KR101474237B1 - Hemostatic Agent based on Silk Fibroin with adhesive and wound healing effect - Google Patents

Abstract

The present invention relates to a hemostatic sponge having an adhesive force containing silk fibroin.
A method for producing a sponge for hemostasis according to the present invention comprises the steps of: removing sericin and impurities from a silkworm cocoons to obtain a silk fibroin solution; adding a polymer to the silk fibroin and mixing the silk fibroin solution to obtain a foamed silk fibroin / And a third step of preparing a sponge for hemostasis by insolubilizing the silk fibroin / polymer mixed aqueous solution prepared in the foam form by lyophilization.
According to the present invention, it is possible to maximize the hemostatic effect that can be used in the human body because it has biodegradability by maintaining appropriate pores and supporting structure, promoting blood coagulation with fast blood absorption, and exhibiting wound healing effect of surrounding tissues A hemostatic sponge is provided.

Description

Technical Field [0001] The present invention relates to a hemostatic agent based on silk fibroin and an adhesive and wound healing effect containing silk fibroin,

The present invention relates to a hemostatic sponge having an adhesive force containing silk fibroin, and more particularly to a sponge for hemostasis using silk fibroin, and more particularly to a hemostatic sponge having silk fibroin, which is excellent in mass productivity, biodegradability, wound regeneration function, bioadhesive function, excellent biocompatibility and biological risk of silk fibroin Thereby increasing the hemostatic effect of the sponge.

Bleeding refers to the phenomenon of blood coming out of blood vessels due to damage of blood vessels. When bleeding occurs, thrombosis is created by the action of platelets and blood coagulation factors, and blood is produced by blocking damaged blood vessels and tissues. If platelets and blood clotting factors are abnormal, they easily bruise or bleed on small wounds and do not stop well.

The blood clotting mechanism is that when blood comes out of blood vessels, platelets in blood are destroyed and thromboplastin is formed, and thrombopressin works with calcium ions in blood to change prothrombin, which is one of plasma proteins, to thrombin.

Thrombin acts on fibrinogen and becomes a thread-like substance called fibrin. This fibrin is entangled in a net shape, and with blood cells in it, it becomes smaller and smaller as time goes by.

These blood clots are indispensable for maintaining life. If there is no effect on the blood, the individual will not be able to coagulate even after a small bleeding, eventually leading to death due to excessive bleeding.

A variety of hemostatic agents are used in the actual medical field. One of them is a sponge-like Tachcomb® Topical which contains thrombin, fibrinogen, collagen and aprotinin, which contains hemostatic coagulation factors in vivo in the hemostatic agent. Thrombin and other styptic agents. The problem of these stomachs contains fibrinogen, thrombin, collagen, etc. extracted from the blood of the human or human body, and there is a risk of mad cow disease, AIDS and other acquired infection, and the manufacturing cost is high and the price is very high.

Surgicel®, Avitene®, and ChitoClot® are also available as hemostatic agents that have blood coagulation effects by absorbing blood. Surgicel® is a gauze-type hemostat that is a major component of oxidized regenerated cellulose, Avitene® is a sponge of microfibrillar collagen and ChitoClot® is a sponge-type topical hemostat made with chitosan for medical use.

However, such a hemostatic agent has a disadvantage that hemostatic effect is insignificant, price is high, biocompatibility is low, or is not absorbed in vivo, and the regeneration effect of bleeding site is small.

Therefore, it is necessary to develop a new hemostatic agent which can provide hemostatic effect and can be supplied at low cost and has excellent biocompatibility and can regenerate bleeding site and prevent rebleeding.

 Related prior arts are disclosed in International Patent Application No. WO 2011/0798336 "Hemostatic sponge" and Korea Patent Publication No. 10-2011-0023399 "Intelligent Fibrin Biocompatible Agent with Excellent Hemostatic Effects and Mechanical Properties ".

However, the above-mentioned prior arts have a problem that a manufacturing process such as the addition of a separate crosslinkable component or a separate antibiotic for antibacterial effect is required to improve the adhesiveness, There is a dot.

It is an object of the present invention to provide a silk fibroin-containing adhesive which increases the hemostatic effect by adhering to peripheral bleeding sites by taking advantage of silk fibroin's mass productivity, biodegradability, wound regeneration function, bioadhesive function, excellent biocompatibility and biological risk And a sponge for hemostasis.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.

The hemostatic sponge of the present invention is characterized by having silk fibroin and having an adhesive force and a hemostatic effect.

The hemostatic sponge has a pore size of 150 to 500 mu m and has a blood absorption ability.

The hemostatic sponge may further comprise a polymer selected from the group consisting of polyvinyl alcohol (PVA), hyaluronic acid (HA), gelatin, chitosan, polyethylene oxide (PEG) .

The method for producing a sponge for hemostasis according to the present invention comprises the steps of: removing sericin and impurities from a silkworm cocoons to obtain a silk fibroin solution; adding a polymer to the silk fibroin, mixing the silk fibroin, And a third step of preparing a sponge for hemostasis by insolubilizing the silk fibroin / polymer mixed aqueous solution prepared in the foam form by lyophilization.

In the first step, the silk fibroin solution contains 6 to 8% of silk fibroin.

In the second step, the polymer is at least one of polyvinyl alcohol (PVA), hyaluronic acid (HA), gelatin, chitosan, polyethylene oxide (PEO), and polyethylene glycol (PEG).

In the second step, the silk fibroin and the polymer are mixed at 6 to 7: 4 to 3.

And the second step further comprises a surfactant or a foaming agent in the foamed silk fibroin / polymer mixed aqueous solution.

The surfactant includes at least one selected from the group consisting of polyethylene glycol, polyethylene oxide, disodium laureth sulfosuccinate, coco betaine, dessyl glucoside, barba amidopropyl betaine, and cocamidopropyl betaine Feature.

In the third step, the insolubilization treatment is characterized in that any one of methanol, ethanol, and propanol is used.

In the third step, the insolubilization treatment is characterized in that it is composed of hydration treatment for 5 to 8 hours at a humidity of 80 to 90% or high temperature treatment for 2 to 4 hours at 80 to 160 ° C.

According to the present invention, it is possible to maximize the hemostatic effect that can be used in the human body because it has biodegradability by maintaining appropriate pores and supporting structure, promoting blood coagulation with fast blood absorption, and exhibiting wound healing effect of surrounding tissues A hemostatic sponge is provided.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a view showing a simplified view of a manufacturing process of a sponge for hemostasis of silk fibroin according to the present invention. FIG.
2 is a SEM photograph of the sponge for hemostasis of silk fibroin of the present invention.
A: Control (ChitoClot®)
B: Example 1 (S-1)
C: Example 2 (S-2)
D: Example 3 (S-3)
FIG. 3 is a graph showing the results of experiments on the equipment and the adhesive performance used for the adhesion test of the silk fibroin-containing hemostatic sponge of the present invention.
4 is a view showing a swelling force test of the sponge for hemostasis with silk fibroin of the present invention and a result of the test.
5 is a view showing an exploded view of the sponge for hemostasis of silk fibroin according to the present invention with the passage of time.
6 is a diagram showing a hemostatic effect of the sponge for hemostasis of silk fibroin according to the present invention.
A: Control (ChitoClot®)
B: Example 1 (S-1)
C: Example 2 (S-2)
D: Example 3 (S-3)
Fig. 7 is a diagram showing bleeding time of the sponge for hemostasis of silk fibroin according to the present invention. Fig.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail, and a detailed description of known functions and configurations that may unnecessarily obscure the gist of the present invention will be omitted.

Silk is a natural polymer protein obtained from silkworm, which is a mass-produced natural polymer protein capable of obtaining silk fibroin through sericin removal process.

These silk fibroin, unlike other synthetic materials, does not cause an immune response and is a polymer approved by the FDA for use as a medical material. It has been proven to be safe as a surgical suture and other medical materials. Due to these advantages, various medical materials using silk fibroin have been developed in recent years.

Silk fibroin is a protein extracted from silkworm and can be mass-produced. Therefore, silk fibroin, which is a biodegradable polymer which is lower in cost than conventional medical hemostatic agents, excellent in wound healing effect, Has both hydrophilic and hydrophobic properties and has adhesion to living tissue. Recently, the use of such properties has led to development of adhesives for body tissue restoration.

By utilizing the advantages of silk fibroin without mass productivity, biodegradability, wound regeneration, bioadhesion, excellent biocompatibility and biological risks, it is possible to develop a new hemostatic agent that overcomes the disadvantages of conventional hemostatic agents.

Accordingly, in the present invention, it is possible to maximize the hemostatic effect that can be used in the human body because it has suitable voids and supporting structure, has fast blood absorption, has an adhesive force, promotes blood coagulation, And to provide a hemostatic sponge capable of controlling the rate of biodegradation according to the crystallization process and the form of the preparation, thereby obtaining an optimal hemostatic effect according to the hemostasis site.

In addition, since the sponge for hemostasis using silk fibroin of the present invention provides sufficient blood absorption capacity and hemostatic effect to replace the local hemostatic agent, it can be effectively used for stopping hemorrhage even in massive hemorrhage due to trauma and organ damage.

The sponge for hemostasis with silk fibroin of the present invention is produced from a step of preparing a stock solution of silk fibroin, a step of mixing and forming voids, and a step of crystallization.

In the step of forming the mixture and the void, a woven method, a salt extraction method, a method using a foaming agent, a freeze drying method, an electrospinning method, and the like can be manufactured by various methods.

Therefore, according to the present invention, it is possible to manufacture a sponge for hemostasis using silk fibroin which has voids capable of absorbing blood and can control the gap by the process of forming the mixing and voids.

Meanwhile, the produced silk fibroin-containing hemostatic sponge may be further subjected to an insolubilization treatment step. In the present invention, the support structure of the sponge for hemostasis of silk fibroin is maintained by treating with methanol, ethanol or propanol, or by hydration or high-temperature treatment.

Hereinafter, the production process of the sponge for hemostasis with silk fibroin according to the present invention will be described in detail.

1. Stage 1: Preparation of silk fibroin solution

Sericin protein and impurities are removed from Bombyx mori.

At this time, the silkworm is immersed in a basic aqueous solution such as sodium carbonate, heated, and then washed with distilled water. The silk fibroin obtained through this process is dissolved in a salt solution such as calcium chloride and lithium bromide.

It is then dialyzed with distilled water to remove ionic components from the solution. At this time, the dialysis time is 36 to 72 hours, and the concentration of the final silk fibroin solution is 6 to 8%.

2. Stage 2: Preparation of silk fibroin / polymer mixed aqueous solution

In order to improve the support structure and adhesion of the hemostatic sponge, a polymer is mixed in a silk fibroin and mixed to prepare a silk fibroin / polymer mixed aqueous solution.

In this case, the weight ratio of silk fibroin and added polymer is 7: 3 to 6: 4, and the ratio may be changed according to the form of the preparation. The polymer may include polyvinyl alcohol (PVA), hyaluronic acid (HA) , Gelatin, chitosan, polyethylene oxide (PEO), and polyethylene glycol (PEG).

The prepared silk fibroin / polymer mixed aqueous solution is rapidly mixed with a foaming machine or a stirrer at 900 to 1500 rpm for 10 to 30 minutes to prepare a foam.

At this time, it is important to maintain a uniform size of foam to maintain a constant gap, and a certain amount of surfactant can be used for this purpose.

The surfactant to be used herein is at least one selected from the group consisting of polyethylene glycol, polyethylene oxide, disodium laureth sulfosuccinate, coco betaine, dessyl glucoside, baba suiamidopropyl betaine, and cocamidopropyl betaine .

When the prepared silk fibroin / polymer mixed aqueous solution is lyophilized for 24 to 36 hours, a sponge for hemostasis with silk fibroin having a certain gap can be obtained. In order to make such a pore structure, a pore may be formed by using a blowing agent or the like after drying.

The foaming agent used herein may be any one selected from a chemical foaming agent and a physical foaming agent.

3. Step 3: Insolubilization of the sponge for hemostasis with silk fibroin

The prepared silk fibroin-containing hemostatic sponge is hydrophilic and easily dissolved in water, so that it is difficult to maintain the structure. Therefore, the sponge is subjected to an insolubilizing process in order to control the decomposition degree in vivo and to maintain the physical properties. The insolubilization process may be performed by immersing or spraying methanol, ethanol or propanol for 3 to 5 minutes or by hydration (exposure to 80 to 90% of humidity, room temperature (20 to 25 ° C) for 5 to 7 hours) Or by treatment with a high temperature (exposure at 80 to 160 ° C for 2 to 4 hours) to maintain the support structure of the sponge for hemostasis of silk fibroin.

In order to maximize the hemostatic effect such as wound area or nose bleeding where there is little bleeding site, it is effective to carry out the partial insolubilization process through the hydration process and the high temperature treatment. In case of hemostasis of a large amount of hemorrhagic region or hemoglobin, , Ethanol, and propanol.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes only and do not limit the scope of protection of the present invention.

<Example 1> Manufacture of sponge S-1 for hemostasis with silk fibroin of the present invention

First, the Bombyx mori was heated in an aqueous solution of sodium carbonate and then washed with distilled water to remove sericin protein and impurities, and silk fibroin was obtained.

The obtained silk fibroin was dissolved in a lithium bromide solution and then dialyzed with distilled water for 40 hours in order to remove the ion component so that the concentration of the final silk fibroin solution was 6%.

In order to improve the support structure and adhesion of the hemostatic sponge, a polymer mixture solution (1% of polyvinyl alcohol (PVA), 6% of gelatin) was added to the silk fibroin solution at a ratio of 7: 3 to prepare a silk fibroin / .

The prepared silk fibroin / polymer mixed aqueous solution was rapidly mixed with 900 rpm for 10 minutes using a stirrer to prepare a foam.

At this time, polyethylene glycol, which is a surfactant, was added to maintain constant pores.

The prepared silk fibroin / polymer mixed aqueous solution was lyophilized for 30 hours. In order to solve the problem that it is difficult to keep the structure easily due to hydrophilicity due to hydrophilicity, it is exposed at a high temperature for 3 hours at 160 ° C to be insolubilized, (Fig. 1). The sponge S-1 for hemostasis of the silk fibroin of the present invention was prepared.

<Example 2> Manufacture of sponge S-2 for hemostasis with silk fibroin of the present invention

First, the Bombyx mori was heated in an aqueous solution of sodium carbonate and then washed with distilled water to remove sericin protein and impurities, and silk fibroin was obtained.

The obtained silk fibroin was dissolved in a lithium bromide solution and then dialyzed with distilled water for 40 hours in order to remove the ion component so that the concentration of the final silk fibroin solution was 6%.

To improve the support structure and adhesion of the hemostatic sponge, a polymer mixture solution (1% polyvinyl alcohol (PVA), 6% hyaluronic acid (HA)) was mixed in the silk fibroin solution at a ratio of 6: / Polymer mixed aqueous solution.

The prepared silk fibroin / polymer mixed aqueous solution was rapidly mixed with 900 rpm for 10 minutes using a stirrer to prepare a foam.

At this time, polyethylene glycol, which is a surfactant, was added to maintain constant pores.

The prepared silk fibroin / polymer mixed aqueous solution was lyophilized for 30 hours. In order to solve the problem that it is difficult to maintain the structure by dissolving in water due to hydrophilicity, it is insolubilized by ethanol for 3 minutes to have a certain void 2 of the present invention having a silk fibroin (Fig. 1).

<Example 3> Production of a sponge S-3 for hemostasis using silk fibroin of the present invention

Except that the silk fibroin-containing hemostatic sponge S of the present invention having a certain pore size was subjected to insolubilization in the hydration method at a humidity of 85% for 8 hours instead of the insolubilization treatment by the tow method, -3 was prepared (Fig. 1).

<Experimental Example 1> Confirmation of pore structure of a sponge for hemostasis containing silk fibroin

In order to confirm the pore structure of the silk fibroin-containing hemostatic sponges (S-1, S-2, S-3) prepared in Examples 1 to 3, they were observed using a scanning electron microscope.

ChitoClot®, a chitosan lyophilized hemostat, was used as a control.

The results are shown in Table 1 and Fig.

Control group S-1 S-2 S-3 Porosity
(Porosity,%) 49.3 ± 16.4 36.0 + - 10.0 69.5 ± 7.1 57.9 ± 9.1

As shown in Table 1 and FIG. 2, the silk fibroin-containing hemostatic sponges (S-1, S-2, and S-3) prepared in Examples 1 to 3 of the present invention were the conventional hemostatic products The pore structure similar to the hemostat ChitoClot® (control) shows that this pore structure can be used as a hemostatic agent.

Especially, in the case of the sponge for hemostasis of the present silk fibroin, it has a size of about 150 ~ 500 μm, and it can be understood that it has a void structure capable of rapidly absorbing the bleeding blood.

<Experimental Example 2> Physical properties and hemostatic effect of sponge for hemostasis with silk fibroin

1) Adhesive power

In order to confirm the adhesive strength of the sponge for hemostasis (S-1, S-2, S-3) containing silk fibroin prepared in Examples 1 to 3, the adhesive force of each sponge was verified using an all purpose physical property measuring machine.

ChitoClot® was used as a control.

① Experimental method

S-1, S-2 and S-3 prepared in Examples 1 to 3 were prepared in a size of 1 x 1 cm and wetted with 20 占 퐇 of saline for 10 minutes. And then bonded to a test vise.

Then, the adhesive strength was verified by dropping the constant force and speed (5 min / min) of the universal physical property measuring device.

② Experimental results

As shown in FIG. 3, in the case of ChitoClot (R), the silk fibroin-containing hemostatic sponges (S-1, S-2 and S-3) , The adhesive strength was 3 ~ 5 times higher than that of the control group.

2) Swelling power

① Experimental method

The sponge for hemostasis with silk fibroin prepared in Examples 1 to 3 was cut into 1x1 cm size and immersed in saline for 3 hours to measure the swelling power.

The swelling power was calculated as follows.

② Experimental results

As shown in FIG. 4, the swelling power was the best in the conventional hemostatic agent ChitoClot®. However, the swelling power of the silk fibroin-containing hemostatic sponges (S-1, S-2, S -3) showed a swelling power of about 5 ~ 15, which is a result that the bleeding blood can be sufficiently absorbed.

3) Degradability

① Experimental method

The degree of decomposition of the silk fibroin-containing hemostatic sponges (S-1, S-2 and S-3) prepared in Examples 1 to 3 was observed at room temperature for 0 hours, 3 hours and 24 hours.

② Experimental results

As shown in FIG. 5, there was no significant change in each group immediately after immersion. However, in the case of the sponge for hemostasis (S-3) containing silk fibroin prepared in Example 3, the silk fibroin dissolved .

However, the difference in insolubility was confirmed in the sponge for hemostasis using gelatin containing silk fibroin prepared in Examples 1 and 2 and the sponge for hemostasis (S-1, S-2) containing silk fibroin insolubilized with ethanol The difference in solubility according to the degree of crystallization has a usability that can be applied to the bleeding site to be applied to the human body.

That is, when the decomposition and dissolution are required for a short time such as nosebleed, crystallization using a hydration method can be used for human body application. In case of internal organs hemorrhage or massive hemorrhage, completely insoluble silk fibroin- It is considered efficient to use a sponge.

4) Hemostatic effect

① Experimental method

To confirm the hemostatic effect of the sponge for hemostasis of silk fibroin prepared in Examples 1 to 3, the hemostatic effect in an animal model was confirmed.

Rats were used as experimental animals, and unilateral inguinal incisions were made to expose femoral artery and femoral vein. Then, the operation was performed to cut the exposed blood vessels using surgical mass. Immediately after the procedure, a 1 × 1 cm sized silk fibroin-containing hemostat was placed on the bleeding site and hemostasis was performed with a force of 5 N for 30 seconds. After 30 seconds, the sponge for hemostasis with silk fibroin was removed to check for bleeding.

② Experimental results

As shown in FIG. 6, hemostasis was completed within 30 seconds after the bleeding site was completely sealed. At this time, the silk fibroin-containing hemostatic sponge prepared in Examples 1 to 3 absorbed blood and observed a clean hemostatic site without accumulation of blood. The black arrow indicates the femoral artery and the white arrow indicates the hemostasis site.

In addition, as shown in FIG. 7, the hemostasis time of the silk fibroin-containing hemostatic sponges manufactured in Examples 1 to 3 was shorter than that of ChitoClot®, which was conventionally commercialized as a hemostatic agent. As shown in FIG. 7, The superior hemostatic effect of the sponge was confirmed.

From the above results, it can be seen that the sponge for hemostasis of a silk fibroin of the present invention is excellent in hemostatic effect and can control the degree of adhesion and decomposition according to the insolubilization process. Therefore, the sponge for hemostasis using silk fibroin of the present invention is excellent in hemostatic effect, has an adhesive ability, and is excellent in biocompatibility and can be used as a hemostatic agent.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the inventions. Examples and experiments may be implemented. The scope of the present invention is defined by the appended claims, and all differences within the scope of the claims are to be construed as being included in the present invention.

Claims (12)

delete delete delete A first step of removing sericin and impurities from the cocoon to obtain a silk fibroin solution;
A second step of adding a polymer to the silk fibroin and mixing and then stirring the silk fibroin to prepare a foamed silk fibroin /
And a third step of preparing a sponge for hemostasis by lyophilizing the silk fibroin / polymer mixed aqueous solution prepared in the foam form and then performing insolubilization treatment,
Wherein the surfactant or the foaming agent is further contained in the foamed silk fibroin / polymer mixed aqueous solution in the second step.
A method for manufacturing a sponge for hemostasis.
5. The method of claim 4,
Wherein the silk fibroin solution contains 6 to 8% silk fibroin in the first step,
A method for manufacturing a sponge for hemostasis.
5. The method of claim 4,
In the second step, the polymer may be at least one selected from the group consisting of polyvinyl alcohol (PVA), hyaluronic acid (HA), gelatin, chitosan, polyethylene oxide (PEO)
A method for manufacturing a sponge for hemostasis.
5. The method of claim 4,
In the second step, the silk fibroin and the polymer are mixed at 6 to 7: 4 to 3,
A method for manufacturing a sponge for hemostasis.
delete 5. The method of claim 4,
The surfactant may be at least one selected from the group consisting of polyethylene glycol, polyethylene oxide, disodium laureth sulfosuccinate, coco betaine, decylglucoside, babaisamidopropyl betaine, and cocamidopropyl betaine.
A method for manufacturing a sponge for hemostasis.
5. The method of claim 4,
Wherein in the third step, the insolubilizing treatment uses any one of methanol, ethanol, and propanol.
A method for manufacturing a sponge for hemostasis.
5. The method of claim 4,
Wherein the insolubilizing treatment in the third step is carried out by any one of hydration treatment for treatment at 80 to 90% humidity for 5 to 8 hours or high temperature treatment at 80 to 160 DEG C for 2 to 4 hours.
A method for manufacturing a sponge for hemostasis.
Which is produced by the production method of any one of claims 4 to 7 or 9 to 11,
Silk fibroin, a polymer and a surfactant or foaming agent,
An adhesive force and a hemostatic function and has a void of 150 to 500 mu m,
Hemostatic sponge.

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