Composition

Abstract

This invention provides a material that utilizes the self-healing properties and / or high bioadhesion of elastin-like peptides. A composition comprising: (Sequence No. 1: X) 1 GGX 2 G) n The G-sequence blocks formed (where X) 1 Same or different, indicating V or L, X 2 Same or different, representing V or L, n represents an integer greater than 4. ) and by (Sequence number 2: VPGX 3 G) m The P-sequence block formed (where X) 3 ) represents any amino acid that is the same or different, and m represents an integer greater than 5. ) elastin-like block peptide sequences and polypeptides having amino groups, and water-soluble polymers having aldehyde groups.

Description

Technical Field

[0001] This invention relates to compositions that can be used in medical devices for the protection of sutures, cut surfaces of organs or tissues, and damaged areas in the body. Background Technology

[0002] In gastrointestinal anastomosis, there is a problem of incomplete suturing, which can lead to leakage of digestive fluids due to rupture of the suture site postoperatively. Incomplete suturing has been reported to occur in 4–21% of cases, and depending on the circumstances, can sometimes cause peritonitis or even death. Furthermore, leakage of bodily fluids such as blood and tissue fluid can occur at the cut surfaces created during organ removal procedures such as pancreas or liver resection. Therefore, the suture site or the aforementioned cut surfaces require protection with a suitable material.

[0003] Fibrin glue is sometimes used as a protective material. This is a paste-like material made by combining fibrinogen with various agents such as thrombin, and is applied to the affected area by coating the liquid. However, the manufacture of fibrin glue uses blood plasma as a raw material, thus raising concerns about the risk of infection and the instability of raw material supply.

[0004] In addition, nonwoven fabric made of polyglycolic acid is sometimes used as a protective material (Non-Patent Document 1). However, since it is not liquid, it cannot freely change shape according to the object being protected.

[0005] Existing technical documents Non-patent literature Non-patent literature 1: Journal of the American College of Surgeons. 2016; 222(1): 59-64 Non-patent literature 2: International Journal of Molecular Sciences, 22, 4104 (12 pages) (2021) Summary of the Invention

[0006] The problem that the invention aims to solve In Non-Patent Literature 2, an elastin-like polypeptide, created by simplifying the amino acid sequence of elastin, self-assembles into nanofibers to form a hydrogel upon heating in water. Furthermore, this gel has been reported to exhibit self-healing (thixotropic) properties, liquefying upon oscillation and re-gelling upon resting. The inventors of this invention believe that by utilizing such an elastin-like polypeptide, it is possible to create a material that can be liquid-coated onto a protected object, gels to strengthen the protected area, and promote healing. However, the self-healing strength and bioadhesion of the elastin-like polypeptide gel alone are insufficient.

[0007] The subject of this invention is to provide a material with high strength and / or bioadhesion after self-repairing by elastin-like peptides.

[0008] Technical solutions for solving the problem The inventors of this invention conducted in-depth research on the aforementioned issues and discovered that if it contains: including (sequence number 1: X) 1 GGX 2 G) n The G-sequence blocks formed (where X) 1 Same or different, indicating V or L, X 2 Same or different, representing V or L, n represents an integer greater than 4. ) and by (Sequence number 2: VPGX 3 G) m The P-sequence block formed (where X) 3 "Same or different" refers to any amino acid, and "m" represents an integer greater than 5. A composition of a polypeptide having an amino group and an aldehyde group, along with an elastin-like block peptide sequence, can solve the aforementioned problem. Based on this discovery, the inventors of this invention conducted further research and completed this invention. That is, this invention includes the following embodiments.

[0009] Item 1. A composition comprising: comprising (Serial No. 1: X) 1 GGX 2 G) n The G-sequence blocks formed (where X) 1 Same or different, indicating V or L, X 2 Same or different, representing V or L, n represents an integer greater than 4. ) and by (Sequence number 2: VPGX 3 G) m The P-sequence block formed (where X) 3 "Same or different" represents any amino acid, and "m" represents an integer greater than 5. (This refers to) elastin-like block peptide sequences containing amino groups, and water-soluble polymers containing aldehyde groups.

[0010] Item 2. The composition as described in Item 1, wherein it is a gel or gel-forming sol composition.

[0011] Item 3. The composition as described in Item 1, wherein the polypeptide comprises amino acid residues having an amino group.

[0012] Item 4. The composition of Item 3, wherein the polypeptide comprises a cross-linked sequence block comprising two or more amino acid residues having an amino group.

[0013] Item 5. The composition of Item 1, wherein the polypeptide comprises a cell-adhesive sequence block.

[0014] Item 6. The composition as described in Item 1, wherein the water-soluble polymer comprises sugar as a structural unit.

[0015] Item 7. The composition as described in Item 6, wherein the water-soluble polymer is a polysaccharide oxide.

[0016] Item 8. The composition as described in Item 6, wherein the water-soluble polymer is oxidized dextran and / or oxidized dextrin.

[0017] Item 9. The composition of Item 1, wherein the polypeptide and the water-soluble polymer are linked by covalent bonds.

[0018] Item 10. A method of manufacturing a composition for manufacturing any one of items 1 to 9, the method comprising: (a) mixing a sol-gel composition of a gel-like composition with a water-soluble polymer having an aldehyde group, wherein the sol-gel composition of the gel-like composition comprises: (Serial No. 1: X) 1 GGX 2 G) n The G-sequence blocks formed (where X) 1 Same or different, indicating V or L, X 2 Same or different, representing V or L, n represents an integer greater than 4. ) and by (Sequence number 2: VPGX 3 G) m The P-sequence block formed (where X) 3 "Same" or "different" represents any amino acid, and "m" represents an integer greater than 5. (This refers to a polypeptide with an amino group that is an elastin-like block peptide sequence.)

[0019] Item 11. A medical device comprising any one of items 1 to 9.

[0020] Item 12. The medical device as described in Item 11, used for protection or adhesion of sutures, cut surfaces of organs or tissues, or injured areas.

[0021] Invention Effects According to the present invention, it is possible to provide a material with high strength and / or bioadhesion after self-repair by utilizing elastin-like peptides. Attached Figure Description

[0022] Figure 1 A schematic diagram showing the aluminum test piece used in the lap shear test in section 1.4.4.

[0023] Figure 2 This indicates the FTIR spectral measurement results in section 2.1.1.

[0024] Figure 3This indicates the SDS-PAGE results from section 2.2.1.

[0025] Figure 4 This indicates the NMR spectral measurement results in section 2.2.2.

[0026] Figure 5 This indicates the results of the recovery behavior test in section 2.2.3.

[0027] Figure 6 This indicates the results of the lap shear test in section 2.2.4.

[0028] Figure 7 This indicates the results of the in vivo gelation assay in section 2.2.5. Detailed Implementation

[0029] In this specification, the expressions “containing” and “including” include the concepts of “containing”, “including”, “substantially constituted by” and “consistent only by”.

[0030] In this specification, amino acid residues in the amino acid sequence are sometimes simply referred to as amino acids or specific amino acid names (valine, leucine, etc.). Additionally, amino acid residues in the amino acid sequence are sometimes represented by single-letter labels for amino acids.

[0031] In one aspect, the present invention relates to a composition comprising: comprising (Serial No. 1: X) 1 GGX 2 G) n The G-sequence blocks formed (where X) 1 Same or different, indicating V or L, X 2 Same or different, representing V or L, n represents an integer greater than 4. ) and by (Sequence number 2: VPGX 3 G) m The P-sequence block formed (where X) 3 "Same or different" refers to any amino acid, and m represents an integer greater than 5. This includes polypeptides with an amino group (sometimes referred to as "the polypeptide of the present invention") and water-soluble polymers with an aldehyde group (sometimes referred to as "the composition of the present invention"). This will be described below.

[0032] The G sequence block is simply an amino acid sequence (X) as indicated by sequence number 1. 1 GGX 2 Repeated sequences of G (X) 1 GGX 2 G) nAny block can be constructed; there are no particular restrictions. G-sequence blocks are typically blocks capable of forming β-sheet structures. G-sequence blocks can endow peptides with the ability to self-assemble into filaments.

[0033] X 1 Same or different, indicating V or L, X 2 Same or different, indicated by V or L. X 1 and X 2 V is preferred.

[0034] n represents an integer of 4 or higher. By making n 4 or higher, the polypeptide of the present invention can form a fibrous self-assembled structure. n is preferably 4 to 20, more preferably 4 to 12, further preferably 4 to 8, and even more preferably 4 to 6.

[0035] The P sequence block is simply an amino acid sequence (VPGX) as shown in sequence number 2. 3 Repeated sequences of G (VPGX) 3 G) m Any block can be constructed; there are no particular restrictions. P-sequence blocks are typically blocks capable of forming β-turn structures. Through P-sequence blocks, the polypeptide possesses a minimum critical solubility temperature (LCST), at which point it will self-assemble and separate from the aqueous phase.

[0036] The LCST (solvent: water, concentration: 0.03 wt%) of the polypeptide of the present invention is, for example, 10 to 30°C, preferably 15 to 25°C.

[0037] X 3 Same or different, representing any amino acid. As X 3 The amino acids shown can be categorized as follows: lysine, arginine, histidine, and other amino acids with basic side chains; aspartic acid, glutamic acid, and other amino acids with acidic side chains; glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and other amino acids with non-electrified polar side chains; alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and other amino acids with non-polar side chains; threonine, valine, isoleucine, and other amino acids with β-branched side chains; and tyrosine, phenylalanine, tryptophan, histidine, and other amino acids with aromatic side chains. 3 The amino acids shown are preferably amino acids other than proline.

[0038] Through X 3 The amino acids shown can regulate LCST. In X 3If the amino acid shown is a hydrophilic amino acid, the LCST will become too high, and therefore is not preferred. From the viewpoint of easily adjusting the LCST to a suitable (physiologically acceptable) temperature (e.g., 30–40°C), the X in the P sequence block is preferred. 3 Part of the amino acids shown are non-aromatic hydrophobic amino acids such as valine and leucine (preferably valine), while the other amino acids are aromatic hydrophobic amino acids such as phenylalanine and tryptophan (preferably phenylalanine). At this time, X in the P sequence block... 3 The number of non-aromatic hydrophobic amino acids shown relative to X 3 The proportion of the total number is, for example, 60-95%, preferably 70-80%, more preferably 75-85%, X in the P sequence block 3 The number of aromatic hydrophobic amino acids shown relative to X 3 The proportion of the total number is, for example, 5-40%, preferably 20-30%, and more preferably 15-25%. Furthermore, in this case, in the P sequence block, X... 3 The aromatic hydrophobic amino acids shown are preferably dispersed as much as possible, for example, preferably in repeating units (VPGX) of 2 to 8, more preferably 3 to 7, more preferably 4 to 6, and even more preferably 5. 3 The proportion of G) with 1 repetition occurs, and X appears. 3 It is a repeating unit of an aromatic hydrophobic amino acid.

[0039] m represents an integer greater than or equal to 5. m is preferably 10 to 100, more preferably 10 to 50, even more preferably 15 to 35, and particularly preferably 20 to 30.

[0040] The elastin-like block peptide sequence only needs to contain G sequence blocks and P sequence blocks, and there are no particular restrictions. The number of G sequence blocks in the elastin-like block peptide sequence is not particularly limited, for example, 1 to 5, preferably 2 to 5, more preferably 2 to 4, further preferably 2 to 3, and particularly preferably 2. The number of P sequence blocks in the elastin-like block peptide sequence is not particularly limited, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and further preferably 1 to 2.

[0041] Segments (between G-sequence segments and P-sequence segments, between G-sequence segments and G-sequence segments, and between P-sequence segments and P-sequence segments) can be directly connected or separated by a connector sequence. Preferably, segments are separated by a connector sequence.

[0042] As the linker sequence, there are no particular limitations as long as it does not significantly reduce the elastin-like block peptide sequence's ability to form fibers through self-assembly. Generally, any amino acid or amino acid sequence can be used without significant restrictions. The amino acid length of the linker sequence is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, further preferably 1 to 8, and even more preferably 2 to 8. Specific examples of the linker sequence include the amino acid sequence shown in sequence number 7: LWLGSG, the amino acid sequence shown in KL, etc. Amino acid sequences obtained by performing one or more (e.g., 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1) amino acid mutations (e.g., substitution, deletion, insertion, addition, preferably substitution, more preferably conservative substitution) on these amino acid sequences can also be used.

[0043] In this specification, "conservative substitution" refers to the substitution of an amino acid by an amino acid with a similar side chain. For example, substitution between amino acids with basic side chains, such as lysine, arginine, and histidine, is considered conservative substitution. Furthermore, substitution between amino acids with acidic side chains, such as aspartic acid and glutamic acid; amino acids with non-electrolyte polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acids with non-polar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acids with β-branched side chains, such as threonine, valine, and isoleucine; and amino acids with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine, is also considered conservative substitution.

[0044] From the viewpoint of fibrous formation capability through self-assembly, the sequence structure of the elastin-like block peptide sequence, starting from the N-terminus, is preferably G-sequence block-P-sequence block-G-sequence block, G-sequence block-P-sequence block-G-sequence block, P-sequence block-G-sequence block, or G-sequence block-P-sequence block. This sequence structure is particularly preferred as G-sequence block-P-sequence block-G-sequence block, or G-sequence block-P-sequence block-P-sequence block-G-sequence block. In these structures, a "-" indicates a direct link (between blocks) or other sequences (e.g., linker sequences).

[0045] The polypeptide of the present invention can have a cell-adhesive sequence block at its end. There are no particular limitations on the cell-adhesive sequence block, and various sequences can be used. The cell-adhesive sequence block can also be a non-cell-selective sequence such as GRGDS (sequence number 9) or RGDS (sequence number 10), or a cell-selective sequence (e.g., REDV (sequence number 22)). Examples of cell-selective sequences include HHH, VVV, TTT, TGA, NNN, KKK, AAA, RRR, YYY, TTT, GAT, GGG, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, LGL, AVG, GHP, GLI, GVG, GPS, SPG, GPP, GIS, GYL, GEK, QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR, PCG, GDV, IGG, CDG, AVA, FLM, GFD, GTP, GPY, VSG, DGR, GIT, GFL, ASG, GCP, NQG, SGL, GGA, PDG, QAL, GLK, GSP, and GEP. GNS, AKG, DGY, TGP, VGP, SLW, AAG, AGA, ARG, GRD, EGF, GSC, PGQ, HSQ, EAP, RGP, PGD, CNI, GFG, GPT, GDQ, KGE, PFI, QGP, SYW, LPGFPGLK (sequence number 11), LPGFPGTP (sequence number 12), GPPGLSGPP (sequence number 13), FPGPPGPP (sequence number 14), LPGLPGPP (sequence number 15), FPGLPGPP (sequence number 16), GPPGPPGSPG (sequence number 17), LPGPPGPP (sequence number 18), FPGSPGFPG (sequence number 19), GSPGLPGTP (sequence number 20), and IGLSGEKG (sequence number 21), etc.

[0046] In the case where the polypeptide of the present invention comprises a cell adhesion sequence block, the cell adhesion sequence is disposed at the end (N-terminus and / or C-terminus) of the polypeptide of the present invention, preferably at the C-terminus. The cell adhesion sequence may be disposed at only one end of the N-terminus and the C-terminus, or at both ends. In a preferred embodiment of the present invention, the cell adhesion sequence is disposed at only one end.

[0047] In the case where the polypeptide of the present invention comprises a cell adhesion sequence block, the elastin-like block peptide sequence and the cell adhesion sequence can be directly linked, or other amino acid sequences (e.g., sequences of 3-30, 7-25, or 10-20 amino acids in length) can exist between them. From the viewpoint of cell adhesion, sequences rich in hydrophilic amino acids are preferred as other amino acid sequences. In other words, it is preferable to arrange a sequence rich in hydrophilic amino acids on the opposite side of the end of the cell adhesion sequence. The sequence rich in hydrophilic amino acids is not particularly limited as long as it has a high proportion of hydrophilic amino acids; for example, amino acid sequences of 3-15 or 5-10 amino acids in length containing 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of hydrophilic amino acids can be listed. From the viewpoint of facilitating the purification of the polypeptide of the present invention, histidine continuous sequences (His-tagged sequences) are preferred as sequences rich in hydrophilic amino acids. Other amino acid sequences preferably contain amino acids having functional groups capable of cross-linking reactions (e.g., lysine, cysteine, etc.).

[0048] The polypeptide of the present invention has an amino group. The amino group can be an amino group present in the amino acids constituting the polypeptide of the present invention, or it can be an amino group added through chemical modification. The polypeptide of the present invention preferably contains amino acid residues having an amino group. Examples of amino acids having an amino group include, for example, lysine, arginine, and histidine. Preferably, the amino acid with a side chain of -NH is included. 3 The substituted amino acid (e.g., alkyl, preferably alkyl with 1 to 8 carbon atoms) is particularly preferred, with lysine being an example.

[0049] It can be assumed that the peptides of the present invention are covalently linked to the water-soluble polymer by reacting the amino groups with the aldehyde groups of the water-soluble polymer (described later) to form chemical bonds, resulting in higher self-healing strength and / or bioadhesiveness. It is known that the peptides of the present invention form self-healing gels, but by linking other polymers thereto, the structure of the gel-forming fibers changes, raising concerns that the self-healing properties may be compromised. However, unexpectedly, even when the peptides of the present invention are combined with the water-soluble polymer, the self-healing properties are not compromised.

[0050] In the case where the polypeptide of the present invention contains an amino acid residue having an amino group, the amino acid residue may be present within the elastin-like block peptide sequence (e.g., within the P-sequence block, within the linker, etc.) or outside the elastin-like block peptide sequence. Preferably, the polypeptide of the present invention may contain a cross-linked sequence block including an amino acid residue having an amino group, in addition to other sequence blocks. The cross-linked sequence block can further improve the strength and / or bioadhesion after self-repair.

[0051] The cross-linked sequence block may contain preferably 2 or more, more preferably 2 to 10, further preferably 2 to 5, and even more preferably 2 to 3 amino acid residues having an amino group. The ratio of the number of amino acid residues in the cross-linked sequence block to the total number of amino acid residues is, for example, 20% or more, preferably 30% or more, more preferably 40% or more, and also, for example, 80% or less, preferably 70% or less, more preferably 60% or less. In a preferred embodiment, the cross-linked sequence block is KAAK (Sequence Number 8).

[0052] In the case where the polypeptide of the present invention contains a cross-linked sequence block, its position is not particularly limited. In one aspect of the present invention, the cross-linked sequence block is preferably disposed on the C-terminal side of the elastin-like block peptide sequence, and in the case where a cell adhesion sequence block is included, it is preferably disposed between the elastin-like block peptide sequence and the cell adhesion sequence block.

[0053] The polypeptides of this invention can be chemically modified polypeptides, provided they do not significantly impair the property of forming thixotropic gels. The presence or absence of the property of forming thixotropic gels can be evaluated according to the examples or existing reports.

[0054] The C-terminus of the polypeptide of the present invention may be a carboxyl group (-COOH) or a carboxylate group (-COO). － Any one of amide (-CONH2) or ester (-COOR).

[0055] Here, the R in ester can be, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1－6 Alkyl groups; such as cyclopentyl, cyclohexyl, etc. (C60) 3－8 Cycloalkyl groups; such as phenyl, α-naphthyl, etc. 6－12 Aryl groups; such as benzyl, phenethyl, etc. phenyl-C 1－2 Alkyl groups; α-naphthylmethyl and other α-naphthyl-C 1－2 Alkyl and other C 7－14 Aryl alkyl groups; pivaloyloxymethyl, etc.

[0056] The carboxyl group (or carboxyl group) other than the C-terminus of the polypeptide of the present invention can be amidated or esterified. As the ester in this case, for example, the C-terminal ester described above can be used.

[0057] Furthermore, the polypeptide of the present invention includes: an amino group (e.g., formyl, acetyl, etc.) protecting the amino group of the N-terminal amino acid residue. 1－6 alkyl acyl and other C 1－6Peptides protected by acyl groups, etc.; peptides whose N-terminal glutamine residues are oxidized by pyroglutamine to form peptides that can be cleaved in vivo; and peptides whose side chains of amino acids are protected by appropriate groups (e.g., formyl, acetyl, etc.). 1－6 alkyl acyl and other C 1－6 Polypeptides protected by acyl groups, etc., or complex proteins such as so-called glycoproteins bound with glycan chains.

[0058] The polypeptides of this invention can be in the form of pharmaceutically acceptable salts of acids or bases. The salt is not particularly limited as long as it is pharmaceutically acceptable, and can be either an acidic salt or a basic salt. Examples of acidic salts include: inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and p-toluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include: alkali metal salts such as sodium and potassium salts; and alkaline earth metal salts such as calcium and magnesium salts.

[0059] The polypeptides of this invention can be in the form of a solvate. The solvent is any pharmaceutically acceptable substance and is not particularly limited; examples include water, ethanol, glycerol, and acetic acid.

[0060] The polypeptides of the present invention can be readily manufactured according to or based on known genetic engineering methods, or according to or based on known chemical synthesis methods. For example, they can be manufactured using PCR, restriction endonuclease digestion, DNA ligation technology, in vitro transcription / translation technology, recombinant protein production technology, solid-phase synthesis technology, peptide linking technology, etc.

[0061] The polypeptides of the present invention can be a single type or a combination of two or more types.

[0062] The concentration of the polypeptide of the present invention in the composition of the present invention, from the viewpoint of gel formation, self-healing properties, etc., is, for example, 0.2 w / v% or more, preferably 0.4 w / v% or more, more preferably 0.5 w / v% or more, and also, for example, 3.0 w / v% or less, preferably 2.0 w / v% or less, more preferably 1.5 w / v% or less, and even more preferably 1.2 w / v% or less.

[0063] Water-soluble polymers are any polymers that have aldehyde groups and are water-soluble; there are no particular restrictions.

[0064] The water solubility is not particularly limited as long as it is sufficient to form a gel-like structure in the composition of the present invention using water as a solvent. For example, the solubility in 100g of water at 23°C is, for example, 1g or more, preferably 2g or more, more preferably 5g or more, and even more preferably 10g or more.

[0065] The viscosity-average molecular weight of the polymer is not particularly limited, for example, it is 5,000 to 300,000. From the viewpoint of self-healing strength and / or bioadhesion, the viscosity-average molecular weight is preferably 10,000 to 200,000, more preferably 15,000 to 150,000, further preferably 20,000 to 100,000, even more preferably 25,000 to 80,000, particularly preferably 30,000 to 60,000, and especially preferably 30,000 to 50,000. The viscosity-average molecular weight can be determined according to the type of polymer using the most suitable method.

[0066] There is no particular limitation on the number of aldehyde groups; for example, it is 0.1 to 100 per 1000 viscosity-average molecular weight of the polymer. This number is preferably 0.5 to 70 per 1000 viscosity-average molecular weight of the polymer, more preferably 1 to 50, further preferably 2 to 30, even more preferably 3 to 20, and particularly preferably 5 to 15. The number of aldehyde groups can be determined according to the type of polymer using the most suitable method. For example, if the polymer is obtained by oxidizing polysaccharides to generate aldehyde groups on sugar residues, the number of aldehyde groups can be calculated by measuring the degree of oxidation.

[0067] There are no particular limitations on the structural units (constituting monomers) of the water-soluble polymer. Preferably, the water-soluble polymer contains sugars as structural units. In this case, the number of sugars (sugar residues) per 100 structural units is preferably 50 or more, more preferably 70 or more, further preferably 80 or more, even more preferably 90 or more, and particularly preferably 95 or more. The water-soluble polymer is particularly preferably a polysaccharide or its oxide.

[0068] Oxides of polysaccharides can be manufactured according to or based on known methods. For example, polysaccharide oxides can be obtained by reacting polysaccharides in the presence of periodate.

[0069] There are no particular limitations on the type of sugar as a structural unit, the structure of the polymer (straight-chain or branched), or the type of bonds between structural units (e.g., in the case of polysaccharides, α bonds, β bonds, and the numbering of carbon atoms related to bond formation). In a preferred embodiment of the invention, the sugar as a structural unit is glucose, the polymer structure is branched, and the type of bonds between structural units is α bonds (including α-1,6 bonds and α-1,4 bonds). Water-soluble polymers are particularly preferred to be oxidized dextran and / or oxidized dextrin.

[0070] The water-soluble polymer is preferably a water-soluble polymer that does not have an electric charge.

[0071] Water-soluble polymers can be a single type or a combination of two or more types.

[0072] The concentration of the water-soluble polymer in the composition of the present invention, from the viewpoint of gel formation, self-healing properties, etc., is, for example, 0.5 w / v% or more, preferably 1.0 w / v% or more, more preferably 1.5 w / v% or more, and even more preferably 2.0 w / v% or more, and also, for example, 10.0 w / v% or less, preferably 7.0 w / v% or less, more preferably 5.0 w / v% or less, and even more preferably 4.0 w / v% or less.

[0073] The compositions of the present invention may contain other ingredients. Examples of other ingredients include, for instance, cells, pharmaceuticals, etc.

[0074] The compositions of the present invention can be in either a dry or wet form, but from the viewpoint of being readily usable as medical devices, a gel-like or gel-forming sol-like composition is preferred. In this case, for example, the compositions of the present invention may contain a covalently linked peptide of the present invention and a water-soluble polymer, and the peptide of the present invention may be capable of self-assembling to form nanofibers. Preferably, the compositions of the present invention contain a covalently linked nanofibers formed from the peptide of the present invention and a water-soluble polymer.

[0075] The compositions of the present invention can be manufactured by various methods. From the viewpoint that the compositions of the present invention are particularly readily available as self-healing gels or gel-forming sols, the compositions of the present invention are preferably manufactured by a method comprising the step of mixing a sol-gelled composition containing the polypeptides of the present invention with a water-soluble polymer having aldehyde groups.

[0076] After dissolving the polypeptide of the present invention in a solvent at a temperature below the LCST (e.g., 0–15°C, 2–10°C), the resulting solution is heated to above the LCST (e.g., 30–50°C, 30–45°C, 30–40°C) and allowed to stand for a certain period of time (e.g., 1–300 hours, preferably 8–120 hours, more preferably 12–80 hours) to obtain a gel-like composition containing the polypeptide of the present invention. Water can be used as the solvent, but a mixture of water and an organic solvent can also be used. In addition, monosaccharides such as sucrose are preferably added to the solvent (e.g., final concentration 2–20 w / v%, 5–15 w / v%).

[0077] Sol-gel compositions of gel-like compositions can be obtained by applying mechanical strain (e.g., blowing, vortexing, ultrasonic stirring, etc.) to the gel-like composition. These sol-gel compositions gel without or with minimal mechanical strain, and therefore can be said to possess gel-forming ability.

[0078] The water-soluble polymer with aldehyde groups is preferably pre-dissolved in a solvent such as water before mixing. In addition, monosaccharides such as sucrose (e.g., final concentration of 2-20 w / v%, 5-15 w / v%) are preferably added to the solvent.

[0079] The ratio of the volume of the sol-gel composition to the volume of the water-soluble polymer solution is, for example, 1 to 10, preferably 2 to 6, and more preferably 3 to 5.

[0080] There are no particular limitations on the mixing method of the sol-gel composition and the water-soluble polymer; for example, it can be blown, swirled, or ultrasonically stirred.

[0081] After mixing, by allowing the mixture to stand at a temperature above LCST for a certain period of time as described above, a gel-like composition of the present invention can be obtained.

[0082] The compositions of the present invention can be used for a variety of purposes, but are particularly preferred for use as medical devices. Therefore, in one aspect, the present invention relates to a medical device comprising the compositions of the present invention (the medical device of the present invention).

[0083] Medical devices are any mechanical instruments intended for the diagnosis, treatment or prevention of diseases in humans or animals, or for the purpose of affecting the structure or function of the human or animal body, and there are no special restrictions.

[0084] There are no particular restrictions on the biological species to be included; examples include mammals such as humans, monkeys, dogs, cats, horses, cattle, pigs, sheep, mice, rats, and rabbits, or vertebrates including them.

[0085] The compositions of the present invention are gel-like or capable of forming a gel, or capable of forming a sol-like state by applying strain, and capable of returning to a gel-like state when the strain is removed. Therefore, the compositions of the present invention are suitable for use as materials that can be applied in a liquid state to a protected object, gelling to strengthen the protected portion while promoting healing. From this viewpoint, the compositions of the present invention are preferably used in medical devices for the protection or adhesion of sutures, cut surfaces of organs or tissues, or injured areas.

[0086] The suture can be located anywhere inside or outside the body, but is preferably located inside the body. More specifically, the suture is for example, a suture of the digestive tract, blood vessels, nerves, bile ducts, pancreatic ducts, ureters, skin, etc., with a preference for a suture of the digestive tract. Protection of the suture is not particularly limited as long as it applies to the suture and its surrounding area; more specifically, it involves physical reinforcement in a way that prevents the suture from breaking. This helps to suppress and prevent incomplete suturing.

[0087] A cut surface is the cut surface created when a part of an organ or tissue is removed. Examples of organs or tissues include the pancreas, liver, kidneys, blood vessels, lungs, thyroid gland, and prostate. Protection of the cut surface is not particularly limited as long as it is applied in a manner that inhibits and prevents leakage of bodily fluids from the cut surface. In one embodiment, a method of patching the cut surface can be considered, for example, by applying the composition of the present invention to the cut surface of an organ such as the spleen, allowing nearby tissues (e.g., adipose tissue, intestines, etc.) to adhere thereto.

[0088] The injury site can be anywhere inside or outside the body. The location of the injury site is based on the aforementioned suture and cut surface. Protection of the injury site is not particularly limited as long as it is applied to the cut surface in a manner that can inhibit and prevent leakage of bodily fluids from the cut surface.

[0089] The compositions of the present invention can be used by applying or spraying them onto the object to be protected in a sol-like form. The compositions of the present invention can gel even under strain conditions that induce movement in the organism, and due to this excellent property, they remain on the object to be protected, thus providing a protective function.

[0090] Example The present invention will now be described in detail based on embodiments, but the present invention is not limited to these embodiments.

[0091] 1. Experimental Procedure 1.1. Preparation of Oxidized Dextran (OD) Dextran (40000. Viscosity-average molecular weight 40000, Wako Pure Chemical Industries, Ltd., Japan) was dissolved in 50 mL of ultrapure water. 1.6 g of sodium periodate (Wako Pure Chemical Industries, Ltd., Japan) was added as an oxidant, and the mixture was stirred in the dark for 3.5 hours. To stop the oxidation reaction, 0.6 mL of glycerol (Wako Pure Chemical Industries, Ltd., Japan) was added, and the mixture was stirred for another hour. The solution was then added to a dialysis membrane (Spectra / Por (registered trademark) 1 Dialysis Membrane MWCO : 6-8000., Spectrum Laboratories, Inc., USA), and dialyzed in 2 L of ultrapure water at 4 °C. The ultrapure water was replaced three times every 24 hours. The solution was then frozen at -80°C and dried using a freeze dryer (FDU-1200., TOKYO RIKAKIKAI CO.,LTD., Japan) to obtain a powdered sample.

[0092] 1.2. Evaluation of OD For dextran and the OD prepared in 1.1., FT-IR measurements were performed using a Fourier transform infrared spectrometer (FT / IR-610, JASCO., Japan). Each sample was weighed with potassium bromide (Wako Pure Chemical Industries, Ltd., Japan) at a ratio of 1:49, mixed using a mortar and pestle, and the mixture was used as the test sample for KBr-based spectroscopic determination.

[0093] 1.2.1. Titration of Hydroxylamine Hydrochloride (HA-HCl) To determine the oxidation degree of the OD prepared in 1.1, titration was performed using HA-HCl. HA-HCl (Tokyo Chemical Industries, Ltd., Japan), NaOH (Nakarai tesque, Inc., Japan), and methyl orange (Wako Pure Chemical Industries, Ltd., Japan) were dissolved in ultrapure water to prepare 0.25M HA-HCl aqueous solution, 1M NaOH aqueous solution, and 0.1 mg / mL methyl orange aqueous solution, respectively. 100 mg of OD was dissolved in 25 mL of HA-HCl aqueous solution and the solution was shaken at room temperature for 3 hours. A few drops of methyl orange aqueous solution were added as an indicator, and titration was performed using NaOH aqueous solution until the color matched that of the HA-HCl solution in which OD was not dissolved. The oxidation degree was calculated using the following formula. 1.3. Preparation of GPG-KR / OD composite hydrogel 1.3.1. Synthesis of polypeptide (GPG-KR) Synthesize the following polypeptide (GPG-KR): The sequence contains a G sequence block consisting of (sequence number 3: VGGVG)5 starting from the N-terminus and (sequence number 4: VPGXG). 25 The sequence of the P-sequence blocks (where X is the same or different, representing V or F) and the G-sequence blocks composed of (Sequence No. 3: VGGVG)5, and the polypeptide (Sequence No. 5) consisting of elastin-like block peptide sequences linked by linker sequences, is further divided into a crosslinking sequence (Sequence No. 8: KAAK) and a cell adhesion sequence (Sequence No. 9: GRGDS) at the C-terminus of the polypeptide (Sequence No. 6). GPG-KR is a polypeptide composed of known amino acid sequences.

[0094] Specifically, the E. coli BLR(DE3) strain was transformed with plasmid DNA encoding the peptide to express the peptide. The peptide was purified by metal ion affinity chromatography using His-tag. The target peptide was confirmed by SDS-PAGE and MALDI-TOF-MS.

[0095] The recovered peptide solution was added to Spectra / Por (registered trademark) 1 Dialysis Membrane MWCO: 6-8000 (Spectrum Laboratories) and dialyzed in 2 L of ultrapure water at 4°C. The ultrapure water was changed at least twice every 1 hour, then twice every 2 hours, and finally once every 1.5 hours, for a total of 5 dialyzes. After dialyzing, the peptide solution was filtered through a syringe fitted with a Minisart (registered trademark) Syringe Filter 0.2 μm (Sartorius Stedim Biotech) to remove insoluble peptides, and then frozen overnight at -80°C. Finally, it was freeze-dried using an FDU-1200 freeze dryer (TOKYORIKAKIKAI) to obtain GPG-KR powder.

[0096] 1.3.2. Preparation of GPG-KR hydrogel Sucrose (Wako Pure Chemical Industries, Ltd., Japan) was dissolved in ultrapure water to prepare a 100 mg / mL sucrose aqueous solution. Using this solution as a solvent, GPG-KR was added under ice bath conditions, and the solution was shaken at 4°C for 6 hours to ensure complete dissolution. The peptide concentration was adjusted to 10 mg / mL using a NanoDrop2000, and the solution was allowed to stand at 45°C for 3 days to gel.

[0097] 1.3.3. Preparation of GPG-KR / OD composite hydrogel The OD prepared in section 1.1. was dissolved in a 100 mg / mL sucrose aqueous solution to prepare a 150 mg / mL OD solution. The GPG-KR gel prepared in section 1.3.2. was liquefied by vortexing and then ultrasonically stirred (40 kHz, 5 min). The two solutions were then mixed at a ratio of GPG-KR solution:OD solution = 4:1, and ultrasonically stirred again (40 kHz, 5 min). The solution was allowed to stand at 45 °C three times to prepare a composite gel. The prepared sample was mixed with a 100 mg / mL sucrose aqueous solution instead of the GPG-KR_OD and OD solutions, and the mixture was designated as GPG-KR.

[0098] 1.4. Evaluation of GPG-KR / OD composite hydrogel 1.4.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) GPG-KR hydrogel and GPG-KR / OD composite hydrogel were dissolved in ultrapure water at 4 °C, and their absorbance at 280 nm was measured using a NanoDrop2000 (Thermo Fisher Scientific Inc., USA) to prepare 20 μM solutions. The molar absorptivity of both the GPG-KR hydrogel and the GPG-KR / OD composite hydrogel was 11380 dm. 3 / (mol·cm).

[0099] Mix 6g of sodium dodecyl sulfate (SDS; Wako Pure Chemical Industries, Ltd., Japan), 15mL of glycerol, 25mg of CBB R-250 (Wako Pure Chemical Industries, Ltd., Japan), 0.91g of tris(hydroxymethyl)aminomethane (SERVA Electrophoresis GmbH, Germany), and 40mL of ultrapure water. Adjust the pH to 7.0 with hydrochloric acid, then add ultrapure water in a final volume of 50mL. This mixture will serve as the Sample Buffer.

[0100] A mixture of 6 μL of Unstained Protein Standard, Broad Range (10-200 kDa) (New England Biolabs, Inc., UK) and 6 μL of Sample Buffer was used as a marker, and a mixture of 18 μL of ELP aqueous solution and 6 μL of Sample Buffer was used as a sample. The mixtures were incubated at 37 °C for 30 minutes. A pre-prepared polyacrylamide gel (e-PAGEL, ET 12.5L) for electrophoresis was then mounted in the electrophoresis tank. As the anode buffer, a 0.1 mol / L tris(hydroxymethyl)aminomethane solution adjusted to pH 8.9 with 1 mol / L hydrochloric acid was added to the electrophoresis tank. As the cathode buffer, 0.1 mol / L tris(hydroxymethyl)aminomethane, 0.1 mol / L tricine (Dojindo laboratories, Japan), and 1.0 w / v % SDS were added to the electrophoresis tank. After loading the markers and samples into the wells of the gel, electrophoresis was performed at 35 mA for approximately 30 minutes. The electrophoresis tank used was an AE-6500 Rapidus mini-plate electrophoresis tank (ATTO CO., Japan). The gel was then removed and immersed in a mixed solution of 250 mL methanol (Kanto Chemical Co., Inc., Japan), 50 mL acetic acid (Kanto Chemical Co., Inc., Japan), 0.5 g CBBR-250, and 200 mL ultrapure water for approximately 1 hour, mixing while inverting the container to achieve staining. The gel was then removed from the solution and immersed in a mixed solution of 180 mL methanol, 50 mL acetic acid, and 170 mL ultrapure water for approximately 2 hours to achieve destaining.

[0101] 1.4.2. Nuclear Magnetic Resonance Spectroscopy (NMR) The properties of hydrogels prepared in deuterated water were determined using a nuclear magnetic resonance spectrometer (Ascend 500·AVANCE NEO, Bruker., USA). 1 H NMR spectroscopy. At this time, a high-resolution magic angle sample rotation (HR-MAS) probe was used, and the cumulative number of times was set to 64.

[0102] 1.4.3. Shear Flowability and Recovery Behavior Test To investigate the recovery behavior of the prepared samples, shear flowability and recovery behavior tests were conducted using a rheometer (Modular Compact Rheometer MCR302, Anton Paar GmbH, Austria). Measurements were performed using a 1° cone plate (25 mm in diameter) at a gap of 0.048 mm. To prevent the samples from drying out during the measurements, Pro-Wipe wiping paper soaked in ultrapure water was placed around the measuring stage and covered with a solvent collection cap. The temperature was then maintained at 37°C using the rheometer's built-in Peltier system.

[0103] After the sample was loaded, in order to restore the structure damaged by the reduction of the fixture, a time dispersion measurement was performed for 1 hour at a fixed strain of 1% and a fixed frequency of 1Hz.

[0104] The recovery behavior of the gel was then evaluated by applying high shear strain (100%) for 60 seconds and low shear strain (0.5%) for 600 seconds in three cycles at a fixed frequency of 1 Hz.

[0105] 1.4.4. Overlap Shear Measurement To investigate the bioadhesion of the prepared specimens, overlap shear tests were performed. The test method followed ASTM standard F2255-05, using an aluminum mold for the test piece and pig small intestine as the adhesion substrate. The mold shape was as follows... Figure 1 As shown.

[0106] Thawed porcine small intestine (Tokyo Shibaura Organs Co., Ltd.) was washed with phosphate-buffered saline (PBS) and cut into 25mm × 30mm pieces. These pieces were then adhered to test pieces using a cyanoacrylate-based adhesive (Toa Synthetic Co., Ltd.). A gel, liquefied by vortexing, was spread 10mm wide on the distal portion of the small intestine, and the test pieces were adhered to each other with overlapping portions of the small intestine. The test pieces were then wrapped with PBS-soaked Pro-Wipe wipes, covered with plastic wrap, and incubated at 37°C for 2 hours. As a comparison, test pieces were also prepared using an existing tissue adhesive (Beriplast (registered trademark)) to adhere the small intestine.

[0107] Lap shear tests were performed using a tensile testing machine (Autograph AGS-50NX, Shimadzu Co., Ltd., Japan). Each specimen was tested three times at a crosshead speed of 5 mm / min. Test results were output as test force (N) and stroke (mm). The maximum test force was converted to shear strength (kPa) using the following formula. 1.4.5. In vivo gelation assay The gelation behavior of the sample in vivo was investigated using an in vivo assay. Black mice anesthetized with 4% isoflurane underwent a midline incision, and a gel (300 μL GPG-KR and 250 μL GPG-KR_OD) that had liquefied through vortex oscillation was injected into a Douglas bag. The mice were maintained in a supine position for 10 minutes after abdominal closure, and allowed free movement after recovery from anesthesia. An in vivo laparotomy was performed again 24 hours later to observe the process.

[0108] 2. Results 2.1. Evaluation of OD 2.1.1. FT-IR Spectroscopy Measurement The results of measuring the FTIR spectrum of the prepared sample are expressed as follows: Figure 2 The OD peak appearing near wavenumber 1730, which was not observed in dextran, originating from the CO double bond, confirms that a portion of the hydroxyl group was oxidized to an aldehyde group.

[0109] 2.1.2. HA-HCl Titration The prepared sample was titrated with HA-HCl, and the titration volume was 9.9 mL. Substituting this into the formula described in 1.2.1, the oxidation degree of OD is 79.9%.

[0110] 2.2. Evaluation of GPG-KR / OD composite hydrogel 2.2.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) The results of SDS-PAGE analysis on the prepared samples are presented as follows: Figure 3 The observations revealed staining bands in GPG-KR at the origin and near 20 kDa of the electrophoretic layer containing the loaded sample. The band at the origin originated from GPG nanofibers that self-assembled to form high molecular weight structures, while the staining band near 20 kDa originated from GPG-KR monomers with a mass of approximately 18 kDa. These staining bands were not observed in GPG-KR_OD. CBB binds to the amino groups of proteins, thus indicating the possibility of chemical bonds forming between the amino groups of GPG-KR and the OD in GPG-KR_OD.

[0111] 2.2.2. Nuclear Magnetic Resonance Spectroscopy (NMR) The results obtained from measuring the NMR spectrum of the prepared sample are expressed as follows: Figure 4 The measurement results confirmed that the peaks at ppm 3.0 and 1.7 for εCH2 and δCH2 of lysine residues from GPG-KR disappeared in GPG-KR_OD.

[0112] 2.2.3. Shear Flowability and Recovery Behavior Test The results of the recovery behavior test on the prepared sample are expressed as follows: Figure 5The results show that GPG-KR and GPG-KR_OD exhibit viscous behavior (G" being higher than G') under 100% high shear strain. However, if the high shear strain is released, the elastic behavior (G' exceeding G" again) is restored. In a 30 mg / mL OD solution, G' is consistently higher than G" and this behavior was not observed. The recovery rate of the storage modulus was calculated by dividing the value of the storage modulus before applying high shear strain by the value of the storage modulus after the third relaxation time. The calculation results show that the recovery rate is 12.9% in GPG-KR and 41.5% in GPG-KR_OD, indicating improved self-healing properties.

[0113] 2.2.4. Overlap Shear Measurement The results of the lap shear test on the prepared sample are expressed as follows: Figure 6 The test results show that GPG-KR_OD exhibits higher shear strength and improved bioadhesion compared to GPG-KR.

[0114] 2.2.5. In vivo gelation test The results of the In vivo gelation test on the prepared samples are presented as follows: Figure 7 No gelation of the GPG-KR sample was confirmed after 24 hours; the material disappeared. The GPG-KR_OD sample gelled after 24 hours, and was observed to adhere to the genitals.

Claims

1. A composition, characterized in that, contain: Includes (Sequence Number 1: X) 1 GGX 2 G) n The G sequence block formed by (sequence number 2: VPGX) 3 G) m The P-sequence block is an elastin-like block peptide sequence containing an amino group; and Water-soluble polymers containing aldehyde groups, Formula (Sequence Number 1: X) 1 GGX 2 G) n In the middle, X 1 Same or different, indicating V or L, X 2 Same or different, representing V or L, where n represents an integer greater than 4. Formula (Sequence Number 2: VPGX) 3 G) m In the middle, X 3 "Same" or "different" represents any amino acid, and "m" represents an integer greater than 5.

2. The composition according to claim 1, characterized in that: It is a gel-like or gel-forming sol-like composition.

3. The composition according to claim 1, characterized in that: The polypeptide contains amino acid residues with an amino group.

4. The composition according to claim 3, characterized in that: The polypeptide comprises a cross-linked sequence block consisting of two or more amino acid residues having an amino group.

5. The composition according to claim 1, characterized in that: The polypeptide contains cell-adhesive sequence blocks.

6. The composition according to claim 1, characterized in that: The water-soluble polymer contains sugars as structural units.

7. The composition according to claim 6, characterized in that: The water-soluble polymer is a polysaccharide oxide.

8. The composition according to claim 6, characterized in that: The water-soluble polymer is oxidized dextran and / or oxidized dextrin.

9. The composition according to claim 1, characterized in that: It comprises a linker of the polypeptide and the water-soluble polymer via covalent bonds.

10. A method for manufacturing a composition for producing any one of claims 1 to 9, characterized in that: This includes the step of (a) mixing the sol-gel composition of the gel-like composition with a water-soluble polymer having aldehyde groups. The sol-gel composition of the gel-like composition contains: Includes (Sequence Number 1: X) 1 GGX 2 G) n The G sequence block formed by (sequence number 2: VPGX) 3 G) m The P-sequence block is an elastin-like block peptide sequence containing an amino group. Formula (Sequence Number 1: X) 1 GGX 2 G) n In the middle, X 1 Same or different, indicating V or L, X 2 Same or different, representing V or L, where n represents an integer greater than 4. Formula (Sequence Number 2: VPGX) 3 G) m In the middle, X 3 "Same" or "different" represents any amino acid, and "m" represents an integer greater than 5.

11. A medical device, characterized in that: The composition comprising any one of claims 1 to 9.

12. The medical device as described in claim 11, characterized in that: Used for protection or adhesion of sutures, cut surfaces of organs or tissues, or damaged areas.

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