Patent Application
20230416682
The present invention relates to a polyrotaxane having a hydroxy group or a sulfo group.
Cell culture technology is widely used in the pharmaceutical and cosmetic fields and regenerative medicine research, and is an important technology. As environments for cell culture, appropriate culture environments, culture solutions, and culture substrates are required. Among them, the culture substrate has a role as a scaffold to which cells adhere, and is required to have properties such as being a surface suitable for cell culture or being able to be processed, having no cytotoxicity, being able to be sterilized or maintaining a sterile state, being not deteriorated under culture conditions, and not interfering with observation with a microscope.
Conventionally, as culture substrates, glass products have been widely used, but polystyrene products are now widely used. However, a culture substrate obtained by molding a polystyrene resin is generally subjected to a surface treatment because the surface thereof is hydrophobic and affinity with cells is low. Examples of the surface treatment include a plasma treatment, a corona discharge treatment, an oxidizing agent treatment, and coating with a hydrophilic substance. As the coating agent, there are many coating agents promoting cell adhesion, and for example, Type I collagen, Type R1 collagen, gelatin, fibronectin, vitronectin, laminin, matrigel, hydroxyapatite, and the like are known. It is also known that coating with water-soluble elastin promotes differentiation induction of vascular smooth muscle cells or elastin reactive cells.
Mesenchymal stein cells are somatic stein cells derived from mesoderm having self-renewal ability and pluripotency into mesenchymal tissues such as osteoblasts, chondrocytes, adipocytes, skeletal muscle cells, and ligament cells. In adult tissues, mesenchymal stein cells are present in connective tissues such as dermis, skeletal muscle, and adipose tissue and mainly in bone marrow stroma, and function to repair connective tissues, maintain homeostasis, and control proliferation and differentiation of hematopoietic stein cells. Differentiation of mesenchymal stein cells is complicatedly controlled by biological and physical factors. For example, it is known that when dexamethasone, β-glycerophosphate, or ascorbic acid is added to a medium for mesenchymal stein cells, differentiation into osteoblasts can be induced, and when dexamethasone, 3-isobutyl-1-methylxanthine, insulin, or indomethacin is added to a medium for mesenchymal stein cells, differentiation into adipocytes can be induced.
In recent years, attention has been paid to the fact that the properties (for example, bulk properties such as hardness, and surface properties of coating) of a culture substrate affect the function of cultured cells, and a study for proliferating cells having a desired function has been reported (Non-Patent Literatures 1 to 3).
However, it is still difficult to finely adjust the bulk properties (hardness or the like) of cell culture substrate in order to obtain cells having a desired function. It is considered that selecting appropriate physical factors is useful to differentiate mesenchymal stein cells into the desired cell lineage. Therefore, an object of the present invention is to provide a coating agent and a culture substrate which can promote differentiation induction of mesenchymal stein cells.
The present invention provides the following [1] to [10].
[1] A coating agent for promoting osteoblast differentiation and/or adipocyte differentiation of mesenchymal stein cells, the coating agent comprising a polyrotaxane represented by Formula (1):
is a cyclodextrin in which at least one hydroxyl group is modified with a group represented by —X—Y, X is a divalent organic group, and Y is a hydroxyl group or a sulfo group.
[2] The coating agent according to [1], wherein the polyrotaxane comprises a cyclodextrin in which 1 to 18 hydroxyl groups are modified with the group represented by —X—Y.
[3] The coating agent according to [1] or [2], in which the polyrotaxane has a number of threaded cyclodextrins of 3 to 220.
[4] A cell culture method for promoting osteoblast differentiation and/or adipocyte differentiation of mesenchymal stein cells, the method comprising:
is a cyclodextrin in which at least one hydroxyl group is modified with a group represented by —X—Y, X is a divalent organic group, and Y is a hydroxyl group or a sulfo group.
[5] The method according to [4], comprising coating the composition comprising a polyrotaxane represented by Formula (1) onto the surface of the substrate.
[6] The method according to [4] or [5], in which the polyrotaxane comprises a cyclodextrin in which 1 to 18 hydroxyl groups are modified with the group represented by —X—Y.
[7] The method according to any one of [4] to [6], in which the polyrotaxane has a number of threaded cyclodextrins of 3 to 220.
[8] A culture substrate comprising a substrate coated with a composition comprising a polyrotaxane represented by Formula (1):
is a cyclodextrin in which at least one hydroxyl group is modified with a group represented by —X—Y, X is a divalent organic group, and Y is a hydroxyl group or a sulfo group.
[9] The culture substrate according to [8], in which the polyrotaxane comprises a cyclodextrin in which 1 to 18 hydroxyl groups are modified with the group represented by —X—Y.
[10] The culture substrate according to [8] or [9], in which the polyrotaxane has a number of threaded cyclodextrins of 3 to 200.
According to the present invention, it is possible to provide a coating agent capable of promoting osteoblast differentiation and/or adipocyte differentiation of mesenchymal stein cells. According to the present invention, it is also possible to provide a substrate (culture substrate) coated with the coating agent.
A first embodiment of the present invention is a coating agent for promoting osteoblast differentiation and/or adipocyte differentiation of mesenchymal stein cells, the coating agent comprising a polyrotaxane represented by Formula (1):
is a cyclodextrin in which at least one hydroxyl group is modified with a group represented by —X—Y, X is a divalent organic group, and Y is a hydroxyl group (—OH) or a sulfo group (—SO3H). At least one of the cyclodextrins is contained in one molecule of the polyrotaxane.
The polyrotaxane represented by Formula (1) includes an axial molecule (linear polymer) and a modified cyclodextrin (CD, cyclic molecule), and the axial molecule threads at least one modified cyclodextrin and is capped at both ends thereof.
The axial molecule is represented by Formula (2) below, has a polyethylene glycol structure at the center of the molecule, and has a poly(meth)acrylate structure capped with a phenyldithioester group at the terminal. In the formula, R1 is a hydrogen atom or a methyl group. The cyclodextrin is threaded to the polyethylene glycol structure portion. Each R1 may be the same as or different from each other. R1 is preferably a methyl group.
The polyethylene glycol structure may include ethylene glycol as a monomer unit and have a molecular length capable of threading at least one cyclodextrin. The number of ethylene glycol units forming the polyethylene glycol structure may be 20 to 1000, and is preferably 85 to 800 and more preferably 100 to 700. In the formula, n may be 10 to 500, and is preferably 43 to 400 and more preferably 50 to 350.
The poly(meth)acrylate structure includes benzyl (meth)acrylate as a monomer unit. In the present specification, the term “(meth)acrylate” means both “acrylate” and “methacrylate”. The present inventors consider that since the poly(meth)acrylate structure has an action of enhancing adhesion to a substrate such as polystyrene, the polyethylene glycol moiety threading the cyclodextrin is separated from the substrate in a loop shape. The number in of benzyl (meth)acrylate units forming the poly(meth)acrylate structure may be 1 to 2000, and is preferably 20 to 1000 and more preferably 30 to 500, per polybenzyl (meth)acrylate at one terminal of the triblock copolymer. Each in may be the same as or different from each other.
The modified cyclodextrin used in the present embodiment is represented by Formula (3) below, and at least one hydroxyl group of glucose constituting the cyclodextrin is modified with —X—Y. The cyclodextrin may be any of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and combinations thereof. A preferred cyclodextrin is α-cyclodextrin. The following is a figure schematically showing a cyclodextrin modified with —X—Y. “—O—X—Y” in the figure indicates that a hydroxyl group in glucose constituting the cyclodextrin is modified with —X—Y. In the figure, only one “—O—X—Y” is shown, but it should not be interpreted restrictively that only one hydroxyl group is modified with —X—Y.
In the polyrotaxane, at least one modified cyclodextrin is threaded by an axial molecule. The number of cyclodextrins per polyrotaxane molecule can be independently determined and need not be uniquely determined by the molecular length of the polyethylene glycol structure. Stoichiometrically, since the 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 to be used. For example, the number of cyclodextrins per polyethylene glycol molecule having a number average molecular weight of 20000 may be 3 to 220, and is preferably 5 to 150, more preferably 5 to 120, and particularly preferably 5 to 100. However, the length (total length when the polyrotaxane has a plurality of cyclodextrins) of the axial molecule of the cyclodextrin in the main chain direction does not exceed the molecular length of polyethylene glycol.
The number of hydroxyl groups modified with —X—Y in the cyclodextrin may be 1 to 18, and is preferably 1 to 10 and more preferably 2 to 8, with respect to one cyclodextrin. When the number of hydroxyl groups modified with —X—Y is within the above range, the effect of promoting adipocyte differentiation and/or osteoblast differentiation of mesenchymal stein cells is more excellent. When the number of hydroxyl groups modified with —X—Y is 10 or less, solubility in an organic solvent such as dimethyl sulfoxide (DMSO) is more excellent. When the number of hydroxyl groups modified with —X—Y is 2 or more, the amount of change in molecular mobility, the chemical composition of the surface, and the amount of change in physicochemical properties (for example, contact angle and zeta potential) of the surface increase. Since the molecular mobility, the chemical composition of the surface, and the physicochemical properties (for example, contact angle and zeta potential) of the surface of the polyrotaxane can be adjusted by the number of hydroxyl groups modified with —X—Y, a polyrotaxane surface having molecular mobility suitable for promotion of adipocyte differentiation and/or osteoblast differentiation of mesenchymal stein cells can be produced.
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, and the organic group may have an oxo group at any position, may be through an oxy group or an imino group, and may be a combination thereof. For example, a combination of a carbon atom having an oxo group and an oxy group forms an ester bond, and a combination of a carbon atom having an oxo group and an imino group forms an amide bond. The alkylene group having a plurality of oxy groups is also referred to as 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 a methylene group, an ethylene group, a propylene group, a butylene group, a pentylene group, a hexylene group, a heptylene group, an octylene group, a nonanylene group, and a decylene group; alkenylene groups such as a propynylene group, a butenylene group, a pentenylene group, a hexenylene group, a heptenylene group, an octenylene group, a nonenylene group, and a decenylene group; and alkynylene groups such as a propargyl group, a butynylene group, a pentynylene group, a hexynylene group, a heptynylene group, an octynylene group, a nonylene group, and a decynylene group. An organic group having an oxo group, an oxy group, or an imino group preferably forms an ester bond, a carbonate bond, or a urethane bond together with an oxygen atom derived from the hydroxyl group of the cyclodextrin. Examples of the organic group having an oxo group, an oxy group, or an imino group include carbonyl alkylene groups such as a carbonyl methylene group (—C(═O)CH2—); alkylene carbonyl groups such as a methylenecarbonyl group (—CH2C(═O)—); carbonylaminoalkylene groups such as a carbonylaminomethylene group (—C(═O)NHCH2—); alkylenecarbonylamino groups such as a methylenecarbonylamino group (—CH2C(═O)NH—); carbonylaminoalkylene groups such as a carbonylaminoethylene group (—C(═O)NHCH2CH2—), carbonylaminoalkylene oxyalkylene groups such as a carbonylaminoethyleneoxyethylene group (—C(═O)NHCH2CH2OCH2CH2—); and oxyalkylene groups such as an oxypropylene group (—OCH2CH2CH2—).
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 aspects in which 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.
The polyrotaxane includes a structure in which an axial molecule threads at least one cyclodextrin. Each cyclodextrin can move along the main chain direction of the axial molecule and rotate about the main chain of the axial molecule. Such structural properties are referred to as molecular mobility. The molecular mobility can vary, for example, according to the number of cyclodextrins, the number of substituents modified to glucose constituting the constituting cyclodextrin.
The molecular mobility can be evaluated by coating the surface of a culture substrate, then measuring the static contact angle of the coating surface using a contact angle meter, and using a droplet method and a captive bubble method. For the evaluation of molecular mobility, for example, methods described in Soft Matter, 2012, 8, 5477-5485 (Ji-Hun Seo et al.), Adv. Healthcare Mater. 2015, 4, 215-222, and the like may be referred to. Specifically, the contact angle hysteresis value of the coating surface can be calculated from a difference between the contact angle of water (contact angle of water in the atmosphere) measured by the droplet method and the contact angle of water (contact angle of water in water) determined from the contact angle of air bubbles measured by the captive bubble method. As a control, comparison with the effect on a culture substrate coated with DMSO may be conducted.
The molecular mobility can be adjusted by changing the number of threaded cyclodextrins and/or the number of hydroxyl groups modified with —X—Y in the cyclodextrin. For example, when the number of threaded cyclodextrins is 3 to 120, it can be more suitable for promoting adipocyte differentiation and/or osteoblast differentiation of mesenchymal stein cells. When the number of threaded cyclodextrins is large, the cyclodextrin is easily purified by reprecipitation.
The content of the polyrotaxane may be 0.0005 to 5 mass %, preferably 0.01 to 1 mass %, and more preferably 0.02 to 0.5 mass %, based on the mass of the coating agent.
The coating agent of the present embodiment may comprise a solvent and an optional additive in addition to the above-described 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 an additive include an antioxidant.
The coating agent of the present embodiment is used for coating a surface of a substrate that can be used as a culture substrate. The substrate that can be used as a culture substrate may be a substrate well known to those skilled in the art. Examples of the material for the substrate include glass, polystyrene, polypropylene, polyethylene, polyolefin, polycarbonate, and an acrylic block copolymer (BCF).
According to the coating agent of the present embodiment, by adjusting the number of hydroxyl groups modified with —X—Y in glucose constituting the cyclodextrin, it is possible to adjust promotion of adipocyte differentiation and/or osteoblast differentiation of mesenchymal stein cells without depending on the bulk properties of the substrate itself. More specifically, as adipocyte differentiation progresses, the amount of lipid droplets accumulated in cells increases. Lipid droplets accumulated in cells can be stained with Oil Red O dye to evaluate the degree of lipid droplet accumulation in cells. Adipocyte differentiation of mesenchymal stein cells may be determined by the expression level of a differentiation marker gene (for example, PPARγ, C/EBPα, and aP2), which is an index indicating adipocyte differentiation. In osteoblast differentiation, as the differentiation progresses, bone nodules are formed (also called calcification). The bone nodules can be stained with Alizarin Red S to evaluate the extent of calcification. Osteoblast differentiation of mesenchymal stein cells may be determined by the expression level of a differentiation marker gene (for example, RUNX2, alkaline phosphatase, osteocalcin, osteopontin, bone sialoprotein, and Type I collagen), which is an index indicating osteoblast differentiation.
The polyrotaxane can be produced with reference to Examples, and may be produced as follows. Hydroxyl groups at both ends of polyethylene glycol having a desired length are converted into leaving groups (for example, halogenation, methanesulfonylation, or toluenesulfonylation), and etherified with phenylalaninol to obtain a diamine. The obtained diamine is mixed with a cyclodextrin to obtain a pseudo-rotaxane. At this time, the number of threaded cyclodextrins can be adjusted by adjusting the amount of cyclodextrin per diamine molecule. Subsequently, the mixture is reacted with CPADB and DMT-MM to cap the cyclodextrin so as not to be removed from the axial molecule, and then benzyl methacrylate (corresponding to a (meth)acrylate structure) is introduced at both terminals as an anchoring segment by a reversible addition-fragmentation chain transfer polymerization reaction (RAFT polymerization reaction).
A second embodiment of the present invention is a cell culture method for promoting adipocyte differentiation of mesenchymal stein cells, the method comprising: culturing mesenchymal stein cells on a surface of a substrate coated with a composition comprising a polyrotaxane represented by Formula (1).
In the present embodiment, as the “polyrotaxane represented by Formula (1)”, those described in the first embodiment can be referred to, and as the “composition comprising a polyrotaxane represented by Formula (1)”, the coating agent described in the first embodiment can be used.
In the cell culture method of the present embodiment, as the culture substrate, a substrate coated with a composition comprising a polyrotaxane represented by Formula (1) is used. Cells are cultured by adhering the cells to the surface of the coated substrate.
The culture substrate is obtained by applying a coating comprising a polyrotaxane represented by Formula (1) to a substrate. The substrate may be a substrate well known to those skilled in the art. Examples of the material for the substrate include glass, polystyrene, polypropylene, polyethylene, polyolefin, polycarbonate, and an acrylic block copolymer (BCF). The substrate may be a commercially available glass substrate or plastic substrate.
In the cell culture method of the present embodiment, a medium is put on the coating surface provided on the substrate so that the cells are immersed, and the cells to be cultured are seeded and cultured. The medium may be replaced with a new medium as necessary. A step of injecting a medium for proliferation before differentiating the cells to proliferate the seeded cells may be provided. In this case, after the cells are proliferated until a sufficient number of cells is obtained, the medium for proliferation is replaced with a medium for differentiation. As the medium for proliferation and the medium for differentiation, a medium well known to those skilled in the art can be used.
As a medium for adipocyte differentiation of mesenchymal stein cells, for example, Mesenchymal Stein Cell Adipogenic Differentiation Medium 2 (C-28016) available from PromoCell GmbH (Heidelberg, Germany) can be used.
The cell culture environment can be arbitrarily set under the conditions well known to those skilled in the art.
Adipocyte differentiation of mesenchymal stein cells means differentiation of mesenchymal stein cells into adipocytes. As adipocyte differentiation progresses, the amount of lipid droplets accumulated in cells increases. The effect of promoting adipocyte differentiation of mesenchymal stein cells can be evaluated by cell staining with Oil Red O (staining of lipid droplets in cells). The effect may be determined by measuring a change in the expression level of a differentiation marker. Examples of the differentiation marker of adipocyte differentiation include PPARγ, C/EBPα, and aP2. When differentiation is statistically significantly promoted as compared with the case of using a culture substrate in which a substrate (made of glass or polystyrene) is coated with an unmodified polyrotaxane (for example, PRX-PBzMA described below), it can be determined that the effect of promoting adipocyte differentiation of mesenchymal stein cells is exhibited.
The cell culture method of the present embodiment may comprise coating the composition comprising a polyrotaxane represented by Formula (1) onto the surface of the substrate.
The composition comprising a polyrotaxane represented by Formula (1) can be coated on a substrate. The coating method is not particularly limited, and examples thereof include casting, spin coating, gravure coating, die coating, knife coating, bar coating, blade coating, and roll coating. A preferred coating method is casting.
A third embodiment of the present invention is a cell culture method for promoting osteoblast differentiation of mesenchymal stein cells, the method comprising: culturing mesenchymal stein cells on a surface of a substrate coated with a composition comprising a polyrotaxane represented by Formula (1).
In the present embodiment, as the “polyrotaxane represented by Formula (1)”, those described in the first embodiment can be referred to, and as the “composition comprising a polyrotaxane represented by Formula (1)”, the coating agent described in the first embodiment can be used.
In the cell culture method of the present embodiment, as the culture substrate, a substrate coated with a composition comprising a polyrotaxane represented by Formula (1) is used. Cells are cultured by adhering the cells to the surface of the coated substrate.
The culture substrate is obtained by applying a coating comprising a polyrotaxane represented by Formula (1) to a substrate. The substrate may be a substrate well known to those skilled in the art. Examples of the material for the substrate include glass, polystyrene, polypropylene, polyethylene, polyolefin, polycarbonate, and an acrylic block copolymer (BCF). The substrate may be a commercially available glass substrate or plastic substrate.
In the cell culture method of the present embodiment, a medium is put on the coating surface provided on the substrate so that the cells are immersed, and the cells to be cultured are seeded and cultured. The medium may be replaced with a new medium as necessary. A step of injecting a medium for proliferation before differentiating the cells to proliferate the seeded cells may be provided. In this case, after the cells are proliferated until a sufficient number of cells is obtained, the medium for proliferation is replaced with a medium for differentiation. As the medium for proliferation and the medium for differentiation, a medium well known to those skilled in the art can be used.
As a medium for osteoblast differentiation of mesenchymal stein cells, for example, Mesenchymal Stein Cell Osteogenic Differentiation Medium (C-28013) available from PromoCell GmbH (Heidelberg, Germany) can be used.
The cell culture environment can be arbitrarily set under the conditions well known to those skilled in the art.
Osteoblast differentiation of mesenchymal stein cells means differentiation of mesenchymal stein cells into osteoblasts. As osteoblast differentiation progresses, bone nodules are formed (also called calcification). The effect of promoting osteoblast differentiation of mesenchymal stein cells can be evaluated by cell staining with Alizarin Red S (bone nodules are stained). The effect may be determined by measuring a change in the expression level of a differentiation marker. Examples of the differentiation marker of osteoblast differentiation include RUNX2, alkaline phosphatase, osteocalcin, osteopontin, bone sialoprotein, and Type I collagen. When differentiation is statistically significantly promoted as compared with the case of using a culture substrate in which a substrate (made of glass or polystyrene) is coated with an unmodified polyrotaxane (for example, PRX-PBzMA described below), it can be determined that the effect of promoting osteoblast differentiation of mesenchymal stein cells is exhibited.
The cell culture method of the present embodiment may comprise coating the composition comprising a polyrotaxane represented by Formula (1) onto the surface of the substrate.
The composition comprising a polyrotaxane represented by Formula (1) can be coated on a substrate. The coating method is not particularly limited, and examples thereof include casting, spin coating, gravure coating, die coating, knife coating, bar coating, blade coating, and roll coating. A preferred coating method is casting.
A fourth embodiment of the present invention is a culture substrate comprising a substrate coated with a composition comprising a polyrotaxane represented by Formula (1).
In the present embodiment, as the “polyrotaxane represented by Formula (1)”, those described in the first embodiment can be referred to, and as the “composition comprising a polyrotaxane represented by Formula (1)”, the coating agent described in the first embodiment can be used. As the coating method, the method described in the second or third embodiment can be referred to.
The coated substrate (culture substrate) of the present embodiment is particularly suitable as a culture substrate for promoting adipocyte differentiation and/or osteoblast differentiation of mesenchymal stein cells.
According to the culture substrate of the present embodiment, by adjusting the number of hydroxyl groups modified with —X—Y in glucose constituting the cyclodextrin, it is possible to adjust differentiation (promotion of adipocyte differentiation and/or osteoblast differentiation) of mesenchymal stein cells and proliferation (promotion or suppression) of mesenchymal stein cells without depending on the bulk properties of the substrate itself.
Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited thereto. The abbreviations used in the examples are conventional abbreviations well known to those skilled in the art, and the meanings of some abbreviations are shown below.
A method for synthesizing a polyrotaxane is shown below.
Polyethylene glycol having a number average molecular weight of 20000 (25.0 g, 1.25 mmol) and TEA (5.3 mL, 37.5 mmol) were dissolved in anhydrous THF (130 mL), MsCl (2.0 mL, 25.0 mmol) was added dropwise, and the mixture was stirred at 23° C. After 5 hours, the reaction solution was filtered, the filtrate was precipitated with diethyl ether, and the precipitate was collected as a solid. The obtained precipitate was dried under reduced pressure to obtain α,ω-bismesyl polyethylene glycol (21.1 g, yield: 84%).
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. To this mixed liquid, α,ω-bismesyl polyethylene glycol (20.0 g, 0.992 mmol) was added, and the mixture was stirred at 23° C. After 24 hours, the reaction solution was filtered, the filtrate was precipitated with diethyl ether, and the precipitate was collected 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%).
Bis(2-amino-3-phenylpropyl)polyethylene glycol (10.1 g, 0.499 mmol) was dissolved in water (50 mL), a saturated aqueous solution (380 mL) of αCD (55.2 g, 56.6 mmol) was added thereto, and the mixture was stirred at 23° C. After 19 hours, the reaction solution was centrifuged to collect a precipitate, and freeze-dried for 9 days to obtain a pseudo-polyrotaxane as a crude product.
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 hydrous DMSO, centrifuged and lyophilized for 9 days to obtain the polyrotaxane PRX-CPADB (11.9 g, 2-step yield: 18%) as a powder. The structure of polyrotaxane PRX-CPADB was confirmed by 1H-NMR (solvent: DMSO-d6). The number of threaded αCD was determined by 1H-NMR (solvent: D2O).
1H-NMR (500 MHz, DMSO-d6) 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).
Polyrotaxane PRX-CPADB (1.50 g, 13.9 μmop 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 mixed liquid. After degassing by 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 the polyrotaxane PRX-PBzMA (3.13 g, yield: 98%). The number in of benzyl methacrylate units forming the polybenzyl methacrylate structure was determined by 1H-NMR (solvent: DMSO-d6).
1H-NMR (500 MHz, DMSO-d6) 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).
Polyrotaxane PRX-PBzMA (200 mg, 0.895 μmop 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 the solution, and the mixture was further stirred at 23° C. for 1 day. Next, the reaction solution was purified by dialysis for 4 days. The product was freeze-dried for 7 days to obtain NH2—RX (176 mg, yield: 73%) as a powder. The number of amino groups was determined by 1H-NMR analysis (solvent: DMSO-d6).
1H-NMR (500 MHz, DMSO-d6) 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).
Polyrotaxane PRX-PBzMA (0.15 g, 0.671 μmop was dissolved in anhydrous DMSO (1.31 mL), sodium hydroxide (28.7 mg, 0.716 mmol) and iodomethane (15 μL, 0.239 mmol) were added thereto, and the mixture was stirred at 23° C. After 1 hour, methanol was added to stop the reaction, and the mixture was dialyzed for 3 days to obtain a polyrotaxane (115 mg, yield: 75%) of Comparative Example 1. The number of methyl groups was determined by 1H-NMR analysis (solvent: DMSO-d6).
1H-NMR (500 MHz, DMSO-d6) 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, H2, H3, H4, H5, and H6 protons of αCD, and —OCH3 of αCD), 4.44 (m, OH6 of αCD), 4.57-5.10 (m, H1 of αCD and —CH2-Ph of PBzMA), 5.49 (m, OH3 of αCD), 5.64 (m, OH2 of αCD), and 7.26 (m, aromatics of PBzMA).
Polyrotaxane PRX-PBzMA (200 mg, 0.895 μmop was dissolved in anhydrous DMSO (10 mL) and CDI (130 mg, 0.804 mmol) was added to the solution. After stirring at 23° C. for 1 day, 2-(2-aminoethoxy)ethanol (0.80 mL, 8.04 mmol) was added to the solution, and the mixture was further stirred at 23° C. for 1 day. Next, the reaction solution was purified by dialysis for 4 days. The product was freeze-dried for 7 days to obtain OH—PRX (194 mg, yield: 74%) as a powder. The number of OH groups was determined by 1H-NMR analysis (solvent: DMSO-d6).
1H-NMR (500 MHz, DMSO-d6) δ 0.42-0.95 (m, —CH(—CH3)-CH2- of PBzMA), 1.45-2.02 (m, —CH(—CH3)-CH2- of PBzMA), 3.14 (m, —O—CO—NH-CH2-CH2-O-CH2-CH2-0H), 3.17-4.70 (m, PEG backbone, H2, H3, H4, H5, and H6 protons of α-CD, OH6 of α-CD, and —O—CO—NH—CH2-CH2-O-CH2-CH2-OH), 4.86 (m, —CH2-Ph of PBzMA, H1 of α-CD), and 7.26 (m, aromatics of PBzMAH).
Polyrotaxane PRX-PBzMA (150 mg, 0.671 μmol) was dissolved in anhydrous DMSO (3.0 mL) and then NaOH powder (65.1 mg, 1.63 mmol) and 1,3-propane sultone (66.3 mg, 0.543 mmol) were added to the solution. After stirring at 23° C. for 1 day, the mixture was dialyzed and purified for 5 days. The product was freeze-dried for 7 days to obtain SO3H—PRX (155 mg, yield: 68%) as a powder. The number of sulfo groups (—SO3H) was determined by 1H-NMR analysis (solvent: DMSO-d6).
1H-NMR (500 MHz, DMSO-d6) δ=0.33-0.95 (m, —CH(—CH3)-CH2- of PBzMA),1.41-2.12 (m, —CH(—CH3)-CH2- of PBzMA and —O-CH2-CH2-CH2-SO3Na), 2.92-4.23 (m, PEG backbone, H2, H3, H4, H5, and H6 protons of α-CD, and —O-CH2-CH2-CH2-SO3Na), 4.86 (m, —CH2-Ph of PBzMA, H1 of α-CD), 7.26 (m, aromatics of PBzMA).
Data of the obtained polyrotaxane are shown in Table 1.
The obtained polyrotaxane was dissolved in DMSO to produce a coating agent. The obtained coating agent was cast on TCPS.
2. Analysis of Surface Chemical Composition
(1) Elemental Composition of Surface
The chemical composition of the polyrotaxane surface was analyzed by TOF-SIMS (PHI NanoTOF II, ULVAC-PHI) with 30 keV Bi3++ primary ions. The analysis field is 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 with an energy of 1486.6 eV. The diameter of the analysis region was 400 μm, and the elemental composition was calculated from the average of three points on each surface.
(2) Measurement of Contact Angle and Zeta Potential
The coatings comprising polyrotaxane of Examples 1 and 2 and Comparative Examples land 2 were applied to the surface of polystyrene for cell culture (TCPS). The static contact angle of the coating surface was measured using a contact angle meter (trade name: DM-501, manufactured by Kyowa Interface Science Co., Ltd. by both of a droplet method and a captive bubble method. The contact angle hysteresis value of each surface was calculated from a difference between the contact angle of water (contact angle of water in the atmosphere) measured by the droplet method and the contact angle of water (contact angle of water in water) determined from the contact angle of air bubbles measured by the captive bubble method. All measurement values were obtained on four different surfaces, and an average value of three different points on each surface was recorded. It is known that a difference in contact angle hysteresis indicates a difference in molecular mobility between surfaces.
The contact angle and the contact angle hysteresis of the polyrotaxane surfaces of Reference Example 1, Examples 1 and 2, and Comparative Examples 1 and 2 are shown in Table 3. The contact angle of water in the atmosphere on the polyrotaxane surface was in a range of 80 to 100°, and it was confirmed that wettability with the TCPS surface (contact angle: 74°) surface was changed by the polyrotaxane coating. The contact angles of water in the atmosphere on the polyrotaxane surfaces of Examples 1 and 2 and Comparative Examples 1 and 2 were slightly smaller than the contact angle of the polyrotaxane 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 tended to be different, and a significant difference was observed between the polyrotaxane surfaces.
The polyrotaxane surface has molecular mobility derived from the interlocking structure between αCD and the polyethylene glycol chain and exhibits unique behavior in the hydrated state. For example, it is known that the molecular mobility upon hydration of the polyrotaxane surface is related to (1) a result obtained from dissipation energy loss (QCM-D) measured in water using a quartz crystal microbalance with dissipation and (2) a result of the contact angle hysteresis measured by a difference between a contact angle of water in air and a contact angle of water obtained from a contact angle of bubbles in water. Since the value of the contact angle hysteresis changed depending on the type of surface functional group on each coating surface using Examples 1 and 2 and Comparative Examples 1 and 2, functional group modification of the hydroxyl group of the cyclodextrin is considered to be useful for adjusting molecular mobility.
The zeta potential of the polyrotaxane surface was measured using quartz flow cell for a flat plate sample using an electrophoretic light scattering spectrometer (ELSZ-2, Otsuka Electronics Co., Ltd.). The mobility of electroosmosis on the surface was analyzed using monitoring particles (Otsuka Electronics Co., Ltd.) in 10 mM PBS to calculate the zeta potential on the surface. All measurements were performed on three different surfaces.
The zeta potentials of the polyrotaxane surfaces of Reference Example 1, Examples 1 and 2, and Comparative Examples 1 and 2 are shown in Table 3. The polyrotaxane surface of Comparative Example 1 had the least negative charge, and Example 2 had the most negative charge of all surfaces. While the amino group should be positively charged at pH 7.4, the polyrotaxane surface of Comparative Example 1 was negatively charged. This is considered to be due to the negative charge of the underlying substrate (TCPS) of the coating and the negative charge of the unmodified PRX surface.
(3) Fibronectin Adsorption on Polyrotaxane Surface (Micro BCA Assay)
In order to examine fibronectin adsorption on the polyrotaxane surface, a PBS solution of human fibronectin (100 μg/mL) was incubated at 37° C. for 3 hours on each of the surfaces of Reference Example 1, Examples 1 and 2, and Comparative Examples 1 and 2. Each well was washed three times with PBS to remove unadsorbed fibronectin. Adsorbed fibronectin was extracted by adding 5% SDS and a 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 concentrations were measured using a protein assay kit (Micro BCA (trade name), Thermo Scientific) with human fibronectin standards according to the manufacturer's instructions.
The results are shown in
2. Cell Response on Polyrotaxane Surface
(1) Human Mesenchymal Stein Cell (hMSC) Adhesion and Proliferation on Polyrotaxane Surface
In order to evaluate initial adhesion and proliferation of hMSCs on each polyrotaxane surface, cells were seeded on each surface at a concentration of 6.0×103 cells/cm2 and cultured at 37° C. for 3 days in a humidified atmosphere containing 5% CO2 using a medium for hMSC proliferation BulletKit. The morphology of the cells was observed using a phase contrast microscope (BZ-X700, KEYENCE CORPORATION), the adhered cells were removed from the substrate by treatment with a trypsin/EDTA solution, and the number of hMSCs adhered to each surface was measured at intervals of one day using a hemocytometer.
The results are shown in
(2) Osteoblast Differentiation of hMSC on Polyrotaxane Surface
In order to induce osteoblast differentiation (osteogenic differentiation), hMSCs were seeded on each polyrotaxane surface at a density of 2.4×104 cells/cm2, and cultured for 5 days at 37° C. in a humidified atmosphere containing 5% CO2 using a medium for hMSC proliferation BulletKit until the density of cells became excessive confluent. The medium for proliferation was replaced with a medium for osteoblast differentiation, and the cells were incubated for 14 days. The medium was replaced with a fresh medium every 3 to 4 days. As a medium for osteoblast differentiation, Mesenchymal Stein Cell Osteogenic Differentiation Medium (C-28013) available from PromoCell GmbH (Heidelberg, Germany) was used.
After 14 days of differentiation induction, the cells were stained with Alizarin Red S to evaluate the extent of calcification of hMSC. The cells were washed twice with PBS and fixed by treatment with a 4% paraformaldehyde solution for 10 minutes at 23° C. The cells were washed twice with MilliQ water and stained with an Alizarin Red S solution for 10 minutes at 23° C. After removal of the stain solution, the cells were washed four times with MilliQ water and observed with a microscope. The stained area was calculated using ImageJ software. All measurement values were obtained on three different surfaces, and an average value of four different points on each surface was taken as an average value of each surface.
The results are shown in
(3) Adipocyte Differentiation of hMSC on Polyrotaxane Surface
In order to induce adipocyte differentiation, hMSCs were seeded on each polyrotaxane surface at a density of 1.0×104 cells/cm2, and cultured for 5 days at 37° C. in a humidified atmosphere containing 5% CO2 using a medium for hMSC proliferation BulletKit. The medium for proliferation was replaced with a medium for adipocyte differentiation, and the cells were cultured for 15 days. The medium was replaced with a fresh medium every 3 to 4 days. As a medium for adipocyte differentiation, Mesenchymal Stein Cell Adipogenic Differentiation Medium 2 (C-28016) available from PromoCell GmbH (Heidelberg, Germany) was used.
After 15 days of differentiation induction, the cells were stained with Oil Red O to evaluate lipid droplet accumulation in cells. The cells were washed twice with PBS and fixed by treatment with a 4% paraformaldehyde solution for 10 minutes at 23° C. The cells were washed twice with PBS and washed once with 60% 2-propanol. The cells were stained with an Oil Red O solution for 20 minutes at 23° C. 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 measurement values were obtained on three different surfaces, and an average value of three different points on each surface was taken as an average value of each surface.
The results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-196323 | Nov 2020 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/042832 | 11/22/2021 | WO |
