KR102029926B1 - 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 method of manufacturing the same, and more particularly, to a hydrogel using pullulan, including calcium chloride and thrombin, having an excellent blood coagulation ability and in the form of an injectable hydrogel. The present invention relates to a hydrogel hemostatic agent using a pullulan which is manufactured and easy to handle, and has a fluid.

Description

Hydrogel hemostatic agent using pullulan and its manufacturing method {HYDROGEL HEMOSTATIC MATERIAL COMPRISING PULLULAN AND MANUFACTURING METHOD THEREOF}

The present invention relates to a hydrogel hemostatic agent using pullulan and a method of manufacturing the same, and more particularly, to a hydrogel using pullulan, including calcium chloride and thrombin, having an excellent blood coagulation ability and in the form of an injectable hydrogel. The present invention relates to a hydrogel hemostatic agent using a pullulan which is manufactured and easy to handle, and has a fluid.

Hemostasis means that when a blood vessel is injured and blood comes into contact with an external foreign substance, various blood coagulation factors present in platelets or plasma cause a blood clot through a complicated path to stop the bleeding of the wound.

When blood vessels are damaged by trauma or disease and bleeding occurs, blood vessel contraction is first performed to stop the bleeding, and then platelet plugs are formed by adhesion and aggregation of platelets, which are primary hemostatic processes. Through activation of coagulation factors, fibrin is made and a hemostatic stopper is formed to complete the hemostasis of the bleeding site.

To explain the representative mechanism of action of the blood coagulation factors involved in the secondary hemostasis process, first, thromboplastin is released into the plasma from platelets destroyed at the time of bleeding, acting on the inactive prothrombin present in the plasma, and prothrombin It acts to convert thrombin into an active state. Subsequently, the active thrombin catalyzes the reaction of hydrolyzing soluble fibrinogen in the blood and converting it into insoluble fibrin, and the fibrin is entangled with each other to fix the platelet stopper more tightly, resulting in a bleeding hemostatic process.

However, in the case of such a hemostasis process, hemostasis may be sufficiently effective for mild bleeding due to a light wound. However, in case of excessive bleeding during surgery or emergency, the bleeding is limited enough to stop the bleeding. This additional need is often reflected.

Such a hemostatic method includes a mechanical method, a cold temperature method, and a chemical method, and the like, and a typical chemical method is to treat a bleeding site with a blood coagulant to stop the bleeding.

The commercialized products of hemostatic agents, which are currently medical preparations using chemical methods, include Surgicel ^® , Gelfoam ^® , Floseal ^® and Avitene ^® .

Surgicel ^® is an absorbent knit made of oxidized regenerated cellulose, which has a high acidity and acts as a bacteriostatic agent and is absorbed after 7-14 days. However, there are reports of low effectiveness in arterial bleeding, pain relief, sneezing and neurotoxicity due to low pH in body film formation and rejection of foreign bodies, and in rheinological applications.

Gelfoam ^® is a hemostatic agent in the form of a porous foam in which formaldehyde is formed into a sponge phase by means of thermal polymerization foaming in purified gelatin. It is inexpensive and is used as an absorbent hemostatic agent during surgery and trauma and is completely absorbed by tissue within 4-6 weeks of use. However, if arterial bleeding or bleeding is continued, the pressure may not be securely attached, and bleeding may continue, and there is a disadvantage in that the formation of a capsular form and a hematoma due to a foreign body reaction.

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

Avitene ^® is prepared as partial hydrochloric acid salt from bovine corium collagen. This releases coagulation factors that attack platelets to form fibrous masses. It is used when general methods such as controlling capillary bleeding or ligation are ineffective or impossible during surgery. However, it can be easily crushed, do not fall easily attached to the forceps, easy to operate, low adsorption power is not high hemostatic effect.

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

On the other hand, since hydrogels made of poly (2-hydroxyethyl methacrylate) (PHEMA) were developed by Wichterle in the 1960s, their application in the field of biomaterials has increased due to their hydrophilicity and biocompatibility. Hydrogel refers to a structure having a three-dimensional hydrophilic polymer network structure that can contain a large amount of water or biological fluids. The network structure of such a hydrogel is formed by various factors such as chemical bonding or physical cohesion and is then thermodynamically stable after being swollen in aqueous solution. Hydrogels have a flexible strength and high water content, and thus are similar to living tissues and are widely used in medicine or pharmacy.

In particular, injectable systems using hydrogels can design highly efficient delivery systems at desired sites with simple mixing. In addition, its fluidity is suitable for use in filling missing areas, so it is expected to be applied in various fields.

In order to utilize the hydrogel as an injectable material, it needs to be fluidized in vitro, like a fluid, and gelated by physical or chemical stimulation after injection. When hydrogels are formed by entanglement of molecules or secondary bonds including ionic bonds, hydrogen bonds, and hydrophobic interactions, they are called reversible or physical hydrogels. Physical hydrogels are generally non-uniform, with a form in which the middle or ends of the chain are temporarily bonded. Interactions that maintain these physical hydrogels are reversible and can collapse when physical conditions (temperature, pH, concentration, additives, ionic strength, etc.) change.

However, one of the biggest limitations in the model of gelation at the time of injection is that it is strongly influenced by the concentration of various types of gel precursors and crosslinkers. This may result in biomedical constraints caused by the crosslinking agent, and may cause effects such as modification by the crosslinking agent or precursors, resulting in changes in strength and unwanted mechanical properties.

Therefore, in order to overcome these weaknesses, researches on various aspects such as using materials extracted from natural substances have recently been conducted in the selection of chemical crosslinking agents. In particular, horseradish peroxidase (horseradish), an enzyme extracted from horseradish peroxidase (HRP) is one of the many materials of interest.

Horseradish peroxidase (HRP) reacts with tyramine and hydrogen peroxide (H ₂ O ₂ ) to act as a crosslinking agent, and is known as a crosslinking agent with excellent biodegradability and biocompatibility. The reaction proceeds in two stages. In the first step, HRP is oxidized by hydrogen peroxide water, and in the second step, HRP oxidizes phenol groups in tyramine to form an intermediate. As a result, the crosslinking point is generated to form a network structure. An important factor here is that the mechanical strength and the hydrogel formation time can be easily controlled by the concentrations of H ₂ O ₂ and HRP.

There is a need for a hemostatic agent that can be applied to a hemostatic agent by a hydrogel and has a fluid form that is easy to handle and exhibits excellent coagulation ability through a hemostatic factor.

Patent Document 1 KR 10-1624625

The technical problem to be solved by the present invention is applied to the bleeding site in which excessive bleeding occurs during surgery or wound has excellent blood coagulation ability due to the rapid release of blood coagulation factor, easy to handle and flowable pullul in the form of injection hydrogel It is an object of the present invention to provide a hydrogel hemostatic agent using an egg and a method of manufacturing the same.

The characteristic structure of this invention for achieving the objective of this invention mentioned above, and realizing the characteristic effect of this invention mentioned later is as follows.

Method for preparing a hydrogel hemostatic agent using pullulan according to the present invention is (a) 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TEMPO), sodium bromide (NaBr) and sodium hypo Introducing a carboxyl group into pullulan using a chloride solution; (b) Tyramine, N-hydroxysuccinimide (NHS), and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide in pullulan to which the carboxyl group obtained in step (a) is introduced; Introducing tyramine using EDC); (c) preparing a hydrogel by adding horseradish peroxidase (HRP), hydrogen peroxide (H ₂ 0 ₂ ), thrombin and calcium chloride (CaCl ₂ ) to the tyramine-substituted pullulan obtained in step (b). Characterized in that it comprises a step.

In the step (a), the pullulan has a weight average molecular weight of 50,000 to 250,000 g / mol, characterized in that it comprises 1 to 50 parts by weight based on 100 parts by weight of the total composition of the hydrogel hemostatic agent.

In step (b), the substitution rate of tyramine is characterized in that 1 to 15%.

In step (c), the concentration of HRP is included in an amount of 0.1 to 5 parts by weight based on 100 parts by weight of the total composition, and the concentration of hydrogen peroxide (H ₂ 0 ₂ ) is 1 to 30 parts by weight based on 100 parts by weight of the total composition. It is characterized by including.

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

To the primary solution, a secondary solution having 30% hydrogen peroxide (H ₂ O ₂ ) was added to a solution containing 1 to 50 parts by weight based on 100 parts by weight of the total composition to react for 5 seconds to 2 minutes. It characterized in that it comprises a step of producing a hydrogel.

The thrombin is included in 100 to 500 μg per mL of PBS, the calcium chloride is characterized in that it comprises 3 to 15 mM per mL of PBS.

In the step (c) it is characterized in that it further comprises at least one component selected from gelatin, chitosan, hyaluronic acid, alginate and cellulose derivative.

Step (c) is characterized in that it further comprises one or more selected from fibrinogen, vitamin C, vitamin K, collagen, gelatin and microgel foam (gelfoam) as a hemostatic material.

Hydrogel using pullulan prepared according to the production method of the present invention is characterized in that the viscosity of 10,000 to 25,000 cP at room temperature, the adhesive strength of 10 to 30 kPa.

Hydrogel hemostatic agent using pullulan according to the present invention is manufactured in the form of injectable hydrogel has an easy to handle and fluid effect, at the same time to introduce a blood coagulation factor that promotes hemostasis, by setting the optimum concentration to show these effects It is effective to increase the coagulation ability of the hemostatic agent.

In addition, it is effective to exhibit excellent biocompatibility and biodegradability by using flulan, which is a biological polymer derived from a living body, without causing tissue immune reaction in vivo.

In addition, it is suitable for tissue form and easy to apply wounds due to the nature of the gel, and has a certain mechanical strength when the gel is formed has the effect of maintaining the hemostasis effect while maintaining a certain form in vivo.

In addition, by providing an appropriate blending ratio of horseradish peroxidase (HRP) and hydrogen peroxide included in the gelation step, it is possible to optimize the gel formation capacity and gel formation time.

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

DETAILED DESCRIPTION The following detailed description of the invention refers to the accompanying drawings that show, 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 shapes, structures, and characteristics described herein may be embodied in other embodiments without departing from the spirit and scope of the invention with respect to one embodiment. In addition, it is to be understood that the location or arrangement of individual components within each disclosed embodiment may be changed without departing from the spirit and scope of the invention. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the present invention, if properly described, is defined only by the appended claims, along with the full range of equivalents to which such claims are entitled. Like reference numerals in the drawings refer to the same or similar functions throughout the several aspects.

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 implement the present invention.

Hydrogel hemostatic agent using pullulan according to the present invention is composed of pullulan having biodegradability, non-toxicity and biocompatibility as a main component, and reacts with tyramine and hydrogen peroxide (H ₂ O ₂ ) to serve as a crosslinking agent. It is prepared using horseradish peroxidase (HRP), which is known as a crosslinking agent with excellent biodegradability and biocompatibility.

Pullulan (pullulan) is an extracellular production of Aureobasidum pullulans (Aureobasidum pullulans), mainly three glucose is α-1, 3 bonded maltotriose is α-1, 6 bond It is formed of linear glucan repeatedly bonded to. Detailed structural studies have shown that pullulan is not formed by only repeating structures of maltotriose units, but contains several percent of maltoterrane, and the end of the molecule is composed of glucose and a structure different from dextran. Pulran with this structure is odorless and amorphous white powder, and has no problem with various toxicity and mutagenicity test results and is approved as a food additive at home and abroad. Compared to other polysaccharides, it is easy to dissolve in water and has low viscosity, but has strong adhesiveness and elongation property, and is a stable neutral solution. In addition, the viscosity of the aqueous solution is relatively low in temperature dependence and has a characteristic of being less affected by various salts and pH. As an example, in the case of titanium and boron ions, which are considered to form chelates with -OH groups in pullulan molecules with a viscosity of 250 to 300 cp (1% aqueous solution, 25 ° C, 30 rpm) of aqueous solution of pullulan with various metal ions Except for any increase in viscosity or no gel is formed. It is a polymer material with excellent adhesiveness and biodegradable property that is completely magnetized by many microorganisms in structure and finally converted to carbon gas and water.

Hydrogel production method using pullulan as a raw material and HRP as a crosslinking agent includes i) introducing a carboxyl group to pullulan through a TEMPO reaction, ii) an EDC reaction step for introducing tyramine to pullulan, and iii) a modified Preparing a hydrogel by reacting pullulan with horseradish peroxidase and hydrogen peroxide solution.

1 is a view showing a step of the TEMPO reaction for introducing a carboxyl group to pullulan in accordance with the present invention, Figure 2 is a view showing an EDC reaction step for introducing tyramine to the pullulan introduced carboxyl group, Figure 3 It is a figure which shows the manufacturing process of a hydrogel.

1 to 3, the method for preparing a hydrogel hemostatic agent using pullulan according to the present invention is (a) 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TEMPO), Introducing a carboxyl group to pullulan using sodium bromide (NaBr) and a 15% sodium hypochloride solution; (b) Tyramine, N-hydroxysuccinimide (NHS), and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide in pullulan to which the carboxyl group obtained in step (a) is introduced; Introducing tyramine using EDC); (c) To the tyramine-substituted pullulan obtained in step (b) was added horseradish peroxidase (232 unit / mg, HRP), 30% hydrogen peroxide (H ₂ 0 ₂ ), thrombin and calcium chloride (CaCl ₂ ) Reacting to produce a hydrogel is characterized in that it comprises a.

In the step (a), the pullulan may have a weight average molecular weight of 50,000 to 250,000 g / mol, preferably 100,000 to 200,000 g / mol. In addition, the hydrogel hemostatic agent may be included in an amount of 1 to 50 parts by weight, and preferably 5 to 50 parts by weight, based on 100 parts by weight of the total composition. In the case of having a range higher than the above range, the viscosity of the hydrogel becomes too high in the living body, causing gelation to take a long time, delaying the hemostasis time, and having a range lower than the range, may decrease the tissue adhesiveness of the hydrogel. have.

HRP requires a tyramine group to act as a crosslinking agent, but since tyramine groups do not exist in pullulan, introduction of tyramine groups in pullulan is necessary to use pullulan. In the modification of pullulan, considering the mechanical strength and gel formation time of the hydrogel, the substitution rate of tyramine in step (b) is about 1 to 15%, preferably about 4 to 10%, more Preferably a substitution rate of 6.4% is appropriate.

In the step (c) for preparing a hydrogel, the concentration of HRP is included in an amount of 0.1 to 5 parts by weight based on 100 parts by weight of the total composition, and the concentration of hydrogen peroxide (H ₂ 0 ₂ ) is included in 100 parts by weight of the total composition. It may be included in an amount of 1 to 30 parts by weight.

In addition, the step (c) may be divided into two stages of tyramine-substituted pullulan, HRP, thrombin (92.6 units / mg) and calcium chloride at room temperature for 20 minutes to 1 hour, preferably 30 minutes to pH 6 to 8, preferably dissolved in PBS at pH 7 to prepare a primary solution; And a second solution prepared by dissolving tyramine-substituted pullulan and 30% hydrogen peroxide (H ₂ O ₂ ) in PBS at a pH of 6 to 8, preferably at pH 7, to the primary solution for 5 seconds to Reacting for 2 minutes characterized in that it comprises the step of preparing a hydrogel.

HRP oxidized by hydrogen peroxide water forms an intermediate by oxidizing the phenol group in the tyramine group introduced into pullulan, which results in the formation of a crosslinking point to form a network structure, thereby forming an injection hydrogel.

The thrombin may comprise 100 to 500 μg per mL of PBS, and the calcium chloride may comprise 3 to 15 mM per mL of PBS. Below this range, hemostasis is less effective, and higher values can cause economic losses.

In addition to the pullulan in the step (c) is characterized in that it further comprises at least one component selected from gelatin, chitosan, hyaluronic acid, alginate and cellulose derivatives.

In addition to the thrombin and calcium chloride in step (c) is characterized in that it further comprises one or more selected from fibrinogen, vitamin C, vitamin K, collagen, gelatin and microgel foam (gelfoam).

Hydrogel using pullulan prepared according to the production method of the present invention may have a viscosity of 10,000 to 25,000 cP at room temperature. In addition, the adhesive strength is characterized in that 10 to 30 kPa. When the above range is satisfied, the hydrogel hemostatic agent may be prevented from being separated or lost from the hemostatic part. If it has a range higher than the viscosity range, there is a problem for ease of operation during surgery, on the contrary, if it has a range lower than the above range, a problem occurs in tissue adhesion and storage.

In addition, since the hydrogel hemostatic agent of the present invention can be used in an injectable, spray, gel, liquid, and aerosol form, it is preferable to have the above viscosity and adhesive strength range.

Hereinafter, preferred examples are provided to help understanding of the present invention, but the following examples are merely to illustrate the present invention, and the scope of the present invention is not limited to the following examples.

<Example>

1. Introduction of carboxyl group to pullulan

For the modification of pullulan, 2,2,6,6-tetramethyl-1-piperidinyloxy, free radicals (TEMPO) and sigma-aldrich were purchased from Aldrich first to introduce carboxyl groups into pullulan. Sodium bromide (NaBr) and sodium hypochloride solution purchased from (Sigma-Aldrich) were used. The pullulan is added to the tertiary distilled water together with TEMPO and NaBr and stirred sufficiently at 1 ° C. After adding 15% NaOCl solution, the pH of the solution was slowly added while adding about 0.5 M NaOH solution while maintaining the pH of 4 M HCl at 9.5. Thereafter, methanol is added to terminate the reaction, and then neutralized with 4 M HCl. Thereafter, the solution was precipitated with acetone, and then the precipitation process was repeated two or three times to remove unreacted material, and the solution was dried under reduced pressure to prepare pullulan having a carboxyl group introduced therein (FIG. 1).

2. Introduction of tyramine to pullulan

In order to introduce a tyramine group into the pullulan introduced carboxyl group prepared above, tyramine purchased from Fluka, N-hydroxysuccinimide (NHS) purchased from Wako and 1 purchased from TCI The reaction was carried out using -ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC).

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

3. Preparation of Hydrogel Hemostatic Agent

Horseradish peroxidase (232 unit / mg, HRP) purchased from Amresco, a tyramine-substituted pullulan prepared previously, hydrogen peroxide (H ₂ 0 ₂ ), thrombin (from Daejung) 92.6 units / mg) and calcium chloride were added to dissolve at pH 7 for 30 minutes at room temperature, and then reacted within 60 seconds by adding a solution of 30% hydrogen peroxide and tyramine-substituted pullulan. (FIG. 3).

Experimental Example

Experimental Example 1 Attenuated total reflection Confirmation of pullulan modification via FT-IR

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

Experimental Example 2 Confirmation of Pullulan Modification by NMR

1) ¹³ C-NMR Spectrum Analysis

FIG. 5 shows ¹³ C-NMR spectra of pullulan and modified pullulan. FIG. 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 peak of carboxylic acid and carboxylic salt form is shown at 175 ppm through which the primary alcohol is oxidized by the TEMPO reaction and is present in the carboxyl group.

2) ¹ H-NMR spectral analysis

FIG. 6 shows a ¹ H-NMR spectrum of modified pullulan. FIG. Referring to FIG. 6, ¹ H-NMR was used to confirm the introduction of tyramine after the EDC-NHS reaction (BRUKER BIOSPIN / AVANCE 400, 400 MHz FT-NMR, Bruker, Germany). The benzene characteristic peak of tyramine is shown in the vicinity of 7 ppm through which the tyramine is introduced by the EDC-NHS reaction. In addition, as compared to The integration of the hydrogen found in C _2, C ₃ confirmed the substitution rate of tyramine.

Experimental Example 3 Molecular weight analysis through gel permeation chromatography

FIG. 7 is a GPC data of pullulan and modified pullulan, and a water-soluble gel permeation chromatograph system (GPC) (Breeze System, USA) was used to examine the molecular weight change of pullulan during the modification. Referring to Figure 7, it can be seen that the initial molecular weight of 180,000 Mw pullulan is reduced to 97,667 Mw after the TEMPO reaction, 22,355 Mw after the EDC-NHS reaction.

Experimental Example 4 Hydrogelization Time Analysis

8 is a view showing the gelation time according to the pullulan and HRP concentration. When the concentration of pullulan was 0.15 g / ml, it was confirmed that gelation was performed within 5 seconds when the concentration of HRP was 3 mg / ml or more. In addition, when the concentration of HRP is 3 mg / ml, it was confirmed that the gelation is made within 5 seconds when the concentration of pullulan is 0.15 or more.

Experimental Example 5 Rheological Characterization

9 is a view showing the rheological properties of the hydrogel according to the pullulan concentration through the rheometer. Thermo Scientific HAAKE MARS II Rheometer (Thermo Scientific HAAKE MARS II Rheometer, Thermo, Germany) was used to determine the rheological properties of the hydrogels prepared by the process of the present invention. Rheometers are used to measure rheological properties (material viscosity, modulus, stress, shear rate, etc.) of solutions, emulsions, suspensions, and thermosets, as well as polymeric materials. By imposing flows and deformations, physical structures and properties of materials can be determined.

When the concentration of HRP is equal to 3.0 mg / ml, by controlling the concentration of pullulan as the storage elastic modulus increases as the concentration of pullulan (0.3 g / ml, 0.4 g / ml, 0.5 g / ml) increases, It was confirmed that a hydrogel having a desired storage modulus can be made.

Experimental Example 6 Swelling ratio analysis according to pullulan concentration

10 is a view showing the degree of crosslinking density through the swelling ratio according to the pullulan concentration. Hydrogel is injected into an aluminum pan 1 cm in diameter and 0.5 cm in height to make a hydrogel of a certain size, and then it is placed in 50 ml PBS solution (pH 7.2, 37 ℃) for 1, 3, 5, 10 and 15 minutes. The weight was measured. Swelling ratio was calculated by the following equation.

Ws: Weight of swelled hydrogel.

Wd: Weight of lyophilized hydrogel.

When pullulan concentration was 0.3 g / ml and HRP concentration was 3 mg / ml, the swelling ratio with time was the highest.

Experimental Example 7 Of pullulan hydrogel in vitro  Biodegradability Assessment

In vitro biodegradation behavior for determining the biodegradation behavior of the prepared hydrogel hemostatic agent was shown as shown in FIG. The sample was placed in a 50 mL cornical tube, followed by adding 25 mL phosphate buffered saline (PBS, pH 7.4) solution to a shaking bath (90 rpm, 37 ° C) and hydrogel over time (0, 5, 12, 24 h). The biodegradation behavior of was observed. Specimens collected over time were placed in a new 10 mL cornical tube and lyophilized and weighed. As the concentration of pullulan (0.3, 0.4, 0.5 g / mL) increased, biodegradation behavior was observed in vitro after 5 hours, and the weight loss rate was 30.4%, 24.4%, and 17.1%, respectively. After that, 72.7%, 70.5%, and 62.9% were confirmed, respectively. After 24 hours it was observed that all samples had degraded.

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

In the preparation of the hydrogel according to the present invention, a blood coagulation test was performed for each concentration including thrombin and calcium chloride. After 300 μL of the hemostatic agent of each concentration was adjusted to 37% on a thermostatic bath, 300 μL of platelet-poor-plasma (PPP) obtained by centrifuging blood at 3000 rpm or higher was added to the watch plate solution. Since the time when the thrombus is generated was measured.

TABLE 1 TABLE 2

As shown in Table 1, the result of 6s at 67 μg / 300μL for thrombin is shown to be an efficient concentration, and as shown in Table ₂ , when the concentration is too high or too low, the hemostatic effect is decreased. It was confirmed that.

Therefore, when preparing the hydrogel in the step (c) according to the present invention, thrombin may include 100 to 500 μg per 1 mL of PBS, the calcium chloride may comprise 3 to 15 mM per 1 mL of PBS. If you fall outside of this range, the hemostatic effect drops and hemostasis can take too long.

Experimental Example 9 Of the prepared hydrogel hemostatic agent Erythrocyte hemolysis experiment

A erythrocyte hemolysis experiment was performed to measure the amount of residual erythrocytes after blood coagulation, and the blood coagulation ability was confirmed by absorbance of hemoglobin from hemolyzed erythrocytes. The control group was a blood coagulation reaction without introducing a hydrogel sample. After a certain time, 5 mL of distilled water was added to the sample to stop the coagulation reaction, and the supernatant obtained was dispensed into a 96 well plate to measure the absorbance of hemoglobin. When the blood coagulation progresses a lot, since the amount of residual red blood cells is small, the amount of hemoglobin released through hemolysis is small, so that hemoglobin can be observed through the absorbance intensity of hemoglobin.

As shown in FIG. 12, when the hydrogel carrying the hemostatic agent was used as compared with the control group, the absorbance was lower, and the lower the concentration of the pullulan, the lower the hydrogel.

Experimental Example 10 Of the prepared hydrogel hemostatic agent i n vivo  Hemostatic experiment

Figure 13 is a photograph showing a hemostatic animal experiment using the SD rat. Anesthesia for Zoletil ^® 50 and Rompun ^® was administered to the SD rat by anesthesia in a 4: 1 ratio. When the SD rat was anesthetized, hair was removed from the abdomen and tilted at an angle of 45 ° to the operating table. After disinfecting the abdominal hair removal site with povidone iodine solution, the incision was about 20 to 30 mm using a blade No. 10 in the center, and the skin and peritoneum were peeled off to raise both sides to expose the liver. After placing the pre-weighed filter paper under the liver, using a blade No. 10 exposed 3 mm above the stopper, a 3 mm deep wound is placed on the left lobe of the liver. When bleeding begins, 0.5 ml of hydrogel is introduced to hemostasis. The time was measured. When hemostasis was obtained, the blood absorbed filter paper and hydrogel were weighed to calculate the amount of bleeding.

Figure 14 is a photograph showing the results of pullulan concentration of hemostatic animal experiments using the SD rat. As shown in Table 3, the hydrogel hemostatic agent prepared differently with pullulan concentration of 0.3 g / ml, 0.4 g / ml, and 0.5 g / ml was used.

TABLE 3

15 is a graph showing the amount of bleeding in the pullulan concentration by the result of using the SD rat. In the case of bleeding, the control group showed 1,149.9 mg, pullulan 0.3, 0.4, and 0.5 g / ml of bleeding at 651.2, 334.3, and 273.3 mg, respectively, and 0.5 g / ml showed the greatest hemostatic effect.

16 is a graph showing the bleeding time in the pullulan concentration by the result of using the SD rat. The hemostasis time of the control group was about 130 seconds, pullulan 0.3, 0.4, 0.5 g / ml was confirmed that the hemostasis is done within 61.5, 50.1, 10.5 seconds, respectively, 0.5 g / ml was confirmed that the hemostatic effect is the greatest there was. The lower the concentration of pullulan, the lower the hemostasis of 45 ° and the initial hemostasis did not appear.

17 is a view showing the results of histological observation of hemostatic sites of liver treated with pullulan hydrogel through H & E staining. The nucleus of the cell was blue, and the cytoplasm was stained red, and red blood cells were well entangled in the wound, as the wound was red. 18 is a diagram showing the results of histological observation of hemostatic sites of liver treated with pullulan hydrogel through carstairs staining. This staining method stains fibrin red and platelets blue. It was confirmed that fibrin was well formed because the wound was red.

Although the present invention has been described by specific embodiments such as specific components and the like, but the embodiments and the drawings are provided to assist in a more general understanding of the present invention, the present invention is not limited to the above embodiments. For those skilled in the art, various modifications and variations can be made from these descriptions.

Accordingly, the spirit of the present invention should not be limited to the above-described embodiments, and all of the equivalents or equivalents of the claims, as well as the appended claims, fall within the scope of the spirit of the present invention. I will say.

Claims (12)

(a) introducing a carboxyl group to pullulan using a 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TEMPO), sodium bromide (NaBr) and sodium hypochloride solution;
(b) Tyramine, N-hydroxysuccinimide (NHS), and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide in pullulan to which the carboxyl group obtained in step (a) is introduced; Introducing tyramine using EDC); And
(c) preparing a hydrogel by adding horseradish peroxidase (HRP), hydrogen peroxide (H ₂ 0 ₂ ), thrombin and calcium chloride (CaCl2) to the tyramine-substituted pullulan obtained in step (b). In a method for producing a hydrogel hemostatic agent using pullulan, comprising a,
In step (c), the concentration of HRP is 3 to 5 parts by weight and the concentration of hydrogen peroxide (H ₂ 0 ₂ ) based on 100 parts by weight of the total composition, 1 to 30 parts by weight,
Step (c) comprises dissolving tyramine-substituted pullulan, HRP, thrombin and calcium chloride in PBS at pH 6-8 for 20-40 minutes at room temperature to prepare a primary solution; And adding a secondary solution of 30% hydrogen peroxide (H ₂ O ₂ ) to the primary solution to react for 5 seconds to 30 seconds to prepare a hydrogel.
The thrombin is contained in 100 to 500 μg per mL of PBS, the calcium chloride is hydrogel hemostatic preparation using pullulan containing 3 to 15 mM per mL of PBS.
The method of claim 1,
In the step (a), the pullulan has a weight average molecular weight of 50,000 to 250,000 g / mol, hydrogel using pullulan, characterized in that it comprises 1 to 50 parts by weight based on 100 parts by weight of the total composition of the hydrogel hemostatic agent Method of manufacturing hemostatic agent.
The method of claim 1,
In step (b), the substitution rate of tyramine is a method for producing a hydrogel hemostatic agent using pullulan, characterized in that 1 to 15%.
delete delete delete delete The method of claim 1,
The method of preparing a hydrogel hemostatic agent using pullulan, characterized in that it further comprises at least one component selected from gelatin, chitosan, hyaluronic acid, alginate and cellulose derivative in the step (c).
The method of claim 1,
The method of manufacturing a hydrogel hemostatic agent using pullulan, characterized in that it further comprises one or more selected from fibrinogen, vitamin C, vitamin K, collagen, gelatin and microgel foam (gelfoam) as the hemostatic material in step (c).
A hydrogel hemostatic agent using pullulan prepared according to any one of claims 1 to 3 and 8 to 9.
The method of claim 10,
The hydrogel hemostatic agent is a hydrogel hemostatic agent using pullulan, characterized in that the viscosity of 10,000 to 25,000 cP at room temperature.
The method of claim 10,
The hydrogel hemostatic agent is a hydrogel hemostatic agent using pullulan, characterized in that the adhesive strength of 10 to 30 kPa.

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