CN109893678B - Rectorite-chitin nanogel composite hemostatic material and preparation method thereof - Google Patents
Rectorite-chitin nanogel composite hemostatic material and preparation method thereof Download PDFInfo
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- CN109893678B CN109893678B CN201910151155.8A CN201910151155A CN109893678B CN 109893678 B CN109893678 B CN 109893678B CN 201910151155 A CN201910151155 A CN 201910151155A CN 109893678 B CN109893678 B CN 109893678B
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Abstract
The invention discloses a rectorite-chitin nanogel composite hemostatic material and a preparation method thereof. Dissolving chitin powder in a sodium hydroxide/urea system to obtain a chitin aqueous solution; then, the rectorite is evenly dispersed in the chitin solution to obtain suspension. Stirring the suspension at a high speed by a dispersion machine, dialyzing by ultrapure water, and freeze-drying to obtain the rectorite-chitin nanogel composite hemostatic material. The cytotoxicity experiment and the blood coagulation experiment show that when the weight ratio of the chitin powder to the rectorite powder is 1: 1-3, the rectorite-chitin nano gel composite hemostatic material is non-toxic, the hemostatic speed is faster than that of the rectorite and the chitin nano gel, and the composite hemostatic material has high popularization value. The preparation method disclosed by the invention is mild in reaction conditions, green and environment-friendly, low in cost, simple in preparation, capable of realizing one-time large-scale preparation and wide in application potential in the field of biomedicine.
Description
Technical Field
The invention relates to a rectorite-chitin nanogel composite hemostatic material and a preparation method thereof, belonging to the field of natural polymers and also belonging to the fields of chemistry and medicine.
Background
Uncontrolled massive bleeding is a major cause of death in war, automobile accidents, and other accidents. The hemostatic material Quikclot taking zeolite as a raw material saves hundreds of people in a battlefield, however, the zeolite can emit a large amount of heat in the hemostatic process, so that secondary damage to wounds is easily caused, and the application of the Quikclot is greatly limited. Commercial hemostats Celox, Combat Gauze, HemCon, FastAct Seraseal, etc. are expensive and not suitable for large-scale use. Therefore, the development of a hemostatic material with low side effect, low price, easy availability and good hemostatic effect is an urgent problem to be solved.
Chitin is a polysaccharide second to cellulose in quantity on earth, and is a nitrogen-containing natural organic high molecular compound with the largest quantity except protein in nature. The chitin has good biocompatibility and has the functions of promoting wound healing and stopping bleeding. Studies have shown that chitin binds red blood cells while aggregating platelets more than chitosan (y.okamoto, r.yano, k.miyatake, i.tomohiro, y.shigemasa and s.miniam, Carbohydrate polymers,2003,53, 337-. The current report mainly adopts chitin powder with larger particle size, the chitin is prepared into nano gel, the specific surface area of the nano gel can be increased, blood can be rapidly absorbed in the hemostasis process, and erythrocytes and aggregated platelets can be better combined, so the hemostasis effect can be better exerted. However, the hemostatic speed of chitin nanogels is not ideal. Clay substances have good coagulation effects because of the negative charge on their surfaces and the fact that they bind to plasma proteins and activate the intrinsic coagulation mechanism, but pure clay substances such as rectorite have certain biological toxicity and are not biologically safe as hemostatic agents (j.i. dawson and r.o. oreffo, advanced materials,2013,25, 4069-materials 4086.).
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a rectorite-chitin nanogel composite hemostatic material, which not only solves the problem of high toxicity of single rectorite, but also solves the problem of low hemostatic speed of rectorite and chitin nanogel.
The technical scheme provided by the invention is as follows:
a preparation method of a rectorite-chitin nanogel composite hemostatic material comprises the following steps: adding chitin powder into an aqueous solution containing 6-12 wt% of NaOH and 2-8 wt% of urea, and circularly freezing to obtain a chitin aqueous solution with uniform dissolution, wherein the content of the chitin is 0.5-10 wt%; uniformly dispersing rectorite powder into a chitin aqueous solution to obtain a suspension, wherein the weight ratio of the chitin powder to the rectorite powder is 1: 1-3; then stirring the suspension at a speed of more than 5000r/min to obtain a colloidal emulsion; dialyzing the obtained colloidal emulsion in ultrapure water to neutrality, and finally freeze-drying to obtain the rectorite-chitin nano-gel composite hemostatic material.
Preferably:
the NaOH content was 8 wt% and the urea content was 4 wt%.
The temperature of the circulating refrigeration is-10 to-40 ℃.
The number of times of the cyclic freezing is 2 or more.
The stirring time is 5-90 min.
The stirring speed is 6000 to 20000 r/min.
The cut-off molecular weight of dialysis paper used for dialyzing the colloidal emulsion is 8000-14000.
A rectorite-chitin nanogel composite hemostatic material is prepared by the preparation method.
The chitin powder of the invention can be the epidermis of crustaceans such as shrimps, crabs and the like and various insects, and can also be one or more of the skeletons of mollusks such as cuttlefish, shellfish and the like and the chitin in the cell walls of fungi. Before use, the product is purified by known techniques, such as acid treatment to remove calcium salt, alkali treatment to remove protein, oxidation treatment to decolorize, etc.
The method comprises the steps of firstly, circularly freezing and dissolving chitin powder in a NaOH and urea system to obtain a chitin aqueous solution, then forming aggregated nanogel by self-assembling molecular chains in the chitin aqueous solution due to the increase of the system temperature in the process of high-speed stirring by a dispersion machine, and simultaneously uniformly dispersing the aggregated nanogel due to the action of high-speed shearing. Under high-speed stirring, the chitin and the rectorite powder can be uniformly mixed, and the formed chitin nanogel is tightly adhered to the surface of the rectorite through the action of hydrogen bonds and electric charges, so that the uniform and good-biocompatibility rectorite-chitin nanogel composite hemostatic material is prepared.
According to the invention, chitin and rectorite are compounded for the first time to prepare nano-level gel through physical change, and the formed chitin nano-gel is adhered to the surface of the rectorite through the action of hydrogen bonds or positive and negative charges, so that the chitin is tightly combined with the rectorite with the same blood coagulation effect to form the composite hemostatic material of rectorite-chitin nano-gel without biological toxicity. The preparation method disclosed by the invention is mild in reaction conditions, green and environment-friendly, low in cost, simple in preparation, capable of realizing one-time large-scale preparation and wide in application potential in the field of biomedicine.
Drawings
FIG. 1 is a transmission electron microscope image of a rectorite-chitin nanogel composite hemostatic material; wherein, fig. 1(a) is a transmission electron micrograph of sample 4 with a chitin/rectorite weight ratio of 1/1, and fig. 1(b) is a transmission electron micrograph of sample 5 with a chitin/rectorite weight ratio of 3/1.
Fig. 2 is a bar graph of table 2.
Detailed Description
The technical solutions of the present invention will be specifically described below by examples, but these specific examples do not limit the scope of the present invention in any way.
Example 1
Adding 2 parts by weight of chitin powder into 98 parts by weight of aqueous solution containing 8 wt% of NaOH and 4 wt% of urea, and circularly freezing for 2 times at-30 ℃ to dissolve the chitin powder into uniform aqueous solution of chitin; stirring the chitin aqueous solution with a dispersion machine at 10000r/min for 30min to obtain colloidal emulsion; finally, the colloidal emulsion is dialyzed in ultrapure water for 6 days to neutrality by using dialyzing paper with the molecular weight cutoff of 8000-14000, and finally, the chitin nanogel material, namely the sample 8 is obtained by freeze drying.
Example 2
Adding 2 parts by weight of chitin powder into 92 parts by weight of aqueous solution containing 8 wt% of NaOH and 4 wt% of urea, circularly freezing for 2 times at-30 ℃ to dissolve the chitin powder into uniform aqueous solution of chitin, and uniformly dispersing 6 parts by weight of rectorite powder into the aqueous solution of chitin to obtain suspension; stirring the suspension at 10000r/min for 30min by a dispersion machine to obtain colloidal emulsion; finally, the colloidal emulsion is dialyzed in ultrapure water for 6 days by using dialyzing paper with the molecular weight cutoff of 8000-.
Example 3
Adding 2 parts by weight of chitin powder into 94 parts by weight of aqueous solution containing 8 wt% of NaOH and 4 wt% of urea, and circularly freezing for 2 times at-30 ℃ to dissolve the chitin powder into uniform aqueous solution of chitin; uniformly dispersing 4 parts by weight of rectorite powder into a chitin aqueous solution to obtain a suspension; stirring the suspension at 10000r/min for 30min by a dispersion machine to obtain colloidal emulsion; finally, the colloidal emulsion is dialyzed in ultrapure water for 6 days to neutrality by using dialyzing paper with the molecular weight cutoff of 8000-.
Example 4
Adding 2 parts by weight of chitin powder into 96 parts by weight of aqueous solution containing 8 wt% of NaOH and 4 wt% of urea, and circularly freezing for 2 times at-30 ℃ to dissolve the chitin powder into uniform aqueous solution of chitin; uniformly dispersing 2 parts by weight of rectorite powder into a chitin aqueous solution to obtain a suspension; stirring the suspension at 10000r/min for 30min by a dispersion machine to obtain colloidal emulsion; finally, the colloidal emulsion is dialyzed in ultrapure water for 6 days by using dialyzing paper with the molecular weight cutoff of 8000-.
Example 5
Adding 2 parts by weight of chitin powder into 97.33 parts by weight of aqueous solution containing 8 wt% of NaOH and 4 wt% of urea, and circularly freezing for 2 times at-25 ℃ to dissolve the chitin powder into uniform aqueous solution of chitin; uniformly dispersing 0.67 weight part of rectorite powder into a chitin aqueous solution to obtain a suspension; stirring the suspension at a high speed for 30min at 8000r/min by using a dispersion machine to obtain colloidal emulsion; finally, the colloidal emulsion is dialyzed in ultrapure water for 6 days by using dialyzing paper with the molecular weight cutoff of 8000-.
Example 6
Adding 2 parts by weight of chitin powder into 97.67 parts by weight of aqueous solution containing 8 wt% of NaOH and 4 wt% of urea, and circularly freezing for 2 times at-30 ℃ to dissolve the chitin powder into uniform aqueous solution of chitin; uniformly dispersing 0.33 weight part of rectorite powder into a chitin aqueous solution to obtain a suspension; stirring the suspension at 10000r/min for 30min by a dispersion machine to obtain colloidal emulsion; finally, the colloidal emulsion is dialyzed in ultrapure water for 6 days to neutrality by using dialyzing paper with the molecular weight cutoff of 8000-.
Example 7
Adding 2 parts by weight of chitin powder into 97.83 parts by weight of aqueous solution containing 8 wt% of NaOH and 4 wt% of urea, and circularly freezing for 2 times at-30 ℃ to dissolve the chitin powder into uniform aqueous solution of chitin; uniformly dispersing 0.17 weight part of rectorite powder into a chitin aqueous solution to obtain a suspension; stirring the suspension at 10000r/min for 30min by a dispersion machine to obtain colloidal emulsion; finally, the colloidal emulsion is dialyzed in ultrapure water for 6 days to neutrality by using dialyzing paper with the molecular weight cutoff of 8000-.
Example 8
Adding 2 parts by weight of chitin powder into 96 parts by weight of aqueous solution containing 8 wt% of NaOH and 5 wt% of urea, and circularly freezing for 2 times at-30 ℃ to dissolve the chitin powder into uniform aqueous solution of chitin; uniformly dispersing 2 parts by weight of zeolite powder into a chitin aqueous solution to obtain a suspension; stirring the suspension at high speed for 30min at 12000r/min by using a dispersion machine to obtain colloidal emulsion; finally, the colloidal emulsion is dialyzed in ultrapure water for 6 days by using dialyzing paper with the molecular weight cutoff of 8000-14000 to be neutral, and finally, the zeolite-chitin nanogel composite hemostatic material with the chitin/zeolite ratio of 1/1, namely the sample 10, is obtained by freeze drying.
Example 9
Adding 2 parts by weight of chitin powder into 96 parts by weight of aqueous solution containing 10 wt% of NaOH and 3 wt% of urea, and circularly freezing for 2 times at-30 ℃ to dissolve the chitin powder into uniform aqueous solution of chitin; uniformly dispersing 2 parts by weight of montmorillonite powder into a chitin aqueous solution to obtain a suspension; stirring the suspension at 10000r/min for 30min by a dispersion machine to obtain colloidal emulsion; finally, the colloidal emulsion is dialyzed in ultrapure water for 6 days to neutrality by using dialyzing paper with the molecular weight cutoff of 8000-14000, and finally, the montmorillonite-chitin nanogel composite hemostatic material with the chitin/montmorillonite ratio of 1/1, namely the sample 11, is obtained by freeze drying.
Example 10
Adding 2 parts by weight of chitin powder into 96 parts by weight of aqueous solution containing 8 wt% of NaOH and 4 wt% of urea, and circularly freezing for 2 times at-30 ℃ to dissolve the chitin powder into uniform aqueous solution of chitin; uniformly dispersing 2 parts by weight of bentonite powder into chitin aqueous solution to obtain suspension; stirring the suspension at 10000r/min for 30min by a dispersion machine to obtain colloidal emulsion; finally, the colloidal emulsion is dialyzed in ultrapure water for 6 days to neutrality by using dialyzing paper with the molecular weight cutoff of 8000-14000, and finally, the bentonite-chitin nanogel composite hemostatic material with the chitin/bentonite ratio of 1/1, namely the sample 12, is obtained by freeze drying.
Example 11
Adding 2 parts by weight of chitin powder into 96 parts by weight of aqueous solution containing 8 wt% of NaOH and 4 wt% of urea, and circularly freezing for 2 times at-30 ℃ to dissolve the chitin powder into uniform aqueous solution of chitin; uniformly dispersing 2 parts by weight of silicon dioxide powder into chitin aqueous solution to obtain suspension; stirring the suspension at a high speed of 15000r/min for 30min by using a dispersion machine to obtain a colloidal emulsion; finally, the colloidal emulsion was dialyzed in ultrapure water with dialyzing paper having a molecular weight cut-off of 8000-.
Example 12
Adding 2 parts by weight of chitin powder into 96 parts by weight of aqueous solution containing 8 wt% of NaOH and 4 wt% of urea, and circularly freezing for 2 times at-30 ℃ to dissolve the chitin powder into uniform aqueous solution of chitin; uniformly dispersing 2 parts by weight of kaolin powder into a chitin aqueous solution to obtain a suspension; stirring the suspension at 10000r/min for 20min by a dispersion machine to obtain colloidal emulsion; finally, the colloidal emulsion is filtered to be neutral by ultrapure water, and finally, the kaolin-chitin nanogel composite hemostatic material with the chitin/kaolin weight ratio of 1/1, namely the sample 14, is obtained by freeze drying.
Example 13: toxicity test
The in vitro cytotoxicity test of the composite material MTT is to detect the cell compatibility of the material by culturing cells on the surface of the material or in the leaching liquor of the material. The leaching liquor is prepared by adding low-sugar DMEM culture solution (leaching medium) into a leaching medium with the material mass to leaching medium volume ratio of 0.2g/mL according to ISO standard, and culturing in an incubator at 37 ℃ for 72 h. A cell suspension prepared from L929 cells in the logarithmic growth phase was seeded in 96-well plates (100 ul/well) at a density of 1X 103 per well. Adding 75ul of complete culture medium and 25ul of material leaching liquor, adding the complete culture medium with the equivalent serum content of 75% into the 0 hole and the negative control hole, and setting 6 multiple holes. And when the cells are cultured for 1d, 2d and 3d, adding 5mg/ml MTT into each experimental hole, continuously culturing for 4h, pouring out the original culture solution, washing with PBS, adding DMSO into each hole, shaking for 10-15 min, measuring the absorbance value of the cells at 490nm wavelength on an enzyme linked immunosorbent assay, wherein the OD value is in direct proportion to the number of the cells, and evaluating the influence of the materials on the cell proliferation capacity on MTT cytotoxicity. The experimental result shows that the cell survival rate of the rectorite-chitin nano gel composite hemostatic material with the weight ratio of chitin to rectorite of 1/1 is more than 90 percent.
TABLE 1 results of cytotoxicity experiments
| Sample/cell viability (%) | 24h | 48h | 72h |
| |
100 | 100 | 100 |
| Pure chitin nano gel | 114 | 96 | 113 |
| The weight ratio of chitin to rectorite is 12/1 | 118 | 105 | 107 |
| The weight ratio of chitin to rectorite is 6/1 | 115 | 102 | 113 |
| The weight ratio of chitin to rectorite is 3/1 | 131 | 122 | 128 |
| The weight ratio of chitin to rectorite is 1/1 | 125 | 100 | 102 |
| The weight ratio of chitin to rectorite is 1/2 | 132 | 85 | 80 |
| The weight ratio of chitin to rectorite is 1/3 | 129 | 90 | 101 |
| Pure rectorite | 43 | 37 | 39 |
Example 14: clotting time test
The in vitro coagulation time of the chitin nanogel is determined by a test tube method. Collecting clean test tubes, adding 40mg of hemostatic material powder, spreading the powder on the bottom of the test tube, keeping the temperature in 37 deg.C water bath for 1 hr, adding 1m L fresh anticoagulated rabbit blood into the test tubes, and adding 20 μ L CACl into each test tube2Solutions, the tubes were tilted every 30s until the tubes were slowly inverted and blood stopped, clotting times were recorded on a stop-second chart, and 6 replicates were run for each sample. The results show that the mean in vitro coagulation time of pure chitin nanogel is 302s, the mean in vitro coagulation time of a blank control group without the addition of the hemostatic material is 534s, and the weight ratio of chitin/rectorite is 1/1-3, the mean in vitro coagulation time of the rabbit blood added with the rectorite-chitin nanogel composite hemostatic material is far lower than the mean in vitro coagulation time of the rabbit blood added with the pure rectorite and the pure chitin gel.
TABLE 2 in vitro clotting time of rabbit blood
The technical solutions of the present invention have been described in detail with reference to the specific embodiments, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention and are made by those skilled in the art are deemed to be equivalent substitutions within the scope of the present invention.
Claims (8)
1. A preparation method of a rectorite-chitin nanogel composite hemostatic material is characterized by comprising the following steps: adding chitin powder into an aqueous solution containing 6-12 wt% of NaOH and 2-8 wt% of urea, and circularly freezing to obtain a chitin solution uniformly dissolved, wherein the content of the chitin is 0.5-10 wt%; uniformly dispersing rectorite powder into a chitin aqueous solution to obtain a suspension, wherein the weight ratio of the chitin powder to the rectorite powder is 1: 1-3; then stirring the suspension at a speed of more than 5000r/min to obtain a colloidal emulsion; dialyzing the obtained colloidal emulsion in ultrapure water to neutrality, and finally freeze-drying to obtain the rectorite-chitin nano gel-based composite hemostatic material.
2. The method of claim 1, wherein: the NaOH content was 8 wt% and the urea content was 4 wt%.
3. The method of claim 1, wherein: the temperature of the circulating refrigeration is-10 to-40 ℃.
4. The method of claim 1, wherein: the number of times of the cyclic freezing is 2 or more.
5. The method of claim 1, wherein: the stirring time is 5-90 min.
6. The method of claim 1, wherein: the stirring speed is 6000 to 20000 r/min.
7. The method of claim 1, wherein: the cut-off molecular weight of dialysis paper used for dialyzing the colloidal emulsion is 8000-14000.
8. A rectorite-chitin nanogel composite hemostatic material is characterized in that: prepared by the preparation method of any one of claims 1 to 7.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1789312A (en) * | 2005-12-16 | 2006-06-21 | 武汉大学 | Chitosan/rectorite nano composite materials and method for preparing the same |
| CN107141494A (en) * | 2017-06-13 | 2017-09-08 | 武汉大学 | A kind of preparation method of chitin nanogel |
| CN108159480A (en) * | 2017-12-14 | 2018-06-15 | 华南理工大学 | A kind of preparation method and application of injectable polysaccharide clay plural gel |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1789312A (en) * | 2005-12-16 | 2006-06-21 | 武汉大学 | Chitosan/rectorite nano composite materials and method for preparing the same |
| CN107141494A (en) * | 2017-06-13 | 2017-09-08 | 武汉大学 | A kind of preparation method of chitin nanogel |
| CN108159480A (en) * | 2017-12-14 | 2018-06-15 | 华南理工大学 | A kind of preparation method and application of injectable polysaccharide clay plural gel |
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