JPH11169703A - Thermally reversible hydrogel forming composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable addition of functions such as biocompatibility, cell bonding property and the like by setting ratio of an initial content of a physiologically active substance in a gel to a content of a physiologically active substance in the gel which has been immersed in an enormously excessive amount of water at a specific ratio or higher. SOLUTION: A thermally reversible hydrogel forming composition containing an initial content (A) of a physiologically active substance is turned into a sol at a temperature lower than a sol-gel transition temperature and injected into a laboratory dish and is allowed to gel at a temperature higher than the sol-gel transition temperature, and the dish containing a gel is immersed in distilled water having a temperature higher than the sol-gel transition temperature. Then, the dish containing the gel is taken out from underwater and excessive water is removed, and then when content of the physiologically active substance (B) in the gel is measured, ratio of these contents (B/A) is set at 80% or higher. By such processes, precipitation of cells and the like and damages of the cells are effectively suppressed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a thermoreversible (gel) hydrogel-forming composition which reversibly changes between a hydrogel state and an aqueous solution state (sol) in response to a temperature change. The present invention relates to a thermoreversible hydrogel-forming composition containing a physiologically active substance capable of exhibiting physiological activity.

More specifically, the present invention relates to a "gelling type at elevated temperature" in which a sol state is formed at a temperature lower than the first sol-gel transition temperature and a gel state is formed at a temperature higher than the first transition temperature. And a thermoreversible hydrogel-forming composition having the characteristics of

[0003] The thermoreversible hydrogel-forming composition of the present invention is particularly suitable for use as a carrier for cell (tissue) culture, a wound dressing, a bioadhesive, etc., by utilizing the properties of the gelling type when heated. It is possible.

[0004]

2. Description of the Related Art As a thermoreversible hydrogel,
Agar and gelatin gels are well known. An aqueous solution of agar or gelatin loses fluidity upon cooling to form a jelly-like hydrogel, and exhibits a thermoreversible sol-gel transition of a type which returns to an aqueous solution by heating, that is, a gelling type at the time of cooling.

[0005] Contrary to these hydrogels, an aqueous solution of methylcellulose, a polysaccharide derivative, is known as a type exhibiting a gelling property upon heating, which becomes a hydrogel upon heating. However, since the aqueous solution of methyl cellulose gels only at a high temperature of 45 ° C. or higher, it has little practical utility value, and the characteristics of the gelling type at the time of temperature rise have hardly been utilized.

On the other hand, some nonionic surfactants exhibit a gel-type thermoreversible sol-gel transition when the aqueous solution is heated. For example, Pluronic F-127 (trade name, BASF Wyandotte Chemica) in which polyethylene oxide is bonded to both ends of polypropylene oxide
ls Co.) is known to be a hydrogel at about 20 ° C. or higher and an aqueous solution at a lower temperature (for example, B. Chu, Langmuir, 11, 4).
14-421 (1995)).

However, this material (Pluronic F)
In the case of -127), there was a problem that a hydrogel was formed only at a high concentration of about 20% by weight or more, and thus the water content in the produced hydrogel was low.

When this material is used, about 20 wt.
%, And gel is dissolved when water is further added to the gel even when the gel is kept at a temperature higher than the gelation temperature (that is, the hydrogel is water-soluble). There was a problem. Such a phenomenon is a serious disadvantage that, for example, when the gel is used as a wound dressing, the hydrogel dissolves and disappears in the wound surface due to exudate secreted from the wound surface. Connect.

[0009]

SUMMARY OF THE INVENTION An object of the present invention is to provide a thermoreversible hydrogel-forming composition which has solved the above-mentioned drawbacks of the conventional thermoreversible hydrogel-forming composition.

Another object of the present invention is to provide a thermoreversible hydrogel-forming composition capable of favorably retaining a physiologically active substance in a gel.

It is still another object of the present invention to provide a thermoreversible hydrogel-forming composition capable of maintaining good activity of a physiologically active substance held in a gel.

[0012]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that a composition containing a specific thermoreversible hydrogel-forming polymer can suitably hold a physiologically active substance itself in the hydrogel. In addition, it has been found that the gel can substantially retain the original function of the physiologically active substance in the gel.

[0013] The thermoreversible hydrogel-forming composition of the present invention is based on the above findings, and more specifically, contains at least a hydrogel-forming polymer, water and a physiologically active substance; A temperature (first sol-gel transition temperature) of 5 ° C. or more and 40 ° C. or less, a substantially water-insoluble hydrogel state at a temperature higher than the sol-gel transition temperature, and the sol-gel transition temperature A thermoreversible hydrogel-forming composition that exhibits reversible water solubility at a lower temperature; and in the water-insoluble hydrogel state, the initial content (A) of the physiologically active substance in the gel; The ratio (B / A) to the content (B) of the physiologically active substance in the gel after immersion in a large excess of water is 80% or more.

According to the present invention, the composition further comprises at least a hydrogel-forming polymer, water and a physiologically active substance;
Further, there is provided a thermoreversible hydrogel-forming composition having at least first and second sol-gel transition temperatures.

[0015] The composition containing the thermoreversible hydrogel-forming polymer of the present invention having the above-mentioned constitution not only can suitably hold the physiologically active substance itself in the hydrogel, but also
Since the original function of the physiologically active substance can be substantially retained in the gel, by immobilizing the physiologically active substance in the gel, it can be widely used for various uses.

The present inventors have already found that the sol-gel transition temperature is 5 ° C. or more and 40 ° C. or less, a sol is formed at a temperature lower than the transition temperature, and a gel is formed at a temperature higher than the transition temperature.
A thermoreversible hydrogel-forming composition of a gelling type at elevated temperature characterized in that the gel is substantially insoluble in water has been developed (JP-A-5-262882). It has also been proposed to add a low molecular weight bioactive substance to the thermoreversible hydrogel-forming composition to impart various bioactivity to the hydrogel (Japanese Patent Application No. 8-29572, WO
95/15152).

However, as compared with these conventional gelling-type thermoreversible hydrogel-forming compositions at elevated temperatures,
In the hydrogel formed based on the thermoreversible hydrogel-forming composition of the present invention described above, even a relatively low-molecular-weight physiologically active substance can easily pass through the polymer network in the hydrogel, or easily. Since diffusion to the outside of the hydrogel is more effectively suppressed, the hydrogel containing a physiologically active substance can maintain the physiological activity for a longer period of time.

When the sol-gel transition temperature of the gelling type thermoreversible hydrogel-forming composition at the time of the above-mentioned prior application is set at around room temperature, the sol, cell culture temperature and body temperature (37 ° C.) can be obtained at room temperature or lower. A cell (tissue) culture carrier (Japanese Unexamined Patent Publication No. 6-141851) which takes in cells in a liquid at room temperature, raises the temperature to 37 ° C. to form a gel, and allows cells to be cultured three-dimensionally in the gel. A wound dressing (WO 95/07719) which can be applied at room temperature to a wound surface and gelled at body temperature (37 ° C.) to protect the wound surface.

However, the thermoreversible hydrogel-forming composition of the present invention contains a physiologically active substance as compared with the conventional gelling-type thermoreversible hydrogel-forming composition at the time of temperature rise. It has properties superior to these conventional hydrogels in terms of affinity for living bodies such as adhesion.

In addition, the conventional gelling type thermoreversible hydrogel-forming composition at elevated temperature is always in a liquid state at a temperature higher than the freezing point below the sol-gel transition temperature, so its use is limited. There was a case. For example, when storing cultured cells or tissues, it is advantageous to store the cells at as low a temperature as possible in order to keep their metabolic activity low. However, if the temperature is lower than the freezing point, the cells may be damaged by freezing. Therefore, a temperature higher than the freezing point and as low as possible is desirable. However, the conventional gelling type thermoreversible hydrogel-forming composition at elevated temperature,
Since the liquid is always liquid at a temperature higher than the freezing point below the sol-gel transition temperature, cells and the like sediment, and physical damage due to collision between cells or between cells and the container wall during transportation may be caused by cells and the like. In some cases.

On the other hand, when the thermoreversible hydrogel-forming composition having the first and second sol-gel transition temperatures of the present invention is used, the temperature is lower than the second sol-gel transition temperature. Since it can be in a gel state, it becomes possible to effectively suppress sedimentation of the above-described cells and the like and damage to the cells.

[0022]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
In the following description, "%" and "parts"
Is based on weight (ie, weight% and parts by weight) unless otherwise specified.

(Hydrogel-forming polymer) The "hydrogel-forming polymer" which can be used in the present invention is an aqueous solution or aqueous dispersion of the polymer in a specific temperature range of 5 ° C or more and 40 ° C or less. It has the property that it becomes a hydrogel at a temperature higher than the above temperature, and that it becomes a sol or liquid at a temperature lower than this temperature. In this specification, the “specific temperature” is referred to as (first) “sol-gel transition temperature”.

In the case where the thermoreversible hydrogel-forming composition of the present invention is once in a sol state at a temperature lower than the above (first) sol-gel transition temperature, and is again in a gel state when further cooled, it is herein referred to. The transition temperature at this time is referred to as “second sol-gel transition temperature”.

(Sol-gel transition temperature) In the present invention,
The measurement of the sol-gel transition temperature of the sample is described in the literature (H. Yoshiok
a et al., Journal of Macromolecular Science, A31
(1), 113 (1994)).

That is, the dynamic elastic modulus of a sample at an observation frequency of 1 Hz is measured by gradually changing the temperature (from a low temperature side to a high temperature side, or from a high temperature side to a low temperature side at 1 ° C./1 minute). The temperature at the point where the storage elastic modulus (G ′, elastic term) and the loss elastic modulus (G ″, viscous term) intersect is referred to as a sol-gel transition temperature.
The state of G ″ <G ′ is defined as a gel. The sol-gel transition temperature at the time of temperature rise and the time of temperature fall (for example, 2 ° C. or more in absolute value)
If different, the intermediate temperature is taken as the sol-gel transition temperature. In measuring the sol-gel transition temperature, the following measurement conditions can be suitably used.

<Measurement conditions of dynamic elastic modulus> Measuring equipment: trade name = stress control type rheometer-CSL
500, Carri-Med's sample solution (or dispersion) concentration (as the concentration of “hydrogel-forming polymer”): 10 (weight)% Sample solution amount: about 0.8 g Shape and dimensions: Acrylic parallel disk (diameter 4.0 cm), gap 600 μm.

Applied stress: within the linear region.

(Substantially water-insoluble) The thermoreversible hydrogel-forming composition of the present invention becomes a substantially water-insoluble hydrogel state at a temperature higher than its sol-gel transition temperature. The gel does not dissolve substantially, even when subsequently immersed in a large amount of water. The properties of the hydrogel can be confirmed, for example, as follows.

That is, 0.15 g of the hydrogel-forming composition of the present invention is dissolved in 1.35 g of distilled water at a temperature lower than the above-mentioned sol-gel transition temperature (for example, under ice-cooling).
An aqueous solution having a thickness of about 1.5 mm was prepared by preparing an aqueous solution of 0 W%, injecting the aqueous solution into a plastic petri dish having a diameter of 35 mm, and heating the solution to a temperature Th (for example, 37 ° C) higher than the sol-gel transition temperature. After the gel is formed in the petri dish, the weight (f gram) of the whole petri dish including the gel is measured. Next, the whole petri dish containing the gel was washed for 250 minutes.
After standing in water in ml at the above temperature Th for 10 hours, the weight (g gram) of the whole petri dish containing the gel is measured to evaluate whether or not the gel is dissolved from the gel surface. At this time, in the hydrogel-forming composition of the present invention,
The weight loss rate of the gel, that is, (f−g) / f is
It is preferably at most 5.0%, more preferably at most 1.0% (particularly at most 0.1%).

The aqueous solution of the thermoreversible hydrogel-forming composition of the present invention may have a temperature (T) higher than the above sol-gel transition temperature.
h), after immersion in a large amount of water (at a volume ratio of about 0.1 to 100 times that of the gel) at the temperature Th, for a long period of time (for example, about 3 months). The gel does not dissolve.

The temperature (Th) higher than the above-mentioned sol-gel transition temperature is preferably 5 ° C. or more higher than the “sol-gel transition temperature”, and more preferably 10 ° C. or more. .

(Copolymer) The hydrogel-forming polymer that can be used in the present invention is not particularly limited as long as it has the above-mentioned properties. A copolymer obtained by binding a hydrophilic polymer and having a molecular weight of 100,000 or more is preferably used.

Here, the copolymer having a molecular weight of 100,000 or more is
At a temperature lower than the cloud point (for example, 2 ° C. or lower in absolute value), when the aqueous solution was subjected to ultrafiltration using an ultrafiltration membrane having a molecular weight cut off of 100,000 (YM100 manufactured by Amicon),
A substance that is not substantially filtered. More specifically, using an ultrafiltration membrane having a molecular weight cut off of 100,000 under the following conditions,
When ultrafiltration is performed in distilled water, the amount of polymer detected in the filtrate is 10% or less (more preferably 5% or less) of the entire polymer.

<Ultrafiltration method> A hydrogel-forming polymer was dissolved in distilled water at a temperature lower than its (first) sol-gel transition temperature at a concentration of 0.5 wt%, and the molecular weight cutoff was 10%.
Using a 10,000 ultrafiltration membrane (YM100 manufactured by Amicon)
Ultrafiltration is performed under a pressure of kg / cm2 until the amount of the filtrate becomes 1/2, and the filtrate is collected. "Substantially not filtered" means that when the polymer concentration in the filtrate is quantified, the concentration is 0.05 wt% or less (further 0.025 wt% or less).

(Cloud point) The cloud point means that a transparent polymer aqueous solution (concentration: 1 wt%) is gradually (for example, at about 1 ° C./minute).
It is the temperature at which white turbidity occurs when heated. In the present invention, the hydrogel-forming polymer (first)
In order to make the sol-gel transition temperature 5 ° C. to 40 ° C., the cloud point is desirably 5 ° C. to 40 ° C. That is, a polymer whose aqueous solution has a cloud point dissolves in water at a temperature lower than the cloud point, but becomes insoluble in water at a temperature higher than the cloud point and precipitates from water.

(Polymer Having Cloud Point) Examples of the polymer whose aqueous solution has a cloud point include poly-N-isopropylacrylamide, poly-Nn-propylacrylamide,
Poly-N-cyclopropylacrylamide, poly-N,
Poly-N-substituted acrylamide derivatives such as N-diethylacrylamide, poly-N-acryloylpiperidine, poly-N-acryloylpyrrolidine, poly-N, N-ethylmethylacrylamide, poly-N-isopropylmethacrylamide, poly-N-cyclopropyl Poly N-substituted methacrylamide derivatives such as methacrylamide,
Polyalkylene oxide such as polypropylene oxide, polyvinyl methyl ether, polyvinyl alcohol
And partially acetylated products.

The above polymer compound may be used alone or may be obtained by copolymerization with another monomer. As the monomer to be copolymerized, any of a hydrophilic monomer and a hydrophobic monomer can be used. Generally, the cloud point of a poly (N) -substituted (meth) acrylamide derivative increases when copolymerized with a hydrophilic monomer, and decreases when copolymerized with a hydrophobic monomer. Therefore, a polymer having a desired cloud point can also be obtained by selecting them.

(Hydrophilic monomer) As the hydrophilic monomer,
N-vinylpyrrolidone, vinylpyridine, acrylamide, methacrylamide, N-methylacrylamide,
Hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxymethyl methacrylate, hydroxymethyl acrylate, acrylic acid having an acidic group, methacrylic acid and salts thereof, vinyl sulfonic acid, styrene N, N-dimethylaminoethyl methacrylate having a basic group, such as sulfonic acid, N, N
-Diethylaminoethyl methacrylate, N, N-dimethylaminopropylacrylamide and salts thereof, but are not limited thereto.

(Hydrophobic monomer) On the other hand, examples of the hydrophobic monomer include ethyl acrylate, methyl methacrylate, and n
Acrylate and methacrylate derivatives such as -butyl methacrylate and glycidyl methacrylate;
Examples include, but are not limited to, N-substituted alkyl methacrylamide derivatives such as -n-butyl methacrylamide, vinyl chloride, acrylonitrile, styrene, vinyl acetate, and the like.

(Hydrophilic polymer) Examples of the hydrophilic polymer in the present invention include methyl cellulose, dextran, polyethylene oxide, polyvinyl alcohol, and the like.
Poly N-vinyl pyrrolidone, polyvinyl pyridine, polyacrylamide, polymethacrylamide, poly N-methylacrylamide, polyhydroxymethyl acryle
, Polyacrylic acid, polymethacrylic acid, polyvinyl sulfonic acid, polystyrene sulfonic acid and salts thereof,
Examples thereof include poly N, N-dimethylaminoethyl methacrylate, poly N, N-diethylaminoethyl methacrylate, poly N, N-dimethylaminopropyl acrylamide, and salts thereof. Further, various water-soluble biopolymers can also be used as the hydrophilic polymer in the present invention. For example, gelatin, albumin, globulin,
Proteins and peptides such as fibrinogen, insulin, glucagon, starch, agarose, glyco-
Polysaccharides such as gen, hyaluronic acid and heparin, RNA and DNA
And nucleic acids.

(Mechanism of thermoreversible sol-gel transition) The hydrogel-forming polymer of the present invention is a fluid aqueous solution at a low transition temperature, and loses fluidity at a high transition temperature to become a hydrogel. Figure 2 shows a thermogelling type thermoreversible sol-gel transition. According to the findings of the present inventors, the mechanism is presumed as follows.

That is, at a temperature lower than the cloud point of the polymer portion having a cloud point in the copolymer, both the polymer portion having the cloud point and the hydrophilic polymer portion are water-soluble. Completely dissolves in water. However, when the temperature of the aqueous solution is raised to a temperature higher than the cloud point, the polymer portion having the cloud point becomes water-insoluble and aggregates to cause intermolecular association. On the other hand, since the hydrophilic polymer portion maintains water solubility even at a temperature higher than the cloud point, it prevents aggregation between the polymer portions having the cloud point from leading to macroscopic phase separation, and provides a stable hydrogel. Is formed.

(Content of Polymer Having Cloud Point) In the hydrogel-forming polymer used in the present invention, the aqueous solution is combined with the polymer (A) having the cloud point and the hydrophilic polymer (B). The content of (A) in the resulting copolymer is
It is preferably in the range of 10 to 90% by weight (more preferably, 30 to 70% by weight). When the content of the polymer (A) having a cloud point is less than 10% by weight, the cohesive force between the polymer portions having the cloud point is insufficient, so that it is difficult to form a hydrogel even at a temperature higher than the cloud point. The tendency increases. On the other hand, when the content of the polymer (A) exceeds 90% by weight, the cohesive force between the polymer parts having a cloud point is too strong, and the entire system undergoes macroscopic phase separation (significant gel syneresis). And the tendency to obtain a stable hydrogel becomes stronger.

The present inventors have found that the aqueous solution of the hydrophilic polymer portion (B) has a hydrophilic polymer at a temperature lower than the cloud point of the polymer (A) having a cloud point due to, for example, hydrogen bonding. When a polymer compound (eg, gelatin, agarose, or the like) that generates cohesive force is used, a temperature lower than the first sol-gel transition temperature is used in addition to the first sol-gel transition temperature. Has a second sol-gel transition temperature in the region of
The heat which becomes a gel state at a temperature lower than the second sol-gel transition temperature and becomes a sol state at a temperature higher than the second sol-gel transition temperature and lower than the first sol-gel transition temperature. It has been found that a reversible hydrogel-forming composition can be obtained. From the viewpoint of suppressing the metabolism of cells and the like and storing in a gel, such a second sol-gel transition temperature is usually 0 ° C or more and 37 ° C or less (furthermore, 0 ° C or more and 30 ° C or less).
The following is preferred.

(Two Sol-Gel Transition Temperatures) A system (gel-forming composition) having two sol-gel transition temperatures has not heretofore been known. Thus, the mechanism by which a certain system has “two sol-gel transition temperatures” is estimated as follows according to the findings of the present inventors.

That is, at a temperature higher than the first sol-gel transition temperature, as described above, the polymer portion having a cloud point becomes water-insoluble and aggregates to cause intermolecular association. Since the hydrophilic polymer portion maintains water solubility even at a temperature higher than the cloud point, it prevents aggregation between the polymer portions having the cloud point from leading to macroscopic phase separation, and a stable hydrogel is formed. Is done.

On the other hand, at a temperature lower than the second sol-gel transition temperature, intermolecular association occurs between the hydrophilic polymers, and the polymer portion having a cloud point becomes water-soluble at a low temperature. Reversal forms a stable hydrogel.

In a temperature range lower than the first sol-gel transition temperature and higher than the second sol-gel transition temperature, between the polymer portions having a cloud point and between the hydrophilic polymer portions. Since no interaction force occurs, the hydrogel-forming polymer is assumed to be in a fluid aqueous state.

As a system in which cohesive force is generated between molecules at low temperature,
For example, when a cohesive force based on a helix-coil transition is used, as in gelatin, the sol-gel transition behavior shows hysteresis, and the sol-gel transition temperature may not coincide between when the temperature rises and when the temperature falls. This is because the helix-coil transition is a kinetically long process. In this case, for convenience, the intermediate temperature between the sol-gel transition temperature at the time of temperature rise and the sol-gel transition temperature at the time of temperature decrease is determined by the sol-gel of this system.
It can be considered as the gel transition temperature.

(Coupling Mode of Copolymer) Among the hydrogel-forming polymers usable in the present invention, the aqueous solution is obtained by bonding a polymer (A) having a cloud point and a hydrophilic polymer (B). The bonding mode of the resulting copolymer is not particularly limited.
Examples of the bonding mode include a block copolymer of A and B, a graft copolymer in which side chain B is bonded to main chain A, and a graft copolymer in which side chain A is bonded to main chain B. Style.

(Production Method of Copolymer) A block copolymer of a polymer having a cloud point and a hydrophilic polymer may be prepared by, for example, preparing a functional group (hydroxyl group, carboxyl group, amino group, isocyanate group) which is reactive to both in advance. ), And a plurality of these groups are combined by a chemical reaction.

In general, the methods for synthesizing the graft copolymer include 1) a method utilizing a chain transfer reaction of the polymer, and 2) introduction of a functional group which can be split into free radicals into the backbone polymer, from which polymerization is carried out. There are known a method of initiating, 3) a method of initiating ionic polymerization from a trunk polymer, and the like. Although the graft copolymer of the present invention can be obtained by these methods,
From the viewpoint of controlling the degree of polymerization of the side chain, one polymerizable functional group is introduced into a polymer chain having a cloud point and copolymerized with a monomer that gives a hydrophilic polymer; It is advantageous to introduce one polymerizable functional group into the polymer chain and copolymerize it with a monomer that gives a polymer having a cloud point.

(Physiologically Active Substances) The physiologically active substances are a general term for organic substances and inorganic ions which are involved in minute amounts and affect minute living phenomena operated by living organisms. In the present invention, the molecular weight is 1,000 or more, more preferably 3,000 or more (particularly,
000 or more) can be suitably used.
The bioactive substance may be a single molecule or a polymer. If necessary, two or more physiologically active substances may be used. In such an embodiment, at least one of the two or more physiologically active substances has a molecular weight of 1,000 or more. Is preferred.

Specific examples of the biopolymer which is one embodiment of the physiologically active substance usable in the present invention include, for example, collagen, gelatin, albumin, globulin and fibrino-protein.
Examples include proteins and peptides such as gene, insulin and glucagon, polysaccharides such as starch, glycogen, hyaluronic acid, cellulol and heparin, and nucleic acids such as RNA and DNA.

(Method of Containing Physiologically Active Substance) The method for incorporating the above-mentioned physiologically active substance into the thermoreversible hydrogel-forming composition is not particularly limited.

The simplest method of such a content is to mix an aqueous solution or aqueous dispersion of a physiologically active substance with an aqueous solution of a hydrogel-forming polymer at a temperature lower than its sol-gel transition temperature. Is mentioned. After the mixing,
By raising the temperature of the mixed system to a temperature higher than the sol-gel transition temperature, the physiologically active substance is taken in and held in the three-dimensional network structure in which the hydrogel-forming polymer is formed by the hydrophobic interaction. Here, when the molecular weight of the physiologically active substance is less than 1,000, the tendency that the physiologically active substance once retained in the network structure easily passes through the three-dimensional network structure and diffuses is increased, and the hydrogel is physiologically active. It becomes difficult to maintain the activity for a long time.

On the other hand, when the above-mentioned physiologically active substance is a polymer having a large molecular weight, the aqueous phase of the physiologically active substance and the aqueous phase of the hydrogel-forming polymer are liable to undergo phase separation. Difficulty of mixing uniformly tends to increase.

The present inventors have conducted intensive studies and found that a physiologically active substance can be bonded to a hydrogel-forming polymer.
It has been found that this is an extremely effective method in that the physiologically active substance is contained in the thermoreversible hydrogel-forming composition.

According to such a method of binding a physiologically active substance to a hydrogel-forming polymer, problems such as diffusion and elution from a hydrogel can be prevented even for a low-molecular-weight physiologically active substance. However, in this case, the bioactivity may be impaired by binding the low molecular weight bioactive substance to the hydrogel-forming polymer. From such a point, it is preferable that the molecular weight of the physiologically active substance used in the present invention is 1,000 or more. The reason is that, when the bioactive substance is a polymer, even if some of the functional groups are used for binding to the hydrogel-forming polymer, the site that expresses the bioactivity remains while maintaining that activity. This is presumed to be due to a high possibility of performing.

As a method for bonding the physiologically active substance and the hydrogel-forming polymer, covalent bonding is the strongest, and is therefore preferable. On the other hand, when the physiologically active substance is a polymer electrolyte, a charge having an opposite sign may be introduced into the hydrogel-forming polymer, and the hydrogel-forming polymer may be bound as an interpolymer complex via electrostatic interaction. For example, when the physiologically active substance is a polyanion such as heparin, forming a polyion complex by setting a polymer block having a cloud point or a hydrophilic polymer block in the hydrogel-forming polymer as a polycation. Can be.

As a method of covalently binding a physiologically active substance to a hydrogel-forming polymer, for example, a reactive functional group capable of binding with a functional group in the physiologically active substance is added to the hydrogel-forming polymer. There is a method in which a polymer is introduced into a polymer block having a cloud point or a hydrophilic polymer block, and both are bonded to each other. Alternatively, a polymerizable functional group (for example, an acryloyl group) may be introduced into the physiologically active substance, and copolymerized with a monomer that gives a hydrogel-forming polymer. Furthermore, a mixture of a physiologically active substance and a hydrogel-forming polymer (preferably, a mixed aqueous solution) can be irradiated with radiation to introduce a crosslinked structure between the two molecules.

(Content of Physiologically Active Substance) The content (or introduction rate) of the physiologically active substance is within a range in which the hydrogel can exhibit a physiologically active function and the thermoreversible sol-gel transition behavior is not impaired. There are no particular restrictions. Usually, the weight of the contained physiologically active substance is defined as C, as a range in which the physiological activity can be sufficiently exhibited and the physical properties of the hydrogel are not significantly affected.
Assuming that the weight of the hydrogel-forming polymer is D, the weight A of the physiologically active substance is the ratio C / (C + D) to the entire hydrogel-forming polymer including the physiologically active substance (C + D).
Is preferably in the range of 0.1 to 70 wt%, more preferably 0.5 to 10 wt% (particularly 1 to 5 wt%).

(Retention of Physiologically Active Substance) As described above, the thermoreversible hydrogel-forming composition of the present invention exhibits a hydrogel state substantially insoluble in water at a temperature higher than the first sol-gel transition temperature. In this water-insoluble hydrogel state, the initial content of the bioactive substance in the gel is defined as (A), and the content of the bioactive substance after immersing the gel in a large excess of water is defined as (A). In the case of B), the content ratio (B / A) is preferably 80% or more.
From the viewpoint of maintaining the physiological activity for a longer period, the content ratio (B / A) is preferably 90% or more, and more preferably 95% or less.

The initial content (A) and the content after immersion (B) can be suitably measured by the following method.

<Method of Measuring Content of Physiologically Active Substance> 1.5 g of a thermoreversible hydrogel-forming composition containing a physiologically active substance having an initial content (A) was prepared at a temperature lower than the first sol-gel transition temperature. A sol state is poured into a plastic petri dish having a diameter of 35 mm, gelled at a temperature higher than the sol-gel transition temperature, and a petri dish containing the gel in 250 ml of distilled water at a temperature 5 ° C. higher than the sol-gel transition temperature. Immerse. After allowing to stand still at a temperature 5 ° C. higher than the sol-gel transition temperature for 1 hour, taking out a petri dish containing the gel from water and removing excess water, the content of the physiologically active substance in the gel ( Measure B). The content of the physiologically active substance may be determined by quantifying the physiological activity of the physiologically active substance by a biological or biochemical method (for example, cell growth function, cell differentiation function, enzyme activity, etc.), Spectroscopic techniques (eg, elemental analysis, IR,
NMR, ultraviolet-visible absorption analysis, liquid chromatography, etc.). For details of such biological, biochemical, physicochemical or spectroscopic methods, see the literature (eg, Laboratory for Experimental Biology,
1-17, 1982-1985, Maruzen Co., Ltd.).

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

[0068]

EXAMPLE 1 1 g of gelatin (bovine bone, Wako Pure Chemical Industries, Ltd.) was added to distilled water 9
g at 37 ° C., and 17 mg of N-acryloyl succinimide (Kokusan Chemical Co., Ltd.) was added and reacted at 37 ° C. for 4 days to obtain an aqueous solution of polymerizable gelatin having a polymerizable group introduced into gelatin. Was.

2.0 g of N-isopropylacrylamide
Was dissolved in 390 g of distilled water, the aqueous polymerizable gelatin solution obtained above was added to an aqueous solution obtained above, and the mixture was heated to 70 ° C. under a nitrogen stream, and 1 mL of a 10 wt% ammonium persulfate (APS) aqueous solution and N, N, N 0.1 mL of ', N'-tetramethylethylenediamine (TEMED) was added. The polymerization was carried out for 4 hours while maintaining the temperature at 27 ° C. under a nitrogen stream.

The reaction solution after the above reaction was mixed with distilled water 1.
5 L was added, and the aqueous solution was concentrated to 300 mL at room temperature (27 ° C.) using an ultrafiltration membrane (HP-01, manufactured by Amicon). The concentrated solution was diluted by adding 1.5 L of distilled water, and ultrafiltration was performed again to 300 mL. This dilution and concentration operation was further repeated four times (total five times) to remove unreacted substances and low molecular weight substances.

By freeze-drying the final concentrate as described above, 1.74 g of a gelatin-bound hydrogel-forming polymer was obtained. When the gelatin content in the hydrogel-forming polymer was determined by elemental analysis (C, H, and N), it was 43 wt%.

Distilled water was added to 0.50 g of the resulting gelatin-bound hydrogel-forming polymer to make the whole 10 g,
It was dissolved at room temperature (27 ° C.) to obtain a homogeneous aqueous solution having a concentration of 5 wt%. The dynamic viscoelastic behavior of this aqueous solution was observed with a stress-controlling rheometer (CSL-500, manufactured by Carri-Med), and when heated at a rate of 1 ° C./min, the storage modulus G ′ at 35 ° C. Became a hydrogel exceeding the loss elastic modulus G ″ (first sol-gel transition temperature = 35 ° C.). After the hydrogel was heated to 45 ° C., 1 ° C. /
When cooled at a rate of minutes, it became a sol at 35 ° C (sol-
Gel transition temperature = 35 ° C). When the sol was further cooled at a rate of 1 ° C./min, it gelled at 14 ° C. (sol-gel transition temperature at the time of cooling = 14 ° C.). After further cooling to 5 ° C, 1 ° C /
Heating at a rate of minutes resulted in a sol at 28 ° C. (sol-gel transition temperature at elevated temperature = 28 ° C.). Thus, the second sol-gel transition temperature was 21 ° C (as an intermediate temperature between 14 ° C and 28 ° C).

Example 2 1 g of gelatin (manufactured by bovine bone, Wako Pure Chemical Industries, Ltd.) was mixed with distilled water 9
g was dissolved at 37 ° C., and 17 mg of N-acryloylsuccinimide (Kokusan Chemical Co., Ltd.) was added and reacted at 37 ° C. for 4 days to introduce a polymerizable group into gelatin to obtain an aqueous solution of polymerizable gelatin.

N-isopropylacrylamide 14.8
g and n-butyl methacrylate 0.76 g in ethanol
5.5.7 g of polyethylene glycol dimethacrylate (PDE-6000, NOF Corporation).
An aqueous solution obtained by dissolving 2 g in 89.2 g of distilled water and 10 g of an aqueous solution of the above-mentioned polymerizable gelatin were added thereto.
10% by weight ammonium persulfate (APS)
1 mL of an aqueous solution and 0.1 mL of N, N, N ', N'-tetramethylethylenediamine (TEMED) were added. While maintaining the temperature at 70 ° C. under a nitrogen stream, the addition of the above-mentioned APS aqueous solution and TEMED was repeated 5 times every 30 minutes to carry out polymerization. The reaction solution was cooled to room temperature and mixed with 3 L of distilled water at 4 ° C. with stirring to dissolve the reaction product. The aqueous solution was treated with an ultrafiltration membrane (HP-01, manufactured by Amicon) at 4 ° C. for 300 minutes.
Concentrated to mL. Dilute the concentrate with 3 L of distilled water,
Ultrafiltration and concentration were performed again at 4 ° C to 300 mL. This dilution and concentration operation was further repeated four times (total five times) to remove unreacted substances and low molecular weight substances.

The obtained final concentrate was freeze-dried to obtain 13.8 g of a thermoreversible hydrogel-forming composition-forming polymer having gelatin bonded thereto. Distilled water was added to 0.75 g of the resulting gelatin-bonded thermoreversible hydrogel-forming composition-forming polymer to make a total of 10 g, which was dissolved under cooling (4 ° C.) to give a uniform aqueous solution having a concentration of 7.5 wt%. did. When this aqueous solution was heated, a hydrogel was formed at room temperature, and the sol-gel transition temperature was determined by measuring dynamic viscoelasticity. The aqueous solution should be at
At a temperature lower than 5 ° C., it was always liquid and did not gel.

Comparative Example 14.8 g of N-isopropylacrylamide and 0.76 g of n-butyl methacrylate were added to 135.7 of ethanol.
g of polyethylene glycol dimethacrylate (PDE-6000, NOF Corporation)
An aqueous solution obtained by dissolving 6.2 g in 89.2 g of distilled water is added,
The mixture was heated to 70 ° C. under a nitrogen stream, and 1 mL of a 10 wt% ammonium persulfate (APS) aqueous solution and N, N, N ′, N ′
-Tetramethylethylenediamine (TEMED) 0.1
mL was added.

While maintaining the temperature at 70 ° C. in a nitrogen stream, the above A
The addition of the PS aqueous solution and TEMED was repeated 5 times every 30 minutes to polymerize. The reaction solution was cooled to room temperature and mixed with 3 L of distilled water at 4 ° C. with stirring to dissolve the reaction product. The aqueous solution is subjected to an ultrafiltration membrane (HP-01, manufactured by Amicon) at 4 ° C.
And concentrated to 300 mL. 3 L of distilled water in the concentrate
For ultrafiltration again at 4 ° C. to 300 mL. This dilution and concentration operation is performed 4 more times (5 times in total).
Unreacted substances and low molecular weight substances were repeatedly removed. The final concentrate was freeze-dried to obtain 13.8 g of a thermoreversible hydrogel-forming composition-forming polymer.

Distilled water was added to 0.75 g of the resulting thermoreversible hydrogel-forming composition-forming polymer to give a total of 10 g.
And dissolved under cooling (4 ° C.) to obtain a uniform aqueous solution having a concentration of 7.5 wt%. When this aqueous solution was heated, a hydrogel was formed at room temperature, and the sol-gel transition temperature was determined by measuring dynamic viscoelasticity. The aqueous solution was always liquid at a temperature of 0 ° C. or higher and lower than 15 ° C., and did not gel.

Example 3 The hydrogel-forming polymers obtained in Example 2 and Comparative Example were sterilized with ethylene oxide gas (ethylene oxide gas concentration: 20%, temperature: 55 ° C., 4 hours).
Each hydrogel-forming polymer was cooled (4
C.) and dissolved in RPMI1640 medium (manufactured by Gibco) containing 10% fetal bovine serum to obtain a uniform aqueous solution having a concentration of 7.5 wt%.

In each of the media (two types) obtained above, normal human lung-derived fibroblasts (trade name: NHL)
F, manufactured by Takara Shuzo Co., Ltd.) was dispersed under cooling (4 ° C.) (cell concentration: 2 × 10 ⁴ cells / mL), and the temperature was raised to 37 ° C. to gel. When cultured in a 5% carbon dioxide incubator at 37 ° C. for 3 days, when the hydrogel-forming polymer of Example 2 was used, fibroblast proliferation was observed. When the gel-forming polymer was used, fibroblast proliferation was not observed. The presence or absence of the proliferation was determined by observation with an optical microscope and SDI method (succinate dehydrogenase activity measuring method). For details on the optical microscope observation and SDI method used here, refer to the literature (for example,
Experimental Biology Course, Vol. 1-17, 1982-1985, Maruzen Co., Ltd.).

Example 4 The hydrogel-forming polymers obtained in Example 2 and Comparative Example were sterilized with ethylene oxide gas and cooled (4 ° C.)
Then, each was dissolved in RPMI1640 medium (manufactured by Gibco) containing 10% fetal bovine serum to obtain a uniform aqueous solution having a concentration of 10 wt%.

In 0.1 mL of this medium, 100 C3H / He mouse islets of Langerhans (La Islet) cells were dispersed under cooling (4 ° C.), and the temperature was raised to 37 ° C. to gel.
RPMI containing 10% fetal bovine serum in hydrogel containing Lajishima
3 mL of 1640 medium was added, the cells were cultured in a 5% carbon dioxide incubator at 37 ° C. for 30 days, and the insulin concentration in the culture solution changed every two days was measured. When the hydrogel-forming polymer of Example 2 was used, the insulin concentration was 5.8 m in the early stage of the culture.
U / mL, maintained a high value of 5.6 mU / mL after 30 days (that is, Lajit cell maintained its activity).
On the other hand, when the hydrogel-forming polymer of the comparative example was used, the insulin concentration was 3.5 mU / mL in the early stage of the culture,
After 30 days, the value changed to a low value of 1.0 mU / mL.

As described above, by including gelatin having a cell adhesion factor in a thermoreversible hydrogel-forming composition, the insulin secretion ability of cell tissues (Lajishima) is expressed in the hydrogel for a long period of time. It became clear that it could be maintained.

[0084]

As described above, according to the present invention, the sol-gel transition temperature (first sol-gel transition temperature) contains at least a hydrogel-forming polymer, water and a physiologically active substance. 5 ° C. or higher and 40 ° C. or lower, a heat-insoluble hydrogel state at a temperature higher than the sol-gel transition temperature, and reversibly water-soluble at a temperature lower than the sol-gel transition temperature. A reversible hydrogel-forming composition; and in the water-insoluble hydrogel state, the initial content (A) of the bioactive substance in the gel, and the gel after being immersed in a large excess of water. A thermoreversible hydrogel-forming composition, wherein the ratio (B / A) to the content (B) of the physiologically active substance in the composition is 80% or more.

According to the present invention, the composition further comprises at least a hydrogel-forming polymer, water and a physiologically active substance;
Further, there is provided a thermoreversible hydrogel-forming composition having at least first and second sol-gel transition temperatures.

When the thermoreversible hydrogel-forming composition of the present invention is used, various physiologically active functions (eg, biocompatibility, cell adhesion, etc.) can be added to the conventional gelled thermoreversible hydrogel-forming composition at elevated temperature. , Cell proliferation ability, cell differentiation ability, etc.). Therefore, applications utilizing these physiologically active functions, for example, cell (tissue) culture equipment, wound dressing, bioadhesive, DDS
It can be used without particular limitation as a base material for use, an embolic agent, a joint lubricating agent, and the like.

Further, the thermoreversible hydrogel-forming composition of the present invention can be provided with a gelling property at the time of cooling to the conventional gelling type thermoreversible hydrogel-forming composition at the time of heating. Therefore, it is possible to bring innovative products to the biochemistry or medical fields as described above.

Claims (7)

[Claims] 1. A sol-gel transition temperature (first sol-gel transition temperature) of at least 5 ° C. and not more than 40 ° C., comprising at least a hydrogel-forming polymer, water and a physiologically active substance. A thermoreversible hydrogel-forming composition which becomes a substantially water-insoluble hydrogel state at a temperature higher than the sol-gel transition temperature, and which is reversibly water-soluble at a temperature lower than the sol-gel transition temperature. And in the water-insoluble hydrogel state, the initial content (A) of the bioactive substance in the gel, and the content (B) of the bioactive substance in the gel after immersion in a large excess of water. )
A thermoreversible hydrogel-forming composition having a ratio (B / A) of at least 80%. 2. The biologically active substance having a molecular weight of 1,000.
The thermoreversible hydrogel-forming composition according to claim 1, which is the above. 3. The thermoreversible hydrogel-forming composition according to claim 1, wherein the physiologically active substance is bound to the hydrogel-forming polymer. 4. The thermoreversible hydrogel-forming composition according to claim 1, wherein the physiologically active substance is a biopolymer. 5. The method according to claim 1, wherein the second sol-gel transition temperature is lower than the first sol-gel transition temperature and the second sol-gel transition temperature is not lower than 0 ° C. and not higher than 37 ° C.
A gel state at a temperature lower than the second sol-gel transition temperature, and higher than the second sol-gel transition temperature, and the first sol-gel transition The thermoreversible hydrogel-forming composition according to claim 1, which is in a sol state at a temperature lower than the temperature. 6. At least a hydrogel-forming polymer, water and a physiologically active substance; and at least a first sol-gel transition temperature and a second sol-gel transition temperature lower than the first sol-gel transition temperature. A thermoreversible hydrogel-forming composition having a sol-gel transition temperature of 7. A substantially water-insoluble hydrogel state at a temperature higher than the first sol-gel transition temperature, and reversibly water-soluble at a temperature lower than the sol-gel transition temperature; 7. A gel state at a temperature lower than the sol-gel transition temperature of; and a sol state at a temperature range higher than the second sol-gel transition temperature and lower than the first sol-gel transition temperature. The thermoreversible hydrogel-forming composition according to the above.