WO2010103887A1 - 自己組織化ペプチドおよび高強度ペプチドゲル - Google Patents
自己組織化ペプチドおよび高強度ペプチドゲル Download PDFInfo
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- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/08—Materials for coatings
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/145—Hydrogels or hydrocolloids
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28047—Gels
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- C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
- C10M—LUBRICATING COMPOSITIONS; USE OF CHEMICAL SUBSTANCES EITHER ALONE OR AS LUBRICATING INGREDIENTS IN A LUBRICATING COMPOSITION
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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Definitions
- the present invention relates to a self-assembled peptide capable of forming a high-strength peptide gel, and a peptide gel formed from the peptide.
- Collagen gel is generally used as a scaffold (cell scaffold) used in research and actual treatment in the field of regenerative medicine.
- collagen gel is derived from animals, there is a risk of unknown infectious diseases. Scaffolds derived from chemically synthesized materials exist as a means to relieve the anxiety of this unknown infection. Examples of such a material include self-assembling peptides as disclosed in Patent Document 1 or Patent Document 2.
- 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. .
- the self-assembled peptide gel of Patent Document 1 has a problem that transparency is insufficient in a neutral region.
- the present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a peptide gel having sufficient mechanical strength for practical use and a self-assembling peptide capable of forming the peptide gel. is there.
- the self-assembling peptide has 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
- 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, of which 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.
- b 3 and b 4 in the amino acid sequence are hydrophobic amino acid residues.
- b 1 to b 6 are all hydrophobic amino acid residues.
- b 1 to b 6 in the amino acid sequence are each independently an alanine residue, a valine residue, a leucine residue, or an isoleucine residue.
- d is an alanine residue, valine residue, leucine residue, or isoleucine residue in the amino acid sequence.
- the self-assembling peptide is RLDLRLALLLRLDLR (SEQ ID NO: 1), RLDLRLLLRLLDLR (SEQ ID NO: 2), RADRLRLALLLDLR (SEQ ID NO: 6), RLDLRRLALLLRLDAR (SEQ ID NO: 7), RADLRLLLRLLDLR (SEQ ID NO: 8), RADRLRL SEQ ID NO: 9), RLDLRLALLLDLR (SEQ ID NO: 10), or RLDLRLLARLDLR (SEQ ID NO: 11) is a peptide consisting of the amino acid sequence.
- the self-assembling peptide is a peptide consisting of an amino acid sequence of RLDLRLALRLLDLR (SEQ ID NO: 1) or RLDLRLLLLRLDLR (SEQ ID NO: 2).
- a modified peptide is provided.
- the modified peptide is a peptide in which the N-terminal amino group and / or the C-terminal carboxyl group of the self-assembling peptide is modified, and has a self-assembling ability.
- an amino acid sequence containing RGD is added to the N-terminal amino group and / or C-terminal carboxyl group.
- a peptide gel is provided.
- the peptide gel is formed from an aqueous solution containing the self-assembling peptide and / or the modified peptide.
- the aqueous solution further contains an additive.
- the additive comprises a pH adjuster, amino acids, vitamins, saccharides, polysaccharides, alcohols, polyhydric alcohols, dyes, physiologically active substances, enzymes, antibodies, DNA, and RNA. It is at least one selected from the group.
- the peptide gel was run at a compression rate of 0.05 mm / s under a temperature condition of 22 ° C.
- a cell culture substrate contains at least one selected from the group consisting of the self-assembling peptide, the modified peptide, and the peptide gel.
- a method for producing a sterile peptide is provided.
- the method for producing the aseptic peptide includes a step of sterilizing the self-assembled peptide and / or modified peptide at 100 ° C. or higher under pressure.
- a method for producing an article coated with a peptide gel includes a step of freezing the peptide gel, Thawing the frozen product to obtain a peptide sol; Coating at least a portion of the surface of the article to be coated with the peptide sol, and re-forming a peptide gel from the peptide sol.
- a peptide gel having a practical mechanical strength can be obtained.
- FIG. 1 It is a schematic diagram explaining the distance between the peptides consisting of the sequence of n-RADARAAAARADAR-c.
- the thick line in the main chain connecting the N-terminal and C-terminal in the figure indicates a peptide bond.
- 2 is a graph showing the results of a compression test of the peptide gel of Example 1.
- A Photograph when the peptide gel of Example 1 is sandwiched with tweezers
- (c) The peptide gel of Comparative Example 1 is sandwiched with tweezers It is a picture when it is. It is a graph which shows the result of the compression test of the peptide gel of Example 2.
- (A) is a mass spectrometric result of a trade name “PuraMatrix TM ” (manufactured by 3D Matrix) before sterilization, and (b) is a trade name “PuraMatrix TM ” (3D Matrix) after sterilization. (Made by company).
- (A), (b), and (c) are a photograph when the gel is transferred to a petri dish, a photograph of a frozen gel, and a petri dish with the entire surface uniformly coated with a peptide gel.
- (A), (b), and (c) are a photograph when the gel is transferred to a slide glass, a photograph of a frozen gel, and a photograph of a slide glass that is uniformly coated on the entire surface with a peptide gel, respectively. .
- 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.
- 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).
- gel refers to a viscoelastic substance having both viscous and elastic properties.
- 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.
- 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.
- the self-assembling peptide of the present invention comprises 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
- 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, at least of which 5 are hydrophobic amino acid residues
- c 1 and c 2 are acidic amino acid residues
- d is a hydrophobic amino acid residue.
- a self-assembled peptide has a ⁇ -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, and hydrogen bonds acting between the hydrophilic surfaces It is considered that a plurality of peptides are spontaneously assembled by an interaction such as an interaction between ions and a hydrophobic interaction acting between hydrophobic surfaces. Therefore, in conventional self-assembling peptides, it is very important to have hydrophilic amino acids and hydrophobic amino acids alternately and in equal proportions (see, for example, Patent Document 1).
- the self-assembling peptide of the present invention has basic amino acid residues (positions 1, 5, 9, and 13) every other residue, centering on the hydrophobic amino acid residue at position 7. And an amino acid sequence of 13 residues having acidic amino acid residues (positions 3 and 11) at symmetrical positions in the N-terminal direction and the C-terminal direction. That is, one feature of the self-assembling peptide of the present invention is that it does not have hydrophilic amino acids and hydrophobic amino acids alternately. Another feature of the self-assembling peptide of the present invention is that it does not have hydrophilic amino acid residues and hydrophobic amino acid residues in equal proportions.
- the self-assembling peptide of the present invention has four basic amino acid residues and two acidic amino acid residues at a predetermined symmetrical position with the hydrophobic amino acid residue at position 7 as the center, Another feature is that both the N-terminal and C-terminal amino acid residues are basic amino acid residues.
- the self-assembling peptide of the present invention has an excellent self-assembling ability, and can form a peptide gel that is more excellent in mechanical strength than conventional ones.
- 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.
- 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.
- b 1 to b 6 are uncharged polar amino acid residues and / or hydrophobic amino acid residues, and at least 5 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.
- 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.
- 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.
- b 1 to b 6 are all hydrophobic amino acid residues. This is because the self-assembling peptide preferably forms a ⁇ -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 is excellent in solubility in water and can form a high-strength peptide gel can be obtained.
- 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.
- d is a hydrophobic amino acid residue.
- d is a hydrophobic amino acid residue and has a predetermined symmetrical structure
- the self-assembling peptide can form a gel having better mechanical strength than conventional peptide gels. It is done.
- 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.
- the side chain length of the amino acid on the hydrophilic surface side of the ⁇ 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 ⁇ sheet structure is preferably complementary in order to obtain an electrostatic attractive force suitable for self-assembly. It is.
- 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.
- 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 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.
- 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.
- the “neutral region” refers to a region having a pH of 6 to 8, preferably 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 method of the Raininger is performed by a program available on, for example, the website of EMBL WWW Gateway to Isoelectric Point Service (http://www.embl-heidelberg.de/cgi/pi-wrapper.pl).
- aqueous solution containing the self-assembling peptide can form a peptide gel with excellent mechanical strength.
- a self-assembling peptide aqueous solution (preferably 0.2-5 w / v%, more preferably 0.2-2 w / v%, particularly preferably 0.2-1 w / v%, most preferably 0.3 to 0.8 w / v%) using a jig with a tip having a diameter of 3.2 mm and a curvature radius of 1.6 mm under a temperature condition of 22 ° C., and a compression speed of 0.05 mm / s
- the absolute value L (g / s) of the change in load per unit time in the approximate straight line of the measured value from the beginning of compression is preferably 0.03 g.
- the compression test is performed as described in the examples below, for example, a viscoelasticity measuring device (manufactured by TA Instruments Inc.), a product made of stainless steel jig (manufactured by TA Instruments Inc.) Number "RSA III").
- n-RLDLRLALRLLDLR-c SEQ ID NO: 1
- n-RLDLRLLLLRLDLR-c SEQ ID NO: 2
- n-RADLRLALRLLDLR-c SEQ ID NO: 6
- n-RLDLRLALLRLDA-c SEQ ID NO: 7
- n-RADLRLLLRLLDLR-c SEQ ID NO: 8
- n-RADLRLLLRLDA-c SEQ ID NO: 9
- n-RLDLRLALLLDLR-c SEQ ID NO: 10
- n-RLDLRLLARLDLR-c SEQ ID NO: 11
- the self-assembling peptide can be manufactured by any appropriate manufacturing 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 modified peptide of the present invention is a peptide obtained by subjecting the self-assembling peptide to any modification as long as it has the ability to self-assemble.
- the site where the modification is performed may be the N-terminal amino group of the self-assembling peptide, the C-terminal carboxyl group, or both.
- any appropriate modification can be selected as long as the obtained modified peptide has a self-organizing ability.
- 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.
- the peptide after introduction is a peptide in which any amino acid is added to the N-terminal and / or C-terminal of the self-assembling peptide. Included in modified peptides. 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.
- the added peptide (modified peptide) as a whole may not have the characteristics of the self-assembling peptide. Specifically, when an arbitrary amino acid is added and the sequence in the N-terminal direction and the sequence in the C-terminal direction are asymmetric about the 7-position hydrophobic amino acid sequence, the hydrophobic amino acid is equal to the hydrophilic amino acid. In some cases, it has a ratio. Even in such a case, since the self-assembling peptide has an extremely excellent self-assembling ability, the added peptide to which any amino acid is added can also form a peptide gel with excellent mechanical strength. .
- the number of amino acid residues constituting the modified peptide after introduction is preferably 14 to 200, more preferably 14 to 100, still more preferably 14 to 50, It is preferably 14 to 30, and most preferably 14 to 20. This is because if the number of amino acid residues exceeds 200, the self-assembling ability of the self-assembling peptide may be impaired.
- the type and position of the amino acid to be introduced can be appropriately set according to the use of the modified peptide.
- hydrophobic amino acids and hydrophilic amino acids are introduced alternately from the N-terminal and / or C-terminal arginine residues (hydrophilic amino acids) of the self-assembling peptide.
- amino acids to be introduced include, for example, REDV, EILDV, YEKPGSPPREVVPRPRPGV, KNNOKSEPLIGRK, YIGSR, RNIAELLKDI, RYVVLPRPCFCFEKGMNYTVR, IKVAV, PDSGR, and RGD, including G sequences such as G sequence, G RGDF, RGDT, RGDA, RGD, and RGDS) and the like; nuclear transfer signals such as PPKKRKVV, PAAKRVKLD, PQPKKKKP, and QRKRQK, etc .; And the like. These sequences may be introduced alone or as a combination of a plurality of sequences.
- the introduced amino acid sequence and the self-assembling peptide may be linked via one or more arbitrary amino acids between them.
- the above modification can be performed by any appropriate method depending on the type and the like.
- the aqueous solution containing the modified peptide can form a peptide gel with excellent mechanical strength.
- the modified peptide aqueous solution (preferably 0.2-5 w / v%, more preferably 0.2-2 w / v%, particularly preferably 0.2-1 w / v%, most preferably 0 .3 to 0.8 w / v%) under a temperature condition of 22 ° C., using a spherical jig having a tip of 3.2 mm in diameter and a curvature radius of 1.6 mm at a compression speed of 0.05 mm / s.
- the absolute value L (g / s) of the load change amount per unit time in the approximate straight line of the measured value from the beginning of compression (after 8 to 10 seconds) is preferably 0.03 g / s.
- a gel of 0.035 g / s or more, more preferably 0.04 g / s or more can be formed.
- the peptide gel of the present invention comprises the self-assembling peptide and / or the modified peptide (hereinafter, the “self-assembling peptide and / or the modified peptide” may be referred to as “the peptide of the present invention”). It is formed from the aqueous solution containing.
- the peptide of the present invention spontaneously assembles in an aqueous solution to form a fibrous molecular assembly having a nanometer-scale width, so-called nanofibers, mainly due to electrostatic interaction between the nanofibers. It is estimated that a gel is formed by forming a three-dimensional network structure.
- the peptide of the present invention contained in the aqueous solution may be only one type or two or more types.
- the aqueous solution may further comprise any suitable additive in addition to the peptide of the present invention and water.
- the aqueous solution may contain insoluble matter such as cells.
- the concentration of the peptide of the present invention in the aqueous solution is preferably 0.2 to 5 w / v%, more preferably 0.2 to 2 w / v%, particularly preferably 0.2 to 1 w / v%, and most preferably 0. .3 to 0.8 w / v%.
- concentration is within this range, a peptide gel having excellent mechanical strength can be obtained.
- good cell viability can be obtained.
- additives 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, sodium carbonate; amino acids; vitamin A, vitamin B group, vitamin C, vitamin D, vitamin Vitamins such as E and its derivatives; saccharides such as monosaccharides, disaccharides and oligosaccharides; polysaccharides such as hyaluronic acid, chitosan and hydrophilized cellulose; alcohols such as ethanol, propanol and isopropanol; glycerin and propylene glycol Examples include polyhydric alcohols; 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
- 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.
- PBS phosphate buffered saline
- DMEM Dulbecco's modified Eagle medium
- sodium hydroxide 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.
- the pH of the aqueous solution before and after dissolving the peptide of 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. Further, when the pH is within this range, the peptide is hardly decomposed under high temperature and pressure conditions, and therefore, high-pressure steam sterilization such as autoclave can be performed. As a result, a sterile peptide gel can be easily obtained.
- the cells any appropriate cells can be selected 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 peptide gel of the present invention is a compression test conducted at a compression speed of 0.05 mm / s using a jig having a spherical shape with a tip of 3.2 mm in diameter and a curvature radius of 1.6 mm under a temperature condition of 22 ° C.
- the unit is preferably 0.03 g / s or more, more preferably 0.035 g / s or more, particularly preferably 0.04 g / s or more.
- the absolute value L (g / s) of the amount of change in load per hour is shown.
- the peptide gel having the above-mentioned mechanical strength is, for example, preferably 0.2 to 5 w / v%, more preferably 0.2 to 2 w / v%, particularly preferably 0.2 to 1 w of the peptide of the present invention.
- / V% most preferably formed from an aqueous solution containing at a concentration of 0.3-0.8 w / v%.
- the peptide gel of the present invention has a visible light transmittance of preferably 50% or more, more preferably 70% or more, particularly preferably 90% in a cell having an optical path length of 10 mm, measured at an absorbance of 380 nm to 780 nm. That's it.
- the peptide gel having visible light transmittance described above can be formed, for example, from an aqueous solution containing the peptide of the present invention at a concentration of 0.2 to 2 w / v%.
- the decrease rate (%) of visible light transmittance after leaving the peptide gel in a sealed state at room temperature for a long period (for example, 2 months) (100- (visible light transmittance after storage / before storage)
- the visible light transmittance x 100) is preferably 30% or less, more preferably 20% or less, and particularly preferably 10% or less.
- Such a peptide gel having a high visible light transmittance has advantages such as easy cell observation by a fluorescence microscope or the like when used as a cell culture substrate.
- the visible light transmittance can be measured using, for example, a UV / VIS measuring device.
- the peptide gel can be formed by any appropriate method. Typically, the peptide gel can be formed by allowing an aqueous solution containing at least one peptide of the present invention to stand.
- the temperature or time for standing is not particularly limited as long as the peptide of 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 to 50 ° C., preferably 15 to 45 ° C.
- Preferred uses of the self-assembled peptides, modified peptides, and peptide gels of the present invention include, for example, cell culture substrates; cosmetics such as skin care products and hair care products; Drugs for decubitus, bone filler, cosmetic injection, ophthalmic surgery aid, artificial vitreous, artificial lens, joint lubricant, eye drops, DDS base material, hemostatic agent, etc .; moisturizing agent; desiccant A coating agent for medical devices such as contact lenses.
- the cell culture substrate of the present invention contains at least one of the self-assembling peptide, the modified peptide, and the peptide gel. Since the cell culture substrate of the present invention is formed from self-assembled peptides and / or modified peptides obtained by chemical synthesis, safe cell culture is possible without contamination with pathogens and the like. Moreover, since the gel formed from the peptide of the present invention is transparent in the neutral region and excellent in mechanical strength, the cell culture substrate of the present invention is excellent in visibility and operability during cell culture. .
- the peptide of 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.
- the cells to be cultured When culturing on a cell culture substrate, the cells to be cultured can be placed on a peptide gel containing the peptide of the present invention that has already been formed.
- the peptide or peptide aqueous solution of the present invention and cells 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 can be contained in the aqueous solution (mixture), pH, etc.) and formation method are as described in Section D above.
- any appropriate cells can be selected 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.
- 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.
- the method for producing a sterile peptide of the present invention includes a step of sterilizing the self-assembled peptide and / or modified peptide at 100 ° C. or higher under pressure. These peptides are typically subjected to sterilization treatment in the form of an aqueous peptide solution or a peptide gel formed from the aqueous peptide solution.
- the pH of the aqueous peptide solution is preferably 5 to 9, more preferably 5.5 to 8, and particularly preferably 6.0 to 7.5.
- the peptide of this invention of a sterilized state can be obtained.
- the peptide aqueous solution and peptide gel are as described in the above section D.
- any appropriate sterilization method can be adopted as the sterilization method.
- a method of sterilization with high-temperature and high-pressure saturated steam can be preferably used.
- the pressure during autoclave sterilization is preferably 0.122 to 0.255 MPa, and more preferably 0.152 to 0.233 MPa.
- the sterilization temperature is preferably 105 to 135 ° C, more preferably 110 to 125 ° C.
- the sterilization time is preferably 1 to 60 minutes, more preferably 3 to 40 minutes, and particularly preferably 5 to 30 minutes.
- Autoclave sterilization can be performed using a commercially available autoclave apparatus.
- the method for producing an article coated with a peptide gel of the present invention comprises a step of freezing the peptide gel (freezing step), and a step of thawing the frozen product to obtain a peptide sol ( A melting step), a step of coating at least a part of the surface of the article to be coated with the peptide sol (coating step), and a step of re-forming a peptide gel from the peptide sol (gelation step).
- the method may further include an optional step as necessary.
- the bond between peptide molecules is broken and the three-dimensional network structure constituting the gel is disrupted, so that a sol in which peptide molecules are uniformly dispersed in an aqueous solution can be obtained.
- a sol in which peptide molecules are uniformly dispersed in an aqueous solution can be obtained.
- the peptide gel can be suitably frozen by placing it under a temperature condition of ⁇ 10 ° C. or lower.
- any appropriate freezing means such as a home or commercial freezer, liquid nitrogen can be selected.
- the frozen peptide gel can be stored frozen for an arbitrary period until it is subjected to the thawing step.
- the concentration of the additive is preferably a concentration that does not adversely affect the re-formation of the gel in the gelation step.
- the concentration can be appropriately set according to the type, concentration, etc. of the peptide, but is usually preferably a low concentration.
- the final concentration is preferably 50 mM or less, more preferably 40 mM or less.
- the final concentration is preferably 5 mM or less, more preferably 4 mM or less.
- the final concentration is preferably 0.5 ⁇ PBS or less, more preferably 0.3 ⁇ PBS or less. Further, in the case of pharmacopoeia, the final concentration is preferably 0.5% by weight or less, more preferably 0.4% by weight or less.
- the melting temperature can be set to any appropriate temperature as long as the frozen material obtained in the freezing step melts to form a sol. Melting may be performed at a constant temperature, or may be performed stepwise at different temperatures. There is no limitation on the melting rate and time, and melting may be performed gradually or rapidly. For example, it can be suitably melted by placing a frozen peptide gel under a temperature condition of 5 to 70 ° C., preferably 15 to 45 ° C.
- any appropriate means can be selected.
- Specific examples of the melting means include a water bath, an oil bath, and a thermostatic bath.
- the frozen peptide gel may be melted while applying vibrations to such an extent that no bubbles are generated in the sol.
- Examples of the method for applying vibration include shaking a frozen peptide gel or sol, and irradiating them with ultrasonic waves.
- any appropriate method can be adopted as a coating method.
- Specific examples include a dispenser application method, a dipping method, a bar coater method, a method for developing a sol on the surface of an article to be coated by centrifugal force, and causing the sol to flow by inclining the article to be coated to be developed on the surface of the article to be coated.
- a method is mentioned.
- various bonds between peptide molecules are sufficiently cleaved, the viscosity is remarkably reduced, and the peptide molecules are sufficiently dispersed to form a uniform layer on the surface of the article to be coated. Can do.
- any appropriate article can be adopted as the article to be coated.
- Examples thereof include containers such as tubes and bottles, cell culture instruments such as multiwell dishes and petri dishes, and plates such as slide glasses.
- the article to be coated is formed of an arbitrary material such as glass, plastic, or metal.
- the gel reforming conditions are not limited as long as the peptide gel is reformed, and can be appropriately set according to the type and concentration of the peptide. Since the peptide of the present invention has the ability to self-assemble, it can self-assemble and spontaneously re-form a gel by setting to appropriate conditions.
- an article whose surface is coated with the peptide sol may be allowed to stand.
- the standing temperature is preferably 15 ° C. or higher, more preferably 25 to 45 ° C.
- the standing time is preferably 1 minute or longer, more preferably 5 minutes or longer.
- the thickness of the peptide gel re-formed in the gelation step can be, for example, 1 ⁇ m or more, and preferably 1 ⁇ m to 1 cm.
- Example 1 A self-assembling peptide consisting of the amino acid sequence of SEQ ID NO: 1 shown in Table 1 was synthesized by Fmoc solid phase synthesis. Subsequently, the N-terminal was acetylated and the C-terminal was amidated by a conventional method to obtain modified peptide 1 ([CH 3 CO] -RLDLRLALRLLDLR- [NH 2 ]).
- the obtained modified peptide 1 was dissolved in 0.1% by weight sodium hydrogen carbonate solution so as to be 0.2, 0.4, and 0.6 w / v%, respectively, to obtain a peptide solution.
- FIG. 2 shows the results of the compression test.
- FIG. 2 shows the relationship between the load applied to the apparatus and the time when the jig is pressed against the sample. The greater the inclination of the approximate line, the higher the mechanical strength. Therefore, when the absolute value of the slope of the approximate straight line (the amount of change in load per unit time) of the measured value from the beginning of compression (after 8 to 10 seconds) is L, the greater the value of L, the greater the mechanical strength Becomes higher.
- the absolute value L of the load change amount per unit time from the start of compression to about 10 seconds after the compression test of the peptide gel 1 of 0.4 w / v% is 0.0476 g / s.
- the peptide gel 1 When the peptide gel 1 of 0.4 w / v% was sandwiched and carried with tweezers, as shown in FIG. 3 (a), the peptide gel 1 had sufficient strength for sandwiching and excellent operability. It was.
- Example 2 A modified peptide 2 ([CH 3 CO] -RLDLRLLLLRLDLR- [NH 2 ]) was obtained in the same manner as in Example 1 except that the amino acid sequence of SEQ ID NO: 2 was adopted instead of the amino acid sequence of SEQ ID NO: 1. 0.2, 0.4, and 0.6 w / v% peptide gel 2 (liquid phase: DMEM) was formed in the same manner as in Example 1 except that modified peptide 2 was used instead of modified peptide 1. .
- the mechanical strength of the obtained peptide gel was measured in the same manner as in Example 1. The results are shown in FIG. As shown in FIG. 4, the absolute value L of the amount of change in load per unit time in the compression test of 0.4 w / v% peptide gel 2 was 0.0423 g / s.
- the peptide gel 2 When the peptide gel 2 of 0.4 w / v% was sandwiched and carried with tweezers, as shown in FIG. 3 (b), the peptide gel 2 had sufficient strength for sandwiching and excellent operability. It was.
- Example 3 A modified peptide 3 ([CH 3 CO] -RLDLRLALLRLDLRL- [NH 2 ]) was obtained in the same manner as in Example 1 except that the amino acid sequence of SEQ ID NO: 3 was adopted instead of the amino acid sequence of SEQ ID NO: 1. 0.2 and 0.4 w / v% peptide gel 3 (liquid phase: DMEM) was formed in the same manner as in Example 1 except that modified peptide 3 was used instead of modified peptide 1.
- the mechanical strength of the obtained peptide gel was measured in the same manner as in Example 1. The results are shown in FIG. As shown in FIG. 5, the absolute value L of the amount of change in load per unit time in the above compression test of 0.4 w / v% peptide gel 3 was 0.0336 g / s.
- a modified peptide c1 ([CH 3 CO] -RASARADARADARASA- [NH 2 ]) was obtained in the same manner as in Example 1 except that the amino acid sequence of SEQ ID NO: 4 was adopted instead of the amino acid sequence of SEQ ID NO: 1. 0.2, 0.4, and 0.6 w / v% peptide gel c1 (liquid phase: DMEM) was formed in the same manner as in Example 1 except that modified peptide c1 was used instead of modified peptide 1. .
- the mechanical strength of the obtained peptide gel was measured in the same manner as in Example 1. The results are shown in FIG. As shown in FIG. 6, the absolute value L of the amount of change in load per unit time in the above compression test of 0.4 w / v% peptide gel c1 was 0.0143 g / s.
- a modified peptide c2 ([CH 3 CO] -RASARADARASARADA- [NH 2 ]) was obtained in the same manner as in Example 1 except that the amino acid sequence of SEQ ID NO: 5 was adopted instead of the amino acid sequence of SEQ ID NO: 1.
- 0.2 and 0.4 w / v% peptide gel c2 (liquid phase: DMEM) was formed in the same manner as in Example 1 except that modified peptide c2 was used instead of modified peptide 1.
- the mechanical strength of the obtained peptide gel was measured in the same manner as in Example 1. The results are shown in FIG. As shown in FIG. 7, the absolute value L of the load change amount per unit time in the above compression test of 0.4 w / v% peptide gel c2 was 0.0167 g / s.
- Example 4 A modified peptide 4 ([CH 3 CO] -RGDNRLDLRRLRLLDLR- [NH 2 ]) was obtained in the same manner as in Example 1 except that the amino acid sequence of SEQ ID NO: 12 was adopted instead of the amino acid sequence of SEQ ID NO: 1. 0.2, 0.4, and 0.6 w / v% peptide gel 4 (liquid phase: DMEM) was formed in the same manner as in Example 1 except that modified peptide 4 was used instead of modified peptide 1. .
- the mechanical strength of the obtained peptide gel was measured in the same manner as in Example 1. The results are shown in FIG. As shown in FIG. 8, the absolute value L of the amount of change in load per unit time in the compression test of 0.4 w / v% peptide gel 4 was 0.0618 g / s.
- the peptide of the present invention can form a peptide gel having higher mechanical strength than the self-assembled peptide of the comparative example. Moreover, since the peptide gel of this invention has high mechanical strength as shown in FIG. 3, it turns out that it is very excellent in operativity. Furthermore, since the peptide of the present invention can form a peptide gel with sufficient mechanical strength at a low peptide concentration, it is advantageous in terms of cost.
- Example 5 A cell suspension containing mouse myoblasts (L6) at a cell concentration of 2.0 ⁇ 10 6 cells / ml and an aqueous peptide solution containing the modified peptide 1 at 1.0 w / v% in a volume ratio of 3: 2 (cell suspension: aqueous peptide solution).
- the obtained mixture (cell concentration: 1.2 ⁇ 10 6 cells / ml, peptide concentration: 0.4 w / v%) was placed in a cell culture insert (BD Falcon, product number “353096”) at room temperature.
- the peptide gel was formed by leaving still for 1 minute.
- Example 6 Cell culture and DNA quantification were performed in the same manner as in Example 5 except that modified peptide 2 was used instead of modified peptide 1.
- the cell growth rate was 140% after 1 day, 160% after 2 days, 250% after 4 days, and 250% after 4 days, with the passage of days.
- Example 7 The modified peptide 1 was dissolved in a sodium carbonate solution to prepare a 0.5 w / v% aqueous peptide solution (final concentration of sodium carbonate: 2.75 mM).
- the peptide aqueous solution was sterilized at 121 ° C. for 20 minutes using an autoclave apparatus (manufactured by Sanyo Electric Co., Ltd., product number “MLS3020”) to obtain a peptide gel.
- the gel and a cell suspension obtained by suspending mouse NIH3T3 cells in DMEM medium were mixed by pipetting at a volume ratio of 2: 1 (gel: cell suspension).
- each of the obtained cell-gel mixture was added to 5 cell culture inserts (product number “353096” manufactured by BD Falcon), and the cell culture insert was added to 1 mL of 10% calf serum-containing DMEM medium.
- a 24-well plate product number “3820-024” manufactured by AGC Techno Glass Co., Ltd.
- the cell concentration in the cell-gel mixture was 1.45 ⁇ 10 5 cells / 100 ⁇ L.
- cell culture was performed in a 37 ° C. incubator in the presence of 5% CO 2 .
- the cell proliferation rate was measured using the trade name “Cell Counting Kit 8” (manufactured by Dojin Chemical Co., Ltd.). As a result, as shown in FIG. 9, the cell proliferation rate increased with the passage of the culture days.
- the specific procedure for measuring the cell proliferation rate is as follows. That is, 1 mL of the medium in the well was replaced with 1 mL of a new medium, 100 ⁇ L of Cell Counting Kit 8 solution was added, and the cell culture insert was placed in the well and incubated at 37 ° C. for 2 hours. After the incubation, 100 ⁇ L of the medium that had permeated the gel in the cell culture insert was transferred to a 96-well plate, and the absorbance of the medium at 450 nm was measured using a plate reader. The cell proliferation rate in each culture day was determined with the absorbance of the sample after 0 days from the start of culture as 100.
- the peptide gel of the present invention has biocompatibility and can be suitably used as a cell culture substrate.
- Example 8 The modified peptide 1 was dissolved in a sodium carbonate solution to prepare a 0.5 w / v% aqueous peptide solution (final concentration of sodium carbonate: 4.5 mM). The pH of the aqueous peptide solution was in the neutral range. The peptide aqueous solution was sterilized at 121 ° C. for 20 minutes using an autoclave apparatus (manufactured by Sanyo Electric Co., Ltd., product number “MLS3020”).
- Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry MALDI-TOF
- a time-of-flight mass spectrometer manufactured by Bruker, product number “autoflex III”
- Example 3 Sterilization treatment and mass spectrometry (MALDI-TOF-MS) were carried out in the same manner as in Example 8 except that the trade name “PuraMatrix TM ” (manufactured by 3D Matrix) was used instead of the peptide aqueous solution of modified peptide 1. went. The results are shown in FIG.
- the peptide of the present invention is not substantially degraded by sterilization. Therefore, the peptide of the present invention can be sterilized by sterilization.
- FIG. 11 it can be seen that the product name “PuraMatrix TM ” (manufactured by 3D Matrix Co., Ltd.) has undergone peptide degradation by sterilization. This is presumably because the trade name “PuraMatrix TM ” (manufactured by 3D Matrix) is an acidic peptide aqueous solution.
- Example 9 The modified peptide 1 was dissolved in a sodium carbonate solution to prepare a 0.8 w / v% aqueous peptide solution (final concentration of sodium carbonate: 4.5 mM). The obtained peptide aqueous solution was allowed to stand at 22 ° C. for 2 hours to form a peptide gel. The gel was randomly transferred to a glass petri dish ( ⁇ 6 cm). A photograph at this time is shown in FIG. As shown in FIG. 12 (a), the gel contained bubbles and was hard, so that the petri dish could not be uniformly coated.
- the above petri dish was placed in a freezer at ⁇ 20 ° C. together with the gel to freeze the gel.
- a photograph of the frozen gel is shown in FIG.
- the petri dish was taken out of the freezer, the gel was melted while shaking the petri dish at room temperature, and the obtained sol was spread over the entire bottom surface of the petri dish. By allowing the petri dish to stand in that state, the gel was re-formed. Thus, a petri dish having the entire bottom surface uniformly coated with the peptide gel was obtained.
- a photograph of the petri dish is shown in FIG.
- Example 10 A slide glass in which the peptide gel was uniformly coated on the entire surface was obtained in the same manner as in Example 9 except that a slide glass was used instead of the glass petri dish.
- FIGS. 13 (a), (b), and (c) are a photograph of the gel transferred to a slide glass, a photograph of a frozen gel, and a photograph of a slide glass with the entire surface uniformly coated with a peptide gel, respectively. Shown in
- the self-assembling peptide 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.
- SEQ ID NO: 1 is a self-assembling peptide of the present invention.
- SEQ ID NO: 2 is a self-assembling peptide of the present invention.
- SEQ ID NO: 3 is a modified peptide of the present invention.
- SEQ ID NO: 4 is a peptide that is not a self-assembling peptide of the present invention.
- SEQ ID NO: 5 is a peptide that is not a self-assembling peptide of the present invention.
- SEQ ID NO: 6 is a self-assembling peptide of the present invention.
- SEQ ID NO: 7 is a self-assembling peptide of the present invention.
- SEQ ID NO: 8 is a self-assembling peptide of the present invention.
- SEQ ID NO: 9 is a self-assembling peptide of the present invention.
- SEQ ID NO: 10 is a self-assembling peptide of the present invention.
- SEQ ID NO: 11 is a self-assembling peptide of the present invention.
- SEQ ID NO: 12 is a modified peptide of the present invention.
Abstract
Description
アミノ酸配列:a1b1c1b2a2b3db4a3b5c2b6a4
(該アミノ酸配列中、a1~a4は、塩基性アミノ酸残基であり;b1~b6は、非電荷極性アミノ酸残基および/または疎水性アミノ酸残基であり、ただし、そのうちの少なくとも5個は、疎水性アミノ酸残基であり;c1およびc2は、酸性アミノ酸残基であり;dは、疎水性アミノ酸残基である。)
好ましい実施形態においては、上記アミノ酸配列中、b3およびb4が、疎水性アミノ酸残基である。
好ましい実施形態においては、上記アミノ酸配列中、b1~b6がすべて、疎水性アミノ酸残基である。
好ましい実施形態においては、上記アミノ酸配列中、b1~b6が、それぞれ独立してアラニン残基、バリン残基、ロイシン残基、またはイソロイシン残基である。
好ましい実施形態においては、上記アミノ酸配列中、dがアラニン残基、バリン残基、ロイシン残基、またはイソロイシン残基である。
好ましい実施形態においては、上記自己組織化ペプチドは、RLDLRLALRLDLR(配列番号1)、RLDLRLLLRLDLR(配列番号2)、RADLRLALRLDLR(配列番号6)、RLDLRLALRLDAR(配列番号7)、RADLRLLLRLDLR(配列番号8)、RADLRLLLRLDAR(配列番号9)、RLDLRALLRLDLR(配列番号10)、または、RLDLRLLARLDLR(配列番号11)のアミノ酸配列からなるペプチドである。
好ましい実施形態においては、上記自己組織化ペプチドは、RLDLRLALRLDLR(配列番号1)、または、RLDLRLLLRLDLR(配列番号2)のアミノ酸配列からなるペプチドである。
本発明の別の局面によれば、修飾ペプチドが提供される。該修飾ペプチドは、上記自己組織化ペプチドのN末端アミノ基および/またはC末端カルボキシル基が修飾されたペプチドであって、自己組織化能を有する。
好ましい実施形態においては、上記N末端アミノ基および/またはC末端カルボキシル基に、RGDを含むアミノ酸配列が付加されている。
本発明のさらに別の局面によれば、ペプチドゲルが提供される。該ペプチドゲルは、上記自己組織化ペプチドおよび/または上記修飾ペプチドを含む水溶液から形成される。
好ましい実施形態においては、上記水溶液がさらに添加物を含む。
好ましい実施形態においては、上記添加物が、pH調整剤、アミノ酸類、ビタミン類、糖類、多糖類、アルコール類、多価アルコール類、色素、生理活性物質、酵素、抗体、DNA、およびRNAからなる群より選択される少なくとも一つである。
好ましい実施形態においては、上記ペプチドゲルを、22℃の温度条件下で、先端が直径3.2mm、曲率半径1.6mmの球状であるジグを用い、0.05mm/sの圧縮速度で行った圧縮試験において、圧縮開始から8~10秒後までの測定値の近似直線における単位時間当たりの荷重の変化量の絶対値L(g/s)が0.03g/s以上である。
本発明のさらに別の局面によれば、細胞培養用基材が提供される。該細胞培養用基材は、上記自己組織化ペプチド、上記修飾ペプチド、および上記ペプチドゲルからなる群より選択される少なくとも一つを含む。
本発明のさらに別の局面によれば、無菌ペプチドの製造方法が提供される。該無菌ペプチドの製造方法は、上記自己組織化ペプチドおよび/または修飾ペプチドを、加圧条件下、100℃以上で滅菌する工程を含む。
本発明のさらに別の局面によれば、ペプチドゲルでコーティングされた物品の製造方法が提供される。該ペプチドゲルでコーティングされた物品の製造方法は、上記ペプチドゲルを凍結する工程、
該凍結物を融解してペプチドゾルを得る工程、
コーティング対象物品の表面の少なくとも一部を該ペプチドゾルでコーティングする工程、および
該ペプチドゾルからペプチドゲルを再形成する工程を含む。
(1)本明細書において、「自己組織化ペプチド」とは、溶媒中において、ペプチド分子同士の相互作用を介して自発的に集合するペプチドをいう。相互作用としては、特に限定されず、例えば、水素結合、イオン間相互作用、ファンデルワールス力等の静電的相互作用、疎水性相互作用が挙げられる。1つの実施形態において、自己組織化ペプチドは、室温の水溶液(例えば、0.4w/v%のペプチド水溶液)中において、自己組織化してナノファイバーまたはゲルを形成し得る。
(2)本明細書において、「ゲル」とは、粘性的な性質と弾性的な性質とを併せ持つ粘弾性物質をいう。
(3)本明細書において、「親水性アミノ酸」は、アルギニン(Arg/R)、リシン(Lys/K)、ヒスチジン(His/H)等の塩基性アミノ酸、アスパラギン酸(Asp/D)、グルタミン酸(Glu/E)等の酸性アミノ酸、チロシン(Tyr/Y)、セリン(Ser/S)、トレオニン(Thr/T)、アスパラギン(Asn/N)、グルタミン(Gln/Q)、システイン(Cys/C)等の非電荷極性アミノ酸を含む。上記括弧内のアルファベットはそれぞれ、アミノ酸の三文字表記および一文字表記である。
(4)本明細書において、「疎水性アミノ酸」は、アラニン(Ala/A)、ロイシン(Leu/L)、イソロイシン(Ile/I)、バリン(Val/V)、メチオニン(Met/M)、フェニルアラニン(Phe/F)、トリプトファン(Trp/W)、グリシン(Gly/G)、プロリン(Pro/P)等の非極性アミノ酸を含む。上記括弧内のアルファベットはそれぞれ、アミノ酸の三文字表記および一文字表記である。
本発明の自己組織化ペプチドは、下記のアミノ酸配列からなる。
アミノ酸配列:a1b1c1b2a2b3db4a3b5c2b6a4
(上記アミノ酸配列中、a1~a4は、塩基性アミノ酸残基であり;b1~b6は、非電荷極性アミノ酸残基および/または疎水性アミノ酸残基であり、ただし、そのうちの少なくとも5個は、疎水性アミノ酸残基であり;c1およびc2は、酸性アミノ酸残基であり;dは、疎水性アミノ酸残基である。)
n-RLDLRLALRLDLR-c(配列番号1)
n-RLDLRLLLRLDLR-c(配列番号2)
n-RADLRLALRLDLR-c(配列番号6)
n-RLDLRLALRLDAR-c(配列番号7)
n-RADLRLLLRLDLR-c(配列番号8)
n-RADLRLLLRLDAR-c(配列番号9)
n-RLDLRALLRLDLR-c(配列番号10)
n-RLDLRLLARLDLR-c(配列番号11)
本発明の修飾ペプチドは、自己組織化能を有する限りにおいて、上記自己組織化ペプチドに任意の修飾を施したペプチドである。修飾が行われる部位は、上記自己組織化ペプチドのN末端アミノ基であってもよく、C末端カルボキシル基であってもよく、その両方であってもよい。
本発明のペプチドゲルは、上記自己組織化ペプチドおよび/または上記修飾ペプチド(以下、「上記自己組織化ペプチドおよび/または上記修飾ペプチド」を「本発明のペプチド」と称する場合がある)を含む水溶液から形成される。本発明のペプチドが水溶液中で自発的に集合して、ナノメートルスケールの幅を有する繊維状の分子集合体、いわゆるナノファイバーを形成し、該ナノファイバー間に働く静電的相互作用を主因として三次元網状構造を形成することにより、ゲルが形成されると推測される。該水溶液中に含まれる本発明のペプチドは、1種類のみであってもよく、2種類以上であってもよい。該水溶液は、本発明のペプチドおよび水に加えて、任意の適切な添加物をさらに含み得る。また、該水溶液は、細胞等の不溶物を含んでいてもよい。
本発明の自己組織化ペプチド、修飾ペプチド、およびペプチドゲルの好ましい用途としては、例えば、細胞培養用基材;スキンケア用品、ヘアケア用品等の化粧品;じょくそう製剤、骨充填剤、美容形成用注入剤、眼科用手術補助剤、人工硝子体、人工水晶体、関節潤滑剤、点眼剤、DDS基材、止血剤等の医薬品;湿潤用保水剤;乾燥剤;コンタクトレンズ等の医療機器へのコーティング剤が挙げられる。
本発明の細胞培養基材は、上記自己組織化ペプチド、修飾ペプチド、およびペプチドゲルの少なくとも一つを含む。本発明の細胞培養基材は、化学合成によって得られる自己組織化ペプチドおよび/または修飾ペプチドから形成されるので、病原体等が混入することなく、安全な細胞培養が可能である。また、上記本発明のペプチドから形成されたゲルは、中性領域で透明、かつ、力学的強度に優れ得るので、本発明の細胞培養基材は、細胞培養時の視認性および操作性に優れる。
本発明の無菌ペプチドの製造方法は、上記自己組織化ペプチドおよび/または修飾ペプチドを、加圧条件下、100℃以上で滅菌する工程を含む。これらのペプチドは、代表的には、ペプチド水溶液または該ペプチド水溶液から形成されたペプチドゲルの形態で滅菌処理に供される。該ペプチド水溶液のpHは、好ましくは5~9、さらに好ましくは5.5~8、特に好ましくは6.0~7.5である。このようなpHであれば、100℃以上の温度条件で滅菌してもペプチド分解が実質的に生じないので、無菌状態の本発明のペプチドが得られ得る。ペプチド水溶液およびペプチドゲルについては、上記D項に記載のとおりである。
本発明のペプチドゲルでコーティングされた物品の製造方法は、上記ペプチドゲルを凍結する工程(凍結工程)、該凍結物を融解してペプチドゾルを得る工程(融解工程)、コーティング対象物品の表面の少なくとも一部を該ペプチドゾルでコーティングする工程(コーティング工程)、および該ペプチドゾルからペプチドゲルを再形成する工程(ゲル化工程)を含む。該方法は、必要に応じて、任意の工程をさらに含んでもよい。本発明のペプチドゲルを凍結融解することにより、ペプチド分子間の結合が切断されてゲルを構成する三次元網目構造が崩壊するので、ペプチド分子が水溶液中に均一に分散したゾルが得られ得る。この均一性の高いゾルでコーティング対象物品の表面の少なくとも一部をコーティングしてから該ゾルをゲル化することにより、該物品の表面をペプチドゲルで均一にコーティングすることができる。
凍結条件は、ペプチドゲルが凍結する限りにおいて、任意の適切な条件が採用され得る。凍結温度は、ペプチドゲルが凍結する温度以下であればよい。凍結速度にも制限はなく、徐々に冷凍してもよく、急速冷凍してもよい。例えば、ペプチドゲルを-10℃以下の温度条件下に置くことで好適に凍結することができる。
融解温度は、上記凍結工程で得られた凍結物が融解してゾルを形成する温度であれば、任意の適切な温度に設定され得る。一定の温度で融解してもよく、異なる温度で段階的に融解してもよい。融解速度および時間に制限はなく、徐々に融解してもよく、急速に融解してもよい。例えば、5~70℃、好ましくは15~45℃の温度条件下に凍結したペプチドゲルを置くことで好適に融解を行い得る。
コーティングする方法としては、任意の適切な方法が採用され得る。具体例としては、ディスペンサー塗布方式、浸漬方式、バーコータ方式、遠心力によりゾルをコーティング対象物品の表面に展開する方式、コーティング対象物品を傾けることによりゾルを流動させてコーティング対象物品の表面に展開する方式が挙げられる。上記ゾルにおいては、ペプチド分子間の種々の結合が十分に切断されて粘度が著しく低下しており、また、ペプチド分子が十分に分散しているので、コーティング対象物品の表面に均一な層を形成し得る。
ゲルの再形成条件(温度、時間等)は、ペプチドゲルが再形成される限り制限はなく、ペプチドの種類および濃度等に応じて適切に設定され得る。本発明のペプチドは、自己組織化能を有するので、適切な条件に設定することにより、自己集合してゲルを自発的に再形成し得る。
Fmoc固相合成法により、表1に記載の配列番号1のアミノ酸配列からなる自己組織化ペプチドを合成した。次いで、常法により、N末端をアセチル化し、C末端をアミド化して、修飾ペプチド1([CH3CO]-RLDLRLALRLDLR-[NH2])を得た。
22℃の条件下、先端が球状(直径:3.2mm、曲率半径:1.6mm)であるステンレススチール製ジグ(ティー・エイ・インスツルメント社製)を取り付けた粘弾性測定装置(ティー・エイ・インスツルメント社製、製品番号「RSA III」)を用いて、0.05mm/s(s=秒)の速度でゲルを圧縮することにより、力学的強度を測定した。
配列番号1のアミノ酸配列の代わりに配列番号2のアミノ酸配列を採用したこと以外は実施例1と同様にして、修飾ペプチド2([CH3CO]-RLDLRLLLRLDLR-[NH2])を得た。修飾ペプチド1の代わりに修飾ペプチド2を用いたこと以外は実施例1と同様にして0.2、0.4、および0.6w/v%のペプチドゲル2(液相:DMEM)を形成した。
配列番号1のアミノ酸配列の代わりに配列番号3のアミノ酸配列を採用したこと以外は実施例1と同様にして、修飾ペプチド3([CH3CO]-RLDLRLALRLDLRL-[NH2])を得た。修飾ペプチド1の代わりに修飾ペプチド3を用いたこと以外は実施例1と同様にして0.2および0.4w/v%のペプチドゲル3(液相:DMEM)を形成した。
配列番号1のアミノ酸配列の代わりに配列番号4のアミノ酸配列を採用したこと以外は実施例1と同様にして、修飾ペプチドc1([CH3CO]-RASARADARADARASA-[NH2])を得た。修飾ペプチド1の代わりに修飾ペプチドc1を用いたこと以外は実施例1と同様にして0.2、0.4、および0.6w/v%のペプチドゲルc1(液相:DMEM)を形成した。
配列番号1のアミノ酸配列の代わりに配列番号5のアミノ酸配列を採用したこと以外は実施例1と同様にして、修飾ペプチドc2([CH3CO]-RASARADARASARADA-[NH2])を得た。修飾ペプチド1の代わりに修飾ペプチドc2を用いたこと以外は実施例1と同様にして0.2および0.4w/v%のペプチドゲルc2(液相:DMEM)を形成した。
配列番号1のアミノ酸配列の代わりに配列番号12のアミノ酸配列を採用したこと以外は実施例1と同様にして、修飾ペプチド4([CH3CO]-RGDNRLDLRLALRLDLR-[NH2])を得た。修飾ペプチド1の代わりに修飾ペプチド4を用いたこと以外は実施例1と同様にして0.2、0.4、および0.6w/v%のペプチドゲル4(液相:DMEM)を形成した。
マウス筋芽細胞(L6)を2.0×106cells/mlの細胞濃度で含む細胞懸濁液と、上記修飾ペプチド1を1.0w/v%で含むペプチド水溶液とを、容積比3:2(細胞懸濁液:ペプチド水溶液)で混合した。得られた混合物(細胞濃度:1.2×106cells/ml、ペプチド濃度:0.4w/v%)をセルカルチャーインサート(BD Falcon社製、製品番号「353096」)に入れて室温でおよそ1分間静置することにより、ペプチドゲルを形成させた。該ゲルをセルカルチャーインサートごと1mLの10%子牛血清含有DMEM培地が入った組織培養用24ウェルプレート(AGCテクノグラス社製、製品番号「3820-024」)のウェルにセットした。次いで、5%CO2存在下37℃インキュベーターで細胞培養を行った。培地交換は培養開始から2日後に一度だけ行った。培養開始から、1、2、および4日後にゲルを取り出して、商品名「CyQUANT(登録商標) Cell Proliferation Assay Kit *for cells in culture* *1000 assays*」(インビトロジェン社製、製品番号「C7026」)を用いてDNA定量を行い、細胞増殖率を算出した。細胞増殖率は、培養開始直後を100%とした場合に、1日後では150%、2日後では180%、4日後では310%であり、日数の経過に伴って細胞数が増加していた(算出結果はn=3の平均)。
修飾ペプチド1の代わりに修飾ペプチド2を用いたこと以外は実施例5と同様にして、細胞培養およびDNA定量を行った。細胞増殖率を算出したところ、細胞増殖率は、培養開始直後を100%とした場合に、1日後では140%、2日後では160%、4日後では250%であり、日数の経過に伴って細胞数が増加していた(算出結果はn=3の平均)。
上記修飾ペプチド1を炭酸ナトリウム溶液に溶解し、0.5w/v%のペプチド水溶液(炭酸ナトリウムの終濃度:2.75mM)を調製した。該ペプチド水溶液にオートクレーブ装置(三洋電機社製、製品番号「MLS3020」)を用いて、121℃、20分の滅菌処理を行い、ペプチドゲルを得た。該ゲルとマウスNIH3T3細胞をDMEM培地に懸濁した細胞懸濁液とを、容積比2:1(ゲル:細胞懸濁液)でピペッティングにより均一となるように混合した。得られた細胞‐ゲル混合物をセルカルチャーインサート(BD Falcon社製、製品番号「353096」)5個に100μLずつ加え、該セルカルチャーインサートを1mLの10%子牛血清含有DMEM培地が入った組織培養用24ウェルプレート(AGCテクノグラス社製、製品番号「3820-024」)のウェルにセットした。このとき、細胞‐ゲル混合物中の細胞濃度は、1.45×105cell/100μLであった。次いで、5%CO2存在下37℃インキュベーターで細胞培養を行った。培養開始から0日後(2時間後)、1日後、3日後、および5日後に商品名「Cell Counting Kit 8」(同仁化学社製)を使用して細胞増殖率を測定した。その結果、図9に示すとおり、培養日数の経過に伴って細胞増殖率が増加していた。
上記修飾ペプチド1を炭酸ナトリウム溶液に溶解し、0.5w/v%のペプチド水溶液(炭酸ナトリウムの終濃度:4.5mM)を調製した。該ペプチド水溶液のpHは、中性領域であった。該ペプチド水溶液にオートクレーブ装置(三洋電機社製、製品番号「MLS3020」)を用いて121℃、20分の滅菌処理を行った。滅菌処理前後のペプチド水溶液に含まれるペプチド分子の質量を飛行時間型質量分析装置(Bruker社製、製品番号「autoflexIII」)を用い、マトリックス支援レーザー脱離イオン化飛行時間型質量分析法(MALDI-TOF-MS)により調べた。結果を図10に示す。
修飾ペプチド1のペプチド水溶液の代わりに商品名「PuraMatrixTM」(スリー・ディー・マトリックス社製)を用いたこと以外は実施例8と同様にして滅菌処理および質量分析(MALDI-TOF-MS)を行った。結果を図11に示す。
上記修飾ペプチド1を炭酸ナトリウム溶液に溶解し、0.8w/v%のペプチド水溶液(炭酸ナトリウムの終濃度:4.5mM)を調製した。得られたペプチド水溶液を22℃で2時間静置してペプチドゲルを形成した。該ゲルを無造作にガラス製シャーレ(φ6cm)に移した。このときの写真を図12(a)に示す。図12(a)に示されるとおり、ゲルには気泡が入っており、硬いためにシャーレを均一にコーティングすることができなかった。
ガラス製シャーレの代わりにスライドガラスを用いたこと以外は実施例9と同様にして、ペプチドゲルが表面全体に均一にコーティングされたスライドガラスを得た。ゲルをスライドガラスに移した時の写真、凍結したゲルの写真、およびペプチドゲルで表面全体が均一にコーティングされたスライドガラスの写真をそれぞれ、図13(a)、(b)、および(c)に示す。
配列番号2は、本発明の自己組織化ペプチドである。
配列番号3は、本発明の修飾ペプチドである。
配列番号4は、本発明の自己組織化ペプチドではないペプチドである。
配列番号5は、本発明の自己組織化ペプチドではないペプチドである。
配列番号6は、本発明の自己組織化ペプチドである。
配列番号7は、本発明の自己組織化ペプチドである。
配列番号8は、本発明の自己組織化ペプチドである。
配列番号9は、本発明の自己組織化ペプチドである。
配列番号10は、本発明の自己組織化ペプチドである。
配列番号11は、本発明の自己組織化ペプチドである。
配列番号12は、本発明の修飾ペプチドである。
Claims (16)
- 下記のアミノ酸配列からなる自己組織化ペプチド。
アミノ酸配列:a1b1c1b2a2b3db4a3b5c2b6a4
(該アミノ酸配列中、a1~a4は、塩基性アミノ酸残基であり;b1~b6は、非電荷極性アミノ酸残基および/または疎水性アミノ酸残基であり、ただし、そのうちの少なくとも5個は、疎水性アミノ酸残基であり;c1およびc2は、酸性アミノ酸残基であり;dは、疎水性アミノ酸残基である。) - 前記アミノ酸配列中、b3およびb4が、疎水性アミノ酸残基である、請求項1に記載の自己組織化ペプチド。
- 前記アミノ酸配列中、b1~b6がすべて、疎水性アミノ酸残基である、請求項1または2に記載の自己組織化ペプチド。
- 前記アミノ酸配列中、b1~b6が、それぞれ独立してアラニン残基、バリン残基、ロイシン残基、またはイソロイシン残基である、請求項1から3のいずれかに記載の自己組織化ペプチド。
- 前記アミノ酸配列中、dがアラニン残基、バリン残基、ロイシン残基、またはイソロイシン残基である、請求項1から4のいずれかに記載の自己組織化ペプチド。
- RLDLRLALRLDLR(配列番号1)、RLDLRLLLRLDLR(配列番号2)、RADLRLALRLDLR(配列番号6)、RLDLRLALRLDAR(配列番号7)、RADLRLLLRLDLR(配列番号8)、RADLRLLLRLDAR(配列番号9)、RLDLRALLRLDLR(配列番号10)、または、RLDLRLLARLDLR(配列番号11)のアミノ酸配列からなるペプチドである、請求項1から5のいずれかに記載の自己組織化ペプチド。
- RLDLRLALRLDLR(配列番号1)、または、RLDLRLLLRLDLR(配列番号2)のアミノ酸配列からなるペプチドである、請求項1から6のいずれかに記載の自己組織化ペプチド。
- 請求項1から7のいずれかに記載の自己組織化ペプチドのN末端アミノ基および/またはC末端カルボキシル基が修飾されたペプチドであって、自己組織化能を有する修飾ペプチド。
- 前記N末端アミノ基および/またはC末端カルボキシル基に、RGDを含むアミノ酸配列が付加されている、請求項8に記載の自己組織化能を有する修飾ペプチド。
- 請求項1から7のいずれかに記載の自己組織化ペプチドおよび/または請求項8もしくは9に記載の修飾ペプチドを含む水溶液から形成される、ペプチドゲル。
- 前記水溶液がさらに添加物を含む、請求項10に記載のペプチドゲル。
- 前記添加物が、pH調整剤、アミノ酸類、ビタミン類、糖類、多糖類、アルコール類、多価アルコール類、色素、生理活性物質、酵素、抗体、DNA、およびRNAからなる群より選択される少なくとも一つである、請求項11に記載のペプチドゲル。
- 22℃の温度条件下で、先端が直径3.2mm、曲率半径1.6mmの球状であるジグを用い、0.05mm/sの圧縮速度で行った圧縮試験において、圧縮開始から8~10秒後までの測定値の近似直線における単位時間当たりの荷重の変化量の絶対値L(g/s)が0.03g/s以上である、請求項10から12のいずれかに記載のペプチドゲル。
- 請求項1から7のいずれかに記載の自己組織化ペプチド、請求項8または9に記載の修飾ペプチド、および請求項10から13のいずれかに記載のペプチドゲルからなる群より選択される少なくとも一つを含む、細胞培養用基材。
- 請求項1から7のいずれかに記載の自己組織化ペプチドおよび/または請求項8もしくは9に記載の修飾ペプチドを、加圧条件下、100℃以上で滅菌する工程を含む、無菌ペプチドの製造方法。
- 請求項10から13のいずれかに記載のペプチドゲルを凍結する工程、
該凍結物を融解してペプチドゾルを得る工程、
コーティング対象物品の表面の少なくとも一部を該ペプチドゾルでコーティングする工程、および
該ペプチドゾルからペプチドゲルを再形成する工程
を含む、ペプチドゲルでコーティングされた物品の製造方法。
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SG2011064680A SG174857A1 (en) | 2009-03-09 | 2010-02-12 | Self-assembling peptide and peptide gel with high strength |
JP2010521242A JP4620804B2 (ja) | 2009-03-09 | 2010-02-12 | 自己組織化ペプチドおよび高強度ペプチドゲル |
EP10750643.8A EP2407479B2 (en) | 2009-03-09 | 2010-02-12 | Self-assembling peptide and peptide gel with high strength |
US13/255,457 US8729032B2 (en) | 2009-03-09 | 2010-02-12 | Self-assembling peptide and peptide gel with high strength |
CN201080011134.3A CN102348717B (zh) | 2009-03-09 | 2010-02-12 | 自组装肽和高强度肽凝胶 |
KR1020117023114A KR101702677B1 (ko) | 2009-03-09 | 2010-02-12 | 자기 조직화 펩티드 및 고강도 펩티드 겔 |
HK12105222.5A HK1164889A1 (zh) | 2009-03-09 | 2012-05-29 | 自組裝肽和高强度肽凝膠 |
US13/929,279 US20140161753A1 (en) | 2009-03-09 | 2013-06-27 | Self-Assembling Peptide and Peptide Gel with High Strength |
US14/226,341 US8951974B2 (en) | 2009-03-09 | 2014-03-26 | Self-assembling peptide and peptide gel with high strength |
US14/594,503 US20150125611A1 (en) | 2009-03-09 | 2015-01-12 | Self-assembling peptide and peptide gel with high strength |
US14/798,953 US20150315242A1 (en) | 2009-03-09 | 2015-07-14 | Self-assembling peptide and peptide gel with high strength |
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US13/929,279 Continuation US20140161753A1 (en) | 2009-03-09 | 2013-06-27 | Self-Assembling Peptide and Peptide Gel with High Strength |
US14/226,341 Continuation US8951974B2 (en) | 2009-03-09 | 2014-03-26 | Self-assembling peptide and peptide gel with high strength |
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CN (1) | CN102348717B (ja) |
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WO2019142886A1 (ja) | 2018-01-19 | 2019-07-25 | 株式会社メニコン | 免疫原性組成物 |
US11472841B2 (en) | 2018-01-19 | 2022-10-18 | Menicon Co., Ltd. | Immunogenic composition |
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KR20110134440A (ko) | 2011-12-14 |
US20150125611A1 (en) | 2015-05-07 |
JP4620804B2 (ja) | 2011-01-26 |
US8729032B2 (en) | 2014-05-20 |
EP2757108A1 (en) | 2014-07-23 |
EP2407479B2 (en) | 2017-08-23 |
JP2015131845A (ja) | 2015-07-23 |
EP2407479A4 (en) | 2012-07-04 |
KR101702677B1 (ko) | 2017-02-03 |
EP2407479A9 (en) | 2012-06-13 |
US20150315242A1 (en) | 2015-11-05 |
SG174857A1 (en) | 2011-11-28 |
TWI465461B (zh) | 2014-12-21 |
US8951974B2 (en) | 2015-02-10 |
JP2010280719A (ja) | 2010-12-16 |
TW201036990A (en) | 2010-10-16 |
JP6159359B2 (ja) | 2017-07-05 |
US20120058066A1 (en) | 2012-03-08 |
JPWO2010103887A1 (ja) | 2012-09-13 |
HK1164889A1 (zh) | 2012-09-28 |
EP2757108B1 (en) | 2016-08-31 |
CN102348717A (zh) | 2012-02-08 |
EP2407479B1 (en) | 2014-04-16 |
EP2407479A1 (en) | 2012-01-18 |
US20140161753A1 (en) | 2014-06-12 |
JP5727737B2 (ja) | 2015-06-03 |
CN102348717B (zh) | 2015-11-25 |
US20140286888A1 (en) | 2014-09-25 |
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