Phosphazene-based polymer for tissue adhesion, a method for preparing the same, and use thereof

Abstract

The present invention relates to a phosphazene-based polymer comprising an amino acid ester, polyethylene glycol, a group comprising a functional group, and a catechol group linked directly or by a linker to a part of or an entire functional group, in a predetermined ratio. In addition, the present invention relates to a preparation method thereof, and a tissue-adhesive composition comprising the same as an active ingredient.

Claims

1. A phosphazene-based polymer comprising a catechol group, wherein, on a phosphorous atom of a polyphosphazene backbone represented by the following Formula 1, a first moiety of an amino acid ester represented by the following Formula 2; a second moiety of polyethylene glycol represented by the following Formula 3; a third moiety comprising a functional group; and a fourth moiety comprising a catechol group linked directly or by a linker to a part of or an entire functional group of the third moiety: ##STR00003## wherein, the first moiety, the second moiety, the third moiety, and the fourth moiety are present in a molar ratio of a:b:c:d, respectively; wherein a is 55 mol % to 77 mol %; b is 5 mol % to 30 mol %; a sum of c and d is 10 mol % to 20 mol %; and the c:d ratio is in a range of 1:0.1 to 1:1; and wherein, in Formulas 1, 2, and 3, R.sub.1 is C.sub.1-6 alkyl, C.sub.1-6 alkenyl, or C.sub.6-10 aryl-C.sub.1-6 alkyl; R.sub.2 is hydrogen, methyl, isopropyl, 1-methylpropyl, 2-methylpropyl, thiomethyl, methylthioethyl, benzyl, hydroxylbenzyl, or 2-indolylmethyl; R.sub.3 is C.sub.1-6 alkyl; n is an integer of 3 to 100,000; and p is an integer of 1 to 20.

2. The phosphazene-based polymer of claim 1, wherein the functional group is a hydroxy group or a carboxyl group.

3. The phosphazene-based polymer of claim 1, wherein R.sub.1 is methyl, ethyl, propyl, butyl, benzyl, or 2-propenyl; and R.sub.3 is methyl.

4. The phosphazene-based polymer of claim 1, further comprising a fifth moiety, in which at least one functional substance selected from the group consisting of a substance capable of regulating decomposition rate of a polymer, a substituent comprising an ionic group capable of regulating decomposition rate, a substituent capable of cross-linking, an additional compound capable of inducing tissue adhesion, a physiologically active substance, and a composite material formed by linear connection of two or more substances among them is linked directly or by a linker to a part of or an entire functional group of the third moiety.

5. The phosphazene-based polymer of claim 1, wherein the phosphazene-based polymer comprising a catechol group has a weight average molecular weight of 15,000 to 37,000, and is represented by the formula of poly[isoleucine ethyl ester].sub.a(mino methoxypolyethylene glycol 750).sub.b(amino ethylsuccinate or amino ethyladipic acid).sub.c(amino ethyldopamine).sub.dphosphazene].sub.n; wherein, in the above formula, a is from 1.20 to 1.36; b is from 0.37 to 0.53; c is from 0.13 to 0.26; d is from 0.03 to 0.13; a+b+c+d is 2; and n is an integer from 3 to 100,000.

6. A preparation method of the phosphazene-based polymer of claim 1, comprising: a first step of reacting poly(dichlorophosphazene) of Formula 4 with an (amino acid)(C.sub.1-6 alkyl)ester of Formula 5; a second step of reacting the reaction mixture obtained from the previous step by adding amino(C.sub.1-6 alkoxy)polyethylene glycol and amino(C.sub.1-6 alkanol); a third step of reacting the reaction mixture of the previous step by further adding a amino(C.sub.1-6 alkoxy)polyethylene glycol solution dropwise; a fourth step of reacting the product obtained from the previous step with C.sub.1-6 alkanedioic acid or an anhydride thereof, and dimethylaminopyridine; and a fifth step of reacting the product obtained from the previous step with di(C.sub.1-6 alkyl)carbodiimide, hydroxysuccinimide, and dopamine: ##STR00004## wherein, in Formulas 4 and 5, R.sub.1, R.sub.2, and n are as defined in claim 1.

7. The preparation method of claim 6, wherein the first to third steps are carried out in a tetrahydrofuran solution in the presence of triethylamine.

8. The preparation method of claim 6, wherein the first step is carried out for 24 hours to 60 hours while increasing a temperature from a range of 80 C. to 50 C. to a range of 10 C. to 50 C.

9. The preparation method of claim 6, wherein the second and third steps are independently carried out at 35 C. to 60 C. for 24 hours to 60 hours.

10. The preparation method of claim 6, further comprising a step of filtering the resulting reaction mixture; concentrating the filtrate under reduced pressure; dissolving in methanol; and dialyzing the resulting concentrate with methanol and water after the reaction of the third step.

11. The preparation method of claim 6, wherein the fourth step is carried out in a dry tetrahydrofuran solution at 35 C. to 60 C. for 24 hours to 60 hours.

12. The preparation method of claim 6, further comprising a step of filtering the obtained reaction mixture; concentrating the filtrate under reduced pressure; dissolving in methanol; and dialyzing the resulting concentrate with methanol and water after the reaction of the fourth step.

13. The preparation method of claim 6, wherein the fifth step is carried out in a dry dimethylformamide solution at 10 C. to 35 C.

14. The preparation method of claim 6, wherein the fifth step is carried out by sequentially adding di(C.sub.1-6 alkyl)carbodiimide, hydroxysuccinimide, and dopamine while reacting for 10 minutes to 60 minutes, 6 hours to 24 hours, and 24 hours to 72 hours, respectively.

15. The preparation method of claim 6, wherein the (amino acid)(C.sub.1-6 alkyl)ester is isoleucine ethyl ester; the amino(C.sub.1-6 alkoxy)polyethylene glycol is amino methoxypolyethylene glycol; the amino(C.sub.1-6 alkanol) is aminoethanol; the C.sub.1-6 alkanedioic acid or the anhydride thereof is succinic acid, glutaric acid, adipic acid, or an anhydride thereof; and the di(C.sub.1-6 alkyl)carbodiimide is diisopropylcarbodiimide.

16. A tissue-adhesive composition comprising a phosphazene-based polymer comprising a catechol group of claim 1 as an active ingredient.

17. The tissue-adhesive composition of claim 16, wherein the composition is used for wound healing, adhesion of surgical tissue, or hemostasis.

18. The tissue-adhesive composition of claim 16, wherein the composition is converted from a sol form to a gel form due to body temperature.

Description

BRIEF DESCRIPTIONS OF THE DRAWINGS

(1) FIG. 1 is a photograph showing a sol-gel transition behavior depending on a temperature change of the catechol group-containing phosphazene-based polymer of the present invention.

(2) FIG. 2 is a graph showing a change in viscosity due to a temperature change of the catechol group-containing phosphazene-based polymer of the present invention.

(3) FIG. 3 is a photograph showing a decrease in the amount of gel of the catechol group-containing phosphazene-based polymer of the present invention according to the flow of time.

(4) FIG. 4 is a photograph showing a tissue-adhesion ability of the catechol group-containing phosphazene polymer of the present invention in a rat model with liver puncture.

(5) FIG. 5 is a graph showing a decrease in the amount of bleeding due to the catechol group-containing phosphazene-based polymer of the present invention in a rat model with liver puncture.

(6) FIG. 6 is a graph showing cytotoxicity of the catechol group-containing phosphazene-based polymer of the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENT

(7) Hereinbelow, the present invention will be described in detail with accompanying exemplary embodiments. However, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present invention.

(8) <Identification of Compounds>

(9) In the exemplary embodiments below, the elemental analysis of carbon, hydrogen, and nitrogen was carried out using Perkin-Elmer's C, H, and N analyzers in the Advanced Analysis Center at the Korea Institute of Science and Technology (KIST), in order to identify synthesized polymers. In addition, the nuclear magnetic resonance spectrum with hydrogen and phosphorous was measured by using a Varian Gemini-300, and the average molecular weight (M.sub.w) was measured though gel permeation chromatography using a Waters 1515 pump and a 2410 differentiation refractometer.

Example 1: Preparation of Poly[(Isoleucine Ethyl Ester)1.21(Amino Methoxypolyethylene Glycol 750)0.51(Amino Ethylsuccinate)0.22(Aminoethyl Dopamine)0.06Phosphazene]n

(10) Step 1: Dry isoleucine ethyl ester hydrochloride (IleOEt.HCl, 9.79 g, 50.04 mmol) was dissolved in 100 mL of anhydrous tetrahydrofuran (THF), followed by addition of triethylamine (24.41 g, 175.17 mmol). Poly(dichlorophosphazene) (4.00 g, 34.52 mmol) dissolved in anhydrous tetrahydrofuran (50 mL) was added dropwise to the mixed solution in an acetone-dry ice bath at 60 C., and then gradually raised to room temperature, thereby reacting the resultant for 48 hours.

(11) Step 2: After confirmation of the progress of the reaction in Step 1 while confirming .sup.31P-NMR, dry aminoethanol (0.62 g, 10.35 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL), and triethylamine was added thereto so that the resultant was added to the above reactant. Thereafter, a solution, in which triethylamine (4.21 g, 30.20 mmol) was added to dry amino methoxypolyethylene glycol (6.47 g, 8.63 mmol) having a molecular weight of 750, which was dissolved in anhydrous tetrahydrofuran (50 mL), was immediately added dropwise, and then the reaction was carried out at room temperature for 24 hours and at 40 C. to 50 C. for 24 hours.

(12) Step 3: Thereafter, a solution, in which triethylamine (2.10 g, 15.10 mmol) was added to dry amino methoxypolyethylene glycol (3.24 g, 4.331 mmol) having a molecular weight of 750, which was dissolved in anhydrous tetrahydrofuran (50 mL), was additionally added dropwise to the reactant of Step 2, and then the reaction was further carried out at room temperature for 24 hours and at 40 C. to 50 C. for 24 hours.

(13) The solution in which the reaction was completed was filtered in order to remove the produced triethylamine hydrochloride, and the reaction filtrate was concentrated under reduced pressure until only a small amount of solvent remained. The concentrated solution was dissolved in a small amount of methanol, placed in MWCO 12000 Membrane (Spectrum Laboratories, Inc.), dialyzed against methanol at room temperature for 5 days, and then dialyzed once more against distilled water for 5 days. Thereafter, the resultant was lyophilized, thereby obtaining a polyphosphazene polymer [NP(IleOEt).sub.1.21(AMPEG750).sub.0.51(aminoethanol).sub.0.28].sub.n (7.21 g), which contains isoleucine ethyl ester, amino methoxypolyethylene glycol, and aminoethanol.

(14) Step 4: The polyphosphazene polymer [NP(IleOEt).sub.1.21(AMPEG750).sub.0.51(aminoethanol).sub.0.28].sub.n (11.32 g, 6.16 mmol), which was obtained from Step 3, was dissolved in tetrahydrofuran (200 mL), and then reacted at room temperature for 8 hours using 2 equivalents of succinic anhydride (1.23 g, 12.31 mmol) and 2 equivalents of dimethylaminopyridine (1.51 g, 12.31 mmol). The reaction filtrate was concentrated under reduced pressure, dissolved in a small amount of methanol, dialyzed against methanol at room temperature for 5 days, and then dialyzed against distilled water at 4 C. for 5 days. Thereafter, the resultant was lyophilized, thereby obtaining a polyphosphazene polymer [NP(IleOEt).sub.1.21(AMPEG750).sub.0.51(aminoethylsuccinate).sub.0.28].sub.n (10.58 g), which contains isoleucine ethyl ester, amino methoxypolyethylene glycol, and aminoethylsuccinate.

(15) Step 5: [NP(IleOEt).sub.1.21(AMPEG750).sub.0.51(aminoethylsuccinate).sub.0.28].sub.n (4.5 g, 1.88 mmol) obtained from Step 4 was dissolved in dimethylformamide (90 mL), and 2 equivalents of diisopropylcarbodiimide (1.20 g) dissolved in anhydrous dimethylformamide (20 mL) were then added thereto. After 30 minutes, hydroxysuccinimide (0.43 g, 3.75 mmol) was likewise dissolved in dimethylformamide (15 mL), and added to the resultant. The reaction was then carried out at room temperature for 1 day. Thereafter, 2 equivalents of dopamine hydrochloride (0.84 g, 3.75 mmol) and diisopropylethylamine (2.27 g, 7.50 mmol) in dry DMF were added, and then reacted at room temperature for 48 hours. The reaction filtrate was placed in MWCO 12-14000 Membrane, dialyzed against distilled water at 4 C. for 5 days, and the resultant was lyophilized, thereby obtaining a final product [NP(IleOEt).sub.1.21(AMPEG750).sub.0.51(aminoethylsuccinate).sub.0.22(aminoethylsuccinateDN).sub.0.06].sub.n (4.3 g).

(16) Nuclear Magnetic Resonance Spectrum with Hydrogen (CDCl.sub.3, ppm): 0.7 to 1.1 (b, NHCH(CH(CH.sub.3)CH.sub.2CH.sub.3)OCH.sub.2CH.sub.3), 1.1 to 1.3 (b, NHCH(CH(CH.sub.3)CH.sub.2CH.sub.3)OCH.sub.2CH.sub.3), 1.4 to 1.8 (b, NHCH(CH(CH.sub.3)CH.sub.2CH.sub.3)OCH.sub.2CH.sub.3), 1.9 (s, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2O.sub.2C(CH.sub.3)CCH.sub.2), 2.5 to 2.7 (b, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2COOH), 2.9 to 3.2 (b, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2), 3.4 (s, NH(CH.sub.2CH.sub.2O).sub.17CH.sub.3), 3.4 to 3.8 (b, NH(CH.sub.2CH.sub.2O).sub.17CH.sub.3), 3.9 to 4.3 (b, NHCH(CH(CH.sub.3)CH.sub.2CH.sub.3)OCH.sub.2CH.sub.3, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2), 6.4 (s, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2) 6.6 (s, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2) 8.7 (s, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2)

(17) Average molecular weight (M.sub.w): 28,184

Example 2: Preparation of Poly[(Isoleucine Ethyl Ester)1.35(Amino Methoxypolyethylene Glycol 750)0.39(Amino Ethylsuccinate)0.14(Aminoethyl Dopamine)0.12Phosphazene]n

(18) Step 1: Dry isoleucine ethyl ester hydrochloride (IleOEt. HCl, 22.46 g, 114.76 mmol) was dissolved in 300 mL of anhydrous tetrahydrofuran (THF), followed by addition of triethylamine (63.98 g, 459.05 mmol). Poly(dichlorophosphazene) (9.00 g, 77.66 mmol) dissolved in anhydrous tetrahydrofuran (100 mL) was added dropwise to the mixed solution in an acetone-dry ice bath at 60 C., and then gradually raised to room temperature, thereby reacting the resultant for 48 hours.

(19) Step 2: After confirmation of the progress of the reaction in Step 1 while confirming .sup.31P-NMR, dry aminoethanol (1.58 g, 25.89 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL), and triethylamine was added thereto so that the resultant was added to the above reactant. Thereafter, a solution, in which triethylamine (13.35 g, 95.78 mmol) was added to dry amino methoxypolyethylene glycol (35.92 g, 47.89 mmol) having a molecular weight of 750, which was dissolved in anhydrous tetrahydrofuran (200 mL), was immediately added dropwise, and then the reaction was carried out at room temperature for 24 hours and at 40 C. to 50 C. for 24 hours.

(20) Step 3: Thereafter, a solution, in which triethylamine (4.45 g, 31.93 mmol) was added to dry amino methoxypolyethylene glycol (11.97 g, 15.96 mmol) having a molecular weight of 750, which was dissolved in anhydrous tetrahydrofuran (80 mL), was additionally added dropwise to the reactant of Step 2, and then the reaction was further carried out at room temperature for 24 hours and at 40 C. to 50 C. for 24 hours.

(21) The solution in which the reaction was completed was filtered in order to remove the produced triethylamine hydrochloride, and the reaction filtrate was concentrated under reduced pressure until only a small amount of solvent remained. The concentrated solution was dissolved in a small amount of methanol, placed in MWCO 12000 Membrane (Spectrum Laboratories, Inc.), dialyzed against methanol at room temperature for 5 days, and then dialyzed once more against distilled water for 5 days. Thereafter, the resultant was lyophilized, thereby obtaining a polyphosphazene polymer [NP(IleOEt).sub.1.35(AMPEG750).sub.0.39(aminoethanol).sub.0.26].sub.n (23.46 g), which contains isoleucine ethyl ester, amino methoxypolyethylene glycol, and aminoethanol.

(22) Step 4: The polyphosphazene polymer [NP(IleOEt).sub.1.35(AMPEG750).sub.0.39(aminoethanol).sub.0.26].sub.n (23.46 g, 15.56 mmol), which was obtained from Step 3, was dissolved in tetrahydrofuran (400 mL), and then reacted at room temperature for 8 hours using 2 equivalents of succinic anhydride (2.60 g, 25.97 mmol) and 2 equivalents of dimethylaminopyridine (3.17 g, 25.97 mmol). The reaction filtrate was concentrated under reduced pressure, dissolved in a small amount of methanol, dialyzed against methanol at room temperature for 5 days, and then dialyzed against distilled water at 4 C. for 5 days. Thereafter, the resultant was lyophilized, thereby obtaining a polyphosphazene polymer [NP(IleOEt).sub.1.35(AMPEG750).sub.0.39(aminoethylsuccinate).sub.0.26].sub.n (23.50 g), which contains isoleucine ethyl ester, amino methoxypolyethylene glycol, and aminoethylsuccinate.

(23) Step 5: [NP(IleOEt).sub.1.35(AMPEG750).sub.0.39(aminoethylsuccinate).sub.0.26].sub.n (9.5 g, 4.12 mmol) obtained from Step 4 was dissolved in dimethylformamide (180 mL), and 2 equivalents of diisopropylcarbodiimide (1.96 g, 8.23 mmol) dissolved in anhydrous dimethylformamide (40 mL) were then added thereto. After 30 minutes, hydroxysuccinimide (0.94 g, 8.23 mmol) was likewise dissolved in dimethylformamide (30 mL), and added to the resultant. The reaction was carried out at room temperature for 1 day. Thereafter, 2 equivalents of dopamine hydrochloride (1.56 g, 8.23 mmol) and diisopropylethylamine (2.87 g, 16.46 mmol) in dry DMF were added, and then reacted at room temperature for 48 hours. The reaction filtrate was placed in MWCO 12-14000 Membrane, dialyzed against distilled water at 4 C. for 5 days, and the resultant was lyophilized, thereby obtaining a final product [NP(IleOEt).sub.1.35(AMPEG750).sub.0.39(aminoethylsuccinate).sub.0.14(aminoethylsuccinateDN).sub.0.12].sub.n (9.6 g).

(24) Nuclear Magnetic Resonance Spectrum with Hydrogen (CDCl.sub.3, Ppm): 0.7 to 1.1 (b, NHCH(CH(CH.sub.3)CH.sub.2CH.sub.3)OCH.sub.2CH.sub.3), 1.1 to 1.3 (b, NHCH(CH(CH.sub.3)CH.sub.2CH.sub.3)OCH.sub.2CH.sub.3), 1.4 to 1.8 (b, NHCH(CH(CH.sub.3)CH.sub.2CH.sub.3)OCH.sub.2CH.sub.3), 1.9 (s, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2O.sub.2C(CH.sub.3)CCH.sub.2), 2.5 to 2.7 (b, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2COOH), 2.9 to 3.2 (b, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2), 3.4 (s, NH(CH.sub.2CH.sub.2O).sub.17CH.sub.3), 3.4 to 3.8 (b, NH(CH.sub.2CH.sub.2O).sub.17CH.sub.3), 3.9 to 4.3 (b, NHCH(CH(CH.sub.3)CH.sub.2CH.sub.3)OCH.sub.2CH.sub.3, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2), 6.4 (s, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2) 6.6 (s, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2) 8.7 (s, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2)

(25) Average Molecular Weight (M.sub.w): 35,121

Example 3: Preparation of Poly[(Isoleucine Ethyl Ester)1.21(Amino Methoxypolyethylene Glycol 750)0.51(Amino Ethylsuccinate)0.20(Aminoethyl Dopamine)0.08Phosphazene]n

(26) Step 1: Dry isoleucine ethyl ester hydrochloride (IleOEt.HCl, 9.79 g, 50.04 mmol) was dissolved in 100 mL of anhydrous tetrahydrofuran (THF), followed by addition of triethylamine (24.41 g, 175.17 mmol). Poly(dichlorophosphazene) (4.00 g, 34.52 mmol) dissolved in anhydrous tetrahydrofuran (50 mL) was added dropwise to the mixed solution in an acetone-dry ice bath at 60 C., and then gradually raised to room temperature, thereby reacting the resultant for 48 hours.

(27) Step 2: After confirmation of the progress of the reaction in Step 1 while confirming .sup.31P-NMR, dry aminoethanol (0.62 g, 10.35 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL), and triethylamine was added thereto so that the resultant was added to the above reactant. Thereafter, a solution, in which triethylamine (4.21 g, 30.20 mmol) was added to dry amino methoxypolyethylene glycol (6.47 g, 8.63 mmol) having a molecular weight of 750, which was dissolved in anhydrous tetrahydrofuran (50 mL), was immediately added dropwise, and then the reaction was carried out at room temperature for 24 hours and at 40 C. to 50 C. for 24 hours.

(28) Step 3: Thereafter, a solution, in which triethylamine (2.10 g, 15.10 mmol) was added to dry amino methoxypolyethylene glycol (3.24 g, 4.331 mmol) having a molecular weight of 750, which was dissolved in anhydrous tetrahydrofuran (50 mL), was additionally added dropwise to the reactant of Step 2, and then the reaction was further carried out at room temperature for 24 hours and at 40 C. to 50 C. for 24 hours.

(29) The solution in which the reaction was completed was filtered in order to remove the produced triethylamine hydrochloride, and the reaction filtrate was concentrated under reduced pressure until only a small amount of solvent remained. The concentrated solution was dissolved in a small amount of methanol, placed in MWCO 12000 Membrane (Spectrum Laboratories, Inc.), dialyzed against methanol at room temperature for 5 days, and then dialyzed once more against distilled water for 5 days. Thereafter, the resultant was lyophilized, thereby obtaining a polyphosphazene polymer [NP(IleOEt).sub.1.19(AMPEG750).sub.0.55(aminoethanol).sub.0.25].sub.n (7.21 g), which contains isoleucine ethyl ester, amino methoxypolyethylene glycol, and aminoethanol.

(30) Step 4: The polyphosphazene polymer [NP(IleOEt).sub.1.21(AMPEG750).sub.0.51(aminoethanol).sub.0.25].sub.n (11.32 g, 6.16 mmol), which was obtained from Step 3, was dissolved in tetrahydrofuran (200 mL), and then reacted at room temperature for 8 hours using 2 equivalents of succinic anhydride (1.23 g, 12.31 mmol) and 2 equivalents of dimethylaminopyridine (1.51 g, 12.31 mmol). The reaction filtrate was concentrated under reduced pressure, dissolved in a small amount of methanol, dialyzed against methanol at room temperature for 5 days, and then dialyzed against distilled water at 4 C. for 5 days. Thereafter, the resultant was lyophilized, thereby obtaining a polyphosphazene polymer [NP(IleOEt).sub.1.21(AMPEG750).sub.0.51(aminoethylsuccinate).sub.0.28].sub.n (10.58 g), which contains isoleucine ethyl ester, amino methoxypolyethylene glycol, and aminoethylsuccinate.

(31) Step 5: [NP(IleOEt).sub.1.21(AMPEG750).sub.0.51(aminoethylsuccinate).sub.0.2].sub.n (2.93 g, 1.22 mmol) obtained from Step 4 was dissolved in dimethylformamide (80 mL), and 2 equivalents of diisopropylcarbodiimide (0.78 g) dissolved in anhydrous dimethylformamide (20 mL) were then added thereto. After 30 minutes, hydroxysuccinimide (0.28 g, 2.44 mmol) was likewise dissolved in dimethylformamide (15 mL), and added to the resultant. The reaction was then carried out at room temperature for 1 day. Thereafter, 2 equivalents of dopamine hydrochloride (0.55 g, 2.44 mmol) and diisopropylethylamine (1.48 g, 4.88 mmol) in dry DMF were added, and then reacted at room temperature for 48 hours. The reaction filtrate was placed in MWCO 12-14000 Membrane, dialyzed against distilled water at 4 C. for 5 days, and the resultant was lyophilized, thereby obtaining a final product [NP(IleOEt).sub.1.21(AMPEG750).sub.0.51 (aminoethylsuccinate).sub.0.20(aminoethylsuccinateDN).sub.0.08].sub.n (3.03 g).

(32) Nuclear Magnetic Resonance Spectrum with Hydrogen (CDCl.sub.3, Ppm): 0.7 to 1.1 (b, NHCH(CH(CH.sub.3)CH.sub.2CH.sub.3)OCH.sub.2CH.sub.3), 1.1 to 1.3 (b, NHCH(CH(CH.sub.3)CH.sub.2CH.sub.3)OCH.sub.2CH.sub.3), 1.4 to 1.8 (b, NHCH(CH(CH.sub.3)CH.sub.2CH.sub.3)OCH.sub.2CH.sub.3), 1.9 (s, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2O.sub.2C(CH.sub.3)CCH.sub.2), 2.5 to 2.7 (b, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2COOH), 2.9 to 3.2 (b, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2), 3.4 (s, NH(CH.sub.2CH.sub.2O).sub.17CH.sub.3), 3.4 to 3.8 (b, NH(CH.sub.2CH.sub.2O).sub.17CH.sub.3), 3.9 to 4.3 (b, NHCH(CH(CH.sub.3)CH.sub.2CH.sub.3)OCH.sub.2CH.sub.3, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2), 6.4 (s, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2) 6.6 (s, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2) 8.7 (s, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2)

(33) Average Molecular Weight (M.sub.w): 16,729

Example 4: Preparation of Poly[(Isoleucine Ethyl Ester)1.26(Amino Methoxypolyethylene Glycol 750)0.42(Amino Ethyladipic Acid)0.285(Aminoethyl Dopamine)0.035Phosphazene]n

(34) Steps 1 to 3: The reaction was carried out in the same manner as in Example 1.

(35) Step 4: The reaction was carried out in the same manner as in Step 4 of Example 1 except that adipic anhydride was used instead of succinic anhydride, thereby obtaining a polyphosphazene polymer [NP(IleOEt).sub.1.26(AMPEG750).sub.0.42(aminoethyladipate).sub.0.32].sub.n, which contains isoleucine ethyl ester, amino methoxypolyethylene glycol, and aminoethyladipate.

(36) Step 5: [NP(IleOEt).sub.1.26(AMPEG750).sub.0.42(aminoethyladipate).sub.0.32].sub.n (0.4 g, 0.16 mmol) obtained from Step 4 was dissolved in dimethylformamide (100 mL), and 2 equivalents of diisopropylcarbodiimide (78 mg, 0.33 mmol) dissolved in anhydrous dimethylformamide (20 mL) were then added thereto. After 30 minutes, hydroxysuccinimide (37.8 mg, 0.33 mmol) was likewise dissolved in dimethylformamide (20 mL), and added to the resultant. The reaction was then carried out at room temperature for 1 day. Thereafter, 2 equivalents of dopamine hydrochloride (62.2 mg, 0.33 mmol) and diisopropylethylamine (114.3 g, 0.66 mmol) in dry DMF were added, and then reacted for 48 hours. The reaction filtrate was placed in MWCO 12-14000 Membrane, dialyzed against distilled water at 4 C. for 5 days, and the resultant was lyophilized, thereby obtaining a final product [NP(IleOEt).sub.1.26(AMPEG750).sub.0.42(aminoethyladipate).sub.0.285(aminoethyladipateDN).sub.0.035].sub.n (0.5 g).

(37) Nuclear Magnetic Resonance Spectrum with Hydrogen (CDCl.sub.3, ppm): 0.7 to 1.1 (b, NHCH(CH(CH.sub.3)CH.sub.2CH.sub.3)OCH.sub.2CH.sub.3), 1.1 to 1.3 (b, NHCH(CH(CH.sub.3)CH.sub.2CH.sub.3)OCH.sub.2CH.sub.3), 1.4 to 1.8 (b, NHCH(CH(CH.sub.3)CH.sub.2CH.sub.3)OCH.sub.2CH.sub.3), 1.9 (s, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2O.sub.2C(CH.sub.3)CCH.sub.2), 2.5 to 2.7 (b, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CH.sub.2CH.sub.2COOH), 2.9 to 3.2 (b, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2), 3.4 (s, NH(CH.sub.2CH.sub.2O).sub.17CH.sub.3), 3.4 to 3.8 (b, NH(CH.sub.2CH.sub.2O).sub.17CH.sub.3), 3.9 to 4.3 (b, NHCH(CH(CH.sub.3)CH.sub.2CH.sub.3)OCH.sub.2CH.sub.3, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2), 6.4 (s, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2) 6.6 (s, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2) 8.7 (s, NHCH.sub.2CH.sub.2OCOCH.sub.2CH.sub.2CH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2C.sub.6H.sub.5(OH).sub.2)

(38) Average Molecular Weight (M.sub.w): 22,410

Example 5: Sol-Gel Change of Phosphazene-Based Polymer Containing Catechol Group According to Temperature Change

(39) The catechol group-containing phosphazene-based polymers according to Examples 1 to 4 were each dissolved in phosphate-buffered saline (pH 7.4) at 4 C. at a concentration of 10 wt %. Thereafter, the resultants were placed in a chamber of a viscometer (Brookfield DV-III+ Rheometer) equipped with the TC-501 automatic water distiller, and a sol-gel transition behavior was observed by setting a shear rate to 0.1 to 1.7 per second, and by raising a temperature by 0.04 C. per minute.

(40) In FIG. 1, the state of the catechol group-containing phosphazene-based polymer at 25 C. and 37 C. according to Example 1 of the present invention was shown as photographs. As shown in FIG. 1, it was confirmed that the polymer of Example 1 of the present invention existed in a form where a solution is flowing at room temperature, but existed in a gel form at body temperature due to phase transition.

(41) The viscosities of the polymers of Examples 1 to 4 of the present invention according to temperature are shown in FIG. 2, and the gel properties thereof are shown in Table 1 below.

(42) TABLE-US-00001 TABLE 1 Maximum gel Maximum gel temperature strength Polymer Structure ( C.) (Pa .Math. s) Example 1 [NP(IleOEt).sub.1.21(AMPEG).sub.0.51(SA).sub.0.22(SADN).sub.0.06].sub.n 45 894 Example 2 [NP(IleOEt).sub.1.35(AMPEG).sub.0.39(SA).sub.0.14(SADN).sub.0.12].sub.n 36 463 Example 3 [NP(IleOEt).sub.1.21(AMPEG).sub.0.51(SA).sub.0.20(SADN).sub.0.08].sub.n 48 738 Example 4 [NP(IleOEt).sub.1.26(AMPEG).sub.0.42(AA).sub.0.255(AADN).sub.0.035].sub.n 45 1131

(43) In Table 1 above, the maximum gel temperature indicates a temperature at which the viscosity of the polymer aqueous solution reaches the peak point, and the maximum gel strength indicates a viscosity measured at the maximum gel temperature above.

(44) As shown in Table 1, even for the polymers (Examples 1 to 3) composed of the same substituents, it was confirmed that these have different maximum gel temperature and strength depending on the ratio of each substituent.

(45) On the other hand, as shown in FIG. 2, the viscosity of all polymers of Examples 1 to 4 of the present invention started to increase at a temperature in the range of 25 C. to 35 C., which is below body temperature; that is, the polymers started to gelate. However, as shown in Table 1, each maximum viscosity and the temperature indicating the same were different depending on the polymer. This shows that it is possible for the polymers to exhibit sol-gel transition behavior within the desired range because the sol-gel transition can be regulated by changing the type of substituent and its composition from the phosphazene-based polymer.

Example 6: Change in Gel Amount of Phosphazene-Based Polymer Containing Catechol Group Depending on Time

(46) In order to confirm the change in gel amount depending on time, the catechol group-containing phosphazene-based polymers according to Examples 1 and 2 of the present invention were each dissolved in phosphate-buffered saline (pH 7.4) at 4 C. at a concentration of 10 wt %. Thereafter, 200 L of the resultants were injected into mice's backs. After the day of injection, day 1, day 3, day 7, and day 14, respectively, these mice were sacrificed in order to obtain the gel, and the gel was visually observed to evaluate the reduction rate.

(47) As shown in FIG. 3, it was confirmed that although the amount of gel decreased with the lapse of time after injection into the body, the gel injected into the body effectively remained in the injected tissues even after 2 weeks.

Example 7: Tissue-Adhesion Ability of Phosphazene-Based Polymer Hydrogel Containing Catechol Group

(48) In order to directly confirm a tissue-adhesion ability of the catechol group-containing phosphazene-based polymer according to the present invention, a hole was made on mice's livers to induce blood to flow out. Thereafter, the catechol group-containing phosphazene-based polymer solution according to Example 1 of the present invention was directly treated at the bleeding site, and the change in blood outflow amount was observed. Morphology of the damaged mice's livers treated with the catechol group-containing phosphazene-based polymer solution according to Example 1 of the present invention is shown in FIG. 4. In addition, the bleeding volume from the mice's livers treated with the catechol group-containing phosphazene-based polymer solution according to Examples 1 and 2 of the present invention was measured, and is shown in FIG. 5.

(49) As shown in FIG. 5, it was confirmed that the bleeding volume was remarkably reduced when the damaged mice's livers were treated with the catechol group-containing phosphazene-based polymer solution according to Examples 1 and 2 of the present invention, compared with a control group in which the damaged mice's livers were not treated with any substance. This result indicates that the catechol group-containing phosphazene-based polymers according to the present invention have excellent tissue-adhesion ability, thereby effectively preventing bleeding from the wound site.

Example 8: Cytotoxicity of Phosphazene-Based Polymer Containing Catechol Group

(50) In order to ensure stability for in vivo application, cytotoxicity of the catechol group-containing phosphazene-based polymer according to the present invention was measured. Specifically, the polymer according to Example 1 of the present invention was treated with NIH3T3 cell line at a concentration of 100 M to 10,000 M, and cell viability thereof was observed. The result is shown in FIG. 6. NIH3T3 cells not treated with polymers are used as a control group. As shown in FIG. 6, even if the concentration of the polymer increased to 10,000 M, the cell viability exhibited a high value of 85% or more. This result indicates that the polymers according to the present invention do not show toxicity to cells, and thus can be applied to the human body.