A bioactive hydrogel and preparation method thereof

Abstract

The present invention provides a bioactive hydrogel and a preparation method thereof, wherein the preparation method comprises the following steps: preparing thiolated carboxymethyl chitosan, mixing the chitosan with four-arm polyvinyl alcohol norbornene, adding a photoinitiator and deionized water to the mixture, mixing well, preparing a hydrogel precursor solution, subjecting the hydrogel precursor solution to ultraviolet irradiation, preparing a hydrogel matrix, and loading the hydrogel matrix with a drug to prepare a bioactive hydrogel. The hydrogel is gelled by a photopolymerization method, which has the advantages of being fast and convenient; the prepared hydrogel has good biocompatibility and is safe to use, and the hydrogel has a certain mechanical strength, and when acting on a wound, the viscosity is moderate, and the wound will not be damaged secondary when it is replaced and torn, and the hydrogel has the effects of stopping bleeding, antibacterial, promoting cell proliferation and tissue regeneration, and can accelerate wound healing, effectively solving the problems existing in the hydrogel in the prior art.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogels, and in particular relates to a bioactive hydrogel and a preparation method thereof. Background Art

[0002] Skin wound healing is a complex and dynamic process of tissue repair and regeneration after skin injury. This process includes four consecutive and overlapping stages: hemostasis, inflammation, cell proliferation, and tissue regeneration. Compared with traditional wound dressings such as gauze, cotton pads, and synthetic fibers, the new wound dressings currently used, such as semipermeable membranes, hydrocolloids, and hydrogels, allow oxygen and water vapor to pass through, hindering bacterial invasion. Combined with the loading of antibacterial and anti-inflammatory drugs, the new wound dressings have good biological activity and are more conducive to promoting wound healing. Among them, hydrogels have a microstructure similar to that of the extracellular matrix. At the same time, due to their strong hydrophilicity, they can absorb wound exudates to maintain a moist environment around the wound, thereby reducing the risk of wound infection and promoting wound healing, which has attracted much attention.

[0003] Polyethylene glycol (PEG) has good water solubility, biocompatibility and other properties, and is easy to functionalize. Functionalized PEG uses the active groups it contains to couple with drug molecules (including protein drugs and organic small molecule drugs), or other biomolecules and biomaterials through covalent bonds to exert the biological activity of drugs or other biomolecules. PEG-based hydrogels have been widely used in the biomedical field in the past few decades, and are also commonly used in wound dressings, such as hemostatic dressings, antibacterial dressings, and drug-loaded dressings. However, the following problems still exist during the application process:

[0004] (1) The cross-linking substances added during the gel preparation process may produce cytotoxicity and are difficult to remove, affecting the biocompatibility of the hydrogel. For example, the cross-linking agents, initiators, or catalysts (such as organic reagents, metal substances, etc.) used in the preparation process are difficult to remove by simple physical methods, resulting in inhibition of cell proliferation and migration, and poor biocompatibility.

[0005] (2) PEG-based hydrogels have poor mechanical properties. When used in wound dressings, hydrogels should have an elastic modulus comparable to that of the skin and conform to the mechanical conditions of the wound site. However, pure PEG-based hydrogels are brittle, easy to break, and have weak adhesion, which greatly limits their clinical applications such as wound dressings.

[0006] (3) The gel has a relatively single function and limited application scenarios. Common wound types include sterile incisions, potentially contaminated wounds, and contaminated wounds. Currently, most hydrogel designs lack universality. One gel only works on one type of wound and cannot be used to repair multiple types of wounds.

[0007] In order to accelerate the entire process of wound healing and avoid further bleeding or infection during the healing period, it is very important to develop multifunctional and biocompatible wound dressings with hemostatic, anti-oxidant, antibacterial, anti-inflammatory, and healing properties to repair skin damage. Summary of the invention

[0008] In view of the above problems existing in the prior art, the present invention provides a bioactive hydrogel and a preparation method thereof. The hydrogel is gelled by a photopolymerization method, which has the advantages of being fast and convenient. The prepared hydrogel has good biocompatibility and is safe to use. The hydrogel has a certain mechanical strength and moderate viscosity when applied to a wound. The hydrogel will not cause secondary damage to the wound when it is replaced or torn off. The hydrogel has the effects of stopping bleeding, resisting bacteria, and promoting cell and tissue regeneration, which can accelerate wound healing and effectively solve the problems existing in the hydrogel in the prior art.

[0009] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0010] A method for preparing a bioactive hydrogel comprises the following steps:

[0011] Thiolated carboxymethyl chitosan is prepared, mixed with four-arm polyethylene glycol norbornene, a photoinitiator and deionized water are added to the mixture, mixed evenly to obtain a hydrogel precursor solution, the hydrogel precursor solution is subjected to ultraviolet irradiation to prepare a hydrogel matrix, and the hydrogel matrix is ​​loaded with drugs to obtain a bioactive hydrogel.

[0012] In the above scheme, thiolated carboxymethyl chitosan is mixed with four-arm polyethylene glycol norbornene, and a hydrogel polymer is obtained through a thiol-ene photoclick chemistry reaction between the two. The elastic modulus of the hydrogel is consistent with that of human skin, and can be used to prepare wound dressings, which are suitable for a variety of different wound types. The thiol-ene photoclick chemistry reaction process makes the preparation process of the hydrogel have the advantages of simple preparation process and faster gelation.

[0013] Further, thiolated carboxymethyl chitosan is prepared by the following method:

[0014] (1) mixing thioglycolic acid and a catalyst, adding N,N-dimethylformamide thereto, and then stirring and reacting for more than 10 hours to obtain a reaction solution;

[0015] (2) preparing a carboxymethyl chitosan aqueous solution, then adding the reaction solution in step (1) thereto, stirring the reaction in a dark environment for more than 10 hours, then dialyzing the reaction product, and drying it to obtain thiolated carboxymethyl chitosan.

[0016] In the above scheme, carboxymethyl chitosan solution and thioglycolic acid are used as raw materials, and the carboxyl group of thioglycolic acid is activated under the action of a catalyst, so that it reacts with the amino group of carboxymethyl chitosan to obtain thiolated carboxymethyl chitosan grafted with multiple thiol groups. The raw materials that do not participate in the reaction are removed by dialysis to obtain a product with higher purity. The chemical structure of the prepared thiolated carboxymethyl chitosan is as follows:

[0017]

[0018] Since carboxymethyl chitosan is a polymer containing multiple segments of the above structure, during the thiolation reaction, thiolation occurs in at least one segment or all segments of the carboxymethyl chitosan. Therefore, X is limited to -H or

[0019] Furthermore, the catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0020] Furthermore, the mass percentage concentration of four-arm polyethylene glycol norbornene in the hydrogel precursor solution is 4-12%, the mass percentage concentration of the thiolated carboxymethyl chitosan is 1.5-8%, the mass percentage concentration of the initiator is 0.005-5%, and the rest is deionized water.

[0021] Furthermore, the molar ratio of the thiol group in the thiolated carboxymethyl chitosan to the double bond in the four-arm polyethylene glycol norbornene is 1-3:2-4.

[0022] Furthermore, the molecular weight of the thiolated carboxymethyl chitosan is 50,000-100,000.

[0023] Further, the photoinitiator includes at least one of I2959, LAP and I1173.

[0024] Furthermore, the drug is a protein drug or an antibiotic drug.

[0025] Furthermore, the drug is loaded by soaking and swelling.

[0026] A bioactive hydrogel is prepared by the above method.

[0027] The beneficial effects produced by the present invention are:

[0028] 1. In the present invention, thiol groups are introduced into chitosan to obtain thiolated carboxymethyl chitosan, which not only retains the excellent biocompatibility, hemostasis, antibacterial property, biodegradability, bioadhesion and antioxidant capacity of chitosan itself, but also can be used as a cross-linking agent to adjust the mechanical properties of polyethylene glycol-based hydrogel after reacting with polyethylene glycol norbornene. By adjusting the usage ratio of each component, a hydrogel matching the elastic modulus of human skin is obtained.

[0029] 2. The hydrogel in the present application is completed by homopolymerization double bond cleavage and ultraviolet light-induced step-growth thiol-ene photoclick chemistry. Photoclick chemistry is a process in which a photoinitiator absorbs light energy to generate free radicals, and then activates the groups on the polymer chain into free radicals to undergo chemical reactions, thereby forming a covalent cross-linked network. This process occurs quickly and has time and space controllability. At the same time, compared to traditional chain growth, the step-growth reaction can form a more uniform network, and because the double bond addition of four-arm polyethylene glycol norbornene is homopolymerization, less reactive oxygen is produced, which can effectively reduce oxidative stress, avoid protein carbonylation, and reduce damage to protein drugs.

[0030] 3. The hydrogel matrix in the present application has a mesh structure inside and has a certain viscoelasticity, which can play a certain physical blocking role, cooperate with the coagulation effect of carboxymethyl chitosan, and achieve the purpose of rapid coagulation and hemostasis of the wound; the hydrogel in the present application adopts a homopolymerization addition cross-linking method to reduce the generation of reactive oxygen, the cation of carboxymethyl chitosan has antioxidant and antibacterial effects, and the good antifouling properties of PEG-based hydrogels also play a certain antibacterial effect. The combination of the above factors may inhibit the generation of inflammatory factors; the three-dimensional structure of the hydrogel in the present application is similar to the structure of the extracellular matrix, which can promote tissue regeneration; in the present application, the hydrogel matrix is ​​loaded with drugs with coagulation, cell proliferation and other effects, which can also assist the hydrogel matrix to improve hemostasis, anti-inflammatory, promote cell regeneration, promote tissue regeneration, and accelerate wound healing. It can be applied to different types of wound surfaces. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the grafted hydrogen nuclear magnetic resonance spectrum of thiolated carboxymethyl chitosan in Example 1;

[0032] Figure 2 It is a state diagram of the hydrogel precursor solution before and after gelation;

[0033] Figure 3 This is a diagram showing the adhesion of the hydrogel on the skin;

[0034] Figure 4 This is the result of the hydrogel blood compatibility experiment;

[0035] Figure 5 This is a diagram of the active agent state of cells after the hydrogel extract is used for cell culture;

[0036] Figure 6 is the antibacterial ability diagram of the hydrogel;

[0037] Figure 7 This is a diagram showing the treatment of full-thickness skin defect wounds with hydrogel.

[0038] Figure 8 This is a diagram showing the treatment of infected wounds using hydrogel;

[0039] Fig. 9 This is a diagram showing the treatment of scald wounds using hydrogel. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention, that is, the embodiments described are only part of the embodiments of the present invention, rather than all of the embodiments.

[0041] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0042] It should be noted that relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0043] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0044] Example 1

[0045] A bioactive hydrogel, the preparation method of which comprises the following steps:

[0046] (1) Synthesis of thiolated carboxymethyl chitosan

[0047] 1 mL of 10 mol / L thioglycolic acid analytical grade chemical was added to a mixture of 0.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.3 g of N-hydroxysuccinimide, and a sufficient amount of N,N-dimethylformamide was added, and the mixture was reacted on a magnetic stirrer for 12 h to activate the carboxyl group; 1 g of carboxymethyl chitosan (MW = 50000 g / mol) was weighed into a conical flask, and 40 mL of ultrapure water was added. After complete dissolution by ultrasonication or stirring, activated thioglycolic acid was added, and the mixture was placed on a magnetic stirrer in the dark to react for 12 h to obtain a thiolated carboxymethyl chitosan product and raw materials that were not completely reacted; the obtained product was placed in a dialysis bag of appropriate size for product purification to remove unreacted raw materials. After the dialysis was completed, the purified product was precooled at -20°C for 12 h, and finally freeze-dried in a vacuum desiccant to obtain purified thiolated carboxymethyl chitosan;

[0048] (2) Preparation of four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel precursor solution

[0049] 5 mg of four-arm polyethylene glycol norbornene, 3.75 mg of thiolated carboxymethyl chitosan, 6.75 μg of photoinitiator I2959, and deionized water were mixed to form 100 μL of a hydrogel precursor solution, wherein the molar ratio of double bonds to thiol groups was 1:2;

[0050] (3) In situ gelation at the wound site

[0051] Add 40 μL of hydrogel precursor solution to the full-thickness wound and irradiate with 365 nm ultraviolet light for 5 minutes to complete the gelation process; load 10 μL of 20 μg / mL epidermal growth factor (EGF) and 10 μL of 5 μg / mL basic fibroblast growth factor (bFGF), and after the hydrogel state is stable, use a transparent dressing to ensure that the hydrogel does not fall off easily.

[0052] Example 2

[0053] A bioactive hydrogel, the preparation method of which comprises the following steps:

[0054] (1) Synthesis of thiolated carboxymethyl chitosan

[0055] 1 mL of 10 mol / L thioglycolic acid analytical grade chemical was added to a mixture of 0.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.3 g of N-hydroxysuccinimide, and a sufficient amount of N,N-dimethylformamide was added, and the mixture was reacted on a magnetic stirrer for 12 h to activate the carboxyl group; 1 g of carboxymethyl chitosan (MW = 50000 g / mol) was weighed into a conical flask, and 40 mL of ultrapure water was added. After complete dissolution by ultrasonication or stirring, activated thioglycolic acid was added, and the mixture was placed on a magnetic stirrer in the dark to react for 12 h to obtain a thiolated carboxymethyl chitosan product and raw materials that were not completely reacted; the obtained product was placed in a dialysis bag of appropriate size for product purification to remove unreacted raw materials. After the dialysis was completed, the purified product was precooled at -20°C for 12 h, and finally freeze-dried in a vacuum desiccant to obtain purified thiolated carboxymethyl chitosan;

[0056] (2) Preparation of four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel precursor solution

[0057] 5 mg of four-arm polyethylene glycol norbornene, 1.875 mg of thiolated carboxymethyl chitosan, 6.75 μg of photoinitiator I2959 and deionized water were mixed to form 100 μL of a hydrogel precursor solution, wherein the molar ratio of the double bonds in the four-arm polyethylene glycol norbornene to the thiol groups in the thiolated carboxymethyl chitosan was 1:4;

[0058] (3) In situ gelation at the wound site

[0059] Add 40 μL of hydrogel precursor solution to the full-thickness wound and irradiate with 365 nm ultraviolet light for 5 minutes to complete the gelation process; load 10 μL of 20 μg / mL epidermal growth factor (EGF) and 10 μL of 5 μg / mL basic fibroblast growth factor (bFGF), and after the hydrogel state is stable, use a transparent dressing to ensure that the hydrogel does not fall off easily.

[0060] Example 3

[0061] A bioactive hydrogel, the preparation method of which comprises the following steps:

[0062] (1) Synthesis of thiolated carboxymethyl chitosan

[0063] 1 mL of 10 mol / L thioglycolic acid analytical grade chemical was added to a mixture of 0.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.3 g of N-hydroxysuccinimide, and a sufficient amount of N,N-dimethylformamide was added, and the mixture was reacted on a magnetic stirrer for 12 h to activate the carboxyl group; 1 g of carboxymethyl chitosan (MW = 50000 g / mol) was weighed into a conical flask, and 40 mL of ultrapure water was added. After complete dissolution by ultrasonication or stirring, activated thioglycolic acid was added, and the mixture was placed on a magnetic stirrer in the dark to react for 12 h to obtain a thiolated carboxymethyl chitosan product and raw materials that were not completely reacted; the obtained product was placed in a dialysis bag of appropriate size for product purification to remove unreacted raw materials. After the dialysis was completed, the purified product was precooled at -20°C for 12 h, and finally freeze-dried in a vacuum desiccant to obtain purified thiolated carboxymethyl chitosan;

[0064] (2) Preparation of four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel precursor solution

[0065] 5 mg of four-arm polyethylene glycol norbornene, 5.625 mg of thiolated carboxymethyl chitosan, 6.75 μg of photoinitiator I2959, and deionized water were mixed to form 100 μL of a hydrogel precursor solution, wherein the molar ratio of double bonds to thiol groups was 3:4;

[0066] (3) In situ gelation at the wound site

[0067] Add 40 μL of hydrogel precursor solution to the full-thickness wound and irradiate with 365 nm ultraviolet light for 5 minutes to complete the gelation process; load 10 μL of 20 μg / mL epidermal growth factor (EGF) and 10 μL of 5 μg / mL basic fibroblast growth factor (bFGF), and after the hydrogel state is stable, use a transparent dressing to ensure that the hydrogel does not fall off easily.

[0068] Example 4

[0069] A bioactive hydrogel, the preparation method of which comprises the following steps:

[0070] (1) Synthesis of thiolated carboxymethyl chitosan

[0071] 1 mL of 10 mol / L thioglycolic acid analytical grade chemical was added to a mixture of 0.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.3 g of N-hydroxysuccinimide, and a sufficient amount of N,N-dimethylformamide was added, and the mixture was reacted on a magnetic stirrer for 12 h to activate the carboxyl group; 1 g of carboxymethyl chitosan (MW = 100000 g / mol) was weighed into a conical flask, and 40 mL of ultrapure water was added. After complete dissolution by ultrasonication or stirring, activated thioglycolic acid was added, and the mixture was placed on a magnetic stirrer in the dark to react for 12 h to obtain a thiolated carboxymethyl chitosan product and raw materials that were not completely reacted; the obtained product was placed in a dialysis bag of appropriate size for product purification to remove unreacted raw materials. After the dialysis was completed, the purified product was precooled at -20°C for 12 h, and finally freeze-dried in a vacuum desiccant to obtain purified thiolated carboxymethyl chitosan;

[0072] (2) Preparation of four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel precursor solution

[0073] 5 mg of four-arm polyethylene glycol norbornene, 1.875 mg of thiolated carboxymethyl chitosan, 6.75 μg of photoinitiator I2959, and deionized water were mixed to form 100 μL of a hydrogel precursor solution, wherein the molar ratio of double bonds to thiol groups was 1:4;

[0074] (3) In situ gelation at the wound site

[0075] Add 40 μL of hydrogel precursor solution to the full-thickness wound and irradiate with 365 nm ultraviolet light for 5 minutes to complete the gelation process; load 10 μL of 20 μg / mL epidermal growth factor (EGF) and 10 μL of 5 μg / mL basic fibroblast growth factor (bFGF), and after the hydrogel state is stable, use a transparent dressing to ensure that the hydrogel does not fall off easily.

[0076] Example 5

[0077] A bioactive hydrogel, the preparation method of which comprises the following steps:

[0078] (1) Synthesis of thiolated carboxymethyl chitosan

[0079] 1 mL of 10 mol / L thioglycolic acid analytical grade chemical was added to a mixture of 0.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.3 g of N-hydroxysuccinimide, and a sufficient amount of N,N-dimethylformamide was added, and the mixture was reacted on a magnetic stirrer for 12 h to activate the carboxyl group; 1 g of carboxymethyl chitosan (MW = 100000 g / mol) was weighed into a conical flask, and 40 mL of ultrapure water was added. After complete dissolution by ultrasonication or stirring, activated thioglycolic acid was added, and the mixture was placed on a magnetic stirrer in the dark to react for 12 h to obtain a thiolated carboxymethyl chitosan product and raw materials that were not completely reacted; the obtained product was placed in a dialysis bag of appropriate size for product purification to remove unreacted raw materials. After the dialysis was completed, the purified product was precooled at -20°C for 12 h, and finally freeze-dried in a vacuum desiccant to obtain purified thiolated carboxymethyl chitosan;

[0080] (2) Preparation of four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel precursor solution

[0081] 5 mg of four-arm polyethylene glycol norbornene, 3.75 mg of thiolated carboxymethyl chitosan, 6.75 μg of photoinitiator I2959, and deionized water were mixed to form 100 μL of a hydrogel precursor solution, wherein the molar ratio of double bonds to thiol groups was 1:2;

[0082] (3) In situ gelation at the wound site

[0083] Add 40 μL of hydrogel precursor solution to the full-thickness wound and irradiate with 365 nm ultraviolet light for 5 minutes to complete the gelation process; load 10 μL of 20 μg / mL epidermal growth factor (EGF) and 10 μL of 5 μg / mL basic fibroblast growth factor (bFGF), and after the hydrogel state is stable, use a transparent dressing to ensure that the hydrogel does not fall off easily.

[0084] Example 6

[0085] A bioactive hydrogel, the preparation method of which comprises the following steps:

[0086] (1) Synthesis of thiolated carboxymethyl chitosan

[0087] 1 mL of 10 mol / L thioglycolic acid analytical grade chemical was added to a mixture of 0.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.3 g of N-hydroxysuccinimide, and a sufficient amount of N,N-dimethylformamide was added, and the mixture was reacted on a magnetic stirrer for 12 h to activate the carboxyl group; 1 g of carboxymethyl chitosan (MW = 100000 g / mol) was weighed into a conical flask, and 40 mL of ultrapure water was added. After complete dissolution by ultrasonication or stirring, activated thioglycolic acid was added, and the mixture was placed on a magnetic stirrer in the dark to react for 12 h to obtain a thiolated carboxymethyl chitosan product and raw materials that were not completely reacted; the obtained product was placed in a dialysis bag of appropriate size for product purification to remove unreacted raw materials. After the dialysis was completed, the purified product was precooled at -20°C for 12 h, and finally freeze-dried in a vacuum desiccant to obtain purified thiolated carboxymethyl chitosan;

[0088] (2) Preparation of four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel precursor solution

[0089] 5 mg of four-arm polyethylene glycol norbornene, 5.625 mg of thiolated carboxymethyl chitosan, 6.75 μg of photoinitiator I2959, and deionized water were mixed to form 100 μL of a hydrogel precursor solution, wherein the molar ratio of double bonds to thiol groups was 3:4;

[0090] (3) In situ gelation at the wound site

[0091] Add 40 μL of hydrogel precursor solution to the full-thickness wound and irradiate with 365 nm ultraviolet light for 5 minutes to complete the gelation process; load 10 μL of 20 μg / mL epidermal growth factor (EGF) and 10 μL of 5 μg / mL basic fibroblast growth factor (bFGF), and after the hydrogel state is stable, use a transparent dressing to ensure that the hydrogel does not fall off easily.

[0092] Example 7

[0093] A bioactive hydrogel, the preparation method of which comprises the following steps:

[0094] (1) Synthesis of thiolated carboxymethyl chitosan

[0095] 1 mL of 10 mol / L thioglycolic acid analytical grade chemical was added to a mixture of 0.5 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 0.3 g of N-hydroxysuccinimide, and a sufficient amount of N,N-dimethylformamide was added. The mixture was reacted on a magnetic stirrer for 12 h to activate the carboxyl group; 1 g of carboxymethyl chitosan (MW = 50000 g / mol) was weighed into a conical flask, and 40 mL of ultrapure water was added. After complete dissolution by ultrasonication or stirring, activated thioglycolic acid was added, and the mixture was reacted on a magnetic stirrer in the dark for 12 h to obtain the thiolated carboxymethyl chitosan product and the raw materials that were not completely reacted; the obtained product was placed in a dialysis bag of appropriate size for product purification to remove the unreacted raw materials. After the dialysis was completed, the purified product was precooled at -20 ° C for 12 h, and finally freeze-dried in a vacuum desiccant to obtain purified thiolated carboxymethyl chitosan;

[0096] (2) Preparation of four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel precursor solution

[0097] 5 mg of four-arm polyethylene glycol norbornene, 3.75 mg of thiolated carboxymethyl chitosan, 6.75 μg of photoinitiator I2959, and deionized water were mixed to form 100 μL of a hydrogel precursor solution, wherein the molar ratio of double bonds to thiol groups was 1:2;

[0098] (3) In situ gelation at the infected wound site

[0099] Add 40 μL of hydrogel precursor solution to the infected wound and irradiate with 365 nm ultraviolet light for 5 minutes to complete the gelation process; load 10 μL of 20 μg / mL doxycycline hydrochloride, and after the hydrogel state is stable, use a transparent dressing to ensure that the hydrogel does not fall off easily.

[0100] Example 8

[0101] A bioactive hydrogel, the preparation method of which comprises the following steps:

[0102] (1) Synthesis of thiolated carboxymethyl chitosan

[0103] 1 mL of 10 mol / L thioglycolic acid analytical grade chemical was added to a mixture of 0.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.3 g of N-hydroxysuccinimide, and a sufficient amount of N,N-dimethylformamide was added, and the mixture was reacted on a magnetic stirrer for 12 h to activate the carboxyl group; 1 g of carboxymethyl chitosan (MW = 50000 g / mol) was weighed into a conical flask, and 40 mL of ultrapure water was added. After complete dissolution by ultrasonication or stirring, activated thioglycolic acid was added, and the mixture was placed on a magnetic stirrer in the dark to react for 12 h to obtain a thiolated carboxymethyl chitosan product and raw materials that were not completely reacted; the obtained product was placed in a dialysis bag of appropriate size for product purification to remove unreacted raw materials. After the dialysis was completed, the purified product was precooled at -20°C for 12 h, and finally freeze-dried in a vacuum desiccant to obtain purified thiolated carboxymethyl chitosan;

[0104] (2) Preparation of four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel

[0105] 5 mg of four-arm polyethylene glycol norbornene, 3.75 mg of thiolated carboxymethyl chitosan, 6.75 μg of photoinitiator I2959 and deionized water were mixed into 100 μL of hydrogel precursor solution, wherein the molar ratio of double bonds to thiol groups was 1:2; gelation was performed under ultraviolet light irradiation, and after gelation, 10 μL of 20 μg / mL epidermal growth factor (EGF) and 10 μL of 5 μg / mL basic fibroblast growth factor (bFGF) were loaded;

[0106] (3) Application on burn wounds

[0107] Place the hydrogel on the burn wound, and then use a transparent dressing to ensure that the hydrogel does not fall off easily.

[0108] Example 9

[0109] A bioactive hydrogel, the preparation method of which comprises the following steps:

[0110] (1) Synthesis of thiolated carboxymethyl chitosan

[0111] 1 mL of 10 mol / L thioglycolic acid analytical grade chemical was added to a mixture of 0.5 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 0.3 g of N-hydroxysuccinimide, and a sufficient amount of N,N-dimethylformamide was added. The mixture was reacted on a magnetic stirrer for 12 h to activate the carboxyl group; 1 g of carboxymethyl chitosan (MW = 50000 g / mol) was weighed into a conical flask, and 40 mL of ultrapure water was added. After complete dissolution by ultrasonication or stirring, activated thioglycolic acid was added, and the mixture was reacted on a magnetic stirrer in the dark for 12 h to obtain the thiolated carboxymethyl chitosan product and the raw materials that were not completely reacted; the obtained product was placed in a dialysis bag of appropriate size for product purification to remove the unreacted raw materials. After the dialysis was completed, the purified product was precooled at -20 ° C for 12 h, and finally freeze-dried in a vacuum desiccant to obtain purified thiolated carboxymethyl chitosan;

[0112] (2) Preparation of four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel

[0113] 5 mg of four-arm polyethylene glycol norbornene, 3.75 mg of thiolated carboxymethyl chitosan, 100 μg of photoinitiator I2959 and deionized water were mixed into 100 μL of hydrogel precursor solution, wherein the molar ratio of double bonds to thiol groups was 1:2; gelation was performed under ultraviolet light, and after gelation was completed, 10 μL of 12 nM tissue factor (TF) was loaded;

[0114] (3) Application to bleeding wounds

[0115] The hydrogel is placed at the bleeding wound to stop bleeding by utilizing the physical barrier effect of the hydrogel and the exogenous rapid coagulation pathway of TF.

[0116] Test example

[0117] 1. Nuclear magnetic resonance spectroscopy analysis

[0118] The thiolated carboxymethyl chitosan prepared in Example 1 of the present invention was subjected to nuclear magnetic resonance spectroscopy analysis. The analysis results of the MestReNova software are shown in FIG. Figure 1 As shown, the characteristic peak of thiol groups appeared at 2.9 ppm, indicating the successful grafting of thiol groups.

[0119] 2. State changes of four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel

[0120] During the preparation of four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel, the changes in the physical state of the hydrogel were recorded before and after UV irradiation, such as Figure 2 As shown, it can be clearly seen that the precursor solution is a liquid state that transforms into a solid hydrogel.

[0121] 3. Skin adhesion ability

[0122] Directly apply the prepared hydrogel to human skin, such as Figure 3 As shown, the hydrogel also has a certain degree of viscosity when inverted, and can have a good fit with the skin.

[0123] 4. In vitro biocompatibility testing

[0124] After the hydrogel was incubated with rabbit whole blood and centrifuged, the results were shown in Figure 4 The results showed that the hydrogel did not cause a large number of red blood cells to rupture, that is, it had good blood compatibility. In addition, cell experiments verified the good cell compatibility of the four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel.

[0125] Mouse skin fibroblast cell line L929 was inoculated into a blank well plate, cultured with hydrogel extract for 3 days, and then microscopically observed after rhodamine cytoplasmic staining. Figure 5 The experimental results showed that the cells had strong refractive index, full cytoplasm, strong proliferation ability, no obvious changes in cell state, high activity and good cell compatibility.

[0126] 5. Antibacterial ability

[0127] The good antibacterial ability of the four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel was verified by the resistance experiment against Escherichia coli and Staphylococcus aureus. 5 CFU / mL of bacterial solution was added to the hydrogel and incubated in a bacterial incubator for 4 hours. The supernatant after washing was spread on the bacterial culture medium and observed and counted after overnight incubation. Figure 6 As shown, the experimental results showed that compared with the control group, the hydrogel group achieved complete antibacterial effect.

[0128] 6. In vivo biocompatibility testing

[0129] The in vivo biocompatibility and healing ability of four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel were evaluated by three animal wound models. The control group was PBS solution, the commercial group was liquid dressing, and the experimental group was four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel. The wound size, wound infection (suppuration) degree and wound healing time were used as indicators for evaluation. Figure 7 As shown in the figure, the healing diagram of the full-thickness wound shows that the hydrogel group started healing earlier, and its healing-promoting ability was better than that of the PBS group, and even slightly better than that of the commercial group, indicating that the four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel has an excellent healing-promoting effect on ordinary wounds; Figure 8As shown in the figure, when Staphylococcus aureus was added to the wound site to simulate the infection of the wound, obvious pus appeared in the wound site of the commercial group and the PBS group, while the wound of the hydrogel group remained clean and healed steadily, indicating that the four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel has excellent antibacterial, anti-inflammatory, and healing effects on infected wounds; Fig. 9 As shown, the burn wound model shows that the wounds in the hydrogel group healed faster from the initial stage compared with the other two groups, and the scar area at the final healing site was small and shallow. Therefore, the four-arm polyethylene glycol norbornene / thiolated carboxymethyl chitosan hydrogel in the present invention, as a high-water content wound dressing, is also suitable for burn wounds.

Claims

1. A method for preparing a bioactive hydrogel for wound surface, characterized in that: The following steps are involved: preparing thiolated carboxymethyl chitosan, mixing it with four-arm polyethylene glycol norbornene, adding a photoinitiator and deionized water to the mixture, mixing well to obtain a hydrogel precursor solution, subjecting the hydrogel precursor solution to ultraviolet irradiation to prepare a hydrogel matrix, and loading the hydrogel matrix with drugs to obtain a bioactive hydrogel; The mass percentage concentration of four-arm polyethylene glycol norbornene in the hydrogel precursor solution is 4-12%, the mass percentage concentration of the thiolated carboxymethyl chitosan is 1.5-8%, the proportion of the initiator is 0.005-5%, and the rest is deionized water; the molar ratio of the thiol group in the thiolated carboxymethyl chitosan to the double bond in the four-arm polyethylene glycol norbornene is 1-3:2-4; the molecular weight of the thiolated carboxymethyl chitosan is 50,000-100,000; the photoinitiator is I2959; The wounds are full-thickness skin incisions, infected wounds and scald wounds.

2. The method for preparing a bioactive hydrogel for wound surface according to claim 1, characterized in that: Thiolated carboxymethyl chitosan was prepared by the following method: (1) mixing thioglycolic acid and a catalyst, adding N,N-dimethylformamide thereto, and then stirring and reacting for more than 10 hours to obtain a reaction solution; (2) preparing a carboxymethyl chitosan aqueous solution, then adding the reaction solution in step (1) thereto, stirring the reaction in a dark environment for more than 10 hours, then dialyzing the reaction product, and drying it to obtain thiolated carboxymethyl chitosan.

3. The method for preparing a bioactive hydrogel for wound surface according to claim 2, characterized in that: The catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

4. The method for preparing a bioactive hydrogel for wound surface according to claim 1, characterized in that: The drug is a protein drug or an antibiotic drug.

5. The method for preparing a bioactive hydrogel for wound surface according to claim 1, characterized in that: The drug is loaded by soaking and swelling.

6. A bioactive hydrogel for wound surface, characterized in that: The method according to any one of claims 1 to 5 is adopted.

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