JP6084352B2 - High strength peptide gel - Google Patents

Description

  The present invention relates to a high-strength peptide gel.

  It is known that the extracellular matrix affects cell proliferation, differentiation, cell-cell and cell-tissue interactions, and promotes important signal transduction. Furthermore, by culturing cells in a three-dimensional environment rather than two-dimensional culture using conventional culture dishes, an optimized extracellular matrix / cell microenvironment that approximates in vivo can be obtained, and complex tissue reconstruction can be achieved. It is thought that the transplantation effect is amplified as possible.

  Animal-derived materials such as collagen and Matrigel are used for three-dimensional cell culture. However, animal-derived materials have problems such as immunogenicity and infectious diseases. Synthetic scaffolds such as PLA, PLGA, and calcium phosphate are also used for three-dimensional cell culture in order to remove such a risk of infection. However, these synthetic scaffolds are not excellent in cell proliferation. Examples of such a material include self-assembling peptides as disclosed in Patent Document 1 or Patent Document 2. However, since the scaffold (peptide gel) obtained from the self-assembling peptide of Patent Document 1 or 2 has insufficient mechanical strength, for example, there is a problem of operability such as collapse when pinched with tweezers. .

  In recent years, a method of creating a cell sheet and attaching it to the periphery of the heart has been developed. Patent Document 3 discloses a myocardial stem / progenitor cell transplantation method in which a self-assembled nanopeptide applied to the surface of a myocardial infarction site is gelled. However, with this method, it is difficult to produce a transplant sheet having a thickness necessary for repairing the myocardium of a large animal.

US Pat. No. 5,670,483 International Publication No. 2007/000979 Pamphlet JP 2009-90031 A

  The present invention has been made to solve the above problems, and an object of the present invention is to provide a peptide gel having sufficient mechanical strength for practical use.

The peptide gel of the present invention is formed from an aqueous solution containing a self-assembling peptide containing a reactive group that forms a covalent bond by photocrosslinking and / or an amino acid having a linker structure that can be bound by interaction between biomolecules.
In a preferred embodiment, the reactive group that forms a covalent bond by photocrosslinking is a reactive group that can be covalently bonded to the amino group of the self-assembled peptide.
In a preferred embodiment, the amino acid that can be bound by the covalent bond and / or intermolecular interaction is at least one selected from the group consisting of azidophenylalanine, biotin-linked lysine, and benzolylphenylalanine.
In a preferred embodiment, a reactive group capable of covalently bonding with the amino group of the self-assembling peptide is introduced at the C-terminus of the self-assembling peptide.
In a preferred embodiment, the self-assembling peptide is DLRLLDLALDLRLD (SEQ ID NO: 1), DLRLLDLLLDLRLD (SEQ ID NO: 5), DARLDLALDLRLD (SEQ ID NO: 6), DLRLDLLLDLRAD (SEQ ID NO: 7), DARLDLLLDLRLRLD (SEQ ID NO: 8), DARLDLLLLDLLRAD (SEQ ID NO: 8). It is a peptide consisting of the amino acid sequence of SEQ ID NO: 9), DLRLDALLDLLRD (SEQ ID NO: 10), or DRLLDLLADLRLD (SEQ ID NO: 11).
In a preferred embodiment, the self-assembling peptide consists of the following amino acid sequence:
Amino acid sequence: a ₁ b ₁ c ₁ b ₂ a ₂ b ₃ db ₄ a ₃ b ₅ c ₂ b ₆ a ₄
(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 a preferred embodiment, the self-assembling peptide is RLDLRLALLRLLDLR (SEQ ID NO: 4), RLDLRLLLLRLDLR (SEQ ID NO: 12), RADRLRLRLLDLR (SEQ ID NO: 13), RLDLRRLALLLRLDAR (SEQ ID NO: 14), RADLRLLLRLLDLR (SEQ ID NO: 15), RADRLRL SEQ ID NO: 16), RLDLRLALLLDLR (SEQ ID NO: 17) or RLDLRLLARLDLR (SEQ ID NO: 18) is a peptide consisting of the amino acid sequence.
According to another aspect of the present invention, a cell culture substrate is provided. The cell culture substrate includes the peptide gel.
According to still another aspect of the present invention, a carrier for drug transport / sustained release is provided. The drug transport / sustained release carrier includes the peptide gel.

  According to the present invention, a peptide gel with higher mechanical strength can be obtained. Since the peptide gel of the present invention achieves high strength by bonding peptides without using a reagent or an organic solvent, the peptide gel can be used for various applications without being influenced by these.

2 is a schematic diagram for explaining an observation method of the peptide gel obtained in Example 1. FIG. 2 is a photograph showing the meniscus shape of the peptide gel obtained in Example 1. FIG. 2 is a photograph showing the peptide gel obtained in Example 2. 4 is a photograph showing the meniscus shape of the peptide gel obtained in Example 3. FIG. 4 is a graph showing the results of a bromophenol blue sustained release test of the peptide gel obtained in Example 1. FIG. It is a graph which shows the result of the sustained release test of the phenol red of the peptide gel obtained in Example 1, and the peptide gel obtained in Example 2. FIG. It is the microscope picture in the gel after culture | cultivating HEK293 cell in the peptide gel obtained in Example 1 for 3 days. It is the microscope picture in the gel after culture | cultivating the mouse | mouth 3T3 cell in the peptide gel obtained in Example 2 for 3 days.

A. Definition of Terms (1) In this specification, “self-assembling peptide” refers to a peptide that spontaneously assembles in a solvent through interaction between peptide molecules. The interaction is not particularly limited, and examples thereof include a hydrogen bond, an ionic interaction, an electrostatic interaction such as van der Waals force, and a hydrophobic interaction. In one embodiment, self-assembling peptides can self-assemble to form nanofibers or gels in aqueous solutions at room temperature (eg, 0.4 w / v% aqueous peptide solution).
(2) In this specification, “gel” refers to a viscoelastic substance having both viscous and elastic properties.
(3) In the present specification, “hydrophilic amino acid” means basic amino acids such as arginine (Arg / R), lysine (Lys / K), histidine (His / H), aspartic acid (Asp / D), glutamic acid Acidic amino acids such as (Glu / E), tyrosine (Tyr / Y), serine (Ser / S), threonine (Thr / T), asparagine (Asn / N), glutamine (Gln / Q), cysteine (Cys / C) ) And other uncharged polar amino acids. The alphabets in parentheses are the three-letter code and single-character code for amino acids, respectively.
(4) In the present specification, “hydrophobic amino acid” means alanine (Ala / A), leucine (Leu / L), isoleucine (Ile / I), valine (Val / V), methionine (Met / M), Nonpolar amino acids such as phenylalanine (Phe / F), tryptophan (Trp / W), glycine (Gly / G) and proline (Pro / P) are included. The alphabets in parentheses are the three-letter code and single-character code for amino acids, respectively.

B. Peptide gel The peptide gel of the present invention is a self-comprising self-comprising amino acid having a reactive group that forms a covalent bond by photocrosslinking and / or a linker structure that can be bound by interaction between biomolecules (hereinafter also referred to as an amino acid having binding ability). It is formed from an aqueous solution containing an organized peptide. In the peptide gel of the present invention, self-assembling peptides spontaneously assemble in an aqueous solution to form a fibrous molecular assembly having a nanometer-scale width, so-called nanofibers, and static electricity acting between the nanofibers. It is presumed that a gel is formed by forming a three-dimensional network structure mainly due to electrical interaction. The peptide gel of the present invention has a self-organization that forms the nanofibers by a reactive group that forms a covalent bond by photocrosslinking of amino acids contained in the self-assembled peptide and / or a linker structure that can be bound by interaction between biomolecules. It is considered that the organized peptides and / or the nanofibers are further bound to each other. Thereby, it is considered that a peptide gel with higher mechanical strength can be obtained.

  Only one type of peptide may be contained in the aqueous solution, or two or more types of peptides may be used. Moreover, the self-assembling peptide which does not contain the amino acid which has the said binding ability may be included. The aqueous solution may further contain any appropriate additive in addition to the peptide and water used in the present invention. The aqueous solution may contain insoluble matter such as cells.

  The peptide concentration in the aqueous solution is preferably 0.1 to 10 w / v%, more preferably 1 to 5 w / v%. When the concentration is within this range, a peptide gel having excellent mechanical strength can be obtained. In addition, when used as a cell culture substrate, good cell viability can be obtained. With the peptide gel of the present invention, a peptide gel with higher mechanical strength can be obtained without increasing the peptide concentration.

B-1. Self-assembling peptide comprising a reactive group that forms a covalent bond by photocrosslinking and / or an amino acid having a linker structure that can be bound by the interaction between biomolecules. Including. By using such a self-assembling peptide, in addition to self-assembly by electrostatic interaction between peptides, covalent bonds by photocrosslinking and / or bonds by biological interactions can be formed, A peptide gel having high mechanical strength can be formed. Moreover, since these bonds can be formed without using a reagent or an organic solvent, the peptide gel of the present invention does not need to take these effects into consideration and can be applied to various uses.

  In the self-assembling peptide used in the present invention, the position at which an amino acid having binding ability is introduced is not particularly limited. For example, the amino acid may be introduced only at one end of the self-assembling peptide, may be introduced at both ends of the self-assembling peptide, or may be introduced into the sequence. Moreover, the amino acid which has the said binding ability may be contained only 1 type in the self-assembling peptide, and may be contained 2 or more types.

B-1-1. Amino acid having binding ability The amino acid having binding ability may be any amino acid having a linker structure capable of binding by a reactive group and / or biomolecular interaction that forms a covalent bond by photocrosslinking. Things can be used.

  Any appropriate amino acid having a reactive group that forms a covalent bond by photocrosslinking can be used. The self-assembling peptide used in the present invention contains a hydrophobic amino acid residue and a hydrophilic amino acid residue as described later. Therefore, for example, when an amino acid having binding ability is included in the sequence of a self-assembling peptide, and the amino acid that should be at the position where the amino acid is to be inserted is a hydrophilic amino acid residue, the hydrophilic amino acid is shared by photocrosslinking A reactive group that forms a bond and / or a linker structure that can be bonded by an interaction between biomolecules may be used.

  The reactive group that forms a covalent bond by photocrosslinking forms a covalent bond with the amino group at the N-terminal of the self-assembling peptide or the side chain of the amino acid residue constituting the peptide by irradiation with ultraviolet light. The bond between the reactive group that forms a covalent bond by photocrosslinking and the side chain of the amino group or amino acid residue is highly reactive and stable even in the absence of light. Furthermore, since photocrosslinking by the reactive group has high sensitivity to light, it is not necessary to consider damage due to ultraviolet irradiation.

  There is no restriction | limiting in particular as a reactive group which forms a covalent bond by photocrosslinking, For example, an arylazide group, a diazirine group, a benzophenone group etc. are mentioned. Among them, amino acids having an aryl azide group and a benzophenone group are used as a reactive group that forms a covalent bond by photocrosslinking because it is easily available and can form a chemical bond such as a covalent bond without using a reagent. Is preferred.

  The reactive group that forms a covalent bond by photocrosslinking contained in the amino acid having the binding ability is preferably a reactive group capable of covalently bonding to an amino group contained in the self-assembled peptide. By having such a reactive group, the N-terminal amino group of the self-assembling peptide and / or the amino group contained in the amino acid constituting the self-assembling peptide can bind to each other and / or Bonds can be formed between nanofibers formed by self-assembled peptides, and a peptide gel with high mechanical strength can be obtained.

  The reactive group capable of covalently bonding with an amino group contained in the self-assembling peptide is preferably introduced on the C-terminal side of the self-assembling peptide. By introducing a reactive group capable of covalently bonding to an amino group at the C-terminus of the self-assembled peptide, a peptide gel with higher mechanical strength can be obtained. Generally, in the β sheet structure formed by self-assembled peptides, the amino group and carbonyl group of each peptide are hydrogen-bonded. It is considered that a reactive group capable of covalently bonding with an amino group by photocrosslinking is introduced at the C-terminus of the self-assembling peptide, thereby forming a covalent bond with the N-terminal amino group of another self-assembling peptide. It is done. Thereby, it is considered that a bond between self-assembled peptides and / or between nanofibers formed by the self-assembled peptides becomes stronger, and a peptide gel with higher mechanical strength can be obtained.

  As the amino acid having a reactive group that forms a covalent bond by photocrosslinking, azidophenylalanine and benzoylphenylalanine are preferably used from the viewpoint of cost.

  There is no restriction | limiting in particular as interaction between biomolecules, For example, interactions, such as between protein-protein, receptor-ligand, and antigen-antibody, are mentioned. Specifically, an avidin-biotin interaction can be mentioned. As the amino acid having a linker structure that can be bound by the interaction between biomolecules, biotin-linked lysine is preferable from the viewpoints of cost and high affinity and binding stability.

  In the case of binding by interaction between biomolecules, an amino acid having a linker structure derived from both biomolecules may be used, or only an amino acid having a linker structure derived from one biomolecule may be used. When only an amino acid having a linker structure derived from one biomolecule is used, by adding the other biomolecule to the aqueous solution used to form the peptide gel, the self-assembled peptide and / or the self-assembled peptide A bond between the formed nanofibers may be formed. When using a self-assembling peptide containing biotin-conjugated lysine to form a bond between self-assembling peptides and / or nanofibers formed by the self-assembling peptide, avidin is further added to the aqueous solution containing the self-assembling peptide. Is added, a strong bond is formed by the interaction between biotin and avidin contained in the self-assembled peptide, and a peptide gel with higher mechanical strength is obtained. The amount of the biomolecule added to the aqueous solution can be appropriately set according to the number of linker structures contained in the peptide and the amount of peptide used.

  There is no particular limitation on the portion into which the amino acid having a linker structure that can be bound by the interaction between biomolecules is introduced. It is preferable that the amino acid is introduced at the terminal from the viewpoint that production is easy and a chemical bond such as a covalent bond can be formed without using a reagent.

B-1-2. Self-assembling peptide Any self-assembling peptide may be used as long as it contains an amino acid having the binding ability described above. In general, self-assembled peptides constituting nanofibers have a β-sheet structure consisting of a surface on which hydrophilic side chains are arranged and a surface on which hydrophobic side chains are arranged in an aqueous solution. It is considered that a plurality of peptides are spontaneously assembled by an interaction such as a hydrogen bond acting between them, an ionic interaction, and a hydrophobic interaction acting between hydrophobic surfaces. Therefore, the self-assembling peptide constituting the nanofiber includes, for example, those having hydrophilic amino acids and hydrophobic amino acids alternately and in an equal ratio. As the self-assembling peptide, a peptide having an amino acid sequence of 10 to 20 residues is used.

  The amino acid constituting the self-assembling peptide may be an L-amino acid or a D-amino acid. 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.

  Preferably, the self-assembling peptide used in the present invention is a peptide having a 13-residue amino acid sequence, in which a hydrophobic amino acid and a hydrophilic amino acid are alternately located around a 7-position hydrophobic amino acid residue. It consists of an amino acid sequence, and includes an amino acid having the above-mentioned binding ability at any position in the sequence. That is, the self-assembling peptide used in the present invention has hydrophilic amino acids at positions 1, 3, 5, 9, 11, and 13 and hydrophobic amino acids at positions 2, 4, 6, 7, 8, 10, and 12. The amino acid sequence is preferably a 13-residue amino acid sequence, and an amino acid having the above-mentioned binding ability is included at any position of the sequence. In the self-assembling peptide, the three amino acid residues at positions 6 to 8 are successively converted to hydrophobic amino acid residues. Thus, it is estimated that the hydrophobic region formed in the center of the amino acid sequence can contribute to the formation of a peptide gel having excellent strength due to the hydrophobic interaction and the like. The hydrophobic amino acid residue and the hydrophilic amino acid residue contained in the self-assembling peptide may be the same or different. An amino acid having binding ability can be introduced into any part of the amino acid sequence described above.

  The hydrophilic amino acid residues are preferably arginine residues, lysine residues, histidine residues, aspartic acid residues, glutamic acid residues, tyrosine residues, serine residues, threonine residues, asparagine residues, glutamine residues. , A cysteine residue.

  The hydrophobic amino acid residue is preferably an alanine residue, leucine residue, isoleucine residue, valine residue, methionine residue, phenylalanine residue, tryptophan residue, glycine residue, proline residue, more preferably Is a leucine residue, alanine residue, valine residue or isoleucine residue, particularly preferably a leucine residue or an alanine residue. This is because these amino acids are easily available.

The self-assembling peptides used in another preferred embodiment of the present invention are exemplified below.
n-DLRLLDLALDLRLD-c (SEQ ID NO: 1)
n-DLRLDLLLDLLRLD-c (SEQ ID NO: 5)
n-DARLDLALDLRLD-c (SEQ ID NO: 6)
n-DLRLLDALDLRAD-c (SEQ ID NO: 7)
n-DARLDLLLLDLLD-c (SEQ ID NO: 8)
n-DARLDLLLLDLRAD-c (SEQ ID NO: 9)
n-DLRLLDALLDRLD-c (SEQ ID NO: 10)
n-DLRLDLLADLRLD-c (SEQ ID NO: 11)

In another preferred embodiment of the present invention, the self-assembling peptide consists of the following amino acid sequence.
Amino acid sequence: a ₁ b ₁ c ₁ b ₂ a ₂ b ₃ db ₄ a ₃ b ₅ c ₂ b ₆ a ₄
(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.) By using such self-assembling peptides, A peptide gel with higher mechanical strength can be obtained. An amino acid having binding ability can be introduced into any part of the amino acid sequence described above.

  The self-assembling peptide has a basic amino acid residue (positions 1, 5, 9, and 13) and an acidic amino acid residue (positions 3 and 11) every other residue, centered on the hydrophobic amino acid residue at position 7. ) At a symmetrical position in the N-terminal direction and C-terminal direction. That is, one feature of the self-assembling peptide is that it does not have hydrophilic amino acids and hydrophobic amino acids alternately. Another feature of the self-assembling peptide is that it does not have hydrophilic amino acid residues and hydrophobic amino acid residues in equal proportions. Further, the self-assembling peptide has four basic amino acid residues and two acidic amino acid residues at a predetermined symmetrical position centering on the 7-position hydrophobic amino acid residue, and has an N-terminal And the C-terminal amino acid residue are both basic amino acid residues. In general, it is disadvantageous for the 7-position to be a hydrophobic amino acid residue, which is disadvantageous for the formation of a β-sheet structure, and the ratio of hydrophilic amino acids to hydrophobic amino acids is unequal. It has been thought to adversely affect organizational ability. However, the self-assembling peptide has an excellent self-assembling ability, and can form a peptide gel that is more excellent in mechanical strength than in the past. The reason for this effect is not clear, but in addition to the 7th position being a hydrophobic amino acid, it has two more basic amino acid residues than acidic amino acid residues, and By having an amino acid residue at a specific position, it is considered that the electrostatic attractive force and the electrostatic repulsive force work between the peptide molecules in an excellent balance while maintaining the ability to form a β sheet structure. It is done.

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. In the above amino acid sequence, when b ₃ and b ₄ positioned at the 6th and 8th positions are hydrophobic amino acid residues, the 3 amino acid residues at the 6th to 8th positions become the hydrophobic amino acid residues in succession. . Thus, it is estimated that the hydrophobic region formed in the center of the amino acid sequence can contribute to the formation of a peptide gel having excellent strength due to the hydrophobic interaction and the like.

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, particularly preferably 5 or more of them are leucine residues, and most preferably all are leucine residues. This is because a self-assembled peptide that can form a peptide gel having excellent solubility in water and high mechanical strength can be obtained.

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. As described above, when d is a hydrophobic amino acid residue and has a predetermined symmetrical structure, it is considered that the self-assembling peptide can form a gel having better mechanical strength than conventional peptide gels. It is done. The reason why such an effect is achieved is not clear, but the self-assembling peptide of the present invention is such that the amino acid residue d at the 7-position is a hydrophobic amino acid residue, so This is presumed to be because the overlap of the film becomes constant and a highly uniform aggregated state can be formed.

  d is preferably an alanine residue, a valine residue, a leucine residue, or an isoleucine residue. In this case, the side chain length of the amino acid on the hydrophilic surface side of the β sheet structure formed by the self-assembling peptide can be non-complementary, but the self-assembling peptide can exhibit excellent self-organizing ability, Furthermore, it is possible to form a peptide gel that is superior in mechanical strength than conventional ones. This is an effect significantly different from the conventional finding that the side chain length of the amino acid on the hydrophilic surface side of the β sheet structure is preferably complementary in order to obtain an electrostatic attractive force suitable for self-assembly. It is. Here, “the side chain length is complementary” means the number of atoms mainly involved in the side chain length of a pair of amino acid residues (for example, basic amino acid residue and acidic amino acid residue) that exert an interaction. It means that the sum is constant. For example, FIG. 1 is a schematic diagram illustrating the distance between peptides when the side chain lengths are non-complementary. As shown in FIG. 1, the sum (7) of the number of atoms mainly involved in the side chain length of the alanine residue-arginine residue pair surrounded by the dotted line is the aspartic acid residue-arginine residue pair surrounded by the solid line. Smaller than the sum of the number of atoms mainly involved in the side chain length (9).

  The total charge in the neutral region of the amino acid residues contained in the self-assembling peptide is substantially +2. That is, the self-assembling peptide does not cancel out the positive charge and the negative charge derived from the side chain of the amino acid residue contained in the peptide in the neutral region. In addition, since the N-terminal and C-terminal amino acid residues are both basic amino acid residues, the self-assembling peptide of the present invention has, for example, electrostatic repulsion in addition to electrostatic attraction between peptides. It is presumed that a stable gel can be formed without precipitation in the neutral region because the excessive balance is not substantially generated by maintaining the delicate balance. In the present specification, the “neutral region” refers to a region having a pH of 6 to 8, and preferably a pH of 6.5 to 7.5.

  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).

The self-assembling peptides used in another preferred embodiment of the present invention are exemplified below.
n-RLDLRLALRLLDLR-c (SEQ ID NO: 4)
n-RLDLRLLLLRLDLR-c (SEQ ID NO: 12)
n-RADLRLALRLLDLR-c (SEQ ID NO: 13)
n-RLDLRLALLRLDA-c (SEQ ID NO: 14)
n-RADLRLLLRLLDLR-c (SEQ ID NO: 15)
n-RADLRLLLRLDA-c (SEQ ID NO: 16)
n-RLDLRALLRLLDLR-c (SEQ ID NO: 17)
n-RLDLRLLARLDLR-c (SEQ ID NO: 18)

  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.

B-2. Other Components The additive contained in the peptide gel of the present invention can be appropriately selected depending on the use of the peptide gel, the type of peptide contained, and the like. Specific examples of additives include pH adjusters such as sodium hydroxide, potassium hydroxide, hydrochloric acid, phosphoric acid, sodium bicarbonate; amino acids; vitamin A, vitamin B group, vitamin C, vitamin D, vitamin E and its Vitamins such as derivatives; sugars such as monosaccharides, disaccharides and oligosaccharides; polysaccharides such as hyaluronic acid, chitosan and hydrophilic cellulose; alcohols such as ethanol, propanol and isopropanol; polyhydric alcohols such as glycerin and propylene glycol Class; pigments such as phenol red; hormones, cytokines (hematopoietic factors, growth factors, etc.), physiologically active substances such as peptides; enzymes; antibodies; DNA; RNA; Only one type of additive may be added, or two or more types may be added in combination. The concentration of the additive in the aqueous solution can be appropriately set according to the purpose, the use of the peptide gel, and the like.

  By using an additive containing an amino group such as a physiologically active substance, an enzyme, or an antibody, or an additive bound to a biomolecule that can be bound to a linker structure that can be bound by the interaction between biomolecules of a self-assembling peptide These additives can be chemically bound to the interior and / or surface of the peptide gel. Thereby, an additive and a peptide gel can be tied more firmly. When such additives are used, it is preferable to add these additives to the peptide gel before forming a covalent bond by a photocrosslinking reaction and / or a bond by an interaction between biomolecules.

  Specific examples of aqueous solutions containing additives include phosphate buffered saline (PBS), various buffers such as Tris-HCl, cell culture media such as Dulbecco's modified Eagle medium (DMEM), sodium hydroxide, An aqueous solution whose pH is adjusted with hydrochloric acid, sodium hydrogen carbonate, or the like.

  The aqueous solution may have any appropriate pH depending on the purpose. For example, the pH of the aqueous solution before and after dissolving the peptide used in the present invention is preferably 5 to 9, more preferably 5.5 to 8, and particularly preferably 6.0 to 7.5. If it is this range, the peptide gel excellent in mechanical strength can be obtained. Furthermore, when the aqueous solution contains cells, good cell viability can be obtained.

  Any appropriate cells can be selected as the cells depending on the purpose and the like. The cell may be an animal cell or a plant cell. Specific examples of cells include chondrocytes, myoblasts, bone marrow cells, fibroblasts, hepatocytes, cardiomyocytes and the like.

  The peptide gel can be formed by any suitable method. Typically, the peptide gel can be formed by allowing an aqueous solution containing at least one peptide used in the present invention to stand. The temperature or time for standing is not particularly limited as long as the peptide used in the present invention self-assembles to form a gel, and can be appropriately set according to the purpose of use of the gel, the type, concentration, etc. of the peptide. . The time for standing is usually 1 minute or longer, preferably 3 minutes or longer, more preferably 5 minutes or longer. The temperature is usually 4-50 ° C, preferably 15-45 ° C.

C. Uses of Peptide Gels Preferable uses of the peptide gel of the present invention include, for example, cell culture substrates; protein detection substrates; skin care products, hair care products and other cosmetics; decubitus preparations, bone fillers, and cosmetic injections. , Ophthalmic surgery aids, artificial vitreous, artificial lenses, joint lubricants, eye drops, DDS base materials, hemostatic agents, etc .; moisturizing agents; desiccants; coating agents for medical devices such as contact lenses Can be mentioned.

  The substrate for protein detection is obtained, for example, by coating the surface of an arbitrary material with the peptide gel of the present invention so as to have an arbitrary thickness (for example, about 0.01 to 1 mm). A small amount of a solution containing a protein whose type or expression level is unknown is spotted on the surface of the protein detection substrate. Here, as explained in the above section B-2, the peptide gel of the present invention and the amino group of the protein can form a covalent bond by a photocrosslinking reaction. Therefore, by performing a photocrosslinking reaction after spotting an unknown protein, the self-assembled peptide and the unknown protein can be bound more firmly. Subsequently, the protein type and expression level can be identified by performing an arbitrary protein identification step.

D. Cell culture substrate The cell culture substrate of the present invention contains the peptide gel. Since the cell culture substrate of the present invention is formed from a self-assembled peptide obtained by chemical synthesis, safe cell culture is possible without contamination with pathogens and the like. In addition, since the gel formed from the peptide used in the present invention is transparent in the neutral region and can be excellent in mechanical strength, the cell culture substrate of the present invention has high visibility and operability during cell culture. Excellent.

  In the cell culture substrate, the peptide used in the present invention is self-assembled inside the cell culture substrate to form a three-dimensional network structure. Therefore, not only the culture on the cell culture substrate but also the culture in the cell culture substrate is possible.

  When culturing on a cell culture substrate, the cells to be cultured can be placed on the already formed peptide gel of the present invention and cultured. When culturing in a cell culture substrate, the peptide or aqueous peptide solution used in the present invention and the cell or cell suspension can be mixed, and a peptide gel can be formed from the mixture and cultured.

  The liquid phase of the peptide gel can be replaced with a desired culture solution by solvent replacement. The solvent replacement can be performed using, for example, a trade name “Cell Culture Insert” or the like. Details of the peptide gel (peptide concentration, types of additives that the aqueous solution (mixture) can contain, pH, etc.) and formation method are as described in Section C above.

  Any appropriate cell can be selected as the cell to be cultured according to the purpose and the like. The cell may be an animal cell or a plant cell. Specific examples of cells include chondrocytes, myoblasts, bone marrow cells, fibroblasts, hepatocytes, cardiomyocytes and the like. The culture medium and culture conditions can be appropriately selected according to the type of cells to be cultured, the purpose, and the like.

  Since the cell culture substrate of the present invention is excellent in biocompatibility and safety, it can be suitably used, for example, in three-dimensional cell culture in the field of regenerative medicine.

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

(Reference Example 1)
A self-assembling peptide (DLRLDLALDLLRLD-FN ₃ ) in which azidophenylalanine (FN ₃ ) is introduced into the C-terminus of the self-assembling peptide consisting of the amino acid sequence of SEQ ID NO: 1 by the Fmoc synthesis method using ResPep (manufactured by Intavis) ), A self-assembling peptide in which azidophenylalanine (FN ₃ ) is introduced at the N-terminus of the self-assembling peptide consisting of the amino acid sequence of SEQ ID NO: 1 (FN ₃ -DLRLLDLALDLRLD), a self-assembling peptide of the amino acid sequence of SEQ ID NO: 2 Then, a self-assembling peptide (DLRLDL-FN ₃ -LDLRLD) in which phenylalanine at position 7 has an azide group was synthesized. Peptide production was confirmed using TOF-MS mass spectrometry (Applied Sciences).

(Reference Example 2)
A self-assembled peptide (DLRLDLALDRLRLD-KBio) in which biotin-linked lysine (KBio) is introduced at the C-terminus and biotin-linked at the N-terminus in the same manner as in Reference Example 1 except that biotin-conjugated lysine is used instead of azidophenylalanine. A self-assembling peptide (KBio-DLRLLDLALDLRLD) in which lysine (KBio) is introduced, a self-assembling peptide having the amino acid sequence of SEQ ID NO: 3, wherein biotin is bound to lysine at position 7 (DLRLDL) -KBio-LDLRLD) was synthesized.

(Reference Example 3)
A self-assembling peptide (DLRLDLALDLLRLD-FBzo) in which benzolylphenylalanine is introduced at the C-terminus and benzolylphenylalanine is introduced at the N-terminus in the same manner as in Reference Example 1 except that benzoylphenylalanine is used instead of azidophenylalanine. The self-assembled peptide (FBzo-DLRLLDLALDLRLD) and the self-assembled peptide in which benzoylphenylalanine was introduced into the sequence (DLRLDL-FBzo-LDLRLD) were synthesized.

(Reference Example 4)
A self-assembling peptide (RLDLRLALRLLDLR-FN ₃ ) in which azidophenylalanine (FN ₃ ) is introduced into the C-terminal of the self-assembling peptide consisting of the amino acid sequence of SEQ ID NO: 4 by Resmop synthesis using ResPep (manufactured by Intavis) ) Was synthesized. Peptide production was confirmed using TOF-MS mass spectrometry (Applied Sciences).

(Reference Example 5)
In the same manner as in Reference Example 1, a self-assembling peptide consisting of the amino acid sequence of SEQ ID NO: 2 (DLRLDLFLDLRLLD), a self-assembling peptide consisting of the amino acid sequence of SEQ ID NO: 19 (FDRLLDLALDLRLD), and a self-organization consisting of the amino acid sequence of SEQ ID NO: 20 Peptide (DLRLLDALDLRLDF) was synthesized.

[Example 1]
Each self-assembled peptide obtained in Reference Example 1 was dissolved in DMSO, and then deionized water was added so that the peptide concentration was 1 w / v% to obtain a peptide gel. The obtained peptide gel was irradiated with ultraviolet rays at ² J / scm ² (conditions under a normal fluorescent lamp) for 12 hours (integrated light amount: 80 mJ / cm ² ) to obtain the peptide gel of the present invention.

The obtained peptide gel was put into the same amount tube, and it leaned diagonally to the container with a shallow bottom as shown in FIG. 1, and left still for 30 minutes. After standing, the obtained gel was photographed from directly above (in the direction of the arrow in FIG. 1), and the hardness of the gel was confirmed by the shape of the meniscus. The mechanical strength of the obtained peptide gel was confirmed by the shape of the concave meniscus of each peptide gel. The photograph of the peptide gel after each stationary is shown in FIG. A peptide gel using a self-assembling peptide (DLRLLDLALDLRLD-FN ₃ ) in which azidophenylalanine (FN ₃ ) is introduced at the C-terminus is introduced into the tube 5 in FIG. 2, and azidophenylalanine (FN ₃ ) is introduced at the N-terminus. A peptide gel using a self-assembling peptide (FN ₃ -DLRLLDLALDLRLD) was introduced into the tube 3 in FIG. 2, and a self-assembling peptide (DLRLDL-FN ₃ -LDLRLD) in which azidophenylalanine (FN ₃ ) was introduced into the sequence ) Corresponds to each of the tubes in FIG.

  The peptide gel obtained in Example 1 was practically sufficiently strong because the gel was hardly inclined even when it was leaned diagonally.

(Comparative Example 1)
A peptide gel was obtained in the same manner as in Example 1 except that the self-assembled peptide obtained in Reference Example 5 was used. In the same manner as in Example 1, the mechanical strength of the obtained peptide gel was confirmed. The photograph of the peptide gel after each stationary is shown in FIG. A peptide gel using the self-assembling peptide of SEQ ID NO: 2 is in the tube of 2 in FIG. 2, and a peptide gel using the self-assembling peptide of SEQ ID NO: 19 is in the tube of 4 in FIG. Peptide gels using self-assembled peptides correspond to the 6 tubes in FIG.

  The peptide gel obtained in Comparative Example 1 is different from the peptide gel obtained in Example 1 only in that phenylalanine in each amino acid sequence does not have a reactive group that forms a covalent bond by photocrosslinking. The peptide gel obtained in Comparative Example 1 was higher in fluidity than the peptide gel obtained in Example 1.

[Example 2]
Each self-assembled peptide obtained in Reference Example 2 was dissolved in DMSO, and then deionized water was added so that the peptide concentration would be 1 w / v% to obtain a peptide gel. To the obtained peptide gel, 0.1 ml of a 10 mg / ml solution of egg white-derived avidin was added to obtain the peptide gel of the present invention.

  FIG. 3 shows a photograph of a peptide gel using a self-assembling peptide (DLRLDL-KBio-LDLRLD) in which biotin-conjugated lysine is introduced at position 7 of the amino acid sequence. The obtained peptide gel became a massive peptide gel after one day. The obtained peptide gel had hardness (mechanical strength) that can be manipulated with a spatula.

[Example 3]
Each self-assembled peptide obtained in Reference Example 3 was dissolved in DMSO, and then deionized water was added so that the peptide concentration was 1 w / v%, to obtain a peptide gel. The obtained peptide gel was irradiated with ultraviolet rays at 8 J / scm ² (conditions under a normal fluorescent lamp) for 1 hour (integrated light amount: 28.8 kJ / cm ² ) using an ultraviolet lamp (TRANSILLUMINATOR manufactured by UVP). The peptide gel of the present invention was obtained.

  The obtained peptide gel was put into a tube and allowed to stand for 1 minute in an inverted state, and the mechanical strength of the obtained peptide gel was confirmed by the shape of the concave meniscus of each peptide gel. The photograph which put each peptide gel in the tube is shown in FIG. Fig. 4 (a) shows the state of the gel in the tube immediately after being inverted, and Fig. 4 (b) shows the state of the gel in the tube after standing for 1 minute in the inverted state. A self-assembling peptide (DLRLLDLALDLRLD-FBzo) having benzoylphenylalanine introduced at the C-terminus is 3 in FIG. 4, and a self-assembling peptide (FBzo-DLRLLDLALDLRLD) having benzoylphenylalanine introduced at the N-terminus is in FIG. 2. Self-assembling peptides (DLRLDL-FBzo-LDLRLD) into which benzoylphenylalanine is introduced at the 7th position of the amino acid sequence correspond to 1 in FIG. As shown in FIG. 4 (b), the peptide gel obtained in Example 3 had a large volume of hydrogel remaining on the upper base even after standing, and a peptide gel with practically sufficient strength was obtained. .

(Comparative Example 2)
A peptide gel was obtained in the same manner as in Example 3 except that the self-assembled peptide obtained in Reference Example 5 was used. In the same manner as in Example 3, the mechanical strength of the obtained peptide gel was confirmed. The photograph of the peptide gel after each stationary is shown in FIG. A peptide gel using the self-assembling peptide of SEQ ID NO: 19 is in the tube of 4 in FIG. 2, and a peptide gel using the self-assembling peptide of SEQ ID NO: 2 is in the tube of 5 in FIG. Peptide gels using self-assembled peptides correspond to the 6 tubes in FIG.

  The peptide gel obtained in Comparative Example 2 is different from the peptide gel obtained in Example 3 only in that phenylalanine in each amino acid sequence does not have a reactive group that forms a covalent bond by photocrosslinking. The peptide gel obtained in Comparative Example 2 was higher in fluidity than the peptide gel obtained in Example 3.

[Example 4]
Each self-assembled peptide obtained in Reference Example 4 was dissolved in DMSO, and then deionized water was added so that the peptide concentration was 1 w / v% to obtain a peptide gel. The obtained peptide gel was irradiated with ultraviolet rays at 8 J / scm ² (conditions under a normal fluorescent lamp) for 1 hour (integrated light amount: 28.8 kJ / cm ² ) using an ultraviolet lamp (TRANSILLUMINATOR manufactured by UVP). The peptide gel of the present invention was obtained.

  The obtained peptide gel was put into a tube and allowed to stand for 1 minute in an inverted state, and the mechanical strength of the peptide gel obtained from the shape change of the peptide gel obtained in Example 4 was confirmed. Even when the peptide gel obtained in Example 4 was inverted, it did not fall off, and a peptide gel with practically sufficient strength was obtained.

[Test Example 1: Drug sustained release test]
According to the method of Nagai et al. (J. Cont. Rel. 115, 2006, 18-25), the peptide gel of the present invention was tested for sustained drug release. A DMSO solution (peptide concentration: 3 w / v%) of a self-assembling peptide in which azidophenylalanine (FN ₃ ) was introduced at the C-terminus obtained in Reference Example 1 and an aqueous solution containing bromophenol blue were used in a volume ratio of 1: 2. To obtain a peptide gel in which bromophenol blue was dispersed. 600 μl of buffer solution was added to the upper layer of the obtained peptide gel, and after 0.5, 1, 2, 4, 8 hours, the absorbance of the upper buffer solution at 590 nm was measured with Biophotometer Plus (manufactured by Eppendorf) Measured with The concentration calculated from the absorbance at each time is shown in FIG. The sustained release rate from the peptide gel of the present invention was almost the same as that of hydrogel RADA16 (gel using self-assembled peptide in which hydrophilic peptide and hydrophobic peptide are alternately arranged) disclosed in Nagai et al. It was.

[Test Example 2]
A peptide gel in which phenol red was dispersed was obtained in the same manner as in Test Example 1 except that phenol red was used instead of bromophenol blue. After 0.5, 1, 2, 4, and 8 hours, the absorbance at 405 nm of the upper buffer solution of the obtained peptide gel was measured with Biophotometer Plus (Eppendorf). The concentration calculated from the absorbance at each time is shown in FIG. 6 (plot in FIG. 6). The sustained release rate from the peptide gel of the present invention was comparable to hydrogel RADA16 (a gel using a self-assembled peptide in which hydrophilic and hydrophobic amino acids are alternately arranged) disclosed in Nagai et al. It was.

[Test Example 3]
Instead of the self-assembling peptide in which azidophenylalanine was introduced at the C-terminus, a DMSO solution of the self-assembling peptide obtained by introducing biotin-conjugated lysine at the N-terminus obtained in Reference Example 2 (peptide concentration: 3 w / v%) The absorbance of the upper buffer solution was measured in the same manner as in Test Example 2 except that was used. The concentration calculated from the absorbance at each time is shown in FIG. 6 (Δ plot in FIG. 6). The sustained release rate from the peptide gel of the present invention was comparable to hydrogel RADA16 (a gel using a self-assembled peptide in which hydrophilic and hydrophobic amino acids are alternately arranged) disclosed in Nagai et al. It was.

[Test Example 4: Cell culture test]
100 μl of DMSO was added to the self-assembled peptide (2 mmol) with azidophenylalanine introduced into the C-terminus obtained in Reference Example 1 and mixed well to obtain a mixed solution. 20 μl of the mixed solution was added per well to a 96-well tissue culture microplate (Asahi Glass Co., Ltd.). Further, 180 μl of medium DMEM (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was allowed to stand in a CO ₂ incubator (manufactured by Astech) for 30 minutes to gel and equilibrate, and the supernatant was removed. In order to remove DMSO in the medium, the addition and recovery of the medium were repeated several times. HEK293 cells were seeded in a well and cultured in a CO ₂ incubator for 3 days. FIG. 7 shows a photograph of the peptide gel after culturing. When the peptide gel after the culture was observed with a microscope, cells were proliferating in the gel (portion surrounded by the middle line in FIG. 7), and the survival of the cells was confirmed.

[Test Example 5]
Instead of the self-assembling peptide introduced with azidophenylalanine at the C-terminus, the self-assembling peptide introduced with biotin-conjugated lysine at the N-terminus obtained in Reference Example 2 was used, and instead of HEK293 cells, mouse 3T3 cells Cells were cultured in the same manner as in Test Example 4 except that was used. FIG. 8 shows a photograph of the peptide gel after culturing. When the cultured peptide gel was observed with a microscope, cells were proliferating in the gel (portion surrounded by the middle line in FIG. 8), and the survival of the cells was confirmed.

  As can be seen from the results of Test Examples 1 to 5, the peptide gel of the present invention has higher strength than the conventional peptide gel, but the sustained release of the drug and cell culture are preferably used in the same manner as the conventional peptide gel. be able to.

  The peptide gel of the present invention can be applied to regenerative medicine, drug delivery systems, cosmetics, artificial vitreous bodies, hemostatic agents, cosmetic injections, bone filling, joint lubricants, wet water retention materials and the like.

Claims (4)

D LRLDLALDLRLD (SEQ ID NO: 1), DLRLDLLLDLRLD (SEQ ID NO: 5), DARLDLALDLRLD (SEQ ID NO: 6), DLRLDLALDLRAD (SEQ ID NO: 7), DARLDLLLDLRLD (SEQ ID NO: 8), DARLDLLLDLRAD (SEQ ID NO: 9), DLRLDALLDLRLD (SEQ ID NO: 10), DRLLDLLADLRLD (SEQ ID NO: 11), RLDLRLALLRLLDLR (SEQ ID NO: 4), RLDLRLLLLRLDLR (SEQ ID NO: 12), RADRLRLALLLDLR (SEQ ID NO: 13), RLDLRRLALLDRAR (SEQ ID NO: 14), RADLRLLLLRLDLR (SEQ ID NO: L) LLDLRLRLD (LDRLL) (SEQ ID NO: 17) or RLDLRL At least one amino acid selected from the group consisting of azidophenylalanine , biotin-linked lysine and benzoylphenylalanine is introduced at the N-terminal or C-terminal of the self-assembling peptide consisting of the amino acid sequence of LARLDLR (SEQ ID NO: 18) A peptide gel formed from an aqueous solution containing a peptide. The peptide gel according to claim 1, wherein the azidophenylalanine and / or benzolylphenylalanine is introduced at the C-terminus of the self-assembling peptide. A cell culture substrate comprising the peptide gel according to claim 1 or 2 . Comprising the peptide gel according to claim 1 or 2, drug transport, the sustained release carrier.