KR102145097B1 - Hemostatic-capable thrombin-binding biodegradable PLGA mesh and method for manufacturing thereof - Google Patents

Abstract

The present invention relates to a biodegradable PLGA hemostatic mesh for absorbing and hemostasis obtained by grafting thrombin on the surface of a biodegradable polymer, poly(lactic-co-glycolic acid), and a method for producing the same.

Description

Hemostatic-capable thrombin-binding biodegradable PLGA mesh and method for manufacturing thereof

The present invention relates to a hemostatic mesh used in surgery and a method of manufacturing the same.

The biggest issue in soft tissue materials is large-scale bleeding due to severe damage to organs. Biodegradable nonwoven fabric made of medical polyester is a medical device that has been widely used in the treatment of pneumothorax in chest surgery. Therefore, it is widely used, and the most problematic disease is laceration in the field of hepatobiliary pancreas. Injuries caused by traffic accidents or strong external shocks cause serious damage to internal soft tissue organs such as the liver and pancreas. The best way to minimize serious organ damage from these large lacerations is to suture the damaged area to prevent excessive bleeding and preserve the organ. Treatments for lacerations of organs are classified according to the appearance of the damaged organ (blunt or sharp) and the grade of lesions (I-V). In the case of Grade I/II, so far, treatment is performed by surgical methods such as sutures and cauterization. However, the method still has a limitation in preventing bleeding of widely torn soft tissue. In this case, a nonwoven fabric made of biodegradable polyester is used to finally hemostatic the sutured area.

Among the developed hemostatic agents, products such as Tacosyl ^® and Greenplast ^® , which are widely used in various atmospheres, are evaluated as products with excellent hemostatic effects, including coagulation factors such as fibrin and thrombin. But is easy to crumble due to the properties of the material itself if the taco Room ® and the difficulties of a bleeding apply the blood is not easily absorbed by the green plast ^® is instantaneous hemostasis include, but are effective excessive bleeding, this is significantly lowered tackiness have. As a biological factor, the coagulation of platelets by Greenplast ^® is very important for hemostasis, but as an emergency medical finding, compression of the affected area by the use of absorbent substances is also a very important means of physical hemostasis.

In the end, hydrophobic biodegradable polyester mesh or nonwoven fabric is made hydrophilic and the introduction of biological blood coagulation factors means the advent of next-generation products that complement all the shortcomings of currently used products.

As a result of the present inventors' efforts to solve the above problem, the THR-PLGA mesh manufactured by attaching thrombin after inducing a functional group on the surface by treating plasma with a tough and biodegradable lactic acid-glycolic acid copolymer (PLGA) In vitro and in vivo experiments were found to have a very excellent hemostatic effect, and the present invention was completed.

The present invention is to solve the problems of the prior art, and to provide a method of manufacturing a THR-PLGA hemostatic mesh having a shorter blood coagulation time and excellent hemostatic effect than conventional hemostatic materials.

Another object of the present invention is to provide the THR-PLGA hemostatic mesh.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention comprises the steps of inducing a hydrophilic functional group by treating a plasma on the surface of a poly(lactic-co-glycolic acid) (PLGA) mesh, including thrombin in the hydrophilized PLGA mesh by treating the plasma. It provides a method for producing a biodegradable PLGA hemostatic mesh for absorbing and hemostasis of a wide range of bleeding, including the step of dropping a solution and washing and drying after the reaction is completed.

The present invention also provides a biodegradable PLGA hemostatic mesh for absorbing and hemostasis obtained by grafting a coagulation factor onto the surface of a biodegradable polymer, poly(lactic-co-glycolic acid) (PLGA).

The present invention relates to a THR-PLGA (THR-PLGA) mesh coated with thrombin and a method for manufacturing the same, characterized in that thrombin is electrostatically coated on a plasma-treated poly (lactic-co-glycolic acid) (PLGA) surface. , It has an excellent effect in providing a nonwoven fabric for hemostasis during surgery.

In addition, the THR-PLGA mesh of the present invention can reduce blood coagulation time by converting the surface properties of PLGA, which is a hydrophobic polymer, to hydrophilicity.

1 shows a process of inducing a hydrophilic reactor on the surface of PLGA by reacting a PLGA mesh in a plasma generator chamber.
FIG. 2 shows amide and NH ₂ functional groups by FTIR-ATR analysis to confirm that thrombin is bound to the PLGA mesh surface.
3 shows the results of XPS analysis of PLGA and THR-PLGA, and it was confirmed that thrombin was bound to the PLGA mesh surface from the observation that an N1s peak of 400 eV corresponding to the nitrogen of thrombin was observed only in THR-PLGA.
FIG. 4 shows that thrombin was bound to the PLGA mesh surface from the observation of 399.6 and 401 eV peaks corresponding to C=N and NC=O binding in the N1s high-resolution XPS analysis of THR-PLGA.
5 shows the measurement results of the active concentration of thrombin bound to the surface of THR-PLGA.
6 shows the results of measuring FXIIIa activity of rat plasma dropped on THR-PLGA.
Figure 7 shows the blood clotting time of PLGA and THR-PLGA attached to the damaged tail of rat.
8 is an electron microscope photograph of blood coagulation morphology observed from THR-PLGA with different amounts of thrombin binding.

Biodegradable PLGA hemostatic mesh for absorbing and hemostasis of the present invention for achieving the above object;

Inducing a hydrophilic functional group by treating plasma on the surface of a poly(lactic-co-glycolic acid) (PLGA) mesh, dropping a solution containing thrombin on the hydrophilized PLGA mesh, and washing after completing the reaction and It can be prepared through the step of drying.

The present inventors can perform sufficient adhesion and hemostasis even in body fluids and blood-existing areas of the body, in particular, can absorb a large amount of moisture compared to other biodegradable polymers, and a biodegradable medical hemostatic mesh capable of self-degradation in the body Research efforts were made to develop. As a result, it was confirmed that the surface of PLGA was plasma-treated to induce a hydrophilic functional group, and thrombin was grafted to be used to effectively adhere, coat, and hemostasis of living tissue.

As confirmed in the following examples, the PLGA hemostatic mesh prepared according to the manufacturing method of the present invention can easily control the concentration of thrombin bound to the PLGA surface as much as the effective amount of thrombin concentration acting as a hemostatic agent, and FXIIIa activity also in the hemostatic effect. In addition to increasing the blood coagulation time was significantly shortened in the animal model showed an excellent effect (see Fig. 7).

According to an embodiment of the present invention, the biodegradable PLGA hemostatic mesh has a thrombin activity range of 0.5 to 3.5 IU/cm ² per unit area of the PLGA hemostatic mesh, which is a biodegradable polymer, It may be preferably 1.0 to 3.0 IU/cm ² , most preferably 1.5 to 2.5 IU/cm ² .

According to an embodiment of the present invention, the plasma surface modification method may be performed for 20 to 40 seconds at a pressure of 50 mTorr or less and a power condition of 20 to 50 W.

In the plasma-treated PLGA of the present invention, the hydrophilic functional group may include any one or more hydrophilic functional groups selected from the group consisting of a carbonyl group, a hydroxyl group, a carboxyl group and an amine group.

According to another embodiment of the present invention, the present invention is a biodegradable PLGA hemostasis for absorption and hemostasis obtained by grafting thrombin on the surface of a biodegradable polymer, poly(lactic-co-glycolic acid) (PLGA). It relates to the mesh.

Hereinafter, preferred embodiments are presented to aid in understanding the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

1. Materials and methods

1-1. Induce the reactor on the PLGA surface

In order to induce the reactor to the PLGA, a mesh of 1.0 × 1.0 cm ² was prepared and reacted in an RF plasma generator chamber for 50 mTorr, 20 watt, and 30 seconds. Thereafter, exposure to the atmosphere for 10 minutes to induce various reactors such as hydroxyl and carboxyl groups in addition to carbonyl groups on the surface of the PLGA (FIG. 1).

1-2. Thrombin immobilized PLGA (THR-PLGA) manufacturing

A thrombin solution was prepared to introduce a coagulation factor to the surface of the PLGA in which the reactor was induced. Dilute Tris-HCl 1 M (pH 7.4) with tertiary distilled water (DW) to make 20 mM, and treat it for 15 minutes at 121°C in an autoclave. The thrombin solution is thrombin from bovine plasma 0.29 mg 20 mM tris- A stock solution of 80 IU/ml was prepared by dissolving in 2 ml of HCl, and diluted as necessary.

Add 100 μL of thrombin solution with activities of 1, 2, 3, 4, 5, 6, 7, 8 IU to PLGA (1.0 × 1.0 cm ² ) in which the carbonyl group prepared in 1-1 was induced, and then 4 After 24 hours, the reaction was performed at °C and washed with sterilized DW 3 to 5 times, and freeze-dried at -80 °C and 5 mTorr for 24 hours (FIG. 1).

1-3. Material surface analysis

The chemical composition of THR-PLGA was confirmed using Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR; Nicolet iS50, Fisher Scientific, USA). Spectra measured in the range of 600 to 4000 cm ^-1 . The atomic composition of THR-PLGA was analyzed by X-ray photo electron spectroscopy (PHI 5000 VersaProbe, ULVAC PHI, Japan). As analysis conditions, a 15 kV, 24.5 W monochromatic X-ray beam (photoelectron energy = 1486.6 eV) obtained from an aluminum anode was used. The wettability of THR-PLGA was confirmed by measuring the water contact angle. After dropping 10 μL of water droplets on the material surface, the angle between the material surface and the water droplets was checked using OCA 40 (Dataphysics, Germany).

1-4. Evaluation of thrombin activity of THR-PLGA

In order to measure the activity of thrombin introduced into PLGA, a chromogenic substrate assay was performed. 1, 2, 3, 4, 5, 6, 7, 8 IU THR-PLGA (1.0 × 1.0 cm ² ) treated with 20 mM tris-HCl was immersed in 906.25 μL, and 1.9 mM N-(p-Tosyl)-Gly-Pro-Arg p-nitroanilide acetate salt solution was added 93.75 μL. Was added to adjust the concentration to 0.2 mM. After incubation at room temperature for 20 minutes, the absorbance was measured at 405 nm using a microplate reader. The thrombin activity of each THR-PLGA was calculated as the change in absorbance over time.

1-5. Coagulation Factor XIIIa (FXIIIa) activity analysis

The activity of FXIIIa was measured using plasma isolated from blood of rats (Sprague Dawley, 6-9 weeks, 150-200g). After disinfecting the rat's chest with iodine, blood was collected from the heart using a 5 mL syringe, and the collected blood was collected in a vacuum sodium citrate blood collection tube and inverted 10 times. Blood was transferred to a 1.5 mL tube within 1 hour and centrifuged at 1300 rcf for 10 minutes to separate plasma. FXIIIa in rat plasma was measured using Factor XIIIa Activity Assay Kit (Catalog # K522-100, BioVision). THR-PLGA treated with thrombin for each concentration was cut into 6 mm in diameter and placed in a 96-well plate. 100 μL of a mixed solution of 25 μL FXIIIa activation buffer, 25 μL reaction buffer, 48 μL detection buffer, and 2 μL probe was added dropwise to the well in which THR-PLGA was placed, and 25 μL of plasma was added and mixed well. After reacting at room temperature for 20 minutes to activate FXIIIa by thrombin, 100 μL of the supernatant was measured for absorbance at 340 nm with a microplate reader. The activity of FXIIIa for each sample was calculated as the change in absorbance over time.

1-6. Animal hemostasis model

To evaluate the actual hemostatic time of THR-PLGA in vivo, 15 rats (Sprague Dawley, 6-9 weeks, 150-200g) were used. Before surgery, Zoletil (Virbac Laboratories, Carros, France), Rompun® (Bayer Korea Ltd, Seoul, Korea) was anesthetized and the tail was disinfected with iodine. A 2 cm part was cut from the end of the rat tail and bleeding was induced. PLGA and THR-PLGA were attached to the bleeding tail, and the blood clotting time was visually observed. After the blood coagulation experiment, THR-PLGA and PLGA were freeze-dried, and the condition of the coagulated blood in the sample was observed using an electron microscope (SEM).

2. Results

2-1. Surface Characteristics-Confirmation of the covalent bond between thrombin and PLGA (Figs. 2 to 4)

The chemical composition of the surface of the thrombin-bound PLGA sample is shown in FIG. 2. As a result of FTIR-ATR analysis, 2950 cm ^-1 , 1750 cm ^-1 , and 1150 cm ^-1 were observed in the PLGA sample, and this confirmed the specific constituent bindings of PLGA, CH, C=O, and CO. In THR-PLGA, since thrombin binds to PLGA, amide I (1650 cm ^-1 ), amide II (1550 cm ^-1 ) and NH ₂ (3000 ~3500 cm ^-1 ) functional groups could be additionally observed in the graph.

3 shows the XPS analysis results of PLGA and THR-PLGA samples. Carbon and oxygen, the constituents of the PLGA sample, were observed at 284.6 and 532.4 eV, corresponding to C1s and O1s, respectively. The THR-PLGA sample was able to observe an N1s peak of 400 eV corresponding to the nitrogen of thrombin. Also, in the high-resolution spectrum of THR-PLGA. In addition, in the N1s high-resolution XPS analysis of THR-PLGA, 399.6 and 401 eV peaks corresponding to C=N and N-C=O bonds were observed (FIG. 4). Proteins can bind a carbonyl group on a solid substrate through an imine reaction. This is seen as a result of the formation of covalent bonds through the imine reaction of the carbonyl group and thrombin amine group generated in PLGA by plasma treatment.

2-2. Checking the activity of thrombin fixed to THR-PLGA and the measurement result of activity (Fig. 5)

5 shows the measurement results of the active concentration of thrombin bound to the surface of THR-PLGA. The measurements were 0.28±0.12, 0.76±0.24, 1.25±0.21, 1.67±0.21, 1.88±0.21, 2.50±0.21, 2.78±0.12, and 3.47±0.43 IU, respectively. This result indicates the actual activity of thrombin bound to THR-PLGA when 1, 2, 3, 4, 5, 6, 7, 8 IU of thrombin solution was reacted with PLGA, and the effective value of thrombin concentration acting as a hemostatic agent Was obtained.

2-3. Analysis of FXIIIa activity in rat plasma-THR-PLGA's blood coagulation ability and THR with specific concentration in animal experiments ₂ -PLGA, THR ₄ -PLGA, THR ₈ -Reason for using PLC (Fig. 6)

6 shows the results of measuring FXIIIa activity of rat plasma dropped on THR-PLGA. Plasma of THR-PLGA sample showed positive control and higher FXIIIa activity than PLGA. In particular, the highest FXIIIa activity was observed in THR ₈ -PLGA. This result is due to the effect of thrombin on the blood coagulation mechanism. Blood coagulation factor XIII (FXIII) is known to be involved in the final stage of the blood clotting process. FXIII is composed of a "A" unit that acts as a catalyst and a "B" unit that acts as a transport. Thrombin cuts FXIII into active form (FXIIIa) into A and B units. The active form of FXIIIa is known to stabilize the fibrin structure by acting on fibrin to form cross-links between fibrin molecules. Therefore, FXIIIa plays an important role in the hemostasis process, and since the activity was increased by THR-PLGA, it proved the hemostatic effect.

Another phenomenon was found from the results of FXIIIa activity analysis in rat plasma. Interestingly, the FXIIIa activity of rat plasma increased in proportion to the thrombin activity of 0.28, 0.76, 1.25, and 1.67 IU. However, THR-PLGA with thrombin at 1.88, 2.50, 2.78, and 3.47 IU significantly decreased the rate of increase in FXIIIa activity. This is thought to be related to the threshold of thrombin activity and the amount of FXIII in the plasma. In addition, based on this data, THR ₂ -PLGA, THR ₄ -PLGA, and THR ₈ -PLGA having thrombin activity of 0.76±0.24, 1.67±0.21, and 3.47±0.43 IU were used in animal model experiments.

2-4. Results of confirming the hemostatic effect of THR-PLGA in an animal model (Fig. 7)

Blood clotting time was measured from an animal model to see the hemostatic effect of THR-PLGA on the living body. Figure 7 shows the blood clotting time of PLGA and THR-PLGA samples attached to the damaged tail of rats. The rat model without hemostasis took more than 6 minutes (391 seconds) to stop bleeding. The hemostasis of PLGA, THR ₂ -PLGA, THR ₄ -PLGA, and THR ₈ -PLGA was 248, 168, 116 and 103 seconds, respectively, which took less than 3 minutes. THR ₈ -PLGA showed the fastest hemostatic effect, and the difference in coagulation time was not large with THR ₄ -PLGA.

2-5. Blood coagulation morphology confirmation result by electron micrograph (Fig. 8)

Each 1 mL of blood collected from the rat's heart was added dropwise to PLGA, THR ₂ -PLGA, THR ₄ -PLGA, THR ₈ -PLGA, and the blood clotting time of THR ₈ -PLGA measured in 2-4 was 100 seconds. After reacting for a while, PLGA, THR ₂ -PLGA, THR ₄ -PLGA, and THR ₈ -PLGA were taken out from the blood and washed three times with PBS. The washed sample was frozen at -80 °C and then freeze-dried to confirm with an electron microscope (FIG. 8). Red blood cells and platelets were observed in the thrombus formed on the PLGA, and the fibrin net structure was not observed. In the blood coagulated by THR-PLGA, it was confirmed that a large amount of platelets and red blood cells were trapped in the fibrin net structure. In all THR-PLGAs, fibrin nets were formed, and blood cells and platelets were adhered around them. Among them, it was confirmed that the most red blood cells were aggregated in THR ₈ -PLGA.

In the present invention, it was confirmed by FTIR-ATR and XPS analysis that the carbonyl group and thrombin induced by plasma treatment on PLGA covalently bonded through the imine reaction. In addition, the activity of thrombin bound to the PLGA surface was measured and quantified, and the activity of FXIIIa, and THR-PLGA functionality with blood coagulation ability was confirmed through animal experiments. In particular, it was confirmed that the higher the concentration of thrombin, the shorter the blood clotting time, and based on the rapid decrease in the blood clotting time from 1.67 IU or higher, THR-PLGA coated with thrombin of 1.67 IU or more will show appropriate efficiency as a hemostatic agent. I think.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects.

Claims (6)

Inducing a carbonyl group as a hydrophilic functional group by treating plasma on the surface of a poly(lactic-co-glycolic acid) (PLGA) mesh; And
Including the step of forming a covalent bond through an imine reaction of dropping a solution containing thrombin on the surface of the PLGA mesh from which the carbonyl group is derived,
A method of manufacturing a biodegradable PLGA hemostatic mesh for absorbing and hemostasis of extensive bleeding.
The method of claim 1,
The biodegradable PLGA hemostatic mesh has an active range of thrombin per unit area of 0.5 to 3.5 IU/cm ² , a method for producing a biodegradable PLGA hemostatic mesh.
The method of claim 1,
The plasma treatment is carried out for 20 to 40 seconds under a pressure of 50 mTorr or less and a power condition of 20 to 50 W, a method of producing a biodegradable PLGA hemostatic mesh.
delete As a hydrophilic functional group on the surface, a carbonyl group is derived PLGA (poly(lactic-co-glycolic acid)) mesh; And
Containing thrombin covalently bonded to the surface of the PLGA mesh from which the carbonyl group was derived through an imine reaction,
Biodegradable PLGA hemostatic mesh for absorption and hemostasis of extensive bleeding.
The method of claim 5,
The biodegradable PLGA hemostatic mesh having an active range of thrombin per unit area of 0.5 to 3.5 IU/cm ² , a biodegradable PLGA hemostatic mesh.

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