Composition containing polycationic triblock copolymer, polyanionic polymer and physiologically active peptide

Abstract

[Problem] To provide a physiologically active peptide-loaded stable composition for injection into living bodies. [Solution] A composition containing a triblock copolymer represented by formula (I), a polyanionic polymer and a physiologically active peptide:
CNR-PEG-CNR(I) in the formula, CNR moieties are each independently a polymer segment containing a repeating unit that contains, as a part of a pendant group, a cyclic nitroxide radical bonded to a main polymer chain via a linking group that contains at least one amino group, and PEG is a segment that contains poly(ethylene glycol).

Claims

1. A composition comprising a triblock copolymer represented by formula (II), a polyanionic polymer and a physiologically active peptide: ##STR00008## in the formula, L.sub.1 groups are linking groups that may be the same as, or different from, each other, L.sub.2 groups are each independently a C.sub.1-6 alkylene-NH(C.sub.1-6 alkylene)q- group, with q being an integer of 0 or 1, wherein in at least one of the L.sub.2 groups q=1, R groups are each independently such that at least 50% of the total number (n) of R groups are residues of cyclic nitroxide radical compounds selected from the group consisting of 2,2,6,6-tetramethylpiperidin-1-oxyl-4-yl groups, 2,2,5,5-tetramethylpyrrolidin-1-oxyl-3-yl groups, 2,2,5,5-tetramethylpyrrolin-1-oxyl-3-yl groups, 2,4,4-trimethyl-1,3-oxazolidin-3-oxyl-2-yl groups, 2,4,4-trimethyl-1,3-thiazolidin-3-oxyl-2-yl groups and 2,4,4-trimethyl-imidazolidin-3-oxyl-2-yl groups, with the remaining R groups, when present, being hydrogen atoms, halogen atoms or hydroxyl groups, terminal H groups may, in some cases, each independently be substituted by groups selected from among arylthiocarbonylthio groups, alkylthiocarbonylthio groups, alkoxythiocarbonylthio groups and sulfanyl groups, m is an integer between 20 and 5,000, and each instance of n is independently an integer between 3 and 1,000.

2. The composition according to claim 1, wherein the L.sub.1 groups are each independently selected from the group consisting of single bonds, S(CH2).sub.c- groups, S(CH2).sub.cCO groups, (CH2).sub.cS groups, CO(CH2).sub.cS groups, m- or p-phenylene groups, m- or p-xylylene groups and alkylene groups, c is an integer between 1 and 5, with these linking groups able to be in the opposite direction from that shown in formula (II) in cases where the linking groups are not directionally equivalent, R is selected from among groups represented by the following formulae: ##STR00009## in the formulae, R is a methyl group, and at least 80% of the total number (n) of R groups are groups represented by the formula shown above.

3. The composition according to claim 1, wherein the polyanionic polymer is one or more types selected from the group consisting of poly(acrylic acid), poly(methacrylic acid), poly(sulfonic acid), polyanionic polysaccharides and anionic proteins.

4. The composition according to claim 1, wherein the physiologically active peptide is selected from the group consisting of enzyme proteins, antigenic proteins, antibodies, cytokines, peptide hormones and antimicrobial peptides.

5. The composition according to claim 1, wherein the ratio of a total anionic charge relative to a total cationic charge from the triblock copolymer, the polyanionic polymer and the physiologically active peptide is between 10:1 and 1:10 in an aqueous solution.

6. The composition according to claim 1, which is present as polyion complex micelles having an average particle diameter, as measured by a dynamic light scattering (DLS) method, of 10 to 300 nm in an aqueous solution.

7. The composition according to claim 1, which forms a gel according to changes in the ionic strength in the aqueous solution and/or changes in temperature.

8. A gel-forming medical composition comprising a polyion complex formed from the composition according to claim 1, and a pharmaceutically acceptable diluent or excipient.

9. A triblock copolymer represented by formula (II) ##STR00010## in the formula, L.sub.1 groups are linking groups that may be the same as, or different from, each other, L.sub.2 groups are each independently a C.sub.1-6 alkylene-NH(C.sub.1-6 alkylene)q- group, with q being an integer of 0 or 1, wherein in at least one of the L.sub.2 groups q=1, R groups are each independently such that at least 50% of the total number (n) of R groups are residues of cyclic nitroxide radical compounds selected from the group consisting of 2,2,6,6-tetramethylpiperidin-1-oxyl-4-yl groups, 2,2,5,5-tetramethylpyrrolidin-1-oxyl-3-yl groups, 2,2,5,5-tetramethylpyrrolin-1-oxyl-3-yl groups, 2,4,4-trimethyl-1,3-oxazolidin-3-oxyl-2-yl groups, 2,4,4-trimethyl-1,3-thiazolidin-3-oxyl-2-yl groups and 2,4,4-trimethyl-imidazolidin-3-oxyl-2-yl groups, with the remaining R groups, when present, being hydrogen atoms, halogen atoms or hydroxyl groups, terminal H groups are each substituted by groups selected from among arylthiocarbonylthio groups, alkylthiocarbonylthio groups, alkoxythiocarbonylthio groups and sulfanyl groups, m is an integer between 20 and 5,000, and each instance of n is independently an integer between 3 and 1,000.

10. The triblock copolymer according to claim 9, wherein the L.sub.1 groups are each m- or p-phenylene groups, m- or p-xylylene groups or alkylene groups.

11. The composition according to claim 9, wherein the polyanionic polymer is one or more types selected from the group consisting of poly(acrylic acid), poly(methacrylic acid), poly(sulfonic acid), polyanionic polysaccharides and anionic proteins.

12. The composition according to claim 9, wherein the ratio of a total anionic charge relative to a total cationic charge from the triblock copolymer, the polyanionic polymer and the physiologically active peptide is between 10:1 and 1:10 in an aqueous solution.

13. A gel-forming medical composition comprising a polyion complex formed from the composition according to claim 9, and a pharmaceutically acceptable diluent or excipient.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a diagram showing the results of size exclusion chromatography (SEC) measurements and .sup.1H NMR spectral measurements for the PCMS-b-PEG-b-PCMS triblock copolymer obtained in Production Example 1.

(2) FIG. 2 is a diagram showing the results of SEC measurements and .sup.1H NMR spectral measurements for the PMNT-b-PEG-b-PMNT triblock copolymer obtained in Production Example 2.

(3) FIG. 3 shows photographs relating to diagrams showing the state of gelation of the polyion complex micelles prepared in Production Example 4.

(4) FIG. 4 indicates the results of Experiment 1, and is a diagram showing the hydrogen peroxide production capacity of a polyion complex that encapsulates D-amino acid oxidase.

(5) FIG. 5 is a diagram showing particle diameter measurement results for the protein-encapsulating PIC micelles obtained in Production Examples 5 to 7 and the PIC micelles obtained in Comparative Example 1. In this diagram, relate to FITC-Insulin-encapsulating PIC micelles, relate to FITC-BSA-encapsulating PIC micelles, and .box-tangle-solidup. relates to FITC-GOD-encapsulating PIC micelles. .circle-solid. relates to PIC micelles that contain no protein.

(6) FIG. 6 shows the results of fluorescence intensity measurements for a FITC-Insulin-encapsulating PIC micelle solution and a FITC-Insulin aqueous solution in a FITC-Insulin solution, for the same protein concentration.

(7) FIG. 7 shows the results of fluorescence intensity measurements for a FITC-BSA-encapsulating PIC micelle solution and a FITC-BSA aqueous solution in a FITC-BSA solution, for the same protein concentration.

(8) FIG. 8 shows the results of fluorescence intensity measurements for a FITC-GOD-encapsulating PIC micelle solution and a FITC-GOD aqueous solution in a FITC-GOD solution, for the same protein concentration.

(9) FIG. 9 shows the results of protein release measurements from RIG in Experimental Example 2. .circle-solid. relate to FITC-Insulin-encapsulating RIG, .square-solid. relate to FITC-BSA-encapsulating RIG, and relate to FITC-GOD-encapsulating RIG.

(10) FIG. 10 shows photographs that indicate the results of an in vivo protein release evaluation test from RIG in Experimental Example 3.

(11) FIG. 11 shows photographs that indicate the results of a RIG in vivo retention evaluation test in Experimental Example 3.

(12) FIG. 12 shows photographs that indicate the results of a protein in vivo retention evaluation test in Experimental Example 4.

(13) FIG. 13 shows gelation behavior according to changes in the temperature and ionic strength of a (DAO-containing) PIC micelle solution in Experimental Example 5.

(14) FIG. 14 shows gelation behavior according to changes in the temperature and ionic strength of a (DAO-free) PIC micelle solution in Experimental Example 5.

(15) FIG. 15 is a diagram showing the results of SEC measurements and .sup.1H NMR spectral measurements for the Br-PEG-Br obtained in Production Example 14.

(16) FIG. 16 is a diagram showing the results of SEC measurements and .sup.1H NMR spectral measurements for the CTA-PEG-CTA obtained in Production Example 15.

(17) FIG. 17 is a diagram showing the results of SEC measurements and .sup.1H NMR spectral measurements for the PCMS-b-PEG-b-PCMS triblock copolymer obtained in Production Example 16.

(18) FIG. 18 is a diagram showing the results of SEC measurements and .sup.1H NMR spectral measurements for the PMNT-b-PEG-b-PMNT triblock copolymer obtained in Production Example 17.

(19) FIG. 19 is a diagram showing particle diameter measurement results for the polyion complex micelles obtained in Production Example 18.

EXAMPLES

(20) The present invention will now be explained in greater detail through the use of specific examples, but the present invention is in no way limited to these specific examples.

Synthesis of Polychloromethylstyrene-b-Poly(Ethylene Glycol)-b-Polychloromethylstyrene (PCMS-b-PEG-b-PCMS) Triblock Copolymer

(21) The PCMS-b-PEG-b-PCMS was synthesized according to Synthesis Scheme 1 shown below:

(22) ##STR00003##

(23) Poly(ethylene glycol) having a thiol group at both terminals (HS-PEG-SH) (Mn: 10,000; 0.164 mmol, 1.64 g) was added to a reaction vessel. Next, a procedure involving evacuating the reaction vessel to a vacuum and blowing in nitrogen gas was repeated 3 times so as to form a nitrogen atmosphere in the reaction vessel. A solution of azobisisobutyronitrile/toluene (0.164 mmol/16 ml) and a solution of chloromethylstyrene (12.3 mmol, 1.74 mL) were added to the reaction vessel, heated to 60 C. and stirred for 24 hours. A white powder was obtained by washing the reaction mixture 3 times with diethyl ether, which is a good solvent for a polychloromethylstyrene homopolymer, and then freeze-drying in benzene. The quantity recovered was 2.07 mg, which was a yield of 96.5%. The results of size exclusion chromatography (SEC) measurements and .sup.1H NMR spectral measurements for the obtained PCMS-b-PEG-b-PCMS triblock copolymer are shown in FIG. 1.

Synthesis of Triblock Polymer Containing 2,2,6,6-tetramethylpiperidin-1-oxyl-4-yl (TEMPO) (PMNT-b-PEG-b-PMNT)

(24) PCMS-b-PEG-b-PCMS (Mn: 13,052; 1.8 g, 0.138 mmol) was added to a reaction vessel. Next, 4-amino-TEMPO (2.36 g, 13.8 mmol) was dissolved in 20 mL of dimethyl sulfoxide (DMSO), added to the reaction vessel, and stirred for 24 hours at room temperature. Following completion of the reaction, the reaction solution was added to a dialysis membrane (Spectra/Por molecular weight cut-off size 3,500 Spectrum Medical Industries Inc., Houston Tex.), and dialyzed with 2 L of methanol. The methanol was replaced 8 times, every 2 hours, after which the reaction solution was evaporated and freeze-dried in benzene. The yield was 65.6%.

(25) It was found from .sup.1H NMR measurements that 100% of the chloromethyl groups had reacted and TEMPO had been introduced (see FIG. 2).

Design of Polyion Complex Micelles

(26) The powdered PMNT-b-PEG-b-PMNT triblock polymer was dissolved in a 0.1 M aqueous solution of HCl, the amino groups on the PMNT chains were completely protonated, and the aqueous system was freeze-dried and recovered. Next, the PMNT-b-PEG-b-PMNT triblock polymer and poly(acrylic acid) (PAA; Mn: 5,000) were each dissolved in a phosphoric acid buffer solution (0.1 M, pH 6.28) so as to prepare an aqueous polycationic PMNT-b-PEG-b-PMNT solution and an aqueous anionic PAA solution, each having a concentration of 5 mg/ml. In addition, D-amino acid oxidase was dissolved in a phosphoric acid buffer solution (0.1 M, pH 6.28), thereby adjusting the concentration to 0.2 mg/ml. The D-amino acid oxidase was added to the aqueous PAA solution under stirring. PIC micelles were then produced by adding the aqueous PMNT-b-PEG-b-PMNT triblock polymer solution dropwise under stirring to the mixed solution containing the PAA and the D-amino acid oxidase. Here, the PIC micelles were produced in such a way that the PMNT-b-PEG-b-PMNT:PAA molar ratio (r) was 1:1, under conditions whereby the quantity of DAO was changed to 0 ml, 0.2 ml, 0.4 ml or 0.6 ml. (Molar ratio (r)=[number of moles of activated carboxyl groups in PAA]/[number of moles of activated amino groups in PMNT-b-PEG-b-PMNT]). The zeta potential of the obtained PIC micelles was measured. The results are shown in Table 1. When the average particle diameter of the obtained PIC micelles was measured by dynamic light scattering (DLS), it was confirmed that the PIC micelles were unimodal particles having an average particle diameter of 59-67 nm.

(27) TABLE-US-00001 TABLE 1 Particle diameter and zeta potential of polyion complex micelles PDI DAO Particle (polydispersity (0.2 mg/ml) diameter index) z-potential (1) 0 ml 66.7 nm 0.258 +1.22 (2) 0.2 ml 63.0 nm 0.251 +0.654 (3) 0.4 ml 66.6 nm 0.251 +1.54 (4) 0.6 ml 59.5 nm 0.241 +0.395

Design of Injectable Gel

(28) 5 mg/ml of each PIC micelle solution prepared in Production Example 3 was condensed by centrifugal evaporation, thereby adjusting the ionic strength to 150 mM, and gelation experiments were carried out in a water bath at a temperature of 37 C. using a test tube inversion method. Photographs relating to diagrams showing the experimental results (the state of gelation) are shown in FIG. 3. From these diagrams, it was confirmed that an irreversible gel was formed at an ionic strength of 150 mM and a temperature of 37 C.

Evaluation of Hydrogen Peroxide Production Capacity of D-Amino Acid Oxidase

(29) PIC micelles to which 0.4 ml (0.2 mg/ml) of D-amino acid oxidase was added ((3) in table 1 and FIG. 3) were evaluated in terms of hydrogen peroxide production capacity using a peroxidase/o-dianisidine evaluation method. The D-amino acid oxidase was condensed by centrifugal evaporation until the concentration was 5 g/ml, and evaluated through a comparison with control PIC micelles in which the amount of free DAO and D-amino acid oxidase was 0 ml. The results are shown in FIG. 4.

(30) This diagram shows that free D-amino acid oxidase and D-amino acid oxidase-encapsulating PIC micelles exhibit different behavior in terms of hydrogen peroxide production capacity. This is thought to be because the D-amino acid oxidase is incorporated into the PIC micelles through electrostatic interactions. In addition, it can be understood that PIC micelles in which D-amino acid oxidase is encapsulated or loaded slowly release hydrogen peroxide, and are therefore materials that can prevent an initial burst of a drug.

Production Examples 5 to 7

Preparation of PIC Micelles Encapsulating Fluorescein Isothiocyanate (FITC)-Labeled Proteins

(31) The powdered PMNT-b-PEG-b-PMNT triblock polymer was dissolved in a 0.1 M aqueous solution of HCl, the amino groups on the PMNT chains were completely protonated, and the aqueous system was freeze-dried and recovered.

(32) Next, the protonated PMNT-b-PEG-b-PMNT triblock polymer and poly(acrylic acid) (PAA; Mn: 5,000) were each dissolved in a phosphoric acid buffer solution (50 mM, pH 6.2), thereby preparing a polycationic PMNT-b-PEG-b-PMNT aqueous solution and a polyanionic PAA aqueous solution each having a concentration of 5 mg/ml.

(33) To 1.621 mL of the aqueous PAA solution prepared in the manner described above, the following materials were added:

(34) i) 70 L of an aqueous solution obtained by dissolving 1492 g/mL of fluorescein isothiocyanate-labeled insulin (FITC-Insulin) in a phosphate-buffered physiological saline solution (PBS),

(35) ii) 51 L of an aqueous solution obtained by dissolving 2030 g/mL of FITC-labeled bovine serum albumin (FITC-BSA) in a phosphoric acid buffer solution (50 mM, pH 6.2), or iii) 63 L of an aqueous solution obtained by dissolving 1650 g/mL of FITC-labeled glucose oxidase (FITC-GOD) in a phosphate-buffered physiological saline solution (PBS) and stirred. Next, 10 mL of the aqueous solution of the PMNT-b-PEG-b-PMNT triblock polymer was added dropwise under stirring to the mixed solutions of PAA and FITC-proteins under ice cooling, 309 L, 328 L and 316 L respectively of a phosphoric acid buffer solution (50 mM, pH 6.2) was added, thereby preparing PIC micelles encapsulating FITC-Insulin, FITC-BSA and FITC-GOD respectively.

(36) Here, the PIC micelles were prepared so that the PMNT-b-PEG-b-PMNT:PAA molar ratio (r) was 1:1 (molar ratio (r)=[number of moles of activated carboxyl groups in PAA]/[number of moles of activated amino groups in PMNT-b-PEG-b-PMNT]).

(37) The particle diameters of the obtained FITC-protein-encapsulating PIC micelles, as measured by dynamic light scattering (DLS), are shown in FIG. 5 and Table 2.

(38) TABLE-US-00002 TABLE 2 Average particle diameters and polydispersity indices of protein-encapsulating PIC micelles and PIC micelles (PdI) Average particle diameter (nm) PdI FITC-Insulin-encapsulating 66.01 0.261 PIC micelles FITC-BSA-encapsulating 56.34 0.239 PIC micelles FITC-GOD-encapsulating 62.19 0.247 PIC micelles PIC micelles 42.95 0.172

(39) The results of fluorescence intensity measurements of the obtained FITC-Insulin-encapsulating PIC micelle solution, FITC-BSA-encapsulating PIC micelle solution and FITC-GOD-encapsulating PIC micelle solution are shown in FIGS. 6, 7 and 8. In addition, the FITC-protein concentration in each FITC-protein-encapsulating PIC micelle solution and the fluorescence intensities of the FITC-protein aqueous solutions at the same concentrations are also shown in FIGS. 6, 7 and 8. Quenching caused by fluorescence resonance energy transfer (FRET) was confirmed in each FITC-protein-encapsulating PIC micelle solution, which suggested that insulin, bovine serum albumin and glucose oxidase were encapsulated in the PIC micelles.

Preparation of PIC Micelles not Encapsulating Proteins

(40) The powdered PMNT-b-PEG-b-PMNT triblock polymer was dissolved in a 0.1 M aqueous solution of HCl, the amino groups on the PMNT chains were completely protonated, and the aqueous system was freeze-dried and recovered.

(41) Next, the protonated PMNT-b-PEG-b-PMNT triblock polymer and poly(acrylic acid) (PAA; Mn: 5,000) where each dissolved in a phosphoric acid buffer solution (50 mM, pH 6.2), thereby preparing a polycationic PMNT-b-PEG-b-PMNT aqueous solution and a polyanionic PAA aqueous solution each having a concentration of 5 mg/ml.

(42) PIC micelles were prepared by adding 10 mL of an aqueous solution of the PMNT-b-PEG-b-PMNT triblock polymer dropwise under stirring and under ice cooling to 1.621 mL of the aqueous PAA solution prepared in the manner described above. Here, the PIC micelles were prepared so that the PMNT-b-PEG-b-PMNT:PAA molar ratio (r) was 1:1 (the molar ratio (r) is as defined above). The particle diameters of the obtained PIC micelles, as measured by dynamic light scattering (DLS), are shown in FIG. 5 and Table 2.

Preparation of FITC-Protein-Encapsulating Redox Injectable Gels (RIG)

(43) 11 mL of each of the FITC-protein-encapsulating PIC micelle solutions obtained in Production Examples 5 to 7 were condensed to 1 mL by means of centrifugal evaporation. A FITC-Insulin-encapsulating RIG, a FITC-BSA-encapsulating RIG and a FITC-GOD-encapsulating RIG were prepared by placing 300 L of each of the condensed solutions in a 1.5 mL microtube and heating in a constant temperature bath at a temperature of 37 C.

Evaluation of Release of Proteins from Redox Injectable Gels (RIG)

(44) 150 L of PBS was added to microtubes containing the protein-encapsulating RIGs obtained in Production Examples 8 to 10, and the obtained mixtures were incubated at 37 C. and 100 rpm using a shaker. At the sampling point, 100 L of the supernatant liquid was extracted and 100 L of fresh PBS was added. The fluorescence intensity of the extracted supernatant liquid was measured using a plate reader, and the quantity of protein released was calculated from the fluorescence intensity. These results are shown in FIG. 9.

Preparation of HiLyte Fluor 647-Labeled PIC Micelles Encapsulating Indocyanine Green (ICG)-Labeled Bovine Serum Albumin (BSA)

(45) The powdered PMNT-b-PEG-b-PMNT triblock polymer was dissolved in a 0.1 M aqueous solution of HCl, the amino groups on the PMNT chains were completely protonated, and the aqueous system was freeze-dried and recovered.

(46) Next, the protonated PMNT-b-PEG-b-PMNT triblock polymer and poly(acrylic acid) (PAA; Mn: 5,000) where each dissolved in a phosphoric acid buffer solution (50 mM, pH 6.2), thereby preparing a polycationic PMNT-b-PEG-b-PMNT aqueous solution and a polyanionic PAA aqueous solution each having a concentration of 5 mg/ml.

(47) 205 L of an aqueous solution obtained by dissolving 1000 g/ml of indocyanine green (ICG)-labeled bovine serum albumin (ICG-BSA) in a phosphate buffered physiological saline solution (PBS) was added under stirring to 2.76 mL of the aqueous PAA solution prepared in the manner described above. Next, ICG-BSA-encapsulating HiLyte Fluor 647-PIC micelles were prepared by adding a mixed solution consisting of 10 mL of an aqueous solution of the PMNT-b-PEG-b-PMNT triblock polymer and 0.3 mL of an aqueous solution obtained by dissolving 1.67 mg/mL of the HiLyte Fluor 647-labeled PMNT-b-PEG-b-PMNT triblock polymer in a phosphoric acid buffer solution (50 mM, pH 6.2) dropwise under stirring and under ice cooling to the next solution of PAA and ICG-BSA, and then adding 335 L of a phosphoric acid buffer solution (50 mM, pH 6.2). Here, the PIC micelles were prepared so that the PMNT-b-PEG-b-PMNT PAA molar ratio (r) was 1:1 (the molar ratio (r) is as defined above).

Evaluation of In Vivo Release of Proteins from Redox Injectable Gels (RIG) and Evaluation of In Vivo Retention of RIGs

(48) 22 mL of the ICG-BSA-encapsulating HiLyte Fluor 647-PIC micelle solution obtained in Production. Example 11 was condensed to 2 mL by centrifugal evaporation. 200 L of the condensed solution was subcutaneously injected into BALB/c-nu mice. The release of BSA from RIGs and RIG retention were evaluated by imaging a labeled fluorescent dye using an IVIS Spectrum. A fluorescence filter having an excitation wavelength of 745 nm and a fluorescence wavelength of 800 nm was used when imaging ICG-BSA, and a fluorescence filter having an excitation wavelength of 640 nm and a fluorescence wavelength of 680 nm was used when imaging HiLyte Fluor 647-RIG. The results are shown in FIGS. 10 and 11. It was confirmed that RIG-encapsulated BSA remained at the administration site for 14 days and was slowly released. In addition, it was confirmed that the RIG remained at the administration site for 1 month or longer.

Evaluation of In Vivo Retention of Proteins

(49) 200 L of an aqueous solution obtained by dissolving 100 g/mL of ICG-BSA in a phosphate-buffered physiological saline solution (PBS) was subcutaneously injected into BALB/c-nu mice. BSA Retention was evaluated by imaging a labeled fluorescent dye (ICG) using an IVIS Spectrum. The imaging was carried out using a fluorescent filter having an excitation wavelength of 745 nm and a fluorescence wavelength of 800 nm. The results are shown in FIG. 12. It was confirmed that in cases where BSA was administered in isolation, the BSA disappeared from the administration site after 1 day.

Preparation of D-Amino Acid Oxidase (DAO)-Encapsulating PIC Micelles

(50) The powdered PMNT-b-PEG-b-PMNT triblock polymer was dissolved in a 0.1 M aqueous solution of HCl, the amino groups on the PMNT chains were completely protonated, and the aqueous system was freeze-dried and recovered.

(51) Next, the protonated PMNT-b-PEG-b-PMNT triblock polymer and poly(acrylic acid) (PAA; Mn: 5,000) were each dissolved in a phosphoric acid buffer solution (100 mM, pH 6.2), thereby preparing a polycationic PMNT-b-PEG-b-PMNT aqueous solution and a polyanionic PAA aqueous solution each having a concentration of 5 mg/ml.

(52) 347 L of an aqueous solution obtained by dissolving 1 mg/mL of D-amino acid oxidase (DAO) in a phosphoric acid buffer solution (100 mM, pH 6.2) was added under stirring to 2.76 mL of the aqueous PAA solution prepared in the manner described above. Next D-amino acid oxidase (DAO)-encapsulating PIC micelles were prepared by adding 20 mL of an aqueous solution of the PMNT-b-PEG-b-PMNT triblock polymer dropwise under stirring and under ice cooling to a mixed solution of PAA and the D-amino acid oxidase (DAO). Here, the PIC micelles were prepared so that the PMNT-b-PEG-b-PMNT:PAA molar ratio (r) was 1:1 (molar ratio (r)=[number of moles of activated carboxyl groups in PAA]/[number of moles of activated amino groups in PMNT-b-PEG-b-PMNT]).

Preparation of D-Amino Acid Oxidase (DAO)-Encapsulatingredox Injectable Gel (RIG)

(53) 10 mL of the encapsulating PIC micelle solution obtained in Production Example 12 was condensed to 1.5 mL by centrifugal evaporation. 300 l of the condensed solution was diluted to 502.5 l by adding aqueous NaCl solutions having different concentrations, and DAO (D-amino acid oxidase)-encapsulating PIC micelle solutions were prepared, so as to have NaCl concentrations of 0 mM, 150 mM, 300 mM, 500 mM and 1000 mM and PIC concentrations of 20 mg/ml.

Gelation Behavior Caused by Changes in Ionic Strength

(54) The D-amino acid oxidase (DAO)-encapsulating RIG produced in Production Example 13 was placed in a 300 l cell, the transmittance at 600 nm was measured using a UV-VIS apparatus, and gelation was observed when the temperature was gradually increased. The results are shown in FIG. 13. It was confirmed that gelation was facilitated as the ionic strength increased. In addition, with regard to a DAO-free PIC micelle solution, D-amino acid oxidase (DAO)-free PIC micelles (corresponding to a system in which the D-amino acid oxidase of Production Example 12 is not added) were prepared in the same way as the PIC micelles and preparation example described in Production Example 12, and the gelation behavior caused by changes in the ionic strength of the micelle solution was evaluated using the method of Production Example 13. The results of transmittance measurements obtained by altering the temperature and salt concentration are shown in FIG. 14.

Synthesis of Br-PEG(Poly(Ethylene Glycol))-Br

(55) The Br-PEG-Br was synthesized according to Synthesis Scheme 2 shown below:

(56) ##STR00004##

(57) A poly(ethylene glycol) having a hydroxyl group at both terminals (OH-PEG-OH) (Mn: 10,000; 50 g) was dehydrated by means of vacuum drying at 110 C. for 12 hours. Next, 200 ml of THF was added, 10 ml (16 mmol) of butyl lithium and 25 g of dibromoxylene were added thereto, and a reaction was allowed to progress at 50 C. for 24 hours, thereby obtaining Br-PEG-Br, which was brominated at both terminals. The obtained polymer was purified by being precipitated in 2-propanol and vacuum dried. The results of size exclusion chromatography (SEC) measurements and .sup.1H-NMR spectral measurements for the obtained Br-PEG-Br are shown in FIG. 15.

Synthesis of CTA (Chain Transfer Agent)-PEG-CTA (Chain Transfer Agent) (Synthesis of PEG Having a Dithiophenyl Ester at Both Terminals)

(58) The CTA-PEG-CTA was synthesized according to Synthesis Scheme 3 shown below:

(59) ##STR00005##

(60) 2.4 ml of carbon disulfide was added to 50 ml of THF. Next, magnesium benzothiobromide was obtained by gradually adding 6.7 ml (20 mmol) of benzyl magnesium bromide under ice cooling and allowing a reaction to progress. The target CTA (Chain Transfer Agent)-PEG-CTA (Chain Transfer Agent) was obtained by dissolving 50 g of the Br-PEG-Br synthesized in Production Example 14 in 200 mL of THF, adding the entire quantity of the prepared magnesium benzothiobromide, and allowing a reaction to progress at 60 C. for 24 hours. The obtained CTA-PEG-CTA was purified by being precipitated in 2-propanol and vacuum dried. The results of size exclusion chromatography (SEC) measurements and .sup.1H-NMR spectral measurements for the obtained polymer are shown in FIG. 16.

Synthesis of polychloromethylstyrene-b-poly(ethylene glycol)-b-polychloromethylstyrene (PCMS-b-PEG-b-PCMS) Triblock Copolymer

(61) The PCMS-b-PEG-b-PCMS was synthesized according to Synthesis Scheme 4 shown below:

(62) ##STR00006##

(63) The target PCMS-b-PEG-b-PCMS was obtained by adding 10 g of the CTA-PEG-CTA synthesized in Production Example 15, 60 mg of azobisisobutyronitrile (AIBN) to 200 mL of toluene in a nitrogen atmosphere, adding 15 mL of chloromethylstyrene (CMS) and stirring at 60 C. for 24 hours. The obtained polymer was purified by being precipitated in 2-propanol and vacuum dried. The results of size exclusion chromatography (SEC) measurements and .sup.1H-NMR spectral measurements for the obtained PCMS-b-PEG-b-PCMS are shown in FIG. 17.

Synthesis of triblock copolymer containing 2,2,6,6-tetramethylpiperidin-1-oxyl-4-yl (TEMPO) (PMNT-b-PEG-b-PMNT)

(64) The PMNT-b-PEG-b-PMNT was synthesized according to Synthesis Scheme 5 shown below:

(65) ##STR00007##

(66) The target PMNT-b-PEG-b-PMNT was obtained by dissolving 5 g of the PCMS-b-PEG-b-PCMS synthesized in Production Example 16 and 7.7 g of 4-amino TEMPO in DMSO (dimethyl sulfoxide) and stirring at 50 C. so as to allow a reaction to progress. The obtained PMNT-b-PEG-b-PMNT was purified by being precipitated in 2-propanol and vacuum dried. The results of size exclusion chromatography (SEC) measurements and .sup.1H-NMR spectral measurements for the obtained polymer are shown in FIG. 18.

Preparation of Polyion Complex Micelles

(67) 100 mg of the PMNT-b-PEG-b-PMNT triblock copolymer produced in Production Example 17 was dissolved in methanol, and 17.2 mg of PAAc (poly(acrylic acid)) dissolved in water was added to the obtained methanol solution. Next, polyion complex micelles were prepared by dialyzing this solution with water. When the average particle diameter of the obtained polyion complex micelles was measured by dynamic light scattering (DLS), it was confirmed that the polyion complex micelles were unimodal particles having an average particle diameter of 31 nm. (See FIG. 19)