CN111375085B

CN111375085B - Fluid hemostatic gel and preparation method thereof

Info

Publication number: CN111375085B
Application number: CN201811611438.8A
Authority: CN
Inventors: 曹加花; 崔志明; 李睿智; 李亦晨
Original assignee: Imeik Technology Development Co ltd
Current assignee: Imeik Technology Development Co ltd
Priority date: 2018-12-27
Filing date: 2018-12-27
Publication date: 2022-02-01
Anticipated expiration: 2038-12-27
Also published as: CN111375085A

Abstract

The invention provides a fluid hemostatic gel and a preparation method thereof. The fluid hemostatic gel comprises a cross-linked chitosan or derivative thereof, a cross-linked water-soluble protein or polypeptide, and a lubricant. The hemostatic gel does not contain a blood coagulation factor, is in a flowable ointment shape, has proper swelling degree, can be directly used without any time-consuming reconstruction step, and has the advantages of short hemostatic time, high wound healing speed, convenience and rapidness in use, degradability in vivo, good biocompatibility and the like.

Description

Fluid hemostatic gel and preparation method thereof

Technical Field

The invention relates to the field of medicines, in particular to a fluid hemostatic gel and a preparation method thereof.

Background

Wound bleeding is difficult to avoid in daily life, and although minor trauma is not fatal enough, it can be life threatening if blood loss reaches 30% or more of the total blood volume of the human body in a short period of time. Most of death cases in emergencies such as wars, natural disasters, traffic accidents and the like are caused by excessive blood loss, so that local and effective rapid hemostasis is of great importance in the process of rescuing injured people. Local hemostasis is the first step of wound treatment, and an effective hemostasis means can reduce unnecessary casualties, and has a very important effect on saving the life of a patient. At present, most of the hemostatic gel at home and abroad appears in the forms of sponge, fiber, powder, film and the like, and single-component biological hemostatic dressings are taken as main materials, although the products can meet the requirements of general wound hemostasis to a certain extent, the products are easy to adhere to wounds, the hemostatic time is long, the rapid healing of the wounds is not facilitated, and the hemostatic gel is not convenient and rapid to use. Therefore, the research of a novel degradable hemostatic material with short hemostatic time, fast wound healing speed and convenient and fast use becomes a current research focus.

Chinese patent CN105536029A discloses a preparation method of chitosan porous hemostatic sponge, and the hemostatic sponge has good hemostatic, antibacterial, anti-inflammatory and wound healing promoting effects, but the single-component hemostatic sponge is easy to adhere to a wound, has a long hemostatic time and has certain limitations.

Chinese patent CN104189941A discloses a chitosan gel hemostatic material and a preparation method thereof, which is obtained by compounding chitosan, a cross-linking agent and thrombin, and can rapidly stop bleeding in vivo and promote wound healing, but the chitosan gel hemostatic material is in a xerogel state, and has poor adhesiveness and no fluidity after absorbing moisture at a wound, and is not beneficial to wound sealing. In addition, currently marketed floreal and Surgiflo are effective hemostatic products as thrombin-containing hemostatic compositions, but they have poor adhesion, are not suitable for wounds with high blood flow, and are applied after mixing two components before use, which brings great inconvenience to clinical rapid hemostasis.

On the basis of the gelatin product Floseal in the prior art, the Chinese patent CN103889447A researches a hemostatic composition with improved adhesion and hemostatic property, which is more convenient in the actual use process, but the main component of the hemostatic composition exerting hemostatic property is thrombin.

Chinese patent CN104349797A has studied a dry composition which can spontaneously form a uniform paste without mechanical mixing after adding a suitable amount of an aqueous medium, but the composition also contains a blood coagulation factor such as thrombin.

Chitosan is a straight-chain macromolecular compound formed by connecting N-acetyl-D-glucosamine monomers through beta-l, 4-glycosidic bonds, is the only basic polysaccharide existing in the biological world in a large amount, has a basic composition unit of glucosamine, is distributed with a plurality of hydroxyl groups and amino groups on a macromolecular chain of chitosan, and has more active properties. The chitosan is nontoxic, has good biocompatibility, biodegradability and anticoagulation, and has the functions of promoting wound healing, antisepsis, antibiosis and the like, and is concerned as a medical material. In addition, chitosan has a variety of derivatives due to the chitosan2-NH in the structure₂And 6-OH can be used as two reaction sites in the chitosan, and the chitosan can be modified through the two reaction sites. For example, 2-NH₂Carboxymethyl chitosan obtained after carboxymethylation with-OH at the 6-position is a water-soluble chitosan derivative, is also a chitosan derivative which is most widely applied, and is currently applied to aspects such as adhesion prevention, hemostasis, joint cavity visco-elastic supplement and the like.

Albumin, fibrin, collagen-like protein, fibroin, elastin and the like belong to common proteins, and are widely applied to the field of medicines or modified products thereof. The collagen hydrolysis modified product gelatin has the characteristics of low cost, good biocompatibility, weak antigenicity, easy absorption of degradation products, no inflammatory reaction and the like, and has wide application in the medical and health fields of burns, wounds, eye and cornea diseases, cosmetology, orthopedics, hard tissue repair, wound surfaces, hemostasis and the like.

Disclosure of Invention

[ problem ] to

The invention aims to provide a fluid hemostatic gel and a preparation method thereof. The fluid hemostatic gel does not contain a blood coagulation factor, is in a flowable ointment shape, has proper swelling degree, can be directly used without any time-consuming reconstruction step, and has the advantages of short hemostatic time, high wound healing speed, convenience and rapidness in use, degradability in vivo, good biocompatibility and the like. It can be used in the amount suitable for the wound of different tissue parts. After the fluid hemostatic gel is injected to a use part, the fluid hemostatic gel can be rapidly filled in a wound bleeding part in a fluid state, can rapidly absorb wound moisture, concentrates wound plasma, forms a good blood coagulation environment, accelerates blood coagulation, can adapt to clinical complicated wound hemostasis, and can be widely applied as a medical hemostatic material.

[ solution ]

One aspect of the present invention provides a fluid hemostatic gel comprising a crosslinked chitosan or derivative thereof, a crosslinked water-soluble protein or polypeptide, and a lubricant.

The fluid hemostatic gel with both flow property and water absorption property is prepared by adopting the cross-linked chitosan or the derivative thereof and the cross-linked water-soluble protein or polypeptide as main matrixes singly or together and adding the lubricant as an auxiliary matrix.

Wherein the mass ratio of the cross-linked chitosan or the derivative thereof to the cross-linked water-soluble protein or polypeptide is 0.2-2: 1, and the mass ratio of the lubricant to the sum of the cross-linked chitosan or the derivative thereof and the cross-linked water-soluble protein or polypeptide is 0.01-1: 1.

The molecular weight range of the chitosan or the derivatives thereof is 10-500 kDa; the deacetylation degree range is 60-99%. Preferably, the chitosan or derivative thereof may be, for example, carboxymethyl chitosan.

The cross-linked chitosan or the derivative thereof can exist in the form of particles, the particle size range of the cross-linked chitosan or the derivative thereof is 100-2000 mu m, and the cross-linked chitosan or the derivative thereof is a cross-linked product.

The particle size range of the water-soluble protein or polypeptide is 50-500 mu m.

The water-soluble protein or polypeptide is one or more selected from albumin, fibrin, collagen-like protein, silk protein, gelatin or elastin, and modified products of these proteins, but the present invention is not limited thereto. Preferably, the water-soluble protein or polypeptide may be, for example, gelatin.

The cross-linked water-soluble protein or polypeptide is a product obtained by cross-linking the water-soluble protein or polypeptide.

Alternatively, the crosslinked chitosan or derivative thereof and the crosslinked water-soluble protein or polypeptide may be present in the form of a complex gel.

The lubricant can be selected from water-soluble macromolecular compounds, such as polyethylene glycol, polypropylene glycol, higher fatty alcohol (C6-C10), soluble cellulose, algal polysaccharides, alginic acid, sodium hyaluronate, etc.

In addition, the fluid hemostatic gel further comprises a buffer solution, wherein the buffer solution is a phosphate buffer solution, and the mass ratio of the buffer solution to the lubricant is 5-30: 1.

In another aspect, the present invention provides a method for preparing a fluid hemostatic gel, which comprises the following steps:

a) preparing crosslinked chitosan or a derivative thereof by crosslinking chitosan or a derivative thereof;

b) preparing a cross-linked water-soluble protein or polypeptide by cross-linking a water-soluble protein or polypeptide;

c) and mixing the cross-linked chitosan or the derivative thereof and the cross-linked water-soluble protein or polypeptide with a lubricant to obtain the fluid hemostatic gel.

Because the human body contains more lysozyme, glucolase, protease, active oxygen cluster free radicals and other active substances, the chitosan or derivatives thereof and water-soluble protein or polypeptide can be degraded into small molecular monomers to different degrees. Therefore, chitosan or its derivatives and water-soluble protein or polypeptide are usually cross-linked chemically to increase the half-life and prolong the degradation time in human body.

The particle size range of the water-soluble protein or polypeptide is 50-500 mu m. Preferably, the water-soluble protein or polypeptide may be, for example, gelatin.

In particular, said step a) may comprise the steps of:

a1) adding chitosan or its derivative into sodium hydroxide water solution to obtain chitosan or its derivative alkaline water solution;

a2) adding a cross-linking agent to the chitosan or derivative alkaline aqueous solution; and

a3) reacting the mixture obtained in step a2) to obtain the cross-linked chitosan or derivative thereof.

In the step a1), the molar concentration of the sodium hydroxide aqueous solution is 0-0.5 mol/L, and the mass concentration of the chitosan or chitosan derivative alkaline aqueous solution is 0.5-10.0%.

In step a2), the crosslinking agent may be a multifunctional compound, including, for example, a multi-epoxy compound, a multi-alcohol compound, a multi-amine compound, a multi-aldehyde compound, a multi-acid compound, and the like, but the present invention is not limited thereto. Specifically, the crosslinking agent may be, for example, 1, 4-butanediol diglycidyl ether, 1,2,7, 8-diepoxyoctane, glutaraldehyde, or the like.

In addition, the mass ratio of the cross-linking agent to the chitosan or the derivative thereof in the alkaline aqueous solution is 0.001-0.05: 1.

In the step a3), the reaction conditions are as follows: the reaction temperature is 20-60 ℃, the reaction time is 18-24 h, and the mixture is kept stand for reaction.

In addition, the step a) may further include the steps of:

a4) adjusting the pH value of the crosslinked chitosan or the derivative thereof obtained in the step a3) to about 6.8-7.6; and

a5) crushing the product of the step a4) into particles with the particle size range of 100-2000 mu m, and freeze-drying.

Wherein, in the step a4), the pH value is adjusted by using 0.1mol/L hydrochloric acid solution.

In particular, said step b) may comprise the steps of:

b1) preparing a water-soluble protein or polypeptide water solution, and adjusting the pH value of the system to 4.0-6.0;

b2) adding a catalyst into the water-soluble protein or polypeptide water solution, and adjusting the pH value of the system to 4.0-6.0;

b3) reacting the mixture obtained in step b2) to obtain the cross-linked water-soluble protein or polypeptide.

In the step b1), the mass concentration of the water-soluble protein or polypeptide aqueous solution is 0.5-10.0%, and 0.1mol/L hydrochloric acid solution is adopted to adjust the pH of the system.

Wherein, in the step b2), the pH of the system is still adjusted by using 0.1mol/L hydrochloric acid solution.

Further, the catalyst may be water-soluble carbodiimide including 1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide methiodide, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, and the like, but the present invention is not limited thereto. In addition, the mass ratio of the catalyst to the water-soluble protein or polypeptide in the solution is 0.002-0.02: 1.

Wherein, in the step b3), the reaction conditions are as follows: the reaction temperature is 2-10 ℃, the reaction time is 15-24 h, and stirring reaction is carried out.

In addition, the step b) may further include the steps of:

b4) purifying the cross-linked water-soluble protein or polypeptide obtained in step b3) to remove residual catalyst, and lyophilizing.

In the step b4), the crosslinked water-soluble protein or polypeptide solution is firstly kept stand for 30min before purification, the supernatant is discarded, then deionized water is added, the mixture is stirred for 30min at the speed of 200r/min, and the operations are repeated for 5-25 times.

In particular, said step c) may comprise the steps of:

c1) dissolving a lubricant in a phosphate buffer solution with the pH value of 6.6-7.0 to prepare a lubricant aqueous solution;

c2) dissolving the cross-linked chitosan or derivative thereof and the cross-linked water-soluble protein or polypeptide in the aqueous lubricant solution obtained in step c 1); and

c3) homogenizing the mixture obtained in the step c2) to obtain the fluid hemostatic gel.

In the step C1), the lubricant comprises polyethylene glycol, polypropylene glycol, C6-C10 fatty alcohol, soluble cellulose, algal polysaccharide, alginic acid, sodium hyaluronate and the like, and the mass concentration of the prepared lubricant aqueous solution is 0.5-10%.

In the step c2), the mass ratio of the cross-linked chitosan or the derivative thereof to the cross-linked water-soluble protein or polypeptide is 0.2-2: 1, and the mass ratio of the lubricant to the sum of the cross-linked chitosan or the derivative thereof and the cross-linked water-soluble protein or polypeptide is 0.01-1: 1.

Wherein, in the step c3), the homogenization treatment is carried out in a roller type mixer.

d) preparing a composite gel of cross-linked water-soluble protein or polypeptide and chitosan or derivatives thereof; and

e) and mixing the composite gel with a lubricant to obtain the fluid hemostatic gel.

In particular, said step d) may comprise the steps of:

d1) sequentially dissolving water-soluble protein or polypeptide and chitosan or derivatives thereof in water to prepare a water-soluble protein or polypeptide and chitosan or derivatives composite aqueous solution;

d2) adding a cross-linking agent to the composite aqueous solution obtained in step d 1); and

d3) reacting the mixture obtained in step d2) to obtain the cross-linked water-soluble protein or polypeptide and chitosan or derivatives thereof composite gel.

Wherein, in the step d1), the mass concentration of the water-soluble protein or polypeptide in the composite aqueous solution is 0.5-5.0%, and the mass concentration of the chitosan or the derivative thereof is 0.5-5.0%.

In the step d2), the cross-linking agent can be 1, 4-butanediol diglycidyl ether, 1,2,7, 8-diepoxyoctane, glutaraldehyde or the like, and the mass ratio of the cross-linking agent to the sum of the water-soluble protein or polypeptide and the chitosan or derivative thereof in the composite aqueous solution is 0.01-0.1: 1.

Wherein, in the step d3), the reaction conditions are as follows: the reaction temperature is 5-40 ℃, the reaction time is 0-24 h, and the mixture is kept stand for reaction.

In addition, the step d) may further include the steps of:

d4) adjusting the pH value of the composite gel obtained in the step d3) to be about 6.8-7.6; and

d5) crushing the obtained substance in the step d4) into particles with the particle size range of 100-2000 mu m, and freeze-drying.

Wherein, in the step d4), the pH value is adjusted by using 0.1mol/L hydrochloric acid solution or 0.1mol/L sodium hydroxide solution.

In particular, said step e) may comprise the steps of:

e1) dissolving a lubricant in a phosphate buffer solution with the pH value of 6.8-7.2 to prepare a lubricant aqueous solution;

e2) dissolving the cross-linked water-soluble protein or polypeptide and chitosan or derivative thereof composite gel in the aqueous lubricant solution obtained in step e 1);

e3) homogenizing the mixture obtained in the step e2) to obtain the fluid hemostatic gel.

In the step e1), the lubricant comprises polyethylene glycol, polypropylene glycol, C6-C10 fatty alcohol, soluble cellulose, algal polysaccharide, alginic acid, sodium hyaluronate and the like, and the mass concentration of the aqueous solution of the lubricant is 0.5-10%.

In the step e2), the mass ratio of the lubricant to the crosslinked water-soluble protein or polypeptide to the chitosan or derivative composite gel is 0.01-1: 1.

Wherein, in the step e3), the homogenization treatment is carried out in a roller type mixer.

[ advantageous effects ]

The invention adopts a chemical method to crosslink chitosan or derivatives thereof and water-soluble protein or polypeptide, then compounds the crosslinked product and a lubricant, and prepares the fluid hemostatic gel by a gel blending method. The invention plays a synergistic role between chitosan or a derivative thereof and water-soluble protein or polypeptide, promotes blood coagulation by adhering and aggregating erythrocytes on one hand, and provides a physical structure to aggregate blood platelets on the other hand, and can fill wounds and swell to play a role in compression hemostasis. Therefore, the composite hemostatic material can achieve a quick hemostatic effect only through physical hemostatic action without adding blood coagulation factors, and is a composite hemostatic material with remarkable hemostatic performance and wound healing performance.

The fluid hemostatic gel is in a flowable soft paste shape, can be directly used without any time-consuming reconstruction step, and has the advantages of certain fluidity, proper swelling degree, short hemostatic time, high wound healing speed, convenience and rapidness in use, degradability in vivo, good biocompatibility and the like. The wound size of accessible different tissue positions is injected the back to the position of use with right amount of fluid hemostatic glue, can fill in wound bleeding department with the fluid state rapidly to absorb wound moisture fast, concentrate the surface of a wound plasma, form good blood coagulation environment, the blood coagulation is accelerated, but wide application in medical material preparation technical field.

Drawings

FIG. 1 is a graph showing the effect of coagulation in vitro by a fluid hemostatic gel according to the present invention (a photograph of fresh blood of a rabbit containing heparin, b photograph of a treatment with a conventional pure gelatin hemostatic material, c photograph of a treatment with a fluid hemostatic gel according to example 8 of the present invention).

Fig. 2 is a graph showing the hemostatic effect of the fluid hemostatic gel according to the present invention after being applied to a rabbit wound (a photograph of a rabbit wound; b photograph of a rabbit treated with a pure gelatin hemostatic material; c photograph of a rabbit treated with a fluid hemostatic gel according to example 8 of the present invention).

Detailed Description

The present invention is further illustrated by the following examples, which do not limit the scope of the present invention.

The experimental methods in the examples, in which specific conditions are not specified, are generally performed under the conditions described in the manual and the conventional conditions, or under the conditions recommended by the manufacturer; the materials, reagents and the like used, unless otherwise specified, are commercially available.

Example 1: preparation of crosslinked carboxymethyl chitosan

Weighing 0.8g of sodium hydroxide, and dissolving in 185mL of water to obtain 0.1mol/L sodium hydroxide solution; weighing 15.0g of carboxymethyl chitosan (the deacetylation degree is 90.1%, and the molecular weight is 20kDa), dissolving in the sodium hydroxide solution, and stirring for 1h to obtain a carboxymethyl chitosan solution with the mass fraction of 7.5%; adding 0.075g of 1, 4-butanediol diglycidyl ether into the carboxymethyl chitosan solution, and stirring for 0.5 h; adjusting the reaction temperature to 20-60 ℃, and standing for 20 hours to obtain crosslinked carboxymethyl chitosan; adding 200mL of 0.1mol/L hydrochloric acid solution into the crosslinked carboxymethyl chitosan to perform acid-base neutralization reaction, and adjusting the pH value of the crosslinked carboxymethyl chitosan to about 6.8-7.6 by using the 0.1mol/L hydrochloric acid solution; transferring the neutralized crosslinked carboxymethyl chitosan to a homogenizing disperser (AD500S-H, Shanghai-Noni instruments and meters Co., Ltd.), and pulverizing to obtain particles with a particle size of 200 μm; transferring the crushed crosslinked carboxymethyl chitosan into a freeze dryer (VFD-1000, Beijing Bo Yi kang laboratory instruments Co., Ltd.), and freeze-drying for 36-48 h until water is completely removed, wherein the yield is not lower than 90%.

Example 2: preparation of crosslinked carboxymethyl chitosan

Weighing 0.8g of sodium hydroxide, and dissolving in 185mL of water to obtain 0.1mol/L sodium hydroxide solution; weighing 15.0g of carboxymethyl chitosan (the deacetylation degree is 90.1%, and the molecular weight is 20kDa), dissolving in the sodium hydroxide solution, and stirring for 1h to obtain a carboxymethyl chitosan solution with the mass fraction of 7.5%; adding 0.15g of 1, 4-butanediol diglycidyl ether into the carboxymethyl chitosan solution, and stirring for 0.5 h; adjusting the reaction temperature to 20-60 ℃, and standing for 20 hours to obtain crosslinked carboxymethyl chitosan; adding 200mL of 0.1mol/L hydrochloric acid solution into the crosslinked carboxymethyl chitosan to perform acid-base neutralization reaction, and adjusting the pH value of the crosslinked carboxymethyl chitosan to about 6.8-7.6 by using the 0.1mol/L hydrochloric acid solution; transferring the neutralized crosslinked carboxymethyl chitosan to a homogenizing disperser (AD500S-H, Shanghai-Noni instruments and meters Co., Ltd.), and pulverizing to obtain particles with a particle size of 200 μm; transferring the crushed crosslinked carboxymethyl chitosan into a freeze dryer (VFD-1000, Beijing Bo Yi kang laboratory instruments Co., Ltd.), and freeze-drying for 36-48 h until water is completely removed, wherein the yield is not lower than 90%.

Example 3: preparation of crosslinked gelatin

Weighing 30.0g of gelatin particles (the particle size is 500 mu m, the pH value is 5.5), dissolving the gelatin particles in 570mL of water, and stirring for 0.5h to obtain a gelatin solution with the mass fraction of 5%; adjusting the pH value of the system to 5.0-5.5 by using 0.1mol/L hydrochloric acid solution, adding 0.3g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride into the gelatin water solution, and continuously adjusting the pH value of the system to 5.0-5.5 by using 0.1mol/L hydrochloric acid solution; adjusting the reaction temperature to 2-10 ℃, and reacting for 15h under the stirring condition to obtain crosslinked gelatin; transferring the reaction solution to a 3L beaker, adding 2L of water, reacting for 30min under the stirring condition, standing for 30min, removing supernatant, and repeating the above cleaning operation for more than 15 times; and transferring the gelatin solution to a VFD-1000 freeze dryer of Beijing Bo Yi kang laboratory instruments Co., Ltd, and freeze-drying for 36-48 h until water is completely removed, wherein the yield is not lower than 70%.

Example 4: preparation of crosslinked gelatin

Weighing 30.0g of gelatin particles (the particle size is 500 mu m, the pH value is 5.5), dissolving the gelatin particles in 570mL of water, and stirring for 0.5h to obtain a gelatin solution with the mass fraction of 5%; adjusting the pH value of the system to 5.0-5.5 by using 0.1mol/L hydrochloric acid solution, adding 0.6g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride into the gelatin water solution, and continuously adjusting the pH value of the system to 5.0-5.5 by using 0.1mol/L hydrochloric acid solution; adjusting the reaction temperature to 2-10 ℃, and reacting for 15h under the stirring condition to obtain crosslinked gelatin; transferring the reaction solution to a 3L beaker, adding 2L of water, reacting for 30min under the stirring condition, standing for 30min, removing supernatant, and repeating the above cleaning operation for more than 15 times; and transferring the gelatin solution to a VFD-1000 freeze dryer of Beijing Bo Yi kang laboratory instruments Co., Ltd, and freeze-drying for 36-48 h until water is completely removed, wherein the yield is not lower than 70%.

Example 5: preparation of crosslinked gelatin/carboxymethyl chitosan composite gel

Weighing 10g of gelatin particles (the particle size is 500 mu m, the pH value is 5.5) and 5g of carboxymethyl chitosan (the deacetylation degree is 90.1%, the molecular weight is 20kDa), sequentially dissolving in 185mL of water, and stirring for 2h to obtain a gelatin/carboxymethyl chitosan composite solution; adding 0.3g of glutaraldehyde into the gelatin/carboxymethyl chitosan composite solution, and uniformly stirring; adjusting the reaction temperature to 5-40 ℃, and standing for 5 hours to obtain crosslinked gelatin/carboxymethyl chitosan composite gel; adjusting the pH value of the reacted crosslinked gelatin/carboxymethyl chitosan composite gel to about 6.8-7.6 by using 0.1mol/L hydrochloric acid solution or 0.1mol/L sodium hydroxide solution; transferring the neutralized crosslinked gelatin/carboxymethyl chitosan composite gel to an AD500S-H homogenizing disperser of Haoney instruments and meters Co., Ltd, and pulverizing to obtain particles with a particle size of 200 μm; transferring the crushed crosslinked gelatin/carboxymethyl chitosan composite gel into a VFD-1000 freeze dryer of Beijing Bo Yi kang experiment instrument limited, and freeze-drying for 36-48 h until water is completely removed, wherein the yield is not lower than 85%.

Example 6: preparation of crosslinked gelatin/carboxymethyl chitosan composite gel

Weighing 10g of gelatin particles (the particle size is 500 mu m, the pH value is 5.5) and 5g of carboxymethyl chitosan (the deacetylation degree is 90.1%, the molecular weight is 20kDa), sequentially dissolving in 185mL of water, and stirring for 2h to obtain a gelatin/carboxymethyl chitosan composite solution; adding 0.45g of glutaraldehyde into the gelatin/carboxymethyl chitosan composite solution, and uniformly stirring; adjusting the reaction temperature to 5-40 ℃, and standing for 5 hours to obtain crosslinked gelatin/carboxymethyl chitosan composite gel; adjusting the pH value of the reacted crosslinked gelatin/carboxymethyl chitosan composite gel to about 6.8-7.6 by using 0.1mol/L hydrochloric acid solution or 0.1mol/L sodium hydroxide solution; transferring the neutralized crosslinked gelatin/carboxymethyl chitosan composite gel to an AD500S-H homogenizing disperser of Haoney instruments and meters Co., Ltd, and pulverizing to obtain particles with a particle size of 200 μm; transferring the crushed crosslinked gelatin/carboxymethyl chitosan composite gel into a VFD-1000 freeze dryer of Beijing Bo Yi kang experiment instrument limited, and freeze-drying for 36-48 h until water is completely removed, wherein the yield is not lower than 85%.

Example 7: preparation of fluid hemostatic gel

Weighing 7.5g of polyethylene glycol, dissolving in 112.5g of phosphate buffer (pH 6.6) to prepare a polyethylene glycol solution with the mass fraction of 6.25%; weighing 9.0g of the crosslinked carboxymethyl chitosan prepared in the example 1, and 21.0g of the crosslinked gelatin prepared in the example 3, dissolving the crosslinked carboxymethyl chitosan and the crosslinked gelatin in the polyethylene glycol aqueous solution, and stirring for 3 hours to obtain a mixture of polyethylene glycol, the crosslinked carboxymethyl chitosan and the crosslinked gelatin; the mixture is transferred to a roller type mixer (MR-03U, Qi Co., Ltd. in USA) for homogenization treatment for 2h, and the fluid hemostatic gel is obtained.

Example 8: preparation of fluid hemostatic gel

Weighing 7.5g of polyethylene glycol, dissolving in 112.5g of phosphate buffer (pH 7.0), and preparing into 6.25% polyethylene glycol solution; weighing 30g of the crosslinked gelatin/carboxymethyl chitosan composite gel prepared in the embodiment 6, dissolving the crosslinked gelatin/carboxymethyl chitosan composite gel in a polyethylene glycol aqueous solution, and stirring for 3 hours to obtain a mixture of polyethylene glycol and the crosslinked gelatin/carboxymethyl chitosan composite gel; the mixture is transferred to a roller type mixer (MR-03U, Qi Co., Ltd. in USA) for homogenization treatment for 2h, and the fluid hemostatic gel is obtained.

Experimental example 1: in vitro coagulation performance study of fluid hemostatic gel

Weighing 5mL of the fluid hemostatic gel sample in the example 8, and filling the fluid hemostatic gel sample into a 10mL pre-filled and sealed syringe; injecting 1mL of rabbit fresh blood (collected in a heparin tube in advance) into a glass culture dish, and injecting 1mL of fluid hemostatic gel right above the fresh blood; meanwhile, 1mL of pure gelatin hemostatic material is injected into another glass culture dish containing 1mL of fresh rabbit blood as a control group; observing the in vitro coagulation effect (as shown in figure 1), the fluid hemostatic gel is found to rapidly absorb blood and condense and coagulate within 15s, while the pure gelatin hemostatic material is not completely coagulated after absorbing blood and still has fluidity after being inclined.

Experimental example 2: research on animal wound hemostasis effect of fluid hemostatic gel

Weighing and recording the experimental New Zealand white rabbits, and performing compound anesthesia treatment on the white rabbits with 0.5mg/kg of chlorthalidone and 0.5mg/kg of veterinary fast-sleeping New II injection; removing hair and skin of anesthetized white rabbits along the groin, cutting a wound of about 4cm, exposing an artery and a vein, carefully separating the artery and the vein, separating the artery and the vein from each other, and preparing a same wound model at each of the left leg and the right leg; clamping an artery at the proximal end by using a hemostatic forceps, pricking the artery by using a needle, freely spraying blood for 5s, quickly smearing the fluid hemostatic gel prepared in the example 8 at the wound of the right leg, and smearing a pure gelatin hemostatic material at the wound of the left leg as a control group; after the wound is lightly pressed by cotton cloth and is kept stand for 2min, the fluid hemostatic gel on the surface of the wound is removed by washing with deionized water, the hemostatic effect of the wound is observed (as shown in fig. 2), and the wound coated with the pure gelatin hemostatic material still has an obvious bleeding phenomenon, while the wound coated with the fluid hemostatic gel in the embodiment 8 has no obvious bleeding phenomenon.

Claims

1. A fluid haemostatic gel comprising cross-linked chitosan or a derivative thereof, a cross-linked water-soluble protein or polypeptide, and a lubricant,

wherein the mass ratio of the cross-linked chitosan or the derivative thereof to the cross-linked water-soluble protein or polypeptide is 0.2-2: 1, and the mass ratio of the lubricant to the sum of the cross-linked chitosan or the derivative thereof and the cross-linked water-soluble protein or polypeptide is 0.01-1: 1;

the crosslinked chitosan or the derivative thereof exists in the form of particles, and the particle diameter thereof ranges from 100 to 2000 [ mu ] m, and

the cross-linked water-soluble protein or polypeptide exists in a particle form, and the particle size range of the cross-linked water-soluble protein or polypeptide is 50-500 mu m;

the fluid hemostatic gel is prepared by a preparation method comprising the following steps:

c) mixing the crosslinked chitosan or derivative thereof and the crosslinked water-soluble protein or polypeptide with a lubricant;

the step a) comprises the following steps:

a1) adding the chitosan or the derivative thereof into a sodium hydroxide aqueous solution to prepare a chitosan or derivative alkaline aqueous solution;

a3) reacting the mixture obtained in step a2) to obtain the cross-linked chitosan or derivative thereof;

a4) adjusting the pH value of the crosslinked chitosan or the derivative thereof obtained in the step a3) to 6.8-7.6; and

a5) crushing the product obtained in the step a4) into particles with the particle size range of 100-2000 mu m, and freeze-drying;

in the step a1), the molar concentration of the sodium hydroxide aqueous solution is 0-0.5 mol/L, the mass concentration of the chitosan or the derivative thereof alkaline aqueous solution is 0.5-10.0%, and the molecular weight range of the chitosan or the derivative thereof is 10-500 kDa; the deacetylation degree is 60-99%,

wherein in the step a2), the cross-linking agent is one or more selected from 1, 4-butanediol diglycidyl ether, 1,2,7, 8-diepoxyoctane or glutaraldehyde, and the mass ratio of the cross-linking agent to the chitosan or the derivative thereof in the alkaline aqueous solution is 0.001-0.05: 1,

2. The fluid hemostatic gel of claim 1, wherein,

the water-soluble protein or polypeptide is one or more selected from albumin, fibrin, collagen-like protein, silk protein, gelatin and elastin;

the lubricant is one or more selected from polyethylene glycol, polypropylene glycol, C6-C10 fatty alcohol, soluble cellulose, algal polysaccharide and sodium hyaluronate;

the chitosan or derivative thereof is carboxymethyl chitosan, and

the fluid hemostatic gel further comprises a buffer solution, wherein the buffer solution is a phosphate buffer solution, and the mass ratio of the buffer solution to the lubricant can be 5-30: 1.

3. The fluid hemostatic gel of claim 1, wherein the step b) comprises the steps of:

b3) reacting the mixture obtained in step b2) to obtain the cross-linked water-soluble protein or polypeptide,

wherein, in the step b1), the mass concentration of the water-soluble protein or polypeptide aqueous solution is 0.5-10.0%, and 0.1mol/L hydrochloric acid solution is adopted to adjust the pH value of the system,

wherein, in the step b2), 0.1mol/L hydrochloric acid solution is adopted to adjust the pH of the system, the catalyst is selected from 1-cyclohexyl-2-morpholine ethyl carbodiimide p-toluenesulfonate, 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide methyl iodide salt or 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, and the mass ratio of the catalyst to the water-soluble protein or polypeptide in the solution is 0.002-0.02: 1,

wherein, in the step b3), the reaction conditions are as follows: the reaction temperature is 2-10 ℃, the reaction time is 15-24 h, stirring and reacting are carried out, and

the step c) comprises the following steps:

c3) homogenizing the mixture obtained in step c2),

wherein in the step C1), the lubricant is one or more selected from polyethylene glycol, polypropylene glycol, C6-C10 fatty alcohol, soluble cellulose, algal polysaccharide and sodium hyaluronate, and the mass concentration of the prepared lubricant aqueous solution is 0.5-10%,

wherein in the step c2), the mass ratio of the cross-linked chitosan or the derivative thereof to the cross-linked water-soluble protein or polypeptide is 0.2-2: 1, and the mass ratio of the lubricant to the sum of the cross-linked chitosan or the derivative thereof and the cross-linked water-soluble protein or polypeptide is 0.01-1: 1,

4. The fluid haemostatic gel of claim 3, wherein in step a4), the pH is adjusted with 0.1mol/L hydrochloric acid solution,

the step b) further comprises the following steps:

b4) purifying the cross-linked water-soluble protein or polypeptide obtained in step b3) to remove residual catalyst, lyophilizing,