Method for producing porous substrate comprising bioabsorbable polymer that contains heparin, porous substrate comprising bioabsorbable polymer that contains heparin, and artificial blood vessel

Abstract

The present invention aims to provide a method for producing a porous substrate containing a bioabsorbable polymer and heparin in a simple manner without use of a surfactant, a porous substrate containing a bioabsorbable polymer and heparin, and an artificial blood vessel. The present invention provides a method for producing a porous substrate containing a bioabsorbable polymer and heparin, including: a solution preparing step of preparing a heparin-bioabsorbable polymer solution having heparin uniformly dispersed therein and a bioabsorbable polymer dissolved therein, using the bioabsorbable polymer, the heparin, a solvent 1 that is a poor solvent having a lower solvency for the bioabsorbable polymer, a solvent 2 that is a good solvent having a higher solvency for the bioabsorbable polymer and is incompatible with the solvent 1, and a common solvent 3 compatible with the solvent 1 and the solvent 2; a precipitating step of cooling the heparin-bioabsorbable polymer solution to precipitate a porous body containing the bioabsorbable polymer and the heparin; and a freeze-drying step of freeze-drying the porous body containing the bioabsorbable polymer and the heparin to provide a porous substrate containing the heparin.

Claims

1. A method for producing a porous substrate containing a bioabsorbable polymer and heparin, comprising: a solution preparing step of preparing a heparin-bioabsorbable polymer solution having heparin uniformly dispersed therein and a bioabsorbable polymer dissolved therein, using the bioabsorbable polymer, the heparin, a solvent 1 that is a poor solvent having a lower solvency for the bioabsorbable polymer, a solvent 2 that is a good solvent having a higher solvency for the bioabsorbable polymer and is incompatible with the solvent 1, and a common solvent 3 compatible with the solvent 1 and the solvent 2; a precipitating step of cooling the heparin-bioabsorbable polymer solution to precipitate a porous body containing the bioabsorbable polymer and the heparin; and a freeze-drying step of freeze-drying the porous body containing the bioabsorbable polymer and the heparin to provide a porous substrate containing the heparin.

2. The method for producing a porous substrate containing heparin according to claim 1, wherein two or more common solvents 3 are used in combination, and a pore size of the resulting porous body is controlled by adjusting a mixing ratio between the two or more common solvents 3.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows cross-sectional electron micrographs of porous substrates obtained in Example 1 and Comparative Example 1.

(2) FIG. 2 shows a photograph of the porous substrates obtained in Example 1 and Comparative Example 1 after toluidine blue staining.

(3) FIG. 3 shows IR spectra of the porous substrates obtained in Example 1 and Comparative Example 1.

DESCRIPTION OF EMBODIMENTS

(4) The embodiments of the present invention are described in more detail with reference to examples. The present invention however is not limited to these examples.

Example 1

(5) At a room temperature of 25° C., 0.25 g of a L-lactide-ε-caprolactone copolymer (molar ratio: 50:50) was mixed with a mixed solution containing: 0.3 mL of water having heparin (Wako Pure Chemical Industries, Ltd., JIS guaranteed reagent) dissolved therein at a concentration of 7200 units/mL as the solvent 1; 2.0 mL of methyl ethyl ketone as the solvent 2; and 1.0 mL of acetone as the common solvent 3. Thus, a non-uniform solution not dissolving the L-lactide-ε-caprolactone copolymer was obtained. The heparin in the non-uniform solution did not precipitate and formed stable micelles.

(6) Subsequently, the obtained non-uniform solution was put in a glass tube having a diameter of 3.3 mm and heated at 60° C. to give a solution containing the heparin uniformly dispersed therein and the L-lactide-ε-caprolactone copolymer dissolved therein.

(7) The obtained uniform solution was then cooled to 4° C. or −24° C. in a freezer to precipitate a porous body containing the L-lactide-ε-caprolactone copolymer and heparin.

(8) The obtained porous body was immersed in an ethanol bath (50 mL) at 4° C. or −24° C. for 12 hours, and then immersed in a water bath (50 mL) at 25° C. for 12 hours for washing.

(9) Thereafter, the porous body was freeze-dried at −40° C. to give a cylindrical porous substrate having a diameter of 3.0 mm and a height of 15 mm.

Example 2

(10) A porous substrate was produced as in Example 1 except that a solvent mixture of 0.5 mL of acetone (common solvent 3-1) and 0.5 mL of ethanol (common solvent 3-2) was used as the common solvent 3.

(11) The heparin in the obtained non-uniform solution did not precipitate and formed stable micelles.

Example 3

(12) At a room temperature of 25° C., 0.25 g of an L-lactide-ε-caprolactone copolymer (molar ratio: 50:50) was mixed with a mixed solution containing: 0.2 mL of water having heparin (Wako Pure Chemical Industries, Ltd., JIS guaranteed reagent) dissolved therein at a concentration of 7200 units/mL as the solvent 1; 2.5 mL of methyl ethyl ketone as the solvent 2; and 0.8 mL of acetone and 0.2 mL of ethanol as the common solvent 3. Thus, a non-uniform solution not dissolving the L-lactide-ε-caprolactone copolymer was obtained. The heparin in the obtained non-uniform solution did not precipitate and formed stable micelles.

(13) Subsequently, the obtained non-uniform solution was heated at 60° C. to give a uniform solution containing the heparin uniformly dispersed therein and the L-lactide-ε-caprolactone copolymer dissolved therein.

(14) A fluorine-coated stainless steel rod-shaped body having a diameter of 0.6 mm was placed in a glass tube having an inner diameter of 1.1 mm. The uniform solution was poured into the gap between the rod-shaped body and the glass tube. The uniform solution in this state was cooled to −30° C. in a freezer to precipitate a porous body containing the L-lactide-ε-caprolactone copolymer and heparin around the rod-shaped body. The obtained porous body was immersed in an ethanol bath (50 mL) at −30° C. for 12 hours, and then immersed in a water bath (50 mL) at 25° C. for 12 hours for washing.

(15) Thereafter, the porous body was freeze-dried at −40° C. to give a tubular porous body.

(16) Polyglycolide and polylactide were separately dissolved into hexafluoroisopropanol to prepare a hexafluoroisopropanol solution having a polyglycolide concentration of 10% by weight and a hexafluoroisopropanol solution having a polylactide concentration of 10% by weight.

(17) The rod-shaped body with the tubular porous body therearound was used as a collector electrode. The hexafluoroisopropanol solutions were discharged onto the surface of the rod-shaped body using an electrospinning device. At this time, the hexafluoroisopropanol solutions prepared above were charged into two different nozzles, and discharged while rotating the rod-shaped body and reciprocating the nozzles multiple times. Thus, an ultrafine fiber nonwoven fabric layer was formed.

(18) The electrospinning was performed under the conditions of a voltage of −20 kV and a nozzle size of 23 G.

(19) Finally, the rod-shaped body was pulled out to give a tubular artificial blood vessel having an outer diameter of about 1,090 μm and an inner diameter of about 610 μm.

Comparative Example 1

(20) A porous substrate was obtained as in Example 1 except that water without heparin was used as the solvent 1.

(21) (Evaluation)

(22) The porous substrates obtained in the examples and the comparative example were evaluated by the following methods.

(23) (1) Identification of Heparin with Electron Microscope

(24) FIG. 1 shows electron micrographs obtained by cutting each of the cylindrical porous substrates obtained in Example 1 and Comparative Example 1 and photographing an area near the center of the cross section at a magnification of 10,000 times.

(25) In the porous substrate obtained in Example 1, as shown in FIG. 1(a), particles that appear to be heparin were adhered to the surface of the porous body containing the L-lactide-ε-caprolactone copolymer. Such particles were not observed in the porous substrate obtained in Comparative Example 1 shown in FIG. 1(b).

(26) (2) Identification of Heparin by Toluidine Blue Staining

(27) FIG. 2 shows a photograph of the porous substrates obtained in Example 1 and Comparative Example 1, each stained with toluidine blue.

(28) As shown in FIG. 2, the porous substrate obtained in Comparative Example 1 was not stained at all (on the left in the photograph), while the porous substrate obtained in Example 1 was stained dark bluish purple as a whole, indicating the presence of heparin (on the right in the photograph).

(29) (3) Identification by Infrared Spectroscopy

(30) FIG. 3 shows IR spectra of the porous substrates obtained in Example 1 and Comparative Example 1.

(31) As shown in FIG. 3, the IR spectrum of the porous substrate obtained in Example 1 has a peak at 1259 cm.sup.−1 (peak surrounded by a dotted line) which appears to be derived from heparin. This peak is not found in the IR spectrum of the porous substrate obtained in Comparative Example 1.

INDUSTRIAL APPLICABILITY

(32) The present invention can provide a method for producing a porous substrate containing a bioabsorbable polymer and heparin in a simple manner without use of a surfactant, a porous substrate containing a bioabsorbable polymer and heparin, and an artificial blood vessel.