KR20210025918A - Improved hydrogel hemostatic agent manufacturing method, and hemostatic agent thereof - Google Patents

Abstract

The present invention relates to a hydrogel hemostatic agent using pullulan and alginic acid, and a method for manufacturing the same. More specifically, the present invention relates to: an improved hydrogel hemostatic agent which contains calcium chloride and thrombin in hydrogel, in which pullulan and alginic acid are contained, thereby having excellent blood clotting ability and being effective for wound healing at the same time; and a manufacturing method thereof.

Description

Improved hydrogel hemostatic agent manufacturing method and hemostatic agent therefor TECHNICAL FIELD [Improved hydrogel hemostatic agent manufacturing method, and hemostatic agent thereof]

The present invention relates to a method for preparing an improved hydrogel hemostatic agent and a hemostatic agent according thereto, and more particularly, to a hydrogel containing pullulan and alginic acid, including calcium chloride and thrombin, having excellent blood coagulation ability and at the same time treating wounds. It relates to a method for producing a hydrogel hemostatic agent effective in.

Bleeding means that blood comes out of the blood vessels, and the blood vessels that make up the whole body circulate the blood in the blood vessels throughout the body by the pressure of the heart. It is produced, meaning that blood leaks out of the wound. Bleeding, where blood is drained out of blood vessels, can occur in everyday injuries and medical practices such as surgery. Excessive bleeding due to a large wound or penetration of bacteria or viruses into the exposed blood vessels due to the wound can lead to death. Therefore, in order to prevent blood loss and protect exposed blood vessels effectively by clotting the blood quickly, Hemostasis is needed.

Hemostasis means to stop bleeding in the wound area by causing a blood clot through a complex path when blood comes into contact with an external foreign substance due to a wound in a blood vessel. In the case of excessive bleeding that occurs in an emergency situation, it is common to additionally require the use of an effective hemostasis method.

There are largely a mechanical method, a cold-heat method, and a chemical method as a hemostasis method. Among these, a typical chemical method is to stop the bleeding by treating the bleeding area with a blood coagulant.

Currently, studies on hemostatic agents, which are medical preparations using chemical methods, have been actively conducted from a long time ago. Among them, since the hydrogel made of poly(2-hydroxyethyl methacrylate) (PHEMA) was developed by Wichterle in the 1960s, its hydrophilicity and biocompatibility have been applied in the field of biomaterials. Hydrogel refers to a structure that has a three-dimensional hydrophilic polymer network that can contain a large amount of water or biological fluids due to its characteristics. The network network structure of the hydrogel is formed by various factors, such as chemical bonding or physical aggregation, and is thermodynamically stable after swelling in an aqueous solution. Hydrogels have flexible strength and high moisture content, so they are similar to living tissues and are widely used in medicine or pharmaceutical fields.

In particular, in the injection-type system using hydrogel, it is possible to design a highly efficient delivery system to a desired site with only simple mixing. In addition, its fluidity is a method that is expected to be applied in various fields as it has properties suitable for use in filling defective areas.

In order to use the hydrogel as an injection material, it has fluidity like a fluid outside the body, and it needs to be gelled by physical or chemical stimulation after injection. When a hydrogel is formed by entanglement of molecules or secondary bonds including ionic bonds, hydrogen bonds, and hydrophobic interactions, it is called a reversible or physical hydrogel. Physical hydrogels are generally not homogeneous, and are temporarily bonded to the middle or end of the chain. The interactions that maintain these physical hydrogels are reversible and can collapse when 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 greatly influenced by the concentration of various types of gel precursors and crosslinking agents. This may cause biomedical restrictions due to the crosslinking agent, and may cause changes in strength and unwanted mechanical properties while exhibiting effects such as modification by crosslinking agents or precursors. Thus, in order to overcome these weaknesses, studies in various fields such as the use of substances extracted from natural substances in the selection of chemical crosslinking agents have been conducted in recent years.

For example, hemostatic compositions in dry storage-stable form comprising biocompatible, biodegradable, dry stable granular material are known from WO 98-008550 or WO 2003-007845. These products have been successfully applied in the art for hemostasis, and commercially the trademark Floseal® is a powerful and versatile hemostatic agent consisting of a granular gelatin matrix swollen in a thrombin-containing solution to form a flowable paste. Yes.

As another example, Korean Patent Registration No. 10-1814841 discloses a) providing a first component including a dry formulation of a coagulation inducing agent; b) providing a second component comprising a dry preparation of a biocompatible polymer suitable for use in hemostasis; c) c1) filling the final container with the first component and the second component to obtain a dry mixture in the final container, or c2) the first component and the second component in the final container. To obtain a blend, the first component or the second component is provided to the final container and the second component or the first component is added to obtain the first component and the second component. Providing the final container in a combined form, d) finishing the final container with a storable pharmaceutical device containing the first component and the second component in a combined form as a dry and stable hemostatic composition. Disclosed is a method for preparing a dry and stable hemostatic composition containing.

Korean Patent Registration No. 10-1103423 discloses a bioinjectable tissue adhesive hydrogel and its biomedical use. More specifically, it relates to a bioinjectable tissue adhesive hydrogel comprising waveguide or a derivative thereof, wherein the bioinjectable tissue adhesive hydrogel is in situ by an enzymatic reaction occurring in the body unlike the conventional tissue adhesive hydrogel. By inducing crosslinking to form a hydrogel, it provides an in situ-forming tissue-adhesive hydrogel with superior biocompatibility. At the same time, it has excellent biocompatibility and mechanical strength due to hybridization of synthetic and natural polymers. It is mentioned that it provides excellent tissue adhesion.

As described above, the necessity of developing a hemostatic agent excellent in hemostatic effect, biocompatibility, and economic efficiency is continuously required. Accordingly, the present invention uses horseradish peroxidase (HRP), an enzyme extracted from horseradish, as a hydrogel. It was completed in order to provide an improved hemostatic agent with added wound healing effect, further including alginic acid, which is a natural polymer, and is easy to handle by applying to hemostatic agents.

An object of the present invention is to solve the problems and technical problems of the prior art as described above.

An object of the present invention is to provide biodegradability in vivo by being applied to a bleeding site, and to provide excellent hemostatic efficacy.

Furthermore, it is an object of the present invention to provide effective wound healing ability and adhesiveness on the wound surface.

Therefore, it is an object to improve the usability of the technology by providing wound healing ability in use even in surgical procedures such as endoscopy and laparoscopic procedures.

In order to achieve the object of the present invention as described above and to realize the characteristic effects of the present invention to be described later, the characteristic configuration of the present invention is as follows.

A method of preparing an improved hydrogel hemostatic agent according to an embodiment of the present invention is as follows.

(a) introducing a carboxyl group to pullulan; (b) preparing an alginic acid aqueous solution by adding alginic acid to distilled water; (c) mixing and stirring the pullulan and the alginic acid aqueous solution, and then introducing tyramine into the side chains of pullulan and alginic acid; (d) dialysis of pullulan and alginic acid into which the tyramine was introduced and freeze-drying to prepare an improved pullulan; And (e) reacting the improved pullulan with horseradish peroxidase (HRP), hydrogen peroxide solution (H ₂ 0 ₂ ), thrombin and calcium chloride (CaCl ₂ ) to prepare a hydrogel; A method of preparing a hydrogel hemostatic agent is provided.

An improved hydrogel hemostatic agent manufactured according to the manufacturing method according to an embodiment of the present invention is provided.

There is an effect of providing an improved hydrogel hemostatic agent including pullulan and alginic acid according to the present invention.

The improved hydrogel hemostatic agent according to the present invention has an effect of optimizing the blood coagulation ability of the hemostatic agent by introducing a blood coagulation factor that promotes hemostasis, and setting an optimal concentration that exhibits the effect. In addition, it has excellent bio-stability and bonding properties, including natural polymers.

The improved hydrogel hemostatic agent according to the present invention, including alginic acid, has an effect of providing effective wound healing ability and adhesion on a wound surface with an excessive amount of exudate.

The improved hydrogel hemostatic agent according to the present invention is manufactured in the form of an injection-type hydrogel so that it is easy to handle and has an effect of improving fluidity.

1 is a diagram showing a step of a TEMPO reaction for introducing a carboxyl group into pullulan according to the present invention.
2 is a diagram showing an EDC reaction process for introducing tyramine into pullulan and alginic acid into which a carboxyl group has been introduced.
3 is a diagram showing a manufacturing process of a hydrogel.
4 is a diagram showing the gelation time according to the HRP concentration.
5 is a diagram showing the rheological properties of a hydrogel according to HRP concentration through a rheometer.
6 is a diagram showing the degree of crosslinking density through the swelling ratio according to the HRP concentration.
7 is a view showing the in vitro biodegradation evaluation of the hydrogel according to the improved concentration of pullulan.
8 is a diagram showing an experiment for hemolysis of red blood cells using an improved pullulan hydrogel.
9 is a diagram showing a hemostatic animal experiment using SD rats.
10 is a photograph showing the results of the improved pullulan concentration of the hemostatic animal experiment using SD rats.
11 is a graph showing the amount of bleeding in the result of improved pullulan concentration using SD rats.
12 is a result of histological observation of the hemostatic site of the liver treated with the improved pullulan hydrogel through H&E staining.
13 is a result of histological observation of the hemostatic site of the liver treated with the improved pullulan hydrogel through carstairs staining.
14 is a graph showing the rate of reduction in the size of a wound on a skin wound area treated with an improved pullulan hydrogel.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The detailed description of the present invention described below refers to the accompanying drawings, which illustrate specific embodiments in which the present invention may be practiced. These embodiments are described in detail sufficient to enable a person skilled in the art to practice the present invention. It should be understood that the various embodiments of the present invention are different from each other, but need not be mutually exclusive. For example, specific shapes, structures, and characteristics described herein may be implemented in other embodiments without departing from the spirit and scope of the present invention in relation to one embodiment. In addition, it should 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 present invention. Accordingly, the detailed description to be described below is not intended to be taken in a limiting sense, and the scope of the present invention, if appropriately described, is limited only by the appended claims, along with all ranges equivalent to those claimed by the claims. Like reference numerals in the drawings refer to the same or similar functions over several aspects.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings in order to enable those of ordinary skill in the art to easily implement the present invention.

The hydrogel hemostatic agent using pullulan according to the present invention has pullulan having biodegradability, non-toxicity and biocompatibility as its main component, and _{reacts with tyramine and hydrogen peroxide (H 2} O ₂ ) to act as a crosslinking agent. It is manufactured using horseradish peroxidase (HRP), which is known as a crosslinking agent with excellent biodegradability and biocompatibility. Furthermore, including alginic acid, it is possible to provide excellent biocompatibility and aid in the growth of granulation tissues, thereby providing a wound treatment effect at the same time.

Pullulan (pullulan) is a substance produced extracellularly by Aureobasidum pullulans, mainly maltotriose α-1, 3 binding to 3 glucose, α-1, 6 binding It is formed of linear glucans that are repeatedly bonded to each other. By detailed structural studies, it has been found that pullulan is not formed only with a repeating structure of only maltotriose units, but contains several% maltoteraose, and that the end of the molecule is glucose and has a structure different from that of dextran. Pullulan with this structure is a tasteless, odorless, amorphous white powder that has no problem with the results of various toxicity and mutagenicity tests, so it has been approved as a food additive at home and abroad. In addition, it is a polysaccharide substance that is easy to dissolve in water and has low viscosity compared to other polysaccharides, but has strong adhesion and stretchy properties, and is a stable neutral solution that can be easily applied in various ways. In addition, the viscosity of the aqueous solution has a relatively low temperature dependence and is less affected by various salts and pH. As an example, in the case of titanium and boron ions, which are considered to form a chelate with -OH groups in the pullulan molecule, the viscosity of the pullulan aqueous solution in which various metal ions coexist (1% aqueous solution, 25°C, 30 rpm) is 250 to 300 cp. Except in any case, neither increase in viscosity nor gel formation. In addition, it is a high-molecular material with excellent adhesion properties and biodegradable properties that are completely magnetized by many microorganisms due to its structure and finally converted into carbonized gas and water.

In the case of alginic acid, alginic acid, which is contained in seaweed such as kelp, is a polysaccharide that exhibits high-quality dietary fiber functions. This is also known as seaweed mountain (海草酸). Alginic acid is a polymer of two types of uronic acid and has a polymerization degree of 80 and a molecular weight of about 1,500. Alginic acid is precipitated when brown algae washed with dilute sulfuric acid is extracted from dilute alkaline hot water and the extract is made acidic. Alginic acid has the carboxy group (COOH-) of uronic acid in its molecule, so it exhibits the properties of an acid, and is usually treated as a sodium salt. Alginic acid was thought to consist of only manluronic acid until 1957, but recently it was discovered that L-gluronic acid is also a constituent of alginic acid. The structure is that hundreds of manluronic acid and L-gluronic acid are linked by β-1,4 bonds. In addition, there are places where only manluronic acid or only L-gluronic acid is long bonded in the molecule. The M/G ratio (the ratio of the abundance of manluronic acid and L-gluronic acid) varies depending on the type of seaweed. A low M/G ratio forms a hard and brittle gel, and a high M/G ratio forms a more elastic gel. Alginic acid is mostly present in combination with potassium, sodium and calcium. Recently, it has been found that alginic acid combines with various metals (Co,Cu,Cd,Zn, etc.) to generate biopolymers (gel) naturally, and that the synthesized material has ion-exchange ability.This biopolymer is a heavy metal in the environment. In addition to the ability to adsorb etc., it can be synthesized into biodegradable polymers with various properties by controlling the crosslinking method or additives.

In addition, an important characteristic of alginic acid is that it is biocompatibility and is helpful in the growth of granulation tissue, and is particularly included in medical hydrogels as biodegradable polymers, etc., and provides excellent biocompatibility. This is because it is a three-dimensional hydrophilic polymer derived from natural products that contains a large amount of water in its structure, and alginic acid has a hydrated structure similar to the extracellular matrix, which is a component of living tissue. to be. Therefore, in the present invention, in addition to the hydrogel including pullulan, the wound healing effect of the hydrogel is maximized by including alginic acid. In general, since the physical properties of alginic acid change according to the weight% and molecular weight of the total composition, it is important to consider the appropriate weight% and molecular weight of alginic acid when preparing the hydrogel.

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 largely in two stages. HRP is oxidized by hydrogen peroxide in the first stage, and HRP oxidized in the second stage oxidizes the phenol group in tyramine to form an intermediate. The resulting material creates a crosslinking point to form a network structure. An important factor at this time is that the mechanical strength and hydrogel formation time can be easily controlled by the concentration of _{H 2} O _{2 and HRP.}

Accordingly, the improved hydrogel according to the present invention is provided with a method of manufacturing using pullulan and alginic acid as raw materials and horseradish peroxidase (HRP) as a crosslinking agent.

i) introducing a carboxyl group into pullulan using a TEMPO reaction, ii) preparing an alginic acid aqueous solution by adding alginic acid to distilled water, iii) an EDC reaction for introducing tyramine into pullulan into which the carboxyl group has been introduced and alginic acid Step, iv) a step of preparing a hydrogel by reacting pullulan and alginic acid into which tyramine has been introduced with horseradish peroxidase and an aqueous hydrogen peroxide solution.

In more detail, the method for preparing an improved hydrogel hemostatic agent according to the present invention includes the steps of: (a) introducing a carboxyl group into pullulan; (b) preparing an alginic acid aqueous solution by adding alginic acid to distilled water; (c) mixing and stirring the pullulan and the alginic acid aqueous solution, and then introducing tyramine into the side chains of pullulan and alginic acid; (d) dialysis and lyophilization of pullulan and alginic acid into which the thyramine has been introduced to prepare an improved pullulan; And (e) reacting the improved pullulan with horseradish peroxidase (HRP), hydrogen peroxide solution (H ₂ 0 ₂ ), thrombin and calcium chloride (CaCl ₂ ) to prepare a hydrogel.

Introducing a carboxyl group to pullulan in the step (a) according to the present invention is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), sodium bromide (NaBr) and sodium Hypochloride (NaOCl) may be mixed in distilled water as a solvent and introduced through a substitution reaction. This is to introduce a tyramine to pullulan, to introduce a carboxylic acid first, and then to sequentially introduce a tyramine group to the carboxyl group.

In the step (a) according to the present invention, pullulan is provided, including 1 to 50 parts by weight based on 100 parts by weight of the total composition of the hydrogel hemostatic agent. Preferably, it may be included in an amount of 5 to 50 parts by weight. If the range is higher than the above range, the viscosity of the hydrogel is too high in the body, so gelation takes a long time to delay the hemostasis time, and if the range is lower than the above range, the tissue adhesion of the hydrogel may be degraded. have. Therefore, it is possible to solve this problem by providing 1 to 50 parts by weight.

In the step (b) according to the present invention, alginic acid is provided, including 1 to 80 parts by weight based on 100 parts by weight of the total composition of the hydrogel hemostatic agent. When prepared as a hydrogel within the above range, it is possible to provide a wound healing effect of alginate hydrogel for wound treatment while optimizing mechanical properties. Furthermore, if it is dry, it can be easily removed from the wound while making up for the disadvantage of sticking too hard to the wound.

In addition, the alginic acid includes a D-manuronic acid group and an L-gluronic acid group, and the weight ratio of the D-manluronic acid group and the L-gluronic acid group is 40 to 70:60 to 30. As described above, the M/G ratio (ratio of the abundance of manluronic acid and L-gluronic acid) in alginic acid differs depending on the type of seaweed. A low M/G ratio forms a hard and brittle gel, and a high M/G ratio forms a more elastic gel. Therefore, by providing the 40 to 70: 60 to 30, it is possible to provide a balanced rigidity and elasticity.

The introduction of tyramine in the step (c) according to the present invention includes tyramine, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), and N-hydroxysuccinimide (NHS). EDS-NHS reaction can be carried out using, and a side chain can be substituted. The introduction of tyramine means a crosslinking point that connects polymers. Therefore, it means that the gel formation time and mechanical strength of the hydrogel can be adjusted according to the degree of substitution of the introduced tyramine group. Furthermore, it goes without saying that it can affect the functional exertion of the hydrogel through mechanical properties.

After the introduction of tyramine in the step (c) according to the present invention, the tyramine substitution rate in the side chains of pullulan and alginic acid is provided at 1 to 15%. Horseradish peroxidase (HRP) requires a tyramine group in order to act as a crosslinking agent, but pullulan and alginic acid do not have a tyramine group. Therefore, in order to use pullulan and alginic acid, it is necessary to introduce a thyramine group into pullulan. In consideration of the mechanical strength of the hydrogel and the gel formation time, the substitution rate of tyramine may be provided with about 1 to 15%, and preferably about 4 to 10%.

A method for preparing an improved hydrogel hemostatic agent having a weight average molecular weight of 10,000 to 750,000 is provided for pullulan in which the side chain is substituted with tyramine according to the present invention. More specifically, 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. In the case of alginic acid contained in the improved pullulan, the weight average molecular weight may be 10,000 to 500,000 g/mol, and preferably 100,000 to 300,000 g/mol. If the range is higher than the above range, since the viscosity of the hydrogel increases, the ease of operation thereof may decrease, and a result that is not uniform in the manufacturing process may be produced. Conversely, if it has a low range, there may be a problem in that sufficient mechanical strength of the hydrogel is not secured.

In order to prepare the hydrogel according to the present invention, the concentration of the peroxidase (HRP) is 0.1 to 5 parts by weight, and the hydrogen peroxide solution (H ₂ 0 ₂ ) is 1 to 100 parts by weight of the total composition in step (c). It is provided including 30 parts by weight. Hydrogel can control the physicochemical properties selected from gelation time, decomposition time, mechanical strength, or moisture content by controlling the concentration of peroxidase (HRP) and hydrogen peroxide. HRP, oxidized by hydrogen peroxide, oxidizes the phenol group in the thyramine group introduced into the improved pullulan to form an intermediate, thereby forming a crosslinking point to form a network structure, thereby forming an injection-type hydrogel. That is, it is possible to carry out a crosslinking reaction using a bio-friendly horseradish peroxidase, which is advantageous in natural products, as a crosslinking agent. Within the above range, it is possible to easily control the gelation time and mechanical strength of the hydrogel, and to provide an in situ hydrogel having mechanical strength and excellent stability in the body. Therefore, it can be provided in the form of a liquid formulation that is easy for the user to manipulate outside the body.

By dialysis and lyophilization of pullulan and alginic acid into which the tyramine is introduced in step (d) according to the present invention, an improved pullulan containing alginic acid can be prepared.

In the mixing of the step (e) according to the present invention, the improved pullulan is _{reacted with horseradish peroxidase (HRP), thrombin and calcium chloride (CaCl 2} ), at room temperature of pH 6 to 8, for 20 minutes to 1 A first solution was prepared by dissolving in a phosphate buffer solution (PBS) for a period of time, and a second solution of 30% hydrogen peroxide solution (H ₂ O ₂ ) was added to the first solution, followed by reacting for 5 seconds to 2 minutes. It can be provided to make a gel.

That is, it can be carried out in two steps, improved pullulan, horseradish peroxidase (HRP), thrombin (92.6 units/mg) and calcium chloride (CaCl ₂ ) at room temperature for 20 minutes to 1 hour, preferably 30 Prepare a first solution by dissolving in a phosphate buffer solution (PBS) of pH 6 to 8, preferably pH 7, for a minute, pullulan substituted with tyramine in the first solution and 30% hydrogen peroxide solution (H ₂ 0 ₂ ) At room temperature in a phosphate buffer solution (PBS) having a pH of 6 to 8, preferably pH 7, and reacting for 5 seconds to 2 minutes to prepare a hydrogel.

Thrombin per 1 mL of the phosphate buffer solution (PBS) according to the present invention may contain 100 to 500 μg, and the calcium chloride per 1 mL of the phosphate buffer solution (PBS) may contain 3 to 15 mM. If it is lower than this numerical range, the hemostatic effect decreases, and if it is high, it may cause economic loss.

In the step (e) according to the present invention, at least one or more of gelatin, chitosan, hyaluronic acid, alginate and cellulose derivatives may be further included, and fibrinogen, vitamin C, vitamin K, collagen, gelatin and microgel foam as hemostatic substances It may further include at least one or more selected from (gelfoam), but is not limited thereto.

There is provided a hydrogel hemostatic agent using the improved pullulan prepared according to the manufacturing method according to the present invention.

In this case, the hydrogel hemostatic agent may have a viscosity of 10,000 to 25,000 cP at room temperature and an adhesive strength of 10 to 30 kPa. When the above range is satisfied, the hydrogel hemostatic agent provides an effective effect on wound treatment while preventing the phenomenon of being separated or lost from the hemostatic site. If it has a range higher than the viscosity range, there arises a problem with ease of operation during surgery, whereas if it has a lower range than the above range, tissue adhesion and storage problems occur, and the efficiency of wound treatment is also reduced.

In addition, the hydrogel hemostatic agent of the present invention may be used in an injection type, a spray type, a gel type, a liquid type, and an aerosol type, and is preferably provided in an injection type. Accordingly, it is possible to provide an effect of improving the usability of the technology by easily providing it to a wound surface or laparoscopic surgery.

In addition, by providing the viscosity and adhesive strength range, it is possible to utilize more effectively.

Hereinafter, preferred examples are presented to aid in the understanding of the present invention, but the following examples are only illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

<Example>

1. Step of introducing a carboxyl group to pullulan

For the modification of pullulan, 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TEMPO) and sigma-Aldrich purchased from Aldrich to introduce a carboxyl group into pullulan first. A solution of sodium bromide (NaBr) and sodium hypochloride purchased from (Sigma-Aldrich) was used. Put pullulan together with TEMPO and NaBr in tertiary distilled water and sufficiently agitate at 1°C. Thereafter, while adding a 15% NaOCl solution, 4M HCl was slowly added to the solution, while maintaining the pH at 9.5, while adding a 0.5 M NaOH solution, the mixture was stirred for about 120 minutes. After that, methanol was added to terminate the reaction, and then neutralized with 4M HCl. Then, after the solution was precipitated with acetone, the precipitation process was repeated 2 to 3 times to remove unreacted substances, and then the solution was dried under reduced pressure to prepare pullulan into which a carboxyl group was introduced. A process for this is illustrated in FIG. 1.

2. Step of preparing an aqueous alginic acid solution

First, 1 to 10% by weight of alginic acid was added to tertiary distilled water and sufficiently stirred for about 3 to 5 hours, and the resulting solution was prepared in an aqueous alginic acid solution.

3. Preparation of improved pullulan by introducing tyramine to alginic acid and pullulan

After mixing and stirring the previously prepared carboxyl group-introduced pullulan and alginic acid aqueous solution, tyramine purchased from Fluka and N-hydroxysuccin purchased from Wako are The reaction was carried out using mid (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) purchased from TCI.

First, the carboxyl group-introduced pullulan and alginic acid aqueous solution are sufficiently stirred for about 3 to 5 hours, and then tyramine, EDC, and NHS are added to the solution and stirred for about 12 hours while maintaining the pH at 4.7. After neutralizing the prepared solution with 4M HCl, put it in a semi-dialysis membrane and dialyzed for 4 days in 100mM NaCl solution, 25% ethanol, and distilled water for about 2 days, 1 day, and 1 day, respectively. The dialysis-completed solution was freeze-dried to prepare an improved pullulan sample containing alginic acid into which tyramine was introduced. The process for this is shown in FIG. 2.

4. Preparation of improved hydrogel hemostatic agent

Horseradish peroxidase (232unit/mg, HRP) purchased from Amresco (232unit/mg, HRP), hydrogen peroxide (H ₂ 0 ₂ ) purchased from Daejung, thrombin in the previously prepared tyramine-substituted pullulan and alginic acid (92.6 units/mg) and calcium chloride were added and dissolved at pH 7 for 30 minutes at room temperature, and then a solution of 30% hydrogen peroxide and improved pullulan substituted with tyramine was added to react within 60 seconds. A hydrogel hemostatic agent was prepared. The manufacturing process for this is illustrated in FIG. 3.

<Experimental Example>

Experimental Example 1 Analysis of hydrogelation time

4 is a diagram showing the gelation time according to the HRP concentration. According to the results, as the concentration of HRP increased, the gelation time decreased. In this case, the improved concentration of pullulan was provided as 0.3 g/ml. In this case, the concentration of HRP provided 200 to 900 units/ml, and in this case, it was confirmed that gelation was performed within 5 seconds when the concentration of HRP was 692 units/ml or more.

Experimental Example 2 Rheological Characteristics Analysis

Thermo Scientific HAAKE MARS II Rheometer (Thermo Scientific HAAKE MARS II Rheometer, Thermo, Germany) was used to determine the rheological properties of the hydrogel prepared according to the Examples. The rheometer used is a device that measures rheological properties (viscosity, elastic modulus, stress, shear rate, etc.) of solutions, emulsions, suspensions, and thermosetting materials as well as polymer materials. By imposing flow and deformation, it is possible to grasp the structure and properties of a material in a physical way. However, the results showing the rheological properties (storage modulus) of the hydrogel by providing the HRP concentration at 346, 519, 692 and 865 units/ml are shown in FIG. 5.

Experimental Example 3 Analysis of the swelling ratio according to the improved pullulan concentration

After making a hydrogel of a certain size by injecting the hydrogel prepared according to the example into an aluminum pan having a diameter of 1 cm and a height of 0.5 cm, put it in 50 ml PBS solution (pH 7.2, 37°C) to 1, 3, 5, After 10 or 15 minutes, the weight was measured. The swelling ratio was calculated by the following equation.

Ws: This is the weight of the swelled hydrogel.

Wd: This is the weight of the lyophilized hydrogel.

Figure 6 shows the degree of crosslinking density through the swelling ratio according to the HRP concentration. However, the HRP concentration was provided at 173, 346, and 519 units/ml.

Experimental Example 4 of improved pullulan hydrogel in vitro Biodegradability evaluation

In vitro biodegradation behavior was confirmed to determine the biodegradation behavior of the hydrogel hemostatic agent prepared according to the Examples. After putting the sample in a 50 mL cornical tube, add 25 mL phosphate buffered saline (PBS, pH 7.4) solution and put it in a shaking bath (90 rpm, 37℃) to hydrogel according to time (0, 5, 12, 24 h). The biodegradation behavior of was observed. Specimens recovered over time were placed in a new 10 mL cornical tube, lyophilized, and then weighed. However, the concentration of pullulan was provided at 0.3, 0.4, and 0.5 g/mL.

As the concentration of improved pullulan (0.3, 0.4, 0.5 g/mL) increased, when the biodegradation behavior in the in vitro environment was observed after 6 hours, the weight reduction rate was 20.1%, 18.2%, and 16.0%, respectively. After 24 hours, it was confirmed that they were 50.0%, 48.1%, and 46.5%, respectively. After 10 days, the weight reduction rate was 75.0%, 73.6%, and 71.8%, respectively, and after 20 days, it could be observed that all samples were degraded. The results for this are shown in FIG. 7.

Experimental Example 5 PRT experiment according to thrombin and calcium chloride concentration

In the preparation of the hydrogel prepared according to the Example, a blood coagulation capacity test was performed by concentration including thrombin and calcium chloride. After 300 μL was added to the watch dish on the constant temperature water bath in which each concentration of hemostatic agent was adjusted to 37%, 300 μL of platelet-poor-plasma (PPP) obtained by centrifuging the blood at 3000 rpm or higher was added to the solution on the watch dish. Thereafter, the time at which the blood clot was generated was measured. The results for this are shown in Tables 1 and 2.

Trombin(㎍/300μL) Time(s) 20 16 40 10 60 6 80 5 100 5

CaCl ₂ /(mol/L) Time(s) 0.003 227 0.006 206 0.009 108 0.012 134 0.015 166

As shown in Table 1, in the case of thrombin, the result of 6 seconds at 60 μg/300 μL appears to be the most efficient concentration.As shown in Table ₂ , when the concentration of CaCl 2 is too high or too low, the hemostatic effect decreases, but 0.009 mol/L It was confirmed that it is the most efficient at 108 seconds.

Experimental Example 6 Red blood cell hemolysis experiment of the prepared hydrogel hemostatic agent

In order to measure the amount of red blood cells remaining after blood clotting, an erythrocyte hemolysis experiment was conducted, and the blood clotting ability was confirmed through the absorbance of hemoglobin from the hemolytic red blood cells. The control group is a case in which a blood coagulation reaction proceeds without introducing a hydrogel sample. In the case of Experimental Example 6, a hydrogel hemostatic agent prepared according to the example was provided, but the concentration of pullulan was provided at 0.3, 0.4, and 0.5 g/mL.

After a certain period of time, 5 mL of distilled water is added to the sample to stop the blood coagulation reaction, and the obtained supernatant is dispensed into a 96 well plate to measure the absorbance of hemoglobin. If blood coagulation proceeds a lot, the amount of hemoglobin released through hemolysis is small because the amount of residual red blood cells is small, so the hemostatic ability can be observed through the intensity of the absorbance of hemoglobin. The results for this are shown in FIG. 8.

Experimental Example 7 in vivo Animal model experiment

1) of the hydrogel hemostatic agent prepared according to the example in vivo hemostasis experiment

9 is a photograph showing a hemostatic animal test method using SD rats. SD rats were ^{anesthetized by administering an animal anesthetic mixed with Zoletil ®} 50 and Rompun ^® in a ratio of 4:1, and then, when SD rats were anesthetized, the hairs on the abdomen were removed and fixed at a 45° angle to the operating table. After disinfecting the abdominal hair removal site with povidone iodine solution, an incision was made about 20 to 30 mm using a No. 10 blade in the center, and the skin and peritoneum were peeled off and raised in both sides to expose the liver. After placing a pre-weighed filter paper under the liver, a wound with a depth of 3 mm is placed on the left lobe of the liver using a No.10 blade exposed 3 mm above the stopper, and 0.5 ml of the improved hydrogel hemostatic agent is prepared when bleeding begins. The time of introduction and hemostasis was measured. When hemostasis was reached, the amount of bleeding was calculated by weighing the filter paper and hydrogel absorbing blood.

FIG. 10 is a photograph showing the results of adjusting the pullulan concentration (0.3, 0.4, 0.5 g/mL) of pullulan prepared in a hemostatic animal experiment using SD rats.

11 is a graph showing the amount of bleeding in the results of improved pullulan concentrations (0.3, 0.4, 0.5 g/mL) using SD rats. As for the amount of bleeding, it was confirmed that bleeding of 1,080.6 mg in the control group, 580.6, 300.3, and 231.7 mg in the case of improved pullulan 0.3, 0.4, and 0.5 g/ml, respectively, and the hemostatic effect was the greatest in the case of 0.5 g/ml. there was.

In addition, the results of histological observation of the hemostatic site of the liver treated with the improved pullulan hydrogel prepared under the conditions described in FIG. 12 through H&E staining are shown in FIG. 12. The nucleus of the cell is stained blue and the cytoplasm is red, but seeing that the wound area is red, it can be confirmed that the red blood cells are well entangled in the wound area.

In addition, the results of histological observation of the hemostatic site of the liver treated with the improved pullulan hydrogel prepared under the conditions described in FIG. 13 through carstairs staining are shown in FIG. 13. This staining method dyes fibrin red and platelets blue, and it was confirmed that fibrin was well formed by seeing that the wound was red.

2) In vivo wound healing ability test of the prepared hydrogel hemostatic agent

SD rats were ^{anesthetized by administering an veterinary anesthetic mixed with Zoletil ®} 50 and Rompun ^® in a ratio of 4:1. When SD rats were anesthetized, hairs on the trunk between the forelimbs and hind limbs were removed. After disinfection with povidone-iodine solution, a 2 cm-diameter full-thick wound was formed on both sides. Using a transparent OHP film, draw along the wound area to measure the size of the wound, and after injecting improved pullulan hydrogel to the wound area, when crosslinking was completed, the wound area and its surroundings were covered with Tegaderm ^TM to protect it (as a control group). Use sterile gauze). After that, it was lifted every 2 days, and the wound area was drawn using an OHP film to check the wound healing behavior over time.

14 is a graph comparing the extent to which the wound is healed by measuring the size of the wound on the skin treated with the improved pullulan hydrogel prepared according to the example over time with the control group. However, in this case, the hydrogel was prepared and used at 0.4 g/ml of fluran, 0.05 g/ml of alginic acid, and 519 units/ml of HRP.

In the above, the present invention has been described by specific matters such as specific elements and limited embodiments and drawings, but this is provided only to help a more general understanding of the present invention, and the present invention is not limited to the above embodiments. , Anyone having ordinary knowledge in the technical field to which the present invention pertains can make various modifications and variations from these descriptions.

Therefore, the spirit of the present invention is limited to the above-described embodiments and should not be defined, and all modifications that are equally or equivalent to the claims as well as the claims to be described later fall within the scope of the spirit of the present invention. I would say.

Claims (15)

(a) introducing a carboxyl group to pullulan;
(b) preparing an alginic acid aqueous solution by adding alginic acid to distilled water;
(c) mixing and stirring the pullulan and the alginic acid aqueous solution, and then introducing tyramine into the side chains of pullulan and alginic acid;
(d) dialysis and lyophilization of pullulan and alginic acid into which the tyramine has been introduced to prepare an improved pullulan; And
(e) reacting the improved pullulan with horseradish peroxidase (HRP), hydrogen peroxide (H202), thrombin and calcium chloride (CaCl2) to prepare a hydrogel; of an improved hydrogel hemostatic agent comprising: Manufacturing method. The method of claim 1,
Introducing a carboxyl group to pullulan in the step (a),
Preparation of improved hydrogel hemostatic agent by reacting and replacing pullulan with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), sodium bromide (NaBr) and sodium hypochloride (NaOCl) Way. The method of claim 1,
In the step (a), pullulan is an improved method for producing an improved hydrogel hemostatic agent comprising 1 to 50 parts by weight based on 100 parts by weight of the total composition of the hydrogel hemostatic agent. The method of claim 1,
In step (b), alginic acid is an improved method for producing an improved hydrogel hemostatic agent comprising 1 to 80 parts by weight based on 100 parts by weight of the total composition of the hydrogel hemostatic agent. The method of claim 1,
In the step (b), the alginic acid includes a D-manluronic acid group and an L-gluronic acid group, and the weight ratio of the D-manluronic acid group to the L-gluronic acid group is 40 to 70: 60 to 30. The method of claim 1,
In the step (c), the introduction of tyramine,
A method for preparing an improved hydrogel hemostatic agent by reacting and substituting with tyramine, N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) . The method of claim 1,
After introducing the tyramine in the step (c), the substitution rate is 1 to 15% of the improved method for producing an improved hydrogel hemostatic agent. The method of claim 1,
The method for producing an improved hydrogel hemostatic agent having a weight average molecular weight of 10,000 to 750,000 of the pullulan improved in step (c). The method of claim 1,
In step (e), the concentration of the peroxidase (HRP) is 0.1 to 5 parts by weight, and the hydrogen peroxide solution (H ₂ 0 ₂ ) is 1 to 30 parts by weight based on 100 parts by weight of the total composition. . The method of claim 1,
The reaction of step (e) is,
The improved pullulan was mixed with horseradish peroxidase (HRP), thrombin, and calcium chloride (CaCl ₂ ), and dissolved in a phosphate buffer solution (PBS) for 20 minutes to 1 hour at room temperature with a pH of 6 to 8. To prepare a solution,
An improved method for producing an improved hydrogel hemostatic agent to prepare a hydrogel by adding a second solution containing 30% hydrogen peroxide (H ₂ O _{2) to the first solution and reacting for 5 seconds to 2 minutes.} The method of claim 10,
Thrombin per 1 mL of the phosphate buffer solution (PBS) contains 100 to 500 μg, and the calcium chloride (CaCl ₂ ) per mL of phosphate buffer solution (PBS) is 3 to 15 mM. The method of claim 1,
The method for producing an improved hydrogel hemostatic agent further comprising at least one or more of gelatin, chitosan, hyaluronic acid, alginate, and cellulose derivatives in step (e). The method of claim 1,
The method for producing an improved hydrogel hemostatic agent further comprising at least one or more selected from fibrinogen, vitamin C, vitamin K, collagen, gelatin, and gelfoam as hemostatic substances in step (e). An improved hydrogel hemostatic agent prepared according to any one of claims 1 to 13. The method of claim 14,
The hydrogel hemostatic agent is an improved hydrogel hemostatic agent having a viscosity of 10,000 to 25,000 cP at room temperature.

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