CN114732474A

CN114732474A - Halloysite-melanin surface codeposition hemostatic bandage and preparation method thereof

Info

Publication number: CN114732474A
Application number: CN202210484304.4A
Authority: CN
Inventors: 张毅; 黄宗旺
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2022-05-06
Filing date: 2022-05-06
Publication date: 2022-07-12

Abstract

The invention discloses a halloysite-melanin surface codeposition hemostatic bandage and a preparation method thereof. The halloysite-melanin surface codeposition hemostatic bandage is prepared by sequentially codepositing natural melanin particles and halloysite nanotubes on the surface of a hemostatic bandage substrate only through simple impregnation, and the halloysite-melanin surface codeposition hemostatic bandage is simple in preparation process, has synergistic high-efficiency hemostatic performance with the halloysite nanotubes and the natural melanin particles, and has good biological safety.

Description

Halloysite-melanin surface codeposition hemostatic bandage and preparation method thereof

Technical Field

The invention belongs to the technical field of functional hemostatic materials, and particularly relates to a halloysite-melanin surface codeposition hemostatic bandage and a preparation method 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 current common hemostatic materials are 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-infection and pro-complex hydrogel adhesive for less tissue and close. biomacromolecules 2020,21,1243 and 1253), but the application of the chitosan hemostatic material is limited because the chitosan hemostatic material is difficult to degrade and easily causes 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, tannic acid-loaded mesoporous silica for rapid homology and antibacterial activity, Biomaterials science.2018,6,3318-3331), but the potential metabolic toxicity and difficulty in cleaning powder particles at wounds limit the application thereof. 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, calcium ions released by the zeolite-based hemostatic material can activate platelets to accelerate the coagulation process (Liang, Y., Xu, C., Liu F., et al., experiment heat input of zeolite in a haemostasis tissue and ACS Applied Materials interface 2019,11,23848 and 23857), but the severe exothermic effect of the zeolite-based hemostatic material can 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 existing hemostatic material, the invention aims to provide a halloysite-melanin surface codeposition hemostatic bandage and a preparation method thereof.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

a halloysite-melanin surface codeposition hemostatic bandage is prepared by codeposition of natural melanin particles and halloysite nanotubes on the surface of hemostatic bandage base material in sequence.

Further, the mass ratio of the natural melanin particles to the halloysite nanotubes is 0.005-0.1: 1.

further, the natural melanin granules are extracted from at least one of animal hair, feathers, fungi, and bacteria.

Further, the hemostatic bandage base material is selected from one of medical gauze, medical sponge, silk fiber and cotton and linen gauze.

The invention also provides a preparation method of the halloysite-melanin surface codeposition hemostatic bandage, which comprises the following steps:

(1) firstly, immersing a hemostatic bandage substrate in a trihydroxymethyl aminomethane buffer solution, adjusting the pH to 8-9, and performing ultrasonic dispersion pretreatment;

(2) adding the natural melanin particles into the step (1), stirring in a water bath, ultrasonically dispersing, and standing to obtain a hemostatic bandage deposited on the surface of the natural melanin particles;

(3) and (3) finally, placing the hemostasis bandage deposited on the surface of the natural melanin particle in the step (2) in a halloysite nanotube solution, alternately dipping the front side and the back side of a water bath for multiple times, and standing to obtain the halloysite-melanin surface codeposition hemostasis bandage.

Further, in the step (1), the concentration of the tris buffer solution is 8-12mM, and the ultrasonic dispersion time is 0.5-2 h.

Further, in the step (2), the mass concentration of the natural melanin granules is 0.1-1 wt%, the water bath temperature is 45-55 ℃, and the standing time is 12-24 h.

Further, in the step (3), the mass concentration of the halloysite nanotube solution is 10-30 wt%, the water bath temperature is 45-55 ℃, the number of times of alternate impregnation of the front and back surfaces is 3-6, the impregnation time is 1-2h each time, and the standing time is 12-24 h.

The halloysite nanotube can be obtained by carrying out conventional treatment on halloysite raw ore through water washing, ultrasonic stirring, multiple times of centrifugal separation and the like, and the further preferable size is 400-600 nm; the natural melanin granules can be extracted from animal hair, feathers, fungi, bacteria, etc., 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 enzymolysis or ultrasound, washing with water, centrifuging, and drying, etc. (refer to The emulsification of The biological structures of natural animal Journal of The Royal Society Interface,2018,15,20180045), and a more preferable size is 200 to 400 nm. The treatment means of the halloysite nanotubes and the natural melanin particles in the invention are the conventional processes, and are not described herein again.

Compared with the prior art, the invention has the advantages that:

(1) according to the halloysite-melanin surface codeposition hemostatic bandage, the natural melanin particles and the halloysite nanotubes are sequentially deposited on the surface of the base material, and rich surface polar groups (mainly carboxyl and hydroxyl) of the natural melanin particles can effectively enhance the adhesion effect of the halloysite nanotubes and the surface of the base material, so that the problem of falling-off of hemostatic active components is avoided; the deposited natural melanin particles can promote the adhesion and aggregation of platelets and thrombin protein, the halloysite active component particles distributed on the surface of the hemostatic bandage can fully contact blood components to activate a blood coagulation cascade reaction, and the two generate a synergistic effect to accelerate a blood coagulation process.

(2) The halloysite-melanin surface codeposition hemostatic bandage not only provides attachment and support sites for blood-stopping active component particles, but also can form a physical barrier effect on blood flow at a wound, and has the characteristic of easy bandaging and use.

(3) The halloysite-melanin surface codeposition hemostatic bandage is simple in preparation process, and the halloysite nanotubes and natural melanin particles have synergistic high-efficiency hemostatic performance and good biological safety.

Drawings

FIG. 1 is an SEM image of a conventional Medical gauze bandage (Medical gauze BDs; comparative example 4); among them, the Medical gauze BDs fiber surface is smooth and has strip-shaped folds.

FIG. 2 is an SEM image of halloysite-melanin surface codeposition hemostatic bandages (HNTs-MGs BDs; example 1); wherein, the HNTs-MGs BDs have rough surfaces, and a large amount of halloysite nanotube particles are deposited on the outermost layer of the surface.

FIG. 3 is a bar graph of hemostasis time for different hemostatic materials; wherein: blank control group (NC), halloysite-melanin surface codeposition hemostatic bandage (HNTs-MGs BDs; example 1), halloysite-melanin composite powder (HNTs-MGs; comparative example 3), natural melanin granules (MGs; comparative example 2), halloysite nanotubes (HNTs; comparative example 1), common Medical gauze bandage (Medical gauze BDs; comparative example 4).

Detailed Description

The present invention will be further described with reference to the following specific embodiments and 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 halloysite nanotube raw ore is dissolved in 800mL of deionized water (mass ratio of 1: 80), magnetically stirred for 8 hours and filtered. And (3) suspending the halloysite nanotube obtained by suction filtration in 100mL of deionized water, naturally settling for 10min, and taking supernatant for centrifugal separation at the rotating speed of 8000 rpm. And (3) drying the precipitate obtained by centrifugation in a drying oven at 60 ℃ for 12 hours to obtain the halloysite nanotube with the particle size range of 400-600 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 were added to 40mL of phosphate buffer (pH 6.8), 0.4g of Dithiothreitol (DTT) was added thereto, and the mixture was mixed well under N₂Shaking at 37 deg.C for 12h under atmosphere. Add 1mL of 20mg/mL proteinase K and 0.2g DTT and continue shaking for 12 h. 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 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. And after the enzymolysis reaction is completed, standing for 12 hours at 37 ℃, centrifuging, discarding the supernatant, washing for 3 times by using deionized water, and drying at 50 ℃ to obtain natural melanin particles with the particle size range of 200-400 nm.

The purchased medical gauze was immersed in 100mL of 10mM tris buffer solution, the pH was adjusted to 8.5, and sonication was performed for 1 hour. Adding the natural melanin particles into the solution with the concentration of 0.1 wt%, stirring for 24h at the temperature of 50 ℃ in a water bath, and carrying out ultrasonic treatment for 10min at the interval of 3h, wherein the ultrasonic power is 200w, and the frequency is 40 kHz. Standing for 12h at 50 ℃ in a water bath, taking out the medical gauze, and washing the fallen melanin particles with deionized water to obtain the hemostatic gauze deposited on the melanin surface. Deionized water is used for preparing a halloysite nanotube solution with the mass concentration of 20 wt%, ultrasonic dispersion is carried out for 30min, mechanical stirring is carried out for 2h, and the operation is repeated for 3 times to obtain the halloysite nanotube solution with uniform dispersion. The obtained melanin hemostatic gauze was immersed in a halloysite nanotube solution at 50 ℃ in a water bath with the upper side facing the front, and this operation was repeated 3 times with the front turned down with forceps every 1 hour. Then, standing for 12h to ensure that halloysite nanotubes (the mass ratio of the natural melanin particles to the HNTs) are fully deposited on the surface of the gauze. And (3) taking out the gauze, washing the fallen halloysite nanotube particles with deionized water, and drying in an oven at 50 ℃ to obtain the halloysite-melanin surface codeposition hemostatic bandage.

Example 2

30g of halloysite nanotube raw ore is dissolved in 800mL of deionized water (mass ratio of 3: 80), magnetically stirred for 8 hours and filtered. And (3) resuspending the halloysite nanotube obtained by suction filtration in 300mL of deionized water, naturally settling for 10min, and taking supernatant to perform centrifugal separation at the rotating speed of 8000 rpm. And (3) drying the precipitate obtained by centrifugation in an oven at 80 ℃ for 10 hours to obtain the halloysite nanotube with the particle size range of 400-600 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 feathers or hair were added to 40mL of phosphate buffer (pH 6.8), 0.4g of Dithiothreitol (DTT) was added thereto, and the mixture was mixed well under N₂Shaking at 37 deg.C for 12h under atmosphere. Add 1mL of 20mg/mL proteinase K and 0.2g DTT and continue shaking for 12 h. Centrifuging at 8000rpm, washing with deionized water for 3 times, and removing supernatant to obtain precipitate. The resulting precipitate was combined with 0.4mL proteinase K, 32mg DTT and 16mL phosphorusResuspend and shake in acid buffer for 12 h. 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. And after the enzymolysis reaction is completed, standing for 12 hours at 37 ℃, centrifuging, discarding the supernatant, washing for 3 times by using deionized water, and drying at 50 ℃ to obtain natural melanin particles with the particle size range of 200-400 nm.

The purchased medical gauze was immersed in 100mL of 12mM tris buffer solution, the pH was adjusted to 8, and sonication was performed for 1 hour. Adding natural melanin granules into the solution with the concentration of 1 wt%, stirring for 24h at the temperature of 50 ℃ in a water bath, and carrying out ultrasonic treatment for 10min at intervals of 2h, wherein the ultrasonic power is 100w, and the frequency is 40 kHz. Standing for 24h at 50 ℃ in a water bath, taking out the medical gauze, and washing the fallen melanin particles with deionized water to obtain the hemostatic gauze deposited on the melanin surface. Deionized water is used for preparing a halloysite nanotube solution with the mass concentration of 10 wt%, ultrasonic dispersion is carried out for 30min, mechanical stirring is carried out for 2h, and the operation is repeated for 3 times to obtain the halloysite nanotube solution with uniform dispersion. The obtained melanin hemostatic gauze was immersed in a halloysite nanotube solution at 50 ℃ in a water bath, with the upward side being the front side, and the front side was turned downward with tweezers every 1h, and this operation was repeated 6 times. Then, standing for 24h to ensure that halloysite nanotubes (the mass ratio of the natural melanin particles to the HNTs) are fully deposited on the surface of the gauze. And (3) taking out the gauze, washing the fallen halloysite nanotube particles with deionized water, and drying in an oven at 48 ℃ to obtain the halloysite-melanin surface codeposition hemostatic bandage.

Comparative example 1

20g of halloysite nanotube raw ore is dissolved in 800mL of deionized water (mass ratio of 1: 40), magnetically stirred for 8 hours and filtered. And (3) suspending the halloysite nanotubes obtained by suction filtration in 100mL of deionized water, naturally settling for 10min, and taking supernatant for centrifugal separation at the rotating speed of 8000 rpm. And (3) drying the precipitate obtained by centrifugation in an oven at 60 ℃ for 12 hours to obtain the halloysite nanotube with the particle size range of 400-600 nm.

Comparative example 2

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 feathers or hairs were added to 40mL of phosphate buffer (pH 6.8), 0.4g of Dithiothreitol (DTT) was added thereto, and the mixture was mixed well under N₂Shaking at 37 deg.C for 12h under atmosphere. Add 1mL of 20mg/mL proteinase K and 0.2g DTT and continue shaking for 12 h. 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 12 h. 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. And after the enzymolysis reaction is completed, standing for 12 hours at 37 ℃, centrifuging, discarding the supernatant, washing for 3 times by using deionized water, and drying at 50 ℃ to obtain natural melanin particles with the particle size range of 200-400 nm.

Comparative example 3

10g of halloysite nanotube raw ore is dissolved in 800mL of deionized water (mass ratio of 1: 80), magnetically stirred for 8 hours and filtered. And (3) suspending the halloysite nanotubes obtained by suction filtration in 100mL of deionized water, naturally settling for 10min, and taking supernatant for centrifugal separation at the rotating speed of 8000 rpm. And (3) drying the precipitate obtained by centrifugation in a drying oven at 60 ℃ for 12 hours to obtain the halloysite nanotube with the particle size range of 400-600 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 feathers or hair were added to 40mL of phosphate buffer (pH 6.8), 0.4g of Dithiothreitol (DTT) was added thereto, and the mixture was mixed well under N₂Shaking at 37 deg.C for 12h under atmosphere. Add 1mL of 20mg/mL proteinase K and 0.2g DTT and continue shaking for 12 h. Centrifuging at 8000rpm, washing with deionized water for 3 times, and removing supernatant to obtain precipitate. The resulting precipitate was mixed with 0.4mL eggThe protease K and 32mg DTT were added to 16mL of phosphate buffer, resuspended, and shaken for 12 h. 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. And after the enzymolysis reaction is completed, standing for 12 hours at 37 ℃, centrifuging, discarding the supernatant, washing for 3 times by using deionized water, and drying at 50 ℃ to obtain natural melanin particles with the particle size range of 200-400 nm.

50mL of 12mM Tris buffer solution was prepared, pH was adjusted to 8, and sonication was performed for 1 h. Adding 100mg natural melanin granules into the solution, magnetically stirring for 2 h-ultrasonic dispersion for 10min at the temperature of 50 ℃ in a water bath, and repeating the operation for 3 times, wherein the ultrasonic power is 100w and the frequency is 40 kHz. 20g of HNTs nanoparticles were dissolved in 50mL of deionized water and stirred in a water bath at 50 ℃ for 12 h. Mixing the natural melanin particle solution and the HNTs solution in equal volume (the mass ratio of the natural melanin particles to the HNTs is 0.5%), performing ultrasonic dispersion for 10 min-magnetically stirring for 2h in a water bath at 50 ℃, and repeating the operation for 6 times, wherein the ultrasonic power is 100w, and the frequency is 40 kHz. Standing the obtained HNTs-melanin composite hemostatic powder for 24h, and drying in an oven at 50 ℃ to obtain the halloysite-melanin composite hemostatic powder.

Comparative example 4

A sample of a control group of conventional Medical gauze bandages (Medical gauze BDs) was prepared by taking commercially available conventional Medical gauze bandages, sterilizing the gauze bandages by ultraviolet irradiation for 2 hours, and cutting the gauze bandages into small disks having a diameter of 1.2cm while operating in an aseptic environment.

The hemostatic performance of the hemostatic material is evaluated by 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 hemostatic performance is evaluated by applying the same amount of the material prepared in example 1 and the materials prepared in comparative examples 1-3 to the surface of the wound. As shown in fig. 3, the halloysite-melanin composite powder prepared in comparative example 3 has significant statistical advantages in hemostatic time, compared to Halloysite Nanotubes (HNTs) of comparative example 1 and natural melanin particles (MGs) of comparative example 2, indicating that the halloysite and natural melanin composite powder can synergistically improve hemostatic performance; compared with the halloysite-melanin composite powder (HNTs-MGs) of the comparative example 3, the halloysite nanotube-melanin surface codeposition hemostatic bandage (HNTs-MGs BDs) of the example 1 has shorter hemostatic time, and has the advantages of convenient dressing and easy operation; compared with a blank control group (NC) and a common Medical gauze bandage group (Medical gauze BDs; comparative example 4), the HNTs-MGs BDs present remarkable hemostatic performance advantages. In conclusion, the halloysite-melanin surface codeposition hemostatic bandage has good usability and synergistic and efficient hemostatic performance.

Claims

1. A halloysite-melanin surface codeposition hemostatic bandage is characterized in that: the halloysite-melanin surface codeposition hemostatic bandage is formed by sequentially codepositing natural melanin particles and halloysite nanotubes on the surface of a hemostatic bandage base material.

2. The halloysite-melanin surface codeposition hemostatic bandage of claim 1, wherein: the mass ratio of the natural melanin particles to the halloysite nanotubes is 0.005-0.1: 1.

3. the halloysite-melanin surface co-deposition hemostatic bandage of claim 1, wherein: the natural melanin granules are extracted from at least one of animal hair, feathers, fungi, and bacteria.

4. The halloysite-melanin surface co-deposition hemostatic bandage of claim 1, wherein: the hemostatic bandage base material is selected from one of medical gauze, medical sponge, silk fiber and cotton and linen gauze.

5. A method of making a halloysite-melanin surface co-deposition hemostatic bandage as claimed in any one of claims 1 to 4, comprising the steps of:

(3) and (3) finally, placing the hemostatic bandage deposited on the surface of the natural melanin particles in the step (2) into a halloysite nanotube solution, alternately dipping the front side and the back side of the water bath for multiple times, and standing to obtain the halloysite-melanin surface codeposition hemostatic bandage.

6. The method of claim 5, wherein: in the step (1), the concentration of the trihydroxymethyl aminomethane buffer solution is 8-12mM, and the ultrasonic dispersion time is 0.5-2 h.

7. The method of claim 5, wherein: in the step (2), the mass concentration of the natural melanin granules is 0.01-1 wt%, the water bath temperature is 45-55 ℃, and the standing time is 12-24 h.

8. The method of claim 5, wherein: in the step (3), the mass concentration of the halloysite nanotube solution is 10-30 wt%, the water bath temperature is 45-55 ℃, the number of times of alternate impregnation of the front and back surfaces is 3-6, the impregnation time is 1-2h each time, and the standing time is 12-24 h.