A hydrogel-based hemostatic sealant and preparation method thereof

Abstract

The present invention relates to the technical field of hemostatic materials, and in particular to a hydrogel-based hemostatic sealant and a preparation method thereof, comprising: step S1, obtaining an ethanol solution of 3-methyl-3H-diaziridine-3-propionic acid; step S2, obtaining a 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution; step S3, obtaining an activated 3-methyl-3H-diaziridine-3-propionic acid solution; step S4, obtaining a mixed solution; step S5, obtaining a solution after an amidation reaction; step S6, obtaining a first dialysis solution and a second dialysis solution; step S7, obtaining sponge-like 3-methyl-3H-diaziridine-3-propionic acid-chitosan; step S8, obtaining a prepolymer solution; step S9, photocuring the prepolymer solution under a preset irradiation condition for a preset fifth time length t5 to obtain a hydrogel, and using the hydrogel as a hydrogel-based hemostatic sealant. The present invention has biocompatibility while achieving the effects of being easy to carry and use, and being able to stop bleeding quickly and efficiently.

Description

Technical Field

[0001] The present invention relates to the technical field of hemostatic materials, and in particular to a hydrogel-based hemostatic sealant and a preparation method thereof. Background Art

[0002] Incompressible massive bleeding is one of the main causes of death. War, traffic accidents and natural disasters often cause serious incompressible bleeding. More than 30% of traumatic deaths worldwide are caused by excessive blood loss, and more than half of these deaths occur before emergency treatment. For patients suffering from incompressible massive bleeding, every minute is important. If the bleeding cannot be stopped in time, they may die of bleeding in a few minutes or even less. Traditional suturing methods are not suitable for hemostasis of incompressible visceral tissues. Therefore, it is necessary to develop a hemostatic agent with the characteristics of biocompatibility, easy to carry and use, and fast and efficient hemostasis. The design of this type of sealant needs to have fast curing and good tissue adhesion properties, so that the tissue can be covalently bonded to provide strong sealing. However, at this stage, commercial hemostatic sealants cannot have all of the above characteristics at the same time, and the strong tissue adhesion of the sealant needs to be achieved by covalent bonding. At present, most sealants are attached to the tissue surface in the form of non-covalent bonding, and their tissue adhesion is weak, and they cannot seal the bleeding wound well. This is the technical limitation of hemostatic sealants at this stage.

[0003] Chinese Patent Publication No.: CN108525016A discloses a PEG hydrogel based on rapidly degradable chemical bonds and its preparation method and application. The hydrogel is formed by connecting polyethylene glycol derivative 1 (polyethylene glycol amino group) with polyethylene glycol derivative 2 (polyethylene glycol succinimide ester) and polyethylene glycol derivative 3 (polyethylene glycol aldehyde group) through chemical bonds 1 and chemical bonds 2 respectively; chemical bond 1 is a β-carbonylamide bond; chemical bond 2 is a Schiff base bond. Although the PEG hemostatic sealing hydrogel in this scheme has a fast gelation speed and can be quickly formed on the surface of an object; it can be quickly degraded in the body and on the surface of the body; it has an excellent hemostatic effect; it has good biocompatibility; and it can be quickly cleared, but this scheme is not suitable for scenes with incompressible massive bleeding, and is only applicable to drug sustained-release materials; tissue engineering scaffolds; medical sponges; organ hemostatic sealants; surface coatings for medical implants; epidermal hemostatic sealing coatings; coatings for burn treatment; and materials for preventing tissue adhesion. It is impossible to achieve the effect of being easy to carry and use and being able to quickly and efficiently stop bleeding while having biocompatibility. Summary of the invention

[0004] To this end, the present invention provides a hydrogel-based hemostatic sealant and a preparation method, which are used to overcome the problem that most sealants in the prior art are adhered to the tissue surface in the form of non-covalent connection, have weak tissue adhesion, cannot seal bleeding wounds well, and cannot achieve the effects of easy carrying and use, rapid and efficient hemostasis, etc. while having biocompatibility, and cannot be suitable for the timely control of incompressible massive bleeding.

[0005] To achieve the above object, in one aspect, the present invention provides a hydrogel-based hemostatic sealant, wherein the preparation materials of the hydrogel-based hemostatic sealant include:

[0006] 3-Methyl-3H-diaziridine-3-propionic acid, anhydrous ethanol, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, deionized water, chitosan acetic acid solution.

[0007] Furthermore, in the preparation materials of the hydrogel-based hemostatic sealant:

[0008] The molar ratio of 3-methyl-3H-diaziridine-3-propionic acid to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1;

[0009] The mass ratio of 3-methyl-3H-diaziridine-3-propionic acid to anhydrous ethanol is M1, and is set to 0.01≤M1≤0.4;

[0010] The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to deionized water is M2, and is set to 0.01≤M2≤0.3;

[0011] The chitosan mass concentration in the chitosan acetic acid solution is C1, and is set to 9 mg / mL≤C1≤20 mg / mL.

[0012] In another aspect, the present invention further provides a method for preparing a hydrogel-based hemostatic sealant, comprising:

[0013] Step S1, adding 3-methyl-3H-diaziridine-3-propionic acid to anhydrous ethanol according to the mass ratio M1 of 3-methyl-3H-diaziridine-3-propionic acid to anhydrous ethanol to obtain an ethanol solution of 3-methyl-3H-diaziridine-3-propionic acid;

[0014] Step S2, dissolving 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride in deionized water according to a mass ratio M2 of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride to deionized water to obtain a 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution;

[0015] Step S3, adding the ethanol solution of 3-methyl-3H-diaziridine-3-propionic acid to the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and stirring in the dark for a first preset time t1 to obtain an activated 3-methyl-3H-diaziridine-3-propionic acid solution;

[0016] Step S4, adding the activated 3-methyl-3H-diaziridine-3-propionic acid solution to the chitosan acetic acid solution for mixing, and adding 5% (w / v) sodium hydroxide solution to adjust the pH value of the mixed solution to 5.0 to obtain a mixed solution;

[0017] Step S5, heating the mixed solution in a light-proof water bath at a preset water bath temperature Ta, the duration of the light-proof water bath heating being a second preset duration t2, and stirring the mixed solution at a preset stirring speed Va during the light-proof water bath heating to obtain a solution after an amidation reaction;

[0018] Step S6, transferring the solution after the amidation reaction into a dialysis bag, dialyzing it in a first preset environment for a third preset time t3 to obtain a first dialysis solution, and dialyzing the first dialysis solution in a second preset environment for a fourth preset time t4 to obtain a second dialysis solution;

[0019] Step S7, freeze-drying the second dialysis solution under preset freeze-drying conditions to obtain sponge-like 3-methyl-3H-diaziridine-3-propionic acid-chitosan;

[0020] Step S8, dissolving the sponge-like 3-methyl-3H-diaziridine-3-propionic acid-chitosan in water to obtain a prepolymer solution;

[0021] Step S9, photocuring the prepolymer solution for a preset fifth time period t5 under preset irradiation conditions to obtain a hydrogel, and using the hydrogel as a hydrogel-based hemostatic sealant.

[0022] Furthermore, in the step S3, the first preset time t1 refers to the duration of light-shielded stirring, 25 mins≤t1≤35 mins.

[0023] Furthermore, in the step S4, the volume ratio of the activated 3-methyl-3H-diaziridine-3-propionic acid solution to the chitosan acetic acid solution is V1, and is set to 0.5≤V1≤1.5.

[0024] Further, in the step S5, the second preset time duration t2 refers to the duration of the light-proof water bath heating, which is set to 24h≤t2≤72h, the preset stirring speed Va refers to the speed at which the mixed solution is stirred during the light-proof water bath heating, which is set to 400rpm≤Va≤800rpm, and the light-proof water bath heating temperature is Ty, which is set to Ty=25°C.

[0025] Furthermore, in step S6, the first preset environment is set to an environment in which the ratio of anhydrous ethanol to deionized water is 1:1, and the anhydrous ethanol and deionized water in the first preset environment are replaced every 12 hours, and the third preset time t3 is set to 24 hours, and the second preset environment is set to a deionized water environment, and the deionized water in the second preset environment is replaced every 12 hours, and the fourth preset time t4 is set to 72 hours.

[0026] Furthermore, in step S7, the preset freeze-drying condition is set to freeze-drying for 48 hours under vacuum conditions.

[0027] Further, in the step S8, the concentration of the aqueous solution of 3-methyl-3H-diaziridine-3-propionic acid-chitosan in the prepolymer solution is C2, and is set to 30 mg / mL≤C2≤50 mg / mL.

[0028] Furthermore, in step S9, the preset irradiation condition is set to light with a wavelength of 365 nm, and the irradiation intensity Q of the light is set to 20 mW / cm 2 ≤Q≤100 mW / cm 2 ,The preset fifth time duration t5 is the irradiation time duration, which is set to 5s≤t5≤60s.

[0029] Compared with the prior art, the present invention has the beneficial effect that, in the method, 3-methyl-3H-diaziridine-3-propionic acid is dissolved in anhydrous ethanol in step S1 to achieve preliminary dissolution preparation of 3-methyl-3H-diaziridine-3-propionic acid, thereby improving the uniformity and efficiency of subsequent reactions; and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is dissolved in deionized water in step S2 to obtain a 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution, thereby ensuring that 1-ethyl-( The activation effect of 3-dimethylaminopropyl) carbodiimide hydrochloride in the subsequent reaction helps to promote the occurrence of the amidation reaction. In step S3, the dissolved 3-methyl-3H-diaziridine-3-propionic acid is added to the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution and stirred to obtain an activated 3-methyl-3H-diaziridine-3-propionic acid solution to prepare for the subsequent combination with chitosan. In step S4, the activated acid solution is mixed with the chitosan acetic acid solution, and then 5% (w / v) 2-nitropropionic acid is added. ) sodium hydroxide is used to adjust the pH value of the solution to 5.0 to make the reaction more complete and to facilitate the subsequent amidation reaction. Step S5 is performed under light-proof stirring under water bath heating conditions to perform an amidation reaction to generate a solution after the amidation reaction. Step S6 is performed through a dialysis step to remove low molecular weight substances and impurities that may remain in the reaction to obtain a purified product solution, thereby providing a high-purity material for subsequent processing. Step S7 is performed through a freeze-drying treatment to obtain a spongy 3-methyl-3H-diaziridine-3-propionic acid-chitosan, thereby effectively removing moisture and facilitating subsequent storage and use. Step S8 is performed to dissolve the spongy 3-methyl-3H-diaziridine-3-propionic acid-chitosan in water to form a prepolymer solution, thereby providing a basis for the final photocuring step. Step S9 is performed through a photocuring treatment to convert the prepolymer solution into a hydrogel-based hemostatic sealant, thereby enabling rapid hemostasis. The method uses a series of fine steps to achieve the preparation of an efficient and portable biocompatible hemostatic sealant, which is particularly suitable for emergency medical scenarios involving incompressible massive bleeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the process for preparing the hydrogel-based hemostatic sealant of this embodiment;

[0031] Figure 2 This is the H-NMR spectrum of the sponge-like 3-methyl-3H-diaziridine-3-propionic acid-chitosan of this embodiment;

[0032] Figure 3 The ultraviolet-visible light absorption spectrum of the sponge-like 3-methyl-3H-diaziridine-3-propionic acid-chitosan of this embodiment;

[0033] Figure 4This is a schematic diagram of the result of the prepolymer solution of this embodiment being converted into a hydrogel under ultraviolet irradiation;

[0034] Figure 5 This is a schematic diagram of the rheological test and gel time results of Example 1 of this embodiment;

[0035] Figure 6 This is a schematic diagram of the bursting pressure results of Example 1 of this embodiment;

[0036] Figure 7 This is a schematic diagram of the in vitro biocompatibility results of Example 1 of this embodiment;

[0037] Figure 8 This is a schematic diagram comparing the antibacterial properties of the hydrogel-based hemostatic sealants of Example 1 of this embodiment;

[0038] Fig. 9 This is a schematic diagram comparing the healing performance of the hydrogel-based hemostatic sealant of Example 1 of this embodiment;

[0039] Fig.10 This is a schematic diagram of the results of applying Example 1 of this embodiment in a rabbit heart puncture model. DETAILED DESCRIPTION

[0040] In order to make the objects and advantages of the present invention more clearly understood, the present invention is further described 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.

[0041] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the protection scope of the present invention.

[0042] It should be noted that, in the description of the present invention, terms such as "up", "down", "left", "right", "inside" and "outside" indicating directions or positional relationships are based on the directions or positional relationships shown in the drawings. This is merely for the convenience of description and does not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it cannot be understood as a limitation on the present invention.

[0043] A hydrogel-based hemostatic sealant, wherein the preparation materials of the hydrogel-based hemostatic sealant include: 3-methyl-3H-diaziridine-3-propionic acid, anhydrous ethanol, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, deionized water, and chitosan acetic acid solution.

[0044] Specifically, in the preparation materials of the hydrogel-based hemostatic sealant:

[0045] The molar ratio of 3-methyl-3H-diaziridine-3-propionic acid to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:1;

[0046] The mass ratio of 3-methyl-3H-diaziridine-3-propionic acid to anhydrous ethanol is M1, and is set to 0.01≤M1≤0.4;

[0047] The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to deionized water is M2, and is set to 0.01≤M2≤0.3;

[0048] The chitosan mass concentration in the chitosan acetic acid solution is C1, and is set to 9 mg / mL≤C1≤20 mg / mL.

[0049] See also Figure 1 As shown, it is a schematic diagram of the process of preparing the hydrogel-based hemostatic sealant of this embodiment, and the method comprises:

[0050] Step S1, adding 3-methyl-3H-diaziridine-3-propionic acid to anhydrous ethanol according to the mass ratio M1 of 3-methyl-3H-diaziridine-3-propionic acid to anhydrous ethanol to obtain an ethanol solution of 3-methyl-3H-diaziridine-3-propionic acid;

[0051] Step S2, dissolving 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride in deionized water according to a mass ratio M2 of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride to deionized water to obtain a 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution;

[0052] Step S3, adding the ethanol solution of 3-methyl-3H-diaziridine-3-propionic acid to the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and stirring in the dark for a first preset time t1 to obtain an activated 3-methyl-3H-diaziridine-3-propionic acid solution;

[0053] Step S4, adding the activated 3-methyl-3H-diaziridine-3-propionic acid solution to the chitosan acetic acid solution for mixing, and adding 5% (w / v) sodium hydroxide solution to adjust the pH value of the mixed solution to 5.0 to obtain a mixed solution;

[0054] Step S5, heating the mixed solution in a light-proof water bath at a preset water bath temperature Ta, the duration of the light-proof water bath heating being a second preset duration t2, and stirring the mixed solution at a preset stirring speed Va during the light-proof water bath heating to obtain a solution after an amidation reaction;

[0055] Step S6, transferring the solution after the amidation reaction into a dialysis bag, dialyzing it in a first preset environment for a third preset time t3 to obtain a first dialysis solution, and dialyzing the first dialysis solution in a second preset environment for a fourth preset time t4 to obtain a second dialysis solution;

[0056] Step S7, freeze-drying the second dialysis solution under preset freeze-drying conditions to obtain sponge-like 3-methyl-3H-diaziridine-3-propionic acid-chitosan;

[0057] Step S8, dissolving the sponge-like 3-methyl-3H-diaziridine-3-propionic acid-chitosan in water to obtain a prepolymer solution;

[0058] Step S9, photocuring the prepolymer solution for a preset fifth time period t5 under preset irradiation conditions to obtain a hydrogel, and using the hydrogel as a hydrogel-based hemostatic sealant.

[0059] Specifically, the method is applied to the preparation process of a hydrogel-based hemostatic sealant, which is suitable for scenes of incompressible massive bleeding. While having biocompatibility, it is easy to carry and use, and can quickly and efficiently stop bleeding. The method comprises step S1 in which 3-methyl-3H-diaziridine-3-propionic acid is dissolved in anhydrous ethanol to achieve preliminary dissolution preparation of 3-methyl-3H-diaziridine-3-propionic acid, thereby improving the uniformity and efficiency of subsequent reactions. Step S2 in which 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is dissolved in anhydrous ethanol to achieve initial dissolution preparation of 3-methyl-3H-diaziridine-3-propionic acid. The 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution is obtained by dissolving the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride in deionized water to ensure the activation of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride in the subsequent reaction, which helps to promote the occurrence of the amidation reaction. The dissolved 3-methyl-3H-diaziridine-3-propionic acid is added to the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution and stirred in step S3 to obtain an activated 3-methyl-3H-diaziridine-3-propionic acid solution, which is prepared for the subsequent combination with chitosan. The 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution is added to the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution and stirred in step S4 to obtain an activated 3-methyl-3H-diaziridine-3-propionic acid solution. The activated acid solution is mixed with the chitosan acetic acid solution, and then 5% (w / v) sodium hydroxide is added to adjust the pH of the solution to 5.0 to make the reaction more complete and facilitate the subsequent amidation reaction. Step S5 is performed under light-proof stirring under water bath heating conditions to generate an amidation reaction solution. Step S6 is performed through a dialysis step to remove low molecular weight substances and impurities that may remain in the reaction to obtain a purified product solution, providing a high-purity material for subsequent processing. Step S7 is performed through freeze drying to obtain a sponge-like 3-methyl-3H-dihydro-1-nitropropene. Aziridine-3-propionic acid-chitosan can effectively remove moisture and facilitate subsequent storage and use. Through step S8, the sponge-like 3-methyl-3H-diaziridine-3-propionic acid-chitosan is dissolved in water to form a prepolymer solution, which provides a basis for the final photocuring step. Through the photocuring treatment of step S9, the prepolymer solution is converted into a hydrogel-based hemostatic sealant, which can quickly stop bleeding. This method uses a series of fine steps to achieve the preparation of an efficient and portable biocompatible hemostatic sealant, which is particularly suitable for emergency medical scenarios for treating incompressible massive bleeding.

[0060] Specifically, in step S3, the first preset time t1 refers to the duration of light-shielded stirring, 25 mins≤t1≤35 mins.

[0061] It can be understood that the present embodiment does not limit the speed of the light-proof stirring. Those skilled in the art can freely set it according to the actual situation as long as the ethanol solution of the 3-methyl-3H-diaziridine-3-propionic acid and the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution are fully mixed. For example, the speed of the light-proof stirring can be set to 400 rpm.

[0062] Specifically, in the step S4, the volume ratio of the activated 3-methyl-3H-diaziridine-3-propionic acid solution to the chitosan acetic acid solution is V1, and is set to 0.5≤V1≤1.5.

[0063] Specifically, in step S5, the second preset time t2 refers to the duration of the light-proof water bath heating, which is set to 24h≤t2≤72h. The preset stirring speed Va refers to the speed at which the mixed solution is stirred during the light-proof water bath heating, which is set to 400rpm≤Va≤800rpm. The light-proof water bath heating temperature is Ty, which is set to Ty=25°C.

[0064] Specifically, in step S6, the first preset environment is set to an environment in which the ratio of anhydrous ethanol to deionized water is 1:1, and the anhydrous ethanol and deionized water in the first preset environment are replaced every 12 hours, and the third preset time t3 is set to 24 hours, and the second preset environment is set to a deionized water environment, and the deionized water in the second preset environment is replaced every 12 hours, and the fourth preset time t4 is set to 72 hours.

[0065] Specifically, in step S7, the preset freeze-drying condition is set to freeze-drying for 48 hours under vacuum conditions.

[0066] Specifically, in step S7, before freeze-drying, the second dialysis solution is first placed in a refrigerator at -20°C for 12 hours until it is completely frozen, and then the second dialysis solution is freeze-dried under vacuum conditions.

[0067] Specifically, in the step S8, the concentration of the aqueous solution of 3-methyl-3H-diaziridine-3-propionic acid-chitosan in the prepolymer solution is C2, and is set to 30 mg / mL≤C2≤50 mg / mL.

[0068] Specifically, in step S8, when the sponge-like 3-methyl-3H-diaziridine-3-propionic acid-chitosan is dissolved in water, the prepolymer solution refers to a solution in an unpolymerized state before being polymerized into a hydrogel, and the water refers to deionized water.

[0069] Specifically, in step S9, the preset irradiation condition is set to 365 nm UV light, and the irradiation intensity Q of the UV light is set to 20 mW / cm 2 ≤Q≤100mW / cm 2 ,The preset fifth time duration t5 is the irradiation time duration, which is set to 5s≤t5≤60s.

[0070] Specifically, the preparation method of the hydrogel-based hemostatic sealant in this embodiment is implemented as follows: Embodiment 1:

[0071] Step S1, adding 1.32 mL of 3-methyl-3H-diaziridine-3-propionic acid to 37.5 mL of anhydrous ethanol according to the mass ratio M1 of 3-methyl-3H-diaziridine-3-propionic acid to anhydrous ethanol to obtain an ethanol solution of 3-methyl-3H-diaziridine-3-propionic acid;

[0072] Step S2, dissolving 2.71 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride in 22.5 mL of deionized water according to the mass ratio M2 of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride to deionized water to obtain a 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride solution;

[0073] Step S3, adding the 37.5 mL ethanol solution of 3-methyl-3H-diaziridine-3-propionic acid to the 22.5 mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and stirring for 30 mins in a dark environment to obtain an activated 3-methyl-3H-diaziridine-3-propionic acid solution;

[0074] Step S4, adding the activated 3-methyl-3H-diaziridine-3-propionic acid solution to 50 mL of a chitosan acetic acid solution having a mass fraction of 1% (w / v) to mix, and adding a 5% (w / v) sodium hydroxide solution to adjust the pH value of the mixed solution to 5.0 to obtain a mixed solution;

[0075] Step S5, heating the mixed solution in a light-proof water bath at 25° C. for 48 hours, and stirring the mixed solution at a preset stirring speed of 400 rpm during the light-proof water bath heating to obtain a solution after amidation reaction;

[0076] Step S6, transferring the solution after the amidation reaction into a dialysis bag, dialyzing it in a first preset environment for 24 hours, replacing the anhydrous ethanol and deionized water in the first preset environment every 12 hours to obtain a first dialysis solution, dialyzing the first dialysis solution in a second preset environment for 72 hours, replacing the deionized water in the second preset environment every 12 hours to obtain a second dialysis solution;

[0077] Step S7, freeze-drying the second dialysis solution under preset freeze-drying conditions for 48 hours to obtain sponge-like 3-methyl-3H-diaziridine-3-propionic acid-chitosan;

[0078] Step S8, dissolving 50 mg of the sponge-like 3-methyl-3H-diaziridine-3-propionic acid-chitosan in 1 mL of water to obtain 1 mL of a prepolymer solution with a concentration of 50 mg / mL;

[0079] Step S9, at 30 mW / cm 2 The prepolymer solution is photocured under an irradiation condition of , and the prepolymer solution after photocuring for 9 seconds is used as a hydrogel-based hemostatic sealant.

[0080] Specifically, this embodiment 1 adopts a female New Zealand white rabbit heart bleeding model. During the operation, gauze or filter paper is used to collect blood until the bleeding stops. The time from the start of bleeding to the cessation of bleeding is recorded with a stopwatch, and it is used as the rabbit heart hemostasis time. The amount of bleeding collected after the bleeding stops is calculated by weighing, and it is used as the rabbit heart blood loss. Whether it can be gelled refers to whether the preparation result can achieve a gel effect. This embodiment uses a rheological test to determine whether it can be gelled. In the rheological test, the storage modulus of the solution is monitored under ultraviolet light to see whether it can be greater than the loss modulus. If the storage modulus is greater than the loss modulus, it means that gel can be formed, otherwise it means that gel cannot be formed. The gel time refers to the length of time it takes to form a gel. This embodiment starts timing from turning on the ultraviolet light. When the storage modulus of the prepolymer solution is equal to the loss modulus, the gel time is calculated. The timing ends when the measurement is taken, and the time obtained by timing is used as the gel time. The adhesion strength refers to the adhesion strength of the hydrogel-based hemostatic sealant. In this embodiment, two plastic sheets adhered with pig casings are adhered together with the hydrogel-based hemostatic sealant, and then a lap shear test is performed on a universal testing machine. When the sealant is disconnected from the pig casing, it is recorded as the lap shear stress at this time. The lap shear stress is divided by the adhesion area to obtain the adhesion strength of the hydrogel-based hemostatic sealant, which is used as the adhesion strength. The bursting pressure refers to the bursting pressure that the hydrogel-based hemostatic sealant can withstand. In this embodiment, when the hydrogel-based hemostatic sealant is used to block a 2 mm small hole on a pig heart tube, the corresponding pressure when the blockage is broken is the bursting pressure corresponding to the hydrogel-based hemostatic sealant, which is used as the bursting pressure.

[0081] Specifically, the preparation method of Examples 2-8 is the same as that of Example 1, except that the reaction conditions and components are different. The reaction conditions and components of Examples 1-8 are shown in Table 1, and the test results of Examples 1-8 are shown in Table 2.

[0082] Table 1:

[0083]

[0084] Table 2:

[0085]

[0086] Specifically, it can be concluded from the data in Table 1 and Table 2 that the rabbit heart hemostasis time and rabbit heart blood loss obtained in Example 1 using the hydrogel-based hemostatic sealant and preparation method of the present invention can meet the emergency medical scenario of dealing with incompressible massive bleeding, while the rabbit heart hemostasis time and rabbit heart blood loss obtained in Examples 2-8 without using the hydrogel-based hemostatic sealant and preparation method of the present invention cannot be used in emergency medical scenarios for dealing with incompressible massive bleeding.

[0087] See also Figure 2 As shown, it is the nuclear magnetic hydrogen spectrum of the sponge-like 3-methyl-3H-diaziridine-3-propionic acid-chitosan of this embodiment, wherein:

[0088] The methyl proton peak of the diazirine group appeared at a chemical shift of 0.90 ppm, and the proton peaks of the two methylene groups in 3-methyl-3H-diazirine-3-propionic acid appeared at 1.99-2.19 ppm and 1.56 ppm, respectively, indicating that the diazirine group was successfully grafted onto the molecular chain of chitosan.

[0089] See also Figure 3 As shown, it is the ultraviolet visible light absorption spectrum of the sponge-like 3-methyl-3H-diaziridine-3-propionic acid-chitosan of this embodiment, wherein:

[0090] The absorption peak of the diazirine group appeared at 347nm, which was related to the transition of the electrons of the diazirine group from the non-bonding n orbital to the anti-bonding π* orbital. Therefore, this also shows that the diazirine group was successfully introduced into the chitosan molecular chain after chemical modification.

[0091] See also Figure 4 As shown, it is a schematic diagram of the result of the prepolymer solution of this embodiment being converted into a hydrogel under ultraviolet irradiation, wherein:

[0092] The sponge-like 3-methyl-3H-diaziridine-3-propionic acid-chitosan was dissolved in deionized water to obtain a prepolymer solution (concentration of 50 mg / ml), which was then irradiated with 365nm UV light for 30 s to induce cross-linking of the carbon-nitrogen covalent bonds on the chitosan chain, ultimately obtaining a hydrogel-based hemostatic sealant that was rapidly photocured and photoactivated with strong tissue adhesion.

[0093] See also Figure 5 As shown, it is a schematic diagram of the rheological test and gel time results of Example 1 of this embodiment, wherein:

[0094] The prepared sponge-like 3-methyl-3H-diaziridine-3-propionic acid-chitosan was dissolved in deionized water to obtain a prepolymer solution with a concentration of 50 mg / ml. The prepolymer solution was applied to rheological tests to detect its gelation process and gelation time. The study found that after the prepolymer solution was irradiated with ultraviolet light, its storage modulus increased rapidly and exceeded its loss modulus after 9s, which indicates that the prepolymer solution can achieve the transition from solution to gel in a short time.

[0095] See also Figure 6 As shown, it is a schematic diagram of the bursting pressure results of Example 1 of this embodiment, wherein:

[0096] The hydrogel-based hemostatic sealant has the ability to withstand pressures up to 198 mmHg, which is significantly higher than normal human systolic blood pressure (90-140 mmHg) and the burst pressure of commercially available fibrin glue (65 mmHg).

[0097] See also Figure 7 As shown, it is a schematic diagram of the in vitro biocompatibility results of Example 1 of this embodiment, wherein:

[0098] The hydrogel-based hemostatic sealant obtained in Example 1 was tested for in vitro compatibility using NIH3T3 cells. 100 μL NIH3T3 (3×10 4 The cell suspension of 100 μg / mL (100 μg / mL) was added to a 96-well plate, and after culturing for 12 hours in a humid environment at 37°C, the hydrogel-based hemostatic sealant obtained in Example 1 was added to the wells containing the cells, and then the 96-well plate was further cultured in a humid environment at 37°C for 72 hours, and stained with a Live / Dead stain every 24 hours to observe the number and activity of living cells. It was found that the sealant had no obvious toxicity to the cells and the cells continued to proliferate within 3 days. The cell survival rate of the hemostatic sealant prepared by the prior art was greater than 90%, which was consistent with the cell survival rate of the hydrogel-based hemostatic sealant obtained in Example 1 prepared by the present invention, indicating that the hydrogel-based hemostatic sealant obtained in Example 1 had excellent cell compatibility.

[0099] See also Figure 8 As shown, it is a schematic diagram of the comparison of the antibacterial properties of the hydrogel-based hemostatic sealants of Example 1 of this embodiment, wherein:

[0100] The culture dish colony map on the right is the culture dish colony map of Example 1, and the culture dish colony map on the left is the culture dish colony map of the comparative example. When obtaining the culture dish colony map of Example 1, Example 1 and 5×10 3 / mL of Escherichia coli was co-cultured for 1 hour and then plated. When the culture plate colony map of the comparative example was obtained, 5×10 3 / mL of Escherichia coli was cultured for 1 hour and then plated. The number of colonies in the culture dish colony map of Example 1 was 21, and the number of colonies in the culture dish colony map of the comparative example was 542. Example 1 had an obvious antibacterial effect.

[0101] See also Fig. 9 As shown, it is a schematic diagram of the healing performance comparison of the hydrogel-based hemostatic sealant of Example 1 of this embodiment, wherein:

[0102] The one on the right is the healing result graph of Example 1, and the one on the left is the healing result graph of traditional gauze. When obtaining the healing result graph of Example 1, Example 1 was applied to mouse liver hemostasis, and H&E staining was performed on the mouse liver tissue one day after surgery. When obtaining the traditional gauze healing result graph, traditional gauze was applied to mouse liver hemostasis, and H&E staining was performed on the mouse liver tissue one day after surgery. Among them, the healing result graph of Example 1 shows that the mouse liver tissue was basically healed 1 day after surgery, while the traditional gauze healing result graph shows that the mouse liver tissue still has a large area of ​​unhealed gaps 1 day after surgery.

[0103] See also Fig.10 As shown, it is a schematic diagram of the results of the application of Example 1 of this embodiment in a rabbit heart puncture model, wherein:

[0104] After the rabbit was generally anesthetized, the left ventricle of the rabbit's heart was punctured with a syringe needle (1.2 mm in diameter), and 0.5 mL of the precursor solution was immediately used to seal the cardiac puncture hole. In the cardiac puncture bleeding model, fibrin glue could not stop the bleeding, while the hydrogel-based hemostatic sealant obtained in Example 1 could stop the jet-like bleeding in 42 seconds, with a blood loss of 455 mg.

[0105] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

Claims

1. A hydrogel-based hemostatic sealant, characterized in that: The preparation method of the hydrogel-based hemostatic sealant comprises: Step S1, adding 1.32 mL of 3-methyl-3H-diaziridine-3-propionic acid to 37.5 mL of anhydrous ethanol to obtain an ethanol solution of 3-methyl-3H-diaziridine-3-propionic acid; Step S2, dissolving 2.71 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in 22.5 mL of deionized water to obtain a 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution; Step S3, adding the 37.5 mL ethanol solution of 3-methyl-3H-diaziridine-3-propionic acid to the 22.5 mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and stirring for 30 mins in a dark environment to obtain an activated 3-methyl-3H-diaziridine-3-propionic acid solution; Step S4, adding the activated 3-methyl-3H-diaziridine-3-propionic acid solution to 50 mL of a chitosan acetic acid solution having a mass volume fraction of 1% (w / v) to mix, and adding a 5% (w / v) sodium hydroxide solution to adjust the pH value of the mixed solution to 5.0 to obtain a mixed solution; Step S5, heating the mixed solution in a light-proof water bath at 25° C. for 48 hours, and stirring the mixed solution at a preset stirring speed of 400 rpm during the light-proof water bath heating to obtain a solution after amidation reaction; Step S6, transferring the solution after the amidation reaction into a dialysis bag, dialyzing it in a first preset environment for 24 hours, replacing the anhydrous ethanol and deionized water in the first preset environment every 12 hours to obtain a first dialysis solution, dialyzing the first dialysis solution in a second preset environment for 72 hours, replacing the deionized water in the second preset environment every 12 hours to obtain a second dialysis solution; Step S7, freeze-drying the second dialysis solution under preset freeze-drying conditions for 48 hours to obtain sponge-like 3-methyl-3H-diaziridine-3-propionic acid-chitosan; Step S8, dissolving 50 mg of the sponge-like 3-methyl-3H-diaziridine-3-propionic acid-chitosan in 1 mL of water to obtain 1 mL of a prepolymer solution with a concentration of 50 mg / mL; Step S9, at 30 mW / cm 2 The prepolymer solution is photocured under an irradiation condition of , and the prepolymer solution after photocuring for 9 seconds is used as a hydrogel-based hemostatic sealant.

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