Scaffold base material for cell culture and scaffold for cell culture

Abstract

To provide a scaffold base material for cell culture which enables the binding of biofunctional molecules, is moldable into various shapes, and is capable of retaining the molded shape.SOLUTION: A scaffold base material for cell culture comprises a polymer having a repeating unit represented by formula (1) and, optionally, a repeating unit represented by formula (2), as well as a biodegradable polyester, wherein the content of the polymer is 1 to 300 pts.mass per 100 pts.mass of the biodegradable polyester (where, the symbols in the formulas below are as defined in the specification).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a scaffold material for cell culture to which a biofunctional molecule can be bound. The present invention also relates to a scaffold material for cell culture to which a biofunctional molecule is bound. [Background technology]

[0002] In recent years, with the progress of stem cell research such as ES cells and iPS cells, there has been active research into regenerative medicine, which uses cultured cells to treat defective or diseased areas. The cells used in regenerative medicine must be cultured to the required amount before transplantation and kept in the defective area. Therefore, a known treatment method is to place cultured cells together with a flat or three-dimensionally formed biodegradable scaffold in the defective area. As the cells grow in the body, the scaffold gradually degrades in the body, and finally the scaffold disappears, and the defective area becomes a tissue filled with cells, completing the treatment.

[0003] Humans are composed of a wide variety of cell types, and each of these cell types differentiates and matures in different environments while interacting with the extracellular matrix, biofunctional molecules, etc. in the body. Therefore, when culturing cells outside of a body, culturing using a scaffold carrying the extracellular matrix or biofunctional molecules appropriate for the cell type is known to have various effects, such as more efficient cell proliferation and improved differentiation efficiency, compared to conventional culture using polystyrene as a scaffold.

[0004] For example, Non-Patent Document 1 reports a technology in which epidermal growth factor bound to 3,4-dihydroxyphenylalanine is immobilized on the surface of an inorganic material and NRK49F cells are cultured on the surface to improve the proliferation effect. However, inorganic materials are difficult to use as materials for regenerative medicine because they do not decompose in vivo.

[0005] For example, Non-Patent Document 2 reports a technique in which the initial adhesion amount of human gingival fibroblasts is improved by hydrolyzing polylactic acid with an aqueous sodium hydroxide solution to carboxylate the surface, and then immobilizing fibronectin through a condensation reaction. However, since polylactic acid is hydrolyzed, the strength of the scaffold may be impaired depending on the treatment time.

[0006] For example, Non-Patent Document 3 reports a technique in which a caprolactone-cyclic depsipeptide copolymer bound to a REDV peptide is cast onto a stainless steel mandrel and then placed in the abdominal aorta of a rat, forming an endothelial layer with vascular endothelial cells attached. However, the depsipeptide unit bound to the REDV peptide contains an amide bond. Therefore, if it is desired to increase the REDV peptide content, it is necessary to increase the ratio of the depsipeptide unit, which may result in a decrease in biodegradability.

[0007] Therefore, in the field of cell culture, there has been a demand for biodegradable scaffolds to which biofunctional molecules can be attached, which can be molded into various shapes, and which can maintain the obtained shapes. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Advanced Science, 2021, Vol. 8, issue 16, 2100961 [Non-Patent Document 2] JOURNAL OF BIOMATERIALS APPLICATIONS Volume 21, July 2006, 33-47 [Non-Patent Document 3] Biomacromolecules, 2020, 21, 3092-3101 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a scaffold material for cell culture to which biofunctional molecules can be bound, which can be molded into various shapes, and which can maintain those shapes. Another object of the present invention is to provide a scaffold for cell culture in which biofunctional molecules are bound to the scaffold material for cell culture. [Means for solving the problem]

[0010] The present invention capable of achieving the above object is as follows. [1] Equation (1):

[0011] [ka]

[0012] (In the formula, * represents a bonding position, a represents 0 or 1; A represents a divalent organic group. X is a halogen atom, an amino group, a sulfanyl group, an azide group, or a group represented by any one of the formulas (1a) to (1f):

[0013] [ka]

[0014] (wherein * represents a bond position, and R 1 represents a hydrogen atom or a methyl group. and m represents a number from 1 to 300. and optionally having a repeating unit represented by formula (2):

[0015] [ka]

[0016] (wherein * represents a bond position, and n represents a number from 0 to 2,700. and a ratio of n / m is within the range of 0 / 100 to 99 / 1; and Biodegradable polyester A scaffold substrate for cell culture comprising: A scaffold for cell culture, wherein the content of the polymer is 1 to 300 parts by mass per 100 parts by mass of the biodegradable polyester.

[0017] [2] The scaffold substrate for cell culture according to [1] above, wherein the biodegradable polyester is polycaprolactone. [3] The scaffold for cell culture according to [1] or [2] above, wherein the polymer has a number average molecular weight of 1,000 to 300,000.

[0018] [4] A scaffold for cell culture comprising a scaffold base material for cell culture according to any one of [1] to [3] above, to which a biofunctional molecule is bound. [5] The scaffold for cell culture according to any one of [1] to [3] above, and A biofunctional molecule having a functional group capable of reacting with X in formula (1). A method for producing a scaffold for cell culture according to the above item [4], comprising mixing the above. Effect of the Invention

[0019] The scaffold base material for cell culture of the present invention can be bound to biofunctional molecules, can be molded into various shapes, and can maintain those shapes. Therefore, by binding biofunctional molecules to the scaffold base material for cell culture of the present invention, a scaffold for cell culture can be produced. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The scaffold substrate for cell culture of the present invention has the formula (1):

[0021] [ka]

[0022] and optionally having a repeating unit represented by formula (2):

[0023] [ka]

[0024] and the ratio of n / m is within the range of 0 / 100 to 99 / 1 (hereinafter, sometimes abbreviated as "polymer of the present invention"); Biodegradable polyester Herein, the term "scaffold substrate for cell culture" refers to a substrate used for forming a scaffold for cell culture.

[0025] In this specification, the "scaffold substrate for cell culture of the present invention" may be abbreviated as the "substrate of the present invention." In addition, in this specification, the "repeating unit represented by formula (1)" may be abbreviated as the "repeating unit (1)." Repeating units, polymers, monomers, and groups represented by other formulas may also be abbreviated as the "repeating unit (1)."

[0026] <Polymer of the present invention> The polymer of the present invention may be used alone or in combination of two or more. When the polymer of the present invention is a copolymer, the polymer of the present invention may be any of a random copolymer, a block copolymer, an alternating copolymer, or a copolymer having at least two structures selected from the group consisting of a random copolymer structure, a block copolymer structure, and an alternating copolymer structure.

[0027] The definitions of the symbols in formulas (1) and (2) will be explained below in order. The "*" in the formulas of this specification represents a bond position, not a carbon atom. Therefore, the "*-" in the formulas of this specification represents a single bond. "**-" and "-***" also represent a single bond, just like "*-".

[0028] In formula (1), a represents 0 or 1. Here, "a is 0" means that "-O-CO-A" in formula (1) does not exist, and the polymer main chain and X in formula (1) are directly bonded.

[0029] In formula (1), A represents a divalent organic group. A is preferably an alkylene group having 1 to 18 carbon atoms, **-(CH2) b2 -(CH2CH2O) b3 -(CH2) b4 -*** (wherein ** represents the bonding position to X in formula (1), *** represents the bonding position to carbonyl (CO) in formula (1), b2 represents an integer of 0 to 5, b3 represents an integer of 1 to 20, and b4 represents an integer of 1 to 5), or **-CO-(CH2) b5 -*** (wherein ** represents the bonding position to X in formula (1), *** represents the bonding position to carbonyl (CO) in formula (1), and b5 represents an integer of 1 to 5). In this specification, the "alkylene group" may be either linear or branched. In addition, in the above formula, "b2 is 0" means "(CH2) b2 -" means it does not exist.

[0030] A is more preferably an alkylene group having 1 to 6 carbon atoms, **-(CH2CH2O) b3 -(CH2) b4 -*** (wherein ** represents the bonding position to X in formula (1), *** represents the bonding position to carbonyl (CO) in formula (1), b3 represents an integer of 1 to 10, and b4 represents an integer of 1 to 3), or **-CO-(CH2) b5 -*** (wherein ** represents the bonding position to X in formula (1), *** represents the bonding position to carbonyl (CO) in formula (1), and b5 represents an integer of 1 to 3).

[0031] X in formula (1) (hereinafter sometimes referred to as "functional group X") is a halogen atom, an amino group (*-NH2), a sulfanyl group (*-SH), an azide group (*-N=N + =N -), or formula (1a) to formula (1f):

[0032] [ka]

[0033] (wherein * represents a bond position, and R 1 represents a hydrogen atom or a methyl group. The group represented by any one of the following formulas:

[0034] The functional group X, which is a halogen atom, can react with a carboxy group, etc. The functional group X, which is an amino group, can react with a carboxy group, an active ester group, and the like.

[0035] The functional group X, which is a sulfanyl group, is capable of reacting with the group (1a). The functional group X, which is group (1a), is capable of reacting with a sulfanyl group.

[0036] The functional group X, which is an azide group, can react with the groups (1c) to (1e). The functional group X, which is group (1b), can react with group (1e) or group (1f).

[0037] The functional group X, which is any one of the groups (1c) to (1e), is capable of reacting with an azide group. The functional group X, which is group (1e) or group (1f), can react with group (1b).

[0038] From the viewpoint of ease of production of the polymer of the present invention and ease of binding the base material of the present invention to a biofunctional molecule, the functional group X is preferably a halogen atom, an amino group, an azide group, group (1a), or group (1d), more preferably an azide group, group (1a), or group (1d), and even more preferably an azide group.

[0039] In formula (1) and formula (2), m and n respectively represent the number of repeating units. The values ​​of m and n are actually average values ​​and may each be a decimal number.

[0040] m is a number from 1 to 300, preferably a number from 5 to 250, and more preferably a number from 10 to 200. n is a number from 0 to 2,700, preferably from 0 to 2,400, and more preferably from 20 to 2,100. Here, "n is 0" means that the repeating unit (2) is not present. The ratio of n / m is within the range of 0 / 100 to 99 / 1, preferably within the range of 0 / 100 to 98 / 2, and more preferably within the range of 5 / 95 to 80 / 20.

[0041] When m is smaller than 1, the polymer of the present invention does not contain the functional group X, and a biofunctional molecule cannot be bound to the substrate of the present invention. When m is larger than 300, more functional groups X than necessary are exposed on the surface of the substrate of the present invention, and unreacted functional groups are exposed on the surface, which may cause cytotoxicity, and may make it difficult to produce the polymer of the present invention.

[0042] n can be arbitrarily set according to the function to be imparted to the final substrate of the present invention. For example, by decreasing n, the amount of functional group X exposed on the surface of the substrate of the present invention can be increased, and by increasing n, the hydrolysis rate of the polymer of the present invention can be suppressed.

[0043] The polymer of the present invention is preferably a homopolymer consisting of repeating unit (1) or a copolymer consisting of repeating unit (1) and repeating unit (2). Here, "a homopolymer consisting of repeating unit (1)" means a homopolymer in which all repeating units consist of repeating unit (1), and "a copolymer consisting of repeating unit (1) and repeating unit (2)" means a copolymer in which all repeating units consist of repeating unit (1) and repeating unit (2).

[0044] The polymer of the present invention preferably has the formula (p1):

[0045] [ka]

[0046] (wherein a, A, X, m and n are as defined above, R 1 represents a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, and R 2 is a hydrogen atom or *-CO-R 3 (In the formula, * represents a bonding position, and R 3 represents an alkyl group having 1 to 18 carbon atoms. It is a polymer represented by the formula:

[0047] When the polymer (p1) is a copolymer, the polymer (p1) is a random copolymer, a block copolymer, an alternating copolymer, or a copolymer having at least two structures selected from the group consisting of a random copolymer structure, a block copolymer structure, and an alternating copolymer structure.Therefore, for convenience, the formula (p1) is written as the structural formula of a block copolymer, but the polymer (p1) is not limited to a block copolymer.

[0048] In the present specification, the "alkyl group" may be either a straight chain or a branched chain. R in formula (p1) 1 is preferably a hydrogen atom or an alkyl group having 1 to 8 carbon atoms, and more preferably a hydrogen atom. R in formula (p1) 2 is preferably a hydrogen atom or *-CO-R 3 (In the formula, * represents a bonding position, and R 3 represents an alkyl group having 1 to 8 carbon atoms.) and more preferably a hydrogen atom.

[0049] From the viewpoint of ease of production of the polymer of the present invention, the number average molecular weight of the polymer of the present invention is 1,000 to 300,000, more preferably 5,000 to 280,000, and further preferably 10,000 to 260,000. This number average molecular weight can be calculated by gel permeation chromatography (GPC).

[0050] The content of the polymer of the present invention in the substrate of the present invention must be 1 to 300 parts by mass relative to 100 parts by mass of the biodegradable polyester. The content is preferably 2 to 275 parts by mass, more preferably 3 to 250 parts by mass. If the content is more than 300 parts by mass, the substrate of the present invention may become soft and may be deformed by a small external stress, or the obtained scaffold may be damaged during various operations during cell culture. If the content is less than 1 part by mass, the amount of functional group X exposed on the surface of the substrate of the present invention may decrease, the amount of biofunctional molecules bound to the scaffold may decrease, and the effect of the biofunctional molecules may not be sufficiently imparted to the obtained scaffold.

[0051] The polymer of the present invention can be produced by a known method or a method similar to the method described in the synthesis examples below.

[0052] For example, the polymer of the present invention in which X is a halogen atom can be produced by (i) first producing a monomer for the polymer in the same manner as in Synthesis Example 1, and then (ii) using the monomer obtained in (i) above, by a method similar to Synthesis Example 2-1. For example, the polymer of the present invention in which X is an azide group can be produced by the same method as in Synthesis Example 3-1.

[0053] For example, a polymer of the present invention in which a is 1, A is a divalent organic group, and X is an azide group, an amino group, a group (1a), or a group (1d) can be produced by a method similar to that of Synthesis Example 3-7 to Synthesis Example 3-11.

[0054] For example, the polymer of the present invention in which a is 1, A is a divalent organic group, and X is a sulfanyl group can be produced by (i) first producing the polymer of the present invention in which a is 0 and X is a chlorine atom in the same manner as in Synthesis Example 2-1, (ii) then using the polymer (1) obtained in (i) above and a carboxylic acid having a sulfanyl group protected by a protecting group (e.g., trimethylsilyl group) as starting materials, producing the polymer in which a is 1, A is a divalent organic group, and X is a protected sulfanyl group in the same manner as in Synthesis Example 3-7, and (iii) then removing the protecting group.For example, when a trimethylsilyl group is used as the protecting group, the protecting group can be removed by using tetrabutylammonium fluoride.

[0055] For example, a polymer of the present invention in which a is 1, A is a divalent organic group, and X is any of the groups (1b) to (1f) can be produced by (i) first producing a polymer of the present invention in which a is 0 and X is a chlorine atom in the same manner as in Synthesis Example 2-1, and then (ii) using the polymer of the present invention obtained in (i) above and a carboxylic acid having any of the groups (1b) to (1f) as starting materials, in the same manner as in Synthesis Example 3-7.

[0056] <Biodegradable polyester> The biodegradable polyester is not particularly limited as long as it is a generally known biodegradable polyester, and examples thereof include polyglycolic acid, polylactic acid, poly(lactic acid-glycolic acid), polycaprolactone, polyethylene succinate, polybutylene succinate, polybutylene succinate adipate, polyhydroxypropionate, polyhydroxybutyrate, etc. These biodegradable polyesters may be used alone or in combination of two or more. Among these, polyglycolic acid (PGA), polylactic acid (PLA), poly(lactic acid-glycolic acid) (PLGA), and polycaprolactone (PCL), which are highly biocompatible, are preferred, polylactic acid and polycaprolactone are more preferred, and polycaprolactone is even more preferred. The polylactic acid may be any of the L-form, D-form, and DL-form.

[0057] The biodegradable polyester may be a commercially available product or may be appropriately synthesized by a known method. The molecular weight of the biodegradable polyester is not particularly limited as long as it is within a range generally used in various molding methods described below.

[0058] From the viewpoint of availability of biodegradable polyesters, the number average molecular weight of the biodegradable polyester is preferably 1,000 to 200,000, and more preferably 10,000 to 100,000. This number average molecular weight can be calculated by gel permeation chromatography (GPC).

[0059] <Shape of the substrate of the present invention> The shape of the substrate of the present invention is not particularly limited as long as it is a shape that is generally used as a scaffold for cell culture, such as a film, a fiber, a porous body, etc.

[0060] For example, the polymer of the present invention and a biodegradable polyester are mixed in a predetermined weight ratio, dissolved in an organic solvent, and the resulting solution is applied to a substrate, thereby obtaining the substrate of the present invention in the form of a film.

[0061] The method for forming the film-shaped substrate of the present invention is not particularly limited as long as it is a method generally known as a method for forming a film, and examples thereof include a casting method, a dip coating method, a bar coating method, a spray coating method, a die coating method, a flow coating method, a curtain coating method, and a spin coating method.

[0062] The material of the substrate onto which the solution is applied is not particularly limited, but examples thereof include plastics such as polystyrene, polyethylene, and polycarbonate, and glass.

[0063] For example, it is possible to mix the polymer of the present invention and a biodegradable polyester in a predetermined weight ratio, dissolve the mixture in an organic solvent, and use the resulting solution to obtain the substrate of the present invention in the form of a fiber.

[0064] The method for forming the fiber-shaped substrate of the present invention is not particularly limited as long as it is a method known as a method for obtaining general biodegradable polyester microfibers or nanofibers, and examples thereof include a fiber fusion method, a needle punching method, a melt-blowing method, and an electrospinning method.

[0065] For example, it is possible to mix the polymer of the present invention and a biodegradable polyester in a predetermined weight ratio, dissolve the mixture in an organic solvent, and use the resulting solution to obtain the substrate of the present invention in a porous form.

[0066] The method for forming the porous substrate of the present invention is not particularly limited as long as it is a method known as a method for obtaining a general biodegradable polyester porous body, and examples thereof include a freeze-drying method, a foaming method, a phase separation method, and a porogen leaching method.

[0067] <Scaffolds for cell culture> By binding a biofunctional molecule to the substrate of the present invention, a scaffold for cell culture can be obtained that is endowed with the function of the biofunctional molecule. Therefore, the present invention also provides a scaffold for cell culture in which a biofunctional molecule is bound to the substrate of the present invention (hereinafter sometimes abbreviated as "scaffold of the present invention"). In the scaffold of the present invention, the substrate of the present invention and the biofunctional molecule are preferably bound by a covalent bond.

[0068] The biofunctional molecule is not particularly limited as long as it is a generally known compound that has an effect on a living body, and examples thereof include proteins, peptides, growth factors, nucleic acids, hormones, and cytokines. In this specification, "protein" means a compound in which 50 or more amino acids are bound, and "peptide" means a compound in which less than 50 amino acids are bound. Only one type of biofunctional molecule may be used, or two or more types may be used in combination.

[0069] The protein is not particularly limited as long as it is a protein that is generally used in the culture of animal cells, etc., and examples thereof include structural proteins such as collagen and elastin, cell adhesive proteins such as fibronectin, vitronectin, laminin, fragmented laminin, and fibrinogen, and glycosaminoglycans such as hyaluronic acid, chondroitin sulfate, heparan sulfate, and keratan sulfate.

[0070] The peptide is not particularly limited as long as it is a commonly known peptide, and examples thereof include RGD peptide, which is a cell adhesion peptide, REDV peptide, which is a vascular endothelial cell adhesion peptide, IKVAV peptide, which is a neuronal maturation peptide, and peptides that mimic the effects of growth factors.

[0071] The growth factor is not particularly limited as long as it is a growth factor that is generally used in the culture of animal cells, etc., and examples thereof include transforming growth factor (TGF), insulin-like growth factor (IGF), activin A, epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), bone morphogenetic protein (BMP), platelet-derived growth factor (PDGF), transforming growth factor (TGF), hepatocyte growth factor (HGF), etc.

[0072] <Method of manufacturing scaffold for cell culture> The present invention also provides a method for producing the scaffold of the present invention, which comprises mixing the scaffold base material of the present invention with a biofunctional molecule having a functional group (hereinafter sometimes referred to as "functional group Y") capable of reacting with X in formula (1).

[0073] As the biofunctional molecule having a functional group Y, a commercially available product may be used, or one synthesized by a known method may be used.

[0074] The functional group Y is preferably a carboxy group, an active ester group, a sulfanyl group, an azide group, or any one of the groups (1a) to (1f), more preferably a sulfanyl group, an azide group, or the group (1d), and even more preferably the group (1d).

[0075] Examples of the active ester group include groups represented by any one of the following formulae (3a) to (3c) (in the following formulae, * represents the bonding position).

[0076] [ka]

[0077] Functional group X, which is a halogen atom, can react with functional group Y, which is a carboxy group, to form a covalent bond. Functional group X, which is an amino group, can react with functional group Y, which is a carboxy group or an active ester group, to form a covalent bond.

[0078] Functional group X, which is a sulfanyl group, can react with functional group Y, which is group (1a), to form a covalent bond. The functional group X, which is group (1a), can react with the functional group Y, which is a sulfanyl group, to form a covalent bond.

[0079] The functional group X, which is an azide group, can react with the functional group Y, which is any one of the groups (1c) to (1e), to form a covalent bond. Functional group X, which is group (1b), can react with functional group Y, which is group (1e) or group (1f), to form a covalent bond.

[0080] The functional group X, which is any one of the groups (1c) to (1e), can react with the functional group Y, which is an azide group, to form a covalent bond. The functional group X, which is group (1e) or group (1f), can react with the functional group Y, which is group (1b), to form a covalent bond.

[0081] The formation of a covalent bond by the reaction between the functional group X in the base material of the present invention and the functional group Y in the biofunctional molecule is preferably carried out by dissolving the biofunctional molecule having the functional group Y in water and immersing the base material of the present invention in the obtained aqueous solution.

[0082] The concentration of the aqueous solution of the biofunctional molecule having the functional group Y varies depending on the chemical reaction to be carried out, but is preferably 0.01 μM to 10 mM.

[0083] The temperature of the mixture of the base material of the present invention and the aqueous solution of the biofunctional molecule having functional group Y (i.e., the reaction temperature between functional group X and functional group Y) varies depending on the chemical reaction to be carried out, but is preferably 30 to 80°C, more preferably 30 to 50°C.

[0084] The time for which the base material of the present invention is immersed in the aqueous solution of the biofunctional molecule having functional group Y (i.e., the reaction time between functional group X and functional group Y) varies depending on conditions such as reaction temperature, but is preferably about 5 to 24 hours.

[0085] After the reaction between the functional group X and the functional group Y, the aqueous solution of the biofunctional molecule having the functional group Y is removed, and the scaffold of the present invention is obtained by washing with water several times and drying. The obtained scaffold of the present invention may be subjected to UV sterilization or the like. EXAMPLES

[0086] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these.

[0087] [Molecular weight measurement by gel permeation chromatography (GPC) analysis] The molecular weight was measured using Shimadzu Corporation's product name "Prominence," column: Polymer Laboratory's PL gel (product name "MIXED-B"), column temperature: 40°C, sample concentration: 0.2 mass%, injection amount: 100 μL, eluent: 11.5 mM LiBr in dimethylformamide solution, flow rate: 0.6 mL / min, detector: differential refractometer (RI), and standard: polymethyl methacrylate.

[0088] [Synthesis Example 1] Synthesis of monomer (m1)

[0089] [ka]

[0090] 2-Chlorocyclohexanone (16 g, 121 mmol) and dichloromethane (200 ml) were added to a recovery flask, and the mixture was cooled to 0° C. A mixture of metachloroperbenzoic acid and water (38.4 g, content of metachloroperbenzoic acid in the mixture: 70 mass %, amount of metachloroperbenzoic acid: 157 mmol) was added, and the temperature was raised to 30° C. and stirred for 24 hours. After the reaction was completed, the reaction mixture was cooled to 0° C., and an aqueous solution of sodium thiosulfate was added dropwise. After the addition was completed, the mixture was warmed to room temperature, and the precipitated solid was filtered off. The obtained filtrate was washed with an aqueous solution of sodium thiosulfate and then with a saturated aqueous solution of sodium hydrogen carbonate.

[0091] Saturated sodium bicarbonate was then added to the organic phase, and the mixture was stirred at 35°C for 4 hours. After stirring was completed, the aqueous phase was removed, and the organic phase was washed with saturated aqueous sodium chloride solution. The organic phase was dehydrated with magnesium sulfate, and the solvent was removed under reduced pressure. The monomer was purified by silica gel column chromatography (eluent: hexane / ethyl acetate = 4:1) to obtain monomer (m1). The yield was 66%.

[0092] Monomer (m1) 1 H NMR(CDCl3,400MHz)δ 1.8-2.2ppm(m,6H,-O-CH2- CH 2- CH 2- CH 2-), 4.2-4.3ppm (m, 1H, -O- CH 2-), 4.6ppm(ddd,1H,J=2.0,7.6,12.8, -O- CH 2-), 4.8ppm(dd,1H,J=2.8,4.4,-CO- CH Cl-)

[0093] [Synthesis Example 2-1] Synthesis of polymer (2-1) Monomer (m1) (6.5 g, 43.7 mmol) obtained in Synthesis Example 1, ε-caprolactone (11.65 g, 102 mmol), 0.1 M tin 2-ethylhexanoate in dehydrated toluene solution (1.44 mL, amount of tin 2-ethylhexanoate: 14.4 μmol), and dehydrated toluene (18 g) were added to a two-necked eggplant flask, and the mixture was heated to 60° C. Azeotropic dehydration was carried out for 1 hour at a reduced pressure of 1000 Pa while flowing nitrogen gas at a flow rate of 100 mL / min. After the azeotropic dehydration, dehydrated toluene (18 g) was added and the same operation was carried out, and the reaction mixture was then heated to 120° C. and stirred for 48 hours. After the reaction was completed, the obtained polymer was dissolved in toluene, and the obtained solution was dropped into methanol and decanted to obtain polymer (2-1) (a random copolymer in which a in formula (1) is 0, X is a chlorine atom, m is 49, and n in formula (2) is 107). The yield was 68%.

[0094] Polymer (2-1)1 H NMR(CDCl3,400MHz)δ 1.4-2.1ppm(m,12H,-CHCl- CH 2- CH 2- CH 2-CH2-, -CO-CH2- CH 2- CH 2- CH 2-CH2-), 2.3-2.4ppm(m,2H,-CO- CH 2-CH2-), 3.7ppm(t,2H,J=6.4Hz), 4.0-4.1ppm(m,2H,-CH2- CH 2-O-), 4.1-4.2 ppm (m, 2H, -CH2- CH 2-O-), 4.2-4.3 ppm (m, 1H, -CO- CH Cl-CH2-)

[0095] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (2-1) were as follows: Mn=34,573 Mw=63,520 Mw / Mn=1.84

[0096] [Synthesis Example 2-2] Synthesis of polymer (2-2) Polymer (2-2) (a random copolymer in which a in formula (1) is 0, X is a chlorine atom, m is 50, and n in formula (2) is 50) was obtained by carrying out the same operation as in Synthesis Example 2-1, except that monomer (m1) (1.0 g, 6.73 mmol) obtained in Synthesis Example 1, ε-caprolactone (0.77 g, 6.73 mmol), 0.1 M tin 2-ethylhexanoate solution in dehydrated toluene (1.35 mL, amount of tin 2-ethylhexanoate: 135 μmol), and dehydrated toluene (1.8 g) were added to a two-necked eggplant flask. The yield was 56%.

[0097] Polymer (2-2) 1 H NMR(CDCl3,400MHz)δ 1.3-2.0ppm(m,12H,-CHCl- CH 2- CH 2- CH2-CH2-, -CO-CH2- CH 2- CH 2- CH 2-CH2-), 2.3-2.4ppm(m,2H,-CO- CH 2-CH2-), 3.6ppm(t,2H,J=6.4Hz), 4.0-4.1ppm(m,2H,-CH2- CH 2-O-), 4.1-4.2 ppm (m, 2H, -CH2- CH 2-O-), 4.2-4.3 ppm (m, 1H, -CO- CH Cl-CH2-)

[0098] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (2-2) were as follows: Mn=33,621 Mw=60,518 Mw / Mn=1.80

[0099] [Synthesis Example 2-3] Synthesis of polymer (2-3) The same operation as in Synthesis Example 2-1 was carried out except that the monomer (m1) (3.34 g, 22.5 mmol) obtained in Synthesis Example 1, a 0.1 M solution of tin 2-ethylhexanoate in dehydrated toluene (0.23 mL, 23 μmol), and dehydrated toluene (3.3 g) were added to a two-necked eggplant flask, to obtain polymer (2-3) (a homopolymer in formula (1) where a is 0, X is a chlorine atom, m is 96, and n is 0 in formula (2)). The yield was 69%.

[0100] Polymer (2-3) 1 H NMR(CDCl3,400MHz)δ 1.4-2.2ppm(m,6H,-CHCl- CH 2- CH 2- CH 2-CH2-), 3.5ppm(t,2H,J=6.4Hz), 4.1-4.2ppm(m,2H,-CH2- CH 2-O-), 4.2-4.3 ppm (m, 1H, -CO- CH Cl-CH2-)

[0101] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (2-3) were as follows: Mn=14,506 Mw=29,501 Mw / Mn=2.03

[0102] [Synthesis Example 2-4] Synthesis of polymer (2-4) Polymer (2-4) (a random copolymer in which a in formula (1) is 0, X is a chlorine atom, m is 50, and n in formula (2) is 502) was obtained by carrying out the same operation as in Synthesis Example 2-1, except that monomer (m1) (1.0 g, 6.73 mmol) obtained in Synthesis Example 1, ε-caprolactone (7.68 g, 67.3 mmol), 0.1 M dehydrated toluene solution of tin 2-ethylhexanoate (0.74 mL, 74 μmol), and dehydrated toluene (8.7 g) were added to a two-necked eggplant flask. The yield was 68%.

[0103] Polymer (2-4) 1 H NMR(CDCl3,400MHz)δ 1.3-2.0ppm(m,12H,-CHCl- CH 2- CH 2- CH 2-CH2-, -CO-CH2- CH 2- CH 2- CH 2-CH2-), 2.3-2.4ppm(m,2H,-CO- CH 2-CH2-), 3.6ppm(t,2H,J=6.4Hz), 4.0-4.1ppm(m,2H,-CH2- CH 2-O-), 4.1-4.2 ppm (m, 2H, -CH2- CH 2-O-), 4.2-4.3 ppm (m, 1H, -CO- CH Cl-CH2-)

[0104] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (2-4) were as follows: Mn=78,190 Mw=131,359 Mw / Mn=1.68

[0105] [Synthesis Example 2-5] Synthesis of polymer (2-5) Polymer (2-5) (a random copolymer in which a in formula (1) is 0, X is a chlorine atom, m is 52, and n in formula (2) is 990) was obtained by carrying out the same operation as in Synthesis Example 2-1, except that monomer (m1) (1.0 g, 6.73 mmol) obtained in Synthesis Example 1, ε-caprolactone (15.4 g, 13.4 mmol), 0.1 M tin 2-ethylhexanoate in dehydrated toluene solution (1.4 mL, 140 μmol), and dehydrated toluene (16.4 g) were added to a two-necked eggplant flask. The yield was 71%.

[0106] Polymer (2-5) 1 H NMR(CDCl3,400MHz)δ 1.3-2.0ppm(m,12H,-CHCl- CH 2- CH 2- CH 2-CH2-, -CO-CH2- CH 2- CH 2- CH 2-CH2-), 2.3-2.4ppm(m,2H,-CO- CH 2-CH2-), 3.6ppm(t,2H,J=6.4Hz), 4.0-4.1ppm(m,2H,-CH2- CH 2-O-), 4.1-4.2 ppm (m, 2H, -CH2- CH 2-O-), 4.2-4.3 ppm (m, 1H, -CO- CH Cl-CH2-)

[0107] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (2-5) were as follows: Mn=138,227 Mw=239,133 Mw / Mn=1.73

[0108] [Synthesis Example 2-6] Synthesis of polymer (2-6) Polymer (2-6) (a random copolymer in which a in formula (1) is 0, X is a chlorine atom, m is 47, and n in formula (2) is 2,006) was obtained by carrying out the same operation as in Synthesis Example 2-1, except that monomer (m1) (1.0 g, 6.73 mmol) obtained in Synthesis Example 1, ε-caprolactone (30.7 g, 269 mmol), 0.1 M dehydrated toluene solution of tin 2-ethylhexanoate (0.74 mL, 74 μmol), and dehydrated toluene (8.7 g) were added to a two-necked eggplant flask. The yield was 72%.

[0109] Polymer (2-6) 1 H NMR(CDCl3,400MHz)δ 1.3-2.0ppm(m,12H,-CHCl- CH 2- CH 2- CH 2-CH2-, -CO-CH2- CH 2- CH 2- CH 2-CH2-), 2.3-2.4ppm(m,2H,-CO- CH 2-CH2-), 3.6ppm(t,2H,J=6.4Hz), 4.0-4.1ppm(m,2H,-CH2- CH 2-O-), 4.1-4.2 ppm (m, 2H, -CH2- CH 2-O-), 4.2-4.3 ppm (m, 1H, -CO- CH Cl-CH2-)

[0110] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (2-6) were as follows: Mn=251,724 Mw=443,034 Mw / Mn=1.76

[0111] [Synthesis Example 2-7] Synthesis of polymer (2-7) Polymer (2-7) (a random copolymer in which a in formula (1) is 0, X is a chlorine atom, m is 200, and n in formula (2) is 400) was obtained by carrying out the same operation as in Synthesis Example 2-1, except that monomer (m1) (1.0 g, 6.73 mmol) obtained in Synthesis Example 1, ε-caprolactone (1.54 g, 13.5 mmol), 0.1 M dehydrated toluene solution of tin 2-ethylhexanoate (50 μL, 5 μmol), and dehydrated toluene (2.5 g) were added to a two-necked eggplant flask. The yield was 57%.

[0112] Polymer (2-7) 1 H NMR(CDCl3,400MHz)δ 1.3-2.0ppm(m,12H,-CHCl- CH 2- CH 2- CH 2-CH2-, -CO-CH2- CH 2- CH 2- CH 2-CH2-), 2.3-2.4ppm(m,2H,-CO- CH 2-CH2-), 3.6ppm(t,2H,J=6.4Hz,- CH 2-OH), 4.0-4.1 ppm (m, 2H, -CH2- CH 2-O-), 4.1-4.2 ppm (m, 2H, -CH2- CH 2-O-), 4.2-4.3 ppm (m, 1H, -CO- CH Cl-CH2-)

[0113] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (2-7) were as follows: Mn=87,314 Mw=167,643 Mw / Mn=1.92

[0114] [Synthesis Example 2-8] Synthesis of polymer (2-8) Polymer (2-8) (a random copolymer in which a in formula (1) is 0, X is a chlorine atom, m is 13, and n in formula (2) is 31) was obtained by carrying out the same operation as in Synthesis Example 2-1, except that monomer (m1) (1.0 g, 6.73 mmol) obtained in Synthesis Example 1, ε-caprolactone (1.54 g, 13.5 mmol), 1-octanol (26.3 mg, 0.20 mmol), 0.1 M tin 2-ethylhexanoate in dehydrated toluene solution (0.2 mL, 20 μmol), and dehydrated toluene (2.5 g) were added to a two-necked eggplant flask. The yield was 56%.

[0115] Polymer (2-8) 1 H NMR(CDCl3,400MHz)δ 1.3-2.0ppm(m,12H,-CHCl- CH 2- CH 2- CH 2-CH2-, -CO-CH2- CH 2- CH 2- CH 2-CH2-), 2.3-2.4ppm(m,2H,-CO- CH 2-CH2-), 3.6ppm(t,2H,J=6.4Hz,- CH 2-OH), 4.0-4.1 ppm (m, 2H, -CH2- CH 2-O-), 4.1-4.2 ppm (m, 2H, -CH2- CH 2-O-), 4.2-4.3 ppm (m, 1H, -CO- CH Cl-CH2-)

[0116] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (2-8) were as follows: Mn=12,997 Mw=15,986 Mw / Mn=1.23

[0117] [Synthesis Example 3-1] Synthesis of polymer (3-1) The polymer (2-1) (10 g) obtained in Synthesis Example 2-1 and N,N-dimethylformamide (75 mL) were added to a recovery flask, and the polymer (2-1) was dissolved at room temperature. Sodium azide (1.86 g, 28.6 mmol) was added to the recovery flask, and the mixture was heated to 30° C. and stirred for 24 hours. After the reaction was completed, the solvent was removed by distillation under reduced pressure, ethyl acetate was added to dissolve the polymer, and the resulting solution was washed with water and then with a saturated aqueous sodium chloride solution. The resulting organic phase was dehydrated with magnesium sulfate, and the solvent was removed by distillation under reduced pressure to obtain polymer (3-1) (a in formula (1) is 0, X is an azide group, m is 50, and n in formula (2) is 107, a random copolymer). The yield was 95%.

[0118] Polymer (3-1) 1 H NMR(CDCl3,400MHz)δ 1.3-1.9ppm(m,12H,-CHN3- CH 2- CH 2- CH 2-CH2-, -CO-CH2- CH 2- CH 2- CH 2-CH2-), 2.3-2.4ppm(m,2H,-CO- CH 2-CH2-), 3.6ppm(t,2H,J=6.4Hz,- CH 2-OH), 3.8-3.9 ppm (m, 1H, -CO- CH N3-CH2-), 4.0-4.1ppm(m,2H,-CH2- CH 2-O-), 4.1-4.3 ppm (m, 2H, -CH2- CH 2-O-)

[0119] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (3-1) were as follows: Mn=34,016 Mw=67,623 Mw / Mn=1.92

[0120] [Synthesis Example 3-2] Synthesis of polymer (3-2) Polymer (3-2) (a homocopolymer in which a in formula (1) is 0, X is an azide group, m is 96, and n in formula (2) is 0) was obtained by carrying out the same operation as in Synthesis Example 3-1, except that polymer (2-3) (500 mg) synthesized in Synthesis Example 2-3, N,N-dimethylformamide (10 mL), and sodium azide (263 mg, 4.04 mmol) were added to a recovery flask. The yield was 95%.

[0121] Polymer (3-2) 1 H NMR(CDCl3,400MHz)δ 1.5-1.9ppm(m,6H,-CHN3- CH 2- CH 2- CH 2-CH2-), 3.6ppm(t,2H,J=6.4Hz,- CH 2-OH), 3.8-3.9 ppm (m, 1H, -CO- CH N3-CH2-), 4.1-4.2ppm(m,2H,-CH2- CH 2-O-)

[0122] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (3-2) were as follows: Mn=15,332 Mw=28,622 Mw / Mn=1.87

[0123] [Synthesis Example 3-3] Synthesis of polymer (3-3) The same operation as in Synthesis Example 3-1 was carried out except that polymer (2-4) (1 g) obtained in Synthesis Example 2-4, N,N-dimethylformamide (20 mL), and sodium azide (60 mg, 0.92 mmol) were added to the recovery flask, to obtain polymer (3-3) (a random copolymer in which a in formula (1) is 0, X is an azide group, m is 50, and n in formula (2) is 504). The yield was 96%.

[0124] Polymer (3-3) 1H NMR(CDCl3,400MHz)δ 1.3-1.9ppm(m,12H,-CHN3- CH 2- CH 2- CH 2-CH2-, -CO-CH2- CH 2- CH 2- CH 2-CH2-), 2.3-2.4ppm(m,2H,-CO- CH 2-CH2-), 3.6ppm(t,2H,J=6.4Hz,- CH 2-OH), 3.8-3.9 ppm (m, 1H, -CO- CH N3-CH2-), 4.0-4.1ppm(m,2H,-CH2- CH 2-O-), 4.1-4.3 ppm (m, 2H, -CH2- CH 2-O-)

[0125] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (3-3) were as follows: Mn=77,516 Mw=130,227 Mw / Mn=1.68

[0126] [Synthesis Example 3-4] Synthesis of polymer (3-4) The same operation as in Synthesis Example 3-1 was carried out except that polymer (2-5) (1 g) obtained in Synthesis Example 2-5, N,N-dimethylformamide (20 mL), and sodium azide (32 mg, 0.49 mmol) were added to the recovery flask, to obtain polymer (3-4) (a random copolymer in formula (1) where a is 0, X is an azide group, m is 52, and n is 998 in formula (2)). The yield was 95%.

[0127] Polymer (3-4) 1 H NMR(CDCl3,400MHz)δ 1.3-1.9ppm(m,12H,-CHN3- CH 2- CH 2- CH 2-CH2-, -CO-CH2- CH 2- CH 2- CH2-CH2-), 2.3-2.4ppm(m,2H,-CO- CH 2-CH2-), 3.6ppm(t,2H,J=6.4Hz,- CH 2-OH), 3.8-3.9 ppm (m, 1H, -CO- CH N3-CH2-), 4.0-4.1ppm(m,2H,-CH2- CH 2-O-), 4.1-4.3 ppm (m, 2H, -CH2- CH 2-O-)

[0128] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (3-4) were as follows: Mn=139,712 Mw=241,702 Mw / Mn=1.73

[0129] [Synthesis Example 3-5] Synthesis of polymer (3-5) Polymer (3-5) (a random copolymer in which a in formula (1) is 0, X is an azide group, m is 41, and n in formula (2) is 2009) was obtained by carrying out the same operation as in Synthesis Example 3-1, except that polymer (2-6) (1 g) obtained in Synthesis Example 2-6, N,N-dimethylformamide (20 mL), and sodium azide (17 mg, 0.26 mmol) were added to a recovery flask. The yield was 95%.

[0130] Polymer (3-5) 1 H NMR(CDCl3,400MHz)δ 1.3-1.9ppm(m,12H,-CHN3- CH 2- CH 2- CH 2-CH2-, -CO-CH2- CH 2- CH 2- CH 2-CH2-), 2.3-2.4ppm(m,2H,-CO- CH 2-CH2-), 3.6ppm(t,2H,J=6.4Hz,- CH 2-OH), 3.8-3.9 ppm (m, 1H, -CO- CHN3-CH2-), 4.0-4.1ppm(m,2H,-CH2- CH 2-O-), 4.1-4.3 ppm (m, 2H, -CH2- CH 2-O-)

[0131] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (3-5) were as follows: Mn=257,281 Mw=452,815 Mw / Mn=1.76

[0132] [Synthesis Example 3-6] Synthesis of polymer (3-6) The same operation as in Synthesis Example 3-1 was carried out except that the polymer (2-7) (1 g) obtained in Synthesis Example 2-7, N,N-dimethylformamide (20 mL), and sodium azide (207 mg, 3.2 mmol) were added to the recovery flask, thereby obtaining polymer (3-6) (a random copolymer in which a in formula (1) is 0, X is an azide group, m is 199, and n in formula (2) is 403). The yield was 94%.

[0133] Polymer (3-6) 1 H NMR(CDCl3,400MHz)δ 1.3-1.9ppm(m,12H,-CHN3- CH 2- CH 2- CH 2-CH2-, -CO-CH2- CH 2- CH 2- CH 2-CH2-), 2.3-2.4ppm(m,2H,-CO- CH 2-CH2-), 3.6ppm(t,2H,J=6.4Hz,- CH 2-OH), 3.8-3.9 ppm (m, 1H, -CO- CH N3-CH2-), 4.0-4.1ppm(m,2H,-CH2- CH 2-O-), 4.1-4.3 ppm (m, 2H, -CH2- CH 2-O-)

[0134] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (3-6) were as follows: Mn=88,253 Mw=169,446 Mw / Mn=1.92

[0135] [Synthesis Example 3-7] Synthesis of polymer (3-7) 11-azido-3,6,9-trioxaundecanoic acid (66.6 mg, 0.29 mmol) and dimethyl sulfoxide (1 mL) were added to the eggplant flask, and 11-azido-3,6,9-trioxaundecanoic acid was dissolved. Then, sodium hydrogen carbonate (28.6 mg, 0.29 mmol) was added to the eggplant flask, and the mixture was stirred at room temperature for 30 minutes. Next, a dimethyl sulfoxide solution (0.8 mL) of the polymer (2-1) (100 mg) obtained in Synthesis Example 2-1 was added to the eggplant flask, and the mixture was heated to 80° C. and stirred for 6 hours. After the reaction was completed, ethyl acetate was added, and the mixture was washed with water and then with a saturated aqueous sodium chloride solution. The obtained organic phase was dehydrated with magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain polymer (3-7) (a in formula (1) is 1, A is **-(C2H5O)3-CH2-*** (wherein ** is the bonding position with the azide group and *** is the bonding position with the carbonyl (CO)), X is an azide group, m is 49, and n in formula (2) is 110, a random copolymer). The yield was 88%.

[0136] Polymer (3-7) 1 H NMR(CDCl3,400MHz)δ 1.3-1.8ppm(m,12H,-CHO- CH 2- CH 2- CH 2-CH2-, -CO-CH2- CH 2- CH 2- CH 2-CH2-), 2.2-2.3ppm(m,2H,-CO- CH 2-CH2-), 3.3-3.4 ppm (m, 2H, - CH 2-N3), 3.6-3.8ppm(m,10H,-O- CH 2- CH 2-O-,- CH 2-CH2-N3), 4.0-4.1ppm(m,2H,-CH2- CH 2-O-), 4.1-4.2 ppm (m, 2H, -CH2- CH 2-O-), 4.2-4.3 ppm (m, 2H, -CO- CH 2-O-), 5.0-5.1ppm (m, 1H, - CH O-)

[0137] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (3-7) were as follows: Mn=44,832 Mw=83,574 Mw / Mn=1.86

[0138] [Synthesis Example 3-8] Synthesis of polymer (3-8) 6-azidohexanoic acid (44.9 mg, 0.29 mmol) and dimethyl sulfoxide (1 mL) were added to the eggplant flask, and after dissolving 6-azidohexanoic acid, sodium hydrogen carbonate (28.6 mg, 0.29 mmol) was added to the eggplant flask, and the mixture was stirred at room temperature for 30 minutes. Next, a dimethyl sulfoxide solution (0.8 mL) of the polymer (2-1) (100 mg) obtained in Synthesis Example 2-1 was added to the eggplant flask, and the mixture was heated to 80°C and stirred for 6 hours. After the reaction was completed, ethyl acetate was added, and the mixture was washed with water and then with a saturated aqueous sodium chloride solution. The obtained organic phase was dehydrated with magnesium sulfate, and the solvent was distilled off under reduced pressure. Finally, dichloromethane was added and decantation was performed using methanol to obtain polymer (3-8) (a random copolymer in formula (1) where a is 1, A is a pentamethylene group (*-(CH2)5-*), X is an azide group, m is 49, and n is 110 in formula (2)). The yield was 80%.

[0139] Polymer (3-8) 1 H NMR(CDCl3,400MHz)δ 1.2-1.9ppm(m,18H,-CHO- CH2- CH 2- CH 2-CH2-, -CO-CH2- CH 2- CH 2- CH 2-CH2-, - CH 2- CH 2- CH 2-CH2-N3), 2.2-2.3ppm(m,2H,-CO- CH 2-CH2-), 2.4-2.5ppm(m,2H,-CO-CH2-), 3.2-3.3ppm(m,2H,- CH 2-N3), 4.0-4.1ppm (m, 2H, -CH2- CH 2-O-), 4.1-4.2 ppm (m, 2H, -CH2- CH 2-O-), 4.9-5.0 ppm (m, 1H, - GIVE -)

[0140] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (3-8) were as follows: Mn=48,241 Mw=95,244 Mw / Mn=1.97

[0141] [Synthesis Example 3-9] Synthesis of polymer (3-9) N-(tert-butoxycarbonyl)-β-alanine (108 mg, 0.57 mmol) and dimethylformamide (2 mL) were added to the eggplant flask to dissolve N-(tert-butoxycarbonyl)-β-alanine, and then sodium hydrogen carbonate (57.2 mg, 0.571 mmol) was added to the eggplant flask, and the mixture was stirred at room temperature for 30 minutes. Next, a dimethylformamide solution (1.6 mL) of the polymer (2-1) (200 mg) obtained in Example 2-1 was added to the eggplant flask, and the mixture was heated to 80° C. and stirred for 6 hours. After the reaction was completed, the solvent was distilled off under reduced pressure, dichloromethane was added, and decantation was performed using cold methanol to obtain a polymer with a protected amino group. This was dissolved in dichloromethane (1.5 mL), and trifluoroacetic acid (3.3 mL) was added to the resulting solution, and the mixture was stirred at 0° C. for 30 minutes. After the reaction was completed, the mixture was decanted with hexane to obtain polymer (3-9) (a random copolymer in which a in formula (1) is 1, A is *-(CH2)2-*, X is an amino group, m is 49, and n in formula (2) is 114) in a yield of 92%.

[0142] Polymer (3-9) 1 H NMR(CD3OD):1.3-1.9ppm(m,12H,-CHO- CH 2- CH 2- CH 2-CH2-, -CO-CH2- CH 2- CH 2- CH 2-CH2-), 2.2-2.3ppm(m,2H,-CO- CH 2-CH2-), 2.8-2.9 ppm (m, 2H, - CH 2-NH2), 3.1-3.2ppm(m,2H,- CH 2-CH2-NH2), 3.5-3.6ppm(m,2H,- CH 2-OH), 4.0-4.1 ppm (m, 2H, -CH2- CH 2-O-), 4.1-4.2 ppm (m, 2H, -CH2- CH 2-O-), 5.0-5.1ppm (m, 1H, - CH O-)

[0143] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (3-9) were as follows: Mn=40,637 Mw=74,366 Mw / Mn=1.86

[0144] [Synthesis Example 3-10] Synthesis of polymer (3-10) The same procedure as in Synthesis Example 3-8 was carried out except that 4-maleimidobutyric acid (65.4 mg, 0.36 mmol) was used instead of 6-azidohexanoic acid, to obtain polymer (3-10) (a random copolymer in which a in formula (1) is 1, A is a trimethylene group (*-(CH2)3-*), X is a group (1a), m is 45, and n in formula (2) is 115). The yield was 50%.

[0145] Polymer (3-10) 1 H NMR(CDCl3,400MHz)δ 1.3-1.9ppm(m,14H,-CHO- CH 2- CH 2- CH 2-CH2-, -CO-CH2- CH 2- CH 2- CH 2-CH2-,- CH 2-CH2-N-), 2.2-2.3ppm(m,2H,-CO- CH 2-CH2-), 2.5-2.6ppm(m,2H,-O-CO- CH 2-), 3.8-3.9ppm(m,2H,-NH-CO-CH2- CH 2-) 4.0-4.1ppm (m, 2H, -CH2- CH 2-O-), 4.1-4.2 ppm (m, 2H, -CH2- CH 2-O-), 5.0-5.1ppm (m, 1H, - GIVE -), 6.7ppm(s,2H,- CH = CH -)

[0146] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (3-10) were as follows: Mn=42,293 Mw=76,550 Mw / Mn=1.81

[0147] [Synthesis Example 3-11] Synthesis of polymer (3-11) The same procedure as in Synthesis Example 3-8 was carried out except that DBCO-Acid (Tokyo Chemical Industry Co., Ltd., 0.29 mg, 0.29 mmol) was used instead of 6-azidohexanoic acid, to obtain polymer (3-11) (a in formula (1) is 1, A is **-CO-(CH2)2-*** (wherein ** represents the bonding position with X in formula (1) and *** represents the bonding position with carbonyl (CO) in formula (1)), X is group (1d), m is 45, and n in formula (2) is 110, a random copolymer). The yield was 63%.

[0148] Polymer (3-11) 1 H NMR(CDCl3,400MHz)δ 1.3-1.9ppm(m,13H,-CHO- CH 2- CH 2- CH 2-CH2-, -CO-CH2- CH 2- CH 2- CH 2-CH2-, -N-CO- CH 2-), 2.2-2.3ppm (m, 2H, -CO- CH 2-CH2-), 2.5-2.6ppm(m,1H,-O-CO- CH 2-), 3.7-3.8ppm (m, 1H, -N- CH 2-C=) 4.0-4.1ppm (m, 2H, -CH2- CH 2-O-), 4.1-4.2 ppm (m, 2H, -CH2- CH 2-O-), 5.0-5.1ppm (m, 2H, - GIVE -,-N- CH 2-C=), 7.2-7.7ppm(m,8H,- CH = CH -)

[0149] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (3-11) were as follows: Mn=44,929 Mw=83,568 Mw / Mn=1.86

[0150] [Synthesis Example 3-12] Synthesis of polymer (3-12) Polymer (3-12) (a random copolymer in which a in formula (1) is 0, X is an azide group, m is 13, and n in formula (2) is 31) was obtained in a yield of 94% by carrying out the same procedure as in Synthesis Example 3-6, except that polymer (2-8) (1g) obtained in Synthesis Example 2-8 was used instead of polymer (2-7) obtained in Synthesis Example 2-7.

[0151] Polymer (3-12) 1 H NMR(CDCl3,400MHz)δ 1.3-1.9ppm(m,12H,-CHN3- CH 2- CH 2- CH 2-CH2-, -CO-CH2- CH 2- CH 2- CH 2-CH2-), 2.3-2.4ppm(m,2H,-CO- CH 2-CH2-), 3.6ppm(t,2H,J=6.4Hz,- CH 2-OH), 3.8-3.9 ppm (m, 1H, -CO- CH N3-CH2-), 4.0-4.1ppm(m,2H,-CH2- CH 2-O-), 4.1-4.3 ppm (m, 2H, -CH2- CH 2-O-)

[0152] The number average molecular weight (Mn), weight average molecular weight (Mw), and polydispersity (Mw / Mn) of the polymer (3-12) were as follows: Mn=14,023 Mw=16,968 Mw / Mn=1.21

[0153] [Synthesis Example 4] Synthesis of DBCO-RGD GRGDS peptide (Genscript, 14.4 mg, 0.029 mmol, hereinafter referred to as "RGD peptide") and dehydrated DMSO (1 mL) were added to a vial to dissolve the RGD peptide. A dimethyl sulfoxide solution (77 mM, 0.5 mL) of DBCO-PEG4-NHS Ester (Tokyo Chemical Industry Co., Ltd.) and diisopropylethylamine (25 μL, 0.15 mmol) were added to the vial, the vial was capped, and the mixture was stirred at room temperature for 24 hours. After the reaction was completed, the mixture was purified using a gel filtration column (Sephadex g-10, exclusion limit: 700 Da, eluent: ion-exchanged water) to obtain DBCO-RGD (RGD peptide having group (1d)). The yield was 82%.

[0154] DBCO-RGD 1 H NMR(D2O,400MHz)δ 1.4-2.2ppm(m,4H,-CH- CH 2- CH 2-CH2), 2.3-2.5 ppm (m, 2H, -NH- CH 2-CH2-), 2.5-2.8ppm (m, 4H, - CH 2-COOH,-NH- CH 2-CH2-), 2.9-3.1ppm(m,4H,-CH2- CH 2-CO-, -CH2- CH 2-CO-), 3.1-3.3 ppm (m, 2H, - CH 2-NH-C(NH)-), 3.5-3.9ppm(m,22H,- CH 2-O- CH 2- CH 2-, -O- CH 2-CH2-CO-,- CH 2-OH,-NH- CH 2-CO-), 4.1-2.0 ppm (m, 1H, -NH- CH (CH2CH2)-CO-), 4.5-4.8ppm(m,2H,-NH- CH (CH2COOH)-CO-,-NH- CH (CH2OH)-COOH), 7.0-7.5ppm(m,8H,- CH = CH - CH = CH -)

[0155] [Comparative Example 1-1] Polymer film molding The polymer (3-1) (1 g) obtained in Synthesis Example 3-1 was dissolved in 1,3-dioxolane (8.1 g) and propylene glycol-1-monomethyl ether-2-acetate (0.9 g) as a solvent to prepare a polymer solution.

[0156] The obtained polymer solution was spin-coated on a 1 cm x 1 cm glass substrate using a spin coater (ACT-220AII, manufactured by Active Co., Ltd.) The obtained coating film was then heated at 90°C for 10 minutes in the air and then cooled at room temperature to form a polymer film.

[0157] [Comparative Example 1-2] Polymer film molding A polymer film was formed in the same manner as in Comparative Example 1-1, except that polycaprolactone (Mn=80,000, 0.2 g) and the polymer (3-1) (0.8 g) obtained in Synthesis Example 3-1 were used.

[0158] [Example 1-1] Polymer film molding A polymer film was formed in the same manner as in Comparative Example 1-1, except that polycaprolactone (Mn=80,000, 0.3 g) and the polymer (3-1) (0.7 g) obtained in Synthesis Example 3-1 were used.

[0159] [Example 1-2] Polymer film molding A polymer film was formed in the same manner as in Comparative Example 1-1, except that polycaprolactone (Mn=80,000, 0.5 g) and the polymer (3-1) (0.5 g) obtained in Synthesis Example 3-1 were used.

[0160] [Example 1-3] Polymer film molding A polymer film was formed in the same manner as in Comparative Example 1-1, except that polycaprolactone (Mn=80,000, 0.67 g) and the polymer (3-1) (0.33 g) obtained in Synthesis Example 3-1 were used.

[0161] [Examples 1-4] Polymer film molding A polymer film was formed in the same manner as in Comparative Example 1-1, except that polycaprolactone (Mn=80,000, 0.8 g) and the polymer (3-1) (0.2 g) obtained in Synthesis Example 3-1 were used.

[0162] [Examples 1-5] Polymer film molding A polymer film was formed in the same manner as in Comparative Example 1-1, except that polycaprolactone (Mn=80,000, 0.89 g) and the polymer (3-1) (0.11 g) obtained in Synthesis Example 3-1 were used.

[0163] [Examples 1-6] Polymer film molding A polymer film was formed in the same manner as in Comparative Example 1-1, except that polycaprolactone (Mn=80,000, 0.94 g) and the polymer (3-1) (0.06 g) obtained in Synthesis Example 3-1 were used.

[0164] [Examples 1-7] Polymer film molding A polymer film was formed in the same manner as in Example 1-2, except that the polymer (3-3) obtained in Synthesis Example 3-3 was used instead of the polymer (3-1) obtained in Synthesis Example 3-1.

[0165] [Example 1-8] Polymer film molding A polymer film was formed in the same manner as in Example 1-2, except that the polymer (3-4) obtained in Synthesis Example 3-4 was used instead of the polymer (3-1) obtained in Synthesis Example 3-1.

[0166] [Examples 1-9] Polymer film molding A polymer film was formed in the same manner as in Example 1-2, except that the polymer (3-5) obtained in Synthesis Example 3-5 was used instead of the polymer (3-1) obtained in Synthesis Example 3-1.

[0167] [Examples 1-10] Polymer film molding A polymer film was formed in the same manner as in Example 1-2, except that the polymer (3-6) obtained in Synthesis Example 3-6 was used instead of the polymer (3-1) obtained in Synthesis Example 3-1.

[0168] [Examples 1-11] Polymer film molding A polymer film was formed in the same manner as in Example 1-2, except that the polymer (3-2) obtained in Synthesis Example 3-2 was used instead of the polymer (3-1) obtained in Synthesis Example 3-1.

[0169] [Examples 1-12] Polymer film molding A polymer film was formed in the same manner as in Example 1-2, except that the polymer (3-7) obtained in Synthesis Example 3-7 was used instead of the polymer (3-1) obtained in Synthesis Example 3-1.

[0170] [Examples 1-13] Polymer film molding A polymer film was formed in the same manner as in Example 1-2, except that the polymer (3-8) obtained in Synthesis Example 3-8 was used instead of the polymer (3-1) obtained in Synthesis Example 3-1.

[0171] [Examples 1-14] Polymer film molding A polymer film was formed in the same manner as in Example 1-2, except that the polymer (2-2) obtained in Synthesis Example 2-2 was used instead of the polymer (3-1) obtained in Synthesis Example 3-1.

[0172] [Example 1-15] Polymer film molding A polymer film was formed in the same manner as in Example 1-2, except that the polymer (3-9) obtained in Synthesis Example 3-9 was used instead of the polymer (3-1) obtained in Synthesis Example 3-1.

[0173] [Example 1-16] Polymer film molding A polymer film was formed in the same manner as in Example 1-2, except that the polymer (3-10) obtained in Synthesis Example 3-10 was used instead of the polymer (3-1) obtained in Synthesis Example 3-1.

[0174] [Example 1-17] Polymer film molding A polymer film was formed in the same manner as in Example 1-2, except that the polymer (3-11) obtained in Synthesis Example 3-11 was used instead of the polymer (3-1) obtained in Synthesis Example 3-1.

[0175] [Example 1-18] Polymer film molding A polymer film was formed in the same manner as in Example 1-2, except that the polymer (3-12) obtained in Synthesis Example 3-12 was used instead of the polymer (3-1) obtained in Synthesis Example 3-1.

[0176] [Example 1-19] Polymer film molding A polymer film was formed in the same manner as in Example 1-2, except that polylactic acid (Mn=30,000) was used instead of polycaprolactone.

[0177] Table 1 shows the types of biodegradable polyesters and polymers of the present invention used in Comparative Examples 1-1 and 1-2 and Examples 1-1 to 1-19, as well as the amounts of each used.

[0178] [Table 1]

[0179] [Experimental Example 1] The polymer films obtained in any of Comparative Examples 1-1 and 1-2, Examples 1-1, 1-2, 1-4, and Examples 1-7 to 1-19 were immersed in water at 40°C for 1 hour, and then the surfaces were scratched with a pipette at a force of 700 mg. The presence or absence of scratches was confirmed visually. The results are shown in Table 2.

[0180] [Table 2]

[0181] As shown in Table 2, scratches were observed in the polymer films of Comparative Example 1-1 and Comparative Example 1-2. If the scaffold for cell culture is scratched during various operations before cell seeding, the cells cultured in that area cannot receive the effect of the surface-modified biofunctional molecules, and the scaffold is unsuitable as a scaffold for cell culture. It was found that the polymer films obtained in Examples 1-1, 1-2, 1-4, and 1-7 to 1-19 are resistant to external stress and are not scratched. Therefore, the scaffold for cell culture obtained from these polymer films is less likely to be scratched during various operations during the cell culture process, and is more likely to exhibit the effect of the biofunctional molecules bound to its surface.

[0182] [Comparative Example 2-1] Preparation of polymer solution A polymer solution was prepared by dissolving the polymer (3-1) (1.2 g) obtained in Synthesis Example 3-1 using 1,3-dioxolane (7 mL) and hexafluoroisopropanol (3 mL) as solvents. The viscosity of the obtained polymer solution at 24°C was 44 mPa s.

[0183] [Comparative Example 2-2] Preparation of polymer solution A polymer solution was prepared by dissolving the polymer (3-1) (2.4 g) obtained in Synthesis Example 3-1 using 1,3-dioxolane (7 mL) and hexafluoroisopropanol (3 mL) as solvents. The viscosity of the obtained polymer solution at 24°C was 425 mPa s.

[0184] [Example 2-1] Preparation of polymer solution A polymer solution was prepared in the same manner as in Comparative Example 2-1, except that polycaprolactone (Mn=80,000, 0.96 g) and the polymer (3-1) (0.24 g) obtained in Synthesis Example 3-1 were used. The viscosity of the obtained polymer solution at 24°C was 471 mPa s.

[0185] [Example 2-2] Preparation of polymer solution A polymer solution was prepared in the same manner as in Comparative Example 2-1, except that polycaprolactone (Mn=80,000, 0.77 g) and the polymer (3-1) (0.23 g) obtained in Synthesis Example 3-1 were used. The viscosity of the obtained polymer solution at 24°C was 325 mPa s.

[0186] [Example 2-3] Preparation of polymer solution A polymer solution was prepared in the same manner as in Comparative Example 2-1, except that polycaprolactone (Mn=80,000, 0.5 g) and the polymer (3-1) (0.5 g) obtained in Synthesis Example 3-1 were used. The viscosity of the obtained polymer solution at 24°C was 139 mPa s.

[0187] [Experimental Example 2] The polymer solutions obtained in Comparative Example 2-1, Comparative Example 2-2, and Examples 2-1 to 2-3 were electrospun under the following conditions to produce nanofibers, and the nanofibers obtained were observed under a scanning electron microscope to measure their fiber diameters. The results are shown in Table 3.

[0188] Spinning conditions Equipment: Nanofiber electrospinning unit NEU-11-TEA-2622 manufactured by Kato Tech Co., Ltd. Nozzle: 21G needle Target: Aluminum foil Target distance: 100mm Spinning solution discharge rate: 10μL / min Applied voltage: 7.0~9.0kV Spinning environment temperature: 25.0℃ Humidity: 30% or less Spinning time: 3 minutes

[0189] [Table 3]

[0190] As shown in Table 3, nanofibers were not obtained from the polymer solutions of Comparative Example 2-1 and Comparative Example 2-2. On the other hand, nanofibers with fiber diameters of 221 to 341 nm were obtained from the polymer solutions of Examples 2-1 to 2-3. In electrospinning, the viscosity of the polymer solution is one of the important factors, and although the viscosity of the polymer solution of Comparative Example 2-2 at 24°C (425 mPa·s) was equal to or greater than the viscosity of the polymer solution of Example 2-2 at 24°C (325 mPa·s), nanofibers were not obtained from the polymer solution of Comparative Example 2-2. Therefore, it is important for the production of nanofibers to mix the biodegradable polyester and the polymer in the weight ratio of the present invention.

[0191] [Example 3-1] Production of a scaffold for cell culture to which a biofunctional molecule is bound The polymer film obtained in Example 1-3 was placed in the well of a 24-well plate (AGC Technoglass), and 0.5 mL of the aqueous solution of DBCO-RGD (concentration: 97.5 μM) obtained in Synthesis Example 4 was added thereto. The polymer film was immersed in the aqueous solution at 40° C. for 24 hours, and then washed with water and dried to produce a scaffold for cell culture bound with a biofunctional molecule (RGD peptide).

[0192] [Example 3-2] Production of a scaffold for cell culture to which biofunctional molecules are bound A scaffold for cell culture to which a biofunctional molecule (RGD peptide) was bound was produced by carrying out the same procedure as in Example 3-1, except that the polymer film obtained in Example 1-4 was used.

[0193] [Example 3-3] Production of a scaffold for cell culture bound to a biofunctional molecule A scaffold for cell culture to which a biofunctional molecule (RGD peptide) was bound was produced by carrying out the same procedure as in Example 3-1, except that the polymer film obtained in Example 1-5 was used.

[0194] [Example 3-4] Production of a scaffold for cell culture bound to a biofunctional molecule A scaffold for cell culture to which a biofunctional molecule (RGD peptide) was bound was produced by carrying out the same procedure as in Example 3-1, except that the polymer film obtained in Example 1-6 was used.

[0195] [Experimental Example 3] Cell culture The scaffold for cell culture produced in any one of Examples 3-1 to 3-4 was placed in a well of a 24-well plate, and NIH3T3 cells suspended in Dulbecco's modified Eagle medium (DMEM) containing 10% by mass of fetal bovine serum (FBS) were cultured at 1 × 10 4 cells / cm 2 The cells were seeded in 500 μL of each well of a 24-well plate so that the cells were cultured at 37 ° C. in a CO2 incubator for 24 hours. After that, the medium in each well was removed, the scaffold for cell culture was washed with phosphate-buffered saline (PBS), and the scaffold for cell culture was transferred to another well of a 24-well plate. Next, 500 μL of cell counting kit-8-containing culture solution was added to the well containing the scaffold for cell culture, and the resulting mixture was left to stand at 37 ° C. for 3 hours in a CO2 incubator. After 3 hours, 100 μL of the culture solution from each well was transferred to a well of a 96-well plate, its absorbance (450 nm) was measured, and the number of adherent cells was calculated. The results are shown in Table 4.

[0196] [Table 4]

[0197] As shown in Table 4, the number of adherent cells tends to increase as the content of polymer (3-1) increases relative to 100 parts by mass of biodegradable polyester (polycaprolactone). The scaffold used in Experimental Example 3 is bound to RGD peptide, a cell adhesive peptide. Therefore, the above tendency indicates that the amount of RGD peptide increases with the increase in the content, resulting in an increase in the number of adherent cells.

[0198] From the results of this experimental example, it was confirmed that a scaffold useful for cell culture can be produced by binding a biofunctional molecule to the scaffold base material for cell culture of the present invention. Furthermore, since no damage or the like was observed on the scaffold in this experimental example, it was confirmed that the scaffold for cell culture of the present invention can be used for cell culture while maintaining its shape. [Industrial Applicability]

[0199] By using the substrate of the present invention, a scaffold for cell culture to which a biofunctional molecule is bound (i.e., the scaffold of the present invention) can be obtained. The scaffold of the present invention is useful for cell culture.

Claims

1. Formula (1): 【Chemistry 1】 (In the formula, * represents a bonding position. a represents 0 or 1; A represents a divalent organic group; X is a halogen atom, an amino group, a sulfanyl group, an azide group, or a group represented by formula (1a) to formula (1f): 【Chemistry 2】 (wherein * represents a bond position, and R １ represents a hydrogen atom or a methyl group. and m represents a number from 1 to 300. and optionally having a repeating unit represented by formula (2): 【Chemistry 3】 (wherein * represents a bond position, and n represents a number from 0 to 2,700. and the ratio of n / m is in the range of 0 / 100 to 99 / 1; and Biodegradable polyester A scaffold substrate for cell culture comprising: A scaffold material for cell culture, wherein the content of the polymer is 1 to 300 parts by mass per 100 parts by mass of the biodegradable polyester.

2. 2. The scaffold for cell culture according to claim 1, wherein the biodegradable polyester is polycaprolactone.

3. 3. The scaffold for cell culture according to claim 1, wherein the number average molecular weight of the polymer is 1,000 to 300,000.

4. A scaffold for cell culture comprising the scaffold base material for cell culture according to claim 1 or 2 to which a biofunctional molecule is bound.

5. The scaffold substrate for cell culture according to claim 1 or 2, and A biofunctional molecule having a functional group capable of reacting with X in formula (1) 5. A method for producing a scaffold for cell culture according to claim 4, comprising mixing

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