VINYL ALCOHOL-AMINO ACID ESTER COPOLYMER

Abstract

The present invention provides a vinyl alcohol-amino acid ester copolymer that is excellent in properties such as cell culture properties, biocompatibility, antibacterial properties, and solubility in organic solvents, and is applicable to the medical field, the biomaterial field, and the agricultural field (medical and life science fields). Provided is a vinyl alcohol-amino acid ester copolymer including a unit represented by the following formula (1) and a unit represented by the following formula (2):

##STR00001##

where, in the formula (2), R.sup.1 represents a hydrocarbon group that may optionally have a substituent, R.sup.2 and R.sup.3 each independently represent a hydrogen atom or a hydrocarbon group, and R.sup.1 and R.sup.2, or R.sup.2 and R.sup.3, may optionally be bonded together to form a cyclic structure.

Claims

1. A vinyl alcohol-amino acid ester copolymer comprising a unit represented by the following formula (1) and a unit represented by the following formula (2): ##STR00006## where, in the formula (2), R.sup.1 represents a hydrocarbon group that may optionally have a substituent, R.sup.2 and R.sup.3 each independently represent a hydrogen atom or a hydrocarbon group, and R.sup.1 and R.sup.2, or R.sup.2 and R.sup.3, may optionally be bonded together to form a cyclic structure.

2. The vinyl alcohol-amino acid ester copolymer according to claim 1, wherein the content of the unit represented by the formula (1) is 0.1 to 99.5 mol %.

3. The vinyl alcohol-amino acid ester copolymer according to claim 1, wherein the content of the unit represented by the formula (2) is 0.1 to 99.5 mol %.

4. The vinyl alcohol-amino acid ester copolymer according to claim 1, further comprising a unit represented by the following formula (3): ##STR00007## wherein R.sup.4 represents a hydrogen atom or a C1-C20 alkyl group.

5. The vinyl alcohol-amino acid ester copolymer according to claim 4, wherein the content of the unit represented by the formula (3) is 0.5 to 90 mol %.

6. The vinyl alcohol-amino acid ester copolymer according to claim 1, wherein, in the unit represented by the formula (2), R.sup.1 represents divalent hydrocarbon group that may optionally have a substituent, and R.sup.2 and R.sup.3 each represent a hydrogen atom or an alkyl group.

Description

DESCRIPTION OF EMBODIMENTS

[0088] The present invention is further described in detail below with reference to examples. The present invention should not be limited to these examples.

Example 1

[0089] One part by weight of a raw material PVA (PVA-1, degree of saponification: 98.5, degree of polymerization: 300) was added to 49 parts by weight of DMSO (concentration: 2% by weight) and dissolved therein.

[0090] Subsequently, 4.4 parts by weight of L-tyrosine methyl and 6 parts by weight of dilithium tetra-t-butylzincate (TBZL, solvent: tetrahydrofuran, concentration: 13% by weight) were added, and the mixture was stirred at 30° C. for 6 hours to perform transesterification.

[0091] Thereafter, reprecipitation in tetrahydrofuran was performed, followed by washing with tetrahydrofuran, whereby a product (vinyl alcohol-amino acid ester copolymer) was obtained.

Example 2

[0092] A product (vinyl alcohol-amino acid ester copolymer) was obtained as in Example 1 except that the stirring time in the transesterification was changed from “6 hours” to “24 hours”.

Example 3

[0093] A product (vinyl alcohol-amino acid ester copolymer) was obtained as in Example 1 except that the amount of L-tyrosine methyl added was changed from “4.4 parts by weight” to “8.9 parts by weight”.

Example 4

[0094] A product (vinyl alcohol-amino acid ester copolymer) was obtained as in Example 1 except that the amount of L-tyrosine methyl added was changed from “4.4 parts by weight” to “17.7 parts by weight”.

Example 5

[0095] A product (vinyl alcohol-amino acid ester copolymer) was obtained as in Example 4 except that the stirring time in the transesterification was changed from “6 hours” to “24 hours”, and reprecipitation and washing were performed with water.

Example 6

[0096] A product (vinyl alcohol-amino acid ester copolymer) was obtained as in Example 5 except that the raw material PVA (PVA-1, degree of saponification 98.5, degree of polymerization 300) was replaced with a raw material PVA

[0097] (PVA-3, degree of saponification 98.5, degree of polymerization 600).

Example 7

[0098] A product (vinyl alcohol-amino acid ester copolymer) was obtained as in Example 5 except that the raw material OVA (PVA-1, degree of saponification 98.5, degree of polymerization 300) was replaced with a raw material OVA (PVA-4, degree of saponification 98.5, degree of polymerization 1,000).

Example 8

[0099] One part by weight of a raw material poly(vinyl butyral) (PVB, vinyl alcohol unit content: 34 mol %, acetal unit content: 65 mol %, vinyl acetate unit content: 1 mol %, degree of polymerization: 1,700) was added to 19 parts by weight of DMSO (concentration: 5% by weight) and dissolved therein. Subsequently, 2.5 parts by weight of L-tyrosine methyl and 0.25 parts by weight of dilithium tetra-t-butylzincate (TBZL, concentration: 13% by weight) were added, and the mixture was stirred at 30° C. for 6 hours to perform transesterification.

[0100] Thereafter, reprecipitation in water was performed, followed by washing with hot water, whereby a product (vinyl butyral-amino acid ester copolymer) was obtained.

Example 9

[0101] A. product (vinyl alcohol-amino acid. ester copolymer) was obtained as in Example 1 except that the L-tyrosine methyl was replaced with 4-CEP (ethyl 4-piperidinecarboxylate), the amount of TBZL (concentration: 13% by weight) added was changed from “6 parts by weight” to “10 parts by weight”, and the stirring time was changed from “6 hours” to “24 hours”.

Example 10

[0102] A product (vinyl alcohol-amino acid ester copolymer) was obtained as in Example 9 except that the amount of 4-CEP (ethyl 4-piperidinecarboxylate) added was changed from “4.4 parts by weight” to “17.7 parts by weight”.

Example 11

[0103] A product (vinyl alcohol-amino acid ester copolymer) was obtained as in Example 10 except that the raw material PVA (PVA-1, degree of saponification: 98.5, degree of polymerization: 300) was replaced with a raw material PVA (PVA-3, degree of saponification: 98.5, degree of polymerization: 600).

Example 12

[0104] A product (vinyl alcohol-amino acid ester copolymer) was obtained as in Example 10 except that the raw material EVA (PVA-1, degree of saponification: 98.5, degree of polymerization: 300) was replaced with a raw material PVA (PVA-4, degree of saponification: 98.5, degree of polymerization: 1,000).

Example 13

[0105] A product (vinyl alcohol-amino acid ester copolymer) was obtained as in Example 8 except that the L-tyrosine methyl was replaced with 4-CEP (ethyl 4-piperidinecarboxylate).

Reference Example

[0106] A product (vinyl alcohol-amino acid ester copolymer) was obtained as in Example 1 except that the L-tyrosine methyl was replaced with 2-CEP (ethyl 2-piperidinecarboxylate), the amount of TBZI, (concentration: 13% by weight) added was changed from “6 parts by weight” to “10 parts by weight”, and the stirring time was changed from “6 hours” to “24 hours”.

Comparative Example 1

[0107] A vinyl alcohol-vinyl acetate copolymer (PVA-1) having a degree of saponification of 98.5 and a degree of polymerization of 300 was used.

Comparative Example 2

[0108] A vinyl alcohol-vinyl acetate copolymer (PVA-2) having a degree of saponification of 88.0 and a degree of polymerization of 600 was used.

Comparative Example 3

[0109] A vinyl alcohol-vinyl amine copolymer (amine-modified PVA-1, vinyl amine unit content: 12 mol %) having a degree of saponification of 99.5 and a degree of polymerization of 1,000 was used.

Comparative Example 4

[0110] A vinyl alcohol-vinyl amine copolymer (amine-modified PVA-2, vinyl amine unit content: 6 mol %) having a degree of saponification of 99.5 and a degree of polymerization of 500 was used.

(Evaluation)

[0111] The products obtained above were evaluated by the following methods. Table 1 shows the results.

(1) .sup.1H-NMR measurement

[0112] The obtained product was subjected to .sup.1H-NMR measurement to measure the component ratio (vinyl alcohol unit, amino acid ester unit, acetal unit, vinyl acetate unit, and vinyl amine unit) of the product. .sup.1H-NMR measurement was performed at 60° C. using JEOL JNM-ECX500 and a DMSO-d.sub.6 solution (1 wt/vol %).

(2) Evaluation of Cell Culture Properties

[0113] The obtained product was dissolved in DMSO and then dried to apply the product to the bottom of a 96-well plate. Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS) was used as medium. A cell line for mouse fibroblasts (NIH/3T3 cells) was seeded at 1 ×10.sup.4 cells per well and incubated in a 5% CO.sub.2/37° C. incubator. Thereafter, the initial adhesion on day 1 of culture, and the proliferation over time and the adhesion maintenance on day 4 of culture were evaluated according to the following criteria.

[0114] The number of living cells after culture was determined using a disposable hemocytometer (produced by WakenBtech Co., Ltd). The ratio of the number of living cells after culture to the number of seeded cells was calculated. The cell culture properties were evaluated in accordance with the following criteria based on the obtained ratio of the number of living cells.

Initial Adhesion (day 1)

[0115] ○○○ (Excellent): The ratio of the number of living cells after culture to the number of seeded cells was 1.5 or higher.
○○ (Very good): The ratio of the number of living cells after culture to the number of seeded cells was 1.0 or higher and lower than 1.5.
○ (Good): The ratio of the number of living cells after culture to the number of seeded cells was 0.5 or higher and lower than 1.0.
Δ (Fair): The ratio of the number of living cells after culture to the number of seeded cells was 0.2 or higher and lower than 0.5.
x (Poor): The ratio of the number of living cells after culture to the number of seeded cells was lower than 0.2.

Proliferation Over Time and Adhesion Maintenance (day 4)

[0116] ○○○ (Excellent): The ratio of the number of living cells after culture to the number of seeded cells was 3.0 or higher.
○○ (Very good): The ratio of the number of living cells after culture to the number of seeded cells was 2.5 or higher and lower than 3.0.
○ (Good): The ratio of the number of living cells after culture to the number of seeded cells was 2.0 or higher and lower than 2.5.
Δ (Fair): The ratio of the number of living cells after culture to the number of seeded cells was 1.0 or higher and lower than 2.0.
x (Poor):The ratio of the number of living cells after culture to the number of seeded cells was lower than 1.0.

(3) Biocompatibility Evaluation

[0117] The biocompatibility was evaluated by performing ATP assay one day and six days after culture in the above evaluation of cell culture properties. The medium was removed from the wells after culture and washed with a phosphate buffer. An ATP extraction reagent (Intracellular ATP assay kit ver. 2, produced by TOYO B-Net Co., Ltd.) in an amount of 100 μl was added, pipetted five times, and left to stand for five minutes at room temperature. ATP was then extracted. A 1,010-μL sample was taken from the ATP extraction. solution, and 100 μL of an ATP luminescent reagent (the same kit) was added and stirred. The amount of luminescence of the sample was measured using Mithras LB940 (produced by Berthold).

[0118] Cytotoxicity was calculated by the following equation based on the measured amount of luminescence, and evaluated in accordance with the following criteria.

Cytotoxicity=(Amount of luminescence at six days after culture)/(Amount of luminescence at one day after culture)

x 100
○ (Good): A cytotoxicity of lower than 30%
x (poor): A cytotoxicity of 30% or higher

(4) Thrombus Adhesion

[0119] The obtained product was dissolved in DMSO, and the solution was sprayed to the inner surface of a PET test tube (inner diameter 10 mm×length 100 mm) (control) and dried. Into the PET tube was added 1 mL of human blood.

[0120] The mouth of the tube was sealed, and the tube was inverted to wet the entire inner surface of the PET tube with blood.

[0121] The tube was left to stand upright at room temperature for about 4 hours. After coagulation of the stationary blood was confirmed, the blood was centrifuged in a centrifuge (1500 G×5 minutes), and the degree of adhesion of blood (portion coated with a pink or pale red ultrathin film) on the inner surface of the headspace of the PET tube was observed.

[0122] Based on comprehensive assessment of the intensity of redness and the adhesion area, the thrombus adhesion was relatively evaluated as “○ (Good)”, “Δ (Fair)”, or “x (Poor)”, in order from lower to higher adhesion.

(5) Solubility in Organic Solvents

[0123] Toluene (70° C.) was added to the products obtained in Examples 1 to 7 and 9 to 12 and Comparative Examples 1 to 4 (PVA-type products), and butanol (room. temperature) was added to the products obtained in Examples 8 and 13 (PVB-type products). The concentration of each product was adjusted to 1.0% by mass. Thereafter, the solutions were stirred for 6 hours at 500 rpm. The solubility was evaluated in accordance with the following criteria based on the content of undissolved matter.

○(Good): The content of undissolved matter was less than 20% by mass.
x (Poor): The content of undissolved matter was 20% by mass or more.

TABLE-US-00001 TABLE 1 Exam- Exam- Exam- Exam- Exam- Exam- Exam- Exam- Exam- ple 1 ple 2 ple 3 ple 4 ple 5 ple 6 ple 7 ple 8 ple 9 Raw material PVA-1 1 1 1 1 1 — — — 1 (parts by PVA-2 — — — — — — — — — weight) PVA-3 — — — — — 1 — — — PVA-4 — — — — — — 1 — — PVB — — — — — — — 1 — Amine-modified — — — — — — — — — PVA-1 Amine-modified — — — — — — — — — PVA-2 DMSO 49 49 49 49 49 49 49 19 49 L-tyrosine methyl 4.4 4.4 8.9 17.7 17.7 17.7 17.7 2.5 — 4-CEP (ethyl 4- — — — — — — — — 4.4 piperidinecarboxylate) TB2L (13% by weight/THF) 6 6 6 6 6 6 6 0.25 10 Production Reaction temperature (° C.) 30 30 30 30 30 30 30 30 30 process Reaction time (hours) 6 24 6 6 24 24 24 6 24 Unit Vinyl alcohol unit 89.3 76.5 80.5 77.5 63.5 70.5 79.5 32 47.5 content Amino acid ester unit 9.2 22 18 21 35 28 19 2 51 (mol %) Acetat unit 0 0 0 0 0 0 0 65 0 Vinyl acetate unit 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1 1.5 Vinyl amine unit 0 0 0 0 0 0 0 0 0 Evaluation Cell culture Initial adhesion ◯◯ ◯◯ ◯◯ ◯◯ ◯ ◯◯ ◯◯ ◯◯◯ ◯◯◯ properties (day 1) Proliferation over ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯◯◯ ◯◯◯ time and adhesion maintenance (day 4) Biocompatability O ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ Thrombus adhesion Δ Δ Δ Δ ◯ ◯ Δ ◯ ◯ Solubility Toluene ◯ ◯ ◯ ◯ ◯ ◯ ◯ — ◯ organic (70° C.) solvents Butanol — — — — — — — — — Compar- Compar- Compar- Compar- ative ative ative ative Exam- Exam- Exam- Exam- Exam- Exam- Exam- Exam- ple 10 ple 11 ple 12 ple 13 ple 1 ple 2 ple 3 ple 4 Raw material PVA-1 1 — — — 1 — — — (parts by PVA-2 — — — — — 1 — — weight) PVA-3 — 1 — — — — — — PVA-4 — — 1 — — — — — PVB — — — 1 — — — — Amine-modified — — — — — — 1 — PVA-1 Amine-modified — — — — — — — 1 PVA-2 DMSO 49 49 49 19 — — — — L-tyrosine methyl — — — — — — — — 4-CEP (ethyl 4- 17.7 17.7 17.7 2.5 — — — — piperidinecarboxylate) TB2L (13% by weight/THF) 10 10 10 0.25 — — — — Production Reaction temperature (° C.) 30 30 30 30 — — — — process Reaction time (hours) 24 24 24 6 — — — — Unit Vinyl alcohol unit 36.5 43.5 50.5 31 98.5 86 87.5 93.5 content Amino acid ester unit 62 55 48 3 0 0 0 0 (mol %) Acetat unit 0 0 0 65 0 0 0 0 Vinyl acetate unit 1.5 1.5 1.5 1 1.5 12 0.5 0.5 Vinyl amine unit 0 0 0 0 0 0 12 6 Evaluation Cell culture Initial adhesion ◯◯◯ ◯◯◯ ◯◯◯ ◯◯◯ X Δ X X properties (day 1) Proliferation over ◯◯◯ ◯◯◯ ◯◯◯ ◯◯ X X X X time and adhesion maintenance (day 4) Biocompatability ◯ ◯ ◯ ◯ X X X X Thrombus adhesion ◯ ◯ ◯ ◯ X X X X Solubility Toluene ◯ ◯ ◯ — ◯ ◯ ◯ — organic (70° C.) solvents Butanol — — — ◯ — — — ◯

INDUSTRIAL APPLICABILITY

[0124] The present invention can provide a vinyl alcohol-amino acid ester copolymer that is excellent in properties such as cell culture properties, biocompatibility, antibacterial properties, and solubility in organic solvents, and is applicable to the medical field, the biomaterial field, and the agricultural field (medical and life science fields).