl is first pre-acylated to strictly shield the side chain amino group to obtain a biocompatible polymer pre-acylation product, and then the carboxyl on the polymer chain is activated to couple with the amino reagent containing disulfide bond to form an amide bond, and reduction can obtain the corresponding thiol-modified biocompatible polymer derivative. The present application can avoid the three-dimensional cross-linked by-products formed by the amide bond coupling of the amino group itself, and can also avoid the potential risks brought by the hydrazine reagent in the prior art, and has higher safety.

[0009] In order to achieve the above purpose, the present application realizes the following technical scheme:

[0010] The first aspect of the present application provides a thiolated biocompatible polymer derivative, which comprises the following structural units in the molecular chain structure: a general formula (I) structural unit, a general formula (II) structural unit, and a general formula (III) structural unit.

[0011] , 、

[0012] ;

[0013] wherein G is a residue of a biocompatible polymer compound, R1 is selected from an alkylene group, a substituted alkylene group, or an aromatic group, R2 is selected from an alkylene group or an aromatic group, and R3 is a carboxyl group or a carboxyl salt;

[0014] The biocompatible polymer compound contains both an amino group and a carboxyl group; and the residue contains both an amino group and a carboxyl group.

[0015] The amino group of the biocompatible polymer compound is first acylated to form a pre-acylated product (incomplete pre-acylation) containing the general formula (II) structural unit, and then the carboxyl group (derived from the carboxyl group of the biocompatible polymer compound and the carboxyl group introduced by pre-acylation) is coupled by an amide bond to introduce a thiol group to form the general formula (I) structural unit and the general formula (III) structural unit, respectively, thereby obtaining the thiolated biocompatible polymer derivative comprising the general formula (I) structural unit, the general formula (II) structural unit, and the general formula (III) structural unit in the molecular chain structure.

[0016] Further, the pre-acylation rate for forming the general formula (II) structural unit by pre-acylation is 40% to 95%.

[0017] Further, the pre-acylation rate for forming the general formula (II) structural unit by pre-acylation is 40% to 95%.

[0018] Further, the thiol content of the thiolated biocompatible polymer derivative is 0.1 mmol / g to 1.0 mmol / g.

[0019] Further, the thiol content of the thiolated biocompatible polymer derivative is 0.1 mmol / g to 1.0 mmol / g.

[0020] Further, the thiol content of the thiolated biocompatible polymer derivative is 0.1 mmol / g to 1.0 mmol / g.

[0021] Further, the biocompatible polymer compound is selected from one or more of gelatin, genetically recombined gelatin, and genetically recombined human collagen; and the weight average molecular weight of the biocompatible polymer compound is 50 kDa to 2200 kDa, particularly preferably 50 kDa to 300 kDa, and more preferably 50 kDa to 200 kDa.

[0022] The second aspect of the present application provides a preparation method of the above-mentioned thiolated biocompatible polymer derivative, comprising the following steps:

[0023] S1, dissolving the biocompatible polymer compound containing amino and carboxyl in an aqueous solution, adjusting the system to weak alkaline conditions, adding a diacid anhydride compound, maintaining the weak alkaline conditions, and performing a pre-acylation reaction between the side chain amino groups of the biocompatible polymer compound and the diacid anhydride compound; in the reaction process, the amino groups of the biocompatible polymer compound react with the diacid anhydride to form amide bonds and introduce side chain carboxyl groups or carboxyl salts, and a pre-acylated product is obtained by purification;

[0024] The pre-acylated product has a structural unit of general formula (II);

[0025] S2, dissolving the pre-acylated product in an aqueous solution, adding an amino reagent containing a disulfide bond and a first activator, adjusting the system to a first weak acid condition and maintaining it, adding a second activator, adjusting the system to a second weak acid condition after activation is completed, and performing an amidation reaction to generate an intermediate product containing amide bonds and coupled with a disulfide bond; in the reaction process, the side chain carboxyl groups of the pre-acylated product are activated by the activator in the aqueous solution under weak acid conditions, and then react with the amino reagent containing a disulfide bond to generate amide bonds and couple with a disulfide bond;

[0026] Subsequently, a reducing agent is added to the system to reduce the disulfide bond, and a thiolated biocompatible polymer derivative containing active thiol groups is obtained after purification, which has a molecular chain structure comprising structural units of general formula (I) to general formula (III).

[0027] Further, the concentration of the biocompatible polymer compound dissolved in the aqueous solution in S1 is controlled to be 5% w / v to 15% w / v, preferably 10% w / v to 15% w / v;

[0028] The concentration of the pre-acylated product dissolved in the aqueous solution in S2 is controlled to be 5% w / v to 10% w / v, preferably 5% w / v to 8% w / v.

[0029] Further, the diacid anhydride compound in S1 is an aliphatic diacid anhydride or an aromatic diacid anhydride with a carbon chain length of C2-C15, preferably a diacid anhydride with a carbon chain length of C4-C8, and particularly preferably one or more of succinic anhydride, glutaric anhydride, adipic anhydride, maleic anhydride, pimelic anhydride, and suberic anhydride.

[0030] Further, the molar feed ratio of the diacid anhydride compound to the side chain amino groups of the biocompatible polymer compound in S1 is 0.2 to 1.5:1, preferably 0.4 to 1.0:1. Therefore, the above-mentioned pre-acylation rate refers to the degree of substitution of amino groups in the biocompatible polymer compound by diacid anhydride.

[0031] Further, the temperature of the pre-acylation reaction in S1 is 25-50°C, and the time is 0.5-3h.

[0032] Preferably, the temperature of the pre-acylation reaction is 30-40°C, and the time is 1-2h.

[0033] The weak alkaline condition is pH = 7.0-10.0, and particularly preferably pH = 8.0-9.5.

[0034] Further, the first activator in S2 is N-hydroxysulfosuccinimide (Sulfo-NHS), and the second activator is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI).

[0035] Further, the disulfide bond-containing amino reagent in S2 has the following general formula:

[0036] ;

[0037] wherein R1 and R4 are each independently selected from an alkylene group, a substituted alkylene group or an aromatic group having a carbon chain length of C1-C15; and R5 is an active group that does not react with an amino group, such as R5 cannot be a carboxyl group, but can be a hydroxyl group or an amino group, etc.; that is, the disulfide bond-containing amino reagent is a disulfide bond-containing diamine or a disulfide bond-containing monoamine.

[0038] Further, the disulfide bond-containing amino reagent is selected from one or more of the following: cystamine (CAS No. 51-85-4), cystamine dihydrochloride (CAS No. 56-17-7), cystamine sulfate (CAS No. 16214-16-7), 3, 3'-dithiodipropylamine (CAS No. 463-22-9), 4,4'-dithiodibutylamine, 6,6'-dithiodihexylamine, 2-[(3-aminopropyl)disulfanyl]ethylamine, 2,2'-dithiodianiline (CAS No. 1141-88-4), 4,4'-diaminodiphenyl disulfide (CAS No. 722-27-0), bis(2-amino-3-methylphenyl) disulfide (CAS No. 86749-03-3), 2-aminoethyl-2'-hydroxyethyldisulfide (CAS No. 15579-01-8), bis(2-amino-1-methylethyl) disulfide (methylcysteamine, CAS No. 4390-10-7), bis(2-aminopropyl) disulfide hydrochloride (CAS No. 2453-35-2), 2-aminoethyldisulfanyl ethylamine, and N-tert-butoxycarbonyl-cystamine.

[0039] Preferably, the disulfide bond-containing amino reagent is a disulfide bond-containing diamine having the following general formula:

[0040] .

[0041] More preferably, the disulfide-containing amino reagent is selected from one or more of cystamine, cystamine dihydrochloride, 3, 3'-dithiodipropylamine.

[0042] Further, the reducing agent in S2 is selected from one or more of tris (2-carboxyethyl) phosphine or its hydrochloride, glutathione (GSH); preferably tris (2-carboxyethyl) phosphine hydrochloride (TCEP·HCl).

[0043] Further, in S2, the total amount of carboxyl groups in the pre-acylated product is calculated based on the total molar amount of the carboxyl groups in the molecular chain structure of the pre-acylated product and the carboxyl groups or carboxyl salts introduced by pre-acylation,

[0044] The molar ratio of the second activating agent, the first activating agent, and the total amount of carboxyl groups in the pre-acylated product is 0.1-2 : 0.1-1 : 1, preferably the molar ratio is 0.4-1.2 : 0.2-0.5 : 1;

[0045] The molar ratio of the disulfide-containing amino reagent in S2 and the total amount of carboxyl groups in the pre-acylated product is 1-5 : 1, preferably the molar ratio is 1-2 : 1;

[0046] The molar ratio of the reducing agent and the total amount of carboxyl groups in the pre-acylated product is 1-4 : 1, preferably the molar ratio is 1-2 : 1; the more the amount of reducing agent, the faster the reduction speed, and the conventional amount is maintained in the present application.

[0047] Further, in S2, the temperature of the amidation reaction is 25-50°C, and the time is 0.2-24h, preferably the temperature of the amidation reaction is 30-40°C, and the time is 0.5-24h.

[0048] Further, in S2, the temperature of the reduction reaction is 25-50°C, and the time is 0.5-5h, preferably the temperature of the reduction reaction is 30-40°C, and the time is 2-4h.

[0049] Further, in S2, the pH of the first weakly acidic condition is 4.0-6.5, and the pH of the second weakly acidic condition is 3.0-7.0; preferably, in S2, the pH of the first weakly acidic condition is 4.5-5.0, and the pH of the second weakly acidic condition is 4.0-6.0.

[0050] The third aspect of the present application provides an injectable in-situ cross-linked hydrogel mimicking extracellular matrix, which is constructed by the thiolated biocompatible polymer derivative or the thiolated biocompatible polymer derivative prepared by the preparation method described above, and the construction method is to form disulfide bond cross-linking by spontaneous oxidation of the thiol in the molecular chain structure of the thiolated biocompatible polymer derivative under physiological compatible conditions and / or to form cross-linking by high-efficiency click chemistry reaction with a multi-functional electrophilic or nucleophilic cross-linking agent.

[0051] Further, the multi-functional electrophilic or nucleophilic cross-linking agent has one or more of the following functional groups in the molecular structure: aldehyde group, acrylate, acrylamide, maleimide group, vinyl sulfone group, norbornene group, thiol group. For example, the cross-linking agent can be α, β-unsaturated (methyl) acrylate compound, α, β-unsaturated (methyl) acrylamide compound, norbornene compound, maleimide compound, vinyl sulfone compound, aldehyde group compound, thiol compound, etc. The process of spontaneous oxidation or high-efficiency click chemistry reaction with the cross-linking agent is shown in the following formula (I) structure unit, wherein G and R1 in the formula (I) are defined as above; the autoxidation is under the condition of an oxidizing agent such as oxygen and / or hydrogen peroxide; R Figure 11 and R 1 and R 2 are independently at least two polyethylene glycol derivatives (such as two-armed, three-armed, four-armed, eight-armed or more-armed polyethylene glycol derivatives), collagen, gelatin, hyaluronic acid, chitosan, sodium alginate, chondroitin sulfate and other biological polymer materials. In the present application, (methyl) acrylate means acrylate or methacrylate, and the like.

[0052] The fourth aspect of the present application provides the use of the injectable in-situ cross-linked hydrogel mimicking extracellular matrix described above in the preparation of a biological and medical preparation.

[0053] Further, the biological and medical preparation includes one or more of a tissue engineering scaffold, a drug controlled release carrier, a 3D biological printing ink, and a cell encapsulation material, which is suitable for the biomedical field such as skin wound repair, cartilage defect filling, nerve conduit bridging or myocardial tissue regeneration.

[0054] The process of forming the thiolated biocompatible polymer derivative of the present application is different from the high-reactivity hydrazine reagent used in the prior art. The present application can avoid the amide bond coupling side reaction of the carboxyl and amino groups, strictly shield the side reaction by pre-acylating the amino group of the biocompatible polymer compound, and then use a lower-reactivity amino reagent containing disulfide bond as the amino reagent to couple with the carboxyl group of the biocompatible polymer compound and the carboxyl group generated by pre-acylation, so that the prior art Figure 1The path of the middle picture fork is smooth, and the thiolated biocompatible polymer derivative can be successfully prepared after reduction.

[0055] In the present application, pre-acylation refers to the process of introducing side chain carboxyl groups while coupling the side chain amino groups of the biocompatible polymer compound with a diacid anhydride to form an amide bond, which not only avoids the side reaction of amide bond coupling between the amino groups and the carboxyl groups of the biocompatible polymer compound itself to form a three-dimensional cross-linked byproduct, but also increases the number of side chain carboxyl groups available for subsequent thiolation modification; the pre-acylation rate refers to the percentage (%) of the side chain amino groups of the biocompatible polymer compound that are pre-acylated. However, complete pre-acylation of the side chain amino groups, although it can strictly avoid the side reaction of amide bond coupling between the amino groups and the carboxyl groups of the biocompatible polymer compound itself, according to experiments, complete pre-acylation can damage the water solubility of the thiolated biocompatible polymer derivative, seriously affecting its application. The possible reason is that, compared to the structural unit of general formula (I), the structural unit of general formula (III) is coupled with thiol through a longer side chain segment, the hydrophobicity is increased, and thus the overall water solubility of the product is damaged. Therefore, the pre-acylation rate is an important parameter that needs to be accurately controlled, and it needs to be acylated enough to avoid the side reaction of amide bond coupling between the amino groups and the carboxyl groups of the biocompatible polymer compound itself as much as possible, but it is not completely acylated to avoid damage to the water solubility of the product. The reaction principle of the biocompatible polymer compound containing both amino and carboxyl groups and the amino reagent containing disulfide bond to form the thiolated biocompatible polymer derivative is shown in Figure 2 .

[0056] Beneficial technical effects: The present application adopts a pre-acylation strategy for the biocompatible polymer compound containing both amino and carboxyl groups to avoid the formation of a three-dimensional cross-linked byproduct by amide bond coupling between the amino groups and the carboxyl groups of the biocompatible polymer compound itself, thereby ensuring the safe and reliable use of an amino reagent containing disulfide bond to introduce active thiol groups by amide bond coupling, avoiding the potential risk of using a hydrazine-containing reagent, and further improving drug safety; at the same time, using disulfide bond as a protective precursor, active thiol groups are introduced by amide bond coupling, avoiding the self-oxidation of active thiol groups during the reaction process, and ensuring the high activity of thiol groups in the final product;

[0057] The present application combines a high-concentration preparation method and has the advantage of large-scale production, further expanding its industrial application potential; the thiolated biocompatible polymer derivative of the present application can realize rapid in-situ cross-linking through mild spontaneous oxidation or click chemistry reaction, forming a mechanically stable injectable in-situ cross-linking hydrogel that simulates the extracellular matrix, effectively solving the shortcomings of (recombinant) gelatin and recombinant human collagen, such as insufficient mechanical properties, difficulty in accurately controlling the degradation rate, and inability to provide long-term stable support for tissue repair, etc., and is suitable for various biomedical needs such as skin wound repair and cartilage defect filling, without the risk of virus residues and immune rejection, with strong universality and broader application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 Reaction principle diagram for coupling reaction of carboxyl-containing biocompatible polymer compound in prior art with hydrazide reagent or amino reagent;

[0059] Figure 2 Reaction principle diagram for reaction of biocompatible polymer compound containing amino and carboxyl groups in the application with amino reagent containing disulfide bond to form thiolated biocompatible polymer derivative.

[0060] Figure 3 Trend chart of different pre-acylation rates with anhydride feeding amount in Example 1;

[0061] Figure 4 NMR spectrum of gelatin pre-acylation product with different pre-acylation rates in Example 1;

[0062] Figure 5 NMR comparison spectrum of gelatin, gelatin pre-acylation product with pre-acylation rate of 95% and thiolated modified derivative of high pre-acylated gelatin in Example 7;

[0063] Figure 6 Correspondence chart of different activation pH and thiol content of thiolated modified derivative of high pre-acylated gelatin prepared in Example 9;

[0064] Figure 7 Correspondence chart of different amount of carboxyl activator (Sulfo-NHS / EDCI) and thiol content of thiolated modified derivative of high pre-acylated gelatin prepared in Example 10;

[0065] Figure 8 Correspondence chart of different amount of amino reagent and thiol content of thiolated modified derivative of high pre-acylated gelatin prepared in Example 11;

[0066] Figure 9 Microscope observation chart of injectable in-situ crosslinking hydrogel simulating extracellular matrix as stem cell adherent growth matrix in Example 17;

[0067] Figure 10 Chart of detecting number of live cells inside in Example 18 in which bone marrow mesenchymal stem cells are embedded in injectable in-situ crosslinking hydrogel simulating extracellular matrix to proliferate;

[0068] Figure 11 Process of structure unit of general formula (I) in the application undergoing spontaneous oxidation or high-efficiency click chemistry reaction with crosslinking agent. DETAILED DESCRIPTION

[0069] Clearly, the described embodiments are only some, but not all, embodiments of the present application. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the application, its application, or uses, nor is it intended to limit the scope of applicability in any way. Based on the description of the embodiments in the application, all other embodiments that can be obtained by a person of ordinary skill in the art without creative work are within the scope of protection of the application.

[0070] Unless otherwise specifically indicated, the numerical values set forth in the examples herein are not limited to the preferred ranges of the application. Techniques, methods, and protocols known to those of ordinary skill in the relevant art can not be discussed in detail, but should be considered well within the scope of the disclosure, where appropriate. In all examples shown and discussed herein, any specific value should be interpreted as merely illustrative and not as a limitation. Thus, other examples of the exemplary embodiments can have different values. It should be noted that, for example, values expressed as "in the range of a-b", "between a-b", do not include the end point values a and b; values expressed as "a-b", "is a-b", include the end point values a and b.

[0071] In addition, it should be noted that the use of the words "first", "second", and the like to define the activator, weakly acidic conditions, and the like, is merely for the convenience of distinguishing the materials and system environment used in each step, and the above words have no special meaning unless otherwise stated, and therefore cannot be understood as limiting the scope of protection of the application.

[0072] The experimental methods in the following examples without specific conditions are generally determined according to the national standard; if there is no corresponding national standard, they are carried out according to the general standard requirements or general methods.

[0073] In the present application, the w / v symbol means mass percentage by volume, which has the same meaning as g / 100 mL.

[0074] Example 1

[0075] The present example is the preparation of a gelatin pre-acylation product, which includes the following steps:

[0076] Accurately weigh 5.00 g of type A gelatin (300 bloom), add purified water to make up to 50.0 mL, stir in a 35℃ constant temperature water bath until completely dissolved, to prepare a clear and transparent gelatin solution with a concentration of 10% w / v;

[0077] The gelatin solution was adjusted to pH 8.5 using 5 M sodium hydroxide solution. Succinic anhydride was added in an amount of 0.2 to 1.5 equivalents (eq) relative to the amino content in the gelatin to carry out a preacylation reaction under continuous stirring. 5 M sodium hydroxide solution was continuously added dropwise throughout the reaction to maintain the weakly alkaline system at pH 8.0 to 9.5. The preacylation reaction was carried out in a 35°C water bath with continuous stirring for 3 h.

[0078] After the reaction was completed, the pH of the reaction system was adjusted back to about 7.0 with 3 M hydrochloric acid, and stirring was continued for 5 min to ensure the system was homogeneous. Then, the reaction solution was transferred to a dialysis bag (molecular weight cutoff 8000 Da), and purified by dialysis with purified water at 35°C. The dialysis solution was changed every 4 h, and dialysis was continued for 2 days. Finally, the solution in the dialysis bag was collected and freeze-dried to obtain a white flocculent solid, which is the gelatin preacylated product.

[0079] The chemical structure of the gelatin preacylated product in this case is shown in formula (Ⅳ):

[0080] , where G represents the residues of type A gelatin.

[0081] HPLC analysis of the preacylated gelatin product revealed no small molecule impurity peaks, indicating high purity of the preacylated gelatin product.

[0082] The trend of preacylation rate of gelatin preacylation products with the amount of acid anhydride added is as follows: Figure 3 As shown, the content of side-chain amino groups in the gelatin preacylated products is characterized by the preacylation rate, which is determined by the 2,4,6-trinitrobenzenesulfonic acid (TNBS) method. The results show that through quantitative reaction, approximately 19%-100% of the amino groups in gelatin are preacylated and modified into carboxyl groups.

[0083] Nuclear magnetic resonance hydrogen spectroscopy was used to detect the gelatin preacylation products. 1 H-NMR): using deuterated DMSO as a solvent, Figure 3 A series of gelatin preacylization products with different preacylization rates were subjected to NMR analysis, and the resulting spectra are shown below. Figure 4 As shown, by Figure 4It can be seen that the characteristic peaks of gelatin side chain alkyl hydrogen (-CH2-, -CH3) at δ 0.8-2.2 ppm have no significant change in peak shape, but the relative proportion of peak area shows a downward trend; the characteristic peaks of lysine ε-methylene hydrogen (ε-CH2 directly connected to -NH2) at δ 2.6-2.9 ppm gradually weaken with the increase of pre-acylation rate, and the peak almost disappears when the pre-acylation rate reaches 90%, and completely disappears at 100% pre-acylation; the characteristic signals at δ 2.4-2.6 ppm are closely related to succinic anhydride modification, when the pre-acylation rate reaches 19%, two weak single peaks at δ ~2.35 and δ ~2.45 appear in this interval, corresponding to the hydrogen signal of -CH2-CH2- group beside the amide bond introduced by succinic anhydride, and with the increase of pre-acylation rate, the intensity of these two characteristic peaks continues to increase, and the peak shape also gradually becomes sharp from blunt.

[0084] Example 2

[0085] The present case is the preparation of a thiolated biocompatible polymer derivative, which uses a low pre-acylation rate gelatin pre-acylation product for thiolation modification, including the following steps:

[0086] S1, precisely weigh 5.00 g of the gelatin pre-acylation product prepared in Example 1 (pre-acylation rate of 19%), add purified water to make up to 100.0 mL, and place in a 35°C constant temperature water bath to stir until completely dissolved, to prepare a clear and transparent solution with a concentration of 5% w / v;

[0087] S2, add 1.285 g of cystamine dihydrochloride and 0.310 g of N-hydroxysulfosuccinimide (Sulfo-NHS) to the above solution in sequence, and stir until completely dissolved; then, adjust the pH of the system solution to 4.3 with 6 M hydrochloric acid; under continuous stirring, add 0.602 g of 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole hydrochloride (EDCI), and maintain the pH of the system at 4.7±0.05 with 3 M hydrochloric acid during the reaction, and continuously stir for 30 min; after activation is complete, adjust the pH of the system to 6.0 with 5 M sodium hydroxide solution, and continuously stir at 35°C overnight to complete the amidation reaction between the carboxyl group and the amino group of cystamine to generate amide bonds and couple to introduce disulfide bonds;

[0088] Subsequently, 3.271 g of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP·HCl) was added to the reaction system and stirred for 5 min to ensure complete dissolution. The pH of the system was gradually adjusted to 6.0 using 5 M sodium hydroxide solution, and the reaction was continued for 3 h under these conditions to fully reduce the disulfide bonds introduced into the molecule to active thiol groups. However, after the reaction, gelatinous lumps were still found in the reaction system. This was because the preacylation rate was too low. During the amidation reaction, after the abundant carboxyl groups on the gelatin molecular chain were activated by Sulfo-NHS / EDCI, in addition to reacting with cystamine, some carboxyl groups also underwent intramolecular or intermolecular crosslinking with unpre-acylated free amino groups on the same molecule or adjacent molecular chains, forming amide bond network crosslinking byproducts. These covalent crosslinking network byproducts have stable structures and will not be destroyed by TCEP in the subsequent reduction steps; therefore, the final product cannot be dissolved.

[0089] Example 3

[0090] This case study describes the preparation of thiolized biocompatible polymer derivatives. Thiolization modification was performed using gelatin pre-acylated products with moderate pre-acylation rates, including the following steps:

[0091] S1. Accurately weigh 5.00 g of the gelatin preacylated product prepared in Example 1 (preacylation rate of 38%), add purified water to make up to 100.0 mL, place in a 35℃ constant temperature water bath and stir until completely dissolved to obtain a clear and transparent solution with a concentration of 5% w / v.

[0092] S2. Add 1.361 g of cystamine dihydrochloride and 0.328 g of N-hydroxythiosuccinimide (Sulfo-NHS) sequentially to the above solution and stir until completely dissolved. Then, adjust the pH of the solution to 4.3 with 6 M hydrochloric acid. While stirring continuously, add 0.637 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and maintain the pH of the system at 4.7±0.05 with 3 M hydrochloric acid during the reaction. Activate the system by stirring continuously for 30 min. After activation, adjust the pH of the system to 6.0 with 5 M sodium hydroxide solution and stir continuously at 35°C overnight to complete the amidation reaction between the carboxyl group and the amino group of cystamine to form an amide bond and couple introduce a disulfide bond.

[0093] Subsequently, 3.466 g of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP·HCl) was added to the reaction system and stirred for 5 min to completely dissolve it. The pH of the system was gradually adjusted to 6.0 with 5 M sodium hydroxide solution, and the reaction was continued to be stirred for 3 h under this condition to fully reduce the disulfide bonds introduced into the molecule to active thiol groups. After the reaction was completed, the system remained clear and transparent, indicating that the disulfide bonds introduced into the molecular structure were fully reduced to active thiol groups.

[0094] The pH of the system solution after the adjustment reaction was 3.5, and the solution was transferred to a dialysis bag (with a molecular weight cut-off of 14 kDa). The dialysis was performed using a 2wt% sodium chloride solution with a pH of 3.3, and the dialysis solution was replaced every 4 hours. The dialysis was continued for 3 days. Subsequently, the dialysis was performed using a 0.9wt% sodium chloride solution with a pH of 3.3 and pure water with a pH of 3.3, respectively, for 1 day each. Finally, the solution in the dialysis bag was collected, and a white flocculent solid was obtained after freeze-drying, which was the prepared thiolated derivative of the medium pre-acylated gelatin.

[0095] The derivative molecule structure of the present case simultaneously has the following three structural units of formula (V) to formula (VII):

[0096] ; wherein G is the residue of the type A gelatin.

[0097] The derivative of the present case was detected by HPLC, and the content of small molecular impurities in the derivative was less than 10 ppm. The active thiol content of the derivative was about 0.43 mmol / g detected by the improved Ellman method.

[0098] Example 4

[0099] The present case is the preparation of a thiolated biocompatible polymer derivative. The thiolated modification is performed on a pre-acylated product of medium pre-acylation rate gelatin, which includes the following steps:

[0100] S1, precisely weigh 5.00 g of the pre-acylated product of gelatin prepared in Example 1 (with a pre-acylation rate of 58%), add purified water to make up to 100.0 mL, and stir in a 35°C constant temperature water bath until completely dissolved to prepare a clear and transparent solution with a concentration of 5% w / v;

[0101] S2, add 1.438 g of cystamine dihydrochloride and 0.347 g of N-hydroxysulfosuccinimide (Sulfo-NHS) to the above solution in sequence, and stir until completely dissolved. Then, adjust the pH of the system solution to 4.3 with 6 M hydrochloric acid. Under continuous stirring, add 0.673 g of 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole hydrochloride (EDCI), and maintain the pH of the system at 4.7±0.05 with 3 M hydrochloric acid during the reaction. Stir for 30 min to activate. After activation, adjust the pH of the system to 6.0 with 5 M sodium hydroxide solution, and continue stirring at 35°C overnight to complete the amidation reaction between the carboxyl group and the amino group of cystamine to generate amide bonds and couple the introduction of disulfide bonds.

[0102] Subsequently, 3.66 g of tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCl) was added to the reaction system, stirred for 5 min to completely dissolve it, and the pH of the system was gradually adjusted to 6.0 with a 5 M sodium hydroxide solution, and the reaction was continued to stir under this condition for 3 h to fully reduce the disulfide bond introduced in the molecule to active thiol. After the reaction was completed, the system remained clear and transparent, indicating that the disulfide bond introduced in the molecular structure was fully reduced to active thiol;

[0103] The pH of the solution in the reaction system after adjustment was adjusted to 3.5, and it was transferred to a dialysis bag (with a molecular weight cut-off of 14 kDa). Purification dialysis was performed using a 2wt% sodium chloride solution with a pH of 3.3, and the dialysate was changed every 4 h, and the dialysis was continued for 3 days. Subsequently, a 0.9wt% sodium chloride solution with a pH of 3.3 and pure water with a pH of 3.3 were used for dialysis for 1 day each. Finally, the solution in the dialysis bag was collected and freeze-dried to obtain a white flocculent solid, which was the prepared thiolated derivative of medium pre-acylated gelatin.

[0104] The derivative molecule structure of this case contains the same structural units as Example 3.

[0105] The derivative of this case was detected by HPLC, and the content of small molecular impurities in the derivative was less than 10 ppm. The active thiol content of the derivative was about 0.45 mmol / g detected by the improved Ellman method.

[0106] Example 5

[0107] This case is the preparation of a thiolated biocompatible polymer derivative. The thiolated modification is performed on a pre-acylated gelatin product with a medium pre-acylation rate, including the following steps:

[0108] S1, precisely weigh 5.00 g of the pre-acylated gelatin product prepared in Example 1 (with a pre-acylation rate of 79%), add purified water to make up to 100.0 mL, and place it in a 35°C constant temperature water bath to stir until completely dissolved, to prepare a clear and transparent solution with a concentration of 5% w / v;

[0109] S2. Add 1.514 g of cystamine dihydrochloride and 0.365 g of N-hydroxythiosuccinimide (Sulfo-NHS) sequentially to the above solution and stir until completely dissolved. Then, adjust the pH of the solution to 4.3 with 6 M hydrochloric acid. While stirring continuously, add 0.709 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and maintain the pH of the system at 4.7±0.05 with 3 M hydrochloric acid during the reaction. Activate the system by stirring continuously for 30 min. After activation, adjust the pH of the system to 6.0 with 5 M sodium hydroxide solution and stir continuously at 35°C overnight to complete the amidation reaction between the carboxyl group and the amino group of cystamine to generate an amide bond and introduce a disulfide bond through coupling.

[0110] Subsequently, 3.855 g of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP·HCl) was added to the reaction system and stirred for 5 min to completely dissolve it. The pH of the system was gradually adjusted to 6.0 with 5 M sodium hydroxide solution, and the reaction was continued to be stirred for 3 h under this condition to fully reduce the disulfide bonds introduced into the molecule to active thiol groups. After the reaction was completed, the system remained clear and transparent, indicating that the disulfide bonds introduced into the molecular structure were fully reduced to active thiol groups.

[0111] After adjusting the pH of the reaction system solution to 3.5, the solution was transferred to a dialysis bag (molecular weight cutoff of 14 kDa) and purified by dialysis with a 2 wt% sodium chloride solution at pH 3.3. The dialysis solution was changed every 4 hours for 3 consecutive days. Subsequently, the solution was changed to a 0.9 wt% sodium chloride solution at pH 3.3 and pure water at pH 3.3 for 1 day each. Finally, the solution in the dialysis bag was collected and freeze-dried to obtain a white flocculent solid, which is the thiol-modified derivative of the prepared moderately preacylated gelatin.

[0112] The structural units contained in the derivative molecular structure in this case are the same as those in Example 3.

[0113] HPLC analysis of the derivative in this case revealed that the content of small molecule impurities was below 10 ppm. The content of active thiol groups in the derivative, determined using a modified Ellman method, was approximately 0.47 mmol / g.

[0114] Example 6

[0115] This case study describes the preparation of thiolized biocompatible polymer derivatives. Thiolization modification was performed using gelatin pre-acylated products with high pre-acylation rates, including the following steps:

[0116] S1, precisely take 5.00 g of the gelatin pre-acylation product prepared in Example 1 (the pre-acylation rate is 89%), add purified water to make up to 100.0 mL, place in a 35°C constant temperature water bath and stir until completely dissolved to prepare a clear and transparent solution with a concentration of 5% w / v;

[0117] S2, add 1.553 g of cystamine dihydrochloride and 0.374 g of N-hydroxysulfosuccinimide (Sulfo-NHS) to the above solution in sequence, stir until completely dissolved; then, adjust the pH of the system solution to 4.3 with 6 M hydrochloric acid; under continuous stirring, add 0.727 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), and maintain the pH of the system at 4.7±0.05 with 3 M hydrochloric acid during the reaction, and continue stirring for 30 min; after the activation is completed, adjust the pH of the system to 6.0 with 5 M sodium hydroxide solution, and continue stirring at 35°C overnight to complete the amidation reaction between the carboxyl group and the amino group of cystamine to generate an amide bond and couple to introduce a disulfide bond;

[0118] Then, add 3.953 g of tris(2-carboxyethyl) phosphine hydrochloride (TCEP·HCl) to the reaction system, stir for 5 min to completely dissolve, gradually adjust the pH of the system to 6.0 with 5 M sodium hydroxide solution, and continue stirring under this condition for 3 h to fully reduce the disulfide bond introduced in the molecule to active thiol groups. After the reaction is completed, the system remains clear and transparent, indicating that the disulfide bond introduced in the molecular structure is fully reduced to active thiol groups, and after the carboxyl group on the gelatin molecular chain is activated by Sulfo-NHS / EDCI, it only reacts with cystamine and does not crosslink with the unmodified free amino group on the same molecule or adjacent molecular chain.

[0119] Adjust the pH of the solution after the reaction to 3.5, transfer it to a dialysis bag (with a molecular weight cut-off of 14 kDa), and purify it by dialysis using a 2wt% sodium chloride solution with a pH of 3.3, replacing the dialysate every 4 h, and continuing dialysis for 3 days; then, sequentially replace it with a 0.9wt% sodium chloride solution with a pH of 3.3 and purified water with a pH of 3.3, each for 1 day; finally, collect the solution in the dialysis bag, freeze-dry it, and obtain a white flocculent solid, which is the prepared thiol-modified derivative of high pre-acylated gelatin.

[0120] The derivative molecule structure of this case contains the same structural units as Example 3.

[0121] The derivative of this case was detected by HPLC, and the content of small molecule impurities in the derivative was less than 10 ppm. The active thiol content of the derivative was about 0.48 mmol / g by the improved Ellman method.

[0122] Example 7

[0123] This case study describes the preparation of thiolized biocompatible polymer derivatives. Thiolization modification was performed using gelatin pre-acylated products with high pre-acylation rates, including the following steps:

[0124] S1. Accurately weigh 5.00 g of the gelatin preacylated product prepared in Example 1 (preacylation rate of 95%), add purified water to make up to 100.0 mL, place in a 35℃ constant temperature water bath and stir until completely dissolved to obtain a clear and transparent solution with a concentration of 5% w / v.

[0125] S2. Add 1.572 g of cystamine dihydrochloride and 0.379 g of N-hydroxythiosuccinimide (Sulfo-NHS) sequentially to the above solution and stir until completely dissolved. Then, adjust the pH of the solution to 4.3 with 6 M hydrochloric acid. While stirring continuously, add 0.736 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and maintain the pH of the system at 4.7±0.05 with 3 M hydrochloric acid during the reaction. Activate the system by stirring continuously for 30 min. After activation, adjust the pH of the system to 6.0 with 5 M sodium hydroxide solution and stir continuously at 35°C overnight to complete the amidation reaction between the carboxyl group and the amino group of cystamine to form an amide bond and introduce a disulfide bond through coupling.

[0126] Subsequently, 4.001 g of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP·HCl) was added to the reaction system and stirred for 5 min to completely dissolve it. The pH of the system was gradually adjusted to 6.0 with 5 M sodium hydroxide solution, and the reaction was continued to be stirred for 3 h under this condition to fully reduce the disulfide bonds introduced into the molecule to active thiol groups. After the reaction was completed, the system remained clear and transparent, indicating that the disulfide bonds introduced into the molecular structure were fully reduced to active thiol groups, and that the carboxyl groups on the gelatin molecular chain, after being activated by Sulfo-NHS / EDCI, only reacted with cystamine and did not undergo intramolecular or intermolecular crosslinking with unmodified free amino groups on the same molecule or adjacent molecular chains.

[0127] After adjusting the pH of the reaction system solution to 3.5, the solution was transferred to a dialysis bag (molecular weight cutoff of 14 kDa) and purified by dialysis using a 2 wt% sodium chloride solution at pH 3.3. The dialysis solution was changed every 4 hours for 3 consecutive days. Subsequently, the solution was changed to a 0.9 wt% sodium chloride solution at pH 3.3 and pure water at pH 3.3 for 1 day each. Finally, the solution in the dialysis bag was collected and freeze-dried to obtain a white flocculent solid, which is the thiol-modified derivative of the prepared highly preacylated gelatin.

[0128] The structural units contained in the derivative molecular structure in this case are the same as those in Example 3.

[0129] The HPLC detection of the derivative of the present case showed that the content of small molecular impurities in the derivative was less than 10 ppm. The active sulfhydryl content of the derivative was about 0.49 mmol / g detected by the improved Ellman method.

[0130] The nuclear magnetic resonance comparison spectrum of the thiolated modified derivative of gelatin, gelatin pre-acylation product with a pre-acylation rate of 95%, and high pre-acylated gelatin of the present case is shown in Figure 5 As shown in the figure, compared with unmodified gelatin, the ¹H NMR spectrum of the gelatin pre-acylation product showed significant characteristic changes: the relative proportion of the characteristic peak area of the alkyl hydrogen (-CH2-, -CH3) of the side chain of gelatin at δ 0.8-2.2 ppm showed a downward trend; the characteristic peak of lysine ε-methylene hydrogen (ε-CH2 directly connected to -NH2) at δ 2.6-2.9 ppm almost disappeared; two new characteristic single peaks appeared at δ 2.4-2.6 ppm. Further, the spectrum characteristics of the thiolated modified derivative of high pre-acylated gelatin changed further: the characteristic peak at δ 2.4-2.6 ppm changed from a single peak to a clear triple peak; the characteristic multiple peaks corresponding to the -CH2-CH2-S-S- methylene hydrogen in the cystamine molecule appeared at δ 2.9-3.2 ppm.

[0131] Example 8

[0132] The present case is the preparation of a thiolated biocompatible polymer derivative, which uses a completely pre-acylated gelatin pre-acylation product for thiolated modification, including the following steps:

[0133] S1, precisely weigh 5.00 g of the gelatin pre-acylation product prepared in Example 1 (pre-acylation rate of 100%), add purified water to make up to 100.0 mL, and stir in a 35°C constant temperature water bath until completely dissolved to prepare a clear and transparent solution with a concentration of 5% w / v;

[0134] S2, add 1.591 g of cystamine dihydrochloride and 0.3835 g of N-hydroxysulfosuccinimide (Sulfo-NHS) to the above solution in sequence, and stir until completely dissolved; then, adjust the pH of the system solution to 4.3 with 6 M hydrochloric acid; under continuous stirring, add 0.749 g of 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole hydrochloride (EDCI), and maintain the pH of the system at 4.7±0.05 with 3 M hydrochloric acid during the reaction, and continuously stir for 30 min; after activation, adjust the pH of the system to 6.0 with 5 M sodium hydroxide solution, and continuously stir at 35°C overnight to complete the amidation reaction between the carboxyl group and the amino group of cystamine to generate amide bonds and couple to introduce disulfide bonds;

[0135] Subsequently, 4.05 g of tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) was added to the reaction system, stirred for 5 min to completely dissolve it, the system pH was gradually adjusted to 6.0 with 5 M sodium hydroxide solution, and the reaction was continued to stir for 3 h under this condition to fully reduce the disulfide bond introduced in the molecule to active thiol group. After the reaction was completed, there was a gel block in the system which could not be reduced to solution by the reducing agent, indicating that the solubility of the gelatin pre-acylated thiol-modified derivative prepared when the pre-acylation rate (100%) was too high was poor, which may be related to the hydrophobic aggregation of the derivative when the pre-acylation rate is too high.

[0136] Example 9

[0137] The present case is the preparation of thiolated biocompatible polymer derivatives, using gelatin pre-acylated products with high pre-acylation rate for thiol modification, and optimizing the pH of carboxyl activation in S2 (i.e. the pH of the first weak acid condition and the second weak acid condition), including the following steps:

[0138] S1, precisely weigh 5 portions of 5.00 g of gelatin pre-acylated product prepared in Example 1 (pre-acylation rate of 95%), add purified water to make up to 100.0 mL, and stir in a 35°C constant temperature water bath until completely dissolved to prepare a clear and transparent solution with a concentration of 5% w / v;

[0139] S2, add 1.572 g of cystamine dihydrochloride to each of the above solutions, and then add 0.379 g of N-hydroxysulfosuccinimide (Sulfo-NHS) respectively, and stir until completely dissolved; Subsequently, adjust the pH of the 5 groups of solutions to 3.5-5.5 (first weak acid condition) with 6 M hydrochloric acid: pH=3.5 for the first group, pH=3.75 for the second group, pH=4.6 for the third group, pH=5.0 for the fourth group, and pH=5.5 for the fifth group;

[0140] Under continuous stirring, add 0.736 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) to the solutions of the 5 groups with different pH, and maintain the system pH at 4-6 (second weak acid condition) with 3 M hydrochloric acid during the reaction, which are: pH=4.0 for the first group, pH=4.5 for the second group, pH=5.0 for the third group, pH=5.5 for the fourth group, and pH=6 for the fifth group, and continue to stir for 30 min;

[0141] After the activation is completed, adjust the system pH to 5.0 with 5 M sodium hydroxide solution, and continue to stir at 35°C overnight to complete the amidation reaction between the carboxyl group and the amino group of cystamine to generate amide bond and couple to introduce disulfide bond;

[0142] Subsequently, 4.001 g of tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCl) was added to each reaction system, stirred for 5 min to completely dissolve, and the pH of the system was gradually adjusted to 5.0 with 5 M sodium hydroxide solution, and the reaction was continued to stir for 3 h under this condition. The disulfide bond introduced in the molecule was fully reduced to active thiol. After the reaction was completed, each system remained clear and transparent, indicating that the disulfide bond introduced in the molecular structure was fully reduced to active thiol, and after the carboxyl group on the gelatin molecular chain was activated by Sulfo-NHS / EDCI, it only reacted with cystamine and did not react with the unmodified free amino group on the same molecule or adjacent molecular chain to form intramolecular or intermolecular crosslinking;

[0143] The pH of the solution after adjusting the reaction system was adjusted to 3.5, and then transferred to a dialysis bag (with a molecular weight cut-off of 14 kDa). Purification dialysis was performed using a 2wt% sodium chloride solution with a pH of 3.3, and the dialysate was replaced every 4h. The dialysis was continued for 3 days. Subsequently, a 0.9wt% sodium chloride solution with a pH of 3.3 and pure water with a pH of 3.3 were used for dialysis for 1 day each. Finally, the solution in the dialysis bag was collected and freeze-dried to obtain a white flocculent solid, which was the thiolated modified derivative of the high pre-acylated gelatin prepared in the 5 groups.

[0144] The derivative molecule structure of the case contains the same structural units as Example 3.

[0145] The derivative of the case was detected by HPLC, and the content of small molecule impurities in the derivative was less than 10 ppm. The active thiol content of the derivative was detected by the improved Ellman method, which was 0.39 mmol / g, 0.42 mmol / g, 0.45 mmol / g, 0.46 mmol / g and 0.44 mmol / g, respectively. The relationship between the activated pH and the thiol content of the thiolated modified derivative of the high pre-acylated gelatin prepared is shown in Figure 6 .

[0146] Example 10

[0147] In this case, the preparation of a thiolated biocompatible polymer derivative, the thiolated modification of a high pre-acylation rate gelatin pre-acylation product was carried out, and the amount of activator in S2 was optimized, including the following steps:

[0148] S1, precisely weigh 9 portions of 5.00 g of gelatin pre-acylation product prepared in Example 1 (pre-acylation rate of 95%), add purified water to make up to 100.0 mL, and stir in a 35°C constant temperature water bath until completely dissolved to prepare a clear and transparent solution with a concentration of 5% w / v;

[0149] S2, to each of the above solutions, 1.572 g of cystamine dihydrochloride was added, and then a corresponding amount (0.05 eq to 0.5 eq relative to the total amount of carboxyl groups) of N-hydroxysulfosuccinimide (Sulfo-NHS) was added: 0.100 g for group 1, 0.152 g for group 2, 0.227 g for group 3, 0.303 g for group 4, 0.455 g for group 5, 0.530 g for group 6, 0.606 g for group 7, 0.682 g for group 8, and 0.760 g for group 9, and stirring was performed until complete dissolution; then, the pH of the solution of group 5 was adjusted to 4.3 with 6 M hydrochloric acid;

[0150] Under continuous stirring, a corresponding amount (0.11 eq to 1.1 eq relative to the total amount of carboxyl groups) of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) was added to each of the solutions of group 9: 0.236 g for group 1, 0.360 g for group 2, 0.540 g for group 3, 0.718 g for group 4, 0.897 g for group 5, 1.03 g for group 6, 1.178 g for group 7, 1.330 g for group 8, and 1.472 g for group 9, and the pH of the system was maintained at 4.7 ± 0.05 with 3 M hydrochloric acid during the reaction, and stirring was performed for 30 min;

[0151] After the activation was completed, the pH of the system was adjusted to 5.0 with 5 M sodium hydroxide solution, and the reaction was continued overnight at 35 °C under continuous stirring, so as to complete the amidation reaction between the carboxyl groups and the amino groups of cystamine to generate amide bonds and to couple and introduce disulfide bonds;

[0152] Subsequently, 4.001 g of tris(2-carboxyethyl) phosphine hydrochloride (TCEP-HCl) was added to each of the reaction systems, and stirring was performed for 5 min until complete dissolution, the pH of the system was gradually adjusted to 5.0 with 5 M sodium hydroxide solution, and the reaction was continued under stirring for 3 h under this condition, so as to fully reduce the disulfide bonds introduced in the molecule to active thiol groups; after the reaction was completed, each of the systems was clear and transparent, indicating that the disulfide bonds introduced in the structure of the molecule were fully reduced to active thiol groups, and after the carboxyl groups on the gelatin molecular chain were activated by Sulfo-NHS / EDCI, only the cystamine reacted, and no intramolecular or intermolecular crosslinking occurred with the free amino groups on the same molecule or adjacent molecular chains that were not modified;

[0153] The pH of the system solution after the adjustment reaction was 3.5, and the solution was transferred to a dialysis bag (with a molecular weight cut-off of 14 kDa). The dialysis was performed using a 2 wt% sodium chloride solution with a pH of 3.3, and the dialysis solution was replaced every 4 hours. The dialysis was continued for 3 days. Subsequently, the dialysis was performed using a 0.9 wt% sodium chloride solution with a pH of 3.3 and pure water with a pH of 3.3, respectively, for 1 day each. Finally, the solution in the dialysis bag was collected, and a white flocculent solid was obtained after freeze-drying, which was the thiolated modified derivative of the high pre-acylated gelatin prepared in the 9 groups.

[0154] The derivative molecular structure of the present case contains the same structural units as those of Example 3.

[0155] The derivative of the present case was detected by HPLC, and the content of small molecular impurities in the derivative was less than 10 ppm. The active thiol content of the derivative was detected by the improved Ellman method, which was 0.12 mmol / g, 0.22 mmol / g, 0.30 mmol / g, 0.415 mmol / g, 0.56 mmol / g, 0.63 mmol / g, 0.7 mmol / g, 0.67 mmol / g, and 0.64 mmol / g, respectively. The corresponding relationship between the amount of different carboxyl activating agents (Sulfo-NHS / EDCI) and the thiol content of the thiolated modified derivative of high pre-acylated gelatin prepared is shown in Figure 7 .

[0156] Example 11

[0157] The present case is the preparation of a thiolated biocompatible polymer derivative. The thiolated modification is performed on the pre-acylated product of high pre-acylation rate gelatin. The amount of amino reagent containing disulfide bonds in S2 is optimized, including the following steps:

[0158] S1, precisely weigh 4 portions of 5.00 g of the pre-acylated product of gelatin prepared in Example 1 (pre-acylation rate of 95%), add purified water to make up to 100.0 mL, and place in a 35°C constant temperature water bath for stirring until completely dissolved, to prepare a clear and transparent solution with a concentration of 5% w / v;

[0159] S2, add corresponding amounts (2.0 eq-5.0 eq relative to the total amount of carboxyl groups) of cystamine dihydrochloride to the above 4 groups of solutions: 3.144 g for the first group, 4.716 g for the second group, 6.288 g for the third group, and 7.86 g for the fourth group, and stir until completely dissolved. Then, add 0.606 g of N-hydroxysulfosuccinimide (Sulfo-NHS) to each group, and stir until completely dissolved. Subsequently, adjust the pH of the 5 groups of solutions to 4.3 using 6 M hydrochloric acid.

[0160] Under continuous stirring, corresponding amount (0.11 eq~1.1 eq relative to total carboxyl group) of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) was added into each of the 9 groups of solutions: 0.236 g for the 1st group, 0.360 g for the 2nd group, 0.540 g for the 3rd group, 0.718 g for the 4th group, 0.897 g for the 5th group, 1.03 g for the 6th group, 1.178 g for the 7th group, 1.330 g for the 8th group, and 1.472 g for the 9th group, and the pH of the system was maintained at 4.7±0.05 with 3 M hydrochloric acid during the reaction, and the activation was continued for 30 min under continuous stirring;

[0161] After the activation was completed, the pH of the system was adjusted to 5.0 with 5 M sodium hydroxide solution, and the reaction was continued overnight at 35°C under continuous stirring to complete the amidation reaction between the carboxyl group and the amino group of cystamine and to introduce an amide bond and a disulfide bond through coupling;

[0162] Subsequently, 4.001 g of tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl) was added into each of the reaction systems, and stirring was continued for 5 min to completely dissolve the tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl), and the pH of the system was gradually adjusted to 5.0 with 5 M sodium hydroxide solution, and the reaction was continued for 3 h under the above conditions to reduce the disulfide bond introduced in the molecule to an active thiol group. After the reaction was completed, each of the systems remained clear and transparent, indicating that the disulfide bond introduced in the structure of the molecule was fully reduced to an active thiol group, and after the carboxyl group on the gelatin molecule chain was activated by Sulfo-NHS / EDCI, it only reacted with cystamine and did not react with the free amino group on the same molecule or adjacent molecule chain to cause intramolecular or intermolecular crosslinking.

[0163] The pH of the solution after the reaction was adjusted to 3.5, and the solution was transferred into a dialysis bag (with a molecular weight cut-off of 14 kDa), and purified dialysis was performed using a 2wt% sodium chloride solution with a pH of 3.3, and the dialysis solution was replaced every 4 h, and the dialysis was continued for 3 days. Subsequently, a 0.9wt% sodium chloride solution with a pH of 3.3 and pure water with a pH of 3.3 were used for dialysis for 1 day each. Finally, the solution in the dialysis bag was collected, and a white flocculent solid was obtained after freeze-drying, which was the thiolated modified derivative of the high pre-acylated gelatin prepared in the 4th group.

[0164] The derivative molecule structure of the present case contains the same structural units as those of Example 3.

[0165] The derivatives of the present case were detected by HPLC, and the content of small molecular impurities in the derivatives was less than 10 ppm. The active thiol content of the derivatives was 0.79 mmol / g, 0.85 mmol / g, 0.92 mmol / g and 0.99 mmol / g respectively by using the improved Ellman method. The corresponding relationship between the amount of different amino reagents and the thiol content of the thiol-modified gelatin pre-acylation product prepared by using the amino reagent containing disulfide bond as the amino reagent is shown in Figure 8 .

[0166] Example 12

[0167] The present case is the preparation of a thiolated biocompatible polymer derivative, and the thiol-modified modification is carried out on the pre-acylated product of gelatin with medium pre-acylation rate. The type of reducing agent in S2 is screened, including the following steps:

[0168] S1, precisely weigh 4 portions of 5.00 g of the gelatin pre-acylated product prepared in Example 1 (pre-acylation rate of 79%), add purified water to make up to 100.0 mL, and place in a 35°C constant temperature water bath for stirring until completely dissolved, to prepare a clear and transparent solution with a concentration of 5% w / v;

[0169] S2, add 1.514 g of cystamine dihydrochloride to the above 4 groups of solutions in turn, and stir until completely dissolved; then add the corresponding amount (0.25 eq to 0.4 eq relative to the total amount of carboxyl groups) of N-hydroxysulfosuccinimide (Sulfo-NHS): 0.365 g for the first group, 0.437 g for the second group, 0.510 g for the third group and 0.583 g for the fourth group, and stir until completely dissolved; then, adjust the pH of the system solution to 4.3 with 6 M hydrochloric acid;

[0170] Under continuous stirring, add the corresponding amount (0.55 eq to 0.88 eq relative to the total amount of carboxyl groups) of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI): 0.709 g for the first group, 0.848 g for the second group, 0.991 g for the third group and 1.132 g for the fourth group, and stir until completely dissolved. Maintain the pH of the system at 4.7±0.05 with 3 M hydrochloric acid during the reaction, and continuously stir for 30 min; after activation, adjust the pH of the system to 6.0 with 5 M sodium hydroxide solution, and continuously stir at 35°C overnight;

[0171] Subsequently, 2.15 g of dithiothreitol (DTT) was added to the reaction system, stirred for 5 min to completely dissolve, and the pH of the system was gradually adjusted to 8.5 with a 5 M sodium hydroxide solution, and the reaction was continued to be stirred for 3 h under this condition to fully reduce the disulfide bond introduced in the molecule into an active thiol group. After the reaction was completed, each group of systems remained clear and transparent;

[0172] The pH of the solution after reaction of the 4 groups was adjusted to about 3.5, and was transferred to a dialysis bag (with a molecular weight cut-off of 14 kDa). Purification dialysis was performed using a 2wt% sodium chloride solution with pH=3.3, and the dialysis solution was replaced every 4 h. The dialysis was continued for 3 days. When dialysis was performed to the second day, a large amount of white flocculent or even gel was precipitated from the solution. This may be because the hydrophobicity of the gelatin is enhanced after pre-acylation modification, and DTT is oxidized into a dimer, the hydrophobicity is also enhanced and aggregation occurs to form small particles, which are directly precipitated or adsorbed on the surface of the gelatin, inducing protein aggregation and precipitation, resulting in poor solubility of the derivative. In addition, the solution environment mutation induced by dialysis and the excessive use of DTT exacerbate protein precipitation, further reducing the solubility of the derivative.

[0173] Example 13

[0174] The present case is the preparation of a thiolated biocompatible polymer derivative. A gelatin pre-acylation product with a high pre-acylation rate is used for thiolation modification, and the types of reducing agents in S2 are screened, including the following steps:

[0175] S1, 4 portions of 5.00 g of the gelatin pre-acylation product prepared in Example 1 (with a pre-acylation rate of 95%) were accurately weighed, and purified water was added to make up to 100.0 mL. The solution was stirred to completely dissolve in a 35°C constant temperature water bath to prepare a clear and transparent solution with a concentration of 5% w / v;

[0176] S2, 1.572 g of cystamine dihydrochloride was added to each of the above 4 groups of solutions and stirred to completely dissolve. Then, a corresponding amount (0.25 eq to 0.4 eq relative to the total amount of carboxyl groups) of N-hydroxysulfosuccinimide (Sulfo-NHS) was added: 0.379 g for the first group, 0.455 g for the second group, 0.530 g for the third group, and 0.606 g for the fourth group, and stirred to completely dissolve. Subsequently, the pH of the system solution was adjusted to 4.3 with a 6 M hydrochloric acid solution;

[0177] Under continuous stirring, add corresponding amount (0.25 eq~0.4 eq relative to total carboxyl group) of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI): 0.736 g for Group 1, 0.897 g for Group 2, 1.03 g for Group 3, and 1.178 g for Group 4, stir until completely dissolved, and maintain the system pH=4.7±0.05 with 3 M hydrochloric acid during the reaction, and continuously stir for 30 min of activation; after the activation is completed, adjust the system pH=5.0 with 5 M sodium hydroxide solution, and continuously stir at 35°C overnight;

[0178] Subsequently, add 2.15 g of dithiothreitol (DTT) to the reaction system, stir for 5 min to completely dissolve, gradually adjust the system pH=8.5 with 5 M sodium hydroxide solution, and continue to stir under this condition for 3 h to fully reduce the disulfide bond introduced in the molecule to active thiol groups. After the reaction is completed, each group of systems remains clear and transparent;

[0179] Adjust the pH of the solution after reaction of Group 4 to about 3.5, transfer to a dialysis bag (with a molecular weight cut-off of 14 kDa), and purify by dialysis using a 2wt% sodium chloride solution with pH=3.3. The dialysis solution is replaced every 4 h, and the dialysis is continued for 3 days. At the time of dialysis for the second day, a large amount of white flocculent or even gel is precipitated in the solution, which is consistent with the results of Example 12, indicating that the stability of the thiolated modified derivative of high pre-acylated gelatin has no significant correlation with the pre-acylation rate of gelatin. The core problem is that the type of reducing agent, DTT, can cause oxidative polymerization and induce protein aggregation, resulting in decreased solubility of the derivative. Therefore, DTT is not suitable for the thiolation modification process of gelatin after pre-acylation in the present application.

[0180] Example 14

[0181] This example is the preparation of a thiolated biocompatible polymer derivative. A high pre-acylation rate of recombinant collagen protein pre-acylation product is used for thiolation modification, including the following steps:

[0182] S1, Preparation of High Pre-acylated Recombinant Human Collagen Protein

[0183] Accurately weigh 5.00 g of recombinant human type III collagen (M w =50kDa), add purified water to make up to 50.0 mL, and stir in a 35°C constant temperature water bath until completely dissolved to prepare a clear and transparent solution with a concentration of 10% w / v;

[0184] The pH of the above gelatin solution was adjusted to 8.5 using a 5M sodium hydroxide solution, and 1.0 equivalent (eq) of succinic anhydride relative to the amino content of the recombinant human collagen was added for pre-acylation under continuous stirring; 5M sodium hydroxide solution was continuously added during the entire reaction process to maintain the system pH at 8.0-9.5, and the pre-acylation reaction was carried out in a 35°C water bath under continuous stirring for 3h;

[0185] After the reaction was completed, the reaction system pH was adjusted to 7.0 using 3M hydrochloric acid, and stirring was continued for 5 min to ensure uniformity of the system; then the reaction solution was transferred to a dialysis bag (molecular weight cutoff 8000 Da), and purified water was used for dialysis, with the dialysis solution being replaced every 4h, and the dialysis being continued for 2 days;

[0186] Finally, the solution in the dialysis bag was collected, freeze-dried, and a white flocculent solid was obtained, which was high pre-acylated recombinant human collagen, and the pre-acylation rate was measured to be 95%.

[0187] S2, thiolation modification of high pre-acylated recombinant human collagen

[0188] 5.00 g of the above high pre-acylated modified recombinant human collagen was precisely weighed, purified water was added to make up to 100.0 mL, and the solution was stirred to complete dissolution in a 35°C constant temperature water bath to prepare a clear transparent solution with a concentration of 5% w / v;

[0189] To the above solution, 0.586 g of cystamine dihydrochloride and 0.085 g of N-hydroxysulfosuccinimide (Sulfo-NHS) were added in sequence and stirred to complete dissolution; then, the solution pH was adjusted to 4.3 using 6M hydrochloric acid; 0.175 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) was added under continuous stirring, and the system pH was maintained at 4.7±0.05 using 3M hydrochloric acid during the reaction, and the stirring was continued for 30 min; after the activation was completed, the system pH was adjusted to 6.0 using 5M sodium hydroxide solution, and the reaction was continued under continuous stirring at 35°C overnight to complete the amidation reaction between the carboxyl group and the cystamine amino group;

[0190] Then, 0.746 g of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added to the reaction system, stirred for 5 min to completely dissolve; the system pH was gradually adjusted to 6.0 using 5M sodium hydroxide solution, and the stirring was continued under this condition for 3h to fully reduce the disulfide bonds introduced in the molecule to active thiol groups;

[0191] The solution after reaction is adjusted to pH 3.5 after dilution with purified water to 1%, and is transferred to a dialysis bag (with a molecular weight cut-off of 14 kDa), and is purified by dialysis using a sodium chloride solution with pH = 3.3 and a concentration of 2 wt%, and the dialysis solution is replaced every 4 h, and the dialysis is continued for 3 days; then, the dialysis is successively replaced with a sodium chloride solution with pH = 3.3 and a concentration of 0.9 wt% and purified water with pH = 3.3, each for 1 day; finally, the solution in the dialysis bag is collected, and is freeze-dried to obtain a white flocculent solid, which is the prepared thiolated derivative of the highly pre-acylated recombinant human collagen.

[0192] The derivative molecule structure of the present case simultaneously has three structural units as shown in the following formulas (VIII) to (X):

[0193] ; wherein G is a recombinant human type III collagen residue.

[0194] The derivative of the present case is detected by HPLC, and the content of small molecular impurities in the derivative is less than 10 ppm. The content of active thiol groups in the derivative is about 0.13 mmol / g, which is detected by the improved Ellman method.

[0195] Example 15

[0196] The present case is the preparation of an injectable in-situ cross-linked hydrogel simulating the extracellular matrix, which comprises the following steps:

[0197] 0.09 g of the thiolated derivative of the highly pre-acylated gelatin prepared in Example 7 is weighed, dissolved in 3 mL of a 5 mM phosphate buffer (pH 7.2), and heated and stirred in a 40°C water bath until completely dissolved to obtain a clear and transparent solution. The pH of the above solution is adjusted to 7.2-7.4 by using 5 M sodium hydroxide;

[0198] Another functional cross-linking agent (polyethylene glycol diacrylate) 0.06 g is dissolved in 1 mL of a 5 mM phosphate buffer (pH = 7.2) to obtain a clear and transparent cross-linking agent solution;

[0199] The above two solutions are mixed uniformly, and are placed in a 37°C water bath for 1 min-5 min, and the viscosity of the solution gradually increases, and a hydrogel is formed within 5 min.

[0200] Example 16

[0201] The present case is the cytotoxicity detection of an injectable in-situ cross-linked hydrogel simulating the extracellular matrix

[0202] Take the freshly prepared hydrogel from Example 15, add 10 mL of cell culture medium (DMEM, 10% fetal bovine serum), and extract by shaking in a 37°C shaker for 72 h. Take the supernatant and add L929 fibroblasts (1×10⁻⁶ cells) to it. 4 Cell viability was detected by MTT assay (referring to GB / T 16886.5-2017 / ISO 10993-5:2009 standard) after culturing in 96-well plates (100 μL / well) for 24 h. The results are shown in Table 1.

[0203] Table 1. Cytotoxicity test results of injectable in-situ crosslinked hydrogels mimicking extracellular matrix

[0204]

[0205] As shown in Table 1, the survival rate of L929 fibroblasts in the 25%-100% extract groups was greater than 70%, indicating that the prepared injectable in-situ cross-linked hydrogel that mimics the extracellular matrix is ​​non-cytotoxic.

[0206] Example 17

[0207] This case study demonstrates the use of an injectable, in-situ cross-linked hydrogel, mimicking the extracellular matrix, as a substrate for stem cell adherent growth.

[0208] Weigh 0.3 g of the highly preacylated gelatin thiolated modified derivative prepared in Example 7, dissolve it in 10 mL of 0.5 mM phosphate buffer (pH 7.2), filter to remove bacteria, and then adjust the pH to 7.2-7.4 with sterile sodium hydroxide.

[0209] Take another 0.2 g of functional crosslinking agent (polyethylene glycol diacrylate), dissolve it in 3.33 mL of 0.5 mM phosphate buffer (pH=7.2), filter to sterilize and obtain a sterile crosslinking agent solution.

[0210] Mix the two solutions thoroughly and dispense into 12-well plates (1 mL / well). Incubate at 37°C for 30 min to allow for complete cross-linking and hydrogel formation. Add 2 mL of cell culture medium (DMEM, 12% fetal bovine serum) and 1×10⁻⁶ ppm to each well. 5 One bone marrow mesenchymal stem cell (BMSC) was cultured for one month, with the culture medium changed every 2-3 days.

[0211] Microscopic observation results as follows Figure 9 As shown, stem cells rapidly adhered and spread on the hydrogel surface within 24 hours, with a spreading speed superior to that of the control group's two-dimensional cell culture plate. Continuous culture for one month revealed a gradual increase in the number of stem cells, and the morphology and activity of the stem cells were superior to those of the control group, indicating that the hydrogel has good biocompatibility and is suitable for cell adhesion and proliferation.

[0212] Example 18

[0213] This case is the proliferation detection of bone marrow mesenchymal stem cells embedded in an injectable in-situ cross-linked hydrogel simulating extracellular matrix

[0214] Take 0.3 g of the high pre-acylated gelatin thiol-modified derivative prepared in Example 7, dissolve it in 10 mL of 0.5 mM phosphate buffer (pH=7.2), filter sterilize, and adjust the pH to 7.2-7.4 with 5M sterile sodium hydroxide.

[0215] Take another 0.2 g of the functional cross-linking agent (polyethylene glycol diacrylate), dissolve it in 3.33 mL of 0.5 mM phosphate buffer (pH=7.2), filter sterilize to obtain a sterile cross-linking agent solution.

[0216] Count the bone marrow mesenchymal stem cells (BMSCs) after digestion, take 1.2×10 5 Centrifuge the bone marrow mesenchymal stem cells (BMSCs) in a centrifuge tube, discard the supernatant, and then transfer the above two sterile solutions to the centrifuge tube and mix evenly by blowing, and then sub-pack into a 12-well plate (1 mL / well), and stand in a 37°C cell incubator for 15 min for complete cross-linking to form a hydrogel. Then add 2 mL of cell culture medium (DMEM, 12% fetal bovine serum) per well for continuous culture for 1 month, and replace the culture medium every 2-3 days. During this period, the number of living cells inside the hydrogel was detected at different time points using the CCK8 method, and the results are shown in Figure 10 As shown in the figure, the number of stem cells in the hydrogel continues to increase with the extension of the culture time, indicating that the porous structure inside the hydrogel can be used for the delivery of nutrients and metabolic products, and can support the survival and proliferation of the cells inside, and can be used as an excellent matrix for three-dimensional cell culture.

[0217] Comparative Example 1

[0218] The preparation process of the derivative in this case is the same as that in Example 3, except that in S1, the A-type gelatin is not subjected to a pre-acylation reaction, and a 5% w / v clear and transparent solution of the A-type gelatin is directly used for S2. After 3h of reduction reaction, it is found that there are still a large number of gel blocks in the system, which is because during the amide coupling reaction stage, due to the rich carboxyl groups on the gelatin molecular chain, after activation by Sulfo-NHS / EDCI, a part of them will react with the free amino groups on the same molecule or adjacent molecule chain to form intramolecular or intermolecular cross-linking, forming an amide network. This covalent cross-linking network structure is stable and will not be destroyed by TCEP in the subsequent reduction step, so the gel cannot be dissolved.

[0219] Comparative Example 2

[0220] The derivative preparation process of the case is the same as that of Example 14, except that in S1, the recombinant human type III collagen is not pre-acylated, and a clear and transparent recombinant human collagen solution with a concentration of 10% w / v is directly prepared from the recombinant human type III collagen. After 3 hours of reduction reaction in S2, it is found that there are still some gel blocks in the system. This is because, in the amidation reaction stage, due to the rich carboxyl groups on the molecular chain of the recombinant human collagen, after activation by Sulfo-NHS / EDCI, a part of them will also cross-link with the free amino groups on the same molecule or adjacent molecular chain to form a stable amide bond network. The covalent cross-linking network structure is stable and cannot be destroyed by TCEP in the subsequent reduction step, so the gel cannot be dissolved.

[0221] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can make equivalent replacements or changes within the technical scope disclosed by the present application according to the technical solution and inventive concept of the present application, which should be covered within the protection scope of the present application.

Claims

1. A thiolated biocompatible polymer derivative, characterized in that, The molecular chain structure comprises the following general formula (I) structural unit, general formula (II) structural unit and general formula (III) structural unit: 、 、 ； Wherein G is the residue of a biocompatible polymer compound, R1 is selected from alkylene, substituted alkylene or aromatic group, R2 is selected from alkylene or aromatic group, and R3 is carboxyl or carboxyl salt; The biocompatible polymer compound contains both amino and carboxyl groups; the residue contains both amino and carboxyl groups; The amino group of the biocompatible polymer compound is first acylated to form a pre-acylated product containing the general formula (II) structural unit, and then the carboxyl group is coupled to introduce a thiol group through an amide bond to form the general formula (I) structural unit and the general formula (III) structural unit, respectively, to ultimately form the thiolated biocompatible polymer derivative.

2. The thiolated biocompatible polymer derivative according to claim 1, characterized in that, The pre-acylation rate for forming the general formula (II) structural unit through pre-acylation is 40% to 95%.

3. The thiolated biocompatible polymer derivative according to claim 2, characterized in that, The pre-acylation rate for forming the general formula (II) structural unit through pre-acylation is 60% to 95%.

4. The thiolated biocompatible polymer derivative according to any one of claims 1 to 3, characterized in that, The thiol content of the thiolated biocompatible polymer derivative is 0.1 mmol / g to 1.0 mmol / g.

5. The thiolated biocompatible polymer derivative according to claim 4, characterized in that, The thiol content of the thiolated biocompatible polymer derivative is 0.3 mmol / g to 0.9 mmol / g.

6. The thiolated biocompatible polymer derivative according to claim 5, characterized in that, The thiol content of the thiolated biocompatible polymer derivative is 0.4 mmol / g to 0.7 mmol / g.

7. The thiolated biocompatible polymer derivative according to any one of claims 1 to 3, characterized in that, The biocompatible polymer compound is selected from one or more of gelatin, genetically recombined gelatin, and genetically recombined human collagen.

8. A method for preparing a thiolated biocompatible polymer derivative, characterized in that, The method is suitable for preparing the thiolated biocompatible polymer derivative of any one of claims 1 to 7, and comprises the following steps: S1, dissolving a biocompatible polymer compound containing amino and carboxyl groups in an aqueous solution, adjusting the system to weak alkaline conditions, adding a diacid anhydride compound, maintaining the weak alkaline conditions, and performing pre-acylation to obtain a pre-acylated product; S2, dissolving the pre-acylated product in an aqueous solution, adding an amino reagent containing a disulfide bond and a first activator, adjusting the system to a first weak acid condition and maintaining it, adding a second activator, completing the activation, adjusting the system to a second weak acid condition, and performing an amidation reaction to generate an intermediate product containing an amide bond and coupled to introduce a disulfide bond; Subsequently, a reducing agent is added to the system to perform a reduction reaction, and a thiolated biocompatible polymer derivative containing active thiol groups is obtained after purification.

9. The method for preparing the thiolized biocompatible polymer derivative according to claim 8, characterized in that, In S1, the concentration of the biocompatible polymer compound dissolved in the aqueous solution is controlled to be 5% w / v to 15% w / v; in S2, the concentration of the pre-acylated product dissolved in the aqueous solution is controlled to be 5% w / v to 10% w / v.

10. The method for preparing the thiolized biocompatible polymer derivative according to claim 9, characterized in that, In S1, the concentration of the biocompatible polymer compound dissolved in the aqueous solution is controlled to be 10% w / v to 15% w / v; in S2, the concentration of the pre-acylated product dissolved in the aqueous solution is controlled to be 5% w / v to 8% w / v.

11. The method for preparing the thiolized biocompatible polymer derivative according to claim 8, characterized in that, In S1, the diacid anhydride compound is an aliphatic diacid anhydride or an aromatic diacid anhydride with a carbon chain length of C2-C15; In S2, the first activator is N-hydroxysuccinimide, and the second activator is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride.

12. The method for preparing the thiolized biocompatible polymer derivative according to claim 8, characterized in that, The molar ratio of the dianhydride compound to the side chain amino group of the biocompatible polymer compound in S1 is 0.2-1.5:1; The temperature of the pre-acylation reaction in S1 is 25-50°C, and the time is 0.5-3h; The weak alkaline condition in S1 is pH=7.0-10.

0.

13. The method for preparing the thiolized biocompatible polymer derivative according to claim 12, characterized in that, The molar ratio of the dianhydride compound to the side chain amino group of the biocompatible polymer compound in S1 is 0.4-1.0:1; The temperature of the pre-acylation reaction in S1 is 30-40°C, and the time is 1-2h; The weak alkaline condition in S1 is pH=8.0-9.

5.

14. The method for preparing the thiolized biocompatible polymer derivative according to claim 8, characterized in that, The dithio-containing amino reagent in S2 has the following general formula: ； wherein R1 and R4 are independently selected from alkylene, substituted alkylene or aromatic group with carbon chain length of C1-C15; R5 is an active group that does not react with amino group; The reducing agent in S2 is selected from one or more of tris(2-carboxyethyl)phosphine or its hydrochloride, glutathione.

15. The method for preparing the thiolized biocompatible polymer derivative according to claim 14, characterized in that, The dithio-containing amino reagent is selected from one or more of cystamine, cystamine dihydrochloride, cystamine sulfate, 3,3'-dithiodipropylamine, 4,4'-dithiodibutylamine, 6,6'-dithiodihexylamine, 2-[(3-aminopropyl)disulfanyl]ethylamine, 2,2'-dithiodianiline, 4,4'-diaminodiphenyl disulfide, bis(2-amino-3-methylphenyl) disulfide, 2-aminoethyl-2'-hydroxyethyldisulfide, bis(2-amino-1-methylethyl) disulfide, bis(2-aminopropyl) disulfide hydrochloride, 2-aminoethyldisulfanyl ethylamine, N-tert-butoxycarbonyl-cystamine. The reducing agent in S2 is tris(2-carboxyethyl)phosphine hydrochloride.

16. The method for preparing the thiolized biocompatible polymer derivative according to claim 8, characterized in that, The total amount of carboxyl groups in the pre-acylation product in S2 is calculated based on the total molar amount of the carboxyl groups in the molecular chain structure of the pre-acylation product itself and the carboxyl groups or carboxyl salts introduced by pre-acylation, The molar ratio of the second activating agent, the first activating agent and the total amount of carboxyl groups in the pre-acylation product is 0.1-2:0.1-1:1; The molar ratio of the dithio-containing amino reagent and the total amount of carboxyl groups in the pre-acylation product in S2 is 1-5:

1.

17. The method for preparing the thiolized biocompatible polymer derivative according to claim 16, characterized in that, The molar ratio of the second activating agent, the first activating agent and the total amount of carboxyl groups in the pre-acylation product is 0.4-1.2:0.2-0.5:1; The molar ratio of the dithio-containing amino reagent and the total amount of carboxyl groups in the pre-acylation product in S2 is 1-2:

1.

18. The method for preparing the thiolized biocompatible polymer derivative according to claim 8, characterized in that, The temperature of the amidation reaction in S2 is 25-50°C, and the time is 0.2-24h; The temperature of the reduction reaction in S2 is 25-50°C, and the time is 0.5-5h; The pH of the first weak acid condition in S2 is 4.0-6.5, and the pH of the second weak acid condition is 3.0-7.

0.

19. The method for preparing the thiolized biocompatible polymer derivative according to claim 18, characterized in that, The temperature of the amidation reaction in S2 is 30-40°C, and the time is 0.5-24h; The temperature of the reduction reaction in S2 is 30-40°C, and the time is 2-4h; The pH of the first weak acidic condition in S2 is 4.5-5.0, and the pH of the second weak acidic condition is 4.0-6.

0.

20. An injectable in situ crosslinkable hydrogel that mimics the extracellular matrix, characterized in that, The thiolated biocompatible polymer derivative of any one of claims 1-7, or the thiolated biocompatible polymer derivative prepared by the preparation method of any one of claims 8-19, is constructed by spontaneous oxidation of the thiol groups in the molecular chain structure of the thiolated biocompatible polymer derivative to form disulfide bond crosslinking and / or high-efficiency click chemistry reaction crosslinking with a multi-functional electrophilic or nucleophilic crosslinking agent under physiological conditions.

21. The ECM-mimicking injectable in situ cross-linking hydrogel of claim 20, wherein, The multi-functional electrophilic or nucleophilic crosslinking agent has one or more of the following functional groups in the molecular structure: aldehyde group, acrylate, acrylamide, maleimide group, vinyl sulfone group, norbornene group, thiol group.

22. Use of the injectable in situ crosslinkable hydrogel mimicking extracellular matrix according to any one of claims 20-21 in the preparation of a biomedical preparation.

23. Use of the injectable in situ cross-linking hydrogel according to claim 22 for the preparation of a biomedical formulation, characterized in that, The biomedical preparation comprises one or more of a tissue engineering scaffold, a drug controlled release carrier, a 3D bioprinting ink, and a cell encapsulation material.

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