KR20190071453A - Hydrogel hemostatic material comprising pullulan and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a hydrogel hemostatic agent using pullulan and a manufacturing method thereof. More particularly, the present invention relates to a hydrogel hemostatic agent which has excellent blood coagulation ability by including calcium chloride and thrombin in the hydrogel using pullulan and which utilizes pullulan manufactured in the form of an injectable hydrogel, thereby being easy to handle and move; and a manufacturing method thereof.

Description

[0001] HYDROGEL HEMOSTATIC MATERIAL COMPRISING PULLULAN AND MANUFACTURING METHOD THEREOF [0002]

The present invention relates to a hydrogel hemostatic agent using pullulan and a method for preparing the same, and more particularly, to a hydrogel hemostatic agent using pullulan which has excellent blood coagulability including calcium chloride and thrombin in a hydrogel using pullulan, A hydrogel hemostatic agent using a pullulan which is easy to handle and fluid, and a method for producing the hemostatic agent.

Hemostasis means that blood vessels are injured and blood comes into contact with foreign matter, and various blood coagulation factors present in the platelets or plasma cause a clot through the complicated pathway to stop hemorrhage of the wound.

When blood vessels are damaged by trauma or disease and bleeding occurs, blood vessel constriction is first performed to stop the bleeding, followed by formation of a platelet plug by adhesion and coagulation of platelets, which is a primary hemostatic process, Through the activation of coagulation factors, fibrin is formed and a hemostasis plug is formed and complete hemostasis of bleeding site is achieved.

To explain the typical mechanism of action of blood coagulation factors involved in the second hemostatic process, thromboplastin is released into the blood plasma at the time of hemorrhage and acts on the inactive prothrombin present in plasma, To thrombin in the active state. Subsequently, active thrombin catalyzes the conversion of soluble fibrinogen in the blood into a fibrin that is insoluble, and the fibrin clumps together to fix the platelet plug more tightly, resulting in a hemostatic process that stops bleeding.

However, in the case of such a hemorrhage in the body, the hemorrhage can be sufficiently exerted for mild hemorrhage due to mild wound. However, in the case of excessive hemorrhage occurring during surgical operation or emergency, there is a limit in stopping the hemorrhage sufficiently. There are many cases in which additional requirements are required.

Such hemostatic methods include a mechanical method, a cold method, and a chemical method. In chemical methods, hemostasis is stopped by treating a blood coagulant or the like at a bleeding site.

Currently, Surgicel ^® , Gelfoam ^® , Floseal ^®, and Avitene ^{® are} among the commercialized products of hemostatic agents, which are chemical preparations.

Surgicel ^® is an absorbent knit made from oxidized and regenerated cellulose. It has a high acidity and acts as an antimicrobial with hemostasis and absorbed after 7-14 days. However, it has been reported that low blood pressure results in pain, sneezing and neurotoxicity in arterial bleeding, in the formation of body fluids, in rejection of foreign substances, and in rhinological applications.

Gelfoam ^® is a porous foam-type hemostatic agent made by sprinkling formaldehyde in refined gelatin through a thermal polymerization foaming process. It is low cost and used as an absorbable hemostat during surgery and trauma and is completely absorbed in the tissue within 4-6 weeks after use. However, if arterial bleeding or hemorrhage continues, hemorrhage may continue due to pressure failure, and there is a disadvantage in that a foreign body reaction may lead to the formation of a membrane and hematoma.

Floseal ^® is a high viscosity gel consisting of a mixture of Bovine gelatin and thrombin. It can be used when there is irregular and damp surface mucosa and platelet dysfunction. However, it is expensive and can cause adhesion, and there is a side effect of causing foreign body reaction, mucous membrane fusion and inflammatory reaction.

Avitene ^® is made from bovine corium collagen with partial hydrochloric acid salt. This releases the clotting factor that attacks the platelets and forms a fibrous mass. It is used when the usual way of controlling capillary hemorrhage during surgery or ligation is ineffective or impossible. However, it can be easily broken, and it is not easily fallen on the forceps, so it is not easy to operate, and the attraction force is low and the hemostatic effect is not high.

As described above, although currently commercially available absorbent packing is sold at a high price, the hemostatic effect is not sufficient, or the regeneration effect of the mucous membrane is often insignificant. Therefore, there is a continuing need to develop a new hemostatic agent that is inexpensive, has a hemostatic effect, biocompatibility, and regeneration of the mucous membrane and can prevent rebleeding.

On the other hand, since the development of a hydrogel made of poly (2-hydroxyethyl methacrylate) (PHEMA) by Wichterle in the 1960s, its application in the field of biomaterials has been increased due to its hydrophilic and biocompatibility. Hydrogel refers to a structure having a three-dimensional hydrophilic polymer network that can contain a large amount of water or biological fluids in nature. The network structure of these hydrogels is formed by various factors such as chemical bonding or physical agglomeration, and is thermodynamically stable after swelling in an aqueous solution. Because of its flexible strength and high water content, hydrogels are similar to living tissues and are widely used in medical or pharmaceutical fields.

In particular, the injection system using the hydrogel is capable of designing a highly efficient delivery system at a desired site by simple mixing. The fluidity is also expected to be applied in various fields because it has properties suitable for filling defects.

In order to utilize the hydrogel as an injection-type material, it has fluidity like a fluid in vitro and needs to be gelated by physical or chemical stimulation after injection. When hydrogels are formed by molecular entanglement or by secondary bonds including ionic bonds, hydrogen bonds, and hydrophobic interactions, they are called reversible or physical hydrogels. Physical hydrogels are generally not uniform, and the intermediate part or end of the chain is a temporary bond. The interactions that hold these physical hydrogels are reversible and can disintegrate as physical conditions (temperature, pH, concentration, additives, ionic strength, etc.) change.

However, the biggest limitation in the model of gelation during injection is that it is strongly influenced by the concentration of various types of gel precursors and crosslinking agents. This may result in biomedical constraints caused by crosslinking agents and may lead to changes in strength and changes in unwanted mechanical properties, such as modifying by crosslinking agents or precursors.

In order to overcome these drawbacks, in recent years, various studies have been conducted on the selection of chemical cross-linking agents, such as the use of substances extracted from natural substances. In particular, researches on horseradish, an enzyme extracted from wasabi peroxidase, HRP) is one of the many notable materials.

Western horseradish peroxidase (HRP) acts as a crosslinking agent by reacting with tyramine and hydrogen peroxide (H ₂ O ₂ ), and is known as a crosslinking agent having excellent biodegradability and biocompatibility. The first step is the oxidation of HRP by hydrogen peroxide, and the second step, oxidized HRP, forms an intermediate by oxidizing the phenol group in the tyramine. The resultant material creates a network point by creating a bridge point. In this case, the mechanical strength and hydrogel formation time can be easily controlled by the concentration of H ₂ O ₂ and HRP.

There is a need for a hemostatic agent that has a handleable and fluid form through application of such a hydrogel to a hemostatic agent and exhibits excellent blood coagulation ability through a hemostatic agent.

Patent Document 1: KR 10-1624625

The technical problem to be solved by the present invention is to provide a blood coagulation ability due to rapid release of blood coagulation factor applied to a hemorrhage site where excessive bleeding occurs during surgery or wound, And a method for producing the hydrogel hemostatic agent.

In order to accomplish the objects of the present invention as described above and achieve the characteristic effects of the present invention described below, the characteristic structure of the present invention is as follows.

The process for preparing a hydrogel hemostatic agent using pullulan according to the present invention comprises the steps of (a) reacting 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TEMPO), sodium bromide (NaBr) Introducing a carboxyl group into pullulan using a chloride solution; (b) reacting the carboxyl group-introduced pullulan with N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide EDC) to introduce tyramine; (c) preparing a hydrogel by adding horseradish peroxidase (HRP), hydrogen peroxide (H ₂ O ₂ ), thrombin and calcium chloride (CaCl ₂ ) to the tyramine substituted pullulan obtained in the step (b) The method comprising the steps of:

In the step (a), the pullulan has a weight average molecular weight of 50,000 to 250,000 g / mol, and is contained in an amount of 1 to 50 parts by weight based on 100 parts by weight of the total composition of the hydrogel hemostatic agent.

The substitution ratio of tyramine in step (b) is 1 to 15%.

In the step (c), the concentration of the HRP is 0.1 to 5 parts by weight based on 100 parts by weight of the total composition, and the concentration of the hydrogen peroxide solution (H ₂ O ₂ ) is 1 to 30 parts by weight .

The step (c) comprises dissolving tyramine substituted pullulan, HRP, thrombin and calcium chloride in PBS at pH 6 to 8 for 20 minutes to 1 hour at room temperature to prepare a first solution; And

To the solution containing pullulan at a concentration of 1 to 50 parts by weight based on 100 parts by weight of the total composition, a secondary solution of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) was added to the primary solution, and the reaction was performed for 5 seconds to 2 minutes Thereby producing a hydrogel.

The thrombin is contained in an amount of 100 to 500 μg per 1 mL of PBS, and the calcium chloride is contained in an amount of 3 to 15 mM per 1 mL of PBS.

And further comprises at least one component selected from the group consisting of gelatin, chitosan, hyaluronic acid, alginate and cellulose derivatives in the step (c).

In the step (c), the hemostatic material further comprises at least one selected from fibrinogen, vitamin C, vitamin K, collagen, gelatin, and gelfoam.

The hydrogel using pullulan produced according to the production method of the present invention has a viscosity at room temperature of 10,000 to 25,000 cP and an adhesive strength of 10 to 30 kPa.

The hydrogel hemostatic agent using pullulan according to the present invention is manufactured in the form of injection-type hydrogel and is easy to handle and fluid. At the same time, blood coagulation factors for promoting hemostasis are introduced, It has an effect of increasing the blood coagulation ability of the hemostatic agent.

In addition, there is an effect of exhibiting excellent biocompatibility and biodegradability without causing a tissue immune response in vivo by using pluran, a natural polymer derived from a living body.

In addition, there is an effect that the wound is suitable for the tissue type, the wound is easy to apply due to the nature of the gel, and the gel has a certain degree of mechanical strength when formed so as to maintain the shape for a certain period of time in the living body.

In addition, gel forming ability and gel formation time can be optimized by providing an appropriate blending ratio of horseradish peroxidase (HRP) and hydrogen peroxide contained in the gelation step.

1 is a view showing a process of a TEMPO reaction for introducing a carboxyl group into pullulan according to the present invention.
2 is a view showing an EDC reaction step for introducing tyramine into pullulan in which a carboxyl group is introduced.
3 is a view showing a process of manufacturing a hydrogel.
4 is a diagram showing ATR-FTIR spectra of pullulan and modified pullulan.
5 is a diagram showing ¹³ C-NMR spectra of pullulan and modified pullulan.
Figure 6 shows the ¹ H-NMR spectrum of the modified pullulan.
7 shows GPC data of pullulan and modified pullulan.
8 is a graph showing the gelation time according to the pullulan concentration.
9 is a graph showing the rheological characteristics of the hydrogel according to the pullulan concentration through a rheometer.
10 is a graph showing the degree of crosslinking density by the swelling ratio according to the pullulan concentration.
11 is a diagram showing an in vitro biodegradability evaluation according to the concentration of pullulan.
12 is a diagram showing an erythrocyte hemolysis experiment using pullulan hydrogel.
13 is a diagram showing an experiment of a hemostatic animal using SD rats.
14 is a photograph showing the results of pullulan concentration in a hemostatic animal experiment using an SD rat.
Fig. 15 is a graph showing a graph of hemorrhages in the results of pullulan concentration using SD rats.
16 is a graph showing bleeding time in the results of pullulan concentration using SD rats.
FIG. 17 is a histological observation result of H & E staining of a hemostatic site of liver treated with pullulan hydrogel.
FIG. 18 shows the result of histological observation of carotid staining of a hemostatic site of liver treated with pullulan hydrogel.

The following detailed description of the invention refers to the accompanying drawings, which illustrate, by way of illustration, specific embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. It should be understood that the various embodiments of the present invention are different, but need not be mutually exclusive. For example, certain features, structures, and characteristics described herein may be implemented in other embodiments without departing from the spirit and scope of the invention in connection with an embodiment. It is also to be understood that the position or arrangement of the individual components within each disclosed embodiment may be varied without departing from the spirit and scope of the invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is to be limited only by the appended claims, along with the full scope of equivalents to which such claims are entitled, if properly explained. In the drawings, like reference numerals refer to the same or similar functions throughout the several views.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings, so that those skilled in the art can easily carry out the present invention.

The hydrogel hemostatic agent using pullulan according to the present invention contains pullulan having biodegradability, non-toxicity and biocompatibility as main components and reacts with tyramine and hydrogen peroxide (H ₂ O ₂ ) to act as a crosslinking agent (HRP), a biodegradable and biocompatible cross-linking agent.

Pullulan is a material produced by extracellular production of Aureobasidum pullulans, and is mainly composed of three glucose-linked α-1, 3-linked maltotriose α-1, 6-linked And is formed of straight-chain glucan repeatedly bonded to each other. Detailed structure studies have revealed that pullulan is composed not only of a repeating structure of maltotriose units, but of maltoterolose in a few percent, and the end of the molecule is glucose and has a structure different from that of dextran. Pullulan with this structure is tasteless and amorphous white powder and has no problem about various toxicity and mutagenicity test results and is approved as a food additive both at home and abroad. It is a polysaccharide substance which is easy to be dissolved in water and low in viscosity compared to other polysaccharides but is easy to apply for various applications because of its strong adhesive properties and stretching properties like yarn and stable neutral solution. The viscosity of aqueous solution is relatively low in temperature dependence and is less influenced by various salts and pH. As an example, in the case of titanium or boron ions which are believed to form a chelate with the -OH group in the pullulan molecule at a viscosity of 250 to 300 cp (1% aqueous solution, 25 ° C, 30 rpm) of the aqueous solution of pullulan in which various metal ions coexist The viscosity is not increased or the gel is not formed in any case. It is a polymer material with biodegradable properties that is completely magnetized by many microorganisms in structure and finally converted into carbonized gas and water.

The method for preparing hydrogel using pullulan as a raw material and HRP as a crosslinking agent comprises the steps of i) introducing a carboxyl group into pullulan through TEMPO reaction, ii) EDC reaction step for introducing tyramine to tyramine, and iii) And a step of reacting pullulan with horseradish peroxidase and hydrogen peroxide water to prepare a hydrogel.

FIG. 1 is a view showing a process of TEMPO reaction for introducing a carboxyl group into pullulan according to the present invention, FIG. 2 is a view showing an EDC reaction process for introducing tyramine into pullulan in which a carboxyl group is introduced, and FIG. Fig. 3 is a view showing a manufacturing process of a hydrogel. Fig.

1 to 3, a method for preparing a hydrogel hemostatic agent using pullulan according to the present invention comprises: (a) reacting 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TEMPO) Introducing a carboxyl group into pullulan using sodium bromide (NaBr) and 15% sodium hypochloride solution; (b) reacting the carboxyl group-introduced pullulan with N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide EDC) to introduce tyramine; (c) Horseradish peroxidase (232 units / mg, HRP), 30% hydrogen peroxide (H ₂ O ₂ ), thrombin and calcium chloride (CaCl ₂ ) were added to the tyramine substituted pullulan obtained in the step (b) And reacting the mixture to prepare a hydrogel.

In the step (a), the pullulan may have a weight average molecular weight of 50,000 to 250,000 g / mol, and preferably 100,000 to 200,000 g / mol. The hydrogel hemostatic agent may be contained in an amount of 1 to 50 parts by weight, preferably 5 to 50 parts by weight, based on 100 parts by weight of the total composition of the hydrogel hemostatic agent. If the viscosity of the hydrogel is higher than the above range, the viscosity of the hydrogel becomes too high in the living body, and the gelation takes a long time to delay the hemostasis. If the range is lower than the above range, have.

In order for HRP to function as a crosslinking agent, a tyramine group is required, but since a tyramine group is not present in pullulan, introduction of a tyramine group into pullulan is required to use pullulan. In the modification of pullulan, considering the mechanical strength of the hydrogel and the gel formation time, the substitution ratio of tyramine in step (b) is suitably about 1 to 15%, preferably about 4 to 10% Preferably, a replacement rate of 6.4% is appropriate.

The concentration of the HRP in the step (c) for producing the hydrogel is 0.1 to 5 parts by weight based on 100 parts by weight of the total composition, and the concentration of the hydrogen peroxide solution (H ₂ O ₂ ) is 100 parts by weight 1 to 30 parts by weight per 100 parts by weight of the composition.

The step (c) may be carried out in two steps. Tyramine substituted pullulan, HRP, thrombin (92.6 units / mg) and calcium chloride are added at room temperature for 20 minutes to 1 hour, 8, preferably pH 7, to prepare a first solution; And a secondary solution prepared by dissolving tyramine substituted pullulan and 30% aqueous hydrogen peroxide (H ₂ O ₂ ) in PBS at pH 6 to 8, preferably pH 7, in the primary solution, And reacting the mixture for 2 minutes to prepare a hydrogel.

HRP oxidized by aqueous hydrogen peroxide oxidizes the phenol groups in the thylamine groups introduced into pullulan to form intermediates, thereby forming crosslinking sites to form a network structure, thereby forming an injectable hydrogel.

The thrombin may be contained in an amount of 100 to 500 μg per 1 mL of PBS, and the calcium chloride may be contained in an amount of 3 to 15 mM per 1 mL of PBS. Lower than this range may result in a lower hemostatic effect, and higher blood pressure may lead to economic loss.

In the step (c), in addition to pullulan, at least one selected from the group consisting of gelatin, chitosan, hyaluronic acid, alginate and cellulose derivatives is further contained.

In step (c), the hemostatic material may further include at least one selected from the group consisting of fibrinogen, vitamin C, vitamin K, collagen, gelatin, and gelfoam in addition to thrombin and calcium chloride.

The hydrogel using pullulan prepared according to the preparation method of the present invention may have a viscosity of 10,000 to 25,000 cP at room temperature. It is also characterized in that the adhesive strength is 10 to 30 kPa. When the above range is satisfied, it is possible to prevent the hydrogel hemostatic agent from being detached or lost from the hemostatic site. When the viscosity range is higher than the above range, there arises a problem in ease of operation during operation. On the contrary, when the range is lower than the above range, there is a problem in tissue adhesion and storage stability.

In addition, the hydrogel hemostatic agent of the present invention may have a viscosity and an adhesive strength range because it can be used in the form of a spray, spray, gel, liquid and aerosol.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following examples.

<Examples>

1. Introduction of a carboxyl group into pullulan

For the modification of pullulan, 2,2,6,6-tetramethyl-1-piperidinyloxy purchased from Aldrich, free radical (TEMPO) and Sigma-Aldrich Sodium bromide (NaBr) and sodium hypochloride solution purchased from Sigma-Aldrich were used. Pullulan is mixed with TEMPO and NaBr in tertiary distilled water and stirred well at 1 ° C. After adding 15% NaOCl solution, the pH of the solution was gradually added with 4 M HCl and kept at 9.5, and the mixture was stirred for about 120 minutes while adding 0.5 M NaOH solution. After that, methanol was added to terminate the reaction, followed by neutralization with 4 M HCl. After the solution was precipitated with acetone, the precipitate was repeated 2-3 times to remove unreacted materials, and the solution was dried under reduced pressure to prepare pullulan having a carboxyl group (FIG. 1).

2. Step of introducing pullulan into tyramine

Tyramine purchased from Fluka, N-hydroxysuccinimide (NHS) purchased from Wako Co., Ltd., and 1 (1) purchased from TCI in order to introduce a tyramine group into the prepared carboxyl group-introduced pullulan, -Ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC).

First, the modified pullulan is added to the third distilled water and stirred sufficiently for about 3 to 5 hours. Tyramine, EDC and NHS are then added to the solution, and the mixture is stirred for about 12 hours while maintaining the pH at 4.7. The resulting solution was neutralized with 4 M HCl and dialyzed against 100 mM NaCl solution, 25% ethanol and distilled water for about 2 days, 1 day and 1 day, respectively, for 4 days. The dialyzed solution was lyophilized to prepare a sample into which tyramine was introduced (FIG. 2).

3. Preparation of hydrogel hemostatic agent

To the above-prepared thylamin-substituted pullulan, horseradish peroxidase (232 unit / mg, HRP) purchased from Amresco, hydrogen peroxide (H ₂ O ₂ ) purchased from Daejung Co., 92.6 units / mg) and calcium chloride were added and dissolved at pH 7 for 30 minutes at room temperature. Then, a solution in which 30% hydrogen peroxide and thylamin-substituted pullulan were dissolved was added thereto and reacted within 60 seconds to finally prepare hydrogel hemostatic agent (Fig. 3).

<Experimental Example>

Experimental Example 1 Attenuated total reflection Fullerene modification confirmation by FT-IR

4 is a diagram showing ATR-FTIR spectra of pullulan and modified pullulan. Referring to FIG. 4, attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR, IRAffinity-1S, Shimadzu) was used to confirm the presence of pullulan after the TEMPO reaction and the EDC reaction according to the present invention. The peak at 1600 cm ^-1 after the TEMPO reaction indicated that the -OH group oxidized to the OC = O group (carboxylated pullulan). On the other hand, there was no change in peak after EDC-NHS reaction because of the small amount of tyramine introduced. The introduction of tyramine was confirmed by NMR.

Experimental Example 2 Verification of Pullulan Modification by NMR

1) &lt; ¹³ &gt; C-NMR spectrum analysis

5 is a diagram showing ¹³ C-NMR spectra of pullulan and modified pullulan. Referring to FIG. 5, ¹³ C-NMR was used to confirm the modification after the TEMPO reaction (BRUKER BIOSPIN / AVANCE 400, 400 MHz FT-NMR, Bruker, Germany). The characteristic peaks of the carboxylic acid and carboxylate salt forms were observed at 175 ppm, indicating that the primary alcohol was oxidized by the TEMPO reaction to exist as a carboxyl group.

2) ¹ H-NMR spectrum analysis

6 is a view showing the ¹ H-NMR spectrum of the modified pullulan. Referring to FIG. 6, ¹ H-NMR was used for confirming the introduction of tyramine after the EDC-NHS reaction (BRUKER BIOSPIN / AVANCE 400, 400 MHz FT-NMR, Bruker, Germany). The peak of benzene characteristic of tyramine was observed at around 7 ppm, confirming that tyramine was introduced by EDC-NHS reaction. In addition, the substitution ratio of tilamine was confirmed by comparing with the integral ratio of hydrogen appearing in C ₂ and C ₃ .

Experimental Example 3 Molecular weight analysis by gel permeation chromatography

FIG. 7 shows a water soluble gel permeation chromatographic system (GPC) (Breeze System, USA) as a GPC data of pullulan and modified pullulan in order to examine changes in the molecular weight of the pullulan during the modification step. Referring to FIG. 7, it can be seen that the initial 180,000 Mw of pullulan decreased to 97,667 Mw after the TEMPO reaction and to 22,355 Mw after the EDC-NHS reaction.

Experimental Example 4 Hydrogelation time analysis

8 is a graph showing gelation time according to pullulan and HRP concentration. When the concentration of pullulan was 0.15 g / ml, when the concentration of HRP was more than 3 mg / ml, gelation was observed within 5 seconds. When the concentration of HRP was 3 mg / ml, when the concentration of pullulan was 0.15 or more, it was confirmed that the gelation occurred within 5 seconds.

Experimental Example 5 Rheological properties analysis

9 is a graph showing the rheological characteristics of the hydrogel according to the pullulan concentration through a rheometer. A Thermo Scientific HAAKE MARS II Rheometer (Thermo Scientific HAAKE MARS II Rheometer, Thermo, Germany) was used to characterize the rheological properties of the hydrogel prepared by the method of the present invention. The Rheometer used is a device for measuring the rheological properties (viscosity, elastic modulus, stress, shear rate, etc.) of solutions, emulsions, suspensions and thermosetting materials as well as polymeric materials. Flow and deformation can be imposed to identify the structure and properties of the material in a physical way.

When the concentration of HRP was 3.0 mg / ml, the storage elastic modulus was increased as the concentration of pullulan increased (0.3 g / ml, 0.4 g / ml, 0.5 g / ml) It was confirmed that a hydrogel having a desired storage elastic modulus can be produced.

Experimental Example 6 Swelling ratio analysis with pullulan concentration

10 is a graph showing the degree of crosslinking density by the swelling ratio according to the pullulan concentration. After hydrogel was injected into an aluminum pan with a diameter of 1 cm and a height of 0.5 cm to prepare a hydrogel of a predetermined size, it was placed in 50 ml of PBS solution (pH 7.2, 37 ° C) for 1, 3, 5, The weight was measured. The swelling ratio was calculated as follows.

Ws: The weight of the swelled hydrogel.

Wd: Weight of lyophilized hydrogel.

The swelling ratio showed the highest with time when the pullulan concentration was 0.3 g / ml and the HRP concentration was 3 mg / ml.

Experimental Example 7 Of pullulan hydrogel in vitro  Biodegradability evaluation

The biodegradation behavior of hydrogel hemostat was investigated in vitro to determine the biodegradation behavior of the hydrogel hemostat prepared. Samples were placed in a 50 mL cornical tube and placed in a shaking bath (90 rpm, 37 ° C) with 25 mL of phosphate buffered saline (PBS, pH 7.4) Biodegradation behavior was observed. The recovered samples were transferred to a new 10 mL cornical tube, lyophilized and weighed. As the concentration of pullulan increased (0.3, 0.4 and 0.5 g / mL), the weight loss rate was 30.4%, 24.4% and 17.1%, respectively, when the biodegradation behavior was observed in the in vitro environment after 5 hours. And 72.7%, 70.5% and 62.9%, respectively. After 24 hours, it was observed that all the samples were decomposed.

Experimental Example 8 PRT experiments according to thrombin and calcium chloride concentration

In the hydrogel preparation according to the present invention, blood coagulation ability tests were carried out at various concentrations including thrombin and calcium chloride. After adding 300 μL of the hemostatic agent at 37% in each temperature, the blood was centrifuged at 3000 rpm or more, and 300 μL of platelet-poor-plasma (PPP) was added to the solution on the watch plate. The time at which the thrombus was generated was then measured.

[Table 1] [Table 2]

As shown in Table ₂ , when the concentration of CaCl ₂ is too high or too low, the hemostatic effect is reduced. When the concentration is 0.009375 M, 96 s is most effective Respectively.

Accordingly, when preparing the hydrogel in step (c), thrombin may be contained in an amount of 100 to 500 μg per 1 mL of PBS, and the calcium chloride may be contained in an amount of 3 to 15 mM per 1 mL of PBS. Out of this range, it can take too long to stop bleeding and bleed.

Experimental Example 9 Of the manufactured hydrogel hemostat Erythrocyte hemolysis experiment

In order to measure the amount of residual erythrocytes after coagulation of blood, erythrocyte hemolysis experiments were carried out to confirm blood coagulation ability through the absorbance of hemoglobin from hemolyzed red blood cells. The control group is a case where the blood coagulation reaction proceeds without introducing the hydrogel sample. After a certain period of time, 5 mL of distilled water is added to the sample to stop the blood clotting reaction, and the resulting supernatant is dispensed into a 96-well plate to measure the absorbance of hemoglobin. Since the amount of residual red blood cells is small when the blood coagulation proceeds, the amount of hemoglobin released through the hemolysis is small, so that the hemostatic ability can be observed through the absorbance intensity of hemoglobin.

As shown in FIG. 12, when the hydrogel containing hemostatic agent was used, the absorbance was lower than that of the control group, and the hydrogel having lower concentration of pullulan was found to have lower absorbance.

Experimental Example 10 Of the manufactured hydrogel hemostat i n vivo  Hemostasis experiment

13 is a photograph showing a hemostatic animal experiment using an SD rat. SD rats were anesthetized by administering anesthetics mixed with Zoletil ^® 50 and Rompun ^® in a ratio of 4: 1. After the anesthetization of the SD rats was completed, the abdomen hairs were removed and fixed at 45 ° to the operating table. After disinfection of the abdomen epidermal region with povidone-iodine solution, the skin and peritoneum were dissected using a bladder 10 at the center, and the skin and the peritoneum were peeled and the liver was elevated to the both sides. After placing the pre-weighed filter paper under the liver, a wound of depth 3 mm was placed on the left lobe using a No. 10 blade exposed 3 mm above the plug. When the bleeding started, 0.5 ml of hydrogel was introduced to cause hemostasis The time was measured. After hemostasis, the amount of bleeding was calculated by weighing the filter paper and hydrogel that absorbed the blood.

14 is a photograph showing the results of pullulan concentration in a hemostatic animal experiment using an SD rat. As shown in Table 3 below, a hydrogel hemostatic agent prepared by varying the concentration of pullulan at 0.3 g / ml, 0.4 g / ml and 0.5 g / ml was used.

[Table 3]

FIG. 15 is a graph showing the hemorrhages in the results of pullulan concentration using SD rats. In bleeding, bleeding of 651.2, 334.3, and 273.3 mg was observed in 1,149.9 mg of the control group and 0.3, 0.4 and 0.5 g / ml of pullulan, respectively, and 0.5 g / ml showed the greatest hemostatic effect.

16 is a graph showing bleeding time in the results of pullulan concentration using SD rats. The hemostasis time was about 130 seconds in the control group and 61.5, 50.1, and 10.5 seconds in the pullulan 0.3, 0.4, and 0.5 g / ml, respectively. It was found that the hemostatic effect was greatest at 0.5 g / ml there was. It seems that the lower the concentration of pullulan is, the more the flow is made at 45 ° and the initial hemostasis is not achieved. If the concentration is higher, the hemostasis is effective because the initial hemostasis is not performed

FIG. 17 is a diagram showing the result of histological observation of hemostatic site of liver treated with pullulan hydrogel through H & E staining. FIG. The nucleus of the cell was blue, the cytoplasm was reddish, and the wound area was red. As a result, it was confirmed that red blood cells were well intertwined with the wound area. 18 is a diagram showing the result of histological observation of carotid staining of a hemostatic site of liver treated with pullulan hydrogel. This staining method stained fibrin red and platelets blue. The wound area was red, indicating that fibrin was well formed.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, Those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Therefore, the spirit of the present invention should not be construed as being limited to the above-described embodiments, and all of the equivalents or equivalents of the claims, as well as the following claims, I will say.

Claims (12)

(a) introducing a carboxyl group into pullulan using 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TEMPO), sodium bromide (NaBr) and sodium hypochloride solution;
(b) reacting the carboxyl group-introduced pullulan with N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide EDC) to introduce tyramine; And
(c) preparing a hydrogel by adding horseradish peroxidase (HRP), hydrogen peroxide (H ₂ O ₂ ), thrombin and calcium chloride (CaCl ₂ ) to the tyramine substituted pullulan obtained in the step (b) Wherein the method comprises the steps of:
The method according to claim 1,
In the step (a), the pullulan has a weight average molecular weight of 50,000 to 250,000 g / mol, and 1 to 50 parts by weight per 100 parts by weight of the total composition of the hydrogel hemostatic agent. A method for producing a hemostatic agent.
The method according to claim 1,
Wherein the substitution ratio of tyramine in step (b) is 1 to 15%.
The method according to claim 1,
Wherein the concentration of the HRP in the step (c) is 0.1 to 5 parts by weight based on 100 parts by weight of the total composition.
The method according to claim 1,
(C) the concentration of the hydrogen peroxide (H ₂ 0 ₂₎ at step process for producing a hydrogel hemostatic agent using pullulan characterized in that it comprises an amount of 1 to 30 parts by weight based on 100 parts by weight of the total composition.
The method according to claim 1,
The step (c) comprises dissolving tyramine substituted pullulan, HRP, thrombin and calcium chloride in PBS at pH 6 to 8 for 20 minutes to 1 hour at room temperature to prepare a first solution; And
Adding a second solution of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) to the primary solution and allowing the reaction to proceed for 5 seconds to 2 minutes to prepare a hydrogel; &Lt; / RTI &gt;
The method according to claim 6,
Wherein the thrombin is contained in an amount of 100 to 500 μg per 1 mL of PBS, and the calcium chloride is contained in an amount of 3 to 15 mM per 1 mL of PBS.
The method according to claim 1,
Wherein the step (c) further comprises at least one component selected from the group consisting of gelatin, chitosan, hyaluronic acid, alginate and cellulose derivatives.
The method according to claim 1,
Wherein the hemostatic material further comprises at least one selected from fibrinogen, vitamin C, vitamin K, collagen, gelatin, and gelfoam in step (c).
A hydrogel hemostatic agent using pullulan prepared according to any one of claims 1 to 9.
11. The method of claim 10,
Wherein the hydrogel hemostatic agent has a viscosity of 10,000 to 25,000 cP at room temperature.
11. The method of claim 10,
Wherein the hydrogel hemostatic agent has an adhesive strength of 10 to 30 kPa.

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