WO2016208673A1 - コラーゲン様構造を有する重合ペプチド及びゲル - Google Patents
コラーゲン様構造を有する重合ペプチド及びゲル Download PDFInfo
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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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
- A61L15/325—Collagen
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
Definitions
- the present invention relates to a polymerized peptide composed of a collagen-like peptide, a gel containing the polymerized peptide, a polymerized peptide thin film, and a production method and use thereof.
- Collagen has a triple helical structure in which the three chains of a peptide having a primary structure amino acid sequence (Xaa and Yaa represent any amino acid residue) consisting of repeating the basic unit of-(Xaa-Yaa-Gly)- It is a general term for proteins that form, and is a major component of the extracellular matrix that exists between cells of animal tissues. It is abundant in animal connective tissue and constitutes the skeletal structure of the tissue. There are 28 reported human collagen proteins. On the other hand, a motif having physiological activity is incorporated in the amino acid sequence of the primary structure.
- Non-patent Document 1 when tissue collagen is exposed to blood during tissue damage such as a wound, the integrin ⁇ 2 ⁇ 1 receptor and glycoprotein VI (GPVI) Activating the blood coagulation system triggered by the binding of platelets in the blood having a receptor to the wound site as a trigger, and has activities such as hemostasis and promotion of wound healing (Non-patent Document 1).
- GPVI glycoprotein VI
- gelatin produced by heating and extracting collagen collected from livestock, fish, etc. with water by utilizing the strength and physiological activity of collagen is used as a capsule in medicines and medical materials that apply hemostasis and pressure ulcers. It is widely used as a raw material for cosmetics, as a cosmetic raw material, and a food material.
- Natural collagen has not only an action on platelets via the integrin ⁇ 2 ⁇ 1 receptor and the like, but also various activities such as adhesion of cells having discoidin receptor, syndecan receptor and fibronectin (Non-patent Document 1). ). Therefore, with natural collagen, it is substantially difficult to obtain and use collagen having an activity selective to a desired physiological activity.
- Patent Documents 1 and 2). focusing on these characteristics of collagen, non-natural collagen-like peptides having a collagen-like structure and activity are produced, and application to research materials and medical materials has been attempted.
- Collagen is useful as a medical material, but its use on humans is at risk of allergies and zoonotic diseases. Therefore, the use of collagen-like peptides such as artificial collagen or artificial gelatin as medical materials has been studied. However, conventional collagen-like peptides have not always been sufficient in terms of physical strength, properties such as denaturation upon heating and inability to be processed into sheets.
- the present invention provides medical materials and research materials that can imitate or newly impart the structure / physical properties and physiological functions of collagen and ensure safety by making them completely artificial products. It is produced as a polymerized peptide by oxidative crosslinking of a collagen-like peptide having a triple helical structure consisting of three chains of peptide chains containing a plurality of cysteine (Cys) residues.
- the material containing this polymerized peptide can be used after being processed into a hydrogel and a sheet, and by incorporating a functional amino acid sequence present on a biopolymer such as an integrin binding sequence, a specific physiological function such as cell adhesion Can be granted.
- the present invention provides a method for producing the hydrogel and the sheet-like processed product, and uses as a composition for cell culture, wound covering, regenerative medical material, research material and the like using these.
- the present invention is a polymerized peptide, Having a triple-stranded peptide having a triple helical structure as a repeating unit, polymerized by oxidative crosslinking, Each peptide chain constituting the three-chain peptide may be the same or different from each other, Each peptide chain consists of a triple helix-forming peptide group having a repeating structure of at least 5 times with-(Xaa-Yaa-Gly)-as the basic unit, and at least 2 residues within 10 residues from each of the amino terminus and carboxy terminus.
- Xaa and Yaa are each independently a proline (Pro or P) residue, a hydroxyproline (Hyp or O) residue, an arginine (Arg or R) residue, a lysine (Lys or K) residue, a valine ( Val or V) residue, leucine (Leu or L) residue, isoleucine (Ile or I) residue, serine (Ser or S) residue, threonine (Thr or T) residue, alanine (Ala or A) residue Group, glycine (Gly or G) residue, phenylalanine (Phe or F) residue, methionine (Met or M) residue, glutamic acid (Glu or E) residue, aspartic acid (Asp or D) residue, asparagine ( Asn or N) residue, glutamine (Gln or Q)
- the peptide chain is (i) a peptide chain comprising at least one triple helix-forming peptide group and at least one cross-linking peptide group, (ii) a peptide chain comprising at least one peptide group for forming a triple helix and at least one peptide group for forming a cross-linking, and at least one peptide group having a physiologically active motif; and (iii) a peptide comprising at least one peptide group for forming a triple helix and a peptide group for forming a bridge, and at least one peptide group in which a side chain of an amino acid residue is bound to a motif having physiological activity via a linker
- the peptide chain may be at least one selected from the group consisting of chains.
- the polymerized peptide of the present invention may be a polymerized peptide having a structural unit of a trimeric peptide formed from three peptide chains represented by the following formula (I) and oxidatively crosslinked.
- a 1 , A 2 and A 3 may be the same or different and are each independently a peptide chain represented by the following formula (II):
- a 1 , A 2 and A 3 form a trimer having a triple helical structure, and may be cross-linked by a disulfide bond by a cysteine (Cys) residue contained in each peptide chain,
- the trimer is polymerized by oxidative crosslinking at the Cys disulfide bond.
- R 1 and R 4 each have an amino terminus and a carboxy terminus, each independently a peptide group consisting of any 2 to 10 amino acid residues including at least 2 Cys residues
- Z is (i) a peptide group consisting only of a repeating structure having-(Xaa-Yaa-Gly)-as a basic unit; (ii) a peptide group having a structure having a repeating unit of-(Xaa-Yaa-Gly)-and a physiologically active motif, or (iii) It has a structure repeating as a basic unit of-(Xaa-Yaa-Gly)-, and has physiological activity through a linker in the side chain of at least one amino acid residue contained in the peptide chain
- At least one selected from R 2 and R 3 are each independently a peptide group containing a structure that repeats continuously in units of-(Xaa-Yaa-
- the peptide chain may be represented by the following formula (III).
- R 5 and R 6 are each an amino terminus and a carboxy terminus, each independently being a peptide group consisting of any 2 to 10 amino acid residues including at least 2 Cys residues, p, q, and r are all integers of 0 or more, the sum of p, q, and r is 3 or more, and s is an integer of 1 or more.
- the physiological activity may be a specific binding activity for a biopolymer.
- the binding motif of the ligand having specific binding activity to the biopolymer is a binding motif for a collagen receptor selected from integrin, discoidin domain receptor (DDR) or heparan sulfate proteoglycan (HSPG), or derived from fibronectin.
- DDR discoidin domain receptor
- HSPG heparan sulfate proteoglycan
- the binding motif for the integrin is -Gly-Phe-Hyp-Gly-Glu-Arg-
- the binding motif for the discoidin domain receptor is -Gly-Val-Met-Gly-Phe-Hyp-
- the binding motif amino acid for heparan sulfate proteoglycan The sequence is -Lys-Gly-His-Arg-Gly-Phe-
- the binding motif for fibronectin-derived integrin ⁇ v ⁇ 3 may be -Arg-Gly-Asp-.
- the peptide chain may be selected from peptides having an amino acid sequence selected from SEQ ID NOs: 1-7, 10-18, 23 and 24.
- the peptide chain is an amino acid in which one or several amino acids are deleted, substituted or added to the amino acid sequence of a peptide having an amino acid sequence selected from SEQ ID NOs: 1 to 7, 10 to 18, 23 and 24 May consist of an array.
- the peptide chain may be selected from peptides having an amino acid sequence selected from SEQ ID NOs: 1, 2, 5-7, 10-18 and 23.
- polymerized peptide In the invention of the polymerized peptide, one or several amino acids are deleted, substituted or added to the amino acid sequence of the peptide having an amino acid sequence selected from SEQ ID NOs: 1, 2, 5-7, 10-18 and 23. May consist of different amino acid sequences.
- the peptide chain having the physiologically active motif is the side chain of the Lys residue of the peptide group, with the integrin-binding motif derived from fibronectin using Bis (NHS) PEG 5 (bis (succinimidyl) penta (ethylene glycol)) as a linker. In some cases.
- polymerized peptide In the invention of the polymerized peptide, There is a case of a copolymerized peptide produced by combining two or more of the motifs having physiological activity.
- the present invention also provides a method for producing the polymerized peptide, -(Xaa-Yaa-Gly)-as a basic unit, which has a repeating structure of at least 5 times and contains at least 2 cysteine (Cys) residues within 10 residues from each of the amino terminus and carboxy terminus
- a step of dissolving a peptide chain which may be different, in a solvent at a temperature higher than the denaturation temperature, Forming a triple-stranded peptide having a triple helical structure consisting of three of the peptides by self-assembly by cooling;
- a manufacturing method is provided.
- Xaa and Yaa are each independently a proline (Pro or P) residue, a hydroxyproline (Hyp or O) residue, an arginine (Arg or R) residue, or a lysine (Lys or K) residue.
- the proline residue may be modified with an amino group or a fluorine atom, and an N-isobutyl group glycine residue may be used at the Xaa position and the
- the present invention provides a polymerized peptide produced by the production method.
- the present invention provides a gelling agent comprising at least one of the above-mentioned polymerized peptides.
- the present invention provides a gel containing at least one of the polymerized peptides.
- the gel of the present invention may be a gel containing one, two, three, four or five of the polymerized peptides.
- the gel of the present invention may be a gel containing a polymerized peptide in which at least two kinds of three-chain peptides are combined with the three-chain peptide as a polymerization unit.
- the gel of the present invention may be a gel containing a polymerized peptide that is a combination of two, three, four, or five of the three-chain peptides.
- the gel of the present invention may be a gel further containing at least one of three-chain peptides not containing a cysteine residue.
- the gel of the present invention may be a gel produced by oxidative polymerization of at least one of three-chain peptides.
- the gel of the present invention may be a gel produced by oxidative polymerization of one, two, three, four, or five types of three-chain peptides.
- the gel of the present invention comprises a mixture of a three-chain peptide composed of a peptide chain containing a cysteine residue and a three-chain peptide composed of a peptide chain not containing a cysteine residue, followed by oxidative polymerization. It may be a manufactured gel.
- each peptide chain constituting the three-chain peptide includes a peptide chain having a different number of cysteine residues for controlling the stiffness of the gel and a peptide chain having a bioactive motif. It may be a multifunctional gel.
- the peptide chain for controlling the hardness of the gel may be a peptide chain in which the number of cysteine residues contained in the vicinity of the N-terminal and C-terminal of the peptide chain is different.
- the number of the cysteine residues in the peptide chain for controlling the hardness of the gel is selected from 1, 2 or 3 near the N-terminal and C-terminal, respectively, near the N-terminal and the C-terminal. , May be the same or different.
- the gel is a hydrogel and may be used as a substrate for culturing cells selectively.
- the present invention provides a polymerized peptide thin film produced by drying the gel.
- the present invention also provides a regenerative medical material comprising the polymerized peptide thin film.
- the regenerative medical material may be a composition for promoting wound healing.
- the schematic diagram of the polymeric peptide of the collagenous peptide of this invention, the gel containing this, and the polymeric peptide thin film is represented.
- Chromatogram of synthesized peptide by reversed-phase high performance liquid chromatography (RP-HPLC) analysis Chromatogram of CC2-GPOGPR (short). Chromatogram of synthesized peptide by reversed-phase high performance liquid chromatography (RP-HPLC) analysis: C2-GPOGPR) (short) chromatogram. Chromatogram of synthesized peptide by reversed-phase high performance liquid chromatography (RP-HPLC) analysis: Soluble GPOGPR short (short) chromatogram. Results of CD spectrum measurement of the synthesized peptide chain at 4, 37, and 80 ° C .: This shows the CD spectrum of YCC2.
- results of CD spectrum measurement of the synthesized peptide chain at 4, 37 and 80 ° C . CD2 CD spectrum is shown. Results of CD spectrum measurement of the synthesized peptide chain at 4, 37 and 80 ° C .: The CD spectrum of CC1 is represented. Results of CD spectrum measurement of the synthesized peptide chain at 4, 37, and 80 ° C .: represents the CD spectrum of CCC2. Results of CD spectrum measurement of the synthesized peptide chain at 4, 37 and 80 ° C .: Shows the CD spectrum of CCCC2. Results of CD spectrum measurement of the synthesized peptide chain at 4, 37 and 80 ° C .: The CD spectrum of YCC2-Scr is shown.
- YCC2 ⁇ 225 is the result of changing the temperature. This shows the result of measuring ⁇ 225 of YCC2-Scr while changing the temperature. It represents the result of quantifying the amount of SH of each peptide over time in the polymerization reaction of CCCC2, CCC2, CC2, CC1, YCC2 and YCC2-Scr.
- the comparison result of the gel formation ability of each peptide in the hydrogel formed with YCC2, CC2, CC1, C2, CCC2, CCCC2, YCC2-Scr and Y2 is represented.
- Fig. 4 represents the thermal stability measurements of hydrogels in the hydrogels formed with CCCC2, CCC2, CC2 and YCC2.
- the produced polymerized peptide thin film is represented. Left: Inner diameter 6 mm, Peptide amount 1 mg, Support PVDF membrane Inside: Inner diameter 10 mm, Peptide amount 3 mg, Support nylon mesh Right: Inner diameter 6 mm, Peptide amount 1 mg, Support PVDF membrane
- the external view at the time of rehydrating a polymeric peptide thin film is represented. Measurement results of CD spectrum at 4, 37 and 80 ° C.
- CD spectrum of YCC2-GFOGER CD spectrum of YCC2-GFOGER. Measurement results of CD spectrum of synthesized peptide with bioactive motif at 4, 37 and 80 ° C .: CD spectrum of Soluble-GFOGER. Measurement results of CD spectrum at 4, 37 and 80 ° C. of the peptide incorporating the synthesized bioactive motif: The CD spectrum of YCC2-GVMGFO at 4, 37 and 80 ° C. is shown. Measurement results of CD spectra of synthesized peptides incorporating bioactive motifs at 4, 37 and 80 ° C .: CD spectra of Soluble-GVMGFO. Measurement result of CD spectrum at 4, 37 and 80 ° C.
- FIG. 5 shows the results of comparison of human fibroblast adhesion to type I collagen (Collagen I; positive control) on a gel formed with a polymerized peptide of a collagen-like peptide having a GFOGER sequence motif (Pol-GFOGER).
- Collagen I Collagen I; positive control
- GPOGPR motifs of FAK phosphorylation signals via integrins when using gels (Pol-GFOGER) formed from polymerized peptides of collagen-like peptides with GFOGER sequence motifs
- the result of Western blot is shown.
- Collagen I Collagen I
- GPOGPR motif GPOGPR motif of FAK phosphorylation signaling via integrin when using gel
- Poly-GFOGER polymerized peptide of collagen-like peptide with GFOGER sequence motif Comparison with: represents the result of comparing the effects of FAK on phosphorylation. Comparison of changes in the number of adherent cells with changes in the amount of binding motifs in the polymerized peptide (Pol-GFOGER) of a collagen-like peptide having a GFOGER sequence motif compared to collagen I (Collagen I) and GPOGPR motif (Pol-GPOGPR) A microscopic observation diagram showing the results is shown.
- FIG. 2 shows a microscopic observation of the adhesion of human fibroblasts compared to type I collagen (Collagen I), soluble GFOGER (Soluble GFOGER), soluble GVMGFO (Soluble GVMGFO) and soluble KGHRGF (Soluble KGHRGF).
- the observation figure in a microscope showing the evaluation result of the inhibitory effect of EDTA and heparin with respect to the human fibroblast adhesion of the polymerized peptide (Each Pol-GFOGER and Pol-KGHRGF) incorporating GFOGER and KGHRGF is shown.
- the result of having measured the cell number about the inhibitory effect of EDTA and heparin shown by FIG. 15B is represented.
- the observation figure in a microscope showing the examination result of the cell adhesion of the gel by the polymerization peptide which has various content ratios of the some binding motif which combined GFOGER sequence motif (GFO) and GVMGFO sequence motif (GVM) is shown.
- the photograph figure showing the result of the cell adhesion assay using Using a type I collagen as a positive control and a polymer gel containing no CCC2-GFOGER (short) as a negative control, adhesion and aggregation of human skin fibroblasts to the gel were observed with a fluorescence microscope.
- Human skin fibroblast cells that adhere to the gel prepared by changing the composition ratio of the two components to 9: 1, 7: 3, 5: 5, 3: 7, and 1: 9 Aggregation was evaluated.
- FIG. 6 is a photographic view of the dorsal subcutaneous tissue 10 days after transplanted with a collagen-like polymerized peptide thin film under the back of a mouse and stained with hematoxylin-eosin and observed with a microscope.
- FIG. 4 is a photograph of a photograph obtained by transplanting a collagen-like polymerized peptide thin film subcutaneously on the back of a mouse and observing the subcutaneous tissue on the back 30 days after staining with hematoxylin-eosin. PVDF was used as a support for the purpose of confirming the implantation site because it did not show biodegradability.
- polymerized peptide of collagen-like peptide The three-chain peptide according to the present invention is generally also referred to as a collagen-like peptide.
- the “collagen-like peptide” is a non-natural peptide or polypeptide having a repeating structure having a basic unit of — (Xaa-Yaa-Gly) — as in natural collagen. Three chains were self-assembled in a solvent to form a helical structure, thereby forming a three-chain peptide, or a three-chain peptide having the helical structure was further crosslinked. Refers to a peptide or polypeptide.
- Xaa and Yaa are each independently a proline (Pro or P) residue, a hydroxyproline (Hyp or O) residue, an arginine (Arg or R) residue, a lysine (Lys or K) residue, Valine (Val or V) residue, leucine (Leu or L) residue, isoleucine (Ile or I) residue, serine (Ser or S) residue, threonine (Thr or T) residue, alanine (Ala or Ala ) Residue, glycine (Gly or G) residue, phenylalanine (Phe or F) residue, methionine (Met or M) residue, glutamic acid (Glu or E) residue, aspartic acid (Asp or D) residue, Selected from asparagine (Asn or N) residue, glutamine (Gln or Q) residue, histidine (His or H) residue, tryptophan (Trp or W) residue or tyrosine (
- one of the embodiments of the present invention is a polymerized peptide obtained by polymerizing a three-chain peptide, that is, a collagen-like peptide (see FIG. 1).
- the polymerized peptide of the present collagen-like peptide has the following configuration.
- a polymerized peptide comprising: Having a triple-stranded peptide having a triple helical structure as a repeating unit, polymerized by oxidative crosslinking, Each peptide chain constituting the three-chain peptide may be the same or different from each other, and has a repeating structure of at least 5 times with-(Xaa-Yaa-Gly)-as a basic unit, A polymerized peptide comprising at least two cysteine (Cys) residues within 10 residues from each of an amino terminus and a carboxy terminus.
- [Xaa and Yaa are each independently a proline (Pro or P) residue, a hydroxyproline (Hyp or O) residue, an arginine (Arg or R) residue, a lysine (Lys or K) residue, a valine ( Val or V) residue, leucine (Leu or L) residue, isoleucine (Ile or I) residue, serine (Ser or S) residue, threonine (Thr or T) residue, alanine (Ala or A) residue Group, glycine (Gly or G) residue, phenylalanine (Phe or F) residue, methionine (Met or M) residue, glutamic acid (Glu or E) residue, aspartic acid (Asp or D) residue, asparagine ( Asn or N) residue, glutamine (Gln or Q) residue, histidine (His or H) residue, tryptophan (Trp or W) residue or tyrosine (Tyr or Y)
- the “peptide chain” is a peptide or polypeptide having a primary structure necessary for forming a triple helical structure by three peptide chains, and includes a Cys residue for forming an oxidative bridge. It may be expressed as “single-chain peptide”.
- the number of Cys residues contained within 10 residues from the N-terminus and C-terminus of the peptide chain may be independently the same or different, and may be 2 or more, 3 residues As described above, it may be 4 residues or more and 5 residues or more.
- the peptide groups from each of the amino terminus and carboxy terminus containing at least 2 cysteine (Cys) residues are within 9 residues, within 8 residues, other than within 10 residues. It may be within 7 residues, within 6 residues, within 5 residues, within 4 residues or within 3 residues.
- polymerized peptide refers to a peptide obtained by polymerizing a “collagen-like peptide” via a disulfide bond formed by, for example, oxidative crosslinking between cysteine residues contained in the collagen peptide. .
- the degree of polymerization of the polymerized peptide of the present invention is 2 or more, and is not particularly limited as long as it is a degree of polymerization that can form a gel containing the polymerized peptide of the present invention, preferably a hydrogel, but the average degree of polymerization is less than 100, 100 It may be ⁇ 500, 500 ⁇ 1000, 1000 ⁇ 5000, 5000 ⁇ 10000 or 10000 or more.
- the repetitive structure having-(Xaa-Yaa-Gly)-as a basic unit in a collagen-like peptide has a different minimum number of repeats depending on the amino acid residue constituting it.
- 4-fluoroproline is contained in this basic unit, it is known that a stable triple helical structure can be formed if it is a repeating unit of 5 or more times (Sakakibara S. (1973) Biochem. Biophys. Acta, 303, 198-202).
- the collagen-like peptide forms a stable triple helical structure.
- the collagen-like peptide forms a more stable triple helical structure.
- the collagen-like peptide forms a more stable triple helical structure.
- the collagen-like peptide forms a more stable triple helical structure.
- the collagen-like peptide forms a more stable triple helical structure.
- the collagen-like peptide forms a more stable triple helical structure.
- the collagen-like peptide forms a more stable triple helical structure.
- the collagen-like peptide forms a more stable triple helical structure.
- the number of repetitions having-(Xaa-Yaa-Gly)-as the basic unit is 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, or 10 times or more There are cases.
- an amino acid is L-form.
- the amino acids herein include 20 kinds of L-amino acids known to be used for translation of proteins generally used in molecular biology, as well as modified amino acid residues well known in the art, such as 4-amino acids.
- hydroxylproline is 3-hydroxyproline or 4-hydroxy-L-proline, and is represented by “Hyp” in three letter code and “O” in one letter code.
- the homotrimer refers to a trimer composed of three peptides having the same primary structure.
- a heterotrimer is a trimer consisting of two peptides with the same primary structure and one peptide with a primary structure different from the peptide, or three peptides with different primary structures. Is a trimer.
- the denaturation temperature refers to a temperature at which half of the triple helical structure of the collagenous peptide of the present specification is transferred to a random coil.
- the denaturation temperature is measured by a known method such as a higher-order structure analysis by circular dichroism spectrum (CD spectrum) measurement (Patent Documents 1 and 2), but also by other known methods not limited thereto. It can be measured.
- the collagen-like peptide according to the present invention can be produced by a known peptide chemical synthesis method using a commercially available amino acid and not limited thereto (Patent Document 1).
- a nucleic acid sequence encoding a desired amino acid sequence is produced, this is incorporated into an expression vector, a recombinant expression vector is prepared by a known method, and introduced into an appropriate host such as a microorganism such as Escherichia coli to transform. Is made. Since the recombinant peptide chain is produced by culturing the obtained transformant in an appropriate medium, the recombinant peptide chain used in the present invention is recovered by recovering the recombinant peptide chain produced from the culture. Can be prepared.
- Patent Document 2 EP1014176A2, US6992172, WO2004 / 85473, WO2008 / 103041, etc.
- Such a peptide chain can be obtained by separation and purification using a separation means such as high performance liquid chromatography and used for the production of a three-chain peptide.
- this peptide chain may form a triple-stranded peptide having a triple helical structure by self-assembly in an aqueous solvent at the time of manufacture or purification.
- a collagen-like peptide is produced by incorporating a plurality of cysteine residues into the peptide chain, and is not limited thereto, but is oxidized and crosslinked with an oxidizing agent such as dimethyl sulfoxide (DMSO) or air oxidation.
- DMSO dimethyl sulfoxide
- Polymerized peptides are provided in which the peptides are cross-linked with disulfide bonds.
- a polymerized peptide of a collagen-like peptide produced by this method has improved strength as compared with known collagen-like peptides, and can impart properties that do not denature even when heated in an aqueous solution.
- the oxidizing agent for producing the polymerized peptide of the present invention includes oxygen, iodine, hydrogen peroxide, sodium bromate (sodium bromate), potassium bromate, sodium perborate or perboric acid. Although potassium etc. are mentioned, it is not limited to these.
- the confirmation of the formation of a cross-link by the disulfide bond of the present invention can be confirmed, for example, by quantifying the residual thiol group after the oxidative cross-linking reaction with an Elman reagent.
- a polymerized peptide thin film with improved strength as described below is provided.
- a polymer of collagen-like peptide that does not impart physiological activity or has been imparted by incorporating a motif having physiological activity into the peptide chain and / or binding a peptide having a motif having physiological activity to the side chain of the peptide chain A polymerized peptide having the physiological activity, a gel containing the polymerized peptide, and a thin film containing the polymerized peptide can be produced.
- a motif to be incorporated into a collagen-like peptide a polymerized peptide having a physiological activity selective to a desired physiological activity can be imparted, unlike natural collagen.
- the polymer of the collagen-like peptide which does not provide bioactivity can be manufactured by not incorporating the peptide which has these bioactivity.
- the polymerized peptide of this embodiment may be described as the following configuration.
- the peptide chain is (i) a peptide chain comprising at least one triple helix-forming peptide group and at least one cross-linking peptide group, (ii) a peptide chain comprising at least one peptide group for forming a triple helix and at least one peptide group for forming a cross-linking, and at least one peptide group having a physiologically active motif; and (iii) a peptide comprising at least one peptide group for forming a triple helix and a peptide group for forming a bridge, and at least one peptide group in which a side chain of an amino acid residue is bound to a motif having physiological activity via a linker
- the configuration of the polymerized peptide of this embodiment may be described as the following configuration.
- the polymerized peptide comprising: A polymerized peptide comprising a structural unit of a trimeric peptide formed from three peptide chains represented by the following formula (I), which is oxidized and crosslinked.
- a 1 , A 2 and A 3 may be the same or different and are each independently a peptide chain represented by the following formula (II):
- a 1 , A 2 and A 3 form a trimer having a triple helical structure, and may be cross-linked by a disulfide bond by a cysteine (Cys) residue contained in each peptide chain,
- the trimer is polymerized by oxidative crosslinking at the Cys disulfide bond.
- R 1 and R 4 are each an amino terminus and a carboxy terminus, each independently being a peptide group consisting of any 2 to 10 amino acid residues including at least 2 Cys residues
- Z is (i) a peptide group consisting only of a repeating structure having-(Xaa-Yaa-Gly)-as a basic unit; (ii) a peptide group having a structure having a repeating unit of-(Xaa-Yaa-Gly)-and a physiologically active motif, and (iii) It has a structure repeating as a basic unit of-(Xaa-Yaa-Gly)-, and has physiological activity via a linker on the side chain of at least one amino acid residue contained in the peptide chain
- a peptide group formed by binding a motif sequence At least one selected from the group consisting of R 2 and R 3 are each independently a peptide group containing a structure that repeats continuously in units of-(Xa
- motif refers to a small structural portion found in the amino acid sequences of various proteins, and refers to amino acid sequences that are very well conserved locally in natural proteins. It refers to the amino acid sequence of a motif involved in the functional expression of a protein, and is sometimes referred to as “motif sequence” or “sequence motif”.
- Polymerized peptide of a collagen-like peptide that does not impart a specific physiological activity is a polymer of the collagen-like peptide that is a polymerized peptide that does not impart a specific physiological activity.
- the polymerized peptide that does not impart the specific physiological activity is constituted by a peptide chain composed of a repeating sequence having-(Xaa-Yaa-Gly)-as a basic unit and a peptide group containing a plurality of Cys residues.
- a collagen-like peptide having a triple helix structure are formed by forming a collagen-like peptide having a triple helix structure, and the collagen-like peptide is cross-linked and polymerized by a disulfide bond, and more specifically has the structure described below.
- the collagen-like peptide constituting the polymerized peptide of the present invention may be a homotrimer or a heterotrimer.
- a polymer peptide thin film can be used as a medical material for preventing adhesion of an organ and a suture material from a gel containing the polymerized peptide of the present invention or a place where a specific physiological activity is not imparted.
- the gel or polymerized peptide thin film containing the polymerized peptide of the present invention is used as a medical material for preventing adhesion of an organ, for example, it is placed between the organ and the skin at the time of surgery or between the organ and the organ. Thus, adhesion between the organ and the skin or other organs can be prevented. Since the placed medical material of the present invention is gradually solubilized or degraded by phagocytic cells such as peptidases and macrophages present in the living body and disappears, no re-operation is required to remove the medical material. The burden on the patient's prognosis is small and the quality of life (QOL) is improved.
- QOL quality of life
- the medical material of the present invention is an artificially produced collagen-like peptide, unlike natural collagen, there is little risk of virus infection or microbial infection. Further, since it is cross-linked with a disulfide bond and does not denature even when heat-treated, it can be heated for sterilization, for example.
- Collagen-like peptide polymerization peptide consisting of a peptide chain incorporating a biologically active motif Incorporating a biologically active motif into the peptide chain or binding to the single-chain side chain via a linker
- a polymerized peptide of a collagen-like peptide having a desired physiological activity, a regulated activity intensity, and / or a combination of a plurality of physiological activities can be produced.
- a collagen-like peptide polymer incorporating a bioactive motif in a peptide chain has the following constitution.
- Polymer of collagen-like peptide”, The peptide chain is A peptide chain comprising at least one peptide group for forming a triple helix, at least one peptide group for forming a crosslink, and at least one peptide group having a physiologically active motif.
- the peptide chain may be represented by the following formula (III).
- R 5 and R 6 are each an amino terminus and a carboxy terminus, each independently being a peptide group consisting of any 2 to 10 amino acid residues including at least 2 Cys residues, p, q and r are all integers of 0 or more, the sum of p, q and r is 3 or more, and s is an integer of 1 or more.
- the collagen-like peptide constituting the polymerized peptide of the present invention may be a homotrimer or a heterotrimer.
- the physiological activity may be a specific binding activity to a biopolymer.
- biopolymer refers to proteins, polypeptides, peptides, nucleic acids, and fragments thereof possessed by mammals including humans.
- Specific binding activity to a biopolymer includes, but is not limited to, adhesion or binding activity of various cells to a collagen receptor and adhesion or binding activity of various cells to fibronectin.
- a specific motif possessed by a ligand that selectively binds to various receptors present on cell membranes is known as a specific binding activity for biopolymers.
- the binding motif for example, by incorporating a binding motif for a collagen receptor selected from integrin, discoidin domain receptor (DDR) or heparan sulfate proteoglycan (HSPG) at the time of production of the peptide chain, the binding motif can be selectively used.
- DDR discoidin domain receptor
- HSPG heparan sulfate proteoglycan
- a receptor-selective cell adhesiveness having binding activity is imparted, and a research material or a medical material can be provided.
- VWF motif other than the above, VWF motif, SPARC / BM-40 / osteonectin motif, aegyptin motif, LAIR-1 motif, GPVI motif, PEDF motif, Hsp47 motif, HLA motif, HLA super motif, zinc finger C2H2 type motif , Cytochrome b (N-terminal) / b6 / petB motif, immunoglobulin domain motif, WD domain G- ⁇ repeat motif, PDZ domain motif, leucine rich repeat motif, protein kinase domain motif, PH domain motif, EGF-like domain motif, Reverse transcriptase (RNA-dependent DNA polymerase) motif, Ank repeat motif, NADH-ubiquinone / plastoquinone (complex I) motif, EF hand motif, retroviral aspartyl protease motif, 7-transmembrane receptor (rhodopsin family) Mochi
- cell adhesion motif examples include, for example, RGD sequence, LDV sequence, REDV sequence, YIGSR sequence, PDSGR sequence, RYVVLPR sequence, LGTIPG sequence, RNIAEIIKDI sequence, IKVAV sequence, LRE sequence, DGEA sequence, and HAV sequence. Each sequence can be mentioned.
- the target cell for the physiological activity is a cell having a receptor that binds the motif as a binding ligand.
- Specific examples include cells such as fibroblasts, hepatocytes, undifferentiated chondrocytes, muscle cells, platelets, neutrophils, macrophages, Schwann cells, keratinocytes, or epithelial cells.
- motifs other than the above for example, recorded in PROSITE (http://www.expasy.ch/prosite/) and PRINTS (http://bioinf.man.ac.uk/dbbrowser/PRINTS/PRINTS.html)
- PROSITE http://www.expasy.ch/prosite/
- PRINTS http://bioinf.man.ac.uk/dbbrowser/PRINTS/PRINTS.html
- the binding motif for the integrin is -Gly-Phe-Hyp-Gly-Glu-Arg-
- the binding motif for the discoidin domain receptor is -Gly-Val-Met-Gly-Phe-Hyp-
- the binding motif for heparan sulfate proteoglycan is -Lys-Gly-His-Arg-Gly-Phe- can be mentioned, but is not limited thereto.
- the polymerized peptide of the present invention is used as a research substrate having a desired selective activity
- research aimed at elucidating the mechanism of the intracellular signal transduction system of cells that bind to the substrate Can be used for
- a gel or polymerized peptide thin film containing the polymerized peptide of the present invention can be placed on a wound site, a surgical wound at the time of surgery, or the cornea or retina.
- Gel or polymerized peptide thin film enhances the migration and binding of fibroblasts to a wound site or surgical wound, for example, by incorporating the binding motif in which a receptor present on the cell membrane of fibroblasts has binding activity. It can be used for promoting wound treatment that promotes healing of wounds or surgical wounds, or as a medical material for cornea or retina regeneration.
- a polymer of a collagen-like peptide in which a side chain of a peptide chain is bound to a motif having a physiological activity via a linker A collagen-like peptide having a side chain of a peptide chain bound to a motif having a physiological activity via a linker
- the polymer has the following configuration.
- the peptide chain may be represented by the following formula (III).
- R 5 and R 6 are each an amino terminus and a carboxy terminus, each independently being a peptide group consisting of any 2 to 10 amino acid residues including at least 2 Cys residues, p, q, and r are all integers of 0 or more, the sum of p, q, and r is 3 or more, and s is an integer of 1 or more.
- the collagen peptide constituting the polymerized peptide of the present invention may be a homotrimer or a heterotrimer.
- the physiological activity may be a specific binding activity for a biopolymer.
- a fibronectin integrin binding motif is incorporated or bound at the time of production of a peptide chain or after the production of a polymerized peptide, so that the receptor has a selective binding activity to the motif.
- a research material or medical material to which cell adhesion is imparted can be provided.
- the binding motif for fibronectin-derived integrin ⁇ v ⁇ 3 includes, but is not limited to, -Arg-Gly-Asp-.
- VWF motif other than the above, VWF motif, SPARC / BM-40 / osteonectin motif, aegyptin motif, LAIR-1 motif, GPVI motif, PEDF motif, Hsp47 motif, HLA motif, HLA super motif, zinc finger C2H2 type motif , Cytochrome b (N-terminal) / b6 / petB motif, immunoglobulin domain motif, WD domain G- ⁇ repeat motif, PDZ domain motif, leucine rich repeat motif, protein kinase domain motif, PH domain motif, EGF-like domain motif, Reverse transcriptase (RNA-dependent DNA polymerase) motif, Ank repeat motif, NADH-ubiquinone / plastoquinone (complex I) motif, EF hand motif, retrovirus aspartyl protease motif, 7-transmembrane receptor (rhodopsin family) Mochi Etc. The but not limited thereto.
- cell adhesion motif examples include, for example, RGD sequence, LDV sequence, REDV sequence, YIGSR sequence, PDSGR sequence, RYVVLPR sequence, LGTIPG sequence, RNIAEIIKDI sequence, IKVAV sequence, LRE sequence, DGEA sequence, and HAV sequence. Each sequence can be mentioned.
- the target cell for the physiological activity is a cell having a receptor that binds the motif as a binding ligand.
- Specific examples include, but are not limited to, fibroblasts, hepatocytes, undifferentiated chondrocytes, muscle cells, platelets, neutrophils, macrophages, Schwann cells, keratinocytes, and epithelial cells.
- motifs other than the above for example, recorded in PROSITE (http://www.expasy.ch/prosite/) and PRINTS (http://bioinf.man.ac.uk/dbbrowser/PRINTS/PRINTS.html)
- PROSITE http://www.expasy.ch/prosite/
- PRINTS http://bioinf.man.ac.uk/dbbrowser/PRINTS/PRINTS.html
- a gel containing the polymerized peptide of the present invention or a polymerized peptide thin film can be placed on a wound site or a surgical wound at the time of surgery.
- Gel or polymerized peptide thin film enhances migration and binding of fibroblasts to a wound site or surgical wound, for example, by incorporating a binding motif in which a receptor present on the cell membrane of fibroblasts has binding activity, It can be used as a medical material for promoting wound healing that promotes healing of wounds or surgical wounds.
- polymerized peptide incorporating or binding multiple types of bioactive motifs As an aspect of the present invention, a peptide chain incorporating the bioactive motif and / or a peptide chain binding the bioactive motif By using them in combination, polymerized peptides having activities in which the respective physiological activities are selectively combined and the activity intensity of each activity being adjusted to a desired ratio can be mentioned.
- Polymerized peptides having this combination activity have a desired activity by mixing the collagen-like peptides having the respective activities so as to achieve the desired activity, after adjusting the mixing ratio and then polymerizing by oxidative crosslinking. Peptides can be produced. Using this polymerized peptide, a gel containing this polymerized peptide and a polymerized peptide thin film can be produced by the production method described above.
- the motif having physiological activity used in combination includes, but is not limited to, a binding motif for integrin, a binding motif for discoidin, a binding motif for heparan sulfate, and an RGD motif.
- a binding motif for a collagen receptor selected from integrins, discoidin domain receptors (DDR) or heparan sulfate proteoglycans (HSPG), or an RGD motif of fibronectin is used at the time of peptide chain production or polymerization. Incorporation after peptide production can provide a research material or a medical material in which a receptor-selective cell adhesion having a selective binding activity is imparted to the binding motif.
- DDR discoidin domain receptors
- HSPG heparan sulfate proteoglycans
- VWF motif other than the above, VWF motif, SPARC / BM-40 / osteonectin motif, aegyptin motif, LAIR-1 motif, GPVI motif, PEDF motif, Hsp47 motif, HLA motif, HLA super motif, zinc finger C2H2 type motif , Cytochrome b (N-terminal) / b6 / petB motif, immunoglobulin domain motif, WD domain G- ⁇ repeat motif, PDZ domain motif, leucine rich repeat motif, protein kinase domain motif, PH domain motif, EGF-like domain motif, Reverse transcriptase (RNA-dependent DNA polymerase) motif, Ank repeat motif, NADH-ubiquinone / plastoquinone (complex I) motif, EF hand motif, retroviral aspartyl protease motif, 7-transmembrane receptor (rhodopsin family) Mochi
- cell adhesion motif examples include, for example, RGD sequence, LDV sequence, REDV sequence, YIGSR sequence, PDSGR sequence, RYVVLPR sequence, LGTIPG sequence, RNIAEIIKDI sequence, IKVAV sequence, LRE sequence, DGEA sequence, and HAV sequence. Each sequence can be mentioned.
- the target cell for the physiological activity is a cell having a receptor that binds the motif as a binding ligand.
- Specific examples include, but are not limited to, fibroblasts, hepatocytes, undifferentiated chondrocytes, muscle cells, platelets, neutrophils, macrophages, Schwann cells, keratinocytes, and epithelial cells.
- motifs other than the above for example, recorded in PROSITE (http://www.expasy.ch/prosite/) and PRINTS (http://bioinf.man.ac.uk/dbbrowser/PRINTS/PRINTS.html)
- PROSITE http://www.expasy.ch/prosite/
- PRINTS http://bioinf.man.ac.uk/dbbrowser/PRINTS/PRINTS.html
- Gelling agent, gel, hydrogel and polymerized peptide thin film containing polymerized peptide of collagen-like peptide of the present invention When the polymerized peptide is produced in an aqueous solvent, it is produced as a hydrogel of the polymerized peptide of the present invention and water. That is, the polymerized peptide of the present invention has a use as a gelling agent.
- the hydrogel of the present invention can be used as a regenerative medical material such as a post-surgical organ adhesion-preventing agent, a hemostatic agent, a wound healing promoter, and a cornea or retina regenerating material.
- a regenerative medical material such as a post-surgical organ adhesion-preventing agent, a hemostatic agent, a wound healing promoter, and a cornea or retina regenerating material.
- the hydrogel can be dried to produce a polymerized peptide thin film that is a sheet-like film.
- This polymerized peptide thin film can be used, for example, as a regenerative medical material such as a cell sheet that can be rehydrated at the time of use and transplanted into the body such as the above-mentioned wound healing promoting material, artificial cornea or artificial myocardium.
- the polymerized peptide thin film of the present invention can be stored for a long period of time at room temperature by drying as compared with a hydrogel composed of a polymerized peptide before drying, and the interaction between peptide molecules is changed. Even if it adds, stronger intensity
- the gel of the present invention is produced by forming the triple-stranded peptide as a heterotrimer, forming a triple helical structure, and then oxidatively polymerizing with an oxidizing agent to form a gel.
- the triple-stranded peptide is homotrimeric.
- a gel containing at least one kind of peptide chain is produced by forming a triple helical structure as a monomer, and then mixing a plurality of types of homotrimers, followed by oxidative polymerization with an oxidizing agent and gelling. be able to.
- the preparation of the gel obtained by cross-linking and polymerizing a plurality of types of homotrimers described above will be described in more detail. It may be a gel produced by polymerization and gelation.
- This gel composed of multiple types of three-chain peptides is composed of, for example, a three-chain peptide composed of a peptide chain for controlling the hardness of the gel and a peptide chain having a bioactive motif Triple chain peptides can be combined to control the stiffness of the gel and / or adjust the expression of bioactivity.
- gel stiffness refers to the robustness of a gel against degradation by phagocytic cells such as macrophages in vivo.
- the hardness of this gel is influenced by the degree of cross-linking polymerization of the gel and the difference in the branched structure of the cross-linked peptide chain. For example, a hard gel is formed when many crosslinks by disulfide bonds are contained in the gel.
- Examples of the single-chain peptide used in the plurality of types of three-chain peptides include peptide chains in which the number of cysteine residues contained in the single-chain peptide is different in order to control the hardness of the gel. It is done.
- each peptide chain is heated and then slowly cooled to form a triple-stranded peptide having a triple helical structure composed of each peptide chain, and these three-chain peptides are mixed at a predetermined ratio, and then A gel containing a multifunctional polymerized peptide can be produced and used by cross-linking polymerization with an oxidizing agent and gelation.
- Polymerized peptide gels prepared by blending peptides containing many cysteine residues at a high rate tend to be slow in biodegradability in vivo, while blending peptides containing few cysteine residues at a high rate.
- the gel containing the prepared polymerized peptide exhibits faster biodegradability.
- the activity expression intensity of the bioactive motif of the peptide chain can be adjusted. Therefore, by adjusting the degree of crosslinking contained in the gel, It is considered that the hardness of the gel and the expression intensity of the physiological activity can be controlled.
- a peptide chain containing three, two or one cysteine residues in the vicinity of both the N-terminus and the C-terminus is used, and the cysteine residues are located at both ends.
- the combination ratio of peptide chains containing 3 cysteine residues and peptide chains containing 2 cysteine residues or 2 cysteine residues, and 2 peptide chains each containing 2 cysteine residues at each end is 10 parts by mass. % Or less, and by preparing a gel containing less than 70%, preferably 50% or less as a mass ratio of the peptide chain containing one cysteine residue at a time, as a medical material Can be used.
- cysteine residue in the gel is replaced with the peptide chain.
- a gel containing a polymerized peptide containing a peptide chain containing one peptide at each end of the peptide chain containing less than 30%, preferably 10% or less, can be used.
- These peptide chains can be used by combining a peptide chain not containing a cysteine residue with a peptide chain having a cysteine residue.
- Examples of peptide chains containing three, two or one cysteine residues near both the N-terminus and C-terminus are CCC2-GPOGPRGP (short) (SEQ ID NO: 15), CC2-GPOGPR (short) (SEQ ID NO: 23), C2-GPOGPR (short) (SEQ ID NO: 24) and the like.
- Examples of peptide chains that do not contain cysteine residues include Soluble GFOGER (SEQ ID NO: 19), Soluble GVMGFO ⁇ (SEQ ID NO: 20), Soluble KGHRGF (SEQ ID NO: 21), Soluble GPOGPR (short) (SEQ ID NO: 25), etc. Is mentioned.
- the combination of the three-chain peptide composed of the peptide chain that controls the hardness of this gel and the three-chain peptide composed of the peptide chain having the bioactive motif are mixed and mixed, and polymerized by the above method.
- a peptide By forming a peptide, it is possible to produce a multifunctional polymerized peptide in which the hardness of the gel in the body of the polymerized peptide, the type of physiological activity, and the strength of its expression are controlled. Then, by adjusting the hardness of the polymerized peptide and / or the type and amount of the active expression motif in vivo, for example, the biodegradation rate after in vivo transplantation can be adjusted or controlled.
- a gel containing a polymerized peptide using various peptide chains can be produced according to the use for which the gel of the present invention is desired. That is, for example, artificial organ materials, such as artificial blood vessels, that should not be biodegradable are mixed with many peptides with a composition containing many cysteine residues to produce gels, while organ adhesion after surgery For materials that should be biodegraded after remaining in the living body for a predetermined period of time, such as preventives and wound healing promoters, the gel containing the polymerized peptide of the present invention is produced with fewer cysteine residues. .
- the polymerized peptide of the present invention is selectively used by incorporating a motif having a desired physiological activity or binding via a linker.
- a gel, hydrogel or polymerized peptide thin film having physiological activity can be produced.
- a gel, hydrogel or polymerized peptide thin film having no particular physiological activity in other words, having low irritation and high safety in a living body is produced. be able to.
- the polymerized peptide imparted with a selective physiological activity can be obtained by, for example, incorporating or binding the aforementioned binding motif for the collagen receptor into the peptide chain.
- selective binding activity can be imparted to cells having a collagen receptor in a hydrogel or polymerized peptide thin film.
- hydrogels or polymerized peptide thin films as a scaffold, cells with selective binding activity accumulate, and the tissue around the position where the hydrogel or polymerized peptide thin film is placed due to the activity of cytokines or chemokines released by these cells And / or physiological activity, such as a wound healing promotion effect, can be provided to a cell.
- it can be used as a medical material having high selectivity for platelets, fibroblasts and / or corneal cells, preferably a regenerative medical material, and more preferably a regenerative medical material for promoting wound healing.
- a medical material having high selectivity for platelets, fibroblasts and / or corneal cells preferably a regenerative medical material, and more preferably a regenerative medical material for promoting wound healing.
- binding motif examples include a binding motif for a collagen receptor selected from integrin, discoidin domain receptor (DDR) or heparan sulfate proteoglycan (HSPG), or a binding motif for integrin ⁇ v ⁇ 3 derived from fibronectin.
- DDR discoidin domain receptor
- HSPG heparan sulfate proteoglycan
- the binding motif for integrin is -Gly-Phe-Hyp-Gly-Glu-Arg-
- the binding motif for discoidin domain receptor is -Gly-Val-Met-Gly-Phe-Hyp-
- heparan sulfate -Lys-Gly-His-Arg-Gly-Phe- can be used as the binding motif amino acid sequence for proteoglycan
- the binding motif for fibronectin-derived integrin ⁇ v ⁇ 3 includes, but is not limited to, -Arg-Gly-Asp- Not.
- Resin CTC 2-chlorotrityl chloride
- CD Circular dichroism
- DDR discoidin domain receptor
- ECM extracellular matrix
- FAK Focal adhesion kinase
- HDF human skin fibroblasts
- HRP Horseradish peroxidase
- HSPG heparan sulfate proteoglycan
- PEDF Pigment epithelium-derived factor
- SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- vWF von Bill brand factor
- amino acid sequence is represented by a single letter code commonly used by those skilled in the art. However, “O” represents a 4-hydroxyproline residue, “Ac-” represents an acetylated N-terminus, “—NH 2 ” represents an amidated carboxy terminus, and “ ⁇ A” represents ⁇ -alanine. Represents a residue.
- the peptide chain has a repeating sequence 10 to 17 times of the basic unit of-(Xaa-Yaa-Gly)-forming a triple helical structure.
- the basic sequence was designed to be polymerized by incorporating Cys residues at or near both NC ends and forming a disulfide bridge (-S-S-) by oxidation of the side chain thiol group (-SH).
- -S-S- disulfide bridge
- -SH side chain thiol group
- GVMGFO of SEQ ID NOs: 13, 17 and 20 and KGHRGF of SEQ ID NOs: 14, 18 and 21 are integrin receptor, discoidin receptor (DDR) and heparan sulfate proteoglycan, respectively.
- HSPG collagen binding motif of the receptor
- RGQOGVMGFO and KGHRGFSGL are binding motifs for vWF and PEDF, respectively.
- Y2 SEQ ID NO: 8
- YCC2-Scr which is considered to have a random coil structure because it does not have a repetitive sequence of (Pro-Hyp-Gly) were designed (SEQ ID NO: 9) .
- Fmocated amino acids Fmoc-Arg (Pbf) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Gly-OH, Fmoc-Pro-OH and Fmoc-Tyr (tBu) -OH are Novabiochem (Merck Millipore Corporation , USA). Wang resin was also purchased from Novabiochem. Other reagents used were those of grades higher than commercially available special grades. CTC resin was purchased from Peptide Institute, Inc. (Osaka). In preparation of the Ellman reagent, DTNB was dissolved in 50 mM phosphate buffer (pH 8.4) so that the concentration was 9 mg / ml.
- a circular dichroism dispersometer J-820 (JASCO Corporation) was used for CD spectrum measurement of peptides.
- a UV-visible spectrophotometer V-630 (JASCO Corporation) was used for UV absorbance measurement of the Fmoc group.
- TaKaRa Thermal Cycler (Takara Bio Inc., Shiga) was used to control the temperature of peptide refolding.
- Stainless steel balls (diameter 1.5 mm, Funabane Seiko, Hyogo) were used to determine gel formation.
- Cell culture dishes include Nunc Cell-Culture / Petri Dishes (9 cm dishes), Nunc MicroWell 96-Well Microplates (96-well plates), Nunc Cell-Culture Treated Multidishes (6-well plates) (Thermo Fisher Scientific, Inc., USA) ) was used.
- a laser scanning confocal microscope FV1000 (Olympus Corporation, Tokyo) was used for cell observation.
- LAS-3000 (Fuji Film Co., Ltd., Tokyo) was used.
- FIGS. 2A to 2D The results of RP-HPLC analysis of each peptide of YCC2, CC2, CC1, and C2 are shown in FIGS. 2A to 2D (results of RP-HPLC analysis of other peptides are not shown).
- Table 2 shows the retention time of each peptide.
- mrw represents the residue average molecular weight
- c represents the peptide concentration (mg / ml)
- l represents the cell length (cm).
- the thermal stability of the triple helix structure was confirmed by monitoring ⁇ mrw, 225 with temperature change. Measurement conditions are 4 to 75 ° C or 4 to 90 ° C, cell length 0.05 mm, measurement wavelength 225 mm, data acquisition interval 0.5 ° C, temperature gradient 18 ° C / hour, sensitivity standard (100 mm), response 2 seconds, bandwidth 1 nm. In the region where the signal decreased linearly, the triple helix structure was judged to be completely random coiled.
- results of CD spectrum measurement of peptides The results of CD spectrum measurement at 4 ° C, 37 ° C and 80 ° C for each synthesized peptide are shown in Figs. 3A to 3G. 4A and 4B show the results of measuring the temperature change of the CD spectral intensity ( ⁇ 225) at 225 nm for YCC2 and YCC2-Scr. From these measurement results, all the peptides except YCC2-Scr showed a positive cotton effect near 225 nm only at 4 ° C and 37 ° C, and thus formed a triple helix structure below 37 ° C. It was confirmed to be in a denatured state.
- collagen-like peptides except YCC2-Scr form a triple helix regardless of the terminal sequence and internal sequence, and YCC2-Scr does not form a triple helix structure, but has a polyproline type II conformation. It was thought that it was taking.
- a gel composed of a polymerized peptide obtained by polymerizing a collagen-like peptide was prepared by oxidizing and cross-linking the peptide having the triple helical structure with DMSO as an oxidizing agent. The progress of the polymerization reaction due to oxidative crosslinking was confirmed by measuring the residual thiol group.
- a peptide solution having a concentration of 1.11% (w / v) was prepared using degassed water and placed in a PCR tube. This was heated at 80 ° C. for 5 minutes and then allowed to stand overnight at 4 ° C. DMSO was added to this solution to prepare a peptide solution having a final concentration of 10% DMSO and a peptide of 1% (w / v). Mineral oil was filled to cover the surface. Mineral oil was removed from the peptide solution for each measurement to prepare a peptide solution for oxidation monitoring.
- SH group thiol group
- FIG. 5 shows the change in the ratio of the amount of SH with the passage of time when the amount of SH of each peptide at 0 hour is 100%.
- CCCC2 gelled in 12 hours, CCC2 in 24 hours, and CC2 and YCC2 in 74 hours. Therefore, quantitative data for the subsequent time zones are not measured.
- the percent decrease rate of SH amount was almost the same for each peptide.
- the ratio of the amount of SH in CC1 and YCC2-Scr that did not form a gel after 74 hours was about 0%. From the above results, it was shown that the oxidation of many peptides was almost 100% after 74 hours. When 3 days had passed, the sample that did not form a gel was determined to be a peptide that did not gel.
- FIG. 6 shows the results of evaluation of gel forming ability at 1% (w / v) concentration of each peptide.
- the white arrow indicates the position of the stainless sphere. From this result, gel formation was observed in gels having two or more Cys residues near both sides of the N-terminal and C-terminal (see CC2, CC1, and C2). In addition, no gel was formed with the peptide having a random coil structure (see YCC2 and YCC2-Scr). This indicates that the triple helix structure is important for gel formation.
- Table 4 shows the time required for gel formation at 1% (w / v).
- YCC2 ⁇ Preparation of polymerized peptide thin film and evaluation of strength> YCC2 was used for the preparation of the polymerized peptide thin film.
- a peptide solution having a concentration of 1.11% (w / v) was prepared using degassed water and placed in a PCR tube. This was heated at 80 ° C. for 5 minutes and then allowed to stand overnight at 4 ° C. DMSO was added to this solution to prepare a final concentration 10% DMSO, 1.0% (w / v) peptide solution.
- This peptide solution was transferred onto a non-water-absorbing substrate (parafilm, silicon-processed hole glass, Teflon (registered trademark) plate).
- a PVDF membrane or nylon mesh was placed on the non-water-absorbing substrate as a support. Gelation was carried out by allowing to stand for 3 days at room temperature under humid conditions (10% DMSO). Then, it switched to drying conditions and was dried at room temperature for 3 days. As the combination of the non-water-absorbing base and the support, the combination of a Teflon (registered trademark) plate and a nylon mesh was the best.
- FIG. 10 shows the results of obtaining a rehydrated polymerized peptide hydrogel. Since the hydrogel obtained by rehydration has less water retention than the hydrogel before drying, the peptide density was high and a hydrogel with high strength could be produced.
- a laminated polymer peptide thin film was produced by the following method. 1) In the same manner as in Example 4, the collagen-like peptide was dissolved in degassed H 2 O (22.2 mg / ml), heated, and then slowly cooled to form a triple helix. 2) Add DMSO (10% DMSO, 20 mg / ml peptide), oxidize by standing for 4 days in a closed container filled with 10% DMSO on a support (diameter 1 cm circle), hydrogel Formed. 3) Dried and dehydrated in air.
- FIGS. 11A to 11F The measurement results of the CD spectrum are shown in FIGS. 11A to 11F.
- Each peptide exhibited a positive cotton effect at around 225 nm only at 4 ° C and 27 ° C, so it was confirmed that it formed a triple helix structure below 37 ° C and could be denatured at 80 ° C. .
- This result also shows that the formation of a triple helix structure at 37 ° C. or lower is not related to the presence or absence of a Cys residue in the primary structure of the peptide chain.
- Example 3 a gel containing a polymerized peptide of a peptide chain incorporating these physiologically active motifs was prepared, and selective cell binding activity evaluation using cultured cells described below was performed. used.
- D-MEM high glucose, D-MEM low glucose and 0.5% trypsin / EDTA are from Wako Pure Chemical Industries, Ltd. (Osaka), FBS, penicillin streptomycin (100 x) are from Invitrogen (Thermo Fisher Scientific corporation ) Purchased from.
- Human breast adenocarcinoma (MDA-MB-231) was purchased from ATCC and human skin fibroblasts (HDF) were purchased from Cell Applications.
- MDA-MB-231 is cultured in DMEM high glucose (10% FBS, 100 Units / ml penicillin, 100 ⁇ g / ml streptomycin), HDF is DMEM low glucose (10% FBS, 100 Units / ml penicillin, 100 ⁇ g / ml ml streptomycin) and maintained.
- DMEM high glucose 10% FBS, 100 Units / ml penicillin, 100 ⁇ g / ml streptomycin
- HDF DMEM low glucose
- the cells were treated with 0.05% trypsin / EDTA, and the cells were collected in a 15 ml tube and centrifuged at 1200 rpm for 4 minutes. After removing the supernatant, the cells were recovered by suspending them in the culture medium and incubating at 37 ° C. for 20-30 minutes. This cell suspension was used for cell adhesion assay and Western blotting.
- Collagen-like peptides containing Cys residues are dissolved in degassed 0.05% (v / v) TFA aqueous solution, heated to 300 ⁇ g / ml at 80 ° C for 5 minutes, and then gradually cooled to 4 ° C at 1 ° C / min. A triple helix was formed by storing overnight at 4 ° C. This was diluted to 30 ⁇ g / ml to 30% MOPS buffer (pH 7.8) (Sigma-Aldrich: Sigma-Aldrich Japan GK, Tokyo), and 15% methanol, and 50 ⁇ l / well was added to a 96-well plate. Added and allowed to dry overnight.
- adherent cells were fixed with 4% (v / v) p-formaldehyde / phosphate buffer (pH 7.4) (Wako Pure Chemical Industries, Ltd.) Stained with 2% crystal violet / MeOH (Wako Pure Chemical Industries, Ltd.). The stained cells were observed using a laser scanning confocal microscope FV1000 (Olympus Corporation).
- SDS sample buffer 50 mM Tris / HCl (pH 6.7), 2% SDS, 10% glycerol, 1 ⁇ g / ml pepstatin A, 1 ⁇ g / ml leupeptin, 1 mM PMSF, 2 mM NEM, 1 mM Na 3 VO 4 , 10 mM NaF
- the prepared SDS-PAGE sample was quantified for protein by the BCA method. Each sample was added to 10% (w / v) acrylamide gel at 30 ⁇ g / well and separated by SDS-PAGE.
- the separated protein was transferred to a nitrocellulose membrane (GE Healthcare). Then, it was immersed in a 5% (w / v) blocking solution prepared using skim milk (Wako Pure Chemical Industries, Ltd.) and TBS (50 mM Tris ⁇ HCl (pH 7.4), 150 mM NaCl) overnight. Blocked.
- MDA-MB-231 caused cell adhesion dependent on internal sequence to Pol-GFOGER. Furthermore, this cell adhesion was inhibited by EDTA and recovered by Mg 2+ , indicating that this cell adhesion is an integrin-dependent cell adhesion. Therefore, YCC2 can be used as a scaffold for cell culture, and at the same time, receptor-specific cells targeted by incorporating various receptor-binding motifs of natural collagen into the internal sequence of YCC2. It has been shown that adhesion can be induced.
- FIG. 13A shows the results of Western blotting of FAK and pFAK (pTyr397) of cultured cells.
- MDA-MB-231 sensitively sensed the integrin ligand, and the number of adherent cells tended to increase logarithmically with the integrin ligand content.
- the number of adherent cells can be controlled by adjusting the amount of the ligand contained in the polymerized peptide, and various cell culture environments can be reproduced.
- Collagen with a molecular weight of about 300 kDa has one integrin binding site
- YCC2-GFOGER contains one integrin binding site in a triple helix peptide of about 12 kDa.
- the same mass of collagen and a mixed polymerized peptide of YCC2 and YCC2-GFOGER are coated, it is considered that when the content of YCC2-GFOGER is about 4%, an integrin-binding site similar to collagen is included.
- FIG. 15A shows the results of observation after fixing the cells, staining the cells, and culturing the cells.
- FIGS. 15B and 15C show the results of Pol-GFOGER and Pol-KGHRGF in the triple helix sequence.
- a hydrogel having a triple helix structure characteristic of collagen as a basic structure can be prepared. Since the hydrogel-forming ability of the peptide of the present invention does not depend on the amino acids in the triple helix sequence, various amino acid sequences satisfying (Xaa-Yaa-Gly) n can be incorporated into the sequence forming the triple helix. is there. Specifically, sequence-specific cell adhesion is induced by incorporating GFOGER, which is an integrin binding sequence, GVMGFO, which is a binding sequence for DDR, or KGHRGF, which is a binding sequence for HSPG, into the sequence forming the triple helix.
- GFOGER is an integrin binding sequence
- GVMGFO which is a binding sequence for DDR
- KGHRGF which is a binding sequence for HSPG
- the peptide of the present invention is excellent in that a ligand can be quantitatively mixed by copolymerizing peptides containing various sequences.
- the peptide of the present invention and the gel thereof can be provided as a tool for controlling behavior such as cell spreading, proliferation, and differentiation.
- the collagen-like peptide hydrogel produced using the peptide of the present invention is processed into a sheet shape, so that it can be used as an anti-adhesion material for organs after surgery, a suture material and a wound dressing material, and suitable on this sheet. By culturing cells, it becomes possible to produce cell sheets that can be transplanted into the body, such as an artificial cornea or an artificial myocardium.
- CCC2-KGHRGF SEQ ID NO: 18
- the polymerized peptide using CCC2-KGHRGF was diluted to 30 ⁇ g / ml, coated on a 96-well plate overnight, the solution was removed, and 1 mg / ml BS dissolved in 5 mg / ml NaHCO 3 aqueous solution.
- PEG polymerized peptide using CCC2-KGHRGF
- a peptide chain for controlling the gel hardness As a peptide chain for controlling the gel hardness, a three-chain peptide composed of CCC2-GPOGPR (short), CC2-GPOGPR (short) and C2-GPOGPR (short) peptides, and a peptide chain having a bioactive motif
- the polymer peptide gel produced by oxidative cross-linking is combined with a three-chain peptide composed of CCC2-GFOGER (short), and the effect on the cell aggregation activity of human skin fibroblasts is evaluated. did.
- CCC2-GPOGPR (short) (SEQ ID NO: 10), CC2-GPOGPR (short) (SEQ ID NO: 23) or C2-GPOGPR (short) (SEQ ID NO: 24) were dissolved in degassed ultrapure water, respectively. After heating at o C for 5 minutes, the mixture was allowed to stand at 4 o C overnight to form a triple helix. These were mixed at various ratios, DMSO was added to a final concentration of 10%, and 50 ⁇ L each was added to a 96-well plate.
- a 3-chain peptide composed of CCC2-GFOGER (short) (SEQ ID NO: 16) at a weight ratio of 1% was added to all peptide mixtures, and those not added were defined as Ligand-free.
- the plate was allowed to stand in a container filled with 10% DMSO for 4 days or longer to gel the peptide solution. These gels were immersed in cell culture medium overnight to dilute DMSO and used in the following experiments using cells. Type I collagen was used as a positive control.
- HDF Human skin fibroblasts
- FIG. 18A shows the result of comparing the adhesion to human skin fibroblasts with a gel containing a polymerized peptide produced by mixing with a 3-GFOGER (short) 3-chain peptide and then gelling.
- Experimental method 9 1, 7: 3 of two different three-chain peptides among the three-chain peptides composed of CCC2-GPOGPR (short), CC2-GPOGPR (short) and C2-GPOGPR (short), respectively , 5: 5, 3: 7 or 1: 9 combined in a solvent, and mixed in a solvent, and in the same way as in the above experiment, a 3-chain peptide composed of CCC2-GFOGER (short) at a weight ratio of 1% Was added, and a gel was formed by oxidative crosslinking to polymerize the three-chain peptide, and the gelation ability and cell aggregation of human skin fibroblasts adhering to this gel were evaluated in the same manner as in the above experiment. did.
- C2-GPOGPR (short) is a gel with a composition ratio of 7: 3, 5: 5, 3: 7, 1: 9, and CCC2 (short): C2-GPOGPR (short) In the gel having a composition ratio of 3: 7 and 1: 9, gelation did not occur even when DMSO, which is an oxidative crosslinking agent, was added.
- mice were sacrificed 10 and 30 days after transplantation, tissues were removed, fixed with 10% formalin solution, paraffin sections were prepared, hematoxylin-eosin stained, and histological analysis was performed under an optical microscope .
- the gel or polymerized peptide thin film containing the polymerized peptide of the present invention can be used for preventing adhesions to organs, suture materials, wound sites by placing it between the organ and the skin at the time of surgery or between the organ and the organ. It was shown that it can be used as a medical material such as a covering material and a wound healing promoter.
- the indwelled medical material of the present invention is gradually solubilized or degraded by phagocytic cells such as peptidases and macrophages present in the living body and disappears, so that no reoperation is required to remove the medical material. Therefore, the burden on the patient's prognosis is small, and it has the merit of improving QOL (Quality of Life).
- the medical material of the present invention is an artificially produced collagen-like peptide, unlike natural collagen, there is little risk of virus infection or microbial infection. Further, since it is cross-linked with a disulfide bond and does not denature even when heat-treated, it has an advantage that it can be heated for sterilization, for example.
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Abstract
Description
三重らせん構造を有する3本鎖ペプチドを繰り返し単位として有し、酸化架橋で重合され、
前記3本鎖ペプチドを構成する各ペプチド鎖は、同一であっても又は相互に異なっていてもよく、
各ペプチド鎖は、-(Xaa-Yaa-Gly)-を基本単位として少なくとも5回の繰り返し構造を有する三重らせん形成用ペプチド基と、アミノ末端及びカルボキシ末端の各々から10残基以内に少なくとも2残基のシステイン(Cys)残基を含む架橋形成用ペプチド基とを有する重合ペプチドを提供する。
[Xaa及びYaaは、それぞれ独立して、プロリン(Pro又はP)残基、ヒドロキシプロリン(Hyp又はO)残基、アルギニン(Arg又はR)残基、リシン(Lys又はK)残基、バリン(Val又はV)残基、ロイシン(Leu又はL)残基、イソロイシン(Ile又はI)残基、セリン(Ser又はS)残基、トレオニン(Thr又はT)残基、アラニン(Ala又はA)残基、グリシン(Gly又はG)残基、フェニルアラニン(Phe又はF)残基、メチオニン(Met又はM)残基、グルタミン酸(Glu又はE)残基、アスパラギン酸(Asp又はD)残基、アスパラギン(Asn又はN)残基、グルタミン(Gln又はQ)残基、ヒスチジン(His又はH)残基、トリプトファン(Trp又はW)残基又はチロシン(Tyr又はY)残基から選択され、プロリン残基はアミノ基又はフッ素原子で修飾されていてもよく、Xaa位及びYaa位にはN-イソブチル基グリシン残基を用いてもよい。]
前記ペプチド鎖が、
(i) 少なくとも1つの前記三重らせん形成用ペプチド基と少なくとも1つの架橋形成用ペプチド基からなるペプチド鎖、
(ii) 少なくとも1つの前記三重らせん形成用ペプチド基と少なくとも1つの架橋形成用ペプチド基、及び生理活性を有するモチーフを有する少なくとも1つのペプチド基を含むペプチド鎖、及び、
(iii) 少なくとも1つの前記三重らせん形成用ペプチド基と架橋形成用ペプチド基、及び、アミノ酸残基の側鎖にリンカーを介して生理活性を有するモチーフを結合させた少なくとも1つのペプチド基を含むペプチド鎖
からなる群から選択される少なくとも1つであるペプチド鎖である場合がある。
下記式(I)で表される3本のペプチド鎖から形成される三量体ペプチドの構造単位を有し、酸化架橋されてなる重合ペプチドの場合がある。
A1, A2及び A3は、同一又は相違してもよく、それぞれ独立して下記式(II)で表されるペプチド鎖であり、
A1, A2及び A3は、三重らせん構造を有する三量体を形成し、各々のペプチド鎖に含まれるシステイン(Cys)残基によってジスルフィド結合で架橋されていてもよく、
該三量体は、前記Cysのジスルフィド結合で酸化架橋により重合される。
R1及びR4は、各々アミノ末端及びカルボキシ末端を有し、相互に独立して少なくとも2残基のCys残基を含む任意の2~10残基のアミノ酸残基からなるペプチド基であり、
Zは、
(i) -(Xaa-Yaa-Gly)-を基本単位とする繰り返し構造のみからなるペプチド基、
(ii) -(Xaa-Yaa-Gly)-を基本単位として繰り返す構造及び生理活性を有するモチーフを有するペプチド基、又は、
(iii) -(Xaa-Yaa-Gly)-を基本単位として繰り返す構造を有し、かつ、該ペプチド鎖に含まれる少なくとも1残基のアミノ酸残基の側鎖にリンカーを介して生理活性を有するモチーフ配列を結合させてなるペプチド基、
から選択される少なくとも1つであり、
R2及びR3は独立して-(Xaa-Yaa-Gly)-を単位として連続して繰り返す構造を含むペプチド基であり、
-(Xaa-Yaa-Gly)-を基本単位とする繰り返し構造における繰り返し回数は、R2、Z及びR3の各々は0回以上であり、R2、Z及びR3で合計して3回以上であり、
mは1以上の整数である。)]
前記ペプチド鎖は、下記式(III)で表される場合がある。
R5及びR6は、各々アミノ末端及びカルボキシ末端であり、相互に独立して少なくとも2残基のCys残基を含む任意の2~10残基のアミノ酸残基からなるペプチド基であり、
p、q及びrは、いずれも0以上の整数であり、p、q及びrの合計は3以上であり、sは1以上の整数である。]
前記生理活性は生体高分子に対する特異的な結合活性である場合がある。
前記生体高分子に対する特異的な結合活性を有するリガンドの結合モチーフは、インテグリン、ジスコイジンドメイン受容体(DDR)若しくはヘパラン硫酸プロテオグリカン(HSPG)から選択されるコラーゲン受容体に対する結合モチーフ、又はフィブロネクチン由来のインテグリンαvβ3に対する結合モチーフの少なくとも1つから選択される重合ペプチドの場合がある。
前記インテグリンに対する結合モチーフは-Gly-Phe-Hyp-Gly-Glu-Arg-、ジスコイジンドメイン受容体に対する結合モチーフは-Gly-Val-Met-Gly-Phe-Hyp-、ヘパラン硫酸プロテオグリカンに対する結合モチーフアミノ酸配列は-Lys-Gly-His-Arg-Gly-Phe-であり、フィブロネクチン由来のインテグリンαvβ3に対する結合モチーフは-Arg-Gly-Asp-である場合がある。
前記ペプチド鎖は、配列番号1~7、10~18、23及び24から選択されるアミノ酸配列を有するペプチドから選択される場合がある。
前記ペプチド鎖は、配列番号1~7、10~18、23及び24から選択されるアミノ酸配列を有するペプチドのアミノ酸配列に対して1個若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなる場合がある。
前記ペプチド鎖は、配列番号1、2、5~7、10~18及び23から選択されるアミノ酸配列を有するペプチドから選択される場合がある。
前記ペプチド鎖は、配列番号1、2、5~7、10~18及び23から選択されるアミノ酸配列を有するペプチドのアミノ酸配列に対して1個若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなる場合がある。
前記生理活性を有するモチーフを有するペプチド鎖は、フィブロネクチン由来のインテグリン結合モチーフがBis(NHS)PEG5(bis(succinimidyl)penta(ethylene glycol))をリンカーとして、前記ペプチド基のLys残基の側鎖のアミノ基に結合されてなる場合がある。
前記生理活性を有するモチーフの2種類以上を組み合わせて製造される共重合ペプチドの場合がある。
-(Xaa-Yaa-Gly)-を基本単位として少なくとも5回の繰り返し構造を有し、アミノ末端及びカルボキシ末端の各々から10残基以内に少なくとも2残基のシステイン(Cys)残基を含む相互に異なっていてもよいペプチド鎖を変性温度以上の温度で溶媒に溶解する工程、
冷却することにより自己集合により前記ペプチドの3本からなる三重らせん構造を有する3本鎖ペプチドを形成する工程、
該3本鎖ペプチドを構造単位として、酸化架橋で重合させる工程、
を含む製造方法を提供する。
該製造方法において、Xaa及びYaaは、それぞれ独立して、プロリン(Pro又はP)残基、ヒドロキシプロリン(Hyp又はO)残基、アルギニン(Arg又はR)残基、リシン(Lys又はK)残基、バリン(Val又はV)残基、ロイシン(Leu又はL)残基、イソロイシン(Ile又はI)残基、セリン(Ser又はS)残基、トレオニン(Thr又はT)残基、アラニン(Ala又はA)残基、グリシン(Gly又はG)残基、フェニルアラニン(Phe又はF)残基、メチオニン(Met又はM)残基、グルタミン酸(Glu又はE)残基、アスパラギン酸(Asp又はD)残基、アスパラギン(Asn又はN)残基、グルタミン(Gln又はQ)残基、ヒスチジン(His又はH)残基、トリプトファン(Trp又はW)残基又はチロシン(Tyr又はY)残基から選択され、プロリン残基はアミノ基又はフッ素原子で修飾されていてもよく、Xaa位及びYaa位にはN-イソブチル基グリシン残基を用いてもよい。
ゲルが、ヒドロゲルであり、細胞を細胞選択的に培養するための基材として使用される場合がある。
再生医療材料は、創傷治癒促進用組成物である場合がある。
本発明に係る3本鎖ペプチドは、一般に、コラーゲン様ペプチドともいわれる。本明細書において、「コラーゲン様ペプチド」とは、非天然のペプチド又はポリペプチドであって、天然のコラーゲンと同様に、-(Xaa-Yaa-Gly)-を基本単位とする繰り返し構造を有するペプチド鎖の3本が、溶媒中で自己集合して、らせん構造を形成することにより、3本鎖ペプチドを形成させたもの、又は、該らせん構造を有する3本鎖ペプチドを、さらに、架橋させたペプチド又はポリペプチドを言う。ただし、前記Xaa及びYaaは、それぞれ独立して、プロリン(Pro又はP)残基、ヒドロキシプロリン(Hyp又はO)残基、アルギニン(Arg又はR)残基、リシン(Lys又はK)残基、バリン(Val又はV)残基、ロイシン(Leu又はL)残基、イソロイシン(Ile又はI)残基、セリン(Ser又はS)残基、トレオニン(Thr又はT)残基、アラニン(Ala又はA)残基、グリシン(Gly又はG)残基、フェニルアラニン(Phe又はF)残基、メチオニン(Met又はM)残基、グルタミン酸(Glu又はE)残基、アスパラギン酸(Asp又はD)残基、アスパラギン(Asn又はN)残基、グルタミン(Gln又はQ)残基、ヒスチジン(His又はH)残基、トリプトファン(Trp又はW)残基又はチロシン(Tyr又はY)残基から選択され、プロリン残基はアミノ基又はフッ素原子で修飾されていてもよく、Xaa位及びYaa位にはN-イソブチル基グリシン残基を用いてもよい。
三重らせん構造を有する3本鎖ペプチドを繰り返し単位として有し、酸化架橋で重合され、
前記3本鎖ペプチドを構成する各ペプチド鎖は、同一であっても又は相互に異なっていてもよく、-(Xaa-Yaa-Gly)-を基本単位として少なくとも5回の繰り返し構造を有し、アミノ末端及びカルボキシ末端の各々から10残基以内に少なくとも2残基のシステイン(Cys)残基を含むことを特徴とする、重合ペプチド。
[Xaa及びYaaは、それぞれ独立して、プロリン(Pro又はP)残基、ヒドロキシプロリン(Hyp又はO)残基、アルギニン(Arg又はR)残基、リシン(Lys又はK)残基、バリン(Val又はV)残基、ロイシン(Leu又はL)残基、イソロイシン(Ile又はI)残基、セリン(Ser又はS)残基、トレオニン(Thr又はT)残基、アラニン(Ala又はA)残基、グリシン(Gly又はG)残基、フェニルアラニン(Phe又はF)残基、メチオニン(Met又はM)残基、グルタミン酸(Glu又はE)残基、アスパラギン酸(Asp又はD)残基、アスパラギン(Asn又はN)残基、グルタミン(Gln又はQ)残基、ヒスチジン(His又はH)残基、トリプトファン(Trp又はW)残基又はチロシン(Tyr又はY)残基から選択され、プロリン残基はアミノ基又はフッ素原子で修飾されていてもよく、Xaa位及びYaa位にはN-イソブチル基グリシン残基を用いてもよい。]
前記ペプチド鎖に生理活性を有するモチーフを組み込む、及び/又は、ペプチド鎖の側鎖に生理活性を有するモチーフを有するペプチドを結合させることにより、該生理活性を有する重合ペプチド、該重合ペプチドを含有するゲル及び該重合ペプチドを含有する薄膜を製造することができる。コラーゲン様ペプチドに組み込むモチーフを選択することにより、天然のコラーゲンと相違し、所望の生理活性に選択的な生理活性を有する重合ペプチドを付与できる。また、これらの生理活性を有するペプチドを組み込まないことにより、生理活性を付与しないコラーゲン様ペプチドの重合体を製造できる。
前記「1.コラーゲン様ペプチドの重合ペプチド」に記載の重合ペプチドであって、
前記ペプチド鎖は、
(i) 少なくとも1つの前記三重らせん形成用ペプチド基と少なくとも1つの架橋形成用ペプチド基からなるペプチド鎖、
(ii) 少なくとも1つの前記三重らせん形成用ペプチド基と少なくとも1つの架橋形成用ペプチド基、及び生理活性を有するモチーフを有する少なくとも1つのペプチド基を含むペプチド鎖、及び、
(iii) 少なくとも1つの前記三重らせん形成用ペプチド基と架橋形成用ペプチド基、及び、アミノ酸残基の側鎖にリンカーを介して生理活性を有するモチーフを結合させた少なくとも1つのペプチド基を含むペプチド鎖
からなる群から選択される少なくとも1つであることを特徴とする、重合ペプチド。
前記重合ペプチドであって、
下記式(I)で表される3本のペプチド鎖から形成される三量体ペプチドの構造単位を有し、酸化架橋されてなることを特徴とする、重合ペプチド。
A1, A2及び A3は、同一又は相違してもよく、それぞれ独立して下記式(II)で表されるペプチド鎖であり、
A1, A2及び A3は、三重らせん構造を有する三量体を形成し、各々のペプチド鎖に含まれるシステイン(Cys)残基によってジスルフィド結合で架橋されていてもよく、
該三量体は、前記Cysのジスルフィド結合で酸化架橋により重合される。
R1及びR4は、各々アミノ末端及びカルボキシ末端であり、相互に独立して少なくとも2残基のCys残基を含む任意の2~10残基のアミノ酸残基からなるペプチド基であり、
Zは、
(i) -(Xaa-Yaa-Gly)-を基本単位とする繰り返し構造のみからなるペプチド基、
(ii) -(Xaa-Yaa-Gly)-を基本単位として繰り返す構造及び生理活性を有するモチーフを有するペプチド基、及び、
(iii) -(Xaa-Yaa-Gly)-を基本単位として繰り返す構造を有し、かつ、該ペプチド鎖に含まれる少なくとも1残基のアミノ酸残基の側鎖にリンカーを介して生理活性を有するモチーフ配列を結合させてなるペプチド基、
からなる群から選択される少なくとも1つであり、
R2及びR3は独立して-(Xaa-Yaa-Gly)-を単位として連続して繰り返す構造を含むペプチド基であり、
-(Xaa-Yaa-Gly)-を基本単位とする繰り返し構造における繰り返し回数は、R2、Z及びR3の各々は0回以上であり、R2、Z及びR3で合計して3回以上であり、
mは1以上の整数である。)]
本発明の態様の一つは、前記コラーゲン様ペプチドの重合体であって、特定の生理活性を付与しない重合ペプチドである。該特定の生理活性を付与しない重合ペプチドは、-(Xaa-Yaa-Gly)-を基本単位とする繰り返し配列と、複数のCys残基を含むペプチド基からなるペプチド鎖によって構成され、該ペプチド鎖の3本が三重らせん構造を有するコラーゲン様ペプチドを形成し、該コラーゲン様ペプチドがジスルフィド結合で架橋重合された重合ペプチドであり、より具体的には以下に記載の構成を有する。
前記ペプチド鎖は、
少なくとも1つの前記三重らせん形成用ペプチド基と少なくとも1つの架橋形成用ペプチド基からなるペプチド鎖であることを特徴とする。
生理活性を有するモチーフを、前記ペプチド鎖に組み込む、又は前記一本鎖の側鎖にリンカーを介して結合させることにより、所望の生理活性有する、活性強度を調節した、及び/又は、複数の生理活性を組み合わせたコラーゲン様ペプチドの重合ペプチドを製造できる。
生理活性を有するモチーフを組み込んだコラーゲン様ペプチドの重合体は、以下の構成を有する。
前記「1.コラーゲン様ペプチドの重合体」に記載の構成を有する重合ペプチドであって、
前記ペプチド鎖は、
少なくとも1つの前記三重らせん形成用ペプチド基と少なくとも1つの架橋形成用ペプチド基、及び生理活性を有するモチーフを有する少なくとも1つのペプチド基からなるペプチド鎖であることを特徴とする。
前記ペプチド鎖は、下記式(III)で表される場合がある。
R5及びR6は、各々アミノ末端及びカルボキシ末端であり、相互に独立して少なくとも2残基のCys残基を含む任意の2~10残基のアミノ酸残基からなるペプチド基であり、
p、q及びrは、いずれも0以上の整数であり、p、q及びrの合計は3以上であり、sは1以上の整数である。]
ペプチド鎖の側鎖にリンカーを介して生理活性を有するモチーフを結合させたコラーゲン様ペプチドの重合体は、以下の構成を有する。
前記「1.コラーゲン様ペプチドの重合体」に記載の構成を有する重合ペプチドであって、
前記ペプチド鎖は、
-(Xaa-Yaa-Gly)-を基本単位として繰り返す構造を有し、かつ、該ペプチド鎖に含まれる少なくとも1残基のアミノ酸残基の側鎖にリンカーを介して生理活性を有するモチーフを結合させてなるペプチド基である場合がある。
前記ペプチド鎖は、下記式(III)で表される場合がある。
R5及びR6は、各々アミノ末端及びカルボキシ末端であり、相互に独立して少なくとも2残基のCys残基を含む任意の2~10残基のアミノ酸残基からなるペプチド基であり、
p、q及びrは、いずれも0以上の整数であり、p、q及びrの合計は3以上であり、sは1以上の整数である。]
前記生理活性は生体高分子に対する特異的な結合活性である場合がある。
本発明の態様として、前記の生理活性モチーフを組み込んだペプチド鎖及び/又は前記の生理活性モチーフを結合させたペプチド鎖を組み合わせて使用することにより、それぞれの生理活性を選択的に組み合わせた活性を有し、各活性の活性強度を所望の割合に調整した重合ペプチドが挙げられる。
前記重合ペプチドは、水溶媒中で製造される場合に、本発明の重合ペプチドと水とのヒドロゲルとして製造され、即ち、本発明の重合ペプチドは、ゲル化剤としての用途を有する。
本発明の重合ペプチドは、前記に説明のとおり、所望の生理活性を有するモチーフを組み込む又はリンカーを介して結合させることにより、選択的な生理活性を有するゲル、ヒドロゲル又は重合ペプチド薄膜を製造することができる。あるいは、生理活性を有するモチーフを組み込まない又は結合させないことにより、特段の生理活性を有さない、換言すれば、生体で刺激性が小さく、安全性の高いゲル、ヒドロゲル又は重合ペプチド薄膜を製造することができる。
アミノ酸残基(全てL体)
Arg(R):アルギニン
Asp(D):アルパラギン酸
Cys(C):システイン
Gln(Q):グルタミン
Glu(E):グルタミン酸
Gly(G):グリシン
His(H):ヒスチジン
Hyp(O):4-ヒドロキシプロリン
Lys(K):リジン
Pro(P):プロリン
Tyr(Y):チロシン
Ile(I):イソロイシン
Leu(L):ロイシン
Met(M):メチオニン
Phe(F):フェニルアラニン
Ser(S):セリン
Val(V):バリン
Fmoc: 9-フルオレニルメトキシカルボニル
tBu: tert-ブチル
Trt:トリフェニルメチル(トリチル)
Pbf: 2,2,4,6,7-ペンタメチルジヒドロベンゾフラン-5-スルホニル
Boc: tert-ブトキシカルボニル
CTC:塩化2-クロロトリチル
BCA:ビシンコニン酸
BSA:ウシ血清アルブミン
CHCA:α-シアノ-4-ヒドロキシケイ皮酸
DCM:ジクロロメタン
DIC: N,N'-ジイソプロピルカルボジイミド
DIEA: N,N-ジイソプロピルエチルアミン
DMAP: N,N-ジメチルアミノピリジン
DMF: N,N-ジメチルホルムアミド
DMSO:ジメチルスルホキシド
DTNB: 5,5'-ジチオビス-2-ニトロ安息香酸
DTT:ジチオトレイトール
EDT:エタンジチオール
EDTA:エチレンジアミン四酢酸
FBS:ウシ胎児血清
HOBt: 1-ヒドロキシベンゾトリアゾール
MeCN:アセトニトリル
MeOH:メタノール
MOPS: 3-モルホリノプロパンスルホン酸
NEM: N-エチルマレイミド
PBS:リン酸緩衝液
PMSF:フェニルメチルスルホニルフルオリド
SDS:ドデシル硫酸ナトリウム
TBS:トリス緩衝生理食塩水
TFA:トリフルオロ酢酸
TMSO:テトラメチレンスルホキシド
RP-HPLC:逆相高速液体クロマトグラフィー
MALDI-TOF-MS: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計
CD:円偏光二色性
DDR:ジスコイジンドメイン受容体
ECM:細胞外マトリックス
FAK:接着斑キナーゼ (focal adhesion kinase)
HDF:ヒト皮膚線維芽細胞
HRP:ホースラディッシュペルオキシダーゼ
HSPG:ヘパラン硫酸プロテオグリカン
PEDF:色素上皮由来因子
SDS-PAGE:ドデシル硫酸ナトリウム-ポリアクリルアミドゲル電気泳動
vWF:フォン・ビルブランド因子
材料及び方法
ペプチド合成
本実施例で下記表1に記載の配列番号1~22のペプチドを固相合成し、使用する。
固相合成にはPD-10 empty column (GE Healthcare、米国) 又はLibra Tube (株式会社ハイペップ研究所、京都)を用いた。ペプチドの分析にはHPLC C20A シリーズ(株式会社島津製作所、京都)を用い、カラムはCOSMOSIL 5C18 AR-II (ナカライテスク株式会社、京都)を用いた。ペプチドの精製にはLC 2000 Plus シリーズ(日本分光株式会社、東京)を用い、カラムはCOSMOSIL 5C18 AR-II (ナカライテスク株式会社)又はCadenza CD-C18 (インタクト株式会社、京都)を用いた。ペプチドの質量分析にはAutoflex-III (Bruker Corporation、ドイツ)を用いた。ペプチドのCDスペクトル測定には円二色性分散計J-820 (日本分光株式会社)を用いた。Fmoc基のUV吸光度測定には紫外可視分光光度計V-630 (日本分光株式会社)を用いた。ペプチドのリフォールディングの温度制御にはTaKaRa Thermal Cycler (タカラバイオ株式会社、滋賀)を用いた。ゲル形成の判定にはステンレス球(直径1.5 mm、有限会社舟辺精工、兵庫)を用いた。細胞培養皿にはNunc Cell-Culture/Petri Dishes (9 cm ディッシュ)、Nunc MicroWell 96-Well Microplates (96 ウェルプレート)、Nunc Cell-Culture Treated Multidishes (6 ウェルプレート) (Thermo Fisher Scientific, Inc.、米国)を用いた。細胞の観察にはレーザー走査型共焦点顕微鏡FV1000 (オリンパス株式会社、東京)を用いた。ウエスタンブロッティングの可視化にはLAS-3000 (富士フィルム株式会社、東京)を用いた。
ペプチドは、当業者に慣用のFmoc合成法(特開2005-263784号公報)により、合成した。
ペプチドの精製にはRP-HPLC (LC 2000 Plusシリーズ、日本分光株式会社)を用いて、0.05% (v/v) TFA-H2Oと0.05% (v/v) TFA-MeCNの直線濃度勾配により溶出させた。カラム径を20 mm、カラム温度を60℃、流速を5 ml/分、検出波長を220 nmとした。カラムはCOSMOSIL 5C18 AR-II (ナカライテスク株式会社)又はCadenza CD-C18 (インタクト株式会社)を用いた。
合成したペプチドを精製した後、その純度を確かめるためにRP-HPLC により分析した。ペプチドは移動相に0.05% TFA/H2O と0.05% TFA/MeCN を用いた直線濃度勾配により溶出させ、Soluble KGHRGF の分析時の濃度勾配は0.05% TFA/MeCN 0~30%/30分、30~90%/5分とし、その他のペプチドの分析時の濃度勾配は0.05% TFA/MeCN 10~40%/30min、40~90%/5分とした。また、カラム温度は60℃、測定波長は220nm とした。
合成し、精製した上記の各ペプチドの質量分析をMALDI-TOF MSにより行った。各ペプチドの質量の計算値と実測値を表3に示す。測定サンプルを調製するときのマトリックスにはCHCAを用いた。この結果から、すべてのペプチドは目的としたペプチドであることが確認された。
ペプチドのコンフォメーションの確認による三重らせん構造の確認
ペプチドの構造をCDスペクトル(円二色性分散計J-820、日本分光株式会社)により確認した。ペプチド粉末を、脱気した0.05% TFA/H2Oに溶解し、1 mg/mlの溶液を調製した。80℃で5分間加熱後、4℃にて一晩静置してリフォールディングさせた溶液を測定サンプルとして調製した。CDスペクトルは、温度4、37、80℃、セル長0.05 cm、感度standard (100 mdeg)、測定波長250~190 nm、データ取り込み間隔0.2 nm、走査速度50 nm/分、レスポンス0.5秒、バンド幅1 nm、積算回数4回の条件で測定した。得られたシグナル(θobs)を残基平均モル楕円率(θmrw)として示した。θmrwを以下の式により算出した。
合成したそれぞれのペプチドについて、4℃、37℃及び80℃におけるCDスペクトルを測定した結果を図3A~図3Gに示す。また、YCC2及びYCC2-Scrについて、225 nmのCDスペクトル強度(θ225)の温度変化測定を行った結果を図4A及び図4Bに示す。これらの測定結果から、YCC2-Scrを除くすべてのペプチドが4℃及び37℃においてのみ225 nm付近で正のコットン効果を示したため、37℃以下で三重らせん構造を形成しており、80℃では変性状態であることが確認された。C2のCDスペクトル測定は未実施であるが、同様の三重らせん骨格を有するすべてのペプチドにおいて三重らせん形成が見られたことから、C2も同様に三重らせんを形成していると考えられた。一方、YCC2-Scrは測定したすべての温度において225 nm付近に正のコットン効果が見られた。225 nmにおけるθ225の温度変化測定の結果より、CC2では75℃付近で熱変性が起こっている様子が観察された一方、YCC2-Scrでは1次関数的な減衰が見られた。これらのことから、YCC2-Scrを除くコラーゲン様ペプチドは末端配列及び内部配列によらず三重らせんを形成しており、YCC2-Scrは三重らせん構造は形成しないものの、ポリプロリンII型の立体構造をとっていると考えられた。
上記三重らせん構造を有するペプチドを、酸化剤であるDMSOで酸化架橋させることにより、コラーゲン様ペプチドを重合させた重合ペプチドからなるゲルを作製した。残存チオール基を測定することにより、酸化的架橋による重合反応の進行を確認した。
脱気した水を用いて、1.11% (w/v)濃度のペプチド溶液を調製し、PCRチューブに入れた。これを80℃で5分間加熱後、4℃で一晩静置した。この溶液にDMSOを加えて終濃度10% DMSO、ペプチド1% (w/v)のペプチド溶液を調製した。表面を覆うようにミネラルオイルを充填した。ペプチド溶液から測定毎にミネラルオイルを取り除いて酸化モニター用のペプチド溶液を調製した。エルマン試薬は、DTNBを50 mM リン酸緩衝液(pH8.4)に9 mg/mlとなるように調製した。最終濃度が、ペプチド0.167 mg/ml、33 mM リン酸緩衝液(pH8.4)、Ellman試薬1.5 mg/mlの条件で5分間静置した後に吸光度を測定した。DMSOを加えた時間を始点(0時間)として時間経過に伴う吸光度の変化を記録した。Ellman試薬はペプチドのもつチオール基(以下「SH基」と記載)の量に依存して5-メルカプト-2-ニトロ安息香酸(λmax = 412 nm、ε = 1.55×104)を生成するため、412 nmにおける吸光度を測定した。測定後、997 nmと900 nmにおける吸光度を測定して光路長補正を行った。
脱気した水を用いて、1.11% (w/v)又は1.66% (w/v)濃度のペプチド溶液を調製し、PCRチューブに入れた。これをポリメラーゼ連鎖反応(PCR)機(TaKaRa PCR Thermal Cycler、タカラバイオ株式会社)にて80℃で5分間加熱後、4℃で一晩静置した。この溶液にDMSO及び水を加えて10% DMSO、0.1~1.5% (w/v)濃度のペプチド溶液を調製した。液面を覆うようにミネラルオイルを充填し、室温で一定時間静置してゲル化した。その後、ステンレス球をゲルに保持できるかどうかでゲル形成を判定した。
各ペプチドの1% (w/v)濃度でのゲル形成能を評価した結果を図6に示す。白色矢印はステンレス球の位置を示す。この結果から、N末端及びC末端の両側近傍にCys残基を2つ以上もつゲルではゲル形成が観察された(CC2と、CC1及びC2とを参照)。また、ランダムコイル構造を有するペプチドではゲルが形成されなかった(YCC2とYCC2-Scrとを参照)。このことから、ゲル形成に三重らせん構造が重要であることが示された。また、1% (w/v)でのゲル形成に要した時間を表4に示す。
1% (w/v)濃度においてゲル化したペプチド(CCCC2、CCC2、CC2及びYCC2)を用いて、室温、3日間の酸化条件下でのゲル化必要濃度を評価した結果を表5に示す。
ヒドロゲルの還元処理に対する安定性の測定
Cys残基を NC 両末端近傍に2個以上持つペプチドをDMSOで酸化することによってヒドロゲルが形成されることが示された。そこで、還元処理によってジスルフィド架橋を切断することでゲルは崩壊するのかを検証した。作製したヒドロゲル(30 μl)に100 μlの還元溶液(100 mM DTT、50 mM リン酸緩衝液(pH8.4))あるいは水を加えた。最終濃度がペプチド濃度2.3 mg/ml、2.3% DMSO 23 mM DTT、12 mMリン酸緩衝液(pH8.4)あるいは 2.3 mg/ml、2.3% DMSOとなった。その後、それぞれを約90 回/分の頻度で100μl/回の容量を黄色チップ(~200μl)を用いてピペッティングした。図7にその結果を示す。
作製したヒドロゲルにステンレス球を載せた後、恒温装置に移し、35℃~95℃までの間のさまざまな温度で10分間静置した。ステンレス球の沈降によってゲルが崩壊したかを判定した。
1% (w/v)のペプチドを用いて作製したヒドロゲルの熱安定性を測定した結果を図8に示す。この結果から、CCCC2、CCC2又はYCC2から形成されたヒドロゲルは95℃以下ではステンレス球の沈降はみられなかった。CC2から形成されたヒドロゲルは85~90℃でステンレス球が沈降を開始し、95℃で完全に沈降した。したがって、ジスルフィド結合の数に比例して、三重らせん構造が安定化し、ペプチドの変性温度が高くなったと考えられる。
重合ペプチド薄膜の作製にはYCC2を用いた。脱気した水を用いて、1.11% (w/v)濃度のペプチド溶液を調製し、PCRチューブに入れた。これを80℃で5分間加熱後、4℃で一晩静置した。この溶液にDMSOを加えて終濃度10% DMSO、1.0% (w/v)ペプチド溶液を調製した。このペプチド溶液を非吸水基盤(パラフィルム、シリコン加工したホールガラス、テフロン(登録商標)板)上に移した。この時、支持体としてPVDF膜又はナイロンメッシュを非吸水基盤上に設置した。湿潤条件(10% DMSO)の室温で3日間静置することでゲル化させた。その後、乾燥条件に切り換えて、室温で3日間乾燥させた。非吸水基盤と支持体との組み合わせは、テフロン(登録商標)板とナイロンメッシュとの組み合わせが最も優れていた。
YCC2ペプチドから形成された重合ペプチド薄膜を作製したコラーゲン様重合ペプチド薄膜は、非常に透明度が高かった(図9)。
上記方法で作製した重合ペプチド薄膜に非吸水フィルム上で水を添加した。数分間水和させた後、重合ペプチド薄膜が吸収しなかった水を回収除去した。これにより、再水和した重合ペプチドヒドロゲルを得た。
再水和させた重合ペプチドのヒドロゲルを得た結果を図10に示す。再水和で得たヒドロゲルは、乾燥前のヒドロゲルより保水量が少ないため、ペプチド密度が高くなり、強度の高いヒドロゲルを作製することができた。
CCC2-GFOGER(short)(配列番号11)で製造した重合ペプチドを使用して、積層した重合ペプチド薄膜を以下の方法で製造した。
1) 前記実施例4と同様に、コラーゲン様ペプチドを脱気したH2Oに溶解し (22.2 mg/ml)、加熱後、徐冷することにより三重らせんを形成した。
2) DMSOを添加 (10% DMSO、20 mg/mlペプチド)し、支持体 (直径1 cm円)上、10% DMSOを充填した密閉容器中で約4日静置することで酸化し、ヒドロゲルを形成させた。
3) 大気中で乾燥し脱水した。
4) 2)と同様に調製した10% DMSO、20 mg/mlペプチド溶液を積層し、10% DMSOを充填した密閉容器中で約4日酸化した。
5) 大気中で乾燥し脱水した。
この乾燥させた重合ペプチド積層薄膜を再水和し、支持体から切り離すことにより、又は、支持体から切り離した後に再水和させることにより、強度がより向上した重合ペプチド薄膜を製造した。
生理活性を有するモチーフを組み込んだペプチド鎖のコラーゲン様ペプチドの重合ペプチドの製造
YCC2-GFOGER(配列番号12)、YCC2-GVMGFO(配列番号13)及びYCC2-KGHRGF(配列番号14)、並びに、これらの各ペプチドの対照として、各ペプチドのN末端及びC末端の両端近傍にCys残基を含まないSoluble GFOGER(配列番号19)、Soluble GVMGFO(配列番号20)及びSoluble KGHRGF(配列番号21)を、実施例2と同様の方法で合成し、その各ペプチド溶解液のCDスペクトルを測定することにより、三重らせん構造の構築を確認した。
細胞培養
D-MEM高グルコース、D-MEM低グルコース及び0.5%トリプシン/EDTAは和光純薬工業株式会社(大阪)から、FBS、ペニシリン・ストレプトマイシン(100 x)はInvitrogen(Thermo Fisher Scientific corporation)から購入した。ヒト乳腺癌(MDA-MB-231)はATCCから、ヒト皮膚線維芽細胞(HDF)はCell Applications から購入した。MDA-MB-231は、DMEM高グルコース(10% FBS、100 Units/ml ペニシリン、100 μg/ml ストレプトマイシン)で培養し、HDFはDMEM低グルコース(10% FBS、100 Units/ml ペニシリン、100 μg/ml ストレプトマイシン)で培養し、維持した。細胞接着アッセイ及びウエスタンブロッティングに用いる場合は、細胞を0.05%トリプシン/EDTAで処理し、細胞を15 ml チューブに回収して1200 rpmで4分間遠心した。上清を除去した後、細胞を培養培地で懸濁し、37℃で20~30分間インキュベートすることで回復した。この細胞懸濁液を細胞接着アッセイ及びウエスタンブロッティングに用いた。
I型コラーゲン(株式会社高研、東京)及びBSA (Jackson Immuno Research Laboratories Inc.、米国)を、30 μg/mlの水溶液として50 μl/wellを96ウェルプレート(Nunc MicroWell 96-Well Microplates)に添加して2晩乾燥させた。Cys残基を含まないコラーゲン様ペプチドは1~3 mg/mlの水溶液とし、95℃で5分間加熱した後、4℃で1晩保存することで三重らせんを形成させた。これを30 μg/mlに希釈し、50 μl/wellを96ウェルプレートに添加して2晩乾燥させた。Cys残基を含むコラーゲン様ペプチドは脱気した0.05% (v/v) TFA水溶液に溶解し、300 μg/mlとして80℃で5分間加熱した後、1℃/分で4℃まで徐冷し、4℃で1晩保存することで三重らせんを形成させた。これを30 mM MOPS緩衝液(pH7.8) (Sigma-Aldrich:シグマ アルドリッチ ジャパン合同会社、東京)、15%メタノールとなるように30 μg/mlに希釈し、50 μl/wellを96ウェルプレートに添加して2晩乾燥させた。異なるリガンドを組み込んだ重合ペプチドをコートするときは、各ペプチドを脱気した0.05% (v/v) TFA水溶液に溶解して(1 mg/ml)、80℃で5分間加熱した後、1℃/分で4℃まで徐冷し、4℃で1晩保存することで三重らせんを形成させた。これらのペプチドを様々な割合で混合するとともに、20% (v/v) DMSO、総ペプチド濃度が0.3 mg/mlとなるように希釈し、ペプチド溶液上にミネラルオイルを重層し、Ellman試験によってチオールが検出されなくなるまで室温で酸化させた。このペプチド溶液をMilliQ水で30 μg/mlに希釈し、50 μl/wellを96ウェルプレートに添加して2晩風乾した。
ペプチド又はタンパク質をコートした96ウェルプレートを60~70℃で熱変性させた5 mg/ml BSAでブロッキングし、37℃で1~2時間インキュベートした。BSA溶液を除いてPBS (10 mM リン酸緩衝液 (pH 7.4), 137 mM NaCl, 2.7 mM KCl) (Sigma Aldrich)で洗浄し、MDA-MB-231細胞懸濁液(40-60 x 103 cells/well)又はHDF細胞懸濁液(4-10 x 103 cells/well)を添加して37℃で30~180分間インキュベートした。その後、培地とともに浮遊細胞を除去してPBSで洗浄の後、4% (v/v) p-ホルムアルデヒド/リン酸バッファー(pH7.4) (和光純薬工業株式会社)で接着細胞を固定し、2%クリスタルバイオレット/MeOH (和光純薬工業株式会社)で染色した。染色細胞はレーザー走査型共焦点顕微鏡FV1000 (オリンパス株式会社)を用いて観察した。
ペプチド又はタンパク質をコートした6ウェルプレートを60~70℃で熱変性したBSA(5 mg/ml)でブロッキングし、37℃で1~2時間インキュベートした。BSA溶液を除去し、PBSで洗浄の後、MDA-MB-231細胞懸濁液(50 x 103 cells/well)を添加し、37℃で90分間インキュベートした。培地を除去した細胞にSDS サンプルバッファー(50 mM Tris・HCl (pH 6.7), 2% SDS, 10%グリセロール、 1 μg/ml ペプスタチンA、 1 μg/ml ロイペプチン、 1 mM PMSF、2 mM NEM、1 mM Na3VO4、10 mM NaF)を添加し、細胞を溶解した。これを1.5 mlチューブに回収し、95℃で5分間加熱したものをSDS-PAGEサンプルとした。調製したSDS-PAGEサンプルはBCA法により、タンパク質を定量した。10% (w/v)アクリルアミドゲルに各サンプルを30 μg/well添加し、SDS-PAGEにより分離した。分離したタンパク質をニトロセルロース膜(GE Healthcare)に転写した。その後、スキムミルク(和光純薬工業株式会社)と、TBS (50 mM Tris・HCl (pH 7.4)、150 mM NaCl)とを用いて調製した5% (w/v)ブロッキング溶液に浸漬して1晩ブロッキングした。1次抗体希釈液(1 : 1000)(抗FAKマウス抗体(Merck Millipore Corporation、米国)、抗pFAK (pTyr397)ウサギ抗体(Invitrogen)、又は抗β-アクチンマウス抗体(Sigma Aldrich)を添加して2~3時間静置した。1次抗体を除去して、ニトロセルロース膜をTBST(0.1% Tween-20)に浸して10~20分間振盪した。これを3回繰り返した後、2次抗体希釈液(1 : 1000)(抗マウス抗体ヤギ抗体-HRP複合体(プロメガ株式会社、東京)、抗ウサギ抗体ヤギ抗体-HRP複合体(Santa Cruz Biotechnologies Inc.、米国))を加えて、30分間静置した。2次抗体を除去し、ニトロセルロース膜をTBS-Tに浸して10~20分間振盪した。これを3回繰り返した後、Western blotting KitPlus (Pierce; サーモフィッシャーサイエンティフィック株式会社)を用いて発色し、LAS-3000 (富士フィルム株式会社)により可視化して定量した。
BSA、I型コラーゲン、YCC2重合ペプチド(Pol-GPOGPR)、YCC2-GFOGER重合ペプチド(Pol-GFOGER)及びリガンドを含み、重合ペプチドを形成しないペプチド(Soluble GFOGER)を96ウェルプレートにコートし、MDA-MB-231を培養したときの細胞接着を観察し、比較した(図12A及び図12B)。YCC2及びYCC2-GFOGERは還元状態で三重らせんを形成させた後、塩基性バッファーで希釈することで酸化条件とした。これによって96ウェルプレート上でペプチドを酸化し、ジスルフィド架橋させた。
BSA、I型コラーゲン、YCC2-GFOGER重合ペプチド(Pol-GFOGER)及びYCC2重合ペプチド(Pol-GPOGPR)を96ウェルプレートにコートし、MDA-MB-231を90分間培養した。このとき、YCC2及びYCC2-GFOGERは還元状態で三重らせんを形成させた後、塩基性バッファーで希釈することで酸化条件とした。これによって96ウェルプレート上でペプチドを酸化し、ジスルフィド架橋することでコートした。培養した細胞のFAK及びpFAK (pTyr397)のウエスタンブロッティングの結果を図13Aに示す。また、FAKのバンド強度に対するpFAK (pTyr397)のバンド強度を画像解析ソフトウェアImage Jにより解析し、数値化した結果を図13Bに示す。Pol-GPOGPR上で培養した細胞ではFAKのリン酸化が見られなかったが、Pol-GFOGER上で培養した細胞ではI型コラーゲン上で培養した細胞と同程度のリン酸化が観察された。この結果から、本ペプチド配列中にインテグリンのリガンドを組み込むことで、インテグリンを介した細胞接着を誘導するだけでなく、細胞内シグナルの入力が可能であることが示された。なお、インテグリンだけでなく、DDRやHSPGのリガンドのモチーフを組み込んだ場合においても受容体特異的なシグナルの入力が可能であると考えられた。
BSA、I型コラーゲン、熱変性させたI型コラーゲン(ゼラチン)及び様々な重量比で混合したYCC2とYCC2-GFOGERの重合ペプチドを96ウェルプレートにコートし、このプレート上でMDA-MB-231を37℃で5% CO2条件下で180分間培養した。YCC2とYCC2-GFOGERは還元状態で三重らせんを形成させた後、様々な重量比で混合し、塩基性バッファーで希釈することにより酸化し、共重合させ、96ウェルプレートにコートした。細胞を培養した後、接着細胞を固定し、染色し、観察した結果を図14Aに示す。また、接着細胞数を計数し、比較した結果を図14B及び図14Cに示す。
BSA、I型コラーゲン、YCC2重合ペプチド(Pol-GPOGPR)、YCC2-GFOGER重合ペプチド(Pol-GFOGER)、YCC2-GVMGFO重合ペプチド(Pol-GVMGFO)、YCC2-KGHRGF重合ペプチド(Pol-KGHRGF)、Soluble GFOGER、Soluble GVMGFO及びSoluble KGHRGFを96ウェルプレートにコートし、このプレート上でHDFを37℃、5% CO2条件下で60分間培養した。このとき、YCC2-GFOGER、YCC2-GVMGFO及びYCC2-KGHRGFは還元状態で三重らせんを形成させた。その後、塩基性バッファーで希釈し、酸化条件で96ウェルプレート上で酸化し、重合化してコートした。細胞培養後、細胞を固定、染色して観察した結果を図15Aに示す。また、Pol-GFOGER及びPol-KGHRGFへの細胞接着がEDTA、又は三重らせん配列中のKGHRGFに対して特異的に結合することが知られているヘパリンによって阻害されるかどうかを検討した。この結果を図15B及び図15Cに示す。
YCC2-GFOGERとYCC2-GVMGFOとを様々な割合で混合し、共重合させた重合ペプチドをコートした96ウェルプレート上でHDFを培養したときの細胞接着の様子と、接着した細胞数を計数した結果を図16A及び図16Bに示す。
次に、生理活性を有するモチーフを、ペプチド鎖の側鎖にリンカーを介して結合させたコラーゲン様ペプチドの重合ペプチドとこれを含むゲルを作製し、ヒト線維芽細胞の接着性を評価した。
CCC2-KGHRGF(配列番号18)を、前記実施例2と同様の方法で合成した。このCCC2-KGHRGFを1.11 mg/mlで三重らせん形成後、10%までDMSOを加えて、上にミネラルオイルを重層し、室温で1週間程度酸化・重合させた。次に、CCC2-KGHRGFを使用した重合ペプチドを30 μg/mlに希釈して96ウェルプレートに1晩コートし、溶液を除去して、5 mg/ml NaHCO3 水溶液に溶解した1 mg/ml BS(PEG)5(Pierce社製、カタログ番号No.21581)を加え、室温で1時間程度反応させることにより、リンカーを結合させた。次に、10 mM エタノールアミン(EA)、又は、5 mg/ml NaHCO3水溶液に溶解した100 μg/ml RGD pep(配列番号22)を加え、室温で1時間程度反応させることにより、CCC2-KGHRGFに結合させたリンカーに結合させた。
前記RGDモチーフを結合させたゲルに、5 mg/ml BSAを加えて1時間程度ブロッキングした。次にヒト線維芽細胞 (HDF) 懸濁液を加えて37℃で1時間程度培養後、浮遊している細胞を培地とともに除去し、PBSで洗浄した。接着した細胞を4% p-ホルムアルデヒドで固定後、2%クリスタルバイオレット染色、水で洗浄し、風乾後、顕微鏡で観察することにより、この重合ペプチドにリンカーを介して結合させたRGDモチーフへのHDFの結合性を評価した。
実施例5と同様にKGHRGFにリンカーを結合させていない重合ペプチドのゲル(KGHRGD)は、HDFの接着性を示した。一方、このKGHRGFにリンカーを結合させたペプチドの重合ペプチド(KGHRGF-Linker)は、HDFの接着性を示さなかった。このリンカーにさらに、RGDモチーフを結合させた重合ペプチド(KGHRGF-RGD)では、HDFの接着性を示した。また、対照として使用したRGDモチーフを有する可溶性ペプチド(soluble RGD)を使用した場合には、HDFの結合性を示さなかった(図17)。
ゲルの硬さの制御用ペプチド鎖としてCCC2-GPOGPR (short)、CC2-GPOGPR (short)及びC2-GPOGPR (short)の各ペプチドより構成される3本鎖ペプチドと、生理活性モチーフを有するペプチド鎖であるCCC2-GFOGER(short)で構成される3本鎖ペプチドとを組み合わせて、酸化架橋して製造される重合ペプチドのゲルを作製し、ヒト皮膚線維芽細胞の細胞凝集活性に与える影響を評価した。
最初に、ペプチド鎖CCC2-GPOGPR (short)で構成される3本鎖ペプチドにヒト皮膚線維芽細胞接着能を有する少量のCCC2-GFOGER(short)で構成される3本鎖ペプチドを添加して、架橋重合して作製したゲルでのヒト皮膚線維芽細胞の接着性及び細胞間相互作用に基づく細胞凝集性を評価した。
CCC2-GPOGPR (short)(配列番号10)、CC2-GPOGPR (short)(配列番号23)又はC2-GPOGPR (short)(配列番号24)をそれぞれ脱気した超純水に溶解し、80oCで5分間加熱した後、4oCで一晩静置することで三重らせんを形成させた。これらを様々な割合で混合し、終濃度10%となるようにDMSOを添加し、96ウェルプレート中に50 μLずつ加えた。このとき、すべてのペプチド混合液に対して重量比1%のCCC2-GFOGER (short)(配列番号16)で構成される3本鎖ペプチドを添加し、添加していないものをLigand-freeとした。このプレートを10% DMSOで満たした容器中で4日間以上静置することでペプチド溶液をゲル化させた。これらのゲルは細胞培養培地に一晩浸すことでDMSOを希釈し、以下の細胞を用いた実験に使用した。なお、I型コラーゲンを陽性対照とした。
コラーゲン、CCC2-GPOGPR (short)の3本鎖ペプチドのみを架橋重合して製造されるゲル(ligand free)、及び、CCC2-GPOGPR (short)の3本鎖ペプチドと重量比1%のCCC2-GFOGER (short)の3本鎖ペプチドとを混合後、ゲル化して製造される重合ペプチドを含有するゲルでのヒト皮膚線維芽細胞に対する接着性を比較した結果を図18Aに示した。
次に、システイン残基数が異なるペプチド鎖を用い、各ペプチド鎖より構成される3本鎖ペプチドの各種構成比のゲルで、重量比1%のCCC2-GFOGER (short)の3本鎖ペプチドを含むゲルについて、そのゲル化能及び細胞接着能を比較、評価した。
CCC2-GPOGPR (short)、CC2-GPOGPR (short)及びC2-GPOGPR (short)のそれぞれより構成される3本鎖ペプチド中の2種の異なる3本鎖ペプチドを9:1、7:3、5:5、3:7又は1:9の配合比率で組み合わせて溶媒中で混合し、さらに、上記実験と同様に重量比1%のCCC2-GFOGER (short)より構成される3本鎖ペプチドを添加した後に、酸化架橋して3本鎖ペプチドを重合させたゲルを作製し、ゲル化能、及びこのゲルに接着するヒト皮膚線維芽細胞の細胞凝集性を上記実験と同様の方法で評価した。
CC2-GPOGPR (short):C2-GPOGPR (short)が7:3と5:5、3:7、1:9の構成比率のゲル、及びCCC2 (short):C2-GPOGPR (short)が3:7と1:9の構成比率のゲルでは、酸化架橋剤であるDMSOを添加してもゲル化しなかった。
ゲル薄膜を再水和したゲルを生体に移植し、生体中における挙動を検討し、評価した。
1.ゲル薄膜の製造と再水和
CCC2-GPOGPR (short)(配列番号10)で構成される3本鎖ペプチドとSoluble GPOGPR (short)(配列番号25)で構成される3本鎖ペプチドとを1:1の割合で混合後、DMSOで酸化架橋することにより重合ペプチドを含むゲルを製造した。このゲルを乾燥し、室温で1週間以上保存し、使用直前に生理食塩水で再水和したゲルを使用した。このとき、生分解を受けないPVDF(ポリフッ化ビニリデン)膜を移植位置のマーカーとして使用するための支持体として製造した後、下記のin vivo埋込実験に使用した。
コラーゲン様重合ペプチド薄膜をC57BL6マウス(雄性、8週齢)の背部皮下に移植した。移植実験では、イソフルランによる吸入麻酔下、マウスの背部を除毛後2cm程度の皮膚切開を加えた。切開部よりモスキート鉗子を挿入して皮下ポケットを作製し、コラーゲン様重合ペプチド薄膜をこの皮下ポケットに挿入して移植操作を完了し、ナイロン糸を用いて縫合閉鎖した。
Claims (12)
- 重合ペプチドであって、
三重らせん構造を有する3本鎖ペプチドを繰り返し単位として有し、酸化架橋で重合され、
前記3本鎖ペプチドを構成する各ペプチド鎖は、同一であっても又は相互に異なっていてもよく、
各ペプチド鎖は、-(Xaa-Yaa-Gly)-を基本単位として少なくとも5回の繰り返し構造を有する三重らせん形成用ペプチド基と、アミノ末端及びカルボキシ末端の各々から10残基以内に少なくとも2残基のシステイン(Cys)残基を含む架橋形成用ペプチド基とを有することを特徴とする、重合ペプチド。
[Xaa及びYaaは、それぞれ独立して、プロリン(Pro又はP)残基、ヒドロキシプロリン(Hyp又はO)残基、アルギニン(Arg又はR)残基、リシン(Lys又はK)残基、バリン(Val又はV)残基、ロイシン(Leu又はL)残基、イソロイシン(Ile又はI)残基、セリン(Ser又はS)残基、トレオニン(Thr又はT)残基、アラニン(Ala又はA)残基、グリシン(Gly又はG)残基、フェニルアラニン(Phe又はF)残基、メチオニン(Met又はM)残基、グルタミン酸(Glu又はE)残基、アスパラギン酸(Asp又はD)残基、アスパラギン(Asn又はN)残基、グルタミン(Gln又はQ)残基、ヒスチジン(His又はH)残基、トリプトファン(Trp又はW)残基又はチロシン(Tyr又はY)残基から選択され、プロリン残基はアミノ基又はフッ素原子で修飾されていてもよく、Xaa位及びYaa位にはN-イソブチル基グリシン残基を用いてもよい。] - 請求項1に記載の重合ペプチドであって、
前記ペプチド鎖は、
(i) 少なくとも1つの前記三重らせん形成用ペプチド基と少なくとも1つの架橋形成用ペプチド基からなるペプチド鎖、
(ii) 少なくとも1つの前記三重らせん形成用ペプチド基と少なくとも1つの架橋形成用ペプチド基、及び生理活性を有するモチーフを有する少なくとも1つのペプチド基を含むペプチド鎖、及び、
(iii) 少なくとも1つの前記三重らせん形成用ペプチド基と架橋形成用ペプチド基、及び、アミノ酸残基の側鎖にリンカーを介して生理活性を有するモチーフを結合させた少なくとも1つのペプチド基を含むペプチド鎖
からなる群から選択される少なくとも1つであることを特徴とする、重合ペプチド。 - 請求項2に記載の重合ペプチドであって、
前記生理活性を有するモチーフの2種類以上を組み合わせて製造されることを特徴とする、共重合ペプチド。 - 請求項1に記載の重合ペプチドの製造方法であって、
(Xaa-Yaa-Gly)を基本単位として少なくとも5回の繰り返し構造を有し、アミノ末端及びカルボキシ末端の各々から10残基以内に少なくとも2残基のシステイン(Cys)残基を含む相互に異なっていてもよいペプチド鎖を溶媒に溶解する工程、
自己集合により前記ペプチドの3本からなる三重らせん構造を有する3本鎖ペプチドを形成する工程、
該3本鎖ペプチドを繰り返し単位として有し、酸化架橋で重合させる工程、
を含むことを特徴とする、製造方法。
[Xaa及びYaaは、それぞれ独立して、プロリン(Pro又はP)残基、ヒドロキシプロリン(Hyp又はO)残基、アルギニン(Arg又はR)残基、リシン(Lys又はK)残基、バリン(Val又はV)残基、ロイシン(Leu又はL)残基、イソロイシン(Ile又はI)残基、セリン(Ser又はS)残基、トレオニン(Thr又はT)残基、アラニン(Ala又はA)残基、グリシン(Gly又はG)残基、フェニルアラニン(Phe又はF)残基、メチオニン(Met又はM)残基、グルタミン酸(Glu又はE)残基、アスパラギン酸(Asp又はD)残基、アスパラギン(Asn又はN)残基、グルタミン(Gln又はQ)残基、ヒスチジン(His又はH)残基、トリプトファン(Trp又はW)残基又はチロシン(Tyr又はY)残基から選択され、プロリン残基はアミノ基又はフッ素原子で修飾されていてもよく、Xaa位及びYaa位にはN-イソブチル基グリシン残基を用いてもよい。] - 請求項1~3のいずれか1項に記載の重合ペプチドの少なくとも1種を含むことを特徴とする、ゲル化剤。
- 請求項1~3のいずれか1項に記載の重合ペプチドの少なくとも1種を含むことを特徴とする、ゲル。
- 請求項1~3のいずれか1項に記載の3本鎖ペプチドを重合単位として、少なくとも2種の3本鎖ペプチドを組み合わせた重合ペプチド含むことを特徴とする、請求項6に記載のゲル。
- システイン残基を含まない3本鎖ペプチドの少なくとも1種をさらに含むことを特徴とする、請求項6又は7に記載のゲル。
- 請求項6~8のいずれか1項に記載のゲルであって、
ゲルが、ヒドロゲルであり、選択的な細胞を培養するための基材として使用されることを特徴とする、ゲル。 - 請求項6~9のいずれか1項に記載のゲルを乾燥させて製造される、重合ペプチド薄膜。
- 請求項6~9のいずれか1項に記載のゲル又は請求項10に記載の重合ペプチド薄膜を含むことを特徴とする、再生医療材料。
- 請求項11に記載の再生医療材料であって、
創傷治癒促進用組成物であることを特徴とする、再生医療材料。
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019103981A1 (en) * | 2017-11-22 | 2019-05-31 | Geltor Inc. | Methods and systems for engineering collagen |
JP2020054341A (ja) * | 2018-10-01 | 2020-04-09 | 株式会社片岡製作所 | 細胞培養器具および細胞培養器具の製造方法 |
WO2020071332A1 (ja) * | 2018-10-01 | 2020-04-09 | 株式会社片岡製作所 | 細胞培養器具および細胞培養器具の製造方法 |
US11655441B2 (en) | 2019-04-26 | 2023-05-23 | Kataoka Corporation | Cell culture base, cell culture vessel, method for culturing cells, method for producing cells, method for producing cell culture base, and method for producing cell culture vessel |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3867354A4 (en) * | 2018-10-16 | 2022-07-27 | Board of Regents, The University of Texas System | COMPOSITIONS AND METHODS FOR PRODUCTION OF TUMOR ORGANOIDS |
DE102019207859A1 (de) * | 2018-12-21 | 2020-06-25 | Gelita Ag | Synthetische und rekombinant hergestellte Kollagenpeptide mit biologischer Wirksamkeit |
CN115698054A (zh) | 2020-09-02 | 2023-02-03 | 科拉-基因制药公司 | 包含肽的组合物及其用途 |
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CN114404605A (zh) * | 2022-01-29 | 2022-04-29 | 陕西未来多肽生物科技有限公司 | 一种维c金-胶原肽纳米复合材料及其应用 |
WO2023239678A1 (en) * | 2022-06-06 | 2023-12-14 | Collagen Matrix, Inc. | Collagen-impregnated devices and methods for treatment of cancer |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5841559A (ja) * | 1980-10-03 | 1983-03-10 | ドクタ−・ル−ラント・ナハフオルガ−・ゲ−エムベ−ハ− | コラ−ゲン質包帯材 |
JPH0680935A (ja) * | 1992-06-18 | 1994-03-22 | Flamel Technol | 架橋可能な新規コラーゲン誘導体、その製造方法および生体材料の製造へのその応用 |
JPH08504463A (ja) * | 1992-12-16 | 1996-05-14 | フラメル・テクノロジー | 新規なコラーゲン誘導体、これらを製造する方法、および生体材料を製造することへのこれらの応用 |
JP2013074936A (ja) * | 2011-09-29 | 2013-04-25 | Fujifilm Corp | 血管内皮細胞遊走用足場 |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4407767A (en) | 1979-10-31 | 1983-10-04 | Monsanto Company | Drawing and beaming a weftless warp of yarns |
DE69940802D1 (de) | 1998-12-23 | 2009-06-10 | Fujifilm Mfg Europe Bv | Silberhalogenidemulsionen, die rekombinante gelatineartige Proteine enthalten |
US6992172B1 (en) | 1999-11-12 | 2006-01-31 | Fibrogen, Inc. | Recombinant gelatins |
EP1845107B1 (en) | 2003-03-28 | 2019-05-29 | FUJIFILM Manufacturing Europe B.V. | Drug delivery system comprising RGD-enriched gelatine-like proteins with enhanced cell binding |
JP5601437B2 (ja) | 2004-02-16 | 2014-10-08 | 株式会社テクノネットワーク四国 | コラーゲン様構造を有するポリペプチド |
CN100391974C (zh) * | 2006-01-09 | 2008-06-04 | 浙江理工大学 | 一种重组胶原蛋白及其合成和表达纯化方法 |
US8198047B2 (en) | 2007-02-21 | 2012-06-12 | Fujifilm Manufacturing Europe B.V. | RGD containing recombinant gelatin |
EP3044233A4 (en) | 2013-09-09 | 2017-07-19 | Commonwealth Scientific and Industrial Research Organisation | Modified bacterial collagen-like proteins |
-
2016
- 2016-06-23 CN CN201680032893.5A patent/CN107735405B/zh active Active
- 2016-06-23 EP EP16814442.6A patent/EP3315509B1/en active Active
- 2016-06-23 US US15/579,286 patent/US10851152B2/en active Active
- 2016-06-23 WO PCT/JP2016/068667 patent/WO2016208673A1/ja active Application Filing
- 2016-06-23 JP JP2017524969A patent/JP6455862B2/ja active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5841559A (ja) * | 1980-10-03 | 1983-03-10 | ドクタ−・ル−ラント・ナハフオルガ−・ゲ−エムベ−ハ− | コラ−ゲン質包帯材 |
JPH0680935A (ja) * | 1992-06-18 | 1994-03-22 | Flamel Technol | 架橋可能な新規コラーゲン誘導体、その製造方法および生体材料の製造へのその応用 |
JPH08504463A (ja) * | 1992-12-16 | 1996-05-14 | フラメル・テクノロジー | 新規なコラーゲン誘導体、これらを製造する方法、および生体材料を製造することへのこれらの応用 |
JP2013074936A (ja) * | 2011-09-29 | 2013-04-25 | Fujifilm Corp | 血管内皮細胞遊走用足場 |
Non-Patent Citations (4)
Title |
---|
BARTH, DIRK: "The Role of Cystine Knots in Collagen Folding and Stability, Part II. Conformational Properties of (Pro-Hyp-Gly)n Model Trimers with N- and C-Terminal Collagen Type III Cystine Knots", CHEM. EUR. J., vol. 9, no. 15, 28 July 2003 (2003-07-28), pages 3703 - 3714, XP055341145 * |
BOUDKO, SERGEI: "Nucleation and Propagation of the Collagen Triple Helix in Single-chain and Trimerized Peptides: Transition from Third to First Order Kinetics", J. MOL. BIOL., vol. 317, 2002, pages 459 - 470, XP004468980 * |
See also references of EP3315509A4 * |
WEGENER, HENRIK: "The cysteine rich region of type VII collagen is a cystine knot with a new topology", J. BIOL. CHEM., vol. 289, no. 8, 2014, pages 4861 - 4869, XP055341153 * |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019103981A1 (en) * | 2017-11-22 | 2019-05-31 | Geltor Inc. | Methods and systems for engineering collagen |
GB2582108A (en) * | 2017-11-22 | 2020-09-09 | Geltor Inc | Methods and systems for engineering collagen |
EP3713953A4 (en) * | 2017-11-22 | 2021-08-25 | Geltor, Inc. | METHODS AND SYSTEMS FOR GROWING COLLAGEN |
GB2582108B (en) * | 2017-11-22 | 2022-08-17 | Geltor Inc | Methods and systems for engineering collagen |
JP2020054341A (ja) * | 2018-10-01 | 2020-04-09 | 株式会社片岡製作所 | 細胞培養器具および細胞培養器具の製造方法 |
WO2020071332A1 (ja) * | 2018-10-01 | 2020-04-09 | 株式会社片岡製作所 | 細胞培養器具および細胞培養器具の製造方法 |
JP2020156518A (ja) * | 2018-10-01 | 2020-10-01 | 株式会社片岡製作所 | 細胞培養器具および細胞培養器具の製造方法 |
US11655441B2 (en) | 2019-04-26 | 2023-05-23 | Kataoka Corporation | Cell culture base, cell culture vessel, method for culturing cells, method for producing cells, method for producing cell culture base, and method for producing cell culture vessel |
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US20180319867A1 (en) | 2018-11-08 |
EP3315509A4 (en) | 2019-01-09 |
US10851152B2 (en) | 2020-12-01 |
CN107735405B (zh) | 2022-04-08 |
CN107735405A (zh) | 2018-02-23 |
EP3315509B1 (en) | 2021-02-17 |
JP6455862B2 (ja) | 2019-01-23 |
JPWO2016208673A1 (ja) | 2018-04-19 |
EP3315509A1 (en) | 2018-05-02 |
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