Polyrotaxane having an amino group

Abstract

The present invention provides a polyrotaxane which is represented by formula (1). (In the formula, R1 represents a hydrogen atom or a methyl group; m represents a number from 1 to 2,000; n represents a number from 10 to 500; formula (2) represents a cyclodextrin wherein at least one hydroxyl group is modified by a group that is represented by -X-NH2; and X represents a divalent organic group.)

Description

Technical Field

[0001] The present invention relates to a polyrotaxane having an amino group.

Background Art

[0002] Cell culture technology is widely used in the fields of pharmaceuticals and cosmetics, and regenerative medicine research, and is an important technology. As the environment for cell culture, an appropriate culture environment, culture medium, and culture substrate are required. Among them, the culture substrate has the role of a scaffold to which cells adhere, and has characteristics such as being a surface suitable for cell culture or being processable, having no cytotoxicity, being able to be sterilized or maintaining a sterilized state, not deteriorating under culture conditions, and not interfering with observation under a microscope.

[0003] Conventionally, glass products have been widely used as culture substrates, but currently polystyrene products are widely used. However, a culture substrate formed of polystyrene resin has a hydrophobic surface and low affinity for cells, so it is generally surface-treated. Examples of the surface treatment include plasma treatment, corona discharge treatment, oxidizing agent treatment, and coating with a hydrophilic substance. Many coating agents promote cell adhesion. For example, type I collagen, type IV collagen, gelatin, fibronectin, vitronectin, laminin, matrigel, hydroxyapatite, etc. are known. It is also known that coating with water-soluble elastin promotes the induction of differentiation of vascular smooth muscle cells or elastin-responsive cells.

[0004] Mesenchymal stem cells are somatic stem cells derived from the mesoderm that have the ability of self-renewal and multi-differentiation potential into mesenchymal tissues such as osteoblasts, chondrocytes, adipocytes, skeletal muscle cells, and ligament cells. In adult tissues, they exist in connective tissues such as the dermis, skeletal muscle, and adipose tissue, and mainly in the bone marrow stroma, and function in the repair of connective tissues, maintenance of homeostasis, and regulation of the proliferation and differentiation of hematopoietic stem cells. The differentiation of mesenchymal stem cells is complexly regulated by biological factors and physical factors. For example, it is known that when dexamethasone, β-glycerophosphate, and ascorbic acid are added to the culture medium of mesenchymal stem cells, they can be induced to differentiate into osteoblasts, and when dexamethasone, 3-isobutyl-1-methylxanthine, insulin, and indomethacin are added, they can be induced to differentiate into adipocytes.

[0005] In recent years, it has been noted that the characteristics of the culture substrate (for example, bulk properties such as hardness, and surface properties of the coating) affect the functions of cultured cells, and research on growing cells with desired functions has been reported (Non-Patent Documents 1 to 3).

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0007] However, in order to obtain cells with desired functions, it is still difficult to finely adjust the bulk properties (such as hardness) of cell culture substrates. It is considered useful to select appropriate physical factors to differentiate mesenchymal stem cells into desired cell lineages. Therefore, an object of the present invention is to provide a coating agent and a culture substrate that can promote the induction of differentiation of mesenchymal stem cells.

Means for Solving the Problems

[0008] The present invention provides the following [1] to

[15] . [1] A polyrotaxane represented by formula (1).

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[10] The method according to [9], comprising coating a composition containing the polyrotaxane represented by the formula (1) on the surface of a substrate.

[11] The method according to [9] or

[10] , wherein the polyrotaxane contains a cyclodextrin modified with a group represented by -X-NH having 1 to 18 hydroxyl groups 2 wherein X is a divalent organic group.]

[12] The method according to any one of [9] to

[11] , wherein the number of penetrations of cyclodextrin in the polyrotaxane is 3 to 220.

[13] A culture substrate comprising a substrate coated with a composition containing the polyrotaxane represented by the formula (1). [Chemical formula] [wherein, R 1 is a hydrogen atom or a methyl group, m is 1 to 2000, n is 10 to 500, [Chemical formula] is a cyclodextrin modified with a group represented by -X-NH having at least one hydroxyl group 2 wherein X is a divalent organic group.]

[14] The culture substrate according to

[13] , wherein the polyrotaxane contains a cyclodextrin modified with a group represented by -X-NH having 1 to 18 hydroxyl groups 2 wherein X is a divalent organic group.]

[15] The culture substrate according to

[13] or

[14] , wherein the number of penetrations of cyclodextrin in the polyrotaxane is 3 to 200. [Advantages of the Invention]

[0009] According to the present invention, it is possible to provide a coating agent capable of suppressing osteoblast differentiation or promoting adipogenic differentiation of mesenchymal stem cells. Further, according to the present invention, it is also possible to provide a substrate (culture substrate) coated with the coating agent.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Modes for Carrying Out the Invention

[0011] <First Embodiment> The first embodiment of the present invention is a polyrotaxane represented by formula (1).

Chemical formula

[0012] The polyrotaxane represented by formula (1) is composed of an axle molecule (linear polymer) and a modified cyclodextrin (CD, cyclic molecule). The axle molecule penetrates through at least one modified cyclodextrin and is capped at both ends thereof.

[0013] The axle molecule is represented by the following formula (2), has a polyethylene glycol structure at the center of the molecule, and has a poly(meth)acrylate structure capped with a phenyl dithioester group at the ends. In the formula, R 1 is a hydrogen atom or a methyl group. The cyclodextrin penetrates through the polyethylene glycol structure portion. Each R 1 may be the same as or different from each other. As R 1 , a methyl group is preferred. [Chemical formula]

[0014] The polyethylene glycol structure may contain ethylene glycol as a monomer unit and have a molecular length capable of penetrating at least one cyclodextrin. The number of ethylene glycol units forming the polyethylene glycol structure may be from 20 to 1000, preferably from 85 to 800, and more preferably from 100 to 700. In the formula, n may be from 10 to 500, preferably from 43 to 400, and more preferably from 50 to 350.

[0015] The poly(meth)acrylate structure contains benzyl (meth)acrylate as a monomer unit. In this specification, the term "(meth)acrylate" means both "acrylate" and "methacrylate". The inventors believe that since the poly(meth)acrylate structure has the effect of enhancing the adhesion to a substrate such as polystyrene, the polyethylene glycol portion passing through the cyclodextrin is in a state of being looped away from the substrate. The number m of benzyl (meth)acrylate units forming the poly(meth)acrylate structure may be from 1 to 2000, preferably from 20 to 1000, and more preferably from 30 to 500, per polybenzyl (meth)acrylate at one end of the triblock copolymer. Each m may be the same as or different from each other.

[0016] The modified cyclodextrin used in this embodiment is represented by the following formula (3), and at least one of the hydroxyl groups of the constituent glucose is -X-NH 2 modified. The cyclodextrin may be any of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and combinations thereof. The preferred cyclodextrin is α-cyclodextrin. The following is a schematic diagram of cyclodextrin modified with -X-NH 2 In the figure, "-O-X-NH 2 " indicates that the hydroxyl group in glucose constituting the cyclodextrin is modified with -X-NH 2 . In the figure, although only one "-O-X-NH 2 " is shown, it should not be construed as being limited to only one hydroxyl group being modified with -X-NH 2 .

Chemical formula

[0017] In the case of polyrotaxane, at least one modified cyclodextrin is penetrated by an axial molecule. The number of cyclodextrins per molecule of polyrotaxane can be determined independently and does not necessarily have to be uniquely determined by the molecular length of the polyethylene glycol structure. Also, stoichiometrically, since cyclodextrin can include two units of ethylene glycol which is a repeating unit of polyethylene glycol, the number of cyclic molecules has an upper limit depending on the molecular weight of the linear polymer used. For example, the number of cyclodextrins per molecule of polyethylene glycol having a number average molecular weight of 20,000 may be 3 to 220, preferably 5 to 150, more preferably 5 to 120, and particularly preferably 5 to 100. However, the length of the axial molecule of cyclodextrin in the main chain direction (when the polyrotaxane has a plurality of cyclodextrins, the total of the lengths) does not exceed the molecular length of polyethylene glycol.

[0018] -X-NH in cyclodextrin 2 The number of hydroxyl groups modified with may be 1 to 18, preferably 1 to 10, and more preferably 2 to 6 per one cyclodextrin. -X-NH 2 When the number of hydroxyl groups modified with is within the above range, the effect of promoting adipogenic differentiation of mesenchymal stem cells is more excellent, and the effect of suppressing osteoblastic differentiation is also more excellent. -X-NH 2 When the number of hydroxyl groups modified with is 10 or less, cytotoxicity is less likely to occur. Also, -X-NH 2 When the number of hydroxyl groups modified with is 2 or more, the amount of change in molecular mobility, the surface chemical composition, and the amount of change in the surface physicochemical properties (for example, contact angle, zeta potential) become large. -X-NH 2 Since the molecular mobility, the surface chemical composition, and the surface physicochemical properties (for example, contact angle, zeta potential) of polyrotaxane can be adjusted by the number of hydroxyl groups modified with, a polyrotaxane surface having a molecular mobility suitable for promoting adipogenic differentiation and / or suppressing osteoblastic differentiation of mesenchymal stem cells can be produced.

[0019] X is a divalent organic group and is not particularly limited. Examples of the organic group include an alkylene group having 1 to 10 carbon atoms, an alkenylene group having 1 to 20 carbon atoms, and an alkynylene group having 1 to 20 carbon atoms. It may have an oxo group at any position, may be via an oxy group or an imino group, or may be a combination thereof. For example, combining a carbon atom having an oxo group with an oxy group forms an ester bond, and combining a carbon atom having an oxo group with an imino group forms an amide bond. An alkylene group having a plurality of oxy groups is also called an oxyalkylene group and includes, for example, a polyoxyethylene group having 1 to 10 carbon atoms and a polyoxypropylene group having 1 to 10 carbon atoms. Specific examples of the organic group include alkylene groups such as methylene group, ethylene group, propylene group, butylene group, pentylene group, hexylene group, heptylene group, octylene group, nonanylene group, and decylene group; alkenylene groups such as propynylene group, butenylene group, pentenylene group, hexenylene group, heptenylene group, octenylene group, nonenylene group, and decenylene group; and alkynylene groups such as propargyl group, butynylene group, pentynylene group, hexynylene group, heptynylene group, octynylene group, nonylene group, and decynylene group. As the organic group having an oxo group, an oxy group, or an imino group, it is preferable that an ester bond, a carbonate ester bond, or a urethane bond is formed together with an oxygen atom derived from a hydroxyl group of cyclodextrin. Examples of the organic group having an oxo group, an oxy group, or an imino group include a carbonylalkylene group such as a carbonylmethylene group (-C(=O)CH 2 -), an alkylene carbonyl group such as a methylene carbonyl group (-CH 2 C(=O)-), an alkylene carbonylamino group such as a methylene carbonylamino group (-CH 2 C(=O)NH-), a carbonylaminomethylene group (-C(=O)NHCH 2 -), a carbonylaminoalkylene group such as a carbonylaminoethylene group (-C(=O)NHCH 2 CH 2 -), a carbonylaminoethyleneoxyethylene group (-C(=O)NHCH 2 CH 2 OCH 2 CH2 -) and other carbonylaminoalkyleneoxyalkylene groups; carbonylaminoethylene poly(oxyethylene) groups (-C(=O)NHCH 2 CH 2 (OCH 2 CH 2 ) t -) (for example, in the formula, t is from 2 to 1000.) and other carbonylaminoethylene poly(oxyalkylene) groups; oxypropylene groups (-OCH 2 CH 2 CH 2 -) and other oxyalkylene groups.

[0020] The modified cyclodextrin is preferably represented by formula (4). In the formula, -C(=O)NH-Xa- corresponds to X in formula (3) and corresponds to one of the embodiments where X has an oxo group and an imino group. Xa is an organic group having 1 to 9 carbon atoms, preferably an alkylene group having 2 to 9 carbon atoms, and more preferably a polyoxyethylene group having 2 to 8 carbon atoms.

Chemical formula

[0021] The polyrotaxane has a structure in which at least one cyclodextrin penetrates the axle molecule. Each cyclodextrin can move along the main chain direction of the axle molecule and can rotate around the main chain of the axle molecule. Such structural characteristics are called molecular mobility. Molecular mobility can vary, for example, according to the number of cyclodextrins and the number of substituents modified on the glucose constituting the cyclodextrin.

[0022] Molecular mobility can be measured using a contact angle meter for the static contact angle of the coating surface after coating the surface of the culture substrate, and can be evaluated using the droplet method and the captive bubble method. For the evaluation of molecular mobility, reference may be made to, for example, the methods described in Soft Matter, 2012, 8, 5477-5485 (Ji-Hun Seo et al.), Adv. Healthcare Mater. 2015, 4, 215-222, etc. Specifically, the contact angle hysteresis value of the coating surface can be calculated from the difference between the contact angle of water measured by the droplet method (contact angle of water in air) and the contact angle of water obtained from the contact angle of the bubble measured by the captive bubble method (contact angle of water in water). As a control, it may be compared with the effect on the culture substrate coated with DMSO.

[0023] Molecular mobility can be adjusted by changing the number of penetrations of cyclodextrin and / or the number of hydroxyl groups modified with -X-NH in cyclodextrin. For example, when the number of penetrations of cyclodextrin is 3 to 120, it may be suitable for promoting adipogenic differentiation and / or suppressing osteogenic differentiation of mesenchymal stem cells. When the number of penetrations of cyclodextrin is large, it becomes easier to purify by reprecipitation. 2

[0024] <Second Embodiment> The second embodiment of the present invention is a coating agent containing a polyrotaxane represented by the formula (1). In this embodiment, the "polyrotaxane represented by the formula (1)" can be referred to as that described in the first embodiment.

[0025] The content of the polyrotaxane may be 0.0005 to 5% by mass, preferably 0.01 to 1% by mass, and more preferably 0.02 to 0.5% by mass based on the mass of the coating agent.

[0026] ​The coating agent according to this embodiment may contain a solvent and optional additives in addition to the above-mentioned polyrotaxane. Examples of the solvent include dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), N,N-dimethylformamide (DMF), methanol, 2-propanol, chloroform, and methylene chloride. Examples of such additives include antioxidants and the like.

[0027] The coating agent according to this embodiment is used to coat the surface of a substrate that can be used as a culture substrate. The substrate that can be used as a culture substrate may be well-known to those skilled in the art. Examples of the substrate material include glass, polystyrene, polypropylene, polyethylene, polyolefin, polycarbonate, acrylic block copolymer (BCF), and the like.

[0028] According to the coating agent of this embodiment, in the glucose constituting cyclodextrin, by adjusting the number of hydroxyl groups modified with -X-NH 2 it is possible to adjust the differentiation of mesenchymal stem cells (promotion of adipogenic differentiation and suppression of osteoblastic differentiation) without depending on the bulk properties of the substrate itself. More specifically, as adipogenic differentiation progresses, the amount of intracellular lipid droplet accumulation increases. The intracellular lipid droplets accumulated can be stained with Oil Red O dye, and the degree of intracellular lipid droplet accumulation can be evaluated. The adipogenic differentiation of mesenchymal stem cells may be determined by the expression level of a differentiation marker gene (for example, PPARγ, C / EBPα, aP2) that is an indicator of adipogenic differentiation. In osteoblastic differentiation, as differentiation progresses, bone nodules are formed (also called mineralization). The bone nodules can be stained with Alizarin Red S dye, and the degree of mineralization can be evaluated. The osteoblastic differentiation of mesenchymal stem cells may be determined by the expression level of a differentiation marker gene (for example, RUNX2, alkaline phosphatase, osteocalcin, osteopontin, bone sialoprotein, type I collagen) that is an indicator of osteoblastic differentiation.

[0029] The polyrotaxane can be produced with reference to the examples and may be produced as follows. The hydroxyl groups at both ends of polyethylene glycol having a desired length are converted into leaving groups (for example, halogenation, methanesulfonylation, toluenesulfonylation), and etherified with phenylalaninol to obtain a diamine. The obtained diamine is mixed with cyclodextrin to obtain a pseudorotaxane. At this time, the penetration number of cyclodextrin can be adjusted by adjusting the amount of cyclodextrin with respect to one molecule of diamine. Subsequently, after reacting with CPADB and DMT-MM to cap the cyclodextrin so that it does not come off from the axle molecule, benzyl methacrylate (corresponding to the (meth)acrylate structure) is introduced at both ends as an anchoring segment by reversible addition-fragmentation chain transfer polymerization reaction (RAFT polymerization reaction).

[0030] <Third Embodiment> The third embodiment of the present invention is a cell culture method for promoting the adipogenic differentiation of mesenchymal stem cells, which includes culturing mesenchymal stem cells on the surface of a substrate coated with a composition containing the polyrotaxane represented by formula (1).

[0031] In this embodiment, the "polyrotaxane represented by formula (1)" can be referred to as that described in the first embodiment, and the "composition containing the polyrotaxane represented by formula (1)" can use the coating agent described in the second embodiment.

[0032] In the cell culture method according to this embodiment, as a culture substrate, a substrate coated with a composition containing the polyrotaxane represented by formula (1) is used. The cell culture is carried out by adhering the cells to the surface of the coated substrate.

[0033] The culture substrate is obtained by applying a coating containing a polyrotaxane represented by the formula (1) to a substrate. The substrate may be well-known to those skilled in the art. Examples of the material of the substrate include glass, polystyrene, polypropylene, polyethylene, polyolefin, polycarbonate, acrylic block copolymer (BCF), and the like. The substrate may be a commercially available glass substrate or plastic substrate.

[0034] In the cell culture method according to this embodiment, a culture medium is poured onto the coating surface applied on the substrate so that the cells are immersed, and the cells to be cultured are seeded and cultured. The culture medium may be replaced with a new culture medium as necessary. Further, a step of pouring a growth medium before differentiating the cells to grow the seeded cells may be provided. In this case, after growing until a sufficient number of cells are obtained, the growth medium is replaced with a differentiation medium. As the growth medium and the differentiation medium, media well-known to those skilled in the art can be used.

[0035] As the culture medium for adipogenic differentiation of mesenchymal stem cells, for example, Mesenchymal Stem Cell Adipogenic Differentiation Medium2 (C-28016) manufactured by PromoCell GmbH (Heidelberg, Germany) can be used.

[0036] The cell culture environment can be arbitrarily set under conditions well-known to those skilled in the art.

[0037] Adipogenic differentiation of mesenchymal stem cells means the differentiation of mesenchymal stem cells into adipocytes. As adipogenic differentiation progresses, the amount of lipid droplet accumulation in cells increases. The adipogenic differentiation promoting effect of mesenchymal stem cells can be evaluated by cell staining with Oil Red O (lipid droplets in cells are stained). It may also be determined by measuring changes in the expression levels of differentiation markers. For example, differentiation markers for adipogenic differentiation include PPARγ, C / EBPα, and aP2. If differentiation is promoted statistically significantly compared to the case of using a culture substrate coated with unmodified polytaxane (for example, PRX-PBzMA described later) on a substrate (made of glass or polystyrene), it can be determined that it has an adipogenic differentiation promoting effect on mesenchymal stem cells.

[0038] The cell culture method according to this embodiment may include coating a composition containing a polytaxane represented by formula (1) on the surface of the above substrate.

[0039] The composition containing the polytaxane represented by formula (1) can be coated on a substrate. The coating method is not particularly limited, and examples include casting, spin coating, gravure coating, die coating, knife coating, bar coating, blade coating, roll coating, and the like. A preferred coating method is casting.

[0040] <Fourth Embodiment> The fourth embodiment of the present invention is a cell culture method for suppressing osteogenic differentiation of mesenchymal stem cells, which includes culturing mesenchymal stem cells on the surface of a substrate coated with a composition containing a polytaxane represented by formula (1).

[0041] In this embodiment, the "polytaxane represented by formula (1)" can refer to that described in the first embodiment, and the "composition containing the polytaxane represented by formula (1)" can utilize the coating agent described in the second embodiment.

[0042] In the cell culture method according to this embodiment, as a culture substrate, a substrate coated with a composition containing a polyrotaxane represented by the formula (1) is used. The cell culture is carried out by adhering cells to the surface of the coated substrate.

[0043] The culture substrate is a substrate coated with a coating containing a polyrotaxane represented by the formula (1). The substrate may be well-known to those skilled in the art. Examples of the material of the substrate include glass, polystyrene, polypropylene, polyethylene, polyolefin, polycarbonate, acrylic block copolymer (BCF), and the like. The substrate may be a commercially available glass substrate or plastic substrate.

[0044] In the cell culture method according to this embodiment, a medium is poured onto the coated surface applied on the substrate so that the cells are immersed, the cells to be cultured are seeded, and cultured. The medium may be replaced with a new medium as necessary. Also, a step of pouring a growth medium before differentiating the cells to grow the seeded cells may be provided. In this case, after growing until a sufficient number of cells are obtained, the growth medium is replaced with a differentiation medium. As the growth medium and the differentiation medium, media well-known to those skilled in the art can be used.

[0045] As the medium for osteogenic differentiation of mesenchymal stem cells, for example, Mesenchymal Stem Cell Osteogenic Differentiation Medium (C-28013) from PromoCell GmbH (Heidelberg, Germany) can be used.

[0046] The cell culture environment can be arbitrarily set under conditions well-known to those skilled in the art.

[0047] Osteogenic differentiation of mesenchymal stem cells means the differentiation of mesenchymal stem cells into osteoblasts. As osteogenic differentiation progresses, bone nodules are formed (also called mineralization). The osteogenic differentiation promoting effect of mesenchymal stem cells can be evaluated by cell staining with alizarin red S (bone nodules are stained). It may also be determined by measuring changes in the expression levels of differentiation markers. For example, differentiation markers for osteogenic differentiation include RUNX2, alkaline phosphatase, osteocalcin, osteopontin, bone sialoprotein, and type I collagen. If differentiation is promoted statistically significantly compared to the case of using a culture substrate coated with unmodified polytaxane (for example, PRX-PBzMA described later) on a substrate (made of glass or polystyrene), it can be determined that it has an osteogenic differentiation promoting effect on mesenchymal stem cells.

[0048] The cell culture method according to this embodiment may include coating a composition containing a polytaxane represented by formula (1) on the surface of the substrate.

[0049] The composition containing the polytaxane represented by formula (1) can be coated on the substrate. The coating method is not particularly limited, and examples include casting, spin coating, gravure coating, die coating, knife coating, bar coating, blade coating, roll coating, and the like. A preferred coating method is casting.

[0050] <Fifth Embodiment> The fifth embodiment of the present invention is a culture substrate including a substrate coated with a composition containing a polytaxane represented by formula (1).

[0051] In this embodiment, for the "polytaxane represented by formula (1)", reference can be made to that described in the first embodiment, and for the "composition containing the polytaxane represented by formula (1)", the coating agent described in the second embodiment can be used. Also, for the coating method, reference can be made to the method described in the third embodiment.

[0052] The coated substrate (culture substrate) according to this embodiment is particularly suitable as a culture substrate for promoting the adipogenic differentiation of mesenchymal stem cells and / or suppressing the osteogenic differentiation thereof.

[0053] According to the culture substrate of this embodiment, by adjusting the number of hydroxyl groups modified with -X-NH 2 in the glucose constituting cyclodextrin, it becomes possible to adjust the differentiation (promotion of adipogenic differentiation and / or suppression of osteogenic differentiation) and proliferation (promotion or suppression) of mesenchymal stem cells without depending on the bulk properties of the substrate itself.

Examples

[0054] Hereinafter, the present invention will be described in more detail using examples. However, the present invention is not limited thereto. In addition, the abbreviations used in the examples are common abbreviations well-known to those skilled in the art, and the meanings of some abbreviations are shown below. αCD: α-cyclodextrin CDI: Carbonyldiimidazole CPADB: 4-Cyanopentanoic acid dithiobenzoate DMEM: Dulbecco's Modified Eagle Medium DMF: N,N-Dimethylformamide DMSO: Dimethyl sulfoxide DMT-MM: 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride EDTA: Ethylenediaminetetraacetic acid FBS: Fetal bovine serum IBMX: 3-Isobutyl-1-methylxanthine α-MEM: α-Minimum Essential Medium MeOH: Methanol MsCl: Methanesulfonyl chloride PBS: Phosphate Buffered Saline PEG: Polyethylene glycol TCPS: Tissue culture polystyrene TEA: Triethylamine THF: Tetrahydrofuran 1 H-NMR: Proton nuclear magnetic resonance spectrometry

[0055] 1. Synthesis of polyrotaxane (Example 1) The method for synthesizing polyrotaxane is shown below. [Chemical formula]

[0056] Step 1: Synthesis of α,ω-bismesyl polyethylene glycol Polyethylene glycol with a number average molecular weight of 20,000 (25.0 g, 1.25 mmol) and TEA (5.3 mL, 37.5 mmol) were dissolved in anhydrous THF (130 mL), and MsCl (2.0 mL, 25.0 mmol) was added dropwise, followed by stirring at 23°C. After 5 hours, the reaction solution was filtered, and the filtrate was precipitated with diethyl ether to recover the precipitate as a solid. The obtained precipitate was dried under reduced pressure to obtain α,ω-bismesyl polyethylene glycol (21.1 g, yield: 84%).

[0057] Step 2: Synthesis of bis(2-amino-3-phenylpropyl) polyethylene glycol L-phenylalaninol (1.49 g, 9.85 mmol) and sodium hydride (0.971 g, 60% mineral oil) were dissolved in anhydrous DMF (86 mL) under a nitrogen atmosphere. α,ω-Bismesyl polyethylene glycol (20.0 g, 0.992 mmol) was added to this mixture, followed by stirring at 23°C. After 24 hours, the reaction solution was filtered, and the filtrate was precipitated with diethyl ether to recover the precipitate as a solid. The obtained precipitate was dried under reduced pressure to obtain bis(2-amino-3-phenylpropyl) polyethylene glycol (11.8 g, yield: 59%).

[0058] Step 3: Synthesis of pseudo-polyrotaxane Bis(2-amino-3-phenylpropyl) polyethylene glycol (10.1 g, 0.499 mmol) was dissolved in water (50 mL), and a saturated aqueous solution (380 mL) of αCD (55.2 g, 56.6 mmol) was added, followed by stirring at 23 °C. After 19 hours, the precipitate was collected by centrifuging the reaction solution, and lyophilized for 9 days to obtain pseudo-polyrotaxane as a crude product.

[0059] Step 4: Synthesis of polyrotaxane PRX-CPADB CPADB (5.50 g, 19.7 mmol) and DMT-MM (5.50 g, 19.9 mmol) were dissolved in methanol (500 mL), and the pseudo-polyrotaxane obtained above was added to the reaction solution at 23 °C, followed by stirring. After 1 day, the crude product was washed with methanol, reprecipitated with water-containing DMSO, centrifuged, and lyophilized for 9 days to obtain polyrotaxane PRX-CPADB (11.9 g, two-step yield: 18%) as a powder. The structure of polyrotaxane PRX-CPADB was 1 confirmed by 1H-NMR (solvent: DMSO-d 6 6). Also, the number of αCD penetrations was 1 determined by 1H-NMR (solvent: D 2 2O). 1 1H-NMR (500 MHz, DMSO-d 6 6) δ 3.12 - 3.91 (m, PEG backbone and H2, H3, H4, H5, and H6 protons of αCD), 4.43 (m, OH6 of αCD), 4.80 (m, H1 of αCD), 5.49 (m, OH3 of αCD), 5.65 (m, OH2 of αCD), 7.17 (t, aromatics of phenylalanyl group), 7.25 (t, aromatics of phenylalanyl group), 7.52 (t, aromatics of CPADB group), 7.70 (t, aromatics of CPADB), and 7.91 (t, aromatics of CPADB).

[0060] Step 5: Synthesis of polyrotaxane (PRX-PBzMA) of Reference Example 1 Polyrotaxane PRX-CPADB (1.50 g, 13.9 μmol) was dissolved in anhydrous DMSO (12 mL), and benzyl methacrylate (1.71 g, 9.70 mmol) and 4,4'-azobis(4-cyanovaleric acid) (1.55 mg, 5.54 μmol) were added to this mixture. After degassing by the FPT cycle (Freeze-Pump-Thaw cycle), the mixture was stirred at 70°C. After 1 day, the crude product was precipitated with diethyl ether and dried under reduced pressure to obtain polyrotaxane PRX-PBzMA (3.13 g, yield: 98%). The number m of benzyl methacrylate units forming the polybenzyl methacrylate structure was determined by 1 1H-NMR (solvent: DMSO-d 6 6). 1 1H-NMR (500 MHz, DMSO-d 6 6) δ 0.40 - 0.95 (m, -CH(- CH3 )-CH2- of PBzMA), 1.43 - 2.10 (m, -CH(-CH3)- CH2 - of PBzMA), 3.17 - 4.02 (m, PEG backbone and H2, H3, H4, H5, and H6 protons of αCD), 4.44 (m, OH6 of αCD), 4.80 (m, H1 of αCD), 4.86 (m, - CH2 -Ph of PBzMA), 5.49 (m, OH3 of αCD), 5.66 (m, OH2 of αCD), and 7.26 (m, aromatics of PBzMA).

[0061] Step 6a: Synthesis of polyrotaxane (NH 2 -PRX) of Example 1 [Chemical formula] Polyrotaxane PRX-PBzMA (200 mg, 0.895 μmol) was dissolved in anhydrous DMSO (10 mL), and then CDI (104 mg, 0.643 mmol) was added to the solution. After stirring at 23 °C for 1 day, ethylenediamine (0.43 mL, 6.43 mmol) was added to this solution, and the mixture was further stirred at 23 °C for 1 day. Subsequently, the reaction solution was purified by dialysis for 4 days. The product was lyophilized for 7 days to obtain NH 2 -PRX (176 mg, yield 73%). The number of amino groups was determined by 1 1H-NMR analysis (solvent: DMSO-d 6 ). 1 1H-NMR (500 MHz, DMSO-d 6 ) δ = 0.38 - 0.92 (m, -CH(- CH3 )-CH2- of PBzMA), 1.47 - 2.02 (m, -CH(-CH3)- CH2 - of PBzMA), 3.00 (m, -O-CO-NH- CH2 -CH2-NH2), 3.15 - 4.55 (m, PEG backbone, H2, H3, H4, H5, and H6 protons of α-CD, and OH6 of α-CD), 4.86 (m, - CH2 -Ph of PBzMA, H1 of α-CD), and 7.26 (m, aromatics of PBzMA).

[0062] Step 6b: Synthesis of polyrotaxane (CH 3 -PRX) of Comparative Example 1

Chemical formula

[0063] Step 6c: Synthesis of polyrotaxane (OH-PRX) of Comparative Example 2

Chemical formula

[0064] Engineering 6d: Synthesis of the polyrotaxane (SO 3 H-PRX) of Comparative Example 3

Chemical formula

[0065] The data of the obtained polyrotaxane are shown in Table 1. Figure 1 is a diagram comparing the 1H-NMR spectra of the polyrotaxanes of Reference Example 1, Example 1, and Comparative Examples 1 to 3. The characteristic peaks in each spectrum are indicated in gray. The numbers in parentheses indicate the number of functional group modifications per cyclodextrin. 1 H-NMR spectra are compared. The characteristic peaks in each spectrum are shown in gray. The numbers in parentheses indicate the number of functional group modifications per cyclodextrin.

Table 1

[0066] The obtained polyrotaxane was dissolved in DMSO to produce a coating agent. The obtained coating agent was cast on TCPS.

[0067] 2. Analysis of surface chemical composition (1) Elemental composition of the surface The chemical composition of the polyrotaxane surface was analyzed by TOF-SIMS (PHI NanoTOF II, ULVAC-PHI) using 30 keV Bi3 primary ions. The analysis field of view was 100×100 μm. The elemental composition of the surface was determined by X-ray photoelectron spectroscopy (Al-Kα, Thermo Fisher Scientific, East Grinstead) using monochromatized X-ray radiation having an energy of 1486.6 eV. The diameter of the analysis area was 400 μm, and the elemental composition was calculated from the average of three points on each surface. ++ The mass spectra obtained by TOF-SIMS measurement are shown in Figures 2 to 3. A characteristic peak at m / z = 85 was observed on the surfaces other than TCPS. This peak is the methacrylate group (C

[0068] TOF-SIMS measurement are shown in Figures 2 to 3. On the surfaces other than TCPS, a characteristic peak at m / z = 85 was observed. This peak is the methacrylate group (C 4 H 5 O 2 -) was suggested to be present, and it was speculated that the TCPS surface was coated with a triblock copolymer containing a PBzMA structure. Also, for the surfaces of Example 1 and Comparative Example 2, a characteristic peak at m / z = 42 suggesting the presence of an N-containing group (CNO - ) was detected. From the results of the XPS analysis shown in Table 2, the elemental compositions of nitrogen and oxygen were significantly higher on the NH 2 -PRX surface (Example 1) and OH-PRX (Comparative Example 2) than on other surfaces.

Table 2

[0069] (2) Measurement of Contact Angle and Zeta Potential The surfaces of polystyrene for cell culture (TCPS) were coated with the polyrotaxanes of Example 1 and Comparative Examples 1 to 3. Using a contact angle meter (trade name: DM-501, manufactured by Kyowa Interface Science Co., Ltd.) and software, the static contact angles of the coated surfaces were measured by both the droplet method and the captive bubble method. The contact angle hysteresis value of each surface was calculated from the difference between the contact angle of water measured by the droplet method (contact angle of water in air) and the contact angle of water measured by the captive bubble method (contact angle of water in water). All measured values were obtained from four different surfaces, and the average values of three different points on each surface were recorded. The difference in contact angle hysteresis is known to indicate the difference in molecular mobility between surfaces.

[0070] Table 3 shows the contact angles and contact angle hysteresis of the polyrotaxane surfaces of Reference Example 1, Example 1, and Comparative Examples 1 to 3. The contact angle of water in air on the polyrotaxane surface was in the range of 80 to 100°, and it was confirmed that the wettability changed from the TCPS surface (contact angle: 74°) due to the polyrotaxane coating. The contact angle of water in air on the polyrotaxane surfaces of Example 1 and Comparative Examples 1 to 3 was slightly smaller than the contact angle of the polyrotaxane surface of Reference Example 1, and there was no significant difference between the polyrotaxane surfaces. On the other hand, the contact angle of water in water measured using the captive bubble method in water showed different trends, and a significant difference was observed between the polyrotaxane surfaces.

[0071] The polyrotaxane surface has molecular mobility derived from the interlocked structure between αCD and polyethylene glycol chains and exhibits unique behavior in the hydrated state. For example, the molecular mobility of the polyrotaxane surface upon hydration is related to (1) the result obtained from the dissipation energy loss (QCM-D) measured in water using a quartz crystal microbalance with dissipation, and (2) the result of contact angle hysteresis measured by the difference in the contact angle of water obtained from the contact angle of water in air and the contact angle of air bubbles in water. On each coating surface using Example 1 and Comparative Examples 1 to 3, since the value of contact angle hysteresis changed depending on the type of surface functional group, it is considered that the functional group modification of the hydroxyl group of cyclodextrin is useful for adjusting molecular mobility.

[0072] Also, the zeta potential of the polyrotaxane surface was measured using an electrophoretic light scattering spectrometer (ELSZ-2, Otsuka Electronics) with a quartz flow cell for flat samples. The mobility of electroosmosis on the surface was analyzed using monitoring particles (Otsuka Electronics) in 10 mM PBS to calculate the zeta potential on the surface. All measurements were performed on three different surfaces.

[0073] The zeta potentials of the polyrotaxane surfaces of Reference Example 1, Example 1, and Comparative Examples 1 to 3 are shown in Table 3. The polyrotaxane surface of Example 1 had the smallest negative charge, and Comparative Example 3 was the most negatively charged among all the surfaces. Although the amino group should be positively charged at pH 7.4, the polyrotaxane surface of Example 1 was negatively charged. This is considered to be due to the negative charge of the substrate (TCPS) of the coating base and the negative charge of the unmodified PRX surface.

Table 3

[0074] (3) Fibronectin adsorption on the polyrotaxane surface (Micro BCA assay) To examine the adsorption of fibronectin onto the polyrotaxane surface, a PBS solution (100 μg / mL) of human fibronectin was incubated at 37 °C for 3 hours on each surface of Reference Example 1, Example 1, and Comparative Examples 1 to 3. Each well was washed three times with PBS to remove non-adsorbed fibronectin. Adsorbed fibronectin was extracted by adding a 5% SDS and 0.1 N NaOH aqueous solution and incubating at 37 °C for 1 hour according to the method described in J. Biomater. Sci. Polym. Ed., 2017, 28, 986-999 or ACS Biomater. Sci. Eng., 2018, 4, 1591-1597. The fibronectin concentration was measured using a protein assay kit (Micro BCA (trademark), manufactured by Thermo Scientific) together with a human fibronectin standard according to the manufacturer's instructions.

[0075] The results are shown in Fig. 4. The most fibronectin was adsorbed on the surface of Example 1. Fibronectin is negatively charged, and it was considered that a large amount of fibronectin was adsorbed due to electrostatic interaction with the positively charged amino groups. There was no significant difference in the amount of fibronectin adsorbed on the polyrotaxane surfaces of Comparative Examples 1 to 3 compared to Reference Example 1.

[0076] 2. Cellular response on the polyrotaxane surface (1) Adhesion and proliferation of human mesenchymal stem cells (hMSCs) on the polyrotaxane surface To evaluate the initial adhesion and proliferation of hMSCs on each polyrotaxane surface, cells were seeded on each surface at a concentration of 6.0×10 3 cells / cm 2 and cultured using a hMSC growth medium BulletKit at 37 °C for 3 days in a humidified atmosphere containing 5% CO 2 . The cell morphology was observed using a phase-contrast microscope (BZ-X700, KEYENCE). The adhered cells were detached from the substrate by treatment with a trypsin / EDTA solution, and the number of hMSCs adhered to each surface was measured using a hemocytometer at 1-day intervals.

[0077] The results are shown in Fig. 5. On the first day after seeding, no significant difference in cell density was observed among the surfaces of each polyrotaxane. On the third day of seeding, the cell density on the surface of Example 1 was the lowest.

[0078] (2) Osteogenic differentiation of hMSCs on the polyrotaxane surface To induce osteogenic differentiation (osteogenic differentiation), hMSCs were seeded on each polyrotaxane surface at a density of 2.4×10 4 cells / cm 2 , and using the hMSC growth medium BulletKit, the cells were cultured at 37 °C for 5 days in a humidified atmosphere containing 5% CO 2 until the cell density became over-confluent. The growth medium was replaced with the osteogenic differentiation medium, and the cells were incubated for 14 days. The medium was replaced with fresh medium every 3 - 4 days. As the osteogenic differentiation medium, Mesenchymal Stem Cell Osteogenic Differentiation Medium (C-28013) from PromoCell GmbH (Heidelberg, Germany) was used.

[0079] Fourteen days after induction of differentiation, the cells were stained with alizarin red S to evaluate the degree of calcification of hMSCs. The cells were washed twice with PBS and fixed by treatment with a 4% paraformaldehyde solution at 23 °C for 10 minutes. The cells were washed twice with MilliQ water and stained with an alizarin red S solution at 23 °C for 10 minutes. After removing the staining solution, the cells were washed four times with MilliQ water and observed under a microscope. The stained area was calculated using ImageJ software. All measured values were obtained from three different surfaces, and the average value of four different points on each surface was taken as the average value of each surface.

[0080] The results are shown in Fig. 6. After the induction of osteogenic differentiation, the cell morphology changed from an elongated shape to a square shape, and the cell density increased especially on the surface of Comparative Example 3. To evaluate the osteoid nodule formation of hMSCs cultured on each surface, calcium nodules were stained with alizarin red S on the 14th day after the induction of osteoblast differentiation. Fig. 7 is a graph comparing (A) a stained photograph with alizarin red S and (B) the stained area on each polyrotaxane surface. A significant difference was observed in the stained area, which was the largest on the most negatively charged surface (Comparative Example 3), the smallest on the least negatively charged surface (Example 1), and moderate on the other surfaces (Reference Example 1, Comparative Examples 1-2).

[0081] (3) Adipogenic differentiation of hMSCs on the polyrotaxane surface To induce adipogenic differentiation, hMSCs were seeded on each polyrotaxane surface at a density of 1.0×10 4 cells / cm 2 and cultured at 37 °C for 5 days in a humid atmosphere containing 5% CO 2 using the hMSC growth medium BulletKit. The growth medium was replaced with the adipogenic differentiation medium, and the cells were cultured for 15 days. The medium was replaced with fresh medium every 3-4 days. As the adipogenic differentiation medium, Mesenchymal Stem Cell Adipogenic Differentiation Medium2 (C-28016) from PromoCell GmbH (Heidelberg, Germany) was used.

[0082] Fifteen days after the induction of differentiation, the cells were stained with oil red O to evaluate the accumulation of lipid droplets in the cells. The cells were washed twice with PBS and fixed by treatment with a 4% paraformaldehyde solution at 23 °C for 10 minutes. The cells were washed twice with PBS and once with 60% 2-propanol. The cells were stained with the oil red O solution at 23 °C for 20 minutes and washed once with 60% 2-propanol. Finally, the cells were washed twice with PBS and observed in PBS using a microscope. The stained area was calculated using ImageJ software. All measurements were obtained on three different surfaces, and the average value of three different points on each surface was taken as the average value of each surface.

[0083] The results are shown in Fig. 8. Intracellular lipid droplet formation associated with adipogenic differentiation was observed on all surfaces, and an increase in lipid droplets was seen over the culture period. Fig. 9 is a graph comparing (A) a stained photograph with Oil Red O and (B) the stained area on each polyrotaxane surface. The stained areas on the surfaces of Example 1 and Comparative Examples 2 to 3 were larger than those on the surfaces of Reference Example 1 and Comparative Example 1. The stained area with Oil Red O tended to increase with an increase in contact angle hysteresis. Since the difference in contact angle hysteresis indicates the difference in molecular mobility of the surface, the molecular mobility of the polyrotaxane surfaces of Example 1 and Comparative Examples 2 to 3 in particular is considered to be more suitable for promoting adipogenic differentiation.

Claims

1. A polyrotaxane represented by formula (1). 【Chemical 1】 [wherein, R １ is a hydrogen atom or a methyl group, m is from 1 to 2000, n is from 10 to 500, [Chemical Formula 2] is a cyclodextrin modified with a group represented by -X-NH ２ wherein X is a divalent organic group.]

2. A coating agent containing the polyrotaxane represented by formula (1). [Chemical 3] [Wherein, R １ is a hydrogen atom or a methyl group, m is from 1 to 2000, n is from 10 to 500, and [Chemical Formula 4] is a cyclodextrin modified with a group represented by -X-NH ２ wherein X is a divalent organic group.]

3. The polyrotaxane contains cyclodextrin modified with a group represented by 1 to 18 hydroxyl groups -X-NH ２ The coating agent according to claim 2, which contains cyclodextrin modified with a group represented by ２ .

4. The coating agent according to claim 2 or 3, wherein the number of penetrations of cyclodextrin in the polyrotaxane is 3 to 220.

5. A cell culture method for promoting adipogenic differentiation of mesenchymal stem cells, comprising culturing mesenchymal stem cells on the surface of a substrate coated with a composition containing the polyrotaxane represented by formula (1). 【Chemical Formula 5】 [wherein, R １ is a hydrogen atom or a methyl group, m is from 1 to 2000, n is from 10 to 500, 【Chemical Formula 6】 is a cyclodextrin modified with a group represented by -X-NH ２ wherein X is a divalent organic group.]

6. The method according to claim 5, comprising coating the surface of the substrate with a composition containing the polyrotaxane represented by formula (1).

7. The polyrotaxane contains cyclodextrin modified with a group represented by 1 to 18 hydroxyl groups -X-NH ２ The method according to claim 5 or 6, wherein the cyclodextrin is modified with a group represented by ２ .

8. The method according to any one of claims 5 to 7, wherein the number of penetrations of cyclodextrin in the polyrotaxane is 3 to 220.

9. A cell culture method for suppressing osteogenic differentiation of mesenchymal stem cells, comprising culturing mesenchymal stem cells on the surface of a substrate coated with a composition containing the polyrotaxane represented by formula (1). 【Chemical Formula 7】 [wherein, R １ is a hydrogen atom or a methyl group, m is from 1 to 2000, n is from 10 to 500, and 【Chemical 8】 is a cyclodextrin modified with a group represented by -X-NH ２ wherein X is a divalent organic group.]

10. The method according to claim 9, comprising coating the surface of the substrate with a composition containing the polyrotaxane represented by formula (1).

11. The polyrotaxane contains cyclodextrin modified with a group represented by 1 to 18 hydroxyl groups of -X-NH ２ The method according to claim 9 or 10, wherein the cyclodextrin is modified with a group represented by ２ .

12. The method according to any one of claims 9 to 11, wherein the number of penetrations of cyclodextrin in the polyrotaxane is 3 to 220.

13. A culture substrate comprising a substrate coated with a composition containing the polyrotaxane represented by formula (1). 【Chemical Formula 9】 [wherein, R １ is a hydrogen atom or a methyl group, m is from 1 to 2000, n is from 10 to 500, and 【Chemical Formula 10】 is a cyclodextrin in which at least one hydroxyl group is modified with a group represented by -X-NH ２ wherein X is a divalent organic group.]

14. The polyrotaxane contains cyclodextrin modified with a group represented by 1 to 18 hydroxyl groups -X-NH ２ The culture substrate according to claim 13, wherein the cyclodextrin is modified with a group represented by ２ .

15. The culture substrate according to claim 13 or 14, wherein the number of penetrations of cyclodextrin in the polyrotaxane is 3 to 200.

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