CN107137756B - A kind of preparation method of shear thickening hemostatic material - Google Patents

Abstract

The invention discloses a preparation method of a shear thickening hemostatic material, comprising the following steps: S1. Dissolving chitosan, adding lauryl aldehyde to react, adjusting pH, and reacting for a certain period of time; S2. Dissolving the product in S1 with cyano group Sodium borohydride reaction; after the reaction is completed, adjust pH, wash, dry and pulverize; S3. Dissolve the pulverized product in S2 and mix with the shear thickening solution to obtain the shear thickening hemostatic material. Aiming at the characteristics of fast blood flow and high pressure in arterial hemorrhage wounds, the invention proposes to use shear thickening liquid to block arterial hemorrhage wounds by referring to the principle of "liquid bulletproof vest". In addition, the present invention combines the hydrophobically modified chitosan with hemostatic function and the solution with shear thickening function, thereby obtaining a multifunctional hemostatic material which can not only block hypertensive wounds, but also promote wound blood coagulation. At the same time, the material for arterial hemostasis provided by the invention has remarkable effect, is convenient to process and has low cost.

Description

A kind of preparation method of shear thickening hemostatic material

technical field

The invention belongs to the technical field of hemostatic materials, and more particularly, relates to a preparation method of a shear-thickening hemostatic material.

Background technique

Chitosan hemostatic materials are obtained by deacetylating chitin, which is widely present in nature. It has good biocompatibility, certain hemostatic and bacteriostatic effects, and is widely used in the design of hemostatic materials. Among them, hydrophobically modified chitosan Sugar has a strong effect on blood cells, and it can be prepared into a foam spray to achieve a very effective hemostatic effect.

Shear thickening liquid is a kind of nanoparticle solvent in the state of solid-liquid mixing. In the range of a certain shear rate, the viscosity increases with the increase of shear rate, which shows that it has a strong effect on shearing. Resistance effect. When the shear force is large enough, these particles are actually "locked" to each other, and even exhibit solid-like mechanical properties. The principle of shear thickening is now widely used in protective materials such as liquid body armor.

According to the design idea of material shear thickening, the hemostatic material is a discontinuous shear thickening liquid, which has excellent impact resistance, fluid-solid conversion under impact conditions, and better effect on the ejected arterial blood flow. Impact resistance, and the hydrophobically modified chitosan hemostatic material itself has the function of rapid hemostasis. The shear-thickening material is compounded with hydrophobically modified chitosan to obtain a multifunctional hemostatic material that can not only block hypertensive wounds, but also promote wound blood coagulation.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a preparation method of a shear thickening hemostatic material according to the deficiencies in the prior art.

The object of the present invention is achieved through the following technical solutions:

The invention provides a preparation method of a shear thickening hemostatic material, comprising the following steps:

S1. Dissolve chitosan in acetic acid and ethanol solution, add lauryl aldehyde to react, adjust pH, and react for a certain period of time;

S2. react the product in S1 with sodium cyanoborohydride; adjust pH after completion of the reaction, wash, dry and pulverize;

S3. Dissolve the pulverized product in S2 and mix with the shear thickening solution to obtain the shear thickening hemostatic material.

Based on the knowledge of shear-thickening materials, the understanding of human autologous coagulation process, and the knowledge of bionics, the invention proposes to combine shear-thickening materials with chitosan materials to prepare a kind of fast hemostasis and hemostasis Excellent material. Existing shear-thickening materials are mostly used in the preparation of products with high impact strength, and the characteristics of shear-thickening materials can be applied to block arterial bleeding wounds, and such materials have not yet been involved in the field of hemostatic materials. The N-alkylated hydrophobically modified chitosan complexed with it can combine with or adhere to certain components in the blood to form a network-like clot instead of fibrin. The advantage of this is that the hemostasis process can be independent In the autologous coagulation process and the artificial control and enhancement of the coagulation process. The hydrophobic segment of N-alkylated hydrophobically modified chitosan can generate a hydrophobic binding force with the hydrophobic part of the cell membrane, and the ionized chitosan has a positive charge and can generate electrostatic attraction force with the cell membrane to generate adsorption. This enables the material to aggregate blood cells like fibrin and cause blood to clot.

Preferably, the molecular weight of the chitosan is 19k~31kDa, and the degree of deacetylation is 75~85%.

Preferably, the solvent used for dissolving chitosan is acetic acid or ethanol.

Preferably, the mass volume ratio of chitosan and solvent is 1:(90~110).

Preferably, the shear stress generated by the shear thickening hemostatic material in S3 at a shear rate of 0.1s ^-1 -1000s ^-1 is between 100 and 1000Pa, and the preparation of the shear thickening solution can use nano-silica It can be obtained by reacting with acetic acid or ethyl acetate or polyethylene glycol solution, reacting micron-sized particle PMMA with mineral oil or silicone oil and surfactant to form a suspension liquid, and acting on starch and water. Taking starch solution as an example, the mass fraction of starch solution is 53%~57%, and the mass ratio of chitosan to starch is 1:200~1:500.

Preferably, the molar ratio of the chitosan unit to the lauryl aldehyde functional group in S1 is 100:1~10:1, the reaction pH is 4.5~5.5, the reaction time is 3~5h, and the reaction temperature is room temperature.

Preferably, sodium cyanoborohydride is added to S2 so that the molar ratio of chitosan units to sodium cyanoborohydride is (4~15):(2~5), the reaction temperature is room temperature, and the reaction time is 11~13h. The hydrophobically modified chitosan with grafting ratio of 0.24% to 8.43% was obtained.

Preferably, in S2, the pH is adjusted to 9.5-10.5, and 70%, 80%, and 100% ethanol aqueous solutions are sequentially used to wash to neutrality.

Finally, the resulting shear-thickening arterial hemostatic gel is a pure white paste.

The present invention also protects the hemostatic material prepared by the above preparation method.

Compared with the prior art, the present invention has the following beneficial effects:

The hemostatic material provided by the invention can generate a considerable shear stress in a short time to resist the impact of arterial blood, block the bleeding wound of the artery, stop the bleeding quickly, and can form a gel-like protective film on the wound surface after the hemostasis is completed. , thereby shortening the bleeding time and reducing the amount of bleeding. At the same time, its hemostasis process can be independent of the autologous coagulation process, and it is effective for patients taking anticoagulant drugs such as aspirin or clopidogrel, or patients with platelet dysfunction and hemophilia.

Description of drawings

Figure 1 is a scanning electron microscope image of N-alkylated hydrophobically modified chitosan provided by the present invention, from left to right are the internal structure, surface structure and overall appearance structure of N-alkylated hydrophobically modified chitosan.

Figure 2 is an observation diagram of blood coagulation after the N-alkylated hydrophobically modified chitosan provided by the present invention is mixed with blood.

Figure 3 is a product diagram of the obtained shear thickening arterial hemostatic gel provided by the present invention.

Fig. 4 is the result diagram of shear stress generated under the rheometer of the obtained product provided by the present invention.

Figure 5 is a diagram showing the effect of the obtained product on red blood cells observed under a scanning electron microscope provided by the present invention.

FIG. 6 is a comparison diagram of the toxicity test of the negative control group, zeolite, chitosan, the obtained product, and the positive control group provided by the present invention.

Fig. 7 is the comparison chart of the hemostasis time of zeolite, chitosan and the obtained product provided by the present invention.

FIG. 8 is a comparison chart of the bleeding volume of zeolite, chitosan and the obtained product provided by the present invention.

FIG. 9 is a comparison diagram of the zeolite, chitosan, gauze and the obtained product provided by the present invention in a rat femoral artery hemostasis model test.

Detailed ways

The present invention will be further described below with reference to specific embodiments and accompanying drawings, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

Example 1: Preparation of Shear Thickening Arterial Hemostatic Gel

1. Three preparation methods of N-alkylated hydrophobically modified chitosan, the main bearer of hemostatic function, are provided:

1. 4g chitosan (molecular weight is 19k~31kDa, deacetylation degree is 75%~85%) is dissolved in 220mL 0.2mol/L acetic acid solution, stirred at room temperature to dissolve, and then 150mL ethanol is added. After it was mixed evenly, lauraldehyde was added and the molar ratio of chitosan unit and lauraldehyde functional group was controlled to be 10:1, the reaction pH was adjusted to 5.1, and the reaction was carried out at room temperature for 4 hours. Sodium cyanoborohydride was slowly added dropwise so that the molar ratio of chitosan units to sodium cyanoborohydride was 1:3. The reaction was carried out at room temperature for 12h. After the reaction was completed, the pH was adjusted to 10 with sodium hydroxide. The precipitate was collected by filtration, and then washed with 70%, 80%, and 100% aqueous ethanol solution until neutral. Freeze-dried, pulverized. The obtained product was mixed with starch and dissolved in 2% acetic acid solution to obtain a 0.8wt% polymer solution, and the final starch mass fraction was 55%, and the mass ratio of chitosan to starch was 1:500. The grafting rate of hydrophobically modified chitosan was about 8.43% by elemental analysis and substitution degree determination.

2. 4g chitosan (molecular weight 19k~31kDa, deacetylation degree 75%~85%) was dissolved in 220mL 0.2mol/L acetic acid solution, stirred at room temperature to dissolve, and then 150mL ethanol was added. After it was mixed evenly, lauraldehyde was added and the molar ratio of chitosan unit and lauraldehyde functional group was controlled to be 20:1, the reaction pH was adjusted to 5.1, and the reaction was carried out at room temperature for 4 hours. Sodium cyanoborohydride was slowly added dropwise so that the molar ratio of chitosan units to sodium cyanoborohydride was 1:3. The reaction was carried out at room temperature for 12h. After the reaction was completed, the pH was adjusted to 10 with sodium hydroxide. The precipitate was collected by filtration, and then washed with 70%, 80%, and 100% aqueous ethanol solution until neutral. Freeze-dried, pulverized. The obtained product was mixed with starch and dissolved in 2% acetic acid solution to obtain 0.8wt% polymer solution, and the final starch mass fraction was 55%, and the mass ratio of chitosan to starch was 1:400. The grafting rate of hydrophobically modified chitosan was about 5.06% by elemental analysis and substitution degree determination.

3. 4g chitosan (molecular weight 19k~31kDa, deacetylation degree 75%~85%) was dissolved in 220mL 0.2mol/L acetic acid solution, stirred at room temperature to dissolve, and then 150mL ethanol was added. After it was mixed evenly, lauraldehyde was added and the molar ratio of chitosan unit and lauraldehyde functional group was controlled to be 100:1, the reaction pH was adjusted to 5.1, and the reaction was carried out at room temperature for 4 hours. Sodium cyanoborohydride was slowly added dropwise so that the molar ratio of chitosan units to sodium cyanoborohydride was 1:3. The reaction was carried out at room temperature for 12h. After the reaction was completed, the pH was adjusted to 10 with sodium hydroxide. The precipitate was collected by filtration, and then washed with 70%, 80%, and 100% aqueous ethanol solution until neutral. Freeze-dried, pulverized. The obtained product was mixed with starch and dissolved in 2% acetic acid solution to obtain 0.8wt% polymer solution, and the final starch mass fraction was 55%, and the mass ratio of chitosan to starch was 1:200. The graft ratio of hydrophobically modified chitosan was about 0.24% by elemental analysis and substitution degree determination.

2. The preparation of shear thickening solution can use nano-silicon dioxide and acetic acid or ethyl acetate or polyethylene glycol solution reaction, micron-sized particle PMMA and mineral oil or silicone oil and surfactant to react into suspension liquid, starch and water effect and other methods.

Taking starch as an example, hydrophobically modified chitosan with a grafting rate of 0.24% ~ 8.43% was dissolved in 2% acetic acid solution to obtain a polymer solution of 0.2% ~ 1 wt%, which was uniformly mixed with the starch solution to make the final polymer solution. The mass fraction of starch is 53%~57%, and the mass ratio of chitosan to starch is 1:200~1:500. The obtained product is a shear thickening arterial hemostatic gel, the gel is pure white paste, and the shear stress generated by the shear thickening hemostatic material at a shear rate of 0.1s ^-1 -1000s ^-1 is 100~ 1000Pa, as shown in Figures 1 and 3.

Example 2: Coagulation observations of shear thickening arterial hemostatic gel mixed with red blood cells

Prepare the whole blood for anticoagulation, add the unmodified chitosan solution and the shear-thickening gel prepared in Example 1 into the transparent plastic reagent tube containing the red blood cell suspension in a volume ratio of 1:1, Mix with a shaker, invert the sample, and observe whether it becomes a gel or a solution after mixing.

Figure 2 shows the coagulation observation after mixing the hydrophobically modified chitosan and blood. It can be seen that the viscosity of the shear-thickening arterial hemostatic gel provided by the present invention is greatly enhanced compared with ordinary chitosan after mixing with blood.

Example 3: Using a rheometer to detect the shear stress generated when the product resists high shear rates

The shear stress generated when the shear rate of the obtained product is 0.1s ^-1 -1000 ^-1 under the rheometer, because the reference to the literature shows that the shear rate of human arterial blood generally falls in the range of 50-1000s ^-1 , The generated shear stress can generally fall in the interval of 100-1000Pa. Drawing the results, it can be seen that the product exhibits shear thickening effect within the shear rate range of arterial blood. Take the three implementation cases cited above as an example, see Figure 4.

Example 4 Scanning electron microscope observation of the effect of the obtained product on red blood cells

As shown in Figure 5, the effects of the obtained product and pure chitosan on erythrocyte aggregation and morphology were observed by SEM. Comparing the two groups, it can be clearly seen that the obtained product can tightly bind the red blood cells together, the red blood cells are completely deformed, and after aggregation, the layers are superimposed to form blood clots, while the pure chitosan has no obvious effect on the red blood cells.

Example 5 Co-culture with human cells, observation of shear thickening arterial hemostatic gel

The results are shown in Figure 6. The zeolite, chitosan, and shear-thickening arterial hemostatic gel were co-cultured with fibroblasts (3T3). It can be seen that the product has minimal cytotoxicity.

Example 6 Thromboelastography (TEG) detection after blending shear thickening arterial hemostatic gel with whole blood

Thromboelastography (TEG) is a function of time, reflecting the process of clot formation and changes in strength. Compared with APTT, PT and FT, TEG can fully reflect the whole process of coagulation by providing more detailed coagulation information. TEG test results can provide four parameters of the coagulation process: (1) reaction time R, the time required from initiation of coagulation to the formation of fibrin; (2) coagulation time K, the process of dynamic clot formation; (3) α angle , the speed of fibrin crosslinking; (4) depict the maximum amplitude (MA) in the graph, the maximum intensity of the blood clot.

Table 1 The detection value of shear thickening arterial hemostatic gel and chitosan on whole blood coagulation

polymer solution R (min) K (min) α (deg) MA (mm) normal range 5–10 1–3 53–72 50–70 Negative control is normal saline that does not affect coagulation 6.2 1.7 66.2 58.1 Chitosan 11.8↑ 3.3↑ 50.8↓ 56.8 Shear Thickening Arterial Hemostatic Gel 3.0↓ 1.6 67.7 67.1

In the whole process of chitosan coagulation, the R value was 11.8, which exceeded the normal value, which indicated that chitosan would prolong the time required to form fibrin, and the blood clot dynamic time of chitosan was correspondingly prolonged. The rate of protein cross-linking decreased, suggesting that chitosan affects the structure of fibrinogen. However, in the shear-thickening hemostatic gel group, the R value decreased, and the time from coagulation to fibrinogen formation decreased, indicating that the shear-thickening hemostatic gel can greatly shorten the time to form fibrin and the formation of blood clots. .

Example 7 SD rat femoral artery hemostasis model to evaluate the hemostatic efficacy of the composite sponge

The zeolite, chitosan, gauze and the obtained product provided by the present invention are shown in Fig. 9 through the rat femoral artery hemostasis model test to test the hemostatic effect. As can be seen from Fig. 9, the hemostatic sponge provided by the present invention has a hemostatic effect. Time is fast.

The shear thickening arterial hemostatic gel was applied to the animal hemostasis model. It was observed that after the hemostatic sponge was in contact with the blood on the cut surface, it could quickly concentrate the blood, and after the hemostasis was completed, the material was uncovered and it was found that the material could form a layer of gel with the wound surface. The protective film (shown in Figure 9) can shorten the bleeding time and reduce the amount of bleeding, and its effect is obviously better than that of zeolite, chitosan and gauze.

Claims (8)

1. A method of preparing a shear-thickening hemostatic material, comprising the steps of:

s1, dissolving chitosan, wherein the molecular weight of the chitosan is 19 k-31 kDa, the deacetylation degree is 75-85%, adding lauraldehyde for reaction, adjusting the pH value, and reacting for a certain time;

s2, reacting the product in the S1 with sodium cyanoborohydride; after the reaction is finished, adjusting the pH value, washing, drying and crushing;

s3, dissolving the crushed product in the step S2, and mixing the dissolved product with a shear thickening solution to obtain the shear thickening hemostatic material;

the shear thickening solution in the S3 is a starch solution, the mass fraction of the starch solution is 53-57%, and the mass ratio of chitosan to starch is 1: 200-1: 500.

2. The method according to claim 1, wherein the solvents used for dissolving the chitosan are acetic acid and ethanol, and the mass-to-volume ratio of the chitosan to the solvents is 1: (90-110).

3. The method according to claim 2, wherein the chitosan is dissolved in the acetic acid solution and then the ethanol solution is added.

4. The method of claim 1, wherein the hemostatic material that is shear-thickened in S3 is 0.1S^-1～1000s^-1The shear stress generated at the shear rate is 100 to 1000 Pa.

5. The preparation method according to claim 1, wherein the molar ratio of the chitosan unit to the lauraldehyde functional group in S1 is 100: 1-10: 1, the reaction pH is 4.5-5.5, the reaction time is 3-5 h, and the reaction temperature is room temperature.

6. The preparation method according to claim 1, wherein sodium cyanoborohydride is added in S2 so that the molar ratio of chitosan units to sodium cyanoborohydride is (4-15): (2-5), the reaction temperature is room temperature, and the reaction time is 11-13 h.

7. The preparation method according to claim 1, wherein the pH value in S2 is adjusted to 9.5-10.5, and the solution is washed to be neutral by 70%, 80% and 100% ethanol aqueous solution in sequence.

8. A hemostatic material prepared by the method of any one of claims 1 to 7.

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