KR101891688B1 - Method for preparing heparin immobilized polymer material and heparin immobilized polymer material prepared by the same - Google Patents

Abstract

The present invention relates to a method for producing a polymer material into which heparin is introduced and a polymer material produced using the polymer material. More particularly, the present invention relates to a method for producing a polymer material, Grafting the material into the form of an acrylic polymer of a dimer or greater, and introducing heparin simultaneously or subsequently with the grafting step; And a polymer material into which heparin is introduced, in which an acryl-based polymer produced by such a method and a heparin-bound dimer or higher is grafted onto a polymeric material.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a method for producing a polymer material having heparin incorporated therein and a polymer material using the polymer material,

TECHNICAL FIELD The present invention relates to a method for producing a polymeric material for tissue engineering in which heparin is introduced for the sustained release of a cell growth factor and prevention of thrombosis using a radiation technique, and a technique for providing the polymeric material prepared therefrom.

Heparin is a sulfated polysaccharide belonging to the glucose aminoglycan (GAG) family, and has a wide variety of biological activities. The chemical structure of heparin varies widely, and the structural specificity of the two anionic functional groups determines the various activities. These biological activities are related to the interaction and binding affinity of heparin with various proteins.

The interaction between protein and heparin is due to ionic bonding. Proteins with cationic groups and amine groups form ion pairs with carboxyl groups with anions of heparin. In addition, heparin is widely used as an anticoagulant with anticoagulant function due to the mutual affinity with antithrombin III (AT III) which interferes with serine protease in the course of blood coagulation. Bone morphology protein (BMP) and vascular endothelial growth factor (VEGF) bind to heparin-introduced biomaterials with these characteristics to induce the release of bovine or vascular regeneration There is a lot going on.

However, in the conventional method of introducing heparin on the surface of biomaterials, there is a problem in that the introduction rate of heparin is low due to the high toxicity of residual chemicals and uneven surface treatment in the case of the chemical reaction of the substance. On the other hand, Japanese Patent Application No. 2000-025595 discloses a technique of coating accelerated gas ions on the surface of a polymer material and then coating an aqueous solution of heparin. In such a case, however, an ion irradiation device and then a laser light irradiation device are added There is a problem that the process becomes complicated.

Therefore, heparin is bound to a polymer material through graft polymerization without using chemical additives such as catalyst, initiator and crosslinking agent by using radiation to regulate protein adsorption concentration, and osteogenic protein, vascular endothelial cell growth factor It is expected that the polymeric materials for tissue engineering will be widely applied in related fields.

Accordingly, one aspect of the present invention is to provide a method of binding heparin to a polymeric material without chemical additives.

Another aspect of the present invention is to provide a polymeric material in which heparin is bound to a polymeric material without deleterious chemical additives and which can be widely used for tissue engineering.

According to one aspect of the present invention, there is provided a method of manufacturing a polymer electrolyte membrane, comprising: supporting a polymer material in a solution in which an acrylic monomer is dissolved; Irradiating 0.1 to 100 kGy of radiation to graft the acrylic monomer to the polymer material in the form of an acrylic polymer having a dimer or higher; And introducing heparin concurrently with or subsequent to the grafting step, wherein the heparin-introduced polymer material is introduced.

According to another aspect of the present invention, there is provided a polymer material into which heparin is introduced, in which an acryl-based polymer having a heparin-bonded dimer or more grafted onto a polymer material is introduced as the polymer material produced by the production method of the present invention.

According to another aspect of the present invention, there is provided a polymer material into which heparin is introduced, in which an acryl-based polymer having a heparin-bound dimer or higher is grafted onto a polymer material.

According to the present invention, there is provided a polymer scaffold for tissue engineering, wherein heparin is introduced for the sustained release of a cell growth factor and prevention of thrombosis using a radiation irradiation technique without using chemicals harmful to human and natural environment.

Figure 1 schematically illustrates a process for preparing heparin-AEMA-PCL (a) heparin-AAm-PCL nanofibers using radiation.
FIG. 2 shows an SEM image of heparin-acrylamide grafted PCL nanofibers to confirm the results according to heparin concentration.
Fig. 3 shows the ATR-FTIR spectra for the AEME-grafted film (a) and the fiber (b) according to the dose of radiation.
Figure 4 shows the ATR-FTIR spectra for AEME-grafted PLC nanofibers (a) and heparin-immobilized PLC nanofibers (b).
Figure 5 shows the ATR-FTIR spectra of (a) AAm-grafted film and (b) fiber for 0 (pure PCL), 0.01, 0.1, and 0.2 M core concentration.
FIG. 6 shows ATR-FTIR of heparin-acrylamide grafted PCL nanofibers at gamma ray doses of 0.5, 1.0 and 3.0 kGy.
Figure 7 shows the ATR (weight average molecular weight) of acrylamide grafted PCL nanofibers (a) - (b) and heparin-acrylamide grafted PCL nanofibers (c) - (d) at acrylamide concentrations of 0.5, -FTIR.
FIG. 8 shows changes in the water contact angle with heparin-AAM-graft-PCL nanofibers according to the irradiation dose.
Fig. 9 shows changes in graft yield of (a) fluorescein staining of AEMA-grafted film and fiber, and (b) AEMA-grafted film and (c) fiber, according to the dose of radiation. (NA: non-analysis, *: Corresponds to a p> 0.05, no-mark:
Fig. 10 shows fluorescein staining ((a) - (d)), TBO staining ((e) - (h)) and uptake amount (i) of TBO in each nanofiber scaffold. (*: PCL-related, ‡: Statistical significance of H-1AEP (p <0.05))

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiments of the present invention can be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below.

According to the present invention, by using free radicals as a result of ionization of an acrylic monomer having a polymer scaffold and an amine group by using a radiation, graphene polymerization is carried out without chemical additives such as catalyst, initiator and crosslinking agent, To provide a support for tissue engineering polymers capable of releasing osteogenic proteins, vascular endothelial growth factors, and the like.

According to the present invention, there is provided a method of manufacturing a polymer electrolyte fuel cell, comprising the steps of: supporting a polymer material in a solution in which an acrylic monomer is dissolved; Irradiating 0.1 to 100 kGy of radiation to graft the acrylic monomer to the polymer material in the form of an acrylic polymer having a dimer or higher; And introducing heparin simultaneously or subsequently with the grafting step. The present invention also provides a method for producing a heparin-introduced polymeric material.

The radiation is preferably gamma ray ( ⁶⁰ Co) or electron beam.

The acrylic monomer that can be applied to the present invention is preferably at least one monomer selected from 2-aminoethyl methacrylate (AEMA) and acrylamide (AAm).

On the other hand, the polymer material may be at least one selected from the group consisting of polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), silicones, polyurethane (PU) and polyethylene And may be, but not limited to, a polyester of polycaprolactone (PCL).

Further, the shape of the material is not particularly limited, and may be a support made of a film, a fiber, a porous nonwoven fabric, or the like.

According to one aspect of the present invention, there is provided a method of preparing a polymer material into which heparin is introduced, the method comprising: supporting a polymer material on a monomer solution of an acrylic monomer in an alcohol; Irradiating 10 to 100 kGy of radiation to graft the acrylic monomer to the polymer material in the form of an acrylic polymer of a dimer or higher; And a step of binding the polymer material grafted with the acrylic polymer to a heparin solution containing 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide, NHS (N-hydroxysuccinimide) and heparin to bind heparin .

The alcohol solvent of the acrylic monomer solution is preferably ethanol because the acrylic monomer is well soluble in ethanol but is not limited thereto and the alcohol may be at least one selected from the group consisting of other alcohol groups .

The acrylic monomer is preferably 2-aminoethyl methacrylate (AEMA).

When the amount of the acrylic monomer in the monomer solution is less than 5% by weight, the acrylic monomer may not be graft bonded to the surface of the polymer material, and when the amount of the acrylic monomer is less than 5% by weight, On the other hand, when the weight percentage of the acrylic monomer exceeds the above range, the acrylic monomers are bonded to each other rather than being grafted to the surface of the polymer material by radiation. More preferably, the monomer solution contains 10 to 20% by weight of an acrylic monomer.

The step of supporting the polymeric material on the monomer solution dissolved in the alcohol in the acrylic monomer may be carried out for, for example, 1 minute to 60 minutes, preferably 10 minutes to 30 minutes.

After the polymer material is supported on the monomer solution, the grafting step is performed by irradiating 5 to 50 kGy of radiation to graft the acrylic monomer to the polymer material in the form of an acrylic polymer having a dimer or more. When the radiation dose is less than 5 kGy, there is a problem that the acrylic monomer is not graft bonded to the surface of the polymer material. When the radiation dose is more than 50 kGy, the acrylic monomer is not grafted to the surface of the polymer material by radiation, There is a problem that a homopolymerization is formed. Preferably from more than 5 to 50, more preferably from 10 to 50, and even more preferably from 15 to 25 kGy.

Thereafter, a step of binding the polymer material grafted with the acrylic polymer onto a heparin solution containing 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide, NHS (N-hydroxysuccinimide) and heparin to bind heparin .

The heparin solution preferably contains 2 to 20 mg of heparin. When the heparin is less than 2 mg, the grafting efficiency is low due to insufficient amount of heparin binding to the acrylic monomer. , The process economy is not favorable due to the presence of residual heparin combined with an acrylic monomer.

The step of supporting heparin by binding the acrylic polymer-grafted polymer material to a heparin solution containing 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide, NHS (N-hydroxysuccinimide) For 5 to 60 minutes, and preferably for 15 to 30 minutes.

Here, the polymer material may be in the form of film or fiber, and the form thereof is not particularly limited. The fibers may be, for example, micro fibers having an average diameter of 1,000 to 2,000 nm or nanofibers having an average diameter of 300 to 800 nm.

After the step of supporting on the monomer solution and carrying out the step of binding heparin by carrying it on the heparin solution and washing the unreacted material after the step of performing the subsequent steps, washing with an appropriate washing solution composed of distilled water, MES, PBS, etc. , Followed by a subsequent drying step in the washing step.

According to another aspect of the present invention, there is provided a method of preparing a polymer material into which heparin is introduced, comprising: loading a polymer material into an aqueous solution containing an acrylic monomer, a Mohr's salt, and heparin; And irradiating 0.1 to 10 kGy radiation to graft an acrylic polymer having a heparin-bound dimer or more to the polymer material.

The aqueous solution containing the acrylic monomer, Mohr's salt and heparin preferably contains 0.5 to 10% by weight of the acrylic monomer, and when the amount of the acrylic monomer in the monomer solution is less than 0.5% by weight, the surface of the polymeric material of the acrylic monomer There is a problem in that the grafting efficiency is low. When it exceeds 10% by weight, the main reaction is carried out by forming a homopolymerization polymer between the acrylic monomers, which lowers the graft efficiency. More preferably, the monomer solution contains 0.5 to 5% by weight of an acrylic monomer.

The acrylic monomer is preferably acrylamide (AAm).

The aqueous solution preferably contains heparin in an amount of 1 to 20 mg / ml. When heparin is less than 1 mg / ml, there is a problem that the binding ratio of acrylamide to monomer is lowered on the surface of the polymer material due to low heparin concentration , And when it exceeds 20 mg / ml by weight, there is a problem that the amount of heparin remaining after binding with acrylamide increases, resulting in a large economic loss. More preferably, the aqueous solution contains 1 to 10 mg / ml of heparin.

Further, it is preferable that the aqueous solution contains 0.01 to 0.5 M of the mother salt, and when the concentration of the moor salt is low as in the case of the mother salt of less than 0.01 M, the reaction of the acrylic monomers to form the polymer increases, There is a problem that the monomer is reduced, and when it exceeds 0.5 M, there is a problem due to the toxicity of the remaining maw salt which participates in the reaction. More preferably, the aqueous solution contains 0.01 to 0.4 M of a parent salt.

The step of supporting the polymer material in an aqueous solution containing the acrylic monomer, Mohr's salt and heparin may be carried out for, for example, 5 minutes to 60 minutes, preferably 5 minutes to 50 minutes.

Wherein the grafting is a step of irradiating 0.1 to 10 kGy of radiation to graft an acrylic polymer having a heparin-bonded dimer or higher on a polymer material, wherein the grafting step is a step of irradiating the polymeric material with an acryl monomer, There is a problem in binding with heparin because it does not lift onto the surface of the material, and when it exceeds 10 kGy, the chemical structure of heparin is decomposed or deformed by gamma rays. Preferably 0.5 to 5 kGy, and more preferably 0.5 to 3 kGy.

By this step, the heparin and the acrylic monomer are grafted in a single step in the form of an acrylic polymer over a dimer of heparin immobilized on a polymeric support, wherein the polymer can be a dimer, a trimer, a tetramer, or a polymer .

The polymer material may be in the form of a film or a fiber, and the shape of the polymer material is not particularly limited. Preferably, the polymer material is in a fiber form, for example, micro fibers having an average diameter of 1000 to 1500 nm or an average diameter of 300 to 900 nm nanofiber.

After performing the grafting step, washing with an appropriate cleaning solution consisting of distilled water, MES, PBS, etc. may be further performed to clean unreacted materials, followed by drying the cleaning step Can be performed.

According to the present invention, there is provided a polymeric material prepared by the above-described method for producing a polymeric material into which heparin is introduced according to the present invention, and heparin grafted with an acrylic polymer or a grafted onto the polymeric material,

The heparin-introduced polymer material obtained by the present invention exhibits excellent hydrophilicity because the water contact angle is 60 ° or less, for example, 40 ° or less, 20 ° or less, or 10 ° or less.

Further, according to the present invention, it is possible to obtain a polymer material into which heparin is introduced, in which an acryl-based polymer grafted with heparin or the like is grafted onto a polymer material, without using harmful chemical components.

Hereinafter, the present invention will be described more specifically by way of specific examples. The following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited thereto.

Example

One. Materials and methods

1.1 material

Polycaprolactone (PCL), 2-aminoethylmethacrylate (AEMA), acrylamide (AAm), fluorescamine, boric acid, and Mohr's salt were purchased from Sigma- Purchased from Aldrich (St. Louis, Mo., USA). N, N-dimethylformamide (DMF) and sodium dodecyl sulfate (SDS) were purchased from SHOWA (Tokyo, Japan) . All other reagents and solvents used in the experiment were used without further purification.

1.2 PCL  Manufacture of films and nanofibers

(1) PCL film

To prepare the PCL film, 13 wt% PCL was dissolved in a THF solvent and dissolved in a 50 mL glass vial for 6 hours at room temperature using a magnetic stirrer. 30 ml of the dissolved PCL solution was solvant cast on a 148 cm ² glass petri dish and dried in a hood for 3 days to prepare a film.

(2) PCL nanofibers

To prepare PCL nanofibers, 13 wt% PCL was mixed with a mixed solvent of THF and DMF (7: 3, v / v) in a 30 mL glass vial and dissolved for 6 hours at room temperature using a magnetic stirrer. The dissolved PCL solution was injected into a 12 ml plastic syringe (Norm ject, Henke Sass Wolf, Germany) connected with 21 G needle (NanoNC, Korea) and injected with 10 ml of syringe pump (ESR-200RD, NanoNC, Korea) And an aluminum foil was wound around a drum collector to conduct electrospinning. The electrospinning conditions were such that a voltage of 11.3 kV was applied to the syringe needle through a DC voltage power supply (NNC-60K-2 mA, NanoNC, Korea) and spun at a release rate of 2 ml / hr for 5 hours. The rotation speed of the drum collet was 200 rpm, and the distance between the collet and the needle was set to 15 cm. The radiated PCL nanofibers were completely dried at room temperature for 48 hours to remove residual solvent. The average diameter of the nanofibers thus obtained was about 800 nm.

2. Preparation of heparin-incorporated polymeric materials using radiation

Manufacturing example  1: Fixed heparin using radiation AEMA Graft PCL  Produce

A 100 ml glass vial was used to irradiate PCL nanofibers prepared by electrospinning and AEMA, an acrylic monomer having an amine group, on the surface of the film.

1, 5, 7, and 10 wt% AEMA were dissolved in methanol and dissolved at room temperature using a magnetic stirrer. 7 ml of each of the above AEMA solutions was loaded into 8 ml vials to carry PCL films and / or nanofibers of 1x5 cm ² size. Of ⁶⁰ Co gamma rays crew on the supported PCL films and nanofibers to 10 kGy / hr dose rate a (ACEL type C-1882, Korea Atomic Energy Research Institute) at room temperature were 5, 15, 25 kGy. After irradiation, the film and / or nanofibers were transferred to a 50 ml conical tube to remove unreacted AEMA and washed with distilled water for 48 hours. The washed film was dried at room temperature for 24 hours, and the nanofibers were lyophilized for 3 days.

3-dimethylaminopropyl (EDC) was added to 0.1 M MES buffer solution (pH 5.07, 5 mg / ml) to introduce 2 mg / ml heparin into the AEMA-PCL support obtained in the above (1) -carbodiimide), NHS (N-hydroxysuccinimide) and heparin, and stirred for 12 hours. In order to remove unreacted heparin, the cells were washed with MES and PBS for 24 hours and then lyophilized for 3 days using a freeze dryer. 1 (a) shows a general schematic diagram of the present process for the production of heparin-introduced AEMA-PCL.

Manufacturing example  2: Fixed heparin using radiation AAm Graft PCL  Produce

To introduce heparin onto the surface of PCL nanofibers and / or films prepared by electrospinning, AAm, an acrylic monomer with heparin and amine groups, was placed in a 100 ml glass vial and irradiated.

First, 0.01, 0.1, and 0.2M Mohr's salt, 0.5, 1.0, 3.0% AAm and 2, 10, and 20 mg / ml heparin were added to the glass vials in the third distilled water, To prepare a mixed solution. 7 ml of the mixed solution was placed in an 8 ml glass vial to carry a 1 x 5 cm ² PCL film and / or nanofibers. A crew of ⁶⁰ Co gamma rays (ACEL type C-1882, Korea Atomic Energy Research Institute) in the supported PCL film and / or nano-fiber at room temperature to a dose 10 kGy / hr were 0.5, 1.0, 3.0 kGy. After irradiation, the film and / or nanofibers were transferred to a 50 ml conical tube to remove unreacted components and washed with distilled water for 48 hours. The washed films were dried at room temperature for 24 hours and the nanofibers were lyophilized for 3 days. The overall schematic diagram of this process for the preparation of AAm-PCL with heparin incorporated is shown in Figure 1 (b).

3. Scanning electron microscope analysis of heparin-incorporated polymer materials

According to the present invention, a scanning electron microscopic analysis was carried out to confirm the morphological structure change of heparin and acrylamide-introduced fibrous scaffold according to heparin concentration through gamma ray.

The results are shown in FIG. 2. The results are shown in FIG. 2 (a), 2 (b), and 2 (b) with heparin applied at 2 mg / ml, will be. In the PCL fiber support before heparin and acrylamide were introduced, as shown in FIG. 2, there was no significant change in the surface of the PCL fiber strands before and after the introduction of acrylamide, but it was confirmed that the fibers were loosened in the PCL fiber into which acrylamide was introduced there was. This increase in hydrophilicity due to acrylamide is expected to increase the cell adhesion and increase the possibility of application to artificial blood vessels because the cells can penetrate into the loosened fibers. As the concentration of heparin increased, it was confirmed that there was no significant difference in fiber thickness within the error range due to heparin and acrylamide introduced.

4. Attenuated total reflection Fourier-transform infrared spectroscopy (FIR) analysis.

Bruker Tensor 37, Bruker AXS, Inc., Germany) to determine the chemical properties and reactors of the heparin-AEMA-PCL and heparin-AAm-PCL surfaces. Films and nanofibers are cut to a size of 5x5 mm ² in number of injections absorbance mode (Absorbance mode) is 64 times, the wavelength range of 600 ~ 4000 cm ^-1, resolution is measured under the condition of 6 cm- ^1.

(1) heparin-introduced AEMA -g- PCL  Chemical structure analysis

1) Analysis of chemical structure according to dose of radiation

To investigate the chemical structure of grafted AEMA on the surface of PCL film and nanofibers due to the increase of radiation dose, total reflection infrared spectroscopy (ATR-FTIR) analysis was carried out. A is ^1, and 1640 cm ^-1 peak absorption increases PCL ^surface-I did not have amine peak (peak amine) of AEMA, nanofibers in the 3800 cm with increasing radiation dose, as FIG. 3, the radiation dose increases the film, as shown in It was confirmed that the amount of grafted AEMA was increased. As the surface area and radiation dose of film and nanofiber increased, the amount of AEMA introduced increased.

2) AEMA  Analysis of surface change by concentration

The AEMA-PCL nanofibers and the AEMA-PCL nanofibers were grafted with increasing concentrations of AEMA at 0, 1, 5, and 7% of radiation dose of 25 kGy and EDC / NHS chemically reacted with heparin heparin) for introducing the result of check for a heparin--AEMA PCL nanofibers by ATR-FTIR, as a 7% AEMA concentration as shown in 4 grafted PCL nanofibers 3800 cm ^{- 1} and 1640 cm ^-1 peak AEMA free amine group, heparin-AEMA-PCL, heparin-introduced heparin carboxyl group, which was not detected in AEMA-PCL nanofibers, The amount of heparin introduced was increased.

(2) heparin-introduced AAm  -g- PCL  Chemical structure analysis

1) Analysis of chemical structure according to dose of radiation

(ATR-FTIR) was conducted to confirm the chemical properties and chemical functional groups of the acrylamide-introduced PCL film and the fiber support. In Figure acrylamide is in the 3600-3400 cm ^-1 in the introduced PCL NH stretching peak (peak stretching), amide at 1660 cm ^-1 C = O peak band, 1610 cm ^-1, as shown in 5 amide Ⅰ bend peak Newly created. As the amount of moor salt increased, the intensity of the peak increased, indicating that a larger amount of acrylamide was introduced. This is probably because the parent salt inhibited homopolymerization of acrylamide and induced the reaction with PCL.

On the other hand, surface analysis was performed by ATR-FTIR measurement of the heparin-introduced films and fibers. As a result of measurement of ATR-FTIR according to dose, the absorbance at 3700-3000 cm ^-1 was decreased by decreasing amine group as irradiation dose increased as shown in FIG. 6, It was confirmed that the absorbance increases at 1700-1500 cm ^-1 due to the formation of groups.

2) AAm  Analysis of surface change by concentration

ATR-FTIR measurement of heparin-AAm-PCL fiber was performed according to the concentration of acrylamide. As shown in FIG. 7, as the amount of AAm increased, the absorbance of peaks at 3700-3000 cm -1 and 1700-1550 cm -1 increased, indicating that the graft amount of heparin and acrylamide was AAm And it was confirmed that it increased with concentration.

5. Contact angle  (Water contact angle) analysis

The contact angle was measured using a contact angle meter (Phoenix-300m, surface electrooptics Ltd, Korea) to confirm the degree of hydrophilization of heparin-AEMA-PCL and heparin-AAm-PCL surface. After the double-sided tape was attached to the slide glass, the film and the nanofibers were cut to a size of 1 x 1 cm ² . After placing the distilled water in the center of the prepared film and nanofibers so that the distilled water could be dropped, purified distilled water was injected into the syringe and dropped to the surface of the nanofiber by 10 ul. Ten photographs were taken per second for 10 seconds, and contact angles were measured through the obtained photographs.

The contact angle analysis was carried out to confirm the degree of hydrophilization of the PCL film and the fiber support into which the acrylamide as the hydrophilic monomer was introduced. PCL without acrylamide was expected to not absorb water because it had a hydrophobic surface, and when acrylamide was introduced, it was expected that water would be absorbed with a low contact angle by the hydrophilic acrylamide. However, as shown in FIG. 15 (a), the contact angle of PCL film was 84.434, 87.063, 93.256, and 93.558 ° as the concentration of mower salt increased to 0, 0.01, 0.1 and 0.2 M, respectively. As the concentration of mower leaves increased, the contact angle increased and all the films retained their shape without absorbing the water even over time, which is considered to be due to the introduction of amines, the surface characteristics of the film and the two dimensional structure. On the other hand, in the fiber, as shown in FIG. 15 (b), it was confirmed that as the amount of maw salt increases, the contact angle increases and the hydrophilicity increases.

The contact angle of the PCL fiber support without gamma irradiation was 86.277 ° and the hydrophilic surface was not absorbed over time, whereas the PCL fiber support with acrylamide had 46.956, 44.680, 39.771 °, And a hydrophilic surface over which water was completely absorbed over time. The contact angle on the surface of AEMA using gamma ray was measured and it was confirmed that water was absorbed more slowly than acrylamide. Water is absorbed rapidly from acrylamide because unlike AEMA, acrylamide is introduced by forming a polyacrylamide hydrogel layer on the surface. Next, water content was measured to confirm the introduction of acrylamide.

To investigate the effect of radiation doses on heparin-AAm-g-PCL nanofibers, experiments were conducted according to the dose of radiation. As shown in FIG. 8, it was confirmed that as the irradiation dose increases, the amount of acrylamide introduced increases. In addition, the slope of each contact angle was calculated. As a result, the slope was decreased as the dose increased, and the contact angle decreased with increasing AAm concentration.

6. Graft rate  (Graft yield) measurement.

Yield measurement was performed to confirm the amount of AEMA grafted by radiation. The dried film and nanofibers were weighed before irradiation, and the dried film and nanofibers were weighed after irradiation. Using the equation (1), the yields from the weight before irradiation (w1) and the weight after irradiation (w2) were measured (Journal of radiation industry, 2008, 2 (1), 1-7).

(1)Yield (%) =

(One)

Fluorescamine staining was performed by reacting with amine group to identify the amine group at the end of AEMA introduced on the PCL surface to which the AEMA was introduced with increasing radiation dose. As shown in FIG. 9, even when the dose of radiation is increased, the modified amount of AEMA is similar to that of PCL film as a control group because the surface area is narrow. On the other hand, it was confirmed that the graft is increased as the radiation dose increases because the fluorescence of green fluorescence increases as the dose of radiation increases.

7. Fluorescein  Dyeing and Toluidine blue  O ( TBO ) Dyeing and quantification

(1) Fluorescein staining was carried out to identify the amine groups of AEMA and AAm introduced on the surface of AEMA-PCL and AAm-PCL. The film and nanofibers were prepared by using a biopsy punch of 3 mm ² , then immersed in ethanol for 1 minute, and immersed in distilled water for 5 minutes to prepare a 2 ml EP tube. Each of the films and nanofibers was immersed in 200 μl of borate buffer (0.2 M boric acid, pH 9.2), and then 50 μl of a solution of fluorescein prepared by dissolving it in acetone at a concentration of 4 mg / ml in DMSO was added (Biomacromolecules, 2008, 9 , 1772-1781). The mixture was stirred for 1 minute using a voltex and placed on a slide glass and observed at λex 392 nm and λem 480 nm using a Leica Microsystems GmBh (DM1400B, Germany). The heparin-AEMA-PCL and heparin-AAm-PCL were stained and quantified using TBO to identify the carboxyl groups of heparin introduced on the surface. Add 500 μl TBO solution (60 mg NaCl, and 12 mg toluidine blue O chloride in 0.01 M HCl 30 ml) to the 24-well plate and mix with heparin-AEMA-BC for 6 hours or more. And washed until it did not come out. The washed samples were dissolved in a mixture of 0.1 M NaOH and ethanol at 1: 4 (v / v) and then measured at 530 nm using a UV-vis spectrometer (PowerWave XS, Biotek, USA).

 (2) To analyze the staining of AEMA-g-PCL nanofibers with heparin according to AEMA concentration, AEMA was reacted with an amine group to identify the amine group at the end of AEMA introduced on the PCL surface into which the AEMA was introduced. Lt; RTI ID = 0.0 &gt; fluorescein &lt; / RTI &gt; As shown in FIG. 10, green fluorescence does not appear in PCL, but green fluorescence appears in AEMA-PCL. Fluorescein staining changes depending on irradiation doses can be seen in many differences.

(3) To confirm the heparin-mediated carboxyl group on the AEMA-PCL surface introduced with heparin by the EDC / NHS reaction, the cells were stained with polyurilidine blue O (TBO). As shown in FIG. 10, the amount of heparin bound can be determined according to the degree of blue color of TBO. Since PCL is hydrophobic, TBO is stained and can not be stained after washing. Heparin-AEMA-PCL showed more TBO than PCL because of heparin carboxyl group. In addition, as shown in FIG. 10, TBO was quantified as 4.62 ± 1.64 mM / mg and 7.35 ± 0.32 mM / mg at 5% and 7% AEMA, respectively. I could confirm.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, It will be obvious to those of ordinary skill in the art.

Claims (16)

Carrying a polymeric material in an aqueous solution comprising an acrylic monomer, Mohr's salt and heparin; And
A step of irradiating 0.1 to 10 kGy of radiation to graft an acrylic polymer having a heparin-bound dimer or more to the polymer material
Wherein the heparin is introduced into the polymer material.
The method for producing a polymeric material according to claim 1, wherein the acrylic monomer is at least one monomer selected from 2-aminoethyl methacrylate (AEMA) and acrylamide (AAm).
The method of claim 1, wherein the polymeric material is selected from the group consisting of polycaprolactone (PCL), polyester of polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), silicones PU) and polyethylene (PE), wherein the heparin is introduced.
delete delete delete delete delete delete The method for producing a polymeric material according to claim 1, wherein the aqueous solution contains 0.5 to 10% by weight of an acrylic monomer.
The method of claim 1, wherein the aqueous solution contains heparin in an amount of 1 to 20 mg / ml.
The method of producing a polymer material according to claim 1, wherein the aqueous solution contains 0.01 to 0.5 M of a parent salt.
2. The method of claim 1, wherein the grafting is performed by irradiating with radiation of 0.5 to 5 kGy.
The method for producing a polymer material according to claim 1, wherein the polymer material is in the form of a film or a fiber.
A polymeric material produced by any one of claims 1 to 3 and 10 to 14, wherein an acryl-based polymer having a heparin-bound dimer or higher is grafted onto the polymeric material, The introduced polymeric material. delete

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