阅读说明：本技术 一种用于快速止血和伤口修复的海绵及其制备方法 (Sponge for rapid hemostasis and wound repair and preparation method thereof ) 是由 陈景帝 曹淑君 徐敢 杨一凡 于 2021-09-14 设计创作，主要内容包括：本发明公开了一种用于快速止血和伤口修复的海绵及其制备方法,该海绵以壳聚糖和氧化石墨烯为海绵的骨架材料,将单宁酸加入其中冻干成前体海绵,经过氨气的处理形成,单宁酸羟基作为氢供体,壳聚糖的氨基作为氢受体进行二次交联,形成丰富的氢键,使海绵在水中或血液中不会溶解和分散。该海绵具备较好的抗压性能、抗菌和抗氧化性以及快速止血效果,同时生物相容性良好,制备成本低,有望成为一种治疗快速止血和伤口修复的潜在材料。(The invention discloses a sponge for rapid hemostasis and wound repair and a preparation method thereof. The sponge has good compression resistance, antibacterial and antioxidant properties and rapid hemostasis effect, and is good in biocompatibility and low in preparation cost, and is expected to become a potential material for treating rapid hemostasis and wound repair.)

1. A method for preparing a sponge for rapid hemostasis and wound repair, which is characterized by comprising the following steps: the preparation method comprises the steps of taking chitosan and graphene oxide as framework materials of the sponge, adding tannic acid into the framework materials, freeze-drying the mixture to form precursor sponge, and treating the precursor sponge with ammonia gas to form the sponge.

2. The method of claim 1, wherein: the method comprises the following steps:

(1) adding graphene oxide into deionized water, and performing ultrasonic treatment until the graphene oxide is completely dispersed;

(2) adding chitosan and acetic acid, stirring to form a uniform solution, adding a cross-linking agent, and cross-linking at normal temperature for 4-8 h;

(3) adjusting the pH value of the solution obtained in the step (2) to 3.5, adding tannic acid, and stirring for half an hour;

(4) injecting the solution obtained in the step (3) into a mold, and placing the mold at 4 ℃ for 1 h, 20 ℃ below zero for 4 h and 80 ℃ below zero for 12 h to form a solid A;

(5) freeze-drying the solid A obtained in the step (4) in a vacuum freeze dryer to remove water in the sample;

(6) and (3) placing the molded sample subjected to freeze drying in the step (5) in a sealed ammonium carbonate environment at 45 ℃ for 12 h, and then placing the molded sample in a fume hood for 2-3 d to remove redundant ammonia gas, thereby finally obtaining the chitosan/graphene oxide/tannin sponge.

3. The method of claim 2, wherein: the mass ratio of the usage amount of the acetic acid in the step (2) to the chitosan is 1: 1 mL/g, and the concentration of acetic acid is 17.5 mol/L.

4. The method of claim 2, wherein: the cross-linking agent in the step (2) is prepared by mixing EDC and NHS according to the mass ratio of 1: 2.

5. The method of claim 2, wherein: the mass ratio of the cross-linking agent to the graphene oxide is 0.15: 0.04.

6. the method of claim 2, wherein: and (3) chitosan: and (3) graphene oxide: the mass ratio of the added tannic acid is 0.8: 0.04: 0.04 to 0.12.

7. The method of claim 2, wherein: the ammonium carbonate is added in the step (6) so that ammonia gas is generated by the ammonium carbonate at the temperature of 45 ℃.

8. A sponge for rapid hemostasis and wound repair prepared by the method of any one of claims 1 to 7.

Technical Field

The invention belongs to the field of material preparation, and particularly relates to a sponge for rapid hemostasis and wound repair and a preparation method thereof.

Background

Skin is the first line of defense to protect the human body from harmful environmental influences. When skin is injured, the wound quickly stops bleeding and closes, and the injured skin quickly regenerates, again acting as a barrier. However, wound exudate contains a large amount of bacteria, inflammatory factors, proteases and free radicals, thus slowing the rate of wound healing. Skin wound healing is a complex process involving hemostasis, inflammation, proliferation and remodeling. Although various biomaterials have been developed in recent years to accelerate wound healing, wound healing materials not only have rapid hemostatic, antibacterial and healing-promoting functions, but also need to be able to suppress the overproduction of free radicals and the long-term secretion of proinflammatory cytokines in wound tissue, as well as easy manufacturing and low cost. Furthermore, uncontrolled bleeding and infection are major causes of traumatic death. The hemostatic material can rapidly stop bleeding and reduce blood loss, thereby gaining time for rescue and having important significance for saving the life and safety of people. Sponge is an important hemostatic material, and is widely used for rapid hemostasis due to its high liquid absorption rate and aggregation of blood cells/platelets.

The invention provides a special sponge material, which has good compression resistance, antibiosis, inoxidizability and rapid hemostasis effect, simultaneously has good biocompatibility and low preparation cost, and is expected to become a potential material for treating rapid hemostasis and wound repair.

Disclosure of Invention

The invention provides a preparation method of a sponge for rapidly stopping bleeding and healing wounds, which is relatively simple, takes chitosan and graphene oxide as framework materials of the sponge, and cross-links tannic acid to form a precursor sponge for avoiding hydrolysis. Through ammonia gas treatment, hydroxyl of tannic acid is used as a hydrogen donor, and amino of chitosan is used as a hydrogen acceptor to carry out secondary crosslinking to form rich hydrogen bonds, so that the sponge cannot be dissolved and dispersed in water or blood, and the prepared sponge has a good pore structure, can absorb a large amount of blood, has good compression resistance and antibiosis, and can prevent further damage to a wound from the outside, and in order to realize the purpose, the invention adopts the following technical scheme:

a sponge for rapid hemostasis and wound repair is prepared by taking chitosan and graphene oxide as skeleton materials of the sponge, adding tannic acid into the skeleton materials, and treating with ammonia gas to obtain the sponge for rapid hemostasis and wound repair.

The preparation method of the sponge comprises the following steps:

(1) adding graphene oxide into deionized water, and performing ultrasonic treatment until the graphene oxide is completely dispersed;

(2) adding chitosan and acetic acid, stirring to form a uniform solution, adding a cross-linking agent, and cross-linking at normal temperature for 4-8 h;

(3) adjusting the pH value of the solution obtained in the step (2) to 3.5, adding tannic acid, and stirring for half an hour;

(4) injecting the solution obtained in the step (3) into a mold, and placing the mold at 4 ℃ for 1 h, 20 ℃ below zero for 4 h and 80 ℃ below zero for 12 h to form a solid A;

(5) freeze-drying the solid A obtained in the step (4) in a vacuum freeze dryer to remove water in the sample;

(6) and (3) placing the molded sample subjected to freeze drying in the step (5) in a sealed excessive ammonium carbonate environment at 45 ℃ for 12 h, and then placing the molded sample in a fume hood for 2-3 d to remove excessive ammonia gas, so as to finally obtain the chitosan/graphene oxide/tannin sponge.

Further, the mass ratio of the dosage of the acetic acid in the step (2) to the chitosan is 1: 1 mL/g, and the concentration of acetic acid is 17.5 mol/L.

Further, the crosslinking agent in the step (2) is EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide) mixed in a mass ratio of 1: 2.

Further, the mass ratio of the dosage of the cross-linking agent to the graphene oxide is 0.15: 0.04.

further, the ratio of chitosan: and (3) graphene oxide: the mass ratio of the added tannic acid is 0.8: 0.04: 0.04 to 0.12.

Further: the mass of ammonium carbonate in the step (6) is 10 g.

Further: the ammonium carbonate is added in the step (6) so that ammonia gas is generated by the ammonium carbonate at the temperature of 45 ℃.

The chitosan has excellent hemostasis, antibiosis and biocompatibility, contains abundant amino groups on molecular chains, and can be used as an intermediate molecule to form chemical crosslinking with other substances with carboxyl groups through chemical crosslinking and form strong hydrogen bonds with pyrocatechol-like substances.

Graphene Oxide (GO) is prepared by oxidizing graphite with strong acid, and the surface of the graphene oxide contains various active groups, so that the graphene oxide has good hydrophilicity and a larger specific surface area. The graphene oxide sponge dressing with the functions of rapidly absorbing wound exudate, stopping bleeding, resisting bacteria and the like is obtained by combining physical and chemical hybridization crosslinking and uniformly dispersing the graphene oxide sponge dressing in natural polysaccharide macromolecules through a freeze drying technology, wherein the graphene oxide sponge dressing has the function of causing strong platelet aggregation and hemostasis.

The tannic acid contains natural cross-linking agent of pyrogallol, has dopamine-like structure, can adhere to tissue around wound, and directly interacts with protein in blood to achieve hemostatic effect. In order to prepare the sponge with better mechanical property, tannic acid is added for secondary crosslinking, so that the freeze-dried sponge is formed by treating ammonia gas, hydroxyl of the tannic acid is used as a hydrogen donor, and amino of chitosan is used as a hydrogen acceptor for secondary crosslinking to form rich hydrogen bonds.

The invention has the following remarkable advantages: the sponge provided by the invention takes chitosan and graphene oxide as framework materials, and tannic acid is crosslinked to form precursor sponge. Through ammonia gas treatment, hydroxyl of tannic acid is used as a hydrogen donor, and amino of chitosan is used as a hydrogen acceptor to carry out secondary crosslinking to form rich hydrogen bonds.

The method makes full use of the characteristics of chitosan and graphene oxide and utilizes the cross-linking of amino and carboxylIn combination, the solubility of the chitosan in the acid water is mainly used to cause protonation (-NH) of amino group₃ ⁺) In (1), the positive charge of the protonated amino group is neutralized by ammonia to form an amino group (-NH)₂) The sponge and the tannic acid-pyrogallol structure form strong hydrogen bonds, so that the prepared sponge cannot be hydrolyzed, and the sponge has the effect of quickly absorbing blood so as to achieve the aim of quickly stopping bleeding.

The rat liver injury model shows that the prepared sponge has potential application prospect in the aspect of rapid hemostasis.

Compared with other hemostatic and wound healing materials, the synthesized sponge has the following advantages:

(1) the synthesized composite sponge has antibacterial property and oxidation resistance.

(2) The prepared sponge has a good pore structure.

(3) The prepared sponge is treated by ammonia gas after being added with tannic acid, and has the performance of difficult hydrolysis.

(4) Chitosan, graphene oxide and tannic acid all have hemostatic properties, and the prepared sponge has a quick hemostatic effect.

(5) Low cost and simple operation.

Drawings

FIG. 1 is a diagram of a prepared sponge sample;

FIG. 2 is a graph of sponge dissolution performance;

FIG. 3 is a graph of the mechanical properties of four groups of sponges prepared;

FIG. 4 is an electron micrograph of four groups of sponges prepared;

FIG. 5 is a graph of porosity of four sets of sponges prepared;

FIG. 6 is an infrared analysis chart of the sponge components and four sets of sponges;

FIG. 7 is a plot of the inhibition circles for E.coli and S.aureus for four groups of sponges;

FIG. 8 is a bar graph of absorbance after incubation of four groups of sponges with E.coli and Staphylococcus aureus;

FIG. 9 is a diagram showing the formation of colonies on a plate after four groups of sponges are cultured with Escherichia coli and Staphylococcus aureus and then diluted and inoculated with bacterial liquid;

FIG. 10 is an electron micrograph of four groups of sponge blood cells adhering thereto;

figure 11 is a graph of wound repair in four groups of sponges and rats.

Detailed Description

In order to make the aforementioned and other features and advantages of the invention more comprehensible, embodiments accompanied with figures are described in detail below. The method of the present invention is a method which is conventional in the art unless otherwise specified.

Example 1 (without tannic acid)

(1) Taking 40 mL of deionized water, adding 0.04 g of graphene oxide, and carrying out ultrasonic treatment until the graphene oxide is completely dispersed;

(2) adding 0.8g of chitosan and 0.8 mL of acetic acid (17.5 mol/L) into the solution, stirring to form a uniform solution, adding a crosslinking agent EDC/NHS (0.05/0.1 g), and crosslinking for 4 hours at normal temperature;

(3) adjusting the pH value of the solution obtained in the step (2) to 3.5;

(4) injecting the solution obtained in the step (3) into a mold, and placing the mold in a refrigerator at 4 ℃ for 1 h, a refrigerator at-20 ℃ for 4 h and a refrigerator at-80 ℃ for 12 h to form a solid A;

(5) freeze-drying the solid A obtained in the step (4) in a vacuum freeze dryer to remove water in the sample;

(6) and (3) placing the molded sample subjected to freeze drying in the step (5) in a sealed excessive ammonium carbonate environment at 45 ℃ for 12 h, then placing the molded sample in a fume hood for 2 d to remove redundant ammonia gas, and finally obtaining chitosan/graphene oxide/tannin sponge with the serial number of CS/GO.

Example 2

(1) Taking 40 mL of deionized water, adding 0.04 g of graphene oxide, and carrying out ultrasonic treatment until the graphene oxide is completely dispersed;

(2) adding 0.8g of chitosan and 0.8 mL of acetic acid (17.5 mol/L) into the solution, stirring to form a uniform solution, adding a crosslinking agent EDC/NHS (0.05/0.1 g), and crosslinking for 4 hours at normal temperature;

(3) adjusting the pH value of the solution obtained in the step (2) to 3.5, adding 0.04 g of tannic acid into the solution, and stirring for half an hour;

(4) injecting the solution obtained in the step (3) into a mold, and placing the mold at 4 ℃ for 1 h, 4 h at-20 ℃ and 12 h at-80 ℃ to form a solid A;

(5) freeze-drying the solid A obtained in the step (4) in a vacuum freeze dryer to remove water in the sample;

(6) placing the formed sample subjected to freeze drying in the step (5) in a sealed excessive ammonium carbonate environment at 45 ℃ for 12 h, then placing the formed sample in a fume hood for 2 d to remove redundant ammonia gas, and finally obtaining chitosan/graphene oxide/tannin sponge with the serial number CS/GO/TA₁。

Example 3

(1) Taking 40 mL of deionized water, adding 0.04 g of graphene oxide, and carrying out ultrasonic treatment until the graphene oxide is completely dispersed;

(2) adding 0.8g of chitosan and 0.8 mL of acetic acid (17.5 mol/L) into the solution, stirring to form a uniform solution, adding a crosslinking agent EDC/NHS (0.05/0.1 g), and crosslinking for 4 hours at normal temperature;

(3) adjusting the pH value of the solution obtained in the step (2) to 3.5, adding 0.08 g of tannic acid into the solution, and stirring for half an hour;

(4) injecting the solution obtained in the step (3) into a mold, and placing the mold at 4 ℃ for 1 h, 4 h at-20 ℃ and 12 h at-80 ℃ to form a solid A;

(5) freeze-drying the solid A obtained in the step (4) in a vacuum freeze dryer to remove water in the sample;

(6) placing the formed sample subjected to freeze drying in the step (5) in a sealed excessive ammonium carbonate environment at 45 ℃ for 12 h, then placing the formed sample in a fume hood for 2 d to remove redundant ammonia gas, and finally obtaining chitosan/graphene oxide/tannin sponge with the serial number CS/GO/TA₂。

Example 4

(1) Taking 40 mL of deionized water, adding 0.04 g of graphene oxide, and carrying out ultrasonic treatment until the graphene oxide is completely dispersed;

(2) adding 0.8g of chitosan and 0.8 mL of acetic acid (17.5 mol/L) into the solution, stirring to form a uniform solution, adding a crosslinking agent EDC/NHS (0.05/0.1 g), and crosslinking for 4 hours at normal temperature;

(3) adjusting the pH of the solution obtained in the step (2) to 3.5, adding 0.12 g of tannic acid into the solution, and stirring for half an hour;

(4) injecting the solution obtained in the step (3) into a mold, and placing the mold at 4 ℃ for 1 h, 4 h at-20 ℃ and 12 h at-80 ℃ to form a solid A;

(5) freeze-drying the solid A obtained in the step (4) in a vacuum freeze dryer to remove water in the sample;

(6) placing the formed sample subjected to freeze drying in the step (5) in a sealed excessive ammonium carbonate environment at 45 ℃ for 12 h, then placing the formed sample in a fume hood for 2 d to remove redundant ammonia gas, and finally obtaining chitosan/graphene oxide/tannin sponge with the serial number CS/GO/TA₃。

Comparative example (No ammonia treatment)

(1) Taking 40 mL of deionized water, adding 0.04 g of graphene oxide, and carrying out ultrasonic treatment until the graphene oxide is completely dispersed;

(2) adding 0.8g of chitosan and 0.8 mL of acetic acid (17.5 mol/L) into the solution, stirring to form a uniform solution, adding a crosslinking agent EDC/NHS (0.05/0.1 g), and crosslinking for 4 hours at normal temperature;

(3) adjusting the pH of the solution obtained in the step (2) to 3.5, adding 0.12 g of tannic acid into the solution, and stirring for half an hour;

(4) injecting the solution obtained in the step (3) into a mold, and placing the mold at 4 ℃ for 1 h, 4 h at-20 ℃ and 12 h at-80 ℃ to form a solid A;

(5) and (5) freeze-drying the solid A obtained in the step (4) in a vacuum freeze-drying machine, and removing water in the sample to obtain the product sponge.

FIG. 1 is a diagram of four sets of sponges prepared in example 1, example 2, example 3 and example 4, from which it can be seen that the sponges prepared have a three-dimensional cake-like structure 1.5 cm in diameter and are dark gray in color.

FIG. 2 is a graph comparing the solubility properties of comparative example (left) and example 2 (right), and it can be seen that the sponge not treated with ammonia gas was dissolved in water, whereas the sponge of example 2 treated with ammonia gas was not changed in form in water.

FIG. 3 is a graph of the compression properties of four sets of sponges prepared in example 1, example 2, example 3 and example 4, from which it can be seen that CS/GO/TA when the compression reaches 50%₂(0.93N) and CS/GO/TA₃(0.78N) compressive force ratio CS/GO (1.15N) and CS/GO/TA₁(1.12N) group reduction, CS/GO/TA₁The compression force of (c) is slightly lower than that of the CS group. Experimental results show that the tannin increases the flexibility of the sponge and reduces the compression performance of the sponge.

FIG. 4 is an electron micrograph of four sets of sponges prepared in example 1, example 2, example 3 and example 4, from which it can be seen that the sponges have an interconnected porous structure, similar to the cellular structure formed by the sponges.

FIG. 5 is the porosity, CS/GO/TA, of the four sponge groups prepared in example 1, example 2, example 3 and example 4₁，CS/GO/TA₂And CS/GO/TA₃The porosities of (a) are 95.91%, 96.97%, 90.49% and 84.31, respectively, it can be seen that an increase in TA content reduces the porosity of the sponge, increasing the density and strength of the sponge.

Fig. 6 is an infrared absorption spectrum of four groups of sponges prepared in example 1, example 2, example 3 and example 4, and the formation of amide bonds in the sponges can be seen from the infrared absorption spectrum.

FIG. 7 is a plot of the inhibition zones for four sponges prepared in examples 1, 2, 3 and 4, CS/GO/TA₁，CS/GO/TA₂And CS/GO/TA₃Placing the sponge in a flat plate coated with bacterial liquid, culturing at 37 ℃ for 12 h to obtain a bacteriostatic circle diagram, wherein the bacteriostatic circle of the CS/GO group is not obvious in two groups of experiments of staphylococcus aureus and escherichia coli, and the CS/GO/TA is₁And CS/GO/TA₂Compared with the CS/GO group bacteriostatic zone, the bacteriostatic zone of escherichia coli is not obvious, and the bacteriostatic zone of staphylococcus aureus is more obvious. CS/GO/TA₃The size of the inhibition zone of the group is obviously increased compared with the inhibition zones of other groups.

FIG. 8 is a histogram of absorbance of four groups of sponges cultured with E.coli and Staphylococcus aureus bacteria for 12 h, the two bacteria were measured for absorbance at 600 nm and adjusted to 0.01, the sponges were inoculated therein and co-cultured at 37 ℃ for 12 h, from which it can be seen that the absorbance of Staphylococcus aureus and E.coli was significantly increased in the group without sponges. As the amount of tannin added was increased, the absorbance of E.coli and S.aureus in CS/GO/TA sponges was gradually reduced compared to the blank.

FIG. 9 is a graph showing colony formation after incubation of four groups of sponges prepared in example 1, example 2, example 3 and example 4 with bacterial solution, which were co-cultured, coated on a plate and cultured for 12 hours, and diluted 10 after incubation of the sponge for 12 hours⁷The antibacterial effect of the sponge is further verified by double inoculation on a flat plate. It can be seen from the figure that the four groups of sponges inhibited the proliferation of Staphylococcus aureus and Escherichia coli gradually increasing with the addition of tannic acid.

FIG. 10 shows the adhesion of blood cells to four sponges, which have a large amount of blood cells adhered to the surface of the sponges in an irregular aggregation, relative to the gauze group. The addition of the tannic acid is increased, hydrogen bonds formed between the tannic acid and the chitosan are increased, so that pores of the sponge are compact, the sponge can rapidly absorb blood cells in blood plasma, the blood cells are accumulated on the surface of a wound, and the coagulation of the blood on the surface of the wound is further promoted.

FIG. 11 shows four sponges used in the wound healing of rats, with the CS/GO/TA sponges healing faster than the blank and CS/GO sponges after 7 days of treatment. After 14 days, the healing rate of the CS/GO/TA sponge treatment group wound surface reaches more than 90 percent.

The above description is only for the preparation method of the present invention, and all equivalent changes and modifications made according to the claims of the present invention should be covered by the present invention.