JP3151665B2 - Biopolymer / polyallylamine complex and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a biopolymer / polyallylamine complex and a method for producing the same. This complex can be used for various uses in the biochemical and medical fields.

[0002]

2. Description of the Related Art Polyallylamine has a positive charge,
A linear olefin polymer containing a primary amino group in the side chain containing a highly chemically reactive primary amino group. It is soluble in water and is positively charged in water. It has strong hygroscopicity and water absorbency. Polyallylamine has excellent water solubility in the low pH range, so it can be uniformly mixed with nonionic polymers such as polyvinyl alcohol, and also mixed with aqueous solutions of cationic water-soluble polymers such as polyethyleneimine. it can.
Since a series of polyallylamine compounds become insoluble in water in an alkaline pH range, they are usually stored in an acidic solution to improve solubility.

[0003] Such polyallylamine is known as a material for promoting cell proliferation.
No. 28 discloses a method for producing a cell culture bed base material which is rich in durability and has excellent living cell adhesion and cell culture function by applying polyallylamine to various substrate surfaces. . In this case, a positively charged amino group of the side chain of the polyallylamine molecule is applied to the surface of various substrates to achieve the intended purpose. Since the surface of these substrates is usually negatively charged, polyallylamine is electrostatically ionically bonded to the substrate surface, so that the bond between the two is strong and polyallylamine is considered to be difficult to peel off from the substrate surface. I have.

[0004] In addition, biopolymers attach living cells,
It is known that they can be propagated.

Although polyallylamine and biopolymer each have excellent biological functions, they can be used with high added value in advanced fields such as medical fields. A polyallylamine complex containing the biopolymer of (1), which has both excellent functions of each component or a synergistic combination of each function, has not been obtained.

[0006]

Problems to be Solved by the Invention
The degree of charge of the synthetic polymer, glass, metal, and porous filter paper as various substrates described in JP-A-628 is different depending on the substrate, and not all substrates are sufficiently negatively charged to the same extent. . Therefore, depending on the type of the cell culture bed substrate, a difference in the strength of the interaction between the polyallylamine of the cell culture bed substrate and the substrate occurs, and the cell culture bed comes into contact with the cell culture solution when the polyallylamine coating comes into contact with the cell culture solution. There is a risk of peeling off from the substrate surface, which is a practical problem. There has been a strong demand for the development of a cell culture bed base material that does not peel off from the cell culture bed substrate surface and can achieve the intended purpose.

Further, it is possible to form a film by evaporating an aqueous solution of a positively charged polyallylamine to dryness. However, since this film-like molded product has high hygroscopicity, it is left in the atmosphere when left in an air atmosphere. Since the water vapor contained therein was absorbed and dissolved, it was not possible to prepare a stable film-like molded product in the atmosphere. In addition, polyallylamine itself has poor moldability, it is difficult to prepare a film-like or lump-shaped molded product from polyallylamine alone, and there is no suitable insolubilization treatment method for making polyallylamine water-insoluble. Therefore, it has been extremely difficult to produce physically uniform water-soluble and water-insoluble moldings.

The present invention provides a method for adhering cells by drying and drying a mixed aqueous solution or aqueous dispersion obtained by uniformly mixing a biopolymer and polyallylamine in an aqueous solution or aqueous dispersion state on a substrate. A biopolymer / polyallylamine complex (hereinafter sometimes abbreviated simply as "composite") having excellent growth properties, excellent physical properties such as strength, and good moldability, and an efficient use of the complex. An object of the present invention is to provide a method for economically manufacturing.

[0009]

Means for Solving the Problems The present inventors have attempted to improve the defects of physical properties such as moldability and strength of polyallylamine itself, and to apply it to a cell culture bed substrate by utilizing its functional characteristics. Based on this idea, a second substance has been developed which can be uniformly mixed with polyallylamine at the molecular level, and which has a strong molecular interaction with polyallylamine to form an excellent and uniform complex. Here, "can be uniformly mixed with polyallylamine at the molecular level"
In the process of mixing polyallylamine and the second substance in an aqueous solution state, spreading the mixed aqueous solution on a substrate such as polyethylene and evaporating to dryness, the intermolecular cohesion of the components of the aqueous solution becomes dense, This means that a complex having a uniform structure without phase separation and no domain structure is generated.

The inventors of the present invention have developed a means for obtaining a composite which has both the excellent functions of polyallylamine and the second substance, or the physical properties of the polyallylamine and the second substance which have each of the independent functions in a synergistic manner. As a second substance, a biopolymer has been used and a substrate having good molecular interaction acting between the molecules of both substances has been intensively studied. As a result, they found that a complex of a biopolymer that is an ampholyte and a polyallylamine having a positive charge has excellent physical properties such as strength, and has an action of enhancing attachment and proliferation of living cells. The present invention has been completed.

The biopolymer / polyallylamine complex of the present invention is obtained by mixing a biopolymer and polyallylamine in an aqueous solution or an aqueous dispersion and evaporating and removing water to dryness. A water-soluble and water-insoluble complex obtained by enhancing the intermolecular interaction and improving the intermolecular aggregation state. It can be produced by allowing a biopolymer, which is an amphoteric electrolyte, to interact with a positively charged polyallylamine molecule at the molecular level. To prepare the water-insoluble complex, for example, the water-soluble complex may be treated with a mixed aqueous solution of water and alcohol, an epoxy compound, an aldehyde, or a water-insoluble drug aqueous solution in which both compounds are mixed. .

The biopolymer usable for preparing the complex of the present invention includes a silk protein, wool keratin, collagen, spider silk or marine protein obtained from insect-derived silk protein fibers, for example, silkworm. There is a protein such as a thread. Since these biopolymers are composed of about 20 types of amino acids and are ampholytes having an acidic group such as a carboxyl group and a basic group such as an amino group in the molecule, their charge states are as follows: It changes greatly around the isoelectric point. That is, glutamic acid, a negatively chargeable acidic amino acid residue such as aspartic acid, and lysine, arginine, amphoteric electrolyte containing a positively chargeable basic amino group such as histidine, etc., these biopolymers Changing the environmental pH results in a negative or positive charge.

The polyallylamine used in the present invention is not particularly limited, and may be neutralized or non-neutralized, and has a positive charge and a high chemical reactivity. It is a straight-chain olefin polymer containing a secondary amino group in the side chain, and may be any polymer that is soluble in water and positively charged in water. No. of molecular side chains of polyallylamine
Since the primary amino group has high chemical reactivity, a substituent having various functions can be introduced by chemically modifying this site as desired.

According to the method for producing a complex of the present invention, a biopolymer such as a silk protein and polyallylamine are mixed in an aqueous solution or aqueous dispersion, and water is evaporated to form a film or gel. Various types of composites can be manufactured in powdered or fibrous form. The complex is responsible for cell attachment and
In addition to having the function of enhancing proliferation, this complex can comprehensively immobilize drugs, bioactive substances, antibiotics, enzymes, hormones, living cells, microorganisms, antigens, antibacterial components, antibacterial metals, etc. It can also be used as a carrier for supporting substances.

Polyallylamine hydrochloride (PAA-HC)
l: Nitto Boseki Co., Ltd., trade name) showed no accumulative toxicity, no mutagenicity, and no oral acute toxicity in mice (LD5
0) is 1600 mg / kg, and fish toxicity using medaka is very low since the TLm of 48 hours is 0.50 ppm (Toru Harada, Polymer Processing, Vol. 33, 10
No. 21, 21 (1984)). In addition, silk proteins, wool keratin, and the like are natural biopolymers, and none of these materials show toxicity to living tissues. Therefore, it is considered that the biopolymer / polyallylamine complex has no toxicity to living tissues.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The biopolymers that can be used in the present invention include those described above. For example, as the silk protein fiber, in addition to the silkworm silk obtained from the larva of the silkworm (Bombyx mori), the silkworm silk obtained from the larvae of the tussah silkworm, the heavenly silkworm, the eri silkworm, the helmet silkworm, the shinju silkworm belonging to the wild silkworm, or these fiber products Any of these can be used. Further, as the silk protein, sericin that adheres to the outside of the cocoon fiber formed by silkworm spinning, or silk fibroin fiber obtained by removing the sericin is dissolved in an aqueous solution of neutral salt, and then dialyzed with cellulose. Water-soluble silk fibroin obtained by dialysis using a membrane, or water-soluble silk sericin or water-soluble silk fibroin in a silk gland taken out from a silkworm body can also be used.

When discussing the structure, chemical properties, and physical properties of a biopolymer, it is easy to understand the entirety of the biopolymer by explaining using silk fibroin as the biopolymer.
Therefore, in the present invention, silk fibroin is used as a specific biopolymer, and a complex that can be prepared by mixing this with polyallylamine (hereinafter sometimes referred to as complexing) is taken as an example. Will be described.

Silk fibroin can be prepared as follows by dissolving a silk protein fiber (silk thread). To dissolve the silk thread, a conventionally known neutral salt such as calcium chloride, calcium nitrate, lithium bromide and lithium thiocyanate is used. That is, a silk thread is put into a concentrated neutral salt aqueous solution and heated, the dissolved aqueous solution is put into a cellulose dialysis membrane, both ends are sewn with sutures, and placed in room temperature tap water or pure water for 4 to 5 days for replacement. Then, a pure silk fibroin aqueous solution is obtained by completely removing lithium ions. By spreading this silk fibroin aqueous solution on the surface of a substrate such as a polyethylene membrane, drying by blowing air, and evaporating the water,
A transparent silk fibroin membrane can be prepared. The silk fibroin membrane immediately after drying and solidification dissolves in water.If desired, the silk fibroin membrane is immersed in an organic solvent such as alcohol, which is a poor solvent for silk fibroin, and if treated, the cohesion between silk fibroin molecules is improved. Thus, the silk fibroin membrane becomes easily insoluble in water.

When the silk protein fiber is dissolved in the neutral salt aqueous solution, it is possible to take care not to reduce the molecular weight of the silk protein by limiting the neutral salt concentration, the dissolution temperature and the dissolution time. The neutral salt concentration may be about 8.0 to 9.8 M, and the dissolution temperature may be about 25 to 70 ° C. The dissolution temperature is preferably 60 ° C. or lower. If the dissolution temperature is high, the molecular weight of the silk fibroin decreases, and there is a risk that the high molecularity of the silk fibroin is lost. The dissolution time is preferably set to about 1 to 20 minutes. Among the neutral salts, lithium salts excellent in the dissolving power of silk protein fibers are preferably used. Particularly, lithium bromide is preferable. In the case of lithium bromide of 8 M or more, preferably 8.5 M or more, the silk protein fiber dissolves at 55 ° C. or more for about 15 minutes.

The polyallylamine which can be used in the present invention is obtained by polymerizing allylamine and has the above-mentioned physical properties. For example, the polyallylamine may be a hydrochloride or a free form without hydrochloric acid. It may be. Specific examples include diallyldimethylammonium chloride-sulfur dioxide copolymer, diallyldimethylammonium chloride copolymer, diallylamine hydrochloride-sulfur dioxide copolymer, polyallylamine hydrochloride, and polyallylamine. These are all available from Nitto Boseki Co., Ltd., for example, PAA-HCl-3L, PAA-
HCl-3S, PAA-HCl-10L, PAA-HC
It is commercially available as 1-10S (trade name) or the like.

The complex of the present invention is prepared by mixing a biopolymer such as silk fibroin and polyallylamine in an aqueous solution or aqueous dispersion to form a uniform aqueous solution or aqueous dispersion. By spreading it up and evaporating the water, it can be manufactured as a composite having a uniform composition. That is, an aqueous solution or an aqueous dispersion of each substance of a biopolymer such as silk fibroin and polyallylamine is separately prepared, and then a mixture obtained by uniformly mixing the two is used. Using an aqueous solution or an aqueous dispersion of a mixture of the above, or using one aqueous solution or an aqueous dispersion obtained by dissolving and dispersing one in a liquid and dispersing the other and mixing uniformly. It can be produced by spreading on a substrate such as a polyethylene film or a polystyrene film and evaporating water.

To insolubilize the complex with water, the water-soluble complex is immersed in an aqueous solution of an insolubilizing agent obtained by adding an epoxy compound, an aldehyde, or both compounds to an aqueous solution mixed with an organic solvent such as water or methanol. do it. The epoxy compound, aldehyde, and other reagents can be freely changed according to the intended use as long as they are uniformly mixed in the composition of the mixed aqueous solution of water and methanol. The amount of aldehyde dissolved in a mixture of water and methanol is generally larger than that of an epoxy compound. When an epoxy compound is used, if the content of water is high in a mixed liquid composition of water and methanol, the epoxy group of the epoxy compound reacts with water to easily open the ring. Should be small.

As the epoxy compound, an epoxy compound such as monofunctional epichlorohydrin or bifunctional ethylene glycol glycidyl ether can be used. In addition, any monofunctional, difunctional or polyfunctional epoxy compound can be used. The complex is immersed in an aqueous solution of an insolubilizing agent containing at least one of an epoxy compound or an aldehyde such as glutaraldehyde or formaldehyde in a mixed aqueous solution comprising an alcohol such as methanol and water, thereby obtaining a biological solution such as silk fibroin. The conjugate becomes water-insoluble because cross-links are introduced between the molecule and the polyallylamine.

The degree of insolubilization can be achieved by changing the chemical composition of the aqueous solution of the insolubilized drug. The most desirable agent for the insolubilization treatment is an aqueous solution of the insolubilized agent consisting of chlorooxirane, water, and methanol (5: 10: 85% by volume). The methanol concentration is generally 20-85% by volume.
And preferably in the range of 40 to 70% by volume.

When an aqueous solution of silk fibroin is mixed with an unneutralized aqueous solution of polyallylamine having a nitrogen atom whose molecular side chain is positively charged, if the aqueous solution of silk fibroin is less than a certain concentration, both aqueous solutions are uniformly mixed. By evaporating the mixed aqueous solution to dryness, it is possible to prepare a complex in which the components are uniformly mixed.However, when the aqueous solution of silk fibroin exceeds a certain concentration, the two aqueous solutions cannot be mixed uniformly, resulting in separation and cloudiness. As a result, even if the mixed aqueous solution of both substances is evaporated to dryness, it is not possible to prepare a complex in which the respective components are uniformly mixed. This is because electrostatic repulsion occurs between the silk fibroin and the polyallylamine, and the silk fibroin and the polyallylamine are separated in an aqueous solution state and cannot be mixed uniformly. As a result, a structurally uniform silk fibroin / polyallylamine complex cannot be prepared. In addition, since the isoelectric point of silk fibroin is pH = 3.8 to 4.0, when silk fibroin and polyallylamine exhibiting acidic pH are mixed in an aqueous solution, the pH of silk fibroin becomes equal to or lower than the isoelectric point,
Silk fibroin coagulates, making it impossible to prepare a uniform complex with polyallylamine.

To prepare a polyallylamine complex containing an arbitrary amount of silk fibroin without causing phase separation,
Prior to producing the composite, the aqueous solution of polyallylamine preferably has a pH in the range from about neutral to an alkaline region, and more preferably has a pH in the vicinity of neutral. And silk fibroin are well mixed at the molecular level, and no phase separation occurs over the entire pH range. It is believed that adjusting the pH of polyallylamine to near neutrality improves the compatibility with silk fibroin for the following reasons. In the molecular side chain of polyallylamine, there is a positively charged nitrogen atom, and in the presence of hydrochloric acid,
A negatively charged chloride ion is bonded to the nitrogen atom of the polyallylamine to cancel the charge. Therefore, unneutralized polyallylamine does not cause any interaction with the ampholyte silk fibroin, and when polyallylamine and a certain amount or more of silk fibroin are mixed in an aqueous solution state, both are separated and uniform. It does not form a complex with a simple structure. However, by neutralizing polyallylamine with an alkaline agent, the charge state of the positively charged nitrogen atom is exposed, and this portion and the amino acids such as glutamic acid and aspartic acid that are negatively charged in the molecular side chain of silk fibroin. An ionic bond occurs between the residue portion and a hydrogen bond, a hydrophobic bond is formed between the positively charged side chain portion of polyallylamine and the peptide main chain or molecular side chain of silk fibroin, It is believed that both will become homogeneously mixed in the aqueous solution. If the aqueous solution of polyallylamine is neutralized, the pH of the silk fibroin does not become lower than the isoelectric point even when it is mixed with the aqueous solution of silk fibroin, and the silk fibroin does not coagulate. This is useful for uniformly mixing in an aqueous solution state. As a result, a uniform composite having excellent moldability can be obtained by evaporating the silk fibroin / polyallylamine mixed aqueous solution to dryness.

As described above, prior to preparing the complex of the present invention, if necessary, the pH of polyallylamine is adjusted to near neutrality so that this polyallylamine is mixed with an arbitrary amount of silk fibroin. Become like Since the pH of the aqueous solution of polyallylamine is usually on the acidic side for the sake of convenience of stable storage, it is preferable that the aqueous solution of polyallylamine is neutralized by adding an alkali agent. As the chemical that can be used for the neutral treatment, any conventionally known alkaline chemical can be used. Examples of the alkaline agent include those which exhibit alkalinity when formed into an aqueous solution such as sodium hydroxide, potassium hydroxide, potassium carbonate, potassium nitrate, sodium nitrate, sodium carbonate, sodium acetate, sodium hydrogen carbonate, triethylamine, ammonia, and the like. Any alkali chemical can be used similarly. In the case of a concentrated alkaline aqueous solution, it is preferable to neutralize the aqueous solution sufficiently diluted with water by adding it to polyallylamine.

Of the above alkaline agents, ammonia is most preferred. This is because the mixed aqueous solution of the biopolymer / polyallylamine can be removed in the process of evaporating to dryness, and does not remain in the prepared complex. It is advisable to add a dilute aqueous solution of ammonia dropwise to the aqueous solution of polyallylamine little by little to adjust the pH of the aqueous solution of polyallylamine to around 7 using a pH test paper or a pH meter. An aqueous solution of silk fibroin is added to the aqueous solution of polyallylamine adjusted to pH 7, carefully and gently stirred with a glass rod, allowed to stand at room temperature for a predetermined time, for example, about 15 minutes, spread on a substrate such as a polyethylene film, and blow-dried. A complex can be produced by evaporating water. In the complex thus prepared, the two components are uniformly mixed, and the intermolecular interaction is good. The solubility in water differs depending on the content of silk fibroin. The water-soluble complex can be converted to water-insoluble by performing the following insolubilization treatment, if desired.

As described above, a gelling water-insoluble complex can be prepared by applying a gelling agent consisting of an aqueous solution of an insolubilizing drug, that is, an insolubilizing drug to the complex, as described above. A powdery water-insoluble complex can be prepared according to a freeze-drying method. By such a simple treatment, the molecular aggregation state of silk fibroin is improved, and as a result, silk fibroin is insolubilized. Since the polyallylamine is physically and chemically trapped in the matrix of the insolubilized silk fibroin, the silk fibroin / polyallylamine complex can be insolubilized without using a special crosslinking chemical. In addition, as another insolubilization method, there is a method of electrostatically bonding both substances. In other words, in an aqueous solution, polyallylamine having a positive charge and silk fibroin having a glutamic acid and an aspartic acid residue having a negative charge among the positive and negative charges are formed by ionic bonds, hydrogen bonds, and other chemical bonds. Because of the binding, they are combined and insolubilized. After the insolubilization treatment, polyallylamine and silk fibroin do not elute from the complex.

The complex of the present invention includes a drug, a living cell,
Active ingredients such as antibacterial metal components and antibiotics can be immobilized. The type and amount of the active ingredient that can be fixed to the complex are not particularly limited, and can be arbitrarily determined according to the purpose. The active ingredient is preferably a water-soluble one. To prepare a complex containing the active ingredient, the active ingredient is dissolved or dispersed in a mixed aqueous solution of an aqueous solution of a biopolymer such as silk fibroin (silk protein) and an aqueous solution of polyallylamine after neutralization, After gently and carefully stirring the mixed aqueous solution, the mixed aqueous solution may be spread on a substrate film of polystyrene, polyethylene or the like to evaporate water. When preparing a mixed aqueous solution containing the active ingredient, the preferred concentrations of the biopolymer such as silk protein and polyallylamine are each 0.01 to 10% by weight, more preferably 0.1 to 5% by weight. Biopolymer concentration is 10% by weight
If the concentration is more than 0.01%, the moldability is undesirably reduced, and if the concentration of the biopolymer is less than 0.01% by weight, the concentration is too low. In addition, the efficiency of mixing the two aqueous solutions becomes poor. In addition, it is not preferable to use a dilute aqueous solution of a biopolymer because the evaporating and drying time of the mixed aqueous solution with the polyallylamine aqueous solution becomes longer, and the efficiency of preparation of the target product decreases.

[0031]

Next, the present invention will be described in more detail with reference to examples and comparative examples, but the present invention is not limited to these examples. (1) Types of polyallylamine used in Examples Table 1 shows the names, molecular weights and characteristics of polyallylamine used in the following Examples. The water-soluble cationic polymer polyallylamine shown in Table 1 is commercially available from Nitto Boseki Co., Ltd., and the names of the compounds were all according to the trade names of Nitto Boseki Co., Ltd. Details of these polyallylamines are as follows.

PAS-A-120L is a diallyldimethylammonium chloride-sulfur dioxide copolymer, PAS-H-10L is a diallyldimethylammonium chloride copolymer, and PAS-92 is a diallylamine hydrochloride-sulfur dioxide copolymer. And PAA-
HCl-10L is a polyallylamine hydrochloride,
AA-10C is polyallylamine.

[0033]

[Table 1] (2) The following items were tested in order to examine the structural characteristics of the silk fibroin / polyallylamine complex obtained in the examples.

(2-1) Mechanical properties: The mechanical properties (strength and elongation) of the composite membrane were measured, and the strength and elongation of the sample of the composite membrane at the time of cutting were evaluated. Measurement conditions were as follows: sample length 15 mm and width 2 mm, film thickness 10 μm, tensile speed 4
mm / min, chart speed 500 mm / min,
The chart scale was 200 g, and was measured by a tensile tester (Autograph, type AGS-5D) manufactured by Shimadzu Corporation.

(2-2) Fourier Transform Infrared Absorption Spectrum An absorption spectrum relating to the molecular form of the complex was observed using an FT-IR (Fourier transform infrared absorption spectrum) measuring device manufactured by PerkinElmer. The measurement wave number is 200
0 to 400 cm ^-1 and the number of measurement repetitions was 20 times.

(2-3) Moisture Content and Sample Weight Outflow Rate The composite membrane was immersed in water at 20 ° C., and before and after the treatment, what percentage of water had entered the composite membrane sample (water content), and By immersing the composite membrane in water at 20 ° C. for 24 hours, what percentage of the sample weight flowed out (sample weight outflow rate) was determined by the following equation.

Water content = [(Wb−Wc) / Wc] × 100 (%) Sample weight outflow rate = [(Wa−Wc) / Wa] × 100
(%) However, Wa, Wb, and Wc mean the following, respectively.

Wa: 85 ° C. before immersion in water at 20 ° C.
Weight of sample dried for 3 hours at

Wb: Weight of a sample immersed in water at 20 ° C. for 24 hours and having an equilibrium water absorption.

Wc: air-dried under standard conditions after water absorption, 15 ° C.
Weight of sample dried for 3 hours at

(2-4) Thermal decomposition behavior Differential thermal scanning measurement device (DSC-10, manufactured by Rigaku Corporation)
Using A), the measurement was performed in a nitrogen flow of 200 cc / min at a sample weight of the composite membrane of 2.2 mg, a DSC range of 2.5 mcal / s, and a heating rate of 10 ° C./min. In this measurement, the endothermic peak temperature appearing at 180 ° C. or higher was defined as the thermal decomposition temperature of the sample. (3) Evaluation of antibacterial activity by the inhibitory effect of the composite membrane on the growth of phytopathogenic bacteria As a phytopathogenic bacterium, it is a representative of universal phytopathogenic bacteria. It is a multi-rot rot fungus that commits and is one of the few gram-positive bacteria among plant pathogenic bacteria.
rynebacterium michiganese pv. michiganese).

The antibacterial activity against bacteria in the examples was evaluated by the following method.

Assay for antibacterial activity against bacteria: semi-synthetic wakimoto medium or King B medium 2 maintained at 55 ° C. after heat lysis
5 ml and 2 ml of the test bacterium (concentration: 10 ⁹ / ml) were mixed, and the mixture was poured into a petri dish and solidified into a plate. A sample of the composite membrane of about 1 cm square was placed on this bacterial mixture-mixed plate medium, and the whole sample was brought into close contact with the medium. This is 2
The temperature was kept at 0 to 25 ° C., and the degree of inhibition of bacterial growth in the medium near the test sample was measured at predetermined time intervals by measuring the size of an inhibition circle appearing around the sample and evaluating the result in units of mm. (4) Evaluation of antibacterial activity by inhibitory effect of complex membrane on growth of Escherichia coli The inhibitory effect on growth of Escherichia coli was evaluated by the same method as that for evaluating the inhibitory effect on growth of tomato ulcer pathogenic bacteria. As E. coli, Strain: JM manufactured by PROMEGA
LB medium and LB agar medium were used as mediums. The composition of the LB medium per 100 mL of distilled water was as follows. This medium is composed of 1.0 g of polypeptone, 0.5 g of yeast extract, and 1.0 g of sodium chloride (1.5 g of agar in the case of LB agar medium). 0-7.4
Was adjusted. The specific culture method is as follows.

(I) Escherichia coli was grown overnight in a LB medium while shaking at a culture temperature of 35 ° C.

(Ii) An equal amount of the LB agar medium dissolved at about 60 ° C. and the LB medium containing the proliferated Escherichia coli were mixed and injected into a sterilized container.

(Iii) After the medium was solidified, the test sample was placed on the surface of the medium.

(Iv) After incubation for a certain period of time, the size of the inhibition circle was observed. (5) Attachment / proliferation experiment of insect cells Grace medium (Sigma, G8142) containing 5% of powder body fluid of silkworm (Nippon Agricultural Industry Co., Ltd.), 5% of fetal calf serum (Gibco), and 1 Using a medium containing a mixed antibiotic of 5% penicillin / streptomycin, a culture experiment was performed on Ae cells derived from tussah silkworm or Bm cells derived from silkworm. On the first day after culture, cells were attached to an inverted microscope (Ol
ympus 1MT-2) on the same culture plate,
The four visual fields were observed, the culture plate was swung left and right under the visual fields, and the cells adhered to the bottom surface of the cell culture bed and the cells suspended in the culture solution were visually observed. The ratio of the number of adherent cells to the total number of cells under the visual field was visually measured three times, and the average value was evaluated as the state of cell attachment (hereinafter abbreviated as “cell attachment”). The number of cell cultures was observed on a hemocytometer on the second day. The state of adhesion and proliferation of tussah (Ae) and silkworm (Bm) insect cells on the surfaces of various composite membranes having different silk fibroin contents was observed. (6) Attachment and proliferation test of mouse-derived fibroblasts Mouse-derived fibroblasts (L
929) was observed. L929 used was
It was cloned by Sanford in one of a series of mouse fibroblasts whose primary culture was started by Earle in 1940. L929 Cell N
o. Is RCB0091 (RIKEN). Using MEM medium (Gibco) containing 5% fetal calf serum, HEPES 20 mM (Sigma) and glutamine, 3
Cell culture at 7 ° C., the adhesion state of L929 fibroblasts on the fourth day was examined with an inverted microscope (Olympus). The number of cells at the start of the culture was 1.5 × 10 ⁵ cells / ml. Example 1 Evaluation of Compatibility of Silk Fibroin and Polyallylamine in Aqueous Solution A silk fibroin aqueous solution was prepared by the following method. 2.5g
Silkworm silk from an 8.5 M aqueous solution of lithium bromide at 55 ° C in 20
After complete dissolution in mL, the aqueous solution was put into a dialysis membrane made of cellulose, and replaced with distilled water at 5 ° C. for 5 days to remove impurities to prepare a pure silk fibroin aqueous solution. Distilled water was added to the silk fibroin aqueous solution thus prepared to prepare a stock solution of the silk fibroin aqueous solution so that the absolute dry concentration was 6%. Water was added to this silk fibroin aqueous solution to prepare a 3% silk fibroin aqueous solution.
A predetermined amount of a 3% silk fibroin aqueous solution and 6% of various polyallylamine aqueous solutions were mixed such that the mixing ratio of silk fibroin and polyallylamine was constant based on the weight of the complex to be prepared. It was visually observed whether or not the two types of polymers were properly mixed in the aqueous solution immediately after mixing. Table 2 shows the obtained results.

The compatibility was evaluated by the following three steps.

：: well mixed in a transparent state.

Δ: The solution becomes cloudy without mixing.

X: Silk fibroin and polyallylamine are phase separated in an aqueous solution state. In addition, the solubility in Table 2 was evaluated by visually dissolving the composite film obtained by spreading the mixed aqueous solution on a polyethylene film, drying and drying, and immersing the composite film in water. Things.

Dissolution behavior of composite membrane: A: The composite membrane is well soluble in water.

Δ: Part of the composite membrane is soluble in water.

[0054]

[Table 2] Table 2 shows the results of the compatibility and solubility when various polyallylamines without any neutralization treatment were used. When the ratio of silk fibroin and polyallylamine was changed,
Whether or not each component is separated in an aqueous solution state is simply evaluated in advance. From these results, although different depending on the type of polyallylamine, both are uniformly mixed when the silk fibroin content is about 80 to 90% by weight or less. However, when the pH of the polyallylamine is adjusted to near neutrality, the result differs from the result in Table 2 in that the polyallylamine is uniformly mixed with an arbitrary amount of silk fibroin as described below. Example 2 Preparation method of composite membrane Water was added to a polyallylamine aqueous solution to adjust the pH to 3 to 5, and this aqueous solution was mixed with 2% of polyallylamine.
A 3% silk fibroin (hereinafter sometimes abbreviated as SF) aqueous solution was added so as to contain 5% by weight, and mixed. Spread this mixture on a substrate (polyethylene membrane)
It was blow-dried at 25 ° C. to prepare a composite membrane having a thickness of 100 μm. The polyallylamine used was PAS-A-120L or PAS-trade name manufactured by Nitto Boseki Co., Ltd. shown in Table 1.
H-10L, PAS-92, PAA-HCl-10L,
PAA-10C.

In the case of PAA-10C, this aqueous solution (pH
= 11) When an aqueous solution of silk fibroin was added to the solution, milky turbidity
After standing for 30 minutes, oily PAA-10C precipitated at the bottom of the beaker. Then, in order to compound SF and PAA-10C to form a transparent composite film, the following was devised. Aqueous ammonia (pH = 11, manufactured by Wako Pure Chemical Industries, Ltd.) was added to a 3% aqueous solution of silk fibroin, and further 6% P
AA-10C aqueous solution was added so that the content of polyallylamine was 25% by weight. Mixing was good and both materials did not separate. This mixed solution was spread on a polyethylene film and blown dry at 25 ° C. to prepare a transparent composite film sample. Example 3 FT-IR analysis of composite A composite of silk fibroin and polyallylamine was prepared in the same manner as in Example 1 using polyallylamine shown in Table 1. That is, silk fibroin is contained in the complex.
An aqueous solution of silk fibroin and an aqueous solution of unneutralized polyallylamine were uniformly mixed at a molecular level so as to contain 5% by weight, spread on a polyethylene membrane, and dried and dried to prepare a composite membrane. In order to clarify the molecular structural characteristics of the silk fibroin / polyallylamine complex, a Fourier transform infrared spectrum (FT-
IR) was measured with a measuring device manufactured by PerkinElmer.
The measurement wave number region was 2000 to 400 cm ⁻¹ , and the number of measurement repetitions was 20 times. The number of absorption peak positions was determined, and the peak intensities were shown in four levels: very strong (VS), strong (S), medium (M), and weak (W). Table 3 shows the obtained results. In Table 3, for example, SF / PAA-10C
(75/25) means a silk fibroin aqueous solution having a concentration of 3% and a polyallylamine (P
AA-10C) Aqueous solution is uniformly mixed with silk fibroin and polyallylamine so that the weight ratio of silk fibroin and polyallylamine becomes 75% and 25%, and further, water is appropriately added to the mixed solution to convert silk fibroin and polyallylamine. It means a composite membrane prepared by mixing so that the total concentration of the mixed aqueous solution becomes 1%, then spreading this mixed aqueous solution on a substrate and evaporating to dryness. Other composite membranes in Table 3 refer to membranes prepared in a similar manner. The same applies to the composite membranes described in Tables 4 and 6 below.

[0056]

[Table 3] As is clear from Table 3, in the FT-IR spectrum of the silk fibroin / polyallylamine complex, a unique absorption peak derived from the silk protein and a unique absorption peak derived from the polyallylamine appeared in an overlapping manner. You can see that there is. This means that the complex is composed of silk fibroin molecules and polyallylamine molecules, and that no new chemical bond is formed as a result of the combination of the two. Example 4 Estimation of Molecular Interaction Between Composition Components by DSC A polyallylamine film, a silk fibroin (SF) film, and a silk fibroin / polyallylamine composite film having different contents of the silk fibroin were produced as follows. Table 4
5 types of polyallylamine (PAA-10C,
PAA-HCl-10L, PAS-H-10L, PAS
-A, PAS-92) to prepare a silk fibroin / polyallylamine composite membrane in the same manner as in Example 1.
Further, a film of polyallylamine alone and a film of SF alone were prepared in the same manner.

DSC measurement was performed using the composite membrane prepared as described above, and their thermal behavior was examined. 100
The endothermic peak temperature that appears in the temperature range of not less than ° C. was adjusted.
Table 4 shows the obtained results. In Table 4, the letters attached to the indicated temperatures indicate the form of the endothermic peak, B means a wide peak, and VB means a very wide peak.

[0058]

[Table 4] As is clear from Table 4, the DSC endothermic peak of silk fibroin appears at 280 ° C., but in the silk fibroin / polyallylamine complex, the endothermic peak temperature position slightly changes due to the difference in the content of silk fibroin. This means that between silk fibroin and polyallylamine, hydrogen bond, ionic bond, hydrophobic bond, etc.
It suggests that some physical binding occurs that enhances the cohesive action between the molecules. Example 5 Examination of Insolubilization Treatment Method The composite membranes shown in Table 5 were prepared in the same manner as in Example 1, and the degree of water insolubilization of these composite membranes was evaluated as follows. An aqueous solution prepared from chlorooxirane (also called epichlorohydrin, manufactured by Kanto Chemical Co., Ltd.), water, and methanol (5: 10: 85% by volume) was used as an aqueous solution of an insolubilizing agent for insolubilizing the composite membrane with water. The composite membrane shown in Table 5 was applied to this insolubilized drug aqueous solution.
It was immersed in 5 mL and allowed to stand for 3 minutes. After that, the composite membrane is taken out and further washed with a 50% aqueous methanol solution.
By drying at room temperature, composite membranes having different degrees of water solubility were prepared. The water-insoluble sample of the composite membrane thus prepared was immersed in water, and the dissolved state of the sample was visually observed for 30 minutes. Table 5 shows the obtained results. In Table 5, the dissolution state of the composite membrane was evaluated in the following three stages.

：: Insoluble in water.

△: Immediately after immersion, it is insoluble in water, and gradually begins to dissolve by absorbing water.

×: Dissolves immediately after immersion in water.

[0062]

[Table 5] As is clear from Table 5, any polyallylamine shows high hygroscopicity by itself and has the property of being soluble in water.
However, in the case of the present invention, polyallylamine is ionically bonded to the negatively charged amino acid side chain of silk fibroin of the ampholyte, and when the content of silk fibroin is in the range of 50% by weight or more, the composite membrane becomes water-soluble. Becomes insoluble. However, some polyallylamines may be insoluble even at about 25% by weight. Example 6 Mechanical properties of composite membrane Mechanical properties when dried: 6 mL of 3% silk fibroin aqueous solution
And 4 mL of a 6% aqueous solution of polyallylamine, gently stirred with a glass rod, spread this mixture on the surface of a polyethylene membrane, and evaporated water at room temperature to make 75% by weight and 25% by weight of silk fibroin and polyallylamine. % Of the composite membrane was prepared. The strength and elongation of the composite membrane thus obtained were measured, and the results are shown in Table 6.

[0063]

[Table 6] With respect to the mechanical properties during drying, the silk fibroin membrane has a brittle property of a cut elongation of only 0.6%, but becomes easily stretched by being combined with polyallylamine.

Mechanical properties of the composite membrane when wet: The composite membrane obtained in the same manner as above was placed in water for 24 hours, and after the water reached an equilibrium state, the mechanical properties of this composite membrane were examined. ,
Table 7 shows the obtained results.

[0065]

[Table 7] As is clear from Table 7, the water-absorbed composite membrane has lower strength than the dry composite membrane, but has higher elongation and is more easily stretched. Example 7 Application to Artificial Skin A simple treatment in which the composite membrane obtained in the same manner as in Example 1 was immersed in the same insolubilized mixed aqueous solution as in Example 15 for 15 seconds to remove 98% by weight of water for a long time A hydrogel-like substance to be retained was obtained. When this gel-like substance was applied to the skin as it was and dried, an elastic film was obtained. Example 6
As can be seen from the figure, the silk fibroin / polyallylamine composite membrane in the dry state exhibited characteristics that compensated for the disadvantage of the silk fibroin membrane having a small breaking elongation, and was easily stretched (Table 6). In the inhaled state, the elongation of the composite membrane further increased and became elastic (Table 7).

Silk fibroin itself is used by being embedded in a living body as a surgical material and does not adversely affect living tissues. Polyallylamine is transferred to a culture support for living cells and tissues. Immobilizing material (JP-A-2-181628 and 2-191629)
Therefore, it can be said that the composite of the present invention can be used as a medical material such as a wound dressing. Example 8 Experiment of Proliferating Insect Cells on the Surface of the Composite Membrane Method for Preparing Cell Culture Bed Substrate: As described below, using a 3% silk fibroin aqueous solution and a 6% polyallylamine aqueous solution as stock solutions, the content of silk fibroin (100, 90) ,
(75, 50, 25, 0% by weight) were prepared, and 1.2 mL of the mixed aqueous solution was placed in a cell culture vessel, the culture vessel was covered, and a cell culture bed was prepared. A stock solution of a 6% aqueous solution of polyallylamine was prepared by adding a dilute solution of an aqueous ammonia solution to the aqueous solution of polyallylamine little by little and adjusting the pH of the aqueous solution of polyallylamine to about 7 using pH test paper. Using this as a stock solution, a cell culture bed substrate neutralized as described below was produced.

A separately prepared 3% aqueous solution of silk fibroin and a 6% aqueous solution of polyallylamine were combined with 100%, 90, 75, 50, 25, 0% by weight of silk fibroin.
, And water was appropriately added to the mixed solution to prepare an aqueous solution in which silk fibroin and polyallylamine were mixed so as to have a concentration of 1%. 1.5 mL of the mixed aqueous solution thus prepared was added to a cell culture vessel (24 wells, Becton Dickinson Company, trade name: FALCO
N 3047) and left at room temperature for 30 minutes. The mixed solution was completely removed by a decantation method, and air-dried at 20 ° C. for 1 day to form a complex thin film on the surface of the cell culture vessel. Next, in order to insolubilize the composite membrane with water, 2.5 mL of an aqueous solution of an insolubilized drug consisting of chlorooxirane (also called epichlorohydrin, manufactured by Kanto Chemical Co., Ltd.), water, and methanol (5: 10: 85% by volume) was used. It was applied to this composite membrane and allowed to stand for 3 minutes. Next, after the solution was removed, the solution was air-dried in a standard state, and further dried at 40 ° C. for 30 minutes. After washing with 2 mL of methanol, the mixture was air-dried and dried at 40 ° C. for 15 minutes. For example, 3% silk fibroin aqueous solution and 6%
The amounts of each aqueous solution and water for preparing a composite membrane having a weight ratio of silk fibroin and polyallylamine of 90% and 10% from the aqueous solution of polyallylamine were as follows.

[0068] The silk fibroin polyallyl amount of amine solution 360μl 20μl 820μl silk fibroin / polyallylamine preparation conditions of the composite film are shown in Table 8.

[0069]

[Table 8] Insect cell culture experiment (1): The state of adhesion and proliferation of cells was observed using insect cells. Using a non-neutralized polyallylamine aqueous solution, silk fibroin and polyallylamine were complexed to prepare a cell culture bed substrate.
Attachment state (hereinafter abbreviated as Ae and Bm attachment) and growth state (hereinafter Ae) of tussah (Ae) and silkworm (Bm) insect cells on the cell culture bed surface coated with various complex membranes having different silk fibroin contents. , Bm proliferation), and the number of cells was measured. Table 9 shows the obtained results.

[0070]

[Table 9] Insect cell culture experiment (2): The state of cell attachment and proliferation was observed using insect cells. Silk fibroin and polyallylamine were complexed using an aqueous solution of polyallylamine neutralized by adding an alkaline agent to prepare a cell culture bed base material. Attachment state (hereinafter abbreviated as Ae and Bm attachment) and growth state (hereinafter Ae) of tussah (Ae) and silkworm (Bm) insect cells on the cell culture bed surface coated with various complex membranes having different silk fibroin contents. , Bm proliferation), and the number of cells was measured. Table 10 shows the obtained results.

[0071]

[Table 10]In addition, the number of cell proliferation in Tables 9 and 10 is
10 found by abacus ^-FourShows the number of cells per ml
Therefore, the measured value of the number of cells after culturing was 10^Four
Multiplied by.

As can be seen from Tables 9 and 10, tussah cells (Ae cells) classified as adherent cells proliferated in the biopolymer / polyallylamine composite membrane at a silk fibroin content of 75 to 25% by weight. The condition has improved. Also, the adhesion state was good. Examples of polyallylamine include unneutralized PAS-92 and neutralized PA
SH-10L is particularly good. PA in neutralized state
50% silk fibroin on A-HCl-10L surface
On the surface of the composite membrane of ２５25% by weight, Ae cells died, and granule cells were confirmed in round cells. Bm cells derived from the semi-suspended silkworm, Bombyx mori, showed a good growth state at a silk fibroin content of around 50%. As the polyallylamine, PAA-HCl-10 L in an unneutralized state and PAS-92 in a neutralized state are particularly preferable. In the biopolymer / polyallylamine composite membrane, the proliferation of Ae and Bm cells was better when the neutralized sample was used than when the sample was not neutralized.

The reason why the addition of silk fibroin after the neutralization treatment of polyallylamine increased the number of Ae and Bm insect cells and showed good adhesion was considered as follows. Polyallylamine is a hydrochloride of polyallylamine in the hydrochloric acid pH range, but when the pH is adjusted to the alkali side by adding ammonia water little by little, the hydrochloride is removed, and the polyallylamine having a molecular side chain of NH ₂ . Becomes It is considered that the complex and the amphoteric silk protein molecule are well mixed at the molecular level to produce a complex having excellent compatibility, and thus the adhesion and proliferation of living cells are improved. Example 9 Attachment / proliferation experiment using mouse-derived fibroblasts Mouse-derived fibroblasts (L929) on the surface of the composite membrane
On the fourth day of culture, the state of cell attachment and proliferation was observed, and the number of cells was measured. Table 11 shows the obtained results.

The term “Cont” in Table 11 means the state of cell attachment and proliferation on the surface of a commercially available culture plate.
The number of cell proliferation was determined by using a hemocytometer of 10 ^−4.
In order to indicate the number of cells per ml, the measured value of the number of cells after culturing is a value obtained by multiplying the value in the above table by 10 ⁴ .

[0075]

[Table 11] Cell adhesion state in Table 11: +++ Cells are very strongly attached to the cell container surface.

++ Cells adhere well to cell container surface.

+ Cells are normally attached to the cell container surface.

The cells are not attached to the cell container surface;

Observation results of cell attachment / proliferation state in Table 11: * 1 Some cells float on the surface of the medium. There are no agglomerates.

* 2 Cells float on the surface of the medium. Form aggregates.

* 3 Granular substances are found in the cells, and the contents of the cells are flowing out.

* 4 The cell's byssus extends and sticks in a spindle shape. The adhesion state of the cells is good, and some cells float on the surface of the medium.

* 5 There are many cell proliferation numbers. It adheres well to the cell culture bed surface.

* 6 The cells spread well on the surface of the cell culture substrate, and the number of proliferating cells is large.

From Table 11, on the surface of the composite membrane formed of PAA-HCl-10L and silk fibroin, the number of proliferation of mouse-derived fibroblasts was 90 to 5 for silk fibroin content.
A good value was obtained at 0% by weight. PAS-H-10L and silk fibroin (silk fibroin content around 90% by weight)
It was confirmed that fibroblasts were strongly adhered to the surface of the composite membrane. Example 10 Metal ion adsorption (Ag, Co) on composite 3% silk fibroin aqueous solution 3% and PAA-1 6%
After mixing with 2 mL of 0C aqueous solution and gently stirring, the mixture was spread on a polyethylene membrane and dried and solidified at room temperature to prepare a composite membrane composed of silk fibroin and PAA-10C. To insolubilize in water, the composite membrane was immersed in an aqueous solution of insolubilizing agent for 30 seconds in the same manner as in Example 5, taken out, washed with a 50% by volume aqueous methanol solution, dried at room temperature, and dried with water. Insoluble complex (PAA / SF
Abbreviated).

Metal ions were adsorbed to the obtained water-insoluble composite membrane as follows. First, 33 mL of water
9.7 mg of silver nitrate (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved to prepare a 0.5 mM silver nitrate aqueous solution. In this, potassium nitrate (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 505.6.
The pH of the aqueous solution was adjusted to 11.4 with 1N aqueous ammonia (manufactured by Wako Pure Chemical Industries, Ltd.), and then water was added to prepare a metal ion aqueous solution such that the total amount of the solution became 60 mL. . The above composite membrane was put in the aqueous metal ion solution containing silver ions, allowed to stand at 25 ° C. for 5 hours, taken out, and air-dried at room temperature. The complex in which silver ions are adsorbed and coordinated in this manner (hereinafter referred to as PAA / S
(Abbreviated as F-Ag).

Using cobalt nitrate (manufactured by Wako Pure Chemical Industries, Ltd.) instead of silver nitrate, a water-insolubilized complex (hereinafter, PAA / SF-C) coordinated with cobalt in the same manner as described above.
(abbreviated as o) was prepared. Also, a 3% silk fibroin aqueous solution is spread on a polyethylene membrane, and water is evaporated to prepare a silk fibroin (hereinafter abbreviated as SF) membrane, which is added to a silver or cobalt metal ion aqueous solution at 25 ° C.
Silk fibroin (hereinafter, SF-Ag, SF-C) on which silver or cobalt is adsorbed and coordinated by immersion treatment for 5 hours
(abbreviated as o) was prepared.

The inhibitory effect of these metal ion-adsorbing membranes on the growth of tomato canker bacteria was evaluated. Culture was performed in a dark area covered with a black cloth to evaluate antibacterial properties. Table 12 shows the obtained results.

[0089]

[Table 12] As is clear from Table 12, the antibacterial activity is higher when the metal is coordinated to the silk fibroin / polyallylamine complex than when the metal is coordinated to the silk fibroin itself.

Next, Escherichia coli was used instead of the tomato canker bacterium, and the degree of inhibition of the metal ion-adsorbing membrane on the growth of Escherichia coli was examined. Table 13 shows the obtained results.

[0091]

[Table 13] As is clear from Table 13, it can be seen that all the metal ion-adsorbed membranes of the present invention inhibit the growth of Escherichia coli.

In the same manner as in Example 10, Ag, C
The water-insoluble complex in which Cu or Zn was adsorbed and coordinated in place of o suppressed the growth of tomato scabs and Escherichia coli.

Thus, the complex of the present invention is useful as a carrier for supporting various substances.

In the above embodiment, a typical silk protein such as silk fibroin has been described as a biopolymer, but similar results can be obtained with other biopolymers described above.

[0095]

The biopolymer / polyallylamine complex of the present invention is obtained by complexing a biopolymer such as silk fibroin and polyallylamine under predetermined conditions. It has mechanical properties not found in materials or biochemical characteristics of each material synergistically.

According to the complex of the present invention, living cells such as insect cells and mouse-derived fibroblasts can be efficiently adhered to the surface thereof, and the cells can be grown there. Further, the complex, in addition to the cell attachment and proliferation of biopolymers such as silk fibroin,
It also has the cell growth function of polyallylamine. Therefore, the complex of the present invention can be used for immobilized enzymes, medical materials, carriers for separating biological components, immunoassays, base materials for cell culture beds, and the like.

Since the composite of the present invention is composed of polyallylamine having a positive charge in the side chain and a biopolymer of an ampholyte, an ionic bond is generated between the two molecules and the material has strong molecular cohesion. Therefore, by encapsulating the active ingredient in such a complex, a sustained-release carrier capable of containing a drug, a biologically active substance, or the like can be obtained. (Drug delivery Devic
e) Available as The composite can reliably contain an antimicrobial metal, and can be used as a durable antimicrobial material.

Since the complex of the present invention can incorporate a physiologically active substance such as a cell, an enzyme or a hormone,
It can be used as an artificial organ material that is closer to the function of a living body. Further, the composite of the present invention, an artificial blood vessel,
It can also be used as a biomedical material such as a contact lens, a wound dressing, and a material for intradermal injection.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ Identification code FI A61L 27/00 C12Q 1/18 C12N 5/06 C12N 5/00 E C12Q 1/18 A61L 15/01 (72) Inventor Shoji Hayasaka 1-2, Owashi, Tsukuba, Ibaraki Pref., Ministry of Agriculture, Forestry and Fisheries, Research Institute for Silk and Insect Agriculture (56) References -57562 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C08L 39/00 C08K 5/07 C08L 89/00 C12N 11/00 A61L 15/16 A61L 27/00 C12N 5 / 06 C12Q 1/18 WPI (DIALOG)

Claims (7)

(57) [Claims] (1) silk protein, wool keratin, collagen,
A spider silk, or a biopolymer that is a marine protein, and a neutralized or unneutralized polyallylamine,
A biopolymer / polyallylamine complex comprising polyallylamine having a positive charge in the molecule. 2. A pre SL biopolymer / polyallylamine complex biopolymer / polyallylamine complex according to claim 1, characterized in that with the attached-growth enhancing function of cells. 3. The biopolymer / polyallylamine complex according to claim 1 or 2, which is treated with an aqueous solution of a mixed solution of water and an alcohol with an aqueous solution of a water-insolubilizing drug obtained by mixing an epoxy compound, an aldehyde, or both compounds. , The living body height
Water insoluble biopolymer / poly <br/> Ariruami down member, characterized in that cross-linking between molecules and polyallylamine cross is one that was introduced. 4. A silk protein, wool keratin, collagen,
A spider silk or a biopolymer that is a marine protein, and a neutralized or unneutralized polyallylamine are mixed in an aqueous solution or aqueous dispersion, and then evaporated to dryness to obtain a bioactive substance. A method for producing a biopolymer / polyallylamine complex characterized by obtaining a molecule / polyallylamine complex. 5. A method for preparing a silk protein and a polyallylamine.
The composite membrane is mixed with a mixed aqueous solution of water and alcohol.
Poxy compounds, aldehydes, or a mixture of both
Treated with an aqueous solution of water-insolubilized drug to introduce cross-linking
Wound dressing. 6. A method for preparing a silk protein and a polyallylamine.
The composite membrane is mixed with a mixed aqueous solution of water and alcohol.
Poxy compounds, aldehydes, or a mixture of both
Treated with an aqueous solution of water-insolubilized drug to introduce cross-linking
A cell culture bed substrate having cell attachment and proliferation functions. 7. A method for preparing a silk protein and a polyallylamine.
The composite membrane is mixed with an aqueous solution of water and alcohol.
Poxy compounds, aldehydes, or a mixture of both
Treated with an aqueous solution of water-insolubilized drug to introduce cross-links.
Rusty metal Ion adsorption substrate.