JP2955653B2 - Silk protein / collagen composite and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a silk protein / collagen complex and a method for producing the same. More particularly, the present invention relates to a silk protein of an insect biopolymer and a main protein of a biological connective tissue. The present invention relates to a silk protein / collagen complex which is a material which can be produced by mixing collagen having high utility value as a material in the form of an aqueous solution or aqueous dispersion and has excellent biocompatibility, and a method for producing these.

[0002]

2. Description of the Related Art Silk proteins are insect biopolymers produced by insects such as silkworms, and have been conventionally used as clothing materials. Since silk protein fibers have excellent practical properties such as dyeability, moisture absorption / desorption properties, and mechanical properties, they have been useful as clothing materials. Recently, attention has been paid to the excellent biocompatibility of silk proteins, and they have begun to be used as medical materials in advanced fields. It is said that silk has been used for surgical sutures since the 11th century. It is easy to treat bacteria and hardly causes antigen-antibody reactions even when implanted in the body. Is becoming possible.

It is known that even when a silk fibroin membrane is placed in a vein for 2 weeks by the peripheral vein sample coating method, blood coagulation is not observed on the silk fibroin membrane surface. For this reason, the silk protein membrane hardly causes blood coagulation even when it comes into contact with blood, and can be used as a material having high blood compatibility. In addition, the silk protein membrane can be used as a biomaterial because living cells adhere and grow well on the surface thereof. For example, when cells are cultured by immersing a silk fibroin membrane in a mouse-derived fibroblast suspension containing 10% calf serum, the cells adhere well to the silk fibroin membrane surface,
Proliferation has been confirmed (Japanese Patent Publication No. 4-41595).
No.), it has been expected that useful cells can be cultured efficiently at low cost by using silk fibroin as a substrate for cell culture beds. Thus, silk proteins can be used in the field of cell engineering and immunotechnology.

[0004] On the other hand, collagen is a major protein of animal-derived biological connective tissue, and is distributed in many tissues such as skin, blood vessels, bones, and teeth. This collagen is a natural biopolymer, has excellent biological functions, is widely used as a material for artificial blood vessels and artificial organs, and the mechanism of antigen-antibody reaction is analyzed at the molecular level. Collagen fibers and membranes have been used as sutures because they exhibit relatively excellent strength properties and have good compatibility with living tissues. Collagen is regarded as important as a biomaterial, and gelatin, which is a denatured product of collagen, is an indispensable material as a surface coating material for artificial organ materials. Collagen has excellent affinity for living cells and tissues, so it is also promising as a biomaterial that can promote tissue healing and restore it to a tissue equivalent to itself. In addition, since collagen is involved in thrombus formation originating from platelet adhesion and aggregation, it is also useful in designing a biomaterial having excellent blood compatibility. Collagen can be processed into various shapes such as membranes, threads, sponges, and gels.

[0005] Collagen is composed of three peptide chains having a molecular weight of 100,000, and has a triple helix structure. On both sides of this triple helix structure, there is a telopeptide consisting of several tens of amino acids. Atelocollagen is obtained by removing the telopeptide causing the antigen with a protease or the like.

As described above, the insect biopolymer silk protein and the animal-derived collagen each have excellent biological functions, and thus can be used with high added value in the advanced fields of the medical field. A homogeneous complex of protein and collagen has not been obtained so far.

[0007]

Accordingly, by combining natural materials such as the above-mentioned silk protein and collagen, each of the components has a superior function or a synergistic effect of the individual function. It has been desired to develop a manufacturing technique for obtaining a new biochemical material that has a special purpose.

For example, an aqueous solution of silk fibroin is neutral (p
However, when an aqueous solution of collagen is mixed with this aqueous solution of silk fibroin, the aqueous solution of collagen is acidic, and the pH of the mixed aqueous solution becomes 3.8 to 4.0 or less, as the isoelectric point of silk fibroin. Silk fibroin precipitates. As described above, the conventional technique has a problem that the aqueous solution of silk fibroin and the aqueous solution of collagen cannot be mixed uniformly. That is, when the aqueous solution of silk fibroin and the aqueous solution of collagen are mixed by the conventional method, the aqueous acidic solution of collagen lowers the pH of the mixed aqueous solution, and as a result, the pH of the silk fibroin becomes equal to or lower than the isoelectric point.
Silk fibroin precipitates. Thus, since the silk fibroin aqueous solution and the collagen aqueous solution cannot be mixed uniformly, it was extremely difficult to produce a physically uniform composite membrane. Therefore, when mixing an aqueous solution of silk fibroin and an aqueous solution of collagen, it is a preparation technology that is excellent in yield, efficiency and economy and is easy to handle, and a technology for mixing silk fibroin and collagen at the molecular level. Is desired.

It is an object of the present invention to uniformly mix collagen and silk protein in an aqueous solution or aqueous dispersion state, and to efficiently and economically produce a silk protein / collagen complex from the mixed liquid. is there.

[0010]

The silk protein / collagen complex of the present invention is obtained from an aqueous solution or an aqueous dispersion of collagen and silk protein obtained from an insect-derived silk protein fiber such as silkworm. is there.

[0011] As the silk protein, sericin which adheres to the outside of a cocoon fiber formed by silkworm spiking or silk fibroin fiber obtained by removing the sericin is dissolved in a neutral salt aqueous solution to obtain a cellulose protein. Water-soluble silk fibroin obtained by dialysis using a dialysis membrane, or water-soluble silk sericin or water-soluble silk fibroin in a silk gland taken out from a silkworm body can be used.

The collagen may be a collagen molecule side chain composed of three peptide chains having a molecular weight of 100,000 or atelocollagen (a protein obtained by removing a telopeptide consisting of several tens of amino acids on both sides of a triple helix structure of the collagen). )) Water-soluble or water-dispersible acylated collagen or acylated atelocollagen (neutral pH range) obtained by reacting an acylating agent with the basic amino group of the molecular side chain or the hydroxyl group of the molecular side chain of serine or tyrosine. Water-soluble) can be used. As the acylating agent, it is preferable to use an anhydride of a dibasic acid C ₁ -C _4, or anhydrides of monobasic acids C ₁ -C _5. The acylated atelocollagen is preferably succinylated atelocollagen, myristylated atelocollagen, or a mixture of both.

[0013] As the collagen, atelocollagen obtained by removing telopeptide from collagen derived from animal leather with a protease may be used.

The method for producing a silk protein / collagen complex of the present invention is a method for preparing a membrane or gel from an aqueous solution or an aqueous dispersion of an acylated collagen obtained by acylating collagen or atelocollagen or an acylated atelocollagen and silk protein. And a powdery or fibrous silk protein / collagen complex.

The silk protein / collagen complex can be used for fixing biologically active substances, antibiotics, enzymes, hormones, living cells, microorganisms and the like.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The silk protein / collagen complex of the present invention is composed of a silk protein containing a large amount of basic amino groups such as lysine, arginine, histidine and the like, and a chemically modified collagen or atelocollagen. After preparing a complex from both samples, if desired, a desired degree of crosslinking can be obtained by reacting with a chemical crosslinking agent, for example, an epoxy compound according to a conventional method. Solubility can be controlled as desired.

The silk protein fibers used in the present invention include, besides silkworm silk obtained from larvae of Bombyx mori,
Any of wild silkworm silk obtained from larvae of tussah silk, heaven silkworm, eri silkworm, moth silkworm, shinju silkworm belonging to wild silkworm, or any of these fiber products can be used.

For dissolving silk protein fibers (silk yarn), calcium chloride, calcium nitrate, lithium bromide,
A method is used in which a silk thread is heated in a concentrated solution of a neutral salt such as lithium thiocyanate. Dissolution conditions are based on not significantly reducing the molecular weight of the silk protein. The neutral salt concentration may be about 8.0 to 9.8% by weight (higher is better), and the dissolution temperature may be about 25 to 70 ° C. The dissolution temperature is preferably 60 ° C. or lower. When the melting temperature is high, the molecular weight of the silk protein is reduced, and there is a risk that the high molecularity of the material is lost. The dissolution time is 1-2.
It is preferable to set the time to about 0 minutes. Among the neutral salts, the one that dissolves the silk protein fiber well is a lithium salt that is excellent in the dissolving power of the silk fibroin fiber, and usually lithium bromide is preferable. If the lithium bromide is 8 M or more, preferably 8.5 M or more, the silk protein fiber dissolves at 55 ° C. or more for 15 minutes.

In order to obtain a pure aqueous solution of silk protein from a neutral salt solution in which silk protein fibers are dissolved, an aqueous neutral salt solution of silk protein is put into a dialysis membrane made of cellulose, and both ends are sewn with a sewing thread. Alternatively, it is necessary to completely remove lithium ions by placing in pure water for 4-5 days.

The collagen used in the present invention is obtained, for example, as follows. The dermis of calf skin is finely cut and extracted using a citrate buffer. Separate the extract,
Perform a second extraction with fresh citrate buffer. This was filtered using a glass filter or the like, and then Na ₂ HPO ₄
Dialyze using. The collagen precipitate is collected by centrifugation. After washing with water, redissolve in citrate buffer, Na ₂ HP
Repeat O ₄ dialysis. Soluble collagen is obtained by repeating precipitation and redissolution in this way.

Chemically modified collagen and atelocollagen, which can be produced by acylating, for example, succinylating or myristylating collagen or atelocollagen, have the property of being soluble in water in the neutral pH range. Therefore, even if this chemically modified collagen or atelocollagen is mixed with an aqueous silk protein solution, the silk protein does not coagulate.

Hereinafter, a description will be given taking collagen as an example.
The same is true for atelocollagen. Unmodified collagen, which has not been chemically modified, has its isoelectric point (pH)
Is 9.1 and becomes water-soluble when the isoelectric point is 5 or less. When the lysine side chain of the collagen molecule is succinylated, for example, the isoelectric point becomes 4.57 and the pH of the neutral region
Solubilize without precipitation. That is, soluble collagen is soluble in an acidic aqueous solution, but if the pH of the aqueous solution is increased, fiber regeneration occurs at a pH of 5 or more and precipitates. However, acyl groups such as succinyl groups and myristyl groups are sufficiently introduced into collagen chemically modified by acylation such as succinylation and myristylation,
Alternatively, since the COOH group is added to the introduced molecule, the molecule is in a negative charge-rich molecular environment, and shows water solubility even in a neutral pH range.
The aqueous solution of collagen soluble in the neutral region can be mixed uniformly, or the chemically modified collagen can be added to the aqueous solution of silk protein, or the silk protein can be added and mixed uniformly to the aqueous solution of chemically modified collagen.

Examples of the chemical modification method for dissolving collagen in water include a succinylation reaction in which collagen is reacted with succinic anhydride as a dibasic acid anhydride, and a collagen and monobasic acid anhydride. A conventional reaction such as a myristylation reaction of reacting with myristic anhydride as the above can be used. When the collagen is chemically modified by such a reaction, it becomes soluble in water in a neutral pH range and can be complexed with an aqueous silk protein solution. In addition, since the charge state of collagen changes, the reactivity with blood, the interaction with cells, the HLB of collagen (hydro
lipophilic balance) can be easily controlled. Further, since the platelet adhesion / agglutination reaction can be controlled, the antithrombotic surface can be easily formed.

The acylation treatment such as succinylation treatment and myristylation treatment used in the present invention is carried out as follows. As a soluble organic solvent such as an acylating agent for treating collagen, for example, a succinylating agent and a millstilling agent, dimethylformamide, dimethylsulfoxide, pyridine, and a mixed solvent of glacial acetic acid / acetic acid can also be used. It is. Among these organic solvents, pyridine having a catalytic effect exerts particularly excellent effects. It is also possible to use a mixed solution system in which pyridine which is a reaction catalyst is added to a solvent such as dimethylformamide.
The concentration of the anhydride in the processing solvent is preferably set in the range of 5 to 30% by weight, more preferably 10 to 20% by weight. As the processing conditions, it is desirable to perform the processing in a processing solvent at 30 ° C. or more for 1 hour or more.

In the present invention, an acylating agent such as a succinylating agent or a myristylating agent is used in an amount of 3 to 20% by weight, preferably 5 to 1% by weight on a dry matter basis with respect to collagen.
The reaction is preferably performed in such an amount that 5% by weight is introduced. The acylating agents include anhydrides of C ₁ -C ₄ dibasic acids, for example, C ₁ : diglycolic acid, C ₂ : succinic acid, C ₃ : glutaric acid, or C ₄ : adibic acid; C _1-
Anhydrides of C ₁₅ monobasic acids, e.g., C _1: acetate, C _2: propionate, C _5: caproic acid, C _8: caprylic acid, C _11:
Lauric acid, C ₉ : myristic acid, or C ₁₅ : palmitic acid can be used.

In the present invention, a chemical modifier (acylating agent)
When reacting, in the active site of collagen,
Basic amino acid residues such as lysine, arginine and histidine have strong reactivity with acylating agents such as succinylating agents and myristylating agents. These acylating agents also react with hydroxyl groups on the side chains of serine and tyrosine. However, the higher chemical stability of the bonds is due to the lysine, the basic molecular side chain of these drugs and collagen.
This is the case where arginine, histidine and the like are bound.

In order to immobilize an active ingredient such as an antibiotic on the silk protein / collagen complex of the present invention, for example, the active ingredient is added to an aqueous solution obtained by mixing an aqueous silk protein solution and an aqueous collagen solution before preparing the complex. After dissolving and stirring carefully and gently, it may be spread on a substrate film such as polystyrene or polyethylene to evaporate water. To prepare a silk protein / collagen complex with the active ingredient immobilized,
A silk protein aqueous solution or collagen aqueous solution of any concentration can be used. However, in handling these aqueous solutions, the preferred silk protein concentration is 0.01 to 10% by weight, more preferably 0.1 to 5% by weight. When the silk protein concentration exceeds 10% by weight, the coagulation rate of the aqueous solution becomes difficult to control,
On the other hand, if the content is less than 0.01% by weight, the sample concentration is too dilute,
On the contrary, the amount of the aqueous sample solution is too large, and the efficiency of mixing the aqueous collagen solution becomes poor. Further, it is not preferable to use a dilute aqueous solution of silk protein because the evaporating and drying time of the aqueous solution mixed with the aqueous collagen solution is prolonged and the efficiency of sample preparation is reduced. A collagen aqueous solution having a concentration of 0.1 to 5% by weight can be used. A preferred collagen aqueous solution is 0.1 to 3% by weight. If the content exceeds 5% by weight, the collagen aqueous solution is likely to gel at room temperature, which causes inconvenience in handling during evaporation to dryness.

The type and amount of the active ingredient that can be fixed to the silk protein / collagen complex are not particularly limited.
It can be arbitrarily determined according to the purpose.

A complex obtained by evaporating and drying an aqueous solution of a silk protein solution and an aqueous solution of collagen or an aqueous solution thereof containing an active substance and solidifying it is water-soluble.
When the obtained composite is used in an aqueous solution system, it is dissolved, and therefore it is preferable to change the composite to water-insoluble. for that purpose,
(1) treatment with an agent that forms crosslinks between protein molecules, such as formaldehyde and glutaraldehyde;
(2) Chemical treatment with a bifunctional epoxy compound such as ethylene glycol diglycidyl ether, and (3) treatment with a crosslinking agent such as hexamethylene diisocyanate may be used. In the water insolubilization treatment, an insolubilization treatment with other general reagents or an irradiation treatment with ultraviolet rays may be used as long as sufficient care is taken not to reduce the activity of the active ingredient fixed to the complex. In the above treatments (1) and (2), the degree of insolubilization of the complex can be controlled by adjusting the amount of reagent and changing the degree of reaction.

The succinylation of collagen is carried out, for example, by dissolving succinic anhydride in a solvent such as dimethylformamide and reacting the same, whereby lysine residues of collagen are succinylated, and as a result, the introduced lysine residues are introduced into the molecule. C
An OO group is added. Similarly, when myristic anhydride is reacted with collagen in an organic solvent, a myristyl group is introduced and a COOH group is introduced into the molecule introduced into the lysine residue. The isoelectric point (pH) of the modified collagen that can be prepared by such chemical modification is around 4 to 5, and the collagen aqueous solution does not precipitate near neutrality.

In the case of the silk protein / collagen complex of the present invention, an aqueous solution or aqueous dispersion of each component is separately prepared, and then a mixture obtained by uniformly mixing the two is obtained.
Alternatively, using one aqueous solution or aqueous dispersion in which both components are uniformly mixed together, or one is dissolved and dispersed, and the other is dissolved and dispersed, and then uniformly mixed, is used as a polyethylene membrane, polystyrene. A composite membrane is obtained by evaporating water by spreading on a substrate such as a membrane, and a gel-like composite is obtained by adding a known gelling agent to the composite membrane. A powdery composite is obtained.

[0032]

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. There are many types of silk fibroin. Unless otherwise specified, silk fibroin derived from silkworm larvae was used.

In the following examples, as a plant pathogenic bacterium, which is a representative of universal plant pathogenic bacteria, resistant bacilli are likely to appear, and it is a multicidal rot fungus that commits many plants. Tomato scab ( Corynebacterium mic)
higanese pv. michiganese ).

The antibacterial activity against bacteria and filamentous fungi in the examples was evaluated by the following method. A. Assay for antibacterial activity against bacteria: 25 ml of semi-synthetic armpit or King B medium kept at 55 ° C. after heating and dissolving, and 2 ml of test bacteria (concentration: 10 ^{9 -10} cells / ml) are mixed, and this mixture is poured into a petri dish. And solidified into a flat plate. Place the test sample cut to a length of about 2 cm on this bacterial mixture mixed plate medium, carefully embed both ends of the test sample in the medium with tweezers,
The entire membrane sample was brought into close contact with the medium. This is 20-25
C., and the degree of inhibition of bacterial growth in the medium near the test sample was evaluated in four steps at predetermined intervals according to the following criteria. However, those having a large width of the inhibition zone were indicated by actual measurement values (mm).

++: strong (forms a clear growth inhibition zone of 2 mm or more in width) +: weak (forms an indistinct inhibition zone or a clear inhibition zone of 1 mm or less in width) ±: slight (slight inhibition) -): No antibacterial activity was observed.

B. Assay for antibacterial activity against filamentous fungi: PSA medium maintained at 55 ° C. after heating and dissolving, and 2 ml of a spore solution (concentration: 10 5 ^-6 cells / ml) of the test bacteria were mixed, and the mixture was poured into a petri dish and flattened And observed in the same manner as in the evaluation of the antibacterial activity.

The following items were tested for the purpose of investigating how the sample characteristics changed when the composite was made as in the present invention.

C. Hygroscopicity: The sample was left for 1 week in a thermo-hygrostat adjusted to 20 ° C. and 65% RH, and the weight of the sample in which the amount of absorbed moisture was in an equilibrium state was measured. The moisture absorption (%) was determined from the difference from the weight after drying for 2 hours.

D. Mechanical properties: The mechanical properties (strength and elongation) of the silk protein / collagen composite were measured, and the strength and elongation of the sample at the time of cutting were evaluated. The measurement conditions were a sample length of 10 mm and a width of 2 mm, and a tensile speed of 10 mm / mi.
n, full-scale 250 g, measured by a tensile tester (Autograph, type AGS-5D) manufactured by Shimadzu Corporation.

E. Viscoelastic behavior: Using a rheograph / solid S type manufactured by Toyo Seiki Seisaku-sho, Ltd., heating rate 2 ° C
/ Min, measurement temperature range -30 to 260 ° C, measurement frequency 10
Hz, initial tension 30 gf, sample length 10 mm, sample width 2 m
The viscoelastic behavior was evaluated in m. From this measurement, the mechanical loss peak temperature of the sample was determined.

F. Thermal decomposition temperature: using a differential thermal scanning measurement device (DSC-10A) manufactured by Rigaku Denki Co., Ltd .;
2 mg, DSC range 2.5 mcal / s, heating rate 1
At 0 ° C./min, the measurements were performed in a nitrogen stream at 200 cc / min. The endothermic peak temperature appearing at 200 ° C. or higher in this measurement was defined as the thermal decomposition temperature of the sample.

G. Thermal analysis stability: The change in sample weight during the heating and heating process was measured in a nitrogen atmosphere using a thermogravimetric analyzer (TGA) manufactured by Rigaku Corporation. Sample weight 3m
g, heating rate 10 ° C / min, TGA full scale 5m
The analysis was performed under the condition of g. With the weight before the start of the measurement as 100, what percentage of the weight residual ratio was measured during the heating process was measured.

H. Thermal form stability: The dimensional change of the film-like sample in the temperature rising process was measured under a nitrogen atmosphere using a thermomechanical measuring device (TMA) manufactured by Rigaku Corporation.

Sample length 15 mm and width 2 mm, heating rate 10 ° C./min, sampling time 2 sec, T
The analysis was performed under the conditions of MA range ± 1000 μm. Based on the TMA results measured between room temperature and 350 ° C,
The percentage of shrinkage of the composite film at 180 ° C. compared to the sample length before measurement is evaluated, and this is called thermal form stability.

Example 1 2.5 g of silkworm silk was completely dissolved in 20 ml of an aqueous solution of 8.5 M lithium bromide at 55 ° C., and this aqueous solution was placed in a dialysis membrane made of cellulose and mixed with pure water at 5 ° C. for 5 days. The impurities were removed by substitution 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 such that the absolute dry concentration was 0.3%.

The myristylated atelocollagen was prepared as follows. The dermis of the calf skin is finely cut and
Extraction was performed for 1 to 2 days using a 15 M citrate buffer (pH 3.6). The extract was separated and a second extraction was performed with fresh citrate buffer of the same concentration. This was filtered through a glass filter and dialyzed against 0.02 M Na ₂ HPO ₄ . Collagen precipitate is collected by centrifugation, washed with water, and redissolved in 0.15 M citrate buffer.
Na ₂ HPO ₄ dialysis was repeated. Water-soluble collagen was obtained by repeating such precipitation and redissolution. On both sides of the triple helical structure of the water-soluble collagen thus obtained, there is a telopeptide consisting of several tens of amino acids and causing an antigen. Water-soluble atelocollagen was prepared by selectively digesting the telopeptide of collagen and causing pepsin, a proteolytic enzyme having the action of cleaving intermolecular crosslinks, to act on collagen molecules.

Then, myristic anhydride was dissolved in dimethylformamide as a myristylating agent, and atelocollagen prepared by the above method was added thereto.
The reaction was carried out for more than an hour to prepare a water-soluble myristylated atelocollagen. This was dissolved in distilled water to prepare a myristylated atelocollagen aqueous solution having a concentration of 0.3%.

Further, an aqueous succinylated atelocollagen solution was prepared in the same manner as described above.

To a 0.3% aqueous solution of succinylated or myristylated atelocollagen obtained as described above was added a predetermined amount of a stock solution of 0.3% silk fibroin aqueous solution, and the mixture was gently and thoroughly stirred with a glass rod. Was spread over a polyethylene membrane and the water was allowed to evaporate for more than 12 hours to produce a transparent and flexible silk fibroin / atherocollagen composite membrane. The aqueous solution of each component of the composite membrane was similarly evaporated to dryness to produce a membrane. The above evaporation to dryness was performed at 25 ° C. and 65% RH. I went in.

The film sample obtained in this example is as follows.

CSS: Membranous sample obtained from a 0.3% aqueous solution of succinylated atelocollagen.

SMS: Membranous sample obtained from a 0.3% myristylated atelocollagen aqueous solution.

SF / CMS (1: 1): A film-like sample obtained from a mixed aqueous solution obtained by adding 1 volume part of a 0.3% aqueous solution of silkworm silk fibroin to 1 volume part of a 0.3% myristylated atelocollagen aqueous solution.

SF / CSS (1: 1): A film-like sample obtained from a mixed aqueous solution obtained by adding 1 volume part of a 0.3% aqueous solution of silk fibroin to 1 volume part of a 0.3% aqueous solution of succinylated atelocollagen.

SF / CSS (1: 2): A film-form sample obtained from a mixed aqueous solution obtained by adding 2 parts by volume of 0.3% succinylated atelocollagen aqueous solution to 1 part by volume of 0.3% silkworm silk fibroin aqueous solution.

SF: Membranous sample obtained from 0.3% aqueous solution of silk fibroin.

The above-mentioned silk fibroin membrane, atelocollagen membrane, and silk fibroin / atelocollagen composite membrane were examined for moisture absorption, strength, elongation, and thermal form stability. Table 1 shows the obtained results.

Table 1 ─────────────────────────────────── Sample Moisture absorption (%) Strength (kg / mm ² ) Elongation (%) Thermal form stability (%) ────────────────────────────────── ─ CSS 20.0 20.4 4.2 4.4 CMS 21.3 1.6 9.1 11.0 SF / CMS (1: 1) 12.7 12.4 8.1 5.9 SF / CSS (1: 1) 13 11.9 4.6 1.8 SF / CSS (1: 2) 14.7 2.9 6.6 8.2 SF 9.3 3.3 0.8 2.6 ミ リ As is clear from Table 1, myristylation at 180 ° C At 180 ° C on the TMA curve of atelocollagen, 11
% Shrinkage, while only 4.4% shrinkage occurs with succinylated atelocollagen. This is in good agreement with the fact that thermal molecular mobility increases as the hydrocarbon chain of the acylating agent becomes longer.

As can be seen from Table 1, as the silk fibroin content increases, the amount of shrinkage of the sample decreases as apparent from the TMA curve at 180 ° C. This experimental example indicates an increase in the thermal morphological stability of the silk fibroin / atelocollagen composite membrane of the present invention.

The elongation of the myristylated atelocollagen membrane was twice or more larger than that of the succinylated atelocollagen membrane. The cutting strength of succinylated atelocollagen is higher than that of myristylated atelocollagen. Silk fibroin membranes are extremely brittle because they have a low elongation and cut at only 0.8% in a dry state. Complexing myristylated atelocollagen or succinylated atelocollagen has made it possible to improve the mechanical properties of brittle silk fibroin.

Example 2 4 ml of a 2% aqueous succinylated atelocollagen solution and 4 ml of a 5.1% regenerated silk fibroin aqueous solution were mixed in equal amounts to prepare a total of 8 ml of a mixed aqueous solution. 1 in this
0 mg of tetracycline was uniformly dissolved, mixed for 2 hours until the whole became uniform, the mixed aqueous solution was spread on a polystyrene membrane, and water was evaporated at 25 ° C. for 12 hours. Thus, a silk fibroin / succinylated atelocollagen composite membrane containing tetracycline was prepared. This membrane sample was immersed in a 2% aqueous formalin solution for 1 minute and dried at room temperature to prepare a water-insoluble sample (hereinafter abbreviated as sample).

A mixed aqueous solution of 2 ml of an aqueous 2% silk fibroin solution and 2 ml of an aqueous 2% succinylated atelocollagen solution was heated to 50 ° C. Separately, a 20% ethanol aqueous solution containing 20 mg of rifampicin, which is an antibiotic, was prepared, and 1 ml of this ethanol aqueous solution was added to the above-mentioned mixed aqueous solution. The mixed aqueous solution thus produced was spread on a polyethylene membrane, and water was evaporated and dried at room temperature to prepare a silk fibroin / succinylated atelocollagen composite membrane. This film sample is immersed in a 2% formalin aqueous solution for 30 seconds or 5 minutes to insolubilize it, dried under standard conditions, and dried into two types of film samples (hereinafter abbreviated as samples) (sample is The sample was immersed for 30 seconds and the sample was immersed for 5 minutes). A sample not treated with formaldehyde was used as a control (hereinafter abbreviated as a sample).

The various membrane samples (試 料) thus prepared were placed in a 5 ml glass tube containing 3 ml of distilled water and subjected to a shaking treatment for a certain period of time (0 minute to 5 days).
After the shaking treatment, the sample was taken out and air-dried in a standard state, and the antibacterial property of the sample corresponding to each elapsed time was evaluated.

The inhibitory effect of these samples on the growth of plant pathogenic bacteria was evaluated according to the method described above. Table 2 shows the obtained results. In Table 2, the unit of the numerical value is mm.

Table 2 経 過 Elapsed time Sample Sample Sample Sample (Immersion: 1 (Minute) (immersion: none) (immersion: 30 seconds) (immersion: 5 minutes) ─────────────────────────────── {0 min ++ 34 35 34 20 min ++ 40 min + 60 min ± 22 28 29 1 day 16 20 203 3 days 11 13 155 5 days −67 ─────────────よ う As is clear from Table 2, each sample has a sustained release effect,
In particular, in the case of the sample, there is a high sustained release effect. As for the filamentous fungi, as a result of the test conducted in accordance with the above-described method, the growth inhibiting effect and the sustained release effect of the filamentous fungus were obtained for each sample as in the case of the plant pathogenic bacteria. further,
It showed an inhibitory effect on growth of Mycobacterium tuberculosis, Gram-positive bacteria or Gram-negative bacteria.

Example 3 The silk fibroin / atelocollagen composite membrane used in Example 1 was immersed in a 2% glutaraldehyde aqueous solution for 15 seconds to obtain a hydrogel-like substance having a water content of 98% by weight for a long time. When the obtained gel-like substance was applied to the skin as it was and dried, an artificial skin material comprising the complex of the present invention was obtained.

0.3% silkworm silk fibroin aqueous solution and 0.1%
An aqueous solution of an equal volume mixed with a 3% succinylated atelocollagen aqueous solution was gently stirred, spread on a polystyrene membrane, evaporated to dryness, and solidified to prepare a silk fibroin / succinylated atelocollagen (1: 1) membrane. This sample film was cut into a width of 0.3 mm and a length of 2 cm, and the viscoelastic behavior (DMA) was measured using a rheograph solid S type manufactured by Toyo Seiki Co., Ltd. According to DMA, SF / CS
For S (1: 1), mechanical loss peaks appeared at 86 ° C and 188 ° C. It has been found that the peak at 86 ° C. is based on the thermal molecular motion of succinylated atelocollagen and the peak at 188 ° C. is based on the thermal molecular motion of silk fibroin. From this, SF / CSS (1: 1) has a region based on CSS (succinylated atelocollagen) and a region based on SF (silk fibroin), and as a whole, a uniform complex is obtained. I understand.

Example 4 Silk fibroin, atelocollagen, and a silk fibroin / succinylated atelocollagen composite membrane were prepared in the same manner as in Example 1, and the Fourier transform infrared spectrum (FT) was used to clarify their molecular structural characteristics. -I
R) was measured. FT-IR was measured using a measuring device manufactured by PerkinElmer, and a film-like sample was used. The measurement wave number region was 2000 to 400 cm ⁻¹ , and the number of measurement repetitions was 20 times. Find the number of absorption peak positions,
The peak intensity was shown in three stages: strong (s), medium (m), and weak (w). Table 3 shows the obtained results.

The film sample used in this example is as follows.

SF: Same as in the first embodiment.

CSS: Same as in the first embodiment.

SF / CSS (8: 2): A film-like sample obtained from a mixed aqueous solution obtained by adding 2 parts by volume of 0.3% succinylated atelocollagen aqueous solution to 8 parts by volume of 0.3% silkworm silk fibroin aqueous solution.

SF / CSS (6: 4): A film-like sample obtained from a mixed aqueous solution obtained by adding 4 parts by volume of a 0.3% succinylated atelocollagen aqueous solution to 6 parts by volume of a 0.3% silkworm silk fibroin aqueous solution.

SF / CSS (4: 6): A film-like sample obtained from a mixed aqueous solution obtained by adding 6 parts by volume of a 0.3% succinylated atelocollagen aqueous solution to 4 parts by volume of a 0.3% silkworm silk fibroin aqueous solution.

SF / CSS (2: 8): A film sample obtained from a mixed aqueous solution obtained by adding 8 parts by volume of a 0.3% succinylated atelocollagen aqueous solution to 2 parts by volume of a 0.3% aqueous solution of silkworm silk fibroin.

Table 3 ──────────────────────────────────── Sample Absorption peak wave number (cm ^-1 ) ──────────────────────────────────── SF 1629 (s), 1530 (s), 1447 ( m), 1409 (w), 1263 (w), 1234 (m), 1070 (m), 690 (m), 643 (m), 554 (s) SF / CSS (8/2) 1653 (s), 1545 (s), 1452 (m), 1279 (w), 1240 (m), 1071 (w), 937 (w), 526 (m) SF / CSS (6/4) 1658 (s), 1631 (s ), 1547 (m), 1525 (w), 1282 (w), 1239 (w), 1079 (w), 936 (w), 531 (s) SF / CSS (4/6) 1660 (s), 1635 (s), 1548 (m), 1281 (m), 1242 (m), 1078 (m), 936 (w), 530 (s) SF / CSS (2/8) 1659 (s), 1639 (m) , 1554 (m), 1282 (m), 1240 (m), 1165 (m), 1078 (w), 938 (m), 525 (m) CSS 1657 (S), 1555 (m), 1283 (w) , 1240 (w), 1082 (m), 929 (m), 534 (m) ────────────────────────────── 、 As is clear from Table 3, FT-IR spectrum of silk fibroin / succinylated atelocollagen composite membrane In FIG. 2, an absorption peak based on pure silk protein and an IR spectrum peak derived from pure collagen are overlapped. Therefore, it can be concluded that the molecular morphology is composed of a structure having good compatibility between the silk protein and the collagen.

Example 5 CSS measured under the conditions of TMA range ± 1000 μm,
Tt (° C.) is defined as the intersection of the tangent of the TMA curve in the temperature range of 100 ° C. to 180 ° C. and the tangent of the TMA curve at 180 ° C. to 200 ° C. where the sample length rapidly shrinks on the TMA curve of the CMS. Was examined for Tt value. Table 4 shows the obtained values. In Table 4, Tdsc means a main endothermic peak temperature appearing at 200 ° C. or higher as a result of DSC measurement. This Tdsc corresponds to the thermal decomposition temperature of the sample.

Table 4 {Sample Tt (° C) Tdsc (° C)} ───────────── CSS 176 CMS 166 SF / CSS (1: 1) 193 267 SF / CMS (1: 1) 171 SF / CMS (1: 2) 170 266 SF 284 ──よ う As is clear from Table 4, the thermal dimensional stability of succinylated atelocollagen (CSS) is higher than that of myristylated atelocollagen (CMS). 176 ° C which is 10 ° C higher. The composite of silk fibroin and succinylated atelocollagen had a thermal dimensional stability of about 30.degree. It was confirmed that the molecular interaction between silk fibroin and succinylated atelocollagen was specifically improved.

The above experimental fact that the myristylation reaction increases the thermal molecular mobility of collagen and slightly accelerates the thermal decomposition behavior is based on the fact that the chemical reaction results in the introduction of collagen into the molecular side chains of lysine residues of collagen. The length of the hydrocarbon chain
This is because (CH ₂ ) ₁₂ in myristylation, which is 6 times as large as (CH ₂ ) _{2 in} succinylation.

Example 6 The silk fibroin / atelocollagen composite membrane (SF / CMS (1: 1), SF / CSS (1: 1)) used in Example 1 was used.
1) and SF / CSS (1: 2)), the thermal decomposition behavior was measured using a TGA apparatus under a nitrogen atmosphere.
The sample weight is 3 mg, the measurement temperature range is 0 to 400 ° C, TG
A full scale was 5 mg, and sampling time was 3 seconds. The results obtained are shown in FIG. Here, the vertical axis represents the sample weight remaining rate (%), and the horizontal axis represents the sample temperature (° C.).

As is clear from FIG. 1, in the silk fibroin / atelocollagen composite membrane, myristylated atelocollagen (SF / CMS (1: 1)) was better in the case of succinylated atelocollagen (SF / CSS (1: 1)).
The degree of progress of the thermal decomposition was slower than in the case of 1)), and it was clear that the thermal decomposition was inhibited. In addition, a composite membrane having a high content of succinylated atelocollagen (SF /
CSS (1: 2)) further inhibits the degree of thermal decomposition. This is because the molecular momentum of the succinylation site interacting with silk fibroin was lower than that of the myristylation site, and as a result, the degree of thermal decomposition was suppressed.

[0082]

According to the present invention, by combining a silk protein and a collagen, blood compatibility, antithrombotic properties, adhesion and proliferation of living cells, which are possessed by the natural proteins silk protein and collagen, respectively, Materials for synergistically expressing biochemical properties such as interaction with platelets have been provided. The composite technology made it an excellent material with both functions, and a material with a new function in terms of interaction with blood and cells could be prepared. Furthermore, it compensates for the shortcomings of practical functions unique to silk proteins and collagen, and new possibilities come to appear.

The silk protein / collagen complex of the present invention
As a hemostatic agent, wound wound cover material, material for intradermal injection,
It can be used as a carrier for immobilizing pharmaceuticals, physiologically active substances and antibiotics or a carrier for sustained release thereof.

Since the silk protein / collagen complex of the present invention is a complex comprising an insect-derived silk protein and an animal-derived collagen, it is used as a physiologically active substance, an antibiotic, a pheromone, a hormone, and other pharmaceuticals. When used as a sustained-release carrier, a strong intermolecular interaction occurs between the useful substance and the complex, so that it can be used as a sustained-release carrier that maintains the sustained-release effect of the drug for a long time.

Further, the silk protein / collagen complex of the present invention has the anti-thrombotic and cell-adhering properties of silk fibroin, the low immunogenicity of collagen, the interaction with platelets, and the interaction with cells. Since it also has a function of action, it can be used as an artificial organ material having a function closer to that of a living body by incorporating a substance having a physiological activity such as a cell, an enzyme, or a hormone. Also, hemostatic agents, clinical reagents,
It can be used as biomedical materials such as artificial blood vessels, contact lenses, sustained-release carriers (Drug delivery Devices) that gradually release pharmaceuticals, artificial skin, wound dressings, gels for intradermal injection, and collagen for cell culture substrates. Furthermore, the complex of the present invention has various uses as an implantable material because collagen is degraded in a living body, and can be used as a cut gut of an absorbable suture as well as collagen.

[Brief description of the drawings]

FIG. 1 is a graph showing the thermal decomposition behavior of a silk fibroin / atelocollagen composite membrane of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C08L 89/00-89/06 C08J 3/03 C01C 3/00

Claims (7)

(57) [Claims] (1) The outside of a cocoon fiber produced by silkworm spinning is glued.
Sericin to adhere to or silk silk obtained by removing the sericin.
Dissolve ibroin fiber in neutral salt solution
-Soluble silk obtained by dialyzing dialysis using a cellulose dialysis membrane
Fibroin or silk gland removed from silkworm body
Water-soluble silk sericin or water-soluble silk fibroin
An aqueous solution of 0.01-10% by weight silk protein or
Aqueous dispersion and collagen molecule side chains or atherosclerosis
Reaction of the acylating agent on the chemically reactive active site of the
Water-soluble or water-dispersible acylated collars
0.1 or more consisting of gen or acylated atelocollagen
5% by weight aqueous collagen solution or dispersion
The resulting silk protein / collagen complex. 2. The method according to claim 1, wherein the acylating agent is a dicarboxylic acid having ₁ to ₄ carbon atoms .
It is anhydride or anhydrides of monobasic acids C ₁ -C ₁₅
The silk protein / collagen composite according to claim 1, characterized in that:
body. 3. The method according to claim 1, wherein the acylated atelocollagen is a sugarcane.
Synylated atelocollagen or myristylated ateloco
Lagen or a mixture of both
3. The silk protein / collagen complex according to claim 1 or 2. 4. The collagen according to claim 1, wherein said collagen is derived from animal leather.
Proteolytic enzymes remove telopeptides from collagen
Characterized by using atelocollagen obtained by
A silk protein / collage according to any one of claims 1-3.
Complex. 5. The silk according to any one of claims 1 to 4,
The protein / collagen complex was further insolubilized
Water-insolubilized silk protein / collar
Gen complex. 6. The outside of the cocoon fiber produced by silkworm spinning.
Sericin to adhere to or silk silk obtained by removing the sericin.
Dissolve ibroin fiber in neutral salt solution
-Soluble silk obtained by dialyzing dialysis using a cellulose dialysis membrane
Fibroin or silk gland removed from silkworm body
Water-soluble silk sericin or water-soluble silk fibroin
An aqueous solution of 0.01-10% by weight silk protein or
Aqueous dispersion and collagen molecule side chains or atherosclerosis
Reaction of the acylating agent on the chemically reactive active site of the
Water-soluble or water-dispersible acylated collars
0.1 or more consisting of gen or acylated atelocollagen
5% by weight aqueous collagen solution or dispersion
Into a film, gel, or powder.
Or to obtain a fibrous silk protein / collagen complex
A method for producing a silk protein / collagen complex, which is a feature of the present invention. 7. The silk protein / coller according to claim 6, wherein
Characterized in that the gen complex is further subjected to a water insolubilization treatment
A method for producing a water-insoluble silk protein / collagen complex.