JP6490897B2 - Self-assembled peptide gel - Google Patents

Description

  The present invention relates to a self-assembled peptide gel.

  The self-assembling peptide is dissolved in a solvent such as water, and a pH adjusting agent or salt is added as necessary, so that the self-assembling peptide spontaneously aggregates in the solution to form a fibrous molecular aggregate. The solution gels. More specifically, a low molecular weight self-assembled peptide solution behaves like a polymer solution apparently as the peptide forms a fibrous molecular assembly (hereinafter sometimes referred to as “fiber”). Gelation occurs when cross-linking points are formed between the fibrous molecular aggregates. This gel (hereinafter sometimes referred to as “self-assembled peptide gel”), even if the cross-linked structure is destroyed by physical stimulation, the peptide or fibrous molecular aggregate reassembles after standing still for a while, and again. It has the characteristics of forming a gel.

  Conventionally, for self-assembling peptide gels, application to applications such as cell culture substrates, vitreous substitutes, hemostatic materials, etc. has been studied taking advantage of such characteristics (for example, Patent Document 1). ). However, the mechanical strength of self-assembling peptide gels is lower than that of polymer gels, so it is effective enough for applications that require strength, for example, for carrying cells cultured in gels and for hemostasis at sites with high blood pressure. May not be possible.

  On the other hand, it is known that the mechanical strength of the gel is improved by increasing the concentration of the self-assembling peptide. However, in this case, since a large amount of the raw material peptide is required, the price increases. Furthermore, since the high-concentration solution is an environment in which peptide molecules easily collide, the time required for self-assembly is short, and the viscosity of the solution increases immediately after dissolution, which makes it difficult to produce a gel. is there.

Special table 2008-539257

  The object of the present invention is to provide a self-assembled peptide gel with improved mechanical strength without increasing the peptide concentration.

The gel of the present invention is a gel containing self-assembling peptide and water and formed by self-assembly of the self-assembling peptide. The gel of the present invention is prepared by a production method including mixing the self-assembled peptide and water until a foamy cell dispersion is obtained, and then degassing.
In one embodiment, the self-assembling peptide and water are mixed using a rotation and revolution stirring apparatus in a container having an uneven shape on the inner surface.
In one embodiment, the self-assembling peptide and water are mixed using a stirring blade.
In one embodiment, the self-assembling peptide and water are mixed using a stirring blade in a container having an uneven shape on the inner surface.
In one embodiment, the concentration of the self-assembling peptide is 0.1% to 10.0% by weight.
In one embodiment, the total charge at pH 7.0 of amino acid residues constituting the self-assembling peptide is −3 to −1 or +1 to +3.
In one embodiment, the self-assembling peptide forms a fibrous molecular assembly.
According to another aspect of the present invention, a method for producing a gel is provided. The production method comprises mixing a self-assembling peptide and water to obtain a foamy cell dispersion, defoaming the cell dispersion to obtain a self-assembling peptide solution, the self-organization Standing the peptide solution to self-assemble the self-assembling peptide.
In one embodiment, the self-assembling peptide and water are mixed while applying mechanical shearing force.
In one embodiment, mixing of the self-assembling peptide and water is performed using a rotation and revolution stirrer in a container having an uneven surface on the inner surface.

  According to the present invention, a gel having improved mechanical strength can be obtained by adopting a predetermined production method. More specifically, by adopting a predetermined production method, a gel having improved mechanical strength as compared with the gel obtained by the conventional production method can be obtained without increasing the peptide concentration. In addition, according to the present invention, the amount of peptide necessary to obtain an equivalent mechanical strength can be reduced, which leads to a reduction in gel production cost.

(A)-(m) is a schematic horizontal sectional view of an example of the container which can be applied to a stirring apparatus, respectively. It is a graph which shows IR measurement result.

[A. gel]
The gel of the present invention is a self-assembling peptide gel that includes a self-assembling peptide and water and is formed by self-assembly of the self-assembling peptide. In the present specification, the gel means a viscoelastic substance having both a viscous property and an elastic property. Specifically, a substance that satisfies “G ′> G ″” when the storage elastic modulus G ′ and the loss elastic modulus G ″ are measured by performing dynamic viscoelasticity measurement can be referred to as a gel.

  The gel of the present invention is prepared by a production method that includes mixing a self-assembled peptide and water until a foamy cell dispersion and then degassing. The gel of the present invention was obtained by the conventional production method even when the peptide concentration was the same by being prepared using the self-assembled peptide solution obtained through such specific mixing and defoaming. It can have a higher mechanical strength than a gel. The method for producing the gel of the present invention will be described in detail in Section B.

  The reason why the above effect is obtained is not clear, but is presumed as follows. That is, in the conventional method for producing a self-assembling peptide gel, mixing of bubbles into the gel leads to a decrease in transparency and mechanical strength, and it takes time to remove the mixed bubbles. The self-assembling peptide and water are mixed gently (in other words, without foaming vigorously) using a stirrer, and the peptide solution obtained by the mixing is further degassed as necessary and left to stand. And gelled. In the conventional production method, a substantially transparent peptide solution can be obtained by visual observation, so that it was determined that the self-assembling peptide was completely dissolved by such gentle mixing. However, in actuality, the self-assembled peptide is not completely dissolved, and microscopically, a part of it forms a peptide molecule cluster, and the cluster is incorporated into the fiber, so that the fiber It has an adverse effect on the formation of a three-dimensional network structure. On the other hand, in this invention, the cluster in a solution is fully destroyed by stirring vigorously until a foamy bubble dispersion is obtained. As a result, the peptides are very evenly distributed so that they self-assemble to form fibers that are free or reduced by clusters and thereby reduce fiber elongation and interchain interactions. It is considered that a three-dimensional network structure (as a result, a gel) with high mechanical strength is formed by being promoted.

  The gel of the present invention preferably has a pH of 5 to 8, more preferably 5.5 to 7.5, and even more preferably 6 to 7. When the pH is within this range, hydrolysis of the self-assembling peptide during heating can be avoided, so that it can be subjected to sterilization treatment with heating such as autoclaving. Moreover, cytotoxicity can be reduced.

  The gel of the present invention has a visible light transmittance of preferably 80% or more, more preferably 85% or more, and further preferably 90% or more in a cell having an optical path length of 10 mm, measured at an absorbance of 380 nm to 780 nm. It is. When the visible light transmittance is within this range, when used as a cell culture substrate, observation becomes easier when cells or the like are mixed therein. Moreover, if it is an ophthalmic use, a visual function will not be impaired. Moreover, if it is a hemostatic material use, it is easy to apply a hemostatic material and to confirm the completion of hemostasis while confirming a bleeding site.

[A-1. Self-assembling peptide]
As the self-assembled peptide, any suitable peptide that can spontaneously assemble through the interaction of peptide molecules in an aqueous solution to form a gel can be used. More specifically, it spontaneously assembles through interaction between peptide molecules in an aqueous solution to form a fibrous molecular assembly, and a three-dimensional network structure is developed by the interaction between the molecular assemblies. Peptides that can form a gel can be preferably used. Examples of the interaction between peptide molecules include electrostatic interactions such as hydrogen bonds, ionic interactions, van der Waals forces, and hydrophobic interactions. Moreover, formation of a fibrous molecular assembly can be confirmed by microscopic observation.

  The amino acid constituting the self-assembling peptide may be an L-amino acid or a D-amino acid. L-amino acids are preferred. Moreover, a natural amino acid may be sufficient and a non-natural amino acid may be sufficient. Natural amino acids are preferred because they are available at low cost and facilitate peptide synthesis.

  The total charge of amino acid residues constituting the self-assembling peptide at pH 7.0 is preferably −3 to −1 or +1 to +3, and more preferably −3, −2, +2 or +3. . Thus, the balance between the electrostatic attractive force and the repulsive force suitable for gel formation can be obtained because the positive charge and the negative charge derived from the side chain of the amino acid residue contained in the self-assembling peptide are not offset in the neutral region. This is because, as a result, a transparent and stable gel can be formed in the neutral region. In the present specification, the “neutral region” refers to a region of 6.0 to 8.5, preferably 6.5 to 8.0, and more preferably 7.0.

  The charge of the self-assembling peptide at each pH can be calculated, for example, according to the method of Lehninger (Biochimie, 1979). The Rainer's method can be performed, for example, by a program available on the EMBL WWW Gateway to Isoelectric Point Service website (http://www.embl-heidelberg.de/cgi/pi-wrapper.pl).

Specific examples of the self-assembling peptide that can be preferably used in the present invention include peptides having the amino acid sequence of the following formula (I). By using the peptide, a gel having sufficient mechanical strength can be suitably obtained.
a ₁ b ₁ c ₁ b ₂ a ₂ b ₃ db ₄ a ₃ b ₅ c ₂ b ₆ a ₄ (I)
(In the amino acid sequence, a _{1 to} a ₄ are basic amino acid residues; b _{1 to} b ₆ are uncharged polar amino acid residues and / or hydrophobic amino acid residues, provided that at least 5 are hydrophobic amino acid residues; c ₁ and c ₂ are acidic amino acid residues; d is a hydrophobic amino acid residue.)

In the amino acid sequence, a _{1 to} a ₄ are basic amino acid residues. The basic amino acid is preferably arginine, lysine or histidine, more preferably arginine or lysine. This is because these amino acids are strongly basic. a _{1 to} a ₄ may be the same amino acid residue or different amino acid residues.

In the amino acid sequence, b _{1 to} b ₆ are uncharged polar amino acid residues and / or hydrophobic amino acid residues, and at least five of them are hydrophobic amino acid residues. The hydrophobic amino acid is preferably alanine, leucine, isoleucine, valine, methionine, phenylalanine, tryptophan, glycine or proline. The uncharged polar amino acid is preferably tyrosine, serine, threonine, asparagine, glutamine, or cysteine. This is because these amino acids are easily available.

Preferably, b ₃ and b ₄ are each independently any suitable hydrophobic amino acid residue, more preferably a leucine residue, an alanine residue, a valine residue, or an isoleucine residue, particularly preferably Is a leucine residue or an alanine residue.

Preferably, b _{1 to} b ₆ are all hydrophobic amino acid residues. This is because the self-assembling peptide preferably forms a β-sheet structure and can self-assemble. More preferably, b _{1 to} b ₆ are each independently a leucine residue, an alanine residue, a valine residue, or an isoleucine residue, and more preferably a leucine residue or an alanine residue. In a preferred embodiment, 4 or more of b _{1 to} b ₆ are leucine residues, more preferably 5 or more of them are leucine residues, and more preferably all are leucine residues.

In the amino acid sequence, c ₁ and c ₂ are acidic amino acid residues. The acidic amino acid is preferably aspartic acid or glutamic acid. This is because these amino acids are easily available. c ₁ and c ₂ may be the same amino acid residue or different amino acid residues.

  In the amino acid sequence, d is a hydrophobic amino acid residue. d is preferably an alanine residue, a valine residue, a leucine residue, or an isoleucine residue.

In one preferred embodiment, two of the three consecutive amino acid residues of b ₃ , d, b ₄ are leucine residues and the rest are alanine residues. In this case, any of b ₃ , d, and b ₄ may be an alanine residue. In another preferred embodiment, all three consecutive amino acid residues of b ₃ , d, and b ₄ are leucine residues.

Preferred specific examples of the amino acid sequence of the formula (I) are exemplified below.
n-RLDLRLALRLLDLR-c (SEQ ID NO: 1)
n-RLDLRLLLLRLDLR-c (SEQ ID NO: 2)
n-RADLRLALRLLDLR-c (SEQ ID NO: 3)
n-RLDLRLALLRLDA-c (SEQ ID NO: 4)
n-RADLRLLLRLLDLR-c (SEQ ID NO: 5)
n-RADLRLLLRLDA-c (SEQ ID NO: 6)
n-RLDLRALLRLLDLR-c (SEQ ID NO: 7)
n-RLDLRLLARLDLR-c (SEQ ID NO: 8)

Other self-assembling peptides that can be preferably used in the present invention include peptides described in WO2007 / 000979. That is, a self-assembling peptide having 8 to 32 amino acid residues, which has a polar amino acid residue and a nonpolar amino acid residue, and an acidic amino acid residue and a basic amino acid residue as the polar amino acid residue In the neutral region, the sum of the charge of the acidic amino acid residue and the charge of the basic amino acid residue is -3 to -2, or +2 to +3, and is self-assembled in a neutral aqueous solution. In this case, only the nonpolar amino acid residue can form a β-sheet structure arranged on one side, and the nonpolar amino acid residue is an alanine residue, a valine residue, a leucine residue, or an isoleucine residue. , A self-assembling peptide that is an amino acid residue selected from the group consisting of a methionine residue, a phenylalanine residue, a tryptophan residue, and a glycine residue. Of these, self-assembling peptides consisting of the amino acid sequences exemplified below are preferred.
n-RASARADARASARADA-c (SEQ ID NO: 9)
n-RANARADARANARADA-c (SEQ ID NO: 10)
n-RAAARADAARAAARADA-c (SEQ ID NO: 11)
n-RASARADARADARASA-c (SEQ ID NO: 12)
n-RADARASARASARADA-c (SEQ ID NO: 13)
n-RASARASARASARADA-c (SEQ ID NO: 14)
n-RASARADARASA-c (SEQ ID NO: 15)
n-KASAKAEAKASAKAEA-c (SEQ ID NO: 16)
n-SAEAKAEASAEAKAEA-c (SEQ ID NO: 17)
n-KLSLKLDLKLSL-c (SEQ ID NO: 18)
n-KLALKLDLKLAL-c (SEQ ID NO: 19)

Furthermore, commercially available self-assembling peptides such as the product name “PuraMatrix ^™ ” (manufactured by 3D Matrix) can also be used.

  The self-assembling peptide can be produced by any suitable production method. Examples thereof include a chemical synthesis method such as a solid phase method such as the Fmoc method or a liquid phase method, and a molecular biological method such as gene recombinant expression. The self-assembling peptide can be in the form of any suitable salt during the purification process, but in the present invention, a salt-form self-assembling peptide can also be used.

  The self-assembling peptide may be subjected to any appropriate modification depending on the purpose and the like. The site where the modification is performed is not particularly limited, and examples thereof include an N-terminal amino group, a C-terminal carboxyl group, or both of the self-assembling peptide.

  As the modification, any appropriate modification can be selected as long as the modified peptide has the ability to self-assemble. For example, introduction of protecting groups such as acetylation of N-terminal amino group and amidation of C-terminal carboxyl group; introduction of functional groups such as alkylation, esterification or halogenation; hydrogenation; monosaccharide, disaccharide, oligo Introduction of sugar compounds such as sugars or polysaccharides; introduction of lipid compounds such as fatty acids, phospholipids or glycolipids; introduction of amino acids or proteins; introduction of DNA; introduction of compounds having other physiological activities. Only one type of modification may be performed, or two or more types may be combined. For example, the N-terminus of an added peptide having a desired amino acid introduced at the C-terminus of the self-assembling peptide may be acetylated and the C-terminus amidated.

  When an amino acid or protein is introduced, the number of amino acids to be introduced is preferably 1 to 180, more preferably 1 to 50, still more preferably 1 to 30, particularly preferably 1 to 10, and most preferably 1. ~ 5. If the number of amino acid residues to be introduced exceeds 180, the self-organizing ability may be impaired.

  The concentration of the self-assembling peptide in the gel of the present invention can be appropriately set depending on the composition, use, and the like. The concentration of the self-assembling peptide is preferably 0.1% by weight to 10.0% by weight, more preferably 0.5% by weight to 5.0% by weight, still more preferably 1.0% by weight to 3.0% by weight. %. If it is the density | concentration of the said range, the effect of this invention can be acquired suitably.

[A-2. water]
As the water, purified water such as ion-exchanged water or distilled water can be preferably used.

  The moisture content (%) of the gel of the present invention (= weight of water in the gel / total weight of the gel × 100) can be, for example, 80% to 99.9%, preferably 85% to 99%.

[A-3. Additive]
The gel of the present invention may further contain any appropriate additive as required. Specific examples of the additive include pH adjusters; buffers; isotonic agents; salts; amino acids; vitamins; alcohols; cell culture medium components; These additives may be used alone or in combination of two or more.

  Examples of the pH adjuster include hydrochloric acid, citric acid, acetic acid, sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate and the like.

  Buffers include phosphates such as phosphoric acid, sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate; boric acid, borax Borate salts such as sodium borate and potassium borate; citrate salts such as sodium citrate and disodium citrate; acetates such as sodium acetate and potassium acetate; Tris and HEPES.

  Isotonic agents include chlorides such as sodium chloride, potassium chloride, calcium chloride and magnesium chloride; monosaccharides such as glucose, fructose and galactose; disaccharides such as sucrose, trehalose, maltose and lactose; mannitol, sorbitol and the like Sugar alcohols; and the like.

  As the salts, any appropriate salt other than the additives exemplified above can be used. Examples thereof include sodium sulfate and magnesium sulfate.

  The addition amount of the additive can be set to any appropriate value depending on the purpose and the like.

[B. Method for producing gel]
In the method for producing a gel of the present invention, typically, a self-assembling peptide and water are mixed to obtain a foamy cell dispersion (mixing step), and the obtained cell dispersion is defoamed. The self-assembling peptide solution is obtained (defoaming step), and the obtained self-assembling peptide solution is allowed to stand to cause the self-assembling peptide to self-assemble (gelation step).

[B-1. Mixing process]
In the mixing step, the self-assembling peptide, water and optional additives are mixed to form a foam-like foam dispersion, preferably a uniform fine foam-like foam (like cream, whipped or meringue). Get the body.

  The mixing order is not particularly limited, and all the components can be mixed at once. Alternatively, the self-assembling peptide and water may be premixed and then an additive such as a pH adjuster may be added and further mixed.

  The mixing is preferably performed by stirring while applying a mechanical shearing force. By applying mechanical shearing force, the peptide molecule clusters in water can be sufficiently destroyed.

  Any appropriate stirring device can be used as the mixing means. Preferably, a stirrer capable of stirring with application of mechanical shearing force is used. Specific examples of the stirring device include a mechanical stirrer including stirring blades such as a vertical stirrer, a turbine stirrer, a propeller stirrer, and a helical stirrer. Examples of the types of the stirring blades include pitched paddles, pitched turbines, toothed turbines, three propellers, anchor blades (eg, horseshoe-shaped), ribbon blades, and the like. Further, as a stirring device having no stirring blade, a container rotating type stirrer such as a rotation and revolution stirring device can be used. In the container rotating agitator, a cylindrical container having an uneven shape on the inner surface (for reference, FIGS. 1A to 1M show schematic horizontal sectional views of examples of the container) is preferably applied. The In addition, the said cylindrical container is applicable also to a stirring apparatus provided with a stirring blade.

  In the mixing step, two or more different mixing means may be used in combination as long as a foamy cell dispersion is obtained. For example, you may use combining ultrasonic irradiation and the stirring accompanying provision of the said mechanical shear force. Moreover, you may repeat mixing by the same mixing means twice or more.

  Mixing conditions such as mixing time and rotation speed can be appropriately set according to the peptide concentration, the type of stirring device, and the like.

[B-2. Defoaming process]
In the defoaming step, the obtained cell dispersion is defoamed to obtain a self-assembled peptide solution. In the present invention, defoaming may include deaeration.

  As the defoaming method, any appropriate method such as ultrasonic degassing, vacuum decompression degassing, and centrifugal degassing can be used. Two or more defoaming methods may be combined.

  The defoaming step may also serve as a gelation step described later. Therefore, the self-assembly of the self-assembling peptide may have progressed or completed upon completion of defoaming.

[B-3. Gelation process]
In the gelation step, the obtained self-assembled peptide solution is allowed to stand to self-assemble the self-assembled peptide. Thereby, a self-assembled peptide gel is obtained. It should be noted that the standing may not be complete standing as long as the self-assembling peptide can be self-assembled.

  The time and temperature at which the self-assembling peptide solution is allowed to stand are not particularly limited. The time for standing is usually 1 minute or longer, preferably 3 minutes or longer, more preferably 5 minutes or longer. The temperature at the time of standing is 4-50 degreeC normally, Preferably it is 15-45 degreeC.

[B-4. Other processes]
The method for producing a gel of the present invention may include any appropriate other step as required. Examples of the other steps include purification steps such as filtration, sterilization steps such as high-pressure steam sterilization, radiation sterilization, and dry heat sterilization, and dispensing steps into packaging containers.

  In one embodiment, the sol obtained by applying physical stimulation to the gel obtained through the mixing step, the defoaming step and the gelling step may be gelled again. That is, in the method for producing a gel of the present invention, in addition to the mixing step, the defoaming step, and the gelation step, the gel obtained in the gelation step is physically stimulated to obtain a sol (solation step). And a step of allowing the obtained sol to stand and allowing the self-assembled peptide to self-assemble again (regelation step). If necessary, the sol obtained in the sol formation step may be degassed. According to the method for producing a gel of the present invention, by using the self-assembled peptide solution obtained through the mixing step and the defoaming step, a fiber free from or reduced by peptide molecule clusters is formed. Therefore, even when the fiber and the cross-linked structure between the fibers are destroyed by physical stimulation, a three-dimensional network structure (as a result, a gel) with high mechanical strength in the sol containing the fiber and its fragments Can be formed again.

  In the sol formation step, the physical stimulus is preferably a stimulus that maintains the fibers forming the gel. For example, ultrasonic irradiation, pipetting, vortexing and the like can be mentioned. The sol obtained in the sol formation step is typically in a state where the fluidity is increased as compared with the self-assembled peptide solution obtained in the defoaming step. In one embodiment, the sol comprises fibrous molecular assemblies (fibers) of self-assembled peptides as dispersed particles.

  The regelation step can be performed in the same manner as the gelation step.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by these Examples.

[Storage modulus]
The storage elastic modulus G ′ of the gel was measured using a rotary rheometer (manufactured by TA instruments, product name “ADVANCED RHEOMETER AR 1000”) which is a dynamic viscoelasticity measuring device. Specifically, it is as follows. First, the geometry (aluminum cone, diameter 20 mm, cone angle 1 °, gap 24 μm) for contacting the sample and a thermostat for maintaining the sample stage at a constant temperature were attached to the rheometer. Next, under the measurement conditions of temperature: 37 ° C., torque: 1 μN · m, frequency: 0.5 rad / s to 100 rad / s, a sample was measured three times by the following measurement procedure, and the value at 1 rad / s. Was the storage elastic modulus G ′.
(1) Using a pipette, place 55 μL of sample on the sample stage of the rheometer.
(2) The geometry is moved from the sample stage to a gap of 24 μm and brought into contact with the sample.
(3) Move the geometry slightly to blend with the sample.
(4) Measurement is started 15 seconds after stopping the movement of the geometry (a solvent trap for preventing volatilization of the solvent from the sample is attached during the 15 seconds).

[PH]
The pH was measured using a portable pH meter (manufactured by Horiba, Ltd., product number “B-712”).

[appearance]
The appearance of the obtained gel was visually observed and evaluated according to the following criteria.
Good: Air bubbles and cloudiness are not recognized, and it is transparent.
Poor: Bubbles and / or cloudiness are observed.

[Example 1]
A self-assembled peptide gel having the composition shown in Table 1 was prepared. The specific procedure is as follows. That is, a self-assembling peptide (SPG-178: peptide of SEQ ID NO: 1 in which the N-terminus is acetylated and the C-terminus is amidated) and trehalose dihydrate are weighed, and the side wall as shown in FIG. The container was placed in a container having an uneven shape on the inner surface (hereinafter referred to as “uneven shape container”). Next, water was put into the container and the container was covered. The container was set in a rotation / revolution stirrer (manufactured by Shinky Co., Ltd., product number “ARE-310”) and treated in a stirring mode for 30 seconds.

After adjusting the pH by adding a 100 mM Na ₂ CO ₃ aqueous solution to the obtained mixed solution, the concavo-convex shaped container was set in a rotation / revolution stirrer (product number “ARE-310”, manufactured by Shinky Corporation) and stirred mode For about 2 minutes. As a result, a meringue-shaped cell dispersion was obtained.

  The obtained bubble dispersion is put into a cylindrical container having no irregular shape on the inner surface (the inner horizontal cross-sectional shape is circular), and a rotation and revolution stirring device (product number “ARE-310” manufactured by Shinky Corporation). ) And defoamed in defoaming mode. The container was then degassed and degassed with a vacuum pump under reduced pressure to obtain a self-assembled peptide aqueous solution.

  5 g of the obtained self-assembling peptide aqueous solution was dispensed into a syringe at a time, and the syringe was stoppered, a plunger was attached, placed in a sterile bag, and autoclaved at 121 ° C. for 20 minutes. The self-assembled peptide aqueous solution after autoclaving was naturally cooled to room temperature to obtain a self-assembled peptide gel.

[Comparative Example 1]
A self-assembled peptide gel having the composition shown in Table 1 was prepared. The specific procedure is as follows. That is, self-assembling peptide (SPG-178) and sucrose were weighed and put into a centrifuge tube. Next, water was added, and the mixture was treated for 5 minutes at an output of 500 W using an ultrasonic homogenizer (manufactured by Sonic & Material, Inc., product number “Vibra-Cell VC-505”) and mixed. When the obtained liquid mixture was visually confirmed, it was a substantially transparent liquid as a whole although fine bubbles were mixed in some places.

The obtained liquid mixture was centrifuged at 3000 rpm for 5 minutes to degas, and further degassed under reduced pressure using an aspirator. After adjusting the pH by adding 50 mM Na ₂ CO ₃ aqueous solution to the degassed mixture, 500 W using an ultrasonic homogenizer (Sonic & Material, Inc., product number “Vibra-Cell VC-505”). And mixed for 5 minutes. The mixture was degassed under reduced pressure using an aspirator, thereby obtaining a self-assembled peptide aqueous solution.

  5 g of the obtained self-assembled peptide aqueous solution was dispensed into a syringe. The syringe was then stoppered, a plunger was attached, placed in a sterile bag, and autoclaved at 121 ° C. for 20 minutes. The self-assembled peptide aqueous solution after autoclaving was naturally cooled to room temperature to obtain a self-assembled peptide gel.

[Example 2]
A self-assembled peptide gel having the composition shown in Table 1 was prepared in the same procedure as in Example 1 except that the trehalose dihydrate concentration was changed.

[Example 3]
A self-assembling peptide gel having the composition shown in Table 1 was prepared in the same procedure as in Example 1 except that the concentration of the self-assembling peptide was changed and trehalose dihydrate was removed.

[Example 4]
A self-assembling peptide gel having the composition shown in Table 1 was prepared in the same procedure as in Example 1 except that the concentration of the self-assembling peptide was changed and trehalose dihydrate was removed.

[Comparative Example 2]
A self-assembled peptide gel having the composition shown in Table 1 was prepared in the same procedure as in Comparative Example 1 except that trehalose dihydrate was used instead of sucrose and the concentration was changed.

[Comparative Example 3]
A self-assembled peptide gel having the composition shown in Table 1 was prepared in the same procedure as in Comparative Example 1 except that the self-assembled peptide concentration was changed.

[Comparative Example 4]
A self-assembled peptide gel having the composition shown in Table 1 was prepared in the same procedure as in Comparative Example 1 except that the self-assembled peptide concentration was changed.

Table 1 shows the compositions and properties of the gels obtained in the examples and comparative examples.

  As noted above, all of the example gels have a significantly higher storage modulus than the comparative gel having the same self-assembled peptide concentration.

[Reference Example 1]
For the gel obtained in Example 2 and the gel obtained in Comparative Example 2, a Fourier transform infrared spectrophotometer (manufactured by Perkin Elmer, product name “having a trough plate made of HATR sampling accessory and zinc selenide crystal” IR measurement was performed by the total reflection absorption method under the following conditions using “Spectrum One”). A graph of the IR measurement results is shown in FIG.
<IR measurement conditions>
scan number: 32 times Wave number range: 400-4000 cm ⁻¹
Resolution: 4cm ^-1
scan range / cm ⁻¹ : 4000 650 (400)
OPD velocity: 0.2cm / sec
Measurement temperature: Room temperature Infrared incident angle to zinc selenide crystal: 45 °

In the graph shown in FIG. 2, the peak at 1600 to 1500 cm ⁻¹ is derived from the amide group hydrogen-bonded between the peptides, and corresponds to the content of the β sheet structure in the gel. Therefore, it can be seen from the graph of FIG. 2 that the gel of Example 2 prepared using the rotation and revolution stirring apparatus has a more developed β sheet structure. Since the development of the β sheet structure is advantageous for the formation of nanofibers and the development of the three-dimensional network structure, this result contributes to the improvement of the strength of the self-assembled peptide gel. It strongly suggests that.

  The gel of the present invention can be suitably used in the fields of research and development and medicine.

Claims (8)

Mixing a self-assembling peptide and water to obtain a foamy cell dispersion;
Degassing the cellular dispersion to obtain a self-assembled peptide solution; and
The resulting self-assembled peptide solution on standing, self-assembling peptides to a method for producing a free Mugeru to be self-organizing,
The self-assembling peptides, in said self-assembling peptide solution, Ri Ah in peptide to form molecular aggregates fibrous through the formation of β-sheet structure,
The self-assembling peptide is a self-assembling peptide consisting of 8-32 amino acid residues having a polar amino acid residue and a nonpolar amino acid residue,
The polar amino acid residue includes an acidic amino acid residue and a basic amino acid residue,
Under the environment of pH 7.0, the sum of the charge of the acidic amino acid residue and the charge of the basic amino acid residue is −3 to −2, or +2 to +3,
In a pH 7.0 aqueous solution, when self-assembled, a β-sheet structure is formed in which only the nonpolar amino acid residues are arranged on one side;
The nonpolar amino acid residues are selected from the group consisting of alanine residues, valine residues, leucine residues, isoleucine residues, methionine residues, phenylalanine residues, tryptophan residues and glycine residues;
A method for producing a gel, wherein the N-terminal amino group and / or the C-terminal carboxyl group of the self-assembling peptide may be protected . Mixing a self-assembling peptide and water to obtain a foamy cell dispersion;
Degassing the cellular dispersion to obtain a self-assembled peptide solution; and
Standing the obtained self-assembling peptide solution to self-assemble the self-assembling peptide
A method for producing a gel comprising:
The self-assembling peptide is a peptide that forms a fibrous molecular assembly through formation of a β-sheet structure in the self-assembling peptide solution,
A method for producing a gel, wherein the self-assembling peptide is a peptide having the amino acid sequence of the following formula (I) or a peptide in which the N-terminal amino group and / or the C-terminal carboxyl group thereof are protected.
a ₁ b ₁ c ₁ b ₂ a ₂ b ₃ db ₄ a ₃ b ₅ c ₂ b ₆ a ₄ (I)
(In the above amino acid sequence, a ₁ ~ A ₄ Is a basic amino acid residue; b ₁ ~ B ₆ Is a hydrophobic amino acid residue; c ₁ And c ₂ Is an acidic amino acid residue; d is a hydrophobic amino acid residue. ) Mixing a self-assembling peptide and water to obtain a foamy cell dispersion;
Degassing the cellular dispersion to obtain a self-assembled peptide solution; and
Standing the obtained self-assembling peptide solution to self-assemble the self-assembling peptide
A method for producing a gel comprising:
The self-assembling peptide is a peptide that forms a fibrous molecular assembly through formation of a β-sheet structure in the self-assembling peptide solution,
The method for producing a gel, wherein the self-assembling peptide is selected from a peptide consisting of any one of the amino acid sequences of SEQ ID NOS: 1 to 19, wherein the N-terminal amino group and / or the C-terminal carboxyl group may be protected. The production method according to claim 3 , wherein the self-assembling peptide is selected from peptides consisting of the amino acid sequence of SEQ ID NO: 1 or 18, wherein the N-terminal amino group and / or the C-terminal carboxyl group may be protected. The manufacturing method according to any one of claims 1 to 4, wherein the self-assembling peptide and water are mixed while applying mechanical shearing force. The manufacturing method according to any one of claims 1 to 5, wherein the mixing of the self-assembling peptide and water is performed using a rotation and revolution stirring apparatus in a container having an uneven surface on the inner surface. The manufacturing method in any one of Claim 1 to 6 whose density | concentration of the self-assembling peptide in the said self-assembling peptide solution is 0.1 weight%-10.0 weight%. The manufacturing method in any one of Claim 1 to 7 which manufactures the gel whose visible light transmittance | permeability measured by the light absorbency of 380 nm-780 nm is 80% or more in the cell of optical path length 10mm.