CN107137756B - Preparation method of shear thickening hemostatic material - Google Patents
Preparation method of shear thickening hemostatic material Download PDFInfo
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- CN107137756B CN107137756B CN201710230098.3A CN201710230098A CN107137756B CN 107137756 B CN107137756 B CN 107137756B CN 201710230098 A CN201710230098 A CN 201710230098A CN 107137756 B CN107137756 B CN 107137756B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/08—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/04—Materials for stopping bleeding
Abstract
The invention discloses a preparation method of a shear thickening hemostatic material, which comprises the following steps: s1, dissolving chitosan, adding lauraldehyde to react, adjusting pH, 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; and S3, dissolving the crushed product in the step S2, and mixing the dissolved product with the shear thickening solution to obtain the shear thickening hemostatic material. Aiming at the characteristics of high blood flow speed, high pressure and the like of the arterial bleeding wound, the invention provides the method for plugging the arterial bleeding wound by adopting shear thickening liquid by using the principle of 'liquid body armor'. In addition, the invention compounds the hydrophobic modified chitosan with the hemostatic function and the solution with the shear thickening function, thereby obtaining the multifunctional hemostatic material which can not only plug the hypertension wound, but also promote the blood coagulation of the wound. Meanwhile, the material for artery hemostasis provided by the invention has the advantages of obvious effect, convenience in processing and low cost.
Description
Technical Field
The invention belongs to the technical field of hemostatic materials, and particularly relates to a preparation method of a shear-thickening hemostatic material.
Background
The chitosan hemostatic material is obtained by deacetylating naturally and widely existing chitin, has good biocompatibility and certain hemostatic and bacteriostatic effects, is widely used for designing hemostatic materials, has strong effects on hydrophobically modified chitosan and blood cells, is prepared into a foam spray and can realize very effective hemostatic effects.
The shear thickening fluid is a nano particle solvent in a solid-liquid mixing state, and the viscosity is increased along with the increase of the shear rate in a range of bearing a certain shear speed, so that the resistance effect on the shear action is shown. When the shear force is large enough, the particles are actually "locked" to each other, and even exhibit solid-like mechanical properties. The shear thickening principle is now widely used in protective materials such as liquid body armor.
According to the design concept of material shear thickening, the hemostatic material is a discontinuous shear thickening liquid, has excellent impact resistance, has better impact resistance to ejected arterial blood flow due to flow-solid conversion under an impact condition, and has a quick hemostatic function due to the hydrophobic modified chitosan hemostatic material. The shear thickening material is compounded with the hydrophobic modified chitosan to obtain the multifunctional hemostatic material which can not only block the hypertensive wound but also promote the blood coagulation of the wound.
Disclosure of Invention
The present invention is directed to a method of preparing a shear-thickening hemostatic material in accordance with the deficiencies of the prior art.
The purpose of the invention is realized by the following technical scheme:
the invention provides a preparation method of a shear thickening hemostatic material, which comprises the following steps:
s1, dissolving chitosan in acetic acid and ethanol solution, adding lauraldehyde for reaction, adjusting pH, 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;
and S3, dissolving the crushed product in the step S2, and mixing the dissolved product with the shear thickening solution to obtain the shear thickening hemostatic material.
The invention provides a material which is prepared by combining a shear thickening material and a chitosan material and has the advantages of quick hemostasis and excellent hemostasis effect based on the knowledge of the shear thickening material, the understanding of the human coagulation process and the knowledge of bionics. The existing shear thickening material is mostly applied to preparation of products with high impact strength, the characteristic of the shear thickening material can be just applied to blocking arterial bleeding wounds, and the material does not relate to the field of hemostatic materials. The N-alkylated hydrophobic modified chitosan compounded with the N-alkylated hydrophobic modified chitosan can be combined with certain components in blood or adhered to form a network-shaped clot to replace the action of fibrin, so that the advantage is that the hemostasis process can be independent of the auto-coagulation process and can artificially control and enhance the coagulation process. The hydrophobic chain segment of the N-alkylated hydrophobically modified chitosan can generate a hydrophobic combined acting force with the hydrophobic part of a cell membrane, and the ionized chitosan has positive charge which can generate an electrostatic attraction acting force with the cell membrane to generate adsorption, so that the material can aggregate blood cells like fibrin to coagulate blood.
Preferably, the molecular weight of the chitosan is 19 k-31 kDa, and the deacetylation degree is 75-85%.
Preferably, the solvent used to dissolve the chitosan is acetic acid or ethanol.
Preferably, the mass-to-volume ratio of chitosan to solvent is 1: (90-110).
Preferably, the hemostatic material shear-thickened in S3 is at 0.1S-1-1000s-1The shear stress generated at the shear rate is 100-1000Pa, the shear thickening solution can be prepared by the methods of reacting nano silicon dioxide with acetic acid or ethyl acetate or polyethylene glycol solution, reacting micron-sized particles PMMA with mineral oil or silicone oil and surfactant to form suspension, reacting starch with water and the like. Taking a starch solution as an example, the mass fraction of the 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 lauraldehyde functional group in the 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.
Preferably, sodium cyanoborohydride is added into S2 to ensure 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. Obtaining the hydrophobic modified chitosan with the grafting rate of 0.24-8.43%.
Preferably, the pH value of the S2 is adjusted to 9.5-10.5, and the solution is washed to be neutral by 70%, 80% and 100% ethanol water solution in sequence.
Finally, the obtained shear thickening artery hemostatic gel is pure white paste.
The invention also protects the hemostatic material prepared by the preparation method.
Compared with the prior art, the invention has the following beneficial effects:
the hemostatic material provided by the invention can generate considerable shear stress in a short time to resist the impact of arterial blood, block arterial bleeding wounds and rapidly stop bleeding, and a layer of gel-like protective film can be formed on a wound surface after the hemostasis is finished, so that the bleeding time is shortened and the bleeding amount is reduced.
Drawings
FIG. 1 is a scanning electron microscope image of the N-alkylated hydrophobically modified chitosan provided by the present invention, from left to right, respectively showing the internal structure, surface structure and appearance overall structure of the N-alkylated hydrophobically modified chitosan.
FIG. 2 is a blood coagulation observation picture of the N-alkylated hydrophobically modified chitosan provided by the invention after being mixed with blood.
FIG. 3 is a diagram of the resulting shear-thickened arterial hemostatic gel product provided by the present invention.
FIG. 4 is a graph showing the results of shear stress produced by the resulting product provided by the present invention under a rheometer.
FIG. 5 is a diagram of the effect of the product on erythrocytes observed under a scanning electron microscope provided by the invention.
FIG. 6 is a comparison chart of toxicity tests of the negative control group, zeolite, chitosan, the obtained product and the positive control group provided by the invention.
Figure 7 is a graph comparing the hemostatic time of the zeolite, chitosan and resulting product provided by the present invention.
FIG. 8 is a graph comparing the amount of bleeding of zeolite, chitosan and the resulting product provided by the present invention.
FIG. 9 is a comparison of the zeolite, chitosan, gauze and resulting product provided by the present invention in a rat femoral artery hemostasis model test.
Detailed Description
The present invention will be further described with reference to the following specific examples and drawings, which are not intended to limit the invention in any manner. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the present invention are commercially available.
Example 1: preparation of shear thickening arterial hemostatic gel
The preparation method of the N-alkylated hydrophobically modified chitosan which is mainly used for providing the hemostasis function comprises the following three preparation methods:
1. 4g of chitosan (with the molecular weight of 19 k-31 kDa and the deacetylation degree of 75% -85%) is dissolved in 220mL of 0.2mol/L acetic acid solution, stirred at room temperature to be dissolved, and then 150mL of ethanol is added. And (3) after the raw materials are uniformly mixed, adding lauraldehyde, controlling the molar ratio of the chitosan unit to the lauraldehyde functional group to be 10:1, adjusting the reaction pH to be 5.1, and reacting for 4 hours at room temperature. And slowly dripping sodium cyanoborohydride to ensure that the molar ratio of the chitosan unit to the sodium cyanoborohydride is 1: 3. The reaction was carried out at room temperature for 12 h. After the reaction was completed, the pH was adjusted to 10 with sodium hydroxide. Filtering to obtain precipitate, and washing with 70%, 80% and 100% ethanol water solution respectively to neutrality. Freeze-drying and grinding. The obtained product and starch are mixed and dissolved in 2 percent acetic acid solution to prepare 0.8 percent polymer solution by weight, and the mass fraction of the final starch is 55 percent, and the mass ratio of the chitosan to the starch is 1: 500. The grafting rate of the hydrophobically modified chitosan was about 8.43% by elemental analysis and degree of substitution measurement.
2. 4g of chitosan (with the molecular weight of 19 k-31 kDa and the deacetylation degree of 75% -85%) is dissolved in 220mL of 0.2mol/L acetic acid solution, stirred at room temperature to be dissolved, and then 150mL of ethanol is added. And (3) after the raw materials are uniformly mixed, adding lauraldehyde, controlling the molar ratio of the chitosan unit to the lauraldehyde functional group to be 20:1, adjusting the reaction pH to be 5.1, and reacting for 4 hours at room temperature. And slowly dripping sodium cyanoborohydride to ensure that the molar ratio of the chitosan unit to the sodium cyanoborohydride is 1: 3. The reaction was carried out at room temperature for 12 h. After the reaction was completed, the pH was adjusted to 10 with sodium hydroxide. Filtering to obtain precipitate, and washing with 70%, 80% and 100% ethanol water solution respectively to neutrality. Freeze-drying and grinding. The obtained product and starch are mixed and dissolved in 2 percent acetic acid solution to prepare 0.8 percent polymer solution by weight, and the mass fraction of the final starch is 55 percent, and the mass ratio of the chitosan to the starch is 1: 400. The grafting rate of the hydrophobically modified chitosan was about 5.06% by elemental analysis and degree of substitution measurement.
3. 4g of chitosan (with the molecular weight of 19 k-31 kDa and the deacetylation degree of 75% -85%) is dissolved in 220mL of 0.2mol/L acetic acid solution, stirred at room temperature to be dissolved, and then 150mL of ethanol is added. And (3) after the raw materials are uniformly mixed, adding lauraldehyde, controlling the molar ratio of the chitosan unit to the lauraldehyde functional group to be 100:1, adjusting the reaction pH to be 5.1, and reacting for 4 hours at room temperature. And slowly dripping sodium cyanoborohydride to ensure that the molar ratio of the chitosan unit to the sodium cyanoborohydride is 1: 3. The reaction was carried out at room temperature for 12 h. After the reaction was completed, the pH was adjusted to 10 with sodium hydroxide. Filtering to obtain precipitate, and washing with 70%, 80% and 100% ethanol water solution respectively to neutrality. Freeze-drying and grinding. The obtained product and starch are mixed and dissolved in 2 percent acetic acid solution to prepare 0.8 percent polymer solution by weight, and the mass fraction of the final starch is 55 percent, and the mass ratio of the chitosan to the starch is 1: 200. The grafting rate of the hydrophobically modified chitosan was about 0.24% by elemental analysis and degree of substitution measurement.
Secondly, the shear thickening solution can be prepared by the methods of reacting nano silicon dioxide with acetic acid or ethyl acetate or polyethylene glycol solution, reacting micron-sized particles PMMA with mineral oil or silicon oil and surfactant to form suspension liquid, reacting starch with water and the like.
Taking starch as an example, dissolving hydrophobically modified chitosan with the grafting rate of 0.24-8.43% in 2% acetic acid solution to prepare 0.2-1 wt% of polymer solution, and uniformly mixing the polymer solution with the starch solution to ensure that the final mass fraction of the starch is 53-57%, and the mass ratio of the chitosan to the starch is 1: 200-1: 500. The obtained product is shear thickening artery hemostatic gel which is pure white paste, and the shear thickening hemostatic material is in 0.1s-1-1000s-1The shear stress generated under the shear rate is 100-1000Pa, as shown in figures 1 and 3.
Example 2: coagulation observation result of mixed shear thickening arterial hemostatic gel and red blood cell
Anticoagulated whole blood was prepared, and the unmodified chitosan solution and the shear-thickening gel prepared in example 1 were mixed in a volume ratio of 1: 1, adding the mixture into a transparent plastic reagent tube filled with erythrocyte suspension, uniformly mixing the mixture by using a vibration mixer, inverting the sample, and observing whether the mixture becomes gel or solution after mixing.
Fig. 2 is a blood coagulation observation of the mixture of the hydrophobically modified chitosan and blood, and it can be seen that the viscosity of the shear thickening arterial hemostatic gel provided by the invention is greatly enhanced compared with that of the common chitosan after the shear thickening arterial hemostatic gel is mixed with the blood.
Example 3: the shear stress generated when the product resists high shear rate is detected by a rheometer
The shear rate of the obtained product under a rheometer is 0.1s-1-1000-1The shear stress is generated because the human artery blood shear rate can generally fall between 50 and 1000s as can be known by examining the literature-1The generated shear stress can generally fall in the interval of 100-1000 Pa. The results are graphed and it is shown that the product exhibits shear thickening effect over the range of arterial blood shear rates, as exemplified by the 3 examples given above, see figure 4.
Example 4 scanning Electron microscopy of the Effect of the obtained products on erythrocytes
As shown in fig. 5, the effect of the obtained product and pure chitosan on erythrocyte aggregation and morphology was observed by SEM. The comparison of the two groups of controls can obviously show that the obtained product can tightly combine red blood cells together, the red blood cells are completely deformed, and are superposed layer by layer to form blood clots after being gathered, while the effect of pure chitosan on the red blood cells is not obvious.
Example 5 Co-culture with human cells, Observation of shear thickening arterial hemostatic gels
As shown in FIG. 6, the product was found to be extremely cytotoxic when zeolite, chitosan, shear thickening arterial hemostatic gel and fibroblasts (3T 3) were co-cultured.
Example 6 Thrombelastogram (TEG) assay of shear thickening arterial hemostatic gel blended with Whole blood
Thromboelastography (TEG) is a function of time, reflecting the process of blood clot formation and intensity changes. Compared with the tests APTT, PT and FT, TEG can completely reflect the whole process of coagulation by providing more detailed coagulation information. The TEG test results can provide four parameters of the coagulation process: (1) reaction time R, the time required from initiation of coagulation to formation of fibrin; (2) clotting time K, the process of dynamic clot formation; (3) angle α, speed of fibrin cross-linking; (4) maximum Amplitude (MA) in the plot, maximum intensity of the clot is depicted.
TABLE 1 detection values 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 |
| The negative control is normal saline without affecting blood coagulation | 6.2 | 1.7 | 66.2 | 58.1 |
| Chitosan | 11.8↑ | 3.3↑ | 50.8↓ | 56.8 |
| Shear thickening arterial hemostasisGel | 3.0↓ | 1.6 | 67.7 | 67.1 |
In the whole process of chitosan coagulation, the R value is 11.8, and the value exceeds the normal value, which indicates that the chitosan can prolong the time required for forming fibrin, the blood clot dynamic time of the chitosan is correspondingly prolonged, and the crosslinking speed of the fibrin is reduced, which indicates that the chitosan can influence the structure of fibrinogen. The reduced R value of the shear thickening hemostatic gel group, reduced the time from clotting to fibrinogen formation, indicating that the shear thickening hemostatic gel allows for a substantial reduction in fibrin formation time and clot formation time.
Example 7 SD rat femoral artery hemostasis model evaluation of hemostatic effectiveness of composite sponges
The hemostatic effect of the zeolite, chitosan, gauze and the obtained product provided by the invention tested by a rat femoral artery hemostatic model test is shown in fig. 9, and as can be seen from fig. 9, the hemostatic sponge provided by the invention has rapid hemostatic time.
The shear thickening artery hemostatic gel is applied to an animal hemostatic model process, observation shows that after hemostatic sponge is contacted with section blood, blood can be concentrated rapidly, and after hemostasis is finished, a material is uncovered, and a layer of gelatinous protective film (shown in figure 9) can be formed between the material and a wound surface, so that the bleeding time is shortened, the bleeding amount is reduced, and the effect of the shear thickening artery hemostatic gel is obviously superior to 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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| CN109627499B (en) * | 2018-11-30 | 2020-11-27 | 湖南工业大学 | Preparation method of biomass composite gel with shear thickening effect |
| CN112915254B (en) * | 2021-02-04 | 2022-09-09 | 绍兴百立盛新材料科技有限公司 | Grafted long-alkyl chitosan/sodium tripolyphosphate gel and preparation method and application thereof |
| CN114832148A (en) * | 2022-04-20 | 2022-08-02 | 上海交通大学医学院附属第九人民医院 | Hemostatic material with antibacterial and anti-inflammatory functions and preparation method and application thereof |
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Inventor after: Xue Wei Inventor after: Xu Yuying Inventor after: Zhang Yi Inventor after: Wu Chengguang Inventor after: Ma Xin Inventor before: Xue Wei Inventor before: Xu Yuying Inventor before: Zhang Yi Inventor before: Wu Chengguang Inventor before: Ma Xin |