CN114712549B - Montmorillonite-melanin composite hemostatic powder and preparation method and application thereof - Google Patents
Montmorillonite-melanin composite hemostatic powder and preparation method and application thereof Download PDFInfo
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- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
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- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/046—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
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- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/04—Materials for stopping bleeding
Abstract
The invention discloses montmorillonite-melanin composite hemostatic powder and a preparation method and application thereof, the montmorillonite-melanin composite hemostatic powder is prepared by uniformly dispersing and coating natural melanin particles on the surfaces of montmorillonite particles, and surface groups and charges of the montmorillonite particles and the natural melanin particles can activate platelets and activate coagulation cascade reaction through contact action, so that the hemostatic process is accelerated, and the hemostatic performance of a composite material is synergistically and remarkably improved. The montmorillonite-melanin composite hemostasis powder disclosed by the invention is simple in preparation process, and the montmorillonite particles and the natural melanin particles have synergistic high-efficiency hemostasis performance, and meanwhile, the low-toxicity components endow the montmorillonite-melanin composite hemostasis powder with good biological safety.
Description
Technical Field
The invention belongs to the technical field of functional hemostatic materials, and particularly relates to montmorillonite-melanin composite hemostatic powder and a preparation method and application thereof.
Background
The development and utilization of safe and efficient hemostatic materials are the key to the success of pre-hospital first aid. The main hemostatic components of the existing common hemostatic materials comprise chitosan, silicon dioxide, zeolite and the like. The chitosan hemostatic material has a porous structure and amino groups, can increase the adhesion of platelets and promote blood components such as red blood cells to aggregate at a wound site to form thrombus (Du, X, liu, Y, yan, H, et al, anti-invasive and pro-inflammatory hydrogel adhesive 2020,21, 1243-1253), but the application of the chitosan hemostatic material is limited due to the difficulty in degradation and the possibility of causing in vivo anaphylactic reaction. Mesoporous silica can promote the activation of coagulation factor XII and accelerate coagulation cascade to promote coagulation (Wang, C., zhou, H., niu, H., et al, tangnic acid-loaded mesoporous silica for rapid homology and antibiotic activity, biomaterials science.2018,6, 3318-3331), but the potential metabolic toxicity and the difficulty in cleaning powder particles at wounds limit the application of the mesoporous silica. The zeolite-based hemostatic material can adsorb water in blood at a wound to promote aggregation of platelets and coagulation factors, and at the same time, release calcium ions to activate platelets to accelerate the coagulation process (Liang, y., xu, c., liu f., et al. Experiment heat input of zeolite in hemostatis virus tissue reduction of graphene span. Acs Applied Materials interface.2019, 11, 23848-23857), but its violent exothermic effect may cause secondary injury to the wound. Therefore, there is a need to develop a hemostatic material that combines rapid blood coagulation with biosafety.
Disclosure of Invention
Aiming at the defects of the prior art and hemostatic materials, the invention aims to provide the montmorillonite-melanin composite hemostatic powder and the preparation method and application thereof.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a montmorillonite-melanin composite hemostatic powder is prepared by uniformly dispersing and coating natural melanin particles on the surface of montmorillonite particles.
Further, the mass ratio of the montmorillonite to the natural melanin granules is 5-15.
Further, the natural melanin granules are extracted from at least one of animal hair, feathers, fungi, and bacteria.
The invention also provides a preparation method of the montmorillonite-melanin composite hemostatic powder, which comprises the following steps:
(1) Uniformly dispersing montmorillonite particles in water to obtain a montmorillonite solution;
(2) Uniformly dispersing natural melanin particles into a tris buffer solution, adjusting the pH to 8-9, and uniformly stirring in a water bath to obtain a natural melanin particle solution;
(3) And (3) carrying out water bath ultrasonic stirring and mixing on the montmorillonite solution and the natural melanin particle solution uniformly, and carrying out centrifugal drying to obtain the montmorillonite composite hemostatic powder coated by the natural melanin particles.
Further, in the step (1), the montmorillonite raw ore is ultrasonically stirred and uniformly dispersed in water to obtain a montmorillonite raw ore solution; the crude montmorillonite ore solution is naturally settled and separated for a plurality of times to obtain montmorillonite particles with the particle size of 800-1500 nm.
Further, in the step (1), the mass concentration of the raw montmorillonite ore is 5-15wt%.
Further, in the step (2), the mass concentration of the natural melanin granules is 0.1-1wt%, and the water bath temperature is 45-55 ℃.
Further, in the step (3), the temperature of the water bath is 45-55 ℃, the water bath is stirred by ultrasonic for uniform stirring and then stands for 24-48h, and the centrifugal speed is 8000-10000rpm.
The natural melanin granules of The present invention can be extracted from animal hair, feathers, fungi, bacteria, and The like, and can be obtained by, for example, diluting at least one of animal hair, feathers, fungi, and bacteria in a phosphate buffer, subjecting The diluted animal hair, feathers, fungi, and bacteria to enzymatic hydrolysis or ultrasound, washing with water, centrifuging, and drying, and The like (see The emulsification of natural enzymes Journal of The Royal Society Interface,2018,15, 20180045), and further preferably have a size of 200 to 400nm. The treatment means of the natural melanin granules in the invention are all conventional processes, and are not described herein again.
The invention also provides application of the montmorillonite-melanin composite hemostatic powder as a hemostatic active component for preparing an emergency wound hemostatic material, a hemostatic product, a wound repair material or a wound healing material.
Compared with the prior art, the invention has the advantages that:
(1) The montmorillonite-melanin composite hemostatic powder is prepared by uniformly coating natural melanin particles on the surfaces of montmorillonite particles through simple dispersion in a solution, wherein the montmorillonite has good ion exchange property, large specific surface area and excellent adsorption performance, and can promote the aggregation of erythrocytes and platelets; the natural melanin particles on the surface have rich carboxyl groups, so that the attachment of protein molecules can be enhanced, the adhesion of thrombin protein at wounds can be promoted, and the surface groups and charges of the montmorillonite particles and the natural melanin particles can activate platelets and activate coagulation cascade reaction through contact action, so that the hemostasis process is accelerated, and the hemostasis performance of the composite material is synergistically and remarkably improved.
(2) The montmorillonite-melanin composite hemostatic powder has the advantages of simple preparation process, easy dispersion and suitability for complex and irregular wound surfaces.
(3) According to the montmorillonite-melanin composite hemostatic powder, montmorillonite particles and natural melanin particles have synergistic high-efficiency hemostatic performance, and meanwhile, the low-toxicity components endow the montmorillonite-melanin composite hemostatic powder with good biological safety.
Drawings
FIG. 1 is an SEM image of montmorillonite particles (MMT; comparative example 1) with arrows indicating the lamellar structure and well-defined edge morphology of the montmorillonite.
FIG. 2 is an SEM image of a montmorillonite particle composite hemostatic powder (MMT-MGs; example 1) coated with natural melanin particles, with arrows indicating that the montmorillonite powder surface has a smoother melanin coating layer, distinct from the original sharp-edged topographical features of montmorillonite particles.
FIG. 3 is a bar graph of hemostasis time for different hemostatic materials; wherein: blank control group (NC), natural melanin particle-coated montmorillonite particle composite hemostatic powder (MMT-MGs; example 1), montmorillonite particles (MMT; comparative example 1), natural melanin particles (MGs; comparative example 2), commercial common gauze (Medical gauze; comparative example 3).
Detailed Description
The present invention will be further described with reference to the following specific embodiments and the accompanying drawings, which are intended to make the technical features, scheme flows and innovation points of the present invention more clear. It is to be understood that the present embodiments are illustrative only, and that various modifications and changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.
Example 1
10g of montmorillonite raw ore is taken and dissolved in 800mL of deionized water (mass ratio is 1. And (3) suspending the montmorillonite obtained by suction filtration in 100mL of deionized water, naturally settling for 30s, and discarding the precipitate. Taking the upper suspension, naturally settling for 5min, discarding the supernatant, and baking the precipitated montmorillonite raw ore in an oven at 60 ℃ for 12h to obtain montmorillonite particles with the size range of 800-1500 nm.
Commercially available feathers or hairlines were washed with acetone 3 times, deionized water 3 times, and cut into pieces. The obtained feathers or hair are added to 40mL of phosphate buffer (pH = 6.8), 0.4g of Dithiothreitol (DTT) is added, and the mixture is mixed well under N 2 Shaking at 37 deg.C for 12h under atmosphere. Add 1mL of 20mg/mL proteinase K and 0.2g DTT, and continue shaking for 12h. Centrifuging at 8000rpm, washing with deionized water for 3 times, and removing supernatant to obtain precipitate. The resulting pellet was mixed with 0.4mL proteinase K and 32mg DTT in 16mL phosphate buffer, resuspended, and shaken for 12h. Centrifuging, removing supernatant, adding 6mL of phosphate buffer and 0.12mL of polyethylene glycol octyl phenyl ether, oscillating for 6h, centrifuging, washing with methanol for 3 times and deionized water for 3 times in sequence. The resulting precipitate was suspended with 0.4mL of proteinase K and 32mg of DTT in 16mL of phosphate buffer, and shaken at 37 ℃ for 12 hours, and this operation was repeated 2 times. After the enzymolysis reaction is completed, standing for 12h at 37 ℃, centrifuging, discarding supernatant, washing for 3 times by deionized water, and drying at 50 ℃ to obtain natural melanin particles with the particle size range of 200-400 nm.
Dissolving montmorillonite particles in 5mL of deionized water with the mass concentration of 5wt%, performing ultrasonic dispersion for 10min at room temperature, mechanically stirring for 2h, and alternately repeating for 3 times, wherein the ultrasonic power is 400w, and the frequency is 30kHz; the natural melanin granules were dissolved in 5mL of 10mM Tris buffer solution at a mass concentration of 0.1wt%, adjusted to pH =8, and stirred at 50 ℃ for 6h. Mixing montmorillonite solution and natural melanin granule solution in equal volume, performing water bath at 50 deg.C, performing ultrasonic dispersion for 10 min-mechanically stirring for 1h, and alternately repeating for 6 times, wherein the ultrasonic dispersion power is 100w, and the frequency is 30kHz. Standing for 24h, centrifuging at 8000rpm for 15min, discarding supernatant, and drying precipitate in 50 deg.C oven to obtain natural melanin particle-coated montmorillonite particle composite hemostatic powder.
Example 2
20g of montmorillonite raw ore is taken and dissolved in 800mL of deionized water (mass ratio is 2. And (3) resuspending the montmorillonite obtained by suction filtration in 200mL of deionized water, naturally settling for 30s, and discarding the precipitate. Taking the upper suspension, naturally settling for 5min, discarding the supernatant, and baking the precipitated montmorillonite raw ore in an oven at 80 ℃ for 6h to obtain montmorillonite particles with the size range of 800-1500 nm.
Commercially available feathers or hairlines were washed with acetone 3 times, deionized water 3 times, and cut into pieces. The obtained feathers or hair are added to 40mL of phosphate buffer (pH = 6.8), 0.4g of Dithiothreitol (DTT) is added, and the mixture is mixed well under N 2 Shaking at 37 deg.C for 12h under atmosphere. Add 1mL of 20mg/mL proteinase K and 0.2g DTT, and continue shaking for 12h. Centrifuging at 8000rpm, washing with deionized water for 3 times, and removing supernatant to obtain precipitate. The resulting pellet was mixed with 0.4mL proteinase K and 32mg DTT in 16mL phosphate buffer, resuspended, and shaken for 12h. Centrifuging, removing supernatant, adding 6mL phosphate buffer and 0.12mL polyethylene glycol octyl phenyl ether, shaking for 6h, centrifuging, washing with methanol for 3 times, and washing with deionized water for 3 times. The resulting precipitate was suspended with 0.4mL of proteinase K and 32mg of DTT in 16mL of phosphate buffer, and shaken at 37 ℃ for 12 hours, and this operation was repeated 2 times. After the enzymolysis reaction is completed, standing for 12h at 37 ℃, centrifuging, discarding supernatant, washing for 3 times by deionized water, and drying at 50 ℃ to obtain natural melanin particles with the particle size range of 200-400 nm.
Dissolving montmorillonite particles in 5mL of deionized water, wherein the mass concentration is 15wt%, performing ultrasonic dispersion for 10min at room temperature, mechanically stirring for 2h, and alternately repeating for 6 times, wherein the ultrasonic power is 400w, and the frequency is 40kHz; the natural melanin granules were dissolved in 5mL of 10mM Tris buffer solution at a mass concentration of 1wt%, pH =9 and stirred at 50 ℃ for 6h. Mixing montmorillonite solution and natural melanin granule solution in equal volume, performing water bath at 50 deg.C, performing ultrasonic dispersion for 10 min-mechanically stirring for 1h, and repeating for 6 times alternately, wherein the ultrasonic dispersion power is 200w, and the frequency is 40kHz. Standing for 48h, centrifuging at 10000rpm for 10min, discarding supernatant, and oven drying the precipitate in a 50 deg.C oven to obtain natural melanin particle-coated montmorillonite particle composite hemostatic powder.
Example 3
30g of montmorillonite raw ore is taken and dissolved in 800mL of deionized water (mass ratio is 3. And (3) suspending the montmorillonite obtained by suction filtration in 300mL of deionized water, naturally settling for 30s, and discarding the precipitate. Taking the upper suspension, naturally settling for 5min, discarding the supernatant, and baking the precipitated montmorillonite raw ore in a 60 ℃ oven for 12h to obtain montmorillonite particles with the size range of 800-1500 nm.
Commercially available feather or hair is taken, washed with acetone for 3 times, washed with deionized water for 3 times, and cut into pieces. The obtained feather or hair was added to 40mL of phosphate buffer (pH = 6.8), 0.4g of Dithiothreitol (DTT) was added, and mixed well under N 2 Shaking at 37 deg.C for 12h under atmosphere. Add 1mL of 20mg/mL proteinase K and 0.2g DTT and continue shaking for 12h. Centrifuging at 8000rpm, washing with deionized water for 3 times, and removing supernatant to obtain precipitate. The resulting pellet was mixed with 0.4mL proteinase K and 32mg DTT in 16mL phosphate buffer, resuspended, and shaken for 12h. Centrifuging, removing supernatant, adding 6mL phosphate buffer and 0.12mL polyethylene glycol octyl phenyl ether, shaking for 6h, centrifuging, washing with methanol for 3 times, and washing with deionized water for 3 times. The resulting precipitate was suspended with 0.4mL of proteinase K and 32mg of DTT in 16mL of phosphate buffer, and shaken at 37 ℃ for 12 hours, and this operation was repeated 2 times. After the enzymolysis reaction is completed, standing for 12h at 37 ℃, centrifuging, discarding supernatant, washing for 3 times by deionized water, and drying at 50 ℃ to obtain natural melanin particles with the particle size range of 200-400 nm.
Dissolving montmorillonite particles in 5mL of deionized water, wherein the mass concentration is 10wt%, performing ultrasonic dispersion for 10min at room temperature, mechanically stirring for 2h, and alternately repeating for 3 times, wherein the ultrasonic power is 400w, and the frequency is 40kHz; the natural melanin granules were dissolved in 5mL of 10mM Tris buffer solution at a mass concentration of 0.1wt%, adjusted to pH =8.5, and stirred at 50 ℃ for 6h. Mixing montmorillonite solution and natural melanin granule solution in equal volume, performing water bath at 50 deg.C, performing ultrasonic dispersion for 10 min-mechanically stirring for 1h, and alternately repeating for 6 times, wherein the ultrasonic dispersion power is 100w, and the frequency is 40kHz. Standing for 24h, centrifuging at 10000rpm for 10min, discarding supernatant, and oven drying the precipitate in a 50 deg.C oven to obtain natural melanin particle-coated montmorillonite particle composite hemostatic powder.
Comparative example 1
20g of montmorillonite raw ore is taken and dissolved in 800mL of deionized water (mass ratio is 2. And (3) resuspending the montmorillonite obtained by suction filtration in 200mL of deionized water, naturally settling for 30s, and discarding the precipitate. Taking the upper suspension, naturally settling for 5min, discarding the supernatant, and baking the precipitated montmorillonite raw ore in an oven at 80 ℃ for 6h to obtain montmorillonite particles with the size range of 800-1500 nm.
Comparative example 2
Commercially available feathers or hairlines were washed with acetone 3 times, deionized water 3 times, and cut into pieces. The obtained feathers or hair are added to 40mL of phosphate buffer (pH = 6.8), 0.4g of Dithiothreitol (DTT) is added, and the mixture is mixed well under N 2 Shaking at 37 deg.C for 12h under atmosphere. Add 1mL of 20mg/mL proteinase K and 0.2g DTT, and continue shaking for 12h. Centrifuging at 8000rpm, washing with deionized water for 3 times, and removing supernatant to obtain precipitate. The resulting pellet was mixed with 0.4mL of proteinase K and 32mg of DTT in 16mL of phosphate buffer, resuspended, and shaken for 12 hours. Centrifuging, removing supernatant, adding 6mL phosphate buffer and 0.12mL polyethylene glycol octyl phenyl ether, shaking for 6h, centrifuging, washing with methanol for 3 times, and washing with deionized water for 3 times. The resulting precipitate was suspended with 0.4mL of proteinase K and 32mg of DTT in 16mL of phosphate buffer, and shaken at 37 ℃ for 12 hours, and this operation was repeated 2 times. Standing at 37 deg.C for 12 hr after enzymolysis reaction is completed, centrifuging, removing supernatant, washing with deionized water for 3 times, and oven drying at 50 deg.C to obtain natural melaninParticles, the size of the particles is 200-400 nm.
Comparative example 3
A control group of commercial ordinary gauze (Medical gauze) was prepared by taking commercially available ordinary gauze, sterilizing by ultraviolet irradiation for 1.5h, cutting into small round pieces with a diameter of 1.2cm, and operating in a sterile environment.
The hemostasis performance of the hemostasis material is evaluated by using a mouse liver wound bleeding model, obvious wound bleeding is formed on the surface of the mouse liver by using an operating knife, and the same amount of the material prepared in the example 3 and the materials prepared in the comparative examples 1-3 are pasted on the surface of the wound to evaluate the hemostasis performance. As shown in FIG. 1, the natural melanin particle-coated montmorillonite particle composite hemostatic powder (MMT-MGs; example 1) exhibited shorter hemostatic time and better hemostatic effect than the pure montmorillonite particles (MMT; comparative example 1) and the natural melanin particles (MGs; comparative example 2). Compared with a blank control group (without any hemostatic material) and commercial common gauze (Medical gauze; comparative example 3), the MMT-MGs composite hemostatic powder has obviously shortened hemostatic time, and proves better hemostatic performance.
Claims (8)
1. The montmorillonite-melanin composite hemostatic powder is characterized by comprising the following components in parts by weight: the montmorillonite-melanin composite hemostatic powder is prepared by uniformly dispersing and coating natural melanin particles on the surfaces of montmorillonite particles; the mass ratio of the montmorillonite particles to the natural melanin particles is 5-15.
2. The montmorillonite-melanin composite hemostatic powder according to claim 1, wherein: the natural melanin granules are extracted from at least one of animal hair, feathers, fungi, and bacteria.
3. The method for preparing montmorillonite-melanin composite hemostatic powder according to any one of claims 1 to 2, comprising the steps of:
(1) Uniformly dispersing montmorillonite particles in water to obtain a montmorillonite solution;
(2) Uniformly dispersing natural melanin particles into a tris buffer solution, adjusting the pH to 8-9, and uniformly stirring in a water bath to obtain a natural melanin particle solution;
(3) And (3) uniformly stirring and mixing the montmorillonite solution and the natural melanin particle solution in a water bath by ultrasonic, and centrifugally drying to obtain the montmorillonite composite hemostatic powder coated by the natural melanin particles.
4. The production method according to claim 3, characterized in that: in the step (1), raw montmorillonite ore is ultrasonically stirred and uniformly dispersed in water to obtain raw montmorillonite ore solution; carrying out natural settling separation on the crude montmorillonite ore solution for multiple times to obtain montmorillonite particles with the particle size of 800-1500 nm.
5. The production method according to claim 3, characterized in that: in the step (1), the mass concentration of the montmorillonite solution is 5-15wt%.
6. The production method according to claim 3, characterized in that: in the step (2), the mass concentration of the natural melanin granules is 0.1-1wt%, and the water bath temperature is 45-55 ℃.
7. The production method according to claim 3, characterized in that: in the step (3), the temperature of the water bath is 45-55 ℃, the water bath is subjected to ultrasonic stirring uniformly and then stands for 24-48h, and the centrifugal speed is 8000-10000rpm.
8. Use of the montmorillonite-melanin composite hemostatic powder according to any one of claims 1 to 2 or the montmorillonite-melanin composite hemostatic powder prepared by the preparation method according to any one of claims 3 to 7, characterized in that: the compound is used as a hemostatic active component to prepare emergency wound hemostatic materials, hemostatic products, wound repair materials or wound healing materials.
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