Injectable chondroitin sulfate-based hydrogel as well as preparation method and application thereof

Abstract

The invention provides injectable chondroitin sulfate-based hydrogel as well as a preparation method and application thereof, and belongs to the technical field of medical polymer biomaterials. The hydrogel is prepared by mixing a succinimide functionalized chondroitin sulfate solution and a carboxymethyl chitosan solution and then gelating. The chondroitin sulfate-based hydrogel provided by the invention has good biocompatibility, safety, injectability and stability, and can be used independently or used for filling tissue wounds and promoting rapid healing of the wounds in a drug-loaded manner. Moreover, the chondroitin sulfate-based hydrogel provided by the invention is simple in preparation method, low in cost and wide in potential application range.

Description

Technical Field

[0001] The invention relates to the technical field of medical polymer biomaterials, and in particular to an injectable chondroitin sulfate-based hydrogel and a preparation method and application thereof. Background Art

[0002] In recent years, hydrogels have been used in biomedical fields such as tissue filling, hemostasis, tissue repair and drug delivery due to their high structural designability, good biodegradability, and adjustable mechanical properties. According to different gelation methods, hydrogels can be roughly divided into prefabricated hydrogels and injectable hydrogels. Among them, injectable hydrogels refer to hydrogels that can be gelled by in situ polymerization or sol-gel phase transition. The advantages of injectable hydrogels include minimal damage to the body, suitability for irregular and deep wounds, and simple use, so they have received increasing attention in the biomedical field.

[0003] Chondroitin sulfate (CS) is an acidic mucopolysaccharide present in human and animal connective tissues. It is composed of D-glucuronic acid and N-acetyl-D-galactosamine connected by 1,3 glycosidic bonds to form a disaccharide, and the disaccharides are connected by β-1,4 glycosidic bonds. CS has multiple biological activities such as anti-inflammatory, antioxidant, anti-thrombotic, anti-coagulation and immunomodulatory. In addition, as a natural polysaccharide product, CS exhibits good biocompatibility and biodegradability. In view of the above advantages, hydrogels based on CS have been developed in large quantities for use in biomedical fields such as drug delivery, tissue filling, and tissue repair. However, CS-based hydrogels often have problems such as low stability, low applicability, and toxic cross-linking agent residues in their applications, which limits their application and development.

[0004] Some chondroitin sulfate hydrogel products are provided in the prior art, for example: Chinese invention patent CN115429935A discloses an injectable cross-linked chondroitin sulfate hydrogel, and CN116570554A discloses an injectable gel. However, in the above schemes, the cross-linking force of the hydrogel is, in addition to hydrogen bonds, an imine bond and a carbon-nitrogen Schiff dynamic bond, both of which are dynamic covalent bonds, which are prone to breakage under external stimulation, and are not conducive to improving its stability in the human body; Chinese invention patent CN111499889A discloses a chondroitin sulfate magnesium hyaluronic acid hydrogel, however, the hydrogel has the problem of residual toxic cross-linking agent containing aldehyde groups and difficult biodegradation, and the drug is only loaded in the form of encapsulation, so that the release rate and stability of the drug are affected; Chinese invention patent CN118255909A discloses a drug-loaded chondroitin sulfate-based gel, however, the hydrogel is a prefabricated shaping type hydrogel, which is not suitable for making an injectable reagent, and its application in irregular wounds and deep wounds is limited. Summary of the invention

[0005] In response to the current problems of poor in vivo stability, toxic cross-linking agent residues and poor adaptability to wound shapes of chondroitin sulfate-based hydrogels, the present application provides an injectable, in situ gelling and easy-to-use chondroitin sulfate-based hydrogel, as well as a preparation method and application thereof.

[0006] In one aspect, the present application provides an injectable chondroitin sulfate-based hydrogel, wherein the hydrogel has a cross-linked polymer having a structure as shown in formula (I):

[0007]

[0008] In formula (I), (x+m+n)=100 to 10,000, and (q+w)=100 to 10,000.

[0009] It is understood that the subscripts x, m, n, q, and w in structural formula (I) are all positive integers.

[0010] In one embodiment, the hydrogel further comprises an active substance and / or a drug covalently bonded to any group in the structure shown in formula (I).

[0011] In one embodiment, the active substance and / or drug uses a substance containing an amino group in the chemical structure, which can react with the succinimide group in the structure shown in formula (I) to form a covalent bond, so as to fix the active substance and / or drug in the chemical structure of the cross-linked polymer shown in formula (I), thereby achieving a more stable combination between the polymer and the drug component compared to physical encapsulation or dynamic binding loading, and at the same time is beneficial to the release rate and effect of the drug component, thereby achieving a better sustained release effect.

[0012] Preferably, the active substance and / or drug may be a polypeptide or protein containing an amino group.

[0013] Optionally, the hydrogel can be mixed with drugs and / or pharmaceutically acceptable excipients in the form of physical encapsulation or dynamic combined loading to prepare a hydrogel preparation or composition, which can specifically adopt pharmaceutical components or methods known in the art.

[0014] On the other hand, the present application provides a method for preparing the chondroitin sulfate-based hydrogel, the method comprising:

[0015] S1, esterifying chondroitin sulfate and N-hydroxysuccinimide under the action of a dehydrating agent 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride to obtain succinimide-functionalized chondroitin sulfate;

[0016] S2. The succinimide functionalized chondroitin sulfate obtained in S1 is prepared into a succinimide functionalized chondroitin sulfate solution, and the solution is mixed with the carboxymethyl chitosan solution and then gelled to form the chondroitin sulfate-based hydrogel.

[0017] In one embodiment, the succinimide functionalized chondroitin sulfate prepared in step S1 has a structure as shown in formula (II):

[0018]

[0019] In formula (II), n is ≥100, and m is ≥10.

[0020] Among them, the succinimide functionalized chondroitin sulfate having the above structure makes the structure of chondroitin sulfate contain a certain amount of succinimide group ratio and a specific molecular weight, and the grafting rate of succinimide is closely related to the gelation speed and gelation strength after final mixing with carboxymethyl chitosan. The appropriate amount of succinimide grafting rate can be more replaced by carboxymethyl chitosan during subsequent mixing and gelation, while still retaining part of the succinimide substitution group under chemical kinetics, thereby forming a cross-linked polymer as shown in formula (I).

[0021] In one embodiment, the molecular weight of the succinimide functionalized chondroitin sulfate is 50,000 to 5,000,000, for example, 50,000, 100,000, 200,000, 400,000, 600,000, 800,000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, or 5,000,000.

[0022] In one embodiment, the carboxymethyl chitosan has a structure as shown in formula (II):

[0023]

[0024] Preferably, the molecular weight of the carboxymethyl chitosan is 50,000-5,000,000, for example, 50,000, 100,000, 200,000, 400,000, 600,000, 800,000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, or 5,000,000.

[0025] In one embodiment, in S1, the reaction molar ratio of chondroitin sulfate, 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is 1:(0.1-10):(0.1-10), for example, it can be 1:0.1:0.1, 1:0.2:0.2, 1:0.4:0.1, 1:0.8:0.8, 1:1:1, 1:2:2, 1:4:4, 1:8:8, 1:10:10, 1:2:4, 1:2:8, 1:2:10.

[0026] Preferably, the molar ratio of chondroitin sulfate to NHS is 1:(0.1-9), more preferably 1:(0.5-8), for example 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, more preferably 1:2.

[0027] In one embodiment, the reaction conditions are: stirring the reaction in a PBS buffer solution with a pH value of 4 to 6 for 1 to 60 minutes, and the reaction temperature is 0° C. to 55° C.

[0028] Preferably, the reaction temperature is 20 to 35° C., more preferably 25° C.; the reaction time is 5 to 30 min, more preferably 10 min.

[0029] In one embodiment, after the reaction is completed, the reaction solution is poured into anhydrous ethanol at -30 to -10°C to obtain a precipitate, and the precipitate is filtered, washed, and dried to obtain the succinimide-functionalized chondroitin sulfate.

[0030] Preferably, the temperature of the anhydrous ethanol is -20°C.

[0031] In one embodiment, the volume ratio of the reaction solution to anhydrous ethanol is 1:(0.5-100).

[0032] In one embodiment, after the precipitation is filtered, it is washed with anhydrous ethanol for 1-50 times.

[0033] In one embodiment, the precipitate is washed and then dried in a vacuum dryer for 2 to 24 hours.

[0034] In one embodiment, the succinimide functionalized chondroitin sulfate is frozen and stored after preparation and before dissolution, and the freezing conditions are: frozen and stored at -196°C to 0°C.

[0035] In one embodiment, in S2, the mixing mass ratio of succinimide functionalized chondroitin sulfate and carboxymethyl chitosan is (5-0.5): (0.5-5). For example, it can be (5-1): (1-5), preferably (3-1): (1-3), and can also be 3:1, 2:1, 1:1, 1:2, 1:3, and more preferably 1:1.

[0036] In one embodiment, the solvent used to prepare the solution is selected from one or more of physiological saline, PBS buffer, and ultrapure water, preferably physiological saline.

[0037] In one embodiment, the concentration of the succinimide functionalized chondroitin sulfate solution is 0.1% to 25%, and the concentration of the carboxymethyl chitosan solution is 0.1% to 25%.

[0038] Optionally, the preparation concentration of succinimide functionalized chondroitin sulfate and carboxymethyl chitosan solution can be 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 15.0%, 20% and 25%, etc., and the mixing can be equal volume mixing or mixed in proportion. The mass ratio of succinimide functionalized chondroitin sulfate and carboxymethyl chitosan in the final solution can be the required mass ratio.

[0039] In one embodiment, the mixing method in S2 comprises: mixing the succinimide functionalized chondroitin sulfate solution and the carboxymethyl chitosan solution by extruding them through the same injection port of a syringe.

[0040] For example, the two solutions may be respectively loaded into two syringes of a double-needle syringe and then extruded through the same injection port for mixing.

[0041] In one embodiment, in S2, the gelation time of gelation is 1 to 600 s.

[0042] Preferably, the average gelation time of the chondroitin sulfate-based hydrogel is 10 to 200 s, more preferably 20 to 60 s.

[0043] In one embodiment, the injection mixing process is performed at room temperature.

[0044] In one embodiment, the chondroitin sulfate-based hydrogel is injected smoothly without resistance.

[0045] On the other hand, the present application also provides the injectable chondroitin sulfate-based hydrogel, or the use of the injectable chondroitin sulfate-based hydrogel prepared by the method in the preparation of trauma auxiliary treatment products.

[0046] In one embodiment, the trauma auxiliary treatment product includes fillers for tissue trauma or defects, hemostatic and wound protection products, tissue sealing products, drug delivery products, biological scaffolds, injectable cell delivery materials, tissue engineering materials, three-dimensional cell culture materials, and medical cosmetic filling materials.

[0047] In one embodiment, according to the actual application scenario, conventional additives in the art may be added to the chondroitin sulfate-based hydrogel provided in the present application, or other active ingredients may be added to make it have other active effects.

[0048] In one embodiment, the chondroitin sulfate-based hydrogel has good biocompatibility and biodegradability.

[0049] In one embodiment, the chondroitin sulfate-based hydrogel is suitable for treating and filling irregular and deep wounds.

[0050] In one embodiment, the injectable chondroitin sulfate-based hydrogel is suitable for wounds with a wound depth of not less than 1 mm.

[0051] In one embodiment, the wound is a defective wound, including a soft tissue defect caused by accidental trauma or occurring during surgery.

[0052] Alternatively, the organ may be skin, liver, spleen, kidney, gastrointestinal tract or heart.

[0053] In one embodiment, the method of using the chondroitin sulfate-based hydrogel is as follows:

[0054] For irregular or deep wound tissue, succinimide-functionalized chondroitin sulfate and carboxymethyl chitosan are prepared into corresponding solutions and divided into two needle tubes of a double-needle syringe; the solution is injected into the wound surface or injected into a deep wound, or injected into the defect position of the defective tissue. After waiting for several seconds to tens of seconds, the mixed solution forms a hydrogel in situ to complete the filling and covering of the wound.

[0055] This application has at least the following technical advantages or effects:

[0056] 1. During the development process, the present application found that simply dissolving chondroitin sulfate and carboxymethyl chitosan and then mixing them could not form a gel. However, the present application synthesized succinimide-functionalized chondroitin sulfate by grafting an appropriate amount of succinimide groups into chondroitin sulfate molecules through an esterification reaction, and then dissolved the succinimide-functionalized chondroitin sulfate and carboxymethyl chitosan separately and then mixed them, which could form a hydrogel by in-situ crosslinking through covalent bonds, and the hydrogel was suitable for mixed injection with a syringe. The preparation method of the chondroitin sulfate-based hydrogel is simple, the raw materials are cheap and easy to obtain, and the gelation speed of the chondroitin sulfate-based hydrogel can be adjusted by controlling the number of grafted succinimide groups, and it has the characteristics of being suitable for irregular and deep wounds and simple to use, and can be widely used in the field of trauma management.

[0057] 2. In the preparation and use method of the chondroitin sulfate-based hydrogel provided by the present application, the raw material synthesis and the gel preparation process are simple, the use method is simple, and the application range is wide; moreover, the chondroitin sulfate-based hydrogel contains only two components, the composition is simple, and the process of preparing the chondroitin sulfate-based hydrogel only requires dissolution and injection mixing, and the process is simple. Moreover, the prepared chondroitin sulfate-based hydrogel is simple to use, and does not require complex equipment and complicated operation steps.

[0058] 3. The succinimide-functionalized chondroitin sulfate in the chondroitin sulfate-based hydrogel provided in the present application, on the one hand, is covalently cross-linked with carboxymethyl chitosan to form a hydrogel; on the other hand, the hydrogel structure as shown in formula (I) contains some succinimide groups that are not replaced by carboxymethyl chitosan. While improving the mechanical strength of the gel to a certain extent, the succinimide groups can also form a more stable covalent bond with amino-containing active substances or drugs to achieve slow release of drugs during the degradation of the hydrogel at the wound.

[0059] 4. The chondroitin sulfate-based hydrogel provided in the present application also has good cell compatibility, biodegradability and blood compatibility, is safer to use, has good biocompatibility, and the free NHS after gelation also has good biocompatibility. The hydrogel provided in the present application does not need to add any additional toxic cross-linking agents such as those containing aldehyde groups during the gelation process, and there is no problem of residual toxic cross-linking agents and difficulty in degradation.

[0060] 5. The chondroitin sulfate-based hydrogel prepared and provided in the present application contains chondroitin sulfate having anti-inflammatory and immune-regulating activities, and carboxymethyl chitosan having antibacterial activity. The biological activities of the two components are beneficial to promoting wound healing and are suitable for filling and repairing wound sites.

[0061] 6. The chondroitin sulfate-based hydrogel prepared by the present application has succinimide groups and carboxymethyl chitosan in the cross-linked polymer covalently bonded to chondroitin sulfate by ester bonds, which has better stability than hydrogen bonds or dynamic chemical bonds. In addition to having good biocompatibility and safety, carboxymethyl chitosan also contains a large number of amino groups in its structure, which can be further covalently bonded to unsubstituted succinimide groups to form a stable three-dimensional cross-linked network structure, which is conducive to exerting active efficacy at the wound for a long time; at the same time, the hydrogel has a fast and appropriate gelation time, and can quickly fill the wound when used without blocking the injection port, significantly improving the convenience and utilization rate of use. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0063] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the drawings:

[0064] Figure 1 Figure 1 is a preparation route of chondroitin sulfate-based hydrogel in a typical embodiment of the present invention, wherein Figure A is the CS-NHS synthesis route and Figure B is the CS-CMCTS hydrogel preparation route.

[0065] Figure 2 This is a diagram showing the gelation effect of chondroitin sulfate-based hydrogel in a typical embodiment of the present invention.

[0066] Figure 3 This is a graph showing the rheological behavior of chondroitin sulfate-based hydrogel in a typical embodiment of the present invention.

[0067] Figure 4 This is a photograph of the injectable properties of chondroitin sulfate-based hydrogel in a typical embodiment of the present invention.

[0068] Figure 5 This is a graph showing the cell viability results of treatment with a chondroitin sulfate-based hydrogel extract in a typical embodiment of the present invention.

[0069] Figure 6 This is a diagram showing the HE staining results of chondroitin sulfate-based hydrogel-embedded tissue in a typical embodiment of the present invention. DETAILED DESCRIPTION

[0070] In order to more clearly explain the overall concept of the application, the following is described in detail by way of embodiment. In the following description, a large amount of specific details are provided so as to provide a more thorough understanding of the application. However, it is obvious to those skilled in the art that the application can be implemented without one or more of these details. In other examples, in order to avoid confusion with the application, some technical features well known in the art are not described.

[0071] The technical scheme of the present invention is further described in detail below in conjunction with several preferred embodiments and accompanying drawings. Obviously, the described embodiments are only a part of embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work belong to the scope of protection of the present invention. It should be noted that the experimental methods adopted in the following preparation examples and embodiments, if not otherwise specified, are conventional methods, and the reagents used in the preparation examples and embodiments, if not otherwise specified, are reagents supplied by conventional reagent manufacturers.

[0072] The experimental reagents and experimental instrument information involved in the following examples are shown in Table 1:

[0073] Table 1 Reagent and instrument information

[0074]

[0075] The chemical reaction process of the chondroitin sulfate-based hydrogel preparation process involved in the following embodiments of the present application is as follows: Figure 1 shown.

[0076] Example 1

[0077] This embodiment provides a chondroitin sulfate-based hydrogel, and the preparation method thereof comprises the following steps:

[0078] S1. Preparation of succinimide functionalized chondroitin sulfate, comprising the following steps:

[0079] (1) 0.7 g of chondroitin sulfate (0.15 mmol) was dissolved in 7 mL of PBS buffer, 0.0345 g of NHS (0.30 mmol) and 0.0931 g of EDC (0.60 mmol) were dissolved in 1.5 mL of PBS buffer respectively, and the three solutions were mixed;

[0080] (2) adjusting the pH of the PBS buffer solution to 4-6;

[0081] (3) reacting the mixture at 25°C for 10 min;

[0082] (4) After the reaction is completed, the reaction solution is poured into -20°C anhydrous ethanol to precipitate the product;

[0083] (5) Filter the precipitate and wash it five times with -20°C anhydrous ethanol;

[0084] (6) Drying the washed product in a vacuum dryer for 12 h;

[0085] (7) Store the dried product at -20°C.

[0086] S2. The succinimide functionalized chondroitin sulfate prepared in this example is mixed with another component, chitosan, to prepare an injectable hydrogel. The specific steps are as follows:

[0087] (1) dissolving 0.2 g of succinimide-functionalized chondroitin sulfate in normal saline to prepare a 2% succinimide-functionalized chondroitin sulfate solution; dissolving 0.2 g of carboxymethyl chitosan in normal saline to prepare a 2% carboxymethyl chitosan solution;

[0088] (2) The succinimide functionalized chondroitin sulfate solution and the carboxymethyl chitosan solution were divided into two injection tubes of a double-barreled syringe at a volume ratio of 1:1, and then the two solutions were extruded through the same injection port to mix, thereby obtaining a chondroitin sulfate-based hydrogel.

[0089] Example 2

[0090] This embodiment provides a chondroitin sulfate-based hydrogel, and the preparation method thereof comprises the following steps:

[0091] S1. Preparation of succinimide functionalized chondroitin sulfate, comprising the following steps:

[0092] (1) 0.7 g of chondroitin sulfate (0.15 mmol) was dissolved in 7 mL of PBS buffer, 0.0173 g of NHS (0.15 mmol) and 0.0233 g of EDC (0.15 mmol) were dissolved in 1.5 mL of PBS buffer respectively, and the three solutions were mixed;

[0093] (2) adjusting the pH of the PBS buffer solution to 4-6;

[0094] (3) reacting the mixture at 25°C for 10 min;

[0095] (4) After the reaction is completed, the reaction solution is poured into -20°C anhydrous ethanol to precipitate the product;

[0096] (5) Filter the precipitate and wash it five times with -20°C anhydrous ethanol;

[0097] (6) Drying the washed product in a vacuum dryer for 12 h;

[0098] (7) Store the dried product at -20°C.

[0099] S2. The succinimide functionalized chondroitin sulfate prepared in this example is mixed with another component, chitosan, to prepare an injectable hydrogel. The specific steps are as follows:

[0100] (1) dissolving 0.2 g of succinimide-functionalized chondroitin sulfate in normal saline to prepare a 4% succinimide-functionalized chondroitin sulfate solution; dissolving 0.2 g of carboxymethyl chitosan in normal saline to prepare a 4% carboxymethyl chitosan solution;

[0101] (2) The succinimide functionalized chondroitin sulfate solution and the carboxymethyl chitosan solution were divided into two injection tubes of a double-barreled syringe at a volume ratio of 1:2, and then the two solutions were extruded through the same injection port to mix, thereby obtaining a chondroitin sulfate-based hydrogel.

[0102] Example 3

[0103] This embodiment provides a chondroitin sulfate-based hydrogel, and the preparation method thereof comprises the following steps:

[0104] S1. Preparation of succinimide functionalized chondroitin sulfate, comprising the following steps:

[0105] (1) 0.7 g of chondroitin sulfate (0.15 mmol) was dissolved in 7 mL of PBS buffer, 0.0345 g of NHS (0.30 mmol) and 0.0466 g of EDC (0.30 mmol) were dissolved in 1.5 mL of PBS buffer respectively, and the three solutions were mixed;

[0106] (2) adjusting the pH of the PBS buffer solution to 4-6;

[0107] (3) reacting the mixture at 25°C for 10 min;

[0108] (4) After the reaction is completed, the reaction solution is poured into -20°C anhydrous ethanol to precipitate the product;

[0109] (5) Filter the precipitate and wash it five times with -20°C anhydrous ethanol;

[0110] (6) Drying the washed product in a vacuum dryer for 12 h;

[0111] (7) Store the dried product at -20°C.

[0112] S2. The succinimide functionalized chondroitin sulfate prepared in this example is mixed with another component, chitosan, to prepare an injectable hydrogel. The specific steps are as follows:

[0113] (1) dissolving 0.2 g of succinimide-functionalized chondroitin sulfate in normal saline to prepare a succinimide-functionalized chondroitin sulfate solution with a mass fraction of 8%; dissolving 0.2 g of carboxymethyl chitosan in normal saline to prepare a carboxymethyl chitosan solution with a mass fraction of 8%;

[0114] (2) The succinimide functionalized chondroitin sulfate solution and the carboxymethyl chitosan solution were divided into two injection tubes of a double-barreled syringe at a volume ratio of 1:3, and then the two solutions were extruded through the same injection port to mix, thereby obtaining a chondroitin sulfate-based hydrogel.

[0115] Example 4

[0116] This embodiment provides a chondroitin sulfate-based hydrogel, and the preparation method thereof comprises the following steps:

[0117] S1. Preparation of succinimide functionalized chondroitin sulfate, comprising the following steps:

[0118] (1) 0.7 g of chondroitin sulfate (0.15 mmol) was dissolved in 7 mL of PBS buffer, 0.0690 g of NHS (0.60 mmol) and 0.0931 g of EDC (0.60 mmol) were dissolved in 1.5 mL of PBS buffer respectively, and the three solutions were mixed;

[0119] (2) adjusting the pH of the PBS buffer solution to 4-6;

[0120] (3) reacting the mixture at 25°C for 10 min;

[0121] (4) After the reaction is completed, the reaction solution is poured into -20°C anhydrous ethanol to precipitate the product;

[0122] (5) Filter the precipitate and wash it five times with -20°C anhydrous ethanol;

[0123] (6) Drying the washed product in a vacuum dryer for 12 h;

[0124] (7) Store the dried product at -20°C.

[0125] S2. The succinimide functionalized chondroitin sulfate prepared in this example is mixed with another component, chitosan, to prepare an injectable hydrogel. The specific steps are as follows:

[0126] (1) dissolving 0.2 g of succinimide-functionalized chondroitin sulfate in normal saline to prepare a succinimide-functionalized chondroitin sulfate solution with a mass fraction of 16%; dissolving 0.2 g of carboxymethyl chitosan in normal saline to prepare a carboxymethyl chitosan solution with a mass fraction of 16%;

[0127] (2) The succinimide functionalized chondroitin sulfate solution and the carboxymethyl chitosan solution were divided into two injection tubes of a double-barreled syringe at a volume ratio of 2:1, and then the two solutions were extruded through the same injection port to mix, thereby obtaining a chondroitin sulfate-based hydrogel.

[0128] Example 5

[0129] This embodiment provides a chondroitin sulfate-based hydrogel, and the preparation method thereof comprises the following steps:

[0130] S1. Preparation of succinimide functionalized chondroitin sulfate, comprising the following steps:

[0131] (1) 0.7 g of chondroitin sulfate (0.15 mmol) was dissolved in 7 mL of PBS buffer, 0.0345 g of NHS (0.30 mmol) and 0.1862 g of EDC (1.20 mmol) were dissolved in 1.5 mL of PBS buffer respectively, and the three solutions were mixed;

[0132] (2) adjusting the pH of the PBS buffer solution to 4-6;

[0133] (3) reacting the mixture at 25°C for 10 min;

[0134] (4) After the reaction is completed, the reaction solution is poured into -20°C anhydrous ethanol to precipitate the product;

[0135] (5) Filter the precipitate and wash it five times with -20°C anhydrous ethanol;

[0136] (6) Drying the washed product in a vacuum dryer for 12 h;

[0137] (7) Store the dried product at -20°C.

[0138] S2. The succinimide functionalized chondroitin sulfate prepared in this example is mixed with another component, chitosan, to prepare an injectable hydrogel. The specific steps are as follows:

[0139] (1) dissolving 0.2 g of succinimide-functionalized chondroitin sulfate in normal saline to prepare a succinimide-functionalized chondroitin sulfate solution with a mass fraction of 25%; dissolving 0.2 g of carboxymethyl chitosan in normal saline to prepare a carboxymethyl chitosan solution with a mass fraction of 25%;

[0140] (2) The succinimide functionalized chondroitin sulfate solution and the carboxymethyl chitosan solution were divided into two injection tubes of a double-barreled syringe at a volume ratio of 1:1, and then the two solutions were extruded through the same injection port to mix, thereby obtaining a chondroitin sulfate-based hydrogel.

[0141] Embodiment 6-9

[0142] The preparation methods of Examples 6-9 are substantially the same as those of Example 1, with the only difference being that, after the reaction in Example 1 is completed, the reaction solution is poured into anhydrous ethanol at -20°C, while the temperatures of anhydrous ethanol in Examples 6-9 are -40°C, 0°C, 25°C, and 55°C, respectively. The remaining steps are the same as in Example 1, and the gelling effect of the obtained product after mixing with the carboxymethyl chitosan solution is observed.

[0143] According to the experimental results, the time required for the product precipitation process using -40°C ethanol to prepare succinimide functionalized chondroitin sulfate in Example 6 was too long, and the succinimide functionalized chondroitin sulfate prepared in Examples 7-9 was respectively prepared into a solution and mixed with a carboxymethyl chitosan solution, and it was found that none of them could form a gel. It is speculated that the higher temperature of ethanol during the washing process made the succinimide group in the structure unstable and thus reduced the grafting rate. Therefore, in the preparation method provided in the present application, it is preferred to use -20°C anhydrous ethanol to precipitate the reaction solution.

[0144] Test Example 1

[0145] Taking Example 1 as an example, the feeding molar amount of N-hydroxysuccinimide was used as a variable to evaluate the gelation time of succinimide-functionalized chondroitin sulfate with different succinimide group contents after mixing with carboxymethyl chitosan.

[0146] Evaluation method: The evaluation was performed by the vial flipping method: two solutions were injected into the vial at the same time, and the vial was flipped every 5 seconds until the mixed solution did not flow down with the tilt of the vial. This was considered as the formation of a hydrogel, and the gelation time was recorded. The results are shown in Table 2:

[0147] Table 2

[0148] 4-Hydroxysuccinimide feed molar ratio (NHS / CS) Gelation time 10:1 ＜5s 8:1 5～15s 4:1 20～30s 2:1 40～60s 1:1 65～80s 0.5:1 125～140s

[0149] As shown in Table 2, the gelling time of hydrogels prepared with different succinimide group contents is quite different, wherein the more succinimide group content, the faster the gelling time. Based on this, the gelling time and strength can be regulated by adjusting the content of succinimide groups. Among them, the mixed solution of succinimidyl chondroitin sulfate and carboxymethyl chitosan provided in Example 1 forms a hydrogel within 60 seconds, which can be used as a preferred embodiment. Figure 2 shown.

[0150] Test Example 2

[0151] In this test example, the gelation time of the chondroitin sulfate-based hydrogel prepared in Example 1 was evaluated by a flowmeter.

[0152] Evaluation method: Dissolve succinimidyl chondroitin sulfate and carboxymethyl chitosan separately and load them into two needle tubes of a double-needle syringe. Then mix the two solutions through the same injection needle hole and inject them into the sample disk of the rotational rheometer. Then test the gelation time of the mixed solution under the conditions of frequency 10 Hz, strain 1% and temperature 37 ° C. The results are shown in Figure 3 .

[0153] Performance characterization: Figure 3 As shown in the figure, after 50s, the storage modulus (G") of the mixture begins to be greater than the loss modulus (G'), and the time point when the two moduli are equal is the gelation point. The above results show that the gelation time of the chondroitin sulfate-based hydrogel prepared in Example 1 is 50s.

[0154] Test Example 3

[0155] This test example evaluates the injectability of the chondroitin sulfate-based hydrogel prepared in Example 1.

[0156] Evaluation method: According to the preparation method described in Example 1, succinimidyl chondroitin sulfate was synthesized, and succinimidyl chondroitin sulfate and carboxymethyl chitosan solutions were prepared using physiological saline. First, the two solutions were injected using a syringe to test whether the solutions were squeezed out of the syringe smoothly. Then, the two solutions were respectively loaded into the two needles of a double-needle syringe, and the two solutions were injected through the same injection outlet to evaluate whether the solutions were squeezed out smoothly and whether the outlet was blocked by hydrogel.

[0157] Performance characterization: If the gelling speed is too fast, it will easily lead to blockage of the injection port during the injection process, which is not conducive to its injectability. Figure 4 As shown, the succinimidyl chondroitin sulfate and carboxymethyl chitosan solutions showed good injectability. The two solutions also showed good injectability through the same outlet. It can be seen that the hydrogel provided in Example 1 has a suitable gelation time and will not cause the problem of blocking the injection port.

[0158] Test Example 4

[0159] This test example evaluates the cytotoxicity of the chondroitin sulfate-based hydrogel prepared in Example 1.

[0160] Evaluation method: The hydrogel was prepared according to the preparation method described in Example 1, and the hydrogel was broken into pieces with a volume of about 1 mm. 3The small pieces were cultured in RPMI 1640 medium at 37°C for 12 h. The medium was sterilized using a 0.22 μm filter. L929 cells were cultured at 1×10 4 The cells were seeded into 96-well culture plates at a density of 100 cells / well and cultured in fresh culture medium for 24 hours. The complete culture medium was then replaced with chondroitin sulfate-based hydrogel extraction medium, and cell viability was detected using CCK-8 at 72 hours. The results are shown in Figure 5 .

[0161] Performance characterization: Figure 5 As shown, there was no significant difference in the viability of cells cultured in the chondroitin sulfate-based hydrogel extract and the blank control group, indicating that the chondroitin sulfate-based hydrogel did not show obvious cytotoxicity and did not affect the normal proliferation of cells.

[0162] Test Example 5

[0163] This test example evaluates the inflammatory response of the chondroitin sulfate-based hydrogel prepared in Example 1.

[0164] Evaluation method: According to the preparation method described in Example 1, 0.5 mL of a mixture of succinimidyl chondroitin sulfate solution and carboxymethyl chitosan solution was injected subcutaneously into rats, and tissues of the implantation site and adjacent areas were collected on days 3, 7, and 14, and then initially stabilized with 4% paraformaldehyde for 12 hours. Subsequently, these specimens were embedded in paraffin and sliced ​​to obtain slices with a thickness of 4 μm. These slices were then subjected to H&E staining to observe inflammatory cells and thereby evaluate tissue inflammatory response. The results are shown in Figure 6 .

[0165] Performance characterization: Figure 6 As shown, during the whole process of chondroitin sulfate-based hydrogel implantation, the number of inflammatory cells in the tissue of the implanted part of rat skin had no significant difference compared with that of the blank control group, indicating that the chondroitin sulfate-based hydrogel provided by the present invention did not show obvious inflammatory response.

[0166] Test Example 6

[0167] The ability of the prepared chondroitin sulfate-based hydrogel to promote wound healing after skin cyst excision was evaluated.

[0168] Evaluation method: First, 10 mL of air was injected subcutaneously into SD rats, followed by 5 mL of croton oil, and the rats were kept and observed in an SPF environment to establish a cyst model. After the model was established, the cyst was surgically removed, and then chondroitin sulfate-based hydrogel was injected to fill the defective part of the cyst incision and suture it. The healing ability of chondroitin sulfate-based hydrogel was evaluated by recording and calculating the recurrence rate of the cyst. The experimental results are shown in Table 3:

[0169] Table 3

[0170] Group Blank control Hydrogel Medical gauze Cyst recurrence rate (%) 50 0 40

[0171] Performance characterization: As shown in Table 3, the wound filled with chondroitin sulfate-based hydrogel healed completely within 14 days without cyst recurrence. In contrast, the cyst recurrence rates of untreated rats and SD rats in the medical gauze group were as high as 50% and 40%. The above results show that the chondroitin sulfate-based hydrogel provided by the present invention has a significant effect on promoting the healing of wounds after skin cyst excision.

[0172] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. An injectable chondroitin sulfate-based hydrogel, characterized in that: The hydrogel comprises a cross-linked polymer having a structure as shown in formula (I): In formula (I), (x+m+n)=100 to 10,000, and (q+w)=100 to 10,000.

2. The chondroitin sulfate-based hydrogel according to claim 1, characterized in that The hydrogel further comprises active substances and / or drugs covalently bonded to any group in the structure shown in formula (I).

3. The method for preparing a chondroitin sulfate-based hydrogel according to claim 1 or 2, characterized in that: The method comprises: S1, esterifying chondroitin sulfate and N-hydroxysuccinimide under the action of a dehydrating agent 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride to obtain succinimide-functionalized chondroitin sulfate; S2. The succinimide functionalized chondroitin sulfate obtained in S1 is prepared into a succinimide functionalized chondroitin sulfate solution, and the solution is mixed with the carboxymethyl chitosan solution and then gelled to form the chondroitin sulfate-based hydrogel.

4. The method according to claim 3, characterized in that: In S1, the reaction molar ratio of chondroitin sulfate, N-hydroxysuccinimide and 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride is 1:(0.1-10):(0.1-10); And / or, the reaction conditions are: stirring the reaction in a PBS buffer solution with a pH value of 4 to 6 for 1 to 60 minutes, and the reaction temperature is 0° C. to 55° C.; And / or, after the reaction is completed, the reaction solution is poured into anhydrous ethanol at -30 to -10°C to obtain a precipitate, and the precipitate is filtered, washed, and dried to obtain the succinimide-functionalized chondroitin sulfate.

5. The method according to claim 3, characterized in that: In S2, the mixing mass ratio of succinimide functionalized chondroitin sulfate and carboxymethyl chitosan is (5-0.5): (0.5-5); And / or, the solvent used to prepare the solution is selected from one or more of physiological saline, PBS buffer, and ultrapure water.

6. The method according to claim 3, characterized in that The mixing method in S2 includes: The succinimide functionalized chondroitin sulfate solution and the carboxymethyl chitosan solution were mixed by extruding through the same injection port of a syringe.

7. The method according to claim 3, characterized in that In the S2, the gelation time of gelation is 1 to 600 s.

8. The method according to claim 3, characterized in that The molecular weight of the succinimide functionalized chondroitin sulfate is 50,000-5,000,000; and / or the molecular weight of the carboxymethyl chitosan is 50,000-5,000,000.

9. Use of the injectable chondroitin sulfate-based hydrogel according to claim 1 or 2, or the injectable chondroitin sulfate-based hydrogel prepared by any method of claims 3 to 8 in the preparation of a trauma auxiliary treatment product.

10. The use according to claim 9, characterized in that: The trauma-assisted treatment products include fillers for tissue trauma or defects, hemostasis and wound protection products, tissue sealing products, drug delivery products, biological scaffolds, injectable cell delivery materials, tissue engineering materials, three-dimensional cell culture materials, and medical cosmetic filling materials; And / or, the injectable chondroitin sulfate-based hydrogel is suitable for wounds with a wound depth of not less than 1 mm.

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