KR20230057168A - Haemostatic composition comprising keratin conjugated fibrinogen hydrogel - Google Patents

Abstract

The present invention relates to a hemostatic composition comprising, as an active ingredient, a keratin-binding fibrinogen hydrogel in which keratin and fibrinogen are bound, and a method for using the keratin-binding fibrinogen hydrogel in blood hemostasis. The hemostatic composition comprising the keratin-binding fibrinogen hydrogel according to the above serves to help activate blood coagulation components without loss through physical action.

Description

Haemostatic composition comprising keratin conjugated fibrinogen hydrogel as an active ingredient

The present invention relates to a hemostatic composition comprising a keratin-binding fibrinogen hydrogel as an active ingredient, and more particularly, to a hemostatic composition comprising a keratin-binding fibrinogen hydrogel as an active ingredient, in which keratin and fibrinogen are combined, and a combination of keratin and fibrinogen. It relates to a method of using the keratin-binding fibrinogen hydrogel for blood hemostasis.

Hemostatic agent refers to a medical material that promotes hemostasis of blood when bleeding occurs due to surgery or surgical trauma.

Uncontrolled bleeding is one of the leading causes of death in individuals following trauma. The coagulation system can be divided into primary hemostasis, which is initiated by adhesion of platelets to the base of the extracellular matrix (ECM) of damaged tissue, and secondary hemostasis, which is the subsequent activation of the coagulation cascade.

There are several ways to classify hemostatic biomaterials. Depending on the type of product, powder type, fine fiber type, sponge type, non-woven fabric type, injection type, polymer or fiber type product is mainly used.

Keratins are a group of natural proteins derived from the hair appendage. The earliest use of keratin for blood coagulation was in oriental medicine in the 1500s when pyrolysis hair was used to stop bleeding.

Conventional studies on keratin hemostatic biomaterials are limited to material platforms used for tissue repair, such as hydrogels, commercial keratin dressings, and cotton gauze.

On the other hand, among hemostatic biomaterials, fibrinogen is widely used for blood coagulation and wound healing, but the absorption rate of fibrinogen hemostatic agents is relatively low as a single material, which makes hemostasis difficult.

Republic of Korea Patent Publication No. 10-2017-0007024

The present inventors evaluated the structure, cell behavior and surface morphology of keratin-binding fibrinogen (KRT-FIB)-binding hemostatic biomaterials, and investigated blood absorption and comprehensive hemostasis evaluation. As a result, the keratin-binding fibrinogen can be applied to oral tissue regeneration. It was found out that it is a promising hemostatic biomaterial that can be used, and the present invention was completed.

Accordingly, an object of the present invention is to provide a hemostatic composition comprising a keratin-binding fibrinogen hydrogel as an active ingredient.

Another object of the present invention is to provide a hemostatic use of a keratin-binding fibrinogen hydrogel.

In order to achieve the above object, the present invention provides a hemostatic composition comprising, as an active ingredient, a keratin-binding fibrinogen hydrogel in which keratin and fibrinogen are bound.

In one embodiment of the present invention, the keratin may be derived from human hair.

In one embodiment of the present invention, the keratin and fibrinogen may be combined in a molar ratio of 3 to 6: 1.

In one embodiment of the present invention, the keratin and fibrinogen may be combined in a molar ratio of 6:1.

In one embodiment of the present invention, the keratin-binding fibrinogen hydrogel may be included in an amount of 0.01 to 50% by weight based on the total weight of the hemostatic agent composition.

In addition, in order to achieve the above object, the present invention provides a method of using a keratin-binding fibrinogen hydrogel in which keratin and fibrinogen are combined for blood hemostasis.

In one embodiment of the present invention, the keratin may be derived from human hair.

In one embodiment of the present invention, the keratin and fibrinogen may be combined in a molar ratio of 3 to 6: 1.

In one embodiment of the present invention, the keratin and fibrinogen may be combined in a molar ratio of 6:1.

The hemostatic composition comprising the keratin-binding fibrinogen hydrogel according to the present invention helps to activate blood coagulation components without loss through physical action. In particular, keratin activates platelet adhesion to have a primary hemostatic effect, and the adhesion components, thrombin and fibrinogen, are the physiological hemostasis process and the final stage of hemostasis, and serve to fix tissues.

In addition, the hemostatic composition comprising the keratin-binding fibrinogen hydrogel according to the present invention has the advantage of creating a dressing suitable for a medical environment by customizing the shape of a patient's wound with the density of fibrin and cross-linked keratin.

Therefore, the hemostatic composition comprising the keratin-binding fibrinogen hydrogel according to the present invention has the advantage of shortening the hemostasis time and lowering the treatment cost by eliminating the need for preparation before the procedure, shortening the procedure time and minimizing infection.

Figure 1 shows a schematic diagram of hemostatic agents according to the types of absorbable KRT-FIB biomaterials developed.
Figure 2 shows the results of confirming the formation of hair keratin-fibrin (KF) by the chemical reaction between keratin and fibrinogen through GPC. Figure 2a shows the results of keratin (KRT), fibrinogen (FIB) and keratin-fibrinogen (KF), and the TGA analysis of KFS showed slightly higher thermal stability than FIB. Figure 2b shows the TGA diagrams of KRT, FIB and KF precursors, where each arrow indicates the temperature at which major weight loss begins.
Figure 3 shows digital photographs and SEM images (scale bars = 1000 μm and 200 μm, respectively) of KFS-6 and FIBS at different magnifications.
Figure 4 shows a blood-sucking photograph and contact angle of KFS hemostatic biomaterial and a commercial product (Taqhosil®).
Figure 5 shows the comprehensive hemostasis evaluation results of KFS compared to Tachosil®. Figure 5a is a blood coagulation test, Figure 5b is the BCI of the control group and the sample group, Figure 5c is the absorption rate of the control group and the sample, d of Figure 5 is the hemostatic efficiency control group and the sample group, and all statistical data are Expressed as mean ± standard deviation (n = 3, P < 0.005).
Figures 6a and 6b show live or dead staining images (scale bar = 200 μm) of encapsulated HDF cells, and Figures 6c and 6d show DAPI/Phalloidin ( F-actin) staining shows confocal pictures (scaler bar = 50 μm) of encapsulated HDF cells.

A first embodiment of the present invention provides a hemostatic composition comprising, as an active ingredient, a keratin-binding fibrinogen hydrogel in which keratin and fibrinogen are bound.

In general, hemostatic biomaterials using natural polymers have very high biocompatibility with cells and surrounding tissues, and are less inflammatory. They include gelatin, albumin, cellulose, hyaluronic acid, fibrinogen, alginate, and chitosan.

In particular, fibrinogen is a key protein in the regulation of angiogenesis and bone regeneration. Fibrin hemostats are typically produced by thrombin, fibrinogen, transglutaminase factor XIII and calcium ions to form fibrin.

In order to continuously and sufficiently exert the hemostatic effect, various types of sheet and foam formulations are required. For hemostatic products, collagen, thrombin, gelatin, cellulose and complex formulations are being developed.

However, there are no hemostatic ingredients or formulations in which hair keratin and fibrinogen are cross-linked. Therefore, the absorption rate of the fibrinogen hemostatic agent is relatively low for a single substance, which makes hemostasis difficult. In order to solve this problem, the absorption ability was improved with fibrinogen and keratin.

Here, the present inventors confirmed changes in the fibril structure and evaluated the structural properties of the KRT-FIB hemostatic agent. To test the hemostatic efficacy, the present inventors used a clinically relevant blood source that has high absorbability and surface hydrophilicity to promote wound healing synergistically through capillary network formation.

In addition, the present invention provides insight into the interaction of blood coagulation and platelets through comparison of commercially available fibrinogen-binding Tachosil®. Accordingly, the present invention describes an absorbable human hair keratin-fibrinogen hemostatic agent developed to reduce patient pain and treatment cost and reduce recovery time.

In the present invention, a new absorbent sheet crosslinked with keratin and fibrin polymers that promote blood coagulation and a foam type keratin-binding fibrinogen hydrogel (KRT-FIB) hemostatic agent (KFS) were developed (FIG. 1).

To form a hydrophilic surface of keratin-binding fibrinogen hydrogel hemostatic agent, human hair-derived keratin protein is covalently linked to FIB protein through a facile 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) reaction. .

The resulting KRT-FIB precursor (KF precursor) is cross-linked in the presence of THR to form a porous KFH hydrogel. A KFS hemostatic biomaterial with a KRT:FIB molar ratio of 6:1 was optimized by blood absorption measurement, and the coagulability and surface morphology of the KFS hemostatic biomaterial were investigated.

The excellent absorption capacity, coagulation and low hemolysis rate indicate that the absorbable KFS hemostatic agent is a promising platform for wound healing with high clinical applicability.

In the hemostatic composition according to the first embodiment of the present invention, the keratin may be derived from human hair, but is not limited thereto.

In the hemostatic composition according to the first embodiment of the present invention, the keratin and fibrinogen may be combined in a molar ratio of 3 to 6: 1, but is not limited thereto.

In the hemostatic composition according to the first embodiment of the present invention, the keratin and fibrinogen may be combined in a molar ratio of 6: 1, but is not limited thereto.

The hemostatic composition according to the first embodiment of the present invention contains the keratin-binding fibrinogen hydrogel in an amount of 0.01 to 50% by weight, preferably 0.1 to 20% by weight, more preferably 1 to 10% by weight, based on the total weight of the hemostatic composition. % may include, but is not limited thereto.

The hemostatic composition comprising the keratin-binding fibrinogen hydrogel according to the first embodiment of the present invention may further include a pharmaceutically acceptable diluent.

The diluent is a pharmaceutically acceptable aqueous fluid, such as pharmaceutical grade deionized water or a pharmaceutical grade aqueous solution containing certain ions and/or buffering agents.

The hemostatic composition comprising the keratin-binding fibrinogen hydrogel according to the first embodiment of the present invention may further include an excipient, examples of which include human albumin, mannitol, and sodium acetate, but are not limited thereto no.

The hematopoietic composition comprising the keratin-binding fibrinogen hydrogel according to the first embodiment of the present invention may further include a stabilizer, such as ascorbic acid, sodium ascorbate, other salts of ascorbic acid, or antioxidants. Examples include, but are not limited to.

In addition, as a second embodiment, the present invention provides a method of using a keratin-binding fibrinogen hydrogel in which keratin and fibrinogen are combined for blood hemostasis.

In the method according to the second embodiment of the present invention, the keratin may be derived from human hair, but is not limited thereto.

In the method according to the second embodiment of the present invention, the keratin and fibrinogen may be combined in a molar ratio of 3 to 6: 1, but is not limited thereto.

In the method according to the second embodiment of the present invention, the keratin and fibrinogen may be combined in a molar ratio of 6:1, but is not limited thereto.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

<Example>

1. Materials and Methods

(One). Human hair keratin material extraction

KRT protein was extracted from human hair keratin. A human hair sample (50 g) was washed and washed thoroughly with 70% (v/v) ethanol and distilled water. Washed hair was immersed in a mixture of chloroform and methanol (2:1; v/v) for 24 hours to remove lipids, and then air dried. Human hair keratin was extracted according to previously known methods.

Briefly, degreased hair was treated with 2% (v/v) peracetic acid (Sigma) at 37°C for 24 hours, washed with PBS (phosphate buffered saline), and then 5% for 72 hours while stirring at 300 rpm. Hair (20 g) was treated with Shindai solution containing (v/v) 2-mercaptoethanol, 5 M urea, 2.6 M thiourea and 25 mM Tris pH 8.5.

After the resulting mixture was centrifuged at 4,500 g for 20 minutes, the supernatant was filtered with a filter paper having a pore of 2.5 μm. Using the 5,5'-dithio-bis 138 (2-nitrobenzoic acid) (DTNB) method (Ellman's reagent), the obtained solution was diluted with a 12-14 kDa cutoff dialysis membrane (Spectra /Por®) was used for dialysis.

The resulting solution was freeze-dried to obtain keratin powder. Fibrin gels were prepared using human plasma fibrinogen (FIB) (Merckmillipore, USA) and human plasma thrombin (THR) (Calbiochem, USA).

Dulbecco's Phosphate Buffered Saline (DPBS) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Carbodiimide/N-hydroxysuccinimide (EDC/NHS), Tris buffered saline (TBS), sodium hydroxide (NaOH), and calcium chloride (CaCl ₂ ) were purchased from Sigma-Aldrich. Blood was purchased from Innovative Research (USA) and Tachosil® was purchased from (Baxter, USA).

(2). Instrumentation and analysis

Thermal properties of a series of KRT-FIB precursors (KF precursors) and corresponding hydrogels (KFH hydrogels) were analyzed by thermogravimetric analysis (TGA, SDT) under a nitrogen gas flow and a heating rate of 10 °C/min in the temperature range 20–600 °C. Q600, TA Instruments, USA).

The morphology of the hydrogel was observed with a gold-coated scanning electron microscope (SEM, S-4700, Hitachi, Japan). Molecular weight distribution was determined using Gel Permeation Chromatography (GPC) equipped with a Waters 2414 refractive index detector, HPLC pump and three consecutive Styragel® columns. The eluent was PEO at a flow rate of 1 mL/min.

(3). Succinylation of KRT protein (KRT-COOH)

25 mg of succinic anhydride dissolved in PBS was added dropwise to a solution of KRT (200 mg) dissolved in 100 mL of 50 mM PBS (pH 6-7). The resulting mixture was stirred at room temperature for 1 hour and against distilled water (DW) for 4 hours. The purified KRT-COOH was lyophilized and stored at -20 °C until further use.

(4). Synthesis of KRT-FIB precursor (KF precursor) via EDC/NHS coupling

To synthesize a series of KRT-FIB precursors (KF precursors), different amounts of KRT-COOH (0, 5, 10, 30, 60 mg) were activated in the presence of EDC/NHS. First, 19.25 mg of EDC was added to a solution of KRT-COOH in 100 mL PBS. Then, 22 mg of sulfo-NHS was added to the mixed solution and stirred at room temperature for 15 minutes. 62.5 μL of 2-mercaptoethanol was added dropwise.

Then, the pH of the resulting mixture was adjusted to 7.0 by adding 10X PBS (5 ml), and 100 mg of FIB was added to the mixed solution while stirring at room temperature. The reaction proceeded for 2 hours and was quenched by the addition of Tris.

1N NaOH was added to increase the pH of the quenched solution to 8.0. The resulting KF precursor solution was placed on a dialysis membrane (Spectrum Laboratories Inc., Rancho Dominguez, CA, USA) with MWCO = 25,000 g/mol and dialyzed for 3 days. The purified KF precursor was then lyophilized and stored at -20 °C until further use.

(5). Manufacture of KRT-FIB hemostatic biomaterial (KFS)

A series of KRT-FIB hemostatic agents (KFS-1, 3, 6) were prepared using the corresponding KF precursors (KF-1, 3, 6) with different KRT contents (KF-1, 3, 6). First, each KF solution precursor (20mg/mL), THR (10IU/mL) and CaCl ₂ (2.5mg/mL) were prepared.

After mixing the above two materials, it was freeze-dried at -70 ℃. As a control, an FIB hemostatic agent was prepared according to the same method as the FIB solution (20 mg/mL).

(6). contact angle measurement

Contact angle measurements were performed with a VCA Optima XE at ambient temperature (22-25 °C) and relative humidity (20-40%). To prevent cross-contamination, a dedicated test syringe was used for each test liquid. A drop of 2 μL was formed at the tip of the syringe and carefully deposited on the sample surface.

The syringe was withdrawn, and a static contact angle image of liquid immersion (liqiud depostion) was taken within 3 seconds by a camera coupled to the charge. The contact angle was calculated with the software provided by the supplier. Reported contact angle values are based on 5-8 repetitions.

(7). blood clotting index

Blood coagulation index (BCI) was performed according to the method modified by Shih et al. First, FIBS, KFS, and Tachosil® hemostatic agents were cut into 1.0 × 1.0 × 0.1 cm ³ cubes, put into vials, and preheated at 37° C. for 5 minutes.

Second, 200 μL of blood was immediately dripped onto the dressing. Thirdly, after incubating the sample at 37°C for 2 minutes, a total of 50 mL of deionized water was added and shaken at 50 rpm for 10 minutes to dissolve red blood cells (RBC) that did not adhere to the clot.

The absorbance of the resulting hemoglobin solution sample was measured at 540 nm with a Jasco V-630 spectrophotometer (Jasco Company, JPN). The absorbance of 200 μL of coagulated whole blood in 5.0 mL of deionized water was used for comparison.

Finally, the FIBS, KFS, and Tachosil® samples were calculated using the formula below.

(8). cell viability

A Live/dead assay was used to examine the viability of encapsulated HDF cells using the Live/dead assay. Immediately after the encapsulation of the sample and after 3 days of incubation, the sample was incubated in a calcein-AM/ethidium homodimer solution at 37°C for 30 minutes.

Cell viability was quantified using the calcein-AM/ethidium homodimer Live/dead assay according to the manufacturer's instructions. Cell morphology was examined using a fluorescence microscope (IX71; Olympus Life Life Science; Tokyo, Japan).

(9). Confocal Laser Scanning Microscopy (CLSM)

HDF cells were inoculated into hemostatic biomaterials. After 3 days of culture, the encapsulated cells were fixed and stained with rhodamine-phalloidin (Invitrogen) and 2-(4-amidinophenyl)-1H indole-6-carboxamidine (DAPI) to reveal F-actin filaments and cell nuclei, respectively. visualized.

Fluorescence images were obtained using an LSM 980 with an Airyscan 2 (Carl Zeiss, Jena, Germany). The imaging setup was measured with a 40x objective and an immersion lens with a slice thickness and total thickness of 205 μm.

Full-volume hydrogel images were obtained using fluorescence emission intensity and combined into 3D volumes using image processing software (ZEN black software, Germany).

(10). statistical analysis

Statistical analysis was performed by one-way analysis of variance (ANOVA) with Tukey's post-hoc analysis for significance. A p-value less than 0.05 was considered statistically significant. For rheological and image analysis, statistical significance was analyzed in Origin (Origin Software, San Diego, CA).

2. Experimental results

(One). Synthesis of KRT-FIB precursor (KF)

The human hair-derived KRT protein is first succinylated to introduce a carboxylic acid as a terminal functional group. The carboxyl group of the resulting KRT-COOH enables bonding to the amine group on FIB through the EDC/NHS reaction.

A series of KF precursors were prepared with various molar ratios of KRT and FIB and were abbreviated as KF-1, KF-3 and KF-6 with KRT:FIB in 1:1, 3:1 and 6:1 molar ratios.

GPC analysis showed that the maximum composition ratio of keratin to fibri was determined to be 6:1, and the molecular weight of the synthesized polymer varied (see Fig. 2a). GPC analysis was performed to study the change in molecular weight of the KT precursor during the reaction (see FIG. 2 a).

Thermal stability analysis through thermal analysis: Fibrinogen, keratin, keratin-fibrinogen, 5 mg of the comparative material and 5 mg of the sample were each put into an aluminum cell and flowed at 50 ml/min. The temperature of the comparative material and the sample was changed under the same conditions and heated at 10 °C per minute from room temperature to 600 °C. The temperature difference of the materials was continuously measured, and the change in mid-cooling loss according to the temperature change of the two materials was compared.

Heat was applied from room temperature to 600° C., and the two materials were compared and measured. It was shown that the combined KFS has stable structural properties that change at higher temperatures (see Fig. 2b).

(2). Biomaterial structure and absorption characteristics of KFS-6 with optimized weight ratio

SEM images show that compared to non-porous FIBS, KFS has a highly porous structure (>95% porosity) with a diameter of 10-100 μm (see Fig. 3). These results indicate that the absorption rate of KFS hemostatic biomaterials is highly dependent on the porosity of the hemostatic agent.

Scanning electron micrographs of the pore structure and pore surface showed that the keratin-binding hemostat developed an internal structure with more uniform pores than the conventional fibrin hemostat.

(3). Characterization of Absorbable KFS with Optimized Formulation

The blood absorption capacity was evaluated by measuring the contact angle of the surface. As shown in FIG. 4 , the lower the molar ratio of keratin cross-linking, the lower the contact angle, especially in the KFS-6 sample, as low as 53.96° ± 1.1°.

This excellent hydrophilicity and absorbability is helpful in stopping bleeding and promoting wound healing when used as a hemostatic agent.

The optimized KFS-6 hemostat proved to be superior. The developed hemostatic agent is modified into a hydrophilic surface to improve the property of absorbing liquid, and platelets, red blood cells, and coagulation factors in the blood at the bleeding site are aggregated at the wound site.

(4). In vitro hemostasis and biocompatibility of KFS with optimized formulation

The optimized KFS-3 and KFS-6 samples proved superior. Blood coagulated completely within 2 minutes (see Fig. 5a). As a method for evaluating the hemostatic performance of existing hemostatic products, the coagulation time was measured using blood or a large animal model such as a pig.

Therefore, animal models are widely used in studies on blood coagulation and fibrin degradation. In the present invention, pig blood supplemented with sodium citrate was used to evaluate the hemostatic ability. The hemolytic index was lower than that of the tested hemostatic agent Tachosil® and was dependent on the density of keratin cross-links (see Fig. 5c).

KFS actually provided better hemostatic properties (see Fig. 5d). This shows that keratin is more effective in platelet coagulation than the other tested Tachosil® by forming a porous structure on the surface, increasing absorption and providing better contact (see Fig. 5 f).

Thus, these findings open up potential future directions for the development of new hemostatic materials.

(5). HDF viability in keratin-binding fibrin hemostat cytotoxicity studies

Hemostatic biomaterials are medical materials that come in direct contact with human body fluids, and verification of biological safety through cytotoxicity testing is essential. The cytotoxicity of hemostatic biomaterials was qualitatively evaluated in the culture environment of human dermal fibroblasts (HDF) (see FIG. 6).

Cells encapsulated in KFS hemostat containing FIBS, 2.5 mg/mL CaCl ₂ and 10 unit/mL thrombin showed high viability even after 48 hours of culture.

3. Conclusion

The keratin-binding fibrinogen hydrogel in which keratin and fibrinogen, which are active ingredients of the hemostatic composition according to the present invention, are combined, has improved absorption and hemostatic properties, and its strength is far superior to the fibrin hemostatic biomaterials studied in the prior art, and has various sizes. and has a shape

The hemostatic composition according to the present invention is mainly involved in tissue bonding and helps to activate blood coagulation components without loss through physical action. In particular, keratin activates platelet adhesion to have a primary hemostatic effect, and the adhesion components, thrombin and fibrinogen, are the physiological hemostasis process and the final stage of hemostasis, and serve to fix tissues.

In addition, the hemostatic agent composition according to the present invention has the advantage of creating a dressing suitable for a medical environment by customizing the shape of a patient's wound with the density of fibrin and crosslinked keratin.

Therefore, the hemostatic agent composition according to the present invention has advantages of shortening the hemostasis time and lowering the treatment cost by eliminating the need for preparation before the procedure, shortening the procedure time and minimizing infection.

As above, specific parts of the content of the present invention have been described in detail, and for those skilled in the art, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be clear.

Accordingly, the substantial scope of the invention will be defined by the appended claims and their equivalents. Simple modifications or changes of the present invention can be easily used by those skilled in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Claims (9)

A hemostatic composition comprising, as an active ingredient, a keratin-binding fibrinogen hydrogel in which keratin and fibrinogen are bound.
The hemostatic composition according to claim 1, wherein the keratin is derived from human hair.
The hemostatic composition according to claim 1, wherein the keratin and fibrinogen are combined in a molar ratio of 3 to 6:1.
The hemostatic composition according to claim 3, wherein the keratin and fibrinogen are combined in a molar ratio of 6:1.
The hemostatic composition according to claim 1, wherein the keratin-binding fibrinogen hydrogel comprises 0.01 to 50% by weight based on the total weight of the hemostatic composition.
A method of using keratin-binding fibrinogen hydrogel in which keratin and fibrinogen are combined for blood hemostasis.
7. The method according to claim 6, wherein the keratin is derived from human hair.
The method according to claim 6, wherein the keratin and fibrinogen are combined in a molar ratio of 3 to 6:1.
The method according to claim 6, wherein the keratin and fibrinogen are combined in a molar ratio of 6:1.

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