WO2015133439A1

WO2015133439A1 - Pullulan gel, method for producing same, and use of same

Info

Publication number: WO2015133439A1
Application number: PCT/JP2015/056103
Authority: WO
Inventors: 寿一馬場; 理恵子鈴木
Original assignee: 日本全薬工業株式会社
Priority date: 2014-03-07
Filing date: 2015-03-02
Publication date: 2015-09-11
Also published as: JPWO2015133439A1; JP6211680B2

Abstract

The present invention provides a novel method of utilizing pullulan. For example, the present invention relates to: a pullulan gel which is produced by crosslinking multiple pullulan molecules; a pullulan gel as mentioned above, in which a protein is crosslinked with the pullulan molecules; a dried product of the pullulan gel; a pharmaceutical composition; a protein detection kit; and others. The present invention also relates to: methods respectively for producing these products; and others.

Description

Pull-langer and its production method and use

The present invention relates to pullulan gel and its production method and use.

Pullulan is a water-soluble polysaccharide in which three molecules of glucose are linked by α-1,4 and maltotriose is linked by α-1,6 bonds, and is currently widely used as a food additive and pharmaceutical supplement. .

For example, Patent Document 1 describes a water-soluble conjugate preparation composed of a drug and a water-soluble polysaccharide pullulan for the purpose of targeting the drug to the liver. This conjugate preparation is produced by reacting pullulan with cyanuric chloride and then chemically bonding the cyanurized pullulan and the drug.

Non-Patent Document 1 describes a pullulan nanogel to which hydrophobic cholesterol and a cationic functional group are added. This nanogel is produced by assembling hydrophilic pullulan utilizing the property that hydrophobic cholesterol gathers together in an aqueous solvent.

Japanese Patent Publication “JP 9-124512 A” (published May 13, 1997)

As described above, various utilization methods of pullulan have been studied, but the water-soluble nature of pullulan has been exclusively utilized. It would be desirable to provide new ways to use pullulan.

The pullulan gel according to the present invention is formed by crosslinking a plurality of pullulans.

The present invention is also a method for producing a pullulan gel in which a plurality of pullulans are crosslinked, a step of stirring an aqueous solution containing pullulan and a crosslinking agent represented by the following general formula (2), and the aqueous solution after the step A pullulan gel production method comprising the step of cross-linking by standing.

(In Formula (2), a plurality of R ¹ to R ³ are each independently a chlorine atom, a fluorine atom, a bromine atom or an iodine atom)
The present invention is also a method for producing a pullulan gel in which a plurality of pullulans are crosslinked, a step of stirring an aqueous solution containing pullulan and a crosslinking agent represented by the general formula (2), and the aqueous solution after the steps And a step of cross-linking by stirring in a water-insoluble liquid.

The present invention is also a method for producing a pullulan gel in which a plurality of pullulans are crosslinked, a step of stirring an aqueous solution containing pullulan and a crosslinking agent represented by the general formula (2), and the aqueous solution after the steps A step of emulsifying by emulsifying in a water-insoluble liquid, and a step of performing cross-linking by allowing the emulsified solution to stand, wherein at least one of the aqueous solution and the water-insoluble liquid is: A method for producing pullulan gel comprising an emulsifier is provided.

According to the present invention, a new method for using pullulan can be provided.

In an Example, it is a figure which shows the ground pullulan gel and its particle diameter. In an Example, it is a figure which shows transition of the Der f 2-specific IgG antibody in a rat serum. In an Example, it is a figure which shows the result after pullulan microgel and each process. In an Example, it is a figure which shows the pullulan film. In an Example, it is a figure which shows the protein sustained release property of each pullulan gel film. In an Example, it is a figure which shows the particle diameter of a pullulan microgel.

[Pull Langer and production method]
(Pull Langer)
The pullulan gel according to the present invention is formed by crosslinking a plurality of pullulans. Pullulan is a polysaccharide in which maltotriose, in which three molecules of glucose are α-1,4 linked, is linked by α-1,6 bonds. The average molecular weight of the pullulan constituting the pullulan gel according to the present invention is not particularly limited and may be appropriately selected according to the purpose, but may be, for example, 20 kDa to 1600 kDa. Unless otherwise specified in this specification, “A to B” representing a numerical range means “A or more and B or less”.

In one embodiment of the pullulan gel according to the present invention, the protein, peptide or amino acid may be cross-linked to pullulan.

In the pullulan gel according to the present invention, the structure of the crosslinked portion where a plurality of pullulans are crosslinked is not particularly limited. For example, the plurality of pullulans are crosslinked by a structure represented by the following general formula (1).

In the formula (1), at least two of the plurality of * are bridging points with pullulan.

In one embodiment, pullulan gel is formed by crosslinking only pullulan, in which case * not a crosslinking point with pullulan is a hydroxy group. Specifically, two of the plurality of * are crosslinking points with pullulan, the other * is a hydroxy group, or three of the plurality of * are crosslinking points with pullulan. Contains some cross-linked structure. That is, the pullulan gel includes a structure in which two or more molecules, preferably three or more molecules of pullulan are crosslinked by one crosslinked structure. The pullulan gel may contain a structure represented by the above general formula (1) in which only one of a plurality of * is bonded to the pullulan, that is, does not constitute a bridge. .

In another embodiment, a protein, peptide or amino acid may be further cross-linked to pullulan. The structure of the cross-linked moiety in which the protein, peptide or amino acid is cross-linked to pullulan is not particularly limited. For example, it is cross-linked by the structure represented by the general formula (1). In that case, * that is not a cross-linking point with pullulan is a cross-linking point with protein, peptide or amino acid. That is, the pullulan gel contains a cross-linked structure in which two of the plurality of * are cross-linking points with pullulan, and other * are cross-linking points with proteins, peptides or amino acids.

The pullulan gel according to the present invention has many such crosslinked structures. For this reason, in one embodiment, a large number of pullulans are configured in a network via a large number of cross-linked structures. Note that pullulans are not directly connected to each other. Pullulan is considered to be crosslinked with hydroxy groups at random positions. Therefore, pullulan may have a plurality of crosslinking points in one molecule. In addition, when a protein, peptide or amino acid is cross-linked, the protein, peptide or amino acid may be present inside and on the surface of the pullulan gel.

In many cross-linked structures included in one pullulan gel, there is no need for one type of cross-linking. For example, in a pullulan gel in which proteins are cross-linked, proteins are bound to at least some of the many cross-linked structures. That's fine. That is, in one embodiment, in some of the plurality of crosslinking structures, two of the plurality of * are crosslinking points with pullulan, and the other * is a crosslinking point with proteins, In another part of the structure, two of the plurality of * are cross-linking points with pullulan, and the other * is a hydroxy group. In addition, in the pullulan gel, two proteins and one pullulan are bonded in addition to the above-described crosslinking structure (that is, one * is a crosslinking point with pullulan and two * is a crosslinking point with protein). It may contain a crosslinked structure. In addition to the above-mentioned cross-linking structure, one protein and one pullulan are bonded (that is, one * is a cross-linking point with pullulan, one * is a cross-linking point with protein, and one * May be a crosslinked structure).

When a protein or peptide is cross-linked, it is considered that the amino acid residues constituting the protein or peptide are cross-linked by the hydroxy group of amino acids having a hydroxy group in the side chain (serine, tyrosine, threonine, etc.). It is done. Moreover, the carboxy group of an amino acid (such as glutamic acid and aspartic acid) having a carboxy group in the side chain can also be crosslinked. However, it is not necessarily limited to these amino acids.

Examples of proteins that are cross-linked to pullulan gel include antibodies, antigens, receptors, ligands for receptors, enzymes, ligands for enzymes, hormones, and functional fragments thereof. The protein may be a natural protein or a non-natural protein. Such a pullulan gel in which proteins are crosslinked can be used as, for example, a protein detection kit described later.

As used herein, “peptide” refers to a compound in which two or more amino acids are connected by peptide bonds. “Peptide” also includes oligopeptides and polypeptides.

Examples of peptides that are cross-linked to pullulan gel include fragments other than the functional fragments of the above protein. A pullulan gel in which such a peptide is cross-linked can also be used, for example, as a protein detection kit described later.

The amino acid cross-linked to pullulan gel may be a natural amino acid or a non-natural amino acid. The amino acid is preferably an amino acid having a hydroxy group. Examples of amino acids having a hydroxy group include serine, tyrosine, threonine, hydroxyproline, hydroxylysine, and derivatives thereof. In addition, it can bridge | crosslink also in the carboxy group of an amino acid.

The pullulan gel according to the present invention may be a hydrogel or an organogel. Examples of the organic solvent in the organogel include methanol, ethanol, acetone, or chloroform. As shown in Examples described later, the pullulan gel according to the present invention is stable not only with water but also with an organic solvent such as methanol.

The size and shape of pullulanger are not particularly limited, and may be set as appropriate according to the purpose of use. Examples of the shape include a spherical shape, a rectangular parallelepiped, and a sheet shape. The size may be, for example, a size of several tens of cm squares or a size on the order of μm (a pullulan microgel). Further, the size may be on the order of nm (pullulan nanogel). Further, it may be a sheet having a thickness of 1 μm to 3 mm. In one embodiment, particles having an average particle diameter of 50 nm to 1 mm are preferable. The “average particle diameter” can be calculated as an average particle diameter in terms of volume using a measurement method by a laser diffraction / scattering method.

The pullulan microgel and pullulan nanogel of the present invention can be of a size having needle penetration and can be administered to a living body by injection. As shown in the examples described later, pullulan gel does not show irritation and inflammatory reaction when administered subcutaneously, and can be safely administered to a living body.

The color of the pullulan gel of the present invention (when not impregnated with a colored liquid or a colored drug) varies depending on the production conditions, but is transparent to white, preferably transparent. The pullulan gel of the present invention is preferably uniform in color.

Moreover, the pullulan gel according to the present invention may be impregnated with a drug. Examples of the drug include medical drugs, experimental drugs, vaccines, and the like. For example, in addition to the above-described proteins and peptides, biomolecules such as nucleic acids (DNA, RNA, etc.) and other chemical substances may be mentioned. In pullulan gel impregnated with a drug, the drug is not cross-linked with pullulan and is dissolved or suspended in a liquid (water or an organic solvent) retained by the pullulan gel. The pullulan gel impregnated with a drug can also be used as a material having a sustained release property, and can be suitably used in the medical field, for example, as an adjuvant, a wound dressing, and an anti-adhesion agent.

The pullulan gel according to the present invention can maintain its shape even when subjected to autoclave (121 ° C., 20 minutes). Therefore, when the pullulan gel according to the present invention is applied to a living body or the like, or used for an experiment or the like, sterilization by an autoclave can be performed. When using for an autoclave, it is preferable to carry out the pullulan gel soaked in water. In addition, the pullulan gel of the present invention can maintain its shape even when it is frozen and thawed. Therefore, frozen storage is also possible.

Moreover, the pullulan gel according to the present invention does not substantially contain a toxic substance such as cyanuric chloride used in the production method described later. Moreover, the crosslinked structure itself has almost no toxicity to the living body. Therefore, the pullulan gel according to the present invention can be safely applied to a living body.

The pullulan gel according to the present invention may be a hybrid gel containing a polysaccharide other than pullulan. The polysaccharide is not cross-linked to pullulan but is part of the pullulan gel by being entangled with the network structure of the pullulan constituting the pullulan gel. In the pullulan gel in which the protein is cross-linked, the polysaccharide may be entangled in the protein. The polysaccharide may be present inside and / or outside the pullulan gel. A plurality of molecular polysaccharides are entangled with a plurality of molecular pullulans, so that pullulan and the polysaccharides are stretched in a network in a pullulan gel. Examples of the polysaccharide include hydroxypropyl β-cyclodextrin, dextran, sodium dextran sulfate, β-cyclodextrin, sodium alginate, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, xanthan gum, gum arabic, chitosan , And trehalose, and derivatives thereof. The polysaccharide may be one type or two or more types. Such a hybrid gel has the advantage that it can be gelled by combining a gel that is not gelled with a polysaccharide alone with pullulan. For example, a hybrid gel with hydroxypropyl β-cyclodextrin is expected to have a high adjuvant effect. Further, for example, pullulan gel injected subcutaneously is difficult to decompose, but can be easily decomposed by combining with these polysaccharides. Furthermore, a hybrid gel with β-cyclodextrin can have a sustained release property when impregnated with a low molecular compound.

Moreover, the pullulanger according to the present invention may hold an arbitrary object inside. Examples of the object include a magnetic body. Such pullulan gel can be used for various applications utilizing the properties of the object. For example, when a magnetic substance is held inside, there is a usage method such as protein affinity purification.

(Production method)
<Manufacturing method 1>
In one embodiment of the method for producing pullulan gel, a step of stirring an aqueous solution containing pullulan and a crosslinking agent represented by the following general formula (2), and a step of cross-linking by standing the aqueous solution after the step are performed. Including.

In the formula (2), a plurality of R ¹ to R ³ are each independently a chlorine atom, a fluorine atom, a bromine atom or an iodine atom. The crosslinking agent is preferably cyanuric chloride (the following general formula (3)) in which all of R ¹ to R ³ are chlorine atoms.

The concentration of pullulan contained in the aqueous solution is not particularly limited, but may be, for example, 2% (w / v) to 15% (w / v). The hardness of the pullulan gel can be adjusted by the concentration of pullulan contained in the aqueous solution. The higher the pullulan concentration, the harder the pullulan gel. Further, the concentration of the crosslinking agent contained in the aqueous solution is not particularly limited, but may be, for example, 0.1% (w / v) to 0.3% (w / v). Depending on the concentration of the crosslinking agent contained in the aqueous solution, the hardness of the pullulan gel can be adjusted. The higher the concentration of the crosslinking agent, the harder the pullulan gel. A commercially available thing can be used for a pullulan and a crosslinking agent.

The pH of the aqueous solution is preferably 5.0 to 10.0, more preferably 5.0 to 8.0, still more preferably 6.0 to 8.0, and 6.5 to More preferably, it is 7.5, and it is especially preferable that it is pH 7.0. When pH is too high, it will become a non-uniform cloudy gel which has a part from which hardness differs. When sodium hydroxide is used, pullulan undergoes a chemical reaction and develops (colors) from yellow to tan in a few minutes (see also reference examples described later). This is probably because the structure of the pullulan itself has changed. Therefore, the pullulan gel of the present invention cannot be produced using sodium hydroxide.

The temperature at the time of stirring is preferably 0 ° C. to 45 ° C., and more preferably 0 ° C. to 37 ° C. The stirring speed can be, for example, 10 rpm to 1000 rpm, preferably 100 rpm to 500 rpm. The stirring time can be, for example, 1 minute to 5 hours, preferably 5 minutes to 3 hours. Even if the stirring time is short, pullulan gel can be produced in the step of performing cross-linking described later.

In addition to pullulan and a crosslinking agent, the aqueous solution contains, for example, a pH adjusting agent such as sodium bicarbonate (sodium bicarbonate) or sodium carbonate for adjusting pH, an organic solvent such as acetone for dissolving the crosslinking agent, and the like. May be included.

In one embodiment, a crosslinking agent dissolved in an organic solvent is added to the aqueous pullulan solution and the resulting aqueous solution is stirred. In another embodiment, a powdered crosslinker is added to the aqueous pullulan solution and the resulting aqueous solution is stirred.

By stirring, a crosslinking agent having low solubility in water (cyanuric chloride or the like) can be dissolved, and pullulan and the crosslinking agent can be uniformly present in the solution.

After the stirring and before cross-linking, a step of forming the aqueous solution in a mold having a desired size and shape may be performed.

The temperature at the time of crosslinking can be, for example, −20 ° C. to 45 ° C., preferably −20 ° C. to 37 ° C., more preferably 0 ° C. to 37 ° C., and 0 ° C. to 30 ° C. More preferably. The time for crosslinking may be appropriately set according to the size of the container or mold containing the aqueous solution, etc., and can be, for example, 30 minutes to 18 hours, preferably 30 minutes to 5 hours, and more preferably. Is 1 to 4 hours.

When cyanuric chloride is used as a cross-linking agent, it is considered that a chlorine atom is liberated in water and a hydroxyl group is formed at a position where pullulan is not bonded.

The pullulan gel thus obtained may be cut or pulverized into a desired size and shape. The pullulan gel can be pulverized using a pulverizer, a homomixer, a Waring blender, a mortar, or the like, and can be pulverized to the μm order or, in some cases, the nm order. Further, the obtained pullulan gel can be dried and then pulverized.

Further, it may further include a step of washing the cross-linking agent and pullulan adhering to the pullulan gel. The washing step can be performed using water, for example.

<Manufacturing method 2>
In another embodiment of the method for producing pullulan gel, a step of stirring an aqueous solution containing pullulan and the crosslinking agent represented by the general formula (2), and stirring the aqueous solution in a water-insoluble liquid after the step is performed. Cross-linking. This production method is particularly preferred when producing spherical pullulan microgels and pullulan nanogels.

The step of stirring the aqueous solution containing pullulan and the crosslinking agent is as described above, but the concentration of pullulan contained in the aqueous solution is preferably 4% (w / v) or more.

The step of allowing the aqueous solution to stand may be included before the step of crosslinking. The standing time can be, for example, 1 minute to 60 minutes. The temperature at the time of standing can be, for example, 4 ° C. to 37 ° C. It is preferable to carry out until the aqueous solution is slightly pulled.

The water-insoluble liquid used in the cross-linking step is preferably a liquid at room temperature, and oils such as sunflower oil, olive oil, panacet 810 (registered trademark), and soybean oil; hexane, benzene, toluene, xylene, and Although hydrocarbons, such as ethyl acetate, are mentioned, it is not limited to these. Although the viscosity of oil is not specifically limited, For example, the thing of the range of common vegetable oil can be used. The higher the viscosity of the water-insoluble liquid, the smaller the average particle size of the pullulan gel. From the viewpoint of maintaining the particle size, high viscosity oil is preferred.

The amount of the aqueous solution with respect to the water-insoluble liquid is preferably 1% by volume to 50% by volume, more preferably 1% by volume to 20% by volume, and still more preferably 1% by volume to 10% by volume. . The stirring speed at the time of crosslinking is not particularly limited and may be appropriately set according to the size of the pullulan gel to be produced. For example, the stirring speed can be 100 rpm to 1000 rpm, and preferably 500 rpm to 1000 rpm. The faster the stirring speed, the smaller the average particle size of pullulan gel. The temperature at the time of crosslinking is preferably −20 ° C. to 45 ° C., more preferably −20 ° C. to 37 ° C., and further preferably 0 ° C. to 30 ° C. The stirring time can be, for example, 30 minutes to 5 hours, and preferably 1 hour to 4 hours.

When left standing without stirring, the dispersed pullulans are adsorbed to each other and a spherical gel is not generated, but in this production method, small droplets of an aqueous solution containing pullulan and a crosslinking agent are generated in a water-insoluble liquid, Crosslinking proceeds in these droplets while being dispersed by stirring. Therefore, a smaller spherical pullulan gel (pullulan microgel or pullulan nanogel) is produced.

The method for recovering the produced pullulan gel from the water-insoluble liquid is not particularly limited, but it is preferable to add water before recovery. Thereby, pullulan gel floats in water and adsorption | suction of pullulan gels can be suppressed.

After collection, the pullulan gel may be allowed to stand and cross-linking may proceed further.

<Manufacturing method 3>
In still another embodiment of the method for producing pullulan gel, a step of stirring an aqueous solution containing pullulan and the crosslinking agent represented by the above general formula (2), and after the step, the aqueous solution is stirred in a water-insoluble liquid. And emulsifying the emulsified solution and the step of cross-linking the emulsified solution by standing. At least one of the aqueous solution and the water-insoluble liquid contains an emulsifier. This production method is particularly preferred when producing spherical pullulan microgels and pullulan nanogels.

Examples of the emulsifier include surfactants such as sodium lauryl sulfate, DKS NL-15, and DKS NL-50. The emulsifier may be contained in an aqueous solution containing pullulan and a crosslinking agent, may be contained in a water-insoluble liquid, or may be contained in both. The concentration of the emulsifier contained in the aqueous solution or the water-insoluble liquid is not particularly limited as long as it is within a concentration range that forms a water-in-oil (W / O type) emulsion. Such a concentration range can be appropriately determined by those skilled in the art based on the component ratio between the aqueous solution and the water-insoluble liquid, the temperature, and the like.

The step of allowing the aqueous solution to stand may be included before the step of crosslinking. The standing time can be, for example, 10 minutes to 30 minutes. It is preferable that the temperature at the time of standing is 5 degrees C or less. By setting the temperature to 5 ° C. or lower, the progress of the crosslinking reaction is delayed, it is easy to moderately increase the viscosity, and emulsification is facilitated.

The kind of water-insoluble liquid used in the cross-linking step is the same as in production method 2.

The amount of the aqueous solution with respect to the water-insoluble liquid can be, for example, 1% by volume to 30% by volume. The stirring speed at the time of emulsification is not particularly limited, and may be set as appropriate as long as emulsification can be achieved. For example, the stirring speed may be 100 rpm to 1000 rpm. The faster the stirring speed, the more uniform the emulsion. The temperature at the time of emulsification can be, for example, 0 ° C. to 30 ° C. The stirring time can be, for example, 1 minute to 10 minutes. The temperature at the time of crosslinking can be, for example, 0 ° C. to 30 ° C. Leave to crosslink.

When left without being emulsified, the pullulan once dispersed adsorbs and does not produce a spherical gel, but in this production method, small droplets of an aqueous solution containing pullulan and a crosslinking agent are produced in a water-insoluble liquid, By emulsifying with an emulsifier, crosslinking proceeds in these droplets while being stably dispersed. Therefore, a smaller spherical pullulan gel (pullulan microgel or pullulan nanogel) is produced. You may select what has a desired particle size by centrifugation.

After collection, the pullulan gel may be allowed to stand and cross-linking may proceed further. Alternatively, the emulsifier may be removed by dispersing pullulan gel in a W / O system and repeating centrifugal separation and recovery.

<Manufacturing method 4>
When producing a pullulan gel in which proteins, peptides or amino acids are further crosslinked, the aqueous solution further contains proteins, peptides or amino acids. Therefore, in another embodiment of the method for producing pullulan gel, the step of stirring the aqueous solution containing pullulan and the crosslinking agent represented by the general formula (2) and the protein, peptide or amino acid, and the aqueous solution after the step And carrying out cross-linking. In still another embodiment, a step of stirring an aqueous solution containing pullulan, the crosslinking agent represented by the general formula (2), and a protein, peptide, or amino acid, and stirring the aqueous solution in a water-insoluble liquid after the step. And the step of cross-linking. In yet another embodiment, a step of stirring an aqueous solution containing pullulan, the crosslinking agent represented by the general formula (2), and a protein, peptide, or amino acid, and the aqueous solution in a water-insoluble liquid after the step. A step of emulsifying by stirring and a step of cross-linking by allowing the emulsified solution to stand are included, and at least one of the aqueous solution and the water-insoluble liquid contains an emulsifier.

Specific description is the same as that of manufacturing method 1, manufacturing method 2, and manufacturing method 3, respectively.

The concentration of the protein, peptide or amino acid in the aqueous solution may be appropriately set according to the amount to be cross-linked to the pullulan gel, and may be, for example, 10 μg / mL to 1 mg / mL.

<Manufacturing method 5>
In the case of producing an organogel, the method further includes a step of drying the pullulan gel obtained by the above-described production method and a step of immersing the dried pullulan gel in an organic solvent.

The types of organic solvents are as described above. The drying temperature can be, for example, 4 ° C. to 40 ° C. The drying time may be appropriately set according to the size and thickness of pullulan gel, and can be, for example, 30 minutes to 5 hours. Drying may be performed under atmospheric pressure, or may be performed under reduced pressure or under vacuum. The dipping time may be set appropriately according to the thickness of the gel, and can be set to, for example, 1 minute to 5 hours.

<Manufacturing method 6>
When producing a pullulan gel (hybrid gel) containing a polysaccharide other than pullulan, the polysaccharide may be included in the aqueous solution. The kind of polysaccharide is as above-mentioned. The polysaccharide may be one type or two or more types. The concentration of the polysaccharide in the aqueous solution is not particularly limited, and can be, for example, 1% to 10% (w / v) (total concentration in the case of two or more types).

<Manufacturing method 7>
When producing a pullulan gel in which an arbitrary object is held, for example, the object may be contained in the aqueous solution.

In addition, the production methods 1 to 7 mentioned as the production method of pullulan gel can be appropriately combined. For example, it is possible to produce a pullulan microgel in which an arbitrary object is held and a protein is crosslinked.

In the production method according to the present invention, heating is not necessary, and it can be performed even at a temperature below room temperature. Furthermore, in the production method according to the present invention, crosslinking can be performed near neutrality. Therefore, it is simple and can be produced without denaturing the protein. In the known production method of hydrogels of other polysaccharides (for example, agarose etc.), it is necessary to heat or to make the pH to the alkali side. On the other hand, in the production method of pullulan gel of the present invention, heating is performed. It is excellent in that it is unnecessary and can be produced near neutrality.

[Dried pullulanger and method for producing the same]
(Dried pullulanger)
The dried pullulan gel according to the present invention is a dried product obtained by drying the pullulan gel described above. The size and shape of the dried pullulan product are not particularly limited, and may be set as appropriate according to the purpose of use. The pullulan gel to be dried may be a pullulan gel cross-linked with protein or the like, or a pullulan gel impregnated with a drug. When the pullulan gel is immersed in a liquid such as water, the pullulan gel sucks the liquid and returns to the original pullulan gel.

One embodiment of the dried pullulan gel is a dried pullulan microgel or pullulan nanogel. For example, a pullulan microgel or pullulan nanogel used as a pharmaceutical composition having a sustained release property of a drug described later is dried. Since a dried product has higher storage stability than a gel, for example, when a pullulan microgel or a pullulan nanogel containing a drug is used as a preparation, it can be suitably stored as a dried product.

Another embodiment of the dried pullulan product is a film having a thickness of, for example, 1 μm to 500 μm. A known pullulan film obtained by adding water to a pullulan and heating and press-molding is soluble in water. However, the pullulan gel film according to the present invention is insoluble in water and becomes a sheet-like gel when immersed in water. Therefore, a pullulan gel film in which a protein is crosslinked can be washed by reacting an antibody or an antigen, and can be suitably used for protein detection.

In addition, when the dried pullulan gel is immersed in a liquid in which a desired drug (for example, protein) is dissolved or dispersed, the liquid containing the drug penetrates into the dried pullulan gel and becomes a gel. This gel retains the penetrating drug and does not elute immediately. Therefore, it can be used as a sustained release gel. Therefore, the pullulan gel dried product can be used as a material for producing a sustained-release gel.

∙ Dried protein with a cross-linked protein does not elute even if it absorbs water. Therefore, it can be suitably used for protein detection and the like.

(Production method)
One embodiment of the method for producing a dried pullulan product includes a step of drying the above-described various forms of pullulan gel.

The drying temperature is not particularly limited, and can be, for example, 20 ° C. to 100 ° C. The drying time may be appropriately set according to the size and thickness of pullulan gel, and can be set to, for example, 1 second to 10 days. Drying may be performed under atmospheric pressure, or may be performed under reduced pressure or under vacuum. Alternatively, lyophilization may be performed, for example, at −15 ° C. or lower, preferably −50 ° C. or lower.

[Pharmaceutical composition and production method thereof]
(Pharmaceutical composition)
The pullulan gel according to the present invention can be used as a pharmaceutical composition having sustained drug release (see Examples). Moreover, the pullulan gel which concerns on this invention can be utilized as a pharmaceutical composition which has an adjuvant effect (refer an Example).

The pharmaceutical composition may be used in contact with the skin of a living body, or may be used after being administered in vivo. Gelatin hydrogel (Medgel (registered trademark)) is known as a gel having sustained release properties. However, since gelatin produced from bovine bone is used, there is a possibility that BSE or the like may remain. On the other hand, the pullulan gel of the present application can be safely applied to a living body because there is no such concern.

In one embodiment, the pharmaceutical composition is a pullulan gel in the order of nm to μm (or up to about 1 mm) impregnated with a drug. Such a pharmaceutical composition can be suitably administered in vivo. A preferred example is pullulagel having an average particle diameter of 50 nm to 1 mm. Another preferred example is pullulan gel having an average particle diameter of 5 μm to 200 μm impregnated with a drug. Since pullulan gel having this average particle diameter remains at the administration site when administered subcutaneously, the drug can be applied locally. The pullulan gel according to the present invention acts as an adjuvant as shown in the examples. Therefore, the pharmaceutical composition can be a composition having sustained release of the drug and an adjuvant effect. The pharmaceutical composition can be formulated, for example, as a vaccine.

In another embodiment, the pharmaceutical composition is a pullulan gel on the order of mm to cm impregnated with the drug. Such a pharmaceutical composition is suitably applied to living skin, and can be used as, for example, a wound dressing.

In yet another embodiment, the pharmaceutical composition is a pullulan gel crosslinked with proteins, peptides or amino acids. Such a pharmaceutical composition is considered to have an adjuvant effect as it stays without eluting proteins and is phagocytosed by macrophages and dendritic cells. Moreover, in order to give such a pharmaceutical composition sustained release, the pullulan gel can also be made into a hybrid gel with other polysaccharides that are easily degraded in the body.

Also, the pharmaceutical composition may be bound or impregnated with a substance that stimulates and activates dendritic cells and / or NKT cells. Examples of substances that activate dendritic cells include ligands for Toll-like receptors. Examples of substances that activate NKT cells include α-galactosylceramide (α-GalCer).

(Production method)
In one embodiment of the method for producing a pharmaceutical composition, the steps of drying various forms of pullulan gel produced by the above-mentioned production method, and the step of immersing the dried pullulan gel in a liquid containing the above-mentioned agent. Including.

The pullulan gel method in the order of nm to μm may be produced in a water-insoluble liquid as described above, or may be a pulverized pullulan gel produced in an aqueous solution. From the viewpoint of uniformity, pullulan gel is preferably produced in a water-insoluble liquid. The specific method of the drying step is as described above. The dipping time in the dipping step may be set appropriately according to the size of pullulan gel, etc., and may be, for example, 1 to 10 minutes. Further, the concentration of the drug contained in the solution is not particularly limited, and can be, for example, 10 μg / mL to 5 mg / mL. In addition, since the size of pullulan gel before drying and the size after immersion are almost the same, if the average particle size before drying is adjusted to the desired average particle size, the drug having the desired average particle size Can be obtained. Therefore, for example, if a pull lagel having an average particle diameter of 50 nm to 1 mm before drying is used, a pull lagel having an average particle diameter of 50 nm to 1 mm impregnated with a drug can be obtained.

Further, when a pullulan gel with a protein cross-linked is used as a pharmaceutical composition, the pullulan gel may be manufactured by the above-described manufacturing method with a protein cross-linked.

[Protein detection kit]
The protein detection kit according to the present invention includes a pullulan gel in which a protein, peptide or amino acid is crosslinked to pullulan by the structure represented by the general formula (1) or a dried product thereof.

Examples of the protein include antibodies, antigens, receptors, ligands for receptors, enzymes, ligands for enzymes, hormones, and functional fragments thereof.

The protein detected by the protein detection kit is a protein that interacts with a protein, peptide, or amino acid contained in pullulan gel. For example, in a pullulan gel containing an antibody, an antigen to which the antibody can bind can be detected. On the other hand, in pullulan gel containing an antigen, an antibody capable of binding to the antigen can be detected. Such combinations include, in addition to antibody-antigen, receptor-receptor ligand, enzyme-enzyme ligand, and the like.

Examples of the detection method include an ELISA method and an immunochromatography method.

The pullulan gel according to the present invention has a certain stability with respect to an organic solvent and an acid / alkali solution as shown in Examples. Therefore, it can withstand various operations in protein detection.

The protein detection kit according to the present invention may further include various reagents for immunologically detecting proteins captured by peptides, peptides or amino acids cross-linked to pullulan gel (secondary antibodies, reporter molecules, And / or buffers) and instruments (plates and pipettes), protein detection kit instructions for use, and the like.

[Others]
The pullulan gel film of the present invention can also be used in the food field. For example, since the pullulan film of the present invention is transparent and glossy, it can be used as a food brightening agent, and by coating the surface of the food, the food can be polished. Since pullulan film has edible properties, it can be eaten with food as it is. Moreover, the pullulanger film of this invention can also be utilized as a film for wrapping food. Since the pullulan gel film of the present invention has a low oxygen permeability, the food is prevented from being oxidized and quality deterioration is reduced. Furthermore, the pullulan film of the present invention can be printed on the surface, and a wide range of printing inks can be used. In addition, a colored pullulan film can be prepared by impregnating a pigment or the like into a pullulan gel and drying it. Moreover, various substances can be affixed on the surface of the pullulan film.

[Summary]
The pullulan gel according to the present invention is formed by crosslinking a plurality of pullulans.

In the pullulan gel according to the present invention, the plurality of pullulans are preferably cross-linked by a structure represented by the following general formula (1).

(In Formula (1), at least two of the plurality of * are cross-linking points with pullulan)
In the pullulan gel according to the present invention, a protein, peptide or amino acid may be further cross-linked to pullulan.

In the pullulan gel according to the present invention, the protein may be an antibody, an antigen, a receptor, a ligand for the receptor, an enzyme, a ligand for the enzyme, a hormone, or a functional fragment thereof.

The pullulan gel according to the present invention may be impregnated with a drug.

The pullulan gel according to the present invention may have an average particle diameter of 50 nm to 1 mm.

The pullulan gel according to the present invention may contain a polysaccharide other than pullulan.

The present invention also provides a dried pullulan product obtained by drying the pullulan gel.

(In Formula (2), a plurality of R ¹ to R ³ are each independently a chlorine atom, a fluorine atom, a bromine atom or an iodine atom)
The present invention is also a method for producing a pullulan gel in which a plurality of pullulans are crosslinked, a step of stirring an aqueous solution containing pullulan and a crosslinking agent represented by the above general formula (2), and the aqueous solution after the above steps. And a step of cross-linking by stirring in a water-insoluble liquid.

In the pullulan gel production method according to the present invention, the crosslinking agent is preferably cyanuric chloride.

In the pullulan gel production method according to the present invention, the aqueous solution preferably has a pH of 5.0 to pH 8.0.

In the pullulan gel production method according to the present invention, crosslinking is preferably performed at -20 ° C to 37 ° C.

In the pullulan gel production method according to the present invention, the pullulan gel is further cross-linked with a protein, peptide or amino acid, and the aqueous solution may further contain the protein, peptide or amino acid.

The present invention also provides a pullulan gel produced by the above pullulangel production method.

The present invention is also a method for producing a pharmaceutical composition having sustained release of a drug, the step of drying the pullulan gel produced by the method for producing a pullulan gel containing no protein, and the dried pullulan gel, And a step of immersing in a liquid containing the drug.

In the method for producing a pharmaceutical composition according to the present invention, the pullulan gel may have an average particle size before drying of 50 nm to 1 mm.

The present invention also provides a protein detection kit further comprising a pullulan gel in which a protein, peptide or amino acid is cross-linked to pullulan or a dried product thereof.

Examples will be shown below, and the embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention. Moreover, all the literatures described in this specification are used as reference.

[Example 1: Preparation of pullulan gel-1]
Pullulan (6 g, manufactured by Hayashibara Co., Ltd.) having an average molecular weight of 200 kDa was added to 100 mL of water, and a 5% aqueous sodium bicarbonate solution (2 mL, manufactured by Wako Pure Chemical Industries, Ltd.) was further added to adjust the pH to 7. Acetone (2 mL, Wako Pure Chemical Industries) solution of cyanuric chloride (0.2 g, manufactured by Kanto Chemical Co., Inc.) was added thereto. Since the pH gradually decreased when an acetone solution of cyanuric chloride was added, a 5% sodium bicarbonate solution was appropriately added to maintain pH 7. The aqueous solution was then stirred in ice (0-1 ° C.) for 3 hours at a rate of about 300 rpm. Thereafter, the stirring was stopped, and 15 mL each of the aqueous solution was put into 50 mL tubes and allowed to stand at 37 ° C., 25 ° C., 4 ° C., in ice (0-1 ° C.) and −20 ° C. for 18 hours. A pullulan gel could be prepared (the one placed at −20 ° C. was frozen, so it was naturally thawed at room temperature to confirm gelation).

Also, pullulan gels could be produced in the same manner when the amount of pullulan was 4 g, 10 g and 15 g. The higher the pullulan concentration, the harder the pullulan gel. When the amount of cyanuric chloride was 0.1 g and 0.3 g, pullulan gel could be produced in the same manner. The higher the concentration of cyanuric chloride, the harder the pullulan gel.

In addition, pullulan gel could be produced even when left at −20 ° C. with a pullulan amount of 2 g.

[Example 2: Preparation of pullulan gel-2]
Pullulan (10 g, manufactured by Hayashibara Co., Ltd.) having an average molecular weight of 200 kDa was added to 100 mL of room temperature water and dissolved. Cyanuric chloride (0.2 g, manufactured by Kanto Chemical Co., Inc.) was added thereto and stirred for about 5 minutes. 15 mL of this aqueous solution was placed in five 50 mL tubes, and the pH was adjusted to 3.5, 5.0, 7.0, 8.0, and 10.0, respectively. The pH was 3.5 before adjustment, and pH 5.0, 7.0 and 8.0 were adjusted with 5% aqueous sodium bicarbonate solution, and the pH of the 5% aqueous sodium bicarbonate solution was 8.3. The conditions were adjusted with a 5% aqueous sodium carbonate solution.

These aqueous solutions were further stirred for 5 minutes and then allowed to stand at 25 ° C. for 18 hours. As a result, they were not gelled at pH 3.5, and were transparent and uniform gels at pH 5.0, 7.0, and 8.0. In the case of pH 10.0, gelation was partially seen from the time of pH adjustment, and it became a partially turbid and non-uniform gel.

[Example 3: Preparation of pullulan gel-3]
Pullulan (10 g, manufactured by Hayashibara Co., Ltd.) having an average molecular weight of 200 kDa was added to 100 mL of water, 5% aqueous sodium bicarbonate solution (6 mL) was further added, and powder of cyanuric chloride (0.2 g, manufactured by Nacalai Tesque) was added thereto. . After stirring for 1 minute, an appropriate amount of 5% aqueous sodium bicarbonate solution was added so that the pH was 7.0. Next, this aqueous solution was stirred at a speed of 500 rpm for 5 to 10 minutes at room temperature (about 22 ° C.). Thereafter, stirring was stopped and the mixture was allowed to stand at 4 ° C. for 18 hours, whereby pullulan gel could be produced. If necessary, this pullulan gel was immersed in a glycine solution (5%, manufactured by Ajinomoto Co., Inc.) for 24 hours to mask the remaining active groups.

[Example 4: Production of pullulan gel crosslinked with protein]
Pullulan (2 g, manufactured by Hayashibara), cyanuric chloride (40 mg, manufactured by Nacalai Tesque), and protein having an average molecular weight of 200 kDa were added to 20 mL of water. The proteins used were recombinant Der f 2 (20 mg, in-house manufactured), cat HGF (20 mg, in-house manufactured), cat IFN (10 mg, in-house manufactured), and BSA (20 mg, manufactured by Wako Pure Chemical Industries, Ltd.). By stirring in the same manner as in Example 1 and allowing to stand at 4 ° C., a pullulan gel in which proteins were crosslinked could be produced.

Next, the antigenic activity of Der f 2 was confirmed for pullulan gel crosslinked with Der f 2. As a method, first, the pullulan gel was subjected to centrifugal washing three times, and the mixture was shaken with 5% skim milk at room temperature for 2 hours for blocking. Next, the pullulan gel was washed by centrifugation three times, and HRP-labeled anti-Der f 2 antibody was added, followed by shaking at room temperature for 2 hours. Thereafter, the pullulan gel was subjected to centrifugal washing three times, and an OPD substrate solution was added thereto and allowed to stand for 5 minutes. As a control, the same procedure was performed on pullulan gel that had not been cross-linked with protein. As a result, it was confirmed that only the pullulan gel crosslinked with Der f 2 developed color and retained the antigenic activity in the gel.

[Example 5: Grinding of pullulanger]
A pullulan gel cross-linked with cat HGF was prepared by allowing it to stand at 4 ° C. and at −20 ° C. for freezing. These pullulan gels were pulverized using a homomixer (manufactured by PRIMIX Co., Ltd., model number HOMOMIXER MARK II Model 2.5) under conditions of 1 minute at the maximum rotation speed. The microscope image after pulverization of pullulan gel at 4 ° C. is shown in FIG. A microscopic image after pulverization of pullulan gel at −20 ° C. is shown in FIG.

When the average particle size of each pulverized gel (Horiba, Ltd., model number LA-950V2) was measured, it was 70 μm for the pullulan gel at 4 ° C. and 200 μm for the pullulan gel at −20 ° C. (FIG. 1 ( c)). Further, finer particles can be obtained by changing the grinding device.

Moreover, when each crushed gel was passed through the 23G and 25G injection needles, they could pass through without clogging.

Further, when each pulverized gel was CCB-stained, it was confirmed that the pulverized gel was stained, and it was confirmed that the protein did not leak even when pulverized.

[Example 6: Freezing and thawing of pullulanger]
The pullulan gel prepared by allowing to stand at 4 ° C. in the same manner as in Example 1 was frozen at −20 ° C. After 7 days, thawing was performed, and the original pullulanger was restored.

[Example 7: Production of dried pullulan gel]
In the same manner as in Example 1, 1 mL of pullulan gel prepared by allowing to stand at 4 ° C. was placed in a 3 mL vial, and under the automatic operation conditions of a freeze-drying device (Tokyo Rika Kikai Co., Ltd., model number FDU-2100 + DRC-1100). It was freeze-dried to produce a dried pullulan gel.

[Example 8: Production of protein-impregnated pullulan gel]
The pullulan gel prepared by allowing to stand at 4 ° C. in the same manner as in Example 1 was freeze-dried under the conditions of Example 7 to prepare a dried pullulan gel. To this dried product, 1 mL of water containing recombinant Der f 2 (1 mg) was added and allowed to stand at 25 ° C. for 5 minutes. As a result, the inside of the dried product was immersed in water, the appearance returned to the gel before freeze-drying, and a protein-impregnated pullulan gel could be produced.

[Example 9: Confirmation of antibody production ability using pullulan gel]
(Preparation of administration sample)
Der f 2-Pullane conjugate (Der f 2-P conjugate): Pullulan (2 g, Hayashibara) with an average molecular weight of 200 kDa was added to 100 mL of water, and 5% sodium bicarbonate aqueous solution (2 mL, Wako Pure Chemical Industries, Ltd.) was added. ) Was added and dissolved. To this was added a solution of cyanuric chloride (125 mg, manufactured by Kanto Chemical Co.) in acetone (2 mL, manufactured by Wako Pure Chemical Industries, Ltd.). Since the pH gradually decreased when an acetone solution of cyanuric chloride was added, a 5% sodium bicarbonate solution was appropriately added to maintain pH 7. The aqueous solution was then stirred in ice (0-1 ° C.) for 3 hours at a rate of about 300 rpm. In addition, 100 mg of recombinant Der f 2 was added to this aqueous solution. Thereafter, stirring was continued at 37 ° C. for 18 hours. Glycine (5 g, manufactured by Ajinomoto Co., Inc.) was added to this solution and stirred at 37 ° C. for 2 hours to mask the remaining active groups. From this solution, reagents such as unreacted Der f 2, pullulan and cyanuric chloride were removed using column chromatography and ultrafiltration membrane, and the buffer was exchanged with PBS to obtain a Der f 2-pullulan conjugate sample. . This Der f 2-pullulan conjugate is not bonded to each other and is dissolved in PBS as a pullulan derivative.

Der f 2 impregnated gel and Der f 2+ gel mixing: The pullulan gel prepared by the method of Example 3 was pulverized by the method of Example 5, and this was washed by centrifugation three times with PBS, and then water outside the gel was removed. Recombinant Der f 2 (500 μg) was added to 1 mL of this gel and left standing for 3 hours after stirring as a Der f 2 impregnated gel sample, and Der f 2 (500 μg) added and stirred immediately before administration was Der f 2+ A gel mixed sample was obtained.

Der f 2 alone: 1 mL of recombinant Der f 2 added to PBS at a concentration of 500 μg / mL was used as a Der f 2 single sample.

(Administration)
These samples were administered subcutaneously to the neck of 2 rats in each group (twice: at the start and after 7 days). Blood was collected periodically (starting, 7, 14, 21, and 28 days after start), and the transition of Der f 2 specific IgG in serum was compared with the absorbance value by ELISA.

As a result, no change was observed in Der f 2 specific IgG in the serum in the Der f 2 single administration group, whereas in the Der f 2 impregnated gel administration group and Der f 2+ gel mixed administration group, Der f 2 was not changed. -Serum Der f 2-specific IgG increased as in the pullulan conjugate administration group. It is a known phenomenon that Der f 2 alone administration does not produce antibody but Der f 2-pullulan conjugate administration produces antibody. In this test, Der f 2 was impregnated into pullulan gel and mixed immediately before administration. It was a new discovery that antibodies were produced just by administration. In addition, a general adjuvant showed irritation at the time of administration and inflammation after administration, but pullulan gel was not stimulated at the time of administration to rats, and inflammation after administration was not confirmed.

[Example 10: Preparation of pullulan microgel-1]
The microgelation of pullulan by W / O dispersion was examined. After operating by the method of Example 3 and stirring for 10 minutes and leaving to stand for 18 hours, the standing time was 10 minutes, and after confirming that the crosslinking reaction had progressed to some extent and the solution was in a state of slightly pulling the yarn, The liquid is dropped into 50 mL of oil (olive oil, panacetate 810, or sunflower oil) in the range of 1 mL to 10 mL and dispersed by stirring in the range of 100 rpm to 1,000 rpm, and in the range of 30 minutes to 3 hours. The production of spherical gels of the size was examined.

As a result, spherical pullulan gel could be produced with any oil. Among these, sunflower oil having the highest viscosity is preferable, and a faster rotation speed during stirring tends to be a gel having a smaller particle diameter, and it is more preferable that the rotation speed is 500 rpm to 1,000 rpm. When the amount of the aqueous solution dispersed in the oil was large, a phenomenon in which the dispersed particles adsorbed during stirring was observed, and 2 to 10% by volume was more preferable. Further, when the stirring was stopped in order to recover the spherical gel from the oil, the spherical gels adsorbed to form a lump. This is thought to be because the crosslinking reaction was not completed, but adsorption could be prevented by stopping the stirring after adding about half of the oil water before recovery.

Pullulan (2 g) is dissolved in water (20 mL), 5% aqueous sodium bicarbonate solution (1.5 mL) is added and stirred for 1 minute, cyanuric chloride (40 mg) is added and stirred for 10 minutes (about 500 rpm) and allowed to stand for 10 minutes. I put it. This liquid (2 mL) was dispersed in sunflower oil (50 mL), stirred for 1 hour, water (25 mL) was added, stirring was stopped after 5 minutes, and spherical pullulan microgel in the aqueous layer was recovered. This was allowed to stand at 4 ° C. for 18 hours to complete the crosslinking reaction, followed by centrifugal washing with water (after shaking for 10 minutes, 2,000 rpm, 5 minutes × 3 times). When an equal amount of water was added to the gel particles and frozen (−20 ° C., 3 days) and thawed (25 ° C., spontaneous thawing), the gel particles maintained a spherical shape. Next, when an equal amount of water was added to the gel particles and autoclaved (121 ° C., 20 minutes), the gel particles maintained a spherical shape.

A spherical pullulan microgel having a protein (BSA) cross-linked by the same method was produced, and the results were the same as those of a pullulan microgel not having a protein cross-linked ((a) to (c) in FIG. 3). . In addition, the particle diameter described in FIG. 3 is measured by visual measurement using a scale attached to an optical microscope.

When these gel particles were stained with CBB, only a spherical pullulan microgel having a crosslinked protein was stained.

[Example 11: Production of pullulan film-1]
After operating by the method of Example 3 and stirring for 10 minutes and allowing to stand for 18 hours, the standing time was 30 minutes, and after confirming that the crosslinking reaction had progressed to some extent and the solution was in a state of pulling the yarn, Thinly stretched on the board. This was left to stand at 25 ° C. for 48 hours and air-dried to produce a pullulan film. At the same time, BSA (20 mg, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and a pullulan gel in which the protein was crosslinked was prepared in the same manner. As another form, a protein-impregnated pullulan gel film was prepared by immersing a pullulan film not crosslinked with protein in a BSA solution (1 mg / mL) for 10 minutes and then washing with water for 10 minutes. For comparison, a pullulan film (manufactured by Hayashibara Co., Ltd.) was dissolved in water and stretched thinly on a polypropylene plate, which was left to stand at 25 ° C. for 48 hours and naturally dried to prepare a pullulan film.

The pullulan film was found to be capable of producing a film having a good appearance since the solution is more viscous than the pullulan film and can be stretched thinly and neatly (FIG. 4 (a)). . Moreover, when the pullulan film was immersed in water, it absorbed water and became a sheet-like pullulan gel ((b) of FIG. 4). When this pullulan gel was observed with a microscope, it was found that the thickness was uniform ((c) of FIG. 4).

When the three kinds of pullulan gel films were immersed in the CBB staining solution, the protein-crosslinked pullulan film and the protein-impregnated pullulan film were stained, and these films contained protein (protein impregnation). In the pullulan gel film, it was confirmed that BSA protein entered the gel matrix) (FIG. 5 (a)). When these three kinds of pullulan films were shaken in water for 7 days, the protein-crosslinked pullulan film was not changed at all, and the protein-impregnated pullulan film was gradually decolorized. On the other hand, since the pullulan film was not dyed from the beginning, there was no change, but even when shaken in water for 7 days, it did not dissolve like a commercially available pullulan film. From these results, it can be seen that the protein in the pullulan gel film cross-linked with the protein does not elute because it is chemically bound, and the protein in the protein-impregnated pullulan film is gradually eluted and has a sustained release property. It was understood ((b) of FIG. 5).

When the pullulan film was immersed in methanol, 1M sulfuric acid aqueous solution, 1M sodium hydroxide aqueous solution and chemical resistance was confirmed, the one immersed in 1M sulfuric acid aqueous solution gradually expanded and collapsed after 3 days. No change was observed in the samples immersed in sodium even after 14 days.

[Example 12: Production of pullulan film-2]
Pullulan (10 g, manufactured by Hayashibara) and sodium bicarbonate (400 mg) were added to water (90 mL) and completely dissolved. After cooling to 5 ° C. or lower in ice, add a solution of cyanuric chloride (200 mg, manufactured by Kanto Chemical Co.) in acetone (2 mL) and stir at 5 ° C. or lower until a homogeneous solution is obtained. Pullulan 10% -Cyanuric chloride 2% A varnish was prepared. This was developed on a Lumirror film (T60, film thickness 100 μm, manufactured by Toray Industries, Inc.) with a bar coater (# 80) before gelation and dried at room temperature and normal pressure overnight. The obtained pullulan gel film (film thickness 16 μm) was peeled off and subjected to oxygen permeability measurement. The oxygen transmission rate was measured using a measuring instrument: OX-TRAN 2/21 (manufactured by MOCON), and the measurement conditions were 23 ° C. and 50% RH.

As a result, the oxygen transmission coefficient (cm ³ · mm / m ² · day · atm) of the pullulan film was 0.045. On the other hand, the oxygen transmission coefficient (cm ³ · mm / m ² · day · atm) of the pullulan film produced as a comparison was 0.070.

[Example 13: Preparation of hybrid gel-1]
To 20 mL of water, 1.5 mL of pullulan (2 g, manufactured by Hayashibara) with an average molecular weight of 200 kDa, hydroxypropyl β-cyclodextrin (1 g, manufactured by Wako Pure Chemical Industries) and 5% sodium bicarbonate (manufactured by Wako Pure Chemical Industries) was added. Stir for 1 minute (about 500 rpm). Cyanuric chloride (40 mg, manufactured by Nacalai Tesque) was added thereto, stirred for about 10 minutes (500 rpm), and then allowed to stand at 25 ° C. for 18 hours. As a result, the entire solution portion gelled and became one lump.

[Example 14: Preparation of hybrid gel-2]
To 23 mL of water, pullulan (1.40 g, manufactured by Hayashibara), dextran sodium sulfate (0.60 g, manufactured by Wako Pure Chemical Industries) and sodium bicarbonate (100 mg) were added and completely dissolved. Next, a solution of cyanuric chloride (50 mg, manufactured by Kanto Chemical Co., Inc.) in acetone (1 mL) was added thereto, and the mixture was stirred for 1 minute (300 rpm), and then allowed to stand at room temperature for 24 hours to gel. Water (10 mL) was added and the gel was crushed (13,000 rpm) with a Polytron homogenizer, then poured into 400 mL of ethanol, filtered and vacuum dried (50 ° C.). Subsequently, the mixture was pulverized with Labo Mill Surplus to obtain a powdery pullulan-dextran sodium sulfate hybrid gel (1.72 g).

[Example 15: Preparation of hybrid gel-3]
Chitosan 10 (750 mg, manufactured by Wako Pure Chemical Industries, Ltd.) was dispersed in water (15 mL), 1 M hydrochloric acid (2.9 mL) was added and completely dissolved, and then adjusted to pH 6.0 with a sodium bicarbonate solution. Pullulan (500 mg, manufactured by Hayashibara Co., Ltd.) and water were added thereto to a liquid volume of 25 mL, and then an acetone (0.5 mL) solution of cyanuric chloride (31 mg, manufactured by Kanto Chemical Co., Inc.) was added and stirred for 1 minute (300 rpm). Thereafter, the mixture was allowed to stand at room temperature for 24 hours to gel. The gel was crushed with a spatula, poured into ethanol (300 mL), filtered and vacuum dried (50 ° C.). Subsequently, the mixture was pulverized with a lab mill surplus to obtain a powdery pullulan-chitosan hybrid gel (1.00 g).

[Example 16: Preparation of pullulan microgel-2]
A precursor solution (5 mL) of pullulan 8% -cyanuric chloride 2.5% was prepared in the same manner as in Example 12, and sodium lauryl sulfate (250 mg) was added thereto and gently stirred. Separately, a hexane (30 mL) mixture of DKS NL-15 (2.82 g, manufactured by Daiichi Kogyo Seiyaku) and DKS NL-50 (0.71 g, manufactured by Daiichi Kogyo Seiyaku) was prepared, and In addition, the mixture was vigorously stirred by vortex and emulsified. After allowing to stand at room temperature overnight, the gel precipitate was collected by centrifugation, and the emulsifier was removed by repeating dispersion and centrifugation collection with a hexane / water = 1/1 mixed solution. Finally, the giant particles that were dispersed in water and precipitated at 200 × g were removed to obtain a pullulan microgel. The particle size of the pullulan microgel was measured (LA-950V2, manufactured by Horiba Seisakusho). As a result, it was 8.6 μm (volume basis 90% diameter) (FIG. 6).

[Reference Example 1: Gelation experiment with polysaccharides other than pullulan]
It was tested whether it was possible to make a gel with polysaccharides other than pullulan. As polysaccharides other than pullulan, those soluble in water at room temperature were used. The polysaccharides used were dextran 40,000, dextran 200,000, β-cyclodextrin, hydroxypropyl β-cyclodextrin, sodium alginate, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, xanthan gum, and Arabia There are 11 types of gums. 2 g of each was added to 20 mL of water and dissolved by stirring. About what did not melt | dissolve partially, water was added until it melt | dissolved and 20 mL was extract | collected from there.

1.5 mL of 5% sodium bicarbonate aqueous solution was added to each aqueous solution, 40 mg of cyanuric chloride was added, and after stirring for 1 minute, it was confirmed that the pH was 7 or more. Furthermore, after stirring at about 500 rpm for 10 minutes, it was left to stand at 25 degreeC for 18 hours, and when it was confirmed whether it gelatinized, none was gelatinized.

[Reference Example 2: Gelation-1 using sodium hydroxide solution]
When pullulan (2 g, manufactured by Hayashibara) was dissolved in 20 mL of an aqueous sodium hydroxide solution (0.1 M, 0.5 M), color development (light yellow to tan) was observed. When the pH of the aqueous solution was measured, 0.1M was pH 12.6 and 0.5M was pH 13.5. When an aqueous solution of cyanuric chloride (60 mg, manufactured by Nacalai Tesque) in acetone (400 mL, manufactured by Wako Pure Chemical Industries, Ltd.) was added to each aqueous solution, the portion where the acetone solution of cyanuric chloride was dropped was immediately gelled and evenly stirred. However, when it was confirmed after standing for 18 hours, it became a partially cloudy gel with unevenness. Thus, when sodium hydroxide was used, it was suggested that pullulan itself may undergo some chemical reaction and the structure may have changed.

[Reference Example 3: Gelation-2 using sodium hydroxide solution]
When pullulan (2 g, manufactured by Hayashibara) was dissolved in 20 mL of an aqueous sodium hydroxide solution (0.1 M, 0.5 M), color development (light yellow to tan) was observed. As a control, sodium bicarbonate used in the present invention was also carried out at the same time. When pullulan (2 g, manufactured by Hayashibara Co., Ltd.) was dissolved in 20 mL of an aqueous sodium bicarbonate solution (0.5 M), it was colorless and transparent and did not develop color unlike when sodium hydroxide was used. When the pH of these aqueous solutions was measured, the sodium hydroxide had a pH of 12.2 and 0.5M had a pH of 13.2, and the sodium bicarbonate had a pH of 8.4. Powdered cyanuric chloride (60 mg, manufactured by Nacalai Tesque) was added to each aqueous solution and stirred for 10 minutes. When sodium bicarbonate was used, cyanuric chloride was uniformly dispersed and dissolved, but sodium hydroxide was used. The powdery cyanuric chloride was gelled and became lumpy and remained undissolved. From the influence, after standing for 18 hours, 0.1M did not gel. 0.5M gelled but became a colored gel. One using sodium bicarbonate became a colorless and transparent gel. A gel prepared by dissolving pullulan in an aqueous sodium hydroxide solution was different in appearance from the gel of the present invention using sodium bicarbonate. Therefore, it is considered that the chemical structure of the gel is also different.

The pullulan gel of the present invention can be used as a material in the medical field and research field. It can also be used in the food field.

Claims

¡Pull Langer made by cross-linking multiple pullulans.
The pullulan gel according to claim 1, wherein the plurality of pullulans are crosslinked by a structure represented by the following general formula (1).

(In Formula (1), at least two of the plurality of * are cross-linking points with pullulan)
Furthermore, the pullulan gel according to claim 1 or 2, wherein the protein, peptide or amino acid is cross-linked to pullulan.
The pullulan gel according to claim 3, wherein the protein is an antibody, an antigen, a receptor, a ligand for the receptor, an enzyme, a ligand for the enzyme, a hormone, or any functional fragment thereof.
3. The pullulan gel according to claim 1 or 2, which is impregnated with a drug.
The pullulan gel according to any one of claims 1 to 5, wherein the average particle diameter is 50 nm to 1 mm.
The pullulan gel according to any one of claims 1 to 6, comprising a polysaccharide other than pullulan.
A dried pullulan gel obtained by drying the pullulan gel according to any one of claims 1 to 7.
A method for producing a pullulan gel obtained by crosslinking a plurality of pullulans,
A step of stirring an aqueous solution containing pullulan and a crosslinking agent represented by the following general formula (2);
A method for producing pullulan gel, comprising a step of allowing the aqueous solution to stand after the step and performing crosslinking.

(In Formula (2), a plurality of R 1 to R 3 are each independently a chlorine atom, a fluorine atom, a bromine atom or an iodine atom)
A method for producing a pullulan gel obtained by crosslinking a plurality of pullulans,
A step of stirring an aqueous solution containing pullulan and a crosslinking agent represented by the following general formula (2);
And a step of performing crosslinking by stirring the aqueous solution in a water-insoluble liquid after the step.

(In Formula (2), a plurality of R 1 to R 3 are each independently a chlorine atom, a fluorine atom, a bromine atom or an iodine atom)
A method for producing a pullulan gel obtained by crosslinking a plurality of pullulans,
A step of stirring an aqueous solution containing pullulan and a crosslinking agent represented by the following general formula (2);
A step of emulsifying the aqueous solution by stirring in a water-insoluble liquid after the step;
A step of allowing the emulsified solution to stand and performing crosslinking,
A method for producing pullulan gel, wherein at least one of the aqueous solution and the water-insoluble liquid contains an emulsifier.

(In Formula (2), a plurality of R 1 to R 3 are each independently a chlorine atom, a fluorine atom, a bromine atom or an iodine atom)
The pullulan gel production method according to any one of claims 9 to 11, wherein the crosslinking agent is cyanuric chloride.
The pullulan gel production method according to any one of claims 9 to 12, wherein the aqueous solution has a pH of 5.0 to 8.0.
The method for producing pullulan gel according to any one of claims 9 to 13, wherein the crosslinking is performed at -20 ° C to 37 ° C.
The pullulan gel is further crosslinked with proteins, peptides or amino acids,
The method for producing pullulan gel according to any one of claims 9 to 14, wherein the aqueous solution further contains the protein, peptide or amino acid.
A pullulan gel produced by the pullulan gel production method according to any one of claims 9 to 15.
A method for producing a pharmaceutical composition having sustained release of a drug, comprising:
A step of drying the pullulan gel produced by the pullulan gel production method according to any one of claims 9 to 14,
Dipping the dried pullulan gel in a liquid containing the drug, and a method for producing a pharmaceutical composition.
The method for producing a pharmaceutical composition according to claim 17, wherein the pullulan gel has an average particle size before drying of 50 nm to 1 mm.
A protein detection kit comprising the pullulan gel according to claim 3 or 4 or a dried product thereof.