composition

Abstract

The purpose of the present invention is to provide a material that has higher strength and / or bioadhesion after self-repair and is obtained by using an elastin-like polypeptide.　Provided is a composition comprising: a polypeptide that has an amino group and comprises an elastin-like block peptide sequence including a G sequence block comprising (SEQ ID NO: 1:X1GGX2G)n (wherein: X1's are the same as or different from each other and each independently represent V or L; X2's are the same as or different from each other and each independently represent V or L; and n represents an integer of 4 or more) and a P sequence block comprising (SEQ ID NO: 2:VPGX3G)m (wherein X3's are the same as or different from each other and each independently represent an arbitrary amino acid residue, and m represents an integer of 5 or more); and a water-soluble polymer having an aldehyde group.

Description

composition

[0001] The present invention relates to a composition that can be used in medical devices for use in protecting internal sutures, cut surfaces of organs or tissues, damaged areas, etc.

[0002] In gastrointestinal anastomosis surgery, postoperative suture failure, resulting in leakage of digestive fluids, is a problem. It has been reported that suture failure occurs with a probability of 4-21% and can, in some cases, cause peritonitis and even death. Furthermore, the cut surfaces resulting from organ resections, such as pancreatectomy and hepatectomy, can leak blood, tissue fluids, and other bodily fluids. Therefore, it is necessary to protect the sutures and the cut surfaces with some kind of material.

[0003] Fibrin glue is sometimes used as a protective material. It is made by compounding fibrinogen with several drugs, such as thrombin, to create a paste, which is applied as a liquid to the target area. However, since fibrin glue is manufactured using plasma as a raw material, there are concerns about the risk of infection and unstable supply of raw materials.

[0004] In addition, nonwoven fabrics made of polyglycolic acid are sometimes used as protective materials (Non-Patent Document 1). However, since these are not liquid, they cannot be freely deformed to suit the object to be protected.

[0005] Journal of the American College of Surgeons. 2016; 222(1): 59-64International Journal of Molecular Sciences, 22, 4104(12 pages) (2021)

[0006] Non-Patent Document 2 reports that elastin-like polypeptides, created by simplifying the amino acid sequence of elastin, self-assemble into nanofibers when heated in water to form a hydrogel, and that this gel exhibits self-repairing properties (thixotropy), liquefying when shaken and re-gelling when left to stand. The inventors anticipated that the use of this elastin-like polypeptide would lead to the creation of a material that could be applied in liquid form to the area to be protected and gelled to reinforce the area to be protected while promoting healing. However, the strength and bioadhesion of gels made of elastin-like polypeptides alone after self-repair were insufficient.

[0007] An object of the present invention is to provide a material that has higher strength and / or bioadhesiveness after self-repair using elastin-like polypeptides.

[0008] In view of the above problems, the present inventors have conducted extensive research and have found that (SEQ ID NO: 1:X 1 GGX 2 G) n G sequence block consisting of 1 are the same or different and represent V or L, and X 2 are the same or different and represent V or L, and n represents an integer of 4 or more.) and (SEQ ID NO: 2: VPGX 3 G) m P sequence block consisting of 3 are the same or different and represent any amino acid, and m is an integer of 5 or greater. The inventors have found that the above-mentioned problems can be solved by a composition comprising a polypeptide having an amino group and including an elastin-like block peptide sequence containing an elastin-like block peptide sequence including an amino acid ...

[0009] Item 1. (SEQ ID NO: 1:X 1 GGX 2 G) n G sequence block consisting of 1 are the same or different and represent V or L, and X 2 are the same or different and represent V or L, and n represents an integer of 4 or more.) and (SEQ ID NO: 2: VPGX 3 G)m P sequence block consisting of 3 are the same or different and represent any amino acid, and m represents an integer of 5 or more. A composition comprising a polypeptide having an amino group and comprising an elastin-like block peptide sequence comprising:

[0010] Item 2. The composition according to Item 1, which is a gel or gel-forming sol composition.

[0011] Item 3. The composition according to Item 1, wherein the polypeptide comprises an amino acid residue having an amino group.

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

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

[0014] Item 6. The composition according to Item 1, wherein the water-soluble polymer contains a sugar as a constituent unit.

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

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

[0017] Item 9. The composition according to Item 1, comprising a conjugate between the polypeptide and the water-soluble polymer via a covalent bond.

[0018] Item 10. (a) (SEQ ID NO: 1:X 1 GGX 2 G) n G sequence block consisting of 1 are the same or different and represent V or L, and X 2 are the same or different and represent V or L, and n represents an integer of 4 or more.) and (SEQ ID NO: 2: VPGX 3 G) m P sequence block consisting of 3Item 10. A method for producing the composition according to any one of Items 1 to 9, comprising mixing a solated composition of a gel composition containing a polypeptide having an amino group and an elastin-like block peptide sequence comprising the elastin-like block peptide sequence containing the elastin-like block peptide sequence and an amino acid ...

[0019] Item 11. A medical device comprising the composition according to any one of Items 1 to 9.

[0020] Item 12. The medical device according to Item 11, for use in protecting or adhering sutures, cut surfaces of organs or tissues, or damaged areas.

[0021] According to the present invention, it is possible to provide a material that has higher strength and / or bioadhesiveness after self-repair using elastin-like polypeptides.

[0022] A schematic diagram of the aluminum test piece used for lap shear measurement in 1.4.4. is shown. FTIR spectrum measurement results in 2.1.1. are shown. SDS-PAGE results in 2.2.1. are shown. NMR spectrum measurement results in 2.2.2. are shown. Recovery behavior test results in 2.2.3. are shown. Lap shear measurement results in 2.2.4. are shown. In vivo gelation test results in 2.2.5. are shown.

[0023] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0024] In this specification, amino acid residues in an amino acid sequence may be simply represented by the amino acid or by a specific amino acid name (valine, leucine), etc. In addition, amino acid residues in an amino acid sequence may be represented by a single-letter code for the amino acid.

[0025] In one aspect, the present invention provides a method for the preparation of a nucleic acid sequence comprising the steps of: 1 GGX 2 G) n G sequence block consisting of 1 are the same or different and represent V or L, and X 2 are the same or different and represent V or L, and n represents an integer of 4 or more.) and (SEQ ID NO: 2: VPGX 3 G)m P sequence block consisting of 3 The present invention relates to a composition (sometimes referred to herein as the "composition of the present invention") comprising a polypeptide having an amino group and comprising an elastin-like block peptide sequence containing an elastin-like block peptide sequence comprising: (a) a polypeptide having an amino group containing an elastin-like block peptide sequence comprising: (b) a polypeptide having an amino group containing an elastin-like block peptide sequence comprising: (c) a polypeptide having an amino group containing an elastin-like block peptide sequence comprising: (a) a polypeptide having an amino group containing an elastin-like block peptide sequence comprising: (c ...

[0026] The G sequence block is an amino acid sequence shown in SEQ ID NO: 1 (X 1 GGX 2 G) repeat sequence (X 1 GGX 2 G) n There are no particular limitations on the G sequence block, as long as it is a block consisting of the following. The G sequence block is usually a block that can form a β-sheet structure. The G sequence block can impart the ability to form fibers by self-assembly to the polypeptide.

[0027] X 1 are the same or different and represent V or L, and X 2 are the same or different and represent V or L. X 1 and X 2 is preferably V.

[0028] n represents an integer of 4 or greater. By setting n to 4 or greater, the polypeptide of the present invention can form fibrous self-assemblies. n is preferably 4 to 20, more preferably 4 to 12, even more preferably 4 to 8, and even more preferably 4 to 6.

[0029] The P sequence block is the amino acid sequence shown in SEQ ID NO: 2 (VPGX 3 G) repeat sequence (VPGX 3 G) m There are no particular limitations as long as it is a block consisting of the P sequence block. The P sequence block is usually a block that can form a β-turn structure. The P sequence block gives the polypeptide a lower critical solution temperature (LCST), and at or above the LCST, it self-assembles and phase-separates from an aqueous solution.

[0030] The LCST of the polypeptide of the present invention (solvent: water, concentration: 0.03 wt%) is, for example, 10 to 30°C, preferably 15 to 25°C.

[0031] X 3 X represents any amino acid, whether identical or different. 3 Examples of the amino acid represented by the formula (X) include amino acids having a basic side chain such as lysine, arginine, and histidine; amino acids having an acidic side chain such as aspartic acid and glutamic acid; amino acids having an uncharged polar side chain such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acids having a nonpolar side chain such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acids having a β-branched side chain such as threonine, valine, and isoleucine; and amino acids having an aromatic side chain such as tyrosine, phenylalanine, tryptophan, and histidine. 3 The amino acid represented by is preferably an amino acid other than proline.

[0032] X 3 The LCST can be adjusted by the amino acid represented by X. 3 If the amino acid represented by X in the P sequence block is a hydrophilic amino acid, the LCST becomes too high, which is undesirable. From the viewpoint of easily adjusting the LCST to an appropriate (physiologically acceptable) temperature (e.g., 30 to 40°C), 3 It is preferred that some of the amino acids represented by are non-aromatic hydrophobic amino acids such as valine and leucine (preferably valine), and other amino acids are aromatic hydrophobic amino acids such as phenylalanine and tryptophan (preferably phenylalanine). In this case, X in the P sequence block 3 X to the total number of 3 The ratio of the number of non-aromatic hydrophobic amino acids represented by X in the P sequence block is, for example, 60 to 95%, preferably 70 to 80%, more preferably 75 to 85%. 3 X to the total number of 3The ratio of the number of aromatic hydrophobic amino acids represented by X in the P sequence block is, for example, 5 to 40%, preferably 20 to 30%, more preferably 15 to 25%. 3 It is preferable that the aromatic hydrophobic amino acids represented by the formula (VPGX) are distributed as widely as possible, for example, 2 to 8, preferably 3 to 7, more preferably 4 to 6, and even more preferably 5 repeat units. 3 G) at a rate of one for each iteration, x 3 It is preferred that repeat units in which the amino acid is an aromatic hydrophobic amino acid occur.

[0033] m represents an integer of 5 or greater. m is preferably 10 to 100, more preferably 10 to 50, even more preferably 15 to 35, and particularly preferably 20 to 30.

[0034] The elastin-like block peptide sequence is not particularly limited as long as it contains a G sequence block and a P sequence block. The number of G sequence blocks in the elastin-like block peptide sequence is not particularly limited, but is, for example, 1 to 5, preferably 2 to 5, more preferably 2 to 4, even more 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, but is, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 to 2.

[0035] The blocks (between a G sequence block and a P sequence block, between a G sequence block and a G sequence block, or between a P sequence block and a P sequence block) may be directly linked or may be linked via a linker sequence. Preferably, a linker sequence is interposed between the blocks.

[0036] The linker sequence is not particularly limited as long as it does not significantly reduce the fiber-forming ability of the elastin-like block peptide sequence due to its self-assembly, and generally, any amino acid or amino acid sequence can be used without major limitations. The linker sequence may have a length of, for example, 1 to 20 amino acids, preferably 1 to 15 amino acids, more preferably 1 to 10 amino acids, even more preferably 1 to 8 amino acids, and even more preferably 2 to 8 amino acids. Specific examples of linker sequences include the amino acid sequence represented by SEQ ID NO: 7: LWLGSG and the amino acid sequence represented by KL. Furthermore, amino acid sequences obtained by mutation (e.g., substitution, deletion, insertion, addition, preferably substitution, more preferably conservative substitution) of one or more amino acids (e.g., 1 to 5 amino acids, preferably 1 to 3 amino acids, more preferably 1 to 2 amino acids, and even more preferably 1 amino acid) of these amino acid sequences can also be used.

[0037] As used herein, the term "conservative substitution" refers to the substitution of an amino acid with an amino acid having a similar side chain. For example, substitution between amino acids having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitutions include substitution between amino acids having acidic side chains such as aspartic acid and glutamic acid; amino acids having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acids having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acids having β-branched side chains such as threonine, valine, and isoleucine; and amino acids having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0038] From the viewpoint of fiber-forming ability through self-assembly, the sequence structure of the elastin-like block peptide sequence is preferably, from the N-terminus, G sequence block-P sequence block-G sequence block, G sequence block-P sequence block-P sequence block-G sequence block, P sequence block-G sequence block, or G sequence block-P sequence block. The sequence structure is particularly preferably 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, "-" indicates a direct bond (between blocks) or another sequence (for example, a linker sequence, etc.).

[0039] The polypeptide of the present invention can have a cell adhesive sequence block at its terminus. The cell adhesive sequence block is not particularly limited, and various sequences can be used. The cell adhesive sequence block can be one that does not have cell selectivity, such as GRGDS (SEQ ID NO: 9) or RGDS (SEQ ID NO: 10), or one that has cell selectivity (e.g., REDV (SEQ ID NO: 22)). Sequences with cell selectivity include, for example, 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, G PS, 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, GEP, GN Examples include S, 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 (SEQ ID NO: 11), LPGFPGTP (SEQ ID NO: 12), GPPGLSGPP (SEQ ID NO: 13), FPGPPGPP (SEQ ID NO: 14), LPGLPGPP (SEQ ID NO: 15), FPGLPGPP (SEQ ID NO: 16), GPPGPPGSPG (SEQ ID NO: 17), LPGPPGPP (SEQ ID NO: 18), FPGSPGFPG (SEQ ID NO: 19), GSPGLPGTP (SEQ ID NO: 20), and IGLSGEKG (SEQ ID NO: 21).

[0040] When the polypeptide of the present invention comprises a cell adhesion sequence block, the cell adhesion sequence is located at the termini (N-terminus and / or C-terminus) of the polypeptide of the present invention, preferably at the C-terminus. The cell adhesion sequence may be located at either the N-terminus or the C-terminus, or at both. In a preferred embodiment of the present invention, the cell adhesion sequence is located at only one terminus.

[0041] When the polypeptide of the present invention contains a cell adhesive sequence block, the elastin-like block peptide sequence and the cell adhesive sequence may be directly linked, or another amino acid sequence (e.g., a sequence of 3 to 30, 7 to 25, or 10 to 20 amino acids) may be interposed between them. From the viewpoint of cell adhesiveness, this other amino acid sequence is preferably a hydrophilic amino acid-rich sequence. In other words, it is preferable that the hydrophilic amino acid-rich sequence is located on the opposite side of the terminal end of the cell adhesive sequence. The hydrophilic amino acid-rich sequence is not particularly limited as long as it contains a high proportion of hydrophilic amino acids, and examples include amino acid sequences of 3 to 15 or 5 to 10 amino acids in length that contain hydrophilic amino acids at a ratio of 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. A preferred hydrophilic amino acid-rich sequence is a histidine-stranded sequence (His tag sequence), which facilitates purification of the polypeptide of the present invention. The other amino acid sequence preferably contains an amino acid with a functional group that can be used in a crosslinking reaction (e.g., lysine, cysteine, etc.).

[0042] The polypeptide of the present invention has an amino group. The amino group may be one that is possessed by the amino acid that constitutes the polypeptide of the present invention, or may be one that is added by chemical modification. The polypeptide of the present invention preferably contains an amino acid residue that has an amino group. Examples of amino acids that have an amino group include lysine, arginine, and histidine. The amino acid preferably has a side chain of -NH 3 and particularly preferred is lysine.

[0043] It is believed that the amino group reacts with the aldehyde group of the water-soluble polymer described below to form a chemical bond, thereby covalently linking the polypeptide of the present invention to the water-soluble polymer, thereby further increasing the strength and / or bioadhesiveness after self-repair. The polypeptide of the present invention is known to form a gel with self-repairing properties, and there were concerns that linking another polymer to it would change the structure of the gel-forming fiber and impair its self-repairing properties. However, it was unexpected that the self-repairing properties were not impaired when the polypeptide of the present invention was combined with a water-soluble polymer.

[0044] When 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 a P sequence block or a linker), or may be present outside the elastin-like block peptide sequence. Preferably, the polypeptide of the present invention may contain a crosslinking sequence block containing an amino acid residue having an amino group, in addition to other sequence blocks. The crosslinking sequence block can further increase the strength and / or bioadhesion after self-repair.

[0045] The bridging sequence block preferably contains two or more, more preferably 2 to 10, even more 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 having an amino group to the total number of amino acid residues in the bridging sequence block is, for example, 20% or more, preferably 30% or more, more preferably 40% or more, and for example, 80% or less, preferably 70% or less, more preferably 60% or less. In one preferred embodiment, the bridging sequence block is KAAK (SEQ ID NO: 8).

[0046] When the polypeptide of the present invention contains a crosslinking sequence block, its position is not particularly limited. In one embodiment of the present invention, the crosslinking sequence block is preferably located on the C-terminal side of the elastin-like block peptide sequence, and when the polypeptide of the present invention contains a cell adhesive sequence block, the crosslinking sequence block is preferably located between the elastin-like block peptide sequence and the cell adhesive sequence block.

[0047] The polypeptide of the present invention may be chemically modified as long as its ability to form a thixotropic gel is not significantly impaired. The ability to form a thixotropic gel can be evaluated according to the Examples or previous reports.

[0048] The polypeptide of the present invention has a C-terminus containing a carboxyl group (-COOH), a carboxylate group (-COO － ), amide (-CONH2) or ester (-COOR).

[0049] Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1－6 Alkyl groups; for example, C groups such as cyclopentyl and cyclohexyl 3－8 Cycloalkyl groups such as phenyl and α-naphthyl 6－12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1－2 Alkyl group: α-naphthyl-C such as α-naphthylmethyl 1－2 C such as alkyl group 7－14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0050] In the polypeptide of the present invention, a carboxyl group (or carboxylate) other than that at the C-terminus may be amidated or esterified. In this case, the ester used may be, for example, the C-terminal ester described above.

[0051] Furthermore, in the polypeptide of the present invention, the amino group of the N-terminal amino acid residue is protected by a protecting group (e.g., a C group such as a formyl group or an acetyl group). 1－6 C such as alkanoyl 1－6 those in which the N-terminal glutamine residue that can be generated by cleavage in vivo is pyroglutamated; those in which the substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by an appropriate protecting group (e.g., C group such as formyl group, acetyl group, etc.); 1－6 C such as alkanoyl group 1－6Also included are those protected by an acyl group or the like, or conjugated proteins such as so-called glycoproteins to which sugar chains are bound.

[0052] The polypeptides of the present invention may be in the form of a pharmaceutically acceptable salt with an acid or base. The salt is not particularly limited as long as it is pharmaceutically acceptable, and both acidic and basic salts can be used. 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 paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0053] The polypeptide of the present invention may be in the form of a solvate. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0054] The polypeptides of the present invention can be easily produced according to or in accordance with known genetic engineering techniques or known chemical synthesis techniques, such as PCR, restriction enzyme cleavage, DNA ligation, in vitro transcription / translation, recombinant protein production, solid-phase synthesis, and peptide ligation.

[0055] The polypeptide of the present invention may be one type alone or a combination of two or more types.

[0056] The concentration of the polypeptide of the present invention in the composition of the present invention is, from the viewpoint of gel-forming properties, self-repairing properties, etc., for example, 0.2 w / v% or more, preferably 0.4 w / v% or more, more preferably 0.5 w / v% or more, and 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.

[0057] The water-soluble polymer is not particularly limited as long as it is a polymer that has an aldehyde group and is water-soluble.

[0058] The water solubility of the composition of the present invention is not particularly limited as long as it is water soluble to such an extent that the composition can be gelled using water as a solvent, and the solubility in 100 g of water at 23° C. is, for example, 1 g or more, preferably 2 g or more, more preferably 5 g or more, and even more preferably 10 g or more.

[0059] The viscosity average molecular weight of the polymer is not particularly limited, but is, for example, 5,000 to 300,000. From the viewpoint of strength after self-repair and / or bioadhesiveness, the viscosity average molecular weight is preferably 10,000 to 200,000, more preferably 15,000 to 150,000, even more preferably 20,000 to 100,000, still more preferably 25,000 to 80,000, particularly preferably 30,000 to 60,000, and particularly preferably 30,000 to 50,000. The viscosity average molecular weight can be measured by the most appropriate method depending on the type of polymer.

[0060] The number of aldehyde groups is not particularly limited, but is, for example, 0.1 to 100 per 1000 viscosity-average molecular weight of the polymer. The number is preferably 0.5 to 70, more preferably 1 to 50, even more preferably 2 to 30, even more preferably 3 to 20, and particularly preferably 5 to 15 per 1000 viscosity-average molecular weight of the polymer. The number of aldehyde groups can be measured by the most appropriate method depending on the type of polymer. For example, if the polymer is obtained by a method in which aldehyde groups are generated in sugar residues by oxidizing a polysaccharide, the number of aldehyde groups can be calculated by measuring the degree of oxidation.

[0061] The constituent units (constituent monomers) of the water-soluble polymer are not particularly limited. Preferably, the water-soluble polymer contains a sugar as a constituent unit. In this case, the number of sugars (sugar residues) per 100 constituent units is preferably 50 or more, more preferably 70 or more, even more 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 an oxide thereof.

[0062] The polysaccharide oxide can be produced according to or in accordance with a known method. For example, a polysaccharide oxide can be obtained by reacting a polysaccharide in the presence of a periodate.

[0063] There are no particular limitations on the type of sugar that is the structural unit, the polymer structure (linear or branched), or the type of bond between the structural units (for example, in the case of polysaccharides, α-bonds, β-bonds, the number of carbon atoms involved in bond formation, etc.). In a preferred embodiment of the present invention, the sugar that is the structural unit is glucose, the polymer structure is branched, and the type of bond between the structural units is α-bonds (particularly, α-1,6 bond and α-1,4 bond). The water-soluble polymer is particularly preferably oxidized dextran and / or oxidized dextrin.

[0064] The water-soluble polymer is preferably an uncharged water-soluble polymer.

[0065] The water-soluble polymer may be one type alone or a combination of two or more types.

[0066] From the viewpoint of gel-forming properties, self-repairing properties, etc., the concentration of the water-soluble polymer in the composition of the present invention is, for example, 0.5 w / v% or more, preferably 1.0 w / v% or more, more preferably 1.5 w / v% or more, even more preferably 2.0 w / v% or more, and for example, 10.0 w / v% or less, preferably 7.0 w / v% or less, more preferably 5.0 w / v% or less, even more preferably 4.0 w / v% or less.

[0067] The composition of the present invention may contain other components, such as cells, drugs, etc.

[0068] The composition of the present invention may be in either a dry or wet form, but is preferably a gel or gel-forming sol composition from the viewpoint of immediate use as a medical device, etc. In this case, for example, the composition of the present invention may comprise a conjugate between the polypeptide of the present invention and a water-soluble polymer via a covalent bond, and the polypeptide of the present invention can self-assemble to form nanofibers, and preferably the composition of the present invention comprises a conjugate between the nanofiber made of the polypeptide of the present invention and a water-soluble polymer via a covalent bond.

[0069] The composition of the present invention can be produced by various methods. From the viewpoint that a self-repairing gel or gel-forming sol composition of the present invention can be particularly easily obtained, the composition of the present invention is preferably produced by a method comprising mixing a sol of a gel composition containing the polypeptide of the present invention with a water-soluble polymer having an aldehyde group.

[0070] A gel-like composition containing the polypeptide of the present invention can be obtained by dissolving the polypeptide of the present invention in a solvent at a temperature below the LCST (e.g., 0-15°C, 2-10°C), heating the resulting solution to a temperature above the LCST (e.g., 30-50°C, 30-45°C, 30-40°C), and allowing it to stand for a certain period of time (e.g., 1-300 hours, preferably 8-120 hours, more preferably 12-80 hours). Water is used as the solvent, but a mixture of water and an organic solvent can also be used. It is also preferable that a monosaccharide such as sucrose (e.g., a final concentration of 2-20 w / v%, 5-15 w / v%) be added to the solvent.

[0071] A solated composition of a gel composition can be obtained by applying mechanical strain to the gel composition (for example, by pipetting, vortexing, ultrasonic agitation, etc.) The solated composition gels when the application of mechanical strain is removed or weakened, and therefore can be said to have gel-forming ability.

[0072] The water-soluble polymer having an aldehyde group is preferably dissolved in a solvent such as water before mixing, and the solvent preferably contains a monosaccharide such as sucrose (for example, a final concentration of 2 to 20 w / v%, 5 to 15 w / v%).

[0073] The ratio of the volume of the solated 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.

[0074] The method for mixing the solubilized composition and the water-soluble polymer is not particularly limited, and may be, for example, pipetting, vortexing, ultrasonic stirring, or the like.

[0075] After mixing, the composition of the present invention in a gel form can be obtained by leaving the mixture to stand for a certain period of time at a temperature equal to or higher than the LCST, as described above.

[0076] The composition of the present invention can be used for various purposes, but is particularly suitable for use as a medical device. Therefore, in one aspect, the present invention relates to a medical device (the medical device of the present invention) comprising the composition of the present invention.

[0077] A medical device is a machine, instrument, etc. that is intended to be used for the diagnosis, treatment, or prevention of disease in humans or animals, or to affect the structure or function of the human or animal body, and to that extent, there are no particular restrictions.

[0078] The target organisms are not particularly limited, and examples thereof include mammals such as humans, monkeys, dogs, cats, horses, cows, pigs, sheep, mice, rats, and rabbits, as well as vertebrates including these.

[0079] The composition of the present invention is in a gel state or can form a gel, and can be converted into a sol state by applying mechanical strain and can return to a gel state when the application of mechanical strain is stopped. Therefore, the composition of the present invention can be applied in a liquid state to an object to be protected, and when it gels, it is suitable for use as a material that reinforces the area to be protected and promotes healing. From this perspective, the composition of the present invention is preferably used in medical devices for protecting or adhering internal sutures, cut surfaces of organs or tissues, or damaged areas.

[0080] The sutured portion may be either inside or outside the body, but is preferably an inside sutured portion. More specifically, the sutured portion is, for example, a sutured portion of the digestive tract, blood vessels, nerves, bile duct, pancreatic duct, ureter, skin, etc., and is preferably a digestive tract sutured portion. Protection of the sutured portion is not particularly limited as long as it is applied to the sutured portion and its surroundings, but more specifically, it means physically reinforcing the sutured portion to prevent it from failing. This can suppress or prevent anastomotic failure.

[0081] The cut surface is the cut surface that is produced when a part of an organ or tissue is resected. Examples of organs or tissues include the pancreas, liver, kidney, blood vessels, lung, thyroid gland, and prostate. The protection of the cut surface is not particularly limited as long as it is applied to the cut surface so as to suppress or prevent leakage of body fluids from the cut surface. In one embodiment, the composition of the present invention is applied to the cut surface of an organ such as the spleen, and a nearby tissue (e.g., adipose tissue, intestinal tract, etc.) is attached thereto to form a patch on the cut surface.

[0082] The injured area may be either inside or outside the body. The location of the injured area corresponds to the sutured area and cut surface described above. There are no particular limitations on the protection of the injured area, as long as it is applied to the cut surface so as to suppress or prevent leakage of body fluids from the cut surface.

[0083] The composition of the present invention can be used by applying or spraying it in a sol form onto the object to be protected. The composition of the present invention can gel even under conditions where mechanical strain equivalent to that exerted by the movement of a living body is applied, and this excellent property allows it to remain on the object to be protected and to exert its protective role.

[0084] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0085] 1. Experimental Procedure 1.1. Preparation of Oxidized Dextran (OD) Dextran (Dextran 40,000 (viscosity average molecular weight 40,000), Wako Pure Chemical Industries, Ltd., Japan) was dissolved in 50 mL of ultrapure water, and 1.6 g of the oxidizing agent sodium periodate (Wako Pure Chemical Industries, Ltd., Japan) was added and stirred in a dark room for 3.5 hours. To stop the oxidation reaction, 0.6 mL of glycerol (Wako Pure Chemical Industries, Ltd., Japan) was added and stirred for an additional hour. A dialysis membrane (Spectra / Por®) was used. 1 The solution was placed in a dialysis membrane (MWCO: 6-8,000, Spectrum Laboratories, Inc., USA) and dialyzed against 2 L of ultrapure water at 4°C. The ultrapure water was changed three times every 24 hours. The solution was then frozen at -80°C and lyophilized using a freeze dryer (FDU-1200, Tokyo Rikakikai Co., Ltd., Japan) to obtain a powdered sample.

[0086] 1.2. Evaluation of OD FT-IR measurements were performed on dextran and the OD prepared in 1.1 using a Fourier transform infrared spectrometer (FT / IR-610, JASCO, Japan). Each sample was weighed out and mixed with potassium bromide (Wako Pure Chemical Industries, Ltd., Japan) in a ratio of 1:49 using a mortar and pestle. The resulting mixture was used as the measurement sample, and the spectrum was measured using the KBr method.

[0087] 1.2.1. Hydroxylamine Hydrochloride (HA-HCl) Titration To measure the degree of oxidation 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 0.25 M HA-HCl aqueous solution, 1 M NaOH aqueous solution, and 0.1 mg / mL methyl orange aqueous solution. 100 mg of OD was dissolved in 25 mL of HA-HCl aqueous solution and shaken at room temperature for 3 hours. A few drops of methyl orange aqueous solution were added as an indicator, and the solution was titrated with NaOH aqueous solution until the color matched that of the HA-HCl solution without OD dissolved in it. The degree of oxidation was calculated using the following equation.

[0088]

[0089] 1.3. Preparation of GPG-KR / OD Composite Hydrogel 1.3.1. Synthesis of Polypeptide (GPG-KR) The following polypeptide (GPG-KR): G sequence block consisting of (SEQ ID NO: 3: VGGVG)5 from the N-terminus (SEQ ID NO: 4: VPGXG) 25 A polypeptide (SEQ ID NO: 6) was synthesized, which comprises a polypeptide (SEQ ID NO: 5) containing an elastin-like block peptide sequence, in which a P sequence block consisting of (where X is the same or different and represents V or F) and a G sequence block consisting of (SEQ ID NO: 3: VGGVG)5 are linked via a linker sequence, and a cross-linking sequence (SEQ ID NO: 8: KAAK) and a cell adhesion sequence (SEQ ID NO: 9: GRGDS) are located at the C-terminus of the polypeptide. GPG-KR is a polypeptide with a previously reported amino acid sequence.

[0090] Specifically, E. coli BLR(DE3) was transformed with a plasmid DNA encoding the polypeptide, and the polypeptide was expressed. The polypeptide was purified by metal ion affinity chromatography using a His-tag. The desired polypeptide was confirmed by SDS-PAGE and MALDI-TOF-MS.

[0091] The recovered peptide solution was placed in a Spectra / Por® 1 Dialysis Membrane MWCO: 6-8,000 (Spectrum Laboratories) and dialyzed against 2 L of ultrapure water at 4°C. The ultrapure water was changed twice every hour, then twice every two hours, and once every 1.5 hours, for a total of five dialysis sessions. After dialysis, the peptide solution was filtered through a Minisart® Syringe Filter 0.2 μm (SartoriusStedim Biotech) attached to the syringe to remove insolubilized peptide, and then frozen overnight at -80°C. The resulting solution was then lyophilized using a freeze dryer FDU-1200 (TOKYO RIKAKIKAI) to obtain GPG-KR powder.

[0092] 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 solution. GPG-KR was added to this solution in an ice bath and completely dissolved by shaking at 4°C for 6 hours. 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 form a gel.

[0093] 1.3.3. Preparation of GPG-KR / OD Composite Hydrogel The OD prepared in 1.1 was dissolved in 100 mg / mL sucrose aqueous solution to prepare a 150 mg / mL OD solution. The GPG-KR gel prepared in 1.3.2 was liquefied by vortex shaking and subjected to ultrasonic agitation (40 kHz, 5 min). The two solutions were then mixed in a 4:1 ratio of GPG-KR solution:OD solution, and again subjected to ultrasonic agitation (40 kHz, 5 min). The solution was then left to stand at 45°C for 3 days to prepare a composite gel. The prepared sample is referred to as GPG-KR_OD, and the sample prepared by mixing 100 mg / mL sucrose aqueous solution instead of OD solution is referred to as GPG-KR.

[0094] 1.4. Evaluation of GPG-KR / OD Composite Hydrogels 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 20 μM solutions were prepared by measuring the absorbance at 280 nm using a NanoDrop2000 (Thermo Fisher Scientific Inc., USA). The molar extinction coefficients of both GPG-KR hydrogel and GPG-KR / OD composite hydrogel were 11,380 dm 3 / (mol cm).

[0095] A mixture of 6 g of sodium dodecyl sulfate (SDS; Wako Pure Chemical Industries, Ltd., Japan), 15 mL of glycerol, 25 mg of CBB R-250 (Wako Pure Chemical Industries, Ltd., Japan), 0.91 g of tris(hydroxymethyl)aminomethane (SERVA Electrophoresis GmbH, Germany), and 40 mL of ultrapure water was added, adjusted to pH 7.0 with hydrochloric acid, and then added with ultrapure water to bring the total volume to 50 mL. This was used as the sample buffer.

[0096] 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 18 μL of ELP aqueous solution and 6 μL of Sample Buffer was used as a sample. These were left to stand at 37°C for 30 minutes. A polyacrylamide precast gel, e-PAGEL (ET 12.5 L), for electrophoresis was installed in the electrophoresis chamber. The anode buffer was 0.1 mol / L tris(hydroxymethyl)aminomethane solution adjusted to pH 8.9 with 1 mol / L hydrochloric acid. The cathode buffer was 0.1 mol / L tris(hydroxymethyl)aminomethane, 0.1 mol / L tricine (Dojindo Laboratories, Japan), and 1.0 w / v% SDS. The marker and sample were applied to the gel wells and electrophoresed at 35 mA for approximately 30 minutes in an AE-6500 Rapidus Minislab Electrophoresis Chamber (ATTO Co., Japan). The gel was then removed and stained by immersing it in a mixture of 250 mL of methanol (Kanto Chemical Co., Inc., Japan), 50 mL of acetic acid (Kanto Chemical Co., Inc., Japan), 0.5 g of CBB R-250, and 200 mL of ultrapure water for approximately 1 hour, then inverting the container to mix. The gel was then removed from the solution and destained by immersing it in a mixture of 180 mL of methanol, 50 mL of acetic acid, and 170 mL of ultrapure water for approximately 2 hours.

[0097] 1.4.2. Nuclear Magnetic Resonance Spectroscopy (NMR) of Hydrogels Prepared in Deuterium Oxide 1 H nuclear magnetic resonance spectra were measured using a nuclear magnetic resonance spectrometer (Ascend 500 AVANCE NEO, Bruker, USA) with a high-resolution magic-angle spinning (HR-MAS) probe and 64 accumulations.

[0098] 1.4.3. Shear Flow and Recovery Behavior Tests To investigate the recovery behavior of the prepared samples, shear flow and recovery behavior tests were performed using a rheometer (Modular Compact Rheometer MCR302, Anton Paar GmbH, Austria). Measurements were performed using a 1° cone-plate (25 mm diameter) with a 0.048 mm gap. To prevent the sample from drying out during measurements, Prowipes soaked in ultrapure water were placed around the measurement stage and covered with a solvent trap cover. Furthermore, the temperature was maintained at 37°C using the Peltier system built into the rheometer.

[0099] After loading the sample, time dispersion measurements were performed for 1 hour at a constant strain of 1% and a constant frequency of 1 Hz to recover the structure destroyed by lowering the jig.

[0100] The recovery behavior of the gel was then evaluated by sequentially applying 60 s of high shear strain (100%) and 600 s of low shear strain (0.5%) for three cycles at a constant frequency of 1 Hz.

[0101] Lap shear measurements were performed to examine the bioadhesion of the prepared samples. The test method conformed to ASTM standard F2255-05, and an aluminum mold was used as the test specimen, and a pig small intestine was used as the adhesive substrate. The shape of the mold is shown in Figure 1.

[0102] Thawed porcine small intestine (Shibaura Organ Co., Ltd., Tokyo) was washed with phosphate-buffered saline (PBS) and cut into 25 mm x 30 mm pieces. These were then glued to test specimens using a cyanoacrylate adhesive (Toa Gosei Co., Ltd.). A 10 mm-wide gel liquefied by vortex mixing was applied to the distal end of the small intestine, and the test specimens were glued together so that the small intestine sections overlapped. The test specimens were then wrapped in Prowipes moistened with PBS, wrapped in plastic wrap, and left to stand at 37°C for 2 hours. For comparison, test specimens were also prepared in which the small intestine was glued using a conventional tissue adhesive (Beriplast®).

[0103] Lap shear measurements were performed using a tensile testing machine (Autograph AGS-50NX, Shimadzu Co., Ltd., Japan). Tests were performed three times for each sample at a crosshead speed of 5 mm / min. Test results were output in test force (N) and stroke (mm). The maximum test force was converted to shear strength (kPa) using the following formula:

[0104]

[0105] 1.4.5. In vivo gelation test The gelation behavior of the samples in vivo was investigated by an in vivo test. Black mice were anesthetized with 4% isoflurane and underwent a midline laparotomy, and gels (300 μL for GPG-KR, 250 μL for GPG-KR_OD) liquefied by vortex shaking were injected into the Douglas pouch. After abdominal closure, the mice were kept in a supine position for 10 minutes, and after waking from anesthesia, they were allowed to move freely. After 24 hours, the abdomen was reopened and the progress was observed.

[0106] 2. Results 2.1. Evaluation of OD 2.1.1. FT-IR Spectrum Measurement The results of measuring the FTIR spectrum of the prepared sample are shown in Figure 2. In the OD, a peak appeared at around 1730 wavenumber due to the CO double bond, which was not observed in dextran, confirming that some of the hydroxyl groups had been oxidized to aldehyde groups.

[0107] 2.1.2. HA-HCl titration The prepared sample was titrated with HA-HCl, resulting in a titer of 9.9 mL. By substituting this into the equation described in 1.2.1, the degree of oxidation of OD was found to be 79.9%.

[0108] 2.2. Evaluation of GPG-KR / OD Composite Hydrogels 2.2.1. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Figure 3 shows the results of SDS-PAGE performed on the prepared samples. For GPG-KR, stained bands were observed at the origin of the electrophoretic layer where the sample was loaded and at approximately 20 kDa. The band at the origin of the electrophoretic layer originates from GPG nanofibers that self-assembled to form high molecular weight molecules, while the stained band at approximately 20 kDa originates from GPG-KR monomers, which have a mass of approximately 18 kDa. These stained bands were not observed for GPG-KR_OD. Because CBB binds to the amino groups of proteins, this suggests that in GPG-KR_OD, the amino groups of GPG-KR may form chemical bonds with OD.

[0109] 2.2.2. Nuclear Magnetic Resonance Spectroscopy (NMR) The NMR spectrum of the prepared sample was measured and the results are shown in Figure 4. The measurement results confirmed that the peaks at 3.0 and 1.7 ppm, which are derived from the εCH2 and δCH2 of the lysine residues in GPG-KR, disappeared in GPG-KR_OD.

[0110] 2.2.3. Shear flow and recovery behavior tests Figure 5 shows the results of recovery behavior tests performed on the prepared samples. The measurement results showed that GPG-KR and GPG-KR_OD exhibited viscous behavior, with G" exceeding G', during 100% high shear strain. After the high shear strain was released, they returned to elastic behavior, with G' exceeding G" again. This behavior was not observed in the 30 mg / mL OD solution, where G' always exceeded G" . The recovery rate of the storage modulus was calculated by dividing the value of the storage modulus before the application of high shear strain by the value of the storage modulus after the third relaxation period. The calculated recovery rate was 12.9% for GPG-KR and 41.5% for GPG-KR_OD, indicating improved self-healing properties.

[0111] 2.2.4. Lap shear measurement The results of lap shear measurement of the prepared samples are shown in Figure 6. The measurement results showed that GPG-KR_OD exhibited higher shear strength than GPG-KR, indicating improved bioadhesion.

[0112] 2.2.5. In vivo gelation test The results of an in vivo gelation test on the prepared samples are shown in Figure 7. For GPG-KR, gelation of the sample was not confirmed after 24 hours, and the material had disappeared. For GPG-KR_OD, the sample had gelled after 24 hours, and adhesion to the genitals was observed.

Claims

1. (SEQ ID NO:1:X 1 GGX 2 G) n A G sequence block consisting of 1 are the same or different and represent V or L, and X 2 are the same or different and represent V or L, and n represents an integer of 4 or more.) and (SEQ ID NO: 2: VPGX 3 G) m P sequence block consisting of 3 and m is the same or different and represents any amino acid, and m is an integer of 5 or greater. A composition comprising a polypeptide having an amino group and an elastin-like block peptide sequence comprising the peptide sequence and a water-soluble polymer having an aldehyde group.

2. The composition according to claim 1, which is a gel or gel-forming sol composition.

3. The composition of claim 1, wherein the polypeptide comprises an amino acid residue having an amino group.

4. The composition of claim 3, wherein the polypeptide comprises a cross-linked sequence block containing two or more amino acid residues having an amino group.

5. The composition of claim 1, wherein the polypeptide comprises a cell adhesive sequence block.

6. The composition of claim 1, wherein the water-soluble polymer comprises a sugar as a building block.

7. The composition of claim 6, wherein the water-soluble polymer is a polysaccharide oxide.

8. The composition of claim 6, wherein the water-soluble polymer is oxidized dextran and / or oxidized dextrin.

9. The composition of claim 1, comprising a covalent linkage between said polypeptide and said water-soluble polymer.

10. (a) (SEQ ID NO:1:X 1 GGX 2 G) n A G sequence block consisting of 1 are the same or different and represent V or L, and X 2 are the same or different and represent V or L, and n represents an integer of 4 or more.) and (SEQ ID NO: 2: VPGX 3 G) m P sequence block consisting of 3 and m is an integer of 5 or more.) and a polypeptide having an amino group and including an elastin-like block peptide sequence comprising the amino acid sequence represented by the formula (I) and m is an integer of 5 or more.

11. A medical device comprising the composition according to any one of claims 1 to 9.

12. The medical device according to claim 11, for use in protecting or adhering sutures, cut surfaces of organs or tissues, or damaged areas.

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