Thiolated biocompatible polymer derivatives, methods for their preparation, extracellular matrix mimicking injectable in situ crosslinking hydrogels and uses thereof

Abstract

The present application relates to the technical field of biomedical material modification, and particularly relates to a thiolated biocompatible polymer derivative, a preparation method thereof, an injectable in-situ crosslinking hydrogel simulating extracellular matrix and application thereof; the present application first acylates a biocompatible polymer containing amino groups and carboxyl groups through a pre-acylation strategy, thereby not only avoiding a three-dimensional crosslinking by-product formed by coupling of amide bonds of the biocompatible polymer itself, but also increasing the number of side chain carboxyl groups available for thiolation modification; the pre-acylation rate is accurately adjustable, and the physicochemical properties of the derivative can be optimized; meanwhile, the active thiol groups are introduced by using amide bond coupling as a protective precursor of disulfide bond, which not only avoids self-oxidation of the active thiol groups in the reaction process, thereby ensuring the activity of the thiol groups, but also avoids hydrazine-containing reagents, thereby further improving safety, and the present application has no risk of virus residue and immune rejection, is highly universal, and has a wide application prospect; in combination with a high-concentration preparation method, the present application has the advantage of large-scale production, and further widens the industrial application potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical material modification technology, specifically to thiolized biocompatible polymer derivatives and their preparation methods, and injectable in-situ crosslinked hydrogels that mimic extracellular matrix and their applications. Background Technology

[0002] Biocompatible polymers, such as gelatin, are partially hydrolyzed products of collagen, including type A gelatin prepared by acid hydrolysis and type B gelatin prepared by alkaline hydrolysis. Gelatin is a protein containing 18 amino acids and retains a large amount of the bioactive sequence of collagen. Recombinant human collagen, another example of biocompatible polymers, is obtained through genetic engineering to efficiently express the human collagen gene in host cells (such as E. coli, yeast, and mammalian cells). It has significant advantages such as precise sequence design, high batch-to-batch consistency, good solubility, no risk of viral residue, low immunogenicity, and excellent biocompatibility. Recombinant expression technology can also be used to prepare gelatin analogs, i.e., recombinant gelatin, which has a well-defined amino acid sequence, concentrated molecular weight distribution, and excellent biocompatibility and processability, showing broad potential applications in the biomedical field. However, gelatin, recombinant gelatin, and recombinant human collagen all have significant performance defects. For example, they have good water solubility but low viscosity, are prone to flow and diffusion, have insufficient mechanical properties, and are rapidly degraded and absorbed in vivo, failing to provide long-term stable support. These limitations make them unsuitable for clinical applications such as tissue regeneration scaffolds and wound healing dressings. Chemical modification and cross-linking are effective ways to address these performance defects. Thiolization modification is a crucial aspect of the chemical modification of (recombinant) gelatin and recombinant human collagen. Introducing thiol groups into biocompatible polymers yields derivatives with good biocompatibility and no toxic byproducts, while also avoiding the toxic and adverse reactions of traditional chemical cross-linking agents, thus effectively meeting many clinical application needs.

[0003] Gelatin and collagen, among other biocompatible polymers, share similar molecular structures composed of multiple amino acid chains. Their side chains contain both amino (-NH2) and carboxyl (-COOH) groups, with the carboxyl group being a key target group for chemical modification and typically present in large numbers. For example, type B gelatin contains 28 amino and 118 carboxyl groups per 1000 amino acid residues; type A gelatin has approximately 54 carboxyl groups per 1000 amino acid residues, while its amino group count is similar to that of type B gelatin. However, these zwitterionic biocompatible polymers are generally only soluble in aqueous solutions, limiting the available chemical modification methods.

[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 biocompatible polymers such as gelatin and collagen, whose side chains contain both amino and carboxyl groups, the use of EDCI / (sulfo)-NHS to activate the carboxyl groups of the side chains can effectively generate amide bonds for crosslinking with the amino groups of their own side chains. This has become the most important chemical method for preparing gelatin and collagen crosslinking materials (Z Ghassemi et al., Biopolymers 2018 109(9) e23232; JW Luo et al., Materials Science EngineeringC, 98, 2019, 628-634; DV Bax et al., Acta Biomaterialia, 49, 2017, 218-234; LGu et al., Trends in Biotechnology, 37(5), 2019, 464-491). However, the unavoidable coupling and cross-linking side reaction of the amino groups in the side chains of gelatin and collagen severely restricts the chemical modification of the side chain carboxyl groups by introducing thiol groups through amide bond coupling. The resulting thiolized derivatives have poor water solubility, which limits their application in the biomedical field.

[0006] The current approach to solving the aforementioned problem (the side-chain coupling and cross-linking side reaction of the amino group itself) is to use a highly reactive acylhydrazine reagent (-(CO)NHNH2) to couple with activated biocompatible polymers such as gelatin. Acylhydrazine reagents differ from conventional amino reagents in their pKa; amino reagents have a higher pKa (pH≈8-11), while acylhydrazine reagents, due to their adjacent electron-withdrawing groups, significantly lower the pKa of the acylhydrazine amino group (pH≈2.5-4.5). Therefore, under weakly acidic conditions (pH=4-6), the amino group of the acylhydrazine reagent usually exists in a non-protonated form (-(CO)NHNH2), which can efficiently nucleophilically add to O-acetylisourea intermediates to form an acylhydrazine bond (-(CO)NHNH(CO)-, such as...). Figure 1 The route for forming compound (b) shown in the diagram is several orders of magnitude more reactive than conventional amino reagents. Therefore, the use of acylhydrazine reagents can very effectively avoid competing side reactions of their own side-chain amino groups.

[0007] Based on the above reaction principle, previous studies (XZ Shu. et al, Biomaterials, 2003, 24(21):3825-3834; CN101220090A) have used disulfide-containing dihydrazides as hydrazide reagents to react with activated gelatin, which undergoes a nucleophilic addition coupling reaction to form hydrazide bonds, thus obtaining disulfide-modified gelatin. Then, a reducing agent is used to reduce the disulfide bonds to derivatives with free thiol groups. However, one of the raw materials for disulfide-containing dihydrazide reagents is hydrazine, and the coupled hydrazide bond has the potential to hydrolyze into hydrazine in vivo. Hydrazine is a Group 2B potential carcinogen. With increasingly stringent regulations in the current biomedical field, concerns about the long-term safety of biomaterials cross-linked with hydrazide coupling bonds in vivo have increasingly 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; LB Witkin, AMA Archives of Industrial Health, 1956, 13(1): 34-36; SH Kenyon. et al, Biochemical Pharmacology, 1999, 57(11): 1311-1319; L-ATorres-de la Roche et al., Journal of Clinical Medicine, 11, 2022, 931). Summary of the Invention

[0008] To address the potential risks associated with the use of hydrazide reagents in existing technologies and the technical problem that conventional amino reagents cannot yield thiolated modified derivatives, this invention provides thiolated biocompatible polymeric derivatives and their preparation methods, as well as injectable in-situ crosslinked hydrogels mimicking extracellular matrix and their applications. In preparing thiolated modified derivatives of biocompatible polymers, this invention first pre-acylates the biocompatible polymer containing amino and carboxyl groups to strictly shield the side-chain amino groups, obtaining a pre-acylated biocompatible polymeric product. Then, the carboxyl groups on the polymer chain are activated and can be coupled with an amino reagent containing disulfide bonds via amide bonds. Reduction yields the corresponding thiolated biocompatible polymeric derivative. This invention avoids the three-dimensional crosslinking byproducts formed by amide bond coupling of the amino group itself, and also avoids the potential risks associated with the use of hydrazide reagents in existing technologies, thus offering higher safety.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0010] The first aspect of this invention provides a thiolized biocompatible polymeric derivative whose molecular chain structure simultaneously includes the following general formula (Ⅰ) structural units, general formula (Ⅱ) structural units, and general formula (Ⅲ) structural units:

[0011] , ,

[0012] ;

[0013] Where G is a residue of a biocompatible polymer, R1 is selected from alkylene, substituted alkylene or aromatic groups, R2 is selected from alkylene or aromatic groups, and R3 is a carboxyl group or a carboxyl salt.

[0014] The biocompatible polymer contains both amino and carboxyl groups; the residues contain both amino and carboxyl groups.

[0015] The biocompatible polymer compound first undergoes a pre-acylation reaction to form a pre-acylated product containing a general formula (II) structural unit (incomplete pre-acylation). Then, its carboxyl group (derived from its own carboxyl group and the carboxyl group introduced by pre-acylation) is coupled with a thiol group through an amide bond to form a general formula (I) structural unit and a general formula (III) structural unit, respectively. Finally, a thiolized biocompatible polymer derivative is obtained in which the molecular chain structure simultaneously includes the general formula (I) structural unit, the general formula (II) structural unit, and the general formula (III) structural unit.

[0016] Furthermore, the preacylation rate of the general formula (II) structural unit formed by preacylation is 40% to 95%.

[0017] Furthermore, the preacylation rate of the general formula (II) structural unit formed by preacylation is 60% to 95%.

[0018] Furthermore, the thiol content in the thiolized biocompatible polymer derivative is 0.1 mmol / g to 1.0 mmol / g.

[0019] Furthermore, the thiol-modified biocompatible polymer derivative contains 0.3 mmol / g to 0.9 mmol / g of thiol groups.

[0020] Preferably, the thiol content in the thiolized biocompatible polymer derivative is 0.4 mmol / g to 0.7 mmol / g.

[0021] Furthermore, the biocompatible polymer compound is selected from one or more of gelatin, recombinant gelatin, and recombinant human collagen; 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] A second aspect of the present invention provides a method for preparing the above-mentioned thiolized biocompatible polymeric derivative, comprising the following steps:

[0023] S1. A biocompatible polymer containing amino and carboxyl groups is dissolved in an aqueous solution. The system is adjusted to a weakly alkaline condition. A diacid anhydride compound is added to maintain the weakly alkaline condition. The side-chain amino group of the compound undergoes a preacylation reaction with the diacid anhydride compound. During this reaction, the amino group reacts with the diacid anhydride to form an amide bond and introduces a side-chain carboxyl group or a carboxyl salt. The preacylated product is then purified.

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

[0025] S2. The preacylated product is dissolved in an aqueous solution, an amino reagent containing a disulfide bond and a first activator are added, the system is adjusted to a first weakly acidic condition and maintained, a second activator is added, and after activation is completed, the system is adjusted to a second weakly acidic condition to carry out an amidation reaction to generate an intermediate product containing an amide bond and a disulfide bond is introduced via coupling. During this reaction, the carboxyl group of the preacylated product's own side chain is activated by the activator in the weakly acidic aqueous solution, and then reacts with the amino reagent containing a disulfide bond to generate an amide bond and a disulfide bond is introduced via coupling.

[0026] Subsequently, a reducing agent was added to the system to reduce the disulfide bond. After purification, a thiolized biocompatible polymer derivative containing an active thiol group was obtained. Its molecular chain structure includes the following general formula (I) to general formula (III) structural units.

[0027] Furthermore, 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 preacylated product 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 dicarboxylic acid anhydride compound mentioned in S1 is an aliphatic or aromatic dicarboxylic acid anhydride with a carbon chain length of C2-C15, preferably a dicarboxylic acid 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 octanoic anhydride.

[0030] Further, the molar ratio of the diacid anhydride compound to the side-chain amino group of the biocompatible polymer compound in S1 is 0.2–1.5:1, preferably 0.4–1.0:1. Therefore, the preacylation rate mentioned above refers to the degree to which the amino group in the biocompatible polymer compound is replaced by the diacid anhydride.

[0031] Furthermore, the temperature of the preacylation reaction in S1 is 25℃~50℃ and the time is 0.5h~3h;

[0032] Preferably, the preacylation reaction is carried out at a temperature of 30°C to 40°C for 1 hour to 2 hours.

[0033] The weakly alkaline conditions are pH = 7.0 to 10.0, with pH = 8.0 to 9.5 being particularly preferred.

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

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

[0036] ;

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

[0038] Furthermore, the disulfide-containing amino reagent is selected from 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)dithio]ethylamine, 2,2'-dithiodiphenylamine (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) One or more of the following: 2-aminoethyl-2'-hydroxyethyl disulfide (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-aminoethyl dithioethylamine, and N-tert-butoxycarbonyl-cystamine.

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

[0040] .

[0041] More preferably, the disulfide-containing amino reagent is selected from one or more of cystamine, cystamine dihydrochloride, and 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 carboxyl group amount is calculated based on the total molar amount of the self-carboxyl group in the molecular chain structure of the preacylated product and the carboxyl group or carboxyl salt introduced by preacylation.

[0044] The molar ratio of the second activator, the first activator, and the total carboxyl group in the preacylated product is 0.1–2 : 0.1–1 : 1, preferably 0.4–1.2 : 0.2–0.5 : 1;

[0045] The molar ratio of the disulfide-bonded amino reagent in S2 to the total carboxyl group in the preacylated product is 1 to 5:1, preferably 1 to 2:1.

[0046] The molar ratio of the reducing agent to the total carboxyl group in the preacylated product is 1 to 4:1, preferably 1 to 2:1; a higher amount of reducing agent results in a faster reduction rate, and conventional amounts are sufficient in this invention.

[0047] Furthermore, the amidation reaction in S2 is carried out at a temperature of 25°C to 50°C for a time of 0.2h to 24h, preferably at a temperature of 30°C to 40°C for a time of 0.5h to 24h.

[0048] Furthermore, the reduction reaction in S2 is carried out at a temperature of 25°C to 50°C for a time of 0.5h to 5h, and preferably at a temperature of 30°C to 40°C for a time of 2h to 4h.

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

[0050] The third aspect of this invention provides an injectable in-situ crosslinked hydrogel that mimics the extracellular matrix, constructed from the above-mentioned thiolized biocompatible polymer derivatives or thiolized biocompatible polymer derivatives prepared by the above-mentioned preparation methods. The construction method involves the spontaneous oxidation of thiol groups in the molecular chain structure of the thiolized biocompatible polymer derivatives under physiologically compatible conditions to form disulfide bonds and / or crosslinking with multifunctional electrophilic or nucleophilic crosslinking agents through efficient click chemistry.

[0051] Furthermore, the molecular structure of multifunctional electrophilic or nucleophilic crosslinking agents contains one or more of the following functional groups: aldehyde, acrylate, acrylamide, maleimide, vinyl sulfone, norbornene, and thiol. For example, the crosslinking agent can be α,β-unsaturated (meth)acrylate compounds, α,β-unsaturated (meth)acrylamide compounds, norbornene compounds, maleimide compounds, vinyl sulfone compounds, aldehyde compounds, thiol compounds, etc. Taking the general formula (I) structural unit as an example, the process of spontaneous oxidation or efficient click chemical reaction with the crosslinking agent is as follows: Figure 11 As shown, G and R1 in general formula (Ⅰ) are defined as above; auto-oxidation occurs under conditions of oxidizing agents such as oxygen and / or hydrogen peroxide; R in the crosslinking agent... 1 and R 2 Each of these can be independently identified as one of the following biopolymers containing at least two of the aforementioned functional groups: polyethylene glycol derivatives (e.g., two-armed, three-armed, four-armed, eight-armed, or more-armed polyethylene glycol derivatives), collagen, gelatin, hyaluronic acid, chitosan, sodium alginate, chondroitin sulfate, etc. In this invention, the expression "(meth)acrylate" is used to represent either acrylate or methacrylate, and so on.

[0052] The fourth aspect of the present invention provides the use of the above-described injectable in-situ crosslinked hydrogel, which mimics the extracellular matrix, in the preparation of biopharmaceutical formulations.

[0053] Furthermore, the biomedical preparation includes one or more of tissue engineering scaffolds, drug controlled-release carriers, 3D bioprinting inks, and cell encapsulation materials, and is suitable for biomedical fields such as skin wound repair, cartilage defect filling, nerve conduit bridging, or myocardial tissue regeneration.

[0054] The process for forming thiolized biocompatible polymer derivatives in this invention differs from existing technologies that use highly reactive hydrazide reagents. This invention avoids the side reaction of amide bond coupling between the self-carboxyl group and the amino group. First, the amino group of the biocompatible polymer is pre-acylated to strictly shield against side reactions. After activation, a less reactive disulfide-bonded amino reagent is used as the amino reagent to couple the self-carboxyl group and the pre-acylated carboxyl group of the biocompatible polymer to an amide bond, thus avoiding the side reaction of existing technologies. Figure 1The path marked with a cross in the middle is open, and after reduction, thiolized biocompatible polymer derivatives can be successfully prepared.

[0055] In this invention, pre-acylation refers to the process of introducing a side-chain carboxyl group while the side-chain amino group of a biocompatible polymer reacts with a diacid anhydride to form an amide bond coupling. This avoids the subsequent side reaction of the self-carboxyl group coupling with its own amino group to form a three-dimensional cross-linked byproduct, and at the same time 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 group of the biocompatible polymer that has been pre-acylated. However, although complete pre-acylation of the side-chain amino group can strictly avoid the side reaction of the self-carboxyl group coupling with its own amino group, experiments have shown that complete pre-acylation will impair the water solubility of the subsequent thiolation of the biocompatible polymer derivative, seriously affecting its application. The possible reason is that, compared with the general formula (I) structural unit, the general formula (III) structural unit couples with the thiol group through a longer side chain segment, which increases the hydrophobicity and thus impairs the overall water solubility of the product. Therefore, the pre-acylation rate is an important parameter that needs precise control. Sufficient pre-acylation is required to minimize amide bond coupling side reactions, but complete pre-acylation is not necessary to avoid impairing the product's water solubility. The reaction principle of this invention, which involves reacting biocompatible polymers containing both amino and carboxyl groups with disulfide-containing amino reagents to form thiolized biocompatible polymer derivatives, is as follows: Figure 2 As shown.

[0056] Beneficial technical effects: This invention employs a pre-acylation strategy for biocompatible polymers containing both amino and carboxyl groups to prevent the formation of three-dimensional cross-linked byproducts through amide bond coupling between the amino and carboxyl groups. This ensures the subsequent use of safe and reliable disulfide-containing amino reagents to introduce active thiol groups via amide bond coupling, avoiding the potential risks of hydrazine-containing reagents and further improving drug safety. Simultaneously, using disulfide bonds as a protective precursor and amide bond coupling to introduce active thiol groups avoids the self-oxidation of active thiol groups during the reaction, ensuring high activity of thiol groups in the final product.

[0057] This invention, combined with a high-concentration preparation method, possesses advantages for large-scale production, further expanding its industrial application potential. The thiolized biocompatible polymer derivative of this invention can achieve rapid in-situ cross-linking through mild spontaneous oxidation or click chemistry reactions, forming a mechanically stable injectable in-situ cross-linked hydrogel that mimics the extracellular matrix. This effectively solves the shortcomings of (recombinant) gelatin and recombinant human collagen, such as insufficient mechanical properties, difficulty in precisely controlling degradation rates, and inability to provide long-term stable support for tissue repair. It is suitable for various biomedical needs such as skin wound repair and cartilage defect filling, with no risk of viral residue or immune rejection, strong universality, and broader application prospects. Attached Figure Description

[0058] Figure 1 This is a schematic diagram illustrating the coupling reaction principle of carboxyl-containing biocompatible polymers with acylhydrazine or amino reagents in the prior art.

[0059] Figure 2 This is a schematic diagram illustrating the reaction principle of the present invention, in which biocompatible polymers containing both amino and carboxyl groups react with amino reagents containing disulfide bonds to form thiolized biocompatible polymer derivatives.

[0060] Figure 3 This is a graph showing the trend of different pre-acylation rates with the amount of acid anhydride fed in Example 1;

[0061] Figure 4 The NMR spectra of gelatin preacylization products with different preacylization rates in Example 1 are shown.

[0062] Figure 5 The NMR spectra of gelatin in Example 7, gelatin preacylation product with a preacylation rate of 95%, and thiolized modified derivatives of highly preacylated gelatin are shown.

[0063] Figure 6 This is a graph showing the relationship between different activation pH values ​​and the thiol content of the thiol-modified derivatives of the highly preacylated gelatin prepared in Example 9.

[0064] Figure 7 This is a graph showing the relationship between the amount of different carboxyl activators (Sulfo-NHS / EDCI) used in Example 10 and the thiol content of the thiol-modified derivatives of the prepared highly preacylated gelatin.

[0065] Figure 8 This is a graph showing the relationship between the amount of different amino reagents used in Example 11 and the thiol content of the thiol-modified derivatives of the prepared highly preacylated gelatin.

[0066] Figure 9 This is a microscopic observation of the injectable in-situ cross-linked hydrogel that mimics the extracellular matrix, used as a substrate for stem cell adherence and growth in Example 17.

[0067] Figure 10 Example 18: The number of live cells inside a bone marrow mesenchymal stem cell implanted in an injectable in situ cross-linked hydrogel that mimics the extracellular matrix.

[0068] Figure 11 This refers to the process by which the general formula (Ⅰ) structural unit in this invention undergoes spontaneous oxidation or a highly efficient click chemical reaction with a crosslinking agent. Detailed Implementation

[0069] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0070] Unless otherwise specifically stated, the numerical values ​​set forth in these embodiments do not limit the scope of the invention. Techniques and methods known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques and methods should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values. It should be noted that values ​​expressed, for example, as "within the range of ab" or "between the range of ab," do not include the endpoint values ​​a and b; values ​​expressed as "for ab," "is ab," or "ab" include the endpoint values ​​a and b.

[0071] Furthermore, it should be noted that the use of terms such as "first" and "second" to define activators, weakly acidic conditions, etc., is merely for the purpose of distinguishing the materials and system environment used in each step. Unless otherwise stated, the above terms have no special meaning and therefore should not be construed as limiting the scope of protection of this invention.

[0072] Experimental methods not specified in the following examples are generally performed according to national standards; if there is no corresponding national standard, they are performed according to general standard requirements or general methods.

[0073] In this invention, the % w / v symbol means mass-volume percentage, which has the same meaning as g / 100mL.

[0074] Example 1

[0075] This case study describes the preparation of a pre-acylated gelatin 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, and stir in a constant temperature water bath at 35℃ until completely dissolved to obtain 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 preacylated gelatin 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 δ0.8–2.2 ppm range represents the characteristic peaks of alkyl hydrogens (-CH2-, -CH3) in the gelatin side chain, and the peak shape does not change significantly, but the relative proportion of their peak area shows a decreasing trend. The δ2.6–2.9 ppm range corresponds to the characteristic peak of lysine ε-methylene hydrogen (ε-CH2 directly connected to -NH2). The peak intensity gradually weakens with the increase of preacylation rate. When the preacylation rate reaches 90%, the peak almost disappears, and it disappears completely at 100% preacylation. The characteristic signal change in the δ2.4–2.6 ppm range is closely related to the modification of succinic anhydride. When the preacylation rate reaches 19%, two weak single peaks appear in this range at δ ~2.35 and δ ~2.45, corresponding to the hydrogen signal of the -CH2-CH2- group next to the amide bond introduced by succinic anhydride. As the preacylation rate increases, the intensity of these two characteristic peaks continues to increase, and the peak shape gradually becomes sharper from a blunt state.

[0084] Example 2

[0085] This case study describes the preparation of thiolized biocompatible polymer derivatives. Thiolization modification was performed using gelatin pre-acylated products with low pre-acylation rates, including the following steps:

[0086] S1. Accurately weigh 5.00 g of the gelatin preacylated product prepared in Example 1 (preacylation rate of 19%), 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.

[0087] S2. Add 1.285 g of cystamine dihydrochloride and 0.310 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 0.602 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.

[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 reaction solution was adjusted to 3.5, and the solution was transferred to a dialysis bag (molecular weight cutoff of 14 kDa). The solution was purified by dialysis using a 2 wt% sodium chloride solution with pH 3.3. The dialysis solution was changed every 4 hours for 3 days. Subsequently, the solution was changed to a 0.9 wt% sodium chloride solution with pH 3.3 and pure water with 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.

[0095] The derivative molecule in this case simultaneously possesses three structural units from formula (V) to formula (VII):

[0096] ; where G represents a residue of type A gelatin.

[0097] 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 0.43 mmol / g.

[0098] Example 4

[0099] 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:

[0100] S1. Accurately weigh 5.00 g of the gelatin preacylated product prepared in Example 1 (preacylation rate of 58%), 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.

[0101] S2. Add 1.438 g of cystamine dihydrochloride and 0.347 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 0.673 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 in a disulfide bond.

[0102] Subsequently, 3.66 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.

[0103] The pH of the reaction solution was adjusted to 3.5, and the solution was transferred to a dialysis bag (molecular weight cutoff of 14 kDa). The solution was purified by dialysis using a 2 wt% sodium chloride solution with pH 3.3. The dialysis solution was changed every 4 hours for 3 days. Subsequently, the solution was changed to a 0.9 wt% sodium chloride solution with pH 3.3 and pure water with 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.

[0104] The structural units contained in the derivative molecular structure in this case are the same as those in Example 3.

[0105] 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 0.45 mmol / g.

[0106] Example 5

[0107] 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:

[0108] S1. Accurately weigh 5.00 g of the gelatin preacylated product prepared in Example 1 (preacylation rate of 79%), 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.

[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. Accurately weigh 5.00 g of the gelatin preacylated product prepared in Example 1 (preacylation rate of 89%), 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.

[0117] S2. Add 1.553 g of cystamine dihydrochloride and 0.374 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 0.727 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. 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.

[0118] Subsequently, 3.953 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 for 3 h under this condition to fully reduce the disulfide bonds introduced into the molecule to active thiol groups. After the reaction, 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.

[0119] 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.

[0120] The structural units contained in the derivative molecular structure in this case are the same as those in Example 3.

[0121] 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 0.48 mmol / g.

[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] 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 0.49 mmol / g.

[0130] This case study compares the NMR spectra of gelatin, gelatin preacylization products with a preacylization rate of 95%, and thiol-modified derivatives of highly preacylized gelatin, as shown below. Figure 5 As shown, compared to unmodified gelatin, the ¹H NMR spectrum of the preacylated gelatin product exhibits significant characteristic changes: the relative proportion of the characteristic peak area of ​​alkyl hydrogens (-CH2-, -CH3) in the gelatin side chain at δ0.8-2.2 ppm shows a decreasing trend; the characteristic peak corresponding to the ε-methylene hydrogen of lysine (ε-CH2 directly linked to -NH2) in the δ2.6-2.9 ppm range almost disappears; and two new characteristic singlets appear in the δ2.4-2.6 ppm range. Furthermore, the spectral characteristics of the thiolated modified derivatives of highly preacylated gelatin undergo further changes: the characteristic peak at δ2.4-2.6 ppm changes from a singlet to a distinct triplet; and a characteristic multiplet corresponding to the -CH2-CH2-SS-methylene hydrogen in the cystamine molecule appears in the δ2.9-3.2 ppm range.

[0131] Example 8

[0132] This case study describes the preparation of a thiolized biocompatible polymer derivative. The thiolization modification was performed using a fully pre-acylated gelatin pre-acylated product, and included the following steps:

[0133] S1. Accurately weigh 5.00 g of the gelatin preacylated product prepared in Example 1 (preacylation rate of 100%), 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.

[0134] S2. Add 1.591 g of cystamine dihydrochloride and 0.3835 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 0.749 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.

[0135] Subsequently, 4.05 g of tris(2-carboxyethyl)phosphonic 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 in the molecule to active thiol groups. After the reaction, gel blocks were present in the system and could not be reduced to solution by the reducing agent. This indicates that the gelatin preacylated thiolized modified derivative prepared when the preacylation rate (100%) is too high has poor solubility, which may be related to the hydrophobic aggregation of the derivative when the preacylation rate is too high.

[0136] Example 9

[0137] This case study focuses on the preparation of thiolized biocompatible polymer derivatives. Thiolization modification was performed using gelatin pre-acylated products with high pre-acylation rates. The pH for carboxyl group activation in S2 (i.e., the pH of the first and second weakly acidic conditions) was optimized, including the following steps:

[0138] S1. Accurately weigh 5 portions of 5.00 g each 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.

[0139] S2. Add 1.572 g of cystamine dihydrochloride to each of the above solutions, and then add 0.379 g of N-hydroxythiosuccinimide (Sulfo-NHS), stirring until completely dissolved; then adjust the pH of the five groups of solutions to 3.5-5.5 (first weakly acidic condition) with 6 M hydrochloric acid: Group 1 pH= 3.5, Group 2 pH= 3.75, Group 3 pH= 4.6, Group 4 pH= 5.0, Group 5 pH= 5.5;

[0140] Under continuous stirring, 0.736 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) was added to five groups of solutions with different pH values. During the reaction, the pH of the system was maintained at 4-6 (second weak acid condition) with 3 M hydrochloric acid. The pH values ​​were: Group 1 pH=4.0, Group 2 pH=4.5, Group 3 pH=5.0, Group 4 pH=5.5, and Group 5 pH=6. The system was continuously stirred and activated for 30 min.

[0141] After activation, the pH of the system was adjusted to 5.0 with 5 M sodium hydroxide solution, and the reaction was stirred overnight at 35°C 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.

[0142] Subsequently, 4.001 g of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP·HCl) was added to each reaction system and stirred for 5 min to completely dissolve it. 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 this condition to fully reduce the disulfide bonds introduced into the molecule to active thiol groups. After the reaction, each 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.

[0143] 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 highly preacylated gelatin prepared in 5 groups.

[0144] The structural units contained in the derivative molecular structure in this case are the same as those in Example 3.

[0145] HPLC analysis of the derivatives in this case revealed that the content of small molecule impurities was below 10 ppm. The content of active thiol groups in the derivatives, determined using a modified Ellman method, 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 different activation pH values ​​and the thiol content of the thiol-modified derivatives of the prepared highly preacylated gelatin is shown below. Figure 6 As shown.

[0146] Example 10

[0147] This case study describes the preparation of a thiolized biocompatible polymer derivative. A gelatin pre-acylated product with a high pre-acylation rate was used for thiolization modification. The amount of activator in S2 was optimized, and the process included the following steps:

[0148] S1. Accurately weigh 9 portions of 5.00 g each 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.

[0149] S2. Add 1.572 g of cystamine dihydrochloride to each of the above solutions, and then add the corresponding amounts (0.05 eq to 0.5 eq relative to the total carboxyl group) of N-hydroxythiosuccinimide (Sulfo-NHS): Group 1 0.100 g, Group 2 0.152 g, Group 3 0.227 g, Group 4 0.303 g, Group 5 0.455 g, Group 6 0.530 g, Group 7 0.606 g, Group 8 0.682 g, and Group 9 0.760 g, and stir until completely dissolved; then adjust the pH of the five solutions to 4.3 with 6 M hydrochloric acid;

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

[0151] After activation, the pH of the system was adjusted to 5.0 with 5 M sodium hydroxide solution, and the reaction was stirred overnight at 35°C 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.

[0152] Subsequently, 4.001 g of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP·HCl) was added to each reaction system and stirred for 5 min to completely dissolve it. 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 this condition to fully reduce the disulfide bonds introduced into the molecule to active thiol groups. After the reaction, each 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.

[0153] 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 highly preacylated gelatin prepared in group 9.

[0154] The structural units contained in the derivative molecular structure in this case are the same as those in Example 3.

[0155] HPLC analysis of the derivatives in this case revealed that the content of small molecule impurities was below 10 ppm. The content of active thiol groups in the derivatives, determined using a modified Ellman method, 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 relationship between the amount of different carboxyl activators (Sulfo-NHS / EDCI) and the thiol content of the thiol-modified derivatives of the prepared highly preacylated gelatin is shown below. Figure 7 As shown.

[0156] Example 11

[0157] This case study describes the preparation of a thiolized biocompatible polymer derivative. A gelatin pre-acylated product with a high pre-acylation rate was used for thiolization modification. The amount of disulfide-containing amino reagent in S2 was optimized, including the following steps:

[0158] S1. Accurately weigh 4 portions of 5.00 g each 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.

[0159] S2. Add the corresponding amounts (2.0 eq to 5.0 eq relative to the total carboxyl group) of cystamine dihydrochloride to the above four groups of solutions respectively: 3.144 g for group 1, 4.716 g for group 2, 6.288 g for group 3, and 7.86 g for group 4, and stir until completely dissolved; then add 0.606 g of N-hydroxythiosuccinimide (Sulfo-NHS) to each group and stir until completely dissolved; subsequently, adjust the pH of the five groups of solutions to 4.3 with 6 M hydrochloric acid;

[0160] Under continuous stirring, corresponding amounts (0.11 eq to 1.1 eq relative to the total carboxyl group) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were added to the nine groups of solutions respectively: Group 1 0.236 g, Group 2 0.360 g, Group 3 0.540 g, Group 4 0.718 g, Group 5 0.897 g, Group 6 1.03 g, Group 7 1.178 g, Group 8 1.330 g, and Group 9 1.472 g. During the reaction, the pH of the system was maintained at 4.7 ± 0.05 with 3 M hydrochloric acid, and the system was continuously stirred and activated for 30 min.

[0161] After activation, the pH of the system was adjusted to 5.0 with 5 M sodium hydroxide solution, and the reaction was stirred overnight at 35°C 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.

[0162] Subsequently, 4.001 g of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP·HCl) was added to each reaction system and stirred for 5 min to completely dissolve it. 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 this condition to fully reduce the disulfide bonds introduced into the molecule to active thiol groups. After the reaction, each 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.

[0163] 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 four groups of highly preacylated gelatin.

[0164] The structural units contained in the derivative molecular structure in this case are the same as those in Example 3.

[0165] HPLC analysis of the derivatives in this case revealed that the content of small molecule impurities was below 10 ppm. The content of active thiol groups in the derivatives, determined using a modified Ellman method, was 0.79 mmol / g, 0.85 mmol / g, 0.92 mmol / g, and 0.99 mmol / g, respectively. Using disulfide-containing amino reagents as the amino reagents, the relationship between different amounts of amino reagents and the thiol content of the prepared highly preacylated gelatin modified derivatives is as follows: Figure 8 As shown.

[0166] Example 12

[0167] This case study focuses on the preparation of thiolized biocompatible polymer derivatives. Thiolization modification was performed using gelatin pre-acylated products with moderate pre-acylation rates. The types of reducing agents in S2 were screened, and the process included the following steps:

[0168] S1. Accurately weigh 4 portions of 5.00 g each of the gelatin preacylated product prepared in Example 1 (preacylation rate of 79%), 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.

[0169] S2. Add 1.514 g of cystamine dihydrochloride to each of the above four solutions sequentially and stir until completely dissolved; then add the corresponding amounts (0.25 eq to 0.4 eq relative to the total carboxyl group) of N-hydroxythiosuccinimide (Sulfo-NHS): 0.365 g for group 1, 0.437 g for group 2, 0.510 g for group 3, and 0.583 g for group 4, and stir until completely dissolved; subsequently, adjust the pH of the system solution to 4.3 with 6 M hydrochloric acid;

[0170] Under continuous stirring, add the corresponding amounts (0.55 eq to 0.88 eq relative to the total carboxyl group) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI): 0.709 g for group 1, 0.848 g for group 2, 0.991 g for group 3, and 1.132 g for group 4. Stir until completely dissolved, and maintain the pH of the system at 4.7 ± 0.05 with 3 M hydrochloric acid during the reaction. Activate by continuous stirring for 30 min. After activation, adjust the pH of the system to 6.0 with 5 M sodium hydroxide solution, and react overnight at 35°C with continuous stirring.

[0171] Subsequently, 2.15 g of dithiothreitol (DTT) was added to the reaction system and stirred for 5 min to dissolve it completely. The pH of the system was gradually adjusted to 8.5 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, each system remained clear and transparent.

[0172] The pH of the solutions from the four reactions was adjusted to approximately 3.5, and the solutions were transferred to dialysis bags (molecular weight cutoff of 14 kDa). Purification dialysis was performed using a 2wt% sodium chloride solution at pH 3.3, with the dialysis solution changed every 4 hours for 3 consecutive days. On the second day of dialysis, a large amount of white flocculent material, and even gel-like clumps, precipitated in the solution. This is likely because the pre-acylation modification of gelatin enhances its hydrophobicity, causing DTT to be oxidized into a dimer, further increasing its hydrophobicity and leading to aggregation and formation of tiny particles. These particles precipitate directly or adsorb onto the gelatin surface, inducing protein aggregation and precipitation, resulting in decreased solubility of the derivative. Furthermore, the abrupt change in the solution environment caused by dialysis and the excessive use of DTT exacerbate protein precipitation, further reducing the solubility of the derivative.

[0173] Example 13

[0174] This case study focuses on the preparation of thiolized biocompatible polymer derivatives. Thiolization modification was performed using gelatin pre-acylated products with high pre-acylation rates, and the types of reducing agents in S2 were screened. The process included the following steps:

[0175] S1. Accurately weigh 4 portions of 5.00 g each 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.

[0176] S2. Add 1.572 g of cystamine dihydrochloride to each of the above four solutions sequentially and stir until completely dissolved; then add the corresponding amounts (0.25 eq to 0.4 eq relative to the total carboxyl group) of N-hydroxythiosuccinimide (Sulfo-NHS): 0.379 g for group 1, 0.455 g for group 2, 0.530 g for group 3, and 0.606 g for group 4, and stir until completely dissolved; subsequently, adjust the pH of the system solution to 4.3 with 6 M hydrochloric acid;

[0177] Under continuous stirring, add the corresponding amounts (0.25 eq to 0.4 eq relative to the 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 pH of the system at 4.7 ± 0.05 with 3 M hydrochloric acid during the reaction. Activate by continuous stirring for 30 min. After activation, adjust the pH of the system to 5.0 with 5 M sodium hydroxide solution, and react overnight at 35°C with continuous stirring.

[0178] Subsequently, 2.15 g of dithiothreitol (DTT) was added to the reaction system and stirred for 5 min to dissolve it completely. The pH of the system was gradually adjusted to 8.5 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, each system remained clear and transparent.

[0179] The pH of the solutions after the four reactions was adjusted to around 3.5, and then transferred to dialysis bags (molecular weight cutoff of 14 kDa). Purification dialysis was performed using a 2wt% sodium chloride solution at pH 3.3. The dialysis solution was changed every 4 hours, and dialysis was continued for 3 days. On the second day of dialysis, a large amount of white flocculent material or even gel-like lumps precipitated in the solution, consistent with the results of Example 12. This indicates that the stability of the thiolated modified derivatives of highly preacylated gelatin is not significantly related to the preacylation rate of gelatin. The core issue is that the reducing agent type—DTT—can lead to a decrease in the solubility of the derivatives due to oxidative polymerization and induced protein aggregation. Therefore, DTT is not suitable for the thiolation modification process after gelatin preacylation in this invention.

[0180] Example 14

[0181] This case study focuses on the preparation of thiolized biocompatible polymeric derivatives. It utilizes a pre-acylated product of recombinant collagen with a high pre-acylation rate for thiolization modification, and includes the following steps:

[0182] S1. Preparation of highly preacylated recombinant human collagen

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

[0184] The pH of the gelatin solution was adjusted to 8.5 using a 5M sodium hydroxide solution. Succinic anhydride was added in 1.0 equivalents (eq) of the amino content of the recombinant human collagen under continuous stirring to carry out a preacylation reaction. 5M sodium hydroxide solution was continuously added dropwise throughout the reaction to maintain the pH of the system at 8.0-9.5. The preacylation reaction was carried out in a 35°C water bath with continuous stirring for 3 hours.

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

[0186] Finally, the solution in the dialysis bag was collected and freeze-dried to obtain a white flocculent solid, which is highly preacylated recombinant human collagen, with a preacylation rate of 95%.

[0187] S2, Thiolation modification of highly preacylated recombinant human collagen

[0188] Accurately weigh 5.00 g of the above-mentioned highly preacylated modified recombinant human collagen, 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 5% w / v clear and transparent solution;

[0189] Add 0.586 g of cystamine dihydrochloride and 0.085 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 0.175 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 react overnight at 35°C with continuous stirring to complete the amidation reaction between the carboxyl group and the cystamine amino group.

[0190] Subsequently, 0.746 g of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) was added to the reaction system and stirred for 5 min to dissolve it completely. 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.

[0191] The reaction solution was diluted with purified water to 1% and the pH was adjusted to 3.5. It was then 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 days. Subsequently, the solution was changed to a 0.9 wt% sodium chloride solution at pH 3.3 and then purified 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 highly preacylated recombinant human collagen.

[0192] The derivative molecule in this case simultaneously possesses the following three structural units: formula (VIII) to formula (X):

[0193] ; where G represents recombinant human type III collagen residues.

[0194] 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 0.13 mmol / g.

[0195] Example 15

[0196] This case study describes the preparation of an injectable, in-situ crosslinked hydrogel that mimics the extracellular matrix, including the following steps:

[0197] Weigh 0.09 g of the thiol-modified derivative of the highly preacylated gelatin prepared in Example 7, dissolve it in 3 mL of 5 mM phosphate buffer (pH 7.2), and heat and stir in a 40°C water bath until completely dissolved to obtain a clear and transparent solution. Adjust the pH of the above solution to 7.2-7.4 with 5 M sodium hydroxide.

[0198] Take another 0.06 g of functional crosslinking agent (polyethylene glycol diacrylate) and dissolve it in 1 mL of 5 mM phosphate buffer (pH=7.2) to obtain a clear and transparent crosslinking agent solution;

[0199] Mix the two solutions thoroughly and place them in a 37°C water bath for 1-5 minutes. The viscosity of the solution will gradually increase and a hydrogel will form within 5 minutes.

[0200] Example 16

[0201] This case study involves the cytotoxicity detection of an injectable, in-situ cross-linked hydrogel that mimics 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 study demonstrates the proliferation detection of bone marrow mesenchymal stem cells embedded in an injectable, in-situ cross-linked hydrogel that mimics the extracellular matrix.

[0214] 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 sterilize, and then adjust the pH to 7.2-7.4 with 5M sterile sodium hydroxide.

[0215] 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.

[0216] Bone marrow mesenchymal stem cells (BMSCs) from generation P7 were digested and counted; 1.2 × 10⁻⁶ were collected. 5 Bone marrow mesenchymal stem cells (BMSCs) were centrifuged in centrifuge tubes. After discarding the supernatant, the two sterile solutions were transferred to centrifuge tubes and thoroughly mixed by pipetting. The mixture was then aliquoted into 12-well plates (1 mL / well) and incubated at 37°C for 15 min to fully cross-link and form hydrogels. Subsequently, 2 mL of cell culture medium (DMEM, 12% fetal bovine serum) was added to each well and the cells were cultured for one month, with the culture medium changed every 2-3 days. During this period, the number of viable cells inside the hydrogel was detected using the CCK8 assay at different time points. The results are shown below. Figure 10 As shown, the number of stem cells in the hydrogel increases continuously with the extension of culture time, indicating that the porous structure inside the hydrogel can be used for the delivery of nutrients and metabolites, and can support the survival and proliferation of cells inside, making it an excellent substrate for three-dimensional cell culture.

[0217] Comparative Example 1

[0218] The derivative preparation process in this case is the same as in Example 3, except that: in S1, the type A gelatin was not pre-acylated; instead, a 5% w / v clear and transparent solution of type A gelatin was directly used for S2. After 3 hours of reduction reaction, a large amount of gelatin lumps were still found in the system. This is because during the amidation coupling reaction stage, the abundant carboxyl groups on the gelatin molecular chain, after being activated by Sulfo-NHS / EDCI, not only react with the free amino groups on cystamine, but also undergo intramolecular or intermolecular crosslinking with the free amino groups on the same or adjacent molecular chains of the gelatin, forming an amide bond network. This covalent crosslinking network structure is stable and will not be destroyed by TCEP in the subsequent reduction step, therefore the gel cannot dissolve.

[0219] Comparative Example 2

[0220] The derivative preparation process in this case is the same as in Example 14, except that: in S1, the recombinant human type III collagen was not pre-acylated; instead, a clear and transparent recombinant human collagen solution with a concentration of 10% w / v was directly prepared. This solution was then subjected to S2. After 3 hours of reduction reaction, some gel clumps were still observed in the system. This is because during the amidation reaction, the abundant carboxyl groups on the recombinant human collagen molecular chain, after activation by Sulfo-NHS / EDCI, not only react with cystamine but also undergo intramolecular or intermolecular cross-linking with free amino groups on the same molecule or adjacent molecular chains, forming a stable amide bond network. This covalently cross-linked network structure is stable and will not be destroyed by TCEP in the subsequent reduction step; therefore, the gel cannot dissolve.

[0221] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

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, and the biocompatible polymer compound is selected from one or more of gelatin, genetically recombined gelatin and genetically recombined human collagen; 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, so as to finally form the thiolated biocompatible polymer derivative; The pre-acylation rate for forming the general formula (II) structural unit through pre-acylation is 40% to 95%.

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 60% to 95%.

3. The thiolated biocompatible polymer derivative according to any one of claims 1-2, characterized in that, The thiol group content of the thiolated biocompatible polymer derivative is 0.1 mmol / g to 1.0 mmol / g.

4. The thiolated biocompatible polymer derivative according to claim 3, characterized in that, The thiol group content of the thiolated biocompatible polymer derivative is 0.3 mmol / g to 0.9 mmol / g.

5. The thiolated biocompatible polymer derivative according to claim 4, characterized in that, The thiol group content of the thiolated biocompatible polymer derivative is 0.4 mmol / g to 0.7 mmol / g.

6. A method for preparing a thiolated biocompatible polymer derivative, characterized in that, The thiolated biocompatible polymer derivative is suitable for preparing the thiolated biocompatible polymer derivative as claimed in any one of claims 1 to 5, and comprises the following steps: S1, dissolving a biocompatible polymer compound containing amino and carboxyl groups in an aqueous solution, adjusting the system to be weakly alkaline, adding a diacid anhydride compound, maintaining the weakly alkaline condition, pre-acylating, and purifying 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 activating agent, adjusting the system to be a first weakly acidic condition and maintaining, adding a second activating agent, completing activation, adjusting the system to be a second weakly acidic condition, and performing amidation 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 reduction, and after purification, a thiolated biocompatible polymer derivative containing an active thiol group is obtained.

7. The method for preparing the thiolized biocompatible polymer derivative according to claim 6, 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; and 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.

8. The method for preparing the thiolized biocompatible polymer derivative according to claim 7, 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; and 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.

9. The method for preparing the thiolized biocompatible polymer derivative according to claim 6, 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 activating agent is N-hydroxysuccinimide, and the second activating agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride.

10. The method for preparing the thiolized biocompatible polymer derivative according to claim 6, 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.

11. The method for preparing the thiolized biocompatible polymer derivative according to claim 10, 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.

12. The method for preparing the thiolized biocompatible polymer derivative according to claim 6, 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.

13. The method for preparing the thiolized biocompatible polymer derivative according to claim 12, 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.

14. The method for preparing the thiolized biocompatible polymer derivative according to claim 6, 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.

15. The method for preparing the thiolized biocompatible polymer derivative according to claim 14, 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.

16. The method for preparing the thiolized biocompatible polymer derivative according to claim 6, 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.

17. The method for preparing the thiolized biocompatible polymer derivative according to claim 16, 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.

18. An injectable in situ crosslinking hydrogel that mimics the extracellular matrix, characterized in that, The thiolated biocompatible polymer derivative of any one of claims 1-5, or the thiolated biocompatible polymer derivative prepared by the preparation method of any one of claims 6-17, 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.

19. The ECM-mimicking injectable in situ cross-linking hydrogel of claim 18, 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.

20. Use of the injectable in situ crosslinkable hydrogel mimicking extracellular matrix according to any one of claims 18-19 in the preparation of a biomedical preparation.

21. Use of the injectable in situ cross-linking hydrogel according to claim 20, 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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