JP5264103B2 - Biodegradable graft copolymer with temperature responsiveness - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biodegradable polymer which shows sol-gel transition type temperature responsivity in responsive to temperature when made into an aqueous solution and its manufacturing method. <P>SOLUTION: The temperature-responsive biodegradable polymer has a biodegradable copolymer containing a hydroxy acid unit and an aspartic acid unit as the main chain and a side chain containing a hydrophilic polyether in the main chain and is specifically represented by formula (A) (wherein R<SP>1</SP>a d R<SP>2</SP>are each hydrogen or a methyl group and R is a hydrophilic polyether). <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to a biodegradable graft copolymer that gels in response to temperature and a method for producing the same.

  Many studies have focused on the physicochemical response of stimuli-responsive polymers to changes in temperature, pH, electric field, and chemicals. In particular, temperature-responsive polymers that exhibit a phase transition phenomenon in response to external temperature changes have been widely studied as medical materials such as drug carriers.

  The homopolymers or copolymers of N-isopropylacrylamide (NIPAAm) disclosed by Non-Patent Documents 1 and 2 are one class. Another class is from hydrophobic poly (propylene oxide) as the middle block and hydrophilic poly (ethylene oxide) as the side block, such as, for example, Pluronic (Poloxamer ™) disclosed by Non-Patent Document 3. This is a triblock copolymer. This triblock copolymer exhibits a behavior of transition from a solution state (sol) to a gel state containing a solvent in response to temperature (hereinafter referred to as sol-gel transition). This is because an aqueous copolymer solution having a specific molecular weight and composition range exists as an aqueous solution at a temperature lower than the sol-gel transition temperature, but when the temperature is higher than the transition temperature (for example, when the temperature rises to body temperature), This is a phenomenon in which an insoluble gel is formed by the interaction between polymers. These polymers, which can be converted from sol to gel in situ without external additives, can be used as medical materials that can be administered by injection into the body. At the time of administration and after administration, there is an advantage that an implant of any desired shape can be formed in a living body minimally invasively without the need for surgical treatment. In other words, a gel containing a physiological (pharmacologically) active substance can be easily prepared by injecting a physiologically (pharmacologically) active substance and a polymer solution into the body. It is possible to release. Moreover, the gel which took in the cell easily can be prepared by injecting into the body what suspended the suitable cell in the polymer solution.

  Thus, materials that gel in situ are attracting attention as implantable drug delivery systems that can be implanted in vivo by injection or as matrices for tissue engineering. In order to function as an ideal injectable system, an aqueous solution of the polymer must exhibit a viscosity that is low enough to be injectable under the preparation conditions and must gel rapidly under physiological conditions (around 37 ° C.). When considered as a medical material, the biocompatibility and safety of the polymer are also important issues. For this reason, the material must be decomposed to a molecular weight that is biodegradable, metabolizable, or excreted outside the body without developing toxicity. In addition, during the degradation, it should not induce the stimulation of living tissue by retaining the properties of the hydrogel rich in water content.

  However, poloxamer-type copolymer (Pluronic) is non-biodegradable, and animal experiments have shown that triglycerides and cholesterol increase when an aqueous solution of poloxamer is injected intraperitoneally (Non-patent Document 4).

  Recently, Jeong et al., Poly (ethylene glycol-block- (DL-lactic acid-random-glycolic acid) -block-ethylene glycol), which is biodegradable and gels in situ); (PEG-PLGA-PEG) A triblock copolymer has been reported (see Patent Document 1).

  Non-Patent Document 5 reports a (PLGA-PEG-PLGA) triblock copolymer.

  However, these gels, which were initially transparent, become opaque with hydrolysis and eventually disintegrate. In addition, it has been pointed out that the change of the morphological structure and the generation of the interface or phase may denature proteins in the living body or may cause cell damage in tissue engineering. Furthermore, it has been pointed out that due to its low molecular weight, the hydrogel has low mechanical strength and poor mechanical compatibility with living tissue.

  In the medical field, it is generally known that when an artificial material having mechanical characteristics different from that of an organ in a living body is implanted, a chain reaction occurs due to a difference in mechanical properties in the living body. There is a demand for the development of materials that have the same mechanical properties as internal organs.

  However, of the biodegradable polymers that have been developed in situ and have been gelled in situ, very few have sufficient mechanical strength and biocompatibility after implantation in vivo.

  For example, in Non-Patent Documents 6 to 7, a mesogen group (cholesterol group) is bonded to the end of a branched block copolymer composed of polyethylene glycol (PEG) and poly (L-lactic acid) having a structure branched into eight. It has been reported that polymers made exhibit a sol-gel transition in response to temperature and exhibit high mechanical strength in the gel state.

  However, in order to synthesize this polymer, it is necessary to procure a special polymer called PEG branched into eight, and after polymerizing a cyclic dimer (lactide) of lactic acid using it, a cholesterol derivative is obtained. There is a need for chemical modification, and there are restrictions on the procurement and manufacturing method of raw materials.

  By the way, while maintaining or improving the excellent properties of polylactic acid-based polymers such as poly (L-lactic acid), attempts have been made to expand applications and control physical properties by chemical modification. For example, Non-Patent Documents 8 to 11 describe polymer synthesis techniques such as random and block copolymerization with a cyclic comonomer having a functional group, polymerization reaction using a functional molecule having a hydroxyl group as a starting species, and graft polymerization. Utilizing it, the synthesis of lactic acid copolymers with various molecular forms (random, block, graft) and chemical properties (reactive functional groups, hydrophilicity / hydrophobicity) has been presented.

  Specifically, 1) a depsipeptide-lactic acid random copolymer having a reactive functional group on the side chain (Non-Patent Documents 12 to 15), and 2) a depsipeptide-lactic acid block having a reactive functional group on the side chain. Polymers (Non-Patent Documents 16 to 17), 3) Polylactic acid grafted polysaccharides (Non-Patent Documents 18 to 19), 4) Branched polyether-polylactic acid block copolymers (Non-Patent Documents 20 to 21), etc. It is synthesized.

Thus, various types of polylactic acid polymers have been developed, but they are biocompatible, exhibit a sol-gel transition in response to temperature, and exhibit high mechanical strength in the gel state. At present, biodegradable materials that can be synthesized from easily available materials have not yet been obtained.
US Patent No. 6117949 Bae et al. Makromol. Chem. Rapid Commun., 8, 481-485 (1987) Chen et al. Nature, 373, 49-52 (1995) Malston et al. Macromolecules, 25, 5440-5445 (1992) Wout et. Al, J. Parenteral Sci. & Tech., 46, 192-200 (1992) Doo Sung Lee, Macromol. Rapid Commun. 2001, 22, 587. 55th Annual Meeting of the Polymer Society of Japan Polymer Science Society Proceedings Vol. 55, 1935 (issued on May 10, 2006) The 35th Medical Polymer Symposium Proceedings of the Society of Polymer Science, Japan, 23-24 pages (issued August 1, 2006) Yuichi Oya: Present status and new development of biodegradable polymers. Artificial organ 1999, 28: 582-589 Yuichi Oya, Goro Ouchi: New polylactic acid polymers as biodegradable biomaterials. Polymer processing 1999, 48: 530 Yuichi Oya: Synthesis of a novel biodegradable polymer based on polylactic acid and its application as a biomaterial. Polymer Papers 2002, 59: 484-498 Ouchi Goro, Oya Yuichi: New polylactic acid-based medical materials. Future materials 2002, 2: 30-35. Ouchi T, et al: Macromol Chem Rapid Commun 1993, 14: 825-831. Ouchi T, et al: Macromol Chem Phys 1996, 197: 1833-1833. Ouchi T, et al: J Polym Sci Part A: Polym Chem 1997, 35: 377-383. Ouchi T, et al: J Poly Sci Part A: Poly Chem 1998, 36: 1283-1290. Ouchi T, et al: Designed Monom Polym 2000, 3: 279-287. Ouchi T, et al: J Polym Sci Part A: Polym Chem 2002, 40: 1218-1225. Ohya Y, et al: Macromolecules 1998, 31: 4662-4665. Ohya Y, et al: Macromol Chem Phys 1998, 199: 2017-2022. Nagahama K, et al: Polym J 2006, 38: 852-860. Nagahama K, et al: Macromol Biosc 2006, 6: 412-419.

  An object of the present invention is to provide a biodegradable polymer exhibiting a temperature-responsive sol-gel transition having sufficient mechanical strength in a biocompatible and gel state, and a simple production method thereof. Another object of the present invention is to provide a medical material using the biodegradable polymer.

  As a result of intensive studies to solve the above problems, the present inventor has a biodegradable copolymer containing a hydroxy acid unit (for example, a lactic acid unit, a glycolic acid unit, etc.) and an aspartic acid unit as a main chain, It has been found that biodegradable graft copolymers having side chains containing hydrophilic polyethers can form gels with high mechanical strength in situ in response to temperature. Based on this knowledge, this has been further developed to complete the present invention.

  That is, this invention provides the following temperature-responsive biodegradable polymer and its manufacturing method.

  Item 1. A biodegradable polymer having temperature responsiveness, wherein a main chain is a biodegradable copolymer containing a hydroxy acid unit and an aspartic acid unit, and the main chain has a side chain containing a hydrophilic polyether.

  Item 2. Formula (A):

(Wherein R ¹ and R ² independently represent a hydrogen atom or a methyl group, R represents a hydrophilic polyether, x represents 20 to 1000, y represents 0 to 100, and z represents 2 to 2) 1000, z / (x + y + z) is 0.002 to 0.5, and the arrangement of each unit of x, y and z is not limited to the order of the arrangement.)
Item 2. A biodegradable polymer having temperature responsiveness according to item 1.

  Item 3. Item 3. The biodegradable polymer having temperature responsiveness according to Item 2, wherein the hydrophilic polyether represented by R in the general formula (A) is polyalkylene glycol.

  Item 4. In the general formula (A), the hydrophilic polyether represented by R is represented by the general formula (B):

(In the formula, R ³ represents a methyl group, an ethyl group or a hydrogen atom, and n represents 1 to 500.)
Item 3. A biodegradable polymer having temperature responsiveness according to Item 2, which is a group represented by:

  Item 5. Item 5. The biodegradable polymer having temperature responsiveness according to any one of Items 1 to 4, wherein the number average molecular weight (Mn) is 3,000 to 500,000.

  Item 6. In the general formula (A), x is 50 to 200, y is 0 to 20, z is 5 to 100, x / (x + y + z) is 0.6 to 0.95, z / Item 6. The temperature-responsive biodegradable polymer according to Item 3, 4 or 5, wherein (x + y + z) is 0.05 to 0.4 and n is 40 to 200.

  Item 7. Item 7. The biodegradable polymer having temperature responsiveness according to any one of Items 1 to 6, wherein the ratio (Mw / Mn) of the weight average molecular weight to the number average molecular weight is 1.00 to 5.00.

  Item 8. Item 8. The biodegradable polymer having temperature responsiveness according to any one of Items 1 to 7, wherein the storage elastic modulus of the hydrogel formed at a temperature of 37 ° C. is 50 to 50,000 Pa when the aqueous solution has a concentration of 20 wt%.

  Item 9. Formula (A):

(Wherein R ¹ and R ² independently represent a hydrogen atom or a methyl group, R represents a hydrophilic polyether, x represents 20 to 1000, y represents 0 to 100, and z represents 2 to 2) 1000, z / (x + y + z) is 0.002 to 0.5, and the arrangement of each unit of x, y and z is not limited to the order of the arrangement.)
A temperature-responsive biodegradable polymer represented by the general formula (C):

(Wherein R ¹ , R ² , x, y and z are the same as above)
A carboxylic acid compound represented by the general formula (D):
HO-R (D)
(Wherein R is the same as above)
A production method comprising the condensation reaction of an alcohol represented by the formula:

  Item 10. The medical material containing the biodegradable polymer which has the temperature responsiveness in any one of said items 1-8.

  The biodegradable polymer of the present invention exhibits a temperature-responsive sol-gel transition, and is characterized in that it can be gelled particularly at a temperature between room temperature and body temperature. Moreover, a stable biodegradable hydrogel having sufficiently high mechanical strength is formed in vivo. Therefore, it is possible to provide an excellent in situ gelation system, eliminate the need for surgical treatment, and form an implant with minimal invasiveness by injection. Since physiologically (pharmacologically) active substances and the like can be stably encapsulated and released gradually in the body, an injectable (injectable) drug delivery system can be provided. Furthermore, it can also be used as a scaffold for injectable tissue repair and organ regeneration, or in a cell delivery system for transplanting cells into a defect site of in vivo tissue by injection administration by encapsulating suitable living cells. .

  In addition, the method for producing a temperature-responsive biodegradable polymer of the present invention has a feature that it can be carried out easily and in a high yield by using a commercially available reagent that is relatively easily available.

1. Biodegradable polymer having temperature responsiveness The biodegradable polymer having temperature responsiveness of the present invention has a biodegradable copolymer containing a hydroxy acid unit and an aspartic acid unit as a main chain, and contains a hydrophilic polyether. It is a graft copolymer having a side chain.

  Examples of the hydroxy acid constituting the main chain include lactic acid, glycolic acid, tartaric acid, malic acid, and mevalonic acid, and α-hydroxy acids such as lactic acid and glycolic acid are preferred. Hydroxy acid may contain 1 type (s) or 2 or more types.

  The side chain is formed using the side chain carboxylic acid of the aspartic acid unit constituting the main chain as a foothold. Examples of the side chain include hydrophilic polyethers, for example, those containing alkylene glycols such as ethylene glycol and propylene glycol. It is not limited to these.

  Specific examples of the temperature-responsive biodegradable polymer of the present invention include a general formula (A):

(Wherein R ¹ and R ² independently represent a hydrogen atom or a methyl group, R represents a hydrophilic polyether, x represents 20 to 1000, y represents 0 to 100, and z represents 2 to 2) 1000, z / (x + y + z) is 0.002 to 0.5, and the arrangement of each unit of x, y, and z is not limited to the order of the arrangement. That is, a hydrophilic chain derived from a main chain containing a lactide (lactic acid dimer) unit or glycolide (glycolic acid dimer) unit, a lactic acid unit or glycolic acid unit, and an aspartic acid unit, and a side chain carboxylic acid of aspartic acid. And a side chain containing a functional polyether.

R ¹ and R ² each independently represent a hydrogen atom or a methyl group, preferably R ¹ is a methyl group and R ² is a hydrogen atom. When R ¹ and R ² are methyl groups, the carbon atom to which the methyl group is bonded can be an asymmetric carbon. In the polymer of the present invention, the configuration of the asymmetric carbon may be (R) isomer, (S) isomer, or a mixture thereof.

Here, x, y, and z represent the average number of each unit in the polymer, and are determined from ¹ H-NMR and GPC. Further, as described above, the arrangement of each unit of x, y, and z may be random or block.

  X in the polymer of the present invention is 20 to 1000, preferably 50 to 200, and more preferably 100 to 200. The lactide unit and glycolide unit are composed of a dimer of lactic acid and a dimer of glycolic acid, respectively, and are derived from lactide (a cyclic dimer of lactic acid) and glycolide (a cyclic dimer of glycolic acid), respectively. . A lactide unit is preferred. The lactide unit may be composed of either L-lactic acid and / or D-lactic acid, but is composed of a mixture of D-lactic acid and L-lactic acid in which the main chain is amorphous, in particular, an equivalent mixture of both. What is constructed is preferred.

  y is 0-100, Preferably it is 0-20, More preferably, it is 0-10.

  z is 2-1000, Preferably it is 5-100, More preferably, it is 10-20.

  x / (x + y + z) is 0.5 to 0.99, preferably 0.6 to 0.95, and more preferably 0.8 to 0.9. When x is in this range, suitable temperature responsiveness and biodegradability are imparted to the polymer of the present invention.

  z / (x + y + z) is 0.002 to 0.5, preferably 0.05 to 0.4, and more preferably 0.06 to 0.2. When z is in this range, the temperature-responsive sol-gel transition property and high mechanical strength in the gel state are imparted to the polymer aqueous solution of the present invention by the balance between the hydrophilic side chain and the hydrophobic main chain. .

  Specific examples of the hydrophilic polyether represented by R include, for example, the general formula (B):

(Wherein R ³ represents a methyl group, an ethyl group or a hydrogen atom, and n represents 1 to 500). n is preferably 3 to 100, more preferably 5 to 50.

  The hydrophilic polyether represented by R is not particularly limited as long as it is a chain that exhibits hydrophilicity and satisfies the dehydration property at an appropriate temperature. Specific examples include polyethylene glycol, methoxy- or ethoxy-polyethylene glycol. Preferred is methoxy-polyethylene glycol. In an aqueous solution, this chain dehydrates as the temperature rises, causing aggregation of the hydrophobic main chain polymer and imparting a property showing a temperature-responsive sol-gel transition.

  For the sol-gel transition temperature, decomposition rate, and mechanical strength in the gel state of the polymer of the present invention, the molecular weight of the main chain polymer and side chain polyether in the polymer, the introduction rate of the polyether, etc., should be selected appropriately And can be easily adjusted by adjusting the concentration of the polymer.

  The aqueous liquid of the temperature-responsive biodegradable polymer of the present invention is stored below the gelling temperature and can be administered parenterally by intramuscular, intraperitoneal, subcutaneous or similar injection methods.

  The number average molecular weight (Mn) of the polymer of the present invention is 3,000 to 500,000, preferably 10,000 to 50,000, more preferably 10,000 to 25,000, and the weight average molecular weight (Mw) is It is 3,000 to 1,000,000, preferably 10,000 to 100,000, more preferably 10,000 to 30,000. The ratio of the weight average molecular weight to the number average molecular weight (Mw / Mn), which is an index of molecular weight distribution, is 1.00 to 5.00, preferably 1.05 to 3.00, more preferably 1.05 to 2. .00. The number average molecular weight and the weight average molecular weight can be measured using a known method such as GPC.

  The storage elastic modulus of the hydrogel formed at a temperature of 37 ° C. when the polymer of the present invention is an aqueous solution of 20 wt% is 50 to 50,000 Pa, preferably 100 to 30,000 Pa, more preferably 500 to 30, 000 Pa.

For example, the polymer of the present invention is dissolved in a phosphate buffered saline solution having a pH of 7.4 at a concentration of 20 wt% in a test tube, a hydrogel is formed at 37 ° C., and the weight (initial weight) is measured. The hydrogel was immersed in phosphate buffered saline (pH 37) at pH = 7.4 and allowed to stand for 60 days. The weight of the hydrogel was 0 to 80% with respect to the initial weight (100%) at the time of hydrogel formation. , Preferably 0 to 50%.
2. Method for Producing Temperature Responsive Biodegradable Polymer The polymer of the present invention can be produced, for example, as follows.

(In the formula, Bzl represents a benzyl group, and R ¹ , R ² , R, x, y and z are the same as above.)
The compound represented by the above (1) can be produced according to, for example, T. Ouchi, et. Al., Macromol. Chem. Phys. 197, 1823-1833 (1996), etc. ) Is a commercially available compound.

Subsequently, the compound represented by the above (1) (2) is subjected to ring-opening polymerization using a catalyst (such as tin 2-ethylhexanoate) to obtain a compound represented by the above (3). The side chain benzyl ester is converted to a free carboxylic acid by a catalyst (trifluoromethanesulfonic acid: TFMSA, etc.) or hydrogen reduction (H ₂ , Pd / C, etc.) to convert the carboxylic acid compound represented by (C) above. obtain. This reaction can be carried out using a conventional method.

  Furthermore, by reacting the carboxylic acid compound represented by the above (C) with the alcohol compound represented by the general formula (D) in the presence of a condensing agent (dicyclohexylcarbodiimide: DCC, DMAP, etc.), the above (A) The graft copolymer represented by these is obtained.

In this reaction, the alcohol compound represented by the general formula (D) can be generally used in an amount of 1.2 to 2 equivalents with respect to the carboxylic acid compound represented by the above (C). For example, methylene chloride, tetrahydrofuran, or dimethylformamide can be used as the reaction solvent, and the reaction temperature is about 0 to 60 ° C. The amount of the condensing agent is usually 1.2 to 2 equivalents with respect to the carboxylic acid compound represented by the above (C). For example, with respect to the carboxylic acid compound represented by (C) above, DCC is about 1.2 to 2 equivalents and DMAP is a catalytic amount.
3. Properties and Applications of Temperature Responsive Biodegradable Polymer As described above, the polymer of the present invention has sufficient mechanical strength in a gel state in addition to good biocompatibility, biodegradability and temperature responsiveness. . This is advantageous in that the hydrogel can be given mechanical strength equivalent to that of an organ in the living body.

  Here, the temperature responsiveness generally refers to a property in which an aqueous solution of a compound exhibits a reversible sol-gel transition phase separation behavior with a lower critical solution temperature (LCST) as a boundary. Specifically, when heated to a temperature of LCST or higher, it becomes cloudy and becomes a gel state, and when cooled to a temperature lower than that, it dissolves again and returns to a transparent sol state.

  In this specification, the LCST of a polymer is determined by measuring the change in viscosity of an aqueous polymer solution by a test tube inversion method. Alternatively, it can be determined by a dynamic viscoelasticity test.

  The polymer of the present invention usually has LCST in the range of about 10 to 56 ° C., and LCST can be easily adjusted in this range. In particular, the above properties are suitably exhibited in the case of the compound represented by the general formula (A).

  The polymers of the present invention have a very wide range of applications. In particular, it can be used as a medical material together with a drug. Specifically, when the polymer of the present invention has LCST in the range of 25 to 35 ° C, LCST exists between room temperature (for example, about 10 to 20 ° C) and body temperature (about 35 to 37 ° C). It can be administered into the body by injection in the form of a solution, and a hydrogel can be formed in the body. An aqueous solution of such a polymer is in a solution state near room temperature and is easy to handle at the time of injection. On the other hand, it is insoluble in the vicinity of body temperature, so that it becomes insoluble after administration in the body and suppresses early diffusion of the drug. It is possible to improve the retention of the drug at a specific site. Therefore, it can be suitably used as a biodegradable polymer material in an injectable preparation, particularly a long-lasting injectable preparation. Examples of the administration form include subcutaneous injection and intramuscular injection.

  Furthermore, the polymer of the present invention is excellent in safety because it has no or very low cytotoxicity in its degradation product.

  Furthermore, the polymer of the present invention has good mechanical strength in the gel state. For example, when a polymer aqueous solution having a concentration of 20 wt% is brought to a temperature of 37 ° C., the produced hydrogel has a storage elastic modulus of 50 to 50000 Pa, preferably 500 to 30,000 Pa.

  The blending amount of the polymer in the pharmaceutical composition can be appropriately selected depending on the type of drug used and the like. For example, it may be about 60 to 99.9% by weight with respect to the total weight of the pharmaceutical composition.

  The drug used in the pharmaceutical composition is not particularly limited, but has physiologically active peptides, proteins, other antibiotics, antitumor agents, antipyretic agents, analgesics, anti-inflammatory agents, antitussive expectorants, sedatives, Muscle relaxant, antiepileptic agent, antiulcer agent, antidepressant, antiallergic agent, cardiotonic agent, arrhythmia agent, vasodilator, antihypertensive diuretic, diabetes agent, anticoagulant, hemostatic agent, antituberculosis agent, Hormones, narcotic antagonists and the like.

  The amount of the drug in the pharmaceutical composition of the present invention can be appropriately selected depending on the type of drug. In particular, in the case of a sustained injection, the amount of the drug is determined by the type of drug, the period of sustained release, and the like. For example, when the drug is a peptide, in order to obtain a sustained release preparation of about 1 week to about 1 month, it is usually contained in an amount of about 0.001% to 50% by weight with respect to the total weight of the pharmaceutical composition. Good.

  Further, the polymer of the present invention has temperature responsiveness, biodegradability, and safety to living bodies, and therefore can be used as a tissue adhesion preventing material after surgery. Adhesion can be prevented by covering visceral tissue after operation by application or spraying and isolating it from other living tissue for a certain period of time.

  Furthermore, the polymer of the present invention can be applied as a scaffold for regenerative medicine, a cell culture substrate and the like. As the scaffold, cells, the polymer of the present invention, a culture solution, and the like are mixed in a sol state at a low temperature, and the mixture is gelled into a predetermined shape at a high temperature to be used as a scaffold. As a cell culture substrate, a fibrous or porous substrate having a predetermined three-dimensional shape is impregnated with a liquid mixture containing cells, the polymer of the present invention and a culture solution, and the viscosity or gel is increased at a predetermined temperature. It is also possible to hold regenerated cells in the substrate. As the fibrous or porous substrate, a material having high biocompatibility such as collagen and hydroxyapatite can be used, which is particularly effective for regeneration of cartilage tissue or bone tissue. Furthermore, it is possible to provide a system for transplanting cells into damaged tissue (cell delivery).

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. Hereinafter, the polymer of the present invention is also referred to as a poly [(Glc-Asp) -r-DL-LA] -g-PEG copolymer or simply a graft copolymer.
[Example 1] Synthesis of graft copolymer (Fig. 1)
(1) 470 mg (1.79 mmol) of cyclodepsipeptide [Glc-Asp (OBzl)] and 1100 mg (7.64 mmol) of DL-lactide were placed in a polymerization tube and dried on the vacuum line the previous day. Next, in a glove box filled with nitrogen gas, 3.8 mg (9.4 μmol) of 2-ethylhexanoic acid tin (II) dissolved in 100 μl of THF was added to the polymerization tube, and a two-way cock with a slot was added. Covered. Thereafter, the polymerization tube was taken out from the glove box, connected to a vacuum line, freeze-dried with liquid nitrogen to remove THF, and deaeration and nitrogen replacement were repeated 10 times. Thereafter, the polymerization tube was sealed, immersed in a 160 ° C. oil bath to completely melt the monomer, and then held in a 135 ° C. oil bath for 24 hours to perform a ring-opening copolymerization reaction. After completion of the reaction, the reaction mixture was dissolved in a small amount of chloroform and poured into a large amount of methanol to form a precipitate. The collected precipitate was dried under reduced pressure for 24 hours using a vacuum pump to obtain 1,360 mg of poly {[Glc-Asp (OBzl)]-r-DL-LA}.
(2) 635 mg (190 μmol) of the compound obtained in (1) above was dissolved in 15 ml of trifluoroacetic acid, and 1.7 ml (1.67 mmol) of 1M-TFMSA was added under ice cooling and stirred for 1 hour. . The reaction mixture was then precipitated into a large amount of cold diethyl ether. The obtained precipitate was collected and dried under reduced pressure for 24 hours using a vacuum pump to obtain 430 mg of poly [(Glc-Asp) -r-DL-LA].
¹ H NMR (CDCl ₃ ), δ (ppm); 1.5 (3H, CHCH ₃ ), 4.6 (2H, OCH ₂ CO), 4.9 (2H, CH ₂ COOH), 5.1 (1H , CH).
(3) After dissolving 120 mg (0.22 mmol) of 550 methoxy PEG (n is about 12) in 2 ml of chloroform, 54 mg (0.26 mmol) of dicyclohexylcarbodiimide (DCC) was added as a solid, Stir for 2 hours. Thereafter, 300 mg (16.7 μmol) of poly [(Glc-Asp) -r-DL-LA] and 8 mg (65.6 μmol) of DMAP dissolved in 1 ml of methylene chloride were added and stirred at room temperature for 24 hours. The generated DCUre was removed by filtration, and the collected filtrate was added dropwise to n-hexane / ethanol (7/3) to form a precipitate. The collected precipitate was dried under reduced pressure for 24 hours to obtain 327 mg of poly [(Glc-Asp) -r-DL-LA] -g-PEG copolymer (FIG. 1). FIG. 2 shows ¹ H NMR (CDCl ₃ ) of the compound poly [(Glc-Asp) -r-DL-LA] -g-PEG copolymer.
¹ H NMR (CDCl ₃ ), δ (ppm); 1.5 (3H, CHCH ₃ ), 3.0 (2H, CHCH ₂ ), 3.4 (4H, OCH ₂ CH ₂ ), 4.6 (2H , OCH ₂ CO), 4.9 (2H, CH ₂ COO), 5.1 (1H, CHCH ₃ ).

[Examples 2-3]
By changing the ratio of cyclodepsipeptide [Glc-Asp (OBzl)] and DL-lactide used using the raw materials shown in Table 1 and the molecular weight of methoxy PEG used, the reaction was carried out in the same manner as in Example 1 above. The poly [(Glc-Asp) -r-DL-LA] -g-PEG copolymer of Examples 2-3 was obtained.

[Test Example 1] Molecular weight measurement The number average molecular weight (Mn) and molecular weight distribution (Mw / Mn) of the graft copolymers of Examples 1 to 3 were subjected to gel filtration chromatography (GPC; manufactured by TOSOH, Tosoh GPC-8020 series system). It was measured by. The results are shown in Table 1.

  3 mg of the polymer was dissolved in 0.5 ml of DMF, and this was passed through a 0.2 μm pore filter to remove solids such as dust. Thereafter, the polymer was driven into the apparatus using a syringe.

[Test Example 2] Temperature Responsive Behavior of Poly [(Glc-Asp) -r-DL-LA] -g-PEG Copolymer Aqueous Solution Poly [(Glc-Asp) -r-DL- in Examples 1-3 The temperature-responsive gelation behavior of LA] -g-PEG copolymer aqueous solution was investigated at different concentrations.

  The sol-gel transition was measured at a temperature increase of 1 ° C. per process by the test tube inversion method. A graft copolymer aqueous solution was prepared in a sample tube (inner diameter: 15 mm), and the viscosity change of the 5-25 wt% graft copolymer aqueous solution was observed in a temperature range of 10 ° C. to 60 ° C. This sample tube was immersed in an oil bath for 15 minutes. By tumbling the vial, the sol-gel transition temperature was monitored and if it did not flow for 30 seconds it was considered a gel. This transition temperature was measured with an accuracy of ± 1 ° C. Table 1 shows the phase transition temperature range of the 20 wt% graft copolymer aqueous solution and the properties (whether it is a gel) after the phase transition. In addition, a phase diagram of the poly [(Glc-Asp) -r-DL-LA] -g-PEG copolymer aqueous solution of Examples 1 to 3 was prepared as a function of temperature and graft copolymer concentration (FIG. 3). ).

  In FIG. 3, a gel-like shape is shown in a range surrounded by a curve, a sol state is shown below this, and it is separated (precipitated) into two layers above this. The photographs shown in FIG. 3 are photographs of gel state and sol state of a 20 wt% aqueous solution of the graft copolymer of Example 2. This revealed that the graft copolymer exhibited gelation in response to temperature changes in a specific composition. It was also found that the gelation temperature can be adjusted by changing the copolymer composition (graft chain length, number of grafts).

  In particular, in the graft copolymer of Example 3, gelation at a physiological temperature (for example, body temperature of about 37 ° C.) was particularly prevalent, so that it is useful as a base material for drug and cell delivery purposes. It became clear.

Test Example 3 Dynamic Viscoelasticity Test of Poly [(Glc-Asp) -r-DL-LA] -g-PEG Copolymer Aqueous Solution Poly [(Glc-Asp) -r-DL of Examples 1 to 3 The sol-gel transition phenomenon in an aqueous solution of [LA] -g-PEG copolymer (20 wt%) was examined using a dynamic mechanical rheometer (HAAKE RS600, HAAKE) (FIG. 4).

This aqueous graft copolymer solution was placed between parallel plates having a diameter of 25 mm and a gap interval of 0.5 mm. Data was collected controlling the stress (4.0 dyne / cm ² ) and frequency (1.0 rad / sec). The heating rate was 0.5 ° C./min. In FIG. 4, G ′ represents the storage elastic modulus, and G ″ represents the loss elastic modulus. The storage elastic modulus of the graft copolymer aqueous solution in a sol state at room temperature was about 0.6 Pa, but the storage elastic modulus increased remarkably with increasing temperature. For example, the graft copolymer of Example 2 becomes a hydrogel having a storage elastic modulus of 490 Pa at around 37 ° C., and poly (lactic acid-random-glycolic acid) —a biodegradable gelling polymer reported so far. The storage modulus of polyethylene glycol-poly (lactic acid-random-glycolic acid) triblock copolymer (PLGA-PEG-PLGA) greatly exceeded about 80 Pa.

[Test Example 4]
The graft copolymer of Example 2 was subjected to a dynamic viscoelasticity test in phosphate buffered saline (PBS) (pH = 7.4, ionic strength I = 0.14). The result is shown in FIG. The graft copolymer exhibits a storage elastic modulus of 1095 Pa at around 35 ° C., which is a temperature between room temperature and body temperature, and has a higher ionic strength of the solution. It has been shown. Thus, the hydrogel formed from the graft copolymer of the present invention has a high storage elastic modulus, and is confirmed to be useful as a base material for in situ gelled drug delivery and cell delivery purposes. It was.

[Test Example 5] Poly [(Glc-Asp) -r-DL-LA] -g-PEG Copolymer Gel Degradation Test poly [(Glc-Asp) -r-DL-LA] -g of Example 2 The degradability behavior of the -PEG copolymer gel in phosphate buffered saline (PBS) (pH = 7.4, ionic strength I = 0.14) at 37 ° C. (in vitro) was examined. In a test tube, the graft copolymer was dissolved in phosphate buffered saline having a pH of 7.4 at a concentration of 20 wt%, and a hydrogel was formed at 37 ° C. After measuring this weight (initial weight), phosphate buffered saline (pH 37 ° C.) of pH = 7.4 was added and immersed. The weight of the gel was determined by removing the supernatant liquid every predetermined time and weighing the weight of the gel remaining in the test tube. The weight of the gel was expressed as a relative value (%) when the initial weight at the time of gel preparation was 100% (FIG. 6).

  The weight of the gel increased with time, indicating that the gel swelled due to a decrease in the degree of crosslinking as it degraded. After 14 days, the weight of the gel reached 2.2 times the initial weight at the time of gel preparation. Gel elution started after 14 days and the weight gradually decreased, but the gel state was maintained for 60 days. Thereafter, the weight gradually decreased, and finally the shape of the hydrogel collapsed and dissolved in PBS. From this, it was shown that poly [(Glc-Asp) -r-DL-LA] -g-PEG copolymer exhibits degradation characteristics applicable as an implant-type biomaterial.

The synthesis method of the poly [(Glc-Asp) -r-DL-LA] -g-PEG copolymer of Example 1 is shown. ¹ H NMR of the poly [(Glc-Asp) -r-DL-LA] -g-PEG copolymer of Example 1 is shown. It is a graph which shows the phase diagram regarding the temperature and the density | concentration of the temperature-responsive sol-gel transition phenomenon of the poly [(Glc-Asp) -r-DL-LA] -g-PEG copolymer aqueous solution of Examples 1-3. The temperature dependence of temperature, storage elastic modulus (G ′) and loss elastic modulus (G ″) in the aqueous copolymer solution (20 wt%) and the PLGA-PEG-PLGA aqueous solution (20 wt%) of Examples 1 to 3 It is the shown graph. The graph which shows the temperature dependence response property of a storage elastic modulus in the phosphate buffered saline (PBS) of Example 2 (pH = 7.4, ionic strength I = 0.14, 20 wt%). It is. 3 is a graph showing the degradability behavior of the copolymer of Example 2.

Claims (9)

Formula (A):

(Wherein R ¹ and R ² independently represent a hydrogen atom or a methyl group, R represents a hydrophilic polyether, x represents 20 to 1000, y represents 0 to 100, and z represents 2 to 2) 1000, z / (x + y + z) is 0.002 to 0.5, and the arrangement of each unit of x, y and z is not limited to the order of the arrangement.)
A biodegradable polymer having temperature responsiveness represented by The biodegradable polymer having temperature responsiveness according to claim 1, wherein the hydrophilic polyether represented by R in the general formula (A) is a polyalkylene glycol. In the general formula (A), the hydrophilic polyether represented by R is represented by the general formula (B):

(In the formula, R ³ represents a methyl group, an ethyl group or a hydrogen atom, and n represents 3 to 500.)
The biodegradable polymer having temperature responsiveness according to claim 1, which is a group represented by the formula: The biodegradable polymer having temperature responsiveness according to any one of claims 1 to 3, which has a number average molecular weight (Mn) of 3,000 to 500,000. In the general formula (A), x is 50 to 200, y is 0 to 20, z is 5 to 100, x / (x + y + z) is 0.6 to 0.95, z / The temperature-responsive biodegradable polymer according to claim 3 or 4 , wherein (x + y + z) is 0.05 to 0.4 and n is 40 to 200. The ratio of the weight average molecular weight to the number average molecular weight (Mw / Mn) is 1.00 to 5.00, The biodegradable polymer having temperature responsiveness according to any one of claims 1 to 5. The biodegradable polymer having temperature responsiveness according to any one of claims 1 to 6, wherein the storage elastic modulus of the hydrogel formed at a temperature of 37 ° C when the aqueous solution has a concentration of 20 wt% is 50 to 50,000 Pa. Formula (A):

(Wherein R ¹ and R ² independently represent a hydrogen atom or a methyl group, R represents a hydrophilic polyether, x represents 20 to 1000, y represents 0 to 100, and z represents 2 to 2) 1000, z / (x + y + z) is 0.002 to 0.5, and the arrangement of each unit of x, y and z is not limited to the order of the arrangement.)
A temperature-responsive biodegradable polymer represented by the general formula (C):

(Wherein R ¹ , R ² , x, y and z are the same as above)
A carboxylic acid compound represented by the general formula (D):
HO-R (D)
(Wherein R is the same as above)
A production method comprising the condensation reaction of an alcohol represented by the formula: The medical material containing the biodegradable polymer which has the temperature responsiveness in any one of the said Claims 1-7.

Images