CN118304462B

CN118304462B - Hemostatic material and preparation method thereof

Info

Publication number: CN118304462B
Application number: CN202410741895.8A
Authority: CN
Inventors: 白开元; 杨泽羊; 李雅欣; 翟思远
Original assignee: Anhui Haoyuan Pharmaceutical Co ltd
Current assignee: Anhui Haoyuan Pharmaceutical Co ltd
Priority date: 2024-06-11
Filing date: 2024-06-11
Publication date: 2024-08-16
Anticipated expiration: 2044-06-11
Also published as: CN118304462A

Abstract

The invention belongs to the technical field of medical materials, and particularly relates to a hemostatic material and a preparation method thereof. A hemostatic material mainly comprises a composite nanofiber core layer and a hydrogel outer layer; according to the invention, the natural extract is added into the nanofiber to prepare the core layer, the composite nanofiber core layer is wrapped by the hydrogel, the composite nanofiber and the hydrogel precursor are crosslinked in situ to form a composite structure to play a role in cooperation, firm interface connection is formed between the composite nanofiber and the hydrogel precursor, the strength and toughness of the whole material are improved, meanwhile, the hydrogel itself absorbs heat to bring cool feel to the skin when water is evaporated, the temperature of the skin is effectively reduced, and the hemostatic effect of the subsequent nanofiber core layer is promoted.

Description

Hemostatic material and preparation method thereof

Technical Field

The invention belongs to the technical field of medical materials, and particularly relates to a hemostatic material and a preparation method thereof.

Background

Bleeding from skin wounds is a natural response of the body when it is injured from the outside. The severity of bleeding depends on the depth, size and location of the wound. For minor skin wound bleeding, the body is usually able to self-stanch. However, in severe cases, bleeding can be very rapid and severe, resulting in significant loss of blood; when a large amount of hemorrhagic damage occurs, the inherent hemostatic mechanism of the body is insufficient to stop bleeding in time, so that some techniques are required to achieve rapid hemostasis. Traditional hemostatic methods, such as bandages, tourniquets, gauze, pressure hemostasis, rely primarily on physical occlusion and self-clotting systems to aid hemostasis and healing, but may be faced with additional problems and infection risks. Meanwhile, although surgical hemostasis measures can temporarily bleed tissue, adjuvant therapy still requires a hemostasis accessory. The anti-fibrinolytic drug can inhibit the disintegration of blood clots and improve the sealing effect of wounds; however, a decrease in the activity of the fibrinolytic system in vivo may promote thrombosis and bring about therapeutic side effects. Polysaccharides in commonly used hemostatic materials have the ability to promote hemostasis, accelerate wound healing, and antibacterial properties. In contrast, lack of bond strength and mechanical properties limits the use of polysaccharides in the preparation of hydrogel adhesives. Because of the high mechanical strength of protein biomaterials, protein-based hydrogels have been widely developed as effective materials for hemostasis and tissue healing. However, these natural materials have many problems such as degradability and mechanical strength. Based on the above-mentioned problems, it is extremely necessary to develop a biological hemostatic material which is highly biodegradable and has high mechanical strength.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a hemostatic material and a preparation method thereof.

The technical effects of the invention are realized by the following technical scheme: a hemostatic material mainly comprises a composite nanofiber core layer and a hydrogel outer layer, wherein a natural extract is added into nanofibers to prepare the core layer, then the nanofiber core layer is wrapped by the hydrogel, the nanofibers and a hydrogel precursor are crosslinked in situ to form a composite structure to cooperatively play a role, the nanofibers and the hydrogel precursor form firm interface connection, the strength and toughness of the integral material are improved, meanwhile, the hydrogel itself absorbs heat to bring cool feel to the skin when water evaporates, the temperature of the skin is effectively reduced, and the hemostatic effect of the subsequent nanofiber core layer is promoted.

The invention also provides a preparation method of the hemostatic material, which specifically comprises the following steps:

S1: preparing composite nano fibers;

s2: dissolving chitosan in 1wt% acetic acid solution to obtain 2wt% chitosan solution; dissolving gamma-polyglutamic acid in a phosphate buffer, heating in a water bath to control the temperature to be 32-35 ℃, and filtering with a 0.22 mu m filter membrane to obtain 2wt% gamma-polyglutamic acid solution; dissolving diphenylacetyl disulfide in a dimethyl sulfoxide solution, and uniformly dissolving to obtain a diphenylacetyl disulfide solution with the weight percent of 2 percent; dissolving N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide in a dimethyl sulfoxide solution with the mass volume of 25-30 times, and uniformly dissolving in an ice bath to obtain an activated solution;

S3: continuously dripping the activated solution prepared in the step S2 into the chitosan solution prepared in the step S2 in a stirring process, stirring for 4-6 hours in an ice bath at the temperature of-10-0 ℃, and performing ultrafiltration dialysis on the solution at the temperature of 3-10 kDa to obtain an aminoacylated chitosan solution; mixing the aminoacylated chitosan solution with the gamma-polyglutamic acid solution prepared in the step S2, keeping the temperature of the ice bath at 0-4 ℃, and stirring at 300-500 rpm for 15-25 min to obtain a mixed solution;

S4: suspending the composite nanofiber prepared in the step S1 into deionized water to obtain a composite nanofiber suspension with the concentration of 1wt%, adding the composite nanofiber suspension into the mixed solution prepared in the step S3 with the volume of 1-3 times, and uniformly stirring and mixing at 100rpm under the ice bath control temperature of 4 ℃ to obtain a mixed system;

S5: and (3) adding the diphenylacetyl disulfide solution prepared in the step (S2) into the mixed system prepared in the step (S4), maintaining the temperature in an ice bath at 4 ℃, stirring at 300-500 rpm for 2-4 h, washing with deionized water, and performing vacuum freeze drying at-20 ℃ for 8-12 h to obtain the hemostatic material.

Preferably, the preparation steps of the composite nanofiber in step S1 are as follows:

a1: treating the polylactic acid nanofiber in an argon atmosphere with power of 100-300W for 1-5 min to obtain an activated polylactic acid nanofiber; dissolving sulfate and maleic anhydride in dimethyl sulfoxide solution, and uniformly mixing to obtain an initiation solution; adding activated polylactic acid nanofiber into an initiating solution, heating in a water bath to control the temperature to be 30-60 ℃ for 2-4 h, washing with deionized water, and drying at 60 ℃ for 24h to obtain modified polylactic acid nanofiber;

A2: adding the modified polylactic acid nanofiber prepared in the step A1 into dimethyl sulfoxide solution, and dissolving and mixing uniformly to obtain A1 wt% modified polylactic acid nanofiber solution; dissolving methoxy polyethylene glycol in dimethyl sulfoxide solution, and uniformly mixing to obtain 30-50 mg/mL methoxy polyethylene glycol solution; adding the modified polylactic acid nanofiber solution into EDC/NHS activation solution, maintaining the pH value to be 4.5-6.5, and stirring at the ice bath temperature of 4 ℃ for 1-2 hours to obtain activation nanofiber solution;

A3: continuously adding the activated nanofiber solution prepared in the step A2 into the methoxy polyethylene glycol solution prepared in the step A2 with the volume of 3-5 times in the stirring process, controlling the temperature to be 25-37 ℃, stirring at 100rpm for 3-12 hours, centrifuging, alternately washing with deionized water and PBS buffer solution for two times, and performing vacuum freeze drying at-20 ℃ for 12-18 hours to obtain the grafted modified polylactic acid nanofiber;

a4: adding the composite natural extract into 12wt% ethanol solution to obtain composite natural extract solution with concentration of 2 wt%; and (3) adding the grafted modified polylactic acid nanofiber prepared in the step (A3) into a composite natural extract solution, performing ultrasonic treatment at 30W for 5min, controlling the temperature at 4 ℃ in an ice bath, standing for 8-12 h, centrifuging, filtering, washing with deionized water, and performing vacuum freeze-drying at-30 ℃ for 8-12 h to obtain the composite nanofiber.

Preferably, the polylactic acid nanofiber in the step A1 is prepared and obtained by a conventional technical means electrostatic spinning method; the sulfate is any one of sodium sulfate, potassium sulfate and ferric sulfate; the dosage ratio of the sulfate to the maleic anhydride to the dimethyl sulfoxide solution is 10 mg:15-20 mg:1mL; the ratio of the dosage of the activated polylactic acid nanofiber to the dosage of the initiating solution is 0.5-1 g/10 mL.

Preferably, the EDC/NHS activation solution in step A2 is a solution having a concentration of 5-10 mg/mL prepared from 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in a molar ratio of 1:1 in PBS buffer; the volume ratio of the modified polylactic acid nanofiber solution to the EDC/NHS activation solution is 1:3-5.

Preferably, the compound natural extract in the step A4 is prepared from Echinacea purpurea, garlic, radix Scutellariae and mugwort in a mass ratio of 1:0.8:1.2:1 by conventional technical means; the mass-to-mass ratio of the grafted modified polylactic acid nanofiber to the composite natural extract is 1:0.2-0.5.

Preferably, the molar mass ratio of N-hydroxysuccinimide to 1-ethyl- (3-dimethylaminopropyl) carbodiimide in step S2 is 1:1.

Preferably, the volume usage ratio of the activating solution and the chitosan solution in the step S3 is 1-1.5:1; the volume dosage ratio of the aminoacylated chitosan solution to the gamma-polyglutamic acid solution is 1:1.

Preferably, the volume and the dosage ratio of the diphenylacetyl disulfide solution to the mixed system in the step S5 are 1:1-2.

The beneficial effects of the invention are as follows:

The three-dimensional network structure formed by cross-linking the aminoacylated chitosan and the gamma-polyglutamic acid through disulfide ensures that the hydrogel has remarkable mechanical strength and flexibility, and the flexibility ensures that the hydrogel can be closely attached to irregular wound surfaces, thereby improving the hemostatic effect. The chitosan and the gamma-polyglutamic acid are used as natural biological materials, have good biocompatibility, so that the hydrogel can be well combined with in-vivo tissues, and rejection reaction and inflammatory reaction are reduced. Under the condition of high Reactive Oxygen Species (ROS), disulfide-crosslinked hydrogel is degraded, hemostatic drug is released, and degradation products are harmless to human body. Both chitosan and gamma-polyglutamic acid can promote cell proliferation and tissue regeneration, and accelerate the wound healing process. The chitosan and gamma-polyglutamic acid monomer released after the hydrogel is degraded can further promote cell growth and tissue repair; in addition, the hydrogel has good hygroscopicity, can absorb wound exudates, keep the wound dry, reduce the chance of bacterial infection, and has the moisture retention property which is also beneficial to maintaining the moist environment of the wound, promoting cell migration and regeneration and accelerating the wound healing speed. The amide groups introduced in the aminoacylation process increase the active center of chitosan molecules, so that when the chitosan molecules are combined with gamma-polyglutamic acid through disulfide bonds, the crosslinking reaction is more efficient and uniform, and the mechanical strength and elasticity of the hydrogel are improved due to the increased crosslinking density; the aminoacylated chitosan shows better biocompatibility and solubility in vivo, so that the material is easier to be absorbed and utilized by cells under physiological conditions, thereby reducing immune and inflammatory reactions.

The special structure of the surface of the grafted modified polylactic acid nanofiber and the introduced methoxy polyethylene glycol enhance the hydrophilicity and biocompatibility of the grafted modified polylactic acid nanofiber, are favorable for quickly absorbing blood and promoting the aggregation of platelets on the surface of the fiber, accelerate the process of converting prothrombin into thrombin, further promote the start of a coagulation mechanism and effectively control bleeding. The hydrogel layer constructed by chitosan and gamma-polyglutamic acid can tightly fill irregular space of wound surface by virtue of good fluidity and deformability, so as to form physical barrier and effectively prevent blood loss; the complementation on the structure ensures that the composite material can be more closely attached to the wound surface, and a firm hemostatic barrier is formed at the early stage of wound bleeding. The biological activity of the nanofiber surface is improved through argon treatment and grafting modification, so that cell adhesion and proliferation are facilitated, and foreign body rejection is reduced. The high specific surface area and the three-dimensional network structure of the composite nanofiber provide rich loading sites for the composite natural extract, and the loaded extract can be effectively and slowly released in the wound healing process; the methoxy polyethylene glycol in the grafting modification process not only improves the hydrophilicity of the nanofiber, but also enhances the flexibility of the material, so that the composite nanofiber can still maintain good structural stability and fit degree in a wound environment.

Drawings

In order to more clearly illustrate the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only of the invention and that other drawings can be obtained from them without inventive effort for a person skilled in the art.

FIG. 1 is a toxicity test chart of hemostatic materials prepared in examples 2 and 3 and comparative examples 1 to 3 of the present invention;

FIG. 2 is an SEM image of the composite nanofiber prepared in example 3 of the present invention;

FIG. 3 is an FTIR infrared spectrum of a hemostatic material prepared in example 3 of the present invention;

FIG. 4 is an SEM image of a hemostatic material prepared according to example 3 of the present invention;

FIG. 5 is a chart of ROS dissolution test of hemostatic material made in example 3 of the present invention;

FIG. 6 is a graph showing the biodegradation rate of the hemostatic materials prepared in examples 2 and 3 and comparative examples 1 to 3 according to the present invention;

FIG. 7 is a graph showing the antibacterial property test of the hemostatic materials prepared in examples 1 to 4 and comparative examples 1 to 3 of the present invention.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments; all other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Unless otherwise specified, the materials involved in the present invention are purchased from conventional commercial sources.

Example 1: a hemostatic material mainly comprises a composite nanofiber core layer and a hydrogel outer layer.

The preparation method of the hemostatic material specifically comprises the following steps:

S1: preparing composite nano fibers;

A1: treating the polylactic acid nanofiber in an argon atmosphere with 100W power for 5min to obtain an activated polylactic acid nanofiber; 200mg of potassium sulfate and 300mg of maleic anhydride are dissolved in 20mL of dimethyl sulfoxide solution, and the solution is uniformly dissolved and mixed to obtain an initiation solution; adding 1g of activated polylactic acid nanofiber into 20mL of initiating solution, heating in a water bath, controlling the temperature to be 30 ℃, washing with deionized water for 2 hours, and drying at 60 ℃ for 24 hours to obtain modified polylactic acid nanofiber;

a2: adding the modified polylactic acid nanofiber prepared in the step A1 into dimethyl sulfoxide solution, and dissolving and mixing uniformly to obtain A1 wt% modified polylactic acid nanofiber solution; dissolving methoxy polyethylene glycol in dimethyl sulfoxide solution, and uniformly mixing to obtain 30mg/mL methoxy polyethylene glycol solution; adding 100mL of modified polylactic acid nanofiber solution into 500mL of EDC/NHS (ethylene-propylene-diene monomer/ethylene-diene monomer) activating solution with concentration of 5mg/mL, maintaining pH to be 4.5, and stirring at the temperature of 4 ℃ in an ice bath for 1h to obtain an activating nanofiber solution;

a3: continuously adding the activated nanofiber solution prepared in the step A2 into 1800mL of methoxy polyethylene glycol solution prepared in the step A2 in the stirring process, controlling the temperature to be 25 ℃, stirring at 100rpm for 12h, centrifuging, alternately washing with deionized water and PBS buffer for two times, and performing vacuum freeze drying at-20 ℃ for 12h to obtain grafted modified polylactic acid nanofiber;

A4: adding the composite natural extract into 12wt% ethanol solution to obtain composite natural extract solution with concentration of 2 wt%; adding the grafted modified polylactic acid nanofiber prepared in the step A3 into 10mL of composite natural extract solution, performing ultrasonic treatment at 30W for 5min, controlling the temperature at 4 ℃ in an ice bath, standing for 8h, centrifuging, filtering, washing with deionized water, and performing vacuum freeze drying at-30 ℃ for 8h to obtain composite nanofiber;

S2: dissolving chitosan in 1wt% acetic acid solution to obtain 2wt% chitosan solution; dissolving gamma-polyglutamic acid in a phosphate buffer, heating in a water bath to control the temperature to 32 ℃, and filtering with a 0.22 mu m filter membrane to obtain 2wt% gamma-polyglutamic acid solution; dissolving diphenylacetyl disulfide in a dimethyl sulfoxide solution, and uniformly dissolving to obtain a diphenylacetyl disulfide solution with the weight percent of 2 percent; 2.3g of N-hydroxysuccinimide and 3.1g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide were dissolved in 80mL of dimethyl sulfoxide solution, and dissolved uniformly in an ice bath to obtain an activated solution;

s3: continuously dripping 80mL of the activated solution prepared in the step S2 into 80mL of the chitosan solution prepared in the step S2 in a stirring process, stirring for 6h in an ice bath at 0 ℃, and performing 3kDa ultrafiltration dialysis to obtain an aminoacylated chitosan solution; mixing 160mL of the aminoacylated chitosan solution with 160mL of the gamma-polyglutamic acid solution prepared in the step S2, maintaining the temperature at 0 ℃ in an ice bath, and stirring at 300rpm for 25min to obtain a mixed solution;

S4: suspending the composite nanofiber prepared in the step S1 into deionized water to obtain a composite nanofiber suspension with the concentration of 1wt%, adding 100mL of the composite nanofiber suspension into 100mL of the mixed solution prepared in the step S3, controlling the temperature of the ice bath to be 4 ℃, and stirring and mixing uniformly at 100rpm to obtain a mixed system;

S5: and (2) adding 200mL of the diphenylacetyl disulfide solution prepared in the step (S2) into the 200mL of the mixed system prepared in the step (S4), maintaining the temperature at 4 ℃ in an ice bath, stirring at 300rpm for 4 hours, washing with deionized water, and performing vacuum freeze drying at-20 ℃ for 8 hours to obtain the hemostatic material.

Example 2: a hemostatic material mainly comprises a composite nanofiber core layer and a hydrogel outer layer.

S1: preparing composite nano fibers;

A1: treating the polylactic acid nanofiber in an argon atmosphere at 200W power for 3min to obtain an activated polylactic acid nanofiber; 200mg of sodium sulfate and 360mg of maleic anhydride are dissolved in 20mL of dimethyl sulfoxide solution, and the solution is dissolved and mixed uniformly to obtain an initiation solution; adding 1.8g of activated polylactic acid nanofiber into 20mL of initiating solution, heating in a water bath, controlling the temperature to be 50 ℃, washing with deionized water for 3 hours, and drying at 60 ℃ for 24 hours to obtain modified polylactic acid nanofiber;

a2: adding the modified polylactic acid nanofiber prepared in the step A1 into dimethyl sulfoxide solution, and dissolving and mixing uniformly to obtain A1 wt% modified polylactic acid nanofiber solution; dissolving methoxy polyethylene glycol in dimethyl sulfoxide solution, and uniformly mixing to obtain 40mg/mL methoxy polyethylene glycol solution; adding 100mL of modified polylactic acid nanofiber solution into 300mL of 8mg/mL EDC/NHS activation solution, maintaining pH to 5.5, and stirring at 4 ℃ in an ice bath for 1.5 hours to obtain an activation nanofiber solution;

A3: continuously adding the activated nanofiber solution prepared in the step A2 into 1600mL of methoxy polyethylene glycol solution prepared in the step A2 in the stirring process, controlling the temperature to be 32 ℃, stirring at 100rpm for 10h, centrifuging, alternately washing with deionized water and PBS buffer solution for two times, and performing vacuum freeze drying at-20 ℃ for 16h to obtain grafted modified polylactic acid nanofiber;

a4: adding the composite natural extract into 12wt% ethanol solution to obtain composite natural extract solution with concentration of 2 wt%; adding the grafted modified polylactic acid nanofiber prepared in the step A3 into 20mL of composite natural extract solution, performing ultrasonic treatment at 30W for 5min, controlling the temperature at 4 ℃ in an ice bath, standing for 10h, centrifuging, filtering, washing with deionized water, and performing vacuum freeze drying at-30 ℃ for 10h to obtain composite nanofiber;

S2: dissolving chitosan in 1wt% acetic acid solution to obtain 2wt% chitosan solution; dissolving gamma-polyglutamic acid in a phosphate buffer, heating in a water bath to control the temperature to 34 ℃, and filtering with a 0.22 mu m filter membrane to obtain 2wt% gamma-polyglutamic acid solution; dissolving diphenylacetyl disulfide in a dimethyl sulfoxide solution, and uniformly dissolving to obtain a diphenylacetyl disulfide solution with the weight percent of 2 percent; 2.3g of N-hydroxysuccinimide and 3.1g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide were dissolved in 162mL of dimethyl sulfoxide solution, and dissolved uniformly in an ice bath to obtain an activated solution;

s3: continuously dripping 100mL of the activated solution prepared in the step S2 into 80mL of the chitosan solution prepared in the step S2 in a stirring process, stirring for 5h in an ice bath at the temperature of minus 8 ℃, and performing ultrafiltration dialysis on 8kDa to obtain an aminoacylated chitosan solution; 180mL of the aminoacylated chitosan solution and 180mL of the gamma-polyglutamic acid solution prepared in the step S2 are mixed, the ice bath is kept at the temperature of 2 ℃, and stirring is carried out for 20min at 450rpm, so as to obtain a mixed solution;

S4: suspending the composite nanofiber prepared in the step S1 into deionized water to obtain a composite nanofiber suspension with the concentration of 1wt%, adding 100mL of the composite nanofiber suspension into 300mL of the mixed solution prepared in the step S3, controlling the temperature of the ice bath to be 4 ℃, and stirring and mixing uniformly at 100rpm to obtain a mixed system;

S5: 200mL of the diphenylacetyl disulfide solution prepared in the step S2 is added into the 350mL of the mixed system prepared in the step S4, the ice bath is kept at the temperature of 4 ℃, stirring is carried out at 450rpm for 3h, deionized water is used for washing, and vacuum freeze drying is carried out at the temperature of-20 ℃ for 10h, so that the hemostatic material is obtained.

Example 3: a hemostatic material mainly comprises a composite nanofiber core layer and a hydrogel outer layer.

S1: preparing composite nano fibers;

A1: treating the polylactic acid nanofiber in an argon atmosphere at 240W power for 2min to obtain an activated polylactic acid nanofiber; 200mg of ferric sulfate and 350mg of maleic anhydride are dissolved in 20mL of dimethyl sulfoxide solution, and the solution is uniformly dissolved and mixed to obtain an initiation solution; adding 1.5g of activated polylactic acid nanofiber into 20mL of initiating solution, heating in a water bath, controlling the temperature to 40 ℃, washing with deionized water for 3 hours, and drying at 60 ℃ for 24 hours to obtain modified polylactic acid nanofiber;

A2: adding the modified polylactic acid nanofiber prepared in the step A1 into dimethyl sulfoxide solution, and dissolving and mixing uniformly to obtain A1 wt% modified polylactic acid nanofiber solution; dissolving methoxy polyethylene glycol in dimethyl sulfoxide solution, and uniformly mixing to obtain 40mg/mL methoxy polyethylene glycol solution; adding 100mL of modified polylactic acid nanofiber solution into 400mL of 6mg/mL EDC/NHS activated solution, maintaining pH6, and stirring at 4 ℃ in an ice bath for 1.5h to obtain activated nanofiber solution;

A3: continuously adding the activated nanofiber solution prepared in the step A2 into 1500mL of methoxy polyethylene glycol solution prepared in the step A2 in the stirring process, controlling the temperature to be 35 ℃, stirring at 100rpm for 8h, centrifuging, alternately washing with deionized water and PBS buffer for two times, and performing vacuum freeze drying at-20 ℃ for 15h to obtain grafted modified polylactic acid nanofiber;

A4: adding the composite natural extract into 12wt% ethanol solution to obtain composite natural extract solution with concentration of 2 wt%; adding the grafted modified polylactic acid nanofiber prepared in the step A3 into 15mL of composite natural extract solution, performing ultrasonic treatment at 30W for 5min, controlling the temperature at 4 ℃ in an ice bath, standing for 10h, centrifuging, filtering, washing with deionized water, and performing vacuum freeze drying at-30 ℃ for 10h to obtain composite nanofiber;

S2: dissolving chitosan in 1wt% acetic acid solution to obtain 2wt% chitosan solution; dissolving gamma-polyglutamic acid in a phosphate buffer, heating in a water bath to control the temperature to 33 ℃, and filtering with a 0.22 mu m filter membrane to obtain 2wt% gamma-polyglutamic acid solution; dissolving diphenylacetyl disulfide in a dimethyl sulfoxide solution, and uniformly dissolving to obtain a diphenylacetyl disulfide solution with the weight percent of 2 percent; 2.3g of N-hydroxysuccinimide and 3.1g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide were dissolved in 150mL of dimethyl sulfoxide solution, and dissolved uniformly in an ice bath to obtain an activated solution;

S3: continuously dripping 120mL of the activated solution prepared in the step S2 into 100mL of the chitosan solution prepared in the step S2 in a stirring process, stirring for 5h in an ice bath at the temperature of minus 5 ℃, and performing 5kDa ultrafiltration dialysis to obtain an aminoacylated chitosan solution; mixing 220mL of the aminoacylated chitosan solution with 220mL of the gamma-polyglutamic acid solution prepared in the step S2, maintaining the temperature at 2 ℃ in an ice bath, and stirring at 400rpm for 20min to obtain a mixed solution;

S4: suspending the composite nanofiber prepared in the step S1 into deionized water to obtain a composite nanofiber suspension with the concentration of 1wt%, adding 100mL of the composite nanofiber suspension into 200mL of the mixed solution prepared in the step S3, controlling the temperature of the ice bath to be 4 ℃, and stirring and mixing uniformly at 100rpm to obtain a mixed system;

s5: 200mL of the diphenylacetyl disulfide solution prepared in the step S2 is added into the 300mL of the mixed system prepared in the step S4, the ice bath is kept at the temperature of 4 ℃, stirring is carried out at 400rpm for 3h, deionized water is used for washing, and vacuum freeze drying is carried out at the temperature of-20 ℃ for 10h, so that the hemostatic material is obtained.

Example 4: a hemostatic material mainly comprises a composite nanofiber core layer and a hydrogel outer layer.

S1: preparing composite nano fibers;

A1: treating the polylactic acid nanofiber in an argon atmosphere at 300W power for 1min to obtain an activated polylactic acid nanofiber; 200mg of potassium sulfate and 400mg of maleic anhydride are dissolved in 200mL of dimethyl sulfoxide solution, and the solution is dissolved and mixed uniformly to obtain an initiation solution; adding 2g of activated polylactic acid nanofiber into 20mL of initiating solution, heating in a water bath, controlling the temperature to be 60 ℃, washing with deionized water for 2 hours, and drying at 60 ℃ for 24 hours to obtain modified polylactic acid nanofiber;

A2: adding the modified polylactic acid nanofiber prepared in the step A1 into dimethyl sulfoxide solution, and dissolving and mixing uniformly to obtain A1 wt% modified polylactic acid nanofiber solution; dissolving methoxy polyethylene glycol in dimethyl sulfoxide solution, and uniformly mixing to obtain 50mg/mL methoxy polyethylene glycol solution; adding 100mL of modified polylactic acid nanofiber solution into 300mL of 10mg/mL EDC/NHS (ethylene-propylene-diene monomer/N-vinyl acetate) activated solution, maintaining pH at 6.5, and stirring at 4 ℃ in an ice bath for 2 hours to obtain an activated nanofiber solution;

A3: continuously adding the activated nanofiber solution prepared in the step A2 into 2000mL of methoxy polyethylene glycol solution prepared in the step A2 in the stirring process, controlling the temperature to 37 ℃, stirring at 100rpm for 12h, centrifuging, alternately washing with deionized water and PBS buffer for two times, and performing vacuum freeze drying at-20 ℃ for 18h to obtain grafted modified polylactic acid nanofiber;

A4: adding the composite natural extract into 12wt% ethanol solution to obtain composite natural extract solution with concentration of 2 wt%; adding the grafted modified polylactic acid nanofiber prepared in the step A3 into 25mL of composite natural extract solution, performing ultrasonic treatment at 30W for 5min, controlling the temperature at 4 ℃ in an ice bath, standing for 12h, centrifuging, filtering, washing with deionized water, and performing vacuum freeze drying at-30 ℃ for 12h to obtain composite nanofiber;

S2: dissolving chitosan in 1wt% acetic acid solution to obtain 2wt% chitosan solution; dissolving gamma-polyglutamic acid in a phosphate buffer, heating in a water bath to control the temperature to 35 ℃, and filtering with a 0.22 mu m filter membrane to obtain 2wt% gamma-polyglutamic acid solution; dissolving diphenylacetyl disulfide in a dimethyl sulfoxide solution, and uniformly dissolving to obtain a diphenylacetyl disulfide solution with the weight percent of 2 percent; 2.3g of N-hydroxysuccinimide and 3.1g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide were dissolved in 162mL of dimethyl sulfoxide solution, and dissolved uniformly in an ice bath to obtain an activated solution;

S3: continuously dripping 150mL of the activated solution prepared in the step S2 into 100mL of the chitosan solution prepared in the step S2 in a stirring process, stirring for 4h in an ice bath at the temperature of minus 10 ℃, and performing ultrafiltration dialysis on 10kDa to obtain an aminoacylated chitosan solution; mixing 200mL of the aminoacylated chitosan solution with 200mL of the gamma-polyglutamic acid solution prepared in the step S2, maintaining the temperature at 0 ℃ in an ice bath, and stirring at 500rpm for 15min to obtain a mixed solution;

s5: 200mL of the diphenylacetyl disulfide solution prepared in the step S2 is added into the 400mL of the mixed system prepared in the step S4, the ice bath is kept at the temperature of 4 ℃, the stirring is carried out at 500rpm for 2h, the deionized water is used for washing, and the vacuum freeze drying is carried out at the temperature of-20 ℃ for 12h, so that the hemostatic material is obtained.

Comparative example 1: comparative example 1 the procedure is essentially the same as example 2 except that comparative example 1 uses glutaraldehyde instead of diphenylacetyl disulfide as a cross-linking agent to prepare a hemostatic material.

Comparative example 2: comparative example 2 the procedure is essentially the same as example 2, except that comparative example 2 uses chitosan instead of amidated chitosan.

Comparative example 3: comparative example 3 the procedure was essentially the same as example 3 except that comparative example 3 used polylactic acid nanofibers instead of composite graft modified nanofibers.

Performance test:

Toxicity test: the hemostatic material samples prepared in examples 2 and 3 and comparative examples 1 to 3 and the control group (DMEM medium) were uniformly distributed in 96-well plates in 16 wells per group of samples, then Fibroblasts fibroblasts were diluted to 1×10 ⁵/mL and inoculated in 50 μl in 96-well plates, the temperature was set at 37 ℃,5% concentration of CO ₂ was added after 48 hours of incubation, 50 μl of 5mg/mL of MTT solution was added, the incubation was continued for 4 hours, the culture solution in the wells was aspirated, 200 μl of dimethyl sulfoxide was added to the wells, absorbance (OD value) was measured at 570nm using a microplate reader, and the percent activity [ cell viability (%) = (example OD value/control OD value) ×100% ], and the results were averaged for three total treatment analyses for each sample, as shown in fig. 1.

As can be seen from the results of FIG. 1, the hemostatic material prepared by the present invention is effective in promoting cell activity, has excellent biocompatibility, and does not adversely affect cells; from the results of example 3 and comparative example 3, it is understood that the composite graft modified nanofiber has better biocompatibility than the polylactic acid nanofiber.

And (3) map measurement: the composite nanofiber sample prepared in example 2 was subjected to a scanning electron microscope (sem) and the results are shown in fig. 2; the hemostatic material sample prepared in example 2 was subjected to scanning electron microscopy and the results are shown in fig. 4; the FTIR spectrometer tests the hemostatic material sample pattern and the results are shown in fig. 3.

As can be seen from the results of fig. 2, the composite nanofiber image surface prepared by the present invention forms a complex interwoven structure by grafting and introducing; as can be seen from the results of FIG. 3, the stretching vibration of O-H and N-H shows a larger vibration amplitude at 3200-3500cm ^-1: the disulfide bond successfully introduces the characteristic peak of 1450-1500 cm ^-1; as can be seen from the results of FIG. 4, the hemostatic material prepared by the invention has a large number of pores, provides a larger specific surface area for cell adhesion, and greatly improves the adsorption performance of the material.

ROS solubility test: 0.1mM, 1mM, 10mM and 30mM hydrogen peroxide solution was prepared by using hydrogen peroxide (H ₂O₂) and deionized water, 1mM ferric sulfate solution was prepared, ferric sulfate solution and the above-mentioned hydrogen peroxide solutions of different concentrations were mixed in PBS buffer to obtain ROS solutions of different concentrations, the hemostatic material samples prepared in example 2 were respectively immersed in the above-mentioned ROS solutions of different concentrations, untreated control group and PBS immersed group were set as baseline for comparison, and the dissolution rate (%) = (initial weight-residual weight)/time×100%) was calculated by periodic sampling at 0, 5, 10, 30 and 60min, and the results are shown in FIG. 5.

As can be seen from the results of FIG. 5, the hemostatic material prepared by the present invention can be effectively dissolved and released in a high concentration ROS solution, and can achieve more than 90% dissolution within 10min when the concentration of ROS is 30 mM.

Biodegradability test: three samples of the hemostatic materials prepared in examples 2 and 3 and comparative examples 1 to 3 were taken as samples, each sample being 1g, and a total of 18 samples; samples were added to 30mL of PBS buffer at pH 7.4, placed in a 32 ℃ incubator, sampled on days 1, 3, 7, 14, and the degradation rate (%) = (initial weight-remaining weight)/initial weight×100%) was calculated, and the result is shown in fig. 6.

As can be seen from the results of FIG. 6, the hemostatic material prepared by the method has good biodegradation efficiency, can be degraded to more than 80% in 7 days, and can be completely degraded in 14 days; from the results of example 2 and comparative example 1, it is known that glutaraldehyde is substituted for diphenylacetyl disulfide as a crosslinking agent, which affects the biodegradation efficiency and has poor degradation performance; by; from the results of example 2 and comparative example 2, it is understood that the degradation property of amidated chitosan after crosslinking with gamma-polyglutamic acid is superior to that of chitosan after crosslinking with gamma-polyglutamic acid.

Antibacterial test: fresh bacterial solutions of staphylococcus aureus, escherichia coli and candida albicans were prepared, the bacterial solution concentration was 1×10 ⁸ CFU/mL, hemostatic material samples (1g+19ml deionized water) prepared in examples 1 to 4 and comparative examples 1 to 3 of the present invention were added respectively, the mixed bacterial solution was placed in a constant temperature shaker at 37 ℃ for culturing for 48 hours, the total number of bacterial colonies of the bacterial solution was recorded by a plate colony counting method, the test was repeated 3 times, an average value was taken, the bacterial solution without the sample was taken as a control (all strains used in the present experiment were purchased in the market), and the antibacterial ratio [ antibacterial ratio (%) = (bacterial number of 1-control sample/bacterial number of experimental sample) ×100% ] was calculated, and the result is shown in fig. 7.

As can be seen from the results of fig. 7, the hemostatic material prepared by the present invention can effectively achieve antibacterial effect, and as can be seen from the results of example 2 and comparative example 2, the amidated chitosan has stronger antibacterial effect than chitosan; from the results of example 3 and comparative example 3, it is understood that the composite graft modified nanofiber has a better antibacterial effect than the polylactic acid nanofiber.

Hemostatic performance test: a standard small incision of 1.5cm length was made on simulated skin (pigskin) and connected to an artificial blood flow simulation system, samples of hemostatic material prepared in examples 2,3 and comparative examples 1-3 were pre-treated in 30mM ROS solution for 1min, immediately after the pre-treatment was completed, hemostatic material was applied to the wound model, the flow device was started, simulated blood was allowed to flow through the wound, the temperature change of the wound surface after the material application was monitored using a thermometer, and hemostatic time was recorded, and the test was repeated three times, statistical results were shown in the following table.

Table 1. Hemostatic Performance test results.

As can be seen from the results of table 1, the hemostatic material prepared by the present invention can reduce the temperature of the wound site to a small extent, and the hemostatic performance of the composite grafted modified nanofiber is far better than that of the polylactic acid nanofiber as can be seen from the results of example 3 and comparative example 3; from the results of example 2 and comparative example 2, amidated chitosan was also superior to chitosan in hemostatic performance.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. The preparation method of the hemostatic material is characterized in that the hemostatic material consists of a composite nanofiber core layer and a hydrogel outer layer;

S1: preparing composite nano fibers;

S2: dissolving chitosan in acetic acid solution to obtain chitosan solution; dissolving gamma-polyglutamic acid in a phosphate buffer, heating in a water bath to control the temperature, and filtering with a filter membrane to obtain a gamma-polyglutamic acid solution; dissolving diphenylacetyl disulfide in dimethyl sulfoxide solution, and uniformly dissolving to obtain diphenylacetyl disulfide solution; dissolving N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide in a dimethyl sulfoxide solution, and uniformly dissolving in an ice bath to obtain an activated solution;

S3: continuously dripping the activated solution prepared in the step S2 into the chitosan solution prepared in the step S2 in the stirring process, stirring in an ice bath, and performing ultrafiltration dialysis to obtain an aminoacylated chitosan solution; mixing the aminoacylated chitosan solution and the gamma-polyglutamic acid solution prepared in the step S2, keeping the temperature in an ice bath, and stirring to obtain a mixed solution;

S4: suspending the composite nanofiber prepared in the step S1 into deionized water to obtain a composite nanofiber suspension, adding the composite nanofiber suspension into the mixed solution prepared in the step S3, controlling the temperature by an ice bath, and uniformly stirring and mixing to obtain a mixed system;

S5: adding the diphenylacetyl disulfide solution prepared in the step S2 into the mixed system prepared in the step S4, maintaining the temperature in an ice bath, stirring, washing with deionized water, and performing vacuum freeze drying to obtain a hemostatic material;

The preparation steps of the composite nanofiber in the step S1 are as follows:

a1: carrying out ultrasonic treatment on the polylactic acid nanofiber in an argon atmosphere to obtain an activated polylactic acid nanofiber; dissolving sulfate and maleic anhydride in dimethyl sulfoxide solution, and uniformly mixing to obtain an initiation solution; adding the activated polylactic acid nanofiber into an initiating solution, heating in a water bath to control the temperature, washing and drying to obtain the modified polylactic acid nanofiber;

a2: adding the modified polylactic acid nanofiber prepared in the step A1 into dimethyl sulfoxide solution, and dissolving and mixing uniformly to obtain A1 wt% modified polylactic acid nanofiber solution; dissolving methoxy polyethylene glycol in dimethyl sulfoxide solution, and uniformly mixing to obtain methoxy polyethylene glycol solution; adding the modified polylactic acid nanofiber solution into EDC/NHS activated solution, and stirring under ice bath to obtain activated nanofiber solution;

A3: continuously adding the activated nanofiber solution prepared in the step A2 into the methoxy polyethylene glycol solution prepared in the step A2 in the stirring process, stirring, centrifuging, washing, and performing vacuum freeze-drying to obtain the grafted modified polylactic acid nanofiber;

a4: adding the composite natural extract into an ethanol solution to obtain a composite natural extract solution; and (3) adding the grafted modified polylactic acid nanofiber prepared in the step (A3) into a composite natural extract solution, performing ultrasonic treatment, controlling the temperature by using an ice bath, standing, centrifuging, filtering, washing with deionized water, and performing vacuum freeze drying to obtain the composite nanofiber.

2. The method for preparing a hemostatic material according to claim 1, wherein the polylactic acid nanofiber in the step A1 is prepared by a conventional technical means electrospinning method; the sulfate is any one of sodium sulfate, potassium sulfate and ferric sulfate; the dosage ratio of the sulfate to the maleic anhydride to the dimethyl sulfoxide solution is 10 mg:15-20 mg:1mL; the ratio of the dosage of the activated polylactic acid nanofiber to the dosage of the initiating solution is 0.5-1 g/10 mL.

3. The method for preparing a hemostatic material according to claim 2, wherein the EDC/NHS activating solution in step A2 is a solution having a concentration of 5 to 10mg/mL prepared from 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in a molar ratio of 1:1 in PBS buffer; the volume ratio of the modified polylactic acid nanofiber solution to the EDC/NHS activation solution is 1:3-5.

4. A method of preparing a hemostatic material according to claim 3, wherein the compound natural extract in step A4 is prepared by conventional technical means in a mass ratio of 1:0.8:1.2:1 of echinacea, garlic, baikal skullcap root and mugwort; the mass-to-mass ratio of the grafted modified polylactic acid nanofiber to the composite natural extract is 1:0.2-0.5.

5. The method according to claim 4, wherein the molar mass ratio of N-hydroxysuccinimide to 1-ethyl- (3-dimethylaminopropyl) carbodiimide in step S2 is 1:1.

6. The method of preparing hemostatic material according to claim 5, wherein the ratio of the volume amounts of the activated solution and the chitosan solution in step S3 is 1-1.5:1; the volume dosage ratio of the aminoacylated chitosan solution to the gamma-polyglutamic acid solution is 1:1.

7. The method of preparing hemostatic material according to claim 6, wherein the volume ratio of the diphenylacetyl disulfide solution to the mixed system in step S5 is 1:1-2.

8. A hemostatic material prepared by the method of any one of claims 1-7, wherein the hemostatic material is prepared by steps S1-S5.