CN113975454A

CN113975454A - Preparation and application of mesoporous silica/tannic acid composite hydrogel hemostatic material

Info

Publication number: CN113975454A
Application number: CN202111337187.0A
Authority: CN
Inventors: 金明实; 郭建鹏; 曾娜; 朴书庆; 周铂凯; 郑通
Original assignee: Yanbian University
Current assignee: Yanbian University
Priority date: 2021-11-12
Filing date: 2021-11-12
Publication date: 2022-01-28

Abstract

The invention discloses a preparation method and application of a mesoporous silica/tannin composite hydrogel hemostatic material. The hydrogel hemostatic material with the antibacterial function is obtained by compounding inorganic material mesoporous silica (SMS, SBA-15), tannin with the adhesive property and organic material polyacrylamide, the hemostatic effect of the polyacrylamide hydrogel is improved while the problem of mesoporous silica wound residue is solved, the swelling rate and the mechanical property of the hydrogel are improved, and the adhesive property and the antibacterial property of the hydrogel are improved by adding the tannin. The mesoporous silica/tannin hydrogel has multiple functions of hemostasis, antibiosis and healing promotion, is simple, convenient and quick in preparation method, and has wide application prospect in multifunctional hemostatic and antibacterial materials.

Description

Preparation and application of mesoporous silica/tannic acid composite hydrogel hemostatic material

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to a preparation method and application of a mesoporous silica/tannin composite hydrogel hemostatic material with hemostatic and antibacterial functions.

Background

Uncontrolled bleeding is a major challenge in trauma first aid and surgery and is also a major cause of battlefield death. Rapid and effective treatment of hemorrhagic patients, both in peaceful and emergency trauma cases and on modern battlefields, plays a crucial role in reducing infection and mortality. Death from uncontrolled bleeding, particularly in the early stages of bleeding, accounts for over 30% of deaths in military and civilian trauma centers. More than half of these deaths are due to lack of emergency care. Due to poor pre-hospital medical conditions, massive bleeding can lead to coagulation dysfunction, infection and multiple organ failure. Therefore, the development of various hemostatic materials has become of great importance. The current commonly used hemostatic materials can be divided into three types, namely natural polymers, synthetic polymers and inorganic materials, but the use of a single material still has certain problems. These materials either have undesirable side effects or are less effective at stopping bleeding. Natural high molecular materials, such as sodium alginate, cellulose and gelatin, have the main disadvantages of unstable component ratio, application limitation and possible immunogenicity caused by animal-derived materials; synthetic polymer materials, such as synthetic polyacrylic acids, polyamides, and the like, have been applied to the field of hemostatic materials, and have the advantages of strong adhesiveness and good biocompatibility, and the disadvantages of poor mechanical properties and incapability of being applied to severe bleeding wounds. Compared with the traditional hemostatic materials, the zeolite hemostatic has remarkable effects on rapidly stopping bleeding, stabilizing the wounded, reducing the death rate and the like. However, zeolite hemostats generate a significant amount of heat during use, resulting in a sharp local temperature rise that may cause thermal damage to the tissue. In addition, zeolite remaining in the body causes severe rejection due to poor biocompatibility and degradability. With the progress of medical science, the requirements for hemostatic materials are also continuously increased, and therefore, the development of a multifunctional hemostatic material with remarkable hemostatic activity, good biocompatibility and good antibacterial effect is required.

Disclosure of Invention

The invention aims to compound inorganic mesoporous silica (SMS, SBA-15), Tannic Acid (TA) with adhesive property and Polyacrylamide (PAAM) which is an organic material, solve the problem of wound residue of the mesoporous silica and simultaneously improve the hemostatic effect of polyacrylamide hydrogel, thereby developing a multifunctional hemostatic material with hemostatic, antibacterial and healing promoting functions.

The multifunctional hemostatic material provided by the invention comprises mesoporous silica (SMS, SBA-15) with different shapes, acrylamide, tannic acid, a cross-linking agent and an initiator, and the swelling rate, the mechanical property and the antibacterial property of the material are improved and the hemostatic efficiency is improved by adjusting the proportion of different materials.

The multifunctional hemostatic material provided by the invention has the advantages that the hemostatic performance of the hydrogel is synergistically enhanced through the mesoporous silica and the tannic acid, and the mesoporous silica/tannic acid composite polyacrylamide hydrogel hemostatic material is prepared by adding the mesoporous silica and the tannic acid in the polymerization process of acrylamide.

The mesoporous silica may be SMS and/or SBA-15. The addition amount of the mesoporous silica is 0.6-2.2% (mass percentage) of the dosage of the acrylamide, and the addition amount of the tannic acid is 6.0-10% of the dosage of the acrylamide.

Wherein, the mesoporous silica can be prepared by a sol-gel method. In the embodiment of the invention, ethyl orthosilicate is used as a silicon source, surfactant hexadecene-1-acetic acid and ammonia water are used as catalysts, and spherical mesoporous silica SMS is prepared by a sol-gel method; the cashew-shaped mesoporous silica SBA-15 is prepared by a sol-gel method by taking tetraethoxysilane as a silicon source, adopting a surfactant polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer and hydrochloric acid as a catalyst.

Specifically, the preparation method of the mesoporous silica/tannin composite hydrogel hemostatic material comprises the following steps: dissolving acrylamide in deionized water at room temperature, adding mesoporous silica under stirring, and performing ultrasonic treatment; then transferring the mixture into an ice-water bath, adding tannic acid, a cross-linking agent and an initiator under stirring, reacting for a period of time, and then adding an accelerator; the final mixed solution was transferred to a mold and polymerized to form a hydrogel.

In the preparation method, the cross-linking agent is preferably N, N' -methylene bisacrylamide, and the dosage of the cross-linking agent is 0.2-0.6 percent of the mass of the acrylamide; the initiator is preferably ammonium persulfate, and the using amount of the initiator is 1-4% of the mass of the acrylamide; the accelerator is preferably tetramethylethylenediamine, and the dosage of the accelerator is 20-50 mu L/100g of acrylamide.

Further, after acrylamide is dissolved in deionized water, firstly, magnetic stirring is carried out at room temperature (the rotating speed is preferably 300-500 r/min) for 4-6 hours, then, mesoporous silicon dioxide is added, and then, ultrasonic treatment is carried out for 50-80 minutes; transferring the mixture to an ice water bath, adding tannic acid, a cross-linking agent and an initiator, reacting for 20-30 minutes, adding an accelerator, and stirring for 10-20 minutes; and finally, transferring the mixture into a glass bottle or a polytetrafluoroethylene mold, and polymerizing at 40-60 ℃ to form the mesoporous silica/tannin composite polyacrylamide hydrogel.

To prepare the dry hydrogel, the wet hydrogel can be frozen at-40 ℃ for a period of time and then freeze-dried using an ultra-low temperature freeze-dryer.

Compared with the traditional method, the method has the following advantages:

1. the synthesized silicon dioxide material has a mesoporous structure, a large specific surface area and a capillary absorption effect, and can improve the swelling rate of hydrogel; the surface is negatively charged, and FXII coagulation factors can be activated to carry out intrinsic coagulation; the hydrogel can be used as a physical crosslinking agent to improve the mechanical property of the hydrogel; the abundant phenolic hydroxyl on the surface can form intermolecular hydrogen bonds with amino groups of the hydrogel, so that the wound residue of silicon dioxide is reduced, and the hemostatic performance of the hydrogel is improved.

2. The application is inspired by mussel adhesive organisms, tannic acid is added, and as catechol has a unique structure of two-OH groups on a benzene ring, the tannic acid can form strong hydrogen bonds with a hydrophilic surface, can generate an adhesion effect with body tissues, can quickly seal wounds and provide physical barriers for the wounds, so that the bleeding is quickly stopped; the catechol group can be adsorbed on the surface of the bacterial cell wall to physically kill bacteria, so that the material has a certain antibacterial effect.

3. The organic material based on the polyacrylamide hydrogel has a three-dimensional network structure, can absorb exudate, keep the wound surface wet, protect the wound surface, resist the penetration of external bacteria and reduce the infection chance of the wound; are easily replaceable and may incorporate various drugs and growth factors to promote wound healing.

4. According to the mesoporous silica/tannin composite hydrogel hemostatic material, the swelling rate, the mechanical property and the hemostatic efficiency of hydrogel are improved due to the addition of silica, and the adhesion property and the antibacterial property of hydrogel are improved due to the addition of tannin with an antibacterial effect. The preparation method is simple, convenient and quick, and has wide application prospect in multifunctional hemostatic antibacterial materials.

Drawings

The invention is further illustrated by the following figures and examples:

FIG. 1 is an SEM image of four hydrogels synthesized by an example of the present invention, wherein: (a) PAAM; (b) PAAM-TA; (c) PAAM-TA-SMS; (d) PAAM-TA-SBA-15, two magnifications on the right: the upper panel shows the microscopic morphology of SMS, and the lower panel shows the microscopic morphology of SBA-15.

FIG. 2 shows four hydrogels (PAAM, PAAM-TA, PAAM-TA-SMS, PAAM-TA-SBA-15) and mesoporous SiO synthesized by the present invention₂FT-IR patterns of (SMS) and Tannic Acid (TA).

FIG. 3 is a graph showing the swelling ratios of four hydrogels synthesized by examples of the present invention.

FIG. 4 is a compressive stress-strain plot of the hydrogels tested in the examples, where: (a) four hydrogels compressed 80% stress-strain curves; (b) stress-strain curve of PAAM-TA-SMS hydrogel cyclic compression 60%; (c) stress-strain curve of PAAM-TA-SBA15 hydrogel cyclic compression 60%.

FIG. 5 is a graph (a) of the amount of bleeding and a graph (b) of the time of hemostasis for rat tail-broken hemostasis for the four hydrogels used in the examples.

Fig. 6 is a graph of tail-broken bleeding volume of rats stopped by four hydrogels used in the examples, wherein: (a) a gauze group; (b) a PAAM group; (c) PAAM-TA group; (d) PAAM-TA-SMS group; (e) PAAM-TA-SBA15 group.

Detailed Description

The present invention will be described in detail with reference to specific steps.

Preparation of SMS: the sol-gel method has mild reaction conditions and is used for preparing the spherical mesoporous SiO₂One of the commonly used methods. First, 5.1g of hexadecene-1-acetic acid, a surfactant, was weighed out on a weighing paper, dissolved in 350mL of deionized water by sonication, and then 382mL of an isopropanol solution (99.5%) was added to the solution and heated in a water bath to 60 ℃ with stirring, during which time the mixture was observed to change from milky to transparent. Then 2.5mL ammonia water (not less than 25%) is added into the mixed solution and stirred for 1h at a constant temperature of 60 ℃. And finally, quickly adding 27.07g of silicon source tetraethoxysilane (98 percent) and continuously stirring in a constant-temperature water bath kettle at the temperature of 60 ℃ for 15 hours to finish the reaction. And cooling to room temperature. The centrifugal washing is repeated for a plurality of times, and the powder is obtained after drying in an oven overnight. Finally, placing the white dry powder in a muffle furnace, and calcining at 550 ℃ to obtain mesoporous SiO₂Ball SMS. The synthesis result is shown in the upper graph of the right enlarged view of fig. 1, the particle size distribution is uniform, the dispersibility is good, and the surface has three-dimensional interconnected channels which are distributed in a disordered way.

Preparing SBA-15: firstly, 6g of surfactant polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer is added into 155.6mL of deionized water, 37.5g of hydrochloric acid (36% -38%) is added, and the mixture is stirred for 6 hours to be dissolved. Then 12.8g of silicon source tetraethoxysilane (98 percent) is added, heated for 24 hours at 55 ℃, and then placed in a 100 ℃ oven for reaction for 24 hours. The mixed solution is filtered by suction and dried for 40h at 100 ℃. Soaking the dried product in ethanol (99.5%), adding hydrochloric acid (36% -38%) 8-9 drops, stirring for 3-4hThe product was washed 3 times with ethanol (99.5%) and dried in an oven at 80 ℃ for 8 h. Finally, placing the obtained white dry powder in a muffle furnace, and calcining at 550 ℃ to obtain mesoporous SiO₂Ball SBA-15. The synthesis result is shown in the lower graph in the right enlarged view of fig. 1, and has a cashew-like shape, uniform pore diameter distribution, and densely arranged and uniformly distributed particle surface channels.

③ 10g of acrylamide is dissolved in 24g of deionized water at room temperature. It was transferred to an ice-water bath, and then 54.4mg of N, N' -methylenebisacrylamide and 129mg of ammonium persulfate were added with stirring. Then 20. mu.L of tetramethylethylenediamine were introduced for 10 minutes with stirring. The mixed solution was transferred into a glass bottle (diameter: 20mm) and a polytetrafluoroethylene mold (90 mm. times.60 mm). The reaction was carried out at 40 ℃ for 8 hours to form PAAM hydrogel. To prepare the dry hydrogel, the wet hydrogel was frozen at-40 ℃ for 12h and then freeze-dried with an ultra-low temperature freeze-dryer for 48 h. The PAAM hydrogel synthesis result is shown in (a) in FIG. 1, and has a multi-layer porous structure and low crosslinking density.

(iv) 10g of acrylamide was dissolved in 24g of deionized water at room temperature. 0.14g of mesoporous SiO was added under magnetic stirring at 25 ℃ for 5 hours₂Ball SMS, then sonication for 1 hour. It was transferred to an ice-water bath, and then 0.64g of tannic acid, 54.4mg of N, N' -methylenebisacrylamide and 129mg of ammonium persulfate were added with stirring. Then 20. mu.L of tetramethylethylenediamine were introduced for 10 minutes with stirring. The mixed solution was transferred into a glass bottle (diameter: 20mm) and a polytetrafluoroethylene mold (90 mm. times.60 mm). The reaction was carried out at 40 ℃ for 8 hours to form PAAM-TA-SMS hydrogel. PAAM-TA hydrogels without SMS were synthesized by the same procedure and used as controls. To prepare the dry hydrogel, the wet hydrogel was frozen at-40 ℃ for 12h and then freeze-dried with an ultra-low temperature freeze-dryer for 48 h. The PAAM-TA synthesis result is shown in (b) in FIG. 1, and has multi-layer pores, and the crosslinking density is reduced compared with PAAM. The PAAM-TA-SMS synthesis results are shown in FIG. 1 (c), the hydrogel has relatively coarse pore walls and smaller pore sizes, and the addition of SMS increases the crosslinking density of the hydrogel.

The experimental procedure is as above, changing the added SiO₂The material is SBA-15, the synthesis result is shown in figure 1 (d), the hydrogel has relatively rough pore walls and smaller pore diameters, and the addition of SBA-15 improves the crosslinking density of the hydrogel.

Sixthly, the infrared characterization result is shown in figure 2. 3400cm in SMS sample^-1OH of (2) stretching vibration, 1100cm^-1、800cm^-1The stretching vibration of Si-O-Si of (a) indicates successful synthesis of the silica material. A typical characteristic peak was observed in PAAM samples at 3343cm^-1And 3201cm^-1Is in the presence of NH₂Peak of stretching vibration at 1680cm^-1Has C ═ O stretching peak at 1608cm^-1Is in the presence of NH₂Deformation peak. In the PAAM-TA-SMS composite hydrogel sample, the PAAM is 3343cm^-1And 3201cm^-1NH of (C)₂The expansion peak moves to 3337cm^-1And 3200cm^-1At 1680cm^-1The C ═ O stretching peak at the position moves to 1599cm respectively^-1。NH₂And the shift in C ═ O stretching peaks can be attributed to intermolecular hydrogen bonding interactions between PAAM and SMS.

Seventhly, 0.5g of the hydrogel sample was immersed in 25 ℃ PBS buffer (pH7.4) and swollen until an equilibrium state was reached. After a prescribed time interval, after gently removing excess water with filter paper, the swollen hydrogel was weighed and the swelling ratio was calculated. The specific data is shown in FIG. 3, the swelling ratio of PAAM-TA-SMS is improved by 113% compared with PAAM-TA; compared with PAAM-TA, the swelling ratio of PAAM-TA-SBA15 is improved by 200%. The added mesoporous material has a unique pore channel structure, can generate capillary action, can absorb water so as to improve the swelling rate of the hydrogel, and has a water absorption effect superior to that of SMS due to the larger specific surface area and pore volume of the SBA-15. The addition of the mesoporous silica improves the swelling rate of the polyacrylamide hydrogel, can rapidly swell during hemostasis, and absorbs a large amount of water, so that blood cells are coagulated, and the hemostasis effect of the hydrogel is improved.

For cyclic loading-unloading compression test, cylindrical hydrogel with a diameter of 20mm and a height of 15mm was used. They compress to 80% strain at a rate of 5 mm/min. Before the measurement, the hydrogel was coated with a thin layer of silicone oil to prevent water evaporation during the test. Five replicates were measured per sample. The strength of the adhesion of the hydrogel to the pig skin was measured in terms of the force required to peel the adhered skin. The hydrogel was placed between two pigskins with an adhesive area of 25mm x 20 mm. It was then statically placed for 30 minutes and then subjected to a tensile test. Under ambient conditions, the universal tester pulls the sample to failure at a crosshead speed of 5 mm/min. Each test was repeated at least five times and the average value was reported. Specific results as shown in fig. 4, using the example of PAAM-TA-SMS hydrogel, it was able to withstand uniaxial compression up to 80% strain without any damage and was able to recover fully after unloading, indicating that the material had excellent elasticity. When the compressive strain becomes higher than 60%, the compressive stress of all hydrogels starts to grow exponentially as the segments become more and more compact with compression.

Ninthly, anesthetizing the rat (SD, 0.18-0.25kg) by an intraperitoneal injection method (10% chloral hydrate, 0.03 mL/kg); the middle of the tail part is cut off by surgical scissors, bleeding is carried out for 15s, and a normal bleeding model is ensured. Next, a pre-weighed tube of each sample, wet gauze (5X 5cm saturated with physiological saline) was used²Standard gauze), PAAM-TA-SMS, PAAM-TA-SBA15 blood loss and clotting time were calculated during this procedure by dipping the tail over the bleeding site with minimal pressure. Gauze was used as blank group, PAAM-SMS, and PAAM-SBA-15 were used as material control group. The experimental results are shown in fig. 5 and 6, the tail of the rat is cut off with a surgical scissors, and then the hydrogel is stuck to the bleeding incision. No blood was found to seep from the hydrogel/skin interface, indicating that the hydrogel tightly sealed the wound. The control group (gauze group) had a hemostatic time of 662s, while the PAAM-TA-SBA15 group had a substantially reduced hemostatic time of approximately 192 s. The control group had a bleeding amount of 2318mg, while the PAAM-TA-SMS group had a minimal reduction in bleeding amount to 37 mg. The addition of the mesoporous silica obviously reduces the amount of bleeding, shortens the bleeding stopping time and improves the bleeding stopping effect of the hydrogel material. PAAM-TA-SMS has better hemostatic effect, probably because SMS has spherical structure, has smaller curvature radius compared with SBA-15, has larger contact surface with blood protein, and has better blood coagulation effect.

In (r) E.coli as a representative strain. First, the bacteria were activated, and 2mL of the culture medium and 20. mu.L of the bacteria were added and cultured for 24 hours. The bacteria were then subcultured, after which 100 μ L of medium was added to each well of a 96-well plate, 4 μ L of the sample soak was added to the medium of the first well, and then the sample was double diluted. And adding the sample soaking solution into the first hole, and then fully blowing and beating the sample soaking solution by using a liquid transfer gun to fully and uniformly mix the sample soaking solution with the culture medium. Then 100 mul of diluted bacterial liquid is added into each hole, and the operation is repeated for three times. And finally, placing the 96-well plate into a constant-temperature incubator at 37 ℃ for 16-20h, and observing the result. The experimental results are shown in table 1, and it can be seen that the antibacterial performance of the material is significantly improved after the Tannin (TA) is added, the antibacterial effect of the material is more than 75%, and the material has a good antibacterial effect.

TABLE 1 bacteriostatic ratio of four hydrogels

Claims

1. A hydrogel hemostatic material with an antibacterial function is prepared by adding mesoporous silica and tannic acid in the polymerization process of acrylamide so as to prepare the mesoporous silica/tannic acid composite polyacrylamide hydrogel.

2. The hydrogel hemostatic material of claim 1, wherein the mesoporous silica is SMS and/or SBA-15.

3. The hydrogel hemostatic material according to claim 2, wherein the SMS is spherical mesoporous silica prepared by a sol-gel method using tetraethoxysilane as a silicon source, hexadecene-1-acetic acid as a surfactant, and ammonia water as a catalyst.

4. The hydrogel hemostatic material as claimed in claim 2, wherein the SBA-15 is cashew-shaped mesoporous silica prepared by a sol-gel method with tetraethoxysilane as a silicon source, a surfactant of a triblock copolymer of polyethylene oxide-polypropylene oxide-polyethylene oxide, and hydrochloric acid as a catalyst.

5. The hydrogel hemostatic material according to claim 1, wherein the mesoporous silica is added in an amount of 0.6 to 2.2% by mass of acrylamide, and the tannic acid is added in an amount of 6.0 to 10% by mass of acrylamide.

6. A method of preparing a hydrogel hemostatic material of claims 1-5, comprising the steps of:

1) dissolving acrylamide in deionized water at room temperature;

2) adding mesoporous silica under stirring, and carrying out ultrasonic treatment;

3) transferring the solution obtained in the step 2) into an ice-water bath, adding tannic acid, a cross-linking agent and an initiator under stirring, reacting for a period of time, and then adding an accelerator;

4) transferring the mixed solution obtained in the step 3) into a mold, and polymerizing to form hydrogel.

7. The preparation method of claim 6, wherein acrylamide is added into deionized water in step 1), magnetic stirring is performed at room temperature for 4-6 hours, and ultrasonic treatment is performed for 50-80 minutes after mesoporous silica is added in step 2).

8. The preparation method according to claim 6, wherein the cross-linking agent in step 3) is N, N' -methylenebisacrylamide in an amount of 0.2% to 0.6% by mass based on the mass of acrylamide; the initiator is ammonium persulfate, and the using amount of the initiator is 1-4% of the mass of the acrylamide; the accelerant is tetramethylethylenediamine, and the dosage of the accelerant is 20-50 mu L/100g of acrylamide.

9. The method of claim 6, wherein the hydrogel is polymerized at 40 to 60 ℃ in step 4).

10. The method of claim 6, wherein the wet hydrogel polymerized in step 4) is frozen at-40 ℃ for a period of time and then freeze-dried using an ultra-low temperature freeze-dryer to obtain a dry hydrogel.