WO2024219027A1 - Hydrogel and composition for forming hydrogel - Google Patents
Hydrogel and composition for forming hydrogel Download PDFInfo
- Publication number
- WO2024219027A1 WO2024219027A1 PCT/JP2024/000752 JP2024000752W WO2024219027A1 WO 2024219027 A1 WO2024219027 A1 WO 2024219027A1 JP 2024000752 W JP2024000752 W JP 2024000752W WO 2024219027 A1 WO2024219027 A1 WO 2024219027A1
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- WIPO (PCT)
- Prior art keywords
- hydrogel
- functionalized
- polyethylene glycol
- rada
- aldehyde
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Definitions
- the present invention relates to a hydrogel and a composition for forming a hydrogel.
- hydrogels that contain polymer networks made of biopolymers and biocompatible polymer materials have been attracting attention for use as scaffolding materials for cell culture, etc.
- hydrogels As for such hydrogels, the present inventors have proposed a hydrogel formed from a self-assembling peptide, chitosan, and polyethylene glycol having an aldehyde end (see Non-Patent Documents 1 and 2). Such hydrogels can be synthesized in one pot or in situ, and are useful for various applications such as cell culture.
- the mesh polymer in which chitosan and polyethylene glycol having an aldehyde end, which constitutes the hydrogel described in Non-Patent Documents 1 and 2, are cross-linked with a Schiff base has the problem that it is prone to decomposition over time.
- the present invention has been made in consideration of the above problems, and aims to provide a hydrogel that is stable over a long period of time under cell culture conditions, and a composition for forming the hydrogel.
- the present invention is as follows.
- the hydrophilic polymer is a polymer that exhibits a water content of 50% by mass or more and 99% by mass or less, in terms of the mass ratio of water, when the hydrophilic polymer is allowed to swell in equilibrium with the addition of water.
- the hydrogel described in (1) has an interpenetrating polymer network structure in which a self-assembling peptide and a network polymer interpenetrate each other.
- composition according to (7) which is a three-liquid composition consisting of a first liquid containing a self-assembling peptide, a second liquid containing an aldehyde-functionalized or hydrazide-functionalized hydrophilic polymer, and a third liquid containing an aldehyde-functionalized or hydrazide-functionalized polyethylene glycol.
- the present invention provides a hydrogel that is stable over a long period of time under cell culture conditions, and a composition for forming the hydrogel.
- FIG. 1 shows the 1 H NMR spectrum of aldehyde-functionalized polyethylene glycol (CHO-PEG).
- FIG. 1 shows the time course of degradation rate of hydrogels formed from a network polymer obtained by condensing hydrazide-functionalized hyaluronic acid (HA S -CHD, HA S -AHD, HA L -CHD, HA L -AHD) or carboxymethylchitosan (CMCH) with an aldehyde-functionalized polyethylene glycol (CHO-PEG).
- HA S -CHD hydrazide-functionalized hyaluronic acid
- CMCH carboxymethylchitosan
- FIG. 1 shows the time course of swelling ratio of hydrogels formed from network polymers of hydrazide-functionalized hyaluronic acid (HAs - CHD, HAs - AHD, HAl - CHD, HAl - AHD) or carboxymethylchitosan (CMCH) condensed with aldehyde-functionalized polyethylene glycol (CHO-PEG).
- HAs - CHD hydrazide-functionalized hyaluronic acid
- HAs - AHD HAs - AHD
- HAl - CHD HAl - CHD
- HAl - AHD carboxymethylchitosan
- FIG. 1 shows a hydrogel formed from a network polymer obtained by condensing hydrazide-functionalized hyaluronic acid (HA S -CHD) and aldehyde-functionalized polyethylene glycol (CHO-PEG), a hydrogel having an interpenetrating polymer network formed from the network polymer and RADA16, and the dichroic spectroscopy (CD) spectrum of RADA16.
- HA S -CHD hydrazide-functionalized hyaluronic acid
- CHO-PEG aldehyde-functionalized polyethylene glycol
- FIG. 1 shows the shear rate dependence of stress for a hydrogel formed from a network polymer obtained by condensing hydrazide-functionalized hyaluronic acid (HA 20-50 -AHD) with aldehyde-functionalized polyethylene glycol (CHO-PEG), and a hydrogel having an interpenetrating polymer network formed from the network polymer and RADA16.
- HA 20-50 -AHD hydrazide-functionalized hyaluronic acid
- CHO-PEG aldehyde-functionalized polyethylene glycol
- FIG. 1 shows the shear rate dependence of viscosity of a hydrogel formed by a network polymer obtained by condensing hydrazide-functionalized hyaluronic acid (HA 20-50 -AHD) with aldehyde-functionalized polyethylene glycol (CHO-PEG), and a hydrogel having an interpenetrating polymer network formed by the network polymer and RADA16.
- FIG. 1 shows the shear rate dependence of stress for a hydrogel formed from a network polymer obtained by condensing carboxymethylchitosan (CMCH) with aldehyde-functionalized polyethylene glycol (CHO-PEG), and a hydrogel having an interpenetrating polymer network formed from the network polymer and RADA16.
- CMCH carboxymethylchitosan
- CHO-PEG aldehyde-functionalized polyethylene glycol
- FIG. 1 shows the shear rate dependence of viscosity of a hydrogel formed from a network polymer obtained by condensing carboxymethylchitosan (CMCH) with aldehyde-functionalized polyethylene glycol (CHO-PEG), and a hydrogel having an interpenetrating polymer network formed from the network polymer and RADA16.
- CMCH carboxymethylchitosan
- CHO-PEG aldehyde-functionalized polyethylene glycol
- FIG. 1 shows the cell viability when cells are cultured using a hydrogel formed from a network polymer obtained by condensing hydrazide-functionalized hyaluronic acid (HA 20-50 -AHD) or carboxymethylchitosan (CMCH) with an aldehyde-functionalized polyethylene glycol (CHO-PEG), or a hydrogel having an interpenetrating polymer network structure formed from the network polymer and RADA16.
- FIG. 13 is a graph showing the cell survival rate in the case where the cells were cultured after injection of a hydrogel containing cells, and in the case where the cells were cultured without injection of a hydrogel containing cells.
- the hydrogel contains a self-assembling peptide and a network polymer.
- the network polymer has a hydrophilic polymer block derived from a hydrophilic polymer and a polyethylene glycol block derived from polyethylene glycol.
- the hydrophilic polymer is a polymer that exhibits a water content of 50% by mass or more and 99% by mass or less as the mass ratio of water when equilibrium swelling is achieved by adding water.
- the hydrogel contains a self-assembling peptide.
- the self-assembling peptide functions as a scaffold for cell culture together with a mesh-like polymer described later.
- the self-assembling peptide self-assembles through hydrophobic interactions and hydrogen bonds, and is physically cross-linked into a mesh-like structure.
- the self-assembling peptide and the mesh-like polymer described below interpenetrate with linear independence (orthogonal reaction).
- Such a structure in which two types of mesh-like polymers interpenetrate is called an interpenetrating polymer network structure (IPN structure).
- IPN structure interpenetrating polymer network structure
- the extracellular matrix which has a three-dimensional structure in which molecular chains of various biopolymers are intricately entangled, plays a major role in the regeneration and maintenance of cells.
- a hydrogel is obtained that has a three-dimensional structure similar to the extracellular matrix in the living body and can be used as a pseudo-extracellular matrix.
- Self-organization is a phenomenon in which small molecules autonomously assemble through intermolecular interactions and other factors to form three-dimensional structures. For example, collagen, elastin, amyloid, etc., organize under certain conditions in the presence of water to form fibrous one-dimensional structures. When the fibrous one-dimensional structures further intertwine, a gel with a three-dimensional structure is formed.
- a "self-assembling peptide” is a peptide that can undergo sol-gel transition, losing fluidity from a sol state to a gel state under specific conditions such as temperature, pressure, pH, and ion concentration.
- collagen and the peptides described below that are composed of peptide units containing three types of amino acid residues, namely arginine residues, alanine residues, and aspartic acid residues, are examples of self-assembling peptides.
- the "sol state” refers to a liquid state in which colloidal particles are dispersed in a dispersion medium and have fluidity. In general, a gel that has been fluidized by heating it is in a sol state. "Solation” refers to a change from a gel state to a sol state.
- a "colloid” is a state in which minute particles formed by aggregation of molecules or ions are dispersed in a medium. The minute particles that form a colloid are called “colloid particles.”
- the “gel state” refers to a state in which colloidal particles self-organize in a dispersion medium and lose fluidity. In general, a gel state is a state in which a sol loses fluidity by cooling it. "Gellation” refers to the change from a sol state to a gel state.
- the "sol-gel transition” is a reversible phase transition phenomenon between a sol and a gel. The sol-gel transition generally depends on temperature under isobaric conditions.
- a self-assembling peptide can be composed of a polypeptide in which multiple peptide units are linked to each other through peptide bonds.
- a "peptide unit” is a constituent unit of a self-assembling peptide in the present invention.
- a peptide unit is composed of an oligopeptide in which at least three types of amino acid residues are bonded together by four bonds.
- the peptide units that make up the self-assembling peptide preferably contain three types of amino acid residues: arginine residues (Arg, R), alanine residues (Ala, A), and aspartic acid residues (Asp, D).
- the peptide units constituting the self-assembling peptide may further contain residues of hydrophobic amino acids other than alanine.
- hydrophobic amino acids other than alanine include glycine (Gly, G), proline (Pro, P), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), methionine (Met, M), cysteine (Cis, C), phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W).
- glycine and proline are preferred.
- the above amino acids other than glycine may be in either the D- or L-form.
- the peptide units constituting the self-assembling peptide are preferably units consisting of four amino acid residues essentially including arginine, alanine, and aspartic acid residues. Examples of such peptide units include RADA, RXDA, and RADX. X is a glycine or proline residue.
- a preferred self-assembling peptide is, for example, a peptide in which m RADAs are linked to n RXDAs or RADXs.
- m is an integer between 3 and 6.
- n is 1 or 2.
- m and n satisfy the relationship 2n ⁇ m.
- the combination is preferably 3/1, 4/1, 5/1, 6/1, 5/2, or 6/2.
- the peptide units can be linked in any order.
- the C-terminal peptide unit is RXDA or RADX, or the N-terminal peptide unit is RXDA.
- a peptide in which RADA is repeated p times is also preferred.
- p is an integer between 3 and 8.
- self-assembling peptides include peptides having the following amino acid sequences:
- X is a glycine residue or a proline residue:
- the two or more Xs may be the same amino acid residue or different amino acid residues.
- RXDA-(RADA) 4 (SEQ ID NO: 1) (RADA) 5 (SEQ ID NO: 2) (RADA) 6 (SEQ ID NO: 3) RXDA-(RADA) 3 (SEQ ID NO: 4) (RADA) 3 -RXDA (SEQ ID NO:5) (RADA) 3 -RADX (SEQ ID NO: 6) RXDA-(RADA) 4 (SEQ ID NO:7) (RADA) 4 -RXDA (SEQ ID NO: 8) (RADA) 4 -RADX (SEQ ID NO: 9) RXDA-(RADA) 5 (SEQ ID NO: 10) (RADA) 5 -RXDA (SEQ ID NO: 11) (RADA) 5 -RADX (SEQ ID NO: 12) RXDA-(RADA) 6 (SEQ ID NO: 13) (RADA) 6 -RXDA (SEQ ID NO: 14) (RADA) 6 -RADX (SEQ ID NO: 15) (RXDA) 2
- (RADA) 4 is preferred.
- (RADA) 4 is also referred to as RADA16.
- the self-assembling peptides can be synthesized by known peptide synthesis methods.
- the peptide synthesis method may be a chemical method or a genetic engineering method.
- the synthesis method of the self-assembling peptides is described in various documents (Ishida et. al., Chem. Eur. J. 2019, 25, 13523-13530; Peptide Synthesis and Self-Assembly, A. Aggeli et. al., Chapter First Online: 25 October 2011, Peptide-Based Materials, pp27-69; Developments in p eptide and amide synthesis, Fernando Albericio, Current Opinion in Chemical Biology, Volume 8, Issue 3, June 2004, Page 211-221; Peptide synthesis: Chemical or enzymatic, Electron. J. Biotechnol. , 2007; 10:279-314; etc.).
- the hydrogel may contain one type of self-assembling peptide, or may contain a combination of two or more types of self-assembling peptides.
- the content of the self-assembling peptide in the hydrogel is not particularly limited as long as the desired effect is not impaired.
- the content of the self-assembling peptide in the hydrogel is preferably 0.1% by mass or more and 3.0% by mass or less, and more preferably 0.2% by mass or more and 1.0% by mass or less, relative to the total mass of the hydrogel.
- the content of the self-assembling peptide is within the above range, the formation of a ⁇ -sheet structure as a self-assembling structure is remarkable, the self-repairing property of the hydrogel is good, and cell death is unlikely to occur when cells and the hydrogel are injected while applying a shear force.
- the network polymer is a network polymer having hydrophilic polymer blocks derived from a hydrophilic polymer and polyethylene glycol blocks derived from polyethylene glycol.
- the above hydrazone bond is formed by the reaction between an aldehyde group and a carboxylic acid hydrazide group.
- the reaction that forms the hydrazone bond is a reversible reaction. Therefore, in a hydrogel containing a network polymer, even if the crosslinking portion containing the hydrazone bond in the network polymer is cleaved, the hydrazone bond will naturally be reformed. This allows the hydrogel to exhibit self-repairing properties.
- Angiogenesis is one of the fundamental properties of biological tissues, known as self-repair. For this reason, dynamic self-repairing hydrogels with reversible crosslinks are expected to be a material that provides an attractive environment for angiogenesis.
- a hydrogel with self-repairing properties When a hydrogel with self-repairing properties is used for cell culture, the hydrogel can stably exist for a long period of time under culture conditions while repeatedly being destroyed and repaired, and cell migration and nutrient transport within the hydrogel are promoted. Furthermore, a hydrogel with an IPN structure containing a reversible bond-containing mesh polymer exhibits rapid recovery rheological properties that self-repair mechanical strength through sol-gel transition. As a result, a hydrogel with an IPN structure containing the above-mentioned mesh polymer and self-assembling peptide becomes an injectable gel.
- the network polymer is preferably a reaction product of a hydrazide-functionalized hydrophilic polymer and an aldehyde-functionalized polyethylene glycol, or a reaction product of an aldehyde-functionalized hydrophilic polymer and a hydrazide-functionalized polyethylene glycol. More preferably, the network polymer is a reaction product of a hydrazide-functionalized hydrophilic polymer and an aldehyde-functionalized polyethylene glycol.
- a network polymer may be obtained by reacting a hydrazide-functionalized hydrophilic polymer and an aldehyde-functionalized hydrophilic polymer with an aldehyde-functionalized polyethylene glycol.
- a network polymer may be obtained by reacting a hydrazide-functionalized hydrophilic polymer and an aldehyde-functionalized hydrophilic polymer with a hydrazide-functionalized polyethylene glycol.
- a network polymer may be obtained by reacting a hydrazide-functionalized hydrophilic polymer and an aldehyde-functionalized hydrophilic polymer with an aldehyde-functionalized polyethylene glycol and a hydrazide-functionalized polyethylene glycol.
- a network polymer may be obtained by reacting a hydrazide-functionalized hydrophilic polymer with an aldehyde-functionalized polyethylene glycol and a hydrazide-functionalized polyethylene glycol.
- a network polymer may be obtained by reacting an aldehyde-functionalized hydrophilic polymer with an aldehyde-functionalized polyethylene glycol and a hydrazide-functionalized polyethylene glycol.
- the reaction conditions for forming a network polymer from a hydrazide- or aldehyde-functionalized hydrophilic polymer and a hydrazide- or aldehyde-functionalized polyethylene glycol are not particularly limited.
- the network polymer is produced by mixing a hydrazide- or aldehyde-functionalized hydrophilic polymer with a hydrazide- or aldehyde-functionalized polyethylene glycol at a temperature near room temperature, for example, about 0°C to 40°C.
- the self-assembling peptides self-assemble through hydrophobic interactions and hydrogen bonds, and are physically cross-linked into a mesh-like structure.
- a hydrogel is obtained that contains the self-assembling peptides and the mesh-like polymers in a mutually interpenetrating state.
- Hydrazide functionalization is a modification in which a carboxylic acid hydrazide group (-CO-NH-NH 2 ) is introduced.
- Aldehyde functionalization is a modification in which an aldehyde group (-CHO) is introduced.
- the aldehyde-functionalized polyethylene glycol is a polyethylene glycol derivative having an aldehyde group at both ends of a linear molecular chain.
- the hydrazide-functionalized polyethylene glycol is a polyethylene glycol derivative having a carboxylic acid hydrazide group at both ends of a linear molecular chain.
- the aldehyde-functionalized polyethylene glycol and the hydrazide-functionalized polyethylene glycol have reactive functional groups at both ends of a linear molecular chain. Therefore, in order to form a network polymer, it is necessary that the hydrazide-functionalized hydrophilic polymer has three or more carboxylic acid hydrazide groups in one molecular chain, or that the aldehyde-functionalized hydrophilic polymer has three or more aldehyde groups in one molecular chain.
- the network polymer may contain other blocks other than the hydrophilic polymer block and the polyethylene glycol block, so long as the desired effect is not impaired.
- examples of other blocks include blocks derived from hydrazide- or aldehyde-functionalized peptides; blocks derived from hydrazide- or aldehyde-functionalized polysaccharides that do not fall under the definition of hydrophilic polymers described below; blocks derived from hydrazide- or aldehyde-functionalized silicone resins; blocks derived from hydrazide- or aldehyde-functionalized fluororesins; blocks derived from hydrazide- or aldehyde-functionalized polyester resins; blocks derived from hydrazide- or aldehyde-functionalized polyamide resins; and the like.
- the other blocks are not limited to the above blocks.
- the ratio of other blocks in the network polymer is preferably 20% by mass or less, more preferably 10% by mass or less, and even more preferably 5% by mass or less, based on the mass of the network polymer. It is most preferable that the network polymer does not contain other blocks.
- hydrophilic polymer block and polyethylene glycol block are explained below.
- the hydrophilic polymer block is a block derived from a hydrophilic polymer. More specifically, the hydrophilic polymer block is a block derived from a hydrazide- or aldehyde-functionalized hydrophilic polymer.
- the network polymer may contain different hydrophilic polymer blocks derived from two or more hydrophilic polymers.
- a hydrophilic polymer is a polymer that exhibits a water content of 50% by mass or more and 99% by mass or less as the mass ratio of water when swollen to equilibrium by adding water.
- the equilibrium swelling ratio is calculated from the dry mass Md of the gel and the weight Mw of the gel swollen to equilibrium based on the following formula.
- Mw is the mass of the gel swollen to equilibrium when the gel is swollen to equilibrium in water at 25°C.
- Moisture content (mass%) (Mw - Md) / Mw x 100
- hydrophilic polymer block in the mesh polymer promotes the self-assembly of the self-assembling peptide.
- formation of a ⁇ -sheet structure by the self-assembling peptide is promoted.
- the molecular weight of the hydrophilic polymer block is preferably 100,000 or more and 1,600,000 or less, more preferably 100,000 or more and 700,000 or less, and even more preferably 200,000 or more and 500,000 or less, in terms of weight average molecular weight.
- hydrophilic polymers can be used as the polymer exhibiting the above water content.
- Some examples of polymers exhibiting the above water content include partially saponified polyvinyl alkanoates, polysaccharides containing uronic acid units, gelatin, collagen, fibroin, etc. These hydrophilic polymers are aldehyde- or hydrazide-functionalized according to known methods and used to form a network polymer.
- the partially saponified polyvinyl alkanoate As for the partially saponified polyvinyl alkanoate, the partially saponified polyvinyl acetate is preferred in terms of ease of availability and the above-mentioned water content.
- the water content of the partially saponified polyvinyl alkanoate can be adjusted by adjusting the degree of saponification or the molecular weight of polyvinyl alcohol.
- a partially saponified polyvinyl alkanoate has an alcoholic hydroxyl group.
- an aldehyde group can be introduced into a partially saponified polyvinyl alkanoate by reacting the alcoholic hydroxyl group with a carboxylic acid having an aldehyde group to esterify it, or by etherifying the alcoholic hydroxyl group using a compound having an aldehyde group and a halogen atom by a method such as Williamson's ether synthesis.
- the above method is one example of a method for introducing an aldehyde group into a partially saponified polyvinyl alkanoate.
- the method for introducing an aldehyde group into a partially saponified polyvinyl alkanoate is not limited to the above method.
- Carboxy groups can be introduced into the partially saponified polyvinyl alkanoate by reacting the alcoholic hydroxyl group with a dicarboxylic anhydride, or by condensing and esterifying the alcoholic hydroxyl group with a dicarboxylic acid.
- a dicarboxylic anhydride for example, anhydrides of chain aliphatic dicarboxylic acids such as succinic anhydride, glutaric anhydride, adipic anhydride, and maleic anhydride can be used.
- chain aliphatic dicarboxylic acids such as malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, and fumaric acid can be used.
- Hydrazide groups can be introduced into the partially saponified polyvinyl alkanoate by reacting the carboxyl groups introduced into the partially saponified polyvinyl alkanoate with a dihydrazide compound.
- a dihydrazide compound chain aliphatic dicarboxylic acid dihydrazides such as carbohydrazide, malonic acid dihydrazide, succinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, maleic acid dihydrazide, and fumaric acid dihydrazide can be used.
- the uronic acid unit has a carboxy group. Since the carboxy group is highly reactive, a hydrazide group or an aldehyde group can be introduced into the uronic acid unit by various reactions. With respect to the polysaccharide containing the uronic acid unit, the type of uronic acid and the content of the uronic acid unit are not particularly limited as long as the polysaccharide exhibits the above-mentioned predetermined water content. Specific examples of the uronic acid include glucuronic acid, iduronic acid, and galacturonic acid. The uronic acid may be in the D-form or the L-form. As the uronic acid described above, D-glucuronic acid and L-iduronic acid are preferable.
- the above polysaccharides may contain units derived from other monosaccharides in addition to the uronic acid units.
- specific examples of other monosaccharides include D-glucose, D-galactose, D-mannose, xylose, L-fucose, D-glucosamine, D-acetylglucosamine, D-galactosamine, D-acetylgalactosamine, etc.
- so-called amino acids such as D-glucosamine, D-acetylglucosamine, D-galactosamine, D-acetylgalactosamine, etc. or their derivatives are preferred in terms of the ease of obtaining the above polysaccharides.
- glycosaminoglycans are preferred as the polysaccharides.
- glycosaminoglycans include hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin.
- hyaluronic acid is preferred because of its low inflammation-inducing properties and high affinity with fibroblasts, which is expected to increase collagen production.
- hyaluronic acid is likely to promote the formation of a ⁇ -sheet structure by self-assembling peptides.
- the method of hydrazide-functionalizing or aldehyde-functionalizing a polysaccharide containing uronic acid units is not particularly limited. Hydrazide-functionalizing a polysaccharide containing uronic acid units can be carried out, for example, by reacting the polysaccharide with a dihydrazide compound. According to this reaction, a carboxy group in the uronic acid unit of the polysaccharide and a hydrazide group are condensed, and a group represented by -CO-NH- NH2 is introduced at the end of the side chain of the polysaccharide.
- a chain aliphatic dicarboxylic acid dihydrazide such as carbohydrazide, malonic acid dihydrazide, succinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, maleic acid dihydrazide, and fumaric acid dihydrazide can be used.
- One method for functionalizing a polysaccharide containing uronic acid units with an aldehyde is to react the polysaccharide with a periodate.
- the bond represented by -C(OH)H-C(OH)H- in the uronic acid unit is oxidatively cleaved to generate two aldehyde groups.
- the periodate for example, an alkali metal periodate such as sodium periodate or potassium periodate can be used.
- the amount of carboxylic acid hydrazide groups or aldehyde groups in the hydrazide- or aldehyde-functionalized hydrophilic polymer is not particularly limited as long as the desired effect is not impaired.
- the amount of carboxylic acid hydrazide groups or aldehyde groups in the hydrazide- or aldehyde-functionalized hydrophilic polymer is preferably 20 to 200, more preferably 40 to 100, as the average number per molecule of the hydrophilic polymer.
- the ratio of the mass W HP of the hydrophilic polymer block to the mass W PEG of the polyethylene glycol block, W HP :W PEG is preferably from 20:80 to 80:20, more preferably from 30:70 to 70:30, even more preferably from 40:60 to 60:40, and particularly preferably from 45:55 to 55:45.
- the polyethylene glycol block is a block derived from polyethylene glycol. More specifically, the polyethylene glycol block is a block derived from hydrazide- or aldehyde-functionalized polyethylene glycol.
- the network polymer may contain different polyethylene glycol blocks derived from two or more polyethylene glycols of different molecular weights.
- the molecular weight of the polyethylene glycol block is preferably 2,000 or more and 40,000 or less, and more preferably 2,000 or more and 10,000 or less, in terms of number average molecular weight.
- a method for functionalizing polyethylene glycol with an aldehyde there can be mentioned a method of condensing the terminal hydroxyl group of polyethylene glycol with a carboxylic acid having an aldehyde group to form an ester.
- carboxylic acids having an aldehyde group include terephthalaldehyde acid (4-formylbenzoic acid) and 3-formylbenzoic acid.
- polyethylene glycol can be functionalized with aldehydes by introducing carboxyl groups to both ends of polyethylene glycol and then reducing the carboxyl groups to aldehyde groups by a known method.
- carboxyl groups can be introduced to both ends of polyethylene glycol by reacting the terminal hydroxyl groups of polyethylene glycol with a dicarboxylic acid anhydride, or by condensing the terminal hydroxyl groups with a dicarboxylic acid to form an ester.
- the dicarboxylic acid anhydride for example, anhydrides of chain aliphatic dicarboxylic acids such as succinic anhydride, glutaric anhydride, adipic anhydride, and maleic anhydride can be used.
- chain aliphatic dicarboxylic acids such as malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, and fumaric acid can be used.
- a method for functionalizing polyethylene glycol with hydrazide is to react the carboxyl groups introduced at both ends of polyethylene glycol with a dihydrazide compound.
- a dihydrazide compound aliphatic chain dicarboxylic acid dihydrazides such as carbohydrazide, malonic acid dihydrazide, succinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, maleic acid dihydrazide, and fumaric acid dihydrazide can be used.
- hydrogels described above can be used for a variety of purposes, including as scaffolding materials for cell culture, as well as materials for artificial cartilage, artificial muscle, artificial skin, wound healing agents, and carriers for drug delivery systems.
- composition for forming hydrogel A composition for forming a hydrogel is used to form the hydrogel described above, the composition comprising the self-assembling peptide, an aldehyde- or hydrazide-functionalized hydrophilic polymer to provide a hydrophilic polymer block, and an aldehyde- or hydrazide-functionalized polyethylene glycol to provide a polyethylene glycol block, each of which is described above.
- the self-assembling peptide, the aldehyde- or hydrazide-functionalized hydrophilic polymer that provides the hydrophilic polymer block, and the aldehyde- or hydrazide-functionalized polyethylene glycol that provides the polyethylene glycol block are usually dissolved.
- Solvents contained in the composition include water; saline; cell culture medium; cell culture medium containing serum; etc.
- the composition may be a multi-liquid composition consisting of two or more liquids.
- the liquids contained in the multi-liquid composition are mixed when forming the hydrogel.
- each of the liquids constituting the multi-liquid composition has good stability over time
- a three-liquid composition consisting of a first liquid containing a self-assembling peptide, a second liquid containing an aldehyde-functionalized or hydrazide-functionalized hydrophilic polymer, and a third liquid containing an aldehyde-functionalized or hydrazide-functionalized polyethylene glycol is preferred as the multi-liquid composition.
- the concentration of the self-assembling peptide in the composition is not particularly limited, but is preferably from 0.01% to 3% by mass, more preferably from 0.05% to 1% by mass, and even more preferably from 0.1% to 1% by mass.
- concentration of the aldehyde- or hydrazide-functionalized hydrophilic polymer that gives the hydrophilic polymer block, and the concentration of the aldehyde- or hydrazide-functionalized polyethylene glycol that gives the polyethylene glycol block in the composition are not particularly limited, but are preferably 0.01% by mass or more and 20% by mass or less, and more preferably 0.05% by mass or more and 10% by mass or less.
- the preferred ranges of the concentration of the self-assembling peptide in the first part, the preferred ranges of the concentration of the aldehyde- or hydrazide-functionalized hydrophilic polymer in the second part, and the preferred ranges of the concentration of the aldehyde- or hydrazide-functionalized polyethylene glycol in the third part are the same as the preferred ranges described above.
- Synthesis Example 1 CHO-PEG was synthesized by condensation reaction of formylbenzoic acid (terephthalaldehyde acid) with both terminal hydroxyl groups of polyethylene glycol having a number average molecular weight of 1986.
- the reaction formula is shown below.
- the product was confirmed to be CHO-PEG by 1 H NMR spectrum.
- the 1 H NMR spectrum of the product is shown in FIG. 1. From the 1 H NMR spectrum of the product (CHO-PEG) shown in FIG. 1, the introduction rate of aldehyde groups into both ends of polyethylene glycol was obtained. Specifically, the integral value of the protons corresponding to the alkyl chain of polyethylene glycol was set to 174, and the introduction rate of aldehyde groups into both ends of polyethylene glycol was obtained based on the peak of protons b in FIG. 1, which corresponds to the bonding parts of the functional groups (p-formylphenylcarbonyloxy groups) at both ends. As a result, the introduction rate of aldehyde groups into both ends of polyethylene glycol was 99%.
- Synthesis Example 4 200 mg (0.500 mmol) of sodium hyaluronate (HA S , weight average molecular weight: 500,000 to 700,000, manufactured by Kikkoman Biochemifa Corporation) was added to sodium hyaluronate (HA L ).
- the amount of 1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride (EDC, manufactured by Tokyo Chemical Industry Co., Ltd.) was changed from 397.1 mg (2.07 mmol) to 397.9 mg (2.08 mmol) in the same manner as in Synthesis Example 2, and 222.5 mg of the product (HA L -CHD) was obtained (yield 81.3%).
- the hydrazide conversion rate of the side chain carboxyl group of hyaluronic acid calculated from 1 H NMR was 60%.
- Synthesis Example 5 200 mg (0.500 mmol) of sodium hyaluronate (HA S , weight average molecular weight: 500,000 to 700,000, manufactured by Kikkoman Biochemifa Corporation) was added to sodium hyaluronate (HA L ).
- HA S sodium hyaluronate
- HA L weight average molecular weight: 500,000 to 700,000, manufactured by Kikkoman Biochemifa Corporation
- CMCH solution, a HAs - CHD solution, a HAs -AHD solution, a HAl - CHD solution, and a HAl -AHD solution were prepared using phosphate buffered saline (1x PBS).
- a CHO-PEG solution having a concentration of 3.0% by mass was prepared using phosphate buffered saline (1x PBS).
- CMCH solution 200 ⁇ L of CMCH solution, HA S -CHD solution, HA S -AHD solution, HA L -CHD solution, or HA L -AHD solution was mixed with 100 ⁇ L of CHO-PEG solution in a 1.5 mL tube. The solution in the tube was left to stand for 18 hours to form a hydrogel. 1 mL of 1 ⁇ PBS was added onto the hydrogel in each tube, and the hydrogel was allowed to swell at room temperature for 24 hours.
- the time when the hydrogel was swollen was used as the starting point, and the decomposition rate and swelling rate were measured after 1 day, 6 days, 12 days, 18 days, and 24 days according to the following method. During the 24-day test, the supernatant in the tube was removed once every two days, and 1 mL of 1x PBS was added to the tube.
- the hydrogel was washed twice with 1 mL of 1x PBS. After the washing, the supernatant in the tube was removed, and the weight W swell of the swollen hydrogel in the tube was measured. After measuring W swell , the hydrogel was freeze-dried for 18 hours, and the weight W d of the dried hydrogel was measured. The mass of the raw material of the hydrogel added to the tube was W 0 , and the weight of the salt of 1x PBS contained in the gel was 9 mg/mL, and the decomposition rate and swelling rate were calculated based on the following formula.
- a HA.sub.S -CHD solution, a HA.sub.S -AHD solution, a HA.sub.L- CHD solution, and a HA.sub.L-AHD solution were prepared using phosphate buffered saline (1.times.PBS).
- a CHO -PEG solution having a concentration of 3.0% by mass was prepared using phosphate buffered saline (1.times.PBS).
- a solution of hydrazide-functionalized hyaluronic acid and a solution of aldehyde-functionalized polyethylene glycol were mixed on the stage of a rheometer so that the masses of the hydrazide-functionalized hyaluronic acid and the aldehyde-functionalized polyethylene glycol were the same. Then, the change in the storage modulus G' and the change in the loss modulus G" over time were measured under the following measurement conditions.
- the gel point (seconds) was measured, which is the time when the storage modulus G' exceeded the loss modulus G".
- the measurement results of the gel point are shown in Table 1.
- gelation occurred immediately after mixing the hydrazide-functionalized hyaluronic acid and the aldehyde-functionalized polyethylene glycol, so the gel point could not be measured.
- Table 1 shows that in a hydrazide-functionalized hydrophilic polymer (hydrazide-functionalized hyaluronic acid), the longer the side chain length containing a carboxylic acid hydrazide group, the longer the gelation time.
- a hydrazide-functionalized hydrophilic polymer with a long side chain length containing a carboxylic acid hydrazide group a mixed solution containing the hydrogel raw materials can be injected in liquid form into the area where a hydrogel is to be formed.
- liquid containing the hydrogel raw materials does not gel easily, for example, when forming a hydrogel in situ at a location in a living body where specific cells are to be grown, it is easy to inject the liquid containing the hydrogel raw materials into the living body by injection.
- a hydrogel was formed according to the following method using the hydrazide-functionalized hyaluronic acid (HA S -CHD) obtained in Synthesis Example 2, the aldehyde-functionalized polyethylene glycol (CHO-PEG) obtained in Synthesis Example 1, and RADA16 as a self-assembling peptide.
- the circular dichroism (CD) spectrum of the formed hydrogel was measured.
- a 2% by mass HA S -CHD solution was prepared using phosphate buffered saline (1 ⁇ PBS).
- a 4% by mass CHO-PEG solution was prepared using phosphate buffered saline (2 ⁇ PBS).
- a 1% by mass RADA16 solution was prepared using phosphate buffered saline (1 ⁇ PBS).
- the above three solutions were mixed and the concentrations were adjusted so that the concentration of HA S -CHD in the mixed solution was 1.5% by mass, the concentration of CHO-PEG was 1% by mass, and the concentration of RADA16 in the mixed solution was 0.25% by mass, to form a hydrogel (HA S -CHD/CHO-PEG/RADA16).
- the circular dichroism (CD) spectrum of the formed hydrogel was measured in the wavelength range of 190 to 300 nm using a circular dichroism spectrometer (J-820 model, manufactured by JASCO Corporation).
- CD circular dichroism
- the obtained circular dichroism (CD) spectra are shown in Figure 4.
- the spectrum of "(1) RADA16 solution” is the CD spectrum of RADA16.
- the spectrum of "(2) HAs -CHD/CHO-PEG” is the CD spectrum of a hydrogel formed using HAs - CHD and CHO-PEG.
- the spectrum of "(3) HAs - CHD/CHO-PEG/RADA16” is the CD spectrum of a hydrogel formed using HAs - CHD, CHO-PEG, and RADA16.
- the spectrum of the difference between the CD spectrum of (3) above and the CD spectrum of (2) above is shown in Figure 4 as "(4) Difference between (3) and (2)".
- the spectrum (4) is the difference spectrum between the CD spectrum (3) above and the CD spectrum (2) above, and therefore the spectrum (4) can be said to be the spectrum of RADA16 in a hydrogel (mesh polymer) formed using HA S -CHD and CHO-PEG.
- a peak corresponding to a ⁇ -sheet structure appears in the vicinity of 220 to 230 nm wavelength in the CD spectrum.
- the spectrum of (4) can be said to be the spectrum of RADA16 in a hydrogel (network polymer) formed using HA s -CHD and CHO-PEG. From the above, it is considered that the formation of ⁇ -sheet of RADA16 has progressed in the hydrogel (HA s -CHD/CHO-PEG/RADA16) formed using HA s -CHD and CHO-PEG.
- phosphate buffered saline (1xPBS) was used to prepare a HA S -CHD solution, a HA S -AHD solution, a HA L -CHD solution, and a HA L -AHD solution, each having a concentration of 2% by mass.
- Phosphate buffered saline (1xPBS) was used to prepare a CMCH solution having a concentration of 4% by mass.
- Phosphate buffered saline (1xPBS) was used to prepare a CHO-PEG solution having a concentration of 8% by mass.
- a RADA16 solution having a concentration of 2.5% by mass was also prepared.
- the transparent semicircular hydrogel and the blue semicircular hydrogel were placed in 1x PBS while being in contact with each other to form a circle, and the disk-shaped hydrogel was left at room temperature for 18 hours. After 18 hours, the disk-shaped hydrogel was pulled up with tweezers, and the adhesion state between the transparent semicircular hydrogel and the blue semicircular hydrogel was confirmed, and the self-repairing property was evaluated according to the following evaluation criteria.
- the evaluation results are shown in Table 2.
- -Evaluation criteria- Good The transparent semicircular hydrogel and the blue semicircular hydrogel were firmly adhered to each other.
- Poor The transparent semicircular hydrogel and the blue semicircular hydrogel are weakly or not adhered to each other.
- Table 2 shows that hydrogels with an IPN structure consisting of a network polymer formed by condensing hydrazide-functionalized hyaluronic acid and aldehyde-functionalized polyethylene glycol, and a self-assembling peptide, exhibit good self-healing properties.
- hydrogels consisting of a self-assembling peptide and a mesh polymer formed by condensation of carboxymethylchitosan and aldehyde-functionalized polyethylene glycol also showed self-repairing properties.
- the mesh polymer formed by condensation of carboxymethylchitosan and aldehyde-functionalized polyethylene glycol easily decomposes over time. For this reason, hydrogels consisting of a self-assembling peptide and a mesh polymer formed by condensation of carboxymethylchitosan and aldehyde-functionalized polyethylene glycol cannot maintain self-repairing properties for a long period of time.
- HA 20-50 -AHD solution and CMCH solution were prepared using phosphate buffered saline (1xPBS).
- a CHO-PEG solution with a concentration of 4% by mass was prepared using phosphate buffered saline (2xPBS).
- a RADA16 solution (PuraStat, manufactured by 3D Matrix Co., Ltd.) with a concentration of 2.5% by mass was prepared, sonicated for 30 minutes, and then stirred with a vortex mixer.
- hydrogel with an IPN structure consisting of a network polymer condensed from hydrazide-functionalized hyaluronic acid and aldehyde-functionalized polyethylene glycol, and a self-assembling peptide, showed strong shear-thinning properties.
- HA 20-50 -AHD, CMCH, and CHO-PEG were sterilized by irradiation with ultraviolet light for 20 minutes.
- HA 20-50 -AHD solution and CMCH solution were prepared using phosphate buffered saline (1xPBS).
- a CHO-PEG solution having a concentration of 4% by mass was prepared using phosphate buffered saline (2xPBS).
- a RADA16 solution PuraStat, manufactured by Three-D Matrix Co., Ltd.
- concentration of 2.5% by mass was prepared, sonicated for 30 minutes, and then stirred with a vortex mixer.
- MTT reagent solution was prepared using phosphate buffered saline (1xPBS), and 1 M hydrochloric acid was diluted with 2-propanol to 0.04 mM to prepare an MTT extraction reagent.
- Subconfluent HepG2 cells that had been cultured in advance in an incubator (37°C, 5% CO 2 ) were detached by trypsin treatment, centrifuged (1500 rpm, 5 minutes), and the supernatant was removed.
- 1 mL of DMEM was added, trypan blue staining was performed, and the number of live cells was counted using a hemocytometer.
- 4 mL of DMEM was added, centrifuged, and the supernatant was removed.
- HA 20-50 -AHD solution or CMCH solution was added to the cells, and they were suspended to 2.0 x 10 7 cells/mL.
- the HA 20-50 -AHD solution or CMCH solution in which the cells were suspended, the CHO-PEG solution, the RADA16 solution, and ultrapure water were added to a 1.5 mL tube and mixed to obtain the compositions shown in Tables 5 and 6.
- the tube was then placed in an incubator (37°C, 5% CO 2 ) for 30 minutes to form a hydrogel.
- HA 20-50 -AHD, CMCH, and CHO-PEG were sterilized by irradiation with ultraviolet light for 30 minutes. Then, phosphate buffered saline (1xPBS) was used to prepare HA 20-50 -AHD solution and CMCH solution, each with a concentration of 2% by mass. Phosphate buffered saline (2xPBS) was used to prepare a CHO-PEG solution with a concentration of 4% by mass.
- a RADA16 solution PuraStat, manufactured by 3D Matrix Co., Ltd.
- concentration of 2.5% by mass was prepared, sonicated for 30 minutes, and then stirred with a vortex mixer.
- Subconfluent HepG2 cells that had been cultured in advance in an incubator (37°C, 5% CO 2 ) were detached by trypsin treatment, centrifuged (1500 rpm, 5 minutes), and the supernatant was removed.
- 1 mL of DMEM was added, trypan blue staining was performed, and the number of live cells was measured using a hemocytometer.
- 4 mL of DMEM was added, centrifuged, and the supernatant was removed.
- HA 20-50 -AHD solution or CMCH solution was added to the cells, and they were suspended to 1.0 x 10 7 cells/mL.
- the HA 20-50 -AHD solution or CMCH solution in which the cells were suspended, the CHO-PEG solution, the RADA16 solution, and ultrapure water were added to a tube with a capacity of 1.5 mL to obtain the composition described in Tables 7 and 8, and mixed, and immediately aspirated with a 5 mL syringe and left to stand for 30 minutes to form 400 ⁇ L of hydrogel in the syringe.
- an 18G injection needle was attached to the tip of the syringe, and the hydrogel was injected into a 24-well plate at 200 ⁇ L/well.
- the HA 20-50 -AHD solution or CMCH solution in which the cells were suspended the CHO-PEG solution, the RADA16 solution, and ultrapure water were added to a tube with a capacity of 1.5 mL to obtain the composition described in Tables 7 and 8, and immediately transferred to a 24-well plate and left to stand for 30 minutes to form a hydrogel.
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Abstract
Description
本発明は、ハイドロゲル、及びハイドロゲルを形成するための組成物に関する。 The present invention relates to a hydrogel and a composition for forming a hydrogel.
近年、細胞培養用の足場材料等の用途において、生体高分子や、生体適合性高分子材料からなるポリマーネットワークを含むハイドロゲルが注目されている。 In recent years, hydrogels that contain polymer networks made of biopolymers and biocompatible polymer materials have been attracting attention for use as scaffolding materials for cell culture, etc.
このようなハイドロゲルについて、本発明者らは、自己組織化ペプチドと、キトサンと、アルデヒド末端を有するポリエチレングリコールとから形成されたハイドロゲルを提案している（非特許文献１及び２を参照）。かかるハイドロゲルは、ワンポット又はｉｎ ｓｉｔｕでの合成が可能であり、細胞培養用途等の種々の用途において有用である。
As for such hydrogels, the present inventors have proposed a hydrogel formed from a self-assembling peptide, chitosan, and polyethylene glycol having an aldehyde end (see Non-Patent
しかしながら、後述する試験例１に示されるように、非特許文献１及び２に記載されるハイドロゲルを構成する、キトサンとアルデヒド末端を有するポリエチレングリコールとがシッフ塩基架橋された網目状ポリマーには、経時的に分解しやすい問題がある。
However, as shown in Test Example 1 described later, the mesh polymer in which chitosan and polyethylene glycol having an aldehyde end, which constitutes the hydrogel described in
本発明は、上記の課題に鑑みなされたものであり、細胞培養条件下において長期間に亘って安定なハイドロゲルと、当該ハイドロゲルを形成するための組成物とを提供することを目的とする。 The present invention has been made in consideration of the above problems, and aims to provide a hydrogel that is stable over a long period of time under cell culture conditions, and a composition for forming the hydrogel.
本発明者らは、自己組織化ペプチドと、網目状ポリマーとを含むハイドロゲルにおいて、親水性ポリマーブロックと、ポリエチレングリコールブロックとを有する網目状ポリマーを用い、親水性ポリマーブロックと、ポリエチレングリコールブロックとを、－Ｃ＝Ｎ－ＮＨ－で表されるヒドラゾン結合を含む２価の基により結合させることにより、上記の課題を解決できることを見出し、本発明を完成するに至った。より具体的には、本発明は以下のとおりである。 The inventors have discovered that the above problems can be solved by using a hydrogel containing a self-assembling peptide and a mesh polymer, the mesh polymer having a hydrophilic polymer block and a polyethylene glycol block, and by bonding the hydrophilic polymer block and the polyethylene glycol block with a divalent group containing a hydrazone bond represented by -C=N-NH-, and have thus completed the present invention. More specifically, the present invention is as follows.
（１） 自己組織化ペプチドと、網目状ポリマーとを含むハイドロゲルであって、
網目状ポリマーが、親水性ポリマーに由来する親水性ポリマーブロックと、ポリエチレングリコールに由来するポリエチレングリコールブロックとを有し、
親水性ポリマーブロックと、ポリエチレングリコールブロックとが、－Ｃ＝Ｎ－ＮＨ－で表されるヒドラゾン結合を含む２価の基により結合しており、
親水性ポリマーが、水を加えて平衡膨潤化させた場合に水の質量比率として５０質量％以上９９質量％以下の含水率を示すポリマーである、ハイドロゲル。
(1) A hydrogel comprising a self-assembling peptide and a network polymer,
the network polymer has hydrophilic polymer blocks derived from a hydrophilic polymer and polyethylene glycol blocks derived from polyethylene glycol;
the hydrophilic polymer block and the polyethylene glycol block are bonded to each other via a divalent group containing a hydrazone bond represented by -C=N-NH-,
A hydrogel, wherein the hydrophilic polymer is a polymer that exhibits a water content of 50% by mass or more and 99% by mass or less, in terms of the mass ratio of water, when the hydrophilic polymer is allowed to swell in equilibrium with the addition of water.
（２） 自己組織化ペプチドと、網目状ポリマーとが相互に侵入した相互侵入高分子網目構造を有する、（１）に記載のハイドロゲル。 (2) The hydrogel described in (1) has an interpenetrating polymer network structure in which a self-assembling peptide and a network polymer interpenetrate each other.
（３） 親水性ポリマーブロックが、ウロン酸単位を含む多糖に由来するブロックである、（１）又は（２）に記載のハイドロゲル。 (3) The hydrogel according to (1) or (2), wherein the hydrophilic polymer block is a block derived from a polysaccharide containing uronic acid units.
（４） 多糖が、グリコサミノグリカンである、（３）に記載のハイドロゲル。 (4) The hydrogel according to (3), wherein the polysaccharide is a glycosaminoglycan.
（５） グリコサミノグリカンが、ヒアルロン酸である、（４）に記載のハイドロゲル。 (5) The hydrogel according to (4), wherein the glycosaminoglycan is hyaluronic acid.
（６） 網目状ポリマーが、ヒドラジド官能化親水性ポリマーと、アルデヒド官能化ポリエチレングリコールとの反応物である、（１）～（５）のいずれか１つに記載のハイドロゲル。 (6) The hydrogel according to any one of (1) to (5), wherein the network polymer is a reaction product of a hydrazide-functionalized hydrophilic polymer and an aldehyde-functionalized polyethylene glycol.
（７） （１）に記載のハイドロゲルを形成するための組成物であって、
自己組織化ペプチドと、親水性ポリマーブロックを与える、アルデヒド官能化又はヒドラジド官能化された親水性ポリマーと、ポリエチレングリコールブロックを与える、アルデヒド官能化又はヒドラジド官能化されたポリエチレングリコールとを含む組成物。
(7) A composition for forming the hydrogel according to (1), comprising:
A composition comprising a self-assembling peptide, an aldehyde- or hydrazide-functionalized hydrophilic polymer to provide a hydrophilic polymer block, and an aldehyde- or hydrazide-functionalized polyethylene glycol to provide a polyethylene glycol block.
（８） 自己組織化ペプチドを含む第１液と、アルデヒド官能化又はヒドラジド官能化された親水性ポリマーを含む第２液と、アルデヒド官能化又はヒドラジド官能化されたポリエチレングリコールを含む第３液とからなる３液型の組成物である、（７）に記載の組成物。 (8) The composition according to (7), which is a three-liquid composition consisting of a first liquid containing a self-assembling peptide, a second liquid containing an aldehyde-functionalized or hydrazide-functionalized hydrophilic polymer, and a third liquid containing an aldehyde-functionalized or hydrazide-functionalized polyethylene glycol.
本発明によれば、細胞培養条件下において長期間に亘って安定なハイドロゲルと、当該ハイドロゲルを形成するための組成物とを提供することができる。 The present invention provides a hydrogel that is stable over a long period of time under cell culture conditions, and a composition for forming the hydrogel.
≪ハイドロゲル≫
ハイドロゲルは、自己組織化ペプチドと、網目状ポリマーとを含む。網目状ポリマーは、親水性ポリマーに由来する親水性ポリマーブロックと、ポリエチレングリコールに由来するポリエチレングリコールブロックとを有する。網目状ポリマーにおいては、親水性ポリマーブロックと、ポリエチレングリコールブロックとが、－Ｃ＝Ｎ－ＮＨ－で表されるヒドラゾン結合を含む２価の基により結合している。親水性ポリマーは、水を加えて平衡膨潤化させた場合に水の質量比率として５０質量％以上９９質量％以下の含水率を示すポリマーである。
<Hydrogel>
The hydrogel contains a self-assembling peptide and a network polymer. The network polymer has a hydrophilic polymer block derived from a hydrophilic polymer and a polyethylene glycol block derived from polyethylene glycol. In the network polymer, the hydrophilic polymer block and the polyethylene glycol block are bonded to each other via a divalent group containing a hydrazone bond represented by -C=N-NH-. The hydrophilic polymer is a polymer that exhibits a water content of 50% by mass or more and 99% by mass or less as the mass ratio of water when equilibrium swelling is achieved by adding water.
以下、自己組織化ペプチドと、網目状ポリマーとについて説明する。 The self-assembling peptides and mesh-like polymers are explained below.
＜自己組織化ペプチド＞
ハイドロゲルは、自己組織化ペプチドを含む。ハイドロゲルにおいて、自己組織化ペプチドは、後述する網目状ポリマーとともに、細胞培養を行う際の足場として機能する。自己組織化ペプチドは、疎水性相互作用や水素結合により自己組織化して、網目状に物理架橋される。
<Self-assembling peptides>
The hydrogel contains a self-assembling peptide. In the hydrogel, the self-assembling peptide functions as a scaffold for cell culture together with a mesh-like polymer described later. The self-assembling peptide self-assembles through hydrophobic interactions and hydrogen bonds, and is physically cross-linked into a mesh-like structure.
ハイドロゲルにおいては、自己組織化ペプチドと、後述する網目状ポリマーとが、１次独立（直交反応）性をもって相互に侵入していることが好ましい。２種の網目状ポリマーが相互に侵入しているこのような構造は、相互侵入高分子網目構造（ＩＰＮ構造：Ｉｎｔｅｒｐｅｎｅｔｒａｔｉｎｇ Ｐｏｌｙｍｅｒ Ｎｅｔｗｏｒｋ Ｓｔｒｕｃｔｕｒｅ）と呼ばれている。生体内においては、種々の生体高分子の分子鎖が複雑に絡み合った三次元構造を有する細胞外マトリックスが、細胞の再生及び維持に大きく関与している。自己組織化ペプチドと網目状ポリマーとが相互に侵入することにより、生体における細胞外マトリックスに類似する三次元構造を有する、疑似細胞外マトリックスとして使用し得るハイドロゲルが得られる。 In the hydrogel, it is preferable that the self-assembling peptide and the mesh-like polymer described below interpenetrate with linear independence (orthogonal reaction). Such a structure in which two types of mesh-like polymers interpenetrate is called an interpenetrating polymer network structure (IPN structure). In the living body, the extracellular matrix, which has a three-dimensional structure in which molecular chains of various biopolymers are intricately entangled, plays a major role in the regeneration and maintenance of cells. By interpenetrating the self-assembling peptide and the mesh-like polymer, a hydrogel is obtained that has a three-dimensional structure similar to the extracellular matrix in the living body and can be used as a pseudo-extracellular matrix.
「自己組織化」は、小分子が分子間相互作用等により自律的に集合し、３次元立体構造を形成する現象である。例えば、コラーゲン、エラスチン、アミロイド等は、特定の条件下で、水の存在下に組織化して繊維状の１次元構造を形成する。繊維状の１次元構造がさらに絡み合うことで、３次元立体構造を有したゲルが形成される。 "Self-organization" is a phenomenon in which small molecules autonomously assemble through intermolecular interactions and other factors to form three-dimensional structures. For example, collagen, elastin, amyloid, etc., organize under certain conditions in the presence of water to form fibrous one-dimensional structures. When the fibrous one-dimensional structures further intertwine, a gel with a three-dimensional structure is formed.
本明細書において、「自己組織化ペプチド」とは、ゾル状態から、固有の温度、圧力、ｐＨ、イオン濃度等の条件下で流動性を失ってゲル状態になり得る、ゾル－ゲル転移し得るペプチドである。例えば、アルギニン残基、アラニン残基、及びアスパラギン酸残基からなる３種類のアミノ酸残基を含むペプチド単位からなる後述するペプチドや、コラーゲンは、自己組織化ペプチドに該当する。 In this specification, a "self-assembling peptide" is a peptide that can undergo sol-gel transition, losing fluidity from a sol state to a gel state under specific conditions such as temperature, pressure, pH, and ion concentration. For example, collagen and the peptides described below that are composed of peptide units containing three types of amino acid residues, namely arginine residues, alanine residues, and aspartic acid residues, are examples of self-assembling peptides.
「ゾル状態」とは、コロイド粒子が分散媒中に分散し、流動性を有した液体状態である。一般的には、ゲルを昇温させることによって流動化した状態が、ゾル状態である。
「ゾル化」とは、ゲル状態からゾル状態への変化である。
「コロイド」とは、分子やイオンが凝集した微粒子が、媒質中に分散している状態である。コロイドを形成する微粒子が「コロイド粒子」である。
「ゲル状態」とは、コロイド粒子が分散媒中で自己組織化し、流動性を失った状態である。一般的には、ゾルを冷却させることによって流動性を失った状態が、ゲル状態である。
「ゲル化」とは、ゾル状態からゲル状態への変化である。
「ゾル－ゲル転移」とは、ゾル及びゲルの間の可逆的な相転移現象である。ゾル－ゲル転移は、一般的に、等圧条件下では温度に依存する。
The "sol state" refers to a liquid state in which colloidal particles are dispersed in a dispersion medium and have fluidity. In general, a gel that has been fluidized by heating it is in a sol state.
"Solation" refers to a change from a gel state to a sol state.
A "colloid" is a state in which minute particles formed by aggregation of molecules or ions are dispersed in a medium. The minute particles that form a colloid are called "colloid particles."
The "gel state" refers to a state in which colloidal particles self-organize in a dispersion medium and lose fluidity. In general, a gel state is a state in which a sol loses fluidity by cooling it.
"Gellation" refers to the change from a sol state to a gel state.
The "sol-gel transition" is a reversible phase transition phenomenon between a sol and a gel. The sol-gel transition generally depends on temperature under isobaric conditions.
自己組織化ペプチドは、複数のペプチド単位が互いにペプチド結合で連結したポリペプチドによって構成され得る。本明細書において、「ペプチドユニット」とは、本発明における自己組織化ペプチドの構成単位である。ペプチド単位は、少なくとも３種類のアミノ酸残基が４つ結合したオリゴペプチドからなる。 A self-assembling peptide can be composed of a polypeptide in which multiple peptide units are linked to each other through peptide bonds. In this specification, a "peptide unit" is a constituent unit of a self-assembling peptide in the present invention. A peptide unit is composed of an oligopeptide in which at least three types of amino acid residues are bonded together by four bonds.
自己組織化ペプチドを構成するペプチド単位は、アルギニン残基（Ａｒｇ，Ｒ）、アラニン残基（Ａｌａ，Ａ）、及びアスパラギン酸残基（Ａｓｐ，Ｄ）からなる３種類のアミノ酸残基を含むことが好ましい。 The peptide units that make up the self-assembling peptide preferably contain three types of amino acid residues: arginine residues (Arg, R), alanine residues (Ala, A), and aspartic acid residues (Asp, D).
自己組織化ペプチドを構成するペプチド単位は、さらに、アラニン以外の疎水性アミノ酸の残基を含み得る。アラニン以外の疎水性アミノ酸としては、グリシン（Ｇｌｙ，Ｇ）、プロリン（Ｐｒｏ，Ｐ）、バリン（Ｖａｌ，Ｖ）、ロイシン（Ｌｅｕ，Ｌ）、イソロイシン（Ｉｌｅ，Ｉ）、メチオニン（Ｍｅｔ，Ｍ）、システイン（Ｃｉｓ，Ｃ）、フェニルアラニン（Ｐｈｅ，Ｆ）、チロシン（Ｔｙｒ，Ｙ）、及びトリプトファン（Ｔｒｐ，Ｗ）が挙げられる。これらの疎水性アミノ酸の中では、グリシン及びプロリンが好ましい。グリシン以外の上記のアミノ酸は、Ｄ体であってもＬ体であってもよい。 The peptide units constituting the self-assembling peptide may further contain residues of hydrophobic amino acids other than alanine. Examples of hydrophobic amino acids other than alanine include glycine (Gly, G), proline (Pro, P), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), methionine (Met, M), cysteine (Cis, C), phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W). Among these hydrophobic amino acids, glycine and proline are preferred. The above amino acids other than glycine may be in either the D- or L-form.
自己組織化ペプチドを構成するペプチド単位は、アルギニン残基、アラニン残基、及びアスパラギン酸残基を必須に含む４アミノ酸残基からなる単位であることが好ましい。かかるペプチド単位としては、ＲＡＤＡ、ＲＸＤＡ、及びＲＡＤＸが挙げられる。Ｘは、グリシン残基又はプロリン残基である。 The peptide units constituting the self-assembling peptide are preferably units consisting of four amino acid residues essentially including arginine, alanine, and aspartic acid residues. Examples of such peptide units include RADA, RXDA, and RADX. X is a glycine or proline residue.
好ましい自己組織化ペプチドは、例えば、ｍ個のＲＡＤＡと、ｎ個のＲＸＤＡ又はＲＡＤＸとが連結したペプチドである。ｍは３以上６以下の整数である。ｎは１又は２である。ｍとｎとは、２ｎ＜ｍの関係を満たす。ｍ及びｎの組み合わせをｍ／ｎと記載する場合に、当該組み合わせは、３／１、４／１、５／１、６／１、５／２、又は６／２であることが好ましい A preferred self-assembling peptide is, for example, a peptide in which m RADAs are linked to n RXDAs or RADXs. m is an integer between 3 and 6. n is 1 or 2. m and n satisfy the relationship 2n<m. When a combination of m and n is described as m/n, the combination is preferably 3/1, 4/1, 5/1, 6/1, 5/2, or 6/2.
ｍ個のＲＡＤＡと、ｎ個のＲＸＤＡ又はＲＡＤＸとが連結した自己組織化ペプチドにおいて、各ペプチド単位は任意の順序で連結され得る。ｍ個のＲＡＤＡと、ｎ個のＲＸＤＡ又はＲＡＤＸとが連結した自己組織化ペプチドにおいて、Ｃ末端のペプチド単位がＲＸＤＡ又はＲＡＤＸであるか、Ｎ末端のペプチド単位がＲＸＤＡであることが好ましい。 In a self-assembling peptide in which m RADAs are linked to n RXDAs or RADXs, the peptide units can be linked in any order. In a self-assembling peptide in which m RADAs are linked to n RXDAs or RADXs, it is preferred that the C-terminal peptide unit is RXDA or RADX, or the N-terminal peptide unit is RXDA.
自己組織化ペプチドとしては、ＲＡＤＡがｐ個繰り返されたペプチドも好ましい。ｐは、３以上８以下の整数である。 As a self-assembling peptide, a peptide in which RADA is repeated p times is also preferred. p is an integer between 3 and 8.
自己組織化ペプチドの好ましい具体例としては、以下のアミノ酸配列を有するペプチドが挙げられる。下記の配列において、Ｘはグリシン残基又はプロリン残基である。下記のアミノ酸配列に２以上のＸが含まれる場合、２以上のＸは同一のアミノ酸残基であってもよく、異なるアミノ酸残基であってもよい。
（ＲＡＤＡ）４（配列番号１）
（ＲＡＤＡ）５（配列番号２）
（ＲＡＤＡ）６（配列番号３）
ＲＸＤＡ－（ＲＡＤＡ）３（配列番号４）
（ＲＡＤＡ）３－ＲＸＤＡ（配列番号５）
（ＲＡＤＡ）３－ＲＡＤＸ（配列番号６）
ＲＸＤＡ－（ＲＡＤＡ）４（配列番号７）
（ＲＡＤＡ）４－ＲＸＤＡ（配列番号８）
（ＲＡＤＡ）４－ＲＡＤＸ（配列番号９）
ＲＸＤＡ－（ＲＡＤＡ）５（配列番号１０）
（ＲＡＤＡ）５－ＲＸＤＡ（配列番号１１）
（ＲＡＤＡ）５－ＲＡＤＸ（配列番号１２）
ＲＸＤＡ－（ＲＡＤＡ）６（配列番号１３）
（ＲＡＤＡ）６－ＲＸＤＡ（配列番号１４）
（ＲＡＤＡ）６－ＲＡＤＸ（配列番号１５）
（ＲＸＤＡ）２－（ＲＡＤＡ）５（配列番号１６）
ＲＸＤＡ－ＲＡＤＡ－ＲＸＤＡ－（ＲＡＤＡ）４（配列番号１７）
ＲＸＤＡ－（ＲＡＤＡ）２－ＲＸＤＡ－（ＲＡＤＡ）３（配列番号１８）
ＲＸＤＡ－（ＲＡＤＡ）３－ＲＸＤＡ－（ＲＡＤＡ）２（配列番号１９）
ＲＸＤＡ－（ＲＡＤＡ）４－ＲＸＤＡ－ＲＡＤＡ（配列番号２０）
ＲＸＤＡ－（ＲＡＤＡ）５－ＲＸＤＡ（配列番号２１）
ＲＸＤＡ－ＲＡＤＸ－（ＲＡＤＡ）５（配列番号２２）
ＲＸＤＡ－ＲＡＤＡ－ＲＡＤＸ－（ＲＡＤＡ）４（配列番号２３）
ＲＸＤＡ－（ＲＡＤＡ）２－ＲＡＤＸ－（ＲＡＤＡ）３（配列番号２４）
ＲＸＤＡ－（ＲＡＤＡ）３－ＲＡＤＸ－（ＲＡＤＡ）２（配列番号２５）
ＲＸＤＡ－（ＲＡＤＡ）４－ＲＡＤＸ－ＲＡＤＡ（配列番号２６）
ＲＸＤＡ－（ＲＡＤＡ）５－ＲＡＤＸ（配列番号２７）
ＲＡＤＡ－ＲＸＤＡ－（ＲＡＤＡ）４－ＲＸＤＡ（配列番号２８）
（ＲＡＤＡ）２－ＲＸＤＡ－（ＲＡＤＡ）３－ＲＸＤＡ（配列番号２９）
（ＲＡＤＡ）３－ＲＸＤＡ－（ＲＡＤＡ）２－ＲＸＤＡ（配列番号３０）
（ＲＡＤＡ）４－ＲＸＤＡ－ＲＡＤＡ－ＲＸＤＡ（配列番号３１）
（ＲＡＤＡ）５－（ＲＸＤＡ）２（配列番号３２）
ＲＡＤＸ－（ＲＡＤＡ）５－ＲＸＤＡ（配列番号３３）
ＲＡＤＡ－ＲＡＤＸ－（ＲＡＤＡ）４－ＲＸＤＡ（配列番号３４）
（ＲＡＤＡ）２－ＲＡＤＸ－（ＲＡＤＡ）３－ＲＸＤＡ（配列番号３５）
（ＲＡＤＡ）３－ＲＡＤＸ－（ＲＡＤＡ）２－ＲＸＤＡ（配列番号３６）
（ＲＡＤＡ）４－ＲＡＤＸ－ＲＡＤＡ－ＲＸＤＡ（配列番号３７）
（ＲＡＤＡ）５－ＲＡＤＸ－ＲＸＤＡ（配列番号３８）
ＲＡＤＡ－ＲＸＤＡ－（ＲＡＤＡ）４－ＲＡＤＸ（配列番号３９）
（ＲＡＤＡ）２－ＲＸＤＡ－（ＲＡＤＡ）３－ＲＡＤＸ（配列番号４０）
（ＲＡＤＡ）３－ＲＸＤＡ－（ＲＡＤＡ）２－ＲＡＤＸ（配列番号４１）
（ＲＡＤＡ）４－ＲＸＤＡ－ＲＡＤＡ－ＲＡＤＸ（配列番号４２）
（ＲＡＤＡ）５－ＲＸＤＡ－ＲＡＤＸ（配列番号４３）
ＲＡＤＸ－（ＲＡＤＡ）５－ＲＡＤＸ（配列番号４４）
ＲＡＤＡ－ＲＡＤＸ－（ＲＡＤＡ）４－ＲＡＤＸ（配列番号４５）
（ＲＡＤＡ）２－ＲＡＤＸ－（ＲＡＤＡ）３－ＲＡＤＸ（配列番号４６）
（ＲＡＤＡ）３－ＲＡＤＸ－（ＲＡＤＡ）２－ＲＡＤＸ（配列番号４７）
（ＲＡＤＡ）４－ＲＡＤＸ－ＲＡＤＡ－ＲＡＤＸ（配列番号４８）
（ＲＡＤＡ）５－（ＲＡＤＸ）２（配列番号４９）
（ＲＸＤＡ）２－（ＲＡＤＡ）６（配列番号５０）
ＲＸＤＡ－ＲＡＤＡ－ＲＸＤＡ－（ＲＡＤＡ）５（配列番号５１）
ＲＸＤＡ－（ＲＡＤＡ）２－ＲＸＤＡ－（ＲＡＤＡ）４（配列番号５２）
ＲＸＤＡ－（ＲＡＤＡ）３－ＲＸＤＡ－（ＲＡＤＡ）３（配列番号５３）
ＲＸＤＡ－（ＲＡＤＡ）４－ＲＸＤＡ－（ＲＡＤＡ）２（配列番号５４）
ＲＸＤＡ－（ＲＡＤＡ）５－ＲＸＤＡ－ＲＡＤＡ（配列番号５５）
ＲＸＤＡ－（ＲＡＤＡ）６－ＲＸＤＡ（配列番号５６）
ＲＸＤＡ－ＲＡＤＸ－（ＲＡＤＡ）６（配列番号５７）
ＲＸＤＡ－ＲＡＤＡ－ＲＡＤＸ－（ＲＡＤＡ）５（配列番号５８）
ＲＸＤＡ－（ＲＡＤＡ）２－ＲＡＤＸ－（ＲＡＤＡ）４（配列番号５９）
ＲＸＤＡ－（ＲＡＤＡ）３－ＲＡＤＸ－（ＲＡＤＡ）３（配列番号６０）
ＲＸＤＡ－（ＲＡＤＡ）４－ＲＡＤＸ－（ＲＡＤＡ）２（配列番号６１）
ＲＸＤＡ－（ＲＡＤＡ）５－ＲＡＤＸ－ＲＡＤＡ（配列番号６２）
ＲＸＤＡ－（ＲＡＤＡ）６－ＲＡＤＸ（配列番号６３）
ＲＡＤＡ－ＲＸＤＡ－（ＲＡＤＡ）５－ＲＸＤＡ（配列番号６４）
（ＲＡＤＡ）２－ＲＸＤＡ－（ＲＡＤＡ）４－ＲＸＤＡ（配列番号６５）
（ＲＡＤＡ）３－ＲＸＤＡ－（ＲＡＤＡ）３－ＲＸＤＡ（配列番号６６）
（ＲＡＤＡ）４－ＲＸＤＡ－（ＲＡＤＡ）２－ＲＸＤＡ（配列番号６７）
（ＲＡＤＡ）５－ＲＸＤＡ－ＲＡＤＡ－ＲＸＤＡ（配列番号６８）
（ＲＡＤＡ）６－（ＲＸＤＡ）２（配列番号６９）
ＲＡＤＸ－（ＲＡＤＡ）６－ＲＸＤＡ（配列番号７０）
ＲＡＤＡ－ＲＡＤＸ－（ＲＡＤＡ）５－ＲＸＤＡ（配列番号７１）
（ＲＡＤＡ）２－ＲＡＤＸ－（ＲＡＤＡ）４－ＲＸＤＡ（配列番号７２）
（ＲＡＤＡ）３－ＲＡＤＸ－（ＲＡＤＡ）３－ＲＸＤＡ（配列番号７３）
（ＲＡＤＡ）４－ＲＡＤＸ－（ＲＡＤＡ）２－ＲＸＤＡ（配列番号７４）
（ＲＡＤＡ）５－ＲＡＤＸ－ＲＡＤＡ－ＲＸＤＡ（配列番号７５）
（ＲＡＤＡ）６－ＲＡＤＸ－ＲＸＤＡ（配列番号７６）
ＲＡＤＡ－ＲＸＤＡ－（ＲＡＤＡ）５－ＲＡＤＸ（配列番号７７）
（ＲＡＤＡ）２－ＲＸＤＡ－（ＲＡＤＡ）４－ＲＡＤＸ（配列番号７８）
（ＲＡＤＡ）３－ＲＸＤＡ－（ＲＡＤＡ）３－ＲＡＤＸ（配列番号７９）
（ＲＡＤＡ）４－ＲＸＤＡ－（ＲＡＤＡ）２－ＲＡＤＸ（配列番号８０）
（ＲＡＤＡ）５－ＲＸＤＡ－ＲＡＤＡ－ＲＡＤＸ（配列番号８１）
（ＲＡＤＡ）６－ＲＸＤＡ－ＲＡＤＸ（配列番号８２）
ＲＡＤＸ－（ＲＡＤＡ）６－ＲＡＤＸ（配列番号８３）
ＲＡＤＡ－ＲＡＤＸ－（ＲＡＤＡ）５－ＲＡＤＸ（配列番号８４）
（ＲＡＤＡ）２－ＲＡＤＸ－（ＲＡＤＡ）４－ＲＡＤＸ（配列番号８５）
（ＲＡＤＡ）３－ＲＡＤＸ－（ＲＡＤＡ）３－ＲＡＤＸ（配列番号８６）
（ＲＡＤＡ）４－ＲＡＤＸ－（ＲＡＤＡ）２－ＲＡＤＸ（配列番号８７）
（ＲＡＤＡ）５－ＲＡＤＸ－ＲＡＤＡ－ＲＡＤＸ（配列番号８８）
（ＲＡＤＡ）６－（ＲＡＤＸ）２（配列番号８９）
（ＲＡＤＡ）７（配列番号９０）
（ＲＡＤＡ）８（配列番号９１）
Specific preferred examples of self-assembling peptides include peptides having the following amino acid sequences: In the following sequences, X is a glycine residue or a proline residue: When the following amino acid sequences contain two or more Xs, the two or more Xs may be the same amino acid residue or different amino acid residues.
(RADA) 4 (SEQ ID NO: 1)
(RADA) 5 (SEQ ID NO: 2)
(RADA) 6 (SEQ ID NO: 3)
RXDA-(RADA) 3 (SEQ ID NO: 4)
(RADA) 3 -RXDA (SEQ ID NO:5)
(RADA) 3 -RADX (SEQ ID NO: 6)
RXDA-(RADA) 4 (SEQ ID NO:7)
(RADA) 4 -RXDA (SEQ ID NO: 8)
(RADA) 4 -RADX (SEQ ID NO: 9)
RXDA-(RADA) 5 (SEQ ID NO: 10)
(RADA) 5 -RXDA (SEQ ID NO: 11)
(RADA) 5 -RADX (SEQ ID NO: 12)
RXDA-(RADA) 6 (SEQ ID NO: 13)
(RADA) 6 -RXDA (SEQ ID NO: 14)
(RADA) 6 -RADX (SEQ ID NO: 15)
(RXDA) 2 -(RADA) 5 (SEQ ID NO: 16)
RXDA-RADA-RXDA-(RADA) 4 (SEQ ID NO: 17)
RXDA-(RADA) 2 -RXDA-(RADA) 3 (SEQ ID NO: 18)
RXDA-(RADA) 3 -RXDA-(RADA) 2 (SEQ ID NO: 19)
RXDA-(RADA) 4 -RXDA-RADA (SEQ ID NO: 20)
RXDA-(RADA) 5 -RXDA (SEQ ID NO:21)
RXDA-RADX-(RADA) 5 (SEQ ID NO: 22)
RXDA-RADA-RADX-(RADA) 4 (SEQ ID NO: 23)
RXDA-(RADA) 2 -RADX-(RADA) 3 (SEQ ID NO: 24)
RXDA-(RADA) 3 -RADX-(RADA) 2 (SEQ ID NO: 25)
RXDA-(RADA) 4 -RADX-RADA (SEQ ID NO: 26)
RXDA-(RADA) 5 -RADX (SEQ ID NO: 27)
RADA-RXDA-(RADA) 4 -RXDA (SEQ ID NO: 28)
(RADA) 2 -RXDA-(RADA) 3 -RXDA (SEQ ID NO: 29)
(RADA) 3 -RXDA-(RADA) 2 -RXDA (SEQ ID NO: 30)
(RADA) 4 -RXDA-RADA-RXDA (SEQ ID NO: 31)
(RADA) 5 -(RXDA) 2 (SEQ ID NO: 32)
RADX-(RADA) 5 -RXDA (SEQ ID NO: 33)
RADA-RADX-(RADA) 4 -RXDA (SEQ ID NO: 34)
(RADA) 2 -RADX-(RADA) 3 -RXDA (SEQ ID NO: 35)
(RADA) 3 -RADX-(RADA) 2 -RXDA (SEQ ID NO: 36)
(RADA) 4 -RADX-RADA-RXDA (SEQ ID NO: 37)
(RADA) 5 -RADX-RXDA (SEQ ID NO: 38)
RADA-RXDA-(RADA) 4 -RADX (SEQ ID NO: 39)
(RADA) 2 -RXDA-(RADA) 3 -RADX (SEQ ID NO: 40)
(RADA) 3 -RXDA-(RADA) 2 -RADX (SEQ ID NO: 41)
(RADA) 4 -RXDA-RADA-RADX (SEQ ID NO: 42)
(RADA) 5 -RXDA-RADX (SEQ ID NO: 43)
RADX-(RADA) 5 -RADX (SEQ ID NO: 44)
RADA-RADX-(RADA) 4 -RADX (SEQ ID NO: 45)
(RADA) 2 -RADX-(RADA) 3 -RADX (SEQ ID NO: 46)
(RADA) 3 -RADX-(RADA) 2 -RADX (SEQ ID NO: 47)
(RADA) 4 -RADX-RADA-RADX (SEQ ID NO: 48)
(RADA) 5 -(RADX) 2 (SEQ ID NO:49)
(RXDA) 2 -(RADA) 6 (SEQ ID NO:50)
RXDA-RADA-RXDA-(RADA) 5 (SEQ ID NO:51)
RXDA-(RADA) 2 -RXDA-(RADA) 4 (SEQ ID NO:52)
RXDA-(RADA) 3 -RXDA-(RADA) 3 (SEQ ID NO:53)
RXDA-(RADA) 4 -RXDA-(RADA) 2 (SEQ ID NO:54)
RXDA-(RADA) 5 -RXDA-RADA (SEQ ID NO:55)
RXDA-(RADA) 6 -RXDA (SEQ ID NO:56)
RXDA-RADX-(RADA) 6 (SEQ ID NO:57)
RXDA-RADA-RADX-(RADA) 5 (SEQ ID NO:58)
RXDA-(RADA) 2 -RADX-(RADA) 4 (SEQ ID NO:59)
RXDA-(RADA) 3 -RADX-(RADA) 3 (SEQ ID NO: 60)
RXDA-(RADA) 4 -RADX-(RADA) 2 (SEQ ID NO:61)
RXDA-(RADA) 5 -RADX-RADA (SEQ ID NO:62)
RXDA-(RADA) 6 -RADX (SEQ ID NO:63)
RADA-RXDA-(RADA) 5 -RXDA (SEQ ID NO:64)
(RADA) 2 -RXDA-(RADA) 4 -RXDA (SEQ ID NO: 65)
(RADA) 3 -RXDA-(RADA) 3 -RXDA (SEQ ID NO: 66)
(RADA) 4 -RXDA-(RADA) 2 -RXDA (SEQ ID NO: 67)
(RADA) 5 -RXDA-RADA-RXDA (SEQ ID NO: 68)
(RADA) 6 -(RXDA) 2 (SEQ ID NO:69)
RADX-(RADA) 6 -RXDA (SEQ ID NO: 70)
RADA-RADX-(RADA) 5 -RXDA (SEQ ID NO:71)
(RADA) 2 -RADX-(RADA) 4 -RXDA (SEQ ID NO: 72)
(RADA) 3 -RADX-(RADA) 3 -RXDA (SEQ ID NO: 73)
(RADA) 4 -RADX-(RADA) 2 -RXDA (SEQ ID NO: 74)
(RADA) 5 -RADX-RADA-RXDA (SEQ ID NO: 75)
(RADA) 6 -RADX-RXDA (SEQ ID NO: 76)
RADA-RXDA-(RADA) 5 -RADX (SEQ ID NO: 77)
(RADA) 2 -RXDA-(RADA) 4 -RADX (SEQ ID NO: 78)
(RADA) 3 -RXDA-(RADA) 3 -RADX (SEQ ID NO: 79)
(RADA)4-RXDA-(RADA) 2- RADX (SEQ ID NO: 80)
(RADA) 5 -RXDA-RADA-RADX (SEQ ID NO: 81)
(RADA) 6- RXDA-RADX (SEQ ID NO: 82)
RADX-(RADA) 6 -RADX (SEQ ID NO: 83)
RADA-RADX-(RADA) 5 -RADX (SEQ ID NO: 84)
(RADA) 2 -RADX-(RADA) 4 -RADX (SEQ ID NO: 85)
(RADA) 3 -RADX-(RADA) 3 -RADX (SEQ ID NO: 86)
(RADA) 4 -RADX-(RADA) 2 -RADX (SEQ ID NO: 87)
(RADA) 5 -RADX-RADA-RADX (SEQ ID NO: 88)
(RADA) 6 -(RADX) 2 (SEQ ID NO:89)
(RADA) 7 (SEQ ID NO:90)
(RADA) 8 (SEQ ID NO:91)
以上説明した自己組織化ペプチドの中では、（ＲＡＤＡ）４が好ましい。本明細書においては、（ＲＡＤＡ）４をＲＡＤＡ１６とも記す。 Among the self-assembling peptides described above, (RADA) 4 is preferred. In this specification, (RADA) 4 is also referred to as RADA16.
自己組織化ペプチドは、周知のペプチド合成方法により合成し得る。ペプチドの合成方法は、化学的方法であっても、遺伝子工学的な方法であってもよい。自己組織化ペプチドの合成方法は、各種文献に記載されている（Ｉｓｈｉｄａ ｅｔ．ａｌ．，Ｃｈｅｍ．Ｅｕｒ．Ｊ．２０１９，２５，１３５２３－１３５３０；Ｐｅｐｔｉｄｅ Ｓｙｎｔｈｅｓｉｓ ａｎｄ Ｓｅｌｆ－Ａｓｓｅｍｂｌｙ, Ａ．Ａｇｇｅｌｉ ｅｔ．ａｌ．， Ｃｈａｐｔｅｒ Ｆｉｒｓｔ Ｏｎｌｉｎｅ：２５ Ｏｃｔｏｂｅｒ ２０１１，Ｐｅｐｔｉｄｅ－Ｂａｓｅｄ Ｍａｔｅｒｉａｌｓ， ｐｐ２７－６９；Ｄｅｖｅｌｏｐｍｅｎｔｓ ｉｎ ｐｅｐｔｉｄｅ ａｎｄ ａｍｉｄｅ ｓｙｎｔｈｅｓｉｓ， Ｆｅｒｎａｎｄｏ Ａｌｂｅｒｉｃｉｏ， Ｃｕｒｒｅｎｔ Ｏｐｉｎｉｏｎ ｉｎ Ｃｈｅｍｉｃａｌ Ｂｉｏｌｏｇｙ，Ｖｏｌｕｍｅ ８，Ｉｓｓｕｅ ３，Ｊｕｎｅ ２００４，Ｐａｇｅ ２１１－２２１；Ｐｅｐｔｉｄｅ ｓｙｎｔｈｅｓｉｓ： Ｃｈｅｍｉｃａｌ ｏｒ ｅｎｚｙｍａｔｉｃ， Ｅｌｅｃｔｒｏｎ． Ｊ． Ｂｉｏｔｅｃｈｎｏｌ．, ２００７；１０：２７９－３１４；等を参照）。
The self-assembling peptides can be synthesized by known peptide synthesis methods. The peptide synthesis method may be a chemical method or a genetic engineering method. The synthesis method of the self-assembling peptides is described in various documents (Ishida et. al., Chem. Eur. J. 2019, 25, 13523-13530; Peptide Synthesis and Self-Assembly, A. Aggeli et. al., Chapter First Online: 25 October 2011, Peptide-Based Materials, pp27-69; Developments in p eptide and amide synthesis, Fernando Albericio, Current Opinion in Chemical Biology,
ハイドロゲルは、１種の自己組織化ペプチドを含んでいてもよく、２種以上の自己組織化ペプチドを組み合わせて含んでいてもよい。 The hydrogel may contain one type of self-assembling peptide, or may contain a combination of two or more types of self-assembling peptides.
ハイドロゲルにおける自己組織化ペプチドの含有量は、所望する効果が損なわれない限り特に限定されない。ハイドロゲルにおける自己組織化ペプチドの含有量は、ハイドロゲルの総質量に対して、０．１質量％以上３．０質量％以下が好ましく、０．２質量％以上１．０質量％以下がより好ましい。自己組織化ペプチドの含有量が上記の範囲内であることにより、自己組織化構造としてのβシート構造の形成が顕著であり、ハイドロゲルの自己修復性が良好であり、細胞とハイドロゲルとを剪断力をかけつつインジェクトする際の細胞死が起こりにくい。 The content of the self-assembling peptide in the hydrogel is not particularly limited as long as the desired effect is not impaired. The content of the self-assembling peptide in the hydrogel is preferably 0.1% by mass or more and 3.0% by mass or less, and more preferably 0.2% by mass or more and 1.0% by mass or less, relative to the total mass of the hydrogel. When the content of the self-assembling peptide is within the above range, the formation of a β-sheet structure as a self-assembling structure is remarkable, the self-repairing property of the hydrogel is good, and cell death is unlikely to occur when cells and the hydrogel are injected while applying a shear force.
＜網目状ポリマー＞
網目状ポリマーは、親水性ポリマーに由来する親水性ポリマーブロックと、ポリエチレングリコールに由来するポリエチレングリコールブロックとを有する網目状のポリマーである。
<Network polymer>
The network polymer is a network polymer having hydrophilic polymer blocks derived from a hydrophilic polymer and polyethylene glycol blocks derived from polyethylene glycol.
親水性ポリマーブロックと、ポリエチレングリコールブロックとは、－Ｃ＝Ｎ－ＮＨ－で表されるヒドラゾン結合を含む２価の基により結合している。上記のヒドラゾン結合は、アルデヒド基と、カルボン酸ヒドラジド基との反応により形成される。ヒドラゾン結合の生成反応は可逆的な反応である。このため、網目状ポリマーを含むハイドロゲルにおいて、網目状ポリマー中のヒドラゾン結合を含む架橋部分が開裂したとしても、ヒドラゾン結合が自然に再形成される。このことによって、ハイドロゲルは、自己修復性を示す。 The hydrophilic polymer block and the polyethylene glycol block are bonded by a divalent group containing a hydrazone bond represented by -C=N-NH-. The above hydrazone bond is formed by the reaction between an aldehyde group and a carboxylic acid hydrazide group. The reaction that forms the hydrazone bond is a reversible reaction. Therefore, in a hydrogel containing a network polymer, even if the crosslinking portion containing the hydrazone bond in the network polymer is cleaved, the hydrazone bond will naturally be reformed. This allows the hydrogel to exhibit self-repairing properties.
再生医療における喫緊の課題は、再生臓器の周囲に適切に血液を灌流させ、酸素・栄養の運搬を担う微小血管系を作り出すことである。血管新生は自己修復という生体組織の基本的特性の１つである。このため、可逆的架橋を有する動的自己修復ハイドロゲルは、血管新生のために魅力的な環境を提供する材料として期待されている。 The most pressing issue in regenerative medicine is the creation of a microvascular system that can adequately perfuse blood around regenerated organs and transport oxygen and nutrients. Angiogenesis is one of the fundamental properties of biological tissues, known as self-repair. For this reason, dynamic self-repairing hydrogels with reversible crosslinks are expected to be a material that provides an attractive environment for angiogenesis.
自己修復性を有するハイドロゲルを細胞培養に用いる場合、ハイドロゲルが破壊と修復とを繰り返しつつ培養条件下で長期間に亘って安定して存在できるとともに、ハイドロゲル内での細胞の遊走と、栄養素の運搬とが促進される。さらに、可逆結合を有する網目状ポリマーを含むＩＰＮ構造を有するハイドロゲルは、ゾル－ゲル転移により、機械的強度を自己修復する急速な回復レオロジー特性を示す。その結果、上記の網目状ポリマーと自己組織化ペプチドとを含むＩＰＮ構造を有するハイドロゲルは、インジェクタブルゲルとなる。そして、ＩＰＮ構造を有する上記のハイドロゲルと細胞とを、剪断力をかけつつインジェクトする場合、上記の回復レオロジー特性に基づく剪断応力の吸収緩和によって、細胞死が有意に減少する。回復レオロジー特性に基づく上記の機能には、主にペプチドのβシート構造が示す自己修復性が寄与している。 When a hydrogel with self-repairing properties is used for cell culture, the hydrogel can stably exist for a long period of time under culture conditions while repeatedly being destroyed and repaired, and cell migration and nutrient transport within the hydrogel are promoted. Furthermore, a hydrogel with an IPN structure containing a reversible bond-containing mesh polymer exhibits rapid recovery rheological properties that self-repair mechanical strength through sol-gel transition. As a result, a hydrogel with an IPN structure containing the above-mentioned mesh polymer and self-assembling peptide becomes an injectable gel. When the above-mentioned hydrogel with an IPN structure and cells are injected while applying a shear force, cell death is significantly reduced due to the absorption and relaxation of shear stress based on the above-mentioned recovery rheological properties. The self-repairing properties of the β-sheet structure of the peptide mainly contribute to the above-mentioned functions based on the recovery rheological properties.
網目状ポリマーは、ヒドラジド官能化親水性ポリマーと、アルデヒド官能化ポリエチレングリコールとの反応物であるか、アルデヒド官能化親水性ポリマーと、ヒドラジド官能化ポリエチレングリコールとの反応物であることが好ましい。網目状ポリマーは、ヒドラジド官能化親水性ポリマーと、アルデヒド官能化ポリエチレングリコールとの反応物であることがより好ましい。 The network polymer is preferably a reaction product of a hydrazide-functionalized hydrophilic polymer and an aldehyde-functionalized polyethylene glycol, or a reaction product of an aldehyde-functionalized hydrophilic polymer and a hydrazide-functionalized polyethylene glycol. More preferably, the network polymer is a reaction product of a hydrazide-functionalized hydrophilic polymer and an aldehyde-functionalized polyethylene glycol.
なお、ヒドラジド官能化親水性ポリマー及びアルデヒド官能化親水性ポリマーと、アルデヒド官能化ポリエチレングリコールとを反応させて、網目状ポリマーを得てもよい。或いは、ヒドラジド官能化親水性ポリマー及びアルデヒド官能化親水性ポリマーと、ヒドラジド官能化ポリエチレングリコールとを反応させて、網目状ポリマーを得てもよい。或いは、ヒドラジド官能化親水性ポリマー及びアルデヒド官能化親水性ポリマーと、アルデヒド官能化ポリエチレングリコール及びヒドラジド官能化ポリエチレングリコールとを反応させて、網目状ポリマーを得てもよい。或いは、ヒドラジド官能化親水性ポリマーと、アルデヒド官能化ポリエチレングリコール及びヒドラジド官能化ポリエチレングリコールとを反応させて、網目状ポリマーを得てもよい。或いは、アルデヒド官能化親水性ポリマーと、アルデヒド官能化ポリエチレングリコール及びヒドラジド官能化ポリエチレングリコールとを反応させて、網目状ポリマーを得てもよい。 A network polymer may be obtained by reacting a hydrazide-functionalized hydrophilic polymer and an aldehyde-functionalized hydrophilic polymer with an aldehyde-functionalized polyethylene glycol. Alternatively, a network polymer may be obtained by reacting a hydrazide-functionalized hydrophilic polymer and an aldehyde-functionalized hydrophilic polymer with a hydrazide-functionalized polyethylene glycol. Alternatively, a network polymer may be obtained by reacting a hydrazide-functionalized hydrophilic polymer and an aldehyde-functionalized hydrophilic polymer with an aldehyde-functionalized polyethylene glycol and a hydrazide-functionalized polyethylene glycol. Alternatively, a network polymer may be obtained by reacting a hydrazide-functionalized hydrophilic polymer with an aldehyde-functionalized polyethylene glycol and a hydrazide-functionalized polyethylene glycol. Alternatively, a network polymer may be obtained by reacting an aldehyde-functionalized hydrophilic polymer with an aldehyde-functionalized polyethylene glycol and a hydrazide-functionalized polyethylene glycol.
ヒドラジド官能化又はアルデヒド官能化された親水性ポリマーと、ヒドラジド官能化又はアルデヒド官能化されたポリエチレングリコールとから網目状ポリマーを形成する際の反応条件は特に限定されない。典型的には、例えば、０℃～４０℃程度の室温付近の温度で、ヒドラジド官能化又はアルデヒド官能化された親水性ポリマーと、ヒドラジド官能化又はアルデヒド官能化されたポリエチレングリコールとを混合することで、網目状ポリマーが生成する。 The reaction conditions for forming a network polymer from a hydrazide- or aldehyde-functionalized hydrophilic polymer and a hydrazide- or aldehyde-functionalized polyethylene glycol are not particularly limited. Typically, the network polymer is produced by mixing a hydrazide- or aldehyde-functionalized hydrophilic polymer with a hydrazide- or aldehyde-functionalized polyethylene glycol at a temperature near room temperature, for example, about 0°C to 40°C.
前述のとおり、自己組織化ペプチドは、疎水性相互作用や水素結合により自己組織化して、網目状に物理架橋される。網目状に物理架橋された自己組織化ペプチドの存在下に、網目状ポリマーを形成させることで、相互に侵入した状態で、自己組織化ペプチドと、網目状ポリマーとを含むハイドロゲルが得られる。 As mentioned above, the self-assembling peptides self-assemble through hydrophobic interactions and hydrogen bonds, and are physically cross-linked into a mesh-like structure. By forming a mesh-like polymer in the presence of the self-assembling peptides that are physically cross-linked into a mesh-like structure, a hydrogel is obtained that contains the self-assembling peptides and the mesh-like polymers in a mutually interpenetrating state.
ヒドラジド官能化とは、カルボン酸ヒドラジド基（－ＣＯ－ＮＨ－ＮＨ２）が導入される変性である。アルデヒド官能化とは、アルデヒド基（－ＣＨＯ）が導入される変性である。ここで、アルデヒド官能化ポリエチレングリコールは、直鎖状分子鎖の両末端にアルデヒド基を有するポリエチレングリコール誘導体である。ヒドラジド官能化ポリエチレングリコールは、直鎖状分子鎖の両末端にカルボン酸ヒドラジド基を有するポリエチレングリコール誘導体である。上記のとおり、アルデヒド官能化ポリエチレングリコール及びヒドラジド官能化ポリエチレングリコールは、直鎖状分子鎖の両末端に反応性の官能基を有する。このため、網目状ポリマーが形成されるためには、ヒドラジド官能化親水性ポリマーが１つの分子鎖において３以上のカルボン酸ヒドラジド基を有するか、アルデヒド官能化親水性ポリマーが、１つの分子鎖において３以上のアルデヒド基を有する必要がある。 Hydrazide functionalization is a modification in which a carboxylic acid hydrazide group (-CO-NH-NH 2 ) is introduced. Aldehyde functionalization is a modification in which an aldehyde group (-CHO) is introduced. Here, the aldehyde-functionalized polyethylene glycol is a polyethylene glycol derivative having an aldehyde group at both ends of a linear molecular chain. The hydrazide-functionalized polyethylene glycol is a polyethylene glycol derivative having a carboxylic acid hydrazide group at both ends of a linear molecular chain. As described above, the aldehyde-functionalized polyethylene glycol and the hydrazide-functionalized polyethylene glycol have reactive functional groups at both ends of a linear molecular chain. Therefore, in order to form a network polymer, it is necessary that the hydrazide-functionalized hydrophilic polymer has three or more carboxylic acid hydrazide groups in one molecular chain, or that the aldehyde-functionalized hydrophilic polymer has three or more aldehyde groups in one molecular chain.
なお、本明細書において、網目状ポリマーを構成する各ブロックは、ヒドラゾン結合（－Ｃ＝Ｎ－ＮＨ－）中の炭素－窒素二重結合の部分において区分けされる。例えば、アルデヒド官能化ポリエチレングリコールに由来するポリエチレングリコールブロックは、（＝Ｃ－ＰＥＧＡ－Ｃ＝）として表される。ここで、ＰＥＧＡは、アルデヒド官能化ポリエチレングリコールから２つのアルデヒド基を除いた残基である。また、ヒドラジド官能化ポリエチレングリコールに由来するポリエチレングリコールブロックは、（＝Ｎ－ＮＨ２－ＣＯ－ＰＥＧＨ－ＣＯ－ＮＨ２－Ｎ＝）として表される。ここで、ＰＥＧＨは、ヒドラジド官能化ポリエチレングリコールから２つのカルボン酸ヒドラジド基を除いた残基である。 In this specification, each block constituting the network polymer is distinguished by the carbon-nitrogen double bond in the hydrazone bond (-C=N-NH-). For example, a polyethylene glycol block derived from an aldehyde-functionalized polyethylene glycol is represented as (=C-PEG A -C=), where PEG A is a residue obtained by removing two aldehyde groups from an aldehyde-functionalized polyethylene glycol. Also, a polyethylene glycol block derived from a hydrazide-functionalized polyethylene glycol is represented as (=N-NH 2 -CO-PEG H -CO-NH 2 -N=), where PEG H is a residue obtained by removing two carboxylic acid hydrazide groups from a hydrazide-functionalized polyethylene glycol.
網目状ポリマーは、所望する効果が損なわれない範囲で、親水性ポリマーブロック及びポリエチレングリコールブロック以外の他のブロックを含んでいてもよい。他のブロックとしては、例えば、ヒドラジド官能化又はアルデヒド官能化されたペプチドに由来するブロック；ヒドラジド官能化又はアルデヒド官能化された、後述する親水性ポリマーの定義に当てはまらない多糖に由来するブロック；ヒドラジド官能化又はアルデヒド官能化されたシリコーン樹脂に由来するブロック；ヒドラジド官能化又はアルデヒド官能化されたフッ素樹脂に由来するブロック；ヒドラジド官能化又はアルデヒド官能化されたポリエステル樹脂に由来するブロック；ヒドラジド官能化又はアルデヒド官能化されたポリアミド樹脂に由来するブロック；等が挙げられる。他のブロックは、上記のブロックには限定されない。 The network polymer may contain other blocks other than the hydrophilic polymer block and the polyethylene glycol block, so long as the desired effect is not impaired. Examples of other blocks include blocks derived from hydrazide- or aldehyde-functionalized peptides; blocks derived from hydrazide- or aldehyde-functionalized polysaccharides that do not fall under the definition of hydrophilic polymers described below; blocks derived from hydrazide- or aldehyde-functionalized silicone resins; blocks derived from hydrazide- or aldehyde-functionalized fluororesins; blocks derived from hydrazide- or aldehyde-functionalized polyester resins; blocks derived from hydrazide- or aldehyde-functionalized polyamide resins; and the like. The other blocks are not limited to the above blocks.
網目状ポリマーにおける他のブロックの比率は、網目状ポリマーの質量に対して、２０質量％以下が好ましく、１０質量％以下がより好ましく、５質量％以下がさらに好ましい。網目状ポリマーが、他のブロックを含まないのが最も好ましい。 The ratio of other blocks in the network polymer is preferably 20% by mass or less, more preferably 10% by mass or less, and even more preferably 5% by mass or less, based on the mass of the network polymer. It is most preferable that the network polymer does not contain other blocks.
以下、親水性ポリマーブロック及びポリエチレングリコールブロックについて説明する。 The hydrophilic polymer block and polyethylene glycol block are explained below.
〔親水性ポリマーブロック〕
親水性ポリマーブロックは、親水性ポリマーに由来するブロックである。より具体的には、親水性ポリマーブロックは、ヒドラジド官能化又はアルデヒド官能化された親水性ポリマーに由来するブロックである。網目状ポリマーは、２種以上の親水性ポリマーに由来する、異なる親水性ポリマーブロックを含んでいてもよい。
[Hydrophilic Polymer Block]
The hydrophilic polymer block is a block derived from a hydrophilic polymer. More specifically, the hydrophilic polymer block is a block derived from a hydrazide- or aldehyde-functionalized hydrophilic polymer. The network polymer may contain different hydrophilic polymer blocks derived from two or more hydrophilic polymers.
親水性ポリマーとは、水を加えて平衡膨潤化させた場合に、水の質量比率として５０質量％以上９９質量％以下の含水率を示すポリマーである。平衡膨潤率は、ゲルの乾燥質量Ｍｄと、平衡膨潤化させたゲルの重量Ｍｗとから、下記式に基づいて算出される。Ｍｗは、２５℃で、ゲルを水中で平衡膨潤化させた際の、平衡膨潤化したゲルの質量である。
含水率（質量％）＝（Ｍｗ－Ｍｄ）／Ｍｗ×１００
A hydrophilic polymer is a polymer that exhibits a water content of 50% by mass or more and 99% by mass or less as the mass ratio of water when swollen to equilibrium by adding water. The equilibrium swelling ratio is calculated from the dry mass Md of the gel and the weight Mw of the gel swollen to equilibrium based on the following formula. Mw is the mass of the gel swollen to equilibrium when the gel is swollen to equilibrium in water at 25°C.
Moisture content (mass%) = (Mw - Md) /
網目状ポリマーが上記の親水性ポリマーブロックを含むことにより、自己組織化ペプチドの自己組織化が促進される。特に、自己組織化ペプチドによるβシート構造の形成が促進される。 The inclusion of the hydrophilic polymer block in the mesh polymer promotes the self-assembly of the self-assembling peptide. In particular, the formation of a β-sheet structure by the self-assembling peptide is promoted.
網目状ポリマーが親水性を示すことによる自己組織化ペプチドの自己組織化（構造化）の促進については、各種文献に記載されている（Ｄａｉｄｏｎｅ，Ｉｓａｂｅｌｌａ，Ｍａｒｔｉｎ Ｂ． Ｕｌｍｓｃｈｎｅｉｄｅｒ，Ａｌｆｒｅｄｏ Ｄｉ Ｎｏｌａ，Ａｎｄｒｅａ Ａｍａｄｅｉ， ａｎｄ Ｊｅｒｅｍｙ Ｃ． Ｓｍｉｔｈ．“Ｄｅｈｙｄｒａｔｉｏｎ－Ｄｒｉｖｅｎ Ｓｏｌｖｅｎｔ Ｅｘｐｏｓｕｒｅ ｏｆ Ｈｙｄｒｏｐｈｏｂｉｃ Ｓｕｒｆａｃｅｓ ａｓ ａ Ｄｒｉｖｉｎｇ Ｆｏｒｃｅ ｉｎ Ｐｅｐｔｉｄｅ Ｆｏｌｄｉｎｇ．” Ｐｒｏｃｅｅｄｉｎｇｓ ｏｆ ｔｈｅ Ｎａｔｉｏｎａｌ Ａｃａｄｅｍｙ ｏｆ Ｓｃｉｅｎｃｅｓ ｏｆ ｔｈｅ Ｕｎｉｔｅｄ Ｓｔａｔｅｓ ｏｆ Ａｍｅｒｉｃａ １０４，ｎｏ．３９（２００７）：１５２３０－３５；Ｓｍｉｔａ Ｍｕｋｈｅｒｊｅｅ， Ｐｒａｍｉｔ Ｃｈｏｗｄｈｕｒｙ， ａｎｄ Ｆｅｎｇ Ｇａｉ.“Ｅｆｆｅｃｔ ｏｆ Ｄｅｈｙｄｒａｔｉｏｎ ｏｎ ｔｈｅ Ａｇｇｒｅｇａｔｉｏｎ Ｋｉｎｅｔｉｃｓ ｏｆ Ｔｗｏ Ａｍｙｌｏｉｄ Ｐｅｐｔｉｄｅｓ” Ｔｈｅ Ｊｏｕｒｎａｌ ｏｆ Ｐｈｙｓｉｃａｌ Ｃｈｅｍｉｓｔｒｙ Ｂ ２００９ １１３（２），５３１－５３５；等を参照）。 The promotion of self-assembly (structuring) of self-assembling peptides by the hydrophilicity of mesh polymers has been described in various publications (Daidone, Isabella, Martin B. Ulmschneider, Alfredo Di Nola, Andrea Amadei, and Jeremy C. Smith. "Dehydration-Driven Solvent Exposure of Hydrophobic Surfaces as a Driving Force in Peptide Folding." Proceedings of the National Academy of Sciences of the United States of America 104, no. 39 (2007): 15230-35; Smita Mukherjee, Pramit Chowdhury, and Feng Gai. “Effect of Dehydration on the A ggregation Kinetics of Two Amyloid Peptides” The Journal of Physical Chemistry B 2009 113 (2), 53 1-535; etc.).
親水性ポリマーブロックの分子量は、重量平均分子量として、１００，０００以上１，６００，０００以下が好ましく、１００，０００以上７００，０００以下がより好ましく、２００，０００以上５００，０００以下がさらに好ましい。親水性ポリマーブロックの分子量が小さいほど、官能化された親水性ポリマーと、官能化されたポリエチレングリコールとから網目状ポリマーを形成させる際の、網目状ポリマーの生成速度が速くなる。 The molecular weight of the hydrophilic polymer block is preferably 100,000 or more and 1,600,000 or less, more preferably 100,000 or more and 700,000 or less, and even more preferably 200,000 or more and 500,000 or less, in terms of weight average molecular weight. The smaller the molecular weight of the hydrophilic polymer block, the faster the rate of formation of the network polymer when the network polymer is formed from the functionalized hydrophilic polymer and the functionalized polyethylene glycol.
上記の含水率を示すポリマーとして、公知の種々の親水性ポリマーを用いることができる。上記の含水率を示すポリマーのいくつかの例として、ポリビニルアルカノエートの部分けん化物、ウロン酸単位を含む多糖、ゼラチン、コラーゲン、フィブロイン等が挙げられる。これらの親水性ポリマーは、公知の方法に従い、アルデヒド官能化又はヒドラジド官能され、網目状ポリマーの形成に用いられる。 Various known hydrophilic polymers can be used as the polymer exhibiting the above water content. Some examples of polymers exhibiting the above water content include partially saponified polyvinyl alkanoates, polysaccharides containing uronic acid units, gelatin, collagen, fibroin, etc. These hydrophilic polymers are aldehyde- or hydrazide-functionalized according to known methods and used to form a network polymer.
ポリビニルアルカノエートの部分けん化物について、入手の容易性や、上記の含水率の点で、ポリ酢酸ビニルの部分けん化物が好ましい。ポリビニルアルカノエートの部分けん化物の含水率は、けん化度を調整したり、ポリビニルアルコールの分子量を調整したりすることにより調整できる。 As for the partially saponified polyvinyl alkanoate, the partially saponified polyvinyl acetate is preferred in terms of ease of availability and the above-mentioned water content. The water content of the partially saponified polyvinyl alkanoate can be adjusted by adjusting the degree of saponification or the molecular weight of polyvinyl alcohol.
ポリビニルアルカノエートをアルデヒド官能化又はヒドラジド官能化する方法は特に限定されない。ポリビニルアルカノエートの部分けん化物は、アルコール性水酸基を有する。例えば、アルコール性水酸基と、アルデヒド基を有するカルボン酸とを反応させてエステル化したり、アルコール性水酸基を、アルデヒド基とハロゲン原子とを有する化合物を用いてＷｉｌｌｉａｍｓｏｎのエーテル合成等の方法によりエーテル化したりすることにより、ポリビニルアルカノエートの部分けん化物にアルデヒド基を導入できる。なお、上記の方法は、ポリビニルアルカノエートの部分けん化物にアルデヒド基を導入する方法の一例である。ポリビニルアルカノエートの部分けん化物にアルデヒド基を導入する方法は上記の方法に限定されない。 The method for functionalizing polyvinyl alkanoate with an aldehyde or hydrazide is not particularly limited. A partially saponified polyvinyl alkanoate has an alcoholic hydroxyl group. For example, an aldehyde group can be introduced into a partially saponified polyvinyl alkanoate by reacting the alcoholic hydroxyl group with a carboxylic acid having an aldehyde group to esterify it, or by etherifying the alcoholic hydroxyl group using a compound having an aldehyde group and a halogen atom by a method such as Williamson's ether synthesis. The above method is one example of a method for introducing an aldehyde group into a partially saponified polyvinyl alkanoate. The method for introducing an aldehyde group into a partially saponified polyvinyl alkanoate is not limited to the above method.
アルコール性水酸基と、ジカルボン酸無水物とを反応させたり、アルコール性水酸基と、ジカルボン酸とを縮合させてエステル化したりすることにより、ポリビニルアルカノエートの部分けん化物にカルボキシ基を導入できる。ジカルボン酸無水物としては、例えば、コハク酸無水物、グルタル酸無水物、アジピン酸無水物、マレイン酸無水物等の鎖状脂肪族ジカルボン酸の無水物を用いることができる。ジカルボン酸としては、マロン酸、コハク酸、グルタル酸、アジピン酸、マレイン酸、フマル酸等の鎖状脂肪族ジカルボン酸を用いることができる。 Carboxy groups can be introduced into the partially saponified polyvinyl alkanoate by reacting the alcoholic hydroxyl group with a dicarboxylic anhydride, or by condensing and esterifying the alcoholic hydroxyl group with a dicarboxylic acid. As the dicarboxylic anhydride, for example, anhydrides of chain aliphatic dicarboxylic acids such as succinic anhydride, glutaric anhydride, adipic anhydride, and maleic anhydride can be used. As the dicarboxylic acid, chain aliphatic dicarboxylic acids such as malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, and fumaric acid can be used.
ポリビニルアルカノエートの部分けん化物に導入されたカルボキシ基と、ジヒドラジド化合物とを反応させることにより、ポリビニルアルカノエートの部分けん化物にヒドラジド基を導入できる。ジヒドラジド化合物としては、カルボヒドラジド、マロン酸ジヒドラジド、コハク酸ジヒドラジド、グルタル酸ジヒドラジド、アジピン酸ジヒドラジド、マレイン酸ジヒドラジド、フマル酸ジヒドラジド等の鎖状脂肪族ジカルボン酸ジヒドラジドを用いることができる。 Hydrazide groups can be introduced into the partially saponified polyvinyl alkanoate by reacting the carboxyl groups introduced into the partially saponified polyvinyl alkanoate with a dihydrazide compound. As the dihydrazide compound, chain aliphatic dicarboxylic acid dihydrazides such as carbohydrazide, malonic acid dihydrazide, succinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, maleic acid dihydrazide, and fumaric acid dihydrazide can be used.
次いで、ウロン酸単位を含む多糖について説明する。
ウロン酸単位は、カルボキシ基を有する。カルボキシ基は反応性に富むため、種々の反応によって、ウロン酸単位に、ヒドラジド基やアルデヒド基を導入できる。ウロン酸単位を含む多糖について、ウロン酸の種類や、ウロン酸単位の含有量は、当該多糖が上記の所定の含水率を示す限り特に限定されない。ウロン酸の具体例としては、グルクロン酸、イズロン酸、及びガラクツロン酸が挙げられる。ウロン酸は、Ｄ体であってもＬ体であってもよい。以上説明したウロン酸としては、Ｄ－グルクロン酸及びＬ－イズロン酸が好ましい。
Next, polysaccharides containing uronic acid units will be described.
The uronic acid unit has a carboxy group. Since the carboxy group is highly reactive, a hydrazide group or an aldehyde group can be introduced into the uronic acid unit by various reactions. With respect to the polysaccharide containing the uronic acid unit, the type of uronic acid and the content of the uronic acid unit are not particularly limited as long as the polysaccharide exhibits the above-mentioned predetermined water content. Specific examples of the uronic acid include glucuronic acid, iduronic acid, and galacturonic acid. The uronic acid may be in the D-form or the L-form. As the uronic acid described above, D-glucuronic acid and L-iduronic acid are preferable.
上記の多糖は、ウロン酸単位とともに、他の単糖に由来する単位を含み得る。他の単糖の具体例としては、Ｄ－グルコース、Ｄ－ガラクトース、Ｄ－マンノース、キシロース、Ｌ－フコース、Ｄ－グルコサミン、Ｄ－アセチルグルコサミン、Ｄ－ガラクトサミン、Ｄ－アセチルガラクトサミン等が挙げられる。これらの他の単糖の中では、上記の多糖の入手が容易である点で、Ｄ－グルコサミン、Ｄ－アセチルグルコサミン、Ｄ－ガラクトサミン、Ｄ－アセチルガラクトサミン等の所謂アミノ等又はその誘導体が好ましい。 The above polysaccharides may contain units derived from other monosaccharides in addition to the uronic acid units. Specific examples of other monosaccharides include D-glucose, D-galactose, D-mannose, xylose, L-fucose, D-glucosamine, D-acetylglucosamine, D-galactosamine, D-acetylgalactosamine, etc. Among these other monosaccharides, so-called amino acids such as D-glucosamine, D-acetylglucosamine, D-galactosamine, D-acetylgalactosamine, etc. or their derivatives are preferred in terms of the ease of obtaining the above polysaccharides.
上記の多糖としては、グリコサミノグリカンが好ましい。グリコサミノグリカンとしては、ヒアルロン酸、コンドロイチン硫酸、デルマタン硫酸、へパラン硫酸、及びヘパリンが挙げられる。これらの中では、ハイドロゲルの炎症誘発性の低さや、線維芽細胞との親和性が高く、コラーゲンの産生が高まることが期待できることから、ヒアルロン酸が好ましい。特に、ヒアルロン酸は、自己組織化ペプチドによるβシート構造の形成を促進させやすい。 Glycosaminoglycans are preferred as the polysaccharides. Examples of glycosaminoglycans include hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin. Among these, hyaluronic acid is preferred because of its low inflammation-inducing properties and high affinity with fibroblasts, which is expected to increase collagen production. In particular, hyaluronic acid is likely to promote the formation of a β-sheet structure by self-assembling peptides.
ウロン酸単位を含む多糖を、ヒドラジド官能化又はアルデヒド官能化する方法は特に限定されない。ウロン酸単位を含む多糖のヒドラジド官能化は、例えば、当該多糖と、ジヒドラジド化合物とを反応させることにより、行うことができる。この反応によれば、当該多糖が有するウロン酸単位中のカルボキシ基と、ヒドラジド基とが縮合し、当該多糖の側鎖の末端に、－ＣＯ－ＮＨ－ＮＨ２で表される基が導入される。ジヒドラジド化合物としては、カルボヒドラジド、マロン酸ジヒドラジド、コハク酸ジヒドラジド、グルタル酸ジヒドラジド、アジピン酸ジヒドラジド、マレイン酸ジヒドラジド、フマル酸ジヒドラジド等の鎖状脂肪族ジカルボン酸ジヒドラジドを用いることができる。 The method of hydrazide-functionalizing or aldehyde-functionalizing a polysaccharide containing uronic acid units is not particularly limited. Hydrazide-functionalizing a polysaccharide containing uronic acid units can be carried out, for example, by reacting the polysaccharide with a dihydrazide compound. According to this reaction, a carboxy group in the uronic acid unit of the polysaccharide and a hydrazide group are condensed, and a group represented by -CO-NH- NH2 is introduced at the end of the side chain of the polysaccharide. As the dihydrazide compound, a chain aliphatic dicarboxylic acid dihydrazide such as carbohydrazide, malonic acid dihydrazide, succinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, maleic acid dihydrazide, and fumaric acid dihydrazide can be used.
ウロン酸単位を含む多糖を、アルデヒド官能化する方法としては、当該多糖を、過ヨウ素酸塩と反応させる方法が挙げられる。この方法によれば、ウロン酸単位中の、－Ｃ（ＯＨ）Ｈ－Ｃ（ＯＨ）Ｈ－で表される結合が酸化的に開裂し、２つのアルデヒド基が生成する。過ヨウ素酸塩としては、例えば、過ヨウ素酸ナトリウム、過ヨウ素酸カリウム等の過ヨウ素酸アルカリ金属塩を用いることができる。 One method for functionalizing a polysaccharide containing uronic acid units with an aldehyde is to react the polysaccharide with a periodate. According to this method, the bond represented by -C(OH)H-C(OH)H- in the uronic acid unit is oxidatively cleaved to generate two aldehyde groups. As the periodate, for example, an alkali metal periodate such as sodium periodate or potassium periodate can be used.
ヒドラジド官能化又はアルデヒド官能化された親水性ポリマーにおける、カルボン酸ヒドラジド基又はアルデヒド基の量は、所望する効果が損なわれない限り特に限定されない。ヒドラジド官能化又はアルデヒド官能化された親水性ポリマーにおける、カルボン酸ヒドラジド基又はアルデヒド基の量は、親水性ポリマー１分子中の平均数として、２０以上２００以下が好ましく、４０以上１００以下がより好ましい。 The amount of carboxylic acid hydrazide groups or aldehyde groups in the hydrazide- or aldehyde-functionalized hydrophilic polymer is not particularly limited as long as the desired effect is not impaired. The amount of carboxylic acid hydrazide groups or aldehyde groups in the hydrazide- or aldehyde-functionalized hydrophilic polymer is preferably 20 to 200, more preferably 40 to 100, as the average number per molecule of the hydrophilic polymer.
網目状ポリマーにおける、親水性ポリマーブロックの質量ＷＨＰと、ポリエチレングリコールブロックの質量ＷＰＥＧとの比率は、ＷＨＰ：ＷＰＥＧとして、２０：８０～８０：２０が好ましく、３０：７０～７０：３０がより好ましく、４０：６０～６０：４０がさらに好ましく、４５：５５～５５：４５が特に好ましい。 In the network polymer, the ratio of the mass W HP of the hydrophilic polymer block to the mass W PEG of the polyethylene glycol block, W HP :W PEG , is preferably from 20:80 to 80:20, more preferably from 30:70 to 70:30, even more preferably from 40:60 to 60:40, and particularly preferably from 45:55 to 55:45.
〔ポリエチレングリコールブロック〕
ポリエチレングリコールブロックは、ポリエチレングリコールに由来するブロックである。より具体的には、ポリエチレングリコールブロックは、ヒドラジド官能化又はアルデヒド官能化されたポリエチレングリコールに由来するブロックである。網目状ポリマーは、２種以上の分子量の異なるポリエチレングリコールに由来する、異なるポリエチレングリコールブロックを含んでいてもよい。
[Polyethylene glycol block]
The polyethylene glycol block is a block derived from polyethylene glycol. More specifically, the polyethylene glycol block is a block derived from hydrazide- or aldehyde-functionalized polyethylene glycol. The network polymer may contain different polyethylene glycol blocks derived from two or more polyethylene glycols of different molecular weights.
ポリエチレングリコールブロックの分子量は、数平均分子量として、２，０００以上４０，０００以下が好ましく、２，０００以上１０，０００以下がより好ましい。 The molecular weight of the polyethylene glycol block is preferably 2,000 or more and 40,000 or less, and more preferably 2,000 or more and 10,000 or less, in terms of number average molecular weight.
ポリエチレングリコールをアルデヒド官能化する方法としては、ポリエチレングリコールの末端水酸基と、アルデヒド基を有するカルボン酸とを縮合させてエステル化する方法が挙げられる。アルデヒド基を有するカルボン酸としては、例えば、テレフタルアルデヒド酸（４－ホルミル安息香酸）、３－ホルミル安息香酸等が挙げられる。また、３，３－ジエトキシプロピル基のようなアセタール保護された基を一方の末端に有するエチレングリコール誘導体を開始剤として、エチレンオキシドをアニオン開環重合した後、得られたポリエチレングリコール誘導体において、アセタール保護された基を脱保護することによって、一方の末端がアルデヒド官能化されたポリエチレングリコールが得られる。一方の末端がアルデヒド官能化されたポリエチレングリコールの水酸基末端を、上記の方法などによりアルデヒド官能化することにより、両末端がアルデヒド官能化されたポリエチレングリコールを得ることができる。 As a method for functionalizing polyethylene glycol with an aldehyde, there can be mentioned a method of condensing the terminal hydroxyl group of polyethylene glycol with a carboxylic acid having an aldehyde group to form an ester. Examples of carboxylic acids having an aldehyde group include terephthalaldehyde acid (4-formylbenzoic acid) and 3-formylbenzoic acid. In addition, an ethylene glycol derivative having an acetal-protected group such as a 3,3-diethoxypropyl group at one end as an initiator is used to carry out anionic ring-opening polymerization of ethylene oxide, and the acetal-protected group in the resulting polyethylene glycol derivative is then deprotected to obtain polyethylene glycol having one end functionalized with an aldehyde. By functionalizing the hydroxyl terminal of polyethylene glycol having one end functionalized with an aldehyde by the above-mentioned method or the like, it is possible to obtain polyethylene glycol having both ends functionalized with an aldehyde.
また、ポリエチレングリコールの両末端にカルボキシ基を導入した後、カルボキシ基を周知の方法で還元してアルデヒド基に変換することによっても、ポリエチレングリコールをアルデヒド官能化できる。例えば、ポリエチレングリコールの末端水酸基と、ジカルボン酸無水物とを反応させたり、末端水酸基と、ジカルボン酸とを縮合させてエステル化したりすることにより、ポリエチレングリコールの両末端にカルボキシ基を導入できる。ジカルボン酸無水物としては、例えば、コハク酸無水物、グルタル酸無水物、アジピン酸無水物、マレイン酸無水物等の鎖状脂肪族ジカルボン酸の無水物を用いることができる。ジカルボン酸としては、マロン酸、コハク酸、グルタル酸、アジピン酸、マレイン酸、フマル酸等の鎖状脂肪族ジカルボン酸を用いることができる。 Also, polyethylene glycol can be functionalized with aldehydes by introducing carboxyl groups to both ends of polyethylene glycol and then reducing the carboxyl groups to aldehyde groups by a known method. For example, carboxyl groups can be introduced to both ends of polyethylene glycol by reacting the terminal hydroxyl groups of polyethylene glycol with a dicarboxylic acid anhydride, or by condensing the terminal hydroxyl groups with a dicarboxylic acid to form an ester. As the dicarboxylic acid anhydride, for example, anhydrides of chain aliphatic dicarboxylic acids such as succinic anhydride, glutaric anhydride, adipic anhydride, and maleic anhydride can be used. As the dicarboxylic acid, chain aliphatic dicarboxylic acids such as malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, and fumaric acid can be used.
ポリエチレングリコールをヒドラジド官能化する方法としては、ポリエチレングリコールの両末端に導入されたカルボキシ基と、ジヒドラジド化合物とを反応させる方法が挙げられる。ジヒドラジド化合物としては、カルボヒドラジド、マロン酸ジヒドラジド、コハク酸ジヒドラジド、グルタル酸ジヒドラジド、アジピン酸ジヒドラジド、マレイン酸ジヒドラジド、フマル酸ジヒドラジド等の鎖状脂肪族ジカルボン酸ジヒドラジドを用いることができる。 A method for functionalizing polyethylene glycol with hydrazide is to react the carboxyl groups introduced at both ends of polyethylene glycol with a dihydrazide compound. As the dihydrazide compound, aliphatic chain dicarboxylic acid dihydrazides such as carbohydrazide, malonic acid dihydrazide, succinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, maleic acid dihydrazide, and fumaric acid dihydrazide can be used.
以上説明したハイドロゲルは、細胞培養用の足場材料の他、人工軟骨、人工筋肉、人工皮膚、創傷治癒剤、及びドラッグデリバリーシステム用のキャリア等の材料等、種々の用途に適用可能である。 The hydrogels described above can be used for a variety of purposes, including as scaffolding materials for cell culture, as well as materials for artificial cartilage, artificial muscle, artificial skin, wound healing agents, and carriers for drug delivery systems.
≪ハイドロゲルを形成するための組成物≫
ハイドロゲルを形成するための組成物は、以上説明したハイドロゲルを形成するために用いられる。組成物は、それぞれ前述の、自己組織化ペプチドと、親水性ポリマーブロックを与える、アルデヒド官能化又はヒドラジド官能化された親水性ポリマーと、ポリエチレングリコールブロックを与える、アルデヒド官能化又はヒドラジド官能化されたポリエチレングリコールとを含む。
<Composition for forming hydrogel>
A composition for forming a hydrogel is used to form the hydrogel described above, the composition comprising the self-assembling peptide, an aldehyde- or hydrazide-functionalized hydrophilic polymer to provide a hydrophilic polymer block, and an aldehyde- or hydrazide-functionalized polyethylene glycol to provide a polyethylene glycol block, each of which is described above.
組成物において、通常、自己組織化ペプチドと、親水性ポリマーブロックを与える、アルデヒド官能化又はヒドラジド官能化された親水性ポリマーと、ポリエチレングリコールブロックを与える、アルデヒド官能化又はヒドラジド官能化されたポリエチレングリコールとは溶解している。組成物に含まれる溶媒としては、水；生理食塩水；細胞培地；血清を含む細胞培地；等が挙げられる。 In the composition, the self-assembling peptide, the aldehyde- or hydrazide-functionalized hydrophilic polymer that provides the hydrophilic polymer block, and the aldehyde- or hydrazide-functionalized polyethylene glycol that provides the polyethylene glycol block are usually dissolved. Solvents contained in the composition include water; saline; cell culture medium; cell culture medium containing serum; etc.
組成物は、２種類以上の液からなる多液型の組成物であってもよい。多液型の組成物に含まれる複数の液は、ハイドロゲルを形成する際に混合される。 The composition may be a multi-liquid composition consisting of two or more liquids. The liquids contained in the multi-liquid composition are mixed when forming the hydrogel.
多液型の組成物を構成する各液の経時安定性が良好であることから、多液型の組成物としては、自己組織化ペプチドを含む第１液と、アルデヒド官能化又はヒドラジド官能化された親水性ポリマーを含む第２液と、アルデヒド官能化又はヒドラジド官能化されたポリエチレングリコールを含む第３液とからなる３液型の組成物が好ましい。 Since each of the liquids constituting the multi-liquid composition has good stability over time, a three-liquid composition consisting of a first liquid containing a self-assembling peptide, a second liquid containing an aldehyde-functionalized or hydrazide-functionalized hydrophilic polymer, and a third liquid containing an aldehyde-functionalized or hydrazide-functionalized polyethylene glycol is preferred as the multi-liquid composition.
組成物における、自己組織化ペプチドの濃度は、特に限定されず、０．０１質量％以上３質量％以下が好ましく、０．０５質量％以上１質量％以下がより好ましく、０．１質量％以上１質量％以下がさらに好ましい。 The concentration of the self-assembling peptide in the composition is not particularly limited, but is preferably from 0.01% to 3% by mass, more preferably from 0.05% to 1% by mass, and even more preferably from 0.1% to 1% by mass.
組成物における、親水性ポリマーブロックを与える、アルデヒド官能化又はヒドラジド官能化された親水性ポリマーの濃度、及びポリエチレングリコールブロックを与える、アルデヒド官能化又はヒドラジド官能化されたポリエチレングリコールの濃度は、それぞれ特に限定されず、０．０１質量％以上２０質量％以下が好ましく、０．０５質量％以上１０質量％以下がより好ましい。 The concentration of the aldehyde- or hydrazide-functionalized hydrophilic polymer that gives the hydrophilic polymer block, and the concentration of the aldehyde- or hydrazide-functionalized polyethylene glycol that gives the polyethylene glycol block in the composition are not particularly limited, but are preferably 0.01% by mass or more and 20% by mass or less, and more preferably 0.05% by mass or more and 10% by mass or less.
組成物が３液型組成物である場合、第１液における自己組織化ペプチドの濃度の好ましい範囲と、第２液におけるアルデヒド官能化又はヒドラジド官能化された親水性ポリマーの濃度の好ましい範囲と、第３液におけるアルデヒド官能化又はヒドラジド官能化された前記ポリエチレングリコールの濃度の好ましい範囲とは、上記の好ましい範囲と同様である。 When the composition is a three-part composition, the preferred ranges of the concentration of the self-assembling peptide in the first part, the preferred ranges of the concentration of the aldehyde- or hydrazide-functionalized hydrophilic polymer in the second part, and the preferred ranges of the concentration of the aldehyde- or hydrazide-functionalized polyethylene glycol in the third part are the same as the preferred ranges described above.
以下、本発明を実施例によりさらに詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.
〔合成例１〕
数平均分子量が１９８６であるポリエチレングリコールの両末端水酸基に対しホルミル安息香酸（テレフタルアルデヒド酸）を縮合反応させてＣＨＯ－ＰＥＧを合成した。反応式を以下に記す。
Synthesis Example 1
CHO-PEG was synthesized by condensation reaction of formylbenzoic acid (terephthalaldehyde acid) with both terminal hydroxyl groups of polyethylene glycol having a number average molecular weight of 1986. The reaction formula is shown below.
具体的には、まず、容量３００ｍＬの二口ナスフラスコに、ポリエチレングリコール（数平均分子量：１９８６、シグマアルドリッチ社製）５．０２ｇ（２．５３ｍｍｏｌ）を加えた。ナスフラスコ内に、ジクロロメタン５ｍＬを加えて、ポリエチレングリコールを溶解させた。次いで、ナスフラスコ内にベンゼン３０ｍＬを加えた後、ナスフラスコの内容物を３時間凍結乾燥させた。 Specifically, first, 5.02 g (2.53 mmol) of polyethylene glycol (number average molecular weight: 1986, manufactured by Sigma-Aldrich) was added to a 300 mL two-necked eggplant flask. 5 mL of dichloromethane was added to the eggplant flask to dissolve the polyethylene glycol. Next, 30 mL of benzene was added to the eggplant flask, and the contents of the eggplant flask were freeze-dried for 3 hours.
乾燥終了後、ナスフラスコ内を窒素置換した。窒素雰囲気下で、ナスフラスコ内にテレフタルアルデヒド酸（東京化成工業（株）製）１．５０２６ｇ（１０．０ｍｍｏｌ）、４－ジメチルアミノピリジン（ＤＭＡＰ、富士フイルム和光純薬（株）製）７８．８０ｇ（０．６４５ｍｍｏｌ）、及び超脱水テトラヒドロフラン（ＴＨＦ、富士フイルム和光純薬（株）製）７５ｍＬを加え、これらを溶解させた。次いで、２－メチル－６－ニトロ安息香酸無水物（ＭＮＢＡ、東京化成工業（株）製）４．３９７６ｇ（１２．７７ｍｍｏｌ）と、超脱水ＴＨＦ１５ｍＬを、ナスフラスコ内に加えた。ナスフラスコの溶液を、１８時間撹拌して、ポリエチレングリコールと、テレフタルアルデヒド酸とを反応させた。反応後の溶液を、エバポレーターで、体積が約２５ｍＬになるまで濃縮した。濃縮された溶液を、氷冷されたジエチルエーテル５００ｍＬ中に滴下して、生成物を沈殿させた。桐山ロートと濾紙（Ｎｏ．５）とを用いて、沈殿を濾過した。濾取された沈殿物をＴＨＦで溶解させ回収した。沈殿及び濾過の操作を３回繰り返した。３回目の濾過後、回収した溶液を濃縮し、濃縮後の残渣にベンゼンを加えた。得られたベンゼン溶液を凍結乾燥して生成物（ＣＨＯ－ＰＥＧ）３．９０２ｇ（収率６８．６％）を得た。 After drying, the atmosphere in the eggplant flask was replaced with nitrogen. Under a nitrogen atmosphere, 1.5026 g (10.0 mmol) of terephthalaldehydic acid (Tokyo Chemical Industry Co., Ltd.), 78.80 g (0.645 mmol) of 4-dimethylaminopyridine (DMAP, Fujifilm Wako Pure Chemical Industries Co., Ltd.), and 75 mL of ultra-dehydrated tetrahydrofuran (THF, Fujifilm Wako Pure Chemical Industries Co., Ltd.) were added to the eggplant flask and dissolved. Next, 4.3976 g (12.77 mmol) of 2-methyl-6-nitrobenzoic anhydride (MNBA, Tokyo Chemical Industry Co., Ltd.) and 15 mL of ultra-dehydrated THF were added to the eggplant flask. The solution in the eggplant flask was stirred for 18 hours to react the polyethylene glycol with the terephthalaldehydic acid. The solution after the reaction was concentrated in an evaporator until the volume was about 25 mL. The concentrated solution was dropped into 500 mL of ice-cold diethyl ether to precipitate the product. The precipitate was filtered using a Kiriyama funnel and filter paper (No. 5). The filtered precipitate was dissolved in THF and recovered. The precipitation and filtration procedures were repeated three times. After the third filtration, the recovered solution was concentrated, and benzene was added to the residue after concentration. The resulting benzene solution was freeze-dried to obtain 3.902 g (yield 68.6%) of the product (CHO-PEG).
乾燥終了後、生成物の収量を測定した。また、１Ｈ ＮＭＲスペクトルにより生成物がＣＨＯ－ＰＥＧであることを確認した。生成物の１Ｈ ＮＭＲスペクトルを、図１に示す。図１に示される生成物（ＣＨＯ－ＰＥＧ）の１Ｈ ＮＭＲスペクトルから、ポリエチレングリコールの両末端へのアルデヒド基の導入率を求めた。具体的には、ポリエチレングリコールのアルキル鎖に相当するプロトンの積分値を１７４とし、両末端の官能基（ｐ－ホルミルフェニルカルボニルオキシ基）の結合部に相当する、図１中ｂのプロトンのピークに基づいて、ポリエチレングリコールの両末端へのアルデヒド基の導入率を求めた。その結果、ポリエチレングリコールの両末端へのアルデヒド基の導入率は、９９％であった。 After drying, the yield of the product was measured. The product was confirmed to be CHO-PEG by 1 H NMR spectrum. The 1 H NMR spectrum of the product is shown in FIG. 1. From the 1 H NMR spectrum of the product (CHO-PEG) shown in FIG. 1, the introduction rate of aldehyde groups into both ends of polyethylene glycol was obtained. Specifically, the integral value of the protons corresponding to the alkyl chain of polyethylene glycol was set to 174, and the introduction rate of aldehyde groups into both ends of polyethylene glycol was obtained based on the peak of protons b in FIG. 1, which corresponds to the bonding parts of the functional groups (p-formylphenylcarbonyloxy groups) at both ends. As a result, the introduction rate of aldehyde groups into both ends of polyethylene glycol was 99%.
〔合成例２〕
下記反応式に従い、カルボジヒドラジド（ＣＨＤ）によりヒドラジド官能化されたヒアルロン酸を得た。
Synthesis Example 2
Hyaluronic acid functionalized with hydrazide was obtained by carbodihydrazide (CHD) according to the following reaction scheme.
容量１００ｍＬのビーカーに、ヒアルロン酸ナトリウム（ＨＡＳ、重量平均分子量：５００，０００～７００，０００、キッコーマンバイオケミファ（株）製）２００ｍｇ（０．５００ｍｍｏｌ）と、純水５０ｍＬとを加え、ヒアルロン酸ナトリウムを純水中に溶解させた。次いで、カルボジヒドラジド（ＣＨＤ、東京化成工業（株）製）１．３６４ｇ（１５．１ｍｍｏｌ）と、１－（３－ジメチルアミノプロピル－３－エチルカルボジイミド塩酸塩（ＥＤＣ、東京化成工業（株）製）３９７．１ｍｇ（２．０７ｍｍｏｌ）とを、フラスコ内にこの順で加え、溶解させた。フラスコ内の溶液のｐＨを、１Ｎ塩酸水溶液により４．７５に調整した。その後、１Ｎ塩酸水溶液を必要に応じて添加しながら、フラスコ内容の溶液のｐＨを４．７～４．８に保持した。１Ｎ塩酸の添加を開始してから、２時間経過後に、フラスコ内の溶液のｐＨを、１Ｎ水酸化ナトリウム水溶液により７．０に調整して反応を終了させた。得られた溶液に対して、透析膜を用いて純水に対する透析を行った。透析された溶液を、１８時間かけて回収し、生成物（ＨＡＳ－ＣＨＤ）２１１．５ｍｇ（収率９０．８％）を得た。１Ｈ ＮＭＲより算出された、ヒアルロン酸の側鎖カルボキシ基のヒドラジド化率は５５％であった。 In a 100 mL beaker, 200 mg (0.500 mmol) of sodium hyaluronate (HA S , weight average molecular weight: 500,000 to 700,000, manufactured by Kikkoman Biochemifa Corp.) and 50 mL of pure water were added, and the sodium hyaluronate was dissolved in the pure water. Next, 1.364 g (15.1 mmol) of carbodihydrazide (CHD, manufactured by Tokyo Chemical Industry Co., Ltd.) and 397.1 mg (2.07 mmol) of 1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride (EDC, manufactured by Tokyo Chemical Industry Co., Ltd.) were added to the flask in this order and dissolved. The pH of the solution in the flask was adjusted to 4.75 with 1N aqueous hydrochloric acid. Thereafter, the pH of the solution in the flask was maintained at 4.7 to 4.8 while adding 1N aqueous hydrochloric acid as necessary. After 2 hours had elapsed since the start of the addition of 1N hydrochloric acid, the pH of the solution in the flask was adjusted to 7.0 with 1N aqueous sodium hydroxide solution to terminate the reaction. The obtained solution was dialyzed against pure water using a dialysis membrane. The dialyzed solution was collected over 18 hours to obtain 211.5 mg (yield 90.8%) of the product (HA S -CHD). 1 H The hydrazide ratio of the side chain carboxy groups of hyaluronic acid calculated by NMR was 55%.
〔合成例３〕
下記反応式に従い、アジピン酸ジヒドラジド（ＡＨＤ）によりヒドラジド官能化されたヒアルロン酸を得た。
[Synthesis Example 3]
Hyaluronic acid functionalized with adipic dihydrazide (AHD) was obtained according to the following reaction scheme:
具体的には、カルボジヒドラジド（ＣＨＤ、東京化成工業（株）製）１．３６４ｇ（１５．１ｍｍｏｌ）を、アジピン酸ジヒドラジド（ＡＨＤ、東京化成工業（株）製）２．６９９ｇ（１５．５ｍｍｏｌ）に変えることと、１－（３－ジメチルアミノプロピル－３－エチルカルボジイミド塩酸塩（ＥＤＣ、東京化成工業（株）製）の使用量を、３９７．１ｍｇ（２．０７ｍｍｏｌ）から３９７．４ｍｇ（２．０７ｍｍｏｌ）に変えることとの他は、合成例２と同様にして、生成物（ＨＡＳ－ＡＨＤ）２２３．７ｍｇ（収率９５．５％）を得た。１Ｈ ＮＭＲより算出された、ヒアルロン酸の側鎖カルボキシ基のヒドラジド化率は６０％であった。 Specifically, 1.364 g (15.1 mmol) of carbodihydrazide (CHD, manufactured by Tokyo Chemical Industry Co., Ltd.) was changed to 2.699 g (15.5 mmol) of adipic acid dihydrazide (AHD, manufactured by Tokyo Chemical Industry Co., Ltd.), and the amount of 1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride (EDC, manufactured by Tokyo Chemical Industry Co., Ltd.) was changed from 397.1 mg (2.07 mmol) to 397.4 mg (2.07 mmol), but the same procedure as in Synthesis Example 2 was used to obtain 223.7 mg (yield 95.5%) of product (HA S -AHD). The hydrazide conversion rate of the side chain carboxyl group of hyaluronic acid calculated from 1 H NMR was 60%.
〔合成例４〕
ヒアルロン酸ナトリウム（ＨＡＳ、重量平均分子量：５００，０００～７００，０００、キッコーマンバイオケミファ（株）製）２００ｍｇ（０．５００ｍｍｏｌ）を、ヒアルロン酸ナトリウム（ＨＡＬ、重量平均分子量：８５０，０００～１，６００，０００、キューピー（株）製）２００ｍｇ（０．５００ｍｍｏｌ）に変えることと、カルボジヒドラジド（ＣＨＤ、東京化成工業（株）製）の使用量を、１．３６４ｇ（１５．１ｍｍｏｌ）から１．３７５ｇ（１５．３ｍｍｏｌ）に変えることと、１－（３－ジメチルアミノプロピル－３－エチルカルボジイミド塩酸塩（ＥＤＣ、東京化成工業（株）製）の使用量を、３９７．１ｍｇ（２．０７ｍｍｏｌ）から３９７．９ｍｇ（２．０８ｍｍｏｌ）に変えることとの他は、合成例２と同様にして、生成物（ＨＡＬ－ＣＨＤ）２２２．５ｍｇ（収率８１．３％）を得た。１Ｈ ＮＭＲより算出された、ヒアルロン酸の側鎖カルボキシ基のヒドラジド化率は６０％であった。
Synthesis Example 4
200 mg (0.500 mmol) of sodium hyaluronate (HA S , weight average molecular weight: 500,000 to 700,000, manufactured by Kikkoman Biochemifa Corporation) was added to sodium hyaluronate (HA L The amount of 1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride (EDC, manufactured by Tokyo Chemical Industry Co., Ltd.) was changed from 397.1 mg (2.07 mmol) to 397.9 mg (2.08 mmol) in the same manner as in Synthesis Example 2, and 222.5 mg of the product (HA L -CHD) was obtained (yield 81.3%). The hydrazide conversion rate of the side chain carboxyl group of hyaluronic acid calculated from 1 H NMR was 60%.
〔合成例５〕
ヒアルロン酸ナトリウム（ＨＡＳ、重量平均分子量：５００，０００～７００，０００、キッコーマンバイオケミファ（株）製）２００ｍｇ（０．５００ｍｍｏｌ）を、ヒアルロン酸ナトリウム（ＨＡＬ、重量平均分子量：８５０，０００～１，６００，０００、キューピー（株）製）２００ｍｇ（０．５００ｍｍｏｌ）に変えることと、カルボジヒドラジド（ＣＨＤ、東京化成工業（株）製）１．３６４ｇ（１５．１ｍｍｏｌ）を、アジピン酸ジヒドラジド（ＡＨＤ、東京化成工業（株）製）２．６４８ｇ（１５．２ｍｍｏｌ）に変えることと、１－（３－ジメチルアミノプロピル－３－エチルカルボジイミド塩酸塩（ＥＤＣ、東京化成工業（株）製）の使用量を、３９７．１ｍｇ（２．０７ｍｍｏｌ）から４００．２ｍｇ（２．０９ｍｍｏｌ）に変えることとの他は、合成例２と同様にして、生成物（ＨＡＬ－ＡＨＤ）２２５．１ｍｇ（収率８１．９％）を得た。１Ｈ ＮＭＲより算出された、ヒアルロン酸の側鎖カルボキシ基のヒドラジド化率は５５％であった。
Synthesis Example 5
200 mg (0.500 mmol) of sodium hyaluronate (HA S , weight average molecular weight: 500,000 to 700,000, manufactured by Kikkoman Biochemifa Corporation) was added to sodium hyaluronate (HA L The same procedure as in Synthesis Example 2 was repeated except that the amount of 1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride (EDC, manufactured by Tokyo Chemical Industry Co., Ltd.) was changed from 397.1 mg (2.07 mmol) to 400.2 mg (2.09 mmol), and the amount of 1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride (EDC, manufactured by Tokyo Chemical Industry Co., Ltd.) was changed from 397.1 mg (2.07 mmol) to 400.2 mg (2.09 mmol), to obtain 225.1 mg of the product (HA L -AHD) (yield 81.9%). 1 H The hydrazide ratio of the side chain carboxy groups of hyaluronic acid calculated by NMR was 55%.
〔合成例６〕
ヒアルロン酸ナトリウム（ＨＡＳ、重量平均分子量：５００，０００～７００，０００、キッコーマンバイオケミファ（株）製）２００ｍｇ（０．５００ｍｍｏｌ）を、ヒアルロン酸ナトリウム（ＨＡ２０－５０、重量平均分子量：２００，０００～５００，０００、キューピー（株）製）２００ｍｇ（０．５００ｍｍｏｌ）に変えることと、カルボジヒドラジド（ＣＨＤ、東京化成工業（株）製）１．３６４ｇ（１５．１ｍｍｏｌ）を、アジピン酸ジヒドラジド（ＡＨＤ、東京化成工業（株）製）２．６１３ｇ（１５．０ｍｍｏｌ）に変えることと、１－（３－ジメチルアミノプロピル－３－エチルカルボジイミド塩酸塩（ＥＤＣ、東京化成工業（株）製）の使用量を、３９７．１ｍｇ（２．０７ｍｍｏｌ）から３８３．５ｍｇ（２．００ｍｍｏｌ）に変えることとの他は、合成例２と同様にして、生成物（ＨＡ２０－５０－ＡＨＤ）２３２．１ｍｇ（収率８４．４％）を得た。１Ｈ ＮＭＲより算出された、ヒアルロン酸の側鎖カルボキシ基のヒドラジド化率は７５．５％であった。
Synthesis Example 6
200 mg (0.500 mmol) of sodium hyaluronate (HA S , weight average molecular weight: 500,000 to 700,000, manufactured by Kikkoman Biochemifa Corporation) was added to sodium hyaluronate (HA 20-50 The amount of 1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride (EDC, manufactured by Tokyo Chemical Industry Co., Ltd.) was changed from 397.1 mg (2.07 mmol) to 383.5 mg (2.00 mmol) in the same manner as in Synthesis Example 2, except that the amount of 1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride (EDC, manufactured by Tokyo Chemical Industry Co., Ltd.) was changed from 397.1 mg (2.07 mmol) to 383.5 mg (2.00 mmol), and 232.1 mg of the product (HA 20-50 -AHD) was obtained (yield 84.4%). 1 H The hydrazide ratio of the side chain carboxy groups of hyaluronic acid calculated from NMR was 75.5%.
〔試験例１〕
以下の方法に従って、合成例２～５で得たヒドラジド官能化ヒアルロン酸（ＨＡＳ－ＣＨＤ、ＨＡＳ－ＡＨＤ、ＨＡＬ－ＣＨＤ、及びＨＡＬ－ＡＨＤ）と、合成例１で得たアルデヒド官能化ポリエチレングリコール（ＣＨＯ－ＰＥＧ）とが縮合した網目状ポリマーにより形成されたハイドロゲルの安定性を評価した。また、比較のため、カルボキシメチルキトサン（ＣＭＣＨ）と、合成例１で得たアルデヒド官能化ポリエチレングリコール（ＣＨＯ－ＰＥＧ）とが縮合した網目状ポリマーにより形成されたハイドロゲルの安定性も併せて評価した。
[Test Example 1]
According to the following method, the stability of hydrogels formed from network polymers obtained by condensing hydrazide-functionalized hyaluronic acid (HA S -CHD, HA S -AHD, HA L -CHD, and HA L -AHD) obtained in Synthesis Examples 2 to 5 with aldehyde-functionalized polyethylene glycol (CHO-PEG) obtained in Synthesis Example 1 was evaluated. For comparison, the stability of hydrogels formed from network polymers obtained by condensing carboxymethylchitosan (CMCH) with aldehyde-functionalized polyethylene glycol (CHO-PEG) obtained in Synthesis Example 1 was also evaluated.
まず、リン酸緩衝生理食塩水（１×ＰＢＳ）を用いて、それぞれ濃度１．５質量％である、ＣＭＣＨ溶液、ＨＡＳ－ＣＨＤ溶液、ＨＡＳ－ＡＨＤ溶液、ＨＡＬ－ＣＨＤ溶液、及びＨＡＬ－ＡＨＤ溶液を調製した。また、リン酸緩衝生理食塩水（１×ＰＢＳ）を用いて、濃度３．０質量％のＣＨＯ－ＰＥＧ溶液を調製した。 First, a CMCH solution, a HAs - CHD solution, a HAs -AHD solution, a HAl - CHD solution, and a HAl -AHD solution, each having a concentration of 1.5% by mass, were prepared using phosphate buffered saline (1x PBS). Also, a CHO-PEG solution having a concentration of 3.0% by mass was prepared using phosphate buffered saline (1x PBS).
ＣＭＣＨ溶液、ＨＡＳ－ＣＨＤ溶液、ＨＡＳ－ＡＨＤ溶液、ＨＡＬ－ＣＨＤ溶液、又はＨＡＬ－ＡＨＤ溶液２００μＬと、ＣＨＯ－ＰＥＧ溶液１００μＬとを、容量１．５ｍＬのチューブ内で混合した。チューブ内の溶液を１８時間静置して、ハイドロゲルを形成させた。各チューブ内のハイドロゲル上に１×ＰＢＳを１ｍＬずつ加え、室温で２４時間かけてハイドロゲルを膨潤させた。 200 μL of CMCH solution, HA S -CHD solution, HA S -AHD solution, HA L -CHD solution, or HA L -AHD solution was mixed with 100 μL of CHO-PEG solution in a 1.5 mL tube. The solution in the tube was left to stand for 18 hours to form a hydrogel. 1 mL of 1×PBS was added onto the hydrogel in each tube, and the hydrogel was allowed to swell at room temperature for 24 hours.
ハイドロゲルを膨潤させた時点を開始時として、以下の方法に従い、１日経過後、６日経過後、１２日経過後、１８日経過後、２４日経過後の分解率及び膨潤率を測定した。なお、２４日間の試験中、２日に１回、チューブ内の上澄みを除去し、チューブ内に１×ＰＢＳ１ｍＬを加える操作を行った。 The time when the hydrogel was swollen was used as the starting point, and the decomposition rate and swelling rate were measured after 1 day, 6 days, 12 days, 18 days, and 24 days according to the following method. During the 24-day test, the supernatant in the tube was removed once every two days, and 1 mL of 1x PBS was added to the tube.
分解率及び膨潤率を測定する際には、まず、チューブ内の上澄みを取り除き、次いで、１×ＰＢＳ１ｍＬによるハイドロゲルの洗浄を２回行った。洗浄終了後、チューブ内の上澄みを除去した後、チューブ内の膨潤したハイドロゲルの重量Ｗｓｗｅｌｌを測定した。Ｗｓｗｅｌｌの測定後、ハイドロゲルを１８時間凍結乾燥させて、乾燥後のハイドロゲルの重量Ｗｄを測定した。チューブ内に加えられたハイドロゲルの原料の質量をＷ０とし、ゲル中に含まれる１×ＰＢＳの塩の重量を９ｍｇ／ｍＬとして、以下の式に基づいて、分解率及び膨潤率を算出した。分解率及び膨潤率の経時的な変化を、図２及び図３のグラフに示す。
分解率（％）＝（Ｗ０－Ｗｄ－（Ｗｓｗｅｌｌ×９×１０－３））／Ｗ０×１００
膨潤率（％）＝Ｗｓｗｅｌｌ／Ｗｄ×１００
When measuring the decomposition rate and swelling rate, first, the supernatant in the tube was removed, and then the hydrogel was washed twice with 1 mL of 1x PBS. After the washing, the supernatant in the tube was removed, and the weight W swell of the swollen hydrogel in the tube was measured. After measuring W swell , the hydrogel was freeze-dried for 18 hours, and the weight W d of the dried hydrogel was measured. The mass of the raw material of the hydrogel added to the tube was W 0 , and the weight of the salt of 1x PBS contained in the gel was 9 mg/mL, and the decomposition rate and swelling rate were calculated based on the following formula. The changes over time in the decomposition rate and swelling rate are shown in the graphs of Figures 2 and 3.
Decomposition rate (%) = (W 0 −W d −(W swell ×9×10 −3 ))/W 0 ×100
Swelling ratio (%) = Wswell / Wd × 100
図２及び図３から、カルボキシメチルキトサン（ＣＭＣＨ）と、アルデヒド官能化ポリエチレングリコール（ＣＨＯ－ＰＥＧ）とがシッフ塩基架橋された網目状ポリマーにより形成されたハイドロゲルは、ヒドラジド官能化ヒアルロン酸と、アルデヒド官能化ポリエチレングリコールとがヒドラゾン架橋された網目状ポリマーにより形成されたハイドロゲルよりも、分解しやすく、また膨潤しやすいことが分かる。つまり、ヒドラジド官能化親水性ポリマーと、アルデヒド官能化ポリエチレングリコールとが縮合した網目状ポリマーにより形成されたハイドロゲルは、分解しにくく、また膨潤しにくい。そうすると、ヒドラジド官能化親水性ポリマーと、アルデヒド官能化ポリエチレングリコールとが縮合した網目状ポリマーと、自己組織化ペプチドとにより形成されるＩＰＮ構造を有するハイドロゲルも、分解しにくく、また膨潤しにくいといえる。 From Figures 2 and 3, it can be seen that the hydrogel formed from a network polymer in which carboxymethyl chitosan (CMCH) and aldehyde-functionalized polyethylene glycol (CHO-PEG) are crosslinked by Schiff base crosslinking is more easily degraded and swollen than the hydrogel formed from a network polymer in which hydrazide-functionalized hyaluronic acid and aldehyde-functionalized polyethylene glycol are crosslinked by hydrazone crosslinking. In other words, the hydrogel formed from a network polymer in which a hydrazide-functionalized hydrophilic polymer and an aldehyde-functionalized polyethylene glycol are condensed is less likely to degrade and swell. Therefore, it can be said that the hydrogel having an IPN structure formed from a network polymer in which a hydrazide-functionalized hydrophilic polymer and an aldehyde-functionalized polyethylene glycol are condensed, and a self-assembling peptide is also less likely to degrade and swell.
また、図２及び図３から、ヒドラジド官能化親水性ポリマー（ヒドラジド官能化ヒアルロン酸）の分子量が小さいと、ヒドラジド官能化親水性ポリマーと、アルデヒド官能化ポリエチレングリコールとが縮合した網目状ポリマーを含むハイドロゲルが、特に分解しにくく、膨潤しにくいことが分かる。 Furthermore, from Figures 2 and 3, it can be seen that when the molecular weight of the hydrazide-functionalized hydrophilic polymer (hydrazide-functionalized hyaluronic acid) is small, the hydrogel containing the network polymer formed by condensation of the hydrazide-functionalized hydrophilic polymer and the aldehyde-functionalized polyethylene glycol is particularly resistant to decomposition and swelling.
〔試験例２〕
以下の方法に従って、合成例１で得たアルデヒド官能化ポリエチレングリコール（ＣＨＯ－ＰＥＧ）と、合成例２～５で得たヒドラジド官能化ヒアルロン酸（ＨＡＳ－ＣＨＤ、ＨＡＳ－ＡＨＤ、ＨＡＬ－ＣＨＤ、及びＨＡＬ－ＡＨＤ）とを用いて、以下の条件で、レオメーターのステージ上でハイドロゲルを形成しながら、ハイドロゲルの形成過程における貯蔵弾性率Ｇ’及び損失弾性率Ｇ”を測定した。
[Test Example 2]
According to the following method, hydrogels were formed on the stage of a rheometer using the aldehyde-functionalized polyethylene glycol (CHO-PEG) obtained in Synthesis Example 1 and the hydrazide-functionalized hyaluronic acids (HA S -CHD, HA S -AHD, HA L -CHD, and HA L -AHD) obtained in Synthesis Examples 2 to 5 under the following conditions, while the storage modulus G' and loss modulus G" during the formation of the hydrogel were measured.
具体的には、まず、リン酸緩衝生理食塩水（１×ＰＢＳ）を用いて、それぞれ濃度１．５質量％である、ＨＡＳ－ＣＨＤ溶液、ＨＡＳ－ＡＨＤ溶液、ＨＡＬ－ＣＨＤ溶液、及びＨＡＬ－ＡＨＤ溶液を調製した。また、リン酸緩衝生理食塩水（１×ＰＢＳ）を用いて、濃度３．０質量％のＣＨＯ－ＰＥＧ溶液を調製した。 Specifically, first, a HA.sub.S -CHD solution, a HA.sub.S -AHD solution, a HA.sub.L- CHD solution, and a HA.sub.L-AHD solution, each having a concentration of 1.5% by mass, were prepared using phosphate buffered saline (1.times.PBS).Furthermore, a CHO -PEG solution having a concentration of 3.0% by mass was prepared using phosphate buffered saline (1.times.PBS).
レオメーターのステージ上で、ヒドラジド官能化ヒアルロン酸の溶液と、アルデヒド官能化ポリエチレングリコールの溶液とを、ヒドラジド官能化ヒアルロン酸の質量と、アルデヒド官能化ポリエチレングリコールの質量とが同じ質量となるように混合した。そして、以下の測定条件にて、経時的な貯蔵弾性率Ｇ’の変化と、損失弾性率Ｇ”の変化とを測定した。
－測定条件－
測定装置：Ｔｈｅｒｍｏ Ｓｃｉｅｎｔｉｆｉｃ ＨＡＡＫＥ ＭＡＲＳ ＩＩ（サーモフィッシャー・サイエンティフィック社製）
ゲル体積：２１０μＬ
周波数：１Ｈｚ
歪：δ＝０．０５７１４ｒａｄ
センサー：Ｐ３５ＴｉＬ
温度：２０℃
ギャップ：０．０５２
データ取得：１ｐｏｉｎｔｓ／１０ｓ
A solution of hydrazide-functionalized hyaluronic acid and a solution of aldehyde-functionalized polyethylene glycol were mixed on the stage of a rheometer so that the masses of the hydrazide-functionalized hyaluronic acid and the aldehyde-functionalized polyethylene glycol were the same. Then, the change in the storage modulus G' and the change in the loss modulus G" over time were measured under the following measurement conditions.
- Measurement conditions -
Measurement device: Thermo Scientific HAAKE MARS II (manufactured by Thermo Fisher Scientific)
Gel volume: 210 μL
Frequency: 1Hz
Distortion: δ=0.05714rad
Sensor: P35TiL
Temperature: 20°C
Gap: 0.052
Data acquisition: 1 points/10 seconds
貯蔵弾性率Ｇ’及び損失弾性率Ｇ”の測定結果より、貯蔵弾性率Ｇ’が損失弾性率Ｇ”を上回った時間であるゲル化点（秒）を測定した。ゲル化点の測定結果を表１に示す。なお、ＨＡＳ－ＣＨＤ及びＨＡL－ＣＨＤを用いた場合、ヒドラジド官能化ヒアルロン酸と、アルデヒド官能化ポリエチレングリコールとを混合した直後にゲル化していたため、ゲル化点を測定できなかった。 From the measurement results of the storage modulus G' and the loss modulus G", the gel point (seconds) was measured, which is the time when the storage modulus G' exceeded the loss modulus G". The measurement results of the gel point are shown in Table 1. In the case of using HA S -CHD and HA L -CHD, gelation occurred immediately after mixing the hydrazide-functionalized hyaluronic acid and the aldehyde-functionalized polyethylene glycol, so the gel point could not be measured.
表１によれば、ヒドラジド官能化親水性ポリマー（ヒドラジド官能化ヒアルロン酸）において、カルボン酸ヒドラジド基を含む側鎖の鎖長が長いほど、ゲル化に時間を要することが分かる。つまり、カルボン酸ヒドラジド基を含む側鎖の鎖長が長いヒドラジド官能化親水性ポリマーを用いると、ハイドロゲルを形成したい箇所に、ハイドロゲルの原料を含む混合液を液状で注入することができる。ハイドロゲルの原料を含む液がゲル化しにくいと、例えば、生体内の、特定の細胞を増殖させたい箇所においてｉｎ ｓｉｔｕでハイドロゲルを形成させる場合に、生体内への注射によるハイドロゲルの原料を含む液の注入が容易である。 Table 1 shows that in a hydrazide-functionalized hydrophilic polymer (hydrazide-functionalized hyaluronic acid), the longer the side chain length containing a carboxylic acid hydrazide group, the longer the gelation time. In other words, by using a hydrazide-functionalized hydrophilic polymer with a long side chain length containing a carboxylic acid hydrazide group, a mixed solution containing the hydrogel raw materials can be injected in liquid form into the area where a hydrogel is to be formed. If the liquid containing the hydrogel raw materials does not gel easily, for example, when forming a hydrogel in situ at a location in a living body where specific cells are to be grown, it is easy to inject the liquid containing the hydrogel raw materials into the living body by injection.
〔試験例３〕
合成例２で得たヒドラジド官能化ヒアルロン酸（ＨＡＳ－ＣＨＤ）と、合成例１で得たアルデヒド官能化ポリエチレングリコール（ＣＨＯ－ＰＥＧ）と、自己組織化ペプチドとしてのＲＡＤＡ１６とを用いて、以下の方法に従いハイドロゲルを形成した。形成されたハイドロゲルについて、円二色分光（ＣＤ）スペクトルの測定を行った。
[Test Example 3]
A hydrogel was formed according to the following method using the hydrazide-functionalized hyaluronic acid (HA S -CHD) obtained in Synthesis Example 2, the aldehyde-functionalized polyethylene glycol (CHO-PEG) obtained in Synthesis Example 1, and RADA16 as a self-assembling peptide. The circular dichroism (CD) spectrum of the formed hydrogel was measured.
まず、リン酸緩衝生理食塩水（１×ＰＢＳ）を用いて、濃度２質量％のＨＡＳ－ＣＨＤ溶液を調製した。また、リン酸緩衝生理食塩水（２×ＰＢＳ）を用いて、濃度４質量％のＣＨＯ－ＰＥＧ溶液を調製した。また、リン酸緩衝生理食塩水（１×ＰＢＳ）を用いて、濃度１質量％のＲＡＤＡ１６溶液を調製した。 First, a 2% by mass HA S -CHD solution was prepared using phosphate buffered saline (1×PBS). A 4% by mass CHO-PEG solution was prepared using phosphate buffered saline (2×PBS). A 1% by mass RADA16 solution was prepared using phosphate buffered saline (1×PBS).
上記の３つの溶液を、混合液中のＨＡＳ－ＣＨＤの濃度が１．５質量％となり、ＣＨＯ－ＰＥＧの濃度が１質量％となり、混合液中のＲＡＤＡ１６の濃度が０．２５質量％となるように、混合及び濃度調整して、ハイドロゲル（ＨＡＳ－ＣＨＤ／ＣＨＯ－ＰＥＧ／ＲＡＤＡ１６）を形成した。形成されたハイドロゲルについて、円二色性分散計（Ｊ－８２０型、日本分光（株）製）を用いて、波長範囲１９０～３００ｎｍにおいて、円二色分光（ＣＤ）スペクトルの測定を行った。また、比較のため、濃度０．２５質量％のＲＡＤＡ１６溶液と、混合液中のＨＡＳ－ＣＨＤの濃度が１．５質量％となり、ＣＨＯ－ＰＥＧの濃度が１質量％となるように、ＨＡＳ－ＣＨＤ溶液とＣＨＯ－ＰＥＧ溶液とを混合して得たハイドロゲル（ＨＡＳ－ＣＨＤ／ＣＨＯ－ＰＥＧ）とについても、円二色分光（ＣＤ）スペクトルの測定を行った。 The above three solutions were mixed and the concentrations were adjusted so that the concentration of HA S -CHD in the mixed solution was 1.5% by mass, the concentration of CHO-PEG was 1% by mass, and the concentration of RADA16 in the mixed solution was 0.25% by mass, to form a hydrogel (HA S -CHD/CHO-PEG/RADA16). The circular dichroism (CD) spectrum of the formed hydrogel was measured in the wavelength range of 190 to 300 nm using a circular dichroism spectrometer (J-820 model, manufactured by JASCO Corporation). For comparison, circular dichroism (CD) spectra were also measured for a RADA16 solution with a concentration of 0.25% by mass and a hydrogel (HA S -CHD/CHO-PEG) obtained by mixing a HA S -CHD solution with a CHO-PEG solution so that the concentration of HA S -CHD in the mixed solution was 1.5% by mass and the concentration of CHO-PEG was 1% by mass.
得られた円二色分光（ＣＤ）スペクトルを図４に示す。図４中、「（１）ＲＡＤＡ１６溶液」のスペクトルは、ＲＡＤＡ１６のＣＤスペクトルである。「（２）ＨＡＳ－ＣＨＤ／ＣＨＯ－ＰＥＧ」のスペクトルは、ＨＡＳ－ＣＨＤ及びＣＨＯ－ＰＥＧを用いて形成されたハイドロゲルのＣＤスペクトルである。「（３）ＨＡＳ－ＣＨＤ／ＣＨＯ－ＰＥＧ／ＲＡＤＡ１６」のスペクトルは、ＨＡＳ－ＣＨＤ、ＣＨＯ－ＰＥＧ、及びＲＡＤＡ１６を用いて形成されたハイドロゲルのＣＤスペクトルである。また、上記（３）のＣＤスペクトルと、上記（２）のＣＤスペクトルとの差分のスペクトルを、図４中に、「（４）（３）と（２）との差分」として示す。ここで、（４）のスペクトルは、上記（３）のＣＤスペクトルと、上記（２）のＣＤスペクトルとの差分のスペクトルであるため、（４）のスペクトルは、ＨＡＳ－ＣＨＤ及びＣＨＯ－ＰＥＧを用いて形成されたハイドロゲル（網目状ポリマー）中での、ＲＡＤＡ１６のスペクトルであるといえる。 The obtained circular dichroism (CD) spectra are shown in Figure 4. In Figure 4, the spectrum of "(1) RADA16 solution" is the CD spectrum of RADA16. The spectrum of "(2) HAs -CHD/CHO-PEG" is the CD spectrum of a hydrogel formed using HAs - CHD and CHO-PEG. The spectrum of "(3) HAs - CHD/CHO-PEG/RADA16" is the CD spectrum of a hydrogel formed using HAs - CHD, CHO-PEG, and RADA16. The spectrum of the difference between the CD spectrum of (3) above and the CD spectrum of (2) above is shown in Figure 4 as "(4) Difference between (3) and (2)". Here, the spectrum (4) is the difference spectrum between the CD spectrum (3) above and the CD spectrum (2) above, and therefore the spectrum (4) can be said to be the spectrum of RADA16 in a hydrogel (mesh polymer) formed using HA S -CHD and CHO-PEG.
βシート構造に相当するピークは、ＣＤスペクトルにおいて波長２２０～２３０ｎｍ付近に現れる。図４において、ＲＡＤＡ１６（図４中、（１）ＲＡＤＡ１６溶液）のＣＤスペクトルでは、波長２２０～２３０ｎｍ付近において小さなピークしか存在しない。対して、（４）のスペクトルでは、波長２２０～２３０ｎｍ付近において大きなピークが存在する。上記のとおり、（４）のスペクトルは、ＨＡＳ－ＣＨＤ及びＣＨＯ－ＰＥＧを用いて形成されたハイドロゲル（網目状ポリマー）中での、ＲＡＤＡ１６のスペクトルといえる。以上より、ＨＡＳ－ＣＨＤ及びＣＨＯ－ＰＥＧを用いて形成されたハイドロゲル（ＨＡＳ－ＣＨＤ／ＣＨＯ－ＰＥＧ／ＲＡＤＡ１６）中では、ＲＡＤＡ１６のβシート化が進んでいると考えられる。 A peak corresponding to a β-sheet structure appears in the vicinity of 220 to 230 nm wavelength in the CD spectrum. In FIG. 4, in the CD spectrum of RADA16 ((1) RADA16 solution in FIG. 4), only a small peak exists in the vicinity of 220 to 230 nm wavelength. In contrast, in the spectrum of (4), a large peak exists in the vicinity of 220 to 230 nm wavelength. As described above, the spectrum of (4) can be said to be the spectrum of RADA16 in a hydrogel (network polymer) formed using HA s -CHD and CHO-PEG. From the above, it is considered that the formation of β-sheet of RADA16 has progressed in the hydrogel (HA s -CHD/CHO-PEG/RADA16) formed using HA s -CHD and CHO-PEG.
〔試験例４〕
以下の方法に従って、合成例２～５で得たヒドラジド官能化ヒアルロン酸（ＨＡＳ－ＣＨＤ、ＨＡＳ－ＡＨＤ、ＨＡＬ－ＣＨＤ、及びＨＡＬ－ＡＨＤ）と、合成例１で得たアルデヒド官能化ポリエチレングリコール（ＣＨＯ－ＰＥＧ）と、自己組織化ペプチドとしてのＲＡＤＡ１６とを用いて形成されたハイドロゲルの自己修復性を評価した。また、比較のため、カルボキシメチルキトサン（ＣＭＣＨ）と、合成例１で得たアルデヒド官能化ポリエチレングリコール（ＣＨＯ－ＰＥＧ）と、自己組織化ペプチドとしてのＲＡＤＡ１６とを用いて形成されたハイドロゲルの自己修復性も併せて評価した。
[Test Example 4]
According to the following method, the self-repairing properties of hydrogels formed using the hydrazide-functionalized hyaluronic acids (HAS - CHD, HAS -AHD, HAL - CHD, and HAL - AHD) obtained in Synthesis Examples 2 to 5, the aldehyde-functionalized polyethylene glycol (CHO-PEG) obtained in Synthesis Example 1, and RADA16 as a self-assembling peptide were evaluated. For comparison, the self-repairing properties of hydrogels formed using carboxymethylchitosan (CMCH), the aldehyde-functionalized polyethylene glycol (CHO-PEG) obtained in Synthesis Example 1, and RADA16 as a self-assembling peptide were also evaluated.
まず、リン酸緩衝生理食塩水（１×ＰＢＳ）を用いて、それぞれ濃度２質量％である、ＨＡＳ－ＣＨＤ溶液、ＨＡＳ－ＡＨＤ溶液、ＨＡＬ－ＣＨＤ溶液、及びＨＡＬ－ＡＨＤ溶液を調製した。また、リン酸緩衝生理食塩水（１×ＰＢＳ）を用いて、濃度４質量％のＣＭＣＨ溶液を調製した。また、リン酸緩衝生理食塩水（１×ＰＢＳ）を用いて、濃度８質量％のＣＨＯ－ＰＥＧ溶液を調製した。また、濃度２．５質量％のＲＡＤＡ１６溶液を準備した。 First, phosphate buffered saline (1xPBS) was used to prepare a HA S -CHD solution, a HA S -AHD solution, a HA L -CHD solution, and a HA L -AHD solution, each having a concentration of 2% by mass. Phosphate buffered saline (1xPBS) was used to prepare a CMCH solution having a concentration of 4% by mass. Phosphate buffered saline (1xPBS) was used to prepare a CHO-PEG solution having a concentration of 8% by mass. A RADA16 solution having a concentration of 2.5% by mass was also prepared.
これらの溶液を、ヒドラジド官能化ヒアルロン酸又はカルボキシメチルキトサン、アルデヒド官能化ポリエチレングリコール、及びＲＡＤＡ１６の濃度が表２に記載の濃度になるように、混合及び濃度調整して、ハイドロゲル形成用の混合液を得た。ハイドロゲル形成用の混合液としては、無色透明の液と、染料で着色した青色の液とを調製した。得られた混合液を素早く円筒形状の型に流し込み、透明な円盤状のハイドロゲルと、青色の円盤状のハイドロゲルとを得た。得られたハイドロゲルを切断し、自己修復性試験用の半円形状のハイドロゲルを得た。 These solutions were mixed and adjusted so that the concentrations of hydrazide-functionalized hyaluronic acid or carboxymethyl chitosan, aldehyde-functionalized polyethylene glycol, and RADA16 were as shown in Table 2 to obtain a mixture for forming a hydrogel. A colorless, transparent liquid and a blue liquid colored with a dye were prepared as the mixture for forming the hydrogel. The resulting mixture was quickly poured into a cylindrical mold to obtain a transparent, disk-shaped hydrogel and a blue, disk-shaped hydrogel. The resulting hydrogel was cut to obtain a semicircular hydrogel for the self-healing test.
透明な半円形状のハイドロゲルと、青色の半円形状のハイドロゲルとを、円が形成されるように接触させつつ１×ＰＢＳ中に置き、円盤状のハイドロゲルを室温で１８時間静置した。１８時間経過後、円盤状のハイドロゲルをピンセットで引き上げ、透明な半円形状のハイドロゲルと、青色の半円形状のハイドロゲルとの接着状態を確認し、以下の評価基準に従って自己修復性を評価した。評価結果を表２に記す。
－評価基準－
良：透明な半円形状のハイドロゲルと、青色の半円形状のハイドロゲルとが強固に接着していた。
不良：透明な半円形状のハイドロゲルと、青色の半円形状のハイドロゲルとが弱く接着しているか、接着していない。
The transparent semicircular hydrogel and the blue semicircular hydrogel were placed in 1x PBS while being in contact with each other to form a circle, and the disk-shaped hydrogel was left at room temperature for 18 hours. After 18 hours, the disk-shaped hydrogel was pulled up with tweezers, and the adhesion state between the transparent semicircular hydrogel and the blue semicircular hydrogel was confirmed, and the self-repairing property was evaluated according to the following evaluation criteria. The evaluation results are shown in Table 2.
-Evaluation criteria-
Good: The transparent semicircular hydrogel and the blue semicircular hydrogel were firmly adhered to each other.
Poor: The transparent semicircular hydrogel and the blue semicircular hydrogel are weakly or not adhered to each other.
表２によれば、ヒドラジド官能化ヒアルロン酸とアルデヒド官能化ポリエチレングリコールとが縮合した網目状ポリマーと、自己組織化ペプチドとからなるＩＰＮ構造を有するハイドロゲルが、良好な自己修復性を示すことが分かる。 Table 2 shows that hydrogels with an IPN structure consisting of a network polymer formed by condensing hydrazide-functionalized hyaluronic acid and aldehyde-functionalized polyethylene glycol, and a self-assembling peptide, exhibit good self-healing properties.
他方で、カルボキシメチルキトサンとアルデヒド官能化ポリエチレングリコールとが縮合した網目状ポリマーと、自己組織化ペプチドとからなるハイドロゲルもまた、自己修復性を示した。しかしながら、試験例１で確認されたように、カルボキシメチルキトサンとアルデヒド官能化ポリエチレングリコールとが縮合した網目状ポリマーは、経時的に容易に分解してしまう。このため、カルボキシメチルキトサンとアルデヒド官能化ポリエチレングリコールとが縮合した網目状ポリマーと、自己組織化ペプチドとからなるハイドロゲルは、長期間に亘って自己修復性を維持することはできない。 On the other hand, hydrogels consisting of a self-assembling peptide and a mesh polymer formed by condensation of carboxymethylchitosan and aldehyde-functionalized polyethylene glycol also showed self-repairing properties. However, as confirmed in Test Example 1, the mesh polymer formed by condensation of carboxymethylchitosan and aldehyde-functionalized polyethylene glycol easily decomposes over time. For this reason, hydrogels consisting of a self-assembling peptide and a mesh polymer formed by condensation of carboxymethylchitosan and aldehyde-functionalized polyethylene glycol cannot maintain self-repairing properties for a long period of time.
〔試験例５〕
以下の方法に従って、合成例６で得たヒドラジド官能化ヒアルロン酸（ＨＡ２０－５０－ＡＨＤ）と、合成例１で得たアルデヒド官能化ポリエチレングリコール（ＣＨＯ－ＰＥＧ）と、自己組織化ペプチドとしてのＲＡＤＡ１６とを用いて形成されたハイドロゲルのレオロジー物性を評価した。また、比較のため、カルボキシメチルキトサン（ＣＭＣＨ）と、合成例１で得たアルデヒド官能化ポリエチレングリコール（ＣＨＯ－ＰＥＧ）と、自己組織化ペプチドとしてのＲＡＤＡ１６とを用いて形成されたハイドロゲルのレオロジー物性も併せて評価した。
[Test Example 5]
The rheological properties of a hydrogel formed using the hydrazide-functionalized hyaluronic acid (HA 20-50 -AHD) obtained in Synthesis Example 6, the aldehyde-functionalized polyethylene glycol (CHO-PEG) obtained in Synthesis Example 1, and RADA16 as a self-assembling peptide were evaluated according to the following method. For comparison, the rheological properties of a hydrogel formed using carboxymethylchitosan (CMCH), the aldehyde-functionalized polyethylene glycol (CHO-PEG) obtained in Synthesis Example 1, and RADA16 as a self-assembling peptide were also evaluated.
まず、リン酸緩衝生理食塩水（１×ＰＢＳ）を用いて、それぞれ濃度２質量％である、ＨＡ２０－５０－ＡＨＤ溶液及びＣＭＣＨ溶液を調製した。また、リン酸緩衝生理食塩水（２×ＰＢＳ）を用いて、濃度４質量％のＣＨＯ－ＰＥＧ溶液を調製した。また、濃度２．５質量％のＲＡＤＡ１６溶液（ＰｕｒａＳｔａｔ、（株）スリー・ディー・マトリックス製）を準備し、３０分間超音波処理した後、ボルテックスミキサーにて撹拌した。 First, HA 20-50 -AHD solution and CMCH solution, each with a concentration of 2% by mass, were prepared using phosphate buffered saline (1xPBS). A CHO-PEG solution with a concentration of 4% by mass was prepared using phosphate buffered saline (2xPBS). A RADA16 solution (PuraStat, manufactured by 3D Matrix Co., Ltd.) with a concentration of 2.5% by mass was prepared, sonicated for 30 minutes, and then stirred with a vortex mixer.
これらの溶液及び超純水を、表３及び表４に記載の組成となるようにディスク状の型の中に流し込み、混合した。 These solutions and ultrapure water were poured into a disk-shaped mold and mixed to obtain the compositions shown in Tables 3 and 4.
次いで、内部に水を置いた密閉容器内に上記の型を３０分間室温にて静置し、得られたハイドロゲルを型から取り外した。そして、レオメーターを用いて、以下の測定条件にて剪断速度依存測定を行った。
－測定条件－
ゲル体積：３００μＬ
センサー：ＰＰ２０
ノーマルフォース：１Ｎ
測定モード：ＣＳ／ＣＲフローカーブ，ＣＲ
測定範囲：ｄγ／ｄｔ＝０～８０（１／ｓ）
分布：Ｌｉｎｅａｒ
タイプ：Ｓｔｅｐｗｉｓｅ
時間：８０ｓ
データ取得：８０
ステップ保持時間：１．００ｓ
The mold was then placed in a closed container containing water at room temperature for 30 minutes, and the resulting hydrogel was removed from the mold.The shear rate dependence of the hydrogel was measured using a rheometer under the following measurement conditions.
- Measurement conditions -
Gel volume: 300 μL
Sensor: PP20
Normal force: 1N
Measurement mode: CS/CR flow curve, CR
Measurement range: dγ/dt = 0 to 80 (1/s)
Distribution: Linear
Type: Stepwise
Time: 80s
Data acquisition: 80
Step hold time: 1.00 s
親水性ポリマーとしてヒドラジド官能化ヒアルロン酸を用いたハイドロゲルの応力及び粘度の剪断速度依存性を図５Ａ及び図５Ｂに示す。また、親水性ポリマーの代わりにカルボキシメチルキトサンを用いたハイドロゲルの応力及び粘度の剪断速度依存性を図６Ａ及び図６Ｂに示す。図５Ａ及び図５Ｂと図６Ａ及び図６Ｂとに示すとおり、ヒドラジド官能化ヒアルロン酸を用いたハイドロゲルと、カルボキシメチルキトサンを用いたハイドロゲルとのいずれについても、剪断速度の増加に伴って応力及び粘度が減少するシアシニング性を示したが、前者の方が応力及び粘度が大きく減少した。特に、ヒドラジド官能化ヒアルロン酸とアルデヒド官能化ポリエチレングリコールとが縮合した網目状ポリマーと、自己組織化ペプチドとからなるＩＰＮ構造を有するハイドロゲル（ＨＡ ＩＰＮ ｇｅｌ）は、シアシニング性が強く現れた。 The shear rate dependence of stress and viscosity of hydrogels using hydrazide-functionalized hyaluronic acid as a hydrophilic polymer is shown in Figures 5A and 5B. The shear rate dependence of stress and viscosity of hydrogels using carboxymethylchitosan instead of hydrophilic polymer is shown in Figures 6A and 6B. As shown in Figures 5A and 5B and 6A and 6B, both hydrogels using hydrazide-functionalized hyaluronic acid and hydrogels using carboxymethylchitosan showed shear-thinning properties in which stress and viscosity decreased with increasing shear rate, but the former showed a greater decrease in stress and viscosity. In particular, the hydrogel (HA IPN gel) with an IPN structure consisting of a network polymer condensed from hydrazide-functionalized hyaluronic acid and aldehyde-functionalized polyethylene glycol, and a self-assembling peptide, showed strong shear-thinning properties.
〔試験例６〕
以下の方法に従って、合成例６で得たヒドラジド官能化ヒアルロン酸（ＨＡ２０－５０－ＡＨＤ）と、合成例１で得たアルデヒド官能化ポリエチレングリコール（ＣＨＯ－ＰＥＧ）と、自己組織化ペプチドとしてのＲＡＤＡ１６とを用いて形成されたハイドロゲル中での細胞生存率を評価した。また、比較のため、カルボキシメチルキトサン（ＣＭＣＨ）と、合成例１で得たアルデヒド官能化ポリエチレングリコール（ＣＨＯ－ＰＥＧ）と、自己組織化ペプチドとしてのＲＡＤＡ１６とを用いて形成されたハイドロゲル中での細胞生存率も併せて評価した。
[Test Example 6]
According to the following method, cell viability was evaluated in a hydrogel formed using the hydrazide-functionalized hyaluronic acid (HA 20-50 -AHD) obtained in Synthesis Example 6, the aldehyde-functionalized polyethylene glycol (CHO-PEG) obtained in Synthesis Example 1, and RADA16 as a self-assembling peptide. For comparison, cell viability was also evaluated in a hydrogel formed using carboxymethylchitosan (CMCH), the aldehyde-functionalized polyethylene glycol (CHO-PEG) obtained in Synthesis Example 1, and RADA16 as a self-assembling peptide.
まず、ＨＡ２０－５０－ＡＨＤ、ＣＭＣＨ、及びＣＨＯ－ＰＥＧに紫外線を２０分間照射して滅菌した。次いで、リン酸緩衝生理食塩水（１×ＰＢＳ）を用いて、それぞれ濃度２質量％である、ＨＡ２０－５０－ＡＨＤ溶液及びＣＭＣＨ溶液を調製した。また、リン酸緩衝生理食塩水（２×ＰＢＳ）を用いて、濃度４質量％のＣＨＯ－ＰＥＧ溶液を調製した。また、濃度２．５質量％のＲＡＤＡ１６溶液（ＰｕｒａＳｔａｔ、（株）スリー・ディー・マトリックス製）を準備し、３０分間超音波処理した後、ボルテックスミキサーにて撹拌した。さらに、リン酸緩衝生理食塩水（１×ＰＢＳ）を用いて５ｍｇ／ｍＬ ＭＴＴ試薬溶液を調製するとともに、１Ｍ塩酸を２－プロパノールで０．０４ｍＭに希釈してＭＴＴ抽出用試薬を調製した。 First, HA 20-50 -AHD, CMCH, and CHO-PEG were sterilized by irradiation with ultraviolet light for 20 minutes. Next, HA 20-50 -AHD solution and CMCH solution, each having a concentration of 2% by mass, were prepared using phosphate buffered saline (1xPBS). In addition, a CHO-PEG solution having a concentration of 4% by mass was prepared using phosphate buffered saline (2xPBS). In addition, a RADA16 solution (PuraStat, manufactured by Three-D Matrix Co., Ltd.) having a concentration of 2.5% by mass was prepared, sonicated for 30 minutes, and then stirred with a vortex mixer. Furthermore, a 5 mg/mL MTT reagent solution was prepared using phosphate buffered saline (1xPBS), and 1 M hydrochloric acid was diluted with 2-propanol to 0.04 mM to prepare an MTT extraction reagent.
予めインキュベーター（３７℃、５％ＣＯ２）で培養していたサブコンフルエントのＨｅｐＧ２細胞をトリプシン処理で剥がし、遠心処理（１５００ｒｐｍ、５分間）を行った後、上澄みを除去した。次いで、ＤＭＥＭ１ｍＬを加え、トリパンブルー染色を行い、血球計算板で生細胞数を計測した。次いで、ＤＭＥＭ４ｍＬを加え、遠心処理を行った後、上澄みを除去した。細胞にＨＡ２０－５０－ＡＨＤ溶液又はＣＭＣＨ溶液を加え、２．０×１０７ｃｅｌｌｓ／ｍＬとなるように懸濁した。そして、細胞を懸濁したＨＡ２０－５０－ＡＨＤ溶液又はＣＭＣＨ溶液と、ＣＨＯ－ＰＥＧ溶液、ＲＡＤＡ１６溶液、及び超純水とを、表５及び表６に記載の組成となるように容量１．５ｍＬのチューブ内に加えて混合した後、チューブをインキュベーター（３７℃、５％ＣＯ２）内に３０分間静置し、ハイドロゲルを形成させた。 Subconfluent HepG2 cells that had been cultured in advance in an incubator (37°C, 5% CO 2 ) were detached by trypsin treatment, centrifuged (1500 rpm, 5 minutes), and the supernatant was removed. Next, 1 mL of DMEM was added, trypan blue staining was performed, and the number of live cells was counted using a hemocytometer. Next, 4 mL of DMEM was added, centrifuged, and the supernatant was removed. HA 20-50 -AHD solution or CMCH solution was added to the cells, and they were suspended to 2.0 x 10 7 cells/mL. The HA 20-50 -AHD solution or CMCH solution in which the cells were suspended, the CHO-PEG solution, the RADA16 solution, and ultrapure water were added to a 1.5 mL tube and mixed to obtain the compositions shown in Tables 5 and 6. The tube was then placed in an incubator (37°C, 5% CO 2 ) for 30 minutes to form a hydrogel.
次いで、ハイドロゲルの上部にＤＭＥＭ５００μＬを加え、１日間培養した。培養後、上澄みを除去し、ゲルマッシャーを用いてハイドロゲルを粉砕し、ＭＴＴ抽出用試薬を加えて３７℃にて１０分間超音波処理を行った。次いで、遠心処理（１５００ｒｐｍ、５分間）を行い、上澄みを９６ウェルプレートに１００μＬ／ウェルとなるように加えた。そして、プレートリーダーを用いて波長５７０ｎｍにおける吸光度を測定することにより、細胞生存率を算出した。 Then, 500 μL of DMEM was added to the top of the hydrogel and cultured for one day. After culture, the supernatant was removed, the hydrogel was pulverized using a gel masher, MTT extraction reagent was added, and ultrasonic treatment was performed for 10 minutes at 37°C. Next, centrifugation (1500 rpm, 5 minutes) was performed, and the supernatant was added to a 96-well plate at 100 μL/well. The cell viability was calculated by measuring the absorbance at a wavelength of 570 nm using a plate reader.
各ハイドロゲル中で培養した場合の細胞生存率を図７に示す。図７に示すとおり、ヒドラジド官能化ヒアルロン酸を用いたハイドロゲルと、カルボキシメチルキトサンを用いたハイドロゲルとのいずれについても、高い細胞生存率を示した。 The cell viability when cultured in each hydrogel is shown in Figure 7. As shown in Figure 7, both the hydrogel using hydrazide-functionalized hyaluronic acid and the hydrogel using carboxymethyl chitosan showed high cell viability.
〔試験例７〕
以下の方法に従って、合成例６で得たヒドラジド官能化ヒアルロン酸（ＨＡ２０－５０－ＡＨＤ）と、合成例１で得たアルデヒド官能化ポリエチレングリコール（ＣＨＯ－ＰＥＧ）と、自己組織化ペプチドとしてのＲＡＤＡ１６とを用いて形成されたハイドロゲルとともに細胞をインジェクトする場合の細胞生存率を評価した。また、比較のため、カルボキシメチルキトサン（ＣＭＣＨ）と、合成例１で得たアルデヒド官能化ポリエチレングリコール（ＣＨＯ－ＰＥＧ）と、自己組織化ペプチドとしてのＲＡＤＡ１６とを用いて形成されたハイドロゲルとともに細胞をインジェクトする場合の細胞生存率も併せて評価した。
[Test Example 7]
According to the following method, cell viability was evaluated when cells were injected together with a hydrogel formed using the hydrazide-functionalized hyaluronic acid (HA 20-50 -AHD) obtained in Synthesis Example 6, the aldehyde-functionalized polyethylene glycol (CHO-PEG) obtained in Synthesis Example 1, and RADA16 as a self-assembling peptide. For comparison, cell viability was also evaluated when cells were injected together with a hydrogel formed using carboxymethylchitosan (CMCH), the aldehyde-functionalized polyethylene glycol (CHO-PEG) obtained in Synthesis Example 1, and RADA16 as a self-assembling peptide.
まず、ＨＡ２０－５０－ＡＨＤ、ＣＭＣＨ、及びＣＨＯ－ＰＥＧに紫外線を３０分間照射して滅菌した。次いで、リン酸緩衝生理食塩水（１×ＰＢＳ）を用いて、それぞれ濃度２質量％である、ＨＡ２０－５０－ＡＨＤ溶液及びＣＭＣＨ溶液を調製した。また、リン酸緩衝生理食塩水（２×ＰＢＳ）を用いて、濃度４質量％のＣＨＯ－ＰＥＧ溶液を調製した。また、濃度２．５質量％のＲＡＤＡ１６溶液（ＰｕｒａＳｔａｔ、（株）スリー・ディー・マトリックス製）を準備し、３０分間超音波処理した後、ボルテックスミキサーにて撹拌した。 First, HA 20-50 -AHD, CMCH, and CHO-PEG were sterilized by irradiation with ultraviolet light for 30 minutes. Then, phosphate buffered saline (1xPBS) was used to prepare HA 20-50 -AHD solution and CMCH solution, each with a concentration of 2% by mass. Phosphate buffered saline (2xPBS) was used to prepare a CHO-PEG solution with a concentration of 4% by mass. A RADA16 solution (PuraStat, manufactured by 3D Matrix Co., Ltd.) with a concentration of 2.5% by mass was prepared, sonicated for 30 minutes, and then stirred with a vortex mixer.
予めインキュベーター（３７℃、５％ＣＯ２）で培養していたサブコンフルエントのＨｅｐＧ２細胞をトリプシン処理で剥がし、遠心処理（１５００ｒｐｍ、５分間）を行った後、上澄みを除去した。次いで、ＤＭＥＭ１ｍＬを加え、トリパンブルー染色を行い、血球計算板で生細胞数を測定した。次いで、ＤＭＥＭ４ｍＬを加え、遠心処理を行った後、上澄みを除去した。細胞にＨＡ２０－５０－ＡＨＤ溶液又はＣＭＣＨ溶液を加え、１．０×１０７ｃｅｌｌｓ／ｍＬとなるように懸濁した。そして、細胞を懸濁したＨＡ２０－５０－ＡＨＤ溶液又はＣＭＣＨ溶液と、ＣＨＯ－ＰＥＧ溶液、ＲＡＤＡ１６溶液、及び超純水とを、表７及び表８に記載の組成となるように容量１．５ｍＬのチューブ内に加えて混合し、すぐに５ｍＬシリンジで吸引して３０分間静置することで、シリンジ内で４００μＬのハイドロゲルを形成させた。ハイドロゲルの形成後、シリンジの先端に１８Ｇ注射針を装着し２４ウェルプレートに２００μＬ／ウェルとなるようにハイドロゲルをインジェクトした。比較として、細胞を懸濁したＨＡ２０－５０－ＡＨＤ溶液又はＣＭＣＨ溶液と、ＣＨＯ－ＰＥＧ溶液、ＲＡＤＡ１６溶液、及び超純水とを、表７及び表８に記載の組成となるように容量１．５ｍＬのチューブ内に加えて混合し、すぐに２４ウェルプレートに移して３０分間静置することで、ハイドロゲルを形成させた。 Subconfluent HepG2 cells that had been cultured in advance in an incubator (37°C, 5% CO 2 ) were detached by trypsin treatment, centrifuged (1500 rpm, 5 minutes), and the supernatant was removed. Next, 1 mL of DMEM was added, trypan blue staining was performed, and the number of live cells was measured using a hemocytometer. Next, 4 mL of DMEM was added, centrifuged, and the supernatant was removed. HA 20-50 -AHD solution or CMCH solution was added to the cells, and they were suspended to 1.0 x 10 7 cells/mL. Then, the HA 20-50 -AHD solution or CMCH solution in which the cells were suspended, the CHO-PEG solution, the RADA16 solution, and ultrapure water were added to a tube with a capacity of 1.5 mL to obtain the composition described in Tables 7 and 8, and mixed, and immediately aspirated with a 5 mL syringe and left to stand for 30 minutes to form 400 μL of hydrogel in the syringe. After the hydrogel was formed, an 18G injection needle was attached to the tip of the syringe, and the hydrogel was injected into a 24-well plate at 200 μL/well. For comparison, the HA 20-50 -AHD solution or CMCH solution in which the cells were suspended, the CHO-PEG solution, the RADA16 solution, and ultrapure water were added to a tube with a capacity of 1.5 mL to obtain the composition described in Tables 7 and 8, and immediately transferred to a 24-well plate and left to stand for 30 minutes to form a hydrogel.
次いで、シリンジからインジェクトしたハイドロゲル２００μＬが入ったウェルと、２４ウェルプレート内で形成させたハイドロゲル２００μＬが入ったウェルとに対し、ＤＭＥＭ：ＣＣＫ－８（（株）同仁化学研究所）＝２０：１の混合溶液を５００μＬ／ウェルとなるように加え、遮光下、インキュベーター（３７℃、５％ＣＯ２）で２時間培養した。培養後、上澄みを１００μＬずつ９６ウェルプレートに移し、プレートリーダーを用いて波長４５０ｎｍにおける吸光度を測定することにより、細胞生存率を算出した。 Next, a mixed solution of DMEM:CCK-8 (Dojindo Laboratories, Ltd.) = 20:1 was added to the wells containing 200 μL of hydrogel injected from the syringe and to the wells containing 200 μL of hydrogel formed in the 24-well plate at 500 μL/well, and the cells were cultured in an incubator (37°C, 5% CO 2 ) under light shielding for 2 hours. After the culture, 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance at a wavelength of 450 nm was measured using a plate reader to calculate the cell viability.
インジェクトせずに２４ウェルプレート内で形成させたハイドロゲルにおける細胞生存率を基準として、インジェクト後の細胞生存率を算出した結果を図８に示す。図８に示すとおり、ヒドラジド官能化ヒアルロン酸を用いたハイドロゲルと、カルボキシメチルキトサンを用いたハイドロゲルとのいずれについても、自己組織化ペプチドを加えてＩＰＮ構造を形成させることにより、剪断応力による細胞傷害性は緩和された。但し、その緩和効果は、ヒドラジド官能化ヒアルロン酸を用いたハイドロゲルの方が顕著に高かった。 The results of calculating the cell viability after injection based on the cell viability in the hydrogel formed in a 24-well plate without injection are shown in Figure 8. As shown in Figure 8, for both the hydrogel using hydrazide-functionalized hyaluronic acid and the hydrogel using carboxymethyl chitosan, the cytotoxicity caused by shear stress was alleviated by adding a self-assembling peptide to form an IPN structure. However, the alleviating effect was significantly greater for the hydrogel using hydrazide-functionalized hyaluronic acid.
Claims (8)
前記網目状ポリマーが、親水性ポリマーに由来する親水性ポリマーブロックと、ポリエチレングリコールに由来するポリエチレングリコールブロックとを有し
前記親水性ポリマーブロックと、前記ポリエチレングリコールブロックとが、－Ｃ＝Ｎ－ＮＨ－で表されるヒドラゾン結合を含む２価の基により結合しており、
前記親水性ポリマーが、水を加えて平衡膨潤化させた場合に水の質量比率として５０質量％以上９９質量％以下の含水率を示すポリマーである、ハイドロゲル。 A hydrogel comprising a self-assembling peptide and a network polymer,
the network polymer has a hydrophilic polymer block derived from a hydrophilic polymer and a polyethylene glycol block derived from polyethylene glycol, the hydrophilic polymer block and the polyethylene glycol block being bonded to each other via a divalent group containing a hydrazone bond represented by -C=N-NH-,
A hydrogel, wherein the hydrophilic polymer is a polymer that exhibits a water content of 50% by mass or more and 99% by mass or less, in terms of the mass ratio of water, when the hydrophilic polymer is allowed to swell in equilibrium with water.
前記自己組織化ペプチドと、前記親水性ポリマーブロックを与える、アルデヒド官能化又はヒドラジド官能化された親水性ポリマーと、前記ポリエチレングリコールブロックを与える、アルデヒド官能化又はヒドラジド官能化されたポリエチレングリコールとを含む組成物。 10. The hydrogel-forming composition of claim 1, comprising:
A composition comprising the self-assembling peptide, an aldehyde- or hydrazide-functionalized hydrophilic polymer to provide the hydrophilic polymer block, and an aldehyde- or hydrazide-functionalized polyethylene glycol to provide the polyethylene glycol block.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007535607A (en) * | 2004-04-30 | 2007-12-06 | アドヴァンスド カーディオヴァスキュラー システムズ， インコーポレイテッド | Hyaluronic acid copolymer |
| JP2009507110A (en) * | 2005-09-09 | 2009-02-19 | オタワ ヘルス リサーチ インスティテュート | Interpenetrating network and methods and compositions related thereto |
| JP2015502829A (en) * | 2011-12-22 | 2015-01-29 | オステムインプラント カンパニー リミテッド | Hydrated gel capable of controlling rate of biodegradation and method for producing the same |
| WO2022165416A1 (en) * | 2021-02-01 | 2022-08-04 | The Regents Of The University Of Colorado, A Body Corporate | Compositions and methods for making and using hybrid network hydrogels |
| JP2023508448A (en) * | 2019-12-26 | 2023-03-02 | アラーガン、インコーポレイテッド | Physically Mixed HA-Collagen Dermal Filler |
-
2024
- 2024-01-15 WO PCT/JP2024/000752 patent/WO2024219027A1/en unknown
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007535607A (en) * | 2004-04-30 | 2007-12-06 | アドヴァンスド カーディオヴァスキュラー システムズ， インコーポレイテッド | Hyaluronic acid copolymer |
| JP2009507110A (en) * | 2005-09-09 | 2009-02-19 | オタワ ヘルス リサーチ インスティテュート | Interpenetrating network and methods and compositions related thereto |
| JP2015502829A (en) * | 2011-12-22 | 2015-01-29 | オステムインプラント カンパニー リミテッド | Hydrated gel capable of controlling rate of biodegradation and method for producing the same |
| JP2023508448A (en) * | 2019-12-26 | 2023-03-02 | アラーガン、インコーポレイテッド | Physically Mixed HA-Collagen Dermal Filler |
| WO2022165416A1 (en) * | 2021-02-01 | 2022-08-04 | The Regents Of The University Of Colorado, A Body Corporate | Compositions and methods for making and using hybrid network hydrogels |
Non-Patent Citations (2)
| Title |
|---|
| ABBASI AVAL NEGAR; EMADI RAHMATOLLAH; VALIANI ALI; KHARAZIHA MAHSHID; FINNE-WISTRAND ANNA: "An aligned fibrous and thermosensitive hyaluronic acid-puramatrix interpenetrating polymer network hydrogel with mechanical properties adjusted for neural tissue", JOURNAL OF MATERIAL SCIENCE, KLUWER ACADEMIC PUBLISHERS, DORDRECHT, vol. 57, no. 4, 1 January 2022 (2022-01-01), Dordrecht , pages 2883 - 2896, XP037672993, ISSN: 0022-2461, DOI: 10.1007/s10853-021-06733-0 * |
| ISHIKAWA S., IIJIMA K., MATSUKUMA D., IIJIMA M., OSAWA S., OTSUKA H.: "An interpenetrating polymer network hydrogel with biodegradability through controlling self-assembling peptide behavior with hydrolyzable cross-linking networks", MATERIALS TODAY ADVANCES, vol. 9, 1 March 2021 (2021-03-01), pages 100131, XP093222544, ISSN: 2590-0498, DOI: 10.1016/j.mtadv.2021.100131 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN120000835A (en) * | 2025-04-21 | 2025-05-16 | 南京东万生物技术有限公司 | Hyaluronic acid dressing and its application in the treatment of dermatitis |
| CN120000835B (en) * | 2025-04-21 | 2025-07-11 | 南京东万生物技术有限公司 | Hyaluronic acid dressing and application thereof in treating dermatitis |
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